Small molecule screen for inhibitors of expression from canonical CREB response element-containing promoters

Abstract

The transcription factor CREB (cAMP Response Element Binding Protein) is an important determinant in the growth of Acute Myeloid Leukemia (AML) cells. CREB overexpression increases AML cell growth by driving the expression of key regulators of apoptosis and the cell cycle. Conversely, CREB knockdown inhibits proliferation and survival of AML cells but not normal hematopoietic cells. Thus, CREB represents a promising drug target for the treatment of AML, which carries a poor prognosis. In this study, we performed a high-throughput small molecule screen to identify compounds that disrupt CREB function in AML cells. We screened ~114,000 candidate compounds from Stanford University's small molecule library, and identified 5 molecules that inhibit CREB function at micromolar concentrations, but are non-toxic to normal hematopoietic cells. This study suggests that targeting CREB function using small molecules could provide alternative approaches to treat AML.

Keywords: CREB; novel therapeutics; small molecule screen.

Figure 1. Candidate compound counter-selection

From 114, 124 compounds, 980 were selected for counter-screening against KG-1 CMV cells. This was done in order to discriminate between compounds that only suppressed CREB-driven luciferase activity (grey) and those that non-specifically suppressed luciferase (black) in both AML cell lines. (A) Representative graph of compounds that reduced luciferase activity in both cell lines equally. (B–E) Representative graphs of compounds with an IC₅₀ 2-fold higher in KG-1 CMV cells compared to KG-1 CRE cells (were more potent against KG-1 CRE cells) were selected for further study. From 980 compounds, 23 were selected for further study.

Figure 2. Candidate compound selectivity for CREB-driven vs. non-CREB-driven luciferase expression

(A–F) The 23 compounds with putative selectivity for CREB-driven luciferase expression were tested in upscaled luciferase assays using KG-1 CRE and KG-1 CMV AML cells. In these extended assays, 6 of 23 compounds (STF-017794, STF-038533, STF-046536, STF-046728, STF-055910 and STF-120123) showed very little activity in suppressing non-CREB-driven luciferase expression (white), but were able to suppress CREB-driven luciferase expression (black) with at least three concentrations of compound.

Figure 3. Efficacy of candidate compounds against AML cells in vitro

The efficacy of the 6 candidate compounds inhibiting cell proliferation was tested against two well-characterized AML cell lines as described in Materials and Methods.

Figure 4. Toxicity of candidate compounds to normal bone marrow cells in vitro

To evaluate the potential toxicity of these compounds against non-cancer cells, cultured bone marrow cells from human donors were treated with each of 6 candidate compounds (10 μM) for 72 hours. Doxorubicin (3.8 μM) was used as a positive control. The viable cell count, shown as a percent of viable cells remaining after DMSO (vehicle) treatment, remained > 95% of control for cells treated with STF-017794, STF-038533, STF-046728 and STF-120123.

Figure 5. On-target effects of candidate compound STF-038533

To assess whether compound STF-038533(533) exerted ‘on-target’ effects on validated CREB target genes, the expression of RFC3, Fra-1 and POLD2 were examined following 24 hours of treatment with STF-038533 (10 μM) 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 (*p < 0.05).

Figure 6. High-throughput screening strategy

Promoter schematics for the two KG-1 cell lines generated for use in the high-throughput screen. For both cell lines, a Firefly luciferase gene containing two C-terminal protein destabilizing sequences (hCL1 and h PEST) was used in order to reduce this protein's half-life and facilitate early changes in the expression of this reporter gene. The promoter for CREB-specific expression of luciferase was composed of two ‘CRE’ elements, which are the canonical CREB DNA-binding sequences, placed within −200 of the ATG start site and 5′ to the CMV minimal promoter (KG-1 CRE cells). For non-specific luciferase expression, the full CMV promoter (KG-1 CMV cells) was placed 5′ to the luciferase gene.