Research ArticleCELL BIOLOGY

Maintenance of leukemic cell identity by the activity of the Polycomb complex PRC1 in mice

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Science Advances  07 Oct 2016:
Vol. 2, no. 10, e1600972
DOI: 10.1126/sciadv.1600972
  • Fig. 1 PRC1 activity is required for leukemic cell growth independently of oncogenic Hoxa9 activation.

    (A) Growth curves of R26CreERT2 Ring1a/ Ring1bf/f Lin cells purified from bone marrow and transformed by transduction with lentiviruses expressing the indicated human oncogenic proteins. Full PRC1 inactivation was induced by 4-OHT treatment. Ethanol (EtOH) was used as treatment control. Nontransduced cells were used as nontransformed control (NT). (B) May-Grünwald-Giemsa staining of cells obtained from the experiment presented in (A) at 72 hours after 4-OHT treatment, which shows a differentiated morphology in the absence of PRC1 activity. (C) Methylcellulose colony assays starting from 5000 plated cells, the same presented in (A) at the first and third passages. Quantifications of the number of colonies obtained per plate are presented in the right panels. CFU, colony-forming unit. (D) Green fluorescent protein (GFP) expression in the HOXA9-transformed Lin cells, demonstrating that the loss of PRC1 activity does not induce a silencing of the transduced oncogene expression construct. (E) Relative expression levels of Ring1b, p16, and p19 determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) analyses in the Lin cells shown in (A) and (B), demonstrating both the loss of Ring1b expression upon 72 hours of 4-OHT treatment and the transcriptional activation of the Cdkn2a locus products. Gene expression is normalized to Rpo levels. (F) Western blot analyses with the indicated antibodies on proteins extracted from R26CreERT2 Ring1a−/− Ring1bf/f Lin cells treated with 4-OHT for 72 hours, showing the loss of Ring1b expression and H2AK119Ub deposition and the concurrent accumulation of p16 protein levels. EtOH was used as treatment control. Vinculin is presented as loading control. (G) Relative expression levels of Hoxa9 determined by qRT-PCR analyses in the R26CreERT2 Ring1a−/− Ring1bf/f Lin cells expressing the indicated oncogenic proteins and treated for 72 hours with 4-OHT or EtOH (treatment control). Gene expression is normalized to Rpo levels and to the Hoxa9 levels in nontransduced cells (NT).

  • Fig. 2 PRC1 activity is required for leukemic cell growth independently of p16 and p19 expression.

    (A) Relative expression levels of p16 and p19 determined by qRT-PCR analyses in the Cdkn2a-proficient and Cdkn2a-deficient Lin cells treated for 72 hours with 4-OHT or EtOH (treatment control). Gene expression is normalized to Rpo levels. (B) Western blot analyses with the indicated antibodies on proteins extracted from R26CreERT2 Cdkn2a−/− Ring1a−/− Ring1bf/f Lin cells treated with 4-OHT for 72 hours, demonstrating the loss of Ring1b expression, lack of H2AK119Ub deposition, and absence of p16 expression. EtOH was used as treatment control. Vinculin is presented as loading control. (C) Growth curves of R26CreERT2 Cdkn2a−/− Ring1a−/− Ring1bf/f Lin cells purified from bone marrow and transformed by transduction with lentiviruses expressing the indicated oncogenic proteins. Full PRC1 inactivation in the absence of p16 and p19 transcriptional activation (Cdkn2a−/−) was induced by 4-OHT treatment. EtOH was used as treatment control. Nontransduced cells were used as nontransformed control. (D) May-Grünwald-Giemsa staining of the cells obtained from the experiment presented in (C) at 72 hours after 4-OHT treatment. (E) Methylcellulose colony assays starting from 5000 plated cells, the same presented in (C) at the first and third passages, displayed the transformed phenotype acquired upon expression of the different oncogenes, highlighting a dependency on PRC1 activity independent of Cdkn2a expression. Quantifications of the number of colonies per plate are presented in the right panels. (F) Relative expression levels of Ring1b determined by qRT-PCR analyses in the Lin cells shown in (C) and (D), showing efficient loss of Ring1b expression upon 72 hours of 4-OHT treatment. Gene expression is normalized to Rpo levels. (G) Relative expression levels of Hoxa9 determined by qRT-PCR analyses in the R26CreERT2 Cdkn2a−/− Ring1a−/− Ring1bf/f Lin cells expressing the indicated oncogenic proteins and treated for 72 hours with 4-OHT or EtOH (treatment control). Gene expression is normalized to Rpo levels and used as a nontransformed control on the nontransduced cells.

  • Fig. 3 Conserved genomic association of PRC1 activity in different leukemic cells.

    (A) Cumulative H2AK119Ub ChIP-seq signals among all enriched loci in the R26CreERT2 Cdkn2a−/− Ring1a−/− Ring1bf/f nontreated Lin cells expressing the indicated oncogenic proteins show that H2AK119Ub accumulated to the same genomic loci upon transformation with different oncogenes. Rows are sorted by decreasing normalized intensity centered in the ±40-kb window surrounding the H2AK119Ub peaks. (B) Composite volcano plot showing the significantly differentially regulated genes 72 hours after 4-OHT or EtOH (treatment control) treatment in the R26CreERT2 Cdkn2a−/− Ring1a−/− Ring1bf/f Lin cells expressing the indicated oncogenic proteins. WT, wild type. (C) Percentage of H2AK119Ub-enriched promoters undergoing transcriptional activation (fold change ≥ 4) after loss of PRC1 activity. Expression was determined by RNA-seq analysis in the R26CreERT2 Cdkn2a−/− Ring1a−/− Ring1bf/f Lin cells expressing the indicated oncogenic proteins at 72 hours after the 4-OHT treatment. EtOH was used as control treatment. Red bars represent the percentage of H2AK119Ub-positive promoters (PRC1 direct targets); yellow bars represent the H2AK119Ub-negative promoters (PRC1 indirect targets). TSS, transcription start site. (D) P values of the significantly enriched pathways identified by gene ontology interrogation for the activated genes presented in (C). Left panels represent the functional pathways enriched among the PRC1 direct targets; right panels represent the functional pathways enriched among the PRC1 indirect targets.

  • Fig. 4 Loss of PRC1 activity induces leukemic cell differentiation.

    FACS analyses and quantification of the levels of differentiation markers upon loss of PRC1 activity in the different transformed Lin cells treated with 4-OHT at the indicated time points. CD61 (A) is presented for megakaryocyte differentiation; Ter119 (B) for erythrocytes; B220 (C) for B cells; and CD3e (D) for T cell differentiation.

  • Fig. 5 Loss of H2AK119Ub in MLL-AF9–induced primary leukemia blocks cell growth.

    (A) Methylcellulose colony assays starting from 1000 plated cells isolated from the spleen of a mouse that had developed a primary MLL-AF9 leukemia after intravenous inoculation of the MLL-AF9–transduced R26CreERT2 Cdkn2a−/− Ring1a−/− Ring1bf/f Lin cells. Loss of PRC1 activity was induced by 4-OHT treatment at each methylcellulose passage. Quantifications of the colony number per plate are presented in the bottom panel. (B) Composite volcano plot showing the significantly differentially regulated genes 72 hours after 4-OHT treatment of the R26CreERT2 Cdkn2a−/− Ring1a−/− Ring1bf/f MLL-AF9 primary leukemia cells. EtOH was used as control treatment. (C) Overlaps between the up-regulated genes [fold change (FC) ≥ 4] in the MLL-AF9 preleukemic cells and MLL-AF9 leukemic cells upon loss of PRC1 activity. (D) Percentage of H2AK119Ub-enriched promoters undergoing transcriptional activation (fold change ≥ 4) after loss of PRC1 activity. Expression was determined by RNA-seq analysis in the indicated cells after 72 hours from the 4-OHT treatment. EtOH was used as control treatment. Red bars represent the percentage of H2AK119Ub-positive promoters (PRC1 direct targets); yellow bars represent the H2AK119Ub-negative promoters (PRC1 indirect targets). (E) P values of the significantly enriched pathways identified by gene ontology interrogation among the activated genes presented in (D). Top panel represents the functional pathways enriched among the PRC1 indirect targets; bottom panel represents the functional pathways enriched among the PRC1 direct targets. (F) Western blot analyses for total p53 (p53tot) and p53 phosphorylated (p53P) in the R26CreERT2 Cdkn2a−/− Ring1a−/− Ring1bf/f MLL-AF9 primary leukemia cells 72 hours after EtOH or 4-OHT addition, showing that the loss of PRC1 activity does not activate the p53 pathway. Ultraviolet-irradiated R26CreERT2 Cdkn2a−/− Ring1a−/− Ring1bf/f MLL-AF9 primary leukemia cells were used as p53/p53P-positive control (UV). β-Tubulin is presented as loading control. (G) Western blot analysis for H3K27me3 showing that its global deposition is not affected by the loss of H2AK119Ub in the R26CreERT2 Cdkn2a−/− Ring1a−/− Ring1bf/f MLL-AF9 primary leukemia cells 72 hours after EtOH or 4-OHT addition. Vinculin is presented as loading control. (H) Heat map representing the normalized H3K27me3 ChIP-seq intensities ±10 kb around the summit of H3K27me3-positive promoters identified in R26CreERT2 Cdkn2a−/− Ring1a−/− Ring1bf/f MLL-AF9 primary leukemia cells 72 hours after EtOH or 4-OHT addition.

  • Fig. 6 PRC1 activity is essential for leukemia development both in vitro and in vivo.

    (A) Kaplan-Meier survival curves of NOD/SCID mice intravenously inoculated with 1 × 106 MLL-AF9 primary leukemic cells. Mice were treated with tamoxifen to inactivate the Ring1b allele at 12 days after leukemia transplant (arrow). For mice transplants: oil, n = 4; tamoxifen, n = 4. P value was determined by χ2 test. (B) Kaplan-Meier survival curves of C57BL/6 mice intravenously inoculated with 1 × 106 MLL-AF9 primary leukemic cells. Mice were treated with tamoxifen to inactivate the Ring1b allele at 12 days after leukemia transplant (black arrowhead). For mice transplants: oil, n = 8; tamoxifen, n = 9. The pink arrowheads indicate the weekly tamoxifen injections for the group of mice (n = 5) that show improvement in the survival rate (pink survival curve), demonstrating that the leukemic cells that kill the mice are PRC1-proficient escapee cells. P value was determined by χ2 test. (C and D) H&E staining of spleen (C) and liver tissues (D) collected at day 6 or 30 after injection shows a milder leukemic phenotype in the tamoxifen-treated mice. (E and F) Immunohistochemical analyses of spleen (E) and liver tissues (F) collected at days 6 and 30 after tamoxifen injections, showing the rapid impairment of the active H2AK119Ub deposition in infiltrated leukemic cells compared to the control tissues (6 days after injection; top panels) as a consequence of the acute inactivation of PRC1 activity. Thirty days after tamoxifen injections, the infiltrated leukemic cells display an H2AK119Ub-positive staining (bottom panels), indicating the counterselection of H2AK119Ub-negative cells, with respect to the infiltrated leukemic cells with unexcised Ring1b allele. The far right panels are a magnification of the dashed square areas.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/10/e1600972/DC1

    Supplementary Materials and Methods

    fig. S1. Loss of PRC1 induces a differentiated phenotype in Lin transformed cells.

    fig. S2. Loss of PRC1 induces a differentiated phenotype independently of Cdkn2a expression.

    fig. S3. Specificity check of H2AK119Ub signal.

    fig. S4. Loss of PRC1 activity induces leukemic cell differentiation.

    fig. S5. Loss of PRC1 activity negatively affects the growth of primary leukemia both in vitro and in vivo.

    fig. S6. Bmi1 knockdown does not recapitulate the Ring1a/b-deficient phenotype.

    table S1. ChIP-seq results in Lin and leukemic cells.

    table S2. Genome-wide expression in wild-type, PRC1 Lin, and leukemic cells.

    table S3. Genome-wide expression in primary MLL-AF9 leukemic cells.

    table S4. ChIP-seq results in primary MLL-AF9 leukemic cells.

    References (3640)

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Materials and Methods
    • fig. S1. Loss of PRC1 induces a differentiated phenotype in Lin transformed cells.
    • fig. S2. Loss of PRC1 induces a differentiated phenotype independently of Cdkn2a expression.
    • fig. S3. Specificity check of H2AK119Ub signal.
    • fig. S4. Loss of PRC1 activity induces leukemic cell differentiation.
    • fig. S5. Loss of PRC1 activity negatively affects the growth of primary leukemia both in vitro and in vivo.
    • fig. S6. Bmi1 knockdown does not recapitulate the Ring1a/b-deficient phenotype.
    • Legends for tables S1 to S4
    • References (3640)

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    Other Supplementary Material for this manuscript includes the following:

    • table S1 (Microsoft Excel format). ChIP-seq results in Lin and leukemic cells.
    • table S2 (Microsoft Excel format). Genome-wide expression in wild-type, PRC1 Lin, and leukemic cells.
    • table S3 (Microsoft Excel format). Genome-wide expression in primary MLLAF9 leukemic cells.
    • table S4 (Microsoft Excel format). ChIP-seq results in primary MLL-AF9 leukemic cells.

    Download tables S1 to S4

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