The AAA+ATPase RUVBL2 is essential for the oncogenic function of c-MYB in acute myeloid leukemia

Abstract

Subtype-specific leukemia oncogenes drive aberrant gene expression profiles that converge on common essential mediators to ensure leukemia self-renewal and inhibition of differentiation. The transcription factor c-MYB functions as one such mediator in a diverse range of leukemias. Here we show for the first time that transcriptional repression of myeloid differentiation associated c-MYB target genes in AML is enforced by the AAA+ ATPase RUVBL2. Silencing RUVBL2 expression resulted in increased binding of c-MYB to these loci and their transcriptional activation. RUVBL2 inhibition resulted in AML cell apoptosis and severely impaired disease progression of established AML in engrafted mice. In contrast, such inhibition had little impact on normal hematopoietic progenitor differentiation. These data demonstrate that RUVBL2 is essential for the oncogenic function of c-MYB in AML by governing inhibition of myeloid differentiation. They also indicate that targeting the control of c-MYB function by RUVBL2 is a promising approach to developing future anti-AML therapies.

Fig. 1. Elimination of established AML following RUVBL2 silencing.

a Luminescence signal in shSCR and shRUVBL2 THP1-LUC2 clones, prior to transplantation. b Western blot analysis of RUVBL2 protein in shSCR and shRUVBL2 clones treated with doxycycline for 3 days, or left untreated. c Quantification of RUVBL2 expression in b. Data are normalised to Actin loading control and to untreated shSCR and shRUVBL2 clones. d Bioluminescence imaging examples of NSG recipient mice 10 days after injection with shSCR or shRUVBL2 THP1-LUC2 clones, and before doxycycline treatment, (day 10, top), and 21 days later, after treatment with doxycycline (+DOX) or not (-DOX) (day 31, bottom). Bars for luminescence signal represent photons/s/cm²/steradian. e Graph depicting AML progression, measured by bioluminescence imaging, in untreated shSCR (black dashed lines, n = 3) and shRUVBL2 (black solid lines, n = 6) recipient mice or following doxycycline treatment in shSCR (red dashed lines, n = 3) and shRUVBL2 (red solid lines, n = 6) mice. There was no significant difference in bioluminescence signal between groups subjected to doxycycline treatment and those left untreated, at day 10 after transplantation, and before doxycycline treatment started (P = 0.37 for shSCR and P = 0.58 for shRUVBL2 groups), unpaired Student’s t test. f Survival curve for recipient mice in e. Red line above curves indicates length of doxycycline treatment. Survival curves were significantly different for doxycycline treated and untreated groups for shRUVBL2 mice (P = 0.0009), but not shSCR mice (P = 0.23), logrank (Mantel-Cox) test.

Fig. 2. RUVBL2 inhibition impairs AML progression but not normal hematopoiesis.

a Apoptosis induction in mouse MLL-ENL clones, inducibly expressing control (shSCR) or mouse Ruvbl2-specific (shRuvbl2) shRNA 6 days after doxycycline treatment. Bars and error bars represent means and SD of fold changes in apoptosis (annexin V positive cells) from three independent experiments. *P < 0.05; n.s. not significant (relative to untreated cells), one sample t test. b Survival curves for recipient mice transplanted with shSCR.1 (dashed lines) and shRuvbl2.2 (solid lines) clones, untreated (black lines, n = 2 for shSCR.1 and n = 5 for shRuvbl2.2) or treated with doxycycline (red lines, n = 4 for shSCR.1 and n = 7 for shRuvbl2.2). Red arrow indicates point at which doxycycline treatment started. P = 0.0002 for doxycycline treated versus untreated shRuvbl2 mice, logrank (Mantel-Cox) test. c Apoptosis induction in inducible control (CON) and human RUVBL2(DN) mouse MLL-ENL clones 48 hours after doxycycline treatment. Bars and error bars represent means and SD of fold changes in apoptosis from three independent experiments. *P < 0.05; n.s. not significant (relative to untreated cells), one sample t test. d Survival curves for recipient mice transplanted with CON (dashed lines) and RUVBL2(DN).1 (solid lines) clones, untreated [black lines, n = 3 for CON and n = 6 for RUVBL2(DN).1] or treated with doxycycline [red lines, n = 3 for CON and n = 7 for RUVBL2(DN).1]. Red arrow indicates point at which doxycycline treatment started. P = 0.0005 for doxycycline treated versus untreated RUVBL2(DN) mice, logrank (Mantel-Cox) test. e Percentages of total (hCD2⁺CD45.1⁺), myeloid (hCD2⁺CD45.1⁺CD11b⁺GR1⁺) and B lymphoid (hCD2⁺CD45.1⁺CD19⁺B220⁺) transduced donor cells in the bone marrow and spleen of recipient mice, four months after reconstitution with mouse HPC, transduced with control (CON) or RUVBL2(DN) expressing lentiviral vectors. Bars and error bars are means and SD of percentages from three control and five RUVBL2(DN) mice. n.s. = not significant, unpaired Student’s t test.

Fig. 3. Changes in gene expression profiles following RUVBL2 silencing in AML cells.

a Western blot analysis of RUVBL2 protein expression (upper panel) following doxycycline (DOX) treatment of THP1 cells transduced with inducible RUVBL2-specific shRNA (shRUVBL2) or control shRNA (shSCR). GAPDH (lower panel) was used as a loading control. b Quantification of RUVBL2 protein expression at days 2 and 4 after doxycycline treatment of shRUVBL2 THP1 cells. Bars and error bars are means and SD of three (day 2) and five (day 4) independent experiments. Data are normalised to GAPDH loading control and to untreated shRUVBL2 THP1 cells. ***P < 0.001, one sample t test. c, d Volcano plots of fold gene expression changes in shRUVBL2 THP1 cells following treatment with doxycycline for c 2 and d 4 days. Expression changes greater than 2-fold and P < 0.05 are shown in red, Wald test. e, f qRT-PCR validation of changes in BTG2, MAF and MAFB expression in THP1 cells following RUVBL2 inhibition by e two independent shRNA or f RUVBL2(DN) over-expression. Gene expression 24 hours after the end of puromycin selection is shown, normalised to e shSCR (SCR) or f empty vector (CON) transduced cells. Bars and error bars are means and SD of e five, and f three, independent experiments. *P < 0.05; **P < 0.01 (relative to controls), one sample t test.

Fig. 4. RUVBL2 binds c-MYB and maintains its transcriptional program in AML.

a GSEA of c-MYB repressed (top) and activated (bottom) gene sets, as previously defined [7], in gene expression changes in shRUVBL2 THP1 cells following 2 (left panels) and 4 (right panels) days doxycycline treatment. b Table summarizing GSEA of c-MYB gene sets derived from shRNA [22], CRISPR-mediated [23] and peptidomimetic [6] c-MYB targeting in AML cells, and of a myeloid differentiation gene set [24] derived from global gene expression changes following PMA treatment of THP1 cells [25] and a terminal monocyte differentiation program [26]. c Western blot analysis of mouse IgG and anti-RUVBL2 immunoprecipitates from THP1 cells, following DSG cross-linking, stained with anti-c-MYB (top) and anti-RUVBL2 (bottom). d Western blot analysis of mouse IgG and anti-c-MYB immunoprecipitates from HA-RUVBL2 expressing THP1 cells, following DSG cross-linking, stained with anti-HA (top) and anti-c-MYB (bottom). Representative data c, d from one of three independent experiments.

Fig. 5. Loss of RUVBL2 results in increased binding of c-MYB to repressed target genes and relieves their repression.

a Heatmap showing ChIP signal for c-MYB and histone H3K27ac for the dynamic c-MYB peaks (more than 2-fold changed) following shRNA mediated RUVBL2 silencing in THP1 cells. b Exemplar ChIP-Seq tracks for c-MYB peaks showing more than 1.5-fold (dashed arrows) and 2-fold (solid arrows) increased c-MYB binding (MYB UP) following RUVBL2 silencing. c Box plots showing ChIP-Seq signal for c-MYB and histone H3K27ac for MYB UP peaks (>2-fold) with a significant H3K27ac signal (MYB UP H3K27ac). H3K27ac signal increases significantly upon RUVBL2 silencing, Mann–Whitney U test. d Venn diagram showing overlap between genes whose expression increased (>2-fold, P < 0.05) in shRUVBL2 THP1 cells (RNA-Seq UP Day 4), following 4 days doxycycline treatment, and genes with increased MYB binding following RUVBL2 silencing (>2-fold, ChIP-Seq MYB UP). P-value obtained by hypergeometric test. e GSEA of the 36 genes, with increased expression and MYB binding following RUVBL2 silencing, in previously reported gene expression data from THP1 cells treated with PMA for 24 hours [25]. f Table summarising GSEA of the 36 genes in previously reported gene expression data following siRNA knockdown [25], CRISPR-mediated [23] and peptidomimetic [6] targeting of c-MYB in AML cells.