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[Preprint]. 2024 Apr 8:2024.03.26.586811.
doi: 10.1101/2024.03.26.586811.

The SAGA acetyltransferase module is required for the maintenance of MAF and MYC oncogenic gene expression programs in multiple myeloma

Ying-Jiun C Chen  1   2 , Govinal Badiger Bhaskara  1   2 , Yue Lu  1   2 , Kevin Lin  1   2 , Sharon Y R Dent  1   2
Affiliations

The SAGA acetyltransferase module is required for the maintenance of MAF and MYC oncogenic gene expression programs in multiple myeloma

Ying-Jiun C Chen et al. bioRxiv. .

Update in

Abstract

Despite recent advances in therapeutic treatments, multiple myeloma (MM) remains an incurable malignancy. Epigenetic factors contribute to the initiation, progression, relapse, and clonal heterogeneity in MM, but our knowledge on epigenetic mechanisms underlying MM development is far from complete. The SAGA complex serves as a coactivator in transcription and catalyzes acetylation and deubiquitylation. Analyses of datasets in the Cancer Dependency Map Project revealed many SAGA components are selective dependencies in MM. To define SAGA-specific functions, we focused on ADA2B, the only subunit in the lysine acetyltransferase (KAT) module that specifically functions in SAGA. Integration of RNA-seq, ATAC-seq, and CUT&RUN results identified pathways directly regulated by ADA2B include MTORC1 signaling, MYC, E2F, and MM-specific MAF oncogenic programs. We discovered that ADA2B is recruited to MAF and MYC gene _targets, and that MAF shares a majority of its _targets with MYC in MM cells. Furthermore, we found the SANT domain of ADA2B is required for interaction with both GCN5 and PCAF acetyltransferases, incorporation into SAGA, and ADA2B protein stability. Our findings uncover previously unknown SAGA KAT module-dependent mechanisms controlling MM cell growth, revealing a vulnerability that might be exploited for future development of MM therapy.

Keywords: ADA2B; MAF; MYC; SAGA complex; SANT domain; multiple myeloma; oncogenic gene expression programs.

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Conflict of interest statement

Conflicts of Interest The authors declare no competing interests to disclose.

Figures

Figure 1.
Figure 1.. SAGA-specific ADA2B is a dependency in MM.
A) Components of the SAGA complex. Lysine acetyltransferase (KAT), deubiquitinase (DUB), splicing, core, and transcription factor (TF)-binding modules are shown in pink, yellow, purple, green, and blue, respectively. Components specific to SAGA are indicated in red. Relative positions of components are illustrated based on the previously reported cryogenic-electron microscopy structure of human SAGA (Herbst et al. 2021). B) The mean values of dependency scores and expression of ADA2B across cancer cell lines derived from various lineages found in CRISPR DepMap and Sanger (Score) databases (DepMap 23Q2+ Score, Chronos). C) Top twenty co-dependencies of ADA2B across the same cancer cell lines as in panel B. Components specific to SAGA complex are indicated in red. SAGA KAT module components are indicated in pink. D) Dependency scores of SAGA components in KAT, core, and DUB modules for multiple myeloma cell lines. E) Cell viability assay of Dox-inducible shRNA lines. A one-way Analysis of Variance (ANOVA) was performed to analyze the differences among group means, followed by the Tukey HSD post hoc test to determine whether the mean difference between specific pairs of group are statistically significant. * p < 0.05, not significant (NS) p > 0.05.
Figure 2.
Figure 2.. ADA2B is required for maintenance of oncogenic gene expression programs.
A) Immunoblots showing levels of ADA2B, GCN5, PCAF, and H3K9ac in shNT and shADA2B cells with and without Dox treatment. Histone H3 is included as a loading control. B) Volcano plot of gene expression changes detected by RNA-seq in shADA2B cells relative to shNT cells after 5 days of Dox treatment. Genes with statistically significant differential expression with fold change (FC) ≥ 1.5 include 474 downregulated genes and 502 upregulated genes. C) Top five gene expression pathways identified by GSEA that were enriched in downregulated genes in shADA2B cells relative to shNT cells after 3 and 5 days of Dox treatment. D) Enrichment plots for E2F _targets, MYC _targets v1, and G2/M checkpoint hallmarks. E) Volcano plot of chromatin accessibility changes detected by ATAC-seq in shADA2B cells relative to shNT cells after 5 days of Dox treatment. Statistically significant differential peaks with FC ≥ 1.5 include 5230 decreased peaks and 2357 increased peaks. F) Top five TF motifs enriched in decreased ATAC-seq peaks in shADA2B cells relative to shNT cells after 5 days of Dox treatment. G) Average RPKM (reads per kilobase per million) normalized signal of ATAC-seq peaks associated to all (1688) downregulated genes in shADA2B cells relative to shNT cells after 5 days of Dox treatment. H) Venn diagram depicting the overlap between downregulated genes (1688 genes) and genes with decreased accessibility (8959 genes) in shADA2B cells relative to shNT cells after 5 days of Dox treatment.
Figure 3.
Figure 3.. ADA2B is recruited to promoters of genes involved in tumorigenesis.
A) Heatmaps of FLAG-3xHA-ADA2B peaks, H3K9ac peaks in cells expressing FLAG-3xHA, ATAC-seq peaks in Dox-treated shNT and shADA2B cells, and changes of ATAC-seq peak signals in shADA2B cells relative to shNT cells after 5 days of Dox treatment. Decreased peaks are shown in blue; increased peaks are shown in red. Regions are ranked based on ADA2B signal. FLAG-3xHA-ADA2B signals are normalized to IgG signals. ChIP-seq tracks of MAF, MYC, and Pol II represent FC over input. Metaplots show average normalized signal density for FLAG-3xHA-ADA2B, H3K9ac, and changes in ATAC-seq peaks. B) Pie chart depicting genomic distribution of FLAG-3xHA-ADA2B, H3K9ac in cells expressing FLAG-3xHA, and decreased ATAC-seq peaks in shADA2B cells relative to shNT cells after 5 days of Dox treatment. C) Bar chart showing quantity of decreased and increased ATAC-seq peaks associated to ADA2B-bound genes in shADA2B cells relative to shNT cells after 3 and 5 days of Dox treatment. D) Venn diagram depicting the overlap between ADA2B promoter-bound genes (3169 genes) and downregulated genes (1688 genes) in shADA2B cells relative to shNT cells after 5 days of Dox treatment, identifying 356 ADA2B core _targets. ADA2B core _targets include more than 40 genes involved in cell cycle and cell division, 23 genes in MTORC1 signaling, and known regulators of MM biology. E) Top five upstream regulators of ADA2B core _targets identified by IPA. F) Top five canonical pathways of ADA2B core _targets identified by IPA. G) Cell cycle analysis of sgADA2B and control sgLacZ cells. The percentage of events in each cell cycle stage for three biological replicates is graphed. A one-way Analysis of Variance (ANOVA) was performed to analyze the differences among group means, followed by the Tukey HSD post hoc test to determine whether the mean difference between specific pairs of group are statistically significant. ** p < 0.01, * p < 0.05, not significant (NS) p > 0.05.
Figure 4.
Figure 4.. ADA2B promotes MAF protein levels and MAF expression program.
A) Immunoblots showing levels of MYC, E2F1, and MAF in shNT and shADA2B cells with and without Dox treatment. b-Tub is included as a loading control. B) Transcript levels in FPKM (fragments per kilobase per million) of MAF in shNT and shADA2B cells with and without Dox treatment as determined by RNA-seq. C) Effects of CPTH6 treatment on MM.1S cells. Left: immunoblots showing MAF, MYC, GCN5, PCAF, ADA2B levels in MM.1S cells treated with 10 μM CPTH6 for 3 days in comparison to DMSO vehicle control. Histone H3 is included as a loading control. Right: Cell viability assay of MM.1S cells treated with 10 μM CPTH6. A one-way Analysis of Variance (ANOVA) was performed to analyze the differences among group means, followed by the Tukey HSD post hoc test to determine whether the mean difference between specific pairs of group are statistically significant. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, not significant (NS) p > 0.05. D) Immunoblots of MM.1S cells with or without 10 μM CPTH6 treatment for 3 days and subsequent treatment of DMSO or 10 μM MG132 for 5 hours. Histone H3 is included as a loading control. E) GSEA enrichment plots demonstrating enrichment of MAF gene signature from Zhan et al. 2006 (Zhan multiple myeloma MF up) in the genome-wide expression changes induced by Dox in shADA2B cells compared to shNT cells for 3 and 5 days. F) Heatmap showing expression of selected MAF core _targets in shADA2B cells compared to shNT cells after 3 and 5 days of Dox treatment. Genes directly bound by ADA2B as determined by our CUT&RUN data are highlighted in green.
Figure 5.
Figure 5.. ADA2B directly binds to MAF and MYC _targets.
A) Heatmaps of FLAG-3xHA-ADA2B, MAF, and MYC binding peaks from our CUT&RUN analysis for ADA2B and from published ChIP-seq datasets for MAF and MYC (Lin et al. 2012; Katsarou et al. 2023). Regions are ranked based on ADA2B signal. FLAG-3xHA-ADA2B signals are normalized to IgG signals. ChIP-seq tracks of MAF, MYC, and Pol II represent FC over input. Metaplots show average normalized signal density for FLAG-3xHA-ADA2B, MAF, and MYC peaks. B) Venn diagram depicting the overlaps between ADA2B, MAF, and MYC binding sites (peaks). C) Heatmaps of FLAG-3xHA-ADA2B, MAF, MYC, and ATAC-seq peaks at ADA2B, MAF, and MYC shared binding sites and ADA2B and MYC shared binding sites. FLAG-3xHA-ADA2B signals are normalized to IgG signals. ChIP-seq tracks of MAF, MYC, and Pol II represent FC over input. Changes of ATAC-seq peak signals in shADA2B cells relative to shNT cells after 5 days of Dox treatment are also shown, with decreased peaks are in blue and increased peaks in red. D) Metaplots of FLAG-3xHA-ADA2B, MAF, MYC, and changes of ATAC-seq peaks shown in panel C and Supplemental Figure S6D. E) Binding patterns of FLAG-3xHA-ADA2B, MAF, MYC, Pol II, and H3K9ac at CCND2 locus. Peak signals from ATAC-seq and RNA-seq in shADA2B and shNT cells after 5 days of Dox treatment are also shown. ChIP-seq tracks of MAF, MYC, and Pol II represent FC over input.
Figure 6.
Figure 6.. SANT domain of ADA2B is required for GCN5/PCAF interaction, SAGA incorporation, and ADA2B protein stability.
A) Mapping of CRISPR KO hyper-sensitive regions in ADA2B protein by ProTiler (He et al. 2019a). Higher SVM scores indicate higher essentiality. Illustration of domains in ADA2B protein is shown at the top. B) Sequence of the SANT domain in ADA2B. The three predicted core hydrophobic residues (W70, W90, Y110) are highlighted in green. C) Structure of the SANT domain in ADA2B as predicted by AlphaFold (Jumper et al. 2021; Varadi et al. 2021). The three predicted core hydrophobic residues (W70, W90, Y110) are highlighted in green. AlphaFold generates a per-residue confidence score (pLDDT) ranging from 0 to 100. D) Relative RNA expression of ADA2B, including endogenous and exogenous levels, in MM.1S cell lines expressing FLAG-3xHA, FLAG-3xHA-ADA2B, FLAG-3xHA-ADA2BW70A W90A Y110A, or FLAG-3xHA-ADA2BW70F W90F Y110F as determined by qRT-PCR. A one-way Analysis of Variance (ANOVA) was performed to analyze the differences among group means, followed by the Tukey HSD post hoc test to determine whether the mean difference between specific pairs of group are statistically significant. Not significant (NS) p > 0.05. E) Immunoblots showing levels of FLAG-3xHA-ADA2B and endogenous ADA2B in the same cell lines as in panel C. Histone H3 blot and Ponceau S staining are included as loading controls. F) Immunoblots showing co-IP results for FLAG-3xHA, FLAG-3xHA-ADA2B, FLAG-3xHA-ADA2BW70A W90A Y110A, and FLAG-3xHA-ADA2BW70F W90F Y110F using the same cell lines as in panels C and D. GAPDH is included as a loading control in input samples.
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