Citations

The scaffold protein synectin plays a critical role in the trafficking and regulation of membrane receptor pathways. As platelet-derived growth factor receptor (PDGFR) is essential for hepatic stellate cell (HSC) activation and liver fibrosis, we sought to determine the role of synectin on the PDGFR pathway and development of liver fibrosis. Mice with deletion of synectin from HSC were found to be protected from liver fibrosis. mRNA sequencing revealed that knockdown of synectin in HSC demonstrated reductions in the fibrosis pathway of genes, including PDGFR-β. Chromatin IP assay of the PDGFR-β promoter upon synectin knockdown revealed a pattern of histone marks associated with decreased transcription, dependent on p300 histone acetyltransferase. Synectin knockdown was found to downregulate PDGFR-α protein levels, as well, but through an alternative mechanism: protection from autophagic degradation. Site-directed mutagenesis revealed that ubiquitination of specific PDGFR-α lysine residues was responsible for its autophagic degradation. Furthermore, functional studies showed decreased PDGF-dependent migration and proliferation of HSC after synectin knockdown. Finally, human cirrhotic livers demonstrated increased synectin protein levels. This work provides insight into differential transcriptional and posttranslational mechanisms of synectin regulation of PDGFRs, which are critical to fibrogenesis.

Abstract Cancer has been considered a heterogeneous disease, which mainly arises from the accumulation of genetic mutations, leading to a loss of tumor suppressor genes, as well as to an activation of oncogenes. The epigenetic mechanisms, such as DNA and histone modifications, are processes that regulate gene expression, and an alteration of these can contribute to cancer progression. Methylation on histone 3 lysine 9 (H3K9), in particular di-methyl (H3K9me2), is mainly mediated by the nuclear histone lysine methyltransferase G9a. Preliminary data showed that G9a was overexpressed in aggressive and highly metastatic forms of cancer. However, to date, this pathway has remained unstudied in esophageal adenocarcinoma. Interestingly, epigenetic drugs, such as those that target G9a, have the potential to reverse the adverse effects acquired downstream from genetic mutations, which currently cannot be directly repaired or targeted. In the current study, we utilize BRD4770, an S-adenosylmethionine (SAM) mimetic inhibitor of G9a, to determine the benefits of inhibiting this pathway in the context of esophageal adenocarcinoma. We hypothesize that pharmacological targeting of G9a by BRD4770 inhibits esophageal adenocarcinoma cell growth. To examine G9a-mediated dimethyl histone H3K9 levels in esophageal adenocarcinoma, we performed immunohistochemistry on paraffin-embedded esophageal adenocarcinoma tissues with an H3K9me2 antibody. In order to confirm specificity of BRD4770 in regulating the dimethyl histone H3K9 levels, and thus trimethyl H3K9 levels, in esophageal adenocarcinoma, we treated human esophageal adenocarcinoma cells (SKGT-4) with different doses of BRD4770 at various time points for western blot analysis using antibodies against histone H3K9me2 and H3K9me3. Moreover, we found that treatment of SKGT-4 cells with BRD4770 significantly inhibits cell proliferation by MTS assay, as well as clonogenic survival. Therefore, to determine the mechanism(s) by which BRD4770 inhibits the growth of esophageal adenocarcinoma cells, we utilized Caspase-3, Senescence, and Autophagy Assays. Our data shows that inhibition of G9a by BRD4770 does not induce Caspase 3 activity, while it significantly increases senescence and autophagy. Combined, our results suggest that BRD4770 has an important pharmacological role in regulating methyl histone H3K9 levels that may lead to inhibition of esophageal adenocarcinoma cell growth via an increase in senescence and autophagy. Further studies are focused on in vivo models to understand these effects in more detail, however the fact that an anti-G9a drug antagonizes esophageal adenocarcinoma cell growth raises optimism for its potential in the treatment of this malignancy. Note: This abstract was not presented at the meeting. Citation Format: Mabel G. Perez-Oquendo, Gwen A. Lomberk, Raul A. Urrutia, Sarah Kossak, Navtej Buttar, Sounak Gupta. Treatment with the G9a antagonist, BRD4770, inhibits esophageal adenocarcinoma cell growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-094. doi:10.1158/1538-7445.AM2017-LB-094

Abstract Patient-derived xenografts (PDX) are appearing as a prime approach for preclinical studies despite being insufficiently characterized as a model of the human disease and its diversity. In this work, 29 PDX were obtained from either surgery or endoscopic ultrasound-guided fine needle aspirate of pancreatic adenocarcinoma. The extensive genomic profiling of these pancreatic PDX, revealed two clinically-relevant subtypes having broad similarities with human primary tumors. These subtypes are defined by highly specific DNA methylation and transcriptomic profiles (mRNA, miRNA or lncRNA) but are not distinguishable by exonic mutations or copy number aberrations. Moreover, by specifically analyzing the stroma transcriptome, as defined by the expression of murine transcripts, we found that it is able to stratify the patients with the same efficiency than the analysis of grafted human tumor cells. This finding suggest that transformed pancreatic cells drive the composition of their own stroma. Finally, the multiomics analysis pinpoints novel therapeutic targets, one of which we demonstrate to be an efficient method for treating pancreatic cancer. Overall, we show that PDX are trustworthy pre-clinical models of pancreatic adenocarcinoma including of unresectable tumors. Their multiomics profiling allow the independent analysis of the uncontaminated cancer or stromal compartments and discloses several original therapeutics targets. Citation Format: Remy Nicolle, Yuna Blum, Laetitia Marisa, Celine Loncle, Odile Gayet, Vincent Moutardier, Olivier Turrini, Marc Giovannini, Benjamin Bian, Martin Bigonnet, Marion Rubis, Nabila Elarouci, Lucile Armenoult, Mira Ayadi, Pauline Duconseil, Mohamed Gasmi, Mehdi Ouaissi, Aurélie Maignan, Gwen Lomberk, Jean-Marie Boher, Jacques Ewald, Erwan Bories, Jonathan Garnier, Anthony Goncalves, Flora Poizat, Jean-Luc Raoul, Veronique Secq, Stephane Garcia, Philippe Grandval, Marine Barraud-Blanc, Emmanuelle Norguet, Marine Gilabert, Jean-Robert Delpero, Julie Roques, Ezequiel Calvo, Fabienne Guillaumond, Sophie Vasseur, Raul Urrutia, Aurélien de Reyniès, Nelson Dusetti, Juan Iovanna. Multiomics assessment of the cancer and stromal compartments of patient-derived pancreatic xenografts reveals clinically-relevant subtypes and novel targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4396. doi:10.1158/1538-7445.AM2017-4396

Abstract Pancreatic ductular adenocarcinoma (PDAC) ranks fourth as a cause of cancer death in the USA and is almost universally fatal, with the annual number of deaths equivalent to the number of newly diagnosed cases. Valuable research in the field has revealed genetic aberrations that contribute to PDAC development and progression, with KRAS being one of the most frequent mutations in more than 90% of patient samples. However, to date, any efforts to directly target KRAS have failed in the clinic. Thus, there is indisputably an urgent need to further improve our understanding of molecular mechanisms underlying PDAC development as to identify novel therapeutic targets, including druggable important downstream targets and nodes orchestrated by oncogenic KRAS. In particular, we are interested in epigenetic pathways involved in PDAC development and progression due to the potential reversibility of any alteration, unlike genetic mutation. In the current study, using a cell model that allows inducible expression of mutant KRASG12D, we find that protein levels of the dimethyl-K9H3 histone methyl transferase (HMT), G9a, and its complex partners are increased in response to activation of the oncogenic Kras pathway. Furthermore, the activation of this oncogenic pathway results in the formation of the G9a-GLP-Wiz trimer complex, as determined by affinity protein purification, combined with mass spectrometry. In vivo experiments involving the cross of the Pdx1-CRE/LSL-KRASG12D mice with G9afl/fl animals demonstrate that a loss of the H3K9Me2 mark in the nucleus of exocrine cells is accompanied by a significantly reduced number of PanIN lesions. RNA-Seq experiments from these animals reveal that these mice have reduced levels of typical molecular markers of PanINs. In addition, these experiments show changes in the levels in several genes, which have been previously been shown to synergize with Kras to mediate pancreatic cancer initiation. Congruently, pharmacological inhibition of G9a using BRD4770 displays an inhibitory effect on KRASG12D-induced cell proliferation. Combined, these data provide evidence for a key role of the meK9H3-G9a pathway as a mediator of the oncogenic Kras response and defines a novel point of potential therapeutic intervention for PDAC. Citation Format: Angela Mathison, Ann Salmonson, Brooke Paradise, Mckenna Missfeldt, Juan Iovanna, Daniel Billadeau, Raul Urrutia, Gwen Lomberk. The epigenetic regulator, G9a, is a KRAS-inducible protein and its inactivation inhibits PanIN formation by this oncogene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1391. doi:10.1158/1538-7445.AM2017-1391

Significance We describe the interaction between an intrinsically disordered protein (IDP), NUPR1, and the well-folded C-terminal region of RING1B protein of the Polycomb complex, involving residues Ala33 and Thr68 of the IDP. These observations are significant because they raise the possibility that NUPR1 aids the function of the members of the Polycomb complex, playing an active role in carcinogenesis.

Noonan syndrome (NS; MIM 163950) is an autosomal dominant disorder and a member of a family of developmental disorders termed “RASopathies,” which are caused mainly by gain-of-function mutations in genes encoding RAS/MAPK signaling pathway proteins. Whole exome sequencing (WES) and trio-based genomic triangulation of a 15-year-old female with a clinical diagnosis of NS and concomitant cardiac hypertrophy and her unaffected parents identified a de novo variant in MRAS -encoded RAS-related protein 3 as the cause of her disease. Mutation analysis using in silico mutation prediction tools and molecular dynamics simulations predicted the identified variant, p.Gly23Val-MRAS, to be damaging to normal protein function and adversely affect effector interaction regions and the GTP-binding site. Subsequent ectopic expression experiments revealed a 40-fold increase in MRAS activation for p.Gly23Val-MRAS compared with WT-MRAS. Additional biochemical assays demonstrated enhanced activation of both RAS/MAPK pathway signaling and downstream gene expression in cells expressing p.Gly23Val-MRAS. Mutational analysis of MRAS in a cohort of 109 unrelated patients with phenotype-positive/genotype-negative NS and cardiac hypertrophy yielded another patient with a sporadic de novo MRAS variant (p.Thr68Ile, c.203C>T). Herein, we describe the discovery of mutations in MRAS in patients with NS and cardiac hypertrophy, establishing MRAS as the newest NS with cardiac hypertrophy-susceptibility gene.

AbstractBackgroundKleefstra Syndrome (KS) (MIM# 610253) is an autosomal dominant disorder caused by haploinsufficiency of euchromatic histone methyltransferase‐1 (EHMT1, GLP). EHMT1 (MIM# 607001) encodes a histone methyltransferase that heterodimerizes with EHMT2 (also known as G9a, MIM# 604599), which together are responsible for mono‐ and dimethylation of H3 lysine 9 (H3K9me1 and ‐me2), resulting in transcriptional repression of target genes.MethodsThis report describes an 18‐year‐old woman with intellectual disability, severely limited speech, hypotonia, microcephaly, and facial dysmorphisms, who was found to have a novel de novo single‐base frameshift deletion in EHMT1.ResultsFunctional studies using patient fibroblasts showed decreased H3K9me2 compared to wild‐type control cells, thus providing a rapid confirmatory test that complements molecular studies.ConclusionWhole exome sequencing revealed a novel frameshift deletion in EHMT1 after a lengthy diagnostic odyssey in this patient. Functional testing using this patient's fibroblasts provides proof‐of‐concept for the analysis of variants of uncertain significance that are predicted to impact EHMT1 enzymatic activity.

Loeys-Dietz syndrome (LDS) is a connective tissue disorder characterized by vascular findings of aneurysm and/or dissection of cerebral, thoracic, or abdominal arteries and skeletal findings. We report a case of a novel pathogenic variant in TGFBR2 and phenotype consistent with classic LDS. The proband was a 10-year-old presenting to the genetics clinic with an enlarged aortic root (Z-scores 5-6), pectus excavatum, and congenital contractures of the right 2nd and 3rd digit. Molecular testing of TGFBR2 was sent to a commercial laboratory and demonstrated a novel, likely pathogenic, variant in exon 4, c.1061T>C, p.(L354P). Molecular modeling reveals alteration of local protein structure as a result of this pathogenic variant. This pathogenic variant has not been previously reported in LDS and thus expands the pathogenic variant spectrum of this condition.

Key Clinical MessageA de novo GFAP variant, p.R376W, was identified in a child presenting with hypotonia, developmental delay, and abnormal brain MRI. Following the 2015 ACMG variant classification guidelines and the functional studies showing protein aggregate formation in vitro, p.R376W should be classified as a pathogenic variant, causative for Alexander disease.