Page 91 - Haematologica Vol. 109 - July 2024

ARTICLE - Targeting TNF/IL-17/MAPK in hE2A-PBX1 zebrafish C
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Figure 7. KJ-Pyr-9, OUL35, and CID44216842 effectively alleviate hE2A-PBX1-induced myeloid expansion in zebrafish larvae. (A) Flow- chart illustrating the high-throughput drug screening process conducted in this study. Briefly, embryos were collected and subjected to a 1-hour heat shock at 12 hours post-fertilization (hpf), followed by twice daily 2-hour heat shocks from 1 to 3 days post-fertilization (dpf). Subsequently, zebrafish larvae were soaked in a solution containing small molecule compounds (initial screening concentration was 12 μM), the number of neutrophils was quantified using Sudan Black B (SB) staining at 6 dpf. (B) SB staining of sibling (left panel) and Tg(hsp70:E2A-PBX1-EGFP) (right panel) larvae after treatment with dimethyl sulfoxide (DMSO), 6.17 mM Ara-C (cytarabine), 8 μM KJ-Pyr-9, 20 μM CID44216842 and 24 μM of OUL35 at 6 dpf. (B’) Statistical analysis of the SB+ signals shown in panel (B). The black asterisks indicate statistical difference (N≥18, one-way ANOVA, mean ± standard error of the mean [SEM]; *P<0.05, ***P<0.001). (C) SB staining of hE2A-PBX1 zebrafish larvae after inhibitors treatment (DMSO, 500 μM pomalidomide, 500 μM lenalidomide, and 0.75 μM Y-320 inhibitor, respectively). The caudal hematopoietic tissue (CHT) is enlarged in the red box (original magnification ×200). (C’) Sta- tistical analysis of the SB+ signals in panel (C). The black asterisks indicate statistical difference (N≥18, one-way ANOVA, mean ± SEM; **P<0.01, ****P<0.0001). (D) Real time quantitative polymerase chain reaction (RT-qPCR) analysis showing mRNA expression of mmp13, fosab, hsp27, junb, mmp9, and fosl1a in Tg(hsp70:E2A-PBX1-EGFP) compared to siblings after inhibitor treatment (one-way ANOVA, mean ± SEM; *P<0.05, ***P<0.001, ****P<0.0001). (E) Schematic representation of inhibitors targeting TNF/IL-17/MAPK signaling pathway. TNF: tumor necrosis factor; IL-17: interleukin-17; MAPK: mitogen-activated protein kinase. The AP-1 (activator protein 1) transcription factor is a dimeric complex that contains members of the JUN, FOS, ATF and MAF protein families.
The results showed that TNF and IL-17 inhibitors did not activate phosphorylation levels of Erk, Akt, p-38, and Jnk in the MAPK pathway (Online Supplementary Figure S7D, D’). However, they did decrease the expression of the target gene
mmp13, fosab, mmp9 and fosl1a, and finally alleviate myeloid cell proliferation in hE2A-PBX1 zebrafish (Figure 7C, D). These data suggest that TNF and IL-17 regulate myeloid development by upregulating the expression of target genes, not through
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