KRAS-driven Lung Tumorigenesis Research Articles

Abstract 6151: Loss of GPR54 inhibits tumor growth by suppressing AKT/DDC signaling pathway in KRAS-mutant non-small cell lung cancer

Abstract Therapeutic targeting of KRAS-mutant lung cancers has remained a challenge. There are increasing evidence that KISS1/GPR54 signaling pathway is associated with the development of various cancer. Although KISS1 and GPR54 are expressed in the lung, their biological role in the lung tumorigenesis remains unclear. In this study, using the genetically engineered mouse models, we have identified that loss of Gpr54 inhibited Kras-driven lung tumorigenesis by suppressing pathways in oncogenesis such as the MAPK and AKT pathways and inducing apoptosis. Similarly, genomic inhibition of GPR54 suppressed cell proliferation, decreased ERK and AKT signaling pathway, and increased apoptotic cell death in NSCLC cells. RNA-sequencing analysis revealed that DDC expression and glycolysis gene sets were significantly changed in Gpr54 deficient mice. Interestingly, HIF-1α expression is regulated by GPR54/AKT/DDC signaling pathway in NSCLC cells, leading to glycolytic change. Genomic inhibition of DDC suppressed cell proliferation and enhanced apoptotic cell death in NSCLC cell lines. Also, pharmacological inhibition of GPR54 and DDC lower glucose consumption and lactate production in NSCLC cell lines. Taken together, we demonstrate GPR54, as a potential therapeutic target, suggesting a new strategy to cure for lung cancer patients. Citation Format: Seo Yeon Lee, Yu-Jeong Choi, Hyun-Ha Hwang, Sooyeon Kang, Kangwook Lee, Seong-Gyu Ko. Loss of GPR54 inhibits tumor growth by suppressing AKT/DDC signaling pathway in KRAS-mutant non-small cell lung cancer. [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 6151.

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.

Highlights from Recent Cancer Literature.

Breaking Insights| March 15 2023 Highlights from Recent Cancer Literature Author & Article Information Online Issn: 1538-7445 Print Issn: 0008-5472 ©2023 American Association for Cancer Research2023American Association for Cancer Research Cancer Res (2023) 83 (6): 805–806. https://doi.org/10.1158/0008-5472.CAN-83-6-BI Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Tools Icon Tools Get Permissions Cite Icon Cite Search Site Article Versions Icon Versions Version of Record March 15 2023 Citation Highlights from Recent Cancer Literature. Cancer Res 15 March 2023; 83 (6): 805–806. https://doi.org/10.1158/0008-5472.CAN-83-6-BI Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest Search Advanced Search Although the KRAS-protein interactome has been well defined, the functional significance of these protein interactions in KRAS-mutant lung cancer tumorigenesis is poorly understood. Tang and colleagues examined the effect of candidate KRAS-interacting proteins on lung cancer growth utilizing a multiplexed somatic CRISPR/Cas9-based genome editing screen in a KRASG12D mutant mouse model. They found that wild-type HRAS and NRAS suppressed KRAS-driven lung tumorigenesis in vivo and in vitro through suppression of KRAS-KRAS binding as well as through competition for key downstream signaling pathways such as ERK. Expert Commentary: This study suggests that “RAS paralog imbalance” with decreased expression of wild-type HRAS and NRAS in the setting of mutant KRAS can promote tumorigenesis. Tang R, Shuldiner EG, Kelly M, Murray CW, Hebert JD, Andrejka L, et al. Multiplexed screens identify RAS paralogues HRAS and NRAS as suppressors of KRAS-driven lung cancer growth. Nat Cell Biol 2023;25:159-69. Extravasation of metastatic... You do not currently have access to this content.

Miz1 promotes KRAS-driven lung tumorigenesis by repressing the protocadherin Pcdh10

Targeting KRAS-mutated non-small-cell lung cancer (NSCLC) remains clinically challenging. Here we show that loss of function of Miz1 inhibits lung tumorigenesis in a mouse model of oncogenic KRAS-driven lung cancer. In vitro, knockout or silencing of Miz1 decreases cell proliferation, clonogenicity, migration, invasion, or anchorage-independent growth in mutant (MT) KRAS murine or human NSCLC cells but has unremarkable impact on non-tumorigenic cells or wild-type (WT) KRAS human NSCLC cells. RNA-sequencing reveals Protocadherin-10 (Pcdh10) as the top upregulated gene by Miz1 knockout in MT KRAS murine lung tumor cells. Chromatin immunoprecipitation shows Miz1 binding on the Pcdh10 promoter in MT KRAS lung tumor cells but not non-tumorigenic cells. Importantly, silencing of Pcdh10 rescues cell proliferation and clonogenicity in Miz1 knockout/knockdown MT KRAS murine or human tumor cells, and rescues allograft tumor growth of Miz1 knockout tumor cells in vivo. Miz1 is upregulated in MT KRAS lung tumor tissues compared with adjacent non-involved tissues in mice. Consistent with this, Miz1 is upregulated while Pcdh10 is downregulated in human lung adenocarcinomas (LUAD) compared with normal tissues, and high Miz1 levels or low Pcdh10 levels are associated with poor survival in lung cancer patients. Furthermore, the Miz1 signature is associated with worse survival in MT but not WT KRAS LUAD, and Pcdh10 is downregulated in MT compared to WT KRAS LUAD. Taken together, our studies implicate the Miz1/Pcdh10 axis in oncogenic KRAS-driven lung tumorigenesis.

Lyz2-Cre-Mediated Genetic Deletion of Septin7 Reveals a Role of Septins in Macrophage Cytokinesis and Kras-Driven Tumorigenesis.

By crossing septin7-floxed mice with Lyz2-Cre mice carrying the Cre recombinase inserted in the Lysozyme-M (Lyz2) gene locus we aimed the specific deletion of septin7 in myeloid cells, such as monocytes, macrophages and granulocytes. Septin7 flox/flox :Lyz2-Cre mice show no alterations in the myeloid compartment. Septin7-deleted macrophages (BMDMs) were isolated and analyzed. The lack of Septin7 expression was confirmed and a constitutive double-nucleation was detected in Septin7-deficient BMDMs indicating a defect in macrophage cytokinesis. However, phagocytic function of macrophages as judged by uptake of labelled E. coli particles and LPS-stimulated macrophage activation as judged by induction of TNF mRNA expression and TNF secretion were not compromised. In addition to myeloid cells, Lyz2-Cre is also active in type II pneumocytes (AT2 cells). We monitored lung adenocarcinoma formation in these mice by crossing them with the conditional knock-in Kras-LSL-G12D allele. Interestingly, we found that control mice without septin7 depletion die after 3–5 weeks, while the Septin7-deficient animals survived 11 weeks or even longer. Control mice sacrificed in the age of 4 weeks display a bronchiolo-alveolar hyperplasia with multiple adenomas, whereas the Septin7-deficient animals of the same age are normal or show only a weak multifocal brochiolo-alveolar hyperplasia. Our findings indicate an essential role of Septin7 in macrophage cytokinesis but not in macrophage function. Furthermore, septin7 seems absolutely essential for oncogenic Kras-driven lung tumorigenesis making it a potential target for anti-tumor interventions.

The integrated stress response is tumorigenic and constitutes a therapeutic liability in KRAS-driven lung cancer

The integrated stress response (ISR) is an essential stress-support pathway increasingly recognized as a determinant of tumorigenesis. Here we demonstrate that ISR is pivotal in lung adenocarcinoma (LUAD) development, the most common histological type of lung cancer and a leading cause of cancer death worldwide. Increased phosphorylation of the translation initiation factor eIF2 (p-eIF2α), the focal point of ISR, is related to invasiveness, increased growth, and poor outcome in 928 LUAD patients. Dissection of ISR mechanisms in KRAS-driven lung tumorigenesis in mice demonstrated that p-eIF2α causes the translational repression of dual specificity phosphatase 6 (DUSP6), resulting in increased phosphorylation of the extracellular signal-regulated kinase (p-ERK). Treatments with ISR inhibitors, including a memory-enhancing drug with limited toxicity, provides a suitable therapeutic option for KRAS-driven lung cancer insofar as they substantially reduce tumor growth and prolong mouse survival. Our data provide a rationale for the implementation of ISR-based regimens in LUAD treatment.

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.

Endoplasmic reticulum chaperone GRP78/BiP is critical for mutant Kras-driven lung tumorigenesis.

Lung cancer is the leading cause of cancer mortality worldwide and KRAS is the most commonly mutated gene in lung adenocarcinoma (LUAD). The 78-kDa glucose-regulated protein GRP78/BiP is a key endoplasmic reticulum chaperone protein and a major pro-survival effector of the unfolded protein response (UPR). Analysis of the Cancer Genome Atlas database and immunostain of patient tissues revealed that compared to normal lung, GRP78 expression is generally elevated in human lung cancers, including tumors bearing the KRASG12D mutation. To test the requirement of GRP78 in human lung oncogenesis, we generated mouse models containing floxed Grp78 and Kras Lox-Stop-Lox G12D (KrasLSL-G12D) alleles. Simultaneous activation of the KrasG12D allele and knockout of the Grp78 alleles were achieved in the whole lung or selectively in lung alveolar epithelial type 2 cells known to be precursors for adenomas that progress to LUAD. Here we report that GRP78 haploinsufficiency is sufficient to suppress KrasG12D-mediated lung tumor progression and prolong survival. Furthermore, GRP78 knockdown in human lung cancer cell line A427 (KrasG12D/+) leads to activation of UPR and apoptotic markers and loss of cell viability. Our studies provide evidence that targeting GRP78 represents a novel therapeutic approach to suppress mutant KRAS-mediated lung tumorigenesis.

Selective targeting of KRAS-driven lung tumorigenesis via unresolved ER stress.

Lung cancer with oncogenic KRAS makes up a significant proportion of lung cancers and is accompanied by a poor prognosis. Recent advances in understanding the molecular pathogenesis of lung cancer with oncogenic KRAS have enabled the development of drugs, yet mutated KRAS remains undruggable. We performed small-molecule library screening and identified verteporfin, a yes-associated protein 1 (YAP1) inhibitor; verteporfin treatment markedly reduced cell viability in KRAS-mutant lung cancer cells in vitro and suppressed KRAS-driven lung tumorigenesis in vivo. Comparative functional analysis of verteporfin treatment and YAP1 knockdown with siRNA revealed that the cytotoxic effect of verteporfin was at least partially independent of YAP1 inhibition. A whole-transcriptome approach revealed the distinct expression profiles in KRAS-mutant lung cancer cells between verteporfin treatment and YAP1 knockdown and identified the selective involvement of the ER stress pathway in the effects of verteporfin treatment in KRAS-mutant lung cancer, leading to apoptotic cell death. These data provide novel insight to uncover vulnerabilities in KRAS-driven lung tumorigenesis.

PKCε Is Required for KRAS-Driven Lung Tumorigenesis.

Non-small cell lung cancer (NSCLC) is the most frequent subtype of lung cancer and remains a highly lethal malignancy and one of the leading causes of cancer-related deaths worldwide. Mutant KRAS is the prevailing oncogenic driver of lung adenocarcinoma, the most common histologic form of NSCLC. In this study, we examined the role of PKCϵ, an oncogenic kinase highly expressed in NSCLC and other cancers, in KRAS-driven tumorigenesis. Database analysis revealed an association between PKCϵ expression and poor outcome in patients with lung adenocarcinoma specifically harboring KRAS mutations. A PKCϵ-deficient, conditionally activatable allele of oncogenic Kras (LSL-KrasG12D ;PKCϵ-/- mice) demonstrated the requirement of PKCϵ for Kras-driven lung tumorigenesis in vivo, which was consistent with impaired transformed growth reported in PKCϵ-deficient KRAS-dependent NSCLC cells. Moreover, PKCϵ-knockout mice were found to be less susceptible to lung tumorigenesis induced by benzo[a]pyrene, a carcinogen that induces mutations in Kras. Mechanistic analysis using RNA sequencing revealed little overlap for PKCϵ and KRAS in the control of genes and biological pathways relevant in NSCLC, suggesting that a permissive role of PKCϵ in KRAS-driven lung tumorigenesis may involve nonredundant mechanisms. Our results thus, highlight the relevance and potential of targeting PKCϵ for lung cancer therapeutics. SIGNIFICANCE: These findings demonstrate that KRAS-mediated tumorigenesis requires PKCϵ expression and highlight the potential for developing PKCϵ-targeted therapies for oncogenic RAS-driven malignancies.

The Combination of Loss of ALDH1L1 Function and Phenformin Treatment Decreases Tumor Growth in KRAS-Driven Lung Cancer.

Lung adenocarcinoma cells express high levels of ALDH1L1, an enzyme of the one-carbon pathway that catalyzes the conversion of 10-formyltetrahydrofolate into tetrahydrofolate and NAD(P)H. In this study, we evaluated the potential of ALDH1L1 as a therapeutic target by deleting the Aldh1l1 gene in KrasLA2 mice, a model of spontaneous non-small cell lung cancer (NSCLC). Reporter assays revealed KRAS-mediated upregulation of the ALDH1L1 promoter in human NSCLC cells. Aldh1l1−/− mice exhibited a normal phenotype, with a 10% decrease in Kras-driven lung tumorigenesis. By contrast, the inhibition of oxidative phosphorylation inhibition using phenformin in Aldh1l1−/−; KrasLA2 mice dramatically decreased the number of tumor nodules and tumor area by up to 50%. Furthermore, combined treatment with pan-ALDH inhibitor and phenformin showed a decreased number and area of lung tumors by 70% in the KrasLA2 lung cancer model. Consistent with this, previous work showed that the combination of ALDH1L1 knockdown and phenformin treatment decreased ATP production by as much as 70% in NSCLS cell lines. Taken together, these results suggest that the combined inhibition of ALDH activity and oxidative phosphorylation represents a promising therapeutic strategy for NSCLC.

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.

MiR-301a promotes lung tumorigenesis by suppressing Runx3

BackgroundOur previous report demonstrated that genetic ablation of miR-301a reduces Kras-driven lung tumorigenesis in mice. However, the impact of miR-301a on host anti-tumor immunity remains unexplored. Here we assessed the underlying molecular mechanisms of miR-301a in the tumor microenvironment.MethodsThe differentially expressed genes were identified by using deep sequencing. The immune cell counts, and cytokines expression were analyzed by realtime PCR, immunohistochemistry and flow cytometry. The role of miR-301a/Runx3 in lung tumor was evaluated on cell growth, migration and invasion. The function of miR-301a/Runx3 in regulating tumor microenvironment and tumor metastasis were evaluated in Kras transgenic mice and B16/LLC1 syngeneic xenografts tumor models.ResultsIn this work, we identified 1166 up-regulated and 475 down-regulated differentially expressed genes in lung tumor tissues between KrasLA2 and miR-301a−/−; KrasLA2 mice. Immune response and cell cycle were major pathways involved in the protective role of miR-301a deletion in lung tumorigenesis. Overexpression of the miR-301a target, Runx3, was an early event identified in miR-301a−/−; KrasLA2 mice compared to WT-KrasLA2 mice. We found that miR-301a deletion enhanced CD8+ T cell accumulation and IFN-γ production in the tumor microenvironment and mediated antitumor immunity. Further studies revealed that miR-301a deficiency in the tumor microenvironment effectively reduced tumor metastasis by elevating Runx3 and recruiting CD8+ T cells, whereas miR-301a knockdown in tumor cells themselves restrained cell migration by elevating Runx3 expression.ConclusionsOur findings further underscore that miR-301a facilitates tumor microenvironment antitumor immunity by Runx3 suppression in lung tumorigenesis.

KRAS-Driven Cancers Are Dependent on METTL13-Mediated eEF1A Methylation

Methylation of eEF1A by METTL13 is required for KRAS-driven pancreatic and lung tumorigenesis.

Abstract 809: Afatinib restrains K-RAS driven lung tumorigenesis

Abstract Lung cancer is still the leading cause of cancer deaths worldwide. While it is well documented that patients suffering from epidermal growth factor receptor (EGFR) mutated non-small cell lung cancer (NSCLC) benefit from (EGFR) inhibition, there are still no successful targeted therapies for oncogenic mutations in the V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) gene available. Indeed, based on clinical trials using first generation EGFR tyrosine kinase inhibitors (TKI) it became a doctrine that K-RAS mutations drive resistance to EGFR inhibition in non-small cell lung cancer (NSCLC). Conversely, we demonstrate here that EGFR signaling is engaged in K-RAS driven lung tumorigenesis both in human and in mice. Genetic mouse models revealed that deletion of Egfr quenches mutant K-ras activity and transiently reduces tumor growth. However, EGFR inhibition initiates a resistance mechanism which involves non-EGFR ERBB family members. This escape mechanism not only clarifies the disappointing outcome of first generation TKI in clinical trials, but also suggests therapeutic potential of pan-ERBB inhibitors. Indeed, by using several experimental models, including genetically engineered mouse models, (patient derived-) xenografts and in vitro experiments, we demonstrate that the FDA approved pan-ERBB inhibitor afatinib formidably reduces K-RAS driven lung tumorigenesis, whereas first generation TKI erlotinib and gefitinib remained ineffective in our models. Altogether, our data strongly suggests revisiting the potential of EGFR TKI in clinical trials for the treatment of K-RAS mutated NSCLC, with a particular focus on pan-ERBB inhibitors, which are readily available. Citation Format: Herwig P. Moll, Klemens Pranz, Monica Musteanu, Luka Brcic, Helmut Popper, Judit Moldvay, Balázs Dome, Emilio Casanova. Afatinib restrains K-RAS driven lung tumorigenesis [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 809.

Abstract 5510: Loss of epithelial membrane protein 2 enhances Kras-driven lung tumorigenesis via FAK/AKT pathways

Abstract Epithelial membrane protein-2 (EMP2) belongs to the growth arrest specific-3 (GAS3)/PMP22 family of tetraspan proteins. These family members are involved in the pathogenesis of human diseases, including malignancy. EMP2 is highly expressed in normal lung alveoli; however, its functional role in lung tumor is largely unknown. In this study, we focus a novel function for EMP2 in lung tumor progression. To investigate this possibility, EMP2 knock-out mice were crossed with KrasG12Dmice that carry an oncogenic mutation of the Kras gene (G12D) and develop lung adenocarcinoma. Incidence of lung tumor in KrasG12DEMP2-/- mice was higher than that in KrasG12DEMP2+/+ mice. We also found that loss of EMP2 accelerated lung tumor progression. Then, we investigated the role of EMP2 in lung cancer cell line. Expression of EMP2 was knocked out by CRISPR/Cas9 editing system in H1299 (KrasWT), H358 (KrasMut) and A549 cells (KrasMut). Loss of EMP2 induced that expression of integrin α5 is increased but expression of integrin α6 is decreased. Alteration of integrin expression activated FAK/AKT pathway. These results support the role of EMP2 loss in the control of lung cancer progression with oncogenic Kras. Taken together, these findings establish that EMP2 is a potent suppressor of Kras-driven lung tumorigenesis via FAK/AKT pathway. Citation Format: Ji Yun Jang, Mi Kyung Park, Chang Hoon Lee, Ho Lee. Loss of epithelial membrane protein 2 enhances Kras-driven lung tumorigenesis via FAK/AKT pathways [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 5510.

Abstract 3334: CREB is required for KRAS-driven lung tumorigenesis

Abstract The aberrant constitutive activation of the transcription factor, cyclic-AMP response element- binding (CREB) protein, has been associated with poor prognosis and chemotherapeutic resistance in KRAS-mutated human cancers, one of which includes non-small cell lung cancer (NSCLC). However, the role of CREB in KRAS-driven lung tumorigenesis has not been fully understood. Using adenoviral delivery of Cre specifically to the mouse lung, we generated a new transgenic lung cancer mouse model driven by Kras activating mutation G12D and concomitant with Creb loss (KrasLSL-G12D/+; CrebFlox/Flox). In this study, we used this model to show that a proto-oncogenic transcription factor CREB, is required for KRAS-driven lung tumorigenesis. Loss of Creb (Creb-/-) in the mice harboring Kras-mutated lung tumors significantly suppressed tumor development, and thus, dramatically prolonged mouse survival when compared to either wild (Creb+/+) or heterozygous (Creb+/-) deletion of Creb. Mechanistically, we identified a few novel CREB target gene. These data suggest that CREB is essential for KRAS-driven lung tumorigenesis and that CREB could be a potential target for therapeutic treatment for individuals suffering from KRAS-mutant lung cancers. Citation Format: Jong woo Lee, Frank J. Slack, Eric J. Nestler, Roy S. Herbst, Jaseok Peter Koo. CREB is required for KRAS-driven lung tumorigenesis [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 3334.

Abstract 4067: Toll-like receptor 2 deficiency enhances KRAS-driven lung cancer

Abstract Chronic infection and inflammation have been suggested to have a strong association with lung cancer in clinical and epidemiologic studies. Toll-like receptor 2 (TLR2) mediates inflammatory response and is essential in regulating innate and adaptive immunity. TLR2 has also been reported to play either pro-tumorigenesis or tumor-suppressive role depending on the oncogenic driver mutations and cell types. However, the role of TLR2 in lung cancer, especially in the lung adenocarcinoma harboring KRAS mutations, remains unknown. In an analysis using human lung adenocarcinoma cohort (TCGA RNAseq dataset), our results revealed that KRAS-mutant lung tumors showed lower TLR2 mRNA expression than the paired adjacent normal lung tissue. Furthermore, lung adenocarcinoma patients with lower TLR2 expression displayed worse survival outcome. To functionally test the role of TLR2 during KRAS-driven lung tumorigenesis, we generated a mouse model of lung adenocarcinoma mutated for K-ras, with and without TLR2 inactivation (K-rasG12D/+ TLR2-/- and K-rasG12D/+TLR2+/+, respectively). Interestingly, the tumor number and tumor area significantly increased in K-rasG12D/+ TLR2-/- mice compared to K-rasG12D/+TLR2+/+ mice. In addition, the inflammatory cells in bronchoalveolar lavage (BAL), especially the alveolar monocytes, significantly increased in K-rasG12D/+ TLR2-/- mice. Microdissected lung tumors from K-rasG12D/+ TLR2-/- mice showed significantly higher inflammatory chemokines and cytokines gene expression (Ccl1, Ccl2, Cxcl1, Cxcl2, Cxcl5, Csf1, IL-6, Tnf) than those from K-rasG12D/+TLR2+/+mice. Additionally, Cxcl1 was significantly higher in K-rasG12D/+ TLR2-/- when BAL fluid was analyzed for cytokines/chemokines by Luminex assays. Our results suggest that TLR2 deficiency may promote KRAS-mutation driven lung tumorigenesis through increased cancer-related inflammation. Furthermore, the expression may serve as a prognostic biomarker for which the lower TLR2 expression is associated with worse survival outcome in lung adenocarcinoma patients with KRAS mutation. Citation Format: Chia-Hsin Liu. Toll-like receptor 2 deficiency enhances KRAS-driven lung cancer [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 4067.

TRAF2 and OTUD7B govern a ubiquitin-dependent switch that regulates mTORC2 signalling.

The mechanistic target of rapamycin (mTOR) has a key role in the integration of various physiological stimuli to regulate several cell growth and metabolic pathways. mTOR primarily functions as a catalytic subunit in two structurally related but functionally distinct multi-component kinase complexes, mTOR complex 1 (mTORC1) and mTORC2 (refs 1, 2). Dysregulation of mTOR signalling is associated with a variety of human diseases, including metabolic disorders and cancer. Thus, both mTORC1 and mTORC2 kinase activity is tightly controlled in cells. mTORC1 is activated by both nutrients and growth factors, whereas mTORC2 responds primarily to extracellular cues such as growth-factor-triggered activation of PI3K signalling. Although both mTOR and GβL (also known as MLST8) assemble into mTORC1 and mTORC2 (refs 11, 12, 13, 14, 15), it remains largely unclear what drives the dynamic assembly of these two functionally distinct complexes. Here we show, in humans and mice, that the K63-linked polyubiquitination status of GβL dictates the homeostasis of mTORC2 formation and activation. Mechanistically, the TRAF2 E3 ubiquitin ligase promotes K63-linked polyubiquitination of GβL, which disrupts its interaction with the unique mTORC2 component SIN1 (refs 12, 13, 14) to favour mTORC1 formation. By contrast, the OTUD7B deubiquitinase removes polyubiquitin chains from GβL to promote GβL interaction with SIN1, facilitating mTORC2 formation in response to various growth signals. Moreover, loss of critical ubiquitination residues in GβL, by either K305R/K313R mutations or a melanoma-associated GβL(ΔW297) truncation, leads to elevated mTORC2 formation, which facilitates tumorigenesis, in part by activating AKT oncogenic signalling. In support of a physiologically pivotal role for OTUD7B in the activation of mTORC2/AKT signalling, genetic deletion of Otud7b in mice suppresses Akt activation and Kras-driven lung tumorigenesis in vivo. Collectively, our study reveals a GβL-ubiquitination-dependent switch that fine-tunes the dynamic organization and activation of the mTORC2 kinase under both physiological and pathological conditions.