The Expression and Activation of the NF-B Pathway Correlate with Methotrexate Resistance and Cell Proliferation in Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Although its prognosis continually improves with time, a significant proportion of patients still relapse from the disease because of the leukemia's 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-kappa B) 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 alpha (TNF-alpha) signaling pathway via NF-kappa B 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-kappa B activity and MTX resistance, we genetically modified the cell lines to express luciferase under an NF-kappa B-binding-site promoter. We observed that the fold change in NF-kappa B activity triggered by TNF-alpha (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-alpha-triggered NF-kappa B 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-alpha supplementation) of the NF-kappa B pathway did not alter the MTX resistance of the cell lines significantly, evidencing a complex interplay between MTX and NF-kappa B in ALL.