Divergencias genéticas y proteómicas en la fisiopatogenia de las neoplasias mieloproliferativas (NMP)

Abstract

Philadelphia negative chronic myeloproliferative neoplasms (MPN) are clonal haematopoietic stem cell (HSC) disorders characterized by the proliferation of one or more of the myeloid lineage (i.e. granulocytic, erythoid, megakaryocytic). This study is focused in two MPN, polycythaemia vera (PV) and essential thrombocythemia (ET). Both diseases are associated with the acquired somatic mutation of JAK2 V617F: PV in 90% and ET in 50- 60%. However, JAK2 mutation has not showed to be sufficient to develop any of both diseases, and the first or unique clonal event; and moreover, not enough to explain the phenotype divergence between ET and PV. Aims The aim of this work was to investigate genes and proteins involved in the physiopathogenic events and phenotype divergence between PV and ET, in order to obtain a better knowledge of MPN and for a future application as diagnostic tools or therapeutic targets. Methods We performed a gene and protein screening looking for differences between ET and PV at different levels: gene expression (by microarray expression and RT-PCR), epigenetic profile (methylation microarray) and protein expression (by GEL 2D-DIGE/MS). Differential expression of every gene and/or protein was confirmed by proteomic level, immunohistochemistry (IHC), flow cytometry (FCM) and western blot (WB). Additionally, physiopathogenic role of validated proteins, HSP70, MMP14 and CD44 in MPN was confirmed by functional studies (inhibition assays over HEL cell line and burst formation unit erythroid (BFU-E) cultures from mononuclear peripheral blood cells and bone marrow (BM) HSC). Results Gene expression analysis showed MMP14 and CD44 genes differentially expressed in RTPCR between PV and ET. Protein expression analysis by GEL 2D-DIGE/MS demonstrated three proteins with differential expression: HSPA1A (HSP70), SERPINB1 and LTA4H. Additionally, epigenetic analysis showed ZNF577 gene with a differential methylation profile between PV and ET JAK2 V617F. IHC studies resulted in CD44 and SERPINB1 as useful potential biomarkers. CD44 was over-expressed in ET JAK2 wild type granulocytes of BM, and SERPINB1 was over-expressed in MPN (ET, PV and primary myelofibrosis). Functional studies showed BFU-E growth and viability cell decrease after MMP14 inhibition (Marimastat), CD44 antibody blocker (C7923) and HSP70 inhibitor (KNK437). Of interest, Marimastat and C7923 produced specific erythroid lineage decrease, whereas KNK437 reduced lineage in the same way. However, cultures after KNK437 treatment showed a high percentage of apoptotic erythroid cells, especially in PV. In C7923 assay, inhibition was produced via pMEK and pP38; in Marimastat assay inhibition was produced via pMEK, pP38, pSTAT and pJAK2; and in KNK437 assay inhibition was produced via pMEK and pJAK2 Conclusions CD44 and SERPINB1 are potential biomarkers and could be used as diagnostic tool in ET JAK2 wild type and MPN respectively. Furthermore, CD44, along with MMP14, could be playing a key role in erythroid proliferation by MAPK and JAK-STAT pathway activation. They are attractive therapeutic targets in MPNs. Finally, HSP70 could be playing a key role in physiopathology and phenotypic divergence between PV and ET by JAK-STAT pathway activation, providing higher survival over erythroid lineage. In this way, HSP70 could become a promising therapeutic target in PV, and KNK437 a potential treatment.