Effects of Elevated Lactate in the Bone Marrow Microenvironment during Acute Myeloid Leukemia

Leukemia drives dysfunction of the hematopoietic bone marrow microenvironment (BMME), however, the mechanisms are poorly understood. Loss of normal hematopoiesis accounts for much of the morbidity and mortality (~29% survival at 5 years) of acute myeloid leukemia (AML). To define extracellular metabolomic changes in the bone marrow during disease, we assayed global metabolite levels using liquid chromatography coupled with mass spectrometry (LC-MS). This unbiased analysis demonstrated that lactate levels are elevated in the bone marrow supernatant of AML patients at diagnosis compared to healthy controls (mmol/L = 3.62 vs 1.31, p < 0.05, n = 5). Therefore, we have focused on the effects of lactate in the hematopoietic BMME and on AML cells. We hypothesized that lactate secreted to the BMME by AML cells contributes to bone marrow dysfunction and leukemia progression including loss of normal hematopoiesis. To test this, we used both: (i) a murine model of blast crisis chronic myelogenous leukemia (bcCML), which recapitulates the metabolomic analysis of the human AML BMME, and (ii) transgenic mice with a global knockout of the G-protein-coupled lactate receptor GPR81 that do not display hematopoietic defects. Bone marrow support for hematopoiesis was assayed by utilizing cocultures of murine hematopoietic stem and progenitor cells (HSPCs) maintained in vitro by a monolayer of mesenchymal stem cells (MSCs) and macrophages. Exposure to physiologically-relevant elevated lactate levels resulted in a significant reduction in the ability of HSPCs to form colonies in methylcellulose-containing media (CFU-C =12.17 vs 1.167, p < 0.05, n = 3). Next, we wanted to establish which critical HSPC-supportive cells in the BMME are altered by excess lactate. Elevated lactate levels reduced murine MSC colony-forming ability (area in pixels = 330146 at 0 mmol/L vs 146325-18502 at 10-15 mmol/L, p < 0.05, n = 3). In bcCML, leukemia-associated macrophages (LAMs) were found to overexpress the mannose receptor CD206, and GPR81 signaling contributed to this phenotype in vivo (CD206 MFI = 5784 vs 2633, p < 0.05, n = 3). The contribution of lactate signaling to this phenotype was confirmed by lactate treatment of wild-type or GPR81-/- bone-marrow-derived macrophages in vitro. These data together suggest that lactate regulates multiple components of the HSPC niche to drive leukemia-associated hematopoietic dysfunction. To determine if LAMs adversely affect hematopoiesis, the HSPC support assays were repeated but with the addition of equal numbers of LAMs or normal macrophages to the coculture. Addition of LAMs reduced HSPC colony-forming ability compared to normal macrophages (mean fold-change CFU-C normalized to non-leukemic control = 1.00 vs 0.63, p < 0.0001, n = 4). Furthermore, this effect is reduced when LAMs from bcCML in GPR81-/- mice are added, compared to LAMs from bcCML in wt mice (mean fold-change CFU-C normalized to non-leukemic control = 0.69 vs 0.78, p < 0.05, n = 2). This suggests that GPR81 signaling in the BMME polarizes macrophages to a LAM phenotype that has reduced support for hematopoiesis. Finally, leukemic progression was substantially reduced by mid-stage disease when bcCML was initiated using GPR81-/- leukemic cells (% leukemic cells in bone = 45.54 vs 11.75, p < 0.05, n = 5). To assay the involvement of GPR81 signaling on cancer stem cell ability, serial passaging of bcCML cells in methylcellulose-containing media was performed. The number of viable passages was reduced in GPR81-/- bcCML cells compared to wt bcCML cells suggesting that GPR81-/- cells have less repopulating ability, and that GPR81 functions to maintain an LSC phenotype during AML. This research investigates the role of lactate as a critical driver of AML-induced BMME dysfunction and leukemic progression, thus identifying GPR81 as an exciting and novel therapeutic target for the treatment of this devastating disease. Furthermore, as lactate production is a hallmark of cancer, this mechanism is potentially applicable to multiple malignancies with bone marrow involvement including additional types of leukemia as well as bone marrow metastases of solid tumors.