CRISPR-Cas9 Screening Identifies Therapeutic Targets for AML1-ETO Positive Leukemia

• Published in: Blood 2023-11, Vol.142 (Supplement 1), p.5697-5697
• Main Authors: Guo, Chengcai, Liu, Yu, Rao, Qing, Qiu, Shaowei, Gu, Runxia, Wang, Min, Wang, Jianxiang
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2023-179262

Objectives: Targeted therapy has achieved tremendous success in leukemia therapy over the past few decades, especially in those with fusion proteins, such as t(15;17) which generates PML-RARa and t(9;22) which generates BCR-ABL. However, effective targeted therapy for AML1-ETO-positive leukemia, which arises from one of the most frequent chromosomal translocations t(8;21), remains largely elusive. In this study, we performed CRISPR-Cas9 screening on the AML1-ETO-positive Kasumi-1 cell line using an AML-related sgRNA library to identify potential therapeutic targets that could restore normal differentiation in AML1-ETO-positive AML. Methods: To identify critical genes capable of reversing differentiation blockage in AML1-ETO-driven leukemogenesis, we constructed an AML1-related sgRNA library which focused genes and signaling pathways regulated by AML1-ETO. This sgRNA library consist of (1) coding genes of proteins interacting with AML1, (2) genes activated by AML1-ETO, and (3) genes downregulated upon knockdown of AML1-ETO as observed in CHIP-seq results. This sgRNA library comprises 4130 sgRNAs targeting 929 human genes (~4 sgRNA/gene), along with 100 control sgRNAs. CD13 high and CD13 low populations were collected by flow cytometry at 10th day post-transduction of sgRNA library. The enrichment of gRNAs between CD13 high and CD13 low group was analyzed using the MAGeCK program, and the candidate genes were identified based on the log2 fold change of sgRNA abundance. To make the screening results more reliable, we used CD11b as another screening marker to repeat aforementioned process. We further preformed single candidate gene knockout in Kasumi-1 and SKNO-1 cell line to validate the screening phenotype. The regulatory function of candidate gene in AML1-ETO positive leukemia was explored through various in vitro assays, including cell apoptosis, cell proliferation, morphological evaluation, flow cytometry analysis, quantitative real-time PCR (qPCR), and Western blot. Results: The gene set enriched in the CD13 high group is considered to be positive hit. Silence of these genes may reverse blockage of differentiation, making them potential therapeutic targets for AML1-ETO leukemia. Additionally, genes such as CBFB, MTA2, DOT1L, PRMT1, RUNX1 and KDM1A, which had been previously reported to promote myeloid differentiation when down-regulated, were also significantly enriched in the CD13 high group, these findings demonstrated the credibility of our screening