Castration-resistant Prostate Cancer Models Research Articles

Abstract C034: First in Human phase 1/2a trial of a macrocyclic ATR inhibitor (ATRN-119) in patients with advanced solid tumors

Abstract Background: ATRN-119 is an oral and highly selective nanomolar inhibitor of Ataxia Telangiectasia and Rad3- related (ATR) kinase, an enzyme that is activated by replication stress and DNA damage and prevents the collapse of DNA replication forks into double strand breaks. ATRN-119 is being studied in advanced solid tumors with dysregulated components of the DNA damage response (DDR) based on the hypothesis that these tumors will be more susceptible to ATR inhibition. Single agent ATRN-119 demonstrates increased cytotoxicity in vitro against a broad spectrum of human colon, breast, pancreatic, sarcoma, ovarian and prostate cancer cell lines that harbor genomic alterations of DDR genes. In vivo, ATRN-119 has demonstrated tumor growth inhibition in human castration-resistant prostate cancer and human colon cancer cell line-derived xenografts (CDX) mice models, and in BRCA mutant high grade serous ovarian cancer patient derived xenografts (PDXs). Trial design: This is a first-in-human Phase 1/2a open-label study (NCT04905914) to evaluate safety, tolerability, pharmacokinetics (PK), and preliminary e!cacy of oral daily ATRN-119 in subjects with advanced solid tumors, conducted in 2 parts. Part 1 consists of a 3+3 dose escalation design at up to 6 dose levels to evaluate the tolerability, PK, dose limiting toxicity (DLT) and maximum tolerated dose (MTD) of repeated daily doses of ATRN-119. Enrollment in Part 1 is ongoing. Subjects must be ≥12 years old with advanced solid tumor, have documented DDR mutations, or advanced Merkel cell carcinoma, must have failed at least one prior standard treatment, have ECOG performance status of 0 or 1, with a life expectancy > 3 months. Primary objective is safety/tolerability and secondary objective is efficacy. After determination of the MTD or maximum feasible dose (MFD) in Part 1, Part 2 will enroll up to 30 additional subjects. The trial is currently recruiting across 4 locations in the United States. Clinical trial identification: ClinicalTrials.gov Identifier: NCT04905914 Citation Format: Fiona Simpkins, Reva Schneider, Amit Mahipal, Patricia LoRusso, Crystal Miller, Eric Brown, Mike Carleton, Joachim Gullbo, Nadeem Q Mirza. First in Human phase 1/2a trial of a macrocyclic ATR inhibitor (ATRN-119) in patients with advanced solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C034.

Newly developed preclinical models reveal broad-spectrum CDK inhibitors as potent drugs for CRPC exhibiting primary resistance to enzalutamide.

Androgen-deprivation therapy is a standard treatment for advanced prostate cancer. However, most patients eventually acquire resistance and progress to castration-resistant prostate cancer (CRPC). In this study, we established new CRPC cell lines, AILNCaP14 and AILNCaP15, from LNCaP cells under androgen-deprived conditions. Unlike most pre-existing CRPC cell lines, both cell lines expressed higher levels of androgen receptor (AR) and prostate-specific antigen (PSA) than parental LNCaP cells. Moreover, these cells exhibited primary resistance to enzalutamide. Since AR signaling plays a significant role in the development of CRPC, PSA promoter sequences fused with GFP were introduced into AILNCaP14 cells to conduct GFP fluorescence-based chemical screening. We identified flavopiridol, a broad-spectrum CDK inhibitor, as a candidate drug that could repress AR transactivation of CRPC cells, presumably through the inhibition of phosphorylation of AR on the serine 81 residue (pARSer81 ). Importantly, this broad-spectrum CDK inhibitor inhibited the proliferation of AILNCaP14 cells both in vitro and in vivo. Moreover, a newly developed liver metastatic model using AILNCaP15 cells revealed that the compound attenuated tumor growth of CRPC harboring highly metastatic properties. Finally, we developed a patient-derived xenograft (PDX) model of CRPC and DCaP CR from a patient presenting therapeutic resistance to enzalutamide, abiraterone, and docetaxel. Flavopiridol successfully suppressed the tumor growth of CRPC in this PDX model. Since ARSer81 was found to be phosphorylated in clinical CRPC samples, our data suggested that broad-spectrum CDK inhibitors might be a potent candidate drug for the treatment of CRPC, including those exhibiting primary resistance to enzalutamide.

Therapeutic Resistance Models and Treatment Sequencing in Advanced Prostate Cancer.

Current common treatments for castration-resistant prostate cancer (CRPC) typically belong to one of three major categories: next-generation anti-androgen therapies (NGAT) including enzalutamide, abiraterone acetate, apalutamide, and darolutamide; taxane therapy represented by docetaxel; and PARP inhibitors (PARPi) like olaparib. Although these treatments have shown efficacy and have improved outcomes for many patients, some do not survive due to the emergence of therapeutic resistance. The clinical landscape is further complicated by limited knowledge about how the sequence of treatments impacts the development of therapeutic cross-resistance in CRPC. We have developed multiple CRPC models of acquired therapeutic resistance cell sublines from C4-2B cells. These include C4-2B MDVR, C4-2B AbiR, C4-2B ApaR, C4-2B DaroR, TaxR, and 2B-olapR, which are resistant to enzalutamide, abiraterone, apalutamide, darolutamide, docetaxel, and olaparib, respectively. These models are instrumental for analyzing gene expression and assessing responses to various treatments. Our findings reveal distinct cross-resistance characteristics among NGAT-resistant cell sublines. Specifically, resistance to enzalutamide induces resistance to abiraterone and vice versa, while maintaining sensitivity to taxanes and olaparib. Conversely, cells with acquired resistance to docetaxel exhibit cross-resistance to both cabazitaxel and olaparib but retain sensitivity to NGATs like enzalutamide and abiraterone. OlapR cells, significantly resistant to olaparib compared to parental cells, are still responsive to NGATs and docetaxel. Moreover, OlapR models display cross-resistance to other clinically relevant PARP inhibitors, including rucaparib, niraparib, and talazoparib. RNA-sequencing analyses have revealed a complex network of altered gene expressions that influence signaling pathways, energy metabolism, and apoptotic signaling, pivotal to cancer's evolution and progression. The data indicate that resistance mechanisms are distinct among different drug classes. Notably, NGAT-resistant sublines exhibited a significant downregulation of androgen-regulated genes, contrasting to the stable expression noted in olaparib and docetaxel-resistant sublines. These results may have clinical implications by showing that treatments of one class can be sequenced with those from another class, but caution should be taken when sequencing drugs of the same class.

SPOP Mutations Target STING1 Signaling in Prostate Cancer and Create Therapeutic Vulnerabilities to PARP Inhibitor-Induced Growth Suppression.

Speckle-type POZ protein (SPOP) is important in DNA damage response (DDR) and maintenance of genomic stability. Somatic heterozygous missense mutations in the SPOP substrate-binding cleft are found in up to 15% of prostate cancers. While mutations in SPOP predict for benefit from androgen receptor signaling inhibition (ARSi) therapy, outcomes for patients with SPOP-mutant (SPOPmut) prostate cancer are heterogeneous and targeted treatments for SPOPmut castrate-resistant prostate cancer (CRPC) are lacking. Using in silico genomic and transcriptomic tumor data, proteomics analysis, and genetically modified cell line models, we demonstrate mechanistic links between SPOP mutations, STING signaling alterations, and PARP inhibitor vulnerabilities. We demonstrate that SPOP mutations are associated with upregulation of a 29-gene noncanonical (NC) STING (NC-STING) signature in a subset of SPOPmut, treatment-refractory CRPC patients. We show in preclinical CRPC models that SPOP targets and destabilizes STING1 protein, and prostate cancer-associated SPOP mutations result in upregulated NC-STING-NF-κB signaling and macrophage- and tumor microenvironment (TME)-facilitated reprogramming, leading to tumor cell growth. Importantly, we provide in vitro and in vivo mechanism-based evidence that PARP inhibitor (PARPi) treatment results in a shift from immunosuppressive NC-STING-NF-κB signaling to antitumor, canonical cGAS-STING-IFNβ signaling in SPOPmut CRPC and results in enhanced tumor growth inhibition. We provide evidence that SPOP is critical in regulating immunosuppressive versus antitumor activity downstream of DNA damage-induced STING1 activation in prostate cancer. PARPi treatment of SPOPmut CRPC alters this NC-STING signaling toward canonical, antitumor cGAS-STING-IFNβ signaling, highlighting a novel biomarker-informed treatment strategy for prostate cancer.

Discovery of a highly potent and orally available importin-β1 inhibitor that overcomes enzalutamide-resistance in advanced prostate cancer

Nuclear transporter importin-β1 is emerging as an attractive target by virtue of its prevalence in many cancers. However, the lack of druggable inhibitors restricts its therapeutic proof of concept. In the present work, we optimized a natural importin-β1 inhibitor DD1 to afford an improved analog DD1-Br with better tolerability (>25 folds) and oral bioavailability. DD1-Br inhibited the survival of castration-resistant prostate cancer (CRPC) cells with sub-nanomolar potency and completely prevented tumor growth in resistant CRPC models both in monotherapy (0.5 mg/kg) and in enzalutamide-combination therapy. Mechanistic study revealed that by targeting importin-β1, DD1-Br markedly inhibited the nuclear accumulation of multiple CRPC drivers, particularly AR-V7, a main contributor to enzalutamide resistance, leading to the integral suppression of downstream oncogenic signaling. This study provides a promising lead for CRPC and demonstrates the potential of overcoming drug resistance in advanced CRPC via targeting importin-β1.

SRF inhibitors reduce prostate cancer cell proliferation through cell cycle arrest in an isogenic model of castrate-resistant prostate cancer

ABSTRACT Castrate-resistant prostate cancer (CRPC) is challenging to treat, despite improvements with next-generation anti-androgens such as enzalutamide, due to acquired resistance. One of the mechanisms of such resistance includes aberrant activation of co-factors of the androgen receptor (AR), such as the serum response factor (SRF), which was associated with prostate cancer progression and resistance to enzalutamide. Here, we show that inhibition of SRF with three small molecules (CCG-1423, CCG-257081 and lestaurtinib), singly and in combination with enzalutamide, reduces cell viability in an isogenic model of CRPC. The effects of these inhibitors on the cell cycle, singly and in combination with enzalutamide, were assessed with western blotting, flow cytometry and β-galactosidase staining. In the androgen deprivation-sensitive LNCaP parental cell line, a synergistic effect between enzalutamide and all three inhibitors was demonstrated, while the androgen deprivation-resistant LNCaP Abl cells showed synergy only with the lestaurtinib and enzalutamide combination, suggesting a different mechanism of action of the CCG series of compounds in the absence and presence of androgens. Through analysis of cell cycle checkpoint proteins, flow cytometry and β-galactosidase staining, we showed that all three SRF inhibitors, singly and in combination with enzalutamide, induced cell cycle arrest and decreased S phase. While CCG-1423 had a more pronounced effect on the expression of cell cycle checkpoint proteins, CCG-257081 and lestaurtinib decreased proliferation also through induction of cellular senescence. In conclusion, we show that inhibition of an AR co-factors, namely SRF, provides a promising approach to overcoming resistance to AR inhibitors currently used in the clinic.

Abstract B020: BCL2 expression is enriched in AR-independent advanced prostate cancer

Abstract Background The widespread use of highly potent androgen receptor (AR) inhibitors has led to an increasing emergence of AR-independent castration-resistant prostate cancer (CRPC). AR independence, typically driven by loss of AR expression and/or trans-differentiation to a neuroendocrine or basal phenotype, lacks effective therapeutic strategies. The anti-apoptotic protein BCL2 is upregulated in neuroendocrine tumours and has been proposed as a therapeutic target for this subset of advanced prostate cancers. Objective To characterise AR expression in advanced PC, to evaluate its association with BCL2, to elucidate their clinical significance, and to explore whether BCL2 may represent a valid therapeutic target and/or biomarker. Methods AR and BCL2 expression were immunohistochemically scored in two independent CRPC cohorts (431 biopsies from 247 patients), including 62 matched same-patient castration-sensitive PC (CSPC) biopsies. Scores were associated with retrospectively collected clinical data including overall survival (OS) and response to AR-targeting therapy. BCL2 protein expression was also determined in 28 primary prostate samples (19 patients). Transcriptome data from two independent CRPC cohorts were analysed, including for associations between BCL2 expression and gene expression signatures. A patient-derived xenograft-organoid (PDX-O) CRPC model with high expression of BCL2 was treated with multiple BH3 mimetics. Results and limitations We demonstrate that AR protein loss emerges with castration resistance and occurs in subset of metastatic CRPC (mCRPC) (5%) associating with worse OS (51 vs 18 months from CRPC diagnosis, hazard ratio [HR] 5.52 [2.83-10.78], p<0.001). BCL2 protein expression is enriched in AR-negative mCRPC (p<0.001), associating with worse OS (53 vs 19 months from CRPC diagnosis, HR 2.80 [1.40-5.57], p=0.002) and resistance to AR-targeting therapy (≥50% PSA response: 48% vs 13%), irrespective of AR expression status. BCL2 mRNA expression associates with several key signalling pathways known to promote stemness and lineage plasticity, including EMT and IL6/JAK/STAT3. Furthermore, BCL2 is enriched in basal prostate cells, whose expression profile has been linked with aggressive subtypes of mCRPC, including small cell neuroendocrine CPRC. We show that a high BCL2-expressing PDX-O is resistant to BCL2 inhibition, with functional redundancy between the anti-apoptotic BCL2 family proteins. BCLXL and MCL1 are highly expressed in BCL2-positive mCRPC and may drive resistance to BCL2 targeting. Utilising a post-mortem mCRPC biopsy cohort, we show intra-patient heterogeneity in BCL2 expression. These findings highlight the need for combination therapies. Further studies are required to dissect the interaction between AR and BCL2, and to probe the functional role of BCL2 in this subset of mCRPC. Conclusions BCL2-positivity associates with AR-independence, worse OS, and resistance to AR-targeting in CRPC. Combination strategies are required to optimise response to BCL2 inhibition and to combat heterogeneity. Citation Format: Daniel Westaby, Juan M. Jimenez-Vacas, Ines Figueiredo, Claire Pettinger, Bora Gurel, Denisa Bogdan, Jan Rekowski, Lorenzo Buroni, Antje Neeb, Ruth Riisnaes, Mateus Crespo, Susana Miranda, Ana Ferreira, Daniel Nava Rodrigues, George Seed, Claudia Bertan, Maria de los Dolores Fenor de la Maza, Christina Guo, Juliet Carmichael, Rafael Grochot, Khobe Chandran, Andreas Varkaris, Steven P. Balk, Wei Yuan, Suzanne Carreira, Peter S. Nelson, Michael Haffner, Eva Corey, Johann de Bono, Adam Sharp. BCL2 expression is enriched in AR-independent advanced prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B020.

Abstract B007: New generation mTOR blocker sensitizes prostate cancer models to AR-targeting therapy

Abstract Advanced stage prostate cancers (PCa) can develop bone metastasis, a lethal feature of castration-resistant prostate cancer (CRPC) for which no curative options are currently available. Androgen deprivation therapy (ADT) is the main therapeutic strategy for treating advanced PCa; patients with bone metastasis, however, are frequently developing resistance to ADT, progressing to CRPC. Dysregulation of the mTOR pathway has been linked with AR reactivation and with progression to CRPC, and mutations in targets of the PI3K/Akt/mTOR pathway are frequently found in CRPC patients. This study investigates the effect of synergistic AR and mTOR pathways blockade on therapy resistance in advanced PCa in vitro and in vivo. LNCaP and LAPC4 cells were treated in vitro with AR blockers (apalutamide, enzalutamide), mTOR blockers (rapamycin, everolimus, MLN0128 or Rapalink-1) or a combination of both blockers. Viability was measured in a timecourse from 1 to 10 days after drug administration using CellTiter Acqueous 96. To mimick ADT in vitro, cells were cultured for 7 days in charcoal-stripped serum in medium without dihydroxytestosterone or phenol red before the drug screening. Two already established PDX models of bone-metastatic PCa, BM18 and LAPC9 respectively androgen-dependent and -independent, were used to assess the effect of combined ADT and mTOR-targeting therapy in vivo. Both LNCaP and LAPC4 models showed a higher sensitivity to MLN0128 and Rapalink-1 compared to rapalogs, LAPC4 cells in particular were more sensitive than LNCaP cells to mTOR inhibitors. AR blockers were moderately effective and only at concentrations between 10 and 100 µM, when used alone. However, treatment with sublethal doses of Rapalink-1 (0.001-0.01 µM) sensitized both cell lines to 2- to 3-log fold lower concentrations of apalutamide. In vivo, treatment with Rapalink-1 alone significantly reduced the growth rate of LAPC9 PDX, while co-treatment with ADT and Rapalink-1 resulted in regression in this CRPC model, a result not achievable by either treatment alone. Treatment with Rapalink-1, while effectively reducing cell viability in vitro already when used alone, could sensitize the tested models to AR blockers, at clinically relevant concentrations. In vivo treatment with Rapalink-1 was well-tolerated and sufficient to control the growth of the BM18 PDX model. When used in combination with ADT, it could induce regression of the CRPC model LAPC9. Citation Format: Shuang Li, Federico La Manna, Panagiotis Chouvardas, George Thalmann, Marianna Kruithof-de Julio. New generation mTOR blocker sensitizes prostate cancer models to AR-targeting therapy [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B007.

Abstract B003: EVI1 oncogenic role in non-canonical AR driven lethal prostate cancer

Abstract Androgen receptor (AR) remains active in castration-resistant prostate cancer (CRPC), and AR cistrome can become extensively reprogrammed with disease progression. Recent findings from our group and others have shown an association with non-canonical AR signature with resistance to enzalutamide. However, the complete mechanism is not fully revealed. EVI1 belongs to the zinc-finger transcription factor family, and overexpression of EVI1 is associated with poor patient outcomes. However, its role in the context of resistance to AR-targeted therapy remains unclear. Our preliminary data from ChIP-seq and GSEA analysis shows a significant enrichment of EVI1 target gene-set captured from annotated enzalutamide-resistant AR binding sites. Hypothesis: EVI1 cooperates in regulating the non-canonical AR activity associated with resistance to AR targeted therapy and blocking EVI1 may overcome resistance to target therapy. Methods : Using a PDX model of advanced enzalutamide-resistant CRPC, we performed single-cell ATAC-seq + gene expression multi-omic assay. Next, we determined the enrichment of canonical vs. non-canonical AR signatures across cell populations. Using motif analysis and transcription factor foot printing, we determine EVI1 (MECOM) motif z score and enrichment. Results: Our findings demonstrate enrichment of non-canonical AR signatures in distinct population of cells following exposure to enzalutamide. In addition, the subpopulation of cells enriched for non-canonical AR cistrome also exhibited high EVI1 (MECOM) motif z-scores. Conclusion : Our data suggests a potential link between EVI1 and non-canonical AR signaling. Ongoing mechanistic studies will provide insight mechanism by which EVI1 controls non canonical AR activity and its vulnerabilities. Citation Format: Surendra Gulla, Jonathan E. Bard, David J. VanderWeele, Kathleen Kelly, Remi Adelaiye-Ogala. EVI1 oncogenic role in non-canonical AR driven lethal prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B003.

Abstract A058: Lineage plasticity is associated with an altered MAX cistrome through super-enhancer remodeling

Abstract Background: Roughly 20% of late stage prostate cancer patients display a lineage conversion from typical androgen receptor (AR) driven castrate resistant prostate cancer (CRPC) to a particularly lethal neuroendocrine prostate cancer (NEPC) phenotype as a result of AR pathway inhibition (ARPI). Notably, the mutational landscape of CRPC and NEPC is quite similar, suggesting that epigenetic changes may guide this lineage conversion. Our objective was to explore changes in active epigenetic marks with a particular focus on super-enhancers (SEs) and identify factors that regulate these active regions during lineage plasticity. Methods: We performed ChIP-seq for H3K27ac, H3K4me3 and various TFs in our CRPC and ENZ resistant (ENZR) NEPC-like cell models. We leveraged publicly available H3K27ac data in cell lines as well as PDX models to validate our findings. RNA-seq was integrated with our ChIP-seq results to create a signature which we applied to patient data sets. GIGGLE analysis was used to identify candidate factors at SEs. Results: CRPC and NEPC showed similar active marks at promoters however, enhancer elements were significantly remodeled. We observed extensive remodeling of SEs in our ENZR model compared to CRPC and these unique regions showed greater H3K27ac signals in other ENZR models such as NCI-H660. SE linked genes are associated with stemness pathways and are strongly upregulated as a result of ARPI in vitro, and in vivo. Factor enrichment analysis of these regions showed enrichment for MAX and MYC family binding similar to small cell lung cancer cell lines. ChIPseq for MYC and MAX in our cell models revealed an expansion of MAX binding and contraction of MYC binding in our ENZR model compared to CRPC. In particular, we saw increased binding of MAX at plasticity SEs without MYC suggesting an interaction with MYCL or other factors since MYCN is not expressed in our models. Conclusions: This work improves our understanding of the epigenetic changes that occur during lineage plasticity and ARPI resistance. SE analysis in our models suggests that MAX may be activating these regions with MYCL or independently of the MYC family. MYCL expression is upregulated in NEPC and has higher expression than MYCN suggesting a potential role during lineage plasticity. Citation Format: Maxim Kobelev, Dwaipayan Ganguli, Takeshi Namekawa, Amina Talal, Joshua Scurll, Amina Zoubeidi. Lineage plasticity is associated with an altered MAX cistrome through super-enhancer remodeling [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A058.

Abstract A074: In vivo modeling of castration-resistant prostate cancer in the immunodeficient SRG OncoRat

Abstract Prostate cancer is the second most common cause of cancer-related deaths in American men. When locally targeted approaches fail, androgen deprivation therapy is administered to block androgen stimulated prostate cancer growth signals. Most tumors initially exhibit rapid and dramatic regression following androgen deprivation therapy. However, many patients treated with androgen deprivation therapy will have disease recurrence and develop castration-resistant prostate cancer (CRPC), which is highly metastatic with poor survival rates. To improve clinical options for CRPC, pre-clinical animal models that closely recapitulate the human disease course are an essential component of drug development. SRG OncoRats are highly immunocompromised Rag2/Il2rg double knockouts that lack mature B cells, T cells, and circulating NK cells. To establish an in vivo model of CRPC, we inoculated VCaP cells subcutaneously into the hind-flank of SRG OncoRats and monitored tumor growth. On Day 24 rats underwent orchiectomy and tumor growth was further monitored through Day 46. As seen with androgen deprivation therapy in humans, VCaP tumors initially regressed in the SRG OncoRats. However, VCaP tumors resumed growth 14 days after castration, faithfully modeling the progression of CRPC. After tumor regrowth, animals were enrolled into a therapeutic efficacy study that mimics the current clinical challenges in prostate cancer treatment. Citation Format: R. Grace Walton, Diane Begemann, Cynthia Dunn, Valeriya Steffey, Abigail Ross, Razoanul Haque, Fallon K. Noto. In vivo modeling of castration-resistant prostate cancer in the immunodeficient SRG OncoRat [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A074.

Immune checkpoint B7-H3 is a therapeutic vulnerability in prostate cancer harboring PTEN and TP53 deficiencies.

Checkpoint immunotherapy has yielded meaningful responses across many cancers but has shown modest efficacy in advanced prostate cancer. B7 homolog 3 protein (B7-H3/CD276) is an immune checkpoint molecule and has emerged as a promising therapeutic target. However, much remains to be understood regarding B7-H3's role in cancer progression, predictive biomarkers for B7-H3-targeted therapy, and combinatorial strategies. Our multi-omics analyses identified B7-H3 as one of the most abundant immune checkpoints in prostate tumors containing PTEN and TP53 genetic inactivation. Here, we sought in vivo genetic evidence for, and mechanistic understanding of, the role of B7-H3 in PTEN/TP53-deficient prostate cancer. We found that loss of PTEN and TP53 induced B7-H3 expression by activating transcriptional factor Sp1. Prostate-specific deletion of Cd276 resulted in delayed tumor progression and reversed the suppression of tumor-infiltrating T cells and NK cells in Pten/Trp53 genetically engineered mouse models. Furthermore, we tested the efficacy of the B7-H3 inhibitor in preclinical models of castration-resistant prostate cancer (CRPC). We demonstrated that enriched regulatory T cells and elevated programmed cell death ligand 1 (PD-L1) in myeloid cells hinder the therapeutic efficacy of B7-H3 inhibition in prostate tumors. Last, we showed that B7-H3 inhibition combined with blockade of PD-L1 or cytotoxic T lymphocyte-associated protein 4 (CTLA-4) achieved durable antitumor effects and had curative potential in a PTEN/TP53-deficient CRPC model. Given that B7-H3-targeted therapies have been evaluated in early clinical trials, our studies provide insights into the potential of biomarker-driven combinatorial immunotherapy targeting B7-H3 in prostate cancer, among other malignancies.

The CDK7 inhibitor CT7001 (Samuraciclib) targets proliferation pathways to inhibit advanced prostate cancer

BackgroundCurrent strategies to inhibit androgen receptor (AR) are circumvented in castration-resistant prostate cancer (CRPC). Cyclin-dependent kinase 7 (CDK7) promotes AR signalling, in addition to established roles in cell cycle and global transcription, providing a rationale for its therapeutic targeting in CRPC.MethodsThe antitumour activity of CT7001, an orally bioavailable CDK7 inhibitor, was investigated across CRPC models in vitro and in xenograft models in vivo. Cell-based assays and transcriptomic analyses of treated xenografts were employed to investigate the mechanisms driving CT7001 activity, alone and in combination with the antiandrogen enzalutamide.ResultsCT7001 selectively engages with CDK7 in prostate cancer cells, causing inhibition of proliferation and cell cycle arrest. Activation of p53, induction of apoptosis, and suppression of transcription mediated by full-length and constitutively active AR splice variants contribute to antitumour efficacy in vitro. Oral administration of CT7001 represses growth of CRPC xenografts and significantly augments growth inhibition achieved by enzalutamide. Transcriptome analyses of treated xenografts indicate cell cycle and AR inhibition as the mode of action of CT7001 in vivo.ConclusionsThis study supports CDK7 inhibition as a strategy to target deregulated cell proliferation and demonstrates CT7001 is a promising CRPC therapeutic, alone or in combination with AR-targeting compounds.

Computational drug discovery for castration-resistant prostate cancers through in vitro drug response modeling

Prostate cancer (PC) is the most frequently diagnosed malignancy and a leading cause of cancer deaths in US men. Many PC cases metastasize and develop resistance to systemic hormonal therapy, a stage known as castration-resistant prostate cancer (CRPC). Therefore, there is an urgent need to develop effective therapeutic strategies for CRPC. Traditional drug discovery pipelines require significant time and capital input, which highlights a need for novel methods to evaluate the repositioning potential of existing drugs. Here, we present a computational framework to predict drug sensitivities of clinical CRPC tumors to various existing compounds and identify treatment options with high potential for clinical impact. We applied this method to a CRPC patient cohort and nominated drugs to combat resistance to hormonal therapies including abiraterone and enzalutamide. The utility of this method was demonstrated by nomination of multiple drugs that are currently undergoing clinical trials for CRPC. Additionally, this method identified the tetracycline derivative COL-3, for which we validated higher efficacy in an isogenic cell line model of enzalutamide-resistant vs. enzalutamide-sensitive CRPC. In enzalutamide-resistant CRPC cells, COL-3 displayed higher activity for inhibiting cell growth and migration, and for inducing G1-phase cell cycle arrest and apoptosis. Collectively, these findings demonstrate the utility of a computational framework for independent validation of drugs being tested in CRPC clinical trials, and for nominating drugs with enhanced biological activity in models of enzalutamide-resistant CRPC. The efficiency of this method relative to traditional drug development approaches indicates a high potential for accelerating drug development for CRPC.

Abstract 4943: SP-1-39 as a novel tubulin inhibitor overcomes taxane resistance in castration-resistant prostate cancer model

Abstract Colchicine binding site inhibitors have proven to have potent anti-cancer efficacy in many cancer types. Inhibition of tubulin polymerization, a popular mechanism of action for anti-cancer activities, will be activated once the inhibitor interacts with the colchicine binding site. We have previously developed and reported a potent tubulin inhibitor (SB-216). One of its derivatives, the compound SP-1-39, a pyrimidine dihydroquinoxalinone derivative, has been recently designed to further improve the potency and therapeutic properties. Here, we examined the impact of SP-1-39 on preclinical prostate cancer models in vitro and in vivo. EBI competition assay confirmed that SP-1-39 binds to colchicine-binding site and inhibits the formation of EBI:β-tubulin adduct. The anti-proliferation abilities of SP-1-39 was tested against melanoma, breast cancer, pancreatic cancer, prostate cancer, and paclitaxel-resistant prostate cancer cell lines by MTS assay, and defined IC50 values of 0.2-4 nmol/L. Other in vitro results showed SP-1-39 disrupted microtubule network, induced cell cycle arrest at G2/M phase, and caused apoptosis in paclitaxel-resistant human prostate cancer cells (PC-3/TxR). For in vivo study, 2.5mg/kg SP-1-39 significantly attenuated tumor growth in PC-3/TxR xenograft mouse model, inhibited angiogenesis, and overcame taxane resistance. Our findings demonstrate the preclinical therapeutic efficacy and safety of SP-1-39, and provide support for further exploration of novel tubulin inhibitors as anti-cancer agent for cancer therapy. Citation Format: Rui Wang, Kelli L. Hartman, Satyanarayana Pochampally, Duane D. Miller, Wei Li. SP-1-39 as a novel tubulin inhibitor overcomes taxane resistance in castration-resistant prostate cancer model. [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 4943.

Abstract NG10: Chromatin profiles classify castration-resistant prostate cancers suggesting therapeutic targets

Abstract Untreated prostate cancers rely on androgen receptor (AR) signaling for growth and survival, forming the basis for the initial efficacy of androgen deprivation therapy (ADT). Yet the disease can relapse and progress to a lethal stage termed castration-resistant prostate cancer (CRPC). Reactivation of AR signaling represents the most common driver of CRPC growth, and next-generation AR signaling inhibitors (ARSIs) are now used in combination with ADT as first-line therapy. However, ARSIs can result in selective pressure, thereby generating AR-independent tumors. The transition from AR dependence frequently accompanies a change in a phenotype resembling developmental transdifferentiation or “lineage plasticity”. Neuroendocrine prostate cancer, which lacks a defined pathologic classification, is the most studied type of lineage plasticity. However, most AR-null tumors do not exhibit neuroendocrine features and are classified as “double-negative prostate cancer”, the drivers of which are poorly defined. Lineage plasticity studies in CRPC are limited by the lack of genetically defined patient-derived models that recapitulate the disease spectrum. To address this, we developed a biobank of organoids generated from patient biopsies to study the landscape of metastatic CRPC and allow for functional validation assays. Proteins called transcription factors (TFs) are drivers of tumor lineage plasticity. To identify the key TFs that drive the growth of AR-independent tumors, we integrated epigenetic and transcriptomic data generated from CRPC models. We generated ATAC-seq (assay for transposase-accessible chromatin sequencing) and RNA-seq data from 22 metastatic human prostate cancer organoids, six patient-derived xenografts (PDXs), and 12 derived or traditional cell lines. We classified the 40 models into four subtypes and predicted key TFs of each subtype. Besides the well-characterized AR-dependent (CRPC-AR) and neuroendocrine subtypes (CRPC-NE), we identified two novel AR-negative/low groups, including a Wnt-dependent subtype (CRPC-WNT), driven by TCF/LEF TFs, and a stem cell-like (SCL) subtype (CRPC-SCL), driven by the AP-1 family of TFs. To apply the subtype classification to patient samples, we derived RNA-seq signatures from the organoids and applied them to 366 patient samples from two independent CRPC cohorts. The generated signatures recapitulated the four-subtype classification and revealed that CRPC-SCL is the second most prevalent group. Patients from CRPC-SCL are also associated with shorter time under ARSI treatment compared to CRPC-AR, indicating that the ARSI treatments were less effective for CRPC-SCL patients. Additional chromatin immunoprecipitation sequencing (ChIP-seq) analysis indicated that AP-1 (FOSL1) collaboratively binds with TEAD and transcription coactivators, YAP and TAZ. Knocking down of AP-1 (FOSL1), YAP/TAZ decreased cell growth of CRPC-SCL and showed a decrease of chromatin accessibility at CRPC-SCL-specific open chromatin sites and down-regulation of YAP/TAZ target gene expression. In addition, the expression of AP-1 (FOSL1) decreased upon YAP/TAZ knockdown suggesting a positive feedback loop as well as YAP/TAZ as actional targets in CRPC-SCL. We used two small-molecule inhibitors, verteporfin and T-5224, that act on the YAP/TAZ/AP-1 pathway for their potential use as therapeutics for CRPC-SCL tumors, both inhibited the growth of samples from CRPC-SCL but not CRPC-AR. By overexpressing an AP-1 family gene (FOSL1) in AR-high cells, we observed an increase in chromatin accessibility at CRPC-SCL-specific open chromatin sites as well as significant up-regulation of CRPC-SCL signature genes, suggesting that AP-1 functions as a pioneering factor and master regulator for CRPC-SCL. All this work was recently published in Science (Tang, Xu et al. Science, 2022) where I am the co-first author. In summary, by using a diverse biobank of organoids, PDXs, and cell lines that recapitulate the heterogeneity of metastatic prostate cancer, we created a map of the chromatin accessibility and transcriptomic landscape of CRPC. We validated the CRPC-AR and CRPC-NE subtypes and report two novel subtypes of AR-negative/low samples, CRPC-SCL and CRPC-WNT, as well as their respective key TFs. Additional analysis revealed a model in which YAP, TAZ, TEAD, and AP-1 function together and drive oncogenic growth in CRPC-SCL samples. In addition, we proposed small inhibitors of YAP and TAZ that can potentially be used to treat CRPC-SCL patients. Overall, our results show how the stratification of CRPC patients into four subtypes using their transcriptomes can potentially inform appropriate clinical decisions. Citation Format: Fanying Tang, Duo Xu, Shangqian Wang, Chen Khuan Wong, Alexander Martinez-Fundichely, Cindy J. Lee, Sandra Cohen, Jane Park, Corinne E. Hill, Kenneth Eng, Rohan Bareja, Teng Han, Eric Minwei Liu, Ann Palladino, Wei Di, Dong Gao, Wassim Abida, Shaham Beg, Loredana Puca, Maximiliano Meneses, Elisa de Stanchina, Michael F. Berger, Anuradha Gopalan, Lukas E. Dow, Juan Miguel Mosquera, Himisha Beltran, Cora N. Sternberg, Ping Chi, Howard I. Scher, Andrea Sboner, Yu Chen, Ekta Khurana. Chromatin profiles classify castration-resistant prostate cancers suggesting therapeutic targets. [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 NG10.

Abstract 3989: EVI1 oncogenic role in non-canonical AR driven lethal prostate cancer

Abstract Background: Androgen receptor (AR) remains active in castration-resistant prostate cancer (CRPC), and AR cistrome can become extensively reprogrammed with disease progression. Recent findings from our group and others have shown an association with non-canonical AR signature with resistance to enzalutamide. However, the complete mechanism is not fully revealed. EVI1 belongs to the zinc-finger transcription factor family, and overexpression of EVI1 is associated with poor patient outcomes. However, its role in the context of resistance to AR-targeted therapy remains unclear. Our preliminary data from ChIP-seq and GSEA analysis shows a significant enrichment of EVI1 target gene-set captured from annotated enzalutamide-resistant AR binding sites. Hypothesis: EVI1 cooperates in regulating the non-canonical AR activity associated with resistance to AR targeted therapy and blocking EVI1 may overcome resistance to target therapy. Methods: Using a PDX model of advanced enzalutamide-resistant CRPC, we performed single-cell ATAC-seq + gene expression multi-omic assay. Next, we determined the enrichment of canonical vs. non-canonical AR signatures across cell populations. Using motif analysis and transcription factor foot printing, we determine EVI1 (MECOM) motif z score and enrichment. Results: Our findings demonstrate enrichment of non-canonical AR signatures in distinct population of cells following exposure to enzalutamide. In addition, the subpopulation of cells enriched for non-canonical AR cistrome also exhibited high EVI1 (MECOM) motif z-scores. Conclusion: Our data suggests a potential link between EVI1 and non-canonical AR signaling. Ongoing mechanistic studies will provide insight mechanism by which EVI1 controls non canonical AR activity and its vulnerabilities. Citation Format: Surendra Gulla, Jonathan E. Bard, David J. VanderWeele, Kathleen Kelly, Remi Adelaiye-Ogala. EVI1 oncogenic role in non-canonical AR driven lethal 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 3989.

Abstract 1116: Identifying neuroendocrine prostate cancer drivers using in vivo forward genetics

Abstract Prostate cancer (PC) is the malignancy with the highest incidence worldwide in males. Although localized disease can be effectively treated and usually has a favorable prognosis, progression to metastasis results in high mortality rates as current treatment modalities are not yet curative. The standard of care for advanced PC is hormonal therapy, termed Androgen Deprivation Therapy (ADT). Unfortunately, ADT typically elicits the emergence of a lethal phenotype, termed Castration-Resistant Prostate Cancer (CRPC), which is treatment-resistant and frequently gives rise to a highly aggressive variant with features of neuroendocrine (NE) differentiation, known as Neuroendocrine PC (NEPC). There is no cure for NEPC, thus identifying drivers that lead to its emergence is of paramount importance. We used the Sleeping Beauty (SB) transposon system to perform a forward genetic screening on Genetically-Engineered Mouse Models (GEMMs) of CRPC that are known to develop NEPC under ADT pressure. SB transposon activation generated tumors with an accelerated growth phenotype and increased metastasis incidence compared to non-activated controls. Notably, several tumors presented histological features of NEPC. We sequenced SB transposon integration sites on tumors’ genomes to identify recurrent patterns suggesting genes responsible for phenotype acceleration and NEPC differentiation. Recently, we generated and assembled a set of >100 GEMM-derived tumors, that recapitulate a broad spectrum of PC disease states, to reverse-engineer a network of regulatory proteins. We used this network on RNA-Seq data collected from SB-activated tumors to identify Master Regulator (MR) proteins that are specifically associated with NEPC emergence. Cross-species network analysis of patient-specific MRs activity conservation between patient and our mouse cohorts, aligned NEPC patients to mice tumors with NE features, corroborating our study's clinical relevance. We performed an integrative network analysis that links genomic events as generated by SB transposon integrations to downstream regulatory programs implemented by MR proteins. This analysis prioritized Sirt1 as upstream modulator of NEPC MRs activity. Activation of SIRT1 in human adenocarcinoma PC cells induced NE-like phenotype, while SIRT1 inhibitor in NEPC cells reduced tumor aggressiveness. In summary, we used in vivo models of prostate cancer to elucidate molecular drivers of NEPC, a lethal variant that is treatment-resistant. Integrative network data analysis prioritized Sirt1 as driver of NEPC emergence and was validated in vivo. Citation Format: Alessandro Vasciaveo, Francisca Nunes De Almeida, Min Zou, Matteo Di Bernardo, Andrea Califano, Cory Abate-Shen. Identifying neuroendocrine prostate cancer drivers using in vivo forward genetics [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 1116.

Abstract 6046: Enhanced Acod1 expression in cancer cells promotes immune evasion

Abstract The monoclonal antibody αPD-1 used in immune checkpoint blockade (ICB) therapy has made a substantial impact in clinical outcomes for melanoma, lung, and breast cancer patients. Despite the success of ICB in these contexts, such strategies are not universally effective due to unresponsiveness or development of resistance by the malignant cells. Poor understanding of the molecular mechanisms leading to ICB resistance is a crucial barrier to more effective ICB. Recent studies have demonstrated that the metabolic milieu of the tumor microenvironment (TME) can modulate the anti-tumor immune response. However, the specific mechanisms by which the secretion of cancer cell metabolites into the TME impacts the efficacy of ICB in unresponsive tumors remains poorly understood. To better characterize the factors that influence ICB, we utilized a murine model (Smad4−/−/Pten−/−/Trp53−/−) of castration-resistant prostate cancer (CRPC) that is responsive to αPD-1. Serial culture and passage of residual tumor cells following exposure to αPD-1 treatment into new recipient mice allowed us to develop an ICB-insensitive CRPC cell line. Unbiased metabolic analysis revealed that the αPD-1 resistant cells produced elevated levels of itaconate, a metabolite whose biological functions in epithelial cells remain poorly characterized. Consistent with this finding, the resistant cells display increased expression of aconitate decarboxylase 1 (Acod1), the enzyme responsible for itaconate production. Additionally, we have identified established human epithelial cancer cell lines which express high levels of ACOD1. Interestingly, conditioned media (CM) from the Acod1-high cell lines, but not the Acod1-low lines, restricts the proliferation and activation of naïve CD8+ T cells in vitro. CRISPR-mediated Acod1 reduction in the αPD-1 insensitive cells (Acod1KO) produces CM which promotes increased naïve CD8+ T proliferation and activation, suggesting a link between Acod1 abundance in cancer cells and the activity of cytotoxic T cells. Furthermore, Acod1KO tumors have increased CD8+ tumor infiltrating lymphocytes (TILs) and increased sensitivity towards αPD-1 ICB in vivo. Taken together, these data suggest that cancer cell intrinsic ACOD1 abundance may be predictive of ICB efficacy. Citation Format: James H. Schofield, Joseph Longo, Sean Murphy, Ryan Sheldon, Mark A. Hawk, Emma Albano, Russell G. Jones, Xin Lu, Zachary T. Schafer. Enhanced Acod1 expression in cancer cells promotes immune evasion [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 6046.

Abstract 5773: A forward genetic screen identifies SIRT1 as a driver of neuroendocrine prostate cancer

Abstract Neuroendocrine prostate cancer (NEPC) is a deadly variant of prostate cancer for which no cure is yet available. Up to 20 percent of the patients with castration resistant prostate cancer (CRPC), will progress to NEPC after treatment with standard-of-care therapies, such as abiraterone and enzalutamide, that target the androgen receptor (AR) pathway. However, the molecular mechanisms that drive the emergence of NEPC are largely unknown, contributing to a lack of treatment options for patients with NEPC. We implemented a forward genetic approach to identify novel drivers of NEPC, by using the Sleeping Beauty (SB) transposon-based forward genetic mutagenesis screening system in our Pten-, Trp53-deficient mouse model that is primed to develop NEPC at a low frequency. We generated a mouse strain (NPp53-SB) in which the Nkx3.1 promoter regulates the expression of tamoxifen-inducible Cre specifically in the mouse prostate epithelium, resulting in transposition of the T2Onc2 transposable element and conditional deletion of Pten and Trp53 in mouse prostate epithelium. We observed an increased NEPC incidence in our NPp53-SB mouse model compared to control mice without the transposon T2Onc2, suggesting that the SB-mediated insertional mutagenesis events are driving the development of NEPC. To elucidate the genetic events that are driving the observed NEPC phenotype, we identified the common insertion sites (CISs) of the transposon insertions, which revealed a total of 330 CIS-associated genes. We further performed an integrative analysis using RNA-seq data from our SB cohort, a second mouse dataset and a metastatic CRPC patient cohort, which allowed us to identify the Master Regulator (MR) proteins that are associated with NEPC, as well as the upstream genomic events that modulate these MRs and the emergence of NEPC. Our integrative analysis identified the deacetylase Sirt1 as a candidate driver of NEPC. Using CRISPR activation (CRISPRa) technology in vivo we observed that SIRT1 activation induced an NEPC-like phenotype in the adenocarcinoma LNCaP cell line, while Sirt1 drug-mediated inhibition in an NEPC cell line resulted in less aggressive tumors.In summary, we have developed a Sleeping Beauty forward genetic screen in our mouse model of CRPC, which not only accelerates disease progression, but also increases the incidence of NEPC. Our integrative analysis identified a panel of CIS-associated genes that are likely to be functionally involved in progression to NEPC. In particular, Sirt1 was shown to be a promising candidate, which may shed light on mechanisms that this aggressive disease uses to evade treatments and may inform the development of novel therapies. Citation Format: Francisca Nunes de Almeida, Alessandro Vasciaveo, Min Zou, Matteo Di Bernardo, Andrea Califano, Cory Abate-Shen. A forward genetic screen identifies SIRT1 as a driver of neuroendocrine 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 5773.