儿童癌症模型 z-bo 2020-12-11 548

　　Childhood and adolescent cancer:

　　 Childhood cancer is one of the main causes of death in children and adolescents (0-19 years old), mainly including leukemia, nervous system cancer and sarcoma. In order to better understand the etiology of these diseases, recent genome analysis work has made possible the classification of tumor types based on molecular characteristics, and discovered potential gene drivers and coordinated molecular events that lead to the development of different childhood cancers. Foundation. Most childhood malignancies are mutation-silent and are thought to be caused by a single driver gene, fusion oncoprotein, or structure/copy number changes. In contrast, adult tumors often exhibit a high mutation burden, which may be due to long-term mutation under selective pressure. However, despite these differences, childhood cancer treatments are still largely imitating treatments for adults, and when used in children, they can cause debilitating, long-term side effects. In order to design more precise and targeted treatments to improve the prognosis of childhood cancer patients, it is ultimately necessary to develop a powerful preclinical childhood cancer model that can accurately summarize these diseases. Here, we will discuss how zebrafish as a preclinical model for gene and drug discovery can promote the development of childhood cancer.

　　 Application of zebrafish in cancer research:

　　 The zebrafish was established as a developmental biology model because it is particularly suitable for the development of genetics, embryology and imaging, so that it can discover new mechanisms that control embryogenesis, neurogenesis and organ formation. These characteristics also make zebrafish an attractive model for cancer research, because: (i) transparent zebrafish embryos and adults can directly observe the behavior of tumor cells in the body; (ii) the rapid production of zebrafish animals is genetic and Drug screening provides a highly scalable platform; and (iii) the significant conservation of cancer signaling pathways between fish and humans enables people to identify new molecular mechanisms of tumorigenesis. More than 50 genetically engineered zebrafish models of human cancer have been established, which are very similar to human zebrafish at the histological and/or genomic level. The zebrafish cancer model has accelerated the discovery of new mechanisms driving human cancer and identified new drugs for clinical trials. Many recent reviews have detailed experimental methods for generating different models of leukemia, sarcoma, neuroblastoma, and germ cell tumors. We are concerned about how the genetically engineered zebrafish line specifically simulates childhood and adolescent cancers, which we define as zebrafish tumors that appear within the first 90 days of life.

　　 describes the incidence of zebrafish juvenile (inner circle) and juvenile (outer circle) tumors and the corresponding tumor anatomical location. Zebrafish models have been developed for childhood leukemia, brain tumors, sarcomas, germ cell tumors, and neuroblastomas. Representative histology of these zebrafish models is shown on the right. Histological images come from: (T-cell acute lymphoblastic leukemia), (primitive neuroectodermal tumor of the central nervous system), (embryonic rhabdomyosarcoma), (germ cell tumor) and (neuroblastoma).

　　 Leukemia

　　Acute lymphocytic leukemia (ALL) occurs in the T lymphocytes and B lymphocytes in the bone marrow and is the most common cancer in children. At present, the 5-year survival rate of these children is as high as 90%. Typical treatment methods include chemotherapy and allogeneic bone marrow transplantation for high-risk or recurring cases. Although the survival rate of these patients is high, current treatment methods still have many long-term harmful side effects. For these reasons, current efforts are focused on reducing the toxicity of treatments and developing targeted treatments for high-risk patients and patients with recurrent diseases.

　　 T-cell acute lymphoblastic leukemia (T ALL)

　　 T-ALL accounts for 15% of all cases in children. T-ALL is formed by immature thymocytes in the thymus, which have acquired genetic or epigenetic changes and migrate to the bone marrow, peripheral blood and lymph nodes. Genetic changes in T-ALL patients include translocations, gene fusions, chromosome gains and deletions, and epigenetic abnormalities. Both children and adults T-ALL show frequent changes in the NOTCH, PI3K–AKT, JAK–STAT and RAS pathways. The early zebrafish model of T-ALL recapitulates research in mice and humans, and quickly established a new mechanism for tumor progression and survival of the disease.

　　 Myc-driven T-ALL zebrafish model

　　 The first genetically engineered zebrafish cancer model is classified as T-ALL, in which the rag2 promoter is used to drive the expression of the mouse proto-oncogene Myc in T and B lymphocytes. In the Tg(rag2:mMyc) transgenic model, mosaic expression of Myc induced 6% of animal T-ALL with an average incubation period of 44 days. Myc-driven T-ALL conditional germline transgenic model has been established, using one of three induction methods: (i) injection of Cre mRNA into Tg (rag2-lox-dsRED-lox-EGFP-mMyc) embryos (ii) Tg (Rag2:LDL-EGFP-mMyc) and Tg(hsp70:Cre) fish were crossed; (iii) Tg(rag2:hMYC-ER) fish were treated with 4-hydroxytamoxifen. The increased reproducibility and tumor penetrance in these fine models enable the identification of new genes and/or gene pathways, promote or inhibit the initiation, progression and survival of Myc-driven T-ALL, and discover drugs that inhibit tumorigenesis in vivo .

　　Notch drive T-ALL zebrafish model "In more than 60% of T-ALL cases, the NOTCH signaling pathway is abnormally activated, and it can promote T-ALL tumorigenesis through MYC-dependent and independent pathways. In addition, in the pathogenesis of zebrafish T-ALL, NOTCH1 and Myc act synergistically, because the expression of the constitutively active intracellular domains (NICD) of Myc and NOTCH1 mediated by rag2 enhances T compared to the expression of Myc or NICD alone. -ALL process. The enhanced Notch signal cannot increase the number of leukemia proliferating cells, indicating that Notch controls the proliferation of precancerous cells, while Myc mainly drives the growth and survival of cloned cells. In about 16% of recurrent childhood T-ALL cases, mutations in the Notch pathway are also associated with the activation of the Hedgehog pathway. Ptch1 is a negative regulator of the Hedgehog pathway, and its loss accelerates the occurrence of T-ALL induced by zebrafish notch1, which indicates that the activation of the Hedgehog signaling pathway works in concert with notch1 to drive T cell transformation. These zebrafish models highlight the therapeutic potential of targeting multiple developmental pathways during T-ALL disease progression.

　　 B cell zebrafish model of acute lymphoblastic leukemia (B-ALL)

　　 B-ALL accounts for 85% of all childhood ALL cases. Although B-ALL and T-ALL are indistinguishable in morphology, each disease is characterized by different molecular subtypes. Similar genetic and chromosomal abnormalities exist in both adult and childhood B-ALL, including fusion genes such as BCR-ABL1 and ETV6-RUNX, MLL rearrangements, hyperploidy, and diploidy, albeit at different frequencies. In addition, it was recently discovered that some animals from the Tg (rag2: hMYC) line also suffer from B-ALL tumors, which have different gene expression characteristics from T-ALL tumors. It shows that the transgenic line is actually a mixed model. These studies emphasize the need to incorporate modern comparative carcinogenic methods to verify the exact type and/or subtype of human tumors simulated by the zebrafish line.

　　Zebrafish model of acute myeloid leukemia (AML)

　　Acute myeloid leukemia is defined as the accumulation of immature myeloid cells, which account for at least 20% of the patient’s bone marrow. The 5-year overall survival rate of AML is 64%, but depending on the molecular subtype, the survival rate varies significantly. Unlike other liquid malignancies, some somatic mutations in children with AML are significantly different from those in adult AML patients. The unique feature of childhood acute myeloid leukemia is gene fusion events such as AML1-ETO (also known as RUNX1-RUNX1T1), NUP98-NSD1 and KMT2A-MLLT3. The expression of AML1-ETO driven by the hsp70 promoter and the expression of FLT3-ITD or FLT3-TKD driven by the CMV promoter resulted in the expansion of myeloid cells in early zebrafish embryos. The COX2 inhibitor nimesulide blocking aβ-catenin-mediated increase in myelopoiesis can reverse the hematopoietic defect in AML1-ETO embryos. In addition, treatment with a tyrosine kinase inhibitor (AC220) can make the myeloid expansion in FLT3-ITD (but not FLT3-TKD) disappear, which indicates that there are different therapeutic opportunities to treat seemingly similar genetic abnormalities. These studies show that zebrafish embryos are useful for understanding the basic mechanism of oncogenic fusion genes in hematopoiesis and discovering new methods for the treatment of childhood leukemia.

　　 Brain and central nervous system tumors

　　 Brain tumors and central nervous system tumors are the main causes of childhood cancer-related deaths. Approximately 75% of childhood brain tumors are malignant, and the 5-year overall survival rate is 78%. In addition, surgical removal of tumors, radiotherapy, and systemic chemotherapy can cause long-term complications in the developing brain. Fortunately, significant progress has been made in the genomic characteristics of childhood brain tumors. Specific brain tumor entities with different molecular characteristics have been identified, so that tumors with recurrent genomic characteristics can be classified more accurately. The challenge now is to establish new cell and animal models of brain tumor entities for the discovery of genes and drugs. Zebrafish seems to be a good animal model for detecting gene drivers of OPC-derived childhood tumors. For example, the deletion of nf1a/b or rb1 tumor suppressor genes in zebrafish, usually accompanied by p53 deficiency, will produce brain tumors similar to oligodendrogliomas or embryonic tumors of the central nervous system PNET. Comparative genomic analysis between the Tg (sox10: NRAS) model and the rb1 defect model showed that there was a significant overlap in gene expression changes.

　　Neuroblastoma zebrafish model

　　 The expansion of MYCN is observed in about 20% of neuroblastoma (NB), which is related to a poor prognosis. The dopamine-β-hydroxylase (dβh) promoter was used to direct human MYCN gene expression to the developing peripheral sympathetic nervous system to establish a zebrafish NB model. Low (17%) and high (70%) penetrance NB zebrafish models have been developed to detect synergistic mutations that increase or decrease tumor burden, respectively. The low penetrance NB model Tg (dβh:EGFP-MYCN) expresses the EGFP-MYCN fusion protein, while the high penetrance Tg (dβh:EGFP; dβh:MYCN) model involves the co-integration of EGFP and MYCN genes instead of fusion. In these two models, zebrafish NB tumors are histologically similar to human NB, mainly appearing in the interadrenal gland, which is equivalent to the human adrenal medulla. The low penetrance Tg (dβh:EGFP-MYCN) NB model helps to identify a variety of genes that cooperate with MYCN to promote NB tumorigenesis.

　　Sarcoma

　　 Soft tissue and osteosarcoma account for 7% of childhood cancers. The most common sarcoma in children and adolescents is rhabdomyosarcoma (RMS). It is formed by undifferentiated muscle cells and usually appears in the head/neck, urogenital tract or extremities. There are two main histological subtypes of rhabdomyosarcoma: embryonic type (ERMS) and alveolar type (ARMS). ERMS accounts for 75% of all RMS cases, and its typical feature is loss of heterozygosity at MYOD1, FGFR4, and any 11p15.5 locus or point mutation that encodes the major RAS gtpase gene. In contrast, 80% of ARMS patients showed overexpression of the PAX3–FOXO1 or PAX7–FOXO1 fusion gene. RMS treatment is limited to local resection, radiotherapy and multi-drug chemotherapy, and has remained basically unchanged in the past 50 years. Despite advances in next-generation sequencing and patient molecular typing, the 5-year survival rate of ERMS is still 82%. ARMS The 5-year survival rate is 65%. The zebrafish RMS model helps us understand the causes of these childhood cancers.

　　 fusion-positive sarcoma zebrafish model

　　 Under the background of lack of p53 gene, the CMV promoter was used to express the human PAX3-FOXO1 fusion gene extensively, and a zebrafish ARMS model was established. Therefore, compared with the ERMS model, the origin cell in ARMS is unknown. Nevertheless, these studies have shown that the PAX3-FOXO1 human fusion oncogene is sufficient to transform zebrafish cells into ARMS tumors or CNS-PNETs with unique histological characteristics. PAX3–FOXO1 causes abnormal expression of the transcription factor hes3 in muscle cells, thereby blocking the expression of muscle differentiation markers. For a long time, the expression of human HES3 in zebrafish and muscle progenitor cells inhibits myogenesis, and increased expression of HES3 in RMS patients indicates a poor prognosis. Therefore, this zebrafish model provides important clues for studying the conservative genetic mechanism of PAX3-FOXO1 fusion gene-mediated tumorigenesis. In fact, there is an important progress in the study of fusion-positive sarcomas, that is, the use of zebrafish lines can quickly detect the transformation potential of fusion oncoproteins without knowing the origin of tumor cells in advance.

　　 Conclusion: Compared with adult cancer, childhood cancer is a rare disease. Therefore, many childhood tumor entities do not have relevant cell lines or animal model systems to define disease pathogenesis or test potential treatments. In our opinion, the most important benefit of the zebrafish system for childhood cancer research is the timely and affordable development of rare tumor subtypes for pre-clinical drug discovery, such as leukemia, brain tumors, and brain tumors. NB and sarcoma. In addition, the unique imaging characteristics of zebrafish provide a powerful tool for directly monitoring the behavior of tumor cells during invasion and metastasis and their response to drug treatment. These characteristics are also important for studying adult cancer.

　　 The childhood cancer models of zebrafish and mice are complementary in identifying new mechanisms of childhood tumors. Both of these model systems have conservative developmental and carcinogenic pathways, can be used for patient-derived xenografts (PDXs), and have available genome editing technologies. Zebrafish has unique advantages in scalability, cost and imaging. In contrast, mice have conservative physiological characteristics (body temperature and organ systems) and more precise drug delivery, administration and metabolism.