Aberrant DNA methylation profiles were observed in patients with MM, and treatment with DNMT1i induces apoptosis in MM cells and reduces tumor growth in vivo. (A) Characterization of methylated CpGs in patients with MM compared with BMPCs. (B) Differentially methylated CpGs in patients with MM compared with BMPCs (patients with potentially hypermethylated genome in black rectangle). (C) Average distribution of CpGs that gain methylation in MM compared with BMPCs. Values are presented with SEM. (D-E) Overlap of CpGs that gain methylation and downregulated genes in patients with MM. (F) Average distribution of CpGs that lose methylation in AZA-treated INA-6 MM cells. Statistical analysis was performed with multiple t tests, corrected for multiple comparisons with 2-stage step-up (Benjamini, Krieger, and Yekutieli) by FDR. Values are presented with SEM. (G) GSEA of DNMTs target genes in INA-6 MM cells. (H-I) Representative western blot against DNMT1 in INA-6 (H) and OPM2 (I) after DNMTi treatment. Actin was used as loading control. Data were collected from 3 biological replicates. Corresponding uncropped western blot can be found in supplemental Figure 11. (J-K) Flow cytometry analysis of apoptosis markers in DNMTi-treated INA-6 (J) and OPM2 MM cells (n(per cell line) = 3) (K). Representative flow cytometry gating can be found in supplemental Figure 21. Statistical analysis was performed with 1-way ANOVA. Values are presented with SEM. (L) Schematic representation of the experimental setup used for single-agent DAC in vivo procedure. (M) Percent tumor volume in OPM2 tumor-bearing female Balb/c nu/nu CDX mice after treatment with the DNMT inhibitor DAC for 5 days (nvehicle = 9; nDAC = 9). Statistical analysis: a linear mixed-effects model was fitted to account for random variation across replicates, followed by pairwise comparisons using Fisher least significant difference test. Comparisons were specifically performed between the vehicle group and each treatment dosage. Values are presented with SEM. (N) Body weight of mice monitored over the 9-day DAC treatment period (nDAC = 9). (O) Representative western blot against DNMT1 levels in tumor lysates collected from mice after 5 days of DNMTi treatment. Actin was used as loading control. Data were collected from 3 biological replicates per treatment group. Corresponding uncropped western blot can be found in supplemental Figure 12B. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. Avg, average; CTRL, control; Diff., difference; DM, differentially methylated. Panel 3L was created with BioRender.com. Nylund P. and Garrido-Zabala B. (2025) https://BioRender.com/q2i0r5z.

Aberrant DNA methylation profiles were observed in patients with MM, and treatment with DNMT1i induces apoptosis in MM cells and reduces tumor growth in vivo. (A) Characterization of methylated CpGs in patients with MM compared with BMPCs. (B) Differentially methylated CpGs in patients with MM compared with BMPCs (patients with potentially hypermethylated genome in black rectangle). (C) Average distribution of CpGs that gain methylation in MM compared with BMPCs. Values are presented with SEM. (D-E) Overlap of CpGs that gain methylation and downregulated genes in patients with MM. (F) Average distribution of CpGs that lose methylation in AZA-treated INA-6 MM cells. Statistical analysis was performed with multiple t tests, corrected for multiple comparisons with 2-stage step-up (Benjamini, Krieger, and Yekutieli) by FDR. Values are presented with SEM. (G) GSEA of DNMTs target genes in INA-6 MM cells. (H-I) Representative western blot against DNMT1 in INA-6 (H) and OPM2 (I) after DNMTi treatment. Actin was used as loading control. Data were collected from 3 biological replicates. Corresponding uncropped western blot can be found in supplemental Figure 11. (J-K) Flow cytometry analysis of apoptosis markers in DNMTi-treated INA-6 (J) and OPM2 MM cells (n(per cell line) = 3) (K). Representative flow cytometry gating can be found in supplemental Figure 21. Statistical analysis was performed with 1-way ANOVA. Values are presented with SEM. (L) Schematic representation of the experimental setup used for single-agent DAC in vivo procedure. (M) Percent tumor volume in OPM2 tumor-bearing female Balb/c nu/nu CDX mice after treatment with the DNMT inhibitor DAC for 5 days (nvehicle = 9; nDAC = 9). Statistical analysis: a linear mixed-effects model was fitted to account for random variation across replicates, followed by pairwise comparisons using Fisher least significant difference test. Comparisons were specifically performed between the vehicle group and each treatment dosage. Values are presented with SEM. (N) Body weight of mice monitored over the 9-day DAC treatment period (nDAC = 9). (O) Representative western blot against DNMT1 levels in tumor lysates collected from mice after 5 days of DNMTi treatment. Actin was used as loading control. Data were collected from 3 biological replicates per treatment group. Corresponding uncropped western blot can be found in supplemental Figure 12B. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. Avg, average; CTRL, control; Diff., difference; DM, differentially methylated. Panel 3L was created with BioRender.com. Nylund P. and Garrido-Zabala B. (2025) https://BioRender.com/q2i0r5z.

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