Reprogramming Acute Lymphoblastic Leukemia cell metabolism by inhibition of Carnitine Palmitoyltransferase 1a (Cpt1a) protein.

Introduction. Tumor cells gain a survival and growth advantage by several mechanisms, including the metabolic change known as the “Warburg effect”. In fact, by this profound biochemical alteration malignant cells preferentially use the up-regulated pathways involved in the aerobic glycolysis. It has been reported that Carnitine Palmitoyltransferase 1a (CPT1a), a protein that controls the fatty acid translocation across the mitochondrial membrane may play a fundamental role in this metabolic transformation. Aim of this study is to evaluate the expression of CPT1a in Acute Lymphoid Leukemia (ALL) cells, assessing the functional activity of CPT1a inhibitors, a developmental class of molecules with potential anti-tumor activity. Methods. Proliferation assay: MTT test. Protein expression: Western Blot (WB) analysis. Analysis of the cellcycle and apoptosis levels: Acridine Orange technique and Annexin-V staining. Results. CPT1a expression, as determined by WB analysis, was measured in three T-ALL cell lines, the parental and the derived resistant CEM clones and the MOLT-4. CPT1a expression, detected in ALL cell lines, showed overexpression in clone CEM R. The functional activity of the novel CPT1a inhibitor ST1326 (kindly provided by SigmaTau) and of the well known inhibitor Etomoxir, was then in vitro evaluated. ST1236, at concentrations ranging between 1 and 50 M, induced apoptosis on the MOLT-4 cell line, in a dose and time dependent fashion, as demonstrated at 72 hours by an increase of the subG1 peak from a baseline value of 10% to 37% and 95.8%, at 20 and 50 M, respectively. A lower activity was found in the CEM S cell lines with an increase, at 72 hours, of the subG1 peak from a baseline value of 8.5% to 15.94% and 26.6%, at the same concentrations. Conversely, the CEM R cell line, proved resistant. These data were further confirmed by Annexin V staining. The effects on apoptosis induced by ST1326 were significantly higher (p>0.05) compared to those observed following Etomoxir exposure. Preliminary results on primary ALL blasts, obtained from 5 cases exposed in vitro to 10, 20 and 50 M of ST1326, showed an apoptosis induction (sub-G1 DNA content) from 38.9% ±23.7 (control) to 48.0% ±26.6, 53.5% ±30.6 and 68.9% ±23.4, respectively. The statistical significance (P<0.005) was however obtained only at the highest ST1326 concentration (50 µM). Conclusions. Overall, our results prompt the analysis of CPT1a expression in a large ALL sample population. In addition, the pro-apoptotic activity of ST1326 on ALL cells suggests that further studies aimed at validating CPT1a as a potential target for novel therapeutic approaches in ALL are needed.