THE METHIONINE CYCLE AS A NOVEL THERAPEUTIC TARGET IN NPM1C DRIVEN ACUTE MYELOID LEUKEMIA

Acute myeloid leukemia (AML) is the most common leukemia in adults and its prognosis is usually poor. Patients are still treated with conventional chemotherapy, which induces disease cure in <40% of patients. The main culprit of therapy failure is the genomic and biological heterogeneity of the disease and the lack of specific targeting-approaches. NPM1 is the most frequently mutated gene in AMLs (~30% of cases). Though the role of the NPM1 mutation (NPM1c) in the maintenance of the leukemic state is well characterized, little is known about underlying molecular mechanisms of oncogenesis. Most notably, the mutation causes an aberrant cytoplasmic localization of the protein, which is otherwise a nucleus-cytoplasmic protein mainly localized in the nucleolus and in the nucleoplasm. Unpublished data generated in our lab have shown that ectopic expression of NPM1c in Murine Embryonic Fibroblasts (MEFs) leads to an unbalanced methionine-cycle with decreased ratio between S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), key metabolites of the methionine cycle. Decreased SAM/SAH ratio is known to inhibit cellular methyltransferases activity. Consistently, the majority of NPM1c regulated genes (e.g. HoxA cluster) and enhancers in MEF cells result markedly hypomethylated and overexpressed or active, respectively. Here, we show that SAM exogenous supplementation in NPM1c-MEF cells increases DNA methylation at specific loci and reverts significantly expression and activity of NPM1c-targeted promoters and enhancers. We then investigated the effects of SAM supplementation in NPM1c-AML blasts, and demonstrated that SAM reverts the NPM1c transcriptional signature, promotes monocytic differentiation and cell death in vitro and inhibits AML growth in vivo, suggesting that restoration of a physiological SAM/SAH ratio reverts the malignant phenotype of NPM1c-AMLs. Using engineered AML cells that allow degradation of NPM1c or its ectopic expression, we demonstrated that NPM1c is directly responsible for: i) the cytoplasmic mis-localization of Methionine Adenosyltransferase 2A (MAT2A), the key enzyme that catalyzes SAM production, leading to SAM/SAH ratio unbalance; ii) the SAM/SAH ratio unbalancing; and iii) the sensitivity to SAM treatment. To explore alternative approaches to increase the SAM/SAH ratio, we used Metformin, known to activate adenosylhomocysteinase (SAHH) and reduce SAH levels, and showed that it synergizes with SAM, inhibits growth and promotes monocytic differentiation of NPM1c-AML growth in vitro. In summary, in this PhD project, we demonstrate that the SAM/SAH unbalance is at the basis of the NPM1c-dependency of AML blasts, thus paving the way for the therapeutic use of SAM as specific targeting approach in NPM1c-AMLs.