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Acute myeloid leukemia (AML) is an aggressive hematological malignancy. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) represents a curative treatment option for AML. Alloreactive donor T cells can produce the graft-versus-leukemia (GVL) effect which represents a major therapeutic benefit of allo-HSCT. However, evading from allogeneic immune surveillance, potentially through the downregulation of human leukocyte antigen class II (HLA-II) antigen presentation machinery, can contribute to AML relapse. Lysine-specific demethylase 1 (LSD1) is an emerging epigenetic therapeutic target in AML. The present study aims to explore whether LSD1 inhibition can enhance AML immunogenicity to promote T-cell mediated immune response. The immunological effects of a clinical-stage LSD1 inhibitor bomedemstat (IMG-7289) were examined in both human and murine AML cell models in vitro. The results demonstrated that bomedemstat treatment significantly enhanced the expression of class II transactivator (CIITA). It subsequently led to the upregulation of HLA-DR in certain human AML cell lines when stimulated by IFN-γ. Bomedemstat also markedly upregulated the expression of CD86 in all human AML cell lines tested. The study also demonstrated that bomedemstat treatment significantly increased the production of pro-inflammatory cytokines IL-12 and CXCL-10. In murine AML models, bomedemstat concurrently upregulated the expression of major histocompatibility complex class II (MHC-II) and CD86 in H9M-transformed cells without IFN-γ stimulation. This effect was not observed in MN1-transformed cells. Bomedemstat-treated H9M cells were subsequently shown to induce antigen-dependent T cell activation. Functional assays revealed that bomedemstat treatment sensitized H9M cells to antigen-dependent immune killing effect mediated by CD4+ T cells. In conclusion, the current study demonstrates both phenotypically and functionally that LSD1 inhibition by bomedemstat treatment can enhance AML immunogenicity. This is evident by the increased antigen presentation, co-stimulation and production of inflammatory cytokines. These findings suggest that LSD1 inhibition may be a relevant strategy to pursue as a maintenance therapy after allo-HSCT. / Thesis / Master of Science in Medical Sciences (MSMS) / Acute myeloid leukemia (AML) is an aggressive blood cancer characterized by the rapid growth of abnormal blood cells in the bone marrow. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an important treatment for AML that involves taking blood stem cells from a healthy donor and transplanting them into an AML patient. Meanwhile, immune cells such as T cells from the donor can help destroy leukemia cells. However, AML frequently reappears after allo-HSCT and new therapies are needed to prevent the disease from coming back. The present study investigates whether blocking a protein called lysine-specific demethylase 1 (LSD1) can increase T cells’ ability to identify and kill cancer cells. The results demonstrate that treatment with an LSD1 blocker called bomedemstat can enhance the recognition of AML cells by T cells, thereby enhancing their immune response. These findings suggest that blocking LSD1 is a promising approach to enhance the effectiveness of allo-HSCT.

Biological organisms have evolved complex epigenetic mechanisms to tailor their gene expression programs to specific needs. These adaptations allow cells, that otherwise have identical genomes, to carry out specialized functions. In this work I develop and use data-integrative techniques to examine the mechanisms and consequences of epigenetic processes. To better understand the changes in DNA methylation landscape that accompany breast cancer molecular subtypes, I integrated DNA methylation and gene expression data from 208 breast cancer samples obtained from a Polish population-based case-control study. Using a weighted correlation network approach, I identified gene co-methylation modules and asked if the genes in these modules are preferentially methylated and silenced in a breast cancer subtype-specific manner. This approach identified two non-overlapping gene co-methylation modules. The first module is silenced in Basal breast cancers, while the second is silenced in Luminal B breast cancers. Gene-set enrichment analysis suggests that epigenetic silencing of these modules interferes with processes that maintain cellular differentiation, and that the methylation status of the Luminal B module is associated with disease prognosis. To uncover the determinants of the temporal order of metazoan genome replication, I used a reductionist model of DNA replication to test the ability of hundreds of epigenetic marks to predict replication timing. My work showed that DNA replication timing can be completely predicted from locations of DNase I hypersensitive sites. I further demonstrated the robust emergent character of DNA replication that could be understood without invoking a complex regulatory mechanism. To determine the underlying cause of cell de-differentiation in osteosarcoma, I examined the relationship between microRNA expression and the bone-cell differentiation program. Focusing on the inhibitory role of miR-23a in bone differentiation, I analyzed the effect of its over-expression in osteosarcoma cells. Extensive computational analysis led me to propose that a major mechanism by which miR-23a exerts its effect is by interfering with expression of GJA1, which encodes a gap junction channel essential for intercellular communication and external stimuli sensing in bone cells. Follow-up experiments indicate that GJA1 is sharply up-regulated during bone cell differentiation and that GJA1 inhibition significantly delays the onset of differentiation. Together, this work uses data integrative techniques to provide new insights into the decisive role of epigenetic processes in cellular differentiation. / 2019-04-01T00:00:00Z

Genomic DNA methylation is an epigenetic marker that reflects influences of the environment, aging, and diseases. Although causal mechanisms of these alterations are understudied, the first step to addressing changes in DNA methylation is to map alterations appearing in a particular context. For example, human populations have diverse situational exposures. As an extreme example, the isolated populations of nomadic hunter/gatherer individuals in the Kalahari Desert lack access to most of the conveniences of modern lifestyles. Due to climate and behavioral adaptations of the lifestyle of these isolated populations (acoustic sensitivity to predators, irregular water and food availability), I predicted and demonstrated altered methylation landscapes compared with a non-Khoesan group of southern Africans with more industrialized lifestyles. The sites of differential methylation were assessed for potential functional impact at several example loci. A related project addressed nucleotide variants at interrogated methylation loci. For instance, C to T polymorphisms occurring in some individuals cannot initially be discriminated from loci that have differential cytosine methylation (following bisulfite treatment) in an array-based assay. My solution to this problem was the development of a computational approach to detect loci in methylation array data, which show tiered patterns created by SNP alleles rather than the usual continuum of differential methylation values. This approach was applied to the Kalahari populations and HapMap groups to show the utility of the approach. In the Kalahari populations, post-infancy ages are not recorded. We used functions that utilize DNA methylation to calculate estimates of aging and compared these results with predictions reported by the sample collectors, which were based primarily on interactions with non-nomadic neighbors. I compared the same aging estimates to known ages in the non-Khoesan samples and found correspondence. Although DNA methylation is a good predictor of cellular age, another method is telomere length measurement. To assess a relationship between predictors, I assessed associations in 300 samples between age, DNA methylation, and telomere length. Initial results indicated multiple correlated loci when accounting for gender and ethnicity using a linear model approach. / 2020-07-31T00:00:00Z

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Schizophrenia is a debilitation mental illness characterized by positive symptoms (mania and hallucinations), negative symptoms (flat affect), and cognitive impairments (learning and memory deficits). These symptoms arise from dysfunction of several neurotransmitter systems including the dopaminergic, seratonergic, cholinergic, and glutamatergic pathways. As such, treatment of this disease has been difficult due to the number of systems involved. Various theories dealing with maternal infection, chemical imbalance, genetics, and epigenetics have emerged postulating the origin of the disease. To date, there is no one unifying hypothesis that encompasses all of the behavioral and biological deficits manifested in schizophrenia. A review of the current research suggests a central role of kynurenic acid (KYNA) in all of these theories. As an endogenous antagonist of cholinergic and glutamatergic receptors, KYNA has been shown to mimic the disease when administered exogenously. Additionally, KYNA levels appear to be elevated in the brains of schizophrenics. Understanding how this chemical works and how it becomes elevated in the first place will be key to understanding the pathology of schizophrenia and developing effective treatments.

The fundamental unit of chromatin is the nucleosome, which consists of a core of eight proteins wrapped by DNA. This core is composed of four pairs of histone proteins: H2A, H2B, H3, and H4. The DNA wraps around the protein core ~1.7 times, facilitating compaction of DNA length in the cell. Further, the location of nucleosomes makes genomic elements encoded in the DNA, such as promoters or enhancers, accessible or inaccessible to RNA polymerase and transcription factors. Thus, where nucleosomes are located (or positioned), can play a major role in transcription or other cellular processes. Additionally, histone proteins are frequently post-translationally modified, and these modifications further play a role in cellular processes, and in some cases are even required for specific protein function. What positions nucleosomes, and the downstream results of positioning or post-translational modifications (PTMs) is a topic of prolific study. Nucleosome formation is not random. In vivo it is believed that chromatin remodelers are the primary determinant of where nucleosomes form, while in vitro the DNA itself is the primary determinant. Formation of nucleosomes in vitro is a potent tool to elucidate fundamentals of chromatin. Considering that in vitro nucleosome formation is dependent on free energy, morphology and base composition of the DNA influence the free energy of formation. We found that the ends of linear DNA fragments were much more likely to have in vitro nucleosomes form on them. While this has the potential to bias results, based on our observations we could not find any significant alteration of the overall underlying DNA sequence composition due to the end preference observed. Histone proteins frequently receive the PTMs of methylation or acetylation. Histone methylation is typically indicative of repressed genes, while histone acetylation is typically indicative of active genes. In vivo the addition and removal of methylation and acetylation is highly dynamic. We hypothesized that the histone PTMs of methylation and acetylation also played a role in where nucleosomes formed. Comparing both in vivo and in vitro datasets, we observed strikingly similar patterns of nucleosomes for several histone methylations and acetylations, suggesting that these PTMs do indeed direct nucleosome formation. Upon further investigation, the underlying DNA sequence preferences change when compared to unmodified nucleosomes. This suggests that the genome is encoded to position these marks in locations where they are likely to be needed.

There are about 20,000 genes in the human genome. The lowly nematode worm, C. elegans, has about the same number of genes. How could two organisms that are so different arise from a similar number of genes? The answer is epigenetics, or the factors that help control when and where genes are expressed. There are many layers that comprise the epigenetic control of genes. One of which is the structure or architecture of chromatin. Chromatin is a complex of DNA and proteins. Histone proteins with DNA wrapped around them form the fundamental component of chromatin, the nucleosome. Chromatin exists in two forms, euchromatin and heterochromatin. Euchromatin is made of loosely packed nucleosomes while in heterochromatin nucleosomes are tightly packed. Genome elements are not accessible in heterochromatin but are in euchromatin. In this way chromatin architecture provides a layer of control of genetic expression. Where nucleosome form is a function of several factors including the underlying DNA sequence, and binding competition between histones and other DNA binding proteins. Here we test the ability of various DNA sequences to position and repel histone proteins in C. elegans worms. We find that the 601sequence can position nucleosomes and that the PRS-322 sequence does repel nucleosomes in vivo. Assessing chromatin architecture requires sequences to be aligned to a reference genome, however, there are numerous programs with which to do this. Each program performs this task in a different way. These differences can have a large impact on the downstream analysis of the results. To this end, we have tested various alignment programs to assess how well they align reads to a reference genome. Here we have found that Bowtie2, BWA, and Chromap perform alignments accurately and we suggest using them. As an organism develops its genetic expression changes. This change in expression is often the result of temporally specific genomic elements such as enhancers. Understanding when enhancers are accessible during development can lead to a better understanding of the genetic control needed for development. Here we utilize data gathered at specific developmental stages in C. elegans to elucidate enhancer accessibility. In this work we have furthered the understanding of epigenetic control of expression by quantifying positioning and repelling sequences, testing read mapping programs for accuracy and identifying temporally specific enhancers in developing worms.

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