MRT68921 HCl Inhibits ULK1-induced Leukemia Cell Death And Exerts The Anti-tumor Effect

BackgroundAcute myeloid leukemia is a malignant disease of myeloid hematopoietic stem/ progenitor cells.The abnormal proliferation of primitive myeloid cells in bone marrow and peripheral blood is the main feature.The symptoms of the disease are anemia,hemorrhage,infection and fever,organ infiltration,metabolic abnormality,etc.In most cases,the condition is severe and the prognosis is dangerous.If not treated in time,it can lead to life-threatening.A number of retrospective studies have shown that the five-year survival rate for patients under the age of 60 is about 40%,while for patients over the age of 60,the five-year survival rate is about 5%-15%.With the deepening of the aging of our society,AML has become a serious threat to the life and health of our people.At present,in addition to all trans retinoic acid combined with arsenic in the treatment of acute promyelocytic leukemia has played a revolutionary role,for the treatment of other subtypes of AML,cytarabine and daunorubicin are still the main combination chemotherapy.However,the adverse reactions of chemotherapy drugs are not suitable for elderly patients,which limits the use of standard therapy.Therefore,it is urgent to find new targets for AML treatment and develop new drugs.ObjectiveThe purpose of this project is to study the relationship between the expression of ULK1 in patients with AML and prognosis,and to clarify the role of ULK1 in AML cells,so as to determine whether ULK1 can be used as a new therapeutic target in AML,and lay a theoretical foundation for the subsequent application of ULK1 inhibitors in clinical treatment.Methods1.The expression of ULK1 in patients with AML and its relationship with poor prognosis were analyzed by TCGA database;2.Western blot analysis of ULK1 protein level and q PCR detection of m RNA expression in AML cell lines and primary AML cells;3.AML cell lines were treated with three specific inhibitors of ULK1,SBI-0206965,MRT68921 HCl and MRT67307 HCl,and lentivirus was used to knock down ULK1,to analyze the changes of cell viability stimulated by ULK1inhibition;4.The morphology of cell death induced by ULK1 inhibition was observed under microscope.,Western blot was used to detect the intracellular molecular pathway,and to analyze the changes of autophagy flow after MRT68921 HCl treatment,so as to clarify the molecular mechanism of AML cell death induced by ULK1 inhibition;5.The THP-1 cell line stably transformed into luciferase was constructed.The AML model mouse was established by injecting THP-1 luciferase cells into the tail vein and treated with ULK1 inhibitor MRT68921 HCl.The tumor load,survival rate and invasion degree of tumor cells in spleen,liver and bone marrow were analyzed;6.The blood samples of AML patients were collected,the primary AML cells were isolated,and the changes of cell viability after MRT68921 HCl treatment were analyzed.Results1.The expression of ULK1 and ULK2 in patients with AML is higher than that in normal subjects,and it is related to poor prognosis;2.ULK1 was highly expressed in many leukemic cell lines and primary AML cells,but no obvious expression of ULK2 was detected;3.Three inhibitors of ULK1,MRT68921 HCl,SBI-0206965 and MRT67307 HCl,can induce leukemic cell death,and MRT68921 HCl has the best effect,while knocking down ULK1 can also induce AML cell death;4.MRT68921 HCl induces THP-1 cell death through caspase-3-GSDME pathway,and HL60 cell death through caspase-3-PARP pathway,which has nothing to do with autophagy,ROS and NADPH;5.After MRT68921 HCl treatment of AML model mice,the tumor load in mice was significantly reduced,the survival time of mice was prolonged,and the invasion degree of tumor cells in spleen,liver and bone marrow was effectively inhibited;6.MRT68921 HCl also has killing effect on primary AML cells collected from AML patients.ConclusionDrug inhibitors or gene knock outing ULK1 can induce AML cell death,which has nothing to do with autophagy pathway.ULK1 inhibitor MRT68921 HCl can reduce tumor load,prolong survival time and slow down the invasion of tumor cells in AML model mice,indicating that ULK1 and ULK1 commercial inhibitors have the potential to become new targets and drugs for AML therapy.