Functional Genomic Landscape of Genes with Recurrent Mutations in Multiple Myeloma

During the last two decades, cell lines and patient-derived samples from multiple myeloma (MM) have been extensively profiled for alterations in their genome with the anticipation that those genes with the most recurrent lesions could represent attractive novel therapeutic targets or markers for aggressive disease. Yet for many of these genes, their functional significance for MM cells has not been formally evaluated. With the advent of new CRISPR/Cas9-based functional genomics platforms, it is possible to generate in genome- or subgenome-scale direct quantitative information on the impact that perturbation of these genes exerts on tumor cell survival, proliferation or other phenotypes. We therefore examined the landscape of our CRISPR-based functional genomic data for these recurrently dysregulated genes We specifically curated information from the MMRF CoMMpass study and multiple other publicly available studies, to identify genes which are recurrently identified to harbor nonsynonymous mutation (SNV or indel), DNA copy number loss or gain, or participation in chromosomal translocations. We then examined the patterns of results for these genes in our genome scale CRISPR-based gene-editing studies for loss-of-function in n=18 MM cell lines.

We identified a subset of genes (e.g. FAM46C, CDKN2C, RASA2) which are considered targets for recurrent loss-of-function events and indeed exhibit, for large fractions of the cell lines tested enrichment, of their sgRNAs in CRISPR knock-out studies, consistent with a role of these genes as suppressors of tumor cell survival or proliferation. CRISPR KO of TP53 leads to increased survival/proliferation of only a small minority (2/18 of cell lines tested thus far), which reflects the fact that the overwhelming majority of MM cell lines already harbor LOF events for this gene. Interestingly, a substantial number of genes which have been considered to harbor recurrent LOF events in MM patient samples (e.g. NF1, NF2, CYLD) do not exhibit sgRNA enrichment in CRISPR KO screens in the MM cell lines tested so far. In addition, several other recurrently mutated genes for which their loss- or gain-of-function status had not been previously evaluated with extensive functional studies in MM (e.g. SP140, LTB, EGR1, ATM, PARK2, PRKD2, RAPGEF5, DOCK5, TGDS, TNFAIP8) exhibit in the majority of cell lines tested in in CRISPR knockout studies no significant enrichment or depletion of their sgRNAs. In contrast, PTPN11, CREBBP, EP300, KMT2B, KMT2C, SETD2, SF3B1 and UBR5, are notable examples of recurrently mutated genes which represent dependencies for large fractions of MM cell lines in vitro. These results highlight the value of interpreting results from next generation sequencing studies in the context of information provided by the genome scale by use of functional genomic characterization of available cell line models. We envision that, similar sub-genome scale assays were performed at the level of patient derived samples will also provide direct information about the relevance of some of these genes. In addition, functional studies conducted with context of tumor-microenvironemtn compartment interactions and tumor interface will be needed to evaluate several genes identified in the study.