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. 2012 Mar 27;109(13):5028-33.
doi: 10.1073/pnas.1202258109. Epub 2012 Mar 6.

Polycomb repressive complex 2 is required for MLL-AF9 leukemia

Tobias Neff  1 , Amit U Sinha, Michael J Kluk, Nan Zhu, Mohamed H Khattab, Lauren Stein, Huafeng Xie, Stuart H Orkin, Scott A Armstrong
Affiliations

Polycomb repressive complex 2 is required for MLL-AF9 leukemia

Tobias Neff et al. Proc Natl Acad Sci U S A. .

Abstract

A growing body of data suggests the importance of epigenetic mechanisms in cancer. Polycomb repressive complex 2 (PRC2) has been implicated in self-renewal and cancer progression, and its components are overexpressed in many cancers. However, its role in cancer development and progression remains unclear. We used conditional alleles for the PRC2 components enhancer of zeste 2 (Ezh2) and embryonic ectoderm development (Eed) to characterize the role of PRC2 function in leukemia development and progression. Compared with wild-type leukemia, Ezh2-null MLL-AF9-mediated acute myeloid leukemia (AML) failed to accelerate upon secondary transplantation. However, Ezh2-null leukemias maintained self-renewal up to the third round of transplantation, indicating that Ezh2 is not strictly required for MLL-AF9 AML, but plays a role in leukemia progression. Genome-wide analyses of PRC2-mediated trimethylation of histone 3 demonstrated locus-specific persistence of H3K27me3 despite inactivation of Ezh2, suggesting partial compensation by Ezh1. In contrast, inactivation of the essential PRC2 gene, Eed, led to complete ablation of PRC2 function, which was incompatible with leukemia growth. Gene expression array analyses indicated more profound gene expression changes in Eed-null compared with Ezh2-null leukemic cells, including down-regulation of Myc target genes and up-regulation of PRC2 targets. Manipulating PRC2 function may be of therapeutic benefit in AML.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Inactivation of Ezh2 in vivo is compatible with MLL-AF9 leukemia growth but leads to a less aggressive phenotype. (A) No survival difference between recipients of MLL-AF9–transduced cells with WT or inactivated Ezh2 locus. (B) Western blot analysis demonstrates loss of Ezh2 protein in Ezh2-null leukemic cells in vivo and substantially reduced H3K27me3. (C) Significantly prolonged survival of secondary recipients in the Ezh2-null group. (D) BrdU in vivo cell cycle analysis of leukemic WT and Ezh2-null leukemic spleen cells showing moderate cell cycle alterations in the Ezh2-null group (decrease in S phase). n = 2 (WT) and n = 4 (Ezh2-null). Error bars indicate range. G0/G1: P = 0.0007, S: P = 0.0028. G2/M: not significant (analysis of bone marrow and spleen cells, two-way ANOVA). (E) In vivo up-regulation of p16ink4a in Ezh2-null leukemia by Western blot. (F) In vivo up-regulation of p19arf in Ezh2-null leukemia.
Fig. 2.
Fig. 2.
Analysis of Ezh2-null leukemia. (A) Gene set enrichment analysis reveals enrichment of PRC2 module in Ezh2-null secondary leukemia. (B) Highly significant negative enrichment of Myc module genes in Ezh2-null compared with WT secondary leukemia. NES, normalized enrichment score. (C) Increased infiltration of the kidney in tertiary recipients of WT leukemias compared with recipients of Ezh2-null leukemic cells. (D) Myc protein levels are comparable between tertiary WT and Ezh2-null leukemia.
Fig. 3.
Fig. 3.
ChIP-Seq analysis of primary and secondary WT and Ezh2-null leukemic cells distinguishes class 1 genes that lose H3K27me3 and class 2 genes that retain H3K27me3. (A) Representative plot showing loss of H3K27me3 at the Bmi1 locus (as an example of a class 1 gene) and retention of H3K27me3 at the Hes1 locus (as an example of a class 2 gene). (B) Genes in class 1 showed a significant increase in expression in Ezh2-null cells compared with WT cells. In contrast, there is no significant pattern of expression change in class 2 genes. (C) Genes retaining significant H3K27me3 level (P = 0.005) after inactivation of Ezh2 overlap significantly with documented EZH1 targets (genes bound by EZH1 and genes that retain H3K27me3 in Ezh2-null ES cells).
Fig. 4.
Fig. 4.
Eed is strictly required for MLL-AF9 leukemia. (A) Experimental design to test leukemia maintenance: Replated blast colonies derived from MLL-AF9–transduced LSK cells from Eedflox/flox and WT mice were injected into sublethally irradiated primary recipients. The leukemic cells also carried alleles for MxCre and the YFP-Cre reporter. Leukemic animals were killed and secondary sublethally irradiated recipients were injected with sorted GFP+ cells, (1 × 105 per recipient) harvested from primary recipients. pIpC was administered to inactivate Eed and to activate the YFP reporter, and the survival of mice was assessed. (B) Significantly prolonged survival in recipients of Eedflox/flox cells. (C) Flow cytometric analysis demonstrates predominant outgrowth of unexcised cells. (D) Analysis of Eedflox/flox cells early (3 d after last dose of pIpC) and late (8 d after last dose of pIpC). Early after pIpC, we found efficient deletion in sorted GFP+/YFP+ cells with complete loss of H3K27me3 in three tested leukemias shown here. At the later time point (8 d after the last dose of pIpC), there was outgrowth of heterozygously inactivated cells with significant levels of H3K27me3 in four of four tested leukemias.
Fig. 5.
Fig. 5.
Detailed analysis of Eed-null MLL-AF9 leukemia. (A) In contrast to WT leukemic cells, Eed-null leukemic cells engraft secondary recipients poorly. A total of 1 × 106 sorted GFP+/YFP+ cells from primary recipients were transplanted per mouse. n = 3 for each group and time point (except n = 2 for WT 1 mo time point). Error bars indicate SEM. (B) Cdkn2a gene products are up-regulated in Eed-null MLL-AF9 leukemic cells in vivo. (C) Comparison of expression of Cdkn2a gene products between sorted preleukemic cells with WT, Ezh2-null, and Eed-null genotype. LSK-derived MLL-AF9 colonies from two separate donors were transduced with self-excising Cre vector and GFP+/YFP+ double positive cells were sorted 6 d after deletion. Note more pronounced derepression of p19arf in Eed-null cells. (D) GSEA analysis of class 2 genes, which retain H3K27me3 in Ezh2-null MLL-AF9 AML. This group of genes is not enriched in Ezh2-null vs. WT leukemia (Fig. 3B) but is derepressed and significantly enriched in Eed-null vs. Ezh2-null leukemic cells.
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