Novel Evidence That the Pannexin 1 Channel Is Involved in Adenosine Triphosphate (ATP) Release from Cells for Optimal Mobilization of Hematopoietic Stem Progenitor Cells, and the Pannexin 1 SNP 5 (Rs3020015) T/C Polymorphism Characterizes Poor Mobilizer Status in Patients

Background. Pannexins have been shown to act predominantly as large transmembrane channels connecting the intracellular and extracellular space, allowing the passage of ions and small molecules, such as adenosine triphosphate (ATP), between these compartments. Pannexin 1 channels are involved in the release of ATP from cells and have been shown to be involved in the early stages of the innate immune response through an interaction with the P2X7 purinergic receptor. Recently, we provided evidence that activation of innate immunity in BM after administration of G-CSF produces "sterile inflammation" in the BM microenvironment and activates the pannexin channel to release ATP, which through binding to the P2X7 receptor leads to the mobilization of HSPCs (Leukemia 2018, 32:1920-1931). Corroborating our observation, it has been recently reported that the presence of the Gln460Arg SNP polymorphism within the P2X7 receptor gene in HSPCs, which is always co-inherited with Ala348Thr to form the gain-of-function haplotype 4, resulted in a significant increase in CD34⁺ HSPC mobilization (Leukemia 2018; 32:2724-2726). To shed more light on the novel concept that pannexin-1-released ATP, which interacts with P2X7, plays a crucial role in the egress of HSPCs from BM into peripheral blood (PB), we focused on the role of pannexin 1 in the mobilization process. Hypothesis. We hypothesized that pannexin 1 deficiency would negatively impact mobilization of HSPCs. Materials and Methods. First, we mobilized mice with G-CSF or AMD3100 in the presence of pannexin-1-blocking peptide. Following mobilization, we measured i) the total number of white blood cells (WBCs) and ii) the number of circulating clonogenic colony-forming unit granulocyte/macrophage (CFU-GM) progenitors and Sca-1⁺c-kit⁺lineage^- (SKL) cells circulating in PB. Next, we analyzed five types of polymorphisms in the human pannexin 1 gene (SNP1-Rs1138800 A/C, SNP2-Rs7928030 G/C, SNP3-Rs12294985 C/T, SNP4-Rs127933348 A/G, and SNP5-Rs3020015 T/C) and correlated them with good or poor mobilization status of the patients. Patients in our studies were mobilized in the Bone Marrow Transplant Unit, Warsaw Medical University with G-CSF using a standard mobilization protocol, and poor mobilizers were identified as patients who were not able to mobilize the required number of CD34⁺ cells according to standard criteria. In our studies DNA from patient blood samples was isolated and fragments of DNA amplified and subsequently sequenced. Our patients in the good- and poor-mobilizer groups were matched for age, sex, and basic disorders. Results. We found that mice with blocked pannexin 1 channels mobilized HSPCs significantly less efficiently. More importantly, our patient data revealed that ~60% of patients that turned out to be poor HSPC mobilizers (n=20) displayed the pannexin 1 polymorphism SNP5 (Rs3020015) T/C. This polymorphism was observed in only 1 out of 26 good-mobilizer patients. Conclusions. Our results further support an important role for ATP-mediated purinergic signaling in the mobilization of HSPCs. Moreover, the pannexin 1 SNP5Rs3020015 T/C polymorphism may serve as a diagnostic tool to identify poor mobilizers. This investigation shed more light on the molecular pathways involved in the egress of HSPCs from BM into PB and will help to design better mobilization protocols in the case of poor mobilizers.