One of the first steps towards elucidating the biological function of a putative transcriptional regulator is to ascertain its preferred DNA-binding sequences. This may be rapidly and effectively achieved through the application of a combinatorial approach, one involving very large numbers of randomized oligonucleotides and reiterative selection and amplification steps to enrich for high-affinity nucleic acid-binding sequences. Previously, we had developed the novel combinatorial approach Restriction Endonuclease Protection, Selection and Amplification (REPSA), which relies not on the physical separation of ligand-nucleic acid complexes but instead selects on the basis of ligand-dependent inhibition of enzymatic template inactivation, specifically cleavage by type IIS restriction endonucleases. Thus, no prior knowledge of the ligand is required for REPSA, making it more amenable for discovery purposes. Here we describe using REPSA, massively parallel sequencing, and bioinformatics to identify the preferred DNA-binding sites for the transcriptional regulator SbtR, encoded by the TTHA0167 gene from the model extreme thermophile Thermus thermophilus HB8. From the resulting position weight matrix, we can identify multiple operons potentially regulated by SbtR and postulate a biological role for this protein in regulating extracellular transport processes. Our study provides a proof-of-concept for the application of REPSA for the identification of preferred DNA-binding sites for orphan transcriptional regulators and a first step towards determining their possible biological roles.

Gliomas are the largest category of primary malignant brain tumors in adults, and glioblastomas account for nearly half of malignant gliomas. Glioblastomas are notoriously aggressive and drug-resistant, with a very poor 5 year survival rate of about 5%. New approaches to treatment are thus urgently needed. We previously identified an enzyme of fatty acid metabolism, very long-chain acyl-CoA synthetase 3 (ACSVL3), as a potential therapeutic target in glioblastoma. Using the glioblastoma cell line U87MG, we created a cell line with genomic deletion of ACSVL3 (U87-KO) and investigated potential mechanisms to explain how this enzyme supports the malignant properties of glioblastoma cells. Compared to U87MG cells, U87-KO cells grew slower and assumed a more normal morphology. They produced fewer, and far smaller, subcutaneous xenografts in nude mice. Acyl-CoA synthetases, including ACSVL3, convert fatty acids to their acyl-CoA derivatives, allowing participation in diverse downstream lipid pathways. We examined the effect of ACSVL3 depletion on several such pathways. Fatty acid degradation for energy production was not affected in U87-KO cells. Fatty acid synthesis, and incorporation of de novo synthesized fatty acids into membrane phospholipids needed for rapid tumor cell growth, was not significantly affected by lack of ACSVL3. In contrast, U87-KO cells exhibited evidence of altered sphingolipid metabolism. Levels of ceramides containing 18-22 carbon fatty acids were significantly lower in U87-KO cells. This paralleled the fatty acid substrate specificity profile of ACSVL3. The rate of incorporation of stearate, an 18-carbon saturated fatty acid, into ceramides was reduced in U87-KO cells, and proteomics revealed lower abundance of ceramide synthesis pathway enzymes. Sphingolipids, including gangliosides, are functional constituents of lipid rafts, membrane microdomains thought to be organizing centers for receptor-mediated signaling. Both raft morphology and ganglioside composition were altered by deficiency of ACSVL3. Finally, levels of sphingosine-1-phosphate, a sphingolipid signaling molecule, were reduced in U87-KO cells. We conclude that ACSVL3 supports the malignant behavior of U87MG cells, at least in part, by altering cellular sphingolipid metabolism.

Decreasing the expression of very long-chain acyl-CoA synthetase 3 (ACSVL3) in U87MG glioblastoma cells by either RNA interference or genomic knockout (KO) significantly decreased their growth rate in culture, as well as their ability to form rapidly growing tumors in mice. U87-KO cells grew at a 9-fold slower rate than U87MG cells. When injected subcutaneously in nude mice, the tumor initiation frequency of U87-KO cells was 70% of that of U87MG cells, and the average growth rate of tumors that did form was decreased by 9-fold. Two hypotheses to explain the decreased growth rate of KO cells were investigated. Lack of ACSVL3 could reduce cell growth either by increasing apoptosis, or via effects on the cell cycle. We examined intrinsic, extrinsic, and caspase-independent apoptosis pathways; none were affected by lack of ACSVL3. However, significant differences in the cell cycle were seen in KO cells, suggesting arrest in S-phase. Levels of cyclin-dependent kinases 1, 2, and 4 were elevated in U87-KO cells, as were regulatory proteins p21 and p53 that promote cell cycle arrest. In contrast, lack of ACSVL3 reduced the level of the inhibitory regulatory protein p27. Gamma-H2AX, a marker of DNA double strand breaks, was elevated in U87-KO cells, while pH3, a mitotic index marker, was reduced. Previously reported alterations in sphingolipid metabolism in ACSVL3-depleted U87 cells may explain the effect of KO on cell cycle. These studies reinforce the notion that ACSVL3 is a promising therapeutic target in glioblastoma.

The objective of this study was to identify inhibitors of the enzyme Very Long Chain Acyl‐CoA Synthetase 3 (ACSVL3; SLC27A3). ACSVL3 is present in human glioma cells but not healthy glial cells, and knocking out ACSVL3 in the U87 human glioblastoma cell line has previously been shown to result in slower growth rate, fewer and smaller tumors formed in xenografts, and disrupted Akt signaling. Therefore, an inhibitor of its activity is desired for both understanding its mechanism of action in tumorigenesis and for potential chemotherapeutic usage. Several drugs and compounds that were previously shown to inhibit the homologous enzyme ACSVL1 (SLC27A2) were studied to see if they had similar inhibitory effects on ACSVL3. COS‐1 cells stably expressing ACSVL1 or ACSVL3 were harvested and cell pellets assayed for activation of [1‐14C]stearic acid (C18:0, a preferred ACSVL3 substrate) to its CoA derivative in the absence and presence of inhibitors. We also developed a fluorescence‐based assay using COS‐1 cells stably expressing a chimeric ACSVL1/3 protein containing the regulatory domain of ACSVL1 and the catalytic domain of ACSVL3. The regulatory domain of ACSVL1 conferred the ability to uptake the fluorescent fatty acid analog, C1‐BODIPY‐C12, in a process requiring functional ACSVL3 enzyme activity. Most ACSVL1 inhibitors tested also inhibited ACSVL3 using in vitro acyl‐CoA synthetase assays. These compounds inhibited BODIPY fatty acid uptake in COS‐1 cells expressing chimeric ACSVL1/3 in a dose‐dependent manner. One compound, Grassofermata/CB5, was a significantly more potent inhibitor of stearic acid activation by ACSVL3 than by ACSVL1 and was tested further. CB5 was the most potent inhibitor of stearic acid activation in U87 glioma cells. To assess effects on cell growth, CB5 was added to U87 cells in culture. At a concentration of 3 μM, CB5 drastically slowed cell growth rate but did not lead to increased cell mortality. The effects of CB5 on cell growth were reversible; when CB5 was removed from cells after several days of treatment, they reverted to a faster growth rate. A dose response curve of cell growth to CB5 concentration revealed a narrow therapeutic window. In conclusion, many known inhibitors of ACSVL1 are also potent inhibitors of ACSVL3, and the small molecule CB5 shows promise as a potential chemotherapeutic drug and tool to understand the mechanism(s) underlying the role of ACSVL3 in glioma.Support or Funding InformationKennedy Krieger institutional funds.

Expression of ACSVL3, an acyl‐CoA synthetase that activates long‐ to very long‐chain fatty acids (FA), is elevated in malignant gliomas and in glioma cells. Knocking down ACSVL3 expression in U87 glioma cells by RNA interference decreased their in vitro and in vivo malignant properties. To determine whether the beneficial effects of ACSVL3 depletion were mediated by changes in downstream lipid metabolism, we investigated the metabolic fate of FAs in control and ACSVL3 knockdown (KD) U87 cells. Beta‐oxidation of both C16:0 and C24:0 was decreased in KD cells. There was decreased incorporation of radiolabeled FAs (C16:0, C18:0, C18:1, C18:2n‐6, C20:4n‐6, C22:6n‐3) into phospholipids PI, PS, and PE, but not into PC. ACSVL3 KD had little effect on incorporation of several FAs into most neutral lipids. However, increased incorporation of C20:4n‐6 and C22:6n‐ 3 into triacylglycerol and cholesterol esters was seen in KD cells. ACSVL3 KD altered the total FA profile of U87 cells, and KD cells had decreased rates of de novo FA synthesis from acetate. Thus, depletion of ACSVL3 had multiple effects on U87 glioma cell lipid metabolism. Further study is needed to determine whether any of these changes are critical for switching these cells from a malignant to a more normal phenotype. Supported by NS062043.