CRISPR-Cas9 Screening Identifies Therapeutic Targets for AML1-ETO Positive Leukemia
Authors: Guo Chengcai , Liu Yu , Rao Qing , Qiu Shaowei , Gu Runxia , Wang Min , Wang Jianxiang 
Published: December 2023

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 strategy and results. Clathrin Heavy Chain (CLTC) emerged as one of the top-ranking genes in our study. CLTC is a crucial protein involved in the regulation of clathrin-mediated endocytosis, which plays a critical role in various cellular processes including cell signaling, proliferation, and differentiation. Expression of CD13 and CD16 were upregulated in CLTC knockout cells, further validating our screening results. Besides, morphological analysis displayed a more mature cell morphology with a decrease in nucleus-to-cytoplasm ratio and indented shaped nuclei, suggesting that repression of CLTC promotes terminal differentiation of AML blasts. Cell biology experiments indicated that knockout of CLTC remarkably inhibited the proliferation of Kasumi-1 and SKNO-1 cells, meanwhile, apoptosis of these cells was accelerated. Therefore, these results collectively suggested that the down-regulation of CLTC exhibited significant tumor-suppressive effect in AML1-ETO leukemia. Furthermore, Western blot and RT-qPCR analyses revealed that CLTC is highly expressed in AML1-ETO-positive leukemia, whereas its expression is downregulated upon deletion of AML1-ETO fusion protein. This suggested that CLTC might be regulated by AML1-ETO fusion protein and contribute to its oncogenic activity.
Conclusions: Our results revealed a possible novel mechanism underlying the blockage of myeloid differentiation in AML1-ETO leukemia, and have identified CLTC as a promising therapeutic target for AML1-ETO-driven leukemogenesis.