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POLE germline mutations from the IRRDC and literature

Supplementary Figure S1 (related to Figure 1): PoleP286R mouse model design, validation, and tumor findings. Supplementary Figure S2 (related to Figure 1): PoleS459F mouse model design, validation, and tumor findings. Supplementary Figure S3 (related to Figure 2): WES analysis reveals a stochastic accumulation of mutations in Pole mutant tumors. Supplementary Figure S4 (related to Figure 2): Tumors from Pole mutant mice exhibit mutational signatures found in POLE driven human cancers. Supplementary Figure S5 (related to Figure 2): Exome data from tumor fractions from a single mouse were compared for 3 additional mice. Supplementary Figure S6. Determination of cell of origin for PoleS459F/S459F and PoleS459F/+ mice lymphomas (Related to Figure 3). Supplementary Figure S7. Characterization of T cell malignancies in PoleS459F/S459F and PoleS459F/+ mice (Related to Figure 3). Supplemental Figure S8. Extrinsic effect of 'Group B' malignant T cells on B cell population; (Related to Figure 3).

POLE mutations are a major cause of hypermutant cancers, yet questions remain regarding mechanisms of tumorigenesis, genotype–phenotype correlation, and therapeutic considerations. In this study, we establish mouse models harboring cancer-associated POLE mutations P286R and S459F, which cause rapid albeit distinct time to cancer initiation in vivo, independent of their exonuclease activity. Mouse and human correlates enabled novel stratification of POLE mutations into three groups based on clinical phenotype and mutagenicity. Cancers driven by these mutations displayed striking resemblance to the human ultrahypermutation and specific signatures. Furthermore, Pole-driven cancers exhibited a continuous and stochastic mutagenesis mechanism, resulting in intertumoral and intratumoral heterogeneity. Checkpoint blockade did not prevent Pole lymphomas, but rather likely promoted lymphomagenesis as observed in humans. These observations provide insights into the carcinogenesis of POLE-driven tumors and valuable information for genetic counseling, surveillance, and immunotherapy for patients.Significance:Two mouse models of polymerase exonuclease deficiency shed light on mechanisms of mutation accumulation and considerations for immunotherapy.See related commentary by Wisdom and Kirsch p. 5459

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Many long-term survivors of childhood cancer (LTSCC), individuals at least 5 years post-diagnosis or 2 years post-treatment, experience late- and long-term effects from their treatments, including pain. Yet, pain is poorly understood among LTSCC. The current study aimed to (1a) describe rates and multiple dimensions of pain; (1b) identify patterns of chronic pain; and (2) test correlates of chronic pain in LTSCC. Survivors (n = 140; 48.6% male, Mage = 17.3 years (range = 8–25)) were recruited from across Canada. Between 2017 and 2019, participants completed the Pain Questionnaire, Pain Catastrophizing Scale, Pediatric Quality of Life Inventory, Patient-Reported Outcome Measurement Information System (PROMIS)–Pain Interference, Anxiety, and Depression scales, Child Posttraumatic Stress Scale, the Posttraumatic Stress Disorder Checklist for the DSM-V, and the Cancer Worry Scale. Twenty-six percent of LTSCC reported experiencing chronic pain. Exploratory cluster analysis showed 20% of survivors had moderate to severe chronic pain based on measures of pain intensity and interference. The combination of higher posttraumatic stress symptoms, older current age, more pain catastrophizing, and sex (being female) significantly predicted the presence of chronic pain in logistic regression, χ2 (4, N = 107) = 28.10, p < .001. Higher pain catastrophizing (OR = 1.09; 95% CI = 1.02–1.16), older current age (OR = 1.20; 95% CI = 1.07–1.34), and higher posttraumatic stress (OR = 1.92; 95% CI = 1.01–3.63) were significant predictors of chronic pain. LTSCC should be screened for the presence and magnitude of chronic pain during long-term follow-up visits so appropriate interventions can be offered and implemented. Future research should investigate pain interventions tailored for this population. Findings support regular screening for the presence and magnitude of chronic pain in survivors of childhood cancer in long-term follow-up care.

Although replication repair deficiency, either by mismatch repair deficiency (MMRD) and/or loss of DNA polymerase proofreading, can cause hypermutation in cancer, microsatellite instability (MSI) is considered a hallmark of MMRD alone. By genome-wide analysis of tumors with germline and somatic deficiencies in replication repair, we reveal a novel association between loss of polymerase proofreading and MSI, especially when both components are lost. Analysis of indels in microsatellites (MS-indels) identified five distinct signatures (MS-sigs). MMRD MS-sigs are dominated by multibase losses, whereas mutant-polymerase MS-sigs contain primarily single-base gains. MS deletions in MMRD tumors depend on the original size of the MS and converge to a preferred length, providing mechanistic insight. Finally, we demonstrate that MS-sigs can be a powerful clinical tool for managing individuals with germline MMRD and replication repair–deficient cancers, as they can detect the replication repair deficiency in normal cells and predict their response to immunotherapy. Significance: Exome- and genome-wide MSI analysis reveals novel signatures that are uniquely attributed to mismatch repair and DNA polymerase. This provides new mechanistic insight into MS maintenance and can be applied clinically for diagnosis of replication repair deficiency and immunotherapy response prediction. This article is highlighted in the In This Issue feature, p. 995

POLE mutations are a major cause of hypermutant cancers, yet questions remain regarding mechanisms of tumorigenesis, genotype–phenotype correlation, and therapeutic considerations. In this study, we establish mouse models harboring cancer-associated POLE mutations P286R and S459F, which cause rapid albeit distinct time to cancer initiation in vivo, independent of their exonuclease activity. Mouse and human correlates enabled novel stratification of POLE mutations into three groups based on clinical phenotype and mutagenicity. Cancers driven by these mutations displayed striking resemblance to the human ultrahypermutation and specific signatures. Furthermore, Pole-driven cancers exhibited a continuous and stochastic mutagenesis mechanism, resulting in intertumoral and intratumoral heterogeneity. Checkpoint blockade did not prevent Pole lymphomas, but rather likely promoted lymphomagenesis as observed in humans. These observations provide insights into the carcinogenesis of POLE-driven tumors and valuable information for genetic counseling, surveillance, and immunotherapy for patients. Significance: Two mouse models of polymerase exonuclease deficiency shed light on mechanisms of mutation accumulation and considerations for immunotherapy. See related commentary by Wisdom and Kirsch p. 5459

IntroductionConsiderable observational evidence suggests that cancer online support groups reduce feelings of isolation, depression and anxiety, enhance coping and self-management, and lead to better informed patients. Other studies indicate that cancer online support groups can increase distress. Yet no studies theorise the complex, context-dependent mechanisms by which cancer online support groups generate their—sometimes contrasting—outcomes.Methods and analysisGuided by an integrated knowledge translation approach and the strategy for patient-oriented research, we will conduct a realist review of cancer online support groups in partnership with stakeholders. We will follow Pawson’s five steps and existing quality standards to develop a program theory that explains how cancer online support groups work, for whom and in what circumstances. The specific research questions will be: what positive and negative outcomes have been reported on cancer online support groups? What are the mechanisms that are associated with these outcomes, in which contexts and for whom? Through a rigorous review of relevant scientific and grey literature, as well as ongoing dialogue with stakeholders, a program theory will be developed to explain who benefits from cancer online support groups and who does not, what benefits they derive (or do not), and the factors that affect these outcomes.Ethics and disseminationThe use of secondary data for this review precludes the need for ethical approval. Dissemination will be informed by the knowledge-to-action framework and will consist of tailored knowledge products that are conceived of collaboratively with stakeholders. These will include peer-reviewed publications on how cancer online support groups can be optimised and best practice recommendations to maximise the benefits experienced by people with cancer. These traditional scientific outputs, along with their respective evidence summaries, will be amplified through strategic social media events hosted and promoted by knowledge users.PROSPERO registration numberCRD42021250046.

Mismatch repair (MMR) is a critical defence against mutation, but we lack quantification of its activity on different DNA lesions during human life. We performed whole-genome sequencing of normal and neoplastic tissues from individuals with constitutional MMR deficiency to establish the roles of MMR components, tissue type and disease state in somatic mutation rates. Mutational signatures varied extensively across genotypes, some coupled to leading-strand replication, some to lagging-strand replication and some independent of replication, implying that the various MMR components engage different forms of DNA damage. Loss of MSH2 or MSH6 (MutSα), but not MLH1 or PMS2 (MutLα), caused 5-methylcytosine-dependent hypermutation, indicating that MutSα is the pivotal complex for repairing spontaneous deamination of methylated cytosines in humans. Neoplastic change altered the distribution of mutational signatures, particularly accelerating replication-coupled indel signatures. Each component of MMR repairs 1-10 lesions/day per normal human cell, and many thousands of additional events during neoplastic transformation.HighlightsMMR repairs 1-10 lesions/day in every normal cell and thousands more in tumor cellsMMR patterns and rates are shaped by genotype, tissue type and malignant transformationMSH2 and MSH6 are pivotal for repairing spontaneous deamination of methylated cytosineReplication indels and substitutions vary by leading versus lagging strand and genotype

The growing number of cancer survivors and the high prevalence of cancer-related physical and psychosocial effects pose important and pressing challenges to health systems. The coronavirus disease 2019 (COVID-19) pandemic has led to further stressors on cancer survivors and health care systems. As the pandemic continues to have substantial impact on the world, it is critical to focus attention on the health care needs of cancer survivors. In this commentary, we propose an adoption of essential steps that should be part of a continuous adaptive approach to promote effective cancer survivorship care during ongoing COVID-19 waves and beyond.

Nitrous oxide (N2 O) is frequently used for short anesthesia/analgesia in children undergoing painful or repetitive procedures. Children with acute lymphoblastic leukemia (ALL) require repeated lumbar punctures with direct instillation of intrathecal chemotherapy, usually the anti-folate agent methotrexate, during their treatment. These procedures are frequently performed under anesthesia. Concerns have been intermittently raised about a drug interaction between methotrexate and N2 O that may potentiate the undesirable side effects of methotrexate, including neurotoxicity. However, the clinical evidence consists mainly of isolated case reports leading to a lack of consensus among pediatric anesthetists about the relative risk benefits of using N2 O in children with ALL. In this article, we review the biochemical basis and scientific observations that suggest a significant interaction between N2 O and methotrexate due to their dual inhibition of the key enzyme methionine synthase. The possible role of this interaction in potentiating neurotoxicity in children with cancer is discussed, and arguments and counterarguments about the clinical significance of this largely theoretical relationship are explored. Following comprehensive review of all the available data, we make the case for the circumstantial evidence being sufficiently compelling to prompt a review of practice by pediatric anesthetists and call for a precautionary approach by avoiding the use of N2 O in children receiving concurrent methotrexate.

Key Points Constitutional mismatch repair deficiency syndrome should be considered in children with acute leukemia and characteristic skin lesions. The high mutation burden of CMMRD-related cancers contributes to treatment resistance, necessitating individualized treatment strategies.

Replication repair deficiency (RRD) is the leading cause of hypermutant brain tumours in children. RRD is caused by defects in one of four mismatch repair (MMR) genes and mutations in POLE or POLD1. Such tumours are resistant to common therapeutic agents and animal models are needed to study RRD in vivo and test novel therapies like immune checkpoint inhibitors (ICIs). To model RRD brain tumours specifically, we engineered a Pole mutant mouse model harbouring the S459F mutation (PoleS459F). We combined PoleS459F mice with conditional Msh2 knockout (Msh2LoxP) and Nestin-cre mice. All Nestin-cre+Msh2LoxP/LoxPPoleS459F/+ mice rapidly succumbed to posterior fossa brain tumours between 8.6 and 12.4 weeks. Importantly, tumours exhibited hallmark “ultrahypermutation” (~350 mutations/Mb) and the corresponding signatures characteristic of human combined MMR and POLE-proofreading glioblastoma. Interestingly, Nestin-cre+Msh2LoxP/LoxPPoleS459F/S459F mice failed to establish normal cerebella, suggesting such mutational loads may not support normal brain development. Furthermore, OLIG2-cre+Msh2LoxP/LoxPPoleS459F/+ mice failed to develop tumors. Tumors transplanted into syngeneic vs immunocompromised animals egrafted well orthotopically in the mouse hindbrain but significantly less efficiently when engrafted subcutaneously. Furthermore, immunocompromised and subcutaneous tumors revealed striking differences in mutational burden and clonal architecture, suggestive of nonautonomous immunoediting. Finally, anti-PD1 was sufficient to treat subcutaneously engrafted tumors in immunocompetent animals. This first mouse model of immunocompetent, hypermutant brain tumors can be used to uncover unique characteristics of RRD tumour evolution and allow for immune based therapeutic preclinical testing. Experiments to assess combinational ICIs and other therapeutic interventions in orthotopically transplanted tumors will also be presented.

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Purpose: Methotrexate administration is associated with\ud frequent adverse neurological events during treatment for\ud childhood acute lymphoblastic leukemia. Here, we present\ud evidence to support the role of common drug interactions\ud and low vitamin B12 levels in potentiating methotrexate\ud neurotoxicity.\ud Methods: We review the published evidence and highlight\ud key potential drug interactions as well as present clinical\ud evidence of severe methotrexate neurotoxicity in conjunction\ud with nitrous oxide anesthesia and measurements of\ud vitamin B12 levels among pediatric leukemia patients during\ud therapy.\ud Results: We describe a very plausible mechanism for\ud methotrexate neurotoxicity in pediatric leukemia patients\ud involving reduction in methionine and consequential disruption\ud of myelin production. We provide evidence that a\ud number of commonly prescribed drugs in pediatric leukemia\ud management interact with the same folate biosynthetic\ud pathways and/or reduce functional vitamin B12 levels and\ud hence are likely to increase the toxicity of methotrexate in\ud these patients. We also present a brief case study supporting\ud out hypothesis that nitrous oxide contributes to methotrexate\ud neurotoxicity and a nutritional study, showing that \ud patients.\ud Conclusions: Use of nitrous oxide in pediatric leukemia\ud patients at the same time as methotrexate use should be\ud avoided especially as many suitable alternative anesthetic\ud agents exist. Clinicians should consider monitoring levels\ud of vitamin B12 in patients suspected of having methotrexate-\ud induced neurotoxic effects.

Up to 15% of acute promyelocytic leukemia (APL) patients fail to achieve or maintain remission. We investigated a common G > A polymorphism at position −1377 (rs2234767) in the core promoter of the CD95 cell death receptor gene in 708 subjects with acute myeloid leukemia, including 231 patients with APL. Compared with the GG genotype, carrier status for the −1377A variant was associated with a significantly worse prognosis in APL patients. Carriers were more likely to fail remission induction (odds ratio = 4.22; 95% confidence interval, 1.41-12.6, P = .01), were more likely to die during the first 8 weeks of remission induction therapy (hazard ratio = 7.26; 95% confidence interval, 2.39-22.9, P = .0005), and had a significantly worse 5-year overall survival (odds ratio = 2.14; 95% confidence interval, 1.10-4.15, P = .03). The −1377A variant destroys a binding site for the SP1 transcriptional regulator and is associated with lower transcriptional activity of the CD95 promoter. Identifying patients at high risk of life-threatening events, such as remission induction failure, is a high priority in APL, especially because such events represent a major cause of death despite the introduction of differentiation therapy.

Background/Objectives\ud \ud Adverse neurological events are common (4-20%) during treatment for pediatric acute lymphoblastic leukaemia (ALL) and include seizures, stroke like syndrome and leukoencephalopathy. In addition, chronic neurotoxicity is emerging as a worrying late effect of treatment with long-term survivors experiencing decreased executive function, processing speed and memory function. Survivors are also at increased risk of experiencing learning difficulties, social withdrawal issues and inattention hyperactivity disorders. Methotrexate, an anti-folate chemotherapy agent, is a mainstay of pediatric leukemia treatment regimens globally and is widely implicated as a cause of these neurological side effects. We hypothesise that methotrexate disrupts DNA methylation via effects on S-adenosyl methionine, a key metabolic component that has previously been described to regulate genes involved in myelination.\ud \ud Design/Methods\ud \ud Using both the oligodendrocytic-like cell line MO3.13 and glial cells derived from induced pluripotent stem cells (iPSC) treated with methotrexate, we assayed for changes in DNA methylation and effects on gene expression using whole-genome methylation arrays and RNAseq, respectively. Genes with corresponding methylation and expression changes were selected for further studies of expression by real-time qPCR and assessment of protein levels.\ud \ud Results\ud \ud We identified DNA methylation and corresponding expression changes in genes involved in neurodevelopmental pathways and neurological disorders. Of particular interest was dose-dependent demethylation and increased gene expression of IRS1, a vital component of insulin signalling pathways that is highly expressed in neural tissue and implicated in regulating cognitive performance. We also detected altered DNA methylation within the PLP1 gene, which encodes the most prevalent protein component of myelin. We found that methotrexate treatment in iPSC-derived oligodendrocytes resulted in increased PLP1 methylation associated with a reduction in PLP1 transcript levels as well as PLP1 protein levels.\ud \ud Conclusions\ud \ud Our work provides insight as to the biological mechanisms behind methotrexate-induced neurological side effects for the first time and implicates altered insulin signalling and myelination pathways as a potential causative factor in neurotoxicity. Further work including the use of animal models is warranted for advancing these results towards informing clinical practice.

Abstract 3441 The translocation t(8;21)(q22;q22) is the most common cytogenetic abnormality in acute myeloid leukemia (AML), detected in around 15% of all cases. The translocation fuses the DNA binding domain of AML1 (RUNX1) to the almost full-length ETO (RUNX1T1, MTG8) protein converting a transcriptional modulator essential for hematopoiesis into a constitutive repressor. Expression of AML1/ETO in animal models is not sufficient for leukemogenesis, and further mutations are required. AML1/ETO has been detected in the Guthrie cards of pediatric patients who subsequently developed t(8;21) AML, suggesting that AML1/ETO+ hematopoietic precursor cells remain latent for many years before leukemic transformation. The mechanisms by which additional mutations are acquired are not well characterised. AML1/ETO affects DNA repair gene expression and is associated with lower levels of 8-oxoguanine DNA glycosylase (OGG1) in patient samples and transduced primary CD34+ cells. OGG1 removes oxidatively damaged guanine bases from the DNA strand as an initial step of the base-excision repair pathway. Taken together, these observations suggest the existence of a pre-leukemic, AML1/ETO-expressing hematopoietic precursor which is susceptible to the acquisition of further DNA mutations by virtue of compromised DNA repair function. In order to test this hypothesis we investigated the effect of AML1/ETO expression on susceptibility to spontaneous and exposure driven mutation in TK6 cells. TK6 lymphoblastoid cells have one functional copy of the thymidine kinase (TK) gene, allowing for quantification of mutant cells by functionally screening for the TK−/− phenotype. TK6 cells were lentivirally transduced with the pHR-SINcPPT-SIEW vector containing AML1/ETO; and EGFP as an expression marker. Expression was confirmed by flow cytometry, western blotting and qRT-PCR. Serial dilution assays were performed to generate AML1/ETO+ TK6 clonal populations with AML1/ETO expression varying from 10–150% of that found in Kasumi-1; a t(8;21)+ patient-derived cell line. We also used the PIGA gene as a second mutational target. Loss of this glycosyl-phosphatidylinositol (GPI) anchor results in an absence of GPI-linked proteins from the cell membrane, including CD55 and CD59. Cells that are simultaneously negative for two or more GPI-linked proteins are considered to be PIGA mutants and are detected using fluorochrome-conjugated antibodies. Preliminary mutation results on AML1/ETO+ TK6 clones revealed that the fusion gene conferred an approximate 2 fold higher background TK−/− mutation frequency (MF) (2.1 × 10−6−2.6 × 10−6) compared to controls (0.9 × 10−61.1 × 10−6), and a 2–4 fold higher PIGA MF (Controls: 0.32 × 10−4−1.4 × 10−4 and AML1/ETO+ Clones: 2.7 × 10−4– 5.7 × 10−4), suggesting that AML1/ETO confers a moderate mutator phenotype even without exposure to genotoxic agents. Furthermore, spontaneous MF was proportional to AML1/ETO expression, with higher-expressing clones displaying a higher MF than lower expressers and control cells. When treated with low-dose doxorubicin (a chemotherapeutic anthracyline that induces strand breaks in DNA but can also generate reactive oxygen species (ROS)), AML1/ETO+ TK6 clones had a substantially higher TK−/− MF (0.5 × 10−5– 1.2 × 10−5) in comparison to controls; wild-type TK6 and backbone vector-alone transduced TK6 (1.8x 10−6−3.2 × 10−6) (p We have shown that AML1/ETO confers a mutator phenotype on cells that is associated with downregulation of OGG1, providing a plausible explanation for the particular susceptibility of AML1/ETO+ cells to mutation induction by agents that generate reactive oxygen species. This work suggests a mechanism by which AML1/ETO translocated pre-leukemic hematopoetic cells acquire additional mutations, ultimately leading to leukemic transformation. Disclosures: No relevant conflicts of interest to declare.

Abstract 2426 BACKGROUND The chromosomal rearrangement t(8;21)(q22;q22) encodes the fusion gene AML1/ETO, which is the most common translocation in acute myeloid leukemia (AML). It has an incidence of approximately 15% and a favourable prognosis in comparison to other AML subtypes. Dysregulated angiogenesis in the bone marrow niche environment is predicted to have an important role in leukemia pathogenesis, and several factors have been implicated in this process. Angiopoietin-1 (ANGPT1) is a cytokine involved in hematopoietic stem cell quiescence and regulation of microvessel density within the bone marrow, as well as having a role in transendothelial migration. Moreover, ANGPT1 is upregulated in leukemic blast cells from AML patients. In this study we investigated the role of AML1/ETO as a regulator of ANGPT1 expression, as well as functional implications of ANGPT1 in AML1/ETO-positive AML. METHODS In order to investigate putative AML1/ETO-dependent regulation of ANGPT1, we performed gain-of-function studies using lentiviral gene transfer to ectopically express AML1/ETO in HL-60 and U937 AML cell lines. We also depleted AML1/ETO in the t(8;21)-positive AML cell line Kasumi-1 using fusion gene specific siRNA. Additionally, the functional role of ANGPT1 was studied using targeted RNAi in Kasumi-1 cells. Transcript expression of AML1/ETO and ANGPT1 was analysed by quantitative Real-Time PCR (qRT-PCR) and AML1/ETO protein expression was quantified by western blotting. Angiopoietin-1 protein secretion was determined using enzyme-linked immunosorbent assay (ELISA). We also utilised Matrigel transwell assays to test the effect of ANGPT1 downregulation on the invasive and migratory properties of Kasumi-1. RESULTS In HL-60 and U937 transduced with AML1/ETO, we observed an up to 280 fold increase in ANGPT1 mRNA transcript levels as measured by qRT-PCR, which correlated with an increase in secreted Angiopoietin-1 protein. Conversely, siRNA-mediated AML1/ETO depletion in Kasumi-1 cells significantly decreased ANGPT1 transcript and protein levels after a single electroporation. After three serial electroporations with siRNA, AML1/ETO transcript levels were reduced by 85%, with a concomitant decline in ANGPT1 transcript (>99%) and secreted protein. Preliminary data suggest siRNA targeting of ANGPT1 in Kasumi-1 decreases the invasive ability of these cells, causing a ≥50% reduction in invasion when compared to controls. CONCLUSIONS We have identified a strong correlation between AML1/ETO and ANGPT1 expression, whereby a reduction of AML1/ETO results in a substantial reduction of ANGPT1. Similarly, the introduction of AML1/ETO into myeloid cell lines results in a large upregulation of ANGPT1. Preliminary evidence suggests that a reduction of ANGPT1 reduces the invasive and migratory potential of Kasumi-1. This could have major functional consequences in the bone marrow niche with regards to understanding the AML stem cell and its interaction with the niche environment as well as providing insight into how leukemic cells in the circulation might interact with the vasculature. Disclosures: No relevant conflicts of interest to declare.

Abstract 3368 Translocation (8;21)(q22;q22) is the most common cytogenetic abnormality found in acute myeloid leukemia (AML), presenting in around 15% of all cases and is also reported in lymphoid leukemia. This subtype of AML has a favourable prognosis in contrast to other AML subtypes, however the mechanisms underlying this good response to treatment are not well understood. The translocation between AML1 (RUNX1) and the almost full-length ETO (RUNX1T1, MTG8) gene produces a 752 amino acid fusion protein and changes a transcriptional modulator essential for hematopoiesis into a constitutive repressor. When AML1/ETO (AE) is expressed in animal models, further mutations are required for the development of leukemia. AE has also been detected at a considerable frequency in the Guthrie cards of individuals who developed t(8;21) AML up to twelve years later, suggesting that AE+ hematopoietic precursor cells can remain latent for many years before transformation. AE+ cells have an activated p53 pathway, increased levels of p53 response genes and display an increased susceptibility to DNA damaging agents. AE also decreases the levels of DNA repair genes, including those involved in base-excision repair. This evidence predicts the existence of a pre-leukemic, AE-expressing hematopoietic precursor, which easily acquires additional mutations required for transformation, but that also responds well to treatment. There are multiple alternative forms of AE, the best described being AML1/ETO9a (AE9a), which codes for a truncated version of the protein lacking a significant proportion of ETO. AE9a has been found to be more leukemogenic than AE in mouse models by causing leukemia without the need for additional mutations. Our investigation aims to test the effect of cellular expression of AE or AE9a on mutation, cell death and transformation following exposure to leukemogens and to identify co-operating mutations involved in t(8;21) AML. TK6 lymphoblastoid cells are heterozygous for the TK gene and when exposed to mutagens, can lose their one functional copy and become TK-/-. These mutants can then be selected for and quantified. TK6 were lentivirally transduced with vectors containing EGFP and either AE or AE9a. AE was cloned into the pHR-SINcPPT-SIEW lentiviral transfer vector and a similar AE9a construct acquired. Virus was produced utilising the 293T cell line and TK6 were transduced by a spinfection technique. Expression was confirmed by flow cytometry, western blotting and qRT-PCR. Single clonal populations were generated and fusion gene expression levels of 28% (AE) and 40% (AE9a) relative to AE expression in Kasumi-1 (a t(8;21) AML patient-derived cell line) were achieved. We used these transduced cell line models to determine whether the presence of AE or AE9a affected cell viability and susceptibility to the acquisition of mutations. Spontaneous TK-/- mutants were first purged from the population by treatment with CHAT media. Two weeks post CHAT treatment, AE+ TK6 were shown to acquire a greater number of mutations than controls, both spontaneously and when exposed to doxorubicin (Figure 1). AE+ TK6 had a 2.2 fold higher background mutation frequency than controls, suggesting that AE promotes spontaneous acquisition of mutations. When treated with low-dose doxorubicin, AE+ TK6 had a 2–3 fold higher mutation frequency (3.5 × 10−5) in comparison to controls; wild-type TK6 and backbone transduced TK6 (1.4 × 10−5 and 1.1 × 10−5 respectively). High density SNP array analysis of AE-expressing TK6 has recently identified deletions at 10q25.1 and 3p24.3. The deletion at 3p24.3 potentially affects expression of SGOL1, coding for a protein involved in sister chromatid segregation and PCAF, an acetyl transferase which has been implicated in the regulation of p53. The effect of the deletion on these genes is currently being evaluated. Figure 1. Mutation frequency of wild type TK6, backbone control and AE-expressing TK6 in response to doxorubicin. Figure 1. Mutation frequency of wild type TK6, backbone control and AE-expressing TK6 in response to doxorubicin. Our data show that TK6 expressing AE or AE9a have an enhanced sensitivity to the cytotoxic effects of the anthracycline doxorubicin. This contributes to the understanding of the relatively good prognosis of t(8;21) AML. We have shown that expression of AE in a cell system promotes the acquisition of more mutations than that of non-expressing cells. This provides further understanding as to how the pre-leukemic AE+ hematopoetic clone transforms and results in leukemia. Disclosures: No relevant conflicts of interest to declare.