Abstract B66: Chronic myeloid leukemic cells trigger poly(ADP-ribose) polymerase-dependent cell death in natural killer cells.
Authors: Aurelius Johan , Martner Anna , Riise Rebecca E. , Romero Ana I. , Palmqvist Lars , Brune Mats , Hellstrand Kristoffer , Thorén Fredrik B. 
Published: August 2014

Background and Purpose: Natural killer (NK) cells are commonly dysfunctional in chronic myeloid leukemia (CML) and their status may determine the course of disease. We aimed to define the molecular mechanisms of leukemia-induced NK cell inhibition with focus on the role of CML cell-derived reactive oxygen species (ROS) and the poly(ADP-ribose) polymerase-1 (PARP-1) pathway of cell death (parthanatos).

Methods: Malignant granulocytes from patients with BCR-ABL+ CML were analyzed using flow cytometry for cell surface expression of the oxygen radical-producing NADPH/NOX-2 oxidase (determined by expression of the NADPH oxidase subunit gp91phox). ROS production by CML cells was assessed by chemiluminescence. In co-culture experiments, purified NK cells from healthy donors were exposed to malignant CML cells followed by analysis of NK cell viability. In these experiments, inhibitors of PARP-1 (PJ34 and EB-47) and the caspase cascade (Z-VAD.fmk) were added to determine the mechanisms of cell death in NK cells exposed to CML cells. Translocation of apoptosis-inducing factor (AIF) from mitochondria to nucleus was determined in sorted NK cells by immunoblot technique.

Results: All BCR-ABL+ CML cells showed high cell surface expression of gp91phox, indicating the presence of a NADPH/NOX-2 oxidase. CML cells produced high amounts of ROS upon stimulation with the protein kinase C activator PMA or by the bacterial tripeptide fMLF suggestive of a functional ROS-forming oxidase. Unstimulated CML cells triggered extensive apoptosis-like cell death in co-cultured NK cells, which was prevented by the ROS-degrading enzyme catalase and by the NADPH oxidase/NOX-2 inhibitor diphenylene iodonium. The PARP-1 inhibitor PJ34 (0.5 microM) prevented CML-induced NK cell death at CML/NK cell ratios of 0.125: p&lt;0.05 (using cells from five CML patients); 0.25: p&lt;0.0001 (n=8), 0.5: p&lt;0.0001 (n=6), and 1: p&lt;0.0001 (n=8; analyzed by two-way Anova followed by Bonferroni's multiple correction test). A structurally unrelated PARP-1 inhibitor, EB-47, shared the NK cell-protective properties of PJ34 and EB-47 whereas the pan-caspase inhibitor Z-VAD.fmk failed to rescue NK cells from CML cell-induced death. After CML cell exposure, NK cells expressed JC-1 monomers suggestive of a disrupted mitochondrial membrane potential. AIF, a mitochondrial protein involved in PARP-1 dependent cell death, accumulated in NK cell nuclei after interaction with CML cells.

Conclusions: Our results imply (1) that paracrine ROS production by CML cells followed by NK cell death may constitute a mechanism by which CML cells evade destruction by cytotoxic lymphocytes, and (2) that CML cells trigger NK cell death by inducing PARP-1 dependent parthanatos. These findings may have implications for the design of more efficient immunotherapeutic protocols in CML.

Citation Format: Johan Aurelius, Anna Martner, Rebecca E. Riise, Ana I. Romero, Lars Palmqvist, Mats Brune, Kristoffer Hellstrand, Fredrik B. Thorén. Chronic myeloid leukemic cells trigger poly(ADP-ribose) polymerase-dependent cell death in natural killer cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B66.