Supplementary Table S4. Analysis of ALK and potential ALK-ligand mRNA expression in primary prostate cancer dataset.

Supplementary Table from A Genome-Wide CRISPR Activation Screen Identifies PRRX2 as a Regulator of Enzalutamide Resistance in Prostate Cancer

Androgen receptor pathway inhibitors are the mainstay treatment for advanced prostate cancer, but resistance is common. Here, we used a CRISPR activation screen to identify genes that promote enzalutamide resistance in the metastatic castration-sensitive prostate cancer cell line LNCaP. We found that activation of the TGF-β target gene, PRRX2, promoted enzalutamide resistance. PRRX2 expression is highest in double negative prostate cancer (DNPC) patients and a PRRX2-related gene signature identified a subset of DNPC patients with lower overall survival. PRRX2 expressing cells showed alterations in the CDK4/6/Rb/E2F and BCL2 pathways. Accordingly, CDK4/6 and BCL2 inhibitors sensitized PRRX2-expressing enzalutamide-resistant tumors to enzalutamide. High PRRX2 expression/signature in prostate cancer merit investigation as markers of enzalutamide resistance that could be reversed with CDK4/6 and BCL2 inhibitors. Citation Format: Yara R. Rodriguez, Kenji Unno, Mihai Truica, Zachary R. Chalmers, Young A Yoo, Rajita Vatapalli, Vinay Sagar, Jindan Yu, Barbara Lysy, Huiying Han, Sarki A. Abdulkadir. Genome-wide CRISPR activation screen identifies PRRX2 as an enzalutamide resistance gene in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB082.

Abstract Androgen receptor (AR) pathway inhibitors are the mainstay treatment for advanced prostate cancer, but resistance to therapy is common. Here, we used a CRISPR activation screen in metastatic castration-sensitive prostate cancer cells to identify genes that promote resistance to AR inhibitors. Activation of the TGFβ target gene paired-related homeobox2 (PRRX2) promoted enzalutamide resistance. PRRX2 expression was the highest in double-negative prostate cancer (DNPC), which lack AR signaling and neuroendocrine differentiation, and a PRRX2-related gene signature identified a subset of patients with DNPC with reduced overall survival. PRRX2-expressing cells showed alterations in the CDK4/6/Rb/E2F and BCL2 pathways. Accordingly, treatment with CDK4/6 and BCL2 inhibitors sensitized PRRX2-expressing, castration-resistant tumors to enzalutamide. Overall, PRRX2 was identified as a driver of enzalutamide resistance. The PRRX2 signature merits investigation as a biomarker of enzalutamide resistance in prostate cancer that could be reversed with CDK4/6 and BCL2 inhibitors. Significance: PRRX2 mediates enzalutamide resistance via activation of the E2F and BCL2 pathways, which can be targeted with CDK4/6 and BCL2 inhibitors to reverse resistance.

Background Inflammatory bowel disease (IBD) has been implicated as a risk factor for prostate cancer, however, the mechanism of how IBD leads to prostate tumorigenesis is not known. Here, we investigated whether chronic intestinal inflammation leads to pro-inflammatory changes associated with tumorigenesis in the prostate. Methods Using clinical samples of men with IBD who underwent prostatectomy, we analyzed whether prostate tumors had differences in lymphocyte infiltrate compared to non-IBD controls. In a mouse model of chemically-induced intestinal inflammation, we investigated whether chronic intestinal inflammation could be transferred to the wild-type mouse prostate. In addition, mouse prostates were evaluated for activation of pro-oncogenic signaling and genomic instability. Results A higher proportion of men with IBD had T and B lymphocyte infiltration within prostate tumors. Mice with chronic colitis showed significant increases in prostatic CD45 + leukocyte infiltration and elevation of three pro-inflammatory cytokines—TIMP-1, CCL5, and CXCL1 and activation of AKT and NF-kB signaling pathways. Lastly, mice with chronic colitis had greater prostatic oxidative stress/DNA damage, and prostate epithelial cells had undergone cell cycle arrest. Conclusions These data suggest chronic intestinal inflammation is associated with an inflammatory-rich, pro-tumorigenic prostatic phenotype which may explain how gut inflammation fosters prostate cancer development in men with IBD.

Abstract Neuroendocrine prostate cancer (NEPC) is an aggressive subtype of prostate cancer with poor prognosis, and there is a critical need for novel therapeutic approaches. NEPC is associated with molecular perturbation of several pathways, including amplification of MYCN. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase involved in the pathogenesis of neuroblastoma and other malignancies where it cooperates with N-Myc. We previously identified the first case of ALK F1174C-activating mutation in a patient with de novo NEPC who responded to the ALK inhibitor, alectinib. Here, we show that coactivation of ALK and N-Myc (ALK F1174C/N-Myc) is sufficient to transform mouse prostate basal stem cells into aggressive prostate cancer with neuroendocrine differentiation in a tissue recombination model. A novel gene signature from the ALK F1174C/N-Myc tumors was associated with poor outcome in multiple human prostate cancer datasets. ALK F1174C and ALK F1174C/N-Myc tumors displayed activation of the Wnt/β-catenin signaling pathway. Chemical and genetic ALK inhibition suppressed Wnt/β-catenin signaling and tumor growth in vitro in NEPC and neuroblastoma cells. ALK inhibition cooperated with Wnt inhibition to suppress NEPC and neuroblastoma proliferation in vitro and tumor growth and metastasis in vivo. These findings point to a role for ALK signaling in NEPC and the potential of cotargeting the ALK and Wnt/β-catenin pathways in ALK-driven tumors. Activated ALK and N-Myc are well known drivers in neuroblastoma development, suggesting potential similarities and opportunities to elucidate mechanisms and therapeutic targets in NEPC and vice versa. Significance: These findings demonstrate that coactivation of ALK and N-Myc induces NEPC by stimulating the Wnt/β-catenin pathway, which can be targeted therapeutically.

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology I (MP16)1 Apr 2020MP16-19 MODELING THE IMPACT OF INFLAMMATORY BOWEL DISEASE ON PROSTATE CANCER RISK IN MICE: PRELIMINARY RESULTS OF AN ONGOING STUDY Anuj Desai*, Barbara Lysy, Vinay Sagar, Rajita J Vatapalli, HuiYing Han, Kenji Unno, Yara R Rodriguez, Conor Driscoll, Jenny Ross, Jennifer D Wu, Sarki A Abdulkadir, and Shilajit Kundu Anuj Desai*Anuj Desai* More articles by this author , Barbara LysyBarbara Lysy More articles by this author , Vinay SagarVinay Sagar More articles by this author , Rajita J VatapalliRajita J Vatapalli More articles by this author , HuiYing HanHuiYing Han More articles by this author , Kenji UnnoKenji Unno More articles by this author , Yara R RodriguezYara R Rodriguez More articles by this author , Conor DriscollConor Driscoll More articles by this author , Jenny RossJenny Ross More articles by this author , Jennifer D WuJennifer D Wu More articles by this author , Sarki A AbdulkadirSarki A Abdulkadir More articles by this author , and Shilajit KunduShilajit Kundu More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000841.019AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Recent epidemiological data has implicated inflammatory bowel disease (IBD) as a risk factor for clinically significant prostate cancer (PC). One key driver of malignant transformation may include longstanding chronic inflammation, however, the local effects of gut inflammation on the prostate are unknown. Using a well-established murine model for chronic colitis, we are assessing whether gut inflammation leads to changes in the inflammatory milieu in the prostate of wild-type mice and transgenic prostate cancer-prone Pten-knockout mice. Here, we report preliminary results of a pilot study that is currently being validated in a larger cohort. METHODS: Chronic colitis was induced by administering C57BL/6 mice with 3 cycles of 3% dextran-sodium sulfate (DSS) – treated water for 7 days followed by 14 days of rest. Preliminary histologic analysis has been performed on DSS-treated mice gut and prostate tissue with independent pathologist review. Relative mRNA expression of prostatic inflammatory cytokines was obtained by quantitative real-time PCR. A similar experiment is currently ongoing with cohorts of both wild-type and transgenic Pten knockout mice (n=5-7/group). RESULTS: DSS-treated mice exhibited bloody diarrhea and weight loss with progression of colitis. Histologic analysis of the colon demonstrated marked epithelial erosion and infiltration of granulocytes into submucosa. In the prostate, DSS-treated mice had relatively higher expression of CD45 and cleaved caspase-3 compared to the control. In addition, TNF-alpha mRNA in the prostate was 6-fold higher in treated mice compared to the control. CONCLUSIONS: Preliminary data from this ongoing study suggests that induction of chronic colitis in mice is associated greater immune-cell infiltrate and higher inflammatory cytokine profile in the prostate. We anticipate that ongoing work from this study will better help to elucidate potential mechanisms of prostate tumorigenesis in men with IBD. Source of Funding: SPORE in Prostate Cancer © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e222-e223 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Anuj Desai* More articles by this author Barbara Lysy More articles by this author Vinay Sagar More articles by this author Rajita J Vatapalli More articles by this author HuiYing Han More articles by this author Kenji Unno More articles by this author Yara R Rodriguez More articles by this author Conor Driscoll More articles by this author Jenny Ross More articles by this author Jennifer D Wu More articles by this author Sarki A Abdulkadir More articles by this author Shilajit Kundu More articles by this author Expand All Advertisement PDF downloadLoading ...

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology I (MP16)1 Apr 2020Prostate Cancer: Basic Research & Pathophysiology I (MP16) View All Author Informationhttps://doi.org/10.1097/JU.0000000000000841AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Expand All Advertisement PDF downloadLoading ...

Abstract Potent anti-androgens such as Enzalutamide (Enza) have been approved for the treatment of mCRPC. Despite initial response, nearly all patients eventually develop resistance to Enza. Although a few mechanisms of Enza resistance have been identified, none have been translated into a clinical benefit for recurrent patients. Our goal is to identify new therapeutic targets to overcome Enza resistance. We performed a genome-wide CRISPR activation screen using deactivated Cas9 (dCas9) fused to VP64 transcriptional activator in the androgen-dependent prostate cancer cell line (LNCaP). We used the Calabrese sgRNA library which targets 18,885 genes using 3 different sgRNAs per gene (56,762 sgRNAs). Cells expressing the gRNA library were treated with Enza (20µM) for 7 weeks to allow for enrichment of potential resistance genes. We identified 10 genes which were targeted by at least 2 sgRNAs with a Fold Change (FC) > 4 in the Enza vs control group. Notably, AR was the most enriched gene. AR up-regulation is one of the main drivers of Enza resistance in patients. Pathway enrichment analysis using all the sgRNAs with FC> 5 revealed an over-representation of the RB1, TP53 and TGF-β pathways. Importantly, these pathways are also known to drive Enza resistance. Our screen also identified new candidate genes that have never been linked to Enza resistance. Interestingly, most of these genes are transcription factors. We validated the screen results in vitro. Further analysis of this target genes in vitro and in vivo will allow us to identify novel genes and potential therapeutic targets for Enza resistance. Citation Format: Yara Rodriguez, Rajita Vatapalli, Huyin Han, Kenji Unno, Vinay Sagar, Zachary Chalmers, Barbara Lysy, Young A Yoo, Mihai Truica, Sarki Abdulkadir. Genome-wide CRISPRa screen reveals new drivers of Enzalutamide resistance in 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 4102.

BACKGROUND Recurrence following androgen-deprivation therapy is associated with adverse clinical outcomes in prostate cancer, but the cellular origins and molecular mechanisms underlying this process are poorly defined. We previously identified a population of castration-resistant luminal progenitor cells expressing Bmi1 in the normal mouse prostate that can serve as a cancer cell-of-origin. Here, we investigate the potential of Bmi1-expressing tumor cells that survive castration to initiate recurrence in vivo. METHODS We employed lineage retracing in Bmi1-CreER; R26R-confetti; Ptenf/f transgenic mice to mark and follow the fate of emerging recurrent tumor clones after castration. A tissue recombination strategy was used to rescue transgenic mouse prostates by regeneration as grafts in immunodeficient hosts. We also used a small molecule Bmi1 inhibitor, PTC-209, to directly test the role of Bmi1 in recurrence. RESULTS Transgenic prostate tumors (n = 17) regressed upon castration but uniformly recurred within 3 months. Residual regressed tumor lesions exhibited a transient luminal-to-basal phenotypic switch and marked cellular heterogeneity. Additionally, in these lesions, a subpopulation of Bmi1-expressing castration-resistant tumor cells overexpressed the stem cell reprogramming factor Sox2 (mean [SD] = 41.1 [3.8]%, n = 10, P < .001). Bmi1+Sox2+ cells were quiescent (BrdU+Bmi1+Sox2+ at 3.4 [1.5]% vs BrdU+Bmi1+Sox2- at 18.8 [3.4]%, n = 10, P = .009), consistent with a cancer stem cell phenotype. By lineage retracing, we established that recurrence emerges from the Bmi1+ tumor cells in regressed tumors. Furthermore, treatment with the small molecule Bmi1 inhibitor PTC-209 reduced Bmi1+Sox2+ cells (6.1 [1.4]% PTC-209 vs 38.8 [2.3]% vehicle, n = 10, P < .001) and potently suppressed recurrence (retraced clone size = 2.6 [0.5] PTC-209 vs 15.7 [5.9] vehicle, n = 12, P = .04). CONCLUSIONS These results illustrate the utility of lineage retracing to define the cellular origins of recurrent prostate cancer and identify Bmi1+Sox2+ cells as a source of recurrence that could be targeted therapeutically.

The EPHB4 receptor is implicated in the development of several epithelial tumors and is a promising therapeutic target, including in prostate tumors in which EPHB4 is overexpressed and promotes tumorigenicity. Here, we show that high expression of EPHB4 correlated with poor survival in prostate cancer patients and EPHB4 inhibition induced cell death in both hormone sensitive and castration-resistant prostate cancer cells. EPHB4 inhibition reduced expression of the glucose transporter, GLUT3, impaired glucose uptake, and reduced cellular ATP levels. This was associated with the activation of endoplasmic reticulum stress and tumor cell death with features of immunogenic cell death (ICD), including phosphorylation of eIF2α, increased cell surface calreticulin levels, and release of HMGB1 and ATP. The changes in tumor cell metabolism after EPHB4 inhibition were associated with MYC downregulation, likely mediated by the SRC/p38 MAPK/4EBP1 signaling cascade, known to impair cap-dependent translation. Together, our study indicates a role for EPHB4 inhibition in the induction of immunogenic cell death with implication for prostate cancer therapy.

Small molecules that directly target MYC and are also well tolerated in vivo will provide invaluable chemical probes and potential anti-cancer therapeutic agents. We developed a series of small-molecule MYC inhibitors that engage MYC inside cells, disrupt MYC/MAX dimers, and impair MYC-driven gene expression. The compounds enhance MYC phosphorylation on threonine-58, consequently increasing proteasome-mediated MYC degradation. The initial lead, MYC inhibitor 361 (MYCi361), suppressed in vivo tumor growth in mice, increased tumor immune cell infiltration, upregulated PD-L1 on tumors, and sensitized tumors to anti-PD1 immunotherapy. However, 361 demonstrated a narrow therapeutic index. An improved analog, MYCi975 showed better tolerability. These findings suggest the potential of small-moleculeMYC inhibitors as chemical probes and possible anti-cancer therapeutic agents.

Abstract Introduction: Recurrence is associated with mortality in prostate cancer, but the cellular origins and molecular mechanisms that drive this process have not been well-defined. An emerging model of resistance to androgen deprivation therapy (ADT) in prostate cancer, particularly to the more potent newer drugs, involves lineage plasticity or transdifferentiation. Epigenetic regulators and stem cell reprogramming factors including Sox2 and Ezh2 appear to play important roles in this process. However, there has been a paucity of studies that attempt to specifically follow the fate of malignant prostate cells in regressed tumors following castration in order to understand the cellular processes associated with the emergence of castration-resistant prostate cancer (CRPC). We recently identified a population of luminal progenitors termed castration-resistant Bmi1-expressing cells (CARBs) in the mouse prostate that serve as a prostate cancer cell of origin. Here, we hypothesized that analogous castration-resistant Bmi1-expressing tumor cells (tumor CARBs) exist in prostate cancer and could seed recurrence after ADT. Methods: We adopted lineage retracing strategies using an inducible Bmi1-CreER driver and a multicolor reporter R26R-Confetti allele, which enables lineage-marking and kinetically re-tracing of the emerging recurrent tumor clones within castrated tumors over times in vivo. A tissue recombination strategy was used to rescue transgenic Bmi1-CreER; Ptenf/f mice (BC-Pten) mouse prostates by regeneration as grafts in SCID mice. The ability of tumor CARBs to seed recurrence was determined by lineage retracing methodology of castrated mice after establishing tumor by tamoxifen treatment, which was coupled with immunohistochemistry and Western blotting. We also used a small molecule Bmi1 inhibitor, PTC-209, to directly test the role of Bmi1 in recurrence. Results: Deletion of Pten in Bmi1+ luminal prostate progenitors generates a luminal prostate cancer that regresses upon castration, followed by recurrence over time. Notably, castration induced a transient luminal-to-basal phenotypic switch in regressed tumors with recurrent tumors reverting to a luminal phenotype. Analysis of regressed tumors post-castration showed cellular heterogeneity, with a population of apoptosis-resistant, proliferative, Bmi1-expressing tumor cells that also upregulate expression of the stem cell reprogramming factor Sox2. Expression of Sox2 was maintained in recurrent tumors despite loss of basal cell transdifferentiation. Lineage re-tracing with the R26R-Confetti allele established that Bmi1+ tumor cells in regressed tumors drive recurrence. Furthermore, treatment with the small molecule Bmi1 inhibitor PTC-209 eliminated Bmi1+Sox2+ cells and significantly decreased recurrence. Conclusion: These results shed light on the cellular origins of recurrent prostate cancer and the roles of the epigenetic/stem cell regulators Bmi1 and Sox2 in therapeutic resistance to androgen deprivation. Citation Format: Young A. Yoo, Rajita Vatapalli, Barbara Lysy, Hanlin Mok, Mohamed M. Desouki, Sarki A. Abdulkadir. Castration-resistant Bmi1+Sox2+ cells drive recurrence 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 LB-046.

Identification of defined cell populations with stem/progenitor properties is key for understanding prostate development and tumorigenesis. Here we show that the polycomb repressor protein Bmi1 marks a population of castration-resistant luminal epithelial cells enriched in the mouse proximal prostate. We employ lineage tracing to show that these castration-resistant Bmi1-expressing cells (or CARBs) are capable of tissue regeneration and self-renewal. Notably, CARBs are distinct from the previously described luminal castration-resistant Nkx3.1-expressing cells (CARNs). CARBs can serve as a prostate cancer cell-of-origin upon Pten deletion, yielding luminal prostate tumours. Clonal analysis using the R26R-confetti allele indicates preferential tumour initiation from CARBs localized to the proximal prostate. These studies identify Bmi1 as a marker for a distinct population of castration-resistant luminal epithelial cells enriched in the proximal prostate that can serve as a cell of origin for prostate cancer.