Search

Introduction Lung adenocarcinomas harboring EGFR mutations do not respond to immune checkpoint blockade therapy and their EGFR wildtype counterpart. The mechanisms underlying this lack of clinical response have been investigated but remain incompletely understood. Methods We analyzed three cohorts of resected lung adenocarcinomas (Profiling of Resistance Patterns of Oncogenic Signaling Pathways in Evaluation of Cancer of Thorax, Immune Genomic Profiling of NSCLC, and The Cancer Genome Atlas) and compared tumor immune microenvironment of EGFR-mutant tumors to EGFR wildtype tumors, to identify actionable regulators to target and potentially enhance the treatment response. Results EGFR-mutant NSCLC exhibited low programmed death-ligand 1, low tumor mutational burden, decreased number of cytotoxic T cells, and low T cell receptor clonality, consistent with an immune-inert phenotype, though T cell expansion ex vivo was preserved. In an analysis of 75 immune checkpoint genes, the top up-regulated genes in the EGFR-mutant tumors (NT5E and ADORA1) belonged to the CD73/adenosine pathway. Single-cell analysis revealed that the tumor cell population expressed CD73, both in the treatment-naive and resistant tumors. Using coculture systems with EGFR-mutant NSCLC cells, T regulatory cell proportion was decreased with CD73 knockdown. In an immune-competent mouse model of EGFR-mutant lung cancer, the CD73/adenosine pathway was markedly up-regulated and CD73 blockade significantly inhibited tumor growth. Conclusions Our work revealed that EGFR-mutant NSCLC has an immune-inert phenotype. We identified the CD73/adenosine pathway as a potential therapeutic target for EGFR-mutant NSCLC.

AXL, a TAM (TYRO3, AXL, and MERTK) family receptor tyrosine kinase, is increasingly being recognized as a key determinant of resistance to targeted therapies, as well as chemotherapy and radiation in non–small cell lung cancer (NSCLC) and other cancers. We further show here that high levels of AXL and epithelial-to-mesenchymal transition were frequently expressed in subsets of both treatment-naïve and treatment-relapsed NSCLC. Previously, we and others have demonstrated a role for AXL in mediating DNA damage response (DDR), as well as resistance to inhibition of WEE1, a replication stress response kinase. Here, we show that BGB324 (bemcentinib), a selective small-molecule AXL inhibitor, caused DNA damage and induced replication stress, indicated by ATR/CHK1 phosphorylation, more significantly in TP53-deficient NSCLC cell lines. Similar effects were also observed in large-cell neuroendocrine carcinoma (LCNEC) cell lines. High AXL protein levels were also associated with resistance to ATR inhibition. Combined inhibition of AXL and ATR significantly decreased cell proliferation of NSCLC and LCNEC cell lines. Mechanistically, combined inhibition of AXL and ATR significantly increased RPA32 hyperphosphorylation and DNA double-strand breaks and induced markers of mitotic catastrophe. Notably, NSCLC cell lines with low levels of SLFN11, a known predictive biomarker for platinum and PARP inhibitor sensitivity, were more sensitive to AXL/ATR cotargeting. These findings demonstrate a novel and unexpected role for AXL in replication stress tolerance, with potential therapeutic implications. Implications: These findings demonstrate that the combination of AXL and ATR inhibitors could be a promising therapeutic combination for NSCLC, LCNEC, and other cancers.

We have previously shown that gene expression profiles of oral leukoplakia (OL) may improve the prediction of oral cancer (OC) risk. To identify new targets for prevention, we performed a systematic survey of transcripts associated with an increased risk of oral cancer and overexpressed in OC vs normal mucosa (NM).We used gene expression profiles of 86 patients with OL and available outcomes from a chemoprevention trial of OC and NM. MET expression was evaluated using immunohistochemistry in 120 OL patients, and its association with OC development was tested in multivariable analysis. Sensitivity to pharmacological Met inhibition was tested invitro in premalignant and OC cell lines (n = 33) and invivo using the 4-NQO model of oral chemoprevention (n = 20 mice per group). All statistical tests were two-sided.The overlap of 693 transcripts associated with an increased risk of OC with 163 transcripts overexpressed in OC compared with NM led to the identification of 23 overlapping transcripts, including MET. MET overexpression in OL was associated with a hazard ratio of 3.84 (95% confidence interval = 1.59 to 9.27, P = .003) of developing OC. Met activation was found in OC and preneoplastic cell lines. Crizotinib activity in preneoplastic and OC cell lines was comparable. ARQ 197 was more active in preneoplastic compared with OC cell lines. In the 4-NQO model, squamous cell carcinoma, dysplasia, and hyperkeratosis were observed in 75.0%, 15.0%, and 10.0% in the control group, and in 25.0%, 70.0%, and 5.0% in the crizotinib group (P.001).Together, these data suggest that MET activation may represent an early driver in oral premalignancy and a target for chemoprevention of OC.

// Robert J. Cardnell 1 , Lerong Li 2 , Triparna Sen 1 , Rasha Bara 1 , Pan Tong 2 , Junya Fujimoto 3 , Abbie S. Ireland 4 , Matthew R. Guthrie 4 , Sheila Bheddah 5 , Upasana Banerjee 1 , Nene N. Kalu 1 , You-Hong Fan 1 , Scott J. Dylla 5 , Faye M. Johnson 1,6 , Ignacio I. Wistuba 3 , Trudy G. Oliver 4 , John V. Heymach 1 , Bonnie S. Glisson 1 , Jing Wang 2,4,* and Lauren A. Byers 1,6,* 1 Department of Thoracic/Head and Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 2 Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 3 Department of Molecular Translational Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 4 Department of Oncological Sciences, University of Utah, Huntsman Cancer Institute, Salt Lake City, UT, USA 5 AbbVie StemCentrx, LLC, San Franciso, CA, USA 6 The University of Texas Graduate School of Biomedical Sciences, Houston, TX, USA * Co-senior authors Correspondence to: Lauren A. Byers, email: // Keywords : Alisertib, DLL3, rovalpituzumab tesirine, SCLC, TTF1 Received : July 04, 2017 Accepted : August 14, 2017 Published : September 01, 2017 Abstract Small cell lung cancer (SCLC) is a recalcitrant cancer for which no new treatments have been approved in over 30 years. While molecular subtyping now guides treatment selection for patients with non-small cell lung cancer and other cancers, SCLC is still treated as a single disease entity. Using model-based clustering, we found two major proteomic subtypes of SCLC characterized by either high thyroid transcription factor-1 (TTF1)/low cMYC protein expression or high cMYC/low TTF1. Applying “drug target constellation” (DTECT) mapping, we further show that protein levels of TTF1 and cMYC predict response to targeted therapies including aurora kinase, Bcl2, and HSP90 inhibitors. Levels of TTF1 and DLL3 were also highly correlated in preclinical models and patient tumors. TTF1 (used in the diagnosis lung cancer) could therefore be used as a surrogate of DLL3 expression to identify patients who may respond to the DLL3 antibody-drug conjugate rovalpituzumab tesirine. These findings suggest that TTF1, cMYC or other protein markers identified here could be used to identify subgroups of SCLC patients who may respond preferentially to several emerging targeted therapies.

Purpose: The primary cause of death due to head and neck squamous cell carcinoma (HNSCC) is local treatment failure. The goal of this study was to examine this phenomenon using an unbiased approach. Experimental Design: We utilized human papilloma virus (HPV)-negative cell lines rendered radiation-resistant (RR) via repeated exposure to radiation, a panel of HPV-negative HNSCC cell lines and three cohorts of HPV-negative HNSCC tumors (n = 68, 97, and 114) from patients treated with radiotherapy and subjected to genomic, transcriptomic, and proteomic analysis. Results: RR cell lines exhibited upregulation of several proteins compared with controls, including increased activation of Axl and PI3 kinase signaling as well as increased expression of PD-L1. Additionally, inhibition of either Axl or PI3 kinase led to decreased PD-L1 expression. When clinical samples were subjected to RPPA and mRNA expression analysis, PD-L1 was correlated with both Axl and PI3K signaling as well as dramatically associated with local failure following radiotherapy. This finding was confirmed examining a third cohort using immunohistochemistry. Indeed, tumors with high expression of PD-L1 had failure rates following radiotherapy of 60%, 70%, and 50% compared with 20%, 25%, and 20% in the PD-L1–low expression group (P = 0.01, 1.9 × 10−3, and 9 × 10−4, respectively). This finding remained significant on multivariate analysis in all groups. Additionally, patients with PD-L1 low/CD8+ tumor-infiltrating lymphocytes high had no local failure or death due to disease (P = 5 × 10−4 and P = 4 × 10−4, respectively). Conclusions: Taken together, our data point to a targetable Axl–PI3 kinase–PD-L1 axis that is highly associated with radiation resistance. Clin Cancer Res; 23(11); 2713–22. ©2017 AACR.

Small cell lung cancer (SCLC) accounts for approximately 15% of lung cancer cases with a poor five year survival rate of 6%. The National Cancer Institute has designated SCLC as one of two “recalcitrant” cancers and urges translational research to advance treatment options. In our effort to maximize treatment benefits, we have aimed our investigation at uncovering metabolic differences contributing to disease progression that may be targeted through specific pathway inhibition. Although metabolism is relatively unstudied in SCLC, metabolic reprogramming is recognized as a hallmark of cancer and can be regulated by several different mechanisms. Notably, the transcription factor MYC is overexpressed in 30% SCLC tumors and is known to modulate the balance between two major pathways of metabolism: glycolysis and mitochondrial respiration. While mitochondrial respiration is the preferred source of energy production in terminally differentiated cells, many cancer cells switch to become predominantly glycolytic in an effort to generate energy quickly and produce biomolecules and electron carriers required for proliferation—a phenomena known as the Warburg Effect. Since MYC is expressed in only a subset of highly aggressive SCLC, we applied bimodal separation to both patient and cell line SCLC datasets to establish defined MYC subsets (MYCLow; MYCHigh) and performed gene ontology pathway analysis to genes that were significantly upregulated among the MYC-expressing samples. This revealed that 37% of significantly upregulated genes were linked to metabolic processes and further investigation showed many of those genes were linked to the glycolysis pathway. We also confirmed upregulation of protein expression through reverse phase protein array (RPPA). With glycolysis proving to be increased in MYCHigh samples, we simultaneously characterized pathway utilization in cell lines at baseline and in the presence of a potent glycolysis inhibitor. MYCHigh cell lines exhibited greater glucose consumption and lactate secretion, which was significantly decreased by glycolytic inhibition. Likewise, mitochondrial analysis revealed lower oxygen consumption and ATP production that were further reduced in the presence of the glycolysis inhibitor. Although mitochondrial density was unchanged regardless of MYC expression or glycolytic inhibition, reactive oxygen species (ROS) generation was greatly enhanced in the MYCHigh subset upon suppression of glycolysis. Lastly, SCLC xenografts derived from a MYCHigh SCLC cell line showed significantly slower tumor growth whereas MYCLow derived xenografts exhibited no significant difference in tumor growth. Together, these data provide evidence of metabolic differences among SCLC subsets such that MYC expression induces reliance on glycolysis, which can be targeted for therapeutic intervention. Citation Format: Kasey R. Cargill, C. Allison Stewart, Elizabeth M. Park, Robert J. Cardnell, You Hong Fan, Qi Wang, Lixia Diao, Wai Kin Chan, Philip L. Lorenzi, Jing Wang, Lauren A. Byers. Targeting MYC-enhanced glycolysis in small cell lung cancer [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 2335.

Purpose: Head and neck squamous cell carcinoma (HNSCC) is commonly treated with radiotherapy, and local failure after treatment remains the major cause of disease-related mortality. To date, human papillomavirus (HPV) is the only known clinically validated, targetable biomarkers of response to radiation in HNSCC. Experimental Design: We performed proteomic and transcriptomic analysis of targetable biomarkers of radioresistance in HPV-negative HNSCC cell lines in vitro, and tested whether pharmacologic blockade of candidate biomarkers sensitized cells to radiotherapy. Candidate biomarkers were then investigated in several independent cohorts of patients with HNSCC. Results: Increased expression of several targets was associated with radioresistance, including FGFR, ERK1, EGFR, and focal adhesion kinase (FAK), also known as PTK2. Chemical inhibition of PTK2/FAK, but not FGFR, led to significant radiosensitization with increased G2–M arrest and potentiated DNA damage. PTK2/FAK overexpression was associated with gene amplification in HPV-negative HNSCC cell lines and clinical tumors. In two independent cohorts of patients with locally advanced HPV-negative HNSCC, PTK2/FAK amplification was highly associated with poorer disease-free survival (DFS; P = 0.012 and 0.034). PTK2/FAK mRNA expression was also associated with worse DFS (P = 0.03). Moreover, both PTK2/FAK mRNA (P = 0.021) and copy number (P = 0.063) were associated with DFS in the Head and Neck Cancer subgroup of The Cancer Genome Atlas. Conclusions: Proteomic analysis identified PTK2/FAK overexpression is a biomarker of radioresistance in locally advanced HNSCC, and PTK2/FAK inhibition radiosensitized HNSCC cells. Combinations of PTK2/FAK inhibition with radiotherapy merit further evaluation as a therapeutic strategy for improving local control in HPV-negative HNSCC. Clin Cancer Res; 22(18); 4643–50. ©2016 AACR.

Purpose: Promising results in the treatment of non–small cell lung cancer (NSCLC) have been seen with agents targeting immune checkpoints, such as programmed cell death 1 (PD-1) or programmed death ligand-1 (PD-L1). However, only a select group of patients respond to these interventions. The identification of biomarkers that predict clinical benefit to immune checkpoint blockade is critical to successful clinical translation of these agents. Methods: We conducted an integrated analysis of three independent large datasets, including The Cancer Genome Atlas of lung adenocarcinoma and two datasets from MD Anderson Cancer Center (Houston, TX), Profiling of Resistance Patterns and Oncogenic Signaling Pathways in Evaluation of Cancers of the Thorax (named PROSPECT) and Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination (named BATTLE-1). Comprehensive analysis of mRNA gene expression, reverse-phase protein array, IHC, and correlation with clinical data were performed. Results: Epithelial–mesenchymal transition (EMT) is highly associated with an inflammatory tumor microenvironment in lung adenocarcinoma, independent of tumor mutational burden. We found immune activation coexistent with elevation of multiple targetable immune checkpoint molecules, including PD-L1, PD-L2, PD-1, TIM-3, B7-H3, BTLA, and CTLA-4, along with increases in tumor infiltration by CD4+Foxp3+ regulatory T cells in lung adenocarcinomas that displayed an EMT phenotype. Furthermore, we identify B7-H3 as a prognostic marker for NSCLC. Conclusions: The strong association between EMT status and an inflammatory tumor microenvironment with elevation of multiple targetable immune checkpoint molecules warrants further investigation of using EMT as a predictive biomarker for immune checkpoint blockade agents and other immunotherapies in NSCLC and possibly a broad range of other cancers. Clin Cancer Res; 22(14); 3630–42. ©2016 AACR. See related commentary by Datar and Schalper, p. 3422

Purpose: VEGF pathway inhibitors have been investigated as therapeutic agents in the treatment of non–small cell lung cancer (NSCLC) because of its central role in angiogenesis. These agents have improved survival in patients with advanced NSCLC, but the effects have been modest. Although VEGFR2/KDR is typically localized to the vasculature, amplification of KDR has reported to occur in 9% to 30% of the DNA from different lung cancers. We investigated the signaling pathways activated downstream of KDR and whether KDR amplification is associated with benefit in patients with NSCLC treated with the VEGFR inhibitor vandetanib. Methods: NSCLC cell lines with or without KDR amplification were studied for the effects of VEGFR tyrosine kinase inhibitors (TKI) on cell viability and migration. Archival tumor samples collected from patients with platinum-refractory NSCLC in the phase III ZODIAC study of vandetanib plus docetaxel or placebo plus docetaxel (N = 294) were screened for KDR amplification by FISH. Results: KDR amplification was associated with VEGF-induced activation of mTOR, p38, and invasiveness in NSCLC cell lines. However, VEGFR TKIs did not inhibit proliferation of NSCLC cell lines with KDR amplification. VEGFR inhibition decreased cell motility as well as expression of HIF1α in KDR-amplified NSCLC cells. In the ZODIAC study, KDR amplification was observed in 15% of patients and was not associated with improved progression-free survival, overall survival, or objective response rate for the vandetanib arm. Conclusions: Preclinical studies suggest KDR activates invasion but not survival pathways in KDR-amplified NSCLC models. Patients with NSCLC whose tumor had KDR amplification were not associated with clinical benefit for vandetanib in combination with docetaxel. Clin Cancer Res; 22(8); 1940–50. ©2015 AACR.

Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor that constitutes approximately 14% of all lung cancers diagnosed in the United States. Despite recent advances in treatment options, recurrence is a major challenge and long-term survival remains poor underscoring the importance of investigating mechanisms of treatment resistance and therapeutic strategies. Interestingly, our laboratory has recently uncovered evidence for the classification of SCLC into distinct subtypes, each with unique gene expression patterns and therapeutic vulnerabilities. These subtypes are characterized by differential expression of the transcription factors ASCL1, NEUROD1, or POU2F3. A fourth subtype is negative for all three transcription factors and has prominent expression of immune genes, thus is termed the Inflamed subtype. These subtypes are associated with differences in drug sensitivity, biomarker signatures, and mechanisms of cellular growth. One facet of tumor biology that has not been explored across the subtypes, or in SCLC in general, is the metabolic mechanisms of energy generation. Although metabolic reprogramming from mitochondrial respiration to glycolysis is considered a major hallmark of cancer, increasing evidence suggests that mitochondrial or amino acid dependencies may also contribute to treatment resistance. Therefore, understanding the metabolic demand, particularly among the subtypes, is critical for the development of new therapies. To address this, gene set enrichment analysis was applied to 81 human SCLC tumors categorized by subtype, and revealed differential gene expression correlating to pathways such as fatty acid metabolism in POU2F3 and reactive oxygen species in Inflamed that was confirmed by reverse phase protein analysis (RPPA). Further, we evaluated nutrient requirements, glycolysis dependence, and mitochondrial function in human SCLC cell lines and found differences in glucose uptake and subsequent lactate generation between the subtypes suggesting that glycolysis inhibition may be effective in particular subsets. Based on this, we then tested the efficacy of PFK-158 (glycolysis inhibitor) on cellular proliferation and metabolic function in these cell lines, which revealed variations in susceptibility dependent on subtype classification. Together, this data serves to enhance the current understanding of SCLC mechanisms of growth and provides evidence for metabolic inhibition as a potential therapeutic opportunity. Citation Format: Kasey R. Cargill, Carl M. Gay, Robert J. Cardnell, You-Hong Fan, Qi Wang, Lixia Diao, Jing Wang, Lauren A. Byers. Comprehensive metabolic profiling and vulnerabilities to metabolic inhibitors among small cell lung cancer subtypes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 232.

Background: Small cell lung cancer (SCLC) is characterized by a loss of TP53 and RB1 and by amplification of MYC in about 25% of patients. Our group and others have shown that MYC-driven SCLC cell lines are vulnerable to Aurora Kinase A (AURKA) inhibition. Alisertib, a selective AURKA inhibitor, has also shown clinical efficacy in MYC-positive relapsed SCLC patients in combination with paclitaxel in a phase II trial (NCT02038647). AURKA regulates DNA chromosome alignment during cell division and thus its inhibition results in mitotic stress and subsequently DNA damage. We have previously demonstrated that DNA damage response (DDR) inhibitors synergize with immune checkpoint inhibition in SCLC in vivo models by activating a STING-mediated innate immune response. Based on these findings, we hypothesized that AURKA inhibition may activate the STING pathway and induce PD-L1 expression in SCLC. Methods: We compared AURKA gene expression among a panel of SCLC and NSCLC cell lines and, similarly, in tumor versus normal tissue in SCLC clinical dataset (Sato et al.). In parallel, we explored the landscape of STING and immune genes expression in two SCLC clinical datasets (George et al. and Sato et al.).We tested human and GEMM-derived SCLC cell lines for changes in immune and DDR-related protein expression by Western blot analysis after treatment with the AURKA-i alisertib. Results: AURKA gene expression was higher in SCLC cell lines as compared to NSCLC cell lines (p=0.023) and in SCLC tumors compared to normal tissue (FC=1.6; p0.45, p Citation Format: Carminia M. Della Corte, Liz Lauren Ajpacaja, Robert J Cardnell, Carl M Gay, Lixia Diao, Qi Wang, Kavya Ramkumar, Allison C. Stewart, Rebecca B. Kow, Hannah E Stumpf, You-Hong Fan, Jing Wang, John V Heymach, Lauren A. Byers. The Aurora kinase A-inhibitor, alisertib, is a potential candidate for combination with immunotherapy in small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B064. doi:10.1158/1535-7163.TARG-19-B064

Effective targeted therapies for small-cell lung cancer (SCLC), the most aggressive form of lung cancer, remain urgently needed. Here we report evidence of preclinical efficacy evoked by targeting the overexpressed cell-cycle checkpoint kinase CHK1 in SCLC. Our studies employed RNAi-mediated attenuation or pharmacologic blockade with the novel second-generation CHK1 inhibitor prexasertib (LY2606368), currently in clinical trials. In SCLC models in vitro and in vivo , LY2606368 exhibited strong single-agent efficacy, augmented the effects of cisplatin or the PARP inhibitor olaparib, and improved the response of platinum-resistant models. Proteomic analysis identified CHK1 and MYC as top predictive biomarkers of LY2606368 sensitivity, suggesting that CHK1 inhibition may be especially effective in SCLC with MYC amplification or MYC protein overexpression. Our findings provide a preclinical proof of concept supporting the initiation of a clinical efficacy trial in patients with platinum-sensitive or platinum-resistant relapsed SCLC. Cancer Res; 77(14); 3870–84. ©2017 AACR .

Small cell lung cancer (SCLC) is an aggressive malignancy distinct from non–small cell lung cancer (NSCLC) in its metastatic potential and treatment response. Using an integrative proteomic and transcriptomic analysis, we investigated molecular differences contributing to the distinct clinical behavior of SCLCs and NSCLCs. SCLCs showed lower levels of several receptor tyrosine kinases and decreased activation of phosphoinositide 3-kinase (PI3K) and Ras/mitogen-activated protein (MAP)/extracellular signal–regulated kinase (ERK) kinase (MEK) pathways but significantly increased levels of E2F1-regulated factors including enhancer of zeste homolog 2 (EZH2), thymidylate synthase, apoptosis mediators, and DNA repair proteins. In addition, PARP1, a DNA repair protein and E2F1 co-activator, was highly expressed at the mRNA and protein levels in SCLCs. SCLC growth was inhibited by PARP1 and EZH2 knockdown. Furthermore, SCLC was significantly more sensitive to PARP inhibitors than were NSCLCs, and PARP inhibition downregulated key components of the DNA repair machinery and enhanced the efficacy of chemotherapy. Significance: SCLC is a highly lethal cancer with a 5-year survival rate of less than 10%. To date, no molecularly targeted agents have prolonged survival in patients with SCLCs. As a step toward identifying new targets, we systematically profiled SCLCs with a focus on therapeutically relevant signaling pathways. Our data reveal fundamental differences in the patterns of pathway activation in SCLCs and NSCLCs and identify several potential therapeutic targets for SCLCs, including PARP1 and EZH2. On the basis of these results, clinical studies evaluating PARP and EZH2 inhibition, together with chemotherapy or other agents, warrant further investigation. Cancer Discov; 2(9); 798–811. ©2012 AACR. Read the Commentary on this article by Rosell and Wannesson, p. 769. This article is highlighted in the In This Issue feature, p. 753.

Patients with oral premalignant lesion (OPL) have a high risk of developing oral cancer. Although certain risk factors, such as smoking status and histology, are known, our ability to predict oral cancer risk remains poor. The study objective was to determine the value of gene expression profiling in predicting oral cancer development. Gene expression profile was measured in 86 of 162 OPL patients who were enrolled in a clinical chemoprevention trial that used the incidence of oral cancer development as a prespecified endpoint. The median follow-up time was 6.08 years and 35 of the 86 patients developed oral cancer over the course. Gene expression profiles were associated with oral cancer–free survival and used to develop multivariate predictive models for oral cancer prediction. We developed a 29-transcript predictive model which showed marked improvement in terms of prediction accuracy (with 8% predicting error rate) over the models using previously known clinicopathologic risk factors. On the basis of the gene expression profile data, we also identified 2,182 transcripts significantly associated with oral cancer risk–associated genes (P value < 0.01; univariate Cox proportional hazards model). Functional pathway analysis revealed proteasome machinery, MYC, and ribosomal components as the top gene sets associated with oral cancer risk. In multiple independent data sets, the expression profiles of the genes can differentiate head and neck cancer from normal mucosa. Our results show that gene expression profiles may improve the prediction of oral cancer risk in OPL patients and the significant genes identified may serve as potential targets for oral cancer chemoprevention. Cancer Prev Res; 4(2); 218–29. ©2011 AACR.

Cigarette smoke is the major cause of lung cancer and can interact in complex ways with drugs for lung cancer prevention or therapy. Molecular genetic research promises to elucidate the biological mechanisms underlying divergent drug effects in smokers versus nonsmokers and to help in developing new approaches for controlling lung cancer. The present study compared global gene expression profiles (determined via Affymetrix microarray measurements in bronchial epithelial cells) between chronic smokers, former smokers, and never smokers. Smoking effects on global gene expression were determined from a combined analysis of three independent data sets. Differential expression between current and never smokers occurred in 591 of 13,902 measured genes (P < 0.01 and >2-fold change; pooled data)—a profound effect. In contrast, differential expression between current and former smokers occurred in only 145 of the measured genes (P < 0.01 and >2-fold change; pooled data). Nine of these 145 genes showed consistent and significant changes in each of the three data sets (P < 0.01 and >2-fold change), with eight being down-regulated in former smokers. Seven of the eight down-regulated genes, including CYP1B1 and three AKR genes, influence the metabolism of carcinogens and/or therapeutic/chemopreventive agents. Our data comparing former and current smokers allowed us to pinpoint the genes involved in smoking-drug interactions in lung cancer prevention and therapy. These findings have important implications for developing new targeted and dosing approaches for prevention and therapy in the lung and other sites, highlighting the importance of monitoring smoking status in patients receiving oncologic drug interventions.

The lungs and oral cavity of smokers are exposed to tobacco carcinogens. We hypothesized that tobacco-induced molecular alterations in the oral epithelium are similar to those in the lungs, and thus the oral epithelium may be used as a surrogate tissue for assessing alterations in the lungs. We used methylation-specific PCR to analyze promoter methylation of the p16 and FHIT genes at baseline and 3 months after intervention in 1,774 oral and bronchial brush specimens from 127 smokers enrolled in a randomized placebo-controlled chemoprevention trial. The association between methylation patterns in oral tissues and bronchial methylation indices (methylated sites / total sites per subject) was analyzed in a blinded fashion. At baseline, promoter methylation in bronchial tissue was present in 23% of samples for p16, 17% for FHIT, and 35% for p16 and FHIT; these percentages were comparable to methylation in oral tissue: 19% (p16), 15% (FHIT), and 31% (p16 and FHIT). Data from both oral and bronchial tissues were available for 125 individuals, in whom the two sites correlated strongly with respect to alterations (P < 0.0001 for both p16 and FHIT). At baseline, the mean bronchial methylation index was far higher in patients with oral tissue methylation (in either of the two genes; 39 patients) than in patients without oral tissue methylation (86 patients): 0.53 ± 0.29 versus 0.27 + 0.26 methylation index (P < 0.0001). Similar correlations occurred at 3 months after intervention. Our results support the potential of oral epithelium as a surrogate tissue for assessing tobacco-induced molecular damage in the lungs and thus have important implications for designing future lung cancer prevention trials and for research into the risk and early detection of lung cancer.

The molecular underpinnings that drive the heterogeneity of KRAS-mutant lung adenocarcinoma are poorly characterized. We performed an integrative analysis of genomic, transcriptomic, and proteomic data from early-stage and chemorefractory lung adenocarcinoma and identified three robust subsets of KRAS-mutant lung adenocarcinoma dominated, respectively, by co-occurring genetic events in STK11/LKB1 (the KL subgroup), TP53 (KP), and CDKN2A/B inactivation coupled with low expression of the NKX2-1 (TTF1) transcription factor (KC). We further revealed biologically and therapeutically relevant differences between the subgroups. KC tumors frequently exhibited mucinous histology and suppressed mTORC1 signaling. KL tumors had high rates of KEAP1 mutational inactivation and expressed lower levels of immune markers, including PD-L1. KP tumors demonstrated higher levels of somatic mutations, inflammatory markers, immune checkpoint effector molecules, and improved relapse-free survival. Differences in drug sensitivity patterns were also observed; notably, KL cells showed increased vulnerability to HSP90-inhibitor therapy. This work provides evidence that co-occurring genomic alterations identify subgroups of KRAS-mutant lung adenocarcinoma with distinct biology and therapeutic vulnerabilities. Significance: Co-occurring genetic alterations in STK11/LKB1, TP53, and CDKN2A/B—the latter coupled with low TTF1 expression—define three major subgroups of KRAS-mutant lung adenocarcinoma with distinct biology, patterns of immune-system engagement, and therapeutic vulnerabilities. Cancer Discov; 5(8); 860–77. ©2015 AACR. This article is highlighted in the In This Issue feature, p. 783

The p16INK4a and RASSF1A are tumor suppressor genes frequently inactivated by de novo promoter hypermethylation in non-small cell lung cancer. We studied 119 patients with non-small cell lung cancer (70 stage I/II and 49 stage IIIA) who had undergone surgery with curative intent. The p16INK4a and RASSF1A promoter methylation statuses were determined by methylation-specific PCR. Statistical analyses, all two-sided, were performed to determine the prognostic effect of hypermethylation on various clinical parameters. Hypermethylation of the p16INK4a and RASSF1A promoters was found in 58 (49%) and 46 (39%) tumors, respectively, and 30 tumors (25%) exhibited hypermethylation of both gene promoters. In patients with stage I/II tumors, only p16INK4a promoter hypermethylation was associated with a poor 5-year overall survival rate (P = 0.002). In patients with stage IIIA disease, however, RASSF1A promoter hypermethylation was a stronger predictor of a poor 5-year overall survival rate (P < 0.0001) than p16INK4a promoter hypermethylation. Among the 49 patients with stage IIIA tumors, 16 (89%) of the 18 patients whose tumors showed RASSF1A promoter hypermethylation died within 3 years after surgery, as compared with only 12 (39%) of the 31 patients whose tumors had no RASSF1A promoter hypermethylation (P < 0.0001). Multivariate analysis indicated that RASSF1A promoter hypermethylation was the stronger independent predictor for survival in patients with locally advanced non-small cell lung cancer. Our results indicate that p16INK4a promoter hypermethylation predicts a poor 5-year survival rates for patients with resectable non-small cell lung cancer, particularly for those with early stage tumors, whereas RASSF1A promoter hypermethylation is a profound prognostic predictor for patients with locally advanced non-small cell lung cancer, suggesting an important role of RASSF1A in non-small cell lung cancer progression.

Purpose: Retinoic acid receptor-β2 (RAR-β2) expression is suppressed in oral premalignant lesions and head and neck squamous cell carcinomas (HNSCCs). This study was conducted to determine whether RAR-β2 gene expression in such lesions can be silenced by promoter methylation. Experimental Design: RAR-β2 methylation was analyzed in DNA samples from 22 pairs of primary HNSCC and adjacent normal epithelium, 124 samples of oral leukoplakia, and 18 HNSCC cell lines using methylation-specific PCR. RAR-β2 promoter was methylated in 67, 56, and 53% of HNSCC tumors, HNSCC cell lines, and microdissected oral leukoplakia specimens, respectively. RAR-β2 hypermethylation was confirmed by sodium bisulfite-PCR combined with restriction enzyme digestion analysis and by random cloning and sequencing of bisulfite-treated DNA isolates. Results: Significantly higher RAR-β2 hypermethylation levels were found in tumor tissue compared with adjacent normal tissue (P = 0.002). RAR-β2 methylation in the cell lines was correlated with loss of RAR-β2 expression (P = 0.013) and inversely related to the presence of mutated p53 (P = 0.025). The demethylating agent 5-aza-2′-deoxycytidine (5-aza-CdR) restored RAR-β2 inducibility by all-trans-retinoic acid (ATRA) in some of the cell lines, which posses a methylated RAR-β2 promoter. In some cell lines, this effect was associated with increased growth inhibition after combined treatment with 5-aza-CdR and ATRA. Conclusions: RAR-β2 silencing by methylation is an early event in head and neck carcinogenesis; 5-Aza-CdR can restore RAR-β2 inducibility by ATRA in most cell lines, and the combination of 5-aza-CdR and ATRA is more effective in growth inhibition than single agents.

Background: WEE1 inhibitors (WEEi) are a novel class of small molecule inhibitors that are now under early clinical trials for small cell lung cancer (SCLC). WEE1 is a protein kinase that plays a key role in regulating the G2 checkpoint in response to DNA damage. Moreover, because WEE1 inhibition induces a HR deficient state, dual targeting of WEE1 and PARP (another promising target previously identified in our group) may induce synthetic lethality. In the present study we evaluate the efficacy of AZD1775 alone and with the PARP inhibitor, olaparib; investigate the mechanisms of primary resistance to AZD1775 in SCLC models; and assess the combinatorial efficacy of AZD1775 treatment with the chemotherapy drug, temozolomide (TMZ), that has shown promise in patients with recurrent SCLC and has been included in the National Comprehensive Cancer Network guidelines for standard treatment for SCLC. Results: Combination of AZD1775 with olaparib revealed an additive effect in vitro in 90% of SCLC cell lines. AZD1775 combined with TMZ synergistically decreased viability, increased DNA damage and apoptosis in SCLC cell lines (n=10) irrespective of MGMT status or initial response to AZD1775 alone. However, unlike the sensitive cells, SCLC cell lines showing primary resistance to AZD1775 had an intact DNA repair mechanism (after DNA damage), which may contribute to the resistance mechanism. Proteomic analysis revealed AXL (receptor tyrosine kinase) and phospho-S6K (S240/244) as markers of AZD1775 resistance and treatment with AXL inhibitor, TP0903 (40nM), resensitized the cells to AZD1775. Pre and post-AZD1775 treated samples revealed sustained activation of mTOR pathway in AZD1775 primary resistant lines. We further demonstrated that pre-treatment of the cells with the mTOR inhibitor everolimus sensitized SCLC cells to AZD1775 by causing downregulation of AKT/mTOR pathway. Conclusion: WEE1 inhibitors are currently in clinical trials for SCLC patients. However, as with any targeted therapy, drug resistance is an important barrier to clinical benefit which could be addressed with therapeutic combinations. We show the efficacy of single agent AZD1775 and in combination with olaparib. WEE1 inhibitor, AZD1775 synergizes with TMZ irrespective of MGMT status in all tested in vitro models which warrants further clinical investigation. We also show that the activity of the WEE1 inhibitors might be limited in cancer cells overexpressing of AXL and activated mTOR pathway and that AXL and mTOR inhibition re-sensitized the cells to AZD1775. Our work supports further exploration of the combination of PARP and WEE1 in SCLC and also the possibility of AXL/mTOR inhibition as a mechanism to overcome WEE1 inhibition resistance in SCLC. SCLC is a disease with very limited therapeutic options and no targeted agents with proven benefit; thus the results from this study have clear translational benefit. Citation Format: Triparna Sen, Pan Tong, Lixia Diao, You Hong-Fan, C. Allison Stewart, John V. Heymach, Jing Wang, Lauren A. Byers. WEE1 inhibitor activity correlates to AXL/mTOR expression and exhibits synergy with temozolomide (TMZ) in small cell lung cancer (SCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 298. doi:10.1158/1538-7445.AM2017-298

The PI3K/AKT/mTOR pathway is frequently activated in head and neck squamous cell carcinoma (HNSCC), but pathway inhibition has variable efficacy. Identification of predictive biomarkers and mechanisms of resistance would allow selection of patients most likely to respond and novel therapeutic combinations. The purpose of this study was to extend recent discoveries regarding the PI3K/AKT/mTOR pathway in HNSCC by more broadly examining potential biomarkers of response, by examining pathway inhibitors with a diverse range of targets, and by defining mechanisms of resistance and potential combination therapies. We used reverse-phase protein arrays (RPPA) to simultaneously evaluate expression of 195 proteins; SNP array to estimate gene copy number; and mass array to identify mutations. We examined altered signaling at baseline and after pathway inhibition. Likewise, we examined the activation of the PI3K/AKT/mTOR pathway in HNSCC tumors by RPPA. Cell lines with PIK3CA mutations were sensitive to pathway inhibitors, whereas amplification status did not predict sensitivity. While we identified a set of individual candidate biomarkers of response to pathway inhibitors, proteomic pathway scores did not correlate with amplification or mutation and did not predict response. Several receptor tyrosine kinases, including EGFR and ERK, were activated following PI3K inhibition in resistant cells; dual pathway inhibition of PI3K and EGFR or MEK demonstrated synergy. Combined MEK and PI3K inhibition was markedly synergistic in HRAS-mutant cell lines. Our findings indicate that clinical trials of single-agent PI3K/AKT/mTOR pathway inhibitors in selected populations and of PI3K/EGFR or PI3K/MEK inhibitor combinations are warranted; we plan to conduct such trials. Mol Cancer Ther; 13(11); 2738–50. ©2014 AACR.

Background and Purpose: Genetic damage has been identified at multiple chromosomal sites (i.e., loci) in lung cancer cells. We questioned whether similar damage could be detected in the bronchial epithelial cells of chronic smokers who do not have this disease. Methods: Biopsy specimens from six different bronchial regions were obtained from 54 chronic smokers (40 current smokers and 14 former smokers). The presence of squamous metaplasia and dysplasia (abnormal histologic changes) in the specimens was documented by examination of hematoxylin-eosin-stained sections, and a metaplasia index ([number of biopsy specimens with metaplasia/total number of biopsy specimens] x 100%) was calculated for each subject. Loss of heterozygosity (i.e., loss of DNA sequences from one member of a chromosome pair) involving microsatellite DNA at three specific loci-chromosome 3p14, chromosome 9p21, and chromosome 17p13-was evaluated by means of the polymerase chain reaction. Fisher's exact test and logistic regression analysis were used to assess the data. Reported P values are two-sided. Results: Data on microsatellite DNA status at chromosomes 3p14, 9p21, and 17p13 were available for 54, 50, and 44 subjects, respectively. The numbers of individuals who were actually informative (i.e., able to be evaluated for a loss of heterozygosity) at the three loci were 36 (67%), 37 (74%), and 34 (77%), respectively. DNA losses were detected in 27 (75%), 21 (57%), and six (18%) of the informative subjects at chromosomes 3p14, 9p21, and 17p13, respectively. Fifty-one subjects were informative for at least one of the three loci, and 39 (76%) exhibited a loss of heterozygosity. Forty-two subjects were informative for at least two of the loci, and 13 (31%) exhibited losses at a minimum of two loci. Loss of heterozygosity at chromosome 3p14 was more frequent in current smokers (22 [88%] of 25 informative) than in former smokers (five [45%] of 11 informative) (P = .01) and in subjects with a metaplasia index greater than or equal to 15% (21 [91%] of 23 informative) than in subjects with a metaplasia index of less than 15% (six [46%] of 13 informative) (P = .003). In five informative individuals among nine tested nonsmokers, a loss of heterozygosity was detected in only one subject at chromosome 3p14 (P =.03), and no losses were detected at chromosome 9p21 (P = .05). Conclusions: Genetic alterations at chromosomal sites containing putative tumor-suppressor genes (i.e., 3p14 and the FHIT gene, 9p21 and the p16 gene [also known as CDKN2], and 17p13 and the p53 gene [also known as TP53]) occur frequently in the histologically normal or minimally altered bronchial epithelium of chronic smokers.

Purpose: Small cell lung carcinoma (SCLC) is an aggressive malignancy affecting nearly 30,000 people annually in the United States. We have previously identified elevated PARP1 levels in SCLC and demonstrated in vitro sensitivity to the PARP inhibitors AZD 2281 and AG014699. Here, we evaluate activity of a novel, potent PARP inhibitor, BMN 673, and identify markers of response as a basis for developing predictive markers for clinical application. Experimental Design: Inhibition of SCLC proliferation by BMN 673 was assayed in vitro and effects on tumor growth were measured in SCLC xenograft models. Protein expression and pathway activation was assessed by reverse phase protein array and western blot analysis. PARP inhibition was confirmed using a PAR ELISA. Results: We demonstrate striking, single agent activity of BMN 673 in SCLC cell lines and xenografts, with single agent BMN 673 exhibiting in vivo activity similar to cisplatin. Sensitivity to BMN 673 was associated with elevated baseline expression levels of several DNA repair proteins, whereas greater drug resistance was observed in SCLC models with baseline activation of the PI3K/mTOR pathway. Furthermore, we developed and confirmed these data with a novel “DNA repair score” consisting of a group of 17 DNA repair proteins. Conclusions: Elevated expression of multiple DNA repair proteins, as well as a corresponding “DNA repair protein score,” predict response to BMN 673 in in vitro SCLC models. These observations complement recent work in which PI3K inhibition sensitizes breast cancer models to PARP inhibition, suggesting cooperation between DNA repair and PI3K pathways. Clin Cancer Res; 19(22); 6322–8. ©2013 AACR.

Background: Small cell lung cancer (SCLC), the most lethal form of lung cancer, is characterized by early metastasis and rapid development of drug resistance. SCLC is characterized by ubiquitous loss of TP53 and RB1 and mutually exclusive amplification of the MYC oncogene (CMYC, MYCN, MYCL). The loss of TP53 is thought to render SCLC cells deficient at the G1-S cell cycle checkpoint, thus making these cells completely dependent on the G2-M checkpoint for DNA repair, mainly governed by CHK1 and WEE1. CHK1 is a vital serine-threonine kinase regulating the G2-M checkpoint upon DNA damage. The WEE1 kinase is a regulator of CDK1/2 and inhibition of WEE1 leads to aberrant DNA replication and premature mitosis. In this study we investigated the effect of CHK1 (LY2606368) and WEE1 (AZD1775) inhibitors as therapeutic targets for SCLC. Methods: Protein and gene expression was determined by immunoblot and real-time-RT-PCR respectively. Effect of the drugs on cell viability was determined by CellTiter-Glo. Efficacy of LY2606368 in H69 (chemosensitive) and H69/CR (chemoresistant) models was further determined by in vivo flank tumor regression analysis. The effect of LY2606368 monotherapy was also tested in a spontaneous SCLC genetically engineered mouse model (GEMM) model. The mechanistic studies were conducted by immunoblot analysis, apoptosis assay and flow cytometry. Pre and post-treatment cell lysates were analyzed by reverse phase protein array (RPPA) for biomarker exploration. Result: Higher expression of CHK1 and WEE1 was observed in SCLC patient tumor samples compared to normal lung (p WEE1 inhibitor AZD1775 treatment decreased cell viability (IC50 Significance: The study establishes CHK1 and WEE1 as therapeutic targets in SCLC, a disease for which the standard of care has remained unchanged for >30 years. Together, these results strongly indicate the potency of G2-M cell cycle checkpoint inhibitors as effective targeted therapies in both chemosensitive and chemoresistant patient population. The CHK1 inhibitor, LY2606368, and the WEE1 inhibitor, AZD1775, are both in current Phase 1 clinical trials. The correlative biomarker data from this study will not only indicate the subset of the patient population who are most likely to respond to these treatments, but it will also guide the selection of most effective combination strategies with CHK1 and WEE1 inhibition. This abstract is also being presented as Poster A25. Citation Format: Triparna Sen, Pan Tong, Catherine Allison Stewart, Sandra Cristea, You Hong-Fan, Bonnie S. Glisson, Don L. Gibbons, Julien Sage, Jing Wang, Lauren A. Byers. Exploiting the G2-M cell cycle checkpoint dependency in small cell lung cancer (SCLC) using pharmacological inhibitors of CHK1 and WEE1. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr PR10.

e23222Background: MEK inhibitors have limited success in cancer treatment because tumors circumvent their action. Here, we report broad proteomic analysis of NSCLC cell lines after treatment with t...

Gene expression alterations in response to cigarette smoke have been characterized in normal-appearing bronchial epithelium of healthy smokers, and it has been suggested that adjacent histologically normal tissue displays tumor-associated molecular abnormalities. We sought to delineate the spatial and temporal molecular lung field of injury in smoker patients with early-stage non–small cell lung cancer (NSCLC; n = 19) who were accrued into a surveillance clinical trial for annual follow-up and bronchoscopies within 1 year after definitive surgery. Bronchial brushings and biopsies were obtained from six different sites in the lung at the time of inclusion in the study and at 12, 24, and 36 months after the first time point. Affymetrix Human Gene 1.0 ST arrays were used for whole-transcript expression profiling of airways (n = 391). Microarray analysis identified gene features (n = 1,165) that were nonuniform by site and differentially expressed between airways adjacent to tumors relative to more distant samples as well as those (n = 1,395) that were significantly altered with time up to 3 years. In addition, gene interaction networks mediated by phosphoinositide 3-kinase (PI3K) and extracellular signal–regulated kinase (ERK)1/2 were modulated in adjacent compared with contralateral airways and the latter network with time. Furthermore, phosphorylated AKT and ERK1/2 immunohistochemical expression were significantly increased with time (nuclear pAKT, P = 0.03; cytoplasmic pAKT, P < 0.0001; pERK1/2, P = 0.02) and elevated in adjacent compared with more distant airways (nuclear pAKT, P = 0.04; pERK1/2, P = 0.03). This study highlights spatial and temporal cancer–associated expression alterations in the molecular field of injury of patients with early-stage NSCLCs after definitive surgery that warrant further validation in independent studies. Cancer Prev Res; 6(1); 8–17. ©2012 AACR.

Leukoplakia is the most common premalignant lesion of the oral cavity. Epidermal growth factor receptor (EGFR) abnormalities are associated with oral tumorigenesis and progression. We hypothesized that EGFR expression and gene copy number changes are predictors of the risk of an oral premalignant lesion (OPL) progressing to oral squamous cell carcinoma (OSCC). A formalin-fixed, paraffin-embedded OPL biopsy specimen was collected from each of 162 patients in a randomized controlled clinical trial. We assessed EGFR expression by immunohistochemistry with two methods: a semiquantitative analysis (145 evaluable specimens) and an automated quantitative analysis (127 evaluable specimens). EGFR gene copy number was assessed by fluorescence in situ hybridization (FISH) in a subset of 49 OPLs with high EGFR expression defined by the semiquantitative analysis. We analyzed EGFR abnormalities for associations with OSCC development. High EGFR expression occurred in 103 (71%) of the 145 OPLs and was associated with a nonsignificantly higher risk of OSCC (P = 0.10). Twenty (41%) of 49 OPLs assessed by FISH had an increased EGFR gene copy number (FISH-positive). Patients with FISH-positive lesions had a significantly higher incidence of OSCC than did patients with FISH-negative (a normal copy number) lesions (P = 0.0007). Of note, 10 of 11 OSCCs that developed at the site of the examined OPL were in the FISH-positive group, leaving only one FISH-negative OPL that did so (P < 0.0001). Our data indicate that an increased EGFR gene copy number is common in and associated with OSCC development in patients with OPLs expressing high EGFR, particularly OSCC developing at the site of a high-expression OPL; they also suggest that EGFR inhibitors may prevent oral cancer in patients with OPLs having an increased EGFR gene copy number. Cancer Prev Res; 3(7); 800–9. ©2010 AACR.

Background: Small cell lung cancer (SCLC) is the most lethal form of lung cancer, accounting for around 15% of lung cancers in the United States, currently having no targeted therapies with established efficacy. Using a high-throughput proteomic screen, we demonstrated overexpression of checkpoint kinase 1 (Chk1) in SCLC. Chk1 being the master regulator of the cell cycle in absence of p53, we hypothesize that Chk1 targeting may be effective SCLC because of near ubiquitous loss of p53. In the present study, we tested the effect of Chk1 targeting by shRNA-mediated knockdown and pharmacologic inhibition in an extensive panel of SCLC cell lines and in animal models. Experimental procedures: shRNA-mediated knockdown of Chk1 was done in select human SCLC lines and confirmed at the mRNA and protein level. The effect of Chk1 inhibition by LY2606368 alone and in combination with cisplatin or the PARP1/2 inhibitor talazoparib was tested in 39 SCLC cell lines. Proliferation was assessed by cell titer glo analysis and effect on cell cycle was tested by flow cytometry. Drug sensitivity (IC50) was then correlated with baseline expression level of >190 total or phosphorylated proteins measured by reverse phase protein array (RPPA) to identify potential predictive biomarkers. In vivo effect of the Chk1 inhibitor with or without cisplatin or talazoparib was tested in syngeneic and xenograft model of SCLC. Results: shRNA-mediated targeted Chk1 knockdown significantly decreased the proliferation of SCLC lines confirming Chk1 as a viable target (p90% reduction in volume) which was durable (ongoing response >50 days in 7/9 animals). The combination with cisplatin or talazoparib also resulted in tumor regression and was significantly more effective than either cisplatin or talazoparib alone. Tumor regression following LY2606368 treatment was also observed in cis-resistant H69 xenograft model of SCLC. Conclusion: The study established checkpoint kinase 1 (Chk1) targeting as a novel and active therapeutic strategy for SCLC, a disease for which the standard of care has remained unchanged for >30 years. Chk1 knockdown was sufficient to inhibit proliferation in SCLC cell lines and LY2606368, a Chk1 inhibitor in current clinical trials, showed activity as a single agent and in combination with cisplatin and PARP inhibition in vitro and in vivo. TP53-mutant cells (hallmark of SCLC) rely on Chk1 for cell cycle arrest; hence, inhibition of Chk1 in SCLC likely works by driving cells to mitotic catastrophe and apoptosis. These findings support the further investigation of Chk1 targeting and Chk1 predictive biomarkers in SCLC. Citation Format: Triparna Sen, Pan Tong, C. Allison Stewart, You Hong-Fan, Ying Feng, Jing Wang, Lauren A. Byers. Investigation of Chk1 as a novel therapeutic target for small cell lung cancer (SCLC). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C153.

Introduction The development of more effective treatment strategies for LUAC bearing activating mutations in KRAS is hampered by the biological heterogeneity of KRAS-mutant tumors. The molecular underpinnings that drive this process are poorly characterized. Here, we implemented an integrated approach to the discovery of biologically distinct subsets of KRAS-mutant LUAC and explored their molecular vulnerabilities. Methods Our datasets consisted of 68 KRAS-mutant tumors from TCGA, 88 additional chemo-naive KRAS-mutant LUACs (PROSPECT and Chitale datasets) and 36 platinum-refractory LUACs from the BATTLE-2 clinical trial. Non-negative matrix factorization (NMF) consensus clustering was applied to RNASeq data as previously described. Signature enrichment was assessed using Gene Set Enrichment Analysis (GSEA). Results NMF consensus clustering identified three robust subsets of KRAS-mutant LUAC that were reproducible across diverse clinical datasets of early-stage, chemotherapy-naive and metastatic, chemo-refractory tumors. Distinct KRAS-mutant alleles were not differentially represented in the three subgroups (P = 0.3). In contrast, the subgroups were dominated, respectively, by co-occurring genetic events in STK11/LKB1 (termed the KL subgroup) (P = 1.03e−05), TP53 (KP) (P = 3.8e-06) and low expression of TTF1 coupled with frequent CDKN2A/B inactivation (KC) (P = 0.004 and P = 0.002). Distinct patterns of intracellular signaling were detected in the three subsets. KL tumors showed evidence of LKB1-AMPK pathway inactivation and adaptation to energetic, proteotoxic and oxidative stress, the latter exemplified by near ubiquitous inactivation of KEAP1 and up-regulation of a NRF2-driven antioxidant signature. KP tumors carried a higher somatic mutation load and were characterized by prominent inflammation and up-regulation of several immune checkpoint effector molecules, including PD-L1. KC tumors frequently displayed a GI-like differentiation program, suppression of MTORC1 signaling and elevated wild-type p53 transcriptional output. Using a large panel of KRAS-mutant NSCLC cell lines we detected co-mutation-dependent patterns of drug sensitivity. Specifically, KL cell lines showed enhanced sensitivity to several structurally distinct HSP90 inhibitors. These results were confirmed in panels of isogenic cell lines. Mechanistically, treatment with ganetespib resulted in concurrent degradation of several molecules with established role in supporting the fitness of LKB1-deficient cells. Conclusions Our work identifies three major subsets of KRAS-mutant LUAC - dominated by co-occurring genetic events - with distinct biology and therapeutic vulnerabilities. Citation Format: Ferdinandos Skoulidis, Lauren Byers, Lixia Diao, Vassiliki Papadimitrakopoulou, Pan Tong, Julie Izzo, Carmen Behrens, Humam Kadara, Edwin R. Parra, Jaime Rodriguez-Canales, Jianjun Zhang, Uma Giri, Jayanthi Gudikote, Maria Angelica Cortez, Chao Yang, You Hong Fan, Michael Peyton, Luc Girard, Kevin R. Coombes, Carlo Toniatti, Timothy P. Heffernan, Murim Choi, Garrett M. Frampton, Vincent Miller, John N. Weinstein, Roy S. Herbst, Kwok-Kin Wong, Jianhua Zhang, Padmanee Sharma, Gordon M. Mills, Waun Ki Hong, John D. Minna, James P. Allison, Andrew Futreal, Jing Wang, Ignacio Wistuba, John V. Heymach. Co-occurring genomic alterations define major subsets of KRAS-mutant lung adenocarcinoma (LUAC) with distinct biology and therapeutic vulnerabilities. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 968. doi:10.1158/1538-7445.AM2015-968

Background- Salivary gland cancer (SGC) is a highly heterogenous disease with distinct histological and pathological features. Although subtypes of salivary gland cancer have been extensively characterised based on histological features, very little is known at the proteomic level, as to how and what key proteins and signaling pathways are differentially activated. Currently, there are no approved targeted therapies for SGCs, although there appears to be benefit of androgen receptor (AR) and Her2 blockade in a small subset of molecularly defined patients ( Method- Reverse phase protein array (RPPA) analysis was performed to measure 195 total and/or phosphorylated proteins in matched normal and tumor samples from 75 SGC patients, including 16 salivary duct carcinomas (SDC); 14 mucoepidermoid carcinomas (MEC); 30 acinic cell carcinomas (ACC) and 15 adenocystic carcinomas (ADCC). Differences in protein expression between normal and tumor samples was assessed by paired t test. Results- RPPA analysis showed distinct protein expression and pathway activation between and within subtypes. Using a conservative cutoff to account for multiple testing (FDR increased dependency on glycolysis. Increased expression of other potentially druggable targets such as p-MEK, Akt, vascular endothelial growth factor (VEGFR), and AR were also observed. Consistent with other reports, AR was found at higher levels in tumors (vs. normal tissue) in 62.5% of SDC tumors (fold change = 2.33, p>0.001). Interestingly, AR overexpressing tumors also showed increased activation of EGFR (pY1173) compared to those with low AR expression. ACC tumor tissue expressed higher levels of proteins involved in cell cycle, DNA repair and metabolism. ADCC had higher expression of oncoproteins (c-kit and bcl-2); as well as IGF-binding protein 2 (IGFBP2) and its downstream effector proteins. MEC showed increased expression of EGFR. Conclusion- This study provides an insight into the molecular heterogeneity and enrichment of distinct signaling pathways in the different SGC subtypes. The observed changes in protein expression of EGFR, IGFR and PIK3CA could be driven by genetic aberrations, indeed copy number variations and mutations in several genes including EGFR, IGFR and PIK3CA have been observed in SDC. This study has identified proteins and signaling pathways that could potentially be targeted for the treatment of salivary gland cancer patients. Citation Format: Seema Mukherjee, Yoshitsugu Mitani, Robert Cardnell, You Hong Fan, Lixia Diao, Jing Wang, Adel K. El-Naggar, Lauren Averette Byers. Comprehensive proteomic analysis of salivary gland cancer subtypes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-111. doi:10.1158/1538-7445.AM2015-LB-111

Background: Head and neck squamous cell carcinoma (HNSCC) is the fifth most common cancer. In 2013, there were ∼53,000 newly diagnosed cases and ∼11,000 deaths related to HNSCC in the USA. Overexpression of EGFR is seen in 90% HNSCC; but, only ∼10% of patients treated with the anti-EGFR antibody cetuximab show increased response rates to cetuximab and these eventually gain resistance by poorly-characterized mechanisms. We showed an association between EMT and resistance to EGFR inhibitors in lung cancers (LC) and HNSCC using a 76-gene EMT signature. AXL was identified as a therapeutic candidate linking EMT and drug resistance, showing significantly higher expression in erlotinib resistant cell lines. Other groups have linked AXL to drug resistance in HNSCC, LC and breast cancers. Here we identify signaling pathways that are regulated by AXL, mediate drug resistance, and identify potential therapeutic targets to combine with AXL inhibition. Methods: Using 6 clinical cohorts including The Cancer Genome Atlas (TCGA N = 493) and PROSPECT (N = 142) across 3 cancer types, we identified genes whose mRNA expression was highly correlated with AXL. Protein profiling by reverse phase protein array (RPPA) to analyze total and phospho-proteins in HNSCC cell lines, pre- and post-AXL inhibitor treatment was used to identify pathways altered upon AXL inhibition. The response to AXL inhibition was assayed in HNSCC cell lines by proliferation assays and correlated to mRNA and protein expression. Results: Using gene-expression and RPPA analysis we saw the highest association of AXL with pathways involved in EMT (TGF-β, Rho GTPases), autophagy and immune response. Following treatment with an AXL inhibitor, we observed a decrease in phospho-proteins in the PI3K-AKT pathway, increased expression of markers associated with apoptosis, an epithelial phenotype, and p-EGFR. Using an AXL knockdown model system in HNSCC cell lines, we validated an increase in EGFR signaling (EGFR and p-Erk), epithelial (E-cadherin), apoptotic (cleaved PARP and caspase-7) and DNA repair proteins (RAD51, ku-80 and PARP) and a decrease in Slug, Twist and ZEB-1, indicating that AXL may be directly involved in mediating EMT. AXL knockdown reduced proliferation of HNSCC cell lines and AXL inhibition was able to re-sensitize resistant HNSCC cell lines to erlotinib, an EGFR tyrosine kinase inhibitor. Conclusions: In summary, we identified potential therapeutic targets that are upregulated with AXL expression in HNSCC and LC patient tumors and cell lines. Using AXL inhibitor and knockdown in HNSCC cell lines, we validated biomarkers involved in EMT, EGFR signaling and apoptosis that are altered upon AXL inhibition. AXL inhibition led to an epithelial phenotype in cells and re-sensitized resistant cells to erlotinib. Studies are ongoing to validate the mechanisms of AXL-mediated drug resistance and to identify potential combination treatments that can synergize with AXL-inhibition. Citation Format: Kavitha Balaji, Robert Cardnell, Lixia Diao, Pan Tong, Milena Mak, You Hong Fan, Fatemeh Masrorpour, Steven L. Warner, David J. Bearss, Ignacio Wistuba, Gordon B. Mills, John Heymach, Khandan Keyomarsi, Jing Wang, Lauren Averett Byers. Identification of biomarkers of AXL-mediated drug resistance in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3579. doi:10.1158/1538-7445.AM2015-3579

11002 Background: Little is currently known about how the oncogenotype of LUAC shapes its immune micro-environment. We recently identified that major subsets of KRAS-mutant LUAC defined on the basi...

3030 Background: Promising results in the treatment of NSCLC have been seen with agents targeting immune checkpoints, such as programmed cell death 1 (PD-1) or programmed cell death 1 ligand (PD-L1...

Introduction The molecular underpinnings that beget the considerable biological and clinical heterogeneity of lung adenocarcinoma (AC) driven by oncogenic KRAS are poorly characterized. We hypothesized that significantly co-occurring mutations in components of key cellular tumor suppressor pathways are major determinants of signalling diversification downstream of mutant KRAS. Methods We applied un-supervised hierarchical analysis and classical multi-dimensional scaling to determine patterns of somatic (co)-mutations within a curated set of significantly mutated genes (MutSigCV p value Results Clustering of KRAS-driven lung ACs based on patterns of somatic mutations revealed six major mutation clades. Five clades could be defined by combinations of somatic mutations in only four genes : KRAS, TP53, STK11/LKB1 and KEAP1, whereas a sixth clade appeared more heterogenous and was associated with a higher overall rate of somatic mutation. Co-mutations in STK11/LKB1 and TP53 accounted for higher branches of the cluster dendrogram and were largely non-overlapping in the context of early-stage disease. Integration of co-mutation analysis with unsupervised hierarchical analysis of RNA Seq differential expression data identified 3 major expression clusters, that were significantly enriched for the KRAS;TP53 (KP), KRAS;LKB1 (KL) and KRAS only (K) somatic mutation clades, as well as sub-clusters characterized by the presence of mutations in KEAP1. On the contrary, the distribution of the main KRAS amino acid substitutions (G12C, G12V and G12D) did not differ significantly between the expression clusters, indicating that co-mutations rather than distinct KRAS codon 12 base substitutions are the major driver of biological heterogeneity within KRAS-mutant lung ACs. Re-analysis of publicly available large scale drug sensitivity data based on the proposed sub-classification revealed novel genotype-specific therapeutic vulnerabilities for the KL and KP subgroups including selective sensitivity of KL AC cell lines to MK-2206 ( a pan-AKT inhibitor), BX-795 (a PDK1 and TBK1/IKKϵ inhibitor) and the type II topoisomerase inhibitor etoposide. Selected hits were subsequently validated using pairs of isogenic cell lines. Conclusions Co-mutations in a small number of key tumor suppressor genes define biologically distinct and therapeutically relevant subgroups of KRAS-driven lung AC. Efforts to target KRAS signalling in the context of specific co-mutations may provide a more rational and personalized approach to therapy for this challenging group of lung cancer patients. Citation Format: Ferdinandos Skoulidis, Kevin R. Coombes, Lixia Diao, Pan Tong, Maria A. Cortez, Uma Giri, Chao Yang, You Hong Fan, John N. Weinstein, Vassiliki Papadimitrakopoulou, John D. Minna, Jing Wang, Lauren A. Byers, John V. Heymach. Co-mutations define major subsets of KRAS-driven lung adenocarcinoma with implications for targeted cancer therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1541. doi:10.1158/1538-7445.AM2014-1541

6030 Background: The incidence of oropharyngeal cancer (OPC) has increased 5% per year over the past decade due to a rise in human papilloma virus (HPV)-driven OPC. HPV+ OPC is clinically and molecularly distinct from HPV- head and neck squamous cell carcinomas (HNSCC). However, there are currently no validated therapeutic targets for HPV+ HNSCC. Methods: Reverse phase protein array (RPPA) was performed on HNSCC tumors from The Cancer Genome Atlas to measure cancer-associated pathways and targets. Differences in individual markers and proteomic pathway scores between HPV+ and HPV– tumors were assessed by t-test in the overall group and for the subset of OPC. Results: Significant proteomic differences were observed in 14 HPV+ and 186 HPV- HNSCC. Of 160 proteins, the top marker overexpressed in HPV+ HNSCC was p16, an established clinical biomarker of HPV status (p

8104 Background: All NSCLC patients treated with erlotinib (E) eventually develop resistance. Both the epithelial-mesenchymal transition (EMT) and cancer stem cells may contribute to resistance to E. The Notch pathway plays a key role in maintaining stem cell viability and in EMT. Gamma secretase inhibitors such as RO4929097 (R) inhibit Notch activity by preventing cleavage of Notch to its active form. In this CTEP-sponsored study, primary endpoint of phase I was to determine maximum tolerated dose (MTD) of the combination of E and R; primary endpoint of phase II was response rate. Methods: Eligible pts had incurable NSCLC, measurable disease, ECOG PS 0-1, and adequate laboratory parameters. There were no limits on prior therapy. All pts were required to have a tumor biopsy. Patients received E PO daily on a continuous schedule and R PO daily on days 1-3, 8-10, and 14-17 of a 21 day cycle. Dose cohorts (E/R both in mg) were as follows: 100 /20, 150/20, 150/30, 150/45. Imaging was performed every 6 weeks. Patients could remain on therapy until progression. Results: 21 pts were enrolled between 9/2010 and 3/2012. 5 were not eligible and did not receive treatment. For the 16 treated pts: median age was 61 yrs (40-75), 7 (44%) were male, 14 (88%) had adenocarcinoma. All pts had received prior chemotherapy (1-8 regimens); 7 (44%) had prior E. 3 pts had known EGFR mutations; 2 pts had KRAS mutations. Time on therapy ranged from 20 to 248 days. Median PFS was 42 days. Pts with prior progression on E had a median PFS of 64 days. 1 pt (6%) had a partial response, 4 (19%) had stable disease at 6 weeks. Median overall survival is 6.5 months (1.3 to 24.5+ months). Observed dose-limiting toxicity was hypophosphatemia in 1 pt in 150/45 cohort. Other toxicities included rash, neuropathic pain and nausea. MTD was determined to be 150/45. The study was halted early, after completion of the phase I cohort, as production of R was discontinued by the manufacturer. 12 pts (75%) have adequate tumor tissue for analyses. Biomarker studies are pending. Conclusions: Combination of R and E is safe and feasible in pts with NSCLC. Though development of R has been discontinued, other drugs targeting the Notch pathway are in development (NCI R21CA153017). Clinical trial information: NCT01193881.

6011 Background: Epithelial-mesenchymal transition (EMT) has been associated with EGFR inhibitor resistance in preclinical studies of head and neck squamous cell carcinoma (HNSCC). Recently, we developed an EMT signature that predicts EGFR inhibitor resistance in lung cancer. Using this signature, we explored the association between EMT and drug response in HNSCC, focusing on the tyrosine kinase Axl as a potential therapeutic target. Methods: We conducted an integrated molecular and drug response analysis in HNSCC. A 76-gene EMT signature previously developed and validated in lung cancer was tested in HNSCC cell lines (n>50) and patient tumors from The Cancer Genome Atlas (TCGA) (n=113) and a MDACC cohort (n=105). Reverse phase protein array (RPPA) and proliferation assays were used to measure protein expression and sensitivity to erlotinib and the Axl inhibitors SGI-7079 and TP-0930. Results: The EMT signature identified distinct epithelial and mesenchymal subsets of HNSCC among cell lines and patient tumors. RPPA experiments revealed higher protein levels of the receptor tyrosine kinase Axl, vimentin, and N-cadherin and lower expression of E-cadherin and beta-catenin in mesenchymal HNSCC (p-values 10μM); however, we discovered that mesenchymal HNSCC were highly sensitive to two Axl inhibitors, SGI-7079 and TP-0930 (IC50s ≤1.2μM and 0.2uM, respectively). Conclusions: Our EMT gene expression signature identified discrete epithelial and mesenchymal subgroups of HNSCC. Mesenchymal HNSCC cells expressed higher levels of Axl protein and exhibited sensitivity to Axl inhibition, but resistance to erlotinib. These results highlight differences in drug response between epithelial and mesenchymal cancers and support Axl as a potential therapeutic target and predictive marker of EGFR inhibitor resistance in HNSCC. (Funded in part by 5 P50 CA097007-10)

Background: LKB1 is a tumor suppressor gene that is lost in ∼35% of non-small cell lung cancers (NSCLC), half of which carry concurrent KRAS mutations. When functioning normally, LKB1 plays a critical role in energy sensing and regulates the PI3K/mTOR pathway. We have previously shown that LKB1 loss is associated with greater resistance to certain targeted drugs. Here, we investigate signaling pathways dysregulated in the setting of LKB1 loss, with a particular focus on potential novel therapeutic targets. Methods: Expression of >140 total and phospho-proteins were measured in 109 NSCLC cell lines by reverse phase protein array (RPPA). Differences in protein expression between LKB1 deficient versus intact cell lines were determined by t-test, with correction for multiple testing. IC50s for targeted therapies were determined by MTS assay in cell lines with and without LKB1. For the drug combination assay, nine NSCLC cell lines with and without LKB1 were treated with the IGFR inhibitor OSI-906 plus serial dilutions of the dual mTOR inhibitor OSI-027. Proliferation was measured after 72 hours. Bliss algorithm was used to calculate synergy and fractional inhibition for each plate independently. Further validation was performed using NSCLC cells stably overexpressing LKB1. Results: Loss of LKB1 via mutation or deletion was strongly correlated with LKB1 protein expression (p Conclusions: These studies identify new potential targets in LKB1 deficient NSCLC, which represents a significant portion of lung cancer patients. Specifically, the combination of IGFR and mTOR inhibition demonstrated synergy, supporting further investigation as a therapeutic approach for this molecularly defined subset of NSCLC patients. Citation Format: Lauren Averett Byers, Maria Angelica Cortez, Chao Yang, Jing Wang, Lixia Diao, You Hong Fan, Luc Girard, Adi Gazdar, Ignacio Wistuba, John D. Minna, Matthew O'Connor, Sharon Barr, John V. Heymach. Proteomic profiling of signaling pathways in LKB1 deficient non-small cell lung cancers (NSCLC) identifies novel therapeutic targets including IGF1R pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2403. doi:10.1158/1538-7445.AM2013-2403

Background: Small cell lung cancer (SCLC) is an aggressive malignancy that accounts for 13% of lung cancers in the US and has a 5-year survival rate Methods: Sensitivity of SCLC lines to BMN673 and cisplatin was determined by CellTiter-Glo cell viability assay. Total and phospho-protein expression of 200 markers was measured at baseline and following drug treatment in SCLC cell lines by reverse phase protein array (RPPA). Correlations between baseline protein expression and drug sensitivity were determined by Spearman rank correlation. Modulation of protein expression post treatment was assessed by ANOVA. Results: The novel PARP inhibitor BMN673 had potent in vitro activity in a panel of 12 SCLC cell lines with IC50’s ranging from 1.7-15nM. A higher expression level of several DNA repair proteins was associated with greater BMN673 sensitivity. For example, protein expression levels of ERCC1, DNA PKcs, ATM, FANCD2, and pChk2 were inversely correlated with IC50 values (Rho values -0.93 to -0.81; p≤0.02). In contrast, high baseline expression of phosphorylated Akt (T308) (R=0.91, p=0.005) and pAkt (S473) (R=0.81, p=0.02) were associated with decreased sensitivity to BMN673. We also observed increased phosphorylation of mTOR, Akt, and S6 (p Conclusions: Here we demonstrate significant, single-agent in vitro activity of the PARP inhibitor BMN673 in SCLC and potential predictive markers of response. Interestingly, for the first time we show an inverse correlation between PARP inhibitor sensitivity and PI3K/Akt/mTOR pathway activation. This observation parallels recent data suggesting that PI3K inhibition may sensitize breast cancer to PARP inhibition and suggests a potential coordinated regulation between DNA repair and PI3K pathways. The activity of BMN673 and these candidate response biomarkers will be further investigated in a Phase I cohort expansion of BMN673 in patients with SCLC. Citation Format: Robert J. Cardnell, Ying Feng, Lixia Diao, You Hong Fan, Jing Wang, Yuqiao Shin, John D. Minna, John V. Heymach, Lauren A. Byers. Proteomic profiling identifies PI3K and DNA repair pathways as potential markers of response to PARP inhibitor BMN673 in SCLC. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2489. doi:10.1158/1538-7445.AM2013-2489

The phosphoinositide 3-kinase (PI3K) signaling pathway is the most commonly dysregulated pathway in many human cancers, including non-small cell lung cancer (NSCLC). Therefore, targeting PI3K signaling has become a promising approach in cancer therapy. Although studies have indicated development of resistance to PI3K inhibitors, the molecular mechanism of drug resistance is largely unknown in NSCLC. Here, we sought to identify new potential biomarkers and pathways that confer resistance to PI3K inhibitors in NSCLC. To this end, we used reverse-phase protein array (RPPA) to assess the expression levels and activation status of 137 proteins involved in signaling pathways implicated in lung cancer. Seventy-four NSCLC cell lines were treated with the PI3K inhibitor BAY 80-6946 and lysates were collected for proteomic analysis. First, we evaluted the association between drug sensitivity (IC50) and RPPA expression levels at baseline in sixty NSCLC cell lines. Correlation test was applied to each marker vs drug and Wilcox Rank test was performed to compare the smallest and largest one third of all the cell lines. We found increased phosphorylation and inactivation of pro-apoptotic protein Bad and phospho-LKB1 in the resistant cell lines. An inverse correlation was found for phosphorylation and inactivation of c-Src. Interestingly, activation of EGFR and increased levels of PTCH and PKCα were found in the resistant cell lines. Next, paired t-test was applied to each marker comparing the difference between drug and control treated cells. As expected, treatment with BAY 80-6946, significantly downregulates components of PI3K/mTOR pathway, as illustrated by decreased levels of phospho-Akt (-4.28-fold relative expression) and phospho-S6 ribosomal protein (-9.64-fold), phospho-p70S6K (-3.47-fold) and phospho-mTOR (-1.42-fold). We observed elevated expression of negative regulators of mTOR pathway, LKB1 (1.11-fold), AMPKα (1.12-fold) and phospho-AMPKα (12-fold). On the other hand, our analysis identified new potential pathways of resistance that are activated upon PI3K inhibition. We found increased levels of phospho-EGFR (P Citation Format: Maria A. Cortez, Lauren Averett Byers, You Hong Fan, Lixia Diao, Philip Groth, Julianne Paul, Jing Wang, Uma Giri, Jayanthi Gudikote, Hai Tran, Kevin Coombes, John D. Minna, Ningshu Liu, John V. Heymach. Proteomic analysis reveals Src/Stat and EGFR/MAPK pathways as potential mechanism of resistance to PI3K inhibitors in lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2498. doi:10.1158/1538-7445.AM2013-2498

Background: MEK inhibitors such as BAY86-9766 are a new class of agents that show promise in the treatment of non-small cell lung cancer (NSCLC). The downstream effects of MEK inhibition in NSCLC have not been fully elucidated. We performed a broad proteomic analysis to determine which signaling pathways were modulated by BAY86-9766 treatment and how these pathways correlate with sensitivity. Methods: We treated 109 lung cancer cell lines with MEK inhibitor BAY86-9766 at a concentration of 2200 nM. Drug sensitivity was determined by CellTiter-Glo assay and cell lines were classified as sensitive or resistant based on whether their IC50 values were in the highest or lowest 1/3rd of those tested. Using paired t-tests, we compared pre- versus post-treatment protein levels in the overall group and between the sensitive vs. resistant cell lines. Results: MEK inhibitor BAY86-9766 was effective in reducing phosphorylation of direct downstream signaling molecules pMAPK (p Conclusions: We have performed broad proteomic analysis on cell lines treated with MEK inhibitor BAY86-9766 to determine which pathways are modulated by treatment and how they might relate to sensitivity. We conclude that MEK inhibition with BAY86-9766 may exert some of its effects by suppressing mTOR activity via the LKB1/AMPK pathway, and that the degree of modulation of the AMPK pathway correlates with sensitivity. This mechanism may involve decreased activation of p90RSK by MAPK, which leads to decreased degradation and increased activity of LKB1, and thereby increased AMPK activity and decreased mTOR signaling. This works suggests a rational basis for combinations of targeted agents to overcome resistance, such as combinations of MEK inhibitors with mTOR inhibitors or PI3 kinase inhibitors. Citation Format: Kathryn A. Gold, Lauren A. Byers, You Hong Fan, Lixia Diao, Philip Groth, Julianne Paul, Jing Wang, Uma Giri, Jayanthi Gudikote, Hai T. Tran, Kevin R. Coombes, John D. Minna, Ningshu Liu, John V. Heymach. Proteomic analysis of effects of MEK inhibition with BAY86-9766 on LKB1/AMPK and mTOR pathway in lung cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1045. doi:10.1158/1538-7445.AM2013-1045

10612 Background: LKB1 is a serine/threonine kinase which is mutated in 20-30% of non-small cell lung cancers (NSCLC) and functions as a tumor suppressor by activating AMPK. Loss of LKB1 by point mutation or deletion suppresses AMPK, leading to increased mTOR signaling. We investigated the signaling pathways modulated by LKB1 mutations and by mTOR inhibition in NSCLC. Methods: Protein expression in cell lines was measured by reverse phase protein array. Differences in protein expression at baseline in LKB1 wild-type versus mutant cell lines and the effects of protein modulation by treatment were assessed by ANOVA. Results: LKB1 mutant cell lines had lower expression of phosphorylated AMPK and TSC (p

5524 Background: We have reported that DNA methyltransferase-3B (DNMT3B) gene expression is a strong predictor of OC dvlpt. Using high-throughput DNA methylation profiles of oral preneoplastic lesions (OPL), we identified 86 candidate genes that were hypermethylated in patients (pts) developing OC and suggested that a CpG island methylation phenotype may occur early during OC dvlpt. Our aim was to validate some of the candidate genes and to study the value of LINE1 repetitive element methylation, a surrogate of global DNA methylation, as biomarkers of risk to develop OC. Methods: Validation cohort included 40 pts with a median follow-up of 4.2 years, including 14 pts who developed OC. None of them were used in the discovery phase. Frozen OPL were collected prospectively and subject to DNA extraction. We performed a quantitative analysis of the degree of methylation of LINE1, and AGTR1, FOXI2, PENK, and HOXA9 promoters CpG sites using pyrosequencing. Results: The degree of methylation of AGTR1 (Mann-Whitney P