Using novel small molecule modulators as a tool to elucidate the role of the Myocyte Enhancer Factor 2 (MEF2) family of transcription factors in leukemia.

摘要

The Myocyte Enhancer Factor 2 (MEF2) family of transcription factors are DNA-bound proteins that regulate gene expression via their interaction with co-regulatory proteins. This interaction between MEF2 and its co-factors is necessary for the development and proper function of many mammalian cell types, and is a major factor in the onset and progression of many cancers, specifically leukemia. Understanding the MEF2:co-factor interaction in leukemia, which is often times an epigenetic event, is of utmost importance to those searching for therapies that have high specificity and low chance of side effects.;In the first part of this paper, the structural characteristics of the MEF2 family that allow its interaction with co-regulators is examined. The study revealed that the structure of MEF2 proteins are not majorly altered by the binding of a co-regulator, such as class IIa histone deacetylases (HDACs), Cabin1, or p300, at the protein-protein interface, and are inherently able to be bound while interacting with DNA. This overruled previous notions that the hydrophobic groove, or "binding pocket", of MEF2 was not present unless it was interacting with a co-regulator.;The second, and most involved, study of this paper examines the interaction of MEF2 and class IIa HDACs in leukemia. This study was aided by the availability of a small molecule benzamide compound NKL-30, which we call a MEF2 modulator (MEF2m), that was used as a tool to observe the consequences of inhibiting the protein-protein interaction both in vitro and in vivo. Our MEF2m were able to preferentially decrease the viability of leukemia cells over non-cancerous control B cells. We were also able to show that our compound was able to ablate the MEF2:class IIa HDAC interaction and result in the cytoplasmic shuttling of these co-repressors. Additionally, we were able to show that the expression of the protein NR4A1/Nur77, a MEF2 target gene associated with apoptosis, increased after treatment with NKL-30, likely because of the derepression of MEF2 afforded by the exodus of class IIa HDACs from the nucleus. Finally, we were able to show in a mammalian leukemia model utilizing Nalm6 cells, that mice treated with NKL-30 enjoy an increased lifespan compared to those receiving vehicle control injections.;The third and final part of this work involves the gene expression analysis of B cells that have been treated with our MEF2m. Both healthy and leukemic B cells were treated with our small molecule compound or a solvent control and then data was collected and analyzed using mRNA-seq. We have shown that our MEF2m often preferentially activates gene expression, possibly due to the derepression seen on MEF2 and additional off-target effects. While these differentially regulated genes seen after drug treatment do not fit a single profile or pathway, we do know that the treatment of leukemia cells with our MEF2m usually results in death. Therefore, this data is crucial in our quest for understanding this phenomenon. This work is still ongoing and can be massaged for more data and relationships with the help of future replication and analyses.;In summation, the work presented in this dissertation addresses the possibility of a small molecule compound to specifically target leukemia cells for death over healthy cells due to its ability to bind to MEF2 proteins, disallowing the binding of co-repressors, and resulting in an epigenetic alteration event. The mechanism and consequence of this event, while not yet completely understood, is detailed within the following chapters and the data suggest that we are on the right track toward understanding a way to develop targeted therapies for previously untreatable or high mortality-rate diseases.