Chromatin-Modifying Factors in Zebrafish Models of Rhabdomyosarcoma and Hematopoiesis

Epigenetics, or the reversible and heritable marks of gene regulation not including DNA sequence, encompasses modifications on both the DNA and histones and is as important as the DNA sequence itself. Gene transcription, DNA repair, DNA replication, and the cell cycle are each impacted by the chromatin structure. A variety of enzymes modulate these modifications, and a suite of factors interacts with them to aid in promoting or inhibiting cellular functions. Many of these chromatin-modifying factors are deregulated in cancer, making them novel therapeutic targets. This dissertation describes the identification of an H3K9 histone methyltransferase, SUV39H1, as a suppressor of rhabdomyosarcoma formation in zebrafish. This suppressor is dependent on the methyltransferase domain of the enzyme, ruling out any scaffold effects since this enzyme is a part of a multiprotein complex. SUV39H1-overexpressing and control tumors share many of the same characteristics, including proliferation rate, muscle differentiation state, and tumor growth rate. The tumor suppressive phenotype cannot be rescued by alterations in the downstream muscle program alone. However, SUV39H1-overexpressing fish initiate fewer tumors, which results in the observed suppressive phenotype. This initiation defect occurs between 5 and 7 days of life in the zebrafish, likely by impacting cyclin B1 expression. This dissertation also describes the development of a novel F1 transgenic screening strategy in the zebrafish. This approach was utilized to screen a variety of chromatin-modifying factors for their effects on hematopoietic development. The developed strategy will have future applications as a zebrafish screening tool. Our data suggest that chromatin-modifying factors play an important role in rhabdomyosarcoma and illustrate the use of the zebrafish in discovering genes involved in tumorigenesis and hematopoiesis

Phosphatidylinositol 3-kinase pathway genomic alterations in 60,991 diverse solid tumors informs targeted therapy opportunities.

BackgroundThe phosphatidylinositol 3-kinase (PI3K) pathway is frequently altered in cancer. This report describes the landscape of PI3K alterations in solid tumors as well as co-alterations serving as potential resistance/attenuation mechanisms.MethodsConsecutive samples were analyzed in a commercial Clinical Laboratory Improvement Amendment-certified laboratory using comprehensive genomic profiling performed by next-generation sequencing (315 genes). The co-alterations evaluated included the Erb-B2 receptor tyrosine kinase 2 (ERBB2), ERBB3, ERBB4, RAS, MET proto-oncogene tyrosine kinase (MET), and mitogen-activated protein kinase kinase (MAP2K) genes as well as tumor protein 53 (TP53), estrogen receptor 1 (ESR1), and androgen receptor (AR).ResultsAlterations in any of 18 PI3K-pathway associated genes were identified in 44% of 60,991 tumors. Although single base and insertions/deletions (indels) were the most frequent alterations, copy number changes and rearrangements were identified in 11% and 0.9% of patients, respectively. Overall, the most frequently altered genes were PIK3 catalytic subunit α (PIK3CA) (13%), phosphatase and tensin homolog (PTEN) (9%), and serine/threonine kinase 11 (STK11) (5%). Tumor types that frequently harbored at least 1 PI3K alteration were uterine (77%), cervical (62%), anal (59%), and breast (58%) cancers. Alterations also were discerned frequently in tumors with carcinosarcoma (89%) and squamous cell carcinoma (62%) histologies. Tumors with a greater likelihood of co-occurring PI3K pathway and MAPK pathway alterations included colorectal cancers (odds ratio [OR], 1.64; P < .001), mesotheliomas (OR, 2.67; P = .024), anal cancers (OR, 1.98; P = .03), and nonsquamous head and neck cancers (OR, 2.03; P = .019). The co-occurrence of ESR1 and/or AR alterations with PI3K alterations was statistically significant in bladder, colorectal, uterine, prostate, and unknown primary cancers.ConclusionsComprehensive genomic profiling reveals altered PI3K-related genes in 44% of solid malignancies, including rare disease and histology types. The frequency of alterations and the co-occurrence of resistance pathways vary by tumor type, directly affecting opportunities for targeted therapy

Pan-squamous genomic profiling stratified by anatomic tumor site and viral association

Background: Squamous cell carcinomas (SCC) have diverse anatomic etiologies but may share common genomic biomarkers. We profiled 7,871 unique SCCs across nine anatomic sites to investigate commonality in genomic alterations (GA), tumor mutational burden (TMB), human papillomavirus (HPV) association, and mutational signatures. Methods: Tissue from over 8,100 unique SCC samples originating from nine anatomic sites (anogenital (anus, cervix, penis, vagina, vulva), esophagus, head and neck, lung, and skin) were sequenced by hybrid capture-based comprehensive genomic profiling to evaluate GA and TMB. About 3% of non-cutaneous SCC samples had UV signatures, indicative of potential primary site misdiagnoses, and were filtered from the analysis. Detection of HPV, including high-risk strains 16, 18, 31, 33, and 45, was implemented through de novo assembly of non-human sequencing reads and BLASTn comparison against all viral nucleotide sequences in the NCBI database. Results: The proportion of HPV+ patients by anatomic site varied, with the highest being anal (91%) and cervical (83%). The mutational landscape of each cohort was similar, regardless of anatomic origin, but clustered based on HPV status. The largest differences in GA frequency as stratified by HPV- vs. HPV+ were TP53 (87% vs. 12%), CDKN2A (45% vs. 6%), and PIK3CA (22% vs. 33%). The median TMB in cases originating from HPV-associated sites was similar, regardless of HPV status. Higher median TMB was observed in lung and skin cases, which exhibited significant enrichment of mutational signatures indicative of tobacco- and UV-induced DNA damage, respectively. Conclusions: HPV+ and HPV- SCC populations have distinct genomic profiles and, for the latter, anatomic site is correlated with TMB distribution, secondary to associated carcinogen exposure. As such, biomarkers such as TMB and UV signature can provide unexpected insight into site of origin misdiagnoses and may correlate with benefit from immune checkpoint inhibitors

The Histone Methyltransferase SUV39H1 Suppresses Embryonal Rhabdomyosarcoma Formation in Zebrafish

Epigenetics, or the reversible and heritable marks of gene regulation not including DNA sequence, encompasses chromatin modifications on both the DNA and histones and is as important as the DNA sequence itself. Chromatin-modifying factors are playing an increasingly important role in tumorigenesis, particularly among pediatric rhabdomyosarcomas (RMS), revealing potential novel therapeutic targets. We performed an overexpression screen of chromatin-modifying factors in a KRASG12D-driven zebrafish model for RMS. Here, we describe the identification of a histone H3 lysine 9 histone methyltransferase, SUV39H1, as a suppressor of embryonal RMS formation in zebrafish. This suppression is specific to the histone methyltransferase activity of SUV39H1, as point mutations in the SET domain lacked the effect. SUV39H1-overexpressing and control tumors have a similar proliferation rate, muscle differentiation state, and tumor growth rate. Strikingly, SUV39H1-overexpressing fish initiate fewer tumors, which results in the observed suppressive phenotype. We demonstrate that the delayed tumor onset occurs between 5 and 7 days post fertilization. Gene expression profiling at these stages revealed that in the context of KRASG12D overexpression, SUV39H1 may suppress cell cycle progression. Our studies provide evidence for the role of SUV39H1 as a tumor suppressor

A Fungal Glycosphingolipid Directly Activates Natural Killer T Cells and Rapidly Induces Airways Disease

Aspergillus fumigatusis a saprophytic fungus that is ubiquitous in the environment and commonly associated with allergic sensitization and severe asthma in humans. Although A. fumigatus is recognized by multiple microbial pattern recognition receptors, we identified and synthesized an A. fumigatus glycosphingolipid, asperamide B, that directly activated invariant natural killer T (iNKT) cells in vitro in a CD1d-restricted, MyD88- and dectin-1-independent fashion. Moreover, asperamide B, when loaded into CD1d, directly stained, and was sufficient to activate, iNKT cells. In vivo, asperamide B rapidly induced airway hyperreactivity, a cardinal feature of asthma, by activating pulmonary iNKT cells in an IL-33-ST2-dependent fashion. Asperamide B is thus the first fungal glycolipid found to directly activate iNKT cells. These results extend the range of microorganisms that can be directly detected by iNKT cells to the Kingdom of Fungi, and may explain the effectiveness of A. fumigatus in causing severe chronic respiratory diseases in humans

Tissue expression of PD-L1 mediates peripheral T cell tolerance

Programmed death 1 (PD-1), an inhibitory receptor expressed on activated lymphocytes, regulates tolerance and autoimmunity. PD-1 has two ligands: PD-1 ligand 1 (PD-L1), which is expressed broadly on hematopoietic and parenchymal cells, including pancreatic islet cells; and PD-L2, which is restricted to macrophages and dendritic cells. To investigate whether PD-L1 and PD-L2 have synergistic or unique roles in regulating T cell activation and tolerance, we generated mice lacking PD-L1 and PD-L2 (PD-L1/PD-L2−/− mice) and compared them to mice lacking either PD-L. PD-L1 and PD-L2 have overlapping functions in inhibiting interleukin-2 and interferon-γ production during T cell activation. However, PD-L1 has a unique and critical role in controlling self-reactive T cells in the pancreas. Our studies with bone marrow chimeras demonstrate that PD-L1/PD-L2 expression only on antigen-presenting cells is insufficient to prevent the early onset diabetes that develops in PD-L1/PD-L2−/− non-obese diabetic mice. PD-L1 expression in islets protects against immunopathology after transplantation of syngeneic islets into diabetic recipients. PD-L1 inhibits pathogenic self-reactive CD4+ T cell–mediated tissue destruction and effector cytokine production. These data provide evidence that PD-L1 expression on parenchymal cells rather than hematopoietic cells protects against autoimmune diabetes and point to a novel role for PD-1–PD-L1 interactions in mediating tissue tolerance

Fungal vaccines and immunotherapeutics: current concepts and future challenges

Purpose of review The remarkable advances in modern medicine have paradoxically resulted in a rapidly expanding population of immunocompromised patients displaying extreme susceptibility to life-threatening fungal infections. There are currently no licensed vaccines, and the prophylaxis and therapy of fungal infections in at-risk individuals remains challenging, contributing to undesirable mortality and morbidity rates. The design of successful antifungal preventive approaches has been hampered by an insufficient understanding of the dynamics of the host-fungus interaction and the mechanisms that underlie heterogenous immune responses to vaccines and immunotherapy. Recent findings Recent advances in proteomics and glycomics have contributed to the identification of candidate antigens for use in subunit vaccines, novel adjuvants, and delivery systems to boost the efficacy of protective vaccination responses that are becoming available, and several targets are being exploited in immunotherapeutic approaches. Summary We review some of the emerging concepts as well as the inherent challenges to the development of fungal vaccines and immunotherapies to protect at-risk individuals.ThisworkwassupportedbytheNorthernPortugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) (NORTE-01-0145-FEDER-000013), and the Fundação para a Ciência e Tecnologia (FCT) (contracts IF/00735/ 2014 to A.C., and SFRH/BPD/96176/2013 to C.C).info:eu-repo/semantics/publishedVersio