Metalloproteases Could Be Involved in Erythroid Differenciation in MPN

Several hypotheses have been developed to explain how a unique mutation is related to three different phenotypes in Philadephia negative myeloproliferative neoplasms (MPN). Metalloproteases are a group of proteins involved in matrix remodelling, migration and differentiation processes. Although their role has never been studied in MPN, our group has found a differential expression of Matrix metalloproteinase-14 (MMP14) and CD44 between PV and ET JAK2V617F patients (E. Albizua et al, Ann Hematol. 2011Feb).

The purpose of the present study is to validate MMP14 and CD44 differential gene expression profile between PV and ET using a proteomic-based approach and an in vitro model, with the aim of confirming MMP14 and CD44 as MPN biomarkers as well as novel targeted therapy.

Seventy MPN patients diagnosed by WHO criteria were included in the study: 24 Polycythemia Vera (PV) 24 Essential Thrombocythemia (ET) JAK2V617F, 11 ET JAK2 wild type (JAK2WT) and 11 Primary Myelofibrosis (PMF). In addition 24 healthy donors were used as controls.

Peripheral blood was analyzed by flow cytometry (FCM) using anti-CD44 and anti-CD45 antibodies in 10 PV and 10 ET. Fifty-five bone marrow biopsies (11 PV, 11 ET JAK2V617F, 11 ET JAK2WT, 11 PMF and 11 healthy controls) were processed to perform immunohistochemistry (IHC) with anti-MMP14 and anti-CD44 antibodies (R&D). Finally an in vitro MPN model was performed. Mononuclear cells from 10 MPN (5 PV and 5 ET) and 5 healthy controls were extracted and seeded in Methocult with IL-3, SCF and EPO. MMP14 was inhibited by Marimastat at 50μM, 25μM and 10μM; and CD44, with anti-CD44 antibody at 10mg/ml, 1mg/ml and 0.1mg/ml. Results were analyzed by BFU-E count, viability study by trypan blue and FCM employing anti-CD45, anti-CD41, anti-CD34, anti-CD71, anti-CD44, and Annexin antibodies. BFU-E DNA was extracted to analyze JAK2V617F allele burden by RT-PCR.

Intracellular proteins, including phospho-proteins p38, P-p38, MEK, P-MEK, STAT1, P-STAT1, AKT and P-AKT were studied by cytometric bead array multiplexed bead-based immunoassay (CBAs) technique.

The Mann-Whitney non-parametrical statistical hypothesis test was used to assess the statistical significance of our results.

First of all, FCM of the granulocytes showed a slight over-expression of CD44 in PV population versus ET.

Secondly, IHC also showed MMP14 over-expression in megakaryocites of PV (100% positive patients, 60% positive megakaryocites), and ET (100% positive patients 73% positive megakaryocites) compared to controls (36% positive subjects, 2% positive megakaryocites). Additionally 45% ET JAK2WT were MMP14 positive (20% positive megakaryocites).

Significant differences in inhibition of BFU-E growth and cell proliferation were found comparing cultures either under anti-MMP14, Marimastat (50 μM and 25 μM) or under anti-CD44 (10 mg/ml and 1 mg/ml-) to cultures under no inhibitory treatment (P=0.05 and P=0.037 respectively). FCM of BFU-E cultures pointed to a significant decrease of CD71 (erythroid population) with any of the inhibitors.

Over-expression of CD44 in PV compared to ET was observed and confirmed in BFU-E by FCM.

All phospho-proteins studied halved after anti-CD44 (1 mg/ml) and Marimastat treatments (25 μM).

Our results suggest that MMP14 and CD44 could play a role in erythroid differentiation by JAK-STAT, MAPK and AKT pathways. Differences between ET and PV may be caused by both molecules, which may contribute to phenotypic divergence. Additionally, MMP14 IHC could become a useful diagnostic tool in, at least, 45% of ET JAK2WT. Our results suggest that MMP14 and CD44 could become future therapeutic targets and could help to MPN diagnosis.

No relevant conflicts of interest to declare.