EZH2 Inhibition Overcomes Immunomodulatory Drug (IMiD) Resistance in Multiple Myeloma Cell Lines in a Cereblon Pathway Dependent Manner

Introduction

Immunomodulatory agents (IMiDs) are effective therapies for multiple myeloma (MM), approved for use at all stages of disease. IMiDs function as molecular glues at cereblon (CRBN), part of the CRL4 ^CRBN E3 ubiquitin ligase complex, designating neo-substrates for proteasomal degradation. In myeloma cells the B cell transcription factors Ikaros and Aiolos are degraded, leading to downregulation of IRF4 which is critical for myeloma cell survival. Novel CRBN ligase modulators (CELMoDs) are currently in early phase clinical trials and have demonstrated activity in patients refractory to IMiDs, but resistance inevitably develops over time.

EZH2 is a histone methyltransferase which acts at histone 3 lysine 27 (H3K27) leading to trimethylation (H3K27me3). The aberrant expression of EZH2 leads to abnormal gene repression, contributing to tumour development and progression. EZH2 inhibitors (EZH2i) have been shown to overcome drug resistance in several cancers and we hypothesized that combining EZH2i with IMiDs/CELMoDS would overcome resistance to these agents in MM cell lines.

Methods

MM cell lines KMS11, RPMI and MOLP8 were used as models of IMiD/CELMoD resistance and MM.1S as a control model of sensitivity. The isogenic pair of KMS11 with CRBN knockout (KMS11 ^{CRBN KO}) was established using CRISPR-Cas9. Following concentration/duration optimisation, cell lines were treated with EZH2i (Tazemetostat) 0.25-1µM or DMSO control for 5 days alone, and then in combination with IMiD (Lenalidomide, Len, 0-20 µM, Pomalidomide, Pom, 0-8 µM) or CELMoD (Iberdomide, CC-220, 0-2 µM and Mezigdomide, CC-92480, 0-0.1uM) for a further 5 days. Cell viability was measured using CellTiter Blue® and the level of synergy assessed using SynergyFinder (Bliss model). The effect on apoptosis and cell cycle was examined using flow cytometry after AnnexinV-PI and/or DAPI staining respectively. To elucidate the mechanism of synergy, the effect of the single agents and combinations on key CRBN pathway effectors (CRBN, Ikaros/Aiolos, IRF4) were examined using WB and qPCR. ChIP-PCR was employed to assess the changes in enrichment of H3K27me3 and key MM transcription factors (Ikaros/Aiolos) within the IRF4 promoter region upon EZH2i treatment.

Results

MM.1S cells were sensitive to IMiDs (Len/Pom) and CELMoDS (CC-220/CC-92480) with GI50s of 0.279 µM, 0.08 µM, 2 nM and 0.3 nM respectively. KMS11, MOLP8 and RPMI cell lines were resistant at more than 10x the GI50 in MM.1S. In these models of resistance, the cells express functional CRBN protein, demonstrated by the degradation of neo-substrates Ikaros/Aiolos. However, whilst their degradation results in reduced IRF4 expression and cell death in sensitive cells (e.g. MM.1S), this does not occur in the resistant models suggesting an uncoupling of Ikaros/Aiolos control of IRF4 expression in the resistant setting.

Combining EZH2i with IMiDs/CELMoDs led to a reduction in cell viability (e.g. CC-220 shown in Fig.1A) and demonstrated synergy across resistant cell lines (KMS11, MOLP8 and RPMI). An increase in apoptosis was seen using AnnexinV/PI staining, with no demonstrable change in cell cycle. WB and qPCR results showed a significant reduction in IRF4 mRNA and protein with the combination treatment. The combination of EZH2i and IMiD/CELMoDs did not reduce cell viability or IRF4 expression in the KMS11 ^{CRBN KO} cell line ( Fig.1A), indicating that the observed synergistic effect is CRBN-dependent.

Compared to DMSO control, EZH2i increased IRF4 mRNA, with a reduction of H3K27me3 enrichment observed within the IRF4 promoter region by ChIP-PCR in KMS11 ( Fig.1B). In addition, EZH2i also enhanced Ikaros and Aiolos enrichment within the IRF4 promoter region ( Fig.1B).

Conclusions

Our results suggest that combining EZH2i with IMiDs/CELMoDs can overcome resistance to these agents in MM cell line models, with synergy that is CRBN-dependent. By examining the key components of the CRBN pathway we identified that EZH2i reduced H3K27me3 and increased Ikaros and Aiolos association at the IRF4 promoter, suggesting a possible re-coupling of Ikaros/Aiolos to IRF4 expression, which may be responsible for reinstating IMiD/CELMoD activity, driving the synergistic effect seen.