Molecular Mechanism for Synergistic Anti-Leukemic Activity of Tyrosine Kinase Inhibitors and Glucocorticoids Against Ph+ALL

The tyrosine kinase inhibitors (TKIs) have dramatically altered the management of patients with Philadelphia chromosome-positive ALL (Ph+ALL). In earlier trials of imatinib monotherapy for relapsed or refractory patients with lymphoid blast crisis of CML (CML-LC) and Ph+ALL, complete hematologic remission (CHR) was achieved only in 20%, and relapses occurred in most of the patients within several months. Thereafter, combination induction therapy of imatinib with conventional chemotherapeutic agents profoundly improved the therapeutic outcome in the patients with Ph+ALL. In elderly patients, to reduce the therapy-related toxicities, combination induction therapy of imatinib with glucocorticoids (GCs) followed by imatinib monotherapy was performed. Surprisingly, CHR was obtained in all of the patients with this simple therapy, and median survival from diagnosis was 20 months (Blood 2007). Similarly, induction therapy of dasatinib, a second-generation TKI, combined with GCs achieved CHR in all of the newly diagnosed Ph+ALL patients (Blood 2011). These clinical findings indicate a synergistic anti-leukemic activity of TKIs with GCs in Ph+ALL, but its underlying molecular mechanisms remain totally unknown. Thus, we analyzed synergistic effects of TKIs and dexamethasone (Dex) in a panel of leukemic cell lines derived from Ph+ALL. Indeed, in the presence of 0.5μM of imatinib, IC50 values of Dex were approximately 3-8 times lower than those in the absence of imatinib in the most of Dex-sensitive Ph+ALL cell lines. Since gene expression level of GC receptor (GR; NR3C1) was associated with Dex-sensitivity in Ph+ALL cell lines, we next analyzed the effects of imatinib on gene expression level of NR3C1. Of note, NR3C1 gene expression level was significantly upregulated in the presence of 0.5 μM of imatinib approximately 1.5-7-fold in all of 14 Ph+ALL cell lines except for SK9, which was an imatinib-resistant cell line having a T315I mutation of BCR-ABL, whereas it was unchanged in all of 11 Ph-negative ALL cell lines. Induction of GR was also confirmed by immuno-blotting in representative Ph+ALL cell lines. Moreover, treatment with either dasatinib or nilotinib clearly upregulated the NR3C1 gene expression in the representative Ph+ALL cell lines. Of importance, although gene expression level of NR3C1 was significantly upregulated in the presence of imatinib in the imatinib-sensitive Ph+ALL cell lines, SU-Ph2 and TCCY, it was unchanged in their imatinib-resistant sublines, SU/SR and TCCY/SR, respectively, in which T315I mutation was acquired after the culture with increasing concentrations of imatinib, indicating that upregulation of GR by TKIs in Ph+ALL cell lines was mediated by an inactivation of BCR-ABL. To further verify the downstream pathway of BCR-ABL that is critically involved in the TKI-induced GR upregulation, we treated imatinib-sensitive SU-Ph2 and its imatinib-resistant subline SU/SR with specific inhitors of PI3K (GDC0941, LY294002, and AS606240), JAK2(SD1029), and MAPK(UO126). Among five agents, only UO126 effectively upregulated the NR3C1 gene expression both in SU-Ph2 and in SU/SR, suggesting that TKIs upregulate GR in Ph+ALL cell lines mainly through an inactivation of the MAPK pathway. Previous reports revealed that three promoters, 1A, 1B, and 1C, are mainly involved in the NR3C1 gene expression in ALL cells. We therefore performed real time RT-PCR analysis of the NR3C1 gene using three sets of primers that are specific for exons 1A3, 1B, or 1C. The strongest induction by an imatinib-treatment was observed in the 1A promoter in Ph+ALL cell lines. Finally, since BIM, one of BH3-only pro-apoptotic members of BCL2 family, has been reported to be critically involved in the anti-leukemic activities of both TKIs and GCs, we analyzed BIM expression. Synergistic induction of BIM was confirmed both in mRNA and protein expression levels by a simultaneously treatment of Ph+ALL cell lines with imatinib and Dex. Taken together, these observations in Ph+ALL cell lines indicate that TKIs induce GR expression in Ph+ALL mainly through the MAPK pathway and the 1A promoter of NR3C1 gene by inactivating BCR-ABL and subsequently exert a synergistic anti-leukemic activity with GCs through the induction of BIM.