Understanding the molecular mechanism of a protein-recycling complex in small cell lung cancer treatment resistance.

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PROJECT SUMMARY Small cell lung cancer (SCLC) patients have an initial robust response to combinations of DNA damaging agents (e.g. cisplatin, etoposide, radiotherapy), however, many patients inevitably suffer from relapse and resistant disease. A clear understanding of these resistance mechanisms remains elusive. Consequently, there is a critical need to: (1) understand the mechanisms of therapeutic resistance and (2) develop novel therapeutics. Poly-(ADP)-ribose polymerase enzymes (PARP) protein levels are upregulated in SCLC relative to other lung cancers, and initial studies suggest that this upregulation is associated with increased sensitivity of SCLC to PARP inhibitors (PARPi) in vitro. PARP inhibitors are synthetic lethal with BRCA1/2 mutated homologous recombination (HR) deficient tumors and restoration of HR by BRCA reversion mutations is a known mechanism of PARPi and cisplatin resistance. However, as BRCA1/2 mutations are exceedingly rare in SCLC non-BRCA mechanisms must be operant. We performed a genome-wide CRISPR knockout screen to identify novel mechanisms of PARPi resistance. From subsequent functional validation and clinical genomic correlation, we identified deficiency in an F-box protein coding gene as a putative biomarker of resistance to PARP inhibitors and cisplatin in SCLC that may be present in up to ~20% of relapse patient tumors. Loss of this F-box protein abrogates the function of its corresponding SKP1, CUL1, F-box (SCF) E3 ubiquitin ligase complex. By proximity-dependent biotin identification (BioID) of this F-box protein, we have identified a high confidence interactor with substrate-like behavior for SCF-mediated ubiquitin-proteasomal degradation that is important for regulation of HR and DNA repair. This proposal aims to: (1) determine the mechanism of this specific SCF complex with its substrate to engage the ubiquitin-proteasome pathway; (2) elucidate the impact of this F-box protein on HR, DNA repair, and therapeutic sensitivity to PARPi/cisplatin; and (3) identify synthetic lethal interactions with deficiencies in this F-box protein to provide biologic insight and characterize immediately translatable approaches for relapsed treatment resistant SCLC.