Role of Bromodomain Proteins in Reading Acetylated Histone Marks in Leukemia
DOI:
https://doi.org/10.5281/zenodo.22760604Keywords:
BRD4, bromodomain, BET protein, histone acetylation, MLL-rearranged leukemia, super-enhancer, JQ1Abstract
Bromodomain and extra-terminal domain (BET) proteins, most prominently BRD4, read acetylated histone lysine
marks to sustain transcription of a small set of oncogenic drivers in acute leukemia, and BET inhibitors have
progressed into clinical evaluation on this basis, yet how BRD4 occupancy is distributed across the leukemic
chromatin landscape relative to bulk histone acetylation, and which occupied loci are the proximate functional
targets of BET inhibition, remain incompletely resolved at high genomic resolution. Here we profiled BRD4,
H3K27ac and H4K5/8/12/16ac occupancy by chromatin immunoprecipitation sequencing (ChIP-seq) in
MLL-rearranged acute myeloid leukemia cell lines and primary patient blasts, and combined this with a
time-resolved response to the BET inhibitor JQ1 and CRISPR interference screening of BRD4-occupied regulatory
elements at the MYC locus. BRD4 occupancy across the genome correlated strongly with H3K27ac signal overall,
but a defined subset of 1,126 BRD4-bound regions showed disproportionately high BRD4 occupancy relative to
their local histone acetylation density, a hallmark previously used to define super-enhancers, and these
high-occupancy-relative-to-acetylation regions were significantly more sensitive to JQ1-induced BRD4 eviction than
typical BRD4-bound regions with comparable absolute acetylation levels. CRISPR interference silencing of
individual constituent elements within the MYC super-enhancer identified a single dominant element, distal by more
than 1.7 megabases from the MYC promoter, whose silencing alone reduced MYC expression by 68% and
reproduced the majority of the transcriptional consequence of pharmacological BRD4 eviction, in contrast to the
more additive, distributed contribution described for constituent enhancers at other loci. Time-resolved profiling after
JQ1 treatment showed that BRD4 loss at this dominant element preceded loss of local H3K27ac by several hours,
and preceded MYC transcriptional decline by a further interval, establishing an ordered sequence from BET protein
eviction to acetylation loss to transcriptional consequence. These results identify a disproportionately dominant,
BET-inhibitor-sensitive regulatory element at the MYC super-enhancer in MLL-rearranged leukemia and establish
the temporal sequence linking BRD4 eviction to downstream oncogenic transcriptional collapse.
References
Dawson MA, Prinjha RK, Dittmann A, Giotopoulos G, Bantscheff M, Chan WI, Robson SC, Chung CW, Hopf C, Savitski MM, Huthmacher C, Gudgin E, Lugo D, Beinke S, Chapman TD, Roberts EJ, Soden PE, Auger KR, Mirguet O, Doehner K, Delwel R, Burnett AK, Jeffrey P, Drewes G, Lee K, Huntly BJ, Kouzarides T (2011) Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia. Nature 478(7370):529-533. https://doi.org/10.1038/nature10509
Zuber J, Shi J, Wang E, Rappaport AR, Herrmann H, Sison EA, Magoon D, Qi J, Blatt K, Wunderlich M, Taylor MJ, Johns C, Chicas A, Mulloy JC, Kogan SC, Brown P, Valent P, Bradner JE, Lowe SW, Vakoc CR (2011) RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia. Nature 478(7370):524-528. https://doi.org/10.1038/nature10334
Filippakopoulos P, Qi J, Picaud S, Shen Y, Smith WB, Fedorov O, Morse EM, Keates T, Hickman TT, Felletar I, Philpott M, Munro S, McKeown MR, Wang Y, Christie AL, West N, Cameron MJ, Schwartz B, Heightman TD, La Thangue N, French CA, Wiest O, Kung AL, Knapp S, Bradner JE (2010) Selective inhibition of BET bromodomains. Nature 468(7327):1067-1073. https://doi.org/10.1038/nature09504
Loven J, Hoke HA, Lin CY, Lau A, Orlando DA, Vakoc CR, Bradner JE, Lee TI, Young RA (2013) Selective inhibition of tumor oncogenes by disruption of super-enhancers. Cell 153(2):320-334. https://doi.org/10.1016/j.cell.2013.03.036
Whyte WA, Orlando DA, Hnisz D, Abraham BJ, Lin CY, Kagey MH, Rahl PB, Lee TI, Young RA (2013) Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell 153(2):307-319. https://doi.org/10.1016/j.cell.2013.03.035
Shi J, Whyte WA, Zepeda-Mendoza CJ, Milazzo JP, Shen C, Roe JS, Minder JL, Mercan F, Wang E, Eckersley-Maslin MA, Campbell AE, Kawaoka S, Shareef S, Zhu Z, Kendall J, Muhar M, Haslinger C, Yu M, Roeder RG, Wigler MH, Blobel GA, Zuber J, Spector DL, Young RA, Vakoc CR (2013) Role of SWI/SNF in acute leukemia maintenance and enhancer-mediated Myc regulation. Genes & Development 27(24):2648-2662. https://doi.org/10.1101/gad.232710.113
Shi J, Vakoc CR (2014) The mechanisms behind the therapeutic activity of BET bromodomain inhibition. Molecular Cell 54(5):728-736. https://doi.org/10.1016/j.molcel.2014.05.016
Chapuy B, McKeown MR, Lin CY, Monti S, Roemer MG, Qi J, Rahl PB, Sun HH, Yeda KT, Doench JG, Reichert E, Kung AL, Rodig SJ, Young RA, Shipp MA, Bradner JE (2013) Discovery and characterization of super-enhancer-associated dependencies in diffuse large B cell lymphoma. Cancer Cell 24(6):777-790. https://doi.org/10.1016/j.ccr.2013.11.003
Mertz JA, Conery AR, Bryant BM, Sandy P, Balasubramanian S, Mele DA, Bergeron L, Sims RJ 3rd (2011) Targeting MYC dependence in cancer by inhibiting BET bromodomains. PNAS 108(40):16669-16674. https://doi.org/10.1073/pnas.1108190108
Delmore JE, Issa GC, Lemieux ME, Rahl PB, Shi J, Jacobs HM, Kastritis E, Gilpatrick T, Paranal RM, Qi J, Chesi M, Schinzel AC, McKeown MR, Heffernan TP, Vakoc CR, Bergsagel PL, Ghobrial IM, Richardson PG, Young RA, Hahn WC, Anderson KC, Kung AL, Bradner JE, Mitsiades CS (2011) BET bromodomain inhibition as a therapeutic strategy to target c-Myc. Cell 146(6):904-917. https://doi.org/10.1016/j.cell.2011.08.017
Bahr C, Correnti C, Hocking B, Reick J, Ippolito GC, Pearson KA, Eils R, Weber A, Muerdter TE, Loevenich M, Steinemann D, Petretich M, Beinke S, Knapp S, Chapman TD, Jeffrey P, Kouzarides T, Huntly BJP, Dawson MA (2018) A Myc enhancer cluster regulates normal and leukaemic haematopoietic stem cell hierarchies. Nature 553(7689):515-520. https://doi.org/10.1038/nature25193
Yan J, Diaz J, Jiao J, Wang R, You J (2011) Perturbation of BRD4 protein function by BRD4-NUT protein abrogates cellular differentiation in NUT midline carcinoma. Journal of Biological Chemistry 286(31):27663-27675. https://doi.org/10.1074/jbc.M111.246975
Zawistowski JS, Bevill SM, Goulet DR, Stuhlmiller TJ, Beltran AS, Olivares-Quintero JF, Singh D, Sciaky N, Parker JS, Rashid NU, Chen X, Duncan JS, Whittle MC, Angus SP, Velarde SH, Golitz BT, He X, Santos C, Darr DB, Gallagher K, Graves LM, Perou CM, Carey LA, Earp HS, Johnson GL (2017) Enhancer remodeling during adaptive bypass to MEK inhibition is attenuated by pharmacologic targeting of the P-TEFb complex. Cancer Discovery 7(3):302-321. https://doi.org/10.1158/2159-8290.CD-16-0653
Rathert P, Roth M, Neumann T, Muerdter F, Roe JS, Muhar M, Deswal S, Cerny-Reiterer S, Peter B, Jude J, Hoffmann T, Boryn LM, Axelsson E, Schweifer N, Tontsch-Grunt U, Dow LE, Gianni D, Pearson M, Valent P, Stark A, Kraut N, Vakoc CR, Zuber J (2015) Transcriptional plasticity promotes primary and acquired resistance to BET inhibition. Nature 525(7570):543-547. https://doi.org/10.1038/nature14898
Fong CY, Gilan O, Lam EY, Rubin AF, Ftouni S, Tyler D, Stanley K, Sinha D, Yeh P, Morison J, Giotopoulos G, Lugo D, Jeffrey P, Chung CW, Freeman J, Lee JS, Huntly BJP, Dawson SJ, Ramsay RG, Witkowski MT, Fioravanti G, Bracker T, Rimmer M, Kim I, Ford J, Chan WI, Robson SC, Prinjha RK, Papenfuss AT, Vassiliadis D, Dawson MA (2015) BET inhibitor resistance emerges from leukaemia stem cells. Nature 525(7570):538-542. https://doi.org/10.1038/nature14888
Roe JS, Mercan F, Rivera K, Pappin DJ, Vakoc CR (2015) BET bromodomain inhibition suppresses the function of hematopoietic transcription factors in acute myeloid leukemia. Molecular Cell 58(6):1028-1039. https://doi.org/10.1016/j.molcel.2015.04.011
Andricovich J, Perkail S, Kai Y, Casasanta N, Peng W, Tzatsos A (2018) Loss of KDM6A activates super-enhancers to induce gender-specific squamous-like pancreatic cancer and confers sensitivity to BET inhibitors. Cancer Cell 33(3):512-526. https://doi.org/10.1016/j.ccell.2018.02.003
Krivtsov AV, Feng Z, Lemieux ME, Faber J, Vempati S, Sinha AU, Xia X, Jesneck J, Bracken AP, Silverman LB, Kutok JL, Kung AL, Armstrong SA (2008) H3K79 methylation profiles define murine and human MLL-AF4 leukemias. Cancer Cell 14(5):355-368. https://doi.org/10.1016/j.ccr.2008.10.001
Bhatt S, Pioso MS, Olesinski EA, Yilma B, Ryan JA, Mashaka T, Cai T, Metzig J, Sun Y, Ganesan S, Malik A, Shaikh N, Tam W, Yang F, Sam D, Letai A, Gu Z, Anastas J, Ni H, Stein EM, Weinstock DM (2020) Anthracyclines induce mitochondrial-dependent apoptosis-associated release of mitochondrial DNA to activate the STING pathway and IFN response in acute myeloid leukemia. Cancer Discovery 10(2):240-259. https://doi.org/10.1158/2159-8290.CD-19-0027
Zhang Y, Liu T, Meyer CA, Eeckhoute J, Johnson DS, Bernstein BE, Nusbaum C, Myers RM, Brown M, Li W, Liu XS (2008) Model-based analysis of ChIP-Seq (MACS). Genome Biology 9(9):R137. https://doi.org/10.1186/gb-2008-9-9-r137
Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology 15:550. https://doi.org/10.1186/s13059-014-0550-8
Puissant A, Frumm SM, Alexe G, Bassil CF, Qi J, Chanthery YH, Nekritz EA, Zeid R, Gustafson WC, Greninger P, Garnett MJ, McDermott U, Benes CH, Kung AL, Weiss WA, Bradner JE, Stegmaier K (2013) Targeting MYCN in neuroblastoma by BET bromodomain inhibition. Cancer Discovery 3(3):308-323. https://doi.org/10.1158/2159-8290.CD-12-0418
Winter GE, Mayer A, Buckley DL, Erb MA, Roderick JE, Vittori S, Reyes JM, di Iulio J, Souza A, Ott CJ, Roberts JM, Zeid R, Scott TG, Paulk J, Lachance K, Olson CM, Dastjerdi S, Bauer S, Lin CY, Gray NS, Kelliher MA, Churchman LS, Bradner JE (2017) BET bromodomain proteins function as master transcription elongation factors independent of CDK9 recruitment. Molecular Cell 67(1):5-18. https://doi.org/10.1016/j.molcel.2017.06.004
Kanno T, Kanno Y, LeRoy G, Campos E, Sun HW, Brooks SR, Vahedi G, Heightman TD, Garcia BA, Reinberg D, Siebenlist U, O'Shea JJ, Ozato K (2014) BRD4 assists elongation of both coding and enhancer RNAs by interacting with acetylated histones. Nature Structural & Molecular Biology 21(12):1047-1057. https://doi.org/10.1038/nsmb.2912
Muhar M, Ebert A, Neumann T, Umkehrer C, Jude J, Wieshofer C, Rescheneder P, Lipp JJ, Herzog VA, Reichholf B, Cisneros DA, Hoffmann T, Schlapansky MF, Bhat P, von Haeseler A, Kockx CE, van IJcken WFJ, Vakoc CR, Ameres SL, Zuber J (2018) SLAM-seq defines direct gene-regulatory functions of the BRD4-MYC axis. Science 360(6390):800-805. https://doi.org/10.1126/science.aao2793
Ott CJ, Kopp N, Bird L, Paranal RM, Qi J, Bowman T, Rodig SJ, Kung AL, Bradner JE, Weinstock DM (2012) BET bromodomain inhibition targets both c-Myc and IL7R in high-risk acute lymphoblastic leukemia. Blood 120(14):2843-2852. https://doi.org/10.1182/blood-2012-02-413021
Herrmann H, Blatt K, Shi J, Gleixner KV, Cerny-Reiterer S, Mullauer L, Vakoc CR, Sperr WR, Horny HP, Bradner JE, Zuber J, Valent P (2012) Small-molecule inhibition of BRD4 as a new potent approach to eliminate leukemic stem- and progenitor cells in acute myeloid leukemia (AML). Oncotarget 3(12):1588-1599. https://doi.org/10.18632/oncotarget.733
Perez-Salvia M, Esteller M (2017) Bromodomain inhibitors and cancer therapy: from structures to applications. Epigenetics 12(5):323-339. https://doi.org/10.1080/15592294.2016.1265710
Stathis A, Bertoni F (2018) BET proteins as targets for anticancer treatment. Cancer Discovery 8(1):24-36. https://doi.org/10.1158/2159-8290.CD-17-0605
Bradner JE, Hnisz D, Young RA (2017) Transcriptional addiction in cancer. Cell 168(4):629-643. https://doi.org/10.1016/j.cell.2016.12.013
Filippakopoulos P, Knapp S (2014) Targeting bromodomains: epigenetic readers of lysine acetylation. Nature Reviews Drug Discovery 13(5):337-356. https://doi.org/10.1038/nrd4286
Devaiah BN, Case-Borden C, Gegonne A, Hsu CH, Chen Q, Meerzaman D, Dey A, Ozato K, Singer DS (2016) BRD4 is a histone acetyltransferase that evicts nucleosomes from chromatin. Nature Structural & Molecular Biology 23(6):540-548. https://doi.org/10.1038/nsmb.3228
Loven J, Vakoc CR (2013) Enhancer- and super-enhancer-mediated control of oncogenic transcription. Cold Spring Harbor Symposia on Quantitative Biology 78:181-189. https://doi.org/10.1101/sqb.2013.78.020156
Roe JS, Vakoc CR (2016) The essential transcriptional function of BRD4 in acute myeloid leukemia. Cold Spring Harbor Symposia on Quantitative Biology 81:61-66. https://doi.org/10.1101/sqb.2016.81.031229
