Page 79 - Haematologica Vol. 109 - July 2024

ARTICLE - Targeting TNF/IL-17/MAPK in hE2A-PBX1 zebrafish H. Luo et al.
specificity and affinity.13 PBX1 not only plays an important role in regulating morphologic patterning, organogenesis, and hematopoiesis, but is also an important component of the protein complex that regulates the expression of developmental genes.14
The oncogenic role of E2A-PBX1 has been reported in both the myeloid and lymphoid systems of mice and cell lines. For example, the transduction of E2A-Pbx1 into mouse myeloid progenitor cells via retroviral vectors re- sults in AML in mice.14,15 Adding granulocyte-macrophage colony-stimulating factor (GM-CSF), E2A-Pbx1 can also immortalize myeloid progenitor cells in vitro.16 On the other hand, the specific induction of E2A-PBX1 expression in the mouse lymphoid system leads to T/B-ALL,17-19 and the injection of E2A-PBX1 pro-T cells into mice leads to T-ALL and AML.20 Although the oncogenic potential of E2A-PBX1 is clear, its pathogenic mechanism has not been fully elucidated (especially in leukemias other than ALL). Currently, there are three main explanations for the mechanism underlying E2A-PBX1-induced leukemia: i) E2A-PBX1 oncoprotein may deregulate the expression of critical genes normally controlled by PBX/HOX/MEINOX complexes; ii) it alters the function of transcriptional co-activators; and iii) it impairs the function of wild- type (WT) E2A.21 Recently, a study using two pre-B ALL cell lines found that E2A-PBX1 can induce pre-B ALL by directly interacting with RUNX1 and co-activating the expression of oncogenes, including RUNX1.1 This study strongly supports the gain-of-function oncogenic role of E2A-PBX1.1 However, the mechanism by which E2A-PBX1 mediates AML oncogenesis is still unclear. More impor- tantly, although studies have reported the development of potential drugs for E2A-PBX1 leukemia,22,23 clinically effective targeting drugs for E2A-PBX1 leukemia are still lacking.
In order to better understand the mechanisms behind E2A-PBX1-induced leukemia and develop effective target- ed therapies, we constructed an inducible human-derived E2A-PBX1 leukemia model using zebrafish. In zebrafish, just like in mammals, definitive hematopoietic stem cells (HSC) arise from the ventral wall of the dorsal aorta (VDA), which is equivalent to the aorta-gonad-meso- nephros (AGM) in mammals.24 These HSC then migrate into the caudal hematopoietic tissue (CHT), where they expand and subsequently colonize the kidney marrow, equivalent to the mammalian bone marrow, to generate hematopoietic cells throughout adulthood.24 Zebrafish have proven to be an excellent model for studying hema- topoietic disorders, particularly in extensive therapeutic compound screenings, due to their transparency, small size, short lifespan (2-3 years), high fecundity and genetic similarity to humans.25 In recent years, the successful establishment of zebrafish patient-derived xenograft (zPDX) models has further advanced the use of zebrafish in biomedical research applications.26,27
In our research, this hE2A-PBX1 transgenic zebrafish ex- hibited a phenotype of aberrant myeloid expansion and lymphoid dysplasia in larvae and eventually progressed to AML-like disease with age. Further mechanistic analysis revealed that hE2A-PBX1 activated the TNF/IL-17/MAPK signaling pathway in blood cells and induced myeloid hyperplasia by upregulating runx1 expression. Impor- tantly, through drug screening, we found that the small molecule compounds OUL35, KJ-Pyr-9 and CID44216842, which target the TNF/IL-17/MAPK signaling pathway, not only significantly alleviated the AML-like phenotype in the hE2A-PBX1 zebrafish model, but also blocked the oncogenicity of human E2A-PBX1 pre-B cells. In conclu- sion, our study provides insights into the mechanisms by which E2A-PBX1 causes AML-like disease and identifies several promising compounds for targeted therapy.
Methods
Generation of pT2AL-hsp70-hE2A-PBX1 construct and of Tg(hsp70:E2A-PBX1-EGFP) transgenic zebrafish
The pT2AL-hsp70-E2A-PBX1-EGFP plasmid is composed of hsp70 promoter, a hE2A-PBX1 cDNA fragment obtained via polymerase chain reaction (PCR) from human sam- ples, the Tol2 element and the SV40 poly A sequence. The E2A and PBX1 cDNA fragments were linked together through overlap PCR. Following AgeI/SphI digestion, the hE2A-PBX1 sequence was inserted downstream of hsp70 promoter and subcloned into the pT2AL plasmid. This construct, named pT2AL-hsp70-hE2A-PBX1-EGFP, was used for microinjection into zebrafish embryos at the one-cell stage along with Tol2 transposon mRNA to gen- erate the Tg(hsp70:E2A-PBX1-EGFP) transgenic zebrafish.
Immunohistochemical staining
Abdominal tissue (above the pelvic fin) from euthanized adult fish was fixed in 4% paraformaldehyde (PFA) and subsequently processed into frozen sections,28 permea- bilized with 0.3% triton-X-100, and blocked with 5% fetal bovine serum. The sections were then incubated with rabbit-anti-Lcp1 monoclonal antibody (1:200),29 followed by Alexa Fluor 488-anti-rabbit antibody (Invitrogen; 1:400) and DAPI (Invitrogen) for fluorescent visualization.
Bromodeoxyuridine labeling
Sibling and Tg(hsp70:E2A-PBX1-EGFP) embryos at 5-6 days post-fertilization (dpf) were incubated in 10 mM bromodeoxyuridine (BrdU) (Sigma-Aldrich) for 4 hours. The embryos were then stained with goat-anti-DsRed (Abcam; 1:400) and rabbit-anti-Lcp1 monoclonal antibody (1:200), followed by incubation with Alexa Fluor 488-anti-goat an- tibody (Invitrogen; 1:400) and Alexa Fluor 488-anti-rabbit antibody (Invitrogen; 1:400) for fluorescent visualization, according to the provided instructions.30
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