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

Supplementary Figure S1 and Supplementary Table S1.

Abstract Purpose: Ovarian cancer recurrence with the development of paclitaxel resistance is an obstacle to long-term survival. We showed that secretory leukocyte protease inhibitor (SLPI) is a survival factor for ovarian cancer. We hypothesize that SLPI may antagonize paclitaxel injury. Experimental Design: Differential SLPI induction in response to paclitaxel and in response to stable forced expression of SLPI was shown in A2780-1A9 cells and their paclitaxel-resistant sublines, PTX10 and PTX22, and confirmed with HEY-A8 cells. SLPI-mediated survival was reduced by the MAP/extracellular signal-regulated kinase (ERK) kinase inhibitor, U0126, and a humanized neutralizing monoclonal anti-SLPI antibody, CR012. OVCAR3 xenographs tested the role of CR012 in vivo. Results: SLPI expression was lower in A2780-1A9 ovarian cancer cells than in PTX10 and PTX22, and SLPI was induced by paclitaxel exposure. Stable SLPI expression yielded a proliferation advantage (P = 0.01); expression of and response to SLPI in OVCAR3 cells were abrogated by exposure to CR012. SLPI reduced the paclitaxel susceptibility of 1A9 and HEY-A8 cells (P ≤ 0.05), and SLPI expression did not increase the resistance of PTX10 and PTX22 cells. Both paclitaxel and SLPI overexpression induced ERK activation. Inhibition of MAP/ERK kinase with U0126 increased paclitaxel injury and overcame SLPI-mediated cell protection. It did not reinstate PTX10 sensitivity to paclitaxel, which was associated with AKT activation. Significant inhibition of OVCAR3 xenograft growth was observed with CR012 and paclitaxel, over single agents (P ≤ 0.001). Conclusions: A two-pronged approach confirmed that SLPI overcomes paclitaxel in part through activation of ERK1/2. These results credential SLPI as a molecular target for ovarian cancer and suggest CR012 as a tool for proof of concept. Clin Cancer Res; 16(2); 600–9

Since the initial publication by Towbin and coworkers (1) on the preparation of sodium dodecyl sulfate-polyacrylamide gel replicas, commonly referred to as protein blots, the transfer of proteins from inaccessible gel matrices to readily accessible solid supports such as nitrocellulose or nylon membranes has become widely accepted (2–4). The detection of electrophoretically transferred or blotted proteins has ranged from the selective identification of an individual protein of interest to the general staining of total protein (5). Specific proteins are detected with probes such as antibodies that are either directly radiolabeled or conjugated to an enzyme (6,7). Alternatively, bound and unlabeled antibodies are amplified by a secondary antibody or affinity probe similarly conjugated. The use of biotinylated antibodies followed by avidin or streptavidin enzyme conjugates has become well accepted (8). Indeed, the high affinity interaction between biotin and streptavidin has been further popularized in such diverse methods as generating cRNA (riboprobes) for in situ detection of low-quantity expressed mRNA (9), immunohistochemical analysis of proteins, and high-throughput screening of aptamer microarrays (10).

Uncontrolled fibroblast growth factor (FGF) signaling can lead to human diseases, necessitating multiple layers of self-regulatory control mechanisms to keep its activity in check. Herein, we demonstrate that FGF9 and FGF20 ligands undergo a reversible homodimerization, occluding their key receptor binding sites. To test the role of dimerization in ligand autoinhibition, we introduced structure-based mutations into the dimer interfaces of FGF9 and FGF20. The mutations weakened the ability of the ligands to dimerize, effectively increasing the concentrations of monomeric ligands capable of binding and activating their cognate FGF receptor in vitro and in living cells. Interestingly, the monomeric ligands exhibit reduced heparin binding, resulting in their increased radii of heparan sulfate-dependent diffusion and biologic action, as evidenced by the wider dilation area of ex vivo lung cultures in response to implanted mutant FGF9-loaded beads. Hence, our data demonstrate that homodimerization autoregulates FGF9 and FGF20's receptor binding and concentration gradients in the extracellular matrix. Our study is the first to implicate ligand dimerization as an autoregulatory mechanism for growth factor bioactivity and sets the stage for engineering modified FGF9 subfamily ligands, with desired activity for use in both basic and translational research.

GPNMB is a melanoma-associated glycoprotein that is targeted by the CR011-vcMMAE antibody-drug conjugate (ADC). Previous studies have shown that CR011-vcMMAE induces the apoptosis of GPNMB-expressing tumor cells in vitro and tumor regression in xenograft models. This ADC is currently in clinical trials for melanoma. In the present investigation, a variety of compounds were examined for their ability to increase the expression of GPNMB by cancer cells. These experiments lead to the identification of three distinct groups of compounds that increased GPNMB, some of which were shown to enhance the sensitivity of melanoma cells to CR011-vcMMAE. These data indicate that it may be possible to increase the anticancer activity of CR011-vcMMAE through pharmacological enhancement of GPNMB expression for potential therapeutic benefit.

PURPOSE:Velafermin (recombinant human fibroblast growth factor-20, rhFGF-20) has been shown to reduce the severity and duration of mucositis in preclinical acute (single dose) radiation and chemotherapy/radiation models of oral mucositis. Our present study assessed the impact of velafermin on the severity and duration of oral mucositis that occurred as a consequence of fractionated radiation. EXPERIMENTAL DESIGN:Male Golden Syrian hamsters were exposed to eight doses of radiation (7.5 Gy/dose) to the cheek pouch on days 0, 1, 2, 3, 6, 7, 8 and 9 that resulted in severe mucositis. Velafermin (4 mg/kg intraperitoneally) was administered on days 3 and 9; days 2, 3, 8 and 9; days 3, 4, 9 and 10; or days 4, 5, 10 and 11. RESULTS:Although all velafermin-treated groups showed some reduction in the degree of mucositis relative to the vehicle control, the most significant reduction (p < 0.001) was observed in the groups treated on days 3 and 9 or on days 4, 5, 10 and 11. Further histological analysis of resected buccal mucosa revealed improvements in epithelial tissue degradation, connective tissue degradation and inflammation severity after velafermin treatment. Most notably, velafermin treatment reduced inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor (TNF) production possibly through nuclear factor-kappaB (NF-kappaB) mediation. The detection of increased NF-E2-related factor-2 (NRF-2) expression in the early onset stage of mucositis in the buccal mucosa suggested additional protective benefits from reactive oxygen species (ROS) generated as a consequence of fractionated radiation treatment. CONCLUSION:Thus, velafermin provided therapeutic benefit in a hamster model of oral mucositis induced by fractionated radiation therapy.

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.

BACKGROUND:Arresting or regressing kidney scarring is of major clinical relevance. Platelet-derived growth factor D (PDGF-D) is widely expressed in fibrotic kidneys. Administration of the PDGF-D neutralizing fully human monoclonal antibody CR002 in the acute phase of progressive anti-Thy 1.1 glomerulonephritis reduced glomerular and secondary tubulointerstitial damage.METHODS:Using this model, we now assessed the effects of CR002 (n=15) vs irrelevant control IgG (n=17) administered on days 17, 28 and 35 after disease induction, i.e. after acute glomerular damage had subsided.RESULTS:In vitro, CR002 inhibited the PDGF-D- but not the PDGF-B-induced proliferation of rat renal fibroblasts. Following the first CR002 injection on day 17, exposure to therapeutic levels was maintained until day 49. Proteinuria in the CR002-treated group was transiently reduced between days 49 and 77 (-19 to -23% in comparison with the controls; P<0.05). On day 100, CR002 treatment reduced the number of rats that had doubled their serum creatinine (CR002: 40 vs controls: 71%; P<0.05). Compared with controls, the CR002 animals, on day 100, significantly lowered glomerular expression of vimentin and collagens as well as tubulointerstitial damage scores, interstitial fibrosis, vimentin and cortical PDGF-D mRNA levels.CONCLUSIONS:PDGF-D antagonism, even after the phase of acute glomerular damage, exerts beneficial effects on the course of tubulointerstitial damage, i.e. the final common pathway of most renal diseases.

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).

AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA 691 PXD101 is a novel hydroxamate that potently inhibits the enzymatic activity of histone deacetylases (HDACs) at nanomolar concentration. In the present investigation, the growth-inhibitory activity of PXD101 used alone or in combination with 5-FU, a drug used clinically for the treatment of gastric/gastroesophageal cancer, was examined in vitro . PXD101 monotherapy inhibited the growth of a variety of gastric/gastroesophageal cancer cell lines (n = 10) at sub to low micromolar potency following 3 days of exposure. Cell cycle analysis indicated that PXD101 induced G2/M arrest and cytotoxicity. 5-FU monotherapy exhibited more variable activity than did PXD101, and the potency of 5-FU could in some cases be enhanced by increasing exposure time or using dialyzed serum. Importantly, the combination of PXD101 and 5-FU was found to synergistically inhibit cancer cell growth as determined by CalcuSyn analysis. In mechanistic studies, the expression of thymidylate synthase (TS; an enzyme known to be targeted by 5-FU and whose levels may correlate with 5-FU responsiveness in the clinic), was examined via immunoblotting. In these studies, 5-FU monotherapy was found to form a stable complex with TS, as indicated by a decrease in the mobility of this protein on SDS-PAGE. Work by others indicates this complex represents inactive TS. A timecourse experiment showed that the 5-FU/TS complex formed rapidly and that at early timepoints (2h, 7h of 5-FU exposure) most TS existed in the 5-FU-complexed/inactive form. However, by 24h of 5-FU exposure the level of uncomplexed/active TS returned to baseline, a phenomena that may play a role in the relatively weak growth-inhibitory activity of 5-FU on some cell lines. PXD101 monotherapy was found to strongly decrease TS expression by 24h of exposure, an effect that was maintained for at least 48h. Cells simultaneously exposed to both 5-FU and PXD101 for 48h exhibited little or no detectable uncomplexed/active TS, in contrast to cells treated with 5-FU alone for this amount of time which showed levels of active TS similar to that of untreated cells. Thus, PXD101 apparently overcomes the compensatory mechanisms which lead to enhanced TS synthesis following 5-FU exposure. The results of this study support the clinical evaluation of PXD101 used alone or in combination with 5-FU for the treatment of gastric/gastroesophageal carcinoma.

AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA 3758 Oral mucositis is a debilitating side effect of chemotherapy and/or radiotherapy in the treatment of cancer. Velafermin (rhFGF-20) has been shown to be a novel cytoprotectant of the oral mucosa and to reduce mucositis in preclinical acute (single dose) radiation, as well as chemotherapy/radiation-induced models of oral mucositis. Currently, velafermin is in a Phase II clinical trial for the prevention of severe oral mucositis in patients receiving myeloablative therapy and autologous stem cell transplantation. The aim of our study was to assess the impact of velafermin scheduling on the incidence, severity and duration of oral mucositis that occurs as a consequence of fractionated radiation. In this study, Golden Syrian hamsters were exposed to 8 doses of 7.5 Gy (cumulative dose of 60 Gy) to the cheek pouch on days 0, 1, 2, 3, 6, 7, 8 and 9 resulting in severe mucositis (score >3) by days 14-16. Velafermin was administered as a 4 mg/kg dose intraperitoneally on days 3 and 9; days 2, 3, 8 and 9; days 3, 4, 9 and 10; or days 4, 5, 10 and 11. Our results demonstrated that all of the groups treated with velafermin showed some reduction in the degree of mucositis and a significant increase in weight gain relative to those hamsters treated with the vehicle control alone. A significant reduction (p<0.001) in mucositis was seen in the group treated on days 4, 5, 10 and 11 in the number of days with a score of 3 or higher, as well as within the rank sum analysis of individual daily mucositis scores. Remarkably, a single weekly treatment with velafermin on days 3 and 9 also resulted in a significant reduction (p<0.001) in the number of days with a score of 3 or higher, as well as within the rank sum analysis of individual daily mucositis scores. Both dosing schedules also revealed statistically significant improvements in weight gain. Histology, as well as epithelial degradation, stromal degradation and inflammation severity morphometics are evaluated. All of these data support the continued investigation of velafermin for the reduction of oral mucositis induced by fractionated radiation #

Heterogeneous epithelial populations comprising the thymic environment influence early and late stages of T‐cell development. The processes that regulate the differentiation of thymic epithelium and that are responsible for this heterogeneity are not well understood, although mesenchymal/epithelial interactions are clearly involved. Here, we show that targeted expression by thymocytes of an fibroblast growth factor receptor‐2IIIb (FGFR2IIIb) ligand, FGF10, profoundly alters the differentiation and function of thymic epithelium (TE). Reconstitution of irradiated lck FGF10 mice with normal bone marrow restores normal thymic organization and function, while wild‐type mice reconstituted with lck FGF10 bone marrow recapitulates some of the thymic alterations seen in lck FGF10 mice. We also demonstrate that interference with FGFR2IIIb signaling in the thymus with a soluble FGFR2IIIb dominant‐negative fusion protein leads to precocious reductions in thymic size and cellularity that resemble age‐related thymic involution. These findings indicate that TE compartments are dynamically maintained and that FGF signals are involved in this process. Developmental Dynamics 236:3459–3471, 2007. © 2007 Wiley‐Liss, Inc.

A68 GPNMB is a type IA cell-surface glycoprotein expressed by certain types of cancers including malignant melanoma and glioblastoma. We previously generated an antibody to the extracellular domain of GPNMB, conjugated the antibody to the cytotoxic drug monomethylauristatin E (MMAE) via a protease-sensitive linker to facilitate release of drug following internalization, and found that this antibody-drug conjugate (ADC) effectively targeted GPNMB-expressing tumor cells in vitro and in xenograft models. The ADC, designated CR011-vcMMAE, is currently in Phase I clinical testing. Toward the goal of maximizing the anticancer activity of CR011-vcMMAE, we screened for compounds that increased expression of GPNMB on the surface of tumor cells. Our analyses lead to the identification of compounds falling into two main categories. The first of these categories is comprised of inhibitors of the ERK pathway which strongly increased GPNMB expression in melanoma cells harboring activating mutations in NRAS or BRAF, which account for ~80% of all melanomas. The ERK pathway represents a clinically relevant pathway in oncology drug development, and pharmacological inhibitors of this pathway appear to increase GPNMB expression via a transcriptional mechanism. An examination of a variety of melanoma-associated targets other than GPNMB demonstrated that some, but not all of these other proteins, were also induced by inhibitors of the ERK pathway in melanoma cells and that by way of comparison, GPNMB represented one of the most consistently expressed and strongly induced proteins among those surveyed. As would be predicted, melanoma cells exposed to inhibitors of the ERK pathway were sensitized to the growth-inhibitory effects of CR011-vcMMAE. The second category of compounds that increased GPNMB surface expression did so in both melanoma and glioblastoma cells regardless of NRAS/BRAF mutational status, apparently by enhancing the stability and/or membrane localization of GPNMB. The results presented in this study increase our understanding of GPNMB as an oncology target and provide preclinical support for maximizing the activity of CR011-vcMMAE through use in combination with compounds that enhance the cell-surface expression of GPNMB.

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.