Preclinical Models Of Prostate Cancer Research Articles

Abstract 1629: Prostate cancer RIPTAC™ therapeutics demonstrate activity in preclinical models of Enzalutamide-resistant prostate cancer

Abstract Background: Novel drugs are needed to tackle forms of prostate cancer that demonstrate resistance to hormonal agents. Halda has invented an innovative cancer treatment approach that does not rely on oncogenic drivers. Regulated Induced Proximity Targeting Chimera (RIPTAC™) therapeutics are heterobifunctional small molecules that work via a hold and kill mechanism that has the potential to overcome drug resistance mechanisms. RIPTAC therapeutics function by holding together two proteins, a cancer-specific protein, and a protein with essential function (EP) in a ternary complex, resulting in abrogation of the essential function and subsequent cancer cell death. We exemplify this platform to treat metastatic castration resistant prostate cancer (mCRPC), where the RIPTAC therapeutic utilizes the Androgen Receptor (AR) as a tumor specific protein to selectively inhibit an essential protein involved in transcriptional regulation and provide in vivo efficacy coupled with a therapeutic index. Methods: RIPTACs therapeutics were assayed for their ability to form a ternary complex with AR and the EP using a novel TR-FRET based assay in VCaP prostate cancer cells that harbor AR amplification. Selective apoptosis in ARhigh cells was observed using a Caspase 3/7 Glo assay (Promega). EP pharmacodynamic modulation was ascertained using qRT-PCR and western blotting in both in vitro and in vivo samples. RIPTACs were optimized for oral bioavailability, and tumor ternary complex formation in prostate cancer cell line-derived xenograft models.Results: RIPTAC therapeutics display nanomolar in vitro potency in AR:RIPTAC:EP ternary complex formation, which results in abrogation of the EP function and antiproliferative activity in prostate cancer cell lines, but not in AR-knockout control cells. The prostate cancer RIPTAC therapeutics are active in vitro against clinically relevant AR mutants. Lead molecules utilizing AR as a tumor specific protein are orally bioavailable in multiple preclinical species and induce ternary complex formation in VCaP tumor xenografts grown in mice. We present in vivo data where lead RIPTACs demonstrate significant tumor growth inhibition in several prostate cancer models and induce tumor regressions in VCaP xenografts grown in castrated male mice. Conclusions: Taken together, our in vitro mechanistic data and in vivo PD/efficacy observations in multiple prostate cancer models support further investigation of prostate cancer RIPTAC therapeutics as a novel heterobifunctional therapeutic modality in mCRPC. Citation Format: Xinheng Yu, Kyle J. Eastman, Kanak Raina, Kelli M. Jones, Chris D. Forbes, Abigail Hundt, Marco Garcia, Rebecca Stronk, Katia Howard, Andrew McGovern, Rebekka Chenard, Allison Denny, Mia Forgione, Kyle Bassoli, Ethan Garvin, James J. Mousseau, Hao Li, Madeline P. King, Amit Bhardwaj, Katherine J. Kayser-Bricker, Craig M. Crews. Prostate cancer RIPTAC™ therapeutics demonstrate activity in preclinical models of Enzalutamide-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1629.

Antiandrogen-Equipped Histone Deacetylase Inhibitors Selectively Inhibit Androgen Receptor (AR) and AR-Splice Variant (AR-SV) in Castration-Resistant Prostate Cancer (CRPC).

Epigenetic modification influences androgen receptor (AR) activation, often resulting in prostate cancer (PCa) development and progression. Silencing histone-modifying enzymes (histone deacetylases-HDACs) either genetically or pharmacologically suppresses PCa proliferation in preclinical models of PCa; however, results from clinical studies were not encouraging. Similarly, PCa patients eventually become resistant to androgen ablation therapy (ADT). Our goal is to develop dual-acting small molecules comprising antiandrogen and HDAC-inhibiting moieties that may overcome the resistance of ADT and effectively suppress the growth of castration-resistant prostate cancer (CRPC). Several rationally designed antiandrogen-equipped HDAC inhibitors (HDACi) were synthesized, and their efficacy on CRPC growth was examined both in vitro and in vivo. While screening our newly developed small molecules, we observed that SBI-46 significantly inhibited the proliferation of AR+ CRPC cells but not AR- CRPC and normal immortalized prostate epithelial cells (RWPE1) or normal kidney cells (HEK-293 and VERO). Molecular analysis confirmed that SBI-46 downregulated the expressions of both AR+ and AR-splice variants (AR-SVs) in CRPC cells. Further studies revealed the downregulation of AR downstream (PSA) events in CRPC cells. The oral administration of SBI-46 abrogated the growth of C4-2B and 22Rv1 CRPC xenograft tumors that express AR or both AR and AR-SV in xenotransplanted nude mice models. Further, immunohistochemical analysis confirmed that SBI-46 inhibits AR signaling in xenografted tumor tissues. These results demonstrate that SBI-46 is a potent agent that inhibits preclinical models of CRPC by downregulating the expressions of both AR and AR-SV. Furthermore, these results suggest that SBI-46 may be a potent compound for treating CRPC.

Preclinical models of prostate cancer - modelling androgen dependency and castration resistance in vitro, ex vivo and in vivo.

Prostate cancer is well known to be dependent on the androgen receptor (AR) for growth and survival. Thus, AR is the main pharmacological target to treat this disease. However, after an initially positive response to AR-targeting therapies, prostate cancer will eventually evolve to castration-resistant prostate cancer, which is often lethal. Tumour growth was initially thought to become androgen-independent following treatments; however, results from molecular studies have shown that most resistance mechanisms involve the reactivation of AR. Consequently, tumour cells become resistant to castration - the blockade of testicular androgens - and not independent of AR per se. However, confusion still remains on how to properly define preclinical models of prostate cancer, including cell lines. Most cell lines were isolated from patients for cell culture after evolution of the tumour to castration-resistant prostate cancer, but not all of these cell lines are described as castration resistant. Moreover, castration refers to the blockade of testosterone production by the testes; thus, even the concept of "castration" in vitro is questionable. To ensure maximal transfer of knowledge from scientific research to the clinic, understanding the limitations and advantages of preclinical models, as well as how these models recapitulate cancer cell androgen dependency and can be used to study castration resistance mechanisms, is essential.

Effect of Esomeprazole Treatment on Extracellular Tumor pH in a Preclinical Model of Prostate Cancer by MRI-CEST Tumor pH Imaging.

Novel anticancer treatments target the pH regulating system that plays a major role in tumor progression by creating an acidic microenvironment, although few studies have addressed their effect on tumor acidosis. In this study, we investigated in vivo several proton pump inhibitors (PPIs) targeting NHE-1 (Amiloride and Cariporide) and V-ATPase (Esomeprazole and Lansoprazole) proton transporters in the DU145 androgen-insensitive human prostate cancer model. In cellulo results showed that DU145 are sensitive, with decreasing efficacy, to Amiloride, Esomeprazole and Lansoprazole, with marked cell toxicity both in normoxia and in hypoxia, with almost any change in pH. In vivo studies were performed upon administration of Esomeprazole to assess both the acute and chronic effects, and Iopamidol-based tumor pH imaging was performed to evaluate tumor acidosis. Although statistically significant tumor pH changes were observed a few hours after Esomeprazole administration in both the acute study and up to one week of treatment in the chronic study, longer treatment resulted in a lack of changes in tumor acidosis, which was associated to similar tumor growth curves between treated and control groups in both the subcutaneous and orthotopic models. Overall, this study highlights MRI-CEST tumor pH imaging as a valid approach to monitoring treatment response to PPIs.

Head-up tilt does not enhance prostate tumor perfusion or oxygenation in young rats.

Solid tumors contain hypoxic regions that contribute to anticancer therapy resistance. Thus, mitigating tumor hypoxia may enhance the efficacy of radiation therapy which is commonly utilized for patients with prostate cancer. Increasing perfusion pressure in the prostate with head-up tilt (HUT) may augment prostate tumor perfusion and decrease hypoxia. The purpose of this study was to determine if an increase in the vascular hydrostatic gradient via 70° HUT increases tumor perfusion and decreases tumor hypoxia in a preclinical orthotopic model of prostate cancer. Male Copenhagen rats (n=17) were orthotopically injected with Dunning R-3327 (AT-1) prostate adenocarcinoma cells to induce prostate tumors. After tumors were established, prostate tumor perfusion and hypoxia were measured in rats during level (0°) and 70° HUT positions. To compare the magnitude of the hydrostatic column to that present in humans, ultrasound was used to measure the heart to prostate distance in male human subjects to estimate the prostate vascular hydrostatic pressure with the upright posture. In young rats, no differences were detected in prostate tumor perfusion or prostate tumor hypoxia with 70° HUT versus the level position. However, from the retrospective study, young rats increased prostate vascular resistance to HUT, whereas aged rats lacked this response. Tumor vessels co-opted from existing functional vasculature in young rats may be sufficient to negate increases in perfusion pressure with HUT seen in aged rats. Additionally, in humans, the estimated hydrostatic column at the level of the prostate is five times greater than that of the rat. Therefore, 70° HUT may elicit increases in prostate/prostate tumor blood flow in humans that is not seen in rats.

Efficacy of PACE4 pharmacotherapy in JHU-LNCaP-SM preclinical model of androgen independent prostate cancer

Prostate cancer (PCa) is a complex disease progressing from in situ to invasive or metastatic tumors while also being capable of modulating its androgen dependence. Understanding how novel therapies are working across the different stages of the disease is critical for their proper positioning in the spectrum of PCa treatments. The targeting of proprotein convertase PACE4 (Paired basic Amino Acid-Cleaving Enzyme 4) has been proposed as a novel approach to treat PCa. Animal studies performed on LNCaP xenografts, an androgen-dependent model, already yielded positive results. In this study, we tested PACE4 inhibition on JHU-LNCaP-SM, a newly described androgen-independent model, in cell-based and xenograft assays. Like LNCaP, JHU-LNCaP-SM cells express PACE4 and its oncogenic isoform PACE4-altCT. Using isoform-specific siRNAs, downregulation of PACE4-altCT resulted in JHU-LNCaP-SM growth inhibition. Furthermore, JHU-LNCaP-SM responded to the PACE4 pharmacological inhibitor known as C23 in cell-based assays as well as in athymic nude mice xenografts. These data support the efficacy of PACE4 inhibitors against androgen independent PCa thereby demonstrating that PACE4 is a key target in PCa. The JHU-LNCaP-SM cell line represents a model featuring important aspects of androgen-independent PCa, but it also represents a very convenient model as opposed to LNCaP cells for in vivo studies, as it allows rapid screening due to its high implantation rate and growth characteristics as xenografts.

Preclinical and Clinical Research Models of Prostate Cancer: A Brief Overview.

The incidence and mortality from prostate cancer (PCa) are on the rise which poses a major public health concern worldwide. In this narrative review, we have summarized the characteristics of major in vitro and in vivo PCa models including their utility in developing treatment strategies. Androgens, particularly, testosterone and dihydrotestosterone (DHT) activate the androgen receptor (AR) signaling pathway that facilitates the development and progression of castration resistant PCa. Several enzymes namely, CYP17A1, HSD17B, and SRD5A are essential to furnishing DHT from dehydroepiandrosterone in the classical pathway while DHT is formed from androstanediol in the backdoor pathway. The advancement in delineating the molecular heterogeneity of PCa has been possible through the development of several in vitro and in vivo research models. Generally, tissue culture models are advantageous to understand PCa biology and investigate the efficacy and toxicity of novel agents; nevertheless, animal models are indispensable to studying the PCa etiology and treatment since they can simulate the tumor microenvironment that plays a central role in initiation and progression of the disease. Moreover, the availability of several genetically engineered mouse models has made it possible to study the metastasis process. However, the conventional models are not devoid of limitations. For example, the lack of heterogeneity in tissue culture models and the variation of metastatic characteristics in xenograft models are obviously challenging. Additionally, due to the racial and ethnic disparities in PCa pathophysiology, a new model that can represent PCa encompassing different ethnicities is urgently needed. New models should continue to evolve to address the genetic and molecular complexities as well as to further elucidate the finer details of the steroidogenic pathway associated with PCa.

Novel AR/AR-V7 and Mnk1/2 Degrader, VNPP433-3β: Molecular Mechanisms of Action and Efficacy in AR-Overexpressing Castration Resistant Prostate Cancer In Vitro and In Vivo Models.

Prostate cancer (PCa) relies in part on AR-signaling for disease development and progression. Earlier, we developed drug candidate galeterone, which advanced through phase 2-clinical trials in treating castration-resistant PCa (CRPC). Subsequently, we designed, synthesized, and evaluated next-generation galeterone-analogs including VNPP433-3β which is potently efficacious against pre-clinical models of PCa. This study describes the mechanism of action of VNPP433-3β that promotes degradation of full-length AR (fAR) and its splice variant AR-V7 besides depleting MNK1/2 in in vitro and in vivo CRPC models that stably overexpresses fAR. VNPP433-3β directly engages AR within the cell and promotes proteasomal degradation of fAR and its splice variant AR-V7 by enhancing the interaction of AR with E3 ligases MDM2/CHIP but disrupting AR-HSP90 binding. Next, VNPP433-3β decreases phosphorylation of 4EBP1 and abates binding of eIF4E and eIF4G to 5′ cap of mRNA by depleting MNK1/2 with consequent depletion of phosphorylated eIF4E. Finally, RNA-seq demonstrates modulation of multiple pathways that synergistically contribute to PCa inhibition. Therefore, VNPP433-3β exerts its antitumor effect by imposing 1) transcriptional regulation of AR and AR-responsive oncogenes 2) translational regulation by disrupting mRNA-5′cap-dependent translation initiation, 3) reducing AR half-life through enhanced proteasomal degradation in vitro and AR-overexpressing tumor xenografts in vivo.

Nimbolide enhances the antitumor effect of docetaxel via abrogation of the NF-κB signaling pathway in prostate cancer preclinical models

Prostate cancer is the second most frequent type of cancer that affects men. Docetaxel (DTX) administration is the front-line therapy for patients with advanced prostate cancer and unfortunately, half of these patients develop resistance to DTX which could be due to its ability to activate the NF-κB pathway. The combinational effect of DTX and nimbolide on proliferation, apoptosis, activation of NF-κB, DNA binding ability of NF-κB, and expression of NF-κB-targeted gene products was investigated. The antitumor and antimetastatic effect of DTX or NL alone or in combination was also examined. The co-administration of NL and DTX resulted in a significant loss of cell viability with enhanced apoptosis in DTX-sensitive/resistant prostate cancer cells. NL abrogated DTX-triggered NF-κB activation and expression of its downstream antiapoptotic factors (survivin, Bcl-2, and XIAP). The combination of NL and DTX significantly reduced the DNA binding ability of NF-κB in both cell types. NL significantly enhanced the antitumor effect of DTX and reduced metastases in orthotopic models of prostate cancer. NL abolishes DTX-induced-NF-κB activation to counteract cell proliferation, tumor growth, and metastasis in the prostate cancer models.

Preclinical Assessment of the Combination of PSMA-Targeting Radionuclide Therapy with PARP Inhibitors for Prostate Cancer Treatment.

Prostate specific membrane antigen targeted radionuclide therapy (PSMA-TRT) is a promising novel treatment for prostate cancer (PCa) patients. However, PSMA-TRT cannot be used for curative intent yet, thus additional research on how to improve the therapeutic efficacy is warranted. A potential way of achieving this, is combining TRT with poly ADP-ribosylation inhibitors (PARPi), which has shown promising results for TRT of neuroendocrine tumor cells. Currently, several clinical trials have been initiated for this combination for PCa, however so far, no evidence of synergism is available for PCa. Therefore, we evaluated the combination of PSMA-TRT with three classes of PARPi in preclinical PCa models. In vitro viability and survival assays were performed using PSMA-expressing PCa cell lines PC3-PIP and LNCaP to assess the effect of increasing concentrations of PARPi veliparib, olaparib or talazoparib in combination with PSMA-TRT compared to single PARPi treatment. Next, DNA damage analyses were performed by quantifying the number of DNA breaks by immunofluorescent stainings. Lastly, the potential of the combination treatments was studied in vivo in mice bearing PC3-PIP xenografts. Our results show that combining PSMA-TRT with PARPi did not synergistically affect the in vitro clonogenic survival or cell viability. DNA-damage analysis revealed only a significant increase in DNA breaks when combining PSMA-TRT with veliparib and not in the other combination treatments. Moreover, PSMA-TRT with PARPi treatment did not improve tumor control compared to PSMA-TRT monotherapy. Overall, the data presented do not support the assumption that combining PSMA-TRT with PARPi leads to a synergistic antitumor effect in PCa. These results underline that extensive preclinical research using various PCa models is imperative to validate the applicability of the combination strategy for PCa, as it is for other cancer types.

P597: HIGH DOSE IRON IMPAIRS MALIGNANT B-CELL VIABILITY IN CHRONIC LYMPHOCYTIC LEUKEMIA

Background: Identification of new strategies to improve Chronic Lymphocytic Leukemia (CLL) outcome is fundamental since the disease remains incurable. Malignant B-cells show high levels of reactive oxygen species (ROS), which favors adaptation and survival but also enhances sensitivity to pro-oxidant therapeutics. Redox homeostasis is affected by iron balance since iron excess works as pro-oxidant, and cancer cells rearrange iron trafficking proteins to promote iron uptake. This adaptation might be exploited by exposing malignant cells to iron excess to cause ROS generation, lipid peroxidation, and eventually ferroptosis. We previously demonstrated the feasibility of this strategy in multiple myeloma and prostate cancer pre-clinical models. Aims: We aim at exploiting high dose iron to negatively affect malignant cell survival and increase therapeutic efficacy of current target therapies (BTK and BCL2 inhibitors). We aim at dissecting the molecular mechanisms underlying iron toxicity and at testing whether iron can improve immune dysfunctions. Methods: CLL cell lines (MEC-1, MEC-2 and PCL-12), patients peripheral blood and bone marrow mononuclear cells (PBMC and BMMC) and B lymphocytes purified from patients PB were treated with 300 µM ferric ammonium citrate (FeAC) alone or in combination with the BTK inhibitor ibrutinib (IBR, 5-10 µM) or the BCL2 inhibitor venetoclax (VNTX, 1.25-2.5 nM). We evaluated cell viability by annexin PI staining, lipid peroxidation and ferroptosis by measuring malondialdehyde (MDA) levels and antioxidant gene expression levels by qRT-PCR. We additionally evaluated T cell activation by measuring CD25 expression. MEC-1 xenografts generated in immunodeficient RAG2-/-yc-/- mice were treated with 20 mg/Kg ferric carboximaltose. Results:In vitro, iron treatment impaired cell proliferation in all CLL cell lines analyzed, inducing accumulation of MDA and cell death (p<0.01). In vivo, iron supplementation reduced the amount of MEC-1 cells in bone marrow and spleen of xenograft mice as compared to control saline-treated mice. In patients primary samples, iron induced cell death of leukemic lymphocytes (CD19+CD5+) either as purified cells or within bulk PBMC (p<0.01). Moreover, combination of iron with IBR or VNTX induced cell death at a higher extent compared to each single drug or to iron alone (p<0.01). Leukemic cells in BMMC samples were also iron sensitive and the effect became more evident upon combination with IBR or VNTX (p<0.05). Neither iron nor iron-drug combinations induced cell death in non-malignant cells. Iron also increased CD25 expression in CD8+ T cells and we are currently exploring whether this might indicate that iron can also improve cytotoxic mediated immune response against leukemic cells. As single patient analysis revealed heterogeneity of response, we investigated whether we might predict iron sensitivity by analyzing the antioxidant gene expression profile. All CLL cell lines analyzed showed a lower basal expression of NFE2L2, HMOX1, and GPX4 antioxidant genes than typical iron-resistant cell lines, such as prostate cancer PC-3 cells, suggesting that differences in antioxidant gene expression levels may mediate iron response and that this association is worth to be explored in CLL primary samples to explain heterogeneous iron response. Summary/Conclusion: Our pre-clinical studies suggests that exploiting iron toxicity might be a valuable strategy to improve current target therapies in CLL. Additional studies aimed at distinguishing iron sensitive patients from poor responders will improve the translational potential of this therapeutic approach.

Modeling prostate cancer: What does it take to build an ideal tumor model?

Prostate cancer is frequently characterized as a multifocal disease with great intratumoral heterogeneity as well as a high propensity to metastasize to bone. Consequently, modeling prostate tumor has remained a challenging task for researchers in this field. In the past decades, genomic advances have led to the identification of key molecular alterations in prostate cancer. Moreover, resistance towards second-generation androgen-deprivation therapy, namely abiraterone and enzalutamide has unveiled androgen receptor-independent diseases with distinctive histopathological and clinical features. In this review, we have critically evaluated the commonly used preclinical models of prostate cancer with respect to their capability of recapitulating the key genomic alterations, histopathological features and bone metastatic potential of human prostate tumors. In addition, we have also discussed the potential use of the emerging organoid models in prostate cancer research, which possess clear advantages over the commonly used preclinical tumor models. We anticipate that no single model can faithfully recapitulate the complexity of prostate cancer, and thus, propose the use of a cost- and time-efficient integrated tumor modeling approach for future prostate cancer investigations.

Abstract 4178: PAK4 inhibition improves PD1 blockade immunotherapy in prostate cancer

Abstract Prostate cancers have low immune infiltration and do not respond to PD1 blockade immunotherapy. The objective of this study was to examine the effect of PAK4 inhibition on anti-tumor immune response in preclinical prostate cancer models. C57BL/6J mice (male, 5-8 weeks old) were injected with wild type (WT) or PAK4 knockdown (KD) syngeneic Rm1 tumor cells (2X105 cell/mouse) and treated with or without αPD1 blockade (200μg, BioXCell). Tumor growth was monitored and analyzed using ANOVA of repeated measures (n=6). In a separate experiment, 1 cm RM1 tumors were collected for RNA sequencing (RNASeq) and immunohistochemistry (IHC). RNASeq data were normalized using the variance stabilizing transformation (VST) function as implemented within the DESeq2 package. IHC images were captured with Keyence BZ-X800 and positive staining was calculated using ImageJ. CD8+ lymphocytes depletion was achieved by CD8 antibody injection (200ug, BioXCell). PAK4 inhibitors PF-3758309 (PF) and KPT-9274 (KPT) were evaluated in murine models. PAK4 KD tumors treated with αPD1 antibody (αPD1) showed a significant reduction of tumor growth when compared to growth in WT (P&amp;lt;.05), WT αPD1 (P&amp;lt;.01), and KD (p&amp;lt;.05). Analysis of bulk tumor RNAseq showed that the combination of PAK4 KD with αPD1 was associated with increased CD8a, IFNγ, and PD-L1 expression. IHC with αCD8 verified that PAK4 KD increased CD8+ cell infiltration in mouse tumors. Further, RNAseq showed that PAK4 KD increased the expression of cytokines, chemokines, and genes related to the IFNγ response. These results suggest that PAK4 KD primes the tumor for immune infiltration and improves αPD1 treatment response. The anti-tumor effect of PAK4 KD with αPD1 in mice was CD8-dependent and could be eliminated by CD8+ cell depletion (p&amp;lt;.05). We tested two different PAK4 inhibitors in mice with WT Rm1 tumors. PF is a kinase inhibitor that blocks the function of PAK4. A combination of PF and αPD1 significantly reduced tumor growth when compared to WT (P&amp;lt;.05), WT αPD1 (P&amp;lt;.01), and PF (p&amp;lt;.05). KPT is a dual inhibitor of PAK4 and NAMPT, which reduces the cytosolic protein concentration of PAK4. A combination of KPT and αPD1 significantly reduced tumor growth when compared to WT (P&amp;lt;.01) and WT αPD1 (P&amp;lt;.01). In conclusion, PAK4 inhibition increased immune infiltration and improved αPD1 treatment response in preclinical mouse prostate cancer models. Citation Format: Shengchen Su, Sungyong You, Yanping Wang, Patrick Tamukong, Catherine S. Grasso, Hyung L. Kim. PAK4 inhibition improves PD1 blockade immunotherapy in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4178.

Abstract 2155: PMR-116, a novel inhibitor of ribosome biogenesis with antitumor activity in preclinical models of prostate cancer

Abstract Advanced prostate cancer is characterised by mutations and amplifications of genes involved in regulating protein synthesis. PTEN-loss stimulates activity of the mTOR pathway, while amplification of MYC leads to increased ribosome biogenesis and elevated mRNA translation rate. Our previous work has demonstrated the efficacy of co-targeting ribosome biogenesis, via inhibition of RNA Pol I activity, and 4E-BP1 phosphorylation to suppress prostate cancer growth in vivo in GEMM of PCa and in patient-derived xenografts (Rebello et al., Clinical Cancer Research, 2016; Lawrence et al., European Urology, 2018). In a collaboration with Pimera Inc., we investigated the efficacy of their new lead RNA Pol I inhibitor PMR-116 in models of prostate cancer. PMR-116 is well tolerated in vivo in mice and can be given at 300mg/kg weekly. Using the Hi-MYC mouse model of PCa we show that dosing 6 month old mice once weekly for 4 weeks can decrease the incidence of invasive lesions by up to 85% compared to vehicle control while reverting glands to patterns of low grade intraepithelial neoplasia. PMR-116 rapidly inhibits proliferation in the Hi-MYC model with a 50% decrease in Ki67 observed 12 hours after oral administration. Conversely, PMR-116 showed minimal anti-tumour efficacy in the PTEN-null model of PCa suggesting that elevated MYC signalling may be required for optimal response. To further validate our promising GEMM results in more clinically relevant human-derived models, we used patient-derived xenografts lines we established from multidrug-resistant, metastatic PCa (Lawrence et al, European Urology, 2018). PMR-116 treatment decreased tumour volume in all PDX tested including complete response in a line in which tumour volume decreased by ~90% compared to baseline. We believe this new RNA Pol I inhibitor shows promising results in a wide range of preclinical models and may exert higher efficacy in tumours expressing high levels of MYC. PMR-116 is currently in Phase I dose escalation trial in patient with solid tumours (ACTRN12620001146987). Citation Format: Alisee Huglo, Richard Rebello, Mitchell Lawrence, Gail Risbridger, Denis Drygin, Mustapha Haddach, Katherine Hannan, Ross Hannan, Luc Furic. PMR-116, a novel inhibitor of ribosome biogenesis with antitumor activity in preclinical models of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2155.

Abstract 5471: PRT2527, a novel highly selective cyclin-dependent kinase 9 (CDK9) inhibitor, is active in preclinical models of prostate cancer

Abstract CDK9 is a serine/threonine kinase belonging to the subclass of the transcription associated CDKs. CDK9 complexes with cyclin T and cyclin K, the positive transcription elongation factor b (P-TEFb), and phosphorylates Serine 2 of RNA polymerase II (pSer2 RNAPII) to activate transcription. Consequently, targeting CDK9 could effectively interfere with epigenetic and transcriptional reprogramming and prevent disease progression and treatment resistance in human cancers. Androgen receptor (AR)-dependence in prostate cancer is linked to CDK9 function. CDK9 stabilizes AR-associated proteins, and pharmacological inhibition of CDK9 can inhibit AR, AR variants, and their downstream transcription programs. We evaluated the novel CDK9 inhibitor, PRT2527, in prostate cancer models to evaluate its effects on cell proliferation, stem-like tumor cells, and tumor growth. PRT2527 is a potent inhibitor of CDK9/CyclinT1 complex, and when evaluated at concentration 200 times the biochemical IC50, PRT2527 was highly selective for CDK9 inhibition. We verified in biochemical assays the ability of PRT2527 to suppress pSer2 RNAPII and reduce expression of c-Myc, a common target of CDK9, in a concentration-dependent manner in multiple human prostate cancer cell lines. PRT2527 also inhibited c-Myc-dependent transcription in vitro in luciferase reporter assays. Furthermore, RNA sequencing showed altered expression of several genes with significant enrichment of c-Myc and E2F targets and RNAPII dependent transcription among downregulated genes in PRT2527-treated VCaP cells. In vitro, PRT2527 inhibited the proliferation of androgen-dependent and androgen-independent prostate cancer cell lines (IC50, ≤50 nM). PRT2527 was highly effective (IC50, ≤10 nM) in tumor-spheroid assays in blocking the growth of stem-like tumor cells and significantly suppressed the in vitro growth of tumor organoids from both human cell lines and patient-derived xenografts (PDXs). In mice, PRT2527 IV administration reduced pSer2 RNAPII in tumor xenografts and c-Myc-dependent transcriptional activity in a DU145 luminescence reporter model. PRT2527 administration in mice significantly reduced growth of PDX LuCaP 35 (castration-sensitive, adenocarcinoma) and LuCaP 145.2 (castration-resistant, neuroendocrine) along with the fraction of tumor-initiating stem-like cells in ex vivo assays. PRT2527 reduced pSer2 RNAPII in both PDXs, whereas c-Myc decreased in LuCaP 35 and Sox2 in LuCaP 145.2, relative to basal expression levels. Collectively, our data demonstrate that PRT2527 has potent pharmacodynamic and antitumor activity in multiple models of castration-sensitive and castration-resistant prostate cancer. PRT2527 is advancing into phase 1 studies in solid tumors. Citation Format: Elisa Federici, Gianluca Civenni, Aleksandra Kokanovic, Giada Sandrini, Luca Guarrera, Simone Mosole, Alessia Cacciatore, Valeria Uboldi, Manuel Lessi, Giovanni Papa, Domenico Albino, Elisa Storelli, Jessica Merulla, Andrea Rinaldi, Marco Bolis, Yang Zhang, Kris Vaddi, Peggy Scherle, Bruce Ruggeri, Giuseppina M. Carbone, Carlo V. Catapano. PRT2527, a novel highly selective cyclin-dependent kinase 9 (CDK9) inhibitor, is active in preclinical models of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5471.

Abstract 1345: p300/CBP bromodomain inhibitor CCS1477 enhances the efficacy of immune checkpoint blockade therapy in cancer treatment

Abstract Background: Blockade of programmed death-ligand 1 (PD-L1) by therapeutic antibodies has shown to be a promising strategy in cancer therapy, yet clinical response in many types of cancer is limited. Tumor cells secrete PD-L1 through exosomes or splice variants, which has been described as a new mechanism for the resistance to PD-L1 blockade therapy in multiple cancers. Our previous findings showed that the histone acetyltransferases p300/CBP could be recruited to the promoter of CD274 (encoding PD-L1) to regulate the expression of PD-L1. P300/CBP inhibition abrogated this process and reduced the secretion of exosomal PD-L1 by blocking the transcription of CD274, which combined with the anti-PD-L1 antibody to reactivate T cells function for tumor attack. In this study we have tested the outcome of combined immune checkpoint inhibitors with CCS1477, a novel p300/CBP bromodomain inhibitor, in pre-clinical models of prostate cancer and melanoma. Results: CCS1477 significantly decreased human prostate (DU145/PC-3) and melanoma (A375/SK-MEL-28) cancer cell line PD-L1 expression, as well as exosome secreted PD-L1 after 24 hours treatment. Moreover, consistent with these findings in vitro, CCS1477 (2.5mg//kg) also reduced PD-L1 expression in a syngeneic mouse prostate model (TRAMP-C2) and syngeneic mouse melanoma model (B16-F10). Meanwhile, CCS1477 treatment significantly increased tumor-infiltrating CD8+ and CD4+ T cells in syngeneic mouse models. Monotherapy treatment with CCS1477 or immune checkpoint inhibitors (anti-PD-L1 antibody for prostate cancer or anti-PD-L1 and anti-CTLA4 antibody for melanoma) resulted in significant but modest efficacy. Strikingly, the combination of immune checkpoint inhibitors with CCS1477 showed robust synergistic responses resulting in tumor regression. Mechanistically, CCS1477 treatment induced significant loss of H3K27 acetylation from super-enhancers of CD274, which subsequently induced a significant reduction of PD-L1 expression and exosome secreted-PD-L1. Also, we found that CCS1477 treatment significantly decreased the abundance of circulating and tumor-infiltrating myeloid-derived suppressor cells (MDSCs), which are known to play important roles in tumor immune evasion by inhibiting T cell’s activation. Reduction of MDSCs and exosome secreted-PD-L1 preserved T-cell function and elicited robust immunotherapy response combined with immune checkpoint blockade agents. Conclusions: These observations illuminate a clinically relevant hypothesis for combining CCS1477 with immune checkpoint blockade in the treatment of cancer. Citation Format: Jinghui Liu, Xinyi Wang, Katelyn Jones, Nigel Brooks, Neil Pegg, Xiaoqi Liu. p300/CBP bromodomain inhibitor CCS1477 enhances the efficacy of immune checkpoint blockade therapy in cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1345.

Abstract 421: PLX-3618, a potent, selective monovalent BRD4 degrader demonstrates activity in models of prostate cancer

Abstract Prostate cancer is the second most common cause of cancer related deaths in men in the United States. Pathogenesis is driven by the androgen receptor (AR), which has led to front-line treatment modalities that are based on androgen deprivation therapy (ADT). About 10-20% of all prostate cancers evolve to resist ADT and are classified as castration-resistant prostate cancer (CRPC) indicating the continued need for new treatment options. Bromodomain-containing protein 4 (BRD4) is an acetylated-chromatin associating protein that is involved in transcriptional elongation, mRNA splicing, epigenetic bookmarking, and super-enhancer activity. The BRD4 protein has been shown to both bind and colocalize with AR at androgen response elements (AREs) on chromatin. Furthermore, elevated BRD4 expression is prognostic of increased prostate specific antigen (PSA) levels following radical prostatectomy and is correlated with higher Gleason scores and poor overall survival. The intertwined activity of BRD4 with multiple essential driver mechanisms of prostate cancer suggests it may be a key target for developing novel therapeutics. Using our ultra-high throughput cell-based screening platform, which directly measures degradation of pathogenic proteins upon exposure to diverse chemical libraries, we identified a series of novel monovalent BRD4 degraders that was optimized to produce our lead compound PLX-3618. A cancer cell panel screen for antiproliferative effects of PLX-3618 indicated enhanced sensitivity in subsets of prostate cancer lines. PLX-3618 elicited selective, rapid, and deep degradation of BRD4 protein in prostate cancer cell models, without degrading the closely related BRD2 and BRD3 proteins. Addition of either proteosome or neddylation inhibitors blocked BRD4 degradation indicating a ubiquitin-proteosome system mediated clearance mechanism. Degradation of BRD4 led to sustained multimodal inhibition of the AR pathway and disruption of key oncogene enhancer networks. When compared to pan-BET inhibitors, treatment of select prostate cancer cell lines with PLX-3618 resulted in increased levels of the tumor suppressors p53 and p21, an aberrant DNA damage response, and substantially amplified apoptosis. Finally, in in vivo preclinical models of prostate cancer, PLX-3618 showed far superior efficacy over a pan-BET inhibitor. Taken together, the selective degradation of BRD4 via the potent monovalent degrader PLX-3618 represents a novel strategy in treating prostate cancer. Citation Format: Kenneth Steadman, Gregory S. Parker, Geoffray Leriche, Sarah Fish, Julia Toth, Mary E. Spalding, Elizabeth Daniele, Aleksandar Jamborcic, Xiaoming Li, E Adam Kallel, Farhana Barmare, Kenneth Chng, Erika Green, Michael Hocker, Elliot Imler, Yi Zhang, Peggy A. Thompson, Simon Bailey. PLX-3618, a potent, selective monovalent BRD4 degrader demonstrates activity in models of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 421.

Abstract 3109: Establishment and characterization of a panel of castrate-resistant prostate cancer XPDX models with differential enzalutamide response

Abstract Background: Preclinical models of prostate cancer are challenging to develop and maintain, especially those that grow in a castrated setting while maintaining receptor and antigen expression. To this end, we have established and characterized a panel of XenoSTART patient-derived (XPDX) prostate models using both intact and castrated athymic nude mice. These models, designated ST1273, ST2347, ST4017, and ST4420, were characterized for receptor expression, genomic alterations, and in vivo drug sensitivity to relevant therapies. Methods: XPDX models representing prostate cancer were established from primary (ST1273) or metastatic (ST2347, ST4017, ST4420) biopsy samples implanted into intact athymic nude mice supplemented with exogenous testosterone. Resulting models were passaged and further developed in both intact and castrated athymic nudes until growth stabilization. Resulting models were characterized using genomic analysis, including WES and RNAseq, receptor expression, and in vivo drug sensitivity studies. Models found sensitive to enzalutamide were conditioned to resistance in vivo by chronic drug administration and resulting models (designated STxxxx/EZR) were characterized and compared with parent lines. For in vivo studies, activity of relevant treatments were benchmarked including enzalutamide administered once daily by oral gavage at 50 mg/kg and docetaxel administered by intravenous injection once weekly at 10 mg/kg. In vivo study endpoints included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a %T/C of ≤ 20 versus control was considered sensitive. Results: Each model developed in a castrate or conditioned setting retained similar receptor expression to the parent model including positive AR (2+/3+) and PSMA (2+/3+) staining. Genomic characterization identified a PIK3CA mutation in ST1273 models (PIK3CAE545A), AR mutations in ST2347 (ART878A) and ST4017 (ARH875Y), and a TMPRSS2:ERG fusion in ST4420. In vivo, the ST1273 parent model was found sensitive to enzalutamide (%T/C=11%) but insensitive in castrated mice (%T/C=76%) and the ST1273/EZR model was resistant to enzalutamide in intact (%T/C=56%) or castrated (%T/C=100%) mice. The ST2347 parent model was also found sensitive to enzalutamide (%T/C=19%) but insensitive in castrated mice (%T/C=50%). ST4017 and ST4420 studies in intact and castrated mice are currently underway. All models were sensitive to docetaxel. Conclusion: We have established and characterized a panel of prostate XPDX models using both intact and castrated athymic nude mice and conditioned resistance to enzalutamide by chronic drug administration. These models can be utilized as a valuable tool in better understanding castrate-resistant prostate cancer and in developing novel therapies for enzalutamide-resistant patients. Citation Format: Johnnie Flores, Alyssa Simonson, Dustin Kneifel, Alejandra Diaz, Morgan Harris, Kyriakos Papadopoulos, Amita Patnaik, Drew Rasco, Scott Ulmer, Michael J. Wick. Establishment and characterization of a panel of castrate-resistant prostate cancer XPDX models with differential enzalutamide response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3109.

Abstract 5928: MTA1-targeted interception by natural stilbenes in high-risk prostate cancer

Abstract Active surveillance subpopulation of patients with prostate cancer includes men with varying cancer risk categories of precancerous disease due to prostatic intraepithelial neoplasia (PIN) heterogeneity. Identifying molecular alterations associated with PIN can provide preventable measures through finding novel targets for cancer interception, specifically by active dietary polyphenols with potent biological properties including anti-inflammatory, anti-oxidant, and anticancer effects. A large volume of preclinical evidence shows that natural stilbenes produce anticancer responses in a variety of cell lines and animal models of different types of cancer, including solid tumors as well as hematological malignancies. However, identification of a particular target with clinical relevance is rare. I will discuss the potent MTA1-targeted anticancer effects of natural stilbenes (resveratrol, pterostilbene, and Gnetin C) observed in unique preclinical models of prostate cancer mimicking high-risk premalignant tumors with overexpressed MTA1. We have shown that MTA1 cooperates with PTEN deficiency to accelerate high-grade PIN development through MTA1-associated signaling. We further demonstrated that stilbene supplemented diets lead to more favorable histopathology with decreased severity and number of PIN foci accompanied with reduced proliferation, inflammation, and angiogenesis concomitant to reduction of MTA1-associated signaling, including specific miRNAs. Together, our preclinical data provide strong evidence for the potential of stilbenes as promising natural agents for MTA1-targeted interceptive strategy. Human cancer prevention trials for protecting select patients with high MTA1 from developing into full-fledged disease are urgently needed. This can be one extra forward step towards viable and successful interception/chemoprevention potential for prostate cancer with an implication for precision medicine. Supported by NCI/NIH R15CA216070 to ASL. Citation Format: Anait S. Levenson. MTA1-targeted interception by natural stilbenes in high-risk prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5928.

Abstract 2896: DLL3-targeted T cell engager therapy (HPN328) for neuroendocrine prostate cancer

Abstract Background: Neuroendocrine Prostate Cancer (NEPC) is an aggressive variant of prostate cancer that can arise de novo or emerge during prostate cancer progression as a mechanism of therapy resistance. The development of effective therapies for patients with NEPC is a clinical unmet need. Delta-Like Ligand 3 (DLL3) is a negative regulator of Notch signaling and aberrantly expressed on the cell surface in the majority of NEPC and other small cell carcinomas. HPN328 is a DLL3-targeted Tri-specific T cell Activating Construct (TriTAC) that binds to DLL3 on tumor cells, CD3 on T cells, and human serum albumin. Here, we evaluated the specificity and anti-tumor activity of HPN328 in NEPC preclinical models. Methods: Immunohistochemistry (IHC) was used to evaluate DLL3 expression in prostate cancer preclinical models. We co-cultured DLL3-positive and DLL3-negative tumor cells in vitro with either human PBMCs or T cells and used CellTiter-Glo and live-cell imaging to examine tumor cell viability after the exposure to HPN328. We conducted flow cytometry to examine T cell activation. Patient-derived organoid xenografts were used to determine the anti-tumor activity of HPN328 in vivo and we applied digital spatial profiling (Nanostring) to examine effects on immune cell infiltration. Results: HPN328 suppressed cell growth and induced cell death in DLL3-positive prostate cancer organoid cell line models at a range of 0.7nM - 10nM with the presence of human T cells. Flow cytometry analysis illustrated that T cell activation markers, such as CD25, were upregulated in a dose- and time-dependent manner. In vivo studies demonstrated robust anti-tumor activity of HPN328 in causing tumor regression and stable disease after stopping the treatment in DLL3-positive models, but not in DLL3-negative models. Sustained anti-tumor effects were observed without adverse events in mice. Conclusions: The DLL3-targeted TriTAC HPN328 demonstrates robust and specific anti-tumor activity in DLL3-positive NEPC preclinical models in vitro and in vivo. T cell engagers targeting DLL3 represent a promising therapeutic strategy for NEPC. Acknowledgements: This research is funded by a Helen Trailblazers Award from the Helen Gurley Brown Foundation/DFCI Presidential Initiative (H.B.). Citation Format: Sheng-Yu Ku, Yasutaka Yamada, Patrick Ng, Laura Sun, Himisha Beltran. DLL3-targeted T cell engager therapy (HPN328) for neuroendocrine prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2896.