Development of companion diagnostics for dasatinib-based personalized therapy for T-ALL

项目总结 急性淋巴细胞性白血病(ALL)是儿童最常见的恶性肿瘤，也是主要病因 儿童时期与癌症相关的死亡案例。ALL可发生在两种淋巴谱系中，其中B-ALL和 T-ALL分别占癌症的85%和15%。T-ALL与更具攻击性有关 与B-ALL相比，目前的特点和历史上较差的治疗结果。当前T-ALL 治疗很大程度上依赖于细胞毒性化疗药物。近年来，进一步加剧了 化疗导致了T-ALL治愈率的递增，但很可能有 由于过度的毒性(特别是在复发的情况下)达到了平台期。此外，在 复发的T-ALL，白血病对细胞毒药物有明显的耐药性。不像高风险的B-都是为了 CAR-T等细胞疗法非常有效，目前还没有针对T-ALL的免疫疗法 而复发患者的五年存活率不到25%，令人沮丧。因此，小说 需要分子靶向治疗来改善患者的生存和生活质量 患有T-ALL的儿童。 我们先前观察到，在的T-ALL病例中(43名儿童和21名成年人) 38%的患者在体外对达沙替尼表现出显著的敏感性。尤其是T-ALL敏感的比例 儿童服用达沙替尼的比例明显高于成人(分别为49%和14%)。达沙替尼LC50 (杀死50%白血病细胞的药物浓度)在这些T-ALL病例中与 在BCR-ABL1B-ALL中观察。然而，对达沙替尼敏感的T-ALL病例中没有一例发生ABL 融合也不会对更具ABL特异性的抑制剂产生反应。我们证明了LCK的激活 而其下游靶标的磷酸化水平是导致这种敏感性的原因，以及更多 重要的是，可以用来预测达沙替尼治疗T-ALL的有效性。在.期间 在这个项目中，我们将开发一个配套的诊断面板，可以用来预测对 达沙替尼和波纳替尼以个性化的方式。以下目标将在 建议：目标#1.通过对LCK的磷酸化和激活状态的综合表征 T-ALL细胞中的LC-MS。目的#2.基于PRM-MS和免疫分析的方法的发展 P-LCK、p-CD247和p-ZAP70的快速定量分析。到这个项目完成时，一个 将开发配套的诊断面板，并用细胞培养样本进行验证。这种可行性 部分将使这一个性化诊断测试在PDX小鼠中得到更广泛的验证 第二阶段的模型和患者样本。

PROJECT SUMMARY Acute lymphoblastic leukemia (ALL) is the most common malignancy in children and a leading cause of cancer-related death during childhood. ALL can arise in both lymphoid lineages, with B-ALL and T-ALL accounting for 85% and 15% of this cancer. T-ALL is associated with more aggressive presenting features and historically inferior treatment outcomes compared to B-ALL. Current T-ALL therapy largely relies on cytotoxic chemotherapeutic agents. In recent years, further intensification of chemotherapy has led to incremental increases in the cure rate of T-ALL, but is likely to have reached a plateau due to excessive toxicities (especially in the relapse setting). Moreover, in relapsed T-ALL, leukemia is markedly resistant to cytotoxic drugs. Unlike high-risk B-ALL for which cellular therapy such as CAR-T is highly effective, there are no immunotherapies available for T-ALL and patients with relapsed disease have a dismal five-year survival rate below 25%. Therefore, novel and molecularly targeted therapeutics are needed to improve both survival and quality of life for children with T-ALL. We have previously observed that in 64 T-ALL cases (43 children and 21 adults) profiled thus far 38% showed a striking sensitivity to dasatinib in vitro. In particular, the proportion of T-ALL sensitive to dasatinib is markedly higher in children than in adults (49% vs 14%, respectively). Dasatinib LC50 (drug concentration that kills 50% of leukemia cells) in these T-ALL cases were on par with that observed in BCR-ABL1 B-ALL. However, none of the dasatinib-sensitive T-ALL cases had ABL fusion nor did they respond to a more ABL-specific inhibitor. We demonstrated that LCK activation and phosphorylation level of its downstream targets are responsible for this sensitivity, and, more importantly, can be used to predict the effectiveness of dasatinib treatment in T-ALL cases. During this project, we will develop a companion diagnostic panel that can be used to predict sensitivity to dasatinib and ponatinib in a personalized manner. The following aims will be completed in the proposal: Aim #1. Comprehensive characterization of phosphorylation and activation state of LCK by LC-MS in T-ALL cells. Aim #2. Development of the PRM-MS and immunoassay-based methods for rapid quantitative analysis of p-LCK, p-CD247, and p-ZAP70. By the completion of this project, a companion diagnostic panel will be developed and validated with cell culture samples. This feasibility portion will enable a much more extensive validation of this personalized diagnostic test in PDX mice models and patient samples in Phase II.