Tuneable endogenous mammalian target complementation via multiplexed plasmidbased recombineering

Understanding the quantitative functional consequences of human disease mutations requires silencing of endogenous genes and expression of mutants at close to physiological levels. Changing protein levels above or below these levels is also important for system perturbation and modelling. Fast design optimization demands flexible interchangeable cassettes for endogenous gene silencing and tuneable expression. Here, we introduce ‘TEMTAC’, a multigene recombineering and delivery system for simultaneous siRNA-based knockdown and regulated mutant (or other variant) expression with different dynamic ranges. We show its applicability by confirming known phenotypic effects for selected mutations for BRAF, HRAS, and SHP2

Circulating tumor DNA tracking through driver mutations as a liquid biopsy-based biomarker for uveal melanoma

© The Author(s). 2021 Open Access. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.Background: Uveal melanoma (UM) is the most common intraocular tumor in adults. Despite good primary tumor control, up to 50% of patients develop metastasis, which is lethal. UM often presents asymptomatically and is usually diagnosed by clinical examination and imaging, making it one of the few cancer types diagnosed without a biopsy. Hence, alternative diagnostic tools are needed. Circulating tumor DNA (ctDNA) has shown potential as a liquid biopsy target for cancer screening and monitoring. The aim of this study was to evaluate the feasibility and clinical utility of ctDNA detection in UM using specific UM gene mutations. Methods: We used the highly sensitive digital droplet PCR (ddPCR) assay to quantify UM driver mutations (GNAQ, GNA11, PLCβ4 and CYSTLR2) in cell-free DNA (cfDNA). cfDNA was analyzed in six well established human UM cell lines with known mutational status. cfDNA was analyzed in the blood and aqueous humor of an UM rabbit model and in the blood of patients. Rabbits were inoculated with human UM cells into the suprachoroidal space, and mutated ctDNA was quantified from longitudinal peripheral blood and aqueous humor draws. Blood clinical specimens were obtained from primary UM patients (n = 14), patients presenting with choroidal nevi (n = 16) and healthy individuals (n = 15). Results: The in vitro model validated the specificity and accuracy of ddPCR to detect mutated cfDNA from UM cell supernatant. In the rabbit model, plasma and aqueous humor levels of ctDNA correlated with tumor growth. Notably, the detection of ctDNA preceded clinical detection of the intraocular tumor. In human specimens, while we did not detect any trace of ctDNA in healthy controls, we detected ctDNA in all UM patients. We observed that UM patients had significantly higher levels of ctDNA than patients with nevi, with a strong correlation between ctDNA levels and malignancy. Noteworthy, in patients with nevi, the levels of ctDNA highly correlated with the presence of clinical risk factors. Conclusions: We report, for the first time, compelling evidence from in vitro assays, and in vivo animal model and clinical specimens for the potential of mutated ctDNA as a biomarker of UM progression. These findings pave the way towards the implementation of a liquid biopsy to detect and monitor UM tumors.info:eu-repo/semantics/publishedVersio

Long-term responders on olaparib maintenance in high-grade serous ovarian cancer: Clinical and molecular characterization

Purpose: Maintenance therapy with olaparib has improved progression-free survival in women with high-grade serous ovarian cancer (HGSOC), particularly those harboring BRCA1/2 mutations. The objective of this study was to characterize long-term (LT) versus short-term (ST) responders to olaparib. Experimental Design: A comparative molecular analysis of Study 19 (NCT00753545), a randomized phase II trial assessing olaparib maintenance after response to platinum-based chemotherapy in HGSOC, was conducted. LT response was defined as response to olaparib/placebo > 2 years, ST as < 3 months. Molecular analyses included germline BRCA1/2 status, three-biomarker homologous recombination deficiency (HRD) score, BRCA1 methylation, and mutational profiling. Another olaparib maintenance study (Study 41; NCT01081951) was used as an additional cohort. Results: Thirty-seven LT (32 olaparib) and 61 ST (21 olaparib) patients were identified. Treatment was significantly associated with outcome (P < 0.0001), with more LT patients on olaparib (60.4%) than placebo (11.1%). LT sensitivity to olaparib correlated with complete response to chemotherapy (P < 0.05). In the olaparib LT group, 244 genetic alterations were detected, with TP53, BRCA1, and BRCA2 mutations being most common (90%, 25%, and 35%, respectively). BRCA2 mutations were enriched among the LT responders. BRCA methylation was not associated with response duration. High myriad HRD score (>42) and/or BRCA1/2 mutation was associated with LT response to olaparib. Study 41 confirmed the correlation of LT response with olaparib and BRCA1/2 mutation. Conclusions: Findings show that LT response to olaparib may be multifactorial and related to homologous recombination repair deficiency, particularly BRCA1/2 defects. The type of BRCA1/2 mutation warrants further investigation. (C) 2017 AACR

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Regulation of site-specific liver metastasis by extracellular matrix proteins

Metastatic disease remains the main cause of death from cancer. Few therapeutic options for patients have demonstrated potential in curing metastatic disease. The molecular mechanisms underlying site-specific metastasis and the factors mediating tumor cell homing remain largely unknown. Based on a murine Lewis lung carcinoma tumour model of site-specific metastasis mediated by the expression of the insulin-like growth factor - I receptor (IGF-IR), we identified ECM components that show particular promise in regulating metastasis to a specific site. Specifically, we identified collagen IVα1 and α2 as differentially expressed in liver- and lung-colonizing cells. The overexpression of these genes caused major changes to cell structure and function including differences in cellular morphology, anchorage-independent growth, and resulted in a switch from a lung- to a liver-metastasizing phenotype. These changes were at least in part due to α2-integrin-mediated activation of focal adhesion kinase (FAK) and protection from anoikis. Collagen IV α1 suppression resulted in increased anoikis and decreased tumour cell colonization of the liver, making it an essential and sufficient gene in liver metastasis in our model. Moreover, type IV collagen overexpression resulted in major changes to ECM and ECM-degradation genes decreasing MMP-3, MMP-9, MMP-13, and collagen type III. Uveal melanoma cells with distinct metastatic phenotypes also showed major changes to these genes. Finally, by analyzing human specimens of metastatic disease, collagen IV was shown to be expressed only in metastatic and specifically hepatic metastases when compared to primary tumours and metastases to other organs. Collectively, these findings implicate collagen IV as a clinically relevant marker and potential target against site-specific metastasis to the liver.Le cancer métastatique ne présente que peu d'options thérapeutiques et de ce fait constitue la cause principale de décès chez les patients qui en sont atteints. Par ailleurs, les mécanismes moléculaires responsables de l'atteinte d'organes-cibles de la métastase par les cellules cancéreuses demeurent largement méconnus. La thèse qui suit présente l'identification d'un marqueur moléculaire qui apparait comme étant un facteur crucial dans l'atteinte des organes-cibles par les cellules métastatiques. En effet, la constituante de la matrice extracellulaire collagene IVα1 et α2, semble être exprimée de manière distinctive chez les cellules colonisant le foie et les poumons. Nous basant sur un modèle murin de métastase provenant de carcinome pulmonaire et véhiculée par l'expression de IGF-IR, nous avons identifié des changements majeurs quant à la structure et à la fonction des cellules cancéreuses, suite à la surexpression des gènes du Collagene IVα1 et α2. Ces changements comprennent, entre autre, des différences au niveau de la morphologie et la croissance cellulaire et ont aussi pour résultat la mutation du phénotype métastatique de pulmonaire à hépatique. Ces changements sont en partie conséquence de l'activation de la kinase d'adhésion focale (FAK) et de la protection de l'anoikis. Par contre, la suppression de l'expression du Collagène IV accroit l'anoikis et diminue la colonisation du foie par les cellules cancéreuses. En outre, la surexpression du Collagène IV apporte des changements majeurs au niveau de la matrice extracellulaire et aux gènes de dégradation de celle-ci, diminuant MMP-3, MMP-9, MMP-13 et le collagène III. Les cellules de mélanome uvéale à phénotype métastatique distinct présentent aussi des changements importants quant à l'expression ces gènes. Finalement, après analyse de spécimens humains, le collagène IV semble être exprimé exclusivement au niveau des tumeurs métastatiques et e

COMPUTER-ASSISTED MECHANISTIC EVALUATION OF PERICYCLIC REACTIONS

CAMEO, an interactive computer program that assists in the mechanistic evaluation of organic reactions, is under continued development. It has been significantly expanded by the addition of a module for thermal pericyclic chemistry. Sophisticated predictions of the likelihood and regioselectivity of cycloadditions involving up to 10 (pi) electrons are now possible. The heuristics were based on the frontier molecular orbital method. Consequently, it was necessary to devise efficient algorithms for the prediction of the energies and relative coefficients of frontier molecular orbitals. Sigmatropic processes, including the {2,3} and the {3,3} rearrangements as well as hydrogen and carbon migrations, have also been implemented. In addition, the necessary heuristics for and the incorporation of electrocyclic reactions, cheletropic extrusions, and the reverse Diels-Alder reactions are described. Finally, a general scheme for the treatment of periselectivity is presented