Meis1 supports leukemogenesis through stimulation of ribosomal biogenesis and Myc

Abstract

The homeobox transcription factors HoxA9 and Meis1 are causally involved in the etiology of acute myeloid leukemia. While HoxA9 alone immortalizes cells, cooperation with Meis1 is necessary to induce a full leukemic phenotype. Here, we applied degron techniques to elucidate the leukemogenic contribution of Meis1. Chromatin immunoprecipitation experiments revealed that Meis1 localized mainly to H3K27 acetylated and H3K4 mono-methylated enhancers preactivated by HoxA9. Chromatin association of Meis1 required physical presence of HoxA9 and all Meis1 DNA interactions were rapidly lost after HoxA9 degradation. Meis1 controlled a gene expression pattern dominated by Myc, ribosome biogenesis and ribosomal RNA synthesis genes. While Myc accounted for the cell cycle stimulating effect of Meis1, overexpression of this oncogene alone did not accelerate leukemogenesis. Besides its effect on Myc, Meis1 induced transcription of ribosomal biogenesis genes. This was accompanied by an elevated resistance against inhibition of ribosomal RNA synthesis and translation, but without affecting steady-state protein synthesis. Finally, we demonstrate that HoxA9 and Meis1 proteins are stabilized by post-translational modification. Mutation of HoxA9/Meis1 phosphorylation sites or inhibition of casein kinase 2 lead to rapid protein degradation suggesting a potential pathway for pharmacological intervention.

Figure 1.

Meis1 binds preferentially to enhancers. (This figure is supplemented by the Online Supplementary Figure S1). (A) Strategy to generate primary transformed cell lines for chromatin immunoprecipitation (ChIP) experiments: hematopoietic stem and precursor cells (HSPC) were isolated from animals with a genetic knockout of myeloid granule proteases elastase, proteinase 3, and cathepsin G to allow for efficient ChIP. (B) Expression of ChIP targets and induced degradation of Meis1-FKBP^F36V. Extracts from transformed primary cells were probed by HA-specific western for constitutive expression (left panel) of ChIP targets and for induced degradation of Meis1-FKBP^F36V after addition of dTAG13 (a “proteolysis targeting chimera”; PROTAC) (right panel). (C) Meis1 ChIP is reproducible and peaks can be verified by Meis1 degradation. Integrated genomics viewer (IGV) panels showing Meis1 binding patterns at 2 typical Meis1 loci, Myb and its major enhancer (top panel) and the known Meis1-responsive gene Flt3 (lower panel). Tracks correspond to a replicate obtained with constitutively expressed Meis1 as well as Meis1-FKBP^F36V before and 8 hours after initiation of degradation as labeled. An input track is added as control. (D) Meis1 localizes predominantly to putative enhancer locations. Pie chart of Meis1 peak distribution across functionally annotated genetic elements. Analysis was done for the 10,000 top scoring peaks.

Figure 2.

Meis1 binding sites colocalize with enhancer-typic chromatin modifications. (A) Meis1 co-localizes with enhancer modifications. Meta-gene plots showing the distribution of enhancer-typical chromatin modifications H3K27ac (active enhancer) and H3K4me1 (putative enhancer) around the top 10,000 Meis1 peaks with best reproducibility. The plot is peak-centered and ordered top to down according to Meis1 binding density. (B) Meis1 homes in on enhancer centers. Exemplary integrative genome viewer (IGV) panels demonstrating localization of Meis1 at the center of active enhancer modifications. (C) Meis1 marks typical hematopoietic enhancers. De novo motif search results of sequences +/-150 bp of Meis1 peaks yields putative binding sites for known hematopoietic transcription factors. (D) Distribution of identified binding motifs supports Meis1 or Meis1/HoxA9 composite binding at the center of identified chromatin immunoprecipitation peaks.

Figure 3.

HoxA9 is epistatic to Meis1. (This figure is supplemented by the Online Supplementary Figure S2). (A) Meis1 and Pbx3 bind in a more defined pattern than HoxA9. Integrative genomics viewer (IGV) plots detailing binding of Meis1, Pbx3 and HoxA9 either in cells co-expressing Meis1 (HoxA9 + Meis1) or in cells in the absence of Meis1 (HoxA9_noMeis1). Sharp colocalized peaks for Meis1 and Pbx3 are different from more diffuse HoxA9 binding characteristics. (B) HoxA9 binding does not change after introduction of Meis1. Global comparison of HoxA9 binding in the vicinity of Meis1 peaks in cells transformed by HoxA9 or by HoxA9 in combination with Meis1 as indicated. (C) Meis1 and Pbx3 co-localize in areas of high HoxA9 density. Metagene plots depicting binding intensity of HoxA9, Meis1, and Pbx3 around identified Meis1 peaks. Heatmaps are ordered top to down according to decreasing Meis1 binding strength. Plotted are the 10,000 top-scoring Meis1 peaks as before. (D) Meis1 by itself cannot maintain the transformed state of precursor cells. Upper panel: western blot demonstrating rapid degradation of HoxA9-FKBP^F36V after addition of dTAG13 without affecting Meis1 protein levels. Lower panel: May-Grünwald-Giemsa stained cytospin preparations of hematopoietic stem and precursor cells (HPSC) lines generated after transduction either with HoxA9-FKBP^F36V alone or a combination of HoxA9-FKBP^F36V with Meis1. Cells are shown before and 96 hours (h) after induction of HoxA9 degradation by the addition of dTAG13. Differentiation with generation of mature granulocytic cells and macrophages is observed in both cases. (E) Meis1 rapidly leaves chromatin after HoxA9 degradation. IGV plots demonstrating Meis1 occupancy at Myb and Flt3 loci in the presence of HoxA9 and 2 h after induction of HoxA9 degradation. (F) Meis1 exits from chromatin rather than changes localization after loss of HoxA9. Global Meis1 read density comparison in the presence and after degradation of HoxA9. A global left shift of Meis1 densities combined with a relative maintenance of correlation indicates loss from chromatin rather than redistribution.

Figure 4.

The Meis1 induced gene expression program is dominated by Myc and ribosome biogenesis. (This figure is supplemented by the Online Supplementary Figure S3 and S4 and the Online Supplementary Table S1). (A) The primary Meis1 controlled gene expression program can be determined by nascent RNA sequencing. Overview of experimental strategy. (B) Meis1 expression can be positively induced in a doxycycline (dox)-controlled system. Schematic depiction of an “all-in-one” inducible expression system based on a self-inactivating (SIN) retroviral backbone. LTR: long terminal repeat, self-inactivating after proviral integration; LNGFR: truncated low affinity growth factor receptor displayed on membrane for antibody-based (anti human CD271) cell selection; 2A: viral-derived “self-cleaving” peptide, blocking peptide bond formation during translation and thus allowing expression of two proteins from fusion sequence; rtTA3: reverse tetra/dox-inducibel transactivator of 3^rd generation; IRES: internal ribosomal entry site, puro: puromycin resistance. The western blot shows expression of HA-Meis before and 72 hours (h) after addition of dox in transduced hematopoietic stem and precursor cells (HPSC). Small amounts of full length LNGFR-2A-Meis1 fusion are also visible. The western was done with hot SDS extracts as the dox-inducible Meis1 system was introduced in wild-type (wt) cells. (C) The conditional Meis1 expression constructs are biologically active. The amount of RNA coding for the Meis1 sentinel gene Flt3 was determined by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) in a time series either after induction of Meis1 expression by dox addition or after induction of Meis1-FKBP^F36V degradation by supplementation with dTAG13. In order to demonstrate reversibility inducers were washed out after 48 h and re-added again after 120 h. Values were normalized to actin transcripts and starting amounts before treatment were defined as one unit. (D) Meis1 induces a Myc and ribosome-synthesis dominated gene expression program. Transcription rates in inducible Meis1 cells were determined in the Meis_on (72 h dox-added or dTAG13 absent) and in the Meis_off state (dox-absent or 24 h dTAG13 present) by nascent RNA sequencing. For graphical representation RPKM expression values for each gene bank accession number were added and plotted in Meis_on and Meis_off states. Shown are values (collapsed to individual genes) for all genes with a significant induction defined as log₂(RPKM_{Meis_on}/RPKM_{Meis_off})_dox + log₂(RPKM_{Meis_on}/RPKM_{Meis_off})_dTAG > 1.0. Red dots denote the top 30 outliers in gene expression change. The top 100 expressed accessions are colored orange (aggregated to gene names). Red labels identify genes investigated further. The left inset shows a Venn-diagram displaying overlap of primary Meis1-induced transcripts with HoxA9 targets identified previously by a similar approach. The right inset depicts the top-scoring result of a gene set enrichment analysis demonstrating a strong similarity of the Meis1-induced expression pattern to the known Myc-regulated program.

Figure 5.

Myc controls the proliferative aspect of Meis1 activity (This figure is supplemented by the Online Supplemental Figure S5). (A) Myc, JunB, and Angptl4 are direct targets of Meis1. Transcription rates of Myc, JunB, and Angptl4 were determined by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) on nascent RNA isolated in a time series after induction/degradation of Meis1. The response kinetics suggest direct control by Meis1. (B) Individual expression of single target genes. RT-qPCR after transduction of HoxA9-transformed cells with individual Meis1 target genes demonstrates higher expression than in HoxA9 + Meis1 cells. (C) Myc and Meis1 accelerate cell proliferation. Individual cell lines, transduced as labeled were cultivated under identical conditions and cell proliferation was determined by counting triplicates. *P<0.05 in two-sided t-test. (D) Myc and Meis1 induce cell cycle. Propidium-iodide staining of test cell lines demonstrates more cells in cell cycle (S/M and G2 phases) as consequence of Meis1 or Myc coexpression. Values correspond to averages and standard deviations of triplicate experiments. **P<0.05 in two-sided t-test. (E) Myc increases colony forming cell (CFC) numbers. CFC capacity was tested for all test lines by seeding 5,000 cells in triplicate into semi solid methylcellulose medium and by counting resulting colonies after 4 to 6 days of incubation. Average and standard deviation is given. *P<0.05 in two-sided t-test.

Figure 6.

Myc does not substitute for Meis1. (A) Meis1 does not influence cytokine signaling. Test cell lines were plated in medium supplemented with a serial dilution of cytokines (1-fold =100 ng/mL stem cell factor [SCF] plus 10 ng/mL each of interleukin 3 [IL-3], IL-6, and granulocyte macrophage colony-stimulating factor [GM-CSF]). Proliferation/viability was tested after 72 hours (h) by a standard MTT test in triplicates and plotted relative to the value in 1-fold cytokines that was set to one unit. Averages and standard deviations are plotted. Only Myc-overexpressing cells showed a significant effect (P<0.05, two sided t-test) for some values. (B) Upper panel: Meis1 and Myc differentially retard morphological differentiation. May-Grünwald-Giemsa stained cytospin preparations of primary hematopoietic cells as indicated. If grown in cytokine mix (SCF, IL3, IL6, GM-CSF) HoxA9 transformed cells have myeloid precursor morphology independent of the co-transduced gene. HoxA9 + vector cells are shown as representative example. Switching cytokine substitution to 10 ng/mL G-CSF for 72 h forces cells into differentiation with Meis1 and Myc retarding this process. Lower panel: surface CD117/Kit expression was determined in test lines as indicated. Data were recorded in normal conditions supplemented with four cytokines and 72 h after induction of forced differentiation by replacement of normal cytokine supplementation by G-CSF showing that Meis1 acts stronger than Myc. (C) Myc does not accelerate leukemia development. Kaplan-Meier graph depicting disease free survival of sublethally irradiated syngenic animals transplanted with cell lines transduced as indicated.

Figure 7.

Meis1 boosts ribosomal biogenesis capacity. (A) Ribosomal RNA (rRNA) transcription correlates with Meis1 activity. Total RNA was isolated from cells transformed by HoxA9 and Meis1-FKBP^F36V in a time series after induction of Meis1, degradation, recovery, and a second degradation phase. Concentrations of 18S rRNA were determined by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and are plotted relative to the starting value. (B) Meis1 increases resistance against RNA polymerase I inhibition. Cells transduced as indicated were subjected to treatment with increasing concentrations of the RNA polymerase I inhibitor CX5461 and viability was determined by MTT assay. Values are plotted based on untreated cells set to one unit. Averages and standard deviation of a triplicate are shown. (C) Meis1 cells increases resilience towards puromycin. Experiment done as above. (D) Co-expression of Meis1 or Myc does not alter sensitivity towards doxorubicine. (E) Meis1 and Myc have a minor influence on steady state and phosphorylation levels of ribosomal protein S6. Cells transduced as indicated were lysed, and the equivalent of 40,000 cells were loaded per lane on a SDS PAGE for detection with S6 and phospho-S6 specific antibodies. (F) Meis1 and Myc do not alter steady-state protein synthesis rate. Cells were incubated with O-propargyl-puromycin (OPP) for 30 minutes, fixed and then OPP was conjugated with Alexa-Fluor 488 and chain-terminated translation products were recorded by fluorescence-activated cell sorting.

Figure 8.

Meis1 and HoxA9 stability is controlled by caseine kinase 2-mediated phosphorylation. (A) Meis1 contains a phosphorylation site closely downstream of the Pbx3 binding domain. Schematic depiction. (B) Pbx3 and phosphorylation control Meis1 stability independently. Wild-type Meis1 (Wt-Meis1), a phosphodefective mutant (AKADA) exchanging respective serines against alanines, as well as a phosphomimetic version (DKDD) mimicking modification by introduction of negative charges were transfected with and without Pbx3 in 293T cells. Meis1 stability was recorded by western blot. Meis1 with a deletion of the Pbx3 binding domain was added as additional control. (C) Meis1_AKADA is unstable in primary myeloid cells. Hematopoietic stem and precursor cells (HPSC) were transduced as indicated and the resulting cell lines were tested for Meis1 and HoxA9 expression by western blot. (D) Inhibition of caseine kinase 2 reduces Meis1 and HoxA9 concentrations and phosphomimetic mutants are resistant. Primary cells transduced with wt HoxA9 and either wt-Meis1 or Meis1_DKDD were treated for 4 hours (h) with increasing concentrations of the caseine kinase 2 inhibitor CX4945 as indicated. Cell extracts were probed by western blotting for Meis1 and HoxA9. (E) HoxA9 is regulated by casein kinase 2. Upper panel: schematic location of the known HoxA9 phosphorylation site. Lower panel: western blots made with extracts from cells transduced with phosphomimetic versions of HoxA9 (HoxA9_DGGD) and Meis1 (Meis1_DKDD) probed for HoxA9 and Meis1 after treatment with CX4945. (F) Left panel: densitometric evaluation of protein stability under casein kinase 2 inhibition. Protein amounts of HoxA9 and Meis1 wt and phosphomimetic versions were determined by densitometric analysis and normalization to actin levels corresponding to western blots shown in (D), left and (E). Right panel: introduction of phosphomimetic HoxA9 and Meis1 mutants does not significantly increase overall resistance of cells against casein kinase 2 inhibition. Cells transduced either with wt versions or with phosphomimetic variants of HoxA9 and Meis1 as indicated were subject to varying concentrations of the caseine kinase inhibitor CX4945 for 72 h and viability/proliferation was tested by MTT assay. Relative values are plotted with untreated cells set to one unit.