Page 87 - Haematologica Vol. 109 - July 2024

ARTICLE - Targeting TNF/IL-17/MAPK in hE2A-PBX1 zebrafish H. Luo et al.
leukemia (AML)-like E2A-PBX1 Lyz-DsRed+ cells. N/N, number of zebrafish larvae showing representative phenotype/total num- ber of zebrafish larvae examined. (C) Wright-Giemsa staining of blood cells in recipient KM after AML-like E2A-PBX1 and WT Lyz-DsRed+ transplantation. The red arrows indicate the AML-like myelocytes, the green arrows indicate the blasts and the black arrows indicate lymphocytes in recipients. (C’) Statistical analysis of cell counts in panel (C). The black asterisks indicate statistical difference (one-way ANOVA, mean ± standard error of the mean; *P<0.05, **P<0.01). (D) Survival curves of Tg(hsp70:E2A- PBX1-EGFP) and WT fish up to 90 day after heat shock. The black asterisks indicate statistical difference (log-rank [Mantel-Cox] test; **P<0.01). (E) Imaging of dying Tg(hsp70:E2A-PBX1-EGFP;lyz:DsRed) at 15-month-old after 3-month heat shock, reveals severe myeloid cell invasion.
3-month induction of hE2A-PBX1 (Figure 4B, B’).
In order to clarify the malignancy of myeloid cells in Tg(hsp70:E2A-PBX1-EGFP), we assessed the invasion of myeloid cells in the muscle after 3 month of heat shock in 3-month-old and 1-year-old zebrafish. The results showed a significant invasion of Lcp1+ myeloid cells in adult fish muscle after induction of hE2A-PBX1 expression compared with the control group (Figure 5A; Online Supplementa- ry Figure S5A). Consistent with this, by using transgenic zebrafish, we observed pronounced systemic invasion of Lyz-DsRed+ myeloid cells in 1-year-old fish after 3 months of heat shock (Online Supplementary Figure S5B). We al- so detected substantial Lyz-DsRed+ myeloid cell invasion in the kidneys, liver, and spleen upon dissection of fresh tissues from adult zebrafish (Online Supplementary Figure S5C). In order to further confirm the transplantability of hE2A-PBX1-derived myeloid cells, we transplanted whole KM blood cells from Tg(hsp70:E2A-PBX1-EGFP;lyz:DsRed) zebrafish into immunocompromised adult host foxn1/Casper (Online Supplementary Figure S5D). The results revealed that 2 months after transplantation, compared to Tg(- lyz:DsRed) control donors, hE2A-PBX1-derived AML-like donor cells successfully repopulated lyz-dsred+ cells in the foxn1/Casper recipients (Figure 5B). Blood smears stained with Giemsa also showed a significant increase in myeloid cells and blasts in both peripheral blood (PB) and KM in recipients following AML-like fish KM transplants (Figure 5C, C’). Furthermore, survival analysis indicates that with the prolonged duration of heat shock, there is a gradual increase in mortality among 1-year-old hE2A-PBX1 zebraf- ish, accompanied by more severe systemic granulocytic invasion (Figure 5D, E). Taken together, our findings indicate that the induced expression of hE2A-PBX1 leads to myeloid expansion in adult fish and has the potential to progress to AML-like disease. Additionally, the degree of disease is positively correlated with the expression level of hE2A-PBX1 and the age of the animal.
hE2A-PBX1 induces myeloid expansion and activates TNF/IL-17/MAPK signaling pathway through upregulation of runx1 expression
A recent in vitro study has shown that hE2A-PBX1 can activate oncogenes (including RUNX1), through direct interaction with RUNX1 via PBX1, leading to the devel- opment of pre-B ALL.1 In addition, mutations in RUNX1 have been found to be a frequent cause of AML, T-ALL,
B-ALL, and myelodysplastic syndrome in clinical.41 In order to investigate whether hE2A-PBX1 causes AML-like symptoms are related to its regulation of runx1, we exam- ined the expression level of runx1 in Tg(hsp70:E2A-PBX1- EGFP) after heat shock by WISH and qPCR. The results showed that the expression level of runx1 in transgenic zebrafish at 3 dpf and 5 dpf dpf after heat shock was significantly increased compared to the control group (Figure 6A, A’, B). In order to further investigate the role of Runx1 in hE2A-PBX1-induced myeloid expansion, we crossed Tg(hsp70:E2A-PBX1-EGFP) with the runx1 loss- of-function mutant (runx1w84x) to obtain Tg(hsp70:E2A- PBX1-EGFP); runx1w84x embryos and detected the number of lyz+ granulocytes. The results showed a significant reduction in the number of granulocytes in both the sibling and hE2A-PBX1 transgenic zebrafish after runx1 deletion (Online Supplementary Figure S6A, A’). Addi- tionally, to investigate the dose-dependent relationship between the expression level of runx1 and the number of granulocytes, we used the reported runx1 morpholino (MO) to knock down runx1 gradient (injection concentra- tion: 0.7 mM, 0.5 mM, 0.2 mM) and detected the num- ber of lyz+ granulocytes by WISH. Our results showed that the reduction in granulocytes was similar to that of runx1w84x when injected at a concentration of 0.7 mM (Online Supplementary Figure S6B, B’), while restoring granulocyte numbers to sibling levels when injected at 0.2 mM (Figure 6C, C’), with intermediate levels observed at 0.5 mM (data not shown). These results suggest that hE2A-PBX1 induces myeloid expansion by upregulating Runx1 expression. Further, we found that deletion of one allele of runx1 in hE2A-PBX1 zebrafish (Tg(hsp70:E2A- PBX1; runx1w84x/+) increased survival to a level similar to that of siblings, whereas overexpression (Tg(hsp70:E2A- PBX1-EGFP; hsp70:myc-runx1)) did not further exacerbate mortality in hE2A-PBX1 zebrafish (Online Supplementary Figure S6C). This is possibly because hE2A-PBX1 trans- genic zebrafish already have extremely high levels of runx1 expression, and further elevating runx1 levels may not lead to additional mortality.
In order to further explore the effect of upregulation of runx1 expression on hematopoietic transcription regu- lation in the hE2A-PBX1 transgenic line, we performed RNA sequencing analysis on blood cells of KM from 3-month-old Tg(hsp70:E2A-PBX1-EGFP) after 3-month continuous heat shock. The results showed that the TNF/
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