Abstract 2934: TET2 deficiency accelerates leukemogenesis in the NHD13 mouse model of MDS
Authors: Huang Feiteng , Sun Jie , Chen Wei , Zhang Lei , He Xin , Dong Haojie , Wu Yuhui , Wang Hanying , Li Zheng , Huang Victor , Khaled Samer , Li Ling 
Published: July 2021

Abstract
Myelodysplastic syndromes (MDS) frequently progresses to acute myeloid leukemia (AML). Mechanisms underlying MDS transformation remain unclear. We showed that TET2 deficiency enhances MDS maintenance (PMID: 30146412). Besides frequent mutated in MDS/AML, TET2 levels were decreased during MDS progression (PMID: 23827711). Recently, we analyzed a dataset (GSE19429) and found lower TET2 levels correlated with shortened survival of patients with high-risk MDS. We thus assessed TET2 function in MDS leukemia transformation by deleting TET2 in a NHD13 knock-in mouse model, in which ~30% of mice develop AML. Therefore, we crossed conditional TET2-KO mice with NHD13 transgenic mice. NHD13/TET2-KO (NT) mice developed leukemia with shorter survival than did NHD13/TET2-WT (ctrl) mice (median days, NT: 219 vs. ctrl: 366, n=12, p&lt;0.01). Half of the NT mice developed AML in 30 weeks, while ctrl mice did not. At 20th weeks (pre-leukemia stage), NT mice exhibited splenomegaly and hyper-cellular bone marrow (BM). Flow cytometry analysis of NT BM cells revealed increased lin-c-kit+sca-1- (LK) BM subset (LK%, NT: 3.89% vs. ctrl: 1.96%, n=12, p&lt;0.01) and decreased apoptosis (AV+%, NT: 2.85% vs. ctrl: 7.59%, n=12, p&lt;0.001). To search for acquired genetic lesions due to TET2-KO, we performed whole-exome sequencing of c-kit+ cells from pre-leukemic NT vs. ctrl mice. We focused on mutations newly acquired or those with increased variant allele frequency (VAF, fold change&gt;2) in NT mice and ranked them based on their association with AML prognosis using PRECOG (http://precog.stanford.edu). We selected top three candidates SETD2, ARIH2, TET3 for further analysis. ChIP-qPCR revealed increased 5hmC modification at the mutant loci of these genes, consistent with a finding that the genomic regions with increased 5hmC modification are more subjective to occur mutations (PMID: 28440315). TET3 and SETD2, as tumor suppressors, exhibit frameshift-deletion. ARIH2 represents a single-nucleotide variant (ARIH2K440N). To test if ARIH2 loss-of-function accelerates proliferation, we transduced NHD13 c-kit+ cells with shARIH2 and performed CFC analysis. ARIH2 knockdown increased colony number relative to ctrl (shARIH2: 163±3.7 vs. shCtrl: 95±1.7, n=3, p&lt;0.01). To assess ARIH2K440N function we constructed the corresponding human mutation ARIH2K441N as well as ARIH2R484I, seen in a patient with MDS (PMID: 19620960). We genetically deleted endogenous ARIH2 of K562 cells, then transfected with exogenous ARIH2-WT or ARIH2 mutants respectively. Unlike ARIH2-WT, ectopic expression of ARIH2K441N or ARIH2R484I blocked FGFR1 ubiquitination and degradation, activating STAT5, AKT and ERK1/2 signaling, suggesting that TET2 loss leads to secondly hits, which activate proliferation/survival signaling. Taken together, our finding provides a rationale for enhancing TET2 function to block MDS-malignant transformation.
Citation Format: Feiteng Huang, Jie Sun, Wei Chen, Lei Zhang, Xin He, Haojie Dong, Yuhui Wu, Hanying Wang, Zheng Li, Victor Huang, Samer Khaled, Ling Li. TET2 deficiency accelerates leukemogenesis in the NHD13 mouse model of MDS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2934.