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 摘要平滑肌肉瘤(LMS)是最常见的肉瘤之一，对其治疗具有相当大的挑战。 因为50%的患者发生转移，而目前的化疗只对 少数人。我们已经证明，大多数LMS都有染色体不稳定(CIN)，这在一定程度上是由于 同源重组缺陷和普遍存在的TP53和RB1失活。我们最近的研究表明 这些CIN异常与对DNA蛋白激酶(DNA-PK)的高度依赖有关，DNA-PK-DNA-PK- 与ATM和ATR-一起，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，我们希望开发最大限度地增加固有LMS基因毒性应激的治疗方法，特别是靶向 通过发现更有选择性的方法来靶向DNA-PK途径。为此，目标1确定 相关生物标志物(生化DNA-PK途径激活、DNA损伤反应和基因组 CIN的证据)在给定患者的转移内和不同转移中均可发现。 目标1还确定了部分失活和完全失活TP53突变是否对LMS有不同的影响 CIN和DNA-PK激活水平与靶向治疗的不同预测相关性 DNA-PK和DNA完整性。通过确定DNA-PK激活和DNA损伤的检测是否有效 通过LMS IHC和基因组图谱的验证，AIM 1表明这些检测方法是否可以作为稳健和 为我们随后的结合低剂量的临床试验(目标3b)进行可重复性的相关科学评估 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筛查，在有/没有DNA-PKI、PARPI和阿霉素治疗的情况下进行。这个 CRISPR筛查旨在发现DNA-PK抑制剂的合成致死和抗性机制，以及生物 通过在同一屏幕中执行RNAseq和磷酸蛋白质组分析，可以增强来自这些屏幕的洞察力 LMS条件，并通过鉴定对DNA-PKI或阿霉素获得性耐药的LMS细胞。目标3是 项目1的临床翻译，包括体外试验，小鼠临床前验证和初步的 阿霉素联合DNA-PK抑制的临床试验。在这里，我们测试了一种假设，即非常低剂量的 阿霉素使LMS细胞对DNA-PKI敏感，从而将CIN转化为易感性，同时最小化 非肿瘤细胞毒性。