AbstractPurpose: To assess the efficacy and safety of apalutamide plus goserelin for androgen receptor (AR)–positive unresectable or recurrent/metastatic salivary gland carcinoma. Patients and Methods: This trial was an open-label, single-arm, multicenter phase II study. Patients with histologically confirmed unresectable or recurrent/metastatic salivary gland carcinoma with AR expression were included. The primary endpoint was the overall response rate (ORR) according to RECIST v1.1 by an independent central radiology review in the first 24 response-evaluable (RE) patients who had been observed at least 24 weeks from study initiation (primary RE patients). The efficacy was to be declared when at least 8 of the 24 primary RE patients responded. Results: A total of 31 patients were enrolled. In the first 24 primary RE patients with a median follow-up of 7.4 months, confirmed ORR by independent central radiology review was 25.0% [6/24 patients; 95% confidence interval, 9.8%–46.7%; P = 0.11 (one-sided)], which did not meet the predefined criteria of efficacy. Clinical benefit rate (ORR + rate of stable disease for at least 24 weeks) and median progression-free survival were 50.0% and 7.4 months, respectively. Both median duration of response and overall survival were not reached. Exploratory analyses showed a better ORR of 54.5% (6/11) in patients with AR positivity ≥70% and no history of prior systemic therapy. Grade 3 or higher treatment-emergent adverse events were reported in 35.5% (11/31), which included skin rash, anemia, leukopenia, and cancer pain. Conclusions: Although this study did not meet the predefined efficacy criteria, apalutamide plus goserelin showed clinically meaningful efficacy in a subset of patients with AR-positive salivary gland carcinoma and safety consistent with prior experience in prostate cancer.

FOXA1 is a pioneer transcription factor that is frequently mutated in prostate, breast, bladder, and salivary gland malignancies. Indeed, metastatic castration-resistant prostate cancer (mCRPC) commonly harbour FOXA1 mutations with a prevalence of 35%. However, despite the frequent recurrence of FOXA1 mutations in prostate cancer, the mechanisms by which FOXA1 variants drive its oncogenic effects are still unclear. Semaphorin 3C (SEMA3C) is a secreted autocrine growth factor that drives growth and treatment resistance of prostate and other cancers and is known to be regulated by both AR and FOXA1. In the present study, we characterize FOXA1 alterations with respect to its regulation of SEMA3C. Our findings reveal that FOXA1 alterations lead to elevated levels of SEMA3C both in prostate cancer specimens and in vitro. We further show that FOXA1 negatively regulates SEMA3C via intronic cis elements, and that mutations in FOXA1 forkhead domain attenuate its inhibitory function in reporter assays, presumably by disrupting DNA binding of FOXA1. Our findings underscore the key role of FOXA1 in prostate cancer progression and treatment resistance by regulating SEMA3C expression and suggest that SEMA3C may be a driver of growth and tumor vulnerability of mCRPC harboring FOXA1 alterations.

Supplementary Materials for Apalutamide and Goserelin for Androgen Receptor-Positive Salivary Gland Carcinoma: A Phase 2 Nonrandomized Clinical Trial, YATAGARASU

PDF - 132KB, Effect of AZD5363 on nuclear translocation of AR

Supplementary methods, figure legends, and primer lists.

PDF - 84KB, AZD5363 activity in prostate cancer cell lines in vitro

PDF - 67KB, Cartoon showing development of CRPC and treatment regime

Gene Profiling data of CRPC cells with BRN2 over-expression.

PDF - 138 KB, Effect of AZD5363 on body weight

PDF - 164KB, AZD5363 treatment activates the AR and receptor tyrosine kinases pathways

Supplementary Figure S1. Generation of ENZR Xenografts and Cell Lines. Supplementary Figure S2. AR non-driven ENZR cells display a NE differentiation signature. Supplementary Figure S3. BRN2 expression is associated with PCa progression and a NE phenotype in human tumors. Supplementary Figure S4. BRN2 expression is inversely related to AR activity and is required for ENZ-induced NE marker expression in CRPC. Supplementary Figure S5. BRN2 overexpression induces NE marker expression and reciprocally regulates AR. Supplementary Figure S6. BRN2 activity and markers of NE differentiation are suppressed by androgens in CRPC and ENZR cells. Supplementary Figure S7. SOX2 expression is induced by ENZ and suppressed by androgens. Supplementary Figure S8. BRN2 is required for neuroendocrine marker expression and aggressive growth of CRPC cells in vitro.

Treatment with androgen receptor pathway inhibitors (ARPIs) in prostate cancer leads to the emergence of resistant tumors characterized by lineage plasticity and differentiation toward neuroendocrine lineage. Here, we find that ARPIs induce a rapid epigenetic alteration mediated by large-scale chromatin remodeling to support activation of stem/neuronal transcriptional programs. We identify the proneuronal transcription factor ASCL1 motif to be enriched in hyper-accessible regions. ASCL1 acts as a driver of the lineage plastic, neuronal transcriptional program to support treatment resistance and neuroendocrine phenotype. Targeting ASCL1 switches the neuroendocrine lineage back to the luminal epithelial state. This effect is modulated by disruption of the polycomb repressive complex-2 through UHRF1/AMPK axis and change the chromatin architecture in favor of luminal phenotype. Our study provides insights into the epigenetic alterations induced by ARPIs, governed by ASCL1, provides a proof of principle of targeting ASCL1 to reverse neuroendocrine phenotype, support luminal conversion and re-addiction to ARPIs.

The increased incidence of treatment-emergent neuroendocrine prostate cancer (NEPC) is particularly alarming as this diagnosis is associated with poor prognosis. Despite initial responses to platinum-based chemotherapy, relapses are common and there is no effective second line therapy for NEPC. We previously identified that neuronal transcription factor BRN2 (POU3F2) is a potent driver of neuroendocrine differentiation and an attractive target for NEPC. Utilizing a combination of in silico modeling and X-ray crystallography followed by structure-based lead optimization, we have developed the first potent, specific and orally bioavailable BRN2 inhibitor (B18-94), which inhibits the interaction between BRN2 and DNA. This loss of BRN2 on the chromatin drastically reduces its transcriptional output resulting in downregulation of several known targets in NEPC such as SOX2, ASCL1 and PEG10 . Additionally, B18-94 reduces specifically cell proliferation specifically in multiple NEPC models with no effect on adenocarcinoma and other BRN2 negative prostate cancer models. Importantly, the consistency in the transcriptomic changes driven by B18-94 and or CRISPR/Cas9 mediated BRN2 knockout confirmed the on-target specificity, with both methods of BRN2 inhibition downregulating pathways involved in cellular plasticity and proliferation. Finally, we have demonstrated that B18-94, the first-in-field POU-domain transcription factor inhibitor, significantly reduced tumor growth in several NEPC xenograft models with no observable toxicity, suggesting potential for therapeutic intervention of NEPC.

Cancers adapt to increasingly potent targeted therapies by reprogramming their phenotype. Here we investigated such a phenomenon in prostate cancer, in which tumours can escape epithelial lineage confinement and transition to a high-plasticity state as an adaptive response to potent androgen receptor (AR) antagonism. We found that AR activity can be maintained as tumours adopt alternative lineage identities, with changes in chromatin architecture guiding AR transcriptional rerouting. The epigenetic regulator enhancer of zeste homologue 2 (EZH2) co-occupies the reprogrammed AR cistrome to transcriptionally modulate stem cell and neuronal gene networks—granting privileges associated with both fates. This function of EZH2 was associated with T350 phosphorylation and establishment of a non-canonical polycomb subcomplex. Our study provides mechanistic insights into the plasticity of the lineage-infidelity state governed by AR reprogramming that enabled us to redirect cell fate by modulating EZH2 and AR, highlighting the clinical potential of reversing resistance phenotypes.

Abstract Introduction: Resistance to newly developed androgen receptor pathway inhibitors (ARPIs), such as Enzalutamide (ENZ), rapidly emerges. In particular, a subset of patients who relapse following ARPI therapy their dependence on AR signaling and emerge with neuroendocrine features. These tumors, termed treatment induced small-cell prostate cancer (t-SCPC) or neuroendocrine prostate cancer (t-NEPC), carry an extremely poor prognosis and, to date, treatment remains decades old cytotoxic chemotherapies. Previously, our group identified the neural transcription factor BRN2 as a major clinically relevant driver of SCPC and now report that targeting BRN2 is a promising strategy to prevent neuroendocrine differentiation or treat NEPC.Methods/Results: In silico screening of small molecules was conducted on a model of BRN2 which was later validated with the first-in-field crystal structure of BRN2 DNA binding domain. On the basis of the model, several small molecules were identified that showed direct binding to BRN2 and inhibited its activity. Pharmacokinetic studies measured stability and bioavailability of med-chem optimized lead compound (BRN2i) that significantly reduced tumor growth in multiple xenograft models with no measurable side-effects. In silico modeling predicted a 7Å “closing” in the DBD once it was bound to BRN2i, this shift translated to reduced interaction with DNA by chromatin fractionation and ChIP-seq, thus confirming the mode of action for BRN2i is through loss of DNA binding. Loss of BRN2 binding reduced cell proliferation in tSCPC cell line 42DENZR, de novo SCPC cell line NCI-H660 and NEPC organoids as well as downregulated several known targets like EZH2, ASCL1, SOX2 and PEG10. Down-regulation of these target genes was also measured in the xenograft tumors, confirming on target effect in vivo. Moreover, the specificity of BRN2i was validated with CRISPR/Cas9 mediated knockout of BRN2 with downstream mRNA expression and phenotypic changes. Conclusion: The described work aims to lay the pre-clinical foundation for the integration of BRN2 targeted therapies into the treatment landscape to improve survival for patients suffering from small-cell neuroendocrine prostate cancer. Citation Format: Daksh Thaper, Ravi Munuganti, Adeleke Aguda, Soojin Kim, Shungyu Ku, Sahil Kumar, Sepideh Vahid, Shaghayegh Nouruzi, Olena Sivak, Dwaiyapan Ganguli, Colm Morrissey, Eva Corey, Himisha Beltran, Amina Zoubeidi. Selective inhibition of transcription factor BRN2 as a treatment strategy for Small Cell Prostate Cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3083.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-dependent transcription factor belonging to the type II nuclear receptor family. PPARγ overexpression and activation are important hallmarks of the luminal subtype of muscle invasive bladder cancer (MIBC), where PPARγ has been ascribed an oncogenic role and has been associated with immune exclusion. There is therefore an important need to better understand the mechanisms that regulate PPARγ. A high throughput genome-wide CRISPR knock-out screen in a luminal MIBC cell line was performed to identify endogenous regulators of PPARγ expression. Using CRISPR knock-in technology, the cells were engineered to express GFP and PPARγ proportionally. The top candidates were then individually validated by RNAi and CRISPR gene knockout for their ability to alter PPARγ mRNA and protein levels. The screen highlighted 47 potential regulators of PPARγ expression with a false discovery rate below 1%. These can be grouped in functional clusters of transcription factors and chromatin remodeling enzymes currently known for orchestrating cellular oxidative stress response, detoxification from xenobiotic chemicals or general gene transcription regulation. In this study we developed a powerful screening tool for the characterization of novel factors involved in the expression of key bladder cancer players. In particular, our results revealed the intricated regulatory mechanisms of PPARγ expression exploited by luminal MIBC, thus highlighting the importance of fine-tuning of this nuclear receptor in the biology of the disease.

Bladder cancer is among the top ten most common cancer types in the world, with approximately 550,000 new cases annually. The highest burden of bladder cancer is currently on most developed communities across the world and an estimated 9,000 Canadians are diagnosed with bladder cancer each year. Cisplatin-based Neoadjuvant chemotherapy (NAC) followed by radical cystectomy in patients with muscle-invasive bladder cancer (MIBC) has been shown to improve five-year survival, and is therefore currently the first-line standard of care in patients. However, 60% of patients are inherently resistant to NAC at the time of cystectomy. While several mechanisms of cellular resistance to cisplatin have been proposed, the mechanisms presented thus far still do not offer and effective patient response prediction in the context of MIBC. There is therefore, an urgent and unmet need to determine clinically actionable mechanisms of cisplatin resistance. In order to elucidate mechanisms of resistant to cisplatin, the study presented in this thesis takes advantage of a pooled genome‐wide CRISPR knock‐out library targeting 19,114 protein coding genes with 76,441 synthetic guide RNAs (sgRNAs) which allows for an unbiased screen. Upon completion of the screen the top hit was validated in vitro (CRISPR knockout cell lines), in vivo (mouse models) and in patient tumour tissue samples by immunohistochemistry. A full-scale screen revealed that several genes involved in the pro-apoptotic pathway (such as CASP8, BAX, and TNFSFR10A) and cell cycle regulation have the potential to confer resistance to cisplatin when knocked out. For this study however, we validated the top hit from our screen - Schlafen 11 (SLFN11) - and established that the complete loss of SLFN11 confers a cisplatin resistant phenotype in MIBC. We further established that SLFN11 is involved in the regulation of cell cycle progression upon cisplatin challenge and does so via interactions with Mediator of DNA Damage Checkpoint 1 (MDC1) protein. Overall, the study presented here offers SLFN11 as a potential biomarker to aid in clinical decision making and to anticipate resistance to cisplatin-based NAC in MIBC. Furthermore, targeting SLFN11 associated pathways could allow for the development of combination therapies to be used in conjunction with cisplatin in the future.

Abstract Therapies targeting tumor-fueling androgens have been mainstay treatments of advanced prostate cancer for almost 5 decades. While the androgen receptor (AR) inhibitor enzalutamide (ENZ) prolongs survival of castration-resistant prostate cancer patients (CRPC), ENZ-resistant (ENZR) tumors rapidly recur. Our laboratory has identified that ENZ resistance can occur both in the presence and absence of continued classical AR activity. Lack of AR activity is clinically relevant, as it has been estimated that up to 25% of men who die from advanced CRPC have non-AR driven disease and lethal AR inactive neuroendocrine and anaplastic phenotypes of CRPC emerge after hormone therapy. Recently our group identified the neural transcription factor BRN2 as a major clinically relevant driver of neuroendocrine prostate cancer (NEPC). Hence, using the integrated power of computational drug discovery platform and biologic testing, we identified first-in-field inhibitors for BRN2. First, a homology model of BRN2 protein was generated and used as an input to perform large-scale virtual screening and virtual hits were tested in a series of experiments. Cpd 18-14, a lead BRN2 inhibitor, exhibited profound inhibition of BRN2 in transcriptional assay and binding to the BRN2 in cellular and in vitro assays. Furthermore, Cpd18-14 reduced the expression of neuroendocrine genes SOX2 and NCAM1. Importantly, Cpd18-14 displayed antiproliferative activity specifically in patient-derived BRN2hi NCI-H660 and in house-developed 42DENZRcells while displaying no effect on BRN2low 16DCRPC and LNCaP cells. Further, the most stable binding orientation of Cpd18-14 was determined using molecular dynamics simulations and was used to carry out structure-based lead optimization. As a result, we developed several synthetic derivatives with significant improvement in antiproliferative activity and metabolic stability. Finally, Cpd18-94 reduced the growth of NCI-H660 and 42DENZR NEPC tumors significantly in xenograft models. We anticipate that the developed drug prototypes will lay a foundation for the development of small-molecule therapies capable of combating highly aggressive and lethal form of neuroendocrine tumors. Citation Format: Ravi Munuganti, Daksh Thaper, Adeleke Aguda, Soojin Kim, Olena Sivak, Amina Zoubeidi. Computer-aided discovery of small-molecule inhibitors targeting neural transcription factor BRN2 in neuroendocrine prostate tumors [abstract]. In: Proceedings of the AACR Special Conference on Advancing Precision Medicine Drug Development: Incorporation of Real-World Data and Other Novel Strategies; Jan 9-12, 2020; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_1):Abstract nr 14.

Progression to the malignant state is fundamentally dependent on transcriptional regulation in cancer cells. Optimum abundance of cell cycle proteins, angiogenesis factors, immune evasion markers, etc. is needed for proliferation, metastasis or resistance to treatment. Therefore, dysregulation of transcription factors can compromise the normal prostate transcriptional network and contribute to malignant disease progression.The androgen receptor (AR) is considered to be a key transcription factor in prostate cancer (PCa) development and progression. Consequently, androgen pathway inhibitors (APIs) are currently the mainstay in PCa treatment, especially in castration-resistant prostate cancer (CRPC). However, emerging evidence suggests that with increased administration of potent APIs, prostate cancer can progress to a highly aggressive disease that morphologically resembles small cell carcinoma, which is referred to as neuroendocrine prostate cancer (NEPC), treatment-induced or treatment-emergent small cell prostate cancer. This chapter will review how neuronal transcription factors play a part in inducing a plastic stage in prostate cancer cells that eventually progresses to a more aggressive state such as NEPC.

No targeted therapies exist against aggressive neuroendocrine tumors; hence, these patients are limited to platinum-based chemotherapy that has not advanced in over three decades. [...]