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Effect of the LSD-1 Inhibitor RN-1 on g-globin and Global gene expression during Erythroid Differentiation in Baboons

ABSTRACT: Increased levels of Fetal Hemoglobin reduce the severity of symptoms of patients with sickle cell disease and decrease the risk of death. An affordable, small molecule drug that stimulates HbF expression in vivo would be ideally suited to treat the large numbers of SCD patients that exist worldwide. Our previous work showed that administration of the LSD1 (KDM1A) inhibitor RN-1 to normal baboons increased Fetal Hemoglobin (HbF) and was tolerated over a prolonged treatment period. HbF elevations were associated with changes in epigenetic modifications that included increased levels of H3K4 di-and tri-methyl lysine at the g-globin promoter. While dramatic effects of the loss of LSD1 on hematopoietic differentiation have been observed in murine LSD1 gene deletion and silencing models, the effect of pharmacological inhibition of LSD1 in vivo on hematopoietic differentiation is unknown. The goal of these experiments was to investigate the in vivo mechanism of action of the LSD1 inhibitor RN-1 by 1) determining its effect on g-globin expression in highly purified subpopulations of bone marrow erythroid cells enriched for varying stages of erythroid differentiation isolated directly from baboons treated with RN-1 and also by 2) investigating the effect of RN1 on the global transcriptome in a highly purified population of proerythroblasts. Our results show that RN-1 administered to baboons targets an early event during erythroid differentiation responsible for γ-globin repression and increases the expression of a limited number of genes including genes involved in erythroid differentiation such as GATA2, GFi-1B, and LYN.

ORGANISM(S): Papio anubis

PROVIDER: GSE235633 | GEO | 2023/12/27

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.

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Effect of the LSD-1 Inhibitor RN-1 on g-globin and Global gene expression during Erythroid Differentiation in Baboons

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