Published OnlineFirst December 11, 2014; DOI: 10.1158/1078-0432.CCR-13-3317 Cancer Therapy: Preclinical Clinical Cancer Research Dual Inhibition of Bcr-Abl and Hsp90 by C086 Potently Inhibits the Proliferation of Imatinib-Resistant CML Cells Lixian Wu1,2,3, Jing Yu4, Ruijia Chen1,2, Yang Liu2,3,5, Liguang Lou6,YingWu2,3, Lisen Huang1, Yingjuan Fan2, Pinzhang Gao1, Meijuan Huang7, Yong Wu7, Yuanzhong Chen7, and Jianhua Xu1,2,3 Abstract Purpose: Although tyrosine kinase inhibitors (TKI) such as Results: Biochemical assays with purified recombinant Abl imatinib provide an effective treatment against Bcr-Abl kinase kinase confirmed that C086 can directly inhibit the kinase activity in the mature cells of patients with chronic myelogenous activity of Abl, including WT and the Q252H, Y253F, and leukemia (CML), TKIs probably cannot eradicate the leukemia T315I mutations. Furthermore, we identified C086 as a novel stem cell (LSC) population. Therefore, alternative therapies are Hsp90 inhibitor with the capacity to disrupt the Hsp90 chap- required to target both mature CML cells with wild-type (WT) or erone function in CML cells. Consequently, it inhibited the mutant Bcr-Abl and LSCs. To investigate the effect of C086, a growth of both imatinib-sensitive and -resistant CML cells. derivative of curcumin, on imatinib-resistant cells, we explored Interestingly, C086 has the capacity to inhibit LTC-ICs and to þ þ þ À its underlying mechanisms of Bcr-Abl kinase and heat shock induce apoptosis in both CD34 CD38 and CD34 CD38 protein 90 (Hsp90) function inhibition. cells in vitro. Moreover, C086 could decrease the number of þ þ þ þ þ þ À Experimental Design: Biochemical assays were used to test ABL CD45 ,CD45 CD34 CD38 , and CD45 CD34 CD38 cells kinase activity; fluorescence measurements using recombinant in CML NOD-SCID mice. NHsp90, Hsp90 ATPase assay, immunoprecipitation, and immu- Conclusions: Dual suppression of Abl kinase activity and noblotting were applied to examine Hsp90 function. Colony- Hsp90 chaperone function by C086 provides a new therapeutic forming unit, long-term culture-initiating cells (LTC-IC), and flow strategy for treating Bcr-Abl–induced leukemia resistant to TKIs. cytometry were used to test CML progenitor and stem cells. Clin Cancer Res; 21(4); 833–43. Ó2014 AACR. Introduction chronic-phase chronic myelogenous leukemia (CML; ref. 1). The use of imatinib in patients with CML has led to an enormous The Bcr-Abl kinase inhibitor imatinib is a standard treatment þ reduction in 5-year mortality. Despite outstanding clinical results, for Ph leukemia and has been shown to induce a complete imatinib-resistant leukemia and clinical relapse eventually hematologic and cytogenetic response in most patients with emerge. In fact, upon discontinuation of therapy, the disease aggressively relapses in the majority of patients. The mechanisms 1Department of Pharmacology, School of Pharmacy, Fujian Medical of resistance to imatinib include mutations of the Bcr-Abl kinase University (FMU), Fuzhou, P.R. China. 2Institute of Materia Medica, fi bcr-abl 3 domain, the ampli cation of the gene, and the Fujian Medical University (FMU), Fuzhou, P.R. China. Fuijan Key – Laboratory of Natural Medicine Pharmacology, Fujian Medical Univer- insensitivity of leukemia stem cells (LSC) to imatinib (2 5). sity (FMU), Fuzhou, P. R. China. 4Department of Pathology, School of Clinically observed mutations have been identified within Basic Medicine, Fujian Medical University (FMU), Fuzhou, P.R. China. several regions of the Bcr-Abl kinase domain. In this study, we 5 Department of Pharmacochemistry, School of Pharmacy, Fujian Med- examined three kinase domain variants, Q252H, Y253F, and ical University (FMU), Fuzhou, P.R. China. 6Shanghai Institute of Mate- ria Medica, P.R. China. 7Fujian Institute of Hematology, Union Hospital, T315I, and bcr-abl gene amplification. These variants contain Fujian Medical University (FMU), Fuzhou, P. R. China. several functionally distinct kinase domain regions, including the Note: Supplementary data for this article are available at Clinical Cancer nucleotide-binding P-loop (Q252H and Y253F) and two imati- Research Online (http://clincancerres.aacrjournals.org/). nib mesylate contact residues (Y253F and T315I). L. Wu and J. Yu contributed equally to this article. There is considerable interest in developing alternative Abl kinase inhibitors capable of inhibiting the Bcr-Abl kinase domain Corresponding Authors: Lixian Wu, Fujian Medical University, 88 Jiaotong Road, Fuzhou 350004, Fujian, China. Phone: 86-1825-9000-966; Fax: 86-591- mutants observed in relapsed patients. The second-generation 8356-9307; E-mail: [email protected]; Yuanzhong Chen, Fujian Institute of tyrosine kinase inhibitors (TKI), such as nilotinib and dasatinib, Hematology, Union Hospital, FMU, Fuzhou 350004, Fujian, China. E-mail: are able to override the majority of the mutations conferring [email protected]; and Jianhua Xu, Department of Pharmacology, Insti- resistance to imatinib, with the exception of the T315I mutation tute of Materia Medica, School of Pharmacy, Fuzhou, Fujian 350004, China. (6–11). GNF-2, a selective allosteric Bcr-Abl inhibitor, binds to Phone: 86-591-8356-9307; Fax: 86-591-8356-9307; E-mail: the myristate-binding site of Abl, leading to changes in the [email protected] structural dynamics of the ATP-binding site and rendering Abl doi: 10.1158/1078-0432.CCR-13-3317 unaffected by kinase mutations (12, 13). Another new agent, Ó2014 American Association for Cancer Research. ponatinib (AP24534), is a potent oral TKI that blocks native and www.aacrjournals.org 833 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst December 11, 2014; DOI: 10.1158/1078-0432.CCR-13-3317 Wu et al. Translational Relevance Materials and Methods Reagents Although the discovery of tyrosine kinase inhibitors The PathScan Bcr/Abl Activity Assay Multiplex Western Detec- targeting the Bcr-Abl kinase revolutionized chronic mye- tion and Apoptosis Antibody Sampler Kit were purchased from logenous leukemia therapy, resistant leukemia and clinical Cell Signaling Technology, Inc. StemSpan CC100, StemSpan relapse eventually emerge. The mechanisms of resistance to Serum-Free Expansion Medium (SFEM), EasySep Human Whole imatinib include mutations of the Bcr-Abl kinase domain, Blood CD34-Positive Selection Kit, MethoCult H4434, and Mye- the amplification of the bcr-abl gene, and the insensitivity of loCult H5100 were purchased from STEMCELL Technologies Inc. leukemia stem cells (LSC) to imatinib. There were reports Wild-type (WT) and mutant ABL kinases were purchased from indicating that inhibition of Hsp90 can effectively reduce Millipore Corporation. The Abl kinase substrate was purchased the survival and proliferation of LSCs. Unfortunately, the from Enzo Life Science, Inc. use of Hsp90 inhibitors has been limited due to their significant hepatotoxicity in clinical trials. Here, we iden- tified C086 as a dual inhibitor of Abl kinase activity and Cell culture Hsp90 chaperone function. In addition to the inhibition of The 32D-WT, 32D-T315I, 32D-Q252H, and 32D-Y253F cell kinase activity of Bcr-Abl, the elimination of Bcr-Abl by lines used in this study were constructed as described previously C086 via Hsp90 inhibition provides a new therapeutic (23). The cell lines have been tested and authenticated by real- strategy for treating Bcr-Abl–induced leukemia and other time quantitative PCR, DNA sequencing, mycoplasma detection, cancers resistant to tyrosine kinase inhibitors. and cell vitality detection. Human leukemic K562 cells were obtained from the Cell Bank of the Chinese Academy of Sciences, where they were characterized by DNA fingerprinting, mycoplas- ma detection, and cell vitality detection. These cell lines were mutated Bcr-Abl, including the gatekeeper mutant T315I, which is immediately expanded and frozen. K562 cells were cultured and uniformly resistant to TKIs (14). passaged in RPMI 1640 containing 10% heat-inactivated FBS, 100 Although the discovery of TKIs targeting the Bcr-Abl kinase U/mL penicillin, 100 mg/mL streptomycin, and 2 mmol/L glu- fi dramatically decreased disease burden, these drugs probably tamine (medium A) in a 5% humidi ed CO2 atmosphere at 37 C. cannot eliminate quiescent LSCs, allowing disease relapse. There- Imatinib-resistant K562/G01 cells were purchased from the Insti- fore, it is essential that alternative strategies are developed to target tute of Hematology, Chinese Academy of Medical Sciences, and the LSC population. Peking Union Medical College (Tianjin, China), where they were Based on its chaperone function in regulating many tumor- characterized by FISH analysis for the detection of Bcr-abl gene related client proteins, Hsp90 is a promising target for che- copies, mycoplasma detection, and cell vitality detection. These motherapy (15–18). Gorre and colleagues (19) reported that cell lines were immediately expanded and frozen such that they Bcr-Abl point mutants isolated from patients with imatinib- could be restarted every 3 to 4 months from a frozen vial of the resistant CML remain sensitive to the Hsp90 inhibitors gelda- same batch of cells. The K562/G01 cells were maintained in namycin (GA) and 17-allylaminogeldanamycin (17-AAG). medium A either containing or lacking 4 mmol/L imatinib. Moreover, Peng and colleagues (20) reported that IPI-504, an Hsp90 inhibitor, had a dramatic inhibitory effect on these Tyrosine kinase assay LSCs. This