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

项目摘要 小细胞肺癌（SCLC）患者对DNA损伤的组合有最初的强烈反应， 然而，许多患者不可避免地遭受复发， 抵抗疾病。对这些耐药机制的清楚理解仍然是难以捉摸的。因此，委员会认为， 迫切需要：（1）了解治疗抗性的机制和（2）开发新的 治疗学 与其他肺组织相比，SCLC中的聚（ADP）-核糖聚合酶（PARP）蛋白水平上调 初步研究表明，这种上调与SCLC对肿瘤的敏感性增加有关， 体外PARP抑制剂（PARPi）。PARP抑制剂是与BRCA 1/2突变同源的合成致死性 BRCA逆转突变导致的重组（HR）缺陷型肿瘤和HR恢复是已知的 PARPi和顺铂耐药的机制。然而，由于BRCA 1/2突变在乳腺癌中非常罕见， SCLC非BRCA机制必须是可操作的。 我们进行了全基因组CRISPR敲除筛选，以确定PARPi抗性的新机制。 从随后的功能验证和临床基因组相关性，我们确定了F盒缺陷 蛋白编码基因作为SCLC中PARP抑制剂和顺铂耐药的假定生物标志物， 存在于高达~20%的复发患者肿瘤中。这种F-box蛋白的缺失废除了其功能， 相应的SKP 1、CUL 1、F-box（SCF）E3泛素连接酶复合物。通过邻近依赖性生物素 通过这种F-box蛋白的鉴定（BioID），我们已经鉴定了一种高置信度的与底物样 SCF介导的泛素-蛋白酶体降解的行为，对HR和DNA的调节很重要 修复. 本提案旨在：（1）确定这种特异性SCF复合物与其底物结合的机制 泛素-蛋白酶体途径;（2）阐明这种F-box蛋白对HR，DNA修复和 对PARPi/顺铂的治疗敏感性;和（3）鉴定具有这种缺陷的合成致死相互作用。 F-box蛋白提供生物学洞察力，并表征复发性乳腺癌的立即可翻译方法 难治性小细胞肺癌

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.