SWI/SNF Subunits SMARCA4, SMARCD2 and DPF2 Collaborate in MLL-Rearranged Leukaemia Maintenance

<div><p>Alterations in chromatin structure caused by deregulated epigenetic mechanisms collaborate with underlying genetic lesions to promote cancer. SMARCA4/BRG1, a core component of the SWI/SNF ATP-dependent chromatin-remodelling complex, has been implicated by its mutational spectrum as exerting a tumour-suppressor function in many solid tumours; recently however, it has been reported to sustain leukaemogenic transformation in MLL-rearranged leukaemia in mice. Here we further explore the role of SMARCA4 and the two SWI/SNF subunits SMARCD2/BAF60B and DPF2/BAF45D in leukaemia. We observed the selective requirement for these proteins for leukaemic cell expansion and self-renewal <i>in-vitro</i> as well as in leukaemia. Gene expression profiling in human cells of each of these three factors suggests that they have overlapping functions in leukaemia. The gene expression changes induced by loss of the three proteins demonstrate that they are required for the expression of haematopoietic stem cell associated genes but in contrast to previous results obtained in mouse cells, the three proteins are not required for the expression of c-MYC regulated genes.</p></div

No changes in <i>MYC</i> expression after SMARCA4 knockdown.

<p>(A-B) GSEA plots showing enrichment of the indicated gene sets in genes ranked by Signal2noise metric in SMARCA4 knockdown versus control THP-1 cells. (C) Top: schematic representation of the human <i>MYC</i> locus; coding sequence is marked in green; qPCR primer pairs and their amplification products are represented as regions 1–4. Bottom: Relative mRNA levels of <i>MYC</i> assessed by using the indicated primer pairs. (D-F) Myc rescue experiments of Smarca4 and Jmjd1c KD in MLL-AF9 cells. (D-E) Relative mRNA levels of the indicated genes in cells transduced with the indicated combinations of vectors. e, empty vector; Myc, Myc expression vector; shScr, non-targeting control pMLS-YFP vector; shJmjd1c and shSmarca4, pMLS-YFP vectors targeting Jmjd1c and Smarca4, respectively. (F) Normalised ratios of GFP⁺ YFP⁺ cell percentages between shJmjd1c and shScr samples (left) or shSmarca4 and shScr samples (right) plotted over an 8-day time course starting from day 2 after transduction.</p

Knockdown of SWI/SNF complex subunits impairs growth of human leukaemic cells

<p>(A) Number of THP-1 and MV4-11 cells over a time course starting from day 4 after transduction. Cells were transduced with the indicated pLKO vectors. (B) Western blots showing SMARCA4, SMARCD2 and DPF2 levels in THP-1 cells transduced with shScr or the indicated pLKO vectors. Vinculin was used as a loading control. (C) Number of Jurkat and T-ALL1 cells over a time course starting from day 3 after transduction. Cells were transduced with the indicated pLKO vectors. (D) Relative mRNA levels of <i>SMARCA4</i> in Jurkat and T-ALL1 cells transduced with the indicated pLKO constructs.</p

Inhibition of Smarca4, Smarcd2 or Dpf2 expression leads to apoptosis, cell cycle changes and myeloid differentiation.

<p>(A) Experimental overview for panels B and C. (B) Relative percentage of Annexin V-positive MLL-AF9 cells at days 2, 3 and 4 after transduction with non-targeting control (shScr) or shRNAs targeting <i>Smarcd2</i>, <i>Smarca4</i> and <i>Dpf2</i>. (C) Cell cycle analysis of Smarcd2, Smarca4 and Dpf2-depleted MLL-AF9 cells compared to control cells (shScr). (D) Flow cytometry analysis of c-Kit and Mac1 staining in MLL-AF9 cells transduced with shScr, shSmarca4, shSmarcd2 or shDpf2. (E) Representative images of May-Grünwald-Giemsa stained MLL-AF9 cells transduced with the indicated shRNAs.</p

Gene expression changes upon knockdown of single SWI/SNF complex components in human AML cells.

<p>(A and B) Venn diagrams showing overlap of genes significantly changing down (A), or up (B) (p<0.05) upon SMARCA4, SMARCD2 and DPF2 knockdown in THP-1 cells. (C-D) Gene set enrichment analysis (GSEA) plots showing enrichment of indicated gene sets in genes ranked by Signal2noise metric in SMARCA4 or DPF2 knock-down versus control THP-1 cells.</p

Depletion of single SWI/SNF complex components inhibits AML maintenance.

<p>(A) Mouse MLL-AF9 cell number fold change between day 5 and day 9 after transduction. Cells were transduced with pLKO constructs expressing the indicated shRNAs and selected with puromycin from day 2 after transduction. (B) Relative mRNA levels of <i>Smarca4</i>, <i>Smarcd2</i> and <i>Dpf2</i> in MEFs transduced with the indicated pLKO constructs. (C) Number of colonies generated by MLL-AF9 cells transduced with the indicated pMLS vectors. (D) Relative mRNA levels of <i>Smarca4</i>, <i>Smarcd2</i> and <i>Dpf2</i> in MEFs transduced with the indicated pMLS vectors. (E) Rescue experiments. MLL-AF9 cells were co-transduced with pMLS-YFP carrying shScr, shSmarca4, shSmarcd2 or shDpf2, as indicated, and control pMIGRI (pMIG-Stuffer) or pMIGRI expressing human <i>SMARCA4</i>, <i>SMARCD2</i> or <i>DPF2</i> cDNA. Normalized ratios of GFP⁺/YFP⁺ cell percentages between shSmarca4, shSmarcd2 or shDpf2 and shScr samples are plotted over a time course starting from day 2 after transduction. (F) Competitive proliferation assay of c-Kit-enriched mouse bone marrow cells transduced with the indicated pMLS vectors. (G) Left: Number of colonies generated by LSK cells transduced with the indicated pLKO shRNAs. Right: Absolute numbers of LSK cells with indicated knockdown in liquid culture. (H) Survival curves of sublethally irradiated mice transplanted with 10⁴ MLL-AF9 cells transduced with the indicated pMLS vectors.</p

Distinct subsets within the classically defined CMP and MEP compartments can be segregated on the basis of CD55, CD150 and CD31 expression.

<p><b>A-B.</b> Proposed models of fetal liver myeloid progenitor hierarchy. A. In wild type fetal liver, commitment to the erythroid lineage correlates with downregulation of the <i>Runx1b</i> and <i>Gfi1</i> TFs, plus the cell surface markers CD150, CD31, CD45 and CD48. B. RUNX1-null fetal liver hematopoietic progenitors have impaired Mk and erythroid differentiation (dark blue bars), whereas RUNX1C-null (<i>P1-MRIPV)</i> hematopoietic progenitors have impaired megakaryocyte specification, displaying enhanced erythroid and granulocyte/monocyte commitment (light blue arrows).</p

<i>Runx1-P2</i> reporter expression demarcates mono and bipotent subsets in the CD150⁺ MEP.

<p><b>A-B.</b> Expression of <i>Runx1-P1-GFP</i> and <i>P2-hCD4</i> in <i>P1-GFP</i>::<i>P2-hCD4/+</i> E12.5, E13.5 and E14.5 FL CD41^- CD150⁺ MEPs. A. Representative FACS plots. B. Quantitation of the proportions of <i>P1</i>^<i>-</i> <i>P2</i>^<i>-</i>, <i>P1</i>^<i>+</i> <i>P2</i>^<i>-</i>, <i>P1</i>^<i>+</i> <i>P2</i>^<i>+</i> and <i>P1</i>^<i>-</i> <i>P2</i>^<i>+</i> MEPs as a percentage of total live red blood cell lysed fetal liver cells. N = 3 C. Differential CFU-C activity of wild type, <i>P2-hCD4</i>^<i>-</i> and <i>P2-hCD4</i>^<i>+</i> E14.5 FL CD41^- CD150⁺ MEPs. N = 6 <b>D-E.</b> Lineage output of day 7 OP9 co-cultured wild type, <i>P2-hCD4</i>^<i>-</i> and <i>P2-hCD4</i>^<i>+</i> CD41^- CD150⁺ MEPs. D. Representative FACS plots of TER119 and CD41. E. Proportion of erythroid and megakaryocyte cells. N = 4 <b>F-G.</b> Lineage output of single cultured <i>P2</i>^<i>-</i> and <i>P2</i>^<i>+</i> MEPs in OP9 co-cultures (F) and MethoCult semi-solid myeloid culture medium (G). <b>H-I.</b> Short-term (14 hours) differentiation of <i>P2</i>^<i>-</i> and <i>P2</i>^<i>+</i> CD41^- CD150⁺ MEPs in pro-myeloid liquid culture. H. Representative FACS plots of CD16/32/CD34 (top) and CD150/CD41 (bottom) expression in the LK gate of cultured MEPs. I. Proportions of immunophenotypic MkP, CD41^- CD150^- MEPs and CD41^- CD150⁺ MEPs in short-term cultures. N = 5.</p

<i>Runx1-P2-hCD4</i>^<i>+</i> CMPs have enriched multilineage output.

<p><b>A-B.</b> Expression of <i>Runx1-P1-GFP</i> and <i>P2-hCD4</i> in <i>P1-GFP</i>::<i>P2-hCD4/+</i> E12.5, E13.5 and E14.5 FL CD41^- CD150⁺ CMPs. A. Representative FACS plots. B. Quantitation of the proportions of <i>P1</i>^<i>-</i> <i>P2</i>^<i>-</i>, <i>P1</i>^<i>+</i> <i>P2</i>^<i>-</i>, <i>P1</i>^<i>+</i> <i>P2</i>^<i>+</i> and <i>P1</i>^<i>-</i> <i>P2</i>^<i>+</i> CMPs as a percentage of total live red blood cell lysed fetal liver cells. N = 3 <b>C.</b> Differential CFU-C activity of wild type, <i>P2-hCD4</i>^<i>-</i> and <i>P2-hCD4</i>^<i>+</i> E14.5 FL CD41^- CD150⁺ CMPs. N = 7 <b>D-E.</b> Lineage output of day 7 OP9 co-cultured wild type, <i>P2-hCD4</i>^<i>-</i> and <i>P2-hCD4</i>^<i>+</i> CD41^- CD150⁺ CMPs. D. Representative FACS plots of CD11b, GR1, TER119 and CD41. E. Proportion of granulocyte/monocyte, erythroid and megakaryocyte cells. N = 7 <b>F-G.</b> Lineage output of single cultured <i>P2</i>^<i>-</i> and <i>P2</i>^<i>+</i> CMPs in OP9 co-cultures. F. Table of plating efficiency. G. Ternary plots displaying proportions of granulocyte/monocyte, erythroid and megakaryocyte cells in each positive well for wild type, <i>P2-hCD4</i>^<i>-</i> and <i>P2-hCD4</i>^<i>+</i> CD41^- CD150⁺ CMPs. <b>H-I.</b> Short-term (14 hours) differentiation of <i>P2</i>^<i>-</i> and <i>P2</i>^<i>+</i> CD41^- CD150⁺ CMPs in pro-myeloid liquid culture. H. Representative FACS plots of CD16/32/CD34 (top) and CD150/CD41 (middle and bottom) expression in the LK gate of cultured CMPs. I. Proportions of immunophenotypic MkPs, CD41^- CD150^- MEPs, CD41^- CD150⁺ MEPs, GMPs, CD41^- CD150^- CMPs and CD41^- CD150⁺ CMPs in short-term cultures. N = 3.</p

Impact of the absence of <i>P1</i>-directed <i>Runx1</i> expression on adult hematopoiesis.

<p>(A) Top: Schematic diagrams of the <i>Runx1 WT</i> (top) and <i>P1-GFP</i> (bottom) alleles. Expression of GFP is directed by <i>Runx1</i> promoter <i>P1</i> but expression of <i>Runx1</i> from the <i>P2</i> promoter remains intact. Bottom: Schematic diagram of the experimental design for the investigation of the impact of <i>Runx1 P1</i> deletion on adult hematopoiesis. Peripheral blood, BM, thymus and spleen samples were collected from adult WT, <i>P1-GFP</i> heterozygous (<i>P1-GFP/+</i>) and homozygous (<i>P1-GFP/GFP</i>) adult mice. All samples were analyzed for mature blood cell surface marker expression. In addition, blood samples were subjected to automated cell counts (Sysmex) and CFU-C assays were performed on unfractionated BM. (B) Peripheral blood cell counts of WT, <i>P1-GFP/+</i> and <i>P1-GFP/GFP</i> mice as determined by Sysmex automated cell counting. (C) Numbers of CD3e+ T cells, CD11b+ Gr1+ GM cells and B220+ CD19+ B cells as a proportion of total ACK-lysed blood cells from WT, <i>P1-GFP/+</i> and <i>P1-GFP/GFP</i> mice. (D) CFU-C activity of WT, <i>P1-GFP/+</i> and <i>P1-GFP/GFP</i> unfractionated ACK-lysed BM following culture in pro-myeloid semi-solid methylcellulose-based medium. (n = 4.) (E) Numbers of erythroid lineage (ProE, EryA, EryB and EryC) cells as a proportion of live unfractionated BM cells. (F) Numbers of T cell lineage populations as a proportion of live unfractionated thymus cells. (G) Numbers of CD4 SP and CD8 SP T cells as a proportion of live unfractionated spleen cells. (H) Ratio of splenic CD4 SP T cells to splenic CD8 SP T cells (n = 4).</p