Abstract 4206: Increased responsiveness to ligand stimulation of the STAT pathway at relapse in acute myelogenous leukemia

Background: We have shown that Stat pathway sensitivity to G-CSF and IL-6, measured by the increase in tyrosine phosphorylated Stat3 (pY-Stat3), is associated with clinical outcome in pediatric AML patients. These results support a possible relationship between chemoresponsiveness and ligand-induced Stat3 response. We hypothesized that consistently altered changes in Stat3 signaling between diagnosis and relapse would represent advantageous adaptations that promote chemotherapy resistance in pediatric AML. Methods: 25 sample pairs from initial diagnosis and relapse from pediatric AML patients treated on the COG frontline trial AAML0531 were analyzed. After thawing, ≥80% viability was confirmed by Trypan exclusion. Constitutively phosphorylated Stats (pY-Stat3, pS-Stat3, and pY-Stat5), total Stat3 (TStat3), G-CSF receptor, and gp130 were measured in unstimulated cells. Additionally, cells were stimulated for 15 minutes with 10 or 100 ng/ml G-CSF, or 5 or 50 ng/ml IL-6 with 10 or 100 ng/ml soluble IL-6 receptor, respectively, for measurement of ligand-induced pStats. Data were collected on the LSR II (BD) and analyzed with FCS Express 4 (DeNovo). For the ligand-induced pStats, data are expressed as the fold change in mean fluorescence intensity (ΔMFI) of the stimulated sample compared to the corresponding unstimulated sample. Constitutive pStats, receptors, and TStat3 data are expressed as percent in the positive region, as defined by the upper limit of the signal in the isotype control. The Wilcoxon Signed Rank test was used to test for significant differences in parameters between diagnosis and relapse. Results: 24/25 sample pairs had adequate cell numbers and viability for analysis. At both doses of G-CSF, 21/24 pairs demonstrated an increase in pY-Stat3 ΔMFI. At the lower G-CSF dose, the mean ±SEM ΔMFI increased from 1.41±0.13 to 2.36±0.28 (p=0.0001). At the higher dose, ΔMFI increased from 1.65±0.20 to 2.71±0.33 (p=0.0002). Similarly, at both doses of IL-6, 17/24 pairs demonstrated an increase in pY-Stat3 ΔMFI. At the lower IL-6 dose, the ΔMFI of pY-Stat3 increased from 1.25±0.09 to 1.56±0.16 (p=0.0168). At the higher dose, ΔMFI increased from 1.49±0.17 to 2.01±0.24 (p=0.0051). Only 3/24 pairs showed >15% increase in G-CSF receptor expression, while 10/24 had a >15% increase in gp130 expression. No significant changes were seen in constitutive activity of pStats, or TStat3 expression. Conclusions: Our data demonstrate that ligand-induced Stat3 signaling pathways evolve to become stronger at relapse in pediatric AML. More robust signaling was not due to increased expression of total Stat3 and was rarely associated with increased receptor expression. Our data suggest that ligand-induced Stat pathway activation may be promoting survival in relapsed AML. Our results provide support for further development and evaluation of targeted agents against this pathway in AML. Citation Format: Alexandra M. Stevens, Marcos Ruiz, Michele S. Redell. Increased responsiveness to ligand stimulation of the STAT pathway at relapse in acute myelogenous leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4206. doi:10.1158/1538-7445.AM2014-4206