STK11/LKB1 Mutations and PD-1 Inhibitor Resistance in KRAS-Mutant Lung Adenocarcinoma

Abstract

KRAS is the most common oncogenic driver in lung adenocarcinoma (LUAC). We previously reported that STK11/LKB1 (KL) or TP53 (KP) comutations define distinct subgroups of KRAS-mutant LUAC. Here, we examine the efficacy of PD-1 inhibitors in these subgroups. Objective response rates to PD-1 blockade differed significantly among KL (7.4%), KP (35.7%), and K-only (28.6%) subgroups (P < 0.001) in the Stand Up To Cancer (SU2C) cohort (174 patients) with KRAS-mutant LUAC and in patients treated with nivolumab in the CheckMate-057 phase III trial (0% vs. 57.1% vs. 18.2%; P = 0.047). In the SU2C cohort, KL LUAC exhibited shorter progression-free (P < 0.001) and overall (P = 0.0015) survival compared with KRAS^MUT;STK11/LKB1^WT LUAC. Among 924 LUACs, STK11/LKB1 alterations were the only marker significantly associated with PD-L1 negativity in TMB^{Intermediate/High} LUAC. The impact of STK11/LKB1 alterations on clinical outcomes with PD-1/PD-L1 inhibitors extended to PD-L1-positive non-small cell lung cancer. In Kras-mutant murine LUAC models, Stk11/Lkb1 loss promoted PD-1/PD-L1 inhibitor resistance, suggesting a causal role. Our results identify STK11/LKB1 alterations as a major driver of primary resistance to PD-1 blockade in KRAS-mutant LUAC.Significance: This work identifies STK11/LKB1 alterations as the most prevalent genomic driver of primary resistance to PD-1 axis inhibitors in KRAS-mutant lung adenocarcinoma. Genomic profiling may enhance the predictive utility of PD-L1 expression and tumor mutation burden and facilitate establishment of personalized combination immunotherapy approaches for genomically defined LUAC subsets. Cancer Discov; 8(7); 822-35. ©2018 AACR.See related commentary by Etxeberria et al., p. 794This article is highlighted in the In This Issue feature, p. 781.

Figure 1. STK11/LKB1 co-mutations are associated with inferior objective response rate with PD-1 blockade in KRAS-mutant LUAC

A. Objective response rate (RECISTv1.1) to PD-1 axis blockade in the KL, KP and K-only subgroups in the overall SU2C population (N=173 response-evaluable patients) and in each of the three independent cohorts (MDACC, MSKCC, DFCI/MGH). A two-tailed Fisher’s exact test (computed from a 2×3 contingency table) was used to assess the significance of the association between group membership and best overall response (PR/CR vs SD/PD). B. Objective response rate to nivolumab in the KL, KP and K-only subgroups in the CheckMate-057 international randomized phase III clinical trial (N=24). A two-tailed Fisher’s exact test (computed from a 2×3 contingency table) was used to assess the significance of the association between group membership and best overall response (PR/CR vs SD/PD). C. Waterfall plots illustrating individual patient-level maximal % change in tumor burden from baseline in response to PD-1/PD-L1 inhibition in the SU2C cohort. Only data from response-evaluable patients with measurable disease are graphed.

Figure 2. STK11/LKB1 genetic alterations are associated with shorter progression-free and overall survival with PD-1 blockade among KRAS-mutant LUAC in the SU2C cohort

A. Kaplan-Meier estimates of progression-free survival with PD-1 blockade in the KL, KP, K-only subgroups (left panel) and in the two-group comparison between KRAS^MUT;STK11/LKB1^MUT (KL) and KRAS^MUT;STK11/LKB1^WT LUAC (encompassing KP and K-only tumors) (right panel). Tick marks represent data censored at the last time the patient was known to be alive and without disease progression (date of last radiological assessment). B. Kaplan-Meier estimates of overall survival with PD-1 inhibitors in the KL, KP, K-only subgroups (left panel) and in the two group comparison between KRAS^MUT;STK11^MUT (KL) and KRAS^MUT;STK11/LKB1^WT tumors (right panel). Tick marks represent data censored at the last time the patient was known to be alive.

Figure 3. LKB1 expression by IHC can identify LKB1-deficient LUAC in the absence of STK11/LKB1 alterations

A. LKB1 IHC expression (H-score) in KRAS^MUT;STK11/LKB1^MUT (KL) and KRAS^MUT;STK11/LKB1^WT LUAC. Quantitative IHC using a commercially available LKB1 rabbit monoclonal antibody (clone D60C5F10, Cell Signaling Technology) is technically robust and can identify LKB1-deficient tumors with intact STK11/LKB1 genomic locus (26). (Left panel) KL LUAC (N=12) exhibit absent or minimal cytoplasmic LKB1 staining, whereas KRAS^MUT;STK11/LKB1^WT LUAC (N=34) display variable LKB1 H-score. LUAC were therefore considered LKB1-proficient if they had intact STK11/LKB1 locus and expressed LKB1 by IHC at any level (LKB1 H-score > 0) and LKB1-deficient if they were STK11/LKB1-altered and/or exhibited LKB1 H-score = 0. Representative images of KL and KP LUAC immuno-stained for LKB1 are included (right panel). Staining was performed as previously described (26). B. Kaplan-Meier estimates of progression-free survival (left panel) and overall survival (right panel) with PD-1 blockade in LKB1-deficient (STK11/LKB1-mutant and/or LKB1 H-score = 0; N=61) and LKB1-proficient (STK11/LKB1-wild-type and LKB1 H-score > 0; N=38) KRAS-mutant LUAC.

Figure 4. STK11/LKB1 genomic alterations are enriched in LUAC with intermediate or high TMB that are negative for PD-L1 expression

(A) PD-L1/TMB landscape matrix illustrating the enrichment analysis strategy in 924 LUAC samples with available CGP and PD-L1 expression (FM cohort). Enrichment of individual genomic alterations in PD-L1^Neg; TMB^I/H vs PD-L1^HP; TMB^I/H tumors was assessed using a one-sided Fisher’s exact test. (B) Heatmap of log-odds values reflecting the prevalence of STK11/LKB1 alterations in different cells of the PD-L1/TMB matrix. Alterations in STK11/LKB1 primarily cluster in TMB^I;PD-L1^Neg LUAC. (C) PD-L1 expression in the KL, KP and K-only subgroups in the FM (N=346), SU2C (N=69) and CM-057 (N=44) cohorts. A two-tailed Fisher’s exact test (computed from a 2×3 contingency table) was used to assess the significance of the association between group membership and PD-L1 expression status [PD-L1 positive (≥1%) or negative (0%)]. (D) TMB (Log10) in the KL, KP and K-only subgroups among 346 KRAS-mutant LUAC in the FM cohort.

Figure 5. STK11/LKB1 mutations are a genomic determinant of poor clinical outcome with PD-1 axis blockade in PD-L1 positive non-squamous NSCLC, regardless of KRAS status

A. Objective response rate (RECISTv1.1) to PD-1/PD-L1 inhibitors in STK11/LKB1-mutant and wild-type patients with PD-L1-positive non-squamous NSCLC (≥1%) from MDACC (N=66). PD-L1 expression was assessed using the FDA-approved 22C3 pharmDx assay (Dako). A two-tailed Fisher’s exact test (computed from a 2×2 contingency table) was used to assess the significance of the association between group membership (STK11/LKB1-mutant versus STK11/LKB1 -wild-type) and best overall response (PR/CR vs SD/PD). B. Fractions of PD-L1 low-positive (1%-49%) and PD-L1 high-positive (≥50%) tumors in the STK11/LKB1-mutant and wild-type groups. C. Kaplan-Meier estimates of progression-free survival with PD-1/PD-L1 blockade in STK11/LKB1-mutant and wild-type groups. Tick marks represent data censored at the last time the patient was known to be alive and without disease progression (date of last radiological assessment). D. Kaplan-Meier estimates of overall survival with PD-1 inhibitors in the STK11/LKB1-mutant and wild-type groups. Tick marks represent data censored at the last time the patient was known to be alive.

Figure 6. Stk11 ablation directly promotes primary resistance to PD-L1/PD-1 blockade in immune-competent murine models of Kras-mutant LUAC

Stk11-proficient/deficient isogenic derivatives of the LKR13 (A) and LKR10 (B) cell lines were used in preclinical experiments. Changes in mean (main panels) and individual (insert panels, “spider plots”) subcutaneous tumor volume following treatment with (A) anti-PD-L1 (mIgG1-D265AFc clone 80) or IgG control antibody (LKR13/LKR13KO isogenic pair) and (B) anti-PD-1 monoclonal antibody (clone RMPI-14; BioXCell) or isotype control antibody (clone 2A3; BioXCell) (LKR10/LKR10KO isogenic pair) are graphed. Error bars represent standard error of the mean. Mean tumor volume plots are depicted from the time of randomization to the time that the first mouse in any of the two treatment arms was sacrificed. Spider plots indicate individual tumor volume trajectories for the entire duration of the in vivo experiment (25 days for the LKR13/LKR13KO and 39 days for the LKR10/LKR10KO model). Note that PD-1/PD-L1 blockade blunts the in vivo growth of Stk11/Lkb1-proficient Kras-mutant LUAC, whereas Stk11/Lkb1 knockout renders tumors recalcitrant to PD-1/PD-L1 inhibition. The Mann-Whitney U test was used to compare mean tumor volumes between IgG control and anti-PD-L1/anti-PD-1 treated mice in each syngeneic model. Asterisks denote statistical significance at the P≤0.05 (*) and P≤0.01 (**) level.