The genomic and evolutionary landscapes of anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma is arguably the most lethal human malignancy. It often co-occurs with differentiated thyroid cancers, yet the molecular origins of its aggressivity are unknown. We sequenced tumor DNA from 329 regions of thyroid cancer, including 213 from patients with primary anaplastic thyroid carcinomas. We also whole genome sequenced 9 patients using multi-region sequencing of both differentiated and anaplastic thyroid cancer components. Using these data, we demonstrate thatanaplastic thyroid carcinomas have a higher burden of mutations than other thyroid cancers, with distinct mutational signatures and molecular subtypes. Further, different cancer driver genes are mutated in anaplastic and differentiated thyroid carcinomas, even those arising in a single patient. Finally, we unambiguously demonstrate that anaplastic thyroid carcinomas share a genomic origin with co-occurring differentiated carcinomas and emerge from a common malignant field through acquisition of characteristic clonal driver mutations.

F4/80+Ly6Chigh Macrophages Lead to Cell Plasticity and Cancer Initiation in Colitis.

BACKGROUND & AIMS: Colorectal cancer is a leading cause of cancer death, and a major risk factor is chronic inflammation. Despite the link between colitis and cancer, the mechanism by which inflammation leads to colorectal cancer is not well understood. METHODS: To investigate whether different forms of inflammation pose the same risk of cancer, we compared several murine models of colitis (dextran sodium sulfate [DSS], 2,4,6-trinitrobenzene sulfonic acid, 4-ethoxylmethylene-2-phenyloxazol-5-one, Citrobacter rodentium, Fusobacterium nucleatum, and doxorubicin) with respect to their ability to lead to colonic tumorigenesis. We attempted to correlate the severity of colitis and inflammatory profile with the risk of tumorigenesis in both azoxymethane-dependent and Dclk1/APCfl/fl murine models of colitis-associated cancer. RESULTS: DSS colitis reproducibly led to colonic tumors in both mouse models of colitis-associated cancer. In contrast, all other forms of colitis did not lead to cancer. When compared with the colitis not associated with tumorigenesis, DSS colitis was characterized by significantly increased CD11b+F4/80+Ly6Chigh macrophages and CD11b+Ly6G+ neutrophils. Interestingly, depletion of the CD11b+F4/80+Ly6Chigh macrophages inhibited tumorigenesis, whereas depletion of CD11b+Ly6G+ neutrophils had no effect on tumorigenesis. Furthermore, the macrophage-derived cytokines interleukin-1ß, tumor necrosis factor-α, and interleukin-6 were significantly increased in DSS colitis and promoted stemness of Dclk1+ tuft cells that serve as the cellular origin of cancer. CONCLUSIONS: We have identified CD11b+F4/80+Ly6Chigh macrophages as key mediators of cancer initiation in colitis-associated cancer. Development of new therapies that target these cells may provide an effective preventative strategy for colitis-associated cancer.

Immune-based classification of HPV-associated oropharyngeal cancer with implications for biomarker-driven treatment de-intensification.

BACKGROUND: There is significant interest in treatment de-escalation for human papillomavirus-associated (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) patients given the generally favourable prognosis. However, 15-30% of patients recur after primary treatment, reflecting a need for improved risk-stratification tools. We sought to develop a molecular test to risk stratify HPV+ OPSCC patients. METHODS: We created an immune score (UWO3) associated with survival outcomes in six independent cohorts comprising 906 patients, including blinded retrospective and prospective external validations. Two aggressive radiation de-escalation cohorts were used to assess the ability of UWO3 to identify patients who recur. Multivariate Cox models were used to assess the associations between the UWO3 immune class and outcomes. FINDINGS: A three-gene immune score classified patients into three immune classes (immune rich, mixed, or immune desert) and was strongly associated with disease-free survival in six datasets, including large retrospective and prospective datasets. Pooled analysis demonstrated that the immune rich group had superior disease-free survival compared to the immune desert (HR = 9.0, 95% CI: 3.2-25.5, P = 3.6 × 10-5) and mixed (HR = 6.4, 95% CI: 2.2-18.7, P = 0.006) groups after adjusting for age, sex, smoking status, and AJCC8 clinical stage. Finally, UWO3 was able to identify patients from two small treatment de-escalation cohorts who remain disease-free after aggressive de-escalation to 30 Gy radiation. INTERPRETATION: With additional prospective validation, the UWO3 score could enable biomarker-driven clinical decision-making for patients with HPV+ OPSCC based on robust outcome prediction across six independent cohorts. Prospective de-escalation and intensification clinical trials are currently being planned. FUNDING: CIHR, European Union, and the NIH.

Dysregulated transforming growth factor-beta mediates early bone marrow dysfunction in diabetes.

Diabetes affects select organs such as the eyes, kidney, heart, and brain. Our recent studies show that diabetes also enhances adipogenesis in the bone marrow and reduces the number of marrow-resident vascular regenerative stem cells. In the current study, we have performed a detailed spatio-temporal examination to identify the early changes that are induced by diabetes in the bone marrow. Here we show that short-term diabetes causes structural and molecular changes in the marrow, including enhanced adipogenesis in tibiae of mice, prior to stem cell depletion. This enhanced adipogenesis was associated with suppressed transforming growth factor-beta (TGFB) signaling. Using human bone marrow-derived mesenchymal progenitor cells, we show that TGFB pathway suppresses adipogenic differentiation through TGFB-activated kinase 1 (TAK1). These findings may inform the development of novel therapeutic targets for patients with diabetes to restore regenerative stem cell function.

Practice guidelines for BRCA1/2 tumour testing in ovarian cancer.

The purpose of this document is to provide pre-analytical, analytical and post-analytical considerations and recommendations to Canadian clinical laboratories developing, validating and offering next-generation sequencing (NGS)-based BRCA1 and BRCA2 (BRCA1/2) tumour testing in ovarian cancers. This document was drafted by the members of the Canadian College of Medical Geneticists (CCMG) somatic BRCA Ad Hoc Working Group, and representatives from the Canadian Association of Pathologists. The document was circulated to the CCMG members for comment. Following incorporation of feedback, this document has been approved by the CCMG board of directors. The CCMG is a Canadian organisation responsible for certifying medical geneticists and clinical laboratory geneticists, and for establishing professional and ethical standards for clinical genetics services in Canada. The current CCMG Practice Guidelines were developed as a resource for clinical laboratories in Canada; however, they are not inclusive of all information laboratories should consider in the validation and use of NGS for BRCA1/2 tumour testing in ovarian cancers.

CAP-ACP Workload Model for Advanced Diagnostics in Precision Medicine.

OBJECTIVES: In precision medicine, where oncologic management is tailored to the individual's clinical and genetic profiles, advanced diagnostic testing provides prognostic information and guides management in a growing number of malignancies. There is a need to capture the work pathologists perform to meet this demand by providing medically relevant, timely, and accurate testing results. This work includes not only direct patient consults (interpretation of results and issuing reports) but the administrative and medical oversight as well as the research needed to provide the necessary quality assurance, quality control, direction, and framework for the laboratory. METHODS: An expert panel of Canadian pathologists involved in advanced diagnostics was convened to establish and beta test a model for workload assessment in advanced diagnostics. RESULTS: All aspects of the advanced diagnostics workload were detailed and applied to models based on members' experience, including medical oversight, administration, and the introduction of new testing and platforms. Models for biomarker testing were developed for simple and complex or multiplexed assays, and a detailed model was developed to assess the workload for next-generation sequencing-based assays. CONCLUSIONS: This paper provides the first detailed proposal for capturing an advanced diagnostic workload to enable appropriate pathologist allotment for performing all the steps required to run an advanced diagnostic service.

Does HPV Subtype Predict Outcomes in Head and Neck Cancers?

BACKGROUND: Recently, reanalysis of The Cancer Genome Atlas study demonstrated that human papillomavirus (HPV) genotypes in head and neck cancers other than HPV-16 have inferior survival to HPV-16-positive tumors. We aimed to examine the association of HPV subtypes and survival in a large cohort of patient samples from our institution. METHODS: Fresh frozen primary site biopsy samples were collected either in clinic or at the time of surgery. Patient demographic, staging, and survival data were also collected. Tumors were tested for HPV subtypes by quantitative polymerase chain reaction (qPCR). Univariable and multivariable analyses were performed using Cox proportional hazards regression. RESULTS: 280 patient biopsy samples were collected between 2011 and 2017. Mean ± standard deviation (SD) age was 61.9 ± 11.1 years and most patients (78%) were male. The majority of cancers were of the oral cavity (60%) or oropharynx (25%) and 30% had HPV-positive disease. Median follow-up was 3.76 years and 96/280 patients (34%) developed recurrences. Patients with p16-positive versus negative disease had significantly improved 5-year overall survival (OS, 77.6% vs. 53.3%; p=0.009) and progression-free survival (PFS, 67.3% vs. 41.0%, p=0.006). Similarly improved 5-year OS and PFS were observed for patients with HPV-positive versus negative disease (65.0% vs. 55.0%, p=0.084; 53.3% vs. 43.2%, p=0.072, resp.). Patients with HPV-16 compared to other HPV diseases had worse 5-year OS and PFS (62.1% vs. 88.9%, p=0.273; 49.0% vs. 88.9%, p=0.081, resp.). CONCLUSIONS: In contrast to the data derived from The Cancer Genome Atlas, patients with HPV-16 tumors trended towards decreased PFS and OS compared with tumors driven by other HPV genotypes. Further larger multi-institutional studies are necessary to understand the relationship between other HPV genotypes and survival in head and neck squamous cell carcinomas.

Low expression of NSD1, NSD2, and NSD3 define a subset of human papillomavirus-positive oral squamous carcinomas with unfavorable prognosis.

BACKGROUND: Frequent mutations in the nuclear receptor binding SET domain protein 1 (NSD1) gene have been observed in head and neck squamous cell carcinomas (HNSCC). NSD1 encodes a histone 3 lysine-36 methyltransferase. NSD1 mutations are correlated with improved clinical outcomes and increased sensitivity to platinum-based chemotherapy agents in human papillomavirus-negative (HPV-) tumors, despite weak T-cell infiltration. However, the role of NSD1 and related family members NSD2 and NSD3 in human papillomavirus-positive (HPV+) HNSCC is unclear. METHODS: Using data from over 500 HNSCC patients from The Cancer Genome Atlas (TCGA), we compared the relative level of mRNA expression of NSD1, NSD2, and NSD3 in HPV+ and HPV- HNSCC. Correlation analyses were performed between T-cell infiltration and the relative level of expression of NSD1, NSD2, and NSD3 mRNA in HPV+ and HPV- HNSCC. In addition, overall survival outcomes were compared for both the HPV+ and HPV- subsets of patients based on stratification by NSD1, NSD2, and NSD3 expression levels. RESULTS: Expression levels of NSD1, NSD2 or NSD3 were not correlated with altered lymphocyte infiltration in HPV+ HNSCC. More importantly, low expression of NSD1, NSD2, or NSD3 correlated with significantly reduced overall patient survival in HPV+, but not HPV- HNSCC. CONCLUSION: These results starkly illustrate the contrast in molecular features between HPV+ and HPV- HNSCC tumors and suggest that NSD1, NSD2, and NSD3 expression levels should be further investigated as novel clinical metrics for improved prognostication and patient stratification in HPV+ HNSCC.

TAM family receptors in conjunction with MAPK signalling are involved in acquired resistance to PI3Kα inhibition in head and neck squamous cell carcinoma.

BACKGROUND: Aberrant activation of the phosphatidylinositol 3-kinase (PI3K) pathway is common in many malignancies, including head and neck squamous cell carcinoma (HNSCC). Despite pre-clinical and clinical studies, outcomes from targeting the PI3K pathway have been underwhelming and the development of drug resistance poses a significant barrier to patient treatment. In the present study, we examined mechanisms of acquired resistance to the PI3Kα inhibitor alpelisib (formerly BYL719) in HNSCC cell lines and patient-derived xenografts (PDXs). METHODS: Five unique PDX mouse models and three HNSCC cell lines were used. All cell lines and xenografts underwent genomic characterization prior to study. Serial drug treatment was conducted in vitro and in vivo to develop multiple, clinically-significant models of resistance to alpelisib. We then used reverse phase protein arrays (RPPAs) to profile the expression of proteins in parental and drug-resistant models. Top hits were validated by immunoblotting and immunohistochemistry. Flow cytometric analysis and RNA interference studies were then used to interrogate the molecular mechanisms underlying acquired drug resistance. RESULTS: Prolonged treatment with alpelisib led to upregulation of TAM family receptor tyrosine kinases TYRO3 and AXL. Importantly, a significant shift in expression of both TYRO3 and AXL to the cell surface was detected in drug-resistant cells. Targeted knockdown of TYRO3 and AXL effectively re-sensitized resistant cells to PI3Kα inhibition. In vivo, resistance to alpelisib emerged following 20-35 days of treatment in all five PDX models. Elevated TYRO3 expression was detected in drug-resistant PDX tissues. Downstream of TYRO3 and AXL, we identified activation of intracellular MAPK signalling. Inhibition of MAPK signalling also re-sensitized drug-resistant cells to alpelisib. CONCLUSIONS: We have identified TYRO3 and AXL receptors to be key mediators of resistance to alpelisib, both in vitro and in vivo. Our findings suggest that pan-TAM inhibition is a promising avenue for combinatorial or second-line therapy alongside PI3Kα inhibition. These findings advance our understanding of the role TAM receptors play in modulating the response of HNSCC to PI3Kα inhibition and suggest a means to prevent, or at least delay, resistance to PI3Kα inhibition in order to improve outcomes for HNSCC patients.

Spleen tyrosine kinase expression is correlated with human papillomavirus in head and neck cancer.

OBJECTIVES: Spleen tyrosine kinase (SYK) is a promoter of cell survival in a variety of cell types, including normal and cancerous epithelial cells. We hypothesized that SYK would an important therapeutic target to inhibit for the treatment of HNSCC. MATERIALS AND METHODS: SYK protein abundance in patient tumours was evaluated. SYK protein and mRNA abundance was used to examine patient survival and human papillomavirus (HPV) status. Small-interfering RNAs and gene editing with CRISPR/Cas9 were used to evaluate SYK expression on proliferation in HNSCC cell lines. The potency of SYK inhibitor ER27319 maleate on cellular proliferation was tested using a panel of 28 HNSCC cell lines and in vivo in HNSCC patient-derived xenograft (PDX) models. RESULTS: Moderate to high protein expression of SYK was observed in 24% of patient tumors and high SYK expression was exclusively observed in HPV-positive samples (p < 0.001). SYK inhibition with RNA interference, gene editing or a SYK inhibitor (ER27319) decreased cell proliferation and migration. Treatment of PDXs with ER27319 maleate was observed to reduce tumour burden in vivo in two of three models. CONCLUSIONS: HPV-positive HNSCC harbours high SYK protein levels. We demonstrate that proliferation, migration and overall burden of these tumours can be reduced by genetic or pharmacologic inhibition of SYK. Taken together, these data establish SYK as a therapeutic target for HNSCC.

Disruption of the RICTOR/mTORC2 complex enhances the response of head and neck squamous cell carcinoma cells to PI3K inhibition.

Phosphoinositide 3-kinase (PI3K) is aberrantly activated in head and neck squamous cell carcinomas (HNSCC) and plays a pivotal role in tumorigenesis by driving Akt signaling, leading to cell survival and proliferation. Phosphorylation of Akt Thr308 by PI3K-PDK1 and Akt Ser473 by mammalian target of rapamycin complex 2 (mTORC2) activates Akt. Targeted inhibition of PI3K is a major area of preclinical and clinical investigation as it reduces Akt Thr308 phosphorylation, suppressing downstream mTORC1 activity. However, inhibition of mTORC1 releases feedback inhibition of mTORC2, resulting in a resurgence of Akt activation mediated by mTORC2. While the role of PI3K-activated Akt signaling is well established in HNSCC, the significance of mTORC2-driven Akt signaling has not been thoroughly examined. Here we explore the expression and function of mTORC2 and its obligate subunit RICTOR in HNSCC primary tumors and cell lines. We find RICTOR to be overexpressed in a subset of HNSCC tumors, including those with PIK3CA or EGFR gene amplifications. Whereas overexpression of RICTOR reduced susceptibility of HNSCC tumor cells to PI3K inhibition, genetic ablation of RICTOR using CRISPR/Cas9 sensitized cells to PI3K inhibition, as well as to EGFR inhibition and cisplatin treatment. Further, mTORC2 disruption led to reduced viability and colony forming abilities of HNSCC cells relative to their parental lines and induced loss of both activating Akt phosphorylation modifications (Thr308 and Ser473). Taken together, our findings establish RICTOR/mTORC2 as a critical oncogenic complex in HNSCC and rationalize the development of an mTORC2-specific inhibitor for use in HNSCC, either combined with agents already under investigation, or as an independent therapy.

RB1 Deletion in Retinoblastoma Protein Pathway-Disrupted Cells Results in DNA Damage and Cancer Progression.

Proliferative control in cancer cells is frequently disrupted by mutations in the retinoblastoma protein (RB) pathway. Intriguingly, RB1 mutations can arise late in tumorigenesis in cancer cells whose RB pathway is already compromised by another mutation. In this study, we present evidence for increased DNA damage and instability in cancer cells with RB pathway defects when RB1 mutations are induced. We generated isogenic RB1 mutant genotypes with CRISPR/Cas9 in a number of cell lines. Cells with even one mutant copy of RB1 have increased basal levels of DNA damage and increased mitotic errors. Elevated levels of reactive oxygen species as well as impaired homologous recombination repair underlie this DNA damage. When xenografted into immunocompromised mice, RB1 mutant cells exhibit an elevated propensity to seed new tumors in recipient lungs. This study offers evidence that late-arising RB1 mutations can facilitate genome instability and cancer progression that are beyond the preexisting proliferative control deficit.

The Loss of ATRX Increases Susceptibility to Pancreatic Injury and Oncogenic KRAS in Female But Not Male Mice.

Background: Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in North America, accounting for >30,000 deaths annually. Although somatic activating mutations in KRAS appear in 97% of PDAC patients, additional factors are required to initiate PDAC. Because mutations in genes encoding chromatin remodelling proteins have been implicated in KRAS-mediated PDAC, we investigated whether loss of chromatin remodeler ɑ-thalassemia, mental-retardation, X-linked (ATRX) affects oncogenic KRAS's ability to promote PDAC. ATRX affects DNA replication, repair, and gene expression and is implicated in other cancers including glioblastomas and pancreatic neuroendocrine tumors. The hypothesis was that deletion of Atrx in pancreatic acinar cells will increase susceptibility to injury and oncogenic KRAS. Methods: Mice allowing conditional loss of Atrx within pancreatic acinar cells were examined after induction of recurrent cerulein-induced pancreatitis or oncogenic KRAS (KRASG12D ). Histologic, biochemical, and molecular analysis examined pancreatic pathologies up to 2 months after induction of Atrx deletion. Results: Mice lacking Atrx showed more progressive damage, inflammation, and acinar-to-duct cell metaplasia in response to injury relative to wild-type mice. In combination with KRASG12D, Atrx-deficient acinar cells showed increased fibrosis, inflammation, progression to acinar-to-duct cell metaplasia, and pre-cancerous lesions relative to mice expressing only KRASG12D. This sensitivity appears only in female mice, mimicking a significant prevalence of ATRX mutations in human female PDAC patients. Conclusions: Our results indicate the absence of ATRX increases sensitivity to injury and oncogenic KRAS only in female mice. This is an instance of a sex-specific mutation that enhances oncogenic KRAS's ability to promote pancreatic intraepithelial lesion formation.

Comprehensive gene expression profiling identifies distinct and overlapping transcriptional profiles in non-specific interstitial pneumonia and idiopathic pulmonary fibrosis.

BACKGROUND: The clinical-radiographic distinction between idiopathic pulmonary fibrosis (IPF) and non-specific interstitial pneumonia (NSIP) is challenging. We sought to investigate the gene expression profiles of IPF and NSIP vs. normal controls. METHODS: Gene expression from explanted lungs of patients with IPF (n = 22), NSIP (n = 10) and from normal controls (n = 11) was assessed. Microarray analysis included Significance Analysis of Microarray (SAM), Ingenuity Pathway, Gene-Set Enrichment and unsupervised hierarchical clustering analyses. Immunohistochemistry and serology of proteins of interest were conducted. RESULTS: NSIP cases were significantly enriched for genes related to mechanisms of immune reaction, such as T-cell response and recruitment of leukocytes into the lung compartment. In IPF, in contrast, these involved senescence, epithelial-to-mesenchymal transition, myofibroblast differentiation and collagen deposition. Unlike the IPF group, NSIP cases exhibited a strikingly homogenous gene signature. Clustering analysis identified a subgroup of IPF patients with intermediate and ambiguous expression of SAM-selected genes, with the interesting upregulation of both NSIP-specific and senescence-related genes. Immunohistochemistry for p16, a senescence marker, on fibroblasts differentiated most IPF cases from NSIP. Serial serum levels of periostin, a senescence effector, predicted clinical progression in a cohort of patients with IPF. CONCLUSIONS: Comprehensive gene expression profiling in explanted lungs identifies distinct transcriptional profiles and differentially expressed genes in IPF and NSIP, supporting the notion of NSIP as a standalone condition. Potential gene and protein markers to discriminate IPF from NSIP were identified, with a prominent role of senescence in IPF. The finding of a subgroup of IPF patients with transcriptional features of both NSIP and senescence raises the hypothesis that "senescent" NSIP may represent a risk factor to develop superimposed IPF.

Immunohistochemical Detection of the Retinoblastoma Protein.

The retinoblastoma protein (pRB) plays a key role in proliferative control and genome stability. For these reasons its functions are considered to be tumor suppressive. Its functional status offers critical insight into proliferative control signaling in tissues and in developing malignancies. In this chapter, we outline basic procedures to detect the retinoblastoma protein in formalin fixed, paraffin embedded tissue sections. In addition, we provide protocols to detect phosphorylation levels of pRB in tissues and offer controls to ensure fidelity of measurement. Importantly, these staining methods utilize broadly available reagents and equipment making them accessible to most biomedical research laboratories.

Activating transcription factor 3 promotes loss of the acinar cell phenotype in response to cerulein-induced pancreatitis in mice.

Pancreatitis is a debilitating disease of the exocrine pancreas that, under chronic conditions, is a major susceptibility factor for pancreatic ductal adenocarcinoma (PDAC). Although down-regulation of genes that promote the mature acinar cell fate is required to reduce injury associated with pancreatitis, the factors that promote this repression are unknown. Activating transcription factor 3 (ATF3) is a key mediator of the unfolded protein response, a pathway rapidly activated during pancreatic insult. Using chromatin immunoprecipitation followed by next-generation sequencing, we show that ATF3 is bound to the transcriptional regulatory regions of >30% of differentially expressed genes during the initiation of pancreatitis. Of importance, ATF3-dependent regulation of these genes was observed only upon induction of pancreatitis, with pathways involved in inflammation, acinar cell differentiation, and cell junctions being specifically targeted. Characterizing expression of transcription factors that affect acinar cell differentiation suggested that acinar cells lacking ATF3 maintain a mature cell phenotype during pancreatitis, a finding supported by maintenance of junctional proteins and polarity markers. As a result, Atf3-/- pancreatic tissue displayed increased tissue damage and inflammatory cell infiltration at early time points during injury but, at later time points, showed reduced acinar-to-duct cell metaplasia. Thus our results reveal a critical role for ATF3 as a key regulator of the acinar cell transcriptional response during injury and may provide a link between chronic pancreatitis and PDAC.

Impaired H3K36 methylation defines a subset of head and neck squamous cell carcinomas.

Human papillomavirus (HPV)-negative head and neck squamous cell carcinomas (HNSCCs) are deadly and common cancers. Recent genomic studies implicate multiple genetic pathways, including cell signaling, cell cycle and immune evasion, in their development. Here we analyze public data sets and uncover a previously unappreciated role of epigenome deregulation in the genesis of 13% of HPV-negative HNSCCs. Specifically, we identify novel recurrent mutations encoding p.Lys36Met (K36M) alterations in multiple H3 histone genes. histones. We further validate the presence of these alterations in multiple independent HNSCC data sets and show that, along with previously described NSD1 mutations, they correspond to a specific DNA methylation cluster. The K36M substitution and NSD1 defects converge on altering methylation of histone H3 at K36 (H3K36), subsequently blocking cellular differentiation and promoting oncogenesis. Our data further indicate limited redundancy for NSD family members in HPV-negative HNSCCs and suggest a potential role for impaired H3K36 methylation in their development. Further investigation of drugs targeting chromatin regulators is warranted in HPV-negative HNSCCs driven by aberrant H3K36 methylation.

An RB-EZH2 Complex Mediates Silencing of Repetitive DNA Sequences.

Repetitive genomic regions include tandem sequence repeats and interspersed repeats, such as endogenous retroviruses and LINE-1 elements. Repressive heterochromatin domains silence expression of these sequences through mechanisms that remain poorly understood. Here, we present evidence that the retinoblastoma protein (pRB) utilizes a cell-cycle-independent interaction with E2F1 to recruit enhancer of zeste homolog 2 (EZH2) to diverse repeat sequences. These include simple repeats, satellites, LINEs, and endogenous retroviruses as well as transposon fragments. We generated a mutant mouse strain carrying an F832A mutation in Rb1 that is defective for recruitment to repetitive sequences. Loss of pRB-EZH2 complexes from repeats disperses H3K27me3 from these genomic locations and permits repeat expression. Consistent with maintenance of H3K27me3 at the Hox clusters, these mice are developmentally normal. However, susceptibility to lymphoma suggests that pRB-EZH2 recruitment to repetitive elements may be cancer relevant.

Lack of matrix metalloproteinase 3 in mouse models of lung injury ameliorates the pulmonary inflammatory response in female but not in male mice.

BACKGROUND: The acute respiratory distress syndrome (ARDS) is a complex pulmonary disorder in which the local release of cytokines and chemokines appears central to the pathophysiology. OBJECTIVE: Based on the known role of matrix metalloproteinase-3 (MMP3) in inflammatory processes, the objective was to examine the role of MMP3 in the pathogenesis of ARDS through the modulation of pulmonary inflammation. MATERIALS AND METHODS: Female and male, wild type (MMP3+/+) and knock out (MMP3-/-) mice were exposed to two, clinically relevant models of ARDS including (i) lipopolysaccharide (LPS)-induced lung injury, and (ii) hydrochloric acid-induced lung injury. Parameters of lung injury and inflammation were assessed through measurements in lung lavage including total protein content, inflammatory cell influx, and concentrations of mediators such as TNF-α, IL-6, G-CSF, CXCL1, CXCL2, and CCL2. Lung histology and compliance were also evaluated in the LPS model of injury. RESULTS: Following intra-tracheal LPS instillation, all mice developed lung injury, as measured by an increase in lavage neutrophils, and decrease in lung compliance, with no overall effect of genotype observed. Increased concentrations of lavage inflammatory cytokines and chemokines were also observed following LPS injury, however, LPS-instilled female MMP3-/- mice had lower levels of inflammatory mediators compared to LPS-instilled female MMP3+/+ mice. This effect of the genotype was not observed in male mice. Similar findings, including the MMP3-related sex differences, were also observed after acid-induced lung injury. CONCLUSION: MMP3 contributes to the pathogenesis of ARDS, by affecting the pulmonary inflammatory response in female mice in relevant models of lung injury.