Annamaria Rapisarda

Development of HIF-1 inhibitors for cancer therapy

•u2002 Introduction •u2002 Mechanisms of action of action of HIF‐1 inhibitors ‐u2002 Inhibitors of HIF‐1α mRNA expression ‐u2002 Inhibitors of HIF‐1α protein translation ‐u2002 Inhibitors that affect HIF‐1α degradation pathway ‐u2002 Inhibitors of HIF‐1 binding to DNA ‐u2002 Inhibitors of HIF‐1α transcriptional activity •u2002 Conclusions

Role of the hypoxic tumor microenvironment in the resistance to anti-angiogenic therapies.

Angiogenesis, a key process for the growth of human cancers, has recently been exploited for the development of a novel class of cancer therapeutics that was thought to have wide applications and not to induce resistance in the clinical setting. Indeed, anti-angiogenic therapy has become an important option for the management of several human malignancies. However, a significant number of patients either do not respond to anti-angiogenic agents or fairly rapidly develop resistance. In addition, the benefit of anti-angiogenic therapy is relatively short-lived and the majority of patients eventually relapses and progresses. Several mechanisms of resistance to anti-angiogenic therapy have been recently proposed. The current review focuses on the role of intra-tumor hypoxia as a mechanism of resistance to anti-angiogenic agents and speculates on therapeutic approaches that might circumvent resistance and thereby improve clinical outcome.

Role of the VEGF/VEGFR axis in cancer biology and therapy.

New vessel formation (angiogenesis) is an essential physiological process for embryologic development, normal growth, and tissue repair. Angiogenesis is tightly regulated at the molecular level; however, this process is dysregulated in several pathological conditions such as cancer. The imbalance between pro- and antiangiogenic signaling molecules within tumors creates an abnormal vascular network that is characterized by dilated, tortuous, and leaky vessels. The pathophysiological consequences of these vascular abnormalities include temporal and spatial heterogeneity in tumor blood flow, oxygenation, and increased tumor interstitial fluid pressure. The resultant microenvironment deeply impacts on tumor progression, and also leads to a reduction in therapy efficacy. The discovery of vascular endothelial growth factor (VEGF) as a major driver of tumor angiogenesis has led to efforts to develop novel therapeutics aimed at inhibiting its activity. Anti-VEGF therapy has become an important option for the management of several human malignancies; however, a significant number of patients do not respond to anti-VEGF therapy when used either as single agent or in combination with chemotherapy. In addition, the benefit of antiangiogenic therapy is relatively short lived and the majority of patients relapse and progress. An increasing amount of reports suggest several potential mechanisms of resistance to antiangiogenic therapy including, but not limited to, tumor hypoxia. This chapter discusses the role of the VEGF axis in tumor biology and highlights the clinical application of anti-VEGF therapies elaborating on pitfalls and strategies to improve clinical outcome.

Human Monocytes Undergo Functional Re-programming during Sepsis Mediated by Hypoxia-Inducible Factor-1α

Sepsis is characterized by a dysregulated inflammatory response to infection. Despite studies in mice, the cellular and molecular basis of human sepsis remains unclear and effective therapies are lacking. Blood monocytes serve as the first line of host defense and are equipped to recognize and respond to infection by triggering an immune-inflammatory response. However, the response of these cells in human sepsis and their contribution to sepsis pathogenesis is poorly understood. To investigate this, we performed a transcriptomic, functional, and mechanistic analysis of blood monocytes from patients during sepsis and after recovery. Our results revealed the functional plasticity of monocytes during human sepsis, wherein they transited from a pro-inflammatory to an immunosuppressive phenotype, while enhancing protective functions like phagocytosis, anti-microbial activity, and tissue remodeling. Mechanistically, hypoxia inducible factor-1α (HIF1α) mediated this functional re-programming of monocytes, revealing a potential mechanism for their therapeutic targeting to regulate human sepsis.

Hypoxia Selectively Inhibits Monocyte Chemoattractant Protein-1 Production by Macrophages

Hypoxia, a local decrease in oxygen tension occurring in inflammatory and tumor lesions, modulates gene expression in macrophages. Because macrophages are important chemokine producers, we investigated the regulatory effects of hypoxia on macrophage-derived chemokines. We demonstrated that hypoxia inhibits the production of the macrophage and T lymphocyte chemotactic and activating factor, monocyte chemoattractant protein-1 (MCP-1). Exposure of mouse macrophages to low oxygen tension resulted in the down-regulation of constitutive MCP-1 mRNA expression and protein secretion. Hypoxia inhibitory effects were selective for MCP-1 because the chemokines macrophage inflammatory protein-1β (MIP-1β), RANTES, IFN-γ-inducible protein-10, and MIP-2 were not affected, and MIP-1α was induced. Hypoxia also inhibited, in a time-dependent fashion, MCP-1 up-regulation by IFN-γ and LPS. Moreover, the inhibitory action of hypoxia was exerted on human monocytic cells. MCP-1 down-regulation was associated with inhibition of gene transcription and mRNA destabilization, suggesting a dual molecular mechanism of control. Finally, we found that the triptophan catabolite picolinic acid and the iron chelator desferrioxamine, which mimic hypoxia in the induction of gene expression, differentially regulated the expression of MCP-1. This study characterizes a novel property of hypoxia as a selective inhibitor of MCP-1 production induced by different stimuli in macrophages and demonstrates that down-regulation of gene expression by hypoxia can be controlled at both transcriptional and posttranscriptional levels. Inhibition of MCP-1 may represent a negative regulatory mechanism to control macrophage-mediated leukocyte recruitment in pathological tissues.

Synergystic induction of HIF-1α transcriptional activity by hypoxia and lipopolysaccharide in macrophages

Hypoxia Inducible Factor-1 (HIF-1) is activated by a variety of stimuli, including inflammatory mediators. In this report we investigated the role that bacterial lipopolysaccharide (LPS) and hypoxia play in the regulation of HIF-1-dependent gene expression in macrophages. We report that murine macrophages stimulated with low concentrations of LPS (1-10 ng/ml) expressed significantly higher levels of inducible nitric oxide synthase (iNOS) mRNA when cultured under hypoxic compared to normoxic conditions. Functional studies of the iNOS promoter demonstrated that the synergistic interaction between LPS and hypoxia was mediated, at least in part, by the NFκB and the HIF-1 binding sites. In addition, transient transfection experiments using a Hypoxia Response Element (HRE)-containing plasmid showed that LPS and hypoxia synergistically induced HIF-1-dependent transcriptional activity. Interestingly, LPS did not significantly affect HIF-1α protein levels or HIF-1 DNA binding activity relative to hypoxic induction. HIF-1α, but not HIF-2α, was critical for the synergistic induction of HRE-dependent transcriptional activity in macrophages, as indicated by experiments using siRNA targeting HIF-1α or HIF-2α. Addition of ROS-scavengers completely abrogated the synergistic induction of HIF-1 transcriptional activity by LPS and hypoxia, but neither inhibited HIF-1 transcriptional activity induced by hypoxia alone nor affected HIF-1α protein levels or HIF-1 DNA binding induced by hypoxia alone or hypoxia plus LPS. Taken together, our results demonstrate that LPS and hypoxia act synergistically to induce HIF-1α-transcriptional activity and they emphasize the existence of a cross talk between hypoxic and non-hypoxic signaling pathways in the regulation of macrophages gene expression.

Macrophage Activating Properties of The Tryptophan Catabolite Picolinic Acid

Recent studies have suggested a role for aminoacid catabolites as important regulators of macrophage (Mphi) activities. We reported previously that picolinic acid (PA), a tryptophan catabolite produced under inflammatory conditions and a costimulus with IFNgamma of Mphi effector functions, is a selective inducer of the Mphi inflammatory protein-1alpha (MIP-1alpha) and -1beta (MIPs), two CC-chemokines involved in the elicitation of the inflammatory reactions and in the development of the Th1 responses. In this study, we have investigated the effects of IFNgamma on PA-induced MIPs expression and secretion by mouse Mphi as well as the regulation of MIP-1alpha/beta receptor, CCR5, by both stimuli alone or in combination. We demonstrated that IFNgamma inhibited MIPs mRNA stimulation by PA in a dose-and time-dependent fashion, despite its ability to induce other CC- or CXC chemokines. MIPs mRNA down-regulation was associated with decreased intracellular chemokine expression and secretion and was dependent on both mRNA destabilization and gene transcription inhibition. Moreover, IFNgamma inhibitory effects were stimulus-specific because MIPs induction by PA was either unaffected or increased by the anti-inflammatory cytokines, IL-10 and IL-4, or the pro-inflammatory stimulus, LPS, respectively. In contrast, we found that IFNgamma increased CCR5 basal expression, whereas PA down-regulated both constitutive and IFNgamma-induced CCR5 mRNA and protein levels. These results demonstrate that IFNgamma and PA have reciprocal effects on the production of MIPs chemokines and the expression of their receptor. The concerted action of IFNgamma and PA on MIP-1alpha/beta chemokine/receptor system is likely to be of pathophysiological significance and to represent an important regulatory mechanism for leukocyte recruitment and distribution into damaged tissues during inflammatory responses.

Abstract A102: NCI-60 response profiles of >400 investigational oncology agents: A resource enabling drug and biomarker discovery.

We have acquired >400 investigational oncology agents, comprised primarily of targeted small molecules currently in clinical and/or preclinical anticancer studies. As oncology treatment moves toward personalized targeted therapeutic agents, the NCI-60 human tumor cell line panel is an ideal community-wide tool to further understanding of the disease targets of new agents. The panel includes cell lines from nine tumor types, and is extremely well characterized at the molecular level, with both in-house and crowd-sourced characterization, including exome sequence for mutations, SNPs, DNA methylation, metabolome, mRNA, microRNA, and protein expression. This molecular characterization dataset enables interrogation of patterns of growth inhibition by the investigational drug set looking for characteristics of the cell lines that determine sensitivity. More than 150,000 small molecules, including all (> 100) FDA-approved anticancer drugs and now our acquired set of 400 investigational oncology agents have been screened against the panel for their effects on cell growth. We have used a number of online tools to enable data analysis for this set, including COMPARE (http://dtp.nci.nih.gov/compare/), which provided for the identification of compounds and/or genes that have highly correlated response patterns for any selected ‘seed’ compound. This presentation provides the first public disclosure of the NCI-60 data for this set of novel, targeted, investigational oncology agents. We anticipate that these data will enable comparison between drug sensitivity profiles that could lead to the elucidation of common mechanistic targets or pathways, associations with potential response biomarkers, the confirmation of mechanism of action or identification of novel mechanisms, and the uncovering of unexpected off-target activities. For example, Akt, pI3K, PDK, and mTOR inhibitors, multiple agents targeting one signaling pathway, display strong correlations with one another. Using the allosteric Akt inhibitor MK-2206 as the seed compound, response patterns for the ATP-competitive Akt inhibitors PF-4173640 (0.84), GDC-0068 (0.80), AZD-5363 (0.83), GSK-690693 (0.67), and CCT-128930 (0.69) are highly correlative. Moreover, the NCI-60 response pattern for the androgen receptor modulator AZD-3514 has a 0.77 correlation with the BET bromodomain inhibitor JQ-1, suggesting a commonality of target/pathway for these compounds. Further correlations, associations and hypotheses generated from interrogating the compound response patterns, gene expression profiles, mutations and other characteristics will be presented. Funded by NCI Contract No. HHSN261200800001E.nnCitation Information: Mol Cancer Ther 2013;12(11 Suppl):A102.nnCitation Format: Joel Morris, Mark Kunkel, Eric Polley, Susan Holbeck, Kazimierz Wrzeszczynski, Anne Monks, David Evans, Annamaria Rapisarda, Jerry Collins, Beverly A. Teicher. NCI-60 response profiles of >400 investigational oncology agents: A resource enabling drug and biomarker discovery. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A102.

Abstract C103: Sarcoma cell line sensitivity towards approved oncology drugs and investigational agents identifies distinct patterns of response which can be interrogated with associated gene expression.

Sarcomas represent a heterogeneous group of cancers with significant unmet medical needs. We have examined the response of 64 sarcoma cell lines to treatment with 103 approved oncology drugs (available from The NCI/DTP Open Chemical Repository) and 420 agents in the investigational agents library, using inhibition of proliferation as an endpoint. Cells were exposed to compounds at varying concentrations (10μM to 1.5nM) for 96 h and the effect of compound on cell viability was monitored using Alamar Blue. From curve fitting algorithms we determined the IC50 values of each agent on the cell lines. Adult sarcomas comprise 23% of this sarcoma panel and have a different spectrum of sensitivities to selected agents. They were generally more chemoresistant than the pediatric lines, although they are marginally more sensitive to MEK inhibitors. Synovial tumor cell lines were an exception, being sensitive to several classes including dasatinib and Bcr-abl inhibitors. Ewings tumors tend to be the most sensitive pediatric group responding to Parp-1 and IGF-1R inhibitors. Overall, gene, and to a lesser extent, miRNA profiles from the adult sarcomas were more similar to the profiles of normal, non-tumor cells, than the pediatric tumors, and this lack of genotypic divergence may underlie the insensitive phenotype observed in the sarcoma panel. From an analysis of sensitivity clustering, IGF-1R inhibitors (8), cluster with some of the AKT (8) and ALK (8) inhibitors. None of the cell lines have the EM4L-ALK translocation, thus we investigated the genes associated with sensitivity to these three mechanisms. Dendrograms identified a close relationship between the IGF-1R and AKT inhibitors, based on the gene expression patterns, while the ALK inhibitors were quite distinct, substantiating known pathways of IGFR-1R signaling through AKT, but unrelated to ALK. Genes associated with ALK sensitivity included several from gluconeogenesis, and potential activation of the MYC response. In contrast, genes associated with AKT and IGF1R sensitivity are focused around FOXO1 transcription factor. Interestingly, the PAX-FOXO1 gene fusion is a hallmark of the aggressive alveolar rhabdomyosarcoma which are more sensitive to these agents than embryonal rhabdomyosarcoma. Thus the combination of drug sensitivity data, together with the gene and miRNA profiles may allow correlations in treatment efficacy that may point to new avenues for clinical development in sarcoma. Funded by NCI Contract No. HHSN261200800001E.nnCitation Information: Mol Cancer Ther 2013;12(11 Suppl):C103.nnCitation Format: Anne Monks, David Evans, Thomas Silvers, Rene Delosh, Julie Laudeman, Chad Ogle, Russell Reinhart, Michael Selby, John Connelly, Annamaria Rapisarda, Mark Kunkel, Joel Morris, Kazimierz Wrzeszczynski, Eric Polley, Beverley Teicher. Sarcoma cell line sensitivity towards approved oncology drugs and investigational agents identifies distinct patterns of response which can be interrogated with associated gene expression. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C103.

Abstract 2416: Weekly EZN-2208 (pegylated SN-38) in combination with bevacizumab in patients with refractory solid tumors.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnBackground: Anti-angiogenic therapies such as bevacizumab (Bev) have been shown to up-regulate hypoxia-inducible factor-1α (HIF-1α), a possible mechanism of resistance. Camptothecin analogues, including SN-38, have been shown to reduce the expression and transcriptional activity of HIF-1α in preclinical models. Thus, co-administration of pegylated SN 38 (EZN 2208) could offset the induction of HIF-1α following administration of Bev, resulting in synergistic anti-tumor effects. We are conducting a trial of the combination of EZN 2208 (E) with Bev in patients with refractory solid tumors to determine the safety and tolerability of the combination, and to evaluate modulation of HIF-1α protein in tumor biopsies following administration of study drugs. Methods: Eligible pts have refractory advanced solid tumors that have progressed following standard therapy; ≥ 18 yrs of age; ECOG PS 0-2; adequate organ function. Bev at 5mg/kg is administered on D-7 and D15 for cycle 1 only and on D1, 15 for each subsequent cycle; q28d cycles. E(9mg/kg) is administered on D1, 8, and 15. Tumor biopsies and dynamic contrast enhanced MRI (DCE-MRI) are obtained on D1 prior to EZN administration (7 days post Bev) and on C2D15 post Bev and E. An additional DCE-MRI is also performed at baseline prior to Bev on D-7. Tumor biopsies are analyzed for HIF-1α protein levels using a validated immunoassay, and HIF-1α response genes, Hexokinase 2(HK), vascular endothelial growth factor A(VEGF), pyruvate dehydrogenase kinase, isozyme 1(PDK1), carbonic anhydrase IX(CA9), solute carrier family 2 (GLUT1), are assessed using RTPCR.nnResults: Twelve pts have been enrolled to date; median age 50 (range 27-76 yrs); median # prior therapies 3 (1-6); Dx: Soft tissue sarcoma (4), colorectal cancer (2), parotid gland cancer (1), malignant hurthle cell tumor (1), HCC (1), melanoma (1), gastrointestinal stromal tumor (1), Head and neck cancer (1). Grade 3/4 toxicities (#pts): neutropenia (8), leucopenia (3), lymphopenia (2), hypertension (1). Eight pts are evaluable for response; prolonged stable disease was observed in 2 pts: HCC 16 cycles, desmoplastic round cell tumor 7 cycles. HIF-1α protein and mRNA levels of HIF-1α dependent genes were assessed in 5 pts who had paired biopsies. Reduction in HIF-1α protein levels compared to baseline (post Bev alone) was seen in 4 of 5 pts (range 24% - 64%), with evidence of modulation of mRNA in 3 of 5 pt tumors evaluated. Quantitative analysis of DCE MRI from 3 pts revealed changes in Ktrans and kep maps following drug administration. Correlation of observed changes with tumor response is ongoing.nnConclusions: This clinical trial provides preliminary proof of mechanism demonstrating modulation of HIF-1α levels in tumors following administration of a camptothecin analog, EZN 2208. Accrual is ongoing to establish safety, efficacy, and further proof of target modulation.nnCitation Format: Woondong Jeong, Sook Ryun Park, Annamaria Rapisarda, Michelle Eugeni, Robert Kinders, Alice Chen, Giovanni Melillo, Baris Turkbey, James H. Doroshow, Shivaani Kummar. Weekly EZN-2208 (pegylated SN-38) in combination with bevacizumab in patients with refractory solid tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2416. doi:10.1158/1538-7445.AM2013-2416