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The mutational landscape is shaped by many processes. Genic regions are vulnerable to mutation but are preferentially protected by transcription-coupled repair1. In microorganisms, transcription has been demonstrated to be mutagenic2,3; however, the impact of transcription-associated mutagenesis remains to be established in higher eukaryotes4. Here we show that ID4—a cancer insertion–deletion (indel) mutation signature of unknown aetiology5 characterized by short (2 to 5 base pair) deletions —is due to a transcription-associated mutagenesis process. We demonstrate that defective ribonucleotide excision repair in mammals is associated with the ID4 signature, with mutations occurring at a TNT sequence motif, implicating topoisomerase 1 (TOP1) activity at sites of genome-embedded ribonucleotides as a mechanistic basis. Such TOP1-mediated deletions occur somatically in cancer, and the ID-TOP1 signature is also found in physiological settings, contributing to genic de novo indel mutations in the germline. Thus, although topoisomerases protect against genome instability by relieving topological stress6, their activity may also be an important source of mutations in the human genome. We thank S. Jinks-Robertson for suggesting the traffic light reporter approach; H. Klein for guidance on fluctuation assays; R. van Boxtel for sharing sequencing data for MLH1-KO organoids; A. Bretherick, O. B. Reina and G. Kudla for advice on HygroR re-coding; staff at the IGC core services (L. Murphy, C. Nicol, C. Warnock, E. Freyer, S. Brown and J. Joseph), C. Logan, A. Fluteau, A. Robertson and the staff at Edinburgh Genomics for technical assistance; staff at Liverpool CLL Biobank (funded by Blood Cancer UK) for samples used to generate GEL WGS data; A. Ewing, C.-A. Martin, N. Hastie and W. Bickmore for discussions. Funding for this work: UK Medical Research Council Human Genetics Unit core grants (MC_UU_00007/5 to A.P.J., MC_UU_00007/11 to M.S.T.); Edinburgh Clinical Academic Track PhD programme (Wellcome Trust 204802/Z/16/Z) to T.C.W.; 2021 AACR-Amgen Fellowship in Clinical/Translational Cancer Research (grant number 21-40-11-NADE) to F.N.; a CRUK Brain Tumour Centre of Excellence Award (C157/A27589) to M.D.N.; EKFS research grant (2019_A09), Wilhelm Sander-Stiftung (2019.046.1) to K.A., CRUK programme grant (C20807/A2864) to T.S.; La Caixa Foundation (CLLEvolution-LCF/PR/HR17/52150017, Health Research 2017 Program HR17-00221) to E.C.; E.C. is an Academia Researcher of the Institució Catalana de Recerca i Estudis Avançats of the Generalitat de Catalunya. Edinburgh Genomics is partly supported by NERC (R8/H10/56), MRC (MR/K001744/1) and BBSRC (BB/J004243/1). This research was made possible through access to the data and findings generated by the 100,000 Genomes Project. The 100,000 Genomes Project is managed by Genomics England Limited (a wholly owned company of the Department of Health and Social Care). The 100,000 Genomes Project is funded by the National Institute for Health Research and NHS England. The Wellcome Trust, Cancer Research UK and the Medical Research Council have also funded research infrastructure. The 100,000 Genomes Project uses data provided by patients and collected by the National Health Service as part of their care and support. Peer Reviewed "Article signat per 22 autors/es: Martin A. M. Reijns, David A. Parry, Thomas C. Williams, Ferran Nadeu, Rebecca L. Hindshaw, Diana O. Rios Szwed, Michael D. Nicholson, Paula Carroll, Shelagh Boyle, Romina Royo, Alex J. Cornish, Hang Xiang, Kate Ridout, The Genomics England Research Consortium, Colorectal Cancer Domain UK 100,000 Genomes Project, Anna Schuh, Konrad Aden, Claire Palles, Elias Campo, Tatjana Stankovic, Martin S. Taylor & Andrew P. Jackson "

Heterogeneous upregulation of multiple prosurvival pathways underlies resistance to damage-induced apoptosis in acute lymphoblastic leukemia (ALL) cells despite normal p53 responses. Here, we show that the dual combination of insulin-like growth factor 1 (IGF1)/IGF1 receptor (IGF1/R) and mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibition using AG1024 + U0126 can sensitize apoptosis-resistant ALL cells to ionizing radiation-induced DNA damage irrespective of effect of single pathway inhibition in vitro. This AG1024 + U0126 combination also significantly potentiates the ability of the core chemotherapy compounds vincristine, dexamethasone, and daunorubicin to kill ALL cells in vitro. Evidence of the synergistic action of AG1024 + U0126 in samples with variable basal levels of phosphorylated IGF1/Rβ and ERK1/2 suggested additional targets of this drug combination. Consistent with this, gene expression profiling identified 32 "synergy genes" differentially targeted by IGF1/R + MEK inhibition and, among these, Signal transducer and activator of transcription 6 (STAT6) and platelet-derived growth factor-associated protein 1 (PDAP1) were the most differentially downregulated cluster. Pearson correlation analyses revealed that STAT6 and PDAP1 display significant expression codependency and a common expression pattern linked with other key "synergy" genes, supporting their predicted role in an STAT6-ERK-nuclear factor kappa beta (NF-κB) network. Knockdown studies revealed that loss of STAT6, but not PDAP1, impinges on the cell cycle, causing reduced numbers of viable cells. In combination with daunorubicin, STAT6 loss has an additive effect on cell killing, whereas PDAP1 loss is synergistic, indicating an important role of PDAP1 in the cellular response to this anthracycline. Inhibition of STAT6 or PDAP1 may therefore represent a potential novel therapeutic strategy for resistant ALL by enhancing sensitivity to chemotherapy.

Chronic lymphocytic leukaemia (CLL) cells require microenvironmental support for their proliferation. This can be recapitulated in highly immunocompromised hosts in the presence of T cells and other supporting cells. Current primary CLL xenograft models suffer from limited duration of tumour cell engraftment coupled with gradual T-cell outgrowth. Thus, a greater understanding of the interaction between CLL and T cells could improve their utility. In this study, using two distinct mouse xenograft models, we investigated whether xenografts recapitulate CLL biology, including natural environmental interactions with B-cell receptors and T cells, and whether manipulation of autologous T cells can expand the duration of CLL engraftment. We observed that primary CLL xenografts recapitulated both the tumour phenotype and T-cell repertoire observed in patients and that engraftment was significantly shorter for progressive tumours. A reduction in the number of patient T cells that were injected into the mice to 2-5% of the initial number or specific depletion of CD8+ cells extended the limited xenograft duration of progressive cases to that characteristic of indolent disease. We conclude that manipulation of T cells can enhance current CLL xenograft models and thus expand their utility for investigation of tumour biology and pre-clinical drug assessment., Summary: Manipulation of T cells can enhance current CLL xenograft models, expanding their utility for investigation of tumour biology and pre-clinical drug assessment.

Multiple cancers represent 2.42\% of all human cancers and are mainly double or triple cancers. Many possible causes of multiple malignancies have been reported such as genetic alterations, exposure to anti-cancer chemotherapy, radiotherapy, immunosuppressive therapy and reduced immunologic response. We report a female patient with multiple sclerosis and quadruple cancers of different embryological origin. Patient was diagnosed with stage III (T3, N1a, MO) medullary thyroid carcinoma (MTC), multicentric micropapillary thyroid carcinoma, scapular and lumbar melanomas (Clark II, Breslow II), and lobular invasive breast carcinoma (T1a, NO, MO). All tumors present in our patient except micropapillary thyroid carcinomas were investigated for gene alterations known to have a key role in cancer promotion and progression. Tumor samples were screened for the p16 alterations (loss of heterozygosity and homozygous deletions), loss of heterozygosity of PTEN, p53 alterations (mutational status and loss of heterozygosity) and mutational status of RET, HRAS and KRAS. Each type of tumor investigated had specific pattern of analyzed genetic alterations. The most prominent genetic changes were mutual alterations in PTEN and p53 tumor suppressors present in breast cancer and two melanomas. These co-alterations could be crucial for promoting development of multiple malignancies. Moreover the insertion in 4th codon of HRAS gene was common for all tumor types investigated. It represents frameshift mutation introducing stop codon at position 5 which prevents synthesis of a full-length protein. Since the inactivated RAS enhances sensitivity to tamoxifen and radiotherapy this genetic alteration could be considered as a good prognostic factor for this patient. Ministry of Education, Science and Technological Development of the Republic of Serbia {[}III41031]

Background: Persistently infected cells by herpesvirus (HSV, CMV, EBV) and their dysfunction are associated with different disorders. The immune and endocrine systems are involved in pathogenesis of herpesvirus infection. Changes of IFN-γ, IL-4, DHEAS and cortisol level are a part of the regulatory mechanisms of the immune and endocrine system responses in persistent herpesvirus infection. Objectives: The aim of this study was to identify immunoregulatory mechanisms (clinical manifestations, cytokines level-IFN-γ, et IL-4, hormones level-DHEAS, cortisol, parameters of oxidative metabolism, enzymes of cell injury) and their consequences concerning clinical disease expression (low level of hemoglobin (Hb), leucocitopenia, high level of monocytes, hyper gamaglobulins, low level of NBT-test). Study design: 40 children (1-16 years old) with herpesvirus infection were analyzed. In patients routine laboratory investigations were performed: white blood cells count, lactate dehydrogenize-LDH, creatinin-kinaza- CPK, oxidative metabolism of the blood phagocytes, the ability of phagocytes nitro-blue tetrazolium-NBT reduction and immunoglobulin serum level. Serum levels of IFN-γ, IL-4, DHEAS and cortisol were measured by ELISA test. Normal healthy controls had negative ELISA tests for CMV, HSV and EBV Results: The high level of LDH, CPK, low ability of NBT reduction and hypergammaglobulins were detected. The increases of IFN-γ, IL-4 level, as well as, the decrease of DHEAS and cortisol level were present. The patients with the high level of IFN-γ, had the high level of LDH, CPK, GOT, GPT, the low level of Hb, leucocitopenia and monocytes. Conclusions: Two groups of patients were presented. First with the high level of IFN-γ (70%) and second with the high level of IL-4.The patients in the first group, with the high level of IFN-γ, had anemia, respiratory infections, BHR, labial HSV, stomatitis aphtosa. Vasculitis, anginae recidivans, was clinical manifestation characteristics for in patients in the second group with the high level of IL-4. The high level of LDH, CPK, the low ability of NBT reduction and hypergammaglobulins were detected. The patients with the high levels of IFN-γ, had the high level of LDH, CPK, GOT, GPT, the low level of Hb, leukopenia and monocytosis. The decreased level of DHEAS (50%) and the normal level of cortisol (76%) were present. The low levels of DHEAS followed the IgG high values while leucopenia (78%), was followed by high values of DHEAS. The patients with normal cortisol values had the significant monocytosis (80%) while patients with the low cortisol level had the high IgG (85%) level.

We showed recently that mutation of the hMRE11 gene identified a new ataxia telangiectasia-like disorder (ATLD). In this report we describe the genomic organization of the hMRE11 gene and the analysis of a promoter region that appears to direct the divergent transcription of hMRE11 and the adjacent gene. The characterization of the genomic organization of the hMRE11 gene allowed us to determine the basis of an apparent null hMRE11 allele present in the mother and two patients in one of our two ATLD families. Polymorphic markers in the hMRE11 gene, including the promoter region, provided evidence that the mutated maternal allele was not deleted. An exon by exon search revealed the presence of a missense mutation in exon 15, the effect of which was to create a premature termination codon. Transcripts derived from the mutant allele were found to be subject to nonsense-mediated mRNA decay (NMD). Therefore, this allele was effectively null, because little if any mRNA from it was available for translation. The ATLD patients carrying this protein-truncating hMRE11 mutation have survived because the null allele they inherited from their mother is present with a missense mutation inherited from their father, which is expressed as normal levels of partially functional MRE11 protein. The mutation in the maternal hMRE11 allele of family 2 was also identified in a further unrelated Italian family with ATLD and also found to be subject to NMD.

The paper proposes a practical assessment approach to estimate duration of the product development process incorporating risk management principles. Risk assessment, as a crucial part of risk management process, is a systematic process for estimating the risk level of previously identified and recognized risks. Currently, the best-known relationship in risk assessment is that risk level equals the product of two factors – likelihood and impact. The proposed approach treats these factors as random variables with triangular probability distributions in order to mitigate biases due to subjective risk estimation. Based on a literature review, the approach considers probability functions assigned to estimated values which together with Monte Carlo simulation avoid fixed single-point estimations and deterministic simulation issues. Relating this kind of assessment with previously established risk mapping on the product development process broadens insight about the influence of risk on the process metrics. Since, risk assessment is still mostly approached in terms of individual reasoning, intuition or previous experience, providing a practical method for assessing risks can enhance and facilitate the usability of risk management principles in product development companies. The proposed approach is illustrated using an example, which involves an automated car washing facility product development process.

Paediatric B-precursor ALL is a highly curable disease, however, treatment resistance in some patients and the long-term toxic\ud effects of current therapies pose the need for more targeted therapeutic approaches. We addressed the cytotoxic effect of JQ1,\ud a highly selective inhibitor against the transcriptional regulators, bromodomain and extra-terminal (BET) family of proteins, in\ud paediatric ALL. We showed a potent\ud in vitro\ud cytotoxic response of a panel of primary ALL to JQ1, independent of their prognostic\ud features but dependent on high\ud MYC\ud expression and coupled with transcriptional downregulation of multiple pro-survival\ud pathways. In agreement with earlier studies, JQ1 induced cell cycle arrest. Here we show that BET inhibition also reduced c-Myc\ud protein stability and suppressed progression of DNA replication forks in ALL cells. Consistent with c-Myc depletion and\ud downregulation of pro-survival pathways JQ1 sensitised primary ALL samples to the classic ALL therapeutic agent dexamethasone.\ud Finally, we demonstrated that JQ1 reduces ALL growth in ALL xenograft models, both as a single agent and in combination with\ud dexamethasone. We conclude that targeting BET proteins should be considered as a new therapeutic strategy for the treatment of\ud paediatric ALL and particularly those cases that exhibit suboptimal responses to standard treatment.

Ataxia telangiectasia (A-T) is a human disorder that results in a number of clinical symptoms, including cerebellar degeneration and increased cancer predisposition. Recently the gene that is defective in A-T has been cloned and designated ATM. Here, we describe the production of antisera raised against the approximately 350 kDa ATM protein. Antisera specificity is confirmed by them recognising a approximately 350 kDa polypeptide in wild-type cells but not in A-T cells containing mutations that truncate ATM upstream of the antibody binding sites. We show that ATM is almost exclusively nuclear and is expressed in all cell lines and tissues analysed. However, ATM levels are not regulated in response to u.v. or ionising radiation. These data are consistent with ATM being a component of the DNA damage detection apparatus rather than being an inducible downstream effector of the DNA damage response. In addition, we analyse ATM protein expression in a variety of A-T patients. Strikingly, ATM expression is reduced drastically or absent in all patients analysed, including those predicted to express proteins that should be detected by our antisera. Thus, the A-T phenotype may result not only from mutations that disrupt functional domains of ATM, but also from mutations that destabilise the ATM mRNA or protein. Finally, we report that a group of patients displaying an intermediate A-T phenotype express low levels of apparently full-length ATM. This suggests that the ATM pathway is partially active in these individuals and that there is a correlation between levels of residual ATM expression and disease severity.