Identifying synthetic lethal interactions in castration-resistant prostate cancer using HSP40 and HSP70 inhibitors

Castration-resistant prostate cancer (CRPC) is frequently characterized by elevated expression of alternative nuclear receptors able to at least partially maintain the androgen receptor (AR) transcriptional program. These alternative receptors include ARv7, a ligand binding domain (LBD)-deficient, constitutively active splice variant that correlates with poor prognosis, reduced survival, and resistance to existing LBD-targeted standard of care therapy. As a nuclear hormone receptor, AR exists in a dynamic cycle with molecular chaperones (heat shock proteins, HSPs) HSP40/HSP70/HSP90 which foster proper folding and remodeling of the LBD to bind ligand. Notably, HSP90 inhibitors promote AR degradation and display efficacy in prostate cancer xenograft models. Although it has been shown that LBD-deficient ARv7 functions independently of HSP90, we have observed that loss of the LBD does not abrogate HSP40/HSP70 binding, as ARv7 interacts with HSP40/HSP70 in CRPC cells. As such, targeting either HSP40 (C86) or HSP70 (JG98/JG231) inhibits stability and function of AR/ARv7. However, this is complicated by the fact that over 40 isoforms of HSP40 (DnaJ proteins) and 13 HSP70 paralogs exist in humans. Therefore, we sought to identify the requirements for components of the HSP40/HSP70 network in mediating intracellular stability of AR/ARv7, and determine which chaperones orchestrate sensitivity to HSP40/HSP70 inhibitors. Using a doxycycline-inducible lentiviral proteostasis shRNA library, we identified genes that appear to be required for proliferation of 22Rv1 CRPC cells (expressing endogenous AR and ARv7) distinct from those found in ARv7-deficient LNCaP prostate cancer cells. These include genes from several chaperone families including DnaJA3 (HSP40), HSPA2 (HSP70-2), and HSPD1 (HSP60), which may be unique regulators of ARv7 stability. Synthetic lethal screens using C86 and JG231 were also performed to determine which genes may mediate sensitivity to these chaperone inhibitors. Knockdown of HSP40 isoforms (DnaJA1, DnaJA2), HSP70 paralog HSPA4, or stress factors (ATF6, HSF1) in combination with C86 lead to growth impairment in 22Rv1 cells. DnaJA1, DnaJA2, and DnaJA3 were all found in the cytosol and nucleus of 22Rv1 cells, and in a complex with AR/ARv7 after a biotin-C86 IP, suggesting they be required for AR/ARv7 stability, dimerization, and/or nuclear translocation. Other genes from chaperone families HSP40 (DnaJC7, DnaJC8, DnaJC16), HSP70 (HSPA5, HSPA8, HSPA9), and HSP90/HSP90 co-chaperones (HSP90AB1, Cdc37, PTGES3 [p23]) were found to be important mediators of sensitivity to JG231, revealing targets for possible combination therapies. Taken together, these data help identify HSP40/HSP70 subnetworks regulating AR/ARv7 and those mediating sensitivity to chaperone inhibitors to aid in identifying new drug targets for efficacy in CRPC. Citation Format: Michael A. Moses, Genesis Rivera-Marquez, Isabelle Taylor, Hao Shao, Jason Gestwicki, Jane Trepel, Len Neckers. Identifying synthetic lethal interactions in castration-resistant prostate cancer using HSP40 and HSP70 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2901.