ALBERT

Description: Background DHX9(Asp-Glu-Ala-His box helicase 9) is considered to play a key role in many biological processes and cancer development. However, the effect of DHX9 mutation and the main function is not fully understood in myelodysplastic syndromes (MDS). Objective The objective of this study is to investigate the frequency of DHX9 mutations and their relationship with clinical characteristics in MDS. Methods Next generation sequencing was used to determined the DHX9 mutations and other recurrent genes mutations in 320 MDS patients. The effect of DHX9 mutations or combination with other genes mutations on patients' survival was analyzed. The association of DHX9 mutations with clinical characteristics of MDS was also studied. In addition, the effect of DHX9 on tumor biological features was evaluated in vitro. Results 1. DHX9 mutations were detected in 10.94% of MDS patients(35/320). 42.9% of these mutation were del(c.306_308), 31.4% were splicing mutations (c.674-4A〉G; c.2512+8C〉A) and the rest (25.7%) were missense mutation. In these patients with DHX9 mutations, 24(68.6%) had concomitant occurrence with other mutations such as ANKRD11, TET2, ASXL1, TP53 and U2AF1 (Fig.1A). 2. The patients with DHX9 mutations especially sole mutations had significantly superior survivalcompared with those without mutations (Fig.2B). The patients with DHX9 mutations also had superior survivalcompared with those with genes mutations with poor prognosis, such as DNMT3A/RUNX1/ASXL1, IDH1/TP53/U2AF1, PTPRD/ANKRD11, even those mutations with good prognosis such as TET2 and SF3B1 (Fig.2C, 2D and 2E). 3. We also analyzed the difference in clinical features between the patients with and without DHX9 mutations (Table 1). Obviously, the patients with DHX9 mutations exhibited more severe BM failure and reduced Tc1/Tc2 polarization. 4. The quantitive PCR revealed that the patients with DHX9 mutations had lower expression of DHX9 mRNA than those without DHX9 mutations. The MDS patients had significantly higher expression of DHX9 than normal controls. No difference was observed in DHX9 expression between the MDS patients with DHX9 mutations and normal controls (Fig.2A). 5. In vitro experiments,knockdown of DHX9 induces increased cell apoptosis and inhibits cell growth in myeloid cell lines (SKM-1, K562 and HEL cells) (Fig.2B and 2C). Knockdown of DHX9 significantly enhanced the expression of apoptotic proteins (Fig.2D). Conclusion DHX9 mutations are novel recurrent molecular aberrations in MDS and closely related to bone marrow failure. Figure 1. The distribution of DHX9 mutations and the effect of DHX9 mutations or combination with other genes mutations on patients' survival. Figure 1. The distribution of DHX9 mutations and the effect of DHX9 mutations or combination with other genes mutations on patients' survival. Figure 2. Comparison between patients with or without DHX9 mutations Figure 2. Comparison between patients with or without DHX9 mutations Figure 3. The mRNA expression of DHX9 in MDS and the effect of DHX9 knockdown in myeloid cell lines Figure 3. The mRNA expression of DHX9 in MDS and the effect of DHX9 knockdown in myeloid cell lines Disclosures No relevant conflicts of interest to declare.

Description: Background Increased cell apoptosis mediated by P53 or activated autoimmunity was frequent in low-grade MDS especially in 5q-syndrome and those with trisomy 8. However, MDS clonal cells can gradually acquired apoptosis resistance under different stressed conditions. The mechanism of apoptosis resistance is still not fully understood. The polycomb group protein EZH2 as an anti-apoptosis protein drives tumorigenesis. The association of EZH2 with apoptosis resistance of MDS clonal cells is worth further investigating. Objective The objective of this study is to investigate the role of EZH2 in the mechanism of apoptosis resistance of MDS. Methods The expression of EZH2 protein was analyzed by flow cytometry in 71 MDS patients. EZH2-targeted genes were identified through an integrated analysis of gene expression microarray analysis in MDS patients and cell line. EZH2-targeted genes were confirmed through chromatin immunoprecipitation and luciferase reporter gene assay. Cell functional and metabolic experiments were also performed. Results We observed upregulation of the EZH2 expression in the patients with low-grade MDS especially in the individuals with trisomy 8 although downregulation in the patients with high-grade MDS. Based on the distinct EZH2 expression pattern, we identified an EZH2-targeted gene PSAT1, a serine biosynthetic critical enzyme through an integrated analysis of gene expression microarray analysis in MDS patients and cell line. Functionally, EZH2 can directly activate PSAT1 transcription by binding to its promoter and this effect is independent of its H3K27me3 enzymatic activity in vitro. Expanded sample analysis revealed the overexpression of PSAT1 in MDS patients especially in low-grade MDS. High expression of PSAT1 was associated with severe cytopenias, iron overload and activation of T lymphocytes-mediated autoimmunity. In vitro, overexpression of PSAT1 led to a significant proliferation advantage in the MDS and leukemia cell lines. Overexpression of PSAT1 could also downregulate apoptosis sensitivity to inflammatory cytokines and cytotoxic agents in vitro. However, knockdown of PSAT1 resulted in reduced cell growth capacity and increased the apoptosis sensitivity to cytotoxic agents. Further cancer metabolic experiments showed that overexpression of PSAT1 inhibited the ROS generation and induced an increase in glucose uptake, lactate and ATP production, which may make MDS clonal cells adaptive in the stress-related apoptosis environment. Conclusion EZH2/PSAT1 axis-mediated serine biosynthetic pathway is activated in low-grade MDS, which may confer MDS clonal cells growth advantages and resistance to stress-related apoptosis. Disclosures No relevant conflicts of interest to declare.