Toward pharmacologic therapy for glioblastoma: Characterization of the very long-chain acyl-CoA synthetase 3 (ACSVL3) inhibitor Grassofermata

Malignant brain tumors are among the most aggressive and difficult to treat human cancers. Glioblastomas (World Health Organization grade IV gliomas) are particularly lethal and refractory to treatment. Few drugs exist that are even somewhat effective. Our investigation of the physiologic role of fatty acid (FA) activating enzymes (acyl-CoA synthetase; ACS) identified an ACS that was widely expressed in gliomas but not in normal glial cells. Depletion of this enzyme, ACSVL3 (very long-chain ACS3), by knockdown or knockout decreased the malignant behavior of several glioma cell models including U87MG and Mayo-22 cells both in culture and when grown as xenografts. Hypothesizing that ACSVL3 is a potential therapeutic target in glioma, we conducted a search for inhibitors of this enzyme and found that CB5 (grassofermata) was a promising candidate. Treating U87MG glioma cells with CB5 slowed growth in monolayer culture; the growth rate was similar to that seen in cells in which ACSVL3 was either knocked down or knocked out. CB5 inhibited growth in a dose-dependent manner over a narrow range, and concentrations above 10 M were toxic. Treatment at the lower dose of 3 M inhibited growth of U87MG cells but was reversible, suggesting that this dose was not toxic. CB5- treated U87MG cells exhibited an altered morphology with a larger size and longer projections. In contrast, normal human fibroblasts treated with 10 M CB5, a concentration that was toxic to U87MG cells, showed no effect on either growth rate or morphology. Treating U87MG cells with 3 M CB5 induced differentiation as shown by increased expression of the astrocyte-specific marker glial fibrillary acidic protein (GFAP). In contrast, GFAP levels remained low in ACSVL3 knockdown cells. CB5- treated U87MG cells were less invasive, and thus less malignant, than either untreated cells or ACSVL3 knockout cells when assessed by a scratch wound healing assay. Acute treatment of U87MG cells with 3 M CB5 decreased the ability of these cells to degrade FA of differing chain lengths from 16-24 carbons by {beta}-oxidation, suggesting that decreased ACS enzyme activity contributes at least in part to the drugs mechanism of action. NOD/SCID mice receiving up to 32 mg/kg/day CB5 by intraperitoneal injection showed no obvious side effects, suggesting that the drug was well-tolerated. Xenografts induced by subcutaneous injection of U87MG cells in the flanks of NOD/SCID mice were allowed to grow for 8 days after which half of the mice were treated with 2 mg/kg/day CB5. After 7 days of treatment, xenograft growth slowed in the treated mice and by 12 days tumor size had begun to decrease, suggesting therapeutic efficacy. When a similar study was done using xenografts induced by subcutaneous injection of Mayo-22 cells, which are maintained as subcutaneous tumors in mice rather than in cell culture, the effect of CB5 on tumor growth or weight at sacrifice was not statistically significant. The results of these studies suggest that CB5 may have therapeutic value in malignant glioma. Additional studies using other glioma models and other drugs chemically related to CB5 seem warranted.