Juliann Shih

Large panels of comprehensively characterized human cancer models, including the Cancer Cell Line Encyclopedia (CCLE), have provided a rigorous framework with which to study genetic variants, candidate targets, and small-molecule and biological therapeutics and to identify new marker-driven cancer dependencies. To improve our understanding of the molecular features that contribute to cancer phenotypes, including drug responses, here we have expanded the characterizations of cancer cell lines to include genetic, RNA splicing, DNA methylation, histone H3 modification, microRNA expression and reverse-phase protein array data for 1,072 cell lines from individuals of various lineages and ethnicities. Integration of these data with functional characterizations such as drug-sensitivity, short hairpin RNA knockdown and CRISPR–Cas9 knockout data reveals potential targets for cancer drugs and associated biomarkers. Together, this dataset and an accompanying public data portal provide a resource for the acceleration of cancer research using model cancer cell lines. The original Cancer Cell Line Encyclopedia (CCLE) is expanded with deeper characterization of over 1,000 cell lines, including genomic, transcriptomic, and proteomic data, and integration with drug-sensitivity and gene-dependency data.

Liver cancer has the second highest worldwide cancer mortality rate and has limited therapeutic options. We analyzed 363 hepatocellular carcinoma (HCC) cases by whole-exome sequencing and DNA copy number analyses, and we analyzed 196 HCC cases by DNA methylation, RNA, miRNA, and proteomic expression also. DNA sequencing and mutation analysis identified significantly mutated genes, including LZTR1, EEF1A1, SF3B1, and SMARCA4. Significant alterations by mutation or downregulation by hypermethylation in genes likely to result in HCC metabolic reprogramming (ALB, APOB, and CPS1) were observed. Integrative molecular HCC subtyping incorporating unsupervised clustering of five data platforms identified three subtypes, one of which was associated with poorer prognosis in three HCC cohorts. Integrated analyses enabled development of a p53 target gene expression signature correlating with poor survival. Potential therapeutic targets for which inhibitors exist include WNT signaling, MDM4, MET, VEGFA, MCL1, IDH1, TERT, and immune checkpoint proteins CTLA-4, PD-1, and PD-L1.

We performed integrated genomic, transcriptomic, and proteomic profiling of 150 pancreatic ductal adenocarcinoma (PDAC) specimens, including samples with characteristic low neoplastic cellularity. Deep whole-exome sequencing revealed recurrent somatic mutations in KRAS, TP53, CDKN2A, SMAD4, RNF43, ARID1A, TGFβR2, GNAS, RREB1, and PBRM1. KRAS wild-type tumors harbored alterations in other oncogenic drivers, including GNAS, BRAF, CTNNB1, and additional RAS pathway genes. A subset of tumors harbored multiple KRAS mutations, with some showing evidence of biallelic mutations. Protein profiling identified a favorable prognosis subset with low epithelial-mesenchymal transition and high MTOR pathway scores. Associations of non-coding RNAs with tumor-specific mRNA subtypes were also identified. Our integrated multi-platform analysis reveals a complex molecular landscape of PDAC and provides a roadmap for precision medicine.

Aneuploidy, whole chromosome or chromosome arm imbalance, is a near-universal characteristic of human cancers. In 10,522 cancer genomes from The Cancer Genome Atlas, aneuploidy was correlated with TP53 mutation, somatic mutation rate, and expression of proliferation genes. Aneuploidy was anti-correlated with expression of immune signaling genes, due to decreased leukocyte infiltrates in high-aneuploidy samples. Chromosome arm-level alterations show cancer-specific patterns, including loss of chromosome arm 3p in squamous cancers. We applied genome engineering to delete 3p in lung cells, causing decreased proliferation rescued in part by chromosome 3 duplication. This study defines genomic and phenotypic correlates of cancer aneuploidy and provides an experimental approach to study chromosome arm aneuploidy.

<h2>Summary</h2> Comprehensive multiplatform analysis of 80 uveal melanomas (UM) identifies four molecularly distinct, clinically relevant subtypes: two associated with poor-prognosis monosomy 3 (M3) and two with better-prognosis disomy 3 (D3). We show that <i>BAP1</i> loss follows M3 occurrence and correlates with a global DNA methylation state that is distinct from D3-UM. Poor-prognosis M3-UM divide into subsets with divergent genomic aberrations, transcriptional features, and clinical outcomes. We report change-of-function <i>SRSF2</i> mutations. Within D3-UM, <i>EIF1AX</i>- and <i>SRSF2</i>/<i>SF3B1</i>-mutant tumors have distinct somatic copy number alterations and DNA methylation profiles, providing insight into the biology of these low- versus intermediate-risk clinical mutation subtypes.

Patient-derived xenografts (PDXs) have become a prominent cancer model system, as they are presumed to faithfully represent the genomic features of primary tumors. Here we monitored the dynamics of copy number alterations (CNAs) in 1,110 PDX samples across 24 cancer types. We observed rapid accumulation of CNAs during PDX passaging, often due to selection of preexisting minor clones. CNA acquisition in PDXs was correlated with the tissue-specific levels of aneuploidy and genetic heterogeneity observed in primary tumors. However, the particular CNAs acquired during PDX passaging differed from those acquired during tumor evolution in patients. Several CNAs recurrently observed in primary tumors gradually disappeared in PDXs, indicating that events undergoing positive selection in humans can become dispensable during propagation in mice. Notably, the genomic stability of PDXs was associated with their response to chemotherapy and targeted drugs. These findings have major implications for PDX-based modeling of human cancer.

We describe a comprehensive genomic characterization of adrenocortical carcinoma (ACC). Using this dataset, we expand the catalogue of known ACC driver genes to include PRKAR1A, RPL22, TERF2, CCNE1, and NF1. Genome wide DNA copy-number analysis revealed frequent occurrence of massive DNA loss followed by whole-genome doubling (WGD), which was associated with aggressive clinical course, suggesting WGD is a hallmark of disease progression. Corroborating this hypothesis were increased TERT expression, decreased telomere length, and activation of cell-cycle programs. Integrated subtype analysis identified three ACC subtypes with distinct clinical outcome and molecular alterations which could be captured by a 68-CpG probe DNA-methylation signature, proposing a strategy for clinical stratification of patients based on molecular markers.

Abstract Malignant pleural mesothelioma (MPM) is a highly lethal cancer of the lining of the chest cavity. To expand our understanding of MPM, we conducted a comprehensive integrated genomic study, including the most detailed analysis of BAP1 alterations to date. We identified histology-independent molecular prognostic subsets, and defined a novel genomic subtype with TP53 and SETDB1 mutations and extensive loss of heterozygosity. We also report strong expression of the immune-checkpoint gene VISTA in epithelioid MPM, strikingly higher than in other solid cancers, with implications for the immune response to MPM and for its immunotherapy. Our findings highlight new avenues for further investigation of MPM biology and novel therapeutic options. Significance: Through a comprehensive integrated genomic study of 74 MPMs, we provide a deeper understanding of histology-independent determinants of aggressive behavior, define a novel genomic subtype with TP53 and SETDB1 mutations and extensive loss of heterozygosity, and discovered strong expression of the immune-checkpoint gene VISTA in epithelioid MPM. See related commentary by Aggarwal and Albelda, p. 1508. This article is highlighted in the In This Issue feature, p. 1494

We studied 137 primary testicular germ cell tumors (TGCTs) using high-dimensional assays of genomic, epigenomic, transcriptomic, and proteomic features. These tumors exhibited high aneuploidy and a paucity of somatic mutations. Somatic mutation of only three genes achieved significance-KIT, KRAS, and NRAS-exclusively in samples with seminoma components. Integrated analyses identified distinct molecular patterns that characterized the major recognized histologic subtypes of TGCT: seminoma, embryonal carcinoma, yolk sac tumor, and teratoma. Striking differences in global DNA methylation and microRNA expression between histology subtypes highlight a likely role of epigenomic processes in determining histologic fates in TGCTs. We also identified a subset of pure seminomas defined by KIT mutations, increased immune infiltration, globally demethylated DNA, and decreased KRAS copy number. We report potential biomarkers for risk stratification, such as miRNA specifically expressed in teratoma, and others with molecular diagnostic potential, such as CpH (CpA/CpC/CpT) methylation identifying embryonal carcinomas.

This integrated, multiplatform PanCancer Atlas study co-mapped and identified distinguishing molecular features of squamous cell carcinomas (SCCs) from five sites associated with smoking and/or human papillomavirus (HPV). SCCs harbor 3q, 5p, and other recurrent chromosomal copy-number alterations (CNAs), DNA mutations, and/or aberrant methylation of genes and microRNAs, which are correlated with the expression of multi-gene programs linked to squamous cell stemness, epithelial-to-mesenchymal differentiation, growth, genomic integrity, oxidative damage, death, and inflammation. Low-CNA SCCs tended to be HPV(+) and display hypermethylation with repression of TET1 demethylase and FANCF, previously linked to predisposition to SCC, or harbor mutations affecting CASP8, RAS-MAPK pathways, chromatin modifiers, and immunoregulatory molecules. We uncovered hypomethylation of the alternative promoter that drives expression of the ΔNp63 oncogene and embedded miR944. Co-expression of immune checkpoint, T-regulatory, and Myeloid suppressor cells signatures may explain reduced efficacy of immune therapy. These findings support possibilities for molecular classification and therapeutic approaches.

Tuberous sclerosis complex (TSC) is a rare genetic disease causing multisystem growth of benign tumours and other hamartomatous lesions, which leads to diverse and debilitating clinical symptoms. Patients are born with TSC1 or TSC2 mutations, and somatic inactivation of wild-type alleles drives MTOR activation; however, second hits to TSC1/TSC2 are not always observed. Here, we present the genomic landscape of TSC hamartomas. We determine that TSC lesions contain a low somatic mutational burden relative to carcinomas, a subset feature large-scale chromosomal aberrations, and highly conserved molecular signatures for each type exist. Analysis of the molecular signatures coupled with computational approaches reveals unique aspects of cellular heterogeneity and cell origin. Using immune data sets, we identify significant neuroinflammation in TSC-associated brain tumours. Taken together, this molecular catalogue of TSC serves as a resource into the origin of these hamartomas and provides a framework that unifies genomic and transcriptomic dimensions for complex tumours.

(Cell Reports 18, 2780–2794; March 14, 2017) In the originally published version of this article, the sample names in Figure 5D were incorrectly ordered during assembly of the heatmap. The sample order has now been corrected online, and the corrected figure appears below. The authors regret this error. Also, due to a misunderstanding prior to publication, the TCGA network authors were not properly indexed in PubMed. This has now been corrected online.Figure 5ARID1A Is Hypermethylated and Has Low Expression in the IDH COCA (original)View Large Image Figure ViewerDownload Hi-res image Download (PPT) Integrative Genomic Analysis of Cholangiocarcinoma Identifies Distinct IDH-Mutant Molecular ProfilesFarshidfar et al.Cell ReportsMarch 14, 2017In BriefFarshidfar et al. present The Cancer Genome Atlas (TCGA) marker analysis of cholangiocarcinoma. Through multi-platform analyses, they identify a distinct subtype enriched for IDH mutants. This subtype shows increased mitochondrial and decreased chromatin modifier gene expression, including potential epigenetic silencing of ARID1A. Other IDH-mutant liver cancers molecularly resemble cholangiocarcinoma. Full-Text PDF Open Access

Aneuploidies-whole-chromosome or whole-arm imbalances-are the most prevalent alteration in cancer genomes1,2. However, it is still debated whether their prevalence is due to selection or ease of generation as passenger events1,2. Here we developed a method, BISCUT, that identifies loci subject to fitness advantages or disadvantages by interrogating length distributions of telomere- or centromere-bounded copy-number events. These loci were significantly enriched for known cancer driver genes, including genes not detected through analysis of focal copy-number events, and were often lineage specific. BISCUT identified the helicase-encoding gene WRN as a haploinsufficient tumour-suppressor gene on chromosome 8p, which is supported by several lines of evidence. We also formally quantified the role of selection and mechanical biases in driving aneuploidy, finding that rates of arm-level copy-number alterations are most highly correlated with their effects on cellular fitness1,2. These results provide insight into the driving forces behind aneuploidy and its contribution to tumorigenesis.

Abstract The Krüppel-like family of transcription factors plays critical roles in human development and is associated with cancer pathogenesis. Krüppel-like factor 5 gene (KLF5) has been shown to promote cancer cell proliferation and tumorigenesis and to be genomically amplified in cancer cells. We recently reported that the KLF5 gene is also subject to other types of somatic coding and noncoding genomic alterations in diverse cancer types. Here, we show that these alterations activate KLF5 by three distinct mechanisms: (i) Focal amplification of superenhancers activates KLF5 expression in squamous cell carcinomas; (ii) Missense mutations disrupt KLF5–FBXW7 interactions to increase KLF5 protein stability in colorectal cancer; (iii) Cancer type–specific hotspot mutations within a zinc-finger DNA binding domain of KLF5 change its DNA binding specificity and reshape cellular transcription. Utilizing data from CRISPR/Cas9 gene knockout screening, we reveal that cancer cells with KLF5 overexpression are dependent on KLF5 for their proliferation, suggesting KLF5 as a putative therapeutic target. Significance: Our observations, together with previous studies that identified oncogenic properties of KLF5, establish the importance of KLF5 activation in human cancers, delineate the varied genomic mechanisms underlying this occurrence, and nominate KLF5 as a putative target for therapeutic intervention in cancer. Cancer Discov; 8(1); 108–25. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 1

(Cancer Cell 29, 723–736; May 9, 2016) Due to a miscommunication during the process of transferring this manuscript from our editorial team to Production, the TCGA network authors were not properly indexed in PubMed. This has now been corrected online. We apologize for any confusion or inconvenience that this oversight might have caused. Comprehensive Pan-Genomic Characterization of Adrenocortical CarcinomaZheng et al.Cancer CellMay 09, 2016In BriefZheng et al. perform comprehensive genomic characterization of 91 cases of adrenocortical carcinoma (ACC). This analysis expands the list of driver genes in ACC, reveals whole-genome doubling as a hallmark of ACC progression, and identifies three ACC subtypes with distinct clinical outcome. Full-Text PDF Open Archive

Early-onset gastric cancer, which develops in patients younger than most gastric cancers, is usually detected at advanced stages, has diffuse histologic features, and occurs more frequently in women. We investigated somatic genomic alterations associated with the unique characteristics of sporadic diffuse gastric cancers (DGCs) from younger patients.We conducted whole exome and RNA sequence analyses of 80 resected DGC samples from patients 45 years old or younger in Korea. Patients with pathogenic germline mutations in CDH1, TP53, and ATM were excluded from the onset of this analysis, given our focus on somatic alterations. We used MutSig2CV to evaluate the significance of mutated genes. We recruited 29 additional early-onset Korean DGC samples and performed SNP6.0 array and targeted sequencing analyses of these 109 early-onset DGC samples (54.1% female, median age, 38 years). We compared the SNP6.0 array and targeted sequencing data of the 109 early-onset DGC samples with those from diffuse-type stomach tumor samples collected from 115 patients in Korea who were 46 years or older (late onset) at the time of diagnosis (controls; 29.6% female, median age, 67 years). We compared patient survival times among tumors from different subgroups and with different somatic mutations. We performed gene silencing of RHOA or CDH1 in DGC cells with small interfering RNAs for cell-based assays.We identified somatic mutations in the following genes in a significant number of early-onset DGCs: the cadherin 1 gene (CDH1), TP53, ARID1A, KRAS, PIK3CA, ERBB3, TGFBR1, FBXW7, RHOA, and MAP2K1. None of 109 early-onset DGC cases had pathogenic germline CDH1 mutations. A higher proportion of early-onset DGCs had mutations in CDH1 (42.2%) or TGFBR1 (7.3%) compared with control DGCs (17.4% and 0.9%, respectively) (P < .001 and P = .014 for CDH1 and TGFBR1, respectively). In contrast, a smaller proportion of early-onset DGCs contained mutations in RHOA (9.2%) than control DGCs (19.1%) (P = .033). Late-onset DGCs in The Cancer Genome Atlas also contained less frequent mutations in CDH1 and TGFBR1 and more frequent RHOA mutations, compared with early-onset DGCs. Early-onset DGCs from women contained significantly more mutations in CDH1 or TGFBR1 than early-onset DGCs from men. CDH1 alterations, but not RHOA mutations, were associated with shorter survival times in patients with early-onset DGCs (hazard ratio, 3.4; 95% confidence interval, 1.5-7.7). RHOA activity was reduced by an R5W substitution-the RHOA mutation most frequently detected in early-onset DGCs. Silencing of CDH1, but not RHOA, increased migratory activity of DGC cells.In an integrative genomic analysis, we found higher proportions of early-onset DGCs to contain somatic mutations in CDH1 or TGFBR1 compared with late-onset DGCs. However, a smaller proportion of early-onset DGCs contained somatic mutations in RHOA than late-onset DGCs. CDH1 alterations, but not RHOA mutations, were associated with shorter survival times of patients, which might account for the aggressive clinical course of early-onset gastric cancer. Female predominance in early-onset gastric cancer may be related to relatively high rates of somatic CDH1 and TGFBR1 mutations in this population.

Abstract The PPARG gene encoding the nuclear receptor PPARγ is activated in bladder cancer, either directly by gene amplification or mutation, or indirectly by mutation of the RXRA gene, which encodes the heterodimeric partner of PPARγ. Here, we show that activating alterations of PPARG or RXRA lead to a specific gene expression signature in bladder cancers. Reducing PPARG activity, whether by pharmacologic inhibition or genetic ablation, inhibited proliferation of PPARG-activated bladder cancer cells. Our results offer a preclinical proof of concept for PPARG as a candidate therapeutic target in bladder cancer. Cancer Res; 77(24); 6987–98. ©2017 AACR.

Functional redundancy shared by paralog genes may afford protection against genetic perturbations, but it can also result in genetic vulnerabilities due to mutual interdependency1–5. Here, we surveyed genome-scale short hairpin RNA and CRISPR screening data on hundreds of cancer cell lines and identified MAGOH and MAGOHB, core members of the splicing-dependent exon junction complex, as top-ranked paralog dependencies6–8. MAGOHB is the top gene dependency in cells with hemizygous MAGOH deletion, a pervasive genetic event that frequently occurs due to chromosome 1p loss. Inhibition of MAGOHB in a MAGOH-deleted context compromises viability by globally perturbing alternative splicing and RNA surveillance. Dependency on IPO13, an importin-β receptor that mediates nuclear import of the MAGOH/B-Y14 heterodimer9, is highly correlated with dependency on both MAGOH and MAGOHB. Both MAGOHB and IPO13 represent dependencies in murine xenografts with hemizygous MAGOH deletion. Our results identify MAGOH and MAGOHB as reciprocal paralog dependencies across cancer types and suggest a rationale for targeting the MAGOHB-IPO13 axis in cancers with chromosome 1p deletion. Analysis of paralog gene pairs using data from loss-of-function genetic screens in cancer cells identifies MAGOH and MAGOHB as reciprocal paralog dependencies across cancer types.

The role of PPM1D mutations in de novo gliomagenesis has not been systematically explored. Here we analyze whole genome sequences of 170 pediatric high-grade gliomas and find that truncating mutations in PPM1D that increase the stability of its phosphatase are clonal driver events in 11% of Diffuse Midline Gliomas (DMGs) and are enriched in primary pontine tumors. Through the development of DMG mouse models, we show that PPM1D mutations potentiate gliomagenesis and that PPM1D phosphatase activity is required for in vivo oncogenesis. Finally, we apply integrative phosphoproteomic and functional genomics assays and find that oncogenic effects of PPM1D truncation converge on regulators of cell cycle, DNA damage response, and p53 pathways, revealing therapeutic vulnerabilities including MDM2 inhibition.

The expansion of targeted panel sequencing efforts has created opportunities for large-scale genomic analysis, but tools for copy-number quantification on panel data are lacking. We introduce ASCETS, a method for the efficient quantitation of arm and chromosome-level copy-number changes from targeted sequencing data.ASCETS is implemented in R and is freely available to non-commercial users on GitHub: https://github.com/beroukhim-lab/ascets, along with detailed documentation.Supplementary data are available at Bioinformatics online.

Variation is essential to species survival and adaptation during evolution. This variation is conferred by the imperfection of biochemical processes, such as mutations and alterations in DNA sequences, and can also be seen within genomes through processes such as the generation of antibodies. Recent sequencing projects have produced multiple versions of the genomes of humans and fruit flies (Drosophila melanogaster). These give us a chance to study how individual gene sequences vary within and between species. Here we arranged human and fly genes in orthologous pairs and compared such within-species variability with their degree of conservation between flies and humans. We observed that a significant number of proteins associated with mRNA translation are highly conserved between species and yet are highly variable within each species. The fact that we observe this in two species whose lineages separated more than 700 million years ago suggests that this is the result of a very ancient process. We hypothesize that this effect might be attributed to a positive selection for variability of virus-interacting proteins that confers a general resistance to viral hijacking of the mRNA translation machinery within populations. Our analysis points to this and to other processes resulting in positive selection for gene variation.

Aneuploidy, whole chromosome or chromosome arm copy number imbalance, is a near-universal characteristic of human cancers. We applied methods that define chromosome arm-level aneuploidy and a global cancer aneuploidy score to 10,522 tumors of 33 types in the Cancer Genome Atlas (TCGA). Aneuploidy level was correlated with TP53 mutation, somatic mutation rate, and expression of proliferation genes. Aneuploidy was anti-correlated with expression of immune signaling genes, due to decreased leukocyte infiltrates in high-aneuploidy samples. Although yeast and mammalian models of whole chromosome aneuploidies have been extensively investigated, chromosome arm-level aneuploidies have rarely been modeled. Cancer subtypes are often characterized by tumor specific patterns of these arm-level copy number alterations; for example, squamous cell carcinomas (SCCs) from different tissues of origin (including lung, esophagus, and bladder) have a pattern of chromosome 3p loss and chromosome 3q gain. Our analysis of 495 lung SCCs found chromosome 3p deletion to be the most frequent genomic alteration, occurring in almost 80% of the tumors and covering the entire length of the chromosome arm. Over two-thirds of chromosome 3p genes showed significantly decreased expression in these samples. Without models of chromosome arm-level alterations, the phenotypic effects of specific aneuploidies in cancer, such as 3p deletion, remain unknown. However, recent advances in genome engineering and targeting of endonucleases allow new approaches to generate chromosomal alterations. Here, we used the CRISPR-Cas9 system to delete one copy of chromosome 3p in vitro. We successfully isolated almost 90 clones of immortalized lung epithelial cells with deletion of the 3p arm, with 8 validated by whole genome sequencing. Consistent with patient data, expression of 3p genes was also decreased upon deletion, as well as increased expression of interferon response genes. Phenotypic characterization revealed that cells with chromosome 3p deletion initially proliferated more slowly than their siblings. These chromosome 3p deleted cells had increased G1 arrest but did not undergo increased apoptosis or cell death. Interestingly, after several passages in culture, the proliferation defect was rescued in chromosome 3p deleted cells; genome sequencing and karyotype analyses suggested that this was the result of chromosome 3 duplication. With our cellular model of chromosome arm-level aneuploidy, we uncovered a possible selection mechanism that allows aneuploidy tolerance in vitro. We used genome engineering to model chromosome arm-level deletions, providing a robust model that will address a gap in our understanding of aneuploidy in cancer. aneuploidy, squamous cancers, genome engineering

Abstract Patient-derived xenografts (PDXs) have become a prominent model for studying human cancer in vivo . The underlying assumption is that PDXs faithfully represent the genomic features of primary tumors, retaining their molecular characteristics throughout propagation. However, the genomic stability of PDXs during passaging has not yet been evaluated systematically. Here we monitored the dynamics of copy number alterations (CNAs) in 1,110 PDX samples across 24 cancer types. We found that new CNAs accumulated quickly, such that within four passages an average of 12% of the genome was affected by newly acquired CNAs. Selection for preexisting minor clones was a major contributor to these changes, leading to both gains and losses of CNAs. The rate of CNA acquisition in PDX models was correlated with the extent of both aneuploidy and genetic heterogeneity observed in primary tumors of the same tissue. However, the specific CNAs acquired during PDX passaging differed from those acquired during tumor evolution in patients, suggesting that PDX tumors are subjected to distinct selection pressures compared to those that exist in human hosts. Specifically, several recurrent CNAs observed in primary tumors gradually disappeared in PDXs, indicating that events undergoing positive selection in humans can become dispensable during propagation in mice. Finally, we found that the genomic stability of PDX models also affected their responses to chemotherapy and targeted drugs. Our findings thus highlight the need to couple the timing of PDX molecular characterization to that of drug testing experiments. These results suggest that while PDX models are powerful tools, they should be used with caution.

Abstract Adrenocortical carcinoma (ACC) is a rare neoplasm with a heterogeneous outcome and limited treatment options. To understand its molecular and genomic landscape as a part of The Cancer Genome Atlas (TCGA) project, we performed the genomic, transcriptomic, epigenomic and proteomic profiling of 91 ACCs. We identified potential driving alterations including amplifications (TERT, TERF2 and CDK4), deletions (ZNRF3, CDKN2A and RB1) and point mutations in genes unknown to participate in adrenal disease (RPL22) and genes known to initiate familial syndromes that occasionally include adrenocortical neoplasms (TP53, CTNNB1, PRKAR1A, MEN1). The finding of PRKAR1A expands the catalogue of pathogenic pathways underlying ACC, suggesting of the protein kinase alpha signaling pathway as a potential target for molecular interventions. Novel transcript fusions potentially leading to overactive kinases included EXOSC10-MTOR and PPP1CB-BRE. DNA copy number analysis unveiled prevalent DNA losses leading to hypodiploidy as well as whole genome doubling (WGD) in 51% of ACC. The similar penetrance of loss of heterozygosity before and after WGD suggests a sequential development from hypodiploidy to polyploidy along the doubling in a subset of ACCs, which was endorsed by the worse outcome for WGD samples relative to nonWGD ACCs. An association between TERT expression and WGD was observed, suggesting a role for telomere regulation. These findings present ACC as a model disease for studies of WGD which is a frequent event in many tumor types. Unsupervised clustering of DNA methylation, copy number, gene expression, miRNA expression and protein abundance converged into three classes with specific biological characteristics and a respective median event free survival of 8, 38 and &amp;gt;100 months (p-value 1.7e-13). Comparison of the subtypes suggested additional drivers such as protein kinase C (PKC) phosphorylation and upregulation of a miRNA cluster at chromosome Xq27.3, which complemented the genomic alterations identified in these subtypes. To gain more insights into this rare cancer type, we placed ACC in a broader context of cancer genomic profiles including an array of other cancer types. These analyses revealed interesting shared features, including beta-catenin activation with a subset of endometroid cancer, DNA mismatch repair gene mutational signature with gastrointestinal cancers and a smoking signature with lung cancer. These findings highlight the commonalities between ACC and other lineages of cancer. Taken together, we found Wnt signaling pathway and p53/Rb signaling pathway were the most frequently altered pathways in ACC. Meanwhile, new players surfaced from our analyses including the PKA and PKC pathways. Our results present a comprehensive genomic landscape and refined molecular classification of ACC improve our understanding of its pathogenesis, and will ultimately improve the care of patients. Citation Format: Siyuan Zheng, Andrew D. Cherniack, Ninad Dewal, Richard A. Moffitt, Ludmila Danilova, Bradley A. Murray, Antonio M. Lerario, Tobias Else, Theo A. Knijnenburg, Giovanni Ciriello, Seungchan Kim, Guillaume Assie, Olena Morozova, Rehan Akbani, Juliann Shih, Katherine A. Hoadley, Toni K. Choueiri, Jens Waldmann, Ozgur Mete, Gordon A. Robertson, Matthew Meyerson, Michael J. Demeure, Felix Beuschlein, Anthony Gill, Ana C. Latronico, Maria C. Fragosa, Leslie Cope, Electron Kebebew, Mouhammed A. Habra, Timothy G. Whitsett, Kimberly J. Bussey, William E. Rainey, Sylvia Asa, Jérôme Bertherat, Martin Fassnacht, David A. Wheeler, The Cancer Genome Atlas Research Network, Gary D. Hammer, Thomas J. Giordano, Roel Verhaak. Comprehensive Pan-Genomic characterization of adrenocortical carcinoma. [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 2976. doi:10.1158/1538-7445.AM2015-2976

&lt;div&gt;Abstract&lt;p&gt;The &lt;i&gt;PPARG&lt;/i&gt; gene encoding the nuclear receptor PPARγ is activated in bladder cancer, either directly by gene amplification or mutation, or indirectly by mutation of the &lt;i&gt;RXRA&lt;/i&gt; gene, which encodes the heterodimeric partner of PPARγ. Here, we show that activating alterations of &lt;i&gt;PPARG&lt;/i&gt; or &lt;i&gt;RXRA&lt;/i&gt; lead to a specific gene expression signature in bladder cancers. Reducing PPARG activity, whether by pharmacologic inhibition or genetic ablation, inhibited proliferation of PPARG-activated bladder cancer cells. Our results offer a preclinical proof of concept for PPARG as a candidate therapeutic target in bladder cancer. &lt;i&gt;Cancer Res; 77(24); 6987–98. ©2017 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;Supplementary Methods. Supplementary Figure 1. Somatic alterations in PPARG and RXRA are hallmarks of luminal bladder cancer. Supplementary Figure 2. PPARG pathway is activated by overexpression of RXRA S427F/S427Y, but not other mutant alleles in bladder cancer cells. Supplementary Figure 3. Representation of the effects of ligand-dependent modulation on the PPARG interactome. Supplementary Figure 4. Downregulation of FABP4 protein by treatment of PPARG-activated bladder cancer cell lines by inverse-agonist T0070907. Supplementary Figure 5. Genome engineering scheme for generating reporter cell line. Supplementary Figure 6. Basal expression of FABP4 is reduced by PPARG inverse agonists, but not antagonists. Supplementary Figure 7. Lipid metabolism genes are inhibited by PPARG inverse-agonists. Supplementary Figure 8. PPARG inverse-agonists inhibit proliferation of PPARG activated bladder cancer cell lines in clonogenic assays. Supplementary Figure 9. PPARG inverse-agonists, but not antagonists, inhibit proliferation of PPARG-activated bladder cancer cell lines. Supplementary Figure 10. Somatic alterations in RXRA and PPARG.&lt;/p&gt;

&lt;p&gt;Chemical structures of compounds used in study&lt;/p&gt;

&lt;p&gt;Chemical structures of compounds used in study&lt;/p&gt;

&lt;p&gt;Supplementary Methods. Supplementary Figure 1. Somatic alterations in PPARG and RXRA are hallmarks of luminal bladder cancer. Supplementary Figure 2. PPARG pathway is activated by overexpression of RXRA S427F/S427Y, but not other mutant alleles in bladder cancer cells. Supplementary Figure 3. Representation of the effects of ligand-dependent modulation on the PPARG interactome. Supplementary Figure 4. Downregulation of FABP4 protein by treatment of PPARG-activated bladder cancer cell lines by inverse-agonist T0070907. Supplementary Figure 5. Genome engineering scheme for generating reporter cell line. Supplementary Figure 6. Basal expression of FABP4 is reduced by PPARG inverse agonists, but not antagonists. Supplementary Figure 7. Lipid metabolism genes are inhibited by PPARG inverse-agonists. Supplementary Figure 8. PPARG inverse-agonists inhibit proliferation of PPARG activated bladder cancer cell lines in clonogenic assays. Supplementary Figure 9. PPARG inverse-agonists, but not antagonists, inhibit proliferation of PPARG-activated bladder cancer cell lines. Supplementary Figure 10. Somatic alterations in RXRA and PPARG.&lt;/p&gt;

&lt;p&gt;Table S2: Oligos used in the study&lt;/p&gt;

&lt;p&gt;Figure S1. Super-enhancers near the KLF5 gene are focally amplified in cancer cell lines;Figure S2. The regulatory potential of the KLF5/KLF12 intervening region;Figure S3. Expression level of KLF12, DIS3 and PIBF1 after repression of KLF5 superenhancers;Figure S4. Genomic analysis of KLF5 binding sites in BICR31 cells;Figure S5. Target genes of KLF5 in head and neck squamous cell carcinomas;Figure S6. The effect of KLF5 silencing on H3K27ac profile;Figure S7. Mutation hotspots in KLF5;Figure S8. Mutation profile in FBXW7 in colorectal cancers; Figure S9. Ectopic expression of KLF5 mutants in HEK293T cells; Figure S10. Ectopic expression of KLF5 E419Q in HCC95 cells&lt;/p&gt;

&lt;div&gt;Abstract&lt;p&gt;The Krüppel-like family of transcription factors plays critical roles in human development and is associated with cancer pathogenesis. Krüppel-like factor 5 gene (&lt;i&gt;KLF5&lt;/i&gt;) has been shown to promote cancer cell proliferation and tumorigenesis and to be genomically amplified in cancer cells. We recently reported that the &lt;i&gt;KLF5&lt;/i&gt; gene is also subject to other types of somatic coding and noncoding genomic alterations in diverse cancer types. Here, we show that these alterations activate KLF5 by three distinct mechanisms: (i) Focal amplification of superenhancers activates &lt;i&gt;KLF5&lt;/i&gt; expression in squamous cell carcinomas; (ii) Missense mutations disrupt KLF5–FBXW7 interactions to increase KLF5 protein stability in colorectal cancer; (iii) Cancer type–specific hotspot mutations within a zinc-finger DNA binding domain of KLF5 change its DNA binding specificity and reshape cellular transcription. Utilizing data from CRISPR/Cas9 gene knockout screening, we reveal that cancer cells with &lt;i&gt;KLF5&lt;/i&gt; overexpression are dependent on KLF5 for their proliferation, suggesting KLF5 as a putative therapeutic target.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Significance:&lt;/b&gt; Our observations, together with previous studies that identified oncogenic properties of KLF5, establish the importance of KLF5 activation in human cancers, delineate the varied genomic mechanisms underlying this occurrence, and nominate KLF5 as a putative target for therapeutic intervention in cancer. &lt;i&gt;Cancer Discov; 8(1); 108–25. ©2017 AACR.&lt;/i&gt;&lt;/p&gt;&lt;p&gt;&lt;i&gt;This article is highlighted in the In This Issue feature, p. 1&lt;/i&gt;&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;Table S1: KLF5 E419Q-gained super-enhancers and the nearest genes&lt;/p&gt;

&lt;p&gt;Table S1: KLF5 E419Q-gained super-enhancers and the nearest genes&lt;/p&gt;

&lt;p&gt;Figure S1. Super-enhancers near the KLF5 gene are focally amplified in cancer cell lines;Figure S2. The regulatory potential of the KLF5/KLF12 intervening region;Figure S3. Expression level of KLF12, DIS3 and PIBF1 after repression of KLF5 superenhancers;Figure S4. Genomic analysis of KLF5 binding sites in BICR31 cells;Figure S5. Target genes of KLF5 in head and neck squamous cell carcinomas;Figure S6. The effect of KLF5 silencing on H3K27ac profile;Figure S7. Mutation hotspots in KLF5;Figure S8. Mutation profile in FBXW7 in colorectal cancers; Figure S9. Ectopic expression of KLF5 mutants in HEK293T cells; Figure S10. Ectopic expression of KLF5 E419Q in HCC95 cells&lt;/p&gt;

&lt;p&gt;Table S2: Oligos used in the study&lt;/p&gt;

&lt;div&gt;Abstract&lt;p&gt;The Krüppel-like family of transcription factors plays critical roles in human development and is associated with cancer pathogenesis. Krüppel-like factor 5 gene (&lt;i&gt;KLF5&lt;/i&gt;) has been shown to promote cancer cell proliferation and tumorigenesis and to be genomically amplified in cancer cells. We recently reported that the &lt;i&gt;KLF5&lt;/i&gt; gene is also subject to other types of somatic coding and noncoding genomic alterations in diverse cancer types. Here, we show that these alterations activate KLF5 by three distinct mechanisms: (i) Focal amplification of superenhancers activates &lt;i&gt;KLF5&lt;/i&gt; expression in squamous cell carcinomas; (ii) Missense mutations disrupt KLF5–FBXW7 interactions to increase KLF5 protein stability in colorectal cancer; (iii) Cancer type–specific hotspot mutations within a zinc-finger DNA binding domain of KLF5 change its DNA binding specificity and reshape cellular transcription. Utilizing data from CRISPR/Cas9 gene knockout screening, we reveal that cancer cells with &lt;i&gt;KLF5&lt;/i&gt; overexpression are dependent on KLF5 for their proliferation, suggesting KLF5 as a putative therapeutic target.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Significance:&lt;/b&gt; Our observations, together with previous studies that identified oncogenic properties of KLF5, establish the importance of KLF5 activation in human cancers, delineate the varied genomic mechanisms underlying this occurrence, and nominate KLF5 as a putative target for therapeutic intervention in cancer. &lt;i&gt;Cancer Discov; 8(1); 108–25. ©2017 AACR.&lt;/i&gt;&lt;/p&gt;&lt;p&gt;&lt;i&gt;This article is highlighted in the In This Issue feature, p. 1&lt;/i&gt;&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;Table S6 contains results from the analysis of DNA methylation in SETD2 mutated and BAP1 inactivated samples.&lt;/p&gt;

&lt;p&gt;Table S5 contains detailed miRs and lncRNAs results.&lt;/p&gt;

&lt;p&gt;Table S3 contains the karyotypes of 16 genome-wide LOH MPM cases from the BWH cohort.&lt;/p&gt;

&lt;p&gt;Table S4 includes immune signature and iCluster results.&lt;/p&gt;

&lt;p&gt;Table S7 contains microbe screening results.&lt;/p&gt;

&lt;p&gt;Supplementary materials and methods, as well as supplementary figures S1-S7.&lt;/p&gt;

&lt;p&gt;Table S2 contains BAP1 analysis results, as well as detailed lists of YY1 and IRF8 target genes.&lt;/p&gt;

&lt;p&gt;Table S1 contains cohort description, Master Patient Table and MutSigCV results.&lt;/p&gt;

&lt;div&gt;Abstract&lt;p&gt;Malignant pleural mesothelioma (MPM) is a highly lethal cancer of the lining of the chest cavity. To expand our understanding of MPM, we conducted a comprehensive integrated genomic study, including the most detailed analysis of &lt;i&gt;BAP1&lt;/i&gt; alterations to date. We identified histology-independent molecular prognostic subsets, and defined a novel genomic subtype with &lt;i&gt;TP53&lt;/i&gt; and &lt;i&gt;SETDB1&lt;/i&gt; mutations and extensive loss of heterozygosity. We also report strong expression of the immune-checkpoint gene &lt;i&gt;VISTA&lt;/i&gt; in epithelioid MPM, strikingly higher than in other solid cancers, with implications for the immune response to MPM and for its immunotherapy. Our findings highlight new avenues for further investigation of MPM biology and novel therapeutic options.&lt;/p&gt;Significance:&lt;p&gt;Through a comprehensive integrated genomic study of 74 MPMs, we provide a deeper understanding of histology-independent determinants of aggressive behavior, define a novel genomic subtype with &lt;i&gt;TP53&lt;/i&gt; and &lt;i&gt;SETDB1&lt;/i&gt; mutations and extensive loss of heterozygosity, and discovered strong expression of the immune-checkpoint gene &lt;i&gt;VISTA&lt;/i&gt; in epithelioid MPM.&lt;/p&gt;&lt;p&gt;&lt;i&gt;See related commentary by Aggarwal and Albelda, p. 1508&lt;/i&gt;.&lt;/p&gt;&lt;p&gt;&lt;i&gt;This article is highlighted in the In This Issue feature, p. 1494&lt;/i&gt;&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;Supplementary materials and methods, as well as supplementary figures S1-S7.&lt;/p&gt;

&lt;p&gt;Table S4 includes immune signature and iCluster results.&lt;/p&gt;

&lt;p&gt;Table S5 contains detailed miRs and lncRNAs results.&lt;/p&gt;

&lt;p&gt;Table S6 contains results from the analysis of DNA methylation in SETD2 mutated and BAP1 inactivated samples.&lt;/p&gt;

&lt;p&gt;Table S2 contains BAP1 analysis results, as well as detailed lists of YY1 and IRF8 target genes.&lt;/p&gt;

&lt;p&gt;Table S1 contains cohort description, Master Patient Table and MutSigCV results.&lt;/p&gt;

&lt;p&gt;Table S7 contains microbe screening results.&lt;/p&gt;

&lt;p&gt;Table S3 contains the karyotypes of 16 genome-wide LOH MPM cases from the BWH cohort.&lt;/p&gt;

Chen, Claire1; Shih, Juliann1; Sutton, Jenna1; Cabugao, Paul2; Romero, Arthur1 Author Information

&lt;div&gt;Abstract&lt;p&gt;The &lt;i&gt;PPARG&lt;/i&gt; gene encoding the nuclear receptor PPARγ is activated in bladder cancer, either directly by gene amplification or mutation, or indirectly by mutation of the &lt;i&gt;RXRA&lt;/i&gt; gene, which encodes the heterodimeric partner of PPARγ. Here, we show that activating alterations of &lt;i&gt;PPARG&lt;/i&gt; or &lt;i&gt;RXRA&lt;/i&gt; lead to a specific gene expression signature in bladder cancers. Reducing PPARG activity, whether by pharmacologic inhibition or genetic ablation, inhibited proliferation of PPARG-activated bladder cancer cells. Our results offer a preclinical proof of concept for PPARG as a candidate therapeutic target in bladder cancer. &lt;i&gt;Cancer Res; 77(24); 6987–98. ©2017 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;Malignant pleural mesothelioma (MPM) is a highly lethal cancer of the lining of the chest cavity. To expand our understanding of MPM, we conducted a comprehensive integrated genomic study, including the most detailed analysis of &lt;i&gt;BAP1&lt;/i&gt; alterations to date. We identified histology-independent molecular prognostic subsets, and defined a novel genomic subtype with &lt;i&gt;TP53&lt;/i&gt; and &lt;i&gt;SETDB1&lt;/i&gt; mutations and extensive loss of heterozygosity. We also report strong expression of the immune-checkpoint gene &lt;i&gt;VISTA&lt;/i&gt; in epithelioid MPM, strikingly higher than in other solid cancers, with implications for the immune response to MPM and for its immunotherapy. Our findings highlight new avenues for further investigation of MPM biology and novel therapeutic options.&lt;/p&gt;Significance:&lt;p&gt;Through a comprehensive integrated genomic study of 74 MPMs, we provide a deeper understanding of histology-independent determinants of aggressive behavior, define a novel genomic subtype with &lt;i&gt;TP53&lt;/i&gt; and &lt;i&gt;SETDB1&lt;/i&gt; mutations and extensive loss of heterozygosity, and discovered strong expression of the immune-checkpoint gene &lt;i&gt;VISTA&lt;/i&gt; in epithelioid MPM.&lt;/p&gt;&lt;p&gt;&lt;i&gt;See related commentary by Aggarwal and Albelda, p. 1508&lt;/i&gt;.&lt;/p&gt;&lt;p&gt;&lt;i&gt;This article is highlighted in the In This Issue feature, p. 1494&lt;/i&gt;&lt;/p&gt;&lt;/div&gt;

Almost 90% of tumors are aneuploid and have arm- or whole- chromosome level copy number changes. Arm-level copy number alterations cluster by tumor type, suggesting that specific arm-level changes are influenced by cell type. Systematic methods of generating copy number changes on a particular chromosome have not been tested, leaving the effects of specific aneuploidies in cancer unclear. We used the CRISPR-Cas9 system to delete one copy of chromosome 3p, which is lost in 90% of lung squamous cell carcinomas (SCCs). We have successfully isolated almost 90 immortalized lung cell clones with deletion of the 3p arm, with 8 validated by whole genome sequencing. Consistent with patient data, expression of 3p genes is also decreased upon deletion. Phenotypic characterization revealed that cells with chromosome 3p deletion initially proliferate more slowly than their siblings. 3p deleted cells show increased G1 arrest, but do not undergo increased apoptosis or cell death. We are currently testing 3p loss in combination with other frequent SCC alterations, such as SOX2 and TP63 gain. These studies provide a robust model that will address a gap in our understanding of aneuploidy in cancer by using targeted endonuclease technology to create models of partial aneuploidies.

Abstract A subset of muscle-invasive bladder cancer (BLCA) is typified by PPARG pathway activation. PPARG focal gene amplification occurs in 15% of bladder cancer patients, and similarly, 5% of BLCA patients possess hotspot mutations in the requisite heterodimer partner of PPARG, RXRA (S427F, S427Y). We used genetic perturbation to study the role of PPARG in bladder cancer. Our results show that overexpression of RXRA and PPARG mutant alleles activate expression of PPARG and PPARA target genes in a ligand-independent manner, and that bladder cancer cell lines are dependent on PPARG for viability. These findings suggest that PPARG may be a promising therapeutic target for treatment of bladder cancer. Citation Format: Jonathan T. Goldstein, Craig Strathdee, Fujiko Duke, Juliann Shih, Matthew Meyerson. Validation of PPARG and RXRA as drivers of bladder cancer [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 3627. doi:10.1158/1538-7445.AM2017-3627

Abstract Almost 90% of tumors are aneuploid and have arm- or whole- chromosome level copy number changes. Arm-level copy number alterations cluster by tumor type, suggesting that specific arm-level changes are influenced by cell type. Systematic methods of generating copy number changes on a particular chromosome have not been tested, leaving the effects of specific aneuploidies in cancer unclear. The most frequent genomic alteration in lung squamous cell carcinomas (SCCs) is loss of chromosome 3p. This alteration occurs in 90% of lung SCCs, and covers the entire length of the chromosome arm. Over two-thirds of 3p genes show significantly decreased expression. However, the phenotypic effects of 3p loss in tumorigenesis are not known. We used the CRISPR-Cas9 system to generate double-strand breaks (DSBs) and produce partial aneuploidies of arm-level loss. We have targeted guide RNAs adjacent to centromeric satellite-repeat sequences on chromosome arm 3p. At this location, we induced homologous recombination with a selection cassette and artificial telomere. We have successfully isolated almost 90 clones of immortalized lung epithelial cells with deletion of the 3p arm, with 8 validated by whole genome sequencing. Consistent with patient data, expression of 3p genes is also decreased upon deletion. Phenotypic characterization revealed that cells with chromosome 3p deletion proliferate more slowly than their siblings. 3p deleted cells show increased G1 arrest, but do not undergo increased apoptosis or cell death. We are currently testing 3p loss in combination with other frequent SCC alterations, such as SOX2 and TP63 gain. We have also identified expression changes in trans, outside of chromosome 3p. These studies provide a robust model that will address a gap in our understanding of aneuploidy in cancer by using targeted endonuclease technology to create models of partial aneuploidies. Future work will include investigation of how different chromosomal changes contribute to cancer formation, which will have implications on our understanding of tumorigenesis. Citation Format: Alison M. Taylor, Gavin Ha, Juliann Shih, Xiaoyang Zhang, Joshua M. Francis, Matthew Meyerson. Genome engineering to generate models of chromosome arm-level aneuploidies in lung carcinoma [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 409. doi:10.1158/1538-7445.AM2017-409

Abstract Patient-derived xenografts (PDXs) have become a prominent cancer model system, as they are presumed to faithfully represent the genomic features of primary tumors. Here we monitored the dynamics of copy number alterations (CNAs) in 1,110 PDX samples across 24 cancer types. We observed rapid accumulation of CNAs during PDX passaging, often due to selection of pre-existing minor clones. CNA acquisition in PDXs was correlated with the tissue-specific levels of aneuploidy and genetic heterogeneity observed in primary tumors. However, the particular CNAs acquired during PDX passaging differed from those acquired during tumor evolution in patients. Several CNAs recurrently observed in primary tumors gradually disappeared in PDXs, indicating that events undergoing positive selection in humans can become dispensable during propagation in mice. Importantly, the genomic stability of PDXs was associated with their response to chemotherapy and targeted drugs. These findings have important implications for PDX-based modeling of human cancer. Citation Format: Uri Ben-David, Gavin Ha, Yuen-Yi Tseng, Noah F. Greenwald, Coyin Oh, Juliann Shih, James M. McFarland, Bang Wong, Jesse S. Boehm, Rameen Beroukhim, Todd R. Golub. Patient-derived xenografts undergo mouse-specific tumor evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1028.

Abstract Aneuploidy, whole chromosome or chromosome arm copy number imbalance, is a near-universal characteristic of human cancers. Although yeast and mammalian models of whole chromosome aneuploidies have been extensively investigated, chromosome arm-level aneuploidies have rarely been modeled. Cancer subtypes are often characterized by tumor-specific patterns of these arm-level copy number alterations; for example, squamous cell carcinomas (SCCs) from different tissues of origin (including lung, esophagus, and bladder) have a pattern of chromosome 3p loss and chromosome 3q gain. Our analysis of 495 lung SCCs found chromosome 3p deletion to be the most frequent genomic alteration, occurring in almost 80% of the tumors and covering the entire length of the chromosome arm. Over two-thirds of chromosome 3p genes showed significantly decreased expression in these samples. Without models of chromosome arm-level alterations, the phenotypic effects of specific aneuploidies in cancer, such as 3p deletion, remain unknown. However, recent advances in genome engineering and targeting of endonucleases allow new approaches to generate chromosomal alterations. Here, we used the CRISPR-Cas9 system to delete one copy of chromosome 3p in vitro. We successfully isolated almost 90 clones of immortalized lung epithelial cells with deletion of the 3p arm, with 8 validated by whole-genome sequencing. Consistent with patient data, expression of 3p genes was also decreased upon deletion. Phenotypic characterization revealed that cells with chromosome 3p deletion initially proliferated more slowly than their siblings. These chromosome 3p-deleted cells had increased G1 arrest, but did not undergo increased apoptosis or cell death. Interestingly, after several passages in culture, the proliferation defect was rescued in chromosome 3p-deleted cells; genome sequencing and karyotype analyses suggested that this was the result of chromosome 3 duplication. With our cellular model of chromosome arm-level aneuploidy, we uncovered a possible selection mechanism that allows aneuploidy tolerance in vitro. We used genome engineering to model chromosome arm-level deletions, providing a robust model that will address a gap in our understanding of aneuploidy in cancer. Citation Format: Alison Marie Taylor, Xiaoyang Zhang, Juliann Shih, Gavin Ha, Galen F. Gao, Ashton C. Berger, Andrew D. Cherniack, Rameen Beroukhim, Matthew Meyerson. Genome engineering approaches to generate models of chromosome arm-level cancer aneuploidy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3002.

Abstract Aneuploidy, whole-chromosome or chromosome arm copy number imbalance, is a near-universal characteristic of human cancers. We previously applied methods that define chromosome arm-level aneuploidy and a global cancer aneuploidy score to 10,522 tumors of 33 types in The Cancer Genome Atlas (TCGA). Aneuploidy level was correlated with TP53 mutation, somatic mutation rate, and expression of proliferation genes. Overall aneuploidy was anti-correlated with expression of immune signaling genes, due to decreased leukocyte infiltrates in high-aneuploidy samples. In contrast, some individual chromosome arm alterations correlated with higher expression of immune signaling genes, including deletion of chromosome 3p, 8p, and 17p. Cancer subtypes are often characterized by tumor specific patterns of arm-level copy number alterations; for example, squamous cell carcinomas (SCCs) from different tissues of origin (including lung, esophagus, and bladder) have a pattern of chromosome 3p loss and chromosome 3q gain. Although yeast and mammalian models of whole-chromosome aneuploidies have been extensively investigated, chromosome arm-level aneuploidies have rarely been modeled. However, recent advances in genome engineering and targeting of endonucleases allow generation of large chromosomal alterations. We used the CRISPR-Cas9 system to delete one copy of chromosome 3p in human immortalized lung epithelial cells, validated in 8 clones by whole-genome sequencing. Consistent with patient data, expression of 3p genes was decreased upon deletion, as well as increased expression of interferon response genes. Phenotypic characterization revealed that cells with chromosome 3p deletion initially proliferated more slowly than their siblings. These chromosome 3p-deleted cells had increased G1 arrest but did not undergo increased apoptosis or cell death. Interestingly, after several passages in culture, the proliferation defect was rescued in chromosome 3p-deleted cells; genome sequencing and karyotype analyses suggested that this was partially the result of chromosome 3 duplication. With our cellular model of chromosome arm-level aneuploidy, we uncovered a selection mechanism that allowed aneuploidy tolerance in vitro. We have also grown these cells in spheroid and organoid culture systems to assess the effects of chromosome 3 arm-level aneuploidies on cytokine production and squamous differentiation and are using these systems to identify how chromosome 3p deletion affects drug sensitivity. In conclusion, our genome engineering approach to model chromosome arm-level deletions provides a robust model that will address a gap in our understanding of aneuploidy in cancer. Citation Format: Alison M. Taylor, Sejal Jain, Juliann Shih, Andrew D. Cherniack, Rameen Beroukhim, Matthew Meyerson. Functional models of chromosome arm-level aneuploidies in cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr A44.

Abstract Aneuploidies, defined as whole-arm or whole-chromosome imbalances, are the most prevalent alteration in cancer genomes. However, the extent to which they are enriched due to selection is unclear, against the alternative hypothesis that they are passenger events that are simply highly prone to occur. We developed a novel method, BrISCUT, that identifies loci under selective advantage or disadvantage due to arm-level copy-number alterations by interrogating length distributions of events that are bounded at either the telomere or centromere. These loci were significantly enriched for known cancer driver genes, including genes not detected through analysis of focal copy-number events, and were often lineage-specific. We also formally quantified the role of selection and mechanistic biases in driving aneuploidy, finding that rates of arm-level SCNAs are most highly correlated with selective pressures. These results provide insight into the causes of aneuploidies and their contributions to tumorigenesis.

Abstract Aneuploidy, which we define as whole chromosome or chromosome arm copy number imbalance, is a near-universal characteristic of human cancers. Cancer subtypes are often characterized by tumor specific patterns of chromosome arm copy number alterations; for example, squamous cell carcinomas (SCCs) from different tissues of origin (including lung, head and neck, esophagus, and bladder) have a pattern of chromosome 3p loss and chromosome 3q gain. Although these alterations are frequent, they are not well understood due to difficulty in modeling specific aneuploidy alterations in the matching cell type. However, recent advances in genome engineering allow generation of large chromosomal alterations. We used the CRISPR-Cas9 system to delete one copy of chromosome 3p in human immortalized lung epithelial cells most similar to upper airway basal cells. Deletion of chromosome 3p was validated by whole genome sequencing and karyotyping. Consistent with patient data, expression of 3p genes was decreased upon deletion, as well as increased expression of interferon response genes. Phenotypic characterization revealed that cells with chromosome 3p deletion initially proliferated more slowly than their siblings. These chromosome 3p deleted cells had increased G1 arrest but did not undergo increased apoptosis or cell death. Interestingly, after several passages in culture, the proliferation defect was rescued in chromosome 3p deleted cells. Genome sequencing and karyotype analyses found that evidence of chromosome 3 duplication, transitioning the cell to a state of chromosome 3q gain. We isolated sibling cells with chromosome 3p deletion or chromosome 3q gain and demonstrated that duplication of chromosome 3 could indeed rescue proliferation rates. With our cellular model of chromosome arm-level aneuploidy, we uncovered a selection mechanism that allowed aneuploidy tolerance in vitro, and a possible explanation for joint alteration of both arms of chromosome 3. In conclusion, our genome engineering approach to model chromosome arm-level deletions provides a robust model that will address a gap in our understanding of aneuploidy in cancer. Citation Format: Alison Marie Taylor, Sejal Jain, Juliann Shih, Andrew D. Cherniack, Rameen Beroukhim, Matthew Meyerson. Functional models of chromosome arm aneuploidies in lung squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2135.