Signals of the Inflammatory Microenvironment Promote a Mutation-Associated Functional Dysregulation of the Circulating Megakaryocyte Progenitors of Myelofibrosis

Abstract

Introduction: Myelofibrosis (MF), a clonal disorder of the hemopoietic stem/progenitor cell, is characterized by distinctive abnormalities in megakaryocyte (MK) development. A proliferation of dysplastic MK with disturbance of nuclear/cytoplasmic maturation is observed. In addition to driver mutations in Janus Kinase 2 (JAK2) and Calreticulin (CALR) genes, chronic inflammation has emerged as a key-player in MF pathogenesis. Here, we analyzed the potential contribution of crucial factors of the inflammatory microenvironment (Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α and Tissue Inhibitor of Metalloproteinases (TIMP)-1 to MF dysmegakaryocytopoiesis.Methods: We studied 10 patients with MF at diagnosis while 6 patients had received previous treatment for MF. In all cases, therapies had been discontinued for at least two months before peripheral blood samples collection. To characterize the mutational status, JAK2V617F and CALR exon 9 mutations were assessed by quantitative PCR-based allelic discrimination assay and Next Generation Sequencing approach, respectively. To quantify MK progenitors, circulating CD34+CD41+ cells of MF patients (10 JAK2V617F and 6 CALR mutated) and controls (cord blood (CB); 8 cases) were identified by flow cytometry analysis. To evaluate the pro-inflammatory profile, IL-1β, TNF-α and TIMP-1 were measured in plasma of patients (10 JAK2V617F and 6 CALR mutated) and healthy subjects (10 cases) by ELISA. To determine whether and to what extent crucial factors of the inflammatory microenvironment may affect the in vitro growth of MK progenitors, Colony Forming Units-Megakaryocyte (CFU-MK) growth of circulating CD34+ cells from MF patients (6 JAK2V617F and 6 CALR mutated) and CB (6 cases) was assessed with a collagen-based medium in the presence or absence of TIMP-1, TNF-α and IL-1β (alone or in combination). Pure and mixed CFU-MK were quantified on the basis of CD41 immunostaining.Results: We found that, irrespective of mutational status, IL-1β, TNF-α and TIMP-1 plasma levels were significantly increased in MF patients. The frequency of circulating MF-derived CD34+CD41+ cells was significantly higher than the CB counterparts. However, a significantly higher proportion of cells was associated with CALR compared to JAK2V617F mutated patients.The growth of pure CFU-MK from untreated cells of the JAK2V617F mutated patients was significantly decreased as compared to that of CALR mutated patients and the CB counterparts. Factors alone did not stimulate the CFU-MK growth from CB CD34+ cells. Conversely, IL-1β had stimulatory activity on pure MK colony formation of JAK2V617F, but not CALR, mutated patients and the growth of MK progenitors from MF patients was significantly inhibited by TNF-α, irrespective of mutational status. TIMP-1 was ineffective. Factors in combination did not significantly modify the growth of pure CFU-MK from patients/CB as compared with factors alone. The growth of mixed MK colonies from untreated cells was absent (JAK2V617F mutated patients) or very low (CALR mutated patients and CB). Only TNF-α (alone or in combination (IL-1β+TNF-α and IL-1β+TNF-α+TIMP-1)) increases the growth of mixed MK colonies of patients harboring CALR mutation and CB. Interestingly, factors alone or in combination failed to stimulate the growth of mixed MK colonies from JAK2V617F mutated patients.Conclusions: Taken together these results demonstrate that in MF signals provided by the inflammatory microenvironment promote a mutation-associated functional dysregulation of MK progenitors and suggest a mechanism by which chronic inflammation may play a role in the development/maintenance of the abnormal megakaryocytopoiesis. DisclosuresMartinelli:BMS: Speakers Bureau; MSD: Consultancy; Pfizer: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; Roche: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Genentech: Consultancy; Celgene: Consultancy, Speakers Bureau. Cavo:Janssen-Cilag: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Millennium: Consultancy, Honoraria.