Ps970 Acute Myeloid Leukemia Cells Are Dependent on S100a4 for Growth and Survival

Abstract

Background:Nuclear mislocalization of proteins can interfere with normal cellular function and cooperatively drive tumor development. To understand how this process mediates AML development, we analyzed the nuclear proteome of AML blasts in comparison with normal human CD34+ cells to identify misregulated nuclear proteins. In a preliminary study, we identified S100A4 to be over‐expressed in 73% (11/15) AML patients and mislocalized to the nucleus in AML blasts. S100A4 belongs to the S100 multigene family of calcium‐binding proteins of the EF‐hand type and has been implicated in tumor progression and metastasis in many solid tumours but little is known of its role in hematological malignancy.Aims:To validate S100A4 overexpression and mislocalization in primary AML blasts. To determine the functional role of S100A4 on hematopoietic cell development, growth and survival in normal human CD34+ cells and AML cell lines.Methods:S100A4 expression was determined by western blot. Lentiviral vectors were used to ectopically express S100A4 and shRNA was used to knock down S100A4 expression. Effects on cell growth, survival and differentiation were determined by flow cytometry. To determine S100A4 interacting proteins, S100A4 was co‐immunoprecipitated with its binding partners using Ca+2 enriched conditions from nuclear extracts of ME‐1 cells followed by LC‐MS/MS analysis.Results:Using western blotting, S100A4 protein expression was observed in the nucleus of AML blasts (73%; 24/33) whilst normal CD34+ or CD14+ differentiated monocytic controls have shown only cytosolic expression of S100A4. Upregulated S100A4 is also supported by transcriptome analysis indicating that overexpression arises at least partly at a transcriptional level. An independent dataset (TCGA) supports the overexpression of S100A4 mRNA in AML and suggests that overexpression may confer a poor prognosis (p = 0.0118). To determine whether ectopic expression of nuclear S100A4 can affect the growth and survival of CD34+ cells, we attempted to overexpress nuclear‐targeted S100A4 in normal human hematopoietic cells. Overexpression of nuclear S100A4 could not be demonstrated in transduced CD34+ or in normal differentiated cells (probably due to rapid degradation of ectopically expressed S100A4 in these cells). To examine the importance of S100A4 expression in normal and leukaemic cells we next knocked‐down S100A4 expression. In CD34+ cells we observed no significant effect on the growth or lineage development of these cells suggesting S100A4 is not required for normal hematopoiesis. We next examined the consequences of knocking down S100A4 expression in AML cell lines (NOMO‐1, TF‐1, THP‐1, and OCI‐AML2). S100A4 knockdown severely impaired the growth TF‐1 and OCI‐AML2 (p < 0.05). Significant apoptosis was observed in OCI‐AML‐2, TF‐1, and NOMO‐1 (p < 0.05). Using co‐immunoprecipitation coupled with LC/MS to identify binding partners of S100A4, we identified heterogeneous nuclear Ribonucleoprotein M (hnRNPM) as novel binding partner of S100A4 AML. Interestingly, hnRNPM has been implicated in metastasis in breast cancer through mediating alternative splicing.Summary/Conclusion:We found that S100A4 is mislocalized to the nucleus in AML blasts compared to normal hematopoeitic cells and is essential for AML cells growth and survival. HnRNPM emerged as novel binding partner to S100A4 and we hypothesise that S100A4 mediates its effect through forming a splicing complex with known leukemic splicing factors such as SF3B1 and SRSF2 (also been pulled down with S100A4 co‐IP).