Transcriptomic analysis of CD34⁺ cells in myelofibrosis highlights their role in extracellular matrix dysregulation and marrow fibrosis

Abstract Myelofibrosis (MF) is the most aggressive Ph-negative myeloproliferative neoplasm, characterized by progressive bone marrow (BM) fibrosis and reduced survival (PMID:36416738). Despite noteworthy strides in understanding its molecular bases, the exact pathogenesis of BM fibrosis still remains enigmatic. Abnormal activation of stromal cells induced by proinflammatory cytokines released by the megakaryocytic clone (PMID: 30416700), and proliferation of neoplastic fibrocytes (PMID:32472085) have been pinpointed so far as the main events underlying the increased deposition of reticulin and collagen fibers. We recently demonstrated that, in MF, endothelial cells share with CD34+ cells somatic mutations in key genes for myeloid lineage, suggesting a profound involvement of the microenvironment in disease pathogenesis (PMID:34685741). The role of CD34+ cells as potential drivers of the fibrotic process has not been fully investigated yet, even if it has been demonstrated they could overexpress specific cytokines in pts with higher fibrosis grade (PMID: 36800567). To provide insights in this context, we performed RNASeq analysis on immunomagnetically isolated CD34+ cells from MF pts (pre- PMF, overt-PMF, secondary-MF) to identify the transcriptome differences according to disease subtype, degree of bone marrow fibrosis (G≤1 vs. G>1) and therapy with JAK-inhibitors (therapy naïve, MF-TN vs. MF-JAKi). CD34+ cells from healthy donors (HD) were utilized 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 (GO) and KEGG. Genes were considered differentially expressed when presented at least >2- fold change and p value <0.05. In order to identify the most impactful pathways, a gene ratio cut-off of 0.02 was set as reference. We identified 8353 differentially expressed genes (DEGs) among MF-TN and HD, 583 among pre vs overt PMF, 2425 among MF with fibrosis G > 1 vs. ≤ 1, and 1112 among MF TN vs. JAKi. The results of the top 10 enriched GO pathways among all MF-TN and HD CD34+ cells showed that the biological process changes were in positive regulation of wound healing response, cell-matrix adhesion, cell adhesion mediated by integrin and haemostasis. Intriguingly, when comparing overtPMF vs. prePMF, GO analysis identified among the top 10 enriched pathways: positive regulation of supramolecular fiber organization, ossification and BMP (bone morphogenetic protein) pathway. These results were confirmed when comparing MF with fibrosis G > 1 (overt + secondary MF) vs. ≤ 1. Notably, GO pathway analysis of JAKi vs. TN MF highlighted that pathways dedicated to collagen-containing extracellular matrix/extracellular matrix in general, extracellular matrix structural constituent, and ossification resulted the most differentially enriched. Modification of the transcriptome involving signalling receptor acitvity, including the MAPK cascade, as expected during JAKi-therapy, further strengthen our findings. Evaluation of enriched signaling pathways and their implicated genes shows that integrins β5, α11, α2B, and coagulation factor F7 are significantly upregulated in naïve MF pts in comparison to healthy individuals. Among these, integrin α11 is notably downregulated in response to JAKi. Consistently, COL2A1 (collagen), CEACAM1 (angiogenesis's regulator), NCKAP1 (focal adhesion modulator), and BMP2 resulted strongly upregulated in MF with fibrosis >1 when compared with MF with fibrosis ≤1. Increased expression of BMP8B parallels the increase of fibrosis grade, too. Interestingly, CEACAM1 and BMP2 are downregulated in JAKi MF compared to TN-MF. Comparing primary MF with secondary MF, FHOD1, a regulator of actin microtubules involved in maintaining cell's structure, resulted downregulated, while it is upregulated by JAKi. All the queries of KEGG confirmed the results. To date, transcriptomic data from purified CD34⁺ hematopoietic stem cells in MF remain limited. Our analysis reveals that pathways associated with wound healing, osteogenesis, and extracellular matrix remodeling are differentially expressed in malignant CD34⁺ cells, correlating with the extent of bone marrow fibrosis. Notably, JAKi therapy modulates the expression of several of these genes involved in tissue remodeling and angiogenesis, suggesting a broader impact on disease biology beyond cytokine signaling suppression.