Mihir Doshi

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.

ATXN1L, CIC, and ETS Transcription Factors Modulate Sensitivity to MAPK Pathway Inhibition

Intrinsic resistance and RTK-RAS-MAPK pathway reactivation has limited the effectiveness of MEK and RAF inhibitors (MAPKi) in RAS- and RAF-mutant cancers. To identify genes that modulate sensitivity to MAPKi, we performed genome-scale CRISPR-Cas9 loss-of-function screens in two KRAS mutant pancreatic cancer cell lines treated with the MEK1/2 inhibitor trametinib. Loss of CIC, a transcriptional repressor of ETV1, ETV4, and ETV5, promoted survival in the setting of MAPKi in cancer cells derived from several lineages. ATXN1L deletion, which reduces CIC protein, or ectopic expression of ETV1, ETV4, or ETV5 also modulated sensitivity to trametinib. ATXN1L expression inversely correlates with response to MAPKi inhibition in clinical studies. These observations identify the ATXN1L-CIC-ETS transcription factor axis as a mediator of resistance to MAPKi.

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.

Synergistic interactions with PI3K inhibition that induce apoptosis

Activating mutations involving the PI3K pathway occur frequently in human cancers. However, PI3K inhibitors primarily induce cell cycle arrest, leaving a significant reservoir of tumor cells that may acquire or exhibit resistance. We searched for genes that are required for the survival of PI3K mutant cancer cells in the presence of PI3K inhibition by conducting a genome scale shRNA-based apoptosis screen in a PIK3CA mutant human breast cancer cell. We identified 5 genes (PIM2, ZAK, TACC1, ZFR, ZNF565) whose suppression induced cell death upon PI3K inhibition. We showed that small molecule inhibitors of the PIM2 and ZAK kinases synergize with PI3K inhibition. In addition, using a microscale implementable device to deliver either siRNAs or small molecule inhibitors in vivo, we showed that suppressing these 5 genes with PI3K inhibition induced tumor regression. These observations identify targets whose inhibition synergizes with PI3K inhibitors and nominate potential combination therapies involving PI3K inhibition. DOI: http://dx.doi.org/10.7554/eLife.24523.001

Abstract B18: Modeling renal medullary carcinomas identifies druggable vulnerabilities in SMARCB1-deficient cancers

Renal medullary carcinomas (RMCs) are thought to be driven by the loss of tumor suppressor, SMARCB1. These rare kidney cancers carry a very poor prognosis and primarily affect African American adolescents and young adults with sickle cell trait. From two patients with RMC, we have identified by whole-genome sequencing mechanisms of SMARCB1 loss (e.g., inactivating fusion events involving SMARCB1). We developed in vitro models of primary and relapsed metastatic disease. We performed biochemical and functional studies to conclusively show that RMC is dependent on loss of SMARCB1, similar to rhabdoid tumors and atypical teratoid/rhabdoid tumors. Furthermore, we performed small-molecule screens, pooled CRISPR-Cas9 knockout, and RNAi suppression screens focused on druggable cancer targets. Integration of these orthogonal methods identifies a core set of targets that may provide a rational approach to therapeutic targeting for this rare kidney cancer and other SMARCB1-deficient cancers. Citation Format: Andrew L. Hong, Yuen-Yi Tseng, Bryan D. Kynnap, Mihir B. Doshi, Jeremiah Wala, Gabriel Sandoval, Alanna J. Church, Elizabeth Mullen, Cigall Kadoch, Charles W.M. Roberts, Rameen Beroukhim, Jesse S. Boehm, William C. Hahn. Modeling renal medullary carcinomas identifies druggable vulnerabilities in SMARCB1-deficient cancers [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B18.

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 PR04: Integration of CRISPR-Cas9, RNAi and pharmacologic screens identify actionable targets in a rare cancer

Loss-of-function screening using RNAi technologies over the past decade and more recently with CRISPR-Cas9 technologies have been applied to well-established cancer models. We asked if minimally passaged cancer models would tolerate such screening modalities, particularly perturbations focused on actionable drug targets. We have established a patient derived model, CLF-PED-015-T, as a proof of concept to test this question. After validating that the cell line retains the major genomic, transcriptomic and tumorigenic properties of the tissue it was derived from, we then performed systematic genetic screens using both CRISPR-Cas9 and RNAi to identify potentially actionable vulnerabilities. We then overlapped this with pharmacologic screens. We identified dependencies to CDK4 and XPO1 that spanned all three screens. These dependencies have subsequently validated in an in vivo model. These results suggest use of such technologies at early stages of patient derived model development is feasible. This abstract is also being presented as Poster B14. Citation Format: Andrew L. Hong, Yuen-Yi Tseng, Glenn Cowley, Oliver Jonas, Jaime Cheah, Mihir Doshi, Bryan Kynnap, Coyin Oy, Paula Keskula, Gregory Kryukov, Michael Cima, Robert Langer, Stuart Schreiber, David Root, Jesse Boehm, William Hahn. Integration of CRISPR-Cas9, RNAi and pharmacologic screens identify actionable targets in a rare cancer. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr PR04.

Abstract B38: Developing a functional genomics platform to interrogate rare pediatric cancers

Of pediatric solid tumors, as many as 10% of tumors are categorized as rare. Many of these rare tumors lack standard effective known therapy. The ability to identify vulnerabilities for many rare tumors has been significantly limited by the lack of in vitro and in vivo models. Furthermore, current approaches to study such vulnerabilities are usually limited to a specific compound or target. Our objectives were 1) to develop a platform to collect tumor samples and generate in vitro models and 2) to develop systematic and orthogonal approaches focused on currently known druggable cancer targets to identify vulnerabilities in these difficult to treat cancers. We have developed a proof of concept cell line from a patient who succumbed to progressive undifferentiated sarcoma treated on an aggressive multi-therapy regimen. This cell line, in its early passages, has novel gene fusions that match that of the primary tumor. Furthermore, even at early passages, this cell line was amenable to high throughput functional screens. Using a targeted pooled shRNA screen (employing matched seed controls) and an analogous CRISPR screen we identified dependencies to XPO1 and CDK4. In parallel, compounds against these targets were identified in a small molecule compound screen. These targetable dependencies were further validated in vivo with a micro-dosing device. These observations identify new targets in this rare malignancy. Furthermore, this suggests that the interrogation of patient derived cell lines facilitates the identification of testable therapeutic approaches. Citation Format: Andrew L. Hong, Glenn S. Cowley, Yuen-Yi Tseng, Jaime H. Cheah, Oliver Jonas, Mihir B. Doshi, Bryan D. Kynnap, Coyin Oh, Stephanie Meyer, Paul Clemons, Michael Burger, Francisca Vazquez, Barbara Weir, Gregory V. Kryukov, Alanna Church, Alma Imamovic, Aviad Tsherniak, Craig Bielski, Brian Crompton, Elizabeth Mullen, Charles Roberts, Carlos Rodriguez-Galindo, Katherine A. Janeway, Kimberly Stegmaier, Paul van Hummelen, Robert Langer, Levi A. Garraway, Stuart L. Schreiber, David E. Root, Jesse S. Boehm, William C. Hahn. Developing a functional genomics platform to interrogate rare pediatric cancers. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B38.