ALL1 fusion proteins induce deregulation of EphA7 and ERK phosphorylation in human acute leukemias

By applying a transfection strategy to ectopically express ALL1/AF4 and ALL1/AF9 in K562 cells (Fig. 1A bottommost blot, left lane), we determined the expression level of the genes encoding eight EphA receptors and five ephrin-A ligands in the transfectants. Semiquantitative RT-PCR analysis showed that both ALL1 fusion proteins induced transcription of all EphA receptor genes, whereas they did not exert a noticeable effect on induction of ephrin-A genes (Fig. 1A). To further extend this finding to leukemic cells with ALL1 rearrangement, the SEMK2 and RS4;11 pro-B cell lines harboring the t(4;11) translocation, and the 380 pro-B and 697 pre-B cell lines lacking ALL1 abnormalities, were subjected to similar analysis (Fig. 1B). This analysis showed consistent and differential expression of EphA7 in the t(4;11) leukemic cell lines. Subsequent quantification of EphA7 transcript by application of real-time RT-PCR methodology enabled estimation of the amounts of the transcript in K562 cells transfected with ALL1/AF4 or ALL1/AF9, in SEMK2 and RS4;11 cells to be 0.04 ± 0.01, 0.014 ± 0.006, 1.97 ± 0.6, and 0.68 ± 0.09 (mean ± SD femtogram), respectively. In parallel, the amount of EphA7 RNA in vector-transfected K562, intact 380 and 697 cells was determined to be <0.001 fg (Fig. 1C). These results collectively pointed to EphA7 as a consistently responsive target of ALL1 fusion proteins and prompted further analysis of its up-regulation. To ascertain that the endogenous ALL1 fusion protein produced in leukemic cells carrying an ALL1 abnormality regulates EphA7 transcription, we suppressed ALL1/AF4 produced in SEMK2 cells by applying siRNA methodology. SEMj siRNA generated by us (14) and others (15) is designed to target SEMK2 cell-specific ALL1/AF4 fusion junction; in parallel, MVj siRNA targets the fusion junction produced in other cells (MV4;11), and thus served as a negative control. We first determined the efficiency of SEMj siRNA in suppressing the ALL1/AF4 protein and found ≈80% reduction of the latter, with no effect on the expression level of normal ALL1 or AF4 (Fig. 2A). SEMK2 cells treated with SEMj siRNA were then subjected to semiquantitative RT-PCR analysis to determine the expression levels of EphA7 and HoxA9; the latter is a known target of ALL1 fusion proteins (16) and thus served as a positive control to ascertain the effect of elimination of the ALL1 fusion protein. This analysis demonstrated that the suppression of the ALL1/AF4 in SEMK2 cells attenuated expression of EphA7 and HoxA9, supporting the notion of ALL1 fusion protein-mediated transcriptional regulation of EphA7 (Fig. 2B).

By applying ChIP methodology, we determined the occupancy of HA-tagged ALL1/AF4 and ALL1/AF9 exogenously expressed in K562 cells. The EphA7 genomic regions analyzed by ChIP mapped ≈0.7 kb upstream and ≈0.6 kb downstream from the transcription initiation site and within 3′ noncoding sequence; they were termed regions 1, 2, and 3, respectively (Fig. 3A). This analysis showed the binding of the ALL1/AF4 and ALL1/AF9 chimeric proteins to regions 1 and 2, but not to region 3 (Fig. 3B). Quantitative real-time PCR analysis supported the previous results (Fig. 3C), indicating that EphA7 is a direct target of the ALL1 fusion proteins.

Little is known about the signal transduction pathway(s) in which EphA7 is involved. At first, we examined in K562 cells transfected with full-length EphA7 construct the phosphorylation status of several proteins commonly associated with RTK signal transduction pathways. Consistent with previous results (see Fig. 1A Right), the EphA7 protein was not observed in intact K562 cells but was detected in a dose-dependent manner in cells transfected with the EphA7 construct (Fig. 4A Left). We found no apparent induction of RAF or MEK1/2 phosphorylation, two proteins that are phosphorylated in response to activation of the Ras transduction pathway (Fig. 4A Left). In contrast, we found that overexpressing EphA7 correlated with ERK phosphorylation. This correlation was determined by sequential probing of a Western blot with anti-ERK Ab and subsequently with antibody against phosphorylated-ERK which specifically detects p-ERK (Fig. 4A Left). It was previously shown that MEK1/2 are highly phosphorylated in intact K562 cells (17). Such basic high MEK1/2 phosphorylation may have obscured an additional change in phosphorylation induced by overexpressing EphA7. Therefore, we ectopically expressed EphA7 in the KG1 AML cell line. Here, we found induction of MEK1/2 phosphorylation after expression of recombinant EphA7 (Fig. 4A Right). We also noticed that the MEK1/2 phosphorylation in KG1 cells was accompanied by ERK2 as well as ERK1 phosphorylation (Fig. 4A Right). Because the prominent function of MEK is to phosphorylate ERK (18), our results indicate that EphA7 is positioned upstream of this cascade.

We next determined whether ALL1 fusion proteins induce ERK phosphorylation as EphA7 does. Indeed, induction of ERK phosphorylation was observed in K562 cells transfected with the ALL1/AF4 and ALL1/AF9 constructs (Fig. 4B). To validate that in these transfectants EphA7 is an essential mediator for ERK phosphorylation, SEMK2 cells were treated with siRNAs targeting EphA7 mRNA at two distinct regions within the transcript (siEphA7, -1, and -2 in Fig. 4C). Both siRNAs were shown to down-regulate the mRNA at an efficiency of ≈70% (Fig. 4C Lower). Suppression of EphA7 resulted in strong reduction of ERK1 and ERK2 phosphorylation, with no effect on the amount of total ERK (Fig. 4C Upper). This finding implied that EphA7 is an essential mediator for ERK1/2 phosphorylation in cells expressing ALL1 fusions. Furthermore, we found that ALL1/AF4 knockdown in SEMK2 cells similarly caused reduced phosphorylation of ERK1/2 (Fig. 4D).

We examined the effect of 5-ITU, an inhibitor for ERK phosphorylation (19), on cell proliferation. To ascertain the biochemical effect of 5-ITU, SEMK2 cells with the t(4;11) and producing phosphorylated ERK1/2 (see “−” and “si control” lanes in Fig. 4 C and D) were treated with this compound, and ERK phosphorylation status was determined by Western blot analysis (Fig. 5Top Right). The result indicated inhibition of ERK phosphorylation by 5-ITU. Application of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay indicated reduction in numbers of SEMK2 and RS4;11 cells with the t(4;11) and no diminution in the number of the 380 and 697 control cells (Fig. 5 Left). We also found that ALL1/AF4 knockdown in SEMK2 cells, which caused ERK1/2 inactivation (see Fig. 4D), resulted in ≈25% reduction in the cell number as compared with the number of cells treated with control siRNA (H.N., unpublished results). To determine the cause for the reduction in cell numbers, the 5-ITU-treated cells were subjected to assays determining apoptotic cell death (caspase-3 activity assay and FACS analysis). Increased caspase 3 activity was demonstrated at 24 h in 5-ITU sensitive leukemic cells (SEMK2 and RS4;11 in Fig. 5 Center). In parallel, a high proportion of these cells distributed at the sub-G₁ phase (Fig. 5 Right). These results demonstrated dependency of the leukemic cells producing ALL1/AF4 fusion protein on ERK phosphorylation, with the latter preventing apoptotic cell death.

The human erythroleukemia cell line K562, the AML cell line KG1, and the pro-B acute lymphoblastic leukemia cell line RS4;11 with the t(4;11) abnormality were obtained from American Type Culture Collection (ATCC). The SEMK2 line was provided by Johann Greil. Other acute lymphoblastic leukemia cell lines selected for this study (380, 697) are maintained in our laboratory. All cell lines were grown in RPMI medium 1640 supplemented with 10% FCS.

Total RNA was isolated from transfectant 16 h after transfection by using RNeasy spin-column kits according to the manufacturer's instructions (Qiagen, Valencia, CA). Five micrograms of total RNA was subjected to cDNA synthesis by using SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA). At the end of the reaction, 40 μl of TE (10 mM Tris/1 mM EDTA, pH 7.6) was added, and 1 μl of the reaction (1/60 volume) was used as a template for PCR. The following number of cycles, which allowed semiquantitative comparison, were applied to PCR: 35 cycles for EphA1, -2, -4, -7, and -8 and ephrin-A1, -2, -3, -4, and -5; 40 cycles for EphA3, -5, and -6; 25 cycles for GAPDH; and 32 cycles for HoxA9. Primer sequences are shown in SI Table 1. EphA7 transcript was quantified by using QuantiTect SYBR Green RT-PCR kit (Qiagen) for the amplification of cDNA (1 μl cDNA of a 60-μl reaction, which corresponds to 0.083 μg RNA) and iCycler real-time PCR detection system (Bio-Rad, Hercules, CA) for the detection of PCR product. Cycle threshold (Ct) values determined by using a known amount of EphA7 cDNA (10-fold serial dilutions starting at 100 pg) were used to construct the standard curve. Ct value of the tested cDNA was converted to weight according to the standard curve.

ChIP assay was performed by using the ChIP assay kit from Upstate Biotechnology (Lake Placid, NY). Sixteen hours after transfection with the ALL1 fusion constructs, 5 × 10⁶ cells (K562 transfectants) were cross-linked, washed with PBS, resuspended in 1 ml of SDS lysis buffer, and sonicated to generate ≈200–1,000 bp DNA fragments. One-tenth of a volume of this preparation, i.e., 0.1 ml aliquot containing 25 μg DNA, was used per ChIP. Antibodies used were anti-HA mAb clone F-7 and normal mouse IgG purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Immunoprecipitated chromatin was reverse cross-linked, deprotenized, and resuspended in 50 μl TE (pH 8.0), and 1-μl aliquot of this preparation was used as a template for PCR. Input indicates PCR with 0.05% of total input chromatin, which corresponds to 0.0125 μg DNA. EphA7 primer sequences used in ChIP assay are as follows: region 1, 5′-ATGCAGCGAAATGGAAAACT-3′ (forward) and 5′-AAAAGGGAGTGGGAAAGGAA-3′ (reverse); region 2, 5′-TAGTACCTCAGGCGGGTCAC-3′ (forward) and 5′-TTCCGAGCTCATCGAAGTCT-3′ (reverse); region 3, 5′-TTGTCGTTGGACGTTCACAT-3′ (forward) and 5′-CAATAGCGCCTCATCTGACA-3′ (reverse).

ChIP-enriched DNA was also subjected to real-time PCR analysis essentially as described in Semiquantitative and Real-Time RT-PCR Analysis. Results were calculated as the percentage of input according to the following formula: 100/2^{Ct of ChIP-enriched DNA − Ct of input DNA} (%).