Abstract
Glucocorticoids (GC) have been used for decades in the treatment of B-cell acute lymphoblastic leukemia (B-ALL) in children and adults. Induction of apoptosis is thought to be the principal effector mechanism of GC's action, but recent studies highlight the role of autophagy upstream of apoptotic cell death (Laane et al 2009). Resistance to GCs is a major adverse prognostic factor, however the molecular mechanisms leading to GC resistance are not completely understood. Herein, we sought to elucidate the molecular mechanisms driving GC-resistance in precursor B-cell acute lymphoblastic leukemia cells and in vitro characterize the therapeutic potential of targeted intervention in these mechanisms.
To identify molecular mechanisms involved in GC resistance, we performed gene set enrichment analysis of gene expression profiles GC-sensitive and -resistant B-ALL blasts using publicly available datasets and GenePattern program. Resistant cells exhibited significantly higher expression of MAPK/ERK pathway components (p<.002, FDR=0.13). To validate these findings, we assessed DEX sensitivity in ALL cells with high (SEMK2) or undetectable (RS4;11) activity of MAPK/ERK pathway. SEMK2 cells were resistant to DEX, whereas RS4;11 were highly sensitive to this drug. In GC-resistant cell line SEMK2, inhibition of MEK1 kinase with SEL completely abrogated ERK and p90RSK phosphorylation and increased sensitivity to GC by 1.8-2.6-fold. Similar pattern was observed in primary ALL blasts from 19 of 23 tested patients. Overexpression of a constitutively active MEK mutant in GC-sensitive cells (RS4;11) reversed sensitivity of these cells to DEX. Since GC in leukemic cells induce autophagic cell death, we assessed LC3 processing, MDC staining (a dye of autophagolysosomes) and GFP-LC3 relocalization in cells incubated with either DEX, SEL or combination of drugs. Either drug alone caused only marginal change in the level of these markers, but their combination markedly increased autophagic flux. Since mTORC1 is the critical regulator of autophagy, we assessed the activity of mTORC1 following DEX/SEL co-treatment and found that the combination resulted in a marked decrease of p4E-BP1, an mTORC1 substrate. Finally, to assess whether induction of autophagy is required for the observed synergy between SEL and DEX we used an shRNA approach to silence beclin-1 (BCN1), a gene required for autophagosome formation, and assessed cellular responses to DEX/SEL co-treatment. In control cells transduced with non-targeting shRNA, SEL sensitized cells to DEX, but in BCN1-deficient cells, the synergy of DEX and SEL was markedly decreased. Taken together, we show that MEK1 inhibitor selumetinib enhances DEX toxicity in GC-resistant B-ALL cells. The underlying mechanism of this interaction involves inhibition of mTORC1 signaling pathway and induction of autophagy that leads to apoptotic cell death.
Disclosures
No relevant conflicts of interest to declare.
MEK1 Inhibitor Selumetinib Sensitizes Precursor B-Cell Acute Lymphoblastic Leukemia Cells (B-ALL) to Dexamethasone through Modulation of mTOR Activity and Stimulation of Autophagy
