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

了解蛋白质回收复合物在小细胞肺癌治疗耐药中的分子机制。

• 批准号: 10055266
• 负责人: Benjamin H Lok
• 金额: $ 28.19万
• 依托单位: UNIVERSITY HEALTH NETWORK
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-25 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10055266
• 关键词: Amino Acid Sequence; Antibodies; BRCA1 gene; Behavior; Biochemical; Biological; Biological Assay; Biological Markers; Biotin; CRISPR library; CRISPR screen; CRISPR/Cas technology; CUL1 gene; Cancer Biology; Cancer Etiology; Cancer Patient; Cancer cell line; Cell Line; Cellular Assay; Cessation of life; Cisplatin; Clinical; Clinical Research; Clustered Regularly Interspaced Short Palindromic Repeats; Co-Immunoprecipitations; Code; Complementary DNA; Complex; Custom; DNA Damage; DNA Repair; DNA Repair Pathway; Disease; Disease Resistance; Enzyme Inhibitor Drugs; Enzymes; Etoposide; Exhibits; F Box Domain; F-Box Proteins; FDA approved; Gene Targeting; Genes; Genetic Screening; Genomics; Goals; In Vitro; Knock-out; Laboratories; Lead; Libraries; Malignant Neoplasms; Malignant neoplasm of lung; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Modeling; Molecular; Mutagenesis; Mutate; Mutation; Non-Small-Cell Lung Carcinoma; Pathway interactions; Patient-Focused Outcomes; Patients; Pharmaceutical Preparations; Pharmacotherapy; Phase; Poly(ADP-ribose) Polymerases; Protein Cycling Pathway; Proteins; Radiation therapy; Recurrent disease; Recycling; Regulation; Relapse; Reporter; Reporting; Resistance; Role; Structure; Tertiary Protein Structure; Therapeutic; Ubiquitin; Up-Regulation; Validation; Woman; Work; Xenograft Model; Xenograft procedure; chemotherapy; chromosome 2p loss; functional genomics; genome-wide; homologous recombination; improved; improved outcome; in vivo; inhibitor/antagonist; insight; lung small cell carcinoma; men; multicatalytic endopeptidase complex; novel; novel therapeutics; pre-clinical; recombinational repair; recruit; relapse patients; resistance mechanism; response; restoration; small hairpin RNA; therapeutic development; therapy resistant; treatment response; tumor; ubiquitin-protein ligase; vector

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.

项目总结