Biallelic non-productive enhancer-promoter interactions precede imprinted expression of Kcnk9 during mouse neural commitment.

It is only partially understood how constitutive allelic methylation at imprinting control regions (ICRs) interacts with other regulation levels to drive timely parental allele-specific expression along large imprinted domains. The Peg13-Kcnk9 domain is an imprinted domain with important brain functions. To gain insights into its regulation during neural commitment, we performed an integrative analysis of its allele-specific epigenetic, transcriptomic, and cis-spatial organization using a mouse stem cell-based corticogenesis model that recapitulates the control of imprinted gene expression during neurodevelopment. We found that, despite an allelic higher-order chromatin structure associated with the paternally CTCF-bound Peg13 ICR, enhancer-Kcnk9 promoter contacts occurred on both alleles, although they were productive only on the maternal allele. This observation challenges the canonical model in which CTCF binding isolates the enhancer and its target gene on either side and suggests a more nuanced role for allelic CTCF binding at some ICRs.

GPR68-ATF4 signaling is a novel prosurvival pathway in glioblastoma activated by acidic extracellular microenvironment.

BACKGROUND: Glioblastoma multiforme (GBM) stands as a formidable challenge in oncology because of its aggressive nature and severely limited treatment options. Despite decades of research, the survival rates for GBM remain effectively stagnant. A defining hallmark of GBM is a highly acidic tumor microenvironment, which is thought to activate pro-tumorigenic pathways. This acidification is the result of altered tumor metabolism favoring aerobic glycolysis, a phenomenon known as the Warburg effect. Low extracellular pH confers radioresistant tumors to glial cells. Notably GPR68, an acid sensing GPCR, is upregulated in radioresistant GBM. Usage of Lorazepam, which has off target agonism of GPR68, is linked to worse clinical outcomes for a variety of cancers. However, the role of tumor microenvironment acidification in GPR68 activation has not been assessed in cancer. Here we interrogate the role of GPR68 specifically in GBM cells using a novel highly specific small molecule inhibitor of GPR68 named Ogremorphin (OGM) to induce the iron mediated cell death pathway: ferroptosis. METHOD: OGM was identified in a non-biased zebrafish embryonic development screen and validated with Morpholino and CRISPR based approaches. Next, A GPI-anchored pH reporter, pHluorin2, was stably expressed in U87 glioblastoma cells to probe extracellular acidification. Cell survival assays, via nuclei counting and cell titer glo, were used to demonstrate sensitivity to GPR68 inhibition in twelve immortalized and PDX GBM lines. To determine GPR68 inhibition's mechanism of cell death we use DAVID pathway analysis of RNAseq. Our major indication, ferroptosis, was then confirmed by western blotting and qRT-PCR of reporter genes including TFRC. This finding was further validated by transmission electron microscopy and liperfluo staining to assess lipid peroxidation. Lastly, we use siRNA and CRISPRi to demonstrate the critical role of ATF4 suppression via GPR68 for GBM survival. RESULTS: We used a pHLourin2 probe to demonstrate how glioblastoma cells acidify their microenvironment to activate the commonly over expressed acid sensing GPCR, GPR68. Using our small molecule inhibitor OGM and genetic means, we show that blocking GPR68 signaling results in robust cell death in all thirteen glioblastoma cell lines tested, irrespective of genetic and phenotypic heterogeneity, or resistance to the mainstay GBM chemotherapeutic temozolomide. We use U87 and U138 glioblastoma cell lines to show how selective induction of ferroptosis occurs in an ATF4-dependent manner. Importantly, OGM was not-acutely toxic to zebrafish and its inhibitory effects were found to spare non-malignant neural cells. CONCLUSION: These results indicate GPR68 emerges as a critical sensor for an autocrine pro-tumorigenic signaling cascade triggered by extracellular acidification in glioblastoma cells. In this context, GPR68 suppresses ATF4, inhibition of GPR68 increases expression of ATF4 which leads to ferroptotic cell death. These findings provide a promising therapeutic approach to selectively induce ferroptosis in glioblastoma cells while sparing healthy neural tissue.

Polygenic Risk Score Associates With Atherosclerotic Plaque Characteristics at Autopsy.

BACKGROUND: Polygenic risk scores (PRSs) for coronary artery disease (CAD) potentially improve cardiovascular risk prediction. However, their relationship with histopathologic features of CAD has never been examined systematically. METHODS: From 4327 subjects referred to CVPath by the State of Maryland Office Chief Medical Examiner for sudden death between 1994 and 2015, 2455 cases were randomly selected for genotyping. We generated PRS from 291 known CAD risk loci. Detailed histopathologic examination of the coronary arteries was performed in all subjects. The primary study outcome measurements were histopathologic plaque features determining severity of atherosclerosis, including %stenosis, calcification, thin-cap fibroatheromas, and thrombotic CAD. RESULTS: After exclusion of cases with insufficient DNA sample quality or with missing data, 954 cases (mean age, 48.8±14.7 years; 75.7% men) remained in the final study cohort. Subjects in the highest PRS quintile exhibited more severe atherosclerosis compared with subjects in the lowest quintile, with greater %stenosis (80.3%±27.0% versus 50.4%±38.7%; adjusted P<0.001) and a higher frequency of calcification (69.6% versus 35.8%; adjusted P=0.004) and thin-cap fibroatheroma (26.7% versus 9.5%; adjusted P=0.007). Even after adjustment for traditional CAD risk factors, subjects within the highest PRS quintile had higher odds of severe atherosclerosis (ie, ≥75% stenosis; adjusted odds ratio, 3.77 [95% CI, 2.10-6.78]; P<0.001) and plaque rupture (adjusted odds ratio, 4.05 [95% CI, 2.26-7.24]; P<0.001). Moreover, subjects within the highest quintile had higher odds of CAD-associated cause of death, especially among those aged ≤50 years (adjusted odds ratio, 4.08 [95% CI, 2.01-8.30]; P<0.001). No statistically significant associations were observed with plaque erosion after adjusting for covariates. CONCLUSIONS: This is the first autopsy study investigating associations between PRS and atherosclerosis severity at the histopathologic level in subjects with sudden death. Our pathological analysis suggests PRS correlates with plaque burden and features of advanced atherosclerosis and may be useful as a method for CAD risk stratification, especially in younger subjects.

The USP46 complex deubiquitylates LRP6 to promote Wnt/ß-catenin signaling.

The relative abundance of Wnt receptors plays a crucial role in controlling Wnt signaling in tissue homeostasis and human disease. While the ubiquitin ligases that ubiquitylate Wnt receptors are well-characterized, the deubiquitylase that reverses these reactions remains unclear. Herein, we identify USP46, UAF1, and WDR20 (USP46 complex) as positive regulators of Wnt signaling in cultured human cells. We find that the USP46 complex is similarly required for Wnt signaling in Xenopus and zebrafish embryos. We demonstrate that Wnt signaling promotes the association between the USP46 complex and cell surface Wnt coreceptor, LRP6. Knockdown of USP46 decreases steady-state levels of LRP6 and increases the level of ubiquitylated LRP6. In contrast, overexpression of the USP46 complex blocks ubiquitylation of LRP6 by the ubiquitin ligases RNF43 and ZNFR3. Size exclusion chromatography studies suggest that the size of the USP46 cytoplasmic complex increases upon Wnt stimulation. Finally, we show that USP46 is essential for Wnt-dependent intestinal organoid viability, likely via its role in LRP6 receptor homeostasis. We propose a model in which the USP46 complex increases the steady-state level of cell surface LRP6 and facilitates the assembly of LRP6 into signalosomes via a pruning mechanism that removes sterically hindering ubiquitin chains.

Polygenic Risk Score Associates with Atherosclerotic Plaque Characteristics at Autopsy.

Background: Polygenic risk scores (PRS) for coronary artery disease (CAD) potentially improve cardiovascular risk prediction. However, their relationship with histopathologic features of CAD has never been examined systematically. Methods: From 4,327 subjects referred to CVPath by the State of Maryland Office Chief Medical Examiner (OCME) for sudden death between 1994 and 2015, 2,455 cases were randomly selected for genotyping. We generated PRS from 291 known CAD risk loci. Detailed histopathologic examination of the coronary arteries was performed in all subjects. The primary study outcome measurements were histopathologic plaque features determining severity of atherosclerosis, including %stenosis, calcification, thin-cap fibroatheromas (TCFA), and thrombotic CAD. Results: After exclusion of cases with insufficient DNA sample quality or with missing data, 954 cases (mean age 48.8±14.7; 75.7% men) remained in the final study cohort. Subjects in the highest PRS quintile exhibited more severe atherosclerosis compared to subjects in the lowest quintile, with greater %stenosis (80.3%±27.0% vs. 50.4%±38.7%; adjusted p<0.001) and a higher frequency of calcification (69.6% vs. 35.8%; adjusted p=0.004) and TCFAs (26.7% vs. 9.5%; adjusted p=0.007). Even after adjustment for traditional CAD risk factors subjects within the highest PRS quintile had higher odds of severe atherosclerosis (i.e., ≥75% stenosis; adjusted OR 3.77; 95%CI 2.10-6.78; p<0.001) and plaque rupture (adjusted OR 4.05; 95%CI 2.26-7.24; p<0.001). Moreover, subjects within the highest quintile had higher odds of CAD-associated cause of death, especially among those aged 50 years and younger (adjusted OR 4.08; 95%CI 2.01-8.30; p<0.001). No associations were observed with plaque erosion. Conclusions: This is the first autopsy study investigating associations between PRS and atherosclerosis severity at the histopathologic level in subjects with sudden death. Our pathological analysis suggests PRS correlates with plaque burden and features of advanced atherosclerosis and may be useful as a method for CAD risk stratification, especially in younger subjects. Highlights: In this autopsy study including 954 subjects within the CVPath Sudden Death Registry, high PRS correlated with plaque burden and atherosclerosis severity.The PRS showed differential associations with plaque rupture and plaque erosion, suggesting different etiologies to these two causes of thrombotic CAD.PRS may be useful for risk stratification, particularly in the young. Further examination of individual risk loci and their association with plaque morphology may help understand molecular mechanisms of atherosclerosis, potentially revealing new therapy targets of CAD. Graphic Abstract: A polygenic risk score, generated from 291 known CAD risk loci, was assessed in 954 subjects within the CVPath Sudden Death Registry. Histopathologic examination of the coronary arteries was performed in all subjects. Subjects in the highest PRS quintile exhibited more severe atherosclerosis as compared to subjects in the lowest quintile, with a greater plaque burden, more calcification, and a higher frequency of plaque rupture.

Functional mapping of microRNA promoters with dCas9 fused to transcriptional regulators.

MicroRNAs are small non-coding RNAs that control gene expression during development, physiology, and disease. Transcription is a key factor in microRNA abundance and tissue-specific expression. Many databases predict the location of microRNA transcription start sites and promoters. However, these candidate regions require functional validation. Here, dCas9 fused to transcriptional activators or repressors - CRISPR activation (CRISPRa) and inhibition (CRISPRi)- were targeted to the candidate promoters of two intronic microRNAs, mmu-miR-335 and hsa-miR-3662, including the promoters of their respective host genes Mest and HBS1L. We report that in mouse embryonic stem cells and brain organoids, miR-335 was downregulated upon CRISPRi of its host gene Mest. Reciprocally, CRISPRa of Mest promoter upregulated miR-335. By contrast, CRISPRa of the predicted miR-335-specific promoter (located in an intron of Mest) did not affect miR-335 levels. Thus, the expression of miR-335 only depends on the promoter activity of its host gene Mest. By contrast, miR-3662 was CRISPR activatable both by the promoter of its host gene HBS1L and an intronic sequence in HEK-293T cells. Thus, CRISPRa and CRISPRi are powerful tools to evaluate the relevance of endogenous regulatory sequences involved in microRNA transcription in defined cell types.

Identification of novel genetic variants, including PIM1 and LINC01491, with ICD-10 based diagnosis of pulmonary arterial hypertension in the UK Biobank cohort.

Pulmonary arterial hypertension (PAH) is characterized by remodeling and narrowing of the pulmonary vasculature which results in elevations of pulmonary arterial pressures. Here, we conducted a genome-wide association study (GWAS) using the UK Biobank, analyzing the genomes of 493 individuals diagnosed with primary pulmonary hypertension, based on ICD-10 coding, compared to 24,650 age, sex, and ancestry-matched controls in a 1:50 case-control design. Genetic variants were analyzed by Plink's firth logistic regression and assessed for association with primary pulmonary hypertension. We identified three linked variants in the PIM1 gene, which encodes a protooncogene that has been garnering interest as a potential therapeutic target for PAH, that were associated with PAH with genome wide significance, one (rs192449585) of which lies in the promoter region of the gene. We also identified 15 linked variants in the LINC01491 gene. These results provide genetic evidence supporting the role of PIM1 inhibitors as a potential therapeutic option for PAH.

Association of Incident Cardiovascular Disease With Time Course and Cumulative Exposure to Multiple Risk Factors.

BACKGROUND: The quantitative relationship of incident cardiovascular disease (CVD) to lifetime cumulative risk factor exposure is not well understood. OBJECTIVES: Using CARDIA (Coronary Artery Risk Development in Young Adults) study data, we examined the quantitative associations of cumulative exposure over time to multiple, simultaneously operating risk factors with CVD incidence and the incidence of its components. METHODS: Regression models were developed quantifying the influence of the time course and severity of multiple CVD risk factors, operating simultaneously, on risk of incident CVD. The outcomes were incident CVD and the incidence of its components: coronary heart disease, stroke, and congestive heart failure. RESULTS: Our study included 4,958 asymptomatic adults enrolled in CARDIA from 1985 to 1986 (ages 18 to 30 years) who were followed for 30 years. Risk of incident CVD depends on the time course and severity of a series of independent risk factors, the impact of which is mediated by their effects on individual CVD components after age 40 years. Cumulative exposure (AUC vs time) to low-density lipoprotein cholesterol and triglycerides was independently associated with risk of incident CVD. Of the blood pressure variables, areas under the mean arterial pressure vs time curve and the pulse pressure vs time curve were strongly and independently associated with incident CVD risk. CONCLUSIONS: The quantitative description of the link between risk factors and CVD informs the construction of individualized CVD mitigation strategies, design of primary prevention trials, and assessment of public health impact of risk factor-based interventions.

Impaired Reorganization of Centrosome Structure Underlies Human Infantile Dilated Cardiomyopathy.

BACKGROUND: During cardiomyocyte maturation, the centrosome, which functions as a microtubule organizing center in cardiomyocytes, undergoes dramatic structural reorganization where its components reorganize from being localized at the centriole to the nuclear envelope. This developmentally programmed process, referred to as centrosome reduction, has been previously associated with cell cycle exit. However, understanding of how this process influences cardiomyocyte cell biology, and whether its disruption results in human cardiac disease, remains unknown. We studied this phenomenon in an infant with a rare case of infantile dilated cardiomyopathy (iDCM) who presented with left ventricular ejection fraction of 18% and disrupted sarcomere and mitochondria structure. METHODS: We performed an analysis beginning with an infant who presented with a rare case of iDCM. We derived induced pluripotent stem cells from the patient to model iDCM in vitro. We performed whole exome sequencing on the patient and his parents for causal gene analysis. CRISPR/Cas9-mediated gene knockout and correction in vitro were used to confirm whole exome sequencing results. Zebrafish and Drosophila models were used for in vivo validation of the causal gene. Matrigel mattress technology and single-cell RNA sequencing were used to characterize iDCM cardiomyocytes further. RESULTS: Whole exome sequencing and CRISPR/Cas9 gene knockout/correction identified RTTN, the gene encoding the centrosomal protein RTTN (rotatin), as the causal gene underlying the patient's condition, representing the first time a centrosome defect has been implicated in a nonsyndromic dilated cardiomyopathy. Genetic knockdowns in zebrafish and Drosophila confirmed an evolutionarily conserved requirement of RTTN for cardiac structure and function. Single-cell RNA sequencing of iDCM cardiomyocytes showed impaired maturation of iDCM cardiomyocytes, which underlie the observed cardiomyocyte structural and functional deficits. We also observed persistent localization of the centrosome at the centriole, contrasting with expected programmed perinuclear reorganization, which led to subsequent global microtubule network defects. In addition, we identified a small molecule that restored centrosome reorganization and improved the structure and contractility of iDCM cardiomyocytes. CONCLUSIONS: This study is the first to demonstrate a case of human disease caused by a defect in centrosome reduction. We also uncovered a novel role for RTTN in perinatal cardiac development and identified a potential therapeutic strategy for centrosome-related iDCM. Future study aimed at identifying variants in centrosome components may uncover additional contributors to human cardiac disease.

A Novel COVID-19 Severity Score is Associated With Survival in Patients Undergoing Percutaneous Dilational Tracheostomy.

INTRODUCTION: Tracheostomy in patients with COVID-19 is a controversial and difficult clinical decision. We hypothesized that a recently validated COVID-19 Severity Score (CSS) would be associated with survival in patients considered for tracheostomy. METHODS: We reviewed 77 mechanically ventilated COVID-19 patients evaluated for decision for percutaneous dilational tracheostomy (PDT) from March to June 2020 at a public tertiary care center. Decision for PDT was based on clinical judgment of the screening surgeons. The CSS was retrospectively calculated using mean biomarker values from admission to time of PDT consult. Our primary outcome was survival to discharge, and all patient charts were reviewed through August 31, 2021. ROC curve and Youden index were used to estimate an optimal cut-point for survival. RESULTS: The mean CSS for 42 survivors significantly differed from that of 35 nonsurvivors (CSS 52 versus 66, P = 0.003). The Youden index returned an optimal CSS of 55 (95% confidence interval 43-72), which was associated with a sensitivity of 0.8 and a specificity of 0.6. The median CSS was 40 (interquartile range 27, 49) in the lower CSS (<55) group and 72 (interquartile range 66, 93) in the high CSS (≥55 group). Eighty-seven percent of lower CSS patients underwent PDT, with 74% survival, whereas 61% of high CSS patients underwent PDT, with only 41% surviving. Patients with high CSS had 77% lower odds of survival (odds ratio = 0.2, 95% confidence interval 0.1-0.7). CONCLUSIONS: Higher CSS was associated with decreased survival in patients evaluated for PDT, with a score ≥55 predictive of mortality. The novel CSS may be a useful adjunct in determining which COVID-19 patients will benefit from tracheostomy. Further prospective validation of this tool is warranted.

Exosomes mediated fibrogenesis in dilated cardiomyopathy through a MicroRNA pathway.

Cardiac fibrosis is a hallmark in late-stage familial dilated cardiomyopathy (DCM) patients, although the underlying mechanism remains elusive. Cardiac exosomes (Exos) have been reported relating to fibrosis in ischemic cardiomyopathy. Thus, we investigated whether Exos secreted from the familial DCM cardiomyocytes could promote fibrogenesis. Using human iPSCs differentiated cardiomyocytes we isolated Exos of angiotensin II stimulation conditioned media from either DCM or control (CTL) cardiomyocytes. Of interest, cultured cardiac fibroblasts had increased fibrogenesis following exposure to DCM-Exos rather than CTL-Exos. Meanwhile, injecting DCM-Exos into mouse hearts enhanced cardiac fibrosis and impaired cardiac function. Mechanistically, we identified the upregulation of miRNA-218-5p in the DCM-Exos as a critical contributor to fibrogenesis. MiRNA-218-5p activated TGF-ß signaling via suppression of TNFAIP3, a master inflammation inhibitor. In conclusion, our results illustrate a profibrotic effect of cardiomyocytes-derived Exos that highlights an additional pathogenesis pathway for cardiac fibrosis in DCM.

Transcriptional Dysregulation Underlies Both Monogenic Arrhythmia Syndrome and Common Modifiers of Cardiac Repolarization.

BACKGROUND: Brugada syndrome (BrS) is an inherited arrhythmia syndrome caused by loss-of-function variants in the cardiac sodium channel gene SCN5A (sodium voltage-gated channel alpha subunit 5) in ≈20% of subjects. We identified a family with 4 individuals diagnosed with BrS harboring the rare G145R missense variant in the cardiac transcription factor TBX5 (T-box transcription factor 5) and no SCN5A variant. METHODS: We generated induced pluripotent stem cells (iPSCs) from 2 members of a family carrying TBX5-G145R and diagnosed with Brugada syndrome. After differentiation to iPSC-derived cardiomyocytes (iPSC-CMs), electrophysiologic characteristics were assessed by voltage- and current-clamp experiments (n=9 to 21 cells per group) and transcriptional differences by RNA sequencing (n=3 samples per group), and compared with iPSC-CMs in which G145R was corrected by CRISPR/Cas9 approaches. The role of platelet-derived growth factor (PDGF)/phosphoinositide 3-kinase (PI3K) pathway was elucidated by small molecule perturbation. The rate-corrected QT (QTc) interval association with serum PDGF was tested in the Framingham Heart Study cohort (n=1893 individuals). RESULTS: TBX5-G145R reduced transcriptional activity and caused multiple electrophysiologic abnormalities, including decreased peak and enhanced "late" cardiac sodium current (INa), which were entirely corrected by editing G145R to wild-type. Transcriptional profiling and functional assays in genome-unedited and -edited iPSC-CMs showed direct SCN5A down-regulation caused decreased peak INa, and that reduced PDGF receptor (PDGFRA [platelet-derived growth factor receptor α]) expression and blunted signal transduction to PI3K was implicated in enhanced late INa. Tbx5 regulation of the PDGF axis increased arrhythmia risk due to disruption of PDGF signaling and was conserved in murine model systems. PDGF receptor blockade markedly prolonged normal iPSC-CM action potentials and plasma levels of PDGF in the Framingham Heart Study were inversely correlated with the QTc interval (P<0.001). CONCLUSIONS: These results not only establish decreased SCN5A transcription by the TBX5 variant as a cause of BrS, but also reveal a new general transcriptional mechanism of arrhythmogenesis of enhanced late sodium current caused by reduced PDGF receptor-mediated PI3K signaling.

Endophenotype effect sizes support variant pathogenicity in monogenic disease susceptibility genes.

Accurate and efficient classification of variant pathogenicity is critical for research and clinical care. Using data from three large studies, we demonstrate that population-based associations between rare variants and quantitative endophenotypes for three monogenic diseases (low-density-lipoprotein cholesterol for familial hypercholesterolemia, electrocardiographic QTc interval for long QT syndrome, and glycosylated hemoglobin for maturity-onset diabetes of the young) provide evidence for variant pathogenicity. Effect sizes are associated with pathogenic ClinVar assertions (P < 0.001 for each trait) and discriminate pathogenic from non-pathogenic variants (area under the curve 0.82-0.84 across endophenotypes). An effect size threshold of ≥ 0.5 times the endophenotype standard deviation nominates up to 35% of rare variants of uncertain significance or not in ClinVar in disease susceptibility genes with pathogenic potential. We propose that variant associations with quantitative endophenotypes for monogenic diseases can provide evidence supporting pathogenicity.

Identifying genetic variants associated with the ICD10 (International Classification of Diseases10)-based diagnosis of cerebrovascular disease using a large-scale biomedical database.

OBJECTIVES: To utilize the UK Biobank to identify genetic variants associated with the ICD10 (International Classification of Diseases10)-based diagnosis of cerebrovascular disease (CeVD). BACKGROUND: Cerebrovascular disease occurs because of a complex interplay between vascular, environmental, and genetic factors. It is the second leading cause of disability worldwide. Understanding who may be genetically predisposed to cerebrovascular disease can help guide preventative efforts. Moreover, there is considerable interest in the use of real-world data, such as EHR (electronic health records) to better understand disease mechanisms and to discover new treatment strategies, but whether ICD10-based diagnosis can be used to study CeVD genetics is unknown. METHODS: Using the UK Biobank, we conducted a genome-wide association study (GWAS) where we analyzed the genomes of 11,155 cases and 122,705 controls who were sex, age and ancestry-matched in a 1:11 case: control design. Genetic variants were identified by Plink's firth logistic regression and assessed for association with the ICD10 codes corresponding to CeVD. RESULTS: We identified two groups of SNPs closely linked to PITX2 and LRRTM4 that were significantly associated with CeVD in this study (p < 5 x 10-8) and had a minor allele frequency of > 0.5%. DISCUSSION: Disease assignment based on ICD10 codes may underestimate prevalence; however, for CeVD, this does not appear to be the case. Compared to the age- and sex-matched control population, individuals with CeVD were more frequently diagnosed with comorbid conditions, such as hypertension, hyperlipidemia & atrial fibrillation or flutter, confirming their contribution to CeVD. The UK Biobank based ICD10 study identified 2 groups of variants that were associated with CeVD. The association between PITX2 and CeVD is likely explained by the increased rates of atrial fibrillation and flutter. While the mechanism explaining the relationship between LRRTM4 and CeVD is unclear, this has been documented in previous studies.

The Casein kinase 1α agonist pyrvinium attenuates Wnt-mediated CK1α degradation via interaction with the E3 ubiquitin ligase component Cereblon.

The Cullin-RING ligase 4 E3 ubiquitin ligase component Cereblon (CRBN) is a well-established target for a class of small molecules termed immunomodulatory drugs (IMiDs). These drugs drive CRBN to modulate the degradation of a number of neosubstrates required for the growth of multiple cancers. Whereas the mechanism underlying the activation of CRBN by IMiDs is well described, the normal physiological regulation of CRBN is poorly understood. We recently showed that CRBN is activated following exposure to Wnt ligands and subsequently mediates the degradation of a subset of physiological substrates. Among the Wnt-dependent substrates of CRBN is Casein kinase 1α (CK1α), a known negative regulator of Wnt signaling. Wnt-mediated degradation of CK1α occurs via its association with CRBN at a known IMiD binding pocket. Herein, we demonstrate that a small-molecule CK1α agonist, pyrvinium, directly prevents the Wnt-dependent interaction of CRBN with CK1α, attenuating the consequent CK1α degradation. We further show that pyrvinium disrupts the ability of CRBN to interact with CK1α at the IMiD binding pocket within the CRBN-CK1α complex. Of note, this function of pyrvinium is independent of its previously reported ability to enhance CK1α kinase activity. Furthermore, we also demonstrate that pyrvinium attenuates CRBN-induced Wnt pathway activation in vivo. Collectively, these results reveal a novel dual mechanism through which pyrvinium inhibits Wnt signaling by both attenuating the CRBN-mediated destabilization of CK1α and activating CK1α kinase activity.

Antibiotic Therapy for Culture-Proven Bacterial Overgrowth in Children With Intestinal Failure Results in Improved Symptoms and Growth.

OBJECTIVES: To evaluate symptoms, enteral tolerance, growth, and antibiotic regimens in pediatric intestinal failure (IF) patients after treated with antibiotic therapy for small bowel bacterial overgrowth (SBBO). METHODS: Single-center retrospective review of children 0-18 years with IF with endoscopic cultures demonstrating >10 5 CFU/mL from 2010 to 2017. Symptoms, enteral tolerance, growth, and antibiotic regimens were evaluated at the time of endoscopy and 6 months later. RESULTS: Of 505 patients followed in our intestinal rehabilitation program, 104 underwent upper gastrointestinal endoscopy and 78 had positive duodenal cultures. Clinical data pre- and post-endoscopy were available for 56 patients. Compared to baseline, in the 6 months following targeted antibiotic treatment, children showed significant improvement in emesis or feeding intolerance (58.9% vs 23.2%, P < 0.001), abdominal pain (16.1% vs 7.1%, P = 0.02), high stool output (42.9% vs 19.6%, P = 0.002), and gross GI bleeding (19.6% vs 3.6%, P = 0.003). Mean BMI-for-age z scores increased significantly (-0.03 ± 0.94 vs 0.27 ± 0.82, P = 0.03); however, height-for-age z scores, weight-for-age z scores, and percent of calories from enteral intake were not significantly different after therapy. Antibiotic regimens remained highly variable. CONCLUSIONS: Children with IF and culture-positive SBBO showed significant improvement in symptoms and BMI-for-age z scores after duodenal culture with subsequent targeted antibiotic therapy. Longer follow-up may be needed to detect improvements in linear growth and percent of calories from enteral feeds. Antibiotic regimens remain highly variable. Long-term consequences of chronic antimicrobial therapy, including antimicrobial resistance, remain unknown. Prospective studies focused on standardizing duodenal sampling technique, correlating culture and pathology data, and evaluating antibiotic resistance patterns are needed.