BRCA1 and BRCA2 mutations are associated with genomic instability and poor prognosis of LUAD.

A. Distribution of HRD score, TMB and neoantigen load in patients with wild-type, BRCA1- and BRCA2-mutated lung adenocarcinoma from the TCGA-LUAD cohort (n=566). B. Proportion of immune subtypes. The significance level was calculated by Chi-squared test. C1: wound healing, C2: IFN-g dominant, C3: inflammatory, C4: lymphocyte depleted, C6: TGF-b dominant. C-D. Activity of DNA damage repair and cell cycle-related pathways in wild-type, BRCA1- and BRCA2-mutated patients (TCGA-LUAD cohort). E-F. Survival analysis of BRCA mutations in TCGA-LUAD (E) and OncoSG-LUAD (F) cohorts. G. Survival analysis of BRCA mutations in LUAD patients after ICB treatment. H. Survival analysis based on BRCA1 (left) and BRCA2 (right) expression in patients with ICB treatment. I. Lymphocyte infiltrates in patients with wild-type, BRCA1 and BRCA2 mutations (TCGA-LUAD cohort). J. Mutation frequencies of the oncogenic signaling pathway genes (in-house data).

Single-cell transcriptome analysis and LUAD malignant cells identification.

A. UMAP visualization of cell types in patients with BRCA1/2 mutations (in-house data). B. The number of each cell type and its proportion in different samples, including tumor tissue (T), adjacent normal tissue (N), and peripheral blood (B). C. The bubble plot shows the expression of the markers used for cell type identification. D. The heat map inferred CNVs in epithelial cells, malignant cells, and reference cells. E. Distribution of CNV scores across different cell types on specific chromosomes. F. UMAP visualization of CNV score in epithelial cells and malignant cells. G. UMAP visualization (left) and box plot (right) of tumor upregulated signature score. H. Expression of CEACAM6 in epithelial and malignant cells.

Identification of key programs in BRCA1/2 mutated tumors.

A. Functional enrichment analysis of genes that upregulated in malignant cells relative to normal epithelial cells. B. The heat map of the similarity between pairs of programs is identified based on the NMF algorithm. C. MP score between BRCA1 and BRCA2 mutated malignant cells. D. K-M curves for MP1 score survival analysis. E. Enrichment map of the functions enriched by MP1 and MP2 genes. F. The overlap of in-house MPs and known cancer MPs. G. The heat map shows the activity of known cancer MPs in BRCA1 and BRCA2 mutated malignant cells. H. Volcano diagram of differentially expressed genes between BRCA1 and BRCA2 mutated malignant cells.

Tumor evolutionary analysis of BRCA1 and BRCA2 mutations.

A. Pseudotime analysis of epithelial and malignant cells. The branched trajectory was colored by pseudotime (left) and cell types (right). The pie chart shows the proportion of sample groups in each branch. B. The top 50 cell fate genes and their enriched GO BP terms of BRCA1/2 mutations in malignant2 cell type. C-E. Pseudotime was broken down into 10 bins to smooth gene expression patterns. Average gene module score between BRCA1 and BRCA2 mutation groups for tumor down-regulation (C), MP1 cell cycle G2/M (D), and MP5 stress (E) gene modules. F, J. Cell density distribution based on cell cycle HMG rich versus stress gene modules (F) and MHC class I versus MHC class II gene modules (J) in the same cells between BRCA1 and BRCA2 mutations. Color represents the density of cells. The dotted line represents the median value of the corresponding module score. G. Average gene module score for IFN-α response (top) and MHC class II (bottom) gene signatures. H-I. The activities of representative signatures between BRCA1 and BRCA2 mutations in bin 10 of malignant cells according to pseudotime.

BRCA1/2 mutations are associated with tumor lymphocyte activation.

A. Functional enrichment of differentially expressed genes in malignant cells between BRCA1 and BRCA2 mutation groups. B. GESA of immune response profile between BRCA1 and BRCA2 mutations. Red (blue) indicates the gene set enriched in BRCA2 (BRCA1)-mutated malignant cells. C-D. GESA plot of immune signature (C) and gene module of immune cell type (D) in malignant2 subset. NES: normalized enrichment score. E. Density ridge plot of representative pathways in two types of malignant subset with BRCA1 and BRCA2 mutations. HRR: homologous recombination repair. F. UMAP visualization of cGAS pathway score. G. Mean of pathway activities related to DNA damage repair across two types of malignant cells with BRCA1 and BRCA2 mutations. H. The expression of the representative markers. I. Immune cell activities between BRCA1 and BRCA2 mutations in bin 10 of malignant cells according to pseudotime. J. Lymphocyte activity between BRCA1 and BRCA2 mutations in lymphocyte cells.

T lymphocyte infiltration and TCR clonal expansion analysis.

A-B. UMAP visualization (A) and proportion (B) of T lymphocyte subsets in combined dataset. C-D. Box plot (C) and UMAP plot (D) of representative immune pathways from ImmPort database. E-F. The activity of tissue-specific T cell signatures from Judith Wienke et al. G. Terminal exhaustion marker expression levels. H. UMAP of lymphocyte subsets in in-house dataset. I. Quantification of clonal size across sample types. Clonotypes are ranked by expansion level, including: single (1 cell), small (>1 and <5 cells), medium (>5 and <20 cells), large (>20 and <100 cells), and hyperexpanded (>100 and <500 cells). J. Expanded clonotypes distribution in different samples. K. Proportional and dynamic changes in shared clonotypes (top5 per group) between different samples within the same patient. The colors represent different clonotypes. L. The proportion of clonally expanded cells (≥5 clone size) in BRCA1/2 mutated tumor tissues for specific cell types. M. The sample and cell proportions of the 50 most abundant TCR motifs.

Targeting BRCA1 mutation-related risk genes inhibits tumor growth.

A-B. Survival analysis of BRCA1-mutant signature score in TCGA-LUAD (A) and OAK (B) cohorts. C. Survival analysis of S100A10 (left) and LDHA (right) in TCGA-LUAD cohort according to the expression median. D. The effects of sh-LDHA, S100A10, GAPDH, and vector on cell proliferation were determined by a cell proliferation assay in A549 LUAD cell line. E. The effects of sh-LDHA, S100A10, and GAPDH on protein levels in A549 cell line (The original image is shown in Fig. S12A). F. The molecular perturbations of 4-R genes in A549 cell line at 10 µM concentration (24 h) from the LINCS data resource. NCS: Normalized connectivity score. G. The effects of HDAC inhibitors concentration on cell proliferation in A549 cell line. H. Cell replication levels after treatment with different concentrations of entinostat (left) and vorinostat (right) in LUAD cell lines. I. The protein levels of LDHA, S100A10, and GAPDH after treatment with vorinostat (left) and belinostat (right) in A549 cell line were measured using western blotting (The original image is shown in Fig. S12B). J. The mRNA levels of LDHA and GAPDH after treatment (3 µM) with vorinostat (left) and belinostat (right) in A549 cell line were measured using qPCR. Significance levels were determined by Student’s t-test. For all statistical tests, *P≤0.05; **P≤0.01; ***P≤0.001.