LMO2 and IL2RG synergize in thymocytes to mimic the evolution of SCID-X1 gene therapy-associated T-cell leukaemia

The SCID-X1 disease occurs in males that lack a functional X-linked gene encoding the interleukin 2 receptor subunit gamma (IL2RG) and thus are immuno-deficient (reviewed in Rochman et al.). Gene therapy has been a success in curing SCID-X1 in patients receiving autologous CD34+-bone marrow cells infected with retroviruses expressing IL2RG. This treatment protocol has, however, produced adverse T-cell effects where clonal T-cell leukaemias arose, and four have insertional mutagenesis of the T-cell oncogene LMO2. LMO2 is a T-cell oncogene first discovered via chromosomal translocations in T-cell acute leukaemia (T-ALL) (reviewed in Chambers and Rabbitts). It is unclear if the T-cell neoplasias in the SCID-X1 patients are simply due to insertional activation of the LMO2 gene or reflect synergy between LMO2 and IL2RG. Further, the recurrent involvement of LMO2 in SCID-X1 leukaemias is puzzling as other T-cell oncogenes (for example, TAL1/SCL, HOX11 and LYL1) might equally have been targets. This suggests that specific properties of LMO2 per se are required in these adverse events. The oncogenic potential of IL2RG itself also remains controversial. Although it causes T-cell lymphomas in mice transplanted with virally transduced haematopoetic stem cells, other studies have indicated that IL2RG is not an oncogene. Here we provide evidence that synergy is required between LMO2 and IL2RG proteins specifically in the T-cell lineage to elicit neoplasias and that additional mutations are required such as Notch1 mutations like those in human T-ALL

RAD51 Is a Selective DNA Repair Target to Radiosensitize Glioma Stem Cells.

Patients with glioblastoma die from local relapse despite surgery and high-dose radiotherapy. Resistance to radiotherapy is thought to be due to efficient DNA double-strand break (DSB) repair in stem-like cells able to survive DNA damage and repopulate the tumor. We used clinical samples and patient-derived glioblastoma stem cells (GSCs) to confirm that the DSB repair protein RAD51 is highly expressed in GSCs, which are reliant on RAD51-dependent DSB repair after radiation. RAD51 expression and RAD51 foci numbers fall when these cells move toward astrocytic differentiation. In GSCs, the small-molecule RAD51 inhibitors RI-1 and B02 prevent RAD51 focus formation, reduce DNA DSB repair, and cause significant radiosensitization. We further demonstrate that treatment with these agents combined with radiation promotes loss of stem cells defined by SOX2 expression. This indicates that RAD51-dependent repair represents an effective and specific target in GSCs

Cancer cell killing by target antigen engagement with engineered complementary intracellular antibody single domains fused to pro-caspase3

Many tumour causing proteins, such as those expressed after chromosomal translocations or from point mutations, are intracellular and are not enzymes per se amenable to conventional drug targeting. We previously demonstrated an approach (Antibody-antigen Interaction Dependent Apoptosis (AIDA)) whereby a single anti-β-galactosidase intracellular single chain Fv antibody fragment, fused to inactive procaspase-3, induced auto-activation of caspase-3 after binding to the tetrameric β-galactosidase protein. We now demonstrate that co-expressing an anti-RAS heavy chain single VH domain, that binds to mutant RAS several thousand times more strongly than to wild type RAS, with a complementary light chain VL domain, caused programmed cell death (PCD) in mutant RAS expressing cells when each variable region is fused to procaspase-3. The effect requires binding of both anti-RAS variable region fragments and is RAS-specific, producing a tri-molecular complex that auto-activates the caspase pathway leading to cell death. AIDA can be generally applicable for any target protein inside cells by involving appropriate pairs of antigen-specific intracellular antibodies

EGFRvIII upregulates DNA mismatch repair resulting in increased temozolomide sensitivity of MGMT promoter methylated glioblastoma

The oncogene epidermal growth factor receptor variant III (EGFRvIII) is frequently expressed in glioblastomas (GBM) but its impact on therapy response is still under controversial debate. Here we wanted to test if EGFRvIII influences the sensitivity towards the alkylating agent temozolomide (TMZ). Therefore, we retrospectively analyzed the survival of 336 GBM patients, demonstrating that under standard treatment, which includes TMZ, EGFRvIII expression is associated with prolonged survival, but only in patients with O6-methylguanine-DNA methyltransferase (MGMT) promoter methylated tumors. Using isogenic GBM cell lines with endogenous EGFRvIII expression we could demonstrate that EGFRvIII increases TMZ sensitivity and results in enhanced numbers of DNA double-strand breaks and a pronounced S/G2-phase arrest after TMZ treatment. We observed a higher expression of DNA mismatch repair (MMR) proteins in EGFRvIII+ cells and patient tumor samples, which was most pronounced for MSH2 and MSH6. EGFRvIII-specific knockdown reduced MMR protein expression thereby increasing TMZ resistance. Subsequent functional kinome profiling revealed an increased activation of p38- and ERK1/2-dependent signaling in EGFRvIII expressing cells, which regulates MMR protein expression downstream of EGFRvIII. In summary, our results demonstrate that the oncoprotein EGFRvIII sensitizes a fraction of GBM to current standard of care treatment through the upregulation of DNA MMR

Evidence for intron length conservation in a set of mammalian genes associated with embryonic development

<p>Abstract</p> <p>Background</p> <p>We carried out an analysis of intron length conservation across a diverse group of nineteen mammalian species. Motivated by recent research suggesting a role for time delays associated with intron transcription in gene expression oscillations required for early embryonic patterning, we searched for examples of genes that showed the most extreme conservation of total intron content in mammals.</p> <p>Results</p> <p>Gene sets annotated as being involved in pattern specification in the early embryo or containing the homeobox DNA-binding domain, were significantly enriched among genes with highly conserved intron content. We used ancestral sequences reconstructed with probabilistic models that account for insertion and deletion mutations to distinguish insertion and deletion events on lineages leading to human and mouse from their last common ancestor. Using a randomization procedure, we show that genes containing the homeobox domain show less change in intron content than expected, given the number of insertion and deletion events within their introns.</p> <p>Conclusions</p> <p>Our results suggest selection for gene expression precision or the existence of additional development-associated genes for which transcriptional delay is functionally significant.</p

Randomised, placebo-controlled, phase 3 trial of the effect of the omega-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) on colorectal cancer recurrence and survival after surgery for resectable liver metastases: EPA for Metastasis Trial 2 (EMT2) study protocol

AbstractIntroduction There remains an unmet need for safe and cost-effective adjunctive treatment of advanced colorectal cancer (CRC). The omega-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) is safe, well-tolerated and has anti-inflammatory as well as antineoplastic properties. A phase 2 randomised trial of preoperative EPA free fatty acid 2 g daily in patients undergoing surgery for CRC liver metastasis showed no difference in the primary endpoint (histological tumour proliferation index) compared with placebo. However, the trial demonstrated possible benefit for the prespecified exploratory endpoint of postoperative disease-free survival. Therefore, we tested the hypothesis that EPA treatment, started before liver resection surgery (and continued postoperatively), improves CRC outcomes in patients with CRC liver metastasis.Methods and analysis The EPA for Metastasis Trial 2 trial is a randomised, double-blind, placebo-controlled, phase 3 trial of 4 g EPA ethyl ester (icosapent ethyl (IPE; Vascepa)) daily in patients undergoing liver resection surgery for CRC liver metastasis with curative intent. Trial treatment continues for a minimum of 2 years and maximum of 4 years, with 6 monthly assessments, including quality of life outcomes, as well as annual clinical record review after the trial intervention. The primary endpoint is CRC progression-free survival. Key secondary endpoints are overall survival, as well as the safety and tolerability of IPE. A minimum 388 participants are estimated to provide 247 CRC progression events during minimum 2-year follow-up, allowing detection of an HR of 0.7 in favour of IPE, with a power of 80% at the 5% (two sided) level of significance, assuming drop-out of 15%.Ethics and dissemination Ethical and health research authority approval was obtained in January 2018. All data will be collected by 2025. Full trial results will be published in 2026. Secondary analyses of health economic data, biomarker studies and other translational work will be published subsequently.Trial registration number NCT03428477

Left-Right Function of dmrt2 Genes Is Not Conserved between Zebrafish and Mouse

Background: Members of the Dmrt family, generally associated with sex determination, were shown to be involved in several other functions during embryonic development. Dmrt2 has been studied in the context of zebrafish development where, due to a duplication event, two paralog genes dmrt2a and dmrt2b are present. Both zebrafish dmrt2a/terra and dmrt2b are important to regulate left-right patterning in the lateral plate mesoderm. In addition, dmrt2a/terra is necessary for symmetric somite formation while dmrt2b regulates somite differentiation impacting on slow muscle development. One dmrt2 gene is also expressed in the mouse embryo, where it is necessary for somite differentiation but with an impact on axial skeleton development. However, nothing was known about its role during left-right patterning in the lateral plate mesoderm or in the symmetric synchronization of somite formation. Methodology/Principal Findings: Using a dmrt2 mutant mouse line, we show that this gene is not involved in symmetric somite formation and does not regulate the laterality pathway that controls left-right asymmetric organ positioning. We reveal that dmrt2a/terra is present in the zebrafish laterality organ, the Kupffer’s vesicle, while its homologue is excluded from the mouse equivalent structure, the node. On the basis of evolutionary sub-functionalization and neo-functionalization theories we discuss this absence of functional conservation. Conclusions/Significance: Our results show that the role of dmrt2 gene is not conserved during zebrafish and mous

Cytoplasmic Prep1 Interacts with 4EHP Inhibiting Hoxb4 Translation

embryo development. Interestingly, Prep1 contains a putative binding motif for 4EHP, which may reflect a novel unknown function. development effect. mRNA to the 5′ cap structure. This is the first demonstration that a mammalian homeodomain transcription factor regulates translation, and that this function can be possibly essential for the development of female germ cells and involved in mammalian zygote development