Heparanase (Hpa1) is expressed by tumor cells and cells of the tumor microenvironment and functions to remodel the extracellular matrix (ECM) and regulate the bioavailability of ECM-bound factors that support tumor growth. Heparanase expression is upregulated in human carcinomas, sarcomas, and hematological malignancies, correlating with increased tumor metastasis, vascular density, and shorter postoperative survival of cancer patients, and encouraging the development of heparanase inhibitors as anti-cancer drugs. Among these are heparin/HS mimetics, the only heparanase-inhibiting compounds that are being evaluated in clinical trials. We have synthesized dicarboxylated oxy-heparins (DCoxHs) containing three carboxylate groups per split residue (DC-Hep). The resulting lead compound (termed XII) was upscaled, characterized, and examined for its effectiveness in tumor models. Potent anti-tumorigenic effects were obtained in models of pancreatic carcinoma, breast cancer, mesothelioma, and myeloma, yielding tumor growth inhibition (TGI) values ranging from 21 to 70% and extending the survival time of the mice. Of particular significance was the inhibition of spontaneous metastasis in an orthotopic model of breast carcinoma following resection of the primary tumor. It appears that apart from inhibition of heparanase enzymatic activity, compound XII reduces the levels of heparanase protein and inhibits its cellular uptake and activation. Heparanase-dependent and -independent effects of XII are being investigated. Collectively, our pre-clinical studies with compound XII strongly justify its examination in cancer patients.

Heparanase is the only human enzyme responsible for heparan sulfate (HS) breakdown, an activity that remodels the extracellular matrix (ECM) and strongly drives cancer metastasis and angiogenesis. Compelling evidence implies that heparanase promotes essentially all aspects of the tumorigenic process, namely, tumor initiation, vascularization, growth, metastasis, and chemoresistance. A key mechanism by which heparanase accelerates cancer progression is by enabling the release and bioavailability of HS-bound growth factors, chemokines, and cytokines, residing in the tumor microenvironment and supporting tumor growth and metastasis. The currently available heparanase inhibitors are mostly HS/heparin-like compounds that lack specificity and exert multiple off-target side effects. To date, only four such compounds have progressed to clinical trials, and none have been approved for clinical use. We have generated and characterized an anti-heparanase monoclonal antibody (A54 mAb) that specifically inhibits heparanase enzymatic activity (ECM degradation assay) and cellular uptake. Importantly, A54 mAb attenuates xenograft tumor growth and metastasis (myeloma, glioma, pancreatic, and breast carcinomas) primarily when administered (syngeneic or immunocompromised mice) in combination with conventional anti-cancer drugs. Co-crystallization of the A54 Fab fragment and the heparanase enzyme revealed that the interaction between the two proteins takes place adjacent to the enzyme HS/heparin binding domain II (HBDII; Pro271-Ala276), likely hindering heparanase from interacting with HS substrates via steric occlusion of the active site cleft. Collectively, we have generated and characterized a novel mAb that specifically neutralizes heparanase enzymatic activity and attenuates its pro-tumorigenic effects in preclinical models, paving the way for its clinical examination against cancer, inflammation, and other diseases.

Parkinson’s disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra and the presence of α-synuclein-positive inclusions known as Lewy bodies. Synphilin-1 is a protein of unknown function that interacts with α-synuclein and has been shown to exhibit cytoprotective effects in both in vitro and in vivo models. In this study, we investigated whether synphilin-1 is phosphorylated by pathological CDK5 and explored the consequences of this modification. Pathological activation of CDK5 occurs mainly through its association with the calpain-cleaved protein p25. Although CDK5 inhibition protects against neurodegeneration in pharmacological PD models, we now show that p25 levels are increased in PD brains. Furthermore, we demonstrate that CDK5, in conjunction with p25, directly phosphorylates synphilin-1, mainly at serine 566. This phosphorylation reduces synphilin-1′s interaction with SIAH1, leading to reduced ubiquitination and subsequent accumulation. We also observed that CDK5-phosphorylated synphilin-1 exhibits a reduced ability to interact with PINK1 and to promote basal levels of mitophagy. Consistent with these findings, the phosphorylation-mimicking synphilin-1 S566E shows decreased translocation to mitochondria, and synphilin-1 levels are reduced in the mitochondria of PD brains compared to age-matched controls. Finally, synphilin-1 S566E promotes retraction of neuronal processes. Taken together, our results suggest that phosphorylation by CDK5 disrupts synphilin-1′s interactions with its protein partners, rendering it more toxic and impairing its ability to support mitophagy and maintain neuronal process homeostasis. We hypothesize that phosphorylation of synphilin-1 by CDK5 may contribute to the pathogenesis of PD.

Parkinson’s disease (PD) is a common neurodegenerative disorder marked by progressive loss of dopaminergic neurons in the substantia nigra pars compacta and the accumulation of Lewy bodies, intracellular inclusions enriched in α-synuclein. Synphilin-1 interacts with α-synuclein, localizes to Lewy bodies, and has been implicated in inclusion formation and neuroprotection in cellular and animal models; however, its physiological function in vivo remains poorly defined. Here, we generated and characterized a synphilin-1 knockout (Sph-1 KO) mouse by targeted genetic deletion of the Sph-1 locus and performed a comprehensive phenotyping battery including behavioral testing as well as biochemical, histological, structural, and ultrastructural analyses. Sph-1 KO mice survived to nearly two years of age and showed normal body weight, lifespan, motor performance, learning and memory, anxiety-like behavior, attention, and gross brain morphology. Western blot analyses indicated that levels of α-synuclein and synaptic proteins were largely unchanged. While outer mitochondrial membrane proteins were unaffected, the mitochondrial matrix protein HSP60 was reduced, consistent with altered mitochondrial proteostasis in the absence of synphilin-1. Strikingly, histochemical analyses, magnetic resonance imaging, and electron microscopy revealed early-onset hydrocephalus in Sph-1 KO mice associated with severe loss and disorganization of motile ependymal cilia in the ventricular lining, a cell type that normally expresses high levels of synphilin-1. Ultrastructural and immunohistochemical analyses revealed disrupted ependymal architecture, mislocalization of acetylated α-tubulin to the cytoplasm, cellular swelling, and enlarged, aberrant mitochondria, whereas cortical neurons appeared largely structurally unaffected. Together, these findings identify synphilin-1 as a key regulator of microtubule organization and cytoskeletal/organelle homeostasis in ependymal cells, required to maintain motile ciliogenesis, cerebrospinal fluid flow, and ventricular integrity. This unexpected role for synphilin-1 in ciliated brain epithelia, along with a reduction in the critical mitochondrial chaperone HSP60, broadens our understanding of synphilin-1 biology and provides a new framework for its potential relevance to PD-associated pathology.