Abstract SY42-02: Inactivating STK11/LKB1 genomic alterations are a major driver of primary resistance to PD-1 axis blockade in non-squamous non-small cell lung cancer
Authors: Skoulidis Ferdinandos 
Published: August 2019

Abstract
Despite improvements in overall survival (OS) and clinical responses of unprecedented duration with the use of therapeutic monoclonal antibodies that target PD-1 or PD-L1, the majority of patients with non-small cell lung cancer (NSCLC) fail to respond to PD-1/PD-L1 axis inhibitors. The genomic determinants of PD-1/PD-L1 inhibitor efficacy in NSCLC are incompletely understood and likely key to the development of novel personalized immunotherapy approaches. Through integrative analysis of multiple independent datasets of patients with KRAS-mutant lung adenocarcinoma (LUAC) - the most prevalent oncogenic driver event in LUAC- we identified three major disease subsets defined on the basis of co-occurring genomic events in STK11/LKB1 (the KL subgroup), TP53 (KP) and CDKN2A/B, the latter coupled with low expression of the TTF1 transcription factor (KC) (Skoulidis F et al., Cancer Discovery, 2015). STK11/LKB1 is somatically mutated in ~17% of LUAC and encodes a serine/threonine kinase with an established role in the regulation of cellular metabolism, growth and polarity. Gene set enrichment analysis of RNASeq data from the TCGA LUAC cohort indicated striking de-enrichment of multiple gene expression signatures pertaining to anti-tumor immunity in the KL subgroup, raising the possibility that inactivation of STK11/LKB1 may promote establishment of a non-T-cell inflamed immune microenvironment in lung adenocarcinoma. Indeed, STK11/LKB1-mutant LUAC exhibited significantly lower density of infiltrating CD3+ (P=0.0019) and CD8+ (P=0.0072) T-lymphocytes by automated quantitative immunohistochemistry (IHC) in a tumor microarray encompassing early stage, surgically resected, chemotherapy-naïve tumors with available whole exome sequencing (PROSPECT cohort). In an independent, unbiased analysis of a large dataset of 924 LUAC with comprehensive NGS-based genomic profiling and available tumor cell PD-L1 expression (Foundation Medicine dataset) seeking to identify candidate genomic drivers of immune evasion and immunotherapy resistance, STK11/LKB1 genomic alterations emerged as the only significantly enriched gene in PD-L1Negative;TMBIntermediate/High compared to PD-L1High Positive;TMBIntermediate/High tumors (adjusted P&lt;0.001). Thus, establishment of a “cold” tumor immune microenvironment in STK11/LKB1-mutant NSCLC occurs despite the presence of an intermediate/high TMB. Based on these findings we further hypothesized that STK11/LKB1 genomic alterations may impact clinical responses to PD-1 blockade in lung adenocarcinoma. In order to address this critical question, we assembled five independent cohorts of non-squamous NSCLC patients treated with PD-1 axis inhibitors. Three independent retrospective cohorts were studied from members of the Stand Up to Cancer/American Cancer Society Lung Cancer Translational Dream Team (MD Anderson Cancer Center, Memorial Sloan Kettering Cancer Center, Dana-Farber Cancer Institute/Massachusetts General Hospital), collectively forming the SU2C cohort (N=174). A fourth cohort of 66 patients with PD-L1 positive (≥1%) non-squamous NSCLC (regardless of KRAS status) from MD Anderson Cancer Center (MDACC) was assessed to determine the impact of STK11/LKB1 genomic alterations on clinical outcomes with anti-PD-1/PD-L1 therapy in PD-L1-positive tumors. A fifth independent cohort of 44 patients with KRAS-mutant NSCLC (24 treated with nivolumab and 20 treated with docetaxel) from the CheckMate-057 (CM-057) international phase III randomized controlled trial was also analyzed. Objective response rates to PD-1 blockade differed significantly among KL (7.4%), KP (35.7%) and K-only (28.6%) subgroups (P&lt;0.001) in the SU2C cohort and in patients treated with nivolumab in the CheckMate-057 phase III randomized controlled trial (0% vs 57.1% vs. 18.2%; P=0.047). In the SU2C cohort, KL LUAC exhibited shorter progression-free (P&lt;0.001) and overall (P=0.0015) survival compared with KRASMUT;STK11/LKB1WT LUAC. Because non-mutational mechanisms can also account for STK11/LKB1 inactivation in LUAC, we further assessed expression of STK11/LKB1 by IHC in a subset of tumors for which archival tissue was available. KRASMUT;STK11/LKB1MUT (KL) tumors expressed low to undetectable levels of STK11/LKB1 protein, whereas KRASMUT;STK11/LKB1WT tumors displayed variable levels of STK11/LKB1 expression, with 17.6% having a STK11/LKB1 H-score of zero. Patients bearing STK11/LKB1-deficient tumors (STK11/LKB1MUTor STK11/LKB1WT and STK11/LKB1 H-score zero) exhibited significantly shorter PFS (HR 1.80; 95% CI, 1.15-2.82; P = 0.0094) and OS (HR 2.03; 95% CI, 1.13-3.65; P = 0.016) compared with those harboring STK11/LKB1-proficient tumors (STK11/LKB1WT and STK11/LKB1 H-score &gt; 0). Therefore, quantitative IHC for STK11/LKB1 can capture STK11/LKB1-deficient tumors with intact STK11/LKB1 genomic locus and may enhance the predictive utility of STK11/LKB1 genomic alterations. Importantly, the impact of STK11/LKB1 mutations on clinical outcomes with PD-1/PD-L1 inhibitors extended to PD-L1 positive tumors. Among 66 PD-L1-positive (≥1%) non-squamous NSCLC treated with PD-1/PD-L1 inhibitors, STK11/LKB1-mutated tumors exhibited significantly lower objective response rates to PD-1/PD-L1 blockade compared with NSCLC with intact STK11/LKB1 (ORR 0% vs. 34.5%, P = 0.026) and dramatically shorter PFS (HR 4.76; 95% CI, 2.0-11.1, P = 0.00012, log-rank test) and OS (HR 14.3; 95% CI, 3.4-50.0, P &lt; 0.0001, log-rank test). More recently, we also interrogated the impact of STK11/LKB1 genomic alterations on clinical outcomes with chemo-immunotherapy, the new standard of care for the first line treatment of patients with metastatic non-squamous NSCLC. Among 43 patients with metastatic non-squamous NSCLC (wild-type for EGFR and ALK) that received pemetrexed/carboplatin/pembrolizumab at MDACC. STK11/LKB1 alterations were associated with significantly lower objective response rate (31.3% vs. 65.6%, P=0.035) and shorter progression free survival (HR 2.73; 95% CI 1.15-6.48; P=0.018) compared to STK11/LKB1 wild-type tumors. Finally, in order to establish whether primary resistance to immunotherapy is causally linked with STK11/LKB1 inactivation, we generated Stk11/Lkb1-proficient/deficient isogenic derivatives of the LKR10 and LKR13 Kras-mutant murine LUAC cell lines using CRISPR/Cas9-mediated bi-allelic disruption of the Stk11/Lkb1locus, implanted them into the flanks of syngeneic recipient mice and randomized cohorts of tumor bearing mice to treatment with anti-PD-1 mAb (LKR10), anti-PD-L1 mAb (LKR13) or IgG control. Treatment with PD-1/PD-L1 axis inhibitors potently suppressed Stk11/Lkb1-proficient tumors in both the LKR13 and LKR10 models, whereas Stk11/Lkb1 deficient isogenic tumors were recalcitrant to PD-1 axis blockade. Therefore, in these immune-competent models of KRAS-mutant LUAC, loss of STK11/LKB1 directly promotes PD-1/PD-L1 inhibitor resistance, suggesting a causal role. In agreement with data from human tumors, significantly lower numbers of CD3+CD8+ and CD3+CD8+PD1+ T lymphocytes were present in Stk11/Lkb1-deficient murine tumors compared to their Stk11/Lkb1-proficient counterparts. Taken together, our results identify inactivating mutations in STK11/LKB1 as a highly prevalent genomic determinant of a “cold” tumor immune microenvironment and major driver of de novo resistance to PD-1/PD-L1 inhibitors in non-squamous NSCLC independently of TMB and tumor cell PD-L1 expression. Furthermore, attribution of a large fraction of primary resistance to PD-1 axis blockade in non-squamous NSCLC to inactivating mutations in a single tumor suppressor gene - and therefore potentially to a single molecular mechanism - paves the way for the development of rational, science-driven combination therapeutic strategies in order to restore effective anti-tumor immunity in STK11/LKB1-deficient tumors.
Citation Format: Ferdinandos Skoulidis. Inactivating STK11/LKB1 genomic alterations are a major driver of primary resistance to PD-1 axis blockade in non-squamous non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr SY42-02.