Molecular signature of ETV6-related inherited thrombocytopenia – MOST

ETV6 is an ETS family transcription factor that controls the maintenance of hematopoietic stem and progenitor cells (HSPC) and thrombopoiesis that leads to the formation of blood platelets. Using exome sequencing, we were one of the top three groups in the world to identify pathogenic variations of the ETV6 gene in 6 unrelated European families. Patients harboring pathogenic germline monoallelic mutations in ETV6 present moderate thrombocytopenia and unexpected bleeding. They also have an increased lifetime risk of acquiring, typically pre-B cell leukemia, myeloid malignancies including myelodysplasia. How ETV6 variations negatively affects megakaryocyte (MK) differentiation, platelet production and leukemogenesis is poorly understood. Our previous results support a role for epigenetic changes driving transcriptional dysregulation in ETV6 deprived cells.
Our hypothesis is that, due to ETV6 loss and the resulting epigenetic changes, dysregulated expression of key genes play a crucial role at a pivotal stage of megakaryocytes differentiation leading to proliferation and polyploidization defects and reduced platelet formation.
Presuming that ETV6 mutations would have selective effects at different levels of megakaryopoiesis, we will use single cell (sc) RNA-seq to examine transcriptome heterogeneity of HSPC-derived megakaryocytes and peripheral blood mononuclear cells issued from controls and ETV6 mutation carriers (Workpackage 1). While scRNA-seq alone will indicate about the consequences of ETV6 mutation in subset of cells and will precise the level of differentiation arrest and the gene expression signature linked to this blockage or defect, we could learn about direct, as well as indirect (epigenetic) effects of ETV6 inactivation by combining scRNA-seq with ChIP-seq. ChIP-seq analysis of ETV6 and epigenetic marks in mutated, as well as in wild-type megakaryocytes will complete the description of the different affected gene pathways looking at the responsiveness to ETV6 and epigenetic changes (Workpackage 1).
Patient-derived induced pluripotent stem cells (iPSc) are currently being generated. These cells produced in large quantities will offer the possibility, thanks to genome editing techniques, to verify the function of selected candidate genes and the effects of their modulation in the same genetic background as the patients (Workpackage 2).
The MOST research program will contribute to better understand the physiological and pathological differentiation of MKs in ETV6 related disorder. We expect i) to identify key deregulated genes involved in ETV6-deficient megakaryopoiesis. Those genes may represent potential targets for diagnosis, prognosis or therapy purposes, ii) to demonstrate that dysregulated expression of targeted genes plays a pivotal role at a key stage of megakaryocytes differentiation, iii) to implement iPSC tools, which will be particularly useful to perform functional studies and confirm the contribution of the identified targets.
The MOST program brings together experts in megakaryopoiesis, single cell analysis and iPSc to develop a comprehensive view of the mechanisms leading to a poor prognosis thrombocytopenia. The support provided by the ANR will bring a vision never reported before on the ETV6 related thrombocytopenia and will thus strongly reinforce the development of the research group of the MOST project coordinator.