HDAC Inhibitor Effectively Reverses Stemness Transformation of ETV6::Mecom-Positive Leukemia Cells By Upregulating CDKN1C
Authors: Hao Yuchao , Liu Jing , Zhou Jiayin , Hu Jiong , Yan Jinsong 
Published: December 2024

Introduction
AML with MECOM rearrangement (MECOM-r) is a distinct entity recognized by the World Health Organization, characterized by an aggressive course and poor prognosis. There are two primary pathogenic mechanisms of MECOM-r, one of which is a large number of new fusion proteins transcribed from the promoters of partner genes, such as ETV6::MECOM. To date, 78 cases of ETV6::MECOM have been reported, of which 69% were AML, 18% were MDS, and 10% were CML. Clinicopathological data showed abnormal myeloproliferative disorders in most cases. In addition, patients positive for this fusion gene showed significant resistance to chemotherapy and almost all leukemia chemotherapeutic agents. Relevant research and clinical data suggest that patients with ETV6::MECOM rearrangements have a poor prognosis, and current treatments do not improve their prognosis. And its mechanism in the development of leukemia requires further exploration, the new treatments and therapeutic targets need to be developed.
Methods
By integrating in-house data (n = 71) and six public datasets from the Leucegene project (n = 452), identified three cases of ETV6::MECOM in-frame fusion gene and validated with FISH, Sanger sequencing and Giemsa staining analysis. Followed by bioinformatic assays to estimate the relevance of their expression with leukemogenesis. CCK8 assay to determined cell viability in ETV6::MECOM-positive K562 and 32D cells using different types of drug treatments. Tumorigenicity assays were performed to determine the effects of ETV6::MECOM on colony formation, cell proliferation, the cell cycle and apoptosis (in vitro), and on leukemogenesis or survival of NSG mice xenografted with AML cells (in vivo). Transcriptome sequencing was performed to determine the expression profiles of ETV6::MECOM-regulated genes. Effects of ETV6::MECOM on CDKN1C expression was determined by immunoblot and RT-PCR. Detection of whether CDKN1C mediates the essential function of ETV6-MECOM by dependency assays. Meanwhile, impacts of HDACi on ETV6::MECOM-positive cells were evaluated by CCK8 and flow cytometric assays. Cell activity assay and mouse model to verify whether HDACi affect the pro-cancer function of ETV6::MECOM.
Results
ETV6::MECOM induces a significant stemness expression signature and poor prognosis. ETV6::MECOM-positive cells exhibited more stemness-like properties, as evidenced by reduced colony formation, epithelial-mesenchymal transition (EMT), enhanced cell migration and invasion, reduced spheroidogenicity, and cell cycle arrest at G0. The mechanism is that the rearranged form of ETV6::MECOM upregulates the expression of CDKN1C, a gene related to the maintenance of cell cycle quiescence, and the upregulation of CDKN1C promotes leukemia cell arrest at the G0 phase of the cell cycle, which ultimately promotes stemness transformation of leukemia cells. Introduction of the epigenetic therapeutic agent histone deacetylase inhibitor (HDACi) effectively inhibited CDKN1C expression and stem cell-like properties induced by ETV6::MECOM.
Conclusions
ETV6::MECOM-positive patients commonly present with poor outcomes attributed to refractoriness and relapse. This study revealed that Chidamide, a histone deacetylase inhibitor, effectively reversed the stemness transformation of ETV6::MECOM-positive cells by upregulating CDKN1C expression, providing a promising treatment option for ETV6::MECOM positive patients.