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Characterization of ENZ-resistant cell lines

Clinical characteristics of prostate cancer patients with matched diagnostic (archival) castration-sensitive and metastatic castration-resistant tissue biopsies

IKKB immunohistochemistry validation

16DCRPC cell line variants expressing BCL-2 and IKKB are resistant to ENZ

Purpose:Although enzalutamide (ENZ) has been widely used to treat de novo or castration-resistant metastatic prostate cancer, resistance develops and disease progression is ultimately inevitable. There are currently no approved targeted drugs to specifically delay or overcome ENZ resistance.Experimental Design:We selected several ENZ-resistant cell lines that replicated clinical characteristics of the majority of patients with ENZ-resistant disease. A high-throughput pharmacologic screen was utilized to identify compounds with greater cytotoxic effect for ENZ-resistant cell lines, compared with parental ENZ-sensitive cells. We validated the potential hits in vitro and in vivo, and used knockdown and overexpression assays to study the dependencies in ENZ-resistant prostate cancer.Results:ABT199 (BCL-2 inhibitor) and IMD0354 (IKKB inhibitor) were identified as potent and selective inhibitors of cell viability in ENZ-resistant cell lines in vitro and in vivo which were further validated using loss-of-function assays of BCL-2 and IKKB. Notably, we observed that overexpression of BCL-2 and IKKB in ENZ-sensitive cell lines was sufficient for the emergence of ENZ resistance. In addition, we confirmed that BCL-2 or IKKB inhibitors suppressed the development of ENZ resistance in xenografts. However, validation of both BCL-2 and IKKB in matched castration-sensitive/resistant clinical samples showed that, concurrent with the development of ENZ/abiraterone resistance in patients, only the protein levels of IKKB were increased.Conclusions:Our findings identify BCL-2 and IKKB dependencies in clinically relevant ENZ-resistant prostate cancer cells in vitro and in vivo, but indicate that IKKB upregulation appears to have greater relevance to the progression of human castrate-resistant prostate cancer.

List of antibodies used for Western Blot analyses

Emergence of ENZ resistance can be prevented by BCL-2 or IKKB inhibitor in vivo and is associated with IKKB upregulation

BCL-2 immunohistochemistry validation

Treatment of BCL-2 and IKKB inhibitor decreases cell growth and induces cell death in ENZ-resistant cell lines

Compound full list of two libraries

Pharmacological screens identify BCL-2 and IKKB inhibitor that specifically targets ENZ-resistant cell lines

List of primers used for qPCR analyses

Purpose: Although enzalutamide (ENZ) has been widely used to treat de novo or castration-resistant metastatic prostate cancer, resistance develops and disease progression is ultimately inevitable. There are currently no approved targeted drugs to specifically delay or overcome ENZ resistance. Experimental Design: We selected several ENZ-resistant cell lines that replicated clinical characteristics of the majority of patients with ENZ-resistant disease. A high-throughput pharmacologic screen was utilized to identify compounds with greater cytotoxic effect for ENZ-resistant cell lines, compared with parental ENZ-sensitive cells. We validated the potential hits in vitro and in vivo, and used knockdown and overexpression assays to study the dependencies in ENZ-resistant prostate cancer. Results: ABT199 (BCL-2 inhibitor) and IMD0354 (IKKB inhibitor) were identified as potent and selective inhibitors of cell viability in ENZ-resistant cell lines in vitro and in vivo which were further validated using loss-of-function assays of BCL-2 and IKKB. Notably, we observed that overexpression of BCL-2 and IKKB in ENZ-sensitive cell lines was sufficient for the emergence of ENZ resistance. In addition, we confirmed that BCL-2 or IKKB inhibitors suppressed the development of ENZ resistance in xenografts. However, validation of both BCL-2 and IKKB in matched castration-sensitive/resistant clinical samples showed that, concurrent with the development of ENZ/abiraterone resistance in patients, only the protein levels of IKKB were increased. Conclusions: Our findings identify BCL-2 and IKKB dependencies in clinically relevant ENZ-resistant prostate cancer cells in vitro and in vivo, but indicate that IKKB upregulation appears to have greater relevance to the progression of human castrate-resistant prostate cancer.

Introduction: Enzalutamide is a recently approved potent androgen receptor antagonist for prostate cancer both in the pre- and post-docetaxel settings. Its action relies upon 3 defined mechanisms of action: 1) blocking testosterone binding to the androgen receptor (AR), 2) preventing nuclear translocation of AR, and 3) inhibiting AR-DNA binding. However, despite its utility in prolonged overall survival in both these settings, disease relapse is inevitable, suggesting that resistance to the drug is somehow being acquired and is of vital concern. Methods: Using a combination of high-throughput functional genomic (kinome-shRNA) and chemical genomic (pharmacological inhibitor) screens, along with various in vitro / in vivo assays (proliferation, apoptosis, necrosis, soft agar growth, etc.) we have identified the IkB kinase (IKK) / IkB / NFkB signaling axis as important for acquisition of, and continued resistance to, Enzalutamide. Results: Using a panel of Enzalutamide-resistant and -sensitive cell lines, we show that targeting this signaling axis will selectively kill Enzalutamide-resistant cells while not affecting the proliferation of sensitive cells. Moreover, our data shows that the pharmacological inhibitors and shRNAs are effective even without Enzalutamide administration, negating any potential drug-drug interaction issues. Finally, we have found that our cells are resistant to Enzalutamide in an AR-V7-independent manner, suggesting that NFkB signaling is acting in a novel manner to bring about the resistance phenotype. Conclusions: Through in vitro and in vivo experiments, we provide evidence that targeting activators of NFκB signalling can be a strategy to help patients with Enzalutamide-resistant prostate cancer. Note: This abstract was not presented at the meeting. Citation Format: Sujeeve Jeganathan, Amina Zoubeidi, Martin Gleave, Brad G. Wouters, Anthony M. Joshua. Using functional and chemical genomics to identify mechanisms of Enzalutamide resistance in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 732. doi:10.1158/1538-7445.AM2015-732