Increased Responsiveness to Ligand Stimulation of the STAT Pathway At Relapse in Acute Myelogenous Leukemia

Acute myeloid leukemia (AML) is a very heterogeneous disease and changes in characteristics such as immunophenotype and mutation profiles are known to occur between diagnosis and relapse. Alterations in signaling pathways are likely to occur as well, but have not yet been reported. Our lab has recently shown that Stat3 signaling profiles are associated with outcome in pediatric AML patients. Specifically, patients whose blasts were sensitive to both G-CSF and IL-6, measured by increased tyrosine phosphorylation of Stat3 (pY-Stat3), had significantly improved event-free and overall survival compared to those whose blasts were resistant to one or both of the tested ligands. These results support the importance of ligand responsiveness within the STAT pathway in relation to chemoresponsiveness in AML. We hypothesized that comparison of STAT signaling pathways in paired diagnostic and relapse samples would identify changes that are likely to represent strengthening of pathways involved in chemoresistance.

Twenty two sample pairs from initial diagnosis and relapse from pediatric AML patients treated on the Children's Oncology Group protocol AAML0531 have been analyzed for this study. After thawing, ≥80% viability was confirmed by Trypan blue stain. Constitutively phosphorylated STATs (including pY-Stat3, pS-Stat3, and pY-Stat5), total Stat3 (TStat3), pY418-Src, G-CSF receptor, and gp130 were measured in unstimulated cells. Additionally, cells were stimulated for 15 minutes with 10 or 100ng/ul G-CSF, or 5 or 50ng/ul IL-6 with 10 or 100 ng/ul 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 (DMFI) of the stimulated sample compared to the corresponding unstimulated sample. Constitutive pStats, receptors, TStat3, and pY418-Src data are expressed as percent positive compared to isotype control. The Wilcoxon Signed Rank test was used to test for significant differences in parameters between diagnosis and relapse.

Of the 22 sample pairs thawed, 21 had adequate cell numbers and viability for analysis. Increases in ΔMFI of pY-Stat3 between diagnosis and relapse were seen in response to both doses of G-CSF. At the lower dose, ΔMFI increased from 1.45±0.14 to 2.30±0.33 (p=0.0023), with increases demonstrated in 16/21 pairs. At the higher dose level, ΔMFI increased from 1.70±0.20 to 2.68±0.38 (p=0.0019) with increases in 16/21 pairs. Similarly, in response to the lower IL-6 dose, the ΔMFI of pY-Stat3 increased from 1.29±0.10 to 1.58±0.18 (p=0.0183), with increases in 14/21 pairs while at the higher dose, ΔMFI increased from 1.53±0.20 to 2.07±0.27 (p=0.0079) with increases in 12/21 pairs. Evaluated parameters that did not change significantly between diagnosis and relapse included G-CSF receptor expression (74.09% to 80.16; p=0.1526), gp130 expression (51.63% to 60.52%; p=0.0569), constitutive activity of pYStat3 (23.60% to 26.63%; p=0.0962), and TStat3 expression (35.32% to 43.96%; p=0.053). Though the changes were small, pY418-Src was found to be significantly increased between paired samples (0.90% to 2.58%; p=0.0011).

STAT pathway signaling patterns evolve between diagnosis and relapse in pediatric AML. Our data demonstrate that ligand-induced Stat3 signaling pathways evolve to become stronger at relapse. This suggests that the resistance seen to chemotherapy in relapsed patients may be in part due to the increased activity of this anti-apoptotic pathway in response to growth factors and cytokines present in the bone marrow niche. The increases in activity within the STAT pathway at relapse provide support for further development and evaluation of targeted agents against this pathway in relapsed patients with AML.

No relevant conflicts of interest to declare.