Search

Background: Irreversible allosteric KRAS p.G12C inhibitors (KG12Ci) such as sotorasib and adagrasib have revolutionized the therapeutic landscape of advanced KG12C-mutant NSCLC, however individual responses are heterogeneous and curtailed by innate and adaptive/acquired resistance. Molecular determinants of KG12Ci efficacy in NSCLC are poorly defined. We dissected the impact of major KG12C co-mutations and explored the effects of less prevalent co-alterations on the clinical activity of KG12Ci in the largest treated cohort to date of patients (pts) with advanced NSCLC. Key findings were validated in preclinical KG12C NSCLC models. Methods: Baseline clinico-genomic features and clinical outcome data from pts with stage IV KG12C NSCLC (ECOG PS 0-2) treated with single-agent KG12Ci were collected retrospectively from 20 centers in the US and Europe. The Kaplan-Meier method was used to estimate PFS and OS and differences were assessed with the log-rank test. Hazard ratios (HR) and their 95% CI were estimated using a Cox proportional hazards model stratified for clinical co-variates. The impact of selected co-alterations on sotorasib efficacy was assessed in syngeneic (C57BL/6) KG12C NSCLC models. Results: 411 eligible pts were included in the study. Median age was 68 years, 77% of pts had received both platinum-based chemotherapy and PD-(L)1 inhibitors and 35% had brain metastases. 83% of pts received sotorasib. ORR with KG12Ci was 32.4% (95% CI, 27.9-37.1), PFS was 5.1m (95% CI, 4.5-5.6) and OS was 10.2m (95% CI, 8.4-12.1). Co-alterations in KEAP1, SMARCA4 and CDKN2A/B were each associated with significantly shorter PFS (KEAP1: 2.8m vs 5.5m, HR 2.50, P Conclusions: Co-mutations in KEAP1, SMARCA4 and CDKN2A/2B define subgroups of KG12C NSCLC pts with markedly distinct outcomes with KG12Ci monotherapy. Tailoring of KG12C inhibitor-anchored therapeutic strategies and patient stratification should take into account the co-mutation status of individual tumors. Citation Format: Marcelo V. Negrao, Haniel A. Araujo, Giuseppe Lamberti, Alissa J. Cooper, Teng Zhou, Neal Akhave, Lukas Delasos, J Kevin Hicks, Mihaela Aldea, Gabriele Minuti, Jacobi Hines, Jacqueline V. Aredo, Michael J. Dennis, Turja Chakrabarti, Susan Scott, Paolo Bironzo, Matthias Scheffler, Petros Christopoulos, So Yeon Kim, Sarah Goldberg, Ying Ni, Blerina Resuli, Lorenza Landi, Shu-Chi Tseng, Mizuki Nishino, Dwight Owen, Collin Blakely, Giannis Mountzios, Catherine A. Shu, Christine Bestvina, Marina Garassino, Kristen Marrone, Jhanelle Gray, Sandip Pravin Patel, Amy L. Cummings, Heather A. Wakelee, Jurgen Wolf, Giorgio V. Scagliotti, Federico Cappuzzo, Fabrice Barlesi, Pradnya Patil, Don L. Gibbons, Funda Meric-Bernstam, J Jack Lee, John V. Heymach, David S. Hong, Rebecca S. Heist, Mark M. Awad, Ferdinandos Skoulidis. Molecular determinants of KRAS p.G12C inhibitor efficacy in advanced NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3431.

Neuroendocrine carcinomas (NECs) are clinically aggressive carcinomas commonly arising from the respiratory and gastrointestinal tracts, typically categorized as large-cell neuroendocrine carcinomas (LCNECs) or small cell carcinomas (most commonly small cell lung cancer (SCLC)). Clinically, pulmonary LCNECs (pLCNECs) mirror the course common to SCLC - initial response followed by rapid and insurmountable resistance to one-size-fits-all approaches. Recently, SCLC has been subdivided into four subtypes with unique vulnerabilities, three of which are defined by the transcription factors ASCL1, NEUROD1, and POU2F3, while a fourth group exhibits an inflamed signature. We hypothesize that pLCNEC may be similarly classified into molecularly distinct subsets with unique therapeutic vulnerabilities - a fundamental step toward personalized medicine. We applied our SCLC 1300 gene signature to pLCNEC patient tumors and, as in SCLC, found three distinct subtypes determined by differential expression of ASCL1, NEUROD1, and POU2F3, but with a unique fourth subtype marked by expression of the transcription factor YAP1. Unlike in treatment-naïve SCLC, where YAP1 is absent, YAP1 expression clearly defines pLCNEC as two, roughly equal subsets with the YAP1-low tumors encompassing tumors expressing the other three transcription factors. Conversely, YAP1-high pLCNEC is more mesenchymal and inflamed, and less neuroendocrine (NE), reminiscent of inflamed SCLC. Additionally, YAP1-high status is associated with smoking exposure (P Citation Format: C. Allison Stewart, Lixia Diao, Yuanxin Xi, Runsheng Wang, Kavya Ramkumar, B. Leticia Rodriguez, Benjamin B. Morris, Li Shen, Bingnan Zhang, Yan Yang, Azusa Tanimoto, Veronica Y. Novegil, Luisa M. Solis Soto, Pedro F. Simoes da Rocha, Natalie Vokes, Don L. Gibbons, Michael Frumovitz, Junya Fujimoto, Jing Wang, Bonnie Glisson, Lauren A. Byers, Carl M. Gay. YAP1 in relapsed pulmonary high-grade neuroendocrine carcinomas (NEC) is associated with CDKN2A loss, intact RB1, EMT and therapeutic vulnerability to MEK1 and CDK4/6 inhibition. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4525.

Background: Compared to other well-studied biomarkers, the relationship between overall tumor burden and immune-checkpoint inhibitor (ICI) efficacy is poorly understood. Methods: We identified patients with stage IV NSCLC without EGFR, ALK or ROS1 alterations treated on the LONESTAR protocol with nivolumab + ipilimumab with formal 3-month radiographic response evaluation. Response was annotated using RECIST 1.1. Manual segmentation of primary and measurable metastatic lesions on pre-treatment CT scans was performed, and tumor volume was calculated from these measurements. Tumor burden was defined as the involvement status, volume, or lesion counts within specific organs (lungs, pleura, lymph nodes, liver, adrenals, bone, and soft tissue) and across whole body. We explored the impact of baseline tumor burden on ICI response and its association with other clinical features. Results:152 patients were included; patients with progressive disease (PD; n=50) at 3 months were compared to all others (n=102). Overall disease volume correlated to tumor volume at the primary lesion (spearman rho=0.69), lymph node (rho=0.45), soft tissue (rho=0.23), lung metastases (rho=0.22), and pleura (rho=0.20); overall lesion counts correlated to lesion counts in lymph nodes (rho=0.61), lung metastases (rho=0.45), and bone (rho=0.16). On univariate analysis, overall tumor volume (P=2e-53) and lesion counts (P=2e-51) had the strongest association with PD, outperforming PD-L1 (P=0.02), ECOG performance status (PS, P=7e-16), and clinical variables including prior treatment and smoking status. PD correlated with higher disease burden in all individual organs except bone; adrenal metastases inversely correlated with PD. Volumetric features complemented PD-L1 and other clinical risk factors to predict PD; in a composite model (ClinVol) AUC=0.82, sensitivity=66%, specificity=93%, while PD-L1 alone yielded an AUC=0.62, sensitivity=94%, specificity=28%. Similar trends were observed for subgroup analyses stratified by brain metastasis status or smoking history, in which volumetric measures increased the power of PD-L1 to predict PD by a consistent AUC increase of ~0.2. Upon subgroup analysis stratified by PD-L1, volumetric models showed robust stratification of PD, with AUC of 0.97, 0.81, 0.81 respectively for PD-L1 Conclusion: Higher baseline tumor burden was associated with increased likelihood of disease progression on combination ICI therapy. ClinVol modeling improves predictive accuracy of 3-month progression. Citation Format: Muhammad Aminu, Natalie I. Vokes, Maliazurina B. Saad, Hui Li, Lingzhi Hong, Mohamed S. Mohamed, John Boom, Pingjun Chen, Mehmet Altan, Saumil Gandhi, Stephen Swisher, Mara B. Antonoff, Jenny V. Pozadzides, George Blumenschein Jr, Don L. Gibbons, Tina Cascone, Yasir Y. Elamin, Xiuning Le, Marcelo V. Negrao, Ferdinandos Skoulidis, Anne S. Tsao, Janet Tu, J.Jack Lee, Jianjun Zhang, John V. Heymach, Jia Wu. Tumor volumetric analysis to correlate disease burden with response to dual immune checkpoint blockade in metastatic NSCLC. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4393.

Background: Immune checkpoint inhibitors (ICIs) as monotherapy (ICI-mono) or with chemotherapy (ICI-chemo) are standard first-line treatment in NSCLC patients lacking targetable driver mutations. Biomarkers to identify patients at risk for early progression on ICI-mono or those who would maximally benefit from upfront ICI-chemo have not been defined. Methods: We queried the GEMINI database to identify metastatic NSCLC patients without targetable EGFR/ALK alterations who were treated with ICI-mono or ICI-chemo. Mutational profiling was performed on tissue or blood using targeted NGS. Outcome measures were defined as clinical progression free survival (PFS) or early progressive disease (PD) rate (defined as rate of 3-month progression), and their association with variables was assessed via Cox Proportional Hazards regression (PFS) or logistic regression (early PD). Predictive deep learning models were used to integrate clinicopathological factors and genomic profile. Results: 735 patients were included in this study, 269 treated with ICI-chemo and 466 with ICI-mono; 446 were treated in the first-line setting. TP53 was the most frequently altered gene (60%), followed by KRAS (37%), AR (21%), and STK11 (19%). In ICI-mono patients, alterations in STK11, ERBB2, ARID1A and CDK6 were associated with a higher likelihood of early PD; only STK11 was associated with early PD (29% vs 17%, P = 0.04) on ICI-chemo. In all patients, low PD-L1 expression and high disease burden (stage IVb and liver metastases) associated with early PD, but there were borderline significant treatment effects in favor of ICI-chemo in never smokers and patients with liver metastases and stage IVb. Shorter PFS was observed in the ICI-chemo group who had CDKN2A alterations vs wild type (median PFS: 5.1 vs 9.0 months; HR: 1.72; P = 0.01). A subgroup analysis of patients with CDKN2A alterations demonstrated preferentially worse outcomes in ICI-chemo compared to ICI-mono, with the best PFS achieved in the ICI-mono treated patients with CDKN2A point mutation. Integration of clinicogenomic features into a multivariate model with feature selection to predict early PD demonstrated a predictive performance of AUC 0.73 (vs PD-L1 alone, AUC 0.60) in the ICI-mono group, driven by liver metastases, stage IVb disease, PD-L1 expression, and STK11 alterations. These features were less predictive in ICI-chemo-treated patients, indicating a protective effect against early PD in these patients from combination chemoimmunotherapy. Conclusions: Low PD-L1, high disease burden, and STK11 alterations are markers of early PD on ICI-mono, and patients with these features may particularly benefit from upfront combination treatment with ICI-chemo to protect against early progression. Citation Format: Lingzhi Hong, Muhammad Aminu, Xuetao Lu, Maliazurina B. Saad, Pingjun Chen, Waree Rinsurongkawong, Amy Spelman, Yasir Y. Elamin, Marcelo V. Negrao, Ferdinandos Skoulidis, Carl M. Gay, Tina Cascone, Mara B. Antonoff, Boris Sepesi, Jeff Lewis, Don L. Gibbons, Ara A. Vaporciyan, Xiuning Le, J.Jack Lee, Sinchita Roy-Chowdhuri, Mark J. Routbort, John V. Heymach, Jia Wu, Jianjun Zhang, Natalie I. Vokes. Genomic and clinical predictors of early disease progression and chemoimmunotherapy benefit in advanced NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 964.

Introduction: Atypical adenomatous hyperplasia (AAH) is the only recognized preneoplasia of lung adenocarcinoma, which can progress to adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA) and eventually to invasive adenocarcinoma (ADC). A more complete understanding of the early carcinogenesis of lung cancer is critical for lung cancer early detection and interception. However, studying these lung cancer precursors is challenging because these lesions are often insufficient for molecular and immune profiling. Artificial intelligence (AI)-based studies on H&E histopathology images, termed pathomics, have achieved substantial progress in revealing heterogeneous phenotypic characteristics of various cancers. However, pathomics on lung precancerous progression and their correlation with genomic features remain underexplored. Methods: We curated FFPE H&E slides from two ethnic groups, including the Caucasian cohort containing 46 lesions with 170 regions of interest (ROI) (74 AAH, 10 AIS, 21 MIA, and 65 ADC) and the Asian cohort containing 128 lesions with 369 ROIs (59 AAH, 84 AIS, 77 MIA, and 149 ADC). We adopted the expert-in-the-loop strategy to develop a deep learning pipeline to segment and annotate the cells within ROI into three categories: epithelial, lymphocyte, and other. Next, we measured the ratio and density of epithelial cells and lymphocytes inside each ROI as pathomics features. Finally, we interrogated ROI-level features and examined their correlation with molecular and immune features in the Asian cohort. Results: We observed a progressive increase in the ratio and density of epithelial cells and a progressive decrease in the ratio and density of lymphocytes defined by the AI model from AAH to AIS, MIA, and ADC, consistent with the same trends defined by T cell receptor (TCR) sequencing and multiplex immunofluorescence (mIF). When correlating pathomics features with molecular/immune features, the epithelial cell ratio exhibited prominent positive correlations with the frequency of allelic imbalance (rho=0.588, p=4.71e-13) and nonsynonymous mutation burden (rho=0.453, p=1.03e-7). In contrast, the lymphocyte ratio showed a notable negative correlation with copy number variation burden (rho=-0.412, p=1.61e-6). Conclusion: Employing AI tools to analyze HE images of lung precancerous lesions, we revealed that molecular and immune evolution during early lung carcinogenesis is consistent with the results from complicated, time-consuming, and expensive molecular/immune profiling, which requires a large number of tissue specimens, highlighting the potential of pathomics in the study of cancer biology, particularly in diseases having limited tissue specimens. Citation Format: Pingjun Chen, Frank Rojas, Xin Hu, Junya Fujimoto, Alejandra Serrano, Bo Zhu, Lingzhi Hong, Rukhmini Bandyopadhyay, Muhammad Aminu, Maliazurina B. Saad, Morteza Salehjahromi, Sheeba J. Sujit, Neda Kalhor, Harvey I. Pass, Andre L. Moreira, Ignacio I. Wistuba, Don L. Gibbons, John V. Heymach, Luisa M. Solis Soto, Jianjun Zhang, Jia Wu. Pathomics reveals the molecular and immune evolution from lung preneoplasia to invasive adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5443.

The epithelial-to-mesenchymal transition (EMT) is a dynamic epigenetic reprogramming event that occurs in a subset of tumor cells and is an initiating step toward invasion and distant metastasis. The process is reversible and gives plasticity to cancer cells to survive under variable conditions, with the acquisition of cancer stem cell–like characteristics and features such as drug resistance. Therefore, understanding survival dependencies of cells along the phenotypic spectrum of EMT will provide better strategies to target the spatial and temporal heterogeneity of tumors and prevent their ability to bypass single-inhibitor treatment strategies. To address this, we integrated the data from a selective drug screen in epithelial and mesenchymal KRAS/p53 (KP)-mutant lung tumor cells with separate datasets including reverse-phase protein array and an in vivo shRNA dropout screen. These orthogonal approaches identified AXL and MEK as potential mesenchymal and epithelial cell survival dependencies, respectively. To capture the dynamicity of EMT, incorporation of a dual fluorescence EMT sensor system into murine KP lung cancer models enabled real-time analysis of the epigenetic state of tumor cells and assessment of the efficacy of single agent or combination treatment with AXL and MEK inhibitors. Both two- and three-dimensional culture systems and in vivo models revealed that this combination treatment strategy of MEK plus AXL inhibition synergistically killed lung cancer cells by specifically targeting each phenotypic subpopulation. In conclusion, these results indicate that cotargeting the specific vulnerabilities of EMT subpopulations can prevent EMT-mediated drug resistance, effectively controlling tumor cell growth and metastasis. Significance: This study shows that a novel combination of MEK and AXL inhibitors effectively bypasses EMT-mediated drug resistance in KRAS/p53-mutant non–small cell lung cancer by targeting EMT subpopulations, thereby preventing tumor cell survival.

Background: Our understanding of the immunopathology of early-stage NSCLC is still limited. While neoadjuvant immunotherapeutic strategies have recently shown anti-tumor effects in resectable NSCLC, their mechanisms remain inadequately understood. Here, we explore immune programs that inform of tumor immunity and response to neoadjuvant chemotherapy and chemoimmunotherapy in localized NSCLC. Methods: Targeted immune gene sequencing using the HTG Precision Immuno-Oncology panel was performed in localized NSCLCs from three cohorts based on treatment: naïve (n=190), neoadjuvant chemotherapy (n=38) and neoadjuvant chemoimmunotherapy (n=21). Three tumor immune microenvironment (TIME) phenotypes (inflamed, cold, excluded) were derived based on CD8+ T cell infiltration. Signatures of immune cell abundance and immune genes were statistically compared based on tumoral PD-L1 expression, immune phenotypes, associated with pathological response, and were cross-compared across the three cohorts. Results: PD-L1 positive tumors exhibited increased signature scores for various lymphoid and myeloid cell subsets (both, p Conclusions: Our findings highlight immune gene programs that may underlie host tumor immunity and response to neoadjuvant chemotherapy and chemoimmunotherapy in early-stage NSCLC. Citation Format: Pedro Rocha, Jiexin Zhang, Raquel Laza-Briviesca, Alberto Cruz-Bermúdez, Katsuhiro Yoshimura, Carmen Behrens, Apar Pataer, Edwin Parra-Cuentas, Cara Haymaker, Junya Fujimoto, Stephen Swisher, John Heymach, Don L. Gibbons, J Jack Lee, Boris Sepesi, Tina Cascone, Luisa M. Solis, Mariano Provencio, Ignacio I. Wistuba, Humam Kadara. Distinct immune gene programs associated with host tumor immunity, neoadjuvant chemotherapy and chemoimmunotherapy in resectable NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6152.

Background: We have previously demonstrated that the presence of a proinflammatory peripheral cytokine milieu correlates with high levels of circulating neutrophils at the time of surgery and reduced overall survival and recurrence-free survival. In this study, we hypothesized that functional immune features or states in circulation may indicate early tumor recurrence when assessed longitudinally. We also investigated whether potential deficiencies in peripheral immune functionality at the time of lung cancer resection could identify correlates with subsequent outcome. Methods: We performed flow cytometry and luminex profiling of blood samples collected from patients with stage I-IIIA resected NSCLC (n=150) and enrolled on the Immunogenomic profiling of NSCLC (ICON) prospective protocol. Patient characteristics include 75 adenocarcinomas, 30 squamous and 12 mixed or other histologies. Only patients who underwent primary cancer resection without neoadjuvant therapy were included. At a median follow up of 18.2 months, 37 patients had disease recurrence. Blood was collected at the time of primary lung cancer resection, at 4 weeks, and 4 months thereafter with PBMCs utilized for flow cytometry and plasma for cytokine assessment. Changes in cytokines were assessed by normalizing to baseline levels. Results: Larger tumors as well as advanced clinical and pathological stages were associated with higher frequencies of proliferating Ki67+CD4+ and Ki67+CD8+ T cells in circulation at time of resection, suggesting an activated circulating immune response. We identified novel strong correlations in the plasma between soluble BTLA and Tim3 (r= 0.87, p=1.74e-140), PD1 and CD80 (r=0.72, p=6.41e-74) and moderate correlations between soluble PD1 and PDL1 (r=0.39, p=8.41e-18). Efforts are ongoing to determine the association of specific circulating immune states with the presence of these soluble receptors. CD8+Tim3+ T cells (FC=5.2%, p=0.028) and CTLA4+NK cells (FC=47.7%, p=0.002) as well as CTLA4+Tregs (FC=25.2%, p=0.05) were found to be increased in circulation at pre-recurrence time points (either 4 weeks or 4 months) relative to resection. This suggests the emergence of a suppressive cell type as well as induction of specific checkpoint receptors on effector cells that correlate with tumor recurrence. Finally, we identified a cytokine signature associated with recurrence by testing three sets within the ICON cohort with a training set AUC = 0.76 and the test set AUC=0.72, which was validated in a third set of patients yielding an AUC = 0.76. Conclusions: We identified circulating immune features associated with initial tumor size and overall stage as well as unique associations among soluble proteins. Increased presence of potentially inhibited or suppressed CD8+ T cells and NK cells as well as Tregs are suggestive of mechanisms of immune suppression relative to tumor recurrence. Citation Format: Younghee Lee, Daniel McGrail, Hai Tran, Mayra E. Vasquez, Carlos Ramos, Alexandre Reuben, Ara A. Vaporciyan, Annikka Weissferdt, Chantale Bernatchez, Tina Cascone, Ignacio I. Wistuba, Jianjun Zhang, John Heymach, Marcelo V. Negrao, Don L. Gibbons, Boris Sepesi, Cara L. Haymaker. Circulating biomarkers are associated with recurrence following complete resection of non-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 1670.

Introduction: Differences in the host immune environment are thought to mediate heterogeneous treatment responses in non-small cell lung cancer (NSCLC). Unlike individual platform analyses, integrative analysis of multi-platform profiling allows for the discovery of novel interactions that expand our understanding of the disease. Utilizing the ImmunogenomiC prOfiling of NSCLC patient cohort (ICON), a prospective multi-omics protocol of operable early-stage NSCLC tumors with integrated immune, genomic, and clinical data, we hypothesized that multi-platform analyses would identify differences in the immune-genomic landscape that are associated with disease recurrence. Methods: Tumor and tumor-adjacent uninvolved lung was collected at resection; blood was collected before and after surgery. Tissue samples underwent WES, RNAseq, TCR sequencing (TCRseq), multiplex immunofluorescence (mIF), and RPPA profiling; tissue and blood (PBMC) samples were analyzed by flow cytometry. An integrated, inter-modality network was built using Spearman correlations between measurement pairs from different data modalities. Multivariate analysis was performed to adjust for stage and histology. Results: A total of 89 treatment-naïve patients with Stage 1-3 resected NSCLC (Squamous: 19; Non-squamous: 70) and 24 months of follow-up were analyzed (recurrence N = 24; no recurrence N = 65). The data network includes over 4,000 measurements linked by over 50,000 correlations. InfoMap, a community detection approach, was used to extract sub-network modules, which were used to contextualize the results of multivariate analysis. Tumors from patients with recurrence demonstrated decreased immune cell infiltration and activation including decreased cytotoxic CD8 T-cells (CD8+PD1+; fold-change (FC) = 0.898, p = 0.018; flow cytometry), decreased T-cell clonality (FC = 0.954, p = 0.017; TCRseq), and decreased tumor-associated macrophages (CD68+PD-L1+; FC = 0.426, p = 0.011; mIF). Furthermore, circulating CD8+ICOS+ activated T cells were decreased in patients with recurrence suggesting an impaired systemic anti-tumor immune response (FC = 0.552, p = 0.042; PBMC Flow). Finally, tumor-adjacent uninvolved lungs showed distinct T-cell phenotypes with accumulation of inactive CD8 T-cells (CD8+PD1-TIM3-) in patients with recurrence and increased populations of activated CD8 T-cells (CD8+PD1+) in patients without recurrence. Conclusion: Integrative multi-omic analysis suggests preserved anti-tumor immune surveillance in patients who are disease-free after 2 years from surgical resection with curative intent for treatment of NSCLC relative to patients with disease recurrence. Further analysis is ongoing to interrogate genomic and immune variables that are associated with disease recurrence. Citation Format: Neal Akhave, Stephanie Schmidt, Alexandre Reuben, Tina Cascone, Jianhua Zhang, Jun Li, Junya Fujimoto, Lauren A. Byers, Beatriz Sanchez-Espiridion, Lixia Diao, Jing Wang, Lorenzo Federico, Marie-Andree Forget, Daniel J. McGrail, Annikka Weissferdt, Shiaw-Yih Lin, Younghee Lee, Carmen Behrens, Ignacio I. Wistuba, Andrew Futreal, Ara Vaporciyan, Boris Sepesi, John V. Heymach, Chantale Bernatchez, Cara Haymaker, Jianjun Zhang, Christopher A. Bristow, Marcelo V. Negrao, Don L. Gibbons. Integrated multi-platform profiling of early-stage non-small cell lung cancer identifies relationship between disease recurrence and decreased native immune response in treatment-naïve resected NSCLC [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 619.

Lung cancer is the leading cause of cancer-related death worldwide due to the ability of cancer cells to metastasize. Therefore, it is essential to expand our current knowledge of the biological processes that contribute to metastasis to guide the discovery of novel therapeutic modalities. Epithelial-to-mesenchymal transition (EMT) is a mechanism for metastasis, which changes polarized epithelial cells into mesenchymal cells with enhanced motility and invasiveness. High expression of the Zinc finger E-box binding homeobox 1 (ZEB1) transcription factor is correlated to poor outcomes in cancer, including therapeutic resistance and EMT-mediated metastasis. ZEB1 has a predicted molecular weight of 125kDa; however, multiple groups have reported discrepancies in the observed molecular weight (approximately 190-250kDa). This has been attributed to dimerization mediated by post-translational modifications (PTMs). Therefore, we performed mass spectrometry and identified a novel PTM - K811 acetylation - that may regulate ZEB1 dimerization and function. To define the role of ZEB1 acetylation, we generated ZEB1 acetyl mimetic (K811Q) and deficient (K811R) mutants in the 393P lung adenocarcinoma cell line. We hypothesize that ZEB1 acetylation regulates dimerization and protein stability to promote lung adenocarcinoma progression and metastasis. Overexpression of each ZEB1 mutant fused with GFP demonstrated nuclear localization consistent with wild-type (WT) ZEB1. Interestingly, we determined that the acetyl-deficient mutant (125kDa) exhibits a decreased half-life compared to WT and acetylated ZEB1, suggesting that disruption of acetylation hinders dimerization and protein stability. However, the acetyl-mimetic mutant (250kDa) protects ZEB1 from proteasomal degradation and promotes migration and invasion in vitro compared to ZEB1 acetyl-deficient. We further established that the control GFP vector grew as non-invasive in 3D culture, but ZEB1 (WT and acetylation) colonies displayed an elongated invasive response associated with Rac1 and Fak signaling activation. Conversely, disruption of acetylation produced rounded invasive structures associated with RhoA expression. Rac1/RhoA small GTPases are key regulators of the mesenchymal or amoeboid mode of migration respectively as well as the transition between each mode (MAT and AMT). Rac1 inhibitor and RhoA inhibitor markedly reduced EMT characteristics in the ZEB1 acetyl-mimetic and acetyl-deficient mutants, respectively, suggesting that EMT may contribute to MAT. Combined, our results suggest that PTM regulates ZEB1 to promote its stability and progression of lung adenocarcinoma via EMT. Future studies are focused on further understanding the role of ZEB1 acetylation in vivo. However, the fact that acetylation contributes to ZEB1-mediated metastasis demonstrates its potential for use in the treatment of lung cancer. Citation Format: Mabel G. Perez-Oquendo, Michelle Barton, Don L. Gibbons. Regulation and function of ZEB1 dimerization in lung adenocarcinoma progression and metastasis [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 2477.

Lung adenocarcinoma (LUAD) is the most commonly diagnosed histological subtype of lung cancer. While earlier work has underscored genomic and immune alterations in LUAD, the roles of individual cell populations in early-stage human LUAD evolution in space remain unknown. Here, we provide a detailed cellular atlas of early-stage LUAD and its spatial ecosystem along the peripheral lung. We performed single-cell RNA sequencing of 186,916 cells including enriched epithelial fractions from five early-stage LUADs with fourteen multi-region normal lung tissues of defined spatial proximities from the primary LUADs. We show that major epithelial and immune cellular lineages, states, and transcriptomic features geospatially and progressively evolve across normal regions and with increasing LUAD proximity. Analysis of 70,030 lung epithelial cells unraveled diverse lineage trajectories, transcriptional lineage plasticity programs underlying KRAS-mutant cells, and intratumoral heterogeneity within single sites. T regulatory cell programs including multiple immune checkpoints increased in tissues with closer proximity to LUADs, in sharp contrast to signatures of CD8+ cytotoxic T cells, antigen presentation by macrophages, and inflammatory dendritic cells. We found that some spatial signatures (e.g. a B cell signature score) were increased along the pathologic spectrum of normal lung, preneoplastic lesions, and matched invasive LUADs. LUAD cell-cell communication networks were enriched with ligand-receptor interactions involving CD24, LGALS9 and TIM3 immune checkpoints, including crosstalk between CD24 antigen in LUAD epithelial cells and SIGLEC10 in myeloid subsets. CD24 was markedly increased in preneoplasias relative to normal lung and further in LUAD, and its expression was highly positively correlated with immunosuppressive phenotypes. These data provide an atlas of cellular states and phenotypes underlying early-stage LUAD evolution in space, and a scalable resource for identification of targets for early treatment. Citation Format: Ansam Sinjab, Guangchun Han, Warapen Treekitkarnmongkol, Kieko Hara, Patrick Brennan, Minghao Dang, Dapeng Hao, Ruiping Wang, Enyu Dai, Hitoshi Dejima, Jiexin Zhang, Elena Bogatenkova, Beatriz Sanchez-Espiridion, Kyle Chang, Danielle R. Little, Samer Bazzi, Linh Tran, Kostyantyn Krysan, Carmen Behrens, Dzifa Duose, Edwin R. Parra, Maria Gabriela Raso, Luisa M. Solis, Junya Fukuoka, Jianjun Zhang, Boris Sepesi, Tina Cascone, Lauren A. Byers, Don L. Gibbons, Jichao Chen, Seyed Javad Moghaddam, Edwin J. Ostrin, Daniel G. Rosen, John V. Heymach, Paul Scheet, Steven Dubinett, Ignacio I. Wistuba, Junya Fujimoto, Christopher S. Stevenson, Avrum E. Spira, Linghua Wang, Humam Kadara. Resolving the spatial and cellular architecture of lung adenocarcinoma by multi-region single-cell sequencing [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 130.

CD8+ T lymphocytes in tumors often become dysfunctional, prompting cancer immune escape and immunotherapy resistance. Restoring T-cell anti-tumor immunity remains an unmet challenge. Here, we show that histamine receptor H1 (HRH1) is highly expressed and tightly associated with T-cell dysfunction in human cancers. Histamine, the HRH1 ligand, is also increased in human and mouse tumors. HRH1 is predominantly expressed on tumor-associated macrophages (TAMs) and is activated by cancer cell-secreted histamine. The histamine/HRH1 axis in TAMs confers CD8+ T-cell dysfunction via increasing membrane localization of V-domain Ig suppressor of T cell activation (VISTA), an inhibitory immune checkpoint that promotes immunotherapy resistance. Blocking HRH1, genetically, or by anti-allergy drugs, reversed immunosuppressive TAMs, activated T-cells, and inhibited tumor growth in vivo. HRH1-targeting anti-allergy drugs more efficaciously enhanced immunotherapy response than anti-VISTA antibodies. Markedly, allergy-induced histamine also activates HRH1 on macrophages to induce T-cell dysfunction and immunotherapy resistance, which were reversed by the over-the-counter anti-allergy drug. Taken together, tumor cell-derived and allergy-released histamines activate HRH1 on TAMs to concede immunosuppression, pro-tumor function, and immunotherapy resistance, which can be negated by inexpensive and safer anti-allergy drugs. Citation Format: Hongzhong Li, Yi Xiao, Jun Yao, Xiangliang Yuan, Xuedong Yin, Yuan Zhang, Yohei Saito, Hideo Yagita, Don Gibbons, Dihua Yu. Blocking histamine receptor H1 on macrophages with anti-allergy drugs restores T-cell anti-tumor immunity [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 2794.

The advent of PD-1/PD-L1 agents has transformed the therapeutic landscape of many advanced cancers, including non-small cell lung cancer (NSCLC). However, our understanding of the genomic biomarkers underlying effective treatment response remain limited. Here we provide updated results from our ongoing effort, the Stand Up To Cancer Lung (SU2C-Lung)/Mark Foundation EXTOLConsortium, a multi-institution collaboration to expand our understanding of the molecular determinants of immunotherapy response in NSCLC. Comprising a set of nearly 400 patients, this cohort enables evaluation of both genomic and transcriptomic factors associated with checkpoint blockade response. In addition to validating previously known associations including TMB and neoantigen burden, we examined transcriptional predictors associated with response. We generated a list of differentially expressed genes with respect to best overall response (BOR), and performed dimensionality reduction using semi-supervised Bayesian Non-Negative Factorization (ssBNMF). We identified 3 distinct clusters with strong sample membership. Characterization of these subtypes revealed varying levels of immune infiltrate, histologic composition, and response rates to checkpoint blockade. Of these three subtypes, two were associated with low response rates to PD-1/PD-L1 blockade, suggesting the existence of distinct avenues toward resistance. To further characterize these transcriptional subtypes, we used ssBNMF marker genes to classify publicly available NSCLC samples from The Cancer Genome Atlas (TCGA), along with gene expression from a smaller cohort of large-cell neuroendocrine (LCNE) samples. Histologic composition showed good concordance with our SU2C samples, and redemonstrated observations within our smaller SU2C cohort of distinct immuno-suppressive and immuno-depleted milieus associated with resistance. Citation Format: Monica B. Arniella, Arvind Ravi, Justin Gainor, Chip Stewart, Sam Freeman, Mark Awad, Patrick Forde, Valsamo Anagnostou, Brian Henick, Jonathan W. Riess, Don Gibbons, Nathan Pennell, Vamsidhar Velcheti, Ignaty Leshchiner, Jaegil Kim, Subba Digumarthy, Mari Mino-Kenudson, John Heymach, Nir Hacohen, Naiyer Rizvi, Roy Herbst, Victor E. Velculescu, Julie Brahmer, Kurt Schalper, Pasi Jänne, Jedd Wolchok, Alice Shaw, Gad Getz, Matthew D. Hellman. An SU2C-Mark Foundation Lung collaborative update: integrative genomics identifies distinct transcriptional states associated with checkpoint blockade resistance [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 LB197.

The novel coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Severely symptomatic COVID-19 is associated with lung inflammation, pneumonia, and respiratory failure, thereby raising concerns of elevated risk of COVID-19-asociated mortality among lung cancer patients. Angiotensin converting enzyme 2 (ACE2) is the major receptor for SARS-CoV-2 entry into lung cells. Yet, the single-cell expression landscape of ACE2 and other SARS-CoV-2-related genes in pulmonary tissues of lung cancer patients remains unknown. To fill these voids, we leveraged our ongoing efforts in single-cell transcriptomic analysis of 186,916 cells including a large number of epithelial cells (n=70,030) derived from 5 LUADs and 14 matching uninvolved normal lung tissues, to delineate expression levels and cellular distribution of ACE2 and SARS-CoV-2 priming proteases TMPRSS2 and TMPRSS4. Single-cell RNA sequencing of 186,916 cells revealed epithelial-specific expression of ACE2, TMPRSS2 and TMPRSS4. Analysis of 70,030 LUAD- and normal-derived epithelial cells showed that ACE2 levels were highest in normal alveolar type 2 (AT2) cells and that TMPRSS2 was expressed in 65% of normal AT2 cells. Conversely, expression of TMPRSS4 was highest and most frequently detected (75%) in malignant lung cells. ACE2-positive cells co-expressed genes implicated in lung pathobiology, including COPD-associated HHIP, and the scavengers CD36 and DMBT1. Notably, the viral scavenger DMBT1 was significantly positively correlated with ACE2 expression in AT2 cells. In conclusion, we describe normal and tumor lung epithelial populations that express SARS-CoV-2 receptor and proteases, as well as major host defense genes, and that thus comprise potential treatment targets for COVID-19 particularly among lung cancer patients. Citation Format: Guangchun Han, Ansam Sinjab, Kieko Hara, Warapen Treekitkarnmongkol, Patrick Brennan, Kyle Chang, Elena Bokatenkova, Beatriz Sanchez-Espiridion, Carmen Behrens, Luisa M. Solis, Boning Gao, Luc Girard, Jianjun Zhang, Boris Sepesi, Tina Cascone, Lauren A. Byers, Don L. Gibbons, Jichao Chen, Seyed Javad Moghaddam, Edwin J. Ostrin, Paul Scheet, Junya Fujimoto, Jerry Shay, John V. Heymach, John D. Minna, Steven Dubinett, Ignacio I. Wistuba, Christopher S. Stevenson, Avrum E. Spira, Linghua Wang, Humam Kadara. Single-cell expression landscape of SARS-CoV-2 receptor ACE2 and host proteases in human lung adenocarcinoma [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 702.

Introduction: Despite the recent approval of immune checkpoints inhibitors (ICI) as a treatment option in the extensive-stage small cell lung cancer (SCLC) setting, survival has not significantly changed in the last decades. Recent scientific efforts have led to the identification of 4 major subtypes defined by expression of three transcription factors: ASCL1 (SCLC-A), NEUROD1 (SCLC-N), POU2F3 (SCLC-P) with the fourth subtype characterized by increased expression of immune genes (Inflamed subtype - SCLC-I). Transcriptomic results, along with recent immunotherapy trials, suggest that modulation of tumor immune microenvironment (TIME) could potential be critical to achieving clinical responses in a subset of patients, hence a comprehensive study of the TIME in SCLC is imperative. Here we report the feasibility of a multiplex immunofluorescence (mIF) methodology to analyze the TIME in SCLC. Methods: FFPE sections from surgically resected SCLC (N=4, one representative case across all SCLC subtypes) were identified from the ICON Project at UT MD Anderson Cancer Center. We used mIF to identify and quantify immune markers grouped into two 6-antibody panels: Panel 1: cytokeratin (CK, AE1/AE3), CD3, CD8, PD-1, PD-L1 and CD68; Panel 2: CK, CD20, granzyme B, FOXP3, CD45RO, and CD57. Finally, genomic (WES), transcriptomic (RNA sequencing) and proteomic (RPPA) data from these cases were integrated with the mIF data. Results: SCLC molecular subtypes (SCLC-A, N, P, I) were classified using transcriptomic and proteomic data. Analysis of TIME unveils a higher immune cell infiltration within SCLC-I subtype compared with the other cases representing, immune “cold” SCLC subtypes. SCLC-I subtype showed a 2-13 folder higher (range) immune cell density than SCLC-A, N and P subtypes (measured as a median of cell density). Specifically, T cells (CD3+) (695 and 242 cells/mm2, for SCLC-I and the median for the other subtypes respectively), T cytotoxic cells (CD3+ CD8+) (206 and 105), activated T cells (CD3+ CD8+ granzyme+) (20 and 2), antigen experienced ‘like' T cells (CD3+ PD-1+) (17 and 0), memory T cells (CD3+ CD45RO+) (328 and 91) and macrophages (CD68+) (773 and 57). PD-L1 expression in malignant cells did not show significant differences within the 4 SCLC subtypes. However, PD-L1 expression in macrophages was significantly higher in the SCLC-I subtype, suggesting an increase of IFN-gamma in the TIME. Conclusions: TIME study show the use of mIF in SCLC tumors to be feasible, and could potentially provide key information towards the identification of SCLC patients that could benefit from ICI. For the first time we complemented transcriptomic data from SCLC tumors with mIF analysis unveiling the complex interplay of the host immune response and malignant cells. Our preliminary results warrant further studies to explore the role of TIME in immunotherapeutic response in SCLC. Citation Format: Pedro Rocha, C. Allison Stewart, Edwin Parra, Luisa M. Solis, Carl M. Gay, Robert J. Cardnell, Naohiro Uraoka, Alejandro Francisco-Cruz, Hitoshi Dejima, Yuanxin Xi, Lixia Diao, Jing Wang, Marcelo V. Negrao, Jianjun Zhang, Ignacio Wistuba, Don L. Gibbons, Lauren A. Byers. Multiplex immunofluorescence (mIF) reveals differences in tumor immune microenvironment between molecularly-defined subsets of 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 2758.

Background: Tumor genomic information from tissue has been the standard of practice for identifying actionable molecular alterations. The same genomic profiling is also widely available by a non-invasive blood test from circulating tumor DNA (ctDNA). We hypothesized that patients with advanced non-small cell lung cancer (NSCLC) with actionable oncogenic driver mutations with either low or high variant allelic frequency (%VAF) identified by ctDNA would have similar clinical benefits after treatment with targeted therapies. Methods: In this retrospective study (UTMDACC IRB-approved protocol PA16-0061), patients with a targetable mutation identified by ctDNA NGS assay who received an FDA-approved therapy were identified in our GEMINI database. All potential patients were individually reviewed and information confirmed with review of their electronic medical records. Blood based genomic profiling was completed by NGS of ctDNA with a panel of up to 70 cancer-related genes at a CLIA-certified lab (Guardant360, Guardant Health, Redwood City, CA). The %VAF was reported for each mutation identified. We defined low group with VAF of 0.01% to 0.99% and high group with VAF > 1%. Tumor measurements were assessed using RECIST V1.1. For comparisons analysis, Kaplan-Meier estimator was used to estimate progression free survival (PFS) with Mantel-Cox and un-paired t-test was used for tumor responses. Results: Eighty-nine patients with advanced NSCLC treated with an FDA approved therapy based on ctDNA genomic profiling were identified and the results are summarized in table below: LOW %VAFHIGH %VAFP valuegroup (n=39)group (n=50)Gender (Female/Male)20/1928/22Age (median, range)54(27-85)63(29-89)Targetable mutations:EGFR2246ALK114MET exon 1420ROS120BRAF20Line of therapy (median, range)2(1-7)2(1-7)ALL PatientsTumor response (mean, range)-37.23% (-100 to +38)-31.08% (-100 to +59)0.42PFS (median, range)215 (27 – 848) days280 (43 – 894) days0.70EGFR onlyTumor response (mean, range)-24.86% (-100 to +16)-23.54% (-100 to +59)0.88PFS (median, range)189 (57 – 848) days293 (43-894) days0.23 Conclusions: In this analysis of patients who were treated with an FDA approved therapy based on genomic results from ctDNA profiling, there were no differences in either tumor response (P value = 0.42) or PFS (P value = 0.70) between those patients with low and high ctDNA as assessed by %VAF of identified targetable mutation. Similar findings were also observed in the analysis of the EGFR only subgroup. This study reveals patients with low %VAF received comparable clinical benefits as those with high %VAF and no deleterious harm was observed from treating patients with low %VAF with appropriate targeted therapy. Citation Format: Hai T. Tran, Vincent K. Lam, Mayra Vasquez, Yasir Elamin, Victoria Raymond, Lingzhi Hong, George Blumenschein, Brett Carter, Richard Lanman, Mehmet Altan, Ann Tsao, Lauren Byers, Don Gibbons, Jianjun Zhang, John V. Heymach. Comparable clinical benefits between low and high variant allelic frequency in NSCLC patients treated with targeted therapies based on molecular alterations from ctDNA NGS [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 787.

PD-1/PD-L1 checkpoint blockade has been a landmark advance for many patients suffering from advanced non-small cell lung cancer (NSCLC). However, detailed biomarkers of response beyond tumor mutational burden (TMB) are still poorly understood. As part of the effort to elucidate these additional signatures, we describe our progress on the Stand Up to Cancer Lung (SU2C-Lung) cohort. Initial characterization of exomes recapitulates mutational and copy number profiles seen in The Cancer Genome Atlas (TCGA) project. To better define expression subtypes using RNA sequencing, we performed non-negative matrix factorization (NMF) across an aggregated set of publicly available NSCLC expression data (including adenocarcinoma, squamous cell carcinoma, and large cell neuroendocrine histologies), and demonstrate good concordance in the SU2C-Lung cohort between this expression-based classifier and clinically annotated histology. To gain further insight into how immune cell infiltrates vary across our cohort, we additionally tested two common deconvolution algorithms, EPIC and CIBERSORT. While these two methods agree for some prominent cell types, such as B cells and CD4 T cells, discrepancies in minor infiltrating components such as NK cells may suggest a limit to the inference of rare subpopulations from bulk sequencing data.Finally, we describe a novel approach for determining single-gene predictors of response. Using the method, which is based on comparison of top single transcriptional features identified from random bootstraps of the full cohort as compared to a set of background shuffles, we are able to show that we remain powered for discovery of RNA response biomarkers despite the typically burdensome toll of multiple hypothesis correction at genome wide scale. Acknowledgment: Supported by Stand Up To Cancer-American Cancer Society Lung Cancer Dream Team Translational Research Grant SU2C-AACR-DT17-15. Citation Format: Monica Arniella, Arvind Ravi, Chip Stewart, Sam Freeman, Mark Awad, Patrick Forde, Valsamo Anagnostou, Brian Henick, Jonathan Riess, Don Gibbons, Nathan Pennell, Vamsidhar Velcheti, Ignaty Leshchiner, Jaegil Kim, Subba Digumarthy, Mari Mino-Kenudson, John Heymach, Nir Hacohen, Naiyer Rizvi, Roy Herbst, Victor Velculescu, Julie Brahmer, Kurt Schalper, Pasi Jänne, Jedd Wolchok, Alice Shaw, Justin Gainor, Matthew Hellmann, Gad Getz. Integrative genomic analysis of checkpoint blockade in lung cancer: A multi-institution SU2C collaborative [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 5902.

PDXs recapitulate histologic features, gene expression patterns, and genomic alterations in human primary tumors, and thus have emerged as robust preclinical models for drug development, molecular characterization of cancers, identification of biomarkers, and strategic development of precision therapy. We tested multiple combinations of small molecule targeted drugs selected on the basis of known NSCLC vulnerabilities for efficacy in NSCLC PDXs with known genotypes. We determined in vivo treatment responses to single agent and combination therapies for pathway targeted therapeutic agents, including the MEK inhibitor trametinib, the MDM2 inhibitor KRT-232, the BCL2/BCL-XL inhibitor navitoclax, and their combinations in 8-23 molecularly annotated NSCLC PDX models. Mice (n=3-5/group) were enrolled into treatment individually when tumors reached 200 mm3 in size, and were treated 5 days/week for 3 weeks. Tumor growth was monitored 2-3 times/week. We used the following criteria to determine treatment responses: 1) Tumor Regression (or partial response): tumor regression ≥ -30% based on tumor volume changes calculated by AUC0-21day or at day 21 after treatment start when compared with baseline (beginning of treatment at day 0); 2) Tumor Growth Inhibition (or stable disease): Tumor growth was significantly suppressed when compared with control (P < 0.05), but no tumor regression was observed, or tumor regression was less than -30% based on tumor volume changes calculated by AUC0-21day or at day 21 after treatment start when compared with baseline; 3) Resistance: Tumor volume changes calculated by AUC0-21day was not significantly different from control group (P>0.05). Our results showed that KRT-232 alone resulted in 15.8% (3/19) tumor regression and 26.3% (5/19) tumor growth inhibition, all in TP53 wild type PDXs. Trametinib alone induced 10% (2/20) tumor regression and 50% (10/20) growth inhibition, respectively. 80% (8/10) of KRAS mutant PDXs responded to trametinib treatment with tumor regression (1/10) or growth inhibition (7/10). Combination therapies of trametinib plus KRT-232 and trametinib plus navitoclax led to improved in vivo anticancer activity over single agent activity in a subset PDX models with KRAS mutations. Tumor regression was observed in 26% (6/23) and 50% (5/10) of trametinib plus KRT-232 and trametinib plus navitoclax treatment groups, respectively. Navitoclax alone did not induce tumor regression in 8 PDX models tested, and navitoclax plus KRT-232 did not lead to significant improvement in activity over single agents in 11 PDXs. Our results show that combination therapies of trametinib plus KRT-232 or navitoclax result in improved efficacy in a subgroup of NSCLC PDX models with KRAS mutations. Clinical trials with these targeted drug combinations in NSCLC are warranted. Citation Format: Xiaoshan Zhang, Ran Zhang, Huiqin Chen, Li Wang, Chenghui Ren, Shuhong Wu, Min Jin Ha, Jeffrey Morris, Yuanxin Xi, Jing Wang, Don L. Gibbons, John V. Heymach, Funda Meric-Bernstam, John Minna, Stephen G. Swisher, Jack A. Roth, Bingliang Fang. Novel targeted combination therapies active in KRAS mutant non-small cell lung cancer (NSCLC) identified using patient-derived xenografts (PDX) [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 557.

Lung adenocarcinoma (LUAD) is the most frequently diagnosed histological subtype of lung cancer and accounts for most smoking-related cancer deaths, warranting strategies for early intervention. Earlier work revealed genome-wide aberrations in LUADs and the adjacent premalignant field, known as “field carcinogenesis”, that are pertinent to LUAD pathogenesis. Yet, we still poorly understand the cellular and molecular architecture of LUAD and its nearby “field”. To fill this void, we performed, using the 10X Genomics system and NovaSeq 6000 platform, single-cell RNA sequencing (scRNA-seq) analysis of 4 early-stage resected LUADs as well as 11 matched normal lung tissues with differing spatial proximity from the tumors. We first analyzed 15,739 cells from a LUAD and matched tumor-adjacent and -distant normal tissues from patient 1. The resulting fraction of EPCAM+ cells from this analysis was approximately 4%. For deeper single-cell resolution of LUAD, we thus performed scRNA-seq of separately sorted/enriched epithelial cells (EPCAM+; n = 50,883) and non-epithelial cells (EPCAM-; n= 91,093) from patients 2, 3 and 4, each with an early-stage LUAD and three matched spatially-distributed normal-appearing tissues. Overall, we achieved on average approximately 150,000 reads and 1,956 genes detected per cell. Divergent populations of malignant and non-malignant cells and multiple epithelial and immune subsets clustered in an overall spatially modulated pattern according to proximity to the tumor. Hierarchical clustering revealed multiple distinct populations of airway lineage cells, including alveolar type 1 (AT1), alveolar type 2 (AT2), AT1/AT2 bipotential cells, club, goblet, basal and ciliated cells, with cells from the resected LUAD present in some but not all clusters. By analysis of epithelial-enriched fractions, we were also able to identify and interrogate rare cell subpopulations including CFTR-expressing ionocytes. By inference of copy number variation along with analysis of key oncogene expression patterns, we also identified epithelial cells that represent potential tumor cells-of-origin. Spatial reprogramming of field carcinogenesis encompassed immune cell populations including tumor-exclusive FOXP3+ regulatory T cells with marked over-expression of the major immune checkpoints CTLA4 and TIGIT. In contrast, for at least some of the cases, tumors were nearly devoid of natural killer cells, and abundance of CD8+ T cells dampened with increasing proximity to the tumor. Our single-cell surveys offer insights into novel cues in LUAD pathogenesis. Efforts are underway to interrogate the single-cell epithelial and immune landscape of additional LUADs and matched nearby normal-appearing lung, including those from non-smokers, to better understand the evolution of the disease and, thus, identify a low-hanging fruit of targets for early management of this fatal malignancy. Citation Format: Ansam Sinjab, Guangchun Han, Warapen Treekitkarnmongkol, Patrick Brennan, Kieko Hara, Kyle Chang, Elena Bogatenkova, Beatriz Sanchez-Espiridion, Carmen Behrens, Jianjun Zhang, Boris Sepesi, Tina Cascone, Don L. Gibbons, Jichao Chen, George Eapen, Edwin J. Ostrin, Junya Fujimoto, Avrum E. Spira, Paul A. Scheet, Ignacio I. Wistuba, Linghua Wang, Humam Kadara. A single-cell transcriptomic atlas of lung adenocarcinoma and adjacent normal-appearing tissue [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 1518.

Therapeutic resistance limits effective treatment of non-small cell lung cancer (NSCLC) and a better understanding of mechanisms contributing to resistance and strategies to overcome these are urgently needed. AXL, a TAM family receptor tyrosine kinase, has emerged as a key determinant of intrinsic and acquired resistance to chemotherapy, radiation and targeted therapies in NSCLC and other cancers, through its roles in mediating epithelial-mesenchymal transition (EMT) and immune escape. We previously showed that AXL may also play a role in DNA damage repair and that AXL overexpression mediated primary as well as acquired resistance to inhibitors of WEE1, a replication stress response kinase, in small cell lung cancer. In the present study, we further investigated the role of AXL in replication stress response. We found that, in NSCLC cell lines, AXL inhibition with the selective small-molecule AXL inhibitor BGB324 caused replication stress checkpoint activation, as indicated by increased RPA32 hyper-phosphorylation and ATR-mediated CHK1 phosphorylation. We further screened ATR inhibitors, VX-970 and AZD6738, in a panel of 20 NSCLC cell lines and correlated drug sensitivity with baseline expression of over 200 phosphorylated and total proteins, measured by reverse phase protein array. Notably, AXL was one of the biomarkers of resistance to the ATR inhibitor VX-970 (rho=0.52, p In conclusion, these findings suggest that AXL may play a novel and unexpected role in regulating replication stress. Furthermore, our results show that targeting AXL sensitizes NSCLC cell lines with primary resistance to ATR inhibitors and that AXL/ATR inhibitor combinations could be useful in treating platinum- and PARP inhibitor-resistant SLFN11-low tumors. Citation Format: Kavya Ramkumar, Pan Tong, You-Hong Fan, David Peng, John V. Heymach, Don L. Gibbons, Jing Wang, Lauren A. Byers. Targeting AXL sensitizes non-small cell lung cancer to ATR inhibitors by enhancing replication stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 276.

Background: Although immune checkpoint blockade has been successfully utilized in treating patients with non-small cell lung cancer (NSCLC), the benefit of immunotherapy for patients with advanced EGFR-mutant (EGFRm) NSCLC has been limited. Data from IMpower150 trial’s subgroup analysis suggests modulation of the tumor immune microenvironment (TME) with antiangiogenic agents and chemotherapy might enhance response to anti-PD-1/PD-L1 blockade in EGFRm lung cancers. We aim to gain a deep understanding of EGFRm NSCLC TME as the first step to make EGFRm amenable to immune therapy. Methods: We queried two cohorts of resected stage I-III lung adenocarcinomas: PROSPECT (94 tumors) and Immune Genomic Profiling of NSCLC (ICON, 76 tumors). In the PROSPECT cohort (14 EGFRm vs 80 WT), we compared 10 immune markers by IHC (PD-L1, PD-1, CD3, CD4, CD8, CD45RO, CD57, CD68, FoxP3 and Granzyme B) and 44 immune regulator genes’ expression levels and pathway signatures from microarray data. We subsequently validated the observation of differentially expressed genes in the ICON cohort (15 EGFRm vs 61 WT) using IF, RNAseq and WES. Results: In the PROSPECT cohort, lower PD-L1 (2.2 vs 10.4 H-score, p Conclusion: Our analysis showed that EGFR-mutant NSCLCs demonstrate a PD-L1 low, GzmB low, IFN-gamma low immune phenotype, suggesting a suppressive tumor microenvironment. We found that the CD8+ T cells were present in the tumor, but likely suppressed functionally by the negative regulators in the TME. These results direct future analysis of suppressive immune cell populations (CD4+ subpopulation, macrophages and dendritic cells) in EGFRm lung cancers, and represent an initial step for rationale combination of immune therapy to modulate the suppressed TME, which might lead to enhanced treatment efficacy to benefit patients with EGFR-mutant lung cancers. Citation Format: Xiuning Le, Marcelo V. Negrao, Alexandre Reuben, Won-Chul Lee, Edwin Parra, Jun Li, Tatiana Karpinets, Carmen Behrens, Boris Sepesi, Ara Vaporiciyan, Jack Roth, Cara Haymaker, Emily Roarty, Jianhua Zhang, Chantale Bernatchez, Jianjun Zhang, Ignacio Wistuba, Don Gibbons, John Heymach. Characterization of the tumor immune microenvironment in treatment-naïve EGFR-mutant NSCLC uncovers a low IFN-gamma suppressive immune phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5028.

Metastatic breast cancer is the most dreadful malignant disease that accounts for the majority of cancer-related deaths worldwide among women. A number of studies have shown that the tumor microenvironment (TME) plays a crucial role in regulating metastasis. It is therefore imperative to understand the dynamic interactions between cancer cells and their microenvironment to examine the molecular interaction and to effectively target cancer cells. TME comprises a variety of cells including immune cells which can influence tumor survival, growth and metastasis. Tumor-infiltrating lymphocytes (TILs), in particular, the CD8 T lymphocytes, has emerged as a promising prognostic marker for immunotherapy in a variety of cancers. However, the key molecular factors that regulate the cross-talk between tumor cells and CD8 T lymphocytes and its impact on metastatic traits in breast cancer is still inconclusive. Platelets are crucial components of the tumor microenvironment that are known to modulate tumor promotion and metastasis. The contribution of platelets and platelet secreted molecules are also carefully examined in metastasis of various cancers. The primary objective of this study is to investigate the role of CD8 T lymphocytes and platelets in breast tumor progression using isogenic tumor lines that form identical primary tumors but differ in their ability to develop metastasis. Citation Format: Robiya Joseph, Rama Soundararajan, Suhas Vasaikar, Fei Yang, Sevinj Isgandarova, Lin Tian, Monika Haemmerle, Barbara Mino, Tieling Zhou, Geraldine Vidhya Raja, Esmeralda Ramirez Pena, Petra Den Hollander, Neeraja Bhangre, Crystal Shin, Melisa Martinez, Jaime Rodriguez Canales, Jeffrey Chang, Anil Sood, Ignacio Ivan Wistuba, Don L. Gibbons, Jeffrey M. Rosen, Ghanashyam Acharya, Navin Varadarajan, Xiang H. Zhang, Sendurai A. Mani. Regulation of metastasis by CD8 T lymphocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3761.

Introduction: immune checkpoint inhibitors have revolutionized the treatment of non-small cell lung cancer (NSCLC), but only ~15% of patients will achieve durable clinical benefit. Our group and others have focused on characterizing the interaction between tumor genomic characteristics and immune microenvironment. Genes such as PTEN, CTNNB1, STK11 and KEAP1 have been associated with intra-tumor immune depletion, but a broader understanding of other key driver genes of NSCLC and their interplay with the immune microenvironment is currently lacking. To prospectively delineate the genomic and immune landscape of NSCLC, we launched the ICON Project (Immunogenomic Profiling of NSCLC). Methods: we prospectively collected tissue specimens from patients with stage I-IIIA NSCLC undergoing surgical resection. Samples were subjected to extensive immune-genomic profiling, including whole-exome and RNA sequencing, multiplex immunofluorescence (mIF), and flow cytometry. Results: from 2016-2018, 150 patients were accrued, with 78 samples having undergone genomic profiling. Most patients had stage I-II disease (74%) and non-squamous histology (70%). Most commonly mutated genes were TP53, EGFR, KRAS, CDKN2A, STK11, and mean TMB was 7.6 mut/Mb, which is comparable to TCGA (6.9mut/Mb, p=ns). Unsupervised clustering according to expression of CD8+ T cell related genes segregated tumors into immune-enriched (clusters 1 and 3) and immune-depleted (clusters 2 and 4) subgroups. We validated these findings through flow cytometry (CD8+GzB+ T cells) and mIF (CD8), with both methods showing CD8+ T cell infiltration to be enriched in clusters 1 and 3. Cluster 1 included squamous and non-squamous histology, tumors with epithelial and mesenchymal signatures, and high TMB. Cluster 2 included exclusively squamous histology with predominant mesenchymal tumor signature, high TMB, and was enriched for TP53 mutations. Cluster 3 had predominantly non-squamous histology, epithelial signature, and low TMB. Cluster 4 had exclusively non-squamous histology, predominant epithelial signature, low TMB, and was enriched for KEAP1 mutations. In addition, PD-L1 expression (by mIF) was negatively correlated with TGF-beta and WNT/Beta-catenin signaling pathway, as well as high EGFR and FGFR2 expression on malignant cells. Conclusion: CD8+ T-cell gene signature identifies immunosuppressed subgroups of lung cancer, especially among squamous cell carcinomas with a mesenchymal signature, that may be associated with resistance to immune-based therapies. TGF-beta and Wnt/beta-catenin pathways are associated with an immunosuppressed tumor microenvironment, and may be potential novel targets to restore anti-tumor immunity in lung cancers with low PD-L1 expression. Citation Format: Marcelo V. Negrao, Tatiana Karpinets, Jun Li, Alexandre Reuben, Cara Haymaker, Kyle G. Mitchell, Junya Fujimoto, Chi-Wan Chow, Edwin R. Parra, Lorenzo Federico, Jianhua Zhang, Ara A. Vaporciyan, Chantale Bernatchez, Tina Cascone, Boris Sepesi, Ignacio I. Wistuba, John V. Heymach, Jianjun Zhang, Don L. Gibbons, ICON Team. Immunogenomic profiling identifies a subgroup of squamous cell lung cancers with immunosuppressed tumor microenvironment and correlates TGF-beta and Wnt/beta-catenin signaling as predictive of low PD-L1 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1176.

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 .

The microRNA-200 (miR-200) family restricts epithelial–mesenchymal transition (EMT) and metastasis in tumor cell lines derived from mice that develop metastatic lung adenocarcinoma. To determine the mechanisms responsible for EMT and metastasis regulated by this microRNA, we conducted a global liquid chromatography/tandem mass spectrometry analysis to compare metastatic and nonmetastatic murine lung adenocarcinoma cells which had undergone EMT because of loss of miR-200. An analysis of syngeneic tumors generated by these cells identified multiple novel proteins linked to metastasis. In particular, the analysis of conditioned media, cell surface proteins, and whole-cell lysates from metastatic and nonmetastatic cells revealed large-scale modifications in the tumor microenvironment. Specific increases were documented in extracellular matrix (ECM) proteins, peptidases, and changes in distribution of cell adhesion proteins in the metastatic cell lines. Integrating proteomic data from three subproteomes, we defined constituents of a multilayer protein network that both regulated and mediated the effects of TGFβ. Lastly, we identified ECM proteins and peptidases that were directly regulated by miR-200. Taken together, our results reveal how expression of miR-200 alters the tumor microenvironment to inhibit the processes of EMT and metastasis. Cancer Res; 71(24); 7670–82. ©2011 AACR.

Background Advances in molecular analyses of tumor tissues based on high-throughput technologies can contribute to a better prediction of disease prognosis and the efficacy of immunotherapies. We investigated the GED related to immune response in a large cohort of NSCLC. Methods Using low-input, high-throughput HTG EdgeSeq assay, we examined expression of a panel of 549 immune response-related genes (28 are categorized as immune function genes) in FFPE tumor tissues of 226 surgically resected, stage I to III NSCLCs (137 adenocarcinomas [ADC] and 89 squamous cell carcinomas [SqCC]) with annotated clinical, pathological characteristics. Molecular data were available on mutations of EGFR and KRAS (cases=126), STK11, PIK3CA and TP53 by whole exome sequencing (n=180), and TAICs density detected by IHC (CD3, CD4, CD68, CD8, FOXP3, GZB, PD-L1 and PD-1) (n=145). We associated GED to 12 clinical, pathological or molecular characteristics including age, gender, smoking history, histology, stage, size, lymph node status and gene mutations of tumors and to patients' outcome (recurrence free survival [RFS] and overall survival [OS]). Results Univariate analysis showed upregulations of 82 genes in ADC and 89 in SqCC by comparing each other (P≤0.001). In ADC, 23 GEDs [FDR (false discover rate) ≤5%] associated with ever smoking history (8 up- and 15 down-regulated). Some genes were identified to be associated with mutations of EGFR, KRAS, STK11, PIK3CA and P53 genes. GEDs were identified in the tumors with TAIC high density by IHC. In ADC, 192 genes were down- and 191 up-regulated; while among the 28 immune function genes 21 down- and, only the gene APP (in all 8 high density TAICs) up-regulated. In SqCC, 121 genes down- and 112 up-regulated; while in the 28 genes 17 were down- and only the gene CAP up-regulated slightly in the high FOXP3 tumors. Cox proportional hazard model using FDR 5% in multi-testing showed that in ADC treated with adjuvant chemotherapy high-expression in 10 genes such as CD52, CD74 and HLA-DRA having a beneficial and 5 genes such as HRT2 and SERPINB2 having worse effect on OS; in the ADC patients who did not receive adjuvant therapy, high-expression of PIK3CD showed beneficial and, TTK and CXCL8 showed detrimental effect on OS; no gene was found to be associated with RFS in ADC and with OS and RFS in SqCC. Conclusion Gene expression profiling analysis of a panel of immune-related genes using low-input mRNA from FFPE tumor tissues revealed a number of genes associating to tumor characteristics, gene mutation patterns and TAIC infiltration in NSCLC. Interestingly, almost of immune function genes were down-regulated in the tumor with TAIC high density in NSCLC. (Supported in part by grant CPRIT RP160668) Citation Format: Ximing Tang, Wei Lu, Jiexin Zhang, Carmen Berhens, Edwin R. Parra, John Wineman, Jianjun Zhang, Don Lynn Gibbons, Mark Koeppel, BJ Kerns, Mark Stern, Boris Sepesi, Jack J. Lee, Ignacio I. Wistuba. Gene expression difference (GED) revealed immune function gene down-regulation as tumor-associated inflammatory cell (TAIC) infiltration in microenvironment in non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4054.

Background: The immunocheckpoint represents a group of membrane proteins expressing on immune cells, composed of multiple co-inhibitory and co-stimulatory pathways. They are involved in eliminating unwanted substances while maintaining self-tolerance, which play a crucial role in immunomodulation. Malignant cells (MCs), on the other hand, often tends to take advantage of immunocheckpoints, for instance, by activating inhibitory checkpoint pathways in situ, to evade immune responses. Therefore, cancer immunotherapies, which target key immune checkpoints to restore effective anti-tumor immune function, emerge as a revolutionary strategy during recent years. The aim of this study was analyze a full range of checkpoint available to investigate their role in the microenvironment and their frequent co-expression in MCs and tumor infiltrated lymphocytes (TILs) in non-small cell lung carcinomas. Material and Methods: 4 µm-thick histology sections obtained from formalin-fixed paraffin-embedded (FFPE) tissues from a TMA containing 256 NSCLCs (adenocarcinomas, ADC=156; squamous cell carcinomas, SCC=100); were used for multiplex immunofluorescence (mIF) analyses. Nine immune markers including PD-L1, B7-H3, B7-H4, IDO-1, ICOS, OX-40, LAG3, TIM3, VISTA, CD3 and CD20 was optimized for immunofluorescence. Two mIF immunocheckpoints panels were composed included seven antibodies as follows: Panel 1: AE1/AE3, PD-L1, B7-H3, B7-H4, IDO1, VISTA, and CD3; and Panel 2: AE1/AE3, ICOS, LAG3, TIM3, OX40, CD3, and CD20. For quantitative image analysis of mIF markers, the stained TMA slides were scanned using the Polaris™ multispectral microscope (Perkin Elmer) and analyzed using the inForm™ 2.3.1 software. Results: In this preliminary report using the median density in MCs and TILs expressing those markers, we identified overall higher expression of immunocheckpoints in SCC than ADC. The higher density co-expression by MCs (AE/AE3+) was B7-H3 + IDO-1+ following by B7-H3+PD-L1+ expression when compared with the other markers (P~0.001) in the panel 1. Furthermore, we observed higher densities of TILs (CD3+) expressing IDO-1+ as well as B7-H3+ and high densities of TILs (CD3+) co-expressing B7-H3+PD-L1+ when compared the other (P~0.001) markers in the same panel. In panel 2, we observed higher densities of TILs (CD3+) expression ICOS+ than the other markers (P~0.001). Conclusions: Here, we describe a mIF platform with computational image processing workflows, enabling simultaneous evaluation of 12 biomarkers to FFPE tissue section. These data highlight digital image processing pipelines to the community for examining immune complexity of immunocheckpoints expression in MCs and TILs to improve biomarker discovery and assessment. Citation Format: Edwin Roger Parra, Mei Jiang, Jiexin Zhang, Carmen Behrens, Jack Lee, John Victor Heymach, Marie-Andrée Forget, Cara Haymaker, Chantale Bernatchez, Cesar Moran, Jianjun Zhang, Don Gibbons, Ignacio Wistuba. Immunofluorescence profiling of co-expression of multiple immune checkpoints in malignant and tumor infiltrated lymphocytes in non-small cell lung carcinomas using image analysis system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3633.

Background: The clinical efficacy observed with PD-1/PD-L1 inhibitors in non-small cell lung carcinoma (NSCLC) has prompted to characterize the immune response in lung tumors treated with chemotherapy. The aim of this study was to determine the changes of the immune microenvironment in surgically resected NSCLCs from patients who received and did not receive neo-adjuvant chemotherapy. Methods: We studied formalin-fixed and paraffin embedded (FFPE) tumor tissues from 112 stage II/III resected NSCLC, including 61 chemotherapy-naïve (adenocarcinoma, ADC=33; squamous cell carcinoma, SCC=28) and 51 chemotherapy-treated (ADC=31; SCC=20) tumors. mIF was performed using the Opal 7-color fIHC Kit™, scanning in the Vectra™ multispectral microscope and analyzed using the inForm™ software (Perkin Elmer, Waltham, MA). The markers studied were grouped in two 6-antibody panels: Panel 1, AE1/AE3 pancytokeratins, PD-L1 (clone E1L3N), CD3, CD4, CD8 and CD68; and Panel 2, AE1/AE3, PD1, Granzyme B, FOXP3, CD45RO and CD57. Results: Positive PD-L1 expression (>5%) in malignant cells (MCs) was detected in 48% (n=53/112) of NSCLCs. Overall, chemotherapy-treated tumors showed significantly higher percentages of MCs expressing PD-L1 (median, 19.52%) than chemotherapy-naïve cases (median, 1.54%; P=0.008). Higher densities of lymphocytes expressing CD3+ (P=0.021), CD4+ (P=0.05), CD57 (P Conclusions: Neo-adjuvant chemotherapy-treated NSCLCs exhibited higher levels of PD-L1 expression and T cell subsets regulation compared to chemotherapy-naïve tumors, suggesting that chemotherapy activates specific immune response mechanisms in lung cancer. These results provide an inclination towards the design of clinical trials combining neo-adjuvant chemotherapy and immunotherapy prior to surgical resection of locally advanced NSCLC. (Supported by CPRIT MIRA and UT Lung SPORE grants, and MD Anderson Moon Shot Program). Citation Format: Edwin Roger Cuentas, Carmen Behrens, Jaime Rodriguez-Canales, Mei Jiang, Apar Pataer, Arelene Correa, Stephen Swisher, Boris Sepesi, Annikka Weissferdt, Neda Kalhor, Jiexin Zhang, Jack Lee, John Heymach, Cesar Moran, Jianjun Zhang, Don Gibbons, Ignacio Wistuba. Neoadjuvant chemotherapy influence changes of the immune response in non-small cell lung carcinomas immune response in non-small cell lung carcinomas [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 2934. doi:10.1158/1538-7445.AM2017-2934

Although strategies incorporating immune checkpoint inhibition, e.g. PD-1/PD-L1 blockade, are achieving unprecedented successes and increasingly becoming incorporated into standard of care regimens for cancer patients, high rates of resistance still limit the potential efficacy. Therapeutic improvement requires a thorough understanding of the biological process of resistance. To date there have been few studies reporting mechanisms of resistance to PD-L1 blockade. We have explored the resistance mechanisms to functional PD-L1 loss in preclinical lung cancer models by using pharmacological and genetic approaches (PD-L1 blocking antibody treatment or CRISPR/Cas9-mediated deletion of PD-L1 on tumor cells). The molecular and immune profiles of the tumor microenvironment were evaluated in mutant K-ras/p53 (KP) GEM lung cancer models and multiple immunocompetent syngeneic models (both KP and Lewis lung cancer). Additionally, to determine the applicability of the results to patients with lung cancer, we analyzed 259 patient tumor specimens with IHC staining and evaluated the immune markers in TCGA datasets (adenocarcinoma and squamous) and the MD Anderson PROSPECT dataset. We observed that lung tumors gained resistance to anti-PD-L1 antibody treatment over time, and that the up-regulation of CD38 on tumor cells accounted for the treatment resistance. We also observed the same resistance mechanism caused by CD38 up-regulation in PD-L1 KO mice bearing PD-L1 KO Lewis lung tumors generated with the CRISPR/Cas9 system. Manipulation of CD38 on a panel of lung cancer cell lines, demonstrated that CD38 inhibits CD8+ T cell proliferation, antitumor cytokine secretion, and tumor cell killing capability in vitro and in vivo. Furthermore, to test whether CD38 blockade might be therapeutically efficacious to counter anti-PD-L1 resistance, we applied the combination therapy of anti-CD38 and anti-PD-L1 in lung cancer animal models and demonstrated dramatic therapeutic benefit on primary tumor growth and metastasis. Bioinformatic analyses of the patient tumor databases revealed a strong correlation between CD38 expression and an immune suppressive inflammatory signature. Finally, in 259 lung cancer specimens, 18.5% of cases exhibited positive staining for CD38 on tumor cells. Based upon our studies, we conclude that the up-regulation of CD38 on tumor cells is a major mechanism of resistance to anti-PD-L1 therapy, and that CD38 is a novel immune checkpoint that inhibits CD8+ T cell function. The blockade of CD38 and PD-L1 is a rational combination to prevent immune resistance and increase the response rate for lung cancer patients. Citation Format: Limo Chen, Lixia Diao, Yongbin Yang, Xiaohui Yi, Jaime Rodriguez, Youhong Fan, Leticia Rodriguez, Jared Fradette, Christin Ungewiss, Jonothan Roybal, Jingfen Zhu, Jing Wang, Lauren Byers, Stephen Ullrich, Ignacio Wistuba, John Heymach, Xiao-Feng Qin, Don Gibbons. CD38 blockade overcomes the immune resistance to anti-PD-L1 therapy by boosting CD8 T cell response [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 567. doi:10.1158/1538-7445.AM2017-567

Metastatic lung cancer is the leading cause of cancer related death in the world and novel approaches are necessary to elucidate specific genes that drive lung cancer progression and metastasis. Lung cancers commonly demonstrate genetic alteration of potent drivers like Kras, p53 or EGFR accompanied with hundreds of low frequency gene aberrations which are a mix of key driver events and nascent passenger mutations. We established a robust screening platform to selectively identify these functionally critical “driver” genes in lung cancers from a prioritized list of candidates selected by a multi-level cross-species comparison of our published high confidence transcriptome data from genetically-engineered mouse models and genomic data of human lung cancers from TCGA, focusing on elevated gene expression and/or gene amplification. We identified 225 putative candidate driver genes which were used to construct a lentiviral based cDNA expression library with unique molecular barcoding of individual cDNAs. Using a non-metastatic syngeneic mouse lung cancer model we generated individually transduced stable over-expressing lines for each gene. These candidate lines were used for a unique in vivo positive selection screen to identify functional metastasis drivers. We transplanted pools of 20 cDNA expressing lines into syngeneic mice and observed for primary tumor growth and occurrence of metastases in lungs and other organs. Metastatic drivers were identified by the relative enrichment of the unique barcode sequences in the genomic DNA from metastatic lesions over primary tumors. We have identified both known (e.g. MYC) and several novel (e.g. THRA, TMEM106B, GNAS) potential oncogenic and metastatic drivers. We validated our top hits for their individual in vivo metastatic potencies by gain-of-function/loss-of-function studies and identified TMEM106B as one of the primary drivers of metastasis. TMEM106B is a transmembrane protein localizing to the lysosomes and has been shown to drive expression of lysosomal genes. We hypothesize that elevated levels of TMEM106B is able to drive the expression and secretion of lysosomal enzymes thus making cancer cells hyper invasive and metastatic. We found strong correlation for expression of TMEM106B with several lysosomal genes in a panel of 1016 human lung cancers and are currently performing in depth studies to understand these mechanisms of TMEM106B driven metastasis. We are also analyzing the clinical relevance of TMEM106B based upon their expression pattern and prognostic utility across TCGA and other public datasets, along with in-house patient samples and tissue microarrays of resected and biopsy specimens. Identification of such novel players will significantly advance the field of cancer target discovery by identifying new drug targets and biomarkers, essential for effective treatment options for lung cancer patients. Citation Format: Samrat Kundu, Caitlin Grzeskowiak, Chad J. Creighton, Kenneth L. Scott, Don L. Gibbons. Identifying TMEM106B as a novel metastasis driver in non-small cell lung cancers through an in vivo gain-of-function screen. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 688.

About 221,200 new patients will be diagnosed with lung cancer and ∼158,040 will succumb to this disease in the United States in 2015. Non-small cell lung cancer (NSCLC) has a 17.4% overall 5-year survival, with metastasis contributing to the vast majority of deaths. Analyzing NSCLC tumors spontaneously arising in KrasLA1/+; P53R172HG/+ (KP) mice, we identified Musashi-2 (MSI2) protein, a stem cell-associated factor that is regulates mRNA translation, as upregulated in the metastasis-competent mouse cell lines. Importantly, MSI2 shRNA depletion in either mouse or human NSCLC cells decreased invasion in Matrigel in vitro and decreased metastasis upon orthotopic injection 129Sv immunocompetent mice in vivo. Mechanistically, by both overexpressing Msi2 cDNA in 393p murine NSCLC and shRNA depleting MSI2 in four independent mouse and human NSCLC cell lines, we defined MSI2 as a driver of a partial epithelial-mesenchymal transition (EMT) program in NSCLC cells. In support of EMT, MSI2 increases the expression of the Snail and Slug pro-EMT transcription factors, as well as the mesenchymal protein vimentin (VMN). MSI2 also downregulates expression of the extracellular matrix component fibronectin (FN1) and tight junction proteins Claudin-3, 5 and 7. However, MSI2 also inhibits protein expression of Zeb-1 and Zeb-2, while sustaining expression of E-cadherin (E-Cad), associated with epithelial identity. Moreover, MSI2 represses NOTCH-1 and upregulates VEGFR2 at the mRNA and protein levels. This is of interest, as NOTCH-1 has been shown to regulate VEGFR2 in angiogenic signaling. We found that knockdown of NOTCH-1 in MSI2-depleted mouse and human NSCLC cells rescued the loss of VEGFR2 expression, suggesting MSI2 increases VEGFR2 expression in a NOTCH-1 dependent manner. Additionally, siRNA of VEGFR2 in the highly metastatic NSCLC cell line 344sq significantly decreased Matrigel invasion, but had only a limited effect in 344sq derivative lines with stable depletion of MSI2, and human NSCLC experiments are ongoing. Together, these results indicate a possible role of MSI2/NOTCH-1/VEGFR2 axis in NSCLC, and suggest that MSI2 connects cancer cell stemness, EMT, and angiogenesis in lung cancer metastasis. Citation Format: Alexander Kudinov, Alexander Deneka, Anna Nikonova, Ilya Serebriiskii, Tim N. Beck, Qi Cai, Brian L. Egleston, Emmanuelle Nicolas, Hossein Borghaei, Don Gibbons, Jonathan Kurie, Erica A. Golemis, Yanis Boumber. Musashi-2 (MSI2) drives TGFBR1/SMAD3 dependent partial EMT and supports VEGFR2 expression and metastasis of human and mouse NSCLC cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1584.

In the decades since the introduction of platinum-etoposide, treatment of small cell lung cancer (SCLC) remains virtually unchanged. Data from our lab and others have identified novel targets now being investigated in clinical trials. However, further characterization of the molecular heterogeneity within SCLCs will be critical to identify subsets with specific therapeutic vulnerabilities and change the treatment paradigm, similar to what has occurred in non-small cell lung cancer (NSCLC). Here we report expression analyses of 2578 microRNAs (miRs) in 62 SCLC cell lines, followed by unsupervised clustering to identify three distinct miR subtypes. Using ANOVA, we then identified differences in mRNA and protein expression (by reverse phase protein array), as well as drug response, which corresponded to miR expression profiles. The first subtype is characterized by low expression of miR-200, a family with known function in epithelial to mesenchymal transition (EMT) and previously implicated in cisplatin resistance. miR-200 low lines have significantly higher EMT scores (using our published EMT signature), as well as increased sensitivity to the polo-like kinase 1 (PLK-1) inhibitor GW843682X (p = 0.007), the sensitivity to which is associated with EMT in NSCLC. In addition, this subtype possesses increased expression of c-MYC (p = 1.03 e-05), a known repressor of miR-200. A second miR subtype is defined by reduced expression of miR-378, a family whose expression predicts cisplatin sensitivity in NSCLC. This subtype is also typified by increased expression of Wee1 (p = 5.91 e-04), a putative target in SCLC, as well as increased sensitivity to inhibitors of protein phosphatase 1D (PPM1D), tropomyosin receptor kinase A (TrkA) and B-Raf (p = 0.009, 0.011 and 0.032, respectively). The third subtype is distinguished by reduction in several miRs including miRs-216 and -217, a family with a putative role as tumor suppressors in pancreatic adenocarcinoma. Intriguingly, this subtype shows an increase in multiple proteins associated with immune checkpoint pathways, such as programmed death ligand 1 (PDL-1), cluster of differentiation 40 (CD40) and MHC class I polypeptide-related sequence A (MICA) (p = 6.38 e-04, 0.016 and 0.039, respectively) suggesting a group that may have enhanced response to immune-checkpoint inhibition. These subtypes are divided almost equally among the tested lines, constituting 34%, 32% and 34%, respectively. This is the first report of SCLC heterogeneity at the miR level and provides preliminary evidence regarding differences in mRNA and protein expression, as well as drug sensitivity between SCLC with distinct miR profiles. Integrated analysis across miRs, mRNA, protein and drug response represents a powerful tool for identifying candidate biomarkers for drug response, novel molecular targets and insights into the biological heterogeneity across SCLC. Citation Format: Carl M. Gay, Samrat Kundu, Lixia Diao, Bonnie S. Glisson, Jing Wang, John V. Heymach, Lauren A. Byers, Don L. Gibbons. Unique microRNA signatures in small cell lung cancer correlate with distinct protein expression profiles and drug response. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-170.

PURPOSE: Lung squamous cell carcinoma (LUSC) accounts for 20-30% of non-small cell lung cancers (NSCLCs). There are limited treatment strategies for LUSC in part due to our inadequate understanding of the molecular underpinnings of the disease. We sought to perform whole-exome sequencing (WES), comprehensive immune profiling and clinicopathological analysis of early-stage LUSCs to increase our understanding of the pathobiology of this malignancy. METHODS: Matched pairs of surgically resected stage I-III LUSCs and normal lung tissues (n = 108) were analyzed by WES. Immunohistochemistry and image analysis-based profiling of 10 immune markers was done on a subset of LUSCs (n = 91). Associations among mutations, immune markers and clinicopathological variables were statistically examined using ANOVA and Fisher tests. Cox proportional hazards regression models were used for statistical analysis of clinical outcome. RESULTS: This early-stage LUSC cohort displayed an average of 209 exonic mutations per tumor. Fourteen genes exhibited significant enrichment for mutation: TP53, MLL2, PIK3CA, NFE2L2, CDH8, KEAP1, PTEN, ADCY8, PTPRT, CALCR, GRM8, FBXW7, RB1 and CDKN2A. Among mutated genes associated with poor recurrence-free survival, MLL2 mutations predicted poor prognosis in both TP53 mutant and wild type LUSCs. We also found that in treated patients, FBXW7 and KEAP1 mutations were associated with poor response to adjuvant therapy, particularly in TP53-mutant tumors. Analysis of mutations with immune markers revealed that ADCY8 and PIK3CA mutations were associated with markedly decreased tumoral PD-L1 expression, LUSCs with PIK3CA mutations exhibited elevated CD45ro levels and CDKN2A-mutant tumors displayed an up-regulated immune response. CONCLUSION: Our findings pinpoint mutated genes that may impact clinical outcome as well as personalized strategies for targeted immunotherapies in early-stage LUSC. Citation Format: Murim Choi, Humam Kadara, Jiexin Zhang, Edwin Parra Cuentas, Jaime Rodriguez Canales, Stephen G. Gaffney, Zi-Ming Zhao, Carmen Behrens, Junya Fujimoto, Chi-Wan Chow, Neda Kalhor, Cesar Moran, David Rimm, Stephen Swisher, Don L. Gibbons, John V. Heymach, Edward Kaftan, Jeffrey Townsend, Thomas J. Lynch, Joseph Schlessinger, J. Jack Lee, Richard Lifton, Roy S. Herbst, Ignacio I. Wistuba. Mutation and immune profiles in early-stage lung squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 142.

PURPOSE: Lung adenocarcinomas (LUADs) lead to the preponderance of deaths attributable to lung cancer. We performed whole-exome sequencing (WES), comprehensive immune profiling and clinicopathological analysis of LUADs to better understand the molecular pathogenesis of this disease and identify clinically relevant molecular markers. METHODS: We performed WES of 108 paired surgically resected stage I-III LUADs and normal lung tissues using the Illumina HiSeq 2000 platform. Additionally, ten immune related markers (PD-L1, PD-1, CD3, CD4, CD8, CD45ro, CD57, CD68, FOXP3 and Granzyme B) were profiled by imaging-based immunohistochemistry in a subset of LUADs (n = 92). Associations among mutations, immune markers and clinicopathological variables were analyzed using ANOVA and Fishers Exact tests. Cox proportional hazards regression models were employed for multivariate analysis of clinical outcome. RESULTS: LUADs in this cohort exhibited an average of 243 coding mutations per tumor. We identified 28 genes with significant enrichment for mutation. SETD2-mutant LUADS exhibited relatively poor recurrence-free survival (RFS) and mutations in STK11 and ATM were associated with poor RFS in KRAS-mutant tumors. EGFR, KEAP1 and PIK3CA mutations were predictive of poor response to adjuvant therapy. Immune marker analysis demonstrated that PD-L1 expression was increased in smoker compared to non-smoker LUADs and, along with other immune markers, was positively correlated with somatic mutation burden. Moreover, immune marker levels including PD-L1 were elevated in TP53-mutant LUADs. In contrast, STK11 and U2AF1 mutant tumors exhibited a suppressed immune response and LUADs with PIK3CA mutations exhibited markedly decreased tumoral PD-L1 expression. CONCLUSION: Our study highlights mutations that may impact clinical outcome and personalized strategies for immune-based therapy of early-stage LUAD patients. Citation Format: Humam Kadara, Murim Choi, Jiexin Zhang, Edwin Parra Cuentas, Jaime Rodriguez Canales, Stephen Gaffney, Zi-Ming Zhao, Carmen Behrens, Junya Fujimoto, Chi-Wan Chow, Neda Kalhor, Cesar Moran, David Rimm, Stephen G. Swisher, Don L. Gibbons, John V. Heymach, Edward Kaftan, Jeffrey Townsend, Thomas J. Lynch, Joseph Schlessinger, J. Jack Lee, Richard Lifton, Ignacio I. Wistuba, Roy S. Herbst. Whole-exome sequencing and immune profiling of early-stage lung adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 89.

Either tumor cell epithelial-mesenchymal transition (EMT) or immunosuppression plays the very important role in metastasis during cancer progression. How these distinct processes are co-opted has remained obscure. To address this question, we have generated a cohort of epithelial and mesenchymal cell lines with manipulation of microRNA-200/ZEB1 (an EMT regulatory axis) in KrasLA1/+p53R172HΔg/+ spontaneous mouse model of lung cancer and performed tumor immune cell profiling and functional analysis. The data demonstrates that the microRNA-200 repression and ZEB1 activation target PD-L1 on tumor cells, leading to immunosuppression and metastasis by CD8+ T cell dysfunction, and that marked accumulation of regulatory T cells and myeloid-derived suppressor cells in mesenchymal tumors compared with epithelial tumors. Furthermore, by analyzing two large independent datasets (TCGA and PROSPECT) from lung cancer patients, our study demonstrates that EMT is highly associated with a distinct inflammatory tumor microenvironment, showing the elevation of multiple immune checkpoints such as PD-L1, PD-1, TIM-3, BTLA, CTLA-4 and B7-H3, high levels of tumor-infiltrating Foxp3+ regulatory T cells and immunosuppressive cytokines such as CXCL12, CCL2 and CCL18. The data discovered a link between EMT and immunosuppression, indicating that EMT causes immunosuppression, thereby metastasis. On the other hand, the immunosuppressive microenvironment transforms and/or maintains cancer cells in a mesenchymal state, therefore, cancer cells get the potential/power to metastasize. Immune suppressive cells are able to produce a diverse array of EMT inducers. For instance, T regulatory cells produce TGF-β;, IL-6, IL-10, and TNF-α;. Other immune suppressive cells such as myeloid-derived suppressor cells, tumor-associated macrophages, and tumor-associated neutrophils can also produce strong EMT inducers. Both human and mouse data convincingly established that EMT and immunosuppression form a double positive feed-forward loop, synergizing to promote tumor development. However, when the tumor-bearing mice were treated with anti-PD-L1 antibody, the mesenchymal tumors demonstrated greater sensitivity to the immunotherapy. This is a striking result because we have reported that the mesenchymal subpopulation accounts for aggressive growth, invasion, and metastasis. Since previous studies suggested that some chemotherapeutic agents can induce EMT, we likely find a way, targeting the loop of EMT and immunosuppression, to turn cancer's strengths into targetable weaknesses by combining EMT-induced chemotherapeutics and immunotherapeutics to reduce/prevent tumor metastasis. Citation Format: Limo Chen, Yanyan Lou, John Heymach, Ignacio Wistuba, Stephen Ullrich, Xiao-Feng Qin, Don Gibbons. Targeting the EMT-immunosuppression loop in lung cancer as a strategy to prevent metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr PR01.

Introduction: Lung cancer is the leading cause of cancer-related death, primarily due to distant metastatic disease. Metastatic cancer cells undergo an epithelial-to-mesenchymal transition (EMT) regulated by a double-negative feedback loop between the microRNA-200 (miR-200) family and Zeb1, but the precise mechanisms of Zeb1-dependent EMT in promoting malignancy remain largely undefined. While the cell-intrinsic effects of EMT are important for tumor progression, the reciprocal dynamic crosstalk between mesenchymal cancer cells and the extracellular matrix (ECM) is equally critical in regulating invasion and metastasis. This study investigates the collaborative effect of EMT and ECM in the metastatic process. Methods: Bioinformatic analysis of TCGA dataset was done to correlate ECM-associated gene expression with EMT gene signature scores. Western blotting and qPCR analysis of epithelial and mesenchymal lung cancer cell lines were performed to determine expression levels of collagen, LOX, and LOXL2. Lung tumor tissues from non-metastatic KrasG12D and metastatic KrasG12D;p53R172H mutant mice were analyzed by immunohistochemistry, Masson's trichrome staining, and second harmonics generation for collagen, LOX, and LOXL2 expression as well as collagen fiber organization. Syngeneic primary tumors generated by subcutaneous injection of murine lung cancer cell lines were analyzed in a similar fashion. Promoter and 3′-UTR luciferase reporter assays were performed to determine direct regulation LOXL2 and LOX by Zeb1 and miR-200, respectively. Results: Our results reveal increased collagen deposition in metastatic tumor tissues as a direct consequence of amplified collagen gene expression in Zeb1-activated mesenchymal lung cancer cells. Additionally, collagen fibers in metastatic lung tumors exhibit greater linearity and organization as a result of collagen crosslinking by the lysyl oxidase (LOX) family of enzymes. Expression of the LOX and LOXL2 isoforms is directly regulated by miR-200 and Zeb1, respectively, and their upregulation in metastatic tumors and mesenchymal cell lines is coordinated to that of collagen. Functionally, LOXL2, as opposed to LOX, is the principle isoform driving lung cancer metastasis by crosslinking and stabilizing insoluble collagen deposition in primary tumor tissues. Conclusions: Our study demonstrates that mesenchymal lung cancer cells metastasize by modulating the compositional and structural properties of the ECM through LOXL-mediated collagen crosslinking and deposition. We are the first to validate direct regulation of LOX and LOXL2 by the miR-200/Zeb1 axis, delineate collagen as a prognostic marker for lung cancer, and identify LOXL2 as a potential therapeutic target against tumor progression. Citation Format: David H. Peng, Pan Tong, Lauren A. Byers, Jing Wang, Chad J. Creighton, Don L. Gibbons. Zeb1 induces LOXL2-mediated collagen stabilization and deposition in the extracellular matrix to drive lung cancer invasion and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A27.

s: AACR Special Conference on Tumor Metastasis; November 30-December 3, 2015; Austin, TX Metastatic lung cancer is the leading cause of cancer related mortality in the world. Insights on specific genetic causes are limited and novel approaches are necessary to elucidate specific genes that drive lung cancer progression and metastasis. Extensive genomic and transcriptomic profiling of human lung cancers by consortia like ‘The Cancer Genome Atlas’ (TCGA) and the ‘International Cancer Genome Consortium’ (ICGC) has revealed a complex genetic landscape marked by abundant aberrations. In addition to acquiring mutations or amplification of well documented drivers like Kras, p53 or EGFR, lung cancers also demonstrate an enrichment of hundreds of low frequency gene aberrations which are a mix of key driver mutations and nascent passenger alterations caused by progressive genomic instability that could have definite role in cancer progression and metastasis. Our goal was to establish a robust screening platform to selectively identify these functionally critical “driver” genes in lung cancers. To identify a prioritized list of potential candidates we performed a multi-level cross-species comparison of our published high confidence transcriptome data from genetically-engineered mouse models of lung cancers and human lung cancer genomic data from TCGA, focusing on elevated gene expression and/or gene amplification. We identified an enriched list of 225 putative driver genes in lung cancers which were used to construct a lentiviral based cDNA expression library with unique molecular barcoding of individual cDNAs. Using a non-metastatic syngeneic mouse lung cancer model we generated individually transduced stable over-expressing lines for each of the 225 genes. These candidate lines were then entered into parallel in vitro and in vivo screens. In vitro testing includes cell invasion assays in a 96-well format, with positive hits defined as those scoring above 3x standard deviations relative to the control cells expressing mCherry. Positive hits were revalidated using standard 24-well invasion assays and also studied for growth phenotypes in 3D cultures. To selectively identify genes with functional role in metastasis, we performed a unique in vivo positive selection screen to test individual and combinatorial effects of lung cancer driver aberrations. For this we used pools of 20 cDNA expressing lines transplanted into syngeneic mice and observed for primary tumor growth and occurrence of metastases in lungs and other organs. Metastatic lesions and primary tumors were collected and genomic DNA from these was used to identify the unique individual barcodes by NGS barcode enrichment analysis. Metastatic drivers were identified by the relative enrichment of the unique barcode sequences for all the respective pool cDNAs in the metastatic tissue. We have identified both known (e.g. MYC) and several novel (e.g. THRA, TMEM106B, GNAS) potential oncogenic and metastatic drivers from the pooled screen which are currently being revalidated for their individual in vivo metastatic potencies. We are performing gain-of-function/loss-of-function studies for the identified driver genes to determine the pathways which they regulate. We are also analyzing the clinical relevance of these genes based upon their expression pattern and prognostic utility across TCGA and other public datasets, along with in-house patient samples and tissue microarrays of resected and biopsy specimens. Our unique high through-put approach will significantly advance the field of cancer target discovery by identifying new drug targets and biomarkers, essential for novel effective treatment options for lung cancer patients. Citation Format: Samrat T. Kundu, Caitlin Grzeskowiak, Chad J. Creighton, Kenneth L. Scott, Don L. Gibbons. A novel in vivo gain-of-function screen for metastasis drivers in non-small cell lung cancers. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B25.

Better understanding of the biophysical and biochemical cues of the tumor extracellular matrix environment that influence metastasis may have important implications for new cancer therapeutics. Initial exploration into this question has used naturally derived protein matrices that suffer from variability, poor control over matrix biochemistry, and inability to modify the matrix biochemistry and mechanics. Here, we report the use of a synthetic polymer-based scaffold composed primarily of poly(ethylene glycol), or PEG, modified with bioactive peptides to study murine models of lung adenocarcinoma. In this study, we focus on matrix-derived influences on epithelial morphogenesis of a metastatic cell line (344SQ) that harbors mutations in Kras and p53 (trp53) and is prone to a microRNA-200 (miR-200)–dependent epithelial–mesenchymal transition (EMT) and metastasis. The modified PEG hydrogels feature biospecific cell adhesion and cell-mediated proteolytic degradation with independently adjustable matrix stiffness. 344SQ encapsulated in bioactive peptide-modified, matrix metalloproteinase–degradable PEG hydrogels formed lumenized epithelial spheres comparable to that seen with three-dimensional culture in Matrigel. Altering both matrix stiffness and the concentration of cell-adhesive ligand significantly influenced epithelial morphogenesis as manifest by differences in the extent of lumenization, in patterns of intrasphere apoptosis and proliferation, and in expression of epithelial polarity markers. Regardless of matrix composition, exposure to TGF-β induced a loss of epithelial morphologic features, shift in expression of EMT marker genes, and decrease in mir-200 levels consistent with EMT. Our findings help illuminate matrix-derived cues that influence epithelial morphogenesis and highlight the potential utility that this synthetic matrix-mimetic tool has for cancer biology. Cancer Res; 72(22); 6013–23. ©2012 AACR.

Promising results in the treatment of non-small cell lung cancer (NSCLC) have been seen with immunomodulatory agents targeting immune checkpoints, such as programmed cell death 1 (PD-1) or programmed cell death 1 ligand (PD-L1). However, only a select group of patients respond to these interventions. The identification of biomarkers that predict clinical benefit to immune-based approaches, such as immune checkpoint blockade, is critical to successful translation of these agents. By using integrated gene array analysis in three large independent NSCLC patient datasets along with immunohistochemistry study (IHC), reverse phase protein array (RPPA) and in vivo animal study, we demonstrated that epithelial-mesenchymal transition (EMT) is highly associated with a profound inflammatory tumor microenvironment in lung adenocarcinoma. Our data revealed immune activation and simultaneous development of multiple immune suppressive mechanisms, including elevation of immune checkpoints such as PD-L1, PD-L2, PD-1, TIM-3, BTLA and CTLA-4, as well as an increase in tumor infiltrating CD4+Foxp3+ regulatory T cells, IL-6 and indoleamine 2,3-dioxygenase (IDO) in lung adenocarcinomas with a mesenchymal phenotype. Our data suggested that EMT might represent a potential biomarker to select the patients who will benefit from immune checkpoint blockade agents and other immunotherapies in NSCLC and possibly other cancers. Note: This abstract was not presented at the meeting. Citation Format: Yanyan Lou, Lixia Diao, Parra Cuentas Edwin Roger, Warren L. Denning, Limo Chen, Youhong Fan, Jaime Rodriguez, Lauren Byers, Jing Wang, Vassiliki Papadimitrakopoulou, Behrens Carmen, Ignacio I. Wistuba, Patrick Hwu, John V. Heymach, Don L. Gibbons. Epithelial-mesenchymal transition is associated with a profound inflammatory tumor microenvironment in lung adenocarcinoma. [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 3364. doi:10.1158/1538-7445.AM2015-3364

Traditional pathological divisions of lung cancer are giving rise to molecular classifications that identify unique drivers of tumor malignancy. Of these molecular classifications, KRAS represents about 30% of all lung adenocarcinomas. Our group has recently investigated if co-mutations within KRAS-mutated lung tumors can aid in identifying a unique therapeutic strategy for patient treatment. Given that tumors exert a strong influence over the immune system and the recent successes in immune targeting via checkpoint blockade therapy in various cancers, including lung, we hypothesized that co-mutational subgroups of KRAS-mutant NSCLC elicit distinct immune profiles and support a rationale for evaluating these subgroups in conjunction with appropriate immunotherapies for further clinical testing. To investigate this phenomenon, we performed a supervised analysis of 86 immune-related genes from The Cancer Genome Atlas data set in lung adenocarcinomas with KRAS mutations and TP53 or STK11(LKB1) co-mutations. The results from our analysis revealed that KRAS-mutant NSCLC with LKB1 (KL) and TP53 (KP) co-mutations have distinct immune profiles. KP tumors have an increase in (1) PD-L1 and PD-L2; (2) co-stimulation via CD40, CD80, CD86; (3) antigen presentation HLA-I, HLA-II, and CD1; (4) FOXP3 and CD4, indicating T regulatory populations; (5) CD33, CD11b, and CD11c, which are potentially suppressive myeloid cell populations. We conclude that KRAS co-mutations can create significantly different immune microenvironments; KRAS + TP53 (KP) mutant tumors may specifically benefit from checkpoint blockade, depletion of immunosuppressive myeloid and T regulatory populations, but could support de novo anti-tumor T cell expansion due to high expression of co-stimulatory molecules and HLA-I and HLA-II expression. Conversely, KRAS + LKB1 (KL) mutant tumors have an immunosuppressed or inert phenotype and may potentially benefit from adoptive cell transfer of anti-tumor T cells in conjunction with or in lieu of checkpoint blockade. Citation Format: Warren L. Denning, Lixia Diao, Ferdinandos Skoulidis, Yanyan Lou, Lauren Byers, Jing Wang, John Weinstein, Don Gibbons, John Heymach. Defined co-mutation subgroups of KRAS-mutated NSCLC display distinct immune profiles. [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 456. doi:10.1158/1538-7445.AM2015-456

Approximately 220,000 new patients were diagnosed with lung cancer and ∼160,000 died from this disease in the United States in 2014. Non-small cell lung cancer (NSCLC) has a 5 year survival rate of approximately 16%, with most deaths associated with distant metastasis. To gain a better insight into the regulation of metastasis, we studied metastasis-competent versus low metastasis-potential cell lines derived from NSCLC tumors of KrasLA1/+; P53R172HG/+ (KP) mice. Initial screening showed consistent and statistically significant upregulation of Musashi-2 (MSI2) in highly metastatic cells on both mRNA and protein level. Furthermore, we established statistically significant elevation of MSI2 protein expression in 123 human NSCLC tumor specimens versus normal lung tissue (p MSI2 is an RNA binding protein that regulates mRNA translation and is upregulated and functionally important in hematologic malignancies including CML and AML. MSI2 knockdown in four independent metastatic murine and human NSCLC cell lines very significantly decreased invasion in vitro but did not significantly change cell proliferation or survival. In orthotropic lung injection of mouse NSCLC cells into the lungs of immunocompetent 129Sv mice, MSI2 depletion dramatically decreased invasion of mediastinal lymph nodes and abrogated metastasis. Reverse-phase protein array (RPPA) screening indicated that Msi2 depletion significantly affected the expression of multiple proteins associated with epithelial-mesenchymal transition (EMT) and control of cell-cell attachments, including the extracellular matrix component fibronectin (FN1) and the tight junction protein claudin-7 (CLDN7). These and additional targets were validated in all four NSCLC model systems. MSI2 dependent control of FN1 expression was mediated at the level of mRNA induction, while CLDN7 expression was strikingly upregulated (10-fold) at the protein but not mRNA level, nominating CLDN7 as a putative direct MSI2 target. Parallel evaluation of a series of candidate direct MSI2 translational targets indicated MSI2 depletion downregulated translation of TGF-β receptor (TGF-βRI) and SMAD3. Morphologically, MSI2 depleted cells were marked by a greater degree of cell-cell attachment, potentially explaining their decreased invasive capacity. Based on this work, we propose that MSI2 supports TGF-βRI dependent EMT signaling and downregulates tight junction controls in a subset of metastatic NSCLC, and may influence tumor response to inhibitors targeting the TGF-β pathway. Citation Format: Alexander Kudinov, Alexander Deneka Deneka, Anna Nikonova, Young-Ho Ahn, Xin Liu Liu, Ilya Serebriiskii, Andrey Efimov, Dong-Hua Yang, Mark Andrake Andrake, Emmanuelle Nicolas, Brian Egleston, Hossein Borghaei, Don Gibbons, Jonathan Kurie, Erica Golemis, Yanis Boumber. Musashi-2 (MSI2) activates TGF-β signaling and inhibits CLDN7 to promote non-small cell lung cancer (NSCLC) metastasis. [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 4098. doi:10.1158/1538-7445.AM2015-4098

Background: Programmed cell death ligand 1 (PD-L1) is a major immune checkpoint protein that mediates anti-tumor immune suppression and response. Indeed, the importance of PD-L1 based immune suppression is highlighted by the advent of anti-PD-L1 immunotherapies that have moved to the forefront of lung cancer treatment. A better understanding of the “profile” of PD-L1 expression and its interplay with immune cells will provide important insights into lung cancer pathogenesis, and thus, improve immunotherapeutic strategies targeting this important immune checkpoint protein. The goal of this study was to investigate the correlation between immunohistochemical (IHC) expression of PD-L1 and the density and nature of tumor infiltrating immune cells in surgically resected lung adenocarcinomas, and correlate those profiles with clinical and pathological variables including patient outcome. Methods: We examined 146 stages I-III lung adenocarcinomas using whole tissue sections obtained from formalin-fixed and paraffin-embedded tissues. PDL-1 expression on tumor cells and density of inflammatory cells expressing PD-1, CD3, CD4, CD8, CD45RO, CD57, CD68, Granzyme B and FOXP3 were evaluated by automated IHC and quantified using image analysis in intra-tumoral (IT) and peri-tumoral (PT) compartments. PD-L1 expression was scored as positive if at least 5% of tumor cells showed membranous staining, and an immune-score (IMS) using CD8/CD4/CD68 was devised. KRAS and EGFR mutation status were available. Results: Thirty-four (23%) of 146 tumors had positive levels of PD-L1 IHC expression in malignant cells. Higher levels of PD-L1 expression were detected in tumors with solid histology pattern compared with other histologies (P = 0.034), and in lifetime smokers compared with non-smokers (P Conclusions: In lung adenocarcinoma, PD-L1 expression correlates with less differentiated tumors and higher level of tumoral immune infiltrate. We developed a lung adenocarcinoma IMS which when combined with PD-L1 expression significantly correlates with patient outcome when analyzed in surgically resected tumors specimens. (Supported by grants UT-Lung SPORE P50CA70907 and CPRIT RP120713). Citation Format: Edwin R. Parra, Carmen Behrens, Jaime Rodriguez-Canales, Heather Lin, Barbara Mino, Jorge Blando, Yanyan Lou, Don L. Gibbons, John V. Heymach, Stephen G. Swisher, Annikka Weissferdt, Neda Kahlor, Julie Izzo, J. Jack Lee, Humam Kadara, Cesar Moran, Ignacio Wistuba. High programmed cell death ligand 1 expression and low immune infiltrate score correlate with worse outcome in patients with lung adenocarcinoma. [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 4256. doi:10.1158/1538-7445.AM2015-4256

Background: Small cell lung cancer (SCLC) is the most aggressive form of lung cancer, accounting for 14% of lung cancers. It is associated with poor outcomes and few effective treatments. Identification of novel therapeutic targets is imperative for improving treatment outcomes. In our previous work we identified several DNA repair proteins including check point kinase 1 (Chk1) and poly (ADP-ribose) polymerase 1 (PARP1) that are overexpressed in SCLC and are high priority candidate targets. TP53-mutant cells (a hallmark of SCLC) rely on Chk1 to arrest the S and G2 phases of the cell cycle. Hence, inhibition of Chk1 in p53 mutant cells abrogates the S and G2 checkpoints driving such cells to mitotic catastrophe and apoptosis. In this study we tested the in vitro efficacy of Chk1 inhibitors as single agents and in combination with a PARP1 inhibitor and cisplatin. Experimental design: SCLC cell lines [human cell lines plus those from a patient derived xenograft (PDX) and genetically engineered mouse models (GEMMs)] were treated with Chk1 inhibitors (SCH900776, LY2603618, LY2606368) alone and in combination with olaparib (PARP inhibitor) or cisplatin. Cell proliferation was measured by Cell Titer Glo assay. Drug sensitivity (IC50) was correlated with baseline expression levels of >190 total or phosphorylated proteins measured by reverse phase protein array (RPPA) to identify potential predictive markers. Result: Single-agent Chk1 suppression decreased proliferation of SCLC cells in a dose-dependent manner. SCLC lines exhibited varied levels of sensitivity to the Chk1 inhibitors, with greatest single agent activity observed with LY2606368 (IC50 2-30nM). LY2606368 also showed appreciable single agent activity in PDX and GEMM-derived cell lines (IC50 2-7nM). Single agent activity was also observed with the other Chk1 inhibitors (SCH900776 IC50 0.3-10uM; LY2603618 IC50 0.2-10uM). Combination of LY2606368 with olaparib and cisplatin revealed an additive effect. Proteomic analysis revealed an association between Chk1 inhibitor sensitivity and elevated basal expression of pro-apoptotic proteins (cleaved PARP, Bax, and Caspase 3; p Conclusion: Identification and characterization of novel therapeutic targets is essential to improve efficacy of SCLC therapy. In this preliminary study we tested Chk1 inhibitors as potential therapies in SCLC and provide evidence that targeting Chk1 could be a promising therapeutic strategy for SCLC. Further preclinical and clinical investigation is needed to confirm the molecular mechanism and in vivo outcome of Chk1 targeting. Acknowledgement: 1. 2013 NCI Cancer Clinical Investigator Team Leadership Award, P30 CA016672 (Byers) 2. UT MD Anderson Small Cell Lung Cancer Working Group and Abell Hangar Foundation Distinguished Professor Endowment (Glisson). Citation Format: Aly A. Valliani, Triparna Sen, Fatehmeh Masrorpour, Lixia Diao, Robert J. Cardnell, Jing Wang, Bonnie S. Glisson, Helen Piwnica-Worms, Don L. Gibbons, Lauren A. Byers. Check point kinase 1 (Chk1) targeting as a novel therapeutic strategy in small cell lung cancer (SCLC). [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 5316. doi:10.1158/1538-7445.AM2015-5316

Large-scale profiling efforts by consortia such as The Cancer Genome Atlas (TCGA) are revealing the complexity of cancer genomes, which are comprised of causal “driver” aberrations and many biologically neutral “passengers”. Most cancers acquire one or more well-studied high frequency driver events that promote tumor growth (e.g., mutations/copy number changes in KRAS, TP53, EGFR, MYC). Much less is known about the thousands of low frequency gene aberrations and their contribution to cancer progression, particularly metastasis, which is the primary cause of cancer-related mortality. Comprehensive biological assessment of low frequency metastasis drivers is difficult given their large number and the fact that their activity may be influenced by the specific biological context of a given cancer such as tissue type, microenvironment, and the host immune system. We sought to address these challenges in the context of lung cancer, which presents as metastatic disease in approximately 65% of patients and carries a 5-year survival rate of 35,000 sequence verified human gene clones (2) a molecular barcoding strategy that permits simultaneous DNA tagging of gene clones through multi-fragment DNA recombineering for (3) pooled functional screening in vivo to identify metastasis drivers that work alone or in combination. We used these technologies to build gene libraries based on oncogenomics-guided integrations of mutant KRAS-specific gene signatures derived from mouse and human TCGA lung cancer datasets. The resulting barcoded libraries were delivered to non-metastatic lung cancer cells expressing oncogenic KRAS and then implanted into immune competent mice. Animals were sacrificed at maximal tumor burden and resulting primary tumors and metastases subjected to barcode enrichment analysis by next generation sequencing to identify gene aberrations enriched within metastatic lesions. Our screening approach has identified known (MYC and SNAI2) and many novel (e.g., MBIP and CCNE1) potent drivers of lung cancer growth and metastasis currently under mechanistic and pre-clinical evaluation. These efforts are revealing new pathways contributing to lung cancer aggression and our ultimate goal is to translate these findings into the care of metastatic lung cancer patients who have few treatment options. We have also scaled these efforts across other screening platforms, functionalizing thousands of aberrations across diverse cancer types. Together these systems reveal the highest priority targets to enroll in deep mechanistic biology studies and drug discovery and development programs. Citation Format: Caitlin L. Grzeskowiak, Rosalba Minelli, Ping Wu, Samrat Kundu, Don L. Gibbons, Kenneth L. Scott. High-throughput functional screening for metastasis drivers of lung cancer. [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 955. doi:10.1158/1538-7445.AM2015-955

The microRNA-200 family (miR-200) has been shown to be a master regulator of epithelial-to-mesenchymal transition (EMT), partially through a double-negative feedback loop with the transcription repressor Zeb1, allowing cells to acquire an invasive and metastatic phenotype. Using our mutant Kras and p53 murine model of metastatic lung adenocarcinoma, we are investigating the role of the Zeb1/miR-200 axis in regulating cytoskeletal reorganization, a characteristic requirement of cell motility and invasion. Functional assays demonstrated differential actin reorganization within cell lines with different miR-200 levels based upon the extracellular matrix composition, leading us to hypothesize that miR-200 regulates the activation of signaling pathways that mediate a pro-invasive cytoskeletal reorganization. Based upon the importance of cell-matrix interactions in producing cellular activation we identified the Src pathway as a critical mediator of invasion upon miR-200 repression in murine and human lung cancer cell lines. Pharmacologic inhibition of Src significantly reduces in vitro cell migration and invasion in Boyden chamber assays and in 3D cell cultures, which can be mimicked using Integrin β1 blockade. Biochemical analysis of this Src inhibition reveals a significant reduction in the phosphorylated FAK and cortactin levels, two downstream targets of Src. TGFβ stimulation is a strong EMT inducer, producing miR-200 repression and Src activation in our models, which can be blocked biochemically using Src inhibitors, with associated phenotypic changes in 2D and 3D cultures. This data suggests that miR-200 regulates the cell-matrix contact (e.g. integrins), acting through the Src pathway and modulating central players in actin dynamics to promote actin reorganization and invasion. Citation Format: Christin Ungewiss, Jonathon D. Roybal, David H. Peng, Don L. Gibbons. The microRNA-200/Zeb1 axis regulates ECM-dependent Src signaling, cytoskeletal changes and cancer cell invasion. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B44. doi:10.1158/1538-7445.CHTME14-B44

Lung cancer is the deadliest cancer in the United States due primarily to the development of metastatic disease, resulting in a 16% five year overall survival. Regrettably response rates are poor with the current modalities used to treat patients, which implies that a greater understanding of the biology of invasion and metastasis is indispensable. It is thought that the epithelial-mesenchymal transition (EMT) plays a crucial role in regulating invasion, migration, and metastasis. The microRNA-200 family (miR-200) is a key maintenance factor of the epithelial phenotype by regulating EMT and metastatic potential, although the mechanistic details of this regulation are incompletely understood. Recent research has demonstrated that miRNAs are capable of regulating the target mRNAs that coordinate intracellular signaling pathways. Using our mutant Kras and p53 murine model of metastatic lung adenocarcinoma, we have shown through Reverse Phase Protein Array (RPPA) profiling that high levels of miR-200 in poorly metastatic lung tumor cells can suppress cancer-promoting signaling molecules, such as Signal Transducer and Activator of Transcription 3 (STAT3). In contrast, highly metastatic lung tumor cells with low miR-200 expression have STAT3 activation and increased migratory and invasive properties. Utilizing pharmacologic approaches to inhibit STAT3 in highly metastatic lung tumor cells decreased their ability to migrate and invade. Our data suggests that the STAT3 pathway is under the control of miR-200 and inhibition in metastatic lung cancer could provide benefit to lung cancer patients as a potential treatment option. Citation Format: Jonathon D. Roybal, David H. Peng, Christin Ungewiss, Pijus K. Mandal, John S. McMurray, Lauren A. Byers, Don L. Gibbons. The role of the microRNA-200 family in the regulation of tumor cell signaling and metastasis in lung cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B36. doi:10.1158/1538-7445.CHTME14-B36

Tumor microenvironment plays a very important role in regulating the cell behavior within a tissue, largely by interacting with the extracellular matrix (ECM). Recently, our lab has developed a novel ex vivo lung model that forms perfusable tumor nodules, which mimic human lung cancer histopathology and protease secretion patterns better than tumor cells grown on a petri dish. In this study, we have compared the genome-wide gene expression profiles (Affymetrix 430_2) and protein arrays (Reverse phase protein array or RPPA) of 2D (monolayer) culture versus our ex vivo 3D cell culture using 393p cells, derived from mice lung cancer. Expression array data showed a set 1314 gene probes differentially expressed between monolayer 2D culture and 3D ex vivo cell culture. Gene Ontology (GO) analysis of differentially expressed genes showed the upregulation of several genes associated with extracellular matrix (ECM1, BMP15, FMOD, cathepsin, MMP9, MMP10), cell signaling (VEGFA, TGFB2, FGF20), and immune response (C1, C3, MHC class II, IL1α, IL1RA, IL13, TNF receptor superfamily, CXCL2). Selected genes were also validated using real time PCR. We found 46 proteins with significant (p Citation Format: Dhruva K. Mishra, Chad Creighton, Yiqun Zhang, Don L. Gibbons, Jonathan M. Kurie, Min P. Kim. Gene expression analysis of perfusable nodules grown on ex vivo 3D model shows effects on extracellular matrix, cell signaling and immune response. [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 1508. doi:10.1158/1538-7445.AM2013-1508

We have previously developed an in vivo mouse model of metastatic lung adenocarcinoma by combining a mutant p53 allele (p53R175HΔG) with a KrasG12D allele (KrasLA1). From the tumors arising in KrasLA1/+; p53R172HΔG/+ mice, we have established lung adenocarcinoma cell lines (KP cells) with high (344SQ) or low (393P) metastatic potential. The metastatic potential of KP cells is governed by Zeb-1 and the microRNA-200 family, which interact through a double-negative feedback loop. Given that metastatic KP cells exhibit the capacity to form polarized epithelial spheres in Matrigel and undergo epithelial-to-mesenchymal transition (EMT) in response to extracellular cues, we hypothesized that stem cell factors serve as drivers of tumor cell invasion and metastasis in this model. We examined the expression of 13 stem cell factors in metastatic and non-metastatic KP cell lines using qPCR and found that Musashi-1 (Msi-1) was more highly expressed in metastatic KP cells, which was confirmed by western analysis. Msi1 is an RNA-binding protein that plays a central role during nervous system development, stem cell maintenance, and tumorigenesis. High levels of Msi1 have been reported in several malignancies including brain tumors, colon and breast cancer, and associate with poor survival in patients with breast and colon cancer. We have used a panel of human lung cancer cell lines and found Msi-1 protein to be expressed in lung adenocarcinoma and squamous carcinoma cell lines. Further analysis revealed that Msi-1 expression is up-regulated by Zeb-1 and down-regulated by miR-200, raising the possibility that Msi-1 functions as a downstream mediator of the Zeb-1/miR-200 axis, which we are currently investigating by carrying out Msi-1 loss-of-function and gain-of-function experiments and by analyzing human tissue microarrays to determine whether Msi-1 has prognostic value in patients. Our data suggests that Msi-1 is a candidate driver of tumor progression in NSCLC. Citation Format: Yanis Boumber, Alexander Kudinov, Xin Liu, Young-Ho Ahn, Jaime Rodriguez, Ignacio Wistuba, Don Gibbons, Jonathan Kurie. The role of musashi-1 in tumor progression in non-small cell 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 2690. doi:10.1158/1538-7445.AM2013-2690

Lung cancer is one of the deadliest cancers in the United States due primarily to the development of metastatic disease, resulting in a 16% five year overall survival. Unfortunately response rates are poor with the current modalities used to treat patients, which implies that a greater understanding of the biology of invasion and metastasis is essential. It is thought that the epithelial-mesenchymal transition (EMT) plays a critical role in regulating invasion, migration, and metastasis. The microRNA-200 family (miR-200) is a key maintenance factor for the epithelial phenotype by regulating EMT-inducing transcription factors such as Zeb1, and subsequent metastatic potential, although the mechanistic details of this regulation are incompletely understood. Recent research has demonstrated that miRNAs are capable of regulating the target mRNAs that coordinate intracellular signaling pathways. Using our mutant Kras and p53 murine model of metastatic lung adenocarcinoma, we have shown through reverse phase protein array (RPPA) profiling that in poorly metastatic lung tumor cells, high miR-200 levels can suppress cancer-promoting signaling molecules, such as Signal Transducer and Activator of Transcription 3 (STAT3). In contrast, highly metastatic lung tumor cells with low miR-200 expression have STAT3 activation and increased migratory and invasive properties. Utilizing siRNA approaches or pharmacological inhibition of STAT3 in highly metastatic lung tumor cells decreased their ability to migrate and invade. Our data suggests that the STAT3 pathway is under the control of miR-200/Zeb1 axis and could provide a potential therapeutic target in lung cancer patients. Citation Format: Jonathon D. Roybal, David H. Peng, Christin Ungewiss, Pijus K. Mandal, John S. McMurray, Lauren A. Byers, Don L. Gibbons. The role of the microRNA-200 family in the regulation of tumor cell signaling and metastasis in lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C57.

Lung cancer is the leading cause of all cancer-related deaths, accounting for 160,000 deaths per year in the US, primarily caused by metastasis. Understanding the molecular mechanisms leading to metastasis will identify new therapeutic targets for improved treatment strategies. Using our mutant Kras and p53 murine model of metastatic lung adenocarcinoma, we have shown that loss of microRNA-200 family (miR-200) expression is necessary and sufficient to drive an epithelial-to-mesenchymal transition (EMT), allowing cells to acquire an invasive and metastatic phenotype necessary for metastasis. Cell motility is characterized by the reorganization of the actin cytoskeleton, for which we are currently investigating the role of miR-200. Cortactin is a scaffold protein known to play a central role in different signaling pathways involved in the formation of pro-invasive cell structures by recruiting actin-associated proteins and proteases leading to the degradation of the extracellular matrix (ECM). Maturation of cell protrusions requires the phosphorylation of cortactin by upstream tyrosine kinases such as src and arg. Functional assay of cell behavior on 2D agarose demonstrates actin reorganization within our cell lines with different miR-200 levels. Biochemical analysis of cell lines from our model and human lung cancer cells shows that metastatic cells (low-miR-200) have increased levels of phosphorylated cortactin compared to the non-metastatic cells (high miR-200), leading us to hypothesize that miR-200 regulates the biochemical activation of signaling pathways that mediate cortactin phosphorylation and a pro-invasive cytoskeletal reorganization. Inhibition of src using pharmacologic inhibitors and an siRNA approach significantly reduces phosphorylated cortactin levels, inhibits in vitro cell migration and invasion, and decreases the invasiveness of cells grown in 3D, demonstrating the importance of this pathway in invasion. Furthermore, our study demonstrates that the pro-survival PI3K pathway can be inhibited using cortactin siRNA, underlining the multi-faceted role of cortactin in tumor progression. Citation Format: Christin Ungewiss, Jonathon D. Roybal, David H. Peng, Don L. Gibbons. microRNA-200 control of tumor cell cytoskeletal changes and invasion. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C56.