ALBERT

Description: As the source of all cells in the developing embryo proper, embryonic stem cells (ESC) bear the unique responsibility to prevent mutations from being propagated throughout the entire organism and the germ line. It is likely for this reason that ESC and induced pluripotent stem cells (iPSC) maintain a dramatically lower mutation frequency than cultured somatic cells. Multiple mechanisms for this enhanced genomic surveillance have been proposed, including hypersensitivity of DNA damage response signaling pathways and increased activity of error-free DNA repair pathways, such as homologous recombination. However, the effect of loss of function of DNA repair pathways in these cells remains poorly understood. The Fanconi Anemia (FA) pathway is a DNA repair pathway that is required for the repair of DNA interstrand crosslink damage and also promotes repair of DNA double-strand breaks by homologous recombination . Genetic defects in this pathway cause a disease characterized by bone marrow failure and extreme cancer incidence. Several recent studies have revealed that the FA pathway is required for efficient somatic cell reprogramming to iPSC and suggest that FA cells undergo cell death during this process. Another recent study found that the growth of FA patient-specific iPSC was attenuated with a G2/M arrest when compared to control iPSC, suggesting that these cells arrest upon failed DNA repair. In this study, we sought to determine the effects of acute loss of function of the FA pathway in iPSC through the generation of FA patient-derived iPSC with inducible complementation of the defective FA gene. Fibroblasts were cultured from skin biopsies of multiple FA patients and transduced with a lentiviral vector expressing the complementing FA gene product under DOX-inducible control. Cells were then reprogrammed to iPSC using episomal transfection. These cells formed iPSC colonies only when reprogramming was carried out in the presence of DOX, confirming that the FA pathway is required for efficient reprogramming. Once cell lines were obtained, DOX-dependent FA functionality was verified based on FANCD2 monoubiquitination and nuclear focus formation after treatment with DNA damaging agents. We then cultured the iPSC for extended periods of time in the presence and absence of DOX. Interestingly, the cultures underwent profound cell death and cell cycle arrest within 7 days of DOX-withdrawal and completely failed to expand after one passage. EdU cell cycle analysis confirmed cell cycle arrest in the G2/M phase. Furthermore, cleaved caspase 3 staining confirmed that the number of apoptotic cells increased by 3-fold in the -DOX culture. Despite these effects, cells cultured in both the presence and absence of DOX formed teratomas in nude mice, thus indicating the maintenance of full differentiation capacity in the absence of the FA pathway. In order to determine the mechanisms underlying G2/M arrest and cell death, expression of p53 and its target genes was detected by both western blot analysis and qRT-PCR. Only a slight increase in p53 activation was observed by 7 days post DOX-withdrawal. Furthermore, knockdown of p53 resulted in rescue from apoptosis to normal levels but not rescue from cell cycle arrest. Increased ATM and ATR DNA damage sensor kinase activities were also detected in –DOX cells, concominant with increased phosphorylation of the ATM-target Chk2 and reduced abundance of the G2/M checkpoint protein CDC25A. These results reveal hyperactive DNA damage responses upon FA loss which may underlie the attenuated cell cycle progression of FA-iPSC independent of p53. Remarkably, effects in this FA model system appear equivalent to those responsible for the depletion of HSC in the bone marrow of FA patients. Thus, iPSC models may be useful for future studies of the mechanisms underlying FA stem cell arrest and for the development of therapeutics that alleviate these phenotypes. Disclosures No relevant conflicts of interest to declare.

Description: The Fanconi Anemia (FA) DNA Repair pathway functions through homologous recombination for error-free repair of DNA interstrand crosslinks. Loss of function of this pathway causes a complex genetic disease that is characterized by congenital abnormalities, bone marrow failure (BMF), and extreme incidence of squamous cell carcinomas. BMF is caused by exhaustion of hematopoietic stem and progenitor cells (HSPCs) and is nearly 100% penetrant by age 40 in FA patients, indicating a profound sensitivity of HSPCs to FA pathway deficiency. In contrast, stem cells in other rapidly regenerating tissues, such as the skin and intestine, are not similarly exhausted. Interestingly, squamous epithelium is highly prone to transformation while intestinal epithelium is not. In order to explore the developmental origins of such striking tissue-specific phenotypes in FA patients, we have generated induced pluripotent stem cell (iPSC) lines conditional for FA pathway function (cFA-iPSCs) and used them to derive FA-proficient and deficient in vitro models of diverse tissues. FA patient cells are refractory to reprogramming. To circumvent this defect and prevent the selection of FA-resistant iPSC clones, fibroblasts from 2 FANCA patients were inducibly complemented with a FANCA transgene under the control of a tetracycline-inducible promoter and then were reprogrammed to iPSC. In this way, the FA pathway was functional throughout reprogramming and could then be turned on or off in established iPSC lines by the addition or withdrawal of doxycycline (DOX) to the media. Here, we describe the effect of FA pathway loss on iPSCs, and present preliminary data on iPSC-derived equivalents of three lineages: hematopoietic, squamous, and intestinal. First, functional consequences of FA pathway loss on iPSC pluripotency and self-renewal were examined. Upon withdrawal of DOX from the culture media, the complementing FA transgene was effectively silenced, resulting in loss of FA pathway function within 7 days. FA-deficient iPSCs maintained normal expression of OCT-3/4 and NANOG and formed teratomas in NSG mice, indicating that pluripotency was maintained. However, profound cell cycle arrest and apoptosis were observed under normal in vitro culture conditions within 7 days of DOX-withdrawal, and the iPSCs failed to expand by 2-3 passages. Thus, we concluded that iPSCs require an intact FA pathway for self-renewal in vitro. Mechanistic studies of FA pathway-deficient iPSCs revealed a 10-fold increase in gH2AX foci in the G2-M phase of the cell cycle. This correlated with activated DNA damage response signaling through ATR and CHK1. Inhibition of CHK1 completely restored the growth of FA-deficient iPSCs to that of their FA-proficient counterparts through a remarkable rapid bypass of the G2-M checkpoint. Unexpectedly, cells maintained in CHK1 inhibitor for over 40days accrued few karypotypic abnormalities (

Description: Abstract 394 Background: Long-term low molecular weight heparin (LMWH) is the current standard for treatment of venous thromboembolism (VTE) in cancer patients. Whether treatment strategies should vary according to individual risk of VTE recurrence remains unknown. We have derived a clinical prediction rule that stratifies VTE recurrence risk in patients with cancer-associated VTE. The derivation model includes 4 independent predictors (sex, primary tumor site, stage and prior VTE). The score sum ranges between −3 and +3 points. Patients with a score ≤ 0 had low risk (≤4.5%) for recurrence and patients with a score above 1 had a high risk (≥ 19%) for VTE recurrence. Subsequently, we applied and validated the rule in an independent set of 819 patients from 2 randomized controlled trials comparing LMWH to warfarin for VTE treatment in cancer patients. In the current study we aim to externally validate our clinical prediction rule with an independent population of patients with cancer-associated VTE followed at the Thrombosis clinics of two tertiary Canadian centres. Methods: We conducted a retrospective cohort study of patients with cancer and VTE diagnosed and/or followed at the Thrombosis Clinic of the Victoria Hospital (London, Canada) from January 2006 to December 2010; and the Thrombosis Unit of the Ottawa Hospital (Ottawa, Canada) from January 2009 to December 2011. We included data from adult patients with active malignancy and objectively diagnosed acute pulmonary embolism (PE) or deep venous thrombosis (DVT) of the lower extremity (above knee), upper extremity and neck veins, or unusual site thrombosis. The primary outcome measure was VTE recurrence during the first six months of anticoagulation. Results: 353 patients fulfilled our inclusion criteria and were included in the study. There were 149 males, and the overall population had a median age of 64 years (range: 18 – 95). One hundred and twenty-three patients had lower extremity DVT, 93 had PE and 57 had both. The remaining 80 patients had either upper extremity/neck DVT (n = 55) or unusual site thrombosis (n = 25). 77 patients had a prior history of VTE. The most common primary tumour site was gastrointestinal, followed by the lung. Of the 304 patients with solid tumours, 230 (75.7%%) had TNM greater than I. Two hundred and ninety-three (83.0%) patients were treated with longterm low molecular weight heparin (LMWH) only and 60 (17.0%) with warfarin (VKA). VTE recurrence occurred in 44 of 353 patients (12.4%). When we evaluated VTE recurrence risk per site, there was no significant difference: London 13 of 90 and Ottawa 31 of 263 [RR=1.23 (95%CI= 0.671 – 2.237; p=0. 507)]. In addition, there was no significant benefit with the use of LMWH (37 of 293) over VKA (7 of 60) in the risk of recurrence [RR=0.92 (95%CI= 0.433 – 1.973; p= 0.8379)]. When we applied our clinical prediction rule (Table 1) in the entire study population, recurrent VTE occurred in 12 of 204 (5.8%) patients stratified as low risk probability and in 32 of 149 (21.4%) patients stratified as high risk probability (Table 2). Conclusions: Our prediction rule has been adequately validated to now be used in prospective trials of treatment. Future trials evaluating novel treatment strategies for high risk patients are warranted. Disclosures: No relevant conflicts of interest to declare.