A Cell-Based High-Throughput Screening for Inducers of Myeloid Differentiation.
ABSTRACT: Recent progress of genetic studies has dramatically unveiled pathogenesis of acute myeloid leukemia (AML). However, overall survival of AML still remains unsatisfactory, and development of novel therapeutics is required. CCAAT/enhancer binding protein ? (C/EBP?) is one of the crucial transcription factors that induce granulocytic differentiation, and its activity is perturbed in human myeloid leukemias. As its reexpression can induce differentiation and subsequent apoptosis of leukemic cells in vitro, we hypothesized that chemical compounds that restore C/EBP? expression and/or activity would lead to myeloid differentiation of leukemic cells. Using a cell-based high-throughput screening, we identified 2-[(E)-2-(3,4-dihydroxyphenyl)vinyl]-3-(2-methoxyphenyl)-4(3H)-quinazolinone as a potent inducer of C/EBP? and myeloid differentiation. Leukemia cell lines and primary blast cells isolated from human patients with AML treated with ICCB280 demonstrated evidence of morphological and functional differentiation, as well as massive apoptosis. We performed conformational analyses of the high-throughput screening hit compounds to postulate the spatial requirements for high potency. Our results warrant a development of novel differentiation therapies and significantly affect care of patients with AML with unfavorable prognosis in the near future.
Project description:The CCAAT enhancer-binding protein ? (C/EBP?) plays an important role in myeloid cell differentiation and in the enhancement of C/EBP? expression/activity, which can lead to granulocytic differentiation in acute myeloid leukemia (AML) cells. We found that styryl quinazolinones induce upregulation of C/EBP? expression, and thereby induce myeloid differentiation in human myeloid leukemia cell lines. We screened a series of active styryl quinazolinones and evaluated the structure?activity relationship (SAR) of these small molecules in inducing C/EBP? expression-thereby prompting the leukemic cells to differentiate. We observed that compound 78 causes differentiation at 3 ?M concentration, while 1 induces differentiation at 10 ?M concentration. We also observed an increase in the expression of neutrophil differentiation marker CD11b upon treatment with 78. Both the C/EBP? and C/EBP? levels were found to be upregulated by treatment with 78. These SAR findings are inspiration to develop further modified styryl quinazolinones, in the path of this novel differentiation therapy, which can contribute to the care of patients with AML.
Project description:Mutation or epigenetic silencing of the transcription factor C/EBP? is observed in ?10% of patients with acute myeloid leukemia (AML). In both cases, a common global gene expression profile is observed, but downstream targets relevant for leukemogenesis are not known. Here, we identify Sox4 as a direct target of C/EBP? whereby its expression is inversely correlated with C/EBP? activity. Downregulation of Sox4 abrogated increased self-renewal of leukemic cells and restored their differentiation. Gene expression profiles of leukemia-initiating cells (LICs) from both Sox4 overexpression and murine C/EBP? mutant AML models clustered together but differed from other types of AML. Our data demonstrate that Sox4 overexpression resulting from C/EBP? inactivation contributes to the development of leukemia with a distinct LIC phenotype.
Project description:Nowadays, drug resistance still represents a major obstacle to successful acute myeloid leukemia (AML) treatment and the underlying mechanism is not fully elucidated. Here, we found that high expression of c-Myc was one of the cytogenetic characteristics in the drug-resistant leukemic cells. c-Myc over-expression in leukemic cells induced resistance to chemotherapeutic drugs, enhanced colony formation capacity and inhibited cell differentiation induced by all-trans retinoic acid (ATRA). Meanwhile, inhibition of c-Myc by shRNA or specific c-Myc inhibitor 10058-F4 rescued the sensitivity to cytotoxic drugs, restrained the colony formation ability and promoted differentiation. RT-PCR and western blotting analysis showed that down-regulation of C/EBP? contributed to the poor differentiation state of leukemic cells induced by c-Myc over-expression. Importantly, over-expression of C/EBP? could reverse c-Myc induced drug resistance. In primary AML cells, the c-Myc expression was negatively correlated with C/EBP?. 10058-F4, displayed anti-proliferative activity and increased cellular differentiation with up-regulation of C/EBP? in primary AML cells. Thus, our study indicated that c-Myc could be a novel target to overcome drug resistance, providing a new approach in AML therapy.
Project description:The transcription factor C/EBP? is required for granulocytic differentiation of normal myeloid progenitors and is frequently inactivated in acute myeloid leukemia (AML) cells. Ectopic expression of C/EBP? in AML cells suppresses proliferation and induces differentiation suggesting that restoring C/EBP? expression/activity in AML cells could be therapeutically useful. Unfortunately, current approaches of gene or protein delivery in leukemic cells are unsatisfactory. However, "drug repurposing" is becoming a very attractive strategy to identify potential new uses for existing drugs. In this study, we assessed the biological effects of candidate C/EBP?-mimetics identified by interrogation of the Connectivity Map database. We found that amantadine, an antiviral and anti-Parkinson agent, induced a monocyte-macrophage-like differentiation of HL60, U937, Kasumi-1 myeloid leukemia cell lines, as indicated by morphology and differentiation antigen expression, when used in combination with suboptimal concentration of all trans retinoic acid (ATRA) or Vit D3. The effect of amantadine depends, in part, on increased activity of the vitamin D receptor (VDR), since it induced VDR expression and amantadine-dependent monocyte-macrophage differentiation of HL60 cells was blocked by expression of dominant-negative VDR. These results reveal a new function for amantadine and support the concept that screening of the Connectivity Map database can identify small molecules that mimic the effect of transcription factors required for myelo-monocytic differentiation.
Project description:While acute myeloid leukemia (AML) comprises many disparate genetic subtypes, one shared hallmark is the arrest of leukemic myeloblasts at an immature and self-renewing stage of development. Therapies that overcome differentiation arrest represent a powerful treatment strategy. We leveraged the observation that the majority of AML, despite their genetically heterogeneity, share in the expression of HoxA9, a gene normally downregulated during myeloid differentiation. Using a conditional HoxA9 model system, we performed a high-throughput phenotypic screen and defined compounds that overcame differentiation blockade. Target identification led to the unanticipated discovery that inhibition of the enzyme dihydroorotate dehydrogenase (DHODH) enables myeloid differentiation in human and mouse AML models. In vivo, DHODH inhibitors reduced leukemic cell burden, decreased levels of leukemia-initiating cells, and improved survival. These data demonstrate the role of DHODH as a metabolic regulator of differentiation and point to its inhibition as a strategy for overcoming differentiation blockade in AML.
Project description:Leukemias exhibit a dysregulated developmental program mediated through both genetic and epigenetic mechanisms. Although IKZF2 is expressed in hematopoietic stem cells (HSCs), we found that it is dispensable for mouse and human HSC function. In contrast to its role as a tumor suppressor in hypodiploid B-acute lymphoblastic leukemia, we found that IKZF2 is required for myeloid leukemia. IKZF2 is highly expressed in leukemic stem cells (LSCs), and its deficiency results in defective LSC function. IKZF2 depletion in acute myeloid leukemia (AML) cells reduced colony formation, increased differentiation and apoptosis, and delayed leukemogenesis. Gene expression, chromatin accessibility, and direct IKZF2 binding in MLL-AF9 LSCs demonstrate that IKZF2 regulates a HOXA9 self-renewal gene expression program and inhibits a C/EBP-driven differentiation program. Ectopic HOXA9 expression and CEBPE depletion rescued the effects of IKZF2 depletion. Thus, our study shows that IKZF2 regulates the AML LSC program and provides a rationale to therapeutically target IKZF2 in myeloid leukemia.
Project description:Myb is a key regulator of hematopoietic progenitor cell proliferation and differentiation and has emerged as a potential target for the treatment of acute leukemia. Using a myeloid cell line with a stably integrated Myb-inducible reporter gene as a screening tool we have previously identified Celastrol, a natural compound with anti-tumor activity, as a potent Myb inhibitor that disrupts the interaction of Myb with the co-activator p300. We showed that Celastrol inhibits the proliferation of acute myeloid leukemia (AML) cells and prolongs the survival of mice in an in vivo model of AML, demonstrating that targeting Myb with a small-molecule inhibitor is feasible and might have potential as a therapeutic approach against AML. Recently we became aware that the reporter system used for Myb inhibitor screening also responds to inhibition of C/EBP?, a transcription factor known to cooperate with Myb in myeloid cells. By re-investigating the inhibitory potential of Celastrol we have found that Celastrol also strongly inhibits the activity of C/EBP? by disrupting its interaction with the Taz2 domain of p300. Together with previous studies our work reveals that Celastrol independently targets Myb and C/EBP? by disrupting the interaction of both transcription factors with p300. Myb, C/EBP? and p300 cooperate in myeloid-specific gene expression and, as shown recently, are associated with so-called super-enhancers in AML cells that have been implicated in the maintenance of the leukemia. We hypothesize that the ability of Celastrol to disrupt the activity of a transcriptional Myb-C/EBP?-p300 module might explain its promising anti-leukemic activity.
Project description:C/EBPs are a family of transcription factors that regulate growth control and differentiation of various tissues. We found that C/EBP? is highly upregulated in a subset of acute myeloid leukemia (AML) samples characterized by C/EBP? hypermethylation/silencing. Similarly, C/EBP? was upregulated in murine hematopoietic stem/progenitor cells lacking C/EBP?, as C/EBP? mediates C/EBP? suppression. Studies in myeloid cells demonstrated that CEBPG overexpression blocked neutrophilic differentiation. Further, downregulation of Cebpg in murine Cebpa-deficient stem/progenitor cells or in human CEBPA-silenced AML samples restored granulocytic differentiation. In addition, treatment of these leukemias with demethylating agents restored the C/EBP?-C/EBP? balance and upregulated the expression of myeloid differentiation markers. Our results indicate that C/EBP? mediates the myeloid differentiation arrest induced by C/EBP? deficiency and that targeting the C/EBP?-C/EBP? axis rescues neutrophilic differentiation in this unique subset of AMLs.
Project description:Transcription factor C/EBP? is a master regulator of myelopoiesis and its inactivation is associated with acute myeloid leukemia. Deregulation of C/EBP? by microRNAs during granulopoiesis or acute myeloid leukemia development has not been studied. Here we show that oncogenic miR-182 is a strong regulator of C/EBP?. Moreover, we identify a regulatory loop between C/EBP? and miR-182. While C/EBP? blocks miR-182 expression by direct promoter binding during myeloid differentiation, enforced expression of miR-182 reduces C/EBP? protein level and impairs granulopoiesis in vitro and in vivo. In addition, miR-182 expression is highly elevated particularly in acute myeloid leukemia patients with C-terminal CEBPA mutations, thereby depicting a mechanism by which C/EBP? blocks miR-182 expression. Furthermore, we present miR-182 expression as a prognostic marker in cytogenetically high-risk acute myeloid leukemia patients. Our data demonstrate the importance of a controlled balance between C/EBP? and miR-182 for the maintenance of healthy granulopoiesis.C/EBP? is a critical transcription factor involved in myelopoiesis and its inactivation is associated with acute myeloid leukemia (AML). Here the authors show a negative feedback loop between C/EBP? and miR-182 and identify this miRNA as a marker of high-risk AML.
Project description:The transcription factor CCAAT enhancer binding protein ? (C/EBP?) is a master regulator in granulopoiesis and is frequently disrupted in acute myeloid leukemia (AML). We have previously shown that C/EBP? exerts its effects by regulating microRNAs (miRs) such as miR-223 and miR-34a. Here, we confirm miR-30c as a novel important target of C/EBP? during granulopoiesis. Thus, wild-type C/EBP?-p42 directly upregulates miR-30c expression, whereas C/EBP?-p30, found in AML, does not. miR-30c is downregulated in AML, especially in normal karyotype AML patients with CEBPA mutations. An induced C/EBP? knockout in mice leads to a significant downregulation of miR-30c expression in bone marrow cells. We identified NOTCH1 as a direct target of miR-30c. Finally, a block of miR-30c prevents C/EBP?-induced downregulation of Notch1 protein and leads to a reduced CD11b expression in myeloid differentiation. Our study presents the first evidence that C/EBP?, miR-30c, and Notch1 together play a critical role in granulocytic differentiation and AML, and particularly in AML with CEBPA mutations. These data reveal the importance of deregulated miRNA expression in leukemia and may provide novel biomarkers and therapeutic targets in AML.