P348: mrk-560 and dexamethasone are synergistic in the treatment of t-cell acute lymphoblastic leukemia

Background: NOTCH1 activating mutations are found in the majority of T-cell acute lymphoblastic leukemia (T-ALL) cases and inhibition of NOTCH1 activation is therefore a potential therapeutic option in T-ALL. Gamma-secretase inhibitors have been shown to block NOTCH1 activation, but are also associated with dose-limiting toxicity, preventing their use for T-ALL treatment. We have recently shown that a more selective inhibition of the gamma-secretases with PSEN1-selective γ-secretase inhibitors, such as MRK-560, retains a strong anti-leukemia effect with minimal gastrointestinal toxicities. Aims: We wanted to investigate if MRK-560 would show synergy with currently used chemotherapeutic drugs, such as dexamethasone, vincristine and doxorubicin, and if it could be safely combined with chemotherapy for treating T-ALL patients. Methods: Dose response curves were performed in DND-41 and SUPT-1 T-ALL cell lines, which were treated with DMSO/MRK-560 alone or in combination with different concentrations of chemotherapy. Apoptosis assay was conducted with annexin-V/PI staining. Human primary T-ALL samples were transduced with a luciferase vector to obtain luciferase positive xenografts in immunodeficient NSG mice that could be used for in vivo treatment. Mice were treated for 3 weeks (5 days on – 2 days off) with vehicle, dexamethasone (5mg/kg, IP), MRK-560 (15mg/kg, IP) or the combination. Leukemia progression was followed by weekly blood sampling and bioluminescent imaging after injection of luciferin. Results: Treatment with MRK-560 increased the sensitivity to dexamethasone in NOTCH1-dependent T-ALL cell lines (DND-41 and SUPT-1) and we documented a strong synergy between these drugs. The combination between MRK-560 and doxorubicin/vincristine showed only an additive effect. Furthermore, we demonstrated that treatment with MRK-560 and dexamethasone increased apoptosis 2.5 fold compared to single dexamethasone treatment. Dexamethasone acts by activating the glucocorticoid receptor (NR3C1) which activates several target genes including BIM, an inducer of apoptosis. Mechanistically, we found that mRNA and protein levels for NR3C1 and BIM were significantly more upregulated after combination treatment compared to single drug treatment. This was associated with strong downregulation of HES-1 levels. These data indicate that MRK-560 synergizes with dexamethasone via downregulation of HES-1 (downstream of NOTCH1), resulting in enhanced glucocorticoid signaling with higher BIM expression levels and apoptosis. Finally, we also studied the synergy between MRK-560 and dexamethasone in patient-derived xenograft models of T-ALL. Bioluminescent imaging showed significant reduction in leukemia progression in combination treatment compared to single treatments. Furthermore, combination treatment (41 days) significantly prolonged survival compared to single MRK-560 (34 days), dexamethasone (30 days) or placebo (16 days) treatment. Summary/Conclusion: We have shown that the PSEN1-selective gamma-secretase inhibitor MRK-560 can be safely combined with chemotherapy for the treatment of T-ALL patients thereby demonstrating a synergistic effect between MRK-560 and dexamethasone. Mechanistically, MRK-560 acts in a similar way as broad spectrum gamma-secretase inhibitors, but does not show gastrointestinal toxicity.