Hiromasa Takeda

Synergistic Effect of Olaparib with Combination of Cisplatin on PTEN-Deficient Lung Cancer Cells

PARP enzyme plays a key role in the cellular machinery responsible for DNA damage repair. PTEN is a tumor-suppressor gene deactivating PI3K downstream of EGFR signaling. We hypothesize that PTEN-deficient lung cancer cells suppressed DNA damage signaling and that the absence of PTEN can sensitize these cells to a concurrent treatment of a DNA-damaging agent (cisplatin) and a PARP inhibitor (olaparib). To investigate the effect of olaparib and cisplatin on PTEN-deficient lung tumors, two EGFR-mutant (deletion in exon19) non–small cell lung cancer (NSCLC) cell lines, PC-9 (PTEN wild-type) and H1650 (PTEN loss), were used. We transfected intact PTEN gene into H1650 cells (H1650PTEN+) and knocked down PTEN expression in the PC-9 cells (PC-9PTEN−) using short hairpin RNA (shRNA). Combination of cisplatin with olaparib showed a synergistic effect in vitro according to the combination index in H1650 cells. Restoration of PTEN in the H1650 cells decreased sensitivity to the combination. Ablation of PTEN in PC-9 cells increased sensitivity to olaparib and cisplatin. We also examined the effectiveness of cisplatin and olaparib in a xenograft model using H1650 and PC-9PTEN− cells. The combination of cisplatin with olaparib was more effective than each agent individually. This effect was not observed in a xenograft model using H1650PTEN+ and PC-9 cells. Mechanistic investigations revealed that PTEN deficiency caused reductions in nuclear RAD51 and RPA focus formation and phosphorylated Chk1 and Mre11. Thus, genetic inactivation of PTEN led to the suppression of DNA repair. Mol Cancer Res; 11(2); 140–8. ©2012 AACR.

JAK2-related pathway induces acquired erlotinib resistance in lung cancer cells harboring an epidermal growth factor receptor-activating mutation

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, such as gefitinib and erlotinib, are effective for non‐small cell lung cancer with activating EGFR mutations. However, even in patients with an initial dramatic response to such a drug, acquired resistance develops after 6–12 months. A secondary mutation of T790M in EGFR and amplification of the MET gene account for this resistance; however, the mechanism(s) of approximately 30% of acquired resistance cases remain unknown. We established an erlotinib‐resistant lung cancer cell line named PC‐9/ER3 that harbors an EGFR mutation after continuously exposing PC‐9 cells to erlotinib. PC‐9/ER3 cells were 136‐fold more resistant to erlotinib than the parental cells. Although the PC‐9/ER3 cells did not carry the T790M mutation or MET amplification and had similar levels of phosphorylated (p) STAT3, pJAK2 increased in the resistant cells. It was found in the present study that 3–12 h of exposure to erlotinib in both cell lines did not affect pJAK2 expression, but did result in increased pSTAT3 expression. pAkt in PC‐9/ER3 cells was less suppressed than in PC‐9 cells, although pEGFR and pMAPK were markedly suppressed in both cell lines. The combined treatment of erlotinib plus a JAK2 inhibitor (JSI‐124) suppressed pAkt in PC‐9/ER3 cells. Similarly, the combination of erlotinib plus JSI‐124 or siRNA against JAK2 restored sensitivity to erlotinib in PC‐9/ER3 cells. The combination of erlotinib plus JSI‐124 was also effective for reducing PC‐9/ER3 tumors in a murine xenograft model. Our results suggest that the activation of JAK2 partially accounts for acquired erlotinib resistance.(Cancer Sci, doi: 10.1111/j.1349‐7006.2012.02363.x, 2012)

Vandetanib is effective in EGFR-mutant lung cancer cells with PTEN deficiency.

The effectiveness of vandetanib, an agent that targets RET, VEGFR and EGFR signaling, against EGFR-mutant lung cancer cells with PTEN loss was investigated. Two EGFR mutant non-small cell lung cancer (NSCLC) cell lines, PC-9 (PTEN wild type) and NCI-H1650 (PTEN null), were used. We transfected an intact PTEN gene into H1650 cells and knocked down PTEN expression in PC-9 cells using shRNA. The effectiveness of gefitinib and vandetanib was assessed using a xenograft model. While PC-9 cells were more resistant to vandetanib than gefitinib, H1650 cells were more sensitive to vandetanib than gefitinib. Both gefitinib and vandetanib suppressed the activation of EGFR and MAPK in H1650 cells, although phosphorylated AKT levels were not affected. In an H1650 cell xenograft model, vandetanib was also more effective than gefitinib. Although PTEN-transfected H1650 cells did not show restoration of sensitivity to gefitinib in vitro, the xenograft tumors responded to gefitinib and vandetanib. Knockdown of PTEN in PC-9 cells caused resistance to gefitinib. In conclusion, vandetanib might be effective in NSCLC with EGFR mutations that lack PTEN expression. The contribution of PTEN absence to vandetanib activity in NSCLC cells harboring EGFR mutations should be further examined.

Chemopreventive effects of gefitinib on nonsmoking-related lung tumorigenesis in activating epidermal growth factor receptor transgenic mice

Twenty-five percent of all lung cancer cases are not attributable to smoking. Epidermal growth factor receptor (EGFR) mutations, which are involved in approximately 50% of nonsmoker lung cancer, are positively correlated with responsiveness to gefitinib, and inversely correlated with smoking history. Activating EGFR mutations play a critical role in the carcinogenesis of nonsmoking-related lung cancer. To investigate the chemopreventive effects of gefitinib on nonsmoking-related lung cancer, we generated transgenic mice expressing EGFR L858R in type II pneumocytes constitutively using the surfactant protein-C promoter. The transgenic mice invariably developed atypical adenomatous hyperplasia at age 4 weeks and multifocal adenocarcinoma of varying sizes at age 7 weeks. Notably, the expression levels of phosphorylated and total ErbB2, ErbB3, and thyroid transcription factor-1 were elevated in the transgenic mice compared with wild-type controls at age 3 weeks. Administration of gefitinib to 3-week-old transgenic mice for 1 week before carcinogenesis reduced the amount of phosphorylated EGFR in the lungs of the mice to the baseline level. Gefitinib (5 mg/kg/d; n = 5, 5, and 15) or vehicle (n = 5, 5, and 15) was administered to transgenic mice from age 3 to 8, 13, and 18 weeks, respectively. The numbers of lung tumors in the control and gefitinib-treated groups were 1.75, 5.8, 10.2, and 0 (P < 0.05), respectively. No fatal toxic events occurred in either group, and gefitinib inhibited tumorigenesis completely in this mouse model. These results suggest the utility of molecular targeted chemoprevention against nonsmoking-related lung cancer.

Abstract 2048: PTEN deficient lung cancer cells are sensitive to the combination of olaparib with cisplatin through suppressing DNA damage signaling.

PARP inhibition in combination with DNA-damaging agents is one of the most promising new therapeutic approaches to cancer. PTEN is a tumor-suppressor gene deactivating PI3K downstream of EGFR signaling. In H1650 cells harboring EGFR deletion mutation in exon 19 with PTEN loss, we reported that synergistic effects of a PARP inhibitor (olaparib) and a DNA-damaging agent (cisplatin) were observed in vitro and in vivo (#4686, AACR 2012). We further clarified how PTEN loss affected DNA damage signaling. H1299 and A549 are lung cancer cells harboring wild type PTEN. Antagonistic effects of olaparib with cisplatin were shown in H1299 and A549 cells according to combination index (CI). Sensitivity to a PARP inhibitor was reported to be associated with a defect in Mre11 expression. H1650 cells exhibited lower levels of Mre11 compared with PTEN-transfected H1650 cells (designated H1650PTEN+ cells). To address how lower expression levels of Mre11 affects the synergism between cisplatin and olaparib, Mre11 expression vector was transfected into H1650 cells (designated H1650Mre11+ cells). CIs were 0.23, 0.20, 0.57 and 0.29 when concentration ratios of cisplatin and olaparib were designed to be molar ratios of 1:1, 1:2, 1:3 and 1:5, respectively, in H1650 cells, while CIs were 0.76, 0.91, 0.97 and 0.77 in H1650Mre11+ cells. Although these CIs in H1650Mre11+ were somewhat elevated compared with those observed in original H1650 cells, these results indicated that lower levels of Mre11 alone could not be the sole reason for the synergism. On the other hand, PTEN has other nuclear functions, including transcriptional regulation of the RAD51 gene, whose product is essential for homologous recombination (HR) repair of DNA breaks. RPA is displaced from single stranded DNA by RAD51 to initiate HR. There were no significant differences of RAD51 and RPA levels by Western blotting. Meanwhile, PTEN deficiency resulted in significant reduction in RAD51 and RPA focus formation after drug-exposure or irradiation compared to H1650PTEN+ cells although γH2AX was similarly increased in both cells. As RPA binds to single stranded DNA, the RPA focus could be a marker for end resection at the double stranded DNA ends. Thus, inactivation of PTEN might lead to suppression of DNA damage signaling, leading to the lower levels of end resection and, hence, less RPA focus formation. As shown above, reduced levels of Mre11 alone could not provide a sufficient explanation for this, though Mre11 is involved in the molecular mechanisms of the end resection. The combination of cisplatin with olaparib in PTEN deficient lung tumors might be pursued in clinical trials although further investigations should be required to clarify whether and to what extent the molecular events including Mre11, RAD51 and RPA are responsible for the synergic effect. Citation Format: Daisuke Minami, Nagio Takigawa, Hiromasa Takeda, Minoru Takata, Nobuaki Ochi, Eiki Ichihara, Akiko Hisamoto, Katsuyuki Hotta, Mitsune Tanimoto, Katsuyuki Kiura. PTEN deficient lung cancer cells are sensitive to the combination of olaparib with cisplatin through suppressing DNA damage signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2048. doi:10.1158/1538-7445.AM2013-2048

Abstract 4686: Synergistic effect of olaparib (AZD2281) with combination of cisplatin on PTEN-deficient lung cancer cell lines

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Poly(ADP-ribose) polymerase (PARP) enzyme plays a key role in the cellular machinery responsible for DNA damage repair. Although inhibition of this enzyme using a pharmacological inhibitor is postulated to enhance the cytotoxicity of DNA-damaging cytotoxic chemotherapy, this has not been profoundly studied in lung cancer. PTEN (phosphatase and tensin homologue deleted on chromosome ten) is a tumor-suppressor gene deactivating PI3K downstream of EGFR (epidermal growth factor receptor) signaling. Approximately 2% to 9% of non-small cell lung cancers (NSCLC) are considered to have PTEN loss, and a recurrent gross-mutation of the PTEN gene is identified in lung cancer with deficient DNA double-strand break repair. We hypothesize that there is a cellular threshold for error-free DNA-repair in lung cancer cells and that the absence of PTEN can sensitize these cells to a concurrent treatment of a DNA-damaging agent (cisplatin) and a PARP inhibitor (olaparib). To investigate the effect of olaparib and cisplatin on PTEN deficient lung tumors, two EGFR mutant (deletion in exon19) NSCLC cell lines, PC-9 (PTEN wild type) and NCI-H1650 (PTEN loss) were used. Two additional cell lines with PTEN loss, prostate cancer cell line (PC-3) and glioblastoma cell line (U-87MG) were also examined. We transfected intact PTEN gene into H1650 cells and knocked down PTEN expression in the PC-9 cells using shRNA. Antiproliferative activity was determined by MTT assay in terms of 50% inhibitory concentration (IC50) values. Olaparib alone was not effective for H1650 cells and other PTEN-deficient cells. IC50 values ranged from 6 to 40 μM for olaparib and from 0.2 to 2 μM for cisplatin. Combination of cisplatin with olaparib showed a synergistic effect in vitro according to the combination index (CI). CIs were 0.23, 0.20, 0.57 and 0.29 when concentration ratios of cisplatin and olaparib were designed to be molar ratios of 1:1, 1:2, 1:3 and 1:5, respectively. Restoration of PTEN in the H1650 cells decreased sensitivity to olaparib and cisplatin (CI >1). Ablation of PTEN in PC-9 cells increased sensitivity to olaparib and cisplatin (CI <1). We also examined the effectiveness of the cisplatin (5 mg/kg/week) and olaparib (50 mg/kg/day) in a xenograft model using H1650 cells. The combination of cisplatin with olaparib was more effective than each agent individually. This effect was not observed in a xenograft model using PTEN transfected H1650 cells. Mechanistic investigations revealed that PTEN-deficiency caused a reduction in radiation-induced nuclear RAD51 focus formation and activation of Chk1 S345. Thus, genetic inactivation of PTEN led to suppression of DNA repair. The combination of cisplatin with olaparib in PTEN deficient lung tumors should be further pursued. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4686. doi:1538-7445.AM2012-4686

Abstract 3681: Vascular endothelial growth factor receptor tyrosine kinase inhibitor inhibited mutated epidermal growth factor receptor-driven tumors ex vivo and in vivo

We previously established a non-smoking-related lung cancer mouse model employing an epidermal growth factor receptor (EGFR) L858R transgenic mouse using an SP-C promoter. The mice developed pulmonary atypical adenomatous hyperplasia (AAH), multifocal adenocarcinoma, and died due to multiple invasive lung tumors at 4, 7, and approximately 40 weeks of age, respectively. We demonstrated the marked chemopreventive effect of gefitinib, an EGFR tyrosine kinase inhibitor, in a non-smoking-related lung cancer mouse model and presented a novel concept of molecular targeted chemoprevention of lung cancer in non-smokers. In this study, we focused on the expression of vascular endothelial growth factor receptor (VEGFR) -2 and vascular endothelial growth factor (VEGF) in a non-smoking-related lung cancer mouse model. Surprisingly, we found that VEGFR-2 and EGFR were activated in the transgenic mice at 3 weeks of age by Western blotting, before the histological detection of AAH and adenocarcinoma. And fluorescent immunostaining of lung tissues in transgenic mice showed activation of VEGFR-2 occurred in tumor cells themselves or cells in an initial stage of carcinogenesis, not only in vascular endothelial cells. We also found that VEGFR-2 was activated in the lung cancer cell lines (PC-9, H3255) harboring EGFR mutation, and that EGFR-L858R transfected 293T cells expressed more phosphorylated VEGFR-2 than non-transfected 293T cells. EGFR-L858R-transfected 293T cells were more sensitive than non-transfected 293T cells to cediranib (Recentin), a pan-VEGFR tyrosine kinase inhibitor. In this mouse model, cediranib (5 mg/kg/day, 5 days/week, 12 weeks, p.o.) inhibited surface lung tumors (long axis diameter >1 mm, 0 ± 0.4 versus 4.9 ± 3.2, p If VEGFR-2 is activated during an early phase of carcinogenesis due to EGFR mutations, these data suggest that inactivation of VEGFR-2 may be effective in preventing carcinogenesis of non-smoking-related lung cancer. These findings provide t the new concept of VEGFR-2 role in chemoprevention. Recentin (cediranib) is a trademark of the AstraZeneca group of companies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3681. doi:10.1158/1538-7445.AM2011-3681

Abstract 4487: Effect of everolimus on lung tumorigenesis in transgenic mice carrying activating EGFR mutation

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The mammalian target of rapamycin (mTOR) plays a major role in the regulation of protein translation, cell growth, and metabolism. Alterations of the mTOR signaling pathway are common in cancer, and thus mTOR is being actively pursued as a therapeutic target. Everolimus is an orally available rapamycin analogue showing anticancer effects on renal cell carcinoma and used in clinical. Furthermore, in preclinical studies, everolimus shows anticancer effects in various cancer cell lines and xenograft models, including lung cancer. However, the effect of mTOR inhibitors on non-small cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) mutation remains unclear. Our present study was carried out to investigate the preclinical efficacy of everolimus in NSCLC harboring EGFR mutation. Growth inhibition was measured by MTT assays. RPC-9 cells that acquired T790M mutation of EGFR gene were established from parental PC-9 cells harboring exon 19 del mutation (delE746-A750) in our laboratory (Cancer Res 67, 2007). The drug concentration required to inhibit the growth of tumor cells by 50% (IC50) for 96 h exposure was used to evaluate the effectiveness of everolimus. Protein expression was determined by Western blotting. The IC50s were 2 μM for PC-9 cells and 4 μM for RPC-9 cells. RPC-9 cells that showed a 400-fold resistance to gefitinib compared with parental PC-9 cells remained the sensitivity to everolimus. Phosphorylated mTOR and phosphorylated S6 were suppressed by treatment with everolimus (2 μM), while phosphorylated AKT was increased presumably due to negative feedback of mTOR inhibition. We generated transgenic mice expressing the L858R mutation of EGFR driven by the SP-C promoter (Cancer Res 69, 2009). In the absence of treatment, the transgenic mice developed atypical adenomatous hyperplasia at age 4 weeks and adenocarcinoma at 6 to 7 weeks of age, and died due to tumor progression at 25 to 40 weeks. To investigate the effect of everolimus on lung tumors induced by an activating EGFR mutation in vivo, the transgenic mice were treated with oral everolimus (10 mg/kg/day, n = 13) or vehicle alone (n = 14) from 5 to 20 weeks of age. The number of lung tumors (long axis exceeding 1 mm) in the everolimus-treated group and control group was 1.9 ± 0.9 and 9.4 ± 3.2 (log-rank test, p<0.001), respectively. Phosphorylated S6 was suppressed during everolimus treatment. To evaluate the efficacy of everolimus on survival in EGFR transgenic mice, everolimus (10 mg/kg/day, n=12) or vehicle (n=12) was administered orally from 5 weeks of age. The median survival time in the everolimus-treated group (58 weeks) was significantly longer than that in the control group (33 weeks) (log-rank test, p<0.001). In conclusion, these preclinical data suggest that everolimus may be effective for chemoprevention and treatment of lung tumors induced by an activating EGFR gene mutation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4487. doi:10.1158/1538-7445.AM2011-4487

Abstract 724: Role of ERK reactivation mediated by Src in acquired resistance to gefitinib in non-small cell lung cancer with EGFR mutation

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: Non-small cell lung cancer (NSCLC) harboring some mutations in epidermal growth factor receptor (EGFR) often shows dramatic responses to EGFR tyrosine kinase inhibitors (EGFR-TKIs). Although the problem of acquired resistance to EGFR-TKI is widely recognized and has grown in clinical situation, some mechanisms have been elucidated. 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was known as tobacco specific nitrosamine. To clarify the resistant mechanisms to EGFR-TKIs associated with smoking, we established novel gefitinib-resistant NSCLC cells using gefitinib and NNK. Materials and Methods: The EGFR mutant (frame deletion in exon 19) NSCLC cell line, PC-9, was treated with gefitinib and NNK. The drug concentration required to inhibit the growth of tumor cells by 50% (IC50) was evaluated by MTT assay. Protein expression was determined by immunoblotting using specific antibodies. Extracted mRNA and DNA were analyzed by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and by sequencing with specific primers, respectively, to determine gene amplification and mutation. Some specific small interfering RNA (siRNA) was used to down-regulate the target gene expression. Result: IC50s of the established resistant cells (PC9-GR) to gefitinib ranged from 2 to 4 µM, which was a 100- to 200-fold decrease in gefitinib sensitivity compared with parental PC-9 cells. The resistant mechanisms in PC9-GR cells were different from T790M mutation, RAS mutation, MET amplification, phosphatase and tensin homolog (PTEN) loss or the insulin-like growth factor-1 receptor (IGF-1R) signaling. Although gefitinib promptly suppressed phosphorylations of EGFR, Akt and the mitogen-activated protein/extracellular signal-regulated kinase (ERK), only ERK was reactivated 6 hours after treatment with gefitinib in PC9-GR cells but not in parental PC-9 cells. the mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) inhibitor (U0126) significantly suppressed ERK reactivation and also inhibited cell growth. These results suggest ERK reactivation play a key role in this resistant mechanism. Furthermore, we found the combination of gefitinib with Src inhibitor (dasatinib) suppressed ERK reactivation in PC9-GR cells and significantly inhibited cell growth. In addition, the combination of gefitinib with Src-specific siRNA brought improvement of gefitinib sensitivity in PC9-GR cells. Conclusion: ERK reactivation mediated by Src participated in the novel gefitinib-resistant mechanism. Src inhibition might be a new therapeutic strategy to overcome an acquired gefitinib-resistant NSCLC with EGFR mutation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 724. doi:10.1158/1538-7445.AM2011-724

Abstract 621: High-dose gefitinib overcomes the resistance to gefitinib related to hepatocyte growth factor

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Molecular targeted agents are being a mainstay in non-small cell lung cancer. Dramatic responses to the epidermal growth factor receptor (EGFR) -tyrosine kinase inhibitor (TKI) are observed in advanced non-small cell lung cancer (NSCLC), especially with EGFR mutation. Different from cytotoxic agents, it remains unclear whether EGFR-TKI exerts its anti-tumor effect dose-dependently. Gefitinib could not show survival benefit in previously treated unselected patients with NSCLC in a randomized phase III trial, whereas erlotinib did. The difference is considered resulting from the fact that erlotinib was dosed at its maximum-tolerated dose (MTD), while gefitinib was dosed at about one third of its MTD. To investigate the dose-dependency of gefitinib in the cells harboring mutated EGFR, we compared two doses of gefitinib (15 or 50 mg/kg) using PC-9 (adenocarcinoma of lung) xenografts. Both doses of gefitinib reduced tumors. However, the tumors regrew in the low-dose group after several weeks of treatment, whereas did not in the high dose group. This result indicates that gefitinib might have the dose-dependency in the tumors harboring mutated EGFR. We resected tumors at 10 weeks after gefitinib treatment. The removed tumors did not acquired T790M mutation and MET amplification. Interestingly, the cells (PC-9/RX) that were derived from the xenograft tumors treated with low-dose gefitinib remained the sensitivity to gefitinib. Thus, we investigated hepatocyte growth factor (HGF) as the environmental factor. Tumor tissues treated with gefitinib were positively stained with anti-mouse HGF. Mouse HGF mRNA and protein expression in the tumor tissues treated with gefitinib were significantly high when compared to those treated with vehicle alone, and similar results were obtained by ELISA. We confirmed that mouse HGF induced resistance to gefitinib in an ex vivo experiment. To elucidate the role of HGF in vivo, PC-9 xenografts were treated with gefitinib in combination with rabbit anti-HGF antibody. The combination of gefitinib with anti-HGF antibody inhibited tumor growth partially compared with the low-dose gefitinib alone. Taken together, HGF might induce resistance to low-dose of gefitinib treatment in an in vivo xenograft model before the tumor cells themselves acquired the resistant genetic changes. In conclusion, higher-dose of gefitinib might prevent resistance to gefitinib related to HGF and produce longer response duration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 621.