Marcos J. Ruiz

Stat3 signaling in acute myeloid leukemia: ligand-dependent and -independent activation and induction of apoptosis by a novel small-molecule Stat3 inhibitor

Acute myeloid leukemia (AML) is an aggressive malignancy with a relapse rate approaching 50%, despite aggressive chemotherapy. New therapies for AML are targeted at signal transduction pathways known to support blast survival, such as the Stat3 pathway. Aberrant activation of Stat3 has been demonstrated in many different malignancies, including AML, and this finding is frequently associated with more aggressive disease. The objectives of this study were: (1) to characterize Stat3 signaling patterns in AML cells lines and primary pediatric samples; and (2) to test the efficacy and potency of a novel Stat3 inhibitor in inducing apoptosis in AML cells. We found that Stat3 was constitutively activated in 6 of 7 AML cell lines and 6 of 18 primary pediatric AML samples. Moreover, constitutively phosphorylated Stat3 was frequent in samples with normal karyotype but uncommon in samples with t(8;21). Most cell lines and primary samples responded to G-CSF stimulation, although the sensitivity and magnitude of the response varied dramatically. Our novel small-molecule Stat3 inhibitor, C188-9, inhibited G-CSF-induced Stat3 phosphorylation, induced apoptosis in AML cell lines and primary samples, and inhibited AML blast colony formation with potencies in the low micromolar range. Therefore, Stat3 inhibition may be a valuable strategy for targeted therapies for AML.

FACS analysis of Stat3/5 signaling reveals sensitivity to G-CSF and IL-6 as a significant prognostic factor in pediatric AML: a Children's Oncology Group report

Signal transducer and activator of transcription 3 (Stat3) and Stat5 are critical signaling intermediates that promote survival in myeloid leukemias. We examined Stat3 and Stat5 activation patterns in resting and ligand-stimulated primary samples from pediatric patients with acute myeloid leukemia. Phosphorylated Stats were measured by FACS before and after stimulation with increasing doses of granulocyte-colony stimulating factor or IL-6. We also measured positive and negative regulators of Stat signaling, and we compared the variation in multiple parameters to identify biologic relationships. Levels of constitutively phosphorylated Stats were variable and did not correlate with survival. In terms of induced phospho-Stats, 15 of 139 specimens (11%) phosphorylated Stat3 in response to moderate doses of both granulocyte-colony stimulating factor and IL-6. Compared with groups that were resistant to 1 or both ligands, this pattern of dual sensitivity was associated with a superior outcome, with a 5-year event-free survival of 79% (P = .049) and 5-year overall survival of 100% (P = .006). This study provides important and novel insights into the biology of Stat3 and Stat5 signaling in acute myeloid leukemia. Patterns of ligand sensitivity may be valuable for improving risk identification, and for developing new agents for individualized therapy.

Ligand-induced STAT3 signaling increases at relapse and is associated with outcome in pediatric Acute Myeloid Leukemia: A Report from the Children' s Oncology Group

Signal Transducer and Activator of Transcription 3 (STAT3) responses to ligands correlate with outcome in pediatric acute myeloid leukemia (AML),[1][1] suggesting a possible relationship between chemotherapy response and the STAT3 pathway. We hypothesized that consistently altered changes in STAT3

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

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC 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 Childrens 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.

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

Abstract 2908: Multiparameter FACS analysis of Stat3 and Stat5 signaling in pediatric AML samples

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 Children9s 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 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