G9a inhibition leads to induction of apoptosis in MM cells and reduction of tumor growth in vivo. (A) H3K9me2 enrichment in MM.1S MM cells (n = 1238), annotation of significant peak and distribution in genome features and (B) distance between peak to annotated TSSs. (C) RNA-seq data from patients with MM (logFC > 1.5; −log10P > 1.3 compared with tPCs; nMM = 3 and ntPC = 3). Data collected from the BPC. Statistical test: Wald test with Benjamini-Hochberg procedure to control the FDR. (D) GSEA of downregulated genes in patients with MM. (E-F) Overlap between genes enriched for H3K9me2 and downregulated in patients with MM. (G-H) Representative western blot against H3K9me1 and H3K9me2 in INA-6 (G) and OPM2 (H) after 9 days of G9ai treatment. Total histone H4 was used as loading control. Data were collected from 3 biological replicates. Corresponding uncropped western blot can be found in supplemental Figures 9 and 10. (I-J) Flow cytometry analysis of apoptosis markers in G9ai-treated INA-6 (I) and OPM2 MM cells (J) (n(per cell line) = 3). Statistical analysis was performed with 1-way analysis of variance (ANOVA). Values presented with SEM. Representative flow cytometry gating can be found in supplemental Figures 17 and 18. (K) Schematic representation of the experimental setup used for single-agent A366 in vivo treatment. (L) Percent tumor volume in OPM2 tumor-bearing female Balb/c nu/nu CDX mice after treatment with the G9ai A366 over 9 days (nvehicle = 9; nA366 = 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 presented with SEM. (M) Body weight of mice monitored over the 9-day A366 treatment period (nA366 = 9). (N) Representative western blot against H3K9me1/2 levels in tumor lysates collected from mice after 9 days of G9ai treatment. Total histone H4 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 12A. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. CTRL, control; FCm fold change; FDR, false discovery rate; PI, propidium iodide; tPC, tonsillar plasma cell; TSS, transcription start site; UTR, untranslated region. Panel 2K was created with BioRender.com. Nylund P. and Garrido-Zabala B. (2025) https://BioRender.com/q2i0r5z.
Figure 2.

G9a inhibition leads to induction of apoptosis in MM cells and reduction of tumor growth in vivo. (A) H3K9me2 enrichment in MM.1S MM cells (n = 1238), annotation of significant peak and distribution in genome features and (B) distance between peak to annotated TSSs. (C) RNA-seq data from patients with MM (logFC > 1.5; −log10P > 1.3 compared with tPCs; nMM = 3 and ntPC = 3). Data collected from the BPC. Statistical test: Wald test with Benjamini-Hochberg procedure to control the FDR. (D) GSEA of downregulated genes in patients with MM. (E-F) Overlap between genes enriched for H3K9me2 and downregulated in patients with MM. (G-H) Representative western blot against H3K9me1 and H3K9me2 in INA-6 (G) and OPM2 (H) after 9 days of G9ai treatment. Total histone H4 was used as loading control. Data were collected from 3 biological replicates. Corresponding uncropped western blot can be found in supplemental Figures 9 and 10. (I-J) Flow cytometry analysis of apoptosis markers in G9ai-treated INA-6 (I) and OPM2 MM cells (J) (n(per cell line) = 3). Statistical analysis was performed with 1-way analysis of variance (ANOVA). Values presented with SEM. Representative flow cytometry gating can be found in supplemental Figures 17 and 18. (K) Schematic representation of the experimental setup used for single-agent A366 in vivo treatment. (L) Percent tumor volume in OPM2 tumor-bearing female Balb/c nu/nu CDX mice after treatment with the G9ai A366 over 9 days (nvehicle = 9; nA366 = 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 presented with SEM. (M) Body weight of mice monitored over the 9-day A366 treatment period (nA366 = 9). (N) Representative western blot against H3K9me1/2 levels in tumor lysates collected from mice after 9 days of G9ai treatment. Total histone H4 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 12A. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. CTRL, control; FCm fold change; FDR, false discovery rate; PI, propidium iodide; tPC, tonsillar plasma cell; TSS, transcription start site; UTR, untranslated region. Panel 2K was created with BioRender.com. Nylund P. and Garrido-Zabala B. (2025) https://BioRender.com/q2i0r5z.

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