Page 93 - Haematologica Vol. 109 - July 2024

ARTICLE - Targeting TNF/IL-17/MAPK in hE2A-PBX1 zebrafish H. Luo et al.
Figure 8. KJ-Pyr-9, OUL35 and CID44216842 effectively inhibit the oncogenicity of human E2A-PBX1-associated pre-B-lineage acute lymphoblastic leukemia. (A) Schematic representation of xenograft development using human B-lineage acute lympho- blastic leukemia (B-ALL) E2A-PBX1(+) RCH-ACV cells in zebrafish larvae. Briefly, embryos were injected with 600 fluorescently labeled RCH-ACV cells into the posterior cardinal vein (PCV) or dorsal aorta (DA) at 3 days post-fertilization (dpf), followed by treatment with small molecules at 24 hours post-injection (hpi). The pre-B ALL cells injected in runx1w84x embryos were monitored by imaging at 12 hpi, 24 hpi, 72 hpi and 96 hpi. (B) RCH-ACV cells were calculated after xenografting into runx1w84x zebrafish at 12 hpi, 24 hpi, 72 hpi and 96 hpi. (B’) Statistical analysis of the RCH-ACV cell number after injecting in panel (B). The black asterisks indicate statistical difference (N≥11, one-way ANOVA, mean ± standard error of the mean; **P<0.01, ****P<0.0001). (C) RCH-ACV cells were calculated after xenografting into runx1w84x zebrafish, followed by treatment with dimethyl sulfoxide (DMSO), 6.17 mM Ara-C (cytarabine), 8 μM KJ-Pyr-9, 20 μM CID44216842 and 24 μM OUL35 at 96 hpi. (C’) Statistical analysis of the RCH-ACV cell number with drug treatment after injecting in panel (C). The black asterisks indicate statistical difference (N≥10, one-way ANOVA, mean ± standard error of the mean; ***P<0.001, ****P<0.0001).
the activation of the MAPK signaling pathway (Figure 7E).
KJ-Pyr-9, OUL35 and CID44216842 effectively inhibit the oncogenicity of human E2A-PBX1-associated pre-B lineage acute lymphoblastic leukemia
Considering that hE2A-PBX1 can promote pre-B ALL by upregulating RUNX1 expression, we further verified whether KJ-Pyr-9, OUL35, and CID44216842 could also alleviate hu- man pre-B-ALL with E2A-PBX1. We treated RCH-ACV cells with cytarabine, KJ-Pyr-9, OUL35, and CID44216842 for 60 hours, followed by assessing cell proliferation using the CCK- 8 assay and apoptosis with the TUNEL assay. The results demonstrated that cytarabine, as well as these three small molecule compounds, significantly inhibited the proliferation of RCH-ACV cells and increased apoptosis in these cells (Online Supplementary Figure S8A, B). This suggests that the E2A-PBX1+ B-ALL cell line (RCH-ACV) is sensitive to these three small molecule inhibitors. We stained RCH-ACV with the lipophilic dye Dil and microinjected it at a concentration of 600 cells per embryo into posterior cardinal vein (PCV) or DA sites in immunodeficient runx1w84x fish at 3 dpf (Figure 8A). Drug treatment was performed on the day after injection (24 hpi), and the number of leukemia cells was counted at 12-96 hpi. Our findings revealed that DMSO-treated ze- brafish showed rapid expansion of RCH-ACV cells at 24-96 hpi (Figure 8B), whereas treatment with OUL35, KJ-Pyr-9, and CID44216842 inhibited the high-speed expansion of RCH-ACV cells (Figure 8C). These data suggest that KJ- Pyr-9, OUL35 and CID44216842 can effectively inhibit the oncogenicity of human E2A-PBX1-associated pre-B ALL.
Discussion
In summary, we developed a hE2A-PBX1 transgenic zebrafish line that can induce myeloid expansion and eventual pro- gression to AML-like hemogram. We found that hE2A-PBX1 induces upregulation of the runx1-activated TNF/IL-17/MAPK signaling pathway. Through high-throughput drug screening and validation, we identified compounds KJ-Pyr-9, OUL35 and CID44216842 that can alleviate hE2A-PBX1-induced AML-like disease in zebrafish as well as the human pre-B ALL. Our study highlights the potential of these compounds as targeted
drugs for the future clinical treatment of E2A-PBX1 leukemia. Clinically, hE2A-PBX1 primarily induces ALL, but in our hE2A-PBX1 zebrafish model, it causes myeloid leukemia. There are two possible explanations for this: i) impaired E2A function: E2A plays a critical role in regulating early B-cell development and can enhance B-cell-specific gene transcription even in non-lymphoid cells.1,48 The fusion gene hE2A-PBX1 disrupts the normal structure and function of WT E2A, resulting in E2A dosage and functional defects. This disruption leads to aberrant B-cell development and differentiation. Notably, in our hE2A-PBX1 transgenic fish, the endogenous e2a structure remains intact with normal expression levels, indicating a distinction from human cases. ii) Expression distribution of hE2A-PBX1 in different blood lineages: in patients, the expression of hE2A-PBX1 is controlled by the endogenous E2A promoter, implying that lymphocytes may exhibit higher expression levels of hE2A-PBX1 compared to other blood cells. The lymphoid lineage, with relatively higher expression of hE2A-PBX1, might be more prone to leukemic transformation. In our transgenic fish, hE2A-PBX1 was expressed at the initial stage of hematopoiesis (HSPC cells) through heat shock induction. This led to elevated expression of Runx1 in HSPC, ultimately allowing HSPC to acquire a propensity for myeloid differentiation and the potential for myeloid expansion. It is important to note that the induction of both lymphoid and myeloid leukemogenesis by hE2A-PBX1 is strongly correlated with heightened Runx1 activity, as supported by our findings and previous studies.1,49 Inhibiting the ab- normal activation of RUNX1 and its downstream pathways shows promise as a potential shared therapeutic strategy for treating E2A-PBX1 multitype leukemia.
Based on previous structural and molecular functional studies, there are three main explanations for the potential mechanisms underlying hE2A-PBX1-induced pre-B leuke- mia:21 i) The fusion gene hE2A-PBX1 impairs the function of WT E2A; ii) hE2A-PBX1 oncoprotein may deregulate the expression of critical genes normally controlled by PBX/ HOX/MEINOX complexes; and iii) hE2A-PBX1 alters the function of transcriptional co-activators, such as histone acetyltransferases p300 and CREB-binding protein (CBP). These three factors may coordinate and regulate the oc- currence of hE2A-PBX1 pre-B leukemia. On the one hand,
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