Targeting the leukemia cell metabolism by the Carnitinepalmitoyltransferase1a (CPT1a) inhibition: functional pre-clinical effects in Acute Myeloid Leukemias.

Introduction. Cancer cells are characterized by perturbations of their metabolic processes, such as the acquisition of a "proglycolitic phenotype" (Warburg effect) and/or an enhanced glutamine catabolism. A novel class of drugs, aimed at targeting the metabolic pathways, are nowadays under investigation. Since the CarnitinePalmitoylTransferase1a (CPT1a) is a protein that catalyzes the first step of fatty acid oxidation by loading long chain fatty acyl groups onto carnitine, transporting them through the mitochondrial membrane, we aimed in this study at evaluating the anti-leukemia effect of the CPT1a inhibition. Particularly, we evaluated the activity of two CPT1a-inhibitors, the well known Etomoxir and the novel ST1326 (kindly provided by Sigma-Tau), on the proliferation and apoptosis of Acute Myeloid Leukemia (AML) leukemia cell lines and in primary cells obtained from AML patients. Methods. The cytotoxic effects of ST1326 on AML cell lines (HL-60, HL-60/MX2, U937, K562) and on primary AML were evaluated by MTT test. The drug concentration inducing 50% cell killing (IC50) was calculated from the doseresponse curve. The expression of CPT1 in AML cell lines was evaluated by western blot analysis. Flow cytometry Acridine-Orange technique and AnnexinV binding assay were used to examine cell cycle changes and apoptosis. Results. The CPT1a expression was preliminary demonstrated in the AML cell lines. Subsequently, we evaluated the activity of ST1326 on AML models, demonstrating at increasing concentration of ST1326, a dose- and time-dependent cell growth arrest, caused by mitochondrial damage and apoptosis induction. The HL-60 cell line, following 72 hours of ST1326 exposure, showed an increase of the subG1 peak from a baseline value of 9.9% to 22.3%, 49.6%, 58.1% and 80.9% at 1, 5, 10 and 50 M, respectively. Similarly, the U937 and HL60/MX2 proved to be highly sensitive to ST1326 (IC50: 8.2 and 8.8 M), while the K562 cell line was resistant (IC50 n.d.). ST1326 resulted significantly more effective compared to Etomoxir (subG1 peak in HL60 at 72 hours remained unchanged: 10.5% at 50 M Etomoxir). The activity of ST1326 was further determined on 12 primary AML samples and a pro-apoptotic activity of ST1326 was observed in all AML samples: AnnexinV positive cells significantly increased at 72-96 hours from 23.27%±13.63 (control) to 36.59%±19.97 (P=0.23), 40.51%±18.66 (P=0.0074), 43.43%±19.81 (P=0.0071) and 75.30%±11.52 (P=0.00018) in the presence of 5, 10, 20 and 50 M of ST1326, respectively. Conclusions. ST1326 shows high in vitro pro-apoptotic activity on AML models and on primary cells, prompting further studies by molecular inhibition of metabolic pathways in leukemia treatment. Studies are ongoing to evaluate the expression of CPT1a, according to the AML clinical and biological characterization, and to define mechanisms underlying this activity.