The anaplastic lymphoma kinase (ALK) is chromosomally rearranged in a subset of certain cancers, including 2% to 7% of non–small cell lung cancers (NSCLC) and ∼70% of anaplastic large cell lymphomas (ALCL). The ALK kinase inhibitors crizotinib and ceritinib are approved for relapsed ALK+ NSCLC, but acquired resistance to these drugs limits median progression-free survival on average to ∼10 months. Kinase domain mutations are detectable in 25% to 37% of resistant NSCLC samples, with activation of bypass signaling pathways detected frequently with or without concurrent ALK mutations. Here we report that, in contrast to NSCLC cells, drug-resistant ALCL cells show no evidence of bypassing ALK by activating alternate signaling pathways. Instead, drug resistance selected in this setting reflects upregulation of ALK itself. Notably, in the absence of crizotinib or ceritinib, we found that increased ALK signaling rapidly arrested or killed cells, allowing a prolonged control of drug-resistant tumors in vivo with the administration of discontinuous rather than continuous regimens of drug dosing. Furthermore, even when drug resistance mutations were detected in the kinase domain, overexpression of the mutant ALK was toxic to tumor cells. We confirmed these findings derived from human ALCL cells in murine pro-B cells that were transformed to cytokine independence by ectopic expression of an activated NPM–ALK fusion oncoprotein. In summary, our results show how ALK activation functions as a double-edged sword for tumor cell viability, with potential therapeutic implications. Cancer Res; 75(14); 2916–27. ©2015 AACR.
ALK is a receptor tyrosine kinase expressed in the developing nervous system (1, 2). Chromosomal rearrangements in subsets of several cancers result in constitutive expression and activation of its C-terminal kinase domain, which prolifically turns on oncogenic signaling pathways, including JAK3/STAT3, MEK/ERK, and PI3K/AKT (3–6). Addiction of tumor cells to ALK activity in laboratory models and promising activity of ALK inhibition in early-phase clinical trials have made it an intensive target for TKI development in recent years. ALK originally was cloned from the recurrent t(2;5)(p23;q35) chromosomal rearrangement found in anaplastic large-cell lymphoma (ALCL), an uncommon tumor derived from mature T cells (7). The resulting NPM–ALK fusion kinase and other less common ALK fusions drive malignant growth in ∼70% of ALCL cases. Though identified in ALCL 20 years ago, ALK has only become a target for intensive drug development since 2007, when its presence was discovered in a subset of non–small cell lung cancer (NSCLC). Here, activation is again through chromosomal rearrangement creating an expressed and activated fusion kinase, most commonly EML4-ALK (8, 9).
Such rearrangements are present in 2% to 7% of NSCLC cases and led to a 57% response in phase 1 evaluation of the first-in-class ALK TKI crizotinib in relapsed ALK+ NSCLC patients (10). The drug won accelerated limited FDA approval in 2011 and subsequently promoted longer progression-free survival than chemotherapy as first or second-line treatment for metastatic ALK+ NSCLC (11, 12). The second-generation inhibitor ceritinib (LDK378) was approved recently for crizotinib-resistant ALK+ NSCLC patients based on a 58% response rate in phase 1, including 56% response in patients with prior crizotinib exposure (13), and several other ALK TKIs are under development (14). ALCL, meanwhile, which makes up 2% to 5% of non-Hodgkin lymphoma (NHL), is the most common T-cell NHL in children and the third most common in adults. Although ALK+ ALCL has better response to chemotherapy than other peripheral T-cell lymphomas (15), poor prognosis of relapsed or refractory patients and its predilection for younger populations have prompted clinical investigation of ALK tyrosine kinase inhibition in both adults and children with the disease (16, 17).
Studies of resistance to crizotinib in NSCLC identify target-dependent mechanisms such as kinase-domain mutations and fusion-ALK copy-number gains in 34% to 46%; resistant cases also show frequent activation of known alternate NSCLC drivers such as c-KIT, EGFR, or KRAS, both with and without concurrent ALK mutations, whereas many cases are resistant for unclear reasons (14, 18, 19). Here, we studied resistance to ALK-kinase inhibition in ALK+ ALCL using both crizotinib and ceritinib. In contrast to findings in lung cancer, our study identified no ability of ALCL cells to bypass the driver kinase by activation of alternate pathways. Instead, cells upregulated NPM–ALK expression through genomic amplification and other mechanisms, suggesting a nearly absolute addiction to ALK-kinase activity. Strikingly, however, withdrawal of ALK inhibitor from resistant cells causes rapid growth arrest followed by entry into apoptosis, which promoted regression of tumors in vivo.
Materials and Methods
Cell lines and reagents
All lines except FL5.12 were purchased from DSMZ, authenticated by STR-fingerprinting (last tested 11/2013) and mycoplasma tested by PlasmoTest kit (Invivogen; REP-PT1). Culture media for Karpas-299, SU-DHL-1, MAC-2A: RPMI 1640, 10% fetal bovine serum (FBS), penicillin/streptomycin (P/S); SUP-M2: same but 20% FBS; Phoenix: DMEM, 10% FBS, P/S. FL5.12 cells (gift of Hans-Guido Wendel, Memorial Sloan Kettering Cancer Center, New York, NY): RPMI 1640, 10% FBS, P/S, ±10% WeHi-3B supernatant and murine IL3 (400 ρmol/L; eBioscience).
Cell Signaling Technology: ALK (#3333S), pALK-Y1604 (3341S), AKT (#4685S), pAKT-S473 (#4060S), ERK1/2 (#4695S), pERK1/2-T202/Y204 (#4370S), STAT3 (#4904S) and pSTAT-Y705 (#9145S). Sigma: horseradish peroxidase-conjugated anti-rabbit secondary (#A0545), horseradish peroxidase-conjugated anti-mouse secondary (#A9917), α-tubulin (#T9026).
Crizotinib (PF-02341066; #S1068) was purchased from Selleck Chemicals. Ceritinib (LDK378) kindly provided by Novartis.
Long insert whole genome sequencing
Long insert whole genome sequencing was carried out as described (20), utilizing genomic DNA extracted using the AllPrep DNA/RNA Mini Kit (QIAgen #80204).
Nexin reagent (Millipore #4500-0455) by the manufacturer's instructions using the Guava EasyCyte flow cytometer.
Cell viability assays
Cells were seeded at 3,000 per well with serial dilutions of indicated inhibitors. Viability was assessed at 72 hours unless otherwise indicated with Cell Titer Glo reagent (Promega #G7573) by the manufacturer's protocol. Luminescence measured by BioTek Synergy HT plate reader. IC50s were calculated with nonlinear curve-fit regression in GraphPad Prism version 6.
Proliferation assays and imaging
Cells were plated at 100,000/mL on day 0 and counted using Trypan-blue exclusion. Cells were stained with Hoechst (1:5,000; Invitrogen #33342) and imaged using the DeltaVision Core system (Applied Precision) equipped with an Olympus IX71 microscope, a 40× objective (NA 1.35) and a 20× objective (NA 0.70), and a cooled charge-coupled device camera (CoolSNAP HQ2; Photometrics). Images were acquired with softWoRx v1.2 software (Applied Science).
Protein extraction, quantification, and immunoblotting
Cells were seeded at 500,000/mL and incubated as indicated. Proteins were extracted using RIPA (50 mmol/L Tris-HCL pH 8.0, 150 mmol/L NaCl, 1% NP40, 0.5% sodium deoxycholate, and 0.1% SDS), 7× phosphatase inhibitor (Roche #04693159001), Phosphatase Halt (Thermo #78428), and Triton X-100. Proteins were quantified using the BCA Assay (Thermo #23225) with 30 μg loaded per lane for Western blotting. All blots were developed using autoradiography film (GeneMate #F-9023-8×10) after incubation with antibodies indicated above.
Genomic DNA was extracted by AllPrep DNA/RNA Mini Kit (QIAgen #80204). Copy-number probes for NPM1 exon 1 (#Hs01219772_cn), NPM intron 11 (#Hs06724352_cn), ALK intron 1 (#Hs05853206_cn), ALK intron 23 (#Hs04555433_cn), and TERT (#4401633) were purchased from Life Technologies, and assays were conducted per the manufacturer's instructions. Copy-number analysis was by Applied Biosystems Prism 7000 Sequence Detection System. Data were normalized to the housekeeping gene (TERT) and analyzed using the 2−ΔΔCt method.
RNA isolation by RNeasy Mini Kit (QIAgen #74106); cDNA generated by Taqman Reverse Transcriptase Kit (Roche; #N808-0234) on a Bio-Rad T100 Thermal Cycler. qRT-PCR employed an Applied Biosystems Prism 7000 Sequence Detection System with Taqman probes (Life Technologies): NPM–ALK (#Hs03024829_ft), CDKN2A (#Hs00923894_m1), and GAPDH as endogenous control (#Hs02758991_g1). Data were analyzed using the 2−ΔΔCt method.
Targeted selection and sequencing of the expressed NPM1–ALK translocation locus
cDNA was synthesized from 100 ng total RNA using the Nugen Ovation RNA-Seq System v2. Presence of NPM–ALK confirmed by PCR giving a 429bp product (Kapa Biosystem's HiFi Readymix; #KK1006) using previously published primers flanking the breakpoint (21). NPM1–ALK fusion cDNA was then amplified using the QIAgen Long Range PCR Kit (#206401) and custom primers (F1: GTCCGCCTTCTCTCCTACCT, R1: TTGGCACAAAACAAAACGTG) flanking the breakpoint, encompassing 391bp of NPM1 and 1804bp of ALK cDNA. Size selection and purification of the PCR product using Bio-Rad's Freeze ‘N’ Squeeze DNA gel extraction spin columns (#4106139) followed by Beckman Coulter Agencourt AMPure XP Bead purification (#A63880). A total of 50 ng of each PCR product was fragmented to 300bp using the Covaris E210 sonicator, and libraries were constructed using Kapa Biosystems' Hyper Kit (#KK8504) following the manufacturer's protocol. Libraries were equimolarly pooled and sequenced on the Illumina MiSeq for paired 84bp reads using Illumina's MiSeq Reagent Kit v3 (#MS-102-3001). FASTQ files generated from the Illumina MiSeq were aligned against build 37 of the human reference genome using the Burrows-Wheeler Alignment (BWA) tool (22). Following alignment, .sai files were used to create .sam (sequence alignment map) files, which were used to create binary sequence (.bam) files using SAMtools (23). PCR duplicates were flagged for removal using Picard (http://picard.sourceforge.net), and base quality scores were recalibrated using Genome Analysis Toolkit (GATK; ref. 24). Comparisons within each cell line family were performed to identify point mutations and small indels using three somatic callers, including Seurat (25), MuTect (26), and Strelka (27) as well as Sanger sequencing.
Transfections, infections, and selection
Phoenix packaging cells were seeded at 700,000 cells/mL for 16 hours, to which, a cocktail of DMEM, X-treme GENE 9 DNA transfection reagent (Roche #06365787001) and 1 μg MIG-NPM–ALK was added drop-wise. This mixture was incubated for 48 hours to allow production of viral supernatant. A total of 100,000 murine pro-B 5-12 cells were then resuspended in 600 μL of syringe-filtered viral supernatant mixed with 150 μL of a 5× infection solution (WeHi-3B, Polybreen, and interleukin-3). This was repeated a further three times with at least 6 hours between each repeat to allow viral supernatant to reach maximum titer. Cells were then plated in RPMI 1640 media supplemented with 10% FBS, P/S, 10% WeHi-3B supernatant, and interleukin-3 for 24 hours and assessed by FACS (using the Guava EasyCyte flow cytometer) for GFP levels as a mark of initial infection. Cytokine withdrawal was carried out by washing cells in RPMI 1640 media supplemented with 10% FBS and P/S four times and subsequently plating them in this cytokine-free media with 1:1,000 DMSO or the indicated ceritinib concentrations. FACS plots were analyzed using FlowJo version 10.
All mouse experiments were approved by the University of Arizona Animal Care and Use Committee (protocol no. 12-377). Mice were maintained under specific pathogen-free conditions, and food and water were provided ad libitum.
In vivo dependence
Severe combined immunodeficiency (SCID) mice were injected with 2 × 106 K299-CR1000 cells in 1:1 Matrigel and sterile saline in a total volume of 100 μL subcutaneously into the lower flank. These mice were divided immediately to two groups for treatment with ceritinib or vehicle by oral gavage. Ceritinib was formulated freshly before each dosing as a solution in 0.5% MC (methylcellulose)/0.5% Tween 80 as described (28). Because of the requirement for ALK inhibition for K299-CR1000 cells in vitro, dosing began on the day of flank injections 2 hours before hand and continued daily.
Up-front intermittent versus continuous dosing
SCID mice were injected with 2 × 106 Karpas-299 parental cells as above. After tumors reached ∼500 mm3, the mice were split into seven cohorts (n = 3) and were treated continuously with vehicle, continuously with ceritinib (at either 33.33 or 50 mg/kg) or intermittently with the same concentrations of ceritinib using a ‘4 weeks on, 2 weeks off’ schedule.
Two-tailed Student t test was carried out for all expression data using the GraphPad t test calculator and verified using the SPSS Statistics software from IBM, with P < 0.05 considered statistically significant with a 95% confidence interval. Apoptosis data were calculated using the Mann–Whitney test in GraphPad where P = 0.05 was considered statistically significant with a 95% confidence interval.
TKI resistance and dependence are invariably coselected in ALK+ ALCL
NPM–ALK-driven ALCL cell lines Karpas-299, SU-DHL-1, and SUP-M2 were highly sensitive to crizotinib and ceritinib, with the latter drug having 3 to 5× greater potency (Fig. 1A; Supplementary Table S1). We selected for resistance to both inhibitors independently through serial plating in increasing drug concentrations (Supplementary Fig. S1A). Cells derived from Karpas-299 able to grow in 1,000 nmol/L crizotinib (hereafter K299-CR1000) strikingly when washed out of ALK inhibitor and reassessed for drug sensitivity showed viability stimulation by both inhibitors (Fig. 1B). Stimulation was an on-target effect, indicated by the stimulation at lower concentration by the more potent inhibitor. All resistant clones emerging from selections demonstrated similar findings [Fig. 1C, lines named for parent, drug used in selection (L for ceritinib/LDK378), and top nanomolar concentration]. When washed out of inhibitor and plated in drug-free media, resistant cells halted growth, suggesting ALK inhibition had become a requirement for viability (Fig. 1D–E). Plated without ALK inhibitor, resistant lines appeared shrunken and involuted, consistent with onset of apoptosis, while those in either ALK inhibitor appeared viable similar to parent cells growing without drug (Fig. 1F; Supplementary Fig. S1B). Flow cytometry confirmed entry into apoptosis by resistant cells plated in drug-free media, in contrast to those maintained in either ALK inhibitor (Fig. 1G). ALK+ ALCL cells therefore reliably develop ALK-inhibitor dependence at the same time they develop resistance, suggesting selection for a process with paradoxical effects on viability.
NPM–ALK upregulation drives both resistance and dependence
To investigate the resistant-dependent phenotype, we performed long-insert whole-genome sequencing (LI-WGS; ref. 20) of the Karpas-299 parent line and the K299-CR1000 subclone. Mapping of reads against the reference genome (hg19) showed amplification in the subclone of the portions of the NPM and ALK loci that are translocated to form NPM–ALK (Fig. 2A). Copy-number assays demonstrated gain consistent with genomic amplification specifically of the fusion locus but not the uninvolved portions of either gene (Fig. 2B). This generated more than 50-fold increased expression of NPM–ALK mRNA (Fig. 2C). The additional resistant-dependent subclones also had increased expression relative to their corresponding parent lines (Fig. 2D), ranging from 1.5× for SUP-LR150 to 12.0× for K299-LR150. Of these, only K299-LR150 had clear evidence of NPM–ALK copy-number gain (Supplementary Fig. S2), demonstrating increased expression results also from other mechanisms. NPM–ALK protein levels also were elevated in subclones (Fig. 2E). This is seen at NPM–ALK's full-length position (80 kDa) and at a prominent high molecular weight aggregate band known to result from elevated NPM–ALK protein levels (29). Activating ALK auto-phosphorylation, highly drug sensitive in parent lines, was drug resistant in subclones during TKI exposure. STAT3 activation previously was shown to be required for ALK-mediated lymphomagenesis (30). In addition, the gene-expression signature of clinical cases is most similar to that of a STAT3 survival signature (31). We found all resistant subclones preserved STAT3 phosphorylation during ALK inhibitor exposure in marked contrast to the drug-sensitive parent lines (Fig. 2E). Subclones withdrawn from drug showed increase in ALK and STAT3 phosphorylation but, interestingly, slight but noticeable decline in total STAT3, suggesting feedback repression of STAT3 expression. In sum, increased ALK expression drives resistance associated with preserved STAT3 activation but leads to increased ALK signaling when drug is withdrawn. This combined with the rescue of cell viability by ALK inhibition (Fig. 1) supports a conclusion that ALK overdose is the reason for growth arrest and apoptosis upon inhibitor withdrawal.
We also performed both Sanger and targeted deep sequencing of the NPM–ALK fusion locus (Supplementary Table S2). Neither K299-CR1000 nor K299-LR150 cells developed any second-site kinase-domain mutations. Resistance in these lines therefore was driven purely by increased NPM–ALK levels. The same may be true of DHL1-CR500, which acquired only R1192P, a germline predisposition allele for neuroblastoma with no known role in TKI resistance (32). Intriguingly, both SUP-CR500 and SUP-LR150 developed mutations with known roles in TKI resistance (respectively I1171S, seen only by Sanger sequencing, and F1174L, seen by both targeted and Sanger sequencing; Supplementary Table S2; refs. 33–36). The fact that these lines also developed ALK upregulation leading to TKI dependence to avoid signaling overdose further illustrates the intense pressure for these systems to acquire target-dependent resistance. Moreover, a treatment strategy designed to exploit ALK overdose such as discontinuous dosing may be at least temporarily successful even when drug-specific kinase-domain mutations also have begun to arise.
ALK overdose also impairs the fitness of cytokine-dependent pro-B cells
To replicate resistance-dependence in an independent system, we employed the FL5.12 line, murine pro-B cells that rapidly cease proliferating and undergo apoptosis without IL3 (37). We created NPM–ALK-dependent FL5.12 lines through retroviral introduction of NPM–ALK with GFP coexpression followed by IL3 withdrawal. We hypothesized simultaneous IL3 withdrawal and ALK-inhibitor exposure would require progressively higher NPM–ALK levels to permit survival in the presence of drug. Figure 3A shows representative percentage GFP enrichment in one of three replicate experiments 4 days after IL3 withdrawal and incubation in 0, 10, 20, 50, 150, or 500 nmol/L ceritinib. Confirming NPM–ALK dependence, cells withdrawn in the two highest concentrations always died, whereas the others proliferated only as 100% GFP+ lines. In addition, cells infected with empty vector invariably perished after IL3 withdrawal (not shown). Flow-cytometry analysis showed progressively brighter GFP coexpression in the transformed cells selected and maintained in higher ceritinib concentrations (Fig. 3B), and qPCR confirmed increased NPM–ALK mRNA (Fig. 3C). By immunoblot, we observed progressively higher NPM–ALK total protein and phosphorylation, showing again selection for increased ALK expression and activity to permit survival of NPM–ALK-dependent cells during TKI exposure (Fig. 3D). We then washed cells selected and maintained in 50 nmol/L ceritinib out of drug, plated them in drug-free media, and compared their proliferation with the same cells maintained in drug. The cells deprived of drug had significantly reduced proliferation (Fig. 3E). Therefore, an independent system confirmed both selection for increased NPM–ALK expression to mediate TKI resistance and the negative impact on cell fitness of this increased expression in TKI absence.
Cells return NPM–ALK to basal levels to permit growth without ALK inhibition
To identify factors permitting resistant-dependent cells to resume growth without ALK inhibition, we attempted to derive lines with this phenotype in vitro. Invariably, plating resistant cells at <106/mL without ALK inhibitor resulted in complete death of the culture plate (data not shown). At concentrations ≥106/mL, however, we eventually isolated clones from resistant-dependent lines able to again grow in drug-free media (Supplementary Fig. S1, range 4–56 days). Designated RS for resensitized (K299-CRS, K299-LRS, SUP-CRS, and SUP-LRS), these lines lacked viability stimulation by either ALK inhibitor (Fig. 4A). Proliferation assays showed ability to grow again without drug but resensitization to both ALK inhibitors in all RS lines (Fig. 4B). The RS lines, like parent cells, were less apoptotic in drug-free media than in either ALK inhibitor (Fig. 4C). These clones therefore lost ability to proliferate in ALK TKIs as they regained ability to grow without drug. [Note: ALK TKI resistance mutations detected in SUP-CRS and SUP-LRS (Supplementary Table S2), which likely shifted their viability curves compared with parent cells in Fig. 4A, were not sufficient to restore proliferation or avoid apoptosis during drug exposure.] LI-WGS of K299-CRS showed no genomic amplification of NPM–ALK (Fig. 4D), and copy-number assays showed K299-CRS and K299-LRS have the NPM–ALK translocation at the same copy-number as the original parent (Fig. 4E). The RS subclones all returned NPM–ALK mRNA (Fig. 4G) and protein (Supplementary Fig. S3) to approximately the same levels as those in the corresponding original parent cells. Return of ALK activity to baseline therefore appears necessary for resistant subclones to resume proliferation in an absence of an ALK inhibitor. Simultaneous resensitization to drug that results from this return to baseline strongly validates the conclusion that ALK overdose drives both resistance to and dependence on TKIs.
We also re-selected K299-CRS cells for crizotinib resistance and obtained a secondary resistant clone able to grow at 1,000 nmol/L (K299-CR1000-2). Strikingly, the resistance selection took just as long as the original selection of Karpas-299 cells and again resulted in NPM–ALK copy-number gain, increased mRNA expression, and the resistant-dependent phenotype (Supplementary Fig. S4). Again no mutations were acquired within the ALK kinase domain (Supplementary Table S2). This further demonstrates the great difficulty ALK+ ALCL cells have in gaining an ALK-independent means of survival.
ALK overdose prevents tumor engraftment without TKI treatment in vivo and permits prolonged tumor control through discontinuous dosing
Our data suggest withdrawal of drug from ALK+ ALCL patients with TKI resistance due to target upregulation may promote tumor regression due to ALK overdose. We therefore xenografted K299-CR1000 cells to SCID mice, immediately began treatment with vehicle or ceritinib (50 mg/kg, n = 5 per group), and followed tumor volume (Fig. 5A). No vehicle-treated animals experienced tumor engraftment, whereas three of five ceritinib animals did. On day 62, we discontinued ceritinib treatment, resulting in tumor regression in all engrafted mice (Fig. 5B). Tumors began growing again, however, after about 25 days. We followed them until the tumor burden of one mouse approached 2,000 mm3, at which point we took tumor samples for in vitro culture and then resumed ceritinib therapy. We successfully cultured the cells from two of the three mice. Strikingly, cells from both animals had all the characteristics of the in vitro RS subclones (Fig. 4), including no viability stimulation by either ALK TKI (Fig. 5C), ability to proliferate in vehicle but not either ALK inhibitor (Fig. 5D), loss of the NPM–ALK copy-number gain (Fig. 5E), and return of NPM–ALK mRNA expression to the baseline of the original Karpass-299 parent line (Fig. 5F). Consistent with resensitization, ceritinib resumption halted the growth of tumors in all three mice, with two experiencing tumor reduction over the next 22 days when the experiment ended (Fig. 5B). The third tumor began growing again after 12 days despite the ongoing ceritinib therapy. These results show proof of principle that discontinuous dosing in vivo can take cells through full cycles of ALK TKI dependence and sensitivity and can prolong tumor control in host animals bearing tumors with resistance driven by ALK overexpression. Up-front comparison of intermittent versus continuous in SCID mice engrafted with parental Karpas-299 xenografts did not generate relapses in any group at effective treatment doses (Supplementary Fig. S5), however, showing a need to interrogate ALK expression in resistant tumors before attempting discontinuous dosing (see Discussion).
ALK-kinase inhibition is an important new treatment modality for the subset of NSCLC cases containing ALK rearrangements, but median progression-free survival has ranged from 7 to 10 months during treatment with crizotinib or ceritinib (10, 13, 38). Target-independent resistance is common, as target-dependent mechanisms such second-site kinase-domain mutations are detectable in fewer than half of resistant samples and even then activation of alternate signaling may also be present (14, 18, 19). The small number of ALK+ ALCL patients treated to date with ALK TKIs has not permitted determination of typical progression-free survival, and resistant clinical samples are not yet available for assessment (16, 17). In this study, we have attempted to predict resistance pathways using laboratory-based selections, an approach that has identified clinically relevant reasons for targeted drug failure in numerous cancer types (39–42).
Our results suggest a much stronger addiction to ALK signaling in ALCL than observed in NSCLC. Multiple-independent selections performed with both crizotinib and ceritinib resulted in upregulation of NPM–ALK to permit survival in the presence of inhibitor, and there was no clear evidence of alternate signaling able to bypass it. Even re-selection of cells already selected for resistance once, but which lost resistance in order to avoid the toxic effects of ALK overdose, again generated ALK upregulation (Supplementary Fig. S4). All resistant lines preserved ALK activation in the presence of inhibitor, though preservation was stronger in some than in others (K299 subclones vs. the others in Fig. 2E), whereas STAT3 activation was strongly preserved in all. It is therefore possible additional mechanisms preserving STAT3 activation arose in some cases, though this was not observed after lines were reselected for ability to grow without inhibitor. Either way, we find the effect of ALK signaling intensity on cell survival exists as an inverted U-shaped curve, which we represent schematically in Fig. 6. NPM–ALK overexpression associated with genomic amplification in the case of resistant clones derived from Karpas-299 and without amplification in other clones. The consistent results with Karpas-299 suggest this line might have a subpopulation with NPM–ALK amplification at baseline. Such population is then most able to upregulate expression from a tolerable prosurvival baseline to the levels necessary to overcome high concentrations of inhibitor. Even in the resistant NPM–ALK-amplified K299-CR1000 clone, however, a subpopulation without the amplification appears to persist, because it reemerges upon selection for ability to grow again without inhibitor. Another possibility is that the amplification arises extra-chromosomally (e.g., as a double-minute chromosome), which is then lost through chromosomal segregation during selection for growth without inhibitor. Either way, the overexpression without amplification in other resistant clones show additional genetic and/or epigenetic mechanisms can arise to drive overexpression. Gene-expression profiling, unbiased interrogation of signaling through phosphoproteomics, and subsequent functional studies are needed to fully determine these mechanisms and also the mechanisms by which ALK-overdose leads to cell death.
Consistent and reproducible selection for increased NPM–ALK in multiple models of ALK+ ALCL we believe may be therapeutically exploitable, with potential application to additional malignancies in which ALK has an established or potential role (4). Therapeutic strategies of inhibitor withdrawal from patients with resistant tumors and up-front schedules of intermittent dosing are approaches carrying far less toxicity and expense than almost any other strategy aimed at overcoming resistance. Our results (Fig. 5) support the former in cases when ALK overexpression is suspected or, preferably, can be specifically detected. Unlike similar results reported recently for BRAF-driven melanoma (43); however, we did not prolong control in the up-front setting with intermittent dosing (Supplementary Fig. S5). These results, however, do not fully interrogate the strategy since no relapses were observed, which is out of line with expected patient experience, especially in those receiving ALK inhibitors as salvage therapy. Major progress toward producing a phenotypically accurate murine model of ALK+ ALCL came recently with demonstration that CRISPR/CAS9-mediated chromosomal breaks can generate NPM–ALK in murine fibroblasts (44). The same report showed retroviral introduction of guide strands generating breaks for EML4–ALK into mouse lungs generated a highly accurate model of ALK+ NSCLC. Efforts are underway by several groups including ours to apply these techniques in generation of a new model for ALK+ ALCL. Evaluation of up-front intermittent dosing and discontinuous dosing in the setting of acquired resistance when such a system becomes available are justified by our findings. It also will permit studies of the mechanisms of ALK-overdose toxicity and of non–amplification-driven NPM–ALK overexpression in a physiologically accurate immunocompetent model system.
Important questions raised by our data include those of broader applicability. In lung cancer, EML4-ALK copy-number gain and increased expression are reported in association with crizotinib resistance (18, 19). There are recent case reports of ALK+ NSCLC patients who became refractory to crizotinib obtaining secondary responses upon re-challenge after a period off the drug (45, 46). Both patients, however, received chemotherapy upon initial crizotinib discontinuation, so we do not know if ALK overdose was present or might have led to spontaneous regressions if they had been observed without therapy. Another case series found continuing crizotinib treatment despite progression led to higher performance status than discontinuation (47), but no study has addressed the question of whether therapy interruption may benefit some patients based on the particular resistance mechanism(s) present in their tumors. In other cancers with known or potential ALK activation, evaluation of ALK inhibition is at early stages with results largely unreported. We encourage careful evaluation for changes in ALK signaling dosage during ALK inhibitor therapy in ongoing and planned trials. Patients who come off therapy and subsequently experience regression should be assessed for the potential role of ALK overdose in these findings.
In sum, ALK activity can exist as a double-edged sword for cancer cells. Those tumors that are most addicted to its activities for continued proliferation correspondingly are most prone to select for higher expression to bypass its inhibition. Interruption of inhibition would then provide a new way of prolonging tumor control as the other edge of the sword comes into play.
Disclosure of Potential Conflicts of Interest
S.D. Puvvada has received speakers bureau honoraria from Seattle Genetics and is a consultant/advisory board member for Pharmacyclics. No potential conflicts of interest were disclosed by the other authors.
Conception and design: A.D. Amin, L. Cuyugan, J.H. Schatz
Development of methodology: A.D. Amin, W.S. Liang, M.J. Groysman, J. Adkins, Y.A. Lussier, J.H. Schatz
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A.D. Amin, S.S. Rajan, W.S. Liang, P. Pongtornpipat, M.J. Groysman, E.O. Tapia, T.L. Peters, L. Cuyugan, J. Adkins, L.M. Rimsza, J.H. Schatz
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): A.D. Amin, W.S. Liang, M.J. Groysman, Y.A. Lussier, J.H. Schatz
Writing, review, and/or revision of the manuscript: A.D. Amin, M.J. Groysman, L.M. Rimsza, Y.A. Lussier, S.D. Puvvada, J.H. Schatz
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A.D. Amin, P. Pongtornpipat, Y.A. Lussier
Study supervision: A.D. Amin, J.H. Schatz
Other (assistance in obtaining drug): S.D. Puvvada
The authors thank Emanuela Colombo (University of Milano, Milan, Italy) for generously sending the pMIG-NPM–ALK plasmid to us. The authors also thank Denise Olivas-Castillo and Shobana Sekar (Translational Genomics Research Institute, Phoenix, AZ) for their technical expertise for the targeted selection and sequencing experiments. The authors especially thank Bethany Skovan, Gillian Paine-Murrieta, and Erica Sontz (Experimental Mouse Shared Service, University of Arizona, Tucson, AZ) for their help and support in conducting any in vivo studies.
This work was supported by the NIH/NCI (1R01CA190696-01; J.H. Schatz), University of Arizona Bio5 Institute and Cancer Center (J.H. Schatz), the Lymphoma Research Foundation (J.H. Schatz), and Translational Genomics Research Institute (W.S. Liang, L. Cuyugan, J. Adkins).
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.