The Effets Of Epigenetic Drugs On Glioblastoma And Their Mechanisms

Part ? Targeting Super-Enhancer-associated Transcriptional Addiction through CDK7 Inhibition is a Novel Therapeutic Strategy against GBMIntroduction Glioblastoma(GBM)is the most common and aggressive brain tumor in adults1.Numerous targeted therapeutic agents for treating adult GBM so far mostly show at best modest and infrequent efficacy.Tumor heterogeneity and plasticity have been recognized as major causes for such therapeutic inefficacy.GBM cells within the same tumor could display distinct gene expression patterns,generating various subpopulations of cells that carrying different druggable targets and exhibiting different response to therapeutic approaches.GBM cells could also rapidly modulate their genome or transcriptome to develop adaptive responses to chemotherapy and targeted therapy,resulting in oncogenic driver shift and drug resistance.Therefore,there is an urgent need of novel therapeutic strategies that could potentially overcome these problems.Here a covalent CDK7 inhibitor THZ1 was identified as one of the top potent agents in our anti-GBM screening with a collection of epigenetic modulating small molecules.We further demonstrated that CDK7 inhibition through either THZ1 or genetic approaches(CRISPR-Cas9 and RNA interference)effectively disrupted GBM growth both in vitro and in vivo.The mechanism of its anti-GBM effects was further investigated.Purpurse 1? Validation of anti-GBM effects of THZ1 both in vitro and in vivo 2? To investigate the anti-GBM effects by targeting CDK7 throuth CRISPR-Cas9 and RNA interference.3? To investigate the mechanism of anti-GBM by targeting CDK7Methods 1? Epigenetic drug screen on GBM through MTS assay 2?To investigate the ani-GBM effects of targeting CDK7 through Celltiter-Glo and Celltiter-Blue assay.3? To investigate the anti-GBM effects of THZ1 in vivo 4? To investigated the anti-GBM effects of targeting CDK7 through CRISPR-Cas9 or RNA interference 5? To investigate the proliferation inhibition effects of THZ1 on GBM through Ed U assay 6? To investigate the caspase3/7 activation effects of THZ1 on GBM through Caspase-Glo assay7? To investigate the cytotoxicity of THZ1 on GBM through Cytotox-Glo assay 8? To investigate apoptosis induction effects of THZ1 on GBM through Annexin-V/DAPI assay.9? To investigate the anti-GBM mechanism of THZ1 through q PCR,Western Blot,RNAseq and Ch IPseq analysis.10?Progonosis analysis of CDK7 m RNA expression in clinical databases.Results 1.Identification of THZ1 as a potent inhibiter of GBM cells in vitro 2.THZ1 inhibits growth of GBM cells in vivo 3.Targeting CDK7 by CRISPR-Cas9 or RNA interference inhibits growth of GBM cells 4.THZ1 causes cell cycle arrest,shuts off proliferation and induces apoptosis of GBM cells 5.THZ1 inhibits global gene transcription and preferentially targets super-enhancer-associated genes in GBM cells 6.Functional validation of THZ1-sensitive super-enhancer-associated genes in GBM cells 7.CDK7 is a prognostic marker of glioma and GBMConclusion 1?Our study demonstrates targeting CDK7-associated transcriptional addiction could be an effective therapeutic strategy against GBM 2?CDK7 is a prognosis marker for GBM and glioma patients.Part ? Enhanced efficacy of Panobinostat combined with JQ1 or BEZ235 in glioblastomaIntroduction Epigenetic mechanisms are increasingly recognized as a major factor contributing to pathogenesis of cancer including glioblastoma.Histone deacetylases(HDACs)are overexpressed and mutated in various solid and hematologic malignancies and play key roles in tumorigenesis.Various histone deacetylase inhibitors(HDACIs),such as panobinostat,vorinostat and VPA et al.,have shown potent efficacy against GBM in preclinical studies.Although the main mechanism of anti-cancer activity for HDACIs has not yet been found,multiple biological pathways have been suggested,including the induction of cell cycle arrest,differentiation,apoptosis,autophagic cell death,generation of reactive oxygen species,inhibition of angiogenesis and DNA damage repair(DDR).While the results from pre-clinical studies are encouraging,early clinical trials showed only modest benefit.Therefore,further work is urgently required to develop more effective combination strategies with HDACi for GBM treatment.In this study,we performed a combined drug screen of panobinostat with BET inhibitors or PI3K/m TOR signaling inhibitors and found that panobinostat also has synergistical effects with BET inhibitor or PI3K/m TOR inhibitor in GBM models.And their mechanisms were investigated further.Purpurse 1? Indentification of drugs which have synergistic effects with panobinostat against GBM 2? To investigate the synergistical mechanism of combined drugs with panobinostat.Methods 1?Combined drug screen with panobinostat on GBM through Celltiter-Glo assay 2?Validation of the synergistical effects of panobinostat with identified drugs in 1.3?To investigate the proliferation inhibition effects of combined drugs on GBM through Ed U assay 4? To investigate the caspase3/7 activation effects of combined drugs on GBM through Caspase-Glo assay 5? To investigate the cytotoxicity of combined drugs on GBM through Cytotox-Glo assay 6? To investigate apoptosis induction effects of combined drugs on GBM through Annexin-V/DAPI assay.7? To investigate the anti-GBM mechanism of combined drugs through q PCR,Western Blot,RNAseqResults 1.Cotreatment with Panobinostat and BEZ235 or BET inhibitors suppress cell growth synergistically in GBM cells 2.Cotreatment with Panobinostat and BEZ235 or JQ1 markedly inhibit cell proliferation and induce apoptosis in GBM cells 3.Cotreatment with Panobinostat and JQ1 commonly downregulate oncogenic gene expression 4.Cotreatment with Panobinostat and JQ1 commonly upregulate tumor suppressors and tumor metabolic pathwaysConclusion 1.Combinational treatment with panoninostat and BEZ235 has synergistical efficacy against GBM cells.2.Combinational treatment with panoninostat and JQ1 has synergistical efficacy against GBM cells.