Elucidating anti-angiogenic tyrosine kinase inhibitor-induced vascular dysfunction

项目摘要 虽然酪氨酸激酶抑制剂（TKI）在治疗恶性肿瘤中是高度有效的，但是它们中的>60%已经被用于治疗恶性肿瘤。 据报道，导致不良心血管结局。此外，抗血管生成TKI，如舒尼替尼， 优先诱导微血管毒性，随后是心功能障碍。然而，我们对 TKI诱导的血管毒性（TKI-VT）的潜在致病机制部分受到以下因素的阻碍： 对人类患病血管组织进行分子和细胞分析的途径有限。因此，没有有效的 已经开发了预防或治疗这些否则会危及生命的心血管并发症的策略。 该项目将利用患者特异性诱导多能干细胞（iPSC）衍生的心脏周细胞 (PCs)和内皮细胞（EC），以了解TKI-VT的分子和细胞基础， 为患有TKI诱导的心血管疾病的癌症患者提供个性化治疗。目标1： 将采用单一培养和芯片上血管（VoC）共培养系统来表征舒尼替尼诱导的细胞 iPSC-PC和iPSC-EC之间的类型特异性细胞毒性特征和异常细胞串扰， 有助于TKI-VT。重叠上调和下调的差异表达基因（DEG） 在两种细胞类型中由舒尼替尼触发的基因突变将作为大规模可药物靶筛选的候选基因。 在目标2中，将在舒尼替尼治疗的受试者中进行CRISPR干扰/激活（CRISPRi/a）存活筛选。 使用定制的慢病毒sgRNA文库靶向目标1中鉴定的重叠DEG的iPSC-PC。顶部 CRISPRi和CRSIPRa机器中的10个命中基因将进行基于结构的虚拟筛选（SBVS） 以发现可以减轻iPSC-VoC中TKI-VT表型的候选化合物。在Aim 3中，3D iPSC- 工程化血管组织（EVT）和小鼠模型将用于验证 候选化合物对TKI-VT在更生理环境中的作用。产生的离体血浆蛋白质组 人全血灌注的3D iPSC-EVT将与患者中鉴定的那些相关联，以发现可靠的 疾病相关生物标志物预测个体癌症患者对TKI-VT的易感性。研究与 在K99阶段的职业发展培训计划，在吴博士和Ky的指导下，以及 专家跨学科咨询委员会，将为沈博士提供先进的干细胞知识 生物学、血管生物学、心脏肿瘤学、CRISPR技术和生物工程。的发展 CRISPRi/a和SBVS筛查平台（K99）和3D iPSC-EVT（R 00）将使他能够进行疾病诊断， 特别是在心脏肿瘤学和一般血管疾病方面的建模和药物发现研究。新 在K99/R 00职业发展奖期间获得的技能和经验，结合沈博士之前的 血管生物学的专业知识，将有助于他过渡到一个独立的职业生涯进行基础和 心脏肿瘤学的转化研究，特别关注血管方面。

PROJECT SUMMARY Although tyrosine kinase inhibitors (TKIs) are highly potent in treating malignancies, >60% of them have been reported to lead to adverse cardiovascular outcomes. Moreover, anti-angiogenic TKIs, such as sunitinib, preferentially induce microvascular toxicity followed by cardiac dysfunction. However, our knowledge of the underlying pathogenic mechanisms of TKI-induced vascular toxicity (TKI-VT) has been hampered partially by the limited access to human diseased vascular tissues for molecular and cellular analysis. As such, no effective strategies have been developed to prevent or treat these otherwise life-threatening cardiovascular complications. This proposed project will leverage patient-specific induced pluripotent stem cell (iPSC)-derived cardiac pericytes (PCs) and endothelial cells (ECs) to understand the molecular and cellular basis of TKI-VT and discover personalized therapies for cancer patients who are suffering TKI-induced cardiovascular disease. In Aim 1, both monoculture and vessel-on-chip (VoC) coculture systems will be employed to characterize sunitinib-induced cell type-specific cytotoxicity profiles and aberrant cellular crosstalk between iPSC-PCs and iPSC-ECs that can contribute to TKI-VT. Overlapping upregulated and downregulated differentially expressed genes (DEGs) triggered by sunitinib in both cell types will serve as the candidate genes for large-scale druggable target screens. In Aim 2, CRISPR interference/activation (CRISPRi/a) survival screens will be performed in sunitinib-treated iPSC-PCs using a customized lentiviral sgRNA library targeting the overlapping DEGs identified in Aim 1. Top 10 hit genes in CRISPRi and CRSIPRa machineries will be subjected to structure-based virtual screens (SBVS) to discover candidate compounds that can mitigate TKI-VT phenotypes in iPSC-VoCs. In Aim 3, 3D iPSC- engineered vascular tissues (EVTs) and a mouse model will be used to validate the mitigation efficacy of candidate compounds on TKI-VT in a more physiological setting. The ex vivo plasma proteome generated from human whole blood-perfused 3D iPSC-EVTs will be correlated with those identified in patients to discover reliable disease-relevant biomarkers in predicting individual cancer patients’ susceptibility to TKI-VT. The research and career development training plans during the K99 phase, under the mentorship of Drs. Wu and Ky, as well as an expert interdisciplinary advisory committee, will provide Dr. Shen with advanced knowledge in stem cell biology, vascular biology, cardio-oncology, CRISPR technology, and bioengineering. The development of CRISPRi/a and SBVS screen platforms (K99) and 3D iPSC-EVTs (R00) will enable him to conduct disease modeling and drug discovery research in cardio-oncology specifically and vascular disease in general. The new skills and experience gained during this K99/R00 career development award, combined with Dr. Shen’s prior expertise in vascular biology, will facilitate his transition to an independent career conducting basic and translational research in cardio-oncology with a particular focus on the vascular aspect.