Ian Paul

Obatoclax induces Atg7-dependent autophagy independent of beclin-1 and BAX/BAK

Direct pharmacological targeting of the anti-apoptotic B-cell lymphoma-2 (BCL-2) family is an attractive therapeutic strategy for treating cancer. Obatoclax is a pan-BCL-2 family inhibitor currently in clinical development. Here we show that, although obatoclax can induce mitochondrial apoptosis dependent on BCL-2 associated x protein/BCL-2 antagonist killer (BAX/BAK) consistent with its on-target pharmacodynamics, simultaneous silencing of both BAX and BAK did not abolish acute toxicity or loss of clonogenicity. This is despite complete inhibition of apoptosis. Obatoclax dramatically reduced viability without inducing loss of plasma membrane integrity. This was associated with rapid processing of light chain-3 (LC3) and reduction of S6 kinase phosphorylation, consistent with autophagy. Dramatic ultrastructural vacuolation, not typical of autophagy, was also induced. Silencing of beclin-1 failed to prevent LC3 processing, whereas knockout of autophagy-related (Atg)7 abolished LC3 processing but failed to prevent obatoclax-induced loss of clonogenicity or ultrastructural changes. siRNA silencing of Atg7 in BAX/BAK knockout mouse embryonic fibroblasts did not prevent obatoclax-induced loss of viability. Cells selected for obatoclax resistance evaded apoptosis independent of changes in BCL-2 family expression and displayed reduced LC3 processing. In summary, obatoclax exhibits BAX- and BAK-dependent and -independent mechanisms of toxicity and activation of autophagy. Mechanisms other than autophagy and apoptosis are blocked in obatoclax resistant cells and contribute significantly to obatoclaxs anticancer efficacy.

PARP inhibition induces BAX/BAK-independent synthetic lethality of BRCA1-deficient non-small cell lung cancer.

Evasion of apoptosis contributes to both tumourigenesis and drug resistance in non‐small cell lung carcinoma (NSCLC). The pro‐apoptotic BCL‐2 family proteins BAX and BAK are critical regulators of mitochondrial apoptosis. New strategies for targeting NSCLC in a mitochondria‐independent manner should bypass this common mechanism of apoptosis block. BRCA1 mutation frequency in lung cancer is low; however, decreased BRCA1 mRNA and protein expression levels have been reported in a significant proportion of lung adenocarcinomas. BRCA1 mutation/deficiency confers a defect in homologous recombination DNA repair that has been exploited by synthetic lethality through inhibition of PARP (PARPi) in breast and ovarian cells; however, it is not known whether this same synthetic lethal mechanism exists in NSCLC cells. Additionally, it is unknown whether the mitochondrial apoptotic pathway is required for BRCA1/PARPi‐mediated synthetic lethality. Here we demonstrate that silencing of BRCA1 expression by RNA interference sensitizes NSCLC cells to PARP inhibition. Importantly, this sensitivity was not attenuated in cells harbouring mitochondrial apoptosis block induced by co‐depletion of BAX and BAK. Furthermore, we demonstrate that BRCA1 inhibition cannot override platinum resistance, which is often mediated by loss of mitochondrial apoptosis signalling, but can still sensitize to PARP inhibition. Finally we demonstrate the existence of a BRCA1‐deficient subgroup (11–19%) of NSCLC patients by analysing BRCA1 protein levels using immunohistochemistry in two independent primary NSCLC cohorts. Taken together, the existence of BRCA1‐immunodeficient NSCLC suggests that this molecular subgroup could be effectively targeted by PARP inhibitors in the clinic and that PARP inhibitors could be used for the treatment of BRCA1‐immunodeficient, platinum‐resistant tumours. Copyright

Voltage dependent anion channel-1 regulates death receptor mediated apoptosis by enabling cleavage of caspase-8.

BackgroundActivation of the extrinsic apoptosis pathway by tumour necrosis factor related apoptosis inducing ligand (TRAIL) is a novel therapeutic strategy for treating cancer that is currently under clinical evaluation. Identification of molecular biomarkers of resistance is likely to play an important role in predicting clinical anti tumour activity. The involvement of the mitochondrial type 1 voltage dependent anion channel (VDAC1) in regulating apoptosis has been highly debated. To date, a functional role in regulating the extrinsic apoptosis pathway has not been formally excluded.MethodsWe carried out stable and transient RNAi knockdowns of VDAC1 in non-small cell lung cancer cells, and stimulated the extrinsic apoptotic pathway principally by incubating cells with the death ligand TRAIL. We used in-vitro apoptotic and cell viability assays, as well as western blot for markers of apoptosis, to demonstrate that TRAIL-induced toxicity is VDAC1 dependant. Confocal microscopy and mitochondrial fractionation were used to determine the importance of mitochondria for caspase-8 activation.ResultsHere we show that either stable or transient knockdown of VDAC1 is sufficient to antagonize TRAIL mediated apoptosis in non-small cell lung cancer (NSCLC) cells. Specifically, VDAC1 is required for processing of procaspase-8 to its fully active p18 form at the mitochondria. Loss of VDAC1 does not alter mitochondrial sensitivity to exogenous caspase-8-cleaved BID induced mitochondrial depolarization, even though VDAC1 expression is essential for TRAIL dependent activation of the intrinsic apoptosis pathway. Furthermore, expression of exogenous VDAC1 restores the apoptotic response to TRAIL in cells in which endogenous VDAC1 has been selectively silenced.ConclusionsExpression of VDAC1 is required for full processing and activation of caspase-8 and supports a role for mitochondria in regulating apoptosis signaling via the death receptor pathway.

Implications for Powering Biomarker Discovery Studies

This study examined variations in gene expression between FFPE blocks within tumors of individual patients. Microarray data were used to measure tumor heterogeneity within and between patients and disease states. Data were used to determine the number of samples needed to power biomarker discovery studies. Bias and variation in gene expression were assessed at the intrapatient and interpatient levels and between adenocarcinoma and squamous samples. A mixed-model analysis of variance was fitted to gene expression data and model signatures to assess the statistical significance of observed variations within and between samples and disease states. Sample size analysis, adjusted for sample heterogeneity, was used to determine the number of samples required to support biomarker discovery studies. Variation in gene expression was observed between blocks taken from a single patient. However, this variation was considerably less than differences between histological characteristics. This degree of block-to-block variation still permits biomarker discovery using either macrodissected tumors or whole FFPE sections, provided that intratumor heterogeneity is taken into account. Failure to consider intratumor heterogeneity may result in underpowered biomarker studies that may result in either the generation of longer gene signatures or the inability to identify a viable biomarker. Moreover, the results of this study indicate that a single biopsy sample is suitable for applying a biomarker in non-small-cell lung cancer.

Apical Schwannoma Presenting as Harlequin Syndrome

Fig 3. Aa 12-month history of unilateral facial flushing when she exercised in the gym; only the left side of her face became red (Fig 1). Physical examination at rest was normal. There was no detectable difference in the sweat pattern on her hands or face and no signs of Horner’s syndrome. These symptoms and signs are consistent with Harlequin syndrome. First reported in 1988 by Lance and colleagues [1], this syndrome occurs because of autonomic neuropathy to the side that lacks flushing. Varying causes have been reported. In this patient, a chest roentgenogram revealed a well-defined mass in the right apex medially (Fig 2). Magnetic resonance imaging of the thorax showed a right upper thoracic paravertebral mass. Thoracoscopic excision and histologic examination revealed a World Health Organization grade I schwannoma (Fig 3; hematoxylin and eosin stain, 20 magnification). The patient has recovered well and is considering left-sided thoracoscopic sympathectomy to restore facial symmetry.

BRCA1 expression and efficacy of vinorelbine in malignant mesothelioma.

e13541 Background: Malignant mesothelioma is an aggressive tumor refractory to current therapies and this may be due to intrinsic apoptosis resistance. Vinorelbine has been shown to exhibit useful clinical activity in phase II trials and showed a trend to improved survival as front-line monotherapy in the MS01 phase III study. BRCA1 has been reported to regulate sensitivity to microtubule poisons; however its involvement in regulating apoptosis in Mesothelioma has not been investigated. We have hypothesised that loss of BRCA1 confers resistance to vinorelbine induced apoptosis. METHODS Dose-response curves were generated by vialight assay and caspase-3 activity determined by luminescence assay. Three resistant cell lines were generated by increasing exposure to vinorelbine. Cells were transfected with 10nM non-targeting siRNA or BRCA1/Caspase8 siRNA. The percentage of apoptotic cell population was determined by PI staining and measurement of hypodiploidy. RESULTS Vinorelbine induced cytotoxicity correlated with BRCA1 expression level in a panel of 6 mesothelioma cell lines. We then investigated the effect BRCA1 silencing on vinorelbine sensitivity. The downregulation blocked caspase-3 activation, PARP cleavage and the percentage of subG1 cell population. Moreover, when cells were selected for resistance to vinorelbine, this was associated with a reduction in BRCA1 expression compared to parental cells. BRCA1 mRNA expression was not altered as shown by gene expression analysis, suggesting a post-transcriptional regulation mechanism. Data obtained in BAX/BAK double negative cells show that vinorelbine mediates toxicity irrespective of a functional mitochondrial apoptosis pathway, however caspase 8 can be activated in these cells; furthermore silencing of Caspase8 induced resistance to apoptosis by vinorelbine. We are addressing the prevalence of BRCA1 negativity in primary mesotheliomas including vinorelbine responders vs non-responders to delineate its potential as a predictive biomarker and will present these results. CONCLUSIONS Our data highlight the BRCA1 as a candidate predictive biomarker for vinorelbine induced apoptosis suggesting a potential utility in personalising therapy with this agent.

The Rab27A effector MYRIP as a regulator of survival in non-small cell lung cancer cells.

e13537 Background: Personalising therapy for non-small cell lung cancer has been validated as an effective treatment paradigm. Somatic gene alterations confer sensitivity to inhibition of growth survival pathways irrespective of underlying resistance to chemotherapeutic agents, and enables efficient activation of BCL-2 family-dependent pro-apoptotic signalling. In platinum resistant cancers this signalling is blocked, however the intrinsic pathway remains sensitive to exogenous BH3 death signals. METHODS Microarray profiling coupled to focused RNAi screening was used to reveal those resistance genes involved in derepression of proapoptotic BCL-2 family proteins. Analysis of the effect of MYRIP expression on survival was generated from studying 6 NSCLC datasets from Gene Expression Omnibus which were paired with survival time and status information. The toxic effect of MYRIP depletion on NSCLC and non-cancerous lung cells was determined by cell viability and clonogenic assays. Apoptotic induction was examined by caspase and PARP cleavage experiments, and fractionation of mitochondrial proteins. FACS analysis was used to examine effects on cell cycle. RESULTS MYRIP/Slac2c, a gene hitherto associated with vesicle trafficking but not apoptosis, was overexpressed in platinum resistant NSCLC cells. In particular, cisplatin resistant cells expressed a heavier protein isoform of MYRIP which appeared to be associated with the mitochondria. Silencing of MYRIP induced BIM and BAX/BAK-dependent mitochondrial apoptosis which was selective for NSCLC, but not non-cancerous lung cells. In addition, loss of MYRIP induced S-phase arrest. CONCLUSIONS Overexpression of MYRIP is an independent, poor prognostic factor suggesting that targeting these genes may be therapeutically relevant. In summary, MYRIP exhibits a previously unknown survival function associated with suppression of pro-apoptotic BCL-2 family proteins and maintenance of cancer cell proliferation and survival.

Abstract B30: Phosphorylation of c‐jun N terminal kinase (JNK) regulates induction of mitochondrial apoptosis by pro‐suvival BCL‐2 antagoinist obatoclax

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.