The Effect Of Abivertinib,A Novel BTK Inhibitor,and Its Mechanism Research In Acute Myeloid Leukemia

BackgroundAcute myeloid leukemia(AML)is the most common malignant tumor of the hematopoietic system,which accounts for about 70% of adult acute leukemia.It is characterized by malignant clonal proliferation of hematopoietic stem cells and precursor cells.Although with the continuous improvement of clinical and laboratory technology,the prognosis of AML has improved significantly,but currently about 70% of AML patients cannot survive more than 5 years after diagnosis.In recent years,key molecular inhibitors targeting hematological malignancies disease processes,such as IDH1 / 2,BCL2,and FLT3 inhibitors,have been increasingly used in the blood system and have achieved ascendant results.Among them,Ibrutinib,which targeting Bruton tyrosine kinase(BTK)has been approved by the FDA in patients of CLL/SLL,MCL and WM.However,the effect and mechanism of treatment involved BTK inhibitors in AML are not completely clear and needs to be continuously explored.ObjectiveBone marrow and PB mononuclear cell specimens from healthy donors and AML patients were collected respectively,and the expression of BTK m RNA was detected by q-PCR.Based on the result,the differences of BTK expression level was analyzed between healthy people and AML patients,as well as BTK expression and AML prognosis.Investigate the inhibitory effect of a novel BTK inhibitor,Abivertinib on AML cells and its potential mechanism.Explore the synergistic inhibitory effect and mechanism of Abivertinib and HHT or BCL2 inhibitor Venetoclax on AML cells.Prove the safety and effectiveness of the combined therapy in AML-xenograft experiments.Methods1.Collected bone marrow blood from AML patients and peripheral blood samples from healthy donors,isolated mononuclear cells,collected cell clumps from AML cell lines,extracted total RNA,and detected BTK m RNA expression.Patients' information was collected including clinical diagnosis time,age,gender,peripheral white blood cell count at the time of onset,hemoglobin amount,platelet count,bone marrow primordial cell ratio,chromosome karyotype,gene mutation type,treatment plan,treatment effect,survival time.Differences in BTK expression between healthy donors and AML patients were analyzed.AML patients were grouped according to BTK expression levels.KaplanMeier method and log-rank test were used to draw and compare survival time curves.2.Detected the inhibitory effect of Abivertinib,a novel BTK inhibitorits on AML cells by MTS assay.Explored the apoptosis and cell cycle blocking effect induced by Abivertinib by flow cytometry.The sensitivity differences on AML cells of Abivertinib and traditional BTK inhibitor Ibrutinib were compared.At the same time,the AML cells treated with Abivertinib were collected,and changes in apoptosis-related proteins,cyclerelated proteins,BTK and its downstream pathway proteins,and tumor proliferationrelated signaling pathway proteins were verified by western blot.After AML cells were conducted by gene interference(RNAi)technology,the sensitivity changes of AML cells to Abivertinib were observed to confirm its mechanism.3.AML cell lines and blasts were treated with Abivetinib,HHT alone or in combination,the growth inhibitory effects were measured and the combined index was calculated.Collected AML cells treated with single or combined agents followed by flow cytometry and western blot to detect the apoptosis and cycle arrest induced by drugs.Measured the changes of proteins of BTK and tumor proliferation related pathway.4.Bone marrow mononuclear cell specimens from 6 AML patients treated with VA(Venetoclax + azacitidine)were collected for RNA-sequence sequencing.Three patients achieved complete response(CR)and 3 patients did not(NR).Analyzed the differentially expressed genes based on the results.5.AML cell lines and blasts were treated with Abivetinib,Venetoclax alone or in combination,the growth inhibitory effects were measured and the combined index was calculated.Collected AML cells treated with single or combined agents followed by flow cytometry and western blot to detect the apoptosis and cycle arrest induced by drugs.Confirm the changes of BCR signaling pathways after agents' exposure by western blot.After gene interference(RNAi)treatment of AML cells,the synergistic changes of the two drugs in AML cells were observed to determine the combination mechanism.6.Therapeutic effect of Abivertinib combined with HHT or Venetoclax on AML animal models.AML cells(MV4-11-luc,MOLM-13 and THP-1-luc)were injected into NSG mice via tail vein to induce AML.The mice were randomly divided into groups followed by therapy of placebo,Abivertinib,HHT or Venetoclax and Abivertinib combined with HHT or Venetoclax treatment.The tumor burden in mice was detected by fluorescence image or measurement of human-CD45 in peripheral blood weekly,and the body weight of the mice was recorded every 3 days.Mice were sacrificed when the mice were paralyzed,and bone marrow,peripheral blood,and spleen cells were collected to detect the expression of human-CD45.The survival time of each group of mice was recorded for survival analysis.The degree of spleen and bone marrow tumor cell infiltration in mice was detected by immunohistochemistry.Results1.The expression level of BTK in 271 normal karyotype AML(CN-AML)patients and AML cell lines was higher than that in healthy people.However,further survival analysis results showed that the overall survival time(OS)of AML patients with high BTK expression were not statistically different from those with low expression.2.The novel BTK inhibitor Abivertinib can significantly inhibit the growth of AML cells,especially those carried FLT3-ITD mutation,and its effect is better than Ibrutinib;Abivertinib could induce apoptosis of AML cells,activate the cleavage band of caspase family and down-regulated the expression of mitochondrial family anti-apoptotic protein BCL2 / BCL-XL;Abivertinib can down-regulate the expression of checkpoint proteins CDK2,CDK4 and CDK6 in G0 / G1 phase,blocking AML cell cycle in G0 / G1.In addition,Abivertinib could inhibit AML progression by inhibiting BTK and its downstream signaling pathways and impair FLT3,PI3 K and other BTK-independent pathways.3.Abivertinib combined with HHT could significantly inhibit the growth of AML cells,the combined index was less than 1,and the synergistic effect is good.Compared with the single drug group,the apoptotic AML cells in combination group were significantly increased.The expression of Cleaved Caspase3,7 and Cleaved PARP in the apoptotic family was up-regulated,and the down-regulation of BCL2 / BCL-XL was also detected in the combined group.Abivertinib could cooperate with HHT to down-regulate PI3 K and its downstream signaling pathways.4.RNA-sequence of bone marrow mononuclear cell samples from 6 AML patients(3 CR and 3 NR)with VA treatment,and analysis of the results showed that the expression of BTK in non-responding patients was significantly higher than that in patients with complete response.However the expression of other members of the Tec family was not statistically different.5.Abivertinib combined with Venetoclax can significantly inhibit the growth of AML cells.Compared with single drugs,Abivertinib significantly increased the apoptosis of AML cells.The expression of Cleaved Caspase3 and Cleaved PARP in the apoptotic family was significantly increased,while the expression of MCL-1 was down-regulated.In the combination group,the AML cell cycle was arrested in the G0 / G1 phase,and the cell cycle node protein CDK family protein was significantly down-regulated.The synergy between Abivertinib and Venetoclax in AML disappeared when the expression of BTK in AML cells was impaired,indicating that this synergistic effect is BTKdependent.6.Abivertinib combined with HHT or Venetoclax can significantly reduce leukemia burden in AML-xenograft mice,reduce leukemia infiltration in the spleen and bone marrow of mice,and prolong the survival time of mice compared with single drugs.ConclusionIn this study,we confirmed that BTK was highly expressed in CN-AML,but did not indicate the bad prognosis of AML patients.A novel BTK inhibitor Abivertinib could inhibit the progression of AML cells via BTK-dependent and-independent manner,and its effect is better than Ibrutinib.Moreover,Abivertinib combined with chemotherapy drugs HHT or BCL2 inhibitor Venetoclax could synergistically inhibit the growth of AML cells,induce the apoptosis and block the cell cycle of AML cells.At the same time,in vivo results have confirmed that the combination of drugs could effectively decrease the leukemia burden of AML mice and prolong the survival time of AML mice.