Combinatorial inhibition of G9a and DNMTs demonstrates an enhanced effect on cell viability, induces apoptosis, and reduces the abundance of MM-associated oncogenes. (A-B) Normalized expression of PAX5 and DCN in MGUS and MM compared with NPC, analyzed with GEO2R (GSE6477). Statistical analysis was performed with Kruskal-Wallis test. Values presented with SEM. (C-D) Normalized expression of PLEKHO1 and DENND2D in MGUS, MM, and rMM compared with NPC, analyzed with GEO2R (GSE6477). Statistical analysis was performed with Kruskal-Wallis test. Values presented with SEM. (E-F) Selection of normalized expression of (MAS5) IPO13 and PTPRT genes from CD138+ normal BMPCs, MGUS, patients with smMM, MM, and PCL (U133 Plus 2.0; Affymetrix GSE5900). Statistical analysis was performed with 1-way ANOVA in panel E and Kruskal-Wallis test in panel F. Values are presented with SEM. (G-K) Normalized (MAS5) PAX5, DCN, PLEKHO1, IPO13, and PTPRT expression-based survival data from BM-collected CD138+ from patients with MM (GSE9782). Statistical analysis was performed with log-rank test. (L-O) Response-based (nnon-response = 126; nresponse = 113) normalized expression of (MAS5) GATA4, DCN, PLEKHO1, and IPO13 genes from BM samples of patients with myeloma before treatment with bortezomib or dexamethasone. Statistical analysis was performed with Student t test in panels G,J and Mann-Whitney U test in panels H-I. Values are presented with SEM. (P) Representative western blot against H3K9me2, DNMT1, G9a, c-Myc, IRF4, and ɣH2AX. Actin and total histone H4 were used as loading controls. Data were collected from 3 biological replicates. The corresponding uncropped western blot figures can be found in supplemental Figure 13. (Q-S) Flow cytometry analysis of apoptosis markers on single- and combinatorially treated MM cell lines INA-6 (Q), OPM2 (R), and KMS-28PE (n = 3) (S). Statistical analysis was performed with 1-way ANOVA. Values are presented with SEM. Representative flow cytometry gating can be found in supplemental Figures 24, 26, and 28. (T) A-366 and AZA Bliss scores calculated from the effect of the drug combination on viability in INA-6 cells. Bliss score >0 indicates synergy. (U) GSEA shows gain of expression of genes associated with apoptosis, which become upregulated only with the combinatorial therapy in INA-6 cells. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. Ctrl, control; NPC, normal plasma cell; NR, nonresponsive; R, responsive; rMM, relapsed MM.
Figure 6.

Combinatorial inhibition of G9a and DNMTs demonstrates an enhanced effect on cell viability, induces apoptosis, and reduces the abundance of MM-associated oncogenes. (A-B) Normalized expression of PAX5 and DCN in MGUS and MM compared with NPC, analyzed with GEO2R (GSE6477). Statistical analysis was performed with Kruskal-Wallis test. Values presented with SEM. (C-D) Normalized expression of PLEKHO1 and DENND2D in MGUS, MM, and rMM compared with NPC, analyzed with GEO2R (GSE6477). Statistical analysis was performed with Kruskal-Wallis test. Values presented with SEM. (E-F) Selection of normalized expression of (MAS5) IPO13 and PTPRT genes from CD138+ normal BMPCs, MGUS, patients with smMM, MM, and PCL (U133 Plus 2.0; Affymetrix GSE5900). Statistical analysis was performed with 1-way ANOVA in panel E and Kruskal-Wallis test in panel F. Values are presented with SEM. (G-K) Normalized (MAS5) PAX5, DCN, PLEKHO1, IPO13, and PTPRT expression-based survival data from BM-collected CD138+ from patients with MM (GSE9782). Statistical analysis was performed with log-rank test. (L-O) Response-based (nnon-response = 126; nresponse = 113) normalized expression of (MAS5) GATA4, DCN, PLEKHO1, and IPO13 genes from BM samples of patients with myeloma before treatment with bortezomib or dexamethasone. Statistical analysis was performed with Student t test in panels G,J and Mann-Whitney U test in panels H-I. Values are presented with SEM. (P) Representative western blot against H3K9me2, DNMT1, G9a, c-Myc, IRF4, and ɣH2AX. Actin and total histone H4 were used as loading controls. Data were collected from 3 biological replicates. The corresponding uncropped western blot figures can be found in supplemental Figure 13. (Q-S) Flow cytometry analysis of apoptosis markers on single- and combinatorially treated MM cell lines INA-6 (Q), OPM2 (R), and KMS-28PE (n = 3) (S). Statistical analysis was performed with 1-way ANOVA. Values are presented with SEM. Representative flow cytometry gating can be found in supplemental Figures 24, 26, and 28. (T) A-366 and AZA Bliss scores calculated from the effect of the drug combination on viability in INA-6 cells. Bliss score >0 indicates synergy. (U) GSEA shows gain of expression of genes associated with apoptosis, which become upregulated only with the combinatorial therapy in INA-6 cells. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. Ctrl, control; NPC, normal plasma cell; NR, nonresponsive; R, responsive; rMM, relapsed MM.

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