A functional genomic approach to actionable gene fusions for precision oncology.

Fusion genes represent a class of attractive therapeutic targets. Thousands of fusion genes have been identified in patients with cancer, but the functional consequences and therapeutic implications of most of these remain largely unknown. Here, we develop a functional genomic approach that consists of efficient fusion reconstruction and sensitive cell viability and drug response assays. Applying this approach, we characterize ~100 fusion genes detected in patient samples of The Cancer Genome Atlas, revealing a notable fraction of low-frequency fusions with activating effects on tumor growth. Focusing on those in the RTK-RAS pathway, we identify a number of activating fusions that can markedly affect sensitivity to relevant drugs. Last, we propose an integrated, level-of-evidence classification system to prioritize gene fusions systematically. Our study reiterates the urgent clinical need to incorporate similar functional genomic approaches to characterize gene fusions, thereby maximizing the utility of gene fusions for precision oncology.

Global impact of somatic structural variation on the DNA methylome of human cancers.

BACKGROUND: Genomic rearrangements exert a heavy influence on the molecular landscape of cancer. New analytical approaches integrating somatic structural variants (SSVs) with altered gene features represent a framework by which we can assign global significance to a core set of genes, analogous to established methods that identify genes non-randomly targeted by somatic mutation or copy number alteration. While recent studies have defined broad patterns of association involving gene transcription and nearby SSV breakpoints, global alterations in DNA methylation in the context of SSVs remain largely unexplored. RESULTS: By data integration of whole genome sequencing, RNA sequencing, and DNA methylation arrays from more than 1400 human cancers, we identify hundreds of genes and associated CpG islands (CGIs) for which the nearby presence of a somatic structural variant (SSV) breakpoint is recurrently associated with altered expression or DNA methylation, respectively, independently of copy number alterations. CGIs with SSV-associated increased methylation are predominantly promoter-associated, while CGIs with SSV-associated decreased methylation are enriched for gene body CGIs. Rearrangement of genomic regions normally having higher or lower methylation is often involved in SSV-associated CGI methylation alterations. Across cancers, the overall structural variation burden is associated with a global decrease in methylation, increased expression in methyltransferase genes and DNA damage response genes, and decreased immune cell infiltration. CONCLUSION: Genomic rearrangement appears to have a major role in shaping the cancer DNA methylome, to be considered alongside commonly accepted mechanisms including histone modifications and disruption of DNA methyltransferases.

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.

A Pan-Cancer Proteogenomic Atlas of PI3K/AKT/mTOR Pathway Alterations.

Molecular alterations involving the PI3K/AKT/mTOR pathway (including mutation, copy number, protein, or RNA) were examined across 11,219 human cancers representing 32 major types. Within specific mutated genes, frequency, mutation hotspot residues, in silico predictions, and functional assays were all informative in distinguishing the subset of genetic variants more likely to have functional relevance. Multiple oncogenic pathways including PI3K/AKT/mTOR converged on similar sets of downstream transcriptional targets. In addition to mutation, structural variations and partial copy losses involving PTEN and STK11 showed evidence for having functional relevance. A substantial fraction of cancers showed high mTOR pathway activity without an associated canonical genetic or genomic alteration, including cancers harboring IDH1 or VHL mutations, suggesting multiple mechanisms for pathway activation.

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.