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

项目总结 小细胞肺癌(SCLC)患者最初对DNA损伤组合有强烈的反应 药物(如顺铂、依托泊苷、放射治疗)，然而，许多患者不可避免地复发和 抵抗力强的疾病。对这些耐药机制的清楚理解仍然难以捉摸。因此， 迫切需要：(1)了解治疗耐药的机制和(2)开发新的 治疗学。 小细胞肺癌中多聚(ADP)核糖聚合酶(PARP)蛋白水平相对于其他肺组织的上调 癌症，初步研究表明，这种上调与小细胞肺癌对 PARP抑制剂(PARPI)在体外。PARP抑制剂与BRCA1/2突变同源物合成致死 重组(HR)缺陷肿瘤和通过BRCA逆转突变恢复HR是已知的 PARPI与顺铂耐药机制的研究然而，由于BRCA1/2突变在 SCLC非BRCA机制必须有效。 我们进行了全基因组CRISPR基因敲除筛选，以确定PARPI耐药的新机制。 从随后的功能验证和临床基因组相关性中，我们确定了F-box的缺陷 蛋白编码基因可能是小细胞肺癌对PARP抑制剂和顺铂耐药的生物标志物 出现在高达20%的复发患者肿瘤中。这种F-box蛋白的丢失会使其功能丧失 相应的Skp1、CUL1、F-box(SCF)E3泛素连接酶复合体。由邻近依赖的生物素 这个F-box蛋白的鉴定(BioID)，我们已经鉴定出一个与底物样的高置信度相互作用子 SCF介导的泛素-蛋白酶体降解行为对HR和DNA的调节很重要 修理。 本建议旨在：(1)确定这种特定的SCF络合物与其底物接合的机制 泛素-蛋白酶体途径；(2)阐明该F-box蛋白对HR、DNA修复和 对PARPI/顺铂的治疗敏感性；以及(3)确定合成致命性相互作用与这方面的缺陷 F-box蛋白为复发患者提供生物学洞察力并鉴定可立即翻译的方法 耐药小细胞肺癌。

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.