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1.
Cell ; 172(1-2): 90-105.e23, 2018 01 11.
Article in English | MEDLINE | ID: mdl-29249359

ABSTRACT

R-2-hydroxyglutarate (R-2HG), produced at high levels by mutant isocitrate dehydrogenase 1/2 (IDH1/2) enzymes, was reported as an oncometabolite. We show here that R-2HG also exerts a broad anti-leukemic activity in vitro and in vivo by inhibiting leukemia cell proliferation/viability and by promoting cell-cycle arrest and apoptosis. Mechanistically, R-2HG inhibits fat mass and obesity-associated protein (FTO) activity, thereby increasing global N6-methyladenosine (m6A) RNA modification in R-2HG-sensitive leukemia cells, which in turn decreases the stability of MYC/CEBPA transcripts, leading to the suppression of relevant pathways. Ectopically expressed mutant IDH1 and S-2HG recapitulate the effects of R-2HG. High levels of FTO sensitize leukemic cells to R-2HG, whereas hyperactivation of MYC signaling confers resistance that can be reversed by the inhibition of MYC signaling. R-2HG also displays anti-tumor activity in glioma. Collectively, while R-2HG accumulated in IDH1/2 mutant cancers contributes to cancer initiation, our work demonstrates anti-tumor effects of 2HG in inhibiting proliferation/survival of FTO-high cancer cells via targeting FTO/m6A/MYC/CEBPA signaling.


Subject(s)
Antineoplastic Agents/pharmacology , Brain Neoplasms/drug therapy , Glioma/drug therapy , Glutarates/pharmacology , Leukemia/drug therapy , Signal Transduction/drug effects , Adenosine/analogs & derivatives , Adenosine/metabolism , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/metabolism , Animals , Antineoplastic Agents/therapeutic use , CCAAT-Enhancer-Binding Proteins/metabolism , Cell Line, Tumor , Glutarates/therapeutic use , HEK293 Cells , Humans , Jurkat Cells , Mice , Proto-Oncogene Proteins c-myc/metabolism , RNA Processing, Post-Transcriptional
2.
Cell ; 168(4): 629-643, 2017 02 09.
Article in English | MEDLINE | ID: mdl-28187285

ABSTRACT

Cancer arises from genetic alterations that invariably lead to dysregulated transcriptional programs. These dysregulated programs can cause cancer cells to become highly dependent on certain regulators of gene expression. Here, we discuss how transcriptional control is disrupted by genetic alterations in cancer cells, why transcriptional dependencies can develop as a consequence of dysregulated programs, and how these dependencies provide opportunities for novel therapeutic interventions in cancer.


Subject(s)
Neoplasms/drug therapy , Neoplasms/genetics , Animals , DNA Methylation , Epigenesis, Genetic , Gene Expression Regulation, Neoplastic , Homeostasis , Humans , Neoplasms/pathology , Transcription Factors/metabolism , Transcription, Genetic
3.
Cell ; 171(7): 1573-1588.e28, 2017 Dec 14.
Article in English | MEDLINE | ID: mdl-29224777

ABSTRACT

There is considerable evidence that chromosome structure plays important roles in gene control, but we have limited understanding of the proteins that contribute to structural interactions between gene promoters and their enhancer elements. Large DNA loops that encompass genes and their regulatory elements depend on CTCF-CTCF interactions, but most enhancer-promoter interactions do not employ this structural protein. Here, we show that the ubiquitously expressed transcription factor Yin Yang 1 (YY1) contributes to enhancer-promoter structural interactions in a manner analogous to DNA interactions mediated by CTCF. YY1 binds to active enhancers and promoter-proximal elements and forms dimers that facilitate the interaction of these DNA elements. Deletion of YY1 binding sites or depletion of YY1 protein disrupts enhancer-promoter looping and gene expression. We propose that YY1-mediated enhancer-promoter interactions are a general feature of mammalian gene control.


Subject(s)
Enhancer Elements, Genetic , Promoter Regions, Genetic , YY1 Transcription Factor/metabolism , Animals , CCCTC-Binding Factor/metabolism , Embryonic Stem Cells/metabolism , Humans , Mice
4.
Cell ; 164(1-2): 293-309, 2016 Jan 14.
Article in English | MEDLINE | ID: mdl-26771497

ABSTRACT

Large-scale genomic studies have identified multiple somatic aberrations in breast cancer, including copy number alterations and point mutations. Still, identifying causal variants and emergent vulnerabilities that arise as a consequence of genetic alterations remain major challenges. We performed whole-genome small hairpin RNA (shRNA) "dropout screens" on 77 breast cancer cell lines. Using a hierarchical linear regression algorithm to score our screen results and integrate them with accompanying detailed genetic and proteomic information, we identify vulnerabilities in breast cancer, including candidate "drivers," and reveal general functional genomic properties of cancer cells. Comparisons of gene essentiality with drug sensitivity data suggest potential resistance mechanisms, effects of existing anti-cancer drugs, and opportunities for combination therapy. Finally, we demonstrate the utility of this large dataset by identifying BRD4 as a potential target in luminal breast cancer and PIK3CA mutations as a resistance determinant for BET-inhibitors.


Subject(s)
Algorithms , Breast Neoplasms/genetics , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Cell Cycle Proteins , Cell Line, Tumor , Class I Phosphatidylinositol 3-Kinases , Cluster Analysis , Drug Resistance, Neoplasm , Gene Dosage , Gene Expression Profiling , Genome-Wide Association Study , Humans , Linear Models , Nuclear Proteins/genetics , Phosphatidylinositol 3-Kinases , Transcription Factors/genetics
5.
Cell ; 161(4): 774-89, 2015 May 07.
Article in English | MEDLINE | ID: mdl-25957685

ABSTRACT

We have ablated the cellular RNA degradation machinery in differentiated B cells and pluripotent embryonic stem cells (ESCs) by conditional mutagenesis of core (Exosc3) and nuclear RNase (Exosc10) components of RNA exosome and identified a vast number of long non-coding RNAs (lncRNAs) and enhancer RNAs (eRNAs) with emergent functionality. Unexpectedly, eRNA-expressing regions accumulate R-loop structures upon RNA exosome ablation, thus demonstrating the role of RNA exosome in resolving deleterious DNA/RNA hybrids arising from active enhancers. We have uncovered a distal divergent eRNA-expressing element (lncRNA-CSR) engaged in long-range DNA interactions and regulating IgH 3' regulatory region super-enhancer function. CRISPR-Cas9-mediated ablation of lncRNA-CSR transcription decreases its chromosomal looping-mediated association with the IgH 3' regulatory region super-enhancer and leads to decreased class switch recombination efficiency. We propose that the RNA exosome protects divergently transcribed lncRNA expressing enhancers by resolving deleterious transcription-coupled secondary DNA structures, while also regulating long-range super-enhancer chromosomal interactions important for cellular function.


Subject(s)
B-Lymphocytes/metabolism , Exosome Multienzyme Ribonuclease Complex/metabolism , Gene Expression Regulation , RNA, Long Noncoding/metabolism , Animals , Embryonic Stem Cells/metabolism , Enhancer Elements, Genetic , Genomic Instability , Heterochromatin/metabolism , Immunoglobulin Class Switching , Immunoglobulin Heavy Chains/genetics , Mice , Regulatory Sequences, Nucleic Acid
6.
Cell ; 159(7): 1538-48, 2014 Dec 18.
Article in English | MEDLINE | ID: mdl-25483776

ABSTRACT

Activation-induced cytidine deaminase (AID) initiates both somatic hypermutation (SHM) for antibody affinity maturation and DNA breakage for antibody class switch recombination (CSR) via transcription-dependent cytidine deamination of single-stranded DNA targets. Though largely specific for immunoglobulin genes, AID also acts on a limited set of off-targets, generating oncogenic translocations and mutations that contribute to B cell lymphoma. How AID is recruited to off-targets has been a long-standing mystery. Based on deep GRO-seq studies of mouse and human B lineage cells activated for CSR or SHM, we report that most robust AID off-target translocations occur within highly focal regions of target genes in which sense and antisense transcription converge. Moreover, we found that such AID-targeting "convergent" transcription arises from antisense transcription that emanates from super-enhancers within sense transcribed gene bodies. Our findings provide an explanation for AID off-targeting to a small subset of mostly lineage-specific genes in activated B cells.


Subject(s)
Cytidine Deaminase/metabolism , Enhancer Elements, Genetic , Genomic Instability , Transcription, Genetic , Animals , B-Lymphocytes/metabolism , Humans , Immunoglobulin Class Switching , Mice , Transcription Initiation Site
7.
Cell ; 157(2): 369-381, 2014 Apr 10.
Article in English | MEDLINE | ID: mdl-24703711

ABSTRACT

Chromosomal rearrangements without gene fusions have been implicated in leukemogenesis by causing deregulation of proto-oncogenes via relocation of cryptic regulatory DNA elements. AML with inv(3)/t(3;3) is associated with aberrant expression of the stem-cell regulator EVI1. Applying functional genomics and genome-engineering, we demonstrate that both 3q rearrangements reposition a distal GATA2 enhancer to ectopically activate EVI1 and simultaneously confer GATA2 functional haploinsufficiency, previously identified as the cause of sporadic familial AML/MDS and MonoMac/Emberger syndromes. Genomic excision of the ectopic enhancer restored EVI1 silencing and led to growth inhibition and differentiation of AML cells, which could be replicated by pharmacologic BET inhibition. Our data show that structural rearrangements involving the chromosomal repositioning of a single enhancer can cause deregulation of two unrelated distal genes, with cancer as the outcome.


Subject(s)
Chromosomes, Human, Pair 3 , DNA-Binding Proteins/genetics , Enhancer Elements, Genetic , GATA2 Transcription Factor/genetics , Gene Expression Regulation, Neoplastic , Leukemia, Myeloid, Acute/genetics , Myelodysplastic Syndromes/genetics , Proto-Oncogenes/genetics , Transcription Factors/genetics , Cell Line, Tumor , Chromosome Inversion , Humans , MDS1 and EVI1 Complex Locus Protein , Promoter Regions, Genetic , Transcriptional Activation , Translocation, Genetic
8.
Cell ; 153(2): 320-34, 2013 Apr 11.
Article in English | MEDLINE | ID: mdl-23582323

ABSTRACT

Chromatin regulators have become attractive targets for cancer therapy, but it is unclear why inhibition of these ubiquitous regulators should have gene-specific effects in tumor cells. Here, we investigate how inhibition of the widely expressed transcriptional coactivator BRD4 leads to selective inhibition of the MYC oncogene in multiple myeloma (MM). BRD4 and Mediator were found to co-occupy thousands of enhancers associated with active genes. They also co-occupied a small set of exceptionally large super-enhancers associated with genes that feature prominently in MM biology, including the MYC oncogene. Treatment of MM tumor cells with the BET-bromodomain inhibitor JQ1 led to preferential loss of BRD4 at super-enhancers and consequent transcription elongation defects that preferentially impacted genes with super-enhancers, including MYC. Super-enhancers were found at key oncogenic drivers in many other tumor cells. These observations have implications for the discovery of cancer therapeutics directed at components of super-enhancers in diverse tumor types.


Subject(s)
Antineoplastic Agents/pharmacology , Azepines/pharmacology , Enhancer Elements, Genetic , Mediator Complex/metabolism , Neoplasms/genetics , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Transcription, Genetic/drug effects , Triazoles/pharmacology , Cell Cycle Proteins , Cell Line, Tumor , Chromatin , Gene Expression Regulation, Neoplastic/drug effects , Genome-Wide Association Study , Humans , Mediator Complex/antagonists & inhibitors , Multiple Myeloma/genetics , Nuclear Proteins/antagonists & inhibitors , Transcription Elongation, Genetic , Transcription Factors/antagonists & inhibitors
9.
Cell ; 154(3): 569-82, 2013 Aug 01.
Article in English | MEDLINE | ID: mdl-23911322

ABSTRACT

Heart failure (HF) is driven by the interplay between regulatory transcription factors and dynamic alterations in chromatin structure. Pathologic gene transactivation in HF is associated with recruitment of histone acetyl-transferases and local chromatin hyperacetylation. We therefore assessed the role of acetyl-lysine reader proteins, or bromodomains, in HF. Using a chemical genetic approach, we establish a central role for BET family bromodomain proteins in gene control during HF pathogenesis. BET inhibition potently suppresses cardiomyocyte hypertrophy in vitro and pathologic cardiac remodeling in vivo. Integrative transcriptional and epigenomic analyses reveal that BET proteins function mechanistically as pause-release factors critical to expression of genes that are central to HF pathogenesis and relevant to the pathobiology of failing human hearts. This study implicates epigenetic readers as essential effectors of transcriptional pause release during HF pathogenesis and identifies BET coactivator proteins as therapeutic targets in the heart.


Subject(s)
Heart Failure/metabolism , Transcription Factors/metabolism , Transcription, Genetic , Animals , Cardiomegaly/genetics , Cardiomegaly/metabolism , Chromatin , Disease Models, Animal , Epigenesis, Genetic , Heart , Heart Failure/drug therapy , Heart Failure/genetics , Humans , Mice , Mice, Inbred C57BL , Myocytes, Cardiac/metabolism , Protein Structure, Tertiary , Rats , Transcription Factors/antagonists & inhibitors , Transcription Factors/chemistry , Transcriptome
10.
Mol Cell ; 78(6): 1096-1113.e8, 2020 06 18.
Article in English | MEDLINE | ID: mdl-32416067

ABSTRACT

BET bromodomain inhibitors (BBDIs) are candidate therapeutic agents for triple-negative breast cancer (TNBC) and other cancer types, but inherent and acquired resistance to BBDIs limits their potential clinical use. Using CRISPR and small-molecule inhibitor screens combined with comprehensive molecular profiling of BBDI response and resistance, we identified synthetic lethal interactions with BBDIs and genes that, when deleted, confer resistance. We observed synergy with regulators of cell cycle progression, YAP, AXL, and SRC signaling, and chemotherapeutic agents. We also uncovered functional similarities and differences among BRD2, BRD4, and BRD7. Although deletion of BRD2 enhances sensitivity to BBDIs, BRD7 loss leads to gain of TEAD-YAP chromatin binding and luminal features associated with BBDI resistance. Single-cell RNA-seq, ATAC-seq, and cellular barcoding analysis of BBDI responses in sensitive and resistant cell lines highlight significant heterogeneity among samples and demonstrate that BBDI resistance can be pre-existing or acquired.


Subject(s)
Drug Resistance, Neoplasm/genetics , Proteins/antagonists & inhibitors , Triple Negative Breast Neoplasms/drug therapy , Animals , Antineoplastic Agents/pharmacology , Azepines/pharmacology , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Chromosomal Proteins, Non-Histone/metabolism , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Mice , Mice, Inbred NOD , Nuclear Proteins/metabolism , Proteins/metabolism , Signal Transduction/drug effects , Transcription Factors/metabolism , Triazoles/pharmacology , Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/metabolism
11.
Cell ; 151(1): 56-67, 2012 Sep 28.
Article in English | MEDLINE | ID: mdl-23021215

ABSTRACT

Elevated expression of the c-Myc transcription factor occurs frequently in human cancers and is associated with tumor aggression and poor clinical outcome. The effect of high levels of c-Myc on global gene regulation is poorly understood but is widely thought to involve newly activated or repressed "Myc target genes." We report here that in tumor cells expressing high levels of c-Myc the transcription factor accumulates in the promoter regions of active genes and causes transcriptional amplification, producing increased levels of transcripts within the cell's gene expression program. Thus, rather than binding and regulating a new set of genes, c-Myc amplifies the output of the existing gene expression program. These results provide an explanation for the diverse effects of oncogenic c-Myc on gene expression in different tumor cells and suggest that transcriptional amplification reduces rate-limiting constraints for tumor cell growth and proliferation.


Subject(s)
Gene Expression Regulation, Neoplastic , Neoplasms/genetics , Proto-Oncogene Proteins c-myc/metabolism , Cell Line, Tumor , Cell Proliferation , Enhancer Elements, Genetic , Humans , Neoplasms/pathology , Promoter Regions, Genetic , Transcription, Genetic
12.
Cell ; 150(4): 673-84, 2012 Aug 17.
Article in English | MEDLINE | ID: mdl-22901802

ABSTRACT

A pharmacologic approach to male contraception remains a longstanding challenge in medicine. Toward this objective, we explored the spermatogenic effects of a selective small-molecule inhibitor (JQ1) of the bromodomain and extraterminal (BET) subfamily of epigenetic reader proteins. Here, we report potent inhibition of the testis-specific member BRDT, which is essential for chromatin remodeling during spermatogenesis. Biochemical and crystallographic studies confirm that occupancy of the BRDT acetyl-lysine binding pocket by JQ1 prevents recognition of acetylated histone H4. Treatment of mice with JQ1 reduced seminiferous tubule area, testis size, and spermatozoa number and motility without affecting hormone levels. Although JQ1-treated males mate normally, inhibitory effects of JQ1 evident at the spermatocyte and round spermatid stages cause a complete and reversible contraceptive effect. These data establish a new contraceptive that can cross the blood:testis boundary and inhibit bromodomain activity during spermatogenesis, providing a lead compound targeting the male germ cell for contraception.


Subject(s)
Azepines/pharmacology , Contraceptive Agents, Male/pharmacology , Nuclear Proteins/antagonists & inhibitors , Triazoles/pharmacology , Animals , Azepines/chemistry , Blood-Testis Barrier , Contraceptive Agents, Male/chemistry , Female , Humans , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Models, Molecular , Nuclear Proteins/chemistry , Protein Structure, Tertiary , Sperm Count , Sperm Motility/drug effects , Spermatozoa/drug effects , Testis/cytology , Testis/drug effects , Triazoles/chemistry
13.
Cell ; 150(3): 575-89, 2012 Aug 03.
Article in English | MEDLINE | ID: mdl-22863010

ABSTRACT

The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. Our study implicates five networks of kinases that regulate the switch to polyploidy. Moreover, we find that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. An integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora kinase A (AURKA). We further find that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in acute megakaryocytic leukemia (AMKL) blasts and displayed potent anti-AMKL activity in vivo. Our findings provide a rationale to support clinical trials of MLN8237 and other inducers of polyploidization and differentiation in AMKL.


Subject(s)
Azepines/pharmacology , Drug Discovery , Leukemia, Megakaryoblastic, Acute/drug therapy , Megakaryocytes/metabolism , Polyploidy , Pyrimidines/pharmacology , Small Molecule Libraries , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/analogs & derivatives , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , Animals , Aurora Kinase A , Aurora Kinases , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Humans , Leukemia, Megakaryoblastic, Acute/genetics , Megakaryocytes/cytology , Megakaryocytes/pathology , Mice , Mice, Inbred C57BL , Protein Interaction Maps , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , rho-Associated Kinases/metabolism
14.
Cell ; 146(6): 904-17, 2011 Sep 16.
Article in English | MEDLINE | ID: mdl-21889194

ABSTRACT

MYC contributes to the pathogenesis of a majority of human cancers, yet strategies to modulate the function of the c-Myc oncoprotein do not exist. Toward this objective, we have targeted MYC transcription by interfering with chromatin-dependent signal transduction to RNA polymerase, specifically by inhibiting the acetyl-lysine recognition domains (bromodomains) of putative coactivator proteins implicated in transcriptional initiation and elongation. Using a selective small-molecule bromodomain inhibitor, JQ1, we identify BET bromodomain proteins as regulatory factors for c-Myc. BET inhibition by JQ1 downregulates MYC transcription, followed by genome-wide downregulation of Myc-dependent target genes. In experimental models of multiple myeloma, a Myc-dependent hematologic malignancy, JQ1 produces a potent antiproliferative effect associated with cell-cycle arrest and cellular senescence. Efficacy of JQ1 in three murine models of multiple myeloma establishes the therapeutic rationale for BET bromodomain inhibition in this disease and other malignancies characterized by pathologic activation of c-Myc.


Subject(s)
Antineoplastic Agents/pharmacology , Drug Discovery , Multiple Myeloma/drug therapy , Proto-Oncogene Proteins c-myc/antagonists & inhibitors , Animals , Antineoplastic Agents/chemistry , Azepines/chemistry , Azepines/pharmacology , Benzodiazepines/chemistry , Benzodiazepines/pharmacology , Cell Line, Tumor , Disease Models, Animal , Humans , Mice , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Structure, Tertiary , Proto-Oncogene Proteins c-myc/genetics , Transcriptional Activation/drug effects , Triazoles/chemistry , Triazoles/pharmacology
15.
Genes Dev ; 32(11-12): 849-864, 2018 06 01.
Article in English | MEDLINE | ID: mdl-29907650

ABSTRACT

Activating JAK2 point mutations are implicated in the pathogenesis of myeloid and lymphoid malignancies, including high-risk B-cell acute lymphoblastic leukemia (B-ALL). In preclinical studies, treatment of JAK2 mutant leukemias with type I JAK2 inhibitors (e.g., Food and Drug Administration [FDA]-approved ruxolitinib) provided limited single-agent responses, possibly due to paradoxical JAK2Y1007/1008 hyperphosphorylation induced by these agents. To determine the importance of mutant JAK2 in B-ALL initiation and maintenance, we developed unique genetically engineered mouse models of B-ALL driven by overexpressed Crlf2 and mutant Jak2, recapitulating the genetic aberrations found in human B-ALL. While expression of mutant Jak2 was necessary for leukemia induction, neither its continued expression nor enzymatic activity was required to maintain leukemia survival and rapid proliferation. CRLF2/JAK2 mutant B-ALLs with sustained depletion or pharmacological inhibition of JAK2 exhibited enhanced expression of c-Myc and prominent up-regulation of c-Myc target genes. Combined indirect targeting of c-Myc using the BET bromodomain inhibitor JQ1 and direct targeting of JAK2 with ruxolitinib potently killed JAK2 mutant B-ALLs.


Subject(s)
Janus Kinase 2/genetics , Janus Kinase 2/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/physiopathology , Animals , Antineoplastic Agents/pharmacology , Azepines/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Gene Knockdown Techniques , Humans , Male , Mice , Mutation , Nitriles , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Pyrazoles/pharmacology , Pyrazoles/therapeutic use , Pyrimidines , RNA Interference , Receptors, Cytokine/genetics , Transcriptome , Triazoles/pharmacology
16.
Mol Cell ; 67(1): 5-18.e19, 2017 Jul 06.
Article in English | MEDLINE | ID: mdl-28673542

ABSTRACT

Processive elongation of RNA Polymerase II from a proximal promoter paused state is a rate-limiting event in human gene control. A small number of regulatory factors influence transcription elongation on a global scale. Prior research using small-molecule BET bromodomain inhibitors, such as JQ1, linked BRD4 to context-specific elongation at a limited number of genes associated with massive enhancer regions. Here, the mechanistic characterization of an optimized chemical degrader of BET bromodomain proteins, dBET6, led to the unexpected identification of BET proteins as master regulators of global transcription elongation. In contrast to the selective effect of bromodomain inhibition on transcription, BET degradation prompts a collapse of global elongation that phenocopies CDK9 inhibition. Notably, BRD4 loss does not directly affect CDK9 localization. These studies, performed in translational models of T cell leukemia, establish a mechanism-based rationale for the development of BET bromodomain degradation as cancer therapy.


Subject(s)
Cyclin-Dependent Kinase 9/metabolism , Nuclear Proteins/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Transcription Elongation, Genetic , Transcription Factors/metabolism , Adaptor Proteins, Signal Transducing , Animals , Antineoplastic Agents/pharmacology , Cell Cycle Proteins , Cyclin-Dependent Kinase 9/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dose-Response Relationship, Drug , Female , Gene Expression Regulation, Leukemic , HCT116 Cells , HEK293 Cells , Humans , Jurkat Cells , Mice, Inbred NOD , Mice, SCID , Mice, Transgenic , Multiprotein Complexes , Nuclear Proteins/genetics , Peptide Hydrolases/genetics , Peptide Hydrolases/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Protein Stability , Proteolysis , RNA Polymerase II/metabolism , Time Factors , Transcription Elongation, Genetic/drug effects , Transcription Factors/genetics , Transfection , Ubiquitin-Protein Ligases , Xenograft Model Antitumor Assays
17.
Proc Natl Acad Sci U S A ; 119(3)2022 01 18.
Article in English | MEDLINE | ID: mdl-35031563

ABSTRACT

Drugs that block the activity of the methyltransferase EZH2 are in clinical development for the treatment of non-Hodgkin lymphomas harboring EZH2 gain-of-function mutations that enhance its polycomb repressive function. We have previously reported that EZH2 can act as a transcriptional activator in castration-resistant prostate cancer (CRPC). Now we show that EZH2 inhibitors can also block the transactivation activity of EZH2 and inhibit the growth of CRPC cells. Gene expression and epigenomics profiling of cells treated with EZH2 inhibitors demonstrated that in addition to derepressing gene expression, these compounds also robustly down-regulate a set of DNA damage repair (DDR) genes, especially those involved in the base excision repair (BER) pathway. Methylation of the pioneer factor FOXA1 by EZH2 contributes to the activation of these genes, and interaction with the transcriptional coactivator P300 via the transactivation domain on EZH2 directly turns on the transcription. In addition, CRISPR-Cas9-mediated knockout screens in the presence of EZH2 inhibitors identified these BER genes as the determinants that underlie the growth-inhibitory effect of EZH2 inhibitors. Interrogation of public data from diverse types of solid tumors expressing wild-type EZH2 demonstrated that expression of DDR genes is significantly correlated with EZH2 dependency and cellular sensitivity to EZH2 inhibitors. Consistent with these findings, treatment of CRPC cells with EZH2 inhibitors dramatically enhances their sensitivity to genotoxic stress. These studies reveal a previously unappreciated mechanism of action of EZH2 inhibitors and provide a mechanistic basis for potential combination cancer therapies.


Subject(s)
DNA Damage/genetics , DNA Damage/physiology , Enhancer of Zeste Homolog 2 Protein/genetics , Enhancer of Zeste Homolog 2 Protein/metabolism , Transcriptional Activation , CRISPR-Cas Systems , Cell Line, Tumor , DNA Repair/genetics , DNA Repair/physiology , Epigenesis, Genetic , Gene Expression Regulation, Neoplastic , Gene Knockout Techniques , Hepatocyte Nuclear Factor 3-alpha/genetics , Hepatocyte Nuclear Factor 3-alpha/metabolism , Humans , Male , Prostatic Neoplasms, Castration-Resistant/genetics , Prostatic Neoplasms, Castration-Resistant/metabolism
18.
PLoS Genet ; 18(8): e1010376, 2022 08.
Article in English | MEDLINE | ID: mdl-35994477

ABSTRACT

The class I histone deacetylases are essential regulators of cell fate decisions in health and disease. While pan- and class-specific HDAC inhibitors are available, these drugs do not allow a comprehensive understanding of individual HDAC function, or the therapeutic potential of isoform-specific targeting. To systematically compare the impact of individual catalytic functions of HDAC1, HDAC2 and HDAC3, we generated human HAP1 cell lines expressing catalytically inactive HDAC enzymes. Using this genetic toolbox we compare the effect of individual HDAC inhibition with the effects of class I specific inhibitors on cell viability, protein acetylation and gene expression. Individual inactivation of HDAC1 or HDAC2 has only mild effects on cell viability, while HDAC3 inactivation or loss results in DNA damage and apoptosis. Inactivation of HDAC1/HDAC2 led to increased acetylation of components of the COREST co-repressor complex, reduced deacetylase activity associated with this complex and derepression of neuronal genes. HDAC3 controls the acetylation of nuclear hormone receptor associated proteins and the expression of nuclear hormone receptor regulated genes. Acetylation of specific histone acetyltransferases and HDACs is sensitive to inactivation of HDAC1/HDAC2. Over a wide range of assays, we determined that in particular HDAC1 or HDAC2 catalytic inactivation mimics class I specific HDAC inhibitors. Importantly, we further demonstrate that catalytic inactivation of HDAC1 or HDAC2 sensitizes cells to specific cancer drugs. In summary, our systematic study revealed isoform-specific roles of HDAC1/2/3 catalytic functions. We suggest that targeted genetic inactivation of particular isoforms effectively mimics pharmacological HDAC inhibition allowing the identification of relevant HDACs as targets for therapeutic intervention.


Subject(s)
Histone Deacetylase 1 , Histone Deacetylase Inhibitors , Acetylation , Histone Deacetylase 1/genetics , Histone Deacetylase 1/metabolism , Histone Deacetylase 2/genetics , Histone Deacetylase 2/metabolism , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/genetics , Histone Deacetylases/metabolism , Humans , Protein Isoforms/genetics , Protein Isoforms/metabolism
19.
Blood ; 139(19): 2983-2997, 2022 05 12.
Article in English | MEDLINE | ID: mdl-35226736

ABSTRACT

Despite advances in the field, chronic graft-versus-host-disease (cGVHD) remains a leading cause of morbidity and mortality following allogenic hematopoietic stem cell transplant. Because treatment options remain limited, we tested efficacy of anticancer, chromatin-modifying enzyme inhibitors in a clinically relevant murine model of cGVHD with bronchiolitis obliterans (BO). We observed that the novel enhancer of zeste homolog 2 (EZH2) inhibitor JQ5 and the BET-bromodomain inhibitor JQ1 each improved pulmonary function; impaired the germinal center (GC) reaction, a prerequisite in cGVHD/BO pathogenesis; and JQ5 reduced EZH2-mediated H3K27me3 in donor T cells. Using conditional EZH2 knockout donor cells, we demonstrated that EZH2 is obligatory for the initiation of cGVHD/BO. In a sclerodermatous cGVHD model, JQ5 reduced the severity of cutaneous lesions. To determine how the 2 drugs could lead to the same physiological improvements while targeting unique epigenetic processes, we analyzed the transcriptomes of splenic GCB cells (GCBs) from transplanted mice treated with either drug. Multiple inflammatory and signaling pathways enriched in cGVHD/BO GCBs were reduced by each drug. GCBs from JQ5- but not JQ1-treated mice were enriched for proproliferative pathways also seen in GCBs from bone marrow-only transplanted mice, likely reflecting their underlying biology in the unperturbed state. In conjunction with in vivo data, these insights led us to conclude that epigenetic targeting of the GC is a viable clinical approach for the treatment of cGVHD, and that the EZH2 inhibitor JQ5 and the BET-bromodomain inhibitor JQ1 demonstrated clinical potential for EZH2i and BETi in patients with cGVHD/BO.


Subject(s)
Bronchiolitis Obliterans , Enhancer of Zeste Homolog 2 Protein , Germinal Center , Graft vs Host Disease , Proteins , Animals , B-Lymphocytes/drug effects , B-Lymphocytes/metabolism , B-Lymphocytes/pathology , Bronchiolitis Obliterans/genetics , Bronchiolitis Obliterans/metabolism , Bronchiolitis Obliterans/pathology , Chronic Disease , Enhancer of Zeste Homolog 2 Protein/antagonists & inhibitors , Enhancer of Zeste Homolog 2 Protein/genetics , Enhancer of Zeste Homolog 2 Protein/metabolism , Enzyme Inhibitors/pharmacology , Germinal Center/drug effects , Germinal Center/pathology , Graft vs Host Disease/drug therapy , Graft vs Host Disease/genetics , Graft vs Host Disease/pathology , Humans , Mice , Proteins/metabolism , Transcriptome
20.
Nature ; 543(7644): 270-274, 2017 03 09.
Article in English | MEDLINE | ID: mdl-28241139

ABSTRACT

Recurrent chromosomal translocations producing a chimaeric MLL oncogene give rise to a highly aggressive acute leukaemia associated with poor clinical outcome. The preferential involvement of chromatin-associated factors as MLL fusion partners belies a dependency on transcription control. Despite recent progress made in targeting chromatin regulators in cancer, available therapies for this well-characterized disease remain inadequate, prompting the need to identify new targets for therapeutic intervention. Here, using unbiased CRISPR-Cas9 technology to perform a genome-scale loss-of-function screen in an MLL-AF4-positive acute leukaemia cell line, we identify ENL as an unrecognized gene that is specifically required for proliferation in vitro and in vivo. To explain the mechanistic role of ENL in leukaemia pathogenesis and dynamic transcription control, a chemical genetic strategy was developed to achieve targeted protein degradation. Acute loss of ENL suppressed the initiation and elongation of RNA polymerase II at active genes genome-wide, with pronounced effects at genes featuring a disproportionate ENL load. Notably, an intact YEATS chromatin-reader domain was essential for ENL-dependent leukaemic growth. Overall, these findings identify a dependency factor in acute leukaemia and suggest a mechanistic rationale for disrupting the YEATS domain in disease.


Subject(s)
Gene Expression Regulation, Neoplastic , Leukemia/genetics , Leukemia/metabolism , Protein Domains , Transcription, Genetic , Transcriptional Elongation Factors/chemistry , Transcriptional Elongation Factors/metabolism , Animals , CRISPR-Cas Systems , Cell Line, Tumor , Cell Proliferation , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Epigenesis, Genetic , Gene Editing , Genome/genetics , Histone-Lysine N-Methyltransferase/metabolism , Humans , Leukemia/pathology , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Mice , Myeloid-Lymphoid Leukemia Protein/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Proteolysis , RNA Polymerase II/metabolism , Transcription Elongation, Genetic , Transcription Factors/chemistry , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptional Elongation Factors/genetics
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