TET2 Deficiency Increases the Competitive Advantage of Hematopoietic Stem Cells through Upregulation of Thrombopoietin Receptor Signaling

Ten-Eleven Translocation-2 (TET2) mutations drive the expansion of mutant hematopoietic stem cells (HSCs) in clonal hematopoiesis (CH). However, the precise mechanisms by which TET2 mutations confer a competitive advantage to HSCs remain unclear. Here, through an epigenetic drug screen, we discovered that inhibition of disruptor of telomeric silencing 1-like (DOT1L), a H3K79 methyltransferase, selectively reduced the fitness of Tet2 knockout (Tet2KO) hematopoietic stem and progenitor cells (HSPCs). Mechanistically, we found that TET2 deficiency increased H3K79 dimethylation and expression of Mpl, which encodes the thrombopoietin receptor (TPO-R). Correspondingly, TET2 deficiency was associated with a higher proportion of primitive Mpl-expressing (Mpl+) cells in the HSC compartment. Importantly, inhibition of Mpl expression or the signaling downstream of TPO-R was sufficient to reduce the competitive advantage of murine and human TET2-deficient HSPCs. Our findings demonstrate a critical role for aberrant TPO-R signaling in TET2 mutation-driven CH and uncover potential therapeutic strategies against this condition. For RNA-seq, wildtype and Tet2 knockout (KO) HPCHOXB4 cells were cultured in growth medium supplemented with 0.01% DMSO or SGC0946 at 1uM for seven days prior to harvest. Total RNA was extracted from cells using the Qiagen RNeasy plus kit (Qiagen, #74134) and quantified using a Nanodrop spectrophotometer. All samples had a RIN value greater than 9. Samples were submitted to Novogene Corporation for sequencing analysis. Library construction, sequencing, and processing of sequencing data were done by Novogene following the standard pipeline. For H3K79me2 ChIP-seq, wildtype and Tet2 knockout (KO) HPCHOXB4 were cultured in growth medium supplemented with DMSO (0.01%) or SGC0946 (1uM) for seven days prior to harvest. Cells were fixed with 1% formaldehyde (Thermo Scientific, #410730050) according to the Active Motif ChIP cell fixation protocol. Fixed cell pellets containing 5 millon cells per sample were submitted to Active Motif for Ch-IP reactions and downstream data processing and analyses. For scRNA-seq, Whole bone marrow cells were isolated from 15 to 20-week-old Tet2WT and Tet2KO male littermates following red blood cell depletion. Cells from each donor were transplanted into four lethally irradiated 8-week-old CD45.2 Tet2WT recipients at 1x106 cells per animal. Following four weeks of engraftment, mice were subject to pinometostat (60mg/kg) or vehicle (10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline) treatment through subcutaneous injection twice a day (2 animals per cell type per treatment group) for 21 days.