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The main objective of this experiment was to determine and study the effects of combining two epigenetic modifiers, 5-azacyticidine (5-AzaC) and SB939, on a RM-1 murine prostate cancer cell model. The effectiveness of this combination on prostate cancer cells has not been previously studied. The study was implemented on ex vivo cell models to gain a better understanding of the true effects of the combination therapy on prostate cancer cells. Two variations of the combination therapy were tested in this study, each with different concentrations of SB939 (100nm and 200nm).To determine the effectivity of the combination therapy on prostate cancer cells, three factors were measured: cell proliferation, cancer testis antigen (CTA) expression, and apoptosis rate. To measure cell proliferation, a cell proliferation assay was conducted, and absorption rate was measured through a 450 nm wavelength. CTA expression was measured through a quantitative polymerase chain reaction (quant-PCR). For this study, the expression rates of five CTAs were measured (TEX15, CEP55, CCNA1, P1A, SPA17). Apoptosis rate was measured through an Annexin-V assay, in which two markers, Annexin-V and 7-AAD, were used.We found that SB939 combined with 5-AzaC show highest efficacy compare to each drug alone in terms of inhibiting tumor cell proliferation, as well as inducing tumor cells apoptosis and enhancing tumor cell immunogenicity by the induction of the expression of CTAs. This combination proved to be effective in combating murine prostate cancer cells, and can potentially be effective within in vivo models due to its high toxicity to these cancer cells, and its ability to render prostate cancer more immunogenic.

INTRODUCTION Decreased expression of highly immunogenic cancer-testis antigens (CTA) might help tumor to achieve low immunogenicity, escape immune surveillance and grow unimpeded. Our aim was to evaluate CTA expression in tumor and normal tissues and to investigate possible means of improving the immune response in a murine prostate cancer (CaP) model by using the combination of epigenetic modifier 5-azacitidine (5-AzaC) and immunomodulator lenalidomide. No study to date has examined the effect of this combination on the prostate cancer or its impact on antigen-presenting cells (APC). MATERIALS AND METHODS Gene microarrays were performed to compare expression of several CTA in murine prostate cancer (RM-1 cells) and normal prostate. RM-1 cells were treated with 5-AzaC and real-time PCR was performed to investigate the expression of several CTA. Western blotting was used to determine whether expression of CTA-specific mRNA induced by 5-AzaC resulted in increase in the corresponding protein. Effect of the epigenetic agents and immunomodulators was assessed on dendritic cells (DC) using flow cytometry, ELISA and T-cell proliferation assay. RESULTS Gene arrays demonstrated decreased expression of 35 CTA in CaP tissue compared to normal prostate. 5-AzaC treatment of RM-1 prostate cancer cells upregulated the expression of all 13 CTA tested in a dose-dependent fashion. DC were treated with 5-AzaC and lenalidomide and the expression of surface markers MHC Class I, MHC Class II, CD80, CD86, CD 205, and CD40 was increased. Combination of 5-AzaC and lenalidomide enhances the ability of DC to stimulate T-cell proliferation in mixed leukocyte reaction. Secretion of IL-12 and IL-15 by DC increased significantly with addition of 5-AzaC or 5-AzaC and lenalidomide. CONCLUSIONS Decreased expression of CTA by prostate cancer may be a means of escaping immune monitoring. Combination of epigenetic modifications and immunomodulation by 5-AzaC and lenalidomide increased tumor immunogenicity and enhanced DC function and may be used in the treatment of advanced prostate cancer. Prostate 77: 361-373, 2017. © 2016 Wiley Periodicals, Inc.

Prostate cancer is one of the leading causes of cancer deaths, with no curative treatments once it spreads. Alternative therapies, including immunotherapy, have shown limited efficacy. Dendritic cells (DC) have been widely used in the treatment of various malignancies. DC capture antigens and move to the lymphoid organs where they prime naive T cells. Interaction between DC and T cells are most active in lymph nodes and suppression of DC trafficking to lymph nodes impairs the immune response. In this work, we aimed to study trafficking of DC in vivo via various routes of delivery, to optimize the effectiveness of DC-based therapy. A DC labeling system was developed using 1,1'-dioctadecyltetramethyl indotricarbocyanine Iodine for in vivo fluorescent imaging. DC harvested from C57B/6 mice were matured, labeled, and injected intravenously, subcutaneously, or intratumorally, with or without antigen loading with whole tumor lysate, into C57B/6 mice inoculated with RM-1 murine prostate tumor cells. Signal intensity was measured in vivo and ex vivo. Signal intensity at the tumor site increased over time, suggesting trafficking of DC to the tumor with all modes of injection. Subcutaneous injection showed preferential trafficking to lymph nodes and tumor. Intravenous injection showed trafficking to lungs, intestines, and spleen. Subcutaneous injection of DC pulsed with whole tumor lysate resulted in the highest increase in signal intensity at the tumor site and lymph nodes, suggesting subcutaneous injection of primed DC leads to highest preferential trafficking of DC to the immunocompetent organs.

Ataxia Telangiectasia and Rad3-related (ATR) and its downstream effector Checkpoint Kinase 1 (CHK1) are central to the protection of stalled replication forks.Specific targeting of the ATR is synthetically lethal with multiple cancer-associated changes including oncogenic stress and defects in the DDR pathway and represents an emerging strategy to treat a broad spectrum of cancers. Atrin Pharmaceuticals has rationally designed a novel series of conformationally constrained macrocyclic ATR inhibitors with higher potency and selectivity than other ATR inhibitors currently in clinical development. An in-depth characterization of the ATRN series identified ATRN-119 as our lead compound with an in vitro enzyme IC50 of 20 nM and inhibition of ATR substrate CHK1 Ser345 phosphorylation in cells at an IC50 of 5 nM. ATRN-119 inhibits ATR in cells at much lower concentrations and demonstrates higher selectivity (>2000) for ATR over other closely-related PIK-kinases like ATM, DNA-PK and mTOR. Additionally, ATRN series have favorable ADME properties with increased water solubility and metabolic stability in human serum of up to 4 hours. Oral dosing of ATRN-119 showed significant antitumor effects in human pancreatic and colon cancer xenografts and in orthotopic ovarian Patient Derived Xenograft (PDX) tumors. Notably, hematological analysis of mice treated with daily oral dosing of ATRN-119 indicated no thrombocytopenia, anemia or neutropenia up to 4 weeks of treatment. Exploratory multiple high dosing toxicity studies in rats and dogs indicate significant exposure and good tolerability with lack of anemia or neutropenia. In a series of in-vitro cell viability assays, three dimensional organoid cultures and in-vivo combinationstudies, ATRN-119 showed significant synergism with various PARP inhibitors as well as restoration of PARPi sensitivity in PARPi resistant tumors. Our data suggests a new generation of highly potent and selective ATR inhibitors with favorable safety profile and a broad clinical therapeutic potential either as monotherapy, in combination with PARP inhibitors or as a synthetic lethal approach with key DDR mutations. Citation Format: Sahithi Pamarthy, Dansu Li, Ekaterine Goliadze, Tina Gill, Lanqi Jia, Erin George, Laura R. Butler, Ryan L. Ragland, Michel Afargan, Fiona A. Simpkins, Eric J. Brown, Oren Gilad. Highly specific macrocyclic ATR inhibitors for the targeted treatment of a broad spectrum of cancers showing lack of anemia or neutropenia in pre-clinical animal models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3498.

Decreased expression of highly immunogenic cancer-testis antigens (CTA) might help tumor to achieve low immunogenicity, escape immune surveillance and grow unimpeded. Our aim was to evaluate CTA expression in tumor and normal tissues and to investigate possible means of improving the immune response in a murine prostate cancer (CaP) model by using the combination of epigenetic modifier 5-azacitidine (5-AzaC) and immunomodulator lenalidomide. No study to date has examined the effect of this combination on the prostate cancer or its impact on antigen-presenting cells (APC).Gene microarrays were performed to compare expression of several CTA in murine prostate cancer (RM-1 cells) and normal prostate. RM-1 cells were treated with 5-AzaC and real-time PCR was performed to investigate the expression of several CTA. Western blotting was used to determine whether expression of CTA-specific mRNA induced by 5-AzaC resulted in increase in the corresponding protein. Effect of the epigenetic agents and immunomodulators was assessed on dendritic cells (DC) using flow cytometry, ELISA and T-cell proliferation assay.Gene arrays demonstrated decreased expression of 35 CTA in CaP tissue compared to normal prostate. 5-AzaC treatment of RM-1 prostate cancer cells upregulated the expression of all 13 CTA tested in a dose-dependent fashion. DC were treated with 5-AzaC and lenalidomide and the expression of surface markers MHC Class I, MHC Class II, CD80, CD86, CD 205, and CD40 was increased. Combination of 5-AzaC and lenalidomide enhances the ability of DC to stimulate T-cell proliferation in mixed leukocyte reaction. Secretion of IL-12 and IL-15 by DC increased significantly with addition of 5-AzaC or 5-AzaC and lenalidomide.Decreased expression of CTA by prostate cancer may be a means of escaping immune monitoring. Combination of epigenetic modifications and immunomodulation by 5-AzaC and lenalidomide increased tumor immunogenicity and enhanced DC function and may be used in the treatment of advanced prostate cancer. Prostate 77: 361-373, 2017. © 2016 Wiley Periodicals, Inc.

common precursor of testosterone (T) and dihydrotestosterone (DHT). We reported 5a-androstane-3b,17b-diol (3b-diol), which is inactive androgen metabolized from DHT, can be converted back to DHEA and finally to DHT. In this pathway, 3b-hydroxysteroid dehydrogenase (3bHSD) is the key enzyme for the conversion from DHEA and epiandrosterone (epiAND) to DHT, and Rui et al. reported abiraterone inhibited 3b-HSD in CRPC. We examined the effect of CYP17 inhibitors on the intracrine back-conversion pathway induced by 3b-diol in prostate cancer cells. METHODS: LNCaP cells were incubated in the presence of 3bdiol or DHEA 10nmol/L with or without CYP17 inhibitors (abiraterone, ketoconazole, or orteronel) 10nmol/L. In all experiments, LNCaP cells were incubated without any androgens and CYP17 inhibitors as a control (CTRL). After incubation, PSA levels in the media were determined by enzyme immunoassay, and androgen levels in media were measured by LC-MS/MS. RESULTS: By adding DHEA or 3b-diol in media, PSA levels were increased (p 1⁄4 0.013 and p < 0.001, respectively). 3b-diol highly activated AR about five-times compared to DHEA. Inhibition of PSA secretion by abiraterone, ketoconazole and orteronel was found in the presence of DHEA (p < 0.001, p 1⁄4 0.002, and p 1⁄4 0.017, respectively). 3b-diol-induced PSA accumulation in media was inhibited by abiraterone, not ketoconazole or orteronel. By adding 3b-diol in medium, DHEA, androstenediol (A-diol), androstendione (A-dione), T, and DHT levels were increased compared to CTRL. In the media with 3b-diol and abiraterone, A-dione, T, DHT, and 5a-androstanedione (5a-A-dione) levels were decreased, DHEA, 5-diol, and epiAND levels increased compared to 3b-diol only. CONCLUSIONS: AR was highly activated by 3b-diol compared to DHEA through the conversion of 3b-diol to DHT by way of epiAND and 5a-A-dione, which abiraterone blocked by inhibiting 3b-HSD. Abiraterone has a potential to inhibit the intracrine back-conversion pathway.

INTRODUCTION AND OBJECTIVES: The apoptotic and suppressive effects of prostate cancer on immune-competent cells are well known. The aim of this study was to evaluate the genome-wide effects of prostate cancer on dendritic cells (DC), the most effective antigenpresenting cells, in a murine prostate cancer model. METHODS: Dendritic cells were compared to DC incubated with RM-1 murine prostate cancer cells. Affymetrix Mouse 430 v2 Array was used for analysis containing 45000 probes. Quantitative polymerase chain reaction (qPCR) was performed to validate changes in the endothelin axis. RNAs from murine prostate cancer and normal murine prostate were also compared. RESULTS: There was a significant increase in the expression of cell death inducing genes in DC incubated with prostate cancer cells, including multiple caspases. There was also a down regulation of proinflammatory genes including a number of interleukins and interferons. A change in the expression of endothelin receptors was seen as well. Considering the role of the endothelin axis in DC functioning, we used qPCR to further evaluate the condition of the endothelin axis in DC under prostate cancer influence. There was a significant decrease in the expression of ET-1 (4.50 1.33 fold) and ETA receptor (2.67 0.13), which are involved in DC survival and pro-inflammatory function. There was also an increase in the expression of ETB receptor (3.42 0.14) involved in DC apoptosis. Genome-wide comparison of RNAs derived from murine prostate cancer and murine prostate further demonstrated decreased expression of apoptotic genes by prostate cancer cells. CONCLUSIONS: Prostate cancer induces a wide variety of changes in DC including decreased expression of genes responsible for pro-inflammatory function and survival. Alterations of the endothelin axis may be one reason why tumor escapes immune monitoring.