OC 27.2 - Mechanistic studies of runt-related transcription factor-1 (RUNX1)-deficient megakaryopoiesis and its correction by RepSox (RS), a TGFβ-receptor-1 inhibitor

Background: Heterozygous defects in RUNX1 are causative of familial platelet disorder with associated myeloid malignancy (FPDMM). Previously, we characterized the defects in short hairpin (sh) RNA-RUNX1-megakaryocytes (shRX-Mks) derived from adult CD34+-hematopoietic stem and progenitor cells (HSPCs). We found that the TGFβ-receptor-1 inhibitor, RS, can correct shRX-Mk/platelet defects.

Aims: To study the mechanisms of RUNX1-deficient megakaryopoiesis and its correction by RS.

Methods: Single-cell (sc) RNA-sequencing and analysis of control shNT-(nontargeting), or shRX-(targeting RUNX1) Mks differentiated from CD34+-HSPCs with or without RS exposure.

Results: Clustering identification and visualization of transcriptional heterogeneity in shNT- vs. shRX-Mks ± RS by dimensionality reduction showed two clusters recovered with RS exposure (Figure 1, Clusters 1 and 2). There was a new population created in shRX-Mks, which almost disappeared with RS (Figure 1, Cluster 3). Differential-expressed (DE)-gene analyses in shRX-Mks vs. shRX-Mks+RS (123-up and 66-down among 748) compared to DE-genes in the shNT-Mks vs. shNT-Mks+RS comparison (0-up and 11-up among 873), suggesting that RS impacts gene expression depending on RUNX1 expression level (Figure 2). Over half of the 20 most highly-expressed-genes in shRX-Mks are categorized as “tumor-suppressors” (Figure 2, yellow-boxed). Many upregulated genes in shRX-Mks were overexpressed in acute myeloid leukemia, including these tumor suppressor genes and also immune-response-related genes (Figure 2, green-boxed). RS exposure downregulated most of these overexpressed genes and increased the expression of many Mk-related genes (Figure 2, arrows), especially the transcriptional factor ETS1 (Figure 2, red-boxed).

Conclusion(s): scRNA-sequencing of shRX-Mks suggests important pathways by which RUNX1 haploinsufficiency can increase leukemic risk in FPDMM and lead to Mk/platelet defects. RS reversed many of these DE-genes. RS also increased ETS1 expression. ETS1 complexes with RUNX1 and is central to megakaryopoiesis. We propose that increased ETS1 expression by shRX-Mks following RS treatment is how RS compensates for RUNX1 deficiency, corrects the Mk/platelet axis and may be a therapeutic target for FPDMM.