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1379 Ebf1 Is Involved in Primitive Myelopoiesis and Contributes in Leukemia Pathogenesis

Program: Oral and Poster Abstracts
Session: 602. Myeloid Oncogenesis: Basic: Poster I
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Acute Myeloid Malignancies, AML, Translational Research, Diseases, Myeloid Malignancies
Saturday, December 9, 2023, 5:30 PM-7:30 PM

Rongtao Xue, MD1*, Junwei Lian, PhD2*, Jinping He, MD1*, Tong Lin, MD1*, Yiyue Zhang, PhD2* and Qifa Liu, MD3

1Department of Hematology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China
2Southern China University of Technology, Guangzhou, China
3Nanfang Hospital, Southern Medical University, Guangzhou, China

Acute myeloid leukemia (AML) is caused by the abnormal proliferation and differentiation of a clonal population of myeloid progenitors. EBF1 is a key transcriptional factor in regulating hematopoiesis and has been linked to acute lymphoblastic leukemia (ALL). However, the relationship between AML and EBF1 mutation has not yet been studied. Recent research has shown that deleting EBF1 results in myeloid-biased progenitors and decreased HSC quiescence and repopulation capacity. This suggests that EBF1 is involved in earlier hematopoiesis and has additional functions beyond B-cell committed development.

Our clinical center conducted whole-exome sequencing on 231 cases of acute myeloid leukemia and identified four patients with an Ebf1 mutation. This corresponds to a mutation frequency of 1.73% and the mutations were identified as p.Q477H (c.1434G>T), p.S513P (c.1537T>C), p.C534F (c.1601GT), and p.P570A (c.1708C>G). Data from the cBioPortal database showed that AML patients with Ebf1 deep deleted mutation had shortened survival. To better understand the role of Ebf1 in AML pathogenesis, we generated an ebf1a-deficient zebrafish mutant. Further research showed that the ebf1a deficiency caused neutrophilia during primitive hematopoiesis. BrdU staining in Tg(coronin1a: GFP) labeled myeloid progenitors was significantly higher in ebf1a-deficient larvae than in wild-type larvae, indicating that ebf1a deficiency led to increased proliferating myeloid progenitors which would take responsibility for the neutrophilia in early-developing larvae.

We conducted experiments using four RNA variants (p.Q477H, p.S513P, p.C534F, and p.P570A) injected into ebf1a deficiency zebrafish larvae to verify their pathogenicity. Our findings showed that while EBF1 p.Q477H and p.S513P caused a decrease in neutrophils, p.C534F resulted in neutrophilia, indicating that it might be a loss-of-function mutation. To further understand the mechanism, we introduced EBF1 p.C534F to AML cell culture, and CCk8 and cell cycle analysis showed increased proliferation rates and disturbed cell cycle balance, leading to increased proliferation of AML cells of EBF1-C534F mutation. The cells bearing this mutation showed a higher rate of proliferation, likely due to a disturbance in the balance of the cell cycle. These findings provide valuable insight into the potential pathogenetic mechanism of EBF1 p.C534F.

Our investigation into the cause of ebf1 deficiency disrupting normal hematopoiesis led us to perform bulk RNA-sequencing with sorted myeloid-progenitors labeled by Tg:(coronin1a-GFP) in 30hpf zebrafish larvae. We found that leukocyte differentiation and myeloid cell cycle-related genes such as lyz, lygl1, coro1a, lcp1, cebpa, spi1b, mpx, irf8, il6, il34, etc were upregulated in ebf1a deficiency zebrafish. Additionally, anxa1c, anxa1b, Cish, and nlrc3l, which have been reported to suppress lymphoid proliferation but increase myeloid expansion, were down-regulated. Go enrichment analysis showed that the significant genes were associated with hematopoietic disorders including AML and MDS. To address the issue of disturbed cell cycle balance caused by EBF1 deficiency and loss-of-function mutation, we sought to suppress the G1-S transition via CDK inhibitor. We treated EBF1-C534F, EBF1-WT, and control AML cell culture with CDK4 inhibitor: AMG925. We were surprised to find that the CDK4 inhibitor sufficiently suppressed the proliferation and reversed the prolonged S phase caused by EBF1 mutation.

We investigated the myeloid-bias function of Ebf1 deficiency and the potential pathogenesis of EBF1 mutations of AML from the development view and were surprised to find CDK inhibitors as a potential drug for EBF1-deficient AML patients.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH