Sorafenib enhances anti-leukemia effect via promoting macrophage-NK cell immune crosstalk in FLT3-ITD+ AML patients receiving allo-HSCT

Abstract

Abstract Sorafenib maintenance therapy has been shown to reduce relapse in FLT3-ITD+ AML patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT), while the mechanism remains unclear. Given the low tumor burden in patients with complete remission (CR) post-transplantation, we hypothesized that sorafenib might improve outcomes by promoting the graft-versus-leukemia effect in patients post-transplantation. To understand how sorafenib affects immune reconstitution post-transplantation, bone marrow aspirates were collected from FLT3-ITD+ AML patients who did or did not receive sorafenib maintenance. Included patients were in CR at day 30 post-transplantation. Patients were divided into sorafenib and control groups. For the sorafenib group, sorafenib maintenance was started from day 30 post-transplantation and continued until at least day 90 post-transplantation. For the control group, sorafenib or other FLT3 inhibitors were not used. Bone marrow aspirates were collected at day 30 (T1) and day 90 (T2) post-transplantation. Mononuclear cells were separated and processed for single-cell RNA sequencing. Cells were annotated into 6 major clusters: T cell, NK cell, Myeloid, B cell, Erythroid, and Megakaryocyte. At T2, both groups had higher T cell proportions than at T1. Compared to the control group, the sorafenib group had an increased proportion of NK cells at T2. NK cells were annotated into 5 subtypes: NK_Naive, NK_CD56_bright, NK_CD56_dim_GZMH, NK_CD56_dim_FCGR3A, and NK_CD56_dim_IFNG. Pseudotime analysis revealed 3 distinct NK cell evolution trajectories. Cells from the sorafenib group were enriched in trajectory 2 (NK_Naive → NK_CD56_bright → NK_CD56_dim_IFNG), while those from the control group were enriched in trajectory 1 (NK_Naive → NK_CD56_bright → NK_CD56_dim_GZMH). Enrichment analysis showed that NK_CD56_dim_IFNG enriched pathways like IFN-γ production, inflammatory response, and NK cell-mediated cytotoxicity. Flow cytometry results also showed that patients receiving sorafenib maintenance had a higher proportion of CD16+ CD56dim NK cells and IFN-γ+ NK cells. These data suggest that sorafenib maintenance enhances NK cell reconstitution and promotes its cytokine secretion and cytotoxicity. To explore the mechanism by which sorafenib improves NK cell function, NK cells from healthy donors were separated and treated with sorafenib ex vivo. Results showed that sorafenib did not enhance the expression of functional markers like CD107a, IFN-γ, Granzyme B, and Perforin in NK cells. These results led us to hypothesize that sorafenib might promote NK cell function indirectly. Cell communication analyses showed that sorafenib enhanced communication between macrophages and NK cells, especially in the IL-18 signaling pathway. In vitro experiments showed that sorafenib treatment enhanced the transcription and secretion of IL-18 in macrophages. Also, patients and mice receiving post-transplantation sorafenib maintenance had increased IL-18 serum levels. Sorafenib treatment significantly elevated IFN-γ levels in NK cells co-cultured with macrophages, while this effect was diminished by anti-IL-18 neutralizing antibodies. These results indicate that sorafenib promotes NK cell cytotoxicity and cytokine secretion via macrophage-NK cell IL-18 immune crosstalk. We next explored the mechanism by which sorafenib promotes IL-18 transcription and secretion in macrophages. ScFEA analyses indicated increased glycolysis and decreased oxidative phosphorylation in macrophages within the sorafenib group, which was validated by Seahorse assay. As a key modulator of glycolysis, immunoblotting results showed an increased proportion of dimer and monomer forms and a decreased proportion of tetramer form of PKM2 in macrophages treated with sorafenib. Immunoprecipitation-mass spectrometry and CUT&Tag results revealed that sorafenib increased the binding of PKM2 and RelA, and their binding to the promoter region of the IL18 gene. The PKM2 activator, DASA-58, diminished the effect of sorafenib on NK cells and macrophages. These data indicate that sorafenib enhances IL-18 secretion and transcription by promoting PKM2-RelA binding. Taken together, our work reveals that post-transplantation sorafenib maintenance increases glycolysis in macrophages, promoting PKM2-RelA binding, resulting in increased transcription and secretion of IL-18, eventually promoting the expansion of the IFN-γ+ NK cell subset.