A Subpopulation of Blasts with Attenuated p38MAPK Response Is Seen in Virtually All AML Patients and AML Cell Lines and Is Defined By Cells with Augmented Lipid-Associated Anti-Oxidant Defense

• Published in: Blood 2014-12, Vol.124 (21), p.785-785
• Main Authors: Hole, Paul S., Davies, Sara, Munje, Chinmay R, Kreuser, Sandra, Hills, Robert K., Omidvar, Nader, Knapper, Steven, Burnett, Alan K., Tonks, Alex, Darley, Richard L.
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V124.21.785.785

The serine/ threonine kinase, p38MAPK is activated by phosphorylation in response to a variety of cellular stresses including oxidative stress. Prolonged p38MAPK activation drives cell-cycle arrest and apoptosis; and in HSC activation of p38MAPK leads to a loss of reconstituting capacity (Ito et al, Nat.Med. 2006;12:446-451). In cancer, p38MAPK responses are often attenuated and cancer models suggest that this is a necessary adaptation for transformation (Dolado et al, Cancer Cell 2007;11:191-205). Previously we have shown that 60% of acute myeloid leukemia (AML) patients constitutively generate significantly more extracellular reactive oxygen species (ROS) than normal hematopoietic CD34+ cells (Hole et al, Blood 2013;122:3322-3330). Despite this, AML blasts showed low or absent p38MAPK phosphorylation; even in patients generating high levels of ROS. Here we examine p38MAPK activation at the single cell level in primary AML blasts using flow cytometry. We challenged AML blasts with a dose of hydrogen peroxide (H2O2) sufficient to completely activate p38MAPK in normal CD34+ cells (1 mM for 30 min), where the threshold for activation was defined as the 95th percentile of basal p38MAPK activation in unstimulated cells. Attenuated responses to H2O2 were seen in 14/15 (93%) of patients; where 16-95% of the total blast population failed to activate p38MAPK. These non-responding cells are hereafter termed “Δpp38MAPK cells” and were absent in normal CD34+ cells (p < 0.01; Figure 1). Examination of a panel of 6 AML cell lines showed that each of the lines contained Δpp38MAPK cells at different frequencies: MV4-11 (10%); HL60 (10%); KG-1 (15%), U937 (30%), NB4 (50%), THP-1 (65%). Further analysis showed that Δpp38MAPK cells were not distinguished by cell cycle phase, immunophenotype or reduced viability in either cell lines or AML blasts. These data suggest that nearly all AML patients harbor a population of blasts which have developed resistance to p38MAPK activation. We reasoned that failure to respond could arise either through defective p38MAPK signaling or because of enhanced anti-oxidative protection in a subpopulation of cells. To investigate the latter, we labelled cells with the lipophilic oxidation probe, C11 -BODIPY or the cytosolic oxidant probe, CM-DCFDA and monitored the oxidative response to H2O2 at the single cell level in the AML cell lines: KG-1, MV4-11 and THP-1. In each case C11 -BODIPY oxidation exactly matched the heterogeneous profile of p38MA