Abstract B30: Phosphorylation of c-jun N terminal kinase (JNK) regulates induction of mitochondrial apoptosis by pro-suvival BCL-2 antagoinist obatoclax
Authors: McCoy Francis G.P. , Paul Ian , Hurwitz Jane L. , O'Hagan Barry , Odrzywol Krzysztofa , Murray James T. , McKerr George , Fennell Dean A. 
Published: June 2012

Background: Failure to induce apoptosis is a major limitation of conventional chemotherapy for lung cancer. Resistance to apoptosis contributes to this chemoresistant phenotype. New therapeutic strategies are therefore required to bypass block in apoptosis. Obatoclax (obx) is a small molecule inhibitor of pro-survival BCL-2 family proteins currently being clinically evaluated in both small-cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). However, little is known regarding the molecular mechanisms that might underlie sensitivity and resistance to obx. We are addressing this important question by exploring the pharmacodynamics of obx in NSCLC.

Methods: To isolate mitochondria harvested cells were homogenized in ice-cold isolation buffer and the resulting homogenate subjected to differential centrifugation to isolate the mitochondrial fraction. To conduct shRNA transfections, shRNA plasmids targeted against BAX, BAK, BIM and beclin1 were purchased from SA Biosciences (Frederick, MD, USA). H460 cells were transfected with each plasmid and cells were subjected to at least two rounds of selection to create stable clones. For transmission electron microscopy, cells were fixed in double strength TEM fixative (4% paraformaldehyde, 2.5% glutaraldehyde) in 0.1M sodium cacodylate buffer. Samples were analyzed using standard EM procedures. Specific hVps34 Activity was measured in cells deprived of amino acids for 2 hrs in EBSS. Cells were lysed and subjected to immunoprecipitation with anti-hVps34 antibody, and immunocomplexes were assayed for hVps34 lipid kinase activity.

Results: shRNA downregulation of BAX/BAK (H460shBAX/BAK) inhibited obx induced caspase 9/3 cleavage, PARP activation, and mitochondrial outer membrane permeabilization. However obx was equally effective in reducing clonogenic survival, and short term viability (48–72 hrs). Obx induced prolific cytoplasmic vacuolation evident by TEM, and processing of LC3I to LC3II was evident in NSCLC H460 and H1975 cells, and SCLC H196 and H146 cells suggesting induction of autophagy. H460shBAX/BAK clones also displayed unaltered LC3 processing. To determine whether LC3 processing following obx was regulated by beclin1, stable knockdown was acomplished using shRNA. 3 clones were selected and exhibited reduced hVpS34 activity, however obatoclax induced equal clonogenic survival and LC3 processing compared to NT shRNA controls. Neither LC3 nor PARP processing was inhibited by 3MA or wortmannin. Also we observed loss of beclin1 expression following obx. Activation of JNK and ERK have been implicated in beclin1 indpendent mitophagy, while JNK is also a known activator of BAX and BIM. Obx induced phosphorylation of JNK but not ERK, and inhibition of pJNK blocked PARP cleavage. Also, stable knockdown of BIM by shRNA did not significantly alter obx EC50 values. In H727 NSCLC cells resistant to obx induced apoptosis, JNK phosphorylation and LC3II formation was observed but to a significantly lesser extent than sensitive cells.

Summary: JNK phosphorylation regulates obatoclax induced apoptosis, and this activity is reduced in NSCLC cells resistant to obx. LC3 processing was independent of beclin1, thus the roles of Atg5 and 7 in regulating obx induced LC3 processing and cell fate are ongoing and will be presented.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B30.