A CRISPR-mediated gene activation approach to uncover gene networks driving proteasome inhibitor resistance in Multiple Myeloma

Multiple myeloma (MM) is the second most common haematological malignancy arising from terminally differentiated plasma cells within the bone marrow, characterised by serum monoclonal gammopathy, immune suppression and bone disorders. Even though in the last decade MM patients' outcomes have been notably improved by the approval of numerous agents, MM still remains an incurable disease. Since MM cells are characterised by an abnormal immunoglobulin overproduction, they depend on the precise regulation of the protein degradation system, namely the Ubiquitin Proteasome System (UPS). As a consequence, proteasome inhibitors (PIs) are extensively used in combined therapeutic regimens with immunomodulatory agents, monoclonal antibodies and histone deacetylase inhibitors. Nevertheless, in the long run, most patients experience drug refractoriness and relapse. Therefore, further efforts are needed to uncover novel strategies that can improve the management of the early disease and of relapsed/refractory MM patients. Here, to identify genes that may confer resistance to PI Carfilzomib (CFZ), we carried out a genome-wide CRISPR activation screening in a MM cell line, employing the Synergistic Activation Mediator (SAM) system coupled with a pooled human sgRNAs library. Combining next-generation sequencing (NGS) results with data obtained from single-cell cloning, we found 13 putative target genes in common and focused our attention on three target genes that provided different levels of CFZ resistance to cells: OAF, ZUFSP and ZNF774. Our preliminary experiments provide three lines of evidence suggesting that the sgRNA-mediated transactivation of OAF, ZUFSP or ZNF774 is sufficient to support CFZ resistance. Indeed, even if this effect is not really appreciable immediately after CFZ treatment, we showed that MM cells overexpressing the aforementioned genes: 1) recover significantly faster from the cytotoxic effect of CFZ; 2) are progressively enriched in cell competition assays, following CFZ treatment; 3) generate clones of increased number and size in methylcellulose clonogenic assays in the presence of CFZ. Overall, our preliminary results suggest that novel genes as OAF, ZUFSP and ZNF774 might be involved in CFZ resistance. Future studies will be crucial to confirm our findings and to shed light on the mechanisms implicated.