Identification of a Novel Epigenetic Regulator That Acts As a Tumor Suppressor in the Development of MLL AML Leukemic Stem Cells
Authors: Yi Hangyu , Leung Halina , Holik Aliaksei , Voli Florida , Asselin-Labat Marie-Liesse , Wang Jenny Yingzi 
Published: October 2019

Abstract
Acute myeloid leukemia (AML) is still a deadly form of leukemia due to frequent relapse caused by the persistence of drug-resistant leukemic stem cells (LSCs). We have previously demonstrated a crucial role for β-catenin signaling in regulating LSCs and identified GPR84 as an important β-catenin regulator in the maintenance of mixed-lineage leukemia (MLL) LSCs (Wang et al., Science 2010; Dietrich et al., Blood 2014). Hence, targeting LSCs by pharmacological inhibition of GPR84/β-catenin signaling represents a promising therapeutic approach.
In collaboration with a pharmaceutical company that has developed a novel GPR84 antagonist (GP), we investigated the effect of GP in MLL pre-leukemic stem cell (pre-LSC) function. GP (20 μM) significantly inhibited the colony forming ability of MLL pre-LSCs (P < 0.0001) but had little effect on normal hematopoietic stem cells. Quantitative RT-PCR and western blot analysis confirmed GP-induced downregulation of GPR84 target genes, including Hoxa5, Hoxa7 and Meis1a, indicating GP-induced inhibition of GPR84 signaling.
To further examine the mechanism of GPR84 inhibition on MLL pre-LSCs, we evaluated several epigenetic regulators (i.e. JMJD1c and EZH2) known to promote leukemogenesis (Zhu et al., ‎J Clin Invest 2016; Tanaka et al., Blood 2012). Western blot analysis showed that inhibition of GPR84 signaling did not alter the expression of JMJD1c or EZH2. However, we observed a significant increase in the expression of a novel and not-yet-characterized histone demethylase (HD) in AML. To investigate the role of HD in AML leukemogenesis, we overexpressed HD in MLL pre-LSCs and subsequent serial replating assay showed a marked reduction in colony forming ability (P < 0.005), indicating impaired self-renewal in vitro. Consistent with our in vitro observations, in vivo transplantation in syngeneic mice revealed a significant delay in leukemia onset and increase in mouse survival (P < 0.001). We next performed western blot analysis to examine the demethylase activity of HD, and our data revealed that HD overexpression caused a substantial reduction in global histone 3 lysine 36 dimethylation (H3K36me2), an epigenetic mark normally associated with transcriptional activation and elongation. In order to identify genes regulated by HD through demethylation of H3K36me2, we performed H3K36me2 ChIP-seq on HD overexpressing MLL pre-LSCs. Our analysis identified several genes including anti-apoptotic protein Mcl-1 and angiogenic receptor Nrp1, which are known to be involved in AML leukemogenesis, with decreased H3K36me2 mark on both the transcriptional start site and gene body. Subsequent western blot analysis confirmed the decreased expression of both Mcl-1 and Nrp1 in HD overexpressing pre-LSCs. Given the prominent roles of anti-apoptosis and angiogenesis in the development of hematologic malignancies such as leukemia, we are currently evaluating these mechanisms caused by HD overexpression in an important subtype of AML.
Taken together, our study identifies a novel histone demethylase that acts downstream of GPR84 signaling to function as a potent tumor suppressor in the development of MLL LSCs.

Disclosures
No relevant conflicts of interest to declare.