Abstract LB-118: Characterization of lineage vs. context-dependent essential genes in multiple myeloma using CRISPR-Cas9 genome editing

Abstract

Multiple Myeloma (MM) remains incurable in part due to an incomplete understanding of the genes critically responsible for MM cell survival and proliferation. We reasoned that CRISPR/Cas9-based functional genomics could identify genes essential for survival or proliferation of MM cells and define candidate therapeutic targets. We transduced Cas9-expressing RPMI-8226 and MM.1S cells with lentiviral genome-scale GeCKO pooled libraries of sgRNAs. After culture of these cell lines for 2-12 weeks without treatment we identified, based on next generation sequencing, genes with significantly depleted sgRNAs (4-6 sgRNAs/gene, >2-fold average depletion, FDR<0.05, using MAGECK algorithm) in Cas9+ cells compared to initial sgRNA plasmid pools, baseline cultures, or isogenic parental Cas9-negative cells. These results were confirmed for each cell line with a 2nd independent genome-wide analysis and with a focused sgRNA library containing a subset of candidates defined by the genome-wide analyses. We compared these results with data from our in-house or publicly available CRISPR/Cas9 gene editing studies, involving a total of 175 cell lines from other hematologic malignancies and solid tumors. We next integrated our functional data for these genes with their: a) Achilles Heel shRNA data; b) gene expression (RNASeq); c) gene copy number status; d) mutation status; e) proximity to superenhancers; and f) altered expression in MM cells co-cultured with bone marrow stromal cells. We applied a series of statistical tests (e.g. Wilcoxon rank) to identify genes with significantly higher rank in sgRNA or shRNA depletion in MM vs. non-MM lines and stringently identified 50+ candidate genes with preferential dependency in MM. Prominent examples included: transcription factors (e.g. IRF4, CCND2, MAF, NFKB1, NFKB2, RELA, RELB); other NF-κB-related genes (e.g. IKBKB); PIM2; regulators of protein homeostasis; and several other known or biologically plausible dependencies which are under further evaluation. In >90% of MM patients, the candidate MM-preferential essential genes are not mutated. Some of these genes are overexpressed in MM vs. non-MM cell lines (CCLE dataset) or patient-derived samples (Broad/MMRF vs. TCGA datasets); induced by BMSC co-culture; proximal to superenhancers; or correlating with clinical outcome. However, these molecular results were not sufficient, collectively or individually, to identify this cohort of MM-preferential essential genes independently of the CRISPR functional data. Targeting lineage-specific dependencies (e.g. ER or AR in breast or prostate Ca, respectively) has provided major clinical benefit in some tumors; while context-specific dependencies are a cornerstone of molecularly-guided individualized treatments. Therefore, by identifying lineage- and context-dependent essential genes for MM, our functional genomic studies in molecularly annotated MM vs. non-MM cell lines provide an attractive framework towards designing novel therapies for MM. Citation Format: Geoffrey M. Matthews, Ricardo de Matos Simoes, Yiguo Hu, Michal Sheffer, Olga Dashevsky, Eugen Dhimolea, Paul J. Hengeveld, Brian J. Glassner, Sara Gandolfi, Megan A. Bariteau, Quinlan Sievers, Benjamin L. Ebert, Franciska Vazquez, Aedin Culhane, Constantine S. Mitsiades. Characterization of lineage vs. context-dependent essential genes in multiple myeloma using CRISPR-Cas9 genome editing [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 LB-118. doi:10.1158/1538-7445.AM2017-LB-118

Network Controllability Analysis of Three Multiple-myeloma Patient Genetic Mutation Datasets

“…A gene is called survivability-essential for a cell if knocking it out leads to cell death [28,29]. Through the CRISPR-Cas9-based genome editing analysis, a wide range of results became recently available for disease-specific survivability-essential gene, see [30,31,32]: these are genes whose knocking out leads to cell death in diseased cells, but appears neutral in healthy cells. The concept of disease-specific essential genes is immediately amenable to investigations for potential drug targeting.…”

“…This is the objective of our analysis, described in Section 4. The list of 70 multiple myeloma-specific essential genes we used is from [32,33,34] and is shown in Table 2.…”