Signaling Responses to Stroma-Derived Soluble Factors Are Associated with Outcome and with Expression of Microenvironment-Related Genes in Pediatric AML

Pediatric AML has a relapse rate approaching 40%, indicating that resistance to chemotherapy remains a critical problem. We are focused on identifying and overcoming environment-mediated mechanisms of resistance. We previously reported that the sensitivity of G-CSF- and IL-6-induced STAT3 signaling was significantly associated with outcome in pediatric AML patients. In this follow-up study, we used conditioned medium (CM) from HS5 stromal cells as a more physiological stimulus to evaluate the capacity of primary AML cells to activate signaling pathways. We studied 111 diagnostic bone marrow samples from patients who enrolled on Children's Oncology Group (COG) AML treatment studies, AAML03P1 or AAML0531, and provided informed consent to bank bone marrow. The same chemotherapy backbone was used in both trials. All samples had at least 70% viability after thawing. Thawed cells were divided into aliquots for stimulation with 50% CM, 10 ng/ml G-CSF, or 5 ng/ml IL-6. Unstimulated cells were used for isotype controls and for determination of basal signaling levels. Following 15 min stimulation, cells were fixed and processed for FACS analysis of CD45, pY-STAT3, pY-STAT5, pERK1/2, and pAKT. Responses were expressed as the fold change in mean fluorescence intensity for stimulated cells over unstimulated cells (ΔMFI). The ΔMFIs for pY-STAT3 and pY-STAT5 varied between 0.1 and 31.8. The ΔMFIs for pERK1/2 and pAKT rarely exceeded 2. Samples with a robust response to one stimulus generally responded robustly to the others. The Pearson correlation coefficient (r) for CM-induced pY-STAT3 v. G-CSF-induced pY-STAT3 was 0.8511 (p 1.96 (p=0.034). Previously we found that higher inducible pY-STAT3 was associated with significantly better EFS (Redell, et al, Blood, 2013; Long, et al, Oncotarget, 2017). In this study, no cut point for CM-induced pY-STAT3 ΔMFI distinguished patients with good or poor EFS. Cytogenetics and FLT3/ITD were not significantly different for groups above and below the ΔMFI cut points. The finding that robust STAT5 signaling is associated with inferior EFS sugge