LKB1-deficient Non-small Cell Lung Cancer Research Articles

Hyper-Interferon Sensitive influenza induces adaptive immune responses and overcomes resistance to anti-PD-1 in murine non-small cell lung cancer.

Despite recent advances in immunotherapy with immune checkpoint inhibitors (ICI), many patients with non-small cell lung cancer (NSCLC) fail to respond or develop resistance after an initial response. In situ vaccination (ISV) with engineered viruses has emerged as a promising antigen-agnostic strategy that can both condition the tumor microenvironment (TME) and augment anti-tumor T cell responses to overcome immune resistance. We engineered a live attenuated viral vaccine, Hyper-Interferon Sensitive virus (HIS), by conducting a genome-wide functional screening and introducing eight interferon (IFN)-sensitive mutations in the influenza genome. Compared to wild-type (WT) influenza, HIS replication was attenuated in immunocompetent hosts, enhancing its potential as a safe option for cancer therapy. HIS ISV elicited robust yet transient type I IFN responses in murine NSCLCs, leading to an enrichment of polyfunctional effector Th1 CD4 and cytotoxic CD8 T cells into the tumor. HIS ISV demonstrated enhanced anti-tumor efficacy compared to WT in multiple syngeneic murine models of NSCLC with distinct driver mutations and varying mutational burden. This efficacy was dependent on host type 1 IFN responses and T lymphocytes. HIS ISV overcame resistance to anti-PD-1 in LKB-1 deficient murine NSCLC, resulting in improved overall survival and enduring systemic tumor-specific immunity. These studies provide compelling evidence to support further clinical evaluation of HIS as a novel 'off-the-shelf' ISV strategy for patients with NSCLC refractory to ICI.

Abstract 4450: MYC upregulates MCT4 expression and promotes metabolic reprogramming and enhanced lactate utilization in LKB1-deficient NSCLC

Abstract STK11, which encodes the serine/threonine kinase LKB1, is the second most commonly altered tumor suppressor in non-small cell lung cancer (NSCLC). LKB1 loss is frequently co-mutated with KRAS and associated with primary resistance to immunotherapy, which is partially due to MCT4-dependent lactate secretion. However, the mechanisms driving MCT4 elevation and metabolic reprogramming remains unclear. We hypothesized that increased MYC expression, which is associated with LKB1 loss, plays a pivotal role in metabolic alterations, promoting lactate incorporation and maintaining redox homeostasis. To explore the impact of lactate on tumor cells with LKB1 loss, we performed metabolic profiling on KRAS mutant (K) murine lung cancer cells with or without LKB1 knockout (KL) treated with either glucose or lactate as the carbon source. PLS analysis indicated that there was no notable difference between K and KL cells after glucose treatment. However, we observed significant changes in metabolites between K and KL cells when treated with lactate. Differentially altered metabolites included TCA-related components such as acetyl-CoA and glutamate, indicating enhanced lactate utilization in KL tumor cells. We also noticed that KL cells showed increased oxidative phosphorylation (OXPHOS) with the addition of lactate, with increased GSH/GSSH and NADPH/NADP+ ratios, suggesting that, as compared to LKB1-intact cells, LKB1-deficient cells had an enhanced ability to utilize OXPHOS and maintain redox homeostasis with lower levels of ROS generation when utilizing lactate as an energy source. However, in KL cells, MYC knockout (KO) or MCT4 KO partially abolished the lactate-induced OXPHOS and increased ROS. Moreover, [U-13C]lactate tracing revealed that isotopologues were significantly enriched in TCA components in KL cells, and this was reversed by either MYC KO or MCT4 KO, suggesting that MYC and MCT4 were critical for lactate reutilization in KL tumor cells. We observed that MYC could directly bind to the promoter region and transcriptionally activate MCT4 expression, and restoring MCT4 in MYC KO cells enhanced lactate incorporation and decreased ROS levels. Finally, we injected KL murine tumor cells with or without MYC/MCT4 KO into mice and infused [U-13C]lactate to detect the isotopologues distribution in vivo. KL tumors showed significantly enhanced lactate incorporation as compared to K tumors, while MYC KO or MCT4 KO reversed these phenotypes. Collectively, our data indicates that in LKB1-deficient tumors, upregulation of MYC promotes MCT4 expression and increases lactate incorporation and utilization, and that can be inhibited by targeting MYC or MCT4. These findings provide insight into the mechanisms driving the metabolic reprogramming and aggressive phenotype of KRAS-mutant LKB1-deficient tumors and identify a novel therapeutic strategy for this recalcitrant subgroup. Citation Format: Yu Qian, David Molkentine, Yifan Kong, Qian Huang, Chendong Yang, Alissa Poteete, Alvaro P. Guimaraes, Peixin Jiang, Ferdinandos Skoulidis, Alexandre Reuben, John D. Minna, Ralph J. DeBerardinis, John V. Heymach. MYC upregulates MCT4 expression and promotes metabolic reprogramming and enhanced lactate utilization in LKB1-deficient NSCLC [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 4450.

Abstract 4032: ATRA induces an interferon response to reprogram the immunosuppressive tumor microenvironment and overcomes resistance to immune checkpoint inhibition in murine models of LKB1-deficient non-small cell lung cancer

Abstract Lung cancer remains the deadliest form of cancer, claiming 1.8 million lives worldwide in 2020. While treatment options have improved with the advent of immune checkpoint inhibitors (ICI), many patients do not respond to immunotherapy or develop resistance following initial response. A significant subset of non-small cell lung cancer (NSCLC) patients harbors somatic co-mutations in Kirsten rat sarcoma virus (KRAS) and Liver kinase 1 [LKB1, also known as serine/threonine kinase 11 (STK11)] genes, whose tumors are characterized by an immunosuppressive tumor microenvironment (TME) that are resistant to ICI. Our studies revealed that all-trans retinoic acid (ATRA), a metabolite derived from vitamin A, sensitized a murine model of NSCLC with increased tumor mutational burden (Kras G12D P53-/-Lkb1-/-; KPL-3M) to PD-1 blockade. The ATRA and anti-PD-1 combination therapy improved local and systemic T cell activation and generated systemic tumor-specific immunity. To understand ATRA-mediated anti-tumor effects, we performed single cell RNA sequencing (scRNA-seq) of KPL-3M murine tumors with or without 6 daily ATRA treatments. scRNA-seq analysis revealed a reduction of neutrophils as well as an enrichment in T cell and natural killer (NK) cell populations in the TME. Furthermore, scRNA-seq analysis revealed elevated expression of interferon (IFN) downstream genes in multiple immune subpopulations in the TME. Preliminary ex vivo studies indicated that ATRA increases NOX2 levels, intracellular reactive oxygen species (ROS), and IFN signaling in the neutrophils in KPL-3M tumors. Our findings suggest that ATRA may reshape the TME by activating IFN signaling in multiple cell subtypes to sensitize resistant tumors to ICI immunotherapy. Citation Format: William P. Crosson, Rui Li, Ramin Salehi-Rad, Raymond J. Lim, Jensen Abascal, Bitta P. Kahangi, Edgar Perez Reyes, Michael Oh, Camelia Dumitras, Nico Edgar, Ryan Chew, Rashel Jocobo, Adam Tfayli, Zhe Jing, Kostyantyn Krysan, Linh Tran, Steven M. Dubinett, Bin Liu. ATRA induces an interferon response to reprogram the immunosuppressive tumor microenvironment and overcomes resistance to immune checkpoint inhibition in murine models of LKB1-deficient non-small cell lung cancer [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 4032.

Abstract 6664: In situ vaccination with hyper-interferon sensitive influenza induces adaptive immune response and sensitizes murine non-small cell lung cancer to PD-1 blockade

Abstract Despite recent advances in immunotherapy with immune checkpoint inhibitors (ICI), many patients with non-small cell lung cancer (NSCLC) fail to respond or develop resistance after an initial response. The exclusion of T cells from the tumor or the presence of a dysfunctional T cell compartment within the tumor microenvironment (TME) constitute two central hallmarks of resistance to ICI in patients with NSCLC. Seminal studies have identified that loss of LKB1 in KRAS-mutant NSCLC drives resistance to ICI, potentially by suppressing STING, thereby leading to dysregulation of the interferon (IFN) signaling. There is a pressing demand for the development of innovative immune therapies designed to bolster anti-tumor T cell responses to overcome resistance to ICI. In situ vaccination (ISV) with oncolytic viruses has emerged as a promising, antigen-agnostic strategy capable of conditioning the TME and amplifying anti-tumor T cell responses. We engineered a live attenuated viral vaccine, Hyper-Interferon Sensitive virus (HIS), by conducting a genome-wide functional screening and introducing eight interferon (IFN)-sensitive mutations in the influenza A genome. We utilized multiple syngeneic murine models of NSCLC with distinct driver mutations [KrasG12D/P53+/−/Lkb1−/− (KPL); KrasG12D/P53+/− (KP); and KrasG12D (K)] and varying tumor mutational burden to assess the safety and efficacy of intratumoral HIS virotherapy. Compared to wild-type (WT) influenza, HIS replication was attenuated, enhancing its potential as a safe option for cancer therapy. HIS ISV elicited robust yet transient type I IFN responses in murine NSCLCs, leading an influx of polyfunctional effector Th1 CD4 and cytotoxic CD8 T cells into the tumor. HIS ISV demonstrated enhanced anti-tumor efficacy compared to WT in multiple syngeneic murine models of NSCLC. This efficacy was dependent on host type 1 IFN responses and T lymphocytes. HIS ISV synergized with anti-PD-1 to eradicate immune resistant LKB-1 deficient NSCLC in a subset of mice, resulting in improved overall survival and enduring systemic tumor-specific immunity. These studies provide compelling evidence to support further clinical evaluation of HIS as a novel ‘off-the-shelf’ ISV strategy for patients with NSCLC refractory to ICI. Citation Format: Ramin Salehi-Rad, Yushen Du, Tianhao Zhang, Yuan Shi, Bin Liu, Sun Ren. In situ vaccination with hyper-interferon sensitive influenza induces adaptive immune response and sensitizes murine non-small cell lung cancer to PD-1 blockade [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 6664.

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.

Abstract 2124: ATRA mediated inhibition of myeloid-derived suppressor cells overcomes resistance to immune checkpoint inhibition in murine models of LKB1-deficient non-small cell lung cancer

Abstract Lung cancer remains the deadliest form of cancer, claiming the lives of 1.8 million worldwide in 2020. While treatment options have improved with the advent of immune checkpoint inhibitors (ICI), many patients do not respond to immunotherapy or develop resistance following initial response. A significant subset of patients harbor somatic mutations in both Kirsten rat sarcoma virus (KRAS) and Liver kinase 1 [LKB1, also known as serine/threonine kinase 11 (STK11)] genes. LKB1 loss has been identified as a driver of primary resistance to ICI therapy. Tumors from patients that possess these mutations are characterized by an increase in the population of myeloid derived suppressor cells (MDSCs). MDSCs are thought to play a critical role in promoting tumor progression by blocking the tumor killing function of cytotoxic T cells in the tumor microenvironment (TME). Our studies revealed all-trans retinoic acid (ATRA), a metabolite derived from vitamin A, sensitized murine models of non-small cell lung cancer (NSCLC) with LKB1 and KRAS co-mutations to PD-1 blockade. Combination therapy with anti-PD-1 and ATRA improved local and systemic T cell proliferation and generated systemic tumor-specific immunity. We observed that ATRA augmented anti-PD-1 efficacy in additional murine NSCLC models. We demonstrated that ATRA suppresses tumor growth through the inhibition of immunosuppressive MDSCs in the TME, and preliminary data indicates that ATRA increases intracellular reactive oxygen species (ROS) and IFN signaling in MDSC. Our findings implicate MDSCs as a potential mediator of immunosuppression in Lkb1-deficient NSCLC and provide a rationale for utilizing ATRA in combination with anti-PD-1 therapy in patients with Lkb1-deficient NSCLC refractory to ICIs. * B. Liu and S. M. Dubinett contributed equally to this work Citation Format: William P. Crosson, Rui Li, Ramin Salehi-Rad, Raymond J. Lim, Jensen W. Abascal, Bitta P. Kahangi, Edgar Perez Reyes, Camelia Dumitras, Zhe Jing, Kostyantyn Krysan, Linh M. Tran, Bin Liu, Steven M. Dubinett. ATRA mediated inhibition of myeloid-derived suppressor cells overcomes resistance to immune checkpoint inhibition in murine models of LKB1-deficient non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2124.

Abstract 4208: In situ vaccination of non-small cell lung cancer with CXCL9/10-engineered dendritic cells combined with PD-1 blockade

Abstract Lung cancer is the most common cause of cancer death worldwide with approximately 85% of patients having non-small cell lung cancer (NSCLC). Checkpoint blockade immunotherapy has evolved the current treatment landscape with robust and durable responses in approximately 20% of patients with progressive, locally advanced or metastatic NSCLC, as well as in treatment-naïve advanced disease. Inefficient tumor antigen presentation, diminished T cell infiltration into tumor and LKB1-inactivating mutations contribute to the mechanisms of resistance to programmed death-ligand 1 (PD-L1) or programmed cell death protein 1 (PD-1) blockade in NSCLC. LKB1 deficiency also facilitates an immunosuppressive environment that promotes tumor growth. One approach to overcome this immunosuppressive tumor microenvironment (TME) is to utilize in situ vaccination with gene-modified functional antigen presenting cells (APCs) to enhance tumor antigen presentation and promote tumor-specific T cell activation. It has been shown that the chemokines CXCL9 and CXCL10 (CXCL9/10) are important signaling molecules secreted by CD103+ dendritic cells (DCs) to orchestrate the anti-tumor immunity. Thus, we hypothesize that intratumoral (IT) injection of CXCL9/10-secreting DCs (CXCL9/10-DCs) can potentiate the response to anti-PD-1 monotherapy in LKB1-deficient NSCLC by promoting antigen presentation and inducing T cell infiltration. Our preliminary data shows that CXCL9/10-DC injection modulates systemic immunity and generates anti-tumor responses that are augmented by the combination treatment with anti-PD1. In an in vivo murine model of Lkb1-null lung cancer bearing high tumor mutational burden (TMB), CXCL9/10-DC facilitates recruitment of effector T cells into the TME and tumor-specific T cell activation. These studies will enhance our understanding of anti-tumor immunity mediated by in situ DC vaccination and facilitate the improvement of novel vaccine therapies. B. Liu and S. M. Dubinett contributed equally to this work. Citation Format: Raymond J. Lim, Ramin Salehi-rad, Linh M. Tran, Camelia Dumitras, Jensen W. Abascal, William P. Crosson, Tejas S. Patel, Samantha Man, Rui Li, Kostyantyn Krysan, Bin Liu, Steven M. Dubinett. In situ vaccination of non-small cell lung cancer with CXCL9/10-engineered dendritic cells combined with PD-1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4208.

Inactivation of AMPK Leads to Attenuation of Antigen Presentation and Immune Evasion in Lung Adenocarcinoma.

Mutations in STK11 (LKB1) occur in 17% of lung adenocarcinoma (LUAD) and drive a suppressive (cold) tumor immune microenvironment (TIME) and resistance to immunotherapy. The mechanisms underpinning the establishment and maintenance of a cold TIME in LKB1-mutant LUAD remain poorly understood. In this study, we investigated the role of the LKB1 substrate AMPK in immune evasion in human non-small cell lung cancer (NSCLC) and mouse models and explored the mechanisms involved. We addressed the role of AMPK in immune evasion in NSCLC by correlating AMPK phosphorylation and immune-suppressive signatures and by deleting AMPKα1 (Prkaa1) and AMPKα2 (Prkaa2) in a KrasG12D -driven LUAD. Furthermore, we dissected the molecular mechanisms involved in immune evasion by comparing gene-expression signatures, AMPK activity, and immune infiltration in mouse and human LUAD and gain or loss-of-function experiments with LKB1- or AMPK-deficient cell lines. Inactivation of both AMPKα1 and AMPKα2 together with Kras activation accelerated tumorigenesis and led to tumors with reduced infiltration of CD8+/CD4+ T cells and gene signatures associated with a suppressive TIME. These signatures recapitulate those in Lkb1-deleted murine LUAD and in LKB1-deficient human NSCLC. Interestingly, a similar signature is noted in human NSCLC with low AMPK activity. In mechanistic studies, we find that compromised LKB1 and AMPK activity leads to attenuated antigen presentation in both LUAD mouse models and human NSCLC. The results provide evidence that the immune evasion noted in LKB1-inactivated lung cancer is due to subsequent inactivation of AMPK and attenuation of antigen presentation.

Enhanced Vulnerability of LKB1-Deficient NSCLC to Disruption of ATP Pools and Redox Homeostasis by 8-Cl-Ado.

Loss-of-function somatic mutations of STK11, a tumor suppressor gene encoding LKB1 that contributes to the altered metabolic phenotype of cancer cells, is the second most common event in lung adenocarcinomas and often co-occurs with activating KRAS mutations. Tumor cells lacking LKB1 display an aggressive phenotype, with uncontrolled cell growth and higher energetic and redox stress due to its failure to balance ATP and NADPH levels in response to cellular stimulus. The identification of effective therapeutic regimens for patients with LKB1-deficient non-small cell lung cancer (NSCLC) remains a major clinical need. Here, we report that LKB1-deficient NSCLC tumor cells displayed reduced basal levels of ATP and to a lesser extent other nucleotides, and markedly enhanced sensitivity to 8-Cl-adenosine (8-Cl-Ado), an energy-depleting nucleoside analog. Treatment with 8-Cl-Ado depleted intracellular ATP levels, raised redox stress, and induced cell death leading to a compensatory suppression of mTOR signaling in LKB1-intact, but not LKB1-deficient, cells. Proteomic analysis revealed that the MAPK/MEK/ERK and PI3K/AKT pathways were activated in response to 8-Cl-Ado treatment and targeting these pathways enhanced the antitumor efficacy of 8-Cl-Ado. IMPLICATIONS: Together, our findings demonstrate that LKB1-deficient tumor cells are selectively sensitive to 8-Cl-Ado and suggest that therapeutic approaches targeting vulnerable energy stores combined with signaling pathway inhibitors merit further investigation for this patient population.

Abstract 88: 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 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, metastases, and resistance to standard chemotherapy as well as immunotherapy. 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 survival 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 found that KL TDCLs were much sensitive to HCQ-induced cell death compared with 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 the increased sensitivity of KL TDCLs to Trametinib by autophagy ablation, we performed metabolomic profiling of KL TDCLs with Trametinib, HCQ, or combination treatment. We 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. Also, the combination treatment significantly reduced the mitochondrial membrane potential, basal respiration, and ATP production in the KL TDCLs compared with the single agents. However, these effects were not observed in KP TDCLs. Similarly, we found that LKB1-mutant human lung cancer cell lines were much more sensitive to the combination treatment than LKB1 WT cells. Finally, we performed in vivo tumor assay using allograft mouse models and GEMMs to validate our in vitro observations. We found anti-tumor synergistic effects of the combination treatment in KL tumor growth, with no such effect in KP tumor growth. Taken together, our observation suggests that autophagy upregulation in Lkb1-deficient tumors cause resistance to Trametinib treatment by maintaining energy homeostasis for cell survival. Therefore, a combination of autophagy and MEK inhibition could be a novel therapeutic strategy to specifically treat LKB1-deficient NSCLC. Citation Format: Vrushank Dharmesh Bhatt, Taijin Lan, Wenping Wang, Khoosheh Khayati, Eduardo Cararo-Lopes, Jianming Wang, Jerry Kong, Akash Raju, Xuefei Luo, Wenwei Hu, Xiaoyang Su, Eileen White, Jessie Yanxiang Guo. Autophagy inhibition sensitizes liver kinase b1 (LKB1)-deficient kras-driven lung tumors to MEK inhibitor trametinib [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 88.

Autophagy Inhibition Sensitizes Liver Kinase B1 (LKB1)‐Deficient Kras‐Driven Lung Tumors to MEK Inhibitor Trametinib

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, metastases and resistance to standard chemotherapy as well as immunotherapy. 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 found that KL TDCLs were much sensitive to HCQ-induced cell death compared with KP TDCLs. Furthermore, a combination treatment of HCQ with mitogen-activated protein kinase kinase (MAPKK/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, or combination treatment. We 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, we found that the combination treatment significantly reduced mitochondrial membrane potential, basal respiration as well as ATP production in KL TDCLs compared with the single agents. However, these effects were not observed in KP TDCLs. Similarly, we found that LKB1-mutant human lung cancer cell lines were much more sensitive to the combination treatment than LKB1 WT cells. Finally, we performed in vivo tumor assay using allograft mouse models and GEMMs to validate our in vitro observations. We found anti-tumor synergistic effects of the combination treatment in KL tumor growth, with no such effect in KP tumor growth. Taken together, our observations suggest that autophagy upregulation in Lkb1-deficient tumors cause resistance to Trametinib treatment by maintaining energy homeostasis for cell survival. Therefore, a combination of autophagy and MEK inhibition could be a novel therapeutic strategy to specifically treat LKB1-deficient NSCLC.

Inhibition of Granulocytic Myeloid-Derived Suppressor Cells Overcomes Resistance to Immune Checkpoint Inhibition in LKB1-Deficient Non-Small Cell Lung Cancer.

LKB1 inactivating mutations are commonly observed in patients with KRAS-mutant non-small cell lung cancer (NSCLC). Although treatment of NSCLC with immune checkpoint inhibitors (ICI) has resulted in improved overall survival in a subset of patients, studies have revealed that co-occurring KRAS/LKB1 mutations drive primary resistance to ICIs in NSCLC. Effective therapeutic options that overcome ICI resistance in LKB1-mutant NSCLC are limited. Here, we report that loss of LKB1 results in increased secretion of the C-X-C motif (CXC) chemokines with an NH2-terminal Glu-Leu-Arg (ELR) motif in premalignant and cancerous cells, as well as in genetically engineered murine models (GEMM) of NSCLC. Heightened levels of ELR+ CXC chemokines in LKB1-deficient murine models of NSCLC positively correlated with increased abundance of granulocytic myeloid-derived suppressor cells (G-MDSC) locally within the tumor microenvironment and systemically in peripheral blood and spleen. Depletion of G-MDSCs with antibody or functional inhibition via all-trans-retinoic acid (ATRA) led to enhanced antitumor T-cell responses and sensitized LKB1-deficent murine tumors to PD-1 blockade. Combination therapy with anti-PD-1 and ATRA improved local and systemic T-cell proliferation and generated tumor-specific immunity. Our findings implicate ELR+ CXC chemokine-mediated enrichment of G-MDSCs as a potential mediator of immunosuppression in LKB1-deficient NSCLC and provide a rationale for using ATRA in combination with anti-PD-1 therapy in patients with LKB1-deficient NSCLC refractory to ICIs. SIGNIFICANCE: These findings show that accumulation of myeloid-derived suppressor cells in LKB1-deficient non-small cell lung cancer can be overcome via treatment with all-trans-retinoic acid, sensitizing tumors to immunotherapy.

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.

Upregulation of programmed death ligand 1 by liver kinase B1 and its implication in programmed death 1 blockade therapy in non-small cell lung cancer

AimsLiver kinase B1 (LKB1) deficiency is associated with reduced expression of programmed death ligand 1 (PD-L1) and inferior clinical outcomes of PD-1/PD-L1 blockade in non-small cell lung cancer (NSCLC). This study aimed to investigate the mechanism by which LKB1 regulates PD-L1 expression and its role in programmed death 1 (PD-1) blockade therapy in NSCLC. Main methodsThe impact of LKB1 on PD-L1 was assessed by western blot, qRT-PCR and immunohistochemistry in NSCLC. Activators/inhibitors of AMPK and NRF2 were applied to explore the mechanisms underlying the regulation of PD-L1 by LKB1. Efficiency of combined application of metformin and PD-1 blockade was evaluated in immunocompetent C57BL/6 mice. Key findingsA remarkable positive correlation between LKB1 and PD-L1 expression was demonstrated in NSCLC tissues. Knockdown of LKB1 decreased PD-L1 in TC-1 cells, whereas overexpression of LKB1 increased PD-L1 in A549 cells. We further characterized that AMPK mediated the upregulation of PD-L1 by LKB1. Inhibition of AMPK or NRF2 markedly reduced PD-L1 in LKB1-intact NSCLC cells. In contrast, activation of AMPK or NRF2 reversed PD-L1 expression in LKB1-deficient NSCLC cells. Combined administration of metformin and anti-PD-1 antibody efficiently inhibited the growth of LKB1-intact tumors, whereas no obvious suppression was observed in LKB1-deficient tumors. SignificanceThese findings demonstrated that LKB1 upregulates PD-L1 expression in NSCLC by activating the AMPK and KEAP1/NRF2 signaling. Activation of LKB1-AMPK with metformin improves the therapeutic effect of PD-1 blockade in NSCLC with wild-type LKB1.

Discovery of 2,6-Dimethylpiperazines as Allosteric Inhibitors of CPS1.

Carbamoyl phosphate synthetase 1 (CPS1) is a potential synthetic lethal target in LKB1-deficient nonsmall cell lung cancer, where its overexpression supports the production of pyrimidine synthesis. In other cancer types, CPS1 overexpression and activity may prevent the accumulation of toxic levels of intratumoral ammonia to support tumor growth. Herein we report the discovery of a novel series of potent and selective small-molecule inhibitors of CPS1. Piperazine 2 was initially identified as a promising CPS1 inhibitor through a high-throughput screening effort. Subsequent structure-activity relationship optimization and structure-based drug design led to the discovery of piperazine H3B-616 (25), a potent allosteric inhibitor of CPS1 (IC50 = 66 nM).

Abstract 2710: Depletion of CXCR2-dependent myeloid-derived suppressor cells (MDSCs) overcomes anti-PD-1 resistance in a murine model of LKB1-deficient non-small cell lung cancer (NSCLC) with high mutational load

Abstract Checkpoint inhibitors such as PD-L1/PD-1 blockade have rapidly integrated into the paradigm of NSCLC treatment. However, a majority of patients do not benefit from monotherapy with checkpoint inhibitors. High tumor mutational burden (TMB), along with pre-existing intratumoral T cell infiltration and baseline high PD-L1 expression, predicts response to checkpoint blockade. Furthermore, a recent retrospective study identified LKB1 alterations as the most prevalent genomic driver of resistance to PD-1 axis inhibitors in KRAS-mutant lung adenocarcinoma. In this study, we investigate the mechanisms underlying LKB1-mediated immunosuppression in NSCLC. We show that loss of LKB1 in human bronchial epithelial cells (HBECs) and NSCLC cells leads to increased secretion of CXCR2 ligands, including CXCL1, CXCL2, CXCL3, CXCL5 and CXCL8. These CXCR2 ligands are also elevated in LKB1-deficient tumors from patient-derived xenografts and genetically-engineered murine models. We find abundant tumor infiltrating MDSCs in murine Lkb1-deficient NSCLC, consistent with the capacity for CXCR2 ligands to recruit MDSCs. MDSCs mediate potent immune suppressive activities at multiple levels including release of immunosuppressive cytokines, recruitment of regulatory T cells (Tregs), inhibition of CD8 T cell tumor infiltration and upregulation of PD-L1 expression. Although MDSC depletion activates interferon gamma signaling and decreases systemic Tregs in murine KrasK12D;Tp53-/-;Lkb1-/- (KPL) tumors, it does not sensitize KPL tumors to anti-PD-1 therapy. One of the major challenges in the preclinical assessment of lung cancer immunotherapy is that the commonly utilized murine models lack the mutational burden of human NSCLC. To assess this combination therapy in the context of a mutational burden that more accurately reflects the clinical disease, we generated tumors with high TMB by exposing KPL cells in vitroto the tobacco carcinogen N-methyl-N-nitrosourea. In the context of high TMB, MDSC depletion demonstrates remarkable anti-tumor effects in combination with anti-PD-1 therapy. Finally, we delineate the regulation of CXCR2 ligands by LKB1 which is dependent on the MARK-mediated NF-κB pathway. In conclusion, we find that LKB1 deficiency leads to increased CXCR2 ligand production and tumor infiltrating MDSCs. MDSC depletion enhances the efficacy of anti-PD-1 blockade in LKB1-deficient tumors bearing high TMB. Citation Format: Rui Li, Ramin Salehi-Rad, Stephanie Ong, Milica Momcilovic, Bin Liu, Raymond Lim, Linh Tran, Ziling Huang, Zhe Jing, Manash Paul, Kostyantyn Krysan, Stacy Park, John Minna, Michael Teitell, David Shackelford, Steven Dubinett. Depletion of CXCR2-dependent myeloid-derived suppressor cells (MDSCs) overcomes anti-PD-1 resistance in a murine model of LKB1-deficient non-small cell lung cancer (NSCLC) with high mutational load [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 2710.

Abstract 2716: Autophagy modulates lipid metabolism in liver kinase B1 (LKB1)-deficient Kras-driven lung tumorigenesis

Abstract Tumor suppressor liver kinase B1 (LKB1) activates 5’-adenosine monophosphate protein kinase (AMPK) to maintain energy homeostasis. LKB1 mutations are the third most frequent mutation detected in non-small cell lung cancer (NSCLC). Compared to p53 mutation, mutations in LKB1 causes more aggressive tumor growth and resistance to chemotherapy as well as immunotherapy. Thus, identifying a novel target to improve LKB1-deficient NSCLC treatment is urgently needed. Autophagy degrades and recycles macromolecules for cells to survive starvation. Using genetically engineered mouse models for NSCLC with Kras activation and LKB1 loss (KL), we found that autophagy (Atg7) deficiency prevents tumor initiation, reduced tumor growth and extended the mouse lifespan compared to mice bearing Atg7 wild-type (WT) tumors. Most importantly, tumor growth inhibition by Atg7 deficiency was much more dramatic in KL than Kras/p53 (KP) lung tumors. Furthermore, we generated mouse tumor-derived cell lines (TDCLs) and found that Atg7 null TDCLs were more sensitive to starvation-induced cell death than Atg7 WT TDCLs, which can be rescued by palmitate supplementation, indicating that autophagy is required to maintain free fatty acid (FA) level for cells to survive starvation. We further performed 13C glucose tracing study and found that Atg7 deletion increased de novo FA synthesis and reduced FAs elongation. Fasnall, a selective FA synthase inhibitor, inhibited Atg7 null cell proliferation. Treatment with Orlistat, a lipase inhibitor, did not impact cell proliferation in nutrient-rich conditions but accelerated starvation-induced Atg7 null cell death, indicating that lipase-medicated lipid degradation compensates for autophagy loss to provide free FAs in starvation. A carnitine palmitoyltransferase I inhibitor, Etomoxir treatment, accelerated the starvation-induced Atg7 null cell death and reduced oxygen consumption rate (OCR) of Atg7 null cells in starvation, compared with Atg7 WT cells, indicating that autophagy-deficient cells rely more on FA oxidation for energy production in nutrient deprivation. To probe the significance of autophagy in human lung cancer cell lines with LKB1 mutation, we knocked down ATG7 using siRNA. ATG7 knockdown (KD) inhibited clonogenic survival of LKB1 mutant cells in starvation, but had no effect in LKB1 WT cells. Supplementation of glutamine or palmitate rescued the survival of LKB1 mutant cells with ATG7 KD in starvation and maintained OCR, demonstrating that KRAS-driven human lung cancer cells with mutant LKB1 rely on autophagy to maintain mitochondrial metabolism to survive energy crises. Taken together, our results demonstrate that autophagy modulates lipid metabolism for KL cells to survive metabolic stress. Thus, a combination of autophagy inhibition with interruption of lipid metabolism could be a novel therapeutic strategy to specifically treat LKB1-deficient NSCLC. Citation Format: Vrushank D. Bhatt, Khoosheh Khayati, Zhixian Hu, Xiaoyang Su, Jessie Yanxiang Guo. Autophagy modulates lipid metabolism in liver kinase B1 (LKB1)-deficient Kras-driven lung tumorigenesis [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 2716.

Autophagy modulates lipid metabolism in Liver Kinase B1 (LKB1)‐deficient Kras‐driven lung tumorigenesis

Tumor suppressor liver kinase B1 (LKB1) activates 5′‐adenosine monophosphate protein kinase (AMPK) to maintain energy homeostasis. LKB1 mutations are the third most frequent mutation detected in non‐small cell lung cancer (NSCLC). Compared to p53 mutation, mutations in LKB1 causes more aggressive tumor growth and resistance to chemotherapy as well as immunotherapy. Thus, identifying a novel target to improve LKB1‐deficient NSCLC treatment is urgently needed. Autophagy degrades and recycles macromolecules for cells to survive starvation. Using genetically engineered mouse models for NSCLC with Kras activation and LKB1 loss (KL), we found that autophagy (Atg7) deficiency prevents tumor initiation, reduced tumor growth and extended the mouse lifespan compared to mice bearing Atg7 wild‐type (WT) tumors. Most importantly, tumor growth inhibition by Atg7 deficiency was much more dramatic in KL than Kras/p53 (KP) lung tumors. Furthermore, we generated mouse tumor‐derived cell lines (TDCLs) and found that Atg7 null TDCLs were more sensitive to starvation‐induced cell death than Atg7 WT TDCLs, which can be rescued by palmitate supplementation, indicating that autophagy is required to maintain free fatty acid (FA) level for cells to survive starvation. We further performed 13C glucose tracing study and found that Atg7 deletion increased de novo FA synthesis and reduced FAs elongation. Fasnall, a selective FA synthase inhibitor, inhibited Atg7 null cell proliferation. Treatment with Orlistat, a lipase inhibitor, did not impact cell proliferation in nutrient‐rich conditions but accelerated starvation‐induced Atg7 null cell death, indicating that lipase‐medicated lipid degradation compensates for autophagy loss to provide free FAs in starvation. A carnitine palmitoyltransferase I inhibitor, Etomoxir treatment, accelerated the starvation‐induced Atg7 null cell death and reduced oxygen consumption rate (OCR) of Atg7 null cells in starvation, compared with Atg7 WT cells, indicating that autophagy‐deficient cells rely more on FA oxidation for energy production in nutrient deprivation. To probe the significance of autophagy in human lung cancer cell lines with LKB1 mutation, we knocked down ATG7 using siRNA. ATG7 knockdown (KD) inhibited clonogenic survival of LKB1 mutant cells in starvation but had no effect in LKB1 WT cells. Supplementation of glutamine or palmitate rescued the survival of LKB1 mutant cells with ATG7 KD in starvation and maintained OCR, demonstrating that KRAS‐driven human lung cancer cells with mutant LKB1 rely on autophagy to maintain mitochondrial metabolism to survive energy crises. Taken together, our results demonstrate that autophagy modulates lipid metabolism for KL cells to survive metabolic stress. Thus, a combination of autophagy inhibition with interruption of lipid metabolism could be a novel therapeutic strategy to specifically treat LKB1‐deficient NSCLC.Support or Funding InformationThis research has been supported by the research funds from K22 CA190521 (NIH) grant, American Cancer Society Early Investigator Pilot Award, Lung Cancer Research Foundation grant and startup funding from Rutgers Cancer Institute of New Jersey.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 5147: Lactate transporters are a potential therapeutic target for LKB1-deficient lung cancers

Abstract The serine/threonine kinase LKB1 is mutated and inactivated in 20-30% of lung adenocarcinoma representing the second most commonly altered tumor suppressor in non-small cell lung cancer (NSCLC), and to date there are no effective targeting strategies for tumors bearing this mutation. Therefore, new therapeutic approaches are urgently needed for LKB1-deficient tumors. LKB1 activates AMPK, the master sensor of cellular energy and because of this, many of the best known functions of LKB1 are attributed to its ability to control metabolic alterations. Our laboratory and others have demonstrated that loss of LKB1 can promote enhanced glucose metabolism and a shift from aerobic to anaerobic respiration. A result of enhanced glycolysis is an elevated lactate production even under normoxic conditions. It has been reported that lactate-mediated extracellular acidification is a key factor mediating tumor cell invasion and metastasis. We found that LKB1-deficient NSCLC tumors significantly upregulate lactate transporters and, because these tumors exhibit an immunologically inert phenotype, we propose that inhibition of lactate transporters could represent a rational strategy to inhibit tumor growth and enhance immune responses in LKB1 mutant NSCLC. To characterize lactate transporter expression, a panel of NSCLC cell lines was stably transduced to overexpress LKB1 and with shRNA targeting LKB1. We analyzed gene expression of lactate transporters in TCGA dataset of lung adenocarcinoma (LUAD). We validated these results by qPCR and western blot analysis of expression levels of MCT1, MCT4 and MCT14 in human LKB1-intact and deficient cells and in LKB1 KO murine NSCLC cell lines that were generated using CRISPR/Cas9 in a KRASG12D mutant background. To study lactate transporter expression in tumors in vivo, we generated syngeneic NSCLC mouse models via s.c. injection of LKB1-intact and KO murine cells in immunocompetent mice and analyzed the protein expression levels of MCT4 in these tumor samples. The analysis of TCGA LUAD dataset revealed a significant upregulation of lactate transporter SLC16A14 (MCT14) gene expression levels in LKB1-deficient tumors compared with LKB1 wild-type (p<0.001). In vitro, NSCLC LKB1-deficient cells (A549, H460 and H2030) showed increased RNA and protein expression of MCT4 and MCT14 compared to cells where a copy of LKB1 was introduced. In vivo, KRASG12D mutant LKB1 KO tumors from syngeneic mouse models significantly showed an upregulation of MCT4 protein expression compared with KRASG12D mutant LKB1 wild type tumors (p<0.0001). In conclusion, LKB1-deficient NSCLC showed higher levels of glycolysis and significantly elevated expression of lactate transporters in vitro and in vivo compared to LKB1-wild type NSCLC. Additional studies are ongoing to determine the efficacy of lactate transporter inhibition in LKB1-mutant NSCLC. Citation Format: Irene Guijarro, Alissa Poteete, Pan Tong, Jing Wang, John Heymach. Lactate transporters are a potential therapeutic target for LKB1-deficient lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5147.

Abstract 2408: LKB1 and KEAP1/NRF2 pathways cooperatively promote glutamine dependence and vulnerability to glutaminase inhibitors in KRAS-mutant lung adenocarcinoma

Abstract KRAS is the most commonly mutated oncogenic driver in non-small cell lung cancer (NSCLC) and other solid tumors. Recently we conducted an integrative analysis and found three major subgroups of KRAS-mutated cancer defined by co-occurring genomic events with distinct biology, molecular vulnerabilities, and therapeutic sensitivities. One of these genes, the serine/threonine kinase STK11 (LKB1), represents the second most commonly altered tumor suppressor in NSCLC and there are currently no treatment strategies tailored for LKB1-deficient NSCLC. KRAS-mutant/LKB1-deficient (KL) tumors are characterized by high co-occurrence of KEAP1 mutational inactivation. Inactivation of KEAP1 protects cells against REDOX stress via upregulation of NRF2 target genes, in part by production of glutathione. We evaluated the effects of blocking glutamine metabolism using an isogenic series of NSCLC cell lines harboring mutations in STK11 and KEAP1. Through sequential silencing or overexpression of LKB1, KEAP1, or NRF2 we demonstrated that glutaminase inhibitors (GLSi) can block cell proliferation while increasing energetic and REDOX stress specifically in LKB1 deficient cells with hyperactivation of the KEAP1/NRF2 pathway driven by KEAP1 mutations (KLK subtype). In KLK models, overexpression of LKB1 or KEAP1 partially reduced GLSi sensitivity, while siRNA-mediated down-regulation of NRF2 showed a similar effect. Furthermore, the combination of LKB1 add back coupled with down-regulation of NRF2 conferred even greater resistance to GLSi. To confirm the LKB1/KEAP1-driven response to GLSi, we performed in vivo experiments examining the response of subcutaneous xenografts of an A549 isogenic series; A549 (KLK), A549 LKB1 add back (KK) or A549 KEAP1 add back (KL); to GLS inhibition. These experiments demonstrated that GLSi impaired tumor growth in A549 (KLK) tumors, exhibiting significant statistical differences compared with the vehicle group from 18 days of treatment. Conversely, GLS inhibition did not significantly affect the growth of A549/LKB1 (KK) or A549/KEAP1 (KL) tumors. Collectively, our data indicate that in KLK tumors both pathways, LKB1 and KEAP1/NRF2, cooperatively drive a glutamine-addicted metabolic program, making KLK tumors selectively vulnerable to GLSi treatment. These findings have immediate clinical implications and support the future clinical testing of GLS inhibitors in KLK NSCLC. Citation Format: Ana Galan-Cobo, Piyada Sitthideatphaiboon, Xiao Qu, Jeffrey J. Kovacs, Alissa Poteete, Pan Tong, Sungnam Cho, Varsha V. Gandhi, Ferdinandos Skoulidis, Jing Wang, Timothy P. Heffernan, John V. Heymach. LKB1 and KEAP1/NRF2 pathways cooperatively promote glutamine dependence and vulnerability to glutaminase inhibitors in KRAS-mutant lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2408.