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UBC Theses and Dissertations

A model of acquired resistance to the novel AR-NTD inhibitor ralaniten reveals resistance occurs via a selective metabolism pathway Obst, Jonathon

Abstract

Background: Inhibition of the androgen receptor (AR) is the mainstay treatment for advanced prostate cancer. While initially effective, the disease ultimately progresses to metastatic castration-resistant prostate cancer, which is lethal. Ralaniten specifically targets the AR N-terminal domain (NTD). This allows for sustained inhibition in the context of constitutively active AR-splice variants and mutations which often drive resistance to current therapies. Ralaniten acetate is the pro-drug of ralaniten, and the first AR-NTD inhibitor to enter clinical trials (NCT02606123, ESSA Pharma, Inc.). Here we generated and characterized a model of acquired ralaniten resistance to aid in the development of next generation AR-NTD inhibitors. Methods: A resistant cell line (LNCaP-RALR) was created by serially passaging parental LNCaP cells in the presence of ralaniten. Resistant cells were challenged with ralaniten and existing AR antagonists to assess growth and sensitivity of the resistant line in vitro and in vivo. LNCaP-RALR cells were subject to global microarray profiling, and findings were validated using qRT-PCR, western blot and functional assays. Results: Ralaniten displayed antitumour activity in LNCaP but not LNCaP-RALR xenografts due to its reduced ability to block AR mediated gene transcription. Interestingly, LNCaP-RALR cells retained sensitivity to antiandrogens and AR knockdown by targeted siRNA, implying growth remains driven by AR signalling. Interrogation of microarray data revealed candidate genes (UGT2B family) associated with ralaniten resistance. These genes are involved in drug metabolism (glucuronidation), and knockdown of UGT2B isoforms was sufficient to restore sensitivity to ralaniten in resistant cells. LC/MS and ion spectra data from serum samples collected from patients, revealed that ralaniten is glucuronidated in humans. EPI-045 (which is resistant to glucuronidation) significantly inhibited AR mediated transcription and proliferation in LNCaP-RALR cells - both in vitro and in vivo. Conclusions: We have generated a model of acquired ralaniten resistance, and demonstrated that selective modification of ralaniten can reduce its glucuronidation. LNCaP-RALR cells remain dependent upon AR signalling, and are sensitive to both EPI-045 and antiandrogens used clinically. This work highlights the potential for combination or sequential therapy following ralaniten resistance, and provides a model with which to test next generation AR-NTD inhibitors.

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