WO2020154716A1 - Compositions and methods for treating prostate cancer - Google Patents

Abstract

The invention provides novel therapeutically methods and pharmaceutical compositions for treating prostate cancer with increased survival rate and improved treatment outcome. Methods and compositions of the invention can be used to prevent, delay, and/or reduce drug resistance and to increase, restore and/or prolong the effective treatment of prostate cancer with anti-androgen compounds.

Description

COMPOSITIONS AND METHODS FOR TREATING PROSTATE CANCER

Priority Claims and Related Applications

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 62/797,210, filed January 26, 2019, the entire content of which is incorporated herein by reference for all purposes.

Technical Field of the Invention

[0002] The invention generally relates to novel therapeutic methods and pharmaceutical compositions for treating cancer. More particularly, the invention relates to a novel approach to addressing drug resistance in prostate cancer treatment.

Background of the Invention

[0003] Prostate cancer (PCa) is the most common form of cancer and the second leading cause of cancer death among men in the United States. (Centers for Disease Control and Prevention http://www.cdc.gov/cancer/dcpc/data/men.htm.) Approximately one in seven men will be diagnosed with prostate cancer during his lifetime, and about one in 38 men will die of the disease with an estimated 307,000 deaths worldwide in 2012. (Merseburger, et al. 2015 Ther Adv Urol 7, 9-21.)

[0004] The current standard treatment for advanced prostate cancer is androgen deprivation therapy (ADT), which may suppress prostate cancer progression by reducing androgen biosynthesis or by preventing androgen from binding to the androgen receptor. (Chang, et al. 1988 Science 240, 324-326; Heinlein, et al. 2004 Endocr Rev 25, 276-308; Chang, et al. 2014 Oncogene 33, 3225-3234; Niu, et al. 2010 Oncogene 29, 3593-3604.)

[0005] A newly developed anti-androgen compound, Enzalutamide (Enz), was shown to suppress castration resistant prostate cancer (CRPC) and could further extend patients overall survival by an average of 4.8 months. (Scher, et al. , 2012 N Engl JMed 367, 1187-1197.)

[0006] Unfortunately, most ADT, including Enz, may fail after 1-2 years treatment due to development of castration resistance (EnzR). (Miyamoto, et al. 2005 Nature clinical practice. Oncology 2, 236-237; Miyamoto, et al. 2004 The Prostate 61, 332-353.) The detail mechanism, however, remains unclear.

[0007] Several mechanisms have been developed to account for the development of Enz- resistance. For example, Korpal et al. found that the ARF876L mutation might confer the resistance to Enz, and Liu et al. found that AKR1C3 might enhance the Enz-resi stance via increasing the cellular androgen concentration. Interestingly, Arora et al. found that

glucocorticoid receptor might also contribute to the Enz-resi stance via replacing AR to activate some survival related signals. However, among many potential mechanisms to induce the Enz- resistance, the Enz-induced AR splicing mutant ARv7 received the most attention. A recent clinical survey clearly suggested that CRPC patients received ADT with Enz had increased ARv7, and ARv7 expression was higher in patients who developed Enz-resistance. The detailed mechanisms on how Enz-induced ARv7 can lead to Enz resistance and how to overcome Enz- resistance to further suppress CRPC remain to be further elucidated. (Korpal, et al. 2015 Cancer discovery 3, 1030-1043 (2013); Liu, et al. Cancer research 75, 1413-1422; Arora, et al. 2013 Cell 155, 1309-1322; Antonarakis, et al. 2014 The New England journal of medicine 371, 1028- 1038.)

[0008] Currently, there is not an effective way to delay, reduce or reverse such drug resistance, which has seriously limited the overall effectiveness and treatment outcome of anti androgen therapy. There remains an urgent need for novel and improved approaches that effectively address these issues.

Summary of the Invention

[0009] The invention is based in part of the discovery of a unique approach to treatment of prostate cancer, particularly drug resistance prostate cancer. Methods and compositions of the invention can be used to prevent, delay, reduce and/or reverse drug resistance and to increase, restore and/or prolong the effective treatment of prostate cancer with anti-androgen compounds.

[0010] As is disclosed herein, SIP-ceramide axis is altered significantly (with increased SIP yet decreased ceramide) in the Enz-resistant CRPC (EnzR) cells, and targeting this Enz-altered SIP-ceramide axis with SPHK1 inhibitors or tetrahydrocannabinol (THC, the active component of marijuana) can re-sensitize the Enz-R cells to further suppress Enz-R cell growth. [0011] As disclosed herein, Enz alters the SIP-ceramide rheostat to induce the Enz resistance, and targeting this SIP-ceramide rheostat with THC or Fingolimod/SKI2 all resulted in delaying the Enz resistance development and increasing the Enz sensitivity to further suppress the EnzR cells growth. Mechanism dissection revealed that Enz functions via modulating the ARv7-altered miRNAxx signaling to decrease the SPHKAP expression, which then leads to increase SIP production via increasing SPHK1 activity. The

Enz/ARv7/miRNAxx/SPHKAP/SPHKl axis-increased SIP then results in increasing Enz resistance via altering the methuosis, a rare cell death in the EnzR cells. Preclinical studies using multiple in vitro EnzR cell lines and in vivo mouse models demonstrated that targeting this newly identified Enz-altered SIP-ceramide rheostat with THC or Fingolimod/SKI2 all resulted in increasing the Enz sensitivity to further suppress the EnzR cells growth.

[0012] In one aspect, the invention generally relates to a pharmaceutical composition comprising: a first compound of Formula (I)

or a pharmaceutically acceptable salt, ester or pro-drug thereof, and a second compound selected from an inhibitor of SPHK and an activator of ceramide, wherein each of the first and second compounds is present in an amount such that the pharmaceutical composition is effective in the treatment of prostate cancer, or a related disease or condition thereof, in a mammal, including a human, and a pharmaceutically acceptable carrier.

[0013] In yet another aspect, the invention generally relates to a method for treating prostate cancer, or a related disease or condition thereof, comprising administering to a subject in need thereof a pharmaceutical composition disclosed herein.

[0014] In yet another aspect, the invention generally relates to a method for treating prostate cancer, or a related disease or condition thereof, comprising administering to a subject in need thereof a first compound of Formula (I)

or a pharmaceutically acceptable salt, ester or pro-drug thereof, and a second compound which is an inhibitor of SPHK or an activator of ceramide, in amounts effective in the treatment of prostate cancer, or a related disease or condition thereof, in a mammal, including a human, wherein the subject has been detected of the development of drug resistance to the first compound.

[0015] In yet another aspect, the invention generally relates to a method for treating prostate cancer, or a related disease or condition thereof, comprising: administering to a subject in need thereof a first compound of Formula (I)

or a pharmaceutically acceptable salt, ester or pro-drug thereof, in an amount effective in the treatment of prostate cancer or a related disease or condition thereof in a mammal, including a human; monitoring the subject to detect a development of drug resistance to the first compound; upon the subject being detected of the development of drug resistance to the first compound, administering to the subject a second compound selected from an inhibitor of an inhibitor SPHK and an activator of ceramide, in an amount effect to reduce or eliminate drug resistance to the first compound; and monitoring the subject to detect a level of drug resistance to the first compound.

[0016] In yet another aspect, the invention generally relates to a method for treating a drug resistance in connection with a prostate cancer treatment, comprising administering to a subject in need thereof a compound which is an inhibitor of SPHK or an activator of ceramide, in an amount effective to prevent, delay, reduce or reverse resistance to a treatment of prostate cancer, or a related disease or condition thereof in a mammal, including a human.

Brief Description of the Drawings

[0017] FIG. 1. SIP signal is activated in EnzR cells. (A) The RNA-seq was performed to compare the different genes expressions in EnzR and EnzS cells. The genes which showed significant difference were picked up and uploaded for the gene ontology analysis. The results showed that the most of the genes were involved in the different metabolism processes. (B) The list of the genes which showed significant change in EnzR cells was analyzed by Pathway Commons pathway analysis. The results showed that the SIP pathway is significantly altered in EnzR cells. (C) The Gene Set Enrichment Analysis (GSEA) was performed based on the RNA- seq results and results showed that Sphingolipid related pathway is highly activated in EnzR cells. (D) The genes expression which were involved in sphingolipid metabolism were increased in EnzR cells. (E) The SIP and Ceramide levels in EnzRl&4 and EnzSl&4 cells were analyzed by Elisa assay (SIP) and HLPC (ceramide). (F) The EnzSl cells were pre-treated with/without (w/o) 1 uM SIP for 3 days and then the Enz efficacy was analyzed by MTT. (G) The SIP levels in EnzR patients’ plasma were analyzed. For E, data are presented as mean ± SD, * p< 0.05, ** p< 0.005, N.S, not significant by t-test for two groups or ANOVA for more than two groups.

[0018] FIG. 2. Targeting SIP signal with different compounds can reverse the EnzR. (A) The EnzRl cells were treated w/o 2 uM Fingolimod and the Enz efficacy was analyzed by MTT.

(B) The EnzRl cells were treated w/o lOuM SKI2 and the Enz efficacy was analyzed by MTT.

(C) The EnzR4 cells were treated w/o 2 uM Fingolimod, and Enz efficacy was analyzed by MTT. (E-F) The EnzSl, EnzRl and EnzR3 cells were treated with different does of SKI2 (E) and Fingolimod (F) for 24hrs, the cell viabilities were analyzed by MTT assay. (G-H) The EnzRl (G) and EnzR4 (H) cells were treated w/o 20uM ceramide and the Enz efficacy was analyzed by MTT assay. (I) The EnzRl cells were treated w/o lOuM CBD and the Enz efficacy was analyzed by MTT assay. (J) The EnzRl cells were treated w/o 6uM THC and the Enz efficacy was analyzed by MTT assay. (K-L) EnzR4 cells were treated w/o lOuM CBD (K) or 9uM THC. (N) The EnzRl cells were treated w/o different does of THC and CBD, the p-SPHKl level were analyzed. (P-R) The EnzSl cells were treated w/o Enz, SKI2 or CBD for 1 month.

And then the Enz efficacy on the cells with different kinds of treatments was analyzed by MTT assay.

[0019] FIG. 3. The SPHK1 activity is up-regulated in EnzR cells. (A)The SPHK1, SPHK2, CERK and ACER1 expressions were analyzed in EnzRl and EnzSl cells by WB. (B) The SPHKs activity was analyzed in EnzRl and EnzSl cells. (C) The EnzSl cells were treated w/o lOuM Enz for 10 days, and then the SPHKs activity was analyzed. (D) The EnzRl cells were treated w/o SPHK1 inhibitor PF543 or SPHK2 inhibitor ABC294640, and the Enz efficacy were examined. (E) The SPK1 p-S225 level in EnzRl and EnzSl cells were examined by WB. (F) The SPHK1 was knocked down by two shRNAs in EnzR4 cells, and then the Enz efficacy was examined by MTT assay on day 2&4. For B, C and D, data are presented as mean ± SD, * p< 0.05, ** p< 0.005, N.S, not significant by t-test for two groups or ANOVA for more than two groups.

[0020] FIG. 4. Low expression of SPHKAP in EnzR cells promote SPHK1 activity and SIP production. (A) Western blot analysis to detect SPHKAP protein level in EnzRl and EnzSl cells. (B) The EnzSl cells were treated with lOuM Enz for different time points and the SPHKAP level was examined. (C) The PDX-133-4C tumors were treated with 30mg/kg Enz for 20 days (I.P injection each other day). The SPHKAP level was examined by IHC. (D)The SPHK activities in EnzS-plKO and EnzS-sh-SPHKAP cells were analyzed. (E) The SIP levels in EnzS-plKO and EnzS-sh-SPHKAP cells were analyzed. (F) The Enz efficacy on EnzR-pWPI and EnzR-OE- SPHKAP cells was analyzed by MTT assay. (G) The Enz efficacy on EnzS-Plko and EnzS-sh- SPHKAP cells was analyzed by MTT assay. (H) The EnzSl -shSPHKAP cells were treated with CBD or Fingolimod, and the Enz efficacy were analyzed by MTT assay. (I) The mRNA levels of SPHKAP in different stages of PCa samples were analyzed. The data is from TCGA database.

(J) The SPHKAP protein levels in PCa tissues and normal prostate tissues were analyzed. The data is from The Human Protein Atlas. (K) We applied the tissue micro-array to analyze the SPHKAP protein levels in PCa samples with the different gleason scores. For C and D, data are presented as mean ± SD, * p< 0.05, ** p< 0.005, N.S, not significant by t-test for two groups or ANOVA for more than two groups. [0021] FIG. 5. ARv7 suppresses SPHKAP expression via miRNA. (A) EnzSl cells were infected with ARv7 and ARF876L cDNA and the SPHKAP levels were examined by WB. (B) The ARv7 was knocked down in EnzRl cells and SPHKAP level were examined by WB. (C) The SIP levels in EnzSl -pWPI, EnzSl -ARE876L and EnzSl -ARv7 cells were analyzed. (D)

The ARv7 was knocked down in EnzRl cells and SIP levels were examined. (E) The mRNA level of SPHKAP was analyzed in EnzSl and EnzRl cells. (F) The enrichment of SPHKAP mRNA in Ago2 complex was analyzed in EnzRl and EnzSl cells by RNA-IP assay. (G) The potential miRNAs which can target SPHKAP were screened out from four different miRNA databases. The overlapped miRNAs were picked up for further identification. (H) The candidate miRNAs expressions were examined in EnzSl and EnzRl cells by Qpcr.

[0022] FIG. 6. Targeting SIP signal in EnzR cells induce methuosis. (A) EnzRl and EnzSl cells were treated w/o SKI2 or THC, the cell morphologies were observed. (B) EnzR4 and EnzS4 cells were treated w/o Fingolimod and CBD, the cell morphologies were observed. (C) The EnzRl cells were treated w/o Z-VAD, chloroquine or EIPA, the SKI2 efficacy was analyzed by MTT assay. (E) The lucifer yellow absorption assay to identify the macropinocytosis in EnzRl cells treated w/o Fingolimod or SKI2. (F) The EnzRl cells were treated with SKI2 for 24 hrs. And then the cells were incubated with LY for 30mins. The IF images showed that lucifer yellow locate inside the vacuoles. (G) The EnzRl -GFP-Rab7 cells were treated with lOuM SKI2 for 24hrs. The IF images showed that GFP-Rab7 locate on the membrane of the vacuoles. (H) The SIP downstream genes activities, including Racl, mTOR and ERK, were analyzed by WB. (I) The Racl target genes expression were analyzed by qPCR in EnzRl and EnzSl cells.

[0023] FIG. 7. Schematic depiction of Enz/ARv7/SPHKAP/SlP pathway Enz treatment suppress SPHKAP protein level through miRNA. In consequence, SPHKl activity was induced after Enz treatment, which can enhance SlP:Ceramide ration to promote Enz resistance. To conquer EnzR, different compounds were applied (Fingolimod and SKI2 to reduce SIP level, THC and CBD to reduce SIP and increase ceramide level) to interrupt the abnormal S1P- ceramide rheostat, which could induce methuosis. Methuosis could further suppress the EnzR cells proliferation.

Detailed Description of the Invention [0024] The invention provides a unique approach to treatment of prostate cancer, particularly drug resistance prostate cancer. The novel therapeutic methods and compositions provided herein can benefit prostate cancer patients in terms of increased survival rate and improved treatment outcome. Methods and compositions of the invention can be used to prevent, delay, reduce and/or reverse drug resistance and to increase, restore and/or prolong the effective treatment of prostate cancer with anti-androgen compounds.

[0025] Enzalutamide, an anti-androgen agent, has been successfully used as the last line therapy to extend lives of CRPC patients. After prostate cancer patients develop CRPC, the current standard therapy to further suppress CRPC involves either docetaxel (Doc)- chemotherapy or ADT with either using Enz to prevent androgens binding to AR or using abiraterone (ABI) to further suppress the androgen synthesis in renal. Unfortunately, many patients still develop Enz resistance after an average of 4.8 months response to Enz. (Scher, et al. 2012 N Engl J Med 367 , 1187-1197; Dhingra, et al. 2013 Mini Rev Med Chem 13, 1475-1486.)

[0026] The mechanism by which CRPC patients received Enz develop EnzR remains not fully understood.

[0027] ADT with Enz treatment can further suppress the CRPC cell growth to extend patients survivals, yet eventually Enz-resi stance may still be developed. Recent studies demonstrated several mechanisms that could confer to the Enz resistance. The first mechanism that was reported to drive the Enz resistance is activation of GR signaling. (Arora, et al. 2013 Cell 155, 1309-1322.) By inhibiting AR activity with ADT-Enz, GR level is up regulated in a subset of PCa cells due to relief of AR-mediated feedback repression of GR expression. The activated GR could replace some AR roles during development of Enz resistance. Importantly, Arora et al. found the GR agonist dexamethasone was sufficient to confer Enz resistance, whereas a GR antagonist could restore Enz sensitivity. By overexpressing IL6, Liu et al. found that LNCaP cells might acquire the Enzalutamide resistance that may involve the activation STAT3 signaling to promote the AR transactivation and knocking down STAT3 might then result in increasing the Enzalutamide sensitivity in the LNCaP cells. Importantly, adding the STAT3 inhibitor AG490 may then reverse the Enzalutamide resistant in Enzalutamide resistant LNCaP cells, and adding Niclosamide to suppress the STAT3-AR axis could also suppress Enzalutamide resistant cells invasion and migration. Among all of the possible mechanisms, the increase of ARV7 is most convincible one to confer to Enz resistance. Results from in vitro and in vivo mouse model using the CRPC VCaP cells revealed that addition of Enz increased the expression of constitutively active ARv7. Importantly, the Enz-enhanced ARv7 could then transactivate AR target genes to promote CRPC progression in an androgen-independent manner that Enz is no longer able to function as AR antagonist. The clinical survey also suggested that patients with ARv7 might have poor response to ADT-Enz treatment, and ADT-Enz treatment might enhance ARv7 expression. (Yemelyanov, et al. 2012 Cell Cycle 11, 395-406; Sharifi 2014 The New England journal of medicine 370, 970-971; Liu, et al. 2014 The Prostate 74, 201-209; Hu, et al. 2009 Cancer Res 69, 16-22; Lai, et al. 2013 The American journal of pathology 182, 460-473; Antonarakis, et al. 2014 The New England journal of medicine 371, 1028-1038.)

[0028] To overcome the Enz resistance, targeting ARV7 is one of the best strategies. Wang et al. found Enz might be able to function via altering the lncRNA- a/a/7 expression to better interact with splicing factor SRSF1 to increase the ARv7 expression. Knocking-down lncRNA- Malatl with Malatl-siRNA suppressed the Enz-induced ARv7 expression in the Enz-resistant PCa cells, and adding Malatl could then induce ARv7 that can then enhance the development of Enz resistance. By applying the AR degradation enhancer ASC-J9, Yamashita et al. showed that ARV7 expression in 22RV1 cells can be significantly suppressed, which suggested that using ASC-J9 is a potential therapeutic to overcome the Enz resistance. Actually, Wang et al. found out that ASC-J9 do result in a significant reduction of Enz-resistant CRPC cell growth.

Furthermore, results from Chou et al. also found that Cisplatin (and its analog Carboplatin (with less side effects)) could also function via degrading ARv7 (and AR) to further suppress the Enz resistant CRPC cell growth. Liu et al. showed that another FDA approved drug, Niclosamide, can degrade ARV7 expression and re-sensitize the Enz-R cells to Enz treatment. Although in preclinical studies, all of these compounds or treatments showed the significant effect to reduce the Enz resistance, none of them have been approved by FDA to be applied in clinical use. Thus, effectively overcoming Enz resistance remains an unmet medical need. (Wang, et al.“Anti androgen Enzalutamide promotes expression of Androgen receptor splicing variant 3 through Malatl to accelerate the development of Enzalutamide-resistance” (submitted 2015). Yamashita, et al. 2012 Neoplasia 14, 74-83; Liu, et al. 2014 Clinical cancer research : an official journal of the American Association for Cancer Research 20, 3198-3210.)

[0029] The impact of SIP-ceramide axis to the Enz resistance is not yet fully studied. Recent reports showed that sphingosine 1 -phosphate (SlP)-ceramide axis might play key roles in the tumor progression, with SIP promoting cell proliferation and maintain the cell survival, and ceramide playing the opposite role to induce the cell apoptosis and autophagy. Brizuela et al. also found that targeting the SIP with decreasing the SIP lyase could suppress PCa cell growth, and using the SPHK1 inhibitor-Fingolimod to target the SIP could alter the radiation therapy sensitivity to further suppress PCa growth. In contrast, targeting ceramide with up-regulating the ceramidase could then alter the radiation sensitivity to suppress the PCa. (Maceyka, et al. Trends in cell biology 22, 50-60 (2012); Subei, et al. CNS drugs 29, 565-575 (2015); Morad, et al.

Cancer 13, 51-65 (2013); Saddoughi, et al. Advances in cancer research 117, 37-58 (2013); An, et al. The Journal of biological chemistry 275, 288-296 (2000); Calise, et al. Biochimica et biophysica acta 1823, 439-450 (2012); Pettus, et al. Biochimica et biophysica acta 1585, 114- 125 (2002); Yabu, et al. Cell death and differentiation 22, 258-273 (2015); Pattingre, et al.

Autophagy 5, 558-560 (2009); Brizuela, et al. Molecular cancer therapeutics 11, 1841-1851 (2012). Pchejetski, et al. Cancer research 70, 8651-8661 (2010). Mahdy, et al. Molecular therapy : the journal of the American Society of Gene Therapy 17, 430-438 (2009).

[0030] The results disclosed herein indicated that the interruption of the SIP-Ceramide biostat can result in the resistance to the Enz treatment. The mechanistic study revealed for the first time that Enz could control the SIP-Ceramide biostat through degrading SPHKAP and activating SPHK1 activity. By re-balancing the Sl-ceramide biostat, the autophagy and apoptosis can be triggered after Enz treatment and reduce the Enz-R cell survival. Importantly, our study showed that by using certain compounds, some of which are FDA approved, such as fmgolimod and THC, it is possible to re-sensitize the Enz-R cells to Enz. Such results provide an immediate and convenient strategy to prevent the patients’ death from Enz resistant prostate cancer.

[0031] SIP is the key sphingolipid that maintains survival of cancer cells. Ceramide is another important sphingolipid that show the counter effect to SIP. The rheostat (biostat) between SIP and ceramide controls the cell proliferation and death. In prostate cancer, studies have shown that regulation SIP-ceramide by changing the different enzymes could be a potential strategy to reduce the tumor growth. In particular, in irradiation resistant prostate cancer, by decreasing SIP level or increasing ceramide level, the cells can be re-sensitized to IR. Such evidence suggested that SIP-ceramide biostat is also important to different kinds of treatment. In prostate cancer, however, there has been no evidence that showed SIP-ceramide biostat also contributes to the resistance to the anti-androgen treatment. The disclosed study is the first to reveal that targeting this pathway can be a good strategy to reverse the Enz resistance.

Significantly, SPHK1 inhibitors are available for clinical use or are under clinical investigation.

[0032] Additionally, THC, which is the active component of marijuana, can enhance the ceramide level to re-balance the SIP-ceramide biostat. By using these FDA approved

compounds and others under development, a promising and practical approach to overcome Enz resistance has been discovered.

Scheme 1. SIP & Enz-R

Enzalutamide

SPHK1 Activity

S1P Level increase

S1PR activation

Enzalutamide resistance [0033] Thus, the present invention enables a novel approach to suppress the Enz-resistant CRPC progression and to extend survival of CRPC patients. In certain embodiments of the invention, as discussed in details herein, a combination of a SPHK1 inhibitor or an activator of ceramide ( e.g ., THC) with Enz may be used at the beginning of therapy. In certain other embodiments, sequential treatment with Enz therapy at the beginning may be followed by addition of a SPHK1 inhibitor or an activator of ceramide (e.g., THC) at or after confirmation of EnzR (e.g., when decreased PSA start to rise again).

[0034] In one aspect, the invention generally relates to a pharmaceutical composition comprising: a first compound of Formula (I)

or a pharmaceutically acceptable salt, ester or pro-drug thereof, and a second compound selected from an inhibitor of SPHK and an activator of ceramide, wherein each of the first and second compounds is present in an amount such that the pharmaceutical composition is effective in the treatment of prostate cancer, or a related disease or condition thereof, in a mammal, including a human, and a pharmaceutically acceptable carrier.

[0035] In certain embodiments, each of the first and second compounds is present in an amount such that the pharmaceutical composition is effective in reducing, reversing or delaying the onset of or delaying the development of drug resistance.

[0036] In certain embodiments, each of the first and second compounds is present in an amount such that the pharmaceutical composition is effective in reducing drug resistance. In certain embodiments, each of the first and second compounds is present in an amount such that the pharmaceutical composition is effective in reversing drug resistance. In certain embodiments, each of the first and second compounds is present in an amount such that the pharmaceutical composition is effective in delaying the onset of drug resistance. In certain embodiments, each of the first and second compounds is present in an amount such that the pharmaceutical composition is effective in delaying the development of drug resistance.

[0037] The first and second compounds may be present in the pharmaceutical composition at any suitable ratios. For example, the weight ratio of the first compound to the second compound is from about 10 : 1 to about 1 : 10 (e.g., from about 7 : 1 to about 1 : 7, from about 5 : 1 to about 1 : 5, from about 3 : 1 to about 1 : 3, from about 2 : 1 to about 1 : 2, about 1 : 1).

[0038] The first compound may be present in the pharmaceutical composition in any suitable amount, for example in a unit dosage of, in the range of about 0.1 mg to about 1,000 mg, about 1 mg to about 500 mg, about 1 mg to about 10 mg, about 10 mg to about 250 mg, about 10 mg to about 125 mg, about 10 mg to about 100 mg, about 10 mg to about 75 mg, about 10 mg to about 50 mg, about 10 mg to about 25 mg.

[0039] The second compound may be present in the pharmaceutical composition in any suitable amount, for example in a unit dosage of, in the range of about 0.1 mg to about 1,000 mg, about 1 mg to about 500 mg, about 1 mg to about 10 mg, about 10 mg to about 250 mg, about 10 mg to about 125 mg, about 10 mg to about 100 mg, about 10 mg to about 75 mg, about 10 mg to about 50 mg, about 10 mg to about 25 mg.

[0040] In certain embodiments, the pharmaceutical composition is suitable for one or more of oral administration, intravenous, intramuscular, and subcutaneous administration.

[0041] In certain embodiments, the second compound is selected from the group consisting of SKI2, Safmgol, Fingolimod and tetrahydrocannabinol.

SKI2

[0042] In another aspect, the invention generally relates to a unit dosage form comprising a pharmaceutical composition disclosed herein.

[0043] In certain embodiments, the unit dosage form is in the form of a tablet or capsule suitable for oral administration, or in the form of a liquid solution or suspension suitable for intravenous, intramuscular, or subcutaneous administration.

[0044] In yet another aspect, the invention generally relates to a method for treating prostate cancer, or a related disease or condition thereof, comprising administering to a subject in need thereof a pharmaceutical composition disclosed herein.

[0045] In yet another aspect, the invention generally relates to a method for treating prostate cancer, or a related disease or condition thereof, comprising administering to a subject in need thereof a first compound of Formula (I)

or a pharmaceutically acceptable salt, ester or pro-drug thereof, and a second compound which is an inhibitor of SPHK or an activator of ceramide, in amounts effective in the treatment of prostate cancer, or a related disease or condition thereof, in a mammal, including a human, wherein the subject has been detected of the development of drug resistance to the first compound.

[0046] In certain embodiments, the second compound is administered simultaneously with the first compound, or subsequently after the administration of the first compound. In certain embodiments, the second compound is administered simultaneously with the first compound. In certain embodiments, the second compound is administered subsequently after the administration of the first compound.

[0047] In certain embodiments, the first and/or the second compound is administered orally, intravenously, intramuscularly, or subcutaneously.

[0048] In certain embodiments, the second compound is selected from the group consisting of SKI2, Safmgol, Fingolimod and tetrahydrocannabinol. In certain embodiments, the second compound is SKI2. In certain embodiments, the second compound is Safmgol. In certain embodiments, the second compound is Fingolimod. In certain embodiments, the second compound is tetrahydrocannabinol.

[0049] In certain embodiments, the prostate cancer is castration resistant prostate cancer.

[0050] In certain embodiments, the method further includes administering to the subject one or more other anti-cancer agents.

[0051] In certain embodiments, the one or more other anti-cancer agents comprise a chemotherapeutic agent.

[0052] Exemplary chemotherapeutic agents include Erlotinib (TARCEVA®, Genentech/OSI Pharm.), Bortezomib (VELCADE®, Millennium Pharm.), Fulvestrant (FASLODEX®, AstraZeneca), Sutent (SU11248, Pfizer), Letrozole (FEMARA®, Novartis), Imatinib mesylate (GLEEVEC®, Novartis), PTK787/ZK 222584 (Novartis), Oxaliplatin (Eloxatin®, Sanofi), 5-FU (5-fluorouracil), Leucovorin, Rapamycin (Sirolimus, RAPAMUNE®, Wyeth), Lapatinib

(TYKERB®, GSK572016, Glaxo Smith Kline), Lonafarnib (SCH 66336), Sorafenib (BAY43- 9006, Bayer Labs), and Gefitinib (IRESSA®, AstraZeneca), AG1478, AG1571 (SU 5271;

Sugen), alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analog topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g. , calicheamicin, especially calicheamicin gammall and calicheamicin omegall (Angew Chem. Inti. Ed. Engl. (1994) 33: 183-186); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis,

dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, ADRIAMYCIN^® (doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esonibicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamniprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate,

epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;

demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin;

losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK^® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium;

tenuazonic acid; triaziquone; 2,2' ,2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine;

mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. , TAXOL^® (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE® (Cremophor-free), albumin-engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, 111.), and TAXOTERE^® (doxetaxel; Rhone- Poulenc Rorer, Antony, France); chloranmbucil; GEMZAR^® (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; NAVELBINE^® (vinorelbine);

novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA^®);

ibandronate; CPT-11 ; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the above.

[0053] In yet another aspect, the invention generally relates to a method for treating prostate cancer, or a related disease or condition thereof, comprising: administering to a subject in need thereof a first compound of Formula (I)

or a pharmaceutically acceptable salt, ester or pro-drug thereof, in an amount effective in the treatment of prostate cancer or a related disease or condition thereof in a mammal, including a human; monitoring the subject to detect a development of drug resistance to the first compound; upon the subject being detected of the development of drug resistance to the first compound, administering to the subject a second compound selected from an inhibitor of an inhibitor SPHK and an activator of ceramide, in an amount effect to reduce or eliminate drug resistance to the first compound; and monitoring the subject to detect a level of drug resistance to the first compound.

[0054] In certain embodiments, the method further includes, upon the subject being detected of the development of drug resistance to the first compound, continuing to administer the subject the first compound.

[0055] In certain embodiments, the method further includes, upon the subject being detected of the development of drug resistance to the first compound, halting the administration of the first compound, and upon the subject being detected of a substantial reduction or disappearance of drug resistance, re-starting the administration of the first compound.

[0056] In certain embodiments, the first and/or the second compound is administered orally, intravenously, intramuscularly, or subcutaneously.

[0057] In certain embodiments, the prostate cancer is castration resistant prostate cancer.

[0058] In certain embodiments, the second compound is SKI2, Safmgol, Fingolimod or tetrahydrocannabinol. In certain embodiments, the second compound is SKI2. In certain embodiments, the second compound is Safmgol. In certain embodiments, the second compound is Fingolimod. In certain embodiments, the second compound is tetrahydrocannabinol.

[0059] In yet another aspect, the invention generally relates to a method for treating a drug resistance in connection with a prostate cancer treatment, comprising administering to a subject in need thereof a compound which is an inhibitor of SPHK or an activator of ceramide, in an amount effective to prevent, delay, reduce or reverse resistance to a treatment of prostate cancer, or a related disease or condition thereof in a mammal, including a human.

[0060] In certain embodiments, the resistance is to a compound having Formula (I)

or a pharmaceutically acceptable salt, ester or pro-drug thereof.

[0061] In certain embodiments, the compound is SKI2, Safmgol, Fingolimod or

tetrahydrocannabinol .

[0062] The following examples are meant to be illustrative of the practice of the invention, and not limiting in any way.

Examples

SIP-Ceramide axis is altered significantly in EnzR CRPC cells

[0063] As ARv7 has promising human clinical data to support its contribution to the development of Enz resistance, several new therapeutic approaches via direct targeting the ARv7, including ASC-J9, Resveratrol, Galeterone, UT-155, Niclosamide and ARv-771 have been developed and tested in human clinical trials. However, some of them already failed in their clinical trials and others still need more clinical trials before they can be used to treat the EnzR CRPC patients.

[0064] It was decided to search those FDA-approved drugs that could target the ARv7 downstream targets instead of direct targeting the ARv7. First, the RNAseq assay was applied to compare the whole-transcriptome between EnzSl-C4-2 and EnzRl-C4-2 cells, and picked up those genes whose expression are significantly altered in EnzRl cells (Fold log2>1.8) (FIG. 1A). Next, signal pathway analysis was performed based on the database of Pathways common pathway, and found 21 genes linked to the sphingosine-1 -phosphate (SIP) signals, with most of them are SIP downstream genes that belong to the SlP-receptor-1 (S1PR1) sub-pathways, suggesting that SIP signals are activated in EnzRl cells (FIG. IB).

[0065] To confirm these initial key findings, next performed was the GSEA analysis, and results also found that in the EnzR cells, the sphingolipid pathway is highly activated (FIG. 1C- 1D). As early studies suggested that the SIP signals could act as enhancer to increase the cell proliferation, and in contrast, ceramide, which is precursor of SIP, is the key metabolite to promote the cell death, it was therefore hypothesize that the balance between SIP and ceramide (named as SIP-ceramide rheostat) may play key roles to control the cell fate.

[0066] The above bioinformatic results were further confirmed via direct assaying the concentration of SIP and ceramide. The results revealed that higher SIP concentration was detected in the EnzRl -C4-2 cells as compared to the parental EnzSl-C4-2 cells. In contrast, little change was found for the ceramide concentration in the EnzRl -C4-2 cells vs EnzSl-C4-2 cells (FIG. IE). Similar results were also obtained when EnzRl -C4-2/EnzSl-C4-2 cells were replaced with EnzR4-C4-2B/EnzR4-C4-2B cells (FIG. IE). To link higher level of SIP to the Enz-induced Enz resistance in the EnzR cells, the potential impacts of altering the SIP concentration to the Enz sensitivity were then examined. The results revealed that adding luM SIP led to decrease the Enz sensitivity in the EnzSl-C4-2 cells (FIG. IF).

[0067] Together, results from FIG. 1A-1F suggest that altered SIP-ceramide rheostat with higher SIP concentration was found in the EnzRl cells.

[0068] Human clinical sample survey to confirm that SIP-ceramide rheostat is significantly altered in the CRPC patients received Enz treatment. As shown in FIG. 1G, the ADT, including abiraterone and Enz, increased the SIP levels in patients’ plasma. Interestingly, although there are only 4 Enz treated patients’ samples, the SIP level show most significantly increase in these samples. Table 1. SIP downstream genes are highly activated in Enz-R cells

Re-sensitize the Enz-resistance in the EnzR cells via reversing the SIP-Ceramide rheostat through reducing the SIP concentration with Fingolimod or SKI2

[0069] To test whether targeting the Enz-altered S lP-ceramide rheostat in EnzR cells can lead to reverse Enz resistance, FDA-approved Fingolimod was applied which could suppress the SIP production and S1PR activation to confirm the above results, and results revealed that suppressing SIP led to increase significantly the Enz sensitivity in the EnzRl-C4-2 cells (FIG.

2A). Similar results were also obtained when Fingolimod was replaced with another sphingosine kinase (SPHK) inhibitor SKI2, showing suppressing SIP also led to re-sensitize the Enz- resistance to further suppress the EnzRl-C4-2 cell growth (FIG. 2B). As expected, similar results were also obtained when EnzRl-C4-2 cells were replaced with the EnzR4-C4-2B cells

(FIG. 2c)

[0070] Interestingly, it was found that both Fingolimod and SKI2 could only suppress the cell growth in the EnzRl-C4-2 and EnzR3-CWR22Rvl, and not in the EnzSl-C4-2 cells (FIG. 2E-2F), suggesting that suppressing SIP signals with these two inhibitors is rather specific to the CRPC cells that already developed the Enz resistance.

Re-sensitize the Enz-resistance via reversing the SIP-ceramide rheostat through increasing the ceramide concentration/reducing SIP level with mari juana-CBD/THC

[0071] In addition to alter SIP-ceramide rheostat via reducing the SIP concentration, SIP- ceramide rheostat can also be altered via increasing the ceramide concentration. The

consequences of both approaches can all lead to reduce the“ratio of SIP to ceramide”, which may then lead to alter the cell fate, including Enz sensitivity.

[0072] First found was that direct adding the ceramide (to reduce the SIP-ceramide ratio) led to increase the Enz-sensitivity to further suppress the EnzRl cell growth (FIG. 2G). Similar results were also obtained when the EnzRl -C4-2 cells were replaced with the EnzR4-C4-2B cells (FIG. 2H).

[0073] Instead of adding ceramide, cannabidiol (CBD) or tetrahydrocannabinol (THC) was added. The two active components of marijuana can increase the ceramide concentration in various tumors, to the EnzRl -C4-2 and EnzR4-C4-2B cells and results revealed that adding

CBD or THC in these two EnzR cells also led to re-sensitize the Enz-resistance (FIG. 2I-2K).

Similar results were also obtained when MTT growth assay was replaced with Brdu proliferation assay showing adding CBD or THC or Fingolimod could also lead to re-sensitize the Enz- resistance in the EnzRl -C4-2 and EnzR4-C4-2B cells.

[0074] Interestingly, in addition to function via increasing ceramide level with activating the Cannabinoid receptor (CB1 and CB2) to increase ceramide to alter the ratio of SIP to ceramide, it was uncovered that adding CBD or THC could also function via suppressing SPHK1 activity

(FIG. 2N) to further reduce SIP level in EnzR cells. [0075] Together, results from FIG. 2 suggest that adding marijuana-CBD/THC can also re sensitize the Enz-resi stance via increasing ceramide levels/reducing SIP levels to reduce the ratio of SIP to ceramide to further suppress EnzR cell growth.

Targeting the SIP-ceramide axis can also delay the development of Enz resistance in the parental EnzSl-C4-2 cells

[0076] In addition to suppress the CRPC cells that already developed Enz resistance, it was also interesting to see if targeting the SIP-ceramide axis can also delay the Enz development in CRPC cells that are still sensitive to Enz treatment. The EnzSl-C4-2 cells were pretreated with 5 uM Enz for 1 month, and assayed their Enz sensitivity. The results revealed that these cells sensitivity to 10 uM Enz decreased from 50% to 17%, suggesting that long term treatment of Enz could reduce the PCa cells sensitivity to Enz. (FIG. 2P)

[0077] Next, the Enz was combined with marijuana-CBD or SKI2 to pretreat the EnzSl-C4- 2 cells for 1 month, and then assayed their sensitivity to Enz. The results revealed that adding marijuana-CBD or SKI2 to alter the SIP-ceramide axis can significantly increase the EnzSl-C4- 2 cells sensitivity to Enz as compared to the long-term treatment of Enz only (SKI2-40%, CBD- 35%) (FIG. 2Q-2R).

[0078] Together, results from (FIG. 2P-2R) suggest that that targeting the SIP-ceramide axis with either marijuana-CBD or SKI2 can also delay the development of Enz resistance in the parental EnzSl-C4-2 cells

Mechanism dissection of why Enz can alter the SIP-ceramide rheostat via increasing SIP: via increasing the SPHK1 activity in EnzR cells

[0079] To dissect the mechanism of how Enz can alter the SIP-ceramide rheostat via

increasing SIP, several key enzymes that are involved in the sphingolipid’s metabolism via the RNAseq were searched for. It was found that the mRNA expression of ACER1, SPHK1/2 and CERK increased significantly in EnzRl cells. However, the expression of these four proteins (ACER1, CERK and SPHK1/2) failed to increase in EnzRl cells (FIG. 3A). The SPHKs activity became the focus since it is critical for the SIP generation. As shown in FIG. 3B, the SPHK activity enhanced in the EnzRl cells. To study if Enz can impact the SPHKs activity, EnzSl cells were treated with Enz for 10 days, and found long term treatment with Enz enhanced the SPHKs activity (FIG. 3C), suggesting that Enz can alter SPHKs activity to increase SIP levels that led to alter the SIP-ceramide rheostat.

[0080] SPHKs (including SPHKl and SPHK2) can phosphorylate sphingosine to generate SIP⁹ to increase PCa cell growth and survival, and higher SPHKl can also alter the radiation sensitivity to suppress PCa growth. The SIPRs modulator Fingolimod treatment can suppress SPHKI/SIP/SIPRI axis and can sensitize PCa cells to Radiation. To explore which SPHK (1 or 2) can play the key function to alter the Enz sensitivity, either the SPHKl inhibitor (PF543) or SPHK2 inhibitor (ABC294640) with Enz was added to assay their impact on the Enz sensitivity to suppress the EnzRl-C4-2 cell growth. The results revealed that only PF543 could increase the Enz sensitivity to better suppress the EnzRl-C4-2 cell growth (FIG. 3D), suggesting that SPHKl is the key enzyme to impact the Enz sensitivity.

[0081] The SPHKl serine 225 phosphorylation in EnzSl and EnzRl cells was also analyzed since early reports showed that the p-S225 level is an indicator for SPHKl activity, and found the SPHKl p-S225 levels increased in EnzRl cells, suggesting that SPHKl activity is increased in EnzRl cells. (FIG. 3E). Finally, it was found that suppress SPHKl activity via adding

SPHKl-shRNA could increase significantly Enz sensitivity in EnzRl-C4-2 cells (FIG. 3F).

[0082] Together, the results of FIG. 3A-3F suggest that increase SPHKl activity is the key step to increase the SIP concentration to promote the Enz resistance in the EnzR cells.

Mechanism dissection of why Enz can increase the SPHKl activity in the EnzR cells: via suppressing the SPHKAP expression

[0083] To further study how Enz can alter the activity of SPHKl, searches were done for any factors that can alter the SPHKl activity, and found the SPHKl Interactor, AKAP Domain

Containing protein (SPHKAP) was decreased significantly in EnzR cells and adding Enz can decrease SPHKAP expression in EnzSl cells (FIG, 4A-4B). To further confirm this key finding, the human PDX PCa samples (PDX-133-4C) were then implanted into SCID mice and treated the tumor bearing mice with Enz for 20 days. The results also confirm the in vitro cell lines results showing adding Enz can suppress dramatically the SPHKAP expression in the PDX tumors (FIG. 4C). [0084] Since SPHKAP was identified as a SPHK1 co-regulator, which can suppress SPHK1 activity, studies were performed to see if Enz treatment can alter SPHKAP to change SPHKs activity. Results showed that suppressing the SPHKAP in EnzSl cells resulted in higher SPHKs activity, with increase the SIP levels (FIG. 4D-4E).

[0085] Importantly, it was found that increased the SPHKAP expression via adding the

SPHKAP-cDNA in EnzRl-C4-2 cells also led to increase the Enz sensitivity to suppress the EnzRl-C4-2 cell growth (FIG. 4F). In contrast, decreased SPHKAP expression via adding

SPHKAP-shRNA in the parental EnzSl -C4-2 cells led to significantly decreased the Enz- sensitivity to suppress the cell growth (FIG. 4G).

[0086] As the previous results showed that CBD or Fingolimod can increase Enz sensitivity in EnzRl & EnzR4 cells, it was tested whether both compounds have the same effects on EnzSl - shSPHKAP cells and results showed that treating the EnzSl -shSPHKAP cells with CBD or

Fingolimod can all enhance Enz sensitivity (FIG. 4H).

[0087] Together, results from FIG. 4A-4H suggest that Enz can function via suppressing SPHKAP expression to increase the SPHKl activity to enhance the SIP concentration, which may then lead to increase the Enz resistance.

Human clinical sample survey to support the SPHKAP negative roles to promote the PCa progression.

[0088] To further support the above in vitro cell lines results to the human clinical relevance, clinical survey via TCGA database analysis was performed. The results revealed that SPHKAP mRNA expression is negatively correlated with Gleason score and metastasis status (FIG. 41). Results from The Human Protein Atlas also suggested that SPHKAP protein expression is lower in PCa samples than in the normal prostate tissues (FIG. 4J).

[0089] Results from our human tissue micro-array also demonstrated that SPHKAP protein expression is negatively correlated with Gleason score (FIG. 4K).

[0090] Together, results from multiple human clinical sample surveys (FIG. 4I-4K) all suggest that SPHKAP may play negative roles to promote the PCa progression.

[0091] In summary, the results from multiple in vitro cell lines, in vivo mouse model and human clinical sample surveys all suggest that Enz may function via altering the SPHKAP/SPHK1 axis to change the SIP level to impact the SIP-ceramide rheostat to increase the Enz resistance.

Mechanism dissection of how Enz can suppress SPHKAP expression: via increasing the ARv7 expression

[0092] To further understand the mechanism of how Enz can alter SPHKAP expression, key factors were assayed first, including ARF876L, ARv7, and GR that have been linked to

development of Enz-resistance. The WB data showed that only ARv7 could significantly reduce SPHKAP expression in EnzS cells (FIG. 5A) and knocking down ARv7 can increase SPHKAP expression in EnzR cells (FIG. 5B). The results from the Elisa assay also demonstrated that only adding ARv7 could increase the SIP production (FIG. 5C) and in contrast, suppressing ARv7 in EnzRl-C4-2 cells can significantly decrease SIP concentration (Fig. 5D). The Enz-induced

ARv7 has the most convincing human clinical evidence, which shows CRPC patients who have higher ARv7 have more resistance to Enz therapy and CRPC patients who received Enz had higher ARv7 expression⁸.

[0093] Together, results from FIG. 5A-5D suggest that Enz may function via increasing the ARv7 to suppress SPHKAP expression.

Mechanism dissection of how Enz-increased ARv7 can suppress SPHKAP expression: via alterins the miRNA expression

[0094] To further study how Enz-increased ARv7 can suppress SPHKAP expression, it was first noticed that SPHKAP mRNA expression is not altered significantly in the EnzRl-C4-2 cells, suggesting that Enz may regulate mainly the SPHKAP expression at the protein level

(FIG. 5E). Focus was placed on the miRNAs, since recent studies indicated that miRNAs might play important roles to regulate the gene expression at the protein level.

[0095] By Ago2 IP assay, it was found that in EnzRl-C4-2 cells, the binding of SPHKAP mRNA and Ago2 increased, suggesting that some miRNAs may be involved in the SPHKAP is downregulated SPHKAP in the EnzRl-C4-2 cells (FIG. 5F).

[0096] To explore which miRNAs regulate SPHKAP expression, the potential miRNAs were screened to check which can target SPHKAP from 3 miRNA databases and then compared their expression in EnzSl/4 and EnzRl/4 cells (FIG. 5G). Among all candidates, miR-6731-3p and miR-6832-3p both increase in EnzR (1&4) cells (FIG. 5H). Overexpression of ARv7 in

EnzSl cells can induce both miR-6731-3p and miR-6832-3p.

Mechanism dissection of how Enz/ARv7/miRNA/SPHKAP/SPHKl axis altered S IP -cer amide rheostat can lead to increase the Enz sensitivity: via inducins the methuosis

[0097] To dissect the mechanism of how Enz/ARv7/SPHKAP/SPHKl axis altered S1P- ceramide rheostat can lead to increase the Enz sensitivity, attention was paid to those related cell proliferation and cell death to the Enz treatment. It was noticed that adding Marijuana- CBD/THC, or SKI2/Fingolimod to increase the Enz sensitivity also led to an significant

morphology changes in the EnzRl-C4-2 cells us EnzSl -C4-2 cells: it was noticed an significant inducing cell swelling and visible vacuoles accumulation occurred only in the EnzRl-C4-2, and not in the EnzSl -C4-2 cells (FIG. 6A). Importantly, also observed were similar morphology changes in the EnzR4-C4-2B us EnzS4-C4-2B cells (FIG. 6A).

Together, results from FIG. 6A-B suggest that some selective cell death⁴¹ may be involved.

[0098] To further study what kind of the cell death is induced via altering the SIP-ceramide rheostat in EnzR cells, the inhibitors were applied for apoptosis (zVAD), autophagy

(chloroquine), and macropinocytosis (EIPA), and found only the EIPA could block the SKI2- induced cell death in EnzR cells, suggesting that methuosis may be the cell death induced by SKI2. (FIG. 6C). However, it was also found that all three inhibitors can partially suppress the Marijuana-THC induced cell deaths, suggesting that besides methuosis, THC may also function via multiple signals to repress the EnzR cell growth.

[0099] Importantly, electronic microscope was applied to confirm our findings, and results revealed that combination of Marijuana-THC or SKI2 with Enz could significantly increase the number of macropinosomes, which agrees with above results (FIG. 6A-6C) and suggest that Marijuana-THC or SKI2 may function via altering the methuosis to increase the Enz sensitivity to suppress cell growth in EnzR cells.

[00100] Lucifer yellow (LY) absorption assay was applied in EnzR cells, since the fast absorption the LY is the hallmark of the methuosis, and results revealed that treating with SKI2, Fingolimod, and marijuana-THC all led to enhance the LY absorption (FIG. 6E). Furthermore, the fluorescent microscopic images also showed that LY located inside the vacuoles, suggesting these vacuoles are macropinosomes (FIG. 6F). The GFP-Rab7-EnzRl cells were also

established and after treating the cells with SKI2, GFP-Rab7 located on the membrane of the vacuoles was observed, which confirm these vacuoles derived from macropinosomes, since the early reports showed that Rab7 is the key protein to assemble the macropinosome’s membrane

(FIG. 6G).

[00101] Together, results from FIG. 6A-6G suggest that targeting SIP-ceramide rheostat with marijuana-THC or SKI2, Fingolimod can lead to increase the Enz sensitivity via inducing the methuosis.

Mechanism dissection of how targeting the Enz/ARv7/SPHKAP/SPHKl axis altered SIP-ceramide rheostat SIP-Ceramide can induce cell methuosis

[00102] Finally, to dissect the mechanism of how targeting the Enz/ARv7/SPHKAP/SPHKl axis altered SIP-ceramide rheostat can induce cell methuosis in the EnzR cells, first checked was the downstream signals which can be regulated by SIP. As shown in FIG. 6H, in EnzRl cells,

Racl activity was increased significantly, and several Racl target genes expression were

upregulated in EnzRl cells (FIG. 61). Interestingly, early reports also indicated that Racl is the key protein to regulate the maturation of macropinosomes, and interruption of Racl activity would induce methuosis.

[00103] To further investigate the role of Racl, EnzRl cells were treated with Marijuana- THC, SKI2 or Fingolimod and found out Racl activity was suppressed in EnzR cells, which is consistent with our results. Furthermore, increasing Rac activity can then block/reverse the

Marijuana-THC- or SKI2 -induced cell methuosis.

[00104] Together, results from FIG. 6h-k suggested that targeting SIP-ceramide rheostat could reduce the Racl activity and promote methuosis.

Experimental

Cell culture [00105] C4-2, HEK293Tand PC-3, 22RV1 cell lines were purchased from the American Type

Culture Collection (ATCC, Manassas, VA) and cultured in RPMI 1640 with 10% FBS. C4-2 parental and C4-2 MDVR cell lines were generated Enz-resistant PCa cells (named C4-2MDVR) via chronic culture of CRPC C4-2 cells in media containing increasing Enz (from 10 nM to 30 nM). All cells were maintained in a humidified 5% C0₂ environment at 37°C. All cell lines were cultured in a 5% (v/v) C02 humidified incubator at 37°C. All cell lines have been detected and authenticated as mycoplasma and bacteria free following ATCC's instructions.

Cell Proliferation Assays

[00106] 10000 C4-2, 22RV1 and C4-2 MDVR cells were plated into each well of 24 well- plates, on 0, 2, 4, 6 days, cells were collected, and then MTT assay was performed: 100 pi of 5 mg/ml MTT was added to each well. One set of wells with MTT were included but no cells (control), then incubated for 3 hours at 37 °C in a culture hood, removed media and added 150 mΐ DMSO, covered the plates with foil, agitated the cells on an orbital shaker for 15 min, and then read the absorbance at 570 nm.

Lentivirus packaging and cell transfection

[00107] Sh-ARV7 was constructed into the pLKO.1 lentiviral vector as reported previously.

The pLKO. l sh-SPHKAP together with package and envelope plasmids, psPAX2 and pMD2G, were co-transfected into 293T cells for 48hours to produce the SPHKAP shRNA lentivirus particle soup. Lentivirus soup was then collected and frozen at -80°C for later use in transduction of PCa cells.

RNA extraction and quantitative real-time PCR (qPCR) analysis

[00108] Total RNAs were isolated using Trizol reagent (Invitrogen, Grand Island, NY). One pg of total RNA was subjected to reverse transcription using Superscript III transcriptase (Invitrogen, Grand Island, NY). qRT-PCR was conducted using a Bio-Rad CFX96 system with SYBR green to determine the mRNA expression level of a gene of interest. Expression levels were normalized to GAPDH level. Western blot analysis

[00109] Cells were lysed in RIPA buffer and proteins (20-40 pg) were separated on 8-10% SDS/PAGE gel and then transferred onto PVDF membranes (Millipore, Billerica, MA). After blocking membranes, they were incubated with primary antibodies, HRP-conjugated secondary antibodies, and visualized using ECL system (Thermo Fisher Scientific, Rochester, NY). AR, GAPDH, tubulin antibodies were from Santa Cruz Biotechnology, Inc (Santa Cruz, CA).

SPHKAP antibody was purchased from GeneTex.

Chromatin Immunoprecipitation Assay ( ChIP )

[00110] Cells were crosslinked with 4% formaldehyde for 10 minutes followed by cell collection and sonication with a predetermined power to yield genomic DNA fragments of 300- 1000 bp long. Lysates were precleared sequentially with normal rabbit IgG (sc-2027, Santa Cruz Biotechnology) and protein A-agarose. Anti-ARV7 antibody (2.0 pg) was added to the cell lysates and incubated at 4°C overnight. For the negative control, IgG was used in the reaction. Specific primer sets designed to amplify a target sequence within human SPHKAP promoter; PCR products were analyzed by agarose gel electrophoresis.

Luciferase assay

[00111] PCa cells were plated in 24-well plates and transfected with MMTV-luc containing ARE sequence using Lipofectamine (Invitrogen, Grand Island, NY). After transfection, regular media were added with various DHT concentrations, 0 (ethanol as vehicle control), 1 nM, and 10 nM, and incubated for 48 hrs. pRL-TK was used as internal control. Luciferase activity was measured by Dual -Luciferase Assay (Promega, Madison, WI) according to the manufacturer’s manual.

Sphingolipid measurement by HPLC-Mass spectrum

[00112] The sphingolipids in EnzRl, R4 and EnzSl, S4 cells were extracted as previously described. The measurements were performed by HPLC-Mass spectrum by URMC core-facility.

SPHK1 activity assay [00113] The SPHKs assay kit was purchased from Echelon Bio, Inc. The procedure was followed by the instructions.

In vivo tumorigenesis assay

[00114] C4-2 parental or C4-2 MDVR cells (4 million) were mixed with Matrigel (1 : 1) and injected into the prostates of 6- to 7-week old male SCID mice. Tumor-bearing mice were randomized into four groups and treated as follows:(l) vehicle control (5 /% Tween 80 and 5% ethanol in PBS,i.p.),(2)enzalutamide (25 mg/kg,p.o),(3) Fingolimod (10 mg/kg, i.p.),(4) enzalutamide (25 mg/kg, p.o)+ Fingolimod (10 mg/kg, i.p.). (5 CBD, (6 Enz+ CBD. Tumors were measured using calipers twice a week and tumor volumes were calculated using length*width ²/2. Tumor tissues were harvested after 3 weeks of treatment.

[00115] In this specification and the appended claims, the singular forms "a," "an," and "the" include plural reference, unless the context clearly dictates otherwise.

[00116] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

Methods recited herein may be carried out in any order that is logically possible, in addition to a particular order disclosed.

Incorporation by Reference

[00117] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made in this disclosure.

All such documents are hereby incorporated herein by reference in their entirety for all purposes. Any material, or portion thereof, that is said to be incorporated by reference herein, but which conflicts with existing definitions, statements, or other disclosure material explicitly set forth herein is only incorporated to the extent that no conflict arises between that incorporated material and the present disclosure material. In the event of a conflict, the conflict is to be resolved in favor of the present disclosure as the preferred disclosure. Equivalents

[00118] The representative examples disclosed herein are intended to help illustrate the invention, and are not intended to, nor should they be construed to, limit the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including the examples which follow and the references to the scientific and patent literature cited herein. The examples herein contain important additional information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

What is claimed is: CLAIMS

1. A pharmaceutical composition comprising:

a first compound of Formula (I)

or a pharmaceutically acceptable salt, ester or pro-drug thereof, and

a second compound selected from an inhibitor of SPHK or an activator of ceramide,

wherein each of the first and second compounds is present in an amount such that the pharmaceutical composition is effective in the treatment of prostate cancer, or a related disease or condition thereof, in a mammal, including a human, and a pharmaceutically acceptable carrier.

2. The pharmaceutical composition of claim 1, wherein each of the first and second

compounds is present in an amount such that the pharmaceutical composition is effective in reducing drug resistance.

3. The pharmaceutical composition of claim 1, wherein each of the first and second

compounds is present in an amount such that the pharmaceutical composition is effective in reversing drug resistance.

4. The pharmaceutical composition of claim 1, wherein each of the first and second

compounds is present in an amount such that the pharmaceutical composition is effective in delaying the onset of drug resistance.

5. The pharmaceutical composition of claim 1, wherein each of the first and second

compounds is present in an amount such that the pharmaceutical composition is effective in delaying the development of drug resistance.

6. The pharmaceutical composition of any of claims 1-5, wherein the weight ratio of the first compound to the second compound is from about 10 : 1 to about 1 : 10.

7. The pharmaceutical composition of any of claims 1-6, being suitable for one or more of oral administration, intravenous, intramuscular, and subcutaneous administration.

8. The pharmaceutical composition of any of claims 1-7, wherein the second compound is selected from the group consisting of SKI2, Safmgol, Fingolimod and

tetrahydrocannabinol .

9. The pharmaceutical composition of any of claims 1-8, wherein the second compound is selected from the group consisting of SKI2, Safmgol, Fingolimod and

tetrahydrocannabinol .

10. The pharmaceutical composition of claim 9, wherein the second compound is SKI2.

11. The pharmaceutical composition of claim 9, wherein the second compound is Safmgol.

12. The pharmaceutical composition of claim 9, wherein the second compound is

Fingolimod.

13. The pharmaceutical composition of claim 9, wherein the second compound is

tetrahydrocannabinol .

14. A unit dosage form comprising the pharmaceutical composition of any of claims 1-13.

15. The unit dosage form of claim 14, being in the form of a tablet or capsule suitable for oral administration, or in the form of a liquid solution or suspension suitable for intravenous, intramuscular, or subcutaneous administration.

16. A method for treating prostate cancer, or a related disease or condition thereof,

comprising administering to a subject in need thereof the pharmaceutical composition of any of claims 1-15.

17. A method for treating prostate cancer, or a related disease or condition thereof,

comprising administering to a subject in need thereof a first compound of Formula (I)

or a pharmaceutically acceptable salt, ester or pro-drug thereof, and a second compound which is an inhibitor of SPHK or an activator of ceramide, in amounts effective in the treatment of prostate cancer, or a related disease or condition thereof, in a mammal, including a human, wherein the subject has been detected of the development of drug resistance to the first compound.

18. The method of claim 17, wherein the second compound is administered simultaneously with the first compound.

19. The method of claim 17, wherein the second compound is administered subsequently after the administration of the first compound.

20. The method of any of claims 17-19, wherein the first and/or the second compound is administered orally, intravenously, intramuscularly, or subcutaneously.

21. The method of any of claims 17-20, wherein the weight ratio of the first compound to the second compound is from about 10 : 1 to about 1 : 10.

22. The method of any of claims 17-21, wherein the second compound is selected from the group consisting of SKI2, Safmgol, Fingolimod and tetrahydrocannabinol.

23. The method of claim 22, wherein the second compound is SKI2.

24. The method of claim 22, wherein the second compound is Safmgol.

25. The method of claim 22, wherein the second compound is Fingolimod.

26. The method of claim 22, wherein the second compound is tetrahydrocannabinol.

27. The method of any of claims 17-26, further comprising administering to the subject one or more other anti-cancer agents.

28. The method of claim 27, wherein the one or more other anti-cancer agents comprise a chemotherapeutic agent.

29. The method of any of claims 17-28, wherein the prostate cancer is castration resistant prostate cancer.

30. A method for treating prostate cancer, or a related disease or condition thereof,

comprising:

administering to a subject in need thereof a first compound of Formula (I)

or a pharmaceutically acceptable salt, ester or pro-drug thereof, in an amount effective in the treatment of prostate cancer or a related disease or condition thereof in a mammal, including a human;

monitoring the subject to detect a development of drug resistance to the first compound;

upon the subject being detected of the development of drug resistance to the first compound, administering to the subject a second compound selected from an inhibitor of an inhibitor SPHK and an activator of ceramide, in an amount effect to reduce or eliminate drug resistance to the first compound; and

monitoring the subject to detect a level of drug resistance to the first compound.

31. The method of claim 30, further comprising:

upon the subject being detected of the development of drug resistance to the first compound, continuing to administer the subject the first compound.

32. The method of claim 30, further comprising:

upon the subject being detected of the development of drug resistance to the first compound, halting the administration of the first compound, and

upon the subject being detected of a substantial reduction or disappearance of drug resistance, re-starting the administration of the first compound.

33. The method of any of claims 30-32, wherein the first and/or the second compound is administered orally, intravenously, intramuscularly, or subcutaneously.

34. The method of any of claims 30-33, wherein the prostate cancer is castration resistant prostate cancer.

35. The method of any of claims 30-34, wherein the second compound is SKI2, Safmgol, Fingolimod or tetrahydrocannabinol.

36. A method for treating a drug resistance in connection with a prostate cancer treatment, comprising administering to a subject in need thereof a compound which is an inhibitor of SPHK or an activator of ceramide, in an amount effective to prevent, delay, reduce or reverse resistance to a treatment of prostate cancer, or a related disease or condition thereof in a mammal, including a human.

37. The method of claim 36, wherein the resistance is to a compound having Formula (I)

or a pharmaceutically acceptable salt, ester or pro-drug thereof.

38. The method of claim 36 or 37, wherein the compound is SKI2, Safmgol, Fingolimod or tetrahydrocannabinol .