Lactate As Metabolic Link between Cancer Cells and Tumor Microenvironment in Myelofibrosis Patients
Authors: Giallongo Cesarina , Spampinato Mariarita , La Spina Enrico , Parrinello Nunziatina Laura , Longhitano Lucia , Caltabiano Rosario , Salvatorelli Lucia , Romano Alessandra , Barbato Alessandro , Di Rosa Michelino , Lazzarino Giacomo , Li Volti Giovanni , Di Raimondo Francesco , Tibullo Daniele , Palumbo Giuseppe A. 
Published: November 2020

Introduction Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by hematopoietic/stem cell-derived clonal myeloproliferation leading to cytopenia/cytosis, splenomegaly and bone marrow (BM) fibrosis. The alteration of haematopoiesis associted with BM fibrosis is deeply associated with profound modifications of the BM microenvironment, as a consequence of a defective balance between the vascular niche and the endosteal niche, associated to megakaryocytes, endothelial and mesenchymal stromal cells (MSC) dysfunction through the production of a variety of profibrotic, angiogenic, and pro-inflammatory cytokines, including megakaryocyte-derived PDGF, TGF-beta and osteoprotegerin, IL-6, PDGF, RANTSs, BMP-2, and which can trigger auto-immune mechanisms, chronic inflammation and oxidative stress status. It is well-known that oncogenic lesions can switch the bioenergetics of malignant cells from OXPHOS to glycolysis (Warburg effect) with lactate production, a phenomenon clinically relevant, particularly in PMF where the amount of the enzyme lactate dehydrogenase (LDH) is an established biomarker of leucocyte turnover and an independent biomarker of overall and leukemia-free survival. The shuttling of the main LDH metabolite lactate is implicated in the interplay of cancer cells with neighboring stromal cells which become glycolytic and export lactate. In turn, lactate is taken up by cancer cells and used for oxidative metabolism, to drive angiogenesis in endothelial cells and to inhibit T- cell function. Moreover, there are evidences that lactate may be involved in the promotion of the fibrosis increasing the TGF-beta levels. In this work we aimed to investigate the role of lactate in the BM myelofibrosis. Results. From microarray datasets, we selected 34 PMF patients carrying the JAK2V617F mutation, 28 JAK2 wild-type patients and 16 healthy donors (HD). Our analysis showed that SLC16A1 (MCT1), SLC16A3(MCT2) and SLC16A7 (MCT4) genes were upregulated in patients in respect to healthy donors in a JAK2V617F mutation independent manner. Furthermore, we demonstrated a significant increase of lactate concentration in PB sera from PMF patients compared to HD, associated to higher percentage of circulating granulocyte- and monocyte-myeloid derived suppressor cells (Gr- and Mo-MDSCs), and Treg. Moreover, IDO, LAG3, BTLA, PDL-2, TIM-3 and CD152 levels were significantly increased in PMF sera compaterd to HD ones. To demonstrate that lactate could play a role in driving cancer immune evasion in PMF, healthy peripheral blood mononucleated cells (PBMCs) were incubated in presence of lactate. After 3 days we observed a significant increase of the percentage of Mo-MDSCs, Treg, CD4+PD1+ and CD8+PD1+ lymphocytes. The same results were obtained after incubation of PBMCs with sera from PMF patients. No effects were observed using HD sera. Interestingly, the percentages of Treg and Mo-MDSCs increased after exposure to PMF sera were significantly reduced in presence of the inhibitor of lactate transporter AZD3965. To investigate the role of lactate in the PMF microenvironment, we next exposed healthy MSCs to lactate for 48h. Treated MSCs assumed a CAF-like phenotype increasing expression of aSMA, FAP1 and TGF-beta. Moreover, Masson's trichrome staining showed an increase of collagen deposits in BM-MSCs associated to increased release of IL6, TGF-beta, MMP2, MMP9 and RANTES. Also, exposure to PMF sera induced higher collagen deposits in MSCs and this effect was reverted adding AZD3965. As BM fibrosis is frequently accompanied by osteosclerosis, we also investigated the effects of lactate on ostegenic differentiation of BM-MSCs. After 10 days of treatment with lactate, the BM-MSCs showed a morphological change associated to increased osteogenic gene markers such as BMP2, RUNX2 and SPARC (osteonectin), and higher released levels of calcitonin, BMP-2, MCP-1, sRANKL and osteoprotegerin. Conclusion Our results demonstrate that lactate might be involved in the immune impairment and BM fibrosis of PMF. The inhibition of lactate production and shuttles in myelofibrosis may be a strategy not only to inhibit invasive and metastatic behavior of cancer cells, but also to restore the anti-cancer immune response improving the results of therapy in MF patients.

Disclosures
Romano: Novartis: Honoraria; Takeda: Honoraria. Di Raimondo:Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; GSK: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen, Takeda, Novartis: Honoraria; Celgene: Consultancy, Honoraria; GILEAD, Incyte: Research Funding. Palumbo:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.