Haploinsufficiency for NR3C1 Drives Glucocorticoid Resistance By Inactivation the PI3K/AKT/GSK3β/Bim Pathway in Adult Acute Lymphoblastic Leukemia

Introduction: Although steady improvements to chemotherapeutic treatments have helped cure 80% of childhood B-cell acute lymphoblastic leukemia (B-ALL) cases, chemotherapy has proven to be less effective in treating the majority of adult patients, the equivalent rate for adults remains poor at 30-40%, with relapse representing the leading cause of mortality in both children and adults. Development of chemo-resistance is a crucial factor contributing to relapse, therefore understanding the biological mechanisms underlying this resistance is imperative for discovering innovative treatment strategies. Relapse specific/enriched genetic alterations in the emergence of clones that have gained a selective advantage under the pressure of specific chemotherapeutics. We previously performed longitudinal whole-exome sequencing analysis in diagnosis/relapse pairs from adult patients with B-ALL revealed relapse-specific mutations in NR3C1 gene, encoding the glucocorticoid receptor (GCR), are frequently detected. Furthermore, the mutations of NR3C1 identified in relapsed B-ALL patients all resulted in haploinsufficiency for NR3C1. In this study, we sought to delineate whether reduced expression of NR3C1 could give rise to chemo-resistance in B-ALL and elucidate the mechanism responsible for the resistance.

Methods: We performed clinical sample and ex vivo cell studies to elucidate the relationship between endogenous expression of NR3C1 in ALL cells and the sensitivity to dexamethasone. We further induced enforced expression of NR3C1 in dexamethasone-resistant ALL cells by being transfected with the expression vector of NR3C1. Moreover, we used CRISRP/CAS9 system to knock out NR3C1 gene in dexamethasone- susceptible ALL cell lines. Finally, we performed whole-genome transcriptome sequencing (RNA-seq) and Western-blotting analysis to elucidate the molecular mechanisms.

Results: (1) We examined the correlation of endogenous expression of NR3C1 with the sensitivity to dexamethasone in five human ALL cell lines, Reh, Jurkat, CCRF-CEM, Nalm6 and 6T-CEM. Our data suggested that the susceptibility of ALL cells to dexamethasone was positively correlated with endogenous NR3C1 mRNA and protein level. (2) Enforced expression of NR3C1 in dexamethasone-resistant ALL cells can reverse the resistance; (3) Knockout of NR3C1 gene in dexamethasone- susceptible ALL cells can confer resistance to glucocorticoid. (4) RNA-seq analysis showed that NR3C1 protein involved in the regulation of cell apoptosis, p53 pathway and cell cycle. NR3C1 protein regulated transcription of PI3K-related genes in ALL cells, which diminished PIK3CD transcription and increased mRNA level of PIK3R6, PIK3R5, PIK3R2, AKT3 and GSK3β. In addition, NR3C1 increased transcription of pro-apoptotic genes, BCL2L11 (encoding Bim protein), BMF and BOK, and diminished transcription of anti-apoptotic genes, BCL2 and BAG2. Further Western-blotting confirmed that overexpression of NR3C1 increased protein level of p-AKT (Ser 473), p-GSK3β (Ser 9), Bim and Bad, and diminished protein level of of Bcl-2 and Bcl-xl.

Conclusions: Our results provide, for the first time, evidence that haploinsufficiency for NR3C1 in ALL cells causes resistance specifically to glucocorticoid, which are the backbone of induction therapy, by disrupting the activation of PI3K/AKT/GSK3 signaling pathway and the balance between pre-apoptotic proteins and anti-apoptotic proteins.