ALBERT

Description: The majority of patients with adult acute myeloid leukaemia (AML) that present with an apparently normal karyotype (NK-AML) are grouped together in the “intermediate” risk category and constitute 40-45% of all adult AML patients. Mutations in the FMS-like tyrosine kinase 3 (FLT3) receptor whether it is internal tandem duplication (ITD) of its juxtamembrane domain or point mutations in its kinase domain are one of the most common mutations in NK-AML. The presence of FLT3-ITD mutation in NK-AML results in a more aggressive disease, resistance to therapy and poor survival. Acquired copy neutral loss of heterozygosity (CN-LOH) also referred to as uniparental disomy (UPD) is a common phenomenon of myeloid malignancies where an oncogenic allele is duplicated on the other chromosome. The use of single nucleotide phenotype analysis (SNP-A) karyotyping detects CN-LOH in 20% and 40% of newly diagnosed and relapsed AML respectively. CN-LOH at 6p or at the FLT3 locus 13q associated with a FLT3-ITD mutation in NK-AML results in an even more aggressive disease compared to NK-AML + FLT3-ITD without CN-LOH. It has been proposed that CN-LOH is the result of a homologous recombination (HR) DNA repair event. However, the underlying mechanisms that confer CN-LOH have yet to be determined. To elucidate the mechanisms that produce CN-LOH in NK-AML we determined whether oncogenes such as FLT3-ITD have the propensity to generate CN-LOH through up-regulation of inter chromosomal (between maternal and paternal chromosomes) HR DNA repair. We have showed previously that constitutional FLT3-ITD kinase activity increased reactive oxygen species resulting in elevated double strand breaks. Moreover, FLT3-ITD mutation increases homologous recombination activity through transcriptional augmentation of HR factor, RAD51 expression. Firstly, to evaluate FLT3-ITD induced genomic instability we measured sister chromatid exchanges (SCE) that result from HR mediated chromosome cross-over. It has been shown previously in the pre-leukaemic chromosomal instability disorder, Blooms syndrome is characterised by increased SCE and an increased propensity for CN-LOH. FLT3/ITD primary AML (n=17), MOLM-13 and FLT3-ITD transfected U937 cells show a significant increase in SCE compared to WT FLT3 primary AML cells, U937 cells transfected with empty vector, (10 vs. 6 SCE per metaphase, p

Description: Introduction 5-azacitdine (aza) treatment in myelodysplastic syndrome (MDS) induces a response rate of 40-45% and prognostic factors for response and survival remain still largely unknown. Presence of mutant TET2 has been shown to predict better response to aza and the recently described French Azacitidine prognostic score segregates patients into 3 groups with varying median overall survival. Although a plethora of somatic mutations have been described in MDS, none has been consistently shown to be prognostically important in the context of response to hypomethylating agent(s). Patients and methods To identify the mutation signature associated with aza response, we undertook screening of 24 myeloid genes (splicing, epigenetic, transcription factors, STAG2, TP53) in 66 MDS patients treated with aza at our institution over a period of 2004-2012. Mutation analysis was done by deep (454 FLX) and Sanger sequencing. SNP-6 karyotyping was also performed in a subset to correlate with mutation status. Responses were assessed as per the international working group for MDS criteria. The median age was 67 years (range 36–87 years), median number of courses 7(range 2–42), 79% of patients belonged to int-2/high risk IPSS category. WHO category subtypes were; RA/RARS-2; RCMD-9; RAEB-39; s-AML (evolved from pre-existing MDS) -5, therapy related myeloid neoplasm (t-MDS/t-AML) -8 and CMML-3.IPSS cytogenetic subgroups were, good risk: 22, intermediate: 7, and poor risk: 37. One fourth of patients had received prior therapy, with only two receiving low dose cytarabine. Median time from diagnosis to aza treatment was 8.6 months. The overall response rate (ORR) to Aza was 47% (31/66) with complete response (CR) 17%, partial response (PR) 11%, marrow CR (mCR) 12% and stable disease with hematological improvement (SD-HI) 7%. Results Candidate mutations were seen in 82% (54/66) of patients, with the more than half harboring ≥2 mutations each. The most frequently (≥5 %) mutated genes being ASXL1 (29%), TP53 (23%), TET2 (14%), DNMT3A (12%), SRSF2 (12%), EZH2 (11%), NRAS (8%), U2AF1 (8%), IDH2 (8%), RUNX1 (8%), CCBL (6%) and FLT3-ITD (5%). On univariate analysis presence of EZH2 mutations predicted for a better ORR compared to wild type EZH2 (21% vs. 3%, p

Description: It has been previously reported that cells defective for double strand break (dsb) DNA repair can be selectively targeted for apoptosis through abrogation of their PARP activity. It has also been reported that leukemic cells have inherent defects in dsb DNA repair. Exploitation of DNA repair defects using PARP inhibitors (PI) thus represents a specific and less toxic form of therapy for a number of hematological malignancies. In order to test the efficacy of PI therapy we analysed primary cells from acute myeloid leukemia (AML) patients and the potential for combination therapy with inhibitors of DNA methyltransferase (DNMTi) or histone deacetylase inhibitors (HDACi). We report that from a panel of 12 AML patients, 2 AML patient cells demonstrated abnormal cell cycle profiles and apoptosis in response to the PI, KU-0058948 (1μM). In contrast, normal control cells displayed standard cell cycle profiles and no apoptosis in response to PI. Clonogenic cytotoxicity assays also showed that these PI sensitive AML patient cells exhibited between 45–55% cell survival compared with 100% cell survival in control cells (p

Description: The majority of patients with adult acute myeloid leukaemia (AML) that present with an apparently normal karyotype (NK-AML) are grouped together in the “intermediate” risk category and constitute 40-45% of all adult AML patients. Mutations in the FMS-like tyrosine kinase 3 (FLT3) receptor whether it is internal tandem duplication (ITD) of its juxtamembrane domain or point mutations in its kinase domain are one of the most common mutations in NK-AML. The presence of FLT3-ITD mutation in NK-AML results in a more aggressive disease, resistance to therapy and poor survival. Acquired copy neutral loss of heterozygosity (CN-LOH) also referred to as uniparental disomy (UPD) is a common phenomenon of myeloid malignancies where an oncogenic allele is duplicated on the other chromosome. The use of single nucleotide phenotype analysis (SNP-A) karyotyping detects CN-LOH in 20% and 40% of newly diagnosed and relapsed AML respectively. CN-LOH at 6p or at the FLT3 locus 13q associated with a FLT3-ITD mutation in NK-AML results in an even more aggressive disease compared to NK-AML + FLT3-ITD without CN-LOH. It is has been proposed that CN-LOH is the result of a homologous recombination (HR) DNA repair event. However, the underlying mechanisms that confer CN-LOH have yet to be determined. To elucidate the mechanisms that produce CN-LOH in NK-AML we determined whether oncogenes such as FLT3-ITD have the propensity to generate CN-LOH through production of excessive DNA double strand breaks (DSB) and up-regulation of HR DNA repair. Primary NK-AML (n=17) were initially characterised by SNP-A and DNA sequencing. 12/17 primary NK-AML had FLT3/ITD mutations with a clone size ≥ 25%, but were wild type (WT) for RAS. From this cohort 6/12 possessed CN-LOH with 3/12 having CN-LOH at 13q. 5/17 primary NK-AML were WT for FLT3 and RAS, but had nucleophosmin (NPM) and DNA methyltransferase 1 (DNMT1) mutations. From this cohort, 1/5 primary NK-AML + WT FLT3 had CN-LOH. We showed that primary AML + FLT3/ITD cohort, the MOLM 13 cell line that possesses a FLT3/ITD mutation and WT FLT3 KG-1 cells transfected with a FLT3-ITD expression vector demonstrate increased ROS compared to the WT FLT3 primary NK-AML cohort, the WT FLT3 cell line, U937 and KG-1 transfected with empty vector. Immunofluorescence studies showed that primary AML with FLT3/ITD, MOLM 13 and FLT3-ITD transfected KG-1 cells have increased mobilization of phospho-γH2AX (a DNA DSB intermediate) and RAD51 (a HR factor) to sites of DNA DSB in response to mitomycin C (MMC) induced DNA damage compared to non-mutated FLT3 primary AML cells, cell lines and KG-1 transfected with empty vector (phospho-γH2AX, 29% vs. 21%, p

Description: Recent reports suggest that BrCA1−/− and BrCA2−/− cells can be selectively targeted for cell death through abrogation of their PARP activity. It is postulated that as a result of PARP inhibition, accumulation of single strand DNA breaks (SSB) leads to the replication fork collapse and conversion of SSB to double strand DNA breaks (DSB). The inability of repair defective cells such as BrCA2−/− to repair the DSB would lead to cell death. Exploitation of DNA repair defects using PARP inhibitors (PI) thus represents a more specific and less toxic form of therapy for a number of haematological malignancies. Chromosomal instability (CI) syndromes that have inherent defects in double strand DNA repair also have a uniformly high incidence of transformation to acute leukaemia or lymphoma. In order to test the efficacy of PI therapy we analysed CI cell lines, myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) cell lines and the potential for combination therapy with inhibitors of DNA methyltransferase (DNMTi) or histone deacetylase inhibitors (HDACi). We report that cells from CI syndromes; Blooms syndrome, Fanconi Anaemia (FancD2 and FancA), Ataxia telancgectasia and Nijmegen break syndrome display abnormal cell cycle profiles and excessive apoptosis in response to the PI’s PJ34 (3μM) and EB47 (45μM). In contrast, normal control cells displayed standard cell cycle profiles and no apoptosis in response to PI at equivalent concentrations. Clonogenic cytotoxicity assays showed that CI syndrome cells exhibit between 30–75% cell survival compared with 100% cell survival in control cells (p

Description: Mutations in the FMS-like tyrosine kinase 3 (FLT3) receptor whether it is internal tandem duplication (ITD) of its juxtamembrane domain or point mutations in its kinase domain are one of the most common mutations in normal karyotype acute myeloid leukaemia (NK-AML). The presence of FLT3-ITD mutation in NK-AML results in a more aggressive disease, resistance to therapy and poor survival. Acquired copy neutral loss of heterozygosity (CN-LOH) also referred to as uniparental disomy (UPD) is a common phenomenon of myeloid malignancies where an oncogenic allele is duplicated on the other chromosome. The use of single nucleotide phenotype analysis (SNP-A) karyotyping detects CN-LOH at the FLT3 locus, 13q associated with a FLT3-ITD mutation in NK-AML that results in an even more aggressive disease compared to NK-AML + FLT3-ITD without CN-LOH. It has been proposed that CN-LOH is the result of a homologous recombination (HR) DNA repair event. However, the underlying mechanisms that confer CN-LOH have yet to be determined. To elucidate the mechanisms that produce CN-LOH in NK-AML we developed a Zinc Finger Nuclease (ZFN) inter homologous recombination (iHR) targeting assay. The assay, based on the DR-GFP reporter developed by Pierce et al (1999), (Genes Dev. 13(20):2633-8) relies on the sequential targeting of two overlapping, but disrupted GFP repeat sequences to the adeno associated virus integration site 1 (AAVS1) on separate 19q alleles. Productive recombination between heterologous chromosomes in the region of 19q would result in restoration of GFP expression. The AML cell lines NB4 and THP-1 were transfected with a ZFN specific for AAVS1 integration (CompZr, Sigma) and the PZDonor AAVS1 puromycin derived construct DR1-Puro (Figure 1A,C) that possesses a 5' GFP sequence interrupted by an 18bp recognition sequence for the rare restriction enzyme, I-SCE I. Puromycin selected cells were then transfected with pZDonor derived DR2-Blast (Figure 1B,D) possessing the 3' GFP sequence and a blastocidin resistance gene. Puromycin and blastocidin selected cells were verified for correct integration of the plasmids to separate AAVS1 loci using location specific PCR and FISH, Figure 1F-H. Clones with correct integration were then expanded and transfected with FLT3-ITD, WT FLT3 expressing or empty vectors. Transfected cells were treated with an I-SCE I expression viral supernatant to induce a double strand break in DR1-Puro. Cells were cultured for a further 5 days before FACS analysis. FLT3-ITD transfected cells demonstrated GFP positive cells (inter HR events, 0.15% compared to WT-FLT3 and empty vector transfected cells (0.15% vs 0%, p

Description: Abstract 125 Polycomb gene complex mutations have been implicated in the pathogenesis of myeloid neoplasms. ASXL1, a epigenetic regulator of transcription by recruiting polycomb and trithorax complexes to the chromatin domain and EZH2 (histone methyltransferase) is the catalytic domain of polycomb repressive complex (PRC) 2, causing gene silencing by trimethylation of lysine 27 of histone 3(H3K27). To identify the role of mutations in ASXL1, EZH2 and their interaction with other mutations (P53, ras, c-cbl, IDH 1/2, BRAF), we analysed 63 MDS patients treated with 5-Azacitidine (Aza) at our institution. Mutation analysis was done by deep (454 FLX) and Sanger sequencing. SNP-6 karyotyping was also performed to correlate with mutation status. In 43 patients, samples were also analysed at various time points post aza treatment. The median age was 67 years (range 36–86 years), median number of courses 7(range 2–109), 90% (57/63) of patients belonged to high risk IPSS category. WHO category subtypes were; RA/RARS-1; RCMD-7; RAEB-37; s-AML (evolved from pre-existing MDS) -9, therapy related myeloid neoplasm (t-MDS/t-AML) -7 and CMML-2.IPSS cytogenetic subgroups were, good risk: 19, intermediate: 6, and poor risk: 38. Thirty six patients had chromosome 7 abnormalities either isolated (12/36) or with additional aberrations. The median time from diagnosis to treatment with aza was 7 months (range 1–42). The overall response rate to Aza was 48% (30/63) with a better survival in responders compared with non-responders (17.3 vs 10.2 months p

Description: INTRODUCTION The cohesin complex is a multimeric protein complex reported to have putative roles in post-replicative DNA repair, sister chromatids cohesion and transcriptional regulation. Somatic mutations in the genes that constitute the cohesin complex (NIPBL, SMC1A, SMC3, and HDAC8) give rise to the debilitating disease, Cornelia de Lange Syndrome. The cohesin genes are mutated in several cancers, the highest incidence reported in bladder cancer followed by myeloid cancers such as myelodysplastic syndrome (MDS). Inhibition of PARP (Poly ADP Ribose Polymerase) activity has been shown to selectively target AML patients with DNA repair defects (Gaymes et al 2009). Discovery of mutations that confer sensitivity to PARP inhibitors have the potential to identify candidates for therapeutic intervention. Mutation in genes involved in epigenetic regulation and RNA splicing account for 70% of the mutations reported in MDS. Mutations in cohesion complex have been reported, but have not been systematically studied in a large and uniformly treated patient cohort. To characterise still further the mutations that shape the MDS phenotype, 154 MDS patients that had been screened for myeloid gene mutations using the 22 gene panel (Syed et al 2013) were re-screened for mutations in four of the cohesin complex genes (SMC1A, SMC3, RAD21 and STAG2). The functional consequences of defective cohesin were also studied using shRNA silencing. METHODOLOGY Nextera sequencing was used to sequence all the coding exons of SMC1A, SMC3 and RAD21 genes, whilst STAG2 was sequenced using the Roche 454 platform. To further elucidate the role of cohesin complex in MDS, lentiviral shRNA silencing of STAG2 was achieved in the AML cell line, U937 and effects of knockdown was subsequently determined through functional assays. RESULTS Sequencing of the cohesin complex genes revealed mutations in 6% of MDS patients of which 4% had STAG2, 1% had SMC3 and0.6% patients had SMC1A mutations. None of the patients studied had a RAD21 mutation. All detected mutations were confirmed by independent PCR and Sanger Sequencing. For STAG2 mutations, a constitutional source of DNA was also used to confirm the mutations, (skin biopsy (n=2), T cells (n=3). 5/6of the STAG2 mutationswere nonsense with an average allelic burden of 33% indicative of a heterozygous mutation. Mutations in SMC3 were reported in two patients; a frameshift mutation found in the coiled coiled domain and a substitution in the 3' splice acceptor site. The average allelic burden for both the mutations was 20%. A missense mutation was identified in SMC1A in one patient with an allelic burden of 13%. (3/9) patients mutations in STAG2 gene coexisted with SRSF2 and ASXL1 mutations. The patients with cohesion mutations had a significantly lower progression free survival as compared to patients without mutation (Figure 1A) Lentiviral shRNA silencing of 75% and 66% for STAG2 was achieved in U937 cells using two shRNAs, confirmed both by q-PCR and Western blot. shRNA silencing of STAG2 also resulted in reduced expression of partner cohesin sub-unit, RAD21 confirmed by western blot. Strikingly, treatment of shRNA silenced U937 with PARP (Poly-ADP-Ribose Polymerase) inhibitors resulted in 28% cytotoxic reduction compared to scrambled control (Figure 1B). Furthermore, cytotoxicity was independent of Homologous Recombination DNA repair competency as recruitment of RAD51 to damaged sites was not altered in shRNA silenced STAG2 U937 cells upon challenge with PARP inhibitors. CONCLUSION In this study, cohesin complex mutations were found in 6% of MDS patients with STAG2 being the most prevalent. We also report the co-existence of cohesin mutations with mutations in ASXL1 and SRSF2. The cohesin mutant allelic burden in these patients was lower compared to ASXL1 and SRSF2 mutations suggesting that cohesin mutations co-exist as passenger mutations alongside mutations that drive clonal evolution like ASXL1 as reported previously (Thota et al 2014). The observation that loss of STAG2 confers PARP inhibitor sensitivity is of major clinical importance. Identification of a cohort of MDS/AML patients with cohesin mutations would signify a major development in the identification of candidates for PARP inhibitor therapy. Disclosures Kulasekararaj: Alexion: Consultancy.

Description: Genomic instability is the driving force of disease progression to frank leukemia. Evidence suggests that aberrant repair of double strand breaks (DSB) by non homologous end-joining (NHEJ), a major repair pathway in mammalian cells, can lead to chromosomal instability and cancer. We previously reported significantly increased error-prone NHEJ in preleukemic syndromes, and a variety of myeloid malignancies, and demonstrated that these cells harbor constitutive DNA damage. We postulated that increased NHEJ misrepair may be a response to the increased DNA damage. Here, we have studied a mouse model for myeloproliferative/myelodysplastic syndrome (MPD/MDS) to determine whether the frequency of DNA damage and aberrant NHEJ repair may be an indicator for genomic instability as the disease progresses. Transgenic mice bearing mutant NRAS and BCL-2 driven by the MRP8 promoter, which directs expression of the transgene to committed myeloid progenitors and neutrophils, have a relatively mild phenotype with an increase of immature neutrophils. The BCL2 mice have an increase in marrow blasts, but have normal blood counts. Transgenic mice harboring both mutant NRAS and BCL2 genes results in a disease phenotype morphologically resembling human late MDS (FAB subtypes refractory anemia with excess blasts (RAEB), RAEB in transformation (RAEBt) or chronic myelomonocytic leukaemia (CMML)) with increased marrow blasts. We show that the bone marrow and spleen from the NRAS and BCL2 mice demonstrate an increase in the frequency of NHEJ misrepair activity, compared with normal (FVBN) mice (NRAS: 7.6% vs 3.7%, BCL2: 6.5% vs 3.7%, n=3). Strikingly, the NRAS +BCL2 double transgenic mice show a large and significant increase in NHEJ misrepair activity (19.02%, n=3, p

Description: Abstract 276 In both myelodysplatic syndrome (MDS) and acute myeloid leukaemia (AML), relapse from standard chemotherapeutic intervention is common with only 20–30% enjoying long-term disease-free survival. Allogeneic stem cell transplantation still remains the only curative treatment in MDS/AML, but only in 15–20% of patients. Older patients (〉70 years) that constitute the majority of MDS/AML patients are often resistant to chemotherapy, achieve short lived remission and are not candidates for stem cell transplantation. Given the high number of patients refractory to conventional therapy and the relatively high rate of therapy relapse, efforts have been made in the search for alternative treatment strategies. Inhibition of Poly ADP ribose polymerase (PARP) activity can selectively target cancer cells through exploitation of inherent DNA repair defects. Significant single agent anti-tumour responses coupled with a wide therapeutic index have been influential in moving PARP inhibitors (PARPi) to the clinical arena. MDS/AML is characterized by chromosomal instability (CI) manifesting as deletions, translocations and chromosome losses. Single nucleotide polymorphism arrays (SNPA) karyotyping show that loss of heterozygosity (LOH) and uniparental disomy (UPD) are common in MDS/AML and thus it has been suggested that the underlying cause of this CI is a defect in double strand DNA repair. We have previously shown that 15% of AML and MDS primary patient cells and cell lines are sensitive to PARPi through exploitation of homologous recombination DNA repair defects. To explicate still further the mechanisms that support PARPi sensitivity in MDS/AML we tested for microsatellite instability (MSI) in primary high risk MDS and primary AML for the presence of frameshift mutations in specific DNA repair genes. 13 of 63 (21%) high risk MDS patients possessed MSI (9 MSI-low and 4 MSI-high). Significantly, all 13 MSI positive patients possessed chromosomal abnormalities, both gross and cryptic UPD determined by SNPA, whilst 12 patients (19%) found to be have normal cytogenetics and lacking UPD failed to demonstrate MSI. Moreover, 3 patients with MSI-high and 1 patient with MSI-low possessed a mono-allelic 1bp deletion in the CTiP exon coding microsatellite. 1bp deletion within the coding exon of CTiP would result in an abbreviated gene CTiP transcript in these patients. From a panel of 18 primary AML samples, 5 primary AML demonstrated sensitivity to the PARPi, BMN673 (100nM). Immunocytochemical staining also showed that PARPi sensitive AML cells demonstrated severely reduced rad51 and increased phospho-γH2AX foci formation compared to PARPi insensitive AML cells. This confirmed that BMN673 targeted HR deficiencies in AML PARPi sensitive cells. Strikingly, 2 of the 5 PARPi responders exhibited MSI, with 1 patient displaying a bi-allelic 1bp deletion in MRe11 and 1 patient exhibiting a mono-allelic 1bp deletion in CTiP. MSI was not observed in the 13 PARPi insensitive AML patients. Western blotting analysis identified the loss of mismatch repair proteins MLH-1 and MSH-2 respectively, in the 2 MSI positive primary AML underlying the MSI observed in the AML patients cells. Moreover, Western blotting analysis also revealed aberrant expression of Mre11 and CTiP in these patients. Finally, to confirm the relative contribution of mutant MRe11 and CTiP to PARPi sensitivity, an expression construct of MRe11 missing exons 5 to 7 (δ5–7MRe11) was transfected into the MSI negative and PARPi insensitive cell line, U937. Cytotoxicity assays and immunocytochemical staining revealed that U937 + δ5–7MRe11 demonstrated significant sensitivity to PARPi with concomitant HR DNA repair defects compared to U937 + vector control. Similarly, Si-RNA knockdown of CTiP in U937 also conferred hypersensitivity to PARPi as a result of an abrogation of functional HR DNA repair. In conclusion, we make the unique observation that MSI dependent mutations in genes that are essential for DNA repair signalling confer PARPi sensitivity in myeloid malignancy. Identification of a cohort of MDS/AML patients with MSI would signify a major development in the identification of candidates for PARPi therapy. Disclosures: No relevant conflicts of interest to declare.