Project description:Inactivating mutations in the zinc finger gene PHF6 are seen in approximately 40% of adult T-cell acute lymphoblastic leukemias (T-ALLs) and 3% of adult acute myeloid leukemias (AMLs). The absence of PHF6 mutations in B-cell lineage malignancies has led to the hypothesis that PHF6 may act as a lineage-specific tumor suppressor gene. Here, we demonstrate that PHF6 plays a critical role in regulating B-cell identity in the context of B-cell precursor acute lymphoblastic leukemia (preB-ALL). Transplantation of Phf6 knockout preB-ALL cells (hereafter referred to as Phf6KO cells) into immunocompetent syngeneic recipients resulted in the development of a fully penetrant lymphoma-like disease. Strikingly, the resulting lymphomas showed robust up-regulation of the canonical T-cell marker CD4, suggesting that Phf6KO cells adopt a T-cell program in the context of leukemogenesis. RNA sequencing analysis revealed numerous differentially expressed (DE) genes in Phf6WT and Phf6KO cells, including a significant down-regulation of genes and gene sets involved in pathways important for B-cell development. Chromatin immunoprecipitation followed by high-throughput sequencing analysis revealed that PHF6 co-localizes with H3K27ac signals close to the transcription start sites (TSSs) and enhancer regions of a significant proportion of DE genes. Notably, regions flanking the TSS of DE genes showed significant enrichment for binding sites of several well-described master regulators of B-cell development, including PU.1, EGR-1, EBF-1, NF-kB, TCF3 and TCF12. We found that PHF6 and TCF12 physically interact in preB-ALL cells, suggesting that these factors act synergistically in the establishment and maintenance of B-cell identity. In addition, we found that a human PHF6 mutant T-ALL cell line has an incompletely rearranged IGH locus, strongly suggesting that T-ALL can have a B-cell origin. These findings reveal an essential role for PHF6 in the establishment and maintenance of B-cell identity in preB-ALL by directly activating genes that are crucial for B-cell lineage commitment and maintenance. Collectively, these results indicate that loss of function of PHF6 in preB-ALL leads to an unstable cellular state in which cells acquire alternate developmental programs (such as the T-lineage program) to survive, potentially explaining the apparent absence of PHF6 mutations in human B cell-lineage malignancies.

Project description:Integrative epigenomic analysis identifies biomarkers and therapeutic targets in adult B-acute lymphoblastic leukemia. We performed DNA methylation (HELP array) and gene expression profiling in 215 samples of adult B-lineage acute lymphoblastic leukemia (ALL) and 12 normal preB samples. Adult B-lineage acute lymphoblastic leukemia (B-ALL) is an aggressive disease with <40% long-term survival. Genetic alterations such as BCR/ABL, E2A/PBX1 and MLL rearrangement (tMLL) define distinct B-ALL subtypes, which are associated with poor clinical outcome. It has been shown that these B-ALL subtypes have distinct expression profiles. However, the role of the epigenome in shaping these expression profiles and how the aberrant epigenetic gene regulation contributes to the biological and clinical features of those ALL subtypes is largely unknown. To address this question, we performed genome-wide DNA methylation and gene expression profiling on a large cohort of 215 well-characterized adult B-ALL specimens from the ECOG E2993 phase III clinical trial and a cohort of normal precursor B (preB) cells from 12 healthy bone marrows. The integrative analysis of these profiles led to the identification of key gene networks deregulated at the epigenetic and transcriptional levels within each subtype. In BCR/ABL, we identified a network centered on IL2RA(CD25), which is itself hypomethylated and overexpressed in most BCR/ABL B-ALL and confers poor clinical outcomes. In the tMLL subtype, we uncovered aberrant epigenetic and transcriptional activities that include hypomethylation and upregulation of FLT3 and BCL6. After showing that MLL/AF4 fusion protein binds to these genes as well as other hypomethylated and overexpressed genes in tMLL ALL cells, we showed that a specific BCL6 inhibitor, RI-BPI, kills tumor cells in both tMLL ALL cell lines and patient samples. BCL6 inhibition may therefore represent a novel therapeutic strategy for B-ALL patients with MLL translocations. RUNX1 is a key target gene in MLL-AF4 leukemias and contributes to gene activation by interacting with the AF4-MLL complex. The Mixed Lineage Leukemia 1 protein (MLL1) is an important epigenetic protein that is required for the maintenance of gene activation during development, but is also mutated in a subset of aggressive human leukemias. The most common leukemogenic MLL1 mutations are chromosome translocations that fuse MLL1 in-frame to produce novel fusion proteins. Different MLL1 fusion proteins cause unique leukemias even when they are expressed in the same cell type, suggesting that they function through unique molecular mechanisms. We used ChIP-seq in MLL-AF4 patient cell lines to identify target genes that are involved in leukemogenesis. ChIP-seq using MLLN, AF4, H3K4me3 and H3K79me2 antibodies in RS4;11 cells.

Project description:Integrative epigenomic analysis identifies biomarkers and therapeutic targets in adult B-acute lymphoblastic leukemia. We performed DNA methylation (HELP array) and gene expression profiling in 215 samples of adult B-lineage acute lymphoblastic leukemia (ALL) and 12 normal preB samples. Adult B-lineage acute lymphoblastic leukemia (B-ALL) is an aggressive disease with <40% long-term survival. Genetic alterations such as BCR/ABL, E2A/PBX1 and MLL rearrangement (tMLL) define distinct B-ALL subtypes, which are associated with poor clinical outcome. It has been shown that these B-ALL subtypes have distinct expression profiles. However, the role of the epigenome in shaping these expression profiles and how the aberrant epigenetic gene regulation contributes to the biological and clinical features of those ALL subtypes is largely unknown. To address this question, we performed genome-wide DNA methylation and gene expression profiling on a large cohort of 215 well-characterized adult B-ALL specimens from the ECOG E2993 phase III clinical trial and a cohort of normal precursor B (preB) cells from 12 healthy bone marrows. The integrative analysis of these profiles led to the identification of key gene networks deregulated at the epigenetic and transcriptional levels within each subtype. In BCR/ABL, we identified a network centered on IL2RA(CD25), which is itself hypomethylated and overexpressed in most BCR/ABL B-ALL and confers poor clinical outcomes. In the tMLL subtype, we uncovered aberrant epigenetic and transcriptional activities that include hypomethylation and upregulation of FLT3 and BCL6. After showing that MLL/AF4 fusion protein binds to these genes as well as other hypomethylated and overexpressed genes in tMLL ALL cells, we showed that a specific BCL6 inhibitor, RI-BPI, kills tumor cells in both tMLL ALL cell lines and patient samples. BCL6 inhibition may therefore represent a novel therapeutic strategy for B-ALL patients with MLL translocations. RUNX1 is a key target gene in MLL-AF4 leukemias and contributes to gene activation by interacting with the AF4-MLL complex. The Mixed Lineage Leukemia 1 protein (MLL1) is an important epigenetic protein that is required for the maintenance of gene activation during development, but is also mutated in a subset of aggressive human leukemias. The most common leukemogenic MLL1 mutations are chromosome translocations that fuse MLL1 in-frame to produce novel fusion proteins. Different MLL1 fusion proteins cause unique leukemias even when they are expressed in the same cell type, suggesting that they function through unique molecular mechanisms. We used ChIP-seq in MLL-AF4 patient cell lines to identify target genes that are involved in leukemogenesis.

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PHF6 regulates B-cell identity in acute lymphoblastic leukemia [ChIP-Seq]

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