Credentials

We're blood cancer and zebrafish experts

The founding team comprises three founders who share a common goal to improve the treatment outcome of patients with blood cancers.  Professor Anskar Leung has more than 20 year of experience in research and clinical management of blood cancers and both Dr. Ma and Dr. Sun have more than 10 years of research in zebrafish and cancers.  Their complementary expertise has helped in building the synergism and we are determined to make an impact in the field by enabling rapid drug discovery by both pharmaceutical and academic institutes.

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Publications

Publications authored by the ZeBlast team members in relation to blood cancers and zebrafish.

Author: Dandan Wang 1Lichuan Zheng 1Bowie Yik Ling Cheng 1Chun-Fung Sin 2Runsheng Li 3Sze Pui Tsui 2Xinyu Yi 4Alvin Chun Hang Ma 5Bai Liang He 6Anskar Yu Hung Leung 7Xuan Sun 8

Abstract

Isocitrate dehydrogenase 2 (IDH2) mutations occur in more than 15% of cytogenetically normal acute myeloid leukemia (CN-AML) but comparative studies of their roles in leukemogenesis have been scarce. We generated zebrafish models of IDH2R172K and IDH2R140Q AML and reported their pathologic, functional and transcriptomic features and therapeutic responses to target therapies. Transgenic embryos co-expressing FLT3ITD and IDH2 mutations showed accentuation of myelopoiesis. As these embryos were raised to adulthood, full-blown leukemia ensued with multi-lineage dysplasia, increase in myeloblasts and marrow cellularity and splenomegaly. The leukemia cells were transplantable into primary and secondary recipients and resulted in more aggressive disease. Tg(Runx1:FLT3ITDIDH2R172K) but not Tg(Runx1:FLT3ITDIDH2R140Q) zebrafish showed an increase in T-cell development at embryonic and adult stages. Single-cell transcriptomic analysis revealed increased myeloid skewing, differentiation blockade and enrichment of leukemia-associated gene signatures in both zebrafish models. Tg(Runx1:FLT3ITDIDH2R172K) but not Tg(Runx1:FLT3ITDIDH2R140Q) zebrafish showed an increase in interferon signals at the adult stage. Leukemic phenotypes in both zebrafish could be ameliorated by quizartinib and enasidenib. In conclusion, the zebrafish models of IDH2 mutated AML recapitulated the morphologic, clinical, functional and transcriptomic characteristics of human diseases, and provided the prototype for developing zebrafish leukemia models of other genotypes that would become a platform for high throughput drug screening.

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Author: Yuhan Guo 1Bowie Y L Cheng 2Dandan Wang 2Alvin C H Ma 2Bai-Liang He 2Toni K Man 2May P L Cheung 3Xiangguo Shi 2Nelson K L Ng 2Anskar Y H Leung 4

Abstract

ADP-ribosylation factor-like 4aa (Arl4aa) is a member of the ADP-ribosylation factor family. It is expressed in hematopoietic tissue during embryonic development, but its function was unknown. Zebrafish arl4aa is preferentially expressed in the ventral wall of the dorsal aorta (VDA) at 24 and 36 hpf and in caudal hematopoietic tissue at 48 hpf. Morpholino knockdown and transcription activator-like effector nuclease (TALEN) knockout of arl4aa significantly reduced expression of genes associated with definitive hematopoietic stem cells (HSCs). Golgi complex integrity in VDA was disrupted as shown by transmission electron microscopy and immunostaining of Golgi membrane Giantin. Mechanistically, arl4aa knockdown reduced Notch signaling in the VDA and its target gene expression. Protein expression of NICD was also reduced. Effects of arl4aa knockdown on definitive hematopoiesis could be restored by NICD expression. This study identified arl4aa as a factor regulating initiation of definitive HSCs by maintaining the integrity of Golgi complex and, secondarily, maturation of the Notch receptor.

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Author: Bai-Liang He # 1 2Ning Yang # 1Cheuk Him Man 1Nelson Ka-Lam Ng 1Chae-Yin Cher 1Ho-Ching Leung 1Leo Lai-Hok Kan 1Bowie Yik-Ling Cheng 1Stephen Sze-Yuen Lam 1Michelle Lu-Lu Wang 1Chun-Xiao Zhang 1Hin Kwok 3Grace Cheng 3Rakesh Sharma 3Alvin Chun-Hang Ma 4Chi Wai Eric So 5Yok-Lam Kwong 1Anskar Yu-Hung Leung 1

Abstract

Internal tandem duplication of Fms-like tyrosine kinase 3 (FLT3/ITD) occurs in about 30% of acute myeloid leukemia (AML) and is associated with poor response to conventional treatment and adverse outcome. Here, we reported that human FLT3/ITD expression led to axis duplication and dorsalization in about 50% of zebrafish embryos. The morphologic phenotype was accompanied by ectopic expression of a morphogen follistatin (fst) during early embryonic development. Increase in fst expression also occurred in adult FLT3/ITD-transgenic zebrafish, Flt3/ITD knock-in mice, and human FLT3/ITD AML cells. Overexpression of human FST317 and FST344 isoforms enhanced clonogenicity and leukemia engraftment in xenotransplantation model via RET, IL2RA, and CCL5 upregulation. Specific targeting of FST by shRNA, CRISPR/Cas9, or antisense oligo inhibited leukemic growth in vitro and in vivo. Importantly, serum FST positively correlated with leukemia engraftment in FLT3/ITD AML patient-derived xenograft mice and leukemia blast percentage in primary AML patients. In FLT3/ITD AML patients treated with FLT3 inhibitor quizartinib, serum FST levels correlated with clinical response. These observations supported FST as a novel therapeutic target and biomarker in FLT3/ITD AML.

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Author: Bai-Liang He 1, Xiangguo Shi 1, Cheuk Him Man 1, Alvin C.H. Ma 1 2, Stephen C. Ekker 2, Howard C.H. Chow 1, Chi Wai Eric So 3, William W.L. Choi 4, Wenqing Zhang 5, Yiyue Zhang 5, Anskar Y.H. Leung 1

Abstract

FMS-like tyrosine kinase 3 (FLT3) is expressed in human hematopoietic stem and progenitor cells (HSPCs) but its role during embryogenesis is unclear. In acute myeloid leukemia(AML), internal tandem duplication (ITD) of FLT3 at the juxtamembrane (JMD) and tyrosine kinase (TKD) domains (FLT3-ITD+) occurs in 30% of patients and is associated with inferior clinical prognosis. TKD mutations (FLT3-TKD+) occur in 5% of cases. We made use of zebrafish to examine the role of flt3 in developmental hematopoiesis and model human FLT3-ITD+ and FLT3-TKD+ AML. Zebrafish flt3 JMD and TKD were remarkably similar to their mammalian orthologs. Morpholino knockdown significantly reduced the expression of l-plastin (pan-leukocyte), csf1r, and mpeg1 (macrophage) as well as that of c-myb(definitive HSPCs), lck, and rag1 (T-lymphocyte). Expressing human FLT3-ITD in zebrafish embryos resulted in expansion and clustering of myeloid cells (pu.1+, mpo+, and cebpα+) which were ameliorated by AC220 and associated with stat5, erk1/2, and akt phosphorylation. Human FLT3-TKD (D835Y) induced significant, albeit modest, myeloid expansion resistant to AC220. This study provides novel insight into the role of flt3 during hematopoiesis and establishes a zebrafish model of FLT3-ITD+ and FLT3-TKD+AML that may facilitate high-throughput screening of novel and personalized agents.

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Author: Alvin C H Ma 1Xiangguo Shi 1Bai-Liang He 1Yuhan Guo 1Anskar Y H Leung 2

Abstract

The recent advent of next-generation sequencing (NGS) has greatly accelerated identification of gene mutations in myeloid malignancies at unprecedented speed that will soon outpace their functional validation by conventional laboratory techniques and animal models. A high-throughput whole-organism model is useful for the functional validation of new mutations. We recently reported the use of zebrafish to evaluate the hematopoietic function of isocitrate dehydrogenase 1 (IDH1) and the effects of expressing human IDH1-R132H that is frequently identified in human acute myeloid leukemia (AML), in myelopoiesis, with a view to develop zebrafish as a model of AML. Here, we use IDH1 as an example to describe a comprehensive approach to evaluate hematopoietic gene function and the effects of mutations using zebrafish as a model.

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Author: Xiangguo Shi 1Bai-Liang He 1Alvin C H Ma 1Yuhan Guo 1Yali Chi 2Cheuk Him Man 1Wenqing Zhang 2Yiyue Zhang 2Zilong Wen 3Tao Cheng 4Anskar Y H Leung 1

Abstract

Isocitrate dehydrogenase 1 mutation (IDH1-R132H) was recently identified in acute myeloid leukemia with normal cytogenetics. The mutant enzyme is thought to convert α-ketoglutarate to the pathogenic 2-hydroxyglutarate (2-HG) that affects DNA methylation via inhibition of ten-eleven translocation 2. However, the role of wild-type IDH1 in normal hematopoiesis and its relevance to acute myeloid leukemia is unknown. Here we showed that zebrafish idh1 (zidh1) knockdown by morpholino and targeted mutagenesis by transcription activator-like effector nuclease might induce blockade in myeloid differentiation, as evident by an increase in pu.1 and decrease in mpo, l-plastin, and mpeg1 expression, and significantly reduce definitive hematopoiesis. Morpholino knockdown of zidh2 also induced a blockade in myeloid differentiation but definitive hematopoiesis was not affected. The hematopoietic phenotype of zidh1 knockdown was not rescuable by zidh2 messenger RNA, suggesting nonredundant functions. Overexpression of human IDH1-R132H or its zebrafish ortholog resulted in 2-HG elevation and expansion of myelopoiesis in zebrafish embryos. A human IDH1-R132H-specific inhibitor (AGI-5198) significantly ameliorated both hematopoietic and 2-HG responses in human but not zebrafish IDH1 mutant expression. The results provided important insights to the role of zidh1 in myelopoiesis and definitive hematopoiesis and of IDH1-R132H in leukemogenesis.

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Author: Alvin C H Ma 1Tsz K FungRachel H C LinMartin I S ChungDan YangStephen C EkkerAnskar Y H Leung

Abstract

In a chemical screening, we tested the antiangiogenic effects of fumagillin derivatives and identified fumagillin as an inhibitor of definitive hematopoiesis in zebrafish embryos. Fumagillin is known to target methionine aminopeptidase II (MetAP2), an enzyme whose function in hematopoiesis is unknown. We investigated the role of MetAP2 in hematopoiesis by using zebrafish embryo and human umbilical cord blood models. Zebrafish metap2 was expressed ubiquitously during early embryogenesis and later in the somitic region, the caudal hematopoietic tissue, and pronephric duct. metap2 was inhibited by morpholino and fumagillin treatment, resulting in increased mpo expression at 18 hours postfertilization and reduced c-myb expression along the ventral wall of dorsal aorta at 36 hours postfertilization. It also disrupted intersegmental vessels in Tg(fli1:gfp) embryos without affecting development of major axial vasculatures. Inhibition of MetAP2 in CB CD34(+) cells by fumagillin had no effect on overall clonogenic activity but significantly reduced their engraftment into immunodeficient nonobese diabetes/severe combined immunodeficiency mice. metap2 knock-down in zebrafish and inhibition by fumagillin in zebrafish and human CB CD34(+) cells inhibited Calmodulin Kinase II activity and induced ERK phosphorylation. This study demonstrated a hitherto-undescribed role of MetAP2 in definitive hematopoiesis and a possible link to noncanonical Wnt and ERK signaling.

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Author: A C H Ma 1M I S ChungR LiangA Y H Leung

Abstract

Although aldehyde dehydrogenase (ALDH) activity has become a surrogate of hematopoietic stem and progenitor cells (HSPCs), its function during hematopoiesis was unclear. Here, we examined its role in zebrafish hematopoiesis based on pharmacological inhibition and morpholino (MO) knockdown. Zebrafish embryos were treated with diethylaminobenzaldehyde (DEAB, 1 μmol/l) between 0- and 48 hour-post-fertilization (hpf). MOs targeting aldhs were injected between 1 and 4-cell stage. The effects on hematopoiesis were evaluated at different stages. DEAB treatment between 0 and 18 hpf increased gene expression associated with HSPC (scl, lmo2), erythropoiesis (gata1, α- and β-eHb) and myelopoiesis (spi1) as well as gfp(+) cells in dissociated Tg(gata1:gfp) embryos. The effects were ameliorated by all-trans retinoic acid (1 nmol/l). Definitive hematopoiesis and the erythromyeloid precursors were unaffected. In all, 14 out of 15 zebrafish aldhs were detectable by reverse transcription PCR in 18 hpf embryos, of which only aldh1a2 and aldh16a1 were expressed in sites pertinent to hematopoiesis. Molecular targeting by MOs was demonstrated for 15 aldhs, but none of them, even in combined aldh1a2 and aldh1a3 knockdown, recapitulated the hematopoietic expansion in DEAB-treated embryos. In conclusion, DEAB expands HSPC population during primitive hematopoiesis through inhibition of aldh and retinoic acid synthesis. The specific aldh isoform(s) remains to be determined.

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Author: A C H Ma 1M I S ChungR LiangA Y H Leung

Abstract

Janus kinase 2 (Jak2) transduces signals from hematopoietic cytokines, and a gain-of-function mutation (Jak2(617V>F)) is associated with myeloproliferative diseases, particularly polycythemia vera. In this study, we examined the role of jak2a in zebrafish embryos in knock-down and overexpression studies using morpholinos (MOs) targeting the 5′ untranslated region (UTR) (jak2a(UTR)-MO) and splice-site junction (jak2a(SS)-MO) of jak2a, a Jak inhibitor AG490 and a constitutive-active form of jak2a (jak2a(ca)). At 18 and 24 hours after fertilization (hpf), jak2a is expressed predominantly in the intermediate cell mass (ICM; site of primitive hematopoiesis) of wild-type and chordin morphant embryos (characterized by expansion of ICM). Both jak2a MOs and AG490 reduced gata1(+) (erythroid) cells in Tg(gata1:GFP) embryos, signal transducer and activation of transcription 5 (stat5) phosphorylation, and gene expression associated with early progenitors (scl and lmo2) and erythroid (gata1, alphahe1 and betahe1) and myeloid (spi1 [early] and mpo [late]) lineages. The chordin morphant is associated with increased stat5 phosphorylation, and both jak2a MOs and treatment with AG490 significantly ameliorated ICM expansion and hematopoietic gene up-regulation in these embryos. Injection of plasmid encoding jak2a(ca) significantly increased erythropoiesis and expression of gata1, alphahe1 and betahe1, spi1, mpo, and l-plastin. In conclusion, zebrafish jak2a is involved in primitive hematopoiesis under normal and deregulated conditions.

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Author: A C H Ma 1M I S ChungR LiangA Y H Leung

Abstract

Survivin is an inhibitor of apoptosis and its role in embryonic development is not completely understood. In zebrafish, survivin undergoes gene duplication. Survivin1 (sur1) has been shown to mediate angiogenesis but not hematopoiesis. In this study, we examined survivin2 (sur2) with particular reference to its role in primitive hematopoiesis during zebrafish development. sur2 was expressed predominantly in the intermediate cell mass (ICM, site of primitive hematopoiesis). Morpholino (MO) targeting at intron1-exon2 junction of sur2 significantly reduced green fluorescent protein(+) (erythroid) cell population in transgenic Tg (gata1:gfp) embryos at 18 h post-fertilization (h.p.f.; wild type: 4.49+/-0.15%; Sur2(MO) embryos: 2.22+/-0.12%, P=0.02). Molecular targeting was confirmed by reverse transcription-PCR and MO specificity by successful sur2 mRNA rescue. sur2 MO also downregulated genes associated with hematopoietic stem cells (scl, lmo2), erythroid (gata1, alpha- and beta-embryonic hemoglobins) as well as early (pu.1) and late (mpo, l-plastin) myelomonocytic lineages at 12 and 18 h.p.f. This was associated with an increase in apoptosis in the ICM and alteration of cell-cycle status of erythroid cells. Both effects were caspase dependent. In conclusion, sur2 is important in maintaining hematopoietic stem and lineage committed cells during zebrafish development, by virtue of its antiapoptotic activity in a caspase dependent and cell autonomous fashion.

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Author: Lingge Tu, SIN Chun-fung, Yanan Chen, Fangfang He, Dandan Wang, Hoi Yi Chan, Sze Pui Tsui, Alvin Chu Hang Ma, Xuan Sun, Anskar Leung

Abstract

Internal Tandem Duplication of human FMS-like tyrosine kinase (FLT3-ITD) is one of the most frequent genetic alterations in de novo acute myeloid leukemia (AML) and is often acquired during transformation of myelodysplastic syndrome (MDS) into AML, predicting an unfavorable prognosis. RNA splicing factor mutations (e.g. hot spot mutations in serine and arginine rich splicing factor 2 – SRSF2) are often found in MDS and chronic myelomonocytic leukemia (CMML). However, the clinicopathologic features of co-mutated FLT3-ITD and SRSF2 in MDS/CMML and transformation to AML are presently unclear. Here we investigated whether FLT3-ITD cooperates with SRSF2-P95H to promote leukemogenesis in zebrafish.

Transgenic zebrafish carrying human FLT3-ITD or SRSF2-P95H as driven by Runx1 promoter were generated by Tol2 transgenesis. Tg(Runx1:FLT3ITD) was outcrossed with Tg(Runx1SRSF2P95H) to generate Tg(Runx1:FLT3ITDSRSF2P95H) zebrafish. The presence of human FLT3-ITD or SRSF2-P95H was confirmed through Sanger sequencing, and the expression of each transcript was detected by qRT-PCR in both embryos and adult kidney marrow. Their clinicopathologic features were examined.

At the embryonic stage, the number of leukocytes (l-plastin+) was significantly increased in Tg(Runx1FLT3ITDSRSF2P95H) embryos compared with their single mutant and wildtype (WT) siblings, whereas the abundance of neutrophils (SBB+) was unaffected. At six months of age, Tg(Runx1:FLT3ITDSRSF2P95H) zebrafish showed monocytosis in peripheral blood (PB) (1 out of 4), but not in the single mutant and WT siblings. In kidney marrow (KM), there was expansion of both immature and mature monocytes, and dysplasia of granulopoiesis and erythropoiesis. At 9 months of age, all four Tg(Runx1FLT3ITDSRSF2P95H) zebrafish showed neutropenia and substantial increase in monocytosis. At 1 year of age, one out of the nine Tg(Runx1FLT3ITDSRSF2P95H) zebrafish showed a significant increase in blasts in KM, reminiscent of human AML, and the other eight Tg(Runx1FLT3ITDSRSF2P95H) zebrafish demonstrated phenotypes consistent with human CMML-like diseases. Increased monocytic compartment of KM was confirmed by α-naphthyl acetate esterase sensitive to fluoride inhibition. In addition, both the spleen area and cellularity were expanded in Tg(Runx1FLT3ITDSRSF2P95H) zebrafish compared with Tg(Runx1SRSF2P95H) single mutant and WT siblings. Taken together, our findings demonstrated that FLT3-ITD cooperates with SRSF2-P95H to drive the initiation and progression of CMML and the subsequent transformation to AML. Understanding the molecular underpinnings of these events will shed light to the diverse clinicopathologic features in CMML patients.

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Author: Yanan Chen, Sin Chun-Fung, Lingge Tu, Hoi Yi Chan, Fangfang He, Dandan Wang, Sze Pui Tsui, Alvin, Chun Hang Ma, Xuan Sun, Anskar Leung

Abstract

Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic neoplasm characterized by monocytosis and recurrent gene mutations including those of SRSF2ASXL1TET2RUNX1 and NRAS. Different mutation combinations may explain the diverse clinicopathologic features in this disease, ranging from those of myelodysplastic syndrome (MDS) to myeloproliferative neoplasm (MPN) phenotype and even transformation to acute myeloid leukemia (AML). A zebrafish program was initiated to understand the pathogenetic roles of mutation combinations in CMML and generate animal models for high throughput drug screening. In this study, we reported our findings based on SRSF2 and NRAS mutation combination.

Transgenic zebrafish lines expressing human SRSF2P95H and NRASG12Dmutations (F0), as driven by Runx1promoter, were generated using Tol2 transgenesis. These lines were out-crossed with wildtype (WT) to generate F1. F1 SRSF2P95Hmutant were then crossed with F1 NRASG12D mutant to generate WT, Tg(Runx1:SRSF2P95H), Tg(Runx1NRASG12D) and Tg(Runx1:SRSF2P95HNRASG12D). Expression of mutations was confirmed in embryos and adult kidney marrow (KM) by quantitative RT-PCR and DNA sequencing.

At 3 months, there was no morphologic changes in peripheral blood (PB) or KM in double or single mutant fish. From 6-12 months, there was progressive weight loss in Tg (Runx1SRSF2P95H NRASG12D) fish but not single mutant and WT siblings. In the double mutant fish, the PB showed monocytosis and presence of blasts. KM showed increase in cellularity, immature monocytes and blast population but decrease in erythropoiesis. Neutrophils were dysplastic as evident by hypogranularity, abnormal nuclear configuration and features resembling pseudo Pelgar-Huet anomaly as in human MDS. Increased monocytic compartment in KM was confirmed by non-specific esterase (NSE) which was sensitive to fluoride inhibition. Survival of the Tg(Runx1:SRSF2P95HNRASG12D) was significantly shorter than those of single mutant and WT siblings. Both the double mutant and Tg(Runx1:NRASG12D) showed splenomegaly. Of the 49 Tg(Runx1:SRSF2P95HNRASG12D) adult fish examined, 9 developed MDS/AML with 10-19% KM blasts and 18 developed AML with ≥ KM 20% blasts.

Zebrafish model of CMML based on co-expression of SRSF2 and NRAS mutations recapitulated the diverse clinical repertoire of this disease, ranging from MDS to MPN and leukemic transformation into AML occurred in some animals. The molecular basis underlying the difference in penetrance might account for the diverse clinicopathologic characteristics in patients and it warrants further investigations.

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Author: Yik Ling Bowie ChengBai-Liang HeLaam LiYu Hung Anskar Leung

Abstract

The recent advances in sequencing techniques revealed acute myeloid leukaemia (AML) as a highly genetically heterogeneous disease. Each patient carries unique genetic mutational profile which can predict the clinical outcome. Remarkably, there are common patterns of co-occurring genetic mutations, indicating potential cooperativity between these genes in driving leukaemia. For instance, 6.5% of patients with FLT3 mutant AML also carry IDH1/2 mutations and confers to unfavourable risk. Conventional research method may not keep up with the wealth of newly generated data. Thus this project presented a novel platform based on zebrafish that allows quick evaluation of the impact of different genetic mutational combinations on leukaemogenesis, paving the way for personalised medicine. Embryos at 1-cell stage were injected with combinations of mutations in mRNA forms. Their effect on haematopoiesis was determined by the expression pattern and intensity of myeloid lineage markers pu.1 and mpo at yolk sac at 24 hours post fertilisation (hpf) by whole-mount in situ hybridization (WISH). In addition, neutrophils were quantified by Sudan black B (SSB) staining at caudal hematopoietic tissue (CHT) at 48hpf. Microinjection of FLT3-ITD and IDH1/2 mutations (IDH1 R132H, IDH2 R140Q and IDH2 R172K) individually at increasing dosage (25pg, 50pg, 100pg, 150pg) promoted myeloid expansion dose-dependently as evidenced by increased pu.1 expression at yolk sac at 24hpf and SSB staining at CHT at 48hpf respectively. Remarkably, FLT3-ITD and IDH2 R140Q co-operated to enhance myeloid expansion when co-injected at 25pg each, a dosage that had little effect on pu.1 expression as a single mutation. This was evidenced by 72.2% and 51% of embryos showing enhanced expression of pu.1 at yolk sac at 24hpf and SSB staining at CHT at 48hpf respectively. Our preliminary result proved that this platform is an effective method to study the cooperative effect of mutations. For future works, more combinations of genetic mutations will be studied starting with the most frequently co-occurred ones. Their effect on myeloid lineage markers including pu.1, mpo, l-plastin, mpeg1 and c-myb will be studied.

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Author: Yik Ling Bowie Cheng; Bai Liang He;  Dan Dan Wang; Yu Hung Anskar Leung

Abstract

Background and Objectives: Acute myeloid leukaemia (AML) is a highly genetically heterogenous disease. Patients carry unique genetic mutational profile that affect their clinical outcome. Remarkably, there are common patterns of co-occurring mutations, indicating their potential cooperativity in driving leukaemia. This project presents a novel zebrafish platform that allows quick and traceable evaluation of the impact of different genetic mutation combinations on leukemogenesis and drug response, paving the way for personalized medicine.

Experimental Procedures and Results: Zebrafish embryos were co-injected with combination of mutations FLT3 ITD with IDH2R140Q, IDH2R172K, IDH1R132H and DNMT3AR882H), and their effect on the expression pattern and intensity of different myeloid lineage markers including pu.1 (myeloid progenitor), mpo (macrophage), c-myb (haematopoietic stem cell; HSC) and Sudan Black B staining (neutrophils or granulocytes) were evaluated. Consistent with TCGA database, which showed FLT3 ITD mutation only co-occurred with IDH2R140Q or DNMT3AR882H, overexpression of FLT3 ITD and IDH2R140Q in zebrafish embryo promoted expansion of myeloid progenitors and granulocytes, while co-expression of FLT3 ITD and DNMT3AR882H promoted expansion of HSCs and granulocytes. No combinatory effects were observed between FLT3 ITD and IDH2R172K or IDH1R132H. To establish a zebrafish-based platform for predicting differential drug responses for unique mutational profiles, zebrafish embryos were injected with FLT3 ITD and IDH2R140Q and a list of FDA approved and preclinical drugs were examined for their efficacy on rescuing FLT3 ITD/IDH2R140Q-induced myeloid expansion. Although FLT3 ITD inhibitor was able to rescue FLT3 ITD-induced myeloid expansion, the effect was alleviated if FLT3 ITD and IDH2R140Q were co-expressed, indicating specific mutation patterns affected drug responses. To further validate our findings and study the long-term effect of mutation combinations, transgenic fish lines with FLT3 ITD/IDH2R140Qand FLT3 ITD/IDH2R172K under Runx1 promoter (HSC lineage) were established with Tol2 transgenesis. At 6 months of age, Tg(Runx1:FLT3 ITD, IDH2R140Q)showed increased percentage of myeloid precursors compared to WT and single mutant group.

Conclusion: Our result show that zebrafish can serve as an effective platform to study the cooperative effect of mutations in acute myeloid leukaemia and predict a better option for patients carrying unique genetic mutational profiles, paving the way for personalized medicine.

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Grants

Grants awarded to the ZeBlast core team members.

Project: Centre for Oncology and Immunology
Role:  Programme leader in haematologic malignancy
Year awarded: 2020 (5 years)
Amount(HKD): 501,000,000 (Total Funding Ceiling)

Project: Towards personalized and innovative treatment for acute myeloid leukaemia
Year awarded: 2020 (5 years)
Amount (HKD): 46,667,000

Project title: Modelling myeloproliferative neoplasms (MPNs) in a multi-colour transgenic zebrafish – mechanistic and therapeutic insights.
Year awarded:  2022 (3 years)
Amount (HKD ):  1,363,304.00

Project: Deciphering recurrent mutation combinations in myelodysplasia syndromes using zebrafish – mechanistic and therapeutic insights
Year awarded: 2022 (4 years)
Amount (HKD ): 1,185,561.00

Project: Generation of a novel humanised immunocomprom ised zebrafish model for evaluation of targeted therapy responses in acute myeloid leukemia.
Year awarded: 2020 (2 years)
Amount (HKD ): 150,000

Project: Function of SRSF2 and its mutations in zebrafish haematopoiesis and its relevance in acute myeloid leukaemia.
Year awarded: 2020 (2 years)
Amount (HKD ): 49,500

Project: Modelling TP53-mutated acute myeloid leukaemia in zebrafish – biological and therapeutic implications
Year awarded: 2019 (3 years)
Amount (HKD): 1,498,688

Project: Follistatin as a novel target and biomarker for acute myeloid leukaemia (AML) carrying internal tandem duplication (ITD) of Fms-Like Tyrosine kinase 3 (FLT3).
Year awarded: 2017 (3 years)
Amount (HKD): 1,229,089

Project: Targeting DNA damage and repair mechanism in FLT3-ITD acute myeloid leukaemia (AML) – a mechanistic and therapeutic study (3 years)
Year awarded: 2017 (3 years)
Amount (HKD): 1,200,000

Project: Develop a high throughput platform for personalized treatment of acute myeloid leukaemia
Year awarded: 2016 (3 years)
Amount (HKD): 4,288,400