234 Chapter 7 steroid response in those patients. We attempted to distinguish between the contribution of the GR and MR on steroid-induced cytotoxicity in this specific genetic subgroup, by treating two primary ALL patient samples and one PDX model with different steroids in combination with the MR antagonist RU28318 at a concentration that was minimally cytotoxic by itself but could complete block steroid-induced cell death via the MR. In these samples, we saw a minimal shift in our cell toxicity curves towards resistance as well as minimal reduced expression levels of GR/MR target genes after addition of RU28318, although not significant. This indicates a minimal and subtle involvement of the MR in steroid-induced death in ETV6-RUNX1 rearranged pre-B ALL patients. An explanation for the difference between these patient and PDX samples and our experimental setting may be a lower expression or lower transcriptional activity of NR3C2 compared to NR3C1 in patient leukemic cells and the presence of other more dominant (genetic and/or cellular) factors in these patients. Due to the lack of a functional antibody recognizing NR3C2, we were unable to test this at the protein level. Moreover, no genes have been identified to be specifically regulated by either NR3C1 or NR3C2, prohibiting more specific transcriptional analysis.9,31 Therefore, the contribution of the MR in steroid-induced cytotoxicity in our patient samples remains unclear. In vivo sensitivity to glucocorticoids is an important prognostic factor in the treatment of ALL. In our ALL patient cohort, we did not find an association between basal NR3C1 or NR3C2 mRNA expression levels and event free survival or poor steroid response, as was described before for NR3C1 expression.7 This may be partially explained by other important underlying mechanisms of steroid resistance in pediatric ALL, such as loss of IKZF1 function, epigenetic silencing of the BIM locus, IL7-induced signaling or IL7R signaling mutations.32-34 Furthermore, the median follow up of our cohort was only 26 months, therefore only early events could be analyzed. Since many relapses occur three years after therapy, our results concerning a possible association between NR3C1 and NR3C2 expression levels to event free survival are limited, especially in this relatively small cohort. It is conceivable however, that other crucial processes play a more dominant role in relapse, such as chemotherapy induced mutations.35 In conclusion, in experimental models, the mineralocorticoid receptor (NR3C2) potently induces steroid-induced cell death and hydrocortisone is a potent steroid to initiate this process. However, the contribution of MR-regulated steroid-induced toxicity appears to be minimal or subtle in leukemic patient samples, and the clinical relevance of NR3C2 expression or functionality for patients with acute lymphoblastic leukemia remains to be elucidated.
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