Single-Cell RNA Sequencing Uncovers Abnormal Differentiation of Neutrophil and Megakaryocyte Might Mediated Ruxolitinib Resistance for Patinets with PMF

Background: Primary myelofibrosis (PMF ) is a heterogeneous disorder characterized by bone marrow fibrosis, megakaryocyte hyperplasia, systemic inflammation and extramedullary hematopoiesis. Ruxolitinib (Rux）have shown clinical benefit with reduction of splenomegaly and related symptoms. However, about 50% of patients discontinued ruxolitinib for resistance and/or intolerance，and the mechanism of resistance to ruxolitinib is still unclear. Recently studies found that immune microenvironment and megakaryocyte promote PMF progression, whether the abnormalities of immune microenvironment and megakaryocyte associated with Rux resistance needs to be further elucidated.

M ethods: In this study, 8 participants (Newly Diagnosed PMF =3, Resistance to Rux PMF =3, Healthy Donors =2) were included. Peripheral blood was taken and single-cell transcriptome sequencing technology was used. Combined with pseudo-temporal analysis, SCENIC analysis, Single Cell Lineage Inference Using Cell Expression Similarity and Entropy (SLICE), cellular interaction analysis, gene ontology (GO) and Kyoto Gene and Genome Encyclopedia (KEGG) pathway analysis, Gene Set Variation Analysis (GSVA), L-R receptor coexpression analysis and GEPIA online tool for data analysis, Use R language or Python for data processing.

Results: In PMF patients, the proportion of neutrophils and monocytes was significantly increased, while the proportion of T cells was significantly decreased in Resistance to Rux group (P<0.05). Further subdivision of neutrophils showed that neutrophils were divided into 6 subgroups. Compared with healthy control group, neutrophils increased in Resistance to Rux group and decreased in Neutrophil_1, among which Neutrophil_6 was only enriched in PMF patients. Neutrophil_6 significantly up-regulated tumor necrosis factor (TNF) superfamily product pathway, IL-1 signaling pathway, Toll-like Receptor 4 (TLR4) signaling pathway, peroxide-producing NADPH oxidase activator activity pathway, MAPK pathway and IL-8 product signaling pathway. Neutrophil_4-6 significantly up-regulates MYB, EZH2, SUZ12, YBX1, POLR3G, E2F7, E2F8 and other transcription factors. L-R up-regulation between Neutrophil_4 and Neutrophil_6 and T cells mediated T cell exhaustion by LGALS9/Tim3 relate to resistance to Rux. We have identified a subset of HSCs in PMF that exhibits a bias towards megakaryocytic/platelet differentiation (HSCs_2) in Rux resistant group. This subset is characterized by the upregulation of specific genes, such as GATA1, PRKAR2B, ITGA2B, PLEK, and KLF1.CellPhoneDB revealed that the regulation of platelets-platelets involves CD40L-CD40 ligand receptors. Notably, the heightened reactivity of platelets may be closely associated with the risk of PMF-related thrombosis. Additionally, we observed an upregulation of the MIF-CD74 cytokine ligand receptor between the immune platelet subsets and HSC subsets in Rux resistant group.

Conclusion: In Rux resistant group, Up-regulation of inflammatory and tumor-related signaling pathways promotes the occurrence of PMF inflammation and amplification of malignant clones; Up-regulation of LGALS9 in neutrophil band to receptor TIM3 mediates T cell depletion; the regulation of platelets-platelets in PMF is mediated by CD40L-CD40 ligand receptors; MIF-CD74 might be the relationship between PMF-HSC and PMF-PLT in the context of primary myelofibrosis.