Heterogeneity in Simvastatin-Induced Cytotoxicity in AML Is Related to Differential Ras-Isoprenylation, Rather Than to Blockade of Cholesterol Synthesis.

Cholesterol synthesis is enhanced in blast cells of acute myeloid leukemia (AML) patients, as indicated by high mRNA levels of 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoAR) and LDL receptor (LDLR) and increased LDL uptake. In addition, AML cells exposed to chemotherapeutic drugs acutely increase their cholesterol content, which renders these cells less susceptible to chemotherapy. Statins inhibit HMG-CoAR, the rate-limiting enzyme of the mevalonate pathway. As a result, both the cholesterol synthesis route and the isoprenylation route are blocked. The latter route leads to the production of the isoprenoids farnesyl (FPP) and geranylgeranyl pyrophosphate (GGPP). Small GTP-ases Ras and Rho require farnesyl or geranylgeranyl isoprenylation for their proper function, a process which is catalyzed by farnesyltransferase (FTase) or geranylgeranyltransferase (GGTase). Simvastatin has shown to be cytotoxic to primary (CD34⁺) AML cells in vitro, however, these effects are heterogeneous. The aim of the present study was to elucidate the mechanism of simvastatin-induced cytotoxicity and the heterogeneity therein. This could be extensively studied by making use of cell lines that display a strong heterogeneity in responsiveness to simvastatin.

In 9 myeloid leukemic cell lines we mimicked or rescued the cytotoxic effects of simvastatin with compounds acting on either the cholesterol synthesis route or the isoprenylation route.

In the cell lines, we identified 3 subgroups: sensitive (EC50 3-15 μM: NB4, OCI-M3, HL60), medium sensitive (EC50 16-50 μM: THP-1, U937, K562), and insensitive (EC50 60-100 μM: UT7-GM, TF-1, KG1a) to simvastatin, as determined by assessing cytotoxicity using a chemoluminescence assay. In all cell lines, simvastatin-induced cytotoxicity could completely be rescued by mevalonate, a compound directly downstream of HMG-CoAR. This indicates that the mevalonate pathway is indeed the key pathway involved in statin-induced cytotoxicity. To assess the role of the cholesterol synthesis route, mRNA levels of HMG-CoAR and LDLR were determined following simvastatin-treatment. In all cell lines, HMG-CoAR (1.5-7.8 fold) and LDLR (1.5-3.2 fold) mRNA expression were increased. No relationship existed between the basal or induced mRNA expression levels of both genes and simvastatin-induced cytotoxicity. In addition, squalene, a cholesterol precursor, did not rescue simvastatin-induced cell death. The role of the isoprenylation route, which is linked to the GTP-ases Ras and Rho, was assessed by rescue studies: simvastatin-induced cytotoxicity could especially be rescued by GGPP (68%), but also by FPP (42%), in the sensitive cell lines. Rescue was less pronounced in the insensitive cell lines (12% and 26% by GGPP and FPP, respectively), indicating a difference between the cell lines at the level of isoprenylation. This observation was confirmed by the finding that GGTI-298, a GGTase inhibitor, and to a lesser extent tipifarnib, an FTase inhibitor, were more effective in the sensitive cell lines compared to the insensitive cell lines (60% versus 96% viability at 0.2 μM, p=0.005; and 55% versus 78% viability at 5 μM, p=0.001, respectively). Also the ERK inhibitor U0126, acting downstream of Ras, was more effective in the sensitive cell lines when looking at cell viability (48% versus 70%, p=0.01) and pERK inhibition, as determined by Western blot. This is in line with the observed relationship between simvastatin-induced cytotoxicity and inhibition of pERK. Y27632, an inhibitor of ROCK (downstream target of Rho), and LY294,002, an Akt inhibitor (downstream of Ras), were equally cytotoxic in all cell lines. Currently, experiments are ongoing to obtain more detailed information on the mechanism involved by assessing the activity of Ras, GGTase and FTase, and we will confirm our findings in primary AML cells.

Our data indicate that the Ras-isoprenylation route, rather than the cholesterol synthesis route of the mevalonate pathway is responsible for simvastatin-induced cytotoxicity and the heterogeneous response between AML cells.

Supported by a grant of the Dutch Cancer Society

No relevant conflicts of interest to declare.