Glucocorticoid resistance in two key models of acute lymphoblastic leukemia occurs at the level of the glucocorticoid receptor

Abstract

Glucocorticoids (GCs) specifically induce apoptosis in malignant lymphoblasts and are thus pivotal in the treatment of acute lymphoblastic leukemia (ALL). However, GC-resistance is a therapeutic problem with an unclear molecular mechanism. We generated approximately 70 GC-resistant sublines from a GC-sensitive B- and a T-ALL cell line and investigated their mechanisms of resistance. In response to GCs, all GC-resistant subclones analyzed by real-time polymerase chain reaction (PCR) showed a deficient up-regulation of the GC-receptor (GR) and its downstream target, GC-induced leucine zipper. This deficiency in GR up-regulation was confirmed by Western blotting and on retroviral overexpression of GR in resistant subclones GC-sensitivity was restored. All GC-resistant subclones were screened for GR mutations using denaturing high-pressure liquid chromatography (DHPLC), DNA-fingerprinting, and fluorescence in situ hybridization (FISH). Among the identified mutations were some previously not associated with GC resistance: A484D, P515H, L756N, Y663H, L680P, and R714W. This approach revealed three genotypes, complete loss of functional GR in the mismatch repair deficient T-ALL model, apparently normal GR genes in B-ALLs, and heterozygosity in both. In the first genotype, deficiency in GR up-regulation was fully explained by mutational events, in the second by a putative regulatory defect, and in the third by a combination thereof. In all instances, GC-resistance occurred at the level of the GR in both models.

Levels of glucocorticoid receptor and its ligand determine sensitivity and kinetics of glucocorticoid-induced leukemia apoptosis

“…A very sound mathematical analysis of this issue was performed by Butturini et al 3 Ross et al rightly state that the gold standard for MRD in acute leukemia is relapse. Therefore, studies that simultaneously apply various methods of monitoring MRD and determine which method is best at predicting relapse are strongly needed.…”

An oncogenic role of miR-142-3p in human T-cell acute lymphoblastic leukemia (T-ALL) by targeting glucocorticoid receptor-α and cAMP/PKA pathways

“…Although the mechanism is complicated, GR is considered a fundamental factor for the development of GC resistance. [29][30][31] Owing to the alternative splicing, the GR gene has multiple GR transcript isoforms, including active GRa and inhibitory GRb. 36 The latter can form heterodimer with GRa and inactivate GRa activity.…”

“…This drew our attention for such reasons: (1) GCs are among the most important chemical drugs for T-cell leukemia treatment; 27 (2) GC resistance, the status of insensitivity of leukemic cells to GC, is a therapeutic problem with an incompletely clear molecular mechanism; 28 and (3) downregulation of GR has been believed to contribute to GC resistance. [29][30][31] Therefore, we hypothesized that the expression of GR in leukemic T cells was regulated by miR-142-3p. There are different mRNA transcripts of GR because of alternative splicing.…”

“…Given the critical role of GRa expression in GC resistance, [29][30][31] miR-142-3p might be also involved in GC resistance through regulating GRa. Here, we further hypothesized that miR-142-3p regulated GC resistance in T-leukemic cells.…”

“…To further uncover the link between miR-142-3p and GC resistance, we chose the CCRF/CEM cell line for GC-resistant cell clone screening. 30 After 10 days treatment with 0.1 mM dexamethasone, live cells were diluted into a 96-well plate with 100 cells/well for further selection. Consequently, four highly GC-resistant subclones were selected, in which 490% cells survived from dexamethasone treatment.…”