229 The role of the MR 7 of leukemic cells. We therefore examined the cytotoxic effects of steroid treatment in NR3C1- or NR3C2- expressing Reh cells. In the absence of doxycycline, RehNR3C1 and RehNR3C2 cells were completely refractory to treatment with dexamethasone, prednisolone or hydrocortisone (Figure 2A, Supplemental Figure 2A). Interestingly, doxycycline-induced NR3C1 or NR3C2 expression sensitized Reh cells for all three steroids. Moreover, hydrocortisone appeared to be the most potent cytotoxic steroid in both RehNR3C1 and RehNR3C2 cells. Furthermore, the cytotoxic effect induced by dexamethasone was comparable in cells expressing the GR or the MR. The notable difference in hydrocortisone sensitivity between RehNR3C2 and RehNR3C1 cells to induce cell death is consistent with the superior induction of BIM by hydrocortisone in RehNR3C2 cells (Figure 1C). Combined, these results show that hydrocortisone can induce significant steroid-induced cell death in leukemic cells, either by activation of the MR or the GR. Interestingly, but in contrast to our RTQ-PCR data, dexamethasone induces significant steroid-induced cell death in RehNR3C2 cells, albeit at a slightly higher concentration than in RehNR3C1 cells. To verify the role of the MR in steroid-induced cell death, we treated RehNR3C1 and RehNR3C2 cells with RU28318, a specific MR-antagonist.22 In the absence of steroid treatment, RU28318 was minimally cytotoxic for RehNR3C2 cells (Supplemental Figure 2B). As expected, the treatment with RU28318 did not affect steroid sensitivity of doxycycline-induced RehNR3C1 cells upon treatment with prednisolone, dexamethasone or hydrocortisone. This further confirms the absence of endogenous functional NR3C2 expression in these cells (Figure 2B). RU28318 treatment in RehNR3C2 cells completely inhibited the cytotoxic potential of the MR following steroid treatment. Similar results with MR antagonists Eplerenone and Spironolactone were observed (Supplemental Figure 3A and B). Our results in RehNR3C1 and RehNR3C2 cell lines highlighted a potential role for the MR in steroid-induced cell death in ALL cells. To study the potential clinical relevance of these observations, we determined the relative expression of NR3C1 and NR3C2 in 279 primary ALL patient samples. Overall, the relative expression of NR3C1 was higher than NR3C2 (Figure 3A). After dissecting the cohort according to molecular ALL characteristics, we observed that the expression of NR3C2 in T-ALL patients was relatively low. Of the B-cell precursor acute lymphoblastic leukemia patients, those with an ETV6-RUNX1 fusion gene harbored the highest (relative) expression of NR3C2, as described before.8 This is highly interesting, considering the favorable outcome of this specific leukemic subgroup.23 As a proof of concept, we treated one PDX sample and two patient samples with an ETV6-RUNX1 fusion gene with prednisolone, dexamethasone and hydrocortisone in the presence or absence of RU28318 to measure
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