Citations

Primary cilia serve as cellular antenna for various sensory signaling pathways. However, how the sensory receptors are properly targeted to the ciliary surface remains poorly understood. Here, we show that UBC-9, the sole E2 small ubiquitin-like modifier (SUMO)-conjugating enzyme, physically interacts with and SUMOylates the C terminus of small GTPase ARL-13, the worm orthologue of ARL13B that mutated in ciliopathy Joubert syndrome. Mutations that totally abolish the SUMOylation of ARL-13 do not affect its established role in ciliogenesis, but fail to regulate the proper ciliary targeting of various sensory receptors and consequently compromise the corresponding sensory functions. Conversely, constitutively SUMOylated ARL-13 fully rescues all ciliary defects of arl-13–null animals. Furthermore, SUMOylation modification of human ARL13B is required for the ciliary entry of polycystin-2, the protein mutated in autosomal dominant polycystic kidney disease. Our data reveal a novel but conserved role for the SUMOylation modification of ciliary small GTPase ARL13B in specifically regulating the proper ciliary targeting of various sensory receptors.

Abstract Genetic lesions such as BCR–ABL1, E2A–PBX1, and MLL rearrangements (MLLr) are associated with unfavorable outcomes in adult B-cell precursor acute lymphoblastic leukemia (B-ALL). Leukemia oncoproteins may directly or indirectly disrupt cytosine methylation patterning to mediate the malignant phenotype. We postulated that DNA methylation signatures in these aggressive B-ALLs would point toward disease mechanisms and useful biomarkers and therapeutic targets. We therefore conducted DNA methylation and gene expression profiling on a cohort of 215 adult patients with B-ALL enrolled in a single phase III clinical trial (ECOG E2993) and normal control B cells. In BCR–ABL1-positive B-ALLs, aberrant cytosine methylation patterning centered around a cytokine network defined by hypomethylation and overexpression of IL2RA(CD25). The E2993 trial clinical data showed that CD25 expression was strongly associated with a poor outcome in patients with ALL regardless of BCR–ABL1 status, suggesting CD25 as a novel prognostic biomarker for risk stratification in B-ALLs. In E2A–PBX1-positive B-ALLs, aberrant DNA methylation patterning was strongly associated with direct fusion protein binding as shown by the E2A–PBX1 chromatin immunoprecipitation (ChIP) sequencing (ChIP-seq), suggesting that E2A–PBX1 fusion protein directly remodels the epigenome to impose an aggressive B-ALL phenotype. MLLr B-ALL featured prominent cytosine hypomethylation, which was linked with MLL fusion protein binding, H3K79 dimethylation, and transcriptional upregulation, affecting a set of known and newly identified MLL fusion direct targets with oncogenic activity such as FLT3 and BCL6. Notably, BCL6 blockade or loss of function suppressed proliferation and survival of MLLr leukemia cells, suggesting BCL6-targeted therapy as a new therapeutic strategy for MLLr B-ALLs. Significance: We conducted the first integrative epigenomic study in adult B-ALLs, as a correlative study to the ECOG E2993 phase III clinical trial. This study links for the first time the direct actions of oncogenic fusion proteins with disruption of epigenetic regulation mediated by cytosine methylation. We identify a novel clinically actionable biomarker in B-ALLs: IL2RA(CD25), which is linked with BCR–ABL1 and an inflammatory signaling network associated with chemotherapy resistance. We show that BCL6 is a novel MLL fusion protein target that is required to maintain the proliferation and survival of primary human adult MLLr cells and provide the basis for a clinical trial with BCL6 inhibitors for patients with MLLr. Cancer Discov; 2(11); 1004–23. ©2012 AACR. Read the Commentary on this article by Cimmino and Aifantis, p. 976. This article is highlighted in the In This Issue feature, p. 961

Bone marrow‐derived macrophages (BMMs) treated with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) or macrophage colony‐stimulating factor (M‐CSF), differentiate into GM‐CSF‐induced mouse bone marrow‐derived macrophages (GM‐BMMs) or M‐CSF‐induced mouse bone marrow‐derived macrophages (M‐BMMs), which have an M1 or M2 profile, respectively. GM‐BMMs produce large amounts of proinflammatory cytokines and mediate resistance to pathogens, whereas M‐BMMs produce antiinflammatory cytokines that contribute to tissue repair and remodeling. M‐BMMs stimulated with lipopolysaccharide (LPS) are in an antiinflammatory state, with an IL‐12lowIL‐10high phenotype. However, the regulation of this process remains unclear. Klf10 belongs to the family of Krüppel‐like transcription factors and was initially described as a TGF‐β inducible early gene 1. IL‐12p40 is upregulated in LPS‐stimulated M‐BMMs from Klf10‐deficient mice, but downregulated during Klf10 overexpression. Klf11, another member of the Krüppel‐like factor family, can also repress the production of IL‐12p40. Furthermore, Klf10 binds to the CACCC element of the IL‐12p40 promoter and inhibits its transcription. We have therefore identified Klf10 as a transcription factor that regulates the expression of IL‐12p40 in M‐BMMs.

Abstract Recent molecular and pathologic evidence indicate that high-grade serous carcinomas of the ovary, which are the most common type of epithelial carcinomas, may arise from the distal part of fallopian tubes or tubal carcinoma implants. Oviductal glycoprotein 1 (Ovgp1), a glycosylated protein found in the epithelial cells in the oviduct, is the only specific tubal marker so far. Ovgp1 promoter-driven SV40 large T-antigen transgenic mice develop tumors in the female reproductive organs. To characterize these tumors, we performed immunohistochemical analysis in paraffin-embedded sections. Mice were sacrificed at 8 or 13 weeks old. Macroscopically we observed large tumors in the uterus and cervix and markedly enlarged region in the oviduct. Due to the large tumor in the uterus and cervix, we could not keep the mice longer than 13 weeks. Histologically, there were no major differences in these affected regions between 8 weeks old and 13 weeks old mice. We observed, (i) relatively small ovaries and ovarian follicular hyperplasia, (ii) intact ovarian surface epithelium showing no atypical histology, (iii) atypical epithelial hyperplasia within the oviduct (with endometrioid architectural and cytologic features), (iv) leiomyosarcomatous proliferation in the myometrium, and (v) squamous carcinoma in situ in the cervix or early invasive cervical cancer with extensive papillary architecture. We did not detect metastasis from the tumor to other organs. The atypical abnormalities of the oviduct were more significant in the proximal site to the uterus than the distal site. The proximal oviduct demonstrated weak and spotty p53 expression, beneath the epithelium, similar to p53 signature in the human oviduct. We hypothesized that stem cell components may contribute to the tumor development and that putative cancer stem cells could be identified by stem cell markers. In the oviduct epithelium, Bmi1 expression was not detectable. Cytoplasmic expression of Sox2 was observed in all epithelial cells of the oviduct. There was little correlation between Sox2/Bmi1 staining and p53 staining in the oviduct. Interestingly, Sox2 and Bmi1 staining in the wild-type control mice showed immunoreactive cells interspersed between glandular structures, a pattern similar to previously described label-retaining cells in the endometrium. The expression pattern in Ovgp1-SV40 mice, Bmi1 and Sox2 expression were more obvious in the endometrial stromal area but not in the leiomyosarcoma region, which implies that stem cell components in the endometrial region may support the leiomyosarcoma progression in this mouse model. In conclusion, Ovgp1-SV40 transgenic mouse is a useful model for studies involving fallopian and lower genital tract carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3272. doi:1538-7445.AM2012-3272

Abstract Background: Estrogen receptors are over-expressed in around 70% of breast cancer cases. The genetic changes that occur during aromatase inhibitor (AI) treatment are not well understood and may differ depending upon the patient's response phenotype. Methods: We performed whole genome sequencing (WGS) of matched blood, pre-treatment, and post-treatment biopsy samples from 22 estrogen receptor positive breast cancer patients treated with neoadjuvant aromatase inhibitors. For 5 cases, we performed the whole genome sequencing (WGS) on patients’ matched normal, two pre AI-treatment, and two post AI-treatment DNA isolates from biopsy samples. We validated all putative coding and non-coding somatic mutations using deep sequencing. By comparing the validated somatic mutations from pre- and post- AI treatment biopsy samples, we were able to determine the alterations in the tumor genomes. In every case we defined the clonal architecture of each pair of pre-treatment and post-treatment biopsy samples by comparing the variant allele frequencies from thousands of validated somatic mutations. Results: Comparisons of the two pre AI-treatment biopsy samples from the same patient indicates that the variant allele frequencies of mutations showed high concordances in all 5 cases, 0.74 to 0.95 range of correlation coefficient. Only a small percentage of somatic mutations were detected in one pre-treatment sample and not the other (4.65% overall). In comparing the somatic variations between pre-treatment and matched post-treatment biopsy samples in 22 cases, we found that patients with good clinical response to AI treatment retained known driver mutations only in their pre-treatment tumors. Conversely, those patients with poor clinical response presented new driver mutations in their post-treatment samples. Furthermore, the variant allele frequency for most mutated genes decreased in post AI treatment samples for patients with good AI treatment response; on the contrary, the variant allele frequency increased for patients with poor clinical response. Conclusions: From WGS of matched normal, pre-treatment, and post-treatment biopsy samples, we identified new driver genes mutated in patients with poor clinical response, while patients with good clinical response had lost mutated driver genes in their post-treatment biopsy samples. The genetic landscape revealed by WGS of pre-treatment and post-treatment biopsy samples reveals mutational repertoires are remodeled by AI therapy. This finding suggests deep sequencing of AI treated samples will be necessary to reveal the complete complement of mutations present in a patient's tumor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-423. doi:1538-7445.AM2012-LB-423

Abstract Phospholipase C epsilon (PLCε or PLC∈ 1) is a phosphoinositide-specific enzyme phospholipase C that converts phosphatidylinositol 4,5-biphosphate to two intracellular second messengers, diacylglycerol and inositol 1,4,5-trisphosphate, which regulate protein kinase C activity and calcium mobilization, respectively. These responses are involved in the regulation of cell growth, differentiation and oncogenesis. However, the involvement of PLCε in human carcinogenesis, particularly in esophageal cancer, was not been recognized until a susceptible SNP (rs2274223: A5780G:His1927Arg) was recently identified in a large genome-wide association study (GWAS). This SNP, located within exon 26 and C2 domain of the PLCε gene at chromosome 10q23, leads to a nonsynonymous alteration from CAC (A allele, encoding histidine) to CGC (G allele, encoding arginine), which was associated with an increased risk of both squamous cell carcinoma and esophagus-cardia gastric adenocarcinoma. In the current report, we demonstrate that the presence of the G allele (AG or GG) at this locus leads to increased PLCε mRNA and protein expression in human esophageal cancer tissues and in human esophageal cancer cell lines. The present of G allele also leads o a higher enzyme activity in esophageal cancer cells in vitro. Quantitative analysis of the C2 domain sequences revealed that A:G allelic imbalance is strongly associated with esophageal malignancy. Moreover, the analysis of 10,614 non-cancer subjects from high- and low-risk areas of China demonstrated that the presence of the G allele is strongly associated with moderate to severe esophagitis in the subjects from the high-risk areas of China: 77% of the severe esophagitis individuals in high-risk areas had AG/GG genotypes vs. only 37% of these subjects in low-risk areas (OR 6.03 with 95% CI 1.59-22.9 vs. OR 0.74 with 95% CI 0.33-1.64; p=0.008). These data suggest that the interaction of potential environmental factors with PLCε, particularly in individuals with AG or GG allele, not only exists in high-risk areas for esophageal cancer development in China, but also correlates with the severity of esophagitis. In conclusion, PLCε is likely to play a pivotal role in esophageal carcinogenesis: the presence of the 5780G allele may not only predict a high risk of future esophageal cancer development, but may also participate in esophageal cancer growth and progression by upregulating levels of PLCε mRNA, protein, and enzyme activity, ultimately leading to augmentation of the inflammatory process in esophageal epithelium. Thus, the 5780G allele in PLCε may constitute a promising biomarker for esophageal squamous carcinoma risk stratification, early detection, and progression prediction. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4704. doi:10.1158/1538-7445.AM2011-4704

Providing personalized treatments designed to maximize benefits and minimizing harms is of tremendous current medical interest. One problem in this area is the evaluation of the interaction between the treatment and other predictor variables. Treatment effects in subgroups having the same direction but different magnitudes are called quantitative interactions, whereas those having opposite directions in subgroups are called qualitative interactions (QIs). Identifying QIs is challenging because they are rare and usually unknown among many potential biomarkers. Meanwhile, subgroup analysis reduces the power of hypothesis testing and multiple subgroup analyses inflate the type I error rate. We propose a new Bayesian approach to search for QI in a multiple regression setting with adaptive decision rules. We consider various regression models for the outcome. We illustrate this method in two examples of phase III clinical trials. The algorithm is straightforward and easy to implement using existing software packages. We provide a sample code in Appendix A. Copyright © 2012 John Wiley & Sons, Ltd.

Background Adrenomedullin (ADM) is a newly discovered vasodilating and natriuretic peptide that may play an important role in cardiorenal regulation. Although ADM was originally isolated from human pheochromocytoma, ADM-like immunoreactivity has also been widely detected in various tissues, including the cardiovascular system. Methods and Results In view of reports that ADM circulates in the body and that ADM gene and ADM-like immunoreactivity are present in the heart, the present study was designed to determine the plasma concentration of ADM in healthy subjects and in patients with congestive heart failure (CHF) and to investigate the immunohistochemical presence and localization of ADM in normal and failing human hearts. Plasma ADM concentration was 13.2±2.3 pg/mL in healthy subjects (n=11) and increased to 47.3±6.7 pg/mL in patients with CHF (n=11, P <.05 versus normal). Human cardiac tissues were obtained from five patients with end-stage CHF undergoing cardiac transplantation. Five normal donor hearts that were used for cardiac transplantation served as sources for normal atrial tissues. Normal ventricular myocardium was also obtained by endomyocardial biopsy from the right ventricles of these donor hearts immediately before cardiac transplantation. Positive immunostaining was detected within the myocardia in both atria and ventricles of healthy and severely failing human transplanted hearts and was more intense in the atria than in the ventricles. Although there were no significant differences in the intensity of immunoreactivity between normal and failing atria, ADM immunoreactivity was significantly more intense in the ventricular myocytes from failing hearts compared with normal hearts. Conclusions The present study demonstrates that plasma concentration of ADM is increased in patients with CHF and that ADM is present in the human heart. ADM immunoreactivity is markedly increased in the failing human ventricle, suggesting that ventricular ADM expression may be influenced by the circumstances associated with CHF. This supports a potential role for this newly identified vasoactive and natriuretic peptide, ADM, in the neurohumoral activation that characterizes human CHF.

The present study determined circulating concentrations of atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) in Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) and also investigated the vasorelaxing action of ANP and CNP on isolated contracted aorta. We also defined the vasorelaxing action of a novel and newly synthesized 27-amino acid chimera of ANP and CNP termed vasonatrin peptide (VNP), which we compared with ANP and CNP in WKY rats and SHR. Plasma and urinary cyclic GMP and sodium excretion were also investigated. Plasma ANP was increased in SHR in contrast to no change in circulating CNP. Plasma and urinary cyclic GMP and sodium excretion were no different between WKY rats and SHR. In WKY rats maximal relaxations to VNP in aortic rings without endothelium were greater than those to ANP and CNP. In SHR aortic rings the potency of VNP relaxation was preserved, the actions of ANP were enhanced, and the actions of CNP were markedly impaired. In association with these vasorelaxing actions, these data suggest that (1) circulating CNP is not different in SHR and WKY rats, but the aortic relaxing action of CNP is markedly impaired in SHR; (2) endogenous plasma ANP is significantly increased in SHR without associated increases in plasma or urinary cyclic GMP; (3) there is an increase in aortic relaxation to exogenous ANP in SHR; and (4) VNP has a potent endothelium-independent aortic relaxing action in both WKY rats and SHR.(ABSTRACT TRUNCATED AT 250 WORDS)