WO2018067431A1

WO2018067431A1 - Re-sensitizing drug-resistant cancer cells by combinational therapy

Info

Publication number: WO2018067431A1
Application number: PCT/US2017/054707
Authority: WO
Inventors: Ji-Xin Cheng; Junjie Li
Original assignee: Purdue Research Foundation
Priority date: 2016-10-03
Filing date: 2017-10-02
Publication date: 2018-04-12

Abstract

A combinational therapy that provides at least one agent to inhibit cholesterol esterification pathway to re-sensitizes drug resistant cancer cells. The combinational therapy synergizes the chemotherapy or other therapy effects to the resistant cancer cells. Method of identifying re-sensitizing agent is also provided.

Description

RE-SENSITIZING DRUG-RESISTANT CANCER CELLS BY COMBINATIONAL

THERAPY

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application claims the benefit of U.S. provisional application serial number 62/403,351, filed October 3, 2016, the contents of which is hereby incorporated by reference in its entirety.

FIELD OF INVENTION

[0002] This disclosure is related to a combinational therapy that re-sensitizes cancer cells that are resistant to an existing cancer treatment regimen. Particularly, the combinational therapy provides at least one agent that inhibits cholesterol esterification pathway, which in turn leads to decreased cholesterol ester. The combinational therapy synergizes the chemotherapy or other therapy effects to the resistant cancer cells. Method of identifying re-sensitizing agent is also provided in this disclosure.

BACKGROUND

[0003] This section introduces aspects that may help facilitate a better understanding of the

disclosure. Accordingly, these statements are to be read in this light and are not to be understood as admissions about what is or is not prior art.

[0004] Despite the substantial advances achieved in cancer treatment in the past decades, drug resistance still remains a major impediment to eventually successful control or cure of the diseases. Cancer drug resistance includes primary and acquired resistance, depending on whether resistance occurs prior or after initial therapy. Numerous attempts have been made to decipher the mechanisms underlying cancer resistance. Dysregulated metabolism, mutation of the molecular targets, bypass of the targeted signaling and many other mechanisms have been revealed.

[0005] There are theories that the most common reason for acquisition of resistance to a broad range of anticancer drugs is expression of one or more energy-dependent transporters that detect and eject anticancer drugs from cells, but other mechanisms of resistance including insensitivity to drug-induced apoptosis and induction of drug-detoxifying mechanisms probably play an important role in acquired anticancer drug resistance. Studies on mechanisms of cancer drug resistance have yielded important information about how to circumvent this resistance to improve cancer chemotherapy and have implications for pharmacokinetics of many commonly used drugs.

[0006] Reprogrammed cancer metabolism has been recognized as a hallmark of cancer and an emerging therapeutic target for cancer treatment. Studies have revealed increased glycolysis, glutaminolysis, nucleotide and lipid synthesis in cancer cells. Moreover, chemotherapy-resistant cancer cells have been reported to be associated with distinct metabolic profiles, compared to naive cancer cells. Thus, targeting the metabolic pathway represents a promising strategy to overcome resistance to cancer drug treatment.

[0007] Recent studies have found that targeting glycolytic pathway overcomes resistance to chemotherapies, such as trastuzumab and taxol, in breast cancer. Similarly, overexpression of pyruvate dehydrogenase kinase 3 (PDK3) promotes a metabolic switch from mitochondrial respiration to glycolysis under hypoxia condition and increases drug resistance in cervical and colon cancer. Besides, fatty acid synthase (FASN), a key enzyme in the fatty acid synthesis pathway, was reported to be involved in multiple drug resistance, such as docetaxel/trastuzumab resistance in breast cancer and gemcitabine or radiation resistance in pancreatic cancer.

[0008] Drug development for cancer therapy is a lengthy and costly process. Cancers that are resistant to an existing therapy require imminent attention from the scientific, medical and pharmaceutical field. Identifying drug resistance mechanism in the cancer cells may provide insight on how to combat resistant cancers. Even more challenging, finding a solution to recycle the drugs that become ineffective to the resistant cancer cells will save great amount of resources and may bring down the health care cost to the general public. Therefore, there is an unmet need for developing novel therapies that can reuse existing therapies that are not effective to those resistant cancers.

SUMMARY OF THE INVENTION

[0009] In this disclosure, we studied various aggressive cancer types and their existing therapy, as well as cancer cells resistant to the existing therapy regimen.

[0010] This disclosure provides a combinational therapy to re-sensitize cancer cells that are resistant to an existing cancer treatment regimen. The combinational therapy comprises applying an effective amount of at least one agent to the cancer cells, along with the existing cancer treatment regimen that the cancer cells typically are resistant to in the absence of the agent. The agent re- sensitizes the cancer cells to the existing cancer treatment regimen by inhibiting cholesterol esterification pathway. The combinational therapy treated cancer cells has reduced cholesterol ester level and increased intracellular free cholesterol level that leads to the cancer cells apoptosis.

[0011] This disclosure further discloses a method to identify a compound that re-sensitize cancer cells that are resistant to an existing cancer treatment regimen. The method comprises:

determining the baseline cholesterol ester level in the resistant cancer cells; applying at least one compound along with the existing cancer treatment regimen to the cancer cells; measuring the cholesterol ester level in the cancer cells to identify the compound that reduces the base line cholesterol ester level in resistant cancer cells; and confirming the compound causes the resistant cancer cells' death.

[0012] In one preferred embodiment, the aforementioned combinational therapy is used in said cancer cells selected from the group consisting of prostate cancer, pancreatic cancer, myeloid leukemia, melanoma and lung cancer.

[0013] In one preferred embodiment, the aforementioned combinational therapy uses an agent that down regulates PI3K/AKT/mTOR pathway.

[0014] In one preferred embodiment, the aforementioned combinational therapy uses an agent that is an inhibitor to Acyl-coenzyme A: cholesterol acyltransferase isoform 1 (ACAT-1).

[0015] In one preferred embodiment, the aforementioned combinational therapy uses an agent that downregulates MAPK and NFKB pathways.

[0016] In one preferred embodiment, the agent in the aforementioned combinational therapy is avasimibe.

[0017] In one preferred embodiment, the existing cancer treatment regimen in the combinational therapy is a chemotherapy drug, a targeted therapy, a hormone therapy, or an immunotherapy.

[0018] In one preferred embodiment, the existing cancer treatment regimen in the combinational therapy is gemcitabine for pancreatic cancer, imatinib for chronic myeloid leukemia, or abiraterone/enzalutamide for castration-resistant prostate cancer. [0019] In one preferred embodiment, the combinational therapy comprises an agent to chemotherapy drug ratio of about 5: 1 for avasimibe and germcitabine.

[0020] In one preferred embodiment, the combinational therapy comprises an immunotherapy with PD- 1 antibody for melanoma or lung cancer.

[0021] In one preferred embodiment, the cancer cells' cholesterol ester level is determined by

Raman spectra.

[0022] This disclosure further provides a combinational therapy to synergize the effect of an existing chemotherapy drug to cancer cells. The combinational therapy comprises applying an effective amount of at least one agent to the cancer cells, along with an existing chemotherapy drug, so that the agent inhibits cholesterol esterification pathway in the cancer cells.

[0023] In one preferred embodiment, the agent used in the combination therapy to synergize the existing chemotherapy drug is an inhibitor to Acyl-coenzyme A: cholesterol acyltransferase isoform 1 (ACAT-1).

[0024] In one preferred embodiment, the agent used in the combination therapy to synergize the existing chemotherapy drug down regulates MAPK and NFKB pathways.

[0025] In one preferred embodiment, the agent used in the combination therapy to synergize the existing chemotherapy drug is avasimibe.

[0026] These and other features, aspects and advantages of the present invention will become better understood with reference to the following figures, associated descriptions and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The above and other objects, features, and advantages of the present invention will become more apparent when taken in conjunction with the following description and drawings wherein identical reference numerals have been used, where possible, to designate identical features that are common to the figures.

[0028] Figure 1 CE accumulation in gemcitabine-resistant pancreatic cancer cells. (Fig. a) Representative SRS imaging of gemcitabine- sensitive Mia PaCa-2 cells and gemcitabine- resistant G3K cells. Scale bar: 10 μιη. (Fig. b) Quantification of lipid droplet (LD) amount in Mia PaCa-2 and G3K cells. The results are shown as means + SD, n = 6, *** p < 0.001. (Fig. c) Representative Raman spectra taken from individual LD in Mia PaCa-2 and G3K cells. The spectra were offset to clarify. (Fig. d) Quantification of CE percentage out of total lipids in LDs in Mia PaCa-2 and G3K cells. The results are shown as means + SD, n > 10. (Figs, e, f) Cell viability assay of Mia PaCa-2 and G3K cells treated with (E) gemcitabine or (F) avasimibe at indicated concentrations. The results are shown as means + SD, n = 6.

[0029] Figure 2 Avasimibe and gemcitabine synergistically suppressed pancreatic cancer proliferation in vitro. (Fig. a) Cell viability assay of Mia PaCa-2 cells treated with avasimibe, gemcitabine or combination of avasimibe and gemcitabine at the dose ratio of 5: 1. The results are shown as means + SD, n = 6. (Fig. b) The plot of combination index (CI) versus fractional effect determined by CompuSyn software. The blue dotted line indicated CI = 1.0.

[0030] Figure 3 Avasimibe and gemcitabine synergistically suppressed pancreatic tumor growth in vivo. (Fig. a) Tumor growth curves of mice treated with saline (control), avasimibe (7.5 mg/kg), gemcitabine (50 mg/kg), or combination of avasimibe (7.5 mg/kg) and gemcitabine (50 mg/kg). (Fig. b) Body weight curves of mice treated with saline (control), avasimibe, gemcitabine, or combination of avasimibe and gemcitabine. The results are shown as means + SD, n = 8, * p < 0.05, ** p < 0.01, *** p < 0.001.

[0031] Figure 4 Avasimibe resensitizes pancreatic cancer cells to gemcitabine by

suppressing the Akt activity. (Fig. a) Immunoblotting of β-actin, Akt, and p-Akt in Mia PaCa-2 and G3K cells. (Fig. b) Immunoblotting of β-actin, Akt, and p-Akt in Mia PaCa-2 cells treated with gemcitabine, avasimibe, or combination of both at indicated concentrations for 48 hours. (Fig. c) Immunoblotting of β-actin, SREBP-1, Cav-1, Akt, and p-Akt in Mia PaCa-2 cells treated with avasimibe at indicated concentrations for 48 hours. (Fig. d) Diagram showing the mechanism by which avasimibe overcomes gemcitabine resistance in pancreatic cancer cells.

[0032] Figure 5 CE accumulation in chronic myeloid leukemia (CML). (Fig. a) Raman

Spectral Analysis of leukemia cell lines (Fig. b) Quantification of CE% in leukemia cell lines (Fig. c) SRS Imaging of Ba/F3 Cells, 4X (Fig. d) ImageJ quantification by threshold analysis of LD area fraction from SRS imaging in (c) (Fig. e) Raman spectra of Ba/F3 BCR-ABL variants (Fig. f) Quantification of CE% in LDs from Ba/F3 Cells.

[0033] Figure 6 Avasimibe reduces CE in Ba/F3 BCR-ABL^ cells. (Fig. a) Cells were

treated with avasimibe or DMSO for 48 hours, and the CE percentage was measured using Raman spectral analysis. (Raman spectra were acquired using 5-ps laser excitation for 20 s) (Fig. b) CE percentage from Raman spectral analysis was quantified. [0034] Figure 7 Imatinib (IM) and avasimibe show a significant synergy in K562R cells. (Fig. a) SRS imaging of K562(5X) and K562R(2X) cells (Fig. b) CE% in K562 and K562R LDS (Fig. c) 3D contour plot with colormap of cell lines treated with a 1: 10 constant

combination ratio of imatinib to avasimibe for 72 hours. Viability was measured using the Cell Titer Glo assay, with all viabilities normalized to no inhibitor. (Fig. d) Linear plot of IM, avasimibe, and a 1: 10 constant ratio combination of imatinib to avasimibe treatment over 72 hrs. Viability was measured using the Cell Titer Glo Assay, with all luminescence readouts normalized to the luminescence with no inhibitor.

[0035] Figure 8 Combination of avasimibe and imatinib did not show a synergistic effect in naive K562 cells or BCR-ABL dependent imatinib resistant Ba/F3 BCR-ABLT315I cells. (Figs. 8a, c) 3D Colormap Contour Plot of relative cell viability normalized to no inhibitor of Ba/F3 BCR- ABL¹³¹⁵¹ after 72 hour treatment with a 1: 10 constant combination ratio of imatinib to avasimibe measured by Cell Titer Glo. (Figs, b, d) Linear plot showing the relative cell viabilities of Ba/F3 BCR-ABL¹³¹⁵¹ after 72-hour treatment with avasimibe alone, imatinib alone, and a 1: 10 constant combination ratio of avasimibe to imatinib.

[0036] Figure 9 Combination Index of Imatinib and Avasimibe Combination Therapy.

Combination index was calculated from the cell viability data by the Chou-Talalay Method.

[0037] Figure 10 Imatinib and avasimibe synergistically inhibit K562R tumor xenograft growth in implanted athymic nude mice. (Fig. a) Tumor volume (mm³) measured by a caliper over the course of treatment for the four treatment groups. Significance was measured by

Student's T-Test (*p<0.05, ***p<.001) (Fig. b) Body weight (g) of the mice throughout the course of treatment.

[0038] Figure 11 Avasimibe induces downregulation of the MAPK and NF-κΒ pathways.

(Fig. a) Contour biaxials of pS6 (y-axis) and pCREB (x-axis) gated on pCRKL+ K562R cells examined by mass cytometry. Cells were treated for 0 or 4 hours with 10μΜ avasimibe. (Fig. b) Heatmaps of non-lymphoid CD34⁺ CD38^" cells from normal bone marrow (NBM) (top), peripheral blood from an imatinib- sensitive patient (middle), and peripheral blood from an imatinib-resistant patient without a BCR-ABL kinase domain mutation (bottom). Cells were treated with no inhibitor, ΙμΜ imatinib, 10μΜ avasimibe, or imatinib plus avasimibe at the same concentrations. Imatinib treatment was done for 30 minutes, while avasimibe treatment was done for four hours. Heatmap tile color represents arcsinh ratio of medians normalized to the basal condition for each patient, see Bendall et al. 2011 for details. (Fig. c) Biaxials of p-p65/NF-KB on the x-axis versus p-p38/MAPK on the y-axis in CD34⁺ CD38^" cells from the resistant patient examined by mass cytometry. Each plot represents one of the four stimulation conditions: basal (top left), imatinib (top right), avasimibe (bottom left), and imatinib + avasimibe (bottom right). The contour represents cell density. (Fig. d) The top left plot shows the cell types in the viSNE map from the same experiment as panels (b) and (c), with each gate overlayed over the other and color-coded. The top right plot shows cell density in the viSNE map with red being the most dense and blue being the least dense. Gating was done using the viSNE map. See supplemental figure S2 for surface marker validation. The first set of four plots show p-p65/NF-KB intensity across the four aforementioned conditions (top), the second set shows pCREB (middle), and the third set shows p-p38/MAPK (bottom). The maps are color-coded for marker signal intensity, with red being the maximum intensity.

[0039] Figure 12 CE accumulates in castration-resistant patient-derived xenograft (PDX) model and avasimibe shows effects in enzalutamide-resistant MR49F cells. (Fig. a) SRS images and (Fig. b) Raman spectra in PDX CRPC tumor tissues. (Fig. c) Viability assay of MR49F cells treated with enzalutamide or avasimibe at indicated concentrations.

[0040] Figure 13 Table 1. Synergistic effects of avasimibe and gemcitabine in MiaPaCa2 cells combined at various dose ratios.

DETAILED DESCRIPTION

[0041] While the concepts of the present disclosure are illustrated and described in detail in the figures and the description herein, results in the figures and their description are to be considered as exemplary and not restrictive in character; it being understood that only the illustrative embodiments are shown and described and that all changes and modifications that come within the spirit of the disclosure are desired to be protected.

[0042] Unless defined otherwise, the scientific and technology nomenclatures have the same meaning as commonly understood by a person in the ordinary skill in the art pertaining to this disclosure.

[0043] In the present disclosure the term "about" can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range. [0044] Despite many advances have been achieved, as discussed in the background section regarding metabolic pathways that are involved in the cancer resistance development, one area that is largely ignored is the role of cholesterol metabolism in cancer drug resistance. Recently our research has found an unexpected accumulation of cholesteryl ester (CE) in aggressive types of cancers, such as prostate cancer, pancreatic cancer, colon cancer, lung cancer, and leukemia. US Patent 9,164,084 is hereby incorporated herein for reference entirely. This aberrant accumulation of CE can be detected through Raman Spectra and was found to be induced by the activated PI3K/AKT/mTOR pathway and mediated by the Acyl- coenzyme A: cholesterol acyltransferase isoform 1 (ACAT-1) enzyme. Higher expression of ACAT-1 in human pancreatic cancer tissues was correlated with poor survival rate of patients. Inhibition of ACAT-1 by shRNA knock-down or by a small chemical inhibitor, avasimibe, significantly suppressed tumor growth in subcutaneous xenograft mouse models and dramatically reduced metastatic lesions in distant organs in orthotopic mouse models. Mechanistically, we have shown that inhibition of cholesterol esterification increased intracellular free cholesterol level, which induced endoplasm reticulum stress and eventually led to apoptosis.

[0045] With these data showing AC AT- 1 inhibitor, such as avasimibe, as a promising

therapeutic agent for cancer treatment, we further tested combinations of this metabolic therapy with existing therapies, including chemotherapy, targeted therapy, hormone therapy, and immunotherapy in various types of cancers. Our study demonstrated a strong synergistic effect between avasimibe and gemcitabine in pancreatic cancer and imatinib in chronic myeloid leukemia. Additionally, the synergistic effect of abiraterone/enzalutamide in castration-resistant prostate cancer, and PD-1 antibody in melanoma and lung cancer are expected.

MATERIALS AND METHOD DESCRIPTION Cell lines, patient samples and Chemicals

[0046] Human pancreatic ductal adenocarcinoma cell line Mia PaCa-2 was obtained from the American Type Culture Collection (ATCC). Gemcitabine-resistant G3K cell line was generated from parental Mia PaCa-2 cells by continuous culture with the presence of gemcitabine (1). Mia PaCa-2 cells were cultured in RPMI 1640 + 10% FBS +

Penicillin/Streptomycin. G3K cells were cultured in the same media supplemented with 1 μΜ gemcitabine to maintain the resistance. MOLM14, RCH-ACV, K562, and Kasumi-2 cell lines were obtained from DSMZ and maintained in RPMI medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 0.5% penicillin/streptomycin. Ba/F3 and 32D cells expressing empty vector, BCR-ABL,BCR-ABL^T3151 or BCR-ABL^{kinase dead} were maintained in the same medium. K562R cell lines, which display EVI resistance in the absence of BCR-ABL mutations, were initially generated by culturing naive K562 cells with FGF2 and imatinib. Resistant K562R cells were maintained in 0.5-1 μΜ imatinib.

Enzalutamide-resistant prostate cancer MR49-F cells were cultured in RPMI 1640 supplemented with 10% FBS and 10μΜ enzalutamide. For maintenance, all cells were cultured at 37 °C in a humidified incubator with 5% C0₂ supply. All patient samples were obtained with written consent according to an approved IRB protocol. All CML patient samples had wild-type BCR-ABL. Chemicals including avasimibe, gemcitabine, and imatinib mesylate used in vitro and in vivo studies were purchased from Selleckchem.com.

Cell viability assay and combinational index analysis

[0047] Cell viability was measured by Thiazolyl Blue Tetrazolium Blue (MTT) colorimetric assay (Sigma) or ATP assay Cell Titer Glo™ reagent from Promega. Cells were seeded at day 0 in 96-well plates at same density and incubated overnight. Treatments at indicated concentrations were added at day 1 and incubated for another three days. At day 4, MTT agents were added and absorbance was read at 570 nm with a plate-reader. To determine the combination index, various concentrations of gemcitabine and avasimbie were added with a constant dose-ratio. Combination index was analyzed by the Chou-Tatalay method using CompuSyn software.

In vivo study in xenograft mouse model

[0048] All animal experiments were conducted following protocols approved by Purdue Animal Care and Use Committee (PACUC). 4-6 week-old female athymic nude mice (Envigo) were injected with ~5 x 10⁶ MIA PaCa-2 or K562R cells per mouse. One week after tumor cell inoculation, the mice were divided into four groups, control, avasimibe alone, gemcitabine or imatinib along, and combination of avasimibe and gemcitabine or combination of avasimibe and imatinib, with 8 mice in each group. Avasimibe was administered daily by

intraperitoneal injection at a dose of 7.5 mg/kg, imatinib was administered daily by oral gavage at a dose of 70 mg/kg, and gemcitabine was administrated once every 3 days by intraperitoneal injection at a dose of 50 mg/kg. Tumor volume and body weight were monitored twice every week. Tumor volume was calculated following volume = 1/2 x length x width². Treatment was discontinued when one xenograft reached a volume of 2000 mm³.

Stimulated Raman scattering (SRS) imaging and Raman spectroscopy

[0049] Stimulated Raman scattering (SRS) microscopy was performed with two femto-second laser system. Specifically, a Ti:Sapphire laser (Chameleon Vision, Coherent) with up to 4W (80 MHz, ~ 140 fs pulse width) pumps an optical parametric oscillator (OPO, Chameleon Compact, Angewandte Physik & Elektronik GmbH). The pump and Stokes beams were tuned to 830 nm and 1090 nm, respectively. The pump and Stokes pulse trains were collinearly overlapped and directed into a laser-scanning microscope (FV300, Olympus). A 60X water-immersion objective lens (UPlanSApo, Olympus) was used to focus the laser into a sample. An oil condenser of 1.4 numerical aperture (NA) was used to collect the signal in a forward direction. The typical acquisition time for a 512 x 512 pixels SRL image was 1.12 second. Images were processed using ImageJ.

[0050] Confocal Raman spectral analysis from individual lipid droplets (LDs) were performed as described previously (Slipchenko MN, Le TT, Chen HT, Cheng J-X. High-speed vibrational imaging and spectral analysis of lipid bodies by compound Raman microscopy. J Phys Chem B. 2009;113(21):7681-6. A 5-picosecond laser at 707 nm was used as excitation beam for Raman spectral acquisition. Acquisition time for a typical spectrum from individual LDs was 20 s, with the beam power maintained around 15 mW at sample. For each specimen, at least 10 spectra from individual LDs in different locations or cells were obtained. The spectra were analyzed using software Origin 8.5. The background was removed manually, and peak height was measured.

Mass Cytometry

[0051] Single-cell protein analysis was performed using a CyTOF2 instrument according to previously published procedures. All metal-conjugated antibodies were purchased from Fluidigm. Cells were treated with ΙμΜ imatinib for 30 minutes or 10μΜ avasimibe for 4 hours. Data analysis was performed using Cytobank as described previously. Further analysis was performed using viSNE.

Immunoblotting

[0052] Cells were harvested and lysed in RIPA lysis buffer (Sigma- Aldrich) supplemented with protease and phosphatase inhibitor cocktail. Protein concentration was determined using the Bio-Rad protein assay kit. Protein extraction was subjected to immunoblotting with the antibodies against Akt (Cell Signaling, #4691), p-Akt (Cell Signaling, #4060L), Caveolin-1 (Cell signaling, #3238S), SREBP-1 (Santa Cruz, #sc-13551), and β-actin (Sigma, A5441). β- actin was used as loading control for normalization.

Statistical analysis

[0053] One-way student's t-test or One-way ANOVA test were used for comparisons between groups. Results were represented as means +/+ SEM or as specified. Statistical significance was indicated as * P < 0.05, ** P < 0.01, and *** P < 0.001

EMBODIMENTS AND RESULT

[0054] To test the combinational effects of cholesterol ester inhibitor and an existing cancer treatment therapy to various resistant cancer types, we have performed Raman spectroscopic imaging and spectral analysis, in intro cell viability assay, combination index analysis, and in vivo efficacy test. Furthermore, we conducted experiments to elucidate the possible mechanisms by which the combination therapies excerpt synergistic effects. The following Examples refer to these and other aspects of the invention in more details.

EXAMPLE 1. Cholesterol Ester (CE) accumulation in gemcitabine-resistant pancreatic cancer cells

[0055] In this example, to study whether lipid metabolism was altered in gemcitabine resistant cells, a gemcitabine-resistant cell line G3K was generated from Mia PaCa-2 cells by continuous culture with gemcitabine (15). Stimulated Raman Scattering (SRS) imaging and Raman spectroscopy were used to assess the lipid and cholesterol content in parent MiaPaCa- 2 and gemcitabine-resistant G3K cells. G3K cells were shown to have significantly higher amount of lipid droplets (LDs) than Mia PaCa-2 cells (Fig. la, b). In addition, the percentage of cholesteryl ester (CE) in LDs of G3K cells remains as high as Mia PaCa-2 cells (Fig. lc, d). Considering the increased number of LDs, it indicates that the total amount of CE largely increases in G3K cells, suggesting an opportunity to treat gemcitabine-resistant cancer cells by targeting the increased level of cholesterol esterification. Then an inhibitor of cholesterol esterification, avasimibe, was tested in both Mia PaCa-2 and G3K cells. The results showed that avasimibe effectively suppressed cell viability with similar sensitivity in Mia PaCa-2 and G3K cells with IC50s of 7.0 and 8.85 μΜ, respectively (Fig. le), despite that G3K cells are highly resistant to gemcitabine with IC50s of 1.23 and 36.34 μΜ in Mia PaCa-2 and G3K cells, respectively (Fig. If).

EXAMPLE 2. Synergistic effect between avasimibe and gemcitabine in vitro

[0056] In this example, we have demonstrated that avasimibe, an inhibitor to ACTAl enzyme, synergizes the effect of gemcitabine to cancer cells. Having shown the efficacy of avasimibe in gemcitabine-resistant cells, we further explored the effects of a combination of avasimibe and gemcitabine. Mia PaCa-2 cells were treated with avasimibe, gemcitabine alone, or combination of avasimibe and gemcitabine at concentrations with constant ratios. At a concentration ratio of 5: 1 (avasimibe:gemcitabine), the combinational therapy showed superior inhibitory effects on cell viability, compared to single agent treatments (Fig. 2a). A further analysis of the combination index (CI) between these two compounds suggests a synergistic effect, as indicated by that the value of CIs are below 1.0 at the most

concentrations tested (Fig. 2b). Additionally, the combinational effects were also tested at other concentration ratios, include 10: 1 and 15: 1 (avasimibe:gemcitabine). Combinations at these ratios also showed synergistic effects between avasimibe and gemcitabine (Table 1), and the effects were optimal at the ratio of 5: 1.

EXAMPLE 3. Combination effects of Avasimibe and Gemcitabine in xenograft model

[0057] This example shows combination effects of Avasimibe and Gemcitabine in xenograft model. To further validate the combinational effects of avasimibe and gemcitabine, a xenograft model derived from Mia PaCa-2 cells was used. Avasimibe and gemcitabine were administrated at doses of 7.5 mg/kg and 50 mg/kg, respectively. While avasimibe and gemcitabine alone suppressed tumor growth, combination of these two compounds sigfinicantly amplified this effect by further inducing a regression of tumors after treatment (Fig. 3a). Notably, no obvious treatment associated body weight loss was observed (Fig. 3b). These data suggest a strong synergistic effect between avasimibe and gemcitabine when administrated together as a combinational therapy.

EXAMPLE 4. Avasimibe overcomes gemcitabine-resistance by downregulating Akt pathway

[0058] This example shows avasimibe overcomes gemcitabine-resistance by downregulating Akt pathway. To investigate the potential mechanism by which avasimibe overcomes gemcitabine- resistance, we have performed immunoblotting to evaluate the levels of key signaling pathways. Akt pathway has been known as one of the signaling pathways associated with gemcitabine-resistance in pancreatic cancer. We examined the expression level of Akt and phosphorylated-Akt in gemcitabine-sensitive Mia PaCa-2 cells and gemcitabine-resistant G3K cells. Indeed, expression level of p-Akt largely increased in G3K cells, indicating a correlation between Akt activity and gemcitabine-resistance (Fig. 4a). Consistently, treatment with gemcitabine slightly increased expression of p-Akt, and treatment with avasimibe decreased the expression level of p-Akt. Moreover, combinational treatment of gemcitabine and avasimibe significantly reduced the level of p-Akt compared to single treatments (Fig. 4b). The mechanism by which avasimibe suppresses Akt signaling is proposed to be mediated through SREBP-1. As expected, avasimibe treatment significantly suppressed the expression levels of SREBP-1 in Mia PaCa-2 cells (Fig. 4c). Collectively, these data support that avasimibe overcomes gemcitabine-resistance by increasing cellular free cholesterol level, which inactivates SREBP1 and further downregulated Akt signaling pathway (Fig. 4d).

EXAMPLE 5. Abnormal CE accumulation in chronic myeloid leukemia (CML) is driven by BCR-ABL

[0059] This example shows abnormal CE accumulation in chronic myeloid leukemia (CML) is driven by BCR-ABL. To characterize the lipid metabolism in leukemia cells, Raman spectral analysis was performed on a variety of well-characterized leukemia cell lines, including MOLM14 (AML), RCH-ACV (ALL), Kasumi-2 (ALL), and K562 (CML) cells. An abnormal accumulation of CE was identified in K562 cells, as evidenced by the peak at Raman shift of 702cm^"1 from cholesterol ring vibration (Fig. 5a). Quantitative analysis revealed a 50% level of CE in the lipid droplets of K562 cells, but only around 10% in other cells (Fig. 5b). Considering the correlation between BCR-ABL activation and CE

accumulation in CML, we hypothesized that BCR-ABL drives CE accumulation. To assess whether BCR-ABL was necessary and sufficient to cause CE accumulation, a murine interleukin-3 dependent pro-B cell line Ba/F3 was used. Ba/F3 cells overexpressing BCR- ABL^WT, BCR-ABL¹³¹⁵¹, or empty vector (control) were subjected to SRS imaging to visualize LD accumulation in the three cell lines (Fig. 5c). Ba/F3 cells transfected with empty vector showed no accumulation of LDs, regardless of whether they were stimulated with IL-3 for 48 hours. On the other hand, Ba/F3 BCR-ABL and Ba/F3 BCR-ABL cells had LD accumulation even without IL-3 stimulation (Fig. 5d). Through Raman spectral analysis of these lipid droplets, these LDs were found to be mainly composed of CE (65- 75%) (Fig. 5e-f). The Ba/F3 control cells could not be spectrally analyzed because there were no detectable LDs.

EXAMPLE 6. Treatment with avasimibe was sufficient to remove CE in Ba/F3 BCR- ABL^WT cells

[0060] In this example we are able to show the treatment with avasimibe was sufficient to

remove the accumulated cholesterol ester in the Ba/F3 BCR ABL^WT cells, suggesting the potential of targeting cholesterol metabolism in BCR-ABL driven chronic myeloid lymphoma cells. Detailed description is found in the Brief Description of Drawings section for Figure 6.

EXAMPLE 7. Avasimibe resensitizes BCR-ABL mutation-independent imatinib-resistant CML in vitro

[0061] This example shows avasimibe resensitizes BCR-ABL mutation-independent imatinib- resistant CML in vitro.

[0062] To test whether CE accumulation occurs in BCR-ABL mutation-independent EVI resistant CML, the K562R cell line was established. SRS imaging was used to visualize the lipid droplets in individual K562R cells, as compared to K562. SRS imaging showed noticeable LD accumulation in both cell lines (Fig. 7a). Raman spectral analysis on individual lipid droplets confirmed a high percentage of CE in their LDs (Fig. 7b). To test whether avasimibe could overcome imatinib resistance in CML, K562R cells displaying BCR-ABL mutation- independent resistance were treated with avasimibe and imatinib. The combination of avasimibe and imatinib at a 10: 1 fixed concentration ratio in K562R cells yielded a significant reduction in cell viability at all concentrations tested (Fig. 7c-d)

EXMAPLE 8. Combination treatment of Ba/F3 BCR-ABL^T31SI cells does not yield a synergistic effect.

[0063] This example shows the combination of avasimibe and imatinib did not show a

synergistic effect in naive K562 cells or BCR-ABL dependent imatinib resistant Ba/F3 BCR- ABL cells. Detailed description is found in the Brief Description of Drawings section for Figure 8.

EXAMPLE 9. The combination index (CI) as defined by the Chou-Talalay method indicated a strong synergistic effect between avasimibe and imatinib in K562R cells, but not in K562 or Ba/F3 BCR-ABL^T31SI cells.

[0064] Detailed description for this example is found in the Brief Description of Drawings

section for Figure 7.

EXAMPLE 10. Avasimibe and imatinib synergistically reduce tumor growth in a xenograft mouse model.

[0065] This example shows avasimibe and imatinib synergistically reduce tumor growth in a xenograft mouse model. To confirm the synergy between avasimibe and imatinib in vivo, we used a xenograft mouse model. The combination treatment significantly (p<0.001) reduced tumor growth as compared to the control (DMSO), imatinib, or avasimibe treated groups (Fig. 10a). Moreover, no significant treatment related body weight loss was observed (Fig. 10b). These data suggest that a combination of avasimibe and imatinib could be a promising therapeutic strategy to treat imatinib-resistant CML without BCR-ABL kinase domain mutations.

EXAMPLE 11. Avasimibe induces downregulation of the MAPK and NF-κΒ pathways.

[0066] To understand the mechanism of drug synergy, signaling responses to avasimibe in K562R cells were examined via mass cytometry (CyTOF). Avasimibe treatment led to a substantial reduction in pS6 and pCREB levels (Fig. 11a), suggesting downregulation of the PI3K and MAPK pathways. The effect of combination treatment was tested in normal bone marrow as well as peripheral blood from an imatinib- sensitive patient and an imatinib-resistant CML patient with wild type BCR-ABL. Mass cytometry analysis revealed that CD34⁺ CD38^~cells in the sensitive patient were profoundly sensitive to imatinib treatment, while combination treatment provided minimal additional effect on the levels of eight intracellular signaling markers (Fig. lib). Combination therapy had minimal effect in normal bone marrow. The resistant patient's cells also displayed sensitivity to imatinib as measured by pCRKL levels (canonical downstream target of BCR-ABL). However, in the resistant patient, imatinib treatment led to increased levels of p-p65/NF-KB, p-p38, and pCREB. Combination treatment reversed the effect of imatinib leading to decreased phosphorylation of these signaling effectors (Fig. llb-c). To further understand the effects of imatinib and avasimibe on signaling in CML cells, mass cytometry data was analyzed using the viSNE algorithm that plots every individual cell in on the tSNE axes according to their similarity to neighboring cells. Using viSNE analysis, we found that the pNF-κΒ/ΜΑΡΚ stimulation due to imatinib was not limited to the primitive CD34⁺CD38^~ cell population, but could be generalized to the spectrum of myeloid cells expressing CD34 and CD38, as well as more mature cells of the myeloid lineage including monocytes (Fig. lid). Imatinib-related NF-κΒ activation was not seen, however, in mature T- Cells, B-Cells, or granulocytes, which is to be expected. Combination therapy was also more effective than avasimibe or imatinib monotherapy across the hematopoietic spectrum in the resistant patient, based upon decreased phosphorylation of p-p65/NF-KB, p-p38/MAPK, and pCREB in all cell types with a stimulation response to imatinib (Fig. lid).

EXAMPLE 12. Cholesterol ester (CE) accumulates in castration-resistant patient-derived xenograft

[0067] In this Example, Fig. 12a-b shows Avasimibe has high efficacy in suppressing the viability of enzalutamide-resistant prostate cancer MR49F cells (Fig. 12c), suggesting the potential use of avasimibe to overcome enzalutamide resistance in patients with very late-stage prostate cancer.

[0068] Those skilled in the art will recognize that numerous modifications can be made to the specific implementations described above. The implementations should not be limited to the particular limitations described. Other implementations may be possible.

Claims

Claims:

1. A combinational therapy to re-sensitize cancer cells that are resistant to an existing cancer

treatment regimen, comprising: applying an effective amount of at least one agent and said existing cancer treatment regimen to said cancer cells, wherein said agent re-sensitizes said cancer cells to said existing cancer treatment regimen by inhibiting cholesterol esterification pathway.

2. A method to identify a compound that re-sensitize cancer cells that are resistant to an existing cancer treatment regimen, comprising: determining the baseline cholesterol ester level in said resistant cancer cells; applying at least one compound and the existing cancer treatment regimen to said cancer cells; measuring the cholesterol ester level in said cancer cells to identify the compound that reduces said base line cholesterol ester level in resistant cancer cells; and confirming the compound causes the resistant cancer cells' death.

3. The combinational therapy according to claim 1, wherein said cancer cells has reduced

cholesterol ester level.

The combinational therapy according to claim 1, wherein said cancer cells has increased intracellular free cholesterol level that leads to said cancer cells apoptosis.

The combinational therapy according to claim 1, wherein said cancer cells are selected from the group consisting of prostate cancer, pancreatic cancer, myeloid leukemia, melanoma and lung cancer.

The combinational therapy according to claim 1, wherein said agent down regulates

PI3 K/AKT/mTOR pathway.

7. The combinational therapy according to claim 1, wherein said agent is an inhibitor to Acyl- coenzyme A: cholesterol acyltransferase isoform 1 (ACAT-1).

8. The combinational therapy according to claim 1, wherein said agent down regulates MAPK and NFKB pathways.

9. The combinational therapy according to any of claims 6-8, wherein said agent is avasimibe.

10. The combinational therapy according to claim 1, wherein said existing cancer treatment regimen is a chemotherapy drug, a targeted therapy, a hormone therapy, or an immunotherapy.

11. The combinational therapy according to claim 10, wherein said chemotherapy drug is

gemcitabine for pancreatic cancer, imatinib for chronic myeloid leukemia, or

abiraterone/enzalutamide for castration-resistant prostate cancer.

12. The combinational therapy according to claim 11, wherein said agent and said chemotherapy drug ratio is about 5: 1 for avasimibe to germcitabine.

13. The combinational therapy according to claim 10, wherein said immunotherapy is PD-1 antibody for melanoma or lung cancer.

14. The method according to claim 2, wherein said cholesterol ester level is determined by Raman spectra.

15. A combinational therapy to synergize the effect of an existing chemotherapy drug to cancer cells, comprising applying an effective amount of at least one agent and said existing chemotherapy drug to said cancer cells, wherein said agent inhibits cholesterol esterification pathway.

16. The combinational therapy according to claim 15, wherein said agent is an inhibitor to Acyl- coenzyme A: cholesterol acyltransferase isoform 1 (ACAT-1).

17. The combinational therapy according to claim 15, wherein said agent down regulates MAPK and NFKB pathways.

18. The combinational therapy according to any claims of 15-17, wherein said agent is avasimibe.