Targeting Folate Metabolism In Acute Myelogenous Leukemia

There is increasing evidence that deranged metabolism is an important mechanism of cancer pathogenesis. We conducted multiple genomic analyses of publicly available acute myelogenous leukemia (AML) data sets that revealed a critical role for one carbon and nucleotide metabolism, particularly mitochondrial, in a subset of AML samples. One carbon metabolism is a complex series of pathways involving several amino acids, the synthesis of purines, thymidylate, S-adenosylmethionine, and the support of cellular methylation reactions. SHMT2, MTHFD2, and MTHFD1L are the major enzymes functional in the one carbon folate pathway in the mitochondria. MTHFD2 is a NAD-dependent, mitochondrial methylenetetrahydrofolate dehydrogenase and cyclohydrolase, derived from a similar trifunctional cytoplasmic protein. In the mitochondria, the formyltetrahydrofolate synthetase activity is performed by MTHFD1L. We noted that these enzymes are downregulated with suppression of MYC. Gene set enrichment analysis (GSEA) of cell lines treated with JQ1, a small molecule BET bromodomain inhibitor which suppresses MYC, showed a significant enrichment in genes of the one carbon pool by folate KEGG pathway. We show that treatment of AML cells with JQ1 causes a decrease in MTHFD2 and MTHFD1L levels. This is recapitulated with knockdown of MYC with four shRNAs in multiple AML cell lines. Analysis of ENCODE ChIP-Seq data revealed MYC binding at SHMT2, MTHFD2 and MTHFD1L promoters, which we confirmed with ChIP-qPCR in human AML cell lines. Moreover, Independent component analysis (ICA) of primary AML samples in The Cancer Genome Atlas (TCGA) showed a significant correlation between high MTHFD2 and high MYC expression and a metabolic gene expression signature.

MTHFD2 is differentially expressed in transformed and non-differentiated cells, and is thus an attractive drug target given its limited expression in normal tissues. Knockdown of MTHFD2 with four shRNAs in five AML cell lines caused a decrease in cell proliferation as measured by BrdU incorporation and a decrease in colony formation in methylcellulose. MTHFD2 knockdown also induced myeloid differentiation, as measured by Cd11b expression, morphologic changes and induction of a previously validated AML differentiation gene expression signature. AML cells transduced with MTHFD2-directed shRNAs demonstrated attenuated growth in an orthotopic mouse model of AML at day 15 post-injection. We next deployed a doxycycline inducible shRNA system to demonstrate that shRNAs directed against MTHFD2 cause a decrease in AML burden in mice with established disease as measured by bioluminescence with an increase in survival. Metabolite profiling is currently underway to further elucidate the metabolic consequences of MTHFD2 loss in AML.

In summary, in silico analyses of primary patient AML data sets revealed a subset of AML samples enriched for a metabolic gene expression signature. We demonstrate that MYC is a regulator of the one carbon folate pathway, modulating expression of SHMT2, MTHFD2 and MTHFD1L. In vitro and in vivo data strongly supports a critical role for MTHFD2 in AML pathogenesis and its potential as a new target for AML therapy.