Abstract Obesity expands myeloid progenitors, myelopoiesis and increases the production of monocytes. While weight loss (WL) alleviates aspects of this inflammatory dysregulation, it is not known whether GLP-1 receptor agonists or other traditional modalities of WL differentially modify hematopoietic stem/progenitor cells (HSPCs), hematopoiesis, or inflammatory cell production. To test this, we compared the hematopoietic compartment in lean, obese and weight-reduced mice from tirzepatide treatment and caloric restriction (CR) implemented to match the body weight in both groups. At equal WL, we found CR induced multilineage cytopenias, whereas tirzepatide preserved blood lineages while specifically reducing classical Ly6C hi CCR2 + monocytes. To define the mechanisms underlying these changes we performed single-cell mRNA sequencing of bone marrow HSPCs and mature mononuclear blood cells. CR-HSPCs suppressed gene sets associated with nutrient sensing, proliferation and oxidative phosphorylation (OXPHOS) and exhibited lower inferred cell cycle activity, whereas tirzepatide-HSPCs attenuated these changes. Unlike CR, we found that across progressively differentiated cells from HSPCs to mature blood monocytes, tirzepatide increasingly suppressed OXPHOS and simultaneously shifted the maturation spectrum away from classical monocytes. Following six weeks of tirzepatide withdrawal and weight regain, Ly6C hi CCR2 + monocytes rebounded to levels seen in obese mice. These findings suggest that tirzepatide uncouples WL from the broad hematopoietic suppression seen in CR by preserving progenitor activity but selectively remodeling inflammatory/classical monocytes. We demonstrate that WL modality differentially impacts hematopoietic adaptation and provide evidence that classical monocytes are an effector cell through which tirzepatide may dampen obesity-associated inflammation. Key Points At equivalent weight loss, calorie restriction causes cytopenias and suppresses HSPC cycling, while tirzepatide preserves these parameters Tirzepatide reduces inflammatory monocytes, shifts maturation, decreases OXPHOS genes, and monocytes rebound after drug withdrawal

Background:Mechanical ventricular unloading and systemic circulatory support with left ventricular assist devices (LVADs) enable myocardial recovery in a subset of advanced heart failure (HF) patients, but predictors and mechanisms of recovery are not well understood. Integrating clinical and molecular data may improve identification of patients most likely to recover and uncover biologically relevant targets in HF. Methods:We collected and analyzed left ventricular apical myocardial tissue and clinical data from 208 patients undergoing LVAD implantation across five centers. Pre-implant transcriptomic profiles (22,373 mRNA transcripts) were integrated with 59 clinical variables using supervised machine learning with repeated cross-validation to identify and prioritize features associated with myocardial recovery, defined as a binary outcome based on improvement in left ventricular ejection fraction (LVEF ≥40%) and left ventricular end-diastolic diameter (LVEDD ≤5.9 cm). We also modeled functional (LVEF) and structural (LVEDD) improvement as a continuous outcome without any predefined LVEF and LVEDD pathological thresholds. Feature prioritization was followed by validation in human myocardial tissue and mechanistic interrogation in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Results:Integrative models achieved modest discrimination for myocardial recovery as a binary categorical outcome (maximum mean cross-validated area under the curve 0.73±0.15), identifying clinical features such as HF duration, LVEDD, HF pharmacologic therapy, and device configuration. Leucine-rich repeat neuronal 4C-like ( LRRN4CL ), measured in human myocardium, consistently emerged as a top transcriptomic predictor across both binary and continuous metric models (functional and structural). Higher pre-LVAD LRRN4CL expression was associated with reduced likelihood of myocardial recovery and localized primarily to cardiomyocytes. In iPSC-CMs, LRRN4CL overexpression localized to the sarcoplasmic reticulum, induced transcriptional remodeling characterized by suppression of contractile pathways and activation of stress programs, impaired calcium handling, impaired contraction-relaxation kinetics, and diminished mitochondrial respiratory reserve capacity. Conclusions:Integration of clinical and myocardial transcriptomic data identifies LRRN4CL as a novel marker associated with impaired myocardial recovery following LVAD-mediated ventricular unloading and systemic circulatory support. These findings move beyond predictive modeling, linking integrative computational discovery to cardiomyocyte dysfunction and providing a translational framework for biologically informed risk stratification and therapeutic targeting for myocardial recovery. CLINICAL PERSPECTIVE:What Is New?: Integrative clinical and myocardial transcriptomic modeling identifies LRRN4CL as a novel molecular determinant of structural and functional changes after LVAD-mediated ventricular unloading and enhanced systemic circulatory support. Elevated LRRN4CL expression is associated with adverse remodeling signatures, impaired calcium handling, and stress responses in human iPSC-derived cardiomyocytes. Experimental overexpression of LRRN4CL directly disrupts calcium cycling, contractile performance, and mitochondrial respiration linking molecular signature to functional phenotype. What Are the Clinical Implications?: Identification of LRRN4CL as a marker associated with impaired myocardial recovery supports future efforts toward biologically informed risk stratification for patients undergoing LVAD therapy. LRRN4CL as a marker of cardiac improvement potential may extend beyond advanced HF to earlier stage disease patients and inform prognosis, risk stratification, and response to medical therapies. These findings highlight LRRN4CL -associated pathways as potential therapeutic targets and demonstrate how integrative clinical-transcriptomic approaches can move beyond clinical prediction toward identification of new biologically precise therapeutic targets in HF following a bedside to bench and back approach.

ABSTRACT Inner mitochondrial membrane proteins must be sufficiently hydrophilic to withstand aqueous exposure during translation and transit to the mitochondria. Meanwhile, their transmembrane segments must be sufficiently hydrophobic to stably embed in the lipid membrane. We hypothesized that sequence-level adaptations evolved to balance these constraints. Here, we integrate structure-informed evolutionary analyses of mitochondrial proteins with atomistic simulations and cell-based experiments to identify aliphatic-to-threonine substitutions (ATS) as a potential solution to these constraints. With high statistical confidence, this transmembrane segment-specific adaptation is recurrently and convergently observed throughout mitochondrial evolution. Conformational analyses show that threonine interacts with both water and the transmembrane helix backbone, thereby lowering hydrophobicity without destabilizing secondary structure. In the extremely hydrophobic ATP6 protein, reverting threonines to aliphatic residues disrupts mitochondrial targeting, while introducing threonines into a poorly targeted variant improves its mitochondrial localization. These findings have implications for mitochondrial genome evolution, the rational design of membrane proteins, and potentially mitochondrial gene therapy.

Estrogen receptor alpha (ER) is an established oncogenic transcription factor in breast and endometrial cancer; however, more is known about the mechanisms controlling ER behavior in breast cancer, and therapies targeting ER have been much more successful in breast cancer. To address this disparity, we sought to identify genomic features that control ER in endometrial cancer and determine to what extent these factors differ from those in breast cancer. We focused on the locations of estrogen response elements (EREs), ER’s preferred DNA binding motif, throughout the human genome. To identify factors that predict ER genomic binding and effects on target gene expression, we applied machine learning to genomic data for each ERE in Ishikawa cells (ER+ endometrial cancer) and T-47D cells (ER+ breast cancer). Many of these factors, such as chromatin accessibility and histone modifications, were predictive of ER activity in both cell lines. However, the transcription factors that predict ER activity were found to be cell type-specific, including FOXA1 and GATA3 in T-47D cells, and ETV4 and SOX17 in Ishikawa cells. In addition, the features that predict ER binding and effects on gene expression differed, with transcription at EREs in the absence of estrogen being predictive of regulatory activity. To verify our findings in the Ishikawa cells, we performed a CRISPR knockout screen, which confirmed the discovery that SOX17 controls ER activity in endometrial cancer cells. These results identify important genomic features of ER binding and regulatory activity and how these features differ between endometrial cancer and breast cancer cells.

Myofibroblast differentiation, essential for driving extracellular matrix synthesis in pulmonary fibrosis, requires increased glycolysis. Although glycolytic cells must export lactate, the contributions of lactate transporters to myofibroblast differentiation are unknown. In this study, we investigated how monocarboxylate transporters (MCTs) 1 and 4, key pulmonary lactate transporters, influence myofibroblast differentiation and experimental pulmonary fibrosis. Our findings revealed that inhibiting MCT1 or MCT4 using RNA interference or small molecules reduced transforming growth factor-β1 (TGFβ)-stimulated myofibroblast differentiation in lung fibroblasts from healthy donors and patients with idiopathic pulmonary fibrosis. Small-molecule MCT inhibitors also decreased bleomycin-induced pulmonary fibrosis in C57Bl6/N mice aged 10 to 12 weeks. Through bioenergetic analyses, stable isotope tracing, metabolomics, and imaging mass spectrometry in both human cells and mice, we demonstrate that inhibiting lactate transport enhanced oxidative phosphorylation, reduced reactive oxygen species production, and diminished glucose metabolite incorporation into fibrotic lung regions. Furthermore, we introduce VB253, an MCT4 inhibitor, which ameliorates pulmonary fibrosis in both young and aged mice, with comparable efficacy to established antifibrotic therapies. These results underscore the necessity of lactate transport for myofibroblast differentiation, identify MCT1 and MCT4 as promising pharmacologic targets in pulmonary fibrosis, and support further evaluation of lactate transport inhibitors as a therapy for patients with limited treatment options.

Cells enable specialized metabolism by compartmentalizing metabolic pathways into distinct organelles, which requires the membrane transport of metabolites. In melanocytes, the amino acid tyrosine is imported into developing melanosomes for the synthesis of the UV-protective pigment melanin1,2. In spite of extensive biochemical characterization, the identity of the melanosomal tyrosine transporter remains unknown. Here, we identify SLC16A6 as an orphan melanosome-localized metabolite transporter. Genetic screens reveal that SLC16A6 expression is driven by the SOX10-MITF axis, the well-characterized master regulatory program governing melanogenesis and melanosomal homeostasis3,4. By redirecting SLC16A6 to the plasma membrane with an S240A mutation5, we demonstrate that SLC16A6 transports tyrosine, a process competitively inhibited by other bulky amino acids. We further determine that SLC16A6 is sufficient for in vitro melanosomal tyrosine uptake. Genetic depletion of SLC16A6 triggered loss of melanosome biogenesis and function as well as depletion of most melanosomal components. Collectively, these findings establish SLC16A6 as a melanosomal tyrosine transporter that is essential for melanosome biogenesis.

Metabolic-associated steatohepatitis (MASH) involves hepatocyte damage that cannot be explained solely by lipid accumulation. Here, to discover injury-specific pathways, we focused on a gene of uncertain function, EF-Hand Domain Family Member D1 (EFHD1), identified in human genome-wide association studies of liver injury but not liver fat. We show that EFHD1, a Ca2+-dependent actin crosslinker, stabilizes endoplasmic reticulum-mitochondria contact sites (ERMCS), detecting spatiotemporal coincidence of inter-organellar proximity and ER Ca2+ release. During MASH, EFHD1 upregulation drives pathological mitochondrial fragmentation via excessive contact persistence. This structural failure promotes mitochondrial double-stranded RNA escape and activation of a maladaptive antiviral PKR-dependent stress response, a causal relationship also supported by Mendelian randomization in humans. Consequently, inhibiting EFHD1 in human and mouse models blunts hepatocyte damage. These findings identify EFHD1 as a Ca2+-dependent ERMCS stabilizer, reveal a hepatocyte-intrinsic injury pathway, and suggest EFHD1 inhibition as a therapeutic strategy.

FicD regulates Unfolded Protein Response (UPR) through reversible AMPylation and deAMPylation of BiP, an HSP70 chaperone and master regulator of the UPR. FicD activity is regulated by endoplasmic reticulum-stress, catalyzing BiP AMPylation under low stress conditions to hold inactive chaperone in reserve. In stressed cells, FicD deAMPylates BiP, acutely increasing its active pool to assist in protein folding. Variants in UPR machinery, including those in the FicD gene, are linked to hereditary diseases. Despite the known role of FicD in UPR, in-vivo regulation of its activity remains elusive, and identifying metabolites that alter FicD activity could prove useful pharmaceutically. We applied an unbiased high-throughput screening platform, known as Mass spectrometry Integrated with equilibrium Dialysis for the discovery of Allostery Systematically (MIDAS), to identify small molecule metabolites that might regulate FicD activity. MIDAS revealed interactions between FicD and two mevalonate pathway intermediates: geranyl-pyrophosphate and farnesyl-pyrophosphate. Biochemical characterization indicates that both potently inhibit FicD-mediated AMPylation and deAMPylation. The crystal structure of FicD bound to farnesyl-pyrophosphate demonstrates a competitive inhibition mechanism, with the pyrophosphate adopting the alpha and beta phosphate positions of adenosine triphosphate (ATP) and the hydrocarbon chain filling the nucleoside pocket. FicD variants previously appeared as biochemically indistinguishable, yet lead to different human pathologies. We demonstrate farnesyl-pyrophosphate inhibits FicDR374H and FicDR374C variants implicated in causing hereditary spastic paraplegia, but not the FicDR371S variant associated with neonatal diabetes. This study furthers our understanding of FicD inhibitors and distinguishes disease causing variants, providing insight into pharmacological targeting of UPR activity.

Metabolic-associated steatohepatitis (MASH) involves hepatocyte damage that cannot be explained solely by lipid accumulation. Here, to discover injury-specific pathways, we focused on a gene of uncertain function, EF-Hand Domain Family Member D1 (EFHD1), identified in human genome-wide association studies of liver injury but not liver fat. We show that EFHD1, a Ca2+-dependent actin crosslinker, stabilizes endoplasmic reticulum-mitochondria contact sites (ERMCS), detecting spatiotemporal coincidence of inter-organellar proximity and ER Ca2+ release. During MASH, EFHD1 upregulation drives pathological mitochondrial fragmentation via excessive contact persistence. This structural failure promotes mitochondrial double-stranded RNA escape and activation of a maladaptive antiviral PKR-associated stress response, a causal relationship also supported by Mendelian randomization in humans. Consequently, inhibiting EFHD1 in human and mouse models blunts hepatocyte damage. These findings identify EFHD1 as a Ca2+-dependent ERMCS stabilizer, reveal a hepatocyte-intrinsic injury pathway, and suggest EFHD1 inhibition as a therapeutic strategy.

Perturbation of proximal tubule (PT) lipid metabolism fuels the pathological features of acute kidney injury (AKI). We found that AKI induced biosynthesis of lipotoxic ceramides within PTs in humans and mice and that urine ceramides predicted disease severity in children and adults. Mechanistic studies in primary PTs, which included a thermal proteomic profiling screen for ceramide effectors, revealed that ceramides altered assembly of the mitochondrial contact site and cristae-organizing system (MICOS) and respiratory supercomplexes, leading to acute disruption of cristae architecture, mitochondrial morphology, and respiration. These ceramide actions were dependent on the presence of the 4,5-trans double bond inserted by dihydroceramide desaturase 1 (DES1). Genetically ablating DES1 preserved mitochondrial integrity and prevented kidney injury in mice following bilateral ischemia reperfusion. Moreover, novel DES1 inhibitors that are attractive clinical drug candidates phenocopied the DES1 knockouts. These studies describe a new, therapeutically tractable mechanism underlying PT mitochondrial damage in AKI.

Tumor cells must optimize metabolite acquisition between synthesis and uptake from a microenvironment characterized by hypoxia, lactate accumulation, and depletion of many amino acids, including arginine. We performed a metabolism-focused functional screen using CRISPR-Cas9 to identify pathways and factors that enable tumor growth in an arginine-depleted environment. Our screen identified the SLC-family transporter SLC7A5 as required for growth, and we hypothesized that this protein functions as a high-affinity citrulline transporter. Using isotope tracing experiments, we show that citrulline uptake and metabolism into arginine are dependent upon expression of SLC7A5. Pharmacological inhibition of SLC7A5 blocks growth under low-arginine conditions across a diverse group of cancer cell lines. Loss of SLC7A5 reduces tumor growth and citrulline import in a mouse tumor model. We identify a conditionally essential role for SLC7A5 in arginine metabolism, and we propose that SLC7A5-targeting therapeutic strategies in cancer may be effective in the context of arginine limitation.

Heme is a cofactor essential for a multitude of biological reactions. The terminal step of heme synthesis occurs in the mitochondrial matrix which means that heme must be trafficked from there to other locales in the cell. Thus, identifying intracellular heme chaperones is crucial to understanding regulation of global cellular metabolism. The heme-binding protein progesterone receptor membrane component 1 (PGRMC1) has been proposed to function as a chaperone for several biologically active molecules including heme, but its cellular role is not fully understood. Here, we investigate the function of PGRMC1 in heme metabolism. By monitoring intracellular heme location and concentrations in Saccharomyces cerevisiae, we show that mutants lacking damage associated protein 1 (Dap1), the yeast ortholog of PGRMC1, have altered nuclear heme trafficking which can be corrected by complementation with DAP1 or PGRMC1. Biochemical analyses reveal that PGRMC1 co-localizes with known mitochondrial-associated membrane (MAM) proteins and proteomic comparison of interaction partners shows enrichment of MAM-associated proteins and pathways. Metabolomics profiling of wild-type and PGRMC1 knockout cells identifies significant changes of several metabolites, including heme, several amino acids, long chain acyl-carnitine, ethanolamine phosphate, and mevalonic acid. Together, these results provide evidence that PGRMC1 is involved in heme trafficking and homeostasis through MAMs.

Creatine is a critical metabolite used to buffer cellular energy demands in highly energetic tissues such as the brain and muscle. Genetic defects in endogenous creatine synthesis or transport across cellular membranes lead to a common set of phenotypes referred to as Cerebral Creatine Deficiency Syndrome (CCDS). The most common form of CCDS is Creatine Transporter 1 (CT1) Deficiency (CTD). It accounts for ~ 70% of cases and results from loss-of-function mutations in the X-linked gene SLC6A8 . Affected individuals suffer from intellectual disability, autistic-like behaviors, and epilepsy. There are currently no effective therapies for this disorder, but gene therapy has emerged as a potential approach. The two enzymes which comprise the endogenous creatine synthetic pathway (AGAT and GAMT) are selectively expressed by specific cell types throughout the body. However, after synthesized, creatine uptake relies on the protein product of SLC6A8 , CT1, to transport creatine into target cell types. We hypothesized that gene delivery of GATM (encoding AGAT) and GAMT into end-user cell types would bypass the need for CT1, allowing for intracellular synthesis of creatine. We tested this strategy in two human cell types: HEK293T cells and primary fibroblasts. Co-delivery of GATM and GAMT increased internal creatine concentrations by 7.6-fold in HEK293T cells and 12.3-fold in healthy control fibroblasts. We then employed this approach to primary fibroblasts from patients with CTD. This resulted in an up to 11.6-fold increase in intracellular creatine concentrations, far exceeding the intracellular concentration of creatine in healthy control fibroblasts. Importantly, overexpression of AGAT and GAMT resulted in proper targeting of these enzymes to their natural cellular compartment and did not impair the growth of patient fibroblasts. These findings establish gene therapy with GATM and GAMT as a potential strategy for patients with CTD.

Pyruvate occupies a central node in carbohydrate metabolism such that how it is produced and consumed can optimize a cell for energy production or biosynthetic capacity. This has been primarily studied in proliferating cells, but observations from the post-mitotic Drosophila fat body led us to hypothesize that pyruvate fate might dictate the rapid cell growth observed in this organ during development. Indeed, we demonstrate that augmented mitochondrial pyruvate import prevented cell growth in fat body cells in vivo as well as in cultured mammalian hepatocytes and human hepatocyte-derived cells in vitro. We hypothesize that this effect on cell size was caused by an increase in the NADH/NAD+ ratio, which rewired metabolism toward gluconeogenesis and suppressed the biomass-supporting glycolytic pathway. Amino acid synthesis was decreased, and the resulting loss of protein synthesis prevented cell growth. Surprisingly, this all occurred in the face of activated pro-growth signaling pathways, including mTORC1, Myc, and PI3K/Akt. These observations highlight the evolutionarily conserved role of pyruvate metabolism in setting the balance between energy extraction and biomass production in specialized post-mitotic cells.

Ischemic heart disease and acute myocardial infarction (AMI) is a leading cause of morbidity and mortality. Improvements have been made in coronary interventions to restore blood flow, but ischemia/reperfusion (I/R) injury significantly impacts clinical outcomes. We previously reported that activation of percutaneous mechanical unloading of the left ventricle (LV) with a transvalvular axial-flow device simultaneously with reperfusion improves myocardial salvage. However, the underlying mechanisms, potential adjuvant pharmacological interventions and the timing of the use of LV unloading as a cardioprotective approach in AMI are not well understood. This study investigated a) the mechanisms associated with improved myocardial salvage, b) a pharmacological intervention, and c) the timing of LV unloading. Following 90 minutes of ischemia, adult swine were subjected to reperfusion alone, simultaneous unloading with reperfusion, upfront unloading with delayed reperfusion, upfront reperfusion with delayed unloading, or reperfusion with concurrent use of esmolol and milrinone. Compared to controls, the simultaneous group had a 47% increase in myocardial salvage following AMI. This was associated with increased expression of neutrophil degranulation, macrophage activation, iNOS signaling, wound healing, and PPAR signaling. From these pathways, PPARG (peroxisome proliferator-activated receptor gamma) emerged as a potential cardioprotective gene that was uniquely overexpressed in the simultaneously unloaded and reperfused myocardium. Next, we showed PPARG agonism with rosiglitazone reduces mitochondrial oxygen demand in cardiomyocytes and in vivo, improves myocardial salvage following I/R injury in C57BL6/J mice. Thiazolidinediones (TZDs), such as rosiglitazone could be investigated as therapies combined with simultaneous LV unloading and coronary interventions to mitigate reperfusion injury. GRAPHICAL ABSTRACT:

The adenosine A2A receptor (A2AR) is a Class A G protein-coupled receptor (GPCR) that regulates inflammation, glucose metabolism, and energy homeostasis in metabolically active tissues. While the effects of small-molecule ligands and protein interactions with A2AR have been extensively studied, the regulatory influence of endogenous metabolites remains unexplored. To address this gap, we employed the Mass spectrometry Integrated with equilibrium Dialysis for the discovery of Allostery Systematically (MIDAS) platform to screen a library of human metabolites for interactions with A2AR. This approach identified 180 metabolites that interact with A2AR, including allosteric and orthosteric modulators. We characterized the mechanisms of three metabolites previously unreported to interact with A2AR: prostaglandin D2, an allosteric antagonist that fully inhibits receptor signaling, and two orthosteric agonists, S-adenosyl-L-homocysteine and 2′-deoxyadenosine, that fully activate A2AR. Overall, these findings highlight the potential of the MIDAS platform to uncover previously unrecognized metabolite-GPCR interactions for research and therapeutic applications. The adenosine A2A receptor (A2AR) plays a crucial role in regulating inflammation and metabolism, yet the extent to which endogenous metabolites modulate its activity remains unclear. Here, the authors utilized the MIDAS platform to identify metabolites interacting with A2AR, revealing orthosteric and allosteric modulators.

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.