Lactate export inhibition reveals a metabolic vulnerability in myelofibrosis

Abstract

Abstract Myelofibrosis (MF) is a debilitating myeloproliferative neoplasm characterized by progressive bone marrow fibrosis, constitutional symptoms, impaired hematopoiesis, splenomegaly, and an increased risk of leukemic transformation. Although constitutive activation of JAK/STAT signaling is a hallmark of MF pathogenesis, JAK inhibitors fail to significantly modify disease in most patients. This suggests that additional mechanisms contribute to MF progression. We hypothesized that MF cells undergo metabolic reprogramming during disease evolution, creating novel vulnerabilities amenable to therapeutic targeting. To begin testing this, we performed bulk RNA-sequencing on peripheral blood mononuclear cells (PBMCs) from healthy donors and patients with either JAK2V617F or CALR-mutated MF. Both mutation groups exhibited >5,000 differentially expressed genes compared to healthy controls (FDR < 0.05), and gene set enrichment analysis (GSEA) revealed upregulation of hypoxia and glycolysis-associated transcriptional programs. To further evaluate metabolic function, we performed MetaFlux analysis, which revealed significantly increased glycolytic flux and accumulation of (S)-lactate in MF samples compared to controls. These findings highlight a metabolic shift toward anaerobic glycolysis in MF cells. We next investigated whether this shift was associated with specific changes in lactate export machinery. Among monocarboxylate transporter (MCT) family members, only MCT4 (SLC16A3), the principal exporter of lactate in highly glycolytic cells, was consistently upregulated at the RNA level in both JAK2V617F- and CALR-mutated PBMCs. Immunohistochemistry (IHC) confirmed marked overexpression of MCT4 protein in bone marrow biopsies from MF patients compared to healthy donors, suggesting that upregulation of MCT4 may be a conserved and functional feature of MF pathogenesis. To assess the consequences of MCT4 upregulation in vivo, we treated mice developing aggressive MF driven by MPLW515L with VB124, a previously published, potent, and selective MCT4 inhibitor. Although both vehicle- and VB124-treated groups began with comparable disease burden, VB124 treatment led to a significant reduction in the number of circulating clonal malignant cells beginning three weeks after treatment initiation. After four months of continuous therapy, VB124-treated mice exhibited markedly improved overall survival (53% vs. 100%, Log Rank test: P=0.006, n=15 per group), normalized bone marrow cellularity, significantly reduced reticulin fibrosis, and significantly reduced splenomegaly (2.88% vs. 0.43% of body weight, n=15 per group, P < 0.001). These results suggest that MCT4 inhibition not only impairs clonal expansion but also delays disease progression in vivo. We next sought to determine whether the effect of MCT4 inhibition on clonal suppression was cell autonomous. We generated Ba/F3 cells expressing either wild-type MPL (MPLWT) or the oncogenic variant MPLW515L. As expected, MPLWT cells required IL-3 or TPO for survival, while MPLW515L-expressing cells propagated in a cytokine-independent manner. Interestingly, treatment with VB124 had no effect on the proliferation of MPLWT cells in the presence of IL-3 or TPO, but significantly reduced the growth of MPLW515L cells, suggesting that transformation by MPLW515L confers a specific metabolic dependency on lactate export. Ongoing studies are testing the hypothesis that MPLW515L alters NAD+/NADH homeostasis, thereby promoting oxidative damage and decreasing cellular fitness in the setting of impaired lactate export. To explore the clinical relevance of these findings, we transduced primary human hematopoietic stem cells (HSCs) with MPLW515L and assessed colony-forming ability in methylcellulose. Preliminary data suggest that VB124 selectively impairs colony formation of MPLW515L-transduced HSCs while sparing wild-type HSCs. Serial replating assays, a surrogate for stem cell fitness, further support these findings, indicating that MCT4 inhibition may selectively impair the self-renewal capacity of malignant HSCs while sparing normal hematopoiesis. Given that MCT4 inhibitors are advancing into early-phase clinical trials, our findings reveal a novel and targetable metabolic vulnerability in MF. Together, these data provide preclinical evidence that lactate export is a selective requirement for MF cell survival and that pharmacologic MCT4 inhibition may represent a promising disease-modifying therapeutic strategy.