PS984 CANNABINOIDS EXERT A POTENT ANTI‐LEUKEMIC EFFECT AFFECTING SIGNALING PATHWAYS INVOLVED IN TUMOR CELL METABOLISM
Authors: Medrano M. , Velázquez T. Caballero , Hidalgo F. , Zamora R. , García J.A. Bejarano , Cruz E. Carrillo , Rosado I. Valle , Pérez‐Simón J.A. 
Published: June 2019

Background:Cannabinoids exert their activity upon interaction with cannabinoid receptors: CB1, mostly expressed in the central nervous system and CB2, mainly found in hematopoietic cells. We have described the effect of cannabinoid derivatives in multiple myeloma (Barbado et al, 2018). In this work, we present evidence that cannabinoids acts as a cytotoxic agent against acute myeloid leukemia (AML) blasts without affecting normal hematopoietic cells.Aims:Our main goal is to determine the mechanism of action by which cannabinoids exert their antileukemic effect.Methods:Cell viability was determined by MTT assay and flow cytometry analysis of Annexin V/7‐AAD using 6 AML cell lines, primary hematopoietic stem cells (HSC), lymphocytes from healthy donors and 23 AML patients. mRNA expression profile of HL60 AML cells treated with WIN‐55 vs untreated cells was studied by Clariom S Assays Human (Affimetrix) and Transcriptome Analysis Console (TAC) Software (Thermo Fisher). Western blot and immunoflurescence assays were performed to determine protein expression upon WIN‐55 exposure, including endoplasmic reticulum stress protein. TMRE and MitoSOXTM Red was used to determine mitochondrial damage. The contribution of compounds (glycine, serine, N‐Acetyl‐cysteine, 2‐mercaptoethanol, oxaloacetic acid and methyl pyruvate) involved in key metabolic processes affected upon WIN‐55 exposure were also assessed. Glycolytic flux was assessed by ECAR in a XF24 flux analyzer (Seahorse Biosciences). NAD/NADH and ADP/ATP ratio and Hexokinase activity was also measured. Ceramide and other sphingolipids were quantitated by HPLC‐MS/MS and immunohistochemistry. HL60‐xenotransplant NSG mouse model were established, bone marrow (BM) infiltration confirmed by BM aspirate and subsequent flow cytometry, and WIN‐55 or citarabine treatments was administered. The effect of WIN‐55 on normal hematopoiesis of healthy BALB‐C mice was assessed by flow cytometry and hemogram.Results:We confirmed a potent cytotoxic effect of WIN‐55 on AML cell lines while normal HSC remained unaffected. mRNA expression profile using showed that the pathways mainly affected upon WIN‐55 exposure were unfolded protein response (UPR) and XBP1 activates chaperon genes, PI3K‐Akt‐mTOR, VEGFA‐VEGFR2 and Adipogenesis Signaling Pathway. Using WB assays we confirmed that WIN‐55 triggered caspases activation and we detected ceramide accumulation in AML cells. Also studies on p‐PERK, p‐IRE1 and CHOP confirmed an increased endoplasmic reticulum stress upon exposure to cannabinoids. Pathways involved in cell proliferation/survival including p‐JNK, p‐Erk1/2, p‐p38‐MAPK and p‐AKT were affected. An early mitochondrial damage was also observed in leukemic cells upon exposure to the drug. Ceramide synthesis inhibition prevented less than 10% of the WIN‐55 induced‐apoptosis. By contrast, the proapoptotic effect of cannabinoids on AML cells was abolished upon co‐culture with either CB2 receptor antagonists, pancaspase inhibitors, olaparib (a PARP inhibitor) or glycine. Cannabinoids did not affect the viability of HSC from healthy donors neither in vitro not in vivo, resulting in a lack of hematopoietic toxicity in healthy treated mice. Finally, WIN‐55 significantly prolonged the survival of AML xenografted mice.Summary/Conclusion:Cannabinoids have a potent and selective anti‐leukemic effect affecting several signaling and metabolic pathways. The role of PARP is essential in this apoptotic effect, which is mediated by CB2 receptor. Differences in the metabolic requirements between normal and leukemic cells account for this differential effect.