Mesenchymal stromal cell reprogramming and aberrant inflammatory signalling in myelofibrosis: insights from single-cell RNA sequencing

Myelofibrosis (MF) is a myeloproliferative neoplasm driven by mutations that impact the JAK/STAT signaling in blood cells, eventually causing fibrosis, splenomegaly, and reduced lifespan. To better understand MF, we performed scRNAseq profiling of collagenase-digested bone marrow biopsy samples to identify disrupted cellular pathways for potential treatment. Comparative analysis of 9 MF samples vs. 7 normal controls vs. 4 MF patients after allogeneic transplant showed striking differences in cell type composition and transcriptional profiles. Of note, Compositional comparisons identified the non-hematopoietic stromal cell fraction as harboring the biggest differences with, surprisingly, a proportional relative depletion of mesenchymal stromal cells (MSCs) and enrichment of endothelial cells (ECs) in MF vs. normal controls. Concomitant transcriptional differences were observed between MSCs recovered from MF vs. normal control sample, characterised by reduced expression of hematopoietic support factor genes (eg, KITLG, CXCL12, ANGPTL2, NCAM2, CSF1) and increased expression of extracellular matrix (ECM) genes (eg, FN1, COL1A2, COL3A1, DCN, SPARC), features which suggest an explanation for the increased frequency of graft failure following allograft in MF versus other hematologic malignancies. Single-Cell rEgulatory Network Inference and Clustering (SCENIC) indicated transcription factors with either increased (eg, NR2F2, RUNX2, FOXP2) or decreased (eg, CEBPA, FOXC1, and PPARG) transcriptional activity as candidate master transcriptional regulators of the observed MSC reprogramming in MF. Within the hematopoietic fraction, and agnostic of JAK inhibitor treatment status, comparison of MF vs. control cells unexpectedly revealed significant upregulation of interferon response genes, especially in CD14+ monocytes, but also in neutrophils and their progenitors, centred on expression of interferon-stimulated gene factor 3 (ISGF3) genes IRF9 and STAT1/2. Comparison of MF samples according to ruxolitinib treatment status indicated that ruxolitinib induced downregulation of an NFκB-centered inflammatory program, particularly in neutrophils. Finally, ligand-receptor analysis using NicheNet identified TGFB1, IL1B, and TNF as top predicted ligands for induction of the MF signature in MSCs. While myeloid cells were top producers of these factors, their expression did not vary between MF vs. controls, suggesting other mechanisms, such as prolonged retention of ligand in the ECM, are important. In summary, our comparative scRNA analysis of BM cells from MF patients and controls – the largest reported to date – reveals (i) transcriptional reprogramming of MSCs leading to reduced expression of hematopoietic support factor and increased expression of ECM genes; and (ii) increased expression of interferon and NFkB inflammatory pathways in myeloid cells.