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: 10705729
• 负责人: JONATHAN Alfred FLETCHER
• 金额: $ 48.76万
• 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/10705729
• 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; Compensation; Coupled; Cytotoxic Chemotherapy; Cytotoxic agent; DNA; DNA Damage; Data Analyses; Defect; Dependence; Diagnosis; Dose; Doxorubicin; 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; PIK3CG gene; Pathway interactions; Patients; Pharmaceutical Preparations; Phosphotransferases; Poly(ADP-ribose) Polymerase Inhibitor; Pre-Clinical Model; Protein Kinase; Proteins; RB1 gene; Reproducibility; Resistance; Resources; Safety; Science; Specimen; TP53 gene; Testing; Therapeutic; Time; Toxic effect; Validation; 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; protein kinase inhibitor; repaired; resistance mechanism; response; sarcoma; targeted treatment; therapy development; transcriptome sequencing; validation studies

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（leiomyosarcoma，LMS）是最常见的肉瘤之一，是一个相当大的治疗挑战 因为50%的患者发生转移，而目前的化疗仅为患者提供临床益处， 小少数。我们已经表明，大多数LMS具有染色体不稳定性（CIN），这部分是由于 同源重组缺陷和普遍存在的TP 53和RB 1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）的高度依赖有关， 沿着ATM和ATR -是DNA损伤反应（DDR）酶的PI 3 K样激酶家族的成员。 DNA-PK是双链DNA损伤的关键修复机制，从而将CIN维持在遗传毒性以下。 阈值。 在项目1中，我们希望开发最大化内在LMS遗传毒性应激的疗法，特别是靶向 DNA-PK和发现更多的选择性靶向DNA-PK途径的方法。为此，目标1确定 是否存在相关生物标志物（生化DNA-PK途径活化、DNA损伤反应和基因组 CIN的证据）在转移瘤内和来自给定患者的不同转移瘤中均匀发现。 目的1还确定部分与完全失活TP 53突变是否对LMS具有不同的影响 CIN和DNA-PK活化水平，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活化和DNA损伤的测定是否有效， 通过LMS IHC和基因组图谱证明，Aim 1显示了这些测定是否可以用作稳健的和 我们后续（Aim 3b）临床试验结合低剂量 阿霉素和DNA-PK抑制。目标2提供了实现疗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靶向治疗。这些体外研究包括DDR CRISPR-i 和CRISPR-a筛选，其在有/没有DNA-PKi、PARPi和多柔比星处理的情况下进行。的 CRISPR筛选旨在发现DNA-PK抑制剂的合成致死和耐药机制，以及生物学机制。 通过在相同条件下进行RNAseq和磷酸化蛋白质组谱， LMS条件，并通过表征具有对DNA-PKi或阿霉素的获得性抗性的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始 阿霉素联合DNA-PK抑制的临床试验。在这里，我们测试的假设，一个非常低的剂量， 阿霉素使LMS细胞对DNA-PKi敏感，从而将CIN转化为负债，同时最小化 非肿瘤细胞毒性。