The NR4A Orphan Nuclear Receptors Do Not Confer Prednisolone resistance In Pediatric Acute Lymphoblastic Leukemia
Authors: Ariës Ingrid M. , Dungen Rosanna van den , Pieters Rob , Den Boer Monique L. 
Published: October 2019

Abstract

Background
Resistance against the glucocorticoid prednisolone still remains an obstacle for treatment of pediatric precursor B acute lymphoblastic leukemia (BCP-ALL) at initial diagnosis and even more after relapse. The molecular mechanisms behind prednisolone resistance in pediatric BCP-ALL is poorly defined. The NR4A family, consisting of NR4A1 (Nur77), NR4A2 (Nurr1) and NR4A3 (Nor1), are orphan nuclear receptors, which antagonize the glucocorticoid receptor. We hypothesized that upregulated NR4A family expression is responsible for prednisolone resistance in BCP-ALL.

Methods
Newly diagnosed pediatric acute lymphoblastic leukemia patients’ cells were isolated from bone marrow aspirates and only samples with ≥ 90% leukemic blasts were used in the present study. Gene expression microarrays of 178 BCP-ALL patients tested for in vitro prednisolone resistance were analyzed with Limma R Package in the statistical environment R, version 2.15.0. Microarray expression levels were confirmed using qRT-PCR. Nur77, Nurr1 and Nor1 protein expression in primary BCP-ALL patients’ were assessed with reverse phase protein array. Leukemic patients’ cells were transfected with labeled siRNA against NR4A1, NR4A2, and NR4A3, simultaneously, or with labeled siScrl, using the transfection reagent Dharmafect 4. Hereafter, cytotoxicity to prednisolone was determined by the in vitro 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) drug-resistance assay. Furthermore, viability of transfected cells was counted by trypan blue exclusion assay and cells were harvested after 72 hours of culture for RNA and protein isolation. Knockdown was confirmed with qRT-PCR and Western blot.

Results
In this study of 178 precursor BCP-ALL patients we discovered a 3.0-fold (p=0.007) raise in NR4A1, NR4A2, and NR4A3 microarray mRNA expression in in vitro prednisolone resistant compared to sensitive BCP-ALL patients’ cells, which was confirmed by qRT-PCR. In addition, reverse phase protein array identified a 2.7-fold (p<0.001) increased Nurr1 (NR4A1) and Nur77 (NR4A2) protein expression in in vitro prednisolone resistant compared to sensitive BCP-ALL patients’ cells. Nurr1 and Nur77 protein levels were overall 5-fold (p<0.001) higher in BCP-ALL patients compared to normal bone marrow cells. Simultaneous siRNA mediated knockdown of Nur77, Nurr1 and Nor1 in pediatric leukemic patients’ ALL cells decreased leukemic cell survival by 25.4±11.1% (p=0.029), but did not sensitize these cells to prednisolone (n=4).

Conclusion
The NR4A family members are higher expressed in prednisolone resistant ALL patients’ cells. Although literature describes an antagonizing effect of the NR4A family members on the glucocorticoid receptor, we could not find a functional contribution of the NR4A family to prednisolone resistance in BCP-ALL. We furthermore discovered an increased NR4A family expression in leukemic cells of BCP-ALL patients compared to normal bone marrow cells. Targeting of NR4A genes impaired cell survival. However, compensatory mechanisms exist and consequently all three NR4A members need to be targeted simultaneously to diminish cell survival. These data therefore suggest that the NR4A genes are not suitable to reverse prednisolone resistance nor to kill leukemic cells by targeted drugs.

Disclosures:
No relevant conflicts of interest to declare.