Targeting Metabolic Pathways for Leukemia Treatment
Authors: Mirabilii Simone , Ricciardi Maria Rosaria , Allegretti Matteo , Licchetta Roberto , Iacovelli Stefano , Bergamo Paola , Petrucci Maria Teresa , Nicolai Raffaella , Peluso Gianfranco , Foà Robin , Alimena Giuliana , Tafuri Agostino 
Published: October 2019

Abstract
Abstract 1371
Aberrant signal transduction pathways influence metabolic changes in neoplastic cells. It is well established, in fact, that cancer cells are characterized by a pro-glycolytic metabolic phenotype and recent evidences confirmed that also leukemia cells may switch to a non-oxidative metabolism. Among the different substrates used to sustain the anabolic processes and to keep the tricarboxylic acid cycle (TCA) active, the fatty acid oxidation (FAO) may represent an alternative carbon source. The carnitine palmitoyl transferase 1a (CPT1a) catalyzes the first step of FAO, by loading long chain fatty acyl groups onto carnitine, transporting them through the mitochondrial membrane. Moreover, CPT1a has been previously demonstrated to interact with members of the apoptotic machinery, such as Bcl-2 and t-Bid, and its inhibition can cause an accumulation of the toxic metabolite palmitate, resulting in mitochondrial damage and cell death. In diabetic patients the use of a recently discovered antihyperglycemic agent characterized by the selective and reversible inhibition of CPT1a has been reported. Therefore, we aimed in this study to investigate the in vitro anti-leukemic effect of the CPT1a inhibition. Particularly, we evaluated the activity of two CPT1a-inhibitors, the novel ST1326 (kindly provided by Sigma-Tau) and the previously known Etomoxir on the proliferation and apoptosis of leukemia cell lines and primary cells obtained from patients affected by acute leukemias. The drug concentration inducing a 50% cell killing (IC50) was calculated from the dose-response curve. The cytotoxic drug effects on leukemia cell lines (HL-60, HL-60/MX2, U937, K562, CEM S, CEM R, MOLT-4) and primary cells of acute myeloid (AML) and lymphoid (ALL) leukemia were evaluated using the MTT test. Flow cytometry techniques Acridine-Orange staining, AnnexinV binding assay and Chloromethyl-X-Rosamine (CMXRos)/MitoTracker Green (MTGreen) staining were used to examine cell cycle changes, apoptosis and the loss of mitochondrial membrane potential (ΔΨm). We evaluated the activity of ST1326 (1–50 μM) on leukemia models, demonstrating a dose- and time-dependent cell growth arrest, caused by mitochondrial damage and apoptosis induction. In fact, 6 hours of ST1326 exposure were able to induce a dramatic loss of ΔΨm, as observed in MOLT-4 cells (cells affected increased from 15% in the control to 59.7% at 50 μM). Following 72 hours of ST1326 exposure, the same cells showed then an increase in the subG1 peak from a baseline value of 10% to 8.1%, 7.3%, 10.5%, 37% and 95.8% at 1, 5, 10, 20 and 50 μM, respectively (IC50= 39.2 μM). Etomoxir failed to show any activity (data not shown). The myeloid cell lines HL60, HL60/MX2 and U937 were more sensitive to ST1326, showing an IC50 of 11.8, 8.2 and 8.8 μM, respectively. A milder activity was found in the other lymphoid-derived model, the CEM S cell lines (IC50= 71.1 μM). Instead, both the lymphoid CEM R and the myeloid K562 cell lines proved resistant (IC50= n.d.). We then exposed in vitro (72 hours) primary cells from 12 AML and found in all a significant pro-apoptotic activity (AnnexinV positive cells): 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) following 72 hours of exposure to 5, 10, 20 and 50 μM ST1326, respectively. ALL primary samples (6/8 samples), instead, showed a significant (p<0.005) increase of the sub-G1 DNA apoptotic cells only at the higher ST1326 dose (from 38.9% ± 23.7 to 68.9% ± 23.4 of cells at 50 μM). In conclusion, ST1326 shows a high in vitro pro-apoptotic activity on acute leukemia models and on primary cells, especially in AML, prompting further studies to better define this novel approach based on the targeted inhibition of metabolic pathways in leukemia treatment.

Disclosures:
Petrucci: Jansse-Cilag, Celgene: Honoraria. Nicolai:Sigma Tau Pharmaceutical Industries SpA: Employment. Tafuri:Sigma Tau Pharmaceuticals: Research Funding.