title: BET inhibitor resistance emerges from leukaemia stem cells.
abstract: Bromodomain and extra terminal protein (BET) inhibitors are first-in-class targeted therapies that deliver a new therapeutic opportunity by directly targeting bromodomain proteins that bind acetylated chromatin marks. Early clinical trials have shown promise, especially in acute myeloid leukaemia, and therefore the evaluation of resistance mechanisms is crucial to optimize the clinical efficacy of these drugs. Here we use primary mouse haematopoietic stem and progenitor cells immortalized with the fusion protein MLL-AF9 to generate several single-cell clones that demonstrate resistance, in vitro and in vivo, to the prototypical BET inhibitor, I-BET. Resistance to I-BET confers cross-resistance to chemically distinct BET inhibitors such as JQ1, as well as resistance to genetic knockdown of BET proteins. Resistance is not mediated through increased drug efflux or metabolism, but is shown to emerge from leukaemia stem cells both ex vivo and in vivo. Chromatin-bound BRD4 is globally reduced in resistant cells, whereas the expression of key target genes such as Myc remains unaltered, highlighting the existence of alternative mechanisms to regulate transcription. We demonstrate that resistance to BET inhibitors, in human and mouse leukaemia cells, is in part a consequence of increased Wnt/β-catenin signalling, and negative regulation of this pathway results in restoration of sensitivity to I-BET in vitro and in vivo. Together, these findings provide new insights into the biology of acute myeloid leukaemia, highlight potential therapeutic limitations of BET inhibitors, and identify strategies that may enhance the clinical utility of these unique targeted therapies.
authors.affiliation: Cancer Research Division, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia.|Sir Peter MacCallum Department of Oncology, The University of Melbourne, East Melbourne, Victoria 3002, Australia.|Department of Haematology, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia.
authors.forename: Chun Yew
authors.initials: CY
authors.lastname: Fong
authors.affiliation: Cancer Research Division, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia.|Sir Peter MacCallum Department of Oncology, The University of Melbourne, East Melbourne, Victoria 3002, Australia.
authors.forename: Omer
authors.initials: O
authors.lastname: Gilan
authors.affiliation: Cancer Research Division, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia.
authors.forename: Enid Y N
authors.initials: EY
authors.lastname: Lam
authors.affiliation: Bioinformatics Division, The Walter &Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia.|Department of Medical Biology, University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: Alan F
authors.initials: AF
authors.lastname: Rubin
authors.affiliation: Cancer Research Division, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia.
authors.forename: Sarah
authors.initials: S
authors.lastname: Ftouni
authors.affiliation: Cancer Research Division, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia.|Sir Peter MacCallum Department of Oncology, The University of Melbourne, East Melbourne, Victoria 3002, Australia.
authors.forename: Dean
authors.initials: D
authors.lastname: Tyler
authors.affiliation: Cancer Research Division, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia.
authors.forename: Kym
authors.initials: K
authors.lastname: Stanley
authors.affiliation: Cancer Research Division, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia.
authors.forename: Devbarna
authors.initials: D
authors.lastname: Sinha
authors.affiliation: Cancer Research Division, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia.|Sir Peter MacCallum Department of Oncology, The University of Melbourne, East Melbourne, Victoria 3002, Australia.|Department of Haematology, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia.
authors.forename: Paul
authors.initials: P
authors.lastname: Yeh
authors.affiliation: Department of Haematology, Cambridge Institute for Medical Research and Wellcome Trust-MRC Stem Cell Institute, Cambridge CB2 0XY, UK.
authors.forename: Jessica
authors.initials: J
authors.lastname: Morison
authors.affiliation: Department of Haematology, Cambridge Institute for Medical Research and Wellcome Trust-MRC Stem Cell Institute, Cambridge CB2 0XY, UK.
authors.forename: George
authors.initials: G
authors.lastname: Giotopoulos
authors.affiliation: Epinova DPU, Immuno-Inflammation Centre of Excellence for Drug Discovery, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, UK.
authors.forename: Dave
authors.initials: D
authors.lastname: Lugo
authors.affiliation: Epinova DPU, Immuno-Inflammation Centre of Excellence for Drug Discovery, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, UK.
authors.forename: Philip
authors.initials: P
authors.lastname: Jeffrey
authors.affiliation: Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA.
authors.forename: Stanley Chun-Wei
authors.initials: SC
authors.lastname: Lee
authors.affiliation: Cancer Epigenetics DPU, Oncology R&D, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, Pennsylvania 19426, USA.
authors.forename: Christopher
authors.initials: C
authors.lastname: Carpenter
authors.affiliation: Epinova DPU, Immuno-Inflammation Centre of Excellence for Drug Discovery, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, UK.
authors.forename: Richard
authors.initials: R
authors.lastname: Gregory
authors.affiliation: Cancer Research Division, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia.|Sir Peter MacCallum Department of Oncology, The University of Melbourne, East Melbourne, Victoria 3002, Australia.
authors.forename: Robert G
authors.initials: RG
authors.lastname: Ramsay
authors.affiliation: QIMR Berghofer Medical Research Institute, University of Queensland, Brisbane, Queensland 4029, Australia.
authors.forename: Steven W
authors.initials: SW
authors.lastname: Lane
authors.affiliation: Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA.
authors.forename: Omar
authors.initials: O
authors.lastname: Abdel-Wahab
authors.affiliation: Gurdon Institute and Department of Pathology, Tennis Court Road, Cambridge CB2 1QN, UK.
authors.forename: Tony
authors.initials: T
authors.lastname: Kouzarides
authors.affiliation: Cancer Research Division, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia.|Sir Peter MacCallum Department of Oncology, The University of Melbourne, East Melbourne, Victoria 3002, Australia.
authors.forename: Ricky W
authors.initials: RW
authors.lastname: Johnstone
authors.affiliation: Cancer Research Division, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia.|Sir Peter MacCallum Department of Oncology, The University of Melbourne, East Melbourne, Victoria 3002, Australia.
authors.forename: Sarah-Jane
authors.initials: SJ
authors.lastname: Dawson
authors.affiliation: Department of Haematology, Cambridge Institute for Medical Research and Wellcome Trust-MRC Stem Cell Institute, Cambridge CB2 0XY, UK.
authors.forename: Brian J P
authors.initials: BJ
authors.lastname: Huntly
authors.affiliation: Epinova DPU, Immuno-Inflammation Centre of Excellence for Drug Discovery, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, UK.
authors.forename: Rab K
authors.initials: RK
authors.lastname: Prinjha
authors.affiliation: Cancer Research Division, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia.|Sir Peter MacCallum Department of Oncology, The University of Melbourne, East Melbourne, Victoria 3002, Australia.|Bioinformatics Division, The Walter &Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia.|Department of Medical Biology, University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: Anthony T
authors.initials: AT
authors.lastname: Papenfuss
authors.affiliation: Cancer Research Division, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia.|Sir Peter MacCallum Department of Oncology, The University of Melbourne, East Melbourne, Victoria 3002, Australia.|Department of Haematology, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Dawson
mesh_terms: D000818:Animals; D001381:Azepines / Q000494:pharmacology; D001569:Benzodiazepines / Q000494:pharmacology*; D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D002843:Chromatin / Q000378:metabolism; D002999:Clone Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D019008:Drug Resistance, Neoplasm / Q000187:drug effects* / Q000235:genetics; D044127:Epigenesis, Genetic; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D016259:Genes, myc / Q000235:genetics; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D058990:Molecular Targeted Therapy; D014411:Neoplastic Stem Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology*; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014157:Transcription Factors / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D014230:Triazoles / Q000494:pharmacology; D060449:Wnt Signaling Pathway / Q000187:drug effects; D051176:beta Catenin / Q000378:metabolism; D000092468:Bromodomain Containing Proteins
chemical_list: D001381:Azepines; D001569:Benzodiazepines; D018797:Cell Cycle Proteins; D002843:Chromatin; D009687:Nuclear Proteins; D014157:Transcription Factors; D014230:Triazoles; D051176:beta Catenin; C561695:(+)-JQ1 compound; C441289:BRD4 protein, human; D000092468:Bromodomain Containing Proteins; C554645:molibresib