Project Summary/Abstract: Myelofibrosis (MF) is a type of myeloproliferative neoplasm (MPN) that results in progressive scarring of the bone marrow, leading to constitutional symptoms, decreased hematopoiesis, and increased risk of acute leukemia. Symptomatology and disease progression are driven by constitutive genetic activation of the JAK/STAT pathway. Current treatments target JAK/STAT signaling and alleviate symptoms but fail to alter the natural progression of disease. Hyperactive cytokine signaling though JAK/STAT promotes disease progression by stimulating pro-fibrotic signals, such as TGF-β, which converts mesenchymal stromal cells (MSCs) into myofibroblasts. Myofibroblasts deposit extracellular matrix, which perpetuates fibrosis. One treatment goal is to prevent/reverse fibrosis and allow for expansion of normal hematopoietic stem cells (HSCs). However, there are currently no treatments that reproducibly inhibit progression of fibrotic diseases, and new treatment strategies are needed in this field. Lactate, which was canonically considered a waste product of glycolytic metabolism, has recently been shown to play a role in several diseases. Interestingly, fibroblasts increase glycolysis and lactate production when they convert to myofibroblasts. In line with these previous observations, my preliminary data suggests that inhibiting lactate export from fibroblasts by pharmacologically blocking monocarboxylate transporter 4 (MCT4) prevents TGF-β mediated myofibroblast differentiation in a cell autonomous manner. I also demonstrate that MCT4 is dramatically increased in bone marrow samples from myelofibrosis patients compared to non-fibrotic bone marrow samples. Together, these findings led me to hypothesize that lactate export through MCT4 is necessary for TGF-β mediated MSC-to- myofibroblast differentiation and disease progression in MF. To test this hypothesis, I propose three specific aims. First, I will determine the necessity of MCT4 for bone marrow MSC-to-myofibroblast differentiation. This will include pharmacologically manipulating the fate of pyruvate under different nutrient conditions to mechanistically understand how pyruvate-lactate metabolic axis alters MSC and myofibroblast phenotype. Second, I will test the relevance of MCT4 levels and function to MF pathophysiology in vivo. To do this, I will utilize human samples from pre-fibrotic MPN patients and compare them to patients with MF, to test the hypothesis that increased MCT4 expression correlates with fibrotic disease progression. Using a well characterized mouse model of MF, I will test whether MCT4 inhibition alleviates disease and prolongs survival. In my third aim, I will use unbiased single cell RNA-sequencing and 13C-glucose tracing to understand how different signals, such as TGF-β and mechanical tension, alter metabolism and lead to myofibroblast differentiation. This research proposal will determine whether MCT4 is a viable clinical target for myelofibrosis and given the recent development of selective MCT4 inhibitors, there is an opportunity for translation to the clinic and direct impact on patient care.

Project Narrative: Myelofibrosis (MF) is a poorly understood and fatal disease with few treatment options that results from progressive bone marrow scarring. Here, I demonstrate that prevention of lactate export, via inhibition of Monocarboxylate Transporter 4 (MCT4), prevents myofibroblast differentiation, the effector cells of fibrosis. I propose testing how MCT4 inhibition attenuates myofibroblast differentiation in vitro, in human patient samples, and mouse models of myelofibrosis to mechanistically understand how the pyruvate-lactate metabolic axis influences fibrotic disease.