CD34⁺ Hematopoietic Progenitors Display A Tissue-Remodeling Signature Associated With Bone Marrow Fibrosis In Myelofibrosis

Myelofibrosis (MF) is the most aggressive Philadelphia-negative myeloproliferative neoplasm, characterized by excessive proliferation of malignant CD34⁺ hematopoietic stem/progenitor cells, profound alterations of the bone marrow microenvironment, progressive fibrosis, and chronic inflammation . Abnormal activation of stromal cells induced by proinflammatory cytokines released by the megakaryocytic clone , and proliferation of neoplastic fibrocytes have been pinpointed so far as the main events underlying the increased deposition of reticulin and collagen fibers. The role of CD34+ cells as potential drivers of the fibrotic process has not been fully investigated yet, although it has been demonstrated that they could overexpress specific cytokines in patients with higher fibrosis grading (G) [4-6]. Here, we performed RNASeq analysis on immunomagnetically isolated CD34⁺ cells from MF patients (prePMF, overt-PMF, secondary-MF) to identify transcriptome differences according to disease subtype, grading of BM fibrosis (G ≤1 vs. >1), and exposure to JAK-inhibitor therapy (therapy naïve, MF-TN vs MF-JAKi). CD34+ cells from healthy donors (HD) served as controls. Sequencing was performed using Novogene total RNASeq solution on Illumina NextSeq 500/550 platform on a Mid-Output kit V2.5 (300 cycles). Pathway enrichment analysis was performed using GeneOntology and KEGG. Genes were considered differentially expressed when showing a fold change >2 and a P value <0.05. To identify the most impactful pathways, a GeneRatio ratio cut-off of 0.02 was set as a reference. We identified 8353 differentially expressed genes (DEGs) among MF-TN vs HD, 583 among pre vs overt PMF, 2425 among MF with fibrosis G > 1 vs ≤ 1, and 1112 among MF-TN vs. JAKi. The GO pathway analysis between MF-TN patients and healthy controls highlighted alterations in processes related to cilium organization, microtubule dynamics, and cell division. In parallel, differentially expressed genes included markers associated with extracellular matrix remodeling (BMP8B, COL4A2, COL4A5) and coagulation (F7, F8), suggesting the involvement of CD34+ cells in the of the BM microenvironment alterations. Intriguingly, in overt-PMF TN vs pre-PMF TN patients, the GO pathway analysis revealed that immune-inflammatory responses, wound healing, leukocyte migration, and ECM/cell-adhesion pathways displayed the highest differential enrichment. Consistently, DEGs were related to coagulation, platelet activation (F12 GP5, GP6, GP9), ECM-receptor interaction, and the interaction of cell adhesion molecules (CCN1). These results were confirmed by comparing patients with MF-G > 1 vs. ≤ 1. Notably, when comparing MF-TN versus MF-JAKi, GO analysis highlighted that JAKi therapy modulates gene sets involved in immunoinflammatory pathways, cell chemotaxis, and extracellular matrix organization, in addition to cytokine production. Our analysis reveals for the first time that pathways associated with wound healing, osteogenesis, and extracellular matrix remodeling are differentially expressed in CD34⁺ cells from MF and correlate with the grading of BM fibrosis. Notably, JAKi therapy modulates the expression of several genes involved in tissue remodeling and angiogenesis, suggesting that its biological effects extend beyond the suppression of cytokine signalling and may also affect microenvironmental remodeling processes relevant to disease progression.