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D.A. Fennell



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    MO12 - Prognostic and Predictive Biomarkers III (ID 96)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Medical Oncology
    • Presentations: 1
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      MO12.01 - Novel Mechanisms of Sensitivity and Acquired Resistance to HSP90 inhibition by Ganetespib (ID 2739)

      10:30 - 10:35  |  Author(s): D.A. Fennell

      • Abstract
      • Presentation
      • Slides

      Background
      HSP90 is a promising anti-cancer target. Inhibition by the Hsp90 inhibitor ganetespib has shown promising activity with improved survival in patients with metastatic lung adenocarcinoma, and it is now being evaluated in malignant pleural mesothelioma. However, the mechanisms underlying resistance are currently unknown. The aims of this study were to establish the role for mitochondrial apoptosis in mediating the anti-cancer activity of ganetespib, and to also identify mechanisms of acquired resistance to support personalised therapy.

      Methods
      We conducted a functional genetic screen to determine the role of the proapoptotic BAX/BAK proteins using double knockout mouse embryonic fibroblasts (MEFs) shRNA and siRNA. Focused RNAi targeting BH3-only proteins, Caspase 8 and MCL1 was conducted in MSTO-211H, H460 and H23 cell lines. Apoptosis was measured by a Caspase3 activity assay and data were validated by western blot and SubG1 population analysis. Prosurvival Bcl2 family regulation was evaluated by western blot, and MCL1 transcriptional suppression monitored by real-time quantitative PCR and luciferase reporter assay. MCL1 amplification was quantified by genomic real-time PCR. Ganetespib resistant cells were generated by increasing drug exposure. Hsp90 ATP-binding site and Caspase8 were sequenced in both parental and resistant cell lines.

      Results
      Ganetespib required a functional mitochondrial pathway for induction of apoptosis. Interrogation of pro-apoptotic BH3-only proteins revealed a co-operation between BID, BIK and PUMA. Caspase8 activates BID and, when silenced, protected cells from ganetespib. MCL1 was transcriptionally suppressed by ganetespib, and when Mcl-1 downregulation was achieved by siRNA, it was sufficient to induce BID/BIK-dependent apoptosis in MCL1-dependent cells. We observed that MCL1 addicted cells were also more sensitive to ganetespib than non-addicted. In addition, amplification of MCL1 was detected only in ganetespib sensitive cell lines. Ectopic MCL1 was not sufficient to rescue from ganetespib-induced apoptosis. To better understand mechanisms of resistance, we established ganetespib-resistant cell lines. Resistant cells did not select for HSP90 mutations, and these cells conserved on-target suppression of PI3K/AKT, MAPK signalling, upregulation of HSP70, and MCL1 downregulation. However addiction to MCL1 was lost as was block of Caspase8 activation with consequent cross-resistance to TRAIL. PCR of Caspase8 cDNA revealed an acquired structural alteration in the 3’-untranslated region.

      Conclusion
      Here we show that HSP90 inhibition requires engagement of the mitochondrial apoptosis pathway, and involves cooperation of multiple BH3-only proteins with parallel suppression of MCL1. Interestingly, ganetespib may exploit tumour dependence on MCL1; this may be clinically relevant given that MCL1 (1q21.2) amplification correlates with dependence and its gene copy number alteration is one of the most frequent across cancers. Acquired resistance involves selection for loss of dependence on MCL1, and a block in Caspase8 signalling which lies upstream of BID. Failure of ectopic MCL1 overexpression to rescue is indicative of redundant death signalling by ganetespib. Clinical significance of core apoptosis gene expression will be explored and presented in a correlative analysis of the 9090-06 ganetespib monotherapy clinical trial in NSCLC.

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    P1.02 - Poster Session 1 - Novel Cancer Genes and Pathways (ID 144)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.02-008 - hSSB1: an essential regulator of genomic integrity in lung cancer (ID 2045)

      09:30 - 09:30  |  Author(s): D.A. Fennell

      • Abstract

      Background
      Lung cancer remains a leading cause for cancer mortality worldwide. A key feature of lung cancer development is genomic instability resulting from an accumulation of DNA lesions. In normal settings, these DNA lesions, such as double strand breaks and oxidised DNA, are rapidly repaired to prevent cytotoxicity and loss of genetic information. However, the molecular basis for the loss of genome integrity during cancer development remains to be determined. Herein, we have examined the involvement of hSSB1 in maintenance of genome stability and its potential role in lung cancer progression. hSSB1 is a critical component of the repair of DNA double strand breaks. As part of this study, we examined if hSSB1 is also involved in the repair of oxidative stress-induced DNA modifications. The most common nucleotide modification, 8-oxo-7,8-dihydro-guanine (8-oxoG), is repaired by the enzyme OGG1. Failure to repair these modifications results in mismatch mutations which are common in cancer. Therefore, understanding the molecular basis for genomic instability is key to the development of future therapeutics with clinical relevance.

      Methods
      To determine if hSSB1 expression is associated with lung cancer progression, a tissue microarray (TMA) with cores from 550 patients was stained with an anti-hSSB1 antibody. Kaplan-Meier survival curves were generated with the clinical data and patient prognosis was correlated with hSSB1 expression levels. To determine an in vitro association with lung cancer, A549 lung cancer cells were treated with hSSB1 specific siRNA. Cell survival was determined by microscopy and MTT assay. To examine the role of hSSB1 in repair of oxidised DNA, U2OS cells were treated with 500 µM H~2~O~2~. Cells treated with H~2~O~2~ were fixed, stained with antibodies for hSSB1 and 8-oxoG and examined by deconvolution microscopy. Lysates were collected from H~2~O~2~ treated U2OS cells and subjected to immunoprecipitation and Western blot analysis. Genomic DNA isolated from scrambled or hSSB1 siRNA U2OS cells treated with H~2~O~2~ were immobilised on nylon membrane and stained with antibodies for 8-oxoG.

      Results
      Significantly, TMA staining of lung cancer tissues indicated universal overexpression of hSSB1. Survival curves generated from the patient data indicated a poorer prognosis for patients with increased hSSB1 expression versus lower expressing tumours. Interestingly, in vitro inhibition of hSSB1 using siRNA significantly reduced cell survival. These data highlight the potential prognostic value of hSSB1 expression. As oxidative stress is prevalent in lung cancers, we also tested whether hSSB1 is capable of repairing 8-oxoG DNA lesions. Following oxidative DNA damage, hSSB1 localises rapidly to chromatin. hSSB1 also directly interacts with OGG1 to facilitate OGG1 recruitment to chromatin and repair of DNA damage. Importantly, cells lacking hSSB1 display ineffective repair of 8-oxoGs.

      Conclusion
      Our data highlight a potential role for hSSB1 in lung cancer progression and a novel role in maintenance of genome integrity. As tumours have increased genomic instability, elevated hSSB1 expression in lung cancer may enable tumours to cope with genomic instability. Taken together, these data present hSSB1 both as a prognostic marker and a novel therapeutic target.

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    P1.06 - Poster Session 1 - Prognostic and Predictive Biomarkers (ID 161)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.06-054 - Targeting MCL1 amplification in NSCLC through anthracycline-mediated transcriptional suppression (ID 3213)

      09:30 - 09:30  |  Author(s): D.A. Fennell

      • Abstract

      Background
      Targeting oncogene dependency for effective therapy has been one of the most successful strategies for managing metastatic non-small cell lung cancer (NSCLC). Although validating therapeutically tractable oncogenic driver mutations are a major focus, non-driver mutations may also confer dependencies that may also be exploitable. The prosurvival BCL2 protein, MCL1 prevents mitochondrial apoptosis by blocking interaction of proapoptotic BH3 only proteins with their multidomain proapototic counterparts, BAX and BAK. MCL1 is often mutated in cancers, and ranks as one of the most frequently amplified loci at 1q21.2. MCL1 amplified tumours exhibit addiction to this oncogene. Anthracyclines have been shown to transcriptionally suppress MCL1. Phase IIA studies in NSCLC have shown that epirubicin has useful single agent activity in unselected patients, with a significantly greater response rate than that achieved with standard chemotherapy. We therefore set out to evaluate MCL1 addiction in NSCLC, its correlation with anthracyline induced apoptosis and the prevalence of 1q21.2amplification to support a planned 1q21.2 stratified phase II trial in NSCLC, (EORTC-1303-LCG).

      Methods
      RNAi targeting MCL1was conducted in NCI-H460, NCI-H1299, NCI-H28 and NCI-H23 cell lines. Doxorubicin activity was measured by viability assay and apoptosis was assessed by western blot. gDNA from cell lines was obtained by Phenol-Chloroform extraction. The QIAamp DNA FFPE Tissue Kit was used to extract gDNA from FFPE tissues. MCL1 amplification was quantified by real-time PCR with a set of two primers and one probe (minor groove-binding (MGB) hydrolysis probe assay) for the gene of interest MCL1 and the two reference genes CCT3 and H6PD. Tonsil samples were used as a control diploid population.

      Results
      MCL1 silencing efficiently induced apoptosis in a subset of NSCLC cells, however we identified two cell lines that were resistant to MCL1 knockdown (NCI-H1299 and NCI-H28). Doxorubicin efficiently induced apoptosis in MCL1 addicted cells but exhibited significantly less activity in cells that were not addicted. We developed a genomic DNA based quantitative real time PCR assay to evaluate copy number variation (CNV) at the 1q21.2 locus. A clear correlation r[2] >0.91 was observed for 1q21.2 CNV compared with reference Conan Copy Number Analysis Tool (Cancer genome project, Sanger). Increased 1q21.2 copy number was consistently associated with MCL1addiction; however addiction also occurred in cells lacking 1q21.2 CNV, suggesting that MCL1 amplification represents a subset of MCL1 dependence. The concentration of doxorubicin was titrated against MCL1 protein downregulation into therapeutically sub-micromolar concentration range and we observed that MCL1 downregulation occurred coincidently with cleavage of poly-ADP ribose polymerase. We then screened DNA isolated from 19 adenocarcinomas, and identified 1q21.2 CNVs in 36.8%, with high level amplification (CNV >5) in 1q21.2 in 10.5%.

      Conclusion
      Targeting MCL1 addiction in 1q21.2 amplified NSCLC induces apoptosis and this dependence can be exploited by anthracyclines at therapeutically relevant concentrations. Given its significant prevalence in NSCLC, our data suggests that 1q21.2 amplification could be a novel non-driver mutation predictive for anthracycline response.