Studies On Antitumor Mechanism Of BET Inhibitors

Bromodomain and extraterminal domain(BET)family proteins are important acetylation readers.BET family members include bromodomain containing protein(BRD)2,BRD3,BRD4 and bromodomain testis-specific protein(BRDT).These members widely involved in regulating gene expression regarding transcription,DNA repair,immunity,metabolism and signal transduction by identifying acetylated histones or transcription factors via their two special bromodomains,and become the popular targets for tumor therapy.The first BET inhibitor JQ1 was reported in 2010.Up to now,more than ten BET inhibitors have entered into clinical trials and mainly been used for the treatment of tumor and cardiovascular diseases,but no drugs have been approved on the market.The main reasons for limiting the clinical research progress of BET inhibitors include,in the absence of systematic preclinical research data,especially the lack of mechanism study,many clinical trials have been carried out.The regulation of many BET family target genes related to tumor proliferation,survival and cell cycle progress,such as MYC,CDK6 and BCL2,etc.,which has been reported,but is not enough to fully explain the antitumor mechanism of BET inhibitors.Therefore,the elucidation of other potential target genes of BET will help reveal the new functions of BET family members,and provide new possibilities for clinical treatment and combined application of BET inhibitors.Another reason that limits the clinical research was the poor mono-therapeutic effect of BET inhibitors and easy to develop drug resistance.Therefore,the development of new,potent and safe inhibitors is the key to promoting the clinical application of BET inhibitors.In response to the above problems,this dissertation focuses on two parts including BET regulation mechanism and the antitumor effect and mechanism of new BET inhibitors.In order to study the regulatory mechanism of BET,we first used RNA sequencing(RNA-seq)to discover the candidate target genes after BET inhibitor treatment.The results showed that the new BET inhibitor,compound 9,which was developed by our institute,reduced the transcription of indoleamine 2,3-dioxygenase1(IDO1).IDO1 is an immunocheckpoint protein,which can metabolize tryptophan into kynurenine and play an immunosuppressive role,and is a popular target of antitumor drugs.Further,we verified the inhibitory effect of other BET inhibitors on IDO1 and found that different BET inhibitors(JQ1,OTX015 and ABBV-075)could reduce the expression of IDO1 both at protein and m RNA levels in tumor cell lines from different tissues,and this inhibitory effect was in time-and concentration-dependent manners.Since IDO1 expression levels are obviously different in different cells,we used interferon?(IFN-?)to induce on cells that do not express IDO1.It was found that BET inhibitors could also evidently inhibit the expression of IFN-?induced IDO1 both at protein and m RNA levels.BET is mainly involved in the regulation of gene transcription,so we investigated whether BET family members have a direct regulatory effect on IDO1.In SKOV3 cells,we used small interfering RNA(si RNA)to interfere with the main BET family members BRD2,BRD3 and BRD4.We found that the reduction of any of the members could significantly reduce the IDO1 expression at m RNA and protein levels,but did not affect the expression level of other BET family members.Since the IDO1 promoter region has obvious enrichment of histone 3 lysine 27 acetylation(H3K27Ac),it is beneficial to the binding of BET family proteins.Therefore,we speculated that IDO1is likely to be a direct BET family target gene.Through chromatin immunoprecipitation quantitative polymerase chain reaction(Ch IP-q PCR)experiments,we found that the BET inhibitor JQ1 reduced BRD2,BRD3,BRD4,RNA polymerase II(Pol II),and H3 acetylation enrichment levels in IDO1 promoter region,but did not affect the total protein levels in the cell.When IFN-?was added,the enrichment of BRD2,BRD3,BRD4 and Pol II in the IDO1 promoter region was significantly increased,and JQ1 could reverse the increase induced by IFN-?.IDO1normally acts by catalyzing the conversion of tryptophan to kynurenine.Further,we investigated the effect of BET inhibitors on the production of IDO1 metabolite kynurenine.We found that BET inhibitors reduced the production of IDO1 metabolite L-kynurenine.In reducing L-kynurenine level,the same concentration treatment of BET inhibitors JQ1,OTX015 and IDO1 inhibitor NLG919 induced the similar effect.The combination of BET inhibitors and IDO1 inhibitor further reduced the production of L-kynurenine.We also found that under the same treatment conditions,the proliferation and growth of the cells did not change greatly,so it was further proved that the reduction of L-kynurenine production had nothing to do with the proliferation inhibitory activity of the cells.In the established BALB/c nude mouse xenograft model,BET inhibitor ABBV-075 significantly inhibited the growth of Ty-82xenograft tumors,and obviously reduced the m RNA and protein expression levels of IDO1 in tumor xenograft.Therefore,the data indicate that BET inhibitors indeed reduce the expression of IDO1 both in vitro and in vivo,further suggesting that such a reduction might be used as a pharmacodynamic marker to monitor the pharmacological effects of BET inhibitors on IDO1-expressing tumors.In order to obtain BET inhibitors with promising antitumor activity,we cooperated with pharmaceutical chemistry researchers and obtained a new and highly selective BET inhibitor,compound 19 from optimizing a Polo-like kinase 1(PLK1)and BRD4 double target inhibitor BI2536 by removing the PLK1 binding group and retaining the BRD4 binding group.The result of the bromodomain selectivity showed that in 32 proteins screened,Compound 19 could bind bromodomain I(BD1)and bromodomain II(BD2)of all the BET family members including BRD2,BRD3,BRD4 and BRDT,but not other bromodomain-containing proteins except TAF1(2).The result of kinase selectivity showed that compound 19 had no PLK1 inhibition activity and was weak for other 24 kinases inhibition.OTX015 is one of the most widely studied BET inhibitor in clinical trials.We found that compound 19 had stronger cell proliferation inhibitory activity compared with the BET inhibitor OTX015,which has been studied in clinical trials.Among the tumor cell lines tested,MV4-11 was the most sensitive to compound 19.The 50%inhibitory concentration(IC₅₀)of compound 19 was much lower than that of JQ1 and OTX015(3.4 n M,49.1n M and 32.3 n M,respectively).Consistently,Compound 19 had a stronger inhibitory effect on the BET target gene MYC.Compound 19 at 10 n M and OTX015 at 100 n M had almost the same inhibition effect on MYC both at protein and m RNA levels.Similarly,treatment of cells with compound 19 at a low concentration(10 n M)for 24h caused notable G1 phase arrest,which was stronger than that of 100 n M OTX015caused.This cell cycle arrest was in a concentration-dependent manner.The expression level of cyclin-dependent kinase 6(CDK6),a regulatory protein related to the G1 phase,was decreased obviously by compound 19 treatment.In the apoptosis induction experiment,we found that compound 19 concentration-dependently induced apoptosis of MV4-11 cells.Under the same concentration conditions,Compound 19had a significantly stronger apoptosis-inducing effect than OTX015,which induced the production of more spliced forms of poly(ADP-ribose)polymerase(PARP),as well as the spliced forms of cysteinyl aspartate specific proteinase(Caspase)family members(Caspase-3,Caspase-7 and Caspase-8).At the same time,Compound 19 had a more obvious inhibitory effect against the expression of apoptosis-related protein B cell lymphoma 2(BCL2).Consistent with other BET inhibitors,Compound 19 also reduced the expression of IDO1 protein and m RNA and inhibited the production of L-kynurenine.In the study of antitumor activity in vivo,we established a tumor model of MV4-11 in BALB/c nude mice.Compound 19 was administered orally every day for 28 consecutive days.The mice could tolerate the dose well without death.Compound 19 had stronger antitumor activity in vivo than that of OTX015.50 mg/kg compound 19 inhibited the growth of xenograft tumors more strongly than 50 mg/kg or 100 mg/kg OTX015,and the tumor inhibition rates were 95.6%,71.5%and 87.5%.25 mg/kg compound 19 had the same antitumor effect as 100 mg/kg OTX015,and the tumor inhibition rates were 89.7%and 87.5%,respectively.In addition,we also conducted a preliminary study on the pharmacokinetic(PK)characteristics of compound 19.The result showed that compound 19 was different from JQ1 in that it was less distributed in the brain and might cause less neurotoxicity.Studies on PK parameters of different species have shown that the plasma exposure of compound 19increased with the increasement of the dose,and the half-life(T_1/2)of mice was relatively shortest(mice,100 mg/kg,T_1/2=4.4 h;rat,50 mg/kg,T_1/2=6.3 h;dog,3mg/kg,T_1/2=6.6 h).In the first part of this study,we found and confirmed a new target gene IDO1 of BET family,and clarified the regulation mechanism of BET family both in the constitutive and IFN-?-induced IDO1 expression.This work indicates that BET inhibitors may relieve the non-enzymatic immunosuppression activity of IDO1 and promote antitumor immunity by reducing the expression level of IDO1,and provide theoretical possibility for the combination use of BET inhibitors and IDO1 inhibitors.In the second part of this study,we systematically studied the antitumor effect and mechanism of the new BET inhibitor compound 19,which provides a novel,potent,highly selective and safe compound for preclinical BET inhibitor candidate library.