Targeted Inhibition Of DHODH Is Synergistic With BCL2 Blockade In High Grade B Cell Lymphoma With Concurrent MYC And BCL2 Rearrangement

Objective:Diffuse large B cell lymphoma(DLBCL)has the highest incidence rate among human non Hodgkin's lymphoma.Under the first-line R-CHOP rigemen(rituximab plus cyclophosphamide,doxorubicin,vincristine,and prednisone),about 30%of DLBCL patients are not sensitive to the treatment.With the deepening of molecular research,it is found that this kind of DLBCL patients are different from ordinary DLBCL patients,and their tumor cells show the characteristics of increased expression of c-MYC protein or BCL2 protein.Although there are many reasons for the increased expression of c-MYC protein and BCL2 protein,including the abnormality of gene itself,the abnormality of upstream regulation mechanism and the abnormality of degradation mechanism,the most characteristic reason is the overexpression of c-MYC and BCL2 protein caused by gene abnormality,which accounts for about 10%of this kind of lymphoma patients.In 2016,WHO classified DLBCL with this feature as "high-grade B-cell lymphoma with MYC and BCL2 gene translocation".The PFS and OS are only 20-30%in 5 years.Therefore,we need to explore new drugs or treatment options for this kind of lymphoma.Overexpression of MYC gene is an important feature of this kind of lymphoma.C-MYC protein,the product of MYC gene,is an important transcription factor to maintain the survival and proliferation of tumor cells.It regulates hundreds of genes related to biological processes such as cell cycle,mitosis,mitochondrial function,protein synthesis and cell metabolism.Therefore,c-MYC protein can become an important target for tumor therapy.However,due to the lack of substrate binding sites and short half-life,c-MYC protein is undrugable,so the current research on MYC as a therapeutic target is still in the stage of indirect inhibition,including inhibiting the transcription of c-MYC,affecting the connection between c-MYC and transcription targets,interfering with c-MYC cofactors and inhibiting the related signaling pathways of c-MYC.Therefore,the inhibition methods of MYC need to be further explored.Dihydrolactate dehydrogenase(DHODH)is the key enzyme that catalyzes the fourth step of the de novo pyrimidine pathway.It is also the only pyrimidine synthase located in the mitochondrial membrane.It is coupled with the electron transport chain(ETC),which plays an important role in the process of oxidative phosphorylation,The reduction of dihydroorotic acid(DHO)to orotic acid(OA)provides UMPs for downstream thymine and cytosine synthesis,glycogen synthesis,phospholipid metabolism and glycosylation.In recent years,studies have confirmed that DHODH can inhibit a variety of tumors.In the background of MYC gene rearrangement Burkitt lymphoma,DHODH can significantly inhibit c-MYC protein and Burkitt lymphoma.However,it is not clear which way DHODH can inhibit c-MYC,and whether DHODH can repress high-grade B-cell lymphoma.Therefore,exploring the relationship between DHODH and MYC will help to explore new ways to inhibit MYC,and also help to find new therapeutic targets for high-grade B-cell lymphoma.BCL2 protein is another important protein in the survival of high-grade B-cell lymphoma.When MYC overexpression is combined with BCL2 overexpression,the increased BCL2 protein in cells will weaken the apoptosis inducing effect of MYC overexpression,which leads to excessive cell proliferation and apoptosis inhibition.Therefore,BCL2 protein is an important anti apoptotic protein in high-grade B-cell lymphoma,and it can also be a therapeutic target for this kind of tumor.At present,although BCL2 inhibitors have been approved by FDA for the treatment of small B-cell lymphoma,clinical trials have found that the toxicity of BCL2 inhibitors is strong,and the efficacy and safety of BCL2 inhibitors for DLBCL and high-grade B-cell lymphoma still need to be verified.Therefore,the study on the combination of BCL2 inhibitors is helpful to provide an effective and safe treatment for high-grade B-cell lymphoma.Methods:1.Selection of cell lines and verification of cell background:DB and SU-DHL4 cells were selected as model cells of high-grade B-cell lymphoma,and the gene rearrangement of MYC and BCL2 was detected by FISH;the TP53 mutation was detected by sequencing and literature search.2.Confirm the inhibition of DHODH inhibitor on cell line:BRQ(Brequinar)was selected as DHODH inhibitor,the inhibition of different doses of BRQ on DB and SU-DHL4 cells was detected by CCK8,and the IC50 value was calculated;after the BRQ concentration was determined,uridine was selected as the rescue agent of BRQ effect,and the rescue effect and concentration of uridine were confirmed by CCK8.The effects of BRQ on cell cycle,apoptosis and intracellular reactive oxygen species of DB and SU-DHL4 cells were detected by flow cytometry and Western blot;the effects of BRQ on important signaling pathways(PI3K/Akt and NF-?B)and important proteins(c-MYC,BCL2 and BAX)in DB and SU-DHL4 cells were detected by Western blot;the effects of BRQ on MYC gene transcription were detected by PCR.3.Confirm the target of BRQ:using CRISPR technology to knock down the DHODH gene of SU-DHL4 cells.Since DHODH is a survival dependent gene,in order to maintain cell growth,all the knockout cells were cultured in the medium containing 1000?M uridine.Western blot was used to verify the knockout effect and the change of c-MYC protein after removing uridine;CCK8 method was used to detect the effect of DHODH knockout on the viability of SU-DHL4 cells;flow cytometry was used to detect the changes of cell cycle and apoptosis of SU-DHL4 cells after DHODH knockout.4.Confirm that BCL2 inhibitor and DHODH inhibitor have synergistic inhibitory effect and mechanism:Venentoclax was selected as BCL2 inhibitor,the inhibition of different concentrations of venetoclax on the viability of DB and SU-DHL4 cells was detected by CCK8 method,and the IC50 concentration was calculated.The changes of the viability of DB and SU-DHL4 cells under different concentrations of venetoclax combined with BRQ were detected by CCK8 method,and the synergistic index(CI)was calculated.Finally,the concentration of venetoclax was 20 nM and BRQ was 500 nM.The changes of cell cycle,apoptosis and reactive oxygen species in DB and SU-DHL4 cells were detected by flow cytometry;the changes of venetoclax resistance protein MCL-1 and BCL-XL,important survival protein c-MYC and BCL2 and BAX,c-MYC stability regulated kinase JAK2/STAT3 protein in DB and SU-DHL4 cells were detected by Western blot.The changes of MCL-1 and MYC gene expression in DB and SU-DHL4 cells were confirmed by PCR,and the changes of BCL2 and BAX junction and BCL2 and BIM junction in DB and SU-DHL4 cells were detected by immunoprecipitation.5.Further confirm the synergistic mechanism of BCL2 inhibitor and DHODH inhibitor:RNA-seq method was used to detect the changes of gene expression profile of SU-DHL4 cells under the single and double drug effects,and the genes with obvious changes were selected for verification.Western blot and PCR were used to detect the changes of ribosome pathway genes and B-cell tumor transformation genes of DB and SU-DHL4 cells under the single and double drug effects;flow cytometry was used to detect the expression profile of genes To detect the changes of B cell activating protein in DB and SU-DHL4 cells under single and double drug treatment.Results:1.Genetic background verification of DB and SU-DHL4 cells1.1 both DB and SU-DHL4 cells carry translocation of MYC and BCL2.1.2 DB cells carried mutant TP53,and SU-DHL4 cells carried wild-type TP53.2.The effect of DHODH inhibitor BRQ on the function of DLBCL cells2.1 BRQ inhibited the viability of DB and SU-DHL4 cells in a time-dependent and dose-dependent manner.DB cells were relatively insensitive to BRQ,and the inhibitory effect of BRQ could be saved by uridine.2.2 BRQ induce apoptosis of DB and SU-DHL4 cells by activating endogenous pathway,and induce G1/S phase arrest of DB and SU-DHL4 cells by TP53 independent way.BRQ increase reactive oxygen species in DB and SU-DHL4 cells.The changes of BRQ in apoptosis,cycle and reactive oxygen species level can be rescued by uridine.3.The effect of DHODH inhibitor on DLBCL cell survival dependent protein and signal pathway3.1 BRQ inhibit c-MYC protein,but PI3K/Akt pathway and NF-?B pathway are not affected by BRQ.The inhibition of c-MYC protein by BRQ can be rescued by uridine.3.2 BRQ inhibit c-MYC through transcriptional level,which can also be rescued by uridine.4.The effect of DHODH knockdown is similar to that of DHODH inhibitor.4.1 DHODH knockdown inhibit the viability of SU-DHL4 cells,promote G1/S phase arrest and induce apoptosis of SU-DHL4 cells.4.2 DHODH knockdown and uridine removal inhibit c-MYC protein.5.BCL2 inhibitor and DHODH inhibitor synergistically induce apoptosis of DLBCL5.1 Venetoclax inhibit the viability of DB and SU-DHL4 cells.5.2 The combination of venetoclax and BRQ induce apoptosis of DB and SU-DHL4 cells,but the combination of venetoclax and BRQ had no synergistic effect at the level of cycle and reactive oxygen species.6.The combination of BCL2 inhibitor and DHODH inhibitor produces synergistic effect through complementary mechanism.6.1 Venetoclax inhibited the connection between BCL2 protein and BIM protein,but BRQ did not;BRQ could inhibit the transcription of MCL-1 and c-MYC protein,but venetoclax had the opposite effect.6.2 In the levels of JAK/STAT kinase,BCL2 and BCL-XL,single grug or combination group had no inhibitory effect.6.3 The inhibition of DHODH inhibit many genes and proteins related to ribosome biosynthesis pathway,B cell activation and tumor transformation,but venetoclax has little effect on these genes.Conclusion:The inhibition of DHODH inhibit high-grade B-cell lymphoma and become a new therapeutic target of high-grade B-cell lymphoma;targeting DHODH and BCL2 induce apoptosis of high-grade B-cell lymphoma synergistically by influencing different ways and in complementary pathways.The combination of DHODH inhibitor and BCL2 inhibitor will be a promising method for the treatment of relapsed DLBCL in the future.