In humans, genetically diverse forms of muscular dystrophy are associated with a disrupted sarcoglycan complex. The sarcoglycan complex resides at the muscle plasma membrane where it associates with dystrophin. There are six known sarcoglycan proteins in mammals whereas there are only three in Drosophila melanogaster. Using imprecise P element excision, we generated three different alleles at the Drosophila delta-sarcoglycan locus. Each of these deletions encompassed progressively larger regions of the delta-sarcoglycan gene. Line 840 contained a large deletion of the delta-sarcoglycan gene, and this line displayed progressive impairment in locomotive ability, reduced heart tube function and a shortened life span. In line 840, deletion of the Drosophila delta-sarcoglycan gene produced disrupted flight muscles with shortened sarcomeres and disorganized M lines. Unlike mammalian muscle where degeneration is coupled with ongoing regeneration, no evidence for regeneration was seen in this Drosophila sarcoglycan mutant. In contrast, line 28 was characterized with a much smaller deletion that affected only a portion of the cytoplasmic region of the delta-sarcoglycan protein and left intact the transmembrane and extracellular domains. Line 28 had a very mild phenotype with near normal life span, intact cardiac function and normal locomotive activity. Together, these data demonstrate the essential nature of the transmembrane and extracellular domains of Drosophila delta-sarcoglycan for normal muscle structure and function.

Recent advances in detailed molecular understanding of lung cancer including complete genome sequences affords the possibility of developing molecularly targeted therapy for potentially all lung cancers. Currently the Lung Cancer Mutation Consortium (LCMC) has developed an integrated approach and network for identifying lung cancers with specific mutations and funneling these patients into clinical trials targeting the specific mutations identified. However, another approach is to use genome wide and chemical library screens to identify genetic and epigenetic changes that have been created in lung cancer cells during tumor pathogenesis that are absolutely required for the activated oncogenic pathways to function. These are often referred to as “synthetic lethal” changes and represent adaptations the cancer cell has to make to allow the “oncogene addictions” to drive tumor growth and survival. They are present in tumor but not normal cells and thus represent “acquired vulnerabilities” that can be therapeutically targeted. The NCI's “Cancer Target Discovery and Development Network” (CTD2N) exemplifies this combined genetic and pharmacologic approach. A most important subgroup of these vulnerabilities are changes that are required for the continued function and survival of a subpopulation of tumor cells that have acquired tumor initiating and often metastatic and drug resistant characteristics including many of the properties of stem cells that are referred to as cancer stem cells (CSCs) or “cancer initiating cells.” To achieve these goals we have developed a large integrated research platform. We have also: developed assays for identifying lung CSCs, “molecular portraits” (clades) that group lung cancers into subsets of clinical and molecular relevance; genome wide siRNA and chemical library functional screens to test for portrait/clade specificity. From these efforts we have learned that: There are a subset of cells within lung cancers (ranging from 0.1–30% of non small cell lung cancers, NSCLCs) and 50% + of small cell lung cancers, SCLCs) identified by elevated aldehyde dehydrogenase (ALDH) activity that have dramatically enhanced clonogenic, tumorigenic, and self renewal capacity; Patients whose tumors are enriched is such ALDH+ tumor cells have significantly impaired prognosis; The notch pathway (particularly Notch3) and ALDH1A3 are major vulnerabilities in lung CSCs; NSCLCs can be subdivided by mRNA expression profiles into “molecular portraits” (or “clades”) that have relevant clinical, oncogenotype, and drug response phenotypes; Genome wide siRNA and large chemical library screens identify targets that are specific for lung cancer over normal lung epithelial cells; The identified targets show dramatic clade and oncogenotype specificity as well as specificity for lung cancers with different responses to available chemotherapy and targeted therapy for lung cancer; The newly identified vulnerabilities provide coverage of essentially all lung cancers and as such, provide a new functional “vulnerability classification” of lung cancer. All of these findings set the stage for the development of a rational approach to developing therapy targeted at lung cancer acquired vulnerabilities including those in lung CSCs that will include personalizing the therapy for each patient. (Supported by Lung Cancer SPORE P50CA70907, DOD Prospect, CPRIT, and NCI CTD2N CA148225.)

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.

Purpose: Lung cancer is a disease of great oncogenotype complexity (oncogenes and tumor suppressor gene alterations). These alterations can appear in different combinations even within histologically defined lung cancer subtypes. The success of targeted therapy has led to a search for oncogenotype-specific therapies. But, no one therapy fits all oncogenotypes. Here, we investigate whether characterization of oncogene-specific alterations in cellular signaling at single cell level indicate heterogeneity even within cells from the same patient with defined oncogenotype, and whether they can suggest new targets to deal with this heterogeneity. Methods: We compared signaling alterations in single cells for β-CATENIN, SMAD2/3, phospho-STAT3, P65, FOXO1 and phospho-ERK1/2 among a collection of Human Bronchial Epithelial Cells (HBECs) that have been oncogenically transformed with combinations of TP53, K-RAS, and MYC, commonly found alterations in non-small cell lung cancer (NSCLC). We studied ~3000 cells/signaling marker/HBEC oncogenotype variant using immunofluorescence assays and single-cell image analysis (>1M data points). For downstream target identification and validation we utilized gene expression, Western blot and siRNA mediated knockdown assays. We utilized inhibitors to STAT3 and BCL6 in MTT drug sensitivity and colony formation assay in a panel of NSCLC lines. We used xenografted subcutaneous tumors for the in vivo validation of our results. Results: When all three oncogenic changes were present and the HBECs were tumorigenic, we observed STAT3 upregulation and SMAD2/3 downregulation. Interestingly, these STAT3 and SMAD2/3 signaling changes were found to be mutually exclusive in single cells within the transformed HBEC strain. We targeted the STAT3 upregulated subpopulation with the STAT3 inhibitor Stattic. But, Stattic treatment failed to eliminate the SMAD2/3 downregulated subpopulation. To target the SMAD2/3 down-regulated subpopulation, we identified BCL6, a downstream gene of SMAD2/3, as a novel target in transformed HBECs. Next, to test the generality of BCL6 as a target, we studied 5 NSCLC cell lines with various level of BCL6 expression: H1693, H1819, H1993, HCC827 and H2009. Our data suggests that BCL6 can also be a therapeutic target in a subset of NSCLC lines. Then we tested the response of these NSCLC lines to a combination of BBI608 (potent STAT3 inhibitor) and FX1 (BCL6 inhibitor). The combination treatment eliminated more cancer cells than the single treatments alone. Finally, we confirmed the benefit of the combination therapy in H1993 xenografted tumors. Conclusions: We conclude that BCL6 is a new therapeutic target in NSCLC and combination therapy that targets multiple vulnerabilities (Phospho-STAT3 and BCL6) downstream of common oncogenes and tumor suppressors (TP53, K-RAS, and MYC) may provide a potent way to defeat intra-tumor heterogeneity. Citation Format: Dhruba Deb, Satwik Rajaram, Jill E. Larsen, Patrick P. Dospoy, Rossella Marullo, Longshan Li, Kimberley Avila, Leandro Cerchietti, John D. Minna, Lani F. Wu, Steven J. Altschuler. A novel combination therapy targeting BCL6 and phospho-STAT3 defeats intratumor heterogeneity in a subset of non-small cell lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3950. doi:10.1158/1538-7445.AM2017-3950

MYC is one of the most commonly deregulated oncogenes in various cancers, including breast, colorectal and lung. While mutations are rare, we know that MYC is overexpressed and in some cases amplified in these (and other) cancers. Numerous reports have recently demonstrated the utility of various therapeutics in selectively targeting MYC-driven cancers. However, given the lack of consistency across tissue types, particularly lung cancer, a multimodal approach to delineate MYC-dependent lung cancers is required. Our goal is to identify lung cancers that are addicted to MYC, determine their oncogenotype and molecular properties, and identify a biomarker to identify patients with MYC addicted tumors. We studied a large panel of clinically and molecularly annotated NSCLC lines for MYC mRNA, protein expression, and DNA copy number. Functional tests were performed on 17 NSCLC cell lines using three drugs that were recently shown to selectively target MYC-driven cancers. Further, we utilized the dominant negative mini-protein OMOMYC for functional classification. In all cases, effects were monitored by colony forming efficiency (CFE) assays and proliferation assays. OMOMYC results were confirmed via xenograft experiments. Each of the three MYC inhibitors tested elicited a viability response in a subset of the 17 NSCLC cell lines, though the sensitive subset was not significantly similar between any two drugs (highest correlation coefficient of 0.3). In order to determine which, if any, of the drugs truly targeted MYC-driven lung cancers, we stably expressed OMOMYC in all 17 parental NSCLC cells and performed functional assays. 6/17 cell lines were dramatically sensitive to OMOMYC (with up to 100 fold reduction in CFE), compared to 11/17 totally resistant. The viability in the presence of OMOMYC shows a statistically significant correlation with one of the three MYC inhibitors tested, which supports the notion that this sensitive subset represents a truly MYC-dependent class of lung cancers. Surprisingly, there was no correlation between MYC dependence and either MYC mRNA, protein expression or DNA copy number. OMOMYC levels were normalized in all cell lines tested and quantified using qRT-PCR. Additionally, in all cases, exogenous OMOMYC expression led to down regulation of c-Myc target genes as measured by both qRT-PCR and microarray. These data suggest that the observed phenotype was the result of decreased MYC activity. We conclude: there is a subset of NSCLCs that demonstrates dramatic growth inhibition by a single MYC-inhibitor, and these data are phenocopied by the more specific MYC-dominant negative protein, OMOMYC. We are classifying this subset of cancer cell lines, MYC “addicted.” Using the MYC “addicted” vs. non- addicted NSCLC panel, we are testing for gene expression, methylation and mutational differences in order to identify and eventually characterize a biomarker for MYC dependence in lung cancer. Citation Format: Patrick Dospoy, Chunli Shao, Elizabeth McMillan, Michael Peyton, Jill Larsen, Luc Girard, Ignacio Wistuba, Adi F. Gazdar, John D. Minna. Differential MYC dependence in NSCLC identified through pharmacological and genetic MYC inhibition. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr A22.