LKB1 Loss Research Articles

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.

LKB1 and KEAP1/NRF2 Pathways Cooperatively Promote Metabolic Reprogramming with Enhanced Glutamine Dependence in KRAS-Mutant Lung Adenocarcinoma.

In KRAS-mutant lung adenocarcinoma, tumors with LKB1 loss (KL) are highly enriched for concurrent KEAP1 mutations, which activate the KEAP1/NRF2 pathway (KLK). Here, we investigated the biological consequences of these cooccurring alterations and explored whether they conferred specific therapeutic vulnerabilities. Compared with KL tumors, KLK tumors exhibited increased expression of genes involved in glutamine metabolism, the tricarboxylic acid cycle, and the redox homeostasis signature. Using isogenic pairs with knockdown or overexpression of LKB1, KEAP1, and NRF2, we found that LKB1 loss results in increased energetic and redox stress marked by increased levels of intracellular reactive oxygen species and decreased levels of ATP, NADPH/NADP+ ratio, and glutathione. Activation of the KEAP1/NRF2 axis in LKB1-deficient cells enhanced cell survival and played a critical role in the maintenance of energetic and redox homeostasis in a glutamine-dependent manner. LKB1 and the KEAP1/NRF2 pathways cooperatively drove metabolic reprogramming and enhanced sensitivity to the glutaminase inhibitor CB-839 in vitro and in vivo. Overall, these findings elucidate the adaptive advantage provided by KEAP1/NRF2 pathway activation in KL tumors and support clinical testing of glutaminase inhibitor in subsets of KRAS-mutant lung adenocarcinoma. SIGNIFICANCE: In KRAS-mutant non-small cell lung cancer, LKB1 loss results in enhanced energetic/redox stress, which is tolerated, in part, through cooccurring KEAP1/NRF2-dependent metabolic adaptations, thus enhancing glutamine dependence and vulnerability to glutaminase inhibition.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/13/3251/F1.large.jpg.

Abstract 2049: LKB1 regulates BH3-mimetics vulnerability of KRAS mutant non-small cell lung cancer by alternating mitochondrial apoptotic protein interactions

Abstract Over 30% of lung adenocarcinomas harbor KRAS mutation. Despite progress in targeted therapies for specific genetic subsets of lung cancer (e.g. EGFR or ALK), there are no clinically effective targeted therapies for KRAS non-small cell lung cancer (NSCLC). Interpatient heterogeneity of KRAS mutant lung cancers, with large variability in co-occurring mutations, may contribute to its refractory phenotype. Specifically, KRAS mutant lung cancers with concurrent loss of the tumor suppressor LKB1 (KRAS-LKB1) have increased metastatic frequency and resistance to chemotherapy in pre-clinical models, and these patients have poor responses to immune checkpoint inhibitor. Thus, there is a critical need to develop novel therapeutic approaches for this subset of NSCLC. Intrinsic resistance to apoptosis limits the efficacy of therapies targeting KRAS signaling. Previous studies reported BCL-2/BCL-XL + MEK inhibition can increase apoptotic responses of some KRAS mutant cancers. Recently, potent and selective inhibitors of MCL-1 have been developed, creating additional possibilities for targeting apoptotic machinery for cancers that dependent upon MCL-1 for survival. We recently reported that novel MCL-1 inhibitor AMG-176 combined with MEK inhibitor trametinib can induce tumor regression in subsets of KRAS mutant NSCLC pre-clinical tumor models. In addition to commercially available cell lines, we established 20 patient-derived cell line/xenograft mouse model and showed that KRAS-LKB1 cell lines are particularly sensitive to MEK + MCL1 inhibition with high synergy score. Moreover, restoring LKB1 expression in LKB1-/- cell lines hampers the synergy and blocks mitochondrial depolarization and apoptosis. BH3-profiling reveals high dependency on MCL-1 in KRAS-LKB1 cell lines. We reported that trametinib increase intercellular Bim (pro-apoptotic protein) and subsequent loading of Bim onto pro-survival proteins BCL-XL and/or MCL1. MCL1 inhibitor releases Bim from MCL1 and initiates the apoptotic cascade. Interestingly, trametinib induces preferential sequestration of Bim by MCL-1 in KRAS-LKB1 models. Restoration of LKB1 in LKB1-/- cell lines reduces the trametinib-induced Bim:MCL-1 protein-protein interactions. Combined inhibition of MEK + MCL1 caused dramatic tumor regression in LKB1-deficient xenograft mouse models compared to LKB1-restored models. Our results reveal fundamental insights into a novel role for LKB1 in the regulation of mitochondrial apoptosis and will lay a solid pre-clinical foundation for the clinical investigation of the MEK + MCL-1 inhibitor combination. Our data may also support using LKB1 as a genetic biomarker for guiding the selection of BH3 mimetics in targeting KRAS mutant NSCLC. Citation Format: Chendi Li, Audris Oh, Varuna Nangia, Aaron N. Hata. LKB1 regulates BH3-mimetics vulnerability of KRAS mutant non-small cell lung cancer by alternating mitochondrial apoptotic protein interactions [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 2049.

Abstract 3912: The PI3K/mTOR dual inhibitor PF-04691502 shows antitumor activity in Sezary cells and in a xenograft mouse model

Abstract Introduction: Cutaneous T-cell Lymphomas (CTCLs) are extranodal non-Hodgkin’s lymphomas deriving from neoplastic transformation of T lymphocytes that home to the skin. Mycosis fungoides (MF) is the most diffuse form, usually having an indolent course but presenting in some cases as an aggressive disease with a poor prognosis. Sézary syndrome (SS) is a rare and aggressive variant, with erythroderma, leukemia, and lymph node involvement. Despite recent advances, CTCLs are still incurable cancers and the clinical symptoms are highly disabling. Among the landscape of genomic alterations identified in CTCL, we and others have described alterations of the PI3K pathway members (loss of PTEN, LKB1 and PDCD4, and gain of p70S6K). As a consequence, several SS patients show hyper-activation of PI3K/AKT/mTOR pathway. Hence, inhibition of this pathway represents a potential therapeutic choice against CTCL. Objective: In this work, we investigated the therapeutic potential of the dual-PI3K/mTOR inhibitor PF-04691502 (hereafter PF-502) against CTCL. Materials and Methods: PF-502 therapeutic efficacy was evaluated in four CTCL cell lines and in patient-derived SS cells, for its ability to inhibit cell growth, to induce apoptosis and to inhibit chemotaxis toward the chemokine SDF-1. Moreover, to evaluate PF-502 therapeutic effects in vivo we used a xenograft mouse model, consisting of nude mice transplanted with the CTCL-derived HH cell line. Results: PF-502 inhibited the cell growth of all the CTCL cell lines (0.07-0.32 µM, IC50 range), and of tumor cells derived from six SS patients (2.3 µM, mean IC50). The mechanism of action of PF-502 in CTCL cell lines was a combination of apoptosis induction and G1 block, while in SS patients it was mostly based on a strong apoptosis induction. Of note, PF-502 showed an only modest effect in T-lymphocytes from 3 healthy donors. PF-502 inhibited also cell migration toward the chemokine SDF-1, in both the systems. This result might have an important clinical implication because we have recently demonstrated that malignant lymphocytes are activated by the skin microenvironment. Thus, blocking the homing to the skin could affect the disease course. Finally, PF-502 has a robust antitumor activity in the xenograft mouse model, by reducing tumor volume (400 mm3 in treated mice vs 936 mm3 in controls) as well as tumor weight (0.2 g in treated mice vs 0.56 g in controls). Moreover, the Kaplan-Meier curve, in these mice, revealed that PF-502 treatment prolongs survival (P<0.005). Conclusions: Our results strongly support the therapeutic potential of targeting PI3K/AKT/mTOR pathway in CTCL. Citation Format: Antonella Bresin, Cristina Cristofoletti, Francesca Monzo, Elisabetta Caprini, Mauro Helmer Citterich, Alessandra Frezzolini, Alessandro Monopoli, Roberto Benucci, Maria Cantonetti, Enrico Scala, Giandomenico Russo, Maria Grazia Narducci. The PI3K/mTOR dual inhibitor PF-04691502 shows antitumor activity in Sezary cells and in a xenograft mouse model [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 3912.

Abstract 1242: Examining the role of heme and respiratory proteins in the progression of KRAS/LKB1 mutant subtype

Abstract Currently there are few successful targeted therapies for non-small cell lung cancer (NSCLC). Targeting mutations in EGFR, BRAF, ALK etc. provide treatment options only for a defined subset of adenocarcinoma patients leaving majority of adenocarcinoma and squamous carcinoma patients without any options. Activating KRAS mutations are found in 15-30% of all NSCLC patients; however, existing treatment options for KRAS-mutant cancer are not effective in the subset with concomitant loss of LKB1 (kinase that phosphorylates AMPK). Several studies demonstrate the importance of mitochondrial metabolism and oxidative phosphorylation (OXPHOS) in lung tumorigenesis. Components of OXPHOS complexes and markers of mitochondrial biogenesis are highly predictive of reduced overall survival in NSCLC patients. A study using genetically engineered mouse models (GEMMs) for lung cancer (KrasLSLG12D/+Trp53-/- and KrasLSL-G12D/+Lkb1-/-) showed that along with glucose, lactate contributes to the TCA cycle. This highlights the importance of OXPHOS in these models. Also, heme is a prosthetic group for several of these OXPHOS proteins and is involved in oxygen transport and utilization. Hence, we aim to investigate unique therapeutic targets by examining the role of heme and respiratory proteins in the progression of KRAS/LKB1 mutant subtype. We used LSL-KrasG12D LKB1-/- (LSL-KRASG12D; LKBloxP/loxP; LSL-Luciferase (KLLuc)) GEMM model for our study. Briefly, the mice were infected with AdenoCre virus, and sacrificed at different intervals in the progression of the tumors. For histological and immunohistochemical analysis, lung tissues were formalin fixed, paraffin embedded and sectioned (5µm thick). H&E staining showed consistent progression in tumor size over time. We will examine the time-course expression of OXPHOS, heme pathway proteins, and hemoproteins to determine the extent to which this lung tumor subtype depends on heme and respiratory proteins for its progression. Citation Format: Sanchareeka Dey, Sarada Preeta Kalainayakan, Poorva Ghosh. Examining the role of heme and respiratory proteins in the progression of KRAS/LKB1 mutant subtype [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 1242.

Abstract 3223: Overcome LKB1 mutated cancer resistance to anti-PD1 treatment

Abstract KRAS/STK11 (LKB1) mutant lung cancers are a subtype of cancer that respond poorly in the clinic to immunotherapies with shorter progression-free survival and overall survival comparing with other KRAS mutant lung cancer patients. Interestingly, this group of patients have high tumor mutational burden (TMB) comparable with other KRAS mutant lung cancers, which usually is an indicator for better response to anti-PD1 treatment. By using genetically engineered mouse model (GEMMs), we found that LKB1 loss of function can cause high level of nonsynonymous mutations, as a consequence of both replication dependent and independent DNA repair machinery dysfunctions. This LKB1 mutant dependent increased mutational load might further synergize with smoking mutagen exposures. When LKB1 function is defective, the neoantigens are not properly presented to T cells, which is required for proper T cell activation and effector T cells expansion. This will lead to insensitivity to anti-PD1 antibodies in high TMB tumors and tolerance to neoantigens produced by cancer cells. We further demonstrated that using a small molecule drug that impacts on the LKB1 downstream effector pathway, we can reverse this process through activating innate immunity in tumors, which will restore the antigen presentation functions. Combinational treatment of this small molecule with anti-PD1 can reverse the tumor progression in vivo, through releasing the suppressive tumor infiltrating lymphocytes (TILs) in LKB1 mutant lung cancers. Our study not only helps to further our understanding of the mechanism that contributes to immune evasion in high TMB tumors, but also provides a potential solution for LKB1 mutant cancers to overcome resistance to anti-PD1 treatment in the clinic. Citation Format: Jiehui Deng, Aatish Thennavan, Yuanwang Pan, Igor Dolgalev, Ting Chen, Heather Silver, Matthew Harris, Val Pyon, Fei Li, Chelsea Lee, Aristotelis Tsirigos, Eli Rothenberg, Charles M. Perou, Kwok-Kin Wong. Overcome LKB1 mutated cancer resistance to anti-PD1 treatment [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 3223.

Abstract 1174: Tumor-specific EphA2 receptor tyrosine kinase inhibits anti-tumor immunity by recruiting suppressive myeloid populations in non-small cell lung cancer

Abstract Background: Given the success of both targeted and immunotherapies against cancer, there is increasing utility for identifying targeted agents that also promote anti-tumor immunity. EphA2 is a receptor tyrosine kinase that contributes to tumor growth and metastasis in various cancer types and has been identified as a viable target for non-small cell lung cancer (NSCLC). Here, we examine how tumor-specific EphA2 affects the infiltration of different immune populations, the activation or inhibition of those populations, and the cytokine and chemokine milieu in mouse models of NSCLC. Methods: We generated two murine NSCLC cell lines, one derived from Lewis Lung Carcinoma and the other derived from a primary lung tumor harboring a KRAS mutation, as well as p53 and LKB1 loss of function alterations created using CRISPR-Cas9 gene editing. Effects of EphA2 overexpression in these cells were evaluated in both in vitro assays and in vivo models via subcutaneous tumor implantation and tail vein-injected orthotopic tumor formation in immunocompetent, syngeneic mice. Tumor immune infiltrate was assessed by flow cytometry, and cytokine and chemokine RNA expression levels were evaluated using Nanostring’s PanCancer Immune Profiling Panel. Results: Although EphA2 overexpression in these cell lines did not display significant proliferative advantage in vitro in MTT and colony formation assays, it did confer a growth advantage in vivo. Analysis of lung tumor immune infiltrate revealed decreased NK and T cells in the EphA2-overexpressing tumors, as well as increased myeloid populations, such as macrophages and monocytes. Furthermore, T cell activation markers, such as CD25, CD44, and CD69, in both CD4 and CD8 T cells were decreased in the EphA2-overexpressing tumors, while the percentage of regulatory T cells was increased. These changes in T cell activity were accompanied by increased presence of monocyte-derived CD11b+ Ly6C+ Ly6G- cells and Gr1- F4/80+ MHCII+ tumor-associated macrophages (TAMs). Higher infiltration of these myeloid populations coincided with increased monocyte-attracting chemokine and receptor expression, including CCL2, CCL7, CCL8, CCL12, and CCR2 and increased expression of immunosuppressive proteins, including TGFβ and arginase 1. Conclusion: Our studies suggest tumor-specific EphA2 inhibits infiltration and activation of key lymphocytic populations, especially T cells, while recruiting monocytes and likely promoting their transformation into myeloid-derived suppressor cells (MDSCs) and TAMs. Further functional studies are needed to validate the role of these myeloid cells. Elucidation of EphA2’s role in regulating immune recruitment and function will advance our understanding of tumor immune evasion and the potentially beneficial consequences of targeting EphA2 in cancer on the tumor microenvironment. Citation Format: Eileen Shiuan, Wenqiang Song, Shan Wang, Mark Boothby, Jin Chen. Tumor-specific EphA2 receptor tyrosine kinase inhibits anti-tumor immunity by recruiting suppressive myeloid populations in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1174.

Inactivation of Lkb1 in postnatal chondrocytes leads to epiphyseal growth-plate abnormalities and promotes enchondroma-like formation.

Liver serine-threonine kinase B1 (LKB1) is a tumor suppressor that has been linked to many types of tumors. However, the role of LKB1 in cartilaginous tumorigenesis is still poorly understood. In this study, we find that cartilage-specific, tamoxifen-inducible Lkb1 knockout results in multiple enchondroma-like lesions adjacent to the disorganized growth plates. We showed that chondrocytes retain an immature status caused by loss of Lkb1, which may lead to the dramatic expansion of growth-plate cartilage and the formation of enchondroma-like lesions. Additionally, increased mammalian target of rapamycin complex 1 (mTORC1) activity is observed in the Lkb1 conditional knockout (cKO) chondrocytes, and rapamycin (mTORC1 inhibitor) treatment significantly alleviates the expansion of growth-plate cartilage and eliminates the enchondroma-like lesions in Lkb1 cKO mice. Thus, our findings indicate that loss of Lkb1 leads to the expansion of chondrocytes and the formation of enchondroma-like lesions during postnatal cartilage development, and that the up-regulated mTORC1-signaling pathway is implicated in this process. Our findings suggest that modulation of LKB1 and related signaling is a potential therapy in cartilaginous tumorigenesis.-Zhou, S., Li, Y., Qiao, L., Ge, Y., Huang, X., Gao, X., Ju, H., Wang, W., Zhang, J., Yan, J., Teng, H., Jiang, Q. Inactivation of Lkb1 in postnatal chondrocytes leads to epiphyseal growth-plate abnormalities and promotes enchondroma-like formation.

LKB1 orchestrates dendritic cell metabolic quiescence and anti-tumor immunity

Dendritic cells (DCs) play a pivotal role in priming adaptive immunity. However, the involvement of DCs in controlling excessive and deleterious T cell responses remains poorly defined. Moreover, the metabolic dependence and regulation of DC function are unclear. Here we show that LKB1 signaling in DCs functions as a brake to restrain excessive tumor-promoting regulatory T cell (Treg) and Th17 cell responses, thereby promoting protective anti-tumor immunity and maintaining proper immune homeostasis. LKB1 deficiency results in dysregulated metabolism and mTOR activation of DCs. Loss of LKB1 also leads to aberrant DC maturation and production of cytokines and immunoregulatory molecules. Blocking mTOR signaling in LKB1-deficient DCspartially rectifies the abnormal phenotypes of DC activation and Treg expansion, whereas uncontrolled Th17 responses depend upon IL-6-STAT3 signaling. By coordinating metabolic and immune quiescence of DCs, LKB1 acts as a crucial signaling hub in DCs to enforce protective anti-tumor immunity and normal immune homeostasis.

The Effect of LKB1 Activity on the Sensitivity to PI3K/mTOR Inhibition in Non–Small Cell Lung Cancer

IntroductionLiver kinase B1 (LKB1), also called serine/threonine kinase 11 (STK11), is a tumor suppressor that functions as master regulator of cell growth, metabolism, survival, and polarity. Approximately 30% to 35% of patients with NSCLC possess inactivated liver kinase B1 gene (LKB1), and these patients respond poorly to anti–programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) immunotherapy. Therefore, novel therapies targeting NSCLC with LKB1 loss are needed. MethodsWe used a new in silico signaling analysis method to identify the potential therapeutic targets and reposition drugs by integrating gene expression data with the Kyoto Encyclopedia of Genes and Genomes signaling pathways. LKB1 wild-type and LKB1-deficient NSCLC cell lines, including knockout clones generated by clustered regularly interspaced short pallindromic repeats–Cas9, were treated with inhibitors of mechanistic target of rapamycin kinase (mTOR) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and a dual inhibitor. ResultsIn silico experiments showed that inhibition of both mTOR and PI3K can be synergistically effective in LKB1-deficient NSCLC. In vitro and in vivo experiments showed the synergistic effect of mTOR inhibition and PI3K inhibition in LKB1-mutant NSCLC cell lines. The sensitivity to dual inhibition of mTOR and PI3K is higher in LKB1-mutant cell lines than in wild-type cell lines. A higher compensatory increase in Akt phosphorylation after rapamycin treatment of LKB1-deficient cells than after rapamycin treatment of LKB1 wild-type cells is responsible for the synergistic effect of mTOR and PI3K inhibition. Dual inhibition of mTOR and PI3K resulted in a greater decrease in protein expression of cell cycle–regulating proteins in LKB1 knockout cells than in LKB1 wild-type cells. ConclusionDual molecular targeted therapy for mTOR and PI3K may be a promising therapeutic strategy in the specific population of patients with lung cancer with LKB1 loss.

Suppression of STING Associated with LKB1 Loss in KRAS-Driven Lung Cancer.

KRAS-driven lung cancers frequently inactivate TP53 and/or STK11/LKB1, defining tumor subclasses with emerging clinical relevance. Specifically, KRAS-LKB1 (KL)-mutant lung cancers are particularly aggressive, lack PD-L1, and respond poorly to immune checkpoint blockade (ICB). The mechanistic basis for this impaired immunogenicity, despite the overall high mutational load of KRAS-mutant lung cancers, remains obscure. Here, we report that LKB1 loss results in marked silencing of stimulator of interferon genes (STING) expression and insensitivity to cytoplasmic double-strand DNA (dsDNA) sensing. This effect is mediated at least in part by hyperactivation of DNMT1 and EZH2 activity related to elevated S-adenylmethionine levels and reinforced by DNMT1 upregulation. Ectopic expression of STING in KL cells engages IRF3 and STAT1 signaling downstream of TBK1 and impairs cellular fitness, due to the pathologic accumulation of cytoplasmic mitochondrial dsDNA associated with mitochondrial dysfunction. Thus, silencing of STING avoids these negative consequences of LKB1 inactivation, while facilitating immune escape. SIGNIFICANCE: Oncogenic KRAS-mutant lung cancers remain treatment-refractory and are resistant to ICB in the setting of LKB1 loss. These results begin to uncover the key underlying mechanism and identify strategies to restore STING expression, with important therapeutic implications because mitochondrial dysfunction is an obligate component of this tumor subtype.See related commentary by Corte and Byers, p. 16.This article is highlighted in the In This Issue feature, p. 1.

Evading the STING: LKB1 Loss Leads to STING Silencing and Immune Escape in KRAS-Mutant Lung Cancers.

Mutations in STK11 (LKB1) are a major cause of primary resistance to immunotherapy in non-small cell lung cancer. Kitajima and colleagues dissect the underlying mechanism of this immune-resistant phenotype, demonstrating that LKB1 loss leads directly to suppression of stimulator of interferon genes (STING) and insensitivity to cytoplasmic double-strand DNA detection. Therapies that reactivate LKB1 or theSTING pathway may boost anticancer immune response in cancers with resistance to immune-checkpoint blockade.See related article by Kitajima et al., p. 34.

Blood and skin-derived Sezary cells: differences in proliferation-index, activation of PI3K/AKT/mTORC1 pathway and its prognostic relevance

Sézary syndrome (SS) is a rare and aggressive variant of Cutaneous T-Cell Lymphoma characterized by neoplastic distribution mainly involving blood, skin, and lymph-node. Although a role of the skin microenvironment in SS pathogenesis has long been hypothesized, its function in vivo is poorly characterized. To deepen this aspect, here we compared skin to blood-derived SS cells concurrently obtained from SS patients highlighting a greater proliferation-index and a PI3K/AKT/mTORC1 pathway activation level, particularly of mTOR protein, in skin-derived-SS cells. We proved that SDF-1 and CCL21 chemokines, both overexpressed in SS tissues, induce mTORC1 signaling activation, cell proliferation and Ki67 up-regulation in a SS-derived cell line and primary-SS cells. In a cohort of 43 SS cases, we observed recurrent copy number variations (CNV) of members belonging to this cascade, namely: loss of LKB1 (48%), PTEN (39%) and PDCD4 (35%) and gains of P70S6K (30%). These alterations represent druggable targets unraveling new therapeutic treatments as metformin here evaluated in vitro. Moreover, CNV of PTEN, PDCD4, and P70S6K, evaluated individually or in combination, are associated with reduced survival of SS patients. These data shed light on effects in vivo of skin-SS cells interaction underlying the prognostic and therapeutic relevance of mTORC1 pathway in SS.

Control of Treg cell homeostasis and immune equilibrium by Lkb1 in dendritic cells

To balance immunity and tolerance, the endogenous pool of Foxp3+ regulatory T (Treg) cells is tightly controlled, but the underlying mechanisms of this control remain poorly understood. Here we show that the number of Treg cells is negatively regulated by the kinase Lkb1 in dendritic cells (DCs). Conditional knockout of the Lkb1 gene in DCs leads to excessive Treg cell expansion in multiple organs and dampens antigen-specific T cell immunity. Lkb1-deficient DCs are capable of enhancing, compared with wild-type DCs, Treg cell proliferation via cell-cell contact involving the IKK/IKBα-independent activation of the NF-κB/OX40L pathway. Intriguingly, treating wild-type mice with lipopolysaccharide selectively depletes Lkb1 protein in DCs, resulting in Treg cell expansion and suppressed inflammatory injury upon subsequent challenge. Loss of Lkb1 does not obviously upregulate proinflammatory molecules expression on DCs. We thus identify Lkb1 as a regulatory switch in DCs for controlling Treg cell homeostasis, immune response and tolerance.

Co-occurring KRAS mutation/LKB1 loss in non-small cell lung cancer cells results in enhanced metabolic activity susceptible to caloric restriction: an in vitro integrated multilevel approach

BackgroundNon–small-cell lung cancer (NSCLC) is a heterogeneous disease, with multiple different oncogenic mutations. Approximately 25–30% of NSCLC patients present KRAS mutations, which confer poor prognosis and high risk of tumor recurrence. About half of NSCLCs with activating KRAS lesions also have deletions or inactivating mutations in the serine/threonine kinase 11 (LKB1) gene. Loss of LKB1 on a KRAS-mutant background may represent a significant source of heterogeneity contributing to poor response to therapy.MethodsHere, we employed an integrated multilevel proteomics, metabolomics and functional in-vitro approach in NSCLC H1299 isogenic cells to define their metabolic state associated with the presence of different genetic background. Protein levels were obtained by label free and single reaction monitoring (SRM)-based proteomics. The metabolic state was studied coupling targeted and untargeted mass spectrometry (MS) strategy. In vitro metabolic dependencies were evaluated using 2-deoxy glucose (2-DG) treatment or glucose/glutamine nutrient limitation.ResultsHere we demonstrate that co-occurring KRAS mutation/LKB1 loss in NSCLC cells allowed efficient exploitation of glycolysis and oxidative phosphorylation, when compared to cells with each single oncologic genotype. The enhanced metabolic activity rendered the viability of cells with both genetic lesions susceptible towards nutrient limitation.ConclusionsCo-occurrence of KRAS mutation and LKB1 loss in NSCLC cells induced an enhanced metabolic activity mirrored by a growth rate vulnerability under limited nutrient conditions relative to cells with the single oncogenetic lesions. Our results hint at the possibility that energy stress induced by calorie restriction regimens may sensitize NSCLCs with these co-occurring lesions to cytotoxic chemotherapy.

P3.03-28 LKB1 Mutation Status is Associated with Poor Radiation Outcome in Patients with Non-Small Cell Lung Cancer

Previously, we reported that the upregulation of the NRF2/KEAP1 pathway in non-small cell lung cancers (NSCLC) bearing co-mutations in KRAS and LKB1 is critical to maintain redox homeostasis which contribute to radiation resistance in these tumors. Here, we explore the role of the LKB1 mutation as a potential predictor radiation outcome in NSCLC. We retrospectively analyzed the patients undergoing definitive treatment for newly diagnosed NSCLC who were enrolled in the prospectively collected MD Anderson Lung Cancer Moon Shot GEMINI database according to LKB1 mutation status. Cox regression analysis and log-rank tests were used to correlate with radiation outcomes according to LKB1 mutation status and/or KRAS mutation subgroup. We then investigated the mechanisms of radiation resistance in these tumors in preclinical models. Of the 173 patients with stage III NSCLC treated with definitive radiotherapy, with or without chemotherapy were analyzed according to LKB1 mutation status demonstrated that LKB1 mutation had statistically significantly higher rate of loco-regional recurrence, and shorter disease-free survival than in patients with wild type LKB1 (P = 0.013 and P = 0.037, respectively). Moreover, additional loss of either LKB1 or TP53 in KRAS-mutant tumors renders these tumors higher loco-regional recurrence after radiation (P interaction = 0.045). We identified that LKB1 loss is associated with radio-resistance in part by KEAP1/NRF2 pathway activation. Re-expression of LKB1 or NRF2 pathway suppression (via KEAP1 expression) enhanced radiotherapy sensitivity in vivo. Our results suggest that LKB1 mutation is a potential predictive impact on radiation outcome of patients with NSCLC. The upregulation of the KEAP1/NRF2 pathway in NSCLC bearing co-mutations in KRAS and LKB1 is critical to maintain redox homeostasis and determine the sensitivity for radiation.

The Lineage-Defining Transcription Factors SOX2 and NKX2-1 Determine Lung Cancer Cell Fate and Shape the Tumor Immune Microenvironment

The major types of non-small-cell lung cancer (NSCLC)-squamous cell carcinoma and adenocarcinoma-have distinct immune microenvironments. We developed a genetic model of squamous NSCLC on the basis of overexpression of the transcription factor Sox2, which specifies lung basal cell fate, and loss of the tumor suppressor Lkb1 (SL mice). SL tumors recapitulated gene-expression and immune-infiltrate features of human squamous NSCLC; such features included enrichment of tumor-associated neutrophils (TANs) and decreased expression of NKX2-1, a transcriptional regulator that specifies alveolar cell fate. In Kras-driven adenocarcinomas, mis-expression of Sox2 or loss of Nkx2-1 led to TAN recruitment. TAN recruitment involved SOX2-mediated production of the chemokine CXCL5. Deletion of Nkx2-1 in SL mice (SNL) revealed that NKX2-1 suppresses SOX2-driven squamous tumorigenesis by repressing adeno-to-squamous transdifferentiation. Depletion of TANs in SNL mice reduced squamous tumors, suggesting that TANs foster squamous cell fate. Thus, lineage-defining transcription factors determine the tumor immune microenvironment, which in turn might impact the nature of the tumor.

The LKB1-SIK Pathway Controls Dendrite Self-Avoidance in Purkinje Cells

Strictly controlled dendrite patterning underlies precise neural connection. Dendrite self-avoidance is acrucial system preventing self-crossing and clumpingof dendrites. Although many cell-surface moleculesthat regulate self-avoidance have been identified, the signaling pathway that orchestrates it remains poorly understood, particularly in mammals. Here, we demonstrate that the LKB1-SIK kinase pathway plays a pivotal role in the self-avoidance of Purkinje cell (PC) dendrites by ensuring dendritic localization of Robo2, a regulator of self-avoidance. LKB1 is activated in developing PCs, and PC-specific deletion of LKB1 severely disrupts the self-avoidance of PC dendrites without affecting gross morphology. SIK1 and SIK2, downstream kinases of LKB1, mediate LKB1-dependent dendrite self-avoidance. Furthermore, loss of LKB1 leads to significantly decreased Robo2 levels in the dendrite but not in the cell body. Finally, restoration of dendritic Robo2 level via overexpression largely rescues the self-avoidance defect in LKB1-deficient PCs. These findings reveal an LKB1-pathway-mediated developmental program that establishes dendrite self-avoidance.

Abstract 1454: Glutamate dehydrogenase 1 mediates metabolic reprogramming and promotes tumor metastasis through CamKK2-AMPK signaling in LKB1-deficient lung cancer

Abstract Although elevated glutaminolysis has been demonstrated in cancer cells, the precise mechanism by which glutaminolysis is exploited to promote tumor metastasis has not been elucidated. Lung cancer frequently metastasizes and is the leading cause of cancer-related death worldwide. Loss of LKB1 is associated with increased metastasis and poor prognosis in lung cancer, but the development of targeted agents in LKB1-deficient lung cancer is still in its infancy. Our recent study reveal that glutamate dehydrogenase 1, upregulated upon detachment via the transcription factor pleomorphic adenoma gene 1 (PLAG1), provides anti-anoikis and pro-metastatic signals in LKB1-deficient lung cancer. Mechanistically, the GDH1 product alpha-KG activates CamKK2 by enhancing its substrate AMPK binding and triggers CamKK2-mediated AMPK signaling, which contributes to energy production that confers anoikis resistance. The effect of GDH1 on AMPK is evident in LKB1-deficient lung cancer, where AMPK activation predominantly depends on CamKK2. Targeting GDH1 with the small molecule GDH1 inhibitor R162 led to attenuated anoikis resistance and tumor metastasis in an LKB1-deficient lung patient-derived xenograft model. Correlation studies between GDH1 signaling and metastatic cancer progression in lung cancer patients further validated the clinical relevance of our finding. Our study provides insight into the molecular mechanism by which GDH1-mediated metabolic reprogramming of glutaminolysis mediates lung cancer metastasis. This knowledge could be leveraged for the development of effective therapeutic strategies for patients with LKB1-deficient lung cancer. Citation Format: Sumin Kang, Lingtao Jin, Chaoyun Pan, Jaemoo Chun. Glutamate dehydrogenase 1 mediates metabolic reprogramming and promotes tumor metastasis through CamKK2-AMPK signaling in LKB1-deficient 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 1454.

PO-489 Tumour-specific oncogenic signalling activities define sensitivity to combinatorial MEK inhibition and stratify Kras-driven lung cancer

IntroductionWith the advent of molecular diagnostics and availability of targeted therapies, treatment of NSCLC has been improved, especially for tumours driven by mutant EGFR or ALK fusions. However, other frequently mutated genes, including KRAS, TP53 and STK11/LKB1, are still not targetable. Murine models of lung cancer that mimic human disease genomics and pathology can help refine diagnostic strategies, allowing discovery of new therapeutic options, identification of biomarkers and validation of the responses in vivo.Material and methodsPrimary cultures were derived from murine adenosquamous carcinoma (ASC) or adenocarcinoma (AC) tumours driven by mutant Kras and loss of Lkb1 (KL) or Tp53 (KP), and murine lung-derived healthy epithelia. Ex vivo drug sensitivity profiling, biomarker analysis and in vivo response validations were combined to identify NSCLC subtype-specific drug vulnerabilities. To identify biomarkers predictive of response, spatial analyses of murine and human tumours were conducted.Results and discussionsBy performing ex vivo functional screens, we identified histotype- and genotype-selective drug vulnerabilities. Inhibition of MEK, a downstream KRAS effector, caused a selective transient cytostasis in AC cells. However, long-term MEK inhibition was found to be ineffective in all cultures tested due to adaptive reactivation of MAPK and PI3K/AKT signalling. Resistance to MEK inhibitors was associated with activation of subtype-specific receptor tyrosine kinases: ERBB3 activation in KL;ASCs, adaptive ERBB2 and FGFR1 activation in KL;ACs, or adaptive FGFR1 activation in KP;ACs. Furthermore, while combined inhibition of MEK and ERBB kinases induced cell death in KL;ASC and KL;AC but not in KP;AC ex vivo culture, in vivo this combination showed cytotoxic response only in the squamous cell carcinoma (SCC) regions of KL;ASCs. This was in agreement with baseline ERBB activation occurring selectively in SCC tissue. Interestingly, activation of ERBB family receptors was detected in a significant proportion of human NSCLC tumours. Analysis of spatial signalling activities may therefore identify NSCLC patients that could benefit from combinatorial MEK and ERBB inhibition.ConclusionOur study validates the utility of NSCLC cultures to discover drug resistance mechanisms that can be overcome with subtype-specific drug combinations. Importantly, the combination of ex vivo drug response profiling and in situ spatial signalling shows that Kras-driven lung cancers stratify as functional subgroups.