Abstract
Myelofibrosis (MF) originates at the level of the hematopoietic stem/progenitor cell (HSPC), and is associated with profound abnormalities of components of the tumor microenvironment (TME). To directly explore the consequences of the interactions between TME cells and JAK2V617F+ MF HSPCs, we co-cultured normal donor (ND) or MF HSPCs with ND mesenchymal stromal cells (MSCs) and endothelial cells (ECs) (HSPC/TME) for 16 and 144 hours.
We found that: MF HSPC/MSC+EC cell interactions promoted the predominance of JAK2V617F+ MF CD34+ cells, with the number of MF CD45+/CD34+ cells being significantly increased in the HSPC/TME system as compared to MF HSPCs co-cultures with either MSCs or ECs alone in the absence of exogenous cytokines after 144 hours of incubation (p<0.0005). Furthermore, the MF CD34+ cell/TME cell interactions led to a significant increase in MSCs (p<0.005) but not ECs. Although the number of hematopoietic colonies generated from ND CD34+ cells incubated in the HSPC/TME system was 4-fold greater as compared to ND CD34+ cells grown in suspension culture (SusC), a 10-fold increase in MF CD34+ cells was observed from the HSPC/TME system. Genotyping of individual MF colonies assayed following the MF HSPC/TME system revealed a 6-fold increase in the numbers of JAK2V617F+ colonies as compared to a 2-fold increase in JAK2WT colonies. These findings highlight the increased sensitivity of MF JAK2V617F+ HSPCs to interactions with ECs/MSCs as compared to ND and JAK2WT HSPCs within MF CD34+ cells.
MF HSPC/TME interactions resulted in the retention of the marrow repopulating potential of MF HSPCs. Using a xenograft model, 12 weeks after transplantation, we observed splenomegaly and marrow fibrosis in mice having received MF CD34+ cells following incubation with TME cells but not following SusC. The engraftment of human CD45+, CD34+, CD41+, CD33+ and CD3+ cells in recipient mouse spleens and BMs was only increased when cells that had interacted with TME cells served as grafts. The JAK2V617F allele burden in donor cells in recipient mice was not measurable when MF CD34+ cells following SusC were transplanted but was present to a similar degree in BMs of mice having received CD34+ cells from the MF HSPC/TME system or un-manipulated MF CD34+ cell grafts (p>0.05). These data demonstrate that interactions between MF HSPCs and TME cells are necessary to maintain JAK2V617F+ MF HSPC fitness and function.
Inflammatory responses were further upregulated in MF but not ND CD34+ cells following a 16 hours incubation with MSCs and ECs. Gene set enrichment analysis identified statistically significant upregulation of the TNFα/NFkB pathway, inflammatory response, interferon-α response, interferon-γ response, IL2-STATs signaling and allograft rejection signaling. The pronounced increase in these inflammatory pathways occurred exclusively in MF CD34+ cells and was dependent on interactions with the TME cells. Key upregulated genes identified included CCL3, CCL4, S100A8, S100A9, IRF8, CXCR3, ITGB7, CXCL11, CXCL8 and LCN2.
Exposure of MF CD34+ cells to TME cells also led to upregulation of inflammatory responses in MSCs and ECs and transcriptional reprogramming to a pro-tumorigenic phenotype. Specifically, transcripts of critical inflammatory cytokines, including CXCL8, LCN2, VEGF, SCF and S100A9, were increased. Furthermore, increased elaboration of a select number of inflammatory cytokines such as IL-1β, IL-6, CXCL8 and CXCL12 was observed in the supernatants from the MF HSPC/TME system. In addition, WNT/β-catenin signaling, epithelial-mesenchymal transition, TGFβ signaling and hypoxia response pathways were upregulated in MSCs isolated from MF HSPC/TME system. Meanwhile, the allograft rejection pathway as well as the associated transcripts of genes including CLEC12A, CXCR4, CDH9, FCGR1A, ITGAL, SOX11, HLA-DQA1 and CXCL8 were increased in ECs from MF HSPC/TME system.
In conclusion, these data specify the importance of the interplay of MF HSPCs and TME cells in promoting MF HSPC fitness and their ability to serve as fully functional MF-initiating cells. The interaction between MF HSPCs and TME cells results in a cascade of events that leads to the creation of a pro-inflammatory milieu that affects both MF HSPCs and TME cells to perpetuate the MF phenotype. We speculate that therapeutic strategies that uncouple the interplay between MF HSPC and TME cells will be required to change the clinical course of MF patients.
