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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.

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.

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.