Zoya Galcheva-Gargova

Contaminated Heparin Associated with Adverse Clinical Events and Activation of the Contact System

BACKGROUND There is an urgent need to determine whether oversulfated chondroitin sulfate (OSCS), a compound contaminating heparin supplies worldwide, is the cause of the severe anaphylactoid reactions that have occurred after intravenous heparin administration in the United States and Germany. METHODS Heparin procured from the Food and Drug Administration, consisting of suspect lots of heparin associated with the clinical events as well as control lots of heparin, were screened in a blinded fashion both for the presence of OSCS and for any biologic activity that could potentially link the contaminant to the observed clinical adverse events. In vitro assays for the activation of the contact system and the complement cascade were performed. In addition, the ability of OSCS to recapitulate key clinical manifestations in vivo was tested in swine. RESULTS The OSCS found in contaminated lots of unfractionated heparin, as well as a synthetically generated OSCS reference standard, directly activated the kinin-kallikrein pathway in human plasma, which can lead to the generation of bradykinin, a potent vasoactive mediator. In addition, OSCS induced generation of C3a and C5a, potent anaphylatoxins derived from complement proteins. Activation of these two pathways was unexpectedly linked and dependent on fluid-phase activation of factor XII. Screening of plasma samples from various species indicated that swine and humans are sensitive to the effects of OSCS in a similar manner. OSCS-containing heparin and synthetically derived OSCS induced hypotension associated with kallikrein activation when administered by intravenous infusion in swine. CONCLUSIONS Our results provide a scientific rationale for a potential biologic link between the presence of OSCS in suspect lots of heparin and the observed clinical adverse events. An assay to assess the amidolytic activity of kallikrein can supplement analytic tests to protect the heparin supply chain by screening for OSCS and other highly sulfated polysaccharide contaminants of heparin that can activate the contact system.

M402, a novel heparan sulfate mimetic, targets multiple pathways implicated in tumor progression and metastasis.

Heparan sulfate proteoglycans (HSPGs) play a key role in shaping the tumor microenvironment by presenting growth factors, cytokines, and other soluble factors that are critical for host cell recruitment and activation, as well as promoting tumor progression, metastasis, and survival. M402 is a rationally engineered, non-cytotoxic heparan sulfate (HS) mimetic, designed to inhibit multiple factors implicated in tumor-host cell interactions, including VEGF, FGF2, SDF-1α, P-selectin, and heparanase. A single s.c. dose of M402 effectively inhibited seeding of B16F10 murine melanoma cells to the lung in an experimental metastasis model. Fluorescent-labeled M402 demonstrated selective accumulation in the primary tumor. Immunohistological analyses of the primary tumor revealed a decrease in microvessel density in M402 treated animals, suggesting anti-angiogenesis to be one of the mechanisms involved in-vivo. M402 treatment also normalized circulating levels of myeloid derived suppressor cells in tumor bearing mice. Chronic administration of M402, alone or in combination with cisplatin or docetaxel, inhibited spontaneous metastasis and prolonged survival in an orthotopic 4T1 murine mammary carcinoma model. These data demonstrate that modulating HSPG biology represents a novel approach to target multiple factors involved in tumor progression and metastasis.

Overexpression of Merlin in B16F10 mouse melanoma cells reduces their metastatic activity: Role of the cell surface heparan sulfate glycosaminoglycans

Merlin, the protein product of the neurofibromatosis type 2 gene (NF2) acts as a tumor suppressor in mice and humans. In this study, melanoma B16F10 cells were engineered to overexpress the NF2 gene by establishing stable transductants. A cell line overexpressing Merlin (B16F10-M) was generated. When compared to the parental cells, the B16F10-M cells demonstrated differences in their cell surface organization. The overexpressing strain changed its ability to grow in soft agar as well as its cell motility properties. B16F10-M cells were then examined in the in vivo mouse melanoma tumor growth and tumor metastasis models. While tumor growth was marginally affected, the presence of increased Merlin severely reduced the metastastatic ability of the cells. When isolated using specific enzymes with distinct substrate specificity, the cell surface heparan sulfate glycosaminoglycans (HSGAGs) from the overexpressing B16F10-M cells, inhibited the metastatic properties of the parental B16F10 cells. The results obtained provide a causal link between the reorganization/changes to the cell surface HSGAGs by the overexpression of Merlin and the inhibition of the metastatic activity of the mouse melanoma B16F10 cells in vivo.

Abstract 302: Characterization of effects of M402 on EMT in pancreatic ductal adenocarcinoma.

Background: Pancreatic ductal adenocarcinoma (PDA) is an almost uniformly lethal disease. The current treatment for resectable disease consists of surgery and adjuvant gemcitabine therapy. However, for more than 80 percent of the patients with PDA surgery is not an option. For these patients palliative chemotherapy using gemcitabine, or most recently FOLFIRINOX, is considered standard of care. Even with palliative chemotherapy the median overall survival for patients with metastatic PDA is dismal and improved therapeutic strategies are a clear unmet medical need. Heparins may be of interest for the treatment of PDA. They play an important role in tumor progression and metastasis by binding at heparin binding domains, thereby preventing growth factor gradients created along proteoglycan heparan sulfates and thus disrupting signaling. We used a novel heparan sulfate mimetic, M402, which binds to multiple growth factors, adhesion molecules, and chemokines. We previously showed that M402 decreases both epithelial-to-mesynchymal transition and chemotherapy resistance in PDA. Here we further evaluated the underlying mechanisms of M402 in EMT. Results: We studied the effects of M402 on PDA using a genetically engineered mouse model (GEMM) for PDA (LSL-KRAS G12D/+ ; Trp53 LSL-R172H/flox ; pdx-CRE), which recapitulates human PDA. Combination therapy of M402 with gemcitabine significantly prolonged the average survival of the mice when compared to mice treated with gemcitabine monotherapy (87 days versus 78 days) and significantly reduced metastasis and local invasion into the small intestine. These data suggest an effect of M402 on invasiveness and EMT. Gemcitabine treatment increased EMT as determined by staining for E-cadherin and Fsp1 double positive cells. In contrast, M402 treatment resulted in a decrease in E-cadherin and Fsp1 double positive cells in pancreatic tumors. We next sought to determine whether M402 affects cancer cells or stromal cells. In vitro experiments show that cell lines derived from our model respond to M402 by decreasing their invasive behavior in 3D culture and scratch assays. Moreover, we found that M402 augments the gemcitabine effect in vitro. These data suggest that M402 affects both EMT and gemcitabine response. Further identification by whole transcriptome microarray analysis of treated tumors hints towards a role of M402 in affecting multiple signaling pathways involved in the regulation of EMT. These data will undoubtedly lead to better insight into the mechanism of action of M402 and will increase our understanding of the pathways PDA cells use to evade the effects of chemotherapy. M402 is currently being investigated in a phase 1/2 M402 gemcitabine combination study to assess if these findings can be translated into a clinical benefit in pancreatic cancer patients. Conclusion: These data suggest that M402 reduces acquired chemotherapy resistance in a GEMM for PDA by decreasing EMT. Citation Format: Ilse Oosterom, Birgit C. Schultes, Chia Lin Chu, Zoya Galcheva-Gargova, Elma Kurtagic, He Zhou, Jay Duffner, Emile E. Voest, David A. Tuveson, Martijn Lolkema. Characterization of effects of M402 on EMT in pancreatic ductal adenocarcinoma. [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 302. doi:10.1158/1538-7445.AM2013-302

Role of M402, a novel heparan sulfate mimetic, in pancreatic cancer cell invasion and metastasis: Inhibition of the Sonic Hedgehog pathway and heparanase activity.

25 Background: M402 is a novel heparin sulfate mimetic that binds to multiples growth factors, adhesion molecules, and cytokines to inhibit tumor angiogenesis, progression, and metastasis in nonclinical studies. We investigated if M402 could modulate tumor-stroma interactions in pancreatic cancer by inhibiting the Sonic Hedgehog (Shh) pathway as well as inhibit the activity of the extracellular matrix degrading enzyme, heparanase. METHODS Surface plasmon resonance (SPR) was used for analysis of M402 binding to Shh in vitro. A cell based Gli-1 reporter assay was implemented to assess the effect of M402 on Shh signaling. Immunohistochemistry and RT-qPCR were utilized to investigate M402s effect on Shh activity in an orthotopic Capan-2 model in nude mice. The effect of M402 on heparanase activity in vitro and on Capan-2 tumor samples isolated from treated and untreated mice was measured using an HTRF-FRET assay. RESULTS There was specific binding of M402 to Shh in vitro. Additionally, Shh signaling was inhibited in the presence of M402. Immunohistochemistry and RT-qPCR of Capan-2 tumor samples from animals treated with M402 also demonstrated reduction of Shh signaling via Gli, its targeted transcription factor. The degree of inhibition of heparanase activity, as measured in the HTRF assay, was affected by the size, structure, and sulfation pattern of the different heparin sulfate mimetics evaluated. M402 was the most potent inhibitor of heparanse activity in vitro from all compounds tested. In addition, treatment with M402 inhibited heparanase activity in the pancreatic tumor lysates in a dose-dependent manner. CONCLUSIONS M402 was shown in nonclinical studies to modulate tumor-stroma interactions involved in the metastatic, invasive, and desmoplastic pathways by simultaneously inhibiting two distinct pathways: Shh signaling and the activity of heparanase. M402 regulates a variety of polysaccharide-based binding proteins, which provides a rationale for the clinical investigation of M402 in a range of cancers.

Abstract 2269: M402 - A novel heparan sulfate proteoglycan mimetic targeting tumor-host interactions

Heparan sulfate proteoglycans (HSPGs) play important roles in tumorigenesis by mediating tumor-stromal interactions through the presentation of growth factors, cytokines, and chemokines critical for tumor progression, survival and metastasis. M402 is a rationally engineered, non-cytotoxic HSPG mimetic, designed to disrupt tumor-host interactions. M402 binds and inhibits multiple factors including VEGF, FGF2, SDF-1α, and P-selectin. A single 20 mg/kg subcutaneous (s.c.) dose of M402 effectively reduced seeding of B16F10 murine melanoma cells to the lung in a syngeneic experimental metastasis model. Chronic administration of M402, alone or in combination with cisplatin or docetaxel, inhibited spontaneous metastasis of orthotopically implanted 4T1 murine mammary carcinoma in this model. M402 treatment also normalized circulating levels of GR1 + immature myeloid cells and platelet counts in 4T1 metastatic tumor bearing mice. Fluorescently-labeled M402 exhibited selective accumulation in the primary tumor. Immunohistological analyses of primary tumor presented a decrease in microvessel density in M402-treated animals, suggesting anti-angiogenesis may be one of the mechanisms involved in vivo. Importantly, M402, as monotherapy or in combination with chemotherapeutics, also revealed significant survival benefits in this aggressive tumor model. These data demonstrate that targeting HSPG biology may provide a useful approach to attenuate multiple pathways involved in tumor progression and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2269.