SUMMARY Alternative intronic polyadenylation (IPA) can generate truncated protein isoforms with significantly altered functions. Here, we describe 31 dominant-negative, secreted variant isoforms of receptor tyrosine kinases (RTKs) that are produced by activation of intronic poly(A) sites. We show that blocking U1-snRNP can activate IPA, indicating a larger role for U1-snRNP in RNA surveillance. Moreover, we report the development of an antisense-based method to effectively and specifically activate expression of individual soluble decoy RTKs (sdRTKs) to alter signaling, with potential therapeutic implications. In particular, a quantitative switch from signal transducing full-length vascular endothelial growth factor receptor-2 (VEGFR2/KDR) to a dominant-negative sKDR results in a strong anti-angiogenic effect both on directly targeted cells and on naïve cells exposed to conditioned media, suggesting a role for this approach in interfering with angiogenic paracrine and autocrine loops.
Splicing factor hnRNPH drives an oncogenic splicing switch in gliomasThis study reveals two alternative splicing events that contribute to the development of glioma. HnRNPH is shown to control production of a pro-survival splice variant of the death-domain adaptor protein IG20-MADD and the motility-enhancing isoform of the RON receptor tyrosine kinase.
Acute myeloid leukemia (AML) is the most common acute leukemia in adults, with approximately four new cases per 100,000 persons per year. Standard treatment for AML consists of induction chemotherapy with remission achieved in 50 to 75% of cases. Unfortunately, most patients will relapse and die from their disease, as 5-y survival is roughly 29%. Therefore, other treatment options are urgently needed. In recent years, immune-based therapies have led to unprecedented rates of survival among patients with some advanced cancers. Suppression of T cell function in the tumor microenvironment is commonly observed and may play a role in AML. We found that there is a significant association between T cell infiltration in the bone marrow microenvironment of newly diagnosed patients with AML and increased overall survival. Functional studies aimed at establishing the degree of T cell suppression in patients with AML revealed impaired T cell function in many patients. In most cases, T cell proliferation could be restored by blocking the immune checkpoint molecules PD-1, CTLA-4, or TIM3. Our data demonstrate that AML establishes an immune suppressive environment in the bone marrow, in part through T cell checkpoint function.
Infectious disease, commonly caused by bacterial pathogens, is now the world's leading cause of premature death and third overall cause behind cardiovascular disease and cancer. Urinary Tract Infection (UTI), caused by E. coli bacteria, is a very common bacterial infection, a majority in women (85%) and may result in severe kidney failure if not detected quickly. Among hundreds of strains the bacteria, E. coli 0157:H7, is emerging as the most aggressive one because of its capability to produce a toxin causing hemolytic uremic syndrome (HUS) resulting in death, especially in children. In the present study, a project has been undertaken for developing a rapid method for UTI detection in very low bacteria concentration, applying current knowledge of nano-technology. Experiments have been designed for the development of biosensors using nano-fabricated structures coated with elements such as gold that have affinity for biomolecules. A biosensor is a device in which a biological sensing element is either intimately connected to or integrated within a transducer. The basic principle for the detection procedure of the infection is partly based on the enzyme-linked immunosorbent assay system. Anti-E. coli antibody-bound Gold Nanowire Arrays (GNWA) prepared on anodized porous alumina template is used for the primary step followed by binding of the bacteria containing specimen. An alkaline phosphatase-conjugated second antibody is then added to the system and the resultant binding determined by both electrochemical and optical measurements. Various kinds of GNWA templates were used in order to determine the one with the best affinity for antibody binding. In addition, an efficient method for enhanced antibody binding has been developed with the covalent immobilization of an organic linker Dithiobissuccinimidylundecanoate (DSU) on the GNWA surface. Studies have also been conducted to optimize the antibody-binding conditions to the linker-attached GNWA surfaces for their ability to detect bacteria in clinical concentrations.
Current limitations to applications of monoclonal antibody (mAb) targeted isotope generators in radioimmunotherapy include the low mAb labeling yields and the non-specific radiation of normal tissues by non-targeted radioimmunoconjugates (RIC). Radiotoxicity occurs in normal organs that metabolize radiolabeled proteins and peptides, primarily liver and kidneys, or in radiosensitive organs with prolonged exposure to the isotope from the blood, such as the bone marrow. Actinium-225 nanogenerators also have the problem of released alpha emitting daughters. We developed two new bifunctional chelating agents (BCA) in order to address these issues. Thiol-maleimide conjugation chemistry was employed to increase the efficiency of the mAb radiolabelings by up to 8 fold. In addition, one bifunctional chelating agent incorporated a cleavable linker to alter the catabolism of the alpha particle emitting mAb conjugate. This linker was designed to be sensitive to cathepsins to allow release and clearance of the chelated radiometal after internalization of the radioimmunoconjugate into the cell. We compared the properties of the cleavable conjugate (mAb-DOTA-G3FC) to non-cleavable constructs (mAb-DOTA-NCS and mAb-DOTA-SH). The cleavable RIC was able to release 80% of its radioactive payload when incubated with purified cathepsin B. The catabolism of the constructs mAb-DOTA-G3FC and mAb-DOTA-NCS was investigated in vitro and in vivo. RIC integrity was retained at 85% over a period of 136 hours in mouse serum in vivo. Both conjugates were degraded over time inside HL-60 cells after internalization and in mouse liver in vivo. While we found that the rates of degradation of the two RICs in those conditions were similar, the amounts of the radiolabeled product residues were different. The cleavable mAb-DOTA-G3FC conjugate yielded a larger proportion of fragments below 6kDa in size in mouse liver in vivo after 12 hours than the DOTA-NCS conjugate. Biodistribution studies in mice showed that the mAb-DOTA-G3FC construct yielded a higher liver dose and prolonged liver retention of radioactivity compared to the mAb-DOTA-NCS conjugate. The accumulation in the liver seemed to be in part caused by the maleimide functionalization of the antibody, since the non-cleavable mAb-DOTA-SH maleimide-functionalized control conjugate displayed the same biodistribution pattern. These results provide an insight into the catabolism of RICs, by demonstrating that the release of the radioisotope from a RIC is not a sufficient condition to allow the radioactive moiety to clear from the body. The excretion mechanisms of radiolabeled fragments seem to constitute a major limiting step in the chain of events leading to their clearance.
BackgroundEpigenome-wide studies in hepatocellular carcinoma (HCC) have identified numerous genes with aberrant DNA methylation. However, methods for triaging functional candidate genes as useful biomarkers for epidemiological study have not yet been developed.MethodsWe conducted targeted next-generation bisulfite sequencing (bis-seq) to investigate associations of DNA methylation and mRNA expression in HCC. Integrative analyses of epigenetic profiles with DNA copy number analysis were used to pinpoint functional genes regulated mainly by altered DNA methylation.ResultsSignificant differences between HCC tumor and adjacent non-tumor tissue were observed for 28 bis-seq amplicons, with methylation differences varying from 12% to 43%. Available mRNA expression data in Oncomine were evaluated. Two candidate genes (GRASP and TSPYL5) were significantly under-expressed in HCC tumors in comparison with precursor and normal liver tissues. The expression levels in tumor tissues were, respectively, 1.828 and − 0.148, significantly lower than those in both precursor and normal liver tissue. Validations in an additional 42 paired tissues showed consistent under-expression in tumor tissue for GRASP (−7.49) and TSPYL5 (−9.71). A highly consistent DNA hypermethylation and mRNA repression pattern was obtained for both GRASP (69%) and TSPYL5 (73%), suggesting that their biological function is regulated by DNA methylation. Another two genes (RGS17 and NR2E1) at Chr6q showed significantly decreased DNA methylation in tumors with loss of DNA copy number compared to those without, suggesting alternative roles of DNA copy number losses and hypermethylation in the regulation of RGS17 and NR2E1.ConclusionsThese results suggest that integrative analyses of epigenomic and genomic data provide an efficient way to filter functional biomarkers for future epidemiological studies in human cancers.Electronic supplementary materialThe online version of this article (doi:10.1186/s12920-015-0105-1) contains supplementary material, which is available to authorized users.
Background:To assess antivascular effects, and evaluate clinically translatable magnetic resonance imaging (MRI) biomarkers of tumour response in vivo, following treatment with vanucizumab, a bispecific human antibody against angiopoietin-2 (Ang-2) and vascular endothelial growth factor-A (VEGF-A).Methods:Colo205 colon cancer xenografts were imaged before and 5 days after treatment with a single 10 mg kg−1 dose of either vanucizumab, bevacizumab (anti-human VEGF-A), LC06 (anti-murine/human Ang-2) or omalizumab (anti-human IgE control). Volumetric response was assessed using T2-weighted MRI, and diffusion-weighted, dynamic contrast-enhanced (DCE) and susceptibility contrast MRI used to quantify tumour water diffusivity (apparent diffusion coefficient (ADC), × 106 mm2 s−1), vascular perfusion/permeability (Ktrans, min−1) and fractional blood volume (fBV, %) respectively. Pathological correlates were sought, and preliminary gene expression profiling performed.Results:Treatment with vanucizumab, bevacizumab or LC06 induced a significant (P<0.01) cytolentic response compared with control. There was no significant change in tumour ADC in any treatment group. Uptake of Gd-DTPA was restricted to the tumour periphery in all post-treatment groups. A significant reduction in tumour Ktrans (P<0.05) and fBV (P<0.01) was determined 5 days after treatment with vanucizumab only. This was associated with a significant (P<0.05) reduction in Hoechst 33342 uptake compared with control. Gene expression profiling identified 20 human genes exclusively regulated by vanucizumab, 6 of which are known to be involved in vasculogenesis and angiogenesis.Conclusions:Vanucizumab is a promising antitumour and antiangiogenic treatment, whose antivascular activity can be monitored using DCE and susceptibility contrast MRI. Differential gene expression in vanucizumab-treated tumours is regulated by the combined effect of Ang-2 and VEGF-A inhibition.
Introduction: Daratumumab (DARA) is a human IgG1κ monoclonal antibody that binds with high affinity to a unique epitope on CD38. DARA monotherapy has shown promising activity in relapsed and/or refractory multiple myeloma (MM) patients with a median of 5 prior lines of therapy in two clinical studies (Study GEN501; Lokhorst HM. J Clin Oncol. 2014;32 Suppl:abstr 8513 and Study MMY2002; Lonial S. J Clin Oncol. 2015;33 Suppl:abstr LBA8512). The aim of this analysis was to identify proteins indicative of DARA's multiple mechanisms of action (MOA) and potential predictive pharmacodynamic response markers. A broad aptamer based proteomics platform (SomaSCANTM) evaluated clinical serum samples at study entry and during treatment to determine proteins and biological pathways associated with DARA MOA or clinical response. Methods: All patients were treated with 16 mg/kg DARA and whole blood samples were collected at baseline and after 8 weeks of treatment. Blood samples were processed for serum and stored frozen until batch analysis. Profiling of 1129 serum proteins was performed using the SOMAscan assay. Patients were classified based on overall best response: responders (stringent and complete responses, very good partial and partial responses), stable disease (stable disease or minimal response) and non-responders (progressive disease). Differential level testing included application of the Wilcoxon rank sum test and Limma for responder versus non-responder analysis, and ANOVA for repeated-measures with post-hoc test validation, Wilcoxon signed rank and Friedman tests for baseline versus on-treatment analysis. Results: In MMY2002 at baseline, 51 proteins were significantly different between responders and non-responders. Many have known associations with MM or CD38, and interestingly a subset is associated with T-cell biology. We recently observed that DARA induces a multi-factorial T-cell response in patients including CD8+ T-cell expansion and activation, and increased clonality. Proteins differentially expressed between responders/non-responders at baseline included tumor necrosis factor subfamily 8 (TNFSF8/CD30L), TNFSF9/CD137L, macrophage stimulating 1 (MST1), interleukin-1B (IL1B), cadherin 1 (CDH1) and cadherin 3 (CDH3). Protein profiles were evaluated at baseline and at 8 weeks (Cycle 3 Day 1) to study pharmacodynamic changes. Significant treatment-induced changes were identified in 142 proteins. Of particular interest were the 60 proteins that changed differentially over time in responders vs non-responders. Proteins associated with MM tumor load, such as beta-2-microglobulin [B2M], and immunoglobulins decreased in responders and increased in non-responders. Novel MM therapeutic targets such as signaling lymphocyte activation molecule F7 [SLAMF7] (i.e. CS1) and B-cell maturation antigen [BCMA] decreased in responders and increased in non-responders during DARA treatment. Many proteins associated with immune or T-cell response were also significantly changed by DARA treatment, including TNFRSF1B, CD163, TNFRSF25, TLR2, CCL5, IL5RA, FCGR2A, ICOS, Granzyme B, and programmed cell death ligand 1 [PD-L1] many of which were differential between responders and non-responders. Differential level testing of GEN501 samples identified a small set of proteins that were significantly altered by DARA treatment over time. Many of these proteins overlapped with those identified in the MMY2002 analysis, increasing confidence in the statistical results. Conclusions: This exploratory serum proteomic analysis identified proteins that were differentially expressed between responders and non-responders at baseline, including proteins associated with immune response and T-cell biology (TNFSF8/CD30L, TNFSF9/CD137L, IL1B). In addition, significant differential changes in protein expression between responders and non-responders after DARA treatment were seen. Many of these are associated with MM tumor load (BCMA, immunoglobulins, SLAMF7, and B2M) and decreased in responders and increased in non-responders. In addition, proteins related to T-cell activity, immune checkpoints and immune response (TNFRSF1B, CD163, TNFRSF25, TLR2, CCL5, IL5RA, FCGR2A, ICOS, Granzyme B, PD-L1) also showed changes associated with DARA treatment, supporting the recent findings that DARA induces a T-cell response in MM patients that may contribute to clinical response. Disclosures Casneuf: Janssen: Employment. Lysaght:Immuneering Corp: Employment, Other: Stockholder. LeFave:Immuneering Corp.: Employment. Bald:Janssen: Employment. Weiss:Janssen and Millennium: Consultancy; Janssen and Onclave: Research Funding. van de Donk:Janssen Pharmaceuticals: Research Funding; Amgen: Research Funding; Celgene: Research Funding. Lokhorst:Amgen: Honoraria; Janssen: Honoraria, Research Funding; Genmab: Honoraria, Research Funding. Ahmadi:Janssen: Employment.
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