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The anti-myeloma activity of a novel purine scaffold HSP 90 inhibitor PUH 71 is via inhibition of both HSP 90 A and HSP 90 B 1

Background: Heat shock protein 90 (HSP90) inhibitors have emerged as a promising class of anti-cancer drugs in both solid and hematologic malignancies. The HSP90 family includes the cytosolic HSP90 (HSP90AA1), the ER paralogue gp96 (HSP90B1) and the mitochondrial member TRAP1 (HSP90L). We evaluated the in vitro anti-tumor activity and mechanism of action of PU-H71, a novel purine scaffold HSP90 inhibitor in human multiple myeloma cell lines. Methods: Multiple human myeloma cell lines including cells that are resistant to corticosteroids and bortezimab were treated with PU-H71, followed by analysis of cell viability, cell cycle progression and apoptosis, by flow cytometry and caspase 3 immunoblot. Induction of unfolded protein response was studied by XBP-1 s immunoblot. The role of gp96 was further assessed by small hairpin RNA knockdown of gp96 before treatment with PU-H71. Results: PU-H71 has potent in vitro anti-myeloma activity in both drug-sensitive and drug-resistant cell lines. PU-H71 activates the unfolded protein response and induces caspase-dependent apoptosis. The stable gp96 knockdown human myeloma cell line was found to be more resistant to PU-H71 and other HSP90 inhibitors including 17-AAG and 17-DMAG, even though these cells are more sensitive to conventional anti-myeloma drugs. Conclusion: We conclude that PU-H71 is a promising drug for the treatment of myeloma. Our finding further suggests that PU-H71 and the geldanamycin analogues work in part by inhibiting the endoplasmic reticulum gp96 along with the cytosolic HSP90. Introduction Multiple myeloma is a clonal plasma cell malignancy accompanied by characteristic bone lesions, cytopenias, renal insufficiency and immune deficiency. The last decade has witnessed significant advances in anti-myeloma therapy with median survival extending from 2-3 years to over 7 years for patients younger than 50 years [1]. Heat shock protein 90 (HSP90) inhibitors are an emerging class of targeted agents in cancer therapy. HSP90 inhibition would make intuitive sense as anti-tumor therapy in cell types that depend on sustained protein homeostasis for their survival. A number of HSP90 inhibitors have demonstrated anti-myeloma activity in preclinical studies and at least three compounds have been evaluated in Phase I trials for relapsed/refractory myeloma [2-5]. PU-H71 is a novel purine scaffold HSP90 inhibitor that has shown pre-clinical activity in triple negative breast cancer [6], Bcl6 dependent lymphoma [7], hepatocellular carcinoma [8] and myeloproliferative disorders [9]. HSP90 family of proteins are ubiquitous molecular chaperones that are involved in folding, activation, maturation and assembly of many proteins (referred to as HSP90 client proteins or HSP90 clientele) that include essential mediators of signal transduction and cell cycle progression [10]. The mammalian HSP90 family members include the cytosolic HSP90, the HSP90 paralogue gp96 (also known as grp94, endoplasmin, HSP90B1) in the endoplasmic reticulum (ER) and the mitochondrial * Correspondence: susmani@uams.edu; zihai@musc.edu Myeloma Institute for Research & Therapy, University of Arkansas for Medical Sciences, Little Rock, AR, USA Department of Immunobiology & Cancer Immunology, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC, USA Full list of author information is available at the end of the article Usmani et al. Journal of Hematology & Oncology 2010, 3:40 http://www.jhoonline.org/content/3/1/40 JOURNAL OF HEMATOLOGY & ONCOLOGY © 2010 Usmani et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. protein TRAP1. There have been recent significant progresses in the understanding of both the structure and function of gp96. It serves as an obligate master chaperone for multiple Toll-like receptors [11,12] and integrins [13,14], neither of which could function properly in the absence of gp96. More recently, gp96 has been observed to play a critical role in lymphopoeisis in that deletion of gp96 leads to a transitional block from pro-B to pre-B cells and the inability of thymocytes to develop beyond the CD4(-)CD8(-) stage [14]. gp96 also maintains the fidelity of the endoplasmic reticulum protein synthesis by mediating the unfolded protein response (UPR) [15]. It shares ~50% homology at the amino acid level with its cytosolic HSP90 paralogue, with a similar domain organization consisting of an N-terminal ATP-binding domain, a charged middle domain and a C-terminal homodimerization domain [16]. The unfolded protein response (UPR) is a highly conserved eukaryotic protein homeostasis mechanism that is especially important for secretory cell types (e.g., hepatocytes, plasma cells, etc.) [17]. In response to cellular stress, UPR leads to increased ER chaperones such as grp78, gp96 and calreticulin to deal with the increased load of unfolded and nascent proteins in the ER. In response to sustained cellular stress, the UPR activates the apoptotic pathway. It has been previously demonstrated that, a geldanamycin derived HSP90 inhibitor, can activate the unfolded protein response in myeloma cells [18]. Herein, we evaluated the in vitro anti-myeloma activity of PU-H71, a novel purine scaffold HSP90 inhibitor. We also determined if the anti-tumor activity of HSP90 inhibitors is achieved via targeting both cytosolic HSP90 and the endoplasmic reticulum HSP90 paralogue gp96. Materials and methods