Statins are active in acute lymphoblastic leukaemia (ALL): a therapy that may treat ALL and prevent avascular necrosis.

The prognosis for children with acute lymphoblastic leukaemia (ALL) has substantially improved with the use of multi-agent therapy over the last few decades. Unfortunately, two opposing challenges remain in childhood ALL treatment: relapse and toxicity. One in five children with ALL relapse and many of these succumb to disease. One toxicity from ALL therapy that is particularly debilitating is avascular necrosis (AVN). A recent study suggested over 70% of children with ALL develop AVN, including 15–20% who develop symptomatic AVN (Kawedia et al, 2011). While the cause of AVN is multi-factorial, significant high risk factors include high cholesterol and treatment with corticosteroids. An ideal new agent for ALL treatment would be a drug that has single agent activity, can be effectively combined with existing cytotoxics, and can alleviate the toxic effects of existing agents. Statins, a class of cholesterol lowering agents, may fit this model. Statins are effective in preclinical models of a number of malignancies, including acute myeloid leukaemia (Burke & Kukoly, 2008). Pravastatin and simvastatin have been studied in preclinical models, demonstrating the ability to prevent steroid-induced AVN (Iwakiri et al, 2008). Based on these data, clinicians are using statins in patients off-label and multi-institutional cooperative group clinical trials are under development, moving pravastatin forward as an AVN preventing agent (Sala et al, 2007). We investigated six different statins for their effects against ALL in vitro, including their ability to inhibit proliferation, induce apoptosis, and be combined with cytotoxics. Statins used were atorvastatin, simvastatin, pravastatin, and mevastatin (LKT Laboratories, St. Paul, MN, USA), as well as, lovastatin and fluvastatin (Calbiochem, Gibbstown, NJ, USA). The following human ALL cell lines were used for these experiments: precursor-B cell ALL (Reh, Nalm 6, 380, and RS4-11) and precursor-T ALL (Jurkat, Molt4, CEM). All cell lines were purchased from American Type Culture Collection (Manassas, VA, USA) or Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany). In order to determine the activity of statins against ALL cells, the seven different ALL cell lines were treated with six different statins at doses from 1 to 20 μmol/l. Dose ranges were based on other published work treating malignant cell lines with statins, targeting levels obtainable in patients (Burke & Kukoly, 2008; Gbelcova et al, 2008). Response to treatment was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich, St. Louis, MO, USA) as previously described (Teachey et al, 2008). ALL cells were responsive to most agents (Fig 1A–D). Every ALL cell line was sensitive to fluvastatin, lovastatin, atorvastatin, and simvastatin, however, the degree of sensitivity varied between lines. All cell lines were resistant to pravastatin. The majority of cell lines (five of seven) were resistant to mevastatin. Fig 1 Statins inhibit ALL-cell proliferation. ALL cell lines were treated with six different statins for 72 h and proliferation was determined by MTT assay (Panels A–D and data not shown). ALL cells from all lines were sensitive to simvastatin, fluvastatin, ... The majority of statins, including fluvastatin, lovastatin, atorvastatin, and simvastatin are highly lipophilic substances. Pravastatin, in contrast, is hydrophilic (Gbelcova et al, 2008). Mevastatin is only slightly lipophilic (Neuhaus et al, 2002). Lipophilic statins can non-selectively diffuse into most cell types. Hydrophilic statins require active transport to enter a cell, limiting activity primarily to hepatocytes. Mevastatin's permeability lies in between as it can diffuse into some cell types and not others; it is unable to diffuse into lymphocytes (Neuhaus et al, 2002). The differences in response of ALL cells to statins could be explained by lipid solubility. Simvastatin and pravastatin are the only agents proven to reverse AVN in preclinical models (Iwakiri et al, 2008). Other statins could have a similar benefit; however, they are untested. As simvastatin was one of the most potent statins against the ALL cell lines and pravastatin had no activity, we focused on simvastatin, assessing whether it could eliminate ALL cells as opposed to stopping cell growth, using four cells lines (Nalm6, CEM, Jurkat, and Molt4). We found that simvastatin caused cell death by inducing apoptosis in each cell line (Fig 2A, B). In order to confirm that these findings were not limited to cell lines, primary human ALL cells collected from three patients with pre-B ALL were treated with simvastatin, using a bone marrow stromal culture system supplemented with stem cell factor as previously described (Teachey et al, 2006). All three samples were sensitive to simvastatin with a dose response similar to the cell lines, as determined by 7-aminoactinomycin D staining. After treatment with 20 μmol/l simvastatin for 48 h, there were 52–58% fewer viable CD19+ lymphoblasts in treated wells as compared to control. After treatment for 7 d with 5 or 20 μmol/l of simvastatin, there were 70–81% and 94–98% fewer blasts in treated wells as compared with control, respectively. Using this culture system, it cannot formally be excluded that the statins do not have effects on the stromal cells as well. Fig 2 Mechanistic effects of statins on ALL. Cells were cultured with combinations of simvastatin (20 μmol/l) and mevalonolactone (10 μmol/l) for 72 h. Mevalonolactone converts to mevalonate once added to solution. ALL blasts were plated at ... In order to determine whether statins can be effectively combined with cytotoxic chemotherapy, ALL cells from the seven cell lines were tested with simvastatin in combination with three cytotoxic agents used to treat human ALL, vincristine (Hospira Inc., Lake Forest, IL, USA), doxorubicin (Bedford Labs, Bedford, OH, USA), and dexamethasone (American Reagent Inc., Shirley, NY, USA) (Fig 1E, F and data not shown). Using median effects analysis, we found that the majority of combinations were synergistic; however, some were additive in certain cell lines. Very slight antagonism was found when doxorubicin was combined with simvastatin at very high doses in one of six cell lines and when vincristine was combined with simvastatin at lower doses in one of six cell lines. Overall, these data suggest that simvastatin can be safely combined with cell cycle dependent and independent cytotoxic agents. Statins are 3-hydroxyl-3-meythlglutaryl-CoA (HMG-CoA) reductase inhibitors. To determine if the effects of statins against ALL cells were dependent on inhibiting HMG-CoA reductase, ALL cells were treated with simvastatin with and without mevalonate (mevalonic acid, mevalonolactone) (Gbelcova et al, 2008). HMG-CoA reductase converts HMG-CoA into mevalonate as part of the HMG-CoA reductase pathway in cholesterol biosynthesis. Providing mevalonolactone (Sigma-Aldrich), which converts to mevalonate in solution, directly to the ALL cells in vitro bypasses HMG-CoA reductase. Mevalonate was able to reverse the effects of simvastatin on ALL cells (Fig 2A, B). In AML, statins were demonstrated to have some anti-tumour activity through down-regulation of signalling through the Raf/MEK/ERK pathway (Wu et al, 2004). As targeting the Raf/MEK/ERK pathway is effective against ALL in preclinical models and as ERK-mediated phosphorylation of BIM can reduce ALL blast responsiveness to steroids, the effects of statins on ERK signalling were assessed (Rambal et al, 2009). Treatment with simvastatin decreased phosphorylation of ERK in a dose-dependent manner and had no effect on total ERK (Fig 2C). Mevalonate reversed these effects. In summary, statins have preclinical activity against ALL cells and can be effectively combined with chemotherapy. As this class of drug has the potential ability to reverse avascular necrosis, a devastating side effect of ALL therapy, statins hold the unique promise of treating a disease and reversing the side effects of other medications used in therapy. Nevertheless, not all statins are the same, and careful consideration should be made regarding the best agent to move forward in clinical trials.