Discovering chemical modifiers of oncogene-regulated hematopoietic differentiation

Abstract

It has been proposed that inhibitors of an oncogene's effects on multipotent hematopoietic progenitor cell differentiation may change the properties of the leukemic stem cells and complement the clinical use of cytotoxic drugs. Using zebrafish, we developed a robust in vivo hematopoietic differentiation assay that reflects the activity of the oncogene AML1-ETO. Screening for modifiers of AML1-ETO-mediated hematopoietic dysregulation uncovered unexpected roles of COX-2- and beta-catenin-dependent pathways in AML1-ETO function. This approach may open doors for developing therapeutics targeting oncogene function within leukemic stem cells.

Figure 1

Screening for chemical suppressors of AE. Homozygous Tg(hsp:AML1-ETO) fish were crossed with wild-type fish to generate thousands of heterozygous Tg(hsp:AML1-ETO) embryos. These embryos were raised for 12−16 hours post fertilization at which point five embryos were arrayed into each well of the 96-well plates. The compounds from the library were added to the plates. An hour later, the plates were heat-shocked at 40 °C for 1 hour to induce AE expression. At 90 minutes after the heat shock, the embryos were processed for in situ hybridization of gata1. Induced expression of AE resulted in lost of gata1⁺ hematopoietic cells (indicated as purple dots) in the posterior blood islands of the zebrafish embryos. However, the chemical suppressors of AE, such as nimesulide (shown in the figure), antagonized AE's effect, restoring gata1 expression in Tg(hsp:AML1-ETO) embryos.

Figure 2

Nimesulide does not affect AE expression in Tg(hsp:AML1-ETO) embryos. Western blot analysis shows that while rotenone (0.15 μM) inhibits heat-induced expression of AE and Hsp70, neither nimesulide (40 μM) nor trichostatin A (TSA, 0.5 μM) affects heat-induced expression of AE and Hsp70. The expression of Akt is used as a reference for protein loading. At the concentrations used, all three compounds suppress the zebrafish AE phenotype. DMSO is used as the vehicle control. WT+, wild-type embryos with heat treatment. AE-, Tg(hsp:AML1-ETO) embryos without heat treatment. AE+, Tg(hsp:AML1-ETO) embryos with heat treatment. The vertical black line indicates the juxtaposition of lanes that were not contiguous in the original gel.

Figure 3

Cyclooxygenase (COX) inhibitors reverse AE's effects on hematopoietic differentiation. (a) The chemical structures of the selective COX-2 inhibitors nimesulide, NS-398, and the non-selective COX inhibitor indomethacin (INDM). (b) In situ hybridization of gata1. The COX inhibitors restore gata1 expression in Tg(hsp:AML1-ETO) embryos. To induce AE expression, Tg(hsp:AML1-ETO) embryos were incubated at 39 °C for 1 hour at the 16-somite stage (w/ heat). Induced expression of AE results in the loss of gata1 expression as compared to Tg(hsp:AML1-ETO) embryos without heat incubation. Nevertheless, addition of nimesulide (40 μM), NS-398 (25 μM) or INDM (20 μM) reverses AE's effects. All compounds were added to the embryos 1 hour before the heat shock. DMSO was used as the vehicle control. Scale bar, 0.3 mm. (c) In situ hybridization of mpo. NS-398 (25 μM) and INDM (20 μM) antagonize AE's effect, suppressing the accumulation of mpo⁺ granulocytic cells. The antagonism no longer exists in the presence of prostaglandin E2 (20 μM). All compounds were added at the end of heat shock. Scale bar, 0.3 mm.

Figure 4

The hematopoietic phenotype of AE involves induction of the genes that encode both zebrafish COX-2 isoforms. (a) Real-time PCR indicates that ptgs2b is upregulated in the hematopoietic cells of Tg(hsp:AML1-ETO) embryos at 2 hours after heat shock. Total RNA was extracted from the hematopoietic cells isolated from heat-treated wild-type and Tg(hsp:AML1-ETO) embryos at 22 hpf. The expression of ptgs1, ptgs2a, and ptgs2b was evaluated by real-time PCR analysis using two independent primer sets for each gene, and was normalized to gapdh expression. The fold change indicates the ratio of mRNA expression between Tg(hsp:AML1-ETO) and wild-type embryos. ptgs1, 2.09±0.0.91, 1.22±0.08; ptgs2a, 0.62±0.21; 1.04±0.03; ptgs2b, 10.31±1.39, 3.60±1.25 (mean±SEM). (b) Real-time PCR indicates that ptgs2a is upregulated in the hematopoietic cells of Tg(hsp:AML1-ETO) embryos at 40 hpf. ptgs1, 0.26±0.12, 0.28±0.11; ptgs2a, 3.86±0.36; 7.6±0.98; ptgs2b, 1.28±0.39, 1.45±0.81 (mean±SEM). (c) In situ hybridization of gata1. Knockdown of ptgs1, ptgs2a, or ptgs2b partially restored gata1 expression in heat-treated Tg(hsp:AML1-ETO) embryos. Scale bar, 0.3 mm. (d) In situ hybridization of mpo. Knockdown of ptgs2a or ptgs2b suppresses AE-induced mpo expression. Control, non-injected embryos. Scale bar, 0.3 mm.

Figure 5

Erythroid differentiation of human myelogenous leukemia K562 cells is attenuated by AE via a COX-2-dependent mechanism. (a) Real-time PCR indicates that expression of AE causes upregulation of PTGS2 but not PTGS1 in K562 cells. K562 cells containing stable integration of the vector pLRT-GFP (clone B9) or the AE expression vector pLRT-AE (clone D8) were harvested for RNA extraction. The expression of PTGS1 and PTGS2 was evaluated by real-time PCR analysis and normalized to 18S RNA expression. The fold change indicates the ratio of mRNA expression between D8 and B9 cells. PTGS1, 0.962±0.169; PTGS2, 4.798±0.373 (mean±SEM). (b) Erythroid differentiation efficiency as scored by benzidine staining. While AE suppresses erythroid differentiation of K562 cells, inhibition of COX-2 by NS-398 (75 μM) reverses AE's effect. K562 clone B9 (Control) and clone D8 (AE) were treated with either DMSO or NS-398 for 4 days before staining. Control-DMSO, 24.5±0.65; Control-NS-398, 25±0.58; AE-DMSO, 16.25±1.03; AE-NS-398, 25.75±1.89 (mean±SEM). *p<0.01 (two-tailed t-test).

Figure 6

AE activates β-catenin-dependent transcription through COX-2. (a) Expression of AE induces TOPflash luciferase activity, which can be suppressed by overexpression of a dominant-negative form of TCF (dnTCF) or by NS-398 (75 μM). The empty vector (pCS2) or the expression vector for AE (pCS2-AE) was transfected into K562 cells along with TOPflash and pRL-tk which encodes Renilla luciferase for normalizing the transfection efficiency. The results are shown in relative TOPflash luciferase activities after normalization. pCS2/control, 1±0.09; pCS2+dnTCF, 0.73±0.03; pCS2+NS-398, 0.58±0.01; pCS2-AE/control, 12.29±1.09, pCS2-AE+dnTCF,2.93±0.3; pCS2-AE+NS-398, 5.29±0.57 (mean±SEM). *p<0.01 (two-tailed t-test). (b) 16,16-dimethylprostaglandin E2 (dmPGE2) activates β-catenin-TCF-dependent transcription. K562 cells were transfected with TOPflash and pRL-tk. At 4 hours after transfection, PGE2 (20 μM), dmPGE2 (20 μg ml⁻¹) or DMSO was added to the cells. The luciferase activities were measured two days after the transfection. In addition, the dmPGE2-induced TOPflash activity can be suppressed by overexpression of dnTCF. control, 1±0.05; control+dn-TCF, 0.56±0.03; PGE2, 2.44±0.15; PGE2+dnTCF, 0.83±0.1; dmPGE2, 9.06±1.02; dmPGE2+dnTCF, 1.89±0.38 (mean±SEM). *p<0.01 (two-tailed t-test).

Figure 7

The hematopoietic differentiation effects caused by AE are dependent on β-catenin function. (a-b) In situ hybridization of gata1 (a) and mpo (b). Knockdown of either β-catenin1 ctnnb1 MO) or β-catenin2 (ctnnb2 MO) restores gata1 expression and suppresses mpo upregulation in the presence of AE. Control, non-injected embryos. Scale bar, 0.3 mm.