Introduction
Although significant advances have been made in the treatment of acute lymphoblastic leukemia (ALL) especially in children, only 30-40% of adults have a long-term survival [
1]. A major subclass of ALL with a specially poor prognosis in both adults and children is that of Philadelphia chromosome-positive ALL [
2,
3]. The Ph chromosome is generated by a reciprocal t(9;22) translocation. It is found in around 30% of cases of adult ALL and is the hallmark of chronic myeloid leukemia (CML). The deregulated tyrosine kinase activity of the chimeric Bcr/Abl protein in these leukemias phosphorylates a broad range of substrates, many of which are key cellular signal transduction proteins [
4‐
6]. The tyrosine kinase inhibitor imatinib became the first-line therapy in the conventional treatment of CML, with a relatively selective targeting of the ATP binding site of Bcr/Abl [
2,
7‐
10]. However, the emergence of resistance to imatinib remains a major problem especially for those patients with advanced CML (with progression in about 7% of patients after 7 years), or with Ph-positive ALL. This is due to point mutations in the Bcr/Abl kinase domain, including the most frequent T315I and E225K mutations [
4,
6,
7,
11]. Second-generation tyrosine kinase inhibitors, such as nilotinib, dasatinib and bosutinib are capable of targeting the majority of imatinib-resistant mutations, but none of them are effective against leukemia cells harboring the T315I mutation [
11‐
14]. Thus, the need to find a more effective treatment for leukemia patients with this mutation is obvious.
Aurora kinases are key regulators of cell division [
15,
16] and deregulation of this activity can result in aneuploidy and carcinogenesis [
17]. Therefore, they are attractive targets for anticancer therapy [
18,
19]. Several small molecule inhibitors of Aurora kinases with various properties are in clinical trials including PHA-739358 (Danusertib) [
20,
21], MLN8054 [
22] and AZD1152 [
23]. PHA-739358 is a pan-Aurora kinases inhibitor with activity against all Aurora kinase family members (A, B and C) [
24,
25]. Interestingly, and of importance for the potential use of this compound against poor-prognosis ALL, Gontarewicz
et al, using Bcr/Abl constructs transfected into the BaF3 cell line, showed that PHA-739358 is also effective against imatinib-resistant Bcr/Abl mutants including the T315I [
24]. A determination of the crystal structure of the T315I Abl kinase domain in complex with PHA-739358 showed that the drug interacts with the active conformation of Abl kinase [
26]. Currently, preliminary evidence for anti-tumor activity of PHA-739358 has been seen in various advanced refractory cancers, and phase II studies in solid tumors are ongoing [
20].
In this report, we performed preclinical studies in the presence of stroma in vitro as well as in vivo, to explore the application of PHA-739358 for treatment of a variety of primary human acute lymphoblastic leukemia cells including those belonging to the Ph-positive ALL subclass and harboring the T315I mutation. We conclude that PHA-739358 could be considered for the treatment of patients with different subtypes of ALL in combination with other drugs to potentiate its cytostatic and cytotoxic effects.
Discussion
The current study tested the use of PHA-739358 for the treatment of Ph-positive ALL
in vitro and
in vivo. Since PHA-739358 has dual activity against both Bcr/Abl and Aurora kinases, one could expect that the inhibition of Ph-positive ALL would be more profound than that of Ph-negative ALL. However, we could not detect an increased effect on the Ph-positive samples, and Ph-positive samples with or without the T315I mutation did not differ significantly in sensitivity. Our results with the mutants agree with Gontarewicz et al., who reported that PHA-739358 was effective against imatinib-resistant Bcr/Abl mutants including those with the T315I mutation in human and mouse leukemia cell lines as well as in CD34
+ cells from an imatinib-resistant CML patient [
24].
We did notice that for some samples, dose escalation did not result in a proportionally larger response. This effect was quite marked in, for example, Pt2. Although treatment with 500 nM PHA-739358 caused a drop in viability to around 40% in 3 days, a 10-fold increased dose of 5 μM did not increase the percentage of apoptotic cells or decrease the viability. Similarly, a 100-fold difference (50 nM – 5 μM) of drug exposure of UCSF02 did not cause a corresponding increased loss in viability. The lack of dose-proportionality might be due to saturation of the mechanism at low concentrations. Indeed, data from the colony formation assays show that a significant part of the effects of PHA-739358 are due to its growth inhibitory activity, which is seen at a concentration as low as 10 nM.
In other cancers, deletion or mutation of p53 has been shown to result in resistance to the induction of apoptosis. We therefore examined whether any of the ALL samples contained p53 mutations using RT/PCR but none were detected. Only US6 showed lack of an RT/PCR product, suggesting bi-allelic loss of p53 (results not shown). These cells reacted to the drug by accumulation of cells with a DNA content of >4N but the amount of cells with a sub-G1 DNA content was less than BLQ1, which is wild type for p53. Interestingly, in hepatocellular carcinoma cell lines, Benten
et al also found that PHA-739358 exhibits activity against both p53-wild type and mutated cancers [
36].
In initial studies using 8093 murine Bcr/Abl transgenic ALL cells transplanted into C57Bl recipients, we found that, compared to control mice, mice that had been treated with 30 mg/kg/bid i.v. PHA-739358 for 5 days survived significantly longer than controls (MST 25 days; control MST 17 days; results not shown). However, mice relapsed shortly after termination of the treatment. The behavior of the leukemia cells
in vivo was modeled, to some extent, by
in vitro co-culture with stroma. In that system, a 3-day treatment with PHA-739358 caused a significant reduction in cell numbers of Pt2 and UCSF02 and suppressed cell proliferation for 6 days or more, but, consistent with Gontarewicz
et al[
24] cells subsequently resumed proliferation with restored Bcr/Abl activity.
Because of this, we examined the effect of treatment with PHA-739358 in combination with a second drug. Since the primary mechanism of action of PHA-739358 is to inhibit the cell cycle, we combined it with a farnesyltransferase inhibitor, which has a similar molecular target Farnesyltransferase inhibitors were originally developed to prevent Ras oncoprotein prenylation. However, FTIs also inhibit the farnesylation of mitotic proteins CENP-E and CENP-F, which mediate chromosomal capture and alignment [
37,
38], while Aurora kinases phosphorylate CENP-E [
35]. FTIs were in phase II/III clinical trials for treatment of a variety of malignancies, but as single agents their activity was modest and ongoing clinical trials are evaluating the role of FTIs in combination with standard cytotoxic drugs [
39‐
42]. Our results using Ph-positive ALLs with or without the T315I mutation suggest that a combination of PHA-739358 with an FTI may be an alternative useful combination to test. Interestingly, the addition of PHA-739358 to dasatinib and vincristine, two drugs currently in clinical use, also was beneficial in terms of reducing clonogenic potential and cell killing of ALL cells. These results suggest that there may be numerous other drugs that could be combined with this Aurora kinase inhibitor, a possibility that could be rapidly evaluated in model systems such as the one used in the current study.
An international, multicenter phase I study in adult patients with advanced CML and Ph-positive ALL resistant or intolerant to imatinib or second generation of tyrosine kinase inhibitors used three cycles of PHA-739358 as a 3-hour infusion for 7 consecutive days every 2 weeks [
43]. Therefore, we tested the efficacy of treatment with PHA-739358 on human Ph-positive ALL cells with the T315I mutation by administering the drug in 3 cycles of 7 days each, using a drug dose also used by Carpellini and Moll [
31].
In vivo drug treatment was effective in ablation of the tyrosine kinase activity of the Bcr/Abl T315I mutant. While on treatment with PHA-739358, the number of circulating ALL cells was markedly suppressed and all parameters measured, including peripheral blood ALL cell counts, terminal spleen weight and overall survival show that this approach results in significant reduction of leukemia progression, but not in a cure. Based on these
in vivo and
in vitro data, we conclude that PHA-739358 has therapeutic effects against a variety of ALL cells, including Ph + wt, Ph + T315I and Ph- subclasses. However, increasing the dose of drug did not result in a proportional increase in cell killing and discontinuation of treatment allowed the cells to resume proliferation.
Methods
Drugs, reagents and cells
PHA-739358 (Danusertib) was provided by Nerviano Medical Sciences (Nerviano, Italy). Dasatinib was obtained commercially from Toronto Research Chemicals (Toronto, ON, Canada). PHA-739358 and dasatinib were dissolved in DMSO and stored at −80°C. The FTI SCH66336 was obtained from Schering-Plough. A vincristine sulfate solution was obtained from Hospira Worldwide Inc. (Lake Forest, IL, USA). The murine OP9 stromal cell line (CRL-2749) was obtained from the ATCC (Manassas, VA, USA). Human Ph-positive ALL cells included wild-type Bcr/Abl (UCSF02, TXL2), T315I mutants (BLQ1, Pt2) and Ph negative ALL cells (US6, US7, US7R) and were described previously [
46]. US6 was from a Ph-negative ALL patient at diagnosis. The primary cells were passaged in NOD/SCIDγc−/− (NSG) mice (Jackson Labs, Bar Harbor, ME). Leukemia cells harvested from the spleens of these mice were plated on irradiated OP9 feeder layers. 8093 and Bin2 Bcr/Abl P190-expressing transgenic mouse lymphoblastic leukemia cells have been previously described [
47‐
49] and were grown in the presence of E13.5 irradiated mouse embryonic fibroblasts (MEFs). Human leukemia cells were grown in αMEM medium supplemented with 20% FBS, 1% L-glutamine and 1% penicillin/streptomycin (Invitrogen Corporation). Mouse leukemia cells were grown in McCoy’s 5A medium including 15% FBS 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.
Analysis of cell proliferation, apoptosis and DNA content
ALL cells were cultured in a 24-well or 6-well plate at a density of 1x106 cells/ml, in the presence of irradiated OP9 cells or MEFs. Cells were treated with various concentrations of PHA-739358 or SCH66336 in triplicate wells and viability of cells was measured by Trypan blue exclusion assay. Apoptotic cells were assessed by an Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit I (BD Pharmingen, San Diego, USA). Apoptotic cells were defined by double positivity for Annexin V and PI evaluated by flow cytometry (Accuri, Ann Arbor, MI, 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.
Assessment of phosphorylation status of histone H3 by flow cytometry
BLQ1 or US6 cells were treated with 1 μM PHA-739358 for 24 hours or 48 hours, followed by washing and fixing with 70% ethanol for one hour on ice. Cells were blocked with human FcR Blocking Reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) for 10 minutes and incubated with phospho-histone H3 (Ser10) Ab (Cell Signaling Technology, Danvers, MA). After 45 minutes of incubation, cells were washed and incubated with anti-rabbit IgG FITC-conjugated antibody (Jackson ImmunoResearch, West Grove, PA, USA) for 30 minutes. Cells were washed and stained with PI before measuring by flow cytometry.
Western blotting
BLQ1 and UCSFO2 ALL cells were treated with PHA-739358 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 the following antibodies: pY-20-Horseradish peroxidase conjugated (BD-Transduction Laboratories, San Jose, CA), phospho-Src family (Tyr416), Src, phospho-Crkl, phospho-histone H3 and histone H3 (all from Cell Signaling Technology, USA), Bcr (N-20), Crkl (both from Santa Cruz Biotechnology, USA) and Gapdh (Chemicon International, USA) antibodies using standard procedures.
Evaluation of PHA-739358 in vivo
All animal experiments were carried out in concordance with institutional IACUC and NIH guidelines. To evaluate the efficacy of PHA-739358 against Ph+ ALL with the T315I mutation in vivo, 2x106 Pt2 cells were injected into female NSG mice (6 weeks old). Transplanted mice were treated with vehicle solution or PHA-739358 (30 mg/kg, iv, qd, 7 days on and 7 days off x 3 cycles) 7 days after transplantation. Peripheral blood was collected every two weeks after starting treatment and the percentage of leukemia cells was determined by measuring CD10+CD19+ double positive cells by flow cytometry. To further assess the immediate effect of PHA-739358 in vivo, mice that had developed leukemia were injected with PHA-739358 (45 mg/kg). Two hours after injection, spleen and bone marrow cells were collected and the phosphorylation status of histone H3 and Crkl, as well as total phosphotyrosine, were measured by Western blot.
Pt2 or UCSF02 cells (2x104/well) were plated in complete methylcellulose media supplemented with cytokines (MethoCult H4435, Stem Cell Technologies, Vancouver, Canada) and treated with different concentrations of PHA-739358 with or without the FTI SCH66336/Lonafarnib, vincristine or dasatinib, as indicated, in triplicate wells. Colonies consisting of > 40 cells were counted using an inverted microscope at day 10–14.
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) or paired t-test. The value of P < 0.05 was considered to be statistically significant.
Competing interests
JM is employed by Nerviano Medical Sciences. The other authors declare no conflict of interest.
Author’s contributions
FF designed and performed research, collected, analyzed and interpreted data and wrote the manuscript. ML performed research and collected data; SS helped with design of experiments and performed research; JM interpreted data; JG interpreted data; NH designed experiments, analyzed and interpreted data and wrote the manuscript. All authors read and approved the final manuscript.