Expanding the efficacy of asparaginase to solid tumors

该提案的目的是为血液癌症药物的使用提供体内概念验证 门冬酰胺酶（ASNase）在具有应答生物标志物的肝细胞癌（HCC）患者中的作用 这部生物小说天冬氨酸酶具有独特的作用模式，其中药物消耗氨基酸 因此，依赖于血液天冬酰胺的细胞会挨饿， 杀了目前FDA批准的ASNA酶是细菌来源的，这使得它们具有免疫原性，并且它们的免疫原性是由细菌引起的。 引起毒性的谷氨酰胺酶（GLNase）副活性引起严重的药物副作用，其在 成年人了因此，尽管ASNases用于治疗几种癌症类型的巨大潜力，但这些癌症类型的治疗仍然是非常困难的。 药物主要限于治疗小儿急性淋巴细胞白血病（ALL）。使 ASNase疗法是依赖于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 类人ASNase，可减轻免疫应答，并对消除GLNase相关 毒性值得注意的是，毒性研究比较了我们的工程类人ASNase与目前的标准护理， 细菌ASNase（OnCaspar）证明了我们的ASNase的显著改善的安全性。重要的是 安全性的提高与ALL的疗效相当。总之，这些发展使我们有可能 将ASNase疗法扩展到成人癌症患者的实体瘤。对ASNase的反应取决于无/低 表达从头合成天冬酰胺的酶，称为天冬酰胺合成酶（ASNS）。 然而，持久的反应需要癌细胞不能上调ASNS表达， 对天冬酰胺耗竭的反应。上调ASNS表达的能力是由甲基化决定的。 ASNS启动子的一种状态，其中高甲基化阻止表达，低甲基化允许表达。 表情最近对癌细胞系和患者样本的分析显示，许多HCC患者具有 无论是完整的ASNase-应答信号（低ASNS水平，高甲基化启动子）还是部分ASNase-应答信号（低ASNS水平，高甲基化启动子）， 响应特征（低ASNS水平，低甲基化启动子）。具有完整特征的肿瘤细胞 预测单独对ASNase有强烈反应，而那些具有部分签名的人被预测有强烈反应。 较不持久的反应。在初步工作中，我们验证了完整和部分ASNase的预测能力 在几种HCC细胞系上的应答特征。为了增加该观察结果的临床相关性，在目标1中 我们将从退伍军人（在JBVAMC治疗）和主要少数人中收集HCC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。这些患者样品还将用于产生HCC原代细胞系、类器官， PDX模型在目标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HCC细胞系。目标3将把体内研究扩展到患者- Aim 1中产生的衍生细胞系、类器官和PDX，并评估我们预测哪些患者 会对ASNase治疗有反应这些研究的成功将为使用我们的新， 更安全的ASNase。此外，通过利用基于机制的生物标志物，我们将能够识别 那些对ASNase有反应的病人成功也将为测试这部小说提供动力 在其他癌症中，使用这些研究验证的生物标志物进行患者选择。

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.