Driver Of Castration-resistant Prostate Cancer Research Articles

Abstract A043: CDK8/19-regulated transcriptional reprogramming: a druggable driver of castration-resistant prostate cancer

Abstract Androgen deprivation therapy (ADT) is the mainstay of prostate cancer (PCa) treatment. However, most patients with aggressive PCa become resistant to ADT and develop castration-resistant prostate cancer (CRPC). Newer androgen receptor (AR)-targeting CRPC treatments extend survival by only a few months, and metastatic CRPC (mCRPC) remains an incurable disease. Multiple mechanisms of ADT resistance have been identified, among which non-genetic (transcriptional) mechanisms are prominent, due to the high transcriptomic plasticity of PCa and the role of tumor microenvironment (TME) as a determinant of ADT resistance. PCa progression involves changes in the expression of CDK8 and CDK19, enzymatic components of the cyclin-dependent kinase module (CKM), associated with the transcriptional Mediator complex and implicated in transcriptional reprogramming and immune surveillance. We have investigated the roles of CDK8/19 Mediator kinases in CRPC, through bioinformatic analysis of primary PCa and mCRPC and by analyzing the effects of genetic and pharmacological inhibition of CDK8 and CDK19 on tumor growth and gene expression. CDK8 and CDK19 were found to be differentially regulated by androgen, which downregulates CDK8 and upregulates CDK19. Accordingly, CDK8 is decreased and CDK19 increased in primary PCa, where CDK19 is expressed higher than in any other tissues or tumors. However, both CDK19 and CDK8, as well as the other components of the CKM, are upregulated in mCRPC, concordantly with the changes in AR-mediated transcriptional signaling. Selective Mediator kinase inhibitors did not suppress PCa models driven by canonical AR signaling but inhibited in vivo growth of cell line-based and patient-derived xenograft (PDX) models of CRPC. Genetic inactivation of CDK8 and CDK19 suppressed 22Rv1 CRPC tumors in surgically or chemically castrated (but not intact) male NSG mice, whereas the expression of CDK19 or CDK8 reversed this phenotype. Transcriptomic analysis revealed that Mediator kinase inhibition exerted the strongest effects on gene expression in tumors growing in castrated mice, increasing the transcriptomic effects of castration on most of the genes regulated by CDK8/19, while decreasing its effects on some of the genes implicated in PCa progression. A gene signature reflecting Mediator kinase activity in CRPC xenografts correlated with shorter overall survival among mCRPC patients. Systemic treatment with a CDK8/19 inhibitor also affected stromal gene expression; many of these effects were reproduced by Mediator kinase mutagenesis in tumor cells alone, suggesting that Mediator kinases of tumor cells are involved in shaping the TME. Prolonged (up to 300 days) CDK8/19 inhibition induced not only growth inhibition but also shrinkage of CRPC tumors in nude mice (which contain NK cells that are stimulated by CDK8/19 inhibition) and even produced cures in 18-27% of animals. These results warrant the exploration of CDK8/19 Mediator kinase inhibitors for the treatment of the presently incurable CRPC. Citation Format: Jing Li, Thomas Hilimire, Yueying Liu, Lili Wang, Jiaxin Liang, Balazs Gyorffy, Vitali Sikitzhytski, Hao Ji, Li Zhang, Chen Cheng, Xiaokai Ding, Kendall Kerr, Charles Dowling, Gary Schools, Chang-Uk Lim, Eugenia Broude, George Wilding, Michael Lilly, Igor Roninson, Mengqian Chen. CDK8/19-regulated transcriptional reprogramming: a druggable driver of castration-resistant prostate cancer [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 A043.

Abstract A070: Determining gene expression regulated by androgen receptor variant 7 in co-loss of MAP3K7/CHD1 prostate cancer

Abstract Prostate cancer (PCa) is the second leading cancer diagnosis in men. The ACS estimates 268,490 new cases of PCa and 34,500 deaths in 2022. Most patients with local PCa are disease free at the 5-year mark. Patients with metastatic PCa only have a 31% 5-year disease-free survival rate. While these patients may initially respond to androgen deprivation therapy, many will progress to castration-resistant prostate cancer (CRPC) and fail treatment. There is a need to develop new therapies to treat CRPC. The Cramer lab studies the dual deletion of MAP3K7 and CHD1 PCa subtype. In MAP3K7/CHD1-depleted PCa cell lines the ratio of AR splicing variants shifts to enrich for the constitutively active variant AR-v7, a known driver of CRPC. Other studies demonstrated that AR-v7 has unique target genes and can alter canonical AR target gene expression. I hypothesize that gain of AR-v7 signaling activates atypical gene targets which alter the gene expression of MAP3K7/CHD1-depleted PCa. I will determine the chromatin-binding targets and identify AR-v7 specific candidate genes. Patients with loss of MAP3K7 and CHD1 also enrich in the spliceosome protein U2AF2, therefore I will also perform transcriptomics with/without AR-FL and AR-v7 to characterize the larger impacts of AR-variant signaling at the RNA level. To this end, I will develop an inducible knockdown system in dual shMAP3K7/shCHD1 cells targeting AR-FL, AR-v7, and AR-Total. This will provide insight into the role of AR-v7 in two stages of gene expression and identify candidate therapeutic genes/pathways in loss of MAP3K7 and CHD1 PCa. Citation Format: Claire Gillette, Lauren Jillson, Scott Cramer. Determining gene expression regulated by androgen receptor variant 7 in co-loss of MAP3K7/CHD1 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 A070.

Mechanistic Investigation of the Androgen Receptor DNA-Binding Domain and Modulation via Direct Interactions with DNA Abasic Sites: Understanding the Mechanisms Involved in Castration-Resistant Prostate Cancer.

The androgen receptor (AR) is an important drug target in prostate cancer and a driver of castration-resistant prostate cancer (CRPC). A significant challenge in designing effective drugs lies in targeting constitutively active AR variants and, most importantly, nearly all AR variants lacking the ligand-binding domain (LBD). Recent findings show that an AR's constitutive activity may occur in the presence of somatic DNA mutations within non-coding regions, but the role of these mutations remains elusive. The discovery of new drugs targeting CRPC is hampered by the limited molecular understanding of how AR binds mutated DNA sequences, frequently observed in prostate cancer, and how mutations within the protein and DNA regulate AR-DNA interactions. Using atomistic molecular dynamics (MD) simulations and quantum mechanical calculations, we focused our efforts on (i) rationalising the role of several activating DBD mutations linked to prostate cancer, and (ii) DBD interactions in the presence of abasic DNA lesions, which frequently occur in CRPC. Our results elucidate the role of mutations within DBD through their modulation of the intrinsic dynamics of the DBD-DNA ternary complex. Furthermore, our results indicate that the DNA apurinic lesions occurring in the androgen-responsive element (ARE) enhance direct AR-DNA interactions and stabilise the DBD homodimerisation interface. Moreover, our results strongly suggest that those abasic lesions may form reversible covalent crosslinks between DNA and lysine residues of an AR via a Schiff base. In addition to providing an atomistic model explaining how protein mutations within the AR DNA-binding domain affect AR dimerisation and AR-DNA interactions, our findings provide insight into how somatic mutations occurring in DNA non-coding regions may activate ARs. These mutations are frequently observed in prostate cancer and may contribute to disease progression by enhancing direct AR-DNA interactions.

Abstract 425: Selective ubiquitination of androgen receptor and its splice variants inhibits castration resistant prostate cancer

Abstract Introduction: The primary driver of castration-resistant prostate cancer (CRPC) is the Androgen receptor (AR). Drugs such as Abiraterone or Enzalutamide that target the Ligand Binding Domain (LBD) of AR are largely ineffective on AR splice variants (AR-SVs) that lack LBD and retain the N-terminal Domain (NTD) of AR. AR continues to function through other stimuli (ex: growth factors), leading to disease relapse and progression. Moreover, AR-SVs are known to heterodimerize with AR full length (AR-FL). Hence, treatment strategies that target both AR-FL and AR-NTD are needed for the complete remission of CRPC. Through structure-activity relationship (SAR) studies based on naturally occurring Urolithin A (UroA) structure, we have developed a novel small molecule UroBoc that directly binds to the NTD and inhibits CRPC growth. Methods: To analyze the binding of UroBoc to AR-NTD, we performed differential scanning fluorimetry and measured saturation transfer difference (STD) in NMR spectroscopic studies. In addition, we investigated the effect of UroBoc on CRPC by western blotting, immunofluorescence, Immunoprecipitation, and performed Xenograft studies in castrated/non-castrated mice models. Results: UroBoc treatment shifted the melt temperature (ΔTm) of purified NTD by 12-15 °C (median of 13.5 °C) in differential scanning fluorimetry. Moreover, STD effects observed were 30-18%, indicating a significant binding to NTD in our NMR studies. UroBoc inhibits the growth of AR+ CRPC, including enzalutamide resistant cells, at 800nM concentration, which is 40 times lower than that of the parent compound, UroA. At this concentration, UroBoc is non-toxic to normal prostate epithelial cells or other cancer cells. Interestingly, UroBoc also inhibited AR, AR-SV and PSA expressions in AR-FL (LNCaP, C42B) and AR-V7 (22-RV1) CRPC cell lines. Molecular studies suggested that UroBoc selectively promotes ubiquitination and subsequent degradation of AR and AR-V7 by proteasome activation. Remarkably, UroBoc failed to inhibit AKT, mTOR, PTEN and hormonal receptors such as estrogen and progesterone receptors suggesting AR could be a direct target for UroBoc. Further, in vivo studies revealed that oral administration of UroBoc inhibits tumor growth in CRPC in both castrated and non-castrated mice models. Conclusion: Our results suggest that the novel UroBoc selectively degrades AR, AR-SV and overcomes enzalutamide resistance in preclinical CRPC models. We believe performing PK/PD studies will confirm the translation potential of UroBoc as a therapy for CRPC. Citation Format: Ashish Tyagi, Balaji Chandrasekaran, Uttara Saran, Murali K. Ankem, Nikolay Dokholyan, Jyh M. Lin, Arun K. Sharma, Chendil Damodaran. Selective ubiquitination of androgen receptor and its splice variants inhibits castration resistant 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 425.

Role of the runt-related transcription factor (RUNX) family in prostate cancer.

Prostate cancer (PCa) is a very complex disease that is a major cause of death in men worldwide. Currently, PCa dependence on the androgen receptor (AR) has resulted in use of AR antagonists and antiandrogen therapies that reduce endogenous steroid hormone production. However, within two to three years of receiving first-line androgen deprivation therapy, the majority of patients diagnosed with PCa progress to castration-resistant prostate cancer (CRPC). There is an urgent need for therapies that are more durable than antagonism of the AR axis. Studies of runt-related transcription factors (RUNX) and their heterodimerization partner, core-binding factor subunit b (CBFβ), are revealing that the RUNX family are drivers of CRPC. In this review, we describe what is presently understood about RUNX members in PCa, including what regulates and is regulated by RUNX proteins, and the role of RUNX proteins in the tumor microenvironment and AR signaling. We discuss the implications for therapeutically targeting RUNX, the potential for RUNX as PCa biomarkers, and the current pressing questions in the field.

P38 MAPK Inhibition Mitigates Hypoxia-Induced AR Signaling in Castration-Resistant Prostate Cancer.

Simple SummaryProgression of prostate cancer to a castration-resistant state is associated with poor patient outcomes, and new therapeutic targeting approaches are needed. Poorly oxygenated (hypoxic) cancer cells are resistant to many treatment modalities, and it is therefore important that novel therapies also target these cells. Here we show that targeting the p38 MAPK protein kinase can inhibit growth and survival of both well-oxygenated and hypoxic castration resistant prostate cancer cells and prolong survival of tumor bearing mice. p38 MAPK targeting inhibited phosphorylation of the chaperone protein Hsp27 and activity of the androgen receptor. This demonstrates that prostate cancer cells can remain dependent on the p38 MAPK/Hsp27 signaling axis upon progression to castration-resistance, and that hypoxia does not offer protection against targeting this pathway.Background: Aberrant androgen receptor (AR) signaling is a major driver of castration-resistant prostate cancer (CRPC). Tumor hypoxia increases AR signaling and is associated with treatment resistance in prostate cancer. Heat shock protein 27 (Hsp27) is a molecular chaperone that is activated in response to heat shock and hypoxia. Hsp27 has previously been reported to facilitate AR nuclear translocation in a p38 mitogen-activated protein kinase (MAPK) dependent manner in castration-sensitive prostate cancer cell lines. Here, we evaluated the potential for inhibiting p38 MAPK/Hsp27 mediated AR signaling under normoxia and hypoxia in experimental models of CRPC. Methods: We inhibited p38 MAPK with SB203580 in prostate cancer cell lines and measured Hsp27 phosphorylation, AR activity, cell proliferation, and clonogenicity under normoxia and hypoxia. AR activity was measured using an androgen response element driven reporter assay and qPCR to measure expression of AR target genes. Xenograft-bearing mice were treated with SB203580 to measure tumor growth and serum prostate specific antigen (PSA). Results: Our results indicate that p38 MAPK and Hsp27 are activated under normoxia and hypoxia in response to androgens in CRPC cells. p38 MAPK inhibition diminished Hsp27 activation and the hypoxia-mediated increase in AR activity. Additionally, inhibition of p38 MAPK activity decreased proliferation and survival of CRPC cells in vitro and prolonged the survival of tumor-bearing mice. Conclusions: These results suggest that p38 MAPK inhibition may represent a therapeutic strategy to disrupt AR signaling in the heterogeneous CRPC tumor microenvironment.

2,4-dienoyl-CoA reductase regulates lipid homeostasis in treatment-resistant prostate cancer

Despite the clinical success of Androgen Receptor (AR)-targeted therapies, reactivation of AR signalling remains the main driver of castration-resistant prostate cancer (CRPC) progression. In this study, we perform a comprehensive unbiased characterisation of LNCaP cells chronically exposed to multiple AR inhibitors (ARI). Combined proteomics and metabolomics analyses implicate an acquired metabolic phenotype common in ARI-resistant cells and associated with perturbed glucose and lipid metabolism. To exploit this phenotype, we delineate a subset of proteins consistently associated with ARI resistance and highlight mitochondrial 2,4-dienoyl-CoA reductase (DECR1), an auxiliary enzyme of beta-oxidation, as a clinically relevant biomarker for CRPC. Mechanistically, DECR1 participates in redox homeostasis by controlling the balance between saturated and unsaturated phospholipids. DECR1 knockout induces ER stress and sensitises CRPC cells to ferroptosis. In vivo, DECR1 deletion impairs lipid metabolism and reduces CRPC tumour growth, emphasizing the importance of DECR1 in the development of treatment resistance.

Abstract 395: Aberrant HER2 signaling is a therapeutic target in a subset of castration-resistant prostate cancer

Abstract Background: Aberrant HER2 signaling promotes androgen receptor activity and is associated with 18-25% of castration-resistant prostate cancer (CRPC). We present preclinical and clinical data supporting signaling by canonical HER3/HER2 interaction and a constitutively active splice variant of HER2 (d16HER2) as potential oncogenic drivers of CRPC. Further, we provide preclinical evidence that increased HER2 signaling may be targeted with irreversible HER2-targeted tyrosine kinase inhibitors (TKIs) to block tumor growth. Methods: Human-derived VCaP prostate cancer xenografts were serially biopsied prior to castration, at castration-resistance, and after resistance to abiraterone + enzalutamide (Abi/Enza). Biopsies were submitted for RNA sequencing and RT-PCR, immunohistochemistry (IHC), and reverse-phase protein array (RPPA) for validation of gene/protein expression changes. Castration-resistant VCaP and LuCaP-70CR xenografts were treated with reversible EGFR/HER2 TKI lapatinib (100 mg/kg) or irreversible pan-HER TKIs afatinib (20 mg/kg) or neratinib (20 mg/kg). Transcript levels of total HER2 and d16HER2 variant were determined for TCGA and SU2C prostate cancer (PCa) datasets. IHC for phospho-HER2 (Tyr1221/1222) and phospho-HER3 (Tyr1289) were performed on tumor samples from 49 heavily-treated metastatic CRPC (mCRPC) patients and compared to 18 hormone-naïve prostatectomy samples. Results: VCaP xenografts exhibit increased activation of HER3/HER2 signaling and increased expression of d16HER2 with progression through castration and Abi/Enza therapy. Increases in pHER2 and pHER3 were observed by IHC and RPPA in serial biopsies of individual tumors, and increases in total HER2, wtHER2, and d16HER2 transcripts were observed by RT-PCR. Castration-resistant VCaP and LuCaP-70CR xenografts rapidly become resistant to lapatinib within 5 days, while the irreversible pan-HER TKIs inhibited tumor growth for at least 15 days. Analysis of TCGA (mostly hormone-naïve PCa) and SU2C (mCRPC) RNAseq datasets shows enrichment of tumors expressing d16HER2 in mCRPC. pHER3/pHER2 IHC in patient tumor specimens showed 9/49 (18%) mCRPC tumors exhibited concurrent pHER3/pHER2 staining compared to 1/18 (6%) of hormone-naïve PCa. An additional 4/49 (8%) mCRPC tumors stained positive for pHER2 alone, possibly representing d16HER2 activity. Conclusions: HER2 overexpression and d16HER2 expression are associated with PCa progression, castration-resistance, and resistance to second-line hormonal therapy. HER2-targeted therapy with irreversible TKIs is more effective at inhibiting CRPC growth than the reversible TKI lapatinib. Our data indicate a subset of CRPC with aberrant HER2 signaling may be clinically actionable, and further studies to investigate potential treatment with an irreversible pan-HER TKI such as neratinib in biomarker-selected metastatic CRPC patients is warranted. Citation Format: Joshua W. Russo, Xin Gao, David J. Einstein, Glenn J. Bubley, Steven P. Balk. Aberrant HER2 signaling is a therapeutic target in a subset of castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 395.

Abstract 1510: Androgen regulated IL-8 expression in prostate cancer: Insights into tumor cell mediated immunosuppression

Abstract Androgen deprivation therapy (ADT) results in castration-resistant prostate cancer (CRPC) in a significant fraction of patients. We have previously reported that the protein levels of interleukin-8 (IL-8) were inversely correlated with disease progression in men with biochemical recurrent prostate cancer treated with Lenalidomide. Recently, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) were implicated as potential drivers of CRPC. Here we show that IL-8 expression is upregulated as a consequence of ADT and mediates the recruitment of PMN-MDSCs to the tumor microenvironment. We found that IL-8 expression is regulated by both an inflammatory stimulus (NF-kβ mediated) and loss of androgen receptor (AR) signaling following ADT. We confirmed direct binding of both the p65 subunit of NF-kβ and AR to the IL-8 promoter, and their respective effects on promoter activity. The suppressive activity of AR was further supported by a reduction in active transcription markers at the chromatin level surrounding the IL-8 promoter. Accordingly, intratumoral infiltration of PMN-MDSCs correlated with IL-8 expression, and was reduced in IL-8 knockouts. Taken together, these results suggest an innate inflammatory response, loss of AR suppressive activity, and subsequent chemokine upregulation as a potential mechanism that regulates the infiltration of PMN-MDSCs to the tumor microenvironment of CRPC after ADT. These findings open a window of opportunity for therapeutic interventions aiming to improve responses to checkpoint blockade in prostate cancer. Citation Format: Zoila A. Lopez Bujanda, Michael C. Haffner, Matthew G. Chaimowitz, Nivedita Chowdhury, Paula J. Hurley, Angela M. Christiano, Charles G. Drake. Androgen regulated IL-8 expression in prostate cancer: Insights into tumor cell mediated immunosuppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1510.

ARV-110: An oral androgen receptor PROTAC degrader for prostate cancer.

259 Background: The Androgen Receptor (AR) remains the principal driver of castration-resistant prostate cancer during the transition from a localized to metastatic disease. Most patients initially respond to inhibitors of the AR pathway, but the response is often relatively short-lived. The majority of patients progressing on enzalutamide or abiraterone exhibit genetic alterations in the AR locus, either in the form of amplifications or point mutations in the AR gene. Given these mechanisms of resistance, our goal is to eliminate the AR protein using the PROteolysis TArgeting Chimera (PROTAC) technology. Methods: Here we report an orally bioavailable small molecule AR PROTAC degrader, ARV-110, that promotes ubiquitination and degradation of AR. This molecule has been characterized in in vitro degradation and functional assays, and DMPK, toxicology and preclinical efficacy studies. Results: ARV-110 robustly degrades AR in all cell lines tested, with an observed half-maximal degradation concentration (DC50) of ~1 nM. ARV-110 treatment leads to highly selective AR degradation, as demonstrated by proteomic studies. In VCaP cells, PROTAC-mediated AR degradation suppresses the expression of the AR-target gene PSA, inhibits AR-dependent cell proliferation, and induces apoptosis at low nanomolar concentrations. Further, ARV-110 degrades clinically relevant mutant AR proteins and retains activity in a high androgen environment. In mouse xenograft studies, greater than 90% AR degradation is observed at a 1 mg/kg PO QD dose. Significant inhibition of tumor growth and AR signaling has been achieved in LNCaP, VCaP and prostate cancer patient derived xenograft (PDX) models. Notably, ARV-110 demonstrates in vivo efficacy and reduction of AR-target gene expression in a long term, castrate, enzalutamide-resistant VCaP tumor model. Conclusions: In summary, we report preclinical data on an orally bioavailable AR PROTAC degrader, ARV-110, that demonstrates efficacy in multiple prostate cancer models. ARV-110 has completed IND-enabling studies and FIH studies are planned for 1Q2019.

Abstract 3741: Deciphering the regulome of androgen receptor variants in prostate cancer

Abstract Prostate cancer is an androgen-dependent disease. Androgen function is controlled by the androgen receptor (AR), and treatments targeting AR are effective at preventing disease progression. Despite this, resistance and emergence of castration resistant prostate cancer (CRPC) is responsible for virtually all prostate cancer-related death. Constitutively active AR variants (AR-Vs) have been implicated as drivers of CRPC. AR-Vs consist of the unstructured AR NH2-terminal domain (NTD), and the AR DNA binding domain, but lack the ligand binding domain (LBD), which is targeted by all current therapies. While our understanding of proteins that regulate the AR LBD is mature, there is less known about the molecular regulation of the NTD. This represents a gap in knowledge given that the AR NTD is essential for AR/AR-V transcriptional activity. To address this, we used a technique termed Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins (RIME) with a pair of isogenic prostate cancer cell lines that were manipulated via genome engineering to express either full-length AR or AR-V. RIME identified several candidate proteins that interact with the AR NTD including BCL9, a β-catenin transcriptional coactivator. Interrogation of public databases revealed that BCL9 mRNA is overexpressed in metastatic prostate cancer compared with localized disease. Additionally, knockdown of BCL9 in cell lines expressing full-length AR inhibited expression of AR target genes as well as β-catenin target genes. Interestingly, knockdown of BCL9 did not affect these same genes in cell lines expressing AR-Vs, indicating that AR truncation could represent a mechanism of escape from BCL9 regulation. Collectively, these findings identify a new mechanism of AR regulation by BCL9 in prostate cancer. Citation Format: Mark L. Daniel, Theresa E. Hickey, Jason S. Carroll, Wayne D. Tilley, Luke A. Selth, Scott M. Dehm. Deciphering the regulome of androgen receptor variants in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3741.

Abstract 1918: Patient-derived hormone-naive prostate cancer xenograft models reveal GRB10 as an AR-repressed gene driving the development of castration-resistant prostate cancer

Abstract Prostate cancer is the most commonly diagnosed noncutaneous cancer and the leading cause of cancer-related death in North American men. Although androgen-deprivation therapy is initially effective in controlling the growth of hormone-naive prostate cancers (HNPC) in patients, currently incurable castration-resistant prostate cancer (CRPC) inevitably develops. Thus, we aim to identify CRPC driver genes that may provide new targets to enhance CRPC therapy. In this study, patient-derived xenografts (PDXs) of HNPCs that develop CRPC following host castration were examined for changes in expression of genes at various time points after castration using transcriptome profiling analysis; particular attention was given to pre-CRPC changes in expression indicative of genes acting as potential CRPC drivers. Eighty genes were found to be significantly upregulated at the CRPC stage while 7 of them also showed elevated expression prior to CRPC development. Among the latter, Growth Factor Receptor Bound Protein 10 (GRB10) was the most significantly and consistently upregulated gene. Moreover, we found GRB10 expression was elevated in clinical CRPC compared to HNPC in several clinical cohorts. Further investigation suggests that GRB10 is transcriptionally regulated by androgen receptor through an androgen-responsive element located in GRB10's intron. Functionally, we found that GRB10 knockdown markedly reduced prostate cancer cell proliferation and activity of AKT, a well-established CRPC mediator. Also, a positive correlation between AKT activity and GRB10 expression was found in clinical cohorts. In conclusion, GRB10 acts as a driver of CRPC and sensitizes AR pathway inhibitors, and hence GRB10-targeting provides a novel therapeutic strategy for the disease. Citation Format: Jun Hao, Xinpei Ci, Hui Xue, Rebecca Wu, Xin Dong, Fang Zhang, Sifeng Qu, Fan Zhang, Anne M. Haegert, Peter W. Gout, Colin Collins, Martin E. Gleave, Dong Lin, Yuzhuo Wang. Patient-derived hormone-naive prostate cancer xenograft models reveal GRB10 as an AR-repressed gene driving the development of castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1918.

Abstract 5236: ARV-110: An androgen receptor PROTAC degrader for prostate cancer

Abstract The Androgen Receptor (AR) remains the principal driver of castration-resistant prostate cancer during the transition from a localized to metastatic disease. Most patients initially respond to inhibitors of the AR pathway, but the response is often short-lived. The majority of patients progressing on enzalutamide or abiraterone exhibit genetic alterations in the AR locus, either in the form of amplifications or point mutations in the AR gene. Given these mechanisms of resistance, our goal is to eliminate the AR protein using the PROteolysis TArgeting Chimera (PROTAC) technology. Here we report an orally bioavailable small molecule ARV-110 that leads to ubiquitination and degradation of AR. ARV-110 completely degrades AR in all cell lines tested, with an observed 50% degradation concentration (DC50) < 1 nM. PROTAC-mediated AR degradation suppresses the expression of the AR-target genes PSA and FKBP5, inhibits AR-dependent cell proliferation, and induces potent apoptosis in VCaP cells. ARV-110 degrades clinically relevant mutant AR proteins and retains activity in a high androgen environment. In mouse xenograft studies, greater than 90% AR degradation is observed at a 1 mg/kg PO QD dose. Significant inhibition of tumor growth and AR signaling can be achieved in both an intact and castrate setting. Further, ARV-110 demonstrates in vivo efficacy and reduction of oncogenic Erg protein in a long term, castrate, enzalutamide-resistant VCaP tumor model. DMPK and exploratory toxicology studies show robust oral, dose proportional drug exposure in rodent and non-rodent species. In summary, we report preclinical data on ARV-110, an orally bioavailable androgen receptor PROTAC degrader that demonstrates efficacy in enzalutamide-resistant prostate cancer. Citation Format: Taavi Neklesa, Lawrence B. Snyder, Ryan R. Willard, Nicholas Vitale, Kanak Raina, Jennifer Pizzano, Deborah Gordon, Mark Bookbinder, Jennifer Macaluso, Hanqing Dong, Zheng Liu, Caterina Ferraro, Gan Wang, Jing Wang, Craig M. Crews, John Houston, Andrew P. Crew, Ian Taylor. ARV-110: An androgen receptor PROTAC degrader for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5236.

IL-23 secreted by myeloid cells drives castration-resistant prostate cancer.

Patients with prostate cancer frequently show resistance to androgen-deprivation therapy, a condition known as castration-resistant prostate cancer (CRPC). Acquiring a better understanding of the mechanisms that control the development of CRPC remains an unmet clinical need. The well-established dependency of cancer cells on the tumour microenvironment indicates that the microenvironment might control the emergence of CRPC. Here we identify IL-23 produced by myeloid-derived suppressor cells (MDSCs) as a driver of CRPC in mice and patients with CRPC. Mechanistically, IL-23 secreted by MDSCs can activate the androgen receptor pathway in prostate tumour cells, promoting cell survival and proliferation in androgen-deprived conditions. Intra-tumour MDSC infiltration and IL-23 concentration are increased in blood and tumour samples from patients with CRPC. Antibody-mediated inactivation of IL-23 restored sensitivity to androgen-deprivation therapy in mice. Taken together, these results reveal that MDSCs promote CRPC by acting in a non-cell autonomous manner. Treatments that block IL-23 can oppose MDSC-mediated resistance to castration in prostate cancer and synergize with standard therapies.

Patient-derived Hormone-naive Prostate Cancer Xenograft Models Reveal Growth Factor Receptor Bound Protein 10 as an Androgen Receptor-repressed Gene Driving the Development of Castration-resistant Prostate Cancer

BackgroundAlthough androgen deprivation therapy is initially effective in controlling growth of hormone-naive prostate cancers (HNPCs) in patients, currently incurable castration-resistant prostate cancer (CRPC) inevitably develops. ObjectiveTo identify CRPC driver genes that may provide new targets to enhance CRPC therapy. Design, setting, and participantsPatient-derived xenografts (PDXs) of HNPCs that develop CRPC following host castration were examined for changes in expression of genes at various time points after castration using transcriptome profiling analysis; particular attention was given to pre-CRPC changes in expression indicative of genes acting as potential CRPC drivers. Outcome measurements and statistical analysisThe functionality of a potential CRPC driver was validated via its knockdown in cultured prostate cancer cells; its clinical relevance was established using data from prostate cancer patient databases. Results and limitationsEighty genes were found to be significantly upregulated at the CRPC stage, while seven of them also showed elevated expression prior to CRPC development. Among the latter, growth factor receptor bound protein 10 (GRB10) was the most significantly and consistently upregulated gene. Moreover, elevated GRB10 expression in clinical prostate cancer samples correlated with more aggressive tumor types and poorer patient treatment outcome. GRB10 knockdown markedly reduced prostate cancer cell proliferation and activity of AKT, a well-established CRPC mediator. A positive correlation between AKT activity and GRB10 expression was also found in clinical cohorts. ConclusionsGRB10 acts as a driver of CRPC and sensitizes androgen receptor pathway inhibitors, and hence GRB10 targeting provides a novel therapeutic strategy for the disease. Patient summaryDevelopment of castration-resistant prostate cancer (CRPC) is a major problem in the management of the disease. Using state-of-the-art patient-derived hormone-naive prostate cancer xenograft models, we found and validated the growth factor receptor bound protein 10 gene as a driver of CRPC, indicating that it may be used as a new molecular target to enhance current CRPC therapy.

An oral androgen receptor PROTAC degrader for prostate cancer.

381 Background: The Androgen Receptor (AR) remains the principal driver of castration-resistant prostate cancer during the transition from a localized to metastatic disease. Most patients initially respond to inhibitors of the AR pathway, but the response is often short-lived. The majority of patients progressing on enzalutamide or abiraterone exhibit genetic alterations in the AR locus, either in the form of amplifications or point mutations in the AR gene. Given these mechanisms of resistance, our goal is to eliminate the AR protein using the PROteolysis TArgeting Chimera (PROTAC) technology. Methods: Here we report an orally bioavailable small molecule AR PROTAC that leads to ubiquitination and degradation of AR. This molecule has been characterized in in vitro degradation and functional assays, DMPK, toxicology and preclinical efficacy studies. Results: This AR PROTAC completely degrades AR in all cell lines tested, with an observed 50% degradation concentration (DC50) < 1 nM. PROTAC-mediated AR degradation suppresses the expression of the AR-target gene PSA, inhibits AR-dependent cell proliferation, and induces potent apoptosis in VCaP cells. The AR PROTAC degrades all clinically relevant mutant AR proteins and retains activity in a high androgen environment. In mouse xenograft studies, greater than 90% AR degradation is observed at a 1 mg/kg PO QD dose. Significant inhibition of tumor growth and AR signaling can be achieved in both an intact and castrate setting. Further, the AR PROTAC demonstrates in vivo efficacy and reduction of oncogenic Erg protein in a long term, castrate, enzalutamide-resistant VCaP tumor model. DMPK and exploratory toxicology studies show robust oral, dose proportional drug exposure in rodent and non-rodent species. Conclusions: In summary, we report preclinical data on an orally bioavailable AR PROTAC degrader that demonstrates efficacy in enzalutamide-resistant prostate cancer.

Abstract 5637: An oral Androgen Receptor PROTAC degrader for prostate cancer

Abstract The Androgen Receptor (AR) remains the principal driver of castration-resistant prostate cancer during the transition from a localized to metastatic disease. Most patients initially respond to inhibitors of the AR pathway, but the response is often short-lived. The majority of patients progressing on enzalutamide or abiraterone exhibit genetic alterations in the AR locus, either in the form of amplifications or point mutations in the AR gene. Given these mechanisms of resistance, our goal is to eliminate the AR protein using the PROteolysis TArgeting Chimera (PROTAC) technology. Further, we sought to make an orally bioavailable AR PROTAC. Medicinal chemistry efforts yielded a small molecule AR PROTAC that simultaneously binds E3-ubiquitin ligase and AR, thus leading to ubiquitination and degradation of AR. This molecule has been characterized in in vitro and in vivo preclinical studies. Our lead oral AR PROTAC degrades approximately 98% of total AR in all cell lines tested, with 50% degradation concentration (DC50) < 1 nM. AR degradation suppresses the expression of AR-target gene PSA, inhibits cell proliferation, and induces potent apoptosis in VCaP cells. No activity is observed in AR-null cell lines, such as PC-3. While enzalutamide loses its activity in the presence of elevated androgens, AR PROTAC maintains its antiproliferative activity. Further, AR PROTAC is able to degrade all clinically relevant mutant AR proteins. A robust oral bioavailability is observed across multiple species and overall ADME properties are encouraging. Approximately 95% AR degradation is observed in AR-amplified VCaP xenografts at doses as low as 10 mg/kg. Congruent with AR degradation, a dose responsive tumor growth inhibition is observed in AR-dependent xenograft studies. In summary, we report the first orally bioavailable AR PROTAC that robustly degrades AR in vitro and in vivo. Citation Format: Taavi K. Neklesa, Lawrence B. Snyder, Mark Bookbinder, Xin Chen, Andrew P. Crew, Craig M. Crews, Hanqing Dong, Deborah Gordon, Jennifer Macaluso, Kanak Raina, AnnMarie Rossi, Ian Taylor, Nicholas Vitale, Jing Wang, Ryan R. Willard, Kurt Zimmermann. An oral Androgen Receptor PROTAC degrader for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5637. doi:10.1158/1538-7445.AM2017-5637

Aberrant TGF-β Signaling Drives Castration-Resistant Prostate Cancer in a Male Mouse Model of Prostate Tumorigenesis.

The androgen receptor (AR) plays a critical role as a driver of castration-resistant prostate cancer (CRPC). Our previous studies demonstrated that disruption of transforming growth factor-β (TGF-β) signaling via introduction of dominant-negative transforming growth factor-β type II receptor (DNTGFβRII) in the prostate epithelium of transgenic adenocarcinoma of the prostate mice accelerated tumor. This study investigated the consequences of disrupted TGF-β signaling on prostate tumor growth under conditions of castration-induced androgen deprivation in the preclinical model of DNTGFβRII. Our results indicate that in response to androgen deprivation therapy (ADT) the proliferative index in prostate tumors from DNTGFβRII mice was higher compared with prostate tumors from TGFβRII wild-type (WT) mice, whereas there was a reduced incidence of apoptosis in tumors from DNTGFβRII. Protein and gene expression profiling revealed that tumors from DNTGFβRII mice exhibit a strong nuclear AR localization among the prostate tumor epithelial cells and increased AR messenger RNA after ADT. In contrast, TGFβRII WT mice exhibited a marked loss in nuclear AR in prostate tumor acini (20 weeks), followed by a downregulation of AR and transmembrane protease serine 2 messenger RNA. There was a significant increase in nuclear AR and activity in prostate tumors from castrate DNTGFβRII compared with TGFβRII WT mice. Consequential to aberrant TGF-β signaling, ADT enhanced expression and nuclear localization of Smad4 and β-catenin. Our findings support that under castrate conditions, aberrant TGF-β signaling leads to AR activation and β-catenin nuclear localization, an adaptation mechanism contributing to emergence of CRPC. The work defines a potentially significant new targeting platform for overcoming therapeutic resistance in CRPC.

An oral androgen receptor PROTAC degrader for prostate cancer.

273 Background: The Androgen Receptor (AR) remains the principal driver of castration-resistant prostate cancer during the transition from a localized to metastatic disease. Most patients initially respond to inhibitors of the AR pathway, but the response is often short-lived. The majority of patients progressing on enzalutamide or abiraterone exhibit genetic alterations in the AR locus, either in the form of amplifications or point mutations in the AR gene. Given these mechanisms of resistance, our goal is to eliminate the AR protein using the PROteolysis TArgeting Chimera (PROTAC) technology. Further, we sought to make an orally bioavailable AR PROTAC. Methods: Medicinal chemistry efforts yielded a small molecule AR PROTAC that simultaneously binds E3-ubiquitin ligase and AR, thus leading to ubiquitination and degradation of AR. This molecule has been characterized in in vitro and in vivo preclinical studies. Results: Our lead oral AR PROTAC degrades 92-98% of total AR in all cell lines tested, with 50% degradation concentration (DC50) < 1 nM. AR degradation suppresses the expression of AR-target gene PSA, inhibits cell proliferation, and induces potent apoptosis in VCaP cells. No activity is observed in AR-null cell lines, such as PC-3. While enzalutamide loses its activity in the presence of elevated androgens, AR PROTAC maintains its antiproliferative activity. Further, AR PROTAC is able to degrade all clinically relevant mutant AR proteins. A robust oral bioavailability is observed across multiple species and overall ADME properties are encouraging. Approximately 95% AR degradation is observed in AR-amplified VCaP xenografts at doses as low as 10 mg/kg. Congruent with AR degradation, a dose responsive tumor growth inhibition is observed in AR-dependent xenograft studies. Conclusions: In summary, we report the first orally bioavailable AR PROTAC that robustly degrades AR in vitro and in vivo.

Exploitation of Castration-Resistant Prostate Cancer Transcription Factor Dependencies by the Novel BET Inhibitor ABBV-075.

The ability of BET family inhibitor ABBV-075 to inhibit transcription activation downstream of the initiating events of transcription factors like AR and TMPRSS2:ETS fusion proteins provides a promising therapeutic option for CRPC patients who have developed resistance to second-generation antiandrogens. Mol Cancer Res; 15(1); 35-44. ©2016 AACR.