The Folate Cycle Enzyme MTHFR Is a Critical Regulator of Cell Response to MYC-Targeting Therapies
Authors: Su Angela , Ling Frank , Vaganay Camille , Pardieu Bryann , Benajiba Lina , Sodaro Gaetano , Pasanisi Justine , Lin Kevin H , Rutter Justine , Bassil Christopher F. , Antony-Debre Iléana , Alexe Gabriela , Benoist Jean-François , Pikman Yana , Qi Jun , Dombret Herve , Preudhomme Claude , Fenouille Nina , Golan Hava M. , Stegmaier Kimberly , Lobry Camille , Wood Kris C , Itzykson Raphael , Puissant Alexandre 
Published: February 2020

Metabolic rewiring of neoplastic cells engenders metabolic liabilities that can be exploited to design innovative therapeutic strategies, including those to increase the therapeutic index of existing anticancer therapies. We hypothesized that metabolic perturbation may substantially influence cell response to therapies targeting major oncogenes which are involved in active hijacking of neoplastic cell metabolism. In that regard, MYC represents a paradigmatic oncogene as this transcription factor is deregulated in more than 50% of human cancers and reprograms many aspect of cell metabolism. MYC expression is controlled by clusters of super-enhancer genomic regions densely occupied by transcription factors and chromatin regulators ― including BET bromodomain proteins, and CDK7 and CDK9 kinases.
Two cohorts of patients with Acute Myeloid Leukemia, AML (TCGA-LAML, n=198 and GSE14468, n=526) were queried with multiple gene sets in order to reveal a core of metabolic gene signatures, which are connected to the folate cycle, and whose activation was poorly correlated with an active MYC transcriptional program in AML. According to these data, we established that folate cycle disruption upon folic acid starvation consistently enhanced resistance to MYC targeting by BET or CDK7 inhibitors (JQ1, OTX015, THZ1) as well as BRD4-directed shRNAs in a large panel of human AML cell lines harboring a wide variety of genetic alterations (n=7), in MLL-translocated primary patient samples with AML (n=4), and in animals injected with MLL-AF9-positive leukemic cells.
Using an shRNA-based screening approach against enzymes from the folate cycle, we revealed that the knockdown of the rate-limiting enzyme in the folate cycle, 5,10-methylenetetrahydroflate reductase (MTHFR), significantly increased resistance to OTX015 in AML cell lines (n=4) and in animals transplanted with Mthfr-depleted blasts. Previous reports have identified and extensively studied two common genetic variants in the MTHFR gene, C677T and A1298C, encoding two MTHFR enzyme variants with reduced activity in about 10% of Caucasians. We introduced in KG1a cells these two non-synonymous single nucleotide polymorphisms in MTHFR using CRISPR-Cas9, thereby generating isogenic cell lines exhibiting all combinations of variants. Although the clones which are heterozygous for any of the two variants had similar sensitivity to OTX015 as wild-type clones, 677 TT and 1298 CC homozygous KG1a clones were significantly more resistant to OTX015 than their wild-type counterpart, an effect that was alleviated by exogenous overexpression of wild-type MTHFR or supplementation of cells with the end-product metabolite synthesized by MTHFR, 5-CH3 THF. Consistent with these data, MLL-translocated patients displaying a homozygous and compound heterozygous MTHFR genotype for any of the two variants (n=8) responded significantly less to OTX015 than those with wild-type homozygous and heterozygous MTHFR genotypes (n=8). Finally, we established that the loss of a single copy of Mthfr which phenocopies in mice a partial impairment in MTHFR activity caused by non-synonymous single nucleotide polymorphisms on MTHFR, was sufficient to attenuate sensitivity to JQ1 of MLL-AF9-driven leukemias.
Using metabolomics profiling, we pointed out that a major effect of folate cycle disturbance in AML cells is the intracellular accumulation of S-adenosyl-homocysteine, SAH, which is the downstream effector of MTHFR knockdown triggering BET inhibitor resistance. Given that SAH is a potent inhibitor of SAM-dependent methylation reactions, we determined that folate cycle impairment decreases H3K27 and H3K9 methyltransferase activities and subsequent methylation of H3K27 and H3K9 histone marks across the whole genome of AML cells. By combining ChIP- and RNA-sequencing approaches, we demonstrated that decreased methylation levels of H3K27 and H3K9 histone marks upon folate cycle alteration combined with BET inhibition activates SPI1 and IRF / Interferon signaling transcriptional programs. SPI1 knockdown significantly reduced the resistance to OTX015 of AML cells whose MTHFR expression was suppressed or MLL-AF9-transformed Mthfr knockout primary murine cells.
Our data provide a rationale for screening MTHFR polymorphisms and the folate cycle status to exclude patients least likely and nominate those most likely to benefit from MYC-targeting therapies.

Disclosures
Dombret: CELGENE: Consultancy, Honoraria; AGIOS: Honoraria; Institut de Recherches Internationales Servier (IRIS): Research Funding. Stegmaier:Rigel Pharmaceuticals: Consultancy; Novartis: Research Funding.