Katie O'Callaghan

Platelet Matrix Metalloprotease-1 Mediates Thrombogenesis by Activating PAR1 at a Cryptic Ligand Site

Matrix metalloproteases (MMPs) play important roles in normal and pathological remodeling processes including atherothrombotic disease, inflammation, angiogenesis, and cancer. MMPs have been viewed as matrix-degrading enzymes, but recent studies have shown that they possess direct signaling capabilities. Platelets harbor several MMPs that modulate hemostatic function and platelet survival; however their mode of action remains unknown. We show that platelet MMP-1 activates protease-activated receptor-1 (PAR1) on the surface of platelets. Exposure of platelets to fibrillar collagen converts the surface-bound proMMP-1 zymogen to active MMP-1, which promotes aggregation through PAR1. Unexpectedly, MMP-1 cleaves PAR1 at a distinct site that strongly activates Rho-GTP pathways, cell shape change and motility, and MAPK signaling. Blockade of MMP1-PAR1 curtails thrombogenesis under arterial flow conditions and inhibits thrombosis in animals. These studies provide a link between matrix-dependent activation of metalloproteases and platelet-G protein signaling and identify MMP1-PAR1 as a potential target for the prevention of arterial thrombosis.

Turning receptors on and off with intracellular pepducins: new insights into G-protein-coupled receptor drug development.

G-protein-coupled receptors (GPCRs) are a large family of remarkably versatile membrane proteins that are attractive therapeutic targets because of their involvement in a vast range of normal physiological processes and pathological diseases. Upon activation, intracellular domains of GPCRs mediate signaling to G-proteins, but these domains have yet to be effectively exploited as drug targets. Cell-penetrating lipidated peptides called pepducins target specific intracellular loops of GPCRs and have recently emerged as effective allosteric modulators of GPCR activity. The lipid moiety facilitates translocation across the plasma membrane, where pepducins then specifically modulate signaling of their cognate receptor. To date, pepducins and related lipopeptides have been shown to specifically modulate the activity of diverse GPCRs and other membrane proteins, including protease-activated receptors (PAR1, PAR2, and PAR4), chemokine receptors (CXCR1, CXCR2, and CXCR4), sphingosine 1-phosphate receptor-3 (S1P3), the melanocortin-4 receptor, the Smoothened receptor, formyl peptide receptor-2 (FPR2), the relaxin receptor (LGR7), G-proteins (Gαq/11/o/13), muscarinic acetylcholine receptor and vanilloid (TRPV1) channels, and the GPIIb integrin. This minireview describes recent advances made using pepducin technology in targeting diverse GPCRs and the use of pepducins in identifying potential novel drug targets.

Targeting Protease-Activated Receptor-1 with Cell-Penetrating Pepducins in Lung Cancer

Protease-activated receptors (PARs) are G-protein-coupled receptors that are activated by proteolytic cleavage and generation of a tethered ligand. High PAR1 expression has been documented in a variety of invasive cancers of epithelial origin. In the present study, we investigated the contribution of the four PAR family members to motility of lung carcinomas and primary tumor samples from patients. We found that of the four PARs, only PAR1 expression was highly increased in the lung cancer cell lines. Primary lung cancer cells isolated from patient lung tumors migrated at a 10- to 40-fold higher rate than epithelial cells isolated from nonmalignant lung tissue. Cell-penetrating pepducin inhibitors were generated against the first (i1) and third (i3) intracellular loops of PAR1 and tested for their ability to inhibit PAR1-driven migration and extracellular regulated kinase (ERK)1/2 activity. The PAR1 pepducins showed significant inhibition of cell migration in both primary and established cell lines similar to silencing of PAR1 expression with short hairpin RNA (shRNA). Unlike i1 pepducins, the i3 loop pepducins were effective inhibitors of PAR1-mediated ERK activation and tumor growth. Comparable in efficacy with Bevacizumab, monotherapy with the PAR1 i3 loop pepducin P1pal-7 provided significant 75% inhibition of lung tumor growth in nude mice. We identify the PAR1-ERK1/2 pathway as a feasible target for therapy in lung cancer.

Suppression of Arterial Thrombosis without Affecting Hemostatic Parameters with A Cell-Penetrating PAR1 Pepducin

Background— Thrombin-dependent platelet activation is heightened in the setting of percutaneous coronary intervention and may cause arterial thrombosis with consequent myocardial necrosis. Given the high incidence of adverse effects in patients with acute coronary syndromes, there remains an unmet need for the development of new therapeutics that target platelet activation without unduly affecting hemostasis. The thrombin receptor, PAR1, has recently emerged as a promising new target for therapeutic intervention in patients with acute coronary syndromes. Methods and Results— We report the development of a first-in-class intracellular PAR1 inhibitor with optimized pharmacokinetic properties for use during percutaneous coronary intervention in patients with acute coronary syndromes. PZ-128 is a cell-penetrating pepducin inhibitor of PAR1 that targets the receptor–G-protein interface on the inside surface of platelets. The structure of PZ-128 closely resembles the predicted off-state of the corresponding juxtamembrane region of the third intracellular loop of PAR1. The onset of action of PZ-128 was rapid and suppressed PAR1 aggregation and arterial thrombosis in guinea pigs and baboons and strongly synergized with oral clopidogrel. There was full recovery of platelet function by 24 hours. Importantly, PZ-128 had no effect on bleeding or coagulation parameters in primates or in blood from patients undergoing percutaneous coronary intervention. Conclusions— Based on the efficacy data in nonhuman primates with no noted adverse effects on hemostasis, we anticipate that the rapid onset of platelet inhibition and reversible properties of PZ-128 are well suited to the acute interventional setting of percutaneous coronary intervention and may provide an alternative to long-acting small-molecule inhibitors of PAR1.

Heme-binding Protein HRG-1 Is Induced by Insulin-like Growth Factor I and Associates with the Vacuolar H+-ATPase to Control Endosomal pH and Receptor Trafficking

Endocytosis and trafficking of receptors and nutrient transporters are dependent on an acidic intra-endosomal pH that is maintained by the vacuolar H+-ATPase (V-ATPase) proton pump. V-ATPase activity has also been associated with cancer invasiveness. Here, we report on a new V-ATPase-associated protein, which we identified in insulin-like growth factor I (IGF-I) receptor-transformed cells, and which was separately identified in Caenorhabditis elegans as HRG-1, a member of a family of heme-regulated genes. We found that HRG-1 is present in endosomes but not in lysosomes, and it is trafficked to the plasma membrane upon nutrient withdrawal in mammalian cells. Suppression of HRG-1 with small interfering RNA causes impaired endocytosis of transferrin receptor, decreased cell motility, and decreased viability of HeLa cells. HRG-1 interacts with the c subunit of the V-ATPase and enhances V-ATPase activity in isolated yeast vacuoles. Endosomal acidity and V-ATPase assembly are decreased in cells with suppressed HRG-1, whereas transferrin receptor endocytosis is enhanced in cells that overexpress HRG-1. Cellular uptake of a fluorescent heme analogue is enhanced by HRG-1 in a V-ATPase-dependent manner. Our findings indicate that HRG-1 regulates V-ATPase activity, which is essential for endosomal acidification, heme binding, and receptor trafficking in mammalian cells. Thus, HRG-1 may facilitate tumor growth and cancer progression.

Targeting CXCR4 with cell-penetrating pepducins in lymphoma and lymphocytic leukemia

The chemokine receptor CXCR4, which normally regulates stromal stem cell interactions in the bone marrow, is highly expressed on a variety of malignant hematologic cells, including lymphoma and lymphocytic leukemias. A new treatment concept has arisen wherein CXCR4 may be an effective therapeutic target as an adjunct to treatment of hematologic neoplasms with chemo- and immunotherapy. In the present study, we developed pepducins, cell-penetrating lipopeptide antagonists of CXCR4, to interdict CXCL12-CXCR4 transmembrane signaling to intracellular G-proteins. We demonstrate that pepducins targeting the first (i1) or third (i3) intracellular loops of CXCR4 completely abrogate CXCL12-mediated cell migration of lymphocytic leukemias and lymphomas. Stromal-cell coculture protects lymphoma cells from apoptosis in response to treatment with the CD20-targeted Ab rituximab. However, combination treatment with CXCR4 pepducins and rituximab significantly increases the apoptotic effect of rituximab. Furthermore, treatment of mice bearing disseminated lymphoma xenografts with pepducins alone or in combination with rituximab significantly increased their survival. These data demonstrate that CXCL12-CXCR4 signaling can be effectively inhibited by cell-penetrating pepducins, which represents a potential new treatment strategy for lymphoid malignancies.

Matrix Metalloprotease-1a Promotes Tumorigenesis and Metastasis

Background: Mmp1a is the postulated mouse genetic homologue of MMP1, but its functions in cancer are unknown. Results: Endogenous Mmp1a promotes invasion, tumorigenesis, and metastasis of lung cancer and melanoma. Conclusion: Mmp1a is a functional MMP1 homologue in mouse models of tumorigenesis. Significance: This is the first report that directly characterizes a function for Mmp1a in mouse (patho)physiology. Matrix metalloprotease-1 (MMP1), a collagenase and activator of the G protein-coupled protease activated receptor-1 (PAR1), is an emerging new target implicated in oncogenesis and metastasis in diverse cancers. However, the functional mouse homologue of MMP1 in cancer models has not yet been clearly defined. We report here that Mmp1a is a functional MMP1 homologue that promotes invasion and metastatic progression of mouse lung cancer and melanoma. LLC1 (Lewis lung carcinoma) and primary mouse melanoma cells harboring active BRAF express high levels of endogenous Mmp1a, which is required for invasion through collagen. Silencing of either Mmp1a or PAR1 suppressed invasive stellate growth of lung cancer cells in three-dimensional matrices. Conversely, ectopic expression of Mmp1a conferred an invasive phenotype in epithelial cells that do not express endogenous Mmp1a. Consistent with Mmp1a acting as a PAR1 agonist in an autocrine loop, inhibition or silencing of PAR1 resulted in a loss of the Mmp1a-driven invasive phenotype. Knockdown of Mmp1a on tumor cells resulted in significantly decreased tumorigenesis, invasion, and metastasis in xenograft models. Together, these data demonstrate that cancer cell-derived Mmp1a acts as a robust functional homologue of MMP1 by conferring protumorigenic and metastatic behavior to cells.

Dysregulated protease activated receptor 1 (PAR1) promotes metastatic phenotype in breast cancer through HMGA2

As the majority of patients with basal-like breast carcinoma present with invasive, metastatic disease that do not respond to available therapies, it is essential to identify new therapeutic targets that impact invasion and metastasis. Protease-activated receptor 1 (PAR1), a G-protein coupled receptor has been shown to act as an oncogene, but underlying mechanisms are not well understood. Here, we show that ectopic expression of functionally active PAR1 in MCF-7 cells induced a hormone-refractory, invasive phenotype representative of advanced basal-like breast carcinoma that readily formed metastatic lesions in lungs of mice. PAR1 was found to globally upregulate mesenchymal markers, including vimentin, a direct target of PAR1, and downregulate the epithelial markers including E-cadherin, as well as estrogen receptor. In contrast, non-signaling PAR1 mutant receptor did not lead to an invasive, hormone refractory phenotype. PAR1 expression increased spheroid formation and the level of stemness markers and self-renewal capacity in human breast cancer cells. We identified HMGA2 (high mobility group A2) as an important regulator of PAR1-mediated invasion. Inhibition of PAR1 signaling suppresses HMGA2-driven invasion in breast cancer cells. HMGA2 gene and protein are highly expressed in metastatic breast cancer cells. Overall, our results show that PAR1/HMGA2 pathway may present a novel therapeutic target.

HSP90 Inhibition Enhances Antimitotic Drug-Induced Mitotic Arrest and Cell Death in Preclinical Models of Non-Small Cell Lung Cancer

HSP90 inhibitors are currently undergoing clinical evaluation in combination with antimitotic drugs in non-small cell lung cancer (NSCLC), but little is known about the cellular effects of this novel drug combination. Therefore, we investigated the molecular mechanism of action of IPI-504 (retaspimycin HCl), a potent and selective inhibitor of HSP90, in combination with the microtubule targeting agent (MTA) docetaxel, in preclinical models of NSCLC. We identified a subset of NSCLC cell lines in which these drugs act in synergy to enhance cell death. Xenograft models of NSCLC demonstrated tumor growth inhibition, and in some cases, regression in response to combination treatment. Treatment with IPI-504 enhanced the antimitotic effects of docetaxel leading to the hypothesis that the mitotic checkpoint is required for the response to drug combination. Supporting this hypothesis, overriding the checkpoint with an Aurora kinase inhibitor diminished the cell death synergy of IPI-504 and docetaxel. To investigate the molecular basis of synergy, an unbiased stable isotope labeling by amino acids in cell culture (SILAC) proteomic approach was employed. Several mitotic regulators, including components of the ubiquitin ligase, anaphase promoting complex (APC/C), were specifically down-regulated in response to combination treatment. Loss of APC/C by RNAi sensitized cells to docetaxel and enhanced its antimitotic effects. Treatment with a PLK1 inhibitor (BI2536) also sensitized cells to IPI-504, indicating that combination effects may be broadly applicable to other classes of mitotic inhibitors. Our data provide a preclinical rationale for testing the combination of IPI-504 and docetaxel in NSCLC.

Abstract A77: Efficient targeting of HER-2-positive cancers by Antibody-Coupled T cell Receptor (ACTR) engineered autologous T cells

HER2 gene amplification occurs in 20-30% of aggressive breast and gastric cancer diagnoses, often signifying poor prognosis. The current standard of care for HER2 amplified cancer is the HER2 targeting antibody trastuzumab with chemotherapy. In the setting of HER2 amplified breast cancer, the combination of HER2 targeting antibodies trastuzumab and pertuzumab with the chemotherapeutic docetaxel is the frontline therapy. Despite the success of targeting the HER2 pathway, there are still many patients who are refractory or relapse following HER2 targeting regimens. Outside of the setting of HER2 overexpression or gene amplification, targeting HER2-positive cancers has thus far been ineffective. Engineered autologous T-cells, including chimeric antigen receptors (CARs) and high affinity T-cell receptors (TCRs), have gained attention recently due to their potent efficacy, with overall response rates reaching 80% and examples of long lasting remissions, particularly in advanced lymphoma and leukemia. However, initial clinical attempts to target HER2-amplified breast cancer with CAR-T cell therapy met with either acute toxicities or lack of efficacy. The Antibody-Coupled T-cell Receptor (ACTR) platform is a universal, engineered T cell therapy technology developed to mediate anti-tumor activity in combination with tumor-targeting antibodies. The ACTR chimeric construct is composed of the high-affinity CD16 (FCGR3A) V158 variant with the signaling domains from CD3z and 4-1BB. ACTR is designed to engage the Fc domain of human IgG1 antibodies when opsonized to the cognate target cells and deliver an activation signal to the engineered T cells. ACTR T cells combined with either HER2 targeting trastuzumab or pertuzumab exhibited potent cytotoxic activity, cytokine response and proliferation on a HER2 amplified tumor cell lines. ACTR activity was specific to antibody treated cells, and had little activity on HER2 low or negative tumor lines. Furthermore, the effectiveness of ACTR T cells in a non-amplified HER2 (HER2 low) setting was tested in the presence of a combination of trastuzumab and pertuzumab. This multi-antibody combination increased the cytotoxicity by ACTR T cells, whereas trastuzumab or pertuzumab as single antibody combinations with ACTR had little effect, suggesting a potential therapeutic approach for this disease setting with high unmet medical need. Importantly, the ACTR platform decouples the targeting moiety from the engineered T cell allowing for an antibody dose response which may spare cytotoxicity on normal cells. The specific response to HER2 positive tumor cells in the presence of trastuzumab, pertuzumab or the combination of HER2 directed antibodies demonstrates the potential therapeutic activity of ACTR T cells and supports consideration of clinical testing of ACTR T cells in HER2-positive cancers. Furthermore, combining multiple targeting antibodies to achieve greater potency and efficacy is unique to the ACTR technology and demonstrates the potential for such activity across many other tumor targets and tumor targeting antibodies. Citation Format: Casey B. Judge, Rachel DeBarge, Eugene Choi, Katie O9Callaghan, Lindsay Edwards, Birgit Schultes, Seth Ettenberg, Heather A. Huet. Efficient targeting of HER-2-positive cancers by Antibody-Coupled T cell Receptor (ACTR) engineered autologous T cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A77.