靶向蛋白质组学绝对定量在患者来源异种移植物中识别BRCA1亚效等位基因及其他PARP抑制剂耐药机制

Introduction: BRCA1 is a tumor suppressor required for homologous recombination repair (HRR) of DNA double‑strand breaks. BRCA1 loss-of-function mutations cause HRR deficiency and sensitize tumors to platinum-based chemotherapy and PARP inhibitors (PARPi). Standard gene-level testing may miss other mechanisms affecting BRCA1 protein functionality, such as BRCA1 promoter methylation, or other PARPi response molecular determinants, such as hypomorphic protein expression that partially restores HRR, leading to PARPi resistance. Current standard assays suitable for formalin-fixed paraffin-embedded (FFPE) tissue, such as immunohistochemistry, are limited in their ability to characterize BRCA1 protein levels - highlighting the need for new approaches for protein-level quantification. Methods: Targeted proteomics was performed on an Orbitrap Exploris 480 mass spectrometer coupled to an Evosep One nanoLC to quantify BRCA1 peptides spanning the N-terminal RING domain, Exon 11 and C-terminal BRCT domain, enabling BRCA1 hypomorph detection based on domain-specific peptide abundance. The targeted panel assay also included additional DNA repair proteins. This assay was applied to 56 FFPE tumors from 26 patient-derived xenografts (PDX), including 7 samples collected at acquired olaparib resistance. Results: PDX tumours from BRCA1 wild-type, BRCA2-mutant, or PALB2-mutant models (n=10), had a median BRCA1 peptide expression level of 45.8 amol/µg (range 20.7-120.9), consistent with a normal-expression reference range. In contrast, PDX tumors with BRCA1 promoter hypermethylation (n=4) exhibited low BRCA1 peptide levels (< 1.8 amol/µg). At acquired olaparib resistance, tumours from the same models showed a ~25-fold BRCA1 protein increase, consistent with demethylation and restored BRCA1 expression. Among BRCA1-mutant PDX models (n=15), most models exhibited low expression, consistent with loss-of-function mutations (n=8 with < 5 amol/µg). Targeted proteomics further identified seven candidate BRCA1-mutant PDX hypomorphic cases in which at least one BRCA1 domain peptide exceeded the ~5 amol/µg threshold; their BRCA1 peptide levels ranged from 6.2 to 31.7 amol/µg. Six out of seven hypomorph cases (86%) were PARPi resistant. Low 53BP1 expression was observed in four PARPi-resistant PDX models; three of these had known 53BP1 dysregulation or mutation. However, one model showed low 53BP1 despite the absence of a known TP53BP1 mutation, implicating 53BP1 loss as a plausible contributor to resistance. Conclusion: These data support this targeted proteomic assay as a practical approach to distinguish normal from low BRCA1 expression at the protein level, nominate hypomorphic BRCA1 variants linked to PARPi resistance, and identify deficiency in additional resistance biomarkers.