Abstract 1791: Stat3 signaling in acute myeloid leukemia: Ligand-dependent and -independent activation, impact on prognosis, and induction of apoptosis by a novel Stat3 inhibitor

Abstract

Abstract Acute myeloid leukemia (AML) is an aggressive malignancy with a relapse rate approaching 50%, despite very toxic chemotherapy. Newly developed therapies for AML are targeted at signal transduction pathways known to support blast survival. One such potential target is Stat3, a signaling intermediate that is activated by tyrosine phosphorylation in response to a number of cytokines active in myeloid cells, such as G-CSF. Aberrant activation of Stat3 has been demonstrated in many malignancies, including AML, and this finding is frequently associated with more aggressive disease. The objectives of this study were to 1) characterize Stat3 signaling patterns in AML cells lines and primary pediatric AML samples, and 2) to test the efficacy and potency of a novel Stat3 inhibitor in inducing apoptosis in AML cells. We measured tyrosine-phosphorylated Stat3 (pStat3) in 7 AML cell lines by FACS and Western blot. We found that in 6 of the 7 cell lines, at least 20% of cells had pStat3 (by FACS) in the absence of stimulation, and thus were considered constitutively activated. Likewise, 6 of 7 cell lines demonstrated at least a 2-fold increase in the mean fluorescence intensity (MFI) of pStat3 after G-CSF stimulation, and thus were considered responsive to G-CSF. We next analyzed primary pediatric AML samples (from the Children's Oncology Group AML Reference Lab) and normal bone marrow mononuclear cells (BMMCs) for constitutive and G-CSF-induced pStat3. We found 6 of 18 AML samples and 1of 5 BMMCs showed constitutive pStat3 (pStat3+), and 16/18 AML and 3/5 BMMC samples were responsive to G-CSF. Event-free survival analysis revealed that constitutive pStat3 was significantly associated with inferior outcome. Specifically, 4/6 pStat3+ patients died of relapsed or refractory disease, while 2/12 pStat3- patients died (p = 0.019). These results support the hypothesis that aberrant Stat3 activity promotes an aggressive phenotype in pediatric AML. In this regard, we evaluated the effect of a novel small molecule Stat3 inhibitor that has been developed by us, C188-9, to block G-CSF-induced Stat3 phosphorylation and to induce apoptosis in AML cells. The IC50s of C188-9 to inhibit Stat3 activation in AML cell lines were in the range of 4-7 μM, and in primary AML samples the IC50s were in the range of 8-18 μM. For apoptosis studies, AML cell lines and primary samples were treated for 24 hours with the compound, then apoptotic cells were quantified by FACS analysis for annexin V-labeled cells. The EC50s for apoptosis induction were quite variable, ranging from 6 μM to over 50 μM. These results indicate that there is a subset of AML samples that are dependent on Stat3 signaling for survival and are killed by inhibition of Stat3. Our data suggest that aberrant Stat3 activity may be both a valuable new prognostic indicator and an important target for novel therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1791.