Combined Effects of Novel Heat Shock Protein 90 Inhibitor NVP-AUY922 and Nilotinib against Mutant Forms of BCR-ABL

Abstract

NVP-AUY922 (Novartis) is a novel 4,5-diaryloxazole ATP-binding site heat shock protein 90 (HSP90) inhibitor, which has been shown to inhibit the chaperone function of HSP90 and deplete the levels of HSP90 client protein. Combining AUY922 with ABL kinase inhibitors may provide several advantages, such as enhanced efficacy and reducing the potential emergence of new resistant mutations. Treatment with AUY922 has been shown to exert greater potency against BCR-ABL mutants compared with wild type (wt) BCR-ABL. In the present study, we investigated the combined effects of AUY922 and Nilotinib on mutant forms of BCR-ABL-expressing cells. Co-treatment with AUY922 and Nilotinib resulted in significantly more inhibition of growth than treatment with either agent alone in BaF3 cells expressing wt-BCR-ABL and BCR-ABL mutants (M244V, G250E, Q252H, Y253F, E255K, T315A, T315I, F317L, F317V, M351T, H396P). The observed data from the isobologram indicated the synergistic effect of simultaneous exposure to AUY922 and Nilotinib even in BaF3 cells expressing T315I. In contrast, we did not observe enhanced effects of AUY922 and imatinib in T315I BCR-ABL-expressing cells. Co-treatment with 500nM of Nilotinib significantly increased AUY922-induced apoptosis in BaF3 cells expressing T315I. Combined treatment with AUY922 and Nilotinib in BaF3 T315I also associated with more PARP cleavage, resulting from increased activation of caspase-3 and -9 during apoptosis. Following co-treatment of BaF3 T315I with AUY922 and Nilotinib caused more attenuation of phospho-T315I BCR-ABL levels and the downstream signal transducer, including phosho-CrkL, phospho-Stat5, and phospho-Akt. These results demonstrate that Nilotinib appears to inhibit T315I BCR-ABL kinase activity in BCR-ABL structurally compromised by loss of HSP90 chaperone activity. To assess the in vivo efficacy of AUY922 and Nilotinib, athymic nude mice were injected i.v. with mixture of BaF3 cells expressing wild type BCR-ABL and mutant forms of BCR-ABL(M244V, G250E, Q252H, Y253F, E255K, T315A, T315I, F317L, F317V, M351T, H396P). 24 hrs after injection, the mice were divided four groups (5 mice per group), with each group receiving either vehicle, AUY922 (50mg/kg; i.p. two times per week), Nilotinib (30mg/kg; p.o. once every day), AUY922 (50mg/kg; i.p. two times per week) + Nilotinib (30mg/kg; p.o. once every day). Animals treated with either vehicle or Nilotinib-alone died of a condition resembling acute leukemia by 28 days; animals treated with AUY922 alone survived more than 40 days, and those treated with the combination of AUY922 + Nilotinib survived more than 60 days. Histopathologic analysis of vehicle or Nilotinib-alone treated mice revealed infiltration of the spleen and bone marrow with leukemic blasts. In contrast, histopathologic analysis of organs from AUY922 plus Nilotinib-treated mice demonstrated normal tissue architecture and no evidence of residual leukemia. Taken together, these preclinical studies show that the combination of AUY922 and Nilotinib exhibits a desirable therapeutic index that can reduce the in vivo growth of mutant forms of BCR-ABL-expressing cells, including T315I, in an efficacious manner.