טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentWeissmann Marina
SubjectHeparanase Inhibitors and Neutralizing Antibodies in
Hematological Malignancies
DepartmentDepartment of Medicine
Supervisor Professor Israel Vlodavsky
Full Thesis textFull thesis text - English Version


Abstract

Heparanase is the only enzyme in mammals that cleaves heparan sulfate (HS) side chains of heparan sulfate proteoglycans (HSPG) in the extracellular matrix (ECM) and basement membrane underlying epithelial and vascular endothelial cells. This activity results in structural alteration of the ECM, associating with enhanced cellular invasion and metastasis. Heparanase expression is induced in carcinomas, often associating with enhanced tumor metastasis and poor prognosis, thus encouraging the development of heparanase inhibitors as anti-cancer drugs. Unlike carcinomas, the function of heparanase in hematological malignancies (except myeloma) was not investigated in depth. In my thesis, we provide evidence that heparanase is expressed by human follicular and diffused non-Hodgkin's B-lymphomas. Furthermore, we describe for the first time the development of heparanase-neutralizing monoclonal antibodies (mAb 9E8 and mAb H1023) which inhibit cellular invasion and tumor metastasis, the hallmarks of heparanase activity. Utilizing luciferase labelled lymphoma and myeloma cells we show that the heparanase neutralizing mAbs inhibit lymphoma tumor load in mouse bones, associating with reduced cell proliferation and angiogenesis. Notably, we found that some lymphoma cells lack intrinsic heparanase activity but the tumor xenograft shows typical high heparanase activity likely contributed by host cells composing the tumor microenvironment. Thus, the neutralizing mAbs attenuate lymphoma growth by targeting heparanase in the tumor microenvironment. We also show that a synthetic saccharide-based heparanase inhibitor, PG545, is even more efficient in attenuating lymphoma and myeloma growth. Subsequent analyses revealed that unlike other heparin mimetics or heparanase neutralizing antibodies, PG545 affects determinants critical for lymphoma cell proliferation and survival in-vitro. These include induction of p21 gene expression and cell cycle arrest, reduced Myc and Bcl-6 expression and reduced Src and Akt phosphorylation. Consequently, PG545 treatment elicited a prominent apoptotic cell death response evident by propidium iodide (PI) and Annexin V staining, and increased cleaved caspase-3 and -8 and cleaved PARP levels, classical characteristics of apoptotic cell death. Importantly, PG545 did not elicit apoptosis of naïve B cells but rather improved their survival in-vitro. Likewise, PG545 did not affect the survival of adherent carcinoma cells except for U87 glioma cells. Mechanistically, we found that PG545 induces ER stress is both lymphoma and glioma cells and inhibition of ER stress rescued cells from PG545-induced apoptosis. We also found that PG545 activates the NFκB pathway, and inhibitors of NFκB attenuated cell death evoked by PG545. Notably, we found that ER stress response is interconnected with NFκB because inhibitor of PERK, an upstream initiator of ER stress, also inhibits NFκB activation. The activation of ER stress was also evident in lymphoma xenografts treated with PG545. Taken together, these results imply that heparanase supports lymphoma growth, and heparanase inhibitors (mAbs, PG545) attenuate lymphoma growth in pre-clinical mouse models. Lymphoma cells appear to be highly sensitive to PG545, likely due to induction of a strong and persistent ER-stress response, leading to apoptotic cell death. These findings may assist in directing PG545 to specific indication (i.e., lymphomas) as it enters advanced phase III clinical trials.