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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Cancer-associated ASXL1 mutations may act as gain-of-function mutations of the ASXL1-BAP1 complex

ABSTRACT: ASXL1 is the obligate regulatory subunit of a deubiquitinase complex whose catalytic subunit is BAP1. Heterozygous mutations of ASXL1 that result in premature truncations are frequent in myeloid leukemias and Bohring-Opitz syndrome. Here, we demonstrate that truncated ASXL1 proteins confer enhanced activity on the ASXL1-BAP1 complex. Stable expression of truncated, hyperactive ASXL1-BAP1 complexes in a hematopoietic precursor cell line resulted in global erasure of H2AK119Ub, striking depletion of H3K27me3, selective upregulation of a subset of genes whose promoters bore both H2AK119Ub and H3K4me3, and spontaneous differentiation to the mast cell lineage. These outcomes required the catalytic activity of BAP1, indicating these events were downstream consequences of H2AK119Ub erasure. In bone marrow precursors, truncated ASXL1-BAP1 expression cooperated with TET2 loss-of-function to increase differentiation to the myeloid lineage in vivo. We propose that pathological ASXL1 mutations confer gain-of-function on the ASXL-BAP1 complex. ChIP-Seq for H2AK119Ub, H3K4me3, H3K27me3 on EML cells. RNA-Seq on EML cells expressing ASXL1(1-479)+BAP1 and control.

ORGANISM(S): Mus musculus

SUBMITTER: Anjana Rao

PROVIDER: E-GEOD-65555 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Cancer-associated ASXL1 mutations may act as gain-of-function mutations of the ASXL1-BAP1 complex

Project description:ASXL1 is the obligate regulatory subunit of a deubiquitinase complex whose catalytic subunit is BAP1. Heterozygous mutations of ASXL1 that result in premature truncations are frequent in myeloid leukemias and Bohring-Opitz syndrome. Here, we demonstrate that truncated ASXL1 proteins confer enhanced activity on the ASXL1-BAP1 complex. Stable expression of truncated, hyperactive ASXL1-BAP1 complexes in a hematopoietic precursor cell line resulted in global erasure of H2AK119Ub, striking depletion of H3K27me3, selective upregulation of a subset of genes whose promoters bore both H2AK119Ub and H3K4me3, and spontaneous differentiation to the mast cell lineage. These outcomes required the catalytic activity of BAP1, indicating these events were downstream consequences of H2AK119Ub erasure. In bone marrow precursors, truncated ASXL1-BAP1 expression cooperated with TET2 loss-of-function to increase differentiation to the myeloid lineage in vivo. We propose that pathological ASXL1 mutations confer gain-of-function on the ASXL-BAP1 complex.

2015-03-11 | GSE65555 | GEO

Epigenetic targeted therapy of leukemia through a hyper-activated BAP1-ASXL1-mutant axis

Project description:The ASXL1 gene is the human homolog of the Drosophila Asx gene, a core subunit in the BAP1 histone H2A deubiquitinase complex. Mutations of ASXL1 occur in multiple myeloid neoplasms and are uniformly associated with poor prognosis. However, the molecular mechanism through which ASXL1 mutations alter BAP1 activity to drive leukemogenesis remains unclear. Here we demonstrate that cancer-associated frame-shift ASXL1 mutations, which were originally proposed to act as destabilizing loss-of-function mutations, in fact encode truncated stable gain-of-function proteins. Truncated ASXL1 protein stabilizes BAP1, enhances BAP1 complex recruitment to chromatin and promotes the expression of numerous leukemia associated genes. Chemical inhibition of BAP1 rescues these changes in gene expression in leukemic cells and inhibits tumor progression. This study represents a breakthrough advance in our understanding of the molecular mechanisms of ASXL1 mutations in leukemic pathogenesis and identifies small molecular inhibitors of BAP1 function as a potential targeted therapy for leukemia.

2021-03-07 | GSE166305 | GEO

Human HSPCs engineered with truncated ASXL1 spontaneously progress to myeloid malignancy and can be targeted by PRC1 inhibition

Project description:Mutations in additional sex combs like 1 (ASXL1) confer poor prognosis in myeloid malignancies and are also found in pre-malignant clonal hematopoiesis of indeterminate potential (CHIP). However, the mechanisms by which these mutations contribute to disease initiation remain unresolved and therapeutic targeting has remained elusive. Here, we demonstrate that ASXL1 mutations are frequently founding lesions in acute myeloid leukemia (AML) and lead to the expression of a functional, truncated protein. We developed a human disease model that faithfully recapitulates ASXL1-mutant CHIP with increased self-renewal and in vivo competitive advantage that can spontaneously progress to a lethal myeloid malignancy. We determined that truncated ASXL1 leads to global redistribution of the repressive chromatin mark H2AK119Ub, increased chromatin accessibility, and activation of both myeloid and stem cell gene expression programs. Finally, we identified dysregulation of H2AK119Ub as a therapeutic vulnerability and demonstrated that ASXL1-mutant cells are vulnerable to inhibition of the PRC1 complex.

2024-02-07 | GSE190738 | GEO

The effects of mutant ASXL1 and BAP1 expression on hematopoietic cells

Project description:To identify target genes of mutant ASXL1 and BAP1 in hematopoietic cells, we performed RNA-seq using murine c-kit positive cells transduced with ASXL1-MT (MT) or ASXL1-MT-K351R (KR) together with vector or BAP1. Method：Murine c-kit positive bone marrow cells were transduced with ASXL1-MT (MT) or ASXL1-MT-K351R (KR) (coexpressing blastcidin resistant gene) together with vector or BAP1 (coexpressing puromycin resistant gene). After the selection with blasticidin and puromycin for three days, colony-forming cells were collected to extract RNA for RNA-seq analysis.

2018-05-25 | GSE114861 | GEO

Expression of mutant Asxl1 perturbs hematopoiesis and promotes susceptibility to leukemic transformation

Project description:Additional Sex Combs Like 1 (ASXL1) is frequently mutated in myeloid malignancies and clonal hematopoiesis of indeterminate potential (CHIP). Although loss of ASXL1 promotes hematopoietic transformation, there is growing evidence that ASXL1 mutations might confer an alteration of function. Here we identify that physiological expression of a C-terminal truncated Asxl1 mutant in vivo using conditional knock-in (KI) results in myeloid skewing, age-dependent anemia, thrombocytosis, and morphologic dysplasia. Although expression of mutant Asxl1 altered the functions of hematopoietic stem cells (HSCs), it maintained their survival in competitive transplantation assays and increased susceptibility to leukemic transformation by co-occurring RUNX1 mutation or viral insertional mutagenesis. KI mice displayed substantial reductions in H3K4me3 and H2AK119Ub without significant reductions in H3K27me3, distinct from the effects of Asxl1 loss. ChIP-seq analysis demonstrated opposing effects of wildtype and mutant Asxl1 on H3K4me3. These findings reveal that ASXL1 mutations confer HSCs with an altered epigenome and increase susceptibility for leukemic transformation, presenting a novel model for CHIP.

2018-02-28 | GSE111067 | GEO

Bap1 loss results in EZH2 dependent transformation

Project description:BAP1 and ASXL1 interact to form a polycomb deubiquitinase complex that removes monoubiquitin from histone H2A lysine 119 (H2AK119Ub). However, BAP1 and ASXL1 are mutated in distinct cancer types, consistent with independent roles in regulating epigenetic state and malignant transformation. Here we demonstrate that Bap1 loss results in increased trimethylated histone H3 lysine 27 (H3K27me3), elevated Ezh2 expression, and enhanced repression of Polycomb Repressive Complex 2 (PRC2) targets. These findings contrast with the reduction in H3K27me3 seen with Asxl1 loss. Conditional deletion of Bap1 and Ezh2 in vivo abrogates the myeloid progenitor expansion induced by Bap1 loss alone.

2015-10-05 | GSE61360 | GEO

RNA sequencing of zebrafish asxl1+/+ and asxl1-/-

Project description:ASXL1 gene is one of the most frequently mutated genes in malignant myeloid diseases. In patients, ASXL1 mutations are usually heterozygous frameshift or non-sense mutations leading to C-terminal truncation. Here, we generated an endogenous C-terminal truncated Asxl1 mutant in zebrafish which is more comparable to human malignant leukemia patients. Our data showed that at embryonic stage, neutrophil differentiation was explicitly blocked in our mutant. To understand the basis for the impairment of neutrophil differentiation in zebrafish asxl1 mutants, we performed RNA-seq of asxl1 mutants at 3dpf and their littermate controls. Similar with the phenotype we observed, the expression of neutrophil markers were all included in down-regulated genes. Nonetheless, the expression of myeloid progenitor marker and macrophage marker were not impaired in asxl1 mutants. We also found inflammatory cytokine and matrix metalloproteinases were upregulated after mutated asxl1. It suggests that neutrophil deficiency may stimulate the expression of some inflammatory cytokines and enhances the inflammatory responds. Therefore, transcriptome analysis mainly represented the disruption of neutrophil development.

2021-08-04 | GSE142214 | GEO

Mutant ASXL1 induces age-related expansion of phenotypic hematopoietic stem cells through activation of Akt/mTOR pathway

Project description:Somatic mutations of ASXL1 are frequently detected in age-related clonal hematopoiesis (CH). However, how ASXL1 mutations drive CH remains elusive. Using knockin (KI) mice expressing a C-terminally truncated form of ASXL1-mutant (ASXL1-MT), we examined the influence of ASXL1-MT on physiological aging in hematopoietic stem cells (HSCs). HSCs expressing ASXL1-MT display competitive disadvantage after transplantation. Nevertheless, in genetic mosaic mouse model, they acquire clonal advantage during aging, recapitulating CH in humans. Mechanistically, ASXL1-MT cooperates with BAP1 to deubiquitinate and activate AKT. Overactive Akt/mTOR signaling induced by ASXL1-MT results in aberrant proliferation and dysfunction of HSCs associated with age-related accumulation of DNA damage. Treatment with an mTOR inhibitor rapamycin ameliorates aberrant expansion of the HSC compartment as well as dysregulated hematopoiesis in aged ASXL1-MT KI mice. Our findings suggest that ASXL1-MT provokes dysfunction of HSCs, whereas it confers clonal advantage on HSCs over time, leading to the development of CH.

2021-02-08 | MSV000086816 | MassIVE

Downstream Targets of HOXB4 in the primitive hematopoietic progenitor EML Cell line

Project description:Enforced expression of the homeobox transcription factor HOXB4 has been shown to enhance hematopoietic stem cell (HSC) self-renewal and expansion ex vivo and in vivo. In order to investigate the largely unknown downstream targets of HOXB4 in hematopoietic progenitor cells, HOXB4 was constitutively overexpressed in the primitive hematopoietic progenitor cell line, EML. Gene expression differences were compared between KLS (c-Kit+, Lin-, Sca-1+)-EML cells that overexpressed HOXB4 (KLS-EML-HOXB4) to control KLS-EML cells that were transduced with vector alone. ChIP-chip was used to identify promoter regions bound by HOXB4. We overexpressed HOXB4 in EML cells. We isolated 3 separate single cell clones as assessed by Southern Blot Analysis (3 clones for EML-HOXB4 and 3 clones for control EML-GFP cells). RNA was isolated from the KLS (c-Kit+, Lin-, Sca-1+) fraction of each single cell clone population and processed for hybridization to array chips using established lab protocols. Chip-Chip analysis of the three HOXB4 overexpressing clones was performed to identify HOXB4 bound promoters.

2010-04-20 | E-GEOD-20604 | biostudies-arrayexpress

Functional analysis of KAI1

Project description:We sequenced mRNA from mouse EML cell line under Kai1 knockdown or Kai1 overexpressing condition. KAI1 belongs to tetraspanin superfamily and is known as suppressor of tumor metastasis. Examination of mRNA levels in wildtype (WT) and KAI1 knockdown EML cells were generated by deep sequencing, in duplicate, using Illumina GAIIx. Examination of mRNA levels in wildtype (WT) and KAI1 overexpressing EML cells were also generated in single set.

2016-03-31 | E-GEOD-56867 | biostudies-arrayexpress

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