Folate metabolism: a re-emerging therapeutic target in haematological cancers

Abstract

Folate-mediated one carbon (1C) metabolism supports a series of processes that are essential for the cell. Through a number of interlinked reactions happening in the cytosol and mitochondria of the cell, folate metabolism contributes to de novo purine and thymidylate synthesis, to the methionine cycle and redox defence. Targeting the folate metabolism gave rise to modern chemotherapy, through the introduction of antifolates to treat paediatric leukaemia. Since then, antifolates, such as methotrexate and pralatrexate have been used to treat a series of blood cancers in clinic. However, traditional antifolates have many deleterious side effects in normal proliferating tissue, highlighting the urgent need for novel strategies to more selectively target 1C metabolism. Notably, mitochondrial 1C enzymes have been shown to be significantly upregulated in various cancers, making them attractive targets for the development of new chemotherapeutic agents. In this article, we present a detailed overview of folate-mediated 1C metabolism, its importance on cellular level and discuss how targeting folate metabolism has been exploited in blood cancers. Additionally, we explore possible therapeutic strategies that could overcome the limitations of traditional antifolates.

Fig. 1. Haematological cancer cell lines show high sensitivity to methotrexate.

IC50 values for methotrexate across several cell lines as obtained by the publicly available database Genomics of Drug Sensitivity in Cancer (https://www.cancerrxgene.org/). Number in parenthesis represents number of cell lines analysed per each cancer type. ALL, acute lymphoblastic leukaemia; AML, acute myeloid leukaemia; CLL, chronic lymphoblastic leukaemia; CML, chronic myeloid leukaemia; DLBC, diffuse large B-cell lymphoma; MM, multiple myeloma; ACC, adrenocortical carcinoma; BLCA, bladder urothelial carcinoma; LGG, brain lower grade glioma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; COREAD, colon and rectum adenocarcinoma; ESCA, oesophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; KIRC, kidney real clear cell carcinoma; HCC, hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; MB, medulloblastoma; MESO, mesothelioma; NB, neuroblastoma; OV, ovarian serous cystadenocarcinoma; PDAC, pancreatic adenocarcinoma; PRAD, prostate adenocarcinoma; SKCM, skin cutaneous melanoma; SCLC, small cell lung carcinoma; STAD, stomach adenocarcinoma; THCA, thyroid carcinoma; UCEC, uterine corpus endometrial carcinoma.

Fig. 2. An overview of folate-mediated 1C metabolism and its compartmentalisation.

From yeast to mammalian cells 1C units tend to flow clockwise allowing for a complete oxidative/reductive cycle where serine gets oxidised in the mitochondrial compartment and formate gets reduced in the cytosol. Through this intercompartmental cycle folate metabolism supports anabolic reactions such as purine and thymidylate synthesis as well as the methionine cycle. DHF, dihydrofolate; THF, tetrahydrofolate; DHFR, dihydrofolate reductase; MFT, mitochondrial folate transporter; SHTMT1/2, serine hydroxymethyl transferase, cytosolic (1)/ mitochondrial (2); MTHFD1, methylenetetrahydrofolate dehydrogenase 1; MTHFD2(L), methylenetetrahydrofolate dehydrogenase 2 (2-like); MTHFD1L, monofunctional tetrahydrofolate synthase; TYMS, thymidylate synthetase; MTHFR, methylenetetrahydrofolate reductase; GART, phosphoribosylglycinamide formyltransferase; ATIC, 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase; ALDH1L1/2, 10-formyltetrahydrofolate dehydrogenase cytosolic (1)/mitochondrial (2).

Fig. 3. Mechanism of action of methotrexate.

Methotrexate (MTX) enters the cell mainly through the reduced folate carrier (RFC1) and to a lesser extend through receptor-mediated endocytosis via a folate receptor (FR). Upon entry, MTX gets polyglutamated (MTX(Glu)_n) by folylpolyglutamate synthase (FPGS). Polyglutamates of MTX are a superior antifolate agent compared to MTX, capable of highly potent irreversible inhibition of DHFR. Furthermore, MTX induces inhibition of other enzymes like TYMS and GART/ATIC, ultimately blocking de novo thymidylate and purine syntheses. γ-glutamyl hydrolase (γ-GH) (compartmentalised in lysosomes) removes glutamate residues from MTX, while ATP-binding cassette (ABC) transporters assist in the excretion of MTX from the cell.

Fig. 4. Metabolic changes caused by MTX treatment.

MTX by inhibiting de novo purine synthesis causes significant accumulation of AICAR. AICAR is an activator of 5’ AMP-activated protein kinase (AMPK), a master regulator of energy homoeostasis. AMPK signalling promotes catabolic pathways such as glycolysis, autophagy, lipid oxidation and oxidative phosphorylation etc. to augment cellular bioenergetic capacity. Concurrently, AMPK inhibits anabolic processes such as protein and lipid synthesis and ultimately restrains cellular proliferation.

Fig. 5. Hypothesised mechanisms of action of SHMT inhibitors.

a Proposed mechanism of synergy of MTX with SHMT inhibitors. Inhibition of DHFR by MTX decreases intracellular THF, thus, it sensitises cells to SHMT inhibitors. b Proposed mechanism of increased sensitivity to SHMT inhibitors due to MTX resistance. Decreases transport mediated by RFC1, decreased polyglutamation due to decreased activity of FPGS or increased activity of γ-GH are mechanisms that contribute to MTX resistance. Decreased MTX(Glu)n correlates with decreased THF(Glu)n as folate has a similar influx mechanism and polyglutamation pattern. As THF is a substrate for the SHMT reaction, depletion of this substrate leads to increased sensitivity of the cells to the SHMT inhibitors.