AMG510

KRAS inhibition in nonesmall cell lung cancer: Past failures, new findings and upcoming challenges

Francesco Passiglia a,1, Umberto Malapelle b,1, Marzia Del Re c,1, Luisella Righi a, Fabio Pagni d, Daniela Furlan e, Romano Danesi c,
Giancarlo Troncone b, Silvia Novello a,*

a Department of Oncology, University of Turin, S. Luigi Gonzaga Hospital, Orbassano (TO), Italy
b Department of Public Health, University of Naples Federico II, Naples, Italy
c Clinical Pharmacology and Pharmacogenetics Unit, Department of Clinical and Experimental Medicine, University Hospital of Pisa, Italy
d Department of Medicine and Surgery, Pathology, San Gerardo Hospital, University of Milano- Bicocca, 20900 Monza, Italy
e Pathology Unit, Department of Medicine and Surgery, University of Insubria, 21100, Varese, Italy

Received 9 April 2020; received in revised form 8 June 2020; accepted 18 June 2020

Abstract

Despite the high prevalence of Kirsten rat sarcoma (KRAS ) mutations in non-small cell lung cancer (NSCLC), for a long time it has been defined as an ‘undruggable target’, with precision medicine not considered as an adequate approach to treat this subgroup of patients. After several years of efforts, preliminary data from early clinical trials have recently demon- strated that direct pharmacological inhibition of KRAS p.G12C mutation is possible, emerging as an effective targeted treatment for about 10e12% of patients with advanced NSCLC, with potential relevant impact on their long-term survival and quality of life.

1. Introduction

The application of precision medicine is certainly contributing to the relevant increase of life expectancy observed in patients with lung cancer over the past ten years, with a 5-year survival rate of 21.7% nowadays, up from 17.2% a decade ago [1]. The paradigm of precision medicine is based on a ‘biomarker ensemble’ including predictive molecular alterations, hypothesised to play a crucial role in the disease biological pathway; diagnostic assays, used to determine patient’s biomarker status; and therapeutic agents, intended to be effective within the ‘biomarker-positive’etargeted population. The pharmacological inhibition of the epidermal growth factor receptor (EGFR) [2] represented the first example of successful targeted therapy in lung cancer, revealing that individuals with actionable tumour mutations receiving a matched targeted agent live longer and better [3]. The subsequent, accelerated approval, by the Food and Drug Administration (FDA), of the first anaplastic lymphoma kinase (ALK) inhibitor, crizotinib, even in absence of phase III, randomised clinical trials, revolu- tionised the paradigm of cancer drugs development, introducing the innovative concept of biomarker-driven, early clinical trial design and shortening times for reg- ulatory approval. The advent of next-generation sequencing (NGS) technologies and the rigorous appli- cation of personalised medicine to the clinical research program, favoured the development of new effective drugs targeting the proto-oncogene tyrosine-protein kinase-1 (ROS-1) rearrangements and the BRAF-V600 mutations, recently included in the treatment algorithm of advanced nonesmall cell lung cancer (NSCLC) [4]. Several other compounds targeting rare oncogenic drivers (RET, MET, NTRK, HER2) have recently shown a very promising activity and tolerability profile in selected subgroups of patients included in early phase studies; thus, they will be soon available for clinical use. Tumour genotyping has been now incorporated in the clinical management of advanced NSCLC, with upfront routine testing for EGFR, ALK, ROS-1 and BRAF- V600, recommended in all patients with non-squamous histology, to personalise first-line treatment [5]. Addi- tional molecular analysis by NGS may be evaluated as part of broader testing panels, with the final aim of identifying rare actionable alterations to be targeted within clinical trials.

Despite the great interest of the scientific community, for a long time Kirsten rat sarcoma (KRAS ) mutations have offered a very limited and uncertain role as a prognostic or predictive biomarker in patients with lung cancer, and precision medicine was not considered as an adequate approach to treat this subgroup of patients.After several years of efforts, preliminary data from early clinical trials [6,7] have recently demonstrated that direct pharmacological inhibition of KRAS p.G12C mutation is possible, emerging as an additional effective targeted treatment to be offered to a large subset of patients with advanced NSCLC.In this review we briefly summarise the biological basis of KRAS inhibition in NSCLC, report the current status of KRAS mutations detection in the Italian real- word scenario and provide an updated overview of therapeutic strategies, discussing the potential reasons for past failures and analysing the upcoming challenges related to the advent of new targeted agents in clinical practice.

2. KRAS mutations in NSCLC

KRAS represents the most common oncogene driver in human cancer, occurring in about 30% of lung adeno- carcinoma. Differently from other NSCLC molecular alterations, such as EGFR mutations and ALK rear- rangements, the prevalence of KRAS mutations is higher in Western and smokers’ population [8].

The KRAS oncogene encodes a 21 kD monomeric GTPase, implicated in the transduction of the extracel- lular signals to the intracellular one through the acti- vation and inactivation (on/off state) determined by the nucleotide guanosine triphosphate (GTP) binding (Fig. 1) [8]. Mutation in exon 2, 3 and 4 of the KRAS onco- gene leads to a constitutive activation of the mitogen- activated protein kinase (MAPK) pathway. About 90% of KRAS mutations were detected in codon 12 (exon 2), and, in particular in patients with NSCLC, the most frequent allelic variations are the p.G12C (GGT to TGT) and p.G12V (GGT to GTT), resulting from a G:C to T:A transversion as a classical smoking-induced alteration. Specifically for patients with NSCLC, the frequencies of specific KRAS mutated alleles detected is strictly dependent to the mutational mechanism, and, in terms of prognostic value, patients with p.G12V or p.G12C seem to have longer survival than those with other types of mutations [9]. The KRAS biological ac- tivity is a function of its structure, depending on the GTP-binding state. Overall, the most common KRAS- activating mutations reported in codons 12, 13, 61, 117 and 146 cluster around the nucleotide-binding pocket. In particular, the p.G12 is located on the P-loop and is implicated in the nucleotide stabilisation during the activation step, leading to the alteration of both intrinsic and GAP-induced hydrolysis, not altering the rate of nucleotide exchange [10] (Fig. 2).

Recent papers have consistently demonstrated that KRAS mutations are frequently associated to co- occurring genomic alterations in lung cancer [11]. About half of KRAS-mutant NSCLC harboured addi- tional concomitant mutations in key tumour oncogenes, including TP53, STK11, KEAP1 and CDKN2A/B, among those most commonly reported [12]. Each one of these co-mutational partner acts as a crucial determi- nant of both the tumour cells’ intrinsic RAS signalling and the immune composition of the tumour microen- vironment (TME), thus further contributing to molec- ular and clinical heterogeneity of KRAS-driven NSCLC. In detail, KRAS/TP53 co-mutated tumours seem to be associated with a ‘hot’, immune-responsive TME, characterised by high programmed death ligand-1 (PD-L1) expression and tumour mutation burden , as well as high CD8 T-cells infiltration density [13]. Conversely, low PD-L1 expression and CD8 T- cells infiltration have been detected among co- occurring KRAS/STK11/KEAP1emutant NSCLC, thus less likely to respond to immunotherapy [14]. Overall, these data suggest that KRAS-mutant NSCLC is a heterogeneous disease, characterised by different molecular entities requiring further subtyping as a crit- ical determinant of biological behaviour and clinical response to therapeutic strategies.

3. Detection of KRAS mutation in Italy: current real-word scenario

As reported in our recent paper regarding the status of ‘Lung Cancer in Italy’ [15], the predictive molecular biomarkers currently approved by the Italian Health System include EGFR, ALK, ROS1, PD-L1 and, more recently, also BRAF. To date, specifically for patients with NSCLC, the KRAS mutation test has not been endorsed yet as predictive biomarkers for treatment se- lection, thus it is not currently recommended by the Italian Association of Medical Oncology Lung Cancer Guidelines [16]. Despite that, the data obtained from an Italian survey carried out at the end of 2019, involving more than 50 medical oncology/molecular pathology centres specialised in the field of thoracic malignancies, well distributed across the different Italian regions, revealed that 68% of them tested KRAS mutations in patients with metastatic, non-squamous NSCLC, without any significant differences between geographical areas (69% north versus 63% south of Italy). Reflex KRAS molecular testing by pathologists was routinely done in about half of the centres, whereas in the remaining cases it was conducted upon request by the medical oncologist. In the majority of centres, KRAS mutational analysis was performed concomitantly to the EGFR testing, and a detailed molecular report including exon, codon and the specific kind of mutation was produced. The most frequent testing platform adopted to assess KRAS mutations was NGS (54%), followed by Sanger sequencing (27%) and real-time polymerase chain reaction (RT-PCR) (19%). Even with significant difference in terms of reimbursement systems across the different Italian regions, KRAS molecular analysis was regularly reimbursed among the majority (84%) of evaluated centres, including also those not usually per- forming KRAS mutation analysis. These data suggested that the lack of requests may be likely ascribed to the scientific and cultural background of a consistent KRAS, Kirsten rat sarcoma; NSCLC, nonesmall cell lung cancer; ORR, objective response rate; PI3K, phosphoinositide 3-kinase; mTOR, mammalian target of rapamycin; SHP2, Src homology region 2econtaining protein.

Fig. 1. RAS cell signalling pathway diagram. RAS-GTP and RAS-GDP cycling influenced by GEFs/GAPS were graphically represented. RAS, Rat Sarcoma; GTP, Guanosine-triphosphate; GDP, Guanosine-diphosphate; GEFs, Guanine nucleotide exchange factors; GAPS, GTPase-activating proteins.

Fig. 2. Distribution of KRAS mutation reported in the real-word data set of Italian patients with NSCLC. In the upper part of this figure, the 3D distribution of KRAS mutation reported in patients with NSCLC real word data set was showed. The specific frequency of each KRAS mutation was also reported in the pie chart. In addition, the P loop and switch II domain, in which occurs p.G12ep.G13 and p.Q61, respectively, were showed in the overall 3D KRAS protein structure. KRAS, Kirsten rat sarcoma; NSCLC, nonesmall cell lung cancer.

Portion of the Italian medical oncologists, who, to date, did not consider KRAS mutation as a useful biomarker for the clinical management of patients with lung cancer. To assess the real-world distribution of KRAS mu- tations in NSCLC, we carried out a specific survey involving four referral molecular pathology units well distributed across the Italian territory (the University of Turin, University Federico II of Naples, University Bicocca of Milan and University of Varese), all speci- alised in the field of thoracic malignancies. The survey results revealed that KRAS mutations were detected in 212/766 (27.6%) patients with a newly diagnosed meta- static lung adenocarcinoma in 2019. The KRAS p.G12C and p.G12V were the most frequent mutations found in 92/766 (12%) and 37/766 (4.8%) of evaluated patients, thus representing about 43% and 17% of the overall KRAS mutations, respectively. The other mutations found in the tested population included KRAS p.G12D (2.9%), p.G12A (2.2%), p.G13C (1.2%), p.G13D (0.9%), p.Q61H (0.9%), p.G12R (0.5%), p.G12S (0.4%) and p.Q61L (0.3%) (Fig. 2).

Similarly to the EGFR and BRAF mutation status evaluation, also for KRAS, RT-PCR has represented the most used technique in Italy for a long time, followed by pyrosequencing, matrix-assisted laser desorption/ion- isation time-of-flight mass spectrometry and Sanger sequencing [15].The use of NGS has been recently implemented across the different Italian regions; however, besides its increasing spreading, it remains still limited to large- volume molecular pathology laboratories. This hetero- geneous scenario is leading to a different mutation rate and specific variant distribution mainly depending on the limit of detection and the reference range of the implemented techniques [15,17]. With the upcoming advent of p.G12C-directed targeted agents in the clinical setting, the standardisation of diagnostic molecular ap- proaches across the different sites represent a crucial challenge to be adequately addressed in Italy and in the majority of European countries. Taking into account the growing number of clinical relevant biomarkers for patients with NSCLC, including EGFR, BRAF, KRAS and exon 14 MET skipping mutations, as well as ALK, ROS1, RET, and NTRK fusions, a wide spreading of next generation technologies represents the best option to capitalise the biological source of materials and simultaneously obtain the molecular evaluation of all the clinical relevant predictive biomarkers before to plan patient’s first-line treatment strategies.

4. Therapeutic strategies and clinical evidence

4.1. Indirect KRAS inhibition

For a long time KRAS has been considered as an ‘undruggable target’ in lung cancer, considering its high (nanomolar) affinity for the GTP, which requires more potent inhibition than traditional tyrosine kinase in- hibitors, and lacked accessible binding pockets for small molecules [18]. Therefore the development of alternative therapeutic strategies, mostly focused on downstream inhibition and epigenetic approaches, has been pursued (Table 1).

4.1.1. Targeting downstream KRAS effectors

The existence of several feedback loops and interactions with other pathways make KRAS inhibition difficult. However, the simultaneous targeting of different RAS- regulated pathways may represent an effective strategy to treat this subgroup of tumours.The RAF/MEK/ERK pathway is a KRAS down- stream effector and its targeted inhibition has been investigated in clinical trials including a large number of patients with metastatic NSCLC (Table 1). The encouraged activity observed with the addition of the MEK-inhibitor selumetinib to docetaxel in a rando- mised phase II study [19] was not subsequently confirmed in the phase III SELECT-1 trial, including more than 500 pre-treated patients with KRAS-mutant NSCLC [20]. Similarly another MEK inhibitor, trame- tinib, failed to show any survival improvement compared with docetaxel in the same population with NSCLC [21], whereas another phase II study revealed different responses of trametinib plus docetaxel in pa- tients with KRAS p.G12C versus no p.G12C mutation [22]. The disappointing results of these two main treat- ment approaches suggested that indirect KRAS target- ing by MEK downstream pathway inhibition could not to be the right strategy to fight KRAS-driven NSCLC. However, the reason for such failure could be likely ascribed to an underestimation of the biological het- erogeneity derived from the existence of different RAS- mutant isoforms, KRAS-mutant specific alleles or co- occurring mutations, which may significantly influence the therapeutic responses to targeted therapy. This hy- pothesis has been partially proven by preclinical studies revealing that KRAS-specific alleles may significantly influence MEK dependency in preclinical models [23], whereas translational data from the SELECT-1 trials are eagerly awaited to detect differential response pre- diction signals. Another potential explanation to the MEK inhibition failure in NSCLC could be related to the activation of alternative signalling pathways down- stream to RAS as responsible of adaptive resistance occurrence. This hypothesis supported the design of early phase I studies of MEK inhibitors in combination with phosphoinositide 3-kinase (PI3K) or the mamma- lian target of rapamycin (mTOR) inhibitors, which showed preliminary encouraging activity but high-grade toxicities in patients with metastatic disease, thus limiting any further investigation/approval in the clinical setting [24e26]. Finally, Ambrogio et al. [27] revealed that KRAS dimerisation is crucial for several protein functions and that the presence of KRAS wild-type al- leles antagonises KRAS oncogenic activities, emerging as a potential additional mechanism of resistance to MEK inhibitors. The evidence that MEK inhibition may upregulate FAK in KRAS mutant tumours pro- vided the rational to investigate the combination of a RAF/MEK inhibitor, VS-6766, and a FAK inhibitor, defactinib, within a phase I basket trial including mul- tiple tumour types harbouring KRAS mutations. The preliminary results of the study showed an encouraging activity and safety profile of the combination, with 90% disease control rate (DCR) among the 10 included pa- tients with NSCLC, and higher response rate reported in patients with p.G12V mutation, requiring further investigation in larger prospective cohorts [28]. Conversely defactinib monotherapy showed very modest clinical activity in heavily pretreated patients with KRAS-mutated NSCLC [29].

The PI3K/AKT/mTOR pathway is also part of KRAS downstream signalling, and is implicated in metabolic control, immunity, angiogenesis and cardio- vascular homoeostasis, thus its inhibition could have an effective role in KRAS-mutant NSCLC [30]. Unfortu- nately, the results of different clinical studies reported only a limited activity for PI3K/AKT/mTOR inhibitors in NSCLC. The phase II study BASALT-1 investigated the pan-PI3K inhibitor buparlisib in relapsed patients with NSCLC harbouring PI3K pathwayeactivation. The trial was closed for futility at first interim analysis but showed a trend to a longer progression free survival (PFS) in 12 patients harbouring KRAS muta- tions [31]. The mTOR inhibitors revealed promising results, being able to stop the malignant progression in mice and preclinical models of NSCLC with KRAS mutations [32]. However, disappointing results were reported from a clinical trial which enrolled 79 patients with KRAS-mutant NSCLC treated with ridaforolimus, where patients achieved an objective response rate (ORR) of 1% [33].

4.1.2. Epigenetic approaches

Liu et al. [34] demonstrated that KRAS mutations in- crease telomerase activity and telomere length through the activation of the RAS/MEK pathway. The in vitro experiments showed that lung tumourigenesis and chemo-resistance due to KRAS mutations were reduced by telomerase inhibition; therefore targeting telomerase/ telomere could represent a promising therapeutic strat- egy for patients with KRAS-mutant NSCLC. A phase II trial conducted with the telomerase inhibitor imetelstat as maintenance therapy failed to improve PFS in pa- tients with advanced NSCLC responding to first-line chemotherapy (Table 1). However, there was a trend toward an improved median PFS and OS in patients with short telomere length, even if patients were not tested for KRAS mutation status [35].

Zhang et al. [36] examined the inhibitory growth ef- fects of an anti-KRAS ribozyme adenoviral vector (KRbz-ADV) in in vitro and in vivo NSCLC models. Ribozymes (catalytic RNAs, RNA enzymes) are special RNA molecules with site-specific RNA cleavage or ligation activities [37]. The KRbz-ADV targeting codon 12 mutations was able to inhibit human pancreatic cancer cell growth in vitro and specifically cleaved mutant but not wild-type KRAS mRNA [38]. The study demonstrated that KRbz-ADV reduced NSCLC cell growth, reducing tumour growth by 35% in vitro and inducing complete xenograft regression in 65e75% of animals after multiple intratumoural injections of KRbz-ADV.Sunaga et al. [39] investigated if knocking down the mutant KRAS transcript may revert the malignant phenotype of NSCLC using an RNA interference (RNAi). As results, KRAS expression was inhibited, and cell proliferation was reduced. However, tumour- igenicity was not fully abolished and even if the MAPK pathway resulted significantly downregulated, cells showed increased levels of phospho-STAT3, phospho- EGFR and phospho-Akt as a mechanism of resistance. These findings confirmed the complexity of KRAS oncogenic signalling and highlighted that targeting KRAS alone may not be sufficient.

Despite the results on targeted silencing in patients with NSCLC harbouring a KRAS mutation encouraged its clinical application [40,41], further investigations are needed in the gene therapy field. Chromatin-modifying agents, such as the histone deacetylase inhibitor (HDACi), are able to block KRAS signalling, inhibiting gene transcription and proliferation as well as inducing apoptosis in tumour cells, providing useful results for the treatment of several neoplastic diseases [42]. In fact, the HDACi panobinostat has been evalu- ated by in the KRAS-mutant NSCLC A549 cell line, showing reduction of cell proliferation [43]. Yamada [44] et al. evaluated the synergistic efficacy combining a MEK and a HDACi, showing synergistic effects on cell metabolic activity in RAS-mutated lung cancer cells, suggesting that the dual pharmacological block may be promising as a novel therapeutic strategy in specific populations of patients with lung cancer with mutant RAS. A phase II study of carboplatin and paclitaxel with or without vorinostat has been conducted in treatment-na¨ıve patients with NSCLC, unselected for KRAS alteration. Patients receiving vorinostat experi- enced a higher response rate (34% vs 12.5%; p Z 0.02), PFS (6.0 vs 4.1 months; p Z 0.48), and overall survival (OS) (13.0 vs 9.7 months; p Z 0.17) [45] (Table 1).

KRAS post-translational modifications represent another option for KRAS inhibition. To become active, after translation, KRAS is subjected to multistage modifications, being first prenylated by the addition of a farnesyl tail to its carboxyl terminal by the farnesyl transferase (FTase) [46]. These data provided the rational to investigate the FTase inhibitors (FTIs) in patients with KRAS-mutant NSCLC. Tipifarnib and lonafarnib showed in vitro activity in KRAS-mutant lung tumours in mice [47,48]. Because FTIs did not show any clinical activity in NSCLC clinical trials [49e51] (Table 1), they have never been tested in a KRAS mutant cohort of patients. One of the possible explanations for the FTIs failure may be ascribed to existence of different RAS-mutant isoforms, KRAS- mutant specific alleles or co-occurring mutations, which may significantly influence the therapeutic responses to a targeted therapy. This hypothesis has been partially proven by preclinical studies showing as only HRAS- mutant cancer cells were sensitive to FTIs, whereas alternative mechanisms [46,52] seem to regulate mem- brane association and subsequent activation of KRAS/ NRASemutant proteins, therefore working as an adaptive resistance mechanism [53]. In this regard an alternative approach is represented by the use of mimetic drugs such as salirasib, a RAS farnesyl cysteine mimetic drug, which is able to block the KRAS signal- ling interfering with its localisation in the cellular membrane.
In detail, mimetic drugs are able to dislodge KRAS from its membrane-anchoring sites, preventing the activation of its signalling cascades. However, despite some promising preclinical results [54], showing that salirasib inhibited active KRAS in A549 cells [54], early- phase clinical trial results were disappointing [55] (Table 1). Finally, dual inhibition of both FTase and GTTase has been investigated in a phase I trial aiming to block the alternative lipidation of KRAS and NRAS, showing very limited efficacy in the clinical setting.

4.2. Direct KRAS inhibition

A deeper insight into KRAS biology, along with an emerging characterisation of structural protein biochemistry, favoured by an increased access to novel technologies such as X-Ray crystallography, has recently allowed the identification of previously unrec- ognised druggable pockets and targetable domains, drawing new attention on direct inhibition of specific RAS mutant alleles. Among the different innovative treatment strategies, including pan-RAS inhibitors, intracellular monoclonal antibodies and SPH2 in- hibitors alone or in combination, the development of small molecules selectively targeting KRAS p.G12C mutation emerged as the most promising approach and is now reaching advanced stages of clinical investigation. Given the peculiar crystal structure of the KRAS p.G12C subtype and the unique reactivity of the cysteine thiol, these compounds are able to covalently bind the cysteine residue within the region adjacent to the nucleotide-binding pocket, inducing a significant perturbation of protein functional domains and a negative allosteric regulation of RAS signalling in tumour cells [56]. A series of KRAS p.G12C inhibitors, trapping KRAS protein in its inactive conformation have been recently reported to be active in vitro, conferring consistent KRAS-GTP depletion and down- stream KRAS signalling inhibition [57,58]. Particularly ARS-1620, a covalent KRAS p.G12C inhibitor, demonstrated to be highly potent and selective, showing fast and prolonged target occupancy and being able to induce tumour regression in vivo [59]. Canon et al. [60] identified another potent and highly selective molecule, AMG 510, inducing cell proliferation arrest and tumour regression in preclinical models and tumour response in two of four tested patients with advanced NSCLC harbouring KRAS p.G12C mutation. The clinical ac- tivity of this compound has been extensively investigated in a phase I study including 34 pretreated patients with advanced solid tumours, whose preliminary results have been presented at the 2019 American Society of Clinical Oncology meeting [61] and subsequently updated at the 2019 World Conference of Lung Cancer [6]. The AMG
510 administration showed a very promising activity along with a tolerable safety profile in the cohort of 34 patients with metastatic NSCLC harbouring KRAS p.G12C mutation. Particularly among the 23 patients evaluable for tumour response assessment, an ORR of 48% and a DCR of 96% was observed (Table 1). Neither dose-limiting toxicities nor severe treatment-related adverse events (AEs) leading to drug discontinuation have been reported in the whole study population. Importantly the treatment was well tolerated with the majority (about 26%) of treatment-related AEs being grade I and only 9% of grade IIIeIV (mostly anaemia and diarrhoea) [6]. These promising data have immedi- ately led to the fast track designation of AMG-510 by the FDA, whereas the compound is currently under investigation in phase IIeIII clinical trials, including both pretreated and na¨ıve patients with advanced KRAS p.G12Cemutated NSCLC. Similarly another selective KRAS p.G12C small-molecule inhibitor, MRTX849, demonstrated very potent antitumour activity, deter- mining tumour regression in 65% of KRAS p.G12C positive preclinical models including different tumour types [7], thus supporting the design of a currently ongoing phase I study (NCT03785249). As presented at the 2019 AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics (no abstract available), the MRTX849 administration at the dose of 600 mg twice daily induced partial response in 3 of 6 patients with NSCLC and 1 of 4 patients with colorectal cancer affected by metastatic pretreated dis- ease harbouring KRAS p.G12C mutation (Table 1). Overall, the treatment was well tolerated with the ma- jority of patients experiencing grade I AEs, most commonly being nausea and diarrhoea, and only two reporting grade IIIeIV AEs, such as fatigue and appe- tite decrease. Finally, other compounds, such as JNJ- 74699157 or LY3499446, are currently investigated in first-in-human studies enrolling different KRAS p.G12Cemutated tumour types (NCT04006301; NCT04165031), although additional efforts will be required to apply a similar therapeutic approach to other less frequent KRAS molecular subtypes.

Differently from the G12C-covalent agents, pan-RAS inhibitors would allow to treat all RAS mutations car- ried by the tumour [62]. Many compounds have been tested; however the major issue reported was the lack of discrimination between the active and inactive forms of RAS protein and a relevant off-target binding activity, leading to severe in vivo systemic toxicity [63]. Recently, very promising preclinical data were presented at the 2019 AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics, showing a significant activity (no abstract available) of a new pan- RAS inhibitor, BI 1701963, able to selectively bind SOS1, thus leading to KRAS blockade regardless of the specific mutation subtype. Based on these results this compound has been advanced to phase I clinical trial investigation, both as a single agent and in combination with trametinib, in patients with advanced solid tumours harbouring different KRAS mutations (NCT04111458).

4.3. Immune-checkpoint, CDK4/6, and SHP2 inhibition

Tumour cells’ intrinsic RAS signalling has been associ- ated to different immune-modulating effects, including the regulation of PD-L1 expression, CD8 lymphocytes infiltration and myeloid-derived suppressors cell density in the TME [64], with a potential impact on immune- evasion and metastatic processes. KRAS-mutant NSCLC being more common in smokers may be char- acterised by an increased neoantigen load, emerging as a potential marker for immunotherapy efficacy. The recent evidence of a biological link between the onco- genic RAS signalling and PD-L1 mRNA stabilisation further reinforced this translational hypothesis [65]. However, whether KRAS mutation could predict clin- ical response to immune-checkpoint inhibitors remains currently unclear, with real-word studies [66] and meta- analysis [67] showing controversial results in terms of efficacy, but overall suggesting no significant OS dif- ferences between pretreated patients with KRAS- mutated and KRASewild-type NSCLC receiving a sin- gle-agent PD1/PD-L1 inhibitor as the secondethird line treatment. Similarly a subgroup analysis of the KEY- NOTE 189 study [68] has recently demonstrated that the survival increase derived from the addition of immu- notherapy to platinum chemotherapy in non-squamous metastatic NSCLC persisted regardless of the tumour KRAS mutation status. Unfortunately none of these studies evaluated the potential impact of co-occurring mutations, including TP53 or STK11, which may have had an influence on TME composition and patients’ clinical response to the PD-1 blockade [13,14]. More recently Canon et al. [60] observed durable response to
AMG 510 in animals’ tumours harbouring KRAS p.G12C mutation only if they had a functioning immune system, with 9 of 10 immune-competent mice showing complete tumour regression during AMG-510 plus anti- PD1 therapy. The treatment with AMG-510 has been associated with an increased expression of inflammatory cytokines and a prominent T-cell infiltration in the TME [60]. These promising data support the development of immuneetarget combinations strategies which require further investigation in prospective clinical trials including larger cohorts of patients.

Notably, a number of studies are also looking at the combination of immunotherapy and MEK inhibitors, including the phase Ib/II study of avelumab plus bini- metinib, with or without the PARP inhibitor talazoparib (NCT03637491), and the phase Ib/II BATTLE-2 trial (NCT03225664), exploring the combination of pem- brolizumab with the MEK inhibitor trametinib in pa- tients with RAS-mutant advanced solid tumours.

Based on the recent findings of a patient with colo- rectal cancer treated with adoptive T-cell therapy spe- cific for KRAS p.G12D mutation and restricted to the HLA-C8 [69], a phase I ongoing study (NCT03948673) is currently investigating mRNA-5671 alone or in combination with pembrolizumab in advanced solid tumours, including all HLA comers, with the final aim of providing proof of concept of vaccination efficacy in patients with KRAS-mutant cancer.

The biological link between CDK4/6 and KRAS signalling emerging from synthetic lethality screens and preclinical studies [70,71] provided the rational to investigate CDK4/6etargeted inhibition in the subset of patients with KRAS-mutant NSCLC. The efficacy re- sults of abemaciclib and palbociclib, both administered as a single agent within the JUNIPER trial [72] and the UK-MASTER Protocol [73], respectively, were not encouraging, blocking their advancement to clinical setting and suggesting to be cautious with this kind of approach. However, final results based on further mo- lecular subgroups analysis are eagerly awaited. Different phase I/II studies are currently investigating the poten- tial activity of a promising potential combination be- tween two different targeted therapies, including CDK4/ 6- and MEK inhibitors, in patients with KRAS-mutant NSCLC (NCT03170206).

Finally the therapeutic inhibition of the Src homol- ogy region 2 (SH2)-containing protein tyrosine phos- phatase 2 (SHP2) protein represents another promising field of clinical investigation, testing the emerging hy- pothesis of semi-autonomous, SHP2-dependent, RAS signalling pathway activation. Recent preclinical data identified RTK-mediated feedback reactivation of the wild-type RAS pathway as a key mechanism of adaptive resistance to KRAS p.G12Cetargeted inhibition, showing as vertical co-inhibition of SHP2 universally inhibited feedback reactivation, enhancing therapeutic efficacy both in vitro and in vivo models [74]. Both RMC-4630 and TNO155 are new potent, orally bioavailable, selective, SHP2, small-molecule, allosteric inhibitors, currently tested as single agents in first-in- human, dose-finding studies. Particularly the pre- liminary results of the RMC-4630 phase I trial, pre- sented at the 2020 AACR-IASLC International Joint Conference, showed a promising tolerability and activity profile of this agent in the metastatic NSCLC cohort, with a DCR of 67% among 18 evaluable patients with overall KRAS mutations, reaching 75% when consid- ering selected p.G12C-mutated tumours [75]. Based on preclinical data revealing a potential synergy between SHP2 inhibition and other targeted therapies, several phase I studies are currently investigating the activity and safety profile of different combination strategies in KRAS-mutant advanced solid tumours. Particularly a phase I/II study (NCT03989115) is exploring the asso- ciations of RMC-4630 and the MEK inhibitor cobime- tinib in patients harbouring molecular alterations in the KRAS, BRAF or NF1 genes. Another study (NCT04000529) is evaluating TNO155 in combination with the PD-1 inhibitor spartalizumab or the CDK4/6 inhibitor ribociclib in molecularly selected malignancies. An additional phase I/II study (NCT04330664) is looking at TNO155 in combination with MRTX849 in patients with KRAS p.G12Cemutated advanced solid tumours, aiming to enhance the efficacy of direct KRAS inhibition.

5. Conclusion

In summary, recent evidence has revealed that KRAS is neither an irrelevant nor an undruggable target, sug- gesting a promising role for therapeutic inhibition in patients with lung cancer. Several lessons have been learned from the past decades of clinical investigation, when, besides the great interest and efforts made by the scientific community, a limited understanding of KRAS biology and a non-adequate characterisation of the structural protein biochemistry, along with a suboptimal study design, were responsible for the disappointing results of several clinical trials including a large fraction of patients with lung cancer. However, the recent advent of innovative technologies allowed a deeper insight into RAS biology, leading to the identification of novel druggable pockets within the RAS protein and empha- sising the role of molecular subtyping as a critical determinant of the biological behaviour and clinical response to therapeutic targeting. Among the innovative treatments currently undergoing clinical investigation, direct KRAS p.G12C mutation targeting by small- molecule inhibitors, emerged as the most promising approach for clinical setting. The impressive preclinical and clinical activity showed by the first-in-class com- pound, AMG-510, have immediately led to the fast- track designation by the FDA, representing one of the major breakthrough for the clinical lung cancer research in 2019. When these positive premises will be confirmed by prospective ongoing clinical trials including larger cohorts of molecularly selected patients, new effective targeted drugs will be available for the clinical treatment of about 10e12% of patients with NSCLC harbouring a KRAS p.G12C mutation, with a relevant impact on their long-term survival and quality of life. However the 50% ORR with direct KRAS-targeted inhibitors suggests a relevant molecular heterogeneity among the KRAS p.G12C mutant population, with underlying mecha- nisms of innate/acquired resistance requiring further investigation in translational research studies. With a potential effective treatment on the horizon, there is a renewed interest for combination strategies aiming to further enhance the efficacy of KRAS-directed inhibi- tion. A deeper understanding of the complex interplay between oncogenic RAS tumour cell signalling and im- mune TME provided the rational to explore the efficacy and tolerability of immuneetarget combination strate- gies. The use of SHP2 inhibitors, both as a single agent and in combination with other targeted therapies or immunotherapy, is emerging as an alternative promising approach for this subgroup of patients.

Overcoming mechanisms of innate/acquired resis- tance to TKIs, identifying predictive biomarkers for the clinical selection of NSCLC patients, extending tailored approaches to KRAS mutations other than p.G12C, implementing NGS-based detection platforms to in- crease the diagnostic accuracy of molecular analysis both on tissue and plasma samples and warranting equal access to innovative targeted therapies across the different countries represent the most urgent challenges to be addressed in the upcoming years to optimise the clinical management of patients with KRAS-mutated NSCLC.

Conflict of interest statement

F.P. reports receiving consultant’s fee from MSD and Astra Zeneca; U.P. reports receiving speaker bureau/ advisor’s fee from Boehringer Ingelheim, AstraZeneca, Roche, MSD, Amgen and Merck; M.D.R. reports receiving speaker bureau/consultant’s fee from Astellas, Astra Zeneca, Celgene, Novartis, Pfizer, Bio-Rad, Janssen, Sanofi-Aventis and Ipsen; L.R. reports receiving consultant’s fee from Astra Zeneca, Novartis and Boehringer Ingelheim; R.D. reports receiving speaker bureau/advisor’s fee from Ipsen, Novartis, Pfizer, Sanofi Genzyme, AstraZeneca, Janssen, Gilead, Lilly, Gilead and EUSA Pharma; G.T. reports receiving speaker bureau/advisor’s fee from Roche, MSD, Pfizer and Bayer; S.N. declared speaker bureau/advisor’s fee from Eli Lilly, MSD, Roche, BMS, Takeda, Pfizer, Astra Zeneca and Boehringer Ingelheim. The other au- thors have no conflict to declare.

Acknowledgements

The authors would like to acknowledge the Italian medical oncologists and pathologists, specialised in the field of thoracic malignancies, who provided their contribution to the real-word survey.

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