Coyin Oh

Genomic Copy Number Dictates a Gene-Independent Cell Response to CRISPR/Cas9 Targeting

UNLABELLED The CRISPR/Cas9 system enables genome editing and somatic cell genetic screens in mammalian cells. We performed genome-scale loss-of-function screens in 33 cancer cell lines to identify genes essential for proliferation/survival and found a strong correlation between increased gene copy number and decreased cell viability after genome editing. Within regions of copy-number gain, CRISPR/Cas9 targeting of both expressed and unexpressed genes, as well as intergenic loci, led to significantly decreased cell proliferation through induction of a G2 cell-cycle arrest. By examining single-guide RNAs that map to multiple genomic sites, we found that this cell response to CRISPR/Cas9 editing correlated strongly with the number of target loci. These observations indicate that genome targeting by CRISPR/Cas9 elicits a gene-independent antiproliferative cell response. This effect has important practical implications for the interpretation of CRISPR/Cas9 screening data and confounds the use of this technology for the identification of essential genes in amplified regions. SIGNIFICANCE We found that the number of CRISPR/Cas9-induced DNA breaks dictates a gene-independent antiproliferative response in cells. These observations have practical implications for using CRISPR/Cas9 to interrogate cancer gene function and illustrate that cancer cells are highly sensitive to site-specific DNA damage, which may provide a path to novel therapeutic strategies. Cancer Discov; 6(8); 914-29. ©2016 AACR.See related commentary by Sheel and Xue, p. 824See related article by Munoz et al., p. 900This article is highlighted in the In This Issue feature, p. 803.

Patient-derived xenografts undergo mouse-specific tumor evolution

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.

Scalable whole-exome sequencing of cell-free DNA reveals high concordance with metastatic tumors

Whole-exome sequencing of cell-free DNA (cfDNA) could enable comprehensive profiling of tumors from blood but the genome-wide concordance between cfDNA and tumor biopsies is uncertain. Here we report ichorCNA, software that quantifies tumor content in cfDNA from 0.1× coverage whole-genome sequencing data without prior knowledge of tumor mutations. We apply ichorCNA to 1439 blood samples from 520 patients with metastatic prostate or breast cancers. In the earliest tested sample for each patient, 34% of patients have ≥10% tumor-derived cfDNA, sufficient for standard coverage whole-exome sequencing. Using whole-exome sequencing, we validate the concordance of clonal somatic mutations (88%), copy number alterations (80%), mutational signatures, and neoantigens between cfDNA and matched tumor biopsies from 41 patients with ≥10% cfDNA tumor content. In summary, we provide methods to identify patients eligible for comprehensive cfDNA profiling, revealing its applicability to many patients, and demonstrate high concordance of cfDNA and metastatic tumor whole-exome sequencing.Identifying the mutational landscape of tumours from cell-free DNA in the blood could help diagnostics in cancer. Here, the authors present ichorCNA, software that quantifies tumour content in cell free DNA, and they demonstrate that cell-free DNA whole-exome sequencing is concordant with metastatic tumour whole-exome sequencing.

Integrated genetic and pharmacologic interrogation of rare cancers

Identifying therapeutic targets in rare cancers remains challenging due to the paucity of established models to perform preclinical studies. As a proof-of-concept, we developed a patient-derived cancer cell line, CLF-PED-015-T, from a paediatric patient with a rare undifferentiated sarcoma. Here, we confirm that this cell line recapitulates the histology and harbours the majority of the somatic genetic alterations found in a metastatic lesion isolated at first relapse. We then perform pooled CRISPR-Cas9 and RNAi loss-of-function screens and a small-molecule screen focused on druggable cancer targets. Integrating these three complementary and orthogonal methods, we identify CDK4 and XPO1 as potential therapeutic targets in this cancer, which has no known alterations in these genes. These observations establish an approach that integrates new patient-derived models, functional genomics and chemical screens to facilitate the discovery of targets in rare cancers.

Exome Sequencing of African-American Prostate Cancer Reveals Loss-of-Function ERF Mutations

African-American men have the highest incidence of and mortality from prostate cancer. Whether a biological basis exists for this disparity remains unclear. Exome sequencing (n = 102) and targeted validation (n = 90) of localized primary hormone-naïve prostate cancer in African-American men identified several gene mutations not previously observed in this context, including recurrent loss-of-function mutations in ERF, an ETS transcriptional repressor, in 5% of cases. Analysis of existing prostate cancer cohorts revealed ERF deletions in 3% of primary prostate cancers and mutations or deletions in ERF in 3% to 5% of lethal castration-resistant prostate cancers. Knockdown of ERF confers increased anchorage-independent growth and generates a gene expression signature associated with oncogenic ETS activation and androgen signaling. Together, these results suggest that ERF is a prostate cancer tumor-suppressor gene. More generally, our findings support the application of systematic cancer genomic characterization in settings of broader ancestral diversity to enhance discovery and, eventually, therapeutic applications.Significance: Systematic genomic sequencing of prostate cancer in African-American men revealed new insights into prostate cancer, including the identification of ERF as a prostate cancer gene; somatic copy-number alteration differences; and uncommon PIK3CA and PTEN alterations. This study highlights the importance of inclusion of underrepresented minorities in cancer sequencing studies. Cancer Discov; 7(9); 973-83. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 920.

Abstract S1-01: Whole exome and transcriptome sequencing of resistant ER+ metastatic breast cancer

Background: While great strides have been made in the treatment of estrogen receptor-positive (ER+) metastatic breast cancer (MBC), therapeutic resistance invariably occurs. A better understanding of the underlying resistance mechanisms is critical to enable durable control of this disease. Methods: We performed whole exome sequencing (WES) and transcriptome sequencing (RNA-seq) on metastatic tumor biopsies from 88 patients with ER+ MBC who had developed resistance to one or more ER-directed therapies. For 27 of these patients, we sequenced the treatment-naive primary tumors for comparison to the resistant specimens. Tumors were analyzed for point mutations, insertions/deletions, copy number alterations, translocations, and gene expression. Detailed clinicopathologic data was collected for each patient and linked to the genomic information. Results: WES of all metastatic samples demonstrated several recurrently altered genes whose incidence differed significantly from primary, treatment-naive ER+ breast cancers sequenced in the TCGA study (TCGA). These include ESR1 mutations (n=17, 19.3%; 32.86 fold enrichment, q.value Comparing to matched primary samples from the same patient, many alterations were found to be acquired in several cases, including for ESR1, ERBB2, PIK3CA, PTEN, RB1, AKT1, and others. Initial analysis of RNA-seq data from metastatic samples (n=59) allowed classification of individual resistance mechanisms into broader resistance modes based on the observed transcriptional state. Conclusions: We present a genomic landscape of resistant ER+ MBC using WES and RNA-seq. Multiple genes were recurrently altered in these tumors at significantly higher rates than in ER+ primary breast cancer. When compared with matched primary tumors from the same patient, alterations in these and other genes were often found to be acquired after treatment, suggesting a role in resistance to ER-directed therapies and/or metastasis. Potential resistance mechanisms appear to fall into several categories; integrating RNA-seq data may enhance the ability to identify these categories even when genomic alterations are not identified. Multiple clinically relevant genomic and molecular alterations are identified in metastatic biopsies– with implications for choice of next therapy, clinical trial eligibility, and novel drug targets. Citation Format: Cohen O, Kim D, Oh C, Waks A, Oliver N, Helvie K, Marini L, Rotem A, Lloyd M, Stover D, Adalsteinsson V, Freeman S, Ha G, Cibulskis C, Anderka K, Tamayo P, Johannessen C, Krop I, Garraway L, Winer E, Lin N, Wagle N. Whole exome and transcriptome sequencing of resistant ER+ metastatic breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr S1-01.

Abstract B17: Identification of Druggable Targets through Functional Multi-Omics in Renal Medullary Carcinoma

Renal medullary carcinoma is a rare kidney cancer that is primarily seen in adolescent and young adult African American patients with sickle cell trait. Prognosis is poor and treatment options are limited. We have developed several cell line models that recapitulate the primary and relapsed metastatic samples from a patient who succumbed to this disease. We have confirmed by whole exome sequencing that our models have sickle cell trait and loss of heterozygosity of the SMARCB1 loci, both hallmarks of this disease. By RNA-sequencing, we see a lack of SMARCB1 transcription. We have further shown dependency of our models to SMARCB1 re-expression thus suggesting that this cancer is indeed driven by loss of SMARCB1 at a functional level. We performed pooled CRISPR-Cas9 and RNAi loss of function screens and a small molecule screen focused on druggable cancer targets based on our previous work in parallel to a genome-wide pooled CRISPR-Cas9 loss of function screen. Integrating these complementary and orthogonal methods, we identified a number of targets for further validation. These targets, when combined may provide a rational approach to therapeutic targeting for this rare kidney cancer. Citation Format: Andrew L. Hong, Yuen-Yi Tseng, Bryan D. Kynnap, Mihir B. Doshi, Gabriel Sandoval, Coyin Oh, Abeer Sayeed, Gill Shubhroz, Alanna J. Church, Paula Keskula, Anson Peng, Paul A. Clemons, Aviad Tsherniak, Francisca Vazquez, Carlos Rodriguez-Galindo, Katherine A. Janeway, Levi A. Garraway, Stuart L. Schreiber, David E. Root, Elizabeth Mullen, Kimberly Stegmaier, Cigall Kadoch, Charles W.M. Roberts, Jesse S. Boehm, William C. Hahn. Identification of Druggable Targets through Functional Multi-Omics in Renal Medullary Carcinoma [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr B17.

Abstract B39: Genomic copy number alterations introduce a gene-independent viability bias in CRISPR-Cas9 knock-out screens of cancer cell lines

Recent studies have demonstrated the power of CRISPR-Cas9 screening methods for identifying genetic vulnerabilities in cancer cells. As part of a larger effort to generate a comprehensive catalog of vulnerabilities, we performed CRISPR-Cas9 genome-scale loss-of-function screens in 33 cancer cell lines to identify genes essential for proliferation and survival. We found a strong correlation between gene copy number and cell viability after Cas9-targeting. Copy number alterations are extremely prevalent in human cancers and frequently lead to overexpression of driver oncogenes and potential vulnerabilities. Therefore, we sought to identify such genes by investigating the relationship of genomic copy number with essentiality from our screening data. As expected, known oncogenes scored as essential in cell lines harboring amplifications of these genes. However, the scores of all other genes in these amplified regions were also strongly enriched for apparent essentiality, even among unexpressed genes. Furthermore, the infection of cells with sgRNAs targeting Cas9 to non-coding intergenic sequences within regions of high copy number gain also induced this negative effect on cell viability. We observed this effect across multiple different chromosomal structural alterations, including tandem duplications, breakage-fusion-bridge structures, and arm-level gains. More broadly, we found a striking global correlation between cell viability in response to Cas9-targeting and the genomic copy number of the targeted site, even among low-level copy number gain and loss. For example, Cas9-targeting of genes with two copies resulted in, on average, decreased viability relative to Cas9-targeting of genes with only one copy. By examining sgRNAs that target multiple genomic sites, but not within any amplified loci, we found that this cell response to Cas9-targeting correlated strongly with the total number of target sites. Together, these observations indicate that genome targeting by CRISPR-Cas9 elicits a gene-independent anti-proliferative cell response with a severity proportional to the total number of discrete genomic loci targeted. This effect has important practical implications for interpretation of CRISPR-Cas9 screening data and confounds the use of this technology for identification of essential genes in amplified regions. This result illustrates the sensitivity of cancer cells to site-specific DNA damage, which may provide a path to novel therapeutic strategies. Targeting non-essential genes or non-coding intergenic sequences within regions of copy number amplification may reveal cancer-specific vulnerabilities. Citation Format: Robin M. Meyers, Andrew J. Aguirre, Barbara A. Weir, Francisca Vazquez, Cheng-Zhong Zhang, Uri Ben-David, April Cook, Gavin Ha, William F. Harrington, Mihir Doshi, Stanley Gill, Han Xu, Levi D. Ali, Guozhi Jiang, Sasha Pantel, Yenarae Lee, Amy Goodale, Andrew D. Cherniack, Coyin Oh, Gregory Kryukov, Glenn S. Cowley, Levi A. Garraway, Kimberly Stegmaier, Charles W. Roberts, Todd R. Golub, Matthew Meyerson, David E. Root, Aviad Tsherniak, William C. Hahn. Genomic copy number alterations introduce a gene-independent viability bias in CRISPR-Cas9 knock-out screens of cancer cell lines. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B39.

Abstract P3-04-08: The role of HER2 mutations in resistance to endocrine therapy in ER+ breast cancer

Resistance to endocrine therapies in estrogen receptor positive (ER+) metastatic breast cancer is widespread, and understanding the mechanisms whereby these tumors acquire resistance is a critical need. Through whole-exome sequencing of metastatic tumor biopsies from patients with endocrine resistant ER+ metastatic breast cancer, we identified 13 different HER2 mutations, including five in the kinase domain, four in the signaling domain, three in the extracellular domain, and one in the transmembrane region of the protein. Two of the kinase domain mutations (L755S and V777L) have been previously described and shown to be activating and resistant to reversible anti-HER2 targeted therapies; the remaining mutations have not been reported. In several of these patients, whole exome sequencing of a pre-treatment primary tumor did not identify the HER2 mutations seen in the corresponding metastatic tumor, suggesting that they were acquired during therapy. To examine the role of HER2 mutations in endocrine resistance, we generated ER+ breast cancer cell lines (MCF7 and T47D) stably expressing the HER2 mutants observed in our clinical data. Several mutants promoted enhanced growth in charcoal dextran-stripped media, which lacks estradiol and mimics treatment with aromatase inhibitor. In addition, several mutants conferred varying degrees of resistance to fulvestrant and tamoxifen. Taken together, these results suggest that HER2 mutations are associated with acquired resistance to endocrine therapies in patients with ER+ breast cancer. The ability of irreversible anti-HER2 agents as well as other agents that target the HER2 pathway to overcome this resistance is being tested for individual HER2 mutations in vitro . The results from these studies may provide a clinical rationale for therapeutic combination strategies in patients with refractory tumors that have acquired endocrine resistance through HER2 mutations. Citation Format: Nayar U, Cohen O, Oh C, Wagle N. The role of HER2 mutations in resistance to endocrine therapy in ER+ breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-04-08.

Abstract B26: Accelerating prediction of tumor vulnerabilities using next-generation cancer models

The development of new cancer therapeutics requires sufficient genetic and phenotypic diversity of cancer models. Current collections of human cancer cell lines are limited and for many rare cancer types, zero models exist that are broadly available. Here, we report results from the pilot phase of the Cancer Cell Line Factory (CCLF) project that aims to overcome this obstacle by systematically creating next-generation in vitro cancer models from adult and pediatric cancer patients9 specimens and making these models broadly available. We first developed a workflow of laboratory, genomics and informatics tools that make it possible to systematically compare published ex vivo culture conditions for each individual tumor to enable the scientific community to iterate towards disease-specific culture recipes. Based on sample volume and rarity, 4-100 conditions were applied to each sample and all data was captured in a custom Laboratory Information Management System to enhance subsequent predictions. We developed a