HDAC8 Overexpression in Mesenchymal Stromal Cells from JAK2⁺ myeloproliferative Neoplasms: A New Therapeutic Target?

JAK2-V617F mutation in hematopoietic stem cells (HSC) is a common finding in myeloproliferative neoplasms (MPNs). Although alterations in the hematopoietic microenvironment have been described in these entities, information on the functional and genetic characteristics of bone marrow (BM) derived mesenchymal stromal cells (BM-MSC) from JAK2+ MPNs patients is scarce. The aim of the current study was to characterize and compare BM-MSC from 24 MPNs patients with JAK2V617F mutation (14 BM-MSC from essential thrombocythemia-ET and 10 BM-MSC from polycythemia vera-PV) with those from 14 healthy donors-HD. For this purpose BM-MSC expansion, multilineage differentiation, apoptosis, inmunophenotyping, gene expression profiling, RT-PCR and Western Blot analysis were performed.

Compared with HD, BM-MSC from MPNs patients showed similar morphology and differentiation capacity, but an increased proliferation rate with less apoptosis cells. BM-MSC from MPNs expressed comparable levels of CD73, CD44, CD90 and CD166, whereas they were negative for hematopoietic markers. The median expression of CD105 was lower in BM-MSC from MPNs patients (p <.05) when compared with BM-MSC from HD. Gene expression profile of BM-MSCs from 8 JAK2V617F (4 PV/4 TE) patients, and from 10 HD showed a total of 169 genes that were differentially expressed in BM-MSC from MPNs patients compared to HD. RT-PCR was performed in two genes to confirm these results, demonstrating that HDAC8 and CXCL12 genes were up-regulated. To analyze whether these changes in MPNs-MSC conferred an alteration in their functional capacity, co-cultures with CD34⁺ cells from MPNs and BM-MSC were performed. A significant increase in the CFU-GM clonogenic supporting capacity of MPNs-MSC when compared with HD-MSC was observed. To evaluate whether a Histone deacetylase (HDAC) inhibitor could modify the behavior of MPNs-MSC an HDAC8 specific inhibitor, PCI-34051 was used. A decrease in HDAC8 gene (RT-PCR) and protein (WB analysis) expression was observed in BM-MSC from MPNs treated with PCI-34051 at a concentration of 25µM for 48 hours. HDAC8-selective inhibition also induced a cell cycle arrest in the MPNs BM-MSC with an increase of the proportion of apoptotic cells. To assess the impact of this inhibition on the capacity of MPNs-MSC to support hematopoiesis, BM mononuclear cells (BM-MNC) were co-cultured in transwell for 48 hours with PCI-34051-treated and non-treated BM-MSC. After co-culture, cell viability, clonogenic (CFU-GM) assays and TP53 expression were analyzed. A decrease in cell viability (p=0.028) and CFU-GM (p=0.018) was demonstrated when BM-MNC from MPNs had been in culture with MPNs BM-MSC treated with the HDAC8 inhibitor, as well as an increase in TP53 expression.

These results suggest that MPNs-MSC display different proliferative rate, MSC markers, gene expression profile and HDAC8 overexpression compared to HD-MSC. The inhibition of HDAC8 expression by its specific inhibitor decreases the capacity of the stroma to support hematopoietic cells from MPNs patients, suggesting that HDAC8 may be a potential therapeutic target in this setting.