Abstract
Background: Identifying biomarkers that monitor clinical response to cancer therapies is crucial. Tetrathiomolybdate (TM) is a highly specific, high-affinity, orally available copper-binding compound that inhibits CuZn SOD1, leading to robust anti-angiogenic and anti-tumor effects. TM is currently being evaluated in Phase II cancer trials including for relapsed and refractory multiple myeloma (MM). TM’s inhibition of angiogenesis has been attributed to depletion of systemic Cu, which is known to affect multiple key angiogenesis regulators; however, in mice, inhibition of angiogenesis by TM occurred before a measurable decrease in systemic Cu. We have recently shown that in MM, circulating endothelial progenitor cell (EPC) levels serve as a reliable biomarker of disease severity which covaries with tumor load and disease progression. Furthermore, recent evidence from our laboratory and by others indicates that EPCs display evidence of clonality and are genetically related to MM cells. In the present study, the relationship between anti-angiogenic and anti-SOD1 activities of TM were compared by determining its effects on:
circulating endothelial progenitor cells (EPCs) in bonnet macaques (M. radiata); on angiogenesis and SOD1 activity in blood cells from mice and humans.
Methods: TM was manufactured with >99% purity using a proprietary process (choline salt: ATN-224). For animal studies, three female and three male young adult monkeys were treated with TM (0.5 mg/kg, s.c.) daily for 22 days, followed by a 26 day drug holiday, and a second course of drug. Blood was drawn from the cephalic vein, separated by Ficoll, and red cell lysis. EPCs were identified as CD31+/CD133+/CD45− weak cells using 3-color flow cytometry. For SOD activity, blood from normal control subjects was incubated with TM for 5.5 hr at 37°C. SOD activity was measured in 30 μg of protein from treated blood pellets using an SOD Activity Kit.
Results: Administration of TM to the monkeys resulted in a rapid, profound, and reversible decrease in circulating EPCs without significant toxicity. The levels of EPCs increased initially at day 6 in 5 of the 6 subjects, and then decreased to 7% of baseline at day 21. The EPC levels rebounded to normal levels once treatment was discontinued and dropped again after treatment was reinstated. Other hematopoietic parameters (RBC, WBC, platelet counts, and hemoglobin) were not affected by treatment. Although we did not measure blood cell SOD in the macaque study, substantial inhibition of blood cell SOD is associated with hematologic toxicities which did not occur in these animals, suggesting that RBC SOD activity is inhibited at higher doses than those needed for anti-angiogenic activity. TM treatment inhibited SOD activity in blood cell pellets obtained from human controls in a dose dependent manner, with an IC50 3 μM.
Conclusion: These results show, for the first time, that in vivo effects of TM include suppression of circulating EPC levels. Since circulating EPCs are genetically related to tumor cells in MM, at least the anti-MM effects of TM may depend on its suppressive effects on EPCs as well as on angiogenesis in general. Furthermore, EPCs are a sensitive indicator of TM actions since their reversible modulation correlated with drug therapy. Lastly, macaques may represent a reliable model of human response to determine drug effects on EPCs and angiogenesis.
In vivo upregulation of CD95 and CD95L causes synergistic inhibition of angiogenesis by TSP1 peptide and metronomic doxorubicin treatment
