Oncogene

Oncogene. molecules could provide alternate approaches to treat AML. studies revealed that CREB overexpression in AML cells augments their growth rate and confers resistance to apoptosis [2]. In contrast, CREB knockdown inhibited AML cell proliferation and induced apoptosis, but experienced no effect on normal hematopoietic stem cells in mouse transduction/transplantation assays [3]. In addition, the manifestation of this potential drug target is typically much higher in AML cells compared to normal hematopoietic cells, the parental cells of this tumor. This suggests higher reliance on this transcription element for AML cell homeostasis [1, 2, 4]. Given these data, we hypothesize that inhibition of CREB function may represent a novel, effective and targeted approach to treat AML. Previous reports possess described successful disruption of HYRC the association between CREB and its essential transcriptional co-activator CBP [5]. Post-translational changes of CREB including phosphorylation, acetylation, and SUMOylation, are critical for its function [6]. CREB also Sodium lauryl sulfate differentially binds like a homodimer or heterodimer with users of the ATF transcription element family, resulting in differential gene manifestation based on the cellular context [6]. These observations demonstrate that numerous CREB functionalities could be selected for targeted disruption by a small molecule. Luckily, the arrival of high-throughput screening offers facilitated exploration of a wide array of chemical moieties in search of molecules which may disrupt any of these varied processes, actually in the absence of knowledge of which of these processes are most important for cellular homeostasis. Thus, in this study, we performed a small molecule screen in search of compounds capable of disrupting CREB-driven transcription in AML cells. To this end, we screened 114,124 candidate compounds from your compound library available at the Stanford University or college High-Throughput Bioscience Center. This collection was put together from several commercial vendors, including ChemDiv, Specs, and Chembridge, and possesses drug-like qualities [7]. These compounds were first tested for their ability to specifically disrupt CREB-driven expression of a reporter gene in KG-1 cells. Determined compounds that exceeded this initial screen were validated and examined for their ability to selectively kill AML cells, but not normal hematopoietic cells, potency and lower toxicity than doxorubicin. Given the Sodium lauryl sulfate high potency (low IC50 value), selectivity and non-toxicity of compound STF-038533, this molecule was analyzed for its ability to reduce CREB target gene transcription. The and genes each possess CRE elements within 200 bp of their transcription start sites, and previous work supports the importance of CREB in regulating their transcription [8, 12]. RT-PCR data show Sodium lauryl sulfate that this transcription of these genes in KG-1 cells treated with 10 M STF-038533 for 24 hours was reduced compared to DMSO-treated cells. KG-1 cells with CREB knockdown using shRNAs were used as a positive control (Physique ?(Figure55). Open in a separate window Physique 4 Toxicity of candidate compounds to normal bone marrow cells and were examined following 24 hours of treatment with STF-038533 (10 M) and compared to KG-1 cells in which CREB expression was reduced by shRNA(CREB KD). Each of these genes exhibited significantly reduced expression compared to control cells, treated with DMSO (* 0.05). Conversation Recent preclinical reports have exhibited the efficacy of targeting transcription factors in specific cancers [13C16]. The association of CBP with – and -catenin has been targeted using a small molecule, and this strategy was effective against both main and relapsed ALL in mice [13]. Another group exhibited the efficacy of targeting the mutant fusion transcription factor CBP-SMMHC, which drives inv(16)+ AML [15]. The association between menin and MLL fusion proteins, which drives subtypes of both AML and ALL, has also been successfully targeted using a small molecule [14]. These studies demonstrate the potential of transcription factor-directed therapy, and encourage further development of these novel candidate compounds for eventual clinical use. In this study, we employed a high-throughput screening strategy, which yielded five chemically unique compounds, which exhibited encouraging potencies and efficacies and showed little to no toxicity to normal hematopoietic cells compared to doxorubicin. In summary, the data presented here suggest that the development of small molecules that target CREB could lead to novel approaches to treat AML. Even though compounds identified require structure-activity relationship-based optimization, characterization of their specific mechanism(s) of action, and screening prior to their access into clinical trials, our data encourage further studies of the advantages of targeting this transcription factor. METHODS AML cell lines AML cell lines were purchased from ATCC and managed with IMDM (Gibco) supplemented with 10% FBS (Fisher Scientific) and 1% PSG (Gibco). KG-1 is usually a well-characterized human AML cell collection commonly used to study novel therapies for AML. KG-1 cells were established from a patient with.