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Therapeutic Potential of HSP90 Inhibition for Neurofibromatosis Type 2

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Therapeutic Potential of HSP90 Inhibition for Neurofibromatosis Type 2 Published OnlineFirst May 28, 2013; DOI: 10.1158/1078-0432.CCR-12-3167 Clinical Cancer Cancer Therapy: Preclinical Research Therapeutic Potential of HSP90 Inhibition for Neurofibromatosis Type 2 Karo Tanaka1, Ascia Eskin3, Fabrice Chareyre1, Walter J. Jessen6, Jan Manent7, Michiko Niwa-Kawakita8, Ruihong Chen5, Cory H. White2, Jeremie Vitte1, Zahara M. Jaffer1, Stanley F. Nelson3, Allan E. Rubenstein9, and Marco Giovannini1,4 Abstract Purpose: The growth and survival of neurofibromatosis type 2 (NF2)–deficient cells are enhanced by the activation of multiple signaling pathways including ErbBs/IGF-1R/Met, PI3K/Akt, and Ras/Raf/Mek/Erk1/2. The chaperone protein HSP90 is essential for the stabilization of these signaling molecules. The aim of the study was to characterize the effect of HSP90 inhibition in various NF2-deficient models. Experimental Design: We tested efficacy of the small-molecule NXD30001, which has been shown to be a potent HSP90 inhibitor. The antiproliferative activity of NXD30001 was tested in NF2-deficient cell lines and in human primary schwannoma and meningioma cultures in vitro. The antitumor efficacy of HSP90 inhibition in vivo was verified in two allograft models and in one NF2 transgenic model. The underlying molecular alteration was further characterized by a global transcriptome approach. Results: NXD30001 induced degradation of client proteins in and suppressed proliferation of NF2- deficient cells. Differential expression analysis identified subsets of genes implicated in cell proliferation, cell survival, vascularization, and Schwann cell differentiation whose expression was altered by NXD30001 treatment. The results showed that NXD30001 in NF2-deficient schwannoma suppressed multiple path- ways necessary for tumorigenesis. Conclusions: HSP90 inhibition showing significant antitumor activity against NF2-related tumor cells in vitro and in vivo represents a promising option for novel NF2 therapies. Clin Cancer Res; 1–15. Ó2013 AACR. Introduction of a functional neurofibromatosis type 2 (NF2) protein, Vestibular schwannomas account for approximately 5% merlin/schwannomin (4, 5). Although sporadic vestibular to 10% of all tumors inside the skull (1); about 1 out of schwannomas occur in later stages of life, the hallmark of every 100,000 individuals per year develops a vestibular familial NF2 is the development of early-onset, bilateral schwannomas (2). Conventional treatment of this benign vestibular schwannomas, often associated with other cra- tumor includes surgical removal and radiotherapy but to nial and spinal nerves schwannomas, meningiomas, and date no validated chemotherapy is available due to poor ependymomas that frequently requires repeated invasive response to tested interventions (3). Both sporadic and surgeries. Hence, less invasive chemotherapy will be highly familial forms of vestibular schwannomas lack expression beneficial to the patients with NF2 for tumor control and prevention. Merlin is a unique member of the ezrin–radixin–moesin gene family with tumor-suppressing activities (6). Its over- Authors' Affiliations: 1Center for Neural Tumor Research and 2Section on Genetics of Hereditary Ear Disorders, House Research Institute; 3Depart- expression was shown to decrease cell growth concomitant- ment of Human Genetics, University of California, Los Angeles; 4Department ly with cell-cycle arrest and apoptosis (7, 8), whereas of Cell and Neurobiology, University of Southern California, Keck School of 5 its depletion by antisense oligonucleotides resulted in Medicine, Los Angeles; NexGenix Pharmaceuticals, Burlingame, California; 6Informatics, Covance Inc., Princeton, New Jersey; 7Peter MacCallum Can- increased cell proliferation (9). Consistently, mice hetero- cer Institute, Melbourne, Australia; 8Inserm U944, CNRS U7212, Universite zygous for Nf2 inactivation (10), homozygous for tissue- Paris Diderot-Paris 7, Institut Universitaire d'Hematologie, Paris, France; and 9New York University Langone Medical Center, New York, New York specific Nf2 inactivation (11), and transgenic for expression of a human dominant-negative NF2 mutant (12) developed Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). a range of tumors. Merlin is involved in cellular function by providing the link between the actin cytoskeleton and Corresponding Author: Marco Giovannini, House Research Institute, Center for Neural Tumor Research, 2100 West 3rd Street, Los Angeles, multiple membrane-associated proteins, which are essen- CA 90057. Phone: 213-989-6708; Fax: 213-989-6778; E-mail: tial for processing extracellular signals, cell adhesion, and [email protected] cytoskeletal architecture (reviewed in refs. 13, 14). Various doi: 10.1158/1078-0432.CCR-12-3167 biologic pathways were suggested for merlin’s involvement Ó2013 American Association for Cancer Research. in cell proliferation control, including negatively regulation www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst May 28, 2013; DOI: 10.1158/1078-0432.CCR-12-3167 Tanaka et al. The novel small-molecule inhibitor series pochonin and Translational Relevance pochoximes, developed on the radicicol scaffold, were For sporadic neurofibromatosis type 2 (NF2)–deficient shown to be highly potent and specific inhibitors of HSP90 vestibular schwannoma, surgical removal is both an with antiproliferative activity (26, 27). One of the lead effective and sufficient intervention in most cases. How- compounds NXD30001 (pochoximeA) penetrated the ever, patients with NF2 have high risks of developing blood–brain and blood–spinal cord barriers and accumu- cranial, spinal, and peripheral nerve schwannoma and lated in nervous tissues (28), a pre-requisite for treating meningioma at a younger age, the majority of whom tumors in the central/peripheral nervous system including present with hearing loss and deafness. Moreover, a some NF2-related tumors, such as ependymomas. To fur- significant proportion of meningiomas is inoperable. ther characterize the underlying molecular mechanisms of Therefore, development of effective chemotherapy action, we used the next-generation sequencing (NGS) would be most beneficial for overall NF2 tumor preven- approach for assessing the global abundance of transcripts. tion. HSP90 inhibitors have acquired significant impor- Treatment of NF2-deficient cells with NXD30001 in vitro tance in the field of cancer chemotherapy in recent years. resulted in the depletion of multiple signaling molecules In particular, the response of ERBB2 (HER2)-positive implicated in NF2. Concomitantly, in vivo administration of breast cancer has offered promise for the use of HSP90 NXD30001 reduced growth of NF2-deficient tumors, and inhibitors as a novel therapeutic alternative, a strategy further gene expression analysis identified multiple biologic that is currently in clinical trials. Given their partial pathways that may contribute the efficacy of HSP90 inhi- dependence on ErbB signaling, NF2-related tumors are bition against NF2-related tumorigenesis. readily amenable with this strategy. Materials and Methods Reagents The structure of NXD30001 and its formulation have of Rac pathway necessary for Ras transformation (15), and been described elsewhere (27). Details are given in Supple- contact inhibition of growth through interaction with mentary Materials and Methods. CD44 (16). Recently, merlin’s translocation into the nucle- us was shown to suppress tumorigenesis by inhibiting the Cell lines and primary cultures nuclear E3 ubiquitin ligase CRL4 (DCAF1) implicated in À À Mouse Nf2 / embryonic Schwann cells, ESF-FC1801, DNA replication and cell-cycle progression (17). and mouse schwannoma cells expressing the human Sch- Multiple links between merlin and its interacting proteins D(39–121) mutant, 08031-9, were derived in our labo- suggested the activation of various signaling pathways in ratory. An immortalized human schwannoma cells NF2-related tumors, which present a challenge for devel- HEI193 was a gift from Dr. Lim (House Research Institute, oping targeted therapeutics for NF2. HSP90 is a ubiquitous Los Angeles, CA). The human schwannoma and menin- molecular chaperone that is responsible for maintaining a gioma and the mouse Schwann cell (mSC) primary cul- subset of proteins involved in cell proliferation and trans- tures were freshly prepared and used before exceeding formation (18). HSP90 inhibition induces proteasomal 5 passages. Details are given in Supplementary Materials degradation of its client proteins, providing an attractive and Methods. therapeutic strategy that can simultaneously suppress mul- tiple signaling pathways. HSP90 is often found overex- pressed in malignant tumors, and its elevated level was Cell viability and proliferation assay shown to correlate with poor survival among patients (19). MTS-based monolayer cell proliferation assay (Promega) A study showed that the HSP90 complexes in tumor cells and soft agar clonogenic assay were used to test cell viability possess greater affinity to an HSP90 inhibitor 17-AAG, and proliferation at 72 hours and at 3 weeks of drug thereby promising selectivity for targeting tumor cells over administration in vitro, respectively. The drug concentration normal cells (20). There is single report suggesting the that inhibits cell growth by 50% (GI50) was determined efficacy of HSP90 inhibition in NF2 (21). Many known using Prism 5.0 (GraphPad
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