DHODH-driven pyrimidine metabolism is a targetable vulnerability in myeloproliferative neoplasms Free

Metabolic rewiring is a hallmark of malignancy, enabling cancer cells to sustain proliferation and resist apoptosis. The extent to which metabolic alterations contribute to the pathogenesis of JAK2-mutatedmyeloproliferative neoplasms (MPNs) remains incompletely understood. To address this, we conducted integrated metabolomic and transcriptomic analyses of hematopoietic tissues from Jak2^V617F knock-in mice,as well as serum and bone marrow samples from MPN patients. Our data revealed a consistent and significant upregulation of the de novo pyrimidine biosynthesis pathway in both murine and human MPN samples. Metabolite profiling revealed elevated levels of key intermediates, such as orotate and dihydroorotate, accompanied by a marked depletion of glutamine and uridine, indicating increased flux through the pyrimidine biosynthetic pathway to support abnormal cell proliferation.

RNA-seq and GSEA analyses revealed activation of oncogenic pathways, including JAK-STAT, KRAS, and MYC signaling in Jak2^V617F cells, aligning with the observed metabolic phenotype. Protein-level validation confirmed increased expression of phosphorylated CAD (pCAD) and dihydroorotate dehydrogenase (DHODH), two key enzymes regulating the rate-limiting steps in de novo pyrimidine synthesis. Mechanistically, we identified a STAT5–MYC–DHODH regulatory axis: pharmacologic inhibition of STAT5 or MYC reduced DHODH expression, and ChIP-qPCR analysis demonstrated direct MYC binding at the DHODH promoter in JAK2-mutant cells.

To evaluate the therapeutic relevance of this metabolic dependency, we treated Jak2^V617F knock-in mice with Brequinar, a clinically investigated DHODH inhibitor, either alone or in combination with ruxolitinib, a JAK1/2 inhibitor approved for MPNs. DHODH inhibition significantly reduced red blood cell count, hematocrit, and splenomegaly, and decreased the frequency and absolute number of hematopoietic stem and progenitor cells (HSPCs), including MPP and MEP populations in both bone marrow and spleen. Metabolomic analysis following treatment revealed partial restoration of pyrimidine metabolic homeostasis, characterized by reduced orotate levels and increased uridine and glutamine levels. These effects were further enhanced by the combination of Brequinar and ruxolitinib, which together produced a broader suppression of erythropoiesis and progenitor expansion, indicating a synergistic therapeutic strategy.

To assess the translational potential of our findings in a human system, we utilized an induced pluripotent stem cell (iPSC)-derived bone marrow organoid model that recapitulates the 3D architecture and multilineage hematopoiesis of human marrow. CD34⁺ HSPCs from JAK2^V617F-mutant MPN patients were successfully engrafted into the organoid system. Upon treatment with Brequinar, either alone or in combination with ruxolitinib, we observed reduced erythroid differentiation and impaired multilineage output, confirming the functional impact of DHODH inhibition on human MPN cells.

Together, these results demonstrate that JAK2^V617F-driven MPNs are metabolically dependent on elevated pyrimidine biosynthesis to sustain hyperproliferation. We identify DHODH as a critical downstream effector in this metabolic program, transcriptionally regulated via the STAT5–MYC axis. Pharmacologic targeting of DHODH disrupts pyrimidine metabolism, suppresses aberrant hematopoiesis, and enhances the efficacy of JAK inhibition in both murine and human models. These findings position DHODH inhibition as a promising therapeutic approach for the dysregulated pyrimidine metabolism in MPNs, supporting the rationale for combining metabolic and signaling pathway inhibitors in future clinical strategies.