Browsing by Subject "BRD4"

Now showing 1 - 3 of 3
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    Adaptation and innovation of anti-cancer epigenetic targeting therapeutic strategies for the treatment of murine chronic graft vs host disease
    (2021-08) Zaiken, Michael
    Each year in the United States nearly 200,000 patients receive a diagnosis of a hematological cancer. While the treatment of these conditions has improved remarkably over the last few decades, nearly a quarter of these patients are expected to die from their disease. Much of the improvement in treatment comes from advancements in the field of allogenic hematopoietic stem cell transplant (aHSCT), the only curative therapy for the treatment of hematological malignancies. While aHSCT is an incredibly powerful technique, it comes with the major drawback of inducing graft vs host disease (GVHD), the second leading cause of mortality following aHSCT after reoccurrence of the original disease. Chronic GVHD in particular has proven difficult to manage. cGVHD is an auto-immune-like condition that arises as a result of donor-derived immune cells attacking the recipient tissues post-transplant. The current standard of treatment for cGVHD involves broad steroid immunosuppression, however in cases where steroid refractory forms of the disease arise treatment options are extremely limited. Murine modeling of cGVHD has proven to be a highly effective method for the development and preclinical testing of novel therapies. Based on these studies, it has been determined that the germinal center reaction (GCR) forms a critical step in the pathogenesis of cGVHD, as it is necessary for the development of allo-antibodies that drive fibrotic tissue damage which characterizes the condition. As such, the development of novel therapeutics that disrupt this reaction is of particular interest for the treatment of cGVHD. Epigenetic targeting drugs inhibit enzymes involved in chromatin remodeling and changing the epigenetic landscape of cells. Their use in cancer treatment is primarily driven by the drug’s impact on the aberrant epigenome that arises in cancer cells. In the studies presented here, we sought to leverage this strategy for the treatment of cGVHD, not by targeting aberrant epigenomic changes, but instead using these compounds to disrupt cellular processes necessary for the pathogenesis of cGVHD in particular the GCR. To that end, we tested the preclinical efficacy of three different classes of epigenetic targets in the treatment of cGVHD. We first investigated the potential of BET-bromodomain inhibitor JQ1, which inhibits a number of epigenetic reader enzymes, in particular BRD4. We show that while JQ1 has great impact in a fibrotic model of cGVHD that recapitulates pulmonary dysfunction from bronchiolitis obliterans (BOS), its impact in inflammatory models of GVHD is limited. Next, we tested the role of the epigenetic writer EZH2 in the pathogenesis and treatment of cGVHD. We show that not only is EZH2 necessary for the establishment of cGVHD, but that a novel small molecule inhibitor of EZH2, JQ5, is highly effective in treating multiple murine models of the disease. We further demonstrate that while both of these strategies disrupt the GCR, they do so through the regulation of unique sets of genetic targets, which may have important implications for their clinical translation. Finally, we tested the role of the dynamic DNA demethylases TET2 and TET3 in cGVHD. We show that TET3 is critical for the initiation of disease not through inhibition of the GCR, but by changing the reaction to alter the IgG class switching of antibody secreting B cells. In doing so Tet3 loss of function prevents the development of fibrotic pathology while preserving adaptive immune activity. These trials demonstrate not only the preclinical efficacy of these therapeutic targets but show that the targeting of epigenetic regulators of the GCR is a viable avenue for the development of further therapies for the treatment of cGVHD.
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    Computational Analysis on a Novel Set of 1,4,5 tri-Substituted Imidazole Based Bromodomain Inhibitors with Human BRD4
    (2020-05) Jones, Peter
    Bromodomain-Containing Protein 4 (BRD4) is a human transcriptional regulator and member of the N-Terminal Bromodomain and Extra Terminal Domain (BET) family of proteins. BRD4 binds to acetylated chromatin, preserving epigenetic modifications in the chromatin structure and activating the positive transcription elongation factor (p-TEFb) complex. This complex phosphorylates RNA polymerase II and promotes transcription of the immediate downstream genomic element. BRD4 shows promise as a target for anticancer therapies, with most research focusing on a class of drugs known as BET inhibitors. These drugs bind to the active site of BET family proteins, preventing BRD4 specifically from associating with chromatin. However, there is a lack of atomistic understanding regarding the binding of these drugs to BET family members. Many factors which influence the binding affinity of a series of 1,4,5 tri-substituted imidazole-based bromodomain inhibitors bound to the active site of BRD4 have yet to be characterized. Further, the effects of these inhibitors on the structural waters intrinsic to BRD4 remains unclear. Experimental work has suggested that the IC50 of this series of BET inhibitors could be explained in terms of a few specific interactions in the binding site. In this analysis, Free Energy Perturbations (FEP) are used to probe the relative free energy of binding for this set of differentially substituted BET inhibitors. Our working hypothesis is that the chlorine, bromine, and iodine substitutions participate in a halogen bond with the backbone oxygen of Met105 in BRD4, stabilizing the drug in the active site. Further, we propose that substitutions which cannot form this halogen bond, such as fluorine and other non-halogen substitutions, would have a higher free energy of binding. FEP analysis revealed that the chlorinated, brominated, and iodinated substitutions displayed a lower free energy of binding than the other substitutions, with evidence of a halogen bond between the drug and the backbone oxygen of Met105. It was also observed that this set of BET inhibitors displaces several highly coordinated solvent molecules in the active site of BRD4. By contrast, a simulation of BRD4 complexed with JQ1, another known BET inhibitor, does not displace these waters. These results support our hypothesis that a halogen bond is formed between the large halogen substitutions and the protein, increasing the binding affinity for substitutions that can participate in this type of interaction. This halogen bond can be exploited for improving this set inhibitors and designing novel compounds which bind more favorably to BRD4.
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    Functional Proteomics Analysis To Discover And Characterize Oxygen-Dependent Cellular Pathways
    (2019-02) Erber, Luke
    Oxygen and iron homeostasis are a critical components for the maintenance of cellular biology. These metabolites are essential substrates in cellular metabolism, signaling and bioenergetics, thereby inseparably linked to the normal physiology of all metazoans. To adapt to changes in the microenvironment, cells dynamically modulate hypoxia response pathways. Lack of oxygen reduces the post-translational modification proline hydroxylation and altering key transcription factors such as hypoxia-inducible factor 1alpha and prevents its hydroxyproline-dependent degradation. Stabilized HIF proteins activate the expression of hypoxiaresponse genes to sustain growth under hypoxia condition. The studies herein focus on the hypothesis that post-translational modifications of hydroxylation and phosphorylation are a mechanistic link between oxygen and iron availability and the cellular physiological response. This research has focused on the characterization of oxygen and iron-sensing pathways dependent on proline hydroxylation and phosphorylation. Through a system-wide proteomics survey, I identified Brd4 as a novel proline hydroxylation protein substrate in cancer cells. Specific prolyl hydroxylase activity significantly regulates the Brd4-mediated transcriptional function and strongly induced acute myeloid leukemia cell proliferation and apoptosis. This study integrates molecular biology and quantitative proteomics approaches to discover and characterize cellular oxygen and iron-sensing physiology and reveal novel cellular pathways that may have broad impact in cancer biology and metabolic diseases.