Association Between NR3C1 Mutations and Glucocorticoid Resistance in Children With Acute Lymphoblastic Leukemia

Abstract

Treatment outcomes in children with acute lymphoblastic leukemia (ALL) have been improved substantially, with a cure rate exceeding 80% using conventional therapy. However, the outcome for patients with relapsed/refractory ALL remains unsatisfactory, despite the fact that these patients generally receive more intense therapy. Glucocorticoid (GC) resistance is a leading cause of treatment failure and relapse in ALL. Abnormal NR3C1 transcription and/or translation is strongly associated with GC resistance, but the underlying molecular mechanism and the clinical value of NR3C1 alterations with GC resistance in ALL treatment remain unclear. This study applied panel sequencing to 333 newly diagnosed and 18 relapsed ALL samples to characterize the link between NR3C1 and ALL further. We identified NR3C1 mutations in three patients with newly diagnosed ALL (0.9%) and two patients with relapsed ALL (11.1%). Functional analyses revealed that four of these five NR3C1 mutations (p. R477H, p. Y478C, p. P530fs, and p. H726P) were loss-of-function (LoF) mutations. A drug sensitivity test further showed that LoF NR3C1 mutations influence GC resistance. Saturated mutagenesis of hotspot R477 demonstrated the importance of this residue for NR3C1 function. The dominant-negative effect of p. R477C and p. R477S and the non-dominant negative effect of p. R477H and p. Y478C suggests multiple mechanisms underlying GC resistance. Thus, primary or acquired genomic lesions in NR3C1 may play a critical role in GC resistance and contribute to ALL treatment failure and/or relapse.

Keywords: NR3C1; acute lymphoblastic leukemia; drug resistance; glucocorticoid; glucocorticoid receptor.

FIGURE 1

NR3C1 mutations in patients with newly diagnosed and relapsed B-ALL. (A) Identification of NR3C1 mutations by panel sequencing in patients with newly diagnosed (N = 333) and relapsed (N = 18) B-ALL enrolled in CCCG-ALL-2015 in Guangzhou Women and Children’s Medical Center. (B) NR3C1 mutations data from PCGP, cBioPortal, and our hospital were analyzed. NR3C1 mutations spanned the full length of the gene (upper: p. R477H, p. Y478C, p. P530fs, p. I539fs, p. H726P from our hospital; lower: p. S114fs, p. E116Gfs, p. S241fs, p. N242Efs, p. E285*, p.M336fs, p. N337Gfs, p. S395fs, p. L456_C457 > HS, p. C476S, p. R477C, p. R477H, p. L639P, p. L669 > LGQ, p. R714Q form PCGP and cBioPortal; red line, newly diagnosed B-ALL; black line, relapsed B-ALL). (C) Dexamethasone treatment responses were analyzed in patients with newly diagnosed ALL with (N = 3) or without (N = 330) an NR3C1 mutation.

FIGURE 2

Transcriptional activity and effect on GC resistance for various NR3C1 mutations in ALL cells. (A) The activity of NR3C1 mutants and wild-type NR3C1 treated with dexamethasone was tested by glucocorticoid response element (GRE)-reporter assays (black bar, wild-type; orange bar, p. Y478C; green bar, p. I539fs). (B) Cytotoxicity of prednisolone was examined in Nalm6 cells harboring mutant NR3C1 (blue line, EV; brown line, wild-type NR3C1; purple line, p. Y478C; orange line, p. R477H). (C) Cytotoxicity of prednisone was examined in REH cells with mutant NR3C1 (blue line, EV; brown line, wild-type NR3C1; purple line, p. Y478C; orange line, p. R477H.). NR3C1 mutations (p. Y478C and p. R477H) lead to significant resistance to prednisolone in ALL lines. Cells were incubated with drugs for 72 h, and viability was then measured by an MTT assay. Experiments were performed in triplicate and repeated at least three times.

FIGURE 3

Saturation mutagenesis of NR3C1 R477 and its effects on protein function. (A) Twenty R477 mutation types were constructed by saturated mutagenesis. The transcriptional activity of NR3C1 mutations was presented as the −log10 value of relative activity. (B) Wild-type and R477 mutant (p.R477C, p. R477S, p. R477H, and p. Y478C) NR3C1 were co-transfected in different proportions (1:1, 1:2, 1:4, and 1:8). Mutated NR3C1 (p.R477C and p. R477S) showed dominant-negative regulatory effects on the transcriptional activity of wild-type NR3C1, while mutant NR3C1 (p.R477H and p. Y478C) showed non-dominant-negative regulatory effects on the transcriptional activity of wild-type NR3C1. All data are presented as means ± SD of triplicate samples from each independent experiment.

FIGURE 4

NR3C1 mutations contribute to GC resistance by dominant-negative and non-dominant-negative regulatory effects. (A) The expression of apoptosis-related genes in NR3C1 mutants was determined by quantitative PCR. The expression levels of anti-apoptotic genes (BCL2, BCL-XL, MCL1, and BCL-W) were up-regulated in NR3C1 mutants (p.R477C and R477S) by a dominant-negative regulatory effect. Pro-apoptotic genes (BAX, BAK, BOK, BIM, BID, and PUMA) were down-regulated in NR3C1 mutants (p.R477H andp. Y478C) by non-dominant-negative regulation. (B) Model of the mechanism underlying GC resistance caused by NR3C1 mutations.