Induction of apoptosis in imatinib sensitive and resistant chronic myeloid leukemia cells by efficient disruption of bcr-abl oncogene with zinc finger nucleases

K562 (Cell Bank of Shanghai Institute of Cell Biology, Chinese Academy of Sciences) and K562/G01 cells were maintained in RPMI 1640 (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA). The resistant cell line, K562/G01, was screened from K562 by culturing with successively increased concentrations of imatinib for several months [42]. For 32D cells, additional 1 ng/ml of murine IL-3 (PeproTech, USA) was supplemented. 293 T and HepG2 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) contained of 10% fetal bovine serum. All cells were cultured at 37 °C in a 5% CO₂ humidified incubator.

ZFNs were transfeced using the Amaxa Nucleofector II device together with the cell line nucleofector kit V or human CD34⁺ cells nucleofector kit (Lonza, Basel, Switzerland). 1 × 10⁶ cells were collected, resuspended in 100 μl of the pre-mixed nucleofector solution with DNA plasmids and nucleofected with the program T-016 for K562 and K562/G01, E-032 for 32D, and U-008 for CD34⁺ cells. After nucleofection, the cells were immediatly resuspended in 500 μl pre-warmed medium and maintained in a 12-well plate.

ZFP-L and ZFP-R were designed and assembled as described. FokI plasmids, containing the Sharkey mutations and the ELD/KKR obligate heterodimer mutations, were obtained from Addgene (FokI-L: plasmid #37198; FokI-R: plasmid #37199) containing the Sharkey mutations and the ELD/KKR obligate heterodimer mutations. Assembled ZFNs were cloned into pAdTrack-CMV termed ZFN-L and ZFN-R. FLAG tag was added to the N-terminal of pAdTrack-CMV. The homology arms in the donor, containing left arm and right arm, were amplified through PCR from human and mouse genomic DNA. The left arm (sense: 5’-CGGGGTACCCAGCGATGGGGCTTCCGGCG-3′, antisense: 5’-AAGGAAAAAAGCGGCCGCGGGTTCAACTCGGCGTCCTCGTAGTCG-3′) and right arm (sense: 5’-AAGGAAAAAAGCGGCCGCCCGCTTCCTGAAGGACAACCTGATCG-3′, antisense: 5’-GCTCTAGAGCCAGGATTCCCGACAGGACCCATTTTC-3′) were inserted into pAdTrack-CMV vector at KpnI, NotI, and XbaI site to generate the donor plasmid.

The amino acid sequences of ZFP-L and ZFP-R were as follows respectively:

ZFP-L: 5’ LEPGEKPYKCPECGKSFSDCRDLARHQRTHTGEKPYKCPECGKSFSDPGNLVRHQRTHTGEKPYKCPECGKSFSDPGALVRHQRTHTGEKPYKCPECGKSFSDCRDLARHQRTHTGKKTS 3’.

ZFP-R: 5’ LEPGEKPYKCPECGKSFSRSDNLVRHQRTHTGEKPYKCPECGKSFSDCRDLARHQRTHTGEKPYKCPECGKSFSDPGNLVRHQRTHTGEKPYKCPECGKSFSRSDNLVRHQRTHTGKKTS 3’.

The binding sites of ZFN-L and ZFN-R to bcr-abl were as follows respectively: 5′- GGCGTCGACGGCGAGGACGCCGAG-3′.

5’-CTCGGCGTCCTCGCCGTCGACGCC-3’.

The cleavage site of ZFNs is 5’-GACTAC-3′.

Genomic DNA was extracted from the cells treated with ZFNs using the Hipure Tissue DNA Mini Kit (Magen, China). PCR amplification of the region surrounding the ZFNs target site was performed using the PrimeSTAR HS DNA Polymeras (TaKaRa) and 100 ng of genomic DNA as template with primer 5’-GACGCCGAGAAGCCCTTC-3’and 5’-AATCCTCAAAACTCCGGGGG -3′. The PCR products were melted and annealed to form heteroduplex DNA. The annealed DNA was treated with T7 endonuclease 1 (New England BioLabs) for 15 min at 37 °C [37]. Data was analyzed by agarose gel electrophoresis. Ratio of cleaved to uncleaved products was calculated as a measure of frequency of gene disruption and the mutation was also analyzed using next-generation sequencing, as described [43].

K562 cells were transfected with ZFN-L/R and donor vector. After treated for 48 h, the genomic DNA of cells was extracted and then amplified by PCR as described above. Donor DNA was characterized by the NotI site they carried, so the PCR products were analyzed by NotI enzyme digestion with the following reaction system:1 μl NotI, 2 μl 10 × H Buffer, 2 μl 0.1%BSA, 2 μl 0.1% Triton X-100, 1 μg DNA and ddH₂O up to 20 μl at 37 °C. These results were measured by agarose gel electrophoresis.

Western blotting assay was performed as previously described [44]. The primary antibodies were as follows: anti-BCR-ABL, anti-Phospho-BCR-ABL, anti-c-Abl, anti-Phospho-STAT5, anti-STAT5, anti-ERK 1/2, anti-Caspase-3 and anti-PARP were all purchased from Cell Signaling Technology (USA), used at 1:1000 dilution. Anti-FLAG monoclonal antibody (Sigma, USA) was added at a concentration of 1:500 and anti-β-Actin (Zhong Shan Jin Qiao, China) antibody was used at 1:1000 dilution. The expression quantity of each protein was normalized against the β-Actin protein expression using image software.

The treated cells were plated into 96-well plates at a density of 2000 cells per well with 100 μl RPMI 1640 containing 15% fetal bovine serum and cultured at 37 °C in a 5% CO₂ humidified incubator. To prevent the medium evaporation, we added 100 μl PBS to the wells surrounding the sample wells. At indicated time 10 μl of CCK-8 (Solarbo,China) was added to each well then incubated at 37 °C for 3 h. Then the absorbance at 450 nm was measured by micro-plate reader (Eon, BioTeck, USA). Each assay was repeated for five times.

Treated cells were collected and plated (300 cells/well) in 24-well plates with methylcellulose for assessing the colony-forming ability. The number of colonies were counted at 7–14 days later, using an inverted microscope. The colony formation assay were performed for five times.

Cells were collected for immunofluoresence assay, washed 3 times by PBS and coated on slides. The cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 at 37 °C, blocked in 1% BSA with 5% goat serum and incubated primary antibody at 4°C overnight. Next, the cells were incubated with fluorochrome-conjugated secondary antibody (1:200, Zhong Shan Jin Qiao, China) in a dark room at 37 °C for 1 h. Lastly, the nucleus was stained with DAPI.

Samples were obtained from CML patients, who were initially diagnosed with CML and had not undergone any chemotherapy (Table 1), or anemia individuals from the first affiliated hospital of Chongqing Medical University or the second affiliated hospital of Chongqing Medical University. The CD34⁺ cells were performed using Stemsep human CD34 positive selection cocktail and cultured in StemSpan serum-free expansion medium (Stem Cell Technologies, Canada) supplemented with 50 ng/ml SCF, 10 ng/ml IL-6 and 10 ng/ml IL-3 (PeproTech, USA) at 37 °C in 5% CO₂. The study was approved by the ethical committee of Chongqing Medical University.

5–6 weeks old female NOD/SCID mice (n = 5, each group) were selected and received 250 cGy radiation before injection. 2–4 h later, 5 × 10⁶ K562/G01 cells in 200 μl PBS modified by ZFNs or treated with mock were injected intravenously. The weight change and white blood cells count of mice were monitored weekly. Weight loss, mental fatigue, splenomegaly and leukocyte hyperplasia were considered as the signs and symptoms of CML-like disease in mice.Peripheral blood was collected and incubated with the antibody against human CD45 to analyze the proportion of CD45⁺ cells by flow cytometry. All animal experiments were performed in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No.8023, revised 1978) and were conducted with the approval of the Biomedical Ethics Committee of Chongqing Medical University.

The zinc finger nucleases (ZFNs) targeting exon 1 of the bcr-abl gene, which could cause a double-strand break (DSB), were designed and generated following modular assembly approach [45, 46]. Both of the two zinc finger proteins (ZFPs) (designated ZFP-L and ZFP-R) arrays containing four zinc finger domains were assembled using an archive of ZFP DNA-binding modules [47, 48]. Each of ZFPs was coupled with a codon-optimized FokI domain containing mutations that can prevent homodimer formation and enhance the cleavage activity [30], which is termed as ZFN-L and ZFN-R respectively (Fig. 1a). A nuclear localization signal (NLS) was fused to ZFN and a FLAG tag was incorporated to N-terminal of the protein (Fig. 1b). The NLS allows transportation of ZFN protein to the nucleus binding to the targeted DNA. Our goal is to terminate the translation of BCR-ABL through the direct modification of bcr-abl gene sequence, so we built a suitable donor plasmid to trigger the HDR. The donor sequence containing a NotI site, which composed of 8-base, could result in the alteration of the open reading frame and the subsequently premature termination of translation (Fig. 1c).

The applications of gene editing by ZFNs are based on the generation of a site-specific DSB into the interesting sequence. Firstly, we analyzed the expression of ZFNs proteins. The nucleofected K562 cells were harvested at 0 h, 12 h, 24 h, 48 h and 96 h. The result of western blot analysis showed the expression of ZFNs protein can be detected at 12 h after transfection, with a peak at 48 h and diminished at 72 h (Additional file 1: Figure S1A). To demonstrate the nuclear localization of the ZFNs proteins, cells were transfected with ZFN-L and ZFN-R plasmids, together or separately. After 48 h, the amount of ZFNs proteins in the nucleus and cytoplasm were analyzed by western blot respectively. We demonstrated that the engineered ZFNs proteins could localized and expressed in nucleus (Additional file 1: Figure S1B).

Next, to determine whether our ZFNs can introduce DSBs at exon 1 of bcr-abl gene, we transfected K562 cells with ZFN-L, ZFN-R separately or together. By detecting the p53-binding protein 1 (53BP1), which forms foci at DNA damage sites, we can evaluate the formation of DSBs by immunofluorescence [28]. Etoposide treated cells as positive control had a high level of 53BP1 foci (79.2% > 3 foci). We observed a low level of 53BP1-stain foci in nontransduced or pAd-Track transduced K562 cells (5.2% > 3 foci and 7.8% > 3 foci respectively) (Fig. 2a). Cells treated with ZFN-L or ZFN-R showed similar level of 53BP1-stain foci (8.3% > 3 foci and 6.7% > 3 foci respectively) (Additional file 2: Figure S2A). In contrast, a dramatic high level of 53BP1 foci (56.9% > 3 foci) was observed in ZFN-L/R group (Fig. 2a). 53BP1 is recruited to the DSBs sites early in the DNA repair response and regarded as the hallmark of DSB. As shown above, ZFN-L/R can, and only can, cause DSBs in K562 cells. To further confirm this result, we analyzed another DNA damage marker, phosphorylated histone H2AX (γH2AX) by western blot. As shown in Fig. 2b, compared with pAd-Track vector and K562 group, the expression of γH2AX was significantly increased in ZFN-L/R group and no significant difference was observed among other conditions (Additional file 2: Figure S2B). The detection of γH2AX expression further confirmed that the DSBs in K562 cells can be induced by ZFN-L/R. Together, these results demonstrate that ZFN-L/R induces DSBs in K562 cells.

Off-target DNA cleavage by ZFNs can be caused by the formation of homodimers between ZFN-L and ZFN-R [28, 29]. To avoid off-target effects, we adopted FokI nuclease variants that are restricted to form heterodimers as described above [45, 49]. To evaluate the activity of ZFNs at the endogenous target sequence, T7 endonuclease I (T7E1) assay was carried out. A ZFNs-induced DSB is typically repaired by NHEJ, which often contains small insertions or deletions (called “indels”) near the break site. The presence of indels in the K562 cells which treated by ZFNs, is confirmed by the mismatch-sensitive T7E1. Then the percentage of bcr-abl gene modification (47.8%) was quantified using gel imaging station (Fig. 2c). Moreover, genomic sequencing verified that the existence of various deletions and insertions resulting from NHEJ repair of the DSBs induced by activity of the ZFNs (Fig. 2d).

To determine whether the ZFNs and donor can mediate BCR-ABL translation termination, K562 cells were transfected with ZFN-L/R and donor, individually or together.

BCR-ABL translation termination, K562 cells were transfected with ZFN-L/R and donor, individually or together. After 48 h, genomic DNA were collected and amplified by semiquantitative PCR followed by digestion with NotI restriction enzyme. HDR was observed in 60.14% of the bcr-abl gene (Fig. 2e). Genomic DNA cut by NotI was sequenced, results showed a stop codon and an end of translation near the DSB site (Fig. 2f).

We performed western blot to confirm whether the ZFNs system can prevent BCR-ABL translation. In K562 and K562/G01 cells, the quantity of BCR-ABL and the activity of p-BCR-ABL in the group treated with donor and ZFN-L/R were significantly lower than that in groups of blank, ZFN-L, ZFN-R or Donor (Fig. 3a, Additional file 3: Figure S3A). We noticed that there was still a few amount of BCR-ABL expression, presumably because the restriction of plasmid transfection efficiency led to a small number of cells did not be edited (in our experiment, the efficiency of transfection of K562 cells can reach 93%. Data not shown). Considering BCR-ABL activates multiple downstream pathways, we further measured key downstream proteins and found that the phosphorylation levels of STAT5, ERK and CRKL were decreased (Fig. 3b, Additional file 3: Figure S3B). Taken together, these results demonstrated that ZFNs targeting bcr-abl caused attenuation of BCR-ABL, accompanied with decreased phosphorylation of its downstream molecules including STAT5, ERK and CRKL.

To detect the effect of ZFNs on apoptosis of CML cells, we transfected K562 and K562/G01 cells with ZFN-L, ZFN-R, Donor individually or together. Apoptosis was detected by flow cytometry and apoptotic rate significantly increased in donor and ZFN-L/R treated group compared to other groups (Fig. 4a, Additional file 4: Figure S4A). To confirm this effect, we visualized the nuclear morphology changes by DAPI staining. As shown in Fig. 4b, apoptosis characteristic morphological changes, such as condensed and fragmented nuclear, were observed in CML cells treated with ZFN-L/R and donor and no significant changes of nuclear morphology were observed in other groups (Additional file 4: Figure S4B). The presence of BCR-ABL protein makes CML cells resistant to various apoptotic stimuli [50]. Cleaved caspase-3 and PARP as the activation of caspase pathway were measured by western blotting. The result shown that caspase pathway activated in K562 and K562/G01 cells treated with donor and ZFN-L/R (Fig. 4c).

Next, we tested the influence of ZFNs on the proliferation of CML cells. Cell proliferation was measured by CCK-8 assay and colony-forming assay. We found that, since the fourth day, the viability of donor and ZFN-L/R transfected cells was significantly lower than another groups. What’s more, the CML cells in the group treated with donor and ZNF-L/R, from the first day to the fifth day, only had less proliferation (Fig. 4d). The result of colony-forming assay shown that donor and ZNF-L/R co-delivery also inhibited the colony formation ability of CML cells (Fig. 4e, Additional file 5: Figure S5A). Furthermore, to confirm whether the ZFNs system has cytotoxicity on bcr-abl negative cells, we transfected 32D, HepG2 and 293 T cells with the donor and ZFN-L/R expression plasmids and detected the proliferation using CCK-8 assay. The results showed that no significant change was observed in these cells compared with untreated cells (Additional file 5: Figure S5B-D). These results indicated that co-delivery of donor and ZFN-L/R induced apoptosis and suppressed proliferation of imatinib sensitive and resistant CML cells.

To determine whether our ZFNs have effect on bcr-abl in primary leukemia stem/progenitor cells, we collected CD34⁺ cells from CML patients and transfected them with ZFN-L/R and donor. The efficiency of gene modification was measured by PCR-amplification followed by NotI digestion and agarose gel electrophoresis. These results showed that the combination of donor and ZFN-L/R was able to induce gene editing in CD34⁺ cells from CML patients (Fig. 5a). Next, we detected the viability of modified cells by CCK-8 assay and apoptosis through flow cytometry. ZFN-L/R and donor DNA significantly suppressed the proliferation and promoted the apoptosis of CD34⁺ cells from CML patients (Fig. 5b-f), but had no effect on CD34⁺ cells from bcr-abl negative patients who suffered from anemia (Additional file 6: Figure S6A-D).

In order to determine the effects of bcr-abl disruption by ZFNs in vivo, K562/G01 cells with treatments or no treatment were injected into NOD/SCID mice through tail vein. The treatments include transfection with pAd-Track, ZFN-L, ZFN-R, Donor or donor with ZFN-L/R. The white blood cell counts of mice in groups were monitored weekly and the peak number of each mouse was recorded. As shown in Fig. 6a, the peak level of leukocytes ZFN-L/R and donor treated mice reached was significantly lower when compared to other groups. To confirm whether these leukocytes were leukemic or not, peripheral blood was collected from each group and CD45 antigen which expressed on the membrane surface of human leukocytes were detected by FCM. The number of CD45 positive cells indicated the number of K562/G01 cells propagating in murine bone marrow. As shown in Fig. 6b, the donor and ZFN-L/R group had lower level of CD45-positive cells than another groups. These results proved that ZFN-L/R and donor suppressed the proliferation of K562/G01 cells in NOD-SCID mice. Furthermore, mice in group blank, ZFN-L, ZFN-R or Donor developed with more severe hepatomegaly and splenomegaly compared with ZFN-L/R and donor group (Fig. 6c-d, Additional file 7: Figure S7A). There was no mice harboring solid tumor in group treated with donor and ZFN-L/R. In contrast, in another groups there was one to two mice suffered from solid tumor in enterocoelia, buttock or cheek (Fig. 6e, Additional file 7: Figure S7B). Infiltration of leukemic cells in tissues was examined by hematoxylin/eosin (HE) and Wright’s staining, and the BCR-ABL expression was detected via immunofluorescent. Mice treated with ZFN-L/R and donor were observed with less leukemic cell infiltration in the liver and spleen (Fig. 6f, Additional file 7: Figure S7C). By Wright’s staining of bone marrow, a reduced Myeloid: Erythroid ratio was shown in ZFN-L/R and donor group when compared with other groups (Additional file 7: Figure S7D). Moreover, a high level expression of BCR-ABL was detected in cells from livers, spleens, bone marrow or soild tumors from blank, ZFN-L, ZFN-R or Donor treated mice (Fig. 6g or not shown). Kaplan-Meier survival analysis showed that the mice receiving ZFN-L/R and donor treated cells demonstrated significantly longer survival time than another groups (Fig. 6h). In summary, co-delivery of ZFN-L/R and donor was able to inhibit the proliferation and infiltration of K562/G01 cells, ultimately, delay the onset of CML in mice.