Genome-Scale Crispr-Cas9 Knockout Studies Reveal Mutifactorial and Functionally Overlapping Mechanisms of Myeloma Cell Resistance to Proteasome Inhibition

Acquired or de novo resistance to established and investigational therapies represents a major clinical challenge for multiple myeloma (MM) and other neoplasias. Despite extensive efforts, clinically-validated molecular markers that predict for proteasome inhibitor (PSI) resistance in most MM patients remain elusive. This challenge is partly due to limited availability so far of molecular data on MM patients before the start of PSI treatment vs. immediately after resistance to it develops; this challenge may also reflect the heterogeneity of the complex molecular mechanisms regulating MM cell response to PSIs. We hypothesized that resistance to PSIs can be mediated by disruption of several functionally overlapping genes, and that the prevalence of any of these lesions may be too low to detect in datasets available thus far. To examine this latter hypothesis, we performed a genome-wide screen for genes whose loss confers to MM cells resistance against bortezomib, through the use of the CRISPR (clustered regularly interspaced short palindromic repeats)–associated nuclease Cas9 system. Specifically RPMI-8226 MM cells were transduced with lentiviral construct for Cas9 nuclease, followed by lentiviral delivery of a genome-scale pooled library of 123,411 single-guide RNAs (sgRNAs), which selectively align to target sequences at the 5′ constitutive exons of 18,080 genes and direct the Cas9 nuclease to cause double-stranded cleavage and loss of function of the respective gene. From the pool of MM cells transduced with the sgRNA library and treated with bortezomib, treatment-resistant cells were processed for deep sequencing, to identify enriched sgRNAs and their corresponding genes. We identified that loss-of-function of 33 candidate genes is associated with bortezomib resistance. We observed a high level of consistency between independent sgRNAs targeting the same gene, as well as a high rate of hit confirmation across different biological replicates. Notably, this set of candidate bortezomib-resistance genes was distinct from the "hits" we identified through a parallel CRISPR screen on the same cell line for resistance to a different targeted therapy (namely the bromodomain inhibitor JQ1), supporting the ability of this approach to identify treatment-specific resistance genes. These candidate bortezomib-resistance genes have documented or presumed roles in the regulation of extrinsic and intrinsic apoptotic cascades, autophagy, Toll-like receptor and NF-kappaB signaling, aggresome function, heat shock protein expression, chromatin remodeling, nutrient sensing, and tumor suppressor gene networks. Importantly, information from several publically available molecular profiling datasets converge to support the putative clinical relevance of these genes. For instance, gene expression data from tumor cells of bortezomib-naive patients with advanced MM revealed several transcriptional signatures of these candidate genes (defined by low transcript levels for any of the genes in the signature) which correlated with shorter time to disease progression after treatment with bortezomib (p<0.01, log-rank test), but not dexamethasone (p>0.426). Congruent with these findings, the highly bortezomib-responsive clinical setting of newly-diagnosed MM is associated with low cumulative frequency of mutations of these bortezomib-resistance genes (e.g. cumulative mutation rate of 3.9%, 95% confidence interval [CI] 1.25-6.55%). Notably, in other malignancies that are typically PSI-resistant, a higher cumulative frequency of such lesions is observed (average of ~28%, range 0-76%, 95% CI 22.46-32.70%; 57 datasets from 20+ neoplasias examined). In summary, this first application of the CRISPR/Cas9-based technology in MM illustrates its power to interrogate gene function on a genome-wide scale. This approach identifies bortezomib-resistance genes that are associated with pathways linked with the regulation of proteasome inhibitor response. Results from molecularly-annotated clinical samples converge to support a possible role for these genes in bortezomib resistance. This experience supports the value of CRISPR/Cas9-based studies to dissect the molecular mechanisms of treatment resistance in MM and other hematologic neoplasias (* equal contribution of M.S. and Y.H.).