Proteostasis perturbation of N-Myc by HSP70 inhibition improves treatment in neuroendocrine prostate cancer I
N-Myc is a key driver of neuroendocrine tumors, such as neuroblastoma and neuroendocrine prostate cancer (NEPC). However, N-Myc is considered undruggable because it lacks clear binding sites for small molecules. One potential way to circumvent this challenge is to identify and target the protein homeostasis (proteostasis) system(s) that maintain N-Myc levels. Here we developed a novel spontaneously indefinite prostate cancer cell line UCDCaP and its paired castration-resistant line UCDCaP-CR, which exhibits neural lineage plasticity and high levels of N-Myc protein expression. Through rapid immunoprecipitation mass spectrometry assay, we revealed that heat shock protein 70 (HSP70) is a top partner for N-Myc. HSP70 is known to function in protein folding and it also coordinates with the E3 ubiquitin ligase, STIP1 homology and U-box containing protein 1 (STUB1), to regulate oncoprotein degradation. We find that HSP70 binds a conserved 'SELILKR' motif and prevents the access of STUB1 on N-Myc possibly through steric hindrance. When HSP70's dwell time on N-Myc is increased by the conformational change of HSP70, STUB1 is in close proximity with N-Myc and becomes functional to promote N-Myc ubiquitination on the K416 and K419 sites of the bHLH-LZ domain and form the poly ubiquitination chains linked by the K11 and K63 sites. Inhibition of HSP70's ATPase activity by an allosteric inhibitor, JG231, promoted degradation of N-Myc through enhancing STUB1 binding in the nucleus. Notably, JG231 significantly suppressed NEPC tumor growth and limited expression of neuroendocrine related pathways. Guided by a potential bypass route of N-Myc turnover in the cancer cell, we find that dual targeting of Aurora kinase A (AURKA) and HSP70 drastically suppressed N-Myc and tumor growth in NEPC. Overall design: A total of 3 samples were analyzed in this study. The study focused on the NCI-H660 neuroendocrine prostate cancer cell line. Cell cultures were either treated with vehicle (DMSO) or treated with JG231. Total RNA was then isolated (Qiagen RNeasy MiniKit) and then for RNA-sequencing analysis.