Cannabinoids Induce Cell Death in Leukemic Cells through Parthanatos and PARP-Related Metabolic Disruptions

Abstract

BACKGROUNDWe have previously described the antitumor effect of the cannabinoid WIN-55,212-2 (WIN-55) and a set of cannabinoid derivatives (CNB) specific for CB2 in multiple myeloma (Barbado et al, 2018). In AML, we also observed a potent and selective antileukemic effect, affecting signaling and metabolic pathways essential for the viability of tumor cells. Among them, we found an increased stress on the endoplasmic reticulum, mitochondrial damage, and alteration of the metabolism of ceramides, although none of these events turned out to be the main trigger of cell death, since the inhibition of each of them did not prevent the antileukemic effect of CNB.On the other hand, disruption of the mecanisms of DNA repair have been identified as a key oncogeneic event in different solid tumors, and some studies have also suggested that it might be involved in leukemogenesis. More specifically, PARP1 is involved in DNA damage repair. Other functions include the regulation of glycolysis enzymes through the addition of Poly ADP-Ribose (PAR) and the execution of Parthanatos, that occurs whenever PARP-1 becomes over-activated in response to extreme damage inducing nuclear translocation of AIF and depletion of NAD +. OBJECTIVESIn this study we set out to identify the ultimate mechanism that justifies the aforementioned pleiotropic effect of CNB on the metabolism of leukemic cells and their viability. METHODSCell viability was determined by MTT and flow cytometry. The mRNA and / or protein expression profile of AML samples or healthy progenitor cells were studied by qPCR and / or Western blot. Glycolytic flux was studied with the XF Glycolytic Rate Assay (Seahorse Biosciences). NAD + levels and glycolytic enzyme activity were measured using quantification kits. Parylation of different enzymes were confirmed by Co-IP using the corresponding antibodies.Finally, NOD/scid/IL-2R gammae null (NSG) mice were xenotransplanted with HL60-Luciferase cell line. Once the presence of leukemic cells was confirmed, treatment with vehicle, WIN-55 cannabinoid at a dose of 5 mg/kg/day or citarabine (ARA-C) at 50 mg/kg during 5 days was administered. Also we tested the effect of these compounds on normal hematopoiesis by treating healthy BALB-C mice. RESULTSPretreatment of leukemic cells with Olaparib, a PARP1 inhibitor, reversed WIN-55 induced apoptosis by almost 100%. WIN-55 affected the activity of most glycolysis enzymes, with a marked drop of the activity of GAPDH and pyruvate kinase which was reversed by Olaparib pretreatment. Also G6PDH activity was markedly affected upon culture with WIN-55. Co-IP confirmed parylation of these enzymes which was reversed with Olaparib.ECAR data detected by Seahorse also confirmed the drop in glycolytic capacity produced by WIN-55 in leukemic cells which again was reversed upon culture with Olaparib. In addition, the addition of nicotinamide mononucleotide (NAM), a precursor of NAD +, reversed the loss of viability produced by WIN-55. Next, we confirmed that PARP1 levels were significantly higher in leukemic cell lines and in a series of 40 AML patients as compared to healthy hematopoietic stem cells (HSC).Finally, we observed a translocation of AIF to the nucleus, confirming that WIN-55 produces PARTHANATOS.In a murine model we confirmed treatment with WIN-55,212-2 significantly prolonged survival in AML xenograft mice, with disappearance of the leukemic clone in a significant proportion of cases. By contrast, cannabinoids did not affect the viability of hematopoietic stem cells (HSC) in vivo, resulting in a lack of myeloid toxicity in healthy treated mice. CONCLUSIONSWIN-55 exerts a selective antileukemic effect through the overactivation of PARP1, affecting the levels of parylation in enzymes involved in glycolysis and pentose phosphate pathways, leading to the translocation of AIF to the nucleus and to depletion of NAD +, which were reversed through PARP1 inhibition. These effects are not observed in normal HSC. These data are confirmed in murine models in which we confirmed the antileukemic effect of WIN-55 withouth hampering normal hematopoiesis. [Display omitted] DisclosuresPérez-Simón: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.