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Mechanisms controlling the balance between proliferation and self-renewal versus growth suppression and differentiation during normal and leukemic myelopoiesis are not understood. We have used the bi-potent FDB1 myeloid cell line model, which is responsive to myelopoietic cytokines and activated mutants of the granulocyte macrophage-colony stimulating factor (GM-CSF) receptor, having differential signaling and leukemogenic activity. This model is suited to large-scale gene-profiling, and we have used a factorial time-course design to generate a substantial and powerful data set. Linear modeling was used to identify gene-expression changes associated with continued proliferation, differentiation, or leukemic receptor signaling. We focused on the changing transcription factor profile, defined a set of novel genes with potential to regulate myeloid growth and differentiation, and demonstrated that the FDB1 cell line model is responsive to forced expression of oncogenes identified in this study. We also identified gene-expression changes associated specifically with the leukemic GM-CSF receptor mutant, V449E. Signaling from this receptor mutant down-regulates CCAAT/enhancer-binding protein alpha (C/EBPalpha) target genes and generates changes characteristic of a specific acute myeloid leukemia signature, defined previously by gene-expression profiling and associated with C/EBPalpha mutations.

The tumor suppressor Gadd45a was earlier shown to be a repressed target of sustained receptor-mediated ERK1/2 signaling. We have identified Gadd45a as a downregulated gene in response to constitutive signaling from two FLT3 mutants (FLT3-ITD and FLT3-TKD) commonly found in AML, and a leukemogenic GM-CSF receptor trans-membrane mutant (GMR-V449E). GADD45A mRNA downregulation is also associated with FLT3-ITD þ AML. Sustained ERK1/2 signaling contributes significantly to receptor-mediated downregulation of Gadd45a mRNA in FDB1 cells expressing activated receptor mutants, and in the FLT3-ITD þ cell line MV4;11. Knockdown of Gadd45a with shRNA led to increased growth and survival of FDB1 cells and enforced expression of Gadd45a in FDB1 cells expressing FLT3-ITD or GMR-V449E resulted in reduced growth and viability. Gadd45a overexpression in FLT3-ITD þ AML cell lines also resulted in reduced growth associated with increased apoptosis and G 1 /S cell cycle arrest. Overexpression of Gadd45a in FDB1 cells expressing GMR-V449E was sufficient to induce changes associated with myeloid differentiation suggesting Gadd45a downregulation contributes to the maintenance of receptor-induced myeloid differentiation block. Thus, we show that ERK1/2-mediated downregulation of Gadd45a by sustained receptor signaling contributes to growth, survival and arrested differentiation in AML.

Granulocyte-macrophage colony stimulating factor (GM-CSF) promotes the growth, survival, differentiation and activation of normal myeloid cells and is essential for fully functional macrophage differentiation in vivo. To better understand the mechanisms by which growth factors control the balance between proliferation and self-renewal versus growth-suppression and differentiation we have used the bi-potent FDB1 myeloid cell line, which proliferates in IL-3 and differentiates to granulocytes and macrophages in response to GM-CSF. This provides a manipulable model in which to dissect the switch between growth and differentiation. We show that, in the context of signaling from an activating mutant of the GM-CSF receptor β subunit, a single intracellular tyrosine residue (Y577) mediates the granulocyte fate decision. Loss of granulocyte differentiation in a Y577F second-site mutant is accompanied by enhanced macrophage differentiation, accumulation of β-catenin together with activation of Tcf4 and other Wnt target genes. These include the known macrophage lineage inducer, Egr1. We show that forced expression of Tcf4 or a stabilised β-catenin mutant is sufficient to promote macrophage differentiation in response to GM-CSF and that GM-CSF can regulate β-catenin stability, most likely via GSK3β. Consistent with this pathway being active in primary cells we show that inhibition of GSK3β activity promotes the formation of macrophage colonies at the expense of granulocyte colonies in response to GM-CSF. This study therefore identifies a novel pathway through which growth factor receptor signalling can interact with transcriptional regulators to influence lineage choice during myeloid differentiation.

Over the past several years we have used a factor-dependent murine bi-potential cell line, FDB1, in combination with activated mutants of the GM-CSF receptor common beta subunit (hβc) to dissect receptor pathways and gene expression changes that can contribute to myeloid cell growth and differentiation. Of particular interest are the mechanisms by which signalling from the GM-CSF receptor can promote simultaneous granulocyte and macrophage differentiation. In this context we have characterised differentiation of FDB1 cells in response to an activated hβc mutant (FIΔ). For this mutant, which confers growth factor-independent granulocyte-macrophage differentiation in the FDB1 cell line, the presence of a single intracellular tyrosine residue (Y577) is a requirement for granulocyte differentiation. In the absence of this tyrosine residue FDB1 cells expressing FIΔ undergo uni-lineage macrophage differentiation (Brown et al., 2004). Comparative transcriptional profiling of these FDB1 populations has allowed us to identify gene expression changes associated with receptor-induced granulocyte or macrophage differentiation. An examination of the genes selectively associated with macrophage differentiation revealed a potential role for the transcription factor TCF4/TCF7L2, which is a central mediator of the canonical Wnt signalling pathway through its role as the DNA binding co-factor for β-catenin. Over-expression of TCF4 in FDB1 cells could initiate growth arrest and differentiation, under growth-promoting conditions. In FDB1 cells expressing the FIΔ mutant forced TCF4 expression resulted in an increase in the proportion of cells differentiating to macrophages. Further examination of signalling in the FDB1 FIΔ populations demonstrated that stabilisation of β-catenin is associated with the switch to macrophage differentiation. GM-CSF signalling in FDB1 cells also induces transient β-catenin stabilisation during bi-lineage GM differentiation. In FDB1 cells stabilisation of β-catenin and myeloid differentiation is also induced using the GSK3β inhibitor BIO (6-bromoindirubin-3′-oxime). The results using this unique model of receptor-induced GM differentiation are consistent with a role for the β-catenin pathway in regulating the macrophage lineage.

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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<0.001), but was not associated with FLT3-ITD or other high risk cytogenetic groups. Multivariate analysis (including age, wcc, FLT3-ITD, IDH1/2) revealed that methylation of GADD45A CpG1 is an independent predictor of poor survival in AML, overall (OS; HR 2.17, p=0.006: EFS; HR 2.43, p=0.001), and in normal karyotype AML (OS; HR 2.86, p=0.014: EFS; 5.25, p<0.001). Additionally, treatment of AML cell lines and patient blasts with decitabine (5-Aza-deoxycitidine) resulted in induction of GADD45A mRNA selectively in samples with GADD45A hyper-methylation, and this was associated with increased sensitivity to daunorubicin. Conclusions: DNA methylation of the GADD45A proximal promoter marks a large percentage AML patients, including those with with IDH1/2 mutations, and is an independent predictor of poor outcome, particularly in AML patients with normal karyotype. Our biological data shows that induction of GADD45A mRNA expression with decitabine in methylated samples is associated with increased response to cytotoxic agents. Thus GADD45A proximal promoter methylation represents a new biomarker that may provide prognostic information in the heterogeneous normal karyotype AML group. Given that recent clinical trials are combining Azacitidine with chemotherapy and other agents for induction therapy in AML (Blood;118:1472), this may represent a marker to help define patients that will benefit from this approach. Disclosures: Wei: Celgene: Research Funding.

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.