Paul Russell

Mutations truncating the EP300 acetylase in human cancers

The EP300 protein is a histone acetyltransferase that regulates transcription via chromatin remodelling and is important in the processes of cell proliferation and differentiation. EP300 acetylation of TP53 in response to DNA damage regulates its DNA-binding and transcription functions. A role for EP300 in cancer has been implied by the fact that it is targeted by viral oncoproteins, it is fused to MLL in leukaemia and two missense sequence alterations in EP300 were identified in epithelial malignancies. Nevertheless, direct demonstration of the role of EP300 in tumorigenesis by inactivating mutations in human cancers has been lacking. Here we describe EP300 mutations, which predict a truncated protein, in 6 (3%) of 193 epithelial cancers analysed. Of these six mutations, two were in primary tumours (a colorectal cancer and a breast cancer) and four were in cancer cell lines (colorectal, breast and pancreatic). In addition, we identified a somatic in-frame insertion in a primary breast cancer and missense alterations in a primary colorectal cancer and two cell lines (breast and pancreatic). Inactivation of the second allele was demonstrated in five of six cases with truncating mutations and in two other cases. Our data show that EP300 is mutated in epithelial cancers and provide the first evidence that it behaves as a classical tumour-suppressor gene.

The Contribution of Germline BRCA1 and BRCA2 Mutations to Familial Ovarian Cancer: No Evidence for Other Ovarian Cancer-Susceptibility Genes

To establish the contribution of germline BRCA1 and BRCA2 mutations to familial ovarian cancer, we have analyzed both genes in DNA samples obtained from an affected individual in each of 112 families containing at least two cases of epithelial ovarian cancer. Germline mutations were found in 43% of the families; BRCA1 mutations were approximately four times more common than BRCA2 mutations. The extent of family history of ovarian and breast cancers was strongly predictive of BRCA1-mutation status. Segregation analysis suggests that a combination of chance clustering of sporadic cases and insensitivity of mutation detection may account for the remaining families; however, the contribution of other genes cannot be excluded. We discuss the implications for genetic testing and clinical management of familial ovarian cancer arising from the data presented in these studies.

Frequent loss of BRCA1 mRNA and protein expression in sporadic ovarian cancers

Germline mutations in the BRCA1 gene cause inherited susceptibility to breast and ovarian cancers. However, somatic mutations of BRCA1 are rare in sporadic breast and ovarian tumours. To establish whether BRCA1 is altered during the development of sporadic ovarian cancer by mechanisms other than somatic mutation, we have analysed 57 sporadic epithelial ovarian tumours for BRCA1 protein and RNA expression. Reduced or absent protein expression was observed in 90% of tumours. Decreased protein expression was significantly associated with a reduction in the levels of RNA expression. Somatic mutations of BRCA1 and LOH at the BRCA1 locus were detected in 3.5% and 44% of informative tumours, respectively; there was no significant correlation between the levels of protein and RNA expression and the DNA mutation and/or LOH status. Together, these data suggest that expression of BRCA1 is down‐regulated at the level of transcription during the development of sporadic ovarian cancers. Int. J. Cancer 87:317–321, 2000.

Context Dependence of Checkpoint Kinase 1 as a Therapeutic Target for Pancreatic Cancers Deficient in the BRCA2 Tumor Suppressor

Inherited mutations in the tumor suppressor BRCA2 are predisposed to pancreatic adenocarcinomas, which carry activating mutations in the KRAS oncogene in more than 95% of cases, as well as frequent TP53 inactivation. Here, we have established an RNA interference (RNAi) screen to identify genes whose depletion selectively inhibits the growth of cells lacking BRCA2, and then studied the effects of the genetic depletion or pharmacologic inhibition of 1 candidate, the checkpoint kinase 1 (CHK1), in the context of pancreatic cancer. Pharmacologic inhibition of CHK1 using small-molecule inhibitors (CHK1i) reduced cell growth in several cell lines depleted of BRCA2. Unexpectedly, these drugs did not suppress the growth of BRCA2-deficient pancreatic cancer cell lines from humans or gene-targeted mice expressing active Kras and trans-dominant inhibitory mutant Trp53. Remarkably, the expression of KRASG12V and TP53G154V in BRCA2-depleted HEK293 cells was sufficient to render them resistant to CHK1i (but not to mitomycin C or inhibitors of PARP1). CHK1i sensitivity was restored by gemcitabine, an S-phase genotoxin used to treat pancreatic adenocarcinoma. Thus, the growth-suppressive effect of CHK1 inhibition in BRCA2-mutant tumors can be opposed by concurrent KRAS activation and TP53 mutations typical of pancreatic adenocarcinoma, and CHK1i resistance in this setting can be overcome by gemcitabine. Our findings show that approaches that use potential therapeutic targets for cancer identified in synthetic lethal RNAi screens are affected by the genetic context of specific malignancies and combination therapy with other agents. This concept should be taken into account in the ongoing and future development of targeted cancer therapies. Mol Cancer Ther; 10(4); 670–78. ©2011 AACR.

Abstract 700: Investigating KRAS synthetic lethal/co-dependency interactions using siRNA and CRISPR

No molecularly targeted therapy has yet been identified for KRAS mutant cancers. As oncogenic mutations reduce RAS enzymatic activity, classic small molecule approaches are ineffective, hence most work has focussed on drugging RAS-effector pathways. Multiple inhibitors of MEK, RAF and PI3K have been identified but toxicity issues and pathway adaptation have stymied their success against KRAS-driven cancers. An alternative approach is to exploit “non-oncogene addiction” by identifying targets with synthetic lethal or co-dependence interactions with KRAS. A number of siRNA and shRNA screens have identified targets that exhibit differential dependencies between KRAS mutant and KRAS wild-type tumours, but there is poor overlap between the different published studies. This discordance may arise from (1) the noise inherent in using cell line panels differing in much more than their KRAS mutant/wild-type status and (2) the use of RNA interference methodologies driving incomplete knockdown and associated with substantial off-target effects. Next generation screens that exploit both isogenic cell lines and cell line panels, and use a combination of knockdown and knock-out (i.e. CRISPR/Cas9-sgRNA) methodologies, may be better suited for identifying novel targets that withstand validation. However, if we are to detect co-dependence as well as synthetic lethal interactions, screens must be performed under conditions where mutant KRAS alleles are essential for growth. A library of siRNAs targeting proposed KRAS synthetic lethal targets was assembled and screened under conditions where proliferation is dependent on KRAS status. DLD1 cells harbour an activating KRASG13D mutation dispensable for proliferation in 2D, but essential for proliferation under 3D (soft agar) conditions. Knockdown of several targets including KRAS itself, PLK1, TBK1, BCL-XL & RAF1 proved more anti-proliferative under 3D conditions. This screen was extended to a panel of KRAS-mutant colon lines, with varying levels of KRAS sensitivity, where we found the requirement for RAF1 highly correlated with the requirement for KRAS. With the advent of CRISPR we are now able to design sgRNA libraries capable of probing the effect of ‘knocking out’ rather than ‘knocking down’ targets, providing a potentially superior alternative to RNA interference. Data from mouse models indicates RAF1 is required for the initiation of lung cancer by oncogenic KRAS. Although we found good correlation between sensitivity to KRAS and RAF1 depletion, we were unable to unambiguously validate RAF1 as a target in human lung cancer cells using RNA interference methodologies. However, using CRISPR-Cas9, we found complete loss of RAF1 expression was anti-proliferative in A549 cells, supporting the concept of targeting RAF1 in a KRAS mutant lung cancers. These results demonstrate that a more penetrant sgRNA based screening approach may identify novel KRAS synthetic lethal or co-dependent interactions. Citation Format: Simon F. Scrace, Elpida Tsonou, Paul Russell, Julie A. Wickenden, Steffen Lawo, Tim M. Scales, Ceri M. Wiggins, Jonathan D. Moore. Investigating KRAS synthetic lethal/co-dependency interactions using siRNA and CRISPR. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 700. doi:10.1158/1538-7445.AM2015-700

VILBIAM (RIB 154) : Kidnap or Robbery?

The form vilbiam has been long known from the earliest of the Bath curse tablets to have been discovered. It has usually, but with some reservations, been regarded as a female personal name. If so, then, this curse tablet ( RIB 154) is the only one to record the theft of a person. It is suggested here that vilbia , rather than being a personal name, is the term for some form of sharp-pointed object. It is further argued that it may be of Celtic origin and may have a Celtic reflex in Middle Welsh gwlf .

Abstract A25: Synthetic lethal CRISPR-Cas9 screen imply an oncogenic role for FBXW7 mutations in colon cancer

Mutations in tumour suppressors and un-druggable oncogenes dominate the landscape of cancer driver genes. Only a minority of colon cancers have mutations in druggable cancer drivers, such as PIK3CA. Conversely, mutations in tumour suppressors such as APC and TP53 are frequent, as are mutations in the notoriously difficult to drug KRAS target. There is an urgent need for new therapeutics to target tumours driven by these mutations: immune checkpoint approaches are likely to only prove effective in the fraction of patients whose tumours bear high mutation loads, which is colon cancer may be restricted to the minority of mismatch repair deficient cancers. The concepts of non-oncogene addiction and synthetic lethality provide a conceptual framework for finding therapeutic targets in these cancers. We have used arrayed siRNA and pooled CRISPR-Cas9 libraries to screen a panel of isogenic and non-isogenic colon cancer cell lines under conditions designed to increase the cells dependency on oncogenic pathways. This panel contains cell lines with mutations in TP53, APC, KRAS, PIK3CA and/or FBXW7 for which we are aiming to identify co-dependencies that consistently segregate with genotype. Our screens have identified a number of novel targets for which reduced expression imposes specific fitness defects on cells with mutant PIK3CA, mutant TP53 or mutant FBXW7. Few of these targets were identified by both siRNA and CRISPR-Cas9 approaches. Furthermore, isogenic pairs of cell lines have not proved helpful for identifying synthetic lethal targets in the KRAS or PIK3CA genotypes. However, we believe the increased penetrance of the CRIPSR-Cas9 approach has uncovered novel candidate synthetic lethal genes that were not found by RNA interference. Although our current screens are not saturating, we have confirmed several previously reported genetic interactions including the requirements for expression of MDM2 in TP53 wild-type cancers and HK2 in colon cancer lines with KRAS mutations. The screens in the FBXW7 genotype were particularly interesting: in addition to identifying a potential synthetic lethal interaction with a target for which a drug has recently entered the clinic, we also found that FBXW7 itself seemed to be required in many of the colon cancers with FBXW7 mutations. This suggests that FBXW7 may sometimes act as an oncogene, rather than as a tumour suppressor in cancer initiation or progression. Finally, we have attempted to validate some of the candidate synthetic lethal genes identified by RNA interference via ultra-deep CRISPR-CAS9 pooled screening, extending the approach of the Vakoc lab (Nature Biotech. 33, 661-667, 2015). We can confirm this method can highlight functional domains of particular significance to a protein9s function, but note that many-fold drop outs of sgRNAs can also occur where they target multiple sites in the genome, presumably via a DNA damage response. Citation Format: Jonathan D. Moore, Chantelle Hudson, Paul Russell, Gaganpreet Tiwana, David Walter, Ceri M. Wiggins, Joanne Yarker. Synthetic lethal CRISPR-Cas9 screen imply an oncogenic role for FBXW7 mutations in colon cancer [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A25.

Abstract 4620: Wild-type IDH1: A molecular target in IDH1 mutant cancers

Neomorphic mutations targeting R132 of the TCA cycle enzyme, IDH1, have been identified in multiple cancer types and lead to a build up of (R)-2-hydroxyglutarate ((R)-2HG). Several mechanisms have been proposed to account for mutant-IDH1-mediated transformation: due to its structure similarity with alpha-ketoglutarate (α-KG), (R)-2HG can inhibit α-KG-dependent enzymes that act as tumour suppressors such as TET2 or EGLN, (R)-2HG might inhibit electron transport chain function, or rapid (R)-2HG generation may deplete the cellular pool of α-KG leading to depletion of NADPH. Recently, it has also emerged that IDH1 may be required to catalyse a series of back reactions through TCA cycle components (reductive decarboxylation) to facilitate fatty acid synthesis from media glutamine, particularly under hypoxic conditions. According to some reports in the literature, heterodimer formation between mutant an wild-type alleles of IDH1 is important for the production of high levels of (R)-2HG. We were interested in exploring novel ways to target tumour cells bearing mutant IDH1 alleles that were distinct from the obvious opportunity available to identify mutant-specific IDH1 inhibitors. The potential metabolic vulnerabilities of mutant IDH1 cancers raised the possibility that wild-type IDH1 might be essential for tumourigenesis or tumour maintenance in this context. We therefore employed Horizon9s rAAV-mediated homologous recombination gene engineering technology to generate conditional knockouts of the IDH1+ or IDH1R132C alleles in the fibrosarcoma cell line, HT1080. Despite several attempts, we did not recover a correctly targeted conditional knock-out of the IDH1R132C allele. However, we did recover a derivative cell line where the wild-type allele had been converted into a loxP-flanked IDH1R132C allele, and two independent clones where the wild-type allele had been flanked with loxP sites. Interestingly, in these latter two clones, no expression of wild-type IDH1 was detectable. Here we present data characterising the growth of these clones under a variety of culture conditions, including the hypoxic state where IDH1 has been proposed to be important for enabling lipid synthesis from glutamine. We also demonstrate that HT1080 cells that express no detectable levels of wild-type IDH1 retain the ability to be tumorigenic in immuno-compromised mice. These data suggest that the wild-type allele of IDH1 is dispensable for cancer formation. Citation Format: Julie A. Wickenden, Paul Russell, Amy Smith, Tom Henley, Jane Elliott, Dan Gitterman, Mark Stockdale, Christine Schofield, Chris Torrance, Jonathan D. Moore. Wild-type IDH1: A molecular target in IDH1 mutant cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4620. doi:10.1158/1538-7445.AM2014-4620

Laws, Glossaries and Legal Glossaries in Early Ireland

Only rarely has the the study of early Irish law and that of early Irish glossaries come into contact with one another. On the occasions when they have, it has usually been to the benefit of legal studies when a passage of legal text is illuminated by reference to the glossaries. In general terms, the two areas of study have suffered contrasting fortunes. From Thurneysens ground-breaking contributions onwards, study of the legal material has gone from strength to strength. The publication in 1978 of the Binchys Corpus luris Hibernici (CIH) marked another crucial stage; for at last most of the legal material was in print even though in a diplomatic edition (in passing we may note that it also brought by chance an enormous amount of glossarial material into the light of day for the first time). Elsewhere, Binchy made a major contribution in his editions of important texts. Since then the work of subsequent scholars has taken the subject even further. In stark contrast, with one of two exceptions, we are still operating with the same editions of glossaries as Thurneysen would have known, and work on glossaries has not yet progressed far beyond where it was in 1914