Mechanisms of acquired and familial thrombocytosis – Thrombocytosis

The aim of this proposal is to identify the cellular and molecular mechanisms driving sporadic and familial forms of essential thrombocythemia (ET), a disorder of platelet production, through the use of next generation sequencing. Our project is based on the hypothesis that both familial and sporadic ET share common mechanisms, such as those affecting the MPL-TPO signaling axis.
We propose to
1) use cellular and biochemical approaches to sub-classify a cohort of 70 sporadic ET patients devoid of mutations known to drive ET development. The sequence of MPL, JAK2, LNK and CBL genes will first be verified, as the clonality of the granulocyte population in female patients. Our preliminary results identify three ET subgroups: one resembling the JAKV617F-positive patients (normal MPL expression and TPO level in vivo, spontaneous megakaryocytic growth in vitro inhibited by JAK2 inhibitors), and two other subgroups, which both exhibit MPL expression abnormalities but differ by TPO retention in platelets. Analyses of this series of patients will be completed. The mechanisms affecting the MPL-TPO axis in patient samples with MPL expression abnormalities will be investigated by studying the expression of miRNAs targeting MPL and MPL trafficking.
2) compare, by exome sequencing using Next Generation Sequencing approach, the coding sequence of clonal (granulocytes) to matched germ line control (CD3+ lymphocytes) to identify acquired causal mutations. In a few patients, the coding sequence of megakaryocytes will also be compared to granulocytes. Mutations will be validated by Sanger analyses. Their recurrence will be investigated in the ET cohort and other sporadic and familial MPDs collections.
3) apply, in two large families, the same exome sequencing strategy to identify the genetic predisposition to ET development and susceptibility to malignant transformation. Comparison of 5 affected subjects at different steps of the disease and one unaffected will allow identification of a gene important for hematopoietic differentiation. Its involvement in both sporadic and familial MPDs will be investigated.
4) investigate the molecular mechanism of a mutated form of MPL previously identified in familial thrombocytosis (MPLP106L) using cellular, biochemical approaches and also mouse models. These experiments will pave the way for the analysis of mutations (identified in 2 and 3) that affect the MPL-TPO signaling axis. From two selected patient samples, we will establish induced pluripotent stem cell lines (iPSC) to facilitate the analyses of mutations affecting MPL trafficking and their impact on megakaryocytic differentiation.
Identification of novel genes mutated in sporadic and familial ET will provide new approaches for diagnosis and therapeutics, and will fuel basic researches on hematopoiesis