Intratumoral Heterogeneity in EGFR-Mutant NSCLC Results in Divergent Resistance Mechanisms in Response to EGFR Tyrosine Kinase Inhibition

Abstract Non–small cell lung cancers (NSCLC) that have developed resistance to EGF receptor (EGFR) tyrosine kinase inhibitor (TKI), including gefitinib and erlotinib, are clinically linked to an epithelial-to-mesenchymal transition (EMT) phenotype. Here, we examined whether modulating EMT maintains the responsiveness of EGFR-mutated NSCLCs to EGFR TKI therapy. Using human NSCLC cell lines harboring mutated EGFR and a transgenic mouse model of lung cancer driven by mutant EGFR (EGFR-Del19-T790M), we demonstrate that EGFR inhibition induces TGFβ secretion followed by SMAD pathway activation, an event that promotes EMT. Chronic exposure of EGFR-mutated NSCLC cells to TGFβ was sufficient to ind…

CXCR7 Reactivates ERK Signaling to Promote Resistance to EGFR Kinase Inhibitors in NSCLC

Abstract Although EGFR mutant–selective tyrosine kinase inhibitors (TKI) are clinically effective, acquired resistance can occur by reactivating ERK. We show using in vitro models of acquired EGFR TKI resistance with a mesenchymal phenotype that CXCR7, an atypical G protein-coupled receptor, activates the MAPK–ERK pathway via β-arrestin. Depletion of CXCR7 inhibited the MAPK pathway, significantly attenuated EGFR TKI resistance, and resulted in mesenchymal-to-epithelial transition. CXCR7 overexpression was essential in reactivation of ERK1/2 for the generation of EGFR TKI–resistant persister cells. Many patients with non–small cell lung cancer (NSCLC) harboring an EGFR kinase domain mutatio…

β-Catenin Contributes to Lung Tumor Development Induced by EGFR Mutations

Abstract The discovery of somatic mutations in EGFR and development of EGFR tyrosine kinase inhibitors (TKI) have revolutionized treatment for lung cancer. However, resistance to TKIs emerges in almost all patients and currently no effective treatment is available. Here, we show that β-catenin is essential for development of EGFR-mutated lung cancers. β-Catenin was upregulated and activated in EGFR-mutated cells. Mutant EGFR preferentially bound to and tyrosine phosphorylated β-catenin, leading to an increase in β-catenin–mediated transactivation, particularly in cells harboring the gefitinib/erlotinib-resistant gatekeeper EGFR-T790M mutation. Pharmacologic inhibition of β-catenin suppresse…

Integrative genomic and proteomic analyses identify targets for Lkb1 deficient metastatic lung tumors

SummaryIn mice, Lkb1 deletion and activation of KrasG12D results in lung tumors with a high penetrance of lymph node and distant metastases. We analyzed these primary and metastatic de novo lung cancers with integrated genomic and proteomic profiles, and have identified gene and phosphoprotein signatures associated with Lkb1 loss and progression to invasive and metastatic lung tumors. These studies revealed that SRC is activated in Lkb1-deficient primary and metastatic lung tumors, and that the combined inhibition of SRC, PI3K, and MEK1/2 resulted in synergistic tumor regression. These studies demonstrate that integrated genomic and proteomic analyses can be used to identify signaling pathw…

Abstract C75: Overcoming KRAS/LKB1 mutant NSCLC resistance to BET bromodomain inhibitors with gemcitabine or Mcl-1 inhibition

Abstract The purpose of our study was to define a method and mechanism for overcoming the resistance of clinically relevant KRAS-mutant/LKB1-deficient NSCLC cells to the BET-bromodomain inhibitor JQ1. LKB1 (Serine/threonine kinase 11) is mutated with loss of function in conjunction with mutated KRAS in 7-10% of NSCLC. Importantly, KRAS-mutant/LKB1-deficiency is associated with tumor aggressiveness and poor survival in human patients as well as in genetically engineered mouse models. Indeed, although the BET bromodomain inhibitor JQ1 dramatically reduces tumor volume in KRAS mutant mice, it has little effect in KRAS-mutant/LKB1-deficient mice. BET bromodomain proteins are chromatin readers t…

Abstract LB-085: A new role for LKB1 to regulate Heat Shock Protein 90 activity

Abstract Approximately 30% of human non-small cell lung cancer (NSCLC) patients harbor a somatic KRAS mutation resulting, in aberrant activation of downstream signaling pathways that control cell proliferation, cell growth, and cell survival. Importantly, alleles of LKB1, a serine/threonine kinase that functions as a tumor suppressor, are somatically inactivated in ~30% of NSCLCs within KRAS-mutant NSCLC. The loss of LKB1 gives rise to aggressive, highly metastatic, and highly drug resistant tumors. We have previously demonstrated that the inactivation of the tumor suppressor lkb1 rendered mutant kras murine NSCLC resistant to targeted agents including BET bromodomain and kinase inhibitors.…

Activation of the PD-1 Pathway Contributes to Immune Escape in EGFR-Driven Lung Tumors

Abstract The success in lung cancer therapy with programmed death (PD)-1 blockade suggests that immune escape mechanisms contribute to lung tumor pathogenesis. We identified a correlation between EGF receptor (EGFR) pathway activation and a signature of immunosuppression manifested by upregulation of PD-1, PD-L1, CTL antigen-4 (CTLA-4), and multiple tumor-promoting inflammatory cytokines. We observed decreased CTLs and increased markers of T-cell exhaustion in mouse models of EGFR-driven lung cancer. PD-1 antibody blockade improved the survival of mice with EGFR-driven adenocarcinomas by enhancing effector T-cell function and lowering the levels of tumor-promoting cytokines. Expression of m…

Targeting transcriptional addictions in small cell lung cancer with a covalent CDK7 inhibitor.

Small cell lung cancer (SCLC) is an aggressive disease with high mortality, and the identification of effective pharmacological strategies to target SCLC biology represents an urgent need. Using a high-throughput cellular screen of a diverse chemical library, we observe that SCLC is sensitive to transcription-targeting drugs, in particular to THZ1, a recently identified covalent inhibitor of cyclin-dependent kinase 7. We find that expression of super-enhancer-associated transcription factor genes, including MYC family proto-oncogenes and neuroendocrine lineage-specific factors, is highly vulnerability to THZ1 treatment. We propose that downregulation of these transcription factors contribut…

Abstract 968: β-catenin plays an important role in lung tumor development induced by EGFR mutations

Abstract The discovery of somatic mutations in epidermal growth factor receptor (EGFR) and the development of EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, have revolutionized treatment for non-small cell lung cancer (NSCLC). Resistance to TKIs emerges in almost all patients, but currently no effective treatment is available.Therefore, novel strategies to either prevent or overcome resistance are sorely needed. Here we show that β-catenin is essential for development of EGFR mutated lung cancers. We found that β-catenin was upregulated, translocated to the nucleus, and subsequently activated in both EGFR mutated lung cancer cell lines and EGFR mutation driven lung…

Abstract 1126: Efficacy of BET bromodomain inhibition in Kras-positive non-small cell lung cancer.

Abstract Amplification of MYC is one of the most common genetic alterations in lung cancer, contributing to a myriad of phenotypes associated with growth, invasion and drug resistance. Murine genetics has established both the centrality of somatic alterations of Kras in lung cancer, as well as dependency of Kras-dependent tumors on c-Myc function. Unfortunately, drug-like small-molecule inhibitors of KRAS and c-Myc have yet to be realized. The recent discovery in hematologic malignancies that bromodomain inhibition impairs MYC expression and MYC-dependent transcriptional function prompted the possibility of targeting KRAS-driven NSCLC with a potent, prototypical BET bromodomain inhibitor, J…

Oncogenic Deregulation of EZH2 as an Opportunity for Targeted Therapy in Lung Cancer.

Abstract As a master regulator of chromatin function, the lysine methyltransferase EZH2 orchestrates transcriptional silencing of developmental gene networks. Overexpression of EZH2 is commonly observed in human epithelial cancers, such as non–small cell lung carcinoma (NSCLC), yet definitive demonstration of malignant transformation by deregulated EZH2 remains elusive. Here, we demonstrate the causal role of EZH2 overexpression in NSCLC with new genetically engineered mouse models of lung adenocarcinoma. Deregulated EZH2 silences normal developmental pathways, leading to epigenetic transformation independent of canonical growth factor pathway activation. As such, tumors feature a transcrip…

Abstract LB-C21: CXCR7 expression is necessary for the maintenance of mesenchymal phenotype in acquired EGFR TKI resistance in NSCLC

Abstract Activating EGFR mutations in non-small lung cancer (NSCLC) confer sensitivity to reversible EGFR tyrosine kinase inhibitors (TKIs), including gefitinib and erlotinib. Despite promising initial response, acquired resistance develops mediated by the emergence of the secondary T790M mutation or by focal amplification of MET. An epithelial-to-mesenchymal transition (EMT) is clinically linked to NSCLCs with acquired EGFR TKI resistance. The exact mechanisms of EGFR TKI resistance with EMT phenotype remain elusive; therefore, we have engineered EGFR-mutated NSCLC cell lines with mesenchymal phenotype by stably depleting E-Cadherin or by overexpressing Snail or chronically exposing the ce…

Metabolic and Functional Genomic Studies Identify Deoxythymidylate Kinase as a target in LKB1 Mutant Lung Cancer

Abstract The LKB1/STK11 tumor suppressor encodes a serine/threonine kinase, which coordinates cell growth, polarity, motility, and metabolism. In non–small cell lung carcinoma, LKB1 is somatically inactivated in 25% to 30% of cases, often concurrently with activating KRAS mutations. Here, we used an integrative approach to define novel therapeutic targets in KRAS-driven LKB1-mutant lung cancers. High-throughput RNA interference screens in lung cancer cell lines from genetically engineered mouse models driven by activated KRAS with or without coincident Lkb1 deletion led to the identification of Dtymk, encoding deoxythymidylate kinase (DTYMK), which catalyzes dTTP biosynthesis, as synthetica…

Abstract LB-399: Chronic inhibition of mutant EGFR in NSCLC leads to EGFR TKI resistance by TGF-β1 mediated epithelial to mesenchymal transition

Abstract In NSCLC, activating EGFR mutations underlie responsiveness of NSCLCs to reversible EGFR tyrosine kinase inhibitors (TKIs), including gefitinib and erlotinib. Despite initial responses, acquired resistance invariably develops, mediated by the emergence of the secondary T790M mutation and by focal amplification of MET, in approximately 50% and 30% of patients, respectively. The resistance mechanisms for the remaining 20% of cases remain elusive. EGFR TKI-sensitive HCC827 cells were exposed to graded concentrations of erlotinib for 6 months. Approximately 70% of the isolated clones were resistant to erlotinib and harbored MET amplification, and were sensitive to dual EGFR/MET inhibit…

Interleukin-17A Promotes Lung Tumor Progression through Neutrophil Attraction to Tumor Sites and Mediating Resistance to PD-1 Blockade

Abstract Introduction Proinflammatory cytokine interleukin-17A (IL-17A) is overexpressed in a subset of patients with lung cancer. We hypothesized that IL-17A promotes a protumorigenic inflammatory phenotype and inhibits antitumor immune responses. Methods We generated bitransgenic mice expressing a conditional IL-17A allele along with conditional Kras G12D and performed immune phenotyping of mouse lungs, a survival analysis, and treatment studies with antibodies either blocking programmed cell death 1 (PD-1) or IL-6 or depleting neutrophils. To support the preclinical findings, we analyzed human gene expression data sets and immune profiled patient lung tumors. Results Tumors in IL-17:Kras…

ETS-1 Regulates Twist-1 Expression In Non-Small Cell Lung Cancer (NSCLC) Progression And Metastasis

Temporal molecular and biological assessment of an erlotinib-resistant lung adenocarcinoma model reveals markers of tumor progression and treatment response.

Abstract Patients with lung cancer with activating mutations in the EGF receptor (EGFR) kinase, who are treated long-term with tyrosine kinase inhibitors (TKI), often develop secondary mutations in EGFR associated with resistance. Mice engineered to develop lung adenocarcinomas driven by the human EGFR T790M resistance mutation are similarly resistant to the EGFR TKI erlotinib. By tumor volume endpoint analysis, these mouse tumors respond to BIBW 2992 (an irreversible EGFR/HER2 TKI) and rapamycin combination therapy. To correlate EGFR-driven changes in the lung with response to drug treatment, we conducted an integrative analysis of global transcriptome and metabolite profiling compared wit…

Efficacy of BET Bromodomain Inhibition in Kras-Mutant Non–Small Cell Lung Cancer

Abstract Purpose: Amplification of MYC is one of the most common genetic alterations in lung cancer, contributing to a myriad of phenotypes associated with growth, invasion, and drug resistance. Murine genetics has established both the centrality of somatic alterations of Kras in lung cancer, as well as the dependency of mutant Kras tumors on MYC function. Unfortunately, drug-like small-molecule inhibitors of KRAS and MYC have yet to be realized. The recent discovery, in hematologic malignancies, that bromodomain and extra-terminal (BET) bromodomain inhibition impairs MYC expression and MYC transcriptional function established the rationale of targeting KRAS-driven non–small cell lung cance…

Abstract B290: Activation of the PD-1 pathway contributes to immune escape in EGFR-driven lung tumors.

Abstract The recent clinical success of therapeutic blockade of the immune checkpoint Programmed Death (PD)-1 in advanced lung cancer patients suggests that mechanisms of immune escape may contribute to lung tumor pathogenesis. We identified a correlation between Epidermal Growth Factor Receptor (EGFR) pathway activation and a gene signature indicative of immunosuppression manifested by upregulation of PD-1, PD-L1, cytotoxic T lymphocyte antigen-4 (CTLA-4) and multiple tumor-promoting inflammatory cytokines. Accordingly, we identified a decrease in the number of cytotoxic T cells and an increase in markers of T cell exhaustion in genetically engineered mouse models (GEMMs) of EGFR-driven lu…

D-2-hydroxyglutarate produced by mutant IDH2 causes cardiomyopathy and neurodegeneration in mice.

Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) have been discovered in several cancer types and cause the neurometabolic syndrome D2-hydroxyglutaric aciduria (D2HGA). The mutant enzymes exhibit neomorphic activity resulting in production of D2-hydroxyglutaric acid (D-2HG). To study the pathophysiological consequences of the accumulation of D-2HG, we generated transgenic mice with conditionally activated IDH2R140Q and IDH2R172K alleles. Global induction of mutant IDH2 expression in adults resulted in dilated cardiomyopathy, white matter abnormalities throughout the central nervous system (CNS), and muscular dystrophy. Embryonic activation of mutant IDH2 resulted in more pronounced ph…

Abstract 766: Suppression of gefitinib-induced EMT in EGFR mutant NSCLC preferentially selects for acquired T790M

Abstract Activating EGFR mutations in non-small lung cancer (NSCLC) confer sensitivity to reversible EGFR tyrosine kinase inhibitors (TKIs), including gefitinib and erlotinib. Despite promising initial response acquired resistance develops mediated by the emergence of the secondary T790M mutation or by focal amplification of MET. An epithelial-to-mesenchymal transition (EMT) is clinically linked to NSCLCs with acquired EGFR TKI resistance. The exact mechanisms of EGFR TKI resistance with EMT phenotype remain elusive; therefore, we attempted to develop a strategy to prevent the emergence of EGFR TKI resistance with EMT phenotype. In order to mimic the development of acquired EGFR TKI resista…

Loss of p53 Attenuates the Contribution of IL-6 Deletion on Suppressed Tumor Progression and Extended Survival in Kras-Driven Murine Lung Cancer

Interleukin-6 (IL-6) is involved in lung cancer tumorigenesis, tumor progression, metastasis, and drug resistance. Previous studies show that blockade of IL-6 signaling can inhibit tumor growth and increase drug sensitivity in mouse models. Clinical trials in non-small cell lung cancer (NSCLC) reveal that IL-6 targeted therapy relieves NSCLC-related anemia and cachexia, although other clinical effects require further study. We crossed IL-6(-/-) mice with Kras(G12D) mutant mice, which develop lung tumors after activation of mutant Kras(G12D), to investigate whether IL-6 inhibition contributes to tumor progression and survival time in vivo. Kras(G12D); IL-6(-/-) mice exhibited increased tumor…