PROJECT 1: Genomic Vulnerabilities in Leiomyosarcoma (LMS)

项目 1：平滑肌肉瘤 (LMS) 的基因组漏洞

• 批准号: 10493629
• 负责人: JONATHAN Alfred FLETCHER
• 金额: $ 46.35万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-16 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10493629
• 关键词: Address; Aftercare; Biochemical; Biological; Biological Assay; Biological Markers; Biopsy; CRISPR interference; CRISPR screen; CRISPR-mediated transcriptional activation; Cancer Biology; Cell Line; Cells; Chromosomal Instability; Clinical; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Combined Modality Therapy; Coupled; Cytotoxic Chemotherapy; Cytotoxic agent; DNA; DNA Damage; DNA-dependent protein kinase; Data Analyses; Defect; Dependence; Diagnosis; Dose; Doxorubicin; Doxorubicin-DNA Complex; Enzymes; Essential Genes; Evaluation; Event; Family; Genes; Genetic; Genetic Transcription; Genomics; Genotoxic Stress; Goals; Guide RNA; Heterogeneity; In Vitro; Investigational Therapies; Libraries; Mediating; Metastatic Leiomyosarcoma; Minority; Modeling; Molecular; Mus; Mutation; Neoplasm Metastasis; Pathway interactions; Patients; Pharmaceutical Preparations; Phosphotransferases; Pre-Clinical Model; Protein Kinase; Proteins; RB1 gene; Reproducibility; Resistance; Resources; Safety; Science; Specimen; Sum; TP53 gene; Testing; Therapeutic; Time; Toxic effect; Validation; base; chemotherapy; clinical translation; ds-DNA; gene repression; genomic profiles; genotoxicity; homologous recombination; improved; inhibitor; inhibitor therapy; insight; leiomyosarcoma; member; novel strategies; pre-clinical; preclinical study; predicting response; repaired; resistance mechanism; response; sarcoma; targeted treatment; therapy development; transcriptome sequencing

Project 1: Summary/Abstract Leiomyosarcoma (LMS) is one of the most common sarcomas and is a considerable therapeutic challenge because 50% of patients develop metastases for which current chemotherapy provides clinical benefit to only a small minority. We have shown that most LMS have chromosomal instability (CIN), which results in part from defects in homologous recombination and from ubiquitous TP53 and RB1 inactivation. Our recent studies show that these CIN aberrations are associated with hyper-dependency on DNA protein kinase (DNA-PK), which – along with ATM and ATR – is a member of the PI3K-like kinase family of DNA damage response (DDR) enzymes. DNA-PK is a key repair mechanism for double-strand DNA damage, thereby maintaining CIN below genotoxic thresholds. In Project 1, we wish to develop therapies that maximize intrinsic LMS genotoxic stress, particularly be targeting DNA-PK and by discovering more selective ways to target the DNA-PK pathway. To this end, Aim 1 determines whether relevant biomarkers (biochemical DNA-PK pathway activation, DNA damage response, and genomic evidence of CIN) are found homogeneously within a metastasis and in different metastases from a given patient. Aim 1 also determines whether partially vs. completely inactivating TP53 mutations have differing impact on LMS CIN and DNA-PK activation levels and therefore differing predictive relevance with respect to therapies targeting DNA-PK and DNA integrity. By determining if assays of DNA-PK activation and DNA damage are effectively demonstrated by LMS IHC and genomic profiles, Aim 1 shows whether these assays can be used as robust and reproducible correlative science assessments for our subsequent (Aim 3b) clinical trial combining low-dose doxorubicin and DNA-PK inhibition. Aim 2 provides biologic insights needed to achieve efficacy and selectivity for DNA-PK inhibition and other DDR-targeted therapies in LMS. These in vitro studies include DDR CRISPR-i and CRISPR-a screens, which are performed with/without DNA-PKi, PARPi and doxorubicin treatment. The CRISPR screens aim to discover DNA-PK inhibitor synthetic lethals and resistance mechanisms, and the biologic insights from these screens are enhanced by performing RNAseq and phosphoproteome profiles in the same LMS conditions, and by characterizing LMS cells with acquired resistance to DNA-PKi or doxorubicin. Aim 3 is the clinical translation for Project 1, which encompasses in vitro, and murine preclinical validations and an initial clinical trial of doxorubicin coupled with DNA-PK inhibition. Here we test the hypothesis that a very low dose of doxorubicin sensitizes LMS cells to DNA-PKi, thereby converting CIN into a liability while minimizing nonneoplastic cell toxicity.

项目1：摘要/摘要 平滑肌肉瘤（LMS）是最常见的肉瘤之一，是一项巨大的治疗挑战 因为有50％的患者发生转移试剂，目前的化学疗法仅为A提供临床益处 少数。我们已经表明，大多数LMS都有染色体不稳定性（CIN），这部分原因是 同源重组以及无处不在的TP53和RB1失活的缺陷。我们最近的研究表明 这些CIN差与DNA蛋白激酶（DNA-PK）的超依赖性有关，该差异是 - ATM和ATR - 是DNA损伤反应（DDR）酶的PI3K样激酶家族的成员。 DNA-PK是双链DNA损伤的关键修复机制，从而在Genotoxy下保持CIN 阈值。 在项目1中，我们希望开发最大化内在LMS遗传毒性应力的疗法，尤其是针对性 DNA-PK并通过发现靶向DNA-PK途径的更多选择方法。为此，目标1确定 是否相关生物标志物（生化DNA-PK途径激活，DNA损伤反应和基因组 在转移和给定患者的不同转移中发现CIN的证据）。 AIM 1还确定部分与完全失活的TP53突变对LMS有不同的影响 CIN和DNA-PK激活水平，因此与靶向疗法的预测相关性区分了 DNA-PK和DNA完整性。通过确定DNA-PK激活和DNA损伤的测定是否有效 AIM 1由LMS IHC和基因组概况展示，表明这些测定是否可以用作强大和 可重现的正确科学评估对我们随后的（AIM 3B）临床试验，结合了低剂量 阿霉素和DNA-PK抑制。 AIM 2提供了实现效率和选择性所需的生物学见解 用于LMS中的DNA-PK抑制和其他靶向DDR靶向疗法。这些体外研究包括DDR CRISPR-I 和CRISPR-A屏幕，这些屏幕是通过/不使用DNA-PKI，PARPI和阿霉素处理的。这 CRISPR屏幕旨在发现DNA-PK抑制剂合成致死和抗性机制以及生物学 通过在同一的RNASEQ和磷酸蛋白质组曲线上，从这些屏幕中的见解得到增强 LMS条件，并通过表征具有对DNA-PKI或阿霉素的耐药性的LMS细胞。 AIM 3是 项目1的临床翻译，其中包括体外，鼠临床前验证和初始 阿霉素的临床试验与DNA-PK抑制结合。在这里，我们测试了一个非常低剂量的假设 阿霉素将LMS细胞感知到DNA-PKI，从而将CIN转化为责任，同时最小化 非塑性细胞毒性。