Masazumi Nagai

FGFR3 translocations in bladder cancer: differential sensitivity to HSP90 inhibition based on drug metabolism.

Activating mutations and/or overexpression of FGFR3 are common in bladder cancer, making FGFR3 an attractive therapeutic target in this disease. In addition, FGFR3 gene rearrangements have recently been described that define a unique subset of bladder tumors. Here, a selective HSP90 inhibitor, ganetespib, induced loss of FGFR3-TACC3 fusion protein expression and depletion of multiple oncogenic signaling proteins in RT112 bladder cells, resulting in potent cytotoxicity comparable with the pan-FGFR tyrosine kinase inhibitor BGJ398. However, in contrast to BGJ398, ganetespib exerted pleiotropic effects on additional mitogenic and survival pathways and could overcome the FGFR inhibitor–resistant phenotype of FGFR3 mutant–expressing 97-7 and MHG-U3 cells. Combinatorial benefit was observed when ganetespib was used with BGJ398 both in vitro and in vivo. Interestingly, two additional FGFR3 fusion-positive lines (RT4 and SW480) retained sensitivity to HSP90 inhibitor treatment by the ansamycins 17-AAG and 17-DMAG yet displayed intrinsic resistance to ganetespib or AUY922, both second-generation resorcinol-based compounds. Both cell lines, compared with RT112, expressed considerably higher levels of endogenous UGT1A enzyme; this phenotype resulted in a rapid glucuronidation-dependent metabolism and subsequent efflux of ganetespib from SW780 cells, thus providing a mechanism to account for the lack of bioactivity. Implications: Pharmacologic blockade of the molecular chaperone HSP90 represents a promising approach for treating bladder tumors driven by oncogenic gene rearrangements of FGFR3. Furthermore, UDP-glucuronosyltransferase enzyme expression may serve as a predictive factor for clinical response to resorcinol-based HSP90 inhibitors. Mol Cancer Res; 12(7); 1042–54. ©2014 AACR.

The oncology drug elesclomol selectively transports copper to the mitochondria to induce oxidative stress in cancer cells.

Elesclomol is an investigational drug that exerts potent anticancer activity through the elevation of reactive oxygen species (ROS) levels and is currently under clinical evaluation as a novel anticancer therapeutic. Here we report the first description of selective mitochondrial ROS induction by elesclomol in cancer cells based on the unique physicochemical properties of the compound. Elesclomol preferentially chelates copper (Cu) outside of cells and enters as elesclomol-Cu(II). The elesclomol-Cu(II) complex then rapidly and selectively transports the copper to mitochondria. In this organelle Cu(II) is reduced to Cu(I), followed by subsequent ROS generation. Upon dissociation from the complex, elesclomol is effluxed from cells and repeats shuttling elesclomol-Cu complexes from the extracellular to the intracellular compartments, leading to continued copper accumulation within mitochondria. An optimal range of redox potentials exhibited by copper chelates of elesclomol and its analogs correlated with the elevation of mitochondrial Cu(I) levels and cytotoxic activity, suggesting that redox reduction of the copper triggers mitochondrial ROS induction. Importantly the mitochondrial selectivity exhibited by elesclomol is a distinct characteristic of the compound that is not shared by other chelators, including disulfiram. Together these findings highlight a unique mechanism of action with important implications for cancer therapy.

Preclinical activity profile and therapeutic efficacy of the Hsp90 inhibitor ganetespib in triple-negative breast cancer

Purpose: Treatment options for patients with triple-negative breast cancer (TNBC) are largely limited to systemic chemotherapies, which have shown disappointing efficacy in the metastatic setting. Here, we undertook a comprehensive evaluation of the activity of ganetespib, a potent inhibitor of HSP90, in this malignancy. Experimental Design: The antitumor and antimetastatic activity of ganetespib was investigated using TNBC cell lines and xenograft models. Combinatorial drug analyses were performed with chemotherapeutic agents and concomitant effects on DNA damage and cell-cycle disruption were assessed in vitro; antitumor efficacy was assessed in vivo. Metabolic and objective tumor responses were evaluated in patients with metastatic TNBC undergoing ganetespib treatment. Results: Ganetespib simultaneously deactivated multiple oncogenic pathways to potently reduce cell viability in TNBC cell lines, and suppressed lung metastases in experimental models. Ganetespib potentiated the cytotoxic activity of doxorubicin via enhanced DNA damage and mitotic arrest, conferring superior efficacy to the doxorubicin–cyclophosphamide regimen in TNBC xenografts. Ganetespib also promoted mitotic catastrophe and apoptosis in combination with taxanes in vitro, and these effects translated to significantly improved combinatorial activity in vivo. Marked tumor shrinkage of metastatic lung and lymphatic lesions were seen in patients on ganetespib monotherapy. Conclusion: The preclinical activity profile and clinical evidence of tumor regression suggest that ganetespib offers considerable promise as a new therapeutic candidate to target TNBC. Clin Cancer Res; 20(2); 413–24. ©2013 AACR.

Novel indolizine compounds as potent inhibitors of phosphodiesterase IV (PDE4): structure–activity relationship

A series of novel indolizine 2-oxoacetamides were designed and synthesized as PDE4 inhibitors. Preliminary SAR of this new class of compounds revealed key structural features required for high potency. Compounds 1ab and 2a are among the most potent inhibitors of PDE4 with low single nM IC50. Cellular activity was demonstrated by the inhibition of TNFα production from human PBMC with IC50 ranging from 14 to 72 nM. Docking analyses suggest the OH group in 1ab enhance the binding via an H-bond interaction with the PDE4 enzyme.

Establishment of Epithelial Attachment on Titanium Surface Coated with Platelet Activating Peptide.

The aim of this study was to produce epithelial attachment on a typical implant abutment surface of smooth titanium. A challenging complication that hinders the success of dental implants is peri-implantitis. A common cause of peri-implantitis may results from the lack of epithelial sealing at the peri-implant collar. Histologically, epithelial sealing is recognized as the attachment of the basement membrane (BM). BM-attachment is promoted by activated platelet aggregates at surgical wound sites. On the other hand, platelets did not aggregate on smooth titanium, the surface typical of the implant abutment. We then hypothesized that epithelial BM-attachment was produced when titanium surface was modified to allow platelet aggregation. Titanium surfaces were coated with a protease activated receptor 4-activating peptide (PAR4-AP). PAR4-AP coating yielded rapid aggregation of platelets on the titanium surface. Platelet aggregates released robust amount of epithelial chemoattractants (IGF-I, TGF-β) and growth factors (EGF, VEGF) on the titanium surface. Human gingival epithelial cells, when they were co-cultured on the platelet aggregates, successfully attached to the PAR4-AP coated titanium surface with spread laminin5 positive BM and consecutive staining of the epithelial tight junction component ZO1, indicating the formation of complete epithelial sheet. These in-vitro results indicate the establishment of epithelial BM-attachment to the titanium surface.

Abstract C11: The oxidative stress inducer elesclomol requires copper chelation for its anticancer activity

Introduction: Elesclomol [N‐malonyl‐bis (N′‐methyl‐N′‐thiobenzoyl hydrazide)] is a first‐in‐class investigational drug, believed to exert anticancer activity through the elevation of reactive oxygen species (ROS) levels leading to the activation of the mitochondrial apoptosis pathway. The mechanism of ROS induction by elesclomol was previously unknown. Data presented here shows that ROS is generated via chelation and redox cycling of copper. Methods: Formation of a Cu chelate of elesclomol was analyzed by LCMS and single crystal X‐ray diffraction. ROS was measured using DCF‐DA and cytotoxicity assessed using a WST‐8 assay primarily in Ramos human B cell lymphoma and M14 human melanoma cell lines. Cellular levels of free elesclomol and elesclomol‐Cu were determined by LCMS. Redox potential was analyzed by cyclic voltammetry. Results: Elesclomol readily formed a Cu chelate and strongly preferred Cu over zinc, iron or manganese in competition assays. Cu bound elesclomol in a 1:1 molar ratio and the chelate formed a flat rigid structure. The donation of four lone‐pair electrons from elesclomol to Cu(II) yields higher hydrophobicity, which may facilitate greater cell permeability relative to free elesclomol. We evaluated the effect of Cu on both cellular uptake and cytotoxic activity of elesclomol on cultured cancer cells. Elesclomol lost cytotoxicity when applied to cells under Cu‐starved conditions. The presence of Cu was required for elesclomol entry into cells. The cell membrane impermeable Cu chelator BCP blocked both uptake of elesclomol and cytotoxicity, indicating that elesclomol obtains Cu outside the cell and requires it for cellular entry and cytotoxicity. Elesclomol was able to obtain Cu from serum as well as from purified ceruloplasmin, the primary Cu‐binding protein in blood. Elesclomol had poor activity against densely‐plated cancer cells in culture, but became highly potent when extra Cu was added to the medium, indicating that Cu may be limiting under dense culture conditions. We next evaluated the impact of Cu on ROS generation. A cellfree assay system showed that elesclomol‐Cu(II) was capable of efficient generation of ROS via the reduction of Cu(II) to Cu(I). A correlation was observed between redox potential and ROS production for Cu chelates of elesclomol and its analogs. Elesclomol also chelated nickel. However, while elesclomol‐Ni was highly cell permeable, it was inactive for ROS production and cancer cell cytotoxicity. The lack of activity of elesclomol‐Ni can be explained by its low redox potential. The redox potential of elesclomol‐Cu(II) was −333mV, while that of elesclomol‐Ni was −1100mV. Conclusion: Elesclomol chelates Cu outside of cells and enters cells as elesclomol‐Cu(II). At present, our data support the hypothesis that elesclomol generates ROS by redox cycling of Cu(II) to Cu(I), and that this process is necessary for its anticancer activity. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C11.

Abstract 4545: Anticancer activity of elesclomol correlates with low LDH levels and active mitochondrial respiration

Elesclomol is a first-in-class investigational drug that exerts anticancer activity through the elevation of reactive oxygen species (ROS). Our previous studies revealed that elesclomol selectively chelates copper and generates ROS via reduction of Cu(II) to Cu(I). In a Phase 3 trial in metastatic melanoma, the level of baseline lactate dehydrogenase (LDH) in patients emerged as an important prognostic factor for treatment outcomes with elesclomol. Additional investigations were undertaken to evaluate the relation between elesclomol activity and LDH levels. Because the copper redox reactions necessary for elesclomol activity can be influenced by changes in cellular metabolic properties, we investigated whether elesclomol activity varies between normoxic conditions (active mitochondrial respiration, low LDH) and hypoxic conditions (decreased mitochondrial respiration, high LDH). To assess the influence of hypoxic conditions, we evaluated elesclomol activity under several situations in which cells express high levels of Hypoxia Inducible Factor-1α (HIF1α), a transcription factor that induces a complete set of glycolytic enzymes, including LDH, and switches off glycolytic carbon flow to mitochondria. First, an increase in the level of HIF1α protein was observed in M14 melanoma cells at low oxygen level or high cell density. Elesclomol showed decreased cytotoxicity under both of these conditions. Second, we evaluated lines within the same cancer phenotype but showing distinct levels of HIF1α, and found that the high-HIF1α-expressing Caki-2 renal cancer line was resistant to elesclomol, while lower-HIF1α-expressing renal cancer cell lines were sensitive to elesclomol. Third, we assessed HIF1α levels and elesclomol activity in cells treated with CoCl 2 , a chemical mimetic of hypoxia. High levels of HIF1α induction were present in M14 cells treated with CoCl 2 , and elesclomol was significantly less active in these cells. The addition of oxamate, which selectively inhibits LDHA and activates pyruvate entry into mitochondria, restored the activity of elesclomol in the CoCl 2 -treated cells. These results support the hypothesis that elesclomol is more active under normoxic conditions (low LDH) and less active under hypoxic conditions (high LDH), consistent with the clinical findings. The results may be important for identifying patient populations for future clinical development of elesclomol. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4545.

Function of Platelet-Induced Epithelial Attachment at Titanium Surfaces Inhibits Microbial Colonization:

The aim of this study was to evaluate the barrier function of platelet-induced epithelial sheets on titanium surfaces. The lack of functional peri-implant epithelial sealing with basal lamina (BL) attachment at the interface of the implant and the adjacent epithelium allows for bacterial invasion, which may lead to peri-implantitis. Although various approaches have been reported to combat bacterial infection by surface modifications to titanium, none of these have been successful in a clinical application. In our previous study, surface modification with protease-activated receptor 4–activating peptide (PAR4-AP), which induced platelet activation and aggregation, was successful in demonstrating epithelial attachment via BL and epithelial sheet formation on the titanium surface. We hypothesized that the platelet-induced epithelial sheet on PAR4-AP–modified titanium surfaces would reduce bacterial attachment, penetration, and invasion. Titanium surface was modified with PAR4-AP and incubated with platelet-rich plasma (PRP). The aggregated platelets released collagen IV, a critical BL component, onto the PAR4-AP–modified titanium surface. Then, human gingival epithelial cells were seeded on the modified titanium surface and formed epithelial sheets. Green fluorescent protein (GFP)–expressing Escherichia coli was cultured onto PAR4-AP–modified titanium with and without epithelial sheet formation. While Escherichia coli accumulated densely onto the PAR4-AP titanium lacking epithelial sheet, few Escherichia coli were observed on the epithelial sheet on the PAR4-AP surface. No bacterial invasion into the interface of the epithelial sheet and the titanium surface was observed. These in vitro results indicate the efficacy of a platelet-induced epithelial barrier that functions to prevent bacterial attachment, penetration, and invasion on PAR4-AP–modified titanium.

Structure-activity relationship study and discovery of indazole 3-carboxamides as calcium-release activated calcium channel blockers

Aberrant activation of mast cells contributes to the development of numerous diseases including cancer, autoimmune disorders, as well as diabetes and its complications. The influx of extracellular calcium via the highly calcium selective calcium-release activated calcium (CRAC) channel controls mast cell functions. Intracellular calcium homeostasis in mast cells can be maintained via the modulation of the CRAC channel, representing a critical point for therapeutic interventions. We describe the structure-activity relationship study (SAR) of indazole-3-carboxamides as potent CRAC channel blockers and their ability to stabilize mast cells. Our SAR results show that the unique regiochemistry of the amide linker is critical for the inhibition of calcium influx, the release of the pro-inflammatory mediators β-hexosaminidase and tumor necrosis factor α by activated mast cells. Thus, the indazole-3-carboxamide 12d actively inhibits calcium influx and stabilizes mast cells with sub-μM IC50. In contrast, its reverse amide isomer 9c is inactive in the calcium influx assay even at 100μM concentration. This requirement of the specific 3-carboxamide regiochemistry in indazoles is unprecedented in known CRAC channel blockers. The new structural scaffolds described in this report expand the structural diversity of the CRAC channel blockers and may lead to the discovery of novel immune modulators for the treatment of human diseases.

Identification of putative bone anabolic peptides targeting adherent plasma membrane

Bone matrix provides unknown essential cues for osteoblast lineage cells to develop, grow, repair and remodel bones via adherent plasma membrane. Because of its tight sealing with bone matrix in vivo and culture surface in vitro as well, the adherent plasma membrane has been unveiled target of investigation to date. Herein, we report a new approach to explore the adherence plasma membrane of osteoblasts with biofunctional peptide candidates in a bacterial peptide library. To accomplish this, human osteoblast like hFOB 1.19 cells were cultured on porous filter with 8 μm pore through which bacterial peptides were allowed to meet the membrane for affinity selection. The affinity-selected peptides were coated on culture plate to further evaluate their influence on osteoblastic cell adhesion, as well as expressions of osteoblast differentiation markers, alkaline phosphatase and osteocalcin. Finally, the serial screenings identified two prominent active peptides that enhanced the differentiation markers nearly to the same level as a control peptide of bone morphogenetic protein-2. Osteogenic activity is expected for the peptides when immobilized on bone implant surface.