PolQ as a novel therapeutic target in AML

PolQ 作为 AML 的新型治疗靶点

• 批准号: 10322361
• 负责人: Richard T Pomerantz
• 金额: $ 53.58万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-17 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10322361
• 关键词: AML1-ETO fusion protein; Acute Myelocytic Leukemia; Acute leukemia; Adult; BRCA deficient; BRCA1 gene; BRCA2 gene; Biochemical; Biological Assay; CD34 gene; CRISPR/Cas technology; Cells; Cisplatin; Clinical Trials; Cytotoxic agent; DNA Double Strand Break; DNA Repair; DNA Repair Pathway; DNA lesion; DNA-Directed DNA Polymerase; Data; Databases; Development; Disease remission; Dominant-Negative Mutation; Double Strand Break Repair; Drug Kinetics; Drug Targeting; FLT3 gene; Gene Expression; Gene Mutation; Genes; Genetic; Genotoxic Stress; In Vitro; Individual; Induced Mutation; Infection; Knock-out; Laboratories; Left; Leukemic Cell; MLL-AF9; Malignant Neoplasms; Malignant neoplasm of ovary; Mediating; Messenger RNA; Modality; Modeling; Mus; Mutate; Mutation; Mutation Analysis; Normal Cell; PALB2 gene; Pathway interactions; Patients; Pharmaceutical Chemistry; Pharmaceutical Preparations; Pharmacology; Play; Polymerase; Prognosis; Proteins; RAD52 gene; Relapse; Reporting; Resistance; Role; Sampling; Solid Neoplasm; Stem cell transplant; Structure; Testing; The Cancer Genome Atlas; Therapeutic; Toxic effect; Tyrosine Kinase Inhibitor; Western Blotting; Xenograft procedure; acute myeloid leukemia cell; base; brca gene; cBioPortal; cancer cell; chemotherapy; cohort; design; high throughput screening; homologous recombination; improved; inhibitor; leukemia; leukemic stem cell; malignant breast neoplasm; mutant; neoplastic cell; new therapeutic target; novel; novel therapeutic intervention; novel therapeutics; older patient; paralogous gene; prevent; replication stress; response; small hairpin RNA; small molecule inhibitor; standard care; success; targeted treatment; therapeutic evaluation; tool; tumor

Acute myeloid leukemia (AML) is the most common type of acute leukemia among adults with overall poor prognosis. More specific therapies have been developed against FLT3(ITD/TKD)-positive AML (FLT3 tyrosine kinase inhibitors, TKi). Still, complete remissions are rare, and after initial response the leukemia invariably progresses within a few months. AML cells accumulate lethal DNA double-strand breaks (DSBs), but DSB repair pathways maintain their survival. BRCA1/2-mediated homologous recombination (HR) usually plays a major role in DSB repair. However, we reported that leukemias expressing AML1-ETO and PML-RAR, 15- 35% of karyotypic normal AMLs, and TKi – treated FLT3(ITD/TKD)-positive AMLs (39% of karyotypic normal AMLs) are HR-deficient. Proliferation of HR-deficient solid tumor cells depends on DNA polymerase theta (Polθ) encoded by POLQ gene, which promotes a backup DSB repair called microhomology-mediated end-joining (MMEJ). According to cBioPortal database AMLs express the highest levels of POLQ mRNA among all tumors. Western blot analysis detected high levels of Polθ in AML cells when compared to normal counterparts. Altogether, these findings supported by preliminary data suggest that survival of AML cells (especially HR-deficient), which are under constant genotoxic stress, depends on Polθ. Therefore, Polθ may be a novel and promising drug target in AML. Our preliminary data suggest that AML-inducing mutations [FLT3(ITD/TKD) +/- TET2mut, DNMT3Amut, MLL-AF9, AML1-ETO] can modulate the sensitivity of AML cells to the inhibition of Polθ. Therefore, in Aim #1 we will inhibit Polθ (shRNA, mutants and Polq-/- cells) and introduce AML-inducing mutations (retroviral infection, CRISPR/Cas9) to pinpoint mutations, which predispose leukemia cells to be sensitive to Polθ inhibition +/- standard cytotoxic drugs. Using high throughput screening followed by biochemical testing and in vitro cellular sensitivity assays, we identified Polθ inhibitors (Polθi) that specifically kill HR-deficient primary AML cells. In Aim #2 we will further develop Polθi towards novel drug-like entities as targeted therapeutics for AMLs. In Aim #3 we will employ murine AML-like models, primary AML cells and xenografts to test therapeutic potential of inhibition of Polθ combined with (1) other DNA repair inhibitors (RAD52i, PARPi) against HR-deficient AMLs, and (2) standard cytotoxic drugs against HR-proficient AMLs. We anticipate that Polθi developed in these studies will be effective against a wide-range of AMLs and ultimately will be advanced to clinical trials.

急性髓系白血病 (AML) 是成人中最常见的急性白血病类型 预后不良。针对 FLT3(ITD/TKD) 阳性 AML 已开发出更具体的疗法 （FLT3 酪氨酸激酶抑制剂，TKi）。尽管如此，完全缓解是罕见的，并且在最初的缓解之后 白血病总是在几个月内进展。 AML 细胞积累致命的 DNA 双链断裂 (DSB)，但 DSB 修复途径 维持他们的生存。 BRCA1/2 介导的同源重组 (HR) 通常起主要作用 在 DSB 修复中。然而，我们报道说，15-35% 的白血病表达 AML1-ETO 和 PML-RAR 核型正常 AML 和 TKi 治疗的 FLT3(ITD/TKD) 阳性 AML（核型正常 AML 的 39%） 正常的 AML）存在 HR 缺陷。 HR 缺陷的实体瘤细胞的增殖依赖于 DNA 由 POLQ 基因编码的聚合酶 theta (Polθ)，可促进称为 DSB 的备份修复 微同源介导的末端连接（MMEJ）。根据 cBioPortal 数据库 AML 表示 所有肿瘤中 POLQ mRNA 水平最高。 Western blot 分析检测到高水平的 Polθ AML 细胞与正常细胞相比。总而言之，这些发现 初步数据支持表明 AML 细胞（尤其是 HR 缺陷），处于持续的基因毒性应激下，取决于 Polθ。所以， Polθ 可能是 AML 中一个新颖且有前途的药物靶点。 我们的初步数据表明，AML 诱导突变 [FLT3(ITD/TKD) +/- TET2mut， DNMT3Amut、MLL-AF9、AML1-ETO]可以调节 AML 细胞对 Polθ 抑制的敏感性。 因此，在目标#1中，我们将抑制 Polθ（shRNA、突变体和 Polq-/- 细胞）并引入 AML 诱导突变（逆转录病毒感染、CRISPR/Cas9）以精确定位突变， 这使得白血病细胞对 Polθ 抑制+/-标准细胞毒性药物敏感。 使用高通量筛选，然后进行生化测试和体外细胞敏感性 通过分析，我们发现了 Polθ 抑制剂 (Polθi)，可以特异性杀死 HR 缺陷的原发性 AML 细胞。在目标 2 中，我们将进一步开发 Polθi，以新型药物样实体为目标 AML 的治疗方法。 在目标 3 中，我们将采用小鼠 AML 样模型、原代 AML 细胞和异种移植物进行测试 Polθ 抑制与 (1) 其他 DNA 修复抑制剂（RAD52i、 PARPi）针对 HR 缺陷的 AML，以及（2）针对 HR 充分的 AML 的标准细胞毒性药物。 我们预计这些研究中开发的 Polθi 将有效对抗多种 AML 并最终将进入临床试验。