Vulnerabilities In Lung Cancer Research Articles

Abstract 4372: Epithelial-to-mesenchymal transition-driven secretory program underlies vulnerabilities in lung cancer

Abstract Epithelial-to-mesenchymal transition (EMT) is a driving force behind lung adenocarcinoma (LUAD) progression. Although substantial evidence has been attained in the involvement of EMT and its regulatory mechanisms in cancer, few effective EMT-targeted therapies have been available in the clinic thus far. It is urgent to develop new therapeutic strategies for improving patient outcomes. We have identified RAB6A as a master regulator of EMT-driven secretory trafficking, which coordinates cancer cell invasion and immunosuppression in LUAD. We found that RAB6A expression was associated with a poor prognosis in LUAD patients and that RAB6A inhibition in our LUAD models reversed resistance to immune checkpoint inhibition with PD-L1 blockade, an emerging clinical problem. Thus, RAB6A machinery is a therapeutic target of interest in EMT-driven cancers. Screening approaches to identify selective RAB inhibitors are in their infancy, a challenge that can be overcome only by obtaining insights into the regulation of RAB6A. RAB6A is a small GTPase whose activity is controlled by guanine nucleotide exchange factors (GEFs) like RIC1 and RGP1, and GTPase activating proteins (GAPs) like RABGAP1, as well as other effector proteins. While these GEFs, GAPs, and effectors are all known regulators of RAB6A function and are frequently amplified in different cancer types, their specific functions in cancer progression are still unknown. To establish the causal relationship between these proteins and EMT-driven LUAD progression, we utilize human and murine LUAD cell lines that have been characterized with respect to EMT status. Our findings here show that the intervention of RAB6A activity via depletion of these regulators impairs the recruitment of RAB6A to the Golgi. We demonstrate that knocking down the GEFs, RIC1 and RGP1, significantly decreases invasion while having negligible effects on the migrative and proliferative abilities of these cells. RABGAP1 depletion, on the other hand, reduces cell proliferation but has no effects on the migration and invasion of the cells. Consistent with these results, inactivation of RAB6A abolishes the EMT-dependent pro-metastatic secretion. Mechanistically, the EMT activator, ZEB1, upregulates RGP1 via silencing miR-148a that targets this GEF to activate RAB6A. These findings elucidate the regulatory mechanisms underlying the EMT-driven secretory program, providing a foundation for therapeutic intervention to prevent the progression of LUAD. Citation Format: Kevin L. Fulp, Oluwafunminiyi E. Obaleye, Guan-Yu Xiao. Epithelial-to-mesenchymal transition-driven secretory program underlies vulnerabilities in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4372.

Abstract 2932: Computational discovery of paralog dependencies drives target identification in lung cancer

Abstract Lung cancer is the leading cause of cancer-related death in the United States, and worldwide, with the 5-year survival rate of non-small cell lung cancer at 24%. Treatment options are limited, and even in cancers that harbor druggable biomarkers, resistance is near-universal. Identifying novel treatment strategies is essential to improve the outcomes of patients with lung cancer and provide secondary strategies for those who acquire resistance to first-line therapeutics. Identifying targeted therapies based on the genotype or expression pattern of cancers is an ongoing endeavor. In lung cancer, treatment of patients that harbor activating mutations in EGFR with specific EGFR inhibitors has improved progression-free survival rates and extended the lifespan of patients diagnosed with this aggressive cancer, demonstrating the utility of targeted therapies based genetic-based vulnerabilities, or synthetic lethal interactions. A rich source of synthetic lethal genetic interactions are paralogous genes. Paralogs result from gene duplication events through evolutionary history, and many paralogs share both sequence homology and functional redundancy. Co-targeting of some paralogous genes induces a fitness defect that is synergistic compared to individual paralog loss alone. Nearly 70% of genes in the human genome are considered paralogs, and until recent efforts from the Berger lab and others, this buffering effect has caused them to be under-represented in lethality studies. Identifying frequently-inactivated paralog genes in lung cancer will provide a platform to identify new vulnerabilities in lung cancer and other cancer types that share mutational backgrounds. To identify patterns of mutation and expression of paralog genes in lung cancer, we have accessed publicly available data sets for both cell line and patient tumor samples. Using TCGA and CCLE data sources, and a set of 1030 paralog pairs identified previously by the Berger lab as potential drug targets, we have identified paralog genes that harbor high mutational frequencies in lung cancer. Using these candidates, we next asked whether genetic inactivation or transcriptional repression of one paralog influences the essentiality of its paired gene using CERES scores generated by the Cancer Dependency Map. Preliminary results identifies several paralog pairs with distinct dependency profiles in cell lines for which expression of one paralog is lost, including MAGOH/MAGOHB and TLK1/TLK2. Further work to characterize these dependencies in relevant cell line models is ongoing. Additionally, the druggability of these targets will be tested using targeted degradation using the dTAG system. This work will uncover a new set of paralog synthetic lethality targets, and further validate the utility of targeting known synthetic lethal paralogs for the treatment of highly lethal lung cancer. Citation Format: Siobhan O'Brien, Alice H. Berger. Computational discovery of paralog dependencies drives target identification in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2932.

UHRF1 is a mediator of KRAS driven oncogenesis in lung adenocarcinoma

KRAS is a frequent driver in lung cancer. To identify KRAS-specific vulnerabilities in lung cancer, we performed RNAi screens in primary spheroids derived from a Kras mutant mouse lung cancer model and discovered an epigenetic regulator Ubiquitin-like containing PHD and RING finger domains 1 (UHRF1). In human lung cancer models UHRF1 knock-out selectively impaired growth and induced apoptosis only in KRAS mutant cells. Genome-wide methylation and gene expression analysis of UHRF1-depleted KRAS mutant cells revealed global DNA hypomethylation leading to upregulation of tumor suppressor genes (TSGs). A focused CRISPR/Cas9 screen validated several of these TSGs as mediators of UHRF1-driven tumorigenesis. In vivo, UHRF1 knock-out inhibited tumor growth of KRAS-driven mouse lung cancer models. Finally, in lung cancer patients high UHRF1 expression is anti-correlated with TSG expression and predicts worse outcomes for patients with KRAS mutant tumors. These results nominate UHRF1 as a KRAS-specific vulnerability and potential target for therapeutic intervention.

CBX5 loss drives EGFR inhibitor resistance and results in therapeutically actionable vulnerabilities in lung cancer

Although epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFRi) are approved for treating EGFR-mutant lung adenocarcinoma (LUAD), emergence of acquired resistance limits their clinical benefits. Several mechanisms for acquired resistance to EGFRi in LUAD have been identified; however, the molecular basis for this resistance remains unknown in ~30% of LUAD. Chromatin and DNA modifiers and their regulators play important roles in determining response to anticancer therapies. Therefore, to identify nongenetic mechanisms of EGFRi resistance in LUAD, we performed an epigenome-wide shRNA screen targeting 363 human epigenetic regulator genes. This screen identified loss of the transcriptional repressor chromobox homolog 5 (CBX5) as a driver of EGFRi resistance in EGFR-mutant LUAD. Loss of CBX5 confers resistance to multiple EGFRi in both cell culture and mice. We found that CBX5 loss in EGFR-mutant LUAD cells leads to increased expression of the transcription factor E2F1, which in turn stimulates expression of the antiapoptotic gene BIRC5 (survivin). This E2F1-mediated upregulation of BIRC5 in CBX5-knockdown LUAD cells attenuates apoptosis induction following EGFRi treatment. Consistent with these results, knockdown of E2F1 or BIRC5 partly rescues CBX5-knockdown-induced EGFRi resistance in cell culture and mice. EGFRi-resistant LUAD cell lines show reduced CBX5 expression compared to parental lines; however, bromo- and extra-terminal (BET)-domain inhibitors (BETi) restore CBX5 expression in these cells and sensitize them to EGFRi/BETi combination therapy. Similarly, treatment with a BIRC5 inhibitor suppresses growth of EGFRi-resistant LUAD cells. Collectively, these studies identify CBX5 loss as a driver of EGFRi resistance and reveal therapeutic opportunities for treating EGFRi-resistant LUAD.

Integrative metabolomics and transcriptomics analysis reveals novel therapeutic vulnerabilities in lung cancer.

Non-small cell lung cancer (NSCLC) comprises the majority (~85%) of all lung tumors, with lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) being the most frequently diagnosed histological subtypes. Multi-modal omics profiling has been carried out in NSCLC, but no studies have yet reported a unique metabolite-related gene signature and altered metabolic pathways associated with LUAD and LUSC. We integrated transcriptomics and metabolomics to analyze 30 human lung tumors and adjacent noncancerous tissues. Differential co-expression was used to identify modules of metabolites that were altered between normal and tumor. We identified unique metabolite-related gene signatures specific for LUAD and LUSC and key pathways aberrantly regulated at both transcriptional and metabolic levels. Differential co-expression analysis revealed that loss of coherence between metabolites in tumors is a major characteristic in both LUAD and LUSC. We identified one metabolic onco-module gained in LUAD, characterized by nine metabolites and 57 metabolic genes. Multi-omics integrative analysis revealed a 28 metabolic gene signature associated with poor survival in LUAD, with six metabolite-related genes as individual prognostic markers. We demonstrated the clinical utility of this integrated metabolic gene signature in LUAD by using it to guide repurposing of AZD-6482, a PI3Kβ inhibitor which significantly inhibited three genes from the 28-gene signature. Overall, we have integrated metabolomics and transcriptomics analyses to show that LUAD and LUSC have distinct profiles, inferred gene signatures with prognostic value for patient survival, and identified therapeutic targets and repurposed drugs for potential use in NSCLC treatment.

Glucocorticoid receptor triggers a reversible drug-tolerant dormancy state with acquired therapeutic vulnerabilities in lung cancer

The glucocorticoid receptor (GR) regulates gene expression, governing aspects of homeostasis, but is also involved in cancer. Pharmacological GR activation is frequently used to alleviate therapy-related side-effects. While prior studies have shown GR activation might also have anti-proliferative action on tumours, the underpinnings of glucocorticoid action and its direct effectors in non-lymphoid solid cancers remain elusive. Here, we study the mechanisms of glucocorticoid response, focusing on lung cancer. We show that GR activation induces reversible cancer cell dormancy characterised by anticancer drug tolerance, and activation of growth factor survival signalling accompanied by vulnerability to inhibitors. GR-induced dormancy is dependent on a single GR-target gene, CDKN1C, regulated through chromatin looping of a GR-occupied upstream distal enhancer in a SWI/SNF-dependent fashion. These insights illustrate the importance of GR signalling in non-lymphoid solid cancer biology, particularly in lung cancer, and warrant caution for use of glucocorticoids in treatment of anticancer therapy related side-effects.

CSNK1A1, KDM2A, and LTB4R2 Are New Druggable Vulnerabilities in Lung Cancer.

Simple SummaryThe main histological subtypes of lung cancer are small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC). NSCLC is further subdivided into squamous-cell carcinoma (SCC) and adenocarcinoma (AD). Despite the recent introduction of innovative therapies, lung cancer is still the first cause of cancer-related human death, indicating that the discovery of new therapeutic targets is still a compelling need for this disease. In the present work, we performed a functional genomics analysis on different lung cancer histotypes, combining data derived from different omics resources with in vitro validation. Through this approach, we identified and validated CSNK1A1, KDMA2, and LTB4R2 as new druggable vulnerabilities in lung cancer. These results open new possibilities for the development of innovative therapies for lung cancer patients.Lung cancer is the leading cause of cancer-related human death. It is a heterogeneous disease, classified in two main histotypes, small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC), which is further subdivided into squamous-cell carcinoma (SCC) and adenocarcinoma (AD) subtypes. Despite the introduction of innovative therapeutics, mainly designed to specifically treat AD patients, the prognosis of lung cancer remains poor. In particular, available treatments for SCLC and SCC patients are currently limited to platinum-based chemotherapy and immune checkpoint inhibitors. In this work, we used an integrative approach to identify novel vulnerabilities in lung cancer. First, we compared the data from a CRISPR/Cas9 dependency screening performed in our laboratory with Cancer Dependency Map Project data, essentiality comprising information on 73 lung cancer cell lines. Next, to identify relevant therapeutic targets, we integrated dependency data with pharmacological data and TCGA gene expression information. Through this analysis, we identified CSNK1A1, KDM2A, and LTB4R2 as relevant druggable essentiality genes in lung cancer. We validated the antiproliferative effect of genetic or pharmacological inhibition of these genes in two lung cancer cell lines. Overall, our results identified new vulnerabilities associated with different lung cancer histotypes, laying the basis for the development of new therapeutic strategies.

Upregulation of Thr/Tyr kinase Increases the Cancer Progression by Neurotensin and Dihydropyrimidinase-Like 3 in Lung Cancer.

Lung cancer is one of the leading causes of cancer-related death globally, thus elucidation of its molecular pathology is highly highlighted. Aberrant alterations of the spindle assembly checkpoint (SAC) are implicated in the development of cancer due to abnormal cell division. TTK (Thr/Tyr kinase), a dual serine/threonine kinase, is considered to act as a cancer promoter by controlling SAC. However, the mechanistic details of how TTK-mediated signaling network supports cancer development is still a mystery. Here, we found that TTK was upregulated in the tumor tissue of patients with lung cancer, and enhanced tumor growth and metastasis in vitro and in vivo. Mechanistically, TTK exerted a significant enhancement in cancer growth by neurotensin (NTS) upregulation, and subsequently increased the expression of cyclin A and cdk2, which was resulting in the increase of DNA synthesis. In contrast, TTK increased cell migration and epithelial-to-mesenchymal transition (EMT) by enhancing the expression of dihydropyrimidinase-like 3 (DPYSL3) followed by the increase of snail-regulated EMT, thus reinforce metastatic potential and ultimately tumor metastasis. TTK and DPYSL3 upregulation was positively correlated with a poor clinical outcome in patients with lung cancer. Together, our findings revealed a novel mechanism underlying the oncogenic potential effect of TTK and clarified its downstream factors NTS and DPYSL3 might represent a novel, promising candidate oncogenes with potential therapeutic vulnerabilities in lung cancer.

A09 Transcriptional Subtypes Resolve Tumor Heterogeneity and Identify Therapeutic Vulnerabilities in Lung Cancer

A09 Transcriptional Subtypes Resolve Tumor Heterogeneity and Identify Therapeutic Vulnerabilities in Lung Cancer

IBS05.02 Functional Genomic Approaches to Identify Novel Therapeutic Targets in Lung Cancer

Despite the discovery and availability of targeted therapies and immunotherapies, lung cancer remains the leading cause of cancer death worldwide. Importantly, most lung cancer patients are not eligible for targeted therapies because their tumors lack an actionable genomic alteration. Moreover, immunotherapy-based regimens fail to induce treatment responses in a substantial proportion of lung cancer patients (1-3). Therefore, the identification of novel therapeutic modalities remains critical to improving outcomes in lung cancer care. Lung cancer cells may harbor specific genomic or functional alterations that render them vulnerable to particular genetic perturbations (4,5). Discovery of these synthetic lethal interactions may provide opportunities to develop novel classes of therapeutics for this disease. Through systematic analysis of genome-scale loss-of-function datasets (6,7), we identify adenosine deaminase acting on RNA (ADAR or ADAR1) as an essential gene for the survival of a subset of lung cancer cell lines (8). ADAR1-dependent cell lines display increased expression of interferon-stimulated genes. Moreover, activation of type I interferon signaling in the context of ADAR1 deficiency can induce cell lethality in non-ADAR1-dependent cell lines. ADAR deletion causes activation of the cytoplasmic double-stranded RNA sensor, protein kinase R (PKR). Disruption of PKR signaling, through inactivation of PKR or overexpression of either a wild-type or catalytically inactive mutant version of ADAR1, partially rescues cell lethality after ADAR1 loss, suggesting that both catalytic and non-enzymatic functions of ADAR1 may contribute to preventing PKR-mediated cell lethality. Taken together, these data nominate ADAR1 as a potential therapeutic target in lung cancers displaying elevated interferon-stimulated gene expression and underscore the ability of functional genomic approaches to uncover novel genetic vulnerabilities in lung cancer. 1. Gandhi L, Rodriguez-Abreu D, Gadgeel S, Esteban E, Felip E, De Angelis F, et al. Pembrolizumab plus Chemotherapy in Metastatic Non-Small-Cell Lung Cancer. N Engl J Med 2018;378:2078-92 2. Paz-Ares L, Luft A, Vicente D, Tafreshi A, Gumus M, Mazieres J, et al. Pembrolizumab plus Chemotherapy for Squamous Non-Small-Cell Lung Cancer. N Engl J Med 2018;379:2040-51 3. Reck M, Rodriguez-Abreu D, Robinson AG, Hui R, Csoszi T, Fulop A, et al. Pembrolizumab versus Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer. N Engl J Med 2016;375:1823-33 4. Oike T, Ogiwara H, Tominaga Y, Ito K, Ando O, Tsuta K, et al. A synthetic lethality-based strategy to treat cancers harboring a genetic deficiency in the chromatin remodeling factor BRG1. Cancer Res 2013;73:5508-18 5. Zhou Z, Patel M, Ng N, Hsieh MH, Orth AP, Walker JR, et al. Identification of synthetic lethality of PRKDC in MYC-dependent human cancers by pooled shRNA screening. BMC Cancer 2014;14:944 6. Tsherniak A, Vazquez F, Montgomery PG, Weir BA, Kryukov G, Cowley GS, et al. Defining a Cancer Dependency Map. Cell 2017;170:564-76 e16 7. Aguirre AJ, Meyers RM, Weir BA, Vazquez F, Zhang CZ, Ben-David U, et al. Genomic Copy Number Dictates a Gene-Independent Cell Response to CRISPR/Cas9 Targeting. Cancer Discov 2016;6:914-29 8. Gannon HS, Zou T, Kiessling MK, Gao GF, Cai D, Choi PS, et al. Identification of ADAR1 adenosine deaminase dependency in a subset of cancer cells. Nat Commun 2018;9:5450 Functional genomics, Target discovery, Innate immune signaling

Abstract A2-06: An in vivo shRNA functional genomics screen identifies transcriptional and epigenetic regulators that are essential in lung tumorigenesis

Abstract Cancer cells are characterized by the aberrant regulation of signaling pathways that govern response to external stimuli, resulting in uncontrolled cellular proliferation. The genomic alterations that accumulate to cause the malignant growth phenotype can also result in acquired vulnerabilities in the tumor. Loss-of-function screening using pooled short hairpin RNAs (shRNAs) is proving to be a powerful method by which new cancer therapeutic targets can be identified. A major limitation of these studies is that they are usually performed in vitro under low selection pressure, thus potentially missing key vulnerabilities that might be detected in a more clinically relevant setting. Here we tested the use of parallel in vitro and in vivo screens using a mini-library of shRNAs to identify previously unknown acquired vulnerabilities in lung cancer. Using a mini-library of 1062 lentiviral shRNAs targeting nuclear hormone receptors and their coregulators (120 genes total), we screened the lung adenocarcinoma cell line NCI-H1819 for dependency on these genes during in vitro and in vivo growth. Briefly, cells were transduced with the library, allowed to undergo a 4 day period of puromycin selection, and then were either injected subcutaneously into NOD-SCID mice or maintained in 2D tissue culture. Tumors were harvested after ~2 months when reaching a volume of 300 mm3. In a parallel in vitro screen, transduced cells were cultured for 20 population doublings. The relative abundance of individual shRNAs was quantified by next generation sequencing, and candidate essential genes were nominated by filtering for depleted shRNAs that scored in a combination of two statistical methods (t-test and ranked KS-test). We identified six genes required for survival of H1819 in vitro (BRCA1, CCND1, MED1, PHB, HNRNPU, and PELP1), and three genes that were required for tumor survival in vivo, but not in vitro: FOXA1, HDAC1, and NCOR2. None of these genes were mutated by full exome sequencing of H1819, however FOXA1 was found to be co-amplified with NKX2-1 on chromosome 14q. The NKX2-1 gene has previously been identified as one of the most frequent focally amplified genes in lung adenocarcinomas, and we found that FOXA1 is co-amplified with NKX2-1 in 80% of these cases. We also found that NSCLC cell lines and tumor samples have significantly higher FoxA1 expression than normal lung epithelial cells and tissues. Knockdown of Foxa1 in amplified NSCLC cell lines causes significant reduction of clonogenicity, anchorage-independent growth, and in vivo xenograft growth. FoxA1 is a pioneer transcription factor, able to interact with condensed chromatin and recruit other transcription factors to their target genes. Notably, both HDAC1 and NCOR2 are also known to play roles in epigenetic regulation of gene expression. The identification of FOXA1, HDAC1, and NCOR2 as genes that are specifically required for tumor growth in vivo suggests that there are transcriptional and epigenetic programs in lung cancer which depend on microenvironmental cues and/or regulation by paracrine signaling mechanisms. We conclude that our shRNA mini-library screen identifies unsuspected acquired vulnerabilities that are only detected in vivo, and thus new therapeutic targets in lung cancer. Citation Format: Suzie K. Hight, Elizabeth McMillan, Chunli Shao, Luc Girard, Jaime Rodriguez Canales, Michael A. White, Ignacio I. Wistuba, David Mangelsdorf, John D. Minna. An in vivo shRNA functional genomics screen identifies transcriptional and epigenetic regulators that are essential in lung tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-06.

Abstract 4999: Signaling-associated complexes to define targetable vulnerabilities in lung cancer

Abstract Lung cancer is the leading cause of cancer related death in the U.S. Despite successes targeting tyrosine kinase drivers such as EGFR and EML4-ALK, identification of patients who will benefit from emerging targeted therapy regimens remains a challenge. This is exemplified by the disappointing results in the recent Phase III evaluation of ornatuzumab in combination with erlotinib in advanced non-small cell lung cancer (NCT01456324), targeting both MET and EGFR in biomarker-defined patient populations. We have previously shown that proximity ligation assay (PLA) technology can be harnessed to evaluate EGFR pathway activation by annotating the presence of EGFR in complex with its major signaling adaptor GRB2. Presence of these “signaling-associated complexes” correlates with EGFR activity, reveals erlotinib pharmacodynamics and associates with improved outcomes to EGFR-directed therapies. Here, we show that PLA technology can also be harnessed to assess MET signaling activity in lung cancer. We have found that MET is constitutively associated with GRB2 in MET-amplified lung cancer cell lines and this interaction is abrogated by the MET-specific tyrosine kinase inhibitor PHA665,752 (MET TKI) as measured by biochemical approaches and PLA. In parallel experiments, siRNA-mediated knockdown of GRB2 and 100nM MET TKI both lead to dramatic reductions in cell viability, indicating that this interaction is critical in MET-driven cells. Conversely, GRB2 knockdown has no effect in MET-amplified H1648 cells which are also completely resistant to MET TKI. In MET¬-amplified patient-derived xenograft models of lung cancer we observe MET:GRB2 complexes in regions that also stain strongly using pMET(Y1234/5) immunohistochemistry (IHC). However, only a subset of regions exhibiting strong staining for total MET by IHC had MET:GRB2 complexes or pMET. Surprisingly, we have observed a very low frequency (2%) of lung adenocarcinoma patients (N = 169) with MET:GRB2 signaling complexes, even among high MET-expressing tumors, defined by MET IHC. These data suggest that MET:GRB2 is critical in the amplified setting, but may be less important in stromal-induced MET signaling. Consistent with this, GRB2 knockdown in non-amplified A549 and H292 cells fails to impair HGF-induced increases in pAKT and pERK. We are currently conducting mass spectrometry experiments to decipher differences between MET complexes in the amplified vs. ligand-stimulating setting and updated results will be presented. We suggest that only a subset of high MET-expressing cells has evidence of MET in complex with GRB2, potentially explaining the relatively low response rates observed with MET-directed therapies in lung cancer. We believe our approach of combining biochemical, PLA and mass spectrometry-based techniques will also be applicable to the development of improved biomarker strategies for newly emerging targets such as FGFR1. Citation Format: Matthew Smith, Thomas Licata, Yun Bai, Guolin Zhang, Vincent Vuaroqueaux, Heinz-Herbert Fiebig, Eric B. Haura. Signaling-associated complexes to define targetable vulnerabilities in 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 4999. doi:10.1158/1538-7445.AM2015-4999

Abstract 1530: Identification of vulnerabilities in lung cancer via pooled short hairpin RNA screening

Abstract A major goal of cancer research is to identify tumor-specific vulnerabilities by which cancer cells can be selectively killed. The clinical success of targeted therapies along with technological advances in genome-wide profiling have spurred large-scale, on-going efforts to systematically map the molecular landscapes of human cancers. These approaches nominate candidate essential or tumor suppressor genes, but are not able to identify all types of acquired vulnerabilities, such as synthetic lethal relationships between aberrantly regulated pathways. Functional studies are therefore an essential complement to these approaches, serving both to confirm the biological roles of candidate cancer genes, and also to identify other vulnerabilities that are acquired as a consequence of observed genetic alterations. Loss-of-function studies using pooled short hairpin RNAi screening is proving to be a powerful method by which new cancer targets can be identified. In order to identify new vulnerabilities in lung cancer, we have chosen a focused approach using mini-libraries of short hairpins against selected genes of interest. Nuclear hormone receptors and their co-regulators are aberrantly regulated in many cancers, and therapies targeting these receptors are currently used in treatment of breast and prostate cancers. Quantitative PCR expression data across a panel of lung cancer cell lines suggests that nuclear hormone receptors may be useful as prognostic biomarkers in lung cancer patients, but the role of these receptors in lung tumor biology is not fully understood. We have used a lentiviral pooled short hairpin library of 1062 shRNAs against 120 nuclear hormone receptor and co-regulator genes to identify lung cancer-specific vulnerabilities, both in vitro and in vivo. Briefly, cells were transduced with the library, allowed to undergo a period of selection and expansion, and then injected subcutaneously into NOD-SCID mice. Tumors were harvested after reaching a volume of 300 mm3. A parallel in vitro screen was also performed, in which transduced cells were cultured for 20 population doublings. The relative abundance of individual shRNAs was quantified by next generation sequencing, and genes for which 3 or more shRNAs exhibited depletion either in vitro or in vivo were nominated as candidate essential genes. A screen performed in the lung adenocarcinoma cell line H1819 identified 10 genes that were required for in vitro survival, including BRCA1 and PHB. Five genes were also identified as being required in vivo but not in vitro, including HDAC1 and NCOR2. None of these were found to be mutated by exome sequencing of H1819. Validation studies and investigations into the role for each of these genes in lung cancer are currently on-going. We conclude that our shRNA mini-library screen identifies unsuspected synthetic lethal relationships and thus new therapeutic targets in lung cancer, including some that are only detected by in vivo screening. Citation Format: Suzie Hight, Ryan Carstens, Luc Girard, David Mangelsdorf, John D. Minna. Identification of vulnerabilities in lung cancer via pooled short hairpin RNA screening. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1530. doi:10.1158/1538-7445.AM2014-1530

Abstract 1525: Exome sequencing to identify permissive mutations representing acquired vulnerabilities in lung cancer

Abstract When a lung epithelial cell acquires a driver mutation (e.g. in KRAS) it often needs to acquire additional ‘permissive’ genetic alterations to enable the cell to tolerate the oncogenic change. These ‘permissive' alterations represent an ‘acquired vulnerability' and consequently a novel therapeutic target in lung cancers driven by known, but currently un-targetable, driver mutations. Immortalized Human Bronchial Epithelial Cells (HBECs) represent a cell appropriate in vitro model to study the origin and pathogenesis of lung cancer following introduction of defined oncogenic changes. We have previously transformed HBECs to full malignancy by introducing five genetic manipulations (to CDK4, TERT, TP53, KRAS, MYC, BCL2, PTEN and/or STK11). While malignant transformation will be largely driven by the exogenous oncogenotype -i.e. the introduced oncogenic alterations- we hypothesize the cells also must undergo additional somatic ‘permissive' changes to bypass anti-tumor mechanisms. While lung cancers are often highly mutated (mainly due to carcinogens in cigarette smoke), manipulated HBECs should exhibit fewer somatic mutations, providing greater power to identify important somatically acquired mutations that facilitate tumorigenic progression of lung epithelial cells. We have sequenced the exomes of a series of 22 genetically manipulated HBECs derived from two individuals and representing 15 unique oncogenotypes (combinations of 3-5 introduced manipulations). Paired-end sequencing was performed with an Illumina HiSeq 2000 using a VCRome 2.1 liquid capture design ‘exome chip’. The median fold coverage was 114X, resulting in at least 90% of targeted bases are covered at >20-fold. Somatic mutations were called by comparing manipulated HBECs to their immortalized parental cell line. Manipulated HBECs had a median somatic mutation rate of 0.92 mutations/Mb, 7-9-fold lower than observed in lung cancers and tumor cell lines. The median mutation rate increased with the number of introduced manipulations (HBECs with three, four and five manipulations had 0.47, 0.97 and 0.92 mutations/Mb, respectively). Non-synonymous somatic mutations were identified in 470 genes. Thirty-nine genes were mutated in ≥3 HBECs (recurrent mutations) and of these, 35 genes (none currently regarded as ‘driver’ oncogenes and 5 potentially ‘druggable' [DGIdb]) were also mutated in primary lung adenocarcinoma and squamous cell carcinomas (TCGA data). We found the introduction of defined oncogenic alterations in HBECs is accompanied by the acquisition of somatic mutations. Leveraging against public somatic mutation data for lung tumors has allowed us to prioritize mutations for follow up to better identify potential acquired vulnerabilities in lung cancer cells and therefore, novel therapeutic targets. Citation Format: Jill E. Larsen, Caleb F. Davis, Kenneth Huffman, Luc Girard, David A. Wheeler, Richard A. Gibbs, John D. Minna. Exome sequencing to identify permissive mutations representing acquired vulnerabilities in lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1525. doi:10.1158/1538-7445.AM2014-1525

Developmental signalling pathways in lung cancer

Hedgehog, Notch and Wnt signalling are all essential for axial patterning and progenitor cell fates in signalling pathways conserved from flies to humans. Aberrant activation of these pathways is observed in a wide variety of cancers, suggesting that these embryonic signalling pathways contribute in a fundamental way to the evolution and maintenance of a malignant phenotype. Because all three of these pathways participate in lung development, recent studies have begun to explore the connection between lung development, airway epithelial repair and lung cancer. Development, repair and malignant transformation of the neuroendocrine lineage are all accompanied by aberrant Hedgehog pathway activation, whereas Notch and Wnt signalling may be important in other airway cell types. Small molecule targeting of these pathways may provide therapeutic opportunities in lung cancer. The plant-derived alkaloid cyclopamine is a naturally occurring Hedgehog pathway inhibitor that shows therapeutic promise in small cell lung cancer, a highly aggressive neuroendocrine tumour. A more detailed understanding of how embryonic signalling pathways participate in airway epithelial repair and tumourigenesis may reveal more novel therapeutic vulnerabilities in lung cancer.