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Although its prognosis continually improves with time, a significant proportion of patients still relapse from the disease because of the leukemia\u2019s resistance to therapy. Methotrexate (MTX), a folic-acid antagonist, is a chemotherapy agent commonly used against ALL and as an immune-system suppressant for rheumatoid arthritis that presents multiple and complex mechanisms of action and resistance. Previous studies have shown that MTX modulates the nuclear factor kappa B (NF-\u03baB) pathway, an important family of transcription factors involved in inflammation, immunity, cell survival, and proliferation which are frequently hyperactivated in ALL. Using a gene set enrichment analysis of publicly available gene expression data from 161 newly diagnosed pediatric ALL patients, we found the Tumor necrosis factor \u03b1 (TNF-\u03b1) signaling pathway via NF-\u03baB to be the most enriched Cancer Hallmark in MTX-poor-responder patients. A transcriptomic analysis using a panel of ALL cell lines (six B-cell precursor acute lymphoblastic leukemia and seven T-cell acute lymphoblastic leukemia) also identified the same pathway as differentially enriched among MTX-resistant cell lines, as well as in slowly dividing cells. To better understand the crosstalk between NF-\u03baB activity and MTX resistance, we genetically modified the cell lines to express luciferase under an NF-\u03baB-binding-site promoter. We observed that the fold change in NF-\u03baB activity triggered by TNF-\u03b1 (but not MTX) treatment correlated with MTX resistance and proliferation across the lines. At the individual gene level, NFKB1 expression was directly associated with a poorer clinical response to MTX and with both an increased TNF-\u03b1-triggered NF-\u03baB activation and MTX resistance in the cell lines. Despite these results, the pharmacological inhibition (using BAY 11-7082 and parthenolide) or stimulation (using exogenous TNF-\u03b1 supplementation) of the NF-\u03baB pathway did not alter the MTX resistance of the cell lines significantly, evidencing a complex interplay between MTX and NF-\u03baB in ALL.<\/jats:p>","DOI":"10.3390\/genes14101880","type":"journal-article","created":{"date-parts":[[2023,9,27]],"date-time":"2023-09-27T07:38:46Z","timestamp":1695800326000},"page":"1880","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["The Expression and Activation of the NF-\u03baB Pathway Correlate with Methotrexate Resistance and Cell Proliferation in Acute Lymphoblastic Leukemia"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8722-8512","authenticated-orcid":false,"given":"Rafael Renatino","family":"Canevarolo","sequence":"first","affiliation":[{"name":"Centro de Pesquisa Boldrini, Centro Infantil Boldrini, Campinas 13083-210, SP, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nathalia Moreno","family":"Cury","sequence":"additional","affiliation":[{"name":"Centro de Pesquisa Boldrini, Centro Infantil Boldrini, Campinas 13083-210, SP, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1316-3525","authenticated-orcid":false,"given":"Jos\u00e9 Andr\u00e9s","family":"Yunes","sequence":"additional","affiliation":[{"name":"Centro de Pesquisa Boldrini, Centro Infantil Boldrini, Campinas 13083-210, SP, Brazil"},{"name":"Medical Genetics Department, Faculty of Medical Sciences, State University of Campinas, Campinas 13083-970, SP, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,9,27]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2947","DOI":"10.1002\/art.38805","article-title":"Methotrexate inhibits NF-kappaB activity via long intergenic (noncoding) RNA-p21 induction","volume":"66","author":"Spurlock","year":"2014","journal-title":"Arthritis Rheumatol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"178","DOI":"10.1093\/rheumatology\/keu279","article-title":"Methotrexate-mediated inhibition of nuclear factor kappaB activation by distinct pathways in T cells and fibroblast-like synoviocytes","volume":"54","author":"Spurlock","year":"2015","journal-title":"Rheumatology"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"2911","DOI":"10.4049\/jimmunol.167.5.2911","article-title":"Methotrexate suppresses NF-kappaB activation through inhibition of IkappaBalpha phosphorylation and degradation","volume":"167","author":"Majumdar","year":"2001","journal-title":"J. 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