P431: THE ROLE OF SMARCA4 IN HEMATOPOIESIS AND ACUTE MYELOID LEUKEMIA

Background:SMARCA4 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a) is the central ATPase containing enzyme in the SWI/SNF- (switch/sucrose non-fermentable) complex, which influences gene transcription by regulating chromatin accessibility. In solid tumors, SMARCA4 mainly possesses tumor suppressive features. However, in the setting of acute myeloid leukemia (AML), SMARCA4seems to play a different role, acting as a proliferation stimulus for myeloid blasts. Aims: The aim was to investigate the role of SMARCA4 in normal hematopoiesis and in pathogenesis and maintenance of AML to evaluate the prognostic and potential therapeutic relevance of this protein. Methods: We screened 152 bone marrow samples of newly diagnosed adult AML patients compared to 29 healthy bone marrow samples for SMARCA4 expression by using RT-qPCR. SMARCA4 expression was correlated to overall and relapse free survival. We further analyzed proliferation and colony-formation capacity of AML cell lines with lentivirally transduced shRNA-mediated SMARCA4 knockdown or SMARCA4 overexpression induced by lentiviral transduction. Two mouse models were generated: Firstly, to evaluate the impact of SMARCA4 knockout on normal hematopoiesis, a transgenic mouse model with Cre-recombinase mediated conditional knockout of SMARCA4 under the control of an Mx-1 promoter was developed. Blood counts, bone marrow smear, colony formation assays, and RNA expression profiles were analyzed. Secondly, a xenograft mouse model was generated by transplantating the AML cell line MV4-11 with SMARCA4 knockdown or overexpression in NSG mice to analyze overall survival. Results: Expression of SMARCA4 was significantly higher in adult AML samples compared to healthy CD34+ cells (p<0.0001). High SMARCA4 expression was associated with worse overall (p=0.001) and relapse free survival (p=0.0073). SMARCA4 knockdown in AML cell lines resulted in significantly lower proliferation rate in three different AML cell lines compared to controls, whereas SMARCA4 overexpression led to higher proliferation rates. Overall survival of mice transplanted with SMARCA4 overexpressing MV4-11 cells was significantly worse than in the control group, whereas mice transplanted with SMARCA4 knockdown MV4-11 cells lived significantly longer. Transgenic SMARCA4 knockout mice showed significantly lower leukocyte, reticulocyte, and thrombocyte counts and showed lower colony forming capacity compared to Cre-negative control mice. Bone marrow smears showed aplasia of all cell lines in SMARCA4 knockout mice. RNA-sequencing revealed 749 upregulated and 520 downregulated genes with a log fold change ≥ +/- 1 in the bone marrow of SMARCA4knockout mice compared to Cre-negative control mice. Relevant pathways according to REACTOME data base performing over representation analysis included genes of generation of second messenger molecules, cell surface interactions at the vascular wall, and immunoregulatory interactions between lymphoid and non-lymphoid cells. Image:Summary/Conclusion:SMARCA4 is upregulated in primary AML cells and is an unfavorable prognostic marker in AML patients. SMARCA4 overexpression in AML cell lines leads to higher proliferation rate and to a worse overall survival in transplanted mice. In normal hematopoiesis, SMARCA4 knockout leads to decreased cell growth in bone marrow, highlighting the dependence on SMARCA4 for proliferation of hematopoietic (stem) cells.