Preclinical Development of Novel Targeted Therapeutics Against Pediatric Sarcoma

1.IGF1R靶向药物我们以前的研究结果显示IGF1R水平在癌症中有很高的变异(Cao等人，癌症研究68：8039-48,2008)表明癌细胞中的IGF1R水平与对抗IGF1R抗体的抗增殖反应直接相关。高表达IGF1R的癌细胞对IGF1R抗体非常敏感。我们的数据表明，无论在体外还是在体内，肿瘤细胞都高度依赖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AKT信号显著减少。最近，我们在体内和体外建立了IGF1R抗体选择性诱导肿瘤细胞快速死亡的模型系统。我们的结果阐明了通过AKT和BclxL(Mayeenuddin等人，癌基因)介导的抗IGF1R诱导癌细胞死亡的机制。2010)。不表达Bcl2的肿瘤细胞对IGF1R和AKT信号的依赖程度更高，因此更容易受到抗IGF1R诱导的细胞死亡的影响。我们的数据进一步表明IGF1R在肿瘤生长和生存中具有双重功能。我们目前的研究主要集中在IGF1R的耐药机制以及与其他靶向药物的联合应用，以克服耐药，提高IGF1R靶向药物的治疗活性。2.死亡受体靶向药物为了确定具有选择性抗肉瘤活性的新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DR5靶向抗体Drozitumab。我们发现DR5，而不是DR4，在所有横纹肌肉瘤(RMS)细胞表面的高水平持续存在。DR5抗体Drozitumab在体外对大多数RMS细胞株有一定的抑制作用。Caspase-8的表达与Drozitumab的敏感性密切相关，Drozitumab导致死亡诱导的信号复合体的快速组装和caspase-8仅在敏感细胞中的裂解。更重要的是，caspase-8的催化活性是调节Drozitumab敏感性的必要条件和充分条件。此外，Drozitumab对已建立的RMS异种移植瘤具有强大的抗肿瘤活性，其特异性可从体外分析中预测，并且在一半治疗的小鼠中处于无瘤状态。我们的研究首次对死亡受体抗体在RMS中的效力和选择性进行了临床前评估。Drozitumab在体外对大多数表达caspase-8的RMS细胞株有效，在体内可能提供对RMS(Kang Clin)的长期控制。癌症研究报告，2011)。我们目前的工作是通过全基因组RNAi筛选来研究抗性机制。候选基因已经确定，我们正在发现DR5介导的细胞死亡的一种新的调控机制。这项研究可能导致识别与DR5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.