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Proteosomal degradation of NSD2 by BRCA1 promotes leukemia cell differentiation

ABSTRACT: Inhibiton of NSD2 by shRNA induces K562 differentiation via increasing erythroid specfic lineage factors The human myelogenous leukemic cell line, K562 undergoes erythroid differentiation by exposure to hemin. Here, we uncovered NSD2 as an innate erythroid differentiation-related factor through the genome-wide CRISPR library screening and explored the regulatory role of NSD2 during myeloid leukemia cell differentiation. We found that NSD2 stability was disrupted by poly-ubiquitination in differentiated K562 cells. Proteomic analysis revealed interaction between NSD2 and an E3 ubiquitin ligase, BRCA1 which ubiquitylates NSD K292 residue. Depletion of BRCA1 stabilized NSD2 protein and suppressed K562 cell differentiation. Furthermore, BRCA1 protein level was decreased in bone marrow tumor, while NSD2 level was elevated. Surprisingly, among BRCA1 mutation(s) discovered in lymphoma patients, BRCA1 K1183R prevented its translocation into the nucleus and did not reduce NSD2 protein level in hemin-treated K562 cells and eventually disrupted cell differentiation. Our results indicated that regulation of NSD2 stability by BRCA1-mediated ubiquitination as a potential therapeutic target process in multiple myeloma.

ORGANISM(S): Homo sapiens

PROVIDER: GSE144939 | GEO | 2020/03/25

REPOSITORIES: GEO

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Project description:One major mode of action of DNA hypomethylating agents such as decitabine (DAC) is reactivation of gene expression and induction of differentiation. Prompted by observations of differentiation induction in leukemic myeloid cells and upregulation of fetal hemoglobin (HbF) in adult patients suffering from hemoglobinopathies, we aimed at systematically investigating the potential and significance of DAC for in vitro and in vivo induction of erythroid differentiation and HbF expression. In K562 (bcr-abl positive) and HEL (bcr-abl negative) myeloid leukemia cell line models, both with bilineage differentiation potential, erythroid differentiation and hemoglobin synthesis but not megakaryocytic differentiation could be induced by DAC treatment. The induction of erythroid differentiation was accompanied by a specific transcriptome signature that shared common patterns and groups of genes with the changes observed in K562 cells treated with the hemin, a known inducer of erythropoesis. Top regulated groups of transcripts in DAC treated cells were related to erythropoesis and iron metabolism. DAC treatment led to reactivation and upregulation of alpha globin and genes of the beta globin locus including the gamma globins resulting in HbF tetramer protein synthesis in K562 cells. This DAC-induced erythroid differentiation and HbF induction was accompanied by 2-3 fold upregulation of the key erythroid transcription factor GATA1. In contrast, PMA, an inducer of megakaryocytic differentiation, conversely led to a loss of GATA1 expression in K562 cells. Serial HbF measurements in DAC treated AML (n=25) and high-risk MDS (n=15) patients revealed that induction of HbF or persistent elevated HbF levels during treatment can also be detected in a significant proportion of patients in vivo (61,5%, p=0.033). Here, early HbF induction (after 2 courses) was significantly associated with platelet increment (rs=0.534, p=0.027) and strongly predicted neutrophil recovery (rs=0.554, p=0.021). The presence of elevated HbF levels in patients achieving CR upon DAC and exhibiting complete (cytogenetic) clearance of the malignant clone suggests that HbF induction does not occur in cells of the malignant clone but rather in non-malignant, polyclonal erythroid precursor cells. One major mode of action of DNA hypomethylating agents such as decitabine (DAC) is reactivation of gene expression and induction of differentiation. Prompted by observations of differentiation induction in leukemic myeloid cells and upregulation of fetal hemoglobin (HbF) in adult patients suffering from hemoglobinopathies, we aimed at systematically investigating the potential and significance of DAC for in vitro and in vivo induction of erythroid differentiation and HbF expression. In K562 (bcr-abl positive) and HEL (bcr-abl negative) myeloid leukemia cell line models, both with bilineage differentiation potential, erythroid differentiation and hemoglobin synthesis but not megakaryocytic differentiation could be induced by DAC treatment. The induction of erythroid differentiation was accompanied by a specific transcriptome signature that shared common patterns and groups of genes with the changes observed in K562 cells treated with the hemin, a known inducer of erythropoesis. Top regulated groups of transcripts in DAC treated cells were related to erythropoesis and iron metabolism. DAC treatment led to reactivation and upregulation of alpha globin and genes of the beta globin locus including the gamma globins resulting in HbF tetramer protein synthesis in K562 cells. This DAC-induced erythroid differentiation and HbF induction was accompanied by 2-3 fold upregulation of the key erythroid transcription factor GATA1. In contrast, PMA, an inducer of megakaryocytic differentiation, conversely led to a loss of GATA1 expression in K562 cells. Serial HbF measurements in DAC treated AML (n=25) and high-risk MDS (n=15) patients revealed that induction of HbF or persistent elevated HbF levels during treatment can also be detected in a significant proportion of patients in vivo (61,5%, p=0.033). Here, early HbF induction (after 2 courses) was significantly associated with platelet increment (rs=0.534, p=0.027) and strongly predicted neutrophil recovery (rs=0.554, p=0.021). The presence of elevated HbF levels in patients achieving CR upon DAC and exhibiting complete (cytogenetic) clearance of the malignant clone suggests that HbF induction does not occur in cells of the malignant clone but rather in non-malignant, polyclonal erythroid precursor cells.

Project description:We previously characterized zinc finger protein gene HZF1 (ZNF16) and demonstrated its important roles in erythroid and megakaryocytic differentiation of K562 cells by loss-function assay. However its effect in erythroid and megakaryocytic differentiation of hematopoietic stem/progenitor cells (HSPCs) and the mechanisms by which it functions have not been understood. In this study, we detected up-regulation of ZNF16 during erythroid and megakaryocytic differentiation of K562 cells and normal CD34+ HSPCs, and demonstrated that ZNF16 promotes erythroid and megakaryocytic differentiation by gain-of-function and loss-of-function experiments. Gene expression profiling by mRNA array and PCR validation in the K562 transforments with ZNF16 over-expression suggested that cell division cycle-associated 7-like gene (JPO2) and v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog gene (c-KIT) were among the genes regulated possibly by ZNF16. Luciferase reporter assay and Chromatin Immunoprecipitation demonstrated that ZNF16 binds to JPO2 and c-KIT promoters and inhibits their expression in K562 cells. A significant decrease of JPO2 and c-KIT levels was observed during erythroid and megakaryocytic differentiation of K562 and CD34+ cells. The knockdown of either JPO2 or c-KIT partially rescued the differentiation inhibition caused by ZNF16 knockdown. We also found that ZNF16 inhibits c-KIT/c-Raf/MEK/ERK/c-Jun/HEY1 signal pathways, which finally up-regulated expression of GATA1, a central regulator of erthroid and megakaryocyte differentiation. By lentivirus-mediated gene transfer, we demonstrated that enforced expression and knockdown of ZNF16 in HSPCs down-regulated and up-regulated expression of its targets respectively. Our data collectively demonstrate that ZNF16 promotes erythropoiesis and megakaryocytopoiesis via its regulation on JPO2 and c-KIT. 4 samples are analyzed, H4-1 and H4-2 are two stable K562 transductants with ZNF16 over-expression, PC1 and PC2 are stable control K562 transductants. Stable K562 transductants were obtained for RNA extraction and hybridization on an Illumina HumanHT-12 V4.0 expression beadchipe xpression Array platform.

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Proteosomal degradation of NSD2 by BRCA1 promotes leukemia cell differentiation

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