Abstract 3550: Lipid rafts contribute to ligand-induced signaling and survival in pediatric AML
Authors: Narayanan Padmini , Dubrulle Julien , Gerbing Robert B. , Alonzo Todd A. , Stossi Fabio , Redell Michele S. 
Published: August 2018

Abstract
Pediatric acute myeloid leukemia (pAML) is an aggressive malignancy with high mortality due to therapy resistance and relapse. We previously reported that the failure of pAML cells to phosphorylate STAT3 upon G-CSF or IL-6 stimulation was associated with inferior survival. The defect in STAT3 signaling was not due to low expression of receptors or of STAT3. We hypothesized that the signaling failure seen in some pAML samples is not specific for STAT3, but rather a generalized dysfunction due to abnormal lipid raft function. Lipid rafts (LR) are highly ordered plasma membrane microdomains rich in glycosphingolipids and cholesterol. Two types of LR - flat planar and invaginated caveolar - contain different membrane proteins and serve distinct but overlapping cellular functions. Both types of LR organize receptors, downstream mediators, and repressors for efficient signaling. The role of LRs in signaling and clinical outcome is not yet established in pAML. Primary samples (n=95) from the Children's Oncology Group (COG) were stimulated with HS5 stromal cell conditioned medium (HS5CM), which contains IL-6 and dozens of physiological ligands. Basal and induced phospho-STAT3 (pSTAT3), pSTAT5, pAKT and pERK1/2 were measured by intracellular flow cytometry, and the fold change in the mean fluorescence intensity (ΔMFI) was calculated. The HS5CM-induced ΔMFI for pSTAT3 was positively correlated with that for pSTAT5 (p&lt;0.0001, r=0.5), and pAKT (p=0.0002, r=0.4). Samples that failed to increase pSTAT3 failed to activate other pathways, suggesting a generalized signaling dysfunction. In AML cells with intact signaling, disruption of LRs with methylβcyclodextrin markedly reduced HS5CM-induced pSTATs, recapitulating the dysfunctional phenotype. LRs were assessed by immunocytochemistry in 29 primary samples with and without HS5CM stimulation. Choleratoxin B, which binds ganglioside GM-1, stained LRs. Antibodies to caveolin-1 (cav-1) marked caveolar LRs. We also stained for gp130, the signaling subunit of the IL-6 receptor. Images were captured by deconvolution fluorescence microscopy. Expression was quantified by pixel intensity for average of 30 cells per condition. Colocalization of gp130 with LR markers was determined by the Manders Overlap Coefficient (MOC). Intensities and MOCs varied between samples. The MOC of gp130 with cav-1 correlated significantly with pSTAT3 ΔMFI (p=0.02, r=0.43). MOC of gp130 with GM-1 did not correlate with pSTAT3 ΔMFI (p=0.85, r=0.03), suggesting that cav-1 facilitates IL-6/STAT3 signaling. Interestingly, patients whose samples demonstrated basal cav-1 and GM-1 intensities below the median had inferior 5-year event-free survival compared to those with intensities above the median (log-rank p=0.079 for cav-1; p=0.013 for GM-1). Our results suggest that LRs contribute to ligand-induced signaling and possibly to chemotherapy response. Further studies of LR biology in AML are warranted.
Citation Format: Padmini Narayanan, Julien Dubrulle, Robert B. Gerbing, Todd A. Alonzo, Fabio Stossi, Michele S. Redell. Lipid rafts contribute to ligand-induced signaling and survival in pediatric AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3550.