Heparanase Increases Podocyte Survival and Autophagic Flux after Adriamycin-Induced Injury.

The kidney glomerular filtration barrier (GFB) is enriched with heparan sulfate (HS) proteoglycans, which contribute to its permselectivity. The endoglycosidase heparanase cleaves HS and hence appears to be involved in the pathogenesis of kidney injury and glomerulonephritis. We have recently reported, nonetheless, that heparanase overexpression preserved glomerular structure and kidney function in an experimental model of Adriamycin-induced nephropathy. To elucidate mechanisms underlying heparanase function in podocytes-key GFB cells, we utilized a human podocyte cell line and transgenic mice overexpressing heparanase. Notably, podocytes overexpressing heparanase (H) demonstrated significantly higher survival rates and viability after exposure to Adriamycin or hydrogen peroxide, compared with mock-infected (V) podocytes. Immunofluorescence staining of kidney cryo-sections and cultured H and V podocytes as well as immunoblotting of proteins extracted from cultured cells, revealed that exposure to toxic injury resulted in a significant increase in autophagic flux in H podocytes, which was reversed by the heparanase inhibitor, Roneparstat (SST0001). Heparanase overexpression was also associated with substantial transcriptional upregulation of autophagy genes BCN1, ATG5, and ATG12, following Adriamycin treatment. Moreover, cleaved caspase-3 was attenuated in H podocytes exposed to Adriamycin, indicating lower apoptotic cell death in H vs. V podocytes. Collectively, these findings suggest that in podocytes, elevated levels of heparanase promote cytoprotection.

Roneparstat: Development, Preclinical and Clinical Studies.

A growing interest around heparanase and its role in cancer, inflammation and other diseases prompted the identification of specific inhibitors of this enzyme and the exploration of their potential therapeutic role. Roneparstat, a 15-25 kDa N-acetylated and glycol split heparin, is one of the most potent and widely studied heparanase inhibitors. These studies generated a large body of data, which allowed to characterize Roneparstat properties and to endorse its potential therapeutic role. Multiple Myeloma represents the indication that most of the studies, including the phase I clinical trial, addressed. However, Roneparstat antitumor activity activity has been documented in other cancers, and in non-oncological conditions.In addition, assessing Roneparstat activity in different experimental models contributed to understanding heparanase role and the biological factors that may be affected by heparanase inhibition in more detail. Finally, some studies elucidated the molecular mechanisms regulating the enzyme-inhibitor kinetics, thus providing important data for the identification and design of new inhibitors.The objective of this chapter is to provide a comprehensive overview of the most significant studies involving Roneparstat and discuss its potential role in therapy.

Kinetic analysis and molecular modeling of the inhibition mechanism of roneparstat (SST0001) on human heparanase.

Heparanase is a ß-d-glucuronidase which cleaves heparan sulfate chains in the extracellular matrix and on cellular membranes. A dysregulated heparanase activity is intimately associated with cell invasion, tumor metastasis and angiogenesis, making heparanase an attractive target for the development of anticancer therapies. SST0001 (roneparstat; Sigma-Tau Research Switzerland S.A.) is a non-anticoagulant 100% N-acetylated and glycol-split heparin acting as a potent heparanase inhibitor, currently in phase I in advanced multiple myeloma. Herein, the kinetics of heparanase inhibition by roneparstat is reported. The analysis of dose-inhibition curves confirmed the high potency of roneparstat (IC50 ≈ 3 nM) and showed, at higher concentrations, a Hill coefficient consistent with the engagement of two molecules of inhibitor. A homology model of human heparanase GS3 construct was built and used for docking experiments with inhibitor fragments. The model has high structural similarity with the recently reported crystal structure of human heparanase. Different interaction schemes are proposed, which support the hypothesis of a complex binding mechanism involving the recruitment of one or multiple roneparstat chains, depending on its concentration. In particular, docking solutions were obtained in which (i) a single roneparstat molecule interacts with both heparin-binding domains (HBDs) of heparanase or (ii) two fragments of roneparstat interact with either HBD-1 or HBD-2, consistent with the possibility of different inhibitor:enzyme binding stoichiometries. This study provides unique insights into the mode of action of roneparstat as well as clues of its interaction with heparanase at a molecular level, which could be exploited to design novel potential inhibitor molecules.

Novel N-acetyl-Glycol-split heparin biotin-conjugates endowed with anti-heparanase activity.

Heparanase is regarded as a promising target for anticancer drugs and Ronepastat is one of the most promising heparanase inhibitors insert in clinical study for Multiple Myeloma Therapy. To improve its pharmacokinetic/pharmacodynamic profile, as well to have an antidote able to neutralize its activity in case of over dosages or intolerance, a new class of its derivatives was obtained inserting non-carbohydrate moieties of different length between the polysaccharide chain and biotin or its derivatives. In vitro these novel derivatives maintain the anti-heparanase activity without induced toxicity. The newly synthesized compounds retained the ability to attenuate the growth of CAG myeloma tumors in mice with potency similar, or in one case even higher than that of the reference compound Roneparstat as well as inhibited metastatic dissemination (lung colonization) of murine B16-F10 melanoma cells in vivo.

Antitumor efficacy of the heparan sulfate mimic roneparstat (SST0001) against sarcoma models involves multi-target inhibition of receptor tyrosine kinases.

The heparan sulfate (HS) mimic/heparanase inhibitor roneparstat (SST0001) shows antitumor activity in preclinical sarcoma models. We hypothesized that this 100% N-acetylated and glycol-split heparin could interfere with the functions of several receptor tyrosine kinases (RTK) coexpressed in sarcomas and activated by heparin-binding growth factors. Using a phospho-proteomic approach, we investigated the drug effects on RTK activation in human cell lines representative of different sarcoma subtypes. Inhibition of FGF, IGF, ERBB and PDGF receptors by the drug was biochemically and functionally validated. Roneparstat counteracted the autocrine loop induced by the COL1A1/PDGFB fusion oncogene, expressed in a human dermatofibrosarcoma protuberans primary culture and in NIH3T3COL1A1/PDGFB transfectants, inhibiting cell anchorage-independent growth and invasion. In addition, roneparstat inhibited the activation of cell surface PDGFR and PDGFR-associated FAK, likely contributing to the reversion of NIH3T3COL1A1/PDGFB cell transformed and pro-invasive phenotype. Biochemical and histological/immunohistochemical ex vivo analyses confirmed a reduced activation of ERBB4, EGFR, INSR, IGF1R, associated with apoptosis induction and angiogenesis inhibition in a drug-treated Ewing's sarcoma family tumor xenograft. The combination of roneparstat with irinotecan significantly improved the antitumor effect against A204 rhabdoid xenografts resulting in a high rate of complete responses and cures. These findings reveal that roneparstat exerts a multi-target inhibition of RTKs relevant in the pathobiology of different sarcoma subtypes. These effects, likely cooperating with heparanase inhibition, contribute to the antitumor efficacy of the drug. The study supports heparanase/HS axis targeting as a valuable approach in combination therapies of different sarcoma subtypes providing a preclinical rationale for clinical investigation.

Targeting heparanase overcomes chemoresistance and diminishes relapse in myeloma.

In most myeloma patients, even after several rounds of intensive therapy, drug resistant tumor cells survive and proliferate aggressively leading to relapse. In the present study, gene expression profiling of tumor cells isolated from myeloma patients after sequential rounds of chemotherapy, revealed for the first time that heparanase, a potent promoter of myeloma growth and progression, was elevated in myeloma cells that survived therapy. Based on this clinical data, we hypothesized that heparanase was involved in myeloma resistance to drug therapy. In several survival and viability assays, elevated heparanase expression promoted resistance of myeloma tumor cells to chemotherapy. Mechanistically, this enhanced survival was due to heparanase-mediated ERK signaling. Importantly, use of the heparanase inhibitor Roneparstat in combination with chemotherapy clearly diminished the growth of disseminated myeloma tumors in vivo. Moreover, use of Roneparstat either during or after chemotherapy diminished regrowth of myeloma tumors in vivo following therapy. These results provide compelling evidence that heparanase is a promising, novel target for overcoming myeloma resistance to therapy and that targeting heparanase has the potential to prevent relapse in myeloma and possibly other cancers.

Chemotherapy induces expression and release of heparanase leading to changes associated with an aggressive tumor phenotype.

High heparanase expression is associated with enhanced tumor growth, angiogenesis, and metastasis in many types of cancer. However, the mechanisms driving high heparanase expression are not fully understood. In the present study, we discovered that drugs used in the treatment of myeloma upregulate heparanase expression. Frontline anti-myeloma drugs, bortezomib and carfilzomib activate the nuclear factor-kappa B (NF-κB) pathway to trigger heparanase expression in tumor cells. Blocking the NF-κB pathway diminished this chemotherapy-induced upregulation of heparanase expression. Activated NF-κB signaling was also found to drive high heparanase expression in drug resistant myeloma cell lines. In addition to enhancing heparanase expression, chemotherapy also caused release of heparanase by tumor cells into the conditioned medium. This soluble heparanase was taken up by macrophages and triggered an increase in TNF-α production. Heparanase is also taken up by tumor cells where it induced expression of HGF, VEGF and MMP-9 and activated ERK and Akt signaling pathways. These changes induced by heparanase are known to be associated with the promotion of an aggressive tumor phenotype. Importantly, the heparanase inhibitor Roneparstat diminished the uptake and the downstream effects of soluble heparanase. Together, these discoveries reveal a novel mechanism whereby chemotherapy upregulates heparanase, a known promoter of myeloma growth, and suggest that therapeutic targeting of heparanase during anti-cancer therapy may improve patient outcome.