Collaborative Pathways that Lead to Leukemia

导致白血病的协同途径

基本信息

批准号：
10926102
负责人：
Peter Aplan
金额：
$ 82.27万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10926102
关键词：
3&apos Untranslated Regions ABCB1 gene ABL1 gene Acute leukemia Age Months Alleles Animal Model Apoptosis B cell differentiation B-Cell Acute Lymphoblastic Leukemia B-Cell Leukemia B-Lymphocytes Binding Blood CRISPR screen Candidate Disease Gene Cell Line Cells Chemoresistance Clinical Collaborations Complex DHFR gene DNA Resequencing DNA biosynthesis DNA replication fork DNMT3a Development Epigenetic Process Gene Dosage Gene Expression Profile Generations Genes Genetic Genetically Engineered Mouse Genets Genomic DNA Growth Hematopoietic stem cells Human Hunger Immunophenotyping Impairment In Vitro Insertional Mutagenesis Knock-in Knock-out Length Leukemic Cell Licensing Factor Ligands Loss of Heterozygosity Lymphoblastic Leukemia Lymphoma Maintenance Malignant Neoplasms Manuscripts Methotrexate Methylation Modeling Molecular Mouse Strains Mus Mutant Strains Mice Mutate Mutation Myelogenous NOTCH1 gene NU/NU Mouse NUP214 gene NUP98 gene Nature PTPN1 gene Pathway interactions Patients Pattern Phenotype Phosphotransferases Preparation Production Proliferating Proteins Publishing Recurrence Reporting Resistance Sampling T-Cell Leukemia T-Lymphocyte TAL1 gene TCF3 gene TOP1 gene Thymocyte Development Transgenes Transgenic Mice Tumor Suppressor Genes Vincristine acute T-cell lymphoblastic leukemia cell cancer cell cell type exome sequencing experimental study follow-up fusion gene helicase human disease inhibitor leukemia leukemia/lymphoma leukemic transformation mutant novel strategies offspring precursor cell retroviral transduction small hairpin RNA stem cell self renewal theories thymocyte whole genome

项目摘要

We crossed transgenic mice that express a IDH2 R140Q mutation with mice that express an NHD13 fusion; the offspring develop a form of early T cell precursor (ETP) leukemia that resembles the human disease in terms of clinical presentation, immunophenotype, gene expression profile, and collaborative mutations. In terms of molecular mechanism, the IDH2R140Q mutant mice produce the oncometabolite 2HG; consistent with overproduction of 2HG, the leukemic cells show aberrant methylation of genes required for normal thymocyte development. Finally, a specific inhibitor of mutant IDH2 (AG-221) inhibits the growth of these ETP cells in vitro. A manuscript describing these findings was published in FY2021 (PMID: 34321240), and a follow-up manuscript was published in FY2023 (PMID: 36330381). Mini-chromosome maintenance component 2 (Mcm2) is a DNA replication licensing factor that is part of the Mcm2-7 complex which functions as a DNA helicase, unwinding genomic DNA at the replication fork. Not surprisingly, homozygous deletion of Mcm2 is lethal. However, insertion of a cre cassette into the 3' UTR of Mcm2 leads to 50% reduction in Mcm2 protein, and cells with two copies of the cre knock-in allele express only 20-30% as much Mcm2 protein compared to wild-type cells. Despite the diminished Mcm2 protein levels, mice with two copies of the Mcm2cre allele are born at normal Mendelian ratios, are not growth-retarded, and are indistinguishable from wild-type littermates at two months of age. Beginning at 2-3 months of age, the mice become ill, and invariably die from pre-T lymphoblastic leukemia/lymphoma (pre-T LBL). Copy number alteration (CNA) analysis reveals a pattern of gains and losses, predominantly losses 10-1000 kb in length. Notably, there is a recurrent constellation of losses, including biallelic deletions of Pten, Tcf3 (E2a) and Dnmt3a, and mono-allelic deletions of the amino-terminus of Notch1. This constellation of cooperative deletions fits a model (supported by published experiments with Pten, Tcf3, Dnmt3a) in which Dnmt3a deletion leads to increased stem cell self-renewal, Tcf3 deletion blocks thymocyte differentiation, Pten deletion leads to hyperproliferation, and deletion of the amino terminus of Notch1 leads to ligand independent growth. All of these genes except TCF3 are frequently mutated in human T-ALL; NOTCH1 being the single gene most commonly mutated in human T-ALL. Although TCF3 is not frequently deleted in human T-ALL, TCF3 is functionally inactivated by inappropriate expression of TAL1/SCL and LMO1/2 proteins (EMBO J 16:2408-19; Nature Immunol 1:138-44) in 25-50% of human T-ALL patients (Cancer Cell 1:75-87), underscoring the relevance of TCF3 inactivation in human T-ALL. Mice that express a NUP98::HOXD13 (NHD13) transgene develop myeloid, T-cell, and B-cell leukemia. Crossing the NHD13 transgene onto an Mcm2cre/cre background led to B-cell precursor (BCP) ALL in a subset of Mcm2cre/creNHD13+ mice. CNA analysis of these BCP-ALL revealed consistent deletions in Pax5, gains of a region bounded by Nup214 and Abl1, and bi-allelic loss of Ptpn1. The gains of Nup214 and Abl1 led to generation of a Nup214-Abl1 fusion gene, similar to that seen in some human T-ALL and BCP-ALL patients. PTPN1 deletions have not been reported in human BCP-ALL, however, deletions of the closely related PTPN2 co-occur with NUP214-ABL1 fusions, and PTPN2 was identified as a negative regulator of the NUP214-ABL1 kinase (Nat Genet 42:530-5, 2010). This constellation of cooperative losses and gains fits a model in which the NHD13 transgene leads to increased stem cell self-renewal, the Pax5 deletion leads to a block in B cell differentiation, the Nup214-Abl1 fusion leads to hyperproliferation, and the Ptpn1 deletion enforces hyperproliferation. Similar to the findings for T-ALL, these genes and pathways have been highlighted as being important for human BCP-ALL (see review by Mullighan and Hunger, Blood 125:3977-87). Overall, this Mcm2 deficiency leads to a unique mutator phenotype, characterized by copy number gains/losses of 50-1000 kb. A manuscript describing these findings was published in FY2020 (PMID: 31622281). In theory, this mutator phenotype could be used to identify constellations of mutations in other forms of cancer if they lived for 3 months. if Mcm2cre/cre mice could be protected from the highly penetrant pre-T LBL, we might uncover additional malignancies triggered by the Mcm2 deficiency. Therefore, we crossed Mcm2cre/cre mice onto a nu/nu background and demonstrated that these mice are indeed protected from development of pre-T LBL, living a median of 8 months as opposed to 3 months. However, the vast majority of Mcm2cre/cre:nu/nu mice develop B cell ALL, beginning at approximately 9 months of age; a manuscript describing these findings was published in FY 2023 (PMID: 35920299). Given the frequent focal homozygous deletions of Ptpn1 in mice that developed BCP-ALL in the context of an NHD13 fusion, we the NHD13 transgene onto a Ptpn1 knockout background. Over half of the NHD13+/Ptpn1-/- mice developed BCP-ALL, demonstrating a strong genetic collaboration between these two mutations. A manuscript describing these findings is currently in preparation. As a final component to this project, we have generated a panel of Mcm2cre/cre T-ALL cell lines that display the CNA mutator phenotype. Although CRISPR screens are an excellent mechanism to detect phenotypes produced by gene loss, they will not detect phenotypes produced by gene copy number gains. Therefore, we have begun a study to identify copy number loss and gains associated with chemotherapy resistance. Initial results are encouraging, as we have detected methotrexate resistance associated with specific copy number gains of the Dhfr gene, and vincristine resistance associated with focal gains of the Abcb1a (previously known as MDR1) gene.

我们跨越了表达IDH2 R140Q突变的转基因小鼠，并用表达NHD13融合的小鼠。后代发展了早期T细胞前体（ETP）白血病的一种形式，该形式在临床表现，免疫表型，基因表达谱和协作突变方面类似于人类疾病。就分子机制而言，IDH2R140Q突变小鼠产生oncometabolite 2Hg；与2HG过量生产一致，白血病细胞显示出正常胸腺细胞发育所需的基因异常。最后，突变体IDH2（AG-221）的特异性抑制剂抑制了这些ETP细胞在体外的生长。描述这些发现的手稿发表在FY2021（PMID：34321240）中，并在2023财年发表了后续手稿（PMID：36330381）。迷你染色体维护成分2（MCM2）是DNA复制许可因子，是MCM2-7复合物的一部分，它充当DNA解旋酶，可在复制叉处放置基因组DNA。毫不奇怪，MCM2的纯合缺失是致命的。然而，将CRE盒插入MCM2的3'UTR中会导致MCM2蛋白的降低50％，并且与野生型细胞相比，Cre敲打等位基因的两份拷贝仅为20-30％。尽管MCM2蛋白水平降低，但MCM2CRE等位基因的两个副本的小鼠出生于正常的Mendelian比率，并未得到增长，并且与两个月大时的野生型同窝仔不可分割。从2-3个月大时开始，小鼠病就病了，总是死于前淋巴细胞白血病/淋巴瘤（per-t LBL）。拷贝数变化（CNA）分析揭示了损益的模式，主要损失为10-1000 kb。值得注意的是，损失的复发星座，包括PTEN，TCF3（E2A）和DNMT3A的双重缺失，以及Notch1氨基末端的单相关缺失。 This constellation of cooperative deletions fits a model (supported by published experiments with Pten, Tcf3, Dnmt3a) in which Dnmt3a deletion leads to increased stem cell self-renewal, Tcf3 deletion blocks thymocyte differentiation, Pten deletion leads to hyperproliferation, and deletion of the amino terminus of Notch1 leads to ligand independent growth.除TCF3以外的所有基因在人类T-All中经常突变。 Notch1是人类T-ALL中最常见的单个基因。尽管TCF3在人类T-All中不经常被删除，但TCF3在功能上通过不当表达TAL1/SCL和LMO1/2蛋白的表达不当，而TCF3（EMBO J 16：2408-19; Nature Immunol 1：138-44）在25-50％的人类T-All患者中（癌细胞1：75-87），scorcoring in Dyscoring in Dyscoring in Descorcoring conscoring in Underscoring conscore，高的。表达NUP98 :: HOXD13（NHD13）Transgene的小鼠会发展髓样，T细胞和B细胞白血病。将NHD13转基因跨越MCM2CRE/CRENHD13+小鼠的子集，将NHD13转基因跨到MCM2CRE/CRE背景。对这些BCP的CNA分析显示，PAX5的缺失，NUP214和ABL1界定的区域的增长以及PTPN1的BI-平行性丢失。 NUP214和ABL1的收益导致了NUP214-ABL1融合基因的产生，类似于在某些人类T-All和BCP所有患者中看到的基因。在人类BCP-ALL中尚未报道PTPN1缺失，但是，与NUP214-ABL1融合的密切相关的PTPN2 co-COCUR的缺失，PTPN2被鉴定为NUP214-ABL1激酶的负调节剂（NAT Genet 42：530-530-5，2010）。合作损失和收益的该星座符合一个模型，其中NHD13转基因会导致干细胞自我更新增加，PAX5缺失导致B细胞分化的块，NUP214-ABL1融合导致高增生和PTPN1删除效果超增强和PTPN1删除效果。与T-All的发现相似，这些基因和途径被强调为对人类BCP的重要性（请参阅Mullighan and Hunger的评论，血液125：3977-87）。总体而言，这种MCM2缺乏导致了独特的突变器表型，其特征是拷贝数增加/损失50-1000 kb。描述这些发现的手稿发表在2020财年（PMID：31622281）。从理论上讲，如果这种突变型表型可以在其他形式的癌症中识别突变体的星座，则可以使用3个月。如果可以保护MCM2CRE/CRE小鼠免受高度渗透率Pre-T LBL的影响，我们可能会发现MCM2缺乏症引发的其他恶性肿瘤。因此，我们将MCM2CRE/CRE小鼠越过了NU/NU背景，并证明这些小鼠确实受到了Pre-T LBL的发展，中位数为8个月而不是3个月。但是，绝大多数MCM2CRE/CRE：NU/NU小鼠都会从大约9个月大的时候出现B细胞。描述这些发现的手稿发表在2023财年（PMID：35920299）中。鉴于在NHD13融合的情况下，PTPN1经常存在PTPN1的纯合差缺失，因此我们将NHD13转基因转移到PTPN1敲除背景上。 NHD13+/ptpn1 - / - 小鼠的一半以上已经开发了BCP，这表明这两个突变之间存在很强的遗传协作。描述这些发现的手稿目前正在准备中。作为该项目的最终组成部分，我们生成了一个显示CNA突变器表型的MCM2CRE/CRE T-ALL细胞系。尽管CRISPR筛选是检测基因丧失产生的表型的出色机制，但它们将无法检测基因拷贝数增长产生的表型。因此，我们已经开始进行一项研究，以鉴定拷贝数损失和与化学疗法耐药性相关的收益。最初的结果令人鼓舞，因为我们发现与DHFR基因的特定拷贝数获得相关的甲氨蝶呤耐药性以及与ABCB1A（以前称为MDR1）基因的局灶性增长相关的长春新碱耐药性。