WO2017119988A2 - Radioprotection by wnt activation - Google Patents

Abstract

The present invention encompasses the recognition that a multipotent actively cycling intestinal stem cell population exists in the bottom of intestinal crypts and is highly sensitive to irradiation. In some embodiments, the present invention recognizes that proliferation of stem cells in the gut after exposure of a subject to radiation can be beneficial to the subject.

Description

RADIOPROTECTION BY WNT ACTIVATION

GOVERNMENT SUPPORT

[0001] This invention was made with government support under CA013106 and

OD020355 awarded by National Institutes of Health. The government has certain rights in the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application Serial No.

62/265,850, filed on December 10, 2015, the contents of which are herein incorporated by reference in their entirety.

BACKGROUND

[0003] Radiation therapy (RT) is used in the treatment of a broad range of malignancies, but may result in acute and/or chronic toxicities. For example, RT may result in damage to the lining of the intestines, a condition known as radiation enteritis. Radiation enteritis is a major limiting factor in abdominopelvic RT and is also predicted to be a major source of morbidity in the event of nuclear/radiological terrorism.

SUMMARY

[0004] The present disclosure provides, among other things, methods of treatment for subjects suffering from or susceptible to toxicities resulting from radiation exposure. The present disclosure encompasses a recognition that proliferation and expansion of undifferentiated stem cells can mitigate certain toxic effects associated with radiation exposure or therapy. In some embodiments, treatments that induce proliferation of stem cells in the gastrointestinal tract after exposure of a subject to radiation are beneficial to the subject. For example, the present disclosure provides an insight that therapies that induce intestinal stem cell hyperproliferation and/or block stem cell differentiation may be beneficial for a subject suffering from or susceptible to radiation induced gastrointestinal syndrome (RIGS). In some embodiments, the present disclosure provides methods of treating or preventing RIGS in a subject at risk thereof, the method comprising administering an agent that induces intestinal stem cell proliferation and/or blocks stem cell differentiation.

[0005] The present disclosure provides an appreciation that activation of Wnt signaling is beneficial to a subject exposed to radiation. For example, the present disclosure provides an insight that activation of Wnt signaling in the intestine can mitigate RIGS in a subject. In some embodiments, activation of Wnt signaling in the intestine increases proliferation of

undifferentiated stem cells in the intestinal epithelium. In some embodiments, activation of the canonical Wnt signaling pathway (i.e., Wnt/p-catenin pathway) increases proliferation of undifferentiated stem cells in the intestinal epithelium. In some embodiments, the present disclosure provides methods of treating or preventing RIGS in a subject at risk thereof, the method comprising administering an agent that activates and/or increases Wnt signaling. In some embodiments, Wnt signaling may be activated and/or increased directly. In some embodiments, Wnt signaling may be activated and/or increased indirectly. In some

embodiments, Wnt signaling may be activated and/or increased by inhibition or knock down of Adenomatous Polyposis Coli (APC). In some embodiments, Wnt signaling may be activated and/or increased by inhibition or knock down of GSK-3p.

[0006] In some embodiments, the present disclosure provides methods of treating or preventing RIGS in a subject at risk thereof, the method comprising administering an agent that inhibits, reduces expression and/or reduces function of APC. In some embodiments, the present disclosure provides methods of treating or preventing RIGS in a subject at risk thereof, the method comprising administering an agent that inhibits, reduces expression and/or reduces function of GSK-3p. In some embodiments, an agent that reduces the expression, activity and/or function of APC or GSK-3P is an antibody, antibody fragment, aptamer, siRNA, shRNA, DNA/RNA hybrid, antisense oligonucleotide, ribozyme, peptide, peptide mimetic, lipid, or a small molecule.

[0007] In some embodiments, the present disclosure provides methods of treating or preventing RIGS in a subject at risk thereof, the method comprising administering a radiation modifier, wherein the radiation modifier activates Wnt signaling. In some embodiments the radiation modifier attenuates or inhibits APC and/or GSK-3p. In some embodiments, the present disclosure provides methods of treating or preventing RIGS in a subject at risk thereof, the method comprising administering a radiation mitigator, wherein the radiation mitigator activates Wnt signaling. In some embodiments the radiation mitigator attenuates or inhibits APC and/or GSK-3p.

[0008] In some embodiments, a subject for treatment by methods described herein has received or will receive radiation therapy. In some embodiments, a subject for treatment by methods described herein has been or will be exposed to non-therapeutic radiation. In some embodiments, a subject for treatment by methods described herein has been or will be exposed to a radiation dose between about 3 Gy and about 150 Gy.

[0009] In some embodiments, a Wnt-activating agent is delivered before, during or after exposure to ionizing radiation. In some embodiments, a Wnt-activating agent is delivered before, during or after radiation therapy. In some embodiments, a Wnt-activating agent is cleared or otherwise rendered ineffective in vivo after a period of time. In some embodiments, the period of time for clearance and/or inactivation of a Wnt-activating agent is 3 hours, 6 hours, 9 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 72 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months or 6 months. In some embodiments, a Wnt-activating agent is an agent that reduces the expression and/or activity of APC. In some embodiments, a Wnt- activating agent is an agent that reduces the expression and/or activity of GSK-3p. In some embodiments, an agent that reduces the expression and/or activity of APC and/or GSK-3P is cleared or inactivated after a predetermined period of time.

[0010] In some embodiments, the present disclosure provides methods of treating or preventing RIGS in a subject at risk thereof, the method comprising delivering a Wnt-activating agent to a subject in need thereof. In some embodiments, a Wnt-activating agent is delivered in a regulatable manner. In some embodiments, a Wnt-activating agent delivered in sustained, delayed, pulsed or other controlled manner.

[0011] In some embodiments, a subject suffering from or susceptible to RIGS has undergone radiation therapy and/or is undergoing radiation therapy and/or will undergo radiation therapy. In some embodiments, a subject who has received, is receiving or will receive radiation therapy is suffering from cancer. In some embodiments, a subject who has received, is receiving or will receive radiation therapy is suffering from a gastrointestinal or hepatobiliary malignancy.

[0012] In some embodiments, the present disclosure provides methods of treating or preventing chemotherapy induced enteritis. In some embodiments, a subject who has received, is receiving or will receive chemotherapy. In some embodiments, a subject who has received, is receiving or will receive chemotherapy is suffering from cancer. In some embodiments, a subject who has received, is receiving or will receive chemotherapy is suffering is colorectal cancer. In some embodiments, a subject who has received, is receiving or will receive chemotherapy is suffering from a gastrointestinal or hepatobiliary malignancy.

[0013] The present disclosure encompasses a recognition that Wnt-activation may permit dose escalation of radiotherapy and/or chemotherapy. In some embodiments, treatment with a Wnt-activating agent increases the LD50 dose of radiation and/or chemotherapy. In some embodiments, a subject treated with a Wnt-activating agent may receive an increased dose of radiation and/or chemotherapy.

BRIEF DESCRIPTION OF THE DRAWING

[0014] The Drawing included herein, which is composed of the following Figures, is for illustration purposes only not for limitation.

[0015] Figure 1 shows a diagram depicting the hierarchical organization of a small intestine villus. Differentiated cell populations depicted include enterocytes, enteroendocrine cells, and Goblet cells. The intestinal crypt includes highly proliferative transient amplifying cells (TA cells), 4+ 'reserve' stem cells, Lgr5+ intestinal stem cells and niche supporting Paneth cells.

[0016] Figure 2 shows a schematic representing an approach to generate genetically engineered mouse models with inducible shRNA technology. Inducible shRNA mice as depicted can mimic loss or reduction of gene function for any gene in vivo. Cre-dependent tet- transactivator (rtTA) expression that relies on tissue specific promoters, which allows tissue- restricted tetracycline-regulated element (TRE) shRNA expression. Induction of the TRE-driven GFP-miRNA cassette is doxycycline (dox) dependent, and so is inducible and reversible. [0017] Figure 3 illustrates the effects of transient activation of Wnt signaling in vivo.

Doxycycline-induced shApc expression drives Wnt-mediated hyperproliferation and expansion of undifferentiated intestinal stem cells and results in disruption of the crypt-villus axis, whereby stem and progenitor cell, normally restricted to the crypt base, expand and fail to differentiate as they migrate up the villus. Ape restoration induces a rapid and dramatic response with normalization to endogenous Wnt levels and restoration of crypt-villus homeostasis after 4 days of dox withdrawal.

[0018] Figure 4 depicts Whole Abdomen Irradiation (WAI) dosimetry. WAI is performed using a small animal micro-irradiator. Contrast agents and micro-CT technology were employed for real-time animal dosimetry.

[0019] Figure 5 depicts weight loss seen in mice irradiated with increasing doses of whole abdomen irradiation (WAI) . Daily weight was determined for mice treated by WAI in increasing amounts (10 Gy (n=6), 12, Gy (n=6), 14 Gy (n=7), 16 Gy (n=7), and 18 Gy (n=7)). Mean percentage of weight loss was determined over an 8 day period.

[0020] Figure 6 depicts mortality of animals irradiated with increasing doses of whole abdomen irradiation (WAI) at 10 days after irradiation. Doses tested were from 6 Gy up to 18 Gy. The LD50 dose at 10 days was approximately 11 Gy.

[0021] Figure 7 depicts a graph with survival curves of animals treated with increasing amounts of irradiation by WAI. Survival over a 30 day period post- WAI was assessed.

Prolonged survival (through 30 days) was observed in the majority of animals exposed to 10 Gy or less during WAI. Survival steeply declined in animals treated with doses of irradiation greater that 10 Gy during WAI.

[0022] Figure 8 depicts the effect of animal age on survival post WAI. Mortality of 60 day old and 120 day old animals irradiated with increasing doses of whole abdomen irradiation (WAI) was determined after 10 days post- WAI. Older animals exhibited increased sensitivity to WAI. The LD50 dose at 10 days post- WAI was found to be approximately 11 Gy in 60 day old animals and 9 Gy in 120 day old animals. [0023] Figure 9 depicts a schematic of an exemplary experimental timeline for testing the effects of genetic manipulation (through inducible knock down in a genetically engineered mouse model) on irradiation (e.g., WAI).

[0024] Figure 10 depicts effects of transient Wnt signaling activation on irradiation induced weight loss. Ape knockdown (Wnt activated) mice and control mice were treated with WAI in increasing amounts (10 Gy, 12, Gy, and 14 Gy). Mean percentage of weight loss was determined over a two week period. Activation of Wnt signaling by transient knock down of Ape (shAPC), reduced the mean percentage of weight loss over time at all levels of irradiation.

[0025] Figure 11 depicts weight loss seen in genetically engineered mice irradiated with increasing doses of whole abdomen irradiation (WAI). Mice genetically engineered to transiently knockdown BRD4 (shBRD4), APC (shAPC) or control mice were treated by WAI in increasing amounts (10 Gy, 12, Gy, and 14 Gy). Knock down of BRD4 (radiosensitized control) strongly increased weight loss in animals irradiated with 10 Gy and 12 Gy doses. Conversely, knock down of APC expression generally decreased percentage of weight loss relative to control animals.

[0026] Figure 12 depicts effects of transient Wnt signaling activation on animal mortality after whole abdomen irradiation (WAI) was assessed. Mice were treated by WAI at incremental doses from 6 Gy up to 18 Gy and mortality was assessed 10 days after irradiation. Knockdown of APC resulted in an increase in the LD50 dose of irradiation at 10 days post-WAI from approximately 13 Gy (compared to 11 Gy for control animals).

[0027] Figure 13 depicts survival curves of wild-type animals and animals with transient activation of Wnt following irradiation. Specifically, survival of genetically engineered mice that transiently knock down APC (shAPC) and control mice was assessed over a 30 day period following WAI in increasing amounts (10 Gy, 12, Gy, and 14 Gy).

[0028] Figure 14 depicts survival of genetically engineered mice with inducible knockdown: shAPC mice (Wnt activated), shBRD4 mice (radiosensitized control), p53

(radioresistant control) and control mice (shRNA-Renilla (shRNA control)) over a 30 day period following WAI at 10 Gy, 12, Gy, and 14 Gy doses. [0029] Figure 15 shows mini-gut organoid models with engineered shRNA technology.

Figure 15A depicts the effects of Wnt activation (by acute APC loss and restoration). Wnt activation results in a hyperproliferative epithelium with a block in differentiation, while restoration of APC normalizes proliferative indices and induces rapid differentiation. Figure 15B depicts a schematic representation of an experimental timeline to assess the effects of transient genetic manipulation after radiation on organoid cell culture. Exemplary results with shAPC organoids treated with dox post RT demonstrates that Wnt activation increased survival and passage capacity as compared to control organoids.

[0030] Figure 16 depicts effects of Wnt activation in combination with Kras and p53 mutations on irradiation of mini-gut organoid models.

[0031] Figure 17 is related to patient-derived cholangiocarcinoma xenografts. A depicts an exemplary acquisition workflow. B shows that patient derived cholangiocarcinoma xenografts have representative histology. C shows that cholangiocarcinoma PDXs (tumor xenografts) maintain the genetic configuration of the primary tumor.

DEFINITIONS

[0032] This invention is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention is defined by the claims.

[0033] Unless defined otherwise, all terms and phrases used herein include the meanings that the terms and phrases have attained in the art, unless the contrary is clearly indicated or clearly apparent from the context in which the term or phrase is used. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, particular methods and materials are now described. All publications mentioned are hereby incorporated by reference.

[0034] Administration: As used herein, the term "administration" typically refers to the administration of a composition to a subject or system to achieve delivery of an agent (e.g., a Wnt-activating agent) that is, or is included in, the composition. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc.. In some particular embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e. g. intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.

[0035] Agent : In general, the term "agent", as used herein, may be used to refer to a compound or entity of any chemical class including, for example, a polypeptide, nucleic acid, saccharide, lipid, small molecule, metal, or combination or complex thereof. In appropriate circumstances, as will be clear from context to those skilled in the art, the term may be utilized to refer to an entity that is or comprises a cell or organism, or a fraction, extract, or component thereof. Alternatively or additionally, as context will make clear, the term may be used to refer to a natural product in that it is found in and/or is obtained from nature. In some instances, again as will be clear from context, the term may be used to refer to one or more entities that is man- made in that it is designed, engineered, and/or produced through action of the hand of man and/or is not found in nature. In some embodiments, an agent may be utilized in isolated or pure form; in some embodiments, an agent may be utilized in crude form. In some embodiments, potential agents may be provided as collections or libraries, for example that may be screened to identify or characterize active agents within them. In some cases, the term "agent" may refer to a compound or entity that is or comprises a polymer; in some cases, the term may refer to a compound or entity that comprises one or more polymeric moieties. In some embodiments, the term "agent" may refer to a compound or entity that is not a polymer and/or is substantially free of any polymer and/or of one or more particular polymeric moieties. In some embodiments, the term may refer to a compound or entity that lacks or is substantially free of any polymeric moiety.

[0036] Agonist: Those skilled in the art will appreciate that the term "agonist" may be used to refer to an agent condition, or event whose presence, level, degree, type, or form correlates with increased level and/or activity of another agent (i.e., the agonized agent). In general, an agonist may be or include an agent of any chemical class including, for example, small molecules, polypeptides, nucleic acids (e.g., siRNA, shRNA, DNA/RNA hybrid, antisense oligonucleotide, ribozyme, etc.), carbohydrates, lipids, metals, and/or any other entity that shows the relevant activating activity. In some embodiments, an agonist may be direct (in which case it exerts its influence directly upon its target); in some embodiments, an agonist may be indirect (in which case it exerts its influence by other than binding to its target; e.g., by interacting with a regulator of the target, so that level or activity of the target is altered).

[0037] Amelioration: as used herein, the term "amelioration" refers to the prevention, reduction or palliation of a state, or improvement of the state of a subject. Amelioration includes, but does not require complete recovery or complete prevention of a disease, disorder or condition (e.g., radiation injury).

[0038] Animal: as used herein refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans, of either sex and at any stage of development. In some embodiments, "animal" refers to non-human animals, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In some embodiments, an animal may be a transgenic animal, genetically engineered animal, and/or a clone.

[0039] Antagonist: Those skilled in the art will appreciate that the term "antagonist", as used herein, may be used to refer to an agent condition, or event whose presence, level, degree, type, or form correlates with decreased level or activity of another agent (i.e., the inhibited agent, or target). In general, an antagonist may be or include an agent of any chemical class including, for example, small molecules, polypeptides, nucleic acids (e.g., siRNA, shRNA, DNA/RNA hybrid, antisense oligonucleotide, ribozyme, etc.), carbohydrates, lipids, metals, and/or any other entity that shows the relevant inhibitory activity. In some embodiments, an antagonist may be direct (in which case it exerts its influence directly upon its target); in some embodiments, an antagonist may be indirect (in which case it exerts its influence by other than binding to its target; e.g., by interacting with a regulator of the target, so that level or activity of the target is altered).

[0040] Antibody agent: As used herein, the term "antibody agent" refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that includes immunoglobulin structural elements sufficient to confer specific binding. Exemplary antibody agents include, but are not limited to monoclonal antibodies or polyclonal antibodies. In some embodiments, an antibody agent may include one or more constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, an antibody agent may include one or more sequence elements are humanized, primatized, chimeric, etc., as is known in the art. In many

embodiments, the term "antibody agent" is used to refer to one or more of the art-known or developed constructs or formats for utilizing antibody structural and functional features in alternative presentation. For example, embodiments, an antibody agent utilized in accordance with the present invention is in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multi- specific antibodies (e.g., Zybodies®, etc); antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies;

masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals ("SMIPs™ ); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies;, Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc], or other pendant group [e.g., poly-ethylene glycol, etc.]. In many embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR); in some

embodiments an antibody agent is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and/or at least one light chain CDR) that is substantially identical to one found in a reference antibody. In some embodiments an included CDR is substantially identical to a reference CDR in that it is either identical in sequence or contains between 1-5 amino acid substitutions as compared with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), or 100%) sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 96%, 96%, 97%, 98%, 99%), or 100%) sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, an antibody agent is a polypeptide protein having a binding domain which is homologous or largely homologous to an immunoglobulin-binding domain. In some embodiments, an antibody agent is or comprises an antibody-drug conjugate.

[0041] Approximately: As used herein, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%), 4%), 3%), 2%), 1%), or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0042] Biologically Active: as used herein, refers to an observable biological effect or result achieved by an agent or entity of interest. For example, in some embodiments, a specific binding interaction is a biological activity. In some embodiments, modulation (e.g., induction, enhancement, or inhibition) of a biological pathway or event is a biological activity. In some embodiments, presence or extent of a biological activity is assessed through detection of a direct or indirect product produced by a biological pathway or event of interest.

[0043] Cancer: The terms "cancer", "malignancy", "neoplasm", "tumor", and

"carcinoma", are used interchangeably herein to refer to cells that exhibit relatively abnormal, uncontrolled, and/or autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. In general, cells of interest for detection or treatment in the present application include precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and non-metastatic cells. The teachings of the present disclosure may be relevant to any and all cancers. To give but a few, non-limiting examples, in some embodiments, teachings of the present disclosure are applied to one or more cancers such as, for example, hematopoietic cancers including leukemias, lymphomas (Hodgkins and non- Hodgkins), myelomas and myeloproliferative disorders; sarcomas, melanomas, adenomas, carcinomas of solid tissue, squamous cell carcinomas of the mouth, throat, larynx, and lung, liver cancer, genitourinary cancers such as prostate, cervical, bladder, uterine, and endometrial cancer and renal cell carcinomas, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, head and neck cancers, breast cancer, gastro-intestinal cancers (e.g., colorectal cancers) and nervous system cancers, benign lesions such as papillomas, and the like.

[0044] Chemotherapeutic Agent. The term "chemotherapeutic agent", has used herein has its art-understood meaning referring to one or more pro-apoptotic, cytostatic and/or cytotoxic agents, for example specifically including agents utilized and/or recommended for use in treating one or more diseases, disorders or conditions associated with undesirable cell proliferation. In many embodiments, chemotherapeutic agents are useful in the treatment of cancer. In some embodiments, a chemotherapeutic agent may be or comprise one or more alkylating agents, one or more anthracyclines, one or more cytoskeletal disruptors (e.g. microtubule targeting agents such as taxanes, maytansine and analogs thereof, of), one or more epothilones, one or more histone deacetylase inhibitors HDACs), one or more topoisomerase inhibitors (e.g., inhibitors of topoisomerase I and/or topoisomerase II), one or more kinase inhibitors, one or more nucleotide analogs or nucleotide precursor analogs, one or more peptide antibiotics, one or more platinum- based agents, one or more retinoids, one or more vinca alkaloids, and/or one or more analogs of one or more of the following (i.e., that share a relevant anti-proliferative activity). In some particular embodiments, a chemotherapeutic agent may be or comprise one or more of

Actinomycin, All-trans retinoic acid, an Auiristatin, Azacitidine, Azathioprine, Bleomycin, Bortezomib, Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide, Curcumin, Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Etoposide, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Imatinib, Irinotecan,

Maytansine and/or analogs thereof (e.g. DM1) Mechlorethamine, Mercaptopurine, Methotrexate, Mitoxantrone, a Maytansinoid, Oxaliplatin, Paclitaxel, Pemetrexed, Teniposide, Tioguanine, Topotecan, Valrubicin, Vinblastine, Vincristine, Vindesine, Vinorelbine, and combinations thereof. In some embodiments, a chemotherapeutic agent may be utilized in the context of an antibody-drug conjugate. In some embodiments, a chemotherapeutic agent is one found in an antibody-drug conjugate selected from the group consisting of: hLLl -doxorubicin, hRS7-SN-38, hMN-14-SN-38, hLL2-SN-38, hA20-SN-38, hPAM4-SN-38, hLLl-SN-38, hRS7-Pro-2-P-Dox, hMN-14-Pro-2-P-Dox, hLL2-Pro-2-P-Dox, hA20-Pro-2-P-Dox, hPAM4-Pro-2-P-Dox, hLLl- Pro-2-P-Dox, P4/D10-doxorubicin, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, glembatumomab vedotin, SAR3419, SAR566658, BIIB015, BT062, SGN-75, SGN-CD19A, AMG-172, AMG-595, BAY-94-9343, ASG-5ME, ASG-22ME, ASG-16M8F, MDX-1203, MLN-0264, anti-PSMA ADC, RG-7450, RG-7458, RG-7593, RG- 7596, RG-7598, RG-7599, RG-7600, RG-7636, ABT-414, FMGN-853, FMGN-529,

vorsetuzumab mafodotin, and lorvotuzumab mertansine. In some embodiments, a

chemotherapeutic agent may be an antibody-drug conjugate. In some embodiments, a chemotherapeutic agent may be or comprise one or more of farnesyl-thiosalicylic acid (FTS), 4- (4-Chloro-2-methylphenoxy)-N-hydroxybutanamide (CMH), estradiol (E2),

tetramethoxystilbene (TMS), δ-tocatrienol, salinomycin, or curcumin.

[0045] Combination therapy: As used herein, the term "combination therapy" refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents). In some embodiments, the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all "doses" of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, "administration" of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combination composition, or even in a combination compound (e.g., as part of a single chemical complex or covalent entity).

Comparable: As used herein, the term "comparable" refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison therebetween so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0046] Determine: Some methodologies described herein include a step of

"determining." Those of ordinary skill in the art, reading the present specification, will appreciate that such "determining" can utilize or be accomplished through use of any of a variety of techniques available to those skilled in the art, including for example specific techniques explicitly referred to herein. In some embodiments, determining involves manipulation of a physical sample. In some embodiments, determining involves consideration and/or manipulation of data or information, for example utilizing a computer or other processing unit adapted to perform a relevant analysis. In some embodiments, determining involves receiving relevant information and/or materials from a source. In some embodiments, determining involves comparing one or more features of a sample or entity to a comparable reference.

[0047] Dosage form or unit dosage form: Those skilled in the art will appreciate that the term "dosage form" may be used to refer to a physically discrete unit of an active agent (e.g., a therapeutic or diagnostic agent) for administration to a subject. Typically, each such unit contains a predetermined quantity of active agent. In some embodiments, such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen). Those of ordinary skill in the art appreciate that the total amount of a therapeutic composition or agent administered to a particular subject is determined by one or more attending physicians and may involve administration of multiple dosage forms.

[0048] Dosing regimen: Those skilled in the art will appreciate that the term "dosing regimen" may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent (e.g. a Wnt-activating agent) has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which is separated in time from other doses. In some embodiments, individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).

[0049] Expression: As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence {e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and/or 3' end formation); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.

[0050] Ex Vivo: As used herein refers to events that occur in or on tissue from a multicellular organism, such as a human and a non-human animal, in an external environment which resembles the natural conditions of the tissue with a minimum of alterations to the tissue itself.

[0051] Human. In some embodiments, a human is an embryo, a fetus, an infant, a child, a teenager, an adult, or a senior citizen.

[0052] Inhibitory agent: As used herein, the term "inhibitory agent" refers to an entity, condition, or event whose presence, level, or degree correlates with decreased level or activity of a target). In some embodiments, an inhibitory agent may be act directly (in which case it exerts its influence directly upon its target, for example by binding to the target); in some embodiments, an inhibitory agent may act indirectly (in which case it exerts its influence by interacting with and/or otherwise altering a regulator of the target, so that level and/or activity of the target is reduced). In some embodiments, an inhibitory agent is one whose presence or level correlates with a target level or activity that is reduced relative to a particular reference level or activity (e.g., that observed under appropriate reference conditions, such as presence of a known inhibitory agent, or absence of the inhibitory agent in question, etc).

[0053] In vitro: as used herein refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.

[0054] In vivo: as used herein refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).

[0055] Non-human animal: as used herein, the phrase "non-human animal" refers to any vertebrate organism that is not a human. In some embodiments, a non-human animal is a cyclostome, a bony fish, a cartilaginous fish (e.g., a shark or a ray), an amphibian, a reptile, a mammal, and a bird. In some embodiments, a non-human mammal is a primate, a goat, a sheep, a pig, a dog, a cow, or a rodent. In some embodiments, a non-human animal is a rodent such as a rat or a mouse.

[0056] Nucleic acid. As used herein, in its broadest sense, refers to any compound and/or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and/or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. As will be clear from context, in some embodiments, "nucleic acid" refers to an individual nucleic acid residue (e.g., a nucleotide and/or nucleoside); in some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, a "nucleic acid" is or comprises RNA; in some embodiments, a "nucleic acid" is or comprises DNA. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some

embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, a nucleic acid is, comprises, or consists of one or more "peptide nucleic acids", which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention. Alternatively or additionally, in some embodiments, a nucleic acid has one or more phosphorothioate and/or 5'-N-phosphoramidite linkages rather than phosphodiester bonds. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural

nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxycytidine). In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2- aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)- methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a nucleic acid comprises one or more modified sugars (e.g., 2'- fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein. In some embodiments, a nucleic acid includes one or more introns. In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a

complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long. In some embodiments, a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded. In some embodiments a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide. In some embodiments, a nucleic acid has enzymatic activity.

[0057] Patient: As used herein, the term "patient" refers to any organism to which a provided composition is or may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and/or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans). In some embodiments, a patient is a human. In some embodiments, a patient is suffering from or susceptible to one or more disorders or conditions. In some embodiments, a patient displays one or more symptoms of a disorder or condition. In some embodiments, a patient has been diagnosed with one or more disorders or conditions. In some embodiments, the disorder or condition is or includes cancer, or presence of one or more tumors. In some embodiments, a patient is receiving or has received certain therapy to diagnose and/or to treat a disease, disorder, or condition. In some embodiments, a patient is receiving or has received radiation therapy. In some embodiments, a patient was, is or will be exposed to ionizing radiation.

[0058] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" applied to the carrier, diluent, or excipient used to formulate a composition as disclosed herein means that the carrier, diluent, or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0059] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, a pharmaceutical composition may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

[0060] Polypeptide: As used herein refers to any polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and/or produced through action of the hand of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only non- natural amino acids. In some embodiments, a polypeptide may comprise D-amino acids, L- amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide's N-terminus, at the polypeptide's C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic, and/or may comprise a cyclic portion. In some embodiments, a polypeptide is not cyclic and/or does not comprise any cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term "polypeptide" may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides. For each such class, the present specification provides and/or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and/or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family. In some embodiments, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and/or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class). For example, in some embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and/or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%), 98%), or 99%. Such a conserved region usually encompasses at least 3-4 and often up to 20 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. In some embodiments, a useful polypeptide may comprise or consist of a fragment of a parent polypeptide. In some embodiments, a useful polypeptide as may comprise or consist of a plurality of fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to one another than is found in the polypeptide of interest (e.g., fragments that are directly linked in the parent may be spatially separated in the polypeptide of interest or vice versa, and/or fragments may be present in a different order in the polypeptide of interest than in the parent), so that the polypeptide of interest is a derivative of its parent polypeptide.

[0061] Prevent or prevention , as used herein when used in connection with the occurrence of a disease, disorder, and/or condition, refers to reducing the risk of developing the disease, disorder and/or condition and/or to delaying onset of one or more characteristics or symptoms of the disease, disorder or condition. Prevention may be considered complete when onset of a disease, disorder or condition has been delayed for a predefined period of time.

[0062] Protein. As used herein, the term "protein" refers to a polypeptide {i.e., a string of at least two amino acids linked to one another by peptide bonds). Proteins may include moieties other than amino acids {e.g., may be glycoproteins, proteoglycans, etc.) and/or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a

"protein" can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a characteristic portion thereof. Those of ordinary skill will appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means. Polypeptides may contain L-amino acids, D- amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, proteins are antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof.

[0063] Radiation mitigator: as used herein, the term "radiation mitigator" describes agents that are delivered at the time of irradiation and/or after irradiation is complete, but prior to the manifestation of normal tissue injury. In some embodiments, a radiation mitigator is an agent that is delivered during or shortly after irradiation to repopulate a critical cell compartment.

[0064] Radiation modifier or radiation protector: as used herein, the term "radiation modifier" or "radiation protector" describes agents that when present prior to or shortly after radiation exposure alter the response of normal tissues to irradiation. In some embodiments, a radiation modifier is selective in protecting normal tissue from radiation with no or minimal protection conferred on tumor tissue. In some embodiments, a radiation modifier is non-toxic or has minimal toxicity. In some embodiments, a radiation modifier confers protection on tissues that are sensitive to acute and/or late toxicities (e.g., the intestine). For example, a radiation modifier may protect tissues that are dose-limiting and/or responsible for a significant reduction in quality of life (e.g., mucositis, pneumonitis, myelopathy, xerostomia, proctitis,

leukencephalophy, etc.).

[0065] Radiation injury: as used herein, the term "radiation injury" describes any cell, tissue, or organ damage associated with radiation exposure. Examples of radiation injury include, but are not limited to, cerebrospinal injury, lung fibrosis, pneumonitis, hematopoietic injury, gastrointestinal injury, skin injuries and sepsis. In some embodiments, the radiation injury is or includes radiation enteritis.

Reference: As used herein describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and/or comparison to a particular possible reference or control.

[0066] Small molecule: As used herein, the term "small molecule" means a low molecular weight organic and/or inorganic compound. In general, a "small molecule" is a molecule that is less than about 5 kilodaltons (kD) in size. In some embodiments, a small molecule is less than about 4 kD, 3 kD, about 2 kD, or about 1 kD. In some embodiments, the small molecule is less than about 800 daltons (D), about 600 D, about 500 D, about 400 D, about 300 D, about 200 D, or about 100 D. In some embodiments, a small molecule is less than about 2000 g/mol, less than about 1500 g/mol, less than about 1000 g/mol, less than about 800 g/mol, or less than about 500 g/mol. In some embodiments, a small molecule is not a polymer. In some embodiments, a small molecule does not include a polymeric moiety. In some embodiments, a small molecule is not and/or does not comprise a protein or polypeptide (e.g., is not an oligopeptide or peptide). In some embodiments, a small molecule is not and/or does not comprise a polynucleotide (e.g., is not an oligonucleotide). In some embodiments, a small molecule is not and/or does not comprise a polysaccharide; for example, in some embodiments, a small molecule is not a glycoprotein, proteoglycan, glycolipid, etc). In some embodiments, a small molecule is not a lipid. In some embodiments, a small molecule is a modulating agent (e.g., is an inhibiting agent or an activating agent). In some embodiments, a small molecule is biologically active. In some embodiments, a small molecule is detectable (e.g., comprises at least one detectable moiety). In some embodiments, a small molecule is a therapeutic agent. Those of ordinary skill in the art, reading the present disclosure, will appreciate that certain small molecule compounds described herein may be provided and/or utilized in any of a variety of forms such as, for example, crystal forms, salt forms, protected forms, pro-drug forms, ester forms, isomeric forms (e.g., optical and/or structural isomers), isotopic forms, etc. Those of skill in the art will appreciate that certain small molecule compounds have structures that can exist in one or more steroisomeric forms. In some embodiments, such a small molecule may be utilized in accoradance with the present disclosure in the form of an individual enantiomer, diastereomer or geometric isomer, or may be in the form of a mixture of stereoisomers; in some embodiments, such a small molecule may be utilized in accordance with the present disclosure in a racemic mixture form. Those of skill in the art will appreciate that certain small molecule compounds have structures that can exist in one or more tautomeric forms. In some embodiments, such a small molecule may be utilized in accoradance with the present disclosure in the form of an individual tautomer, or in a form that interconverts between tautomeric forms. Those of skill in the art will appreciate that certain small molecule compounds have structures that permit isotopic substitution (e.g., ²H or ³H for H;, ^UC, ¹³C or ¹⁴C for 12C; , ¹³N or ¹⁵N for 14N; ¹⁷0 or ¹⁸0 for 160; ³⁶C1 for XXC; ¹⁸F for XXF; 1311 for XXXI; etc). In some embodiments, such a small molecule may be utilized in accordance with the present disclosure in one or more isotopically modified forms, or mixtures thereof. In some embodiments, reference to a particular small molecule compound may relate to a specific form of that compound. In some embodiments, a particular small molecule compound may be provided and/or utilized in a salt form (e.g., in an acid-addition or base-addition salt form, depending on the compound); in some such

embodiments, the salt form may be a pharmaceutically acceptable salt form. In some

embodiments, where a small molecule compound is one that exists or is found in nature, that compound may be provided and/or utilized in accordance in the present disclosure in a form different from that in which it exists or is found in nature. Those of ordinary skill in the art will appreciate that, in some embodiments, a preparation of a particular small molecule compound that contains an absolute or relative amount of the compound, or of a particular form thereof, that is different from the absolute or relative (with respect to another component of the preparation including, for example, another form of the compound) amount of the compound or form that is present in a reference preparation of interest (e.g., in a primary sample from a source of interest such as a biological or environmental source) is distinct from the compound as it exists in the reference preparation or source. Thus, in some embodiments, for example, a preparation of a single stereoisomer of a small molecule compound may be considered to be a different form of the compound than a racemic mixture of the compound; a particular salt of a small molecule compound may be considered to be a different form from another salt form of the compound; a preparation that contains only a form of the compound that contains one conformational isomer ((Z) or (E)) of a double bond may be considered to be a different form of the compound from one that contains the other conformational isomer ((E) or (Z)) of the double bond; a preparation in which one or more atoms is a different isotope than is present in a reference preparation may be considered to be a different form; etc.

[0067] Subject: As used herein, the term "subject" refers an organism, typically a mammal (e.g., a human, in some embodiments including prenatal human forms). In some embodiments, a subject is suffering from a relevant disease, disorder or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some

embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and/or therapy is and/or has been administered.

[0068] Substantiall . As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0069] Therapeutic agent: As used herein, the phrase "therapeutic agent" in general refers to any agent that elicits a desired pharmacological effect when administered to an organism. In some embodiments, an agent is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population. In some embodiments, the appropriate population may be a population of model organisms. In some embodiments, an appropriate population may be defined by various criteria, such as a certain age group, gender, genetic background, preexisting clinical conditions, etc. In some embodiments, a therapeutic agent is a substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition. In some embodiments, a "therapeutic agent" is an agent that has been or is required to be approved by a government agency before it can be marketed for administration to humans. In some embodiments, a "therapeutic agent" is an agent for which a medical prescription is required for administration to humans. In some embodiments, a therapeutic agent is a Wnt-activating agent.

[0070] Therapeutically effective amount: As used herein, is meant an amount that produces the desired effect for which it is administered. In some embodiments, the term refers to an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and/or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and/or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and/or severity of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition. Those of ordinary skill in the art will appreciate that the term "therapeutically effective amount" does not in fact require successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment. In some embodiments, reference to a therapeutically effective amount may be a reference to an amount as measured in one or more specific tissues (e.g., a tissue affected by the disease, disorder or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those of ordinary skill in the art will appreciate that, in some embodiments, a therapeutically effective amount of a particular agent (e.g. a Wnt-activating agent) or therapy may be formulated and/or administered in a single dose. In some embodiments, a therapeutically effective agent (e.g., a Wnt-activating agent) may be formulated and/or administered in a plurality of doses, for example, as part of a dosing regimen.

[0071] Treat, treatment or treating: as used herein, the term "treat," "treatment," or

"treating" refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and/or reduce incidence of one or more symptoms or features of a particular disease, disorder, and/or condition associated with radiation injury. In some embodiments, treatment may be administered to a subject who does not exhibit signs of radiation injury and/or exhibits only early signs for the purpose of decreasing the risk of developing pathology associated with radiation injury. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0072] The present disclosure provides, among other things, methods of treatment for subjects suffering from or susceptible to toxicities resulting from radiation exposure. The present disclosure encompasses a recognition that proliferation and expansion of undifferentiated stem cells can mitigate certain toxic effects associated with radiation exposure or therapy.

Radiation Exposure and Radiation Therapy

[0073] Humans and animals are highly susceptible to radiation-induced damage resulting in cellular, tissue, organ and systemic injuries. Irradiation of normal tissues can result in a spectrum of side effects including self-limiting acute toxicities, mild chronic symptoms, and severe organ dysfunction. (Citrin, D. et al., (2010) Oncologist 15:360-371.) Radiation-induced damage to cells, tissues, organs and systems can be the result of radiation exposure in the course of a treatment for a disease, such as cancer, or incidental radiation exposure due to a disaster involving release or radiation, such as a nuclear explosion. In accidental radiation exposure, such as a nuclear explosion or a disaster scenario, many victims will suffer from acute radiation syndrome (ARS) to varying degrees. For a disaster scenario, it may be desirable that drugs or treatments be effective when administered at time points following the radiation disaster.

[0074] It is estimated that over 40% of cancer patients will require radiation therapy during management of their disease. Although radiation therapy improves the survival of a significant number of cancer patients, both acute radiation toxicity (that which manifests during a course of clinical radiotherapy or shortly thereafter), and late toxicity (developing months to years after completion of radiotherapy) compromise overall outcomes for successfully treated cancer patients.

[0075] The risk for normal tissue complications in radiation therapy often correlates with the volume of the organ irradiated, dose, fractionation, radiation modifiers, and intrinsic biology. Radiation enteritis is a major limiting factor in abdominopelvic RT and is also predicted to be a major source of morbidity in the event of nuclear/radiological terrorism. Radiation enteritis has been reported in more than 70% of patients who have undergone abdominopelvic RT. (Yang et al., (2013) Curr. Gene Ther. 13 : 305-314.) [0076] In some embodiments, the radiation exposure results in a total body irradiation.

In some embodiments, a subject will receive or has received whole body irradiation. In some embodiments, a subject will receive or has received whole abdomen irradiation. In some embodiments, a subject will receive or has received abdominopelvic RT. Despite development of a number of technologies to target radiation to tumor tissues, normal tissue toxicity remains problematic at least in part because of the intimate relationship between tumors and surrounding normal tissues, as well as the potential for microscopic contamination of normal tissues. In some embodiments, a subject will receive or has received conformational radiotherapy. In some embodiments, a subject will receive or has received intensity-modulated radiotherapy (EVIRT). In some embodiments, a subject will receive or has received proton beam radiotherapy (PBRT). In some embodiments, a subject will receive or has received image-guided radiotherapy. In some embodiments, a subject who has received, is receiving or will receive radiation therapy is suffering from cancer. In some embodiments, a subject who has received, is receiving or will receive radiation therapy is suffering from a gastrointestinal or hepatobiliary malignancy.

[0077] In some embodiments, the radiation is received or administered at a dose sufficient to induce a characteristic associated with acute radiation damage. In some

embodiments, the radiation damage to the subject is chronic or systemic damage. The present disclosure encompasses a recognition that Wnt-activation may permit escalation of the radiation therapy dosing. In some embodiments, when a subject is treated with a Wnt-activating agent, a higher dose of radiation is needed to induce radiation damage (e.g. RIGS). In some

embodiments, treatment with a Wnt-activating agent increases the LD50 dose of radiation. In some embodiments, a subject treated with a Wnt-activating agent may receive an increased dose of radiation.

[0078] In some embodiments, the radiation dose is between about 1 Gy and about 200

Gy. In some embodiments, the radiation dose is between about 3 Gy and about 150 Gy. In some embodiments, the radiation dose is between about 5 Gy and about 100 Gy. In some

embodiments, the radiation dose is about 3 Gy, 4 Gy, 5 Gy, 6 Gy, 7 Gy, 8 Gy, 9 Gy, 10 Gy, 12 Gy, 14 Gy, 15 Gy, 16 Gy, 18 Gy, 20 Gy, 22 Gy, 25 Gy, 30 Gy, 35 Gy, 40 Gy, 45 Gy, 50 Gy, 55 Gy, 60 Gy, 65 Gy, 70 Gy, 75 Gy, 80 Gy, 90 Gy, 100 Gy, 125 Gy or 150 Gy. [0079] With increasing doses of irradiation, symptom onset is generally accelerated and/or severity of symptoms is increased and/or more organ systems may be effective. For example, Acute Radiation Syndromes (ARS) typically follow a generalizable course

characterized by four distinct phases: prodrome, latent, illness and recovery/death, and the severity, duration and/or timing of each phase depends on the irradiation dose. As various tissues exhibit differential sensitivity to radiation, the corresponding ARS associated with each tissue/organ system is also dose dependent, see for example ARS in mice described in Table 1 below.

Table 1 - Acute Radiation Syndromes in mice

* Estimated for dose rate of 100 Gy/hr.

[0080] The dosage and timing associate with ARS vary between organisms. For example, humans develop signs and symptoms of hematologic damage much more slowly than mice. As illustrated in Table 1, peak incidence of death in mice is -8-12 days, whereas the peak incidence of death in humans is at approximately 30 days. Therefore whole body irradiation of mouse models will exhibit symptoms of hematopoietic syndrome prior to showing symptoms of gastrointestinal syndrome. Models of risk of radiation on normal tissue toxicity generally consider volume of the organ irradiated, dose, fractionation, radiation modifiers, and/or intrinsic biology.

[0081] In some embodiments, radiation induces gastrointestinal syndrome in a subject.

The present disclosure encompasses a recognition that proliferation of stem cells in the gastrointestinal tract after exposure of a subject to radiation are beneficial to the subject. For example, the present disclosure provides an insight that therapies that induce intestinal stem cell hyperproliferation and/or block stem cell differentiation may be beneficial for a subject suffering from or susceptible to radiation induced gastrointestinal syndrome (RIGS). RIGS may be characterized by abdominal pain, diarrhea, nausea and/or vomiting and may predispose patients to infection. In some embodiments, the present disclosure provides methods of treating or preventing RIGS in a subject at risk thereof, the method comprising administering an agent that induces intestinal stem cell proliferation and/or blocks stem cell differentiation.

Intestinal Structure

[0082] In some embodiments, the present invention relates to the gastrointestinal tract of a subject. In some embodiments, gastrointestinal tract (GI tract or GIT) is an organ system responsible for consuming and digesting foodstuffs, absorbing nutrients, and expelling waste. The gastrointestinal tract generally consists of, but not is limited to, the mouth, esophagus, stomach and or rumen, intestines (both small and large), cecum (plural ceca), fermentation sacs, and the anus. The gastrointestinal tract may be referred to as divided into upper and lower tracts. The upper gastrointestinal tract generally consists of, but not is limited to, the buccal cavity, pharynx, esophagus, stomach, and duodenum. The lower gastrointestinal tract includes, but is not limited to, most of the small intestine and all of the large intestine.

[0083] In some embodiments, the present invention relates to the intestines (bowel, or gut) of a subject. In human anatomy, the intestine (bowel, or gut) is the segment of the gastrointestinal tract extending from the pyloric sphincter of the stomach to the anus. In humans and other mammals, the intestines generally consists of two segments: the small intestine and the large intestine. In humans, for example, the small intestine may be referred to as further subdivided into the duodenum, jejunum and ileum and the large intestine may be referred to as subdivided into the cecum, colon, rectum, and anal canal. In some embodiments, the present invention relates to the small intestines. The small intestine may provide numerous biological functions, including, aiding digestion (with pancreatic and biliary enzymes/salts), provides an effective barrier against microorganisms and carcinogens present in the lumen, and undergoes rapid self-renewal as persistent aggression induces a high rate of cell death (-200 g/day).

[0084] In some embodiments, the present invention relates to the intestinal villus. The small intestine villus is organized in a hierarchical structure. As depicted in Figure 1, differentiated cell populations of the intestinal villus include enterocytes, enteroendocrine cells, and Goblet cells. The intestinal crypt includes the highly proliferative intestinal stem cells (ISC) (Lgr5+) and niche supporting Paneth cells. Paneth cells are found in close association with the Lgr5+ (Leu-rich repeat- containing G protein-coupled receptor 5-expressing) crypt base columnar (CBC) stem cells at the crypt base. Lgr5+ crypt base columnar stem cells are intercalated with Paneth cells and may continuously generate rapidly proliferating transit- amplifying (TA) cells. Paneth cells are an important source of various niche factors, including epidermal growth factor (EGF), WNT3 A and Notch ligand. Paneth cells also play role in the defense against microorganisms via MyD88-dependent toll-like receptor (TLR) activation. These niche cells also enable the intestine to tailor the output of its stem cell compartment to nutrient availability. Paneth cells respond to calorie restriction by reducing mTOR complex 1

(mTORCl) signalling, initiating a signal cascade that results in a rapid reduction in the size of the LGR5+ stem cell pool.

[0085] The present invention encompasses the recognition that a multipotent actively cycling intestinal stem cell population exists in the bottom of intestinal crypts and is highly sensitive to irradiation. Stem cells reside within restricted tissue microenvironments known as niches. Niches are thought to play an important role in regulating the number of daughter cells that retain stem cell identity and in blocking expansion of the stem cell pool. In some

embodiments, radiation induces loss of intestinal crypts and/or breakdown of the mucosal barrier. Therapeutic regimens and treatments of the present disclosure may mitigate radiation- induced loss of intestinal crypts and/or breakdown of the mucosal barrier. In some

embodiments, acute gastrointestinal syndrome is thought to result from permanent damage to Lgr5+ ISC population. Therapeutic regimens and treatments of the present disclosure may mitigate radiation-induced damage to Lgr5+ ISC population.

[0086] The Wnt pathway output is modulated by co-operative activity of the Hedgehog and bone morphogenic protein (BMP) cascades. In some embodiments, small intestine villi may be characterized by a gradient expression of Wnt and Bmp proteins (with high WNT/low BMP expression at the base, and decreasing WNT/increase BMP expression moving out toward the tip of the villus). As the progenitor cells further decline from the crypt base, the Hedgehog-induced, mesenchyme-to-epithelium BMP signaling promotes differentiation while restraining proliferation. Without being bound by theory, it is thought that Wnt signaling plays a role in the regulation of the intestinal stem cell niche. Abrogation of Wnt signaling results in a complete loss of stem cell proliferation. Moreover, Lgr5, a Wnt target gene, is a validated intestinal stem cell marker. Lineage tracing experiments have demonstrated that Lgr5+ cells are capable of self- renewal and multipotency.

Wnt signaling

[0087] In some embodiments, the present invention relates to activation of Wingless-type

(Wnt) signaling. The present invention encompasses the recognition that Wnt-driven

proliferation is a regulator of epithelial regeneration in mammalian tissues.

[0088] Wnt proteins are a diverse family of secreted lipid-modified signaling

glycoproteins that are approximately 350-400 amino acids in length. Wnt proteins are highly conserved across species and can be found in mice, humans, Xenopus, zebrafish, Drosophila, and many others. Lipid modification of Wnt proteins is typically palmitoylation of cysteines in a conserved pattern of 23-24 cysteine residues. Palmitoylation initiates targeting of the Wnt protein to the plasma membrane for secretion and it allows the Wnt protein to bind its receptor due to the covalent attachment of fatty acids. Wnt proteins also undergo glycosylation, which attaches a carbohydrate in order to insure proper secretion. In Wnt signaling, these proteins act as ligands to activate the different Wnt pathways via paracrine and autocrine routes.

[0089] Wnt signaling begins when one of the Wnt proteins binds to the N-terminal extracellular cysteine-rich domain of a Frizzled (Fz) family receptor. These receptors span the plasma membrane seven times and constitute a distinct family of G-protein coupled receptors (GPCRs). However, to facilitate Wnt signaling, co-receptors may also be required alongside the interaction between the Wnt protein and Fz receptor. Examples include lipoprotein receptor-related protein (LRP)-5/6, receptor tyrosine kinase (Ryk), and ROR2. Upon activation of the receptor, a signal is sent to the phosphoprotein Dishevelled (Dsh), which is located in the cytoplasm. This signal is transmitted via a direct interaction between Fz and Dsh. Dsh proteins are present in all organisms and they all share the following highly conserved protein domains: an amino-terminal DIX domain, a central PDZ domain, and a carboxy-terminal DEP domain. These different domains are important because after Dsh, the Wnt signal can branch off into several different pathways and each pathway interacts with a different combination of the three domains.

[0090] The three best characterized Wnt signaling pathways are the canonical Wnt pathway, the noncanonical planar cell polarity pathway, and the noncanonical Wnt/calcium pathway. As their names suggest, these pathways belong to one of two categories: canonical or noncanonical. The difference between the categories is that a canonical pathway involves the protein β-catenin while a noncanonical pathway operates independently of it.

Canonical Wnt pathway

[0091] The canonical Wnt pathway (or Wnt/p-catenin pathway) is the Wnt pathway that causes an accumulation of β-catenin in the cytoplasm and its eventual translocation into the nucleus to act as a transcriptional coactivator of transcription factors that belong to the TCF/LEF family. Without Wnt signaling, the β-catenin would not accumulate in the cytoplasm since a destruction complex would normally degrade it. This destruction complex includes the following proteins: Axin, adenomatosis polyposis coli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase la (CKla). It degrades β-catenin by targeting it for ubiquitination, which subsequently sends it to the proteasome to be digested. However, as soon as Wnt binds Fz and LRP5/6, the destruction complex function becomes disrupted. This is due to Wnt causing the translocation of the negative Wnt regulator, Axin, and the destruction complex to the plasma membrane. Phosphorylation by other proteins in the destruction complex subsequently binds Axin to the cytoplasmic tail of LRP5/6. Axin becomes de-phosphorylated and its stability and levels are decreased. Dsh then becomes activated via phosphorylation and its DIX and PDZ domains inhibit the GSK3 activity of the destruction complex. This allows β- catenin to accumulate and localize to the nucleus and subsequently induce a cellular response via gene transduction alongside the TCF/LEF (T-cell factor/lymphoid enhancing factor)

transcription factors. APC and 08Κ-3β are regulators of Wnt signaling output via regulating the degradation of β-catenin.

Noncanonical PCP pathway

[0092] The noncanonical planar cell polarity (PCP) pathway is one of the two Wnt pathways that does not involve β-catenin. It does not use LRP-5/6 as its co-receptor and is thought to use NRHl, Ryk, PTK7, or ROR2. As in the canonical Wnt pathway, the PCP pathway is activated via the binding of Wnt to Fz and its co-receptor. The receptor then recruits Dsh, which uses its PDZ and DIX domains to form a complex with Dishevelled-associated activator of morphogenesis 1 (DAAMl). Daaml then activates the small G-protein Rho through a guanine exchange factor. Rho activates Rho-associated kinase (ROCK), which is one of the major regulators of the cytoskeleton. Dsh also forms a complex with racl and mediates profilin binding to actin. Racl activates INK and can also lead to actin polymerization. Profilin binding to actin can result in restructuring of the cytoskeleton and gastrulation.

Noncanonical Wnt/calcium pathway

[0093] The noncanonical Wnt/calcium pathway is the other Wnt pathway that does not stimulate accumulation of β-catenin. Its role is to help regulate calcium release from the endoplasmic reticulum (ER) in order to control intracellular calcium levels. Like other Wnt pathways, upon ligand binding, the activated Fz receptor directly interacts with Dsh and activates specific Dsh-protein domains. The domains involved in Wnt/calcium signaling are the PDZ and DEP domains. However, unlike other Wnt pathways, the Fz receptor also directly interfaces with a trimeric G-protein. This co-stimulation of Dsh and the G-protein can lead to the activation of either PLC or cGMP-specific PDE. If PLC is activated, the plasma membrane component PIP2 is cleaved into DAG and IP3. When IP3 binds its receptor on the ER, calcium is released. Increased concentrations of calcium and DAG can activate Cdc42 through PKC. Cdc42 is an important regulator of ventral patterning. Increased calcium also activates calcineurin and CaMKII.

CaMKII induces activation of the transcription factor NFAT, which regulates cell adhesion, migration, and tissue separation. Calcineurin activates TAK1 and NLK kinase, which can interfere with TCF/B-Catenin signaling in the canonical Wnt pathway. However, if PDE is activated, calcium release from the ER is inhibited. PDE mediates this through the inhibition of PKG, which subsequently causes the inhibition of calcium release.

Integrated Wnt pathway

[0094] The binary distinction of canonical and non-canonical Wnt signaling pathways has come under scrutiny and an integrated, convergent Wnt pathway has been proposed. Some evidence for this was found for one Wnt ligand (Wnt5A). Moreover, evidence for a convergent Wnt signaling pathway, that shows integrated activation of Wnt/Ca2+ and Wnt/B-catenin signaling, for multiple Wnt ligands, was described in mammalian cell lines.

Other pathways

[0095] Along with the pathways, described above, Wnt signaling also regulates a number of other signaling pathways that have not been as extensively elucidated. One such pathway includes the interaction between Wnt and GSK3. During cell growth, Wnt can inhibit GSK3 in order to activate mTOR in the absence of β-catenin. However, Wnt can also serve as a negative regulator of mTOR via activation of the tumor suppressor TSC2, which is upregulated via Dsh and GSK3 interaction. During myogenesis, Wnt uses PA and CREB to activate the genes MyoD and Myf5. Wnt has also been seen to act in conjunction with Ryk and Src to allow for regulation of neuron repulsion during axonal guidance. Wnt regulates gastrulation when CK1 serves as an inhibitor of Rapl-ATPase in order to modulate the cytoskeleton during gastrulation. Further regulation of gastrulation is achieved when Wnt uses ROR2 along with the CDC42 and INK pathway to regulate the expression of PAPC. Dsh can also interact with aPKC, Pa3, Par6, and LG1 in order to control cell polarity and microtubule cytoskeleton development. While these pathways overlap with components associated with PCP and Wnt/Calcium signaling, they are considered distinct pathways because they produce entirely different responses.

Wnt Regulation

[0096] In order to ensure proper functioning, Wnt signaling is constantly regulated at several points along its signaling pathways. For instance, as previously mentioned, Wnt proteins are palmitoylated. The protein porcupine mediates this palmitoylation process, which means that it helps regulate when the Wnt ligand is secreted by determining when it is fully formed.

Secretion of Wnt protein is further controlled with proteins such as wntless and evenness interrupted and complexes such as the retromer complex. Upon secretion, the ligand can also be prevented from reaching its receptor through the binding of certain proteins such as the stabilizers Dally and glypican 3, which inhibit diffusion. At the Fz receptor, the binding of proteins other than Wnt can antagonize signaling. Specific antagonists include Dickkopf (Dkk), Wnt inhibitory factor 1 (WIF-1), secreted Frizzled-related proteins (SFRP), Cerberus, Frzb, Wise, and SOST. All of these constitute inhibitors of Wnt signaling; however, other molecules have been shown to act as activators as well. For example, Norrin and R-Spondin2 have been shown to activate Wnt signaling in the absence of Wnt ligand. Interactions between different Wnt signaling pathways also regulate Wnt signling. As previously mentioned, the Wnt/calcium pathway can inhibit TCF/p-catenin in order to prevent canonical Wnt pathway signaling.

ProstaglandinE2 has been shown to be an essential activator of the canonical Wnt signaling pathway. Interaction of PGE2 with its receptors E2/E4 stabilizes beta catenin through

cAMP/PKA mediated phosphorylation. The synthesis of PGE2 has also been shown to be necessary for Wnt signaling mediated processes like tissue regeneration and control of stem cell population in zebrafish and mouse.

Wnt activating agents

[0097] In some embodiments, the present invention relates to an agent that activates Wnt signaling. In some embodiments, a Wnt-activating agent increases stem cell proliferation and/or blocks stem cell differentiation. In some embodiments, a Wnt-activating agent increases proliferation and/or blocks differentiation of intestinal stem cells. In some embodiments, a therapeutic agent within the context of the present invention increases proliferation and/or blocks differentiation of Lgr5+ intestinal stem cells.

[0098] In some embodiments, a Wnt-activating agent increases or activates Wnt signaling directly. In some embodiments, a Wnt-activating agent increases or activates Wnt signaling indirectly. In some embodiments, Wnt signaling is increased by decreasing the level and/or or activity of APC. In some embodiments, the present invention relates to the reduction of the level or activity of APC. In some embodiments, the present invention relates to removal of the inhibition of Wnt signaling by APC. In some embodiments, the reduction of the level or activity of APC is achieved by delivery, administration or introduction of an agent.

[0099] Wnt activation can be achieved by many different strategies. Glycogen-synthase kinase 3β (GSK-3P) is also a member of the β-catenin destruction complex and a negative regulator of β-catenin like APC. Just as APC inhibition can result in Wnt activation, GSK-3P inhibition can result in activation of Wnt target genes and has been demonstrated to protect intestinal crypt cells from radiation induced apoptosis and increase clonogenic cell survival. A number of GSK-3P inhibitors are clinically available. [0100] In some embodiments, Wnt signaling is increased by decreasing the level and/or or activity of GSK-3p. In some embodiments, the present invention relates to the reduction of the level or activity of GSK-3p. In some embodiments, the present invention relates to removal of the inhibition of Wnt signaling by GSK-3p. In some embodiments, the reduction of the level or activty of GSK-3P is achieved by delivery, administration or introduction of an agent.

[0101] In some embodiments, agents that may be utilized in accordance with the present invention include antibodies, antibody fragments, aptamers, siRNAs, shRNAs, DNA/RNA hybrids, antisense oligonucleotides, ribozymes, peptides, peptide mimetics, lipids, small molecules, etc. In some embodiments, an agent that reduces the expression, activity and/or function of APC or GSK-3P is an antibody, antibody fragment, aptamer, siRNA, shRNA, DNA/RNA hybrid, antisense oligonucleotide, ribozyme, peptide, peptide mimetic, lipid, or a small molecule.

[0102] In some embodiments, RNAi interference is employed to knock down expression of APC and/or GSK-3p. RNA interference operates through a highly conserved mechanism of sequence-specific post-transcriptional gene silencing triggered by the presence of double- stranded RNA (dsRNA). In animals, the RNAi program within somatic tissues is primarily regulated by microRNAs (miRNAs), small non-coding RNAs of 20-25 nucleotides in length transcribed by RNA polymerase II (Lee et al. 2004; Bartel 2009). Mature miRNAs are derived from a longer poly-adenylated primary miRNA (pri-miRNA) transcript (Cai et al. 2004) through a series of cleavage steps. The Drosha/DGCR8 complex first cleaves the pri-miRNA in the nucleus to generate a stem loop pre-miRNA structure with a 2-nucleotide 3' overhang that is exported to the cytoplasm (Lee et al. 2003; Denli et al. 2004; Gregory et al. 2004). The double- stranded pre-miRNA is then cleaved by the ribonuclease Dicer to produce the mature miRNA form (Hutvagner et al. 2001; Bernstein et al. 2001; Hutvagner and Zamore 2002). The miRNA duplex is subsequently separated into single strands and the guide strand is loaded into the RNA- induced silencing complex (RISC). There, it pairs with complementary mRNAs and directs their degradation (Meister et al. 2004; Yekta et al. 2004) or translational silencing (Filipowicz et al. 2008). Other dsRNAs undergo analogous processing by Dicer to generate small interfering RNA (siRNA) effector duplexes that are also incorporated into RISC (Bernstein et al. 2001; Hammond et al. 2001). The most well-studied outcome is post-transcriptional gene silencing, which occurs when the guide strand pairs with a complementary sequence in a messenger RNA molecule and induces cleavage by Argonaute, the catalytic component of the RISC complex

[0103] In some embodiments, an agent that reduces the expression, activity or function of

APC is an shRNA molecule. Description of shRNA that can inhibit APC may be found in Dow et al., 2015, Cell 161, 1539-1552, the entirety of which is herein incorporated by reference.

[0104] In some embodiments, an agent is administered such that approximately l%-5%,

5%-10% 10%-15%, 15%-20%, or 8%-12% intestinal crypt survival is observed in a subject post-exposure to radiation (e.g., post-radiation therapy). Without wishing to be bound by theory, it is estimated that this is the amount of crypt survival necessary for recovery of the GI mucosa and GI tract survival. In some embodiments, a Wnt-activating agent is administered in an amount such that l%-20% intestinal crypt survival is observed after radiotherapy and/or chemotherapy. In some embodiments, a subject treated with a Wnt-activating agent and radiotherapy and/or chemotherapy has l%-20% intestinal crypt survival.

Formulation and Administration of Agents

[0105] In the methods of the invention, a Wnt-activating agent is typically administered to the individual alone, or in compositions or medicaments (e.g., in the manufacture of a medicament for the treatment of radiation toxicity), as described herein. In some embodiments, compositions can be formulated with a physiologically acceptable carrier or excipient to prepare a pharmaceutical composition. In some embodiments, a carrier and/or a composition can be sterile. The formulation should suit the mode of administration.

[0106] Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, sugars such as mannitol, sucrose, or others, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well as combinations thereof. The pharmaceutical preparations can, if desired, be mixed with auxiliary agents (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like), which do not deleteriously react with the active compounds or interference with their activity. In some embodiments, a water-soluble carrier suitable for intravenous administration is used.

[0107] A composition or medicament, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. A composition can be or comprise a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. A composition can be formulated as a suppository, for example utilizing traditional binders and/or carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose, magnesium carbonate, etc.

[0108] A composition or medicament can be formulated in accordance with routine procedures as a pharmaceutical composition adapted for administration to human beings. For example, in some embodiments, a composition for intravenous administration typically is a solution in sterile isotonic aqueous buffer. In some embodiments, a composition may include a solubilizing agent and/or a local anesthetic (e.g., to ease pain at a site of injection). Generally, ingredients may be supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent. Where a composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water. Where a composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0109] Wnt-activating agents can be formulated for use in accordance with the present invention in a neat or neutral form. Alternatively or additionally, in some embodiments, such agents may be formulated in salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, etc. [0110] In some embodiments, Wnt-activating agents may be administered in accordance with the present invention by any appropriate route. In some embodiments, agents are administered intravenously. In some embodiments, agents are administered subcutaneously. In some embodiments, agents are administered by direct administration to a target tissue (e.g., the intestine). Alternatively, agents can be administered orally, parenterally, transdermally, or transmucosally (e.g., orally or nasally). More than one route can be used concurrently, if desired.

[0111] In some embodiments, Wnt-activating agents can be administered alone, or in conjunction with one or more other agents (e.g., other agents that activate Wnt signaling and/or antioxidants). The term, "in conjunction with," indicates that a first agent is administered prior to, at about the same time as, or following another agent.

[0112] In some embodiments, Wnt-activating agents are administered in a therapeutically effective amount (e.g., a dosage amount that, when administered according to a particular regimen, is sufficient to treat a radiation injury, such as by ameliorating and/or delaying onset of one or more symptoms associated with radiation injury, preventing or delaying the onset of radiation injury, and/or also lessening the severity or frequency of symptoms of radiation injury.)

[0113] In some embodiments, a subject who has received, is receiving or will receive radiation therapy is suffering from cancer. In some embodiments, a subject who has received, is receiving or will receive radiation therapy is suffering from a gastrointestinal or hepatobiliary malignancy.

[0114] In some embodiments, the present disclosure provides methods of treating or preventing chemotherapy induced enteritis. In some embodiments, a subject who has received, is receiving or will receive chemotherapy. In some embodiments, a subject who has received, is receiving or will receive chemotherapy is suffering from cancer. In some embodiments, a subject who has received, is receiving or will receive chemotherapy is suffering is colorectal cancer. In some embodiments, a subject who has received, is receiving or will receive chemotherapy is suffering from a gastrointestinal or hepatobiliary malignancy.

[0115] The present disclosure encompasses a recognition that Wnt-activation may permit dose escalation of radiotherapy and/or chemotherapy. In some embodiments, treatment with a Wnt-activating agent increases the LD50 dose of radiation and/or chemotherapy. In some embodiments, a subject treated with a Wnt-activating agent may receive an increased dose of radiation and/or chemotherapy.

[0116] In some embodiments, one or more, in vitro or in vivo assays may be employed to help identify optimal dosage ranges, such as those exemplified below. In some embodiments, a precise dose and/or regimen to be employed may depend on route of administration, and magnitude of the injury, and should be decided according to the judgment of a practitioner and each patient's circumstances. In some embodiments, effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems (e.g., as described by the U.S. Department of Health and Human Services, Food and Drug Administration, and Center for Drug Evaluation and Research in "Guidance for Industry: Estimating Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers", Pharmacology and Toxicology, July 2005.

[0117] In some embodiments, a therapeutically effective amount of an agent can be, for example, more than about 0.01 mg/kg, more than about 0.05 mg/kg, more than about 0.1 mg/kg, more than about 0.5 mg/kg, more than about 1.0 mg/kg, more than about 1.5 mg/kg, more than about 2.0 mg/kg, more than about 2.5 mg/kg, more than about 5.0 mg/kg, more than about 7.5 mg/kg, more than about 10 mg/kg, more than about 12.5 mg/kg, more than about 15 mg/kg, more than about 17.5 mg/kg, more than about 20 mg/kg, more than about 22.5 mg/kg, or more than about 25 mg/kg body weight. In some embodiments, a therapeutically effective amount can be about 0.01-25 mg/kg, about 0.01-20 mg/kg, about 0.01-15 mg/kg, about 0.01-10 mg/kg, about 0.01-7.5 mg/kg, about 0.01-5 mg/kg, about 0.01-4 mg/kg, about 0.01-3 mg/kg, about 0.01-2 mg/kg, about 0.01-1.5 mg/kg, about 0.01-1.0 mg/kg, about 0.01-0.5 mg/kg, about 0.01-0.1 mg/kg, about 1-20 mg/kg, about 4-20 mg/kg, about 5-15 mg/kg, about 5-10 mg/kg body weight. In some embodiments, a therapeutically effective amount is about 0.01 mg/kg, about 0.05 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1.0 mg/kg, about 1.1 mg/kg, about 1.2 mg/kg, about 1.3 mg/kg about 1.4 mg/kg, about 1.5 mg/kg, about 1.6 mg/kg, about 1.7 mg/kg, about 1.8 mg/kg, about 1.9 mg/kg, about 2.0 mg/kg, about 2.5 mg/kg, about 3.0 mg/kg, about 4.0 mg/kg, about 5.0 mg/kg, about 6.0 mg/kg, about 7.0 mg/kg, about 8.0 mg/kg, about 9.0 mg/kg, about 10.0 mg/kg, about 11.0 mg/kg, about 12.0 mg/kg, about 13.0 mg/kg, about 14.0 mg/kg, about 15.0 mg/kg, about 16.0 mg/kg, about 17.0 mg/kg, about 18.0 mg/kg, about 19.0 mg/kg, about 20.0 mg/kg, body weight, or more. In some embodiments, the therapeutically effective amount is no greater than about 30 mg/kg, no greater than about 20 mg/kg, no greater than about 15 mg/kg, no greater than about 10 mg/kg, no greater than about 7.5 mg/kg, no greater than about 5 mg/kg, no greater than about 4 mg/kg, no greater than about 3 mg/kg, no greater than about 2 mg/kg, or no greater than about 1 mg/kg body weight or less.

[0118] In some embodiments, an effective dose for a particular individual may be varied

(e.g., increased or decreased) over time, depending on the needs of the individual.

[0119] In some embodiments, a therapeutically effective amount of a Wnt-activating agent (or composition or medicament containing a Wnt-activating agent or agents may be administered as a one-time dose or administered at intervals, depending on the nature and extent of the radiation injury effects, and on an ongoing basis. Administration at an "interval," as used herein, indicates that the therapeutically effective amount is administered periodically (as distinguished from a one-time dose). The interval can be determined by standard clinical techniques. In some embodiments, an agent is administered bimonthly, monthly, twice monthly, triweekly, biweekly, weekly, twice weekly, thrice weekly, or daily. The administration interval for a single individual need not be a fixed interval, but can be varied over time, depending on the needs and rate of recovery of the individual.

[0120] As used herein, the term "bimonthly" means administration once per two months

(i.e., once every two months); the term "monthly" means administration once per month; the term "triweekly" means administration once per three weeks (i.e., once every three weeks); the term "biweekly" means administration once per two weeks (i.e., once every two weeks); the term "weekly" means administration once per week; and the term "daily" means administration once per day.

[0121] In some embodiments, a Wnt-activating agent is cleared or otherwise rendered ineffective in vivo after a period of time. In some embodiments, the period of time for clearance and/or inactivation of a Wnt-activating agent is 3 hours, 6 hours, 9 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 72 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months or 6 months.

[0122] In some embodiments, methods of the present invention include regulatable delivery of Wnt-activating agents. Dosage forms and compositions of the present invention deliver a Wnt-activating agent or agents according to any of a wide variety of immediate and controlled release profiles. As used herein "immediate release" means that release of the Wnt- activating agent is not significantly delayed by means of a protective coating or need for activation in vivo. "Sustained release" means release of a Wnt-activating agent from a dosage form over a longer period of time than the immediate release time of the same Wnt-activating agent from an equivalent dosage in an immediate release formulation. "Delayed release" means that there is a period of time after the dosage form contacts gastric fluid during which the Wnt- activating agent either is not released or is released at a rate that is not therapeutically effective for the purpose that the drug has been administered to the patient. "Burst delivery" means delivery of most of the Wnt-activating agent over a short period of time, typically less than 30 minutes. "Pulsed delivery" means release of the Wnt-activating agent over two or more time periods separated by a period of time in which either the Wnt-activating agent is not delivered or is delivered at a rate that is not therapeutically effective for the purpose that the drug has been administered to the patient. Burst delivery and pulsed delivery may be coupled with delayed release so that release of the Wnt-activating agent according to that profile begins after a delay period in which the Wnt-activating agent either is not released or is released at a rate that is not therapeutically effective for the purpose that the drug has been administered to the patient. The term "controlled delivery" is used inclusively to mean delivery which may include delayed release; sustained release, including delayed sustained release; burst delivery, including delayed burst delivery; pulsed delivery, including delayed pulsed delivery; and any release other delivery profile other than immediate delivery/release.

[0123] The invention additionally pertains to a pharmaceutical composition comprising a

Wnt-activating agent, as described herein, in a container (e.g., a vial, bottle, bag for intravenous administration, syringe, etc.) with a label containing instructions for administration of the composition for treatment of radiation injury. [0124] In some embodiments, a Wnt-activating agent is delivered or administered to a subject contemporaneously with radiation exposure. In some embodiments, a Wnt-activating agent is delivered or administered subsequent to radiation exposure. In some embodiments, a Wnt-activating agent is delivered or administered at least 1, 3, 6, 12, 24, 36, 48, 72, 120, or 168 hours subsequent to radiation exposure.

[0125] In some embodiments, the present invention relates to treating a subject who may be suffering from or susceptible to symptoms of radiation exposure. In some embodiments, the present invention relates to treating a subject who may be suffering from or susceptible to symptoms of radiation induced gastrointestinal syndrome. In some embodiments, the present invention relates to induction of proliferation of stem cells in the gastrointestinal tract of a subject suffering from or susceptible to symptoms of radiation induced gastrointestinal syndrome. In some embodiments, the subject is suffering from cancer. In some embodiments a subject who may be suffering from or susceptible to symptoms of radiation exposure is also is suffering from a cancer, such as a fibrosarcoma, leiomyosarcoma, pleomorphic sarcoma, liposarcoma, synovial sarcoma, chondrosarcoma, glioblastoma, chordoma, lobular breast carcinoma, T BC breast carcinoma, ER+ breast carcinoma, HER2+ breast carcinoma, ductal breast carcinoma, oral cavity squamous cell carcinoma, pancreatic cystademona, pancreatic intraductal papillary mucinous neoplasm, pancreatic ductal malignancy, uveal melanoma, NS melanoma, acral lentiginous melanoma, nodular melanoma, superficial spreading melanoma, conjunctival melanoma, desmoplastic melanoma, sacromatoid mesotheliaoma, epithelioid mesothelioma, biphasic mesothelioma, osteosarcoma, head and neck cancer, glioblastoma multiforme, sarcoma, adenocarcinoma, or colorectal adenocarcinoma. In some embodiments, the subject is also suffering from colorectal cancer.

[0126] In some embodiments, radiation exposure of a subject may result from radiation therapy. In some embodiments, radiation therapy may be abdominopelvic radiation therapy. In some embodiments radiation exposure of a subject may result from non-therapeutic radiation exposure. In some embodiments, non-therapeutic radiation may be radiation exposure not in the course of a therapy. In some embodiments, non-therapeutic radiation exposures may include natural disasters, non-natural disasters, industrial accidents, acts of terror, otherwise inadvertent exposures to potentially lethal or damaging levels of radiation. [0127] Other features of the invention will become apparent in the course of the following descriptions of exemplary embodiments, which are given for illustration of the invention and are not intended to be limiting thereof.

EXEMPLIFICATION

Example 1. Conditional Wnt activation drives hyperproliferation and expansion of undifferentiated stem cells.

[0128] This example illustrates that conditional activation of Wnt signaling promotes cell proliferation and blocks differentiation in vivo. Moreover, this example illustrates that the effects of activation of Wnt signaling are reversible.

[0129] Mice for targeted and conditional knockdown were produced as described in Dow et al., (2015) Cell, 161 : 1539-1552. Figure 2 shows a schematic representing an approach to generate genetically engineered mouse models with inducible shRNA technology. These shRNA transgenic mice enable conditional and reversible Ape expression by TRE-regulated, GFP-linked short-hairpin RNAs. This mouse model enables confined tet-transactivator (rtTA) expression, which allows tissue-restricted tetracycline-regulated element (TRE) shRNA expression.

[0130] Induction of the TRE-driven GFP-miRNA cassette is doxycycline (dox) dependent it is rapidly inducible, reversible, which was confirmed in vivo via a micro- colonoscopy demonstrating GFP expression. GFP was found to be ubiquitously present within 2 days of dox induction and notably absent 2 days after dox withdrawal (data not shown). This demonstrates that tissue specific induction of shRNA expression in these mouse models can rapidly induce gene knockdown, and further that endogenous gene expression is restore upon reversal.

[0131] In these mice that express a rtTA, doxycycline (dox) administration drives GFP expression, Ape silencing, and thus Wnt activation. Subsequent dox withdrawal results in restoration of endogenous Ape and Wnt expression. It was found that ubiquitous and sustained shApc expression in the intestine leads to Wnt activation, rapid crypt hyperproliferation, and a block in cell differentiation resulting in rapid weight loss and death within 8-10 days. See

Figure 3. Therefore, acute genetic disruption of Ape in the intestine drives hyperproliferation and expansion of undifferentiated progenitor cells. This hyperproliferation disrupts the crypt- villus axis, whereby stem and progenitor cells, normally restricted to the crypt base, expand and fail to differentiate as they migrate up the villus.

[0132] Gene set enrichment analysis (GSEA) performed on Rnaseq transcript profiles of shAPC/Lgr5 tumors compared to the shRNA/Lgr5 "normal" mucosa reveals that significantly dysregulated genes. It was found that shAPC/Lgr5 tumors are enriched or depleted for genes identified in transcriptional signatures of Lgr5+ stem cells (data not shown). For example, Wnt activation upregulates DNA repair genes (e.g., RRM2, MAD2L1, TOP2A, CS K1E, RAD54B, BLM, RAD51, BRCA1, SMC2, EME1, etc.) (data not shown). This confirms that the proliferative cells observed in mice with Wnt activation include undifferentiated stem cells.

[0133] While transient Ape loss results in Wnt-driven crypt hyperproliferation and blocked differentiation, Ape restoration induces a rapid and dramatic response with

normalization to endogenous Wnt levels and restoration of the crypt-villus homeostasis (4-5 days dox withdrawal). See Figure 3. This demonstrates that the in vivo effects of activating Wnt signaling are reversible.

[0134] This example demonstrates that activation of Wnt signaling in the intestine induces hyperproliferation disrupts the crypt-villus axis, by expansion of stem and progenitor cells and further that these in vivo effects are reversible, that normal Wnt signaling and crypt- villus homeostasis can be restored.

Example 2 - Effects of irradiation in vivo.

[0135] This example describes the effects of irradiation in vivo. Specifically this example describes the effects of whole body irradiation (WBI) and targeted, whole abdomen irradiation (WAI) in mice.

[0136] The intestine is a highly proliferative and self-renewing organ that is extremely sensitive to DNA damage. In mice, in the hours after whole body irradiation (<9Gy), extensive p53-mediated apoptosis initially leads to intestinal crypt shrinkage, followed by a burst of proliferation in surviving cells, resulting in transient enlargement of intestinal crypts and subsequent crypt fusion to repopulate the intestine (data not shown). At this dose, there is little or no permanent injury to the intestinal stem cell (ISC) niche. Higher doses of radiation (>12Gy) result in greater p53-mediated apoptosis and almost certain death given a lack of cell proliferation and regeneration, suggesting permanent damage to the ISC niche. See, Metcalfe et al., (2014) Cell Stem Cell, 14: 149-159 and Yu, J. (2013) Transl. Cancer Res., 2: 384-396.

[0137] Previous studies have also demonstrated that actively cycling intestinal stem cells

(ISCs), with a turnover of 24 hours, are highly sensitive to RT with an apoptotic index of 40% at 4 hours. See, Potten & Grant (1998) Br. J. Cancer, 78: 993-1003. Without being bound by theory, this sensitivity is thought to be secondary to pro-apoptotic protein accumulation after genotoxic stress, implicating tumor suppressors p53, ATM, and pro-apoptotic PUMA protein pathways. Interestingly, Lgr5+ crypt based columnar (CBC) ISCs have been reported to be less sensitive to RT than intestine progenitor (transient amplifying (TA) cells) and fully differentiated cells (villus cells). See, Hua et al., (2012) Gastroenterology, 143 : 1266-1276. The relative radi ore si stance of Lgr5+ ISCs is thought to be secondary to enhanced homologous repair, however the mechanism by which this occurs remains to be determined. Lgr5 is a Wnt target gene and a validated intestinal stem cell marker as demonstrated through lineage tracing experiments. As Lgr5+ ISC maintain a low genetic mutation burden through the over 1000 cell divisions in their lifetime, intrinsic and undefined processes are likely critical in the maintenance of the genetic fidelity of these Lgr5+ ISC. Little is known about the molecular processes important for stem cell maintenance, DNA damage repair, and repopulation after RT exposure in the intestine.

[0138] In one experiment, C57BL/6 mice were treated with 10 Gy WBI at age 60 days.

Dosimetry of irradiation was confirmed by CT or fluoroscopy. It was observed that 10 Gy WBI treatment induced weight loss (data not shown).

[0139] A highly reproducible system was developed for whole abdomen irradiation

(WAI) using a small animal micro-irradiator. For this system, a small animal subject (e.g., a mouse or rat) is positioned with a laser coordinate system and irradiated using micro animal irradiator. Contrast agents are administered intravenously, intraperitoneally, and/ or orally, as appropriate, and delineation of irradiation and normal organs is assessed by micro-CT or fluoroscopy. See Figure 4. This system allows for confirmation of positioning and real-time animal dosimetry. [0140] WAI was performed with a 225kV orthovoltage micro animal irradiator (Xrad

225Cx). Post- WAI animals were followed clinically (daily weights, bi weekly complete blood counts, and bi-weekly xylose-absorption assays), observed for survival, and the large and small intestines were harvested for histological analysis.

[0141] Daily weight was determined for mice treated by WAI in increasing amounts (10

Gy (n=6), 12, Gy (n=6), 14 Gy (n=7), 16 Gy (n=7), and 18 Gy (n=7)). Mean percentage of weight loss was determined over an 8 day period is depicted in Figure 5. For animals exposed to 10 Gy WAI, maximal weight loss is observed on day 6, with modest recovery observed on days 7 and 8. All higher doses of WAI exhibit maximal weight loss on day 8, with the maximal mean percentage of weight loss being between 30-40%.

[0142] Mortality of animals irradiated with increasing doses of whole abdomen irradiation (WAI) was assessed. Mice were treated by WAI at incremental doses from 6 Gy up to 18 Gy and mortality was assessed 10 days after irradiation. See Figure 6. The LD50 dose at 10 days post- WAI was approximately 11 Gy. Similar results were found when mortality was assessed at 14 days post- WAI, with a LD50 of approximately 11 Gy (data not shown).

[0143] Animal survival over a 30 day period post- WAI was assessed. Prolonged survival

(through 30 days) was observed in the majority of animals exposed to 10 Gy or less during WAI. Survival steeply declined in animals treated with doses of irradiation greater that 10 Gy during WAI. See Figure 7. Coupling Kaplan-Meier animal survival with the microcolony assay revealed that 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, or 35-40 regenerating crypts per

circumference, or approximately l%-5%, 5%-10% 10%-15%, 15%-20%, or 8%-12% crypt survival, is needed for recovery of the GI mucosa and GI tract survival, (data not shown).

[0144] The effects of animal age on survival post WAI was also assessed. Mortality of

60 day old and 120 day old animals irradiated with increasing doses of whole abdomen irradiation (WAI) was determined after 10 days post- WAI. As shown in Figure 8, older animals exhibited increased sensitivity to WAI. The LD50 dose at 10 days post- WAI was found to be approximately 11 Gy in 60 day old animals and 9 Gy in 120 day old animals.

[0145] Histological analysis of harvested intestines on post- WAI animals was performed to assess the effect of RT on gut epithelial regeneration. Animals were treated with 10 Gy and 12 Gy doses of WAI and stained at 24 hour post-WAI with DAPI (as a nuclei marker) and Cleaved Caspase 3 (as a marker for cell death). Fluorescent microscopy imaging of stained intestinal villa was employed. At 24 hour post-WAI, Cleaved Caspase 3 positive cells were observed in the intestinal crypts, with increased cell death observed in animals treated with 12 Gy compared to 10 Gy. Further, Cleaved Caspase 3 positive cells often had unresolved γ-Η2ΑΧ foci at 24 hours suggesting incomplete DSB repair (data not shown). Animals treated with 10 Gy and 12 Gy doses of WAI were also stained at 48 hour post-WAI with DAPI (as a nuclei marker) and KI67 (a marker for cell survival) and imaged by fluorescent microscopy. Higher KI67 staining was observed in animals 10 Gy WAI-treated animals compared to 12 Gy WAI-treated animals. This histological analysis demonstrates that increasing doses of irradiation increases cell death in the intestinal crypts, with decreasing cell survival (data not shown).

[0146] This example described the effects of irradiation in mice. In particular, this example confirmed the effects of WBI in mice and described a method for targeted irradiation in small animals (WAI). Both WBI and WAI treated animals exhibited dose-dependent weight loss. Further, as demonstrated herein, WAI treated animals showed radiation dose-dependent mortality and decreased survival. These effects are believed to be, at least in part, due to the sensitivity of the intestine to irradiation. Histological analysis demonstrated that increasing doses of WAI irradiation increase cell death in the crypts of the intestinal villa and

correspondingly decrease cell survival.

Example 3 - Activation of Wnt signaling mitigates the negative effects of radiation therapy in vivo.

[0147] This example demonstrates that activation of Wnt signaling after exposure to radiation can mitigate the negative effects of radiation in vivo. As described herein, activation of Wnt signaling after radiation therapy is beneficial in preventing and/or reducing symptoms of radiation induced gastrointestinal syndrome (RIGS). Inactivation of APC, which results in an activation of Wnt signaling, is considered the initiating event in most colorectal cancers.

Moreover, the vast majority of colorectal tumors (-80-90%) contain inactivating mutations in APC (Brannon et al., (2014) Genome Biol., 15: 454). Radiation therapy is frequently used during the course of cancer treatment. It is therefore unexpected that activation of Wnt signaling, which is associated with colorectal cancer, can mitigate the negative effects of radiation therapy.

[0148] Specifically, this example demonstrates that transient activation of Wnt signaling after radiation exposure increases cell proliferation, protects against radiation induced weight loss, decreases mortality and increases overall survival in a mouse model. A schematic of an exemplary experimental timeline is shown in Figure 9. Mice are exposed to varying doses of WAI and then treated with DOX to transiently activate Wnt signaling (e.g., for example by knocking down APC or GSK-3P), and then survival and other metrics of RIGS are closely monitored over time.

[0149] Genetically engineered conditional knockdown mice for different targets including Ape, Ren, Brd4, and p53, were subjected to whole abdomen irradiation (WAI). Post- WAI animals were followed clinically (daily weights, bi-weekly complete blood counts, and biweekly xylose-absorption assays), observed for survival, and the large and small intestines were harvested for histological analysis. Irradiation doses can be adjusted as clinically indicated, for example, adjusted in view of the gut epithelial regenerative response.

[0150] Weight loss after irradiation was evaluated in wild-type control and Ape knockdown (Wnt activated) mice. Animals were treated with WAI in increasing amounts (10 Gy, 12, Gy, and 14 Gy). Activation of Wnt signaling by transient knock down of Ape (shAPC), reduced the mean percentage of weight loss over time at all levels of irradiation. See Figure 10.

[0151] We have found that Brd4 inhibition exacerbates gastrointestinal syndrome and decreases survival of irradiated mice even at 9 Gy, a dose that does not induce lethality in 30 days in wildtype mice (data not shown). Knockdown of Brd4 is therefor used herein, at least in part, as a radiosensitized control. Mice genetically engineered to transiently knockdown BRD4 (shBRD4), APC (shAPC) or control mice were treated by WAI in increasing amounts (10 Gy, 12, Gy, and 14 Gy). Brd4 inhibition/knockdown exacerbates GI syndrome after irradiation (data not shown). Weight was measured in these animals and mean percentage of weight loss was determined over an 12 day period is depicted in Figure 11. We found that knock down of BRD4 strongly increased weight loss in animals irradiated with 10 Gy and 12 Gy doses. Conversely, knock down of APC expression generally decreased percentage of weight loss relative to control animals.

[0152] Effects of transient Wnt signaling activation on animal mortality after whole abdomen irradiation (WAI) was assessed. Wild-type animals and animals that transiently knock down Ape as described in Example 1 were irradiated with increasing doses of WAI. Mice were treated by WAI at incremental doses from 6 Gy up to 18 Gy and mortality was assessed 10 days after irradiation. See Figure 12. Transient Wnt signaling activation (achieved, for example by knock down of APC) resulted in an increase in the LD50 dose of irradiation at 10 days post- WAI from approximately 11 Gy to 13 Gy.

[0153] Survival following irradiation of wild-type animals and animals with transient

Wnt signaling activation over a 30 day period was assessed. Specifically, survival of genetically engineered mice that transiently knock down APC (shAPC) and control mice was assessed over a 30 day period following WAI at 10 Gy, 12, Gy, and 14 Gy doses. At both 10 Gy and 12 Gy WAI doses, activation of Wnt signaling promoted survival of animals (100% survival observed over 30 days), with the effect being highly significant at the 12 Gy dose (p = 0.003). See Figure 13

[0154] Survival of genetically engineered mice with inducible knockdown: shAPC mice

(Wnt activated), shBRD4 mice (radiosensitized control), p53 (radioresistant control) and control mice (shRNA-Renilla (shRNA control)) was assessed over a 30 day period following WAI in increasing amounts (10 Gy, 12, Gy, and 14 Gy). Knock down of Brd4 dramatically decreased survival at both 10 Gy and 12 Gy WAI. Transient activation of Wnt signaling through knock down of APC expression increased survival after 12 Gy and 14 Gy WAI. Interestingly, knock down of p53 after WAI had only modest effects on survival at 12 Gy and 14 Gy WAI, however, initiating p53 knockdown prior to WAI treatment (shP53(preRT)) dramatically increased survival at both 12 Gy and 14 Gy. See Figure 14.

[0155] Histological analysis of harvested intestines on post- WAI animals after dox administration of dox to assess the effects of knock down of Ape and p53 were assessed.

Histological analysis may use markers to detect, for example, DNA damage response (γ-Η2ΑΧ induction), apoptotic cell death (cleaved caspase-3, annexin A5, or TU EL), senescence (β- galactosidase), cellular proliferation (BrdU, Ki67, and PI or 7-aminoactinomycin D), cellular differentiation OLFM4 (ISC), Lysozyme (Paneth cells), Alcian blue (Goblet cells), and Keratin 20 (enterocytes), levels of p53, phoho-p53 (S15), CHK1, CHK2, phospho-CHK2 (T68), ATM, phospho-ATM (SI 981), DNA-PK (marker of non-homologous repair), RAD51 (homologous repair), BRCA1 (homologous repair), Cyclin Dl, Rb, and phospho-Rb by immunoblot, immunofluorescence and/or flow cytometry. Histological analysis showed that at 24 hours post 12 Gy WAI and dox administration, rapid hairpin induction was observed in the intestines, which confirms knock down of target genes. Animals were treated with 12 Gy WAI and 24 hours after WAI were stained with DAPI (as a nuclei marker) and Cleaved Caspase 3 (as a marker for cell death) and KI67 (a marker for cell survival) and imaged by fluorescent microscopy. Stained intestinal villa showed that at 24 hour post- WAI, shAPC animals had increased cell survival and at 48 hour post- WAI shAPC animals exhibited increased cell proliferation (data not shown). Increased cell proliferation was observed in Wnt-activated (shAPC) animals at 3.5 days post radiation therapy (data not shown). Further, animals were assessed 8-9 days post irradiation and transient Wnt activation was shown to promote intestinal recovery (data not shown).

[0156] Ongoing mouse WAI experiments with escalating dose levels will allow us to determine the mechanism of radioprotection/mitigation by transient Wnt activation, using our novel shAPC mouse model as well as our control mice (shRNA-Renilla (shRNA control), shRNA-p53 (radioresistant control), and shRNA-Brd4 (radiosensitize control) to assess the impact of shRNA-APC suppresion. Functional assessment of intestinal absorption will be quantified by the xylose absorption assay.

[0157] This example demonstrated that transient Wnt activation (e.g., by transient Ape inhibition), protects against RIGS after irradiation. Specifically this example demonstrated that transient Wnt activation by transient Ape knockdown after escalating doses of WAI resulted in Wnt-driven gut epithelial regeneration, restoration of body weight, increased crypt regeneration, and increased survival with a shift in the LD50/10 from 11 to 13Gy after WAI. This example demonstrates that pulsed Wnt signaling post-whole abdomen irradiation can abate weight loss, mitigate radiation-induced gastrointestinal syndrome and shift the LD50/10 following WAI, resulting in an overall increase in survival of irradiated mice. [0158] Further, since transient Wnt signaling appears to mitigate radiation induced gastrointestinal syndrome, this method may also be useful in reducing the normal tissue injury from acute radiation exposure, which fits with the "dual-utility' philosophy of the Department of Health and Human Services for developing drugs for terrorism that also have a routine clinical use.

Example 4 - Activation of Wnt signaling mitigates the negative effects of radiation therapy in organoids.

[0159] This example demonstrates that transient Wnt activation can mitigate the negative effects of radiation exposure in organoids, an ex vivo model of organ processes. Closely modeling the mechanisms of Wnt activation on radiation induced gastrointestinal syndrome in vivo is challenging. Intestinal crypt organoid cultures were initially developed to serve as tissue surrogates providing a means to directly interrogate ex vivo responses. Mouse small intestinal organoids have been isolated and have undergone extensive validation.

[0160] Primary small intestinal cells can be propagated as 3 -dimensional, spherical

"organoids" in defined media and extracellular matrix conditions as are known in the art. The combination of growth factors (e.g., R-spondin-1, EGF, and Noggin), antioxidants, vitamins, laminin and collagen IV-rich basement membrane extracts sustain ever-expanding epithelial- derived intestinal organoids, which display all hallmarks of the intestine with respect to architecture, cell-type composition, and self-renewal dynamics. (Sato et al., (2009) Nature, 459: 262-265). There are well-developed and validated protocols for the generation, maintenance, and propagation of small intestinal organoids from mice.

[0161] Small intestinal organoids generated from shApc mice confirm that acute Wnt activation via Ape knock down results in a significant increase in organoid proliferation and a block in differentiation indices. See Figure 15A. Importantly, restoration of Ape expression induces rapid phenotypic reversion with restoration of proliferation and differentiation by 4 days following dox withdrawal, Figure 15A. Therefore, Wnt normalization by Ape re-expression engages a cell intrinsic mechanism to restore tissue homeostasis, such that once aberrantly proliferating cells can restore normal crypt architecture. As these 'mini-gut' organoids can be maintained indefinitely in culture, they represent a unique model to study both drug dose and radiation fractionation.

[0162] Radiation dose escalation experiments are performed on cell irradiator. Post- irradiation experimental and control organoids are monitored for cell viability and propagation efficiency. A schematic representation of an experimental timeline to assess the effects of transient genetic manipulation after radiation on organoid cell culture is depicted in Figure 15B. Figure 15B also shows exemplary results of Wnt activation (shAPC) in irradiated organoids (post RT). Wnt activation (e.g., acute Ape loss) following irradiation increased organoid survival and passage capacity. Further experiments intend to define the temporality and duration of the radioprotective effects of Wnt activation.

[0163] Knock down of APC in Kras and p53 mutant-derived organoids was assessed 24 hours after exposure to 6 Gy radiation therapy or mock radiation therapy. Exemplary results on organoid cultures are shown in Figure 16. As depicted in Figure 16, both control organoids (shRenila) and shAPC irradiated organoids appear smaller with an apparent increase in necrotic cells compared to mock-irradiated samples. KRASG12D mutant irradiated organoids appear also slightly smaller than non-irradiated controls. However, both organoids with loss or mutation of p53 appear to be unaffected by radiation.

[0164] Additional in vivo testing could include the incorporation of chemotherapy to assess GSK-3P inhibition potential use in the setting of chemoradiation therapy. The

effectiveness of the agent will be characterized by determination of the radiation dose reduction factor (DRF). As effective radiation mitigators/ protectors might allow dose escalation the normal tissue complication curve for each agent should be determined with escalating doses of radiation that might be used clinically to ascertain if normal tissue protection occurs at higher radiation doses.

[0165] Dose escalation experiments will be performed on a cell irradiator. Post irradiation experimental and control organoids will be monitored for cell viability and propagation capacity. The minimum effective dose of Wnt-activating agents (e.g., GSK-3P inhibitors) to mitigate radiation enteritis and the optimal timing for Wnt activation relative to radiation dosing/exposure will be determined. [0166] This example demonstrated that Wnt-activation also mitigates the negative effects of radiation exposure in a gut organoid model system. Further, this example demonstrates that gut organoids serve as a useful model for assessing efficacy and timing of Wnt-activating agents relative to irradiation.

Example 5 - Wnt pathway activation in an in vivo cancer model

[0167] This example describes assessing the effects Wnt-activation after irradiation in an in vivo cancer model. In order for radiation mitigators/protectors to be used clinically they should be able to decrease the toxic side effects of radiotherapy and have little or no effect on the anti-tumor efficacy. Generation and characterization of patient derived xenograft are derived from patient specimens, which are de-identified and coded into a centralized and secure database. A gross dissection of the tumor sample will be performed to remove any normal tissue. Tumor specimens will be chopped in to 5mm cubes and incubated in collagenase/ dispase overnight at 37°C. After washing, the now single cell suspension will be divided into aliquots of 1 million cells for orthotopic liver injection into immunodeficient (NOD.Cg-i⁵r c^scld

_//2^tmlWjl_{/SzJ) mi ce}

[0168] In order for radiation mitigators/ protectors to be used clinically they should be able to decrease the toxic side effects of radiotherapy and have no effect on the anti-tumor efficacy. The radioprotective effect of GSK-3P inhibition in pre-established patient derived xenografts of hepatopancreaticobiliary tumors will be assessed. A representative tumor acquisition work flow and validation of tumors and PDX models is shown in Figure 17.

Radioprotective effects may also be determined in select representative NCI-60 cancer cell lines. These cells can be infected with luciferase-green fluorescent protein (GFP) constructs via lentiviral infection permits noninvasive in vivo tumor monitoring and therapeutic response to radiation and/ or chemotherapy. The addition of GFP allows for isolation of a pure population of malignant cells from these complex tumors, which often have a significant admixed murine stromal component given preference for orthotopic implantation. Clonogenic survival assays can be performed from isolated GFP+ cells and tumor volumes can be followed longitudinally through serial ultrasonographic images and bioluminescence signal intensity quantification. For GSK-3P inhibitors to be implemented clinically they must be proven not to protect tumors from radiation, or do so to a much lower extent than the reduction of normal tissue injury. [0169] The radioprotective effect of GSK-3P inhibition will be assessed in pre- established patient derived xenografts of hepatopancreaticobiliary tumors and select

representative NCI-60 cancer cell lines.

[001] Having thus described at least several aspects and embodiments of this invention, it is to be appreciated that various alterations, modifications, and improvements will readily be apparent to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawing are by way of example only and the invention is described in detail by the claims that follow.

EQUIVALENTS

[0170] The articles "a" and "an" as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to include the plural referents. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where elements are presented as lists, (e.g., in Markush group or similar format) it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not in every case been specifically set forth in so many words herein. It should also be understood that any embodiment or aspect of the invention can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification. The publications, websites and other reference materials referenced herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference.

Claims

CLAIMS We Claim:

1. A method of treating a subject suffering from or susceptible to acute radiation syndrome (ARS), the method comprising a steps of :

delivering to a subject suffering from or susceptible to ARS an agent that activates Wnt signaling.

2. The method of claim 1, wherein the subject has received or will receive radiation therapy.

3. The method of claim 1, wherein the subject has been or will be exposed to non- therapeutic radiation.

4. The method of any one of claims 1-3, wherein the subject has or will receive a radiation dose between about 0.7 Gy and about 100 Gy.

5. The method of any one of claims 1-4, wherein the agent decreases level and/or or activity of Adenomatous Polyposis Coli (APC).

6. The method of any one of claims 1-4, wherein the agent decreases level and/or or activity of GSK-3p.

7. The method of any one of claims 1-6, wherein the agent is delivered prior to exposure of subject to radiation.

8. The method of any one of claims 1-6, wherein the agent is delivered concomitantly with exposure of subject to radiation.

9. The method of any one of claims 1-6, wherein the agent is delivered subsequent to exposure of subject to radiation.

10. The method of any one of claims 1-9, wherein Wnt signaling returns to normal levels within a period of time after treatment of the agent has been discontinued, wherein the period of time after treatment is selected from 3 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 6 months, and 1 year.

11. A method of treating a subj ect suffering from or susceptible to radiation induced gastrointestinal syndrome (RIGS), the method comprising a steps of :

delivering to a subject suffering from or susceptible to RIGS an agent that activates Wnt signaling.

12. The method of claim 11, wherein the subject has received or will receive abdominopelvic radiation therapy.

13. The method of claim 11, wherein the subject has been or will be exposed to non- therapeutic radiation.

14. The method of any one of claims 11-13, wherein the subject has or will receive a radiation dose between about 3 Gy and about 150 Gy.

15. The method of any one of claims 11-14, wherein the agent decreases level and/or or activity of APC.

16. The method of any one of claims 11-14, wherein the agent decreases level and/or or activity of GSK-3p.

17. The method of any one of claims 11-16, wherein the agent is delivered prior to exposure of subject to radiation.

18. The method of any one of claims 11-16, wherein the agent is delivered concomitantly with exposure of subject to radiation.

19. The method of any one of claims 11-16, wherein the agent is delivered subsequent to exposure of subject to radiation.

20. The method of any one of claims 11-19, wherein Wnt signaling returns to normal levels within a period of time after treatment of the agent has been discontinued, wherein the period of time after treatment is selected from 3 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 6 months, and 1 year.

21. The method of any one of claims 11-20, wherein the activation of Wnt signaling can be regulated.

22. The method of any one of claims 11-20, wherein the delivery of the agent is controlled.

23. The method of claim 22, wherein the delivery of the agent is pulsed.