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. 2024 Aug 20;25(16):9022.
doi: 10.3390/ijms25169022.

Molecular Signatures of CB-6644 Inhibition of the RUVBL1/2 Complex in Multiple Myeloma

Weijun Yi  1   2 , Sebastian A Dziadowicz  1 , Rachel S Mangano  1   3 , Lei Wang  1 , Joseph McBee  1 , Steven M Frisch  4 , Lori A Hazlehurst  5   6 , Donald A Adjeroh  2 , Gangqing Hu  1   6
Affiliations

Molecular Signatures of CB-6644 Inhibition of the RUVBL1/2 Complex in Multiple Myeloma

Weijun Yi et al. Int J Mol Sci. .

Abstract

Multiple myeloma is the second most hematological cancer. RUVBL1 and RUVBL2 form a subcomplex of many chromatin remodeling complexes implicated in cancer progression. As an inhibitor specific to the RUVBL1/2 complex, CB-6644 exhibits remarkable anti-tumor activity in xenograft models of Burkitt's lymphoma and multiple myeloma (MM). In this work, we defined transcriptional signatures corresponding to CB-6644 treatment in MM cells and determined underlying epigenetic changes in terms of chromatin accessibility. CB-6644 upregulated biological processes related to interferon response and downregulated those linked to cell proliferation in MM cells. Transcriptional regulator inference identified E2Fs as regulators for downregulated genes and MED1 and MYC as regulators for upregulated genes. CB-6644-induced changes in chromatin accessibility occurred mostly in non-promoter regions. Footprinting analysis identified transcription factors implied in modulating chromatin accessibility in response to CB-6644 treatment, including ATF4/CEBP and IRF4. Lastly, integrative analysis of transcription responses to various chemical compounds of the molecular signature genes from public gene expression data identified CB-5083, a p97 inhibitor, as a synergistic candidate with CB-6644 in MM cells, but experimental validation refuted this hypothesis.

Keywords: CB-6644; RUVBL1/2; molecular signatures; multiple myeloma.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Clinical relevance of RUVBL1 expression in MM patients. (A) Comparison of RUVBL1 level between healthy donors and patients at disease stages of MGUS and smoldering. p-value by t-test. (B) Comparison of RUVBL1 level across patients at disease stages of MGUS, MM, and PCL. (C) K–M survival plot for RUVBL1 for ND MM patients from the CoMMpass trial. Patients sorted into the top 25% and others based on RUVBL1 expression (also applied to panels D–G). p-value by log-rank test. (D) K–M survival plot for RUVBL1 for ND MM patients from MAPQ-II (GEO: GSE24080); (E) K–M survival plot for RUVBL1 for ND MM patients from TT2 (GEO: GSE4204); (F) K–M survival plot for RUVBL1 for relapse patients from APEX/SUMMIT (GEO: GSE9782); (G) K–M survival plot for RUVBL1 for previously treated MM patients from TT6 (GEO: GSE57317).
Figure 2
Figure 2
Proliferation suppression by CB-6644 in MM.1S. (A) IC50 of CB-6644 in MM.1S cells measured by CellTiter-Glo® Luminescent Cell Viability Assay (n = 3). (B) Bar graph for the % of AV and PI double-positive cells of MM.1S following 72 h treatment with 120 nM CB-6644 (n = 3). ****: p-value < 0.0001 (t-test). (C,D) Representative flow panels of panel B. Live cells: lower left quadrant; apoptotic cells: lower right quadrant; dead cells: upper right quadrant. Red to blue means higher to lower density. AV: annexin V. PI: propidium iodide.
Figure 3
Figure 3
Molecular pathways affected by CB-6644 in MM cells. (A) MA plot displaying count per million (CPM; log2) and fold change (FC) of expression for the comparisons of CB-6644-treated cells (n = 2) vs. DMSO control cells (n = 3) in MM.1S. Blue: Genes upregulated in expression. Red: Downregulated genes. Light blue: All expressed genes. Shown for data generated in MM.1S, also applying to panels (B,C). (B) Gene ontology enrichment analysis on biological processes for the upregulated (left panel) or downregulated genes (right panel). (C) GSEA of expressed genes sorted by fold change in expression (CB-6644/DMSO) from high (left) to low (right) against MSigDB hallmark gene set “interferon γ response” (blue line) and “E2F targets” (red line). (D) Bubble plot visualization of results from GSEA enrichment analysis of CB-6644-induced expression changes in different cell systems (rows) against MSigDB hallmark gene sets (columns). Color indicates an overall upregulation (blue) or downregulation (red) of the gene set. Circle size indicates significance (FDR q-value). NES: normalized enrichment score. (E) Scatter plot visualization of the significance of transcriptional regulator inference from LISA for genes commonly upregulated (upper panel) or downregulated (lower panel) by CB-6644 in MM.1S and RPMI 8226. In parentheses are cells or tissues where the public ChIP-seq data are sourced from.
Figure 4
Figure 4
CB-6644 induced transformation of accessible chromatin in MM cells. (A) MA plots displaying CPM (log2) and FC of chromatin accessibility for CB-6644 treated (n = 3) vs. DMSO control (n = 3) MM cells. Blue: chromatin-accessible regions increasing in accessibility (“Incr”); red: chromatin-accessible regions decreasing in accessibility (“Decr”); light blue: all chromatin-accessible regions. (B) Distribution in promoter (transcription start sites ±2500 bps; Pro) and non-promoter regions (“Non-pro”) for chromatin-accessible regions sorted by their changes in response to CB-6644 treatment: “I” for increasing, “D” for decreasing, and “N” for no change. (C) IGV genome browser image showing the distribution of Omni-ATAC read density across a genomic region enclosing DUSP22 and IRF4 for samples treated with CB-6644 (blue) or DMSO (red). Y-axis normalized by total library size and adjusted to the same scales. Highlighted in yellow are genomic regions showing a decrease in chromatin accessibility. (D) Volcano plots for differential binding activity vs. the -log10 (p-value) for all TF motifs (dots) from JASPAR. Highlighted DMSO-specific TFs are labeled in red, while CB-6644-specific factors are in blue. (E) Bias-corrected Tn5 signals indicating chromatin accessibility centered on motifs corresponding to ATF4::CEBP (left panel) and IRF4 binding (right panel) for CB-6644-treated cells and DMSO control cells.
Figure 5
Figure 5
Genes associated with a decrease in chromatin accessibility at multiple regulatory regions. (A) IGV genome browser image showing the distribution of Omni-ATAC read density across a genomic region enclosing CDK6 for samples treated with CB-6644 (blue) or DMSO control (red). Highlighted are genomic regions showing a decrease in chromatin accessibility. (B) Empirical cumulative distribution of the expression FC (CB-6644/DMSO) of genes associated with multiple genomic regions that increased (red) or decreased (blue) in chromatin accessibility. Black: all expressed genes. A line shifting to the right indicates an overall increase in expression. p-value by the Kolmogorov–Smirnov (K-S) test. (C) KEGG pathway enrichment analysis for genes associated with multiple genomic regions that increased (blue) or decreased (brown) in chromatin accessibility.
Figure 6
Figure 6
Inference of synergistic compounds with CB-6644 through integrative gene expression analysis. (A) Bubble plot visualization of results from GSEA enrichment analysis of expression changes induced by various chemical compounds in MM cells, as collected from GEO with GSE# indicated in parentheses, against gene sets (rows) that are commonly upregulated or downregulated by CB-6644 in MM.1S and RPMI 8226. Color indicates expression upregulation (blue) or downregulation (red) of the gene sets by the indicated compounds (columns). Circle size indicates FDR q-value. Box: GSEA results detailed in panel B. (B) GSEA of expressed genes sorted by expression FC in response to CB-5083 from high (red spectrum) to low (blue spectrum) against gene sets downregulated or upregulated by CB-6644. (C) % of PI and AV double-positive cells from treatment with 120 nM CB-6644 combined with varying concentrations of CB-5083 in MM.1S cells for 72 h, relative to the basal effect of CB-5083 alone or DMSO alone. *: p < 0.05. ** p < 0.005 (t-test). ns: not significant. n = 3 for each condition. Red and blue arrows correspond to the examples shown in panels (D,E), respectively. (D) Representative flow panel for the % of PI and AV double-positive cells, indicated as the red arrowhead in panel C from CB-6644 treatment (120 nM) relative to DMSO alone (19.43–3.51%). Red to blue means higher to lower density. (E) Representative flow panel for the % of PI and AV double-positive cells, indicated as the blue arrowhead in panel C from the combined treatment of CB-6644 (120 nM) and CB-5083 (200 nM) relative to CB-5083 alone (200 nM) (9.30–3.66%). Live cells: lower left quadrant; apoptotic cells: lower right quadrant; dead cells: upper right quadrant.
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