P1649: gfi1b and lsd1 repress a myeloid differentiation program during induced pluripotent stem cell derived megakaryocyte differentiation

Background: Megakaryocytic development is driven by the transcription factor Growth Factor Independence 1B (GFI1B) and its interaction with the chromatin remodeler Lysine Specific Demethylase 1 (LSD1/KDM1A). GFI1B aberrations are linked to inherited bleeding disorders, resulting in macro-thrombocytopenia, gray platelets and increased expression of CD34. One such mutation, which was previously show to be the disease-causing mutation in a family with gray-platelet syndrome, introduces a premature stop codon (Q287*) in the fifth zinc finger. Aims: Here, we study the effect of the Q287* mutation on gene expression regulation, as well as GFI1B interaction with LSD1, during induced pluripotent stem cells (IPSC) derived megakaryocytic development. Methods: We differentiated IPSCs that contain wildtype GFI1B or GFI1BQ287* into megakaryocytes. During differentiation, megakaryocytes derived from GFI1B wildtype IPSCs were also treated with an LSD1 inhibitor that disrupts GFI1B-LSD1 interaction for 24 hours. The transcriptomic landscape of differentiated IPSCs was explored using single-cell RNA sequencing and downstream computational analysis. We applied a novel filtering strategy, removing cells based on doublet annotation, mitochondrial gene count and exclusive differential expression of ribosomal or mitochondrial genes. Results: After filtering and data integration we retained 14,848 cells. Within this population we were able to annotate six different cell types using single-cell GSEA and publicly available reference datasets. Megakaryocytes and megakaryocyte progenitors made up roughly 75% of all cells differentiated from IPSCs containing wildtype GFI1B. They are characterized by approximately 220 differentially expressed genes (p-value < 0.05, log-fold change > 0.69), including common megakaryocyte markers such as CD41 and CD42b. Strikingly, we also observed a population of myeloid and myeloid-progenitor cells after differentiation towards megakaryocytes. This population specifically expressed common myeloid markers, such as VIM, CD81, and SPI1 as well as approximately 200 other genes (p-value < 0.05, log-fold change > 0.69) and made up around 10% of the entire GFI1B wildtype cell population. This population was expanded two-fold in cells differentiated from GFI1B-wildtype IPSCs treated with the LSD1 inhibitor and three-fold in GFI1BQ287* IPSCs. Notably, in the GFI1BQ287* cell population megakaryocytes were reduced by 2-fold. Pseudotime analysis showed a differentiation trajectory starting at megakaryocytes and going towards myeloid cells. Megakaryocyte and platelet associated genes, such as CD41, CD42b and PF4, are gradually downregulated along this trajectory while myeloid associated genes exhibited a mirrored expression pattern. Thus, LSD1i treated and GFI1BQ287* IPSC-derived cells showed an increase in myeloid marker gene expression after differentiation and downregulation of coagulation-associated genes, hinting at a decreased potential to differentiate into megakaryocytes. Summary/Conclusion: Our data show that GFI1B and LSD1 function to repress a myeloid cell fate during IPSC-induced megakaryocytic development. Disruption of the interaction between GFI1B and LSD1 and a dominant-negative GFI1B mutation results in myeloid differentiation at the expense of megakaryocyte development.