Supplemental Table S2 from Activity of PXD101, a histone deacetylase inhibitor, in preclinical ovarian cancer studies

Background Preclinical data show that belinostat (Bel) is synergistic with carboplatin and paclitaxel in ovarian cancer. To further evaluate the clinical activity of belinostat, carboplatin, and paclitaxel (BelCaP), a phase 1b/2 study was performed, with an exploratory phase 2 expansion planned specifically for women with recurrent epithelial ovarian cancer (EOC). Methods Thirty-five women were treated on the phase 2 expansion cohort. BelCap was given as follows: belinostat, 1000 mg/m 2 daily for 5 days with carboplatin, AUC 5; and paclitaxel, 175 mg/m 2 given on day 3 of a 21-day cycle. The primary end point was overall response rate (ORR), using a Simon 2 stage design. Results The median age was 60 years (range, 39–80 years), and patients had received a median of 3 prior regimens (range, 1–4). Fifty-four percent had received more than two prior platinum-based combinations, sixteen patients (46%) had primary platinum-resistant disease, whereas 19 patients (54%) recurred within 6 months of their most recent platinum treatment. The median number of cycles of BelCaP administered was 6 (range, 1–23). Three patients had a complete response, and 12 had a partial response, for an ORR of 43% (95% confidence interval, 26%–61%). When stratified by primary platinum status, the ORR was 44% among resistant patients and 63% among sensitive patients. The most common drug-related adverse events related to BelCaP were nausea (83%), fatigue (74%), vomiting (63%), alopecia (57%), and diarrhea (37%). With a median follow-up of 4 months (range, 0–23.3 months), 6-month progression-free survival is 48% (95% confidence interval, 31%–66%). Median overall survival was not reached during study follow-up. Conclusions Belinostat, carboplatin, and paclitaxel combined was reasonably well tolerated and demonstrated clinical benefit in heavily-pretreated patients with EOC. The addition of belinostat to this platinum-based regimen represents a novel approach to EOC therapy and warrants further exploration.

Histone deacetylase inhibitors (HDACi) represent a promising new class of anticancer agents. In the current investigation, we examined the activity of the HDACi belinostat in preclinical models of prostate cancer. In vitro proliferation assays demonstrated that belinostat potently inhibited the growth of prostate cancer cell lines (IC(50) < 1.0 microM) and was cytotoxic to these cells. Washout experiments indicated that exposure to belinostat for relatively short periods of time (<12 hr) induced suboptimal growth-inhibition and that cells exposed to 1.0 microM belinostat for 48 hr retained the capacity for regrowth following drug withdrawal, while cells exposed to 4.0 microM belinostat were irreversibly growth-inhibited. Cell cycle analyses demonstrated that belinostat induced G2/M arrest and increased the percentage of cells with subG1 DNA content, thus confirming the growth-inhibitory and cytotoxic effects of this compound. Normal prostate epithelial cells were generally less susceptible to the effects of belinostat than were prostate cancer cells. In an orthotopic prostate cancer tumor model, belinostat inhibited tumor growth by up to 43%. Moreover, metastatic lung lesions were present in 47% of vehicle-treated animals but in none of the animals administered belinostat. Consistent with its observed antimetastatic activity, belinostat inhibited the migration of prostate tumor cells and increased the production of tissue inhibitor of metalloproteinase-1 (TIMP-1) by these cells, the latter effect being replicated by siRNA knockdown of HDAC3. Belinostat also increased the expression of p21 and decreased the expression of potentially oncogenic proteins (mutant p53 and ERG). These results support the clinical evaluation of belinostat for the treatment of prostate cancer.

PURPOSE:Histone deacetylases (HDAC) are involved in the regulation of gene transcription. Aberrant HDAC activity has been associated with tumorigenesis, and, therefore, HDACs are potential targets for the treatment of cancers, including tumors of the central nervous system (CNS). Belinostat is a novel, potent, pan-HDAC inhibitor with antiproliferative activity on a wide variety of tumor cell lines. We studied the cerebrospinal fluid (CSF) penetration of intravenous (IV) belinostat in a non-human primate model as a surrogate for blood:brain barrier penetration.DESIGN:Five adult rhesus monkeys received increasing doses of belinostat (10-60 mg/kg) as a 30-min IV infusion. Serial blood and CSF samples were collected over 48 h. Plasma and CSF concentrations of belinostat were quantified with an LC/MS/MS assay. Pharmacokinetic parameters were calculated using non-compartmental methods, and CSF penetration is expressed as the ratio of the area under the concentration-time curve (AUC) in CSF to the AUC in plasma.RESULTS:Belinostat was cleared rapidly from plasma with a half-life of 1.0 h, a mean residence time of 0.47 h, and a clearance of 425 ml/min/m(2). CSF penetration of belinostat was limited. CSF drug exposure was <1% of plasma drug exposure and <10% of free (non-protein bound) plasma drug exposure.CONCLUSION:IV belinostat is rapidly cleared from plasma and has limited penetration into the CSF.

To investigate the pharmacological properties of the CR011-vcMMAE fully human antibody–drug conjugate (ADC), such as dose titrations, quantitation of the time (days) to complete regression, pharmacokinetics, and schedule dependency. Our prior study characterized a fully human antibody to GPNMB covalently linked to monomethylauristatin E, CR011-vcMMAE, and further demonstrated cell surface staining of melanoma lines susceptible to the immunoconjugate’s cytotoxicity (Clin Cancer Res 2005; 12(4): 1373–1382).

The human HDAC (histone deacetylase) family, a well-validated anticancer target, plays a key role in the control of gene expression through regulation of transcription. While HDACs can be subdivided into three main classes, the class I, class II and class III HDACs (sirtuins), it is presently unclear whether inhibiting multiple HDACs using pan-HDAC inhibitors, or targeting specific isoforms that show aberrant levels in tumours, will prove more effective as an anticancer strategy in the clinic. To address the above issues, we have tested a number of clinically relevant HDACis (HDAC inhibitors) against a panel of rhHDAC (recombinant human HDAC) isoforms. Eight rhHDACs were expressed using a baculoviral system, and a Fluor de Lystrade mark (Biomol International) HDAC assay was optimized for each purified isoform. The potency and selectivity of ten HDACs on class I isoforms (rhHDAC1, rhHDAC2, rhHDAC3 and rhHDAC8) and class II HDAC isoforms (rhHDAC4, rhHDAC6, rhHDAC7 and rhHDAC9) was determined. MS-275 was HDAC1-selective, MGCD0103 was HDAC1- and HDAC2-selective, apicidin was HDAC2- and HDAC3-selective and valproic acid was a specific inhibitor of class I HDACs. The hydroxamic acid-derived compounds (trichostatin A, NVP-LAQ824, panobinostat, ITF2357, vorinostat and belinostat) were potent pan-HDAC inhibitors. The growth-inhibitory effect of the HDACis on HeLa cells showed that both pan-HDAC and class-I-specific inhibitors inhibited cell growth. The results also showed that both pan-HDAC and class-I-specific inhibitor treatment resulted in increased acetylation of histones, but only pan-HDAC inhibitor treatment resulted in increased tubulin acetylation, which is in agreement with their activity towards the HDAC6 isoform.

Platelet-derived growth factor (PDGF)-BB and PDGF-DD mediate mesangial cell proliferation in vitro and in vivo. While PDGF-BB is a ligand for the PDGF alpha- and beta-receptor chains, PDGF-DD binds more selectively to the beta-chain, suggesting potential differences in the biological activities. Signal transduction and regulation of gene expression induced by PDGF-BB and -DD were compared in primary human mesangial cells (HMCs), which expressed PDGF alpha- and beta-receptor subunits. The growth factor concentrations used were chosen based on their equipotency in inducing HMCs proliferation and binding to the betabeta-receptor. Both growth factors, albeit at different concentrations induced phosphorylation and activation of extracellular signal-regulated kinase 1 (ERK1) and ERK2. In addition, PDGFs led to the phosphorylation and activation of signal transducers and activators of transcription 1 (STAT1) and STAT3. HMCs proliferation induced by either PDGF-BB or -DD could be blocked by signal transduction inhibitors of the mitogen-activated protein kinase-, Janus kinase (JAK)/STAT-, or phosphatidyl-inositol 3-kinase pathways. Using a gene chip array and subsequent verification by real-time reverse transcriptase (RT)-polymerase chain reaction, we found that in HMC genes for matrix metalloproteinase 13 (MMP-13) and MMP-14 and, to a low extent, cytochrome B5 and cathepsin L were exclusively regulated by PDGF-BB, whereas no exclusive gene regulation was detected by PDGF-DD. However, at the protein level, both MMP-13 and -14 were equally induced by PDGF-BB and -DD. PDGF-BB and -DD effect similar biological responses in HMCs albeit at different potencies. Rare apparently differential gene regulation did not result in different protein expression, suggesting that in HMCs both PDGFs exert their biological activity almost exclusively via the PDGF beta-receptor.

Purpose: Advanced melanoma is a highly drug-refractory neoplasm representing a significant unmet medical need.We sought to identify melanoma-associated cell surface molecules and to develop aswell as preclinically test immunotherapeutic reagents designed to exploit such targets. Experimental Design and Results: By transcript profiling, we identified glycoprotein NMB (GPNMB) as a gene that is expressed by most metastatic melanoma samples examined. GPNMB is predicted to be a transmembrane protein, thus making it a potential immunotherapeutic target in the treatment of this disease. A fully human monoclonal antibody, designated CR011, was generated to the extracellular domain of GPNMB and characterized for growth-inhibitory activity against melanoma.The CR011monoclonal antibody showed surface staining of most melanoma cell lines by flow cytometry and reacted with a majority of metastatic melanoma specimens by immunohistochemistry. CR011alone didnot inhibit the growthofmelanoma cells. However,when linked to the cytotoxic agent monomethylauristatin E (MMAE) to generate the CR011-vcMMAE antibody-drug conjugate, this reagent now potently and specifically inhibited the growth of GPNMB-positivemelanoma cells in vitro. Ectopic overexpression and small interfering RNAtransfection studies showed that GPNMBexpression is both necessary and sufficient for sensitivity to low concentrations of CR011-vcMMAE. In a melanoma xenograft model, CR011-vcMMAE induced significant dose-proportional antitumor effects, including complete regressions, at doses as low as1.25 mg/kg. Conclusion:These preclinical results support the continued evaluation of CR011-vcMMAE for the treatment of melanoma. Melanoma is a common neoplasm and its incidence is increasing worldwide at a dramatic rate (1). Melanoma accounts for only 4% of skin cancer cases yet causes f79% of all skin cancer deaths. In 2004, an estimated 55,100 Americans were diagnosed with melanoma and f7,910 would die of the disease (2). An increasing frequency of newly diagnosed melanomas, ranging from 3% to 8% annually, has also been observed worldwide (3, 4). Therapeutic options for patients with late-stage melanoma presenting with regional and/or distant metastases are limited. Dacarbazine is the only cytotoxic drug currently approved by the Food and Drug Administration for the treatment of stage IV metastatic melanoma, with a response rate of <15% and a median response duration of 4 to 5 months (5). The majority of polychemotherapy regimens failed to show significant survival benefits (6), nor did the use of adjuvant therapeutic agents such as IFN-a and interleukin 2, which pose severe toxicity (7, 8). The poor efficacy and adverse side effects of available therapies has led to a considerable interest in the development of alternative therapies, such as monoclonal antibodies (mAb), for the treatment of metastatic melanoma (9). Recent advances in genetic engineering have significantly decreased antibody immunogenicity and increased antibody half-life (10, 11). Antibody-based therapeutics, such as Rituxan, Herceptin, and Avastin, have recently enjoyed clinical success in the treatment of some hematopoietic malignancies and solid tumors. Although antibodies that target tumor or its vasculature may be useful in an unconjugated form, it is sometimes advantageous to couple a tumor-targeting antibody to an isotope (e.g., Zevalin and Bexxar) or to a cytotoxic compound (e.g., Mylotarg). This strategy allows for the selective delivery of cytotoxic agents to the tumor with the goal of reducing the toxicity that is often associated with the systemic administration of cytotoxic agents while preserving or enhancing the antitumor activity of these agents. Our genome-wide transcript expression profiling, coupled with a systems biology analysis of human melanoma clinical Cancer Therapy: Preclinical Authors’ Affiliations: CuraGen, Branford, Connecticut; Abgenix, Fremont, California; and Seattle Genetics, Bothell,Washington Received 9/16/05; revised12/9/05; accepted12/14/05. The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely to indicate this fact. Note: K.F.Tse andM. Jeffers contributed equally to this work. Requests for reprints:William J. LaRochelle, CuraGen Corporation, 322 East Main Street, Branford, CT 06405. Phone: 203-871-4288; Fax: 203-315-3301; E-mail: wlarochelle@curagen.com. F2006 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-05-2018 www.aacrjournals.org Clin Cancer Res 2006;12(4) February15, 2006 1373 Research. on May 29, 2017. © 2006 American Association for Cancer clincancerres.aacrjournals.org Downloaded from specimens and cell lines, led to the identification of a tumorassociated protein, called glycoprotein NMB (GPNMB), as a potential target that can be exploited for the treatment of melanoma. GPNMB is predicted to be a 560-amino-acid type I transmembrane protein with closest homology (26% amino acid identity) to the melanocyte/melanoma–specific protein, pMEL17 (12). The normal function of human GPNMB is unknown, and orthologues have been isolated from mouse (DC-HIL; ref. 13), rat (Osteoactivin; ref. 14), and quail (QNR-71; ref. 15). Previous investigations have associated GPNMB expression and function with cancer. GPNMB was first identified as a gene that was differentially expressed among melanoma cell lines with high and low metastatic potential (12) and was subsequently identified as a candidate glioma tumor marker due to its high transcript expression in this tumor type and restricted normal tissue distribution (16). GPNMB expression has also been described in liver cancer, squamous cell lung carcinoma, and soft tissue tumors (17–19). Moreover, ectopic expression of GPNMB in cancer cells increased their in vitro invasiveness and promoted their metastasis in vivo (17, 20). Finally, GPNMB was shown to interact with the surface of endothelial cells (13), a finding that may have implications for GPNMB-expressing melanoma cell transendothelial migration and metastasis. To explore the potential utility of GPNMB as a target for melanoma therapy, fully-human mAbs were generated to this protein. The lead mAb, CR011, was characterized and coupled to the dolastatin-10-related cytotoxic drug monomethylauristatin E (MMAE), a potent inhibitor of mitotic spindle formation (21). The resulting antibody-drug conjugate, designated CR011-vcMMAE, was evaluated for growth-inhibitory activity on melanoma cell lines in vitro and for activity against melanoma xenografts in vivo . The results presented in this study suggest that GPNMB represents a promising target for the identification and treatment of advanced melanoma and that CR011-vcMMAE is worthy of continued therapeutic evaluation. Materials andMethods Cell lines and transfections. M14, UACC-257, and LOXIMVI cell lines were obtained from the National Cancer Institute (Bethesda, MD) and all others from the American Type Culture Collection (Manassas, VA). Cells were maintained in DMEM or RPMI containing 10% fetal bovine serum and penicillin-streptomycin. To establish stable cell lines overexpressing GPNMB, HEK293 cells were transfected with either control vector (pcDNA3.1-V5-His) or this vector containing full-length GPNMB, using LipofectAMINE (Invitrogen, Carlsbad, CA) according to the protocol of the manufacturer. Following selection in medium containing G418 (0.8 mg/mL), individual clones were selected and propagated. Small interfering RNA (siRNA) was used to inhibit GPNMB expression in SK-Mel-2 cells. Cells were transfected with 50 nmol/L of siGENOME SMART pool reagents (Dharmacon, Inc., Chicago, IL), designed to specifically target GPNMB, or siRNA to thymidylate synthase as a negative control, using the OligofectAMINE transfection reagent (Invitrogen) following the instructions of the manufacturer. Reverse transcription-PCR and real-time quantitative PCR. Total RNA was isolated using the RNeasy kit with a DNase digestion step (Qiagen, Inc., Valencia CA). Reverse transcription-PCR (RT-PCR) was done using the OneStep RT-PCR kit (Qiagen) as follows. Reverse transcription: 50jC for 45 minutes and 95jC for 15 minutes for one cycle. PCR: 1 minute at 95jC, 1 minute at 50jC, and 2 minutes at 72jC for 30 cycles with final extension for 10 minutes at 72jC. Products were separated on a 2% agarose/0.33% low melting point agarose gel and visualized by ethidium bromide staining. The integrity of each RNA sample was verified via RT-PCR with primers designed to amplify glyceraldehyde-3-phosphate dehydrogenase. The primers used for amplification are as follows (5V-3V): Real-time quantitative PCR analysis was done with an ABI Prism 7700 Sequence Detection System using TaqMan reagents (PE Applied Biosystems, Foster City, CA). Equal quantities of normalized RNAs were used as a template in PCR reactions for 40 cycles with GPNMB-specific primers to obtain threshold cycle (CT) values. The primers used for amplification are as follows (5V-3V): Forward-TCAATGGAACCTTCAGCCTTA Reverse-GAAGGGGTGGGTTTTGAAG Probe-TET-CTCACTGTGAAAGCTGCAGCACCAG-TAMRA Production and purification of recombinant human GPNMB extracellular domain protein. Oligonucleotide primers were designed to amplify the cDNA encoding the GPNMB extracellular domain (GPNMB-ECD) using a human fetal brain cDNA template. The forward primer included an in-frame BamHI site and the reverse primer contained an in-frame SalI restriction site. The primers used for amplification are as follows (5V-3V): Forward-GGATCCAAACGATTTCATGATGTGCTGGGCAATGAA Reverse-GTCGACCGAGGCTGGGTCTCTGTCAGGAACAGAAAT The PCR product was cloned into the pCR2.1-Topo vector (Invitrogen). The cDNA insert was verified by sequencing and subcloned into the BamHI/XhoI sites of pCEP4 (Invitrogen), which was modified by inserting the murine Ign secretion signal upstream, and a V5-His tag downs