PF554 A GENOME-WIDE CRISPR SCREEN IDENTIFIES TOP2B AS AN ACQUIRED DRUGGABLE VULNERABILITY IN THE CONTEXT OF IMID RESISTANCE

Abstract

Background: Immunomodulatory thalidomide analogues (IMiDs, lenalidomide [LEN] and pomalidomide [POM]) modify the substrate specificity of the CUL4A-DDB1-RBX1 E3 ubiquitin ligase complex through a direct interaction with cereblon (CRBN). In the presence of IMiDs, CUL4ACRBN induces proteasomal degradation of IKZF1 and IKZF3, which are key oncogenic transcription factors in multiple myeloma (MM). Unfortunately, continued IMiD exposure leads to acquired drug resistance in the majority of patients and IMiD-refractoriness conveys poor prognosis. Whereas perturbations of CRBN and directly linked elements of the CUL4A complex are known to mediate IMiD resistance, mechanistic insight downstream of the CUL4ACRBN complex is limited. Aims: We sought to determine the genes, molecular pathways and cellular processes underpinning acquired IMiD resistance and identify novel targets to which IMiD-resistant MM is differentially sensitive that could be exploited for therapeutic gain. Methods: Human MM cell line MM1.S and isogenic LEN-resistant MM1.Sres (MM1.Sres; Zhu et al. Blood 2011; 118–4771–9) were validated for differential IMiD sensitivity by CellTrace™ Violet proliferation assay and propidium iodide staining. Western Blot for IKZF1/3, MYC and IRF4, and 3’RNA-sequencing were compared. The Brunello genome-wide CRISPR library was transduced into Cas9 expressing MM1.S and MM1.Sres cells treated with LEN or POM for 3–8 weeks and genomic DNA sequenced aiming to identify sgRNA enrichment (MM1.S) or loss-of-representation (MM1.Sres). Results: MM1.Sres demonstrated reduced sensitivity to LEN and POM relative to its isogenic counterpart. Acquired IMiD resistance associated with reduced CRBN expression and mitigation of IKZF1/3 degradation following IMiD exposure. Untreated MM1.Sres cells had a distinct gene expression profile relative to LEN-sensitive controls with marked attenuation of the global transcriptional response to IMiD exposure. As expected, the genome-wide resistance CRISPR-Cas9 knockout screen identified enrichment of CRBN and COP9 signalosome (CSN) sgRNAs as major determinants of LEN resistance in MM1.S cells. Conversely, TOP2B and ATXN7 were amongst the most depleted sgRNAs identified in LEN-treated MM1.Sres cells, indicating synthetic lethality between TOP2B/ATXN7 loss and LEN treatment in acquired IMiD resistance. As CUL4A, DDB1, CSN, TOP2B and ATXN7 regulate DNA homeostatic mechanisms including DNA-repair, we hypothesised that IMiD-resistant MM would be sensitized to DNA damaging agents and that these compounds might synergise with IMiDs in LEN-resistant cells. Consistent with this hypothesis, GSEA analysis on the 3’-RNAseq datasets revealed enrichment in DNA repair gene-sets in MM1.Sres cells. Strikingly, MM1.Sres and CRISPR-deleted MM1S CRBN−/− cells displayed greater sensitivity to TOP2B inhibition with etoposide. Moreover, etoposide synergised with LEN in the context of both de novo (RPMI-8226 MM cells) and acquired (MM1.Sres) IMiD resistance. Summary/Conclusion: IMiD-resistant MM is differentially sensitive to genetic depletion or pharmacological antagonism of TOP2B. This phenotype may indicate a more generalised alteration in DNA homeostasis downstream of CRBN loss and IMiD resistance. These findings provide a rationale for the deployment of etoposide-based salvage following IMiD failure and may underpin the activity of infusional, etoposide-based chemotherapy (e.g. DCEP) relapsed / refractory MM.