Combinatorial targeting of metabolic vulnerabilities in t-cell leukemia through NAMPT inhibition

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy that arises from the malignant transformation of T-cell progenitors, accounting for approximately 15% of pediatric and 25% of adult acute lymphoblastic leukemia (ALL) cases. Despite advances in multi-agent chemotherapy, including the use of antimetabolites like methotrexate (MTX), a substantial proportion of patients relapse or develop drug resistance, with limited effective therapeutic alternatives. How metabolic dysregulation in leukemia affects disease progression and resistance to therapy is not well understood. In this study, we uncover a previously underexplored metabolic vulnerability in T-ALL involving the NAD⁺ salvage pathway enzyme nicotinamide phosphoribosyltransferase (NAMPT). NAD⁺ can be synthesized via multiple pathways, including de novo synthesis, the Preiss-Handler Pathway (mediated by NAPRT), and the salvage pathway mediated by NAMPT. We have observed that T-ALL shows a distinct reliance on the salvage pathway compared to all other cancers. Analysis of CRISPR dependency data (DepMap) revealed that T-ALL cells have higher dependence on NAMPT. Furthermore, expression profiles across cancer cell lines showed elevated NAMPT and reduced NAPRT expression in T-ALL cells, indicating the importance of the salvage route for NAD⁺ production. RNA-seq and protein data from T-ALL patient-derived xenografts (PDXs) compared to healthy T-cells from the thymus, further confirmed lower expression of enzymes involved in the de novo and Preiss–Handler pathways and high NAMPT expression, especially in high-risk cases. Functional assays showed that T-ALL cells are highly sensitive to NAMPT inhibition using three structurally different inhibitors, with significantly lower IC₅₀ values compared to other lineages and healthy T-cells. By integration of metabolomics, transcriptomics, and proteomics data, we observed that pharmacological inhibition of NAMPT led to the depletion of NAD⁺ levels, resulting in metabolic disruption, and an aberrant splicing pattern in T-ALL. Importantly, NAMPT inhibition was associated with downregulation of dihydrofolate reductase (DHFR) at both transcript and protein levels. This effect was consistent across multiple T-ALL cell lines, suggesting a mechanistically relevant and selective vulnerability. DHFR is an enzyme that reduces dihydrofolic acid to tetrahydrofolic acid, required for the de novo synthesis of purines, using NADPH as an electron donor, and it is the main target of MTX. Since mutations in DHFR and upregulation of the levels or activity of the enzyme are a known mechanism of resistance to MTX, we examined whether NAMPT inhibition could revert this resistance and enhance MTX cytotoxic effects. We demonstrate that NAMPT inhibition sensitizes T-ALL cells to MTX, significantly enhancing its efficacy both in vitro and in vivo. In vitro combination studies reveal a strong synergy between NAMPT inhibitors and MTX, promoting apoptosis and cell cycle arrest. Mechanistically, this synergy is linked to the dual impairment of folate metabolism: (1) direct DHFR activity inhibition by reducing the levels of DHFR cofactor NADPH, and (2) via DHFR downregulation, converging on a critical bottleneck in nucleotide biosynthesis. In vivo, co-treatment with NAMPT inhibitors and MTX in T-ALL xenograft models leads to significant reduction in tumor progression and engraftment, and prolonged survival compared to the control group and with monotherapies. This effect was validated in a NAMPT knockdown model treated with MTX. Most notably, an MTX-resistant PDX model responded favorably to the combination, with evidence of restored MTX sensitivity and lower leukemic engraftment. These results underscore the translational potential of combining NAMPT inhibitors with conventional chemotherapy to overcome drug resistance. Collectively, our findings highlight that NAMPT is a lineage-specific metabolic dependency in T-ALL. By linking NAD⁺ metabolism to folates pathway regulation, our study uncovers a novel mechanism for overcoming drug resistance that is particularly relevant for high-risk or relapsed T-ALL cases. This work provides a strong preclinical rationale for investigation of NAMPT inhibition in combination with MTX, especially in patients who have developed resistance to standard therapies, with the potential of expanding these findings to other solid and hematological cancers where MTX is established as part of the treatment.