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Letters to the Editor
POM (Figure 1E), reflecting either a difference of potency and/or discrepant substrate specificity between the two IMiD.
The identification of genes that when deleted restore IMiD-sensitivity in MM.1Sres cells was achieved by a loss-of-representation CRISPR screen in 21-day LEN and POM-treated MM.1Sres-Cas9 cells relative to DMSO control (Figure 2A). The dependencies of DMSO-treated MM.1Sres-Cas9 cells partially overlapped and correlated with those of MM.1S-Cas9 cells, suggesting that acquired IMiD-resistance did not globally alter gene dependencies in these cells (Online Supplementary Figure S2A to F). In order to identify selective resentitization mechanisms in the presence of IMiD, the DMSO endpoint was com- pared with the matched timepoint in IMiD-treated MM.1Sres-Cas9 cells. This revealed that deletion of ATXN7, TOP2B, MIER3, YPEL5, MAEA and MED13L sensitized MM.1Sres cells to both LEN and POM (Figure 2B and C). ATXN7 is part of the deubiquitination module of STAGA, a multisubunit entity involved in transcrip- tional regulation and DNA repair.8 MED13L and MIER3 also modulate transcription.9,10 Interestingly, YPEL5 and MAEA co-operate in an E3 ligase complex targeting glu- coneogenesis enzymes.11 However, TOP2B was selected for further study due to its potential tractability as a drug target. TOP2B is an enzyme which resolves topological DNA constraints during replication, transcription and repair.12 In order to validate that TOB2B loss resensitizes to IMiD, competitive proliferation assays were performed in the presence and absence of LEN and POM (Figure 2D). MM.1Sres-Cas9 cells expressing two independent TOP2B sgRNA with a GFP reporter were mixed at a 1:1 ratio with MM.1Sres-Cas9 cells expressing a non-targeting sgRNA with a BFP reporter (Figure 2D to E; Online Supplementary Figure S2G). Consistent with the CRISPR-screen results, a competitive loss of GFP+ relative to BFP+ cells was observed following IMiD treatment (Figure 2E) confirm- ing that TOP2B deletion confers IMiD-sensitivity in MM.1Sres-Cas9 cells. Analogous assays in MM.1S-Cas9 cells revealed that TOP2B loss further sensitized these cells to the anti-tumour effects of IMiD (Figure 2F), indi- cating that TOP2B deletion enhances IMiD activity in both IMiD-naïve and resistant contexts. TOP2B deletion had little or no effect on CRBN expression or subsequent IKZF3 degradation following IMiD treatment (Online Supplementary Figure S3A and B). MYC levels appeared to be modestly more downregulated in IMiD-treated MM.1Sres-Cas9 compared to MM.1S-Cas9 cells while the IMiD-induced downregulation of IRF4 was similar in MM.1Sres-Cas9 compared to MM.1S-Cas9 cells ((Online Supplementary Figure S3C). These observations indicate that the resensitization to IMiD treatment following dele- tion of TOP2B likely does not depend on further effects on the IKZF1/3-IRF4-MYC axis.
Having discovered the IMiD-sensitizing effects of genetic TOP2B depletion in MM.1Sres-Cas9 cells, orthogonal assays using DXZ, a chemical inhibitor of TOP2 that induces selective degradation of TOP2B pro- tein, were used to validate this observation. DXZ also posseses iron-chelating activity and is Food and Drug Administration-approved for prevention of anthracy- cline-induced cardiotoxicity.13 In order to investigate whether DXZ-mediated TOP2B degradation would phe- nocopy genetic deletion, MM.1Sres and MM.1S cells were treated with DXZ alone or in combination with LEN. TOP2B degradation in DXZ-treated MM.1S cells was evident 24 hours (hrs) after drug exposure and remained low in treated cells for up to 72 hrs (Figure 3A) at clinically relevant concentrations.14 Cell cycle analysis
demonstrated that concentrations of DXZ sufficient to induce TOP2B degradation were cytostatic in MM.1S and MM.1Sres cells, with accumulation of cells in SubG1 and >2N, suggesting apoptosis-induction and failure of cytokinesis (Figure 3B; Online Supplementary S3D to F). Combinatorial effects of LEN and DXZ were observed in MM.1S and MM.1Sres cells with DXZ alone inducing cytostasis in MM.1Sres cells (Figure 3C and D). Similar to the effects observed following TOP2B deletion, DXZ did not modulate CRBN expression or alter IKZF3 degrada- tion (Online Supplementary Figure S3G). However, an effect of the LEN and DXZ combination on cMYC, IKZF1, IKZF3 and IRF4 expression (Figure 3E) was evident. These findings may indicate that the effects of LEN and DXZ converge on the IRF4-MYC axis in MM.1Sres cells downstream or in parallel to canonical CRBN-neosub- strate interactions. Subsequently, DXZ combination treat- ments were performed in IMiD-sensitive OPM2 cells and IMiD-resistant RPMI-8226 and JJN3 MM cell lines (Figure 3D). In OPM2 cells, DXZ and LEN alone induced robust growth inhibition with a combinatorial effect observed following treatment with both agents (Figure 3F and G). DXZ induced death of RPMI-8226 and JJN3 cells, with mild additivity in the presence of LEN (Figure 3F, H and I).
CRISPR-based dissection of the genetic dependencies of MM.1S cells provided additional insight into IMiD biology and acquired IMiD resistance. Consistent with the initial description of MM.1Sres,7 downregulation of CRBN expression and attenuation of neosubstrate degra- dation appears to be the major mechanism of IMiD resistance in these cells. Synthetic generation of IMiD resistance using gene deletion in MM.1S cells recapitulat- ed prior studies identifying CRBN and elements of the CSN.5,6 Gene ontology analysis of these hits revealed their importance in transcription-coupled nucleotide excision repair (TC-NER). Moreover, CUL4 and DDB1 have been demonstrated to participate with CSN in DNA repair pathways such as NER and TC-NER.15 IMiD-sensi- tivity in MM.1Sres cells was rescued by knockout of TOP2B, a gene that modulates DNA repair, chromatin stability and gene expression.12 However, LEN did not induce a DNA damage response or synergize with etopo- side in MM.1Sres cells, suggesting that DNA damage induction is not the primary re-sensitization mechanism (Online Supplemnentary Figure S3H and I). Genetic deletion of TOP2B was not lethal to MM.1Sres cells, however these cells were sensitized to IMiD-induced death. This phenotype seemed to be independent of an increase in CRBN activity or expression changes within the IKZF1/3- IRF4-MYC axis. The biology underpinning re-sensitiza- tion of MM.1Sres cells to IMiD through loss of TOP2B remains to be defined.
DXZ had demonstrable anti-MM properties and addi- tional activity in combination with LEN, especially in IMiD-sensitive cells. Since TOP2B deletion did not induce growth inhibition, the single-agent activity of DXZ may depend upon TOP2A inhibition rather than TOP2B degradation or through other effects. Deletion or depletion of TOP2A can have deleterious effects on the growth and/or survival of cancer cells (Online Supplemnentary Figure S3J) but we do not have evidence clearly demonstrating that the anti-MM activity of DXZ is through effects on TOP2A. Given that DXZ did not appear to demonstrably impinge on the IKZF1/3-IRF4- MYC axis, exactly how DXZ confers anti-MM activity, either alone or in combination with IMiD, remains unknown. The IC of DXZ across the MM lines tested
50
spanned from 5 to 20 mM (data not shown), which is sig-
nificantly lower than the peak plasma concentration
2016
haematologica | 2021; 106(7)