Combined Effects of novel Heat Shock Protein 90 Inhibitor NVP-AUY922 and Nilotinib in a Random Mutagenesis Screen.

Abstract 3765

Poster Board III-701

To overcome imatinib resistance, more potent ABL TKIs such as nilotinib and dasatinib have been developed, with demonstrable preclinical activity against most imatinib-resistant BCR-ABL kinase domain mutations, with the exception of T315I. However, imatinib-resistant patients already harbouring mutations have a higher likelihood of developing further mutations under the selective pressure of potent ABL TKIs. The challenge for development of an effective Ph-positive leukemia therapy is therefore to develop an alternative treatment strategy that does not rely solely on kinase domain inhibition but rather results in degradation of the offending BCR-ABL protein regardless of its mutation status. 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. In the present study, we investigated the combined effects of AUY922 and Nilotinib on random mutagenesis for BCR-ABL mutation (Blood, 109; 5011, 2007). We performed a comprehensive drug combination experiment using a broader range of concentrations for AUY922 and Nilotinib. Compared with single agents, combination with AUY922 and Nilotinib was more effective at reducing the outgrowth of resistant cell clones. At the highest concentration of Nilotinib, the mutation spectrum narrowed to T315I and E344V by direct sequencing, whereas, at intermediate concentration of AUY922, the resistant clone was recovered by wild-type BCR-ABL only. No outgrowth was observed in the presence of 2 μM Nilotinib and 20 nM AUY922. To assess the in vivo efficacy of AUY922 and Nilotinib, athymic nude mice were injected s.c. with BaF3 cells expressing random mutagenesis for BCR-ABL mutation. 7 days after injection (average tumor volume, 100 mm³), the mice were divided four groups (5 mice per group), with each group receiving either vehicle, AUY922 (50mg/kg; i.p. weekly), Nilotinib (30mg/kg; p.o. once every day), AUY922 (50mg/kg; i.p. weekly) + Nilotinib (30mg/kg; p.o. once every day). Combination with AUY922 and Nilotinib effectively inhibited tumor growth in mice compared with vehicle- or Nilotinib- or AUY922-treated mice. Histopathologic analysis of tumor tissue from AUY922 plus Nilotinib-treated mice demonstrated an increased number of apoptotic cells detected by TUNEL stain. To investigate combined effects of AUY922 and Nilotinib on primary CML cells, NOD/SCID mice were injected i.v. with bone marrow mononuclear cells from a CML patients with F359V mutation. Treatment with AUY922 and Nilotinib demonstrated a marked segregation of apoptotic cells in both the central bone-marrow cavity and the endosteal surface. Furthermore, treatment with AUY922 and Nilotinib inhibited the phosphorylation of GSK-3β and Stat-3 in the endosteal surface. These results suggest that the combination with a HSP90 inhibitor and ABL TKIs may eliminate the CML initiating cells. Taken together, the present study shows 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.