Ryan Carstens

Inhibition of Cancer Cell Proliferation by PPARγ Is Mediated by a Metabolic Switch that Increases Reactive Oxygen Species Levels

The nuclear receptor peroxisome-proliferation-activated receptor gamma (PPARγ), a transcriptional master regulator of glucose and lipid metabolism, inhibits the growth of several common cancers, including lung cancer. In this study, we show that the mechanism by which activation of PPARγ inhibits proliferation of lung cancer cells is based on metabolic changes. We found that treatment with the PPARγ agonist pioglitazone triggers a metabolic switch that inhibits pyruvate oxidation and reduces glutathione levels. These PPARγ-induced metabolic changes result in a marked increase of reactive oxygen species (ROS) levels that lead to rapid hypophosphorylation of retinoblastoma protein (RB) and cell-cycle arrest. The antiproliferative effect of PPARγ activation can be prevented by suppressing pyruvate dehydrogenase kinase 4 (PDK4) or β-oxidation of fatty acids in vitro and in vivo. Our proposed mechanism also suggests that metabolic changes can rapidly and directly inhibit cell-cycle progression of cancer cells by altering ROS levels.

Functional polyesters enable selective siRNA delivery to lung cancer over matched normal cells.

Significance Ideal cancer therapeutics accurately hit tumors and avoid side effects on healthy cells. We used a patient-derived pair of matched cancer/normal cell lines to discover selective nanoparticles that could deliver a cytotoxic siRNA to kill cancer cells and not normal cells. The finding that cells respond differently to the same nanoparticle has profound implications for gene therapy because cell-type specificity of drug carriers in vivo could alter clinical patient outcomes. Our data suggest that selectivity is an underappreciated reality that should be carefully considered when evaluating drug carriers. The combination of both well-defined molecular targets and nanoparticle delivery to targeted cells is likely required to improve cancer drug accuracy in the clinic. Conventional chemotherapeutics nonselectively kill all rapidly dividing cells, which produces numerous side effects. To address this challenge, we report the discovery of functional polyesters that are capable of delivering siRNA drugs selectively to lung cancer cells and not to normal lung cells. Selective polyplex nanoparticles (NPs) were identified by high-throughput library screening on a unique pair of matched cancer/normal cell lines obtained from a single patient. Selective NPs promoted rapid endocytosis into HCC4017 cancer cells, but were arrested at the membrane of HBEC30-KT normal cells during the initial transfection period. When injected into tumor xenografts in mice, cancer-selective NPs were retained in tumors for over 1 wk, whereas nonselective NPs were cleared within hours. This translated to improved siRNA-mediated cancer cell apoptosis and significant suppression of tumor growth. Selective NPs were also able to mediate gene silencing in xenograft and orthotopic tumors via i.v. injection or aerosol inhalation, respectively. Importantly, this work highlights that different cells respond differentially to the same drug carrier, an important factor that should be considered in the design and evaluation of all NP carriers. Because no targeting ligands are required, these functional polyester NPs provide an exciting alternative approach for selective drug delivery to tumor cells that may improve efficacy and reduce adverse side effects of cancer therapies.

Systematic siRNA Screen Unmasks NSCLC Growth Dependence by Palmitoyltransferase DHHC5

Protein S-palmitoylation is a widespread and dynamic posttranslational modification that regulates protein–membrane interactions, protein–protein interactions, and protein stability. A large family of palmitoyl acyl transferases, termed the DHHC family due to the presence of a common catalytic motif, catalyzes S-palmitoylation; the role of these enzymes in cancer is largely unexplored. In this study, an RNAi-based screen targeting all 23 members of the DHHC family was conducted to examine the effects on the growth in non–small cell lung cancer (NSCLC). Interestingly, siRNAs directed against DHHC5 broadly inhibited the growth of multiple NSCLC lines but not normal human bronchial epithelial cell (HBEC) lines. Silencing of DHHC5 by lentivirus-mediated expression of DHHC5 shRNAs dramatically reduced in vitro cell proliferation, colony formation, and cell invasion in a subset of cell lines that were examined in further detail. The phenotypes were restored by transfection of a wild-type DHHC5 plasmid but not by a plasmid expressing a catalytically inactive DHHC5. Tumor xenograft formation was severely inhibited by DHHC5 knockdown and rescued by DHHC5 expression, using both a conventional and tetracycline-inducible shRNA. These data indicate that DHHC5 has oncogenic capacity and contributes to tumor formation in NSCLC, thus representing a potential novel therapeutic target. Implications: Inhibitors of DHHC5 enzyme activity may inhibit non–small cell lung cancer growth. Mol Cancer Res; 13(4); 784–94. ©2015 AACR.

A subset of nuclear receptors are uniquely expressed in uveal melanoma cells

Uveal melanoma (UM) is recognized as the most common intraocular malignancy and the second most common form of melanoma. Nearly 50% of UM patients develop untreatable and fatal metastases. The 48-member nuclear receptor (NR) superfamily represents a therapeutically targetable group of transcription factors known for their regulation of key cancer pathways in numerous tumor types. Here, we profiled the expression of the 48 human NRs by qRT-PCR across a melanoma cell line panel including 5 UM lines, 9 cutaneous melanoma (CM) lines, and normal primary melanocytes. NR expression patterns identified a few key features. First, in agreement with our past studies identifying RXRg as a CM-specific marker, we found that UM cells also exhibit high levels of RXRg expression, making it a universal biomarker for melanoma tumors. Second, we found that LXRb is highly expressed in both UM and CM lines, suggesting that it may be a therapeutic target in a UM metastatic setting as it has been in CM models. Third, we found that RARg, PPARd, EAR2, RXRa, and TRa expressions could subdivide UM from CM. Previous studies of UM cancers identified key mutations in three genes: GNAQ, GNA11, and BRAF. We found unique NR expression profiles associated with each of these UM mutations. We then performed NR-to-NR and NR-to-genome expression correlation analyses to find potential NR-driven transcriptional programs activated in UM and CM. Specifically, RXRg controlled gene networks were identified that may drive melanoma-specific signaling and metabolism. ERRa was identified as a UM-defining NR and genes correlated with its expression confirm the role of ERRa in metabolic control. Given the plethora of available NR agonists, antagonists, and selective receptor modulators, pharmacologic manipulation of these NRs and their transcriptional outputs may lead to a more comprehensive understanding of key UM pathways and how we can leverage them for better therapeutic alternatives.

Abstract 5757: A new functional classification of lung and breast cancers based on siRNA mediated knockdown of 48 NRs and 72 CoRegulators

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: Nuclear receptors (NRs) and their co-regulators (CoRegs) are master regulators of key transcriptional processes important in cancer pathogenesis and could provide molecular biomarkers for prognosis and personalizing new cancer targeted therapy. Aims and Methods: Our goal is to functionally classify a panel of 54 lung cancer, 20 breast cancer, and several immortalized human lung and breast epithelial cell strains based on their growth and survival response to siRNA mediated knockdown of 48 NRs and 72 CoRegs. Following knockdown of individual NR/CoRegs, changes in growth patterns are quantified and statistical methods are used to identify disease subgroups of similar responders. These subgroups are then integrated with our legacy datasets for clinical demographics, cancer cell oncogenotype and genome wide molecular characterization, and responses to standard and targeted chemotherapy. Results and Conclusions: A diverse, representative panel consisting of lung cancer, breast cancer, and normal epithelial cell lines was tested by siRNA transfection. After optimization to determine transfection conditions, all cell lines were screened using a focused siRNA library targeting all 48 NRs and 72 CoRegs (each gene is targeted by pools of 4 different siRNAs, Qiagen). Screening experiments were performed in triplicate, in 96 well plates, in a five-day MTS assay and assays were repeated 3 times. Correlation between replicates for the same cell line is >0.84 and knockdowns were validated by qRT-PCR. We found: 1) NR/CoReg-specific knockdown could have no effect, increase, or decrease tumor cell viability in a cell line dependent manner. 2) Overall ∼70% of the NRs or CoRegs reduced cell viability in at least one tumor line when their expression was knocked down (examples included NURR1, PRMT1, RXRalpha, BRCA1). 3) NR/CoReg-specific tumor cell toxicity was validated independently by siRNA knockdown combined with a liquid colony formation assay. 4) Knockdown of several NR/CoRegs selectively killed tumor but not normal epithelial cells. 5) Tumor cell lines could be stratified into functionally unique categories based on their changes in growth following NR/CoReg-specific knockdowns. We conclude that siRNA mediated knockdown of a large number of NRs and their CoRegs identifies genes that show tumor cell selective toxicity, and that tumor lines vary in their siRNA response phenotypes. These data strengthen the concept for NR/CoReg targeted therapy of lung and breast cancer and also the need to “personalize” such therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5757. doi:1538-7445.AM2012-5757

Abstract 4779: RUVBL1 and RUBVL2 are chromatin remodelers that represent prognostic and novel therapeutic targets for a subset of non-small cell lung cancers (NSCLCs)

RUVBL1 and RUVBL2 (collectively referred to as RUVBL1/2) are AAA+ ATPases that function in various chromatin remodeling complexes. We found that RUVBL1/2 are overexpressed (n = 224) and prognostic of patient outcome in NSCLC patients (n = 697) who undergo surgical resection (combined TCGA, KMPlot.com, and SPORE P50CA70907 dataset analyses). To assess the importance and biological functions of RUVBL1/2 in NSCLC, we measured cell growth following depletion of RUVBL1/2 in 24 NSCLC lines, representing a spectrum of oncogenotypes and histologies, and 2 normal human bronchial epithelial cell lines (HBECs) using two independent RNAi reagents. Growth inhibitory phenotypes ranged from 19-87% and were “rescued” by exogenous expression of an RNAi-resistant cDNA construct, indicating “on-target” siRNA effects. Four of the 23 NSCLCs were very sensitive (>75% growth inhibition), while 88% of NSCLCs were more growth inhibited than HBECs, indicating a therapeutic window. To establish molecular biomarkers of RUVBL1/2 dependency, we correlated whole exome mutation status, whole transcriptome mRNA levels, and a number of other parameters to sensitivity to RUVBL1/2 depletion. Sensitivity to RUVBL1/2 knockdown did not correlate with the mutation status or expression of any genes. However, sensitivity to RUVBL1/2 depletion did correlate with doubling time. Flow cytometry analysis in NSCLC lines sensitive to RUVBL1/2 KD revealed that RUVBL1/2 depletion resulted in a G2/M arrest (but not apoptosis), and a decrease in cell cycle related transcripts, such as CDKN3, AURKA, CIT and CDC20, whereas resistant cell lines do not exhibit these changes. Initial ChIP-seq analysis suggests that RUVBL1/2 preferentially occupies cell cycle related genes and are depleted in nucleosome free regions (i.e. transcription start sites). These results, combined with results from others demonstrating the necessity of ATP hydrolysis for RUVBL1/29s function(s), indicate that the chromatin remodelers RUVBL1/2 are potential therapeutic targets in a subset of NSCLCs. (Supported by UTSW Green Center Fellowship, CPRIT RP120732, SPORE P50CA70907) Citation Format: Paul M. Yenerall, Rahul Kollipara, Ryan Carstens, Kenneth Huffman, Luc Girard, Jaime Rodriguez, Ignacio Wistuba, David Mangelsdorf, John Minna, Ralf Kittler. RUVBL1 and RUBVL2 are chromatin remodelers that represent prognostic and novel therapeutic targets for a subset of non-small cell lung cancers (NSCLCs). [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 4779. doi:10.1158/1538-7445.AM2015-4779

Abstract IA21: Developing a new functional classification of lung cancer based on tumor acquired vulnerabilities.

We have been comprehensively screening for “vulnerabilities” that have been acquired during the multi-step pathogenesis of lung cancer cells but are not present in normal lung epithelial cells to identify genetic and chemical perturbations that will selectively kill lung cancer. We think many of these have occurred to allow the lung cancers to undergo/tolerate “oncogene addiction.” We tested a sub-panel of 12-15 non-small cell lung cancer (NSCLC) lines that covers the known molecular spectra of lung cancer with genome wide siRNA and large scale chemical library (~250,000 compounds) and natural products in vitro screens to identify “hits” that will kill (suppress the growth of) lung cancer cells but not normal human bronchial epithelial cells and that also only kill a subset of lung cancer cells providing two types of specificity. “Hits” from these broad screens are then tested (including detailed drug concentration curves) across a large panel of lung cancer lines (~100) representing a variety of lung cancer histologic and molecular oncogenotypes. Other versions of these screens include the intensive use of “mini-libraries” each containing 50 – 150 gene targets by siRNAs or shRNAs, or ~200 defined drugs to explore pathways in detail in tests of over 70 NSCLCs. Examples include: nuclear receptors and their co-regulators (120 genes); cancer stem cell pathways (50 genes); chromatin remodelers (75 genes) and identified lung cancer mutated driver oncogenes (175 genes). In addition to the in vitro tests, we have developed in vivo (xenograft) tests where shRNA mini-libraries are introduced into tumor cells at high representation which are grown as xenografts, analyzed by NexGen sequencing and shRNAs identified that drop out or are retained in xenografts compared to in vitro grown cells to identify vulnerabilities that are only detected in the in vivo situation. All of the data are then related to the large legacy molecular datasets associated with the lung cancer lines (including whole exome sequence analyses and genome wide mRNA, copy number variation, methylation, miR expression data and proteomics data). In addition, detailed chemical and pharmacokinetic analyses for favorable drug properties and subsequent chemical modifications also occur for the chemical compounds to progress those towards potential clinical studies. The results of all of these analyses have identified ~300 new chemical compounds and ~300 genetic hits all of which show selectivity for lung cancer over normal lung cells and selectivity for subtypes of lung cancer. The chemical and genes hits are being compared to the tumor molecular information and integrated in turn through a “connectivity map” type of approach – to identify drugs and gene hits involving the same pathways. The molecular correlates of the tumor lines are related to similar molecular changes in patient derived xenografts and patient tumor specimens to provide a connection of the molecular subtype-selective vulnerabilities (“enrollment biomarkers”) between the preclinical response phenotypes and patient tumor specimens. From these data we find lung cancers can be classified into groups (“clades”) that represent functional vulnerabilities to the gene and chemical compound hits and these in turn can be related to molecular abnormalities in tumors. One example of this is our detailed analyses a matched lung adenocarcinoma/normal lung epithelial cell model derived from the same patient which identified three distinct target/response-indicator pairings that are represented a significant frequencies (6-16%) in the lung adenocarcinoma population (Kim et al. Cell 155:552, 2013). These include three totally novel lung cancer selective targeted therapies: NLRP3 mutation/inflammasome activation-dependent FLIP addiction; co-occurring KRAS and LKB1 mutation-driven COPI addiction; and selective sensitivity to a synthetic indolotriazine that is specified by a seven-gene expression signature. Our panel of “hits” provide the opportunity to identify all potential therapeutic targets for lung cancer, while the molecular correlates will allow “personalization” of these new therapies going forward in preclinical and clinical translation. (Supported by NCI SPORE P50CA70907, NCI CTD2N, CPRIT, UTSW CCSG P30CA142543) Citation Format: John D. Minna, Adi Gazdar, Alexander Augustyn, Rebecca Britt, Ryan Carstens, Patrick Dospoy, Boning Gao, Luc Girard, Suzie Hight, Kenneth Huffman, Jill Larsen, Michael Peyton, Chunli Shao, David Mangelsdorf, Rolf Brekken, Ralph Deberardinis, Pei-Hsuan Chen, Carmen Behrens, Lauren Byers, John Heymach, Jack Roth, Ignacio Wistuba, Yang Xie, Caleb Davis, David Wheeler, Richard Gibbs, Edward Marcotte, Joseph Ready, Deepak Nijhawan, Noelle Williams, Steven McKnight, Bruce Posner, John MacMillan, Michael Roth, Michael White. Developing a new functional classification of lung cancer based on tumor acquired vulnerabilities. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr IA21.

Abstract 4619: Protein palmitoylation in non-small cell lung cancer (NSCLC): DHHC5 palmitoyltransferase as a potential therapeutic target

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Post-translational palmitoylation of intracellular proteins is mediated by protein palmitoyltransferases (PAT) belonging to the DHHC family, which share a common catalytic Asp-His-His-Cys (DHHC) motif. According to a genome-wide siRNA screen scoring for growth suppression in over 50 non-small cell lung cancer (NSCLC) cell lines, DHHC family members are strikingly over-represented. Among the tested DHHC palmitoyltransferases, knockdown of DHHC5 showed cell growth inhibition in many cell lines in the initial screen. To further investigate the growth suppression in response to DHHC5 depletion, transient and stable knockdowns of DHHC5 were undertaken in a dozen of NSCLC cell lines identified in the genome-wide screen using siRNAs and lentiviral shRNAs. Such knockdown was also performed in three immortalized human lung bronchial epithelial cell lines (HBECs). The gene expression and protein levels of DHHC5 were analyzed by RT-PCR and immunoblotting, both of which showed efficient knockdown levels. Cell proliferation and anchorage-dependent/independent colony formation assays indicated that DHHC5 knockdown led to cell growth arrest and decreased cell clonogenicity in most of the tested NSCLC cell lines in vitro, but not in the HBECs. H1299, H358 and H2009 cell lines were particularly dependent on DHHC5 expression and the growth-suppression phenotype of DHHC5 stable knockdown H1299/H358/H2009 cells was rescued by overexpression of DHHC5. The catalytically inactive DHHC5 mutant was not able to provide rescue. In vivo tumor formation assays were then carried out by injecting H1299 and H358 control cells and DHHC5 stable knockdown cells subcutaneously into NOD/SCID mice. DHHC5 knockdown led to absence or dramatically decreased tumor xenograft formation. Furthermore, a Tet-On inducible system with TRIPZ shRNA lentiviral delivery was established in H1299. Mouse xenografts using this H1299 Tet-on inducible cell line showed similar results. As DHHC5 function is needed for optimal lung cancer cell growth, it will be important to identify physiologically relevant substrates in order to develop inhibitors of DHHC5 function. Future plans include microarray and protein kinase array analysis to distinguish possible signaling pathways in which DHHC5 may be involved, as well as palmitoyl-protein profiling of normal and knockdown DHHC5 cell lines by mass spectrometry. Citation Format: Hui Tian, Jui-Yun Lu, Chunli Shao, Kenneth Huffman, Ryan Carstens, John D. Minna, Sandra L. Hofmann. Protein palmitoylation in non-small cell lung cancer (NSCLC): DHHC5 palmitoyltransferase as a potential therapeutic target. [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 4619. doi:10.1158/1538-7445.AM2014-4619

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

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA 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 A24: Targeted synthetic lethal screens to identify acquired vulnerabilities in a novel class of ASCL1-dependent neuroendocrine non-small cell lung cancers

Introduction: Gene expression signatures from large cohorts of lung tumors suggest that distinct neuroendocrine (NE) cancers with poor prognosis appear in ~10% of otherwise pathologically unremarkable NSCLCs. However, a complete molecular characterization is lacking because a similar subtype has yet to be identified in a significant number of lung cancer cell lines. Our goal is to develop a pre-clinical model for NSCLC-NE and use it to rationale targeted therapy for this important subtype of lung cancer. We are developing targeted siRNA-based synthetic lethal screens to identify acquired vulnerabilities in lung cancers by using “mini-libraries” of putative cancer stem cell genes (CSC, N = 40), nuclear receptors and their co-regulators (NR/CoReg, N = 120), chromatin remodelers (CR, N = 102), and ASCL1-regulated druggable genes (N = 40). The siRNA libraries were derived from multiple studies while the ASCL1-targeted library was developed from our own ChIP-Seq data. Identifying NSCLC-NE Cell Lines: Using mRNA expression signatures from 207 lung cell lines (NSCLC/SCLC/HBEC) we identified a cluster of NSCLC cell lines (9% of NSCLC) that express genes indicative of a neuroendocrine phenotype (NSCLC-NE). A highly expressed gene in this class of cell lines is the lineage-specific transcription factor ASCL1, which is required for the development of pulmonary neuroendocrine cells. ASCL1 loss-of-function studies demonstrate dramatic cell death compared to controls, suggesting these cells are addicted to ASCL1 for survival. ASCL1 ChIP-Seq Analysis: To better understand the molecular pathogenesis of ASCL1-dependent NSCLC-NEs and identify druggable therapeutic downstream targets, we performed ChIP-Seq analysis on six ASCL1+ and two ASCL1- cell lines. A 125-member “ASCL1-signature” was generated and tested on multiple resected NSCLC datasets (N > 800 patients) for prognostic utility and tumors that exhibited the “ASCL1 signature” had significantly worse prognosis. A target of ASCL1 as determined by ChIP-Seq analysis is the anti-apoptotic regulator BCL2. BCL2 knockdown using siRNA as well as inhibition of BCL2 using a small molecule (ABT-263) results in cell death that is specific to ASCL1+/BCL2+ cells. ASCL1 Upstream Regulation: A separate therapeutic avenue is to determine upstream regulators of ASCL1 and we have demonstrated that ASCL1 participates in a double-negative feedback loop with the MEK/ERK pathway. Pharmacological activation of the MEK/ERK pathway results in loss of ASCL1 mRNA and protein, and induces apoptosis in ASCL1-dependent NSCLC-NE lines. Additionally, siRNA-mediated knockdown of ASCL1 results in reciprocal activation of the MEK/ERK pathway even in the presence of an siRNA targeting MEK1. Combining MEK/ERK activators with ABT-263 results in dramatic induction of apoptosis in NSCLC-NE while sparing normal immortalized HBEC cells suggesting the possibility of a synthetic lethal combination therapy for this subset of tumors. Synthetic Lethal Screens: We have completed synthetic lethal siRNA screens for the CSC, NR/CoReg, and CR mini-libraries using a panel of lung cancer lines. Optimal transfection conditions were established for each line and siRNA pools were used with multiple (n = 9) replicates providing great reproducibility (r > 0.9). From these screens, preliminary data demonstrates that the NSCLC-NE tumor lines have response phenotypes distinct from all other classes of lung cancer. Conclusions: We show that NE gene expression in NSCLC is of clinical relevance, and ASCL1 appears to be a lineage-dependent oncogene for NSCLC-NE. ASCL1 ChIP-Seq and siRNA screen data provide a roadmap for systematically searching for therapeutic targets such as BCL2 while work focused on targeting pathways upstream of ASCL1 also demonstrates clinical potential for NSCLC with neuroendocrine features. Our results indicate that lung cancers driven by ASCL1 have distinct acquired vulnerabilities. (Supported by NCI SPORE P50CA70907, CPRIT) Citation Format: Alexander Augustyn, Mark Borromeo, Tao Wang, Chunli Shao, Patrick Dospoy, Kenneth Huffman, Ryan Carstens, Luc Girard, Carmen Behrens, Ignacio Wistuba, Yang Xie, Jane Johnson, Adi Gazdar, John Minna. Targeted synthetic lethal screens to identify acquired vulnerabilities in a novel class of ASCL1-dependent neuroendocrine non-small cell lung cancers. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr A24.