Multi-omics analysis identifies distinct subtypes with clinical relevance in lung adenocarcinoma harboring KEAP1/ NFE2L2

Abstract

Backgrounds: Lung adenocarcinoma is one of the most common malignant tumors, in which KEAP1-NFE2L2 pathway is altered frequently. The biological features and intrinsic heterogeneities of KEAP1/NFE2L2-mutant lung adenocarcinoma remain unclear. Methods: Multiplatform data from The Cancer Genome Atlas (TCGA) were acquired to identify two subtypes of lung adenocarcinoma harboring KEAP1/NFE2L2 mutations. Bioinformatic analyses, including immune microenvironment, methylation level and mutational signature, were performed to characterize the intrinsic heterogeneities. Meanwhile, initial results were validated by using in silico assessment of common lung adenocarcinoma cell lines, which revealed consistent features of mutant subtypes. Furthermore, drug sensitivity screening was conducted based on public datasets. Results: Two mutant subtypes (P1 and P2) of 89 patients were identified in TCGA. P2 patients had significantly higher levels of smoking and worse survival compared with P1 patients. The P2 subset was characterized by active immune microenvironment and more smoking-induced genomic alterations with respect to methylation and somatic mutations. Validations of the corresponding features in 20 mutant cell lines were achieved. Several compounds which were sensitive to mutant subtypes of lung adenocarcinoma were identified, such as inhibitors of PI3K/Akt and IGF1R signaling pathways. Conclusions: KEAP1/NFE2L2-mutant lung adenocarcinoma showed potential heterogeneities. The intrinsic heterogeneities of KEAP1/NFE2L2 were associated with immune microenvironment and smoking-related genomic aberrations.

Keywords: KEAP1; NFE2L2; lung adenocarcinoma; mutation.

Figure 1

The SNF fused five types of datasets and consensus clustering identifies subsets of KEAP1/NFE2L2-mutant lung adenocarcinoma in patients and cell lines. A. Two subsets of KEAP1/NFE2L2-mutant patients were identified. B. Silhouette values of patient clustering with the k = 2 to 7. C. Two subsets of KEAP1/NFE2L2-mutant cell lines were identified. D. Silhouette values of cell line clustering with the k = 2 to 5.

Figure 2

Survival curves of lung adenocarcinoma patients in TCGA. A. Survival curves of KEAP1/NFE2L2-mutant and wild-type patients (P = 0.212). B. Survival curves of KEAP1/NFE2L2-mutant patient subgroups (P1 and P2) (P = 0.020).

Figure 3

A. The enriched pathways in Hallmark of KEAP1/NFE2L2-mutant P2 patient subgroup. B. The enriched pathways in Hallmark of KEAP1/NFE2L2-mutant C2 cell line subgroup.

Figure 4

Immunological features of lung adenocarcinoma patients in TCGA. A. Comparison of leukocyte fraction stratified by KEAP1/NFE2L2-mutant (P1 and P2) and wild-type patient subgroups (mutant group vs. wild-type group, P = 0.001; P1 group vs. P2 group, P < 0.001). B. Comparison of stromal score calculated by ESTIMATE algorithm stratified by KEAP1/NFE2L2-mutant (P1 and P2) and wild-type patient subgroups (P1 vs. P2 vs. wild-type group, P = 0.005). C. Comparison of immune score calculated by ESTIMATE algorithm stratified by KEAP1/NFE2L2-mutant (P1 and P2) and wild-type patient subgroups (P1 vs. P2 vs. mutant group, P = 0.001). D. Comparison of ESTIMATE score calculated by ESTIMATE algorithm stratified by KEAP1/NFE2L2-mutant. (P1 and P2) and wild-type patient subgroups (P1 vs. P2 vs. wild-type group, P = 0.001). E. Comparison of tumor-infiltrating immune cells stratified by KEAP1/NFE2L2-mutant (P1 and P2) and wild-type patient subgroups based on TIMER database. [mutant group vs. wild-type group: CD4+ T cells (P < 0.001), CD8+ T cells (P = 0.011), B cells (P < 0.001), neutrophils (P < 0.001), dendritic cells (P < 0.001), and macrophages (P = 0.008); P1 group vs. P2 group: B cells (P = 0.017), CD4+ T cells (P = 0.001), CD8+ T cells (P = 0.375), neutrophils (P = 0.002), macrophages (P = 0.113), and dendritic cells (P = 0.006)]. F. Comparison of the number of immunogenic mutations per sample stratified by KEAP1/NFE2L2-mutant (P1 and P2) and wild-type patient subgroups (P1 vs. P2 vs wild-type group, P < 0.001).

Figure 5

Epigenomic features of KEAP1/NFE2L2-mutant subgroups of lung adenocarcinoma patients and cell lines. A. Volcano plot of the global DNA methylation difference between patient mutant subgroups (P1 and P2). B. Volcano plot of the global DNA methylation difference between cell line mutant subgroups (C1 and C2). C. Volcano plot of the smoking-related methylation signatures between patient mutant subgroups (P1 and P2). D. Volcano plot of the smoking-related methylation signatures between cell line mutant subgroups (C1 and C2).

Figure 6

Screened drugs with selective sensitivity toward the KEAP1/NFE2L2-mutant subtypes. A-H. Drugs that selectively killed tumor cells of the C2 subset. I. Drug that selectively killed tumor cells of the C1 subset.