Pathologic TPO-induced TPOR-CSF2RB signaling mediates Down syndrome abnormal myelopoiesis due to GATA1 mutant-dependent CSF2RB overexpression
Transient abnormal myelopoiesis (TAM) is a human myeloid leukemia that occurs in newborns with Trisomy 21, who acquire mutations in the transcription factor GATA1. TAM originates in the fetal liver but spontaneously regresses as hemopoiesis transitions from fetal liver to postnatal bone marrow, unless clonal evolution enables transformation of the TAM clone to Myeloid Leukemia of Down Syndrome. We sought to understand the developmental tissue restriction of TAM. We show that TAM cells are marked by high expression of the cytokine receptor CSF2RB, a direct target of GATA2. Oncogenic GATA1 fails to supress CSF2RB expression. Highly expressed CSF2RB directly interacts with the thrombopoietin receptor (TPOR). Specifically at high TPO levels, activation of JAK-STAT signalling by TPOR-CSF2RB complexes is amplified and distinct from that triggered by TPOR homodimers and drives TAM cell proliferation. CSF2RB-A455D, a previously identified pathogenic gain-of function variant seen in ML-DS, requires interaction with TPOR to induce cytokine-independent cell proliferation. Together, TPOR and CSF2RB jointly exploit TPO produced by the fetal liver to sustain developmentally restricted TAM cell proliferation Overall design: 10x Genomics Chromium Next GEM Single Cell 5' V2 Dual Index Kit  was used to perform scRNAseq on cord blood derived  KIT+ CD34- and KIT+ CD34+ TAM (n=3), Trisomy 21 (n=3), disomic (n=3) cells. CUT&TAG was perfomed in duplicates in CMK cells to measure GATA1s and GATA2 binding as well as H3K27ac. CUT&TAG was performed to measure H3K27ac  in 7 cell lines: CMK,  HEL, SET-2, TF-1, K-562, THP-1, and JURKAT cells 48 hours post lenti delivery of the empty vector or the GATA2 coding sequence. Bulk RNA sequencing was perfomred in triplicates on TF-1 cells expressing the TPOR and empty vector or TPOR and CSF2RB 3 hours post TPO treatment.