Pb1803: smad1 is a tumor suppressor in acute myeloid leukemia with mll rearrangement

Topic: 3. Acute myeloid leukemia - Biology & Translational Research Background: Rearrangements of the Mixed Lineage Leukemia 1 (MLL1) gene in hematopoietic stem and progenitor cells cause leukemia of myeloid and lymphoid origin. MLL1 is a histone lysine-methyltransferase, which methylates H3K4, acts as an epigenetic gene expression regulator and plays an important role in embryonic development and hematopoiesis. H3K4 methylation at promotor regions is associated with active transcription. Due to aberrant function and protein complex formations of MLL1 fusion proteins (MLL-FPs) the expression of many target genes is dysregulated. Upregulation of the leukemogenic homeobox (HOX) family members and Myeloid Ecotropic Viral Integration Site 1 (MEIS1) is a hallmark of MLL rearranged (MLLr) leukemia and critical for leukemia initiation and sustainment. Mothers Against Decapentaplegic Homolog 1 (SMAD1), a transcription factor which is part of the TGF-β and BMP signaling axes, was previously described as a tumor suppressor in Diffuse Large B-cell Lymphoma1 and shown to influence the expression of HOXA92. Aims: We aim to investigate the tumor suppressive function of SMAD1 in MLLr acute myeloid leukemia (AML). Methods: RNA and protein levels of patient samples and cell lines were analyzed using RT-qPCR and Western Blot. MLL-FP knockdown (KD) in MLLr MV4-11 cells was generated with the CRISPR/Cas9 technology. SMAD1 was stably expressed in MV4-11 cells, which express very low levels of SMAD1. MLL fusion genes were introduced into cord blood-derived cells by CRISPR/Cas9 mediated site-directed mutagenesis3. DNA staining and flow cytometry readout were used for cell cycle analysis. An orthotopic xenograft NSG mouse model was used to study the effect of SMAD1 expression in MV4-11 cells in vivo. H3K4 tri-methylation (me3) at the SMAD1 promotor in cells with and without MLLr, was measured by Chromatin Immunoprecipitation (ChIP) coupled with RT-qPCR. Results: The expression of SMAD1 was significantly lower in the bone marrow and the peripheral blood of MLLr AML patients compared to other AML subtypes. Congruently, SMAD1 expression was nearly abolished in cord blood-derived MLLr cells compared to normal cord blood. Vice versa, KD of the MLL-FP in MV4-11 cells led to an increase in SMAD1 mRNA and protein level. Expression of SMAD1 in MV4-11 cells caused a TGF-β-dependent growth disadvantage and G1 cell cycle arrest in vitro. Notably, the expression of SMAD1 decreased HOXA9 and MEIS1 mRNA levels, which was enhanced by TGF-β treatment. Orthotopic transplantation of SMAD1 expressing MV4-11 cells into NSG mice led to reduced engraftment potential in the bone marrow and the spleen compared to control cells (image). ChIP RT-qPCR revealed that H3K4me3 levels at the SMAD1 promotor in MV4-11 cells (0.26 % Input) was lower than in MLL1 WT HL-60 cells (9.8 % Input). Summary/Conclusion: Our data shows that MLL rearrangement leads to loss of SMAD1 expression and that SMAD1, as part of TGF-β signaling, has a tumor suppressive function in AML. We conclude that SMAD1 loss, presumably due to reduced H3K4me levels at the SMAD1 promotor, indirectly contributes to the leukemogenesis of MLLr AML by impacting the regulation of HOXA9 and MEIS1.Keywords: MLL, AML, Smad