Giovanni Melillo

Regulation of the Chemokine Receptor CXCR4 by Hypoxia

Cell adaptation to hypoxia (Hyp) requires activation of transcriptional programs that coordinate expression of genes involved in oxygen delivery (via angiogenesis) and metabolic adaptation (via glycolysis). Here, we describe that oxygen availability is a determinant parameter in the setting of chemotactic responsiveness to stromal-derived factor 1 (CXCL12). Low oxygen concentration induces high expression of the CXCL12 receptor, CXC receptor 4 (CXCR4), in different cell types (monocytes, monocyte-derived macrophages, tumor-associated macrophages, endothelial cells, and cancer cells), which is paralleled by increased chemotactic responsiveness to its specific ligand. CXCR4 induction by Hyp is dependent on both activation of the Hyp-inducible factor 1 α and transcript stabilization. In a relay multistep navigation process, the Hyp–Hyp-inducible factor 1 α–CXCR4 pathway may regulate trafficking in and out of hypoxic tissue microenvironments.

Hypoxia: importance in tumor biology, noninvasive measurement by imaging, and value of its measurement in the management of cancer therapy.

PURPOSE The Cancer Imaging Program of the National Cancer Institute convened a workshop to assess the current status of hypoxia imaging, to assess what is known about the biology of hypoxia as it relates to cancer and cancer therapy, and to define clinical scenarios in which in vivo hypoxia imaging could prove valuable. RESULTS Hypoxia, or low oxygenation, has emerged as an important factor in tumor biology and response to cancer treatment. It has been correlated with angiogenesis, tumor aggressiveness, local recurrence, and metastasis, and it appears to be a prognostic factor for several cancers, including those of the cervix, head and neck, prostate, pancreas, and brain. The relationship between tumor oxygenation and response to radiation therapy has been well established, but hypoxia also affects and is affected by some chemotherapeutic agents. Although hypoxia is an important aspect of tumor physiology and response to treatment, the lack of simple and efficient methods to measure and image oxygenation hampers further understanding and limits their prognostic usefulness. There is no gold standard for measuring hypoxia; Eppendorf measurement of pO(2) has been used, but this method is invasive. Recent studies have focused on molecular markers of hypoxia, such as hypoxia inducible factor 1 (HIF-1) and carbonic anhydrase isozyme IX (CA-IX), and on developing noninvasive imaging techniques. CONCLUSIONS This workshop yielded recommendations on using hypoxia measurement to identify patients who would respond best to radiation therapy, which would improve treatment planning. This represents a narrow focus, as hypoxia measurement might also prove useful in drug development and in increasing our understanding of tumor biology.

Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity

The identification of small molecules that inhibit the sequence-specific binding of transcription factors to DNA is an attractive approach for regulation of gene expression. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that controls genes involved in glycolysis, angiogenesis, migration, and invasion, all of which are important for tumor progression and metastasis. To identify inhibitors of HIF-1 DNA-binding activity, we expressed truncated HIF-1alpha and HIF-1beta proteins containing the basic-helix-loop-helix and PAS domains. Expressed recombinant HIF-1alpha and HIF-1beta proteins induced a specific DNA-binding activity to a double-stranded oligonucleotide containing a canonical hypoxia-responsive element (HRE). One hundred twenty-eight compounds previously identified in a HIF-1-targeted cell-based high-throughput screen of the National Cancer Institute 140,000 small-molecule library were tested in a 96-well plate ELISA for inhibition of HIF-1 DNA-binding activity. One of the most potent compounds identified, echinomycin (NSC-13502), a small-molecule known to bind DNA in a sequence-specific fashion, was further investigated. Electrophoretic mobility shift assay experiments showed that NSC-13502 inhibited binding of HIF-1alpha and HIF-1beta proteins to a HRE sequence but not binding of the corresponding proteins to activator protein-1 (AP-1) or nuclear factor-kappaB (NF-kappaB) consensus sequences. Interestingly, chromatin immunoprecipitation experiments showed that NSC-13502 specifically inhibited binding of HIF-1 to the HRE sequence contained in the vascular endothelial growth factor (VEGF) promoter but not binding of AP-1 or NF-kappaB to promoter regions of corresponding target genes. Accordingly, NSC-13502 inhibited hypoxic induction of luciferase in U251-HRE cells and VEGF mRNA expression in U251 cells. Our results indicate that it is possible to identify small molecules that inhibit HIF-1 DNA binding to endogenous promoters.

Phase I and Pharmacokinetic Study of MS-275, a Histone Deacetylase Inhibitor, in Patients With Advanced and Refractory Solid Tumors or Lymphoma

PURPOSE The objective of this study was to define the maximum-tolerated dose (MTD), the recommended phase II dose, the dose-limiting toxicity, and determine the pharmacokinetic (PK) and pharmacodynamic profiles of MS-275. PATIENTS AND METHODS Patients with advanced solid tumors or lymphoma were treated with MS-275 orally initially on a once daily x 28 every 6 weeks (daily) and later on once every-14-days (q14-day) schedules. The starting dose was 2 mg/m2 and the dose was escalated in three- to six-patient cohorts based on toxicity assessments. RESULTS With the daily schedule, the MTD was exceeded at the first dose level. Preliminary PK analysis suggested the half-life of MS-275 in humans was 39 to 80 hours, substantially longer than predicted by preclinical studies. With the q14-day schedule, 28 patients were treated. The MTD was 10 mg/m2 and dose-limiting toxicities were nausea, vomiting, anorexia, and fatigue. Exposure to MS-275 was dose dependent, suggesting linear PK. Increased histone H3 acetylation in peripheral-blood mononuclear-cells was apparent at all dose levels by immunofluorescence analysis. Ten of 29 patients remained on treatment for > or = 3 months. CONCLUSION The MS-275 oral formulation on the daily schedule was intolerable at a dose and schedule explored. The q14-day schedule is reasonably well tolerated. Histone deacetylase inhibition was observed in peripheral-blood mononuclear-cells. Based on PK data from the q14-day schedule, a more frequent dosing schedule, weekly x 4, repeated every 6 weeks is presently being evaluated.

Topoisomerase I-mediated inhibition of hypoxia-inducible factor 1: mechanism and therapeutic implications.

We have shown previously that the camptothecin analogue topotecan (TPT), a topoisomerase I (Top 1) poison, inhibits hypoxia-inducible factor 1 (HIF-1) transcriptional activity and HIF-1α protein accumulation in hypoxia-treated U251 human glioma cells. In this article, we demonstrate that TPT does not affect HIF-1α protein half-life or mRNA accumulation but inhibits its translation. In addition, we demonstrate that Top 1 is required for the inhibition of HIF-1α protein accumulation by TPT as shown by experiments performed using camptothecin-resistant cell lines with known Top 1 alterations. Experiments performed with aphidicolin indicated that TPT inhibited HIF-1 protein accumulation in the absence of DNA replication. DNA-damaging agents, such as ionizing radiation and doxorubicin, did not affect HIF-1α protein accumulation. Ongoing transcription was essential for the inhibition of HIF-1α protein accumulation by TPT. Our results demonstrate the existence of a novel pathway connecting Top 1-dependent signaling events and the regulation of HIF-1α protein expression and function. In addition, our findings dissociate the cytotoxic activity of TPT from the inhibition of the HIF-1 pathway and raise the possibility of novel clinical applications of TPT aimed at targeting HIF-1-dependent responses.

Targeting hypoxia cell signaling for cancer therapy

Hypoxia, a decrease in oxygen levels, is a hallmark of solid tumors. Hypoxic cells are more resistant to killing by ionizing radiation and chemotherapy, are more invasive and metastatic, resistant to apoptosis, and genetically unstable. Over the last two decades, the discovery of Hypoxia Inducible Factors, a family of transcription factors crucially involved in the response of mammalian cells to oxygen deprivation, has led to the identification of a molecular target associated with hypoxia suitable for the development of cancer therapeutics. These features of solid tumors may offer a unique opportunity for selective therapeutic approaches. A number of strategies targeting hypoxia and/or Hypoxia Inducible Factors (HIF) have been developed over the last several years and will be described. The exponentially growing interest in therapeutic strategies targeting hypoxia/HIF will undoubtedly generate more active compounds for preclinical and clinical development. A rational development plan aimed to validate target inhibition in preclinical models and early clinical trials is essential for a rapid translation of these agents to the treatment of human cancers.

Development of HIF-1 inhibitors for cancer therapy

•  Introduction •  Mechanisms of action of action of HIF‐1 inhibitors ‐  Inhibitors of HIF‐1α mRNA expression ‐  Inhibitors of HIF‐1α protein translation ‐  Inhibitors that affect HIF‐1α degradation pathway ‐  Inhibitors of HIF‐1 binding to DNA ‐  Inhibitors of HIF‐1α transcriptional activity •  Conclusions

Phase I Study of PARP Inhibitor ABT-888 in Combination with Topotecan in Adults with Refractory Solid Tumors and Lymphomas

A phase I trial of ABT-888 (veliparib), a PARP inhibitor, in combination with topotecan, a topoisomerase I-targeted agent, was carried out to determine maximum tolerated dose (MTD), safety, pharmacokinetics, and pharmacodynamics of the combination in patients with refractory solid tumors and lymphomas. Varying schedules and doses of intravenous topotecan in combination with ABT-888 (10 mg) administered orally twice a day (BID) were evaluated. Plasma and urine pharmacokinetics were assessed and levels of poly(ADP-ribose) (PAR) and the DNA damage marker γH2AX were measured in tumor and peripheral blood mononuclear cells (PBMC). Twenty-four patients were enrolled. Significant myelosuppression limited the ability to coadminister ABT-888 with standard doses of topotecan, necessitating dose reductions. Preclinical studies using athymic mice carrying human tumor xenografts also informed schedule changes. The MTD was established as topotecan 0.6 mg/m²/d and ABT-888 10 mg BID on days one to five of 21-day cycles. Topotecan did not alter the pharmacokinetics of ABT-888. A more than 75% reduction in PAR levels was observed in 3 paired tumor biopsy samples; a greater than 50% reduction was observed in PBMCs from 19 of 23 patients with measurable levels. Increases in γH2AX response in circulating tumor cells (CTC) and PBMCs were observed in patients receiving ABT-888 with topotecan. We show a mechanistic interaction of a PARP inhibitor, ABT-888, with a topoisomerase I inhibitor, topotecan, in PBMCs, tumor, and CTCs. Results of this trial reveal that PARP inhibition can modulate the capacity to repair topoisomerase I-mediated DNA damage in the clinic.

Schedule-dependent Inhibition of Hypoxia-inducible Factor-1α Protein Accumulation, Angiogenesis, and Tumor Growth by Topotecan in U251-HRE Glioblastoma Xenografts

We have previously shown that topotecan, a topoisomerase I poison, inhibits hypoxia-inducible factor (HIF)-1α protein accumulation by a DNA damage-independent mechanism. Here, we report that daily administration of topotecan inhibits HIF-1α protein expression in U251-HRE glioblastoma xenografts. Concomitant with HIF-1α inhibition, topotecan caused a significant tumor growth inhibition associated with a marked decrease of angiogenesis and expression of HIF-1 target genes in tumor tissue. These results provide a compelling rationale for testing topotecan in clinical trials to target HIF-1 in cancer patients.

Increased antitumor activity of bevacizumab in combination with hypoxia inducible factor-1 inhibition

Inhibition of hypoxia inducible factor-1 (HIF-1) is an attractive therapeutic strategy to target the tumor microenvironment. However, HIF-1 inhibitors may have limited activity as single agents and combination therapies may be required. We tested the hypothesis that HIF-1 inhibition in a hypoxic-stressed tumor microenvironment, which could be generated by administration of antiangiogenic agents, may result in a more pronounced therapeutic effect. The activity of bevacizumab, either alone or in combination with the HIF-1α inhibitor topotecan, was evaluated in U251-HRE xenografts. Tumor tissue was collected at the end of treatment and changes in tumor oxygenation, angiogenesis, proliferation, apoptosis, HIF-1α levels, HIF-1 target genes, and DNA damage were evaluated. Bevacizumab decreased microvessel-density and increased intratumor-hypoxia, but did not induce apoptosis. Moreover, bevacizumab alone caused a significant increase of HIF-1–dependent gene expression in tumor tissue. Addition of a low dose of daily topotecan to bevacizumab significantly inhibited tumor growth, relative to mice treated with topotecan or bevacizumab alone (P < 0.01). The addition of topotecan to bevacizumab was also associated with profound inhibition of HIF-1 transcriptional activity, significant inhibition of proliferation, and induction of apoptosis. Importantly, DNA damage induced by topotecan alone was not augmented by addition of bevacizumab, suggesting that increased cytotoxic activity did not account for the increased antitumor effects observed. These results strongly suggest that combination of anti–vascular endothelial growth factor antibodies with HIF-1 inhibitors is an attractive therapeutic strategy targeting in the hypoxic tumor microenvironment. [Mol Cancer Ther 2009;8(7):1867–77]