Prolonged Ileus after Colorectal Surgery, a Systematic Review.

BACKGROUND: The development of prolonged post-operative ileus (POI) remains a significant problem in the general surgical patient population. The aetiology of ileus is poorly understood and management options/preventative measures are currently extremely limited. The pathophysiology leading to a post-operative ileus is relatively poorly understood, and there is no validated method to estimate ileus occurrence or duration. Ileus in the post-operative period commonly occurs following major colorectal surgery and leads to painful abdominal distension, vomiting, nutritional deficit, pneumonia, prolonged hospital stays and susceptibility to hospital-acquired infection. An increased hospital stay, the burden of treatment costs and the burden on the health system highlight the importance of future research on finding definitions, preventions and predictions of ileus. METHODS: A systematic literature review was performed to identify randomized controlled trials (RCTs) comparing the rate of ileus on various treatments for prolonged post-operative ileus following colorectal surgery. A confidence evaluation in a meta-analysis were performed using CINeMA. Direct and indirect comparisons of all interventions were simultaneously carried out using a network meta-analysis. The level of certainty was appraised using the Grading of Recommendations, Assessment, Development and Evaluations (GRADE) approach. The method of assessing the risk of bias, the quality assessment, used the Cochrane Risk of Bias 2 tool (RoB2). RESULTS: Among the seven included studies, the majority suffered from considerable within-study bias, affecting the confidence rates of study findings. Heterogeneity and incoherence made the pairwise meta-analysis and ranking of interventions unfeasible. Indirect comparisons were considered unreliable due to this incoherence. CONCLUSIONS: This systematic review, with a confidence evaluation in the network meta-analysis, determined that there is a knowledge gap in the field of study on prolonged ileus following digestive surgery. The current evidence suffers from heterogeneity and incoherence more than imprecision. There is a gap in the data on ileus occurrence in interventional trials for digestive surgery. This could inform clinicians and trialists to better appraise the current literature and plan future trials.

Effectively utilizing publicly available databases for cancer target evaluation.

The majority of compounds designed against cancer drug targets do not progress to become approved drugs, mainly due to lack of efficacy and/or unmanageable toxicity. Robust target evaluation is therefore required before progressing through the drug discovery process to reduce the high attrition rate. There are a wealth of publicly available databases that can be mined to generate data as part of a target evaluation. It can, however, be challenging to learn what databases are available, how and when they should be used, and to understand the associated limitations. Here, we have compiled and present key, freely accessible and easy-to-use databases that house informative datasets from in vitro, in vivo and clinical studies. We also highlight comprehensive target review databases that aim to bring together information from multiple sources into one-stop portals. In the post-genomics era, a key objective is to exploit the extensive cell, animal and patient characterization datasets in order to deliver precision medicine on a patient-specific basis. Effective utilization of the highlighted databases will go some way towards supporting the cancer research community achieve these aims.

Rethinking our approach to cancer metabolism to deliver patient benefit.

Altered cellular metabolism is a major mechanism by which tumours support nutrient consumption associated with increased cellular proliferation. Selective dependency on specific metabolic pathways provides a therapeutic vulnerability that can be targeted in cancer therapy. Anti-metabolites have been used clinically since the 1940s and several agents targeting nucleotide metabolism are now well established as standard of care treatment in a range of indications. However, despite great progress in our understanding of the metabolic requirements of cancer and non-cancer cells within the tumour microenvironment, there has been limited clinical success for novel agents targeting pathways outside of nucleotide metabolism. We believe that there is significant therapeutic potential in targeting metabolic processes within cancer that is yet to be fully realised. However, current approaches to identify novel targets, test novel therapies and select patient populations most likely to benefit are sub-optimal. We highlight recent advances in technologies and understanding that will support the identification and validation of novel targets, re-evaluation of existing targets and design of optimal clinical positioning strategies to deliver patient benefit.

A systematic review of the impact of post-operative oral fluid intake on ileus following elective colorectal surgery.

INTRODUCTION: Ileus (delayed return of bowel function after surgery) is one of the highest priority research questions in modern day colorectal practice. Current Enhanced Recovery After Surgery (ERAS) guidance either does not include a specific recommendation for volume of postoperative oral fluids/foodstuffs or suggests ad-lib fluids. It is unclear if the volume of intake affects ileus rates. This systematic review aimed to determine the optimal fluid volume for patients to consume day one after elective colorectal surgery. METHODS: The literature was searched across seven databases, September 23, 2020. Randomised controlled trials of adults undergoing elective colorectal surgery, comparing oral intake postoperatively were eligible for inclusion. Two blinded reviewers assessed papers with disagreements resolved by a third independent reviewer. Main outcomes were 'resolution of postoperative ileus' and 'length of hospital stay'. Secondary outcomes included vomiting, mortality and complications. RESULTS: Of 2175 screened papers, eight were eligible for inclusion. All studies gave a clear liquid diet postoperatively. The comparison groups followed a traditional nil-by-mouth approach. All studies showed a minor reduction in postoperative ileus and hospital stay in the intervention group, but we are unable to determine the optimal postoperative oral fluid volume. The low number and poor quality of studies was a significant limitation. None of the trials were conducted within an ERAS protocol: only 883 patients were included in total. CONCLUSIONS: From the current literature it is unclear how postoperative oral fluid volume intake affects gastrointestinal function and ileus in elective colorectal surgical patients. This remains an important area for further research.

Disease recurrence after right hemicolectomy in Scotland: Is there rationale to adopt complete mesocolic excision (CME)?

AIMS: Complete mesocolic excision (CME) has been proposed as a way to improve the oncological outcomes in patients with colon cancer. To investigate whether there is rationale for adopting the technique in Scotland, our aim was to define the incidence of disease recurrence following standard right hemicolectomy and to compare this with published CME outcomes. METHODS: Data was collected on consecutive patients undergoing right or extended right hemicolectomy for colonic adenocarcinoma (2012-2017) at three hospitals in Scotland (Raigmore Hospital, Aberdeen Royal Infirmary and Glasgow Royal Infirmary). Emergency or palliative surgery was excluded. Patients were followed up with CT scans and colonoscopy for a minimum of 3 years. RESULTS: 689 patients (M 340, F 349) were included. 30-day mortality was 1.6%. Final pathological stage was Stage I (14%), Stage II (49.8%) and Stage III (36.1%). During follow-up, 10.5% developed loco-regional recurrence and 12.2% developed distant metastases. The 1, 3 and 5-year disease-free survival (DFS) was 94%, 84% and 82% respectively. Primary determinants of recurrence were T stage (p < 0.001), N stage (p < 0.001), apical node involvement (p < 0.001) and EMVI (p < 0.001). When compared to the literature, 30-day mortality was lower than many published series and DFS rates were similar to the largest CME study to date (4 year DFS 85.8% versus 83%). CONCLUSION: The outcomes of patients undergoing right hemicolectomy in Scotland compare favourably with many published CME studies. The technique demands further evaluation before it can be recommended for adoption into routine surgical practice.

Pathological response post neoadjuvant therapy for locally advanced rectal cancer is an independent predictor of survival.

AIM: Neoadjuvant treatment (NaT) for locally advanced rectal cancer prior to surgery has led to improved outcomes. However, the relationship between pathological response to NaT and survival is not entirely clear. The aim of this study was to assess the degree of pathological response to NaT on survival outcomes. METHODS: Clinical and pathological data were collected from a prospectively maintained pathology database between 2005 and 2017. The primary outcome was the overall survival based on pathological response categorized as complete, good partial, partial and minimal. Univariate and multivariate analyses were conducted to identify variables predictive of survival. Cox proportional hazard ratios were used for survival. RESULTS: A total of 596 patients had surgery following NaT for locally advanced rectal cancer. The median follow-up was 4.57 years (interquartile range 2.21-8.15 years). The overall survival for complete pathological response was 75.6% vs. 37.3% for minimal response (P < 0.001). The overall survival at the end of the study in the good partial vs. partial response groups was 58.9% vs. 39% (P < 0.001). On multivariate analysis, the degree of pathological response remains an independent variable for overall and disease-specific survival across all categories. DISCUSSION: In addition to other pathological variables, the degree of pathological response to NaT is an independent predictor for survival outcomes. Future verification of these findings elsewhere could support NaT response being used for adjuvant therapy decision making.

MNK Inhibition Sensitizes KRAS-Mutant Colorectal Cancer to mTORC1 Inhibition by Reducing eIF4E Phosphorylation and c-MYC Expression.

KRAS-mutant colorectal cancers are resistant to therapeutics, presenting a significant problem for ∼40% of cases. Rapalogs, which inhibit mTORC1 and thus protein synthesis, are significantly less potent in KRAS-mutant colorectal cancer. Using Kras-mutant mouse models and mouse- and patient-derived organoids, we demonstrate that KRAS with G12D mutation fundamentally rewires translation to increase both bulk and mRNA-specific translation initiation. This occurs via the MNK/eIF4E pathway culminating in sustained expression of c-MYC. By genetic and small-molecule targeting of this pathway, we acutely sensitize KRASG12D models to rapamycin via suppression of c-MYC. We show that 45% of colorectal cancers have high signaling through mTORC1 and the MNKs, with this signature correlating with a 3.5-year shorter cancer-specific survival in a subset of patients. This work provides a c-MYC-dependent cotargeting strategy with remarkable potency in multiple Kras-mutant mouse models and metastatic human organoids and identifies a patient population that may benefit from its clinical application. SIGNIFICANCE: KRAS mutation and elevated c-MYC are widespread in many tumors but remain predominantly untargetable. We find that mutant KRAS modulates translation, culminating in increased expression of c-MYC. We describe an effective strategy targeting mTORC1 and MNK in KRAS-mutant mouse and human models, pathways that are also commonly co-upregulated in colorectal cancer.This article is highlighted in the In This Issue feature, p. 995.

Screen detection is a survival predictor independent of pathological grade in colorectal cancer. A prospective cohort study.

INTRODUCTION: Patients with screened detected colorectal cancer (CRC) have a better survival than patients referred with symptoms. This may be because of cancers being identified in a younger population and at an earlier stage. In this study, we assess whether screened detected CRC has an improved outcome after controlling for key pathological and patient factors known to influence prognosis. METHOD: This is a cohort study of all CRC patients diagnosed in NHS Grampian. Patients aged 51-75 years old between June 2007 and July 2017 were included. Data were obtained from a prospectively maintained regional pathology database and outcomes from ISD records. All-cause mortality rates at 1 and 5 years were examined. A Cox proportional hazards regression model was used to estimate the effect of screening status, age, gender, Duke stage, tumour location, extramural venous invasion (EMVI) status and lymph node ratio (LNR) on overall survival. RESULTS: Of 1618 CRC cases, 449 (27.8%) were screened and 1169 (72.2%) were symptomatic. Screened CRC patients had improved survival compared to non-screened CRC at 1 year (88.9% vs 83.9% p < 0.001) and 5-years (42.5% vs 36.2%; p < 0.001). On multivariable analysis of patients who had no neoadjuvant therapy (n = 1272), screening had better survival (HR 0.57; 95% CI 0.44-0.74; p < 0.001). EMVI (HR 2.22; CI 1.76 to 2.79; p < 0.001) and tumour location were found to affect outcome. CONCLUSION: Patients referred through screening had improved survival compared with symptomatic patients. Further research could be targeted to determine if screened CRC cases are pathologically different to symptomatic cancers or if the screening cohort is inherently more healthy.

Fadraciclib (CYC065), a novel CDK inhibitor, targets key pro-survival and oncogenic pathways in cancer.

Cyclin-dependent kinases (CDKs) contribute to the cancer hallmarks of uncontrolled proliferation and increased survival. As a result, over the last two decades substantial efforts have been directed towards identification and development of pharmaceutical CDK inhibitors. Insights into the biological consequences of CDK inhibition in specific tumor types have led to the successful development of CDK4/6 inhibitors as treatments for certain types of breast cancer. More recently, a new generation of pharmaceutical inhibitors of CDK enzymes that regulate the transcription of key oncogenic and pro-survival proteins, including CDK9, have entered clinical development. Here, we provide the first disclosure of the chemical structure of fadraciclib (CYC065), a CDK inhibitor and clinical candidate designed by further optimization from the aminopurine scaffold of seliciclib. We describe its synthesis and mechanistic characterization. Fadraciclib exhibits improved potency and selectivity for CDK2 and CDK9 compared to seliciclib, and also displays high selectivity across the kinome. We show that the mechanism of action of fadraciclib is consistent with potent inhibition of CDK9-mediated transcription, decreasing levels of RNA polymerase II C-terminal domain serine 2 phosphorylation, the pro-survival protein Myeloid Cell Leukemia 1 (MCL1) and MYC oncoprotein, and inducing rapid apoptosis in cancer cells. This cellular potency and mechanism of action translate to promising anti-cancer activity in human leukemia mouse xenograft models. Studies of leukemia cell line sensitivity identify mixed lineage leukemia (MLL) gene status and the level of B-cell lymphoma 2 (BCL2) family proteins as potential markers for selection of patients with greater sensitivity to fadraciclib. We show that the combination of fadraciclib with BCL2 inhibitors, including venetoclax, is synergistic in leukemic cell models, as predicted from simultaneous inhibition of MCL1 and BCL2 pro-survival pathways. Fadraciclib preclinical pharmacology data support its therapeutic potential in CDK9- or CDK2-dependent cancers and as a rational combination with BCL2 inhibitors in hematological malignancies. Fadraciclib is currently in Phase 1 clinical studies in patients with advanced solid tumors (NCT02552953) and also in combination with venetoclax in patients with relapsed or refractory chronic lymphocytic leukemia (CLL) (NCT03739554) and relapsed refractory acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) (NCT04017546).

Can Haematology Blood Tests at Time of Diagnosis Predict Response to Neoadjuvant Treatment in Locally Advanced Rectal Cancer?

BACKGROUND: Outcomes in locally advanced rectal cancer are improved by neoadjuvant therapy followed by surgical resection. Some patients respond completely to preoperative treatment. Therefore, predicting the pathological response to preoperative therapy is of clinical importance. Accurate prediction would allow for tailored approaches to neoadjuvant therapy. METHODS: All patients undergoing resection of rectal adenocarcinoma after neoadjuvant therapy between 2006 and 2015 were included in this cohort study. Patients were identified from a prospectively collected database and data were supplemented retrospectively with full blood count at diagnosis. Specimens resected following neoadjuvant therapy were graded according to pathological response. Follow-up data was obtained from the national registry. The primary outcome was complete pathological response. RESULTS: Of 330 patients, 71 (21.5%) responded completely to preoperative therapy. Median age was 66 and 65% were male (n = 215). White cell count (WCC) was the most predictive marker, for predicting pCR; area under the curve (AUC) 0.666. This was higher than neutrophil/platelet ratio (AUC 0.652) or neutrophil/lymphocyte ratios (AUC = 0.437). Kaplan-Meier survival analysis showed those patients with WCC > 8 had poorer survival than those with WCC < 8 (p = 0.009). CONCLUSION: Routinely collected haematology samples at the point of diagnosis can assist in predicting for complete response to neoadjuvant therapy. Although novel biomarkers will have a greater predictive value, this clinically available value test could help to assist in risk stratification of patients using routinely collected laboratory tests.

Is Liver Ultrasound Useful as Part of the Surveillance Strategy following Potentially Curative Colorectal Cancer Resection?

BACKGROUND: Optimal surveillance monitoring following curative resection of colorectal cancer remains unclear. Guidelines recommend computed tomography (CT)-based imaging for the initial 3 years following surgical intervention due to the high rates of local and distant recurrence. However, there is currently limited supporting evidence for this strategy. Our current follow-up practice is to offer annual interval abdominal ultrasound and abdominal/pelvis CT scans starting at 6 and 12 months with the sequence of radiological follow-up remaining at the discretion of each clinician. We aim to establish the additional diagnostic benefit of abdominal ultrasound to CT scans in colorectal cancer surveillance follow-up. METHODS: All patients who underwent colorectal resection with curative intent in our region during a single year were included. Patients were detected from a prospectively collected pathology database and supplemented retrospectively with patient demographics, imaging reports, and mortality data. RESULTS: A total of 243 patients (male n = 135, 55.6%) were included. There was a mortality rate of 31.3% over the study period. Patients who received abdominal ultrasound as their initial imaging modality (n = 64, 26.3%) were significantly older, had less severe disease, and a significantly lower mortality rate when compared to CT -patients (n = 148, 60.9%). All patients with new hepatic disease detected by ultrasound scans had their management discussed in multi-disciplinary team meetings before their next scheduled CT. CONCLUSION: In an era where cross-sectional imaging of colorectal cancer is commonplace, abdominal ultrasound offers additional benefit to CT as a postoperative imaging adjunct for the detection of hepatic disease recurrence.

Prognostic Value of Computed Tomography: Measured Parameters of Body Composition in Primary Operable Gastrointestinal Cancers.

BACKGROUND: Previous reports suggest that body composition parameters can be used to predict outcomes for patients with gastrointestinal (GI) cancers. However, evidence for an association with long-term survival is conflicting, with much of the data derived from patients with advanced disease. This study examined the effect of body composition on survival in primary operable GI cancer. METHODS: Patients with resectable adenocarcinoma of the GI tract (esophagus, stomach, colon, rectum) between 2006 and 2014 were identified from a prospective database. Computed tomography (CT) scans were analyzed using a transverse section at L3 to calculate sex-specific body composition indices for skeletal muscle, visceral fat, and subcutaneous fat. Kaplan-Meier and log-rank analysis were used to compare unadjusted survival. Multivariate survival analyses were performed using a proportional hazards model. RESULTS: The study enrolled 447 patients (191 woman and 256 men) with esophagogastric (OG) (n = 108) and colorectal (CR) (n = 339) cancer. Body composition did not predict survival for the OG cancer patients. Among the CR cancer patients, survival was shorter for those with sarcopenia (p = 0.017) or low levels of subcutaneous fat (p = 0.005). Older age (p = 0.046) and neutrophilia (p = 0.013) were associated with sarcopenia in patients with CR. Tumor stage (p = 0.033), neutrophil count (p = 0.011), and hypoalbuminemia (p = 0.023) were associated with sarcopenia in OG cancer patients. In the multivariate analysis, no single measure of body composition was an independent predictor of reduced survival. CONCLUSION: Sarcopenia and reduced subcutaneous adiposity are associated with reduced survival for patients with primary operable CR cancer. However, in this study, no parameter of body composition was an independent prognostic marker when considered with age, tumor stage, and systemic inflammation.

AluY-mediated germline deletion, duplication and somatic stem cell reversion in UBE2T defines a new subtype of Fanconi anemia.

Fanconi anemia (FA) is a rare inherited disorder clinically characterized by congenital malformations, progressive bone marrow failure and cancer susceptibility. At the cellular level, FA is associated with hypersensitivity to DNA-crosslinking genotoxins. Eight of 17 known FA genes assemble the FA E3 ligase complex, which catalyzes monoubiquitination of FANCD2 and is essential for replicative DNA crosslink repair. Here, we identify the first FA patient with biallelic germline mutations in the ubiquitin E2 conjugase UBE2T. Both mutations were aluY-mediated: a paternal deletion and maternal duplication of exons 2-6. These loss-of-function mutations in UBE2T induced a cellular phenotype similar to biallelic defects in early FA genes with the absence of FANCD2 monoubiquitination. The maternal duplication produced a mutant mRNA that could encode a functional protein but was degraded by nonsense-mediated mRNA decay. In the patient's hematopoietic stem cells, the maternal allele with the duplication of exons 2-6 spontaneously reverted to a wild-type allele by monoallelic recombination at the duplicated aluY repeat, thereby preventing bone marrow failure. Analysis of germline DNA of 814 normal individuals and 850 breast cancer patients for deletion or duplication of UBE2T exons 2-6 identified the deletion in only two controls, suggesting aluY-mediated recombinations within the UBE2T locus are rare and not associated with an increased breast cancer risk. Finally, a loss-of-function germline mutation in UBE2T was detected in a high-risk breast cancer patient with wild-type BRCA1/2. Cumulatively, we identified UBE2T as a bona fide FA gene (FANCT) that also may be a rare cancer susceptibility gene.

E3 ubiquitin ligase HOIP attenuates apoptotic cell death induced by cisplatin.

The genotoxin cisplatin is commonly used in chemotherapy to treat solid tumors, yet our understanding of the mechanism underlying the drug response is limited. In a focused siRNA screen, using an siRNA library targeting genes involved in ubiquitin and ubiquitin-like signaling, we identified the E3 ubiquitin ligase HOIP as a key regulator of cisplatin-induced genotoxicity. HOIP forms, with SHARPIN and HOIL-1L, the linear ubiquitin assembly complex (LUBAC). We show that cells deficient in the HOIP ligase complex exhibit hypersensitivity to cisplatin. This is due to a dramatic increase in caspase-8/caspase-3-mediated apoptosis that is strictly dependent on ATM-, but not ATR-mediated DNA damage checkpoint activation. Moreover, basal and cisplatin-induced activity of the stress response kinase JNK is enhanced in HOIP-depleted cells and, conversely, JNK inhibition can increase cellular resistance to cisplatin and reverse the apoptotic hyperactivation in HOIP-depleted cells. Furthermore, we show that HOIP depletion sensitizes cancer cells, derived from carcinomas of various origins, through an enhanced apoptotic cell death response. We also provide evidence that ovarian cancer cells classified as cisplatin-resistant can regain sensitivity following HOIP downregulation. Cumulatively, our study identifies a HOIP-regulated antiapoptotic signaling pathway, and we envisage HOIP as a potential target for the development of combinatorial chemotherapies to potentiate the efficacy of platinum-based anticancer drugs.

Impact of the Scottish Bowel Cancer Screening Programme on patient and tumour characteristics at a single centre.

AIMS: The Scottish National Bowel Cancer Screening Programme aims to detect asymptomatic colorectal carcinomas and improve outcomes by identifying tumours at an earlier stage. We describe the characteristics of bowel cancers diagnosed through the screening programme since it was established in June 2007 by comparison with colorectal carcinomas from all other referral sources. METHODS: All patients with colorectal cancer discussed by our regional colorectal multidisciplinary team (MDT) from June 2007 to August 2011 were included. Patient and tumour characteristics were collated prospectively from MDT records. The database was then reviewed retrospectively. RESULTS: During the study 209 916 (58%) of 364 759 invitations to participate in screening were accepted yielding 3895 (1.9%) positive results. The 255 (17%) screening-detected (SD) patients and 1232 (83%) other referrals (ORs) were discussed at the MDT within this period. Median age at diagnosis was 65.5 years for SD vs. 71.6 in OR (P < 0.001) with 64% vs. 53% male [SD vs. OR (P < 0.001)]. There were more left-sided tumours in SD (P = 0.005). Tumours were less advanced in SD group (P = 0.02) and more likely to undergo a laparoscopic resection (P = 0.003). Thirty (11.7%) of SD patients were dead at last follow-up compared with 458 (37.2%) of those from other sources (P < 0.001). CONCLUSIONS: This cohort from a centre with an established screening programme supports the effect of screening in detecting earlier stage. Those with screen-detected tumours were more likely to survive than patients from the OR group.

Putting guidelines into practice: a tailored multi-modal approach to improve post-operative assessments.

BACKGROUND: The Scottish Intercollegiate Guideline Network (SIGN) published Postoperative Management in Adults in 2004, advocating post-operative assessments to optimize post-operative care. Our aim was to improve post-operative assessments in a surgical high-dependency unit (HDU). METHODS: A prospective audit of post-operative admissions to surgical HDU over two 4-week periods was performed. Medical and nursing documentations were reviewed. A tailored multi-modal approach targeting specific barriers to change was used to implement changes; education of staff, introduction of designated HDU bleeps and a post-operative assessment pro forma. Re-audit was performed after 6 months. MAIN FINDINGS: The first cycle included 72 patients and the second included 62 patients. Time to assessment improved after changes. Forty-six (74%) patients compared with 27 (37%) patients before were assessed within 4 hours. The number of individual reviews increased and number of reviews due to nursing concerns decreased. Thirty-eight (61%) patients compared with 15 (21%) patients before were assessed through an individual review and one (2%) patient compared with 23 (32%) patients due to nursing concerns. Documentation improved. Documentation of relevant past medical history, medications, allergies, complications and post-operative instructions improved from 2 (3%), 1 (1%), 0, 8 (11%) and 26 (36%), to 18 (29%), 28 (45%), 20 (32%), 18 (29%) and 55 (89%), respectively. Difference between first and second cycles was highly significant throughout (P < 0.001). CONCLUSION: Clinical practice was improved by a tailored multi-modal approach. Educating staff, improving communication and documentation, and re-audit has shown significant improvement. However, further improvements are required to reach best practice.

The Fanconi anaemia components UBE2T and FANCM are functionally linked to nucleotide excision repair.

The many proteins that function in the Fanconi anaemia (FA) monoubiquitylation pathway initiate replicative DNA crosslink repair. However, it is not clear whether individual FA genes participate in DNA repair pathways other than homologous recombination and translesion bypass. Here we show that avian DT40 cell knockouts of two integral FA genes--UBE2T and FANCM are unexpectedly sensitive to UV-induced DNA damage. Comprehensive genetic dissection experiments indicate that both of these FA genes collaborate to promote nucleotide excision repair rather than translesion bypass to protect cells form UV genotoxicity. Furthermore, UBE2T deficiency impacts on the efficient removal of the UV-induced photolesion cyclobutane pyrimidine dimer. Therefore, this work reveals that the FA pathway shares two components with nucleotide excision repair, intimating not only crosstalk between the two major repair pathways, but also potentially identifying a UBE2T-mediated ubiquitin-signalling response pathway that contributes to nucleotide excision repair.

Identification of KIAA1018/FAN1, a DNA repair nuclease recruited to DNA damage by monoubiquitinated FANCD2.

DNA interstrand crosslinks (ICLs) are highly toxic because they block the progression of replisomes. The Fanconi Anemia (FA) proteins, encoded by genes that are mutated in FA, are important for repair of ICLs. The FA core complex catalyzes the monoubiquitination of FANCD2, and this event is essential for several steps of ICL repair. However, how monoubiquitination of FANCD2 promotes ICL repair at the molecular level is unknown. Here, we describe a highly conserved protein, KIAA1018/MTMR15/FAN1, that interacts with, and is recruited to sites of DNA damage by, the monoubiquitinated form of FANCD2. FAN1 exhibits endonuclease activity toward 5' flaps and has 5' exonuclease activity, and these activities are mediated by an ancient VRR_nuc domain. Depletion of FAN1 from human cells causes hypersensitivity to ICLs, defects in ICL repair, and genome instability. These data at least partly explain how ubiquitination of FANCD2 promotes DNA repair.

Biochemical characterisation of the SWI/SNF family member HLTF.

HLTF is highly similar in domain organisation to yeast Rad5. We identify PTIP and RPA70, both involved in DNA replication and DNA repair, as HLTF-interacting proteins although cells depleted of HLTF did not show defects in cellular responses to DNA damage. In vitro, HLTF has ATPase activity and E3 ubiquitin ligase activity with a range of E2 ubiquitin conjugating enzymes. HLTF expression is severely reduced in a range of cancer cells, and we suggest that the HLTF antibodies generated in this study could be used for cancer diagnostic purposes.