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Targeting driver mutations in non-small-cell lung cancer, beyond the usual suspects (EGFR and ALK)

D. Arias1,5, M.A. Flores2, Á. Rodríguez1, J. de Oliveira3, L. Corrales4, J.L. Firvida5, E.S. Santos6, L.E. Raez7, C. Rolfo1

SUMMARY In the last decade, systemic treatment for non-small-cell lung cancer has undergone an unprecedented change because of new targeted and the introduction of immunotherapy. Advances in the under- standing of lung cancer biology have led to the discovery of several oncogenic driver genes and the deve- lopment of drugs that target driver mutations, according to the strategy of ‘personalised ’. The best- known alterations are mutations and anaplastic kinase rearrangements, but the improvement in genomic technologies and the continuous research in this area have led to the identification of new druggable targets. This is a comprehensive overview focused on the development of targeted therapies and their mechanisms of action. (BELG J MED ONCOL 2018;12(5):223-232)

INTRODUCTION mutations and targeted agents. The discovery of epider- During several years, the treatment for advanced non-small- mal (EGFR) mutations and anaplas- cell lung cancer (NSCLC) has been cytotoxic chemothera- tic lymphoma kinase (ALK) gene rearrangements has led to py since the introduction of platinum-based doublets. This the development of EGFR tyrosine kinase inhibitors (TKIs) treatment is still the standard of care in the majority of the and ALK inhibitors, which nowadays are approved in NS- patients, reaching median survivals of 7.9 months.1 Nev- CLC, showing significant survival increase and impact in ertheless, the discovery of key oncogenic alterations and overall response rate (ORR) and progression free surviv- immune checkpoints in the past decade and the develop- al (PFS).2,3 Approximately 60% of patients with NSCLC ment of tailored drugs have changed entirely the treatment have an oncogenic driver mutation, which is more preva- of lung cancer. The implementation of broad-based ge- lent in adenocarcinoma, female patients and non-smokers. nomics and high-throughput sequencing analysis has de- Nowadays, several driver mutations – apart from EGFR and veloped personalised strategies based on oncogenic driver ALK aberrations – have been identified in lung cancer, and

1Phase I - Early Clinical Trials Unit, Antwerp University Hospital & Centre for Oncological Research (CORE), Antwerp, Belgium, 2Oncology Department, Hospital Privado de la Comunidad, Mar del Plata, Argentina, 3Oncology Department, Portuguese Oncology Institute (IPO-Porto), Porto, Portugal, 4Oncology Department, Hospital San Juan de Dios, San Jose, Costa Rica, 5Oncology Department, Complejo Hospitalario Universitario de Ourense, Ourense, Spain, 6Oncology Department, Lynn Cancer Institute, Boca Raton, FL , USA, 7Thoracic Oncology Program, Memorial Cancer Institute, Memorial Health Care System, Pembroke Pines, FL, USA. Please send all correspondence to: C. Rolfo, MD, PhD, Head of Phase I- Early Clinical Trials Unit, Antwerp University Hospital, Wilrijkstraat 10, 2650, Edegem, Belgium, email: [email protected]. Conflict of interest: The authors have nothing to disclose and indicate no potential conflict of interest. Keywords: BRAF, driver mutations, FGFR, KRAS, MET, NSCLC, NTRK, RET, ROS1, targeted therapies.

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Squamous NSCLC Non-squamous NSCLC

EGFR (5%) EGFR (15%)

FGFR1 (20%) ALK (6%) KRAS (25%) FGFR2 (3%) ROS1 (2%) PI3KCA (14%) MET (4%) KRAS (4%) RET (2%) PDGFRA (9%) NTRK (3%) PTEN (19%) BRAF-V600 (2%) Unknown (26%) FGFR1 (2%) HER2 (2%) PI3KCA (1%) >1 mutation (3%) Unknown (33%)

FIGURE 1. Frequencies of driver mutations in squamous and non-squamous NSCLC.

the list of druggable targets that have shown clinical action- that leads ROS1 gene fusions to constitutive activation of ability is increasing. This manuscript is a comprehensive Ras/Raf/MEK/ERK, PI3K/AKT and STAT3 signalling path- overview of genomic alterations and oncogenic drivers be- way is due to chimeric proteins with persistent tyrosine ki- yond EGFR mutations and ALK rearrangements, including nase activity.4,5 c-MET amplification andexon 14 skipping mutation, BRAF and Molecular defined ROS1+ NSCLC shares some similar KRAS mutations, ROS1, RET and NTRK1 fusions and FGFR clinic-pathological features with ALK gene rearrangement alterations. This study aims to discuss the molecular back- patients: young age, never/light-smoking history and ade- ground of every pathway, the current evidence of the new nocarcinoma histology. The ROS1 tyrosine kinase is very inhibitors including ongoing clinical trials and their mech- sensitive to due to a high degree of homolo- anisms of resistance. gy between the ALK and ROS tyrosine kinase domains. Based on this, one phase I study (PROFILE 1001) was do- ROS1 REARRANGEMENT ne, where 50 patients with advanced NSCLC harbouring ROS1 protein is a without known ROS1 fusion were included, in an expansion cohort with ligand, which is involved in anti-apoptotic and pro-surviv- a crizotinib test for ALK-positive NSCLC.6 The ORR was al signalling with carcinogenic properties. This proto-onco- 72% with three complete responses and 33 partial respons- gene is located in chromosome 6q22, and this rearrangement es. The PFS was 19.2 months, with 25 patients still in fol- is found in around 1-2% of NSCLC.4 It has shown to be ex- low-up for progression at the end of the study in 2014, and clusive with other driver mutations such as ALK, EGFR and the OS rate was 85% at twelve months. After these out- KRAS. Many different protein fusion partners have been standing results, the Food and Drug Administration (FDA) described, where CD74 is the commonest. The mechanism approved the use of crizotinib, for patients with the ROS1

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TABLE 1. Ongoing clinical trials targeting KRAS, FGFR and NTRK.

Drug target Tested agent Phase Trial ID number

KRAS II NCT01306045 Selumetinib + vistusertib (TORCMEK trial) I/II NCT02583542 Selumetinib + docetaxel II NCT00890825 + docetaxel II NCT01362296 Trametinib + navitoclax I/II NCT02079740 Trametinib + I NCT02258607 (JUNIPER trial) III NCT02152631 + PD-0325901 I/II NCT02022982 Momelotinib I NCT02258607 Antroquinonol II NCT02047344 + PD-0325901 I/II NCT02039336 (MEK162) + I NCT01859026 FGFR Lucitanib II NCT02109016 II NCT01877083 Lenvatinib + I/II NCT02501096 I NCT02299141 Nintedanib + cisplatin + docetaxel I NCT02225405 II NCT01935336 BGJ398 II NCT02160041 AZD4547 II NCT02117167 ARQ 087 I/II NCT01752920 Erdafinib II NCT02699606 TAS 120 I/II NCT02052778 NTRK (STARTRK-1) I NCT02097810 Entrectinib (STARTRK-2) II NCT02568267 LOXO-101 (NAVIGATE) II NCT02576431 Altiratinib (DCC-2701) I NCT02228811 PLX7486 I NCT01804530 DS-6051b I NCT02279433 TSR-011 I NCT02048488 Sitravatinib (MGCD516) I NCT02219711 II NCT01639508 Merestinib II NCT02920996

translocation; including patients that have received che- showed a PFS of 19.1 months and a 75% of ORR in a sub- motherapy and treatment-naïve patients.6 group of 32 ROS1+ patients.7,8 , an FDA approved Currently, several trials are ongoing in order to test novel drug for treatment of ALK+ NSCLC, is also a type I inhibitor ROS1 inhibitors (Table 1). Entrectinib is an oral multi-kinase of ROS1, and is included in an ongoing phase II trial.9 On the molecule inhibitor of ROS1, ALK and TRK that has been test- other side, single mutations in the ROS1 kinase domain (es- ed in a phase I trial including solid tumours that harbour pecially G2023R and D2033N) have been described as those ROS1, ALK or NTRK rearrangements (STARTRK-1), and responsible for crizotinib resistance. While entrectinib and has shown 75% of partial responses in eight ROS1+ patients. ceritinib have not shown clinical activity against them, lor- Recently, the first results of the phase II trial STARTRK-2 latinib, an ATP-competitive inhibitor of ALK and ROS1, has

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TABLE 2. Ongoing clinical trials targeting ROS1, BRAF, c-MET and RET.

Drug target Tested agent Phase Trial ID Number

BRAF + trametinib II NCT01336634 LY3009120 I NCT02014116 PLX8394 I/IIa NCT02428712 RET Lenvatinib II NCT01877083 II NCT02540824 Cabozantinib II NCT01639508 Ponatinib II NCT01813734 Vantedanib II NCT01823068 Vantedanib + I NCT01582191 c-MET Crizotinib I NCT00585195 I NCT01324479 Capmatinib II NCT2412139 Capmatinib + II NCT01610336 Glesatinib II NCT02544633 Cabozantinib II NCT01639508 Tepotinib + gefitinib I/II NCT01982955 SAR125844 II NCT02435121 + erlotinib II NCT01900652 ROS1 Crizotinib (EUCROSS) II NCT02183870 Entrectinib (STARTRK-2) II NCT02568267 Ceritinib (SIGNATURE) II NCT02186821 I/II NCT01970865 Cabozantinib II NCT01639508

demonstrated clinical activity against D2033N mutation in in lung adenocarcinoma (25%) than in squamous cell car- a phase I trial. Another postulated mechanism of resistance cinoma (4%). Nevertheless, it is frequently associated with is alternative EGFR activation, suggesting a possible bene- smoking and has a lower expression in East Asian patients, fit of dual inhibition (ALK/ROS1+ EGFR) in some patients.8 unlike other alterations as EGFR and ALK. The majority of KRAS mutations occur at exons 2 and 3 involving codons KRAS 12 and 13, with a lower frequency in codon 61. Usually, the KRAS is part of the RAS family of oncogenes, which encodes presence of a KRAS mutation seems to be mutually exclu- four different proteins including KRAS4A and KRAS4B splice sive with other oncogenic drivers, but, recently, studies have variants, HRAS and NRAS. These proteins are guanine tri- demonstrated that EGFR and ALK rearrangement can mutu- phosphate (GTP) kinases whose activating isoforms act as ally coexist with KRAS mutations.10,11 mediators of the mitogen-activated protein kinase (MAPK), Since its discovery, no specific drugs have yet demonstrated JAK/STAT and phosphoinositide 3-kinase pathways, leading efficacy forKRAS mutants. Direct KRAS blocking has prob- to stimulate a complex network involved in regulation of pro- ably failed due to high affinity ofKRAS for GTP/GTD, the ab- liferation, angiogenesis, differentiation and apoptosis.10 KRAS sence of known allosteric sites and the appearance of parallel function depends on the relationship of GTP and guanine di- compensatory pathways.12 The first attempt with farnesyl phosphate (GDT), knowing that GTP activates KRAS and GDT transferase inhibitors, including a phase II testing salirasib, inhibits it and is being regulated by the GTP kinase activity. has not reached significant activity in aKRAS mutated NS- Mutations in the KRAS gene are present in different malig- CLC cohort. Owing to these facts, block of downstream tar- nancies including NSCLC, with a higher prevalence detected gets of the Ras/Raf/MEK/ERK pathway has resulted in a more

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hopeful strategy. Selumetinib is a MEK inhibitor tested in a vorship, motility, invasiveness and protection from apopto- phase II trial combined with docetaxel in advanced KRAS+ sis depending on the cellular context.15,16 NSCLC, increasing ORR and PFS compared with docetaxel The MET pathway is dysregulated in several cancers, hav- monotherapy. Unfortunately, these results weren’t main- ing found three types of alterations: overexpression, ampli- tained in the SELECT-1 phase III trial without differences in fication and mutation.17 Degradation of the MET receptor PFS and OS between the groups. Trametinib, another MEK depends on ubiquitination by a Cbl protein ligase; MET ubiq- inhibitor, has not yet shown benefits in monotherapy com- uitination is voided by a receptor mutation of tyrosine residue pared with pemetrexed or docetaxel and provides a 60% dis- 1003 in the binding domain of E3 ubiquitin-protein ligase ease control rate combined with docetaxel but regardless of c-Cbl, leading to high MET protein levels and stability. These KRAS status. Nowadays, MEK inhibitors become part of com- splice site mutations resulted in MET exon 14 skipping and are binational therapies in KRAS mutant lung cancers. Based on related with the Y1003 location in the juxtamembrane region the preclinical data suggestion that Bcl-XL plus MEK inhi- of MET, which is coded by exon 14. Exon 14-spliced protein bition promote synthetic lethal interaction, trametinib plus voided c-Cbl E3 ligase binding, leading to decreased ubiquiti- navitoclax, a Bcl-2 inhibitor, are being tested in a phase I nation and increase in MET protein levels. Furthermore, MET study.13 New ongoing trials are described in Table 2. Y1003 mutation decreased ubiquitination but also increased Owing to selective cyclin-dependent kinases (CDK) 4 and stability of the MET protein. This splice variant is not restrict- 6, which have an important role in KRAS-driven oncogene- ed to NSCLC (most of them adenocarcinomas) but has also sis, CDK inhibitors are also being tested intending to induce been detected in gastro-intestinal tumors.17,18 MET amplifica- synthetic lethality.10 Abemaciclib was tested in a phase I tri- tion has been reported in 2-4% of NSCLC, while MET exon al showing clinical activity against KRAS mutant patients, 14 skipping has been found in 3-4% of lung adenocarcinoma. achieving 54% DCR compared to 37% of wild-type patients; Increased MET expression is related with higher aggressive- the phase III trial JUNIPER of abemaciclib versus erlotinib ness and acts as a negative prognostic factor.19 in previously treated KRAS patients is ongoing.14 Moreover, Several approaches targeting MET have been investigated, palbociclib, another CDK4/6 inhibitor, has shown preclin- but usually the trials did not select for MET specific patient ical activity combined with MEK inhibitor trametinib in a cohorts.20 MET pathway inhibitors like the HGF antibody fi- phase I/II study.14 KRAS mutations often coexist with PI3K- clatuzumab failed to improve PFS or OS with combination AKT-mTOR pathway and modulate resistance to PI3K inhi- therapy of and gefitinib versus gefitinib alone. bition; nevertheless, , a mTOR inhibitor, failed In a phase II the monovalent antibody had im- to demonstrate a preclinical objective response rate in KRAS proved PFS and OS in MET population, but phase III failed mutated NSCLC patients, but improved PFS and OS. Due to establish this efficacy result in combination with erlotinib. to these results, a phase I trial tested selumetinib combined Possibly part of this lack of benefit is the fact that there is no with MK-2206 (an AKT inhibitor), achieving 23% of objec- standard method for detecting MET related anomalies that tive response. Momelotinib is a JAK/TANK-binding kinase allows adequate selection.21 Crizotinib, a competitive ATP in- 1 inhibitor that has shown synergy combined with MEK in- hibitor selective for ALK and ROS1, was originally developed hibition, and the hypothesis of reduced tumorigenesis is be- as a MET inhibitor and has recently shown antitumor activi- ing investigated in a phase I trial. Future research lines in ty in a phase I trial of NSCLC harbouring MET exon 14 alter- preclinical models probably include glutaminase inhibition, ations, with a response rate as high as 50% in the subgroup combination of mTOR and HSP90 inhibitors, and also FG- with high MET/CEP7 ratio. Other examples are capmatinib, FR1 inhibitors.13 whose results from a phase I trial demonstrated clinical ac- tivity chiefly in patients with high cMET expression; and MET glesatinib, a potent inhibitor of MET and AXL that confirms The MET protein, encoded by the MET proto-oncogene, is a tumour regression in NSCLC models of MET exon 14 skipping. transmembrane receptor tyrosine kinase (RTK) located on Both phase II trials are in progress (Table 2).20 chromosome 7q21–q31 and activated by an endogenous li- MET amplification has also been associated with anti-EGFR gand, the .15 MET is activated when secondary resistance; there have been several phase II stud- the HGF ligand binds to the MET receptor. It promotes MET ies that demonstrated increased efficacy with combined tar- homodimerisation and phosphorylation of tyrosine residues, geted therapy in patients with acquired MET amplification which mediates activation of downstream signalling path- and resistant EGFR mutation.17,20 A phase II trial, where emi- ways including MAPK, PI3K/AKT, Ras-Rac/Rho, NF-kB and betuzumab was combined with erlotinib, found that double phospholipase C, which are involved in cell growth, survi- inhibition was unable to overcome acquired resistance to er-

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lotinib. Recently, capmatinib combined with EGFR inhibitor with KIF5B-RET-fusion. These findings suggest that intrinsic gefitinib has shown increased activity in a group ofEGFR resistance mechanisms to RET inhibition or different efficacy wild-type patients, with a disease control rate of 80% and probably exist depending on fusion partners. Recent molec- an ORR of 30%. Tepotinib plus gefitinib showed a partial ular studies in patients treated with cabozantinib have iden- response and a stable disease in a subgroup of patients with tifiedMDM2 as a mechanism of acquired resistance. Besides higher protein expression, especially in IHC 3+ tumours. RET, cabozantinib also inhibits MET, VEGFR2, ROS1, and This demonstrates a probable relationship between clinical AXL, which could explain a decreased therapeutic window.19 response to MET inhibition and higher levels of amplifica- Although RET-inhibitors have not yet been approved, there tion and protein expression.20 are several clinical trials looking at the efficacy of multiki- nase inhibitors for NSCLC harbouring RET-rearrangements RET (Table 2). Novel compounds have been identified as good RET (rearranged during transfection) is a proto-oncogene candidates. Linn et al. observed encouraging clinical activity located at chromosome 10q11.2, which encodes a family of of , a RET and ALK inhibitor void of VEGFR2/KDR RTK. RET fusions have been reported in 1-2% of NSCLC targeting, in three patients pre-treated with other RET in- and are more common in adenocarcinomas, never smokers hibitors.24 Moreover, in view of the biology of RET signalling, and women. KIF5B-RET driver mutation has been identified combination treatment strategies should be proved. Vande- in approximately 90% of NSCLC with RET rearrangement, tanib was shown to abrogate signalling through the MAPK and fusion partners CCDC6, NCOA4 and TRIM33 represent pathway with effects on effectors of mTor; a phase I study of the remaining 10%.22 plus everolimus showed significant activity in Glial cell-derived neurotrophic factors are the ligand for RET RET-rearranged lung cancers. It appears that MEK and PI3K receptors; the ligand binding leads to activation of the in- inhibitors are good candidates for combination therapies. tracellular kinase domain and to autophosphorylation of tyrosine residues, which acts as a checkpoint for proteins BRAF carrying SRC or phosphotyrosine-binding domains, trans- BRAF is a serine/threonine protein kinase that belongs to the mitting signals to the cell. This leads to activation of intracel- RAF family, which components are involved in the RAS-RAF- lular pathways, including RAS/ERK, PI3K/AKT and STAT3, MEK-ERK signalling pathway. Growth factors, as EGF, cKIT involved in proliferation, differentiation and motility.22 RET or FGF, bind to their respective RTKs leading to dimerisa- gene rearrangements result in formation of fusion proteins tion and autophosphorylation of these receptors and down- capable of activating the ligand independent kinase, which stream activation in parallel of several pathways (RAS, PI3K, leads to increasing function signalling and subsequent neo- mTor).25 RAS stimulates RAF serine-threonine kinases A, B plastic transformation. and C, with MEK as the exclusive target of B-Raf. B-Raf phos- Nowadays, RET-specific inhibitors have not yet been devel- phorylation is involved in MEK and ERK activation, which oped, but since its appearance it was observed that several leads to the increase of several cell mechanisms as growth, multikinase inhibitors, such as vandetanib, and proliferation, differentiation and apoptosis inhibition. If a mu- , supressed growth of RET-rearranged cells, lead- tation on the B-Raf occurs, the continuous activated state of ing to numerous clinical trials looking at the efficacy of the MAPK pathway leads to uncontrolled growth and negative these and other multitargeted tyrosine kinase inhibitors in modulatory feedback signals caused by MEK and ERK activa- RET-rearranged lung cancers, such as cabozantinib, lenvati- tion.25,26 Moreover, it has been suggested that several pathways, nib, dovitinib, ponatinib and alectinib. Drilon et al. enrolled especially PI3K/AKT/mTor, could overcome MAPK inhibitors 26 patients with RET fusion-positive NSCLC in a phase II tri- leading to promotion of oncogenic signals and resistance to al investigating single action of cabozantinib.23 The ORR was MAPK pathway inhibition for cancer therapy. 28% with a median PFS of 5.5 months and a median OS of Mutations on the B-Raf are frequent in human cancer, chief- 9.9 months. The predominant fusion type was KIF5B-RET ly identified in melanoma, hairy cell leukaemia and papil- fusion (62%), with post-hoc analysis suggesting that PFS and lary thyroid cancer. In NSCLC, B-Raf mutations are detected OS were shorter for this subgroup of patients. Recently, the in 1-2% and are highly present in smokers and adenocarci- phase II LURET trial reported results of vandetanib in a co- noma subtype. The most common B-Raf mutation is the va- hort of 17 evaluable patients, with an ORR of 53% and a me- line-to-glutamate substitution at codon 600 (V600), which is dian PFS of 4.7 months.24 The results were better in patients observed in approximately 50% of NSCLC B-Raf mutations; with CCDC6-RET-fusion subtype, with an ORR of 83% and non-V600 mutations include G469A, T599_V600insT and PFS of 8.3 months and only 20% and 2.9 months for those D594N and V600_K601delinsE.26

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Several clinical trials have investigated new BRAF inhibi- As an example, oncogenic NTRK1 fusions have been found in tors. has been tested in a phase II basket trial 3.3% of NSCLC, while NTRK2 and NTRK3 fusions are pres- including V600E mutation-positive non-melanoma cancers, ent in a small percentage (about 1-2%).30,31 and resulted in an ORR of 42% in NSCLC and a median PFS In order to avoid cellular tumour growth, several NTRK in- of 7.3, which is better than the other cohorts. Planchard et al. hibitors have been developed (Table 1). Entrectinib (RX- have evaluated dabrafenib (a BRAF V600E kinase inhibitor) DX-101), a pan-Trk inhibitor named below, has been tested in monotherapy, and in combination with the MEK inhibitor in two phase I trials (STARTRT-1 and ALKA-372-001), show- trametinib (in pre-treated patients) in a phase II trial with a ing antitumor activity in solid tumours but especially in NS- two-stage design, which includes stage IV BRAF V600E-mu- CLC.32 Three of three patients harbouring NTRK aberrations tant NSCLC patients.27,28 The median PFS was 5.5 months achieved partial responses. Due to these results, the FDA with an ORR of 32% and a median duration of response of granted entrectinib the Orphan Drug Designation for treat- 9.6 months with dabrafenib monotherapy. In the combina- ment of Trk-positive NSCLC and colorectal cancer. A phase tion arm, the ORR was 63% and the PFS 9.7 months, demon- II basket trial (STARTRK-2) is recruiting patients harbour- strating improvement in antitumor activity in the indirect ing NTRK fusions. LOXO-101 is a pan-Trk inhibitor with an comparison with dabrafenib monotherapy. The combina- ATP-competitive function that has shown antitumor activi- tion has been approved by the FDA in BRAF V600-mutant ty in preclinical models.30,31 Currently, a phase II basket trial advanced or metastatic NSCLC. Dual blockade MAPK path- (NAVIGATE) is ongoing. Moreover, several drugs are in ear- way could increases the possibilities to overcome resistance ly development stages, for example altiratinib (DCC-2701), a and, theoretically, could be useful in patients with B-Raf- Trk, VEGFR and MET inhibitor that is being tested in a phase non-V600 mutations.27 I study. Other similar agents include PLX-7486 (a pan-Trk in- New BRAF inhibitors are being evaluating in ongoing clin- hibitor), DS6051b (a pan-Trk and ROS1 inhibitor) and TSR- ical trials, most of them in melanoma or colorectal cancer, 011 (a TrkA and ALK inhibitor). including new strategies such as PI3K inhibitors after du- As in other target therapies, mechanisms of resistance can al-blockade progression trying to overcome resistances (Ta- emerge after a period under treatment. A few cases of re- ble 2).27 PLX8394 is being tested in solid tumours harbouring sistance to entrectinib have been reported, and preclinical BRAF mutation in a phase I/IIa trial, and LY3009120 is being studies have shown the presence of two single mutations studied in metastatic BRAF mutated cancers including NS- in tyrosine kinase domain, p.G595R and p.G667C, that oc- CLC. Both of them suggest activity in BRAF mutations with- curred in a dose-dependent way. No data about LOXO-101 out MAPK activation. resistance have been reported, but a second generation NTRK inhibitor called LOXO-195 is ready to be tested during this NTRK year.31 The tropomyosin receptor kinase family includes three trans- membrane proteins named TrkA, TrkB and TrkC , which FGFR are encoded respectively by NTRK1, NTRK2 and NTRK3 The FGFR family includes four transmembrane tyrosine ki- genes.29,30 NTRK members are involved in the development of nase receptors, composed of an intracellular region, a trans- nervous functions by checking cell proliferation, surveillance membrane portion and an extracellular ligand binding and apoptosis. Each of them is activated by ligand binding in domain. This last one is being formed by three immuno- their extracellular domain. Several neurotrophins are known globulin domains that play an important role as a bind- as specific ligands, including (NGF) for ing point for FGFR ligands. In fact, 22 of these ligands (the TrkA, brain-derived growth factor (BDGF) for TrkB and neu- FGFs) have been identified in humans.33 FGFR–FGF binding rotrophin 3 (NT3) for TrkC. Ligand binding to NTRKs pro- achieves FGFR activation due to the presence of heparin sul- motes the activation of several signalling pathways because phate proteoglycans that are involved in stable dimerisation of oligomerisation and phosphorylation of tyrosine do- of this complex. FGFR activation leads to FRS2a phosphor- main. Ras/MAPK pathway is activated by TrkA-NGF bind- ylation, allowing GRB2 recruitment and finally the initia- ing, which modulates cell growth and surveillance, avoiding tion of several intracellular pathways. RAS/MAPK, PI3K/ apoptosis. PI3K/Akt pathway and phospholipase C-γ are reg- AKT/mTOR and DAG-PKC are the main downstream path- ulated by TrkA but also by TrkB, increasing cellular growth ways, and their activation results in the promotion of nucle- and neuronal differentiation.30 NTRK aberrations are, in fact, ar genes that increase cell growth, motility, angiogenesis and related with promoting oncogenic cell potential and especial- invasiveness.34,35 ly with lung cancer aggressive behaviour and poor prognosis. Several FGFR signalling alterations with oncogenic potential,

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KEY MESSAGES FOR CLINICAL PRACTICE

1. The discovery of oncogenic driver mutations, especially involving epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK), has changed the therapeutic landscape of lung cancer.

2. Tyrosine kinase inhibitors targeting EGFR and ALK have demonstrated important anti-tumour activity and clinical improvement, which has led to increased efforts in our search for new targeted therapies.

3. Current available data of genomic alterations beyond EGFR and ALK in NSCLC are presented in this manuscript, including ROS1, BRAF, KRAS, c-MET, RET, NTRK and FGFR.

4. Current clinical trials are testing new targeted drugs against these aberrations, and their mechanisms of action are also included in this review.

as gene amplifications, somatic mutations and chromosom- ib, ponatinib and .37 al translocations, can increase tumour cell growth by FG- Nevertheless, selective FGFR TKIs are being tested, too. An FR upregulation, inhibiting apoptosis. These aberrations are FGFR1-3 inhibitor, NVP-BGJ398, has shown a good safety present in 7% of NSCLCs.34 The most frequent is FGFR1 am- profile in preclinical models, and a phase II trial including plification, located on chromosome 8p12. This gene imbal- patients with FGFR aberrations is ongoing. AZD4547, an- ance promotes ligand-independent signalling. Cihoric et al. other selective FGFR1-3 inhibitor molecule, was tested in 15 demonstrated an increment of FGFR1 amplification in squa- patients with NSCLC in a phase I trial, not reaching the pri- mous lung carcinoma (20% against 2% in adenocarcinoma), mary endpoint.34 Recently, a phase I/II trial of AZD4547 plus in men and smokers, and it could act as a negative prognostic docetaxel has finished, and two randomised phase II trials marker. FGFR2 mutation is found in 3% of NSCLCs, while are recruiting patients. Erdafinib, LY2874455 and ARQ 087 gene fusions occur in 1-2%.35,36 are selective pan-inhibitors tested in respective phase I trials As a targetable pathway, FGFR inhibitors are being devel- with promising results.37 oped, and the majority of them are multitarget inhibitors (Ta- ble 1).35,37 Lucitanib is a non-selective FGFR TKI that inhibits CONCLUSIONS FGFR1-2 and VEGFR1-3. Lucitanib has been tested in a phase During the last years, the improvement of molecular biology I/II trial, which included 76 patients with FGFR alterations, has changed the therapeutic landscape of lung cancer radi- showing an ORR of 28% and PFS of 31 weeks. Results of a cally. The discovery of EGFR mutations and ALK rearrange- phase II trial evaluating lucitanib in NSCLC with FGFR ab- ments, and the development of targeted therapies against errations are ongoing. Dovitinib, a potent PDGFR, VEGFR these alterations, has shown the importance of oncogenic and FGFR inhibitor, was recently tested in a phase II trial in- drivers in a group of patients, increasing the PFS, response cluding 26 NSCLC patients with FGFR1 amplification, but an rate and quality of life. Since then, several potential predictive ORR of 11% was found. Furthermore, a phase I trial inves- biomarkers have been identified, and with the implementa- tigating the combination of dovitinib plus erlotinib in lung tion of new genomic technologies and sequencing analysis, cancer was discontinued because of adverse effects. Lenvati- we are able to typify an important number of genomic aber- nib, another multikinase inhibitor including FGFR1-4, VEG- rations in NSCLC. FR, RET and c-Kit, has shown an improvement in PFS and OR Nowadays, with a number of targetable oncogenic events in a phase II trial in pre-treated NSCLC. Studies including identified in NSCLC, targeted drugs against these pathways lenvatinib as monotherapy or in combination with pembroli- are being tested in several clinical trials with hopeful results. zumab in lung cancer are ongoing. Nintedanib, an inhibitor The incorporation of basket trials, which includes patients of FGFR1-3, VEGFR and PDGFR, is approved in combina- based on molecular profile regardless of tumour histology, tion with docetaxel as second line of NSCLC because of the plays an important role in the research of targeted agents. results of the phase III LUME-Lung 1 trial. An improvement Unfortunately, the success of developing oncogenic drivers of PFS (3.4 vs. 2.7) and OR (10.9 vs. 7.9) has been observed. gave rise to new problems, as acquired resistance to targeted Several trials evaluating nintedanib are ongoing. Other mul- therapy. The role of combinatorial drugs including immuno- titarget inhibitors that are being tested in NSCLC are cediran- therapy instead of sequential treatments can be an import-

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