Nilotinib treatment in mouse models of P190 Bcr/Abl lymphoblastic leukemia

Nilotinib has been reported to be more potent than imatinib in inhibiting the proliferation of Bcr/Abl expressing cells [24‐28]. To study its effectiveness in eliminating lymphoblastic leukemia cells in vitro, we compared 8093 lymphoblastic leukemia cells treated with different concentrations of nilotinib to the same cells treated with 5 μM imatinib. As shown in Fig. 1A, at the start of the drug treatment, all 8093 cells had a viability of >90%. Within 24 hours of treatment, this dropped to less than 45% under all treatment conditions. The effect of nilotinib treatment on cell viability was dose-dependent. 200 nM nilotinib treatment reduced the viability of the 8093 culture from >90% to 18% within 24 hours whereas treatment with 100 nM reduced viability to 28% within 24 hours. A lower dose of 50 nM left about 40% of the cells viable after the same time period. Cell viability was reduced to zero within 72 hours for all three concentrations of nilotinib.

This result showed that nilotinib is very efficient in eradicating a large number of leukemia cells. In comparison, 5 μM imatinib treatment was about as effective as the 200 nM nilotinib treatment (cell viability reduced to 18% and 13%, respectively after 24 hrs). We also compared the effect of nilotinib to that of imatinib in two other independent lines established from two different P190 Bcr/Abl transgenic mice. As shown in Fig. 1B and Fig. 1C, the exact degree of sensitivity differed among the three cell lines, although in all, nilotinib was more effective than imatinib. Overall, we found that nilotinib is 10–25 fold more potent than imatinib, suggesting great potential for in vivo therapy.

The effect of nilotinib has not been evaluated in mouse models of Ph-positive ALL. To examine the effectiveness of nilotinib treatment in vivo, fifteen C57Bl/6J mice were transplanted via a tail vein injection with 10⁴ 8093 cells. Nilotinib treatment (7 mice) or control treatment (8 mice) was started five days after transplantation. The dose of 75 mg/kg daily was chosen based on previous studies using mouse cell lines transfected with Bcr/Abl P210 and transplanted into nude mice [24]. They showed, that at this concentration, the drug was well absorbed and bioavailable for up to 24 hours.

Vehicle treated mice became moribund within 3 weeks of the transplantation. They showed clear symptoms of ALL. Nilotinib-treated mice lived statistically significantly longer as compared with the vehicle-treated mice (p < 0.05)(Fig. 2). This result clearly indicated that nilotinib was very effective in inhibiting the proliferation of the leukemic cells in vivo. However, also five of the seven drug-treated mice died. We ended treatment of the two remaining mice 51 days after the transplant of the leukemic cells, when all vehicle-treated mice had died. At this point both appeared normal. However, these two mice succumbed to leukemia 8 and 14 days later.

In this transplant model, the initiation of leukemia is synchronized and the drug is tested for effect against an initially small number of highly malignant cells. The P190 Bcr/Abl transgenic mice represent a different model of leukemia. The disease has a natural progression, starting with an initial phase in which mice are healthy. On a C57Bl/6J background, mice become overtly sick when they are, on average, 100 days old. To study the effect of nilotinib treatment on this more natural model of advanced stage leukemia, we randomly selected five P190 Bcr/Abl mice showing visible signs of lymphoma and nilotinib treatment of 75 mg/kg daily was started. Remarkably, nilotinib treatment led to a complete regression of the overt lymphomas within six days for all five Bcr/Abl transgenic mice (Fig. 3A). A significant improvement in the health of all five mice was also observed, with increased activity and restored mobility within one week of treatment. We treated the five mice for a total of 30 days (Fig. 3B). Two of the mice that were taken off treatment died 11 days later, whereas three mice survived more than 50 days without visible reoccurrence of the leukemia/lymphoma. Five additional Bcr/Abl transgenic mice were selected upon visible signs of lymphoma and were kept under observation without any treatment. All five mice in the untreated group became moribund within 3–11 days and were sacrificed according to institutional regulations (Fig. 3B).

We analyzed cells from preleukemic, leukemic and control wild type mice for cell surface markers suitable to detect the leukemic cells. CD19 was chosen as a general B cell antigen and AA4.1 as an antigen to distinguish mature B cells (AA4.1^low) from immature B cell precursors (AA4.1^high; ref. [35]). AA4.1^high B cells are very rare in the peripheral blood (PB) of normal mice (Fig. 3C, left panels and Fig. 3D). Whereas in the normal mice, the percentage of CD19⁺ cells in PB was low, the PB of the leukemic animals consisted almost entirely of CD19⁺ cells, of which the majority was AA4.1^high (Fig. 3C, middle panel, Fig. 3D). When these animals were treated for only seven days with nilotinib, the numbers of these CD19⁺AA4.1^high leukemic cells were substantially reduced and other cells re-appeared in the peripheral blood (Fig. 3C, right panel). We also quantitated the numbers of leukemic cells in the PB of the mice. Whereas the PB of preleukemic animals contained low but detectable numbers (mean, around 0.7% of the total WBC), the overtly leukemic animals had large amounts (mean 75% of the total WBC) of such cells in the circulation. Treatment for only 7–8 days with nilotinib had a very significant impact on the leukemic cell numbers, reducing them to levels (0.9% of the total WBC) comparable to that found in a wild type mice (Fig. 3D). Thus, these results clearly showed that nilotinib was also very effective in treating advanced stage leukemia.

During the course of treatment with nilotinib, five out of the seven mice that had been transplanted with the 8093 leukemia cells developed symptoms of lymphoma and were sacrificed. To determine to what extent nilotinib was able to inhibit the Bcr/Abl kinase activity when the mice started showing symptoms of ALL, we sacrificed two of the five mice in the nilotinib treatment group 23 hours after the last nilotinib administration and three within two hours of drug treatment. SDS-SB tissue lysates of lymphomas isolated from the animals were prepared for each of the five animals. We also grew the lymphoblastic leukemia cells from these mice in tissue culture.

Nilotinib acts by inhibiting the tyrosine kinase activity of the Bcr/Abl protein, which is essential for Bcr/Abl mediated oncogenic transformation [4‐6]. As shown in Fig 4A for one representative sample S9, the tyrosine kinase activity of Bcr/Abl was significantly inhibited 2 hours after nilotinib treatment in vivo but was fully active 23 hours after the treatment, as is evident from sample S5 (Fig. 4A) based on immunoblotting of the lysates with an antibody against phosphotyrosine.

In addition, we measured phosphorylation of the Crkl protein, which is a substrate for the Bcr/Abl tyrosine kinase. Tyrosine phosphorylated Crkl is distinguishable from the non-tyrosine phosphorylated form because it has retarded mobility on SDS-PAA gels. The ratio of phosphorylated to non-phosphorylated Crkl thus serves as an independent indicator of Bcr/Abl tyrosine kinase activity. As shown in Fig. 4C, higher levels of phosphorylated Crkl were observed in the samples which showed high levels of Bcr/Abl tyrosine kinase activity. In contrast, in the sample (S9) showing reduction of tyrosine kinase activity, the levels of non-phosphorylated Crkl were higher than those of phosphorylated Crkl. SDS-SB lysates from both the parental cell line 8093 and two cell lines A-5 and A-21 established from randomly selected nilotinib-treated mice were also included for comparison. High levels of tyrosine kinase activity were also observed in these cells (Fig. 4A,C). As controls, we included blotting with antibodies for endogenous Bcr and P190 Bcr/Abl protein, and GAPDH as loading control (Fig. 4B,D).

Amplification of the P210 Bcr/Abl gene has been previously reported to confer Imatinib-resistance in patients [11, 12, 18‐21]. We investigated whether the cell lines A-5 and A-21, isolated from mice that had developed leukemia while on Nilotinib treatment, had BCR/ABL gene amplification as compared to the parental cell line 8093. However, no differences were observed in the gene copy number or protein levels (results not shown and Fig. 4B). Also, mutations in the kinase domain of Abl within Bcr/Abl have been previously reported to confer Imatinib-resistance in CML patients [11, 13‐21] and a recent study showed that certain other mutations in Abl can make cells nilotinib-resistant [29, 30]. However we did not detect any mutations in the Abl ATP binding pocket in DNA from the A-5 and A-21 cell lines isolated from the nilotinib-treated mice or in the parental 8093 cells (not shown).

To investigate whether the cells isolated from the nilotinib-treated mice, A-5 and A-21 had any other cell-inherent mechanism of resistance against nilotinib therapy, we evaluated their in vitro ability to proliferate in the presence of nilotinib. Interestingly, we did not observe any difference in the sensitivity of A-5 and A-21 towards nilotinib as compared to 8093 (Fig. 5). We assessed the viability of the three cell lines during treatment with 20 nM nilotinib both in the presence and absence of stromal support (consisting of a layer of mitotically inactivated mouse embryonic fibroblasts). All three cell lines behaved very similarly: their viability dropped to less than 20% within 48 hours of 20 nM nilotinib treatment. However, we obtained very different results in long-term cultures between cells cultured with and without stroma. Their viability without stroma in the presence of 20 nM nilotinib progressively declined over the course of 3–4 days. By the sixth day, viability was reduced to zero (Fig. 5B). In contrast, though the three cell lines cultured in the presence of irradiated stroma experienced a drastic drop in viability for the initial 4–5 days of treatment, the viability started to improve by the sixth day of treatment. All three cell lines recovered and had a viability of >60% on tenth day of treatment (Fig. 5A). Thus the cells that were obtained after the initial drop in viability were able to proliferate and maintain good viability in the presence of 20 nM nilotinib in vitro.

We next examined a possible mechanism leading to Bcr/Abl-independent resistance to nilotinib. Samantha et al [36] showed that Jak2 is an important target in CML, and the Jak inhibitor AG490 was able to induce apoptosis in cells that expressed imatinib-resistant mutants of Bcr/Abl. Very recently, Wang et al [37] further implicated Jak2 in Bcr/Abl-independent imatinib and nilotinib resistance caused by GM-CSF production by myeloid leukemic cells. Therefore, using the Jak inhibitor AG490, we investigated if Jak2, in addition to its involvement in drug resistance of myeloid leukemia cells, also contributes to resistance development of lymphoid leukemia cells. As shown in Fig. 6A, AG490 treatment significantly decreased the survival of the lymphoid leukemia cells in a dose-dependent manner when these cells were co-cultured with MEFs. Interestingly, AG490 treatment for 48 hours also affected normal function of the feeder layer cells, as the proliferation of non-irradiated MEFs was severely reduced (more than 4 times reduction in the presence of 50 μM AG490; results not shown) compared to treatment with the vehicle DMSO. Treatment of the Bcr/Abl lymphoblastic leukemia cells with AG490 during (Fig. 6B) and after (Fig. 6C) resistance development to nilotinib did not further affect the survival, as compared to its effect on non-resistant leukemia cells (Fig. 6A). Instead, in both experiments, nilotinib-resistant lymphoblastic leukemia cells seemed to also acquire additional resistance to AG490, though in a dose dependent manner, as evidenced by the resumption of growth after an initial drop in viability upon first addition of AG490 (Fig. 6C).

The P190 Bcr/Abl transgenic mouse model has been previously described [33, 34]. On a C57Bl/6J background, average age at death for the f10–f15 generation (n = 127) was 100 days (range 38–265 days). The 8093 lymphoblastic leukemia cell line was established from a P190 Bcr/Abl transgenic mouse on a C57Bl/6J (f11) background as described previously [49]. B-1 and B-2 lymphoblastic leukemia cells have been previously described [50]. Lymphoblastic leukemia cell lines A-5 and A21 were established from nilotinib-treated C57Bl/6J mice transplanted with 8093 cells. The cells were grown in complete lymphoblast medium consisting of McCoy's 5A medium (Life Technologies, Inc., Rockville, MD) supplemented with 15% heat inactivated FCS, 110 mg/L sodium pyruvate, 2 mmol/L L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 10 ng/ml recombinant IL-3 (Calbiochem, San Diego, CA) and 50 μmol/L β-mercaptoethanol in the presence of E14.5 irradiated mouse embryonic fibroblasts (MEFs) [51].

Nilotinib was obtained from Novartis Pharmaceuticals (Basel, Switzerland). AG490 was purchased from Calbiochem (San Diego, USA). The parental lymphoblastic leukemia cell line 8093 and the A-5 and A-21 cell lines were seeded in wells of a 6-well plate (3 × 106 cells/well) either in the presence or absence of E14.5 irradiated MEFs as described [49]. Samples in triplicate wells were treated either with 20, 50, 100, or 200 nM nilotinib or 5 μM imatinib or DMSO as control. In additional pilot experiments, 8093 cells were treated with 100, 75, 50 and 5 μM AG490 while cultured on MEFs. The cell viability in control experiments was consistently above 80%. Drug in the experimental wells was added every second or third day along with the fresh change of medium dependent on proliferation of the treated cells. Aliquots were removed from each individual well and cell viability was determined using the Trypan Blue exclusion method. Viability is expressed as percentage of the number of Trypan Blue excluding cells divided by the number of total cells. In the case of AG490 treatment, viability was measured by propidium iodide uptake using a FACScan (BD Biosciences, San Jose, USA). Each data point is represented as mean ± SEM of triplicate samples.

Fifteen C57Bl/6J mice (male, 6 weeks old, Jackson Lab, Bar Harbor, Maine) were transplanted with 1 × 10⁴ 8093 cells via a tail vein injection. Five days later, mice were randomly selected for vehicle or nilotinib treatment. Eight mice (vehicle group) were fed a mixture of 8 parts peanut butter and two parts vegetable oil and the remaining seven mice (treatment group) were treated with 75 mg of nilotinib/kg body weight added to the same peanut/oil mixture daily. Treatment was stopped 50 days after day 1 of transplantation.

Peripheral blood of preleukemic and overtly leukemic P190 transgenic mice as well as wild-type littermates was examined by flow cytometry using a FACScan (BD Biosystems, Heidelberg, Germany) to identify markers suitable to detect the leukemic cells. Peripheral blood of three additional P190 transgenic animals that had developed overt leukemia/lymphoma was analyzed before and after seven days of treatment with nilotinib as described above. After erythrocyte lysis, cells were stained with antibodies against mouse CD19 and AA4.1 (BD Biosciences, San Jose, CA). In addition, five P190 Bcr/Abl transgenic mice with visible signs of lymphoma were selected at different time points and treated with 75 mg/kg nilotinib as described above. Treatment was continued for 30 days.

Animals that had been transplanted with 8093 cells in the nilotinib-treated group and that started showing signs of ALL were sacrificed either 2 hours or 23 hours after the daily administration of 75 mg/kg of nilotinib. SDS-SB lysates of lymphoma tissue were prepared and lymphoblastic leukemia cell lines were isolated from these mice. Two cell lines, A-5 and A-21, were subsequently used for further experiments. SDS-SB lysates from lymphoma tissues and lymphoblastic leukemia cell lines were run on 7.5% SDS-PAA gels (for detection of phosphotyrosine and Bcr) and 15% SDS-PAA gels (for Crkl detection). Membranes were reacted with PY-20-Horseradish peroxidase (1:2500, BD Transduction Laboratories, CA), Bcr N-20 (1:500, Santa Cruz Biotechnology, CA), Crkl (1:1000, H-62, Santa Cruz Biotechnology), or GAPDH (1:5000, Chemicon International, CA) antibodies using standard procedures.

BCR/ABL gene copy number was assessed using Southern blotting of Bam HI digested genomic DNA isolated from the parental cell line 8093 and the lymphoma derived cell lines A-5 and A-21. To examine the ABL segment in BCR/ABL for mutations, a 417 bp region from the DNA of 8093, A-5 and A-21 was amplified using forward primer 5'-agagatcaaacaccctaacct-3' and reverse primer 5'-gcatttggagtattgctttgg-3' and sequenced. This region includes nucleotides 876–1293 (residues 293–462) of c-Abl (NM_005157) containing point mutations T315, F317, M351, Q252 and H396 detected in human patients [20]. A larger region of 675 bp including both the ATP binding pocket and the activation loop was also amplified and sequenced using primers AN4+ 5'-tggttcatcatcattcaacggtgg-3' and A7- 5'-agacgtcggacttgatggagaact-3' as described by Sacha et al [52].

The Log rank test was used to test the significance of survival. A p-value of less than 0.05 was considered to be significant.