Identification of new risk factors involved in the progression of Philadelphia Negative Chronic Myeloproliferative Neoplasms

Zusammenfassung

Introduction: Myeloproliferative neoplasms are hematological disorders characterized by the clonal expansion of myeloid cell lines in the bone marrow (BM), leading to excessive blood cell production. They are classified as polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF). These three constitute the classic Philadelphia-negative myeloproliferative neoplasms (MPN), as they lack the BCR::ABL1 fusion gene, seen in chronic myeloid leukemia (CML). MPN patients share clinical features like splenomegaly, constitutional symptoms, and a higher risk of thrombotic events. The median survival varies among the subtypes, with MF having the lowest. Mutations in JAK2, CALR, and MPL are key drivers in MPN, activating the JAK/STAT signaling pathway. Inflammation plays a crucial role in the development and progression of MPN, contributing to symptoms, fibrosis, and disease progression. Chronic inflammation can also lead to cardiovascular complications and increase susceptibility to other neoplasms. MiR-146a has been identified as a regulator of the inflammatory NF-κB pathway, with dysregulation linked to various inflammatory diseases. Mice lacking miR-146a (KO) develop a myeloproliferative and fibrotic MF-like phenotype, offering a model to study inflammatory mechanisms in the absence of driver mutations. Understanding the relationship between inflammation and malignant processes is crucial for developing effective treatments for MPN patients. Objectives: To investigate the role of inflammation, specifically the NF-κB pathway, in the progression of MPN. Three specific objectives include studying the impact of MIR146A miR-SNPs on susceptibility and progression to advanced stages of MPN, determining if inhibition of NF-κB and/or JAK/STAT signaling can reverse the MF-like phenotype in aged KO mice, and evaluating if inhibition of JAK2/IRAK1 can prevent the development of MF-like phenotype in younger KO mice. Material and methods: To this end, the relationship between both NF-κB and the JAK/STAT pathways was examined in patients with MPN, as well as in the miR-146a-KO murine model and a cellular model. DNA from patients and controls was collected for genotyping, and plasma cytokines were analyzed. Aged KO mice, exhibiting the phenotype, were treated either with ruxolitinib (JAK1/2 inhibitor), BMS-345541 (IKKα/β inhibitor), both inhibitors in combination, or pacritinib (JAK2/IRAK1 inhibitor) to reverse the phenotype, while younger mice, prior MF-like phenotype, were treated with pacritinib to prevent or delay phenotype development. A complete blood count, plasma cytokine quantification, spleen study (macroscopic, flow cytometry, histological analysis and protein analysis by western blot) and histological study of the femurs BM were performed. The human SET2 cell line was employed as a clonal hematopoiesis model (JAK2V617F). Cultures of these cells were incubated with the same treatments previously described, and protein expression was analyzed by western blot. Additionally, these cells were co-cultured with a stromal line (HS-27A) with the drugs. Collagen (COL1A1) gene and protein expression of HS-27A were analyzed by RT-qPCR and immunofluorescence, respectively. Results: The rs2431697-TT genotype was associated with MF, especially post-PV/TE MF. Patients with this genotype had a lower MF-free survival rate, were more likely to progress to secondary MF, and had elevated levels of inflammatory cytokines. Additionally, miR-146a-KO mice showed increased STAT3 signaling with aging. While all treatments resulted in a reduction of spleen size and a partial restoration of spleen architecture, only NF-κB inhibition led to a reduction of extramedullary hematopoiesis, BM fibrosis and osteosclerosis, along with an attenuation of the exacerbated inflammatory state. However, the combination therapy worsened anemia by inducing BM hypoplasia. Combination therapy and pacritinib reduced NF-κB and JAK/STAT signaling in JAK2-mutated SET-2 cells. Moreover, the combined treatment reduced COL1A1 production in the SET-2+HS-27A co-culture. Pacritinib prevented splenomegaly, BM reticulin fibrosis and osteosclerosis observed in age-matched untreated KO mice. It also avoided the myeloproliferation, loss of splenic architecture, and extramedullary hematopoiesis. Moreover, pacritinib was found to attenuate the pro-inflammatory environment, preventing the increase of inflammatory cytokines (CXCL1 and TNFα) without inducing cytopenias, but rather the opposite. Pacritinib-treated mice exhibited higher platelet counts, while erythrocyte counts were higher in treated mice with longer treatment durations. Finally, pacritinib reduced COL1A1 production in the co-culture model. Conclusions: This thesis project highlights the importance of inflammation in the development and progression of MPN, particularly MF, as a potential therapeutic target. The rs2431697-TT genotype, associated with lower miR-146a levels, stands as a novel prognostic factor for MF progression. In aged mice with MF-like features, inhibition of NF-κB alone or in combination with JAK/STAT reduces inflammation, splenomegaly, and reverses thrombocytopenia and BM fibrosis. JAK2-mutated human cell line studies showed reduced COL1A1 expression. All this support the therapeutic potential in inflammatory diseases. Early dual inhibition of JAK/STAT and NF-κB in young KO mice prevents MF-like symptoms, also supported by cell line results, emphasizing the benefits of early intervention in disease management.