Targeting TNF/IL-17/MAPK pathway in hE2A-PBX1 leukemia: effects of OUL35, KJ-Pyr-9, and CID44216842

Abstract

t(1;19)(q23;p13) is one of the most common translocation genes in childhood acute lymphoblastic leukemia (ALL) and is also present in acute myeloid leukemia (AML) and mixed-phenotype acute leukemia (MPAL). This translocation results in the formation of the oncogenic E2A-PBX1 fusion protein, which contains a trans-activating domain from E2A and a DNA-binding homologous domain from PBX1. Despite its clear oncogenic potential, the pathogenesis of E2A-PBX1 fusion protein is not fully understood (especially in leukemias other than ALL), and effective targeted clinical therapies have not been developed. To address this, we established a stable and heritable zebrafish line expressing human E2A-PBX1 (hE2A-PBX1) for high-throughput drug screening. Blood phenotype analysis showed that hE2A-PBX1 expression induced myeloid hyperplasia by increasing myeloid differentiation propensity of hematopoietic stem cells (HSPC) and myeloid proliferation in larvae, and progressed to AML in adults. Mechanistic studies revealed that hE2A-PBX1 activated the TNF/IL-17/MAPK signaling pathway in blood cells and induced myeloid hyperplasia by upregulating the expression of runx1. Interestingly, through high-throughput drug screening, three small molecules targeting the TNF/IL-17/MAPK signaling pathway were identified, including OUL35, KJ-Pyr-9, and CID44216842, which not only alleviated the hE2A-PBX1-induced myeloid hyperplasia in zebrafish but also inhibited the growth and oncogenicity of human pre-B ALL cells with E2A-PBX1. Overall, this study provides a novel hE2APBX1 transgenic zebrafish leukemia model and identifies potential targeted therapeutic drugs, which may offer new insights into the treatment of E2A-PBX1 leukemia.

Introduction

t(1;19)(q23;p13) is the most common chromosomal translocation in leukemia, causing 5-7% of childhood ALL^1,2 and also AML/MPAL.^3,4 In ALL, E2A-PBX1 is not a primary ALL prognostic factor, but it is associated with poor response to therapy and short remission.⁵ ALL patients with E2A-PBX1 translocation appear to have a high risk of relapse, and this translocation is also associated with known high-risk clinical features, including elevated white blood cell counts at diagnosis and central nervous system leukemia.^6,7 Furthermore, a previous study has shown that the E2A-PBX1 fusion gene can serve as an effective minimal residual disease (MRD) marker following induction chemotherapy, the relapse rate of patients with rapid early conversion to negative for this gene is relatively low.^5,8 Reports based on sporadic cases have indicated that E2A-PBX1-positive patients also exhibit high-risk clinical features, including high cell counts, high serum lactate dehydrogenase levels, and central nervous system (CNS) involvement.³ Although recent intensified treatment regimens and allogeneic hematopoietic stem cell transplantation have improved the prognosis of adult ALL patients with E2A-PBX1,⁹ its systemic toxicity and high cost should not be overlooked.

The E2A-PBX1 protein resulting from t(1;19)(q23;p13) fusion combines the N-terminal transactivation domain of E2A (also known as TCF3) and the C-terminal DNA-binding homologous domain of PBX1.¹⁰ As a major regulator of B-lymphocyte generation, E2A determines the differentiation of B-cell lineages,¹¹ also plays an important role in maintaining the hematopoietic stem cell pool and promoting the maturation of myelo-lymphoid and myelo-erythroid progenitors.¹² PBX1, a TALE (3-amino-acid loop extension) transcription factor, interacts with other members of the HOX family to enhance their DNA-binding 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 results 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 importantly, 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 targeted 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-mesonephros (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 hematopoietic 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 exhibited 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. Importantly, 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 conclusion, 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 samples, 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 generate 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 permeabilized 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 antibody (Invitrogen; 1:400) and Alexa Fluor 488-anti-rabbit antibody (Invitrogen; 1:400) for fluorescent visualization, according to the provided instructions.³⁰

Figure 1.Generation and characterization of transgenic zebrafish expressing human E2A-PBX1 (hE2A-PBX1). (A) Schematic representation of Tg(hsp70:E2A-PBX1-EGFP) expression vector, and Sanger sequencing confirmed the presence of the hE2A-PBX1 sequence. FP: forward primer; pA: poly A termination sequence. (B) F4 generation hE2A-PBX1-transgenic embryos exhibited EGFP expression at 32 hours post-fertilization (hpf). EGFP^- (sibling) and EGFP⁺ (Tg(hsp70:E2A-PBX1-EGFP)) embryos obtained by crossing the stable line Tg(hsp70:E2A-PBX1-EGFP) with wild-type (WT) fish. Tg(hsp70:E2A-PBX1-EGFP) embryos exhibited strong EGFP fluorescence (white arrows). (C) Specific polymerase chain reaction (PCR) amplification of a 231 bp fragment within the hE2A-PBX1 fusion region confirmed the integration of hE2A-PBX1 cDNA sequence into the genomes of Tg(hsp70:E2A-PBX1-EGFP). (D) Real time quantitative PCR (RT-qPCR) analysis showed hE2A-PBX1 mRNA high expression in Tg(hsp70:E2A-PBX1-EGFP) compared to the siblings at 3 days post-fertilization (dpf). The black asterisks indicate statistical difference (Student t tests, mean ± standard error of the mean; ****P<0.0001) (E) Wright-Giemsa staining of whole blood cells from siblings and Tg(hsp70:E2A-PBX1-EGFP) at 5 dpf. Myelocytes are indicated by red arrows in the image. (E’) Statistical analysis of the cell counts in panel (E). The black asterisks indicate statistical difference (one-way ANOVA, mean ± standard error of the mean; *P<0.05). N>150, number of zebrafish larvae. (F) Survival curves of Tg(hsp70:E2A-PBX1-EGFP) and sibling larvae up to 15 days after heat shock. The black asterisks indicate statistical difference (log-rank [Mantel-Cox] test; ****P<0.0001).

TUNEL and immunofluorescence analyses

Transferase dUTP nick end labeling (TUNEL) assay (In Situ Cell Death Detection Kit; Roche) was performed as instructed. The samples were stained with goat-anti-DsRed (Abcam; 1:400) and rabbit-anti-Lcp1 antibody (1:200), followed by incubation with Alexa Fluor 488-anti-goat antibody (Invitrogen; 1:400) and Alexa Fluor 488-anti-rabbit antibody (Invitrogen; 1:400) for fluorescent visualization.

RNA-sequencing analysis

Whole kidney marrow (KM) cell suspensions were isolated from adult WT and Tg(hsp70:E2A-PBX1-EGFP). RNA extraction was performed using TRIZOL (Invitrogen),³⁰ and the extracted RNA samples were sent for library construction and RNA sequencing at Guangzhou Jidio Biotechnology.

Drug and inhibitor treatment

The small molecule inhibitors we screened were randomly selected from TargetMol’s Bioactive Compound Library (Online Supplementary Excel S1). These compounds were all diluted to 20 mM as storage solution. KJ-Pyr-9, OUL35, A-484954, LRRK2-IN-1, CID44216842 were dissolved in dimethyl sulfoxide (DMSO), while L-arginine hydrochloride was dissolved in ddH₂O.

Animal care and xenograft procedure

Animal studies were performed in accordance with the animal research advisory committee of South China University of Technology. At 3 dpf, zebrafish larvae (runx1^w84x) were injected with 600 human leukemic cells per embryo through the yolk sac into the posterior cardinal vein (PCV) or dorsal aorta (DA).^31-33 The embryos were maintained at 35°C. On 1 day post-injection (dpi), zebrafish larvae were treated with the specified drug concentrations. Confocal microscopy was employed from 1 dpi to 4 dpi for image examination.

Statistical analysis

Statistical analysis was performed using GraphPad Prism version 6.000. Survival analysis utilized Kaplan-Meier (K-M) curves and the log-rank (Mantel-Cox) test. Student t tests compared two groups, and one-way analysis of variance (one-way ANOVA) with Tukey’s adjustment was used for multiple group comparisons. A significance level of P<0.05 was applied, and all data were presented as mean ± standard error of the mean.

Results

Generation of an inducible humanized E2A-PBX1 transgenic zebrafish

In order to investigate the effects of humanized E2A-PBX1 on hematopoiesis, we created transgenic zebrafish with hE2A-PBX1 fusion gene expression controlled by the hsp70 promoter. The pT2AL-hsp70-E2A-PBX1-EGFP plasmid with correct sequence (Figure 1A) was obtained after sequencing and microinjected into WT zebrafish to obtain the F0 generation (Online Supplementary Figure S1A). We obtained a diverse range of progeny by crossing the F0 with WT fish and identified a transgenic line (named: Tg(hsp70:E2A-PBX1EGFP)) in the F4 generation, which stably inherited the transgene and exhibited strong EGFP fluorescence. After inducing the transgenic zebrafish with heat shock at 39.5°C, EGFP expression was observed in the whole body of 32 hpf embryos (Figure 1B). In addition, PCR detected a 231 bp human-derived PBX1 target fragment in Tg(hsp70:E2A-PBX1EGFP) transgenic zebrafish (Figure 1C), real time quantitative PCR (RT-qPCR) showed higher hE2A-PBX1 mRNA expression in Tg(hsp70:E2A-PBX1-EGFP) at 3 dpf (Figure 1D). These results demonstrate that we successfully constructed a heritable and stable Tg(hsp70:E2A-PBX1-EGFP) transgenic zebrafish lineage with induced expression of the hE2A-PBX1. Furthermore, qPCR analysis showed consistent expression of endogenous tcf3a and tcf3b (equivalent to human E2A) in both the embryonic tail hematopoietic tissue (CHT) and adult peripheral blood of Tg(hsp70:E2A-PBX1-EGFP) zebrafish (Online Supplementary Figure S1B-D). This suggests that Tg(hsp70:E2A-PBX1-EGFP) zebrafish maintain normal expression of endogenous e2a while overexpressing human E2A-PBX1. Next, to examine the potential effects of the hE2A-PBX1 oncoprotein on blood development in zebrafish, we collected whole blood from zebrafish at 5 dpf after heat shock and performed Giemsa staining. It was found that the number of myeloid cells was significantly increased (Figure 1E, E’), indicating that transient induction of hE2APBX1 expression may disrupt the hematopoiesis in zebrafish. Additionally, survival analysis of Tg(hsp70:E2A-PBX1-EGFP) fish revealed a significant increase in mortality within 15 days following the induction of hE2A-PBX1 expression, in comparison to their siblings (Figure 1F).

Figure 2.Induction of hE2A-PBX1 expression in zebrafish larvae leads to myeloid cell and hematopoietic stem cell expansion. (A) Immunofluorescence staining of Lcp1⁺ cells in siblings (left panel) and Tg(hsp70:E2A-PBX1-EGFP) (right panel) at 5 days post-fertilization (dpf). (A’) Statistical analysis of the Lcp1⁺ signals in panel (A). The black asterisks indicate statistical difference (Student t tests, mean ± standard error of the mean [SEM]; **P<0.01) (B) Whole mount in situ hybridization (WISH) of cebp1, lyz, mpx and mfap4 expressions in Tg(hsp70:E2A-PBX1-EGFP) (right panel) were higher than siblings (left panel) at 5 dpf. The number of lyzDsRed⁺ cells (caudal hematopoietic tissue [CHT] region and Sudan Black B-positive [SB⁺] cells in Tg(hsp70:E2A-PBX1-EGFP) (right) was higher than siblings (left) at 5 dpf. The CHT is enlarged in the red box (original magnification ×200). (B’) Statistical analysis of the positive signals (cebp1, lyz, lyz-DsRed, mpx, SB and mfap4) in panel (B). The black asterisks indicate statistical difference (Student t tests, mean ± SEM; *P<0.05, **P<0.01, ****P<0.0001) (C) WISH of myb expressions in Tg(hsp70:E2A-PBX1-EGFP) (right panel) were higher than siblings (left panel) at 5 dpf. (C’) Statistical analysis of the myb⁺ signals in panel (C). The black asterisks indicate statistical difference (Student t tests, mean ± SEM; *P<0.05). N/N: number of zebrafish larvae showing representative phenotype/total number of zebrafish larvae examined.

hE2A-PBX1 induces abnormal expansion of myeloid cells in zebrafish larvae

In order to further explore the effect of hE2A-PBX1 expression on the development of different hematopoietic lineages, we detected the marker genes of each hematopoietic lineage in Tg(hsp70:E2A-PBX1-EGFP) transgenic zebrafish after heat shock by whole mount in situ hybridization (WISH) and specific staining. The results showed a significant increase in myeloid cells (l-plastin: a myeloid gene that marks granulocytes and macrophages, also known as lcp1) (Figure 2A, A’), which was consistent with the hemogram changes in zebrafish larvae (Figure 1E). Further assays of myeloid terminally differentiated granulocytes and macrophages revealed a significant increase in the number of immature granulocytes (cebp1), mature granulocytes (lyz, mpx, Sudan Black B [SB]) and macrophages (mfap4) in both 3 dpf and 5 dpf (Figure 2B, B’; Online Supplementary S2A, A’). As myeloid cells originate from HSPC;³⁴ we aimed to investigate whether the increased number of myeloid cells was caused by the tendency of HSPC toward the myeloid lineage or if there was an overall increase in HSPC leading to increased differentiation into various lineages? In order to address this question, we examined the changes of HSPC (myb), lymphocytes (rag1, rag2), and erythrocytes (βe1) in Tg(hsp70:E2A-PBX1-EGFP) after heat shock. The results showed that myb⁺ HSPC increased at 5 dpf (Figure 2C, C’), while the number of rag1⁺ and rag2⁺ lymphocytes decreased significantly (Online Supplementary Figure S2B, B’), and βe1⁺ erythrocytes showed no significant change (Online Supplementary Figure S2C, C’). These data suggest that hE2A-PBX1 expression may lead to myeloid expansion in zebrafish by increasing HSPC with myeloid differentiation potential.

In addition, it has been reported that the expression of PBX1 maintains cell proliferation,²¹ while the E2A protein promotes cell differentiation and has anti-proliferation effect.³⁵ Therefore, we investigated whether hE2A-PBX1 influences the proliferation of myeloid and lymphocytes in Tg(hsp70:E2A-PBX1-EGFP). The proliferation and apoptosis of myeloid/lymphocytes in Tg(hsp70:E2A-PBX1-EGFP) fish were examined after heat shock by co-staining with BrdU/TUNEL and Lcp1/Rag2, respectively. The results showed a significant increase in the proportion of proliferating cells among Lcp1⁺ myeloid cells in 5 dpf Tg(hsp70:E2A-PBX1EGFP) compared to the siblings (Figure 3A, 3A’), while the proportion of apoptotic cells showed a significant decrease (Figure 3B, B’). In contrast, Rag2⁺ lymphocytes showed a significant decrease in proliferation (Online Supplementary Figure S3A, A’), with no significant change in apoptotic cells (Online Supplementary Figure S3B, B’). These data suggest that expression of hE2A-PBX1 leads to abnormal myeloid expansion by increasing the myeloid differentiation potential of HSPC on the one hand and inducing myeloid hyperplasia on the other hand.

In order to investigate the potential relationship between myeloid expansion and early mortality in hE2A-PBX1 zebrafish, we treated heat-shocked hE2A-PBX1 fish with drugs capable of reducing myeloid proliferation, namely cytarabine^36-38 and flavopiridol.^39,40 The results showed that, compared to the DMSO-treated control group, both cytarabine and flavopiridol treatments significantly reduce the population of SB⁺ myeloid cells in hE2A-PBX1 fish (Online Supplementary Figure S3C, C’), and increase their survival rate (Online Supplementary Figure S3D). This suggests that myeloid cell proliferation may be one of the contributing factors to the elevated mortality in hE2A-PBX1 zebrafish.

Induction hE2A-PBX1 expression in adult zebrafish leads to an acute myeloid leukemia-like phenotype

In order to analyze whether induced expression of hE2A-PBX1 leads to blood tumors in adult zebrafish, we administered 1-month continuous heat shock to 3-month-old adult fish and collected peripheral blood (PB) cells and kidney marrow (KM) blood cells for Giemsa staining to detect hematological changes. Although 1 month of hE2A-PBX1 induction did not alter the hemogram in PB, it resulted in a significant increase in myeloid cells in KM (Online Supplementary Figure S4A, A’ ). We then extended the heat shock time to 3 months to determine whether the degree of myeloid expansion induced by hE2A-PBX1 is related to the induction time. The results showed that the longer expression of hE2APBX1 not only further exacerbated the myeloid expansion and lymphoid reduction in KM, but also resulted in mild myeloid hyperplasia in PB (Online Supplementary Figure S4B, B’). Since myeloid leukemia is commonly associated with the patient’s age, we induced 3-month continuous expression of hE2A-PBX1 in 1-year-old zebrafish. We found that the AML-like phenotype was more pronounced in aged zebrafish after 3 months of hE2A-PBX1 induced compared to young zebrafish. In addition to the significant increase in myeloid cells and a further decrease in lymphocytes in KM, there was also a notable rise in myeloid blasts, and myeloid cells in PB showed a further increase (Figure 4A, A’; Online Supplementary Figure S4C). Furthermore, we observed that the kidneys of Tg(hsp70:E2A-PBX1-EGFP) were significantly enlarged, and the enlargement worsened with the increase of hE2A-PBX1 expression and age (data not shown), especially in 1-year-old zebrafish with continuous 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 Supplementary 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 also 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 zebrafish, 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.

Figure 3.Abnormal myeloid cell expansion in Tg(hsp70:E2A-PBX1-EGFP) fish caused by proliferation and apoptosis perturbation. (A) Immunofluorescence double staining of Lcp1 and bromodeoxyuridine (BrdU) antibodies reveals a significant increase in myeloid cell proliferation in the caudal hematopoietic tissue (CHT) region of 5 days post-fertilization (dpf) Tg(hsp70:E2A-PBX1-EGFP) larvae (N=22) compared with the siblings (N=17). Lcp1/BrdU double-positive cells are indicated by white arrows. (A’) Statistical analysis of percentage of Lcp1⁺ BrdU⁺ cells in panel (A). The black asterisks indicate statistical difference (Student t tests, mean ± standard error of the mean; *P<0.05). (B) Co-staining of Lcp1 and transferase dUTP nick end labeling (TUNEL) was used to detect the apoptosis in the CHT region of 5 dpf Tg(hsp70:E2A-PBX1-EGFP) larvae (N=22) compared with the siblings (N=24). Lcp1/TUNEL double-positive cells are indicated by white arrows. (B’) Statistical analysis of percentage of Lcp1⁺ TUNEL⁺ cells in panel (B). The black asterisks indicate statistical difference (Student t tests, mean ± standard error of the mean; **P<0.01).

Figure 4.Tg(hsp70:E2A-PBX1-EGFP) adult fish exhibit abnormal myeloid cell expansion which resembles acute myeloid leukemia-like phenotypes. (A) May-Grunwald-Giemsa staining of peripheral blood (PB) cells (upper panels) and kidney marrow (KM) blood cells (lower panels) in 1-year-old wild-type (WT) (left panel) and Tg(hsp70:E2A-PBX1-EGFP) (right panel) adult fish after 3-month heat shock. Red arrows indicate myelocytes, black arrows indicate lymphocytes and green arrows indicate blasts. Original magnification ×400. Blood cell counts of PB and KM were calculated manually based on their morphology. (A’) Statistical analysis of cell counts in panel (A). The black asterisks indicate statistical difference (N=9, one-way ANOVA, mean ± standard error of the mean; *P<0.05, **P<0.01). (B) Kidneys from 1-year-old transgenic fish were enlarged to 1.36-fold in area compared with siblings after 3-month heat shock. (B’) Statistical analysis of the area of kidney in panel (B). The black asterisks indicate statistical difference (N=9, Student t tests, mean ± standard error of the mean; **P<0.01).

Figure 5.Tg(hsp70:E2A-PBX1-EGFP;lyz:DsRed) transgenic cells with induced acute myeloid leukemia-like disease are transplantable. (A) Immunofluorescent staining of the myeloid-specific marker Lcp1 in frozen sections confirmed myeloid cell invasion of the skeletal musculature in 1-year-old wild-type (WT) (left panel) and Tg(hsp70:E2A-PBX1-EGFP) (right panel) adult fish after 3-month heat shock (yellow arrows show invasion areas of myeloid cells into muscle tissue). (B) Direct observation of engrafted acute myeloid leukemia (AML)-like cells. At 60 days post-transplantation (dpt), AML-like E2A-PBX1 Lyz-DsRed⁺ cells could be visualized in foxn1/Casper-recipient kidney marrow (KM). The white arrow indicates the engrafted acute myeloid leukemia (AML)-like E2A-PBX1 Lyz-DsRed⁺ cells. N/N, number of zebrafish larvae showing representative phenotype/total number 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:E2APBX1-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.

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 development 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 examined the expression level of runx1 in Tg(hsp70:E2A-PBX1EGFP) 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 lossof-function mutant (runx1^w84x) to obtain Tg(hsp70:E2APBX1-EGFP); runx1^w84x 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’). Additionally, 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 concentration: 0.7 mM, 0.5 mM, 0.2 mM) and detected the number of lyz⁺ granulocytes by WISH. Our results showed that the reduction in granulocytes was similar to that of runx1^w84x 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:E2APBX1; runx1^w84x/+) increased survival to a level similar to that of siblings, whereas overexpression (Tg(hsp70:E2APBX1-EGFP; hsp70:myc-runx1)) did not further exacerbate mortality in hE2A-PBX1 zebrafish (Online Supplementary Figure S6C). This is possibly because hE2A-PBX1 transgenic 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 regulation 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/IL-17/MAPK signaling pathway was enriched in hE2A-PBX1 zebrafish (Figure 6D), with ten genes (fosl1a, fosab, fosb, mmp13a, junb, hsp27, socs3a, socs3b, mmp9 and jund) were significantly upregulated in these pathways (Figure 6E). Additionally, we microinjected sibling and hE2A-PBX1 transgenic zebrafish with 0.2 mM runx1 MO, and detected the changes of significantly upregulated genes in the TNF/IL-17/MAPK signaling pathway after knockdown of runx1. The results showed that knockdown of runx1 could significantly reverse the high expression of hsp27, junb, socs3b, fosb and fosl1a in hE2A-PBX1 transgenic line (Figure 6F). These data suggest that hE2A-PBX1 can induce myeloid expansion and activate the TNF/IL-17/MAPK signaling pathway through upregulation of runx1 expression.

Figure 6.hE2A-PBX1 activate the TNF/IL-17/MAPK signaling pathway through upregulation of runx1 expression. (A) Whole mount in situ hybridization (WISH) of runx1 expressions in Tg(hsp70:E2A-PBX1-EGFP) (right panel) were higher than siblings (left panel) at 5 days post-fertilization (dpf). The caudal hematopoietic tissue (CHT) is enlarged in the red box (original magnification ×200). (A’) Statistical analysis of the runx1⁺ signals in panel (A). The black asterisks indicate statistical difference (Student t tests, mean ± standard error of the mean [SEM]; *P<0.05). N/N: number of zebrafish larvae showing representative phenotype/total number of zebrafish larvae examined. (B) Real time quantitative polymerase chain reaction (RT-qPCR) analysis showed increased runx1 mRNA expression in Tg(hsp70:E2A-PBX1-EGFP) compared to the sibling controls at 3 dpf. The black asterisks indicate statistical difference (Student t tests, mean ± SEM; ****P<0.0001) (C) Decreased number of lyz⁺ neutrophils in sibling and Tg(hsp70:E2APBX1-EGFP) larvae after injecting 0.2 mM runx1 morpholino (MO) at 3 dpf. The control groups were treated with 0.2 mM random sequence MO. The CHT is enlarged in the red box (original magnification ×200). (C’) Statistical analysis of the lyz⁺ signals shown in panel (C). The black asterisks indicate statistical difference (N≥15, one-way ANOVA, mean ± SEM; ***P<0.001, ****P<0.0001). (D) Top 20 enriched KEGG pathways identified in the analysis of differentially expressed genes (DEG) in KM cells from 6-month-old Tg(hsp70:E2A-PBX1-EGFP) compared to control groups after 3-month heat shock. The TNF/IL-17/MAPK signaling pathway is highlighted by the red arrow. (E) Heatmap of DEG involved in the TNF/IL-17/MAPK signaling pathway between WT and hE2A-PBX1 adult fish. Red and blue represent an increase and decrease gene expression levels, respectively; P<0.05. (F) RT-qPCR analysis showing mRNA expression of fosab, hsp27, junb, mmp9, socs3b, fosb and fosl1a in Tg(hsp70:E2A-PBX1-EGFP) compared to siblings after injecting 0.2 mM runx1 MO at 3 dpf. The control groups were treated with 0.2 mM random sequence MO (one-way ANOVA, mean ± SEM; *P<0.05, **P<0.01, ***P<0.001). TNF: tumor necrosis factor; IL-17: interleukin-17; MAPK: mitogen-activated protein kinase.

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 inhibitors 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 targeting autophagy pathways, LRRK2-IN-1 targeting apoptosis pathways, but also include CID4421684, OUL35 and KJPyr-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 extent as Ara-C (Figure 7B, B’), but OUL35 and CID44216842 exhibited a lower embryo deformity rate (Online Supplementary 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 findings 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 consecutive 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 TNF^42-44 and IL-17^45-47 can activate the MAPK pathway. In order to investigate whether TNF/IL-17 can similarly activate the MAPK signaling in Tg(hsp70:E2APBX1-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.

Figure 7.KJ-Pyr-9, OUL35, and CID44216842 effectively alleviate hE2A-PBX1-induced myeloid expansion in zebrafish larvae. (A) Flowchart 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’) Statistical 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 the activation of the MAPK signaling pathway (Figure 7E).

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 lymphoblastic 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 runx1^w84x embryos were monitored by imaging at 12 hpi, 24 hpi, 72 hpi and 96 hpi. (B) RCH-ACV cells were calculated after xenografting into runx1^w84x 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 runx1^w84x 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).

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 human 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 runx1^w84x 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 zebrafish 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 KJPyr-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 progression 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 abnormal 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 leukemia: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 occurrence of hE2A-PBX1 pre-B leukemia. On the one hand, the insufficient dose of WT E2A affects the regulation of lymphangiogenesis development.^50,51 On the other hand, hE2A-PBX1 induces oncogenic properties, such as cell proliferation, by altering the expression of downstream genes or transcriptional co-activators of PBX1.⁵² In our study, the induction of hE2A-PBX1 expression, along with the regular expression of the endogenous E2A gene, leads to the development of myeloid leukemia. This suggests that the acquired function of hE2A-PBX1, rather than a deficiency of WT E2A, is the primary cause of its carcinogenic potential. However, disruption of the E2A gene may contribute to alterations in lymphoid development. Consistent with this, an unbiased analysis of E2A-PBX1 cistrome in pre-B ALL cells reveals that E2A-PBX1 can interact with RUNX1, resulting in transcriptome alterations, including activation of RUNX1 itself.¹ Moreover, our study revealed aberrant activation of Runx1 in hE2A-PBX1 transgenic fish. Notably, inhibiting Runx1 and its downstream pathway demonstrated significant alleviation in the expansion of leukemic cells in both zebrafish and human cell lines. These findings emphasize that hE2A-PBX1 exerts a significant impact on tumorigenesis by regulating the genome through direct binding with RUNX1. Whether this effect is synergistically facilitated by the inappropriate activation of target genes through the fusion of PBX1 DNA binding motifs remains to be further investigated.

Currently, there are no clinically targeted drugs for the treatment of E2A-PBX1 leukemia, and there are relatively few studies on the treatment of E2A-PBX1-associated AML-like disease and MPAL. In this study, we show that the small molecule compounds KJ-Pyr-9, OUL35 and CID44216842 obtained by hE2A-PBX1 zebrafish leukemia model have great potential. OUL35, recognized as a selective PARP inhibitor that targets PKCδ, a PARP substrate that activates MAPK,^53,54 has demonstrated the ability to protect HeLa cells from ARTD10-induced cell death.⁵⁵ KJ-Pyr-9 is an inhibitor of MYC,⁵⁶ a transcriptional regulatory factor encoded by the common proto-oncogene MYC, which mainly exerts its regulatory effects through binding with MAX protein.⁵⁷ Notably, the expression of max was significantly increased in hE2A-PBX1 zebrafish (data not shown). Literature reports indicate that KJ-Pyr-9 can target MYC to inhibit pleomorphic glioblastoma multiforme (GBM) and significantly reduce the viability of squamous cell carcinoma (SCC) and adenocarcinoma (AC)-derived cells.⁵⁸ CID44216842 acts as an inhibitor of RAS, a key regulator of signaling pathways controlling normal cell growth and malignant transformation. Additionally, CID44216842 has been demonstrated to inhibit stimulus-induced Cdc42 activation in Swiss 3T3 fibroblasts.⁵⁹ While in vitro cellular experiments confirm their efficacy in tumor cell therapy, it is essential to conduct experiments using animal tumor models before progressing to clinical trials. In our study, these three small molecule compounds exhibit the ability to alleviate hE2APBX1-induced AML-like disease in zebrafish as well as pre-B ALL associated with hE2A-PBX1, by targeting the TNF/IL-17/ MAPK pathway downstream of Runx1. However, it is not clear whether these small molecule compounds are applicable to other blood diseases with abnormal RUNX1 activity, and more preclinical animal studies are needed to confirm this. Overall, our findings shed new light on the treatment of E2A-PBX1 and other Runx1-related disorders. Although there is much work to be done, we anticipate that our research will contribute to the development of innovative therapeutic strategies for these diseases.

Footnotes

• Received June 13, 2023
• Accepted February 12, 2024

Correspondence

^*HL and QL contributed equally as first authors

W. Liu liuwei7@scut.edu.cn

W. Zhang mczhangwq@scut.edu.cn

Disclosures

No conflicts of interest to disclose.

Contributions

WL and WZ conceived and supervised the study and edited the article. HL and QL performed experiments and wrote, reviewed, and edited the article. JH, WD, KW, SL, and HW performed the experiments. ZH reviewed and edited the article.

Funding

This work was supported by the National Natural Science Foundation of China (82070163), Guangdong Basic and Applied Basic Research Foundation (2020A1515011218) and Special Fund of Science and Technology Innovation Cultivation for College Students in Guangdong Province (pdjh2020b0048).

Acknowledgments

The authors would like to thank Dr. Huafeng Xie for providing RCH-ACV cells. The authors would like to thank Dr. Kailun Li, Dr. Xiao Fang, Dr. Panpan Zhu, Dr Shuang Zhao and Dr. Zhibin Huang for their helpful suggestions.