The Synergistic Effect And Mechanism Study Of Lsd1inhibitor Ory-1001 And 1，25（OH）₂D₃ On The Differentiation Of Acute Mononuclear Leukemia Cells

Acute monocytic leukemia(AML-M5)is a kind of hematological malignancy with high heterogeneity,characterized by limited differentiation,blocked apoptosis and uncontrolled proliferation of myeloid progenitor cells.Based on the principle that AML-M5 is a kind of disease with blocked differentiation,drugs that can induce the differentiation of leukemia cells are very promising to become a new choice for the treatment of AML-M5.1,25(OH)2D3,an endogenous ligand of vitamin D receptor(VDR),can induce the differentiation of AML-M5 cells,while,clinical trials are restricted by the dose-limiting hypercalcemia.Therefore,reducing the dosage of1,25(OH)2D3 and avoiding the side effects caused by high dosage may be a new strategy for the treatment of AML-M5.At present,the LSD1 inhibitor ORY-1001 developed by Oryzon has entered clinical trials for the treatment of AML,and combined with ATRA has been proposed for the treatment of APL.Although ORY-1001 is effective in treating AML-M5,it has side effects such as anemia and thrombocytopenia.Based on this,our project carried out the study of ORY-1001 combined with 1,25(OH)2D3 to induce the differentiation of acute monocytic leukemia cells.On the one hand,it can reduce the dosage of 1,25(OH)2D3 and avoid the side effects caused by large dosage;on the other hand,it can enhance the curative effect of both.It is expected that on the basis of exploring the synergistic mechanism of the two,we can develop a new clinical medication strategy for the treatment of AML-M5 by inducing differentiation,so as to effectively alleviate the disease of AML-M5 patients.Firstly,we found that ORY-1001 and 1,25(OH)2D3 can induce the differentiation of acute monocytic leukemia cells respectively,which is consistent with previous studies.Furthermore,we detected the ability of ORY-1001 combined with1,25(OH)2D3 to induce the differentiation of AML-M5 cells by flow cytometry and real-time quantitative PCR.For the first time,we found ORY-100 combined with1,25(OH)2D3 synergistic induced the differentiation of AML-M5 cells.Based on this,we further found that interference with LSD1 can also cooperate with 1,25(OH)2D3 to induce the differentiation of AML-M5 cells.The above results indicate that: inhibition or interference of LSD1 combined with 1,25(OH)2D3 synergistic induction of differentiation of acute mononuclear leukemia cells.As 1,25(OH)2D3 plays a biological role by targeting VDR,it gives us an enlightenment: inhibition or interference of LSD1 combined with 1,25(OH)2D3 may induce the differentiation of AML-M5 cells by co-activating VDR.In order to verify this mechanism,we used luciferase reporter gene assay to detect the activation of VDR directly,and real-time quantitative PCR to detect the downstream target genes CYP24A1,HBEGF and FBP1 of VDR indirectly.The results showed that inhibition or interference of LSD1 could induce the differentiation of AML-M5 cells by coactivating VDR with 1,25(OH)2D3 and inducing the expression of its downstream genes.In order to further explore how LSD1 enhances the activation of VDR and whether there is interaction between them.The CO-IP experiment showed that there was interaction between LSD1 and VDR,which was independent of the existence of1,25(OH)2D3.This suggests that LSD1 and VDR belong to the same nuclear transcription complex,which is the basis of inhibiting or interfering with LSD1’s synergism with 1,25(OH)2D3 to activate VDR.Next,to study the level of activation of downstream differentiation pathway.We detected the expression of differentiation pathway related proteins IRF8 at protein and gene levels.The results showed that inhibition or interference of LSD1 combined with1,25(OH)2D3 significantly increased the level of downstream differentiation pathway protein IRF8.In order to evaluate the efficacy of ORY-1001 and 1,25(OH)2D3 in inhibiting the progression of AML-M5,we evaluated the ability of ORY-1001 and1,25(OH)2D3 alone or in combination to induce the differentiation of AML-M5 cells from 5 AML patients,1,25(OH)2D3 and ORY-1001 alone or in combination can induce the differentiation of AML-M5 cells,but the effect of the combination of them is stronger than that of single treatment.In conclusion,from the perspective of inducing the differentiation of acute monocytic leukemia cells,this study proposed the synergistic effect of 1,25(OH)2D3combined with ORY-1001 for the first time,and further elaborated the molecular mechanism of inhibiting or interfering with LSD1 in inducing the differentiation of AML-M5 cells with 1,25(OH)2D3.In cell experiment,it was found that inhibition or interference of LSD1 could induce the differentiation of AML-M5 cells with1,25(OH)2D3.On this basis,further studies found that inhibiting or interfering with LSD1 can induce the differentiation of AML-M5 cells by cooperating with1,25(OH)2D3 to activate VDR and inducing the expression of its downstream genes.In addition,the study also found that LSD1 and VDR belong to the same nuclear transcription complex,which is the basis of inhibiting or interfering with LSD1 to cooperate with 1,25(OH)2D3 to activate VDR.Based on the results of cell experiments,we collected and separated primary AML-M5 cells,and evaluated the differentiation inducing effect of 1,25(OH)2D3 and ORY-1001 alone or in combination on human primary AML-M5 leukemia cells.The following conclusions were obtained:1,25(OH)2D3 and ORY-1001 alone or in combination can induce the differentiation of acute monocytic leukemia cells,but compared with single treatment,the effect of combination of 1,25(OH)2D3 and ORY-1001 is stronger.