C-Myc Circuit in Higher-Risk Myelodysplastic Syndrome

Poster Presentations – 14th International Symposium on Myelodysplastic Syndromes / Leukemia Research 55 S1 (2017) S45–S167

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Fig. 1. (abstract: 164)

Medicine, Medical Faculty Mannheim of the University of Heidelberg, Mannheim, Germany Introduction: Erythroferrone (ERFE) is a regulator of iron homeostasis in the context of hematopoietic stress and erythropoietin (EPO) stimulation as it induces increased iron availability by downregulation of hepcidin. Growth Differentiation Factor 15 (GDF15) is a cytokine belonging to the transforming growth factor-beta superfamily. Specific signaling pathways for GDF15 still remain unknown. However, aberrant expression of GDF15 has been associated with a wide range of pathologies including cellular stress response during inflammation, ischemia, systemic iron metabolism and malignancy. In order to investigate potentially disturbed molecular roles of ERFE and GDF15 in Myelodysplastic Syndromes (MDS), we analyzed their gene expression levels in purified CD71 positive bone marrow cells derived from MDS patients and healthy controls and correlated the differential expression data with clinical parameters and survival. Methods: CD71+ erythroprogenitor cells of a total of n = 148 MDS, n = 18 sAML patients and n = 35 healthy individuals were isolated immunomagnetically via MACS columns (Miltenyi). After total RNA extraction using the AllPrep DNA/RNA Mini kit (Qiagen), cDNA was transcribed from RNA via Quantitect cDNA synthesis kit (Qiagen). Subsequently, ERFE and GDF15 expression was quantified from cDNA by quantitative PCR. Results: As compared to unselected BM or other specific differentiation lineages, ERFE was almost exclusively expressed in the erythropoietic CD71+ compartment. ERFE expression in the CD71+ subset revealed a highly significant overexpression in MDS IPSS-low/int-1-risk (fold change (FC) = 4.3, p < 0.0001), IPSS-int-2/

high-risk (FC = 6.23, p < 0.0001) and sAML (FC = 6.69, p < 0.0001) relative to healthy controls. As compared to healthy controls, GDF15 showed a mean FC = 66 ( p < 0.0001). Interestingly, ERFE expression showed a negative correlation with Hb and positive correlation with blasts. GDF15 levels were positively correlated with transferrin saturation and endogenous EPO levels. Both high ERFE and GDF15 expression levels in CD71+ progenitor cells were significantly associated with superior overall survival (ERFE p = 0.0008, GDF15 p = 0.0069). Conclusion: Highly aberrant overexpression of ERFE and GDF15 in CD71+ erythropoietic progenitor cells suggests an important role for these genes in the dysfunctional erythropoiesis of MDS. The relationship of high ERFE and GDF15 expression with superior survival, especially in low risk MDS patients with no apparent coherence to other established clinical markers warrants further pursuit of ERFE and GDF15 expression profiles in CD71+ BM cells of MDS patients as a possible independent prognostic marker and therapeutic target.

166 RIBOSOMAL PROTEIN L23 NEGATIVELY REGULATES CELLULAR APOPTOSIS VIA THE RPL23/MIZ-1/C-MYC CIRCUIT IN HIGHERRISK MYELODYSPLASTIC SYNDROME L. Wu1, Y. Qi1, X. Li1, C. Chang1, F. Xu1, Q. He1, Y. Zhao1 1 Department of Hematology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, China Ribosomal protein L23 (RPL23) is a component of the 60S ribosomal subunit which is suggested to be a negative regulator of cell

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Poster Presentations – 14th International Symposium on Myelodysplastic Syndromes / Leukemia Research 55 S1 (2017) S45–S167

apoptosis. Previous research has reported that RPL23 overexpression confers apoptotic resistance in CD34+ cells derived from patients with higher-risk myelodysplastic syndrome (MDS) and RPL23 may serve as an independent predictor of prognosis. However, the mechanism underlying RPL23-induced apoptotic resistance in higher-risk MDS patients is poorly understood. In this study, the RNAi knockdown of RPL23 was performed in SKM-1/K562 cells, and subsequent biological function studies suggested that reduced expression of RPL23 suppressed cellular viability by strongly inducing apoptotic cell death and G1/S phase arrest in both cell lines. To further investigate the molecular mechanisms involved in these phenomena, we performed a gene microarray analysis of RPL23-knockdown (KD) and matched control (NC) samples and array data were interpreted by Gene Set Enrichment Analysis (GSEA). The elevated expression of Miz1 and its target molecules p15Ink4b and p21Cip1, as well as the decreased expression of Miz1’s functional repressor, c-Myc was captured due to RPL23 deficiency. GSEA-pathway analysis based on the KEGG

database indicated pathways in cancer (NES = 2.09, nominal p < 0.000) and apoptosis (NES = 1.65, nominal p = 0.033) were highly enriched and the leading edge subsets included Miz1, p21Cip1, cMyc and other apoptosis-related genes. Cells derived from higherrisk MDS patients demonstrated consistent increases in RPL23 and c-Myc expression but decreased Miz1 expression compared with cells from lower-risk patients and normal donors by methods of qRT-PCR and IHC analysis. And the expression of RPL23 and c-Myc mRNA levels demonstrated a strong synthetic relationship in MDS patients (r = 0.703, p < 0.000). In conclusion, expression of Miz-1 decreases in higher-risk MDS patients with overexpression of RPL23, and Miz-1-dependent induction of p15Ink4b and p21Cip1 was subsequently depressed under conditions of higher c-Myc levels. As RPL23 is encoded by a direct target gene of c-Myc, the RPL23/Miz1/c-Myc regulatory circuit may provide a positive feedback loop that links efficient RPL23 expression with c-Myc function to suppress Miz1-induced

Poster Presentations – 14th International Symposium on Myelodysplastic Syndromes / Leukemia Research 55 S1 (2017) S45–S167

Cdk inhibitors and thereby promotes apoptotic resistance happened in higher-risk MDS patients. 167 IGF-IR PROMOTED CLONAL CELL PROLIFERATION IN MYELODYSPLASTIC SYNDROMES THROUGH PI3K AND MAPK PATHWAYS Q. He1, F. Xu1, X. Li1, C.K. Chang1 1 Hematology, Shanghai Jiaotong University Affiliated Sixth People’s Hospital, Shanghai, China IGF-IR (type 1 insulin-like growth factor receptor) is considered to play a key role in many biological processes and cancer development. However, the effect of IGF-IR and the main function is not fully understood in myelodysplastic syndrome. In this study, downregulation of IGF-IR by RNA interference inhibited the proliferation (Figure 1A), promoted cell apoptosis (Figure 1B, C) and reduced the percentage of cells in the S phase in SKM-1 cell line (Figure 1D, E). The gene expression profile analysis from SKM-1 KD cells showed 1,654 differentially expressed genes compared with SKM-1 CTRL

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cells: 954 were downregulated, while 700 genes were upregulated. Pathway analysis showed that 5 significant pathways were affected by downregulated genes in SKM-1 cell line: including PI3K-Akt and MAPK signaling pathway (Figure 2A). GO analysis showed that 5 significant processes were affected by downregulated genes (Figure 2B). Further pathway-net analysis revealed that MAPK, PI3K and RAP1A were described as key pathways attributed by downregulated genes Validated FCM analysis showed that the expression of signaling pathway proteins, including AKT1, p-AKTS473, ERK, p-ERK1/2-T202/Y204, p-STAT3-Y705 and p53, significantly decreased, and the former 4 proteins expression decreased more apparently in the SKM-1 cells with knockdown of IGF-IR compared with the control cells. The expression of CCNB1 increased significantly after knockdown of IGF-IR (Figure 2C). These results indicated that MAPK and PI3K pathways were obviously inhibited, and apoptosis pathway was apparently activated after knockdown of IGF-IR. We also carried out the IGFIR signaling-inhibited experiment to investigate the potential of IGF-IR as therapeutic target of MDS. Suppression of kinase activities of IGF-IR by specific inhibitor PPP resulted in inhibition of cell