WO2014205244A1

WO2014205244A1 - Methods for enhancing effectiveness of medical therapies

Info

Publication number: WO2014205244A1
Application number: PCT/US2014/043227
Authority: WO
Inventors: Marco DEMARIA; Judith Campisi
Original assignee: Demaria Marco; Judith Campisi
Priority date: 2013-06-19
Filing date: 2014-06-19
Publication date: 2014-12-24
Also published as: AR096673A1; TW201536324A

Abstract

Methods are provided herein for enhancing the effectiveness of medical therapies by administering agents that suppress a biological damage response comprising cellular senescence that is induced by the medical therapy administered to a subject. In certain embodiments, a method is provided for ameliorating toxicity of the medical therapy comprising administering an anti-senescent cell agent that destroys or facilitates destruction of the senescent cells. In certain embodiments, methods are provided for ameliorating toxicity of the medical therapy and for inhibiting metastasis of cancer in a subject who has cancer and receives the medical therapy by administering an anti-senescent cell agent that destroys or facilitates destruction of the senescent cells.

Description

METHODS FOR ENHANCING EFFECTIVENESS OF MEDICAL THERAPIES

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is

200201_416WO_SEQUENCE_LISTING.txt. The text file is 34 KB, was created on June 16, 2014 and is being submitted electronically via EFS-Web.

STATEMENT OF GOVERNMENTAL INTEREST

This invention was made with government support under Grant Nos AG09909 and AGO 17242 awarded by the National Institutes of Health. The

Government has certain rights in this invention.

BACKGROUND

Technical Field

Methods for ameliorating toxic side effects of medical therapies are provided herein. Agents used in these methods include agents that suppress a biological damage response.

Description of the Related Art

Cytotoxic and genotoxic therapies are administered to hundreds of thousands of patients each year for treatment of a variety of diseases, most notably, cancers. Cancer includes a broad range of diseases and affects approximately one in four individuals worldwide. In the United States, cancer is the second leading cause of death, accounting for 23% of all deaths. While the five-year relative survival rate for all cancers diagnosed is approximately 68%, treatments and their rates of success vary between cancer types. Even though chemotherapies and radiotherapies are designed to target cancer cells, the therapies can adversely affect normal cells and tissue to an extent that the beneficial effect of the cancer therapy can be significantly compromised. Highly active anti-retroviral therapy administered to men and women who are HIV infected and have developed AIDS has contributed to extending the lifespan and improving the general health of those infected. However, this therapy can also adversely affect normal cell physiology as well. BRIEF SUMMARY

Briefly, provided herein are methods for enhancing the effectiveness of a medical therapy by administering an agent that suppresses a biological damage response, including cellular senescence, which is inducible by the medical therapy. In one embodiment, methods are provided for ameliorating toxicity, such as acute toxicity, caused by a medical therapy in a subject who receives the medical therapy, which medical therapy induces cellular senescence of one or more cells in the subject, wherein the method comprises administering to the subject an agent that selectively destroys or facilitates selective destruction of the one or more senescent cells. In another embodiment, methods are provided for ameliorating toxicity and concomitantly inhibiting metastasis of a cancer in a subject who receives a medical therapy for treatment of the cancer and which medical therapy induces cellular senescence of one or more cells in the subject, which methods comprise administering to the subject an agent that selectively destroys or facilitates selective destruction of the one or more senescent cells. In another embodiment, the method further comprises administering to the subject a second agent that depletes one or more senescence cell-associated molecules produced by a senescent cell.

In one embodiment, a method is provided herein for ameliorating toxicity of a medical therapy in a subject who receives the medical therapy, which medical therapy induces cellular senescence of one or more cells in the subject, said method comprising administering to the subject an agent that selectively destroys or facilitates selective destruction of the one or more of the senescent cells induced by the medical therapy. In a particular embodiment, the toxicity is acute toxicity. In certain embodiments, the medical therapy induces cellular senescence of one or more normal cells. In another particular embodiment, the agent is administered to the subject at least 2 days, 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 10 days, or at least 14 days at least 30 days, at least 60 days, or at least 90 days subsequent to administration of the medical therapy. In a more specific embodiment, the agent is administered to the subject at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 10 days, or at least 14 days subsequent to administration of the medical therapy. In still another embodiment, the medical therapy comprises radiation, a chemotherapy, an anti-viral therapy, or a hormone. In other particular embodiments of the methods described above, the subject has a cancer, is in cancer remission, is at risk of developing a recurrence of a cancer, or has a predisposition for developing a cancer, and the medical therapy comprises an anti-cancer therapy. In a related embodiment, the medical therapy is chemotherapy or radiation and the subject has a cancer. In a specific embodiment, the cancer comprises a solid tumor and in other embodiments, the cancer comprises or a liquid tumor. In still another embodiment, the cancer is metastatic cancer. In yet another certain embodiment, when the subject has a cancer and has received or will receive a stem cell transplant, the medical therapy comprises high dose chemotherapy or high dose radiotherapy or a combination thereof, and in particular embodiments, the stem cell transplant is selected from (a) an autologous stem cell transplant, and (b) an allogenic stem cell transplant. In yet another embodiment, the medical therapy is an anti-viral therapy that is an HIV/AIDS management therapy, and in particular embodiments, the HIV/ AIDS management therapy comprises a highly active antiretro viral therapy (HAART). In still another specific embodiment, a method is provided for ameliorating acute toxicity of a medical therapy in a subject who receives the medical therapy, which medical therapy induces cellular senescence of one or more normal cells in the subject, said method comprising administering to the subject an agent that selectively destroys or facilitates selective destruction of the one or more senescent cells induced by the medical therapy. In certain embodiments of the methods described above and herein, the toxicity comprises acute toxicity comprising energy imbalance, and in a more particular embodiment, the energy imbalance comprises low physical activity. In still another embodiment, the toxicity comprises chronic toxicity. In yet other particular embodiments of the above- described methods, the agent specifically binds to a senescent cell associated antigen and inhibits a function of the antigen, wherein the agent specifically binds to a senescent cell associated antigen and inhibits a function of the antigen, thereby disrupting the integrity of the cell membrane, inhibiting one or more metabolic processes in the cell necessary for cell survival, or disrupting transcription of a gene or translation of a protein necessary for cell survival. In still other particular

embodiments, the agent induces apoptosis of the senescent cells. In yet another embodiment, the agent induces an immune response specific for the senescent cells and which immune response comprises removal of the senescent cells. In more particular embodiments, the agent is a small molecule, polypeptide, peptide, antibody, antigen- binding fragment, peptibody, recombinant viral vector, or a nucleic acid. In another embodiment, the method further comprises administering to the subject a second agent that depletes one or more senescence cell-associated molecules produced by a senescent cell. In a specific embodiment, the second agent is administered after administering the agent that selectively destroys or facilitates selective destruction of the one or more senescent cells induced by the medical therapy. In another specific embodiment, the second agent is administered concurrently with the agent that selectively destroys or facilitates selective destruction of the one or more senescent cells induced by the medical therapy. In still another specific embodiment, the second agent is administered prior to administering the agent that selectively destroys or facilitates selective destruction of the one or more senescent cells induced by the medical therapy. In more particular embodiments, the second agent is a small molecule, polypeptide, peptide, antibody, antigen-binding fragment, peptibody, recombinant viral vector, or a nucleic acid.

In a certain embodiment, a method is provided for inhibiting metastasis of a cancer and for ameliorating toxicity of a medical therapy in a subject who has the cancer and receives the medical therapy, which medical therapy induces cellular senescence of one or more cells in the subject, said method comprising administering to the subject an agent that selectively destroys or facilitates selective destruction of the one or more senescent cells, wherein the medical therapy comprises chemotherapy, radiation therapy, or both chemotherapy and radiation therapy. In another particular embodiment, the medical therapy induces cellular senescence of one or more normal cells. In still another particular embodiment, the toxicity is acute toxicity. In another embodiment, the method further comprises administering to the subject a second agent that depletes one or more senescence cell-associated molecules produced by a senescent cell. In a specific embodiment, the second agent is administered after administering the agent that selectively destroys or facilitates selective destruction of the one or more senescent cells induced by the medical therapy. In another specific embodiment, the second agent is administered concurrently with the agent that selectively destroys or facilitates selective destruction of the one or more senescent cells induced by the medical therapy. In still another specific embodiment, the second agent is administered prior to administering the agent that selectively destroys or facilitates selective destruction of the one or more senescent cells induced by the medical therapy. In more particular embodiments, the second agent is a small molecule, polypeptide, peptide, antibody, antigen-binding fragment, peptibody, recombinant viral vector, or a nucleic acid.

Also provided herein are uses for the agents described herein, which may be used to prepare medicaments for use in ameliorating toxicity and/or inhibiting metastasis of a cancer. Provided herein is a use of an agent that selectively destroys or facilitates selective destruction of one or more senescent cells for ameliorating toxicity of a medical therapy in a subject who receives the medical therapy, which medical therapy induces cellular senescence of one or more cells in the subject. In another embodiment, a use is provided for an agent that selectively destroys or facilitates selective destruction of one or more senescent cells for inhibiting metastasis of a cancer and for ameliorating toxicity of a medical therapy, which is administered to a subject who has a cancer and which medical therapy induces cellular senescence of one or more cells in the subject, wherein the medical therapy comprises chemotherapy, radiation therapy, or both chemotherapy and radiation therapy. In more specific embodiments of the uses described above and herein, the toxicity is acute toxicity. In still another specific embodiment, provided herein is a use of an agent that selectively destroys or facilitates selective destruction of one or more senescent cells for ameliorating acute toxicity of a medical therapy in a subject who receives the medical therapy, which medical therapy induces cellular senescence of one or more normal cells in the subject. In another embodiment, the use of an agent that selectively destroys or facilitates selective destruction of one or more senescent cells further comprises use of a second agent that depletes one or more senescence cell-associated molecules produced by a senescent cell. In a specific embodiment, the agent that selectively destroys or facilitates selective destruction of the one or more senescent cells induced by the medical therapy is for the preparation of a first medicament and the second agent is for the preparation of a second medicament, wherein the second medicament is suitable for administration after the administration of the first medicament. In still another embodiment, the agent that selectively destroys or facilitates selective destruction of the one or more senescent cells induced by the medical therapy is for the preparation of a first medicament and the second agent is for the preparation of a second medicament, wherein the first medicament and the second medicament are suitable for concurrent administration. In yet another specific embodiment, the agent that selectively destroys or facilitates selective destruction of the one or more senescent cells induced by the medical therapy is for the preparation of a first medicament and the second agent is for the preparation of a second medicament, wherein the second medicament is suitable for administration prior to the administration of the first medicament.

In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments. However, one skilled in the art will understand that the invention may be practiced without these details. In other instances, well-known structures have not been shown or described in detail to avoid unnecessarily obscuring descriptions of the embodiments. Unless the context requires otherwise, throughout the specification and claims which follow, the word "comprise" and variations thereof, such as, "comprises" and "comprising" are to be construed in an open, inclusive sense, that is, as "including, but not limited to." In addition, the term "comprising" (and related terms such as "comprise" or "comprises" or "having" or "including") is not intended to exclude that in other certain

embodiments, for example, an embodiment of any composition of matter, composition, method, or process, or the like, described herein, may "consist of or "consist essentially of the described features. Headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

Also, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a non-human animal" may refer to one or more non-human animals, or a plurality of such animals, and reference to "a cell" or "the cell" includes reference to one or more cells and equivalents thereof (e.g., plurality of cells) known to those skilled in the art, and so forth. When steps of a method are described or claimed, and the steps are described as occurring in a particular order, the description of a first step occurring (or being performed) "prior to" (i.e., before) a second step has the same meaning if rewritten to state that the second step occurs (or is performed) "subsequent" to the first step. The term "about" when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range may vary between 1% and 15% of the stated number or numerical range. It should also be noted that the term "or" is generally employed in its sense including "and/or" unless the content clearly dictates otherwise. The term, "at least one," for example, when referring to at least one compound or to at least one composition, has the same meaning and understanding as the term, "one or more."

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1 A and IB show radiation induces persistent senescent cells in pl6-3MR transgenic mice and that GCV treatment leads to depletion of senescent cells and reduction of the level of several SASP (senescence associated secretory phenotype) biomarkers. The transgenic pl6-3MR mice were mock irradiated (Ctrl) or irradiated (IR) (7 Gy whole body X-ray), housed for 3 months, and then treated with vehicle or GCV as described herein. Various tissues were isolated (results here shown are for lung tissue) and measured for rLUC bioluminescence (A) and the abundance of mRNAs encoding the l6INK4a, mRFP, IL-6 and MMP-3 proteins. Results are shown in arbitrary units (AU) after setting Ctrl levels at 1.

Figures 2A - 2C show senescent cells induced in pl6-3MR transgenic mice by irradiation promote primary and metastatic tumor growth. The transgenic pi 6- 3MR mice were mock-irradiated (Ctrl) or irradiated (IR). Three months later, the irradiated mice were treated with vehicle (IR) or GCV (IR+GCV), then injected with fLUC-expressing B16 melanoma cells into the tail veins. Fifteen days later, fLUC bioluminescence of the B16 melanoma cells was measured.

Figure 3 shows full body rLUC luminescence measurements of the B 16 melanoma cells from the mice in Figures 2A-2C. Irradiated mice were moribund at day 15-16 and were sacrificed.

Figures 4A - 4C show that elimination of senescent cells suppresses the development of metastases. The pl6-3MR transgenic mice of Figure 2 were followed for an additional three days (i.e., day 18). The irradiated mice treated with GCV (in which senescent cells were eliminated) eventually developed primary tumors in the lungs (A). But, despite the presence of primary tumors in the lung, the fat and liver tissues remained relatively metastasis free (C). In contrast, irradiated mice not treated with GCV (which retain senescent cells) showed metastatic tumors in the liver and fat tissue (B).

Figures 5A - 5B show that treatment with doxorubicin induces persistent senescent cells in pl6-3MR transgenic mice. The transgenic pl6-3MR mice were mock treated with vehicle (Ctrl) or treated with 10 mg/kg of doxorubicin (DOXO). Various tissues were isolated (liver, heart, lung, kidney, and spleen) and measured for abundance of mRNAs encoding mRFP (A) and pl6INK4a (B) (normalized to actin).

Figure 6 shows that doxorubicin induces persistent senescent cells in pl6-3MR transgenic mice and that GCV treatment leads to depletion of senescent cells and reducing the level of SASP biomarkers, pl6INK4 and mRFP. Skin biopsies were isolated and measured for abundance of l6INK4 and mRFP (normalized to actin).

Results are shown in arbitrary units (AU) after setting Ctrl levels at 1.

Figure 7 shows senescent cells induced in l6-3MR transgenic mice by doxorubicin treatment promoted primary tumor growth. The transgenic pl6-3MR mice were vehicle-treated (Ctrl) or treated with doxorubicin (10 mg/kg). 7 days later, the doxorubicin treated mice were mock treated with vehicle (DOXO) or GCV (DOXO +

GCV), then injected subcutaneously with fLUC-expressing B16 melanoma cells.

Twelve days later, bio luminescence of the B16 melanoma cells was measured.

Figure 8 shows that clearance of senescent cells in doxorubicin treated pi 6-3MR transgenic mice reduced tumor size. The transgenic p 16-3MR mice were vehicle-treated (Ctrl) or treated with doxorubicin (10 mg/kg). 7 days later, the doxorubicin treated mice were mock treated with vehicle (DOXO) or GCV (DOXO +

GCV), then injected subcutaneously with fLUC-expressing B16 melanoma cells.

Twelve days later, primary tumor diameter was measured.

Figure 9 shows that elimination of senescent cells suppresses the multiplicity of K-Ras induced lung tumors as compared to mice in which senescent cells were not cleared or reduced.

Figures 10A-10D provide a listing of an illustrative transgene selectively expressed in senescent cells, the nucleic acid sequence of a pBLUESCRIPT II KS vector containing a pl6^Ink4a promoter-FKBP-caspase-IRES-GFP nucleic acid construct

(SEQ ID NO: l).

Figure 1 lA-1 IF provide a listing of the nucleic acid sequences of Figure 10 with the various vector components and construct components labeled.

Figures 12A-12E show that corticosterone and Cortisol partially suppress the SASP. (A) Senescent X-irradiated with 10 Gy (Sen (XRA)) HCA2 fibroblasts were incubated in medium plus 10% serum containing the indicated concentrations of corticosterone or the highest concentration of DMSO (vehicle control). The cells were given corticosterone or DMSO immediately after irradiation and analyzed 6 days later. The cells were washed and incubated in serum- free medium without corticosterone to generate conditioned media. Conditioned media from pre-senescent (Pre) and control or corticosterone-treated Sen (XRA) cells were analyzed by ELISA for IL-6. (B) Cells were treated, and conditioned media were generated and analyzed as described in (A) except Cortisol was used at the indicated concentrations. (C) Conditioned media were collected from presenescent (PRE) or senescent (XRA) cells that were treated with DMSO, corticosterone (50 nM), or Cortisol (100 nM) as described in (A). The conditioned media were analyzed by antibody arrays. Average signal from PRE and XRA DMSO cells was used as the baseline. Signals higher than baseline are light gray (see +1 on scale to right); signals lower than baseline are dark gray as illustrated by PRE DMSO (see -1 on scale at right). Color intensities represent log₂-fold changes from the average value. The hierarchical clustering relationship between samples is shown as a dendrogram (left). ^Factors significantly (P < 0.05) suppressed by Cortisol. ^Factors significantly suppressed by corticosterone (P < 0.05). (D) Cells were infected with RAS- or MK 6-expressing lentiviruses. After selection, the cells were given DMSO-, 500 nM corticosterone (CI) and 100 nM Cortisol (C2) for 6 days. Conditioned media were generated as previously described and analyzed by ELISA for IL-6.

^Factors significantly different from DMSO treatment (P < 0.05). (E) Cells were treated with 500 nM corticosterone for the indicated intervals (a-d, indicated by the thick lines in the lower panel) before or after X-irradiation (XRA, indicated by the arrow). Conditioned media were prepared and analyzed by ELISA for IL-6 (upper panel). ^Factors significantly different from DMSO treatment (P < 0.05).

Figures 13A1-13G show the effect of glucocorticoids on the SASP depends on the glucocorticoid receptor (GR). (Al) mRNA was extracted from presenescent (Pre) or senescent (Sen (XRA)) HCA2 cells treated with DMSO, 500 nM corticosterone (C I) or 100 nM Cortisol (C2) as described in the legend to Figure 12. (A2) mRNA was extracted from presenescent (Mock) or senescent X-irradiated HCA2 cells treated with DMSO, 500 nM corticosterone, or 100 nM Cortisol as described in Figure 12. Transcripts for IL-5, IL-6, IL-8, MMP-3, IL-l , MCP-2, MCP-3, and GM- CSF were quantified by quantitative PCR (normalized to tubulin). ^Factors

significantly different from DMSO treatment (P < 0.05). (B) mRNA was extracted from Pre and Sen (XRA) cells treated with DMSO, 500 nM corticosterone (CI), or 100 nM Cortisol (C2) as previously described, and transcripts for GR were quantified by

PCR (normalized to tubulin). Although GR mRNA levels tended to be slightly elevated in senescent cells, the increase was not statistically significant. (C) Pre and Sen (XRA) cells treated with DMSO, 500 nM corticosterone, or 100 nM Cortisol as previously described were immunostained for GR 1, 4, and 7 days after X-irradiation. (D) Cells were infected with lentiviruses expressing shRNAs against GFP (control) or GR and selected. Seven days after selection, mRNA was extracted and transcripts for GR were quantified by PCR (normalized to tubulin). (E) Total cell lysates were prepared from the shGFP- and shGR-expressing cells described in (D) and analyzed by western blotting for GR and actin (control). (F) Cells infected with shGFP- or shGR-expressing lentiviruses were X-irradiated and treated immediately thereafter with DMSO, 500 nM corticosterone, or 100 nM Cortisol. Conditioned media were collected 7 days later and analyzed by ELISA for IL-6. (G) Cells were treated as described in (F) except for the addition of RU-486 at the indicated doses. Conditioned media were collected and analyzed by ELISA for IL-6 secretion.

Figures 14A-14C show that glucocorticoids repress IL-l expression. (A) Presenescent (Pre) HCA2 cells were treated with DMSO, 500 nM corticosterone, or 100 nM Cortisol for 24 hours or were induced to senesce by X-irradiation (Sen (XRA)) and given DMSO, corticosterone, or Cortisol immediately thereafter. mRNA was extracted after the indicated intervals, and transcripts for IL-l were quantified by PCR (normalized to tubulin). (B) mRNA extracted from cells described in (A) was used to quantify transcripts for IL-6 (normalized to tubulin). (C) Pre and Sen (XRA) cells, prepared as described in (A), were immunostained for IL-la. Sen (XRA) cells were immunostained 7 days after irradiation.

Figures 15A-15F show that glucocorticoids impair the IL-la/NF-κΒ pathway and suppress the ability of the SASP to induce tumor cell invasion. (A) Total HCA2 cell lysates were prepared from presenescent (Pre) cells, or senescent cells (Sen (XRA)) treated with DMSO, 500 nM corticosterone (CI), or 100 nM Cortisol (C2) in the absence (left panel) or presence (right panel) of recombinant IL-la protein (rIL-la). the lysates were analyzed by western blotting for IRAKI, ΙκΒα, RelA, and actin (control). (B) After irradiation, Sen (XRA) cells were given DMSO, 50 nM

corticosterone, or 100 nM Cortisol. Six days later, the cells were given recombinant IL- la protein at the indicated doses in the presence of the glucorticoids in serum free media. Conditioned media were collected 24 hours later and analyzed by ELISA for IL-6. (C) Nuclear extracts were prepared from Pre cells and Sen (XRA) cells treated with DMSO, 500 nM corticosterone (CI) or 100 nM Cortisol (C2) as described above, and analyzed for NF-κΒ DNA binding activity. (D) Cells were infected with a lentivirus carrying an NF-KB-luciferase reporter construct, irradiated, and allowed to senesce. Immediately after irradiation cells were treated with DMSO, 500 nM corticosterone, or 100 nM Cortisol, plus 0.5 μΜ RU-486 or 2.5 ng mL^"1 IL-la, as indicated. Seven days after irradiation, cells were trypsinized, counted, lysed, and assayed for luciferase activity, which was normalized to cell number. (E) Conditioned media from presenescent (Pre) or senescent (Sen (XRA)) cells that had been treated with corticosterone (CI) or Cortisol (C2) as described in Figure 12 were prepared. The conditioned media were then assayed for ability to stimulate T47D human breast cancer cells to invade a basement membrane, as described in the Experimental Procedures. (F) Nuclear extracts were prepared from Pre cells, and Sen (XRA) treated cells treated with DMSO, 500 nM corticosterone, or 100 nM Cortisol in the absence (left panel) or presence (right panel) of recombinant IL-l protein (rIL-l ) and analyzed for NF-KB DNA binding activity.

Figures 16A-16E: Figure 16A shows IMR-90 fibroblasts that were induced to senesce by X-irradiation (10 Gy; Sen (XRA)) and treated immediately after irradiation with the indicated concentrations of corticosterone or the highest concentration of DMSO (vehicle control) for 7 days. Conditioned media from presenescent (Pre) and the control and glucocorticoid-treated Sen (XRA) cells were analyzed by ELISA for IL-6. Figure 16B shows Sen (XRA) HCA2 cells that were treated with DMSO, 500 nM corticosterone (CI), or 100 nM Cortisol (C2) for 7 days. The percentage of presenescent (Pre) and Sen (XRA) cells that express SA-Bgal were scored (upper panel). A representative field corresponding to each condition is also shown (bottom panels). Figure 16C shows the Pre and Sen (XRA) HCA2 cells described in (B), given BrdU for 24 hours, fixed, and immunostained for nuclear BrdU staining, and then analyzed for the percentage of BrdU-positive cells. Figure 16D shows Pre and Sen (XRA) cells described in (B) immunostained for 53BP1. The percentage of cells with > 2 53BP1 nuclear foci was determined using CELL PROFILER software. At least 200 cells were analyzed per condition. Figure 16E shows the average number of 53BP1 foci from (D), determined using the CELL PROFILER software.

Figures 17A-B: Figure 17A shows presenescent (A) or Sen (Xra) HCA2 cells immunostained for the mineralocorticoid receptor. Sen (XRA) cells were given DMSO, 500 nM corticosterone, or 100 nM Cortisol immediately after irradiation and immunostained 1 or 7 days thereafter. Figure 17B shows Sen (XRA) HCA2 cells that were treated with DMSO, 500 nM corticosterone, or 100 nM Cortisol in the presence or not (-) of RU486, and immunostained for the GR.

Figure 18 mRNA extracted from Pre HCA2 cells treated with DMSO,

500 nM corticosterone, or 100 nM Cortisol for 24 hours and Sen (XRA) HCA2 cells treated with these compounds for 7 days starting immediately after X-irradiation.

mRNA extracts were analyzed for ΙκΒα transcripts by quantitative PCR (normalized to tubulin). The level of ΙκΒα mRNA in DMSO-treated Pre cells was arbitrarily assigned a value of 1.

Figure 19 shows that apigenin treatment partially suppresses SASP. Conditioned media from control (Mock irradiated, DMSO-treated), DMSO-treated (DMSO) or apigenin-treated senescent (Api) cells were analyzed by multiplex ELISA for expression of SASP. Results are shown as fold difference over control (Mock irradiated, DMSO-treated) cells with the vertical axis in log scale.

Figure 20 presents a schematic (top) of a mammary cancer animal model study in which pl6-3MR transgenic mice were injected with MMTV-PyMT cells, followed by treatment with doxorubicin (DOXO) and ganciclovir (GCV). Percent survival of animals was monitored over time (30 days) (bottom).

Figure 21 illustrates the quantity of tumor cells and the location of the tumor cells in pl6-3MR transgenic mice 28 days after injection with MMTV-PyMT cells, followed by treatment with doxorubicin (DOXO) or doxorubicin and ganciclovir (DOXO + GCV).

Figure 22 illustrates metabolic measurements obtained from pl6-3MR transgenic mice that were injected with MMTV-PyMT cells and then treated with (1) doxorubicin (DOXO) or (2) doxorubicin and ganciclovir (GCV) as shown in Figure 20 (top). The measurements were taken 18 days after injection with tumor cells.

* indicates p<0.05.

Figure 23 shows the behavior of the pl6-3MR transgenic mice that were injected with MMTV-PyMT cells and then treated with (1) doxorubicin (DOXO) or (2) doxorubicin and ganciclovir (GCV). The measurements were taken 28 days after injection with tumor cells. * * indicates p<0.01.

Figure 24 presents an exemplary 3MR transgene sequence.

DETAILED DESCRIPTION

Provided herein are methods for ameliorating toxic side effects of a medical therapy by administering an agent that suppresses a biological damage response that is inducible by the medical therapy, and which biological damage response includes induction and establishment of cellular senescence, including senescence of normal cells. Medical therapies, such as cancer chemotherapy, radiation treatment, hormone therapy, and various anti-viral therapies, are intended and designed to target aberrant or abnormal cells that cause the disease, which because of aberrant metabolism, proliferation, repair capacity and/or other physiological and biological properties are presumed to be more sensitive to these therapies. However, these medical therapies, particularly those that are administered systemically, act on normal cells resulting in cell damaging, cytotoxic, and/or genotoxic effects, including inducing cellular senescence. The biological response of the damaged normal cells and tissue to the medical therapy may result in a reduction in the effectiveness of the therapy to treat the underlying disease, for example, by promoting resistance to the medical therapy, producing undesired toxic effects, and/or by exacerbating the underlying disease. The biological response of normal cells that become senescent upon exposure to the medical therapy can cause toxic effects (i.e., also referred to in the art as side effects of the medical therapy) in the subject being treated. As described herein, the toxic effects resulting from induced senescence of normal cells is independent of proximity and interaction with cells targeted by the medical therapy (e.g., tumor cells when the medical therapy is chemotherapy or radiation). Such toxic effects may reduce incentive to initiate a medical therapy, or reduce compliance by the subject to continue the medical therapy, and/or contribute to acute or chronic medical conditions that affect the health and well-being of the treated subject.

In one embodiment, agents that suppress a biological damage response and that are useful in the methods described herein include agents (called herein anti- senescent cell agents) that selectively destroy (kill, clear, remove) one or more senescent cells or that facilitate selective destruction, killing, clearance, or removal of one or more senescent cells. Agents that selectively kill or facilitate selective killing of senescent cells are distinct from those that suppress production and secretion of one or more senescence cell-associated molecules (e.g., cytokines, chemokines, growth factors, and proteases) by senescent cells but that do not kill these cells. As exemplified herein, even after cellular senescence has been established in animals due to exposure to a cancer therapy, removal of senescent cells by an anti-senescent cell agent enhances the efficacy of the therapy to inhibit tumor progression, and significantly reduces metastatic disease, and reduces toxic effects associated with cellular senescence that is induced and established by exposure to the medical therapy.

In other embodiments, two agents that suppress a biological damage response may be used. One agent, such as an anti-senescent cell agent, selectively destroys (kills, clears, removes) one or more senescent cells or facilitate selective destruction, killing, clearance, or removal of one or more senescent cells. The other agent is capable of depleting one or more senescence cell associated molecules that are produced by senescent cells in a statistically or biologically significant manner. Agents that deplete one or more senescence cell associated molecules include those that suppress production and secretion of one or more senescence cell-associated molecules (e.g., cytokines, chemokines, growth factors, and proteases) by senescent cells but that do not kill these cells. These agents are described in greater detail herein.

Methods for Enhancing the Effectiveness of a Medical Therapy

By suppressing the biological damage response that is inducible by the medical therapy, the suppressive agents administered to a subject in need thereof provide enhancement (i.e., improvement) of the effectiveness (i.e., efficacy) of the medical therapy. Administration of an agent that suppresses a biological damage response inducible by the therapy results in an improvement or increase of the medical therapy's therapeutic and/or prophylactic benefit compared with the benefit observed in the absence of administering the agent. In certain embodiments described herein, enhancing the effectiveness of the medical therapy comprises suppressing the deleterious biological and physiological effects (e.g., toxic side effects, cancer metastasis) of the medical therapy.

Methods are provided for enhancing the effectiveness of a medical therapy in a subject who is in need thereof, which method comprises administering to the subject an agent capable of suppressing (i.e., reducing, decreasing, preventing, inhibiting, attenuating) a biological damage response that is inducible by exposure to the medical therapy. In one embodiment, the biological damage response comprises induction and establishment of senescence of cells, including induction and

establishment of senescence of normal cells. Methods are provided for ameliorating toxicity of the medical therapy by administering an agent that destroys or facilitates destruction of senescent cells. When the subject to be treated receives a medical therapy for the treatment of a cancer, methods are provided herein for concomitantly inhibiting metastasis of the cancer and for ameliorating toxicity of the medical therapy by administering an agent that destroys or facilitates destruction of senescent cells. Agents that suppress the biological damage response may be administered to the subject prior to administration of the medical therapy. In other embodiments, agents that suppress the biological damage response may be administered to the subject subsequent to administration of the medical therapy. In certain embodiments, agents may be administered concurrently with the medical therapy. Agents used in the methods described herein include, by way of example, a small molecule, polypeptide, peptide, antibody, antigen-binding fragment, peptibody, recombinant viral vector, or a nucleic acid.

A biological damage response that is inducible by a medical therapy includes a cellular, tissue-related, and/or systemic response of the subject, which response is induced upon exposure of the treated subject to the therapy. The biological damage response inducible by the medical therapies described herein includes, but is not limited to, cellular senescence. A biological damage response may also include a DNA damage response (also called herein and in the art, DDR) or a tumor-promoting response in a subject who has cancer, or combinations of cellular senescence, DDR, and a tumor-promoting response. Medical therapies that induce cellular senescence may induce senescence of normal cells. If the subject has a cancer, the medical therapy may induce senescence of normal cells and tumor cells. In certain instances, when medical therapies induce cellular senescence, the proportion of senescent cells in the subject is increased. Stated another way, the number of senescent cells in the subject is greater than would be present in the subject in the absence of receiving the medical therapy.

Agents useful for suppressing a biological damage response include agents that alter the activity or physiology of a senescent cell in a manner that blunts or reduces (suppresses) the biological damage response. In instances when the induced biological damage response comprises induction and establishment of cellular senescence, useful agents include an anti-senescent cell agent that suppresses the damage response by destroying or facilitating destruction (or clearance, killing, removal) of senescence cells. Accordingly, in a specific embodiment, methods are provided that comprise administering to the subject the medical therapy, which medical therapy induces senescence in one or more cells of the subject; and then administering to the subject an anti-senescent cell agent, which agent selectively destroys or facilitates the selective destruction of the one or more senescent cells. In certain other

embodiments, an anti-senescent cell agent and an agent that depletes one or more senescent cell associated molecules may both be used for suppressing a biological response.

Induction and establishment of cellular senescence by administration of a medical therapy to a subject may lead to toxic effects that provide little or no benefit to treating the underlying disease for which the subject is receiving the medical therapy (also called side effects, toxic side effects, adverse effects, deleterious side effects). As described herein, when a medical therapy induces senescence of cells, including senescence of normal cells, and the senescent cells are then removed (i.e., destroyed, killed), the toxic side effects of the medical therapy are ameliorated (i.e., mitigated, reduced, inhibited, prevented). In particular embodiments, the toxic side effects are acute toxic side effects. In the medical art, acute toxicity typically relates to toxic side effects caused by a medical therapy after a single exposure of the subject to (or administration of) the therapy or after multiple exposures to (or multiple administrations of) the therapy in a short amount of time (e.g., within 24 hours). An acute toxic effect may occur immediately upon administration of the medical therapy, within 4-24 hours, 1 -3 days, 1-5 days, 3-5 days, within 3-7 days, or in certain instances, within 3-14 or 7-14 days after exposure to the therapy. Removal or destruction of senescent cells, such as normal cells in which senescence has been induced by the medical therapy, herein ameliorates acute toxicity, including acute toxicity comprising energy imbalance, of the medical therapy. Acute toxic side effects include but are not limited to gastrointestinal toxicity (e.g., nausea, vomiting, constipation, anorexia, diarrhea), peripheral neuropathy, fatigue, malaise, low physical activity, hematological toxicity (e.g., anemia), hepatotoxicity, alopecia (hair loss), pain, infection, mucositis, fluid retention, dermato logical toxicity (e.g., rashes, dermatitis, hyperpigmentation, urticaria, photosensitivity, nail changes), mouth, gum or throat problems, or any toxic side effect caused by a medical therapy. For example, toxic side effects caused by radiotherapy or chemotherapy (see, e.g., National Cancer Institute web site) may be ameliorated by the methods described herein. Accordingly, in certain embodiments, methods are provided herein for ameliorating (reducing, inhibiting, or preventing occurrence (i.e., reducing the likelihood of occurrence)) acute toxicity or reducing severity of a toxic side effect (i.e., deleterious side effect) of a medical therapy in a subject who receives the medical therapy, which medical therapy induces cellular senescence of cells, including normal cells, in the subject, wherein the method comprises administering to the subject an agent that selectively destroys or facilitates selective destruction of senescent cells. In certain other embodiments, an anti-senescent cell agent and an agent that depletes one or more senescent cell associated molecules may both be used for suppressing a biological response.

In a more specific embodiment, the acute toxicity is an acute toxicity comprising energy imbalance and may comprise one or more of weight loss, endocrine change(s) (e.g., hormone imbalance, change in hormone signaling), and change(s) in body composition. In certain embodiments, an acute toxicity comprising energy imbalance relates to decreased or reduced ability of the subject to be physically active, as indicated by decreased or diminished expenditure of energy than would be observed in a subject who did not receive the medical therapy. By way of non- limiting example, such an acute toxic effect that comprises energy imbalance includes low physical activity. In other particular embodiments, energy imbalance comprises fatigue or malaise.

In other embodiments, methods are provided for ameliorating chronic or long term side effects. Chronic toxic side effects typically result from multiple exposures to or administrations of a medical therapy over a longer period of time.

Certain toxic effects appear long after treatment (also called late toxic effects) and result from damage to an organ or system by the medical therapy. Organ dysfunction (e.g., neurological, pulmonary, cardiovascular, and endocrine dysfunction) has been observed in patients who were treated for cancers during childhood (see, e.g., Hudson et al, JAMA 309:2371-81 (2013)). Without wishing to be bound by any particular theory, by destroying senescent cells, particular normal cells that have been induced to senescence by the medical therapy, the likelihood of occurrence of a chronic side effect may be reduced, or the severity of a chronic side effect may be reduced or diminished, or the time of onset of a chronic side effect may be delayed. Chronic and/or late toxic side effects that occur in subjects who received chemotherapy or radiation therapy include by way of non-limiting example, cardiomyopathy, congestive heart disease, inflammation, early menopause, osteoporosis, infertility, impaired cognitive function, peripheral neuropathy, secondary cancers, cataracts and other vision problems, hearing loss, chronic fatigue, reduced lung capacity, and lung disease.

Production and secretion of senescence cell-associated molecules, including senesnce cell associated polypeptides, may promote tumor progression and/or metastasis and contribute to or cause toxic side effects. Senescence cell-associated polypeptides include those described in greater detail herein and in the art that are components or molecules of a senescence associated secretory phenotype (SASP) of the senescent cell. In certain embodiments of the methods described herein, an agent useful for suppressing a biological damage response includes an agent (also called herein anti- senescent cell agents) that selectively destroys (or kills, removes, clears) one or more senescent cells or that facilitate selective destruction, killing, clearance, or removal of one or more senescent cells. Cell culture studies indicate that senescence is established between approximately 3 to 10 days after exposure to irradiation as evidenced by the time before a SASP was established (see, e.g., Coppe et al, PLoS Biol. 6:2853-68 (2008); Rodier et al, Nature Cell Biol. 11 :973-70 (2009); Laberge et al, Aging Cell l l(4):569-578 (2012). doi: 10.111 l/j. l474-9726.2012.00818.x. Epub 2012 Apr 17)). In particular embodiments, agents useful in the methods described herein include those capable of suppressing, inhibiting, eliminating, or reducing the biological damage response {e.g., cellular senescence) once it has been induced by exposure of cells and tissues to a medical therapy. Accordingly, in one embodiment, such an agent that suppresses this biological damage response is administered subsequent to

administration of the medical therapy. Without wishing to be bound by any particular theory, acute toxic effects associated with cellular senescence may occur during induction of cellular senescence by the medical therapy or after cellular senescence has been established or may occur during induction and after cellular senescence has been established. In certain other embodiments, an anti-senescent cell agent and an agent that depletes one or more senescent cell associated molecules, such as by inhibiting production and/or secretion of senescence cell-associated molecules, {e.g.,

polypeptides) may both be used for suppressing a biological response.

In one embodiment, methods are provided for enhancing the effectiveness of a medical therapy that is a cancer therapy {e.g., irradiation,

chemotherapy). As described herein, a biological damage response induced by cancer therapies, such as radiation and chemotherapy drugs, comprises cellular senescence. The presence of senescent cells promotes tumor progression, which may include promoting tumor growth and increasing size, promoting metastasis, and altering differentiation. When senescent cells are destroyed, tumor progression is significantly inhibited, resulting in tumors of small size and with little or no observed metastatic growth.

A biological damage response inducible by a medical therapy, which includes a cancer therapy {e.g., radiation or chemotherapy) includes expression of senescence cell-associated molecules. As described in the art and in greater detail herein, the phenotype of a senescent cell, such as the phenotype referred to as senescence associated secretory phenotype (SASP), is typified by secretion of numerous cytokines (e.g., inflammatory cytokines), growth factors, extracellular matrix components (ECM) and ECM-degrading enzymes, and proteases, for example. In certain instances, such as in a subject who has cancer, secretion of these factors may be deleterious, for example, by contributing to an undesirable inflammatory response or contributing to toxic side effects.

DNA damaging radiotherapy and chemotherapies can induce a SASP in vivo (see, e.g., Coppe et al, 2008, PLoS Biol. 6:2853-2868), which can have deleterious systemic effects, as well as the ability to stimulate the re-growth of tumor cells that were not eradicated by the anti-cancer therapy. Glucocorticoids suppressed some (e.g., IL-6, IL-l signaling), but not all (e.g., senescence growth arrest), of the factors that comprise the SASP. Glucocorticoids (e.g., corticosterone and Cortisol) may be clinically useful for conditions under which the SASP is thought to be harmful.

An agent that suppresses a biological damage response may be administered prior to (before), subsequent to (after), or concurrently with the medical therapy. In certain embodiments as described herein, when a biological damage response, such as cellular senescence, has occurred and, for example, contributes to or results in toxic side effects, an agent is administered to a subject subsequent to administration of a medical therapy, for example, the initial administration of the agent is at least 2, 3, 4, 5, 6, 7, 8, 10, 14, 30, 60, or at least 90 days or at least between 3-10 days, 10-30 days, 30-60 days or at least between 60-90 days after the subject receives the medical therapy. In a particular embodiment, the agent is administered between 2- 14 days (i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) after administration of the medical therapy. In more particular embodiments, the agent is administered at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 10 days, or at least 14 days subsequent to administration of the medical therapy. The number of doses of the agent will depend upon the physical characteristics of the agent and its biological activity as well as medical considerations, such as the health status of the subject (see additional detail herein). Agents (e.g., as anti-senescent cell agent) that selectively destroy or facilitate selective destruction of a senescent cell may be administered after administration of the medical therapy and, therefore, after induction and establishment of cellular senescence. With respect to a medical therapy regimen that includes more than one cycle of administration of the medical therapy, the agent may be administered after (subsequent to) one or more cycles of therapy, including after each cycle.

Alternative methods for mitigating a biological damage response of a medical therapy that includes induction and establishment of cellular senescence include use of agents that are capable of preventing or inhibiting cells from initiating a damage response or those that are capable of attenuating {i.e., reducing the severity of) the damage response upon exposure of the cells and tissue to a medical therapy. By way of example, these agents may inhibit or prevent induction and establishment of senescent cells, particularly inhibit or prevent induction and establishment of cellular senescence in normal cells. These agents may be administered prior to administration of the medical therapy. In certain embodiments, such an agent is administered for example, at least 1 day, at least 2-6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4-5 weeks, at least 6-8 weeks, or at least 10-12 weeks prior to administration of the therapy. With respect to a medical therapy regimen that includes more than one cycle of administration of the medical therapy, the agent may be administered before (prior to) one or more cycles of therapy, including before each cycle.

Concurrent administration of an agent described herein and the medical therapy meansthat the agent that facilitates destruction of a senescent cell is

administered within 1-24 hours of administration of the medical therapy. Concurrent therapy may comprise overlapping administration of the medical therapy and the agent. In a particular embodiment, the agent is administered concurrently with a portion of the medical therapy. By way of example, administration of the agent may be administered within 1-24 hours of administration of the medical therapy and the agent is continued to be administered after the course of the medical therapy has been completed. By way of example, the agent is first administered within 1-24 hours of administration of the medical therapy, and administration of the agent is continued for an additional 1-10, 2- 10, 3-10, 4-10, 5-10, 6-10, 7-10, 8-10, 9-10 or for longer than ten days (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 days). Stated another way, a portion of the agent which is at least part of the total amount of agent to be delivered is administered concurrently (within 1-24 hours) of administration of the medical therapy. In certain embodiments, the agent may be administered for an extended period of time during which cellular senescence {e.g., exemplified by SASP of the cell) is being established. This therapeutic regimen may result in one or more of the following: (1) prevent induction and thereby prevent establishment of senescence, (2) may destroy senescent cells as they are established, and (3) may alter the secretory phenotype of a senescence cell in a manner that significantly reduces the biological damage, that would otherwise occur in the absence of administering the agent. With respect to a medical therapy regimen that includes more than one cycle of administration of the medical therapy, the agent may be administered concurrent with one or more cycles of therapy, including concurrent administration with the medical therapy administration of each cycle.

Also contemplated herein are methods for enhancing the effectiveness of a medical therapy by administering at least two agents {i.e., two or more agents) that suppress a biological damage response that is inducible by a medical therapy. For convenience, the at least two agents may be called herein a first agent and a second agent and together may suppress a biological damage response in an additive manner or a synergistic manner.

In another embodiment when at least two agents that suppress a biological damage response inducible by a medical therapy are administered, at least one agent is an agent capable of preventing or inhibiting cells from initiating a damage response or is capable of attenuating {i.e., reducing the severity of) the damage response by cells and tissue when exposed to a medical therapy. Such agents include those described herein that prevent {i.e., inhibit; reduce the likelihood of occurrence) senescence of normal cells in the subject. Accordingly, in one embodiment, this agent is administered prior to administration of the medical therapy. At least one additional agent is an anti-senescent cell agent that selectively destroys one or more senescent cells or that facilitates selective destruction, killing, clearance, or removal of one or more senescent cells that exist as a result of exposure to the medical therapy. In one embodiment, the anti-senescent cell agent is administered subsequent to administration of the medical therapy.

A regimen that includes administration of at least two agents may be used when a subject is in need of several cycles of a medical therapy that is a biologically damaging therapy. In one embodiment, the medical therapy is a cancer therapy, such as radiation or chemotherapy or a combination of radiation and chemotherapy. In one embodiment, the agent that inhibits or reduces the likelihood of occurrence of senescence of normal cells in the subject is administered prior to administration of the medical therapy at a time sufficient to permit the agent to suppress the biological damage response, for example, at least 1 day, at least 2-6 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4-5 weeks, at least about 6-8 weeks, or at least about 10-12 weeks prior to administration of the therapy. The anti-senescent cell agent is initially administered at least 2, 3, 4, 5, 6, 7, 8, 10, 14, 30, 60, or at least 90 days after the subject receives the medical therapy, for example, at a time after which cellular senescence has been induced and established. The time points at which each of the first and second agents is administered will depend on the type of therapy and the length of time between each cycle of therapy. In certain particular embodiments, the agent that inhibits or reduces the likelihood of occurrence of senescence of normal cells is administered at least 1 day, at least 2-6 days, or at least about 1 week prior to administration of the medical therapy, and the anti-senescent cell agent is administered at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or 14 days after the subject receives the medical therapy depending on the time interval (also called the gap) between medical therapy cycles.

In another embodiment, methods are provided that comprise administering two agents that suppress a biological damage response (e.g., ameliorating toxicity and/or inhibiting a cancer), wherein one agent (herein called the first agent for convenience) facilitates selective destruction, killing, clearance, or removal of one or more senescent cells (i.e., an anti-senscent cell agent) that exist as a result of exposure to the medical therapy, and another agent (herein called a second agent for

convenience) that depletes one or more senescence cell-associated molecules. Without wishing to be bound by a particular theory, even though senescent cells are directly or indirectly killed or removed by the first agent, an undesired level of senescent cell- associated molecules may be present in the subject, or the medical therapy may continue to cause induction and establishment of senescent cells that causes increased levels of senescent cell-associated molecules, thus, the presence of the second agent contributes to the benefit of the subject by depleting the level of senescent cell- associated molecules.

In a specific embodiment, the second agent that depletes senescent cell- associated molecules is administered to a subject in need thereof after (i.e., subsequent to) administration of the agent that facilitates selective destruction, killing, clearance, or removal of senescent cells. By way of non-limiting example, subsequent to

administering the first agent (i.e., the agent that destroys or facilitates destruction of a senescent cell) according to methods described above, the second agent is administered at least 1 day, at least 2, 3, 4, 5, 6 days, or at least about 1 week, about 2 weeks, or about 3 weeks after administration of the first agent (i.e., the anti-senescent cell agent) is administered.

In another specific embodiment, the second agent that depletes senescent cell-associated molecules is administered to a subject in need thereof before (i.e., prior to) administration of the first agent that facilitates selective destruction, killing, clearance, or removal of senescent cells. By way of non-limiting example, prior to administering the first agent according to methods described above, the second agent is administered at least about 1 day, at least 2, 3, 4, 5, 6 days, or at least about 1 week, about 2 weeks, or about 3 weeks before administration of the first agent (i.e., the anti- senescent cell agent) is administered.

In still another specific embodiment, the first agent (i.e., the anti- senescent cell agent) and the second agent that depletes senescent cell-associated molecules are administered concurrently. Concurrent administration of the first agent and the second agent meansthat each agent is administered within 1-18 hours of administration of the other. In another embodiment, the first and second agents may be administered concurrently initially (i.e., overlapping administration) and then either the first agent or the second agent continues to be adminstered. By way of example, administration of the first and second agents may be administered within 1-24 hours of each other and then either the first agent or the second agent is continued to be administered. By way of example, the administration of the agent that is continued after concurrent administration of both the first and second agents may continue for an additional 1-10, 2-10, 3-10, 4-10, 5-10, 6-10, 7-10, 8-10, 9-10 or for longer than ten days (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 days).

Biological Damage Response

A biological damage response that is inducible (i.e., that is activated, promoted, or stimulated) by a medical therapy includes a cellular, tissue-related, and/or systemic response of the subject that is induced upon exposure of the treated subject to the therapy. The biological damage response that is inducible by the medical therapies described herein includes, but is not limited to, cellular senescence, a DNA damage response (also called herein and in the art, DDR), a tumor-promoting response, and combinations thereof.

A biological damaging response inducible by senescence-inducing medical therapies can cause epigenomic disruption or genomic damage. Eroded telomeres generate a persistent DDR, which initiates and maintains the senescence growth arrest. Many senescent cells also harbor genomic damage at nontelomeric sites, which can generate the persistent DDR signaling needed for the senescence growth arrest. A biological damaging response may comprise cellular senescence in the absence of detectable DDR signaling (see, e.g., Rodier et al, J. Cell Biol. 192:547-56 (2011), and references cited therein). Additionally, ectopic expression of the cyclin- dependent kinase inhibitors (CDKis) that normally enforce the senescence growth arrest, notably p21WAFl and pl6INK4a, may cause senescence without an obvious DDR.

In certain embodiments, normal cells, in addition to cells that the medical therapy is intended to target (e.g., tumor cells), are harmed or damaged by the medical therapy. For example, affected normal cells comprise the microenvironment around, adjacent to, or encompassing the cells and tissue that are the target(s) of the medical therapy. With respect to treating a cancer, medical therapies that comprise radiation or chemotherapy target the tumor cells; however, benign (normal) cells of the micro environment surrounding or adjacent to the tumor (which may be either solid or liquid tumor) may exhibit a medical therapy-induced damage response upon exposure to the therapy. In particular embodiments, regarding methods for ameliorating toxic effects of a medical therapy, normal cells that become senescent due to exposure to the medical therapy and that thereby cause toxic effects in the treated subject are not restricted to normal cells of the tumor microenvironment. As shown herein,

administration of a medical therapy that induces cellular senescence of normal cells causes toxic effects in the treated subject in the absence of tumor cells, and the toxic effects are eliminated or ameliorating by destroying these senescent cells.

Cellular senescence is a stable and essentially permanent arrest of cell proliferation, which is accompanied by extensive changes in gene expression. Many types of cells, both normal cells and tumor cells, undergo senescence in response to stress. As described in the art, the phenotype of a senescence cell, such as the phenotype referred to as senescence associated secretory phenotype (SASP), is typified by secretion of numerous cytokines (e.g., inflammatory cytokines), growth factors, extracellular matrix components (ECM) and ECM-degrading enzymes, and proteases, for example. While proliferative arrest poses a formidable barrier to tumor progression (see, e.g., Campisi, Curr. Opin. Genet. Dev. 21 :107-12 (2011); Campisi, Trends Cell Biol. 11 :S27-31 (2001); Prieur et al, Curr. Opin. Cell Biol. 20: 150-55 (2008)), and molecules secreted by senescent cells can stimulate tissue repair (see, e.g., Adams, Molec. Cell 36:2-14 (2009); Rodier et al, J. Cell Biol. 192:547-56 (2011)), senescent cells also secrete molecules that can cause inflammation (see, e.g., Freund et al, Trends Mol. Med. 16:238-46 (2010); Davalos et al, Cancer Metastasis Rev. 29:273-83 (2010)). Low-level, chronic inflammation is a hallmark of aging tissues, and inflammation is a major cause of, or contributor to, virtually every major age-related pathology, including cancer (Ferrucci et al, 2004, Aging Clin. Exp. Res. 16:240-243; Franceschi et al, 2007, Mech. Ageing Dev. 128: 192-105; Chung et al, 2009, Ageing Res. Rev. 8:18-30;

Davalos et al, 2010, Cancer Metastasis Rev. 29:273-283; Freund et al, 2010, Trends Molec. Med. 16:238-248). Thus, senescent cells, which increase with age and at sites of age-related pathology, might stimulate local chronic inflammation and tissue remodeling, thereby fueling both the degenerative diseases of aging as well as age- related cancer.

A senescent cell may exhibit any one or more of the following characteristics. (1) Senescence growth arrest is essentially permanent and cannot be reversed by known physiological stimuli. (2) Senescent cells increase in size, sometimes enlarging more than twofold relative to the size of nonsenescent

counterparts. (3) Senescent cells express a senescence-associated β-galactosidase (SAP-gal), which partly reflects the increase in lysosomal mass. (4) Most senescent cells express pl6INK4a, which is not commonly expressed by quiescent or terminally differentiated cells. (5) Cells that senesce with persistent DDR signaling harbor persistent nuclear foci, termed DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS). These foci contain activated DDR proteins and are distinguishable from transient damage foci. DNA-SCARS include dysfunctional telomeres or telomere dysfunction-induced foci (TIF). (6) Senescent cells express and may secrete molecules called herein senescent cell-associated molecules, which in certain instances may be observed in the presence of persistent DDR signaling, which in certain instances may be dependent on persistent DDR signaling for their expression. (7) The nuclei of senescent cells lose structural proteins such as Lamin Bl or chromatin-associated proteins such as histones and HMGB1. See, e.g., Freund et al, Mol. Biol. Cell 23:2066-75 (2012); Davalos et al., J. Cell Biol. 201 :613-29 (2013); Ivanov et al, J. Cell Biol. DOI: 10.1083/jcb.201212110, page 1-15; published online July 1, 2013; Funayama et al., J. Cell Biol. 175:869-80 (2006)).

Senescent cell-associated molecules include growth factors, proteases, cytokines {e.g., inflammatory cytokines), chemokines, cell-related metabolites, reactive oxygen species {e.g., H₂0₂), and other molecules that stimulate inflammation and/or other biological effects or reactions that may promote or exacerbate the underlying disease of the subject. Other senescent cell-associated molecules include extracellular polypeptides (proteins) described collectively as the DNA damage secretory program (DDSP) {see, e.g., Sun et al, Nature Medicine published online 5 Aug 2012;

doi: 10.1038/nm.2890). These groupings of senescent cell associated molecules, as described in the art, contain molecules in common and are not intended to describe three separate distinct groupings of molecules. Senescent cell-associated molecules include certain expressed and secreted growth factors, proteases, cytokines, and other factors that may have potent autocrine and paracrine activities (see, e.g., Coppe et al., supra; Coppe et al. J. Biol. Chem. 281 :29568-74 (2006); Coppe et al. PLoS One 5:39188 (2010); Krtolica et al. Proc. Natl. Acad. Sci. U.S.A. 98: 12072-77 (2001);

Parrinello et al, J. Cell Sci. 118:485-96 (2005). The presence of senescent cells can also be determined by detection of these senescent cell-associated molecules.

Without wishing to be bound by theory, the negative effects of senescent cells are believed to be the result of, at least in part, the secretion of pro-inflammatory cytokines, chemokines, growth factors, and proteases that comprise the SASP of a senescent cell (see, e.g., Coppe et al, PLoS Biol. 6:2853-68 (2008)). Senescent cell- associated molecules that comprise the SASP can disrupt normal tissue structure and function and stimulate malignant phenotypes in pre-malignant or non-aggressive cancer cells (see, e.g., Coppe et al, supra; Coppe et al. J. Biol. Chem. 281 :29568-74 (2006); Coppe et al. PLoS One 5 :39188 (2010); Krtolica et al. Proc. Natl. Acad. Sci. U.S.A.

98: 12072-77 (2001); Parrinello et al, J. Cell Sci. 118:485-96 (2005)). ECM associated factors include inflammatory proteins and mediators of ECM remodeling and which are strongly induced in senescent cells (see, e.g., Kuilman et al, Nature Reviews 9:81-94 (2009)). The factors that may have a paracrine effect on cells targeted by medical therapies, such as tumor cells, include extracellular proteins that have elevated expression in a cell after exposure to medical therapies that are genotoxic therapies (see, e.g., Sun et al, Nature Medicine, 18: 1359-1368 (2012)). See also, e.g., Campisi, 2003, Nature Rev. Cancer 3:339-349 Coppe et al., 2010, Annu. Rev. Pathol. 5:99-118

Williams, 1957 ^', Evolution 11 :398-411 Collado et al, 2010, Nature Rev. Cancer 10:51- 57 Beausejour et al, 2006, Nature 443 :404-405 Krizhanovsky et al. , 2008, Cell

134:657-667 Jun et al, 2010, Nature Cell Biol. 12:676-685 Parrinello et al, 2005, J. Cell Sci. 118:485-496 Acosta et al, 2008, Cell 133:1006-1018; Kuilman et al, 2008, Cell 133: 1019-1031 Laberge et al, 2012, Cancer Microenivron. 5:39-44.

Senescence cell-associated molecules include secreted factors which may make up the pro-inflammatory phenotype of a senescent cell (e.g., SASP). These factors include, without limitation, GM-CSF, GROa, GROa,p,y, IGFBP-7, IL-la, IL-6, IL-7, IL-8, MCP-1, MCP-2, MIP-la, MMP-1, MMP-10, MMP-3, Amphiregulin, ENA- 78, Eotaxin-3, GCP-2, GITR, HGF, ICAM-1, IGFBP-2, IGFBP-4, IGFBP-5, IGFBP-6, IL-13, IL-Ιβ, MCP-4, MIF, MIP-3a, MMP-12, MMP-13, MMP-14, NAP2, Oncostatin M, osteoprotegerin, PIGF, RANTES, sgpl30, TIMP-2, TRAIL-R3, Acrp30, angiogenin, Axl, bFGF, BLC, BTC, CTACK, EGF-R, Fas, FGF-7,G-CSF, GDNF, HCC-4, 1-309, IFN-γ, IGFBP-1, IGFBP-3, IL-1 Rl, IL-11, IL-15, IL-2R-a, IL-6 R, I- TAC, Leptin, LIF, MMP-2, MSP-a, PAI-1, PAI-2, PDGF-BB, SCF, SDF-1, sTNF RI, sTNF RII, Thrombopoietin, TIMP-1, tPA, uPA, uPAR, VEGF, MCP-3, IGF-1, TGF- β3, MIP-1 -delta, IL-4, FGF-7, PDGF-BB, IL-16, BMP-4, MDC, MCP-4, IL-10, TIMP- 1 , Fit-3 Ligand, ICAM- 1 , Axl, CNTF, INF-γ, EGF, BMP-6. Additional identified factors, which include those sometimes referred to in the art as senescence messaging secretome (SMS) factors, some of which are included in the listing of SASP

polypeptides, include without limitation, IGF1, IGF2, and IGF2R, IGFBP3, IDFBP5, IGFBP7, PA11, TGF-β, WNT2, IL-1 a, IL-6, IL-8, and CXCR2-binding chemokines. Cell-associated molecules also include without limitation the factors described in Sun et al, Nature Medicine, supra, and include, for example, products of the genes, MMP1, WNT16B, SFRP2, MMP12, SPINK1, MMP10, ENPP5, EREG, BMP6, ANGPTL4, CSGALNACT, CCL26, AREG, ANGPT1, CCK, THBD, CXCL14, NOV, GAL, NPPC, FAM150B, CST1, GDNF, MUCL1, NPTX2, TMEM155, EDN1, PSG9, ADAMTS3, CD24, PPBP, CXCL3, MMP3, CST2, PSG8, PCOLCE2, PSG7, TNFSF15, C17orf67, CALCA, FGF18, IL8, BMP2, MATN3, TFP1, SERPINI 1, TNFRSF25, and IL23A. Senescent cell-associated proteins also include cell surface proteins (or receptors) that are expressed on senescent cells, which include proteins that are present at a detectably lower amount or are not present on the cell surface of a non-senescent cell. Agents That Suppress a Biological Damage Response

Agents that suppress a biological damage response include agents that reduce or inhibit the damage response to the extent that a person skilled in the art recognizes that the suppression is statistically or clinically significant. Agents capable of suppressing a biological damage response include small molecules, polypeptides, peptides, peptibodies, antibodies, antigen binding fragments (i.e., peptides and polypeptides comprising at least one complementary determining region (CDR)), recombinant viral vectors, and nucleic acids.

An agent (which may also be called a therapeutic agent herein) that "selectively" destroys (or kills) or facilitates "selective" destruction (or killing) of a senescent cell is an agent that preferentially (or to a significantly greater degree) destroys or facilitates destruction or facilitates clearance or removal of a senescent cell. In other words, the agent destroys or facilitates destruction of a senescent cell in a biologically, clinically, and/or statistically significant manner compared with its capability to destroy or facilitate destruction of a non-senescent cell. By way of non- limiting example, the agent may directly or indirectly kill a senescent cell by any one or more of the following: disrupting the integrity of the cell membrane; inhibiting one or more metabolic processes in the cell; enhancing or stimulating a signaling pathway that leads to apoptosis or necrosis of the senescent cell; disrupting transcription or translation of genes or proteins, respectively, necessary for cell survival; or binding to the senescent cell to facilitate clearance or removal of the cell, for example, clearance by immune cells.

Agents of interest include those that are activated or that are pro-drugs which are converted to the active form by enzymes that are expressed at a higher level in senescent cells than in non-senescent cells. Other agents of interest include those that bind to proteins on the cell surface of a cell that are present exclusively or at a greater level on senescent cells compared with non-senescent cells. Examples of such proteins include mutant beta actin; beta-actin (ACTB ) protein; drug resistance-related protein LRP; major vault protein (MVP); thyroid hormone binding protein precursor; prolyl 4-hydroxylase, beta subunit precursor (P4HB); chain A, human protein disulfide isomerase (PDI); electron-transfer-flavoprotein, beta polypeptide (ETFP); ATP synthase, H+ transporting, mitochondrial F complex, alpha subunit precursor; cathepsin B; and unnamed protein products, GI: 35655, GI: 158257194; and GI 158259937 (see, e.g., Patent Application Publication No. WO 2009/085216, Table 1, which is incorporated herein by reference in its entirety). In certain embodiments, a therapeutic agent that specifically binds to a senescent cell has at least 2, 4, 8, 10, 50, 100, or 1000 fold greater affinity for a senescent cell than for a non-senescent cell, or in certain embodiments, the agent does not detectably bind to a non-senescent cell. Peptides that specifically bind to senescent cells include 12-amino acid peptides described in PCT Patent Application Publication No. 2009/085216. A protein present at a greater level on a senescent cell than on a non-senescent cell may be a protein that is typically an intracellular protein and not detectable on the cell surface of a non-senescent cell. Other agents that suppress a biological damage response that comprises cellular senescence include those activated by a metabolic process that occurs more frequently or at a higher rate in senescent cells than in non-senescent cells.

As described herein, agents that may be used in combination with an agent that directly or indirectly kills senescent cells include agents that deplete one or more senescent cell-associated molecules (e.g., senescent cell associated polypeptides) produced, expressed, and/or secreted by a senescent cell. Such agents are described in greater detail above. Depletion of one or more senescent cell-associated molecules or reducing the level of a senescent cell-associated molecule present in the subject may result from the capability of the agent to bind to the senescent cell-associated molecule and thereby inhibit or suppress one or more biological activities of the molecule. By way of non-limiting example, such an agent that binds a senescent cell associated molecule may inhibit or suppress interaction or binding to a cell surface receptor, which binding may inhibit triggering a signaling pathway that in the absence of the agent would contribute to the biological damage response. An agent that depletes senescent cell-associated molecules may bind to the molecule thereby forming a complex, resulting in uptake and the removal or excretion of the complex. The agent capable of depleting senescent cell-associated molecules may inhibit or suppress expression or secretion of the molecule from the senescent cell. An example of such an agent that depletes cell-associate molecules is an agent described herein that suppresses some senescent cell associated molecules or factors that comprise the SASP. DNA damaging radiotherapy and chemotherapies can induce a SASP in vivo {see, e.g., Coppe et al, 2008, PLoS Biol. 6:2853-2868), which can have deleterious systemic effects, as well as the ability to stimulate the re-growth of tumor cells that were not eradicated by the anti- cancer therapy. As described herein an example of such agents are glucocorticoids that suppressed some {e.g., IL-6, IL-l signaling), but not all {e.g., senescence growth arrest), of the factors that comprise the SASP. Reducing or decreasing the level of a senescent cell associated molecules would be understood by a person skilled in the art to mean that the level of the senescent cell associated molecules is sufficiently reduced to result in an observable decrease that has a beneficial biological effect to the subject being treated.

By way of example, agents that deplete senescence cell associated molecules include glucocorticoids, such as corticosterone and Cortisol, prednisone, androsterone; flavonoids (e.g., apigenin, luteolin, naringenin); tolazamide;

chlorpropamide; gliclazide; finasteride; norgestrel-(-)-D; estradiol- 17-beta; minoxidil; benfotiamine; calciferol; noscapine, and probucol. As described herein, the

glucocorticoids suppressed some (e.g., IL-6, IL-la signaling), but not all (e.g., senescence growth arrest), of the factors that comprise the SASP. By way of background, glucocorticoids are a class of steroid hormones that includes Cortisol, corticosterone, dexamethasone and related analogs, all of which have wide-ranging tissue-specific effects on metabolism and immune function (see, e.g., Gross and

Cidlowski, 2008, Trends Endocrinol. Metabl. 19:331-339; Zanchi et al, 2010, J. Cell. Physiol. 224:311-315) (see, e.g., Schlossmacher et al. , 2011, J. Endocrinol. 211 : 17-25). Glucocorticoids are believed to suppress inflammation by either inducing immune cell apoptosis, or by activating anti-inflammatory cytokines or repressing genes encoding pro-inflammatory cytokines, respectively. The latter activity is mediated by the ubiquitously expressed glucocorticoid receptor (GR), which exists in multiple isoforms and posttranslationally modified states (see, e.g., Zanchi et al, 2010, J. Cell. Physiol. 224:311-315; Oakley and Cidlowski, 2011, J. Biol. Chem. 286:3177-3184). As described herein, glucocorticoids (e.g., corticosterone and Cortisol) may be clinically useful for conditions under which the SASP is thought to be harmful. For example, as described in detail herein, DNA damaging radiotherapy and chemotherapies can induce a SASP in vivo (see, e.g., Coppe et al, 2008, PLoS Biol. 6:2853-2868), which can have deleterious systemic effects, as well as the ability to stimulate the re-growth of tumor cells that were not eradicated by the anti-cancer therapy.

Agents that suppress a biological damage response comprising cellular senescence include agents described herein that directly or indirectly inhibit secretion and/or expression of a gene product that is important for senescence or that inhibit a biological activity of the gene product. Inhibition of the expression or secretion of the gene product may in turn lead to the death of the senescent cell. Examples of these gene products are provided in the Table below; see also Sun, et al, supra.

Agents that may be used in the methods described herein include, but are not limited to, small organic molecules that suppress a biological damage response, including suppressing cellular senescence. Such small molecules include those that destroy or facilitate destruction or removal or clearance of a senescent cell. Agents that deplete one or more senescent cell-associated molecules also include small molecules. A small molecule compound of interest may be derivatized, either randomly or by SAR, to obtain compounds with improved activity. Small organic molecules typically have molecular weights less than 10⁵ daltons, less than 10⁴ daltons, or less than 10³ daltons.

An agent useful in the methods described herein for enhancing the effectiveness of a medical therapy, which may include ameliorating toxicity of a medical therapy, includes an antibody, or antigen-binding fragment. An antigen- binding fragment may be a fragment prepared from a whole antibody. An antigen- binding fragment also includes a peptide or polypeptide that comprises at least one complementary determining region (CDR). Useful antibodies and antigen-binding fragments include those that specifically bind to a cognate antigen that is overly expressed, selectively expressed, or only expressed by senescent cell compared with a non-senescent, normal cell. The antibody may be an internalising antibody or antigen- binding fragment that is internalized by the senescent cell via interaction with its cognate antigen. An internalizing antibody or antigen-binding fragment may be useful for delivering a cytotoxic agent to the senescent cell. Antibodies that bind to a senescence cell-associated antigen present on the cell surface of a senescent cell may facilitate destruction of the senescent cell when the senescent cell-bound antibody is recognized and bound by an immune cell that removes the senescent cell.

Binding properties of an antibody to its cognate antigen may generally be determined and assessed using immunodetection methods including, for example, an enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, immunoblotting, countercurrent Immunoelectrophoresis, radioimmunoassays, dot blot assays, inhibition or competition assays, and the like, which may be readily performed by those having ordinary skill in the art (see, e.g., Harlow et al, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988)). As used herein, an antibody is said to be "immunospecific," "specific for" or to "specifically bind" to a cognate antigen if it reacts at a detectable level with the antigen or immunogen. Affinities of antibodies and antigen binding fragments thereof can be readily determined using conventional techniques, for example, those described by Scatchard et al. (Ann. N. Y. Acad. Sci. USA 51 :660 (1949)) and by surface plasmon resonance (SPR; BIAcore™, Biosensor, Piscataway, NJ).

The antibodies may be polyclonal or monoclonal, prepared by immunization of animals and subsequent isolation of the antibody, or cloned from specific B cells according to methods and techniques routinely practiced in the art and described herein. A variable region or one or more complementarity determining regions (CDRs) may be identified and isolated from antigen-binding fragment or peptide libraries. An antibody, or antigen-binding fragment, may be recombinantly engineered and/or recombinantly produced.

An antibody may belong to any immunoglobulin class, for example IgG, IgE, IgM, IgD, or IgA and may be obtained from or derived from an animal, for example, fowl (e.g., chicken) and mammals, which include but are not limited to a mouse, rat, hamster, rabbit, or other rodent, a cow, horse, sheep, goat, camel, human, or other primate. For use in human subjects, antibodies and antigen-binding fragments are typically human, humanized, or chimeric to reduce an immunogenic response by the subject to non-human peptides and polypeptide sequences.

The antibody may be a monoclonal antibody that is a human antibody, humanized antibody, chimeric antibody, bispecific antibody, or an antigen-binding fragment (e.g., F(ab')₂, Fab, Fab', Fv, and Fd) prepared or derived therefrom. An antigen-binding fragment may also be any synthetic or genetically engineered protein that acts like an antibody in that it binds to a specific antigen to form a complex. For example, antibody fragments include isolated fragments consisting of the light chain variable region, Fv fragments consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules (scFv proteins), and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region. In certain other embodiments, antibodies are multimeric antibody fragments such as miniantibodies, bispecific and bifunctional antibodies comprising a first Fv specific for an antigen associated with a second Fv having a different antigen specificity, and diabodies and the like. Useful methodologies are described generally, for example in Hayden et al., Curr Opin. Immunol. 9:201-12 (1997); Coloma et al., Nat. Biotechnol. 15:159-63 (1997); U.S. Patent No. 5,910 573); Holliger et al, Cancer Immunol. Immunother. 45: 128-30 (1997); Drakeman et al., Expert Opin. Investig.

Drugs 6:1169-78 (1997); Koelemij et al, J. Immunother. 22:514-24 (1999); Marvin et al, Acta Pharmacol. Sin. 26:649-58 (2005); Das et al, Methods Mol. Med. 109:329-46 (2005).

A minimal recognition unit or other antigen binding fragment may be identified from a peptide library. Such peptides may be identified and isolated from combinatorial libraries {see, e.g., International Patent Application Nos.

PCT/US91/08694 and PCT/US91/04666) and from phage display peptide libraries (see, e.g., Scott et al, Science 249:386 (1990); Devlin et al, Science 249:404 (1990); Cwirla et al, Science 276: 1696-99 (1997); U.S. Pat. No. 5,223,409; U.S. Pat. No. 5,733,731; U.S. Pat. No. 5,498,530; U.S. Pat. No. 5,432,018; U.S. Pat. No. 5,338,665; 1994; U.S. Pat. No. 5,922,545; International Application Publication Nos. WO 96/40987 and WO 98/15833). A peptide that is a minimal recognition unit or a CDR (i.e., any one or more of the three CDRs present in a heavy chain variable region and/or one or more of the three CDRs present in a light chain variable region) may be identified by computer modeling techniques, which can be used for comparing and predicting a peptide sequence that will specifically bind to a polypeptide of interest as described herein (see, e.g., Bradley et al, Science 309:1868 (2005); Schueler-Furman et al, Science 310:638 (2005)).

Antibodies may generally be prepared by any of a variety of techniques known to persons having ordinary skill in the art. Immunogens used to immunize animals and/or to screen for antibodies of desired specificity include proteins isolated from senescent cells that, for example, are present on the cell surface of a senescent cell in greater quantity or having a different conformation than on a non-senescent cell; and senescent cell extracts, including outer membrane preparations, organelles isolated from senescent cells, and the like. Antibodies may also be identified and isolated from human immunoglobulin phage libraries, from rabbit immunoglobulin phage libraries, from mouse immunoglobulin phage libraries, and/or from chicken immunoglobulin phage libraries (see, e.g., Winter et al, Annu. Rev. Immunol. 12:433-55 (1994); Burton et al, Adv. Immunol. 57:191-280 (1994); U.S. Patent No. 5,223,409; Huse et al, Science 246: 1275-81 (1989); Schlebusch et al, Hybridoma 16:47-52 (1997) and references cited therein; Rader et al, J. Biol. Chem. 275: 13668-76 (2000); Popkov et al, J. Mol. Biol. 325:325-35 (2003); Andris-Widhopf et al, J. Immunol. Methods 242: 159-31 (2000)). Antibodies isolated from non-human species or non-human immunoglobulin libraries may be genetically engineered according to methods described herein and known in the art to "humanize" the antibody or fragment thereof.

Useful strategies for designing humanized antibodies may include, for example by way of illustration and not limitation, identification of human variable framework regions that are most homologous to the non-human framework regions of a chimeric antibody (see, e.g., Jones et al, Nature 321 :522-25 (1986); Riechmann et al, Nature 332:323-27 (1988)). A humanized antibody may be designed to include CDR loop conformations and structural determinants of non-human variable regions, for example, by computer modeling, and then comparing the CDR loops and determinants to known human CDR loop structures and determinants (see, e.g., Padlan et al., FASEB 9: 133-39 (1995); Chothia et al, Nature, 342:377-83 (1989)). Computer modeling may also be used to compare human structural templates selected by sequence homology with the non-human variable regions.

Agents such as polypeptides, peptides, peptibodies, antibodies, and antigen binding fragments (i.e., peptides or polypeptides comprising at least one antibody V region) or other agents that specifically to a senescent cell can be linked to (i.e., conjugated to, fused to, or in some manner joined to or attached to) a second agent that selectively destroys or facilitates selective destruction of senescent cells. When delivered to the senescent cell by binding of the agent to the senescent cell, the cytotoxic moiety selectively destroys the senescent cell. If the agent is recombinantly produced, a nucleotide sequence encoding the cytotoxic moiety may be linked in frame to the agent and to one or more regulatory expression sequences to produce a fusion protein comprising the agent and cytotoxic moiety. Such second agents include cytotoxic molecules, including toxins derived from plants and microorganisms, as well as small molecules do not selectively bind to senescent cells in the absence of being linked to the aforementioned antibody, polypeptide, or peptide.

An agent that suppresses a biological damage response includes a peptide-immunoglobulin (Ig) constant region fusion polypeptide, which includes a peptide-IgFc fusion polypeptide (also referred to in the art as a peptibody (see, e.g., U.S. Patent No. 6,660,843)). The peptide may be any naturally occurring or recombinantly prepared molecule. A peptide-Ig constant region fusion polypeptide, such as a peptide-IgFc fusion polypeptide, comprises a biologically active peptide or polypeptide capable of altering the activity of a protein of interest. The Fc polypeptide may also be a mutein Fc polypeptide. Peptides that alter a biological function of a cell, such as the immunoresponsiveness of an immune cell, may be identified and isolated from combinatorial libraries (see, e.g., International Patent Application Nos.

PCT/US91/08694 and PCT/US91/04666) and from phage display peptide libraries (see, e.g., Scott et al, Science 249:386 (1990); Devlin et al, Science 249:404 (1990); Cwirla et al, Science 276: 1696-99 (1997); U.S. Pat. No. 5,223,409; U.S. Pat. No. 5,733,731; U.S. Pat. No. 5,498,530; U.S. Pat. No. 5,432,018; U.S. Pat. No. 5,338,665; 1994; U.S. Pat. No. 5,922,545; International Application Publication Nos. WO 96/40987 and WO 98/15833).

In certain embodiments, an agent that suppresses a biological damage response (e.g., ameliorating toxicity of a medical therapy) is a polynucleotide or oligonucleotide that specifically hybridize to a portion of the genome or mRNA of a cell that is a senescent cell or that is in a disease microenvironment and may be induced to senescence by a biologically damaging (i.e., cell damaging) medical therapy.

Polynucleotides and oligonucleotides are provided that are complementary to at least a portion of a nucleotide sequence encoding a senescent cellular polypeptide of interest (e.g., a short interfering nucleic acid, an antisense polynucleotide, a ribozyme, or a peptide nucleic acid) and that may be used to alter gene and/or protein expression. As described herein, these polynucleotides that specifically bind to or hybridize to nucleic acid molecules that encode a cellular polypeptide may be prepared using the nucleotide sequences available in the art. In another embodiment, nucleic acid molecules such as aptamers that are not sequence-specific may also be used to alter gene and/or protein expression.

Antisense polynucleotides bind in a sequence-specific manner to nucleic acids such as mRNA or DNA. Identification of oligonucleotides and ribozymes for use as antisense agents and identification of DNA encoding the genes for targeted delivery involve methods well known in the art. For example, the desirable properties, lengths, and other characteristics of such oligonucleotides are well known. Antisense technology can be used to control gene expression through interference with binding of polymerases, transcription factors, or other regulatory molecules {see Gee et al, In Huber and Carr, Molecular and Immunologic Approaches, Futura Publishing Co. (Mt. Kisco, NY; 1994)).

Short interfering RNAs may be used for modulating (decreasing or inhibiting) the expression of a gene encoding a senescent cell-associated polypeptide. For example, small nucleic acid molecules, such as short interfering RNA (siRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules may be used according to the methods described herein to modulate the expression of a cellular polypeptide of interest {e.g., a senescent cell associated polypeptide). A siRNA polynucleotide preferably comprises a double-stranded RNA (dsRNA) but may comprise a single-stranded RNA {see, e.g., Martinez et al. Cell 110:563-74 (2002)). A siRNA polynucleotide may comprise other naturally occurring, recombinant, or synthetic single-stranded or double-stranded polymers of nucleotides (ribonucleotides or deoxyribonucleotides or a combination of both) and/or nucleotide analogues as provided herein and known and used by persons skilled in the art.

In a particular embodiment, the polynucleotide or oligonucleotide may be delivered by a recombinant vector in which the polynucleotide or oligonucleotide of interest has been incorporated. In other embodiments, the recombinant viral vector may be a recombinant expression vector into which a polynucleotide sequence that encodes an antibody, an antigen-binding fragment, polypeptide or peptide that is an agent of interest is inserted such that the encoding sequence is operatively linked with one or more regulatory control sequences to drive expression of the polypeptide, antibody, an antigen-binding fragment, or peptide of interest. The recombinant vector or the recombinant expression vector may be a viral recombinant vector or a viral recombinant expression vector. Exemplary viral vectors include, without limitation, a lentiviral vector genome, poxvirus vector genome, vaccinia virus vector genome, adenovirus vector genome, adenovirus-associated virus vector genome, herpes virus vector genome, and alpha virus vector genome. Viral vectors may be live, attenuated, replication conditional or replication deficient, and typically is a non-pathogenic (defective), replication competent viral vector. Procedures and techniques for designing and producing such viral vectors are well known to and routinely practiced by persons skilled in the art. In certain embodiments, the agent that selectively destroys a senescent cell for use in the methods described herein is not an agent that inhibits expression or production of an EXOl enzyme (see, e.g., Int'l Publ. No. WO

2006/018632).

Agents useful in the methods described herein may be identified or screened or characterized by techniques and procedures described herein and in the art. Agents that suppress a biological damage response, including those that destroy or facilitate destruction of senescent cells and those that deplete one or more senescent cell associated molecules, may be identified by in vitro assays that employ a cell line, such as a tumor cell line. The cultured cells can be exposed to a medical therapy and a candidate agent, concurrently or in any order. Such assays may be performed in a matrix (or array) which may include a high throughput screening format. High throughput formats typically comprise automated screening of a large number of candidate agents, which may be available from synthetic or natural product libraries. The candidate agents to be screened may be organized in a high throughput screening format such as using microfluidics-based devices, or a 96-well plate format, or other regular two dimensional array, such as a 384-well, 48-well or 24-well plate format, or an array of test tubes. The format is therefore amenable to automation. An automated apparatus that is under the control of a computer or other programmable controller may be used for one or more steps of the methods described herein. A controller may monitor the results of each step of the method and may automatically alter the testing paradigm in response to those results. Animal models may also be used to identify or characterize agents that suppress a biological response, including those that destroy or facilitate destruction of senescent cells. Animal models may also be used to identify or characterize agents that deplete one or more senescent cell-associated molecules produced by a senescent cell. By way of example, non-human animals, particularly genetically modified non-human animals that comprise a transgene expressed under the control of a senescent cell- specific promoter may be used. By operably (i.e., operatively) linking a senescent cell- specific promoter of a transgene to a nucleic acid sequence encoding a polypeptide of interest (e.g., a detectable label or cytotoxicity-activating molecule), senescent cells within an animal can be monitored in a controlled and user-determined fashion (see Examples herein). An exemplary transgene comprises (1) a senescent cell-specific promoter operatively linked to a polynucleotide encoding (a) at least one detectable label, (b) a cytotoxic agent, (c) a cytotoxicity-activating molecule, (d) an R A, or (e) any combination of (a), (b), (c) and (d); and exhibits a tumor. An exemplary animal model includes a transgene comprising (a) a pl6^Ink4a promoter operatively linked to a polynucleotide sequence encoding a FKBP-caspase fusion polypeptide (pl6-FKBP- caspase transgene) and to a polynucleotide sequence encoding a green fluorescent protein (see, e.g., Baker et al, Nature 479:232-36 (2011), which is incorporated herein by reference in its entirety); or (b) a pl6^Ink4a promoter operatively linked to a polynucleotide sequence encoding a fusion polypeptide comprising a luciferase, a red fluorescent protein, and a truncated herpes simplex virus thymidine kinase (tTK) (pi 6- 3MR transgene), which may be called herein a trimodal fusion protein (3MR). In certain transgenic animals, the luciferase is a renilla luciferase and red fluorescent protein is a monomeric red fluorescent protein.

Any of a number of cytotoxicity-activating molecules may be operably linked to a senescent cell-specific promoter to produce a suitable transgene for use in the animal model. Following its expression in a senescent cell-specific fashion, the cytotoxicity-activating molecule is one that is capable of inducing the controllable killing of the senescent cells in which it is expressed upon administration of an activating agent to the transgenic animal. Illustrative examples of cytotoxicity- activating molecules include herpes simplex virus (HSV) thymidine kinase (TK) polypeptides and FK506 binding protein (FKBP) (or variant thereof)-caspase fusion polypeptide. By way of an additional example, the cytotoxicity-activating molecule encoded by the transgene is a herpes simplex virus (HSV) thymidine kinase (TK) polypeptide (including truncated TK polypeptides) and the activating agent is the pro- drug ganciclovir, which is converted to a toxic moiety that is lethal to the cell in which it is expressed.

Effectiveness of an agent to suppress a biological damage response can be evaluated in such an animal model in which the agent's capability to suppress cellular senescence can be determined. An agent that suppresses cellular senescence may as a consequence inhibit tumor proliferation in the animal model. Tumor proliferation may be determined by tumor size, which can be measured in various ways familiar to a person skilled in the tumor animal model art, such as by palpation or measurement of the volume or area of a tumor (which may be performed postmortem), location(s) of the tumor (e.g., to determine if tumor cells have metastasized from the primary tumor site (i.e., the site where the tumor cells initially colonize). The effect of the therapeutic agent on tumor proliferation may also be evaluated by examining differentiation of the tumor cells.

The transgenic animal models described herein may also be employed for characterizing agents useful in methods for ameliorating toxicity (e.g., acute toxicity) of a medical therapy as described herein. Agents of interest include those that selectively destroy or facilitate selective destruction of senescent cells. Transgenic animals into which a cytotoxicity-activating molecule is operably linked to a senescent cell-specific promoter expressed in a senescent cell-specific fashion may be used. As a positive control, an activating agent that induces cytotoxicity is administered to the transgenic animal. For characterization of a therapeutic agent of interest (i.e., an agent that is capable of destroying (i.e., killing) a senescent cell), is administered instead of the activating agent. Toxic effects in animals may be monitored according to art accepted methods. For example, metabolic parameters in mice or rats may be determined by use of metabolic cages constructed for monitoring several metabolic parameters (e.g., V0₂, VC0₂, food uptake, water uptake, Kcal/hr, and wheel run distance) and which are available from several commercial vendors. Assessments that indicate energy imbalance include changes in respiration as shown by changes of V0₂ and VC0₂; changes in energy expenditure as determined by at least Kcal/hr; level of physical activity, such as wheel run distance, amount of time resting and/or interacting with the the proximal environment and other subjects (e.g., mice or rats sharing a cage). The characteristics of agents of interest may be determined in the presence or absence of an underlying disease condition (e.g., in the presence or absence of malignant tumors).

Senescent cells and senescent cell associated molecules can be detected by techniques and procedures described in the art. For example, senescent cells, including senescent cells obtained from tissues can be analyzed by histochemistry or immunohistochemistry techniques that detect the senescence marker, SA-beta gal (SA- Bgal) (see, e.g., Dimri et al, Proc. Natl. Acad. Sci. USA 92: 9363-9367 (1995)). The presence of the senescent cell-associated polypeptide pl6 can be determined by any one of numerous immunochemistry methods practiced in the art, such as immunob lotting analysis. Expression of nucleic acids encoding senescent cell associated polypeptides, including pl6 mR A, in a cell can be measured by a variety of techniques practiced in the art including quantitative PCR. The presence and level of senescence cell associated molecules, such as senescence cell associated polypeptides (e.g.,

polypeptides of the SASP) can be determined by using automated and high throughput assays, such as an automated Luminex array assay described in the art (see, e.g., Coppe et al., PLoS Biol 6: 2853-68 (2008)). For monitoring a biological damage response that comprises a DNA damage response, the various DNA damage response indicators can be detected, for example, according to the method of Rodier et al, Nature Cell Biol 11 : 973-979 (2009)).

Characterizing an agent that selectively destroys or kills senescent cells, may be determined by a method comprising: contacting (i.e., mixing, combining or in some manner promoting interaction between) senescent cells or quiescent (non- senescent) cells with the agent; and then determining the viability of the senescent cells and the quiescent cells by techniques commonly practiced in the art. Each of the steps of the methods described herein are performed under conditions and for a time sufficient appropriate for each step. Such conditions and time are discussed herein and may be readily determined by persons skilled in the art.

For example, to determine the presence of senescent cells, the level of SASP molecules, such as cytokines (e.g., inflammatory cytokines), growth factors, extracellular matrix components (ECM) and ECM-degrading enzymes, and proteases secreted by each of the senescent cells and the quiescent cells is determined and compared. Various methods of measuring cellular toxicity or cell viability are known in the art, and include, for example, methods for assessing cell membrane integrity (trypan blue or propidium iodide), lactate dehydrogenase assay, MTT or MTS assay, ATP assay, sulforhodamine B assay, and WST assay. In certain embodiments, gross cellular toxicity or cell viablity may be measured by detecting ATP levels.

As described herein, toxic effects in animals may be monitored according to art accepted methods. For example, metabolic parameters in mice or rats may be determined by use of metabolic cages constructed for monitoring several metabolic parameters (e.g., V0₂, VC0₂, food uptake, water uptake, Kcal/hr, and wheel run distance). Behaviors of the animals may also be monitored (e.g., interactions with food and water sources, interaction with a wheel, interaction with habitat, and length of time periods resting (short and long)).

Production of components characteristic of SASP may be measured by a variety of methods. In certain embodiments, the components characteristic of SASP may be measured in medium in which the cells have been cultured. The medium may be conditioned medium, where following treatment of cells with a test agent, cells are washed and incubated in serum- free medium without the presence of the test agent for a period of time to generate conditioned medium. The presence and level of senescence cell associated molecules (e.g., polypeptides of the SASP) can be determined by using automated and high throughput assays, such as an automated Luminex array assay described in the art (see, e.g., Coppe et al, PLoS Biol 6: 2853-68 (2008)). In certain embodiments, components characteristic of SASP are measured using an immunoassay, including, for example, Western blot, ELISA, antibody array, later flow immunoassay, magnetic immunoassay, radioimmunoassay, FACS, and a Surround Optical Fiber Immunoassay (SOFIA). In certain embodiments, the method of identifying or characterizing an agent, such as a small molecule compound, is a high throughput screening method. High throughput methods may also be used for determining the capability of an agent to kill senescent cells. High throughput screening, typically automated screening, of a large number of candidate agents from synthetic or natural product libraries may be used to identify agents. High throughput methods may also be used for characterizing and qualifying agents, including those that are approved or that are pre-clinical compounds. The agents to be analyzed may be organized in a high throughput screening format such as using microfluidics-based devices, or a 96-well plate format, or other regular two dimensional array, such as a 1536 well, 384-well, 48-well or 24- well plate format, or an array of test tubes. The format is therefore amenable to automation. An automated apparatus that is under the control of a computer or other programmable controller may be used for one or more steps of the methods described herein. A controller may monitor the results of each step of the method and may automatically alter the testing paradigm in response to those results. It is apparent to one of skill in the art that a variety of screening formats may be employed, e.g., different test agents may be placed in different vessels or wells, or a plurality of test agents are combined in a single well or vessel, or a combination thereof.

Senescent cells and quiescent (i.e., reversibly non-dividing but non- senescent) cells used in the methods of identifying or characterizing an agent, including a small molecule compound, comprise fibroblasts, including human fibroblasts. Other cell lineages useful for characterizing and identifying agents of interest include but are not limited to epithelial cells and endothelial cells, which may be established cell lines or primary cell cultures. Cells, such as fibroblast cell lines and/or primary fibroblasts, from the same or different species may be used. Agents of interest kill senescent cells but not quiescent cells, proliferating cells, or terminally differentiated (irreversibly non- dividing but non-senescent) cells.

For maintaining viability of cells, including fibroblast and tumor cells, the cells are cultured in media and under conditions practiced in the art for proper maintenance of cells in culture, including media (with or without antibiotics) that contains buffers and nutrients (e.g., glucose, amino acids (e.g., glutamine), salts, minerals (e.g., selenium)) and also may contain other additives or supplements (e.g., fetal bovine serum or an alternative formulation that does not require a serum

supplement; transferrin; insulin; putrescine; progesterone) that are required or are beneficial for in vitro culture of cells and that are well known to a person skilled in the art (see, for example, GIBCO media, INVITROGEN Life Technologies, Carlsbad, CA). Similar to standard cell culture methods and practices, the cell cultures described herein are maintained in tissue culture incubators designed for such use so that the levels of carbon dioxide (typically 5%), humidity, and temperature can be controlled. The cell culture system may also comprise addition of exogenous (i.e., not produced by the cultured cells themselves) cell growth factors, which may be provided, for example, in the media or in a substrate or surface coating. Growth characteristics of the cells for use in the methods described herein may be optimized by altering the composition or type of media, adjusting the amount of one or more nutrients and/or serum, which are procedure with which a skilled artisan is familiar. Persons skilled in the tissue culture art also recognize that conditions employed for routine maintenance of a cell culture (i.e., media, additives, nutrients) may need to be adjusted appropriately for certain manipulations of the cells such as ensuring appropriate confluency and growth properties of cells for the techniques described herein including high throughput screening.

Senescent cells used in methods of identifying or characterizing an agent that selectively destroys scenescent cells or an agent that depletes a senescent cell associated molecule are induced to senescence by exposure to radiation (e.g., X- irradiation), exposure to a chemotherapeutic, or transfection with a nucleic acid construct that expresses one or more proteins that induce senescence, such as oncogenic proteins (e.g., MAPK-6, RAS, MYC, ER , TRK, WNT). In certain embodiments, the senescent cells comprise cells transfected with a construct that expresses MAPK-6 or RAS.

The assay methods described herein further comprises testing the ability of the agent to reduce/suppress/or inhibit the ability of treated senescent cells to stimulate tumor invasion. Tumor cell invasion is one of the hallmarks of metastatic phenotype. The effect of an agent may be evaluated by its ability to selectively kill senescent cells or to facilitiate selective destruction of the senescent cells, or to suppress the ability of SASP to stimulate tumor invasion. Various tumor invasion assays are known in the art, and include, for example, the Boyden chamber assay and

modifications thereof (see, e.g., Albini et al, 1987, Cancer Res. 47:3239; Shaw, 2005, Methods Mol. Biol. 294:97-105; Nicolson, 1982, J. Histochem. Cytochem. 30:214-220; Rapesh, 1989, Invasion & Metastasis 9: 192-208).

A person skilled in the art will readily appreciate that the methods of characterizing and identifying the agents of interest described here may employ equipment, computers (and computer readable medium), and the like that are routinely used for performing steps of the method {e.g., washing, adding reagents and the like) and processing the data. Analytical tools, such as statistical analyses, are also routinely used by the skilled person.

Diseases and Medical Therapies

Diseases and Subjects in Need of Treatment

A subject {i.e., patient) in need of the therapeutic methods described herein is a human or non-human animal. The subject in need of medical therapies that may also cause toxic side effects may exhibit symptoms or sequelae of a disease described herein or may be at risk of developing the disease. Non-human animals that may be treated include mammals, for example, non-human primates {e.g. , monkey, chimpanzee, gorilla, and the like), rodents {e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, swine {e.g., pig, miniature pig), equine, canine, feline, bovine, and other domestic, farm, and zoo animals.

A subject who may receive an agent that suppresses a biological damage response, such as ameliorating toxicity and/or inhibiting metastasis, includes a subject who has a cancer or who is at risk of developing cancer. Subjects who have cancer also include a subject who is in remission (also called cancer remission herein), whether partial or complete. Remission refers to a decrease in or disappearance of signs and symptoms of cancer. In partial remission, some but not all, signs and symptoms of cancer have disappeared. In complete remission, all signs and symptoms of cancer have disappeared and if cancer cells remain, they are not detectable. Subjects who are in remission, either partial or complete, and who have a risk of cancer recurrence may benefit from the methods described herein.

Patients who are at risk of developing a cancer are subjects who have a predisposition to cancer, for example, a genetic predisposition, behavioral

predisposition (i.e., tobacco smoking), and environmental (e.g., exposure to asbestos). Patients with genetic predisposition may have one or more genetic mutations that increase the likelihood that the subject will develop the cancer. By way of example, human genes BRCA1 and BRCA2 belong to a class of genes known as tumor suppressors. Mutation(s) of these genes has been linked to hereditary breast and ovarian cancer. BRCA1 mutations may also increase a woman's risk of developing colon, uterine, cervical, and pancreatic cancer. Certain mutations in BRCA2 also increase the risk of pancreatic cancer as well as stomach cancer, gallbladder and bile duct cancer, and melanoma. Men with certain BRCA1 mutations and/ or BRCA2 mutations also have an increased risk of breast cancer and, and possibly, of pancreatic cancer, testicular cancer, and early-onset prostate cancer. Subjects at risk of developing a cancer also include those who have xeroderma pigmentosum that results from mutations in XPD helicase, which is required for nucleotide excision repair.

As used herein and in the art, the terms cancer or tumor are clinically descriptive terms which encompass diseases typically characterized by cells that exhibit abnormal cellular proliferation. The term cancer is generally used to describe a malignant tumor or the disease state arising from the tumor. Alternatively, an abnormal growth may be referred to in the art as a neoplasm. The term tumor, such as in reference to a tissue, generally refers to any abnormal tissue growth that is characterized, at least in part, by excessive and abnormal cellular proliferation. A tumor may be metastatic and capable of spreading beyond its anatomical site of origin and initial colonization to other areas throughout the body of the subject. A cancer may comprise a solid tumor or may comprise a liquid tumor (e.g., a leukemia).

The methods described herein may be useful for enhancing the effectiveness of a medical therapy that is a cancer therapy (e.g., by reducing or ameliorating toxicity of the medical therapy and/or inhibiting metastasis) in a subject who has any one of the types of tumors described in the medical art. Types of cancers (tumors) include the following: adrenocortical carcinoma, childhood adrenocortical carcinoma, aids-related cancers, anal cancer, appendix cancer, basal cell carcinoma, childhood basal cell carcinoma, bladder cancer, childhood bladder cancer, bone cancer, brain tumor, childhood astrocytomas, childhood brain stem glioma, childhood central nervous system atypical teratoid/rhabdoid tumor, childhood central nervous system embryonal tumors, childhood central nervous system germ cell tumors, childhood craniopharyngioma brain tumor, childhood ependymoma brain tumor, breast cancer, childhood bronchial tumors, carcinoid tumor, childhood carcinoid tumor,

gastrointestinal carcinoid tumor, carcinoma of unknown primary, childhood carcinoma of unknown primary, childhood cardiac (heart) tumors, cervical cancer, childhood cervical cancer, childhood chordoma , chronic myeloproliferative disorders, colon cancer, colorectal cancer, childhood colorectal cancer, extrahepatic bile duct cancer , ductal carcinoma in situ (DCIS), endometrial cancer, esophageal cancer, childhood esophageal cancer, childhood esthesioneuroblastoma, eye cancer, malignant fibrous histiocytoma of bone, gallbladder cancer, gastric (stomach) cancer, childhood gastric (stomach) cancer, gastrointestinal stromal tumors (GIST), childhood gastrointestinal stromal tumors (GIST), childhood extracranial germ cell tumor, extragonadal germ cell tumor, gestational trophoblastic tumor, glioma, head and neck cancer, childhood head and neck cancer, hepatocellular (liver) cancer, hypopharyngeal cancer, kidney cancer, renal cell kidney cancer, Wilms tumor, childhood kidney tumors, Langerhans cell histiocytosis, laryngeal cancer, childhood laryngeal cancer, leukemia, acute

lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), hairy cell leukemia, lip cancer, liver cancer (primary), childhood liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, aids-related lymphoma, burkitt lymphoma, cutaneous t-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, primary central nervous system (CNS) lymphoma, melanoma, childhood melanoma, intraocular (eye) melanoma, Merkel cell carcinoma, malignant mesothelioma, childhood malignant mesothelioma, metastatic squamous neck cancer with occult primary, midline tract carcinoma involving NUT gene, mouth cancer, childhood multiple endocrine neoplasia syndromes, mycosis fungoides, myelodysplasia syndromes, myelodysplasia neoplasms, myeloproliferative neoplasms, multiple myeloma, nasal cavity cancer, nasopharyngeal cancer, childhood nasopharyngeal cancer, neuroblastoma, oral cancer, childhood oral cancer,

oropharyngeal cancer, ovarian cancer, childhood ovarian cancer, epithelial ovarian cancer, low malignant potential tumor ovarian cancer, pancreatic cancer, childhood pancreatic cancer, pancreatic neuroendocrine tumors (islet cell tumors) , childhood papillomatosis , paraganglioma, paranasal sinus cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm , childhood pleuropulmonary blastoma, prostate cancer, rectal cancer, renal pelvis transitional cell cancer, retinoblastoma, salivary gland cancer, childhood salivary gland cancer, ewing sarcoma family of tumors, Kaposi Sarcoma, osteosarcoma,

rhabdomyosarcoma, childhood rhabdomyosarcoma, soft tissue sarcoma, uterine sarcoma, sezary syndrome, childhood skin cancer, nonmelanoma skin cancer, small intestine cancer, squamous cell carcinoma, childhood squamous cell carcinoma, testicular cancer, childhood testicular cancer, throat cancer, thymoma and thymic carcinoma, childhood thymoma and thymic carcinoma, thyroid cancer, childhood thyroid cancer, ureter transitional cell cancer, urethral cancer, endometrial uterine cancer, vaginal cancer, vulvar cancer, Waldenstrom Macroglobulinemia.

Cancers that are liquid tumors are classified in the art as those that occur in blood, bone marrow, and lymph nodes and include generally, leukemias (myeloid and lymphocytic), lymphomas (e.g., Hodgkin lymphoma), and melanoma (including multiple myeloma). Leukemias include for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), and hairy cell leukemia. Cancers that are solid tumors and occur in greater frequency in humans include, for example, melanoma, prostate cancer, testicular cancer, breast cancer, brain cancer, pancreatic cancer, colon cancer, thyroid cancer, stomach cancer, lung cancer, ovarian cancer, Kaposi's sarcoma, skin cancer (including squamous cell skin cancer), renal cancer, head and neck cancers, throat cancer, squamous carcinomas that form on the moist mucosal linings of the nose, mouth, throat, etc.), bladder cancer, osteosarcoma (bone cancer), cervical cancer, endometrial cancer, esophageal cancer, liver cancer, and kidney cancer. The methods described herein are also useful for enhancing the effectiveness of a medical therapy that is a cancer therapy to prevent (i.e., reduce the likelihood of occurrence), inhibit, retard or slow progression of metastatic cancer.

Methods for enhancing the effectiveness of a medical therapy and for ameliorating toxicity of a medical therapy, including acute toxicity, may be

administered to a subject who is infected with HIV, including patients who have developed AIDS. Also as described herein, a subject who has diabetes and receives insulin or a patient who has a condition that is treatable by administering angiotensin as a medical therapy may benefit by receiving an agent that suppresses a biological damage response.

Medical Therapies

Medical therapies that induce, cause, or promote a biological damage response include genotoxic (e.g., DNA damaging) and cytotoxic therapies. Examples of such medical therapies include most therapies used for treating cancers, such as radiation and a wide range of chemicals (i.e., chemotherapies). Radiation and chemotherapies are cytotoxic agents that selectively target cancer cells (i.e., tumor cells) by exploiting differential characteristics of the tumor cell compared with a normal cell. By way of example, differential characteristics and properties of tumor cells include high proliferation rates, hypoxia, aberrant metabolism, less effective repair capacity, and genomic instability.

Radiation therapy comprises use of high-energy radiation to shrink tumors and to kill cancer cells by damaging DNA. Radiation includes X-rays, gamma rays, and charged particles. The radiation may be delivered by a machine outside the body (e.g., external-beam radiation therapy) or the radioactive material placed in the body near cancer cells (i.e., internal radiation therapy, also called brachytherapy, which may be used, for example, in treating breast cancer and prostate cancer). Radiation therapy also includes systemic radiation therapy that uses radioactive substances, such as radioactive iodine (e.g., for treating thyroid cancer), that is administered systemically (for example, parenterally or orally).

Radiation therapy may be given with the intent to cure a cancer, for example, by eliminating a tumor or preventing cancer recurrence, or both. In such instances, radiation therapy may be used alone or in combination with surgery, chemotherapy, or with both surgery and chemotherapy. Radiation therapy may also be administered to have a palliative effect, for example to relieve symptoms (e.g., to shrink tumors of the brain, shrink tumors pressing on the spine or in bone, shrink tumors near the esophagus that interfere with ability to swallow). The appropriate radioactive therapy regimen for the type of cancer, location of a tumor, and for the particular subject (i.e., dependent on age, general health status, etc.) is readily determined by a person skilled in the art. See Lawrence et al, editors. Cancer: Principles and Practice of Oncology. 8^th ed. Philadelphia: Lippincott Williams and Wilkins, 2008.

As described herein, a medical therapy that is capable of inducing a biologically damaging response includes a chemotherapy (which includes a

combination chemotherapy), and which may be referred to as a chemotherapy, chemotherapeutic, or chemotherapeutic drug. Many chemotherapeutics are compounds referred to as small organic molecules. Chemotherapies are widely used for treatment of cancers. As understood by a person skilled in the art, chemotherapy may also refer to a combination of two or more chemotherapeutic molecules that are administered coordinately and which may be referred to as combination chemotherapy. Numerous chemotherapeutic drugs are used in the oncology art and include, without limitation, alkylating agents; antimetabolites; anthracyclines, plant alkaloids; and topoisomerase inhibitors. Alkylating agents include by way of example, cisplatin, carboplatin, oxalaplatin, cyclophosphamide, mechlorethamine, chlorambucil, ifosfamide.

Exemplary antimetabolites include nucleosides antagonists, such as purines (for example, azathioprine, mercaptopurine) and pyrimidines. Other examples of nucleoside antagonists include 5-fluorouracil, 6-mercaptopurine, arabinosylcytosine, capecitabine, clofarabine, cytarabine, dacarbazine, fludarabine, gemcitabine and nelarabine. Vinca alkaloids, include for example, vincristine, vinblastine, vinorelbine, vindesine; taxane and its analogs and derivatives; and podophyllotoxin. Exemplary topoisomerase inhibitors are type I topoisomerase inhibitors such as the camptothecins, for example, irinotecan and topotecan. Other topoisomerase inhibitors are type II topoisomerase inhibitors, for example, amascrine, etoposide, etoposide phosphate, and teniposide, which are semisynthetic derivatives of eipoodophyllotoxins. Cytotoxic antibiotics that are chemotherapeutic agents include without limitation doxorubicin, daunorubicin, valrubicin, idarubicin, epirubicin, bleomycin, plicamycin, and mitomycin. Combination chemotherapies are often referred to by an acronym with which a person skilled in the art will be familiar and may comprise two or more of the chemotherapeutic drugs described above and in the art (e.g., CHOP, ABVD, BEACOPP, CAV, COPP, EPOCH, MACOP-B, MOPP, R-CHOP, FOLFOX, FOL-FIRI, and Stanford V regimens).

Certain chemotherapies are also used for treating other conditions, such as immunological diseases including autoimmune diseases (for example, ankylosing spondylitis, multiple sclerosis, Crohn's disease, psoriasis, psoriatic arthritis, rheumatoid arthritis, and scleroderma).

Other medical therapies that are biologically damaging include antiviral therapies, for example, high active anti-retroviral therapies (HAART) used for treatment of HIV/ AIDS. A HAART regimen may combine three or more different drugs, such as two nucleoside reverse transcriptase inhibitors (NRTIs) and a protease inhibitor (PI); two NRTIs and a non-nucleoside reverse transcriptase inhibitor

(NNRTI); or other combinations. Toxic side effects of HAART depend in part on the particular antiretroviral agents used and have been documented in the medical art. Toxic side effects of anti-retroviral therapies include by way of example,

dermatological conditions, diarrhea, nausea, vomiting, lipodystrophy, diabetes, hyperglycemia, cardiovascular disease, hepatoxicity, hematological conditions (e.g., anemia, neutropenia), mitochondrial toxicity, neurological disorders, insomnia, nightmares, oral ulcers, peripheral neuropathies, and pancreatitis.

Other medical therapies that may induce a biologically damaging response also include hormone therapies, which are generally not genotoxic therapies. By way of example, an angiotensin, Angiotensin II (Ang II) has been reported to promotes vascular inflammation by inducing premature senescence of vascular smooth muscle cells both in vitro and in vivo (see, e.g., Kunieda et al, Circulation 114:953-60 (2006)). Angiotensin is a peptide hormone that causes vasoconstriction and a subsequent increase in blood pressure. Clinical studies have been performed to determine if administering angiotensin to patient with sarcoma would have an anti- tumor effect by constricting blood vessels to the tumor. Insulin has also been described as a hormone that induces cellular senescence.

Medical therapies also include high dose chemotherapies or high dose radiation therapy that is administered to a subject who has a disease, such as cancer, and who is to receive a stem cell transplant (either autologous or allogeneic). By way of example, stem cell replacement therapy has been used for treating aplastic anemia, Hodgkin disease, non-Hodgkin lymphoma, testicular cancer, and leukemias (including acute myelogenous leukemia (AML), acute lymphoblastoic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), therapy-related myelodysplasia (t-MDS) and therapy-related acute myeloid leukemia (t-AML)), and myelodysplasia syndrome.

The term "medical therapy" will be understood to include other terms commonly used in the art, such as medical treatment, therapeutic(s), and the like.

Medical therapy includes a single active ingredient or component, one or more active ingredients or components, or multiple active ingredients or components that are administered to a subject in need to treat or to prevent (i.e., reduce the likelihood of occurrence or recurrence of) a disease or disorder. As described herein, medical therapies that are chemotherapies, such as cancer therapies, may include a single chemotherapeutic agent or may include combinations of two or more chemotherapeutic drugs (also called combination chemotherapy). By way of an additional example, as described herein, HAART is typically a combination or cocktail of three different viral agents.

Assessing Effectiveness of Medical Therapy

Enhancing the effectiveness of a medical therapy results in an improvement or increase of the therapeutic and/or prophylactic benefit compared with the benefit observed in the absence of administering the agent, and as described herein, comprises ameliorating or attenuating (i.e., reducing, decreasing, preventing, inhibiting, suppressing) the deleterious biological and physiological effects of the medical therapy including toxic side effects, and in certain embodiments, metastasis of a cancer. Use of agents that ameliorate one or more toxic side effects resulting from use of a medical therapy that is determined by a person skilled in the medical art necessary or desirable for treating a disease (e.g., cancer) can improve compliance of a subject in need and also influence a subject's decision to proceed with a medical therapy known to have toxic side effects. In addition, by increasing the effectiveness of a medical therapy, the lifetime exposure to the therapy may be decreased, and consequently, biological damage is decreased.

The effectiveness of the methods described herein for ameliorating toxicity, such as acute toxicity, of a medical therapy, may be determined by methods and evaluation methodologies familiar to a person in the clinical and medical arts. To monitor the health status of the subject, physical examination, interviews by clinicians, assessment and monitoring of clinical symptoms, performance of analytical tests, and self-reporting of toxic side effects by the treated subject may be employed to assess the efficacy of an agent that selectively destroys or facilitates selective destruction of senescent cells. By way of example, an acute toxic effect is fatigue, which can be evaluated by physical symptoms, including for example, shortness of breath, heart palpitations, and general lack of energy. Clinical evaluations and performance of diagnostic assays may depend on the particular medical therapy and the known toxic side effects of the medical therapy. Examples of diagnostic assays include assays for determining enzyme levels, such as those indicative of hepatoxicity or cardiovascular disease, with which a skilled person is familiar.

The effectiveness of a medical therapy administered to a subject (i.e., patient) who also receives an agent that suppresses a biological damage response may also be readily be determined by a person skilled in the medical and clinical arts. One or any combination of diagnostic methods, including physical examination, assessment and monitoring of clinical symptoms, and performance of analytical tests and methods described herein, may be used for monitoring the health status of the subject.

Therapeutic and/or prophylactic benefit for subjects to whom the agents are administered, includes, for example, an improved clinical outcome, wherein the object is to prevent or slow or retard (lessen) an undesired physiological change associated with the disease, or to prevent or slow or retard (lessen) the expansion or severity of such disease and/or prevent or slow or retard (lessen) an undesired toxic side effect associated with the medical therapy.

The effectiveness of the medical therapy may include beneficial or desired clinical results that comprise, but are not limited to, abatement, lessening, or alleviation of symptoms that result from or are associated with the disease to be treated; decreased occurrence of symptoms; improved quality of life; longer disease-free status (i.e., decreasing the likelihood or the propensity that a subject will present symptoms on the basis of which a diagnosis of a disease is made); diminishment of extent of disease; stabilized (i.e., not worsening) state of disease; delay or slowing of disease progression; amelioration or palliation of the disease state; and remission (whether partial or total), whether detectable or undetectable; and/or overall survival. The effectiveness of the medical therapy may also mean prolonging survival when compared to expected survival if a subject were not receiving the agent that suppresses a biological damage response. As described herein, agents that selectively destroy senescent cells may also enhance the effectiveness of the medical therapy to treat the underlying disease. When the medical therapy is a cancer therapy, enhancing the effectiveness of the therapy may result in an improvement or increase of the therapeutic and/or prophylactic benefit compared with the benefit observed in the absence of administering the agent. For example, enhancing a medical therapy that is a cancer therapy includes any one or more of reducing the size of the tumor(s), inhibiting tumor progression, inhibiting tumor growth, delaying tumor colonization, and/or inhibiting, preventing, or delaying metastasis of a tumor. Enhancing the effectiveness of the therapy may include preventing, slowing, or decreasing development of resistance of the cancer (i.e., tumor or tumors) to the medical therapy, thereby allowing additional cycles of therapy and/or decreasing the time interval between cycles of therapy.

In addition to the benefits for patients with cancer discussed above, benefits may be provided to subjects who receive high dose chemotherapy and/or high dose radiation followed by autologous or allogenic stem cell replacement therapy. An improved clinical outcome in a subject who receives an agent that suppresses a biological damage response may be assessed by the time (i.e., number of days) for white blood cell recovery. For subjects who receive an allogenic stem cell transplant, improvement in graft versus tumor effect and absence or reduced graft versus host disease compared with subjects who do not receive the agent can indicate enhanced effectiveness of the high dose chemotherapy or high dose radiation.

When the medical therapy is an anti- viral therapy, and similarly as for radiation and chemotherapies for cancer, by suppressing the biological damage response, toxic side effects may be reduced as well as development of resistance to an anti-viral therapy may be reduced, the dose of the medical therapy may be reduced, or the time interval between administration of two doses may be increased, thereby reducing the lifetime exposure to the therapy. Improved clinical outcome is indicated, for example, by amelioration of one or more toxic side effects of the antiviral therapy; by decreasing the time required for complete or partial eradication of the infection; prolonging disease-free status and/or overall survival; maintaining or improving immunological status; or reducing or lessening severity of one or more symptoms of the viral invention. For a person who is infected with HIV, in addition to the above improved clinical outcomes, a more effective anti-viral therapy including HAART may provide stability (i.e., decreasing the rate of decline) or improvement in T cell count; delay or reduce likelihood of occurrence of diseases associated with severe

immunosuppression, such as Kaposi's sarcoma, AIDS related lymphoma, and opportunistic infections (e.g., candidiasis, cryptococcal meningitis, toxoplasmosis; coccidiomycosis; progressive multifocal leukoencephalopathy; HIV-related

encephalopathy; shingles; crytosporidiosis; infections caused by CMV, Mycobacterium including tuberculosis, Herpes simplex virus, human papilloma virus, hepatitis virus B, hepatitis C).

Clinical benefit and improvement or a subject who has diabetes and receives insulin as the medical therapy and who receives an agent that suppresses a biological damage response, may be evaluated by stability of glucose levels. For example, an increase in the length of time between doses of insulin or a decrease in the dose of insulin required to maintain proper glucose levels in patients who receive an agent that suppresses a biological response indicates improved effectiveness of the insulin. Pharmaceutical Compositions

Also provided herein are pharmaceutical compositions comprising any one or more of the agents that suppress a biological damage response resulting from induction and establishment of senescent cells by a medical therapy. A pharmaceutical composition may be a sterile aqueous or non-aqueous solution, suspension or emulsion, which additionally comprises a physiologically acceptable excipient (pharmaceutically acceptable or suitable excipient or carrier) (i.e., a non-toxic material that does not interfere with the activity of the active ingredient). The excipients described herein are merely exemplary and are in no way limiting. An effective amount or therapeutically effective amount refers to an amount of an agent or a composition comprising one or more agents administered to a subject, either as a single dose or as part of a series of doses, which is effective to produce a desired therapeutic effect.

Subjects may generally be monitored for therapeutic effectiveness using assays suitable for the condition being treated or prevented, which assays will be familiar to those having ordinary skill in the art and are described herein. The level of an agent that is administered to a subject may be monitored by determining the level of the agent in a biological fluid, for example, in the blood, blood fraction (e.g., serum), and/or in the urine, and/or other biological sample from the subject. Any method practiced in the art to detect the agent may be used to measure the level of agent during the course of a therapeutic regimen.

When two or more agents (e.g., anti-senescence cell agent and an agent that depletes one or more senescent cell associated molecules) are administered to a subject for treatment of a disease or disorder described herein, each of the agents may be formulated into separate pharmaceutical compositions. A pharmaceutical preparation may be prepared that comprises each of the separate pharmaceutical compositions (which may be referred to for convenience, for example, as a first pharmaceutical composition and a second pharmaceutical composition) comprising each of the first and second agents, respectively. Each of the pharmaceutical compositions in the preparation is administered according to the dosing methods described herein and may the agent that selectively destroys or facilitates selective destruction of senescent cells may be administered before, concurrently, or after administration of an agent that depletes one or more senescent cell associated molecules. Each agent may be administered via the same route of administration or may be administered by different administration routes. Alternatively, two or more agents may be formulated together in a single pharmaceutical composition.

The dose of an agent described herein for enhancing the effectiveness of a medical therapy, which in certain embodiments ameliorates a toxic side effect of the medical therapy, may depend upon the subject's condition, that is, stage of the disease, severity of symptoms caused by the disease, general health status, as well as age, gender, and weight, and other factors apparent to a person skilled in the medical art. Pharmaceutical compositions may be administered in a manner appropriate to the disease to be treated as determined by persons skilled in the medical arts. An appropriate dose and a suitable duration and frequency of administration will be determined by such factors as the condition of the patient, the type and severity of the patient's disease, the particular form of the active ingredient, and the method of administration. Optimal doses of an agent may generally be determined using experimental models and/or clinical trials. The optimal dose may depend upon the body mass, weight, or blood volume of the subject. The use of the minimum dose that is sufficient to provide effective therapy is usually preferred. Design and execution of preclinical and clinical studies for an agent (including when administered for prophylactic benefit) described herein are well within the skill of a person skilled in the relevant art. The optimal dose of an agent may depend upon the body mass, weight, or blood volume of the subject. For example, an amount between 0.01 mg/kg and 1000 mg/kg (e.g., about 0.1 to 1 mg/kg, about 1 to 10 mg/kg, about 10-50 mg/kg, about 50-100 mg/kg, about 100-500 mg/kg, or about 500-1000 mg/kg) body weight.

The pharmaceutical compositions may be administered to a subject in need by any one of several routes that effectively deliver an effective amount of the agent. Such administrative routes include, for example, oral, topical, parenteral, enteral, rectal, intranasal, buccal, by inhalation, sublingual, intramuscular, transdermal, vaginal, rectal, or by intracranial injection, or any combination thereof. Such compositions may be in the form of a solid, liquid, or gas (aerosol). Pharmaceutical acceptable excipients are well known in the

pharmaceutical art and described, for example, in Rowe et al., Handbook of

Pharmaceutical Excipients: A Comprehensive Guide to Uses, Properties, and Safety, 5 ^th Ed., 2006, and in Remington: The Science and Practice of Pharmacy (Gennaro, 21^st Ed. Mack Pub. Co., Easton, PA (2005)). Exemplary pharmaceutically acceptable excipients include sterile saline and phosphate buffered saline at physiological pH. Preservatives, stabilizers, dyes, buffers, and the like may be provided in the pharmaceutical composition. In addition, antioxidants and suspending agents may also be used. In general, the type of excipient is selected based on the mode of administration, as well as the chemical composition of the active ingredient(s). Alternatively, compositions described herein may be formulated as a lyophilizate, or the agent may be encapsulated within liposomes using technology known in the art. Pharmaceutical compositions may be formulated for any appropriate manner of administration described herein and in the art.

A pharmaceutical composition {e.g., for oral administration or delivery by injection) may be in the form of a liquid. A liquid pharmaceutical composition may include, for example, one or more of the following: a sterile diluent such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils that may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents; antioxidants; chelating agents; buffers and agents for the adjustment of tonicity such as sodium chloride or dextrose. A parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. The use of physiological saline is preferred, and an injectable pharmaceutical composition is preferably sterile.

For oral formulations, at least one of the agents described herein can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, and if desired, with diluents, buffering agents, moistening agents, preservatives, coloring agents, and flavoring agents. The agents may be formulated with a buffering agent to provide for protection of the agent from low pH of the gastric environment and/or an enteric coating. An agent included in the compositions may be formulated for oral delivery with a flavoring agent, e.g., in a liquid, solid or semi-solid formulation and/or with an enteric coating.

A composition comprising any one of the agents described herein may be formulated for sustained or slow release. Such compositions may generally be prepared using well known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site. Sustained- release formulations may contain the agent dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane. Excipients for use within such formulations are biocompatible, and may also be biodegradable;

preferably the formulation provides a relatively constant level of active component release. The amount of active agent contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release, and the nature of the condition to be treated or prevented.

In certain embodiments, the pharmaceutical compositions comprising an agent are formulated for transdermal, intradermal, or topical administration.

Pharmaceutical compositions comprising an agent for topical application can be formulated as emulsions. In still other embodiments, an agent is formulated for intranasal or inhalation delivery. In some embodiments, the active ingredient(s) can be formulated with oleaginous bases or ointments to form a semisolid composition with a desired shape. In still other embodiments, an agent is formulated in a pharmaceutical composition as an aerosol for delivery intranasally or by inhalation. Formulation methods and techniques appropriate for different administrative routes are familiar to those skilled in the relevant art.

Medical therapies that are administered as pharmaceutical compositions for treatment of a disease or disorder in a subject are typically administered in formulations and via an administrative route that is described in a product insert if the medical therapy is marketed. If the medical therapy is administered to a subject as part of a clinical trial, compositions comprising the medical therapy and methods for administering the medical therapy are described in a clinical protocol.

Kits with unit doses of one or more of the agents described herein, usually in oral or injectable doses, are provided. Such kits may include a container containing the unit dose, an informational package insert describing the use and attendant benefits of the drugs in treating pathological condition of interest, and optionally an appliance or device for delivery of the composition.

EXAMPLES EXAMPLE 1

PREPARATION OF P 16-3MR TRANSGENIC MICE

To examine the role of senescent cells in cancer, in the risk of developing cancer or in side effects arising after cancer treatment, a transgenic mouse comprising a pl6^Ink4a promoter operative ly linked to a trimodal fusion protein was generated to allow for detection of senescent cells and for selective clearance of senescent cells in those transgenic mice.

The promoter, pl6^Ink4a, which is transcriptionally active in senescent cells but not in non-senescent cells (see, e.g., Wang et al, J. Biol. Chem. 276:48655-61 (2001); Baker et al, Nature, supra) was engineered into a nucleic acid construct. A fragment of the pl6^Ink4a gene promoter (see Figures 5 and 6 providing an exemplary vector and exemplary promoter sequence) was introduced upstream of a nucleotide sequence encoding a trimodal reporter fusion protein. The trimodal reporter protein is termed 3MR and consists of renilla luciferase (rLUC), monomeric red fluorescent protein (mRFP) and a truncated herpes simplex virus thymidine kinase (tTK) (see, e.g., Ray et al, Cancer Res. 64: 1323-30 (2004)). Thus, the expression of 3MR is driven by the pl6^Ink4a promoter in senescent cells only. The polypeptide sequences and the encoding polynucleotides for each of the three proteins are known in the art and are available in public databases, such as GenBank. An exemplary sequence (SEQ ID NO:39) for the 3MR transgene is provided in Figure 24. The 3MR transgene was inserted into a BAC vector using techniques routinely practiced by person skilled in the molecular biology art. The detectable markers, rLUC and mRFP permitted detection of senescent cells by bioluminescence and fluorescence, respectively. The expression of tTK permitted selective killing of senescent cells by exposure to the pro-drug ganciclovir (GCV), which is converted to a cytotoxic moiety by tTK. Transgenic founder animals, which have a C57B16 background, were established and bred using known procedures for introducing transgenes into animals (see, e.g., Baker et al, Nature, supra). The transgenic mice are called pi 6-3MR herein.

EXAMPLE 2

SENESCENT CELLS CAN BE DETECTED AND CLEARED IN TRANSGENIC P 16-3MR MICE

Senescent cells can be detected using a variety of biomarkers, including the strongly upregulated pl6-INK4a tumor suppressor protein (Campisi et al, Nature Rev. Molec. Cell Biol. 8:729-40 (2007)). Using such markers, it was shown that both normal and tumor cells undergo senescence, in mice and humans, after exposure to ionizing radiation or DNA-damaging chemotherapy (Coppe et al., PLoS Biol. 6:2853- 68 (2008); Schmitt et al, Cell 109:335-46 (2002); te Poele et al, Cane. Res. 62: 1876-83 (2002); Le et al, Aging Cell 9:398-409 (2010)). For example, pl6-3MR transgenic mice will accumulate senescent cells when exposed to genotoxins {e.g., ionizing radiation, DNA damaging chemicals), epigenomic toxins {e.g., compounds that perturb histone modifications or DNA methylation), strong mitogenic signals (e.g., activated oncogenes, elevated levels of growth factors, certain hormones). But, as noted herein, one advantage of the pl6-3MR transgenic mice is that they express tTK, which allows for selective killing of senescent cells by administering pro-drug ganciclovir (GCV) to the mice since GCV is converted into a cytotoxin by tTK. Therefore, the clearance of senescent cells in pl6-3MR transgenic mice exposed to radiation was examined after GCV treatment.

Briefly, a group of pl6-3MR transgenic mice were exposed to whole body ionizing radiation (7 Gy X-ray) and a control group of pl6-3MR transgenic mice were mock-irradiated. After three months, the mice were treated with GCV (25 mg/kg) or vehicle only, and then at least two weeks later bioluminescence in tissues was examined after administering the rLUC substrate.

In several tissues, irradiated mice (IR) showed a greater than 2-fold higher bioluminescence than unirradiated mice (Ctrl), indicating that rLUC is expressed three months after radiation exposure and, therefore, the presence of senescent cells is persisting (see Figure 1 A, showing bioluminescence results in lung tissue). Moreover, mice treated with GCV exhibited rLUC expression levels comparable to unirradiated mice, indicating that GCV resulted in elimination of senescent cells (Figure 1 A).

As is known in the literature, senescent cells also secret molecules that can cause inflammation (Freund et al., Trends Mol. Med. 16:238-46 (2010)), which, if chronic, will fuel various pathologies, including cancer (Davalos et al, Cancer

Metastasis Rev. 29:273-83 (2010)) - this is often referred to as senescence-associated secretory phenotype (SASP). For example, IL-6 (interleukin-6) and MMP-3 (matrix metalloproteinase-3) are two prominent SASP components. Hence, R A expression levels of various biomarkers associated with SASP were examined, including pi 6INK4a (p 16), IL-6 and MMP-3. In addition, the level of the mRFP reporter was measured. Figure IB shows that GCV returned pl6INK4a (pi 6), IL-6, MMP-3 and mRFP expression levels to those found in the unirradiated control mice. Furthermore, GCV notably had no detectable effect on expression levels when given to wild-type, non-transgenic C57B16 mice. EXAMPLE 3

CELLULAR SENESCENCE INCREASES THE LIKELIHOOD OF CANCER AND METASTASIS

To examine the role of senescence in contributing to, inducing or increasing the likelihood of tumor formation or growth and metastasis, tumor engraftment was monitored in pl6-3MR transgenic mice that were either depleted of senescent cells and in mice that had senescent cells (naturally developed or induced).

Briefly, 10⁶ B16 mouse melanoma cells, a highly aggressive cell line that is syngeneic with pl6-3MR transgenic mice (C57B16 background), that express firefly luciferase (fLUC, to enable their detection by bio luminescence) were injected into the tail vein of the pl6-3MR transgenic mice approximately three months after being either mock irradiated or irradiated, as described in Example 2. Irradiated mice were treated daily with GCV (25 mg/kg) or vehicle only for 7 days, and then 3 days following the last GCV dose, B16 mouse melanoma cells were injected into the mice. B16 mouse melanoma cells first colonize the lung, where they form primary tumors approximately two weeks after injection, and thereafter metastasize to distal tissues to form secondary tumors in, for example, the pancreas, liver and visceral fat. The biolumninescence markers, fLUC and rLUC are distinguishable because the enzymes use different substrates.

As shown in Figure 2, tumor progression occurred much faster in the irradiated mice as compared to the mock-irradiated mice. Fifteen days after the injection, mock-irradiated (Ctrl) mice had some relatively small lung nodules (see Figure 2A). In contrast, irradiated mice had significantly more primary tumors and, additionally, the animals harbored a large number of metastatic tumors (see Figure 2B) - these animals were moribund between days 15 and 16 after injection. Strikingly, irradiated mice in which senescent cells were cleared after GCV treatment showed much smaller primary tumors and many fewer metastases (see Figure 2C). B16 mouse melanoma cells were detected in the mice -15-18 days post-injection by measuring fLUC biolumenscence. Irradiated mice were moribund at days 15-16 post-injection and sacrificed. Fifteen days after the injection, mock-irradiated (Ctrl) mice and irradiated mice in which senescent cells were cleared after GCV treatment both had relatively low levels of B16 cells as detected by luminescence (see Figure 3). Irradiated mice had significantly larger numbers of B16 cells as detected by luminescence (see Figure 3). On day 18, irradiated mice in which senescent cells were cleared after GCV treatment still showed relatively low levels of B16 cells as did the mock-irradiated control (Ctrl) mice (see Figure 3).

Eighteen days after injecting the B16 melanoma cells, large primary lung tumors were evident in the irradiated mice that received GCV treatment (see Figure 4A). But, despite the presence of tumors in the lungs, the distal organs remained almost devoid of metastases (see Figure 4A; see also Figure 4C showing liver and fat tissue). This was in sharp contrast to irradiated mice not treated with GCV, in which the liver and fat harbored multiple metastatic tumors (see Figure 4B), which were already present by day 15.

Luminescent metastatic nodules were also counted in control, irradiated, and irradiated+GCV treated mice as provided in Table 2. As nodules are difficult to count in fat tissue, metastatic cells were represented as an estimated % of total area of fat. Table 2: Detection of metastatic B16 melanoma cells 18 days after injection

Similar results were observed when the senescent cell accumulation was induced with the chemotherapeutic agent, doxorubicin. Using pl6-3MR mice, treatment doxorubicin (10 mg/kg) induced the persistent presence of senescent cells in tissues, similar to the effects of radiation. Various tissues were isolated (liver, heart, lung, kidney, and spleen) and measured for abundance of mRNAs encoding mRFP and pl6INK4a as markers for senescent cells (see Figures 5 A and 5B, respectively).

Doxorubicin-treated mice consistently expressed higher levels of mRFP and pl6INK4a in all tissues compared to untreated control mice.

Also similar to the effects of radiation, doxorubicin treatment stimulated the growth of B16 melanoma cells that were injected subcutaneously. Again, similar to radiation-treated mice, GCV (which eliminates senescent cells in pl6-3MR mice) substantially reduced the size of B16 melanoma tumors in mice pre-treated with doxorubicin. Briefly, pl6-3MR transgenic mice were treated with vehicle (ctrl) or 10 mg/kg doxorubicin. Seven days after doxorubicin treatment, mice were treated daily with GCV (25 mg/kg) for 7 days or vehicle only. 3 days after the last GCV treatment, 4 x 10⁵ B16 mouse melanoma cells were injected subcutaneously into the pl6-3MR transgenic mice, and mice were sacrificed after 12 days for analysis.

Skin biopsies were collected and measured for abundance of senescent cell biomarkers (pl6INK4a and mRFP mRNAs). As shown in Figure 6, skin biopsies from doxorubicin treated mice showed increased senescence as compared to skin biopsies from untreated control mice, as measured by pl6INK4a and mRFP expression. In contrast, doxorubicin-treated mice in which senescent cells were cleared by GCV treatment showed low levels of pl6INK4a and mRFP expression.

Tumor growth was increased in doxorubicin-treated mice as compared to vehicle-treated control mice (see Figure 7). In contrast, doxorubicin-treated mice in which senescent cells were cleared after GCV treatment showed much smaller primary tumors (see Figure 7). Tumor diameters were also measured and also confirmed that doxorubicin-treated mice in which senescent cells were eliminated by GCV treatment had smaller tumor sizes, and doxorubicin-treated mice had increased tumor sizes (see Figure 8).

Overall, an increase in senescent cell population induced by radiation or doxorubicin correlated with a greatly increased primary tumor size and with metastases (radiation only), but this was largely abrogated when senescent cells were depleted in mice treated with GCV. In other words, these results show that the persistent presence of senescent cells after exposure to a senescence causing stress can promote the growth of primary tumors and will advance the development of metastases. Thus, senescence cell clearance or depletion can delay, prevent, or reduce the risk or likelihood of tumor formation or metastasis.

EXAMPLE 4

SENESCENT CELL CLEARANCE REDUCES LIKELIHOOD

OF K-RAS MEDIATED TUMORIGENESIS

To examine the role of senescence in contributing to, inducing or increasing the likelihood of K-Ras mediated lung tumor formation or growth and metastasis, tumor formation was monitored in INK- ATT AC transgenic mice that were either depleted of senescent cells or have senescent cells (naturally developed or induced).

Briefly, INK-ATTAC (pl6^Ink4a apoptosis through targeted activation of caspase) transgenic mice have an FK506-binding protein (FKBP)-caspase 8 (Casp8) fusion polypeptide under the control of the pl6^Ink4a promoter (see Figure 10 providing a vector sequence for the transgene and Figure 11 providing sequences for components of the transgene including the promoter sequence). In the presence of AP20187, a synthetic drug that induces dimerization of a membrane bound myristoylated FKBP- Casp8 fusion protein, senescent cells specifically expressing the FKBP-Casp8 fusion protein via the pl6^Ink4a promoter undergo programmed cell death (apoptosis) (see, e.g., Baker, Nature, supra, Figure 1 therein). Two founder lines (INK-ATTAC³ and ΓΝΚ- ATT AC⁵) were each bred with the K-rasLAl tumor model. K-rasLAl mice were first developed by Tyler Jacks at M.I.T. {see Johnson, L. et al, Nature 410: 1111-16 (2001). The mice activate a silent K-ras oncogene through a spontaneous recombination event. The mean age of death/sacrifice of K-rasLAl mice is about 300 days as a result of extensive tumor burden. The most frequent organ site is the lung and varying grades of tumors are present as early as six weeks of age from hyperplasia/dysplasia to carcinomas similar to human non-small cell lung cancer. Metastasis to thoracic lymph nodes, kidney and other visceral organs occurs with low frequency. Other organ sites include the thymus (thymic lymphoma) and skin (papillomas). A companion strain (K- ras^LA2) carries an allele that recombines to the activated allele (K-Ras^G12D) 100% of the time.

Two INK- ATT AC : K-RasL A 1 were produced (one for INK-ATTAC line 3 and one for line 5). Beginning at three weeks of age, one half each cohort was treated with 2 mg AP20187/g body weight and the remaining half with vehicle (PBS). Twenty one days after treatment, the mice were sacrificed and tumor multiplicity in lungs was measured. Tumor numbers were found to be significantly reduced in INK-ATTAC3:K- RasLAl and INK-ATTAC5:K-RasLAl transgenic mice that had senescent cells depleted after treatment with AP20187 {see Figure 9). These data suggest that senescent cells support tumor formation that is oncogene-driven. In addition, metastasis and overall survival will be monitored after tumor induction in the presence or absence of pl6-positive cells.

EXAMPLE 5

SENESCENT CELL CLEARANCE REDUCES LIKELIHOOD OF BREAST CANCER

OR SKIN CARCINOGENESIS Similar experiments to those of Example 4 can be performed using doxycycline-mediated expression of HER2 (see, e.g., Yeh et al, J. Clin. Investig.

121 :866-79 (2011); see also Gunther et al, FASEB 16:283-92 (2002)) to examine the role of senescence in contributing to, inducing or increasing the likelihood of breast cancer. For example, founder INK-ATTAC lines are each bred onto a transgenic mouse MMTV-HER2 or a bi-transgenic mouse MMTV-rtT:TetO-HER2 genetic background, wherein doxycycline can be used to induce breast tumor formation subsequent to a senescence inducing factor (e.g., radiation or chemotherapy) used to induce senescent cell accumulation.

Alternatively, INK- ATT AC transgenic mice can be treated with a senescence inducing factor (e.g., radiation or chemotherapy) and then a carcinogen to examine the role of senescence in contributing to, inducing or increasing the likelihood of skin carcinogenesis (see, e.g., Slaga et al, J. Investig. Dermatol. Symp. Proc. 1 : 151-6 (1996)).

EXAMPLE 6

SENESCENT CELL REDUCTION REDUCES LIKELIHOOD OF SIDE EFFECTS

FROM SENESCENCE INDUCING CHEMOTHERAPY

To examine the role of senescence in contributing to, inducing or increasing the likelihood of side effects resulting from, for example, radiation or chemotherapy used to treat cancer that has already developed. Such side effects may include returning or recurring tumor formation or growth and metastasis. Side effects are monitored in pl6-3MR transgenic mice that are either depleted of senescent cells or have senescent cells (naturally developed or induced).

Briefly, tumor cell lines are engineered to express firefly luciferase (fLUC) to enable their detection of tumors and metastases by bioluminescence in a living animal. For example, a B16-fLUC mouse melanoma cell line (PerkinElmer,

Waltham, MA), and an MMTV-PymT:fLUC mammary carcinoma cell line can be used in the experiments described in this example. To prepare the MMTV-PyMT-fLUC cell line, MMTV cells were infected with a lentivirus that contained a sequence that encodes Firefly Luciferase and contained the mammalian puromycin resistance gene. Cells were then selected through puromycin treatment and tested for luminescence.

The MMTV-PymT tumor cells (5 x 10⁵ cells) were injected into a mammary fat pad of each mouse. Small primary tumors formed over a period of one week. Then doxorubicin (DOXO) at 10 mg/kg or vehicle only (PBS) was administered once at Day 7. Beginning three days after mice received DOXO, GCV was then administered 5x daily intraperitoneally at 25 mg/kg, or vehicle only was administered. Four different treatment groups of mice (7 mice per group) included (1) no doxorubicin (vehicle), no GCV (vehicle); (2) doxorubicin, no GCV; (3) no doxorubicin, GCV; and (4) doxorubicin, GCV. Mouse survival was monitored over time (30 days), and the results are presented in Figure 20. Bio luminescence in tissues was examined (after administering the firefly luciferase (fLUC) substrate) to monitor tumor formation (see Figure 21).

Three days after the last administration of GCV or vehicle, mice were housed in metabolic cages (Promethion, Sable Systems International, Las Vegas, NV) for a period of 4 days to monitor food consumption, water consumption, body mass, spontaneous activity and behavior, voluntary exercise, oxygen consumption, and carbon dioxide production. Three days after GCV treatment (day 18 after tumor cell injection), animals were monitored for three nights, and the following nocturnal measurements were obtained: V0₂ (mL/min); VC0₂ (mL/min); food uptake (g); water uptake (g); Kcal/hr; and wheel run distance (m). These data are presented in Table 3 below and in Figure 22. The data represent the average of the three nocturnal measurements.

Table 3

p-value: *<0.05; **<0.01; ***<0.001

Behavior of animals treated doxorubicin or doxorubicin and GCV was assessed by several criteria accepted in the art as behavior metrics. The metrics and data are presented in Table 4 and in Figure 23. The animals in the tumor model that were treated with GCV after being treated with doxorubicin exhibited significantly more active behaviors (see interaction with wheel (WHEEL) and long lounge (LLNGE) and short lounge (SLNGE) in Table 4 and Figure 23). Table 4

WHEEL: Interaction with wheel (>= 1 revolution)

IHOME: Entered habitat (stable mass reading)

THOME: Interaction with habitat (no stable mass reading)

LLNGE: Long lounge (> 60 sec, no non-XY sensor interactions)

SLNGE: Short lounge (5 - 60 sec, no non-XY sensor interactions) p-value: *<0.05; **<0.01; ***<0.001

To determine if differences in the metabolic data and behavioral data resulted from reduction in tumor size and metastasis that was observed in mice treated with doxorubicin and GCV compared with mice treated with doxorubicin only, an experiment was performed in which mice were not injected with tumor cells. Groups of pl6-3MR transgenic mice (5 mice per group) were treated with saline and then seven days after were treated once with doxorubicin (10 mg/kg) or vehicle only as described above. Three days later, GCV was administered 5x daily intraperitoneally at 25 mg/kg, or vehicle only was administered. Three different treatment groups of mice included (1) untreated (NT); (2) doxorubicin, no GCV (DOXO + PBS); (3) doxorubicin and GCV (DOXO + GCV). Mice were housed in metabolic cages for 4 days and monitored as described above. The data are presented in Table 5 and Table 6. Table 5

p-value: *<0.05; **<0.01; ***<0.001

Table 6

p-value: *<0.05; **<0.01; ***<0.001

EXAMPLE 7

SENESCENT CELL REDUCTION REDUCES LIKELIHOOD OF SIDE EFFECTS FROM SENESCENCE INDUCING RADIOTHERAPY To examine the role of senescence in contributing to, inducing or increasing the likelihood of side effects resulting from, for example, radiation or chemotherapy used to treat cancer that has already developed. Such side effects may include returning or recurring tumor formation or growth and metastasis. Side effects are monitored in pl6-3MR transgenic mice that are either depleted of senescent cells or have senescent cells (naturally developed or induced). Briefly, tumor cell lines are engineered to express firefly luciferase (fLUC) to enable their detection of tumors and metastases by bioluminescence in a living animal. In particular, a B16-fLUC mouse melanoma cell line and an MMTV- PymT:fLUC mammary carcinoma cell line are generated. The tumor cells are injected into the mice (i.e., B16 into a tail vein; and MMTV-PymT into a mammary fat pad) and small primary tumors are allowed to form over a period of one to four weeks. Then groups of animals are exposed to non-lethal ionizing radiation (IR) or sham-irradiated. Three days after the last irradiation exposure, GCV is administered 5x daily

intraperitoneally at 25 mg/kg or vehicle only is administered. Four different treatment groups of mice include (1) no IR (sham irradiated), no GCV; (2) IR, no GCV; (3) no IR, GCV; and (4) IR, GCV. Bioluminescence in tissues is examined (after administering the rLUC substrate) to monitor tumor formation and mouse survival is also monitored. In addition, mice may be housed in metabolic cages for 4 days to monitor food consumption, water consumption, body mass, spontaneous activity and behavior, voluntary exercise, oxygen consumption, and carbon dioxide production.

EXAMPLE 8

SCREENING FOR AND CHARACTERIZATION OF COMPOUNDS THAT SELECTIVELY SUPPRESS COMPONENTS OF THE SENESCENCE -ASSOCIATED SECRETORY PHENOTYPE (SASP)

To identify small molecules that potentially suppress the senescence- associated secretory (SASP) phenotype, a screening strategy using normal human fibroblasts that were either quiescent or senescent and a library of compounds that are approved for human use was developed as described in further detail below.

Experimental Procedures:

Cell cultures and regents

HCA2 human neonatal foreskin, IMR-90 human fetal lung fibroblasts and T47D human breast cancer cells were obtained and cultured in 3% 0₂ and 10% C0₂ as previously described (Coppe et al., 2008, PLoS Biol. 6:2853-2868; Rodier et al., 2009, Nature Cell Biol. 11 :973-979; Coppe et al., 2010, PLoS ONE 5:e9188). Cells were induced to senesce by X-irradiation (10 Gy) or lentiviral expression of oncogenic RAS or MAP kinase kinase 6 (MK 6), as described (Coppe et al, 2008, PLoS Biol. 6:2853-2868; Rodier et al, 2009, Nature Cell Biol. 11 :973-979; Freund et a/., 2011, EMBO J. 30: 1536-1548). Pre-senescent and senescent cells had 24-h BrdU labeling indices of >75% and <10% respectively (Rodier et al, 2009, Nature Cell Biol. 11 :973- 979); <10% and >70% respectively stained positive for senescence-associated beta- galactosidase activity (Dimri et al, 1995, Proc. Natl. Acad. Sci. USA 92:9363-9367) (Biovision senescence detection kit). HEK293FT packaging cells (Invitrogen) were used to generate lentiviruses. Corticosterone, Cortisol and RU-486 were obtained from Sigma-Aldrich.

Viral vectors and infection

Lentiviruses encoding oncogenic RAS and MK 6 were described (Coppe et al, 2008, PLoS Biol. 6:2853-2868; Freund et al, 2011, EMBO J. 30: 1536- 1548). Lentiviruses encoding shRNAs against GFP (control) and the GR were purchased from Open Biosystems. The lentiviral NF-κΒ reporter- luciferase construct was purchased from SA Biosciences. Lentiviruses were produced and used as described (Coppe et al, 2008, PLoS Biol. 6:2853-2868; Freund et al, 2011, EMBO J. 30: 1536-1548). To limit side effects of infection, viral titers were adjusted to infect 90% of cells, and cultures were subsequently selected in 1 μg/ml puromycin for 3 days. Initial drug screening

The initial drug screen was performed in a 96-well format using automated liquid handling with a Biomek FX (Beckman Coulter, CA). Senescent cells were plated 24 hours after X-irradiation at 7,500 cells per well in 96-well plates. Six days after plating the senescent cells, the pre-senescent cells were plated at 7,500 cells per well in 96-well plates. Twenty-four hours after pre-senescent plating, both pre- senescent and senescent cells were washed and incubated in low (0.2%) serum for 48 hours to arrest cell proliferation of the pre-senescent cells. Drugs from the Prestwick Chemical Library, which contains 1120 bio-available compounds in DMSO, were given to the cells at 2.5 μΜ in media containing 0.2% serum. Forty-eight hours after compound addition, the medium in each well was removed and frozen for assay by ELISA to quantitate the levels of IL-6. The cells, which remained in the wells after the medium was removed, were lysed and ATP levels were measured (ATPlite 1-step assay, Perkin Elmer, MA) to exclude compounds that lowered IL-6 through toxicity (cell death). Experimental wells in each plate were normalized to plate mean or same- plate DMSO controls for the ELISA and ATP assays, respectively.

Subsequent treatments with glucocorticoids

To validate glucocorticoids as SASP regulators, they were added within 15 min after irradiation (unless otherwise indicated). For cells induced to senesce by MKK6 or RAS overexpression, glucocorticoid treatment started 16 hours after infection. Glucocorticoids were re-added in fresh media every other day. Six days after irradiation or selection, cells were given serum- free DMEM with or without

glucocorticoid for 24 hours; the conditioned media were collected and frozen for ELISAs.

Real-time quantitative PCR

Cells (7,500/well) in 96-well plates were lysed and reverse transcribed using the Cells-To-Ct kit (Ambion). Quantitative PCR was performed using the Roche Universal ProbeLibrary (UPL) and following primer-probe combinations: Tubulin- A (Probe 58; F:5'-CTT CGT CTC CGC CAT CAG-3' (SEQ ID NO:25), R:5'-TTG CCA ATC TGG ACA CCA-3' (SEQ ID NO:26)); IL-6 (Probe 45; F:5'-GCC CAG CTA TGA ACT CCT TCT-3' (SEQ ID NO:27), R:5'-GAA GGC AGC AGG CAA CAC-3' (SEQ ID NO:28)); IL-8 (Probe 72; F:5'-AGA CAG CAG AGC ACA CAA GC-3' (SEQ ID NO:29), R:5'-ATG GTT CCT TCC GGT GGT-3 ' (SEQ ID NO:30)); MMP-3 (Probe 36; F:5'-CAA AAC ATA TTT CTT TGT AGA GGA CAA-3' (SEQ ID NO:31), R: 5'-TTC AGC TAT TTG CTT GGG AAA-3' (SEQ ID NO:32)); GR (Probe 34; F: 5'-GAA AGC CAC GCT CCC TTC-3' (SEQ ID NO:33), R: 5'-AGA CTT AGG TGA AAC TGG AAT TGC T-3' (SEQ ID NO:34)); IL-la (Probe 6; F: 5'-GGT TGA GTT TAA GCC AAT CCA-3' (SEQ ID NO:35), R: 5'-TGC TGA CCT AGG CTT GAT GA-3' (SEQ ID NO:36)); ΙκΒα (Probe 86; F: 5'-GGT GCT GAT GTC AAT GCT CA- 3' (SEQ ID NO:37), R: 5'-ACA CCA GGT CAG GAT TTT GC-3' (SEQ ID NO:38)). Western blotting

Cells were lysed in RIPA buffer. Lysates were sonicated (10 sec), followed by centrifugation. Samples were incubated at 70° C for 10 min, loaded on 4- 15% gradient tris-glycine SDS-polyacrylamide gels (Invitrogen) and separated by electrophoresis. Proteins were transferred to PVDF membranes, blocked in TBST 5% milk for 1 hour at room temperature, and probed overnight at 4° C with primary antibodies in blocking buffer. Membranes were washed in TBST, and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room

temperature. Blots were developed using Western detection substrate (GE Healthcare).

Immunofluorescence

Cells were cultured in 8-well chamber slides, fixed in 4% formaldehyde (Sigma) for 10 min at 4° C and permeabilized in PBS-0.5% Triton for 10 min in 4° C. Slides were blocked for 30 min in 4% goat serum (Invitrogen). Primary antibodies were diluted in blocking buffer and incubated with cells for 1 hour at room temperature. Cells were washed, incubated with secondary antibodies for 30 min at room

temperature, washed and mounted with slow-fade gold (Molecular Probes). Images were acquired using an Olympus BX20 fluorescence microscope with the spotfire software (Diagnostics Instruments) and processed with Photoshop CS (Adobe).

Antibodies

Primary antibodies and dilutions were: anti-GR (SC-8992, Santa Cruz;

1 :500), anti-actin (ab6276, Abeam; 1 :50000), anti-MCR (SC-11412, Santa Cruz;

1 :500), anti-IRAKl (SC-5288, Santa Cruz; 1 :500), anti-ΙκΒα (#9247, Cell Signaling;

1 :500), anti-RelA (SC-109, Santa Cruz; 1 :500), and anti-53BPl (A300-272A, Bethyl;

1 :500). Secondary antibodies used for western analysis were: goat anti-mouse IgG HRP conjugate (#170-5047, BioRad; 1 :5000), and goat anti-rabbit IgG HRP conjugate

(#166-2408, BioRad; 1 :5000). Secondary antibody used for immunostaining was Alexa

Fluor 488 goat anti-rabbit IgG (#A11008, Invitrogen; 1 :750).

NF-KB binding activity and transactivation assays

Nuclear extracts were prepared using the nuclear extract kit (Active Motif), and NF-κΒ DNA binding was determined using the TransAM NF-κΒ p65 kit

(Active Motif). For transactivation assays, cells infected with the NF-κΒ reporter- luciferase lentivirus were lysed in buffer (Promega), and luciferase activity was normalized to cell number, as described (Freund et al, 2011, EMBO J. 30: 1536-1548).

Antibody arrays

Cultures were washed and incubated in serum-free DMEM for 24 hours and the conditioned media were diluted to equivalent cell numbers using DMEM. Antibody arrays from Raybiotech (AAH-CYT-G 1000-8) were used according to the manufacturer's instructions. Arrays were scanned using a GenePix 4200A Professional microarray scanner. Signal intensities were quantitated using LI-COR Odyssey software and normalized to positive controls for each sample, which were then normalized across all samples, as previously described (Freund et al, 2011, EMBO J. 30: 1536-1548).

ELISA

Conditioned media were filtered and stored at -80° C. Cell numbers were determined in every experiment. ELISAs were performed using kits and procedures from PerkinElmer (IL-6 AL223F). Data were normalized and expressed as pg/ml/cell/24h.

Invasion assay

T47D human breast cancer cells (120,000 cells/well) were plated atop a layer of Matrigel in the upper chambers of Transwells (BD Biosciences). The lower chambers contained conditioned media from pre-senescent or senescent HCA2 fibroblasts treated with corticosterone or Cortisol for 10 d. After 18 h, cells that migrated to the underside of the upper chamber filter were stained and counted, as described (Coppe et al, 2008, PLoS Biol. 6:2853-2868; Coppe et al, 2010, PLoS ONE 5:e9188).

Statistical analysis

Error bars on all graphs represent the standard deviation of at least 3 independent measurements. For the antibody array, statistical significance between distributions of signals was evaluated using a two-tailed Student's t-test and assumption of equal variance with three conditioned medium samples per condition. Glucocorticoids Suppress Selected Components of the Senescence- Associated Secretory Phenotype

To identify small molecules that may be potential SASP modulators, a screening strategy that entailed administering compounds to parallel 96-well plates containing human fibroblasts (strain HCA2) that were either quiescent or senescent was developed. The compounds tested comprised the Prestwick Chemical Library, a collection of approximately 1,120 Federal Drug Administration-approved drugs. The compounds were added to duplicate wells at a single concentration (2.5 μΜ). After 48 hours, the medium from each well was removed, and the cells were lysed. ELISAs were used to detect the presence of IL-6 in the medium, a major SASP factor, as an indication of whether a compound suppressed or enhanced the SASP. Cell lysates were assayed for ATP as a surrogate for cell number. The results of the ATP assay allowed elimination highly toxic compounds, or compounds that grossly altered cell number.

Of the 1,120 drugs tested, several suppressed IL-6 secretion without altering ATP levels. These drugs, then, were candidates for having the ability to suppress the SASP without causing cell toxicity or reversing the senescence growth arrest. The candidate drugs included the following: corticosterone, Cortisol, prednisone, androsterone, tolazamide, chlorpropamide, gliclazide, finasteride, norgestrel-(-)-D, estradiol- 17-beta, minoxidil,and benfotiamine. Among these candidates the hormones of the glucocorticoid family, such as corticosterone were the most potent.

To confirm the ability of corticosterone to suppress senescence- associated IL-6 secretion, fresh HCA2 fibroblast cultures were prepared, and senescence was induced by X-irradiation (10 Gy). Under these conditions, cells undergo growth arrest within 24-48 hours, but require 4-5 days before SASP

components are detected in the medium (Coppe et al., 2008, PLoS Biol. 6:2853-2868; Rodier et al, 2009, Nature Cell Biol. 11 :973-979; Coppe et al, 2010, PLoS ONE 5:e9188; Freund et al, 2011, EMBO J. 30: 1536-1548). Varying concentrations of corticosterone were added immediately after irradiation, and the cells were maintained in the drug for 6 days. On the 6^th day, the cells were incubated in serum-free medium with or without corticosterone, the conditioned medium was collected 24 h later, and the medium was assayed for IL-6 by ELISA. Corticosterone decreased IL-6 secretion in a dose-dependent manner (Fig. 12A). At 20 nM, corticosterone reduced IL-6 secretion by approximately 50%; maximal suppression (>90%) was achieved at 500 nM. The ability of corticosterone to suppress IL-6 secretion by senescent cells was not peculiar to HCA2 cells. A similar reduction was observed using another human fibroblast strain (IMR-90 from fetal lung) (Fig. 16A).

Corticosterone is the main GR ligand in rodents and other species;

however, in humans, the main GR ligand is the closely related glucocorticoid Cortisol (Gross and Cidlowski, 2008, Trends Endocrinol. Metab. 19:331-339; Zanchi et al, 2010, J. Cell Physiol. 224:311-315). Therefore, Cortisol was tested for the ability to suppress IL-6 secretion by human fibroblasts induced to senesce by X-irradiation. Cortisol decreased IL-6 secretion in a dose-dependent manner, and was more potent than corticosterone (Fig. 12B). Cortisol reduced senescence-associated IL-6 secretion by 50% at sub-nM concentrations (160-800 pM) and >90% at 100 nM.

To determine whether or not, or to what extent corticosterone or Cortisol suppressed the entire SASP, antibody arrays were used to interrogate the relative secretion of 120 cytokines and growth factors. Presenescent and senescent cells were incubated with 500 nM corticosterone or 100 nM Cortisol (Fig. 12C). Both

glucocorticoids strongly suppressed the secretion of several pro-inflammatory cytokines and chemokines, including IL-6, IL-8, GM-CSF and MCP-2. In addition, they suppressed the secretion of several growth and angiogenic factors such as VEGF.

Neither glucocorticoid suppressed all components of the SASP (Fig. 12C), and thus were selective SASP modulators.

The ability of corticosterone and Cortisol to suppress senescence- associated IL-6 secretion was not limited to cells induced to senesce by X-irradiation. Both glucocorticoids were effective in cells induced to senesce by overexpression of oncogenic RAS or MK 6 (mitogen-activated protein kinase kinase 6) (Fig. 12D), which induce a growth arrest, cell enlargement, senescence-associated β-galactosidase (SA-Bgal) expression and a robust SASP (Coppe et al, 2008, PLoS Biol. 6:2853-2868; Freund et al, 2011, EMBO J. 30: 1536-1548).

The suppression of IL-6 secretion by glucocorticoids required that the steroids be present for an extended period during which the SASP is being established. In irradiated cells, which induces senescence synchronously, the SASP takes 3-4 days, beginning 1-2 days after irradiation, to become established (Coppe et al., 2008, PLoS Biol. 6:2853-2868; Rodier et al, 2009, Nature Cell Biol. 11 :973-979). Pretreating cells with corticosterone prior to inducing senescence by X-irradiation, or treating for only 24 hours immediately following irradiation or after establishment of the SASP (7 days after irradiation), had no effect on IL-6 secretion (Fig. 12E). However, continuous exposure to corticosterone for 7 days after irradiation strongly suppressed IL-6 secretion (Fig. 12E).

In contrast to their effects on the SASP, corticosterone and Cortisol had no effect on the fraction of cells that expressed SA-Bgal (Fig. 16B) or the enlarged senescent morphology. Moreover, neither glucocorticoid reversed the senescence growth arrest. Thus, cells made senescent by X-irradiation and treated with

corticosterone or Cortisol for 7 days maintained their low 24 h BrdU labeling index (Fig. 16C). Furthermore, although the SASP depends on constitutive low level DNA damage response (DDR) signaling (Rodier et al. , 2009, Nature Cell Biol. 11 :973-979) emanating from persistent DNA damage foci (Rodier et al., 2011, J. Cell Sci. 124:68- 81), corticosterone and Cortisol had no effect on the number of persistent DNA damage foci in the nuclei of cells induced to senescent by X-irradiation (Fig. 16D; 16E).

These results demonstrate the feasibility of screening for compounds that selectively reduce the secretion of proteins secreted by senescent cells, including the secretion of pro-inflammatory cytokines. The dual approach of assaying cellular ATP levels to detect substantial cell loss or gain coupled to ELISAs for the prototypical SASP protein IL-6 allowed identification of compounds with potential SASP- suppressing activity, but without gross toxicity or, equally importantly, the ability to reverse the senescence growth arrest. Taken together, the data show that corticosterone and Cortisol decrease the secretion of prominent SASP factors without affecting other prominent senescent phenotypes, including the growth arrest, and were efficacious whether cells were induced to senesce by ionizing radiation or strong mitogenic signals delivered by oncogenic RAS or MAP kinase kinase 6 overexpression. Glucocorticoids Mediate Suppression of SASP Through Glucocorticoid

Receptor

Because most SASP factors are upregulated at the level of mRNA abundance (Coppe et al, 2008, PLoS Biol. 6:2853-2868; Coppe et al, 2010, PLoS ONE 5:e9188), the effects of the glucocorticoids on the mRNA levels of three important SASP factors (IL-6, IL-8, MMP-3) (Figure 13A1) were determined. mRNA was extracted from presenescent (Mock) or senescent X-irradiated HCA2 cells treated with DMSO, 500 n corticosterone, or 100 nm Cortisol as described herein. All three mRNAs were strongly reduced by corticosterone and Cortisol (Fig. 13A1), suggesting that the glucocorticoids act at the level of transcription. In a second experiment, mRNA levels of SASP factors IL-5, IL-6, IL-8, MMP-3, IL-la, MCP-2, MCP-3, and GM-CSF were determined (see Figure 13A2).

Glucocorticoids are ligands for GR isoforms, which, upon ligand binding, translocate to the nucleus where they alter the transcription of numerous genes; most of the physiological effects of glucocorticoids depend on the GR (Gross and Cidlowski, 2008, Trends Endocrinol. Metab. 19:331-339; Zanchi et al, 2010, J. Cell Physiol. 224:311-315; Oakley and Cidlowski, 2011, J. Biol. Chem. 286:3177-3184). GR expression levels were measured for change as a consequence of senescence or addition of corticosterone or Cortisol (Fig. 13B). GR mRNA levels appeared to slightly increase in senescent, relative to presenescent, cells and were unaffected by

glucocorticoid addition. The GR was largely cytoplasmic in presenescent cells, and remained cytoplasmic up to 7 days after the cells were induced to senesce by X- irradiation (Fig. 13C). However, the GR translocated into the nucleus in response to either corticosterone or Cortisol (Fig. 13C), indicating that both these glucocorticoids can activate the GR. In contrast, the related mineralocorticoid receptor, which also binds Cortisol and can physically interact with the GR, remained cytoplasmic after corticosterone or Cortisol addition (Fig. 17A). Thus, corticosterone and Cortisol each specifically induce GR nuclear localization in senescent HCA2 cells.

To determine whether the ability of corticosterone and Cortisol to suppress the expression of selected SASP components was mediated by the GR, RNA interference (RNAi) and lentiviruses that express short hairpin (sh) RNAs designed to deplete cells of the GR were used. Quantitative PCR and western blotting confirmed that two distinct shRNAs reduced GR mRNA and protein levels (Fig. 13D; 13E). GR depletion partially rescued the suppression of IL-6 secretion by corticosterone and Cortisol (Fig. 13F). This partial rescue may be due to incomplete GR depletion by the shRNAs (Fig. 13D; 13E). Consistent with these results, co-treatment of senescent cells with corticosterone or Cortisol plus the glucocorticoid antagonist RU-486 (Cadepond et al, 1997, Annu. Rev. Med. 48: 129-156; Lewis-Tuffm et al, 2007, Molec. Cell Biol. 27:2266-2282) rescued the senescence-associated IL-6 secretion that was suppressed by the glucocorticoids (Fig. 13G). RU-486 blocked this glucocorticoid activity without affecting GR nuclear translocalization (Fig. 17B).

Taken together, these results show that both corticosterone and Cortisol induced GR nuclear translocalization in senescent cells, which suppressed IL-la signaling by inhibiting NF-κΒ DNA binding and transactivation activity. Moreover, because genetic or pharmacological inhibition (RU-486) of the GR rescued the suppression of senescence-associated IL-6 secretion by glucocorticoids, the results suggest the GR is required for the suppressive effects of glucocorticoids in senescent cells.

Glucocorticoids suppress the expression of IL-l , An Upstream SASP Regulator

It has been previously demonstrated that IL-l is a critical upstream regulator of the SASP (senescence-associated IL-6/IL-8 cytokine network) (Orjalo et al, 2009, Proc. Natl. Acad. Sci. USA 106: 17031-17036). IL-la establishes and maintains the SASP by activating the transcription factor nuclear factor-kappa B (NF- KB) (Orjalo et al, 2009, Proc. Natl. Acad. Sci. USA 106: 17031-17036; Freund et al, 2011, EMBO J. 30: 1536-1548), which further stimulates IL-la transcription, thereby establishing a positive feedback loop (Freund et al, 2010, Trends Molec. Med. 16:238- 248). This positive feedback loop leads to increased NF-κΒ activation and, consequently, the transcription of several SASP factors.Therefore, whether

glucocorticoids suppressed the SASP by interfering with IL-la expression was examined.

IL- 1 a mRNA rose rapidly after cells were induced to senesce by X- irradiation (Fig. 14A). When added at the time of irradiation, both corticosterone and Cortisol delayed this rise, as well as the later rise in IL-6 mRNA (Fig. 14A; 14B).

Further, the glucocorticoids continued to suppress IL-la and IL-6 mRNA levels (<10% of control) for at least 7 days after irradiation, at which time the SASP is normally fully developed (Coppe et al, 2008, PLoS Biol. 6:2853-2868; Rodier et al, 2009, Nature Cell Biol. 11 :973-979).

IL-la localizes to both the plasma membrane and the nucleus (Werman et al, 2004, Proc. Natl. Acad. Sci. USA 101 :2434-2439; Orjalo et al, 2009, Proc. Natl. Acad. Sci. USA 106: 10731-10736). Consistent with the suppression of IL-la mRNA levels, corticosterone and Cortisol also suppressed expression of IL-la protein, which was visible as strong nuclear staining in control, but not glucocorticoid-treated, senescent cells (Fig. 14C).

Glucocorticoids Impair the IL-l /NF-κΒ Pathway

To determine whether glucocorticoids suppress the SASP by suppressing

IL-la signaling, the abundance of interleukin-1 receptor-associated kinase 1 (IRAKI) and ΙκΒα, an inhibitor of NF-κΒ were measured. Both these proteins are key components of IL-l /IL-1 receptor (IL-1R) signaling (Perkins, 2007, Nature Rev.

Molec. Cell Biol. 8:49-62; Gottipati et al, 2008, Cell Signal. 20:269-276), and are rapidly degraded after the IL-1R is engaged by IL-la (Perkins, 2007, Nature Rev.

Molec. Cell Biol. 8:49-62; Gottipati et al, 2008, Cell Signal. 20:269-276; Orjalo et al, 2009, Proc. Natl. Acad. Sci. USA 106:17031-17036). IRAKI and ΙκΒα were much less abundant in senescent, compared to presenescent, cells, indicating active IL-1R signaling in senescent cells (Fig. 15 A). The abundance of RelA, an NF-κΒ subunit, was unchanged. Consistent with the suppression of IL-l production and blockade of

IL-1R signaling, corticosterone and Cortisol restored IRAKI and ΙκΒα proteins to near- presenescent levels (Fig. 15 A). Moreover, the glucocorticoids had no effect on ΙκΒα mRNA levels (Fig. 18), suggesting they acted indirectly to reduce protein levels and consistent with their effect on IL-la mRNA levels.

Recombinant IL-l rescued the suppression of IL-6 secretion by corticosterone and Cortisol (Fig. 15B), consistent with the idea that glucocorticoids suppress SASP components such as IL-6 by targeting IL-la/IL-lR signaling. Because

GRs are known to modulate NF-κΒ activity, one potential mechanism by which glucocorticoids might act in this regard is by inhibiting NF-κΒ activity. In support of this model, corticosterone and Cortisol significantly decreased both NF-κΒ DNA binding and transactivation activity in senescent cells (Fig. 15C; 15D, 15F).

Furthermore, co-treatment of senescent cells with either of the glucocorticoids plus RU- 486 (glucocorticoid antagonist) or recombinant IL-l rescued NF-κΒ transactivation activity (Fig. 15D). Without wishing to be bound by theory, glucocorticoids, acting via the GR, appear to suppress the SASP at least in part by preventing establishment of the IL-l /NF-κΒ positive feedback loop that ultimately drives the expression and secretion of SASP components by impairing IL-l expression. Once established, however, the feedback loop appears to be unaffected by glucocorticoids. Thus, the transcriptional landscape that allows establishment of the SASP may differ from the transcriptional landscape that maintains it.

Glucocorticoids Suppress The Ability Of The SASP To Stimulate Tumor Cell Invasion

Senescent cells secrete factors that can stimulate aggressive cancer- associated phenotypes in premalignant or malignant cells (Krtolica et al. , 2001 , Proc. Natl. Acad. Sci. USA 98: 12072-12077; Liu and Hornsby, 2007, Cancer Res. 67:3117- 3126; Coppe et al, 2008, PLoS Biol. 6:2853-2868; Bartholomew et al, 2009, Cancer Res. 69:2878-2886; Coppe et al, 2010, PLoS ONE 5:e9188). Therefore, whether glucocorticoids suppressed the ability of the SASP to stimulate non-aggressive human breast cancer cells (T47D) to invade a basement membrane in Boyden chambers was investigated. Conditioned media prepared from presenescent cells stimulated minimal invasion by T47D cells, whereas media from senescent cells stimulated 4-fold more invasion (Fig. 15E), as expected. Both corticosterone and Cortisol reduced the ability of senescent conditioned media to stimulate T47D invasiveness to near-presenescent levels. Thus, in addition to suppressing the secretion of multiple SASP factors, the glucocorticoids suppressed an important biological property of the SASP.

Flavonoid Apigenin Suppresses Selected Components of the Senescence- Associated Secretory Phenotvpe

Using the screening protocol described previously for identifying potential SASP modulators from a compound library using HCA2 human fibroblasts that were either quiescent (non-senescent; mock-irradiated) or induced to senescence by X-irradiation, flavonoid was also identified, along with glucocorticoids, as being capable of suppressing IL-6 secretion without altering ATP levels. Apigenin (4', 5, 7- trihydorxyflavone) is a naturally occurring plant flavone present in common fruits and vegetables.

To determine whether or to what extent apigenin suppressed the entire

SASP, multiplex ELISA was used to interrogate the relative secretion of 50 cytokines and growth factors. IMR90 fibroblasts were treated with 10 μΜ apigenin or DMSO immediately after irradiation and analyzed 6 days later. Cells were washed and incubated in serum-free media without apigenin to generate conditioned media.

Conditioned media from non-senescent, apigenin- or DMSO-treated irradiated IMR90 cells and control (mock-irradiated, DMSO-treated) cells were analyzed by ELISA for various SASP factors (Figure 19). As shown in Figure 19, apigenin suppressed the secretion of several pro-inflammatory cytokines and chemokines, including, for example, TGFA, MCP3, LIF, ΠΤΝΓβ, IL-6, GROA, and IL-2. Those SASP factors suppressed by less than 2 fold were not considered to be significantly suppressed.

The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet, including but not limited to U.S. Patent Application No. 61/837,089, filed June 19, 2013, are incorporated herein by reference, in their entirety. Aspects of the

embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.

These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible

embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

Claims

CLAIMS We claim the following:

1. A method for ameliorating toxicity of a medical therapy in a subject who receives the medical therapy, which medical therapy induces cellular senescence of one or more cells in the subject, said method comprising administering to the subject an agent that selectively destroys or facilitates selective destruction of the one or more of the senescent cells induced by the medical therapy.

2. The method of claim 1 wherein the toxicity is acute toxicity.

3. The method of claim 1 or claim 2, wherein the medical therapy induces cellular senescence of one or more normal cells.

4. The method of any one of claims 1-3, wherein the agent is administered to the subject at least 2 days, 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 10 days, or at least 14 days at least 30 days, at least 60 days, or at least 90 days subsequent to administration of the medical therapy.

5. The method of claim 4 wherein the agent is administered to the subject at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 10 days, or at least 14 days subsequent to administration of the medical therapy.

6. The method of any one of claims 1-5 wherein the medical therapy comprises radiation, a chemotherapy, an anti-viral therapy, or a hormone.

7. The method of any one of claims 1-6 wherein the subject has a cancer, is in cancer remission, is at risk of developing a recurrence of a cancer, or has a predisposition for developing a cancer, and wherein the medical therapy comprises an anti-cancer therapy.

8. The method of any one of claims 1-7, wherein the medical therapy is chemotherapy or radiation and the subject has a cancer.

9. The method of claim 8, wherein the cancer comprises a solid tumor or a liquid tumor.

10. The method of claim 8 or claim 9, wherein the cancer is metastatic cancer.

11. The method of any one of claims 8-10, wherein the subject has a cancer and has received or will receive a stem cell transplant, and wherein the medical therapy comprises high dose chemotherapy or high dose radiotherapy or a combination thereof.

12. The method of claim 11 wherein the stem cell transplant is selected from (a) an autologous stem cell transplant, and (b) an allogenic stem cell transplant.

13. The method of claim 6 wherein the anti-viral therapy is an HIV/ AIDS management therapy.

14. The method of claim 13 wherein the HIV/ AIDS management therapy comprises a highly active antiretro viral therapy (HAART).

15. The method of any one of claims 1-14 wherein toxicity comprises acute toxicity comprising energy imbalance.

16. The method of claim 15 wherein energy imbalance comprises low physical activity.

17. The method of claim 1 wherein the toxicity comprises chronic toxicity.

18. The method of any one of claims 1-17 wherein the agent specifically binds to a senescent cell associated antigen and inhibits a function of the antigen, thereby disrupting the integrity of the cell membrane, inhibiting one or more metabolic processes in the cell necessary for cell survival, or disrupting transcription of a gene or translation of a protein necessary for cell survival.

19. The method of any one of claims 1-17 wherein the agent induces apoptosis of the senescent cells.

20. The method of any one of claims 1-17 wherein the agent induces an immune response specific for the senescent cells and which immune response comprises removal of the senescent cells.

21. The method of any one of claims 1-20, wherein the agent is a small molecule, polypeptide, peptide, antibody, antigen-binding fragment, peptibody, recombinant viral vector, or a nucleic acid.

22. A method for inhibiting metastasis of a cancer and for ameliorating toxicity of a medical therapy in a subject who has the cancer and receives the medical therapy, which medical therapy induces cellular senescence of one or more cells in the subject, said method comprising administering to the subject an agent that selectively destroys or facilitates selective destruction of the one or more senescent cells, wherein the medical therapy comprises chemotherapy, radiation therapy, or both chemotherapy and radiation therapy.

23. The method of claim 22 wherein the medical therapy induces cellular senescence of one or more normal cells.

24. The method of claim 22 or claim 23 wherein the toxicity is acute toxicity.

25. A method for ameliorating acute toxicity of a medical therapy in a subject who receives the medical therapy, which medical therapy induces cellular senescence of one or more normal cells in the subject, said method comprising administering to the subject an agent that selectively destroys or facilitates selective destruction of the one or more senescent cells induced by the medical therapy.

26. Use of an agent that selectively destroys or facilitates selective destruction of one or more senescent cells for ameliorating toxicity of a medical therapy in a subject who receives the medical therapy, which medical therapy induces cellular senescence of one or more cells in the subject.

27. Use of an agent that selectively destroys or facilitates selective destruction of one or more senescent cells for inhibiting metastasis of a cancer and for ameliorating toxicity of a medical therapy, which is administered to a subject who has a cancer and which medical therapy induces cellular senescence of one or more cells in the subject, wherein the medical therapy comprises chemotherapy, radiation therapy, or both chemotherapy and radiation therapy.

28. Use of either claim 26 or claim 27 wherein the toxicity is acute toxicity.

29. Use of an agent that selectively destroys or facilitates selective destruction of one or more senescent cells for ameliorating acute toxicity of a medical therapy in a subject who receives the medical therapy, which medical therapy induces cellular senescence of one or more normal cells in the subject.

30. The method of any one of claims 1-25 further comprising administering to the subject a second agent that depletes one or more senescence cell- associated molecules produced by a senescent cell.

31. The method of claim 30 wherein the second agent is administered after administering the agent that selectively destroys or facilitates selective destruction of the one or more senescent cells induced by the medical therapy.

32. The method of claim 30 wherein the second agent is administered concurrently with the agent that selectively destroys or facilitates selective destruction of the one or more senescent cells induced by the medical therapy.

33. The method of claim 30 wherein the second agent is administered prior to administering the agent that selectively destroys or facilitates selective destruction of the one or more senescent cells induced by the medical therapy.

34. The method of any one of claims 30-33 wherein the second agent is a small molecule, polypeptide, peptide, antibody, antigen-binding fragment, peptibody, recombinant viral vector, or a nucleic acid.

35. The use of any one of claims 26-29 further comprising use of a second agent that depletes one or more senescence cell-associated molecules produced by a senescent cell.

36. The use of claim 35 wherein the agent that selectively destroys or facilitates selective destruction of the one or more senescent cells induced by the medical therapy is for the preparation of a first medicament and the second agent is for the preparation of a second medicament, wherein the second medicament is suitable for administration after the administration of the first medicament.

37. The use of claim 35 wherein the agent that selectively destroys or facilitates selective destruction of the one or more senescent cells induced by the medical therapy is for the preparation of a first medicament and the second agent is for the preparation of a second medicament, wherein the first medicament and the second medicament are suitable for concurrent administration.

38. The use of claim 35 wherein the agent that selectively destroys or facilitates selective destruction of the one or more senescent cells induced by the medical therapy is for the preparation of a first medicament and the second agent is for the preparation of a second medicament, wherein the second medicament is suitable for administration prior to the administration of the first medicament.