Preclinical Development of Novel Targeted Therapeutics Against Pediatric Sarcoma

1. 我们之前的研究结果揭示了癌症中IGF1R水平的高度变化（Cao等人，Cancer Res. 68: 8039- 48,2008），表明癌细胞中IGF1R水平与抗IGF1R抗体的抗增殖反应之间存在直接关联。表达IGF1R升高的癌细胞对IGF1R抗体非常敏感。我们的数据表明，在体外和体内，肿瘤细胞高度依赖IGF1R的升高来维持高AKT信号。治疗性抗体抑制IGF1R导致IGF1R升高的肿瘤细胞中AKT信号的显著减少。最近，我们在体外和体内发现了一个IGF1R抗体选择性诱导肿瘤细胞快速死亡的模型系统。我们的研究结果阐明了AKT和BclxL介导的抗igf1r诱导的癌细胞死亡机制（Mayeenuddin et al., Oncogene. 2010）。Bcl2未升高的肿瘤细胞对IGF1R和AKT信号的依赖程度更高，因此更容易发生抗IGF1R诱导的细胞死亡。我们的数据进一步显示了IGF1R在肿瘤生长和存活中的双重功能。我们目前的研究主要集中在耐药机制和与其他靶向药物的联合治疗上，以克服IGF1R靶向药物的耐药，提高其治疗活性。2. 为了鉴定对肉瘤具有选择性活性的新型药物和预测反应的生物标志物，我们与Genentech合作研究了一种针对死亡受体DR5的抗体drozitumab。我们发现DR5，而不是DR4，在所有横纹肌肉瘤（RMS）细胞的表面持续保持高水平。DR5抗体drozitumab在体外对大多数RMS细胞系有效。caspase-8的表达与对drozitumab的敏感性之间存在很强的相关性，仅在敏感细胞中诱导死亡诱导信号复合物的快速组装和caspase-8的裂解。更重要的是，caspase-8的催化活性是介导对drozitumab敏感性的必要和充分条件。此外，drozitumab对已建立的RMS异种移植物具有有效的抗肿瘤活性，具有从体外分析预测的特异性，并且在一半的治疗小鼠中无肿瘤状态。我们的研究首次对RMS中死亡受体抗体的效力和选择性进行了临床前评估。在体外，Drozitumab对大多数表达caspase-8的RMS细胞系有效，在体内，可以提供RMS的长期控制（Kang clini）。癌症，2011)。我们目前的工作重点是利用全基因组RNAi筛选研究耐药机制。候选基因已经确定，我们正在发现DR5介导的细胞死亡的新调控机制。这项研究可能会导致与dr5靶向药物协同作用的药物的鉴定。

1. IGF1R targeted agents Our previous results revealed a high degree of variation of IGF1R levels in cancers (Cao et al., Cancer Res. 68: 8039-48, 2008) showed a direct correlation between the levels of IGF1R in cancer cells and the anti-proliferative response to anti-IGF1R antibodies. Cancer cells expressing elevated IGF1R were very sensitive to IGF1R antibody. Our data suggested that tumor cells had a high degree of dependence on elevated IGF1R for maintaining high AKT signaling, both in vitro and in vivo. The inhibition of IGF1R with therapeutic antibodies resulted in a dramatic reduction of AKT signaling in tumor cells with elevated IGF1R. Recently, we identified a model system in which IGF1R antibody selectively induced rapid tumor cell death in vitro and in vivo. Our results illustrate the mechanism of anti-IGF1R-induced cancer cell death mediated via AKT and BclxL (Mayeenuddin et al., Oncogene. 2010). Tumor cells without elevated Bcl2 had a greater degree of dependence on IGF1R and AKT signaling, and thus, more susceptible to anti-IGF1R induced cell death. Our data further showed a dual function for IGF1R in tumor growth and survival. Our currently studies have been focused on the resistance mechanism and combination with other targeted agents to overcome resistance and to enhance therapeutic activity of IGF1R targeted agents. 2. Death receptor targeted agents To identify novel agents with selective activity against sarcoma and biomarkers predictive of responses, we investigated a death receptor DR5 targeted antibody drozitumab in working with Genentech. We show that DR5, but not DR4, persisted at high levels and on the surface of all rhabdomyosarcoma (RMS) cells. DR5 antibody drozitumab was effective in vitro against the majority of RMS cell lines. There was a strong correlation between caspase-8 expression and the sensitivity to drozitumab, which induced the rapid assembly of the death-induced signaling complex and the cleavage of caspase-8 only in sensitive cells. More importantly, caspase-8 catalytic activity was both necessary and sufficient for mediating the sensitivity to drozitumab. Furthermore, drozitumab had potent antitumor activity against established RMS xenografts with a specificity predicted from the in vitro analysis and with tumor-free status in half of the treated mice. Our study provides the first preclinical evaluation of the potency and selectivity of a death receptor antibody in RMS. Drozitumab is effective, in vitro, against the majority of RMS cell lines that express caspase-8 and, in vivo, may provide long-term control of RMS (Kang Clin. Cancer Res. 2011). Our current work is focusing on the resistance mechanism using whole-genome RNAi screening. Candidate genes were identified and we are in the process of discovery of a novel regulatory mechanism of DR5 mediated cell death. This study may lead to the identification of agents that synergize with DR5-targeted agents.