Neogambogic Acid(NGA)induces Apoptosis In FLT3-ITD(+)AML Through Degrading FLT3 And Increasing Intracellular Reactive Oxygen Species Level

Myeloid leukemia includes acute myeloid leukemia(AML)and chronic myeloid leukemia(CML),both of which can carry abnormally mutated oncogenes to produce corresponding oncoproteins,such as drugs that can specifically degrade oncoproteins,will Bring breakthrough progress to treatment.Among patients with acute myeloid leukemia,the proportion of cases carrying FLT3-ITD mutation is about 30%,and many inhibitors have been developed for this target.Inhibitors as a single agent for relapsed and refractory leukemia can achieve rapid and short-term efficacy and remission,and when combined with traditional chemotherapy,can improve the survival rate of patients.However,FLT3 inhibitors are only effective in the short term and soon fail due to the emergence of drug resistance.One of the drug resistance mechanisms includes point mutations in the TKD region of the FLT3 protein itself.In our research,we screened a Tenghuangaceae plant extract neogambogic acid(NGA)has a good anti-tumor effect,including for FLT-ITD + cell lines,we also confirmed that NGA on FLT3-Primary cells of ITD + patients also have a good tumor killing effect,and have little effect on normal human mononuclear cells.We used CCK8,Westernblot,and flow cytometry to prove the value-added inhibition and pro-apoptotic effect of the drug.The subsequent separation of aggregate components and immunofluorescence experiments using the aggregate detection kit confirmed that the drug can promote FLT3 to Aggregates transfer and degrade.In addition,we used the ROS probe DHE and DCFH-DA to detect the NGA-treated FLT3-ITD + cell line and found that the ROS level was increased.Reverse the apoptosis caused by ROS.NGA also has the effect of inducing apoptosis and degrading FLT3 in the cell line Baf3 that stably transfers the drug resistance point mutations D835 V and Y842 C in the TKD region.NGA and the clinical drug bortezomib show a strong synergistic effect and can accelerate FLT3 degradation and increase apoptosis Therefore,it is expected to overcome the current resistance problem of FLT3 inhibitors to a certain extent,and provide new options for the treatment of FLT3-ITD + patients.For patients with chronic myeloid leukemia,resistance to tyrosine kinase inhibitors(TKIs)is an important clinical challenge.There are some CML patients in the chronic phase or accelerated phase that are not effective for TKIS and other treatments.Homoharringtonine(HHT)has been approved for the treatment of these patients,but the underlying mechanism of action is still unclear.In this study,we used MTS and Western blot experiments to confirm that HHT can induce apoptosis in imatinib-resistant K562 R cells and downregulate BCR-ABL protein.CHX half-life experiments show that HHT can induce BCR-ABL protein degradation,and this effect can be reversed by autophagolysosome inhibitors CQ and Baf-A1.Subsequently,we demonstrated in H562 R cells that HHT can induce autophagy production,and silencing the key autophagy proteins ATG5 and Beclin-1 inhibited the degradation and cytotoxic effect of HHT on BCR-ABL protein.We further confirmed by immunoprecipitation that the autophagy receptor p62 /SQSTM1(p62)is involved in HHT-induced autophagy degradation of BCR-ABL.Our conclusion is that HHT induces p62-mediated autophagy in the imatinib-resistant CML cell line K562 R and causes degradation of the BCR-ABL protein.Inducing BCR-ABL protein degradation by autophagy is a new strategy for TKI-resistant CML treatment.Therefore,our research above has innovatively identified two drugs capable of degrading target proteins in two common myeloid leukemias,providing new ideas for treatment and overcoming existing drug resistance problems to some extent.