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) harboring a RANBP2-ALK translocation who progressed while on 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, and the HSP90 inhibitor 17-AAG are both effective in models harboring 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. Cancer Res; 70(24); 10038–43. ©2010 AACR.
Chromosomal translocations involving the anaplastic lymphoma kinase (ALK) have been detected in several human malignancies including in anaplastic large cell lymphomas (ALCL), inflammatory myofibroblastic tumors (IMT), and non–small cell lung carcinomas (NSCLC; refs. 1–3). ALK translocated fusion proteins include the entire ALK kinase domain and lead to constitutive ALK kinase activity and oncogenic transformation both in vitro and in vivo (3). Somatic mutations in the ALK kinase domain, including at position F1174L, have been detected in neuroblastomas and are also transforming in vitro and in vivo (4, 5). These findings have led to preclinical and clinical development of ALK kinase inhibitors including PF-2341066 (crizotinib; ref. 6). ALK kinase inhibitors lead to apoptosis in vitro and tumor shrinkage in mouse models of EML4-ALK NSCLC (7, 8). To date, significant clinical activity, including tumor shrinkage in 60% of patients, has been observed in a phase I trial of crizotinib in EML4-ALK NSCLC (9). Furthermore, clinical efficacy of crizotinib has been observed in an IMT patient harboring an ALK translocation (10). However, despite these dramatic effects, as with other kinase inhibitors, drug resistance (herein termed acquired resistance) to ALK kinase inhibitors is likely to emerge. An understanding of the acquired resistance mechanisms will be important for the development of additional ALK kinase inhibitors and/or combination therapeutic strategies.
The patients were treated in a clinical trial that was sponsored by Pfizer. Tumor biopsies were obtained under an IRB approved protocol. Both patients provided written informed consent. Total RNA was isolated using Trizol (Invitrogen) and purified using RNeasy minielute cleanup kit (Qiagen).
ALK genomic analyses
The ALK kinase domain was sequenced from all of the available specimens. Exon 23 of ALK was amplified from DNA using exon-specific primers, PCR products cloned into a TOPOTA vector (Invitrogen), transformed into bacteria, and individual clones sequenced. The PCR primers and conditions are available upon request. ALK fluorescence in situ hybridization (FISH) was performed using the break apart probes (Vysis LSI ALK Dual Color) as previously described (11).
Expression constructs and cell culture
The full length RANBP2-ALK cDNA from patient A and the EML4-ALK (variant 1) cDNA from the H3122 cell line were cloned into pDNR-Dual (BD Biosciences). The F1174L mutation was introduced using site-directed mutagenesis (Stratagene) with mutant-specific primers according to the manufacturer's instructions and as previously described (12). All constructs were confirmed by DNA sequencing. Retroviral infection and culture of Ba/F3 cell were performed using previously described methods (12). Polyclonal cell lines were established by puromycin selection and subsequently cultured in the absence of interleukin-3 (IL-3). Uninfected Ba/F3 cells or those expressing EGFR delE746_A750 or the JP1536 empty vector were used as controls.
Antibodies and Western blotting
Cell lysis, Western blotting, and immunoblotting were performed as previously described (12). Anti-phospho-ALK (DF53), anti-phospho-Akt (Ser-473), and anti-total-Akt were obtained from Cell Signaling Technology. Total ERK1/2 and phospho-ERK1/2 (pT185/pY187) antibodies were from Invitrogen. Immunoprecipitations were performed using anti-Flag-M2 agarose (Sigma-Aldrich). ALK immunohistochemistry (IHC) was performed using the mouse monoclonal antihuman CD246 (clone: ALK1, DAKO) as previously described (11).
Cell proliferation and growth assays
Crizotinib was provided by Pfizer. TAE684 was synthesized as previously described (7). Growth and inhibition of growth were assessed by MTS assay according to previously established methods (12). All experimental points were set up in 6 to 12 wells and all experiments were repeated at least 3 times.
Results and Discussion
We identified 2 patients with ALK-translocated cancers that developed clinical acquired resistance to crizotinib. Patient A, with IMT, achieved a partial response with crizotinib therapy lasting 8 months but subsequently developed regrowth of several tumor lesions and had these surgically removed (Supplementary Table S1; see ref. 10). Both pre- and posttreatment tumor specimens had evidence of viable tumor, expressed ALK by IHC, and contained an ALK translocation (Fig. 1A). There was no evidence of ALK amplification in the posttreatment tumor (data not shown). This patient's tumor was known to harbor the RANBP2-ALK translocation (10). Sequencing of the entire ALK kinase domain demonstrated that one of the clinically progressing tumor lesions contained a F1174L mutation (Fig. 1B). This was not detected by direct sequencing, or by cloning and sequencing of individual clones, in the 2 other progressing lesions, or by direct sequencing in two other tumors that were clinically stable at the time of surgery (Supplementary Table S1). Furthermore, it was not detected in the pretreatment tumor specimen even by cloning and sequencing of individual clones (Supplementary Table S1). Patient B, with EML4-ALK NSCLC, achieved a partial response with crizotinib treatment but developed acquired resistance following 5 months of therapy. At the time of progression, a liver biopsy of a growing lesion was performed (Supplementary Table S1). EML4-ALK variant 1 was identified by RT-PCR, but no secondary mutations in ALK were detected in the acquired resistance tumor specimen.
We next evaluated the biologic impact of the F1174L mutation. Both RANPB2-ALK and RANBP-ALK F1174L led to IL-3 independent growth of Ba/F3 cells (Fig. 2A) but the growth was faster in the presence of the F1174L mutation. This increased growth rate was mirrored by a greater baseline ALK phosphorylation of RANBP2-ALK F1174L compared with RANBP2-ALK (Fig. 2B) and by increased downstream AKT and ERK 1/2 phosphorylation (Fig. 2C). The RANB2-ALK F1174L cells were significantly more resistant to crizotinib (Fig. 2D) and the F1174L mutation diminished crizotinib-mediated inhibition of ALK signaling and blocked apoptosis (Supplementary Fig. S1A and B). We also introduced the F1174L mutation into the background of EML4-ALK found in NSCLC (3). Similar to RANPB2-ALK, the EML4-ALK F1174L Ba/F3 cells grew faster (Fig. 3A), had a greater baseline ALK phosphorylation (Fig. 3B), and were more resistant to crizotinib growth inhibition than EML4-ALK Ba/F3 cells (Fig. 3C). Consistent with these findings on growth, greater concentrations of crizotinib were required to inhibit ALK phosphorylation in the EML4-ALK F1174L cells compared with those with EML4-ALK alone (Fig. 3B). Collectively, our studies demonstrate that the F1174L mutation imparts both biologic and drug resistance properties to cancers harboring ALK translocations. Furthermore, patients with neuroblastoma harboring the F1174L ALK mutation may have a transient or no clinical benefit from crizotinib treatment using the current dosing schedules (13).
Crizotinib is administered continuously daily (250 mg BID), reaching a median through plasma concentration of 57 nmol/L of free drug and is clinically effective at this dosing in ALK rearranged IMT and NSCLC (9, 10, 13). Our preclinical studies suggest that higher doses of crizotinib could be used to overcome the F1174L-mediated resistance mechanism (Fig. 2D). This could potentially be achieved using intermittent administration of higher doses of crizotinib, to achieve a higher Cmax, sufficient to inhibit ALK phosphorylation in the presence of F1174L. Similarly, some imatinib resistance mutations (including F359V, M244V, Q252H, and E355G), many of which effect the conformational change in ABL, cause a relative drug resistance that can be overcome by higher drug doses in vitro and in some cases clinically by using higher doses of imatinib (14, 15). In order to develop additional therapeutic strategies, we evaluated a structurally unrelated ALK kinase inhibitor, TAE684, and the heat shock protein (HSP) 90 inhibitor 17-AAG in the crizotinib resistant models (7, 16). Although the F1174L mutation slightly increased the IC50 for TAE684 against RANBP-ALK Ba/F3 cells (59 nmol/L with F1174L; 22 nmol/L without), the IC50 was still substantially below the concentrations of crizotinib (IC50 200 nmol/L) required to inhibit growth and ALK phosphorylation in the parental RANBP-ALK Ba/F3 cells (Figs. 2B and D and 4A). Similar findings were observed for EML4-ALK Ba/F3 cells (Fig. 4A and data not shown). The effects of TAE684 on growth were also mirrored at the level of ALK phosphorylation (Fig. 4B). Recent clinical studies have identified antitumor activity of the HSP90 inhibitor IPI-504 in ALK-translocated NSCLC (17). We thus evaluated the effects of the HSP90 inhibitor 17-AAG in models harboring the F1174L mutation (Fig. 4C). Ba/F3 cells with and without F1174L were equally sensitive to 17-AAG in vitro (Fig. 4C). As many ALK inhibitors are in preclinical development and several HSP90 inhibitors currently undergoing clinical development, our findings provide direct therapeutic strategies for patients that develop crizotinib resistance.
The structural basis for the crizotinib resistance of the F1174L mutation is not entirely clear. Examination of the recently published crystal structure of ALK in an inactive conformation reveals that the F1174L mutation is not in direct contact with the ATP-binding pocket, where both crizotinib and TAE684 are expected to bind (Fig. 4D; see refs. 18, 19). Thus, the F1174L mutation is unlikely to confer resistance via direct steric interactions. Crizotinib is known to bind an inactive conformation of MET (20) and a recently released crystal structure in complex with ALK indicates that it binds a distinct inactive conformation in ALK (PDB ID 2XP2). The activating F1174L mutation must promote the active conformation of the kinase and therefore may disfavor binding of crizotinib analogous to some imatinib resistance mutations in ABL (14). A more definitive understanding of the mechanism of resistance, and the differential effect of the mutation on crizotinib versus TAE684, will require detailed binding and structural studies of these inhibitors with the F1174L mutant.
This study highlights the need to study drug resistance mechanisms from cancer patients treated with kinase inhibitors. The molecular, cellular, and structural understanding of drug resistance mechanisms will continue to reveal therapeutic insights for the development of future anticancer therapies.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
This study was supported by NIHR01CA136851 (N.S. Gray and P.A. Jänne) and R01CA135257 (P.A. Jänne) and by the NCI Lung SPORE P50CA090578 (P.A. Jänne and G.I. Shapiro).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
- Received August 11, 2010.
- Revision received October 12, 2010.
- Accepted October 20, 2010.
- ©2010 American Association for Cancer Research.