Abstract
Myelofibrosis (MF) is a myeloproliferative neoplasm (MPN) associated with cytopenias, megakaryocytic hyperplasia, systemic symptoms, progressive bone marrow (BM) fibrosis and extramedullary hematopoiesis (EMH) occurring primarily in the spleen. MF arises from acquired somatic driver mutations (JAK2, CALR, MPL) in hematopoietic stem cells (HSC) as well as co-mutations in epigenetic modifiers, signaling molecules and transcription factors (TF). Prior studies of MPN patient peripheral blood and BM hematopoietic stem and progenitor cells (HSPC) identified an erythroid/megakaryocyte (Er/Mk) differentiation bias. However, transcriptional and epigenetic drivers of MF EMH and the Er/Mk differentiation bias have not been fully characterized. In depth molecular studies of the hematopoietic cells that contribute to MF EMH have not been previously reported.
We compared the cellular, transcriptional and epigenetic landscapes of hematopoietic cells from the spleens of JAK2V617F and/or CALRmut MF patients that had been surgically removed to spleens from healthy individuals. Then, to understand molecular drivers of MF EMH, we compared the transcriptional and epigenetic single cell landscapes of CD34+ MF splenic HSPC and HSPC from healthy BM donors.
We performed Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) on 7 MF spleens for a total of 38,444 cells and 3 healthy spleens for a total of 18,229 cells. We also performed single cell Assay for Transposase-Accessible Chromatin (scATAC-seq) on 5 MF spleens (total 25,610 cells) and 2 healthy spleens (total 9,500 cells).
Using supervised reference mapping as implemented in Seurat, we mapped our CITE-seq spleen and healthy BM datasets to a scRNA-seq Azimuth reference of over 300k BM cells from healthy donors. Additionally, we used a BM sc multiome dataset (ATAC + RNA from NeurIPS) as a bridge dataset to map our scATAC-seq data to the same RNA reference. In our CITE-seq data, we identified expanded HSPC populations in MF spleens (median = 50.2%, IQR = 32.5%) as compared to healthy spleens (median = 0.3%, IQR = 0.1%) indicating extensive MF EMH. Within the HSPC, we identified an expansion of Er/Mk progenitors (Er/MkP) and a contraction of myeloid and lymphoid progenitors indicating an Er/Mk differentiation bias in MF splenic HSPC vs. healthy BM HSPC (Er/MkP, MF: median = 40.9%, IQR = 17.9%, healthy: median= 18.9%, IQR = 5.1%) (Fig 1A). Similar patterns were observed in our scATAC-seq data.
To identify transcriptional drivers of Er/Mk differentiation bias, we performed differential expression analysis comparing MF splenic HSPC to matched populations present in 10 healthy BM donors (2,410 cells). In HSC, GO enrichment analysis on significantly upregulated genes (in at least 3 MF patients) revealed increased expression of genes involved in RNA splicing, chromatin organization and NF-κB signaling. MF erythro-myeloid progenitors (EMP) demonstrated increased expression of genes related to DNA replication and DNA damage repair. Cell cycle analysis revealed a significant increase in the proportion of HSC, lymphoid-primed multi-potent progenitors, and EMP that were actively cycling in MF spleens as compared to healthy BM.
To understand changes in chromatin accessibility driving MF Er/Mk differentiation bias, we identified differentially open and closed chromatin motifs enriched in MF HSC, EMP and early erythroid progenitors (EEP) as compared to equivalent populations from previously published scATAC-seq data of healthy BM CD34+ cells (PMID: 31792411). Across erythroid differentiation trajectory (HSC/EMP/EEP), we identified increased activity of TF motifs including Kruppel-like factor (KLF) family, interferon regulatory factors and specificity proteins (SP) in MF spleen compared to healthy BM (Fig 1B). In contrast, we identified decreased activity of TF motifs in RUNX and GATA families.
Our sc analyses documented expanded HSPC populations in MF spleens with HSPC Er/Mk differentiation bias as compared to healthy BM. Transcriptional and epigenetic analysis of MF Er/Mk differentiation bias revealed extensive dysregulation of gene expression and chromatin accessibility compared to Er/Mk differentiation in healthy BM. These findings provide evidence of the unique transcriptional and epigenetic programs that characterize splenic HSPC which likely contribute to the development of MF EMH and disease progression in advanced MF.
Disclosures
Satija:Nanostring: Consultancy; Kallyope: Consultancy; NYC Pandemic Response Lab: Consultancy; Regeneron: Consultancy; Bristol-Myers Squibb: Consultancy; ImmunAI: Consultancy; Resolve Biosciences: Consultancy. Hoffman:Silence Therapeutics: Consultancy; Novartis: Other: Chair DSMB; Abbvie: Other: Chair DSMB, Research Funding; Ionis: Consultancy; Protagonist Therapeutics: Consultancy; Scholar Rock: Research Funding; Turning Point: Research Funding; Repare: Research Funding; Novartis: Research Funding.
