A New Nonpeptidic Inhibitor of 14-3-3 Induces Apoptotic Cell Death in Chronic Myeloid Leukemia Sensitive or Resistant to Imatinib □ S Manuela Mancini, Valentina Corradi, Sara Petta, Enza Barbieri, Fabrizio Manetti, Maurizio Botta, and Maria Alessandra Santucci Dipartimento di Ematologia e Scienze Oncologiche “Lorenzo e Ariosto Sera ` gnoli,” Universita ` di Bologna, Italy (M.M., S.P., E.B., M.A.S.); and Dipartimento Farmaco Chimico Tecnologico, Universita ` di Siena, Italy (V.C., F.M., M.B.) Received July 8, 2010; accepted October 20, 2010 ABSTRACT Resistance of chronic myeloid leukemia (CML) to tyrosine ki- nase inhibitor imatinib mesylate (IM) is most often due to point mutations in the Bcr-Abl fusion gene. T315I mutation (resulting in substitution of Ile for a Thr residue at the “gatekeeper” position 315) raises particular concern, because it also provides resistance to second-generation kinase inhibitors already ap- proved for clinical use (nilotinib and dasatinib). Much effort is therefore focused on alternative molecular-based strategies. Previous studies proved that binding to 14-3-3 scaffolding proteins leads to cytoplasmic compartmentalization and sup- pression of proapoptotic and antiproliferative signals associ- ated with Bcr-Abl protein kinase, hence contributing to leuke- mic clone expansion. Here we investigated the effect of 14-3-3 inhibition disruption on hematopoietic cells expressing the IM- sensitive wild type Bcr-Abl and the IM-resistant T315I mutation. Using a virtual screening protocol and docking simulations, we identified a nonpeptidic inhibitor of 14-3-3, named BV02, that exhibits a remarkable cytotoxicity against both cell types. c-Abl release from 14-3-3, promoting its relocation to nuclear com- partment (where it triggers transcription of p73-dependent pro- apoptotic genes) and to mitochondrial membranes (where it induces the loss of mitochondrial transmembrane potential) combined with c-Abl enhanced association with caspase 9 (a critical step of sequential caspase activation further contribut- ing to c-Abl pro-apoptotic function) has a prominent role in the effect of BV02 on Bcr-Abl-expressing cells. In conclusion, BV02 may be considered as a treatment option for CML and, in particular, for more advanced phases of the disease that de- veloped IM resistance as a consequence of Bcr-Abl point mutations. Introduction The tyrosine kinase (TK) inhibitor imatinib mesylate (IM) has revolutionized the prognosis of chronic myeloid leukemia (CML), with best complete hematologic and cytogenetic re- sponse rates of 98 and 87%, respectively, after 5 years of fol- low-up (Druker et al., 2006). However, there is growing concern for the development of resistance, most often as a result of the emergence of Bcr-Abl point mutations. At the protein level, Bcr-Abl point mutations may either distort the configuration of Abl kinase, rendering it unable to adopt the inactive conforma- tion to which IM binds, or change the identity of residues that directly contact IM (Weisberg et al., 2007). The greatest thera- peutic challenge is posed by the substitution of highly conserved Thr residue at position 315, controlling the access to a hydro- phobic pocket of the enzymatic active site, by Ile (T315I). In fact, T315I mediates resistance not only to IM but also to second- generation Abl kinase inhibitors highly effective in patients with CML who failed IM therapy (Druker, 2008). The need for new strategies to treat IM-resistant CML has stimulated con- siderable efforts to develop compounds targeting key functional motifs distant from the ATP-binding pocket of Bcr-Abl protein or, alternatively, kinase downstream effectors. 14-3-3 are a highly conserved family of 28- to 33-kDa acidic proteins consisting of seven members (, , ε, , , , and ; This study was supported by the University of Bologna [ex60% funds]; Ministero della Pubblica Istruzione [PRIN], BolognaAIL, Fondazione Monte dei Paschi di Siena; Asinex; Department of Radiation Oncology [Postdoctoral Grant (to M.M.)]; and Department of Biological Sciences, University of Calgary (Calgary, AB, Canada) [Postdoctoral Grant (to V.C.)]. M.M. and V.C. contributed equally to this work. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.110.172536. □ S The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. ABBREVIATIONS: TK, tyrosine kinase; CML, chronic myeloid leukemia; IM, imatinib mesylate; BV02, 2-(1,5-dimethyl-3-oxo-2-phenyl-2,3- dihydro-1H-pyrazol-4-ylcarbamoyl)terephthalic acid; wt, wild type; JNK, c-Jun NH 2 terminal kinase; MD, molecular dynamics; GA, genetic algorithms; MIFs, molecular interactions fields; PI, propidium iodide; IP, immunoprecipitation; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]- 1-propanesulfonic acid; FLICA, fluorochrome-bound inhibitor of caspase. 0022-3565/11/3363-596–604$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 336, No. 3 Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics 172536/3658113 JPET 336:596–604, 2011 Printed in U.S.A. 596 http://jpet.aspetjournals.org/content/suppl/2010/11/01/jpet.110.172536.DC1.html Supplemental material to this article can be found at: at ASPET Journals on May 5, 2016 jpet.aspetjournals.org Downloaded from