Page 90 - Haematologica Vol. 109 - July 2024

ARTICLE - Targeting TNF/IL-17/MAPK in hE2A-PBX1 zebrafish H. Luo et al.
KJ-Pyr-9, OUL35, and CID44216842 effectively alleviate hE2A-PBX1-induced myeloid expansion in zebrafish
In order to identify small molecule compounds that can target hE2A-PBX1, we screened 560 small molecule inhibi- tors using Tg(hsp70:E2A-PBX1-EGFP) zebrafish. After 3 days of heat shock, embryos of Tg(hsp70:E2A-PBX1-EGFP) were collected and treated with drugs on day 4 (initial screening concentration was 12 μM), and then fixed at 6 dpf to detect the number of SB+ granulocytes (Figure 7A). From the initial screening, we obtained seven small molecule inhibitors that significantly reduced the number of SB+ granulocytes in a concentration gradient of 8 μM, 14 μM, 20 μM and 24 μM (Online Supplementary Table S1). Interestingly, these seven drugs not only include L-arginine hydrochloride targeting inflammatory pathways, A-484954 and GLPG1837 target- ing autophagy pathways, LRRK2-IN-1 targeting apoptosis pathways, but also include CID4421684, OUL35 and KJ- Pyr-9 targeting TNF/IL-17/MAPK, which is consistent with our previous finding that hE2A-PBX1 can activate the TNF/ IL-17/MAPK signaling pathway in zebrafish (Figure 6D). In order to compare the effects of these three drugs with the existing chemotherapeutic drug Ara-C (cytarabine), we treated hE2A-PBX1 zebrafish with 6.17 mM Ara-C, 8 μM KJ-Pyr-9, 20 μM CID44216842 and 24 μM of OUL35, respectively. The results showed that all three inhibitors reduced the number of SB+ granulocytes to the same ex-
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tent as Ara-C (Figure 7B, B’), but OUL35 and CID44216842 exhibited a lower embryo deformity rate (Online Supple- mentary Figure S7A). Consistently, intraperitoneal injection of adult fish with cytarabine (8,000 mg/kg), CID44216842 (125 mg/kg), KJ-Pyr-9 (90 mg/kg), and OUL35 (150 mg/kg) for 5 consecutive days significantly reduced the proportion of myeloid cells in the KM (Online Supplementary Figure S7B, B’). Additionally, KJ-Pyr-9, OUL35, and CID44216842 also lowered the blast percentage in the KM. These find- ings indicate that KJ-Pyr-9, OUL35, and CID44216842 can alleviate the myeloid expansion in hE2A-PBX1 fish. Further, by long-term drug treatment (administration for 7 consec- utive days) of hE2A-PBX1 zebrafish at 5 dpf, we found that cytarabine, CID44216842 and OUL35 could significantly improve the survival rate of hE2A-PBX1 zebrafish (Online Supplementary Figure S7C). KJ-Pyr-9 did not improve the death of hE2A-PBX1 zebrafish, which may be due to other side effects caused by long-term treatment of KJ-Pyr-9. It has been reported that TNF42-44 and IL-1745-47 can activate the MAPK pathway. In order to investigate whether TNF/IL-17 can similarly activate the MAPK signaling in Tg(hsp70:E2A- PBX1-EGFP), we treated 4 dpf hE2A-PBX1 embryos with TNF-α inhibitors pomalidomide and lenalidomide, as well as IL-17 inhibitor Y-320. After 48 hours, we examined the activation of the MAPK pathway, expression of downstream target genes, and changes in SB+ myeloid cell numbers.
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