Abstract
Alterations in differentiation pathways contribute to the development of acute myeloid leukemia (AML). Differentiation therapy with all-trans retinoic acid (ATRA) has dramatically altered the treatment of acute promyelocytic leukemia, transforming it from a nearly fatal disease to a curable one. We set out to identify cellular pathways that contribute to AML differentiation, with the goal of identifying new therapeutic targets. We analyzed gene expression data from AML cell lines treated with phorbol 12-myristate 13-acetate (PMA), ATRA, Vitamin D, the BET inhibitor JQ1 and the DOT1L inhibitor EPZ00477, treatments known to induce AML differentiation and impair growth. Folate-mediated one-carbon metabolism was one of only three metabolic pathways altered by these compounds, with expression of MTHFD2 consistently downregulated with each compound.MTHFD2 is an NAD-dependent, bi-functional mitochondrial methylenetetrahydrofolate dehydrogenase and cyclohydrolase. It is differentially expressed in embryonic and transformed tissues and is upregulated in myeloid progenitors. MTHFD2 is the most differentially expressed metabolic enzyme in cancer cells versus normal cells, including normal proliferating cells. We thus investigated the role of MTHFD2 in myeloid malignancy. First, we demonstrated using ChIP-qPCR, MYC knockdown and MYC inhibition with a BET inhibitor, that MYC directly regulates MTHFD2 expression in AML. Knockdown of MTHFD2 with two shRNAs confirmed to have on-target activity, induced myeloid differentiation in AML cell lines, as measured by Cd11b expression, morphologic changes and induction of a previously validated AML differentiation gene expression signature. MTHFD2 knockdown decreased cell growth in AML cell lines, as well as decreased colony formation in methylcellulose in both AML cell lines and primary patient blasts. AML cells transduced with these two MTHFD2-directed shRNAs demonstrated attenuated growth in an orthotopic mouse model of AML. Furthermore, three MTHFD2-directed shRNAs prolonged survival in an MLL-AF9 mouse leukemia model. Additionally, using a doxycycline inducible shRNA system, we demonstrated that two miR30-shRNAs directed against MTHFD2 decreased AML burden in mice with established disease and prolonged survival.To identify biomarkers of response to MTHFD2 suppression, we used single sample Gene Set Enrichment Analysis (ssGSEA) to identify primary patient AML samples enriched for gene expression signatures of folate-mediated one-carbon metabolism and MTHFD2. We found in both independent data sets that the cluster of patients enriched for expression of the one-carbon folate pathway gene signatures was also enriched for patients with FLT3-ITD mutations, a subset of AML with a particularly poor prognosis. In addition, in an shRNA screen targeting 11,194 genes and performed in 216 cancer cell lines, including 17 AML lines, FLT3-ITD was a biomarker of response to MTHFD2 knockdown. We next validated that while MTHFD2 suppression induced measureable differentiation in all six AML cell lines examined, it induced the most robust induction of apoptosis in FLT3-ITD mutant AML. The mitochondrial one-carbon folate pathway is thought to contribute to cellular oxidative balance by providing reducing power in the form of NAD(P)H, and suppression of MTHFD2 was previously shown to increase ROS levels. Indeed, suppression of MTHFD2 led to a marked increase in ROS in the FLT3-ITD positive AML cell lines in which apoptosis was induced.In summary, a decrement in MTHFD2 expression was found at the center of multiple AML perturbations that impair AML growth and induce differentiation. Our data support MTHFD2 as an AML dependency and FLT3-ITD as a potential biomarker of response. We thus provide critical preclinical evidence for targeting of MTHFD2 as a therapeutic strategy in AML. DisclosuresStone:Celgene: Consultancy; Merck: Consultancy. DeAngelo:Celgene: Consultancy; Pfizer: Consultancy; Incyte: Consultancy; Agios: Consultancy; Novartis: Consultancy; Ariad: Consultancy; Bristol Myers Squibb: Consultancy; Amgen: Consultancy. Stegmaier:Novartis Pharmaceuticals: Consultancy.
Highlights
One-carbon folate metabolism is a core pathway altered with compounds active in acute myeloid leukemia (AML) cells We analyzed gene expression signatures of HL-60 cells treated with 1,25-dihydroxy Vitamin D3 (Vitamin D), PMA, and all-trans retinoic acid (Stegmaier et al, 2004), along with gene expression signatures of THP-1 cells treated with the BET bromodomain inhibitor JQ1 (Zuber et al, 2011), and MV4-11 and MOLM-13 cells treated with the DOT1L inhibitor EPZ004777 (Daigle et al, 2011), compounds that cause AML differentiation and death
Given the recent interest in this pathway in cancer metabolism, we focused on its role in AML, with a specific study of methylenetetrahydrofolate dehydrogenase-cyclohydrolase 2 (MTHFD2), a mitochondrial enzyme involved in folate metabolism that is consistently down-regulated across all of the probed datasets and highly differentially expressed in cancer compared with normal cells
The dehydrogenase and cyclohydrolase activities are performed by MTHFD2, whereas the synthetase activities are performed by MTHFD 1-like (MTHFD1L) (Christensen and Mackenzie, 2008)
Summary
In the 1940s, the discovery and application of aminopterin, which was later found to target dihydrofolate reductase (DHFR), a cytoplasmic enzyme involved in one-carbon folate metabolism, yielded the first remission in a child with acute lymphoblastic leukemia (Farber et al, 1948). Other folate derivatives, such as methotrexate, were later developed. Drugs such as 5-fluorouracil and pemetrexed that target thymidylate synthetase, another enzyme involved in one-carbon folate metabolism, were found to be effective therapies for some cancers (Locasale, 2013).
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