Dual inhibition of DNMTs and EZH2 can overcome both intrinsic and acquired resistance of myeloma cells to IMiDs in a cereblon-independent manner

Abstract

Thalidomide and its derivatives, lenalidomide and pomalidomide (also known as IMiDs), have significantly changed the treatment landscape of multiple myeloma, and the recent discovery of cereblon (CRBN) as their direct biological target has led to a deeper understanding of their complex mechanism of action. In an effort to comprehend the precise mechanisms behind the development of IMiD resistance and examine whether it is potentially reversible, we established lenalidomide-resistant (-LR) and pomalidomide-resistant (-PR) human myeloma cell lines from two IMiD-sensitive cell lines, OPM2 and NCI-H929, by continuous culture in the presence of lenalidomide or pomalidomide for 4-6 months, until acquirement of stable resistance. By assessing genome-wide DNA methylation and chromatin accessibility in these cell lines, we found that acquired IMiD resistance is associated with an increase in genome-wide DNA methylation and an even greater reduction in chromatin accessibility. Transcriptome analysis confirmed that resistant cell lines are mainly characterized by a reduction in gene expression, identifying SMAD3 as a commonly downregulated gene in IMiD-resistant cell lines. Moreover, we show that these changes are potentially reversible, as combination of 5-azacytidine and EPZ-6438 not only restored the observed accessibility changes and the expression of SMAD3, but also resensitized the resistant cells to both lenalidomide and pomalidomide. Interestingly, the resensitization process was independent of CRBN. Our data suggest that simultaneous inhibition of DNA methyl transferases and EZH2 leads to an extensive epigenetic reprogramming which allows myeloma cells to (re)gain sensitivity to IMiDs.

Keywords: 5-azacytidine; cereblon; epigenetics; immunomodulatory drugs; multiple myeloma.

Figure 1

(A) qPCR results of the CRBN expression across the IMiD‐sensitive cell lines (OPM2, NCI‐H929: dark bars on the left and right plot, respectively) and their lenalidomide‐ and pomalidomide‐resistant counterparts (dark gray and light gray bars, respectively). There is a significant downregulation of CRBN mRNA in all four cell lines with acquired IMiD resistance, compared to the parental, sensitive cell lines. **P < 0.01, ***P < 0.001, and ****P < 0.0001.(B) Western blot for CRBN, confirming the reduction in CRBN expression at protein level in loss of IMiD sensitivity. (C) Cytospin and immunohistochemical staining for CRBN in OPM2, NCI‐H929, and their IMiD‐resistant counterparts, confirming the significant reduction in CRBN expression in the resistant cells.

Figure 2

(A) Methylation‐specific melting curve analysis for the IMiD‐sensitive and IMiD‐resistant cell lines, showing the absence of promoter DNA methylation of CRBN. The blue curves are the fully unmethylated and fully methylated controls (left and right curve, respectively), and the green lines represent the cell samples. (B, C) ChIP for H3K4me3, H3K27Ac, and H3K27me3 around the transcription site of CRBN for OPM2 and NCI‐H929, respectively, revealed that IMiD‐resistant cell lines retained their activating histone marks (H3K4me3, H3K27Ac) without any significant increase in the repressive mark H3K27me3. (D) Chromatin accessibility values for the probes mapping to CRBN, assessed with AcceSssIble. The difference in the accessibility of the resistant cell lines compared with their parental cell line (ΔAcc) was calculated by subtracting the accessibility value of the paternal cell line from the accessibility value of the resistant cell line for each individual probe. None of the probes covering CRBN showed significant accessibility changes in any of the IMiD‐resistant cell lines. (E, F) Kernel density plot of the combined DNA methylation changes and chromatin accessibility changes observed in the IMiD‐resistant cell lines. The OPM2 IMiD‐resistant cells exhibit primarily a decrease in the global chromatin accessibility when compared with the parental OPM2 (E), while H929 IMiD‐resistant cell lines exhibit a pattern of combined loss of chromatin accessibility with increased DNA methylation (F).

Figure 3

(A) Apoptosis results for OPM2, OPM2‐PR, and resensitized OPM2‐PR. The original OPM2 cells are sensitive to both lenalidomide and pomalidomide, where treatment with 10 μm of either compound led to an apoptotic response after 72 h. In contrast, both drugs fail to induce apoptosis in the pomalidomide‐resistant OPM2‐PR cells. However, after epigenetic pretreatment with 5‐Aza (0.1 μm) and EPZ‐6438 (10 μm) for 48 h and subsequent treatment with either lenalidomide or pomalidomide for 3 days, the apoptotic response to the drugs was restored. (B) Apoptosis measurements for OPM2‐PR without any pretreatment (black bars), with pretreatment only with 0.1 μm of 5‐Aza (green bars), with EPZ‐6438 (blue bars), and with both (red bars). Even though both 5‐Aza and EPZ‐6438 have a slight resensitizing effect as monotherapy, the combination is significantly more effective in restoring IMiD sensitivity to OPM2‐PR cells. *P < 0.05, **P < 0.01, and ***P < 0.001. (C) Heatmaps with all the probes exhibiting either accessibility changes (N = 6844, upper heatmap in blue/yellow) or DNA methylation changes (N = 2102, bottom heatmap in red/green) in OPM2‐PR when compared with OPM2. The majority of the probes (approximately 80%) that exhibited altered chromatin accessibility restored their initial accessibility values after 48 h of treatment with 5‐Aza and EPZ‐6438, while the acquired DNA methylation patterns in the resistant cells were more stable.

Figure 4

(A) The expression of CRBN at mRNA level was not restored after treatment of the resistant cells with 5‐Aza, EPZ‐6438, or their combination, suggesting that the downregulation of CRBN, which is observed in all the IMiD‐resistant cells with acquired resistance, might not have a direct causality to the development of resistance. (B) Western blot of IKZF1 in OPM2, OPM2‐PR, and OPM2‐PR cells after treatment with 5‐Aza and EPZ‐6438. All the cells were either untreated or treated with lenalidomide or pomalidomide (10 μm) for 24 h, at which point cell lysates were isolated. The CRBN‐dependent degradation of IKZF1 after treatment with IMiDs is reduced in OPM2‐PR, but is partly rescued in the resensitized OPM2‐PR cells. (C) Apoptosis response in primary IMiD‐resistant cell lines JJN3, LP1, and RPMI‐8226, showing that the combination of 5‐Aza and EPZ‐6438 – or EZH2 inhibition alone (in the case of JJN3), can also overcome intrinsic resistance to IMiDs. **P < 0.01, ***P < 0.001, and ****P < 0.0001.