Abstract 2908: Multiparameter FACS analysis of Stat3 and Stat5 signaling in pediatric AML samples
Authors: Redell Michele S. , Ruiz Marcos J. , Gerbing Robert B. , Alonzo Todd A. , Tweardy David J. 
Published: June 2012

Pediatric acute myeloid leukemia (AML) is a devastating disease with a relapse rate near 50%. New targeted therapies are entering clinical use, yet the pathways affected by these drugs remain poorly understood. Aberrantly active Stat3 and Stat5 are found in AML and have been associated with chemoresistant disease, likely due to increased pro-survival gene expression. Our hypothesis is that analysis of Stat3/5 activation, and expression levels of key proteins in the pathways, will provide insight into mechanisms of aberrant signaling, and will guide the development of targeted therapies. We performed multiparameter FACS analysis of tyrosine- and serine-phosphorylated Stat3 (pY- and pS-Stat3; n=65) and pY-Stat5 (n=47) in pediatric AML samples from the Children's Oncology Group. Constitutive activation was common and quite variable (median pY-Stat3+ events/sample: 38%, range: 3 – 82%; median pY-Stat5+: 34%, range: 5.9 – 77%). There was non-significant correlation between %pY-Stat3+ and %pY-Stat5+ events in unstimulated samples. Constitutive pS-Stat3 was rare (median 2%). As expected, there was a significant correlation between constitutive %pY-Stat3+ and total Stat3 expression (by MFI), with linear correlation coefficient R=0.411 (p&lt;0.001). Most samples were not responsive to G-CSF or IL-6; 38/65 (58%) failed to show at least a 2-fold increase in the pY-Stat3 MFI after treatment with G-CSF (100 ng/ml), and 42/65 (65%) did not respond to IL-6 (50 ng/ml IL-6 + 100 ng/ml soluble IL-6 receptor (sIL6R)). A pY-Stat3 response to only this high dose of G-CSF was seen in 23% of samples, while 15% responded to 10 ng/ml, and only 1 sample responded to 1 ng/ml G-CSF. The patterns of pY-Stat5 responses to G-CSF and of pY-Stat3 responses to IL-6 were similar. The pY-Stat3 and pY-Stat5 responses to G-CSF, as well as the pY-Stat3 responses to G-CSF and to IL-6, were significantly correlated (R=0.911, p&lt;0.001 and R=0.431, p&lt;0.001, respectively), suggesting overlap of these signaling pathways. Surface G-CSFR expression positively correlated with the GCSF-induced pY-Stat3 response (R=0.333, p=0.005). Interestingly, surface gp130 expression negatively correlated with the IL6-induced response (R=−0.349, p&lt;0.005), meaning samples with robust IL-6 responses tended to have fewer gp130+ cells. Expression of the negative regulators SOCS3 and Shp1 were measured by Western blot (n=46) and normalized to the AML cell line Kasumi-1. SOCS3 expression in primary samples was consistently low, whereas Shp1 expression was up to 26-fold higher in primary samples compared to Kasumi-1 cells. Neither SOCS3 nor Shp1 levels varied in proportion to basal pY-Stat3. In summary, multiparameter FACS analysis of baseline and ligand-induced Stat3 and Stat5 activity provides biologic insight into signaling aberrancies in AML, and this knowledge will promote rational design and testing of new therapies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2908. doi:10.1158/1538-7445.AM2011-2908