BackgroundThe prognosis of patients with recurrent/refractory acute myelogenous leukemia (AML) remains poor and cell-based immunotherapies hold promise to improve outcomes. Natural Killer (NK) cells can elicit an antileukemic response via a repertoire of activating receptors that bind AML surface ligands. NK-cell adoptive transfer is safe but thus far has shown limited anti-AML efficacy. Here, we aimed to overcome this limitation by engineering NK cells to express chimeric antigen receptors (CARs) to boost their anti-AML activity and interleukin (IL)-15 to enhance their persistence.MethodsWe characterized in detail NK-cell populations expressing a panel of AML (CD123)-specific CARs and/or IL-15 in vitro and in AML xenograft models.ResultsCARs with 2B4.ζ or 4-1BB.ζ signaling domains demonstrated greater cell surface expression and endowed NK cells with improved anti-AML activity in vitro. Initial in vivo testing revealed that only 2B4.ζ Chimeric Antigen Receptor (CAR)-NK cells had improved anti-AML activity in comparison to untransduced (UTD) and 4-1BB.ζ CAR-NK cells. However, the benefit was transient due to limited CAR-NK-cell persistence. Transgenic expression of secretory interleukin (sIL)-15 in 2B4.ζ CAR and UTD NK cells improved their effector function in the setting of chronic antigen simulation in vitro. Multiparameter flow analysis after chronic antigen exposure identified the expansion of unique NK-cell subsets. 2B4.ζ/sIL-15 CAR and sIL-15 NK cells maintained an overall activated NK-cell phenotype. This was confirmed by transcriptomic analysis, which revealed a highly proliferative and activated signature in these NK-cell groups. In vivo, 2B4.ζ/sIL-15 CAR-NK cells had potent anti-AML activity in one model, while 2B4.ζ/sIL-15 CAR and sIL-15 NK cells induced lethal toxicity in a second model.ConclusionTransgenic expression of CD123-CARs and sIL-15 enabled NK cells to function in the setting of chronic antigen exposure but was associated with systemic toxicities. Thus, our study provides the impetus to explore inducible and controllable expression systems to provide cytokine signals to AML-specific CAR-NK cells before embarking on early-phase clinical testing.

The identification of new pathways supporting the myelodysplastic syndrome (MDS) primitive cells growth is required to develop targeted therapies. Within myeloid malignancies, men have worse outcomes than women, suggesting male sex hormone–driven effects in malignant hematopoiesis. Androgen receptor promotes the expression of five granulocyte colony-stimulating factor receptor–regulated genes. Among them, CCRL2 encodes an atypical chemokine receptor regulating cytokine signaling in granulocytes, but its role in myeloid malignancies is unknown. Our study revealed that CCRL2 is up-regulated in primitive cells from patients with MDS and secondary acute myeloid leukemia (sAML). CCRL2 knockdown suppressed MDS92 and MDS-L cell growth and clonogenicity in vitro and in vivo and decreased JAK2/STAT3/STAT5 phosphorylation. CCRL2 coprecipitated with JAK2 and potentiated JAK2-STAT interaction. Erythroleukemia cells expressing JAK2V617F showed less effect of CCRL2 knockdown, whereas fedratinib potentiated the CCRL2 knockdown effect. Conclusively, our results implicate CCRL2 as an MDS/sAML cell growth mediator, partially through JAK2/STAT signaling.