Preclinical Development of Novel Targeted Therapeutics Against Pediatric Sarcoma

1. IGF 1 R靶向剂我们先前的结果揭示了癌症中IGF 1 R水平的高度变化（Cao等人，癌症研究68：8039-48，2008）显示癌细胞中IGF 1 R的水平与对抗IGF 1 R抗体的抗增殖应答之间的直接相关性。表达升高的IGF 1 R的癌细胞对IGF 1 R抗体非常敏感。我们的数据表明，肿瘤细胞在体外和体内都高度依赖于升高的IGF 1 R来维持高AKT信号传导。用治疗性抗体抑制IGF 1 R导致IGF 1 R升高的肿瘤细胞中AKT信号传导显著减少。最近，我们确定了一个模型系统，其中IGF 1 R抗体选择性地诱导快速肿瘤细胞死亡在体外和体内。我们的结果说明了通过AKT和BclxL介导的抗IGF 1 R诱导的癌细胞死亡的机制（Mayeenuddin等人，致癌基因2010年）。没有升高的Bcl 2的肿瘤细胞对IGF 1 R和AKT信号具有更大程度的依赖性，因此，更容易受到抗IGF 1 R诱导的细胞死亡。我们的数据进一步显示了IGF 1 R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应用，以克服耐药和增强IGF 1 R靶向药物的治疗活性。2.死亡受体靶向药物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合作研究了死亡受体DR 5靶向抗体drozitumab。我们发现，DR 5，而不是DR 4，坚持在高水平和表面上的所有横纹肌肉瘤（RMS）细胞。DR 5抗体drozitumab在体外对大多数RMS细胞系有效。caspase-8的表达与对drozitumab的敏感性之间存在强相关性，drozitumab诱导死亡诱导信号复合物的快速组装和caspase-8的切割仅在敏感细胞中发生。更重要的是，半胱天冬酶-8的催化活性对于介导对drozitumab的敏感性是必要的，也是足够的。此外，drozitumab对已建立的RMS异种移植物具有有效的抗肿瘤活性，具有体外分析预测的特异性，并且在一半的治疗小鼠中具有无肿瘤状态。我们的研究提供了RMS中死亡受体抗体的效力和选择性的第一个临床前评价。Drozitumab在体外对大多数表达半胱天冬酶-8的RMS细胞系有效，在体内可长期控制RMS（Kang Clin. Cancer Res. 2011）。我们目前的工作是利用全基因组RNAi筛选抗性机制。候选基因已被确定，我们正在发现DR 5介导的细胞死亡的新调控机制。这项研究可能导致识别与DR 5靶向药物协同作用的药物。

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.