Epigenetic reprogramming of T cells: unlocking new avenues for cancer immunotherapy.

T cells, a key component of cancer immunotherapy, undergo a variety of histone modifications and DNA methylation changes since their bone marrow progenitor stages before developing into CD8+ and CD4+ T cells. These T cell types can be categorized into distinct subtypes based on their functionality and properties, such as cytotoxic T cells (Tc), helper T cells (Th), and regulatory T cells (Treg) as subtypes for CD8+ and CD4+ T cells. Among these, the CD4+ CD25+ Tregs potentially contribute to cancer development and progression by lowering T effector (Teff) cell activity under the influence of the tumor microenvironment (TME). This contributes to the development of therapeutic resistance in patients with cancer. Subsequently, these individuals become resistant to monoclonal antibody therapy as well as clinically established immunotherapies. In this review, we delineate the different epigenetic mechanisms in cancer immune response and its involvement in therapeutic resistance. Furthermore, the possibility of epi-immunotherapeutic methods based on histone deacetylase inhibitors and histone methyltransferase inhibitors are under investigation. In this review we highlight EZH2 as the principal driver of cancer cell immunoediting and an immune escape regulator. We have addressed in detail how understanding T cell epigenetic regulation might bring unique inventive strategies to overcome drug resistance and increase the efficacy of cancer immunotherapy.

Human testis-specific Y-encoded protein-like protein 5 is a histone H3/H4-specific chaperone that facilitates histone deposition in vitro.

DNA and core histones are hierarchically packaged into a complex organization called chromatin. The nucleosome assembly protein (NAP) family of histone chaperones is involved in the deposition of histone complexes H2A/H2B and H3/H4 onto DNA and prevents nonspecific aggregation of histones. Testis-specific Y-encoded protein (TSPY)-like protein 5 (TSPYL5) is a member of the TSPY-like protein family, which has been previously reported to interact with ubiquitin-specific protease USP7 and regulate cell proliferation and is thus implicated in various cancers, but its interaction with chromatin has not been investigated. In this study, we characterized the chromatin association of TSPYL5 and found that it preferentially binds histone H3/H4 via its C-terminal NAP-like domain both in vitro and ex vivo. We identified the critical residues involved in the TSPYL5-H3/H4 interaction and further quantified the binding affinity of TSPYL5 toward H3/H4 using biolayer interferometry. We then determined the binding stoichiometry of the TSPYL5-H3/H4 complex in vitro using a chemical cross-linking assay and size-exclusion chromatography coupled with multiangle laser light scattering. Our results indicate that a TSPYL5 dimer binds to either two histone H3/H4 dimers or a single tetramer. We further demonstrated that TSPYL5 has a specific affinity toward longer DNA fragments and that the same histone-binding residues are also critically involved in its DNA binding. Finally, employing histone deposition and supercoiling assays, we confirmed that TSPYL5 is a histone chaperone responsible for histone H3/H4 deposition and nucleosome assembly. We conclude that TSPYL5 is likely a new member of the NAP histone chaperone family.

Epigenetic Reprogramming of the Glucose Metabolic Pathways by the Chromatin Effectors During Cancer.

Glucose metabolism plays a vital role in regulating cellular homeostasis as it acts as the central axis for energy metabolism, alteration in which may lead to serious consequences like metabolic disorders to life-threatening diseases like cancer. Malignant cells, on the other hand, help in tumor progression through abrupt cell proliferation by adapting to the changed metabolic milieu. Metabolic intermediates also vary from normal cells to cancerous ones to help the tumor manifestation. However, metabolic reprogramming is an important phenomenon of cells through which they try to maintain the balance between normal and carcinogenic outcomes. In this process, transcription factors and chromatin modifiers play an essential role to modify the chromatin landscape of important genes related directly or indirectly to metabolism. Our chapter surmises the importance of glucose metabolism and the role of metabolic intermediates in the cell. Also, we summarize the influence of histone effectors in reprogramming the cancer cell metabolism. An interesting aspect of this chapter includes the detailed methods to detect the aberrant metabolic flux, which can be instrumental for the therapeutic regimen of cancer.

Role of the Histone Acetyl Transferase MOF and the Histone Deacetylase Sirtuins in Regulation of H4K16ac During DNA Damage Repair and Metabolic Programming: Implications in Cancer and Aging.

The accurate repair of genomic damage mediated by ionizing radiation (IR), chemo- or radiomimetic drugs, or other exogenous agents, is necessary for maintenance of genome integrity, preservation of cellular viability and prevention of oncogenic transformation. Eukaryotes have conserved mechanisms designed to perceive and repair the damaged DNA quite efficiently. Among the different types of DNA damage, double strand breaks (DSB) are the most detrimental. The cellular DNA DSB response is a hierarchical signaling network that integrates damage sensing and repair with chromatin structural changes that involve a range of pre-existing and induced covalent modifications. Recent studies have revealed that pre-existing histone modifications are important contributors within this signaling/repair network. This chapter discusses the role of a critical histone acetyl transferase (HAT) known as MOF (males absent on the first) and the histone deacetylases (HDACs) Sirtuins on histone H4K16 acetylation (H4K16ac) and DNA damage repair. We also discuss the role of this important histone modification in light of metabolic rewiring and its role in regulating human pathophysiologic states.

Understanding Nutritional Knowledge and Experiences in Families With a Child Newly Diagnosed With Type 1 Diabetes.

Nutrition is an integral part of diabetes management. Caregiver nutritional knowledge has been implicated in glycemic management of youth with type 1 diabetes. This study assessed the nutritional knowledge of parents/caregivers of children newly diagnosed with type 1 diabetes. Findings suggest there is a need for more targeted support and training during the initial diagnosis and a desire for more technology-related resources such as virtual nutritionist-guided grocery shopping visits. Integrating these interventions into routine care for children with type 1 diabetes will help address the current gaps in caregiver nutritional literacy and their ability to provide appropriate care.

Cancer disparities in the context of rurality: risk factors and screening across various U.S. rural classification codes.

PURPOSE: Prior cancer research is limited by inconsistencies in defining rurality. The purpose of this study was to describe the prevalence of cancer risk factors and cancer screening behaviors across various county-based rural classification codes, including measures reflecting a continuum, to inform our understanding of cancer disparities according to the extent of rurality. METHODS: Using an ecological cross-sectional design, we examined differences in cancer risk factors and cancer screening behaviors from the Behavioral Risk Factor Surveillance System and National Health Interview Survey (2008-2013) across rural counties and between rural and urban counties using four rural-urban classification codes for counties and county-equivalents in 2013: U.S. Office of Management and Budget, National Center for Health Statistics, USDA Economic Research Service's rural-urban continuum codes, and Urban Influence Codes. RESULTS: Although a rural-to-urban gradient was not consistently evident across all classification codes, the prevalence of smoking, obesity, physical inactivity, and binge alcohol use increased (all ptrend < 0.03), while colorectal, cervical and breast cancer screening decreased (all ptrend < 0.001) with increasing rurality. Differences in the prevalence of risk factors and screening behaviors across rural areas were greater than differences between rural and urban counties for obesity (2.4% vs. 1.5%), physical activity (2.9% vs. 2.5%), binge alcohol use (3.4% vs. 0.4%), cervical cancer screening (6.8% vs. 4.0%), and colorectal cancer screening (4.4% vs. 3.8%). CONCLUSIONS: Rural cancer disparities persist across multiple rural-urban classification codes, with marked variation in cancer risk factors and screening evident within rural regions. Focusing only on a rural-urban dichotomy may not sufficiently capture subpopulations of rural residents at greater risk for cancer and cancer-related mortality.

Understanding the role of non-Watson-Crick base pairs in DNA-protein recognition: Structural and energetic aspects using crystallographic database analysis and quantum chemical calculation.

Specific recognition of DNA base sequences by proteins is vital for life-cycles of all organisms. In a large number of crystal structures of protein-DNA complexes, DNA conformation significantly deviates from the canonical B-DNA structure. A key question is whether such alternate conformations exist prior to protein binding and one is selected for complexation or the structure observed is induced by protein binding. Non-canonical base pairs, such as Hoogsteen base pairs, are often observed in crystal structures of protein-DNA complexes. We decided to explore whether the occurrence of such non-canonical base pairs in protein-DNA complexes is induced by the protein or is selected from pre-existing conformations. Detailed quantum chemical calculations with dispersion-corrected density functional theory (DFT-D) indicated that most of the non-canonical base pairs with DNA bases are stable even in the absence of the interacting amino acids. However, the G:G Hoogsteen base pair, which also appears in the telomere structure, appears to be unstable in the absence of other stabilizing agents, such as positively charged amino acids. Thus, the stability of many of the non-canonical base pair containing duplexes may be close to the canonical B-DNA structure and hence energetically accessible in the ground state; suggesting that the selection from pre-existing conformations may be an important mechanism for observed non-canonical base pairs in protein-DNA complexes.

Structural insights into histone chaperone Asf1 and its characterization from Plasmodium falciparum.

Asf1 is a highly conserved histone chaperone that regulates tightly coupled nucleosome assembly/disassembly process. We observed that Plasmodium falciparum Asf1 (PfAsf1) is ubiquitously expressed in different stages of the life cycle of the parasite. To gain further insight into its biological activity, we solved the structure of N-terminal histone chaperone domain of PfAsf1 (1-159 amino acids) by X-ray crystallography to a resolution of 2.4 Å. The structure is composed of two beta-sheet to form a beta-sandwich, which resembles an immunoglobulin-like fold. The surface-charge distribution of PfAsf1 is distinct from yAsf1 and hAsf1 although the core-structure shows significant similarity. The crystal-structure indicated that PfAsf1 may exist in a dimeric-state which was further confirmed by solution cross-linking experiment. PfAsf1 was found to specifically interact with Plasmodium histone H3 and H4 and was able to deposit H3/H4 dimer onto DNA-template to form disomes, showing its characteristic histone chaperone activity. We mapped the critical residues of PfAsf1 involved in histone H3/H4 interaction and confirmed by site-directed mutagenesis. Further analysis indicates that histone interacting surface of Asf1 is highly conserved while the dimerization interface is variable. Our results identify the role of PfAsf1 as a mediator of chromatin assembly in Plasmodium falciparum, which is the causative agent of malignant malaria in humans.

Insights on the disruption of the complex between human positive coactivator 4 and p53 by small molecules.

Interaction between human positive coactivator 4 (PC4), an abundant nuclear protein, and the tumor suppressor protein p53 plays a crucial role in initiating apoptosis. In certain neurodegenerative diseases PC4 assisted-p53-dependent apoptosis may play a central role. Thus, disruption of p53-PC4 interaction may be a good drug target for certain disease pathologies. A p53-derived short peptide (AcPep) that binds the C-terminal domain of PC4 (C-PC4) is known to disrupt PC4-p53 interaction. To fully characterize its binding mode and binding site on PC4, we co-crystallized C-PC4 with the peptide and determined its structure. The crystal, despite exhibiting mass spectrometric signature of the peptide, lacked peptide electron density and showed a novel crystal lattice, when compared to C-PC4 crystals without the peptide. Using peptide-docked models of crystal lattices, corresponding to our structure and the peptide-devoid structure we show the origin of the novel crystal lattice to be dynamically bound peptide at the previously identified putative binding site. The weak binding is proposed to be due to the lack of the N-terminal domain of PC4 (N-PC4), which we experimentally show to be disordered with no effect on PC4 stability. Taking cue from the structure, virtual screening of ∼18.6 million small molecules from the ZINC15 database was performed, followed by toxicity and binding free energy filtering. The novel crystal lattice of C-PC4 in presence of the peptide, the role of the disordered N-PC4 and the high throughput identification of potent small molecules will allow a better understanding and control of p53-PC4 interaction.

Analysis of blood stream infections, antibiograms and clinical outcomes in haematological patients with febrile neutropenia: data from a tertiary care haematology institute in India.

Timely administration of appropriate empirical antibiotics in febrile neutropenia is crucial for favourable patient outcomes. There are guidelines in place recommending such antibiotics. However, regional variations and local epidemiological data must be evaluated to tailor the antibiotics for best possible and rational use. In this study, we audited the clinical and microbiological data of febrile neutropenic episodes occurring at a tertiary care haematology institution. Three hundred and ninety-three febrile neutropenic episodes occurring in 123 patients over a 1-year period were analysed for microbial profile, sensitivity and resistance patterns, and finally clinical outcomes. Gram-negative bacilli (GNB) blood stream infections (46.9%) were more prevalent as compared to gram-positive infections (41.9%). Overall mortality due to complicated neutropenic sepsis was 19.5% (24/123 patients). Increased resistance to carbapenems, beta-lactam beta-lactamase inhibitor combinations, aminoglycosides, fluoroquinolones, and cephalosporins were observed. Cefepime and tigecycline resistance were seen in 20% and 15% GNB isolates, respectively. Chest was the most frequent focus of infection, and acute myeloid leukaemia (AML) was the most common underlying disorder which correlated with the likelihood of death (p < 0.01). Multidrug-resistant GNB (esp. Klebsiella sp.) are still most worrisome isolates in neutropenic patients. Single-agent cefepime or piperacillin-tazobactam/tigecycline combination may be considered empirical agents. Chest infections and AML were independent predictors of poor clinical outcome in neutropenic patients. Regular audit of infections and antibiotic susceptibility data is needed to improve clinical outcomes in patients with febrile neutropenia.

Systematic Review of Interventions to Increase Stool Blood Colorectal Cancer Screening in African Americans.

African Americans experience colorectal cancer (CRC) related disparities compared to other racial groups in the United States. African Americans are frequently diagnosed with CRC at a later stage, screening is underutilized, and mortality rates are highest in this group. This systematic review focused on intervention studies using stool blood CRC screening among African Americans in primary care and community settings. Given wide accessibility, low cost, and ease of dissemination of stool-based CRC screening tests, this review aims to determine effective interventions to improve participation rates. This systematic review included intervention studies published between January 1, 2000 and March 16, 2019. After reviewing an initial search of 650 studies, 11 studies were eventually included in this review. The included studies were studies conducted in community and clinical settings, using both inreach and outreach strategies to increase CRC screening. For each study, an unadjusted odds ratio (OR) for the CRC screening intervention compared to the control arm was calculated based on the data in each study to report effectiveness. The eleven studies together recruited a total of 3334 participants. The five studies using two-arm experimental designs ranged in effectiveness with ORs ranging from 1.1 to 13.0 using interventions such as mailed reminders, patient navigation, and tailored educational materials. Effective strategies to increase stool blood testing included mailed stool blood tests augmented by patient navigation, tailored educational materials, and follow-up calls or mailings to increase trust in the patient-provider relationship. More studies are needed on stool blood testing interventions to determine effectiveness in this population.

TCF19 Promotes Cell Proliferation through Binding to the Histone H3K4me3 Mark.

Transcription factor 19 (TCF19) plays critical roles in type 1 diabetes and the maintenance of pancreatic ß cells. Recent studies have also implicated TCF19 in cell proliferation of hepatic carcinoma and non-small cell lung carcinoma; however, the mechanism underlying this regulation remains elusive. At the molecular level, TCF19 contains two modules, the plant homeodomain (PHD) finger and the forkhead-associated (FHA) domain, of unclear function. Here, we show that TCF19 mediates hepatocellular carcinoma HepG2 cell proliferation through its PHD finger that recognizes trimethylated lysine 4 of histone 3 (H3K4me3). W316 of the PHD finger of TCF19 is one of the critical residues eliciting this function. Whole genome microarray analysis and orthogonal cell-based assays identified a large subset of genes involved in cell survival and proliferation that depend on TCF19. Our data suggest that TCF19 acts as a pro-oncogene in hepatocellular carcinoma cells and that its functional PHD finger is critical in cell proliferation.

The cancer-associated, gain-of-function TP53 variant P152Lp53 activates multiple signaling pathways implicated in tumorigenesis.

TP53 is the most frequently mutated tumor suppressor gene in many cancers, yet biochemical characterization of several of its reported mutations with probable biological significance have not been accomplished enough. Specifically, missense mutations in TP53 can contribute to tumorigenesis through gain-of-function of biochemical and biological properties that stimulate tumor growth. Here, we identified a relatively rare mutation leading to a proline to leucine substitution (P152L) in TP53 at the very end of its DNA-binding domain (DBD) in a sample from an Indian oral cancer patient. Although the P152Lp53 DBD alone bound to DNA, the full-length protein completely lacked binding ability at its cognate DNA motifs. Interestingly, P152Lp53 could efficiently tetramerize, and the mutation had only a limited impact on the structure and stability of full-length p53. Significantly, when we expressed this variant in a TP53-null cell line, it induced cell motility, proliferation, and invasion compared with a vector-only control. Also, enhanced tumorigenic potential was observed when P152Lp53-expressing cells were xenografted into nude mice. Investigating the effects of P152Lp53 expression on cellular pathways, we found that it is associated with up-regulation of several pathways, including cell-cell and cell-extracellular matrix signaling, epidermal growth factor receptor signaling, and Rho-GTPase signaling, commonly active in tumorigenesis and metastasis. Taken together, our findings provide a detailed account of the biochemical and cellular alterations associated with the cancer-associated P152Lp53 variant and establish it as a gain-of-function TP53 variant.

A Peptide-PNA Hybrid Beacon for Sensitive Detection of Protein Biomarkers in Biological Fluids.

Specific and rapid detection of proteins in biological fluids poses a challenging problem. In biological fluids, many proteins are present at low concentrations, requiring high affinity and specificity of the beacon-protein interaction. We report the design of a peptide-PNA hybrid beacon that exploits the dimeric nature of a target protein, S100B, a biomarker for brain trauma, to enhance binding affinity and specificity. The complementary base-pairing of the PNA bases brings the two arms of the beacon, one carrying an Alexa tag and the other carrying a Dabcyl moiety, into proximity, thus quenching Alexa fluorescence. Each of the arms carries a sequence that binds to one of the subunits. Binding to the target separates the quencher from the probe lifting the quenching of fluorescence. Enhanced affinity and specificity resulting from simultaneously binding to two sites allowed specific detection of S100B at low-nanomolar concentrations in the presence of serum. The design can be easily adapted for the detection of proteins containing multiple binding sites and could prove useful for rapid and sensitive biomarker detection.

Bi-stability of the master gene regulatory network of the common dendritic precursor cell: Implications for cell differentiation.

In cell lineage commitment decisions, a gene regulatory network (GRN) consisting of a limited number of transcription factors forms the regulatory pivot. Myeloid lineage dendritic cells or DCs are specialized cells having the antigen-presenting ability and are of immense importance in immune surveillance. In this report, we analyze the GRN that governs the lineage commitment of Common DC Progenitor (CDP) cells to conventional dendritic cells (cDC) and plasmacytoid dendritic cells (pDC). We have analyzed the quantitative behavior of the master regulatory circuit of CDP that governs the lineage commitment. Simulations showed that the GRN displays a bi-stable behavior within a range of parameter values. Several transcription factors, PU.1, IRF8, Flt3, and Stat3, whose concentrations vary significantly in the two steady states, appear to be the key players. We hypothesize that the two stable steady states are precursors of cDC and pDC, and the variation of concentration of these key transcription factors in the two states may be responsible for early events in lineage commitment.

Tumor Suppressor p53-Mediated Structural Reorganization of the Transcriptional Coactivator p300.

Transcriptional coactivator p300, a critical player in eukaryotic gene regulation, primarily functions as a histone acetyltransferase (HAT). It is also an important player in acetylation of a number of nonhistone proteins, p53 being the most prominent one. Recruitment of p300 to p53 is pivotal in the regulation of p53-dependent genes. Emerging evidence suggests that p300 adopts an active conformation upon binding to the tetrameric p53, resulting in its enhanced acetylation activity. As a modular protein, p300 consists of multiple well-defined domains, where the structured domains are interlinked with unstructured linker regions. A crystal structure of the central domain of p300 encompassing Bromo, RING, PHD, and HAT domains demonstrates a compact module, where the HAT active site stays occluded by the RING domain. However, although p300 has a significant role in mediating the transcriptional activity of p53, only a few structural details on the complex of these two full-length proteins are available. Here, we present a cryo-electron microscopy (cryo-EM) study on the p300-p53 complex. The three-dimensional cryo-EM density map of the p300-p53 complex, when compared to the cryo-EM map of free p300, revealed that substantial change in the relative arrangement of Bromo and HAT domains occurs upon complex formation, which is likely required for exposing HAT active site and subsequent acetyltransferase activity. Our observation correlates well with previous studies showing that the presence of Bromodomain is obligatory for effective acetyltransferase activity of HAT. Thus, our result sheds new light on the mechanism whereby p300, following binding with p53, gets activated.

Transcription factor 19 interacts with histone 3 lysine 4 trimethylation and controls gluconeogenesis via the nucleosome-remodeling-deacetylase complex.

Transcription factor 19 (TCF19) has been reported as a type 1 diabetes-associated locus involved in maintenance of pancreatic ß cells through a fine-tuned regulation of cell proliferation and apoptosis. TCF19 also exhibits genomic association with type 2 diabetes, although the precise molecular mechanism remains unknown. It harbors both a plant homeodomain and a forkhead-associated domain implicated in epigenetic recognition and gene regulation, a phenomenon that has remained unexplored. Here, we show that TCF19 selectively interacts with histone 3 lysine 4 trimethylation through its plant homeodomain finger. Knocking down TCF19 under high-glucose conditions affected many metabolic processes, including gluconeogenesis. We found that TCF19 overexpression represses de novo glucose production in HepG2 cells. The transcriptional repression of key genes, induced by TCF19, coincided with NuRD (nucleosome-remodeling-deacetylase) complex recruitment to the promoters of these genes. TCF19 interacted with CHD4 (chromodomain helicase DNA-binding protein 4), which is a part of the NuRD complex, in a glucose concentration-independent manner. In summary, our results show that TCF19 interacts with an active transcription mark and recruits a co-repressor complex to regulate gluconeogenic gene expression in HepG2 cells. Our study offers critical insights into the molecular mechanisms of transcriptional regulation of gluconeogenesis and into the roles of chromatin readers in metabolic homeostasis.

Evaluating Water Lead Levels During the Flint Water Crisis.

In April 2014, the drinking water source in Flint, Michigan was switched from Lake Huron water with phosphate inhibitors to Flint River water without corrosion inhibitors. The absence of corrosion control and use of a more corrosive source increased lead leaching from plumbing. Our city-wide citizen science water lead results contradicted official claims that there was no problem- our 90th percentile was 26.8 µg/L, which was almost double the Lead and Copper Rule action level of 15 µg/L. Back calculations of a LCR sampling pool with 50% lead pipes indicated an estimated 90th percentile lead value of 31.7 µg/L (±4.3 µg/L). Four subsequent sampling efforts were conducted to track reductions in water lead after the switch back to Lake Huron water and enhanced corrosion control. The incidence of water lead varied by service line material. Between August 2015 and November 2016, median water lead reduced from 3.0 to <1 µg/L for homes with copper service lines, 7.2-1.9 µg/L with galvanized service lines, and 9.9-2.3 µg/L with lead service lines. As of summer 2017, our 90th percentile of 7.9 µg/L no longer differed from official results, which indicated Flint's water lead levels were below the action level.

Enhanced basepair dynamics pre-disposes protein-assisted flips of key bases in DNA strand separation during transcription initiation.

Localized separation of strands of duplex DNA is a necessary step in many DNA-dependent processes, including transcription and replication. Little is known about how these strand separations occur. The strand-separated E.coli RNA polymerase-promoter open-complex structure showed four bases of the non-template strand, the master base -11A, -7, -6 and +2, in a flipped state and inserted into protein pockets. To explore whether any property of these bases in the duplex state pre-disposes them to flipping, NMR studies were performed on a wild-type promoter in the duplex state. Measurement of relaxation times indicates that a limited number of base pairs, including the flipped ones, have faster opening rates than the rest. Molecular dynamics studies also show an inherently high dynamic character of the -11A:T base pair in the wild-type strand-paired state. In order to explore the role of the RNA polymerase in the flipping process, we have used 2-aminopurine as a fluorescent probe. Slower kinetics of the increase of 2-aminopurine fluorescence was observed with RNA polymerases containing several mutant σ70s. This may be interpreted as the protein playing an important role in enhancing the flipping rate. These results suggest that flipping of -11A, and perhaps other flipped bases observed in the open-complex, is facilitated by its inherent proclivity to open-up with further assistance from the protein, thus leading to a strand-open state. Other DNA-based processes that require strand-separation may use similar pathways for strand separation. We conclude that not only basepair stability, but also dynamics may play an important role in the strand-separation.