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. Author manuscript; available in PMC: 2011 May 1.
Published in final edited form as: Mol Cancer Ther. 2010 Apr 13;9(5):1318–1327. doi: 10.1158/1535-7163.MCT-10-0069

Activity of the Aurora Kinase inhibitor VX-680 against Bcr/Abl positive acute lymphoblastic leukemias

Fei Fei 1, Sonia Stoddart 1, John Groffen 1,2,3,4, Nora Heisterkamp 1,2,3,4
PMCID: PMC2868097  NIHMSID: NIHMS185920  PMID: 20388735

Abstract

The emergence of resistance to tyrosine kinase inhibitors due to point mutations in Bcr/Abl is a challenging problem for Philadelphia-chromosome positive (Ph-positive) acute lymphoblastic leukemia (ALL) patients, especially for those with the T315I mutation, against which neither nilotinib or dasatinib shows significant activity. VX-680 is a pan-Aurora kinase inhibitor active against all Bcr/Abl proteins but has not been extensively examined in preclinical models of Ph-positive ALL. Here, we have tested VX-680 for treatment of Bcr/Abl positive ALL when leukemic cells are protected by the presence of stroma. Under these conditions, VX-680 showed significant effects on primary human Ph-positive ALL cells both with and without the T315I mutation, including ablation of tyrosine phosphorylation downstream of Bcr/Abl, decreased viability and induction of apoptosis. However, drug treatment of human Ph-positive ALL cells for 3 days followed by drug removal allowed the outgrowth of abnormal cells 21 days later, and upon culture of mouse Bcr/Abl ALL cells on stroma with lower concentrations of VX-680, drug-resistant cells emerged. Combined treatment of human ALL cells lacking the T315I mutation with both VX-680 and dasatinib caused significantly more cytotoxicity than each drug alone. We suggest that use of VX-680 together with a second effective drug as first-line treatment for Ph-positive ALL is likely to be safer and more useful than second-line treatment with VX-680 as monotherapy for drug-resistant T315I Ph-positive ALL.

Keywords: Aurora kinase inhibition, dasatinib, stromal support, environmental-mediated drug resistance

Introduction

Philadelphia-chromosome positive (Ph-positive) leukemias are characterized by the presence of a BCR-ABL fusion gene, resulting from a reciprocal translocation between chromosomes 9 and 22. The product of this gene, the Bcr/Abl oncoprotein, is a constitutively active tyrosine kinase that initiates chronic myeloid leukemia (CML) and around 30% of acute lymphoblastic leukemia (ALL) (1, 2). The tyrosine kinase inhibitor imatinib (formerly STI571), which relatively selectively interrupts Bcr/Abl oncogenic signaling, has become the new “gold standard” for treatment of patients with CML (3). However, a subset of patients develops resistance to imatinib due to point mutations within the Abl kinase domain which impair imatinib binding (4-6). The need for overcoming imatinib resistance has led to the development of a second-generation of tyrosine kinase inhibitors including nilotinib and dasatinib (7, 8). Even though these bind to the majority of Abl mutants, neither has significant activity against leukemia cells with the Abl T315I mutation (9). This mutation has been reported to occur in approximately 20% of imatinib-resistant CML patients that had been treated with this drug (7, 10). Leukemias carrying Abl point mutations appear to be rapidly selected out by treatment with such drugs particularly in Ph-positive ALL: these were detected on average 10 months after initiation of kinase inhibitor treatment (11). Because of these reasons, the search for additional treatment for Ph-positive leukemias continues.

Serine/threonine kinases of the Aurora family, including Aur-A, -B and –C, are essential for the control of mitotic processes during cell division (12). Aberrant Aurora kinase activity occurs in a variety of human tumor cells, indicating a role for this family in tumorigenesis (13, 14). VX-680 (MK-0457) is a pan-Aurora kinase inhibitor that induces apoptosis in many human tumor types by blocking cell cycle progression (14). Interestingly, Carter et al (15), using a murine cell line transfected with different Bcr/Abl mutants, reported that VX-680 inhibits the activity of both wild-type and mutated Bcr/Abl proteins, including those with the T315I mutation. VX-680 is able to bind to Abl in a mode that accommodates the substitution of isoleucine for threonine at residue 315, the so-called gatekeeper position (16). Dai et al. (17) also showed that VX-680 is effective in imatinib resistant CML cells and Ba/F3 cells expressing T315I Bcr/Abl. Similar results were observed by Fiskus et al. who found that VX-680 induces cells death in AML and CML cell lines (18).

Surprisingly few other preclinical studies have been performed with VX-680 on Ph-positive leukemias, and of these, most concentrated on CML or on cell line models for CML such as K562. Moreover, studies with existing CML cell lines were performed in the absence of stroma and those using primary patient materials used short-term treatment. However, the drug was used to treat 3 patients with two CML and one Ph-positive ALL with the T315I mutation in a small phase I study (19).

Because of the reported activity on the Bcr/Abl T315I mutant, drugs such as VX-680 may find increased use in the secondary treatment of relapsed Ph-positive ALL, in which drug resistance to imatinib, dasatinib and nilotinib tends to rapidly evolve. However, it remains poorly characterized for this purpose. It is a reason for concern that mutant mice lacking one copy of the Aurora A kinase are more prone to develop malignancies than those who have the normal genetic complement, and treatment of mouse embryonic fibroblasts with VX-680 caused the emergence of hyperdiploid cells (20). For these reasons, we have here explored the effects of long-term VX-680 treatment in both mouse and human Bcr/Abl positive ALL cells in the presence of stroma, to more closely approximate treatment with this drug in vivo.

Dasatinib (Sprycel, formerly BMS-354825; Bristol-Myers Squibb, New York, NY) is a potent, orally bioavailable dual Abl and Src kinase inhibitor that has been developed for overriding imatinib resistance (21). Dasatinib exhibits a 325-fold greater potency than imatinib in inhibiting the activity of non-mutated Bcr/Abl and is active against most imatinib-resistant Bcr/Abl mutations except the T315I mutation (7, 22). We additionally report that relatively high VX-680 concentrations are needed to inhibit wild type and T315I Bcr/Abl, but that a combination with dasatinib in those ALL cells that respond to it is very effective in eradicating the ALL cells.

Materials and methods

Drugs, reagents and cells

VX-680 (Selleck Chemicals LLC, USA), dasatinib (Toronto Research Chemicals, North York, Ontario, Canada), AMN107 (nilotinib, Novartis, Basel, Switzerland), and vincristine (Hospira Inc. Lake Forest, Australia) were diluted in DMSO.

The murine OP9 stromal cell line (CRL-2749) was obtained from the ATCC (Manassas, VA, USA). Human Ph-positive ALL cells (also see Figure 1B) include those expressing wild type Bcr/Abl (UCSF02, TXL2, TXL3, US9) and T315I mutants (BLQ1, Pt2). TXL2 has been described and was from a Ph-positive ALL patient at diagnosis (23). US7 and US7R are Ph-negative ALL cells, however, US7R was isolated from the same patient in relapse phase. Based on this analysis, all human ALL samples expressed the Bcr/Abl P210 or P190 fusion protein (not shown). The primary cells were passaged in (NOD/SCID/IL2R)r2γ−/− mice (Jackson Labs). Leukemia cells harvested from the spleens of these mice were plated on irradiated OP9 feeder layers. The 8093 and Bin2 human Bcr/Abl P190-expressing transgenic mouse lymphoblastic leukemia cells have been previously described (24-26) and were grown in the presence of E13.5 irradiated mouse embryonic fibroblasts (MEFs). Mouse leukemia cells were grown in McCoy’s 5A medium including 15% FBS (Invitrogen Corporation, Carlsband, CA, USA) supplemented with 110 mg/L sodium pyruvate, 1% L-glutamine, 1% penicillin/streptomycin, 10 ng/ml recombinant IL-3 (Invitrogen Corporation, Carlsband, CA, USA) and 50 μmol/L β-mercaptoethanol. Human leukemia cells were grown in αMEM medium supplemented with 20% FBS, 1% L-glutamine and 1% penicillin/streptomycin.

Figure 1.

Figure 1

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Aurora A is highly expressed in primary human lymphoblastic leukemia cells. A, Human samples as indicated were analyzed for Aurora A expression by Western blot. GAPDH serves as a loading control. B, Samples shown in A. normal control and Down’s syndrome were peripheral blood lymphocytes.

In vitro treatment with VX-680, dasatinib, AMN107 and vincristine

Lymphoblastic leukemia cells were seeded in the wells of a 24-well plate or a 6-well plate at a density of 1×106 cells/ml, either in the presence of OP9 cells or E13.5 irradiated MEFs. Cells were treated with drugs as indicated in triplicate wells and viability of cells was measured by Trypan Blue exclusion. In experiments to make drug-resistant 8093 cells, VX-680 or nilotinib (AMN107) was added every second day along with a fresh complete change of medium. Apoptotic cells were measured using an Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit I (BD Pharmingen, San Diego, CA, USA). Apoptotic cells were defined by double positivity for Annexin V and PI assessed by flow cytometry (FACScan, Becton Dickinson, CA, USA). For cell cycle distribution, cells were washed and fixed in 70% ethanol for one hour. Fixed cells were stained with PI and subjected to flow cytometry.

Western blotting

Human leukemia cells (BLQ1 and TXL2 cells) were treated with various concentrations of VX-680 with or without 100 nM dasatinib for 24 hours and lysed in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 5 mM EDTA) containing PMSF, aprotinin, leupeptin, pepstatin A, Na Fluoride and Na Orthovanadate for 30 minutes on ice. Cell extracts were subjected to 8-15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Membranes were reacted with PY-20-Horseradish peroxidase (BD-Transduction Laboratories, San Jose, CA), phospho-Src family (Tyr416), Src, phospho-Stat5 (Tyr694) and Aurora A (all from Cell Signaling Technology, Danvers, MA, USA), Bcr (N-20), Crkl (both from Santa Cruz Biotechnology, CA, USA) and GAPDH (Chemicon International, USA) antibodies using standard procedures.

Giemsa staining

For morphology analysis, cells were cytospun onto glass slides and fixed in methanol for 5 min, dried and stained with Wright-Giemsa (Sigma, St Louis, MO, USA). Cell images were acquired with an Axionplan 2 imaging upright microscope (Carl Zeiss Germany).

Statistical analysis

Statistical analysis was performed with SPSS software. Data were presented as mean ± SD. Statistical significance of differences between groups was evaluated using one-way-ANOVA (post hoc Scheffe test). The value of P < 0.05 was considered to be statistically significant.

Results

VX-680 is cytotoxic for Bcr/Abl positive lymphoblastic leukemia cells including those carrying the T315I mutation

Aurora kinases have abnormally high levels of expression in several cancers (13). To investigate Aurora kinase expression in acute lymphoblastic leukemia, we performed Western blotting using Aurora A antibodies on primary human ALL samples that had been passaged in NOD/SCID mice and subsequently grown on OP9 stromal support. As shown in Fig. 1A, as compared to controls, high levels of Aurora A were present in these leukemia samples. BLQ1, Pt2, TXL2, and UCSF02 (Fig. 1B) are primary human Ph-positive ALLs. 8093 and Bin2 were isolated from the lymph nodes of BCR/ABL transgenic mice and are grown as cell lines in the presence of mouse embryonic fibroblasts. As shown in Fig. 2A, increasing concentrations of VX-680 produced a dose-dependent reduction of cell viability after 72 hrs in all leukemia cells tested including BLQ1 and Pt2 with the T315I mutation. There were differences between the four human samples in response rate, with UCSF02 showing the least response. However, we could not find the correlation between Aurora A expression and response to VX-680. The murine Bcr/Abl leukemia cells were significantly more sensitive than the human leukemia cells to this drug- a dose of 50 nM reduced the viability of these cells to around 50% after a 3-day treatment, whereas a 500 nM dose was needed to achieve the same effect in the human cells.

Figure 2.

Figure 2

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VX-680 shows cytotoxic activity against human and mouse Bcr/Abl lymphoblastic leukemia cells. A, BLQ1 (T315I mutation), Pt2 (T315I mutation), TXL2 (no mutation), UCSF02 (no mutation), 8093 and Bin2 cells (1×106/well) were grown in the presence of stroma. Viable cell counts were performed in triplicate over a 24~72 hr period as indicated. Error bars indicate SD. Results shown are one of two independently performed experiments with similar results. B, BLQ1 and UCSF02 cells were treated with increasing concentrations of VX-680 for 48 hrs. The percentage of apoptotic cells was determined by FACS analysis. C, BLQ1 cells were treated with 1 μM VX-680 and cell cycle distribution was determined by flow cytometry at time points of 24 hrs and 48 hrs.

To examine how changes in viability correlated with degree of apoptosis, we next treated ALL samples with a Bcr/Abl wt and a T315I mutant over a period of 2 days with different amounts of VX-680. As shown in Fig. 2B, VX-680 induced apoptosis both in BLQ1 and UCSF02 cells in a dose-dependent manner. Because Aurora kinase inhibitors were reported to cause endoreduplication and polyploidy (14), we measured cell cycle after a 1 and 2-day treatment with VX-680. Consistent with earlier findings in HeLa cells (14), treatment with 1 μM VX-680 caused a marked accumulation of ALL cells with greater than 4N DNA content (Fig. 2C).

VX-680 potentiates the inhibitory effects of dasatinib in wild type Bcr/Abl positive ALL cells

Dasatinib is the treatment of choice for Ph-positive ALL. We therefore investigated the combined effects of dasatinib and VX-680 in wild-type Bcr/Abl positive ALL cells. As shown in Fig. 3A, co-treatment with 1 μM VX-680 and 100 nM dasatinib caused an impressive 80% decrease in viability, whereas treatment with 1 μM VX-680 or 100 nM dasatinib alone only caused a 40% drop in viability in TXL2 cells over a period of 72 hrs (p < 0.001). Similar effects were observed in UCSF02 cells. We also evaluated the combined effects of VX-680 and dasatinib on apoptosis and cell cycle progression. Consistent with the results of the viability assay, co-treatment with VX-680 and dasatinib induced significantly more cells to undergo apoptosis than treatment with a single drug (Fig. 3B). As shown in Fig. 3C, VX-680 treatment caused accumulation of cells with a greater than 4N DNA content. Compared with treatment with either drug alone, an increase in the numbers of cells with less than N DNA content was seen at 48 hrs. Together, these results show that VX-680 and dasatinib synergize to induce cytotoxic activities in wild-type Bcr/Abl positive ALL cells.

Figure 3.

Figure 3

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VX-680 and dasatinib synergize to induce cytotoxic activity in wild type Bcr/Abl-positive human acute lymphoblastic leukemia cells. A, TXL2 and UCSF02 cells were exposed to 1 μM VX-680 with or without 100 nM dasatinib for 24~72 hrs as indicated after which the percentage of viable cells were counted by trypan blue exclusion. B, TXL2 cells were treated with or without VX-680 and dasatinib for 48 hrs in triplicate. ** p< 0.001 for VX-680 and dasatinib co-treated TXL2 compared to VX-680 or dasatinib alone treated TXL2 cells. Apoptotic cells were defined by flow cytometry as Annexin V and PI double positive cells. C, TXL2 cells were exposed to VX-680 and/or dasatinib and cell cycle distribution was assessed by flow cytometry.

VX-680 inhibits Bcr/Abl kinases in Bcr/Abl positive ALL cells in co-culture with stroma

VX-680 was reported to inhibit both Aurora kinases and the Bcr/Abl kinase in vitro (16), whereas dasatinib targets Bcr/Abl as well as Src-family kinases (27). To evaluate the effect of these drugs on Bcr/Abl when the ALL cells were grown in the presence of protective stroma, we treated BLQ1 and TXL2 cells with different concentrations of VX-680 with or without 100 nM dasatinib for 24 hrs. Fig. 4 shows that treatment with 5 μM VX-680 caused distinct inhibition in total phosphotyrosine, reflecting both direct and indirect substrates of the activated Bcr/Abl tyrosine kinase. We also evaluated the phosphorylation status of Crkl, a direct Bcr/Abl substrate and that of Stat5, a well-known downstream target of Bcr/Abl. As shown in Fig. 4, without drug treatment, the majority of Crkl protein was phosphorylated in both BLQ1 and TXL2. After exposure to increasing concentrations of VX-680 for 24 hrs, Crkl shifted to its non-tyrosine phosphorylated, more rapidly migrating form but 10 μM drug was required to inhibit all Crkl phosphorylation. Co-treatment with 1 μM VX-680 and 100 nM dasatinib in TXL2 cells (Fig. 4, right panel) induced a more significant reduction in phosphotyrosine, p-Crkl, p-Stat 5 and p-Src than treatment with either VX-680 or dasatinib alone, whereas there was no significant effect of combining VX-680 with dasatinib in BLQ1 cells with the T315I mutation.

Figure 4.

Figure 4

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VX-680 eliminates Bcr/Abl kinase activities. BLQ1 (T315I mutation) and TXL2 (no mutation) cells were treated with the indicated concentrations of VX-680 with or without 100 nM dasatinib for 24 hrs. Western blot analysis was done on total lysates with the antibodies indicated to the left. Blots were stripped and reprobed with Bcr (N-20), Src and GAPDH antibodies as loading controls.

Short-term response of leukemia cells to VX-680

As mentioned above (Fig. 2A), after a 3-day treatment, VX-680 at a concentration of 1 μM only killed about 50 % of the human ALL cells co-cultured with stromal support. In the study reported by Giles et al.(19), two CML patients had a clinical response at doses of 20 and 24 mg/m2/hr, reaching maximal plasma concentrations of 1 μM or more, whereas levels of less than 1 μM were reported at 12 and 16 mg/m2/hr. Drug was administered continuously by IV for a period of 5 days, followed by 14-21 day intervals without drug. We therefore wondered what the long-term effect could be of treatment with the drug followed by no treatment. To investigate this, BLQ1 cells were exposed to 1 μM VX-680 for 3 days. After 3 days, the drug was washed out and the cells were cultured without VX-680. From day 3 to day 21 we assessed their viability, numbers and cell cycle distribution. As shown in Fig. 5A (top left panel), after 3 days of VX-680 exposure, viability of the cells decreased to 40~50 %. After removal of the drug, viability of the culture began to gradually increase. On day 15, viability of the culture had reached a level similar to that of the control group and the cells were able to proliferate again (Fig. 4A, lower left panel). Cell cycle analysis indicated that even though we removed the VX-680, a subpopulation of BLQ1 cells was still arrested, with more than 4N DNA content (37 %), compared to cells untreated with VX-680 (22.8 %). On day 21, we compared BLQ1 cells that had never been VX-680 treated with those that had been exposed to the drug 21 days before (BLQ1-VX-Tx). Remarkably, compared to the control cells, a subpopulation of the VX-680 treated BLQ1 cells was much larger (Fig. 5B) than the original cells. Similar results were obtained with Pt2 cells (data not shown).

Figure 5.

Figure 5

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Responses of human ALL cells to short-term VX-680 treatment. A, BLQ1 cells were treated with 1 μM VX-680 for 3 days. After 3 days, the drug was removed from the medium and cells were cultured without VX-680. During this period (day 3 ~ day 21) without drug, viability (upper left), cell numbers (lower left) and cell cycle distribution (right panel) of BLQ1 cells were assessed. B, BLQ1 and BLQ1-VX-Tx cells were cytospun onto glass slides and fixed, dried and stained with Wright-Giemsa on day 21. All images are at 63× magnification. C, BLQ1 and BLQ1-VX-Tx cells were treated with 1.5 μM VX-680 or 5 nM vincristine for 72 hrs. Cell viability was measured by trypan blue exclusion. *p< 0.05 for vincristine-treated BLQ1 compared to vincristine-treated BLQ1-VX-Tx.

We then compared the sensitivity of BLQ1 and BLQ1-VX-Tx to treatment with VX-680. We found that BLQ1-VX-Tx cells were still sensitive to VX-680 (Fig. 5C). We also treated BLQ1 and BLQ1-VX-Tx cells with vincristine, a tubulin depolymerizing agent. Interestingly, BLQ1-VX-Tx cells were more sensitive (p< 0.05) to vincristine than the original BLQ1 cells.

Long-term treatment of leukemia cells with VX-680 in the presence of stroma

We also wished to investigate the effect of long-term continuous treatment of Bcr/Abl-positive ALL cells with VX-680, to address the question if ALL cells would ever emerge that were able to proliferate in the presence of this drug. The relatively low proliferation rate of the human ALL cells precluded their use for this purpose. However, murine Bcr/Abl ALL cells proliferate rapidly on stroma and were therefore suitable to test this. We added VX-680 every second day along with a fresh change of medium to 8093 ALL cells. Consistent with the results in Fig. 2A, 75 nM VX-680 decreased the viability of 8093 cells from 80~90% to 25~30% in the first 3~4 days. However, viability of 8093 cells began to increase gradually thereafter and on the tenth day, viability of the culture recovered to a level similar to that of the control group. Thus, with the support of stroma, the cells were able to proliferate again, even in the presence of concentrations of VX-680 which initially could kill ~70% of cells within first 2~3 days treatment.

We next compared the sensitivity of the original 8093 cells and the VX-680 resistant 8093 cells (8093R) to treatment with 16 nM nilotinib (AMN 107) in the presence of stroma. As reported before (25), 8093 cells were sensitive to nilotinib and it caused a drop in viability to 20~30 % in the first 2~4 days. Similar to 8093 cells, 8093R were initially sensitive to nilotinib (Fig. 6A) but both 8093 and 8093R cells were able to develop stroma-assisted resistance to nilotinib after more prolonged culture. In contrast to the BLQ1-VX-Tx cells (Fig. 5), we found that there was no difference in the cell cycle distribution and size between 8093 and 8093R cells (Fig. 6B, C). We also isolated RNA from VX-680 resistant 8093 cells to sequence the Abl ATP binding domain of Bcr/Abl. However, the region of Abl encoding the ATP binding pocket as well as the activation loop lacked point mutations in the VX-680 resistant cells (data not shown).

Figure 6.

Figure 6

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Reponses of 8093 ALL cells to long-term VX-680 treatment in the presence of stroma. A, Left panel, 8093 cells (3×106/well) were treated with DMSO (■) or with 75 nM VX-680 (▲) in the presence of stroma. Right panel, 8093 or 8093 VX-680 resistant cells (8093R) were treated with DMSO (■) or 16 nM nilotinib (▲, AMN 107). Fresh drug was added every second day with a complete medium change. Viability was assessed by trypan blue exclusion and is expressed as percentage. Each point represents the average of triplicate values ± SD. B, Giemsa staining of 8093 and 8093R cells. All images are at 63× magnification. C, Cell cycle distribution of 8093 and 8093R cells as measured by flow cytometry.

Discussion

In the present study we have examined the activity of the Aurora kinase inhibitor VX-680 against Ph-positive ALL cells that were provided with the protective effect of stromal support, a clinically relevant model. Used as a monotherapy and at a concentration of 1 μM, this drug compares quite favorably with dasatinib in terms of reducing overall ALL cell viability. Moreover, this is a concentration that was reported in the plasma of patients responding to and treated with this drug.

Using the CML cell lines K562 and BV-173 and two fresh CML patient samples but without stroma, Donato et al (28) argued that inhibition of the Bcr/Abl T315I mutant is unlikely to be a major factor in the activity of VX-680 at clinically achievable (up to 5 μM) levels. Our studies support this concept, to some extent. We did notice a reduction in overall phosphotyrosine in the BLQ1 cells with the T315I at 1 μM concentrations. However, because of the non-quantitative nature of the assay used by ourselves and others (Western blotting) this is difficult to translate into clinical effects. The tyrosine phosphorylation of Crkl, a direct substrate of Bcr/Abl, was only blocked at high levels of VX-680. Nonetheless, VX-680 concentrations as low as 1 μM generated a notable reduction in p-Stat5 levels, with a complete elimination of p-Start5 at 5 μM. We speculate that this is an effect of this drug on other kinase activities in these cells, albeit not of Src, of which the tyrosine phosphorylation was unaffected by VX-680. We found that VX-680 reduced viability of the two human T315I-bearing ALL samples somewhat better than that of the two samples lacking the mutation. However, this is unlikely to be correlated with the mutation status. Since the two Ph-positive ALL samples lacking the T315I grow somewhat more slowly than those with the mutation, it appears that the different activity of VX-680 on these cells may reflect the activity of the drug on Aurora kinases and cell cycle.

The only clinical trail published with this drug for the treatment of Ph-positive leukemias used a regimen of a 5-day treatment followed by a 14-21 day non-treatment. Dreier et al (29) studied the long-term fate of colon cancer cell lines after short-term treatment with Aurora kinase inhibitors. After treatment with ZM447439 for 4~7 days, followed by removal of the drug, cells of different sizes with variable numbers of nuclei were recovered, that were able to proliferate. They hypothesized that such cells initially underwent multiple failed divisions in the presence of the Aurora kinase inhibitors and as a result became multinucleated giants. Upon removal of the drug, some of these giant cells were able to divide again in an asymmetrical way, producing giant cells as well as small cells. In our studies, after treatment of BLQ1 cells with 1 μM VX-680 for 3 days, followed the removal of the drug from the culture system, we observed the appearance of ALL cells that were clearly larger than the original cells. Alarmingly, the cells we obtained were able to proliferate. In combination with the reports that VX-680 treatment of mouse embryonic fibroblasts causes the emergence of hyperdiploid cells, we were concerned that these large ALL cells could represent a population with a greater degree of malignancy.

However, when we re-treated these BLQ1 cells with VX-680 they were still sensitive to the drug and showed a greater sensitivity to vincristine. Dreier et al. also noted that the clones that emerged after removal of ZM447439 were not resistant to the drug, and in this study the cells proliferated at similar rates to the parental cell line (29). Walsby et al indicated that after treatment of the NB4 acute myeloid leukemia cell line for 48 hrs with AZD1152-HQPA or ZM447439, the cells showed a reduced clonal growth when re-plated as single cells, indicating that recovery from hyperploidy was impaired (30).

To examine the possibility of environmental-mediated drug resistance to VX-680, we determined the effects of long-term treatment with it on ALL cells in the presence of stroma. Consistent with our previous studies using other drugs (31), we observed the emergence of VX-680 resistant cells. Unlike the short-term treated human ALL cells, these VX-680 resistant cells were similar in size to the parental cells. Similar to the parental cells, they could be made resistant to 16 nM nilotinib but, reassuringly, the development of resistance to this drug was not accelerated. However, these in vitro results suggest that it will not be possible to eradicate all Ph-positive ALL cells when they are in the presence of stroma, regardless of whether in short-term or long-term treatment, if VX-680 is used as monotherapy.

Because VX-680 was reported to inhibit Ph-positive leukemias that have a T315I mutant Bcr/Abl, the option to use this drug as a second-line monotherapy subsequent to the development of drug-resistant ALL is obvious. However, a drug that is specifically targeted to inhibit the Bcr/Abl T315I mutant has been recently reported (30). Based on the impressive cell killing noted in our study by the simultaneous use of VX-680 with dasatinib in Ph-positive ALL cells lacking the T315I, as well as the useful effect of VX-680 on other kinases besides Bcr/Abl, we suggest that first-line therapy of Ph-positive ALL could include the use Aurora kinases inhibitors together with a second drug. A combination of Imatinib with intensive chemotherapy was recently reported to increase the event-free survival of children with Ph-positive ALL (30). Similarly, combinations of VX-680 with different kinase inhibitors such as imatinib, dasatinib, nilotinib or the newly reported AP24534 might be effective to definitively preclude the emergence of drug resistant leukemia cells.

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