PB0387 - Characterising the molecular pathways controlled by ETV6 during megakaryopoiesis

Background: Megakaryopoiesis is a process where haematopoietic stem cells differentiate to megakaryocytes and platelets, which is controlled by different transcription factors. TEL/ETV6, an ETS family transcription factor, that besides its role in haematopoietic stem cell maintenance, has recently been discovered to be associated with autosomal dominant thrombocytopenia. ETV6 mainly functions as a transcriptional repressor through dimerisation via pointed (PNT) domain and interactions with other cofactors via central domain. DNA binding is via the conserved ETS domain where most clinically relevant mutations are found.

Aims: To unravel the underlying molecular functions of ETV6 in megakaryopoiesis

Methods: RNA sequencing analysis was performed on homozygous and heterozygous ETV6 knock out generated using Meg-01, a megakaryocytic leukaemic cell line. Differentially expressed genes were validated by flow cytometry and mass spectrometry.

Results: RNA sequencing analysis of ETV6 knock out cells, comparing to wild type, showed 507 up regulated and 292 down regulated genes, which is consistent with the transcriptional repressive function of ETV6. Genes related to erythrocyte development, such as GYPA and ALAS2, were down regulated, which could be explained by an increased expression of another ETS family transcription factor, FLI1, that is critical in driving megakaryopoiesis. Genes related to cell signalling pathways, such as KIT, CD44, IL1R, IL6R, that are crucial in stem cell identity establishment, were upregulated, which conflicts with FLI1 driven megakaryocyte development. These upregulated RNA’s were validated by flow cytometry. Increased expression of several genes was confirmed using mass spectrometry based proteomics performed on patient platelets with ETV6 mutations (e.g. CD44).

Conclusion(s): This conflict in cellular identity may contribute to abnormal megakaryocyte differentiation and maturation. Findings in this study will contribute to a better understanding of megakaryopoiesis, and identification of mechanisms that drive faulty megakaryopoiesis by ETV6 mutant, which will potentially lead to better treatment of ETV6 related thrombocytopenia.