Animal models to study the role of INK4b loss in human acute myeloid leukemia

研究 INK4b 缺失在人类急性髓系白血病中的作用的动物模型

• 批准号: 8763311
• 负责人: Linda Wolff
• 金额: $ 48.47万
• 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/8763311
• 关键词: Acute Myelocytic Leukemia; Acute leukemia; Affect; Allogenic; Animal Model; Animals; Antigens; Apoptotic; BFU-E; Blast Cell; Blood Platelets; Bone Marrow; Bone Marrow Stem Cell; CD80 gene; CD8B1 gene; CDKN2A gene; Cell Cycle; Cell Lineage; Cells; Characteristics; Chronic Disease; Chronic Myelomonocytic Leukemia; Commit; Cyclin-Dependent Kinase Inhibitor; Data; Defect; Development; Disease; Dysmyelopoietic Syndromes; Erythrocytes; Erythroid; Erythropoiesis; Exons; Family member; Fluorouracil; Frequencies; Gene Deletion; Gene Mutation; Gene Silencing; Genes; Goals; Hematopoiesis; Hematopoietic; Human; Hypermethylation; Immune response; Immunotherapy; Impairment; In Vitro; Investigation; Knock-out; Knockout Mice; Laboratories; Lead; Leukocytes; MEKs; MYB gene; Malignant - descriptor; Megakaryocytes; Modeling; Molecular; Monocytosis; Mus; Mutation; Myelogenous; Myeloid Leukemia; Myeloid Progenitor Cells; Myeloproliferative disease; Patients; Phenylhydrazines; Phosphorylation; Play; Prevalence; Process; Production; Property; Protein Kinase; Proteins; Proto-Oncogene Proteins c-myb; Reporting; Retroviridae; Role; Signal Transduction; Site; Skin Neoplasms; Stem cells; Stream; Stress; Syndrome; System; T cell response; Technology; Tissues; Transactivation; Transcript; Tumor Suppressor Proteins; Wild Type Mouse; base; erythroid differentiation; granulocyte; human GATA1 protein; improved; interest; leukemia; leukemogenesis; macrophage; man; member; novel; peripheral blood; phenylhydrazine; progenitor; proto-oncogene protein Spi-1; research study; response; restoration; sarcoma; self-renewal; soft tissue; stem cell differentiation; tool; tumor; uptake; Acute Myelocytic Leukemia; Acute leukemia; Affect; Allogenic; Animal Model; Animals; Antigens; Apoptotic; BFU-E; Blast Cell; Blood Platelets; Bone Marrow; Bone Marrow Stem Cell; CD80 gene; CD8B1 gene; CDKN2A gene; Cell Cycle; Cell Lineage; Cells; Characteristics; Chronic Disease; Chronic Myelomonocytic Leukemia; Commit; Cyclin-Dependent Kinase Inhibitor; Data; Defect; Development; Disease; Dysmyelopoietic Syndromes; Erythrocytes; Erythroid; Erythropoiesis; Exons; Family member; Fluorouracil; Frequencies; Gene Deletion; Gene Mutation; Gene Silencing; Genes; Goals; Hematopoiesis; Hematopoietic; Human; Hypermethylation; Immune response; Immunotherapy; Impairment; In Vitro; Investigation; Knock-out; Knockout Mice; Laboratories; Lead; Leukocytes; MEKs; MYB gene; Malignant - descriptor; Megakaryocytes; Modeling; Molecular; Monocytosis; Mus; Mutation; Myelogenous; Myeloid Leukemia; Myeloid Progenitor Cells; Myeloproliferative disease; Patients; Phenylhydrazines; Phosphorylation; Play; Prevalence; Process; Production; Property; Protein Kinase; Proteins; Proto-Oncogene Proteins c-myb; Reporting; Retroviridae; Role; Signal Transduction; Site; Skin Neoplasms; Stem cells; Stream; Stress; Syndrome; System; T cell response; Technology; Tissues; Transactivation; Transcript; Tumor Suppressor Proteins; Wild Type Mouse; base; erythroid differentiation; granulocyte; human GATA1 protein; improved; interest; leukemia; leukemogenesis; macrophage; man; member; novel; peripheral blood; phenylhydrazine; progenitor; proto-oncogene protein Spi-1; research study; response; restoration; sarcoma; self-renewal; soft tissue; stem cell differentiation; tool; tumor; uptake

Using p15Ink4b embryonal knock-out mice, our initial studies of p15Ink4b provided preliminary evidence that the gene is a tumor suppressor for myeloid leukemia. Subsequently, we developed an improved model that more closely resembles the disease in man by deleting the gene specifically in myeloid lineage cells. The new model employing the Cre-loxP system allows conditional deletion of the gene. We successfully accomplished the myeloid tissue specific deletion by crossing INK4b(exon 2) floxed mice with LysMCre mice and showed by PCR technology that Ink4b was deleted in common myeloid progenitors. The p15Ink4bfl/fl-LysMcre mice develop progressive monocytosis in the peripheral blood accompanied by increased numbers of myeloid and monocytic cells in the bone marrow. Morphological characteristics of p15Ink4bfl/fl-LysMcre mice most closely resemble a mild myeloproliferative form of chronic myelomonocytic leukemia (CMML). In accord with our findings, hypermethylation of p15INK4b was reported previously in approximately 60% of CMML cases. In the p15Ink4bfl/fl-LysMcre mice spontaneous progression from chronic disease to acute leukemia was not observed. Nevertheless, MOL4070LTR retrovirus integrations provided cooperative genetic mutations resulting in a high frequency of myeloid leukemia in knockout mice. Two common retrovirus insertion sites near c-myb and Sox4 genes were identified and their transcript upregulated in leukemia, suggesting a collaborative role of their protein products with p15Ink4b-deficiency in promoting malignant disease. This animal model demonstrates that p15Ink4b plays an active role in the establishment of preleukemic conditions. In addition, this is the first demonstration that p15Ink4b can act as a tumor suppressor in the presence of a fully functional p16Ink4a locus. Previously, tumor suppressor activity of p15Ink4b was demonstrated by others only in an animal model where both p16INK4a and p15INK4b genes were simultaneously inactivated and the animals developed a wide spectrum of tumors with prevalence of skin tumors and soft tissue sarcomas, but not of myeloid origin. p15INK4b is a member of the INK4 family member of cyclin-dependent kinase inhibitors. However, since INK4b is the only member that is inactivated in acute myeloid leukemia, we have been interested in determining if there is any other function specific to the myeloid lineage that can be assigned to this gene. For these experiments we used the two knock-out models described above. Initial investigations of hematopoiesis in Ink4b- deficient mice showed that they have greater numbers of bi-potent granulocyte-macrophage progenitors (GMP) and this was found to be intrinsic to the cells. Interestingly, Ink4b-deficient granulocyte-macrophage progenitors did not cycle more frequently than wild-type progenitors and showed no differences in apoptotic potential or self-renewal potential. However, Ink4b was shown to affect differentiation of common myeloid progenitor (CMP) cells, resulting in an imbalance of down-stream progenitors. In vitro analysis of progenitor cells from knock-out mice demonstrated that loss of Ink4b causes an increase in GMP and decrease in MEP (megakaryocyte-erythroid progenitor) potential. Based upon the data obtained from the knockout mice, we hypothesized that p15Ink4b is required for efficient erythropoiesis. We have now shown that p15Ink4b promotes erythroid differentiation and suppresses myeloid differentiation of hematopoietic progenitors under normal and stress conditions. It was found that p15Ink4b is expressed more highly in committed megakaryocyte-erythroid progenitors (MEP) than granulocyte-macrophage progenitors (GMP). More importantly, mice lacking p15Ink4b have lower numbers of primitive red cell progenitors and a severely impaired response to 5-fluorouracil and phenylhydrazine induced hematopoietic stress. Introduction of p15Ink4b in multi-potential progenitor cells produced changes at the molecular level, including activation of MEK/ERK signaling, increase GATA-1, EpoR, decrease Pu1 and GATA-2 expression. These changes rendered cells more permissive to erythroid commitment and less permissive to myeloid commitment, as demonstrated by an increase in early BFU-E formation with a concomitant decrease in myeloid progenitors. Our results indicate that p15Ink4b functions in hematopoiesis, by maintaining proper lineage commitment of progenitors and assisting in rapid red blood cell replenishment following stress. Recently, we found that expression of p15Ink4b is strongly induced in a biphasic manner during development and activation of mouse bone marrow-derived DCs (BM-DCs), suggesting an important role for p15Ink4b in DC maturation. Interestingly, myeloid-specific deletion of p15Ink4b in mice resulted in significantly fewer and less mature CD8- conventional DCs (cDCs) as compared to wild type mice. Consistent with this data, ex-vivo generated bone marrow-derived DCs (BM-DCs) from the knockout mice have markedly lower levels of expression of the antigen presenting (MHCII) and the co-stimulatory molecules (CD80, CD86) than wild-type controls. Accordingly, these cells show a reduced ability to uptake antigen and to stimulate allogeneic T-cell responses. The re-expression of p15Ink4b in progenitors results in a restoration of MHCII expression, as well as co-stimulatory molecules, confirming a positive role for p15Ink4b in cDCs development. Furthermore, p15Ink4b expression increases phosphorylation of Erk1/Erk2 protein kinases that leads to an increased transactivation activity of the PU.1 transcription factor, an important regulator of DC development. Taken together, our results indicate a novel role for p15Ink4b in mDC development, and suggest that frequent inactivation of the gene in myeloid malignancies could lead to an inefficient anti-leukemic immune response during leukemogenesis. Our data also have an important translational significance. AML blasts isolated from patients, and differentiated ex-vivo into DCs, represent a powerful immunotherapy tool. However, AML-DCs have reportedly a partially impaired maturation process as compared to DCs from healthy donors. We propose that re-expression of p15INK4b in AML-DCs may overcome some of the limitations of a DC-based immunotherapy for AML patients.

使用P15INK4B胚胎敲除小鼠，我们对P15INK4B的最初研究提供了初步证据，表明该基因是髓样白血病的肿瘤抑制剂。随后，我们开发了一个改进的模型，该模型通过在髓样谱系细胞中删除该基因，与人类中的疾病更相似。采用CRE-LoxP系统的新模型允许有条件地缺失基因。我们通过将Ink4b（外显子2）用lysmcre小鼠跨越Ink4b（外显子2），成功地完成了髓样组织特异性缺失，并通过PCR技术表明Ink4b在普通髓样祖细胞中被删除。 P15INK4BFL/FL-LYSMCRE小鼠在外周血中出现进行性单核细胞增多，并伴随着骨髓中髓样细胞和单核细胞的数量增加。 p15INK4BFL/FL-Lysmcre小鼠的形态特征最类似于慢性脊髓细胞性白血病（CMML）的轻度骨髓增生性形式。根据我们的发现，以前在大约60％的CMML病例中报道了P15INK4B的高甲基化。在P15INK4BFL/FL散液液中，没有观察到从慢性疾病到急性白血病的自发进展。然而，MOL4070LTR逆转录病毒的整合提供了合作基因突变，导致敲除小鼠中髓样白血病的高频率。鉴定了C-MYB和SOX4基因附近的两个常见逆转录病毒插入位点，并在白血病中上调了转录物，这表明其蛋白质产物与p15INK4B缺陷在促进恶性疾病中的协作作用。该动物模型表明，p15INK4B在建立peleukemic条件中起着积极作用。此外，这是第一个证明P15INK4B可以在功能齐全的P16INK4A基因座的情况下充当肿瘤抑制剂。以前，其他人仅在动物模型中证明了p15ink4b的肿瘤抑制活性，在该模型中，P16INK4A和P15INK4B基因同时被灭活，并且动物在肿瘤的广泛范围中，具有皮肤肿瘤的流行和软组织肉瘤，而不是髓细胞。 P15INK4B是Cyclin依赖性激酶抑制剂的Ink4家族成员的成员。但是，由于INK4B是唯一在急性髓细胞性白血病中灭活的成员，因此我们感兴趣地确定是否存在可以分配给该基因的其他功能。对于这些实验，我们使用了上面描述的两个敲除模型。对Ink4b缺乏小鼠中造血的初步研究表明，它们具有更多的双功率粒细胞 - 巨噬细胞祖细胞（GMP），并且发现这对细胞是固有的。有趣的是，Ink4b缺陷型粒细胞 - 巨噬细胞祖细胞的循环频率不如野生型祖细胞更频繁，并且在凋亡潜力或自我更新潜力上没有差异。但是，INK4B被证明会影响常见髓样祖细胞（CMP）细胞的分化，从而导致下游祖细胞的不平衡。对淘汰小鼠的祖细胞的体外分析表明，Ink4b的损失会导致GMP增加，MEP（巨核细胞 - 毛细血管祖细胞）的潜力降低。根据从敲除小鼠获得的数据，我们假设p15ink4b是有效的红细胞生成所必需的。现在，我们已经表明，P15INK4B在正常和应激条件下促进红细胞分化并抑制造血祖细胞的髓样分化。发现与粒细胞 - 巨噬细胞祖细胞（GMP）相比，P15INK4B在承诺的巨核细胞 - 颈骨祖细胞（MEP）中表达得更高。更重要的是，缺乏P15INK4B的小鼠的原始红细胞祖细胞数量较低，对5-氟尿嘧啶和苯基羟酰亚拉津诱导的造血应激的反应严重受损。在多电位祖细胞中引入P15INK4B在分子水平上产生变化，包括激活MEK/ERK信号传导，增加GATA-1，EPOR，降低PU1和GATA-2的表达。这些变化使细胞更具允许的红斑承诺，而对髓样承诺的允许更少，这表明，早期BFU-E形成的增加表明，髓样祖细胞的同时减少。我们的结果表明，P15INK4B在造血中的作用，通过保持祖细胞的适当谱系承诺并在压力后有助于快速红细胞补充。最近，我们发现在小鼠骨髓衍生的DC（BM-DC）的开发和激活过程中，P15INK4B的表达强烈诱导，这表明P15INK4B在DC成熟中起重要作用。有趣的是，与野生型小鼠相比，小鼠p15INK4B的髓样特异性缺失导致较少和更不成熟的常规DC（CDC）。与这些数据一致，从敲除小鼠产生的骨髓衍生的DC（BM-DC）比野生型对照组明显低的抗原表达水平（MHCII）和共刺激分子（CD80，CD86）低。因此，这些细胞表现出降低的吸收抗原和刺激同种异体T细胞反应的能力。 P15INK4B在祖细胞中的重新表达导致MHCII表达的恢复以及共刺激分子，证实了P15INK4B在CDCS开发中的积极作用。此外，P15INK4B表达增加了ERK1/ERK2蛋白激酶的磷酸化，从而导致PU.1转录因子的反式激活活性增加，这是DC发育的重要调节剂。综上所述，我们的结果表明，P15INK4B在MDC发育中起了新的作用，并表明髓样恶性肿瘤中基因的频繁失活可能导致白血病发生期间的抗白血病免疫反应效率低下。我们的数据也具有重要的翻译意义。从患者中分离出来的AML爆炸，并将Ex-Vivo分化为DC，代表了强大的免疫疗法工具。但是，据报道，与健康捐助者的DC相比，AML-DC的成熟过程部分受损。我们建议在AML-DC中重新表达P15INK4B可能会克服针对AML患者的基于DC的免疫疗法的某些局限性。