Marie-Julie Nokin

Chiara Ambrogio

Ernest Nadal
email: esnadal@iconcologia.net

David Santamaria
email: d.santamaria@iecb.u-bordeaux.fr

Targeting Infrequent Driver Alterations in Non-Small Cell Lung Cancer

Keywords: NSCLC, infrequent drivers, targeted therapy, drug resistance

Abstract

The discovery of oncogenic driver mutations led to the development of targeted therapies with non-small cell lung cancer (NSCLC) being a paradigm for precision medicine in this setting. Nowadays, the number of clinical trials focusing on targeted therapies for uncommon drivers is growing exponentially, emphasizing the medical need for these patients. Unfortunately, similar to what is observed with most targeted therapies directed against a driver oncogene, the clinical response is almost always temporary and acquired resistance to these drugs invariably emerges. Here, we review the biology of infrequent genomic actionable alterations in NSCLC as well as the current and emerging therapeutic options for these patients. Mechanisms leading to acquired drug resistance and future challenges in the field are also discussed.

Glossary

Epithelial to mesenchymal transition (EMT): process by which epithelial cells lose their epithelial phenotype and gain mesenchymal properties.

MAPKsignaling pathway: signaling cascade (RAS-RAF-MEK-ERK) that mediates signals from cell surface receptors regulating cell growth, differentiation and survival.

Median progression-free survival (mPFS): length of time during and after the treatment that a patient lives with the disease and does not get worse.

Non-small cell lung cancer (NSCLC): major subtype of lung cancer (accounts for 80-90% of all lung cancers) comprising both squamous neoplasms and adenocarcinoma (LUAD).

Objective/overall response rate (ORR): proportion of patients in a trial whose tumor is significantly reduced by a drug.

The challenge of targeted therapy in NSCLC driven by uncommon genetic alterations

The expanding spectrum of oncogenic driver mutations and clinically available signaling pathway inhibitors had a major impact on non-small cell lung cancer (NSCLC) (see Glossary) patients management in the last decade [START_REF] Rosell | Large-scale screening for somatic mutations in lung cancer[END_REF]. Cancer driver mutations not only initiate the disease but also sustain tumor progression and therefore their inhibition results in a therapeutic benefit. Driver oncogenes are often identified due to their recurrence in patients, a complicated feature when they appear with low prevalence as those covered in this review. Yet the oncogenic nature and the addiction of cancer cells to this set of infrequent drivers have been clearly established as well as their mutually exclusive pattern with other oncogenes. With the exception of BRAF, all belong to the receptor tyrosine kinase (RTK) family and when in the oncogenic form constantly activate essential downstream signaling pathways, including the MAPK (see Glossary), PI3K and JNK, resulting in sustained growth, increased survival and enhanced dissemination. Recently, Food and Drug Administration (FDA) has approved specific inhibitors targeting infrequent NSCLC oncogenic drivers (ROS1, MET, RET, BRAF, and NTRK). Beyond those, promising preliminary data with compounds targeting other emerging driver alterations (e.g. HER2) have been reported [START_REF] Baraibar | Novel drugs targeting EGFR and HER2 exon 20 mutations in metastatic NSCLC[END_REF].

Used as first or latter lines of treatment (after standard chemotherapy and/or immunotherapy), these targeted therapies have greatly increased patients' outcome [START_REF] Planchard | Dabrafenib plus trametinib in patients with previously treated BRAF(V600E)-mutant metastatic non-small cell lung cancer: an open-label, multicentre phase 2 trial[END_REF][START_REF] Drilon | Efficacy of Selpercatinib in RET Fusion-Positive Non-Small-Cell Lung Cancer[END_REF][START_REF] Gainor | Registrational dataset from the phase I/II ARROW trial of pralsetinib (BLU-667) in patients (pts) with advanced RET fusion+ non-small cell lung cancer (NSCLC)[END_REF][START_REF] Hong | Larotrectinib in patients with TRK fusion-positive solid tumours: a pooled analysis of three phase 1/2 clinical trials[END_REF][START_REF] Doebele | Entrectinib in patients with advanced or metastatic NTRK fusion-positive solid tumours: integrated analysis of three phase 1-2 trials[END_REF][START_REF] Drilon | Entrectinib in ROS1 fusion-positive non-small-cell lung cancer: integrated analysis of three phase 1-2 trials[END_REF][START_REF] Wolf | Capmatinib in MET Exon 14-Mutated or MET-Amplified Non-Small-Cell Lung Cancer[END_REF].

Unfortunately, as anticipated by the lessons learned from the clinical management of EGFRmutated and ALK-rearranged LUAD (Box 1), therapeutic resistance eventually appears in most patients and, therefore, advanced NSCLC remains largely incurable (for review see [START_REF] Rotow | Understanding and targeting resistance mechanisms in NSCLC[END_REF]). In the present review, we describe the biological relevance and the therapeutic options of a series of infrequent drivers for which clinical treatments have been approved or are under evaluation. Likewise, the clinical management of on-target resistance is discussed per driver while that due to bypass signaling is collectively addressed given its pan-driver conservation.

ROS1 Biological and clinical features

ROS1 is a conserved orphan RTK initially identified in glioblastoma and was the first chromosomal rearrangement described in NSCLC [START_REF] Rikova | Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer[END_REF]. Since then, several ROS1 breakpoints have been described involving the entire ROS1 kinase domain and accounting for 1-2% of NSCLC. Twenty fusion partners (e.g. SLC34A2, EZR and SDC4) have been reported, among which CD74-ROS1 is the most common [START_REF] Liu | Identification of a novel WNK1-ROS1 fusion in a lung adenocarcinoma sensitive to crizotinib[END_REF][13][START_REF] Lin | Recent Advances in Targeting ROS1 in Lung Cancer[END_REF]. Whether distinct partners are functionally equivalent is unknown, although in vitro data indicates that they impose changes in subcellular localization affecting signaling and oncogenic properties [START_REF] Neel | Differential Subcellular Localization Regulates Oncogenic Signaling by ROS1 Kinase Fusion Proteins[END_REF]. Unlike ALK rearrangements, most of the known ROS1 fusion partners lack constitutive dimerization domains and the mechanism of ROS1 oncogenic activation remains unknown. Along with RET and NTRK, ROS1 rearrangements are associated with younger age at diagnosis, none or low tobacco exposure and generally have adenocarcinoma histology [START_REF] Lin | ROS1 Fusions Rarely Overlap with Other Oncogenic Drivers in Non-Small Cell Lung Cancer[END_REF][START_REF] Wang | RET fusions define a unique molecular and clinicopathologic subtype of non-small-cell lung cancer[END_REF]. Diagnosis of ROS1 fusions by immunohistochemistry (IHC) is the most cost-effective method requiring confirmation by break-apart probe fluorescent in situ hybridization (FISH) or hybrid RNAbased next-generation sequencing (NGS) if the RNA quality is optimal [START_REF] Lindeman | Updated Molecular Testing Guideline for the Selection of Lung Cancer Patients for Treatment With Targeted Tyrosine Kinase Inhibitors: Guideline From the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology[END_REF][START_REF] Davies | Comparison of Molecular Testing Modalities for Detection of ROS1 Rearrangements in a Cohort of Positive Patient Samples[END_REF].

Frontline targeted therapy

ROS1-positive lung cancer seems particularly sensitive to pemetrexed-based chemotherapy [START_REF] Chen | Efficacy of Pemetrexed-Based Chemotherapy in Patients with ROS1 Fusion-Positive Lung Adenocarcinoma Compared with in Patients Harboring Other Driver Mutations in East Asian Populations[END_REF]. The ALK inhibitor crizotinib was supposed to efficiently target ROS1-rearranged tumors given that ROS1 and ALK belong to the insulin receptor superfamily [START_REF] Robinson | The protein tyrosine kinase family of the human genome[END_REF] sharing >80% of amino acid sequence within their ATP-binding sites. Furthermore, crizotinib binds with high affinity to both kinases and inhibiting cell line proliferation. Indeed, this hypothesis was corroborated in the clinic with crizotinib showing marked therapeutic effect in patients with ROS1-rearranged NSCLC [START_REF] Shaw | Crizotinib in ROS1-rearranged non-small-cell lung cancer[END_REF]. Accordingly, crizotinib, first approved for the treatment of ALKrearranged NSCLC, received FDA and European Medicines Agency (EMA) expanded approval in 2016 for ROS1-rearranged NSCLC [START_REF] Shaw | Crizotinib in ROS1-rearranged non-small-cell lung cancer[END_REF][START_REF] Moro-Sibilot | Crizotinib in c-MET-or ROS1-positive NSCLC: results of the AcSe phase II trial[END_REF][24][START_REF] Wu | Phase II Study of Crizotinib in East Asian Patients With ROS1-Positive Advanced Non-Small-Cell Lung Cancer[END_REF][START_REF] Michels | Safety and Efficacy of Crizotinib in Patients With Advanced or Metastatic ROS1-Rearranged Lung Cancer (EUCROSS): A European Phase II Clinical Trial[END_REF] (Figure 1 and Table 1). Similarly, in 2020, entrectinib has obtained FDA and EMA approval in this tumor type [START_REF] Drilon | Entrectinib in ROS1 fusion-positive non-small-cell lung cancer: integrated analysis of three phase 1-2 trials[END_REF][START_REF] Drilon | Safety and Antitumor Activity of the Multitargeted Pan-TRK, ROS1, and ALK Inhibitor Entrectinib: Combined Results from Two Phase I Trials (ALKA-372-001 and STARTRK-1)[END_REF]. In addition, other kinase inhibitors including ceritinib and lorlatinib have been evaluated in ROS1-positive crizotinib naïve NSCLC [START_REF] Lim | Open-Label, Multicenter, Phase II Study of Ceritinib in Patients With Non-Small-Cell Lung Cancer Harboring ROS1 Rearrangement[END_REF][START_REF] Shaw | Lorlatinib in advanced ROS1-positive non-small-cell lung cancer: a multicentre, open-label, single-arm, phase 1-2 trial[END_REF] (Figure 1 and Table 1).

Addressing on-target mechanisms of resistance

The accumulated knowledge after a decade of using RTKi for the treatment of ALK-and EGFR-mutant LUAD (Box 1) anticipated that the selection of on-target mutations within the kinase domain would be the main mechanism of acquired resistance in ROS1 and the rest of infrequent drivers covered in this review. Indeed, several mutations have been reported affecting the solvent-front, the gatekeeper residue, the activation loop or the DFG motifs and all are predicted to prevent drug binding due to steric hindrance. Figure 2A summarizes the different on-target mutations identified and their functional analogy among the different drivers together with ALK and EGFR for comparative purposes.

Fortunately, also guided by the successful clinical management of resistant disease in EGFRand ALK-mutant patients (Box 1), new generation inhibitors have been identified or purposely designed to combat drug resistance. Figure 2B summarizes pre-clinical and clinical data regarding the sensitivities of the most common secondary mutations to a panel of inhibitors for each driver. In the case of ROS1, resistance profiling studies using both in vitro and in vivo models demonstrated that cabozantinib (and its structural analog foretinib) as well as DS-6051b (a novel ATP-competitive ROS1/NTRK inhibitor) markedly inhibited the growth of ROS1 G2032R mutants [START_REF] Davare | Structural insight into selectivity and resistance profiles of ROS1 tyrosine kinase inhibitors[END_REF][START_REF] Davare | Foretinib is a potent inhibitor of oncogenic ROS1 fusion proteins[END_REF][START_REF] Zou | PF-06463922 is a potent and selective next-generation ROS1/ALK inhibitor capable of blocking crizotinib-resistant ROS1 mutations[END_REF][START_REF] Chong | Identification of Existing Drugs That Effectively Target NTRK1 and ROS1 Rearrangements in Lung Cancer[END_REF][START_REF] Katayama | Cabozantinib overcomes crizotinib resistance in ROS1 fusion-positive cancer[END_REF][START_REF] Katayama | The new-generation selective ROS1/NTRK inhibitor DS-6051b overcomes crizotinib resistant ROS1-G2032R mutation in preclinical models[END_REF]. Indeed, several case reports of ROS1-rearranged LUAD patients resistant to crizotinib and ceritinib reported responses to cabozantinib [START_REF] Drilon | A Novel Crizotinib-Resistant Solvent-Front Mutation Responsive to Cabozantinib Therapy in a Patient with ROS1-Rearranged Lung Cancer[END_REF][START_REF] Sun | Lengthy Progression-Free Survival and Intracranial Activity of Cabozantinib in Patients with Crizotinib and Ceritinib-Resistant ROS1-Positive Non-Small Cell Lung Cancer[END_REF]. With similar efficacy, DS-6051b had better toxicity profile [START_REF] Fujiwara | Safety and pharmacokinetics of DS-6051b in Japanese patients with nonsmall cell lung cancer harboring ROS1 fusions: a phase I study[END_REF] and two ongoing clinical trials are evaluating its efficacy in ROS1-rearranged NSCLC i,ii . In addition, repotrectinib (TPX-0005), a selective and highly potent TKI against ROS1, NTRK, and ALK, demonstrated potent anti-proliferative activity against the G2032R and D2033N ROS1 mutations in cellular inhibitory assays and xenografts [START_REF] Drilon | Repotrectinib (TPX-0005) Is a Next-Generation ROS1/TRK/ALK Inhibitor That Potently Inhibits ROS1/TRK/ALK Solvent-Front Mutations[END_REF]. This has been tentatively confirmed in a clinical setting (TRIDENT-1, NCT03093116) reporting that repotrectinib achieved tumor regression in 45% of patients with ROS1-positive NSCLC resistant to prior TKI(s) [START_REF] Ou | OA09 Preliminary Clinical Activity of Repotrectinib (TPX-0005) in Advanced ROS1 Fusion-Positive Non-Small Cell Lung Cancer[END_REF]41]. Interestingly, not all ontarget mutations are equally sensitive to new generation inhibitors and patient stratification will be required. Indeed, each TKI is structurally distinct with varying degrees of activity against the different resistance mutations, giving them unique activity profiles. This is illustrated by lorlatinib that demonstrated promising activity in 40 patients with ROS1positive NSCLC who progressed to crizotinib with an overall response rate (ORR, see Glossary) of 35% and a median progression-free survival (mPFS, see Glossary) of 8.5 months. While patients harboring K1991Q and S1986F ROS1 mutations achieved durable responses to lorlatinib, none of the patients with the more frequent G2032R mutation did benefit [START_REF] Shaw | Lorlatinib in advanced ROS1-positive non-small-cell lung cancer: a multicentre, open-label, single-arm, phase 1-2 trial[END_REF].

NTRK Biological and clinical features

The Neurotrophic Tropomyosin Receptor Kinase (NTRK) genes (NTRK1, 2 and 3) encode TRKA, TRKB and TRKC, respectively. They are activated by a family of four ligands (NGF, BDNF, NT-3 and NT-4) known as neurotrophins that display different affinities for each RTK and play essential roles in the development and function of the nervous system. Activating mutations in NTRK have been identified in NSCLC, yet their oncogenic role remains controversial [START_REF] Harada | Role and relevance of TrkB mutations and expression in non-small cell lung cancer[END_REF]. Similarly, activating splice variants and overexpression have been reported in pan-cancer studies but, undoubtedly, the most frequent aberrations involve intra-and inter-chromosomal gene-fusions implicating NTRK that occur at a frequency below 0.4% in NSCLC. The biology of NTRK oncogenes has been comprehensively reviewed elsewhere [START_REF] Vaishnavi | TRKing down an old oncogene in a new era of targeted therapy[END_REF]44]. Of note, a NTRK fusion was the first non-RAS oncogene identified almost 40 years ago (indicated as OncD in [START_REF] Pulciani | Oncogenes in solid human tumours[END_REF]; for review see [START_REF] Barbacid | On the right TRK: from oncogene discovery to cancer therapeutics[END_REF]). Of the three NTRK, pan-cancerrelated fusions more commonly affect NTRK1 and NTRK3, and 48 different partners have been reported [47]. In lung, the fusions involve NTRK1 and NTRK3 with five and two distinct partners respectively, being ETV6-NTRK3 the most prevalent [47,[START_REF] Farago | Clinicopathologic Features of Non-Small-Cell Lung Cancer Harboring an NTRK Gene Fusion[END_REF]. The NGS-based FoundationOneCDx test has just received FDA-approval for the identification of NTRK fusions.

Frontline targeted therapy

Early phase basket trials of solid tumors harboring NTRK gene rearrangements were performed with first-generation TKIs such as larotrectinib, which is selective for TRKA/B/C, and entrectinib, which targets TRKA/B/C as well as ALK and ROS1. Both compounds have demonstrated marked tumor-agnostic efficacy in pan cancer trials [START_REF] Hong | Larotrectinib in patients with TRK fusion-positive solid tumours: a pooled analysis of three phase 1/2 clinical trials[END_REF][START_REF] Doebele | Entrectinib in patients with advanced or metastatic NTRK fusion-positive solid tumours: integrated analysis of three phase 1-2 trials[END_REF] (Figure 1 and Table 1). Based on these results larotrectinib received accelerated FDA approval for the treatment of TRK fusion-positive cancers in 2018 followed by entrectinib one year later. Both drugs also obtained histology-independent approval in solid tumors harboring NTRK rearrangements by EMA. Ongoing trials may help to better estimate efficacy and duration of response in NSCLC.

Addressing on-target mechanisms of resistance

Next-generation TRK inhibitors, such as selitrectinib and repotrectinib (TPX-0005), have been designed to combat on-target acquired resistance mutations and their activity is being evaluated in phase I/II trials or compassionate use protocols for NTRKaltered cancers. Indeed, selitrectinib was developed as a second-generation TRK inhibitor using modeling studies even before acquired resistance appeared in the clinic. An initial evaluation performed on a first-in-human basis proved its activity against solvent-front and xDFG mutants inducing rapid tumor responses [49]. This has been extended in an ongoing phase I/II trial upon enrolling patients with prior treatment with NTRK inhibitors iii .

Preliminary results indicate an ORR of 45% in patients with mutations affecting the kinase domain [50]. Similarly, repotrectinib evaluation in a phase I/II first-in-human dose-escalation study (NCT03093116) demonstrated efficacy in patients harboring NTRK3 solvent front resistant mutations [START_REF] Drilon | Repotrectinib (TPX-0005) Is a Next-Generation ROS1/TRK/ALK Inhibitor That Potently Inhibits ROS1/TRK/ALK Solvent-Front Mutations[END_REF]. Whether these next-generation TRK inhibitors will exert a similar benefit in resistant LUAD patients with kinase-domain acquired mutations is still unknown.

RET Biological and clinical features

Unlike other RTKs, RET (REarranged during Transfection) does not directly bind to its ligands but requires an additional co-receptor. Indeed, ligands of the glial cell line-derived neurotrophic factor (GDNF) family bind to one of the four receptors of the GDNF family receptor-α (GFRα), which subsequently allow RET dimerization, autophosphorylation and activation [51]. Despite a few studies reporting concomitant genomic alterations (for review see [START_REF] Ferrara | Clinical and Translational Implications of RET Rearrangements in Non-Small Cell Lung Cancer[END_REF]), RET rearrangements are considered mutually exclusive with other alterations in NSCLC indicating an independent carcinogenic role and have been identified in 1-2% of cases. Chromosomal inversions or translocations involving RET results in a juxtaposition of its kinase domain and the coiled coil domain of the partner. Similar to the oncogenic activation of ALK fusions, this coiled coil domain is responsible of the ligand-independent homodimerization and constitutive RET activation [53]. In NSCLC, up to 12 fusion partners (e.g. CCDC6, NCOA4, TRIM33) have been described with the most frequent being KIF5B [54].

Unlike ALK and ROS1 rearrangements, RET fusion genes cannot be adequately detected by IHC due to low sensitivity and highly variable specificity [START_REF] Ferrara | Clinical and Translational Implications of RET Rearrangements in Non-Small Cell Lung Cancer[END_REF]. Therefore, NGS is the most accurate option as FISH and RT-PCR may lead to false negatives due to technical limitations.

Frontline targeted therapy

Similar to ALK and ROS1-positive tumors, durable benefits have been obtained with pemetrexed-based chemotherapy in RET-rearranged NSCLC [START_REF] Drilon | Clinical outcomes with pemetrexed-based systemic therapies in RETrearranged lung cancers[END_REF]. Considering the structural similarities of both the kinase domain and the ATP-binding site with other RTKs [START_REF] Knowles | Structure and chemical inhibition of the RET tyrosine kinase domain[END_REF], several multi-kinase inhibitors (MKIs; e.g. vandetanib, cabozantinib, lenvatinib as well as alectinib, a VEGFR-sparing MKI) were initially used for the treatment of patients bearing RET fusions (for review see [START_REF] Ferrara | Clinical and Translational Implications of RET Rearrangements in Non-Small Cell Lung Cancer[END_REF][START_REF] Drilon | Targeting RET-driven cancers: lessons from evolving preclinical and clinical landscapes[END_REF][START_REF] Bronte | Targeting RET-rearranged non-small-cell lung cancer: future prospects[END_REF]) (Figure 1 and Table 1). Recently, more specific and potent RET inhibitors are under investigation. These include pralsetinib (BLU-667) and selpercatinib (LOXO-292), which have received FDA approval for RET-altered NSCLC [START_REF] Drilon | Efficacy of Selpercatinib in RET Fusion-Positive Non-Small-Cell Lung Cancer[END_REF][START_REF] Gainor | Registrational dataset from the phase I/II ARROW trial of pralsetinib (BLU-667) in patients (pts) with advanced RET fusion+ non-small cell lung cancer (NSCLC)[END_REF][59][START_REF] Solomon | RET Solvent Front Mutations Mediate Acquired Resistance to Selective RET Inhibition in RET-Driven Malignancies[END_REF][START_REF] Evans | P2.03-44 BLU-667 Demonstrates Robust Activity in RET Fusion-Driven Intracranial Tumor Models[END_REF] (Figure 1 and Table 1).

Treatment evolution of RET-driven disease is an illustrative example of the continuous need to design specific and potent inhibitors. New putative oncogenic fusions are being discovered [START_REF] Zhuo | Analysis of MET kinase domain rearrangement in NSCLC[END_REF][START_REF] Reddy | BRAF fusions in clinically advanced non-small cell lung cancer: An emerging target for anti-BRAF therapies[END_REF] that could also require initial treatment with less-efficient MKIs in parallel to the development of specific compounds.

Addressing on-target mechanisms of resistance

The differential response of various on target mutants has also been reported in RET-altered tumors. Whereas most MKIs are ineffective against resistant mutations, both ponatinib [START_REF] Huang | Preclinical Modeling of KIF5B-RET Fusion Lung Adenocarcinoma[END_REF] and AD80 [START_REF] Plenker | Drugging the catalytically inactive state of RET kinase in RET-rearranged tumors[END_REF] showed pre-clinical efficacy against specific mutations (V804 and G810).

Whether this may provide clinical benefit is under evaluation for ponatinib iv . In addition, while pralsetinib displayed inhibitor activity against V804M/L and S904F mutations but failed to inhibit G810 alterations [START_REF] Solomon | RET Solvent Front Mutations Mediate Acquired Resistance to Selective RET Inhibition in RET-Driven Malignancies[END_REF], TPX-0046, a structurally different RET/SRC inhibitor, showed potent in vitro and in vivo activity against the RET solvent-front mutation G810R and is currently tested in a phase I/II trial v .

MET Biological and clinical features

In NSCLC, c-Mesenchymal-Epithelial Transition factor (c-MET) gene rearrangements, fusions and somatic mutations have been identified but are rare [66]. MET overexpression, however, is frequent in unselected NSCLC patients although its clinical value is unclear as this criterion is largely dependent on the antibody assay and the determination of a positive threshold. MET amplification is also a common mechanism of resistance to EGFR-TKIs in NSCLC. Anyhow, increased expression is considered a causative driver only if co-occurring with clinically relevant MET genomic alterations like MET gene amplification or mutations resulting in exon 14 skipping (METΔ14). This alteration results in a MET protein lacking the juxtamembrane domain with extended half-life after ligand stimulation and is considered an oncogenic driver. METΔ14 is predominantly associated to sarcomatoid carcinoma histology [67,[START_REF] Tong | MET Amplification and Exon 14 Splice Site Mutation Define Unique Molecular Subgroups of Non-Small Cell Lung Carcinoma with Poor Prognosis[END_REF] and detected in 2-4% of NSCLC patients while MET amplification affects approximately 2% of LUAD.

Several MET inhibitors, including TKIs and monoclonal antibodies against MET or its ligand HGF have been assessed in NSCLC (for review see [69,[START_REF] Liang | MET Oncogene in Non-Small Cell Lung Cancer: Mechanism of MET Dysregulation and Agents Targeting the HGF/c-Met Axis[END_REF]). Various agents have reached phase III trials and failed in patients with MET alterations, illustrating the need for better stratification upfront. Indeed, a significant fraction of patients display co-existing oncogenes and only a small fraction of highly amplified tumors may actually be bona-fide MET-addicted cases, which are associated to more effective responses to targeted therapies [START_REF] Noonan | Identifying the Appropriate FISH Criteria for Defining MET Copy Number-Driven Lung Adenocarcinoma through Oncogene Overlap Analysis[END_REF].

Frontline targeted therapy

Crizotinib received FDA approval for ALK and ROS1 NSCLC patients but was initially developed as a MET inhibitor [START_REF] Cui | Structure based drug design of crizotinib (PF-02341066), a potent and selective dual inhibitor of mesenchymal-epithelial transition factor (c-MET) kinase and anaplastic lymphoma kinase (ALK)[END_REF]. Significant clinical activity was reported in patients harboring METΔ14 alterations including complete responses. Yet, the ORR of crizotinib in this cohort was lower compared to those achieved with targeted therapies for other NSCLC drivers [START_REF] Drilon | Antitumor activity of crizotinib in lung cancers harboring a MET exon 14 alteration[END_REF] (Figure 1 and Table 1). Neither variations in the mutation type or splice-site region nor the MET copy number appear to be major differentiating response factors, so the underlying causes of the heterogeneous clinical outcome are unknown. Novel and highly selective type Ib MET inhibitors such as tepotinib and capmatinib have been evaluated in phase II trials [START_REF] Wolf | Capmatinib in MET Exon 14-Mutated or MET-Amplified Non-Small-Cell Lung Cancer[END_REF]74] (Figure 1 and Table 1). In general, METΔ14 tumors display lower initial response rates to TKIs compared to other RTK drivers in NSCLC. This could be due to high rate of co-existing genomic alterations inducing both MAPK and PI3K pathway activation resulting in innate/primary resistance [75,76].

Addressing on-target mechanisms of resistance

In the case of MET-resistant disease, mutations like those affecting the Y1230 and D1228 residues may predict response to type II MET inhibitors such as cabozantinib and glesatinib [START_REF] Engstrom | Glesatinib Exhibits Antitumor Activity in Lung Cancer Models and Patients Harboring MET Exon 14 Mutations and Overcomes Mutation-mediated Resistance to Type I MET Inhibitors in Nonclinical Models[END_REF]. Others, including substitutions implicating L1195 or F1200, may not be sensitive to treatment with these compounds as they preclude binding to the DFG-out pocket [75]. In a similar study, 7 out of 20 (35%) METΔ14 patients who progressed to MET TKI had on-target kinase domain mutations (H1094, G1163, L1195, D1228, Y1230) or elevated MET amplification. Cabozantinib has also been shown to be active against the D1228V mutation with concomitant MET amplification [START_REF] Bahcall | Acquired METD1228V Mutation and Resistance to MET Inhibition in Lung Cancer[END_REF].

HER2/ERBB2

Biological and clinical features

Human epidermal growth factor receptor 2 (HER2), also called ERBB2, is one of the four members of the EGFR family. It remains an orphan receptor activated by heterodimerization with other ERBB family members (for review see [START_REF] Yarden | Untangling the ErbB signalling network[END_REF]). In NSCLC, HER2 dysregulations can be caused by gene amplification, protein overexpression and mutations. Despite the efficacy of anti-HER2 agents in breast and gastric cancer, clinical interest on HER2-positive NSCLC was dampened when the first clinical trials evaluating trastuzumab, an anti-HER2 humanized antibody reported negative results [START_REF] Gatzemeier | Randomized phase II trial of gemcitabine-cisplatin with or without trastuzumab in HER2-positive non-small-cell lung cancer[END_REF]81]. In parallel, HER2 kinase domain mutations were identified in NSCLC patients not associated with HER2 amplification/overexpression [START_REF] Shigematsu | Somatic mutations of the HER2 kinase domain in lung adenocarcinomas[END_REF][START_REF] Stephens | Lung cancer: intragenic ERBB2 kinase mutations in tumours[END_REF].

These results were further confirmed [START_REF] Li | HER2 Amplification and HER2 Mutation Are Distinct Molecular Targets in Lung Cancers[END_REF] indicating that HER2 amplification and mutations represent two distinct oncogenic drivers and potential therapeutic targets. These findings, coupled with the encouraging results of TKIs in EGFR mutants, launched the clinical evaluation of anti-HER2 agents specifically in NSCLC patients bearing HER2 mutations (90 % of them are located in exon 20). HER2 exon 20 alterations comprise point mutations (e.g. L755S, V777L and G776C) or, more frequently, insertions [START_REF] Pillai | HER2 mutations in lung adenocarcinomas: A report from the Lung Cancer Mutation Consortium[END_REF]. The most prevalent (over 80%) is a 12 base pair in-frame insertion causing duplication of amino acids YVMA at codon 775 (A775_G776insYVMA) [START_REF] Arcila | Prevalence, clinicopathologic associations, and molecular spectrum of ERBB2 (HER2) tyrosine kinase mutations in lung adenocarcinomas[END_REF]. Mutations in exon 20 are analogous to EGFR exon 20 mutations and lead to HER2 constitutive activation [START_REF] Perera | HER2YVMA drives rapid development of adenosquamous lung tumors in mice that are sensitive to BIBW2992 and rapamycin combination therapy[END_REF][START_REF] Shimamura | Non-small-cell lung cancer and Ba/F3 transformed cells harboring the ERBB2 G776insV_G/C mutation are sensitive to the dual-specific epidermal growth factor receptor and ERBB2 inhibitor HKI-272[END_REF][START_REF] Wang | HER2 kinase domain mutation results in constitutive phosphorylation and activation of HER2 and EGFR and resistance to EGFR tyrosine kinase inhibitors[END_REF]. Overall, HER2 mutations account for 2-4% of the NSCLC cases and tend to be women and light/never smokers with LUAD histology [START_REF] Pillai | HER2 mutations in lung adenocarcinomas: A report from the Lung Cancer Mutation Consortium[END_REF].

Frontline targeted therapy

HER2 targeted therapy is not yet standard of care in NSCLC and, currently, chemotherapies remain the main strategy. Patients with HER2-mutant LUAD, especially YVMA insertion, demonstrated a markedly inferior response to first line pemetrexed-based chemotherapy compared to those harboring ALK/ROS1 rearrangements, which strengthen the need for effective anti-HER2 agents [90]. Retrospectives studies displayed encouraging disease control rates in NSCLC patients bearing HER2 exon 20 insertions treated with HER2-targeted drugs [91,92]. Several clinical studies have been carried out or are ongoing using both anti-HER2 antibodies and small molecules (for review see [START_REF] Baraibar | Novel drugs targeting EGFR and HER2 exon 20 mutations in metastatic NSCLC[END_REF]) (Figure 1 and Table 1). Among these compounds, poziotinib, a pan-HER TKI, has structural features that can circumvent the steric changes induced by exon 20 insertions and increase its affinity compared to larger TKIs [START_REF] Robichaux | Mechanisms and clinical activity of an EGFR and HER2 exon 20selective kinase inhibitor in non-small cell lung cancer[END_REF]. Next-generation TKIs with increased selectivity towards the EGFR and HER2 mutants (such as TAK-788) are being developed to limit the toxicity associated to wild-type EGFR inhibition [94,95]. In addition, trastuzumab and its antibody-drug conjugate, trastuzumabemtansine (T-DM1), showed mixed results in the clinic due in part to the molecular heterogeneity of HER2-altered NSCLC (for review see [START_REF] Baraibar | Novel drugs targeting EGFR and HER2 exon 20 mutations in metastatic NSCLC[END_REF]). Trastuzumab-deruxtecan (DS-8201a) showed promising efficacy among NSCLC patients with HER2 exon 20 mutations [96] (Figure 1 and Table 1). In this context, patients with HER2-mutated NSCLC often develop brain metastases with disease progression and trastuzumab would be inefficient due to its inability to cross the blood-brain barrier. Therefore, monitoring and treatment of CNS metastases may require specific approaches.

BRAF Biological and clinical features

BRAF, together with ARAF and CRAF, constitute the RAF family of serine/threonine kinases, a core component of the MAPK cascade. Since the first evidence in 2002 [START_REF] Davies | Mutations of the BRAF gene in human cancer[END_REF], almost 200 distinct BRAF mutations have been reported in around 8% of cancers. In NSCLC, they represent 1.5-3.5% of the cases and affect the kinase domain, either in the glycine-rich loop in exon 11 or in the activation segment in exon 15, resulting in increased ERK signaling (for review see [98]). Clinical characteristics associated with BRAF mutations in lung cancer vary without obvious segregation in specific parameters, except the significant association with LUAD histology. Most patients with BRAF mutations are smokers, although patients with V600 mutations are more likely to be light/never-smokers compared with patients with non-V600 mutations [START_REF] Leonetti | BRAF in non-small cell lung cancer (NSCLC): Pickaxing another brick in the wall[END_REF][100][101][102]. BRAF mutations as well as HER2 and MET alterations are generally well covered by NGS gene panels. BRAF mutants are classified in three categories [103].

Class 1 (V600) and class 2 (G464, G469, L597 and K601) mutants are mutually exclusive, RASindependent and signal constitutively as active monomers or active dimers, respectively. Class 3 (e.g. G466 and D594) have impaired/null kinase activity and activation of downstream signaling is RAS-and CRAF-dependent and sensitive to ERK-mediated feedback.

In this case, RAS is generally activated by RTK signaling [103]. Notably, expression of an endogenous class 3 BRAF mutant triggers LUAD in mice, indicating that kinase-inactive BRAF isoforms are oncogenic drivers [104].

Frontline targeted therapy

Experience with BRAF-mutant melanoma has heavily influenced the management of NSCLC.

Targeted therapy has evolved from BRAF monotherapy to combined BRAF/MEK inhibition showing superior efficacy and favorable safety profile in BRAF V600E-mutant NSCLC [START_REF] Planchard | Dabrafenib plus trametinib in patients with previously treated BRAF(V600E)-mutant metastatic non-small cell lung cancer: an open-label, multicentre phase 2 trial[END_REF][105][106][107][108][109] (Figure 1 and Table 1). The combination of dabrafenib plus trametinib has received both FDA and EMA approval for the treatment of metastatic NSCLC harboring the BRAF V600E mutation regardless of previous therapies. Another pair of BRAF and MEK inhibitors, encorafenib and binimetinib, is under investigation and has shown encouraging CNS activity in a patient with BRAF V600E-mutated LUAD [110].

Next-generation RAF inhibitors have been reported to be active in BRAF-mutant NSCLC in the clinic (for review see [111]). Notably, the pan-RAF inhibitor PLX8394 selectively disrupts both BRAF homo-and BRAF-CRAF heterodimers [112,113]. Furthermore, a phase I study evaluating ulixertinib, a first-in-class ERK inhibitor, reported partial responses in NSCLC patients bearing both V600 and non-V600 mutations as well as CNS activity [114]. In addition, BRAF-mutant NSCLC is likely to have high level of PD-L1 expression [115]. When treated with immune checkpoint inhibitors, no significant differences in ORR and mPFS were reported among V600E and non-V600E mutants. The optimal therapeutic sequence has yet to be established in both V600 and non-V600 (Box 2) BRAF-mutated LUAD patients.

Signaling bypass activation and other resistance mechanisms

In accordance with EGFR-and ALK-driven NSCLC (Box 1), the development of resistance to targeted therapies in lung cancer bearing infrequent alterations also implicates signaling bypass tracks, although the underlying molecular mechanisms are less understood. The activation of bypass pathways described so far in both preclinical and clinical studies are depicted for each uncommon driver in Figure 3.

Alterations affecting the RAS-MAPK pathway or upstream receptors are the most frequent.

Notably, EGFR [116][117][118][119] and HER2 [120,121] activating mutations or amplification have been reported in ROS1-and MET-altered patients after progressing to crizotinib.

Interestingly, EGFR activation may not only contribute to the resistant phenotype by supporting bypass signaling but also by protein-protein interactions that reactivate druginhibited ROS1 [118]. EGFR signaling has also been identified as a critical adaptive survival mechanism to RET [118,122,123] and NTRK [118] blockade in NSCLC preclinical models. In addition, KRAS and NRAS mutations on codons 12 and 61 have been identified both in preclinical models of resistance to MKIs in RET-altered cells [123] and in MET, NTRK and BRAF-altered patients after progressing to frontline targeted therapies [126,[128][129][130][131][132].

Similarly, MEK1 activating mutations were described in case reports of BRAF V600E NSCLC after progression to dabrafenib and trametinib [133] as well as in NTRK-rearranged patients after TKIs treatment [126]. Furthermore, case-reports highlighted BRAF V600E mutation or amplification in post-treatment biopsies of ROS1, NTRK and METΔ14-altered NSCLC patients [126,129,134,135]. Intriguingly, RTK-RAS-MAPK activating mutations tend to be mutually exclusive in primary tumors, a finding justified due to functional redundancy and lack of selective advantage. However, several studies have demonstrated that synthetic toxicity due to excessive signaling could equally underlie oncogene incompatibility [136][137][138]. In this context, intermittent therapy resulting in excessive signaling proves to be advantageous in drug resistant melanoma harboring co-existing oncogenes [139]. Yet, in NSCLC, this approach could only be applied to certain oncogenic combinations [140]. Additional clinical support from both prospective and retrospective studies is required to validate this therapeutic strategy.

Another frequently altered pathway upon TKI treatment is PI3K-AKT-mTOR. Indeed, activating mutations of PIK3CA have been reported in both 141] and HER2 exon 20-mutated patients [142] after progression to targeted therapies.

Finally, other alterations potentially contributing to drug resistance have been reported such as in preclinical models of ROS1 and MET-altered NSCLC, respectively. Additionally, acquired amplification of MDM2, which encodes the E3 ubiquitinprotein ligase MDM2, has been identified in RET-rearranged cabozantinib-resistant patients Finally, phenotypic changes such as epithelial to mesenchymal transition (EMT) (see Glossary) has been reported in one ROS1 crizotinib-resistant patient lacking resistant ROS1 mutations or alternative pathway mutations [117]. EMT along with acquisition of cancer stem cell-like properties have been identified in both TKI resistant 151]. No cases of histological transformation to a small-cell lung cancer (SCLC), a phenomenon observed in EGFR and ALK TKI-resistant tumors, has been reported so far in the context of uncommon drivers.

Concluding remarks and future perspectives

Alongside EGFR-and ALK-driven NSCLC, patients bearing infrequent drivers display limited benefit from targeted therapies due to the onset of resistance. Despite the growing body of evidence about their clinical management many key knowledge gaps remain (see Outstanding Questions). First, lung cancers genomes display substantial heterogeneity and complex branching evolution that can be influenced by treatment-imposed selective pressure [152][153][154]. The understanding of the subclonal origin of resistance in the context of rare drivers will require deep sequencing studies of either multiregional biopsies or single tumor cells that are currently missing. This information could help develop tailored treatments to tackle resistance by anticipating the emergence of on-target or bypass mechanisms. Interestingly, recent preclinical evidence has demonstrated that enhanced therapeutic benefit and delayed onset of resistance upon multiple low dosing of different nodes within the same pathway [155]. Beside targeted therapies, these patients may benefit from combining chemotherapy and immunotherapy, but further information is needed.

Recent retrospective studies highlighted that immune checkpoint inhibitors may provide an effective option for NSCLC patients with certain infrequent alterations, especially in those associated with smoking such as BRAF mutations [115,156,157]. Future clinical trials should determine the ideal regimen of combination therapies including dosing and nature of targeted agents, anti-angiogenic drugs and/or chemo plus immunotherapy. When defining the ideal clinical regimen caution should be applied if transposing information from other cancer types harboring the same infrequent driver. For example, the extended survival upon combinatorial treatments in BRAF V600E mutant melanoma has also been achieved in LUAD patients. However, the therapeutic response of colorectal carcinoma patients bearing the same alteration was disappointing due to EGFR-mediated intrinsic resistance [158]. In any case, an ideal clinical regimen would require radiological and molecular monitoring of disease progression. Resistance mechanisms could be captured by non-invasive methods like liquid biopsy [119,125,159] (see Outstanding Questions). A recurrent and important problem is the occurrence of brain metastases, thereby requiring the use of compounds with good intracranial activity. In conclusion, a better understanding of the alterations underlying the development of resistance and the design of tailored treatments will allow delaying tumor progression and achieving improved clinical outcomes in NSCLC patients bearing infrequent alterations.

Box 1. Targeting EGFR and ALK alterations in NSCLC

EGFR mutations occur in 15% of NSCLC, being in-frame deletion in exon 19 (19Del) gatekeeper and G1269A mutations the most frequent. In 2014, second-generation ALK inhibitor ceritinib was approved after confirmation of its efficacy in both crizotinib-naïve and crizotinib-resistant patients with L1196M and G1269A/S mutations [172,173]. In approximately half of cases, resistance is caused by emergence of additional ALK mutations, the most common being the solvent-front substitution G1202R. Therefore, two other second-generation ALK inhibitors -alectinib and brigatinib -were granted accelerated FDA approval for ALK-positive patients who had failed on crizotinib treatment, demonstrating superior inhibitory profile against all known 17 secondary ALK mutations [174,175]. Both alectinib and brigatinib were later approved also as first line therapy in treatment-naïve patients, although alectinib has become the preferred agent. Recently, the third-generation ALK/ROS1 inhibitor lorlatinib, specifically designed to target resistance mutations and to penetrate the blood-brain barrier, received accelerated FDA approval for the treatment of patients with ALK-rearranged advanced NSCLC after progression on crizotinib and at least one other ALK inhibitor [176]. The phase III trial CROWN comparing lorlatinib with crizotinib as first-line therapy is currently ongoing vi .

Box 2. BRAF non-V600 mutations in NSCLC

While more than 90% of BRAF-altered melanoma bear a V600 mutation, BRAF-positive NSCLC are equally divided in V600 and non-V600 mutations and, therefore, represent one of the cancers with the higher proportion of non-V600 mutations. The tumor-initiating potential of many of the rare BRAF mutations has yet to be demonstrated in vivo. In addition, data demonstrating clinical activity of MAPK-directed therapies in non-V600 BRAFmutant lung cancers are lacking. Therefore, there is currently no consensus on how to optimally manage patients with class 2 and 3 BRAF mutations. In the EURAF retrospective series that included 6 NSCLC patients with BRAF mutations other than V600E, only one patient (G596V) achieved a partial response to Vemurafenib [105]. These findings are consistent with the statement that class 2 and 3 mutants signal as RAF dimers rather than monomers [103], making them resistant to the BRAF monomer inhibitors. However, in vitro, both class 2 and 3 mutated cell lines are sensitive to MEK inhibition [177]. Additionally, the combination of BRAF and MEK inhibitors enhanced MAPK inhibition and cell proliferation reduction in both class 2 and 3 BRAF-mutant NSCLC preclinical models compared to MEK inhibitor alone [178][179][180][181]. Interestingly, oncogenic signaling triggered by class 3 BRAF mutants depends on a variety of RTKs that should be identified per patient [103].

Alternatively, the inhibition of SHP2, a scaffold protein downstream of RTKs promoting RAS activation, or the use of pan-RTK inhibitors (such as afatinib) could be considered [182].

Incidentally, concomitant SHP2 and BRAF/MEK inhibition showed synergistic effects in a class 3 BRAF-mutant cell line [183]. Additionally, allosteric inhibitors targeting the heterodimer interface between BRAF class 3 mutants with CRAF may represent an alternative approach [184]. Further studies are required to [START_REF] Rosell | Large-scale screening for somatic mutations in lung cancer[END_REF] precise the molecular mechanisms of each BRAF mutants (i.e. homo versus heterodimer), ( 2) define the importance of the additional molecular determinants that may play a role in modulating therapeutic response (i.e. expression level of CRAF and concomitant genetic alterations) and

(3) identify the most promising strategies with potential drug combination to achieve successful clinical response in patients with non-V600 BRAF mutations. [START_REF] Drilon | Targeting RET-driven cancers: lessons from evolving preclinical and clinical landscapes[END_REF]. X and Y axis represent the objective response rate and the median progression-free survival duration, respectively, as reported from phase I and II clinical trials (see main text and Table 1 for details and references). Abbreviations: GK, gatekeeper; SF, solvent-front; AL, activation loop. References [START_REF] Drilon | A Novel Crizotinib-Resistant Solvent-Front Mutation Responsive to Cabozantinib Therapy in a Patient with ROS1-Rearranged Lung Cancer[END_REF][START_REF] Solomon | RET Solvent Front Mutations Mediate Acquired Resistance to Selective RET Inhibition in RET-Driven Malignancies[END_REF]75,117,120,129,[197][198][199][200][201][202][203][204][205][206][207]. Oncogenic drivers and their downstream signaling pathways are represented in blue and green, respectively. On-target resistance mutations as well as histological transformation are indicated (boxes). Protein alterations that have been implicated in the resistance to targeted agents are shown in orange. Potential therapeutic strategies are also highlighted in red.

Outstanding questions

-Is it ethical to have an approved targeted treatment against an uncommon driver and not implement the diagnostic tests as routine to identify the patients?

-Are large randomized clinical trials comparing targeted agents with platinum-based chemotherapy needed in patients with rare oncogenic drivers?

-How could we design and validate combination treatments in the clinic that would outperform single agent therapies? Could we develop in parallel predictive biomarkers for stratifying patients?

-How can we improve the early identification of emergent on-target mutations? Could multiple samplings along the treatment through liquid biopsies and circulating DNA represent an alternative option to monitor resistance onset?

-Could concomitant low dosing therapies targeting different effectors within the RTK-RAS-MAPK pathway be implemented in the clinic?

Outstanding Questions

Highlights

-Lung cancer is responsible of around 1.8 million deaths per year worldwide and is widely the leading cause of cancer-related mortality.

-The development of specific inhibitors is currently extending to less common oncogenic drivers (i.e. ROS1, MET, RET, NTRK, HER2 and BRAF) with an exponential growth of dedicated clinical trials.

-While, individually, each of these drivers appears with low prevalence, altogether, they account for 15% of all lung cancer cases, thereby affecting a large population of patients worldwide.

-Similar to what is observed with most targeted therapies directed against an oncogenic driver, the initial clinical response to targeted kinase inhibitors is almost always temporary and acquired resistance to these drugs invariably emerges, restricting their clinical utility.

-The characterization of intrinsic and acquired resistance relies on tissue or liquid biopsy upon disease progression. However genomic testing is not always capturingthe underlying mechanism of resistance.

-Both on-target and off-target resistance mechanisms to targeted therapies could be tackled in the clinic but the design of the most effective treatment still requires further evaluation.

  Beside EGFR and HER2, other RTKs have been associated to drug resistance in lung cancer. Particularly, MET amplification has been identified in afatinib-resistance cell lines harboring HER2 alterations [124], in pralsetinib resistant RET-rearranged tumors [125] and in entrectinib resistant NTRK-rearranged biopsies [126]. Furthermore, FGFR autocrine pathway activation through FGF1 secretion was shown to be associated with increased sensitivity to FGFR inhibitor in resistant BRAF V600E LUAD [127].

[

  145]. Likewise, the Hippo pathway with its effector YAP has been recognized as a mechanism of resistance to BRAF and MEK targeted therapy in both preclinical and clinical samples bearing BRAF V600 mutations [146]. Yet, given their low prevalence, their clinical significance requires further confirmation. Importantly, the activation of bypass signaling pathways often results in the acquisition of vulnerabilities that could be therapeutically exploited. In a recent study, novel activating MAPK alterations (deletions of several nucleotides in MAP3K1 and MAP2K1 as well as NF1 loss-of-function mutations) were acquired in patients harboring ROS1-rearranged tumors being treated with ROS1 inhibitors. Cells bearing these alterations are resistant to ROS1-TKIs but sensitive to a combination of ROS1 and MEK inhibitors in vitro and in vivo [147]. Importantly, a METΔ14-altered patient who acquired a KRAS gain after crizotinib treatment achieved clinical response upon addition of trametinib [75]. The identification of PIK3CA or PTEN mutations suggests that activation of the PI3K-AKT-mTOR pathway could represent an acquired vulnerability and supports the potential use of mTOR inhibitors for the treatment of resistant NSCLC. In a phase I clinical trial, a BRAF V600E-mutated NSCLC patient who progressed to single-agent dabrafenib and multiple lines of chemotherapy achieved a partial response with the combination vemurafenib plus everolimus, a mTOR inhibitor [148]. Preliminary results from a clinical trial (NCT01582191) combining vandetanib and the mTOR inhibitor everolimus showed significant activity in RETpositive NSCLC [149] as well as CNS responses [150]. Overall, combined inhibition of uncommon drivers and bypass signaling pathway (either with mTOR inhibitors or MEK inhibitors) could be a promising therapeutic strategy that should be assessed in resistant patients lacking on-target resistance mutations.

  or L858R mutation the most frequent. Both first-generation (erlotinib and gefitinib) and secondgeneration (afatinib) inhibitors demonstrated more durable responses than chemotherapy [160-164], receiving FDA-approval as first-line treatments in 2013 and 2014. In almost half of patients treated with these compounds, resistance is mediated by the T790M gatekeeper mutation (equivalent to those described in ROS1, RET and NTRK, Figure 2A). Thirdgeneration EGFR inhibitors such as osimertinib demonstrated impressive response in patients with EGFR T790M-positive NSCLC after progression on early-generation TKIs, receiving FDA-approval in 2017 [165]. Due to its ability to spare wild-type EGFR, inhibit EGFR 19Del/L858R/T790M and CNS penetration, osimertinib is approved as first-line treatment since 2018 [166]. Unfortunately, new resistance mechanisms have emerged like the tertiary C797S mutation affecting the covalent binding of the drug [167, 168] or the development of T790 wild-type clones [169]. ALK rearrangements are observed in 5% of NSCLC, being EML4-ALK the most prevalent. At least 15 EML4-ALK variants have been identified so far, with some expressed as multiple isoforms [170]. All contain the ALK entire intracellular kinase domain (exons 20-29) but differ in the breakpoint with the EML4 gene. First-generation crizotinib received FDA approval in 2011, becoming the first approved ALK inhibitor for NSCLC [171]. Almost one third of crizotinib-treated patients acquire resistance by kinase domain mutations, being L1196M

Figure 1 :

 1 Figure 1: Comparative clinical efficacy of targeted therapies in ROS1, MET, RET, HER2 and BRAF-altered lung cancers. Each circle on the plot represents the efficacy of the indicated inhibitor inpatients for each specific driver (indicated with the different colors). Inspired by Figure 5 in Drilon A. and colleagues [57]. X and Y axis represent the objective response rate

Figure 2 :

 2 Figure 2: Schematic representation of the on-target resistance mutations and activity profile of kinase inhibitors targeting these specific resistance mutations among the different oncogenic drivers in NSCLC. A. The different types of mutations affecting the solvent-front, the gatekeeper residue, the activation loop or the DFG motifs are represented. Most resistance mutations seen in uncommon driver oncogenes have analogous resistance mutations identified in EGFR and ALK. B. Blue boxes indicate kinase inhibitor for which antitumoral activity against the indicated mutants has been reported in preclinical models (preclinical validation). Orange boxes designate kinase inhibitor that failed to show any inhibitory activity in vitro and/or in vivo (preclinical invalidation). Green boxes highlight reports of clinical activity (tumor shrinkage) while red dots indicate agents that failed to achieve an objective response in patients. Asterisks (*) indicate FDA approved drugs.

Figure 3 :

 3 Figure 3: Signaling pathways driving resistance to targeted therapies in ROS1 (A), RET (B) and NTRK (C) fusions-positive NSCLC and MET (D), HER2 (E) and BRAF (F)-mutated NSCLC.

Table 1 : Comparative clinical efficacy of targeted therapies in ROS1, MET, RET, NTRK, HER2 1063 and BRAF-altered lung cancers.

 1

	1064
	Driver	Drug	Clinical trial identifier a /name (phase)	NSCLC, n	ORR, %	mPFS, months	Intracranial disease control, n (%)	FDA approval	Refs
			EUROS1 (retrospective)	32	80	9.1			[24]
		Crizotinib	NCT00585195/PROFILE 1001 (I) NCT01945021 (II)	50 127	72 71.7	19.2 15.9			[22] [25]
			NCT02183870/EUCROSS (II)	34	70	20			[26]
			NCT02034981/AcSé (II)	78	47.2				[23]
		Ceritinib	NCT01964157 (II)	32 (30 crizotinib naïve)	62 (overall) and 67 (naïve)	9.3 (overall) and 19.3 (naïve)	5/8 (63%)		[28]
	ROS1			40 (previously	35 (previously	8.5 (previously	Previously treated:
		Lorlatinib	NCT01970865 (I/II)	treated) and	treated) and 62	treated) and 21	12/24 (50%)		[29]
				21 (TKI	(naïve)	(naïve)	Naïve: 7/11
				naïve)			(64%)
			NCT02097810/STARTRK-1
			(I),
		Entrectinib	NCT02568267/STARTRK-2 (II) and EudraCT 2012-	53	77	19	11/20 (55%)		[8, 27]
			000148-88/ALKA-372-001
			(I)
		Crizotinib	NCT00585195/PROFILE 1001 (I)	65	32	7.3			[73]
		Tepotinib	NCT02864992/VISION (II)	99	46	8.5	6/11 (55%)		[74]
	MET	Capmatinib	NCT02414139/GEOMETRY mono-1 study (II)	69 (previously 28 (naïve) treated) and	41(previously (naïve) treated) and 64	5.4 (previously (naïve) treated) and 12.4	12/13 (92%)		[9]
		Vandetanib	UMIN000010095/LURET (II) NCT01823068 (II)	19 17	47 18	4.7 4.5			[185] [186]
		Cabozantinib	NCT01639508 (II)	25	28	5.5			[187]
		Lenvatinib	NCT01877083 (II)	25	16	7.3			[188]
		Cabozantinib,
		vandetanib,
		sunitinib,
		sorafenib,
		alectinib,	GLORY (retrospective)	53	26	2.3			[54]
		lenvatinib,
		nintedanib,
		ponatinib or
		regorafenib
	RET	RXDX-105	NCT01877811 (I/Ib)	31 (RET inhibitor	19 (0% with KIF5B-RET and 67% with				[189]
				naïve)	non-KIF5B partners)
		Alectinib	UMIN000020628 (II) and NCT03131206 (II)	ongoing
		Pralsetinib	NCT03037385/ARROW (I/II) NCT04222972/AcceleRET (III)	116 (26 naïve) ongoing	65 (overall) and 73 (naïve)		5/9 (56%)		[5]
		Selpercatinib	NCT03157128/LIBRETTO-001 (I/II)	105 (previously treated) and 39 (naïve)	64 (previously treated) and 85 (naïve)	16.5 (previously treated)	10/11 (91%)		[4]
			NCT04194944/LIBRETTO-431 (III)	ongoing
		Larotrectinib	NCT02122913 (I) NCT02576431 (II)	12 ongoing	88 (pan cancer)				[6]
	NTRK	Entrectinib	NCT02097810/STARTRK-1 (I), NCT02568267/STARTRK-2	10	57 (pan cancer)	11.2 (pan cancer)			[7]
			(II) and EudraCT 2012-

Acknowledgements

This work was supported by grants from INCA (Plbio19-190), Siric-BRIO and a Senior Chair Idex-Université de Bordeaux (2016-0396) to D.S.E.N. received support from the Department