From development to disease: Cellular models of hematopoiesis and platelet disorders

This thesis explores the transcriptional regulation of hematopoiesis, with a focus
on megakaryocyte (MK) and platelet development. Many transcription factors
(TFs), such as GFI1B, FLI1, and ETV6, are expressed in both hematopoietic
stem cells (HSCs) and MKs, consistent with the notion that MKs may derive directly
from HSCs. Germline mutations in these TFs are associated with inherited
platelet disorders; however, the mechanisms by which specific variants disrupt
gene regulation and cellular function remain largely unresolved. Studying individual
TF mutants offers a powerful entry point to unravel the downstream consequences of disrupted transcriptional control. Because many of these mutations
do not cause complete loss of function, but rather result in dominant-negative
effects, their impact cannot be predicted based on sequence alone. By modeling
specific variants such as FLI1 p.Met100* or ETV6 p.R369W in hematopoietic
cell systems, we can dissect how these mutants alter gene expression profiles,
protein interactions, and cellular phenotypes. This not only improves our understanding
of disease mechanisms but also informs functional classification of
variants of uncertain significance (VUS), which are frequently encountered in
clinical genetic testing. Moreover, transcriptional profiling of these mutants can
reveal key gene networks and regulatory circuits affected in disease, offering
potential biomarkers or therapeutic targets. As such, functional studies of TF
mutants bridge the gap between genotype and phenotype, and provide mechanistic
insights into how perturbations in lineage-specific transcriptional regulation
lead to hematopoietic dysfunction.
In Chapters 2 and 3, we established two induced pluripotent stem cell (iPSC)
lines from patients with congenital platelet disorders: SANi011-A (heterozygous
FLI1 c.297del p.Met100*) and SANi012-A (heterozygous ETV6 c.1105C>T
p.R369W). Both lines retained pluripotency and trilineage potential. Given
the limitations of patient-derived primary cells, these iPSCs offer a renewable
platform for disease modeling and therapeutic research. Chapter 4 explores
early hematopoietic development through single-cell RNA sequencing of iPSCderived
CD144+/CD31+ cells enriched for hemogenic endothelium. This approach
captured the endothelial-to-hematopoietic transition (EHT) and enabled
comparative analysis of EMP-like and AGM-derived hematopoiesis, facilitating
the identification of wave-specific HE markers. We showed that LSD1 inhibition
arrests EHT upstream of GFI1B expression, revealing distinct temporal
roles for these regulators. Additionally, ectopic GFI1B expression in HE cells
suppressed endothelial gene programs while activating hematopoietic transcriptional
networks. In Chapter 5, we describe a novel heterozygous nonsense FLI1
mutation (FLI1 p.Met100*) in a patient with neonatal thrombocytopenia. The
truncated protein impairs megakaryocyte differentiation and promotes erythroid
gene expression. Immunoprecipitation analysis revealed that the mutant retains
interaction with wild-type FLI1 cofactors, supporting a dominant-negative mode
of action. Chapter 6 describes two patients carrying the ETV6 p.R369W variant
within the ETS domain. This heterozygous missense mutation, associated with
thrombocytopenia, impairs DNA binding and exerts a dominant-negative effect
by sequestering cofactors shared with the wild-type protein. Functional studies
in megakaryocytic cell lines and primary CD34+ progenitors demonstrated
altered transcriptional networks and impaired megakaryocyte differentiation.
30.