New Generations of Tyrosine Kinase Inhibitors in Treating NSCLC with Oncogene Addiction: Strengths and Limitations

Simple Summary This manuscript focuses on improving the treatment of non-small cell lung cancer, with actionable gene alterations. The aim is to understand how the treatment with Tyrosine Kinase Inhibitors (TKIs) can be used and improved. Newer generations of TKIs have better results in controlling the disease and extending patient survival. These drugs also work better in the brain, which is crucial for patients with brain metastases. However, there are challenges. The use of newer TKIs may limit the role of older ones, and resistance to the drugs can emerge. The considerations from this manuscript suggest that understanding the biology of the tumor and the properties of the drugs could help develop new treatment strategies and ultimately benefit patients with this type of lung cancer. Abstract Tyrosine kinase inhibitors (TKIs) revolutionized the treatment of patients with advanced or metastatic non-small cell lung cancer (NSCLC) harboring most driver gene alterations. Starting from the first generation, research rapidly moved to the development of newer, more selective generations of TKIs, obtaining improved results in terms of disease control and survival. However, the use of novel generations of TKIs is not without limitations. We reviewed the main results obtained, as well as the ongoing clinical trials with TKIs in oncogene-addicted NSCLC, together with the biology underlying their potential strengths and limitations. Across driver gene alterations, novel generations of TKIs allowed delayed resistance, prolonged survival, and improved brain penetration compared to previous generations, although with different toxicity profiles, that generally moved their use from further lines to the front-line treatment. However, the anticipated positioning of novel generation TKIs leads to abolishing the possibility of TKI treatment sequencing and any role of previous generations. In addition, under the selective pressure of such more potent drugs, resistant clones emerge harboring more complex and hard-to-target resistance mechanisms. Deeper knowledge of tumor biology and drug properties will help identify new strategies, including combinatorial treatments, to continue improving results in patients with oncogene-addicted NSCLC.


Introduction
Lung cancer remains one of the most prevalent and deadly malignancies worldwide, with non-small cell lung cancer (NSCLC) constituting the majority of cases diagnosed [1].Over the past few decades, significant strides have been made in understanding the molecular underpinnings of NSCLC, leading to the identification of driver gene alterations that have, in turn, transformed the landscape of treatment for this disease.Among these remarkable advancements, the advent of tyrosine kinase inhibitors (TKIs) has emerged as a paradigm-shifting approach, offering newfound hope and extended survival to patients with advanced or metastatic NSCLC harboring specific genetic alterations [2,3].
The landscape of medical oncology has witnessed a revolution triggered by these targeted therapies, as they have the potential to halt the progression of the disease with Cancers 2023, 15, 5079 2 of 27 greater efficacy and fewer adverse effects compared to conventional chemotherapy.Understanding the historical evolution and ongoing developments of TKIs is crucial for both clinicians and researchers in the field of medical oncology.
The inception of TKIs in NSCLC therapy began with the first generation of these agents.Initially designed to target the epidermal growth factor receptor (EGFR), this class of drugs showed unprecedented promise in a subset of NSCLC patients harboring EGFR mutations [4].It was a watershed moment, offering personalized treatment options and substantially improving outcomes for these individuals.However, the first-generation TKIs, such as erlotinib and gefitinib, brought with them their own set of limitations, including the emergence of resistance mechanisms like the p.T790M mutation [5].
Recognizing the need for more potent and selective therapies, researchers swiftly moved forward in the development of subsequent generations of TKIs.These newer iterations promised enhanced specificity for their respective targets and a greater ability to circumvent resistance mechanisms [6].
Despite these remarkable advancements, the utilization of novel generations of TKIs is not without its complexities and challenges.Resistance remains a persistent issue, necessitating ongoing research into more effective treatment strategies [7][8][9].Additionally, the optimal sequencing of these agents and their integration into the treatment landscape of NSCLC requires careful consideration (Figure 1).The identification of predictive biomarkers and the management of adverse effects associated with TKI therapy are also areas of active investigation [10].
Cancers 2023, 15, x 2 of 30 survival to patients with advanced or metastatic NSCLC harboring specific genetic alterations [2,3].The landscape of medical oncology has witnessed a revolution triggered by these targeted therapies, as they have the potential to halt the progression of the disease with greater efficacy and fewer adverse effects compared to conventional chemotherapy.Understanding the historical evolution and ongoing developments of TKIs is crucial for both clinicians and researchers in the field of medical oncology.
The inception of TKIs in NSCLC therapy began with the first generation of these agents.Initially designed to target the epidermal growth factor receptor (EGFR), this class of drugs showed unprecedented promise in a subset of NSCLC patients harboring EGFR mutations [4].It was a watershed moment, offering personalized treatment options and substantially improving outcomes for these individuals.However, the first-generation TKIs, such as erlotinib and gefitinib, brought with them their own set of limitations, including the emergence of resistance mechanisms like the p.T790M mutation [5].
Recognizing the need for more potent and selective therapies, researchers swiftly moved forward in the development of subsequent generations of TKIs.These newer iterations promised enhanced specificity for their respective targets and a greater ability to circumvent resistance mechanisms [6].
Despite these remarkable advancements, the utilization of novel generations of TKIs is not without its complexities and challenges.Resistance remains a persistent issue, necessitating ongoing research into more effective treatment strategies [7][8][9].Additionally, the optimal sequencing of these agents and their integration into the treatment landscape of NSCLC requires careful consideration (Figure 1).The identification of predictive biomarkers and the management of adverse effects associated with TKI therapy are also areas of active investigation [10].This review manuscript will provide a thorough analysis of the strengths and limitations associated with the use of TKIs in NSCLC with oncogene addiction.We will explore the pivotal clinical trials that have shaped the current treatment paradigm and shed light on the emerging therapies currently under investigation.Furthermore, we will delve into the intricate biology underlying the potential strengths and limitations of these agents, unraveling the complex interplay between oncogenic signaling pathways and therapeutic interventions.

Efficacy
Since the advent of the first-generation TKIs, erlotinib and gefitinib, for the treatment of patients with EGFR-sensitizing mutations, an enduring paradigm shift towards precision oncology has guided the development of more potent and specific TKIs to overcome intrinsic and acquired resistance mechanisms responsible to treatment failure (efficacy results of novel generation TKIs in clinical practice are shown in Table 1).
Second-generation TKIs afatinib and dacomitinib improved clinical outcomes compared to platinum-based chemotherapy (PBC) and first-generation TKIs in patients with EGFR-sensitizing mutations [11][12][13][14][15][16].Moreover, in a pooled analysis of the LUX-Lung trials, afatinib was also active in tumors with uncommon EGFR mutations, although the clinical benefit was lower in patients with de novo T790M and exon-20 insertion mutations [17].However, after these molecules entered clinical practice, the occurrence of severe adverse events (AEs), mostly skin rash and diarrhea, due to the inhibition of wild-type EGFR narrowed the therapeutic window that was needed to effectively overcome acquired resistance mechanisms, especially the T790M mutation.To address this shortcoming, the third-generation osimertinib was developed to specifically target the EGFR-T790M mutation while retaining activity against initial activating mutations and selectivity over wild-type EGFR.Osimertinib received its first approval from the Food and Drug Administration (FDA) for the treatment of EGFR-T790M-positive NSCLC based on a 6-month improvement in progression-free survival (PFS) compared to PBC in the AURA3 trial and a hazard ratio (HR) for overall survival (OS) of 0.54 after adjustment for the high crossover rate in the study [18,19].Subsequently, in the FLAURA trial, osimertinib outperformed first-generation TKIs in PFS (18.9 vs. 10.2 months, HR 0.46) and OS (38.6 vs. 31.8months, HR 0.80), regardless of the T790M mutation, with better tolerability, establishing the role of first-line osimertinib as the gold-standard [20,21].Despite these results, the emergence of resistance ultimately leads to treatment failure.Resistance mechanisms are highly complex and multifaceted, including the emergence of the C797S mutation, the loss of T790M, small cell lung cancer (SCLC) transformation, and MET amplification; thus, tumor biopsy upon disease progression should be considered whenever feasible to optimize treatment strategies [22,23].
Although relatively rare, accounting for 2-3% of cases, EGFR-exon 20 insertion mutations confer resistance to TKIs, requiring treatment with PBC.The oral TKI Mobocertinib was active and led to sustained responses in PBC-treated patients with EGFR-exon 20 insertion mutations.Based on these results, despite gastrointestinal and dermatological AEs hampering their clinical utility, mobocertinib was granted FDA accelerated approval [24,25].However, the confirmatory trial EXCLAIM-2 ended prematurely in July 2023 as first-line mobocertinib monotherapy failed to improve PFS compared to PBC [26].Therefore, it is still to be determined whether the approval will remain intact, particularly given the PFS improvement observed with the combination of first-line amivantamab, an EGFR-MET bispecific antibody, and PBC over PBC alone in the phase III PAPILLON trial [27].
In the realm of HER2-mutation-positive NSCLC, phase 2 trials have investigated the role of small molecule TKIs, pyrotinib and poziotinib.These agents showed only modest activity and severe gastrointestinal and cutaneous AEs, owing to EGFR inhibition, that hindered further development [28][29][30].Current research is focusing on novel HER2selective TKIs that lack activity against other HER/ERBB family members, aiming for enhanced activity and improved safety.Notably, in the phase 2 study DESTINY-Lung01 the antibody drug-conjugate (ADC) trastuzumab-deruxtecan (TDXd) yielded durable activity in previously treated patients and was generally well-tolerated, though interstitial lung disease (ILD) required prompt diagnosis and management [31], and the ongoing DESTINY-Lung-04 will determine its superiority over PBC as first-line (NCT05048797).
Following the approval of the first-in-class ALK-TKI, crizotinib [32], there was a compelling need for more potent therapeutic alternatives to overcome resistance.Owing to an inhibitory activity against several crizotinib or ceritinib-resistant ALK mutations, alectinib first improved PFS and intracranial objective response rate (ORR) in crizotinibresistant patients compared to PBC, with an acceptable safety profile [33].Subsequently, the ALEX trial established the superiority of first-line alectinib compared to crizotinib, with a 24-month PFS improvement (HR 0.32) and higher CNS activity (59% vs. 26%) [34,35].Firstline brigatinib also improved long-term outcomes over crizotinib in the ALTA 1L study and stands as a viable treatment option in this setting [36].The third-generation lorlatinib, initially developed to overcome resistance mechanisms responsible for progression to second-generation TKIs, significantly improved PFS (HR 0.28) compared to crizotinib in the CROWN trial [37,38].Treatment with lorlatinib was associated with an acceptable toxicity profile, as grade 3-4 AEs were mostly represented by altered lipid levels [38].Interestingly, some compound mutations that confer resistance to lorlatinib might re-sensitize tumoral cells to crizotinib, making molecularly guided treatment a potentially valuable therapeutic strategy in some cases [39].
In the context of ROS1-fusion-positive NSCLC, crizotinib is associated with a median PFS of approximately 19 months [40], yet treatment failure and CNS progression generally occur within 2 years of treatment [41].Lorlatinib demonstrated activity in crizotinibresistant, ROS1-positive NSCLC in a Phase I-II trial, achieving an ORR of 35% [42].Recently, in a pooled analysis of the phase I-II trials, ALKA-372-001, STARTRK-1, and STARTRK-2 entrectinib achieved an ORR of 67%, with a median duration of response (DoR) of almost 16 months, thereby supporting the choice of this agent for first-line treatment.Although generally well tolerated, severe AEs occurring at a low frequency, including cardiac and CNS AEs, need to be carefully monitored as they might require dose modifications in some instances [43,44].
Persistent efforts in the structural analyses of KRAS, a protein that has been historically deemed "undraggable", paved the way for the development of KRAS G12C -selective inhibitors sotorasib and adagrasib, that, although not in the class of TKIs, are worth mentioning as they have been both approved for clinical use following at least one prior line of systemic therapy.Sotorasib demonstrated activity in the phase I/II CodeBreaK-100 trial, with an ORR of 41%.The most common AEs included diarrhea and elevation in transaminases.However, in the phase III CodeBreaK-200 trial, the PFS improvement was small compared with docetaxel (5.6 vs. 4.5 months, HR 0.66), and OS was similar in the two arms (10.6 vs. 11.3 months) [45].Similarly, adagrasib achieved an ORR of 43% in phase I/II KRYSTAL-1 trial, with durable responses and grade ≥3 AEs in 45% of patients [46,47], while its efficacy as second-line compared to docetaxel and as first-line is currently under investigation (KRYSTAL-12, NCT04685135; KRYSTAL-7, NCT04613596).
Traditionally, MET gene alterations have been treated with crizotinib [48], and other multikinase inhibitors, with limited efficacy and significant toxicity.Selective MET-TKIs capmatinib and tepotinib have revolutionized the treatment landscape for patients with a MET-exon-14-skipping mutation, leading to high and durable responses in both previously treated (ORR 40-51%) and treatment-naïve patients (ORR 56-67%) in the GEOMETRY mono-1 and VISION trials, respectively [49,50].Noteworthy, common AEs associated with these agents include peripheral edema, increased creatinine levels, and gastrointestinal events.Another specific MET-TKI, Savolitinib, was only approved in the People's Republic of China in 2021 [51].Notably, MET-TKIs have yet to receive approval for high-level MET amplification, although preliminary data warrant further investigation, and the ideal methodology for determining the level of amplification and appropriate cutoffs for treatment is still an active area of research.
In RET-fusion positive NSCLC, the RET-selective TKIs selpercatinib and pralsetinib earned approval in 2021, as strong clinical activity was observed in the phase I/II LIBRETTO-001 and ARROW studies, both in treatment-naïve (ORR 84% and 72%, respectively) and previously treated patients (ORR 61% and 59%, respectively) [52,53].Common AEs for selpercatinib include hypertension and increased liver enzymes, while it is crucial to monitor the occurrence of ILD associated with pralsetinib [54,55].
For BRAF-V600E-mutant NSCLC, the combination of oral serine/threonine kinase inhibitors dabrafenib and trametinib obtained significant responses in both the first (ORR 68%) and second (ORR 64%) line in the phase II BRF113928 study, and it is considered standard of care [56].Recently, encorafenib plus binimetinib showed comparable efficacy, with an ORR of 75% in treatment-naïve and 46% in pretreated patients, and this combination might emerge as a new therapeutic option [57].

CNS Activity
As a result of the specific and potent oncogene inhibition with novel TKIs, leading to improved extracranial disease control and prolonged survival, approximately 20-40% of patients ultimately develop CNS metastases.Brain metastases are associated with poor longterm outcomes, and few therapeutic options are available.Conventional local therapies include whole-brain radiation therapy, rarely curative and burdened by neurocognitive toxicity, and stereotactic radiosurgery, whereas poor performance status and disease burden often preclude neurosurgery.Furthermore, few chemotherapy agents have the ability to cross the blood-brain barrier (BBB), while, in contrast, novel generation TKIs have been specifically engineered to improve their intracranial permeability and activity, eventually leading to better intracranial outcomes.
In the natural history of ALK-translocated NSCLC, most patients develop CNS metastases.First-line alectinib or brigatinib have improved intracranial ORR compared to crizotinib, with intracranial ORRs of approximately 80% and durable intracranial responses [34][35][36].In cross-study comparisons, ceritinib showed lower CNS penetration, with an intracranial ORR of 72% observed in the ASCEND-4 trial [63].The third-generation lorlatinib outperformed the intracranial activity of crizotinib in the CROWN trial (83% vs. 23%), with complete responses in 71% of patients treated with lorlatinib.Moreover, the 12-month rate of CNS progression in patients with and without baseline brain metastases was improved in the experimental arm (7% vs. 72% and 1% vs. 18%, respectively) [38,64].
For patients with ROS-rearranged NSCLC, the intracranial activity of entrectinib further enforces its use in treatment-naïve patients, with this agent leading to intracranial ORRs of approximately 80%, alongside a median intracranial PFS of 12.0 months and a median intracranial DoR of 12.9 months [44].
Similarly, both tepotinib and capmatinib have shown intracranial activity in patients with MET exon-14 skipping, and an intracranial ORR of 68% was observed in treatmentnaïve patients treated with capmatinib in the GEOMETRY-Mono-1 study [49,50].
In the LIBRETTO-001 trial, CNS responses with selpercatinib were documented in 85% of patients, regardless of previous systemic treatment and/or radiotherapy, with a median DoR of 9.4 months [71].Pralsetinib also led to an intracranial ORR of 78% in the ARROW trial [55].
Notably, several targeted agents have shown efficacy in patients with leptomeningeal disease, including lorlatinib and alectinib for ALK-positive cancers and selpercatinib in RET-fusion NSCLC [80,81].For those with an EGFR mutation and leptomeningeal disease, osimertinib has demonstrated significant intracranial activity against brain metastases at a dose of 80 to 160 mg daily [82].

EGFR
Beyond osimertinib, several third-generation TKIs have been developed, and three of them (lazertinib, almonertinib, furmonertinib) have already been approved with the same indications of osimertinib in Korea and China [83][84][85][86][87]. Table 2 summarizes their principal features, their main clinical results, and, where available, their approval indications.Similarly to osimertinib, they have proven to be active towards EGFR-T790M resistance mutation and showed superiority to 1st or 2nd generation TKI.Moreover, they share the same risk of developing rare and severe toxicity (ILD and QTc prolongation) and are inactive toward EGFR p.C797S mutation.In the absence of any head-to-head comparison results, the real advantage taken from more similar drugs available in the market will be the potential improvement in the cost-effectiveness of these drugs [88][89][90].To overcome EGFR-C797X mediated resistance, several 4th generation TKIs have been designed and are at difference stages of clinical research (Table S1) [101].The first developed drugs (EAI045, JBJ-04-125-02, BLU-701) were rapidly withdrawn since their activity depends on combination with other drugs or lack of efficacy [102][103][104].In the same line, BLU-945 is also being investigated in combination with osimertinib to improve the activity against EGFR-sensitizing mutations [105].Further, fourth-generation EGFR-TKIs, which have demonstrated proof of activity in cancer models harboring C797S with or without T790M, are being experimented in phase I trials (Table S1) [106][107][108][109][110][111][112][113][114].
With concern on the EGFR and HER2 Exon 20 insertion mutations that are intrinsically resistant to available EGFR-TKIs, different drugs with similar properties are currently evaluated in clinical research (Tables S2 and S3) [115][116][117][118][119][120][121].The main issue in the development of TKIs targeting EGFR or HER2 exon 20 insertions consists of achieving high selectivity over wild-type receptors in order to increase their therapeutic window [114].With regard to uncommon EGFR mutations, few studies are investigating the efficacy of specific TKIs or combinations: phase II trials of furmonertinib (NCT05548348), sutetinib (NCT05168566) and the afatinib/bevacizumab combination (NCT05267288), phase III trial of almonertinib over standard chemotherapy (NCT04951648).

KRAS
The efficacy of KRAS-G12C inhibitors is tempered by the RAS pathway complexity, the concomitant presence of other gene mutations, such as KEAP1, and the acquired secondary mutations on the switch pocket II of KRAS [122,123].New KRAS inhibitors have been designed to inhibit this target more potently and selectively (Table 3 and Table S4).Among them, divarasib and JDQ433 are on more advanced clinical development.Divarasib (GDC-6036) has been designed to inhibit covalently, selectively, and with more potency KRAS G12C compared to sotorasib and adagrasib [124].JDQ443 has been designed to overcome resistance mechanisms observed with other KRAS G12C inhibitors since it acts through a novel binding mechanism that forms novel interactions with KRAS under the switch II pocket, irreversibly trapping KRAS in the inactive, GDP-bound state reaching the residue C12 without interfering with residue H95 [125].Abbreviations: IC50 = Half-maximal inhibitory concentration; OD = once daily; BID = two times a day; nM = nanomolar; ORR = overall response rate; mPFS = median progression-free survival; mDoR = median duration of response; AE(s) = adverse event(s); G = grade; Ph = phase; CT = chemotherapy; ICI immune-checkpoint inhibitor; Tx = treatment.

BRAF and MET
The novel encorafenib and binimetinib combination, already approved for BRAFmutated melanoma, has been investigated in two different phase II trials (ENCO-BRAF, OCEANII) for NSCLC patients.Several trials are experimenting with novel (pan)-RAF inhibitors alone or in combination with MEK, FAK, RAS, or SHP-2 inhibitors in patients with BRAF-V600E solid tumors, including NSCLC refractory to BRAK/MEK-inhibitors or harboring other RAF alterations (BRAF class II and III mutations, RAF gene fusions or amplification) [127] (Table S5).
Despite the development of acquired MET mutations seems to be related to the type of MET-inhibitors, at present, no clinical trial has been designed to investigate the re-sensitiveness of these patients to a novel class of MET TKIs, with the exception of a small phase II trial of capmatinib in crizotinib-resistant NSCLC patients, which showed discouraging results [128].At the state-of-the-art, only the bifunctional anti-EGFR and MET antibody amivantamab have shown modest activity (ORR 17%) in MET-TKI-resistant patients in the Chrysalis trial [129].Preliminary results were presented from the phase I SHIELD trial of elzovantinib, a MET, SRC, and CSF1R inhibitor, in 52 patients with MET dysregulated solid tumors, including 30 patients with MET-altered NSCLC (20 MET-ex14 skipping mutations, 8 MET amplified, 2 other MET mutations) [130].Among the eleven TKI naïve NSCLC patients, the ORR was 36% regardless of dose modifications (Table S6).

Fusion Genes
Novel 3rd and 4th ALK-TKIs are underway in clinical research.APG-2449 is a novel FAK inhibitor and a third-generation ALK/ROS1-TKI that has shown potent activity towards a range of ALK-resistant mutations and brain penetrant capacity in pre-clinical NSCLC tumor models.An ongoing phase I trial is evaluating patients with secondgeneration TKIs-resistant ALK/ROS1-positive NSCLC.Preliminary results have shown an ORR of 28.5% among 14 patients with ALK-TKI refractory NSCLC [131].TPX-0131 and NVL-655 are the 4th generation ALK-Is.The clinical development of TPX-0131 has been withdrawn due to safety issues; meanwhile, phase I/II of NVL-655 is ongoing [132,133] (Table S7).Tables S8 and S9 show the ongoing clinical trials of new ROS1 and NTRK inhibitors (taletrectinib, repotrectinib, NVL-520 among the others for ROS1 and repotrectinib, VMD-928 and XZP-928 for NTRK) with high BBB penetrance and activity towards secondary single or double mutations.
Novel RET inhibitors have been designed to cover acquired resistance mutations while sparing the inhibition of other targets, such as VEGFR2, to augment their therapeutic window (Table 4).

EGFR
After the encouraging results of the Japanese phase III trial NEJ009 [140] and phase II OPAL trial [141], the FLAURA-2 trial confirmed the benefit of combining PBC to the third generation TKI osimertinib in 586 treatment naïve EGFR+ NSCLC patients, as the combination led to a PFS improvement in 8 months (HR 0.62) [142].Table 5 depicts trials of the TKI-CT combination for a selected population of patients (p53 mutant or other tumor suppressor genes, lack of circulating DNA clearance, brain metastases).In the post-TKI setting, the COMPEL phase III trial is investigating the role of adding a TKI to standard second-line chemotherapy in order to prevent CNS progression [143].
Histologic transformation into SCLC has been observed in 3-14% of EGFR+ NSCLC patients treated with first-generation EGFR-TKIs (gefitinib or erlotinib), frequently mediated by p53/RB1 loss of function [144].An ongoing phase II trial is evaluating the combination of olaparib and durvalumab in this setting (NCT04538378).
Another important line of research is represented by the development of bispecific antibodies.In the CHRYSALIS (NCT02609776), the combination of amivantamab plus lazertinib was tested in 20 treatment-naïve Asian patients with EGFR-mutant NSCLC, attaining an ORR of 100%.Interestingly, after a median duration of treatment of 33.5 months, median DOR, PFS, and OS have not yet been reached [145].
The phase 3 MARIPOSA study is further investigating the lazertinib/amivantamab combination versus lazertinib or osimertinib alone in 1074 treatment-naïve NSCLC patients with EGFR-common mutations, and positive results have been preliminarily announced [146].Indeed, in the MARIPOSA2 phase III trial, the combination of amivantamab alone or plus lazertinib with standard PBC has led to a PFS improvement in NSCLC patients harboring common EGFR-mutations after failure of treatment with osimertinib [147].
Among the different TKI-TKI combinations, the major interest is focused on MET inhibition.Alongside the first-line combination of savolitinib plus osimertinib (FLOWERS, NCT05163249; NCT04743505), major efforts are being oriented towards the post-3rd generation TKI setting.Based on the results of preliminary trials with gefitinib plus capmatinib and osimertinib plus savolitinib [148], phase II and III trials have been designed to confirm the efficacy of these combinations [149,150].Moreover, the ORCHARD trial is a biomarkerdirected phase II platform study evaluating the optimal treatment for individual patients with EGFR-mutant NSCLC [151] (Table 5, Table S10).

Other Driver Gene Mutations
Table S11 summarizes ongoing TKI-based combinations with chemotherapy, antiangiogenics, multitargeted drugs, or immune-modulating agents for NSCLC patients with EGFR or HER2 exon 20 insertion mutations.
Several combinations have been designed and are under development in clinical trials to overcome resistance to KRAS inhibitors (Table S12).Indeed, trials have been designed to investigate the MEK-inhibitors and ICI combinations in solid tumors, BRAF or KRAS mutated NSCLC on the basis of preclinical data and case reports suggesting that MEK inhibition can modulate CTLA-4 expression and potentially increase the efficacy of ICI [152,153] (Table S13).
Few clinical trials are ongoing with TKI-based combinations of drugs (TKI plus MEK-I or ICI plus amivantamab), aiming to overcome the occurrence of resistance to MET inhibitors (Table S14).

The Issue of Sequencing Treatments with New Generation TKIs
In the dynamic landscape of targeted therapy for driver gene alterations in cancer, the introduction of novel generations of TKIs has ushered in a new era of treatment paradigms.These advancements have conferred delayed resistance, prolonged overall survival, and enhanced CNS penetration compared to their predecessors [20,64,154,155].This transformative impact has often led to the relocation of these novel agents from later lines of therapy to the front-line treatment setting.While these innovations represent a substantial leap forward in cancer care, they raise a significant and somewhat paradoxical challenge: the potential obsolescence of TKI treatment sequencing and the diminished role of previous TKI generations.
However, this progress comes with its own set of complexities, particularly in terms of toxicity profiles.While the safety and tolerability of these novel agents are generally manageable, they often differ from those of their predecessors, requiring new learning curves for medical oncologists on the management of novel and peculiar adverse events in clinical practice (e.g., cognitive effects with lorlatinib and entrectinib, the management of interstitial lung disease occurring at different rates with different drugs) [59,156].
strategic shift towards the front-line adoption of novel TKIs raises a critical question regarding the sequencing of TKI treatments.Historically, the sequencing of TKIs was a vital aspect of managing drug resistance and optimizing patient outcomes.Patients who developed resistance to an earlier generation of TKI often had the option to switch to a subsequent generation with a different mechanism of action, thereby extending the duration of disease control [18,33].However, the ascendancy of novel TKIs as first-line therapies has effectively closed this avenue.With the superior efficacy of these agents, previous generations have become less relevant in the treatment algorithm, relegating them to a historical perspective rather than a therapeutic option.As such, whereas TKI sequencing from first-to novel generations relegated chemotherapy options at the end of the treatment sequence, anticipating novel TKI generations in the front-line setting made the role of the 'old' chemotherapy options being revived as a necessary second-line treatment option, outside clinical trials [2].
Nonetheless, this paradigm shift underscores the imperative for continuous innovation and adaptation in oncology as the field continues to evolve to meet the ever-changing needs of patients with driver gene alterations.

The Issue of Resistance: Selective Pressure on Resistant Clones
Resistance to cancer therapies is a formidable challenge in the field of medical oncology, and a crucial aspect of this challenge is the selective pressure imposed on resistant clones within heterogeneous tumors [7,157].Molecular heterogeneity, a pervasive feature of most cancers, lies at the heart of acquired resistance development.Tumors, even when sharing similar clinical characteristics, are composed of a mosaic of molecular clones, each characterized by unique genetic and phenotypic traits [158].This inherent diversity within tumors provides fertile ground for the emergence of drug-resistant clones, each endowed with specific survival advantages and resistance mechanisms [7].Particular subgroups referred to as Drug-Tolerant Persister (DTP) cells, have the capacity to endure high-dose treatments [159].Intriguingly, these sub-clones possess distinctive stem cell markers and can adapt their characteristics in response to therapy-induced selective pressure [159].DTPs, along with de novo mutations and preexisting resistance mechanisms, are among the potential strategies employed by cancers to evade the pressures exerted by anticancer drugs [157,158].
The consequence of this molecular diversity is that therapeutic interventions, while initially effective against a subset of tumor cells, inadvertently create an environment conducive to the survival and proliferation of drug-resistant clones.The selective pressure imposed by treatments favors the outgrowth of these resistant populations over time, ultimately leading to therapeutic failure [158].This selective pressure is a dynamic process in which sensitive tumor cells are progressively eliminated, allowing resistant clones to dominate [160].
The role of druggable driver mutations further underscores the complexity of acquired resistance development.In cases where tumors harbor well-defined driver mutations, resistance mechanisms are often intricately linked to these drivers.However, the potency of the therapeutic agent employed can significantly influence the nature of resistance.High-potency drugs, as novel generations of TKIs are, can exert more substantial selective pressure, potentially driving the development of resistance mechanisms that are independent of the drug's primary target [157,161].
A notable example is observed in the treatment of EGFR-mutant lung cancers, where the use of different generations of tyrosine kinase inhibitors (TKIs) leads to distinct resistance patterns [161].First-generation TKIs like gefitinib and erlotinib are associated with a high incidence of the p.T790M resistance mutation.In contrast, second-generation TKIs such as afatinib have a reduced incidence of p.T790M mutations, while third-generation TKIs like osimertinib exhibit even lower rates of EGFR-dependent resistance mecha-nisms [23,162].Instead, under the selective pressure of more potent drugs like osimertinib, alternative resistance mechanisms such MET amplification can become prevalent [163].Similarly, the different structure and potency among first-, second-, and third-generation ALK TKIs lead to a different selection of resistance mutations [164,165].In addition, the sequential use of subsequent generations of TKIs may lead to the emergence of compound resistance patterns [166].
Indeed, under the selective pressure of more potent compounds, as novel generations of TKIs, resistant clones emerge, harboring more complex and hard-to-target resistance mechanisms [7,167].Understanding the interplay between selective pressure, molecular heterogeneity drug potency, binding affinity, and structure is critical for devising effective strategies to overcome drug resistance and improve treatment outcomes in cancer patients.

Conclusions and Future Directions
From the humble beginnings of the first-generation TKIs, exemplified by erlotinib and gefitinib in EGFR-mutant disease, to the cutting-edge third-generation agents such as osimertinib and extending the application of TKI treatment to other oncogene-driven diseases, we have witnessed a remarkable transformation in the therapeutic landscape of NSCLC [168].These targeted therapies have not only prolonged the lives of countless patients but have also provided a blueprint for precision medicine in oncology.
As we conclude our exploration of the strengths and limitations of novel generations of TKIs in NSCLC, it is evident that these agents have significantly improved disease control and survival rates among patients harboring specific genetic alterations.However, it is equally clear that challenges persist, and there is much work yet to be done to optimize their use and expand treatment options.One notable limitation is the development of resistance mechanisms, which underscores the need for ongoing research into novel therapeutic strategies.
One promising avenue lies in the realm of Antibody-Drug Conjugates (ADCs).These innovative biopharmaceuticals combine the specificity of monoclonal antibodies with the cytotoxic potency of chemotherapy drugs (payload), offering a new approach to target oncogenic pathways in NSCLC [169].ADCs have the potential to overcome some of the limitations of TKI therapy, particularly in cases of resistance and heterogeneous tumor populations.To date, different ADCs are being investigated in driver-mutant NSCLCs after the failure of standard TKI treatment [170].In a future perspective, these agents can be designed to target specific driver mutations, either by direct antibodies or even using TKIs as pharmaceutical components (as payload or instead of monoclonal antibody) of the ADCs, providing a level of precision therapy that was previously unthinkable [169].
In addition, combination treatments represent another potential strategy.The intricate biology of NSCLC, characterized by the crosstalk between multiple signaling pathways, necessitates a multifaceted approach to therapy.Combinations of TKIs with chemotherapy agents as recently demonstrated in the FLAURA-2 trial with osimertinib plus platinumdoublet, have shown promise in enhancing antitumor responses and delaying the emergence of resistance [142].Furthermore, rational combinations of TKIs with other targeted therapies, such as MET inhibitors [171], are being actively investigated to address resistance mechanisms and broaden the scope of effective treatment options.

Table 1 .
Efficacy results in registrational trials for 2nd and 3rd generation TKIs and KRAS-inhibitors in clinical practice.

Table 3 .
Summary of the novel KRAS inhibitors most advanced in clinical research.

Table 4 .
Summary of clinical results of novel RET inhibitors.

Table 5 .
Main TKI-based combinations either in tx naïve or previously TKI pretreated EGFRmutant NSCLC.