Background
Anaplastic lymphoma kinase-expressing (ALK
+) T cell lymphoma is an aggressive neoplasm that constitutes 3–5% and 40% of adults and children/adolescents non-Hodgkin lymphoma, respectively [
1]. Wild-type ALK is a receptor protein tyrosine kinase with an expression restricted to neural tissues at an early stage of human development [
2]. It is believed that the phosphorylation and activation of ALK is tightly regulated through a number of yet not fully characterized ligands [
3‐
5]. Approximately 80% of ALK
+ T cell lymphoma cases harbor the nucleophosmin-ALK (
NPM-
ALK) oncogene, which results from the fusion of the
ALK gene on the 2p23 chromosome to the
NPM gene on the 5q35 chromosome [
6]. NPM-ALK lacks an extracellular domain; however, it functions as a constitutively activated cytoplasmic tyrosine kinase that is capable of translocating to the nucleus [
7]. The activation of NPM-ALK induces activation of several downstream signaling pathways including phospholipase C-γ [
8], phosphoinositide 3-kinase (PI-3K)/AKT [
9,
10], Janus kinases/signal transducers and activators of transcription (JAK/STAT) [
11‐
13], and Ras/mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) [
14], all of which are crucial for cell survival and proliferation.
Treatment options for NPM-ALK
+ T cell lymphoma were limited to conventional polychemotherapy such as the CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) regimen. The discovery of the selective, small-molecule ALK inhibitors revolutionized the approach to treat ALK
+ tumors. Crizitonib (PF-2341066) and NVP-TAE684 are two of the earlier first-generation ALK inhibitors [
15,
16]. Although initial responses to selective ALK inhibitors are favorable, about 30% of the NPM-ALK
+ T cell lymphoma cases develop resistance and have multiple relapses leading to disease-related morbidities and mortalities [
17]. In an attempt to avoid this outcome, newer generations of ALK inhibitors were developed. Unfortunately, resistance, relapse, and disease progression also occurred when these inhibitors were used [
18‐
20].
We have previously demonstrated that NPM-ALK is physically associated and reciprocally interacts with IGF-IR; another protein tyrosine kinase with potent oncogenic potential [
21,
22]. This functional relationship appears to enhance the phosphorylation/activation of the two kinases and potentiates their effects on common downstream survival signaling JAK/STAT [
12,
23]. We also found ASP3026, a second-generation ALK inhibitor that has been recently utilized in patients with ALK
+ cancers, capable of overcoming the resistance to crizotinib-induced ALK mutants [
24‐
26]. Notably, development of resistance to ALK inhibition has also been noted in the case of the ASP3026 small molecule inhibitor [
27]. A previous study also reported that IGF-IR constitutive activation could be an important factor contributing to the acquired resistance to ALK inhibitors [
28]. Collectively, these data warrant the search for more refined strategies to overcome these hurdles, and suggest that it is still possible that the utilization of significantly smaller doses of ALK inhibitors in combination with other legitimate targeted therapies, such as IGF-IR inhibitors, may limit significantly the resistance to higher doses of ALK when used alone.
Picropodophyllin (PPP; AXL1717) is a clinically utilized, selective, small molecule inhibitor of IGF-IR [
29‐
33]. It has been shown to be effective in inhibiting various types of cancers including those of the gastrointestinal tract, nasopharynx, liver, lung, ovary, soft tissues, and hematopoietic system including NPM-ALK
+ T cell lymphoma [
21,
34‐
46]. Recently, however, higher doses of PPP have been shown to induce bone marrow toxicity in some patients [
30]. In this paper, we tested the hypothesis that dual suppression of ALK and IGF-IR could represent a superior strategy that significantly improves the effects of the isolated inhibition of each enzyme alone. It is also anticipated that this approach might lead to decreased acquired resistance and eliminate potential unwarranted side effects that may associate the utilization of higher doses.
To achieve our goals, we used low doses of ASP3026 and PPP, and compared their in vitro and in vivo effects alone or combination. Our in vitro data demonstrated that low doses of ASP3026 in combination with PPP act synergistically to exert more pronounced anti-proliferative and apoptotic effects on NPM-ALK+ T cell lymphoma cells than the effects of each drug alone. In a systemic NPM-ALK+ T cell lymphoma mouse model, combined treatment with ASP3026 and PPP was associated with slower tumor growth and longer survival when compared with individual drug treatments. Considering that the two inhibitors have been individually utilized in patients, our data stress the feasibility of the combination strategy to be further tested in the clinic.
Methods
IGF-IR and ALK inhibitors
PPP was dissolved in DMSO for in vitro studies, and in DMSO/vegetable oil (10:1) for in vivo studies. ASP3026 (CT-ASP302; ChemieTek, Indianapolis, IN) was dissolved in DMSO with H2O and HCl (1:1) for in vitro experiments, and in 0.5% methyl cellulose for in vivo experiments.
Cell lines
The NPM-ALK+ T cell lymphoma cell lines Karpas 299, DEL, and SR-786 were purchased from DSMZ (Braunschweig, Germany). Cells were maintained in RPMI 1640 plus 10% FBS, 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin, in a humidified chamber at 37 °C with 5% CO2.
Generation of cells resistant to the ALK inhibitor ASP3026
DEL cells were cultured in increasing concentrations of ASP3026 (0.125–1.0 μM) for 4 months. Acquired resistance to 1.0 μM ASP3026 (DEL-R cells) was determined by an MTS assay as described below.
Antibodies
The following antibodies were used for Western blotting: pALKY1586 (NPM-ALKY646; catalogue number: 3343), IGF-IR (9750), pIGF-IRY1135/1136 (3024), STAT3 (9139), pSTAT3Y705 (4113), PARP (9532), cleaved PARP (5625) (Cell Signaling, Danvers, MA), ALK (M719501-2) (Dako, Carpinteria, CA), β-actin (A-2228) (Sigma-Aldrich, St. Louis, MO). Antibodies used for immunohistochemical staining are described below.
MTS assay
Cell viability was measured using the MTS reagent 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (Promega, Madison, WI). Briefly, cells (1 × 106/mL) were seeded into 96-well plates and treated with relevant drugs after overnight incubation. Thereafter, each well was incubated with 20 μL of MTS reagent for 1 to 4 h at 37 °C in 5% CO2. Absorbance at 490 nm was read using a plate reader.
Isobolographic analysis
We used the concentration-dependent viability (MTS) assay curves to generate isoeffect curves by using isobolograms to determine whether the net effects of PPP and ASP3026 were synergistic, additive, or antagonistic. Concentration-dependent effects were calculated using Excel software (Microsoft, Redmond, WA) for one drug while keeping constant concentrations for the other.
Apoptosis detection
Induction of apoptosis was detected by annexin V-FITC and PI-based flow cytometric analysis using apoptosis detection kit (556547, BD Biosciences). Briefly, cells were simultaneously stained with annexin V-FITC and PI and incubated for 15 min at room temperature in 1× annexin binding buffer. Fluorescence intensity was measured by flow cytometry (BD FACSCalibur system). This assay distinguishes cells that are intact (FITC−/PI−), apoptotic (FITC+/PI−), or necrotic (FITC+/PI+). Analysis was performed using flow cytometry (BD FACSCalibur, BD Biosciences, San Jose, CA). The percentage of cells undergoing apoptosis was quantified by CellQuest software (BD Biosciences).
Cell proliferation
Cell proliferation was measured by a BrdU assay using a standard kit (X1327K1, Exalpha Biologicals, Shirley, MD). Cells were plated at a concentration of 2 × 105 cells/well. Thereafter, 20 μL of BrdU (1:500) were added for 24 h. Plates were centrifuged for 30 min, and 100 μL of anti-BrdU antibody were added for 1 h, followed by 100 μL peroxidase goat anti-mouse IgG conjugate (1:2000) for 30 min. Thereafter, 100 μL of TMB substrate were added for 30 min. Stop solution (50 μL) was added and plates were read at 450/595 nm.
Anchorage independent colony formation
Cells were plated in a methylcellulose-based medium (Methocult H4230; Stemcell Technologies, Vancouver, BC, Canada) mixed in RPMI-1640 (1:4). Harvested cells were mixed in a 1:10 (v/v) ratio with methylcellulose media in a 15 mL conical vial. Tubes were inverted to mix the contents and poured to 6-well plates without any bubbles. The plates were then incubated at 37 °C in a 5% CO2 incubator for approximately 5 days. In order to stain the colonies, p-iodonitrotetrazolium violet was added and incubated for 24 h. Colonies were visualized using the FluorChem 8800 imaging system (Alpha Innotech, San Leandro, CA).
Western blotting
Lysis buffer contained 25 mM HEPES (pH 7.7), 400 mM NaCl, 1.5 mM MgCl2, 2 mM EDTA, 0.5% Triton X-100, 0.1 mM phenylmethylsulfonyl fluoride, 3 mM dithiothreitol, phosphatase inhibitor cocktail (20 mM β-glycerol phosphate, 1.0 mM sodium orthovanadate), and protease inhibitor cocktail (10 μg/mL leupeptin, 2 μg/mL pepstatin, 50 μg/mL antipain, 1× benzamidine, 2 μg/mL aprotinin, 20 μg/mL chymostatin). Further, 50 μg total proteins were electrophoresed on 10% SDS-PAGE, transferred onto nitrocellulose membranes, and probed with primary antibodies, followed by matched secondary antibodies conjugated with horseradish peroxidase (HRP). Protein expression was detected using chemiluminescence and a commercially available kit (GE Healthcare, Piscataway, NJ).
Systemic NPM-ALK+ T cell lymphoma mouse model
In vivo studies were approved by the Institutional Animal Care and Use Committee. Karpas 299 cells permanently expressing firefly luciferase and GFP were generated by using the F-Luc-GFP lentivirus (Capital Biosciences, Rockville, MD) in which humanized firefly luciferase (hLUC) was expressed under the CMV promoter, and GFP with puromycin resistance marker were co-expressed bicistronically under the SV40 promoter. C.B-17 SCID mice (6–8-week-old females) were purchased from Taconic Biosciences (Cambridge City, IN). Moreover, 1.0 × 106 cells were injected via tail vein and allowed to establish disseminated malignant lymphoma for about 3 weeks. To monitor lymphoma, d-luciferin (Gold Biotechnology, St. Louis, MO) was i.p. injected and luciferase signaling was detected using bioluminescence imaging (IVIS Lumina XR imaging system; Caliper Life Sciences, Alameda, CA). Starting from the third week, a low dose of ASP3026 (5 mg/kg/day) was administered once a day by oral gavage for 6 weeks. PPP (20 mg/kg) was administered i.p. every 12 h only for 3 weeks. In a subgroup of mice, CHOP was administered at 1/3 of the standard dose (cyclophosphamide, 13.3 mg/kg; doxorubicin, 1.1 mg/kg, and vincristine, 0.166 mg/kg, were all administered intravenously; whereas prednisone, 0.06 mg/kg, was given once a day by using oral gavage) for five consecutive days at the beginning of the third week. Tumor progression was monitored weekly until death or study conclusion. Kaplan–Meier survival curves were used to determine the efficacy of individual or combined drugs compared to controls. Mice underwent total necropsy, and tumors were fixed in 10% buffered formalin or snap-frozen in liquid nitrogen.
Immunohistochemical staining
Lymphoma tumors from mice were processed by fixation in formalin followed by paraffin embedding. Prior to staining, tumors were deparaffinized in alcohol gradient, washed, and then subjected to antigen retrieval for 45 min in a steamer using Target Retrieval Solution (1×; pH 9.0, Dako). Sections were left for 20 min to cool down to room temperature, washed, and incubated in 3% H2O2 for 15 min to block endogenous peroxidase activity. Sections were then blocked for 30 min at room temperature in serum-free blocking solution (Dako). The primary antibody, diluted in blocking buffer, was added for overnight incubations at 4 °C. Primary antibody dilutions were 1:50 for ALK (M719501-2, Dako) and pSTAT3Y705 (4113, Cell Signaling); and 1:75 for pIGF-IRY1161 (ab39398, Abcam, Cambridge, MA) and Ki-67 (M7240, Dako). Sections were washed three times and incubated for 30 min with the secondary antibody Dako Envision+Link System-HRP. Signals were developed using 3,3’-diaminobenzidine tetrachloride substrate, and hematoxylin was used for counterstaining.
Statistical analysis
In vitro assays were set up in triplicates and the results were expressed as means ± S.D. Statistical significance between the experimental groups were detected by using student’s t test or two-way analysis of variance (ANOVA), where appropriate. All statistical analyses including generation of the Kaplan–Meier curves were performed by using the GraphPad PRISM software (GraphPad Software Inc., San Diego, CA). P < 0.05 was considered statistically significant.
Discussion
In this paper, we used two clinically utilized small molecule inhibitors, PPP and ASP3026, to antagonize the oncogenic kinases IGF-IR and ALK, respectively, in NPM-ALK
+ T cell lymphoma [
24]. Simultaneous treatment with PPP and ASP3026 caused a remarkable decrease in cell viability, proliferation, and anchorage-independent colony formation. The combined treatment regimen was also associated with a marked increase in apoptotic cell death. It is important to emphasize that combined treatment with the two inhibitors induced synergistic effects and was much more pronounced than the treatment with any of these two inhibitors alone. As a possible explanation for these findings, combined treatment abolished the phosphorylation of IGF-IR and NPM-ALK as well as the phosphorylation of their common downstream target STAT3; a major survival-promoting transcription factor in this lymphoma [
11,
12,
48]. When utilized together, PPP and ASP3026 increased significantly the levels of cleaved PARP, which could explain, at least in part, the remarkable increase in apoptosis when the combined regimen was used. We also found that acquired resistance to ALK inhibition could be associated with upregulation of IGF-IR phosphorylation. Of important note is that NPM-ALK
+ T cell lymphoma cells resistant to ALK inhibition remained sensitive to IGF-IR inhibition.
In addition to the in vitro findings, similar in vivo results were observed when a low dose of ASP3026 was simultaneously used with PPP to treat systemically disseminated NPM-ALK
+ T cell lymphoma in mice. The combination treatment regimen was not only more effective in suppressing lymphoma tumor growth, but it also improved the survival of the mice. It is important to emphasize that PPP potentiated the effects of the low dose of ASP3026 despite the fact that PPP was only used for 3 weeks of the entire 6 weeks of treatment. Histologically, combined treatment with PPP and low dose of ASP3026 was associated with expanded areas of tumor cell necrosis, decreased proliferation index as evaluated by Ki-67 staining, and downregulation of phosphorylated IGF-IR and STAT3. The effects induced by combined treatment were more pronounced than the changes noted when mice were treated by PPP or ASP3026 alone. Interestingly, treatment with PPP or ASP3026 alone slightly decreased the nuclear localization of phosphorylated STAT3, and combined treatment caused a much more pronounced decrease in such localization. These observations are consistent with our previous studies in NPM-ALK
+ T cell lymphoma that showed inhibition of IGF-IR by PPP decreased significantly the binding of STAT3 to DNA [
21].
NPM-ALK
+ T cell lymphoma is an aggressive neoplasm. Classical treatments have primarily included combination chemotherapies with CHOP being the most commonly utilized regimen. However, previous studies demonstrated that up to 40% of the patients develop CHOP resistance and eventually relapse. Moreover, patients also die because of CHOP-related complications that are not directly related to their lymphoma [
49]. We have previously noticed that the utilization of CHOP at the standard dose used in mice with subcutaneous xenografts of non-NPM-ALK
+ T cell lymphoma [
50,
51] was associated with pronounced toxicity that frequently caused early expiration of mice with systemic NPM-ALK
+ T cell lymphoma [
26]. Therefore, in the current study, we resorted to treat a subgroup of mice with only 1/3 of the standard concentration of CHOP (CHOP1/3). The growth inhibitory effects of combining PPP and CHOP1/3 in NPM-ALK
+ T cell lymphoma were significantly more pronounced than the effects of CHOP1/3 alone.
More recently, selective ALK small molecule inhibitors have emerged as an alternative approach with enhanced effects as well as with improved safety margin [
52]. Notably, the ALK small molecule inhibitors have been more evaluated in ALK
+ solid tumors such as EML4-ALK
+ non-small cell lung cancer. For instance, crizotinib, the prototype of first-generation ALK inhibitors, was primarily used to treat non-small cell lung cancer patients with ALK expression in their tumors. Although crizotinib initially demonstrated promising effects, subsequent studies highlighted the development of significant resistance as a major hurdle to crizotinib [
53]. In a previous study, we systematically characterized the in vitro effects of ASP3026, an orally available second-generation ALK inhibitor, in NPM-ALK
+ T cell lymphoma cell lines [
26]. We also examined the effects of ASP3026 compared with CHOP in our systemic NPM-ALK
+ T cell lymphoma model in mice. ASP3026 demonstrated remarkable in vitro and in vivo effects and was superior to and safer than CHOP. Moreover, ASP3026 successfully overcame the resistance to crizotinib. However, subsequent studies demonstrated that resistance also develops to ASP3026 because of ALK mutations [
27]. In the current study, we examined the effects of a much lower concentration of ASP3026 than the one we used previously (5 mg/kg/day vs. 30 mg/kg/day) [
26]. Nonetheless, combining PPP with this very low concentration of ASP3026 caused pronounced in vivo inhibitory effects in NPM-ALK
+ T cell lymphoma tumors and improved mice survival as well. These pronounced effects occurred despite the fact that PPP was administered for only 3 weeks out of the entire 6 weeks of treatment.
IGF-IR is a receptor tyrosine kinase with potent oncogenic potential that has been observed in numerous types of cancer including hematological neoplasms [
54]. We have previously demonstrated that IGF-IR is highly expressed in NPM-ALK
+ T cell lymphoma [
21]. Increased expression of IGF-IR in this lymphoma can be attributed, at least in part, to decreased expression of the transcription factors Ikaros isoform 1 (Ik-1) and myeloid zinc finger 1 (MZF1) [
55]. Under the physiological conditions, Ik-1 and MZF1 appear to repress the expression of the
IGF-
IR gene in normal T lymphocytes. Moreover, we have provided evidence to support that IGF-IR and NPM-ALK are physically associated and interact reciprocally to enhance their phosphorylation/activation as well as the activation of their common downstream modulators including STAT3 [
21,
22]. Furthermore, it appears that the association between IGF-IR and NPM-ALK plays critical roles in maintaining the stability of NPM-ALK protein [
22]. In further support of an important role of IGF-IR in the pathogenesis of NPM-ALK
+ T cell lymphoma, our current data show that acquired resistance to ALK inhibition is associated with a remarkable increase in the phosphorylation of IGF-IR. The increase in IGF-IR phosphorylation is an event that probably occurred secondary to the increase in the phosphorylation of NPM-ALK, which was noted in cells resistant to ALK inhibition. Nonetheless, our data show that inhibition of IGF-IR remained to be substantially effective in decreasing the viability of NPM-ALK
+ T cell lymphoma cells resistant to ALK inhibition. Most likely, high levels of phosphorylation of IGF-IR detected in these cells enhanced their dependence on IGF-IR signaling and, as a result, maintained their sensitivity to IGF-IR inhibition. Our results show that treatment of the NPM-ALK
+ T cell lymphoma cell line DEL-R, which was resistant to the ALK inhibitor, with PPP induced downregulation of pIGF-IR and pNPM-ALK. These results are in agreement with our previous findings demonstrating the association and reciprocal interactions through phosphorylation that exist between IGF-IR and NPM-ALK in this lymphoma [
21,
22]. Notably, however, PPP did not only abrogate the phosphorylation of IGF-IR and NPM-ALK in the ALK inhibitor-resistant cells but also downregulated the basal levels of these two oncogenic kinases. Several previous studies have demonstrated similar effects of PPP on the basal levels of IGF-IR [
56‐
58]. The effects of PPP might be attributed to the fact that it, similar to other small molecule kinase inhibitors, induces selective but not entirely specific effects. For instance, one possible mechanism for PPP effects on the basal levels of IGF-IR could be attributed to the MDM2 E2 ligase-induced ubiquitination and subsequent downregulation of IGF-IR expression after treatment with PPP [
56]. Similar to our findings in the ALK-inhibitor-resistant cells, PPP-induced downregulation of IGF-IR was shown to be pronounced in multidrug resistant cancer cells [
58]. On the other hand, we have previously found that the association between IGF-IR and NPM-ALK through Tyr
644 and Tyr
664, located within the C terminus of NPM-ALK, is required to maintain the stability of NPM-ALK protein [
22]. Therefore, the decrease in the basal levels of IGF-IR after treating the ALK inhibitor-resistant cells with PPP could explain the simultaneous decrease in NPM-ALK basal protein levels. Although, future studies are required to further analyze these important observations, our data support that strategies based on exploiting IGF-IR signaling might represent a legitimate approach to overcome ALK resistance.
One of the strategies that has been suggested in previous preclinical studies in epithelial tumors to enhance the effects of ALK inhibitors as well as to avoid the resistance to these inhibitors is to combine ALK inhibition with IGF-IR inhibition [
59‐
61]. To our knowledge, such an approach has never been examined in NPM-ALK
+ T cell lymphoma, the prototype of ALK
+ malignant neoplasms. Moreover, previous studies in NPM-ALK
+ T cell lymphoma as well as ALK
+ lung cancer have shown cross-resistance among various ALK inhibitors [
28,
62,
63]. Combined treatment with low doses of ALK inhibitors in combination with IGF-IR inhibitors might bypass cross-resistance. Our results strongly suggest that combining low doses of ALK inhibitors with IGF-IR inhibitors might represent an effective strategy to successfully eradicate this aggressive lymphoma. Importantly, the utilization of such low concentrations of ALK might help to avoid development of therapeutic resistance as well as to increase the clinical tolerance to these therapeutic agents when used alone at higher concentrations.
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