RETINOID THERAPY OF NEUROBLASTOMA

我们的临床前研究表明，13-顺式维甲酸(13-cis-RA)在体外可以抑制神经母细胞瘤(NBL)细胞的生长，降低MYCN癌基因的表达。这些数据使我们进行了第一阶段和第三阶段的随机试验，结果表明，在强化化疗后给予高剂量、脉冲13-顺式-RA显著提高了高危NBL的存活率。并不是所有的患者都受益于13-cis-RA，我们发现一些临床上对13-cis-RA耐药的患者的NBL细胞株在体外对13-cis-RA和反式RA都具有耐药性。我们发现维甲酸衍生物N-(4-羟基苯基)维甲酰胺(Fenretinide；4-HPR)对耐13-顺式维甲酸的NBL细胞株具有高度的细胞毒作用。这项提议的假设是，4-HPR将通过根除肿瘤细胞，包括那些对13-cis-RA具有耐药性的细胞，进一步提高高危NBL患者的存活率。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NBL细胞株神经酰胺(一种参与凋亡信号传递的脂质)水平的大幅增加，并且(与外源性神经酰胺一样)4-HPR的细胞毒性既涉及细胞凋亡，也涉及坏死。我们将继续这项工作，确定神经酰胺合成酶是由4-HPR诱导的，还是神经酰胺代谢酶下调。为了确定大剂量4-HPR介导的细胞毒性是否是由于神经酰胺增加所致，我们将含有葡萄糖神经酰胺合成酶的诱导性载体导入NBL细胞系，这将使4-HPR处理的细胞中的神经酰胺分流到无毒形式。将鉴定和发展对4-HPR具有耐药性的细胞系，以确定4-HPR的耐药性是通过未能增加神经酰胺对4-HPR的反应还是通过将神经酰胺“分流”到无毒形式来介导的。抑制神经酰胺分流的药物将被评估其增强4-HPR对NBL细胞系的细胞毒性的能力。在体外增强对NBL细胞系的细胞毒性的药物与4-HPR的组合将被测试对成纤维细胞和髓系祖细胞的一般毒性。以及对NBL异种移植的毒性和有效性。将评估4-HPR和增强剂对耐13-顺式维甲酸和化疗药物的NBL细胞株的疗效。该项目与我们正在进行的4-HPR第一阶段试验相结合，将提供数据以优化使用4-HPR对抗NBL的临床试验设计。我们预计，所产生的数据将支持开发一项未来的临床试验，测试4-HPR在清髓治疗和13-顺式RA后给予4-HPR改善高危NBL预后的能力。阐明4-HPR的作用机制将使我们能够确定临床上有用的药物，以增强4-HPR的抗癌活性，以便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.