MAT2A as key regulator and therapeutic target in MLL leukemogenesis

• Applicant: Privatdozentin Corina Schneidawind, Ph.D.
• Subject Area: Hematology, Oncology
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 442788847

The MLL proto-oncogene is a frequent target for chromosomal translocations in a diverse subset of leukemias, which are associated with a poor prognosis despite aggressive therapeutic regimens. Thus, new targeted therapeutic strategies are urgently needed. Epigenetic dysregulation plays a pivotal role in MLL pathogenesis and its mechanistic understanding paves the avenue for the development of new targeted therapies: posttranslational modifications by methylation of histones or promoter regions alter gene expression activity that in turn trigger leukemogenesis. SAM is the universal methyl donor in human cells and is therefore utilized as substrate for different methyltransferases like DOT1L or PRMT5 essential for MLL leukemogenesis. In turn, SAM is synthesized by MAT2A in a reaction using ATP and methionine. MAT2A is already known to be deregulated in different cancer types including T-cell leukemia and colon cancer. Recently, a new inhibitor of MAT2A named PF-9366 has been developed allowing to evaluate the role of MAT2A in MLL leukemogenesis and as potential therapeutic target. For that purpose, we are using our very recently published innovative CRISPR/Cas9-MLLr model based on complete translocations of the MLL and AF4 or AF9 gene, respectively. AF4 and AF9 account for the two most frequent fusion partners in MLL leukemia in children and adults, respectively. Our model is focused on distinct driver translocations leading to immortalization of the MLLr cells already in in vitro culture systems. By reanalyzing publicly available patient data, we were able to find a significantly elevated MAT2A expression in MLLr leukemias compared to other tumor entities, non-MLLr leukemias and healthy controls. In our well established MLLr model, we could confirm the high expression of MAT2A by qPCR amenable to investigate the impact of MAT2A in the MLL leukemogenesis. Following inhibitor treatment with PF-9366 in vitro, the cells will be functionally analyzed: proliferation, apoptosis/necrosis, cell cycle, differentiation, cell lysis, formation of colonies and replating capacity, expression of MLL target genes as well as synergistic anti-tumoral effects in combination with methotrexate or inhibitors against PRMT5 and DOT1L. The anti-leukemic effect of PF-9366 on leukemia initiation and survival will be validated in a pretreatment mouse xenograft model. Finally, PF-9366 will be administered in vivo following transplantation of the CRISPR/Cas9-MLLr cells in immunocompromised mice to evaluate its impact on survival. To understand the underlying pathomechanism of MAT2A, we will also analyze the CRISPR/Cas9-MLLr cells upon PF-9366 treatment regarding open chromatin,methylation status and transcriptome to investigate the mechanistic role of MAT2A and to gain insights into known and novel gene signatures driving MLL leukemogenesis. Our study paves the avenue for clinical application of PF-9366 to improve the treatment of a poor prognosis subset of leukemia.

• DFG Programme: Research Grants