The Anaplastic Lymphoma Kinase (ALK) is an orphan receptor tyrosine kinase, which undergoes post-translational N-linked glycosylation. The catalytic domain of ALK was originally identified in the t(2;5) translocation that produces the unglycosylated oncogenic protein NPM-ALK, which occurs in Anaplastic Large Cell Lymphoma (ALCL). Recently, both germline and somatic activating missense mutations of ALK have been identified in neuroblastoma (NB), a pediatric cancer arising from neural crest cells. Moreover, we previously reported that ALK expression is significantly upregulated in advanced/metastatic NB. We hypothesized that ALK function may depend on N-linked glycosylation and that disruption of this post-translational modification would impair ALK activation, regardless the presence of either gene mutations or overexpression.
We employed tunicamycin to inhibit N-linked glycosylation. The following ALK-positive NB cell lines were used: SH-SY5Y and KELLY (ALK mutation F1174L), UKF-NB3 (ALK mutation R1275Q) and NB1 (ALK amplification). As a control, we used the NB cell lines LA1-5S and NB5 (no ALK expression), and the ALCL cell line SU-DHL1 (NPM-ALK).
Tunicamycin treatment of ALK-positive NB cells resulted in a hypoglycosylated ALK band and in decreased amounts of mature full size receptor. Concomitantly, we observed a marked reduction of mature ALK phosphorylation. On the contrary, tunicamycin had no effects on NPM-ALK phosphorylation in SU-DHL1 cells. Moreover, phosphorylation levels of ALK downstream effectors (AKT, ERK1/2, STAT3) were clearly impaired only in ALK mutated/amplified NB cell lines, whereas no significant reduction was observed in both ALK-negative and NPM-ALK-positive cell lines. Furthermore, inhibition of N-linked glycosylation considerably impaired cell viability only of ALK mutated/amplified NB cells. Finally, the cleavage of the Poly-ADP-ribose-polymerase (PARP) suggested that apoptotic pathways may be involved in cell death.
In this study we showed that inhibition of N-linked glycosylation affects ALK phosphorylation and disrupts downstream pro-survival signaling, indicating that inhibition of this post-translational modification may be a promising therapeutic approach. However, as tunicamycin is not a likely candidate for clinical use other approaches to alter N-linked glycosylation need to be explored. Future studies will assess whether the efficacy in inhibiting ALK activity might be enhanced by the combination of ALK specific small molecule and N-linked glycosylation inhibitors.
Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase aberrantly expressed in neuroblastoma, a devastating pediatric cancer of the sympathetic nervous system. Germline and somatically acquired ALK aberrations induce increased autophosphorylation, constitutive ALK activation and increased downstream signaling. Thus, ALK is a tractable therapeutic target in neuroblastoma, likely to be susceptible to both small-molecule tyrosine kinase inhibitors and therapeutic antibodies–as has been shown for other receptor tyrosine kinases in malignancies such as breast and lung cancer. Small-molecule inhibitors of ALK are currently being studied in the clinic, but common ALK mutations in neuroblastoma appear to show de novo insensitivity, arguing that complementary therapeutic approaches must be developed. We therefore hypothesized that antibody targeting of ALK may be a relevant strategy for the majority of neuroblastoma patients likely to have ALK-positive tumors. We show here that an antagonistic ALK antibody inhibits cell growth and induces in vitro antibody-dependent cellular cytotoxicity of human neuroblastoma-derived cell lines. Cytotoxicity was induced in cell lines harboring either wild type or mutated forms of ALK. Treatment of neuroblastoma cells with the dual Met/ALK inhibitor crizotinib sensitized cells to antibody-induced growth inhibition by promoting cell surface accumulation of ALK and thus increasing the accessibility of antigen for antibody binding. These data support the concept of ALK-targeted immunotherapy as a highly promising therapeutic strategy for neuroblastomas with mutated or wild-type ALK.
neuroblastoma; anaplastic lymphoma kinase; immunotherapy; receptor tyrosine kinase
Activating mutations in the anaplastic lymphoma kinase (ALK) gene were recently discovered in neuroblastoma, a cancer of the developing autonomic nervous system that is the most commonly diagnosed malignancy in the first year of life. The most frequent ALK mutations in neuroblastoma cause amino acid substitutions (F1174L and R1275Q) in the intracellular tyrosine kinase domain of the intact ALK receptor. Identification of ALK as an oncogenic driver in neuroblastoma suggests that crizotinib (PF-02341066), a dual-specific inhibitor of the ALK and Met tyrosine kinases, will be useful in treating this malignancy. Here, we assessed the ability of crizotinib to inhibit proliferation of neuroblastoma cell lines and xenografts expressing mutated or wild-type ALK. Crizotinib inhibited proliferation of cell lines expressing R1275Q-mutated ALK and a cell line with amplified and overexpressed wild-type ALK. By contrast, cell lines harboring F1174L-mutated ALK were relatively resistant to crizotinib. Biochemical analyses revealed that this reduced susceptibility of F1174L-mutated ALK to crizotinib inhibition results from an increased ATP-binding affinity (as also seen in acquired resistance to EGFR inhibitors), and should be surmountable with higher doses of crizotinib and/or with higher affinity inhibitors.
Neuroblastoma is a childhood extracranial solid tumour that is associated with a number of genetic changes. Included in these genetic alterations are mutations in the kinase domain of the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (RTK), which have been found in both somatic and familial neuroblastoma. In order to treat patients accordingly requires characterisation of these mutations in terms of their response to ALK tyrosine kinase inhibitors (TKIs). Here, we report the identification and characterisation of two novel neuroblastoma ALK mutations (A1099T and R1464STOP), which we have investigated together with several previously reported but uncharacterised ALK mutations (T1087I, D1091N, T1151M, M1166R, F1174I and A1234T). In order to understand the potential role of these ALK mutations in neuroblastoma progression, we have employed cell culture-based systems together with the model organism Drosophila as a readout for ligand-independent activity. Mutation of ALK at position 1174 (F1174I) generates a gain-of-function receptor capable of activating intracellular targets such as ERK (extracellular signal regulated kinase) and STAT3 (signal transducer and activator of transcription 3) in a ligand-independent manner. Analysis of these previously uncharacterised ALK mutants and comparison with ALKF1174 mutants suggests that ALK mutations observed in neuroblastoma fall into three classes. These classes are: (i) gain-of-function ligand-independent mutations such as ALKF1174l, (ii) kinase-dead ALK mutants, e.g. ALKI1250T (Schönherr et al., 2011a) and (iii) ALK mutations that are ligand-dependent in nature. Irrespective of the nature of the observed ALK mutants, in every case the activity of the mutant ALK receptors could be abrogated by the ALK inhibitor crizotinib (Xalkori/PF-02341066), albeit with differing levels of sensitivity.
The anaplastic lymphoma kinase (ALK) gene is overexpressed, mutated or amplified in most neuroblastoma (NB), a pediatric neural crest-derived embryonal tumor. The two most frequent mutations, ALK-F1174L and ALK-R1275Q, contribute to NB tumorigenesis in mouse models, and cooperate with MYCN in the oncogenic process. However, the precise role of activating ALK mutations or ALK-wt overexpression in NB tumor initiation needs further clarification.
Human ALK-wt, ALK-F1174L, or ALK-R1275Q were stably expressed in murine neural crest progenitor cells (NCPC), MONC-1 or JoMa1, immortalized with v-Myc or Tamoxifen-inducible Myc-ERT, respectively. While orthotopic implantations of MONC-1 parental cells in nude mice generated various tumor types, such as NB, osteo/chondrosarcoma, and undifferentiated tumors, due to v-Myc oncogenic activity, MONC-1-ALK-F1174L cells only produced undifferentiated tumors. Furthermore, our data represent the first demonstration of ALK-wt transforming capacity, as ALK-wt expression in JoMa1 cells, likewise ALK-F1174L, or ALK-R1275Q, in absence of exogenous Myc-ERT activity, was sufficient to induce the formation of aggressive and undifferentiated neural crest cell-derived tumors, but not to drive NB development. Interestingly, JoMa1-ALK tumors and their derived cell lines upregulated Myc endogenous expression, resulting from ALK activation, and both ALK and Myc activities were necessary to confer tumorigenic properties on tumor-derived JoMa1 cells in vitro.
ALK; neuroblastoma; Myc; tumorigenesis; differentiation
Mutations in the ALK tyrosine kinase receptor gene represent important therapeutic targets in neuroblastoma, yet their clinical translation has been challenging. The ALKF1174L mutation is sensitive to the ALK inhibitor crizotinib only at high doses and mediates acquired resistance to crizotinib in ALK-translocated cancers. We have shown that the combination of crizotinib and an inhibitor of downstream signaling induces a favorable response in transgenic mice bearing ALKF1174L/MYCN-positive neuroblastoma. Here, we investigated the molecular basis of this effect and assessed whether a similar strategy would be effective in ALK-mutated tumors lacking MYCN overexpression. We show that in ALK-mutated, MYCN-amplified neuroblastoma cells, crizotinib alone does not affect mTORC1 activity as indicated by persistent RPS6 phosphorylation. Combined treatment with crizotinib and an ATP-competitive mTOR inhibitor abrogated RPS6 phosphorylation, leading to reduced tumor growth and prolonged survival in ALKF1174L/MYCN-positive models compared to single agent treatment. By contrast, this combination, while inducing mTORC1 downregulation, caused reciprocal upregulation of PI3K activity in ALK-mutated cells expressing wild-type MYCN. Here, an inhibitor with potency against both mTOR and PI3K was more effective in promoting cytotoxicity when combined with crizotinib. Our findings should enable a more precise selection of molecularly targeted agents for patients with ALK-mutated tumors.
ALK; neuroblastoma; crizotinib; mTOR inhibitor; MYCN
Crizotinib, the first FDA-approved ALK inhibitor, showed significant antitumor activity in young patients with anaplastic large-cell lymphoma (ALCL) frequently displaying ALK rearrangement. However, long-term therapeutic benefits of crizotinib are limited due to development of drug resistance. CH5424802—more potent and selective ALK inhibitor—comprises a good candidate for second-line treatment in crizotinib-relapsed patients. The aim of this study was to determine possible mechanisms of resistance to ALK inhibitors that can appear in ALCL patients.
ALK+ ALCL cell lines resistant to crizotinib (Karpas299CR) and to CH5424802 (Karpas299CHR) were established by long-term exposure of Karpas299 cells to these inhibitors. Next, alterations in their sensitivity to ALK, HSP90 and mTOR inhibitors were investigated by cell viability and BrdU incorporation assays and immunoblot analysis.
cDNA sequencing of ALK kinase domain revealed activating mutations—I1171T in Karpas299CR and F1174C in Karpas299CHR. The resistant cells displayed diminished sensitivity to structurally unrelated ALK inhibitors—crizotinib, CH5424802 and TAE684. Nevertheless, CH5424802 and TAE684 were still more potent against the resistant cells than crizotinib. Moreover, Karpas299CR and Karpas299CHR cells remained sensitive to HSP90 or mTOR inhibitors.
Resistance mediated by activating mutations in ALK kinase domain may emerge in ALCL patients during ALK inhibitors treatment. However, more potent second-generation ALK inhibitors, HSP90 or mTOR inhibitors may represent an effective therapy for relapsed ALK+ ALCL patients.
Electronic supplementary material
The online version of this article (doi:10.1007/s00432-014-1589-3) contains supplementary material, which is available to authorized users.
ALK; ALCL; Drug resistance; ALK inhibitors; Crizotinib; CH5424802
In pediatric neuroblastoma (NBL), high anaplastic lymphoma kinase (ALK) levels appear to be correlated with an unfavorable prognosis, regardless of ALK mutation status. This suggests a therapeutic role for ALK inhibitors in NBL patients. We examined the correlation between levels of ALK, phosphorylated ALK (pALK) and downstream signaling proteins and response to ALK inhibition in a large panel of both ALK mutated and wild type (WT) NBL cell lines.
We measured protein levels by western blot and ALK inhibitor sensitivity (TAE684) by viability assays in 19 NBL cell lines of which 6 had a point mutation and 4 an amplification of the ALK gene.
ALK 220 kDa (p = 0.01) and ALK 140 kDa (p = 0.03) protein levels were higher in ALK mutant than WT cell lines. Response to ALK inhibition was significantly correlated with ALK protein levels (p < 0.01). ALK mutant cell lines (n = 4) were 14,9 fold (p < 0,01) more sensitive to ALK inhibition than eight WT cell lines.
NBL cell lines often express ALK at high levels and are responsive to ALK inhibitors. Mutated cell lines express ALK at higher levels, which may define their superior response to ALK inhibition.
Electronic supplementary material
The online version of this article (doi:10.1007/s13402-011-0048-2) contains supplementary material, which is available to authorized users.
ALK; Kinase inhibitor; Neuroblastoma; PHOX2B; TAE684
Activation of the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase is a key oncogenic mechanism in a growing number of tumor types. In the majority of cases, ALK is activated by fusion with a dimerizing partner protein as a result of chromosomal translocation events, most studied in the case of the nucleophosmin–ALK and echinoderm microtubule-associated protein-like 4–ALK oncoproteins. It is now also appreciated that the full-length ALK receptor can be activated by point mutations and by deletions within the extracellular domain, such as those observed in neuroblastoma. Several studies have employed phosphoproteomics approaches to find substrates of ALK fusion proteins. In this study, we used MS-based phosphotyrosine profiling to characterize phosphotyrosine signaling events associated with the full-length ALK receptor. A number of previously identified and novel targets were identified. One of these, signal transducer and activator of transcription 3 (STAT3), has previously been observed to be activated in response to oncogenic ALK signaling, but the significance of this in signaling from the full-length ALK receptor has not been explored further. We show here that activated ALK robustly activates STAT3 on Tyr705 in a number of independent neuroblastoma cell lines. Furthermore, knockdown of STAT3 by RNA interference resulted in a reduction in myelocytomatosis neuroblastom (MYCN) protein levels downstream of ALK signaling. These observations, together with a decreased level of MYCN and inhibition of neuroblastoma cell growth in the presence of STAT3 inhibitors, suggest that activation of STAT3 is important for ALK signaling activity in neuroblastoma.
anaplastic lymphoma kinase; cancer; neuroblastoma; SHP-2; signal transducer and activator of transcription 3 (STAT3)
Activating mutations in the kinase domain of anaplastic lymphoma kinase (ALK) have recently been shown to be an important determinant in the genetics of the childhood tumor neuroblastoma. Here we discuss an in-depth analysis of one of the reported gain-of-function ALK mutations—ALKI1250T—identified in the germ line DNA of one patient. Our analyses were performed in cell culture-based systems and subsequently confirmed in a Drosophila model. The results presented here indicate that the germ line ALKI1250T mutation is most probably not a determinant for tumor initiation or progression and, in contrast, seems to generate a kinase-dead mutation in the ALK receptor tyrosine kinase (RTK). Consistent with this, stimulation with agonist ALK antibodies fails to lead to stimulation of ALKI1250T and we were unable to detect tyrosine phosphorylation under any circumstances. In agreement, ALKI1250T is unable to activate downstream signaling pathways or to mediate neurite outgrowth, in contrast to the activated wild-type ALK receptor or the activating ALKF1174S mutant. Identical results were obtained when the ALKI1250T mutant was expressed in a Drosophila model, confirming the lack of activity of this mutant ALK RTK. We suggest that the ALKI1250T mutation leads to a kinase-dead ALK RTK, in stark contrast to assumed gain-of-function status, with significant implications for patients reported to carry this particular ALK mutation.
The ALK kinase inhibitor crizotinib (PF-02341066) is clinically effective in patients with ALK-translocated cancers, but its efficacy will ultimately be limited by acquired drug resistance. Here we report the identification of a secondary mutation in ALK, F1174L, as one cause of crizotinib resistance in a patient with an inflammatory myofibroblastic tumor (IMT) harbouring a RANBP2-ALK translocation who progressed while crizotinib therapy. When present in cis with an ALK translocation, this mutation (also detected in neuroblastomas) causes an increase in ALK phosphorylation, cell growth and downstream signaling. Furthermore, the F1174L mutation inhibits crizotinib mediated downregulation of ALK signaling and blocks apoptosis in RANBP2-ALK Ba/F3 cells. A chemically distinct ALK inhibitor, TAE684, or the HSP90 inhibitor 17-AAG are both effective in models harbouring the F1174L ALK mutation. Our findings highlight the importance of studying drug resistance mechanisms in order to develop effective clinical treatments for patients with ALK-translocated cancers.
Inflammatory myofibroblastic tumor; Anaplastic lymphoma kinase; kinase inhibitor; drug resistance
Since the original descriptions of gain-of function mutations in anaplastic lymphoma kinase (ALK), interest in the role of this receptor tyrosine kinase in neuroblastoma development and as a potential therapeutic target has escalated. As a group, the activating point mutations in full-length ALK, found in approximately 8% of all neuroblastoma tumors, are distributed evenly across different clinical stages. However, the most frequent somatic mutation, F1174L, is associated with amplification of the MYCN oncogene. This combination of features appears to confer a worse prognosis than MYCN amplification alone, suggesting a cooperative effect on neuroblastoma formation by these two proteins. Indeed, F1174L has shown more potent transforming activity in vivo than the second most common activating mutation, R1275Q, and is responsible for innate and acquired resistance to crizotinib, a clinically relevant ALK inhibitor that will soon be commercially available. These advances cast ALK as a bona fide oncoprotein in neuroblastoma and emphasize the need to understand ALK-mediated signaling in this tumor. This review addresses many of the current issues surrounding the role of ALK in normal development and neuroblastoma pathogenesis, and discusses the prospects for clinically effective targeted treatments based on ALK inhibition.
neuroblastoma; ALK; tyrosine kinase receptor; targeted therapy; crizotinib; drug resistance; point mutations; small molecule inhibitors; combination treatment
Recently, activating mutations of the full length ALK receptor, with two hot spots at positions F1174 and R1275, have been characterized in sporadic cases of neuroblastoma. Here, we report similar basal patterns of ALK phosphorylation between the neuroblastoma IMR-32 cell line, which expresses only the wild-type receptor (ALKWT), and the SH-SY5Y cell line, which exhibits a heterozygous ALK F1174L mutation and expresses both ALKWT and ALKF1174L receptors. We demonstrate that this lack of detectable increased phosphorylation in SH-SY5Y cells is a result of intracellular retention and proteasomal degradation of the mutated receptor. As a consequence, in SH-SY5Y cells, plasma membrane appears strongly enriched for ALKWT whereas both ALKWT and ALKF1174L were present in intracellular compartments. We further explored ALK receptor trafficking by investigating the effect of agonist and antagonist mAb (monoclonal antibodies) on ALK internalization and down-regulation, either in SH-SY5Y cells or in cells expressing only ALKWT. We observe that treatment with agonist mAbs resulted in ALK internalization and lysosomal targeting for receptor degradation. In contrast, antagonist mAb induced ALK internalization and recycling to the plasma membrane. Importantly, we correlate this differential trafficking of ALK in response to mAb with the recruitment of the ubiquitin ligase Cbl and ALK ubiquitylation only after agonist stimulation. This study provides novel insights into the mechanisms regulating ALK trafficking and degradation, showing that various ALK receptor pools are regulated by proteasome or lysosome pathways according to their intracellular localization.
EML4–ALK fusions define a subset of lung cancers that can be effectively treated with anaplastic lymphoma kinase (ALK) inhibitors. Unfortunately, the duration of response is heterogeneous and acquired resistance limits their ultimate efficacy. Thus, a better understanding of resistance mechanisms will help to enhance tumor control in EML4–ALK-positive tumors.
By applying orthogonal functional mutagenesis screening approaches, we screened for mutations inducing resistance to the aminopyridine PF02341066 (crizotinib) and/or the diaminopyrimidine TAE684.
Here, we show that the resistance mutation, L1196M, as well as other crizotinib resistance mutations (F1174L and G1269S), are highly sensitive to the structurally unrelated ALK inhibitor TAE684. In addition, we identified two novel EML4–ALK resistance mutations (L1198P and D1203N), which unlike previously reported mutations, induced resistance to both ALK inhibitors. An independent resistance screen in ALK-mutant neuroblastoma cells yielded the same L1198P resistance mutation but defined two additional mutations conferring resistance to TAE684 but not to PF02341066.
Our results show that different ALK resistance mutations as well as different ALK inhibitors impact the therapeutic efficacy in the setting of EML4–ALK fusions and ALK mutations.
Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs), including crizotinib, are effective treatments in preclinical models and in cancer patients with ALK-translocated cancers. However, their efficacy will ultimately be limited by the development of acquired drug resistance. Here we report two mechanisms of ALK TKI resistance identified from, a crizotinib treated non-small cell lung cancer (NSCLC) patient and in a cell line generated from the resistant tumor (DFCI076), and from studying a resistant version of the ALK TKI (TAE684) sensitive H3122 cell line. The crizotinib resistant DFCI076 cell line, harboured a unique L1152R ALK secondary mutation, and was also resistant to the structurally unrelated ALK TKI TAE684. Although the DFCI076 cell line was still partially dependent on ALK for survival, it also contained concurrent co-activation of epidermal growth factor receptor (EGFR) signalling. In contrast, the TAE684 resistant (TR3) H3122 cell line did not contain an ALK secondary mutation but instead harboured co-activation of EGFR signalling. Dual inhibition of both ALK and EGFR was the most effective therapeutic strategy for the DFCI076 and H3122 TR3 cell lines. We further identified a subset (3/50; 6%) of treatment naïve NSCLC patients with ALK rearrangements that also had concurrent EGFR activating mutations. Our studies identify resistance mechanisms to ALK TKIs mediated by both ALK and by a bypass signalling pathway mediated by EGFR. These mechanisms can occur independently, or in the same cancer, suggesting that the combination of both ALK and EGFR inhibitors may represent an effective therapy for these subsets of NSCLC patients.
Non-small cell lung carcinoma; translocation; kinase inhibitor; drug resistance; mutation
Translocation of the anaplastic lymphoma kinase (ALK) gene is involved in the tumorigenesis of a subset of non-small cell lung carcinomas (NSCLCs) and identifies patients sensitive to ALK inhibitors. ALK copy number changes and amplification, which plays an oncogenic role in tumors such as neuroblastoma, are poorly characterized in NSCLC. We aimed to study the prevalence of ALK copy number changes and their correlation to ALK protein expression, epidermal growth factor receptor (EGFR) status, and clinicopathological data in patients with NSCLC.
ALK status was evaluated by fluorescence in situ hybridization (FISH). Specimens with ALK translocation were studied for echinoderm microtubule-associated protein-like 4 (EML4), KIF5B, and TFG status. ALK expression was assessed by immunohistochemistry. EGFR gene and protein status were evaluated in adenocarcinomas. Survival analysis was performed.
One hundred seven NSCLC cases were evaluated. There were two cases of EML4-ALK translocation and one with an atypical translocation of ALK. Both cases of EML4-ALK translocation had ALK protein expression, whereas in the rest, ALK was undetected. Eleven cases (10%) exhibited ALK amplification and 68 (63%) copy number gains. There was an association between ALK amplification and EGFR FISH positivity (p < 0.0001) but not with prognosis. In conclusion, EML4-ALK translocation is a rare event in NSCLC.
The study reveals a significant frequency of ALK amplification and its association with EGFR FISH positivity in lung adenocarcinomas. Based on these findings, a potential role of ALK amplification in the response to ALK inhibitors alone or combined with EGFR inhibitors in NSCLC merits further studies.
ALK; Non-small cell lung cancer; Amplification; Translocation; Polysomy
Neuroblastoma is a pediatric solid tumor that can be stratified into stroma-rich and stroma-poor histological subgroups. The stromal compartment of neuroblastoma is composed mostly of Schwann cells, and they play critical roles in the differentiation, survival, and angiogenic responses of tumor cells. In certain neuroblastoma cell lines, the coexistence of neuroblastic N-type and substrate-adherent S-type is frequently observed. One such cell line, SK-N-SH, harbors a F1174L oncogenic mutation in the anaplastic lymphoma kinase (ALK) gene. Treatment of SK-N-SH with an ALK chemical inhibitor, TAE684, resulted in the outgrowth of S-type cells that expressed the Schwann cell marker, S100α6. Nucleotide sequencing analysis of these TAE684-resistant (TR) sublines revealed the presence of the ALK F1174L mutation, suggesting their tumor origin, although ALK protein was not detected. Consistent with these findings, TR cells displayed approximately 9-fold higher IC50 values than N-type cells. Also, unlike N-type cells, TR cells have readily detectable phosphorylated STAT3 but weaker phosphorylated AKT. Under coculture conditions, TR cells conferred survival to N-type cells against the apoptotic effect of TAE684. Cocultivation also greatly enhanced the overall phosphorylation of STAT3 and its transcriptional activity in N-type cells. Finally, conditioned medium from TR clones enhanced cell viability of N-type cells, and this effect was phosphatidylinositol 3-kinase dependent. Taken together, these results demonstrate the ability of tumor-derived S-type cells in protecting N-type cells against the apoptotic effect of an ALK kinase inhibitor through upregulating prosurvival signaling.
neuroblastoma; ALK; TAE684; Schwann cells; stromal
Anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase in the insulin receptor superfamily, was initially identified in constitutively activated oncogenic fusion forms – the most common being nucleophosmin-ALK – in anaplastic large-cell lymphomas, and subsequent studies have identified ALK fusions in diffuse large B-cell lymphomas, systemic histiocytosis, inflammatory myofibroblastic tumors, esophageal squamous cell carcinomas and non-small-cell lung carcinomas. More recently, genomic DNA amplification and protein overexpression, as well as activating point mutations, of ALK have been described in neuroblastomas. In addition to those cancers for which a causative role for aberrant ALK activity is well validated, more circumstantial links implicate the full-length, normal ALK receptor in the genesis of other malignancies – including glioblastoma and breast cancer – via a mechanism of receptor activation involving autocrine and/or paracrine growth loops with the reported ALK ligands, pleiotrophin and midkine. This review summarizes normal ALK biology, the confirmed and putative roles of ALK in the development of human cancers and efforts to target ALK using small-molecule kinase inhibitors.
anaplastic large-cell lymphoma; anaplastic lymphoma kinase; esophageal squamous cell carcinoma; glioblastoma; inflammatory myofibroblastic tumor; midkine; neuroblastoma; non-small-cell lung carcinoma; pleiotrophin; targeted cancer therapy; tyrosine kinase inhibitor
The EML4-ALK fusion gene has been detected in ~7% of Japanese non-small cell lung cancers (NSCLC). We determined the frequency of EML4-ALK in Caucasian NSCLCs and in NSCLC cell lines. We also determined whether TAE684, a specific ALK kinase inhibitor, would inhibit the growth of EML4-ALK containing cell lines in vitro and in vivo.
We screened 305 primary NSCLCs (both US (n=138) and Korean (n=167) patients) and 83 NSCLC cell lines using RT-PCR and by exon array analyses. We evaluated the efficacy of TAE684 against NSCLC cell lines in vitro and in vivo.
We detected 4 different variants, including two novel variants, of EML4-ALK using RT-PCR in 8/305 tumors (3%) and in 3/83 (3.6%) NSCLC cell lines. All EML4-ALK containing tumors and cell lines were adenocarcinomas. EML4-ALK was detected more frequently in NSCLC patients who were never or light (< 10 pack years) cigarette smokers compared to current/former smokers (6% vs. 1%; p=0.049). TAE684 inhibited the growth of 1 of 3 (H3122) EML4-ALK containing cell lines in vitro and in vivo, inhibited Akt phosphorylation and caused apoptosis. In another EML4-ALK cell line, DFCI032, TAE684 was ineffective due to co-activation of EGFR and ERBB2. The combination of TAE684 and CL-387,785 (EGFR/ERBB2 kinase inhibitor), inhibited growth and Akt phosphorylation and led to apoptosis in the DFCI032 cell line.
EML4-ALK is found in the minority of NSCLCs. ALK kinase inhibitors alone or in combination may nevertheless be clinically effective treatments for NSCLC patients whose tumors contain EML4-ALK.
Carcinoma, Non-Small-Cell lung; EML4-ALK; ALK; Kinase inhibitor
Activating gene rearrangements of anaplastic lymphoma kinase (ALK) have been identified as driver mutations in non-small-cell lung cancer, inflammatory myofibroblastic tumors, and other cancers. Crizotinib, a dual MET/ALK inhibitor, has demonstrated promising clinical activity in patients with non-small-cell lung cancer and inflammatory myofibroblastic tumors harboring ALK translocations. Inhibitors of driver kinases often elicit kinase domain mutations that confer resistance, and such mutations have been successfully predicted using in vitro mutagenesis screens. Here, this approach was used to discover an extensive set of ALK mutations that can confer resistance to crizotinib. Mutations at 16 residues were identified, structurally clustered into five regions around the kinase active site, which conferred varying degrees of resistance. The screen successfully predicted the L1196M, C1156Y, and F1174L mutations, recently identified in crizotinib-resistant patients. In separate studies, we demonstrated that crizotinib has relatively modest potency in ALK-positive non-small-cell lung cancer cell lines. A more potent ALK inhibitor, TAE684, maintained substantial activity against mutations that conferred resistance to crizotinib. Our study identifies multiple novel mutations in ALK that may confer clinical resistance to crizotinib, suggests that crizotinib's narrow selectivity window may underlie its susceptibility to such resistance and demonstrates that a more potent ALK inhibitor may be effective at overcoming resistance.
crizotinib; EML4-ALK; ENU mutagenesis; gatekeeper; resistance
Survival rates for the childhood cancer neuroblastoma have not substantively improved despite dramatic escalation in chemotherapy intensity. Like most human cancers, this embryonal malignancy can be inherited, but the genetic etiology of familial and sporadically occurring neuroblastoma was largely unknown. Here we show that germline mutations in the anaplastic lymphoma kinase gene (ALK) explain the majority of hereditary neuroblastomas, and that activating mutations can also be somatically acquired. We first identified a significant linkage signal at the short arm of chromosome 2 (maximum nonparametric LOD=4.23 at rs1344063) using a whole-genome scan in neuroblastoma pedigrees. Resequencing of regional candidate genes identified three separate missense mutations in the tyrosine kinase domain of ALK (G1128A, R1192P and R1275Q) that segregated with the disease in eight separate families. Examination of 491 sporadically occurring human neuroblastoma samples showed that the ALK locus was gained in 22.8%, and highly amplified in an additional 3.3%, and that these aberrations were highly associated with death from disease (P=0.0003). Resequencing of 194 high-risk neuroblastoma samples showed somatically acquired mutations within the tyrosine kinase domain in 12.4%. Nine of the ten mutations map to critical regions of the kinase domain and were predicted to be oncogenic drivers with high probability. Mutations resulted in constitutive phosphorylation consistent with activation, and targeted knockdown of ALK mRNA resulted in profound growth inhibition of 4 of 4 cell lines harboring mutant or amplified ALK, as well as 2 of 6 wild type for ALK. Our results demonstrate that heritable mutations of ALK are the major cause of familial neuroblastoma, and that germline or acquired activation of this cell surface kinase is a tractable therapeutic target for this lethal pediatric malignancy.
The echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion gene resulting from an inversion within chromosome 2p occurs in approximately 5% of non-small cell lung cancer and is mutually exclusive with Ras and EGFR mutations. In this study, we have used a potent and selective ALK small molecule inhibitor, NPV-TAE684, to assess the oncogenic role of EML4-ALK in non-small cell lung cancer (NSCLC). We show here that TAE684 inhibits proliferation and induces cell cycle arrest, apoptosis, and tumor regression in two NSCLC models that harbor EML4-ALK fusions. TAE684 inhibits EML4-ALK activation and its downstream signaling including ERK, AKT, and STAT3. We used microarray analysis to carry out targeted pathway studies of gene expression changes in H2228 NSCLC xenograft model after TAE684 treatment and identified a gene signature of EML4-ALK inhibition. The gene signature represents 1210 known human genes, and the top biologic processes represented by these genes are cell cycle, DNA synthesis, cell proliferation, and cell death. We also compared the effect of TAE684 with PF2341066, a c-Met and ALK small molecule inhibitor currently in clinical trial in cancers harboring ALK fusions, and demonstrated that TAE684 is a much more potent inhibitor of EML4-ALK. Our data demonstrate that EML4-ALK plays an important role in the pathogenesis of a subset of NSCLC and provides insight into the mechanism of EML4-ALK inhibition by a small molecule inhibitor.
Neuroblastoma, the most common extra- cranial solid tumor in children, is derived from neural crest cells. Nearly half of patients present with metastatic disease, and have 5-year EFS of less than 50%. New approaches with targeted therapy may improve efficacy without increased toxicity. The current review will evaluate three promising targeted therapies, including 131I-metaiodobenzylguanidine (MIBG), a radiopharmaceutical taken up by the human norepinephrine transporter expressed in 90% of neuroblastomas, immunotherapy with monoclonal antibodies targeting the GD2 ganglioside, expressed on 98% of neuroblastoma cells, and inhibitors of ALK, a tyrosine kinase which is mutated or amplified in approximately 10% of neuroblastoma and expressed on the surface of most neuroblastoma cells. Early phase trials have confirmed the activity of 131I-MIBG in relapsed neuroblastoma, with response rates of about 30%, but the technical aspects of administration of large amounts of radioactivity in young children and the limited access have hindered incorporation into treatment of newly diagnosed patients. Anti-GD2 antibodies have also demonstrated activity in relapsed disease, and a recent phase III randomized trial showed a significant improvement in event-free survival for patients receiving chimeric anti-GD2 (ch14.18) combined with cytokines and isotretinoin after myeloablative consolidation therapy. A recently approved small molecule inhibitor of ALK has promising pre-clinical activity for neuroblastoma, and is currently in phase I and II trials. This is the first agent directed to a specific mutation in neuroblastoma, and marks a new step toward personalized therapy for neuroblastoma. Further clinical development of targeted treatments offers new hope for children with neuroblastoma.
The multitargeted tyrosine kinase inhibitor (TKI) crizotinib is active against ALK translocated non-small-cell lung cancer (NSCLC); however acquired resistance invariably develops over time. ALK mutations have previously been implicated in only a third of resistant tumors. We sought to evaluate alternative mechanisms of resistance and preclinical strategies to overcome these in a cell line driven by EML4-ALK.
We selected the NSCLC cell line NCI-H3122 (H3122: EML4-ALK E13;A20) and derived resistant variants that were able to grow in the presence of 1μM crizotinib. These were analyzed for ALK mutations, sensitivity to crizotinib in combination to other TKIs, and for activation of alternative tyrosine kinases.
All H3122 crizotinib resistant (CR) clones lacked amplification or mutations in the kinase domain of ALK. To evaluate if possible alternative kinases functioned as “bypass” tracks for downstream signaling activation in these resistance cells, we performed of phosho-receptor tyrosine kinase array that demonstrated that CR clones had higher phospho-EGFR signals than H3122 cells before and after exposure to crizotinib. A functional approach of dual ALK TKI (with crizotinib) with combinatory TKI inhibition was used as a secondary screen for possible targets. Crizotinib + erlotinib (reversible EGFR TKI) and crizotinib + afatinib (irreversible EGFR/ERBB2 TKI) were able to inhibit the growth of H3122 CR clones, confirming EGFR activation as a mechanism of resistance. The removal of crizotinib from the culture media re-sensitized CR cells to crizotinib.
We identified activation of EGFR as a mechanism of resistance to crizotinib in preclinical models of ALK translocated NSCLC. If EGFR activation is confirmed as a predominant mechanism of ALK TKI-induced resistance in patient-derived tumors, the use of ALK plus EGFR TKIs could be explored for this important cohort of NSCLCs.
lung cancer; non-small-cell lung cancer; tyrosine kinase; kinase inhibitor; epidermal growth factor receptor; EGFR; EGF; anaplastic lymphoma kinase; ALK; crizotinib; erlotinib; afatinib
Thyroid cancer is the most common endocrine cancer and targeted approaches to treat it pose considerable interest. In this study, we report the discovery of ALK gene mutations in thyroid cancer that may rationalize clinical evaluation of ALK inhibitors in this setting. In undifferentiated anaplastic thyroid cancer (ATC), we identified two novel point mutations in exon 23 of the ALK gene, C3592T and G3602A, with a prevalence of 11.11%, but found no mutations in the matched normal tissues or in well-differentiated thyroid cancers. These two mutations, resulting in L1198F and G1201E amino acid changes, respectively, both reside within the ALK tyrosine kinase domain where they dramatically increased tyrosine kinase activities. Similarly, these mutations heightened the ability of ALK to activate the PI3K/Akt and MAP kinase pathways in established mouse cells. Further investigations demonstrated that these two ALK mutants strongly promoted cell focus formation, anchorage-independent growth, and cell invasion. Similar oncogenic properties were observed in the neuroblastoma-associated ALK mutants K1062M and F1174L, but not in wild-type ALK. Overall, our results reveal two novel gain-of-function mutations of ALK in certain ATCs and they suggest efforts to clinically evaluate the use of ALK kinase inhibitors to treat patients who harbor ATCs with these mutations.
ALK mutation; anaplastic thyroid cancer; anaplastic lymphoma kinase; MAP kinase pathway; PI3K/Akt pathway