ALBERT

Description: Therapy-related acute myeloid leukemia (t-AML) is a rare but fatal complication of cytotoxic therapy. Whereas sporadic cancer results from interactions between intermittent or low-level environmental exposures and multiple low-penetrance alleles, t-AML results from an acute exposure to potent genotoxic agents. Consequently, we hypothesized that only a small number of relatively higher-penetrance variants would predispose towards t-AML and that, therefore, the association signal from these t-AML risk alleles could be detected even in a modest-sized cohort. To test this, we undertook a pilot genome-wide association study to identify genetic variants associated with t-AML. We genotyped nonmalignant DNA from 80 well-characterized t-AML patients of European descent followed at the University of Chicago and 150 healthy controls using the Affymetrix GeneChip Human Mapping 10K array platform. Following the application of quality control filters, our overall call rate was 98.4%. There was no evidence for population stratification or other forms of bias in our data. Using permutation methods, we first assessed the evidence for an excess of disease-genotype associations in our data over what would have been expected by chance at multiple significance thresholds. Even at nominal significance thresholds, we observed a marked enrichment; for example, we identified 15 SNPs at p 〈 0.001, whereas we only expected 5.5, and at p 〈 0.05, we found 329 associations as compared to 278.6 expected by chance. Then, to determine whether this apparent enrichment over chance was significant, we calculated empirical enrichment p-values at each significance threshold. Even at p = 0.05, this enrichment was highly significant (penrich = 0.008), indicating that we have sufficient power to identify genetic variants likely to be truly associated with t-AML susceptibility. At p = 0.001, the enrichment for SNPs associated with t-AML over chance was almost 3-fold. The odds ratio for each SNP at this threshold was 〉 2.0, suggesting that many are likely to be moderately- or highly-penetrant t-AML risk alleles. Nine of the 15 SNPs significant at this threshold are in linkage disequilibrium (LD) with known genes; of these, 4 are intronic. Although none have been previously studied in t-AML, several encode proteins directly implicated in leukemogenesis, including TLE4, a tumor suppressor commonly deleted in a subset of patients with de novo AML; IPMK, a positive regulator of the pro-survival AKT kinase; and the IL-1 family gene cluster, which has been linked to cell proliferation and apoptosis resistance in AML blasts and has also been shown to promote tissue invasion by leukemic cells. To confirm our findings, we validated a subset of top associations in an independent cohort of 70 patients with t-AML and 95 controls. As a complementary approach to this association mapping, we employed a multi-locus method contrasting the extent of LD in cases and controls in 5-SNP sliding windows throughout the genome. We identified a number of plausible candidate genes, including FHIT and ESR1, genes known to be somatically mutated in t-AML. We also performed copy number analysis to identify regions that are recurrently deleted or amplified in cases but not controls. No such alterations were identified. Taken together, these analyses suggest that genetic variants predisposing to cancer are greatly enriched in t-AML as compared to sporadic cancer and can be detected even in a small patient cohort. Given that germline samples from patients with t-AML are extremely scarce, the demonstration that even a low resolution and modest-sized study can yield a compelling list of candidate genes and polymorphic variants is a particularly important step towards the goal of identifying patients at risk for t-AML at the time of their initial diagnosis so that their treatment can be modified to minimize this risk. Furthermore, because cytotoxic therapy is a potent surrogate for the environmental exposures that drive virtually all cancers, t-AML may be a powerful model for the study of gene-environment interactions in cancer. Hence, this study may point towards genetic risk factors not only for t-AML but also a variety of commonly-occurring cancers, given the appropriate environmental exposures.

Description: The p53 tumor suppressor directs the cellular response to many mechanistically distinct DNA-damaging agents and is selected against during the pathogenesis of therapy-related acute myeloid leukemia (t-AML). We hypothesized that constitutional genetic variation in the p53 pathway would affect t-AML risk. Therefore, we tested associations between patients with t-AML (n = 171) and 2 common functional p53-pathway variants, the MDM2 SNP309 and the TP53 codon 72 polymorphism. Although neither polymorphism alone influenced the risk of t-AML, an interactive effect was detected such that MDM2 TT TP53 Arg/Arg double homozygotes, and individuals carrying both a MDM2 G allele and a TP53 Pro allele, were at increased risk of t-AML (P value for interaction is .009). This interactive effect was observed in patients previously treated with chemotherapy but not in patients treated with radiotherapy, and in patients with loss of chromosomes 5 and/or 7, acquired abnormalities associated with prior exposure to alkylator chemotherapy. In addition, there was a trend toward shorter latency to t-AML in MDM2 GG versus TT homozygotes in females but not in males, and in younger but not older patients. These data indicate that the MDM2 and TP53 variants interact to modulate responses to genotoxic therapy and are determinants of risk for t-AML.

Description: 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.

Description: Abstract 2727 Introduction: Chronic lymphocytic leukemia (CLL) is a B-cell lymphoproliferative disease which follows a heterogeneous clinical course. Although the two staging systems, based on clinical parameters, have been effective in stratifying patients into different risk groups, they both fail to identify the early stage patients who will progress rapidly and therefore benefit from an early or more intensive therapy. This is important as most patients present at an early stage of disease. A number of biological markers have been identified to date to aid prediction. These include the IgVH mutational status, CD38 and Zap-70 expression. Although these parameters provide fairly accurate prognostic information, neither marker alone or combined can identify either Binet stage A patients who are going to progress, or the stage B or C patients who may require alternative treatment at the onset. It is therefore important to identify new predictive markers which may provide additional or better prognostic information. The microRNAs are endogenous, non-coding RNAs that play key regulatory roles in a diverse range of pathways, including development, cell proliferation, differentiation and apoptosis. These 18–24 nucleotide single-stranded RNAs are involved in post-transcriptional gene regulation, by binding to complimentary sites in the 3' UTR of messenger RNAs (mRNA), usually resulting in gene silencing. A number of findings early on in the history of microRNAs suggested their potential role in human cancer, and in 2006 Calin et al directly associated de-regulated expression of miR-15 and -16 in the development of CLL. Since this time, a number of microRNAs have been implicated in CLL lymphomagenesis, including miR-92, which is a mature member of the miR-17-92 cluster. The miR-17-92 is located on chromosome 13q31 and shown to act in an oncogenic capacity (oncomiR-1). With respect to CLL, although the expression levels of mature microRNAs of the miR-17-92 cluster have been assessed, the role of such expression in predicting disease outcome has never been examined. Methods and Results: We used qRT-PCR to assess the expression levels of mature microRNAs of the miR-17-92 cluster, relative to normal CD19+ B-cells. We report that, despite being transcribed from the same parental cluster, the expression levels of all mature microRNAs vary, with miR-17-5p and -18 showing significantly higher levels than the other members of the cluster (p =

Description: The stress-inducible transcription factor, NF-κB, plays an important in role in B-cell malignancies, regulating expression of a plethora of genes including those that drive proliferation, apoptosis and survival. Non-canonical ('alternative') NF-κB signalling, resulting in processing of the p100 subunit to p52 and dimerization with RelB, is less well-studied than the canonical pathway. Previous studies on NF-κB signalling, particularly in CLL, showed that the RelA (canonical) subunit is highly expressed and associated with poor outcome. In multiple myeloma and lymphoma, mutations in genes that alter NF-κB activity (NOTCH1, BIRC3, MYD88, TRAF2/3) can promote cell survival, by increasing activation of the CD40 receptor or NIK (NF-κB-inducing kinase), resulting in constitutive non-canonical NF-κB signalling. More recently, a comprehensive study showed that recurrent mutations in TRAF3, BIRC3 and NFKB2, all of which regulate non-canonical NF-κB signalling, also occur in CLL (Puente XS et al, Nature 2015). Taken together, these data suggest that non-canonical NF-κB signalling is important in CLL and that this pathway represents a novel therapeutic target. We hypothesised that receptor-activated proliferation in CLL cells is reliant on non-canonical NF-κB-regulated gene transcription, and that targeting this pathway would decrease CLL cell survival. Since the IKKα kinase phosphorylates p100, which in turn leads to active p52/RelB dimers in the nucleus, it is a key driver of the non-canonical signalling pathway. We used cell line models and patient-derived CLL cells to evaluate a series of selective, first-in-class IKKα inhibitors. Initially, we assessed 20 novel IKKα inhibitors in MEC-1 (CLL-like, BIRC2/3 and TP53 deleted) and Maver-1 (mantle cell lymphoma, TRAF3 deleted) cell lines, as these are representative of B-cell malignancies with constitutive activation of non-canonical NF-κB. Compounds S and U were found to be potent (cell-free IC50 values 〈 50nM) and selective (e.g. 〉50-fold over IKKβ). In growth inhibition studies, compound S inhibited cell proliferation with GI50 values of 4.3 μM (MEC-1) and 2.7 μM (Maver-1) whereas compound U gave GI50 values of 0.47 μM (MEC-1) and 0.5 μM (Maver-1). We then examined selected cases from our CLL cohort. Western blotting of nuclear extracts showed that expression of NF-κB subunits in patient-derived CLL cells was heterogeneous: RelA was constitutively expressed in most cases, and although expression of the non-canonical subunits, p52 and RelB, varied, high levels of p52 were associated with high RelB expression, indicating that non-canonical signalling is active in CLL. The panel of 20 IKKα inhibitors were also tested in viability and apoptosis assays in patient-derived CLL cells, revealing LC50 values ranging from 0.5 μM (Compound U) - 5μM (48 hr exposure). Decreasing cell survival correlated with increased caspase 3/7 activation. To model the tumour microenvironment, we used CD40L-expressing fibroblasts in co-culture with primary CLL cells to stimulate NF-κB signalling and CLL cell proliferation. CD40L stimulation led to increased levels of nuclear RelB and p52 levels after 2-8 hrs co-culture, concomitant with activation of IKKα, as demonstrated by rapid phosphorylation of p100 subunit. Compound S was evaluated in CD40L-stimulated CLL cells where it was shown to inhibit p100 processing and decrease levels of nuclear RelB and p52. Compound S significantly reduced CD40L-stimulated proliferation of CLL cells following 7 day treatment (1μM), in a similar fashion to the clinically-used PI3Kδ inhibitor, Idelalisib, and was also effective at reducing proliferation of TP53-mutated patient-derived CLL cells. Compound S had little or no effect on accumulation of nuclear RelA (p65), indicating selectivity of these inhibitors for IKKα over IKKβ and suggesting that targeting receptor-stimulated IKKα in B cells may avoid 'global' toxicity anticipated by use of IKKβ-targeting therapies. Future studies will examine the gene expression profile following IKKα inhibition in CLL, and also interrogate the underlying genotypes among patients that may drive NF-κB signaling. These data provide proof of concept that targeting non-canonical NF-κB signaling is a valid therapeutic strategy in CLL. Disclosures No relevant conflicts of interest to declare.

Description: The ataxia telangiectasia and RAD3-related (ATR) protein kinase is a component of the cellular DNA damage response pathway and promotes cell survival by signalling repair of collapsed replication forks generated by replication stress. We hypothesised that inhibition of ATR potentiates the anti-leukaemic activity of chain terminating nucleoside analogues used in the treatment of acute myeloid leukaemia (AML). We used VE-821 and its derivative VX-970 (Vertex Pharmaceuticals, Abingdon, UK) as potent and specific inhibitors of ATR kinase activity to examine the effects of ATR inhibition in AML cell lines, primary AML cells and AML xenografts. Co-treatment with 1mM VE-821 did not consistently potentiate the anti-proliferative effects of cytarabine, clofarabine or fludarabine in a panel of AML cell lines. However, there was consistent potentiation of hydroxyurea and gemcitabine in all 7 AML cell lines tested. Treatment with hydroxyurea, which induces replication stress via depletion of dNTPs, resulted in phosphorylation of CHK1, a downstream target of ATR. CHK1 phosphorylation was attenuated when 1mM VE-821 was co-administered with hydroxyurea. Exposure of cells to gemcitabine or hydroxyurea slowed transit through S phase, which was pronounced in combination with VE-821. HL-60 AML cell clones expressing either a constitutively active or inducible shRNA construct targeting ATR had reduced ATR protein expression compared to control cells and were significantly more sensitive to the anti-proliferative effects of gemcitabine and hydroxyurea, but not to cytarabine, clofarabine or fludarabine. The growth inhibitory effects of hydroxyurea and gemcitabine were also significantly potentiated by VE-821 in primary AML patient samples, which included three adult patients with de novo AML and a paediatric patient with therapy-related AML. In contrast, ATR inhibition did not potentiate the inhibitory effects of hydroxyurea or gemcitabine in primary bone marrow cells from healthy donors ex vivo. We next sought to determine whether ATR inhibition potentiated hydroxyurea and gemcitabine in an orthotopic mouse model of AML. MV4-11 AML cells engineered to express firefly luciferase (MV4-11 pSLIEW) were intrafemorally transplanted into immunodeficient Rag2-/- gc-/- mice. Bioluminescent imaging via IVIS Spectrum (PerkinElmer, Buckinghamshire, UK) demonstrated localised femoral engraftment first detectable 4-5 days post-injection, with luciferase signal developing in other parts of the body (liver, ovaries) between days 15 and 18 in untreated mice. Treatment was initiated 7 days post-injection when disease was localised to the femur and prior to emergence of disseminated luciferase signal. Single agent hydroxyurea (250mg per kg, IP days 0-4 and 7-11) conferred some early disease control compared to controls as determined by luciferase total body flux measured on day 14, but this was not statistically significant (p=0.18) and did not affect overall survival (mean 35 days for controls and 37 days for hydroxyurea, p=0.47). Monotherapy with VX-970 (60 mg per kg, orally on days 0-4 and 7-11) also conferred early disease control compared to vehicle-treated mice (p=0.18), and resulted in significantly longer overall survival (mean 40 days, p=0.017). Combination treatment with hydroxyurea and VX-970 did not result in more effective early disease control or improved overall survival compared to monotherapy with either agent. Treatment with gemcitabine monotherapy (100 mg per kg, intraperitoneal injection on days 0, 3, 7 and 10) conferred significant early disease control (p=0.002) and significantly improved overall survival compared to controls (mean survival 73 days, p

Description: The first two authors and last two authors contributed equally. Genome-wide association studies (GWAS) have identified risk loci for Acute Lymphoblastic Leukemia (ALL), Chronic Lymphoblastic Leukemia (CLL) and Non-Hodgkin Lymphoma, however an Acute Myeloid Leukemia (AML) GWAS has not been published to date. We performed a GWAS to identify AML and Myelodysplastic Syndrome (MDS) risk loci using a nested case-control study design in the DISCOVeRY-BMT cohorts which includes almost 2000 AML and MDS patients as cases and 2813 unrelated donors as controls. Genotyping was performed using the Illumina Human OmniExpress BeadChip and imputed using the Haplotype Reference Consortium, yielding 〉 8 million high-quality variants for statistical analysis. Logistic regression models with AML (de novo AML with normal cytogenetics, de novo AML with abnormal cytogenetics and therapy-related AML) and MDS cases and European American healthy donor controls adjusted for age and sex were used to test the association of each SNP with disease status. To identify the strongest association signal with disease we conducted a summary statistic SNP-based association analysis (ASSET) using non-overlapping AML and MDS cases implemented in R statistical software. ASSET uses an exhaustive search for SNPs with small but common pleiotropic effects across groups of traits while accounting for the multiple tests required by the subset search, as well as any shared controls between groups. This approach allowed us to further investigate the heterogeneity within AML subtypes and to gain increased power by pooling subtypes that show pleiotropic effects. ASSET genome wide (GW) significance is defined as P

Description: 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.

Description: Personalised medicine is predicted to significantly improve outcomes for cancer patients, but implementation requires comprehensive genetic characterisation of malignant cells to identify therapeutically exploitable vulnerabilities. Using an isogenic cell model system with CRISPR-inactivated TET2 in HEL acute myeloid leukemia (AML) cells and an orthotopic mouse xenograft model we demonstrate that mutant TET2 allele dosage significantly affects sensitivity to 5-azacitidine hypomethylating therapy in AML, with biallelic mutation conferring hypersensitivity relative to monoallelic mutation. In the presence of 5-azacitidine, cell clones with biallelic TET2 mutation had significantly lower cloning efficiency (P = 3 x 10-3) and proliferation in liquid culture (P 〈 1 x 10-4) compared to isogenic clones with monoallelic TET2 mutation. Mixed populations of monoallelic and biallelicTET2 mutated HEL AML cells were transplanted via intrafemoral injection into Rag2−/−Il2rg−/−129×Balb/c mice, and treatment with 5-azacitidine resulted in significant negative in vivo selection against TET2 null cells relative to cells with monoallelic TET2 mutation (P = 4 x 10-4). Methylation analysis revealed the acquisition of an overall hypermethylation phenotype in TET2 null cells and RNA sequencing identified significant down-regulation of ABCB1 transcript, resulting in concomitant pronounced down-regulation of the MDR1 drug efflux transporter at the protein level. RNA sequencing pathway analysis also identified a global effect on ribosome pathway (KEGG pathway ko03010) transcript levels (Padjusted = 0.002), evidenced by down-regulation of numerous RNA polymerase II components in cells with bi-allelic TET2 mutation compared to cells with monoallelic TET2 mutation. Consistent with our isogenic model data, we characterise biallelic somatic TET2 mutation in a patient with AML that was chemoresistant to anthracycline/cytarabine-based chemotherapy but acutely sensitive to 5-azacitidine, resulting in durable cytomorphological remission. Integration of next generation sequencing, interphase FISH and SNP array analysis of bone marrow at AML presentation, relapse and during remission was used to infer tumour phylogeny which indicated that disease pathogenesis was initiated by a TET2 nonsense mutation (c.2815C〉T, Q939*) with subsequent deletion of the second TET2 allele and a NPM1 mutation (c.863_864ins, TCTG) that arose after the acquisition of bi-allelic TET2 mutation. Furthermore, our data demonstrate that 5-azacitidine treatment almost completely eliminated the TET2/NPM1-mutated clone. 5-azacitidine also induced a modest reduction in ancestral pre-leukemic cells carrying bi-allelic TET2 mutation but negative for the NPM1 mutation, although the majority retained viability and re-acquired the ability to differentiate and recapitulate normal haematopoiesis rendering a cytomorphological remission. These observations suggest that bi-allelic TET2 mutation confers sensitivity to the cytotoxic effects of 5-azacitidine, but that the major effect of 5-azacitidine is the induction of phenotypic re-programming. The frequency of TET2 mutation in primary AML is estimated at 10-20%, with the majority of these being monoallelic. We determined the frequency of TET2 alterations in AML patients presenting with a chromosome 4 abnormality discernible cytogenetically. TET2 copy number and mutational status were determined using high density SNP arrays and gene sequencing, respectively. In a panel of 30 AML cases with a chromosome 4 abnormality, four patients were heterozygous for TET2 mutation (all deletions resulting in reduced copy number) and three patients were homozygous for TET2 mutation (deletion plus base substitution in two cases and homozygous base substitution resulting from uniparental disomy in one case). Furthermore, all seven cases with TET2 mutation were characterised by cytogenetics that included loss or gain of material on chromosome 4. In contrast, only 1 case with a TET2 mutation had a translocation affecting chromosome 4. In summary, our data argue in favour of using 5-azacitidine in patients with biallelic TET2-mutated AML and demonstrate the importance of considering mutant allele dosage in the implementation of personalised medicine for cancer patients. Disclosures Stoelzel: JAZZ Pharmaceuticals: Consultancy; Neovii: Other: Travel funding; Shire: Consultancy, Other: Travel funding. Jackson:Celgene, Amgen, Roche, Janssen, Sanofi: Honoraria. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.