Novel Third-Generation EGFR Tyrosine Kinase Inhibitors And

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Published OnlineFirst February 4, 2019; DOI: 10.1158/0008-5472.CAN-18-1281

Cancer Review Research

Novel Third-Generation EGFR Tyrosine Kinase Inhibitors and Strategies to Overcome Therapeutic Resistance in Lung Cancer Ayesha Murtuza, Ajaz Bulbul, John Paul Shen, Parissa Keshavarzian, Brian D. Woodward, Fernando J. Lopez-Diaz, Scott M. Lippman, and Hatim Husain

Abstract

EGFR-activating mutations are observed in approximately (PF-0647775), and AC0010. However, therapeutic resistance 15% to 20% of patients with non–small cell lung cancer. after the administration of third-generation inhibitors is com- Tyrosine kinase inhibitors have provided an illustrative exam- plex and not fully understood, with significant intertumoral ple of the successes in targeting oncogene addiction in cancer and intratumoral heterogeneity. Repeat tissue and plasma and the role of tumor-specific adaptations conferring thera- analyses on therapy have revealed insights into multiple peutic resistance. The compound osimertinib is a third- mechanisms of resistance, including novel second site generation tyrosine kinase inhibitor, which was granted full EGFR mutations, activated bypass pathways such as MET FDA approval in March 2017 based on targeting EGFR T790M amplification, HER2 amplification, RAS mutations, BRAF resistance. The compound has received additional FDA mutations, PIK3CA mutations, and novel fusion events. approval as first-line therapy with improvement in progres- Strategies to understand and predict patterns of mutagenesis sion-free survival by suppressing the activating mutation are still in their infancy; however, technologies to under- and preventing the rise of the dominant resistance clone. stand synthetically lethal dependencies and track cancer Drug development has been breathtaking in this space with evolution through therapy are being explored. The expan- other third-generation compounds at various stages of devel- sion of combinatorial therapies is a direction forward opment: rociletinib (CO-1686), olmutinib (HM61713), targeting minimal residual disease and bypass pathways nazartinib (EGF816), naquotinib (ASP8273), mavelertinib early based on projected resistance.

Introduction Historical Perspective EGFR-mutant lung cancer has been a model of drug develop- Initially approved as second line therapy in advanced non– ment for oncogene addiction in cancer. The receptor belongs to small cell lung cancer (NSCLC) after chemotherapy, first- the ERBB family of receptors that bind ligands including EGF, generation reversible anti-EGFR tyrosine kinase inhibitors (TKI) TGFa, amphiregulin, epigen, betacellulin, heparin-binding EGF, erlotinib and gefitinib entered clinical guidelines based on a body and epiregulin. Upon ligand binding to EGFR, homodimerization of clinical trials that demonstrated efficacy in some patients after or heterodimerization with other ERBB family members triggers initial lines of chemotherapy (2–6). In 2004, three groups of receptor phosphorylation and activation of downstream signaling researchers identified a subset of patients with NSCLC with through RAS-RAF-MEK-ERK and RAS-PI3K-PTEN-AKT-mTOR activating mutations in the EGFR tyrosine kinase domain (2, 5, activation, leading to proliferation, differentiation, and migration 6). The most common EGFR mutations are exon 19 deletions of cancer cells (Supplementary Fig. S1; ref. 1). Herein, we describe (54%), comprising of five amino acids from codons 746-750, and some of the important lessons learned from an over a decade of substitution of leucine with arginine at codon 858 (L858R, 41%; experience with EGFR inhibitors in lung cancer, drug resistance, ref. 7). Other less common aberrations include G719X (4%), and some of the projections forward with new strategies in first- L861X (1%), S768I (1%) mutations, and exon 20 insertions line clinical management. (2%–9%; ref. 8). Patients with activating EGFR mutations (except exon 20 insertions) have been exquisitely sensitive to first- generation reversible EGFR TKIs, and a true paradigm shift emerged when these compounds showed improved efficacy to fi University of California San Diego, Moores Cancer Center, La Jolla, California. chemotherapy as initial therapy. For the rst time, a solid tumor was treated with oral anticancer therapy in the first-line and Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). improved PFS from approximately 5 months with chemotherapy to 11 months (3–4). The lung cancer clinical community was A. Murtuza and A. Bulbul contributed equally to this article. intellectually inspired, and the adoption of tolerable, precision Corresponding Author: Hatim Husain, University of California San Diego, oriented, oral anticancer therapy emerged to replace standard Moores Cancer Center, 3855 Health Sciences Dr #3011, La Jolla, CA 92093. intravenous chemotherapy for a subset of patients. Phone: 858-534-1590; Fax: 858-822-6186; E-mail: [email protected] Despite revolutionary advancements, patients who were sen- doi: 10.1158/0008-5472.CAN-18-1281 sitive to EGFR TKIs eventually progressed after approximately 9 2019 American Association for Cancer Research. to 11 months. More than 60% of patients develop resistance to

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first- or second-generation inhibitors with an acquired second site of development and include nazartinib (EGFR 816), rociletinib mutation in the ATP binding pocket of EGFR exon 20 leading to a (CO-1686), olmutinib (HM61713), naquotinib (ASP8273), threonine-to-methionine substitution at the amino acid position mavelertinib (PF-06747775), and AC0010 (Supplementary 790 (T790M; ref. 7). This mutation prevents drug binding by Fig. S2; Supplementary Table S1). Following over a decade of increasing the affinity of the ATP binding site for ATP. Second- active drug development, osimertinib is the only third-generation generation, irreversible TKIs including afatinib and dacomitinb inhibitor that is currently approved by the FDA, with data that were developed with intent to overcome these resistant tumor demonstrate significant improvement in clinical endpoints, clones (9, 10). However, despite encouraging in vitro data, clinical including PFS, tolerability with limited wild-type inhibition, and trials with these compounds did not improve overall survival in response with central nervous system (CNS) disease (11, 12). T790M mutant patients likely due to the inability to escalate Other third-generation inhibitors as of yet have not demonstrated dosing because of potent effects on wild-type EGFR at the ther- superiority to osimertinib despite antitumor efficacy, and at this apeutic window (9). Third-generation inhibitors with pyrimidine time it remains unclear where they will be clinically positioned. core structures were created to target the T790M clone with maintained activity against the original exon19del and L858R mutations. Binding at the C797 locus and having selectivity Osimertinib against the T790M mutation, third-generation inhibitors have Osimertinib, a pyrimidine-based compound that binds irre- had responses for patients with T790M resistance after first- versibly to the cysteine-797 residue at the ATP binding site of the generation inhibitors (Fig. 1A–E). First-line data have demon- growth factor receptor, preclinically displayed significant tumor strated superior clinical outcomes compared with first-generation cytoreduction in xenograft and transgenic mice with activity inhibitors with trends toward overall survival improvement (11). against activating mutations exon19del and L858R with and without detectable T790M mutations and less activity for the wild-type EGFR target (13). Clinically, the phase II AURA2 trial Third-Generation TKIs was restricted to EGFR T790M-positive NSCLC patients with The race to discover a next-generation EGFR TKI effective disease progression after initial first line therapy and showed a against resistant tumors has been both illustrative and ground 71% (140/199) objective response with six patients (3%) having breaking. Osimertinib initially gained full FDA approval for complete response and 134 (67%) with partial response (14). Full patients with metastatic EGFR T790M mutant NSCLC after pro- FDA approval was given after the phase III AURA3 study dem- gressing on first- or second-generation EGFR TKIs and has sub- onstrated an overall response rate (ORR) of 71% compared with sequently gained approval in the first-line treatment of EGFR- 31% with platinum-pemetrexed therapy (OR 5.39; P < 0.001) and mutant lung cancer (11, 12). Other third-generation inhibitors a median PFS of 101 months versus 44 months (HR 030; P < designed to target the T790M clone have been at various stages 0001; ref. 12). The most common all grade drug-related adverse

Figure 1. In silico modeling of EGFR C797S interaction with osimertinib in the complex of EGFR derived from its crystal structure (PDB4ZAU) using Chimera software. A, Space ﬁlling hydrophobicity surface model of EGFR with osimertinib. B and C, Osimertinib-EGFR interaction with C797wt (B) or C797S-mutant (C) residues pan-view with relevant residues to TKI resistance G719, T790, and L858 indicated on the model. D and E, Osimertinib covalent binding to the SH-group on C797 showing both the covalent binding (red) and the separation distance to C797 wild type. E, In silico modeling of the most stable rotamer substitution by the less nucleophilic serine side chain at position 797, which cannot undergo covalent bond formation. A higher distance between the C797S and osimertinib closest carbon atom is indicated in yellow.

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events included diarrhea (43%) and rash (40%), however impres- Combinational strategies with osimertinib sively less than 1% were grade three or higher. Interstitial lung Studies evaluating the safety and efficacy of additional targeted disease (ILD) was observed in eight patients and, although rare therapies added to osimertinib are underway and include com- with a mechanism that has not yet been described, did have at binations with agents that target pathways including MET, MAPK, least one fatal event (12, 14). In a larger study of 1,217 patients BCL-2, and JAK activation among others. Based on kinome (ASTRIS, NCT02474355), the investigator-assessed response rate screens and given that upregulation of other oncogenic pathways was 64% at 4 months and safety parameters reflected the original is a common resistance mechanism to tyrosine kinase inhibition, study (15). Pooled analysis has demonstrated a median survival multiple clinical studies are evaluating targets in combination of 26.8 months with second-line osimertinib (16). The success of with osimertinib (20, 21). The TATTON phase Ib study the compound in the second line prompted evaluation in the first- (NCT02143466) involves combinations of osimertinib at line therapy for EGFR-mutant disease (11). increasing doses in combination with either savolitinib (AZD6094, MET inhibitor), selumetinib (MEK inhibitor), or First-line data durvalumab (anti-PDL1 antibody; ref. 20). The evaluation of osimertinib in the first-line space hinged on Early work with a gefitinib-sensitive lung cancer cell line reveal- the hypothesis that suppressing the T790M clone early in treated focal amplification of the MET proto-oncogene in cells that ment course may improve clinical outcomes. The phase III developed resistance to gefitinib. Inhibition of MET signaling FLAURA trial (NCT02296125) directly compared osimertinib in these cells restored their sensitivity to gefitinib and MET ampli- (80 mg daily) with first-generation therapy (either gefitinib 250 fication was detected in 4 of 18 (22%) lung cancer specimens mg or erlotinib 150 mg daily) in a total of 556 patients with that had developed resistance to gefitinib or erlotinib. It was NSCLC with newly diagnosed EGFR exon 19 deletions or L858R found that MET amplification causes gefitinib resistance by driv- mutations. Osimertinib had a superior PFS relative to the erlo- ing ERBB3 (HER3)–dependent activation of PI3K (21). In clinical tinib or gefitinib arm with median progression-free survival of samples obtained after treatment with the third generation inhib- 18.9 months compared with 10.2 months for standard therapy itor osimertinib, plasma circulating tumor DNA analyses have [HR 0.46; 95% confidence interval (CI), 0.37–0.57; P < 0.0001; identified MET copy number gain after first line osimertinib in ref. 11]. The median duration of response was 17.2 months in approximately 19% of patients on the FLAURA trial and 15% after patients treated with osimertinib compared with 8.5 months in second-line osimertinib (22, 23). Updated analysis of TATTON the first-generation TKI cohort. The incidence of grade three or phase Ib study combining osimertinib and the MET TKI savoli- higher toxicities was lower with osimertinib (32%) than stan- tinib showed an ORR of (i) 33% in prior third-generation TKI dard treatment (41%; ref. 11). Although OS data have not yet exposed and (ii) 61% in patients without T790M mutation and matured, the compound has been adopted into first-line with no prior third-generation EGFR-TKI exposed cohorts (24). Second FDA approval. site mutations in the MET oncogene (D1228V) have been seen at resistance on the osimertinib and savolitinib trial demonstrating Role in brain metastasis the dependency on the pathway (25). Grade 3 adverse events Brain recurrence for patients on therapy is an unmet need with were experienced by 50% of patients, with the most common limited therapeutic options and contributed by challenging phar- all-cause events being vomiting (8%), AST/ALT increase (6%), macokinetics, drug efflux transporters, and molecular weight (17). and rash (6%). Savolitinib was discontinued for 30% of patients In preclinical models, osimertinib demonstrated the ability to because of adverse events (24). cross the blood–brain barrier (BBB) with a higher ratio of Osimertinib is also being combined with inhibitors in the unbound tissue concentration to unbound plasma concentration MAPK pathway. Resistance to the third generation EGFR inhibitor and higher substrate activity against permeability glycoprotein tool compound WZ4002 has been seen through amplification of (Pgp) and breast cancer resistance protein (BCRP) efflux trans- ERK through activation of the MAPK1 pathway or downregula- porters as compared with gefitinib (17, 18). Measurement of AUC tion of negative regulators of ERK signaling (26). The MEK tissue to plasma ratios showed osimertinib more highly distrib- inhibitor CI-1040 downregulates ERK1/2 phosphorylation and, uted in the brain (1.7–2.0) than gefitinib (0.21), rociletinib when combined with WZ4002, PC9 WZ4002-resistant cells were (<0.08), and afatinib (<0.36). Data from the BLOOM study responsive to the combination in vitro (27). In post-progression (NCT02228369) suggested encouraging activity with osimertinib patient samples, resistance involving the RAS–MAPK pathway has (160 mg daily) with a median treatment duration of 6 months been seen in patients treated with osimertinib, with acquired and manageable toxicity in patients with leptomeningeal disease; KRAS mutations and V600E BRAF mutations observed after 10 patients (43%) have had intracranial radiologic response and acquired resistance (28). Furthermore, preclinical work demon- 13 patients (56%) with stable disease (SD; ref. 18). Among strated that the concomitant inhibition of EGFR and ERK1/2 by patients in the AURA3 randomized phase III study, the median the MEK inhibitor trametinib prevents ERK1/2 reactivation, CNS PFS was significantly longer with osimertinib than with enhances anti-EGFR–induced apoptosis, and inhibits the emer- chemotherapy (11.7 months vs. 5.6 months; HR 0.32; P ¼ gence of resistance in EGFR-sensitive models known to acquire 0.004). CNS ORR was 70% (21/30) with osimertinib compared resistance via both T790M-dependent and T790M-independent with 31% ORR with chemotherapy pointing to the intracranial mechanisms. Resistance to the combination with trametinib may efficacy of the compound (12). In the first-line FLAURA study, emerge due to AKT/mTOR reactivation (29). In another study events of CNS progression were observed in 12 of 61 patients combining osimertinib with selumetinib in ex vivo primary cell (20%) in the osimertinib group and 26 of 67 (39%) in the cultures obtained from osimertinib plus selumetinib-resistant standard EGFR TKI group (11). CNS response rates in measurable tumors, the Hedgehog pathway was activated and a smoothened brain metastases were 91% with osimertinib and 68% with first- (SMO) inhibitor plus osimertinib and selumetinib inhibited generation EGFR TKIs (19). proliferation and migration of resistant cells (30). These collective

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data form the basis of the study of osimertinib and selumetinib onstrated significant tumor regression using in vitro models (35). (NCT02143466) with some responses seen at interim data anal- Ongoing clinical trials have combined EGF816 100 mg daily with ysis in the phase I study. The most commonly reported grade capmatinib 400 mg twice daily in patients with advanced NSCLC >3 adverse events were diarrhea (21%), dermatitis acneiform with EGFR L858R, ex19del, or T790M in several lines of therapy (15%), and hypokalemia (10%; ref. 20). including T790M negative or treatment naive patients Osimertinib in combination with navitoclax is an ongoing (NCT02335944). The addition of either capmatinib or tepotinib phase Ib trial in patients with disease progression on an anti- after progression on gefitinib has been encouraging (ORR 47% EGFR TKI (NCT02520778). Inhibition of Bcl-2, a prosurvival and 45%, respectively, in MET amplified patients; refs. 39, 40). protein, prompts cells to undergo apoptosis by upregulating and dephosphorylating BIM, a proapoptotic protein from Bcl-2 family Rociletinib (CO-1686) components. ABT263 (navitoclax) is a dual inhibitor of BCL-xL Rociletinib is an irreversible inhibitor of mutant EGFR includ- and Bcl-2, which induces BIM by decreasing the capacity of BCL-xL ing exon 19 deletion, L858R, and T790M binding at the C797 to neutralize BIM and facilitates Bax and Bak to initiate caspases, locus. Enrollment of trials with rociletinib ceased in May 2016 leading to cell death. In a drug screen with EGFR T790M patient after updated results in November 2015 showed a less than derived cell lines, navitoclax in combination with the tool com- expected ORR of 33.9% and PFS of 5.7 months for patients with pound WZ4002 induced significantly more apoptosis compared confirmed T790M-positive NSCLC (41). The FDA formed an with the EGFR inhibitor WZ4002 alone (31). independent expert panel and voted 12–1 against approval of Downstream targeting of the mTOR pathway is being studied in rociletinib based on existing clinical trial data. When retrospec- a phase II trial of sapanisertinib (INK128), a potent mTORC1/2 tively analyzing the profound difference in ORR between April inhibitor, in combination with osimertinib for patients with and November 2015, over half of the patients had unconfirmed advanced EGFR-mutated NSCLC (NCT02503722). Preclinically, partial responses (41). Post-analysis revealed 22 of 69 patients inhibition of TORC1 and TORC2 decreased cell growth, metab- (32%, 15 patients with T790Mþ disease and 7 patients with olism, and angiogenesis. mTOR kinase inhibition via INK128, T790M disease) had brain metastases that developed before which involves ATP-dependent phosphorylation of proteins S6 discontinuing rociletinib likely due to the poor BBB penetration and 4EBP1 downstream of TORC1, leads to selective inhibition of of the compound. In patients who progressed after rociletinib and AKT phosphorylation (32). Novel mTOR mutations have been were subsequently treated with osimertinib, there was favorable seen on EGFR-resistant patient samples, and in mutant cells response with osimertinib in five of nine patients (two PR and continuation of EGFR and mTOR inhibition fully abrogated three SD). Three patients who developed brain metastases on EGFR downstream signaling (33). Additionally, osimertinib rociletinib had demonstrated improved CNS disease responses on has been combined with JAK 1 inhibitors interrupting signaling osimertinib (42). The experience with rociIetinib has demonstrat- of the JAK/STAT pathway in an ongoing second-line study ed that each EGFR inhibitor may have different off-target effects in T790M mutant patients (INCB39110, NCT02917993 and (i.e., IGFR activation leading to hyperglycemia attributed to a AZD4205, NCT03450330). metabolite of rocelitinib and occurring in 22% of patients), A phase I dose-escalation study of osimertinib combined with differential selectivity against the target, and different effects in necitumumab, a humanized EGFR monoclonal antibody, is CNS metastases affecting overall response rate. underway for patients who progressed on previous first-line TKI therapy (NCT02789345). Recent work demonstrates that ampli- Olmutinib (HM61713) fication of EGFR wild-type alleles can confer resistance to EGFR Olmutinib (HM61713) began clinical evaluation in May 2015 TKIs (34). By binding to extracellular domain of the EGFR for T790M-positive mutant NSCLC and was approved for treat- receptor and competing with ligand binding, downsteam signal- ment of in South Korea. In phase II ELUXA trials, 76 patients ing leads to impaired tumor cell growth. included in the phase II expansion after first- or second-generation TKIs received 800 mg/day with median treatment duration of 7.0 Nazartinib (EGF816) months. Of the 71 evaluable patients, 54% demonstrated an EGF816 is an irreversible, mutant-selective EGFR inhibitor that objective response, 90% achieved disease control, mean duration targets L858R, exon 19 deletion, T790M mutations, and is also of response was 8.3 months, and median PFS of 6.9 months (95% being explored for efficacy against exon 20 insertion mutations, CI, 5.5–8.3 months; ref. 43). The most common adverse effects which are intrinsically resistant to first- and second-generation included diarrhea (59%), rash (41%), pruritus (42%), and nausea inhibitors (35). A phase I study of EGF816 showed an ORR of (39%). Further ELUXA trials have been stopped due to incidence 44% and DCR of 91% with a median duration of response of of skin toxicity including toxic epidermolysis necrosis (n ¼ 2, one 9.2 months and grade 3 or 4 adverse events including diarrhea fatal) and Stevens-Johnson syndrome (n ¼ 1; ref. 43). The side- (6%), rash (14%), and anemia (6%; ref. 36). In 14 patients who effect profile of this compound is unfavorable relative to osimer- received EGF816 and then osimertinib, the ORR to osimertinib tinib, which has affected its use although the compound had been was 14% (one CR, one PR, eight SD, four PD) with a median approved in South Korea. continuation of osimertinib therapy for 9 months (37). Documented mechanisms of acquired resistance to EGF816 Naquotinib (ASP8273) include EGFR C797S mutations, deletion in mTOR, BRAF fusions, Not all third-generation compounds have shown superiority to c-MET amplification, and concurrent TP53 and RB1 truncating first- and second-generation inhibitors in the first-line. ASP8273 is mutations associated with small cell transformation (38). It is a small irreversible TKI that binds at C797 and targets the unclear if the prevalence of resistance alterations with this com- common EGFR mutations including T790M while sparing pound differs significantly to osimertinib. Dual inhibition of wild-type EGFR. First-line ASP8273 resulted in an ORR of EGFR and cMET with EGF816 and capmatinib (INC280) dem- 52%, disease control rate (DCR) of 94% and PFS of 11.3

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months (44). Among patients treated as second-line therapy, the inhibits activation by displacing the C-helix. With the addition confirmed ORR was 30% and median PFS for T790M-positive of cetuximab, dimerization is inhibited creating uniform vulner- patients treated was 6.0 months (95% CI, 5.3–9.8 months; ability within the kinase. EGFR del 19 or L858R/T790M/C797S ref. 45). Grade 3 adverse events included diarrhea (4%), hypo- cells are resistant to gefitinib, afatinib, WZ4002, rocelitinib, and natremia (17%), and a grade 2 peripheral neuropathy (n ¼ 1) osimertinib; however, triple mutant tumor cells were partially was noted (45). The decision to discontinue development in sensitive to cetuximab (50). These data provide preclinical ratio- the SOLAR first-line study was based on the recommendation nale for the combination of a next-generation inhibitor in com- of the Independent Data Monitoring Committee comparing bination with an EGFR monoclonal antibody for patients with lower PFS and ORR to the comparators, erlotinib or gefitinib. NSCLC with the triple mutation profile. The development of Differential selectivity with ASP8273 against the original EGFR inhibitors that do not bind C797 will be important for patients activating mutations compared with T790M may have contrib- who develop this resistance. One such compound AC0010 is a uted to the efficacy results compared with first- or second- structurally distinct pyrrolopyrimidine-based irreversible inhibi- generation TKIs in the first-line. tor, and in a xenograft model oral administration of AC0010 at a daily dose of 500 mg/kg resulted in remission of tumors with Mavelertinib (PF-0647775) EGFR-active and T790M mutations for over 143 days with no PF-0647775 is a third-generation EGFR TKI currently being weight loss (51). Objective responses have been observed in studied in patients with mutated EGFR ex19del or L858R with or patients with NSCLC with EGFR T790M mutation on this com- without T790M mutation and has completed dose escalation pound, although it has of yet to be tested after osimertinib for from 25 to 600 mg. RECIST responses observed include 11 PRs C797S resistance. (42.3%, five T790Mþ plasma), six stable disease (23.1%, four Additional EGFR second site mutations have been detected on T790Mþ plasma), two PD (7.7%, one T790Mþ plasma), and six therapy. EGFR-mutant cells in vitro exposed to increasing quan- indeterminate (23.1%, three T790Mþ plasma) after unconfirmed tities of WZ4002 acquired resistance through several additional investigator review. Grade 3 adverse events were seen primarily at second-site EGFR mutations including EGFR L718Q, L844V, greater than 150 mg dose and included diarrhea (12.4%) and skin L798I, L692V, E709K. The full impact and biologic consequences toxicities (30.8%; ref. 46). Further analyses will evaluate the of the second site mutations is not clear at this time. EGFR L718Q efficacy and tolerability of the compound in combination with and L844V have been seen after pyrimidine based inhibitors, palbociclib, an inhibitor of the cyclin-dependent kinases CDK4 however have occurred more frequently after rociletinib and and CDK6 based on preclinical work demonstrating synergy WZ4002 than osimertinib (52). In the clinic, resistance due to (NCT02349633). the L718Q was noted after a patient with an activating EGFR L858R mutation was treated with gefitinib and subsequently with Mechanisms of TKI Resistance with Second osimertinib for T790M progression (53). Additional case reports describe other second site mutations such as G724S, G796D, and Site Mutations in EGFR L792H on osimertinib therapy. The second-generation inhibitor Given that third-generation EGFR inhibitors irreversibly bind afatinib has been responsive to the acquired G724S muta- at the C797 location, mutations at this locus within the EGFR tion (54). E709K and L682V, both activating missense substitu- gene confer resistance and estimated to be present in 7% to 20% tions, were seen after exposure to rociletinib. EGFR L798I is a of cases (Fig. 1A–E; refs. 22, 23). In patients who progress novel mutation observed in cis conformation with T790M that on osimertinib circulating tumor DNA analyses have shown affects the covalent bond formation of rociletinib at C797 (55). that T790M may be retained with C797S acquisition (47). When Although sensitivity of EGFR TKIs to some uncommon mutations C797S and T790M are in a cis conformation on the same allele are known (L861Q, G719X, S768I), further work will be needed to (82%), EGFR TKI single agents or combinations are unable to determine the sensitivity of different anti-EGFR TKIs across a suppress activity. When the mutations are in the trans confor- diversity of second-site EGFR TKI mutations and the impact of mation on different alleles, patients have seen responses to the sequence of therapy on outcome (56). Genome editing using combination of first- and third-generation EGFR TKIs (48, 49). CRISPR-Cas9 technologies has been used to create preclinical C797S and T790M mutations were sensitive in preclinical mod- models for drug testing and will improve our ability to investigate els to a combination of gefitinib with WZ4002, a tool com- effective therapy combinations (57). pound developed as a third-generation TKI (49). There have been case reports of clinical efficacy with third- and first-gener- Mechanisms of Resistance in Alternate ation inhibitors with clinical trials ongoing (NCT03122717). Furthermore, as third-generation compounds have moved to the Bypass Pathways first line, clinical trials revisiting reversible first-generation inhi- A recent neoadjuvant study of erlotinib for locally advanced bitors to combat C797 resistance after third-generation inhibi- resectable EGFR-mutant patients has demonstrated that only a tors are being explored. few patients (3/25 pts) had a major pathologic response (<10 Additional novel drug combinations are being explored to viable tumor cells) after first-generation inhibitors (58). This overcome EGFR C797S resistance. EA1045 is an allosteric EGFR highlights that resistance may occur early with mechanisms of inhibitor that in combination with cetuximab prevents effective resistance that are diverse and independent of the EGFR pathway. EGFR dimerization. This compound has been shown to be Continued cell growth and proliferation despite the reduction effective in mouse models of NSCLC with EGFR (L858R/ and disappearance of T790M mutant clones may signify reliance T790M/C797S) mutation by displacing the regulatory C-helix on a different mechanism of resistance or bypass pathways apart inactive conformation of the kinase (50). The allosteric pocket is from EGFR (Fig. 2; refs. 28, 55). Escape mechanisms following accessible in two subunits of the dimer, and the compound therapy with third-generation EGFR TKIs are heterogeneous and

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Clonal Evolution of EGFR Mutant Lung Cancer on Therapy

Chemotherapy Progression

Osimertinib T790M+/– 2nd site mutation T790M+/– 2nd site mutation (i.e C797S) (i.e C797S, G724S)

Erlotinib eted therapy Geﬁtinib Tar g Afatinib T790M+ against bypass Pathway

Chemotherapy

Osimertinib Progression Progression T790M– Alternate bypass pathways

T790M–

EGFR activating mutation clone ∗MET ampliﬁcation, HGF expression, HER2 ampliﬁcation, Alternate bypass mechanism∗ PIK3CA mutations, PTEN loss, KRAS mutations, NRAS mutations, BRAF mutation, MAPK1/AKT3 overexpression, EGFR T790M mutant clone FGF20FGFR1 loop mutation, IGFR1 activation, RB1/p53 loss, EGFR additional second site mutation (ie C797S) small cell histologic switch, fusion events

Figure 2. Clonal heterogeneity through EGFR-targeted therapy. Visual representation of minimal residual disease and malignant cell progression after anti-EGFR TKIs demonstrating genomic patterns of selection observed in clinical samples through different lines of therapy.

may include EGFR amplification, HER2 amplification, MET a monoclonal anti-IGF1/2 antibody sensitized WZ4002 resis- amplification, hepatocyte growth factor (HGF) expression, tant, PC-9/GR/WR cells to WZ4002 in in vitro and xenograft PIK3CA mutations (E545K and E542K), PTEN loss, KRAS muta- mice (63). FGR receptor (FGFR1) amplification has been seen tions, NRAS mutations, BRAF mutation, MAPK1/AKT3 overex- as an acquired resistance mechanism for first-generation EGFR pression, FGF2-FGFR1 loop mutations, IGF1R activation, fusion TKIs, and a patient who had disease progression on osimertinib events, and RB1/p53 loss associated with histologic switch to had FGFR amplification at re-biopsy with disappearance of the small cell cancer (Fig. 2; Supplementary Fig. S1; refs. 26, 28, 59– EGFR T790M clone (59). Several novel fusions have been 63). Amplification with copy number gain across other ERBB identified following first-, second-, and third-generation com- family receptors have been appreciated in samples post osimerti- pounds including RET fusions, FGFR fusions, and BRAF fusions nib compared with first- and second-generation inhibitors (22, with clinical responses seen in inhibitors that target the fusion 60). Additionally, shorter progression-free survival was identified event (28, 64). The role of polymorphisms influencing resis- in patients with coexisting oncogene drivers including cell-cycle tance profiles or third-generation drug sensitivities have yet to genes (60). Small cell transformation has been associated with remain to be explored. both Rb and p53 inactivation, and mutation in apolipoprotein B The transcriptional state change known as epithelial-to- mRNA editing enzyme, catalytic polypeptide-like (APOBEC) is mesenchymal transition (EMT) is another important resistance frequently seen (61). Patients with shorter time on therapy before mechanism to targeted therapies including EGFR inhibi- progression likely develop resistance that is independent of the tors (65). The precise mechanisms underlying EMT are still EGFR pathway (28). being investigated, but suppression of BIM and upregulation of Upregulated MET has been well characterized, and upregulated AXL, SRC, TGFb, and IFG1R have all been associated with EMT HER2, IGFR, and FGFR have been observed. In preclinical models, andresistancetotargetedtherapy(65–68). Of note small HER2 overexpression or knockout confers resistance or sensitivity molecule inhibitors of the kinase AXL have been shown to respectively in EGFR-mutant PC9, HCC827, and H3255 cells (62). re-sensitize resistant mesenchymal cells to EFGR inhibitors, Furthermore, resistance profiling against WZ4002 has demon- suggesting AXL as a potential therapeutic target in resistant strated activated insulin-like growth factor receptor (IGF1R) NSCLC (67). A clinical trial of DS-1205c, an orally adminis- signaling due to loss of expression of EGFBP3 (27). In preclin- tered AXL inhibitor, is currently in dose escalation in combi- ical studies, IGF1R downregulation via an IGF1R inhibitor and nation with osimertinib (NCT03255083). BGB324 is an AXL

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inhibitor being explored with first-generation EGFR TKIs Immune combinations with antiangiogenic strategies have (NCT02424617). been undertaken and further data is awaited to determine if In addition to the careful molecular characterization of resis- there is true synergy with this approach. Overall, antiangiogenic tant tumors, high-throughput screening with CRISPR-Cas9 tech- therapies in combination with EGFR-TKIs have shown encour- nology is a means to discover additional potential resistance aging synergy but limited overall survival benefit. Preclinical mechanisms (69). The nuclease Cas9 can be directed with great work with VEGF inhibitors including vandetinib or erlotinib/ specificity using a short RNA barcode allowing for the rapid bevacizumab were more efficacious than gefitinib or erlotinib knockout of any gene. These barcodes can be read using next- alone (76). The addition of bevacizumab to erlotinib resulted generation sequencing, and CRISPR knockout screens can be in an improved PFS compared with erlotinib alone as initial highly multiplexed allowing for genome-wide analyses (70). treatment (16 months vs. 10 months; HR 0.41; ref. 77). However, Synthetic lethal approaches have revealed mechanisms that can the phase II study ACCRU (NCT01532089) did not report drive both oncogene and non-oncogene EGFR-resistant NSCLC. an improvement in PFS and OS, and a phase 1/2 study Non-oncogene–dependent mechanisms include a host of factors (NCT02803203) is currently assessing osimertinib and beva- including NF-kB, STAT3, Wnt, src family kinases, and chromatin cizumab as an initial treatment for patients with EGFR-mutant remodeling proteins (71). Genome-wide knockout screens in lung cancers. First-line osimertinib and chemotherapy combi- sensitive and resistant NSCLC cell lines can be conducted with nations have been brought forward with the goal of eradicating various EGFR inhibitors to identify de novo and acquired resistance minimal residual disease based on encouraging OS data with mechanisms that generalize and that are specific to a particular first-generation inhibitors (NEJ009). compound.

Unanswered questions Immune Combination Therapies The pattern of drug development in EGFR-mutant lung cancer Anti-PD-1:PDL1 therapies appear to be of limited benefitin has been a model for tumors driven by dominant oncogenic driver patients with EGFR-mutant NSCLC, and the role of checkpoint genes, changing multiple paradigms in clinical care. The FDA inhibitors in EGFR-mutant disease is poorly understood (72). approval of osimertinib and impressive median overall survival Larger studies with anti-PD1:PDL1 antibodies have not shown marks an important advance with simultaneous targeting of high response rates or improved overall survival in the subset of the EGFR activating mutation and the dominant resistance clone. EGFR-mutant patients. In a meta-analysis in NSCLC, immune Clinical trials are exploring targeted therapies in early stage checkpoint inhibitors did improve OS over docetaxel in the EGFR neoadjuvant and adjuvant settings (NCT 03433469 and NCT wild-type subgroup (n ¼ 1362; HR ¼ 0.66; P < 0.0001) but not in 02511106). Advances in genome sequencing technologies pro- the EGFR-mutant subgroup (n ¼ 186; HR 1.05; P ¼ 0.03; ref. 72). vide an opportunity for the serial measurement of circulating Preclinical data have shown that EGFR activation can upregulate tumor DNA to allow for detailed characterization of EGFR inhib- PD-L1 expression (73). However, even in high expressing PDL1 itor resistance through time and on therapy. The optimal order to EGFR-mutant patients, checkpoint inhibitors have had limited sequence EGFR inhibitors and whether combinations of EGFR clinical benefit and have been associated with toxicity with inhibitors or combinations with other targeted therapies can pneumonitis and transaminitis (NCT02879994). A trial of osi- prevent the development of resistance clones is an active area of mertinib combined with durvalumab (NCT02143466) has been investigation. Effective and tolerable combinations that target halted due to an increase incidence of ILD (26% in pretreated EGFR independent bypass pathways have been informed by patients and 64% in treatment na€ve patients; ref. 20). preclinical data from in vitro and patient derived xenograft exper- Hypotheses exploring the lack of response allude to lower imentation and data on resistance patterns collected from clinical tumor mutation burden (TMB) and a non-PDL1–dependent trials. Well-defined pre-clinical, pharmacokinetic, and pharma- immunosuppressive tumor microenvironment. A recent pub- codynamic testing has been employed with novel drug combina- lication has demonstrated that EGFR-mutated tumors generally tions to identify dose scheduling that allows for tolerable side have a low TMB, and higher TMB predicts worse response to effect profiles of combination therapy. anti-EGFR inhibitors (74). Mutations in p53 have been more Additional third-generation TKIs have been evaluated with common in EGFR-mutant patients with high TMB, and EGFR- mixed success, and current strategies are evaluating the first-line mutant tumors have higher TMB at the time of progression on utility of these compounds, sequencing strategies, drug combina- EGFR-TKI compared with pretreatment. Increased TMB may be tions to target cancer dependencies, and effects on brain metas- due to the emergence of subclonal mutations, and these muta- tases. Accounting for the complexity of resistance pathways, the tions may increase overall TMB but may not confer effective ability to characterize resistance clones through plasma-based immunogenicity (74). genomic strategies may assist in tracking cancer evolution through Using flow cytometry and/or quantitative PCR in cell lines, therapy. Novel technologies such as CRISPR-Cas9 gene editing median CD73 expression was increased 10-fold in EGFR- and integration of RNA sequencing analyses to identify bypass mutant NSCLC. Expression of the immunosuppressive mole- pathway activation will be important to better characterize the cule CD73 and reduced expression of the IFNg mRNA signature diversity of acquired resistance. Further understanding the biol- has been hypothesized to contribute to an immune suppressive ogy of the disease, the evolution and progression of clones on environmentinthissubsetofNSCLC(75).Atrialiscurrently therapy, the pharmacology of each inhibitor, polymorphisms, underway evaluating an anti-CD73 molecule (oleclumab, which affect selection and third-generation TKI drug sensitivity, MEDI9447) in combination with osimertinib and the adeno- and the resistance profiles seen with each compound will inform sine receptor antagonist (AZD4635) in EGFR-mutant NSCLC further clinical trials and may lead to more durable responses to (NCT03381274). targeted therapy.

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Search strategy and selection criteria in Tetraphase Pharmaceuticles and Illumina. H. Husain reports receiving other We identified references for this Review through search of commercial research support from Pfizer, AstraZeneca, and Mirati; has received PubMed (MEDLINE, Web of Science, Cochrane Library, etc.) speakers bureau honoraria from AstraZeneca, Merck, and Bristol Myers Squibb; and is a consultant/advisory board member of Astrazeneca, from 2001 to 2018, using the search terms NSCLC, EGFR, TKI, Boehringer Ingelheim, Foundation Medicine, and Abbvie. No potential osimertinib, resistance, heterogeneity in various combina- conflicts of interest were disclosed by the other authors. tions. We only reviewed papers and abstracts in English and fi primarily focused on recent clinical trials. The nal reference Acknowledgments list was based on originality and relevance to this Review's scope. The authors would like to thanks Soham More and Seti Akhavan for their contributions to this work. Disclosure of Potential Conflicts of Interest J. Paul Shen reports receiving other commercial research support from Received April 27, 2018; revised September 16, 2018; accepted December 17, Idyea Biosciences and has ownership interest (including stock, patents, etc.) 2018; published first February 4, 2019.

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698 Cancer Res; 79(4) February 15, 2019 Cancer Research

Novel Third-Generation EGFR Tyrosine Kinase Inhibitors and Strategies to Overcome Therapeutic Resistance in Lung Cancer

Ayesha Murtuza, Ajaz Bulbul, John Paul Shen, et al.

Cancer Res 2019;79:689-698. Published OnlineFirst February 4, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-18-1281

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