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KRAS as a druggable target in NSCLC: Rising like a phoenix after decades of development failures

Published:February 07, 2020DOI:https://doi.org/10.1016/j.ctrv.2020.101978

      Highlights

      • KRAS mutations are found in 20–25% of NSCLC and are the most commonly detected oncogenic driver.
      • Specific KRAS mutations are characterized by a unique biology, involving preferential activation of different downstream signalling cascades.
      • Not all KRAS mutant NSCLC are KRAS-dependant tumours.
      • The co-mutational landscape affects the immune microenvironment: TP53 mutations lead to hot tumours, STK11 mutations to cold tumours.
      • The predictive value of KRAS in immunotherapy remains uncertain, it may be associated with improved outcomes.
      • After years of frustration, today, KRAS is a promising therapeutic target, with positive preliminary response and safety results for direct irreversible allosteric KRAS-G12C inhibitors.
      • Adoptive T-cell therapy is also being developed but presents significant logistic and financial challenges.

      Abstract

      Cancers of nearly all lineages harbor alterations that deregulate mitogen-activated protein kinase signaling, a crucial signaling pathway for tumor formation and maintenance. Of these, KRAS mutations are the most frequent gain-of-function alterations found in patients with cancer. In particular they represents the most common molecular alteration detected in non-small cell lung cancer (NSCLC) accounting for up to 25% of all oncogenic mutations. They were identified decades ago and prior efforts to target these proteins have been unsuccessful. KRAS mutation profiles (i.e. frequency of specific codon substitutions) in smokers and never-smokers are distinct and not all KRAS alterations are driver mutations. KRAS has evolved from a mutation with possible predictive value to a therapeutic target with great promise. Here, we will discuss the biology of KRAS in lung cancer and its clinical implications in oncology today and in the foreseeable future.

      Keywords

      Background of driver mutations in NSCLC

      Accounting for 18% of all cancer-related deaths, lung cancer is the leading cause of cancer-related mortality worldwide [
      • Bray F.
      • Ferlay J.
      • Soerjomataram I.
      • Siegel R.L.
      • Torre L.A.
      • Jemal A.
      Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.
      ]. It comprises two main subtypes, small-cell and non-small-cell lung cancer (NSCLC), the latter representing over 80% of lung cancers. NSCLC is further divided by histologic subtypes. Adenocarcinoma (ADC) (60%) and squamous cell carcinoma (SCC) (35%) are the most common, each with distinct genomic profiles [
      • Sun S.
      • Schiller J.H.
      • Gazdar A.F.
      Lung cancer in never smokers — a different disease.
      ,
      • Friedlaender A.
      • Banna G.
      • Malapelle U.
      • Pisapia P.
      • Addeo A.
      Next generation sequencing and genetic alterations in squamous cell lung carcinoma: where are we today?.
      ]. Until recently, the prognosis of advanced NSCLC was dire due to limited therapeutic options, with a 5-year survival rate under 5% [
      • Chen Daniel S.
      • Mellman I.
      Oncology meets immunology: the cancer-immunity cycle.
      ].
      The last decade has seen major breakthroughs in the treatment of advanced NSCLC due to the development of immune checkpoint inhibitors [
      • Addeo A.
      • Banna G.L.
      • Metro G.
      • Di Maio M.
      Chemotherapy in combination with immune checkpoint inhibitors for the first-line treatment of patients with advanced non-small cell lung cancer: a systematic review and literature-based meta-analysis.
      ] and the identification of targetable driver mutations. These driver mutations are predominantly identified in ADC. Targeted therapy for these driver mutations (EGFR, ALK, ROS1, BRAF, RET, and TRK inhibitors) has proven to be effective in terms of improved response rates, progression-free survival, and toxicity compared to platinum-based chemotherapy. Recently, potentially effective inhibitors of a hitherto untargetable oncogenic driver mutation in NSCLC, Kirsten Rat Sarcoma (KRAS) have been developed [

      trial A. NCT03600883; 2019.

      ]. KRAS mutations are found in 20–25% of NSCLC and are the most commonly detected oncogenic driver. These were identified in NSCLC decades ago and prior efforts to target these proteins have been unsuccessful. The mutation is almost exclusively detected in ADC [
      • Chen D.S.
      • Irving B.A.
      • Hodi F.S.
      Molecular pathways: next-generation immunotherapy—inhibiting programmed death-ligand 1 and programmed death-1.
      ]. It is interesting to note that the KRAS mutation profiles (i.e. frequency of specific codon substitutions) in smokers and never-smokers are distinct [
      • Dogan S.
      • Shen R.
      • Ang D.C.
      • Johnson M.L.
      • D'Angelo S.P.
      • Paik P.K.
      • et al.
      Molecular Epidemiology of EGFR and KRAS Mutations in 3,026 lung adenocarcinomas: higher susceptibility of women to smoking-related KRAS-Mutant Cancers.
      ]. Furthermore, not all KRAS alterations are driver mutations [
      • Román M.
      • Baraibar I.
      • López I.
      • Nadal E.
      • Rolfo C.
      • Vicent S.
      • et al.
      KRAS oncogene in non-small cell lung cancer: clinical perspectives on the treatment of an old target.
      ]. Here, we will discuss the biology of KRAS in lung cancer and its clinical implications in oncology today and in the foreseeable future.

      KRAS biology

      Wildtype RAS biology. RAS proto-oncogenes encode intracellular guanine nucleotide binding proteins that belong to the GTPase family. Structurally, RAS proteins harbour a catalytic domain and a hypervariable region (HVR). The catalytic domain binds guanine nucleotides and activates signalling while the HVR sequence determines how RAS proteins are localized on the cell membrane where signalling occurs. Monomeric RAS GTPases regulate downstream signalling by switching between the active GTP-bound and inactive GDP-bound states in response to extracellular signals. GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs) regulate the exchange between GTP and GDP [
      • Román M.
      • Baraibar I.
      • López I.
      • Nadal E.
      • Rolfo C.
      • Vicent S.
      • et al.
      KRAS oncogene in non-small cell lung cancer: clinical perspectives on the treatment of an old target.
      ].
      RAS-GTP activates multiple signalling cascades. These include the canonical RAS-RAF-MEK-ERK pathway, a commonly hyperactivated pathway in cancer which controls proliferation via cell-cycle regulation. RAS also activates PI3K-AKT-mTOR signalling, promoting cell survival, as well as the RAL and tumour invasion and metastasis-inducing protein 1 (TIAM1-RAC1) [

      Seger RK. The MAPK signaling cascade. Faseb J. 1995;1995 Jun;9(9):726–35.

      ] pathways, required for RAS-dependent tumour growth and vesicle trafficking/cytoskeletal organisation, respectively [
      • Lambert J.M.
      • Lambert Q.T.
      • Reuther G.W.
      • Malliri A.
      • Siderovski D.P.
      • Sondek J.
      • et al.
      Tiam1 mediates Ras activation of Rac by a PI(3)K-independent mechanism.
      ] (Fig. 1). RAS thus plays an important role in regulating cellular proliferation, differentiation, and apoptosis.
      Figure thumbnail gr1
      Fig. 1The biology of KRAS (a) KRAS gene mutations most commonly occur on chromosome 12. (b) KRAS activation involves a GEF-mediated GDP-GTP exchange allowing the phosphorylation of KRAS-GDP to GTP and leading to effector functions. (c) KRAS signalling involves multiple cellular-proliferation pathways.
      RAS-mutant biology and heterogeneity. Oncogenic RAS mutation commonly involves exons 2 and 3, resulting in impaired GTPase function and constitutively decreasing the conversion from GTP-bound to GDP-bound RAS. This in turn leads to increased downstream signalling. It is important to note that KRAS mutations are heterogeneous and can result in substitutions involving codons 12, 13, or 61 [

      catalogue C. Cosmic; 2019.

      ] (Fig. 2). The most frequent substitution, found in 41% of KRAS-mutant NSCLC, is KRAS G12C. This mutation is commonly identified in patients with a substantial history of smoking. In contrast, KRAS G12D substitutions are more commonly found in tumours from patients with little to no prior history of smoking.
      Figure thumbnail gr2
      Fig. 2location of mutations in KRAS (a) The most common amino-acid substitutions involve codons 12, 13 and 61. (b) Structural location of KRAS substitutions. (c) Structural representation of a KRAS G12 amino-acid substitution.
      Specific KRAS mutations are characterized by a unique biology. For example, while KRAS G12, G13, and Q61 substitutions impair GTP hydrolysis, others mutations such as KRAS A146T (the most prevalent substitution in gastrointestinal malignancies) maintain hydrolysis similar to wild-type KRAS. The A146T substitution favours KRAS-GTP formation by increased nucleotide exchange, leading to lower oncogenic potency [
      • Poulin E.J.
      • Bera A.K.
      • Lu J.
      • Lin Y.-J.
      • Strasser S.D.
      • Paulo J.A.
      • et al.
      Tissue-specific oncogenic activity of KRAS A146T.
      ]. The type of KRAS mutation can also affect downstream signalling. Preclinical analyses of cell lines containing KRAS G12C or KRAS G12V found increased RAS-related protein (RAL) A/B signalling and decreased levels of phosphorylated AKT compared to other KRAS substitutions or wild-type cell-lines [
      • Nadal E.
      • Chen G.
      • Prensner J.R.
      • Shiratsuchi H.
      • Sam C.
      • Zhao L.
      • et al.
      KRAS-G12C mutation is associated with poor outcome in surgically resected lung Adenocarcinoma.
      ]. In contrast, KRAS-G12D-containing cell lines preferably activated the PI3K–AKT pathway (Fig. 3) [
      • Ihle N.T.
      • Byers L.A.
      • Kim E.S.
      • Saintigny P.
      • Lee J.J.
      • Blumenschein G.R.
      • et al.
      Effect of KRAS Oncogene Substitutions on Protein Behavior: Implications for Signaling and Clinical Outcome.
      ,
      • Xu S.L.B.
      • Boris G.H.
      • Chen A.
      • Ni S.
      • Kennedy M.A.
      Structural insight into the rearrangement of the switch I region in GTP-bound G12A K-Ras.
      ,

      Cruz-Migoni A, Canning P, Quevedo CE, Bataille CJR, Bery N, Miller A, et al. Structure-based development of new RAS-effector inhibitors from a combination of active and inactive RAS-binding compounds. Proc Natl Acad Sci USA; 2019.

      ,
      • Zeng M.
      • Lu J.
      • Li L.
      • Feru F.
      • Quan C.
      • Gero T.W.
      • et al.
      Potent and selective covalent quinazoline inhibitors of KRAS G12C.
      ,
      • Pettersen E.F.
      • Goddard T.D.
      • Huang C.C.
      • Couch G.S.
      • Greenblatt D.M.
      • Meng E.C.
      • et al.
      UCSF Chimera—a visualization system for exploratory research and analysis.
      ].
      Figure thumbnail gr3
      Fig. 3KRAS variants and their preferential pathway activation (a) Upon binding with its ligand, activated KRAS wild-type signals the Ral A/B, PI3K and RAF pathways. (b) KRAS G12D preferentially activates the PI3K pathway (red arrow). (c) KRAS G12C preferentially activates the Ral A/B pathway (red arrow). KRAS G12C is the target for direct KRAS inhibitors such as AMG 510. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
      RAS dependency. Two different groups of KRAS-mutant NSCLC have been identified according to their need for mutant KRAS to maintain tumour viability: KRAS-dependent tumours and KRAS-independent tumours. Gene expression profiles of NSCLC cell lines show that KRAS dependence is associated with a well-differentiated epithelial phenotype whereas KRAS independence is correlated with an epithelial-mesenchymal transition (EMT) phenotype. KRAS dependency is characterized by the activation specific pathways and genes, leading to unique disease phenotypes [
      • Román M.
      • Baraibar I.
      • López I.
      • Nadal E.
      • Rolfo C.
      • Vicent S.
      • et al.
      KRAS oncogene in non-small cell lung cancer: clinical perspectives on the treatment of an old target.
      ]. Interestingly, treating KRAS-dependent cells with TGFβ1, a cytokine which induces EMT, reduces KRAS-driven dependence. The presence of EMT thus suggests the loss of KRAS oncogene addiction [
      • Singh A.
      • Greninger P.
      • Rhodes D.
      • Koopman L.
      • Violette S.
      • Bardeesy N.
      • et al.
      A gene expression signature associated with “K-Ras addiction“ reveals regulators of EMT and tumor cell survival.
      ,
      • Kharbanda A.
      • Rajabi H.
      • Jin C.
      • Alam M.
      • Wong K.-K.
      • Kufe D.
      MUC1-C confers EMT and KRAS independence in mutant KRAS lung cancer cells.
      ].
      Co-mutational landscape. KRAS-mutant tumours have distinct co-mutational profiles [
      • Ferrer I.
      • Zugazagoitia J.
      • Herbertz S.
      • John W.
      • Paz-Ares L.
      • Schmid-Bindert G.
      KRAS-Mutant non-small cell lung cancer: from biology to therapy.
      ]. In a recent analysis of 1,078 KRAS-mutant NSCLCs, 53% of tumours harboured at least one additional genomic alteration. The most common mutations involved TP53 (39%), STK11 (20%), and KEAP1 (13%). This was consistent with a separate paper that used RNA sequencing to identify three distinct common co-mutation clusters: (1) STK11, (2) TP53 and (3) CDKN2A/B inactivation with low TTF-1 [
      • Skoulidis F.
      • Byers L.A.
      • Diao L.
      • Papadimitrakopoulou V.A.
      • Tong P.
      • Izzo J.
      • et al.
      Co-occurring genomic alterations define major subsets of KRAS-mutant lung adenocarcinoma with distinct biology, immune profiles, and therapeutic vulnerabilities.
      ].
      The biological behaviour of each of these three subgroups was distinct. Interestingly, STK11 inactivated tumours demonstrated hypoxia inducible factor-1 alpha (HIF1α)-mediated metabolic reprogramming and adaptation to oxidative and endoplasmic reticulum stress, as well as, KEAP1 co-mutations. These tumours expressed low levels PD-L1 and contained few tumour infiltrating lymphocytes (TILs), resulting in a pauci-immune microenvironment. In contrast, TP53-mutant tumours were characterized by high PD-L1 expression, an immune-rich microenvironment with TILs, and immunoediting (Fig. 4) [
      • Skoulidis F.
      • Byers L.A.
      • Diao L.
      • Papadimitrakopoulou V.A.
      • Tong P.
      • Izzo J.
      • et al.
      Co-occurring genomic alterations define major subsets of KRAS-mutant lung adenocarcinoma with distinct biology, immune profiles, and therapeutic vulnerabilities.
      ,
      • Bange E.
      • Marmarelis M.E.
      • Hwang W.-T.
      • Yang Y.-X.
      • Thompson J.C.
      • Rosenbaum J.
      • et al.
      Impact of KRAS and TP53 co-mutations on outcomes after first-line systemic therapy among patients with STK11-mutated advanced non-small-cell lung cancer.
      ].
      Figure thumbnail gr4
      Fig. 4Immune phenotype of KRAS co-mutated subgroups. Left: KRAS/STK11 mutant tumours exhibit a poorly immune tumour micro-environment (cold tumour), with a paucity of CD8 TILs and abundant T-regulator cells. Right: KRAS/TP53 mutant tumours exhibit an immunogenic tumour micro-environment (hot tumour), with abundant CD8 TILs, activated dendritic cells and few T-regulator cells.
      The third KRAS co-mutation cluster, involving CDKN2A/B inactivation, resulted in an increased incidence of TTF-1 negative ADC, suggesting a role in tumour differentiation. It was also associated with mucinous histology, reduced mTORC1 signalling and high wild-type p53-regulated transcripts [
      • Skoulidis F.
      • Byers L.A.
      • Diao L.
      • Papadimitrakopoulou V.A.
      • Tong P.
      • Izzo J.
      • et al.
      Co-occurring genomic alterations define major subsets of KRAS-mutant lung adenocarcinoma with distinct biology, immune profiles, and therapeutic vulnerabilities.
      ].
      Beyond de novo heterogeneity within KRAS-mutant NSCLCs, KRAS mutations can also occur in the setting of resistance to targeted therapy in other oncogene-driven cancers. Thus, the same survey of the co-mutational landscape of KRAS-mutant tumours unsurprisingly found MET amplifications in 15%, EGFR mutations in 1%, BRAF mutations in 1%, and ALK fusions in 0.5% of cases [
      • Scheffler M.
      • Ihle M.A.
      • Hein R.
      • Merkelbach-Bruse S.
      • Scheel A.H.
      • Siemanowski J.
      • et al.
      K-ras mutation subtypes in NSCLC and associated co-occuring mutations in other oncogenic pathways.
      ]. In one series, ALK or EGFR mutant NSCLC with concurrent KRAS mutations responded less well to TKI therapy [
      • Schmid S.
      • Gautschi O.
      • Rothschild S.
      • Mark M.
      • Froesch P.
      • Klingbiel D.
      • et al.
      Clinical Outcome of ALK-Positive Non-Small Cell Lung Cancer (NSCLC) Patients with De Novo EGFR or KRAS Co-Mutations Receiving Tyrosine Kinase Inhibitors (TKIs).
      ] but this remains to be investigated in larger series.

      Predictive role of KRAS

      KRAS mutation and chemotherapy. Several investigators have hypothesized that KRAS is a predictive biomarker of response to chemotherapy. Early preclinical data, for example, showed that the presence of KRAS mutation induced greater sensitivity to pemetrexed, an anti-folate, in NSCLC models [
      • Moran D.M.
      • Trusk P.B.
      • Pry K.
      • Paz K.
      • Sidransky D.
      • Bacus S.S.
      KRAS mutation status is associated with enhanced dependency on folate metabolism pathways in non-small cell lung cancer cells.
      ]. F. Pemetrexed treatment was found to alter KRAS RNA expression, leading to the downregulation of KRAS-driven carcinogenesis. Biologically, this was explained by a greater dependency of KRAS-mutant cells on folate metabolism pathways. Unfortunately, these preclinical results did not translate into increased clinical activity [
      • Sun J.-M.
      • Hwang D.W.
      • Ahn J.S.
      • Ahn M.-J.
      • Park K.
      Prognostic and predictive value of KRAS mutations in advanced non-small cell lung cancer.
      ,
      • Renaud S.
      • Guerrera F.
      • Seitlinger J.
      • Reeb J.
      • Voegeli A.-C.
      • Legrain M.
      • et al.
      KRAS-specific amino acid substitutions are associated with different responses to chemotherapy in advanced non-small-cell lung cancer.
      ]. A retrospective analysis of 1,190 KRAS-mutant NSCLC patients treated with front-line platinum-based doublet chemotherapy concluded that pemetrexed was associated with the shortest progression-free interval compared to other agents such as taxanes [
      • Renaud S.
      • Guerrera F.
      • Seitlinger J.
      • Reeb J.
      • Voegeli A.-C.
      • Legrain M.
      • et al.
      KRAS-specific amino acid substitutions are associated with different responses to chemotherapy in advanced non–small-cell lung cancer.
      ].
      While these disparate results may have been secondary to older series investigating the predictive nature of KRAS mutation as a homogeneous and not a heterogeneous entity, data that looked at response by mutation type was similarly conflicting. For example, in vitro data from Garassino et al. [
      • Garassino M.C.
      • Marabese M.
      • Rusconi P.
      • Rulli E.
      • Martelli O.
      • Farina G.
      • et al.
      Different types of K-Ras mutations could affect drug sensitivity and tumour behaviour in non-small-cell lung cancer.
      ] found that specific KRAS mutations were associated with different sensitivities to chemotherapy. The divergent sensitivity patterns were explained by potential differences in downstream signalling pathways [
      • Garassino M.C.
      • Marabese M.
      • Rusconi P.
      • Rulli E.
      • Martelli O.
      • Farina G.
      • et al.
      Different types of K-Ras mutations could affect drug sensitivity and tumour behaviour in non-small-cell lung cancer.
      ]. KRAS G12D-containing models were resistant to taxanes. Models with KRAS G12V were sensitive to cisplatin and resistant to pemetrexed. Finally, models harbouring KRAS G12C were associated with an increased response to taxanes and pemetrexed, and exhibited a resistance to cisplatin. However, in the same retrospective analysis of 1,190 KRAS-mutant NSCLCs described above, while mutation type did lead to significant differences in responses, these outcomes did not clearly align with prior preclinical data. For example, the cancers that derived the most benefit from taxanes harboured KRAS G12V or G13D (and not KRAS G12C) [
      • Renaud S.
      • Guerrera F.
      • Seitlinger J.
      • Reeb J.
      • Voegeli A.-C.
      • Legrain M.
      • et al.
      KRAS-specific amino acid substitutions are associated with different responses to chemotherapy in advanced non–small-cell lung cancer.
      ].
      KRAS mutation and immunotherapy. Beyond chemotherapy, KRAS mutations have also been associated with checkpoint inhibitor benefit in NSCLC patients. A subgroup analysis of Checkmate 057 [
      • Borghaei H.
      • Paz-Ares L.
      • Horn L.
      • Spigel D.R.
      • Steins M.
      • Ready N.E.
      • et al.
      Nivolumab versus docetaxel in advanced nonsquamous non–small-cell lung cancer.
      ] found improved outcomes with immune checkpoint inhibitor (ICI) in KRAS-mutant NSCLC. In a recent meta-analysis by Kim et al. [
      • Kim J.H.
      • Kim H.S.
      • Kim B.J.
      Prognostic value of KRAS mutation in advanced non-small-cell lung cancer treated with immune checkpoint inhibitors: a meta-analysis and review.
      ], ICIs were compared to docetaxel in pre-treated NSCLC patients, with results stratified by KRAS status. Again, patients with KRAS-mutant tumours derived significant OS benefit from ICI versus chemotherapy; those with wild-type tumours did not exhibit greater OS with ICI. Several other trials have shown an association between KRAS mutation and improved response to ICIs [
      • Torralvo J.
      • Friedlaender A.
      • Achard V.
      • Addeo A.
      The activity of immune checkpoint inhibition in KRAS mutated non-small cell lung cancer: a single centre experience.
      ]. A global, multicenter registry (ImmunoTarget) was designed to retrospectively evaluate the sensitivity of NSCLC patients with a variety of driver mutations. Consistently, KRAS mutation was associated with increased immunotherapy benefit compared to other drivers (i.e EGFR, ALK, ROS1, and RET alterations) [
      • Mazieres J.
      • Drilon A.
      • Lusque A.
      • Mhanna L.
      • Cortot A.B.
      • Mezquita L.
      • et al.
      Immune checkpoint inhibitors for patients with advanced lung cancer and oncogenic driver alterations: results from the IMMUNOTARGET registry.
      ].
      There is a potential biological rationale for a positive interaction between KRAS mutations and ICI benefit. KRAS-mutant NSCLC generally exhibit increased tumour mutation burden (TMB), potentially leading to increased ICI sensitivity [
      • Dong Z.-Y.
      • Zhong W.-Z.
      • Zhang X.-C.
      • Su J.
      • Xie Z.
      • Liu S.-Y.
      • et al.
      Potential predictive value of TP53 and KRAS mutation status for response to PD-1 blockade immunotherapy in lung Adenocarcinoma.
      ]. This is very different from other driver mutations (e.g. ALK fusions) which tend to exhibit low immunogenicity. It has been hypothesized that this stems from the increased incidence of select KRAS mutations in smokers, as smoking is known to be associated with elevated somatic tumoral DNA mutations and a higher TMB [
      • Rizvi N.A.
      • Hellmann M.D.
      • Snyder A.
      • Kvistborg P.
      • Makarov V.
      • Havel J.J.
      • et al.
      Cancer immunology. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer.
      ,
      • Addeo A.
      • Banna G.L.
      • Weiss G.J.
      Tumor mutation burden-from hopes to doubts.
      ].
      Concomitant alterations may affect the immunogenicity of KRAS-mutant tumours. Co-occurring TP53 mutations are associated with enhanced tumour cell proliferation and inflammation, allowing for an immune-rich micro-environment. Recently, a trial found that tumours with co-mutated KRAS/TP53 showed increased expression of PD-L1, high TILs, and remarkable clinical benefit with checkpoint inhibition [
      • Dong Z.-Y.
      • Zhong W.-Z.
      • Zhang X.-C.
      • Su J.
      • Xie Z.
      • Liu S.-Y.
      • et al.
      Potential predictive value of TP53 and KRAS mutation status for response to PD-1 blockade immunotherapy in lung Adenocarcinoma.
      ]. In contrast, co-occurring STK11 mutations are present in roughly 20% of KRAS-mutant NSCLC and can decrease immune surveillance, possibly by modulating the NF-kB pathway. Consistent with this, KRAS-mutant and STK11-mutant are associated with decreased TILs, contributing to the suppression of immune surveillance [
      • Skoulidis F.
      • Byers L.A.
      • Diao L.
      • Papadimitrakopoulou V.A.
      • Tong P.
      • Izzo J.
      • et al.
      Co-occurring genomic alterations define major subsets of KRAS-mutant lung adenocarcinoma with distinct biology, immune profiles, and therapeutic vulnerabilities.
      ,
      • Schabath M.B.
      • Welsh E.A.
      • Fulp W.J.
      • Chen L.
      • Teer J.K.
      • Thompson Z.J.
      • et al.
      Differential association of STK11 and TP53 with KRAS mutation-associated gene expression, proliferation and immune surveillance in lung adenocarcinoma.
      ]. Tumours with an immune-inactive tumour microenvironment have been described “cold” tumours (Fig. 2).
      Concurrent KRAS and STK11 mutations in NSCLC are predictive of primary resistance to ICIs [
      • Skoulidis F.
      • Goldberg M.E.
      • Greenawalt D.M.
      • Hellmann M.D.
      • Awad M.M.
      • Gainor J.F.
      • et al.
      STK11/LKB1 Mutations and PD-1 Inhibitor Resistance in KRAS-Mutant Lung Adenocarcinoma.
      ]. However, this constellation is also a poor prognostic factor in patients treated with chemotherapy [
      • Bange E.
      • Marmarelis M.E.
      • Hwang W.-T.
      • Yang Y.-X.
      • Thompson J.C.
      • Rosenbaum J.
      • et al.
      Impact of KRAS and TP53 co-mutations on outcomes after first-line systemic therapy among patients with STK11-mutated advanced non-small-cell lung cancer.
      ]. Given these results from retrospective data, there is no consensus as to the current clinical implications of these mutations.

      KRAS as a therapeutic target

      Targeted therapy. The development of targeted therapies for KRAS-mutant lung cancers has long been marked by frustration. Earlier trials explored the utility of inhibition of multiple pathways such as RHOA-FAK, RAF-MEK-ERK, PI3K/AKT/mTOR, NF-kB and HSP90. These trials were notable for little activity and substantial toxicity in many cases [
      • Román M.
      • Baraibar I.
      • López I.
      • Nadal E.
      • Rolfo C.
      • Vicent S.
      • et al.
      KRAS oncogene in non-small cell lung cancer: clinical perspectives on the treatment of an old target.
      ]. As no targeted therapies are approved for KRAS-mutant tumours, the current standard of care is thus to follow the same treatment algorithms as in oncogene-negative NSCLC (i.e. to use single-agent immunotherapy or platinum doublet therapy with or without immunotherapy in treatment-naïve patients with metastatic disease) [

      Mullard A. Cracking KRAS. Nat Rev Drug Discov; 2019.

      ,

      Race for undruggable KRAS speeds up. Nat Biotechnol 2019;37:1247-.

      ].
      This has recently begun to change with novel targeted therapies that have been developed to target KRAS G12C. Direct irreversible allosteric inhibition of G12C subverts the native nucleotide preference to favour inactive GDP over active GTP-RAS, and impairs RAF binding and downstream signaling [
      • Ostrem J.M.
      • Peters U.
      • Sos M.L.
      • Wells J.A.
      • Shokat K.M.
      K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions.
      ]. Similarly, selective quinazoline-based compounds and guanosine mimetic inhibitors suppress GTP loading of KRAS G12C and cell proliferation [
      • Zeng M.
      • Lu J.
      • Li L.
      • Feru F.
      • Quan C.
      • Gero T.W.
      • et al.
      Potent and selective covalent quinazoline inhibitors of KRAS G12C.
      ,
      • Xiong Y.
      • Lu J.
      • Hunter J.
      • Li L.
      • Scott D.
      • Choi H.G.
      • et al.
      Covalent guanosine mimetic inhibitors of G12C KRAS.
      ]. A different approach is allele-specific inhibition that results in G12C being trapped in its inactive state [
      • Lito P.
      • Solomon M.
      • Li L.-S.
      • Hansen R.
      • Rosen N.
      Allele-specific inhibitors inactivate mutant KRAS G12C by a trapping mechanism.
      ,
      • Patricelli M.P.
      • Janes M.R.
      • Li L.-S.
      • Hansen R.
      • Peters U.
      • Kessler L.V.
      • et al.
      Selective inhibition of oncogenic KRAS output with small molecules targeting the inactive state.
      ].
      A concern with prior nonspecific inhibitors of KRAS was the risk of off-target toxicity as KRAS proteins share similar binding regions and G-domains with other proteins. While this may pose a challenge as other drugs are developed and tested [
      • McCormick F.
      Progress in targeting RAS with small molecule drugs.
      ], selective KRAS G12C inhibitors are not predicted to result in substantial adverse events as the G12C substitution does not exist in non-malignant tissues.
      Early clinical data has recently been released from the phase I trial of AMG510, a small molecule that irreversibly binds G12C, locking KRAS in its inactive GDP-bound state. The drug has a half-life of 6 h. Among the 13 evaluable pre-treated NSCLC patients, a partial response was achieved in 54% and stable disease in 46%, for a disease control rate of 100%. The safety profile was highly favourable given the absence of dose-limiting toxicity, and few drug-related side-effects. A phase II trial is ongoing [

      M F. Phase 1 study evaluating the safety, tolerability, pharmacokinetics (PK), and efficacy of AMG 510, a novel small molecule KRASG12C inhibitor, in advanced solid tumors. J Clin Oncol 2019;37.

      ]. Another ongoing program targeting KRAS G12C is an open-label phase I/II trial evaluating MRTX849, a similar small-molecule direct irreversible inhibitor. Preclinical results show a 65% response rate in patient-derived xenografts from different tumour types [
      • Christensen J.G.
      • Hallin J.
      • Engstrom L.D.
      • Hargis L.
      • Calinisan A.
      • Aranda R.
      • et al.
      The KRASG12C Inhibitor, MRTX849, provides insight toward therapeutic susceptibility of KRAS mutant cancers in mouse models and patients.
      ].The drug has a 20-hour half-life. A third trial evaluating JNJ-74699157, a direct G12C inhibitor, has just begun recruitment (NCT04006301) (see Table 1).
      Table 1Current small-molecules targeting G12C.
      Drug nameTargetTrial phaseStatusClinical Trials ID
      AMG510G12CI-IIRecruitingNCT03600883
      MRTX849G12CI-IIRecruitingNCT03785249
      JNJ-74699157G12CI-IIRecruitingNCT04006301
      RAS as a cellular therapy target. Murine T-cell receptors highly reactive to specific human tumour-associated antigens can be retrovirally inserted into human lymphocytes in order to augment immune responses [
      • Johnson L.A.
      • Morgan R.A.
      • Dudley M.E.
      • Cassard L.
      • Yang J.C.
      • Hughes M.S.
      • et al.
      Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen.
      ]. As RAS proteins are highly immunogenic, the collection and ex-vivo expansion of TILs, known as adoptive cell therapy, is currently under investigation in KRAS-mutant cancers. Preclinical data support the efficacy of CD8 + TILs against a KRAS G12D-containing colorectal cancer. Consistently, infusion of expanded TILs into a colorectal cancer patient resulted in a prolonged partial response [
      • Renaud S.
      • Guerrera F.
      • Seitlinger J.
      • Reeb J.
      • Voegeli A.-C.
      • Legrain M.
      • et al.
      KRAS-specific amino acid substitutions are associated with different responses to chemotherapy in advanced non-small-cell lung cancer.
      ]. The same group repeated this process on a second patient with a KRAS G12D mutant colorectal cancer, without any response [
      • Tran E.
      • Ahmadzadeh M.
      • Lu Y.-C.
      • Gros A.
      • Turcotte S.
      • Robbins P.F.
      • et al.
      Immunogenicity of somatic mutations in human gastrointestinal cancers.
      ]. Similarly, G12V-reactive CD4 + T-cells were isolated, expanded and shown to demonstrate in vitro efficacy against KRAS G12V-mutant NSCLC cells [
      • Tran E.
      • Ahmadzadeh M.
      • Lu Y.-C.
      • Gros A.
      • Turcotte S.
      • Robbins P.F.
      • et al.
      Immunogenicity of somatic mutations in human gastrointestinal cancers.
      ]. Clinical trials are accruing for KRAS G12D and G12V patients (NCT03190941 and NCT03745326). Such programmes are highly effort-intensive and require tumour tissue collection, harvesting and expansion of TILs, conditioning by means of chemotherapy, and interleukin therapy. Compared to the other standard and investigational therapies discussed in this review (i.e. chemotherapy, immunotherapy, and targeted therapy), adoptive T-cell therapy thus presents unique logistic and financial challenges in the clinic.

      Concluding remarks

      KRAS-mutant lung cancers are highly heterogeneous. Differences in mutation type and co-mutational signatures can modulate tumour biology and response to therapy. While data on response to chemotherapy in conflicting, KRAS mutation has been more consistently associated with a favourable response to immunotherapy in NSCLCs. Conversely, the concurrent mutation of STK11 in KRAS-mutant tumours has been associated with decreased benefit with immunotherapy. Finally, the therapeutic landscape has begun to change with the introduction of direct KRAS G12C inhibitors in the clinic. Preliminary data has shown this strategy to be active and safe in prospective trials.

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