Targeting MAT2A alters the methylation landscape and DNA damage response in Multiple Myeloma, revealing novel combination strategies.

Objectives
Increasing evidence supports a role for epigenetic defects in the disease progression and relapse of the plasma cell malignancy multiple myeloma (MM). However, the mechanisms driving this epigenetic shift in MM have yet to be fully elucidated. The metabolic enzyme methionine adenosyltransferase 2A (MAT2A) is responsible for S-adenosyl methionine (SAM) biosynthesis, which is the main methyl donor for enzymes catalyzing methylation processes. Previously, we showed that high MAT2A expression in MM cells is associated with poor overall survival and MAT2A inhibition using siRNA or the small molecule inhibitor FIDAS-5 reduces MM cell proliferation and survival by inhibiting mTOR-mediated protein synthesis. Moreover, FIDAS-5 was able to improve bortezomib-based treatment. As the producer of SAM, we here aimed to further unravel the underlying mechanisms of action of targeting MAT2A by investigating the impact on the methylation landscape in MM cells.
Methods
We established MAT2A knockdown (KD) cell lines (OPM-2 and AMO-1) using doxycycline inducible lentiviral vectors containing a shRNA against MAT2A. KD efficiency was evaluated on mRNA and protein level using qRT-PCR and Western blot, respectively. In addition, the effect on cell viability, cell cycle, and apoptosis was measured by a CellTiter Glo assay, AnnexinV/7AAD and BrdU stainings, respectively. The impact of MAT2A depletion on DNA methylation and histone methylation was evaluated using Enzymatic Methyl sequencing (EM-seq) and western blot, while the impact on the transcriptome was determined by RNA sequencing (RNA-seq).
Results
Doxycycline inducible stable KD of MAT2A in MM cells using shRNA confirmed the tumor promoting role of MAT2A. Consistent with our previous findings, conditional MAT2A KD resulted in a clear decrease in MM cell viability and growth and a strong increase in apoptosis. Moreover cell cycle analysis confirmed that MAT2A depletion in MM cells results in a G1 phase arrest. On a mechanistic level, MAT2A-depleted cells showed a clear decrease in global DNA methylation levels and significant reduction in the global levels of several repressive histone methylation marks. Gene set enrichment of the differentially methylated regions revealed significant enrichment of genes involved in epithelial-mesenchymal transition and apoptosis. Moreover, RNA-seq upon MAT2A KD identified 449 genes upregulated and 493 genes downregulated, with the genes/pathways affected mainly involved in interferon responses, EF2 targets, MYC targets V1, G2M checkpoint and mTORC1 signalling. In addition, Gene ontology (Go) enrichment analysis revealed ten enriched biological processes related to “DNA replication” and ‘Mitosis’. Regarding its molecular function, only six GO terms were significantly enriched, and the top-ranked molecular function was “four-way junction DNA binding”. Finally, key vulnerabilities were found in DNA damage response mechanisms, resulting in synergistic anti-myeloma activity of the novel MAT2A inhibitor AG-270 in combination with either the DNA damaging agent melphalan or the PRMT5 inhibitor EPZ015938.
Conclusion
In conclusion, our research highlights that targeting MAT2A in MM cells reverts aberrant methylation processes and impairs several pro-tumoral signalling pathways, offering promising avenues for developing more effective combination treatments for MM.