RUNX1 Mutation Leads to Megakaryocyte-Primed Hematopoietic Stem Cell Expansion and Familial Platelet Disorder

Backgrounds: Recently it is shown that there exists a subpopulation of hematopoietic stem cells (HSCs) with relatively high expression of VWF and CD41+ at the apex of the hematopoietic stem cell hierarchy. This subset of HSCs, termed mega-HSCs, can give rise to megakaryocytes and platelets directly by bypassing the traditional trajectory of megakaryocyte development from HSC via MPP and MEP. To date, aside from phenotypic marker and transplantation studies, there has been limited understanding of the mechanisms involved in the regulation of mega-HSCs. Runx1 belongs to the RUNT domain transcription factors, and is a key regulator of hematopoiesis especially for the megakaryocyte and platelet differentiation. Loss of RUNX1 in mice causes thrombocytopenia through a blockage of megakaryocyte maturation. One allele RUNX1 loss-of-function mutation is associated with familial platelet disorder (FPD) with a predisposition to developing leukemia. Here we hypothesize that Runx1 plays a role in regulating mega-HSCs to impact on platelet generation, and correcting RUNX1 mutation that causes FPD can therapeutically rescue thrombocytopenia in a mouse model. Methods: We have examined the hematopoiesis and cellularity of various bone marrow (BM) stem/progenitor populations and peripheral blood (PB) in two mouse models. Firstly, conditional knock-out of Runx1^flox/flox was mediated by Mx1-Cre upon poly I:C induction. Secondly, the tetracycline-inducible RUNX1 S291fsX300 mutation was knock-in at the collagen a1 locus and the mice was crossed to MLL-PTD knock-in. The mutant RUNX1 is only expressed when the mice are fed with doxycycline and the RUNX1 mutant is "corrected" upon doxycycline withdrawn. In the second model, PB and BM were also tracked after a removal of DOX-induced RUNX1 mutation. The LSK CD150+ HSCs from the mouse BM were isolated by FACS sorting, and 10x Genomics' single-cell RNA-seq (scRNA-seq) analyses were performed to define and track HSPCs. We applied the Louvain algorithm to the scRNA-seq data to identify the cell type clusters and annotated the cell types using the marker genes of each cell cluster. MAST was employed to identify the cell-type specific, differentially expressed genes. Results: In the Runx1 KO mice, two weeks after deletion of Runx1 platelet count showed an ~2-fold decrease to 400~600 k/ul in PB. In the LSK CD150+CD48- or the LSK CD34- FLT3- compartment of BM, the CD41+ mega-HSCs increased ~2-3 fold. In the RUNX1 mutant-on model, mutant mice developed thrombocytopenia 16 weeks after DOX induction with the average platelet count dropping to ~600 k/ul and being maintained at this level. In transplant recipients, the RUNX1 mutant-on mice contained drastically increased mega-HSCs compared with RUNX1 mutant-off mice, synchronous with a decrease of the platelet count in PB. Consistent with these observations, sc-RNAseq data show that in Runx1 KO, among the sequenced LSK CD150+ cells ~70% are HSCs and ~15% are MPP2. Among the HSCs, mega-primed HSCs consist ~33% while non-mega primed HSCs are ~67%. The mega-primed HSCs are distinct by virtual of upregulated platelet-related genes and relative dormant cell cycle status. In the RUNX1 mutant-on model, we found that among the sequenced LSK CD150+ cells ~50% are HSCs and ~12% are MPP2. Interestingly, the mega-primed HSCs are significantly increased in RUNX1 mut-on HSCs compared with mut-off HSCs. Similarly, we saw highly upregulated platelet-driven pathways in mega-HSCs in the mutant-on HSCs. Finally, one month after a withdraw of DOX from the RUNX1 mutant expressing mice when the RUNX1 mutant gene became undetectable, the platelet count returned from ~600 to ~1,000 k/ul in PB, a reversal of the thrombocytopenia phenotype caused by the RUNX1 S291fsX300 expression. Importantly, the correction of Runx1 mutation significantly decreased the proportion of mega-HSCs and restored platelet related pathways in this subset of HSCs.

Conclusions: Mega-HSCs contain a high level of platelet-driven gene expression. In addition to its role in regulating the HSC-MPP-MEP mediated megakaryocyte development, RUNX1 is important in regulating mega-HSCs by maintaining proper expression of mega-platelet leaning genes. Correction of RUNX1 mutation that causes FPD can rescue mega-HSC population and revert FPD, providing a rationale for future treatment strategies by gene editing in RUNX1 mutation bearing FPD patients.