T-cell acute lymphoblastic leukemia (T-ALL) is an uncommon aggressive leukemia arising from the malignant transformation of hematopoietic progenitors primed toward T-cell development. T-ALL accounts for approximately 10% to 15% of pediatric and 25% of adult ALL cases, and is characteristically more frequent in male than female.[1,2] Clinical T-ALL symptoms present hematopoiesis defect owing to bone marrow permeation by immature lymphoblasts with a T-cell immunophenotype, high white blood cell count, frequent infiltration into central nervous system, and the mediastinal mass coming from the thymus. In the past decade, with the introduction of intensive chemotherapy, the prognosis of T-ALL has made great progress with curative ratio approaching 90% in children and 70% in adolescents. Despite this progress, the prognosis of patients with primary resistant T-ALL who fail to obtain a complete hematological remission or those whose diseases relapse after a transient initial response remains poor.[5,6] Faced with these clinical challenges, much effort has been made to decipher the molecular events underlying T-ALL transformation, aiming to identify more specific therapeutic targets and develop more effective, less toxic anti-leukemic drugs or drug combinations.
T-ALL pathogenesis is a multi-step process of T progenitor transformation in which accumulating genetic abnormalities block T-cell differentiation and convert normal T progenitors to leukemia. In the last decade, systematic analysis of gene expression profiling has revealed aberrant expression of specific transcription factors resulting from chromosome translocations such as TAL1, LMO, LYL1, HOX11, or others, which defines subgroups of T-ALL with immunophenotypes reflecting thymocyte developmental arrest at different stages. Sequencing approaches focusing on candidate oncogenes or genome-wide sequencing have identified over 100 genes mutated in T-ALL. In this context, constitutive activation of NOTCH1 signaling, resulting from NOTCH1 gain-of-function or NOTCH1 E3 ligase F-box and WD repeat domain-containing 7 (FBW7) loss-of-function mutations,[10,11] is the most prominent oncogenic pathway in T-cell transformation. Deletion of the tumor suppressor genes CDK2A/2B is also observed in over 70% of T-ALL cases. Thus, loss of cell cycle regulators cooperates with constitutive activation of NOTCH1 signaling and constitutes the core oncogenic program in the pathogenesis of T-ALL. Additional mutations, although at lower frequencies, were reported playing crucial roles in T-cell leukemogenesis. Mutations in IL7R, JAK1, and JAK3 genes activate the IL7R/JAK-STAT pathway,[13–15]KRAS or NRAS mutations reinforce oncogenic RAS signaling,[16,17] and loss of PTEN tumor suppressor gene activates PI3K/AKT signaling. More recent genetic landscape of T-ALL also revealed loss-of-function mutations in epigenetic modulators (eg, EZH2, SUZ12, and PHF6), amplification of oncogenes (eg, MYB, MYCN)[20,21] and protein-altering mutations in essential components of the translation machinery (eg, RPL5, RPL10, and RPL22).[22,23] Based on existing genomic data from primary patient specimen, each T-ALL case contains probably over 10 biologically relevant genomic lesions that contribute to the transformation phenotype. An increased understanding of T-ALL biology has stimulated the development of T-ALL animal models and opened opportunities for in vivo functional or pre-clinical studies with a purpose of deciphering molecular pathology of this disease.
We and others have applied a series of animal models to recapitulate human T-ALL. In this regard, not only the gene functional studies for discovery of novel therapeutic targets, but also pre-clinical evaluation of potential therapeutic reagents, are applicable in an in vivo setting, which is often considered superior to the cell line-based studies. The animal models of T-ALL, utilizing mouse and zebrafish, will be reviewed in detail below.
Mouse models are more attractive for its exceptional advantages in comparison to the time-consuming and expensive big animal models. Mice are small size creatures, requiring little food or housing space; they have massive litters of offspring and are easy to handle. Murine T-ALL models have consistent disease manifestations, and become the most prominently used animal resource in this research field.
Xenograft is the transplantation of living cells, tissues or organs from one species to another. Xenograft of human tumor cells into immunodeficient mice is a common technique in pre-clinical oncology studies. This model is more representative of the nature and mutations of human cancer because the cancer carries human genetic information. In T-ALL research, either human T-ALL cell lines or patient-derived primary specimens are introduced into immunocompromised mice (Fig. 1). Although both subcutaneous and intravenous injections of human T-ALL cells are used in setting up xenograft model, tail vein injection is more widely applied in order to mimic the human disease development in the circulatory system.
Non-obese diabetic, severe combined immunodeficiency, interleukin (IL)-2 receptor gamma-deficient mice (NSG) mice were generally used to construct T-ALL models. Human T-ALL cell lines are often compatible with either non-obese diabetic mice/SCID or NSG, whereas primary T-ALL is more restricted to engraft in NSG mice. As a result, NSG mice are lack of mature T cells, B cells or functional natural killer cells, and deficient in cytokine signaling, leading to better engraftment of primary human T-ALL than any other published mouse strains. It is notable that establishment of T-ALL xenograft by intravenous injection often requires irradiation (1–2 gray) prior to engraftment. It is possible that human T-ALL cell dissemination in NSG mice are somewhat affected by residual host innate immune system that would be completely abolished by irradiation. While subcutaneous xenograft develops readily detectable tumor mass, T-ALL mice from intravenous injection show characteristic symptoms such as weight loss, a humped back as well as paralysis of the hind legs. Mice with T-ALL usually develop enlarged spleens, with abundant human CD45+ leukemia cells accumulated in spleen, peripheral blood, bone marrow, liver and even brain. In addition, T-ALL cell lines or human primary T-ALL infected with lentiviral particles expressing luciferase can be experimentally manipulated in NSG mice using bioluminescent imaging system, which detects dissemination of malignant cells expressing bioluminescent or fluorescent tags. Without injury or sacrifice, circulating T-ALL cells as well as a variety of leukemia-associated properties can be visualized dynamically in living mice. In Vivo Imaging System provides a valuable tool to examine disease initiation, progression, and maintenance as well as therapeutic response or resistance.
Xenograft model is of great importance in T-ALL research. First, T-ALL is a hematological malignancy with complicated heterogeneity; no animal models other than xenograft mimic the genuine human disease and they are frequently used in functional studies. For instance, human T-ALL cell lines expressing specific shRNA targeting DEPTOR or JMJD3 were engrafted into NSG (or equivalent immunocompromised) mice and showed a delayed leukemia onset and improved animal survival.[27,28] These experiments clearly manifest the pro-leukemogenic roles of DEPTOR and JMJD3 in T-ALL. In addition, patient-derived xenograft (PDX) is of great help in pre-clinical studies, testing potential novel therapeutic agents or drug combination. AKT inhibitors were shown to reverse glucocorticoid resistance in primary T-ALL engrafted into NSG mice, advancing clinical trials of AKT inhibitor and glucocorticoid combination in resistant patients. Second, establishment of xenograft requires no understanding of precise molecular mechanism underlying T-ALL. For any T-ALL subtype facing clinical challenges, the original patient samples can be analyzed in xenograft model for disease characterization and pre-clinical evaluation of potential therapeutics. Early thymocyte progenitor-ALL (ETP-ALL) is an aggressive subtype of acute lymphoblastic leukemia distinguished by stem-cell-associated and myeloid transcriptional programs. The optimal therapeutic approaches to patients with ETP-ALL are poorly characterized. Maude et al applied 6 patient-derived murine ETP-ALL models and showed the pre-clinical in vivo efficacy of JAK1/2 inhibitor ruxolitinib in treating this disease. Despite tremendous importance in T-ALL research, limitations of xenograft models do exist as the immunosuppressive mice represent a microenvironment that is distinct from human patients who oftentimes retain somewhat active immune activity. To overcome this, normal immune cells are usually infused into mice for pre-clinical immunotherapy studies such as those using programmed death 1 (PD-1) antibody.
Genetic murine model
Genetic T-ALL mice models require in-depth understanding of the molecular mechanism underlying T-cell transformation. Successful establishment of these models is attributable to gene expression profiling, sequencing analysis and vigorous functionality studies. In many cases, genetic murine models manifest human T-ALL symptoms including splenomegaly, leukemia cell accumulation in thymus, lymph node, bone marrow, spleen, and infiltration in liver and brain. Notably, these models develop frank leukemia resulting from genetic abnormalities detected in human patients, thus recapitulating disease onset and progression and providing a great resource for pre-leukemia cell and leukemia stem cell research.
Ras transgenic mice
10% Ras activating mutations were found in T-ALL, suggesting the significant contribution to the aggressive phenotype.[16,17] It becomes evident that endogenous oncogenic Ras is sufficient to initiate transformation by stimulating cell proliferation.[33,34] To evaluate the role of activating K-ras in T-ALL, K-rasG12D allele was knocked into the locus of endogenous K-ras under the control of a floxed stop cassette. The floxed stop cassette can be removed by the presence of the Cre recombinase, which leads to the conditional expression of active K-ras mutation. Heterozygous K-rasG12D mice were crossed with homozygous mice that express Cre recombinant enzyme under the control of Lck promoter to generate offsprings expressing K-rasG12D in T-cell lineage (Fig. 2A). These mice developed T-ALL spontaneously in an average of approximately 180 days after birth, and half of the mice acquired NOTCH1 gain-of-function mutations during the progression of the disease. NOTCH1 mutations identified from human patients collaborated with K-ras activation, accelerated T-ALL onset and decreased animal survival rate. The resulting tumors show “addiction” to NOTCH, providing a further rationale for evaluating NOTCH signaling pathway inhibitors in leukemia. When abrogating NOTCH signaling by expressing dominant-negative MAML (DNMAML) in K-rasG12D mouse model, these mice ultimately developed leukemia; the tumor cells acquired NOTCH1 mutations and subsequently deleted DNMAML. These data reinforce the notion that activated NOTCH1 is a primary and specific driving force in T-cell transformation. Intriguingly, hematopoietic stem cells (HSC) from K-rasG12D mice transduced with MYC, but not myristoylated AKT, developed T-ALL that were γ-secretase inhibitor (GSI)-insensitive and lack of NOTCH1 mutations. These data suggest that MYC expression is of predominant importance downstream of NOTCH1, which switches off the selective pressure for NOTCH1 mutations in K-rasG12D-driven T-ALL when overexpressed.
PTEN null mice
As a notable tumor-suppressor with 10% to 15% loss-of-function mutation in T-ALL, PTEN null mice have been used as an important in vivo model in T-ALL studies.[37,38] PTEN is a phosphatase which can dephosphorylates phosphatidylinositol-3,4,5- trisphosphate (PIP3), a phospholipid substrate of phosphoinositide 3-kinase (PI3K). Inactivation of PTEN accelerates the accumulation of PIP3, and as a consequence, leads to the activation of (PI3K)/AKT pathway, which promotes cellular growth and cell proliferation and contributes to tumorigenesis.
The tumor suppressive role of PTEN in T-ALL was confirmed in PTEN null mice. Guo et al showed that VE-cadherin-cre mediated PTEN deletion in 40% of fetal liver HSCs and their differentiated progeny led to a myeloproliferative disorder, followed by T-ALL. During leukemogenesis, PTEN loss promotes the formation and expansion of leukemia stem cell population through enhancing β-catenin and MYC expression. Similarly, vav-iCre mediated PTEN deletion during early fetal hematopoietic development leads to T-ALL in adult mice with a high penetration/prevalence, thus considered as an excellent murine model. More recently, Mirantes et al showed that conditional deletion of PTEN in CD45-expressing cells exclusively induced the development of T-cell lymphoblastic lymphoma but not myeloid malignancies, despite that CD45, known as a common leukocyte antigen, is expressed in virtually all white cells and in hematopoietic stem cells. Taken together, these findings highlight the prominent and preferential role of PTEN in suppressing T-cell transformation.
Constitutive activation of PI3K/AKT, resulting from PTEN loss, enables tumor cell proliferation, survival and even drug resistance. In the tamoxifen-inducible PTEN knockout background, NOTCH1 collaborates with PTEN loss and accelerates T-ALL. Notably, PI3K/AKT activation resulting from PTEN loss is a prominent cause conferring resistance to glucocorticoid therapy. AKT directly phosphorylates glucocorticoid receptor NR3C1 and blocks its nuclear translocation, thus inactivating NR3C1 transcriptional program and resulting in glucocorticoid resistance. Similarly, NOTCH1-driven T-ALL in PTEN deficiency background showed gamma-secretase inhibitor (GSI) resistance, due to AKT-mediated metabolic reprogramming. Together with other findings, therapeutic targeting of AKT shows great promise in inhibiting leukemia cell growth, eliminating leukemia stem cell or reversing drug resistance.[27,29,43,44]
TAL1 transgenic mice
The TAL1 gene product is a serine phosphoprotein and basic helix-loop-helix transcription factor known to regulate normal and malignant hematopoiesis. TAL1 abnormality was originally identified in a human T-ALL case bearing t(1;14) (p33;q11) translocations resulting in constitutive expression. It becomes more evident that TAL1 activation occurs in 60% of childhood and adult T-ALL cases, whereby mechanisms involve not only chromosome translocation/deletion but also micro-insertions in regulatory region, non-coding RNA-mediated regulation and association with various transcriptional complex. Two laboratories independently demonstrated that TAL1 is sufficient to induce T-cell malignant tumors using a transgenic mouse model inserting a human TAL1 cDNA downstream of the Lck promoter,[46,47] verifying its pathogenic role in human T-ALL (Fig. 2B). To determine the in-depth mechanism(s) of TAL1-induced leukemogenesis, transgenic mice were generated expressing a DNA-binding mutant of TAL1[48,49] and surprisingly revealed unaffected leukemogenesis, raising an interesting notion that TAL1 has a DNA-binding independent function in promoting T-ALL. TAL1 forms a heterodimer with class I bHLH factor E-proteins, LMO2 and LDB1. In these binding partners, E-proteins serve as tumor suppressors in the context of T-ALL. By sequestering E-proteins and thus preventing formation of E-protein complexes, transgenic expression of TAL1 in an E2A or HEB heterozygous background accelerated the disease onset, indicating that suppression of E-protein activity is a predominant mechanism in TAL1-mediated T-cell leukemogenesis. By contrast, TAL1 and LMO1 or LMO2 are commonly co-activated in human T-ALL and, in collaboration with LMO2, TAL1 expression induces more aggressive T-cell malignancies. This TAL1/LMO2 co-expression model was more frequently used in the follow-up studies. It has been demonstrated that maintenance of leukemia-initiating cells (LIC) in this co-expression mouse model relies on NOTCH1 activity and γ-secretase inhibitor is capable of eradicating LIC population. Using TAL1/LMO2 transgenic model, the Kelliher lab recently demonstrated RUNX1 as a collaborating oncoprotein in T-cell leukemia through coordinating TAL1 or NOTCH1 for oncogenic MYB or MYC superenhancer activities. These results support the core transcriptional regulatory networks constructed by TAL1 with other transcriptional factors including LMO2, GATA2, and RUNX1, thus forming an auto-regulatory loop in T-ALL.
NOTCH transgenic mice
NOTCH proteins are ligand-activated transmembrane receptors involved in cell development. NOTCH receptors are cleaved into extra- and intracellular portions when activated by transmembrane ligands from neighbor cells. The NOTCH intracellular domain (ICN) translocates to the nucleus, where it combines with additional nuclear proteins to regulate the transcriptional activation of target genes. NOTCH1 was first identified in human T-cell ALL bearing t(7;9)(q34;q34.3) chromosome translocation, resulting in a fusion gene in which the T cell receptor β promoter drives expression of the 3′ end of the NOTCH1 locus, resulting in constitutive expression of active NOTCH1. In a subsequent report, 50% to 60% of T-cell ALL samples contained point mutations in NOTCH1, implicating the central role of this gene in the pathogenesis of most subtypes of T-ALL. More evidences show that both NOTCH1 and NOTCH3, a distinct NOTCH isoform whose expression is induced by NOTCH1 transcriptional activity, are oncogenic in T-ALL. Given the prominent roles of NOTCH in T-ALL, the Screpanti lab generated Lck promoter-driven ICN3 transgenic mice, and demonstrated activated NOTCH3 leads to nuclear factor kappa-light-chain-enhancer of activated B cells activation and T-cell tumorigenesis. Similarly, a NOTCH1(ic) transgenic mice was generated by overexpressing the intracellular domain of NOTCH1, including the RBP-Jκ–associated module domain, ankyrin repeat, and C-terminal transcriptional activation domain but lacking the C-terminal rich in proline(P), glutamic acid(E), serine(S) and the threonine(T) domain. Based on a Dox-inducible NOTCH1(ic) strain, Sharma et al demonstrate that NOTCH1 contributes to mouse T-cell leukemogenesis through directly inducing the expression of MYC. A more recent report described novel bacterial artificial chromosome (BAC) NOTCH1 transgenic mouse strains that have Cre-inducible expression of the entire human NOTCH1 locus. The NOTCH1 transgene expressing gain-of-function mutants significantly decreases the latency of a mutagenesis system-based spontaneous T-cell leukemia model. In a tetracycline-inducible NOTCH1 mouse model (Top-NOTCH(ic)), NOTCH1 was shown repressing the ARF-mdm2-p53 tumor surveillance network and acute induction of p53 expression induced apoptosis and tumor regression. Allograft of bone marrow cells derived from the Top-NOTCH(ic) transgenic mouse shows that NOTCH(ic) is able to maintain T-ALL tumors even when endogenous MYC expression is extinguished. However, MYC is incapable of maintaining these tumors in the absence of NOTCH(ic), underscoring the prominent role of NOTCH1 but not MYC in NOTCH1-derived T-ALL. In addition, deletion of floxed NOTCH1 promoter/exon 1 sequences, resulting in expression of γ-secretase-cleaved intracellular NOTCH1 proteins, significantly accelerates leukemogenesis in Ikaros-deficient mice. These results suggest that intracellular NOTCH1 or NOTCH3 are potent oncogenic drivers in T-ALL and, very likely, they collaborate with many other leukemia-promoting factors in vivo, conferring neoplastic phenotype.
Genetic models provide a more simplified background for functional study such that critical role of candidates may be revealed otherwise buried in complicated, heterogenous patient sample analysis. On the other hand, limitations exist that too simplified model may not truly represent human diseases because of inter- and intra-tumor heterogeneity. As such, human xenografts, in particular, PDX models are often used for confirmative studies following experiments in genetic models. Furthermore, these NOTCH1 transgenic models are more replaced by ICN1 expressing bone marrow transplant models, which not only manifest 100% T-cell leukemia phenotype sufficient for functional studies but also are less expensive in comparison to maintaining transgenic strains.
Retroviral transduction and hematopoietic stem cell transplant model
Although the transgenic mouse model has a significant contribution to our understanding of T-ALL, these models have obvious shortcomings. It takes enormous time and material to construct plasmids, to screen and generate mice and to maintain transgenic strains. Another convenient approach is to obtain hematopoietic stem cells from mice, manipulate them in vitro through retro- or lentiviral transduction, and transplant them into isogenic recipients (Fig. 3). Murine stem cell virus (MSCV) is the most commonly used replication defective retrovirus in the transplant leukemia model. This rapid and efficient system has been more commonly used in T-ALL studies to supplement research with transgenic animals.
Aberrant NOTCH1 activation was initially identified in rare T-ALLs harboring chromosomal translocation, leading to the expression of a truncated and constitutively active form of NOTCH1. These findings lead to the first NOTCH1-induced transplant model. Retroviruses expressing a constitutively active intracellular form of NOTCH1 were transduced into normal hematopoietic progenitors. These cells were subsequently injected into lethally irradiated mice and all the recipients uniformly develop T-cell leukemia with symptoms resembling human T-ALL. In fact, NOTCH1 is not activated by chromosomal translocations in most T-ALLs, but occurs as a result of activating mutations enhancing NOTCH1 activity. To more faithfully mimic human disease, various NOTCH1 mutants found in human patients were used to infect murine hematopoietic progentiors and monitor leukemogenesis. Only strong NOTCH1 mutant alleles, which bear both HD and rich in proline(P), glutamic acid(E), serine(S) and the threonine(T) domain mutations, are sufficient to induce T-ALL.
Based on the NOTCH1-induced T-ALL model, numerous novel candidates involved in T-cell leukemogenesis have been examined. An oncogenic role of miR-128-3p in T-ALL was evident by accelerated leukemia onset in a NOTCH1-induced T-ALL model. In the same model, the chemokine receptor C-X-C chemokine receptor type 4 was shown essential for the leukemia-initiating cell activity and migratory properties of T-ALL cells. Ectopic expression of somatic gain-of-function mutations of IL7Rα, in combination with a NOTCH1 mutant leads to the development of much more aggressive T-ALL. Similarly, in NOTCH1-driven T-ALL model based on fetal liver hepatic progenitor cell transplantation, microRNA-193b-3p was demonstrated as a tumor suppressor by targeting MYB during malignant T-cell transformation. Loss of ubiquitously transcribed tetratricopeptide repeat, X chromosome accelerates leukemia development in the NOTCH1-induced T-ALL mice, demonstrating this H3K27me3 demethylase as a bona fide tumor suppressor in T-ALL.
The allograft model of T-ALL is also known as secondary transplant; leukemic cells from donor mice are transplanted into wild-type isogenic recipient mice with relative normal host immune system. This model has an advantage over xenograft on maintaining some condition of tumor microenvironment, thus providing an excellent tool in elucidating the underlying mechanism of disease development. Transplantation of primary NOTCH1-ΔE or K-rasG12D leukemia cells at a limiting dilution into secondary recipients revealed enriched leukemia initiating cell (LIC) activity within the CD44+ROSlow fraction due to downregulation of protein kinase C θ (PKC-θ). In addition, this model provides a great resource for pre-clinical assessment of potential anti-T-ALL drugs. In a recent study, HSP90 inhibitor was shown to impede T-ALL leukemia progression in a murine allograft model derived from T-ALL cells in K-rasG12D/Notch1L1601P mice. These findings strongly suggest administration of Hsp90 inhibitors as a potential therapeutic regimen for NOTCH1-addicted T-ALL. Interestingly, NOTCH1-induced T-ALL transplant model was also applied to address the impact of a cancer environment on normal tissue stem cells. Hu et al showed that normal hematopoiesis was suppressed during leukemia development, and suggested reversible inhibition of normal hematopoietic stem cells in a leukemic environment.
Retroviral transduction and hematopoietic stem cell transplant model has been intensively applied as an economical and time-saving approach for T-ALL models, mimicking the initiation and progression of human leukemia. However, considering the incidence of leukemogenesis caused by excess retrovirus of even MSCV empty vector in some cases, the magnitude of virus used in transduction should be carefully calculated. Moreover, random insertion mutagenesis and diverse promoter used in retrovirus vector leading to variation in leukemogenesis should be taken into account. Nevertheless, retroviral transduction/transplantation mouse model has been developed as the most prevailing in vivo approach used in modeling T-ALL.
Zebrafish (Danio rerio) is a tropical freshwater fish with unique characteristics, including high fecundity, rapid development, and transparency. As a vertebrate organism with conserved hematopoietic program, zebrafish has unique experimental strengths for investigation of human leukemia.[75–77] A high degree of genetic and morphological similarity in hematopoiesis between zebrafish and human indicates that zebrafish can provide valuable information about the mechanisms underlying pathogenesis of leukemia. These characters fuel the development of zebrafish models of T-ALL to elucidate the molecular pathogenesis and expedite the pre-clinical investigation of novel therapies.
In the past decade, researchers have developed and optimized multiple methodologies for zebrafish T-ALL model. The first transgenic zebrafish line was created by expressing mouse MYC fused with enhanced green fluorescence protein (EGFP) which was placed under the control of zebrafish lymphatic specific rag2 promoter (Fig. 4A). Enforced MYC expression induces T-ALL in about 7 weeks, with enhanced green fluorescent protein (EGFP+) cells initially in the thymus, then followed by these cells spreading rapidly to skeletal musculature, visceral organs, kidney, and bone marrow, ultimately leading to widely disseminated T-ALL. Gene expression analysis confirmed that the outcome resulted from accelerated expansion of pre-T-lymphoblasts. However, high mortality rates resulting from rapid disease onset preclude their fecundity. To overcome this, conditional transgenic approaches were used to establish stable zebrafish leukemia model. Langenau et al developed a Cre/lox-regulated conditional system in which EGFP-mMyc expression is induced upon Cre mRNA injection. This inducible model was further improved by outbreeding it with the transgenic strain in which the expression of Cre recombinase was under the control of heat shock protein 70 (hsp70) promoter, and the incidence of T-ALL progressed to 81% upon the heat-shock treatment (3 days post-fertilization for 45 minutes at 37°C) (Fig. 4B). In this model, co-expression of Bcl2 accelerated the occurrence of T-lymphoblastic lymphoma but not T-ALL.[82,83] Another inducible model was developed by fusing the human MYC gene with a modified estrogen receptor such that MYC expression is stimulated by 4-hydroxytamoxifen (4-OHT). As a result, 4-OHT efficiently induced T-ALL development and withdrawal of 4-OHT led to tumor regression. In this rag2:MYC-ER transgenic fish, Gutierrez et al demonstrate that MYC activates the expression of AKT by inhibiting the tumor suppressor PTEN, leading to cancer cell proliferation. A spectrum of following up studies has confirmed the importance of AKT pathway in tumorigenesis, which indicates a new therapeutic strategy for T-ALL. In addition to MYC transgenic model, overexpression of intracellular NOTCH1 under the control of the zebrafish rag2 promoter also results in progression to a T-cell lymphoproliferative disease in about 5 months. Co-expression of ICN1 with Bcl2 or MYC dramatically accelerated leukemogenesis.[85,86] Moreover, NOTCH1 was found to induce a significant expansion of pre-leukemic clones and activation of the AKT pathway was acquired in a subset of the clones. These studies confirm the striking genetic similarity of T-ALL between zebrafish and mammal in aspects of morphology and genetics, further validating zebrafish as an excellent resource for T-ALL research.
In view of the large clutches of offspring and the low maintenance costs, zebrafish models are greatly useful for large-scale genetic or chemical screens. Using transgenic fish with T-lymphocyte-specific expression of EGFP, Frazer et al pioneered a phenotype-driven forward-genetic screen in zebrafish, performed chemical mutagenesis and identified multiple lines with a heritable predisposition to T-cell malignancy. Multiple zebrafish mutants were revealed to recapitulate human T-cell neoplasia, which represent important candidates involved in the disease progression. Through high-throughput drug screening, an Food and Drug Administration-approved antipsychotic drug perphenazine was identified as a promising anti-T-ALL agent that synergizes with NOTCH inhibitors. The in-depth mechanism study revealed that perphenazine induced activation of the tumor suppressor protein phosphatase 2A. These results provide evidence that activation of protein phosphatase 2A in combination with anti-NOTCH therapeutics may provide an effective T-ALL therapy.
The successful development of zebrafish T-ALL model has helped to overcome initial skepticism concerning their utility in cancer research. To date, investigators have generated zebrafish models of T-ALL that can be experimentally manipulated to elucidate both the molecular and genetic mechanisms underlying leukemic transformation. The availability of these models has allowed investigators to study the genetic basis of leukemia, from disease initiation to progression and drug resistance. Importantly, zebrafish T-ALL, strikingly similar to those in humans, has provided a unique model system to utilize the imaging and genetic advantages for continued insights into human disease. However, due to their evolutionary distance from humans, results from zebrafish models require further validation in mammalian models, particularly as candidate therapeutics for clinical development. Despite these limitations, maximizing their strengths in combination with the use of mammalian systems, zebrafish models will rapidly expand our understanding of T-ALL pathogenesis and make a significant contribution in leukemia research.
In the past 2 decades, multiple T-ALL animal models have been established based on our understanding of this complicated hematologic malignancy. And in turn, complementary mouse and zebrafish models have helped achieve great success in defining functional roles of novel critical pro- or anti-leukemia genes and gain valuable insights into the biology of T-ALL. Retroviral transduction of nuclear NOTCH1 or NOTCH1 mutants followed by hematopoietic stem cell transplantation, in various genetic backgrounds, has most prominently used in mimicking de novo human T-cell leukemogenesis. A panel of newly identified genes has been shown functionally important in this T-ALL model, whose striking roles are usually further confirmed in PDX T-ALL. Zebrafish models also make significant contributions to functionality experiments in tumorigenesis and treatment resistance. In terms of translational studies, zebrafish models are effective and convenient for screening novel agents for experimental therapeutics. When testing compounds or combinatorial therapy regimes, PDX may offer the best option due to the unique advantage of being able to recapitulate characteristics of human T-ALL and their clinical response to therapy. With these powerful tools, pre-clinical studies using complementary models hold enormous promise in translating findings in basic research into clinic. T-ALL animal models, combined with patient-derived clinical information and large-scale genomic data analysis, will undoubtedly advance our understanding of the mechanisms underlying T-ALL pathogenesis, drug resistance and disease relapse to improve therapeutic strategies.
We thank Liu Laboratory members for critical reading of the manuscript and helpful comments and suggestions.
QY, HS, and HL drafted the manuscript; JW, JJ, and GQ helped revise the manuscript. QY, HS, and JW drew the figures and HL designed the structure of the manuscript. All authors have read and approved the final manuscript.
This work was supported by grants from the National Natural Science Foundation of China (81470332 and 81770177 to HL), Hubei Provincial Foundation for Outstanding Young Scholars (2017CFA072 to HL), and Fundamental Research Funds for Central Universities (2017JYCXJJ029 to HS).
Conflicts of interest
The authors declare that they have no conflicts of interest.
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