Establishment Of Pu.1 And C-myb Zebrafish Leukemia Models For Drug Discovery By High-throughput Screens(HTS)

Background:Myeloid cell play an important role in many biological processes including tissue healing,immune defense and repair of the damaged.Its full function is dependent on the presence of a large number in the blood circulation and specific organ/tissue mature myeloid cells,such as the central nervous system,spleen,lung,liver and skin.Myeloid cells are divided into two categories in vertebrates,namely granulocytes(eosinophils,basophils,neutrophils,mast cells)and monocytes/macrophages(blood macrophages,tissue resident macrophages,dendritic cells).These two types of cells have their own characteristics in morphology and biological functions.Granulocytes and monocytes/macrophages first originated in primitive hematopoietic stage.At this stage only a limited number of red blood cells and myeloid cells were produced.After this phase,mature myeloid cells can be formed by differentiation of hematopoietic stem cells through "myeloid forming" process during the definitive hematopoietic.”Myeloid formed" process refers the process of mesodermal cells specialized to progenitor cells or hematopoietic stem cells,and the process of macrophages and neutrophils cell fate decisions.This is a complex and dynamic process.Transcription factors play a decisive role in regulating this process.Gene mutation or rearrangement of these regulatory factors will lead to hematopoietic stem cells or precursor cells of monoclonal amplification,sparking acute/chronic lymphocytic leukemia(ALL/CLL)or acute/chronic myeloid leukemia(AML/CML).Myeloid leukemia is a malignant clonal disease derived from stem cells or blood precursor cells.Abnormal differentiation,uncontrolled proliferation and apoptosis hindered leaving leukemia cells stagnant at different stages of cell development suppress the normal hematopoiesis.Mutation of hematopoiesis transcription factor may play a key role in the occurrence of leukemia,including MLL,AML1 gene fusion,P53 gene deletions,down-regulated nm23 gene,high BCL-2 gene expression and so on.With the development of medicine,although there are a lot of research in the myeloid development and disease models during the past few decades,but the research on hematopoietic development are mainly carried out on mice.Limitation in the detection of mouse embryonic and fetal period myeloid cells,causing the early hematopoietic gene networks is not clear.At the same time,the mouse model has been a valued drug screening model.It can directly reflect the overall adverse drug reactions and side effects and the results obtained from this model have a important clinical value and application prospect.But the mouse models also have weaknesses in high cost,long cycle and lager sample size.The significant deficiencies required a new vertebrate model to make up for deficiencies of our current knowledge,understand the pathogenesis and eventually objective overcome leukemia.Zebrafish is a vertebrate model,which has been widely used in embryonic development,disease research and drug screening within the past ten years.It first be used in the 1930s for the environmental assessment,the assessment of new drugs for the first time in 1988.In Europe,use zebrafish model to assess environmental and discover drug has been accepted government regulators GLP(Good Laboratory Practice)certification.Zebrafish benefited from its in vitro fertilization,small size,reproductive ability,transparent body and many other advantages,quickly applied to diverse research fields,such as drug screening,human diseases study,developmental biology study,environmental safety study and immunological studies.Similar blood composition and gene regulation mechanisms between zebrafish and human make zebrafish an ideal animal model to study human hematopoietic development.Zebrafish hematopoiesis in adult occurs mainly in renal interstitial,consistent with human bone marrow.Kidney marrow can produce different lineages of blood cells.These blood cells mature in the thymus and liver and into enter circulation subsequently.This process involves blood cell development,differentiation and maturation.Regulatory factors participated in this process is highly conserved with human.More importantly,some studies have shown that mutations or genetic rearrangements of these regulatory factors will lead to hematopoietic stem cell or precursor cell monoclonal amplification,sparking acute/chronic lymphocytic leukemia(ALL/CLL)or acute/chronic myelogenous lineage leukemia(AML/CML).The zebrafish has been described as a disease model animal for new molecular mechanisms of some blood diseases and screening of new drugs.Using zebrafish leukemia model can not only further studys the molecular mechanism of human leukemia but also screening the drug in the form of new small molecule compounds.Although some human leukemia model has been established by constructed corresponding transgenic zebrafish or zebrafish mutant,futher have been used in drug screening process.But there is a great lack of some genes of zebrafish leukemia models,such as c-myb,pu.1,BCR-ABL,TET2.Pu.1(also known as SPi1),first isolated from Friend's erythroleukemia,is a transcriptional factor of the Ets family.PU.1 is a member of Ets-family transcription factors which is indispensable for generating early myeloid progenitors and for the determination of monocytic versus granulocytic lineages during myelopoiesis.Apart from its early function in myelopoiesis,PU.1 is also reported to be involved in leukemogenesis in mice and human.6 It has been reported that PU.1 expression is down-regulated in human acute myeloid leukemia(AML)patients and PU.1 mutations is found in about 7%of the patients in a study involving 126 AML patients.Likewise,reduction of PU.1 activity leads to AML in mice with high frequency.Considering the advantage of zebrafish and the highly homologous between zebrafish and human genes,in this study,we acquired a Pu.l down regulation homozygous mutant suing TILLING(Targeted Induced Local Lesions In Genomes)method.Based on this mutant,we tried to obtain the hematopoietic phenotype in larval as well as adult homozygous mutant and to have a preliminary understanding about the impact of pu.l mutation on zebrafish definitive hematopoiesis.Then according to the phenotype,we tried to choose two kinds of specific drug for treatmet,in order to observe their impact on abnormal hematopoiesis in this pu.1 mutant,which might be the foundation of the future leukemia model establishment and the following high-throughput drug screening.c-MYB is an important transcription factor,but also a proto-oncogene in hematopoiesis,that was first isolated from avian myeloid hyperplasia virus homologous with v-MYB.c-MYB protein contains 3 highly conserved DNA domains:(1)N-terminal DNA binding domain,is capable of binding to specific DNA sequences;(2)transcription activation domain located in the center;(3)C-terminal negative regulatory region containing leucine zipper can inhibit transcriptional activation of genes.c-MYB is an important transcription factor involved in the development and differentiation of hematopoietic system that gradually decrease expression from hematopoietic stem cells and progenitor cells to differentiated more mature cells.It may be involved in the development of a series of human leukemia and other malignancies.All in all,c-MYB plays a key role in the development of blood formation and related diseases.However,in animal models,the underlying mechanism of c-MYB aberrant expression resulting in hematological disorders is still lack of evidence.Also the lack of a survival animal model with c-MYB activation leads to the difficulty of tracking diseases course of a c-MYB-related blood disease.In this study,we investigated the induction of myeloid and lymphoid leukemias in transgenic zebrafish aberrant activation c-myb.Respectively study the zebrafish embryo to adult fish from blood phenotype.And selecte some anti-tumor drugs to test the response of this model.We hope that this model can provide a valuable platform for further investigate the role of c-myb in leukemia pathogenesis and developing new anticancer drugs.This study was divided into two parts.The first part is divided into two chapters,the second part is divided into three chapters:Part 1:Establishment of pu.1 zebrafish leukemia models.Part 1/Chapter 1:Hematopoietic phenotype in pu.1 defects zebrafish embryos and adult.Part 1/Chapter 2:Chemotherapy effects on abnormal hematopoietic in Pu.l defect zebrafish.Our findings document that pu.1G242D mutant zebrafish develop a hematologic disorder that resembles human myeloid preleukemic or leukemic phenotype and can respond to chemotherapy treatment.Thus,pu.1G242D mutant fish provide a valuable animal model system for drug evaluation and in vivo large-scale anti-neoplastic screening.Part 2:Establishment of c-myb zebrafish leukemia models.Part 2/Chapter 1:Identification of abnormal activation of c-myb zebrafish lines.Part 2/Chapter 2:Hematopoietic phenotype and mechanism study in c-myb abnormal activation zebrafish.Part 2/Chapter 3:Clincle therapeutic drugs pharmacology validation in c-myb disease model.In this part,we introduced a c-myb aberrant activation transgenic zebrafish c-myb-gfp,in which three different c-myb transcripts were produced.c-myb aberrant activation zebrafish exhibited granulocytic hyperplasia from the embryonic stages to adulthood.Zebrafish developed acute myeloid leukemia(AML)-like or acute lymphoid leukemia(ALL)-like disorders with age,with characteristic infiltration of leukemic blasts in the blood circulation and other,non-hematopoietic tissues,including gill,muscle,liver,eye,and the central nervous system.These phenotypes resembled human myelodysplastic syndrome,AML and ALL.The hematopoietic abnormalities in c-myb-gfp embryos with c-myb overexpression were reduced by anti-leukemia chemotherapy.This animal model will provide a valuable resource for further studies of the molecular mechanisms underlying c-Myb-associated leukemogenesis,and for exploring potential new anti-leukemic drugs.Part 1:Establishment of pu.1 zebrafish leukemia models.Chapter 1:Hematopoietic phenotype in pu.1 defects zebrafish embryos and adult1.Objects1)Preliminary determination Hematopoietic phenotype in pu.l mutant embryos and adult fish.2)Elucidate the cellular basis of myeloid deficient phenotype in pu.l mutants.3)Establish and identify a new leukemia zebrafish animal model.2.Methods1)Using WISH to detect specific myeloid marker lyz,mpo and cebpl expression in 3dpf and 5dpf zebrafish.Observation the phenotypes of myeloid progenitor cells in embryonic mutant.And apply Sudan Black staining to detect the changes of mature granulocytes in respective period.While taking advantage of myeloperoxidase activity staining to assess neutrophil function.2)Using BrdU labeling and TUNEL experiments to determine whether the mutant myeloid progenitor cell proliferation and apoptosis changes during differentiation3)According to the morphological characteristics of the cells,counting myeloid progenitor cells,neutrophils and macrophages in adult fish.Macrophage phagocytosis containing substances,such as apoptotic cell debris and neutrophils lobulated nuclei and active motion of the particles.Zebrafish myeloid progenitors have none of the above features,but there can be clearly distinguished the red blood cells to myeloid characteristics.3.Results1)Compared with wild-type zebrafish,cebp-1,cebpa,lyz and mpo myeloid markers were significantly increased in PBI region of Pu.1 homozygous mutant at the stage of 3dpf.However,the myeloid specific marker mfap4 and c-fms did not increased in 3dpf embryos.Consistent with this,Sudan black(SB)signals also increased significantly in PBI region of Pu.l homozygous mutant at the stage of 3dpf period.At the same time,Myeloperoxidase activity experiments show that the neutrophil myeloperoxidase activity decreased after pu.l mutat,suggesting that neutrophil function is affected.2)We first monitered the cell death of neutrophils by TUNEL assay,and the result show that neutrophil apoptosis in pu.1 mutants is no significant differences with WT.Then,BrdU incorporation assay was performed for detecting the proliferation status,result showed the increased proliferation of neutrophils in pu.l mutants.3)By the 18 months of age,pu.l mutants displayed about 23%increase of myeloid population and-20%reduction of lymphoid population in the KM shown by manual differential blood cell counts.Cytological analysis revealed that the accumulated myeloid population in the pu.1 mutant KM was largely immature myeloid cells.Moreover,the immature myeloid cells were also found to accumulate in the PB of mutant fish.4.Conclusions1)The result showed an obvious expansion of granulocytes in the CHT of 3 dpf pu.l embryos.Meanwhile,macrophages showed decreased numbers in pu.l embryos.Result revealed that the peroxidase activity is markedly reduced in pu.1 mutant,supporting that the expanded neutrophils are immature.2)Data indicated that the enrichment of myeloid cells in pu.l mutants is result from increased proliferation but not attenuated apoptosis of neutrophils.3)These results indicate that pu.l hypomorphic mutation leads to the expansion of immature myeloid cells in the kidney and the accumulation of immature myeloid cells in the PB,a phenotype resembling the blood phenotypes of myeloid dysplastic syndrome(MDS)or AML in humans.Chapter 2:Chemotherapy effects on abnormal hematopoietic in Pu.l defect zebrafish.1.ObjectsBased on previously observed hematopoietic phenotypes in the Pu.l homozygous mutant,we have targeted selected cytarabine and daunorubicin(cell cycle specific and cell cycle non-specific drug),both of which are common used in hematological malignant disease,to treat larvel Pu.1 mutant,in order to observe whether the abnormal hematopoiesis in zebrafish mutant could be recovered through these drug treatment.2.MethodsMutant embryos and their siblings were incubated with 103 mg/L cytarabine or 30 mg/L daunorubicin at 1 dpf and the number of granulocyte lineage cells was determined by calculating the SB+cells in the CHT at 5 dpf,since the enrichment of granulocytes in pu.1 embryos is evident and easy to calculate in this time point.3.ResultsAs shown in Figure 2,after cytarabine treatment for 4 days,the SB+cells in pu.1 embryos were significant fewer in number than those in the untreated control,while the WT control showed insignificant difference whether treated or not.Interestingly,daunorubicin treatment seemed to have little effect on the number of SB+granulocytes in pu.1 mutant embryos.This result indicates that the anti-proliferation drug cytarabine can induce the remission of the neoplastic proliferation of myeloid cells and relief the leukemic like phenotype in pu.1 mutants,but the apoptosis-promote drug daunorubicin can not.4.ConclusionsOur findings document that pu.1G242D mutant zebrafish develop a hematologic disorder that resembles human myeloid preleukemic or leukemic phenotype and can respond to chemotherapy treatment.Thus,pu.1G242D mutant fish provide a valuable animal model system for drug evaluation and in vivo large-scale anti-neoplastic screening.Part 2:Establishment of c-myb zebrafish leukemia models.Chapter 1 Establishment of c-myb zebrafish leukemia models1.Objects1)We tried to characterize aberrant c-myb expression zebrafish c-myb-gfp through whole mount in situ hybridization and SB staining.2)Clarify the abnormal activation reasonsof c-myb in c-myb-gfp.2.Methods1)we sequenced the modified region around the GFP of the PAC in c-myb-gfp transgenic fish.2)5' and 3'-rapid amplification of cDNA ends(RACE)analysis were performed in 2 dpf c-myb-gfp/c-mybhk=3/hk=3 embryos.3)We further explored c-myb expression levels and patterns in the c-myb-gfp transgenic line by WISH and qPCR using whole larvae and adult kidney3.Results1)We sequenced the modified region around the GFP of the PAC in c-myb-gfp transgenic fish.We found a 485-bp repetitive sequence before the translation initiation site of c-myb,including a 315-bp core promoter region and a 170 bp 5 UTR,which may promote c-myb expression.2)By walking the sequence downstream of this c-myb mini promoter,we found the following c-myb gene is incomplete,which is truncated from intron 10,and followed with 77 bp unkown sequence,ampicillin resistance gene,a piece of pWSMK-T vector sequence,the second repeated 315-bp c-myb core promoter,exon 1 to exon 15 of full length c-myb gene in order.3)Besides the expected c-mybhk=3 form of transcript,another different two c-myb transcripts(named c-myb-WT and c-myb-T1 respectively)were detected in c-myb-gfp/c-mybhk=3/hk=3,which were produced by the modified PAC transgene.According to result of 5'-RACE,we found that all these transcripts shared the same transcription initiation site.When fully-sequenced each RACE product,we found that c-myb-WT was identical to the endogenous wildtype form of c-myb transcript,which produced from the second c-myb core promoter.The 4.3 kb c-myb-T1 comprised with truncated c-myb from exon1 to exon10 and followed by a near full-length c-myb from exon2 to exon 15.c-myb-T1 is supposed to translated into a fusion protein with two c-myb forms conjugated,including the first truncated lack of a complete negative-regulatory region and fused with the second c-myb lack of the first 8 aa.4)c-myb is normally found in the aorta gonad mesoderm(AGM),caudal hematopoietic tissue(CHT)and kidney,where HSCs are located.However,c-myb expression in the c-myb-gfp transgenic reporter line was strongly increased for the same time window and the same region.4.Conclusions1)This data indicated that the region from ampicillin resistance gene to c-myb exonl to exon 10 was duplicated within the PAC in the c-myb-gfp transgenic fish,and the predicted myb mini promoter may be sufficient to drive downstream c-myb gene to transcribe.2)Besides the expected c-mybhk=3 form of transcript,another different two c-myb transcripts(named c-myb-WT and c-myb-T1 respectively)were detected in c-myb-gfp/c-mybhk=3/hk=.3)c-myb gene abnormal activation in c-myb-gfp transgenic zebrafish.Chapter 2 Hematopoietic phenotype and mechanism study in c-myb abnormal activation zebrafish1.Objects1)Preliminary determination Hematopoietic phenotype in c-myb-gfp embryos and adult fish.2)Elucidate the cellular basis of myeloid deficient phenotype in c-myb-gfp embryos.3)Establish and identify a new leukemia zebrafish animal model.4)Elucidate the molecular mechanism of myeloid deficient in c-myb-gfp.2.Methods1)In the embryonic period,WISH experiment was performed by specific myeloid maker(lcp mark labeled early myeloid cells;cebpa,cebpl labeled immature neutral granulocyte;mpo,lyz labeled mature neutrophils;mfap4 to mark mature macrophages)to detect the detailed myeloid hematopoietic changes at different stages of myeloid cells.Elaborate the role of c-myb in myeloid progenitor cells,neutrophil and macrophage cell fate choices in zebrafish.2)Calculating the number of myeloid cells,subtype,stage of differentiation by flow cytometry(FACS)and cytology in adult fish Kidneys and circulation.3)With Brdu antibody staining and TUNEL experiments to determine the proliferation and apoptosis during myeloid cell differentiation.4)Comparison with clinical progress in the overall phenotype,PB/KM blood count.Cross c-myb-gfp with myeloid transgenic zebrafish Tg(lyz:dsRed)and lymphoid transgenic zebrafish Tg(rag2:dsRed)to obtain double transgenic zebrafish.Calculating the ratio of MDS model process into the blast phase in the natural course of the disease.And according to cell morphology,tissue sections to distinguish the type of blast phase cells.5)mRNA samples were collected from and wild-type zebrafish to perform qPCR and found the c-myb downstream gene.3.Results1)We clarified the lineage origin of accumulated myeloid cells in c-myb-gfp fish by WISH using macrophage-specific and granulocyte-specific markers.Expression of the macrophage marker mfap4 was significantly decreased in 3-dpf c-myb-gfp embryos.In contrast,the granulocytic marker lysozyme C(lyz)was elevated in c-myb-gfp embryos.2)KM and PB cells were randomly collected from 3-month and 1-year-old c-mybhyper fish with eumorphism or siblings and analyzed by cytological and blood cell count analyses.There were no significant alterations in peripheral blood constituents in c-mybhyper fish at 3 months or 1 year.However,KM cell counts indicated a significant expansion of the myeloid cell population in 3-month-old c-mybhyper fish,along with a correspoLding reduction in progenitor cells.By 1 year old,the myeloid population had almost doubled,accompanied by obvious reductions in other blood constituents in c-mybhyper fish KM,consistent with myeloid hyperplasia.In accordance with the cytological results,flow cytometry analysis of KM also showed a 2-fold increase in the myeloid cell population and peripheral cytopenia in 1-year-old adult c-mybhyper fish.These results indicated marked myelodysplasia in c-mybhyper fish characterized by elevated cell numbers,enlarged cell size and increased cell granularity.3)The results of BrdU experments indicating that the enrichment of myeloid cells in c-mybhyper fish was the result of increased proliferation.4)The kidneys in 1-year-old c-mybhyper zebrafish appeared swollen,with an enlarged kidney area and almost 4-fold increase in kidney weight compared with control siblings.Livers in c-mybhyper fish were also heavier,with enrichment of invaded SB+granulocytes compared with controls.Abnormal tumor-like or acute leukemic-like phenotypes were occasionally observed among c-mybhyper adult fish from 3 months onwards,including cachexia,exophthalmos,bone bending,abdominal mass and bleeding.c-mybhyper MDS fish could progress to AML and ALL.4.Conclusions1)c-myb aberrant activation resulted in accumulation of abnormal granulocytes in early development.2)c-mybhyper adult fish displayed MDS-like phenotypes.3)Granulocyte expansion in c-mybhyper fish was mainly caused by proliferation perturbation.4)c-mybhyper MDS fish could progress to AML and ALL.Chapter 3:Clincle therapeutic drugs pharmacology validation in c-myb disease model.1.ObjectsZebrafish provide a promising model for drug discovery studies.We therefore evaluated the effects of widely-used anti-leukemic agents in c-mybhyper fish.Cytarabine36 is commonly used to treat AML.They act by inhibiting the cell cycle of accumulated leukemic cells.We also investigated the response of the expanded myeloid population in c-mybhyper embryos to the c-myb-targeting drug,the CDK9 inhibitor flavopiridol.2.Methodsc-mybhyper embryos and their control siblings were incubated with 103 mg/L cytarabine or 30 mg/L daunorubicin at 1 dpf,and the numbers of SB+granulocytic cells were calculated in the CHT at 6 dpf.At the same time,we also incubated c-mybhyper embryos and their control siblings with 0.5 um flavopirdol,and the numbers of SB+granulocytic cells were calculated in the CHT at 7 dpf.3.ResultsFive days treatment with cytarabine significantly reduced the number of SB+granulocytes in c-mybhyper embryos compared with the untreated controls,whereas cytarabine had no effect on SB+cell numbers in control siblings.Flavopiridol significantly reduced the numbers of SB+granulocytes in c-mybhyper embryos compared with the untreated controls,whereas SB+cell numbers in control siblingswere unaffected.c-myb RNA levels were also significantly reduced in c-mybhyper embryos by flavopiridol,compared with untreated fish.These results indicated that clinically-used c-Myb-targeting drug effectively reduced the expanded myeloid population in c-mybhyper fish.4.Conclusions1)Abnormal activation of c-myb induced myeloid dysplasia phenotype can be alleviated by the cell cycle specific anti-drug cytarabine.2)These results indicated that clinically-used c-Myb-targeting drug effectively reduced the expanded myeloid population in c-mybhyper fish.