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Therapeutic strategies to overcome crizotinib resistance in non-small cell lung cancers harboring the fusion oncogene EML4-ALK

Ryohei Katayamaa,b, Tahsin M. Khana,c, Cyril Benesa,b, Eugene Lifshitsa, Hiromichi Ebia,b, Victor M. Riverad, William C. Shakespeared, A. John Iafrateb,e, Jeffrey A. Engelmana,b,1, and Alice T. Shawa,b,c,1

aMassachusetts General Hospital Cancer Center, Boston, MA 02129; bDepartment of Medicine, Harvard Medical School, Boston, MA 02115; cKoch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139; dARIAD Pharmaceuticals, Cambridge, MA 02139; and eDepartment of Pathology, Massachusetts General Hospital, Boston, MA 02114

Edited by Peter K. Vogt, The Scripps Research Institute, La Jolla, CA, and approved March 23, 2011 (received for review December 27, 2010) The echinoderm microtubule-associated protein-like 4 (EML4)- tinib in this disease. The paradigm of acquired TKI resistance has anaplastic (ALK) fusion oncogene represents been seen with other successes, including epi- a molecular target in a small subset of non-small cell lung cancers dermal receptor (EGFR) mutant lung cancers and (NSCLCs). This fusion leads to constitutive ALK activation with v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutant potent transforming activity. In a pivotal phase 1 clinical trial, the melanomas (9–16). Similar to the experience with crizotinib, ALK inhibitor (TKI) crizotinib (PF-02341066) demon- sensitive cancers ultimately develop resistance, usually within 1 y. strated impressive antitumor activity in the majority of patients For example, most patients with EGFR-mutant NSCLC will have with NSCLC harboring ALK fusions. However, despite these remark- disease progression after 9–12 mo of EGFR TKI monotherapy. able initial responses, cancers eventually develop resistance to cri- In about one-half of cases, resistance arises due to the acquisition zotinib, usually within 1 y, thereby limiting the potential clinical of a secondary gatekeeper mutation (T790M) within the EGFR benefit. To determine how cancers acquire resistance to ALK inhib- (13, 15, 16). This mutation leads to substitution of a threonine in itors, we established a model of acquired resistance to crizotinib by the ATP-binding pocket with a bulky methionine residue, which – – exposing a highly sensitive EML4-ALK positive NSCLC cell line to hinders drug binding. In about 10 20% of cases, resistance arises MEDICAL SCIENCES increasing doses of crizotinib until resistance emerged. We found as a result of focal amplification of the MET proto-oncogene (9, that cells resistant to intermediate doses of crizotinib developed 16, 17). Importantly, preclinical modeling of acquired resistance to amplification of the EML4-ALK gene. Cells resistant to higher doses EGFR TKIs has proven invaluable, not only for accurately pre- (1 μM) also developed a gatekeeper mutation, L1196M, within the dicting the resistance mechanisms that emerge in the clinic, but kinase domain, rendering EML4-ALK insensitive to crizotinib. This also for assessing new therapeutic strategies to overcome re- gatekeeper mutation was readily detected using a unique and sistance (12, 16–22). highly sensitive allele-specific PCR assay. Although crizotinib was Recently, one NSCLC patient with acquired resistance to cri- ineffectual against EML4-ALK harboring the gatekeeper mutation, zotinib was described (23). In this patient who relapsed after 5 mo we observed that two structurally different ALK inhibitors, NVP- of treatment, molecular analyses revealed that the resistant tumor TAE684 and AP26113, were highly active against the resistant can- cells harbored two secondary mutations within the kinase domain cer cells in vitro and in vivo. Furthermore, these resistant cells of EML4-ALK, C1156Y, and the gatekeeper mutation L1196M. remained highly sensitive to the Hsp90 inhibitor 17-AAG. Thus, These mutations occurred independently in distinct subclones we have developed a model of acquired resistance to ALK inhibitors of the resistant tumor. Studies of Ba/F3 cells engineered to and have shown that second-generation ALK TKIs or Hsp90 inhib- express EML4-ALK harboring either mutation suggested that itors are effective in treating crizotinib-resistant tumors harboring these mutants were resistant to crizotinib as well as resistant to a secondary gatekeeper mutations. more potent ALK TKI, termed 2,4-pyrimidinediamine derivative (PDD). Thus, strategies to overcome this type of resistance have irst described in 2007, the oncogenic fusion kinase echinoderm not yet been established. In addition, because there are no lab- Fmicrotubule-associated protein-like 4-anaplastic lymphoma oratory models of acquired resistance via this mechanism, it kinase (EML4-ALK) is present in ∼4% of patients with non-small remains unknown if cancers that develop these resistance muta- cell (NSCLC) (1). Chromosomal translocations in- tions remain addicted to ALK kinase activity and will be highly volving ALK also occur in other cancers, including anaplastic sensitive to other therapeutics targeting ALK. large cell and inflammatory myofibroblastic tumors. In this study, we have explored resistance to crizotinib by gen- In all cases, the fusion partner (e.g., EML4) is believed to mediate erating and characterizing a unique cell line model of acquired EML4-ALK ligand-independent oligomerization of ALK, resulting in consti- resistance. In this model of acquired resistance, is fi tutive ALK kinase activation (2–4). In cell line and genetically ampli ed and harbors the L1196M gatekeeper mutation. We engineered mouse models, EML4-ALK serves as a potent onco- show that cells expressing the L1196M mutant form of EML4- genic “driver,” and cancers with this translocation are highly ALK are resistant to crizotinib, remain addicted to ALK signaling, sensitive to ALK kinase inhibition (5, 6). Recently, a tyrosine kinase inhibitor (TKI) targeting ALK, crizotinib (PF-02341066), was examined in a phase 1 trial (7). Among 105 patients with Author contributions: R.K., J.A.E., and A.T.S. designed research; R.K., T.M.K., E.L., H.E., EML4-ALK– V.M.R., W.C.S., and A.J.I. performed research; R.K., C.B., V.M.R., W.C.S., and A.J.I. contrib- positive NSCLC, crizotinib showed remarkable ac- uted new reagents/analytic tools; R.K., T.M.K., C.B., A.J.I., J.A.E., and A.T.S. analyzed data; tivity, with an objective response rate of 56% and a median pro- and R.K., J.A.E., and A.T.S. wrote the paper. gression-free survival of 9.2 mo (7, 8). These results support the The authors declare no conflict of interest. EML4-ALK notion that lung cancers harboring are highly sus- This article is a PNAS Direct Submission. ceptible to ALK-targeted therapies and demonstrate the prop- 1To whom correspondence may be addressed. E-mail: [email protected] or erties of “oncogene addiction” to ALK. [email protected]. fi Although many patients derive substantial clinical bene t, the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. development of drug resistance has curbed the impact of crizo- 1073/pnas.1019559108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1019559108 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 and are highly sensitive to other structurally distinct ALK TKIs cells, we detected a C → A substitution at nucleotide 3586 of as well as to Hsp90 inhibition. On the basis of these results, we EML4-ALK variant 1 (Fig. 2B) that was not detected in the pa- propose two therapeutic strategies for overcoming acquired re- rental cells. This 3586C → A substitution results in a leucine → sistance to crizotinib, particularly when mediated by secondary methionine change within the ALK TK domain, corresponding to mutations within the ALK TK domain. the L1196M gatekeeper mutation previously reported in a patient with acquired resistance to crizotinib (23). We did not detect any Results other TK mutations including the C1156Y substitution, which was Generation and Biochemical Characterization of Crizotinib-Resistant also reported in the same patient (23). Consistent with the notion EML4-ALK Cells. The NSCLC cell line H3122 expresses variant 1 that this L1196M mutation confers resistance to crizotinib, Ba/F3 and is highly sensitive to treatment with crizotinib. To explore cells engineered to express this mutant were also resistant to mechanisms of crizotinib resistance, we generated resistant crizotinib, whereas cells expressing wild-type EML4-ALK were H3122 clones by exposing the sensitive parental cells to increasing sensitive (SI Appendix, Fig. S2). Notably, in the DNA sequence concentrations of crizotinib for 4 mo. We maintained cells with tracings, the peak corresponding to the mutation is approximately intermediate crizotinib resistance, referred to as H3122 CR0.6, in one-half to one-third as high as the wild-type peak (Fig. 2B). This 600 nM of crizotinib. The fully resultant cells, H3122 crizotinib- suggests that only a fraction of the amplified EML4-ALK fusion μ resistant (H3122 CR) cells, were maintained in 1 M of crizotinib. genes acquire the secondary resistance mutation. H3122 CR cells were as resistant to crizotinib as other cancer These resistant models were developed by stepwise increases cell lines that did not harbor ALK gene rearrangements (IC50 > 1 μ A SI Appendix A in drug concentration until fully resistant cells emerged. Because M; Fig. 1 and , Fig. S1 ). In addition, we assessed both amplification and mutation were identified in H3122 CR cells, 704 established cancer cell lines derived from a wide variety of we sought to investigate the temporal relationship between gene tumor types for sensitivity to crizotinib as part of our automated amplification and secondary mutation in acquired crizotinib re- platform to examine drug sensitivity across multiple cell lines sistance. Thus, we analyzed H3122 CR0.6 cells, the intermediate, (24). Whereas H3122 parental cells segregated with the most partially resistant predecessors of H3122 CR cells that were re- sensitive cell lines, the H3122 CR cells segregated with the highly sistant to 600 nM crizotinib. By Western blotting analysis, ALK resistant cell lines (Fig. 1B). Unlike the parental H3122 cells, the protein level and p-ALK were up-regulated in H3122 CR0.6 cells H3122 CR cells maintained ALK phosphorylation in the presence (SI Appendix,Fig.S3). FISH analysis demonstrated amplification of crizotinib (Fig. 1C). Accordingly, AKT and ERK phosphory- of the EML4-ALK in the H3122 CR0.6 cells (Fig. 2A). lation were not suppressed by crizotinib in the resistant cells. However, they did not harbor a secondary ALK resistance muta- Notably, H3122 CR cells also contained higher total protein B levels of EML4-ALK compared with the parental line (Fig. 1C). tion (Fig. 2 ). To determine whether L1196M is present at a low level in a subpopulation of H3122 CR0.6 cells, we developed a fi EML4-ALK Gene Is Amplified and Mutated in H3122 CR Cells. To de- highly sensitive gatekeeper mutation-speci c PCR assay that can termine whether gene amplification underlies the elevated protein detect the L1196M mutation when it represents at least 1% of the level of EML4-ALK in H3122 CR cells, we performed fluores- mutated ALK alleles (SI Appendix,Fig.S4). Using this assay, we cence in situ hybridization (FISH) analysis. Relative to parental detected L1196M in H3122 CR cells and in all 11 clones derived H3122 cells, H3122 CR cells showed an increase in the number of from single H3122 CR cells (SI Appendix, Fig. S5), but not in the rearranged EML4-ALK genes per cell (Fig. 2A). H3122 CR0.6 cells, the parental H3122 cells, or the other NSCLC Because the phosphorylation of EML4-ALK in H3122 CR cells cell lines harboring EML4-ALK (Fig. 2C and SI Appendix, Fig. S5). is not impacted by 1 μM crizotinib (Fig. 1C), we hypothesized These results suggest that the L1196M mutation is present in each that resistant cells might also harbor a mutation within ALK.We cell of the resistant H3122 CR cells and that there is a stepwise prepared cDNA and examined the entire coding sequence of model of acquired resistance involving amplification of the target, EML4-ALK in H3122 parental and resistant cells. In the resistant which confers resistance to 600 nM crizotinib, followed by sec-

A B C H3122 H3122 CR 100 crizotinib (200nM), n=704 crizotinib (6hr) crizotinib(6hr) M M non 30 nM 100 nM 1000 nM non 30 nM 100 nM 300 nM 1000 nM 300 nM pALK of control) 1% (pY1604) 50 ALK pAKT (pS473)

panAKT cell number (% cell number (% 0 <02<0.2 0.2 - 0.5 pERK 0.5 - 0.75 H460 H522 A549 ERK H3122 H1299 BT474 > 0.75 SKBR3 Actin H3122 CR

Fig. 1. H3122 CR cells are resistant to crizotinib. (A) Cell lines were seeded in 96-well plates and treated with 1 μM of crizotinib for 72 h. Cell survival was analyzed using the CellTiter-Glo viability assay. In contrast to the parental H3122 cells, which are highly sensitive to crizotinib (red bar), H3122 CR cells (blue bar) are as insensitive to crizotinib as non-ALK rearranged cell lines. (B) Pie chart representation of the sensitivity of H3122 and H3122 CR cells compared with 704 other human cancer cell lines assessed by an automated cell viability assay to 200 nM crizotinib. Viability at 72 h was calculated as a ratio of viable crizotinib-exposed cells to viable DMSO-treated cells. The color scheme corresponds to the relative inhibitory effect of treatment. The chart depicts the most sensitive cell lines in order of decreasing sensitivity (from top to bottom). (C) H3122 parental and H3122 CR cells were treated with crizotinib at the indicated concentration for 6 h. Cell extracts were immunoblotted to detect the indicated proteins.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1019559108 Katayama et al. Downloaded by guest on September 30, 2021 A

H3122 H3122 CR0.6 H3122 CR

B Forward Reverse C

H3122

H3122 CR0.6 water H2228 H3122 3122 CR MGH006 M 22 CR0.6 H3 H312

H3122 CR

Leu (CTG) Met (ATG) MEDICAL SCIENCES

Fig. 2. EML4-ALK is both amplified and mutated in resistant H3122 CR cells. (A) Dual-color FISH [ALK-N-terminal (green)/ALK-C-terminal (red)] analysis was performed on H3122 parental, H3122 CR0.6, and H3122 CR cells. Arrows indicate the split ALK C-terminal region (EML4-ALK). Both H3122 CR0.6 and H3122 CR cells demonstrate amplification of EML4-ALK. (B) Secondary L1196M (gatekeeper) mutation in resistant H3122 CR cells. Shown are electrophoretograms of EML4-ALK cDNA from H3122 parental, H3122 CR0.6, and H3122 CR cells. The 1903C → A mutation within exon 23 results in a L635M substitution that corresponds to the L1196M gatekeeper mutation of ALK. (C) Detection of the gatekeeper mutation using an L1196M mutation-specific PCR assay. Shown is the amplified 160-bp product in H3122 CR cells after 35 PCR cycles. This product is not detected in parental H3122 or H3122 CR0.6 cells or in other EML4-ALK– positive cell lines (H2228, MGH006).

ondary gatekeeper mutation that renders cells resistant to 1 μM (Fig. 1B). In contrast to crizotinib, NVP-TAE684 treatment of crizotinib. H3122 CR cells suppressed phosphorylation of ALK, AKT, and ERK and induced marked (Fig. 4C and SI Appendix, H3122 CR Cells Are Addicted to ALK. To determine if H3122 CR cells Fig. S6). Similarly, NVP-TAE684 potently suppressed the survival continue to require EML4-ALK for their viability, we used siRNAs SI ALK of Ba/F3 cells expressing the EML4-ALK L1196M mutant ( targeting to knock down EML4-ALK. In both H3122 pa- Appendix A rental and H3122 CR cell lines, ALK-specific siRNA decreased ex- , Fig. S7 ). However, in this system, the potency of pression of EML4-ALK and potently suppressed cell growth (Fig. NVP-TAE684 against mutant EML4-ALK was slightly reduced 3 A and B). As expected, the ALK siRNA had no effect on the relative to native EML4-ALK, with IC50 values of 2.7 versus 1.2 growth of KRAS mutant A549 cells (Fig. 3 A and B). These results nM, respectively. demonstrate that resistant H3122 CR cells expressing EML4-ALK L1196M remain addicted to ALK signaling. NVP-TAE684 and AP26113 Overcome Crizotinib Resistance in H3122 CR A B Cells. Based on the crystal structure of the kinase domain of ALK, H3122 the gatekeeper L1196M mutation causes resistance by steric in- H3122 CR A549 l 100 l ol o terference with crizotinib binding (23). Thus, although resistant to o crizotinib, this mutant ALK may be sensitive to structurally dis- si-contr si-ALK si-contr si-ALK si-ALK si-contr tinct ALK kinase inhibitors. To address this hypothesis, we tested pALK the activity of the ALK inhibitor NVP-TAE684, a 5-chloro-2,4- 50 (pY1604) diaminophenylpyrimidine, in parental and resistant H3122 cells. ALK

As shown in Fig. 4, NVP-TAE684 markedly reduced cell survival of control) cell number (% Actin in both sensitive H3122 and H3122 CR cells, but had little to no 0

– ntl ntl ntl effect on the viability of other, non-ALK dependent cancer cell LK LK LK c c c A A A si- si- si- si- lines (Fig.4A and SI Appendixx, Fig. S1B). Within the panel of 704 si- si- established cancer cell lines assessed in the automated platform H3122 H3122 CR A549 (24), the vast majority were largely insensitive to 200 nM NVP- Fig. 3. Resistant H3122 CR cells are oncogene-addicted. (A) The indicated TAE684; however, 1% of lines displayed marked sensitivity to cell lines were transfected with small interfering RNA (siRNA) against ALK NVP-TAE684. Both the H3122 and the H3122 CR cell lines (siALK) or control siRNA, and cell viability was measured after 72 h. (B)Im- segregate with the highly sensitive cell lines (Fig. 4B). This is in munoblotting of protein lysates 72 h after knockdown showing reduced sharp contrast to the resistance of H3122 CR cells to crizotinib levels of phosphorylated ALK and total ALK in siALK-transfected cells.

Katayama et al. PNAS Early Edition | 3of6 Downloaded by guest on September 30, 2021 H3122 H3122 CR A B C TAE684 (6hr) TAE684 (6hr)

TAE684 (200nM), n=704 100 100 nM non 3 nM 10 nM 30 nM 300 nM 10 nM 30 nM non 3 nM 100 nM 300 nM ntrol)

o pALK (pY1604) ALK 50 pAKT (pS473) panAKT ll number (% ll number (% of c e

c 0 pERK ERK H460 A549 H522 H1299 BT474 H3122 < 0.2 SKBR3 Actin 0.2 - 0.5 H3122 CR 0.5 - 0.75 > 0.75

D E H3122 H3122 CR

AP26113 (6hr) AP26113 (6hr) 3 nM 10 nM 30 nM 100 nM 300 nM 3 nM 10 nM 300 nM non crizotinib (1uM) non 30 nM 100 nM crizotinib (1uM) ) 100 pALK (pY1604)

ALK pAKT (% of control of (% 50 (pS473)

panAKT number 0 pERK cell

ERK H460 H522 A549 H1299 H3122 BT474

SKBR3 Actin H3122 CR

Fig. 4. Two different ALK kinase inhibitors, NVP-TAE684 and AP26113, overcome crizotinib resistance in H3122 CR cells. (A) Cell lines were seeded in 96-well plates and treated with 100 nM of TAE684 for 72 h. Cell survival was analyzed using the CellTiter-Glo viability assay. Both parental H3122 cells (red bar) and H3122 CR cells (blue bar) show marked sensitivity to TAE684. Non-ALK rearranged cell lines (A549, H1299, SKBR3, H522, H460, and BT474 cells) show minimal growth inhibition with TAE684. (B) Pie chart representation of the sensitivity of H3122 and H3122 CR cells compared with 704 other human cancer cell lines assessed by an automated cell viability assay to 200 nM TAE684. Viability at 72 h was calculated as a ratio of viable crizotinib-exposed cells to viable DMSO- treated cells. The chart depicts the most sensitive cell lines in order of decreasing sensitivity (from top to bottom). Both parental H3122 and H3122 CR cells are among the 1% of the most sensitive cell lines to TAE684. (C) Suppression of ALK signaling by TAE684 in resistant H3122 CR cells. H3122 parental and resistant cells were exposed to increasing concentrations of TAE684 for 6 h. Cell lysates were immunoblotted to detect the indicated proteins. (D) Sensitivity of H3122 CR cells to AP26113. Cell lines were seeded in 96-well plates and treated with 300 nM of AP26113 for 72 h. Cell survival was analyzed using the CellTiter-Glo viability assay. Both parental H3122 cells (red bar) and H3122 CR cells (blue bar) show marked sensitivity to AP26113, compared with non-ALK rearranged cell lines. (E) Suppression of ALK signaling by AP26113 in resistant H3122 CR cells. H3122 parental and resistant cells were exposed to increasing concentrations of AP26113 for 6 h. Cell lysates were immunoblotted to detect the indicated proteins.

We also tested a second, structurally distinct ALK kinase in- we observed impressive in vivo activity of both NVP-TAE684 and hibitor AP26113. A new highly selective small molecule inhibitor AP26113, but not of crizotinib, against the H3122 CR cells grown as of ALK, AP26113 exhibits about fivefold greater potency in vitro xenograft tumors in vivo (SI Appendix,Fig.S9). compared with crizotinib (SI Appendix,TableS1A). Like NVP- TAE684, AP26113 was highly active against both sensitive and Hsp90 Inhibitor 17-AAG Overcomes Crizotinib Resistance in H3122 CR resistant H3122 cells, decreasing cell growth (Fig. 4D and SI Ap- Cells. In a recent phase 2 study, the Hsp90 inhibitor IPI-504 pendix, Fig. S1C), suppressing ALK phosphorylation (Fig. 4E), and demonstrated clinical activity in three of three patients with ALK- inducing apoptosis (SI Appendix,Fig.S6). Higher doses of AP26113 positive NSCLC and induced rapid degradation of EML4-ALK in were required to completely suppress ALK in the resistant cells vitro (25). Hsp90 inhibitors also induced tumor regression in a because there were greater levels of ALK/p-ALK as a result of gene genetically engineered mouse model of lung cancer driven by amplification. AP26113 was also active in Ba/F3 cells expressing EML4-ALK (5). Therefore,we evaluated the efficacy of the Hsp90 either native or mutant EML4-ALK (IC50’s were 10 and 24 nM, inhibitor 17-AAG in EML4-ALK L1196M-expressing cells. As respectively) (SI Appendix,Fig.S7B). Consistent with the Ba/F3 shown in Fig. 5A, 17-AAG potently suppressed cell growth in both data, the potency of AP26113 against mutant EML4-ALK was parental H3122 and H3122 CR cells, but not in other non-ALK– slightly reduced relative to native EML4-ALK as determined by its dependent lung cancer lines (SI Appendix,Fig.S1D). However, in capacity to decrease p-ALK in the H3122 and H3122 CR cells, with contrast to NVP-TAE684 and AP26113, 17-AAG also inhibited IC50 values of 7.4 versus 16.8 nM, respectively (SI Appendix,Fig. the viability of SKBR3 and BT474 cell lines, both of which harbor S8). These results suggest that AP26113 can overcome crizotinib HER2 amplification and are known to be sensitive to Hsp90 in- resistance mediated by the gatekeeper L1196M mutation. Indeed, hibition (26, 27). In Ba/F3 cells, 17-AAG was active against both

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1019559108 Katayama et al. Downloaded by guest on September 30, 2021 H3122 H3122 CR as C1156Y. However, this seems unlikely because we observed the A B 17AAG (24hr) 17AAG (24hr) L1196M mutation and not the C1156Y mutation in all of the 100 clones derived from single cells from the H3122 CR cell line. 10 nM 300 nM non 3 nM 30 nM 100 nM non 3 nM 10 nM 30 nM 100 nM 300 nM fi ALK pALK Although ampli cation of is a known oncogenic event (pY1604) in pediatric (34), amplification of ALK fusion ALK 50 oncogenes has not been reported. Analysis of partially resistant pAKT H3122 cells (H3122 CR0.6) that harbor EML4-ALK amplification (pS473) (but no gatekeeper mutation) suggests a step-wise evolution of panAkt acquired resistance involving gene amplification followed by point

ell number (% of ell number (% control) pERK ce 0 mutation. This model is also supported by the observation that ERK only a fraction of the amplified ALK fusion genes (per cell) harbor A549 H522 H460 H3122 H1299 BT474 Actin C SKBR3 L1196M (Fig. 2 ). In other TKI-resistant cancers, target ampli-

H3122 CR fication preceding acquisition of secondary mutations has not Fig. 5. The Hsp90 inhibitor 17-AAG can overcome crizotinib resistance in been reported in vitro or in vivo. It remains unknown if this two- H3122 CR cells. (A) Cell lines were seeded in 96-well plates and treated with step mechanism is unique to EML4-ALK–positive NSCLC or if 10 nM of 17-AAG for 72 h. Cell survival was analyzed using CellTiter-Glo. it is a product of the methodology used to develop resistance in Both parental H3122 cells (red bar) and H3122 CR cells (blue bar) show the laboratory. sensitivity to 17-AAG. (B) Suppression of ALK signaling by 17-AAG in re- The detection of gatekeeper and other acquired mutations in sistant H3122 CR cells. H3122 parental and resistant cells were exposed to resistant tumor samples is clinically important, but can be tech- increasing concentrations of 17-AAG for 6 h. Cell lysates were immuno- nically challenging. The EGFR T790M resistance mutation, for blotted to detect the indicated proteins. example, is often difficult to detect on standard sequence trac- ings and may require more sensitive techniques such as deep native and mutant EML4-ALK to a similar extent, but showed no sequencing for detection (19, 35, 36). This problem may be due activity in parental Ba/F3 cells (SI Appendix,Fig.S7C). 17-AAG to contamination of the tumor specimen with noncancerous cells fl treatment decreased both p-ALK level and ALK protein expres- (e.g., stromal or in ammatory cells). Alternatively, for some tumors, only a small percentage of the EGFR alleles per cell may sion with similar potencies in the parental and resistant cells (Fig. “ ” 5B). These results suggest that Hsp90 inhibition might represent harbor the T790M mutation (termed allelic dilution ), or only a small percentage of resistant cells in the tumor specimen might an alternative therapeutic strategy for overcoming acquired re- fi MEDICAL SCIENCES sistance to crizotinib due to acquisition of a resistance mutation. have T790M-mediated resistance. Notably, the nding of both EML4-ALK amplification and L1196M mutation in the CR cells Discussion suggests that allelic dilution also could ultimately contribute to fi In tumors dependent on a driver kinase such as BCR-ABL or dif culty in detecting resistance mutations in cancers with ac- quired resistance to crizotinib. In this study, we developed an mutant EGFR, secondary mutation within the kinase domain is fi a common mechanism of acquired drug resistance. The most L1196M-mutation speci c PCR assay to detect potentially low levels of the gatekeeper L1196M mutation in partially resistant frequent resistance mutation involves the gatekeeper residue. H3122 CR0.6 cells. This assay is highly sensitive with a detection Amino acid substitutions at this position hinder drug binding and limit of 1% or less (SI Appendix, Fig. S4) and requires only 30 ng thereby confer high-level resistance to many tyrosine kinase genomic DNA. Thus, this assay might serve as the basis for inhibitors. Other mechanisms of acquired drug resistance involve a clinical diagnostic test for L1196M in biopsy samples from gene amplification of the kinase target or activation of alterna- crizotinib-resistant patients. tive signaling pathways to bypass the need for kinase activation EGFR fi In both CML and -mutant lung cancer, resistant tumors (10, 28). Identi cation of the genetic alterations underlying ac- with gatekeeper mutations have proven refractory to second- quired drug resistance has fostered the development of targeted generation, more potent TKIs. One recent report suggests that agents for patients with TKI resistance. For example, two second- ALK — — fi the gatekeeper mutation in might behave similarly; com- generation TKIs and have ef cacy against pared with control Ba/F3 cells expressing native EML4-ALK, cells most of the known -resistant mutations and are approved expressing mutant EML4-ALK L1196M were markedly less sen- for patients with imatinib-resistant chronic myeloid sitive to a potent PDD (23). In contrast, we have found that cri- (CML) (29, 30). However, these drugs are not active against the zotinib resistance mediated by the gatekeeper L1196M mutation EGFR gatekeeper T315I mutation within ABL (31). Similarly in - can be effectively overcome by two different ALK TKIs. Impor- mutant lung cancer, second-generation, irreversible EGFR tantly, one of these—AP26113—is under clinical development inhibitors are being explored to treat cancers that develop re- and is scheduled to begin clinical testing within the year. In H3122 sistance via the gatekeeper T790M mutation (22, 32). CR cells, the improved cellular activity of AP26113 compared EML4-ALK– positive NSCLC represents another tyrosine kinase- with crizotinib is likely based on both its enhanced potency against driven cancer that is highly responsive to TKI therapy. Recently, ALK and its increased activity against L1196M-mutated ALK. two studies have reported the identification of secondary re- In vitro kinase assays revealed that wild-type and L1196M- sistance mutations within the ALK TK domain in patients who mutated ALK showed similar Km values for ATP binding (30.7 relapsed on crizotinib (23, 33). One of the mutations identified and 27.2 μM, respectively). However, crizotinib was about 10-fold was the gatekeeper L1196M substitution, analogous to T315I in less potent against L1196M ALK compared with wild-type ALK ABL EGFR and T790M in (23). In this study, we generated a cell (Ki of 8.2 and 0.7 nM, respectively), consistent with its diminished line model of acquired crizotinib resistance by exposing sensitive, efficacy against the L1196M ALK in cell lines (SI Appendix, Table EML4-ALK–positive H3122 cells to increasing concentrations S1B). In contrast, AP26113 was ∼10-fold more potent against of crizotinib. The resistant cells harbor both amplification of ALK than crizotinib in vitro, and unlike crizotinib, it was similarly EML4-ALK and the same L1196M gatekeeper mutation that was potent against the L1196M mutant (Ki of 0.09 and 0.08 nM, identified in a patient with acquired resistance. Notably, we did respectively). not identify any other ALK mutations in this cell line, including In addition to studying ALK TKIs, we have shown that EML4- C1156Y. Because we did not use more sensitive technologies such ALK harboring the gatekeeper mutation is an Hsp90 client as deep sequencing, we cannot exclude the possibility that a small protein similar to wild-type EML4-ALK and sensitive to Hsp90 fraction of resistant cells harbor distinct secondary mutations such inhibition. Notably, cell lines expressing either native EML4-

Katayama et al. PNAS Early Edition | 5of6 Downloaded by guest on September 30, 2021 ALK or EML4-ALK L1196M were even more sensitive to 17- Reagents. Crizotinib and NVP-TAE684 were purchased from ChemieTek and AAG than two Hsp90-dependent, HER2-amplified breast cancer 17-AAG was purchased from Selleck. AP26113 was provided by ARIAD A Pharmaceuticals that makes AP26113 available to qualified academic labo- lines (Fig. 5 ). Therefore, patients with crizotinib resistance due fi fi ratories free of charge for veri cation and reproduction of the results to an acquired L1196M mutation may derive bene t from two reported in this paper. Each compound was dissolved in DMSO for cell culture different ALK-targeted strategies: more potent ALK TKIs such experiments. Control or ALK siRNA was from Invitrogen, and HiPerfect re- as AP26113 or any of a number of Hsp90 inhibitors already in agent was from Qiagen. clinical trials. Notably, although gatekeeper mutations are par- fi ticularly common in EGFR-mutant lung cancer, the frequency of Isolation of Genomic DNA Preparation and L1196M Mutation-Speci c PCR. – Genomic DNA was isolated from cell pellets with a DNeasy (QIAGEN) L1196M in EML4-ALK positive NSCLC has yet to be estab- according to the manufacturer’s protocol. Exon 23 of ALK was PCR-amplified lished. Because secondary mutations such as the gatekeeper from genomic DNA using Pfu Ultra II (Agilent Technologies) and sequenced mutation may not represent the predominant mechanism of ac- bidirectionally by Sanger dideoxynucleotide sequencing with the primers quired crizotinib resistance, additional studies are needed to described in SI Appendix. ALK-Exon23 or L1196M mutation-specific qPCR elucidate other mechanisms of resistance. The results of these was performed by a LightCycler 480 (Roche Diagnostics) with CYBR Green Master Mix (Roche). Primer sequences are provided in SI Appendix. studies will be critical to selecting the best therapeutic strategies for targeting TKI resistance in the clinic. ACKNOWLEDGMENTS. This study was supported in part by a V Foundation for Cancer Research Translational Grant (to J.A.E. and A.T.S.), by the Charles Materials and Methods W. and Jennifer C. Johnson Koch Institute Clinical Investigator Award (to See SI Appendix for the description of the cell culture conditions, generation A.T.S.), by National Institutes of Health K08 Grant CA120060-01 (to J.A.E.), by the Sig Adler Lung Cancer Research Fund, by the Massachusetts General of H3122 CR cells, survival assays, fluorescence in situ hybridization, immu- Hospital Thoracic Oncology Fund, and by a Japan Society for the Promotion noblotting, apoptosis assay retroviral infection, quantitative RT-PCR (qPCR), of Science Postdoctoral Fellowship for Research Abroad from the Ministry of siRNA transfection, xenograft study, and statistical analyses. Education, Culture, Sports, Science, and Technology of Japan (to R.K.).

1. Soda M, et al. (2007) Identification of the transforming EML4-ALK fusion gene in non- 19. Engelman JA, et al. (2006) Allelic dilution obscures detection of a biologically small-cell lung cancer. Nature 448:561–566. significant resistance mutation in EGFR-amplified lung cancer. J Clin Invest 116: 2. Mano H (2008) Non-solid oncogenes in solid tumors: EML4-ALK fusion genes in lung 2695–2706. – cancer. Cancer Sci 99:2349 2355. 20. Faber AC, et al. (2009) Differential induction of apoptosis in HER2 and EGFR addicted 3. Shinmura K, et al. (2008) EML4-ALK fusion transcripts, but no NPM-, TPM3-, CLTC-, cancers following PI3K inhibition. Proc Natl Acad Sci USA 106:19503–19508. ATIC-, or TFG-ALK fusion transcripts, in non-small cell lung carcinomas. Lung Cancer 21. Guix M, et al. (2008) Acquired resistance to EGFR tyrosine kinase inhibitors in cancer 61:163–169. cells is mediated by loss of IGF-binding proteins. J Clin Invest 118:2609–2619. 4. Takeuchi K, et al. (2009) KIF5B-ALK, a novel fusion oncokinase identified by an 22. Zhou W, et al. (2009) Novel mutant-selective EGFR kinase inhibitors against EGFR immunohistochemistry-based diagnostic system for ALK-positive lung cancer. Clin T790M. Nature 462:1070–1074. Cancer Res 15:3143–3149. 23. Choi YL, et al.; ALK Lung Cancer Study Group (2010) EML4-ALK mutations in lung 5. Chen Z, et al. (2010) Inhibition of ALK, PI3K/MEK, and HSP90 in murine lung – adenocarcinoma induced by EML4-ALK fusion oncogene. Cancer Res 70:9827–9836. cancer that confer resistance to ALK inhibitors. N Engl J Med 363:1734 1739. 6. Soda M, et al. (2008) A mouse model for EML4-ALK-positive lung cancer. Proc Natl 24. McDermott U, et al. (2008) Genomic alterations of anaplastic lymphoma kinase may Acad Sci USA 105:19893–19897. sensitize tumors to anaplastic lymphoma kinase inhibitors. Cancer Res 68:3389–3395. 7. Kwak EL, et al. (2010) Anaplastic lymphoma kinase inhibition in non-small-cell lung 25. Sequist LV, et al. (2010) Activity of IPI-504, a novel heat-shock protein 90 inhibitor, in cancer. N Engl J Med 363:1693–1703. patients with molecularly defined non-small-cell lung cancer. JClinOncol28:4953–4960. 8. Camidge DR, et al. (2010) Clinical activity of crizotinib (PF-02341066), in ALK-positive 26. Chandarlapaty S, et al. (2010) Inhibitors of HSP90 block p95-HER2 signaling in patients with advanced non-small cell lung cancer. Ann Oncol 21(Suppl 8):viii123, -resistant tumors and suppress their growth. Oncogene 29:325–334. mdq518. 27. Zheng FF, et al. (2000) Identification of a geldanamycin dimer that induces the fi 9. Bean J, et al. (2007) MET ampli cation occurs with or without T790M mutations in selective degradation of HER-family tyrosine . Cancer Res 60:2090–2094. fi EGFR mutant lung tumors with acquired resistance to ge tinib or . Proc Natl 28. Engelman JA, Settleman J (2008) Acquired resistance to tyrosine kinase inhibitors Acad Sci USA 104:20932–20937. during cancer therapy. Curr Opin Genet Dev 18:73–79. 10. Engelman JA, Jänne PA (2008) Mechanisms of acquired resistance to epidermal 29. Kantarjian H, et al. (2010) Dasatinib versus imatinib in newly diagnosed chronic-phase tyrosine kinase inhibitors in non-small cell lung cancer. Clin chronic myeloid leukemia. N Engl J Med 362:2260–2270. Cancer Res 14:2895–2899. 30. Saglio G, et al.; ENESTnd Investigators (2010) Nilotinib versus imatinib for newly 11. Flaherty KT, et al. (2010) Inhibition of mutated, activated BRAF in metastatic diagnosed chronic myeloid leukemia. N Engl J Med 362:2251–2259. melanoma. N Engl J Med 363:809–819. ’ 12. Johannessen CM, et al. (2010) COT drives resistance to RAF inhibition through MAP 31. O Hare T, et al. (2005) In vitro activity of Bcr-Abl inhibitors AMN107 and BMS-354825 kinase pathway reactivation. Nature 468:968–972. against clinically relevant imatinib-resistant Abl kinase domain mutants. Cancer Res 13. Kobayashi S, et al. (2005) EGFR mutation and resistance of non-small-cell lung cancer 65:4500–4505. to gefitinib. N Engl J Med 352:786–792. 32. Yap TA, et al. (2010) Phase I trial of the irreversible EGFR and HER2 kinase inhibitor 14. Mok TS, et al. (2009) Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. BIBW 2992 in patients with advanced solid tumors. J Clin Oncol 28:3965–3972. N Engl J Med 361:947–957. 33. Sasaki T, et al. (2010) The neuroblastoma associated F1174L ALK mutation causes 15. Pao W, et al. (2005) Acquired resistance of lung adenocarcinomas to gefitinib or resistance to an ALK kinase inhibitor in ALK translocated cancers. Cancer Res 70: erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med 10038–10043. 2:e73. 34. Chiarle R, Voena C, Ambrogio C, Piva R, Inghirami G (2008) The anaplastic lymphoma fi 16. Turke AB, et al. (2010) Preexistence and clonal selection of MET ampli cation in EGFR kinase in the pathogenesis of cancer. Nat Rev Cancer 8:11–23. mutant NSCLC. Cancer Cell 17:77–88. 35. Kosaka T, et al. (2006) Analysis of receptor gene mutation in 17. Engelman JA, et al. (2007) MET amplification leads to gefitinib resistance in lung patients with non-small cell lung cancer and acquired resistance to gefitinib. Clin cancer by activating ERBB3 signaling. Science 316:1039–1043. Cancer Res 12:5764–5769. 18. Corcoran RB, et al. (2010) BRAF gene amplification can promote acquired resistance 36. Kwak EL, et al. (2005) Irreversible inhibitors of the EGF receptor may circumvent to MEK inhibitors in cancer cells harboring the BRAF V600E mutation. Sci Signal 3: fi – ra84. acquired resistance to ge tinib. Proc Natl Acad Sci USA 102:7665 7670.

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