RETINOID THERAPY OF NEUROBLASTOMA

我们的临床前研究表明，13-顺式维甲酸（13-cis-RA）可以抑制神经母细胞瘤（NBL）细胞的细胞生长和降低MYCN癌基因的表达。 这些数据使我们进行了一项I期和一项随机III期试验，结果表明，在强化化疗后给予高剂量脉冲13-cis-RA显著改善了高危NBL患者的生存率。 并不是所有的患者受益于13-cis-RA，我们发现，一些NBL细胞系来源于临床耐13-cis-RA的患者在体外对13-cis-RA和trans-RA耐药。 我们发现，RA衍生物N-（4-羟基苯基）视黄酰胺（芬维A胺; 4-HPR）对耐13-顺式-RA的NBL细胞系具有高度细胞毒性。该提案的假设是，4-HPR将通过根除肿瘤细胞，包括对13-cis-RA耐药的细胞，进一步改善高危NBL患者的生存率。本项目将探索4-HPR选择性诱导肿瘤细胞死亡的分子机制。 我们发现，4-HPR引起的神经酰胺（脂质参与凋亡信号）在NBL细胞系的水平大幅增加，（像外源性神经酰胺）4-HPR的细胞毒性涉及细胞凋亡和坏死。 我们将继续这项工作，确定是否神经酰胺合成酶诱导4-HPR或神经酰胺代谢酶下调。 为了确定由高剂量4-HPR介导的细胞毒性是否是由于神经酰胺增加，我们将用含有葡糖神经酰胺合酶的诱导型载体转染NBL细胞系，其将4-HPR处理的细胞中的神经酰胺分流成无毒形式。 将鉴定和开发对4-HPR具有耐药性的细胞系，以确定4-HPR耐药性是否是由未能响应4-HPR而增加神经酰胺或将神经酰胺“分流”为无毒形式介导的。 将评价抑制神经酰胺分流的药剂增强4-HPR对NBL细胞系的细胞毒性的能力。 将测试体外对NBL细胞系具有增强的细胞毒性的药剂与4-HPR的组合对成纤维细胞和骨髓祖细胞的一般毒性。以及针对NBL异种移植物的毒性和功效。 4-将评价HPR和增强剂对耐13-顺式-RA和化疗药物的NBL细胞系的功效。 该项目与我们正在进行的4-HPR I期试验相结合，将提供数据以优化采用4-HPR对抗NBL的临床试验设计。 我们预计，产生的数据将支持开发一个未来的临床试验，测试4-HPR的能力，以改善高风险NBL的结果后，清髓治疗和13-顺式RA。 阐明4-HPR的作用机制应使我们能够确定临床上有用的药物，将提高抗癌活性4-HPR在未来的临床试验中进行测试。

Our pre-clinical studies showed that 13-cis-retinoic acid (13-cis-RA) can inhibit cell growth and decrease expression of the MYCN oncogene in neuroblastoma (NBL) cells in vitro. These data led us to conduct a phase I and then a randomized phase III trial which showed that high-dose, pulse 13-cis-RA significantly improved survival for high-risk NBL when given after intensive chemotherapy. Not all patients benefited from 13-cis-RA, and we found that some NBL cell lines derived from patients who were clinically resistant to 13-cis-RA are resistant to 13-cis-RA and trans-RA in vitro. We discovered that the RA derivative N-(4-hydroxyphenyl) retinamide (fenretinide; 4-HPR) is highly cytotoxic for NBL cell lines that are resistant to 13-cis-RA. The hypothesis of this proposal is that 4-HPR will further improve the survival of high-risk NBL patients by eradicating tumor cells, including those cells that are resistant to 13-cis-RA. This project will explore the molecular mechanisms by which 4-HPR selectively induces tumor cell death. We showed that 4-HPR caused large increases in the levels of ceramide (a lipid involved in apoptotic signaling) in NBL cell lines and that (like exogenous ceramide) 4-HPR cytotoxicity involved both apoptosis and necrosis. We will continue this work by determining if ceramide synthetic enzymes are induced by 4-HPR or if ceramide metabolic enzymes are down-regulated. To determine if cytotoxicity mediated by high-dose 4-HPR is due to increased ceramide we will transfect a NBL cell line with an inducible vector containing glucosylceramide synthase, which will shunt ceramide in 4-HPR treated cells to a non-toxic form. Cell lines will be identified and developed that are resistant to 4-HPR to determine if 4-HPR-resistance is mediated by a failure to increase ceramide in response to 4-HPR or by "shunting" the ceramide to non-toxic forms. Agents that inhibit ceramide shunting will be evaluated for their ability to enhance the cytotoxicity of 4-HPR for NBL cell lines. Combinations of agents with 4-HPR that have enhanced cytotoxicity against NBL cell lines in vitro will be tested for general toxicity against fibroblasts and myeloid progenitors. and for toxicity and efficacy against NBL xenografts. 4-HPR and enhancing agents will be evaluated for their efficacy against NBL cell lines that are resistant to 13-cis-RA and chemotherapeutic drugs. This project, in combination with our ongoing phase I trial of 4-HPR, will provide data to optimize clinical trial design employing 4-HPR against NBL. We anticipate that data generated will support developing a future clinical trial testing the ability of 4-HPR to improve outcome for high risk NBL when given after myeloablative therapy and 13-cis-RA. Elucidating mechanisms of action for 4-HPR should allow us to identify clinically useful agents that will enhance the anti-cancer activity 4-HPR for testing in future clinical trials.