P2rx7 in AML blasts orchestrates myeloid differentiation through T cell-mediated visfatin and lymphotoxin signaling

Abstract Introduction:P2X7 receptor (P2RX7) overexpression has been found to be associated with a poor prognosis in human acute myeloid leukemia (AML). Our previous study demonstrated that the overexpression of P2rx7 in murine TIB-49 AML cells promoted stemness and aggressiveness of leukemic blasts, while the deletion/inhibition of P2rx7 in TIB-49 cells prolonged the survival of bearing mice (PMID: 39157874, EHA2024-1113). Methods: Wild-type (WT) C57BL/6 mice were bred and maintained at Shandong Cancer Hospital and Institute. The syngeneic murine AML cell line TIB-49 from American Type Culture Collection was transduced with a lentiviral vector to overexpress P2rx7 (hereafter referred to as TIB-P2rx7high). P2rx7 was knocked out by CRISPR on TIB-49 (with TdTomato) cells (TIB-P2rx7-/-). WT mice were injected intravenously with 1×10⁶ TIB-49, TIB- P2rx7high, or TIB-P2rx7-/- cells, respectively. Bone marrow (BM) cells were harvested from leukemic mice for single-cell RNA sequencing (scRNA-seq, CapitalBio Technology, Beijing, China) when showing leukemic symptoms. Results: The scRNA-seq results revealed that the major cell types in AML BM microenvironment included T cells, neutrophils, B cells, dendritic cells (DCs), NK cells, monocytes, erythroid cells, myeloid progenitors, and megakaryocyte progenitors. Cells from BM of the TIB-P2rx7high-bearing mice (BMC2) showed a downregulated proportion of myeloid progenitors when compared to TIB-49-bearing mice (BMC1), while cells from TIB-P2rx7-/--bearing mice (BMC3) exhibited an upregulated proportion. Differentially expressed genes (DEGs) and KEGG pathway analyses indicated that myeloid progenitors in BMC3 had significantly upregulated pathways, including neutrophil extracellular trap formation, transcriptional misregulation in cancer, and ATP-dependent chromatin remodeling, whereas metabolic pathways like oxidative phosphorylation and reactive oxygen species were downregulated. To further explore the impact of P2rx7 on myeloid cells, we re-clustered all myeloid cells (neutrophils, dendritic cells, monocytes, and myeloid progenitors) into distinct subpopulations. The myeloid cells were classified into neutrophils, tissue-resident macrophages (TRMφ), inflammatory macrophages (iMac), plasmacytoid dendritic cells (pDCs), mast cells, monocyte-derived macrophages (Mo_Mac), inflammatory monocytes (iMo), monocyte-derived dendritic cells (Mo_DC), and granulocyte-monocyte progenitors (GMP). Pseudotime trajectory analysis revealed that GMP cells were the most primitive populations and could differentiate into either neutrophils or monocyte/macrophage-lineage cells. In BMC3, the proportion of GMP was significantly increased, suggesting that P2rx7 may play a role in maintaining the homeostasis of myeloid progenitor differentiation in BM. When P2rx7 gene was disrupted - particularly upon knockout - GMP cells tended to retain their self-renewing state rather than undergo terminal differentiation. Cell communication analysis revealed that altered P2rx7 expression changed the number and strength of interactions between T cells and myeloid cells. In the BMC3, TIB-P2rx7-/- cells established bidirectional communication with T cells via the visfatin signaling pathway, which was absent in BMC1 or BMC2. Additionally, T cells in BMC3 transmitted LT (lymphotoxin) signals to myeloid progenitors, which was either not observed in BMC1 or BMC2. These findings suggested that P2rx7 may influence myeloid progenitor differentiation both directly through cell-cell signaling and indirectly by modulating the T cell-derived LT pathway. Conclusion: Our study demonstrated that P2rx7 played a critical role in regulating myeloid progenitor differentiation within the AML BM microenvironment. P2rx7 deficiency led to an accumulation of primitive GMP cells with impaired differentiation potential, accompanied by upregulation of cancer-related pathways and downregulation of metabolic activity. T cell-associated visfatin and LT pathways among leukemic blasts, T cells and myeloid progenitors were uniquely activated in P2rx7-knockout conditions. These findings suggested that P2rx7 maintained myeloid homeostasis by balancing progenitor self-renewal and differentiation, providing a mechanistic basis for potential therapeutic strategies targeting P2rx7 in AML.