CRISPR Activation Screen for Drivers of MM Cell Proliferation
Authors: Tang Huihui , De Matos Simoes Ricardo , Shirasaki Ryosuke , Dashevsky Olga , Glassner Brian , Downey-Kopyscinski Sondra L. , Gandolfi Sara , Sheffer Michal , Bariteau Megan , Doench John G , Mitsiades Constantine S. 
Published: October 2019

Abstract
CRISPR/Cas9-based gene editing has become a powerful tool for loss-of-function (LOF) studies and has allowed us to systematically interrogate the function of genes regulating the survival and proliferation of multiple myeloma (MM) cells in vitro, in vivo and in the context of treatment resistance (e.g. De Matos Simoes et al., Shirasaki et al., and Gandolfi et al. ASH 2017). We reasoned, however, that important additional information can be obtained from CRISPR-based gain-of-function (GOF) approaches which can achieve transcriptional activation at endogenous genomic loci. We thus performed genome-scale CRISPR activation studies using the dCas9-P65-HSF transcriptional activation system (in which a Cas9 variant lacking nuclease activity [dCas9] confers P65-HSF-mediated activation of genes recognized by sgRNAs against their promoter regions). Specifically, MM.1S cells were transduced with the dCas9-P65-HSF system and pooled lentiviral particles of the Calabrese CRISPR activation sgRNA library, consisting of 2 pooled sgRNA sub-libraries (total of ~110,000 sgRNAs targeting ~18000 genes, at initial coverage of 800 cells/sgRNA). Cells were cultured for 12 weeks and harvested at baseline and various intervals, e.g. 4 and 12 weeks of culture. Next generation sequencing of genomic DNA quantified the abundance of sgRNAs in the tumor cell population and genes were ranked (with rank aggregation algorithms) in terms of their sgRNA enrichment or depletion. These analyses allowed us to observe a series of genes with statistically significant sgRNA enrichment and known or presumed roles in MM biology, including key MM transcription factors such as IRF4, the thalidomide derivative targets IKZF3 and IKZF1, and the co-factor POU2AF1; known oncogenes, e.g. KRAS and MYC; NF-kappaB pathway members, e.g. RELA; and signal transduction regulators, e.g. IGF1R and its downstream effectors IRS1 and AKT2. These results are consistent with the major depletion of sgRNAs for these genes in loss-of-function (LOF) CRISPR knockout studies. However, several other genes with significant sgRNA enrichment in CRISPR activation studies did not exhibit major sgRNA depletion in CRISPR knockout studies, including the B/plasma cell transcription factor POU2F2 (Oct2), for which high protein expression correlates with reduced survival in MM (Toman I. et al 2011); the transcription factor PAX2, the TRAF interacting protein TIFA, or the Toll-like receptor TLR4. Interestingly, significant depletion of sgRNAs was observed for several genes with known or proposed tumor suppressive properties including YAP1 (an oncogene for solid tumors, but reported as tumor suppressor for MM and other blood cancers); the pro-death TNFRSF10A (TRAIL receptor DR4), TP73, CDKN1A, the negative regulator of c-Myc MXI1, or the pro-apoptotic Bcl2 family member BAK1. Depletion or enrichment of sgRNAs for most of the aforementioned genes was detectable by 4 weeks of culture, while more pronounced changes and detection of additional statistically-significant hits was observed in later time-points. For genes with significant sgRNA enrichment in our CRISPR activation study, we examined a series of molecular alterations, including transcript overexpression in MM cell lines or patient-derived samples vs. normal plasma cells, or relapsed/refractory MM vs. earlier disease MM stages; mutational status; correlation of transcript levels with clinical outcome in MM; and extent of open chromatin (based on H3K27Ac chromatin marks) within or proximal to each gene in MM cell lines. Some "hits" from our screen exhibited at least one of these molecular alterations, but most genes harbored no such alterations or their magnitude or frequency ranked outside the top 50-100 genes. These results suggest that CRISPR activation studies can identify important putative regulators of MM biology, which may not be readily detectable based on known annotations of the MM cell genome, transcriptome, or epigenome. Genome-scale CRISPR-based transcriptional activation are an important gain-of-function system to uncover genes which induce vs. suppress tumor cell survival and proliferation, and provide information orthogonal to those yielded by other CRISPR-based approaches that involve loss-of-function interventions. Our use of CRISPR activation allowed us to both validate previously known genes and identify promising new candidate regulators of MM cell biology.

Disclosures
Mitsiades: EMD Serono: Research Funding; Abbvie: Research Funding; Takeda: Other: employment of a relative; TEVA: Research Funding; Janssen/ Johnson & Johnson: Research Funding.