ABSTRACT
MLL-fusions represent a large group of leukemia drivers, whose diversity originates from the vast molecular heterogeneity of C-terminal fusion partners of MLL. While studies of selected MLL-fusions have revealed critical molecular pathways, unifying mechanisms across all MLL-fusions remain poorly understood. We present the first comprehensive survey of protein-protein interactions of seven distantly related MLL-fusion proteins. Functional investigation of 128 conserved MLL-fusion-interactors identifies a specific role for the lysine methyltransferase SETD2 in MLL-leukemia. SETD2 loss causes growth arrest and differentiation of AML cells, and leads to increased DNA damage. In addition to its role in H3K36 tri-methylation, SETD2 is required to maintain high H3K79 di-methylation and MLL-AF9-binding to critical target genes, such as Hoxa9. SETD2 loss synergizes with pharmacologic inhibition of the H3K79 methyltransferase DOT1L to induce DNA damage, growth arrest, differentiation, and apoptosis. These results uncover a dependency for SETD2 during MLL-leukemogenesis, revealing a novel actionable vulnerability in this disease.
Subject(s)
Histone-Lysine N-Methyltransferase/metabolism , Leukemia/metabolism , Myeloid-Lymphoid Leukemia Protein/metabolism , Oncogene Proteins, Fusion/metabolism , Amino Acid Motifs , Cell Differentiation , Cell Line, Tumor , DNA Damage , Histone-Lysine N-Methyltransferase/chemistry , Histone-Lysine N-Methyltransferase/genetics , Humans , Leukemia/genetics , Leukemia/physiopathology , Methylation , Methyltransferases/genetics , Methyltransferases/metabolism , Myeloid-Lymphoid Leukemia Protein/chemistry , Myeloid-Lymphoid Leukemia Protein/genetics , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Oncogene Proteins, Fusion/genetics , Protein BindingABSTRACT
Dissection of complex biological systems requires target-specific control of the function or abundance of proteins. Genetic perturbations are limited by off-target effects, multicomponent complexity, and irreversibility. Most limiting is the requisite delay between modulation to experimental measurement. To enable the immediate and selective control of single protein abundance, we created a chemical biology system that leverages the potency of cell-permeable heterobifunctional degraders. The dTAG system pairs a novel degrader of FKBP12F36V with expression of FKBP12F36V in-frame with a protein of interest. By transgene expression or CRISPR-mediated locus-specific knock-in, we exemplify a generalizable strategy to study the immediate consequence of protein loss. Using dTAG, we observe an unexpected superior antiproliferative effect of pan-BET bromodomain degradation over selective BRD4 degradation, characterize immediate effects of KRASG12V loss on proteomic signaling, and demonstrate rapid degradation in vivo. This technology platform will confer kinetic resolution to biological investigation and provide target validation in the context of drug discovery.
Subject(s)
CRISPR-Cas Systems , Nuclear Proteins/chemistry , Proto-Oncogene Proteins p21(ras)/genetics , Tacrolimus Binding Protein 1A/chemistry , Transcription Factors/genetics , Alleles , Animals , Cell Cycle Proteins , Cell Proliferation , Cytoplasm/metabolism , Dimerization , Gene Knock-In Techniques , HEK293 Cells , Homeostasis , Humans , Ligands , Mice , Mutation , NIH 3T3 Cells , Nuclear Proteins/genetics , Protein Binding , Protein Domains , Proteolysis , Proteomics , Signal Transduction , TransgenesABSTRACT
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 AssaysABSTRACT
Evaluating the engagement of a small molecule ligand with a protein target in cells provides useful information for chemical probe optimization and pharmaceutical development. While several techniques exist that can be performed in a low-throughput manner, systematic evaluation of large compound libraries remains a challenge. In-cell engagement measurements are especially useful when evaluating compound classes suspected to target multiple cellular factors. In this study we used a bioluminescent resonant energy transfer assay to assess bromodomain engagement by a compound series containing bromodomain- and kinase-biasing polypharmacophores based on the known dual BRD4 bromodomain/PLK1 kinase inhibitor BI2536. With this assay, we discovered several novel agents with bromodomain-selective specificity profiles and cellular activity. Thus, this platform aids in distinguishing molecules whose cellular activity is difficult to assess due to polypharmacologic effects.
Subject(s)
Nuclear Proteins/metabolism , Pteridines/chemistry , Transcription Factors/metabolism , Cell Cycle Checkpoints/drug effects , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/metabolism , Cell Survival/drug effects , Drug Design , Fluorescence Resonance Energy Transfer , Fluorescent Dyes/chemistry , HEK293 Cells , Humans , Luminescent Measurements , Nuclear Proteins/antagonists & inhibitors , Protein Binding , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/metabolism , Pteridines/metabolism , Pteridines/toxicity , Transcription Factors/antagonists & inhibitors , Polo-Like Kinase 1ABSTRACT
ATAD2 (ATPase family AAA domain-containing protein 2) is a chromatin regulator harboring an AAA+ ATPase domain and a bromodomain, previously proposed to function as an oncogenic transcription co-factor. Here we suggest that ATAD2 is also required for DNA replication. ATAD2 is co-expressed with genes involved in DNA replication in various cancer types and predominantly expressed in S phase cells where it localized on nascent chromatin (replication sites). Our extensive biochemical and cellular analyses revealed that ATAD2 is recruited to replication sites through a direct interaction with di-acetylated histone H4 at K5 and K12, indicative of newly synthesized histones during replication-coupled chromatin reassembly. Similar to ATAD2-depletion, ectopic expression of ATAD2 mutants that are deficient in binding to these di-acetylation marks resulted in reduced DNA replication and impaired loading of PCNA onto chromatin, suggesting relevance of ATAD2 in DNA replication. Taken together, our data show a novel function of ATAD2 in cancer and for the first time identify a reader of newly synthesized histone di-acetylation-marks during replication.
Subject(s)
ATPases Associated with Diverse Cellular Activities/physiology , DNA Replication , DNA-Binding Proteins/physiology , Epigenesis, Genetic , Histone Code , Acetylation , HEK293 Cells , Histone Deacetylase 1/metabolism , Histones/metabolism , HumansABSTRACT
The CEBPA gene is mutated in 9% of patients with acute myeloid leukemia (AML). Selective expression of a short (30-kDa) CCAAT-enhancer binding protein-α (C/EBPα) translational isoform, termed p30, represents the most common type of CEBPA mutation in AML. The molecular mechanisms underlying p30-mediated transformation remain incompletely understood. We show that C/EBPα p30, but not the normal p42 isoform, preferentially interacts with Wdr5, a key component of SET/MLL (SET-domain/mixed-lineage leukemia) histone-methyltransferase complexes. Accordingly, p30-bound genomic regions were enriched for MLL-dependent H3K4me3 marks. The p30-dependent increase in self-renewal and inhibition of myeloid differentiation required Wdr5, as downregulation of the latter inhibited proliferation and restored differentiation in p30-dependent AML models. OICR-9429 is a new small-molecule antagonist of the Wdr5-MLL interaction. This compound selectively inhibited proliferation and induced differentiation in p30-expressing human AML cells. Our data reveal the mechanism of p30-dependent transformation and establish the essential p30 cofactor Wdr5 as a therapeutic target in CEBPA-mutant AML.
Subject(s)
Antineoplastic Agents/pharmacology , Biphenyl Compounds/pharmacology , Dihydropyridines/pharmacology , Gene Expression Regulation, Neoplastic , Histone-Lysine N-Methyltransferase/antagonists & inhibitors , Leukemia, Myeloid, Acute/metabolism , Myeloid-Lymphoid Leukemia Protein/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Amino Acid Sequence , Animals , CCAAT-Enhancer-Binding Proteins/genetics , CCAAT-Enhancer-Binding Proteins/metabolism , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Histones/genetics , Histones/metabolism , Humans , Intracellular Signaling Peptides and Proteins , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Mice , Molecular Docking Simulation , Molecular Sequence Data , Molecular Targeted Therapy , Mutation , Myeloid-Lymphoid Leukemia Protein/genetics , Myeloid-Lymphoid Leukemia Protein/metabolism , Protein Binding , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Structure, Tertiary , Signal Transduction , Tumor Cells, CulturedABSTRACT
Dendritic cell (DC)-mediated inflammation induced via TLRs is promoted by MAPK-activated protein kinase (MK)-2, a substrate of p38 MAPK. In this study we show an opposing role of MK2, by which it consolidates immune regulatory functions in DCs through modulation of p38, ERK1/2-MAPK, and STAT3 signaling. During primary TLR/p38 signaling, MK2 mediates the inhibition of p38 activation and positively cross-regulates ERK1/2 activity, leading to a reduction of IL-12 and IL-1α/ß secretion. Consequently, MK2 impairs secondary autocrine IL-1α signaling in DCs, which further decreases the IL-1α/p38 but increases the anti-inflammatory IL-10/STAT3 signaling route. Therefore, the blockade of MK2 activity enables human and murine DCs to strengthen proinflammatory effector mechanisms by promoting IL-1α-mediated Th1 effector functions in vitro. Furthermore, MK2-deficient DCs trigger Th1 differentiation and Ag-specific cytotoxicity in vivo. Finally, wild-type mice immunized with LPS in the presence of an MK2 inhibitor strongly accumulate Th1 cells in their lymph nodes. These observations correlate with a severe clinical course in DC-specific MK2 knockout mice compared with wild-type littermates upon induction of experimental autoimmune encephalitis. Our data suggest that MK2 exerts a profound anti-inflammatory effect that prevents DCs from prolonging excessive Th1 effector T cell functions and autoimmunity.
