Roberto Pili

Overall Survival and Updated Results for Sunitinib Compared With Interferon Alfa in Patients With Metastatic Renal Cell Carcinoma

PURPOSE A randomized, phase III trial demonstrated superiority of sunitinib over interferon alfa (IFN-alpha) in progression-free survival (primary end point) as first-line treatment for metastatic renal cell carcinoma (RCC). Final survival analyses and updated results are reported. PATIENTS AND METHODS Seven hundred fifty treatment-naïve patients with metastatic clear cell RCC were randomly assigned to sunitinib 50 mg orally once daily on a 4 weeks on, 2 weeks off dosing schedule or to IFN-alpha 9 MU subcutaneously thrice weekly. Overall survival was compared by two-sided log-rank and Wilcoxon tests. Progression-free survival, response, and safety end points were assessed with updated follow-up. RESULTS Median overall survival was greater in the sunitinib group than in the IFN-alpha group (26.4 v 21.8 months, respectively; hazard ratio [HR] = 0.821; 95% CI, 0.673 to 1.001; P = .051) per the primary analysis of unstratified log-rank test (P = .013 per unstratified Wilcoxon test). By stratified log-rank test, the HR was 0.818 (95% CI, 0.669 to 0.999; P = .049). Within the IFN-alpha group, 33% of patients received sunitinib, and 32% received other vascular endothelial growth factor-signaling inhibitors after discontinuation from the trial. Median progression-free survival was 11 months for sunitinib compared with 5 months for IFN-alpha (P < .001). Objective response rate was 47% for sunitinib compared with 12% for IFN-alpha (P < .001). The most commonly reported sunitinib-related grade 3 adverse events included hypertension (12%), fatigue (11%), diarrhea (9%), and hand-foot syndrome (9%). CONCLUSION Sunitinib demonstrates longer overall survival compared with IFN-alpha plus improvement in response and progression-free survival in the first-line treatment of patients with metastatic RCC. The overall survival highlights an improved prognosis in patients with RCC in the era of targeted therapy.

Digoxin and other cardiac glycosides inhibit HIF-1α synthesis and block tumor growth

A library of drugs that are in clinical trials or use was screened for inhibitors of hypoxia-inducible factor 1 (HIF-1). Twenty drugs inhibited HIF-1-dependent gene transcription by >88% at a concentration of 0.4 μM. Eleven of these drugs were cardiac glycosides, including digoxin, ouabain, and proscillaridin A, which inhibited HIF-1α protein synthesis and expression of HIF-1 target genes in cancer cells. Digoxin administration increased latency and decreased growth of tumor xenografts, whereas treatment of established tumors resulted in growth arrest within one week. Enforced expression of HIF-1α by transfection was not inhibited by digoxin, and xenografts derived from these cells were resistant to the anti-tumor effects of digoxin, demonstrating that HIF-1 is a critical target of digoxin for cancer therapy.

Targeting Tumor Angiogenesis with Histone Deacetylase Inhibitors: the Hydroxamic Acid Derivative LBH589

Purpose: Angiogenesis is required for tumor progression and represents a rational target for therapeutic intervention. Histone deacetylase (HDAC) inhibitors have been shown to have activity against various tumor cell types by inhibiting proliferation and inducing apoptosis both in vitro and in vivo. HDAC inhibitors have also been reported to inhibit angiogenesis. The goal of this study was to characterize the antiangiogenic and antitumor activity of a recently developed HDAC inhibitor, the hydroxamic derivative LBH589. Materials and Methods: To evaluate the antiangiogenesis activity of LBH589, we did cell cycle analysis, cell proliferation, tube formation, invasion assays in vitro, and Matrigel plug assay in vivo. To determine the antitumor activity of LBH589, we established human prostate carcinoma cell PC-3 xenografts in vivo. To evaluate the effect of LBH589 on endothelial signaling pathways, gene expression, and protein acetylation, we did Western blots and reverse transcription-PCR in human umbilical vein endothelial cells (HUVEC). Immunohistochemical analysis was done to evaluate new blood vessel formation in vivo. Results: LBH589 induced acetylation of histone H3 and α-tubulin protein in HUVECs. Histone and nonhistone protein acetylation correlated with induction of G2-M cell cycle arrest, inhibition of HUVEC proliferation, and viability. Noncytotoxic concentrations of LBH589 inhibited endothelial tube formation, Matrigel invasion, AKT, extracellular signal-regulated kinase 1/2 phosphorylation, and chemokine receptor CXCR4 expression. In vivo dosing of mice with LBH589 (10 mg/kg/d) reduced angiogenesis and PC-3 tumor growth. Conclusion: This study provides evidence that LBH589 induces a wide range of effects on endothelial cells that lead to inhibition of tumor angiogenesis. These results support the role of HDAC inhibitors as a therapeutic strategy to target both the tumor and endothelial compartment and warrant the clinical development of these agents in combination with angiogenesis inhibitors.

Class II Histone Deacetylases Are Associated with VHL-Independent Regulation of Hypoxia-Inducible Factor 1α

Hypoxia-inducible factor 1 alpha (HIF-1 alpha) plays a critical role in transcriptional gene activation involved in tumor angiogenesis. A novel class of agents, the histone deacetylase (HDAC) inhibitors, has been shown to inhibit tumor angiogenesis and HIF-1 alpha protein expression. However, the molecular mechanism responsible for this inhibition remains to be elucidated. In the current study, we investigated the molecular link between HIF-1 alpha inhibition and HDAC inhibition. Treatment of the VHL-deficient human renal cell carcinoma cell line UMRC2 with the hydroxamic HDAC inhibitor LAQ824 resulted in a dose-dependent inhibition of HIF-1 alpha protein via a VHL-independent mechanism and reduction of HIF-1 alpha transcriptional activity. HIF-1 alpha inhibition by LAQ824 was associated with HIF-1 alpha acetylation and polyubiquitination. HIF-1 alpha immunoprecipitates contained HDAC activity. Then, we tested different classes of HDAC inhibitors with diverse inhibitory activity of class I versus class II HDACs and assessed their capability of targeting HIF-1 alpha. Hydroxamic acid derivatives with known activity against both class I and class II HDACs were effective in inhibiting HIF-1 alpha at low nanomolar concentrations. In contrast, valproic acid and trapoxin were able to inhibit HIF-1 alpha only at concentrations that are effective against class II HDACs. Coimmunoprecipitation studies showed that class II HDAC4 and HDAC6 were associated with HIF-1 alpha protein. Inhibition by small interfering RNA of HDAC4 and HDAC6 reduced HIF-1 alpha protein expression and transcriptional activity. Taken together, these results suggest that class II HDACs are associated with HIF-1 alpha stability and provide a rationale for targeting HIF-1 alpha with HDAC inhibitors against class II isozymes.

The histone deacetylase inhibitor NVP-LAQ824 Inhibits angiogenesis and has a greater antitumor effect in combination with the vascular endothelial growth factor receptor tyrosine kinase inhibitor PTK787/ZK222584

Chromatin remodeling agents such as histone deacetylase inhibitors have been shown to modulate gene expression in tumor cells and inhibit tumor growth and angiogenesis. Vascular endothelial growth factor (VEGF) and VEGF receptors represent critical molecular targets for antiangiogenesis therapy. In this study, we investigated the biological effect of the histone deacetylase inhibitor NVP-LAQ824 in combination with the VEGF receptor tyrosine kinase inhibitor PTK787/ZK222584 on tumor growth and angiogenesis. We report that treatment with NVP-LAQ824 affected tumor and endothelial cells and was associated with increased histone acetylation, p21 up-regulation, and growth inhibition. In addition, NVP-LAQ824 treatment inhibited the expression of angiogenesis-related genes such as angiopoietin-2, Tie-2, and survivin in endothelial cells and down-regulated hypoxia-inducible factor 1-α and VEGF expression in tumor cells. Combination treatment with NVP-LAQ824 and PTK787/ZK222584 was more effective than single agents in inhibiting in vitro and in vivo VEGF-induced angiogenesis. Endothelial cell proliferation, tube formation, and invasion into the Matrigel plugs were reduced. In mouse models with established subcutaneous prostate (PC3) and orthotopic breast tumors (MDA-MB321), this combination treatment induced 80 to 85% inhibition of tumor growth without overt toxicity. These results suggest that the combination of histone deacetylase inhibitors and VEGF receptor inhibitors may target multiple pathways in tumor progression and angiogenesis and represents a novel therapeutic approach in cancer treatment.

VEGF165 Expressed by a Replication-Deficient Recombinant Adenovirus Vector Induces Angiogenesis In Vivo

To evaluate the concept that localized delivery of angiogenic factors via virus-mediated gene transfer may be useful in the treatment of ischemic disorders, the replication-deficient adenovirus (Ad) vector AdCMV.VEGF165 (where CMV is cytomegalovirus and VEGF is vascular endothelial growth factor) containing the cDNA for human VEGF165, a secreted endothelial cell-specific angiogenic growth factor, was constructed. Human umbilical vein endothelial cells (HUVECs) and rat aorta smooth muscle cells (RASMCs) infected with AdCMV.VEGF165 (5 and 20 plaque-forming units [pfu] per cell) demonstrated VEGF mRNA expression and protein secretion into the supernatant. Furthermore, the conditioned medium from these cells enhanced vascular permeability in vivo. In contrast, neither VEGF mRNA nor secreted protein was found in uninfected HUVECs or RASMCs or in cells infected with the control vector AdCMV.beta gal (where beta gal is beta-galactosidase). Assessment of starved HUVECs at 14 days demonstrated sixfold more cells for AdCMV.VEGF165-infected HUVECs (20 pfu per cell) than for either infected or uninfected control cells. RASMC proliferation was unaffected by infection with AdCMV.VEGF165. When plated in 2% serum on dishes precoated with reconstituted basement membrane (Matrigel), HUVECs infected with AdCMV.VEGF165 (20 pfu per cell) differentiated into capillary-like structures. Under similar conditions, both uninfected HUVECs and HUVECs infected with AdCMV.beta gal did not differentiate. To evaluate the ability of AdCMV.VEGF165 to function in vivo, either AdCMV. VEGF165 or AdCMV.beta gal (2 x 10(10) pfu) was resuspended in 0.5 mL Matrigel and injected subcutaneously into mice. Immunohistochemical staining demonstrated VEGF in the tissues surrounding the Matrigel plugs containing AdCMV.VEGF165 up to 3 weeks after injection, whereas no VEGF was found in the control plugs with AdCMV.beta gal. Two weeks after injection, there was histological evidence of neovascularization in the tissues surrounding the Matrigel containing AdCMV.VEGF165, whereas no significant angiogenesis was observed in response to AdCMV.beta gal. Furthermore, the Matrigel plugs with AdCMV.VEGF165 demonstrated hemoglobin content fourfold higher than the plugs with AdCMV.beta gal. Together, these in vitro and in vivo studies are consistent with the concept that Ad vectors may provide a useful strategy for efficient local delivery of VEGF165 in the treatment of ischemic diseases.

Phase II Randomized, Double-Blind, Placebo-Controlled Study of Tasquinimod in Men With Minimally Symptomatic Metastatic Castrate-Resistant Prostate Cancer

PURPOSE The activity of the novel antitumor agent tasquinimod (TASQ) with S100A9 as a molecular target was investigated in men with metastatic castration-resistant prostate cancer (CRPC) and minimal symptoms. PATIENTS AND METHODS We conducted a randomized, double-blind, placebo-controlled phase II trial in men assigned (at a ratio of two to one) to either oral once-daily TASQ 0.25 mg/d escalating to 1.0 mg/d over 4 weeks or placebo. The primary end point was the proportion of patients without disease progression at 6 months, defined by Response Evaluation Criteria in Solid Tumors Group, Prostate Cancer Working Group (PCWG2), or pain criteria, excluding prostate-specific antigen. RESULTS Two hundred one men (134 assigned to TASQ; 67 to placebo) were evaluable, and baseline characteristics were well balanced. Six-month progression-free proportions for TASQ and placebo groups were 69% and 37%, respectively (P < .001), and median progression-free survival (PFS) was 7.6 versus 3.3 months (P = .0042). In PCWG2 CRPC clinical subgroups, PFS in months was as follows: nodal metastases, 6.1 versus 3.1; bone metastases, 8.8 versus 3.4; and visceral metastases, 6.0 versus 3.0 for patients receiving TASQ versus placebo, respectively. Bone alkaline phosphatase levels were stabilized in the TASQ group, whereas the impact on PSA kinetics was less pronounced. Adverse events (AEs) occurring more frequently in the TASQ arm included GI disorders, fatigue, musculoskeletal pains, and elevations of pancreatic and inflammatory biomarkers. Grade 3 to 4 AEs, including asymptomatic elevations of laboratory parameters, were reported in 40% of patients receiving TASQ versus 10% receiving placebo; deep vein thrombosis (4% v 0%) was more common in the TASQ arm. CONCLUSION TASQ significantly slowed progression and improved PFS in patients with metastatic CRPC with an acceptable AE profile.

Id1 regulates angiogenesis through transcriptional repression of thrombospondin-1

Id proteins are helix-loop-helix transcription factors that regulate tumor angiogenesis. In order to identify downstream effectors of Id1 involved in the regulation of angiogenesis, we performed PCR-select subtractive hybridization on wild-type and Id1 knockout mouse embryo fibroblasts (MEFs). Here we demonstrate that thrombospondin-1 (TSP-1), a potent inhibitor of angiogenesis, is a target of transcriptional repression by Id1. We also show that Id1-null MEFs secrete an inhibitor of endothelial cell migration, which is completely inactivated by depletion of TSP-1. Furthermore, in vivo studies revealed decreased neovascularization in matrigel assays in Id1-null mice compared to their wild-type littermates. This decrease was completely reversed by a TSP-1 neutralizing antibody. We conclude that TSP-1 is a major target for Id1 effects on angiogenesis.

Targeting tumor angiogenesis with histone deacetylase inhibitors

Solid tumor malignancies including breast, lung and prostate carcinomas are considered to be angiogenesis dependent. Tumor angiogenesis is often mediated by hypoxia secondary to tumor growth or by increased oncogenic signaling. Both mechanisms result in increased hypoxia-inducible factor-1 alpha (HIF-1alpha) signaling and its transcriptional target vascular endothelial growth factor (VEGF). Critical to HIF-1alpha signaling are post translational modifications including acetylation mediated by histone acetyltransferases (HATS) and deacetylation by histone deacetylases (HDACs). More recently, HDACs were shown to be up-regulated in response to hypoxia mediating increased HIF-1alpha signaling. HDAC inhibitors represent a new class of anti-cancer therapeutics which show great promise at inhibiting angiogenesis in pre-clinical animal models and early phase clinical trials. This review will discuss the role of HIF-1alpha and VEGF influence on tumor angiogenesis and how HDACs play a critical role in HIF-1alpha transcriptional activity. Furthermore it will also be discussed how targeting HDACs via their inhibition create new avenues in treating solid malignancies by increasing the activity of established and novel therapeutic applications.

Adjuvant sunitinib or sorafenib for high-risk, non-metastatic renal-cell carcinoma (ECOG-ACRIN E2805): a double-blind, placebo-controlled, randomised, phase 3 trial

BACKGROUND Renal-cell carcinoma is highly vascular, and proliferates primarily through dysregulation of the vascular endothelial growth factor (VEGF) pathway. We tested sunitinib and sorafenib, two oral anti-angiogenic agents that are effective in advanced renal-cell carcinoma, in patients with resected local disease at high risk for recurrence. METHODS In this double-blind, placebo-controlled, randomised, phase 3 trial, we enrolled patients at 226 study centres in the USA and Canada. Eligible patients had pathological stage high-grade T1b or greater with completely resected non-metastatic renal-cell carcinoma and adequate cardiac, renal, and hepatic function. Patients were stratified by recurrence risk, histology, Eastern Cooperative Oncology Group (ECOG) performance status, and surgical approach, and computerised double-blind randomisation was done centrally with permuted blocks. Patients were randomly assigned (1:1:1) to receive 54 weeks of sunitinib 50 mg per day orally throughout the first 4 weeks of each 6 week cycle, sorafenib 400 mg twice per day orally throughout each cycle, or placebo. Placebo could be sunitinib placebo given continuously for 4 weeks of every 6 week cycle or sorafenib placebo given twice per day throughout the study. The primary objective was to compare disease-free survival between each experimental group and placebo in the intention-to-treat population. All treated patients with at least one follow-up assessment were included in the safety analysis. This trial is registered with ClinicalTrials.gov, number NCT00326898. FINDINGS Between April 24, 2006, and Sept 1, 2010, 1943 patients from the National Clinical Trials Network were randomly assigned to sunitinib (n=647), sorafenib (n=649), or placebo (n=647). Following high rates of toxicity-related discontinuation after 1323 patients had enrolled (treatment discontinued by 193 [44%] of 438 patients on sunitinib, 199 [45%] of 441 patients on sorafenib), the starting dose of each drug was reduced and then individually titrated up to the original full doses. On Oct 16, 2014, because of low conditional power for the primary endpoint, the ECOG-ACRIN Data Safety Monitoring Committee recommended that blinded follow-up cease and the results be released. The primary analysis showed no significant differences in disease-free survival. Median disease-free survival was 5·8 years (IQR 1·6-8·2) for sunitinib (hazard ratio [HR] 1·02, 97·5% CI 0·85-1·23, p=0·8038), 6·1 years (IQR 1·7-not estimable [NE]) for sorafenib (HR 0·97, 97·5% CI 0·80-1·17, p=0·7184), and 6·6 years (IQR 1·5-NE) for placebo. The most common grade 3 or worse adverse events were hypertension (105 [17%] patients on sunitinib and 102 [16%] patients on sorafenib), hand-foot syndrome (94 [15%] patients on sunitinib and 208 [33%] patients on sorafenib), rash (15 [2%] patients on sunitinib and 95 [15%] patients on sorafenib), and fatigue 110 [18%] patients on sunitinib [corrected]. There were five deaths related to treatment or occurring within 30 days of the end of treatment; one patient receiving sorafenib died from infectious colitis while on treatment and four patients receiving sunitinib died, with one death due to each of neurological sequelae, sequelae of gastric perforation, pulmonary embolus, and disease progression. Revised dosing still resulted in high toxicity. INTERPRETATION Adjuvant treatment with the VEGF receptor tyrosine kinase inhibitors sorafenib or sunitinib showed no survival benefit relative to placebo in a definitive phase 3 study. Furthermore, substantial treatment discontinuation occurred because of excessive toxicity, despite dose reductions. These results provide a strong rationale against the use of these drugs for high-risk kidney cancer in the adjuvant setting and suggest that the biology of cancer recurrence might be independent of angiogenesis. FUNDING US National Cancer Institute and ECOG-ACRIN Cancer Research Group, Pfizer, and Bayer.