Castration-resistant prostate cancer (CRPC) remains highly lethal and in need of novel, actionable therapeutic targets. The pioneer factor GATA2 is a significant prostate cancer (PC) driver and linked to poor prognosis. GATA2 directly promotes androgen receptor (AR) gene expression (both full-length and splice-variant) and facilitates AR binding to chromatin, recruitment of coregulators, and target gene transcription. Unfortunately, there is no clinically applicable GATA2 inhibitor available at the moment. Using a bioinformatics algorithm, we screened in silico 2,650 clinically relevant drugs for a potential GATA2 inhibitor. Validation studies used cytotoxicity assays (MTT), global gene expression analysis, reporter assay, reverse phase protein array analysis (RPPA), and immunoblotting. We examined target engagement via cellular thermal shift assay (CETSA), ChIP-qPCR, and GATA2 DNA-binding assay. We identified the vasodilator dilazep as a potential GATA2 inhibitor and confirmed on-target activity via CETSA. Dilazep exerted anticancer activity across a broad panel of GATA2-dependent PC cell lines in vitro and in a PDX model in vivo. Dilazep inhibited GATA2 recruitment to chromatin and suppressed the cell cycle program, transcriptional programs driven by GATA2, AR, and c-MYC, and the expression of several oncogenic drivers, including AR, c-MYC, FOXM1, CENPF, EZH2, UBE2C, and RRM2, as well as of several mediators of metastasis, DNA damage repair and stemness. In conclusion, we provide, via an extensive compendium of methodologies, proof-of-principle that a small molecule can inhibit GATA2 function and suppress its downstream AR, c-MYC, and other PC-driving effectors. We propose GATA2 as a therapeutic target in CRPC.
Background: Prostate cancer (PC) is the single most common and second-most lethal cancer in men, with over 268,000 estimated cases and over 34,500 estimated deaths in the US in 2022. The Speckle-Type POZ protein (SPOP) mutant subclass of PC accounts for 10% to 15% of all primary PC cases. SPOP is an adaptor for Cullin3/Ring (CUL3-RING)-type E3 ubiquitin ligase complexes and provides substrate specificity. The Cancer Genome Atlas (TCGA) studies show that SPOP is the most frequently mutated gene in primary prostate cancer (PC). Interestingly, PC-associated SPOP mutations are always missense and occur in a heterozygous fashion. The current gap in knowledge is the lack of understanding of the role of wildtype SPOP in PC. Methods: By utilizing prostate specific SPOP knockout (KO) mice, we recently reported increased levels of AR and MYC protein and increased cellular turnover (both proliferation and apoptosis) in the prostate luminal epithelium compared to wildtype prostates. We now characterized these mice for the expression of Cre protein and SPOP mRNA at different age using immunohistochemistry and RNA in situ hybridization. Furthermore, we performed RNA-sequencing analysis in the SPOP knockout mice and matched control littermates. Moreover, we performed RNA-seq in LNCaP, LNCaP-Abl, and RWPE1 cells following SPOP inhibition via siRNA targeting SPOP. Finally, we compared our SPOP inhibition signature from in vitro cell lines and prostate specific SPOP knockout murine model to gain insights about the role of wildtype SPOP protein in the prostate epithelium. Result: Using our Spopfl/fl;PBCre+ model, we observed SPOP floxed cells are rapidly lost and the murine prostate epithelium was repopulated with SPOP wildtype carrying cells. Similarly, knockdown (KD) of SPOP through siRNA treatment in a panel of PC cell lines resulted in a significant reduction in cell viability. These observations suggest that SPOP is important for the normal prostate cell viability. Further transcriptomic profiling of SPOP KO (from transgenic murine model) as well as siSPOP treated in vitro prostate cell lines revealed a significant reduction in the transcriptional activity of the AR. Conclusion: Our data illustrate for the first time a critical role for SPOP in the growth and survival of the prostate epithelium and prostate cancer cell. Our findings further validates SPOP as a important therapeutic target for the treatment of prostate cancer. Citation Format: Kinza Rizwan, Darlene Skapura, Cammy Mason, Cristian Coarfa, Nicholas Mitsiades, Damian Young, Salma Kaochar. SPOP: An essential gene for normal and prostate tumor cells [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 1705.
Background: Prostate cancer (PC) is the single most common and second-most lethal cancer in men, with over 268,000 estimated cases and over 34,500 estimated deaths in the US in 2022. The Speckle-Type POZ protein (SPOP) mutant subclass of PC accounts for 10% to 15% of all primary PC cases. SPOP is an adaptor for Cullin3/Ring (CUL3-RING)-type E3 ubiquitin ligase complexes and provides substrate specificity. The Cancer Genome Atlas (TCGA) studies show that SPOP is the most frequently mutated gene in primary prostate cancer (PC). Interestingly, PC-associated SPOP mutations are always missense and occur in a heterozygous fashion. The current gap in knowledge is the lack of understanding of the role of wildtype SPOP in PC. Methods: By utilizing prostate specific SPOP knockout (KO) mice, we recently reported increased levels of AR and MYC protein and increased cellular turnover (both proliferation and apoptosis) in the prostate luminal epithelium compared to wildtype prostates. We now characterized these mice for the expression of Cre protein and SPOP mRNA at different age using immunohistochemistry and RNA in situ hybridization. Furthermore, we performed RNA-sequencing analysis in the SPOP knockout mice and matched control littermates. Moreover, we performed RNA-seq in LNCaP, LNCaP-Abl, and RWPE1 cells following SPOP inhibition via siRNA targeting SPOP. Finally, we compared our SPOP inhibition signature from in vitro cell lines and prostate specific SPOP knockout murine model to gain insights about the role of wildtype SPOP protein in the prostate epithelium. Result: Using our Spopfl/fl;PBCre+ model, we observed SPOP floxed cells are rapidly lost and the murine prostate epithelium was repopulated with SPOP wildtype carrying cells. Similarly, knockdown (KD) of SPOP through siRNA treatment in a panel of PC cell lines resulted in a significant reduction in cell viability. These observations suggest that SPOP is important for the normal prostate cell viability. Further transcriptomic profiling of SPOP KO (from transgenic murine model) as well as siSPOP treated in vitro prostate cell lines revealed a significant reduction in the transcriptional activity of the AR. Conclusion: Our data illustrate for the first time a critical role for SPOP in the growth and survival of the prostate epithelium and prostate cancer cell. Our findings further validates SPOP as a important therapeutic target for the treatment of prostate cancer. Citation Format: Kinza Rizwan, Darlene Skapura, Cammy Mason, Cristian Coarfa, Nicholas Mitsiades, Damian Young, Salma Kaochar. SPOP loss places the prostate luminal epithelial cells at a selective disadvantage [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 B069.
African Americans have the highest mortality rate and shortest survival of any racial/ethnic group in the US for most cancers. An estimated 29,570 cases of prostate cancer are expected to be diagnosed in AA men in 2020. The average annual incidence rate for African American men is 76% higher than the rate in Caucasian Americans. Prostate cancer is the second leading cause of cancer death in African American men, with an estimated 5,350 deaths expected in 2020, which is the highest of all ethnic groups (2.2X higher than Caucasian men). These disparities are appalling and demand immediate action. While the underlying causes are multifactorial, recent studies demonstrate that African American men are at higher risk of being diagnosed with prostate cancer, have higher Gleason grades, and increased risk of progression after radical prostatectomy (RP), even in equal access settings and after accounting for socioeconomic status. These data strongly argue that there is a significant underlying biological/genetic difference between African Americans and Caucasian American men with prostate cancer. Importantly, they highlight an unmet need in order to deliver personalized therapeutic options and ultimately, improve clinical outcomes. Unfortunately, most preclinical studies are performed in prostate cancer cell lines and patient-derived xenografts (PDXs) isolated from patients of Caucasian origin. At Baylor College of Medicine, we are leveraging the high numbers of minority cancer patients in our GU clinics, to generate and utilize innovative prostate cancer PDX and organoid models of diverse racial/ethnic minority origin, which will broaden our understanding of the molecular basis of the disease as well as expedite the drug discovery process. We will discuss preliminary findings from our ongoing studies in genomic and transcriptomic profiling of African American patients, progress in our development of novel prostate cancer models, and lastly, emerging therapeutic opportunities based on precision oncology. Citation Format: Salma Kaochar, Patricia Castro, Sandra Grimm, Darlene Skapura, Matthew Rodriguez, Maurice Dufilho, Cammy Mason, Maria Machado Heredia, Quentxia Wrighting, Jami Daly, Matthew Robertson, Nora Navone, Jacob Berchuck, Matthew Freeman, Zoltan Szallasi, Albert Dobi, Guiherme Godoy, Michael Scheurer, Heidi Dowst, Susan Hilsenbeck, Martha Mims, Anita Sabichi, Edward Yen, Cristian Coarfa, Michael Ittmann, Nicholas Mitsiades. Development of Novel Models and Identification of Therapeutic Vulnerabilities in Highly Aggressive Prostate Cancer In African American Men [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr IA15.
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