AML associated oncofusion proteins PML-RARA, AML1-ETO and CBFB-MYH11 target RUNX/ETS-factor binding sites to modulate H3ac levels and drive leukemogenesis

Chromosomal translocations are one of the hallmarks of acute myeloid leukemia (AML), often leading to gene fusions and expression of an oncofusion protein. Over recent years it has become clear that most of the AML associated oncofusion proteins molecularly adopt distinct mechanisms for inducing leukemogenesis. Still these unique molecular properties of the chimeric proteins converge and give rise to a common pathogenic molecular mechanism. In the present study we compared genome-wide DNA binding and transcriptome data associated with AML1-ETO, CBFB-MYH11 and PML-RARA oncofusion protein expression to identify unique and common features. Our analyses revealed targeting of oncofusion binding sites to RUNX1 and ETS-factor occupied genomic regions. In addition, it revealed a highly comparable global histone acetylation pattern, similar expression of common target genes and related enrichment of several biological pathways critical for maintenance of AML, suggesting oncofusion proteins deregulate common gene programs despite their distinct binding signatures and mechanisms of action.Peer reviewe

Stealth monoolein-based nanocarriers for delivery of siRNA to cancer cells

While the delivery of small interfering RNAs (siRNAs) is an attractive strategy to treat several clinical con- ditions, siRNA-nanocarriers stability after intravenous administration is still a major obstacle for the development of RNA-interference based therapies. But, although the need for stability is well recognized, the notion that strong stabilization can decrease nanocarriers efficiency is sometimes neglected. In this work we evaluated two stealth functionalization strategies to stabilize the previously validated dioctade- cyldimethylammonium bromide (DODAB):monoolein (MO) siRNA-lipoplexes. The nanocarriers were pre- and post-pegylated, forming vectors with different stabilities in biological fluids. The stealth nanocarriers behavior was tested under biological mimetic conditions, as the production of stable siRNA-lipoplexes is determinant to achieve efficient intravenous siRNA delivery to cancer cells. Upon incubation in human serum for 2 h, by fluorescence Single Particle Tracking microscopy, PEG-coated lipo- plexes were found to have better colloidal stability as they could maintain a relatively stable size. In addi- tion, using fluorescence fluctuation spectroscopy, post-pegylation also proved to avoid siRNA dissociation from the nanocarriers in human serum. Concomitantly it was found that PEG-coated lipoplexes improved cellular uptake and transfection efficiency in H1299 cells, and had the ability to silence BCR-ABL, affecting the survival of K562 cells. Based on an efficient cellular internalization, good silencing effect, good siRNA retention and good col- loidal stability in human serum, DODAB:MO (2:1) siRNA-lipoplexes coated with PEG-Cer are considered promising nanocarriers for further in vivo validation. Statement of Significance This work describes two stealth functionalization strategies for the stabilization of the previously validated dioctadecyldimethylammonium bromide (DODAB):monoolein (MO) siRNA-lipoplexes. These nanocarriers are capable of efficiently incorporating and delivering siRNA molecules to cells in order to silence genes whose expression is implicated in a pathological condition. The main objective was to functionalize these nanocarriers with a coating conferring protection to siRNA in blood without compromising its efficient delivery to cancer cells, validating the potential of DODAB:MO (2:1) siRNA-lipoplexes as therapeutic vec- tors. We show that the stealth strategy is determinant to achieve a stable and efficient nanocarrier, and that DODAB:MO mixtures have a very promising potential for systemic siRNA delivery to leukemic cells.FEDER through POFC-COMPETE and by national funds from FCT I.P. through the strategic funding UID/BIA/04050/2013 (CBMA) and PEst-C/FIS/UI0607/2013 (CFUM) and PTDC/QUI/69795/2006. We thank the support of the Frame Work Program 7 of the European Commission: BIOCAPS (316265, FP7/REGPOT) and Xunta de Galicia, Spain (Agrupamento INBIOMED, Grupo con potencial crecimiento) reference IF/00498/2012, scholarship SFRH/BD/68588/2010. NanoDelivery-I&D em Bionanotecnologia, Lda. for access to their equipment

Plasticity of Lgr5-Negative Cancer Cells Drives Metastasis in Colorectal Cancer

Colorectal cancer stem cells (CSCs) express Lgr5 and display extensive stem cell-like multipotency and self-renewal and are thought to seed metastatic disease. Here, we used a mouse model of colorectal cancer (CRC) and human tumor xenografts to investigate the cell of origin of metastases. We found that most disseminated CRC cells in circulation were Lgr5- and formed distant metastases in which Lgr5+ CSCs appeared. This p

Dioctadecyldimethylammonium:monoolein nanocarriers for efficient in vitro gene silencing

This study describes a novel liposomal formulation for siRNA delivery, based on the mixture of the neutral lipid monoolein (MO) and cationic lipids of the dioctadecyldimethylammonium (DODA) family. The cationic lipids dioctadecyldimethylammonium bromide (DODAB) and chloride (DODAC) were compared in order to identify which one will most efficiently induce gene silencing. MO has a fluidizing effect on DODAC and DODAB liposomes, although it was more homogeneously distributed in DODAC bilayers. All MO-based liposomal formulations were able to efficiently encapsulate siRNA. Stable lipoplexes of small size (100-160 nm) with a positive surface charge (>+45 mV) were formed. A more uniform MO incorporation in DODAC:MO may explain an increase of the fusogenic potential of these liposomes. The siRNA-lipoplexes were readily internalized by human nonsmall cell lung carcinoma (H1299) cells, in an energy dependent process. DODAB:MO nanocarriers showed a higher internalization efficiency in comparison to DODAC:MO lipoplexes, and were also more efficient in promoting gene silencing. MO had a similar gene silencing ability as the commonly used helper lipid 1,2-dioleyl-3-phosphatidylethanolamine (DOPE), but with much lower cytotoxicity. Taking in consideration all the results presented, DODAB:MO liposomes are the most promising tested formulation for systemic siRNA delivery.This work was supported by FEDER through POFC - COMPETE and by national funds from FCT through the projects PEst-C/BIA/UI4050/2011 (CBM.A), PEst-C/FIS/UI0607/2011 (CFUM), and PTDC/QUI/69795/2006, while Ana Oliveira holds scholarship SFRH/BD/68588/2010. Eloi Feitosa thanks FAPESP (2011/03566-0) and CNPq (303030/2012-7), and Renata D. Adati thanks FAPESP for scholarship (2011/07414-0). K. Raemdonck is a postdoctoral fellow of the Research Foundation - Flanders (FWO-Vlaanderen). We acknowledge NanoDelivery-I&D em Bionanotecnologia, Lda. for access to their equipment

Plasticity of Lgr5-Negative Cancer Cells Drives Metastasis in Colorectal Cancer

Colorectal cancer stem cells (CSCs) express Lgr5 and display extensive stem cell-like multipotency and self-renewal and are thought to seed metastatic disease. Here, we used a mouse model of colorectal cancer (CRC) and human tumor xenografts to investigate the cell of origin of metastases. We found that most disseminated CRC cells in circulation were Lgr5− and formed distant metastases in which Lgr5+ CSCs appeared. This plasticity occurred independently of stemness-inducing microenvironmental factors and was indispensable for outgrowth, but not establishment, of metastases. Together, these findings show that most colorectal cancer metastases are seeded by Lgr5− cells, which display intrinsic capacity to become CSCs in a niche-independent manner and can restore epithelial hierarchies in metastatic tumors

War, Taxes and Borders: How Beer Created Belgium

The present-day border between Belgium and the Netherlands traces back to the separation of the Low Countries after the Dutch Revolt (1566-1648) against Spanish rule. The capacity to finance war expenditures played a central role in the outcome of this conflict. Excise taxes on beer consumption were the single largest income source in Holland, the leading province of the Dutch Republic. Beer taxes thus played a crucial role in financing the Dutch Revolt which led to the separation of the Low Countries and, eventually, the creation of Belgium

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly