ALBERT

Description: Abstract 3508 Background: Krüppel-like factor 5 (KLF5) is a transcription factor with regulatory roles in cell growth, survival, and differentiation. Our previous studies have indicated that KLF5 is required for myeloid blood lineage differentiation and is a target of genomic methylation in Acute Myeloid Leukaemia (AML) which is associated with reduced expression. Further, we have shown that treatment of leukaemia cell lines, displaying KLF5 hypermethylation, with demethylating agents is associated with induction of KLF5 expression, supporting an inhibitory role of methylation on expression (Diakiw et al, Leuk Res 36:110). Other recent studies have shown that KLF5 expression is reduced by the presence of a heterozygous genotype at a promoter SNP (rs3812852) and plays a role in pathogenic processes in heart and brain. We investigated both methylation and SNP genotype as mechanisms of KLF5 gene regulation in AML and their relationship to patient outcome. Methods: We analysed 232 diagnostic bone marrow mononuclear cell samples from a retrospective cohort of patients with AML. The genotype of the rs3812852 SNP was determined by Sequenom MassARRAY genotyping. DNA methylation of the KLF5 gene locus (intron 1) was assessed using Sequenom MassARRAY epityper. KLF5 gene expression was determined for 85 of these samples using quantitative real-time PCR. Of the 232 patients, 161 received treatment with induction chemotherapy and were included in survival analysis. Results: We found that 28 (12%) patients were heterozygous (G/A) for the SNP (rs3812852) in the KLF5 promoter, with one patient having the rare homozygous genotype (G/G) and 204 (88%) patients the common homozygous genotype (A/A). KLF5 gene expression correlated with the SNP genotype, with the heterozygous patients displaying significantly reduced KLF5 expression compared to normal controls (CD34 cells from normal donors, p=0.044). Consistent with our previous study on a smaller test cohort we observed that hypermethylation of this region of KLF5 is common and is present in 50% of samples in our larger cohort. AML patients with high methylation of KLF5 had significantly reduced KLF5 expression compared to normal controls and AML patients with low KLF5 methylation (p=0.032 and p=0.048 respectively). The correlation of gene expression with the SNP genotype and KLF5 methylation groups was also true when considered together, as patients with the heterozygous genotype (A/G) and high methylation had lower KLF5 expression than patients with the common homozygous genotype (A/A) and low KLF5 methylation (Figure 1A). Cox multi-variate analysis (variables of age, WCC and mutational status of FLT3-ITD, IDH1/2, NPM1) of survival of patients treated with induction chemotherapy showed that the presence of a G/A genotype at the KLF5 promoter SNP was not an independent predictor of overall survival (p= 0.502). In contrast hypermethylation of KLF5 intron 1 was a highly significant independent predictor of poorer overall survival (median survival 10.4 months versus 20.7 months, HR 1.5, p=0.001, Figure 1B). Importantly, when the heterozygous SNP genotype (A/G) and high methylation status were combined, this group of patients had even poorer OS (median survival 5.2 months, HR 3.6, Figure 1C) and this was significant compared to the group with homozygous SNP genotype (A/A) and low methylation (p=0.002). Conclusions: We have shown that both methylation of KLF5 intron 1 and a heterozygous genotype at a promoter polymorphism are associated with reduced KLF5 expression in AML. KLF5 hypermethylation is an independent predictor of poor prognosis and combined with the heterozygous SNP genotype defines a group with particularly poor survival indicating a connection with KLF5 gene expression and response to treatment. Importantly, as the majority of the effect is associated with hypermethylation of KLF5, these patients (50%) may benefit from treatment with demethylating agents. Disclosures: Wei: Celgene: Research Funding.

Description: BACKGROUND: We have previously shown that one target of hyper-methylation in AML is the promoter of the tumour suppressor and stress-response mediator Growth Arrest and DNA Damage inducible 45A (GADD45A) (GADD45AmeHI; 42% of AML). In mice Gadd45a has recently been shown to play a critical role in HSC stress responses. Gadd45a deficiency leads to enhanced HSC self-renewal, DNA damage accumulation in HSC, increased susceptibility to leukemogenesis, and impairment in HSC apoptosis after genotoxic exposure (Chen et al, Blood 2014). These findings suggest that hypermethylation of the GADD45A gene may play an important role in the altered properties of HSC, leukaemic initiation and progression. Promoter hypermethylation of this gene defines a patient group with poor survival on standard therapy (Perugini et al, Leukaemia 2012). To explore further the molecular basis of the GADD45AmeHI patient group weperformed genetic profiling of diagnosis samples using a Sequenom multiplex mutation panel, or using whole exome sequencing for broader coverage (n=95 patients).Sequenom MassARRAY was used for quantitative detection of GADD45A promoter methylation in patient samples. For a cohort of matched diagnosis and relapse samples we used CpG methylation data for GADD45A determined by ERRBS (Akalin et al, PLoSGenetics 2012). Response to cytotoxic drugs and assessment of drug combinations with 5-Aza-deoxycytidine (decitabine, DAC) and anthracycline (Daunorubicin, DNR) was performed in AML cell lines, and with primary leukemic cell populations. RESULTS: The association of the GADD45AmeHI patient group with poor outcome was validated in an independent AML patient cohort of 48 patients from the Alfred Hospital, Melbourne, Australia (p=0.003; HR3.35). Whole exome sequencing and Sequenom multiplex analysis of 95 AML patients revealed a striking co-occurrence of the GADD45AmeHI phenotype with mutations in IDH1, IDH2, and TET2 (p

Description: Abstract 3473 Introduction/Background: Diamond Blackfan Anaemia (DBA) is a rare bone marrow failure disorder characterised predominantly by severe erythroid (red blood cell) failure. While the causative mutation remains unknown in approximately 50% of DBA patients, a mutation resulting in haploinsufficient expression of one of a number of ribosomal proteins (RPS or RPL) has been identified in the remaining 50% of cases, leading to DBA being classified as a ‘ribosomopathy’(Dianzani & Loreni Haematologica. 2008). Activation of the p53 pathway in response to ribosomal disruptions and nucleolar stress has also been implicated in both animal and cellular models of DBA (Danilova et al. Blood 2008; McGowan et al. Nat. Genet. 2008; Fumagalli et al. Nat. Cell. Bio. 2009). Haploinsufficiency of ribosomal proteins appears to be the major cause of DBA although a recent study (Sankaran VG, et al. J. Clin. Invest. 2012) reported the discovery of mutations in the transcription factor GATA-1, in absence of any known ribosomal protein mutation. While GATA-1 mutations have only been detected in 2 DBA pedigrees to date, this exception to the ‘ribosomopathy’ rule may help identify the link between ribosomal protein disruption and the tissue-specificity of the erythroid defect. Results: We have developed a Doxycycline-inducible shRNA RPS19 knockdown model of DBA in the human erythroleukaemic cell line TF1.8. Two independent shRNA triggers targeting RPS19 (the most common gene affected in DBA patients), and a vector-alone control were introduced into cells. RPS19 knockdown was validated by Q-RTPCR which showed a 5-fold decrease in RPS19 mRNA levels upon the addition of 0.25ug/ml Dox (Figure 1). Expression at the protein level was validated by western blotting. Other ribosomal proteins, for example RPL11, are unaffected. The recent discovery of GATA-1 mutations in 2 unrelated DBA pedigrees led us to investigate whether the expression of GATA-1, or other key erythroid transcription factors, may be affected in our RPS19 DBA cell line model. Q-RTPCR results show decreased expression of two key transcription factors (GATA-1 and Myb) in cells with RPS19 knockdown compared to the controls (Figure 1). Furthermore, the expression of the GATA-1 target gene, FOG-1 is decreased compared to controls. MYBBP1A, a Myb target gene involved in ribosomal stress pathways (Yamauchi et al. Genes. Cells 2008) is increased, while another Myb target, GFI-1b is decreased in this model. Discussion/Conclusions: We have generated an inducible RPS19-knockdown model of DBA in TF1.8 cells, and have showed that several key transcription factors important in erythropoiesis (including GATA-1) display down-regulation associated with RPS19-knockdown. This suggests that changes to GATA-1 levels may be important not only in the rare cases of DBA where GATA-1 is mutated, but also in classical DBA associated with ribosomal protein haploinsufficiency. Based on the observation that GATA-1 associates with and inhibits p53 during normal erythropoiesis (Trainor et al. Blood 2009) we propose that the decreased GATA-1 expression may contribute to activation of the p53 pathway during impaired erythropoiesis in Diamond Blackfan Anaemia. Further studies are being undertaken to test this model using assays for transcription factor activity, which will then be further characterised in a DBA model based on CD34+ cord blood cells infected with our validated RPS19 knockdown constructs. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3540 Background: GADD45A is a tumor suppressor gene that plays cell-type dependent roles in cellular stress, coordinating DNA repair and de-methylation, cell cycle arrest, and pro-apoptotic or pro-survival responses. GADD45A expression is normally rapidly induced in response to radiation and cytotoxic drugs1 and ectopic expression of GADD45A in the M1 leukemic cell line sensitises cells to stress-induced apoptosis in response to a range of genotoxic agents.2 Silencing of GADD45A by promoter methylation is a hallmark of many tumors,3–5 however to date there have been no investigations to determine whether this is associated with response to therapy. In AML, we have shown GADD45A expression is broadly down-regulated6 and here we investigate the mechanism of GADD45A repression in AML and its clinical significance. METHODS: We analysed 131 diagnostic bone marrow mononuclear cell samples from a retrospective cohort of patients with de novo AML. 93 of these patients were treated with induction chemotherapy. Patients 60 years or under were treated with chemotherapy regimens containing idarubicin and high dose cytarabine, and patients older than 60 received idarubicin and standard dose cytarabine. We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Sequenom MassARRAY) to analyze methylation of 4 GADD45A promoter CpG dinucleotides previously shown to be associated with silencing of GADD45A in breast and prostate cancer.4,5 We determined association of CpG hyper-methylation with outcome in our treated patient cohort. For AML cells with GADD45A hyper-methylation, and for those with normal levels of GADD45A promoter methylation, we also determined the response to cytotoxic agents in vitro in the presence and absence of hypo-methylating agents. RESULTS: We observed hyper-methylation of the 4 CpG residues in the proximal promoter of GADD45A in 49 of 131 (37%) de novo AML patients and in 6 AML cell lines. Multivariable analysis showed that methylation of a single CpG (CpG1) was an independent predictor of poor survival in AML overall (median survival 281 days versus 794 days, HR 2.25, p=0.009), in normal karyotype AML (NK-AML) (median survival of 281 versus 793 days, HR 5.77, p=0.03, 〉250 days), and in the elderly patient group (〉60 years, median survival of 218 versus 392 days, HR 2.64, p=0.03)(see Figure 1). Additionally, treatment of AML cell lines and patient blasts with decitabine resulted in selective induction of GADD45A mRNA in samples with GADD45A hyper-methylation, and this was associated with increased sensitivity to daunorubicin. CONCLUSIONS: DNA methylation of the GADD45A proximal promoter is an independent predictor of poor outcome particularly in AML patients with normal karyotype and in the elderly group. Our biological data shows that induction of GADD45A mRNA expression with decitabine in hyper-methylated samples is associated with increased response to cytotoxic agents. Thus GADD45A promoter CpG methylation represents a new biomarker that may provide prognostic information in the heterogeneous NK-AML group, and in elderly patients. Given that recent trials are combining Azacitidine with chemotherapy and other agents for initial induction treatment of AML9, this may represent a marker to help define patients that will benefit from this approach. Elderly patients with GADD45A hyper-methylation may benefit from treatment with hypo-methylating agents which are associated with less toxicity (see Refs 7,8). Disclosures: Wei: Celgene: Honoraria, Research Funding.

Description: Abstract 3731 Chronic myeloid leukemia (CML) is characterized by the presence of the BCR-ABL oncogene which encodes an activated tyrosine kinase. Despite the success of tyrosine kinase inhibitors (TKI), such as imatinib (IM), in CML treatment, a considerable percentage (12–50%) of patients develops resistance to TKIs. This can result in progression to blast crisis (BC), at which stage durable response to any TKI therapy is minimal. It is still unclear how additional genetic lesions, believed to play a major role in the transformation to BC, are generated, but it is likely that the BCR-ABL induction of DNA damage and low-fidelity DNA repair, added to inhibition of apoptosis contributes to this process. The Growth Arrest and DNA Damage 45 (GADD45A, B and G) proteins are tumor suppressors which coordinate cell cycle arrest, DNA repair and apoptosis in response to genotoxic and oncogenic stress. Failure to activate GADD45 expression, e.g. due to the presence of leukemic oncogenes or its promoter methylation, is associated with an attenuated DNA-damage response and impaired drug response. A microarray study showed that GADD45G is in the top 10% of genes downregulated in CML patients in BC compared to those in chronic phase (CP). Given the critical role of GADD45 proteins as key regulators of the cellular stress response, our hypothesis is that GADD45G reduced expression in CML-BC is mediated by BCR-ABL and contributes to the accumulation of mutations seen in BC. Therefore the aims of this work are to determine the mechanism of GADD45G downregulation by BCR-ABL and its functional effects on CML cells. We have confirmed the reduced GADD45G levels in CD34+ cells from BC patients versus CP and normal controls using quantitative RT-PCR. Furthermore, ectopic expression of BCR-ABL in a myeloid cell line resulted in decreased expression of GADD45G and, conversely, BCR-ABL inhibition with IM in several CML cell lines resulted in induction of GADD45G expression. These results confirm GADD45G as a down-modulated target of BCR-ABL. To determine the effect of GADD45G expression on CML cells, a CML cell line was transduced with GADD45G and showed significantly reduced proliferation, G1 cell cycle arrest and decrease in cell viability due to increased apoptosis. Once the GADD45G tumor suppressor effect in CML was confirmed, we investigated the mechanism by which BCR-ABL inhibits its expression. Silencing of GADD45G expression in solid tumors can occur by promoter methylation; therefore, we analyzed the GADD45G promoter methylation status in the presence or absence of BCR-ABL. Bisulfite genomic sequencing of BCR-ABL-positive and –negative hematopoietic cell lines and on peripheral blood mononuclear cells from CML-CP and BC patients, as well as of normal controls, showed no correlation between the presence of BCR-ABL and promoter methylation. Another mechanism possibly employed by BCR-ABL to regulate GADD45G expression is interference with transcription factor (TF) binding to the promoter. Bioinformatics analysis revealed, among several other TF binding sites, the presence of two highly conserved RUNX1/AML1 binding sites on the promoter. The TF RUNX1 is a key controller of hematopoiesis and heterodimerises with CBF-β to regulate gene expression. RUNX1 mutations in AML result in lower GADD45A expression and increase in DNA damage. In CML, RUNX1 was reported as mutated in 〉30% of patients in BC, and was suggested to cooperate with Bcr-Abl in the induction of AML in mice. It has also been shown to be a target of inhibitory phosphorylation by SRC kinases, which display increased activity in CML-BC. We therefore co-transfected a CML cell line with a GADD45G promoter luciferase reporter and both RUNX1 and CBF-β constructs. This resulted in a 10-fold increase in luciferase activity, indicating that RUNX1 is involved in the regulation of GADD45G expression in CML cells. Overall, our results suggest that GADD45G has a tumor suppressor role in CML and that its down-regulation by BCR-ABL could, therefore, contribute to the accumulation of DNA damage and progression to BC. In addition, our data identify RUNX1 as an inducer of GADD45G expression and, together with data from the literature, suggest that RUNX1 mutations or protein inactivation in CML could result in the observed reduced GADD45G expression. We are currently investigating this possibility. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2396 Background: Despite recent advances in understanding the key molecular mechanisms of leukemogenesis, the outcome for patients with Acute Myeloid Leukemia, particularly with a normal karyotype, remains poor. For this large group of patients, genetic alterations in genes such as FLT3, NPM1, CEBPA, IDH1/2, and DNMT3A provide useful prognostic information. However, risk stratification of this group remains only partially resolved and markers of response that can be therapeutically targeted would likely improve outcome for these patients. GADD45A is a tumor suppressor gene that plays cell-type dependent roles in cellular stress coordinating DNA repair and de-methylation, cell cycle arrest, and pro-apoptotic or pro-survival responses (Cancer Ther. 2009;7:268). Methylation of four discrete CpG residues in the proximal promoter of GADD45A is a hallmark of many solid tumours and has been associated with impaired cell stress signalling and reduced drug response (Cancer Res. 2009;69:1527; Oncogene. 2005;24:2705). In AML, GADD45A expression is broadly down-regulated both in normal karyotype and other cytogenetic classes. Down-regulation of GADD45A in AML has been associated with FLT3-ITD (Leukemia. 2009;23:729) and RUNX1 mutations (Satoh et al, Leukemia. 2011;Epub). For those patients without these mutations, the mechanism of GADD45A down-regulation and its prognostic significance remains unknown. We hypothesised that the promoter of GADD45A is methylated in AML and that this methylation is functionally important in patient response. Methods: Using the Sequenom MassARRAY methodology we screened for methylation of four GADD45A promoter CpG dinucleotides (CpG1–4) previously shown to be associated with silencing of GADD45A in breast and prostate cancer, in a retrospective cohort of 222 AML patients collected at diagnosis from the Royal Adelaide Hospital. We then determined association of CpG methylation with outcome and mutation status in our treated patient cohort of 167 patients. In AML cell lines and in primary patient samples we also determined the response to cytotoxic agents in vitro in the presence and absence of demethylating agents. Results: We observed hypermethylation of the CpG1–4 in the proximal promoter of GADD45A in 93 of 222 (42%) of AML patients and in 6 AML cell lines. In the 167 patients treated with standard induction chemotherapy regimes, 61 patients showed methylation of the GADD45A proximal promoter. Of the four CpG residues, methylation of CpG1 was associated with poor overall and event-free survival, in AML overall (Figure 1A) and in normal karyotype AML (Figure 1B). GADD45A CpG1 methylation was significantly associated with IDH1 and IDH2 mutations (p

Description: Granulocyte/macrophage colony-stimulating factor promotes growth, survival, differentiation, and activation of normal myeloid cells and plays an important role in myeloid leukemias. The GM-CSF receptor (GMR) shares a signaling subunit, βc, with interleukin-3 and interleukin-5 receptors and has recently been shown to induce activation of Janus kinase 2 (JAK2) and downstream signaling via formation of a unique dodecameric receptor complex. In this study we use 2 activated βc mutants that display distinct signaling capacity and have differential requirements for the GMR α-subunit (GMR-α) to dissect the signaling pathways associated with the GM-CSF response. The V449E transmembrane mutant selectively activates JAK2/signal transducer and activator of transcription 5 and extracellular signal-regulated kinase (ERK) pathways, resulting in a high level of sensitivity to JAK and ERK inhibitors, whereas the extracellular mutant (FIΔ) selectively activates the phosphoinositide 3-kinase/Akt and IκKβ/nuclear factorκB pathways. We also demonstrate a novel and direct interaction between the SH3 domains of Lyn and Src with a conserved proline-rich motif in GMR-α and show a selective requirement for Src family kinases by the FIΔ mutant. We relate the nonoverlapping nature of signaling by the activated mutants to the structure of the unique GMR complex and propose alternative modes of receptor activation acting synergistically in the mature liganded receptor complex.