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Introduction: Methotrexate (MTX), a folic acid antagonist and nucleotide synthesis inhibitor, is a cornerstone drug used against acute lymphoblastic leukemia (ALL), but its mechanism of action and resistance continues to be unraveled even after decades of clinical use. Methods: To better understand the mechanisms of this drug, we accessed the intracellular metabolic content of 13 ALL cell lines treated with MTX by 1HNMR, and correlated metabolome data with cell proliferation and gene expression. Further, we validated these findings by inhibiting the cellular antioxidant system of the cells in vitro and in vivo in the presence of MTX. Results: MTX altered the concentration of 31 out of 70 metabolites analyzed, suggesting inhibition of the glycine cleavage system, the pentose phosphate pathway, purine and pyrimidine synthesis, phospholipid metabolism, and bile acid uptake. We found that glutathione (GSH) levels were associated with MTX resistance in both treated and untreated cells, suggesting a new constitutive metabolic-based mechanism of resistance to the drug. Gene expression analyses showed that eight genes involved in GSH metabolism were correlated to GSH concentrations, 2 of which (gamma-glutamyltransferase 1 [GGT1] and thioredoxin reductase 3 [TXNRD3]) were also correlated to MTX resistance. Gene set enrichment analysis (GSEA) confirmed the association between GSH metabolism and MTX resistance. Pharmacological inhibition or stimulation of the main antioxidant systems of the cell, GSH and thioredoxin, confirmed their importance in MTX resistance. Arsenic trioxide (ATO), a thioredoxin inhibitor used against acute promyelocytic leukemia, potentiated MTX cytotoxicity in vitro in some of the ALL cell lines tested. Likewise, the ATO+MTX combination decreased tumor burden and extended the survival of NOD scid gamma (NSG) mice transplanted with patientderived ALL xenograft, but only in one of four ALLs tested. Conclusion: Altogether, our results show that the cellular antioxidant defense systems contribute to leukemia resistance to MTX, and targeting these pathways, especially the thioredoxin antioxidant system, may be a promising strategy for resensitizing ALL to MTX. [ABSTRACT FROM AUTHOR]

The role of tumour microenvironment in neoplasm initiation and malignant evolution has been increasingly recognized. However, the bone marrow mesenchymal stromal cell (BMMSC) contribution to disease progression remains poorly explored. We previously reported that the expression of serine protease inhibitor kunitz‐type2 (SPINT2/HAI‐2), an inhibitor of hepatocyte growth factor (HGF) activation, is significantly lower in BMMSC from myelodysplastic syndromes (MDS) patients compared to healthy donors (HD). Thus, to investigate whether this loss of expression was due to SPINT2/HAI‐2 methylation, BMMSC from MDS and de novo acute myeloid leukaemia (de novo AML) patients were treated with 5‐Azacitidine (Aza), a DNA methyltransferase inhibitor. In MDS‐ and de novo AML‐BMMSC, Aza treatment resulted in a pronounced SPINT2/HAI‐2 levels up‐regulation. Moreover, Aza treatment of HD‐BMMSC did not improve SPINT2/HAI‐2 levels. To understand the role of SPINT2/HAI‐2 down‐regulation in BMMSC physiology, SPINT2/HAI‐2 expression was inhibited by lentivirus. SPINT2 underexpression resulted in an increased production of HGF by HS‐5 stromal cells and improved survival of CD34+ de novo AML cells. We also observed an increased adhesion of de novo AML hematopoietic cells to SPINT2/HAI‐2 silenced cells. Interestingly, BMMSC isolated from MDS and de novo AML patients had increased expression of the integrins CD49b, CD49d, and CD49e. Thus, SPINT2/HAI‐2 may contribute to functional and morphological abnormalities of the microenvironment niche and to stem/progenitor cancer cell progression. Hence, down‐regulation in SPINT2/HAI‐2 gene expression, due to methylation in MDS‐BMMSC and de novo AML‐BMMSC, provides novel insights into the pathogenic role of the leukemic bone marrow microenvironment. [ABSTRACT FROM AUTHOR]