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Targetable BET Proteins- and E2F1-Dependent Transcriptional Program Maintains the Malignancy of Glioblastoma

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Targetable BET Proteins- and E2F1-Dependent Transcriptional Program Maintains the Malignancy of Glioblastoma Targetable BET proteins- and E2F1-dependent transcriptional program maintains the malignancy of glioblastoma Liang Xua,1,2, Ye Chena,1, Anand Mayakondaa,1, Lynnette Kohb,c, Yuk Kien Chongb, Dennis L. Buckleyd, Edwin Sandanarajb,c,e, See Wee Limb, Ruby Yu-Tong Lina, Xin-Yu Kea, Mo-Li Huanga,f, Jianxiang Cheng, Wendi Sunc, Ling-Zhi Wanga,h, Boon Cher Goha,h,i, Huy Q. Dinhj, Dennis Kappeia, Georg E. Winterd, Ling-Wen Dinga, Beng Ti Ange,k,l,m, Benjamin P. Bermanj, James E. Bradnerd,n,3, Carol Tangb,g,l,3, and H. Phillip Koefflera,i,o,3 aCancer Science Institute of Singapore, National University of Singapore, 117599 Singapore; bDepartment of Research, National Neuroscience Institute, 308433 Singapore; cSchool of Biological Sciences, Nanyang Technological University, 637551 Singapore; dDepartment of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115; eSingapore Institute for Clinical Sciences, Agency for Science, Technology, and Research, 117609 Singapore; fSchool of Biology and Basic Medical Sciences, Soochow University, 215123 Suzhou, China; gHumphrey Oei Institute of Cancer Research, National Cancer Centre, 169610 Singapore; hDepartment of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, 117600 Singapore; iNational University Cancer Institute, National University Hospital, 119074 Singapore; jCenter for Bioinformatics and Functional Genomics, Biomedical Sciences, Cedars-Sinai Medical Center, David Geffen School of Medicine at University of California, Los Angeles (UCLA), Los Angeles, CA 90048; kDepartment of Neurosurgery, National Neuroscience Institute, 308433 Singapore; lDuke-National University of Singapore Medical School, 169857 Singapore; mDepartment of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, 117593 Singapore; nDepartment of Medicine, Harvard Medical School, Boston, MA 02115; and oDivision of Hematology/Oncology, Cedars-Sinai Medical Center, David Geffen School of Medicine at UCLA, Los Angeles, CA 90048 Edited by Webster K. Cavenee, Ludwig Institute, University of California, San Diego, La Jolla, CA, and approved April 25, 2018 (received for review July 20, 2017) Competitive BET bromodomain inhibitors (BBIs) targeting BET for gene expression, transcriptomic response of GBM cells to proteins (BRD2, BRD3, BRD4, and BRDT) show promising preclinical BBI and the genome-wide occupancy of BET proteins in these activities against brain cancers. However, the BET protein-dependent cells remain poorly studied. Therefore, the mechanism and the glioblastoma (GBM)-promoting transcriptional network remains vulnerability of BET protein dependency in GBM cells need to MEDICAL SCIENCES elusive. Here, with mechanistic exploration of a next-generation be characterized further. chemical degrader of BET proteins (dBET6), we reveal a profound In this study, we report that GBM cells develop both intrinsic and consistent impact of BET proteins on E2F1- dependent transcrip- and acquired resistance to BBIs. By employing genetic and tional program in both differentiated GBM cells and brain tumor- initiating cells. dBET6 treatment drastically reduces BET protein genomic occupancy, RNA-Pol2 activity, and permissive chromatin Significance marks. Subsequently, dBET6 represses the proliferation, self-renewal, and tumorigenic ability of GBM cells. Moreover, dBET6-induced Glioblastoma (GBM) cells develop intrinsic or acquired insensi- degradation of BET proteins exerts superior antiproliferation effects tiveness to BET bromodomain inhibitors (BBIs) yet develop per- compared to conventional BBIs and overcomes both intrinsic and sistent BET protein dependency. Selective degradation of BET acquired resistance to BBIs in GBM cells. Our study reveals crucial proteins by a next-generation chemical compound undermines functions of BET proteins and provides the rationale and therapeutic the BET protein dependency and exerts superior antineoplastic merits of targeted degradation of BET proteins in GBM. effects over inhibition of BET bromodomain. Given the signifi- cant difference between bromodomain dependency and BET glioma | BRD2 | BRD3 | BRD4 | E2F protein dependency in GBM cells, chemically induced degrada- tion of BET proteins serves as a promising strategy to overcome romodomain and extraterminal (BET) family proteins are anticipated clinical BBIs resistance. Breaders for histone lysine acetylation and key coactivators for oncogenic transcriptional programs in cancer (1). BET proteins Author contributions: L.X., Y.C., B.T.A., C.T., and H.P.K. designed research; L.X., Y.C., A.M., L.K., Y.K.C., E.S., S.W.L., R.Y.-T.L., X.-Y.K., J.C., W.S., L.-Z.W., L.-W.D., and C.T. performed (including BRD2, BRD3, BRD4, and BRDT) emerge as prom- research; J.E.B., C.T., and H.P.K. supervised the study; D.L.B., W.S., L.-Z.W., B.C.G., D.K., ising anticancer targets along with a growing list of competitive G.E.W., B.T.A., B.P.B., J.E.B., and C.T. contributed new reagents/analytic tools; L.X., Y.C., BET bromodomain inhibitors (BBIs) (e.g., JQ1, I-BET151, A.M., L.K., Y.K.C., E.S., S.W.L., M.-L.H., L.-Z.W., H.Q.D., B.T.A., B.P.B., C.T., and H.P.K. analyzed data; and L.X., Y.C., A.M., L.K., Y.K.C., E.S., C.T., and H.P.K. wrote the paper. OTX015, and CPI203) (2–5). These BBIs block the acetyl-lysine Conflict of interest statement: J.E.B. is an employee, shareholder, and executive of binding activity of BET proteins and show broad anticancer ef- Novartis Pharmaceuticals. J.E.B. is also a Scientific Founder of Tensha Therapeutics, C4 Ther- fects. However, efficacy of BBIs is counteracted by emergence of apeutics, Syros Pharmaceuticals, SHAPE Pharmaceuticals, and Acetylon Pharmaceuticals. D.L.B. both primary and acquired resistance in various types of cancers is an employee of the Novartis Institutes for BioMedical Research. None of these relationships (6–8). Recent development of small molecules that redirect constitutes a conflict of interest for the present work. The remaining authors declare no conflict of interest. E3 ubiquitin ligases to degrade BET proteins shows promise for This article is a PNAS Direct Submission. the treatment of leukemia, lymphoma, and breast cancer (9–11). Nevertheless, the efficacy of these BET protein degraders against Published under the PNAS license. Data deposition: RNA-seq, and ChIP-seq data generated in this study have been deposited BBI-resistant cancer cells remains to be explored. in the NCBI Gene Expression Omnibus database (accession no. GSE99183). Whole exome Glioblastoma (GBM) is a therapy-refractory tumor with dismal sequencing data are available in the NCBI Sequence Read Archive database (accession no. prognosis that is awaiting new therapeutic innovations (12). SRP107365). Overexpression of BRD2 and BRD4 has been reported in human 1L.X., Y.C., and A.M. contributed equally to this work. GBM (13). To date, despite heterogeneous dose responses across 2To whom correspondence should be addressed. Email: [email protected]. GBM cell lines, BBIs (JQ1, I-BET151, and OTX015) have been 3J.E.B., C.T., and H.P.K. contributed equally to this work. shown to trigger G1 cell cycle arrest and to retard xenografted This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tumor growth, partially through induction of p21 and inhibition of 1073/pnas.1712363115/-/DCSupplemental. Bcl-xL and HOTAIR (4, 13–16). While BET proteins are important www.pnas.org/cgi/doi/10.1073/pnas.1712363115 PNAS Latest Articles | 1of10 Downloaded by guest on September 26, 2021 chemical approaches to selectively deplete BET proteins, we dis- BrdU incorporation robustly and to a greater extent than BBIs covered a persistent growth dependency of GBM cells on BET (Fig. 2A). Different from BBI-induced G1/G0 arrest, dBET6 proteins regardless of their sensitivities to BBIs. dBET6, a CRBN- triggered a marked G2/M arrest of GBM cells, with concordant dependent BET protein degrader, showed superior anti-GBM ac- dysregulation of key cell cycle-related genes (Fig. 2 B and C and SI tivities to conventional BBIs, as well as a strong capacity to over- Appendix,Fig.S3J). However, 24-h treatment of dBET6 did not come BBI resistance. dBET6-mediated depletion of BET proteins induce obvious sub-G1 population and apoptosis (Fig. 2B and SI showed distinct transcriptional and cellular responses compared with Appendix,Fig.S3K and L). Therefore, acute dBET6 treatment bromodomain inhibition. Moreover, dBET6 exerted a profound inhibits GBM cell viability mainly through its antiproliferation impact on RNA-Pol2 function and histone modification. Integrative effect. Remarkably, short-term exposure of dBET6 elicited a ChIP followed by sequencing (ChIP-seq) and RNA-sequencing profound impact on GBM tumor initiation in vivo. In an intra- (RNA-seq) analysis demonstrated E2F-dependent transcriptional cranial xenograft model, pretreatment of U87 cells with dBET6 program and cell cycle-related genes as primary downstream targets for 24 h significantly prolonged murine survival and reduced tu- of BET proteins in both GBM cell lines and patient-derived GBM mor incidence in a dose-dependent manner (Fig. 2D). In vivo spheres. A core dBET6-responsive gene signature was identified limiting dilution assay confirmed further the advantage of dBET6 further to be able to stratify pathological grades and patient prog- over JQ1 to suppress the tumorigenic ability of GBM cells (Fig. nosis in three independent glioma cohorts, strongly suggesting that 2E). These observations prompted us to examine the effect of glioma patients
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