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5-Azacytidine depletes HSCs and synergizes with an anti-CD117 antibody to augment donor engraftment in immunocompetent mice.

ABSTRACT: Depletion of hematopoietic stem cells (HSCs) is used therapeutically in many malignant and nonmalignant blood disorders in the setting of a hematopoietic cell transplantation (HCT) to eradicate diseased HSCs, thus allowing donor HSCs to engraft. Current treatments to eliminate HSCs rely on modalities that cause DNA strand breakage (ie, alkylators, radiation) resulting in multiple short-term and long-term toxicities and sometimes even death. These risks have severely limited the use of HCT to patients with few to no comorbidities and excluded many others with diseases that could be cured with an HCT. 5-Azacytidine (AZA) is a widely used hypomethylating agent that is thought to preferentially target leukemic cells in myeloid malignancies. Here, we reveal a previously unknown effect of AZA on HSCs. We show that AZA induces early HSC proliferation in vivo and exerts a direct cytotoxic effect on proliferating HSCs in vitro. When used to pretreat recipient mice for transplantation, AZA permitted low-level donor HSC engraftment. Moreover, by combining AZA with a monoclonal antibody (mAb) targeting CD117 (c-Kit) (a molecule expressed on HSCs), more robust HSC depletion and substantially higher levels of multilineage donor cell engraftment were achieved in immunocompetent mice. The enhanced effectiveness of this combined regimen correlated with increased apoptotic cell death in hematopoietic stem and progenitor cells. Together, these findings highlight a previously unknown therapeutic mechanism for AZA which may broaden its use in clinical practice. Moreover, the synergy we show between AZA and anti-CD117 mAb is a novel strategy to eradicate abnormal HSCs that can be rapidly tested in the clinical setting.

SUBMITTER: Bankova AK

PROVIDER: S-EPMC8679673 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:In contrast to the short-term (ST) CD34+ stem cells, studies have suggested that long-term (LT) hematopoietic stem cells (HSCs) found in the CD34- stem cell pool have trouble migrating and engrafting when introduced through IV. To understand why these deficiencies exist, we set out to fully elucidate the adhesion mechanisms used by ST and LT-HSCs to migrate to the bone marrow(BM). Specifically focusing on murine ST-HSCs (Flk2-CD34+) and LT-HSCs (Flk2-CD34-), we observed a distinctive expression pattern of BM homing effectors necessary for the first step, namely sialyl Lewis-X (sLex) (ligand for E-selectin), and the second step, namely CXCR4 chemokine receptor (receptor for SDF-1). sLex expression was higher on Flk2-CD34+ ST-HSCs (>60%) compared with Flk2-CD34- LT-HSCs (<10%), which correlated to binding to E-selectin. Higher concentrations of CXCR4 were observed on Flk2-CD34+ ST-HSCs compared with Flk2-CD34- LT-HSCs. Interestingly, the expression of CD26, a peptidase known to deactivate chemokines (ie, SDF-1), was higher on Flk2-CD34- LT-HSCs. Given that both E-selectin-binding and CXCR4-mediated migration are compromised in Flk2-CD34- LT-HSCs, we aimed to enhance their ability to migrate using recombinant human fucosyltransferase 6 (rhFTVI) and the CD26 inhibitor, Dip A (diprotin A). To this end, we observed that although LT-HSCs expressed low concentrations of sLex, they were able to engraft when transplanted into recipient mice. Moreover, although both CD26 inhibition and fucosylation enhanced migration of both HSC populations in vitro, only pretreatment of LT-HSCs with Dip A enhanced engraftment in vivo after transplantation into recipient mice. Remarkably, fucosylation of Flk2-CD34+ ST-HSCs consistently led to their ability to transplant secondary recipients. These data suggest that using fucosylation and Dip A to overcome the molecular disparity in adhesion mechanisms among ST-HSCs and LT-HSCs differentially influences their abilities to migrate and engraft in vivo and promotes the ability of ST-HSCs to engraft secondary recipient mice, the gold standard for testing functionality of LT-HSCs.

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5-Azacytidine depletes HSCs and synergizes with an anti-CD117 antibody to augment donor engraftment in immunocompetent mice.

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