Expanding the efficacy of asparaginase to solid tumors

这项建议的目的是为血癌药物的使用提供体内概念验证。 门冬酰胺酶(ASNase)在肝细胞癌患者中的表达 对这部生物小说。天冬氨酸氨基转移酶有一种独特的作用模式，药物会耗尽氨基酸 血液中的天冬酰胺，结果是依赖血液天冬酰胺的细胞挨饿，最终 被杀了。目前FDA批准的ASNase是细菌来源的，这使它们具有免疫原性，并且它们的 导致毒性的谷氨酰胺酶(GLNase)副反应会导致严重的药物副作用，这些副作用在 成年人。因此，尽管ASNase在治疗几种癌症方面具有巨大的潜力，但这些 药物主要限于治疗儿童急性淋巴细胞白血病(ALL)。使 ASNase疗法是依赖血液天冬酰胺的成年癌症患者的一种选择，我们设计了一种 一种类似人类的ASNase，可减轻免疫反应，并具有高度特异性，可消除与GLNase相关的 毒性。值得注意的是，毒性研究将我们的工程化类人ASNase与当前的护理标准进行了比较 细菌ASNase(Oncaspar)证明我们的ASNase的安全性显著提高。重要的是， 安全性的提高对所有人都有同等的效果。总而言之，这些发展使我们有可能 将ASNase治疗扩大到成人癌症患者的实体肿瘤。对ASNase的反应取决于NO/Low 表达合成天冬酰胺的酶，称为天冬酰胺合成酶(ASNS)。 然而，持久的反应需要癌细胞无法上调ASNS在 对天冬酰胺消耗的反应。上调ASNS表达的能力由甲基化决定 ASNS启动子的状态，其中高甲基化阻止表达，低甲基化允许 表情。最近对癌细胞系和患者样本的分析表明，许多肝癌患者拥有 要么是完整的ASNase反应特征(低ASNS水平，高甲基化启动子)，要么是部分ASNase- 应答信号(低ASNS水平，低甲基化启动子)。具有完整签名的肿瘤细胞是 预计只对ASNase有强烈反应，具有部分签名的人预计有 反应不那么持久。在前期工作中，我们验证了完整和部分ASNase的预测能力 几种肝细胞癌细胞系的反应特征。为了增加这项观察的临床相关性，在目标1 我们将从退伍军人(在JBVAMC接受治疗)和以少数人为主的人身上收集肝癌肿瘤样本 人群(在UIC接受治疗)，并确定这些患者拥有ASNase的倾向- 敏感性生物标志物。这些患者样本还将被用来产生肝癌原代细胞系、有机类化合物、 和PDX型号。在目标2中，使用体外和体内研究，我们将确定使 对ASNase治疗敏感或耐药的肝癌细胞系。AIM 3将把体内研究扩展到患者- 在AIM 1中产生的衍生细胞系、有机化合物和PDX，并评估我们预测哪些患者的能力 会对ASNase疗法有反应。这些研究的成功将为使用我们的新的、 肝细胞癌中更安全的ASNase。此外，通过利用基于机械的生物标志物，我们将能够识别 那些对ASNase有反应的患者。成功也将为检验这部小说提供动力 在其他癌症中，使用这些研究验证的生物标记物进行患者选择。

The goal of this proposal is to provide in vivo proof-of-concept for the use of the blood cancer drug asparaginase (ASNase) in hepatocellular carcinoma (HCC) patients who possess the biomarkers for response to this novel biologic. ASNases have a unique mode of action wherein the drug depletes the amino acid asparagine from the blood, and, as a result, cells that rely on blood asparagine are starved and ultimately killed. The current FDA-approved ASNases are of bacterial origin, which makes them immunogenic, and their toxicity-causing glutaminase (GLNase) side activity causes severe drug side effects, which are exacerbated in adults. Therefore, despite the immense potential of ASNases for the treatment of several cancer types, these drugs are predominately confined to the treatment of pediatric acute lymphoblastic leukemia (ALL). To make ASNase therapy an option for adult patients with cancers that depend on blood asparagine, we engineered a human-like ASNase that mitigates the immune response and is highly specific to eliminate the GLNase-related toxicity. Notably, toxicity studies comparing our engineered human-like ASNase to the current standard-of-care bacterial ASNase (Oncaspar) demonstrated the significantly improved safety of our ASNase. Importantly, the improved safety comes with equivalent efficacy for ALL. Together, these developments make it possible to expand ASNase therapy to solid tumors in adult cancer patients. Response to ASNase is dependent on no/low expression of the enzyme that synthesizes asparagine de novo, called asparagine synthetase (ASNS). However, a durable response requires the inability of the cancer cell to upregulate ASNS expression in response to asparagine depletion. The ability to upregulate ASNS expression is determined by the methylation state of the ASNS promoter, where hypermethylation prevents expression and hypomethylation allows for expression. Recent analysis of cancer cell lines and patient samples reveal that many HCC patients possess either the full ASNase-response signature (low ASNS levels, hypermethylated promoter) or partial ASNase- response signature (low ASNS levels, hypomethylated promoter). Tumor cells with the full signature are predicted to strongly respond to ASNase alone, and those with the partial signature are predicted to have a less durable response. In preliminary work, we verified the predictive power of the full and partial ASNase response signatures on several HCC cell lines. To increase the clinical relevance of this observation, in Aim 1 we will collect HCC tumor samples from Veterans (treated at JBVAMC) and from a predominantly minority population (treated at UIC) and determine the predisposition of these patients to possess the ASNase- sensitivity biomarkers. These patient samples will also be used to generate HCC primary cell line, organoid, and PDX models. In Aim 2, using both in vitro and in vivo studies, we will determine the factor(s) that make HCC cell lines sensitive or resistant to ASNase therapy. Aim 3 will expand the in vivo studies to the patient- derived cell lines, organoids and PDXs generated in Aim 1, and evaluate our ability to predict which patients will respond to ASNase therapy. Success in these studies will supply the proof-of-concept for using our new, safer ASNase in HCC. Moreover, by utilizing mechanistically based biomarkers we would be able to identify those patients that would respond to ASNase. Success will also provide the impetus for testing this novel approach in other cancers, using the biomarkers validated by these studies for patient selection.