KRAS-driven Non-small Cell Lung Cancer Research Articles

CTLA4 blockade abrogates KEAP1/STK11-related resistance to PD-(L)1 inhibitors.

For patients with advanced non-small-cell lung cancer (NSCLC), dual immune checkpoint blockade (ICB) with CTLA4 inhibitors and PD-1 or PD-L1 inhibitors (hereafter, PD-(L)1 inhibitors) is associated with higher rates of anti-tumour activity and immune-related toxicities, when compared with treatment with PD-(L)1 inhibitors alone. However, there are currently no validated biomarkers to identify which patients willbenefit from dual ICB1,2. Here we show that patients with NSCLC who have mutations in the STK11 and/or KEAP1 tumour suppressor genes derived clinical benefit from dual ICB with the PD-L1 inhibitor durvalumab and the CTLA4 inhibitor tremelimumab, but not from durvalumab alone, when added to chemotherapy in the randomized phase III POSEIDON trial3. Unbiased genetic screens identified loss of both of these tumour suppressor genes as independent drivers of resistance to PD-(L)1 inhibition, and showed that loss of Keap1 was the strongest genomic predictor of dual ICB efficacy-a finding that was confirmed in several mouse models of Kras-driven NSCLC. In both mouse models and patients, KEAP1 and STK11 alterations were associated with an adverse tumour microenvironment, which was characterized by a preponderance of suppressive myeloid cells and the depletion of CD8+ cytotoxic T cells, but relative sparing of CD4+ effector subsets. Dual ICB potently engaged CD4+ effector cells and reprogrammed the tumour myeloid cell compartment towards inducible nitric oxide synthase (iNOS)-expressing tumoricidal phenotypes that-together with CD4+ and CD8+ T cells-contributed to anti-tumour efficacy. These data support the use of chemo-immunotherapy with dual ICB to mitigate resistance to PD-(L)1 inhibition in patients with NSCLC who have STK11 and/or KEAP1 alterations.

Abstract 3314: Molecular profiling of covalent menin inhibitor, BMF-219, in KRAS-mutated solid tumors

Abstract Menin is an epigenetic protein that drives oncogenic function through transcriptional control directed by its various cofactors such as MLL1 (KMT2A) and MYC. BMF-219 is a covalent menin inhibitor currently under investigation in a dose-finding study evaluating safety and tolerability in adults with KRAS-driven Non-Small Cell Lung Cancer (NSCLC), Pancreatic Adenocarcinoma (PDAC), or Colorectal Cancer (CRC) (NCT NCT05631574). We previously showed that BMF-219 induces effective killing of solid tumor cells irrespective of their KRAS mutation subtype (AACR 2022, abstract 2665) and perturbs genomic binding of MYC and its binding partner MAX, in acute leukemia cells (ASH Abstract 2021). To probe into the underlying mechanism and the potential pathways engaged, BMF-219-induced effects were evaluated in CRC, PDAC and NSCLC cell lines. Cells treated with relevant concentrations of BMF-219 versus vehicle were assessed for menin and MYC protein levels, along with treatment effects on cell cycle and induction of apoptosis. In addition, bulk RNA-seq analysis was performed on the cell lines treated with BMF-219 in a time course study. BMF-219 induces a dose-dependent decrease in menin protein levels in the solid tumor cell lines tested following 24 hours treatment, with the least impact observed in a KRAS WT cell line. The effect of BMF-219 on c-MYC protein levels were variable and highly cell-type dependent, with the CRC metastatic tumor cell line SW620 exhibiting the highest reduction. Differential expression (DE) analysis of bulk RNA-seq data comparing BMF-219 treated versus vehicle treated cells, identified significant alterations in transcript abundance at 6- and 24-hours post treatment. The magnitude of change in the transcript abundance showed a dose-dependent response without significant alteration of a panel of housekeeping genes. GSEA analysis identified pathways associated with cell cycle, cell division and MYC-regulated were significantly differentially expressed across multiple cell lines. These data suggest similar molecular pathways are altered by BMF-219 across KRAS-mutated solid tumor cells. Additional insights into key pathways and modulators will be reported. Collectively, these data suggest that the functional pathway alterations induced by BMF-219 in solid tumor cells are likely pan-KRAS and are similar across different solid tumor indications. This provides further support for advancing the clinical investigation of BMF-219 in KRAS-driven solid tumors. Citation Format: Amin A. Momin, Tripta Rughwani, Nida Tanataweethum, Kasia Mordec, Raechel Lin, Megan Chin, Daniel Lu, Priyanka Somanath, Mini Balakrishnan, Thomas Butler. Molecular profiling of covalent menin inhibitor, BMF-219, in KRAS-mutated solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3314.

Tissue factor overexpression promotes resistance to KRAS-G12C inhibition in non-small cell lung cancer

The recently approved KRASG12C mutation-specific inhibitors sotorasib and adagrasib (KRASG12C-I) represent a promising therapy for KRASG12C-driven non-small cell lung cancer (NSCLC). However, many eligible patients do not benefit due to intrinsic or acquired drug resistance. Tissue factor (TF) is overexpressed in KRAS-mutated (KRASmut) NSCLC and is the target of the FDA-approved ADC Tivdak. Here, we employed HuSC1-39, the parent antibody of a clinical stage TF-ADC (NCT04843709), to investigate the role of TF in KRASmut NSCLC. We found that patients with TF-overexpression had poor survival, elevated P-ERK/P-AKT activity levels and low immune effector cell infiltration in the tumor. In a panel of KRASG12C cell lines, KRASG12C-I response correlated with suppression of TF mRNA, which was not observed in resistant cells. In the drug resistant cells, TF-overexpression relied on an mTORC2-mediated and proteasome-dependent pathway. Combination treatment of HuSC1-39 or mTORC1/2 inhibitor MTI-31 with KRASG12C-I each produced synergistic antitumor efficacy in cell culture and in an orthotopic lung tumor model. TF-depletion in the resistant cells diminished epithelial mesenchymal transition, reduced tumor growth and greatly sensitized KRASG12C-I response. Moreover, employing immunohistochemistry and coculture studies, we demonstrated that HuSC1-39 or MTI-31 reset the tumor microenvironment and restore KRASG12C-I sensitivity by reshaping an M1-like macrophage profile with greatly enhanced phagocytic capacity toward tumor cell killing. Thus, we have identified the TF/mTORC2 axis as a critical new mechanism for triggering immunosuppression and KRASG12C-I resistance. We propose that targeting this axis with HuSC1-39 or MTI-31 will improve KRASG12C-I response in KRAS-driven NSCLC.

Long non-coding RNA HIF1A-As2 and MYC form a double-positive feedback loop to promote cell proliferation and metastasis in KRAS-driven non-small cell lung cancer

Lung cancer is the leading cause of cancer-related deaths worldwide. KRAS is the main oncogenic driver in lung cancer that can be activated by gene mutation or amplification, but whether long non-coding RNAs (lncRNAs) regulate its activation remains unknown. Through gain and loss of function approaches, we identified that lncRNA HIF1A-As2, a KRAS-induced lncRNA, is required for cell proliferation, epithelial-mesenchymal transition (EMT) and tumor propagation in non-small cell lung cancer (NSCLC) in vitro and in vivo. Integrative analysis of HIF1A-As2 transcriptomic profiling reveals that HIF1A-As2 modulates gene expression in trans, particularly regulating transcriptional factor genes including MYC. Mechanistically, HIF1A-As2 epigenetically activates MYC by recruiting DHX9 on MYC promoter, consequently stimulating the transcription of MYC and its target genes. In addition, KRAS promotes HIF1A-As2 expression via the induction of MYC, suggesting HIF1A-As2 and MYC form a double-regulatory loop to strengthen cell proliferation and tumor metastasis in lung cancer. Inhibition of HIF1A-As2 by LNA GapmeR antisense oligonucleotides (ASO) significantly improves sensitization to 10058-F4 (a MYC-specific inhibitor) and cisplatin treatment in PDX and KRASLSLG12D-driven lung tumors, respectively.

Abstract 5723: An in vivo pharmacogenomics platform replicates and extends biomarkers of therapy response identified via causal inference analysis of clinical data

Abstract Recent development of therapies targeting oncogenes have dramatically improved cancer care for subsets of patients and generated a wave of interest in cancer precision medicine. However, effective targeted therapies are scarce as we have a limited understanding of how drug responses are modulated by tumor genotype. Here, we utilize both human data and in vivo models to identify genetic drivers of therapy response in KRAS-driven non-small cell lung cancer patients treated with chemotherapy. We first present an integrative causal inference analysis of three clinical data resources: (1) the recently released Genomics Evidence Neoplasia Information Exchange, Biopharma Collaboration dataset (GENIE-BPC; n=197), (2) a selected set of patients from the Tempus Clinico-genomic Database (n=330), and (3) an additional cohort from Memorial Sloan Kettering Cancer Center (n=218). Each dataset was first analyzed separately using a causal inference pipeline. Doubly robust estimators for each variant were inferred within a counting process survival analysis model accounting for time-varying treatments and immortal time bias. Meta-analysis of the results from all three cohorts identified three commonly mutated and highly replicable genes with a significant effect on overall survival: KEAP1, SMARCA4, and CDKN2A. As further validation, we used our murine in vivo pharmacogenomics (PGx) platform that can quantify the effects of therapies across thousands of tumors of diverse genotypes. These tumors are initiated de novo in the native microenvironmental context in mice with an intact adaptive immune system. We tested a chemotherapy combination of carboplatin and pemetrexed in mice with KrasG12D-driven lung tumors and inactivation of each of 60 putative tumor suppressors. Treatment led to >75% reduction in tumor sizes relative to tumor suppressor inactivated (matched) vehicle-treated controls. These models identified causal effects for two out of the three candidates above: KEAP1 (resistance) and CDKN2A (sensitive). Moreover, our PGx platform identified additional candidate genes beyond those found using the clinical data, which had insufficient sample size for these rarely mutated genes. Together, we demonstrate how leveraging our PGx platform together with human data within a causal inference framework may improve the stratification of patients by their clinical outcomes, profoundly advancing the promise of precision medicine. Citation Format: David Amar, Erick Scott, Ian P. Winters, Gregory D. Wall, Dmitri A. Petrov, Monte M. Winslow, Joseph Juan, Ian K. Lai, Lafia Sebastian, Edwin A. Apilado, Gabriel Grenot, Vy B. Tran, Charles Rudin, Michael J. Rosen. An in vivo pharmacogenomics platform replicates and extends biomarkers of therapy response identified via causal inference analysis of clinical data. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5723.

Abstract 272: Autophagy and MEK inhibition promotes ferroptosis in liver kinase B1 (Lkb1)-deficient Kras-driven lung tumors

Abstract Tumor suppressor Liver Kinase B1 (LKB1) activates 5’-adenosine monophosphate protein kinase (AMPK) and maintains energy homeostasis in response to energy crises. LKB1 and KRAS are the third most frequent co-mutations detected in non-small cell lung cancer (NSCLC), causing aggressive tumor growth and metastases. Unfortunately, standard treatment with RAS-RAF-MEK-ERK signaling pathway inhibitors has minimal therapeutic efficacy in LKB1-mutant KRAS-driven NSCLC. Thus, identifying a novel treatment for patients harboring co-mutations in LKB1 and KRAS is urgently needed. Autophagy degrades and recycles the building blocks for cancer cells to survive metabolic challenges. Using genetically engineered mouse models (GEMMs), we have previously demonstrated that autophagy compensates for Lkb1 loss for Kras-driven lung tumorigenesis; loss of an autophagy-essential gene Atg7 dramatically impaired tumor initiation and tumor growth in KrasG12D/+;Lkb1−/− (KL) lung tumors. This is in sharp contrast to Lkb1 wild-type (WT) (KrasG12D/+;p53−/− (KP)) tumors that are less sensitive to autophagy gene ablation. To further value our discoveries in clinical translational ability, we treated mouse lung tumor derived cell lines (TDCLs) with FDA-approved autophagy inhibitor hydroxychloroquine (HCQ) and MEK inhibitor Trametinib and found that the combination treatment displayed synergistic anti-proliferative effects in KL TDCLs compared to KP TDCLs. To elucidate the underlying mechanism of increased sensitivity of KL TDCLs to Trametinib by autophagy ablation, we performed metabolomic profiling of KL TDCLs with Trametinib, HCQ, or combination treatment and found that several glycolytic and TCA cycle intermediates, amino acids, and ATP levels were significantly upregulated upon treatment with Trametinib, which were significantly reduced by the combination treatment. In addition, the combination treatment significantly reduced mitochondrial membrane potential, basal respiration, and ATP production in KL TDCLs. In vivo studies using tumor allografts, genetically engineered mouse models (GEMMs) and patient-derived xenografts (PDXs) showed anti-tumor activity of the combination treatment on KL tumors, but not in KP tumors. Moreover, we found increased lipid peroxidation indicative of ferroptosis in KL TDCLs and KL PDX tumors with the combination treatment compared to the single agent treatments. Finally, treatment with a ferroptosis inhibitor rescued the reduced KL allograft tumor growth caused by the combination treatment. Taken together, our observations indicate that autophagy upregulation in KL tumors causes resistance to Trametinib treatment by maintaining energy homeostasis for cell survival and inhibits ferroptosis. Therefore, a combination of autophagy and MEK inhibition could be a novel therapeutic strategy to specifically treat LKB1-deficient KRAS-driven NSCLC. Citation Format: Vrushank Bhatt, Taijin Lan, Wenping Wang, Jerry Kong, Eduardo Cararo Lopes, Khoosheh Khayati, Jianming Wang, Akash Raju, Michael Rangel, Enrique Lopez, Zhixian Sherrie Hu, Xuefei Luo, Xiaoyang Su, Jyoti Malhotra, Wenwei Hu, Sharon R. Pine, Eileen White, Jessie Yanxiang Guo. Autophagy and MEK inhibition promotes ferroptosis in liver kinase B1 (Lkb1)-deficient Kras-driven lung tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 272.

Targeting metabolic vulnerability in mitochondria conquers MEK inhibitor resistance in KRAS-mutant lung cancer.

MEK is a canonical effector of mutant KRAS; however, MEK inhibitors fail to yield satisfactory clinical outcomes in KRAS-mutant cancers. Here, we identified mitochondrial oxidative phosphorylation (OXPHOS) induction as a profound metabolic alteration to confer KRAS-mutant non-small cell lung cancer (NSCLC) resistance to the clinical MEK inhibitor trametinib. Metabolic flux analysis demonstrated that pyruvate metabolism and fatty acid oxidation were markedly enhanced and coordinately powered the OXPHOS system in resistant cells after trametinib treatment, satisfying their energy demand and protecting them from apoptosis. As molecular events in this process, the pyruvate dehydrogenase complex (PDHc) and carnitine palmitoyl transferase IA (CPTIA), two rate-limiting enzymes that control the metabolic flux of pyruvate and palmitic acid to mitochondrial respiration were activated through phosphorylation and transcriptional regulation. Importantly, the co-administration of trametinib and IACS-010759, a clinical mitochondrial complex I inhibitor that blocks OXPHOS, significantly impeded tumor growth and prolonged mouse survival. Overall, our findings reveal that MEK inhibitor therapy creates a metabolic vulnerability in the mitochondria and further develop an effective combinatorial strategy to circumvent MEK inhibitors resistance in KRAS-driven NSCLC.

Inhibition of autophagy and MEK promotes ferroptosis in Lkb1-deficient Kras-driven lung tumors

LKB1 and KRAS are the third most frequent co-mutations detected in non-small cell lung cancer (NSCLC) and cause aggressive tumor growth. Unfortunately, treatment with RAS-RAF-MEK-ERK pathway inhibitors has minimal therapeutic efficacy in LKB1-mutant KRAS-driven NSCLC. Autophagy, an intracellular nutrient scavenging pathway, compensates for Lkb1 loss to support Kras-driven lung tumor growth. Here we preclinically evaluate the possibility of autophagy inhibition together with MEK inhibition as a treatment for Kras-driven lung tumors. We found that the combination of the autophagy inhibitor hydroxychloroquine (HCQ) and the MEK inhibitor Trametinib displays synergistic anti-proliferative activity in KrasG12D/+;Lkb1-/- (KL) lung cancer cells, but not in KrasG12D/+;p53-/- (KP) lung cancer cells. In vivo studies using tumor allografts, genetically engineered mouse models (GEMMs) and patient-derived xenografts (PDXs) showed anti-tumor activity of the combination of HCQ and Trametinib on KL but not KP tumors. We further found that the combination treatment significantly reduced mitochondrial membrane potential, basal respiration, and ATP production, while also increasing lipid peroxidation, indicative of ferroptosis, in KL tumor-derived cell lines (TDCLs) and KL tumors compared to treatment with single agents. Moreover, the reduced tumor growth by the combination treatment was rescued by ferroptosis inhibitor. Taken together, we demonstrate that autophagy upregulation in KL tumors causes resistance to Trametinib by inhibiting ferroptosis. Therefore, a combination of autophagy and MEK inhibition could be a novel therapeutic strategy to specifically treat NSCLC bearing co-mutations of LKB1 and KRAS.

Targeting Asparagine Synthetase in Tumorgenicity Using Patient-Derived Tumor-Initiating Cells.

Reprogramming of energy metabolism is regarded as one of the hallmarks of cancer; in particular, oncogenic RAS has been shown to be a critical regulator of cancer metabolism. Recently, asparagine metabolism has been heavily investigated as a novel target for cancer treatment. For example, Knott et al. showed that asparagine bioavailability governs metastasis in a breast cancer model. Gwinn et al. reported the therapeutic vulnerability of asparagine biosynthesis in KRAS-driven non-small cell lung cancer. We previously reported that KRAS-mutated CRC cells can adapt to glutamine depletion through upregulation of asparagine synthetase (ASNS), an enzyme that synthesizes asparagine from aspartate. In our previous study, we assessed the efficacy of asparagine depletion using human cancer cell lines. In the present study, we evaluated the clinical relevance of asparagine depletion using a novel patient-derived spheroid xenograft (PDSX) mouse model. First, we examined ASNS expression in 38 spheroid lines and found that 12 lines (12/37, 32.4%) displayed high ASNS expression, whereas 26 lines (25/37, 67.6%) showed no ASNS expression. Next, to determine the role of asparagine metabolism in tumor growth, we established ASNS-knockdown spheroid lines using lentiviral short hairpin RNA constructs targeting ASNS. An in vitro cell proliferation assay demonstrated a significant decrease in cell proliferation upon asparagine depletion in the ASNS-knockdown spheroid lines, and this was not observed in the control spheroids lines. In addition, we examined asparagine inhibition with the anti-leukemia drug L-asparaginase (L-Asp) and observed a considerable reduction in cell proliferation at a low concentration (0.1 U/mL) in the ASNS-knockdown spheroid lines, whereas it exhibited limited inhibition of control spheroid lines at the same concentration. Finally, we used the PDSX model to assess the effects of asparagine depletion on tumor growth in vivo. The nude mice injected with ASNS-knockdown or control spheroid lines were administered with L-Asp once a day for 28 days. Surprisingly, in mice injected with ASNS-knockdown spheroids, the administration of L-Asp dramatically inhibited tumor engraftment. On the other hands, in mice injected with control spheroids, the administration of L-Asp had no effect on tumor growth inhibition at all. These results suggest that ASNS inhibition could be critical in targeting asparagine metabolism in cancers.

130 (PB120) - Characterization of KRAS-driven NSCLC cell lines with diverse mutational landscape and assessment of their response to MYC inhibition

Introduction: Lung cancer is the second most common cancer type both in males and females and the leading cause of cancer death worldwide. Inside lung cancer, Non-Small-Cell Lung Cancer (NSCLC) represents around 85% of the cases, where KRAS is the leading mutated oncogene (∼25%) and confers poor prognosis and a high risk of tumor recurrence. Subsets of mutant KRAS NSCLC have been described based on the presence of concurrent mutations in tumor suppressor genes (TSGs), being the most common TP53, KEAP1 and LKB1 (also called STK11).

Race-specific genomic determinants of therapeutic response in African American NSCLC patients.

e21015 Background: African American (AA) individuals are disproportionately affected by lung cancer in terms of incidence and mortality, despite lower tobacco exposure compared to Caucasian Americans (CA). Since molecular profiling is critically important to guide therapy selection in non-small cell lung cancer (NSCLC), we compared treatment-relevant molecular data in a cohort of AA and CA patients with EGFR or KRAS-driven NSCLC. Methods: This is a retrospective study using a clinicogenomic electronic medical record database from health systems in the United States designed to evaluate outcomes in advanced stage NSCLC. Eligibility criteria for analysis included a diagnosis of NSCLC between 2010 and 2020, stage III or IV, available race/ethnicity data, and identification of common KRAS or EGFR activating variant within 60 days of diagnosis. The primary objective was to define the type of EGFR or KRAS activating variant and determine the frequency of co-occurring alterations in NSCLC-associated genes by cohort (AA and CA). Secondary objectives included examining the timing of genetic testing and use of targeted therapy in these cohorts. Results: A total of 642 NSCLC patients with KRAS (15.0% AA; 81.6% CA) and 348 patients with EGFR oncogenic mutations (13.8% AA; 70.1% CA) met inclusion criteria. Mean time from diagnosis to first molecular test result was 20.9 days for AA vs. 19.9 days for CA (p = 0.5). The EGFR G719S variant was more prevalent in the AA cohort (6.3% AA v 0.4%) with no significant differences observed in the most common KRAS or EGFR variants tested (Table). A total of 100 KRAS-mutant patients (16 AA, 86 CA) had a reported result in at least one co-mutation of interest. The most frequent co-mutations were TP53 (81.3% AA v 65% CA, p = 0.92); KEAP1 (18.8% AA v 8.3% CA, p = 0.52); ATM (18.8% AA v 9.5% CA, p = 0.54); and PTEN (0% AA v 8.3% CA, p = 0.5). A total of 35 EGFR-mutant patients (4 AA, 31 CA) had a co-mutation reported in TP53 (75% AA v 65% CA, p = 0.77). EGFR targeted therapy was reported in 47.9% of AA and 52.9% of CA (p = 0.639). In the KRAS-mutant cohort, delivery of immune checkpoint inhibitors (ICIs) was reported in 29.2% of AA and 39.3% of CA (p = 0.08); chemotherapy was reported in 36.5% AA and 45.2% of CA (p = 0.14). Delivery of both ICI and chemotherapy was reported in 13.5% of AA and 21.9% of CA (p = 0.08). Conclusions: In a real-world cohort of NSCLC patients, we found similar frequencies of common KRAS and EGFR variants in AA and CA, but higher rates of EGFR G719S variant in AA patients. A more comprehensive analysis is needed to further evaluate the trends in co-mutations observed in our analysis, to determine the race-specific impact of these alterations in EGFR and KRAS-driven NSCLC.[Table: see text]

Discovery of Small Molecule NSC290956 as a Therapeutic Agent for KRas Mutant Non-Small-Cell Lung Cancer.

HRas-GTP has a transient intermediate state with a “non-signaling open conformation” in GTP hydrolysis and nucleotide exchange. Due to the same hydrolysis process and the structural homology, it can be speculated that the active KRas adopts the same characteristics with the “open conformation.” This implies that agents locking this “open conformation” may theoretically block KRas-dependent signaling. Applying our specificity-affinity drug screening approach, NSC290956 was chosen by high affinity and specificity interaction with the “open conformation” structure HRasG60A-GppNp. In mutant KRas-driven non-small-cell lung cancer (NSCLC) model system, NSC290956 effectively suppresses the KRas-GTP state and gives pharmacological KRas inhibition with concomitant blockages of both the MAPK-ERK and AKT-mTOR pathways. The dual inhibitory effects lead to the metabolic phenotype switching from glycolysis to mitochondrial metabolism, which promotes the cancer cell death. In the xenograft model, NSC290956 significantly reduces H358 tumor growth in nude mice by mechanisms similar to those observed in the cells. Our work indicates that NSC290956 can be a promising agent for the mutant KRas-driven NSCLC therapy.

Resistance to immune checkpoint inhibitors in KRAS-mutant non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) patients with Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation are associated with significant clinical heterogeneity and a poor prognosis to standard NSCLC therapies such as surgical resection, radiotherapy, chemotherapies, and targeted medicines. However, the application of immune checkpoints inhibitors (ICIs) has dramatically altered the therapeutic pattern of NSCLC management. Clinical studies have indicated that some KRAS-mutant NSCLC patients could benefit from ICIs; however, the responses in some patients are still poor. This review intends to elucidate the mechanisms of resistance to immunotherapy in KRAS-driven NSCLC and highlight the TME functions altered by immunoinhibitors, immunostimulators, and cancer metabolism. These metabolic pathways could potentially be promising approaches to overcome immunotherapy resistance.

Targeting Oncogenic KRAS in Non-Small-Cell Lung Cancer.

Simple Summaryv-Ki-ras2 Kirsten rat sarcoma viral oncogene (KRAS) is the most common driver in NSCLC, and targeting oncogenic KRAS is a major challenge in the treatment of non-small-cell lung cancer (NSCLC). While several covalent KRAS G12C inhibitors have emerged as a novel anti-KRAS therapy, the development of combined therapies involving the targeting of oncogenic KRAS plus other targeted drugs is still required given the vast heterogeneity of KRAS-mutated tumors. In this review, we summarize the biological and immunological characteristics of oncogenic KRAS-driven NSCLC and the preclinical and clinical evidence for mutant KRAS-targeted therapies. We also discuss the mechanisms of resistance to KRAS G12C inhibitors and possible therapeutic strategies to overcome this drug resistance.Recent advances in molecular biology and the resultant identification of driver oncogenes have achieved major progress in precision medicine for non-small-cell lung cancer (NSCLC). v-Ki-ras2 Kirsten rat sarcoma viral oncogene (KRAS) is the most common driver in NSCLC, and targeting KRAS is considerably important. The recent discovery of covalent KRAS G12C inhibitors offers hope for improving the prognosis of NSCLC patients, but the development of combination therapies corresponding to tumor characteristics is still required given the vast heterogeneity of KRAS-mutated NSCLC. In this review, we summarize the current understanding of KRAS mutations regarding the involvement of malignant transformation and describe the preclinical and clinical evidence for targeting KRAS-mutated NSCLC. We also discuss the mechanisms of resistance to KRAS G12C inhibitors and possible combination treatment strategies to overcome this drug resistance.

Functional assessment of somatic STK11 variants identified in primary human non-small cell lung cancers.

Serine/Threonine Kinase 11 (STK11) encodes an important tumor suppressor that is frequently mutated in lung adenocarcinoma. Clinical studies have shown that mutations in STK11 resulting in loss of function correlate with resistance to anti-PD-1 monoclonal antibody therapy in KRAS-driven non-small cell lung cancer (NSCLC), but the molecular mechanisms responsible remain unclear. Despite this uncertainty, STK11 functional status is emerging as a reliable biomarker for predicting non-response to anti-PD-1 therapy in NSCLC patients. The clinical utility of this biomarker ultimately depends upon accurate classification of STK11 variants. For nonsense variants occurring early in the STK11 coding region, this assessment is straightforward. However, rigorously demonstrating the functional impact of missense variants remains an unmet challenge. Here we present data characterizing four STK11 splice-site variants by analyzing tumor mRNA, and 28 STK11 missense variants using an in vitro kinase assay combined with a cell-based p53-dependent luciferase reporter assay. The variants we report were identified in primary human NSCLC biopsies in collaboration with the University of Vermont Genomic Medicine group. Additionally, we compare our experimental results with data from 22 in silico predictive algorithms. Our work highlights the power, utility and necessity of functional variant assessment and will aid STK11 variant curation, provide a platform to assess novel STK11 variants and help guide anti-PD-1 therapy utilization in KRAS-driven NSCLCs.

Abstract 2706: Utilizing RNAscope to assess STK11-LoF-dependent transcriptional phenotypes in human NSCLC biopsies and evaluate its potential to predict anti-PD1 therapy response

Abstract Our work demonstrates RNAscope allows simultaneous quantitative assessment of multiple transcripts in situ using formalin fixed paraffin embedded (FFPE) human non-small cell lung cancer (NSCLC) biopsies and suggests this methodology could be used as a rapid surrogate biomarker for genotype/transcriptome profiling stemming for Serine Threonine Kinase 11 (STK11) loss of function (LoF). Recent clinical studies have correlated anti-PD1 therapy resistance in NSCLC with somatic disruption of STK11. Historically, rapid assessment of STK11 status using immunohistochemistry (IHC) has been a challenge. Our work in human NSCLC cell lines suggests that STK11 loss correlates with changes in tumor intrinsic cytokine expression. Several recent studies have suggested such changes may drive immune evasion. We therefore wondered whether cytokine expression profiles from primary human NSCLC biopsies might be used as a reliable biomarker to predict anti-PD1 therapy response. Here we present our findings using multiplexed RNAscope probes on KRAS-driven primary human NSCLC biopsies with and without STK11 loss. This initial work focuses on cytokines identified using our cell culture models as a proof of principle approach. Using CRISPR-Cas9 we engineered paired STK11 KO cell lines derived from 3 independent KRAS-driven human lung adenocarcinomas (NCI-H441, NCI-H1792, NCI-H2009). RNAseq and differential gene expression analysis revealed STK11-loss-dependent increases in IL-6, IL-8 and IL-32 in all three lines. We then created FFPE cell blocks using these cell lines and validated the RNAseq expression changes with multiplexed RNAscope probes in situ. Next, we informatically searched the molecular pathology archives at the UVM medical center and identified primary KRAS-driven human NSCLC biopsies with and without predicted STK11 loss. We then performed multiplexed RNAscope analysis on these samples to quantify IL-6, IL-8 and IL-32 gene expression as a function of tumor genotype in vivo. Our data suggest RNAscope represents a valid methodology to exploit quantitative differential gene expression as a reliable and reproducible surrogate biomarker of somatic STK11 disruption in NSCLC. Our future studies aim to establish whether genotype-dependent cytokine profiles predict anti-PD1 therapy response in KRAS-driven lung adenocarcinomas. Citation Format: Hailey M. Sarausky, Sean M. Lenahan, David Joseph Seward. Utilizing RNAscope to assess STK11-LoF-dependent transcriptional phenotypes in human NSCLC biopsies and evaluate its potential to predict anti-PD1 therapy response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2706.

BCL6 confers KRAS-mutant non-small-cell lung cancer resistance to BET inhibitors.

The bromodomain and extra-terminal domain (BET) proteins are promising therapeutic targets to treat refractory solid tumors; however, inherent resistance remains a major challenge in the clinic. Recently, the emerging role of the oncoprotein B cell lymphoma 6 (BCL6) in tumorigenesis and stress response has been unveiled. Here, we demonstrate that BCL6 was upregulated upon BET inhibition in KRAS-mutant cancers, including non-small-cell lung cancer (NSCLC). We further found that BRD3, not BRD2 or BRD4, directly interacted with BCL6 and maintained the negative autoregulatory circuit of BCL6. Disrupting this negative autoregulation by BET inhibitors (BETi) resulted in a striking increase in BCL6 transcription, which further activated the mTOR signaling pathway through repression of the tumor suppressor death-associated protein kinase 2. Importantly, pharmacological inhibition of either BCL6 or mTOR improved the tumor response and enhanced the sensitivity of KRAS-mutant NSCLC to BETi in both in vitro and in vivo settings. Overall, our findings identify a mechanism of BRD3-mediated BCL6 autoregulation and further develop an effective combinatorial strategy to circumvent BETi resistance in KRAS-driven NSCLC.

Targeted inhibition of glutamine metabolism enhances the antitumor effect of selumetinib in KRAS-mutant NSCLC

Regulated by the tumor microenvironment, the metabolic network of the tumor is reprogrammed, driven by oncogenes and tumor suppressor genes. The metabolic phenotype of tumors of different driven-genes and different tissue types is extremely heterogeneous. KRAS-mutant non-small cell lung cancer (NSCLC) has glutamine dependence. In this study, we demonstrated that glutamine utilization of KRAS-mutant NSCLC was higher than that of KRAS wild-type. CB839, an efficient glutaminase inhibitor, synergized with the MEK inhibitor selumetinib to enhance antitumor activity in KRAS-mutant NSCLC cells and xenografts, and the therapeutic response could be well identified by 18F-FDG PET imaging. Combination therapy induced redox stress, manifesting as a decrease in mitochondrial membrane potential and an increase in ROS levels, and energetic stress manifesting as suppression of glycolysis and glutamine degradation. The phosphorylation of AKT was also suppressed. These effects combined to induce autophagy and thereby caused cancer cell death. Our results suggest that dual inhibition of the MEK-ERK pathway and glutamine metabolism activated by KRAS mutation may be an effective treatment strategy for KRAS-driven NSCLC.

Abstract 5710: Autophagy inhibition sensitizes Liver Kinase B1 (LKB1) - deficient Kras-driven lung tumors to MEK inhibitor Trametinib

Abstract Tumor suppressor liver kinase B1 (LKB1) activates 5'-adenosine monophosphate protein kinase (AMPK) and plays an essential role in maintaining energy homeostasis. LKB1 mutations are the third most frequent mutation detected in non-small cell lung cancer (NSCLC). Compared to p53 mutation, co-mutations in LKB1 with KRAS cause aggressive tumor growth, metastases and resistance to chemotherapy as well as immunotherapy. Thus, identifying a novel target to improve LKB1-deficient Kras-driven NSCLC treatment is urgently needed. Our previous work demonstrates that autophagy inhibition is synthetically lethal in KrasG12D/+;Lkb1−/− (KL) mediated tumorigenesis; in contrast to intact autophagy KL lung tumors, loss of an autophagy-essential gene Atg7 dramatically impaired tumor initiation and tumor growth. This is in sharp contrast to wild-type (WT) Lkb1 (KrasG12D/+;p53−/− (KP)) tumors that are less sensitive to autophagy gene ablation. These observations indicate that LKB1 mutations predispose KRAS NSCLC to autophagy inhibition and that LKB1 mutations could be explored as a predictive biomarker for precision lung cancer therapy. To further value our discoveries in clinical translational ability, we treated mouse lung tumor-derived cell lines (TDCLs) with FDA approved autophagy inhibitor hydroxychloroquine (HCQ) and found that KL TDCLs were much sensitive to HCQ-induced cell death compared with Lkb1-WT KP TDCLs. Furthermore, a combination treatment of HCQ with mitogen-activated protein kinase kinase (MAPKK or MEK) inhibitor Trametinib showed synergistic anti-proliferative effects in KL TDCLs, but not in KP TDCLs. To elucidate the underlying mechanism of increased sensitivity of KL TDCLs to Trametinib by autophagy ablation, we performed metabolomic profiling of KL TDCLs with Trametinib, HCQ alone or in combination treatment. We found that the levels of several glycolytic and TCA cycle intermediates, as well as amino acids, were significantly upregulated upon treatment with Trametinib, which were significantly reduced by the combined treatment with HCQ and trametinib. Moreover, Trametinib treatment led to an increased level of ATP, which was decreased by the combination treatment. Similarly, we observed anti-tumor synergistic effects of the combination treatment in KL allograft tumor growth. In addition, metastases of KL TDCLs to the lung was reduced by the combination treatment compared to the single agents. We further found that in KRAS-mutant human lung cancer cell lines, LKB1-mutant cells were much sensitive to the combination treatment than LKB1-WT cells. Our observations suggest that KL TDCLs and KL human lung cancer cells resist to Trametinib treatment by upregulating autophagy to maintain energy homeostasis for survival. Therefore, combination therapy of autophagy and MEK inhibition could be a novel therapeutic strategy to specifically treat LKB1-deficient NSCLC. Citation Format: Vrushank Dharmesh Bhatt, Khoosheh Khayati, Jerry Kong, Akash Raju, Zhixian Hu, Xiaoyang Su, Jessie Yanxiang Guo. Autophagy inhibition sensitizes Liver Kinase B1 (LKB1) - deficient Kras-driven lung tumors to MEK inhibitor Trametinib [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5710.

Abstract PR07: Combinatorial knockout of Rap1GDS1 and RhoA leads to lethality in KRAS-driven non-small cell lung cancer

Abstract Synthetic lethal-based approaches to targeting KRAS driven cancers have recently been receiving increasing attention. However, as KRAS activates multiple effector pathways, targeting single genes may not be sufficient to fully inhibit KRAS-driven oncogenesis. In addition, genome-wide screens can be limited by high noise and low signal. Furthermore, it is increasingly clear that standard screens based on 2D proliferation can miss critical phenotypes relevant to cancer biology. Therefore, we developed a multistage screening strategy that includes a) identification of KRAS-proximal protein-protein interaction (PPI) networks, b) targeted CRISPR-based screening for pairs of genes that together are synthetic lethal with KRAS, and c) validation in a 3D sphere system using both human and mouse NSCLC cells. We used affinity purification/mass spectrometry (AP/MS) to construct a detailed map of protein-protein interactions centered on KRAS. Based on this network, we designed a CRISPR/Cas9 library targeting pairwise combinations of 119 KRAS-interacting genes. We screened two KRAS-driven non-small cell lung cancer (NSCLC) cell lines (A549 and H23). This screen yielded a large number of gene pairs that synergistically impaired cell growth. We selected the 20 most promising targets for further screening in a panel of 5 KRAS-mutant and 4 KRAS wild-type NSCLC cell lines. From these screens we identified a KRAS-specific synergistic interaction between two genes—Rap1GDS1 and RhoA. Validation in 3D cultures in a panel of cell lines showed that combinatorial Rap1GDS1 and RhoA knockout selectively impairs sphere growth of KRAS-mutant NSCLC cells but has minimal effects on KRAS wild-type NSCLC cell lines or nontransformed lung cells. We demonstrate that this effect is primarily due to the induction of apoptosis. Rap1GDS1 has previously been reported to encode two different isoforms with distinct cellular functions. We observe that only one of the isoforms is involved in the mediation of the KRAS-dependent lethal effect. Current efforts are directed at elucidating the mechanistic basis for the isoform-specific combinatorial lethality between Rap1GDS1 and RhoA. Furthermore, we are actively exploring how to exploit this knowledge to define potential novel therapies for KRAS-driven cancer. Overall, we demonstrate that an integrated strategy combining proteomics, paired CRISPR screens and innovative 3D validation systems can identify novel synthetic lethal combinations. This approach may help identify novel synthetic lethal drug combinations for KRAS and other to-date undruggable oncogenes. This abstract is also being presented as Poster A40. Citation Format: Kaja Kostyrko, Marcus Kelly, Kyuho Han, Edwin Jeng, David Morgens, Michael Bassik, Peter Jackson, Alejandro Sweet-Cordero. Combinatorial knockout of Rap1GDS1 and RhoA leads to lethality in KRAS-driven non-small cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr PR07.