Enzalutamide-resistant Prostate Cancer Research Articles

Oxidative stress in peroxisomes induced by androgen receptor inhibition through peroxisome proliferator-activated receptor promotes enzalutamide resistance in prostate cancer.

Androgen receptor (AR)-targeting therapy induces oxidative stress in prostate cancer. However, the mechanism of oxidative stress induction by AR-targeting therapy remains unclear. This study investigated the mechanism of oxidative stress induction by AR-targeting therapy, with the aim to develop novel therapeutics targeting oxidative stress induced by AR-targeting therapy. Intracellular reactive oxygen species (ROS) was examined by fluorescence microscopy and flow cytometry analysis. The effects of silencing gene expression and small molecule inhibitors on gene expression and cytotoxic effects were examined by quantitative real-time PCR and cell proliferation assay. ROS induced by androgen depletion co-localized with peroxisomes in prostate cancer cells. Among peroxisome-related genes, PPARA was commonly induced by AR inhibition and involved in ROS production via PKC signaling. Inhibition of PPARα by specific siRNA and a small molecule inhibitor suppressed cell proliferation and increased cellular sensitivity to the antiandrogen enzalutamide in prostate cancer cells. This study revealed a novel pathway by which AR inhibition induced intracellular ROS mainly in peroxisomes through PPARα activation in prostate cancer. This pathway is a promising target for the development of novel therapeutics for prostate cancer in combination with AR-targeting therapy such as antiandrogen enzalutamide.

Discovery of novel biphenyl derivatives as androgen receptor degraders for the treatment of enzalutamide-resistant prostate cancer

Second-generation AR antagonists, such as enzalutamide, are the primary therapeutic agents for advanced prostate cancer. However, the development of both primary and secondary drug resistance leads to treatment failures and patient mortality. Bifunctional agents that simultaneously antagonize and degrade AR block the AR signaling pathway more completely and exhibit excellent antiproliferative activity against wild-type and drug-resistant prostate cancer cells. Here, we reported the discovery and optimization of a series of biphenyl derivatives as androgen receptor antagonists and degraders. These biphenyl derivatives exhibited potent antiproliferative activity against LNCaP and 22Rv1 cells. Our discoveries enrich the diversity of small molecule AR degraders and offer insights for the development of novel AR degraders for the treatment of enzalutamide-resistant prostate cancer.

Abstract 6603: A TBX2-driven signaling switch from androgen receptor to glucocorticoid receptor confers enzalutamide resistance in prostate cancer

Abstract Background: Recent studies suggest that glucocorticoid receptor (GR) activation can cause enzalutamide resistance in advanced prostate cancer (PCa) via functional bypass of androgen receptor (AR) signaling. However, the specific molecular mechanism(s) driving this process remain unknown. In an effort to identify drivers of prostate cancer progression, in a previous study, we determined that TBX2, a developmental T-box transcription factor (TF) master regulator, is over-expressed in CRPC and drives bone metastatic progression. A recent report confirmed that TBX2 and GR are two of the four TFs that drive enzalutamide resistance in advanced PCa. Our current study demonstrates that TBX2 with known repressor and activator functions, is the molecular switch that represses AR levels while activating GR expression thereby resulting in the replacement of AR signaling to control tumor growth. Methods: We genetically modulated TBX2 using multiple approaches: a) dominant negative, DN, to block TBX2 (TBX2DN), and b) overexpression, OE, to increase TBX2 expression (TBX2OE), c) shRNA mediated knockdown (shTBX2). RNA-seq was performed, and qRT-PCR, Western blot and immunohistochemical (IHC) analyses were used for validation. Further, we used chromatin immunoprecipitation (ChIP) and site directed mutagenesis (SDM) to confirm TBX2 binding on the AR promoter. We also used co-immunoprecipitation (Co-IP) to determine protein partners of TBX2. Results: Mechanistically, our studies revealed that TBX2 bound to the promoters of both AR and GATA2, an AR coregulator, thereby resulting in a bimodal repression of AR expression. Conversely, TBX2 upregulated GR via direct GR promoter binding and TBX2-GR protein-protein interaction. Together, concurrent repression of the AR and activation of GR resulted in enzalutamide resistance. Importantly, we found that SP2509, an allosteric inhibitor of the demethylase-independent function of LSD1, a TBX2-interacting protein in the COREST complex, can disrupt both TBX2-LSD1 and TBX2-GR protein-protein interactions thereby uncovering a unique mode of SP2509 action in CRPC. Together, our study supports a molecular model of CRPC wherein: 1) TBX2, LSD1 and GR proteins interface with each other, and 2) pharmacological inhibition of LSD1 blocks the TBX2-driven AR-to-GR switch through disruption of TBX2-GR interaction. Conclusions: In summary, our study identifies TBX2 as the molecular switch that drives the AR to GR signaling bypass thereby conferring enzalutamide resistance. Further, our study provides key insights into a potential therapeutic modality for targeting the AR to GR signaling switch via disruption of the TBX2-LSD1 and TBX2-GR protein-protein interactions Citation Format: Sayanika Dutta, Girijesh Kumar Patel, Hamed Khedmatgozar, Daniel Latour, Jonathan Welsh, Manisha Tripathi, Srinivas Nandana. A TBX2-driven signaling switch from androgen receptor to glucocorticoid receptor confers 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 6603.

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 5838: Un-methylation of NUDT21 promotes the development of enzalutamide resistance in prostate cancer by regulation of 3’UTR usage switch

Abstract Enzalutamide, a second-generation anti-androgen drug, is a new treatment option for castration resistant prostate cancer (CRPC) patient. However, almost all CRPC patients develop drug resistance for enzalutamide after six to twelve months of treatment. According to our current findings, 3'UTR usage switches of RNA transcripts were observed in the prostate cancer specimen after acquiring enzalutamide resistance and nudix hydrolase 21 (NUDT21), an alternative polyadenylation (APA) regulator, was overexpressed in the enzalutamide resistant (EnzaR) cells, suggesting its potential role involved in regulation of 3'UTR usage switch. However, the underlying mechanism of how NUDT21 regulates 3'UTR usage switch during the development of enzalutamide resistance is still unclear. Interestingly, a potential mono-methylation (MME) is identified in NUDT21 protein by us, and it has never been examined before. Thus, we hypothesize that MME of NUDT21 may regulate the specificity of 3'UTR usage switch and involve in the development of EnzaR. To test the idea, a monoclonal antibody recognized NUDT21 MME was generated by us. Moreover, protein arginine methyltransferase 7 (PRMT7) was identified to be responsible for adding MME to NUDT21. In addition, androgen treatment increased both expression levels of NUDT21 MME and PRMT7 while decreased the expression level of NUDT21 total form in LNCaP cells via androgen receptor (AR) dependent manner. Next, co-treatment of enzalutamide and PRMT7 inhibitor leaded to increase of un-MME form of NUDT21 and it greatly enhanced the enzalutamide resistance in LNCaP cells. Importantly, binding profile of NUDT21 total form indeed had 3'UTR usage switch from distal to proximal APA site in COUP-TFII transcript after acquiring enzalutamide resistance, while its MME form attenuated this phenomenon. In addition, expression levels of NUDT21 MME and its total form were shown to conversely express in prostate cancer specimens. Finally, un-MME form of NUDT21 promoted the cancer growth in vitro and in vivo while its MME form did not. Taken together, our results suggest that down-regulation of PRMT7 by enzalutamide may lead to decrease of NUDT21 MME level and contribute to develop enzalutamide resistance by causing the 3'UTR usage switch of NUDT21 downstream transcript. Citation Format: Shin-Chih (Kenny) Lin, Shih-Chieh Lin. Un-methylation of NUDT21 promotes the development of enzalutamide resistance in prostate cancer by regulation of 3’UTR usage switch [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 5838.

Abstract 3266: Plk1 promotes Pdcd4 degradation to enhance enzalutamide resistance in prostate cancer

Abstract The androgen receptor (AR) signaling pathway plays critical role in the development of prostate cancer (PCa), including castration-resistant prostate cancer (CRPC). As a result, androgen signaling inhibitors (ASIs) like abiraterone and enzalutamide (ENZ) have emerged as the first-line treatment for CRPC. Nevertheless, the limited success of ASIs underscores the urgency of developing new approaches for managing CRPC patients who have become unresponsive to ASIs. Programmed cell death 4 (Pdcd4) is a tumor suppressor, which is frequently down-regulated during PCa progression. It has been shown that reduced Pdcd4 expression promotes both AR-dependent and AR-independent growth in PCa. In this study, we found that down-regulation of Pdcd4 led to acquisition of ENZ resistance in PCa. We also observed that down-regulation of Pdcd4 resulted in activation of mTORC2 activity. Additionally, knockdown of Rictor, a component of mTORC2 complex, enhanced sensitivity to ENZ treatment in PCa. These findings suggested that activation of mTORC2 due to Pdcd4 loss contributes to ASI resistance in PCa. To understand how Pdcd4 is down-regulated in the PCa, we identified that polo-like kinase (Plk1) phosphorylated Pdcd4 at Ser239. Phosphorylation of Pdcd4 by Plk1 led to ubiquitin-mediated proteasomal degradation of Pdcd4. Over-expression of the phosphomimic Pdcd4(S239D) in 22Rv1 cells promoted tumor cell growth in cultured cells and mice. Taken together, our data suggest that targeting Plk1/Pdcd4/mTORC2 signaling axis holds promise as a strategy to overcome ENZ resistance in PCa. Citation Format: Qing Wang, Qiongsi Zhang, Yanquan Zhang, Xiaoqi Liu, Hsin-Sheng Yang. Plk1 promotes Pdcd4 degradation to enhance 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 3266.

Abstract 1725: Role of PRMT5 in neuroendocrine prostate cancer development

Abstract Background: Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer responsible for an estimated 20-30% of castration resistant prostate cancer (CRPC) deaths. While NEPC can arise de novo, the majority of these cases emerge as treatment-induced NEPC (tNEPC). Our data establish PRMT5:MEP50 as a key epigenetic regulator in NEPC and provide a potential predictive biomarker for tNEPC. Methods: Here we generated an in vitro cell culture model and mouse model to mimic the clinical conditions in androgen signaling inhibitor (ASI) or androgen deprivation treatment (ADT) We developed an in vitro cell culture and in vivo model to mimic the clinical conditions in enzalutamide-resistant prostate cancer. Regulation and role of protein arginine methyltransferase 5 (PRMT5) and its cofactor methylosome protein 50 (MEP50) were determined by treating cells and mice tumors with doxycycline inducible-shRNAs and catalytic inhibitors followed by analysis of cell viability, neurite growth, and effects on neuroendocrine related gene transcription. Pro-NEPC development effects were analyzed by immunofluorescence, immunohistochemical analysis, and western blot. PRMT5:MEP50-dependent methyltransferase activity was further validated by observing histone deposition levels via western blot and ChIP-qPCR. For clinical correlation, PRMT5 and MEP50 expression levels were comprehensively analyzed in both CRPC and NEPC patient prostate tissue samples. Results: Our clinical and computational analyses have identified increased expression of PRMT5:MEP50 in NEPC. Overexpression of PRMT5 and MEP50 in prostate cancer cells is sufficient to induce neuroendocrine differentiation (NED) in prostate cancer cells. Gene depletion of PRMT5 and MEP50 prevented the NE phenotype induced by androgen receptor inhibition and resensitized the prostate cancer cells to ASI/ADT. PRMT5 and MEP50 facilitate chromatin modulation through the dimethylation of H4R3, which is highly correlated with the NED phenotype. Moreover, overexpression of PRMT5 and MEP50 in mouse prostate induces NEPC development, and gene downregulation can prevent NED in a xenograft mouse model. Elevated PRMT5 and MEP50 correlated with increased recurrence in prostate cancer patients receiving ADT and their recurrence probability after the treatment. Conclusion: Our data establish PRMT5:MEP50 as a key epigenetic regulator in NEPC and provide a potential predictive biomarker for tNEPC. Citation Format: Hye Seung Nam, Xuehong Deng, Chang-Deng Hu. Role of PRMT5 in neuroendocrine prostate cancer development [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 1725.

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 5837: Aryl hydrocarbon receptor is critical for enzalutamide-resistant prostate cancer growth

Abstract Therapy resistance is a ubiquitous and major challenge for curing cancer including prostate cancer. As the most prevalent non-skin cancer for men, prostate cancer is responsible for more than 30,000 deaths annually in the U.S. Despite a favorable prognosis at early stages, prostate cancer progressively develop into an incurable castration-resistant stage (CRPC) where prostate cancer grows in a low-androgen environment. Enzalutamide (ENZ) is the first drug approved by FDA for treating CRPC by inhibiting the residual androgen signaling. While ENZ significantly improves overall survival and quality of life, resistance inevitably develops and greatly limits its benefit. To understand ENZ resistance and identify novel therapeutic targets with the hope of overcoming resistance, we performed RNA-seq to analyze mRNA expression profiles from three ENZ-resistant human CRPC cell lines (C4-2BENZR, CWR-R1ENZR and VCaPENZR) and identified the aryl hydrocarbon receptor (AHR) among the 114 commonly upregulated genes. AHR is a ligand-dependent transcription factor that has been shown to drive disease progression in multiple types of cancer. Stable shRNA-mediated knockdown of AHR substantially decreased growth of ENZ-resistant CRPC cells. Our pilot study also showed that AHR knockdown resulted in fewer and smaller CWR-R1ENZR tumors in a xenograft mouse model. To elucidate the underlying mechanism of AHR-mediated cancer growth, gene set enrichment analysis (GSEA) revealed a declining trend of enrichment of the 114 commonly ENZ-upregulated genes among differentially altered genes upon AHR knockdown in ENZ-resistant cells, suggesting that AHR may act as a master regulator of ENZ resistance. Moreover, we found that the Hallmark androgen response gene set is downregulated in AHR-silenced ENZ-resistant cells, implying a role of AHR in sustaining androgen response for ENZ-resistance. Consistently, we performed AHR ChIP-seq and confirmed that AHR bound to promoter regions of several androgen-regulated genes in CWR-R1ENZR cells. In addition, three peroxisome-related Hallmark gene sets are also negatively enriched in AHR-knockdown cells, which provides a rationale for further exploring a potential AHR-dependent connection of peroxisomal function to ferroptosis as a newly emerging mechanism of action for ENZ. In summary, our study suggests that AHR plays a critical role in maintaining ENZ-resistant CRPC growth with the likely involvement of modulating androgen signaling and ferroptosis susceptibility. Citation Format: Chia-Hui Chen, Boyang Jason Wu. Aryl hydrocarbon receptor is critical for enzalutamide-resistant prostate cancer growth [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 5837.

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.

Development of a Double-Stapled Peptide Stabilizing Both α-Helix and β-Sheet Structures for Degrading Transcription Factor AR-V7.

Peptide drugs offer distinct advantages in therapeutics; however, their limited stability and membrane penetration abilities hinder their widespread application. One strategy to overcome these challenges is the hydrocarbon peptide stapling technique, which addresses issues such as poor conformational stability, weak proteolytic resistance, and limited membrane permeability. Nonetheless, while peptide stapling has successfully stabilized α-helical peptides, it has shown limited applicability for most β-sheet peptide motifs. In this study, we present the design of a novel double-stapled peptide capable of simultaneously stabilizing both α-helix and β-sheet structures. Our designed double-stapled peptide, named DSARTC, specifically targets the androgen receptor (AR) DNA binding domain and MDM2 as E3 ligase. Serving as a peptide-based PROTAC (proteolysis-targeting chimera), DSARTC exhibits the ability to degrade both the full-length AR and AR-V7. Molecular dynamics simulations and circular dichroism analysis validate the successful constraint of both secondary structures, demonstrating that DSARTC is a "first-in-class" heterogeneous-conformational double-stapled peptide drug candidate. Compared to its linear counterpart, DSARTC displays enhanced stability and an improved cell penetration ability. In an enzalutamide-resistant prostate cancer animal model, DSARTC effectively inhibits tumor growth and reduces the levels of both AR and AR-V7 proteins. These results highlight the potential of DSARTC as a more potent and specific peptide PROTAC for AR-V7. Furthermore, our findings provide a promising strategy for expanding the design of staple peptide-based PROTAC drugs, targeting a wide range of "undruggable" transcription factors.

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.

Retraction Note: Targeting KDM1B-dependent miR-215-AR-AGR2-axis promotes sensitivity to enzalutamide-resistant prostate cancer.

Retraction Note: Targeting KDM1B-dependent miR-215-AR-AGR2-axis promotes sensitivity to enzalutamide-resistant prostate cancer.

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.

LncRNA SNHG4 promotes prostate cancer cell survival and resistance to enzalutamide through a let-7a/RREB1 positive feedback loop and a ceRNA network

BackgroundProstate cancer threatens the health of men over sixty years old, and its incidence ranks first among all urinary tumors among men. Enzalutamide remains the first-line drug for castration-resistant prostate cancer, however, tumors inevitably become resistant to enzalutamide. Hence, it is of great importance to investigate the mechanisms that induce enzalutamide resistance in prostate cancer cells.MethodsBioinformatic analyzing approaches were used to identified the over-expressed genes in prostate cancer tumor tissues from three GEO datasets. qRT-PCR, western blotting and immunochemistry/In situ hybridization staining assays were performed to assess the expression of SNHG4, RRM2, TK1, AURKA, EZH2 and RREB1. Cell cycle was measured by flow cytometry. CCK-8, plate colony formation and EdU assays were performed to assess the cell proliferation. Senescence-associated β-Gal assay was used to detect the cell senescence level. γ-H2AX staining assay was performed to assess the DNA damages of PCa cells. Luciferase reporter assay and RNA immunoprecipitation assay were performed to verify the RNA-RNA interactions. Chromatin immunoprecipitation assay was performed to assess the bindings between protein and genomic DNA.ResultsWe found that RRM2 and NUSAP1 are highly expressed in PCa tumors and significantly correlated with poor clinical outcomes in PCa patients. Bioinformatic analysis as well as experimental validation suggested that SNHG4 regulates RRM2 expression via a let-7 miRNA-mediated ceRNA network. In addition, SNHG4 or RRM2 knockdown significantly induced cell cycle arrest and cell senescence, and inhibited DNA damage repair and cell proliferation, and the effects can be partially reversed by let-7a knockdown or RRM2 reoverexpression. In vitro and in vivo experiments showed that SNHG4 overexpression markedly enhanced cell resistance to enzalutamide. RREB1 was demonstrated to transcriptionally regulate SNHG4, and RREB1 was also validated to be a target of let-7a and thereby regulated by the SNHG4/let-7a feedback loop.ConclusionOur study uncovered a novel molecular mechanism of lncRNA SNHG4 in driving prostate cancer progression and enzalutamide resistance, revealing the critical roles and therapeutic potential of RREB1, SNHG4, RRM2 and let-7 miRNAs in anticancer therapy.

A Multivalent Peptoid Conjugate Modulates Androgen Receptor Transcriptional Activity to Inhibit Therapy-resistant Prostate Cancer.

Prostate cancers adapt to androgen receptor (AR) pathway inhibitors and progress to castration resistance due to ongoing AR expression and function. To counter this, we developed a new approach to modulate the AR and inhibit castration-resistant prostate cancer (CRPC) using multivalent peptoid conjugates (MPC) that contain multiple copies of the AR-targeting ligand ethisterone attached to a peptidomimetic scaffold. Here, we investigated the antitumor effects of compound MPC309, a trivalent display of ethisterone conjugated to a peptoid oligomer backbone that binds to the AR with nanomolar affinity. MPC309 exhibited potent antiproliferative effects on various enzalutamide-resistant prostate cancer models, including those with AR splice variants, ligand-binding mutations, and noncanonical AR gene expression programs, as well as mouse prostate organoids harboring defined genetic alterations that mimic lethal human prostate cancer subtypes. MPC309 is taken up by cells through macropinocytosis, an endocytic process more prevalent in cancer cells than in normal ones, thus providing an opportunity to target tumors selectively. MPC309 triggers a distinct AR transcriptome compared with DHT and enzalutamide, a clinically used antiandrogen. Specifically, MPC309 enhances the expression of differentiation genes while reducing the expression of genes needed for cell division and metabolism. Mechanistically, MPC309 increases AR chromatin occupancy and alters AR interactions with coregulatory proteins in a pattern distinct from DHT. In xenograft studies, MPC309 produced significantly greater tumor suppression than enzalutamide. Altogether, MPC309 represents a promising new AR modulator that can combat resistant disease by promoting an AR antiproliferative gene expression program.

Downregulation of EZH2 inhibits epithelial-mesenchymal transition in enzalutamide-resistant prostate cancer.

Androgen signaling inhibitors (ASI) have been approved for treatment of metastatic castration-resistant prostate cancer (mCRPC). However, the limited success of ASI in clinic justifies an urgent need to identify new targets and develop novel approaches for treatment. EZH2 significantly increases in prostate cancer (PCa). Little is understood, however, regarding the roles of EZH2 in Enzalutamide-resistant (EnzR) mCRPC. We firstly investigated the levels of EZH2 and the altered pathways in public database which was comprised with primary and metastatic PCa patient tumors. To elucidate the roles of EZH2 in mCRPC, we manipulated EZH2 in EnzR PCa cell lines to examine epithelial-mesenchymal transition (EMT). To dissect the underlying mechanisms, we measured the transcription levels of EMT-associated transcription factors (TFs). We found that EZH2 was highly expressed in mCRPC than that of primary PCa tumors and that EnzR PCa cells gained more EMT characteristics than those of enzalutamide-sensitive counterparts. Further, loss of EZH2-induced inhibition of EMT is independent of polycomb repressive complex 2 (PRC2). Mechanistically, downregulation of EZH2 inhibits transcription of EMT-associated TFs by repressing formation of H3K4me3 to the promotor regions of the TFs. We identified the novel roles of EZH2 in EnzR mCRPC. EnzR PCa gains more EMT properties than that of enzalutamide-sensitive PCa. Loss of EZH2-assocaited inhibition of EMT is PRC2 independent. Downregulation of EZH2 suppresses EMT by impairing formation of H3K4me3 at the promotor regions, thus repressing expression of EMT-associated TFs.