Alexander Augustyn

ASCL1 is a lineage oncogene providing therapeutic targets for high-grade neuroendocrine lung cancers

Significance New advances in the treatment of aggressive neuroendocrine lung cancers are needed to improve survival in patients with this class of tumors. The current treatment approach, which has remained unchanged for the past 30 years, involves combination chemotherapy and radiation. To uncover novel drug targets, we identified the transcriptome of achaete-scute homolog 1 (ASCL1), a transcription factor that is both necessary for the proper development of neuroendocrine cells and essential for the growth and survival of neuroendocrine lung cancers. Analysis of downstream targets of ASCL1 has revealed unique molecular vulnerabilities that can be exploited for future therapeutic use. Aggressive neuroendocrine lung cancers, including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), represent an understudied tumor subset that accounts for approximately 40,000 new lung cancer cases per year in the United States. No targeted therapy exists for these tumors. We determined that achaete-scute homolog 1 (ASCL1), a transcription factor required for proper development of pulmonary neuroendocrine cells, is essential for the survival of a majority of lung cancers (both SCLC and NSCLC) with neuroendocrine features. By combining whole-genome microarray expression analysis performed on lung cancer cell lines with ChIP-Seq data designed to identify conserved transcriptional targets of ASCL1, we discovered an ASCL1 target 72-gene expression signature that (i) identifies neuroendocrine differentiation in NSCLC cell lines, (ii) is predictive of poor prognosis in resected NSCLC specimens from three datasets, and (iii) represents novel “druggable” targets. Among these druggable targets is B-cell CLL/lymphoma 2, which when pharmacologically inhibited stops ASCL1-dependent tumor growth in vitro and in vivo and represents a proof-of-principle ASCL1 downstream target gene. Analysis of downstream targets of ASCL1 represents an important advance in the development of targeted therapy for the neuroendocrine class of lung cancers, providing a significant step forward in the understanding and therapeutic targeting of the molecular vulnerabilities of neuroendocrine lung cancer.

Essential role of aldehyde dehydrogenase 1A3 for the maintenance of non-small cell lung cancer stem cells is associated with the STAT3 pathway

Purpose: Lung cancer stem cells (CSC) with elevated aldehyde dehydrogenase (ALDH) activity are self-renewing, clonogenic, and tumorigenic. The purpose of our study is to elucidate the mechanisms by which lung CSCs are regulated. Experimental Design: A genome-wide gene expression analysis was performed to identify genes differentially expressed in the ALDH+ versus ALDH− cells. RT-PCR, Western blot analysis, and Aldefluor assay were used to validate identified genes. To explore the function in CSCs, we manipulated their expression followed by colony and tumor formation assays. Results: We identified a subset of genes that were differentially expressed in common in ALDH+ cells, among which ALDH1A3 was the most upregulated gene in ALDH+ versus ALDH− cells. shRNA-mediated knockdown of ALDH1A3 in non–small cell lung cancer (NSCLC) resulted in a dramatic reduction in ALDH activity, clonogenicity, and tumorigenicity, indicating that ALDH1A3 is required for tumorigenic properties. In contrast, overexpression of ALDH1A3 by itself it was not sufficient to increase tumorigenicity. The ALDH+ cells also expressed more activated STAT3 than ALDH− cells. Inhibition of STAT3 or its activator EZH2 genetically or pharmacologically diminished the level of ALDH+ cells and clonogenicity. Unexpectedly, ALDH1A3 was highly expressed in female, never smokers, well-differentiated tumors, or adenocarcinoma. ALDH1A3 low expression was associated with poor overall survival. Conclusions: Our data show that ALDH1A3 is the predominant ALDH isozyme responsible for ALDH activity and tumorigenicity in most NSCLCs, and that inhibiting either ALDH1A3 or the STAT3 pathway are potential therapeutic strategies to eliminate the ALDH+ subpopulation in NSCLCs. Clin Cancer Res; 20(15); 4154–66. ©2014 AACR.

ZEB1 drives epithelial-to-mesenchymal transition in lung cancer

Increased expression of zinc finger E-box binding homeobox 1 (ZEB1) is associated with tumor grade and metastasis in lung cancer, likely due to its role as a transcription factor in epithelial-to-mesenchymal transition (EMT). Here, we modeled malignant transformation in human bronchial epithelial cells (HBECs) and determined that EMT and ZEB1 expression are early, critical events in lung cancer pathogenesis. Specific oncogenic mutations in TP53 and KRAS were required for HBECs to engage EMT machinery in response to microenvironmental (serum/TGF-β) or oncogenetic (MYC) factors. Both TGF-β- and MYC-induced EMT required ZEB1, but engaged distinct TGF-β-dependent and vitamin D receptor-dependent (VDR-dependent) pathways, respectively. Functionally, we found that ZEB1 causally promotes malignant progression of HBECs and tumorigenicity, invasion, and metastases in non-small cell lung cancer (NSCLC) lines. Mechanistically, ZEB1 expression in HBECs directly repressed epithelial splicing regulatory protein 1 (ESRP1), leading to increased expression of a mesenchymal splice variant of CD44 and a more invasive phenotype. In addition, ZEB1 expression in early stage IB primary NSCLC correlated with tumor-node-metastasis stage. These findings indicate that ZEB1-induced EMT and associated molecular changes in ESRP1 and CD44 contribute to early pathogenesis and metastatic potential in established lung cancer. Moreover, TGF-β and VDR signaling and CD44 splicing pathways associated with ZEB1 are potential EMT chemoprevention and therapeutic targets in NSCLC.

Small Cell Lung Cancer: Can Recent Advances in Biology and Molecular Biology Be Translated into Improved Outcomes?

Paul A. Bunn Jr., MD, John D. Minna, MD, Alexander Augustyn, PhD, Adi F. Gazdar, MD, Youcef Ouadah, BS, Mark A. Krasnow, MD, PhD, Anton Berns, PhD, Elisabeth Brambilla, MD, Natasha Rekhtman, MD, PhD, Pierre P. Massion, MD, Matthew Niederst, PhD, Martin Peifer, PhD, Jun Yokota, MD, Ramaswamy Govindan, MD, John T. Poirier, PhD, Lauren A. Byers, MD, Murry W. Wynes, PhD, David G. McFadden, MD, PhD, David MacPherson, PhD, Christine L. Hann, MD, PhD, Anna F. Farago, MD, PhD, Caroline Dive, PhD, Beverly A. Teicher, PhD, Craig D. Peacock, PhD, Jane E. Johnson, PhD, Melanie H. Cobb, PhD, Hans-Guido Wendel, MD, David Spigel, MD, Julien Sage, PhD, Ping Yang, MD, PhD, M. Catherine Pietanza, MD, Lee M. Krug, MD, John Heymach, MD, PhD, Peter Ujhazy, MD, PhD, Caicun Zhou, MD, PhD, Koichi Goto, MD, Afshin Dowlati, MD, Camilla Laulund Christensen, PhD, Keunchil Park, MD, PhD, Lawrence H. Einhorn, MD, Martin J. Edelman, MD, Giuseppe Giaccone, MD, PhD, David E. Gerber, MD, Ravi Salgia, MD, PhD, Taofeek Owonikoko, MD, PhD, Shakun Malik, MD, Niki Karachaliou, MD, David R. Gandara, MD, Ben J. Slotman, MD, PhD, Fiona Blackhall, MD, PhD, Glenwood Goss, MD, FRCPC, Roman Thomas, MD, Charles M. Rudin, MD, PhD, Fred R. Hirsch, MD, PhD*

Primary Gastric Melanoma: Case Report of a Rare Malignancy

We report the case of a 64-year-old white male who presented to his primary care physician with complaints of fatigue. Physical exam was unremarkable and laboratory studies revealed profound anemia, for which the patient received a transfusion. Esophagogastroduodenoscopy revealed a bleeding mass in the proximal stomach that was histologically determined to be malignant melanoma, with immunohistochemical staining demonstrating positivity for SOX10, S100, MART-1, and HMG-45. After an extensive dermatological exam no other primary lesion was identified. Whole body positron emission tomography (18-FDG-PET/CT) demonstrated pathologic uptake only in the area of the proximal stomach. For this reason, primary gastric melanoma was suspected in this patient. The patient underwent subtotal gastrectomy with mass excision followed by Roux-en-Y reconstruction. Very few cases of primary gastric melanoma have been reported. We report this case and present diagnostic criteria for primary non-cutaneous melanoma and discuss potential non-surgical therapies.

Surveillance for hepatocellular carcinoma secondary to cardiogenic cirrhosis in patients with congenital heart disease

Sirs: Cyanotic congenital heart diseases (CHD), including tetralogy of Fallot (TOF), were previously fatal diseases. Surgical advancements such as the Blalock–Taussig shunt and the Fontan procedure have improved the outlook of patients diagnosed with CHD, such that patients can expect a 20-year survival rate approaching 90 % [1]. The improved survival of children with CHD is now affecting adult medicine. An estimated 1.3 million adults now live in the USA with CHD and that number is expected to increase by 5 % per year [2]. Patients with CHD are predisposed to developing hepatic dysfunction as a result of either the primary cardiac defect or surgical palliation, or both. As the CHD population ages, particular attention must be paid to hepatic surveillance to identify patients with cardiogenic cirrhosis, as there is an increased incidence of subsequent development of HCC over time. To illustrate the complex and chronic nature of HCC development in patients with congenital heart disease, we present the case of a 24-year-old female born with TOF who underwent three separate cardiac procedures and developed cardiogenic cirrhosis secondary to a regurgitant pulmonic valve. Interval imaging and subsequent biopsy identified a lesion within the right lobe of the liver, consistent with HCC. In addition to this report, we review the relevant literature and provide recommendations for surveillance in the adult CHD patient population. Our patient was born cyanotic with TOF and critical pulmonary artery stenosis dependent on ductal pulmonary blood flow. Repair took the form of three surgeries: emergently after birth, a central shunt was placed to circumvent the stenosis; at age 3, a pulmonary valve homograft was placed, the atrial-septal defect was repaired, and the right pulmonary artery was reconstructed to the level of the hilum; and at age 12, due to significant pulmonary valve regurgitation and sonographic evidence of right ventricle (RV) dysfunction, our patient underwent replacement of the pulmonary homograft with a Synergraft extending into the distal left pulmonary artery. Echocardiography performed 5 years after pulmonic valve replacement demonstrated no evidence of intracardiac shunting, but identified a dilated RV with significantly increased systolic pressures secondary to continued pulmonary valve regurgitation. Magnetic resonance imaging (MRI) of the chest identified a hypokinetic and dilated RV with a regurgitant fraction of 31 %. A diagnosis of decompensated congestive heart failure was made by complete heart catheterization. Following identification of the diagnosis of right heart failure, the patient was administered low-dose diuretics, but lost to follow-up due to socioeconomic reasons. At the age of 24, our patient became symptomatic with new-onset abdominal pain, abdominal ascites, and lower extremity edema. Computed tomography of the abdomen demonstrated heterogenous hepatic nodularity consistent with cardiogenic cirrhosis with features suggesting portal hypertension. Laboratory findings demonstrated no evidence of viral hepatitis or abnormal liver function and, except for an elevated serum alpha-fetoprotein (AFP) level of 99 ng/ml, were within normal limits. In addition, there was no history of alcohol intake or features of metabolic syndrome including hypercholesterolemia, morbid obesity, or diabetes mellitus. Additional dynamic contrasted abdominal MRI demonstrated a 2.0 cm hypervascular mass in the right lobe of the liver with washout on delayed imaging. These imaging findings were consistent with HCC (Fig. 1). Fig. 1 Magnetic resonance images demonstrating evidence of a 2.0 cm nodule in segment 7 at the dome of the liver. The mass displayed arterial enhancement and washout with delayed peripheral enhancement, which is consistent with a diagnosis of hepatocellular ... Segmental resection (segment 7) of the HCC lesion was completed after cardiac catheterization confirmed optimization for operative intervention. Resection was considered the best option due to minimal portal hypertension evident by a platelet count of 186,000. The abdominal ascites and peripheral edema were felt to be secondary to right heart failure and were not seen during operative exploration that followed a fairly aggressive diuretic regimen. Other curative therapies including ablation and liver transplantation were not considered due to location of the liver lesion and lack of insurance, respectively. Intraoperative examination of the liver revealed extensive hepatic cirrhosis and one solitary mass lesion in the right lobe of the liver. Intraoperative central venous pressure (CVP) monitoring confirmed significant right-sided cardiac dysfunction with initial CVP readings of 35 mmHg. The resected tumor specimen was a moderately differentiated HCC with negative margins in the background of a cirrhotic liver (Fig. 2). The patient tolerated the procedure well, was discharged on hospital day 5, and remains free of recurrence more than 2 years after the initial resection. Six months following the operative resection, the patient underwent percutaneous pulmonary valve replacement. Fig. 2 Histopathologic analysis of the surgical specimen. a Moderately differentiated hepatocellular carcinoma with adjacent non-neoplastic liver parenchyma (40× magnification). b Trabecular and solid pattern, 4+ cells thick (100× magnification). ... The majority of cases of HCC arise secondary to chronic liver injury from viral hepatitis B or C, excessive alcohol intake, non-alcoholic fatty liver disease, and more rarely from genetic causes such as hemochromatosis, Wilson’s disease, and others [3]. Due to improved care, patients undergoing CHD repairs as infants have significantly increased survival and no longer succumb to acute cardiac issues; however, these patients can demonstrate increased sequelae of longstanding cardiac insufficiency such as right heart failure with progression to cardiogenic cirrhosis over time. This is evident in the increase of reported cases of cardiogenic cirrhosis as the underlying etiology of HCC over the last two decades [4–8]. Asrani et al. describe four patients with univentricular CHD who underwent Fontan repair with subsequent identification of HCC and suggest cirrhosis develops within 11–15 years post-procedure in patients under the age of 25 years, with an estimated incidence of HCC of 1–5 % per year, similar to other etiologies of chronic liver disease producing cirrhosis [8]. An improvement in CHD survival obviates the need for improved cancer monitoring in this population. Despite surgical resection of HCC, our patient, due to her cirrhosis, maintains a 5-year recurrence rate of approximately 50 % [9]. Liver transplantation is another curative modality for patients with HCC; however, liver shortages in the region where this patient was treated necessitate surgical resection rather than orthotopic liver transplantation as the first curative therapy offered. Guidelines need to be established for HCC surveillance in patients with decompensated congestive heart failure in the setting of repaired congenital heart disease. Although cirrhosis is difficult to distinguish from chronic right heart failure, any consequence of portal hypertension such as thrombocytopenia should be enough to warrant surveillance for HCC. Transient elastography may be a useful modality to screen for cirrhosis in the future, but is currently not widely used outside of the research setting in the majority of high-volume centers. In high-risk patients with established chronic liver disease and cirrhosis, the National Comprehensive Cancer Network recommends abdominal ultrasonography and serum AFP every 6–12 months [10]. The American Association for the Study of Liver Diseases (AASLD) suggests HCC surveillance for patients with chronic liver disease and an annual incidence rate that exceeds 1.5 % for patients with hepatitis C and 0.2 % for patients with hepatitis B [11]. Based on currently available patient data, HCC incidence in patients with CHD who developed cirrhosis is between 1 and 5 %, exceeding the chronic liver disease surveillance cutoff suggested by the AASLD [8]. By extension, annual or bi-annual ultrasonography and serum AFP levels should be performed on patients with previously repaired CHD who demonstrate evidence of hepatic cirrhosis. The patient presented above provides an important lesson in proper health management during the transition from pediatric care to adult care, a shift that all patients with CHD must face. Continuity of care is a critical factor in identifying, preventing, and/or significantly delaying the development of chronic liver disease in this patient population. These patients require multidisciplinary medical management that includes (but is not limited to) cardiologists, hepatologists, and social workers that can help identify barriers to regular treatment access. Due to the expanding adult population of patients with surgically repaired CHD, we are beginning to understand the longterm clinical consequences and unique manifestations of disease secondary to the surgical repair process.

Large Malignant Phyllodes Tumor of the Breast with Metastases to the Lungs

Phyllodes tumors of the breast account for less than 0.5% of breast cancers and present most commonly in women 45 to 49 years old. The importance in managing fibroepithelial lesions lies in distinguishing fibroadenomas, which are benign, from phyllodes tumors, which can be malignant and require complete surgical excision. We report the case of a 56-year-old female who presented with a rapidly enlarging mass in her right breast 18 cm in maximum dimension that completely effaced the breast and distorted the nipple. The patient underwent a successful total mastectomy after core biopsy revealed a diagnosis of phyllodes tumor. Surgical resection is the primary treatment modality; neoadjuvant and adjuvant therapies remain controversial. Here, we report the case of a large malignant phyllodes tumor metastatic to the lungs, review the literature, and discuss diagnostic modalities and adjunct nonsurgical therapies.

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 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.

Abstract A46: Identification of ALDH1A3 as an important biomarker and therapeutic target for non-small cell lung cancer (NSCLC) cancer stem cells (CSCs)

A considerable amount of evidence reveals the important roles of cancer stem cells (CSCs, also called “cancer initiating cells”) in tumor growth, metastasis, relapse and drug resistance. Our group has identified lung CSCs in both lung cancer cell lines and patient tumor samples by their elevated Aldehyde Dehydrogenase (ALDH) activity and demonstrated that NSCLC patients whose tumors are enriched in ALDH+ cells have inferior survival, and that the ALDH+ subpopulations (ranging from 0.5 to 30% of tumor cells) are self renewing, and significnatly more clonogenic and tumorigencic (in xenograft models) than the ALDH− population. To further define the molecular profile of lung CSCs and to identify key pathways involved in lung CSC function we performed genome wide microarray expression profiling of isolated ALDH+ and ALDH− cell populations from 8 non-small cell lung cancer (NSCLC) lines representing a variety of oncogenotypes to determine genes that are differentially expressed in these two populations. Strikingly we found that different lung cancers exhibited different ALDH+ cell population expression profiles but that the ALDH1A3 isozyme was one of the few commonly genes whose expression was elevated. (Previously our group and other investigators in lung and other cancers had found the ALDH1A1 and not ALDH1A3 isozyme to be a marker of the ALDH+ population). ALDH1A3 differential expression was confirmed by q RT PCR and elevated ALDH1A3 protein levels which in turn was correlated with the % of ALDH+ tumor cells in each tumor. ALDH1A3 mRNA levels in (N = 182) resected lung adenocarcinoma samples showe that the highest levels of ALDH1A3 were associated with inferior survival. Of great importance, shRNA mediated knockdown of ALDH1A3 dramatically reduced both NSCLC cell colony formation in vitro and tumor growth in vivo demonstrating the functional importance of ALDH1A3 expression in NSCLCs. However, ectopic expression of ALDH1A3 in NSCLC cells did not enhance lung cancer cell growth and tumor formation, suggesting that ALDH1A3 alone is not sufficient to promote lung cancer progression. We conclude that ALDH1A3 is the predominant ALDH isozyme responsible for ALDH activity in NSCLCs of many oncogenotypes, is of potential prognostic importance, and a new therapeutic target for eliminating NSCLC stem cells.