Collaborative Pathways that Lead to Leukemia

导致白血病的协同途径

• 批准号: 10702444
• 负责人: Peter Aplan
• 金额: $ 59.92万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 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/10702444
• 关键词: 3' Untranslated Regions; ABL1 gene; Acute Myelocytic Leukemia; Acute leukemia; Age-Months; Alleles; Animal Model; Apoptosis; B cell differentiation; B lymphoid malignancy; B-Cell Acute Lymphoblastic Leukemia; B-Cell Leukemia; B-Lymphocytes; Blood; Bone Marrow; Bone Marrow Cells; CD8B1 gene; Candidate Disease Gene; Cells; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Complex; DNA; DNA biosynthesis; DNA replication fork; DNMT3a; Development; Epigenetic Process; Erythroid; 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; Lead; Length; Leukemic Cell; Licensing Factor; Ligands; Loss of Heterozygosity; Lymphoblastic Leukemia; Lymphoma; Maintenance; Malignant - descriptor; Malignant Neoplasms; Manuscripts; Methylation; Modeling; Molecular; Mouse Strains; Mus; Mutant Strains Mice; Mutate; Mutation; Myelogenous; Myeloid Cells; NOTCH1 gene; NU/NU Mouse; NUP214 gene; NUP98 gene; Nature; PTPN1 gene; Pathway interactions; Patients; Pattern; Phenotype; Phosphotransferases; Proteins; Publishing; Recurrence; Reporting; Sampling; Site; T-Cell Leukemia; T-Lymphocyte; TAL1 gene; TCF3 gene; TOP1 gene; Thymocyte Development; Thymus Gland; Transgenes; Transgenic Mice; Transplantation; Tumor Suppressor Genes; V(D)J Recombination; acute T-cell lymphoblastic leukemia cell; acute myeloid leukemia cell; cancer cell; cell type; exome sequencing; experimental study; fusion gene; helicase; human disease; in vivo; inhibitor; leukemia; leukemia initiating cell; leukemia/lymphoma; leukemic transformation; mutant; novel strategies; offspring; precursor cell; progenitor; replication stress; retroviral transduction; small hairpin RNA; stem cell self renewal; theories; thymocyte; tumor; whole genome

Using NP23 mice,we identified frequent acquired mutations in progenitor B1 cell ALL in the Bcor and Jak1/2 genes. Subsequently, we used CRISPR to introduce Bcor mutations in primary WT and NP23 BM cells; these cells were transplanted into recipient mice and verified collaboration between NP23 and Bcor in vivo. The leukemias also acquired Jak pathway mutations, suggesting that NP23, Bcor, and Jak collaborated to produce pro-B1 ALL. These results were published in FY2020 (PMID: 30992267). We previously demonstrated that spontaneous mutations of IDH2 occur in NHD13 leukemias. These mutations occur at R140Q; homologous residues are mutated in human leukemia. We crossed IDH2 R140Q transgenic mice with NHD13 mice; 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). Both NP23 and NHD13 mice develop a wide variety of leukemia at 9-14 months of age. Surprisingly, 100% of the NP23-NHD13 double transgenic mice developed acute myeloid leukemia (AML) within 3 months. The leukemias were characterized by extraordinarily high WBC and replacement of the thymus with AML cells; the percent of malignant myeloid cells in the thymus was often higher than the bone marrow (BM). These findings led to the intriguing hypothesis that the AML in NP23-NHD13 mice arose in the thymus, as opposed to the BM. To investigate this possibility, we transplanted CD4-/CD8- double negative (DN) thymocytes from the thymus of a NP23-NHD13 mouse. All mice developed AML within 26 days, indicating that the AML was aggressive and transplantable, and could be transmitted by DN thymocytes. Fractionating DN thymocytes into DN1-DN4 sub-populations revealed that AML initiating cells were found in the DN1 and DN2 compartments. Taken together, these results demonstrate that NP23-NHD13 thymic progenitors retain myeloid and erythroid potential and are potently leukemogenic, leading to the intriguing hypothesis that some human AML might originate in the thymus. A manuscript describing these findings was published in FY2020 (PMID: 31748606). Ongoing experiments are focused on assessing the leukemic potential of combining Bahcc1 mice (see project 1) with NHD13 transgenic mice. 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 Mendellian 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). Sequencing of 323 independent junctions from 91 tumors revealed that mononucleotide repeats were enriched near the breakpoint junctions, consistent with recent observations that increased replicative stress is associated with DNA DSB at sites of mononucleotide repeats (Cell 174:1127-42, 2018). 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. We are now crossing the Mcm2cre/cre mice onto scid/scid and RAG2 KO backgrounds to determine if mice that lack VDJ recombination will be "protected" from both T and B cell malignancies.

使用 NP23 小鼠，我们发现了 B1 祖细胞 ALL 中 Bcor 和 Jak1/2 基因的频繁获得性突变。随后，我们使用CRISPR在原代WT和NP23 BM细胞中引入Bcor突变；这些细胞被移植到受体小鼠体内，并在体内验证了 NP23 和 Bcor 之间的协作。白血病还获得了 Jak 通路突变，表明 NP23、Bcor 和 Jak 共同产生了 pro-B1 ALL。这些结果已于 2020 财年发布（PMID：30992267）。我们之前证明 IDH2 自发突变发生在 NHD13 白血病中。这些突变发生在 R140Q；同源残基在人类白血病中发生突变。我们将 IDH2 R140Q 转基因小鼠与 NHD13 小鼠杂交； 后代会发展出一种早期 T 细胞前体 (ETP) 白血病，在临床表现、免疫表型、基因表达谱和协同突变方面与人类疾病相似。从分子机制来看，IDH2R140Q突变小鼠产生致癌代谢物2HG；与 2HG 的过量产生一致，白血病细胞表现出正常胸腺细胞发育所需基因的异常甲基化。最后，突变 IDH2 (AG-221) 的特异性抑制剂在体外抑制这些 ETP 细胞的生长。描述这些发现的手稿已于 2021 财年发表（PMID：34321240）。 NP23 和 NHD13 小鼠在 9-14 个月龄时都会患上多种白血病。令人惊讶的是，100%的NP23-NHD13双转基因小鼠在3个月内患上急性髓系白血病(AML)。白血病的特点是白细胞异常高，并且胸腺被 AML 细胞取代。胸腺中恶性骨髓细胞的百分比通常高于骨髓（BM）。这些发现引发了一个有趣的假设，即 NP23-NHD13 小鼠的 AML 出现在胸腺中，而不是骨髓中。为了研究这种可能性，我们从 NP23-NHD13 小鼠的胸腺移植了 CD4-/CD8- 双阴性 (DN) 胸腺细胞。所有小鼠均在 26 天内出现 AML，表明 AML 具有侵袭性和可移植性，并且可以通过 DN 胸腺细胞传播。将 DN 胸腺细胞分为 DN1-DN4 亚群，结果显示在 DN1 和 DN2 区室中发现了 AML 起始细胞。 总而言之，这些结果表明 NP23-NHD13 胸腺祖细胞保留了骨髓和红细胞潜能，并且具有潜在的致白血病作用，从而得出了一些有趣的假设，即某些人类 AML 可能起源于胸腺。描述这些发现的手稿已于 2020 财年发表（PMID：31748606）。正在进行的实验重点是评估将 Bahcc1 小鼠（参见项目 1）与 NHD13 转基因小鼠相结合的白血病潜力。 微型染色体维持成分 2 (Mcm2) 是一种 DNA 复制许可因子，是 Mcm2-7 复合体的一部分，其功能相当于 DNA 解旋酶，在复制叉处解开基因组 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会导致Mcm2蛋白减少50%，并且与野生型相比，具有两个cre敲入等位基因拷贝的细胞仅表达20-30%的Mcm2蛋白细胞。尽管 Mcm2 蛋白水平降低，但具有两个 Mcm2cre 等位基因拷贝的小鼠以正常孟德尔比率出生，不会生长迟缓，并且在两个月大时与野生型同窝小鼠没有区别。从 2-3 个月大开始，小鼠就会生病，并且总是死于前 T 淋巴细胞白血病/淋巴瘤（前 T LBL）。拷贝数改变 (CNA) 分析揭示了增益和丢失的模式，主要是长度为 10-1000 kb 的丢失。值得注意的是，存在一系列反复出现的损失，包括 Pten、Tcf3 (E2a) 和 Dnmt3a 的双等位基因缺失，以及 Notch1 氨基末端的单等位基因缺失。这种协同缺失的组合符合一个模型（已发表的 Pten、Tcf3、Dnmt3a 实验支持），其中 Dnmt3a 缺失导致干细胞自我更新增加，Tcf3 缺失阻碍胸腺细胞分化，Pten 缺失导致过度增殖，而氨基缺失Notch1 的末端导致配体独立生长。除 TCF3 外，所有这些基因在人类 T-ALL 中都经常发生突变； NOTCH1 是人类 T-ALL 中最常见突变的单一基因。尽管 TCF3 在人类 T-ALL 中并不经常缺失，但 25-50% 的 TCF3 会因 TAL1/SCL 和 LMO1/2 蛋白的不当表达而功能失活（EMBO J 16:2408-19；Nature Immunol 1:138-44）人类 T-ALL 患者的研究（Cancer Cell 1:75-87），强调了 TCF3 失活与人类 T-ALL 的相关性。表达 NUP98-HOXD13 (NHD13) 转基因的小鼠会患上骨髓性白血病、T 细胞白血病和 B 细胞白血病。将 NHD13 转基因杂交到 Mcm2cre/cre 背景上会导致 Mcm2cre/creNHD13+ 小鼠子集中出现 B 细胞前体 (BCP) ALL。对这些 BCP-ALL 的 CNA 分析揭示了 Pax5 中的一致缺失、Nup214 和 Abl1 边界区域的增加以及 Ptpn1 的双等位基因丢失。 Nup214 和 Abl1 的增益导致 Nup214-Abl1 融合基因的产生，类似于在一些人类 T-ALL 和 BCP-ALL 患者中看到的情况。 人类 BCP-ALL 中尚未报道 PTPN1 缺失，然而，密切相关的 PTPN2 缺失与 NUP214-ABL1 融合同时发生，并且 PTPN2 被确定为 NUP214-ABL1 激酶的负调节因子 (Nat Genet 42:530- 5，2010）。这种合作损失和收益的组合符合这样一个模型：NHD13 转基因导致干细胞自我更新增加，Pax5 缺失导致 B 细胞分化受阻，Nup214-Abl1 融合导致过度增殖，Ptpn1 缺失强制过度增殖。与 T-ALL 的研究结果类似，这些基因和途径已被强调对人类 BCP-ALL 很重要（参见 Mullighan 和 Hunger 的评论，Blood 125：3977-87）。对 91 个肿瘤的 323 个独立连接点进行测序表明，单核苷酸重复序列在断点连接点附近富集，这与最近的观察结果一致，即复制应激增加与单核苷酸重复位点处的 DNA DSB 相关（Cell 174:1127-42, 2018）。总体而言，这种 Mcm2 缺陷会导致独特的突变表型，其特征是拷贝数增加/丢失 50-1000 kb。描述这些发现的手稿已于 2020 财年发表（PMID：31622281）。理论上，如果其他形式的癌症存活了 3 个月，这种突变表型可用于识别它们的突变群。如果可以保护 Mcm2cre/cre 小鼠免受高渗透性 pre-T LBL 的侵害，我们可能会发现由 Mcm2 缺陷引发的其他恶性肿瘤。因此，我们将 Mcm2cre/cre 小鼠与 nu/nu 背景杂交，并证明这些小鼠确实免受前 T LBL 的发展，中位寿命为 8 个月，而不是 3 个月。然而，绝大多数 Mcm2cre/cre:nu/nu 小鼠在大约 9 个月大时开始出现 B 细胞 ALL。我们现在正在将 Mcm2cre/cre 小鼠与 scid/scid 和 RAG2 KO 背景杂交，以确定缺乏 VDJ 重组的小鼠是否会受到 T 和 B 细胞恶性肿瘤的“保护”。