Gilteritinib Synergizes With FLT3-NKG2D-CAR T In FLT3-mutated Acute Myeloid Leukemia And The Underlying Molecular Mechanism

BackgroundAcute myeloid leukemia(AML)is a kind of hematological malignancies caused by the abnormal proliferation of myeloid hematopoietic progenitor cells,which is characterized as the inhibition of normal hematopoietic function and abnormal extramedullary infiltration.FMS-like tyrosine kinase 3 receptor(FLT3)is the most common gene mutation site in AML,among which,FLT3-ITD mutation occurs in 25-30%of AML patients and is associated with poor prognosis.At present,AML patients carrying FLT3-ITD mutation suffer from a low complete remission(CR)rate with the treatment of traditional chemotherapy regimen,and the 5-year overall survival rate(OS)and 5-year progression-free survival rate(DFS)are 29%and 32%.What’s more,FLT3-ITD mutation is also a major factor indicating the poor prognosis after hematopoietic stem cell transplantation(HSCT).Therefore,there is an urgent need to find out novel and effective ways to treat FLT3mut+-AML.Chimeric antigen receptor T cells(CAR-T cells)is a new treatment regiment of tumor immunotherapy,which has made remarkable achievements in the treatment of hematological malignant tumors.In addition,our research team found that molecular targeted drugs can induce the expression of NK cell activating ligands(NKG2DLs)in solid tumor cells.Therefore,we speculated that the combination of molecular targeting drugs with CAR-T cells will have a better anti-tumor effect than each of the treatment alone.What’s more,we proposed the idea for the first time that the combination of FLT3 inhibitor and FLT3-NKG2D-CAR T cells have a synergistic anti-leukemia effect on FLT3mut+-AML.In this study,we generated a novel dual-target FLT3-NKG2D CAR with the recognition domains of FLT3 and NKG2D sites and the intracellular signal domains of 4-1BB and CD3ζ.After that,we obtained the CAR-T cells by lentivirus transfection.In the following experiments,we explored the anti-tumor effects of the single use of CAR-T cells or the combined application of FLT3 inhibitors.At the same time,we studied the molecular mechanisms of how FLT3 inhibitors induce FLT3mut+-AML cells to express NKG2DL and the possibilities of combining the molecular targeted drugs and CAR-T cell therapy in the treatment of FLT3mut+-AML.Research MethodAML cell lines carrying FLT3 mutation gene were used as a model and FLT3 inhibitor Gilteritinib and FLT3-NKG2D-CAR-T cells generated by FLT3-NKG2D specific CAR virus were performed as the acting factors.cellular immunology and DNA recombination technique were applied to explore the synergistic anti-leukemia effect in the combination therapy of FLT3 inhibitors and dual targeted CAR-T cells in vivo and in vitro,including the immune activation receptors and ligands of NKG2D-NKG2DLs changes and molecular mechanismsResearch Results1.Gilteritinib can inhibit the proliferation of FLT3+AML cells and stimulate the expression of FLT3 and NKG2DLs on the surface of FLT3+AML cells.The difference of mean flourscence indensity(MFI)ratio and protein quantification before and after Gilteritinib application was statistically significant.2.We successfully transferred Lenti-EF1a-FLT3(EB10)CAR-NKG2D-CAR-EGFP fragments into T cells,with a transfection efficiency of(24.9±3.2)%.3.After co-culture of FLT3-NKG2D-CAR T cells and FLT3AML cells and primary AML before and after Gilteritinib application,the levels of released IL-2,IFN-γ and other cytokines were significantly higher than the non-application group,the killing efficiency to tumor cells was obviously better than the non-application group.In vivo experiments,survival time of mice in the Gilteritininb and dual-target CART group was significantly prolonged,P=0.027 compared with the FLT3-NKG2D-CAR T group;P=0.007 compared with the Gilteritinib group.4.After the NF-κB pathway was inhibited,Gilterirtinib induced upregulation of NKG2DLs disappeared,the key player of the NF-κB bypass pathway was further identified by examining the Protein phosphorylation of NF-κB pathway after Gilteritinib treatment.Finally,we verified that the NF-κB bypass pathway is an important pathway for Gilteritinib to regulate NKG2D ligand expression in AML cells by silencing NF-κB2 molecules.ConclusionOur data suggest that high-risk AML patients with FLT3-ITD mutations are likely to benefit from FLT3-NKG2D-CAR T cell therapy.This study shows that FLT3 inhibitor Gilteritinib has a triple role.Firstly,the inhibitor has a direct toxic effect on leukemic cells.Secondly,the inhibitor upregulatesthe expression of FLT3 on the surface of AML cells.Finally,it acts as a "stimulation induction" source,to induce leukemic cells to express immune activators NKG2DLs.Meanwhile,we confirmed that Gilteritinib upregulates NKG2DLs expression mainly through the NF-B bypass.And according to in vitro and in vivo experiments and primary cells,it was confirmed that Gilteritinib can enhance the effect of FLT3-NKG2D-CAR T cells on FLT3+AML,which has the value of basic research and clinical transformation significance.