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：总结/摘要 平滑肌肉瘤（LMS）是最常见的肉瘤之一，是一个相当大的治疗挑战 因为 50% 的患者出现转移，而目前的化疗仅对少数患者提供临床益处 少数。我们已经证明，大多数 LMS 具有染色体不稳定性 (CIN)，部分原因是 同源重组缺陷以及普遍存在的 TP53 和 RB1 失活。我们最近的研究表明 这些 CIN 畸变与对 DNA 蛋白激酶 (DNA-PK) 的过度依赖有关，其中 – 与 ATM 和 ATR 一样，它是 DNA 损伤反应 (DDR) 酶类 PI3K 激酶家族的成员。 DNA-PK 是双链 DNA 损伤的关键修复机制，从而使 CIN 低于基因毒性 阈值。 在项目 1 中，我们希望开发能够最大化内在 LMS 基因毒性应激的疗法，特别是针对 DNA-PK 并发现更有选择性的方法来靶向 DNA-PK 途径。为此，目标 1 确定 是否有相关的生物标志物（生化 DNA-PK 通路激活、DNA 损伤反应和基因组 CIN 的证据）在同一患者的转移灶内和不同转移灶中均发现。 目标 1 还确定部分失活和完全失活的 TP53 突变是否对 LMS 有不同的影响 CIN 和 DNA-PK 激活水平，因此与靶向治疗的预测相关性不同 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 通过 LMS IHC 和基因组图谱证明，目标 1 表明这些测定是否可以用作稳健且可靠的检测方法。 结合低剂量的后续（目标 3b）临床试验的可重复相关科学评估 阿霉素和 DNA-PK 抑制。目标 2 提供实现功效和选择性所需的生物学见解 用于 LMS 中的 DNA-PK 抑制和其他 DDR 靶向疗法。这些体外研究包括 DDR CRISPR-i CRISPR-a 筛选，在有/没有 DNA-PKi、PARPi 和阿霉素治疗的情况下进行。这 CRISPR 筛选旨在发现 DNA-PK 抑制剂合成致死和耐药机制，以及生物制剂 通过在相同的环境中执行 RNAseq 和磷酸化蛋白质组图谱，可以增强从这些筛选中获得的见解。 LMS 条件，并通过表征对 DNA-PKi 或多柔比星获得性耐药的 LMS 细胞。目标 3 是 项目 1 的临床翻译，包括体外和小鼠临床前验证以及初步 阿霉素联合 DNA-PK 抑制的临床试验。在这里，我们测试了一个假设，即非常低的剂量 阿霉素使 LMS 细胞对 DNA-PKi 敏感，从而将 CIN 转化为一种负担，同时最大限度地减少 非肿瘤细胞毒性。