Harshad S. Mahadeshwar

Intratumor heterogeneity in localized lung adenocarcinomas delineated by multiregion sequencing

Cancers are composed of populations of cells with distinct molecular and phenotypic features, a phenomenon termed intratumor heterogeneity (ITH). ITH in lung cancers has not been well studied. We applied multiregion whole-exome sequencing (WES) on 11 localized lung adenocarcinomas. All tumors showed clear evidence of ITH. On average, 76% of all mutations and 20 out of 21 known cancer gene mutations were identified in all regions of individual tumors, which suggested that single-region sequencing may be adequate to identify the majority of known cancer gene mutations in localized lung adenocarcinomas. With a median follow-up of 21 months after surgery, three patients have relapsed, and all three patients had significantly larger fractions of subclonal mutations in their primary tumors than patients without relapse. These data indicate that a larger subclonal mutation fraction may be associated with increased likelihood of postsurgical relapse in patients with localized lung adenocarcinomas. Different mutations are present in different regions of any given lung cancer, and their pattern may predict patient relapse. [Also see Perspective by Govindan] Space, time, and the lung cancer genome Lung cancer poses a formidable challenge to clinical oncologists. It is often detected at a late stage, and most therapies work for only a short time before the tumors resume their relentless growth. Two independent analyses of the human lung cancer genome may help explain why this disease is so resilient (see the Perspective by Govindan). Rather than take a single “snapshot” of the cancer genome, de Bruin et al. and Zhang et al. identified genomic alterations in spatially distinct regions of single lung tumors and used this information to infer the tumors evolutionary history. Each tumor showed tremendous spatial and temporal diversity in its mutational profiles. Thus, the efficacy of drugs may be short-lived because they destroy only a portion of the tumor. Science, this issue p. 251, p. 256; see also p. 169

Characterization of HPV and host genome interactions in primary head and neck cancers

Significance A significant proportion of head and neck cancer is driven by human papillomavirus (HPV) infection, and the expression of viral oncogenes is involved in the development of these tumors. However, the role of HPV integration in primary tumors beyond increasing the expression of viral oncoproteins is not understood. Here, we describe how HPV integration impacts the host genome by amplification of oncogenes and disruption of tumor suppressors as well as driving inter- and intrachromosomal rearrangements. Tumors that do and do not have HPV integrants display distinct gene expression profiles and DNA methylation patterns, which further support the view that the mechanisms by which tumors with integrated and nonintegrated HPV arise are distinct. Previous studies have established that a subset of head and neck tumors contains human papillomavirus (HPV) sequences and that HPV-driven head and neck cancers display distinct biological and clinical features. HPV is known to drive cancer by the actions of the E6 and E7 oncoproteins, but the molecular architecture of HPV infection and its interaction with the host genome in head and neck cancers have not been comprehensively described. We profiled a cohort of 279 head and neck cancers with next generation RNA and DNA sequencing and show that 35 (12.5%) tumors displayed evidence of high-risk HPV types 16, 33, or 35. Twenty-five cases had integration of the viral genome into one or more locations in the human genome with statistical enrichment for genic regions. Integrations had a marked impact on the human genome and were associated with alterations in DNA copy number, mRNA transcript abundance and splicing, and both inter- and intrachromosomal rearrangements. Many of these events involved genes with documented roles in cancer. Cancers with integrated vs. nonintegrated HPV displayed different patterns of DNA methylation and both human and viral gene expressions. Together, these data provide insight into the mechanisms by which HPV interacts with the human genome beyond expression of viral oncoproteins and suggest that specific integration events are an integral component of viral oncogenesis.

Recurrent PTPRB and PLCG1 mutations in angiosarcoma

Angiosarcoma is an aggressive malignancy that arises spontaneously or secondarily to ionizing radiation or chronic lymphoedema. Previous work has identified aberrant angiogenesis, including occasional somatic mutations in angiogenesis signaling genes, as a key driver of angiosarcoma. Here we employed whole-genome, whole-exome and targeted sequencing to study the somatic changes underpinning primary and secondary angiosarcoma. We identified recurrent mutations in two genes, PTPRB and PLCG1, which are intimately linked to angiogenesis. The endothelial phosphatase PTPRB, a negative regulator of vascular growth factor tyrosine kinases, harbored predominantly truncating mutations in 10 of 39 tumors (26%). PLCG1, a signal transducer of tyrosine kinases, encoded a recurrent, likely activating p.Arg707Gln missense variant in 3 of 34 cases (9%). Overall, 15 of 39 tumors (38%) harbored at least one driver mutation in angiogenesis signaling genes. Our findings inform and reinforce current therapeutic efforts to target angiogenesis signaling in angiosarcoma.

The SMARCA2/4 ATPase domain surpasses the bromodomain as a drug target in SWI/SNF mutant cancers: Insights from cDNA rescue and PFI-3 inhibitor studies

The SWI/SNF multisubunit complex modulates chromatin structure through the activity of two mutually exclusive catalytic subunits, SMARCA2 and SMARCA4, which both contain a bromodomain and an ATPase domain. Using RNAi, cancer-specific vulnerabilities have been identified in SWI/SNF-mutant tumors, including SMARCA4-deficient lung cancer; however, the contribution of conserved, druggable protein domains to this anticancer phenotype is unknown. Here, we functionally deconstruct the SMARCA2/4 paralog dependence of cancer cells using bioinformatics, genetic, and pharmacologic tools. We evaluate a selective SMARCA2/4 bromodomain inhibitor (PFI-3) and characterize its activity in chromatin-binding and cell-functional assays focusing on cells with altered SWI/SNF complex (e.g., lung, synovial sarcoma, leukemia, and rhabdoid tumors). We demonstrate that PFI-3 is a potent, cell-permeable probe capable of displacing ectopically expressed, GFP-tagged SMARCA2-bromodomain from chromatin, yet contrary to target knockdown, the inhibitor fails to display an antiproliferative phenotype. Mechanistically, the lack of pharmacologic efficacy is reconciled by the failure of bromodomain inhibition to displace endogenous, full-length SMARCA2 from chromatin as determined by in situ cell extraction, chromatin immunoprecipitation, and target gene expression studies. Furthermore, using inducible RNAi and cDNA complementation (bromodomain- and ATPase-dead constructs), we unequivocally identify the ATPase domain, and not the bromodomain of SMARCA2, as the relevant therapeutic target with the catalytic activity suppressing defined transcriptional programs. Taken together, our complementary genetic and pharmacologic studies exemplify a general strategy for multidomain protein drug-target validation and in case of SMARCA2/4 highlight the potential for drugging the more challenging helicase/ATPase domain to deliver on the promise of synthetic-lethality therapy.

Systematic Epigenomic Analysis Reveals Chromatin States Associated with Melanoma Progression

The extent and nature of epigenomic changes associated with melanoma progression is poorly understood. Through systematic epigenomic profiling of 35 epigenetic modifications and transcriptomic analysis, we define chromatin state changes associated with melanomagenesis by using a cell phenotypic model of non-tumorigenic and tumorigenic states. Computation of specific chromatin state transitions showed loss of histone acetylations and H3K4me2/3 on regulatory regions proximal to specific cancer-regulatory genes in important melanoma-driving cell signaling pathways. Importantly, such acetylation changes were also observed between benign nevi and malignant melanoma human tissues. Intriguingly, only a small fraction of chromatin state transitions correlated with expected changes in gene expression patterns. Restoration of acetylation levels on deacetylated loci by histone deacetylase (HDAC) inhibitors selectively blocked excessive proliferation in tumorigenic cells and human melanoma cells, suggesting functional roles of observed chromatin state transitions in driving hyperproliferative phenotype. Through these results, we define functionally relevant chromatin states associated with melanoma progression.

A Pan-Cancer Compendium of Genes Deregulated by Somatic Genomic Rearrangement across More Than 1,400 Cases

A systematic cataloging of genes affected by genomic rearrangement, using multiple patient cohorts and cancer types, can provide insight into cancer-relevant alterations outside of exomes. By integrative analysis of whole-genome sequencing (predominantly low pass) and gene expression data from 1,448 cancers involving 18 histopathological types in The Cancer Genome Atlas, we identified hundreds of genes for which the nearby presence (within 100 kb) of a somatic structural variant (SV) breakpoint is associated with altered expression. While genomic rearrangements are associated with widespread copy-number alteration (CNA) patterns, approximately 1,100 genes-including overexpressed cancer driver genes (e.g., TERT, ERBB2, CDK12, CDK4) and underexpressed tumor suppressors (e.g., TP53, RB1, PTEN, STK11)-show SV-associated deregulation independent of CNA. SVs associated with the disruption of topologically associated domains, enhancer hijacking, or fusion transcripts are implicated in gene upregulation. For cancer-relevant pathways, SVs considerably expand our understanding of how genes are affected beyond point mutation or CNA.

Abstract 3528: The SMARCA2/4 catalytic activity, but not the bromodomain, is a drug target in SWI/SNF mutant cancers

The SWI/SNF multi-subunit complex modulates chromatin structure through the activity of two mutually exclusive catalytic subunits, SMARCA2 and SMARCA4, which both contain a bromodomain and an ATPase domain. Using RNAi, cancer-specific vulnerabilities have been identified in SWI/SNF mutant tumors, including SMARCA4-deficient lung cancer, however, the contribution of conserved, druggable protein domains to this anticancer phenotype is unknown. Here, we functionally deconstructed the SMARCA2/4 paralog dependence of cancer cells using bioinformatics, genetic and pharmacological tools. We evaluated a potent and selective SMARCA2/4 bromodomain inhibitor (PFI-3) and characterized its activity in chromatin-binding and cell-functional assays focusing on cells with altered SWI/SNF status (e.g. Lung, Synovial Sarcoma, Leukemia, and Rhabdoid tumors). We demonstrated that PFI-3 is a cell-permeable probe capable of displacing ectopically expressed, GFP-tagged SMARCA2-bromodomain from chromatin, yet contrary to target knockdown, the inhibitor failed to display an antiproliferative phenotype. Mechanistically, the lack of pharmacological efficacy was reconciled by the failure of bromodomain inhibition to displace endogenous, full-length SMARCA2 from chromatin as determined by in situ cell extraction, chromatin immunoprecipitation (ChIP) and target gene expression and promoter occupancy studies. Using RNAi and cDNA complementation (bromodomain and ATPase-dead constructs), we identified the catalytic ATPase domain, and not the bromodomain of SMARCA2, as a relevant therapeutic target. Taken together, our complementary genetic and pharmacological studies exemplify a general strategy for bromodomain drug-target validation and in case of SMARCA2/4 highlight the requirement for drugging the more challenging helicase/ATPase domain affording potential synthetic-lethal treatment options to cancer patients with genetically defined alterations in SWI/SNF. Citation Format: Bhavatarini Vangamudi, Thomas Paul, Parantu K. Shah, Maria K. Alimova, Lisa Nottebaum, Xi Shi, Yanai Zhan, Elisabetta Leo, Harshad S. Mahadeshwar, Alexei Protopopov, Andrew Futreal, Trang N. Tieu, Mike Peoples, Alessia Petrocchi, Joseph R. Marszalek, Carlo Toniatti, Timothy P. Heffernan, Dominique Verhelle, Giulio Draetta, Dafydd Owen, Philip Jones, Wylie Palmer, Shikhar Sharma, Jannik N. Andersen. The SMARCA2/4 catalytic activity, but not the bromodomain, is a drug target in SWI/SNF mutant cancers. [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 3528. doi:10.1158/1538-7445.AM2015-3528