The Heparanase Inhibitor PG545 is a Potent Anti-Lymphoma Drug - Mode of Action

Introduction: It is now well recognized that heparanase, an endo-β-D-glucuronidase capable of cleaving heparan sulfate (HS) side chains at a limited number of sites, promotes tumorigenesis by diverse mechanisms. Compelling evidence strongly implies that heparanase is a viable target for cancer therapy, thus encouraging the development of heparanase inhibitors as anti-cancer therapeutics. We have reported recently that heparanase is detectable in ~50% of B-cell lymphomas. Notably, lymphoma tumor growth was found to be highly sensitive to heparanase inhibitors, including heparanase-neutralizing antibodies and HS-mimetics, such as PG545. PG545 was highly effective in lymphoma models as a single agent, thus making this compound attractive for further drug development. Here, we examined the mode of action of PG545 (pixatimod).

Materials and methods: Human lymphoma cell lines were treated with PG545 for various time periods, and analyzed for signal transduction, apoptosis, cell cycle and viability by Western blot analysis, Real-Time PCR, immunofluorescence, propidium iodide and Annexin V staining, FACS analysis, and ATP quantification. Electron microscopy micrographs of PG545 treated cells were taken. Immunohistochemistry was performed on sections derived from control and PG545-treated lymphoma xenografts.

Results and discussion: We found that PG545 exhibits a strong anti-lymphoma effect, reducing lymphoma cell viability and eliciting cell apoptosis. Notably, we found that this anti-lymphoma effect involves ER stress response that was accompanied by increased autophagy. The persistent ER stress evoked by PG545 is held responsible for cell apoptosis because apoptotic cell death was attenuated by an inhibitor of PERK, a molecular effector of ER stress. Importantly, PG545 has no such apoptotic effect on naïve splenocytes, further encouraging the development of this compound as an anti-lymphoma drug. Notably, we found that PG545 also elicits apoptosis in lymphoma cells that are devoid of heparanase activity (i.e., Raji), indicating that the drug also exerts heparanase-independent function(s) that together underlie its high potency as anti-lymphoma compound.

Conclusion: We have characterized a new mechanism by which PG545 restrains tumor growth, namely eliciting persistent ER stress response that leads to apoptotic cell death. ER has been proposed as a potential target for anticancer therapy in several tumor types given that cancer cells appear to use the unfolded protein response (UPR) system to escape cell death. This mechanism, combined with its anti-heparanase activity, may underlie the potent anti-lymphoma effect of PG545.