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Letters to the Editor
Whole genome CRISPR screening identifies TOP2B as a potential target for IMiD sensitization in multi- ple myeloma
Thalidomide analogues (IMiD), such as lenalidomide (LEN) and pomalidomide (POM) have significantly improved survival in patients with multiple myeloma (MM).1 However, many patients relapse despite contin- ued IMiD exposure, and IMiD-resistance remains a sig- nificant clinical problem. IMiD engage Cereblon (CRBN), an adaptor for the CUL4A-DDB1-RBX1 E3 ligase com- plex to promote proteasome-dependent degradation of neosubstrates IKZF1 (Ikaros) and IKZF3 (Aiolos). The degradation of these transcription factors is both directly toxic to MM cells and immunostimulatory to T cells.2–4 Interrogation of the molecular events driving IMiD-medi- ated engagement of CRBN neosubstrates has greatly improved our mechanistic understanding of these agents. Acquired or intrinsic IMiD-resistance may occur through several mechanisms including loss of expression of CRBN and/or associated E3 ligase factors.5,6 However, deeper insight into IMiD-resistance mechanisms may inform novel therapeutic approaches. Here, genome-wide CRISPR-Cas9 screening was employed to characterize resistance mechanisms and resensitization factors in iso- genic IMiD-sensitive and -resistant MM lines. Loss of DNA topoisomerase IIβ (TOP2B) resensitized IMiD- refractory cells to IMiD and its inhibition with the cardio-
protective drug, dexrazoxane (DXZ), potentiated IMiD activity. Collectively, these findings identify TOP2B as a potential new therapeutic target in MM.
LEN-resistant MM1.S (MM.1Sres) cells were previous- ly derived by culturing MM.1S cells in presence of increasing doses of LEN.7 MM.1Sres cells displayed no significant increase in cell death upon prolonged (7 days) LEN exposure, while isogenic MM.1S cells were sensitive to LEN-induced cell death (Figure 1A). MM.1Sres cells also exhibited resistance to the anti-proliferative effects of LEN and POM as demonstrated by CellTrace Violet (CTV) labeling (Figure 1B). As previously reported,7 MM.1Sres cells showed reduced CRBN expression and reduced IKZF3 degradation upon IMiD treatment as compared to MM.1S (Figure 1C).
In order to determine genes and pathways required for IMiD anti-myeloma activity in MM.1S cells, a genome- scale CRISPR knockout screen was performed in MM.1S- Cas9 cells treated with LEN, POM or dimethyl sulfoxide (DMSO) (Figure 1D). Genetic dependencies of MM.1S- Cas9 cells were identified by loss-of-representation of short guide RNA (sgRNA) in (DMSO)-treated cells and identified genes such as MYC, IRF4, IKZF1 and IKZF3 (Online Supplementary Figure S1A). MM.1S-Cas9 cells were dependent on essential processes such as RNA metabolism and DNA-damage related pathways (Online Supplementary Figure S1B and C). Deletion of CRBN and members of the COP9 signalosome (CSN), a 9-protein
A B C
D
Figure 1. Resistance to lenalidomide and pomalidomide is mediated by loss of Cereblon and the COP9 signalosome. (A) Bar plot representing the percentage of propidium iodide negative (PI-, viable) MM.1S and lenlidomide (LEN)-resistant MM1.S (MM.1Sres) MM.1Sres cells treated with dimethyl sulfoxide (DMSO) or LEN (2 mM) for 7 days. Results are aggregated from n=3 independent experiments with two technical replicates per experiment. Error bars represents the mean ± standard error of the mean of the three biological replicates with their respective technical replicates; *P<0.0001. (B) CellTraceTM Violet/propidium iodide pro- liferation assay comparing proliferation of MM.1S and MM.1Sres cells in presence of LEN (2 mM) or pomalidomide (POM) (500 nM) (day 7 timepoint) in com- parison to vehicle (DMSO). Results are representative of three independent experiments. (C) Immunoblot of IKZF3 and Cereblon (CRBN) levels in DMSO-, LEN- (2 mM) or POM- (500 nM) treated MM.1S and MM.1Sres cells for 16 hours (blots are representative of n=3 independent experiments). (D) Schematic represen- tation of the CRISPR genome-scale resistance screen workflow. Approximately 500x106 MM.1Sres-Cas9 cells were transduced with the Brunello genome-wide library, selected with puromycin and then treated with DMSO, LEN (2 μM) or POM (500 nM) for approximately 8 weeks. Genomic DNA was extracted for library preparation and Illumina sequencing. (E) Scatter plot showing hits overlapping between LEN and POM that are significant for adjusted P<0.05 and hits unique to LEN-treated cells significant for adjusted P<0.05.
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