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
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. 
Published: October 2019

Abstract
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 p38MAPK activation in these cells, whereas CM-DCFDA showed only homogeneous responses to H2O2 induction. These data show that Δpp38MAPK cells are defined by an enhanced membrane-associated anti-oxidant capacity and we are currently analyzing this resistant subpopulation to identify the molecules responsible.
To examine whether p38MAPK responsiveness influenced responsiveness to pro-oxidant drugs, we selected KG-1 and THP-1 cells (as representative examples of strong and weak p38MAPK responses respectively) and tested their sensitivity to the pro-oxidant drugs, phenethyl isothiocyanate (PEITC) and buthionine sulfoximine (BSO). We found that the IC50 was higher for THP-1 for both PEITC (KG-1 = 0.6µM; THP-1 = 7.5µM) and BSO (KG-1 = 50µM; THP-1 = 70µM), indicating that the p38MAPK responsiveness limits the effectiveness of pro-oxidant drugs. We next examined whether promoting p38MAPK activation could augment the effects of these pro-oxidants. We used the p38MAPK activator 2-benzylidene-3-(cyclohexylamino)-1-indanone (BCI), which promotes activation of p38MAPK via inhibition of a p38MAPK phosphatase, DUSP1. This compound weakly promoted phosphorylation of p38MAPK in THP-1 cells and consistent with this, had no effect on the efficacy of these compounds in these cells. However, BCI potently activated p38MAPK in KG-1 cells and showed synergy with BSO in this context (CI = 0.3; Figure 2) indicating that where BCI is effective in activating p38MAPK it can promote the effectiveness of pro-oxidant drugs.
In summary, we show for the first time that AML patients almost universally display attenuated p38MAPK responses in all or part of the blast population and we suggest that this trait may be selected for to maintain self-renewing potential under the pro-oxidative conditions found in the leukemic marrow. Further we show that by manipulating p38MAPK activity, we can augment the potency of the pro-oxidant compound BSO.
Figure 1 Figure 1.
Figure 2 Figure 2.

Disclosures
No relevant conflicts of interest to declare.