INVESTIGATION OF THE ROLE OF IMMUNE DYSFUNCTION IN MYELOFIBROSIS PROGRESSION

• Grant number: 25/03925-2
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: August 08, 2025
• End date: August 07, 2026
• Field of knowledge: Biological Sciences - Biology
• Principal Investigator: Lorena Lôbo de Figueiredo Pontes
• Grantee: Mariana Medeiros
• Supervisor: Steven William Lane
• Host Institution: Hemocentro de Ribeirão Preto. Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da USP (HCMRP). Secretaria da Saúde (São Paulo - Estado). Ribeirão Preto , SP, Brazil
• Institution abroad: QIMR Berghofer Medical Research Institute, Australia
• Associated to the scholarship: 23/01821-0 - Characterization of subpopulations of cytotoxic cells in murine models of myeloid leukemia, BP.DR

Abstract

Myelofibrosis (MF) is a myeloproliferative neoplasm driven by mutations such as JAK2V617F, CALR, and MPL. It can be classified based on bone marrow fibrosis (pre-fibrotic or advanced) and clinical profile (cytopenic or proliferative phase). Advanced cases are associated with worse prognosis and a higher risk of progression to acute myeloid leukemia. Treatment includes JAK1/2 inhibitors and cytoreductive agents, but the only curative option is hematopoietic stem cell transplantation, which is feasible for only a few patients due to age and comorbidities. Beyond JAK-STAT-mediated proliferation, cellular interactions within the bone marrow niche contribute to disease pathogenesis, promoting inflammation, fibrosis, and hematopoietic failure. NK cells in MF patients exhibit an immature phenotype, reduced activation, and impaired hematopoietic regulation. Jak2V617F murine models also show NK cell dysfunction. During my PhD, we observed that a hypermature NK profile (CD57+ and CD57+NKp80+) correlates with increased fibrosis, whereas the cytopenic phase displays a high frequency of CD56bright NK cells and low NKG2D expression in high-risk patients. T cells also exhibit dysregulation, with an increase in terminally differentiated phenotypes and immune evasion markers. Immunotherapy has emerged as a promising approach for hematologic malignancies, including PD-1/PD-L1 blockade and CAR-T cells. However, CAR-T strategies for myeloid diseases face challenges due to the lack of specific targets. Moreover, JAK2 inhibitors, such as ruxolitinib, may impair immune function by reducing antitumor immunosurveillance. Understanding how MF and its treatments affect the immune response may reveal novel therapeutic targets.This study aims to identify immune pathways altered during MF progression using Jak2V617F and Jak2-EZH2 murine models. The Jak2-EZH2 model better recapitulates advanced MF, including significant bone marrow fibrosis and cytopenias. Comparisons between primary and chimeric models will help determine whether NK cell dysfunction is intrinsic to hematopoietic cells or influenced by the leukemic microenvironment. The specific objectives include: (1) characterizing the phenotype and function of NK and T cells in Jak2V617F and Jak2-EZH2 models to investigate immune alterations associated with disease progression; (2) identifying therapeutic targets to restore cytotoxic function based on the immune profile observed in murine models and patient samples; and (3) testing immune rescue strategies in vitro and in vivo, using immunomodulators such as IFN-¿ and immune checkpoint inhibitors. For immunophenotypic characterization, flow cytometry analyses will be performed to assess frequency, maturation, and expression of activating/inhibitory receptors on NK and T cells. Functional analysis will include cytotoxicity assays, degranulation, and cytokine production. Chimeric models will be established through bone marrow transplantation to evaluate whether immune alterations are dependent on the leukemic microenvironment. Additionally, single-cell RNA sequencing will be conducted to identify altered molecular pathways. To assess therapeutic strategies, IFN-¿ will be tested in vitro and in vivo. Isolated NK cells will be evaluated for lytic capacity, degranulation, and IFN-¿ production. In mice, IFN-¿ treatment will be analyzed for its impact on peripheral blood counts, splenomegaly, and bone marrow fibrosis. Previous studies suggest that IFN-¿ can reduce the mutant clone and restore immune functions, making it a potential candidate for immune modulation in MF.