Enzalutamide-resistant Prostate Cancer Cells Research Articles

Abstract 4751: The antiviral, Daclatasvir, downregulates Tribbles 2 pseudokinase and reverses enzalutamide resistance in prostate cancer

Abstract Background: FDA-approved enzalutamide is commonly prescribed for advanced prostate cancer. However, enzalutamide-resistant prostate cancer (ERPC) invariably develops, which leads to aggressive, lethal disease. Recently, we found that the Tribbles 2 (TRIB2) pseudokinase is overexpressed in ERPC cells and confers resistance to enzalutamide by promoting lineage plasticity to neuroendocrine differentiation. Though TRIB2 emerged as an excellent molecular target for ERPC, suitable inhibitors are not commercially available for effective targeting. Methods: Compounds were tested using a luciferase-tagged TRIB2 fusion protein-based assay system. Binding of drugs with TRIB2 protein was analyzed by thermal shift assays and by advanced computer-based homology modeling. Degradation of TRIB2 protein was measured by Western blot. Drug effects on re-sensitization of ERPC cells and synergy with enzalutamide were determined through cell viability and apoptosis assays. To gauge the in vivo effects of Daclatasvir (DCV), ERPC tumor xenograft-bearing mice were treated with varying doses of DCV via oral gavage. Tumor growth was calculated by measuring volumes and molecular markers in tumors were analyzed by immunohistochemistry. Results: By designing a luciferase-tagged TRIB2 fusion protein-based assay system, we screened a library of about 1,600 FDA-approved compounds and found that DCV, effectively inhibits the TRIB2-luciferase activity (more than 70% inhibition in 24 hours at 10 micromolar) but does not inhibit the activity of free luciferase. Notably, DCV directly binds to pure TRIB2 protein, leading to its destabilization and a reduction in the half-maximal melting temperature (Tm) from 41oC to 37oC, as confirmed by thermal shift assays. Interestingly, we found that DCV degrades TRIB2 proteins via activation of proteasomes and re-sensitizes ERPC cells to enzalutamide. DCV downregulates the master neuronal transcription factor, BRN2, and the stemness factor, SOX2, and synergizes with enzalutamide at lower, sub-lethal doses to decrease the viability of prostate cancer cells by inducing apoptosis. Finally, DCV was found to effectively inhibit the growth of ERPC tumors and decrease the protein level of TRIB2 in mice when delivered via oral gavage. Conclusion: These findings indicate that DCV effectively downregulates TRIB2 both in vitro and in vivo and suggest that further testing of DCV may help design an innovative therapeutic approach for management of enzalutamide-resistant, aggressive, lethal prostate cancer. Citation Format: Jitender Monga, Rohith Guddeti, Craig Rogers, Shirish Gadgeel, Dhananjay Chitale, Jagadananda Ghosh. The antiviral, Daclatasvir, downregulates Tribbles 2 pseudokinase and reverses enzalutamide resistance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4751.

Abstract 3577: The role of PLXND1 in lineage plasticity and progression to neuroendocrine prostate cancer

Abstract Background: Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer and is commonly associated with therapy resistance and poor prognosis. Treatment-induced NEPC (t-NEPC) predominantly develops by lineage plasticity from adenocarcinoma in response to androgen receptor signaling inhibitors, such as enzalutamide. However, the mechanisms underlying the lineage plasticity and progression to NEPC are unclear. PLXND1 (PlexinD1) is a cellular receptor of the semaphorin family and has important functions in modulating the cytoskeleton and cell adhesion. However, the role of PLXND1 in lineage plasticity to NEPC development and progression is not well established. Methods: We performed transcriptomic analysis on NEPC patients’ cohorts and the enzalutamide-resistant prostate cancer cell lines. Correlation of PLXND1 with neuroendocrine feature genes and AR-targeting genes was determined. Kaplan-Meier method and the Cox proportional hazards model was used to estimate the correlation between PLXND1 expression level and patients’ survival. Immunohistochemistry was used to analyze PLXND1 expression in prostate adenocarcinoma and NEPC. Cell viability was determined in C4-2B MDVR, CWR22Rv1, and H660 cells after knocking down PLXND1 using siRNA or knocking out PLXND1 using CRISPR/CAS9. Organoids viability of patient-derived xenografts was measured after knocking down or knocking out PLXND1. Results: Our analysis on different patient cohorts suggests that PLXND1 is highly expressed in NEPC patients, and the expression of PLXND1 is positively correlated with neuroendocrine feature genes (CHGA, ENO2, SYP, and NCAM1). Kaplan-Meier survival analysis shows that high expression of PLXND1 is significantly associated with the poor prognosis of prostate cancer patients. PLXND1 is overexpressed and negatively regulated by androgen receptor signaling in enzalutamide-resistant cells and prostate cancer patients’ cohorts. Furthermore, downregulation of PLXND1 expression suppressed proliferation of the NEPC cells and viability of the PDX tumor organoids. Conclusions: PLXND1 expression is elevated in NEPC and correlated with the poor survival of prostate cancer patients, suggesting it is a potential molecular indicator and therapy target for NEPC. Citation Format: Bo Chen, Pengfei Xu, Joy C. Yang, Shu Ning, Leyi Wang, Christopher Nip, Allen C. Gao, Chengfei Liu. The role of PLXND1 in lineage plasticity and progression to neuroendocrine prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3577.

Abstract A018: Pathways to enzalutamide resistance: An analysis of proteomic changes in enzalutamide-resistant prostate cancer cells

Abstract Introduction: Enzalutamide is indicated for treatment of castrate-resistant prostate cancer (CRPC), but therapeutic effect is transient, with improvement in survival of approximately 4 months. The mechanisms by which prostate cancer (PCa) cells survive Enzalutamide therapy are poorly understood, but androgen receptor (AR) and non-AR mediated mechanisms have been reported from transcriptome studies. We performed unbiased high resolution proteomic analyses to identify proteins and pathways that are involved in survival and progression in Enzalutamide treatment failure. Materials & Methods: Enzalutamide-resistant MR49F and MR42D cell lines were maintained in standard medium (DMEM with 5% FBS) with Enzalutamide 10µM. Parental LNCaP cells were grown in standard medium. For perturbation, MR49F and MR42D cells were then changed to standard medium with either Enzalutamide 10µM (ENZA) or DMSO (control) for 7 days in. Medium was changed every 48 hours. Protein lysates from 4 biological replicates were labeled using a Tandem Mass Tag (TMT) assay and subjected to mass spectrometry (MS). Protein abundance was measured and significant differences in expression were analysed using a 2-way t-test with a permutation based false detection rate (FDR) of 0.05 and 250 randomisations in the Perseus software suite. MR49F and MR42D were compared to parental LNCaP, and MR49F and MR42D were compared on and off Enzalutamide treatment pressure. Functional biological pathways involved were identified using the DAVID and WebGestalt online pathway analysis tools. Results: MS identified 5364 proteins. Of these, 247 (180 up, 67 down) were differentially expressed by at least 50% in both MR49F and MR42D cell lines relative to parental LNCaP cells. When MR49F and MR42D cell lines were grown with or without Enzalutamide pressure, 5 proteins (4 up, 1 down) were regulated by at least 50% in both cell lines (ANLN, NIN, UQCRB, HIST1H3A and S100P). Pathway analyses with the differentially expressed and regulated proteins identified perturbations in biological pathways with a high representation of pathways related to metabolism, including amino acid and fatty acid metabolism, ascorbate and propanoate metabolism, drug transport and metabolism, and cell adhesion molecules. Conclusions: This unbiased proteomic analysis of Enzalutamide-resistant PCa cells identified proteins and pathways that may be essential for PCa cell survival and progression during Enzalutamide therapy failure. Enzalutamide therapy failure appears to involve some non-AR mediated mechanisms which may be targeted for therapeutic benefit. A better understanding of the mechanisms of Enzalutamide resistance could lead to development of novel therapies for post-Enzalutamide disease progression. Citation Format: Chidi N. Molokwu, Anders Kristensen, Chris Sutton. Pathways to enzalutamide resistance: An analysis of proteomic changes in enzalutamide-resistant prostate cancer cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A018.

Structure-guided design of a selective inhibitor of the methyltransferase KMT9 with cellular activity.

Inhibition of epigenetic regulators by small molecules is an attractive strategy for cancer treatment. Recently, we characterised the role of lysine methyltransferase 9 (KMT9) in prostate, lung, and colon cancer. Our observation that the enzymatic activity was required for tumour cell proliferation identified KMT9 as a potential therapeutic target. Here, we report the development of a potent and selective KMT9 inhibitor (compound 4, KMI169) with cellular activity through structure-based drug design. KMI169 functions as a bi-substrate inhibitor targeting the SAM and substrate binding pockets of KMT9 and exhibits high potency, selectivity, and cellular target engagement. KMT9 inhibition selectively downregulates target genes involved incell cycle regulation and impairs proliferation of tumours cells including castration- and enzalutamide-resistant prostate cancer cells. KMI169 represents a valuable tool to probe cellular KMT9 functions and paves the way for the development of clinical candidate inhibitors as therapeutic options to treat malignancies such as therapy-resistant prostate cancer.

Abstract B119: First-in-class orally active pharmacological inhibitors of TRPV6 for the treatment of advanced prostate cancer

Abstract The highly calcium selective ion channel TRPV6, has pronouced overexpression in many prostate cancers, is a promotor of prostate cancer growth and has been proposed as a prognostic marker in prostate cancer. A novel series of first-in-class TRPV6 inhibitors has been developed by the Queensland Emory Drug Discovery Initiative (QEDDI), a drug discovery and development group aiming to collaborativey translate academic biomedical research into new medicines. The preclinical lead drug candidate QED-203, is orally active and potently inhibits (low nM) calcium influx mediated by TRPV6 as assessed by both Ca2+ probe and electrophysiology assays, and TRPV6-driven NFAT activation as assessed by a luciferase reporter assay. Using RNAseq and qPCR analysis TRPV6 pharmacological inhibition and/or siRNA-mediated silencing was shown to remodel the expression of genes related to both NFAT and WNT signalling, as well as ER stress and the cell cycle. In prostate cancer cell lines that had increased levels of TRPV6, TRPV6 inhibition promoted cell cycle arrest, decreased proliferation and promoted apoptosis. Proliferation of enzalutamide resistant prostate cancer cell lines was also inhibited by QED-203. QED-203 exhibited greater potency over enzalutamide, darolutamide and apalutamide [androgen receptor targeting agents (ARTA)] in prostate cancer cell lines with clinically relevant AR mutations or those with the ARV7 splice variant. QED-203 appeared well suited to once-a-day oral dosing with a high bioavailability and an advantageous pharmacokinetic profile. In preclinical models, QED-203 was well tolerated in rodents and had similar in vivo efficacy to enzalutamide in a castrated LNCaP mouse model of prostate cancer as assessed by tumour growth and PSA levels. Increases in urine calcium levels and gene expression changes consistent with TRPV6 inhibition in isolated xenograft tumours, confirmed QED-203 TRPV6 target engagement in vivo. These studies suggest that the orally active TRPV6 inhibitor QED-203 represents a first-in-class mechanism to treat prostate cancer patients whose tumours have become resistant to current ARTA therapies. Pharmacological inhibitors of TRPV6, could be particularly valuable during the advanced stages of prostate cancer, where there is a clear unmet clinical need. Citation Format: Gregory Monteith, Kimberley Beaumont, Rebecca Pouwer, Rebecca Farrow, Matthew McLachlan, Mei Yeh, Therese Johnson, Raphael Rahmani, Claire Levrier, Hasanthi Wijesekera, Malika Kumarasiri, Grant Stuchbury, Terrie-Anne Cock, Andrew Harvey, Brian Dymock. First-in-class orally active pharmacological inhibitors of TRPV6 for the treatment of advanced 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 B119.

Intracellular metabolic profiling of drug resistant cells by surface enhanced Raman scattering

BackgroundIntracellular metabolic profiling reveals real-time metabolic information useful for the study of underlying mechanisms of cells in particular conditions such as drug resistance. However, mass spectrometry (MS), one of the leading metabolomics technologies, usually requires a large number of cells and complex pretreatments. Surface enhanced Raman scattering (SERS) has an ultrahigh detection sensitivity and specificity, favorable for metabolomics analysis. However, some targeted SERS methods focus on very limited metabolite without global bioprofiling, and some label-free approaches try to fingerprint the metabolic response based on whole SERS spectral classification, but comprehensive interpretation of biological mechanisms was lacking. (95) ResultsWe proposed a label-free SERS technique for intracellular metabolic profiling in complex cellular lysates within 3 min. We first compared three kinds of cellular lysis methods and sonication lysis shows the highest extraction efficiency of metabolites. To obtain comprehensive metabolic information, we collected a spectral set for each sample and further qualified them by the Pearson correlation coefficient (PCC) to calculate how many spectra should be acquired at least to gain the adequate information from a statistical and global view. In addition, according to our measurements with 10 pure metabolites, we can understand the spectra acquired from complex cellular lysates of different cell lines more precisely. Finally, we further disclosed the variations of 22 SERS bands in enzalutamide-resistant prostate cancer cells and some are associated with the androgen receptor signaling activity and the methionine salvage pathway in the drug resistance process, which shows the same metabolic trends as MS. (149) SignificanceOur technique has the capability to capture the intracellular metabolic fingerprinting with the optimized lysis approach and spectral set collection, showing high potential in rapid, sensitive and global metabolic profiling in complex biosamples and clinical liquid biopsy. This gives a new perspective to the study of SERS in insightful understanding of relevant biological mechanisms. (54).

Fulvestrant increases the susceptibility of enzalutamide-resistant prostate cancer cells to NK-mediated lysis

BackgroundEnzalutamide, a next-generation antiandrogen agent, is approved for the treatment of metastatic castration-resistant prostate cancer (CRPC). While enzalutamide has been shown to improve time to progression and extend overall survival...

Peptides disrupting TM4SF3 interaction with AR or AR-V7 block prostate cancer cell proliferation.

Androgen receptor (AR) plays a vital role in the development and progression of prostate cancer from the primary stage to the usually lethal stage known as castration-resistant prostate cancer (CRPC). Constitutively active AR splice variants (AR-Vs) lacking the ligand-binding domain are partially responsible for the abnormal activation of AR and may be involved in resistance to AR-targeting drugs occurring in CRPC. There is increasing consensus on the potential of drugs targeting protein-protein interactions. Our lab has recently identified transmembrane 4 superfamily 3 (TM4SF3) as a critical interacting partner for AR and AR-V7 and mapped the minimal interaction regions. Thus, we hypothesized that these interaction domains can be used to design peptides that can disrupt the AR/TM4SF3 interaction and kill prostate cancer cells. Peptides TA1 and AT1 were designed based on the TM3SF3 or AR interaction domain, respectively. TA1 or AT1 was able to decrease AR/TM4SF3 protein interaction and protein stability. Peptide TA1 reduced the recruitment of AR and TM4SF3 to promoters of androgen-regulated genes and subsequent activation of these AR target genes. Peptides TA1 and AT1 were strongly cytotoxic to prostate cancer cells that express AR and/or AR-V7. Peptide TA1 inhibited the growth and induced apoptosis of both enzalutamide-sensitive and importantly enzalutamide-resistant prostate cancer cells. TA1 also blocked the migration and malignant transformation of prostate cancer cells. Our data clearly demonstrate that using peptides to target the important interaction AR has with TM4SF3 provides a novel method to kill enzalutamide-resistant prostate cancer cells that can potentially lead to new more effective therapy for CRPC.

Critical role of antioxidant programs in enzalutamide-resistant prostate cancer.

Therapy resistance to second-generation androgen receptor (AR) antagonists, such as enzalutamide, is common in patients with advanced prostate cancer (PCa). To understand the metabolic alterations involved in enzalutamide resistance, we performed metabolomic, transcriptomic, and cistromic analyses of enzalutamide-sensitive and -resistant PCa cells, xenografts, patient-derived organoids, patient-derived explants, and tumors. We noted dramatically higher basal and inducible levels of reactive oxygen species (ROS) in enzalutamide-resistant PCa and castration-resistant PCa (CRPC), in comparison to enzalutamide-sensitive PCa cells or primary therapy-naive tumors respectively. Unbiased metabolomic evaluation identified that glutamine metabolism was consistently upregulated in enzalutamide-resistant PCa cells and CRPC tumors. Stable isotope tracing studies suggest that this enhanced glutamine metabolism drives an antioxidant program that allows these cells to tolerate higher basal levels of ROS. Inhibition of glutamine metabolism with either a small-molecule glutaminase inhibitor or genetic knockout of glutaminase enhanced ROS levels, and blocked the growth of enzalutamide-resistant PCa. The critical role of compensatory antioxidant pathways in maintaining enzalutamide-resistant PCa cells was validated by targeting another antioxidant program driver, ferredoxin 1. Taken together, our data identify a metabolic need to maintain antioxidant programs and a potentially targetable metabolic vulnerability in enzalutamide-resistant PCa.

Abstract A026: BMX inhibition reverses HSD3B1-driven resistance in prostate cancer

Abstract Background: Prostate cancer is the second leading cause of cancer-related death in men in the United States and is dependent on androgens and androgen receptor (AR) activation. Androgen deprivation therapy (ADT) is the mainstay treatment, but it as well as next-generation treatments eventually fail. Disease then progresses as castration-resistant prostate cancer (CRPC), which is driven by reactivation of the androgen-AR axis. 3β-hydroxysteroid dehydrogenase-1 (3βHSD1) catalyzes the rate-limiting step for potent androgen synthesis from adrenal-derived precursors, thereby stimulating CRPC. HSD3B1, which encodes for 3βHSD1, has a common missense-encoding germline variation that enhances conversion from adrenal DHEA to potent androgens. Clinical studies demonstrate that this “adrenal-permissive” HSD3B1 genotype confers more rapid progression on ADT, and recent studies show that overall survival is shorter in patients with the adrenal-permissive genotype who are treated with ADT + enzalutamide. The Etk/BMX kinase may be involved in the development and progression of CRPC. We sought to determine whether phosphorylation of 3βHSD1 is required for its activation and that the necessary kinase could be a potential target. Methods: In prostate cancer lines, mouse xenograft models, and fresh patient prostatectomy tissues, mass spectrometry and HPLC were used to identify steroid metabolites. Genetic and pharmacological methods were used to identify inhibition of BMX-mediated 3βHSD1 phosphorylation in CRPC. Results: BMX inhibition suppressed castration-resistant prostate cancer through targeting 3βHSD1 phosphorylation. 3βHSD1 transcription level and enzyme activity were induced in enzalutamide-resistant prostate cancer cell lines. 3βHSD1 and SF-1 transcription levels were positively correlated in prostate adenocarcinoma (TCGA database). BMX inhibition by zanubrutinib suppressed enzalutamide resistance induced 3βHSD1 enzyme activity. Conclusions: BMX promotes CRPC by regulating 3βHSD1 phosphorylation. BMX inhibition has potential as a new therapy to optimize CRPC treatment. Citation Format: Xiuxiu Li, Michael Berk, Christopher Goins, Mohammad Alyamani, Yoon-Mi Chung, Chenyao Wang, Monaben Patel, Nityam Rathi, Ziqi Zhu, Belinda Willard, Shaun Stauffer, Eric Klein, Nima Sharifi. BMX inhibition reverses HSD3B1-driven resistance in 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 A026.

Abstract B023: Targeting noncanonical Wnt5a signaling suppresses the neural lineage network and overcomes enzalutamide resistance in advanced prostate cancer

Abstract INTRODUCTION AND OBJECTIVES Androgen receptor (AR) blockade using antiandrogens is a mainstay for the treatment of castration-resistant prostate cancer (CRPC). However, resistance to antiandrogens occurs unavoidably due to the escaping mechanisms of tumor cells that are not completely understood. Upregulation of non-canonical Wnt ligand Wnt5a is identified from circulating tumor cells from CRPC patients after being treated with enzalutamide. FZD2, the cognate frizzled receptor for Wnt5a, is the most commonly co-upregulated non-canonical Wnt signaling molecule in prostate cancer. In this study, we determined the role of non-canonical Wnt5a/FZD2 to enzalutamide resistance and neural lineage plasticity, and explore the potential of targeting Wnt5a/FZD2 to overcome resistance in advanced prostate cancer. METHODS Wnt5a and FZD2 expression was determined in enzalutamide-resistant C4-2B MDVR cells. Wnt5a and FZD2 expressions were modulated using specific siRNAs. Cell growth, colony formation, and migration were studied in vitro. Transcriptomic analysis was performed on C4-2B MDVR cells treated with FZD2 knocked down; gene program of non-canonical Wnt signaling, hallmark androgen response and AR-V7 associated genes, and neural lineage pathways were analyzed. A novel tRNA bioengineered Wnt5a siRNA was developed to target Wnt5a/FZD2 signaling. The effect of tRNA-siWnt5a on tumor growth, sensitivity to enzalutamide treatment, and neural lineage plasticity was evaluated in vitro and in vivo. RESULTS Wnt5a and FZD2 are highly upregulated in castration-resistant prostate cancer patients, which is verified overexpression in enzalutamide-resistant C4-2B MDVR cells. Knocking down Wnt5a and FZD2 diminishes the enrichment of the non-canonical Wnt signaling pathway. Downregulating Wnt5a and FZD2 expression by specific siRNAs suppresses prostate cancer cell proliferation and resensitized C4-2B MDVR cells to enzalutamide treatment. Suppressing Wnt5a and FZD2 abrogated the enrichment of gene programs regulating cancer cell survival/proliferation, and neural lineage pathways including neuron differentiation and embryonic stem cell markers. Using the bioengineered tRNA-Wnt5a siRNA effectively inhibited the growth of enzalutamide-resistant prostate cancer cells and resensitized tumor cells to enzalutamide treatment in vitro, and resistant CRPC PDX LuCaP35CR tumor growth in vivo. Conclusions Our results suggest that activation of noncanonical Wnt5a/FZD2 signaling confers to enzalutamide resistance and neural lineage plasticity. Targeting the Wnt5a signal could provide benefits for CRPC patients with tumors expressing a high level of Wnt5a, FZD2, and lineage markers, not only enhancing the anti-tumor effects of enzalutamide but suppressing the potential of neuroendocrine differentiation in advanced prostate cancer patients. Citation Format: Shu Ning, Wei Lou, Chengfei Liu, Alan Paul Lombard, Leandro S. D' Abronzo, Neelu Batra, Aiming Yu, Christopher P. Evans, Allen Gao. Targeting noncanonical Wnt5a signaling suppresses the neural lineage network and overcomes enzalutamide resistance in 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 B023.

Androgen receptor blockade resistance with enzalutamide in prostate cancer results in immunosuppressive alterations in the tumor immune microenvironment

BackgroundEmerging data suggest that patients with enzalutamide-treated prostate cancer with increased programmed death-ligand 1 (PD-L1) expression may benefit from anti-PD-L1 treatment. Unfortunately, the Phase III IMbassador250 clinical trial revealed that...

Abstract 1698: Novel role of Tribbles 2 driving treatment-emergent neuroendocrine differentiation in prostate cancer

Abstract Background: Prostate cancer initially responds to androgen receptor (AR) targeted therapies, such as enzalutamide. However, therapy-resistant, incurable disease invariably develops which remains as the major cause for mortality among prostate cancer patients. Molecular mechanism underlying emergence of resistant prostate cancer is yet to be fully characterized. Methods: We developed an in vitro cell culture model to mimic the clinical conditions in enzalutamide-resistant prostate cancer. These cells were transcriptionally profiled for gene expression analysis by Illumina Hi-Seq whole genome gene-expression array. Hits were validated by RT-qPCR and Western blot. For clinical correlation, enzalutamide treated prostate PDX models and surgical tumors (warm autopsy) from prostate cancer patients were comprehensively analyzed. Regulation and role of new hits were determined by treating cells and xenograft tumors with lentiviral-based shRNAs followed by analysis of cell viability, apoptosis, and effects on downstream targets. Pro-cancer effects were analyzed by cell proliferation, invasion, and soft-agar colony formation assays. Results: We identified that the Tribbles 2 (TRIB2) pseudokinase is overexpressed in enzalutamide-resistant prostate cancer (ERPC) cells. Elevated level of TRIB2 was also observed in enzalutamide treated PDX tumors, and in prostate tumors from patients who developed enzalutamide resistant metastatic disease. Interestingly, TRIB2 protein level is especially higher in prostate cancer cells and tumors, where the AR activity is low or absent due to genetic changes. Transfection and overexpression of TRIB2 results in aggressive prostate cancer cell growth and resistance to enzalutamide, apalutamide, darolutamide, and abiraterone. Furthermore, TRIB2 overexpression results in trans-differentiation in prostate cancer cells including neurite outgrowth and adeno-to-neuroendocrine lineage transition. Curiously, we found that TRIB2 downregulates luminal markers (AR, CK8), but upregulates the neuroendocrine markers (CHGA, NSE, SYP). Knockdown of TRIB2 triggers loss of neuroendocrine features, reduces viability of ERPC cells, and sensitize them back to enzalutamide, suggesting that TRIB2 plays a direct role in neuroendocrine differentiation and therapeutic resistance. We also found that TRIB2 upregulates the master neuronal transcription factor, BRN2, and the stemness factor, SOX2, and inhibition of either BRN2 or SOX2 re-sensitizes ERPC cells to enzalutamide. Conclusion: These findings reveal that TRIB2 is a novel driver for lineage plasticity and neuroendocrine differentiation in prostate cancer cells and greatly contributes to enzalutamide resistance. Citation Format: Jitender Monga, Craig Rogers, Shirish Gadgeel, Dhananjay Chitale, Jagadananda Ghosh. Novel role of Tribbles 2 driving treatment-emergent neuroendocrine differentiation in 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 1698.

Abstract 5151: Enzalutamide resistance results in immunosuppressive alterations in prostate tumor immune microenvironment

Abstract Background: Emerging data suggested that enzalutamide-treated prostate cancer patients with increased programmed death-ligand 1 (PD-L1) expression may benefit from anti-PD-L1 treatment. Unfortunately, the Phase III IMbassador250 trial revealed that the combination of atezolizumab (PD-L1 inhibitor) and enzalutamide failed to extend overall survival in patients with castration-resistant prostate cancer (CRPC). The mechanisms underlying treatment failure remain unknown. In this study, we investigated the regulation of immunosuppressive signaling in enzalutamide resistant prostate cancer and characterized the immune infiltrating cells in murine prostate tumors. Methods: The expression of interferon gamma-related genes was determined using qRT-PCR and/or western blotting. Androgen receptor (AR) and AR-V7 levels were downregulated using specific siRNA. Myc-CaP cells were chronically exposed to increasing concentrations of enzalutamide (5-50 μM) for >12 months, and the cells resistant to enzalutamide were referred to as Myc-CaP MDVR. The gene-regulating mechanisms in drug-resistant prostate cancer cells were determined by RNA sequencing analyses. Myc-CaP and Myc-CaP MDVR xenograft tumors were established in FVB mice, and tumor-infiltrating lymphocytes were isolated using Ficoll. The stained immune cells were determined by flow cytometry, and the data were analyzed using Flowjo. Results: Immune-related signaling pathways (interferon alpha/gamma response, T cell activation, and cell chemotaxis pathways) were suppressed in C4-2B MDVR cells. However, PD-L1 expression was highly upregulated and negatively regulated by AR in C4-2B MDVR cells. Enzalutamide treatment decreased CD8+ T cell but increased monocytic myeloid-derived suppressor cell (M-MDSC) population and PD-L1 expression in murine Myc-CaP tumors. Consistently, chemotaxis and immune response-regulating signaling pathways were suppressed, and PD-L1 expression was increased in enzalutamide-resistant Myc-CaP MDVR cells. Notably, MDSC populations were significantly increased in Myc-CaP MDVR orthotopic tumors compared with those in Myc-CaP parental tumors. Co-culturing bone marrow cells with Myc-CaP MDVR cells significantly promoted MDSC differentiation and polarized macrophages from the M1 to M2 phase. Conclusions: Immunosuppressive signaling is activated in enzalutamide resistant prostate cancer cells. The activated immune-related signatures involved in T cells, MDSC, and macrophages may reduce the efficacy of immune checkpoint inhibitors in enzalutamide-resistant prostate cancer. Grant Support: This work was supported in part by grants NIH/NCI R37CA249108 (C, Liu) and R01CA251253 (C, Liu). Citation Format: Pengfei Xu, Joy C. Yang, Bo Chen, Christopher Nip, Jonathan E. Van Dyke, Christopher P. Evans, William J. Murphy, Chengfei Liu. Enzalutamide resistance results in immunosuppressive alterations in prostate tumor immune microenvironment. [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 5151.

Abstract 1157: Uncovering a novel role of peroxisomal β-oxidation in advanced, treatment-resistant prostate cancer

Abstract Background and Aims: Prostate cancer (PCa) is highly reliant on lipids for energy, hence targeting fatty acid oxidation (FAO) is a highly promising approach to the treatment of PCa. Recent work by our group and others have shown that FAO is critical for cell viability and survival, and is one of the key drivers of treatment resistance. Although FAO occurs in both the mitochondria and peroxisomes, the functional relevance and therapeutic exploitability of peroxisomal FAO (perFAO) in PCa has not been extensively explored compared to the mitochondria. Herein, we aim to investigate the therapeutic efficacy of targeting perFAO in clinical prostate tumors and PCa cells, and explore its role in PCa progression. Methods and Results: Pharmacological inhibition of perFAO using the clinical agent, thioridazine, significantly suppressed proliferation, migration, and colony formation of a range of advanced and treatment-resistant PCa cells. Further, we evaluated the therapeutic efficacy of thioridazine in a more clinically relevant setting using patient-derived tissues and showed a significant inhibition of cell proliferation (n = 11, p < 0.05). Next, we performed a bioinformatics analysis of clinical RNAseq datasets composed of primary and metastatic PCa tissues and identified DECR2, the auxiliary enzyme of perFAO, as a key target of perFAO in PCa. DECR2 is overexpressed in metastatic castrate-resistant PCa (CRPC) tissues compared to primary PCa and benign tissues, and is significantly associated with shorter relapse-free survival (p < 0.01). DECR2 knockdown significantly suppressed proliferation and migration of CRPC and enzalutamide-resistant PCa cells, and markedly reduced LNCaP tumor growth in vivo. In contrast, overexpression of DECR2 in LNCaP cells significantly increased tumor growth in vivo. Interestingly, analysis of a published proteomics dataset of PCa cells that are either androgen-dependent or resistant to androgen receptor (AR) inhibitors revealed a significant correlation of peroxisomal genes (MSigDB) with acquired resistance to AR inhibition. Notably, DECR2 overexpression was significantly associated with shorter overall survival (p < 0.05) in a metastatic CRPC cohort treated with AR inhibitors. We further showed that DECR2 overexpression in LNCaP cells significantly reduced sensitivity to AR inhibitor treatments and androgen-deprivation, suggesting a role for perFAO in promoting survival and treatment resistance. Conclusion: Our study has revealed a novel role for perFAO in PCa progression and treatment resistance. Importantly, this study also identified DECR2 as a key regulator of tumor cell proliferation and lipid metabolism, representing a potential novel therapeutic target for advanced, treatment-resistant PCa that currently lacks effective targeted therapies. Citation Format: Chui Yan Mah, An D. Nguyen, Takuto Niijima, Madison Helm, Jonas Dehairs, Feargal J. Ryan, Natalie Ryan, Ian G. Mills, Johannes V. Swinnen, David J. Lynn, Zeyad D. Nassar, Lisa M. Butler. Uncovering a novel role of peroxisomal β-oxidation in advanced, treatment-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1157.

Abstract 3441: A multivalent peptoid conjugate that inhibits therapy-resistant prostate cancer cell proliferation by modulating androgen receptor transcriptional activity

Abstract Prostate cancers adapt to androgen receptor (AR) pathway inhibitors and progress to castration resistance despite the continued expression and function of the AR. We developed a new approach to antagonize the proliferative activity of AR and inhibit castration-resistant prostate cancer (CRPC) growth using multivalent peptoid conjugates (MPCs). Our strategy is to display multiple copies of the AR antagonist, ethisterone, on a peptoid scaffold. Such a multivalent display increases the ligand’s effective local concentration, thus targeting AR with high affinity and affecting AR interactions with coregulators. Here, we report a new potent MPC called MPC309, which displays three ethisterone groups and binds to AR with nanomolar affinity. MPC309 reduces the proliferation of enzalutamide-resistant prostate cancer cells, including those harboring AR splice variants, AR ligand binding mutations, and non-canonical AR gene expression programs. We provide evidence that MPC309 enters cells via macropinocytosis, which facilitates the fluid-phase uptake of extracellular macromolecules. Macropinocytotic uptake of MPC309 enhances cancer cell-specific delivery, thus limiting systemic toxicities. MPC309 displays favorable pharmacological properties and produced significantly greater tumor suppression in xenograft studies than enzalutamide, the standard of care anti-androgen in clinical use. Mechanistically, MPC309 inhibits prostate cancer growth by eliciting a unique gene expression program through alterations in AR chromatin occupancy compared to dihydrotestosterone (DHT) or enzalutamide. Thus, MPC309 represents a novel AR antagonist that activates an AR anti-proliferative program to repress the growth of CRPC and supports further investigation of the MPC compound class for treating CRPC. Citation Format: Justine Habault, Jeffrey A. Schneider, Susan Ha, Rachel Ruoff, Joseph Puccini, Dafna Bar-Sagi, Kwok-Kin Wong, Amina Zoubeidi, Frank Claessens, David R. Wise, Susan K. Logan, Kent Kirshenbaum, Michael J. Garabedian. A multivalent peptoid conjugate that inhibits therapy-resistant prostate cancer cell proliferation by modulating androgen receptor transcriptional activity [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 3441.

Abstract 3069: Targeting noncanonical Wnt5a signaling suppresses the neural lineage network and overcomes enzalutamide resistance in advanced prostate cancer

Abstract Introduction and Objectives: Androgen receptor (AR) blockade using antiandrogens is a mainstay for the treatment of castration-resistant prostate cancer (CRPC). However, resistance to antiandrogens occurs unavoidably due to the escaping mechanisms of tumor cells that are not completely understood. Upregulation of non-canonical Wnt ligand Wnt5a is identified from circulating tumor cells from CRPC patients after being treated with enzalutamide. FZD2, the cognate frizzled receptor for Wnt5a, is the most commonly co-upregulated non-canonical Wnt signaling molecule in prostate cancer. In this study, we determined the role of non-canonical Wnt5a/FZD2 to enzalutamide resistance and neural lineage plasticity, and explore the potential of targeting Wnt5a/FZD2 to overcome resistance in advanced prostate cancer. Methods: Wnt5a and FZD2 expression was determined in enzalutamide-resistant C4-2B MDVR cells. Wnt5a and FZD2 expressions were modulated using specific siRNAs. Cell growth, colony formation, and migration were studied in vitro. Transcriptomic analysis was performed on C4-2B MDVR cells treated with FZD2 knocked down; gene program of non-canonical Wnt signaling, hallmark androgen response and AR-V7 associated genes, and neural lineage pathways were analyzed. A novel tRNA bioengineered Wnt5a siRNA was developed to target Wnt5a/FZD2 signaling. The effect of tRNA-siWnt5a on tumor growth, sensitivity to enzalutamide treatment, and neural lineage plasticity was evaluated in vitro and in vivo. Results: Wnt5a and FZD2 are highly upregulated in castration-resistant prostate cancer patients, which is verified overexpression in enzalutamide-resistant C4-2B MDVR cells. Knocking down Wnt5a and FZD2 diminishes the enrichment of the non-canonical Wnt signaling pathway. Downregulating Wnt5a and FZD2 expression by specific siRNAs suppresses prostate cancer cell proliferation and resensitized C4-2B MDVR cells to enzalutamide treatment. Suppressing Wnt5a and FZD2 abrogated the enrichment of gene programs regulating cancer cell survival/proliferation, and neural lineage pathways including neuron differentiation and embryonic stem cell markers. Using the bioengineered tRNA-Wnt5a siRNA effectively inhibited the growth of enzalutamide-resistant prostate cancer cells and resensitized tumor cells to enzalutamide treatment in vitro, and resistant CRPC PDX LuCaP35CR tumor growth in vivo. Conclusions: Our results suggest that activation of noncanonical Wnt5a/FZD2 signaling confers to enzalutamide resistance and neural lineage plasticity. Targeting the Wnt5a signal could provide benefits for CRPC patients with tumors expressing a high level of Wnt5a, FZD2, and lineage markers, not only enhancing the anti-tumor effects of enzalutamide but suppressing the potential of neuroendocrine differentiation in advanced prostate cancer patients. Citation Format: Shu Ning, Wei Lou, Chengfei Liu, Joy C. Yang, Alan P. Lombard, Leandro S. Abronzo, Neelu Batra, Aiming Yu, Christopher P. Evans, Allen C. Gao. Targeting noncanonical Wnt5a signaling suppresses the neural lineage network and overcomes enzalutamide resistance in advanced 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 3069.

MP20-11 ENZALUTAMIDE RESISTANCE RESULTS IN IMMUNOSUPPRESSIVE ALTERATIONS IN PROSTATE TUMOR IMMUNE MICROENVIRONMENT

You have accessJournal of UrologyCME1 Apr 2023MP20-11 ENZALUTAMIDE RESISTANCE RESULTS IN IMMUNOSUPPRESSIVE ALTERATIONS IN PROSTATE TUMOR IMMUNE MICROENVIRONMENT Pengfei Xu, Joy C. Yang, Bo Chen, Christopher Nip, Jonathan E Van Dyke, Christopher P. Evans, William J. Murphy, and Chengfei Liu Pengfei XuPengfei Xu More articles by this author , Joy C. YangJoy C. Yang More articles by this author , Bo ChenBo Chen More articles by this author , Christopher NipChristopher Nip More articles by this author , Jonathan E Van DykeJonathan E Van Dyke More articles by this author , Christopher P. EvansChristopher P. Evans More articles by this author , William J. MurphyWilliam J. Murphy More articles by this author , and Chengfei LiuChengfei Liu More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003245.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Emerging data suggested that enzalutamide-treated prostate cancer patients with increased programmed death-ligand 1 (PD-L1) expression may benefit from anti-PD-L1 treatment. Unfortunately, the Phase III IMbassador250 trial revealed that the combination of atezolizumab (PD-L1 inhibitor) and enzalutamide failed to extend overall survival in patients with castration-resistant prostate cancer (CRPC). The mechanisms underlying treatment failure remain unknown. In this study, we investigated the regulation of immunosuppressive signaling in enzalutamide resistant prostate cancer and characterized the immune infiltrating cells in murine prostate tumors. METHODS: The expression of interferon gamma-related genes was determined using qRT-PCR and/or western blotting. Androgen receptor (AR) and AR-V7 levels were downregulated using specific siRNA. Myc-CaP cells were chronically exposed to increasing concentrations of enzalutamide (5-50 μM) for >12 months, and the cells resistant to enzalutamide were referred to as Myc-CaP MDVR. The gene-regulating mechanisms in drug-resistant prostate cancer cells were determined by RNA sequencing analyses. Myc-CaP and Myc-CaP MDVR xenograft tumors were established in FVB mice, and tumor-infiltrating lymphocytes were isolated using Ficoll. The stained immune cells were determined by flow cytometry, and the data were analyzed using Flowjo. RESULTS: Immune-related signaling pathways (interferon alpha/gamma response, T cell activation, and cell chemotaxis pathways) were suppressed in C4-2B MDVR cells (p<0.001). However, PD-L1 expression was highly upregulated and negatively regulated by AR in C4-2B MDVR cells. Enzalutamide treatment decreased CD8+ T cell (14.8% vs. 10.3%, p<0.05) but increased monocytic myeloid-derived suppressor cell (M-MDSC) population (23.0% vs. 34.2%, p<0.05) and PD-L1 expression in murine Myc-CaP tumors. Consistently, chemotaxis and immune response-regulating signaling pathways were suppressed, and PD-L1 expression was increased in enzalutamide-resistant Myc-CaP MDVR cells. Notably, MDSC populations were significantly increased in Myc-CaP MDVR orthotopic tumors compared with those in Myc-CaP parental tumors (4.0% vs. 1.6%, p<0.01). Co-culturing bone marrow cells with Myc-CaP MDVR cells significantly promoted MDSC differentiation and polarized macrophages from the M1 to M2 phase. CONCLUSIONS: Immunosuppressive signaling is activated in enzalutamide resistant prostate cancer cells. The activated immune-related signatures involved in T cells, MDSC, and macrophages may reduce the efficacy of immune checkpoint inhibitors in enzalutamide-resistant prostate cancer. Source of Funding: This work was supported in part by grants NIH/NCI R37CA249108 (C, Liu) and R01CA251253 (C, Liu) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e279 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Pengfei Xu More articles by this author Joy C. Yang More articles by this author Bo Chen More articles by this author Christopher Nip More articles by this author Jonathan E Van Dyke More articles by this author Christopher P. Evans More articles by this author William J. Murphy More articles by this author Chengfei Liu More articles by this author Expand All Advertisement PDF downloadLoading ...

PD04-11 MCT INHIBITION RESENSITIZES ENZALUTAMIDE-RESISTANT PROSTATE CANCER CELLS TO ENZALUTAMIDE

You have accessJournal of UrologyCME1 Apr 2023PD04-11 MCT INHIBITION RESENSITIZES ENZALUTAMIDE-RESISTANT PROSTATE CANCER CELLS TO ENZALUTAMIDE Sayani Bhattacharjee, Rebecca Wynn, Puneet Sindhwani, Firas Petros, and Nagalakshmi Nadiminty Sayani BhattacharjeeSayani Bhattacharjee More articles by this author , Rebecca WynnRebecca Wynn More articles by this author , Puneet SindhwaniPuneet Sindhwani More articles by this author , Firas PetrosFiras Petros More articles by this author , and Nagalakshmi NadimintyNagalakshmi Nadiminty More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003228.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Prostate cancers (PCa) exhibit a unique metabolic profile with reliance on different forms of glucose metabolism at different stages of disease progression. Early stage PCa cells use the more efficient TCA cycle, while metastatic PCa cells switch to glycolysis (Warburg effect), leading to the accumulation of lactate. Monocarboxylate transporters (MCTs) play a major role in the export/import of lactate to maintain redox balance. Such metabolic reprogramming can lead to gain-of-function mutations and affect drug sensitivity. MCT expression levels are high in enzalutamide-resistant PCa cells. Hence, we hypothesized that MCT inhibition may be an attractive therapeutic strategy to overcome enzalutamide resistance in PCa cells. METHODS: We analyzed the expression levels of MCTs in PCa tissues using public datasets from Oncomine. We treated C4-2B and 22Rv1 parental and enzalutamide-resistant cells with varying concentrations of MCT inhibitors AR-C155858, AZD3965, or syrosingopine either singly or in combination with enzalutamide and assessed cell survival, cell viability, proliferation, clonogenic ability, survival in 3-D models, and glycolytic activity. We treated mouse xenografts of PCa cells with MCT inhibitors or enzalutamide either singly or in combination and assessed tumor growth. RESULTS: We found that the suppression of MCT activity using the MCT antagonists AR-C155858, AZD3965, or syrosingopine significantly diminished the proliferation, survival, and clonogenic ability of enzalutamide-resistant PCa cells compared with parental cells. MCT inhibition in combination with enzalutamide inhibited the metabolic adaptation of PCa cells. Tumor growth of enzalutamide-resistant PCa cell xenografts was suppressed significantly when treated with a combination of MCT inhibitors with enzalutamide. These findings imply that MCT inhibition may be an effective strategy to counter enzalutamide resistance in PCa. CONCLUSIONS: MCT activation may underlie the development of enzalutamide resistance in PCa and targeting it may represent a viable combinatorial option. Source of Funding: Department of Defense Prostate Prostate Cancer Research Program W81XWH2010794 (PC190332) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e146 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Sayani Bhattacharjee More articles by this author Rebecca Wynn More articles by this author Puneet Sindhwani More articles by this author Firas Petros More articles by this author Nagalakshmi Nadiminty More articles by this author Expand All Advertisement PDF downloadLoading ...

MP20-04 ARHGEF2 REGULATES LINEAGE PLASTICITY PROMOTING NEUROENDOCRINE DIFFERENTIATION AND TREATMENT RESISTANCE IN ADVANCED PROSTATE CANCER

You have accessJournal of UrologyCME1 Apr 2023MP20-04 ARHGEF2 REGULATES LINEAGE PLASTICITY PROMOTING NEUROENDOCRINE DIFFERENTIATION AND TREATMENT RESISTANCE IN ADVANCED PROSTATE CANCER Shu Ning, Wei Lou, Alan Lombard, Masuda Sharifi, Zachary Schaaf, Christopher Evans, and Allen Gao Shu NingShu Ning More articles by this author , Wei LouWei Lou More articles by this author , Alan LombardAlan Lombard More articles by this author , Masuda SharifiMasuda Sharifi More articles by this author , Zachary SchaafZachary Schaaf More articles by this author , Christopher EvansChristopher Evans More articles by this author , and Allen GaoAllen Gao More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003245.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Treatment-emergent neuroendocrine prostate cancer (NEPC) after androgen receptor (AR) targeted therapies has been an aggressive variant of prostate cancer with unfavorable prognosis and limited therapeutic options. This study aims to identify potential neural lineage signature genes and evaluate whether they could serve as early biomarkers to stratify patients for optimal treatment and potential therapeutic targets in advanced prostate cancer. METHODS: We performed transcriptomic analysis on the enzalutamide-resistant prostate cancer cell lines and advanced prostate cancer patient databases to identify neural lineage signature (NLS) genes including ARHGEF2. The correlation of NLS genes with clinicopathologic features was determined. RNA sequencing analysis and viability of tumor organoids were performed on C4-2B MDVR and H660 cells with ARHGEF2 knocking down and gene set enrichment was analyzed. RESULTS: We identified a 95-gene NLS representing the molecular landscape of neural precursor cell proliferation, embryonic stem cell pluripotency and neural stem cell differentiation, which may indicate a spectrum of early and intermediate stages of neuroendocrine differentiation. These NLS genes positively correlate with conventional neuroendocrine (NE) markers such as chromogranin and synaptophysin, and negatively correlate with AR and AR target genes. Differentially expressed NLS genes stratify small cell NEPC from prostate adenocarcinoma, which is closely associated with higher Gleason Score and metastasis status. Higher ARHGEF2 levels correlate with a shortened survival time in NEPC patients. ARHGEF2 regulates neuron differentiation and neural precursor cell proliferation which are significantly enriched in treatment-emergent NEPC patients and H660 cells. Upregulation of ARHGEF2 in both patient and experimental models positively correlates with NE markers. ARHGEF2 knocking down inhibited tumor organoids viability and NE markers in enzalutamide-resistant and NEPC cells. CONCLUSIONS: Our studies suggest that ARHGEF2 activation promotes neural lineage plasticity contributing to the survival and proliferation of enzalutamide-resistance and NEPC cells. ARHGEF2 could serve as a biomarker to stratify advanced prostate cancer patients for better treatment options and a potential therapeutic target in advanced prostate cancer. Source of Funding: NIH CA253605, CA 225836, CA250082, DOD PC180180 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e276 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Shu Ning More articles by this author Wei Lou More articles by this author Alan Lombard More articles by this author Masuda Sharifi More articles by this author Zachary Schaaf More articles by this author Christopher Evans More articles by this author Allen Gao More articles by this author Expand All Advertisement PDF downloadLoading ...