DNMT inhibitors reverse a specific signature of aberrant promoter DNA methylation and associated gene silencing in AML

<b>Background</b>. Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are neoplastic disorders of hematopoietic stem cells. DNA methyltransferase inhibitors (DNMTi), 5-azacytidine (AzaC) and 5-aza-2’-deoxycytidine (Decitabine), benefit some MDS/AML patients. However, the role of DNMTi-induced DNA hypomethylation in regulation of gene expression in AML is unclear.<p></p> <b>Results. </b> We compared the effects of AzaC on DNA methylation and gene expression using whole-genome single-nucleotide bisulfite-sequencing (WGBS) and RNA-sequencing in OCI-AML3 (AML3) cells. For data analysis, we used an approach recently developed for discovery of differential patterns of DNA methylation associated with changes in gene expression, that is tailored to single-nucleotide bisulfite-sequencing data (Washington University Interpolated Methylation Signatures (WIMSi)). By this approach, a subset of genes upregulated by AzaC was found to be characterized by AzaC-induced signature methylation loss flanking the transcription start site. These genes are enriched for genes increased in methylation and decreased in expression in AML3 cells compared to normal hematopoietic stem and progenitor cells. Moreover, these genes are preferentially upregulated by Decitabine in human primary AML blasts, and control cell proliferation, death and development. <p></p> <b>Conclusions.</b> Our WGBS and WIMSi data analysis approach has identified a set of genes whose is methylation and silencing in AML is reversed by DNMTi. These genes are good candidates for direct regulation by DNMTi, and their reactivation by DNMTi may contribute to therapeutic activity. This study also demonstrates the ability of WIMSi to reveal relationships between DNA methylation and gene expression, based on single-nucleotide bisulfite-sequencing and RNA-seq data.<p></p&gt

T-Cell Immunogenicity and Dysfunction in Cancer and Viral Diseases

abstract: CD8+ T-lymphocytes (CTLs) are central to the immunologic control of infections and are currently at the forefront of strategies that enhance immune based treatment of a variety of tumors. Effective T-cell based vaccines and immunotherapies fundamentally rely on the interaction of CTLs with peptide-human leukocyte antigen class I (HLA-I) complexes on the infected/malignant cell surface. However, how CTLs are able to respond to antigenic peptides with high specificity is largely unknown. Also unknown, are the different mechanisms underlying tumor immune evasion from CTL-mediated cytotoxicity. In this dissertation, I investigate the immunogenicity and dysfunction of CTLs for the development of novel T-cell therapies. Project 1 explores the biochemical hallmarks associated with HLA-I binding peptides that result in a CTL-immune response. The results reveal amino acid hydrophobicity of T-cell receptor (TCR) contact residues within immunogenic CTL-epitopes as a critical parameter for CTL-self/nonself discrimination. Project 2 develops a bioinformatic and experimental methodology for the identification of CTL-epitopes from low frequency T-cells against tumor antigens and chronic viruses. This methodology is employed in Project 3 to identify novel immunogenic CTL-epitopes from human papillomavirus (HPV)-associated head and neck cancer patients. In Project 3, I further study the mechanisms of HPV-specific T-cell dysfunction, and I demonstrate that combination inhibition of Indoleamine 2, 3-dioxygenase (IDO-1) and programmed cell death protein (PD-1) can be a potential immunotherapy against HPV+ head and neck cancers. Lastly, in Project 4, I develop a single-cell assay for high-throughput identification of antigens targeted by CTLs from whole pathogenome libraries. Thus, this dissertation contributes to fundamental T-cell immunobiology by identifying rules of T-cell immunogenicity and dysfunction, as well as to translational immunology by identifying novel CTL-epitopes, and therapeutic targets for T-cell immunotherapy.Dissertation/ThesisDoctoral Dissertation Biological Design 201

Twin-width VIII: delineation and win-wins

We introduce the notion of delineation. A graph class $\mathcal C$ is said delineated if for every hereditary closure $\mathcal D$ of a subclass of $\mathcal C$, it holds that $\mathcal D$ has bounded twin-width if and only if $\mathcal D$ is monadically dependent. An effective strengthening of delineation for a class $\mathcal C$ implies that tractable FO model checking on $\mathcal C$ is perfectly understood: On hereditary closures $\mathcal D$ of subclasses of $\mathcal C$, FO model checking is fixed-parameter tractable (FPT) exactly when $\mathcal D$ has bounded twin-width. Ordered graphs [BGOdMSTT, STOC '22] and permutation graphs [BKTW, JACM '22] are effectively delineated, while subcubic graphs are not. On the one hand, we prove that interval graphs, and even, rooted directed path graphs are delineated. On the other hand, we show that segment graphs, directed path graphs, and visibility graphs of simple polygons are not delineated. In an effort to draw the delineation frontier between interval graphs (that are delineated) and axis-parallel two-lengthed segment graphs (that are not), we investigate the twin-width of restricted segment intersection classes. It was known that (triangle-free) pure axis-parallel unit segment graphs have unbounded twin-width [BGKTW, SODA '21]. We show that $K_{t,t}$-free segment graphs, and axis-parallel $H_t$-free unit segment graphs have bounded twin-width, where $H_t$ is the half-graph or ladder of height $t$. In contrast, axis-parallel $H_4$-free two-lengthed segment graphs have unbounded twin-width. Our new results, combined with the known FPT algorithm for FO model checking on graphs given with $O(1)$-sequences, lead to win-win arguments. For instance, we derive FPT algorithms for $k$-Ladder on visibility graphs of 1.5D terrains, and $k$-Independent Set on visibility graphs of simple polygons.Comment: 51 pages, 19 figure

Non-cytolytic control of hepatitis B virus replication and the role of interleukin-12 (IL-12)

Hepatitis B is a non-cytopathic virus that causes major morbidity worldwide. Data from animal models suggest that T lymphocytes can control hepatitis B virus (HBV) replication without killing infected hepatocytes through interferon-γ (IFN-γ). Furthermore, IFN-γ production is regulated by interleukin-12 (IL-12), which not only has a direct anti-viral effect but also promotes T helper-1 type cell- mediated immune responses, which are important in the control of HBV. The aim of this thesis was to investigate non-cytolytic control of HBV in human infection, and the role of virus-specific CD4+ T-cells in the resolution of chronic HBV infection. Furthermore, the role of IL-12 in human HBV infection and the potential of combining anti-viral and immunomodulatory drugs for the treatment of chronic HBV infection was investigated. Activated peripheral blood mononuclear cells (PBMC) from chronically infected HBV carriers reduced cytoplasmic HBV DNA in a liver cell line by releasing IFN-γ, and without killing hepatocytes. Furthermore, recombinant IFN-γ reduced the levels of HBV DNA in naturally infected hepatocytes by between 0.3 to 3 log10 and the level of HBV transcription by up to 71% non-cytolytically. Adoptive transfer of HBcAg-reactive CD4+T cells in bone marrow transplant recipients resulted in an ALT flare and the subsequent resolution of chronic HBV infection through the development of anti-HBs. IL-12 receptor (IL-12R) expression was reduced in chronic HBV infection as measured by flow cytometry. This may be a cause of the Th2 immune responses seen in chronic HBV infection. IL-12R expression could be increased to normal levels by recombinant human IL-12 (rhIL-12) resulting in Th1 effector functions. Combination therapy of lamivudine and IL-12 in chronically infected HBV patients has an enhanced anti-viral effect, which is associated with induction of HBcAg-specific CD4 T-cell reactivity and increased frequency of HBcAg- specific CD4+ T-cells, which produce IFN-γ