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1.
Angew Chem Int Ed Engl ; 63(13): e202319579, 2024 Mar 22.
Article in English | MEDLINE | ID: mdl-38291002

ABSTRACT

A cascade of three enzymes, E1-E2-E3, is responsible for transferring ubiquitin to target proteins, which controls many different aspects of cellular signaling. The role of the E2 has been largely overlooked, despite influencing substrate identity, chain multiplicity, and topology. Here we report a method-targeted charging of ubiquitin to E2 (tCUbE)-that can track a tagged ubiquitin through its entire enzymatic cascade in living mammalian cells. We use this approach to reveal new targets whose ubiquitination depends on UbcH5a E2 activity. We demonstrate that tCUbE can be broadly applied to multiple E2s and in different human cell lines. tCUbE is uniquely suited to examine E2-E3-substrate cascades of interest and/or piece together previously unidentified cascades, thereby illuminating entire branches of the UPS and providing critical insight that will be useful for identifying new therapeutic targets in the UPS.


Subject(s)
Ubiquitin-Conjugating Enzymes , Ubiquitin , Animals , Humans , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitination , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/metabolism , Mammals/metabolism
2.
Mol Cell ; 81(17): 3604-3622.e10, 2021 09 02.
Article in English | MEDLINE | ID: mdl-34358447

ABSTRACT

The transformed state in acute leukemia requires gene regulatory programs involving transcription factors and chromatin modulators. Here, we uncover an IRF8-MEF2D transcriptional circuit as an acute myeloid leukemia (AML)-biased dependency. We discover and characterize the mechanism by which the chromatin "reader" ZMYND8 directly activates IRF8 in parallel with the MYC proto-oncogene through their lineage-specific enhancers. ZMYND8 is essential for AML proliferation in vitro and in vivo and associates with MYC and IRF8 enhancer elements that we define in cell lines and in patient samples. ZMYND8 occupancy at IRF8 and MYC enhancers requires BRD4, a transcription coactivator also necessary for AML proliferation. We show that ZMYND8 binds to the ET domain of BRD4 via its chromatin reader cassette, which in turn is required for proper chromatin occupancy and maintenance of leukemic growth in vivo. Our results rationalize ZMYND8 as a potential therapeutic target for modulating essential transcriptional programs in AML.


Subject(s)
Interferon Regulatory Factors/metabolism , Leukemia, Myeloid, Acute/metabolism , Tumor Suppressor Proteins/metabolism , Cell Cycle Proteins/metabolism , Cell Line , Cell Line, Tumor , Cell Proliferation/genetics , Chromatin/genetics , Enhancer Elements, Genetic/genetics , Gene Expression Regulation, Neoplastic/genetics , Humans , Interferon Regulatory Factors/genetics , Leukemia, Myeloid, Acute/genetics , Nuclear Proteins/metabolism , Promoter Regions, Genetic/genetics , Proto-Oncogene Mas , Transcription Factors/metabolism , Transcription, Genetic/genetics , Tumor Suppressor Proteins/genetics
3.
Leukemia ; 35(5): 1405-1417, 2021 05.
Article in English | MEDLINE | ID: mdl-33542482

ABSTRACT

Translocations of Meningioma-1 (MN1) occur in a subset of acute myeloid leukemias (AML) and result in high expression of MN1, either as a full-length protein, or as a fusion protein that includes most of the N-terminus of MN1. High levels of MN1 correlate with poor prognosis. When overexpressed in murine hematopoietic progenitors, MN1 causes an aggressive AML characterized by an aberrant myeloid precursor-like gene expression program that shares features of KMT2A-rearranged (KMT2A-r) leukemia, including high levels of Hoxa and Meis1 gene expression. Compounds that target a critical KMT2A-Menin interaction have proven effective in KMT2A-r leukemia. Here, we demonstrate that Menin (Men1) is also critical for the self-renewal of MN1-driven AML through the maintenance of a distinct gene expression program. Genetic inactivation of Men1 led to a decrease in the number of functional leukemia-initiating cells. Pharmacologic inhibition of the KMT2A-Menin interaction decreased colony-forming activity, induced differentiation programs in MN1-driven murine leukemia and decreased leukemic burden in a human AML xenograft carrying an MN1-ETV6 translocation. Collectively, these results nominate Menin inhibition as a promising therapeutic strategy in MN1-driven leukemia.


Subject(s)
Leukemia, Myeloid, Acute/genetics , Proto-Oncogene Proteins/genetics , Trans-Activators/genetics , Tumor Suppressor Proteins/genetics , Animals , Cell Line, Tumor , Gene Expression Regulation, Leukemic/genetics , HEK293 Cells , Histone-Lysine N-Methyltransferase/genetics , Humans , Mice , Mice, Knockout
4.
Blood Adv ; 4(13): 3109-3122, 2020 07 14.
Article in English | MEDLINE | ID: mdl-32634241

ABSTRACT

Understanding mechanisms of cooperation between oncogenes is critical for the development of novel therapies and rational combinations. Acute myeloid leukemia (AML) cells with KMT2A-fusions and KMT2A partial tandem duplications (KMT2APTD) are known to depend on the histone methyltransferase DOT1L, which methylates histone 3 lysine 79 (H3K79). About 30% of KMT2APTD AMLs carry mutations in IDH1/2 (mIDH1/2). Previous studies showed that 2-hydroxyglutarate produced by mIDH1/2 increases H3K79 methylation, and mIDH1/2 patient samples are sensitive to DOT1L inhibition. Together, these findings suggested that stabilization or increases in H3K79 methylation associated with IDH mutations support the proliferation of leukemias dependent on this mark. However, we found that mIDH1/2 and KMT2A alterations failed to cooperate in an experimental model. Instead, mIDH1/2 and 2-hydroxyglutarate exert toxic effects, specifically on KMT2A-rearranged AML cells (fusions/partial tandem duplications). Mechanistically, we uncover an epigenetic barrier to efficient cooperation; mIDH1/2 expression is associated with high global histone 3 lysine 79 dimethylation (H3K79me2) levels, whereas global H3K79me2 is obligate low in KMT2A-rearranged AML. Increasing H3K79me2 levels, specifically in KMT2A-rearrangement leukemias, resulted in transcriptional downregulation of KMT2A target genes and impaired leukemia cell growth. Our study details a complex genetic and epigenetic interaction of 2 classes of oncogenes, IDH1/2 mutations and KMT2A rearrangements, that is unexpected based on the high percentage of IDH mutations in KMT2APTD AML. KMT2A rearrangements are associated with a trend toward lower response rates to mIDH1/2 inhibitors. The substantial adaptation that has to occur for 2 initially counteracting mutations to be tolerated within the same leukemic cell may provide at least a partial explanation for this observation.


Subject(s)
Gene Rearrangement , Leukemia, Myeloid, Acute , Histones/metabolism , Humans , Leukemia, Myeloid, Acute/genetics , Methylation , Oncogenes
5.
Epigenetics ; 10(6): 467-73, 2015.
Article in English | MEDLINE | ID: mdl-25923537

ABSTRACT

Polycomblike (Pcl) proteins are important transcriptional regulators and components of the Polycomb Repressive Complex 2 (PRC2). The Tudor domains of human homologs PHF1 and PHF19 have been found to recognize trimethylated lysine 36 of histone H3 (H3K36me3); however, the biological role of Tudor domains of other Pcl proteins remains poorly understood. Here, we characterize the molecular basis underlying histone binding activities of the Tudor domains of the Pcl family. In contrast to a predominant view, we found that the methyl lysine-binding aromatic cage is necessary but not sufficient for recognition of H3K36me3 by these Tudor domains and that a hydrophobic patch, adjacent to the aromatic cage, is also required.


Subject(s)
DNA-Binding Proteins/chemistry , Epigenesis, Genetic , Histone-Lysine N-Methyltransferase/chemistry , Polycomb Repressive Complex 2/chemistry , Polycomb-Group Proteins/chemistry , Binding Sites , DNA-Binding Proteins/genetics , Histone-Lysine N-Methyltransferase/genetics , Humans , Hydrophobic and Hydrophilic Interactions , Methylation , Nuclear Magnetic Resonance, Biomolecular , Polycomb Repressive Complex 2/genetics , Polycomb-Group Proteins/genetics , Protein Binding , Protein Conformation , Protein Structure, Tertiary
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