PolQ as a novel therapeutic target in AML

PolQ 作为 AML 的新型治疗靶点

基本信息

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
项目类别：
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

项目摘要

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.

急性髓样白血病（AML）是成年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开发了更具体的疗法（FLT3酪氨酸激酶抑制剂，TKI）。尽管如此，完全的解雇还是很少的，首先之后反应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修复途径保持其生存。 BRCA1/2介导的同源重组（HR）通常起主要作用在DSB维修中。但是，我们报告说表达AML1-Eto和PML-RAR的白血病，15-35％核型正常AML和TKI - 处理过的FLT3（ITD/TKD） - 阳性AML（占核型的39％正常AML）缺乏HR。 HR缺陷型实体瘤细胞的增殖取决于DNA 由POLQ基因编码的聚合酶theta（polθ），该基因促进了备份DSB维修微型学介导的最终连接（MMEJ）。根据CBIOPORTAL数据库AMLS表达所有肿瘤中的POLQ mRNA水平最高。 Western印迹分析检测到高水平的POLθ 与正常对应物相比，AML细胞。这些发现总共受初步数据支持的支持表明AML细胞的存活（尤其是在恒定的遗传毒性应力下的HR缺陷型）取决于polθ。所以， Polθ可能是AML中新型且有前途的药物靶标。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AIM＃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）以查明突变，易感性白血病细胞对polθ抑制+/-标准细胞毒性药物敏感。使用高吞吐量筛选，然后进行生化测试和体外细胞灵敏度测定法，我们确定了特异性杀死HR缺陷原始AML的polθ抑制剂（POLθI）细胞。在AIM＃2中，我们将进一步发展针对新型药物样实体的Polθi AML的治疗。在AIM＃3中，我们将采用鼠类AML样模型，原代AML细胞和异种移植物进行测试抑制POLθ的治疗潜力与（1）其他DNA修复抑制剂（RAD52I， PARPI）针对HR缺陷型AML，以及（2）针对HR促进AML的标准细胞毒性药物。我们预计在这些研究中开发的Polθi将有效地针对广泛的AML 最终将进入临床试验。