PB1282 - ETV6 knockout leads to impaired megakaryoid gene regulation, which is phenocopied by the ETS-domain mutant ETV6R369W via dominant negative actions

Tuesday, June 25, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Presenting Author(s)

HZ

Huan Zhang

PhD student
Sanquin Research Amsterdam
Amsterdam, Noord-Holland, Netherlands

Co-Author(s)

SR

Simon Rouschop

Master student
Sanquin Research Amsterdam
Amsterdam, Noord-Holland, Netherlands
AS

Arthur Flohr Svendsen

PI
Sanquin Research Amsterdam
Amsterdam, Noord-Holland, Netherlands
Cv

Carmen van der Zwaan

Senior technician
Department of Molecular Hematology, Sanquin Research, Amsterdam, the Netherlands
Amsterdam, Noord-Holland, Netherlands
MH

Marten Hansen

Postdoc
Sanquin Research Amsterdam
Amsterdam, Noord-Holland, Netherlands
Arie J. Hoogendijk

Principal investigator
Molecular Hematology, Sanquin Research, Amsterdam, the Netherlands
Amsterdam, Noord-Holland, Netherlands
JG

Jacques Ghysdael

PI
Institut Curie
Orsay, Ile-de-France, France
EH

Elise Huisman

Dr
Erasmus Medical Centre Rotterdam
Rotterdam, Zuid-Holland, Netherlands
Ev

Emile van den Akker

PI
Sanquin Research Amsterdam
Amsterdam, Noord-Holland, Netherlands

Background: Megakaryopoiesis is a process controlled by temporal expression of different transcription factors. TEL/ETV6 is an ETS family transcription factor that recently has 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 the central domain. DNA binding is via the conserved ETS domain, where most clinically relevant mutations are located.

Aims: Here we aimed to unravel the role of ETV6 and patient-specific mutants thereof in megakaryopoiesis.

Methods: CRISPR-editing was used to generate homozygous knockout and heterozygous ETV6 expressing Meg-01. ETV6R369W mutation was studied through lentiviral expression in both the ETV6 wildtype and knockout clones. Immunoprecipitation, RNA-sequencing and flow cytometry were used to unravel the mechanisms behind this pathology.

Results: Consistent with the repressive function of ETV6, RNA sequencing analysis of ETV6 knockout cells revealed 507 upregulated and 292 downregulated genes compared to wildtype cells. Erythroid genes GYPA, FUT1 and ALAS2, were downregulated, putatively caused by increased expression of FLI1, a critical megakaryopoiesis driver. Signalling pathways genes established in stem cell identity, such as KIT, CD44, IL1R, IL6R, were upregulated and validated by flow cytometry. A conflict in cellular identity may contribute to abnormal megakaryocyte differentiation and maturation. Increased expression of several genes was confirmed by proteomics in platelets of patients bearing ETV6R369W mutation. Lentiviral expression of ETV6R369W in wildtype Meg-01 clones resulted in decreased GYPA expression, with levels comparable to ETV6 knockout clones. Co-immunoprecipitation experiments indicated an interaction between ETV6WT and ETV6R369W, suggestive of dimerization and a possible basis of the dominant negative action.

Conclusion(s): In conclusion, here we find that ETV6 knockout leads to a significant deregulation of genes during megakaryopoiesis, while the data suggests a dominant negative mode of action for ETV6R369W. The mechanistic and molecular insights allow better understanding of the ETV6-mutant-induced thrombocytopenia.