METTL16 promotes liver cancer stem cell self-renewal via controlling ribosome biogenesis and mRNA translation.

BACKGROUND: While liver cancer stem cells (CSCs) play a crucial role in hepatocellular carcinoma (HCC) initiation, progression, recurrence, and treatment resistance, the mechanism underlying liver CSC self-renewal remains elusive. We aim to characterize the role of Methyltransferase 16 (METTL16), a recently identified RNA N6-methyladenosine (m6A) methyltransferase, in HCC development/maintenance, CSC stemness, as well as normal hepatogenesis. METHODS: Liver-specific Mettl16 conditional KO (cKO) mice were generated to assess its role in HCC pathogenesis and normal hepatogenesis. Hydrodynamic tail-vein injection (HDTVi)-induced de novo hepatocarcinogenesis and xenograft models were utilized to determine the role of METTL16 in HCC initiation and progression. A limiting dilution assay was utilized to evaluate CSC frequency. Functionally essential targets were revealed via integrative analysis of multi-omics data, including RNA-seq, RNA immunoprecipitation (RIP)-seq, and ribosome profiling. RESULTS: METTL16 is highly expressed in liver CSCs and its depletion dramatically decreased CSC frequency in vitro and in vivo. Mettl16 KO significantly attenuated HCC initiation and progression, yet only slightly influenced normal hepatogenesis. Mechanistic studies, including high-throughput sequencing, unveiled METTL16 as a key regulator of ribosomal RNA (rRNA) maturation and mRNA translation and identified eukaryotic translation initiation factor 3 subunit a (eIF3a) transcript as a bona-fide target of METTL16 in HCC. In addition, the functionally essential regions of METTL16 were revealed by CRISPR gene tiling scan, which will pave the way for the development of potential inhibitor(s). CONCLUSIONS: Our findings highlight the crucial oncogenic role of METTL16 in promoting HCC pathogenesis and enhancing liver CSC self-renewal through augmenting mRNA translation efficiency.

RNA m6A reader YTHDF2 facilitates precursor miR-126 maturation to promote acute myeloid leukemia progression.

As the most common internal modification of mRNA, N6-methyladenosine (m6A) and its regulators modulate gene expression and play critical roles in various biological and pathological processes including tumorigenesis. It was reported previously that m6A methyltransferase (writer), methyltransferase-like 3 (METTL3) adds m6A in primary microRNAs (pri-miRNAs) and facilitates its processing into precursor miRNAs (pre-miRNAs). However, it is unknown whether m6A modification also plays a role in the maturation process of pre-miRNAs and (if so) whether such a function contributes to tumorigenesis. Here, we found that YTHDF2 is aberrantly overexpressed in acute myeloid leukemia (AML) patients, especially in relapsed patients, and plays an oncogenic role in AML. Moreover, YTHDF2 promotes expression of miR-126-3p (also known as miR-126, as it is the main product of precursor miR-126 (pre-miR-126)), a miRNA that was reported as an oncomiRNA in AML, through facilitating the processing of pre-miR-126 into mature miR-126. Mechanistically, YTHDF2 recognizes m6A modification in pre-miR-126 and recruits AGO2, a regulator of pre-miRNA processing, to promote the maturation of pre-miR-126. YTHDF2 positively and negatively correlates with miR-126 and miR-126's downstream target genes, respectively, in AML patients, and forced expression of miR-126 could largely rescue YTHDF2/Ythdf2 depletion-mediated suppression on AML cell growth/proliferation and leukemogenesis, indicating that miR-126 is a functionally important target of YTHDF2 in AML. Overall, our studies not only reveal a previously unappreciated YTHDF2/miR-126 axis in AML and highlight the therapeutic potential of targeting this axis for AML treatment, but also suggest that m6A plays a role in pre-miRNA processing that contributes to tumorigenesis.

TET2-mediated mRNA demethylation regulates leukemia stem cell homing and self-renewal.

TET2 is recurrently mutated in acute myeloid leukemia (AML) and its deficiency promotes leukemogenesis (driven by aggressive oncogenic mutations) and enhances leukemia stem cell (LSC) self-renewal. However, the underlying cellular/molecular mechanisms have yet to be fully understood. Here, we show that Tet2 deficiency significantly facilitates leukemogenesis in various AML models (mediated by aggressive or less aggressive mutations) through promoting homing of LSCs into bone marrow (BM) niche to increase their self-renewal/proliferation. TET2 deficiency in AML blast cells increases expression of Tetraspanin 13 (TSPAN13) and thereby activates the CXCR4/CXCL12 signaling, leading to increased homing/migration of LSCs into BM niche. Mechanistically, TET2 deficiency results in the accumulation of methyl-5-cytosine (m5C) modification in TSPAN13 mRNA; YBX1 specifically recognizes the m5C modification and increases the stability and expression of TSPAN13 transcripts. Collectively, our studies reveal the functional importance of TET2 in leukemogenesis, leukemic blast cell migration/homing, and LSC self-renewal as an mRNA m5C demethylase.

QKI shuttles internal m7G-modified transcripts into stress granules and modulates mRNA metabolism.

N7-methylguanosine (m7G) modification, routinely occurring at mRNA 5' cap or within tRNAs/rRNAs, also exists internally in messenger RNAs (mRNAs). Although m7G-cap is essential for pre-mRNA processing and protein synthesis, the exact role of mRNA internal m7G modification remains elusive. Here, we report that mRNA internal m7G is selectively recognized by Quaking proteins (QKIs). By transcriptome-wide profiling/mapping of internal m7G methylome and QKI-binding sites, we identified more than 1,000 high-confidence m7G-modified and QKI-bound mRNA targets with a conserved "GANGAN (N = A/C/U/G)" motif. Strikingly, QKI7 interacts (via C terminus) with the stress granule (SG) core protein G3BP1 and shuttles internal m7G-modified transcripts into SGs to regulate mRNA stability and translation under stress conditions. Specifically, QKI7 attenuates the translation efficiency of essential genes in Hippo signaling pathways to sensitize cancer cells to chemotherapy. Collectively, we characterized QKIs as mRNA internal m7G-binding proteins that modulate target mRNA metabolism and cellular drug resistance.

Barriers and shortcomings in access to cardiovascular management and prevention for familial hypercholesterolemia during the COVID-19 pandemic.

Familial hypercholesterolemia (FH) is a hereditary condition caused by mutations in the lipid pathway. The goal in managing FH is to reduce circulating low-density lipoprotein cholesterol and, therefore, reduce the risk of developing atherosclerotic cardiovascular disease (ASCVD). Because FH patients were considered high risk groups due to an increased susceptible for contracting COVID-19 infection, we hypothesized whether the effects of the pandemic hindered access to cardiovascular care. In this review, we conducted a literature search in databases Pubmed/Medline and ScienceDirect. We included a comprehensive analysis of findings from articles in English related and summarized the effects of the pandemic on cardiovascular care through direct and indirect effects. During the COVID-19 pandemic, FH patients presented with worse outcomes and prognosis, especially those that have suffered from early ASCVD. This caused avoidance in seeking care due to fear of transmission. The pandemic severely impacted consultations with lipidologists and cardiologists, causing a decline in lipid profile evaluations. Low socioeconomic communities and ethnic minorities were hit the hardest with job displacements and lacked healthcare coverage respectively, leading to treatment nonadherence. Lock-down restrictions promoted sedentary lifestyles and intake of fatty meals, but it is unclear whether these factors attenuated cardiovascular risk in FH. To prevent early atherogenesis in FH patients, universal screening programs, telemedicine, and lifestyle interventions are important recommendations that could improve outcomes in FH patients. However, the need to research in depth on the disproportionate impact within different subgroups should be the forefront of FH research.

Phosphorylation stabilized TET1 acts as an oncoprotein and therapeutic target in B cell acute lymphoblastic leukemia.

Although the overall survival rate of B cell acute lymphoblastic leukemia (B-ALL) in childhood is more than 80%, it is merely 30% in refractory/relapsed and adult patients with B-ALL. This demonstrates a need for improved therapy targeting this subgroup of B-ALL. Here, we show that the ten-eleven translocation 1 (TET1) protein, a dioxygenase involved in DNA demethylation, is overexpressed and plays a crucial oncogenic role independent of its catalytic activity in B-ALL. Consistent with its oncogenic role in B-ALL, overexpression of TET1 alone in normal precursor B cells is sufficient to transform the cells and cause B-ALL in mice within 3 to 4 months. We found that TET1 protein is stabilized and overexpressed because of its phosphorylation mediated by protein kinase C epsilon (PRKCE) and ATM serine/threonine kinase (ATM), which are also overexpressed in B-ALL. Mechanistically, TET1 recruits STAT5B to the promoters of CD72 and JCHAIN and promotes their transcription, which in turn promotes B-ALL development. Destabilization of TET1 protein by treatment with PKC or ATM inhibitors (staurosporine or AZD0156; both tested in clinical trials), or by pharmacological targeting of STAT5B, greatly decreases B-ALL cell viability and inhibits B-ALL progression in vitro and in vivo. The combination of AZD0156 with staurosporine or vincristine exhibits a synergistic effect on inhibition of refractory/relapsed B-ALL cell survival and leukemia progression in PDX models. Collectively, our study reveals an oncogenic role of the phosphorylated TET1 protein in B-ALL independent of its catalytic activity and highlights the therapeutic potential of targeting TET1 signaling for the treatment of refractory/relapsed B-ALL.

METTL16 drives leukemogenesis and leukemia stem cell self-renewal by reprogramming BCAA metabolism.

N6-methyladenosine (m6A), the most prevalent internal modification in mammalian mRNAs, is involved in many pathological processes. METTL16 is a recently identified m6A methyltransferase. However, its role in leukemia has yet to be investigated. Here, we show that METTL16 is a highly essential gene for the survival of acute myeloid leukemia (AML) cells via CRISPR-Cas9 screening and experimental validation. METTL16 is aberrantly overexpressed in human AML cells, especially in leukemia stem cells (LSCs) and leukemia-initiating cells (LICs). Genetic depletion of METTL16 dramatically suppresses AML initiation/development and maintenance and significantly attenuates LSC/LIC self-renewal, while moderately influencing normal hematopoiesis in mice. Mechanistically, METTL16 exerts its oncogenic role by promoting expression of branched-chain amino acid (BCAA) transaminase 1 (BCAT1) and BCAT2 in an m6A-dependent manner and reprogramming BCAA metabolism in AML. Collectively, our results characterize the METTL16/m6A/BCAT1-2/BCAA axis in leukemogenesis and highlight the essential role of METTL16-mediated m6A epitranscriptome and BCAA metabolism reprograming in leukemogenesis and LSC/LIC maintenance.

Clinical characteristics, risk factors and outcomes of cancer patients with COVID-19: A population-based study.

INTRODUCTION: Cancer patients may be susceptible to poorer outcomes in COVID-19 infection owing to the immunosuppressant effect of chemotherapy/radiotherapy and cancer growth, along with the potential for nosocomial transmission due to frequent hospital admissions. METHODS: This was a population-based retrospective cohort study of COVID-19 patients who presented to Hong Kong public hospitals between 1 January 2020 and 8 December 2020. The primary outcome was a composite endpoint of requirement for intubation, ICU admission and 30-day mortality. RESULTS: The following study consisted of 6089 COVID-19 patients (median age 45.9 [27.8.1-62.7] years; 50% male), of which 142 were cancer subjects. COVID-19 cancer patients were older at baseline and tended to present with a higher frequency of comorbidities, including diabetes mellitus, hypertension, chronic obstructive pulmonary disease, ischemic heart disease, ventricular tachycardia/fibrillation and gastrointestinal bleeding (p < 0.05). These subjects also likewise tended to present with higher serum levels of inflammatory markers, including D-dimer, lactate dehydrogenase, high sensitivity troponin-I and C-reactive protein. Multivariate Cox regression showed that any type of cancer presented with an almost four-fold increased risk of the primary outcome (HR: 3.77; 95% CI: 1.63-8.72; p < 0.002) after adjusting for significant demographics, Charlson comorbidity index, number of comorbidities, past comorbidities and medication history. This association remained significant when assessing those with colorectal (HR: 5.07; 95% CI: 1.50-17.17; p < 0.009) and gastrointestinal malignancies (HR: 3.79; 95% CI: 1.12-12.88; p < 0.03), but not with lung, genitourinary, or breast malignancies, relative to their respective cancer-free COVID-19 counterparts. CONCLUSIONS: COVID-19 cancer patients are associated with a significantly higher risk of intubation, ICU admission and/or mortality.

SOX9 and SOX10 control fluid homeostasis in the inner ear for hearing through independent and cooperative mechanisms.

The in vivo mechanisms underlying dominant syndromes caused by mutations in SRY-Box Transcription Factor 9 (SOX9) and SOX10 (SOXE) transcription factors, when they either are expressed alone or are coexpressed, are ill-defined. We created a mouse model for the campomelic dysplasia SOX9Y440X mutation, which truncates the transactivation domain but leaves DNA binding and dimerization intact. Here, we find that SOX9Y440X causes deafness via distinct mechanisms in the endolymphatic sac (ES)/duct and cochlea. By contrast, conditional heterozygous Sox9-null mice are normal. During the ES development of Sox9Y440X/+ heterozygotes, Sox10 and genes important for ionic homeostasis are down-regulated, and there is developmental persistence of progenitors, resulting in fewer mature cells. Sox10 heterozygous null mutants also display persistence of ES/duct progenitors. By contrast, SOX10 retains its expression in the early Sox9Y440X/+ mutant cochlea. Later, in the postnatal stria vascularis, dominant interference by SOX9Y440X is implicated in impairing the normal cooperation of SOX9 and SOX10 in repressing the expression of the water channel Aquaporin 3, thereby contributing to endolymphatic hydrops. Our study shows that for a functioning endolymphatic system in the inner ear, SOX9 regulates Sox10, and depending on the cell type and target gene, it works either independently of or cooperatively with SOX10. SOX9Y440X can interfere with the activity of both SOXE factors, exerting effects that can be classified as haploinsufficient/hypomorphic or dominant negative depending on the cell/gene context. This model of disruption of transcription factor partnerships may be applicable to congenital deafness, which affects ∼0.3% of newborns, and other syndromic disorders.

RNA N6 -methyladenosine reader YTHDC1 is essential for TGF-beta-mediated metastasis of triple negative breast cancer.

RNA N6 -methyladenosine (m6A) modification and its regulators fine tune gene expression and contribute to tumorigenesis. This study aims to uncover the essential role and the underlying molecular mechanism(s) of the m6A reader YTHDC1 in promoting triple negative breast cancer (TNBC) metastasis. METHODS: In vitro and in vivo models were employed to determine the pathological function of YTHDC1 in TNBC metastasis. To identify bona fide YTHDC1 target RNAs, we conducted RNA-seq, m6A-seq, and RIP-seq, followed by integrative data analysis and validation assays. RESULTS: By analyzing The Cancer Genome Atlas (TCGA) dataset, we found that elevated expression of YTHDC1 is positively correlated with poor prognosis in breast cancer patients. Using a mammary fat pad mouse model of TNBC, YTHDC1 significantly promoted lung metastasis of TNBC cells. Through multiple transcriptome-wide sequencing and integrative data analysis, we revealed dysregulation of metastasis-related pathways following YTHDC1 depletion and identified SMAD3 as a bona fide YTHDC1 target RNA. Depletion of YTHDC1 caused nuclear retention of SMAD3 mRNA, leading to lower SMAD3 protein levels. Loss of YTHDC1 led to impaired TGF-ß-induced gene expression, leading to inhibition of epithelial-mesenchymal transition (EMT) and suppressed TNBC cell migration and invasion. SMAD3 overexpression was able to restore the response to TGF-ß in YTHDC1 depleted TNBC cells. Furthermore, we demonstrated that the oncogenic role of YTHDC1 is mediated through its recognition of m6A as m6A-binding defective mutants of YTHDC1 were unable to rescue the impaired cell migration and invasion of YTHDC1 knockout TNBC cells. CONCLUSIONS: We show that YTHDC1 plays a critical oncogenic role in TNBC metastasis through promoting the nuclear export and expression of SMAD3 to augment the TGF-ß signaling cascade. Overall, our study demonstrates that YTHDC1 is vital for TNBC progression by enhancing TNBC cell survival and TGF-ß-mediated EMT via SMAD3 to enable the formation of distant metastasis and highlights the therapeutic potential of targeting the YTHDC1/m6A/SMAD3 axis for TNBC treatment.

Soluble suppression of tumorigenicity 2 (sST2) for predicting disease severity or mortality outcomes in cardiovascular diseases: A systematic review and meta-analysis.

OBJECTIVES: Soluble suppression of tumorigenicity 2 (sST2) is a member of the interleukin-1 receptor family. It is raised in various cardiovascular diseases, but its value in predicting disease severity or mortality outcomes has been controversial. Therefore, we conducted a systematic review and meta-analysis to determine whether sST2 levels differed between survivors and non-survivors of patients with cardiovascular diseases, and whether elevated sST2 levels correlated with adverse outcomes. METHODS: PubMed and Embase were searched until 23rd June 2021 for studies that evaluated the relationship between sST2 levels and cardiovascular disease severity or mortality. RESULTS: A total of 707 entries were retrieved from both databases, of which 14 studies were included in the final meta-analysis. In acute heart failure, sST2 levels did not differ between survivors and non-survivors (mean difference [MD]: 24.2 ± 13.0 ng/ml; P = 0.06; I 2: 95%). Elevated sST2 levels tend to be associated with increased mortality risk (hazard ratio [HR]: 1.12, 95 %CI: 0.99-1.27, P = 0.07; I 2: 88%). In chronic heart failure, sST2 levels were higher in non-survivors than in survivors (MD: 0.19 ± 0.04 ng/ml; P = 0.001; I 2: 0%) and elevated levels were associated with increased mortality risk (HR: 1.64, 95% CI: 1.27-2.12, P < 0.001; I 2: 82%). sST2 levels were significantly higher in severe disease compared to less severe disease (MD: 1.56 ± 0.46 ng/ml; P = 0.001; I 2: 98%). Finally, in stable coronary artery disease, sST2 levels were higher in non-survivors than survivors (MD: 3.0 ± 1.1 ng/ml; P = 0.005; I 2: 80%) and elevated levels were significantly associated with increased mortality risk (HR: 1.32, 95% CI: 1.04-1.68, P < 0.05; I 2: 57%). CONCLUSIONS: sST2 significantly predicts disease severity and mortality in cardiovascular disease and is a good predictor of mortality in patients with stable coronary artery disease and chronic heart failure.

Development of a multivariable prediction model for severe COVID-19 disease: a population-based study from Hong Kong.

Recent studies have reported numerous predictors for adverse outcomes in COVID-19 disease. However, there have been few simple clinical risk scores available for prompt risk stratification. The objective is to develop a simple risk score for predicting severe COVID-19 disease using territory-wide data based on simple clinical and laboratory variables. Consecutive patients admitted to Hong Kong's public hospitals between 1 January and 22 August 2020 and diagnosed with COVID-19, as confirmed by RT-PCR, were included. The primary outcome was composite intensive care unit admission, need for intubation or death with follow-up until 8 September 2020. An external independent cohort from Wuhan was used for model validation. COVID-19 testing was performed in 237,493 patients and 4442 patients (median age 44.8 years old, 95% confidence interval (CI): [28.9, 60.8]); 50% males) were tested positive. Of these, 209 patients (4.8%) met the primary outcome. A risk score including the following components was derived from Cox regression: gender, age, diabetes mellitus, hypertension, atrial fibrillation, heart failure, ischemic heart disease, peripheral vascular disease, stroke, dementia, liver diseases, gastrointestinal bleeding, cancer, increases in neutrophil count, potassium, urea, creatinine, aspartate transaminase, alanine transaminase, bilirubin, D-dimer, high sensitive troponin-I, lactate dehydrogenase, activated partial thromboplastin time, prothrombin time, and C-reactive protein, as well as decreases in lymphocyte count, platelet, hematocrit, albumin, sodium, low-density lipoprotein, high-density lipoprotein, cholesterol, glucose, and base excess. The model based on test results taken on the day of admission demonstrated an excellent predictive value. Incorporation of test results on successive time points did not further improve risk prediction. The derived score system was evaluated with out-of-sample five-cross-validation (AUC: 0.86, 95% CI: 0.82-0.91) and external validation (N = 202, AUC: 0.89, 95% CI: 0.85-0.93). A simple clinical score accurately predicted severe COVID-19 disease, even without including symptoms, blood pressure or oxygen status on presentation, or chest radiograph results.

Multi-modality machine learning approach for risk stratification in heart failure with left ventricular ejection fraction ≤ 45.

AIMS: Heart failure (HF) involves complex remodelling leading to electrical and mechanical dysfunction. We hypothesized that machine learning approaches incorporating data obtained from different investigative modalities including atrial and ventricular measurements from electrocardiography and echocardiography, blood inflammatory marker [neutrophil-to-lymphocyte ratio (NLR)], and prognostic nutritional index (PNI) will improve risk stratification for adverse outcomes in HF compared to logistic regression. METHODS AND RESULTS: Consecutive Chinese patients referred to our centre for transthoracic echocardiography and subsequently diagnosed with HF, between 1 January 2010 and 31 December 2016, were included in this study. Two machine learning techniques, multilayer perceptron and multi-task learning, were compared with logistic regression for their ability to predict incident atrial fibrillation (AF), transient ischaemic attack (TIA)/stroke, and all-cause mortality. This study included 312 HF patients [mean age: 64 (55-73) years, 75% male]. There were 76 cases of new-onset AF, 62 cases of incident TIA/stroke, and 117 deaths during follow-up. Univariate analysis revealed that age, left atrial reservoir strain (LARS) and contractile strain (LACS) were significant predictors of new-onset AF. Age and smoking predicted incident stroke. Age, hypertension, type 2 diabetes mellitus, chronic kidney disease, mitral or aortic regurgitation, P-wave terminal force in V1, the presence of partial inter-atrial block, left atrial diameter, ejection fraction, global longitudinal strain, serum creatinine and albumin, high NLR, low PNI, and LARS and LACS predicted all-cause mortality. Machine learning techniques achieved better prediction performance than logistic regression. CONCLUSIONS: Multi-modality assessment is important for risk stratification in HF. A machine learning approach provides additional value for improving outcome prediction.

Multi-parametric system for risk stratification in mitral regurgitation: A multi-task Gaussian prediction approach.

BACKGROUND: We hypothesized that a multi-parametric approach incorporating medical comorbidity information, electrocardiographic P-wave indices, echocardiographic assessment, neutrophil-to-lymphocyte ratio (NLR) and prognostic nutritional index (PNI) calculated from laboratory data can improve risk stratification in mitral regurgitation (MR). METHODS: Patients diagnosed with mitral regurgitation between 1 March 2005 and 30 October 2018 from a single centre were retrospectively analysed. Outcomes analysed were incident atrial fibrillation (AF), transient ischemic attack (TIA)/stroke and mortality. RESULTS: This study cohort included 706 patients, of whom 171 had normal inter-atrial conduction, 257 had inter-atrial block (IAB) and 266 had AF at baseline. Logistic regression analysis showed that age, hypertension and mean P-wave duration (PWD) were significant predictors of new-onset AF. Low left ventricular ejection fraction (LVEF), abnormal P-wave terminal force in V1 (PTFV1) predicted TIA/stroke. Age, smoking, hypertension, diabetes mellitus, hypercholesterolaemia, ischemic heart disease, secondary mitral regurgitation, urea, creatinine, NLR, PNI, left atrial diameter (LAD), left ventricular end-diastolic dimension, LVEF, pulmonary arterial systolic pressure, IAB, baseline AF and heart failure predicted all-cause mortality. A multi-task Gaussian process learning model demonstrated significant improvement in risk stratification compared to logistic regression and a decision tree method. CONCLUSIONS: A multi-parametric approach incorporating multi-modality clinical data improves risk stratification in mitral regurgitation. Multi-task machine learning can significantly improve overall risk stratification performance.

Incremental Elevations in TNFα and IL6 in the Human Colon and Procancerous Changes in the Mucosal Transcriptome Accompany Adiposity.

BACKGROUND: Obesity, a risk factor for colorectal cancer, raises systemic levels of proinflammatory mediators. Whether increased levels also reside in the colons of obese individuals and are accompanied by procancerous alterations in the mucosal transcriptome is unknown. METHODS: Concentrations of TNFα, IL1ß, and IL6 in blood and colonic mucosa of 16 lean and 26 obese individuals were examined. Differences in the mucosal transcriptome between the two groups were defined. RESULTS: Plasma IL6 and TNFα were 1.4- to 3-fold elevated in obese subjects [body mass index (BMI) ≥ 34 kg/m2] compared with the lean controls (P < 0.01). Among individuals with BMI ≥ 34 kg/m2 colonic concentrations of IL6 and TNFα were 2- to 3-fold greater than in lean subjects (P < 0.03). In a general linear model, adjusted for NSAID use, colonic IL6 (partial r = 0.41; P < 0.01) and TNFα (partial r = 0.41; P = 0.01) increased incrementally over the entire range of BMIs (18.1-45.7). Regular use of nonsteroidal anti-inflammatory drugs (NSAIDs) was associated with a reduction in colonic IL6 (ß = -0.65, P < 0.02). RNA sequencing (NSAID users excluded) identified 182 genes expressed differentially between lean and obese subjects. The two gene networks most strongly linked to changes in expression included several differentially expressed genes known to regulate the procarcinogenic signaling pathways, NFκB and ERK 1/2, in a pattern consistent with upregulation of each in the obese subjects. CONCLUSIONS: Incremental increases in two major proinflammatory colonic cytokines are associated with increasing BMI, and in the obese state are accompanied by procancerous changes in the transcriptome. IMPACT: These observations delineate means by which an inflammatory milieu may contribute to obesity-promoted colon cancer.

Inhibiting the integrated stress response pathway prevents aberrant chondrocyte differentiation thereby alleviating chondrodysplasia.

The integrated stress response (ISR) is activated by diverse forms of cellular stress, including endoplasmic reticulum (ER) stress, and is associated with diseases. However, the molecular mechanism(s) whereby the ISR impacts on differentiation is incompletely understood. Here, we exploited a mouse model of Metaphyseal Chondrodysplasia type Schmid (MCDS) to provide insight into the impact of the ISR on cell fate. We show the protein kinase RNA-like ER kinase (PERK) pathway that mediates preferential synthesis of ATF4 and CHOP, dominates in causing dysplasia by reverting chondrocyte differentiation via ATF4-directed transactivation of Sox9. Chondrocyte survival is enabled, cell autonomously, by CHOP and dual CHOP-ATF4 transactivation of Fgf21. Treatment of mutant mice with a chemical inhibitor of PERK signaling prevents the differentiation defects and ameliorates chondrodysplasia. By preventing aberrant differentiation, titrated inhibition of the ISR emerges as a rationale therapeutic strategy for stress-induced skeletal disorders.

Reprogramming of Mouse Calvarial Osteoblasts into Induced Pluripotent Stem Cells.

Previous studies have demonstrated the ability of reprogramming endochondral bone into induced pluripotent stem (iPS) cells, but whether similar phenomenon occurs in intramembranous bone remains to be determined. Here we adopted fluorescence-activated cell sorting-based strategy to isolate homogenous population of intramembranous calvarial osteoblasts from newborn transgenic mice carrying both Osx1-GFP::Cre and Oct4-EGFP transgenes. Following retroviral transduction of Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc), enriched population of osteoblasts underwent silencing of Osx1-GFP::Cre expression at early stage of reprogramming followed by late activation of Oct4-EGFP expression in the resulting iPS cells. These osteoblast-derived iPS cells exhibited gene expression profiles akin to embryonic stem cells and were pluripotent as demonstrated by their ability to form teratomas comprising tissues from all germ layers and also contribute to tail tissue in chimera embryos. These data demonstrate that iPS cells can be generated from intramembranous osteoblasts.

Clinical outcomes in pancreatic adenocarcinoma associated with BRCA-2 mutation.

Patients with BRCA-1 and BRCA-2 germ line mutations are at an increased risk of developing pancreatic adenocarcinoma (PAC). In particular, the BRCA-2 mutation has been associated with a relative risk of developing PAC of 3.51. The BRCA-2 protein is involved in repair of double-stranded DNA breaks. Recent reports have suggested that in the setting of impaired DNA repair, chemotherapeutic agents that induce DNA damage, such as platinum-based antineoplastic drugs (platins) and poly(ADP-ribose) polymerase inhibitors (PARP inhibitors), have improved efficacy. However, because of the relative rarity of BRCA-related PAC, studies evaluating such agents in this setting are scarce. Patients with a known BRCA-2 mutation and PAC were retrospectively reviewed. Ten patients with PAC and BRCA-2 mutation were identified. Four patients (40%) were of Ashkenazi Jewish descent. Seven patients (70%) received platinum agents, two (20%) received mitomycin-C, one (10%) received a PARP inhibitor, and seven (70%) received a topoisomerase-I inhibitor. Overall, chemotherapy was well tolerated with expected side effects. Patients with a BRCA-2 mutation and PAC represent a group with a unique biology underlying their cancer. Chemotherapies such as platinum derivatives, mitomycin-C, topoisomerase-I inhibitors, and PARP inhibitors targeting DNA require further investigation in this population. Genetic testing may guide therapy in the future.

BRCA-associated pancreatic cancer: the evolving management.

Pancreatic cancer is one of the deadliest cancers. While BRCA-associated pancreatic cancers are uncommon, the distinctive phenotype of this malignancy may offer unique therapeutic targets. At the 2013 American Society of Clinical Oncology (ASCO) Gastrointestinal Cancers Symposium, a review of the characteristics and outcomes of one large case series (Abstract #278), however, did not reveal a benefit to first-line platinum chemotherapy in the treatment of advanced pancreatic cancer. In another study (Abstract #147), substantial responses were observed in both patients with BRCA2-associated pancreatic cancer treated with the poly-(ADP-ribose) polymerase (PARP) inhibitor ABT-888 (veliparib). We will review these abstracts and our current knowledge of the treatment for patients with BRCA-associated pancreatic cancer. In this group of patients, these new results continue to shape our understanding of this disease.

SOX9 governs differentiation stage-specific gene expression in growth plate chondrocytes via direct concomitant transactivation and repression.

Cartilage and endochondral bone development require SOX9 activity to regulate chondrogenesis, chondrocyte proliferation, and transition to a non-mitotic hypertrophic state. The restricted and reciprocal expression of the collagen X gene, Col10a1, in hypertrophic chondrocytes and Sox9 in immature chondrocytes epitomise the precise spatiotemporal control of gene expression as chondrocytes progress through phases of differentiation, but how this is achieved is not clear. Here, we have identified a regulatory element upstream of Col10a1 that enhances its expression in hypertrophic chondrocytes in vivo. In immature chondrocytes, where Col10a1 is not expressed, SOX9 interacts with a conserved sequence within this element that is analogous to that within the intronic enhancer of the collagen II gene Col2a1, the known transactivation target of SOX9. By analysing a series of Col10a1 reporter genes in transgenic mice, we show that the SOX9 binding consensus in this element is required to repress expression of the transgene in non-hypertrophic chondrocytes. Forced ectopic Sox9 expression in hypertrophic chondrocytes in vitro and in mice resulted in down-regulation of Col10a1. Mutation of a binding consensus motif for GLI transcription factors, which are the effectors of Indian hedgehog signaling, close to the SOX9 site in the Col10a1 regulatory element, also derepressed transgene expression in non-hypertrophic chondrocytes. GLI2 and GLI3 bound to the Col10a1 regulatory element but not to the enhancer of Col2a1. In addition to Col10a1, paired SOX9-GLI binding motifs are present in the conserved non-coding regions of several genes that are preferentially expressed in hypertrophic chondrocytes and the occurrence of pairing is unlikely to be by chance. We propose a regulatory paradigm whereby direct concomitant positive and negative transcriptional control by SOX9 ensures differentiation phase-specific gene expression in chondrocytes. Discrimination between these opposing modes of transcriptional control by SOX9 may be mediated by cooperation with different partners such as GLI factors.