Divergent Clinical and Immunologic Outcomes Based on STK11 Co-mutation Status in Resectable KRAS-Mutant Lung Cancers Following Neoadjuvant Immune Checkpoint Blockade

Abstract

Purpose: Co-mutations of the Kirsten rat sarcoma virus (KRAS) and serine/threonine kinase 11 (STK11) genes in advanced non-small cell lung cancer (NSCLC) are associated with immune checkpoint blockade (ICB) resistance. Although neoadjuvant chemoimmunotherapy is now a standard-of-care treatment for resectable NSCLC, the clinical and immunologic impacts of KRAS and STK11 co-mutations in this setting are unknown.

Experimental design: We evaluated and compared recurrence-free survival of resectable KRAS-mutated NSCLC tumors, with or without co-occurring STK11 mutations, treated with neoadjuvant ICB. Single-cell transcriptomics was performed on tumor-infiltrating T cells from seven KRASmut/STK11wt tumors and six KRAS and STK11 co-mutated (KRASmut/STK11mut) tumors.

Results: Relative to KRASmut/STK11wt tumors, KRASmut/STK11mut exhibited significantly higher recurrence risk. Single-cell transcriptomics showed enhanced oxidative phosphorylation with evidence of decreased prostaglandin E2 signaling and increased IL-2 signaling in CD8+ tumor-infiltrating lymphocytes (TIL) from KRASmut/STK11mut tumors, a finding that was mirrored in KRASwt tumors that relapsed. TILs from KRASmut/STK11mut tumors expressed high levels of molecules associated with tumor residence, including CD39 and ZNF683 (HOBIT).

Conclusions: These divergent T-cell transcriptional fates suggest that T-cell maintenance and residence may be detrimental to antitumor immunity in the context of neoadjuvant ICB for resectable NSCLC, regardless of KRAS mutation status. Our work provides a basis for future investigations into the mechanisms underpinning prostaglandin E2 signaling and IL-2 signaling as they relate to T-cell immunity to cancer and to divergent clinical outcomes in KRASmut/STK11mut NSCLC treated with neoadjuvant ICB.

Figure 1.

Pathologic and clinical outcomes of patients with KRAS-mutant NSCLC treated with neoadjuvant ICB. A, Bar graph presenting the MPR rate for patients with KRAS-mutant disease, based on co-mutation status with STK11. The MPR rate for the whole KRAS-mutant cohort is also included as a dotted line for reference and comparison. B, Waterfall plot depicting percent pathologic regression of primary tumor for our KRAS-mutant cohort that underwent definitive resection, determined by baseline genomic sequencing prior to neoadjuvant ICB. Therefore, two patients with KRAS^mut/STK11^wt disease and one patient with KRAS^mut/STK11^mut disease were not included as they had primary progression precluding definitive resection. C, Swimmer plot summarizing treatment type and clinical outcomes for all patients with KRAS-mutant disease divided by the presence or absence of co-occurring STK11 mutation. Patients with primary progression of disease are denoted in this figure, as well as KEAP1 mutation status. Ipi, ipilimumab; Nivo, nivolumab. D, Kaplan–Meier curves depicting recurrence-free survival for patients based on KRAS mutation status. E, Kaplan–Meier curves depicting recurrence-free survival for the KRAS-mutant cohort based on co-mutation status with STK11.

Figure 2.

Transcriptional profiling of neoadjuvant ICB-treated CD8⁺ TILs in NSCLC based on KRAS and STK11 co-mutation status. A, Refined clustering was performed on 92,525 CD8⁺ T cells from tumor (n = 13), normal adjacent the lung (n = 7) and the previously published tissues for MD043-011, which includes tumor-draining lymph node and a distant brain metastasis. Fourteen distinct clusters were annotated and are marked by color on the Uniform Manifold Approximation and Projection (UMAP) projection. B, Expression of memory, TRM, and T-cell checkpoint markers, including CXCL13 and CD39. C, Relative expression of the top five most differentially expressed genes. Five thousand cells were randomly sampled from each cluster for visualization. D, PCA of cell cluster–level pseudobulk gene expression for individual tumor samples (n = 13), based on co-mutation status. A one-sided permutation test was performed.

Figure 3.

CD8⁺ TILs from co-mutated lung cancers exhibit features consistent with terminal differentiation and metabolic dysfunction. A, Volcano plot showing differential expression of CD8⁺ TILs between KRAS^mut/STK11^mut tumors (left) and KRAS^mut/STK11^wt tumors (right). Each dot represents one gene. A FDR <0.05 was considered significant. B, A waterfall plot of the top 10 significantly upregulated genes enriched in KRAS^mut/STK11^mut (red) and in KRAS^mut/STK11^wt (blue) CD8⁺ TILs. C–E, Violin plots for the expression of TIM-3, LAG3, and TOX2 in CD8⁺ TILs between KRAS^mut/STK11^mut (red) and KRAS^mut/STK11^wt (blue) tumors. Comparisons were performed at the individual cell level using the two-sided Wilcoxon rank-sum test. F–H, Violin plots for the expression of key genes associated with tissue residence, memory, and prostaglandin receptor markers, in CD8⁺ TILs between KRAS^mut/STK11^mut (red) and KRAS^mut/STK11^wt (blue) tumors. Comparisons were performed at the individual cell level using the two-sided Wilcoxon rank-sum test. I, Feature plot for the OXPHOS score on CD8⁺ TILs from KRAS^mut/STK11^mut (top) and KRAS^mut/STK11^wt (bottom) tumor resections. This score ranges from 0–1. J, Violin plot of the OXPHOS score in CD8⁺ TILs based on co-mutation status; a two-sided Wilcoxon rank-sum test was performed. K, Violin plot of the expression of IL-2RG in CD8⁺ TILs between KRAS^mut/STK11^mut (red) and KRAS^mut/STK11^wt (blue) tumors. Comparisons were performed at the individual cell level using the two-sided Wilcoxon rank-sum test. L, Violin plot of the IL-2 signaling in CD8⁺ TILs based on co-mutation status; a two-sided Wilcoxon rank-sum test was performed.