OC 55.3 - FLI1Met100* mutation accelerates megakaryocyte differentiation and attenuates maturation

Tuesday, June 25, 2024

10:00 – 10:15 ICT

Room: 110 A-C

Presenting Author(s)

HZ

Huan Zhang

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

Co-Author(s)

Rv

Roel van Rhijn

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
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: Megakaryocytes and platelets arise from hematopoietic stem and progenitor cells, a process orchestrated by various transcription factors including FLI1, an ETS family transcription factor. The deletion of FLI1, along with other genes, is associated with a bleeding phenotype in Paris-Trousseau Syndrome. FLI1 mutations, predominantly in the ETS DNA-binding domain, have been identified in individuals with inherited thrombocytopenia.

Aims: Here we examined a de novo nonsense heterozygous mutation, FLI1Met100* (c.297del), utilizing the megakaryoblast leukemic line Meg-01. We hypothesized that this mutation produces a truncated protein with a dominant negative effect.

Methods: To explore the mutation’s impact on megakaryopoiesis, we overexpressed both the wild type FLI1 (FLI1WT) and the mutant FLI1Met100* in Meg-01 cells and conducted bulk RNA sequencing and proteomics.

Results: Western blot analysis confirmed the presence of the truncated protein after overexpressing FLI1Met100* in Meg-01 and HEK293T cells. Gene set enrichment analysis indicated that FLI1WT overexpression elevates megakaryocytic markers and suppresses erythroid features, whereas FLI1Met100* upregulates both compared to control. This was confirmed by proteomics. Despite the upregulation of certain megakaryocytic features, a subset of genes crucial for megakaryocyte and platelet function, such as GP6, FN1 and PECAM1 (CD31) and CD44, remained unaffected by FLI1Met100*. Flow cytometry validated these findings, showing enhanced CD41 expression by both FLI1WT and FLI1Met100*. In contrast, the erythroid marker CD235a was diminished by FLI1WT but elevated by FLI1Met100*. CD31 and CD44 expression levels were increased by FLI1WT by FLI1Met100* but were not altered by FLI1Met100*. Moreover, FLI1Met100* led to a marked decrease in cellular adhesion, coupled with an increase in cell size and apoptosis.

Conclusion(s): Collectively, our findings imply that the FLI1Met100* mutation affects megakaryocyte differentiation at the expense of cellular maturation, which may explain aspects of the thrombocytopenia observed in this patient.