Crucial Factors of the Inflammatory Microenvironment Promote Maintenance of the Malignant Hemopoietic Clone of Myelofibrosis By Stimulating Survival and Inhibiting Proliferation of CD34+ stem/Progenitor Cells
Authors: Catani Lucia , Rossi Lara , Sollazzo Daria , Franchini Eugenia , Romano Marco , Zuffa Elisa , Barone Martina , Perricone Margherita , Polverelli Nicola , Ottaviani Emanuela , Martinelli Giovanni , Vianelli Nicola , Palandri Francesca 
Published: November 2019

Abstract
Introduction: Myelofibrosis (MF) is an acquired clonal disorder of the hematopoietic stem/progenitor cell (HSPC). Mutations in JAK2, Calreticulin (CALR) and MPL genes are associated with MF. MF is characterized by a state of chronic inflammation and it is argued that the up-regulated production of proinflammatory cytokines by both HSPCs and the surrounding stromal cells generates a microenvironment that selects for the malignant clone. Interestingly, recent findings showed that HSPCs actively sense pro-inflammatory factors. However, the key players linking inflammation and cancer in MF as well as the underlying pathogenetic mechanism are still to be defined. Extracellular nucleotides (ATP) and selected cytokines, such as Tumor Necrosis Factor (TNF)-alpha or the Tissue Inhibitor of Metalloproteinases-1 (TIMP-1), are crucial factors of the inflammatory microenvironment. Here we investigated the in vitro effects of ATP, TNF-alpha or TIMP-1 on phenotype/function of HSPCs from MF patients.
Methods: Circulating CD34+ HSPCs from MF patients with JAK2V617F (7 cases), CALR (3 cases) and MPL (3 cases) mutations or from cord blood (5 samples) have been phenotypically and functionally characterized at baseline or after in vitro incubation with or without increasing doses of ATP, TNF-alpha or TIMP-1 (alone or in combination) at different time points. The expression of selected hemopoietic/endothelial markers (CD45,CD133, CD41, CD105, KDR, CD184,CD44) has been analyzed on CD34+ cells by flow cytometry. The expression of the TIMP-1 receptor CD63 was also assessed. Immunomagnetically isolated CD34+ cells were stained with Annexin-V/Propidium Iodide and survival/apoptosis were analyzed by flow cytometry. Migration of purified CD34+ cells were also assayed towards a CXCL12 gradient (150ng/mL) in transwell chambers. Clonogenic assay (BFU-E and CFU-GM in vitro growth) of CD34+cells was also tested.
Results: We found that the survival of MF CD34+ cells was strongly stimulated by TNF-alpha (10 ng/mL; 2,9 fold increase) as compared with that of cord blood-derived CD34+ cells (1,7 fold increase). TIMP-1 (100ng/mL) treatment also promotes the survival of MF CD34+ cells. Accordingly, MF patients show increased number and percentages of circulating CD63+CD34+ cells. However, at variance with cord blood-derived CD34+ cells, MF CD34+ cells were more resistant to the citotoxic activity of high dose ATP (1000 nM). Interestingly, when we tested ATP, TNF-alpha and TIMP-1 in combination, the survival of CD34+ cells from MF patients was mainly promoted by the TNF-alpha/TIMP-1 treatment. At variance, ATP addition reduced the TNF-alpha or TIMP-1 pro-survival activity. We also found that TNF-alpha (1,6 fold increase) and TIMP-1 (1,4 fold increase), tested alone, slightly increased the BFU-E formation as compared with that of cord blood- derived CD34+ cells (2,2 and 2,0 fold increase, respectively), without affecting the number of CFU-GM progenitors. By contrast, the number of BFU-E and CFU-GM was significantly reduced when CD34+ cells were incubated in the presence of ATP+TNF-alpha (4,5 and 2,1 fold reduction as compared with untreated cells, respectively), whereas TIMP-1 addition slightly reduced the inhibitory activity of ATP+TNF-alpha (4,0 and 1,8 fold reduction, respectively). Of note, survival and proliferation of MF CD34+ cells were independent from the occurrence of JAK2, MPL or CALR mutations. In addition, despite CXCR4 (CD184) expression of CD34+ cells was significantly increased as compared with the cord blood-derived counterparts, when we analyzed the in vitro migration of CD34+ cells toward a CXCL12 gradient, we found the migration rate of MF CD34+ cells to be highly heterogeneous, ranging from 0,9 to 2,9 fold increase as compared with the corresponding untreated samples. Interestingly, CD34+ cells from 2 out of 3 CALR+patients showed the highest migration capacity.
Conclusions: Here we demonstrated that crucial factors of the inflammatory microenvironment abnormally modulate the HSPC functions of MF and may contribute to the maintenance of the malignant clone. Specifically, TNF-alpha together with TIMP-1 promote the survival of the CD34+ HSPCs of MF, while ATP/TNF-alpha combination reduces their proliferation. Interestingly, the inflammatory factors tested in combination differentially modulate the proliferation of the MF CD34+ cells as compared with the factors alone.

Disclosures
Martinelli: NOVARTIS: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.