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Supplementary Table S6. ChIP-sequencing datasets used in the present study.

Supplementary Figure S1. mRNA levels of PRC2 components in CML and normal samples used in this study. Supplementary Figure S2. Effect of TKI treatment on mRNA levels of PRC2 components on CML cells in vitro. Supplementary Figure S3. Analysis of promoters reprogrammed for H3K27me3 levels at promoters. Supplementary Figure S4. Relationship between changes in levels of H3K27me3 and changes in mRNA expression at H3K27me3 targets. Supplementary Figure S5. EZH2i treatment of CML and normal CD34+ cells. Supplementary Figure S6. EZH2i treatment of CML CD34+CD38- cells (LSCs). Supplementary Figure S7. Validation of EZH2 shRNAs in a CML cell line. Supplementary Figure S8. The effect of treatment of CML or normal CD34+ cells with EZH2i (GSK343; 1000 nM) on the global mRNA levels of H3K27me3 and H3K27me1 targets. Supplementary Figure S9. Both imatinib and EZH2i selectively target upregulation of H3K27me3 target genes. Supplementary Figure S10. Nilotinib, dasatinib and EZH2i selectively target upregulation of H3K27me3 target genes. Supplementary Figure S11. mRNA expression levels for H3K27me3 target genes in HSC and LSCs and their behaviour after TKI treatment. Supplementary Figure S12. mRNA expression levels for H3K27me3 target genes in HPC and LPCs (â‰^ CML CD34+) and their behaviour after EZH2i treatment. Supplementary Figure S13. Primary CML cells engrafted in NSG mice treated in vivo with EZH2i and TKI, alone and in combination. Supplementary Figure S14. Validation of mRNA expression changes determined by Affymetrix GeneChip analysis (E-MTAB-2581, Affymetrix HuGe 1.0 ST). Supplementary Figure S15. PRC2 mis-regulation in the three CML patient samples used for Affymetrix GeneChIP and ChIP-sequencing in this study. Supplementary Figure S16. Determining thresholds for identifying H3K27me3 target genes. Supplementary Figure S17. Validation of seqMiner(3) promoter classifications.

Supplementary Table S2. mRNA expression changes in CML and normal CD34+ cells resulting from treatment of EZHi (GSK343) (EMTAB-2893, MTAB-3552; Affymetrix HuGe 1.0 ST).

Supplementary Table S1. Genome-wide analysis of mRNA expression levels in HPCs, HSCs, LSCs and LPCs (E-MTAB-2581, Affymetrix HuGe 1.0 ST).

Supplementary Table S5. Primary CML and normal samples that were used in this study.

Supplementary Table S4. Comparison of the efficacy of EZH2i with inhibitors of other novel CML drug targets (4-15) in like-for-like assays.

Supplementary Table S2 from Inactivation of HOXA Genes by Hypermethylation in Myeloid and Lymphoid Malignancy is Frequent and Associated with Poor Prognosis

Purpose: The HOX genes comprise a large family of homeodomain-containing transcription factors, present in four separate clusters, which are key regulators of embryonic development, hematopoietic differentiation, and leukemogenesis. We aimed to study the role of DNA methylation as an inducer of HOX gene silencing in leukemia.Experimental Design: Three hundred and seventy-eight samples of myeloid and lymphoid leukemia were quantitatively analyzed (by COBRA analysis and pyrosequencing of bisulfite-modified DNA) for methylation of eight HOXA and HOXB cluster genes. The biological significance of the methylation identified was studied by expression analysis and through re-expression of HOXA5 in a chronic myeloid leukemia (CML) blast crisis cell line model.Results: Here, we identify frequent hypermethylation and gene inactivation of HOXA and HOXB cluster genes in leukemia. In particular, hypermethylation of HOXA4 and HOXA5 was frequently observed (26-79%) in all types of leukemias studied. HOXA6 hypermethylation was predominantly restricted to lymphoid malignancies, whereas hypermethylation of other HOXA and HOXB genes was only observed in childhood leukemia. HOX gene methylation exhibited clear correlations with important clinical variables, most notably in CML, in which hypermethylation of both HOXA5 (P = 0.00002) and HOXA4 (P = 0.006) was strongly correlated with progression to blast crisis. Furthermore, re-expression of HOXA5 in CML blast crisis cells resulted in the induction of markers of granulocytic differentiation.Conclusion: We propose that in addition to the oncogenic role of some HOX family members, other HOX genes are frequent targets for gene inactivation and normally play suppressor roles in leukemia development.

Supplementary Table S1 from Inactivation of HOXA Genes by Hypermethylation in Myeloid and Lymphoid Malignancy is Frequent and Associated with Poor Prognosis