Investigation Of New Drug Therapeutics For Refractory Or Relapsed B-ALL

The funding of this research work was from the NIH R01 leukemia microenvironment project of Professor Yong-Mi Kim from the Los Angeles Children’s Hospital of the University of Southern California.Background.In recent years,the prognosis of childhood acute lymphoblastic leukemia(ALL)has been significantly improved,approaching 90%of the overall survival rate,but the prognosis of adult ALL has not been significantly improved,only less than 50%of the overall survival rate.And refractory and relapsed ALL patients have a very poor prognosis,so the main cause of death in ALL is relapse.This study focuses on the hypothesis that the bone marrow microenvironment induces leukemia drug resistance and the formation of minimal residual leukemia(MRD).The most classic theory is the cell adhesion-mediated chemotherapy resistance(CAM-DR).In addition,integrin α4 is a crucial cell adhesion protein,PI3K/AKT pathway is involved in downstream cell signaling of integrin,and the research of c-Myc in drug resistance genes is relatively mature.Therefore,this project will explore the preclinical effects and mechanisms of two new drugs that are potentially useful for the treatment of refractory and relapsed ALL based on the CAM-DR mechanism.One is the oral integrin α4β1 small molecule antagonist AVA4746,and the other is PI3Kδ-BRD4 dual antagonist SF3535.It is hoped that these two targets will provide new ideas and a theoretical basis for the treatment strategy of refractory and relapsed B-ALL.Methods.In vivo and in vitro experimental studies were conducted through mouse transplantation(PDX)models derived from patients with refractory and relapsed B-cell ALL,and the pre-clinical biological effects of two small molecule antagonists,AVA4746 and SF2535,were evaluated.In the AVA4746 study,(1)using flow cytometry to determine the expression of Integrin α4 on the surface of B-ALL and the changes in its expression after the application of AVA4746,(2)the VLA-4 conformational activation test to evaluate the VLA-4 competitive binding of AVA4746 and VCAM-1,(3)Western Blot method to measure B-ALL overall Integrin α4 protein expression after AVA4746 treatment,(4)Cell adhesion test and de-adhesion test to evaluate AVA4746 to B-ALL and ligand VCAM-1 or stromal cells(5)The effect of AVA4746 on inhibiting blood vessel formation was evaluated by HUVEC cell in vitro angiogenesis test,(6)The effect of AVA4746 on B-The oxidative metabolism ofA LL,(7)in vivo animal experiments to evaluate the bone marrow mobilization effect of AVA4746 on B-ALL cells,and the survival evaluation of combined chemotherapy VDL on B-ALL mice.In the SF2535 study,(1)ChIP-qPCR was used to evaluate the target effect of SF2535 on c-Myc Promoter in B-ALL cells,qPCR to evaluate the effect of SF2535 on c-Myc mRNA transcription levels,and Western Blot to evaluate SF2535 on c-Myc protein levels(2)Evaluate the effect of SF2535 on PI3K/p-AKT through the serum deprivation test and Western Blot method;(3)Through the apoptosis test and Western Blot,to determine if SF2535 has an apoptotic effect and apoptosis on B-ALL and apoptosis pathway,(4)Detect the effect of SF2535 on cell cycle by flow cytometry,(5)determine the effect of SF2535 on cell proliferation by Trypan Blue staining cells counting method,(6)Detect the effect of SF2535 on B-ALL by flow cytometry The effect of cell adhesion molecules and the adhesion test clarified its adhesion effect(7)The tumor reduction effect of SF2535 on B-ALL cells was confirmed through an animal model.Results.(1)B-ALL extensively express integrin α4,PI3K,and BRD4;(2)AVA4746 can strongly and competitively antagonize the combination of VCAM-1 and B-ALL cells;(3)AVA4746 significantly reduces B-ALL cells Integrin α4 expression;(4)AVA4746 can clearly de-adhere B-ALL cells to VCAM-1 ligand;(5)AVA4746 has a potential mobilization effect on minimal residual leukemia in bone marrow;(6)AVA4746 affects vascular endothelial cells in vitro Blood vessel formation ability;(7)AVA4746 can increase the sensitivity of refractory and relapsed B-ALL-PDX mice to chemotherapy;(8)AVA4746 affects the metabolism of B-ALL cells;(9)SF2535 significantly down-regulates refractory and relapsed B-ALL cells C-Myc and phosphorylated(p)-AKT expression;(10)SF2535 obviously induces refractory and relapsed B-ALL cell apoptosis through the endogenous apoptosis pathway;(11)SF2535 can affect cell cycle changes and inhibit cells Proliferation;(12)SF2535 affects the changes of cell adhesion molecules;(13)SF2535 reduces the burden of peripheral blood leukemia in refractory and relapsed B-ALL-PDX mice.Conclusion:AVA4746 antagonizes integrin α4β1/VCAM-1 to make refractory and relapsed B-ALL cells de-adhere to the stromal cells in the bone marrow microenvironment,which affects the cell’s ability to adapt to reactive oxygen species,which in turn increases the effect of B-ALL cells on chemotherapy.Sensitivity and SF2535 by directly targeting PI3Kδ-BRD4 has a synergistic effect on the two signaling pathway sites,reducing AKT that mediates cell survival and adhesion,and the key downstream of c-Myc that mediates cell drug resistance and tumor formation.The expression of signal molecules can cause a strong apoptotic effect on refractory and relapsed B-ALL.Innovation.These two pre-clinical drugs developed based on the CAM-DR theory have a high potential of clinical translation and bring new target strategies to refractory and relapsed B-ALL.