SST0001, a chemically modified heparin, inhibits myeloma growth and angiogenesis via disruption of the heparanase/syndecan-1 axis

Abstract

Purpose: Heparanase promotes myeloma growth, dissemination, and angiogenesis through modulation of the tumor microenvironment, thus highlighting the potential of therapeutically targeting this enzyme. SST0001, a nonanticoagulant heparin with antiheparanase activity, was examined for its inhibition of myeloma tumor growth in vivo and for its mechanism of action.

Experimental design: The ability of SST0001 to inhibit growth of myeloma tumors was assessed using multiple animal models and a diverse panel of human and murine myeloma cell lines. To investigate the mechanism of action of SST0001, pharmacodynamic markers of angiogenesis, heparanase activity, and pathways downstream of heparanase were monitored. The potential use of SST0001 as part of a combination therapy was also evaluated in vivo.

Results: SST0001 effectively inhibited myeloma growth in vivo, even when confronted with an aggressively growing tumor within human bone. In addition, SST0001 treatment causes changes within tumors consistent with the compound's ability to inhibit heparanase, including downregulation of HGF, VEGF, and MMP-9 expression and suppressed angiogenesis. SST0001 also diminishes heparanase-induced shedding of syndecan-1, a heparan sulfate proteoglycan known to be a potent promoter of myeloma growth. SST0001 inhibited the heparanase-mediated degradation of syndecan-1 heparan sulfate chains, thus confirming the antiheparanase activity of this compound. In combination with dexamethasone, SST0001 blocked tumor growth in vivo presumably through dual targeting of the tumor and its microenvironment.

Conclusions: These results provide mechanistic insight into the antitumor action of SST0001 and validate its use as a novel therapeutic tool for treating multiple myeloma.

Figure 1. SST0001 is a potent inhibitor of myeloma growth in vivo

A. SST0001 inhibits recombinant heparanase-mediated digestion of ³⁵S-labeled heparan sulfate in a dose dependent manner. B. SST0001 (30 mg/kg/d, 28 days), delivered by Alzet osmotic pumps inhibited subcutaneous tumor growth in the SCID (RPMI-8226 or MM.1S cells) and syngeneic (MPC-11 cells) models of myeloma. C. Growth of KMS-11 (cells injected subcutaneously, top panel) or RPMI-8226 (tumor fragments implanted subcutaneously, bottom panel) myeloma tumors was inhibited by twice daily subcutaneous injection of SST0001 (120 mg/kg/d, total daily dose) for 22–30 days. *p<0.005 vs controls, by Student’s t test. D. Quantification of human kappa immunoglobulin light chain in murine sera (top panel) and bioluminescent imaging (bottom panel) were used to determine tumor burden in the SCID-hu model of myeloma; mice receiving SST0001 (30 mg/kg/day via Alzet pump) displayed significantly lower tumor burden than control mice as assessed by both measures.

Figure 2. SST0001 inhibits angiogenesis and HGF and VEGF expression in vivo

A. SCID mice bearing subcutaneous myeloma tumors formed by RPMI-8226 or CAG cells were treated with SST0001 or saline for 28 days. After treatment, microvessel density was quantified (left panel) in sections of the tumor tissue using anti-CD34 immunohistochemical analysis (representative images, right panel). SST0001 significantly inhibited tumor vascularity. B. Mice bearing subcutaneous tumors formed by HPSE-high cells were treated with either saline or SST0001 (30 mg/kg/d, delivered by Alzet osmotic pumps) for 28 days. Following euthanasia of animals, the tumors were removed and subjected to immunohistochemical analysis of HGF and VEGF. In tumors from animals treated with SST0001, the intra-tumoral levels of both HGF and VEGF were reduced dramatically as compared to animals treated with saline. C. Levels of VEGF in the conditioned medium from HPSE-low or HPSE-high CAG cells treated with saline or 125 µg/ml SST0001 (6.75µM) were measured by ELISA. SST0001 treatment significantly decreased the accumulation of VEGF in HPSE-high cells (n=3 for each group).

Figure 3. SST0001 inhibits heparanase activity in tumor cells expressing high levels of the enzyme

A. In a cell free system, SST0001 blocked heparanase-mediated digestion of the heparan sulfate chains of partially purified syndecan-1(SDC1). The proteoglycan was incubated with recombinant heparanase (rHPSE) in the presence or absence of SST0001 followed by western blotting. Note that in the presence of SST0001, the molecular size of syndecan-1 is larger than in the absence of the inhibitor. B. HPSE-high cells were treated overnight with increasing concentration of SST0001; the cells were extracted and analyzed by western blotting. SST0001 inhibited heparanase digestion of the heparan sulfate chains of syndecan-1, resulting in the high molecular weight form of syndecan-1. C. The amount of syndecan-1 shed into the conditioned medium of HPSE-low or HPSE-high cells treated with SST0001 (125 µg/mL; 6.75µM) or saline was quantified by ELISA. SST0001 significantly inhibited shedding of syndecan-1 from HPSE-high cells (n=3 for each group). D. Conditioned medium from HPSE-low cells or HPSE-high cells treated with SST0001 (6.75µM) or saline was subjected to western blot analysis of syndecan-1. Results confirm that levels of shed syndecan-1 are reduced following treatment of HPSE-high cells with SST0001. Note also that the molecular size of shed syndecan-1 in conditioned medium from HPSE-high cells treated with SST0001 is also larger than that found in untreated cells, again confirming the ability of the compound to block the activity of heparanase.

Figure 4. SST0001 blocks heparanase-mediated MMP-9 expression and ERK signaling

A. Mice bearing tumors formed by HPSE-high cells injected subcutaneously were treated with SST0001 or saline. Tumors were excised and immunohistochemical analysis for MMP-9 revealed diminished levels of expression within SST0001 treated tumors as compared to tumors from animals treated with saline. B. Conditioned media from HPSE-low cells or HPSE-high cells treated with SST0001 (125µg/ml; 6.75µM) or saline were subjected to gelatin zymography. HPSE-high cells treated with SST0001 had significantly reduced levels of MMP-9 activity in their medium as compared to cells treated with saline. C. ERK signaling was assessed by western blotting of extracts from myeloma cell lines treated SST0001 or saline by western blotting for phosphorylated ERK 1/2. Heparanase inhibition with SST0001 caused a dose-dependent reduction in ERK signaling.

Figure 5. SST0001 in combination with dexamethasone is a potent inhibitor of myeloma tumor growth in vivo

A. Dexamethasone resistant MM.1R tumors were established subcutaneously in SCID mice and mice were injected with either saline, a low dose of SST0001 (60 mg/kg/day, as compared to 120 mg/kg/day used for studies in Fig. 1B), dexamethasone (1 mg/kg/day) or SST0001+dexamethasone for 14 days. Mice receiving combination therapy had significantly smaller tumors compared to control mice (n=8 for each group). B. Murine MPC-11 tumors were established in syngeneic BALB/c mice. Mice were then treated with either saline, SST0001 (60 mg/kg/day), dexamethasone (1 mg/kg/day) or SST0001+dexamethasone for 14 days. At the doses utilized, combination therapy potently inhibited tumor growth and was significantly more effective than single agent therapies when compared to saline treated controls (n=6–10 for each group). In both experiments, the effect of the drug combination was additive as determined using the GLM procedure.

Figure 6. SST0001 inhibits heparanase and its downstream effectors to block myeloma growth and angiogenesis

Heparanase activity enhances expression of VEGF, HGF and MMP-9 and stimulates shedding of syndecan-1, thereby fueling an aggressive myeloma phenotype. Through inhibition of enzyme activity, SST0001 shuts down these multiple pathways that together stimulate myeloma tumor growth.