RUNX1 haploinsufficiency causes a marked deficiency of megakaryocyte-biased hematopoietic progenitor cells: Mechanistic studies and drug correction

Abstract

Patients with familial platelet disorder with a predisposition to myeloid malignancy (FPDMM) harbor germline monoallelic mutations in a key hematopoietic transcription factor RUNX1. Previous studies of FPDMM have focused on megakaryocyte (Mk) differentiation, and platelet production and signaling. However, the effects of RUNX1 haploinsufficiency on hematopoietic progenitor cells (HPCs) and subsequent megakaryopoiesis remains incomplete. To address this issue, we studied induced-pluripotent stem cell (iPSC)-derived HPCs (iHPCs) and Mks (iMks) from both patient-derived lines and a wildtype line modified to be RUNX1 haploinsufficient (RUNX1+/−), each compared to their isogenic wildtype control. All RUNX1+/− lines showed decreased iMk yield and depletion of a Mk-biased iHPC subpopulation. To investigate global and local gene expression changes underlying this iHPC shift, single-cell RNA sequencing was performed on sorted FPDMM and control iHPCs. We defined several cell subpopulations in FPDMM Mk-biased iHPCs. Analyses of gene sets upregulated in FPDMM iHPCs indicated enrichment for response to stress, regulation of signal transduction and response to cytokine gene sets. Immunoblotting studies in FPDMM iMks were consistent with these findings, but also identified augmented baseline c-Jun N-terminal kinase (JNK) phosphorylation, known to be activated by transforming growth factor β1 and cellular stressors. J-IN8 and RepSox, small drugs targeting these pathways, corrected quantitative defects in FPDMM iHPC production. These findings were confirmed in adult human CD34+-derived stem and progenitor cells transduced with lentiviral RUNX1 short-hairpin (sh) RNA to mimic RUNX1+/−. These mechanistic studies of the defect in megakaryopoiesis in FPDMM suggest druggable pathways for clinical management of thrombocytopenia in affected patients.Key pointsRUNX1 haploinsufficiency results in a deficiency of megakaryocyte-biased hematopoietic progenitor cells (HPCs).RUNX1 haploinsufficiency elevates druggable proinflammatory and TGFβR1-related pathways in HPCs.

Germline RUNX1 variation and predisposition to childhood acute lymphoblastic leukemia

“…Non-hematopoietic cells such as HEK293T and HeLa have been used extensively to study RUNX1 function (24,50,51), but one could easily argue against their tissue relevance in the context of RUNX1-related leukemia, which is why we performed extensive experiments to confirm RUNX1 function in T-ALL cell lines and also in human cord blood CD34+ cells. Recent development of gene editing techniques in induced pluripotent stem cell (iPSC) provides an exciting system for this type of work (42,52). For example, CRISPR-Cas9 gRNA mediated homology-directed repair (HDR) or base editing in iPSC system can precisely introduce RUNX1 variant at the endogenous locus and in the presence of WT allele, and the hematopoietic differentiation potential of these engineered progenitor cells can be directly characterized in vitro.…”

Single-cell analysis of megakaryopoiesis in peripheral CD34+ cells: insights into ETV6-related thrombocytopenia

“…Using unsupervised clustering, we identified a total of 15 clusters (Figure 1C), which were present in both controls (Supplemental figure 1A-C). We performed a detailed characterization of each cell cluster based on known cell gene sets (signature) [27][28][29][30] (Figure 1D) and the top differentially expressed genes (DEGs) (Figure 1E; Supplemental Figure 2, Supplemental Table 1).…”