Single-cell analyses reveal aberrant pathways for megakaryocyte-biased hematopoiesis in myelofibrosis and identify mutant clone-specific targets

Abstract

Myelofibrosis is a severe myeloproliferative neoplasm characterised by increased numbers of abnormal bone marrow megakaryocytes that induce progressive fibrosis, destroying the hematopoietic microenvironment. To determine the cellular and molecular basis for aberrant megakaryopoiesis in myelofibrosis, we performed high-throughput single-cell transcriptome profiling of 50,538 hematopoietic stem/progenitor cells (HSPCs), single-cell proteomics, genomics and functional assays. We identified an aberrant pathway for direct megakaryocyte differentiation from the earliest stages of hematopoiesis in myelofibrosis and associated aberrant molecular signatures, including surface antigens selectively expressed by JAK2-mutant HSPCs. Myelofibrosis megakaryocyte progenitors were heterogeneous, with distinct expression of fibrosis and proliferation-associated genes and putative therapy targets. We validated the immunoglobulin receptor G6B as a promising JAK2-mutant clone-specific antigen warranting further development as 2 an immunotherapy target. Our study paves the way for selective targeting of the myelofibrosis clone and more broadly illustrates the power of single-cell multi-omics to discover tumor-specific therapeutic targets and mediators of tissue fibrosis.

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

“…We found that a subpopulation of immunophenotypic CD34 + CD38 -HSPCs express CD41 and are Mkbiased in colony assays. The frequency of adult CD34 + CD38 -CD41 + progenitor cells (~25%) reported here is higher than prior reports in freshly isolated bone-marrow HSPCs 27,78 , likely due to longer time in HSPC expansion culture media containing fms-like tyrosine kinase 3 ligand (FLT3L), thrombopoietin (TPO) and stem cell factor (SCF), which also promotes Mk differentiation. The lentiviral shRNA approach utilized here achieved ~50-70% reduction of RUNX1 levels in terminal adult Mks, and a Mk yield defect as anticipated.…”

Single-cell analysis of bone marrow–derived CD34+ cells from children with sickle cell disease and thalassemia

“…As predicted, patients with SCD appear to show an increased proportion of cells with an erythroid gene-expression program although this did not reach statistical significance ( Figure 1B; supplemental Table 3) (control, 7.86%; thalassemia, 6.97%; and SCD, 9.67%). No expansion of erythroid gene-expressing cells was seen in children with thalassemia, consistent with adherence to a rigorous regular transfusion protocol designed to suppress endogenous erythropoiesis in these patients ( Trajectory analysis was performed using an in-house R package, 20 which allowed the expanded lymphoid progenitors to be subclassified into early B progenitors, 22 expressing high levels of IL7R and IGHM, and late CD24-expressing B progenitors (supplemental Figure 5; supplemental Tables 1-3). These data were corroborated using multiparameter flow cytometry, which showed significant increases in the frequency of CD34 1 CD10 1 CD19 1 cells in these patients (Figure 1C-D; supplemental Figures 7 and 8; supplemental Table 4).…”

“…Analysis of scRNA-seq data using Seurat 18 and AUcell 19 (see supplemental Table 2 for quality control data) showed a marked expansion of CD34 1 B-lymphoid progenitors ( Figure 1A-B) in all 8 children with hemoglobinopathies compared with controls (supplemental Figures 1-6). Cells identified as B-lymphoid progenitors were enriched for expression of lymphoid genes 20,21 including CD79A, CD79B, VPREB1, VPREB3, and EBF1 (supplemental Table 3; area under the curve 0.25 was used for all samples). As predicted, patients with SCD appear to show an increased proportion of cells with an erythroid gene-expression program although this did not reach statistical significance ( Figure 1B; supplemental Table 3) (control, 7.86%; thalassemia, 6.97%; and SCD, 9.67%).…”

The BET inhibitor CPI203 promotes ex vivo expansion of cord blood long-term repopulating HSCs and megakaryocytes

“…In contrast, myeloid, erythroid and lymphoid-related genes 13 , including CSF3R, PRTN3, CA1, CNRIP1, APOC1, TFR2, CD4, CD79A and CD79B, were more highly expressed in vehicle/cytokine conditions. Unexpectedly, three of the most upregulated genes in CPI203/cytokine-expanded cells are strongly associated with megakaryocyte (MK) development (CXCR4 17 , PF4 18 and C6orf25 19 ).…”