ASCL1-mediated transcriptional regulation of RET in neuroendocrine prostate cancer

Resistance to second-generation anti-androgen therapies can cause neuroendocrine prostate cancer (NEPC), an aggressive disease variant, in castration-resistant prostate cancer (CRPC) patients. With low survival outcomes and limited therapies, it is imperative to study the molecular basis of NEPC. We show that the receptor tyrosine kinase RET has elevated activity in aggressive variant prostate cancer cell lines. We also show that RET kinase is crucial for the growth and survival of NEPC cells. We aim to unravel the mechanism of RET activation in NEPC to develop novel approaches to target RET and identify additional drug targets. NEPC can be categorized into two subtypes based on the expression of ASCL1 or NEUROD1, two pro-neuronal transcription factors. We show that RET gene expression strongly correlates to ASCL1 gene expression, but not NEUROD1 gene expression in NEPC patient samples. This data is corroborated by single cell-RNA-sequencing data in NEPC patient samples. Informatics modeling of whole transcriptome sequencing data from patient samples shows that RET and ASCL1 have substantially similar gene network signatures in NEPC, implying that these genes share a gene ecosystem in NEPC. To investigate the relationship between RET and ASCL1, we analyzed publicly available ChIP-sequencing data from LuCaP NEPC PDX models and small cell lung cancer (SCLC) cell lines. NEPC and SCLC are known to have similarities, including disease aggressiveness, expression of neuroendocrine markers, and the presence of ASCL1-positive and NEUROD1-positive subtypes. Our analysis showed that ASCL1 directly regulates RET by binding to RET promoter regions. Hence, we note a similar relationship between RET and ASCL1 in SCLC where ASCL1 regulates RET expression. Using knockdown models, we show that the RET-ASCL1 axis is unidirectional with RET having no impact on ASCL1 expression. To drug this pathway, we aim to focus on cell surface targets such as RET. RET inhibitors are approved for non-small cell lung cancers or thyroid cancers with RET fusions, however, they may induce resistance via mutations. Additionally, they may be less effective in tumors with wild-type RET expression, which is typically seen in NEPC. PROTACs can bypass these drawbacks by degrading the entire protein instead of enzymatically inhibiting it, thus overcoming drug resistance. Additionally, PROTACs have a catalytic mechanism that can cause degradation of several target molecules with one PROTAC molecule, leading to longer elimination of target protein with lower doses. We are using RD-23, a published RET PROTAC based on the RET inhibitor selpercatinib, to investigate its effects on NEPC and SCLC cells. While further studies are needed to stratify patients and develop novel pharmacological interventions, these results highlight the crucial role of ASCL1 in mediating RET signaling in NEPC and SCLC.