Targeting the NFAT:AP-1 transcriptional complex on DNA with a small-molecule inhibitor.

The transcription factor nuclear factor of activated T cells (NFAT) has a key role in both T cell activation and tolerance and has emerged as an important target of immune modulation. NFAT directs the effector arm of the immune response in the presence of activator protein-1 (AP-1), and T cell anergy/exhaustion in the absence of AP-1. Envisioning a strategy for selective modulation of the immune response, we designed a FRET-based high-throughput screen to identify compounds that disrupt the NFAT:AP-1:DNA complex. We screened ∼202,000 small organic compounds and identified 337 candidate inhibitors. We focus here on one compound, N-(3-acetamidophenyl)-2-[5-(1H-benzimidazol-2-yl)pyridin-2-yl]sulfanylacetamide (Compound 10), which disrupts the NFAT:AP-1 interaction at the composite antigen-receptor response element-2 site without affecting the binding of NFAT or AP-1 alone to DNA. Compound 10 binds to DNA in a sequence-selective manner and inhibits the transcription of the Il2 gene and several other cyclosporin A-sensitive cytokine genes important for the effector immune response. This study provides proof-of-concept that small molecules can inhibit the assembly of specific DNA-protein complexes, and opens a potential new approach to treat human diseases where known transcription factors are deregulated.

Structure of a domain-swapped FOXP3 dimer on DNA and its function in regulatory T cells.

The transcription factor FOXP3 is essential for the suppressive function of regulatory T cells that are required for maintaining self-tolerance. We have solved the crystal structure of the FOXP3 forkhead domain as a ternary complex with the DNA-binding domain of the transcription factor NFAT1 and a DNA oligonucleotide from the interleukin-2 promoter. A striking feature of this structure is that FOXP3 forms a domain-swapped dimer that bridges two molecules of DNA. Structure-guided or autoimmune disease (IPEX)-associated mutations in the domain-swap interface diminished dimer formation by the FOXP3 forkhead domain without compromising FOXP3 DNA binding. These mutations also eliminated T cell-suppressive activity conferred by FOXP3, both in vitro and in a murine model of autoimmune diabetes in vivo. We conclude that FOXP3-mediated suppressor function requires dimerization through the forkhead domain and that mutations in the dimer interface can lead to the systemic autoimmunity observed in IPEX patients.

Promoter-associated DNA methylation & expression profiling of genes (FLT 3, EPB41L3 & SFN) in patients with oral squamous cell carcinoma in the Khasi & Jaintia population of Meghalaya, India.

Background & objectives: Oral squamous cell carcinoma is one of the most lethal forms of cancer, and its aetiology has been attributed to both genetic and epigenetic factors working in liaison to contribute to the disease. Epigenetic changes especially DNA methylation is involved in the activation or repression of gene functions. The aim of this study was to investigate the DNA methylation pattern and expression profiling of the promoter regions of FMS-related tyrosine kinase 3 (FLT3), erythrocyte membrane protein band 4.1-like 3 (EPB41L3) and stratifin (SFN) genes in oral cancer within the Khasi and Jaintia tribal population of Meghalaya in North East India. Methods: Quantitative methylation analyses of the selected genes were carried out by MassARRAY platform System, and the relative expression profiling was carried out by real-time polymerase chain reaction. Results: Quantitative methylation results indicated that the level of methylation was significantly higher (hypermethylated) for FLT3 and EPB41L3 and significantly lower (hypomethylated) for SFN in tumour tissues as compared to the adjacent paired normal tissue. Expression profiling was in concurrence with the methylation data whereby hypermethylated genes showed low mRNA level and vice versa for the hypomethylated gene. Interpretation & conclusions: The findings show that hyper- and hypomethylation of the selected genes play a potential role in oral carcinogenesis in the selected Khasi and Jaintia tribal population of Meghalaya. The methylation status of these genes has not been reported in oral cancer, so these genes may serve as promising biomarkers for oral cancer diagnosis as well as in disease monitoring.

Whole genome DNA methylation profiling of oral cancer in ethnic population of Meghalaya, North East India reveals novel genes.

Oral Squamous Cell Carcinoma (OSCC) is a serious and one of the most common and highly aggressive malignancies. Epigenetic factors such as DNA methylation have been known to be implicated in a number of cancer etiologies. The main objective of this study was to investigate physiognomies of Promoter DNA methylation patterns associated with oral cancer epigenome with special reference to the ethnic population of Meghalaya, North East India. The present study identifies 27,205 CpG sites and 3811 regions that are differentially methylated in oral cancer when compared to matched normal. 45 genes were found to be differentially methylated within the promoter region, of which 38 were hypermethylated and 7 hypomethylated. 14 of the hypermethylated genes were found to be similar to that of the TCGA-HNSCC study some of which are TSGs and few novel genes which may serve as candidate methylation biomarkers for OSCC in this poorly characterized ethnic group.

Regulatory T Cell Modulation by CBP/EP300 Bromodomain Inhibition.

Covalent modification of histones is a fundamental mechanism of regulated gene expression in eukaryotes, and interpretation of histone modifications is an essential feature of epigenetic control. Bromodomains are specialized binding modules that interact with acetylated histones, linking chromatin recognition to gene transcription. Because of their ability to function in a domain-specific fashion, selective disruption of bromodomain:acetylated histone interactions with chemical probes serves as a powerful means for understanding biological processes regulated by these chromatin adaptors. Here we describe the discovery and characterization of potent and selective small molecule inhibitors for the bromodomains of CREBBP/EP300 that engage their target in cellular assays. We use these tools to demonstrate a critical role for CREBBP/EP300 bromodomains in regulatory T cell biology. Because regulatory T cell recruitment to tumors is a major mechanism of immune evasion by cancer cells, our data highlight the importance of CREBBP/EP300 bromodomain inhibition as a novel, small molecule-based approach for cancer immunotherapy.

Suppression of TH17-mediated pathology through BET bromodomain inhibition.

Epigenetic control of gene expression is enforced in part through histone modifications. Bromodomain and extra terminal domain (BET) proteins function as crucial chromatin readers, responsible for interpretation of the chromatin code in diverse cellular contexts, ultimately impacting gene transcription. BET proteins can play a major role in inflammation by profoundly affecting the biology of the Thelper 17 (TH17) lineage. We summarize recent studies focusing on BET inhibition as a viable therapeutic alternative for the control of autoimmune diseases driven by aberrant activation of TH17 cells.

Spatial and molecular cues for cell outgrowth during C. elegans uterine development.

The Caenorhabditis elegans uterine seam cell (utse) is an H-shaped syncytium that connects the uterus to the body wall. Comprising nine nuclei that move outward in a bidirectional manner, this synctium undergoes remarkable shape change during development. Using cell ablation experiments, we show that three surrounding cell types affect utse development: the uterine toroids, the anchor cell and the sex myoblasts. The presence of the anchor cell (AC) nucleus within the utse is necessary for proper utse development and AC invasion genes fos-1, cdh-3, him-4, egl-43, zmp-1 and mig-10 promote utse cell outgrowth. Two types of uterine lumen epithelial cells, uterine toroid 1 (ut1) and uterine toroid 2 (ut2), mediate proper utse outgrowth and we show roles in utse development for two genes expressed in the uterine toroids: the RASEF ortholog rsef-1 and Trio/unc-73. The SM expressed gene unc-53/NAV regulates utse cell shape; ablation of sex myoblasts (SMs), which generate uterine and vulval muscles, cause defects in utse morphology. Our results clarify the nature of the interactions that exist between utse and surrounding tissue, identify new roles for genes involved in cell outgrowth, and present the utse as a new model system for understanding cell shape change and, putatively, diseases associated with cell shape change.

Synchronous and symmetric migration of Drosophila caudal visceral mesoderm cells requires dual input by two FGF ligands.

Caudal visceral mesoderm (CVM) cells migrate synchronously towards the anterior of the Drosophila embryo as two distinct groups located on each side of the body, in order to specify longitudinal muscles that ensheath the gut. Little is known about the molecular cues that guide cells along this path, the longest migration of embryogenesis, except that they closely associate with trunk visceral mesoderm (TVM). The expression of the fibroblast growth factor receptor (FGFR) heartless and its ligands, pyramus (pyr) and thisbe (ths), within CVM and TVM cells, respectively, suggested FGF signaling may influence CVM cell guidance. In FGF mutants, CVM cells die before reaching the anterior region of the TVM. However, an earlier phenotype observed was that the two cell clusters lose direction and converge at the midline. Live in vivo imaging and tracking analyses identified that the movements of CVM cells were slower and no longer synchronous. Moreover, CVM cells were found to cross over from one group to the other, disrupting bilateral symmetry, whereas such mixing was never observed in wild-type embryos. Ectopic expression of either Pyr or Ths was sufficient to redirect CVM cell movement, but only when the endogenous source of these ligands was absent. Collectively, our results show that FGF signaling regulates directional movement of CVM cells and that native presentation of both FGF ligands together is most effective at attracting cells. This study also has general implications, as it suggests that the activity supported by two FGF ligands in concert differs from their activities in isolation.

Genome-wide screening of aberrant DNA methylation which associated with gene expression in mouse skin cancers.

Epigenetic alteration of genomic DNA is a common and key process in carcinogenesis. There is considerable evidence indicating that some of the somatic alterations occurring during carcinogenesis in humans also involve the same processes as those observed in mice. Therefore, we analyzed mouse skin cancer tissues induced by the 2-stage carcinogenesis model to identify skin tumor-specific differentially methylated regions (ST-DMRs) during the multistep carcinogenesis process. We have previously identified ST-DMRs using the restriction landmark genomic scanning (RLGS) technique and reported that some of the mouse ST-DMRs were also epigenetically modified in human cancers, such as melanoma, neuroblastoma, and brain tumor. These results encouraged us to pursue global methylation screening in mouse skin carcinogenesis. Using the methylated DNA immunoprecipitation (MeDIP) method combined with the NimbleGen promoter plus CpG island (CpGi) array, we identified 615 ST-DMRs. In combination with global gene expression analysis, 91 of these ST-DMRs were shown to be located on or around the genes differentially expressed between normal skin and tumor tissues, including a candidate human tumor suppressor gene Tfap2e. As observed in human colorectal cancers, Tfap2e was methylated at a CpGi located in intron 3 and downregulated in skin tumors. Our results identified aberrant methylated regions that were associated with gene expression regulation during carcinogenesis, which may indicate critical genetic regions also involved in human carcinogenesis. © 2013 Wiley Periodicals, Inc.

Identification of frequent differentially methylated region in sporadic bladder cancers.

INTRODUCTION: Aberrant methylation levels in the cytosine-phosphate-guanine island (CpGi) region from exon 1 to intron 1 of the zygote arrest 1 (ZAR1) gene have been reported in several types of human cancers, including melanoma, brain tumor, and hepatocellular carcinoma. In the present study, methylation levels at the CpGi of ZAR1 exon 1/intron 1 in bladder cancer specimens were analyzed using mass spectrometry. MATERIALS AND METHODS: Genomic DNA was extracted from 20 sporadic bladder cancers, and the methylation levels at ZAR1 CpGi were quantitatively examined by the MassARRAY EpiTYPER method. RESULT: The methylation levels at specific CpG sites of the ZAR1 CpGi were significantly lower in high-grade bladder cancers than in low-grade tumors. CONCLUSIONS: The results of the present study indicated a decreased methylation level at CpG sites of ZAR1 exon 1/intron 1. CpGi could serve as a biomarker for invasive bladder cancer.

Seasonal variation of heavy metals in water and Cyprinus carpio L. from Umiam Lake reservoir of Meghalaya, India: Potential health risk assessment for human consumption.

The purpose of this study is to assess the seasonal variation of heavy metal concentration in water and fish tissues of common carp (Cyprinus carpio L.) from the Umiam Lake reservoir located in the Ri bhoi district of Meghalaya, India, and to elucidate the possible human health risk of ingesting fish captured from the contaminated lake. Results show significant (p < 0.05) seasonal differences of heavy metal concentrations in the water and different tissues of fish Cyprinus carpio L.. The total concentration of heavy metals in the water exceeds the WHO and BIS standards and thus poses a significant threat to the aquatic flora and fauna of the reservoir. The heavy metal concentrations in fish tissues were tissue-dependent, where the average concentration of heavy metals in all the tissues of Cyprinus carpio L. was in the order of Cr > Pb > Cu > Cd. In addition, the health risk assessment suggests that the heavy metals in the fish muscle from the Umiam Lake reservoir might have adverse effects on human. Therefore, the overall results of the study provide an understanding on the seasonal distribution of heavy metals in water, provide insight on their bioaccumulation in the fish tissues, and highlights the potential health risk for the local population of long-term fish consumption from Umiam Lake reservoir.

Identification of aberrant methylation regions in neuroblastoma by screening of tissue-specific differentially methylated regions.

BACKGROUND: The identification of tissue-specific differentially methylated regions (tDMRs) is key to our understanding of mammalian development. Research has indicated that tDMRs are aberrantly methylated in cancer and may affect the oncogenic process. PROCEDURE: We used the MassARRAY EpiTYPER system to determine the quantitative methylation levels of seven neuroblastomas (NBs) and two control adrenal medullas at 12 conserved tDMRs. A second sample set of 19 NBs was also analyzed. Statistical analysis was carried out to determine the relationship of the quantitative methylation levels to other prognostic factors in these sample sets. RESULTS: Screening of 12 tDMRs revealed 2 genomic regions (SLC16A5 and ZNF206) with frequent aberrant methylation patterns in NB. The methylation levels of SLC16A5 and ZNF206 were low compared to the control adrenal medullas. The SLC16A5 methylation level (cut-off point, 13.25%) was associated with age at diagnosis, disease stage, and Shimada classification but not with MYCN amplification. The ZNF206 methylation level (cut-off point, 68.80%) was associated with all of the prognostic factors analyzed. Although the methylation levels at these regions did not reach statistical significance in their association with prognosis in mono-variant analysis, patients with both hypomethylation of SLC16A5 and hypermethylation of ZNF206 had a significantly prolonged event-free survival, when these two variables were analyzed together. CONCLUSIONS: We demonstrated that two tDMRs frequently displayed altered methylation patterns in the NB genome, suggesting their distinct involvement in NB development/differentiation. The combined analysis of these two regions could serve as a diagnostic biomarker for poor clinical outcome.

Hyperactivation of nuclear factor of activated T cells 1 (NFAT1) in T cells attenuates severity of murine autoimmune encephalomyelitis.

Nuclear factor of activated T cells (NFAT) proteins are a group of Ca(2+)-regulated transcription factors residing in the cytoplasm of resting cells. Dephosphorylation by calcineurin results in nuclear translocation of NFAT and subsequent expression of target genes; rephosphorylation by kinases, including casein kinase 1 (CK1), restores NFAT to its latent state in the cytoplasm. We engineered a hyperactivable version of NFAT1 with increased affinity for calcineurin and decreased affinity for casein kinase 1. Mice expressing hyperactivable NFAT1 in their T-cell compartment exhibited a dramatically increased frequency of both IL-17- and IL-10-producing cells after differentiation under Th17 conditions-this was associated with direct binding of NFAT1 to distal regulatory regions of Il-17 and Il-10 gene loci in Th17 cells. Despite higher IL-17 production in culture, the mice were significantly less prone to myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis than controls, correlating with increased production of the immunomodulatory cytokine IL-10 and enhanced accumulation of regulatory T cells within the CNS. Thus, NFAT hyperactivation paradoxically leads to decreased susceptibility to experimental autoimmune encephalomyelitis, supporting previous observations linking defects in Ca(2+)/NFAT signaling to lymphoproliferation and autoimmune disease.

Requirement for balanced Ca/NFAT signaling in hematopoietic and embryonic development.

NFAT transcription factors are highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by the phosphatase calcineurin, NFAT proteins translocate to the nucleus, where they orchestrate developmental and activation programs in diverse cell types. NFAT is rephosphorylated and inactivated through the concerted action of at least 3 different kinases: CK1, GSK-3, and DYRK. The major docking sites for calcineurin and CK1 are strongly conserved throughout vertebrate evolution, and conversion of either the calcineurin docking site to a high-affinity version or the CK1 docking site to a low-affinity version results in generation of hyperactivable NFAT proteins that are still fully responsive to stimulation. In this study, we generated transgenic mice expressing hyperactivable versions of NFAT1 from the ROSA26 locus. We show that hyperactivable NFAT increases the expression of NFAT-dependent cytokines by differentiated T cells as expected, but exerts unexpected signal-dependent effects during T cell differentiation in the thymus, and is progressively deleterious for the development of B cells from hematopoietic stem cells. Moreover, progressively hyperactivable versions of NFAT1 are increasingly deleterious for embryonic development, particularly when normal embryos are also present in utero. Forced expression of hyperactivable NFAT1 in the developing embryo leads to mosaic expression in many tissues, and the hyperactivable proteins are barely tolerated in organs such as brain, and cardiac and skeletal muscle. Our results highlight the need for balanced Ca/NFAT signaling in hematopoietic stem cells and progenitor cells of the developing embryo, and emphasize the evolutionary importance of kinase and phosphatase docking sites in preventing inappropriate activation of NFAT.

Safety and Efficacy of Dostarlimab in Patients With Recurrent/Advanced Non-small Cell Lung Cancer: Results from Cohort E of the Phase I GARNET Trial.

BACKGROUND: Dostarlimab is an anti-programmed cell death protein-1 antibody being evaluated in recurrent/advanced solid tumors, including non-small cell lung cancer (NSCLC), in the ongoing Phase I, multi-center, open-label, 2-part (dose escalation and cohort expansion) GARNET study (NCT02715284). MATERIALS AND METHODS: Here, we report an interim analysis of patients with recurrent/advanced NSCLC who progressed following platinum-based chemotherapy. Patients received dostarlimab (500 mg IV every 3 weeks [Q3W] for Cycles 1-4, then 1000 mg Q6W) until disease progression or unacceptable toxicity for > 2 years. The primary endpoints were immune-related objective response rate (irORR) per investigator-assessed irRECIST and safety. RESULTS: As of 8, July 2019, 67 patients with recurrent/advanced NSCLC were enrolled and treated with dostarlimab; the majority had programmed death ligand 1 (PD-L1) tumor proportion score (TPS) < 1% (35.8% of patients) or PD-L1 TPS 1%-49% (29.9% of patients); 7.5% had PD-L1 TPS ≥ 50%, and 26.9% had unknown PD-L1 TPS status. Median follow-up was 13.8 months (range: 0.0-22.6). irORR was 26.9%, including 2 complete and 16 partial responses. The median duration of response of 11.6 months (range: 2.8-19.4). Responses were observed in 2 of 24 (16.7%) patients with PD-L1 TPS < 1%, 4 of 20 (20.0%) patients with PD-L1 TPS 1%-49% and 2 of 5 (40.0%) patients with PD-L1 TPS ≥ 50%. Fatigue (4.5%) was the most common Grade ≥ 3 treatment-related treatment-emergent adverse event (TRAE). Immune-related TRAEs (any grade) were observed in 28.4% of patients. CONCLUSION: Dostarlimab demonstrated promising antitumor activity in advanced/recurrent NSCLC that progressed following platinum-based chemotherapy, including across all PD-L1 subgroups, and has an acceptable safety profile.

Genome-wide survey reveals dynamic widespread tissue-specific changes in DNA methylation during development.

BACKGROUND: Changes in DNA methylation in the mammalian genome during development are frequent events and play major roles regulating gene expression and other developmental processes. It is necessary to identify these events so that we may understand how these changes affect normal development and how aberrant changes may impact disease. RESULTS: In this study Methylated DNA ImmunoPrecipitation (MeDIP) was used in conjunction with a NimbleGen promoter plus CpG island (CpGi) array to identify Tissue and Developmental Stage specific Differentially Methylated DNA Regions (T-DMRs and DS-DMRs) on a genome-wide basis. Four tissues (brain, heart, liver, and testis) from C57BL/6J mice were analyzed at three developmental stages (15 day embryo, E15; new born, NB; 12 week adult, AD). Almost 5,000 adult T-DMRs and 10,000 DS-DMRs were identified. Surprisingly, almost all DS-DMRs were tissue specific (i.e. methylated in at least one tissue and unmethylated in one or more tissues). In addition our results indicate that many DS-DMRs are methylated at early development stages (E15 and NB) but are unmethylated in adult. There is a very strong bias for testis specific methylation in non-CpGi promoter regions (94%). Although the majority of T-DMRs and DS-DMRs tended to be in non-CpGi promoter regions, a relatively large number were also located in CpGi in promoter, intragenic and intergenic regions (>15% of the 15,979 CpGi on the array). CONCLUSIONS: Our data suggests the vast majority of unique sequence DNA methylation has tissue specificity, that demethylation has a prominent role in tissue differentiation, and that DNA methylation has regulatory roles in alternative promoter selection and in non-promoter regions. Overall, our studies indicate changes in DNA methylation during development are a dynamic, widespread, and tissue-specific process involving both DNA methylation and demethylation.

Preclinical characterization of dostarlimab, a therapeutic anti-PD-1 antibody with potent activity to enhance immune function in in vitro cellular assays and in vivo animal models.

Inhibitors of programmed cell death protein 1 (PD-1) and its ligand (PD-L1) have dramatically changed the treatment landscape for patients with cancer. Clinical activity of anti-PD-(L)1 antibodies has resulted in increased median overall survival and durable responses in patients across selected tumor types. To date, 6 PD-1 and PD-L1, here collectively referred to as PD-(L)1, pathway inhibitors are approved by the US Food and Drug Administration for clinical use. The availability of multiple anti-PD-(L)1 antibodies provides treatment and dosing regimen choice for patients with cancer. Here, we describe the nonclinical characterization of dostarlimab (TSR-042), a humanized anti-PD-1 antibody, which binds with high affinity to human PD-1 and effectively inhibits its interaction with its ligands, PD-L1 and PD-L2. Dostarlimab enhanced effector T-cell functions, including cytokine production, in vitro. Since dostarlimab does not bind mouse PD-1, its single-agent antitumor activity was evaluated using humanized mouse models. In this model system, dostarlimab demonstrated antitumor activity as assessed by tumor growth inhibition, which was associated with increased infiltration of immune cells. Single-dose and 4-week repeat-dose toxicology studies in cynomolgus monkeys indicated that dostarlimab was well tolerated. In a clinical setting, based on data from the GARNET trial, dostarlimab (Jemperli) was approved for the treatment of adult patients with mismatch repair-deficient recurrent or advanced endometrial cancer that had progressed on or following prior treatment with a platinum-containing regimen. Taken together, these data demonstrate that dostarlimab is a potent anti-PD-1 receptor antagonist, with properties that support its continued clinical investigation in patients with cancer.

Tissue specific differentially methylated regions (TDMR): Changes in DNA methylation during development.

Tissue specific differentially methylated regions (TDMRs) were identified and localized in the mouse genome using second generation virtual RLGS (vRLGS). Sequenom MassARRAY quantitative methylation analysis was used to confirm and determine the fine structure of tissue specific differences in DNA methylation. TDMRs have a broad distribution of locations to intragenic and intergenic regions including both CpG islands, and non-CpG islands regions. Somewhat surprising, there is a strong bias for TDMR location in non-promoter intragenic regions. Although some TDMRs are within or close to repeat sequences, overall they are less frequently associated with repetitive elements than expected from a random distribution. Many TDMRs are methylated at early developmental stages, but unmethylated later, suggesting active or passive demethylation, or expansions of populations of cells with unmethylated TDMRs. This is notable during postnatal testis differentiation where many testis specific TDMRs become progressively "demethylated". These results suggest that methylation changes during development are dynamic, involve demethylation and methylation, and may occur at late stages of embryonic development or even postnatally.

Whole Genome DNA Methylation and Gene Expression Profiling of Oropharyngeal Cancer Patients in North-Eastern India: Identification of Epigenetically Altered Gene Expression Reveals Potential Biomarkers.

Oropharyngeal cancer is a subtype of head and neck squamous cell carcinoma that is associated with unique risk exposures like consumption of smokeless tobacco and areca nut and is highly prevalent in the northeastern region of India, especially Meghalaya. However, the underlying epigenetic and transcriptomic changes in this cancer type is yet to be delineated. We have undertaken a study on genome wide somatic alterations in the DNA methylation and transcriptome in oropharyngeal cancer patients from this region using genome wide techniques in paired tumors and adjacent normal tissues. By using integrative approaches, we have identified 194 epigenetically silenced and 241 epigenetically overexpressed genes in the tumor tissue of these patients. Pathways that are significantly enriched by these genes include the pathways of immune systems, such as the interleukin signaling pathways and Toll-like receptor signaling pathway. Also, osteoclast differentiation pathway was found to be epigenetically upregulated. The pathways enriched by the epigenetically downregulated genes were found to be predominantly those involved in xenobiotic metabolism and keratinization. Two major transcription factors - SPI1 and RUNX1 were identified as epigenetically dysregulated, which further modulates 129 downstream genes. Comparison of our observations with the head and neck cancer data from TCGA revealed distinct DNA methylation and gene expression landscapes which might be specific for oropharyngeal cancer. HPV DNA sequences were not detected in any of the tumor samples in RNA-Seq data. The results obtained in this study might provide improved understanding of the disease.

The immune checkpoint molecules PD-1, PD-L1, TIM-3 and LAG-3 in diffuse large B-cell lymphoma.

Signaling through immune checkpoint receptors may lead to T-cell exhaustion and function as immune escape mechanisms in cancer. For diffuse large B-cell lymphoma (DLBCL), the mechanistic and prognostic importance of these markers on tumor cells and the tumor microenvironment remains unclear. We determined the immunohistochemical expression of PD-1, PD-L1, TIM-3, and LAG-3 on tumor cells and on tumor infiltrating lymphocytes (TILs) among 123 DLBCL patients. TIM-3 showed positive staining on tumor cells in 39% of DLBCL cases and PD-L1 expression was noted in 15% of cases. Both PD-1 and LAG-3 were positive on tumor cells in a minority of DLBCL cases (8.3% and 7.5%, respectively), but were more widely expressed on TILs in a correlated manner. With median follow-up of 44 months (n = 70, range 5-85), 4-year progression-free survival (PFS) and overall survival (OS) rates were significantly inferior among DLBCL patients with high vs low/negative TIM-3 expression (PFS: 23% [95% CI 7% to 46%] vs 60% [95% CI 43% to 74%], respectively, P = 0.008; OS: 30% [95% CI 10% to 53%] vs 74% [95% CI 58% to 85%], respectively, P = 0.006). Differences in OS remained significant when controlling for International Prognostic Index in Cox regression analyses (HR 3.49 [95% CI 1.40-6.15], P = 0.007). In addition, we observed that co-culture of DLBCL cell lines with primed T cells in the presence of anti-LAG-3 and anti-TIM-3 induced potent dose-dependent increases in in vitro cell death via AcellaTox and IL-2 ELISA assays, suggesting potent anti-tumor activity of these compounds.