Targeting the KRAS Pathway in Non-Small Cell Lung Cancer

An overview is presented of the KRAS pathway in lung cancer and related therapeutic strategies under investigation. KRAS is one of the most frequently mutated genes in non-small cell lung cancer (NSCLC). Potential approaches for the treatment of KRAS-mutant NSCLC include direct inhibition of KRAS protein, inhibition of KRAS regulators, alteration of KRAS membrane localization, and inhibition of effector molecules downstream of mutant KRAS.

Abstract

Lung cancer remains the leading cause of cancer-related deaths worldwide. However, significant progress has been made individualizing therapy based on molecular aberrations (e.g., EGFR, ALK) and pathologic subtype. KRAS is one of the most frequently mutated genes in non-small cell lung cancer (NSCLC), found in approximately 30% of lung adenocarcinomas, and is thus an appealing target for new therapies. Although no targeted therapy has yet been approved for the treatment of KRAS-mutant NSCLC, there are multiple potential therapeutic approaches. These may include direct inhibition of KRAS protein, inhibition of KRAS regulators, alteration of KRAS membrane localization, and inhibition of effector molecules downstream of mutant KRAS. This article provides an overview of the KRAS pathway in lung cancer and related therapeutic strategies under investigation.

Implications for Practice:

The identification of oncogene-addicted cancers and specific inhibitors has revolutionized non-small cell lung cancer (NSCLC) treatment and outcomes. One of the most commonly mutated genes in adenocarcinoma is KRAS, found in approximately 30% of lung adenocarcinomas, and thus it is an appealing target for new therapies. This review provides an overview of the KRAS pathway and related targeted therapies under investigation in NSCLC. Some of these agents may play a key role in KRAS-mutant NSCLC treatment in the future.

Introduction

Lung cancer remains the leading cause of cancer-related deaths in the United States and worldwide [1, 2]. Outcomes with platinum-based doublet chemotherapy for first-line treatment of patients with stage IV non-small cell lung cancer (NSCLC) are poor [3], with a clear need to improve treatment outcomes and to individualize treatment strategies for these patients. The interactions between pathologic subtype, with bevacizumab toxicity in squamous carcinoma and pemetrexed efficacy in nonsquamous carcinoma, led to different treatment options based on histology [4, 5]. More recently, the identification of activating epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) rearrangements as predictive biomarkers for treatment of NSCLC led to further personalization of therapy with EGFR tyrosine kinase inhibitors (EGFR-TKIs) erlotinib [6], gefitinib [7], and afatinib [8]; and ALK inhibitors, including crizotinib, ceritinib, and alectinib [9]. Therefore, NSCLC molecular profiling is a key factor in treatment decision making, with a number of emerging oncogenic targets and active targeted agents.

Kirsten rat sarcoma viral oncogene homolog (KRAS) is one of the most frequently mutated genes in NSCLC, after TP53. KRAS mutations are found in multiple other solid and hematologic malignancies, including pancreatic, colon, peritoneal, bile duct, small intestine, and endometrial cancers [10]. KRAS mutations occur in approximately 30% of lung adenocarcinomas [11] and 5% of squamous cell carcinomas. The majority of mutations occur in codons 12 and 13, and less commonly in codon 61 or others [11, 12]. Although transition mutations are more common in colorectal cancers, the transversion mutation G12C is among the most commonly reported in lung cancer; other transversion mutations in lung cancer include G12V, G12A, G12R, and transition mutations G12D and G12S. Mutations in codon 13 are less common but may be functionally distinct (e.g., G13D, G13C). Because of its easy identification and high prevalence, KRAS is an appealing therapeutic target in NSCLC [13]. However, at present, no targeted therapy has been approved for KRAS-mutant NSCLC. This review summarizes the potential targets in KRAS pathway and will provide an overview of potential related targeted drugs investigated for NSCLC treatment.

Materials and Methods

PubMed was searched using the following key words: non-small cell lung cancer, KRAS, treatment, target, pathway, mutation, PI3K, mTOR, BRAF, MEK, MET, Hsp90. The proceedings of American Society of Clinical Oncology annual meetings and the World Conference on Lung Cancer were searched from 2004 to 2016 using the same key words. We also searched http://www.ClinicalTrials.gov to identify ongoing trials using KRAS, PI3K, mTOR, BRAF, MEK, MET, and Hsp90 inhibitors, or using KRAS analysis as a predictive biomarker.

KRAS Pathway Biology

KRAS belongs to the family of rat sarcoma virus (Ras) genes. The Ras genes encode four highly related protein isoforms: HRAS, KRAS4a, KRAS4b, and NRAS [14]. Discoveries made in the 1970s and the early 1980s led to the identification of Ras genes and RAS proteins as key drivers in human tumor pathogenesis [15]. RAS proteins are small guanosine triphosphatases (GTPases) involved in cell survival, cell cycle progression, cell polarity and movement, actin cytoskeletal organization, and vesicular and nuclear transport [16]. They function as guanosine diphosphate (GDP)/guanosine triphosphate (GTP) binary switches [17] and are located at the inner face of the cellular membrane, where they regulate signaling from activated transmembrane receptors (such as EGFR) to cytoplasmic effectors (Figure 1).

Figure 1.

KRAS pathway biology. ∗, incorporation of Ras to the inner cell membrane; ∗∗, posttranslational folding of KRAS.

Abbreviations: GAP, GTPase-activating protein; GDP, guanosine diphosphate; GEF, guanine nucleotide exchange factor, GTP, guanosine triphosphate; ICMT, isorenylcysteine carboxyl methyl transferase; RCE1, RAS-converting enzyme 1.

RAS regulatory proteins are divided into two main classes: guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). GEFs are responsible for the exchange of GDP for GTP and promote the active GTP-bound state of RAS protein, whereas GAPs stimulate GTP hydrolysis, promoting the inactive GDP-bound state of RAS [18]. In addition, posttranslational processing of RAS proteins is an important regulatory factor. The majority of RAS proteins have a C-terminal CAAX tetrapeptide sequence [19]. RAS proteins are first synthesized in the cytosol, where their CAAX motif is recognized by farnesyltransferases, which prenylate RAS (adding a farnesyl isoprenoid lipid). This results in the incorporation of RAS into the inner cell membrane [20, 21] and is followed by proteolytic cleavage of the AAX sequence catalyzed by RAS-converting enzyme-1 (RCE1) and carboxymethylation of the farnesylated cysteine residue catalyzed by isoprenylcysteine carboxyl methyltransferase (ICMT) [22]. These reactions modulate the ability of RAS proteins to incorporate into the inner cell membrane and be activated.

Once activated by extracellular stimuli such as activated EGFR, RAS proteins activate downstream cytosolic effectors including the v-Raf murine sarcoma viral oncogene homolog B (RAF)/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway [23] and the phosphoinositide 3-kinase (PI3K)/v-akt murine thymoma viral oncogene (AKT)/mammalian target of rapamycin (mTOR) pathway [24]. Figure 1 summarizes KRAS pathway biology.

KRAS mutations are found in approximately 30% of lung adenocarcinomas (34% of smokers and 6% of nonsmokers). In lung cancer, they occur most frequently in codon 12 or 13 [25]. Mutant RAS proteins are insensitive to GAP and, therefore, are constitutively activated, inducing persistent stimulus-independent activation of downstream pathways [26], leading to tumor growth, proliferation, and survival. KRAS mutations can occur as transversion mutations (substitution of a purine nucleotide to a pyrimidine or vice versa) or transition mutations (purine to purine or pyrimidine to pyrimidine substitution). Transversion mutations appear more commonly in tumors of current or former smokers, whereas transition mutations are more common in never smokers [27]. G12D is the most common transition seen in never smokers, whereas G12C is the most common transversion seen in smokers [25]. Data regarding differential clinical outcomes for patients with KRAS transition and transversion tumor mutations are conflicting [26–28]. More substantial differences in clinical outcomes of patients with KRAS-mutant NSCLC have been described based on mutation location (i.e., in codon 12 or 13) [29, 30].

KRAS as a Prognostic and Predictive Biomarker

KRAS mutations were initially described as a negative prognostic factor in NSCLC. A meta-analysis showed inferior survival for NSCLC with KRAS mutations (hazard ratio [HR], 1.35; 95% confidence interval [CI], 1.16–1.56) [31]. However, the prognostic significance of KRAS mutations remains unclear because studies examining the role of KRAS as a prognostic biomarker have been heterogeneous with contradictory results. Randomized controlled trials of adjuvant chemotherapy have provided large cohorts for the study of KRAS as a prognostic marker. Whereas Eastern Cooperative Oncology Group 4592, National Cancer Institute of Canada Clinical Trials Group JBR.10, European Early Lung Cancer cohort, and Cancer and Leukemia Group B9633 trials did not find any prognostic impact of KRAS status, the International Adjuvant Lung Cancer Trial showed a negative effect of KRAS on overall survival (OS) and disease-free survival (DFS) [27]. The Lung Adjuvant Cisplatin Evaluation (LACE)-Bio group conducted a pooled analysis of patients enrolled in four randomized trials of adjuvant chemotherapy and did not find KRAS mutation to be a prognostic factor for OS (p = .09) or DFS (p = .13) [31]. A recent pooled analysis of 1,362 patients with early- or late-stage NSCLC participating in EGFR TKI trials reported no prognostic impact of KRAS genotype on survival [32]. This analysis excluded patients with EGFR mutations from the KRAS wild-type (WT) population. An initial sample of 677 patients with advanced NSCLC with KRAS mutations suggested inferior survival for those with codon 13 mutations compared with those with codon 12 mutations (1.1 vs. 1.3 years; p = .0009), but this was not validated in a subsequent cohort [30].

KRAS has also been studied as a potential predictive biomarker for response to chemotherapy. The LACE-Bio group did not find any significant impact of KRAS mutational status on adjuvant chemotherapy efficacy (interaction p = .99) [31]. In a separate study, the LACE-Bio group evaluated the predictive value of KRAS/TP53 mutational status and response to chemotherapy. The group found no significant difference in progression-free survival (PFS), relapse-free survival (RFS), and OS in patients with both mutations compared with patients with wild-type KRAS/TP53, KRAS mutant, or TP53 mutant tumors who received either adjuvant, concurrent, or second-line chemotherapy [32]. Furthermore, previous studies have suggested that KRAS mutations correlate with resistance to EGFR-TKI or anti-EGFR antibodies [33]. Two meta-analyses have demonstrated lower response rates but no impact on more meaningful outcomes like survival, with EGFR TKI in patients with KRAS-mutant NSCLC. A recent pooled analysis of four placebo-controlled studies demonstrated variability in outcomes with EGFR TKI based on mutation subtype. Survival benefit was seen in those with G12D/S mutations, with the potential for harm suggested in those with G12C/V mutations. To date, there are insufficient data to support the clinical utility of KRAS to predict the efficacy of EGFR TKIs [32, 34–36]. Given the poor outcome in KRAS mutant colorectal cancer with EGFR antibodies cetuximab and panitumumab, similar results were expected in NSCLC. However, in the cetuximab plus chemotherapy in patients with advanced non-small cell lung cancer (FLEX) study, the addition of cetuximab to chemotherapy did not affect OS, PFS, or response rate (RR) in KRAS mutant NSCLC compared with KRAS wild-type NSCLC [29].

Implications of Concurrent Mutations

Concurrent mutations may play a prognostic role in patients with lung cancer with KRAS mutations. For example, in a study of 92 patients with lung cancer, EML4-ALK fusion and concurrent KRAS mutation were associated with poor prognosis. Median OS in patients with ALK rearrangements was 57.1 months, but was 10.7 months in patients with alterations in both ALK and KRAS [34].

In a study of 239 patients with KRAS-mutant lung cancer, 333 concomitant mutations were found, with a median of 7 concurrent aberrations per patient. TP53 mutations (39%) appeared most frequently alongside KRAS mutations, followed by STK11 (30%), KEAP1 (24%), RBM10 (15%), and PTPRD (15%). Concomitant mutations in KRAS and either STK11 or KEAP1 were associated with shorter survival [35]. Additional coexisting mutations were identified in a similar study of 4,507 patients, including genomic alterations in MET, FGFR1, HER2, PIK3CA, DDR2, PTEN, CTNNB1, BRAF, and EGFR [36].

Skoulidis et al. [37] have defined three subsets of KRAS-mutant lung cancer with concurrent mutations in STK11/LKB1, TP53, and CDKN2A/B. These mutations are often acquired over time, especially with chemotherapy treatment. Preclinical data suggest that KEAP1 inactivation and LKB1 mutation may result in PD-1 inhibitor resistance and HSP90 inhibitor sensitivity. In lung cancer, concurrent KRAS and TP53 mutations may be associated with increased efficacy of immune checkpoint inhibitors and correlated with longer RFS. Additional prospective studies are required to further validate these findings. The CDKN2A/B subtype is associated with distinct pathological characteristics such as invasive mucinous histology, low TTF1 expression, and suppressed mTORC1 signaling [37].

Targeting KRAS

Because of its high mutation frequency in NSCLC, KRAS is an appealing target. To date, however, the development of direct KRAS inhibitors has been challenged by the complexity of KRAS biochemistry. The high affinity of GTP for KRAS [38] as well as the limited number of accessible active binding sites may also explain the difficulty in developing specific KRAS inhibitors [39].

Inhibition of Ras Expression

Although direct targeting of the KRAS protein has not yielded positive results to date, alternative methods to block KRAS activity have been studied. For example, decreasing KRAS protein synthesis via downregulation of KRAS gene expression has been investigated using antisense oligonucleotides targeting specific messenger RNA sequences to block the translation of mRNA to protein. These oligonucleotides could be administered via plasmid or viral vectors, through liposomal transfection or systemic intravenous administration. In vitro testing has shown tumor growth inhibition in cell lines as well as in preclinical mouse models [40–42]. However, progress has been limited because of the lack of efficient delivery, uptake, and gene silencing, and also because epithelial-to-mesenchymal transition of tumors can change their sensitivity to antisense oligonucleotides [43]. Attempts to move small interfering RNA into the clinic are ongoing, as is the further development of specific KRAS inhibitors such as PHT-7390 and -7389.

Interference With Ras Posttranslational Modification

KRAS may be inhibited by hindering its ability to incorporate into the cell membrane, involving multiple enzymes such as farnesyltransferase and geranylgeranyltransferase, RCE1, and ICMT (Fig. 1). The first farnesyltransferase inhibitors (FTIs) developed, such as tipifarnib and lonafarnib, were well tolerated but demonstrated poor clinical activity [44, 45]. Although this lack of efficacy was thought to be due to the alternative prenylation of KRAS by geranylgeranyltransferases, dual inhibition of farnesyltransferase and geranylgeranyltransferase did not show any benefit [46]. Salirasib, a second-generation FTI [47], showed promising results in vitro and in murine models [48, 49] but failed to show clinical benefit in patients with lung cancer [50]. This lack of efficacy could be explained by alternative prenylation of KRAS by geranylgeranyltransferase or alternative mechanisms of resistance such as KRAS gene amplification or off-target effects. Furthermore, several RCE1 and ICMT inhibitors have been developed and show promising results in vitro and in murine models, but further optimization is needed before these drugs enter human trials [51, 52].

Inhibition of Ras Function

Another potential mechanism to inhibit KRAS is via direct competitive inhibition. Some small-molecule inhibitors of KRAS have been identified [53], including development of potential irreversible inhibitors of KRAS G12C mutant protein [54, 55]. Although these new drugs seem to be effective in vitro, there is a need for further clinical studies and new drugs with increased affinity [56].

Inhibition of GEF or activation of GAP proteins are other potential ways to inhibit KRAS, because GEF promotes the active state of KRAS and GAP promotes the inactive state of KRAS. Although the three-dimensional structure of GTPase-GEF complexes have been described [57], no direct inhibitors of Ras-GEF have been developed yet [19]. However, peptides have been designed to competitively bind KRAS at the same location as SOS (a Ras-GEF protein) [58–60]. So far, these molecules have only been tested in vitro. Moreover, whereas GAP promotes the inactive state of KRAS, these proteins are inactive in mutant KRAS tumors. Thus, attempts have been made to develop small molecules to enhance GAP activity against KRAS mutant proteins. However, the development of GAP modulators is challenging because oncogenic mutations disturb the active site of KRAS [61], and agents that stimulate activity are more challenging to develop than inhibitory compounds.

Finally, a further way to inhibit KRAS signaling is via the inhibition of downstream effectors such as PI3K, mTOR, BRAF, and MEK. Table 1 summarizes the ongoing clinical trials targeting these pathways using KRAS genotype as a predictive biomarker.

Table 1.

Ongoing trials selected by KRAS status

Targeting PI3K

PI3K is a cytoplasmic molecule downstream of KRAS and is part of the PI3K/AKT/mTOR pathway. It is a site of convergence of multiple pathways and its regulation is thus complex. PI3KCA mutations are found in approximately 2% of NSCLC and lead to cellular proliferation and survival independently of KRAS activation [62]. The PI3K pathway can also be upregulated through PTEN loss [63], amplification of upstream tyrosine kinase receptors, or via oncogenic KRAS mutation [64]. Novel PI3K inhibitors (e.g., BKM120, GDC0941, and XL147) have been developed and showed initial promising results in phase I clinical trials [65–67]. They are currently being investigated in phase II clinical trials for patients with advanced NSCLC harboring PI3KCA mutations (NCT01297491, NCT01493843). However, although oncogenic KRAS activity requires downstream PI3K activity [68, 69], preliminary data suggest that KRAS-mutant tumors are insensitive to PI3K inhibitors as single-agent therapy [70, 71]. Thus inhibition of the PI3K pathway alone appears insufficient for the treatment of KRAS-mutant NSCLC, likely given the multiple alternative downstream pathways activated by KRAS and potential crosstalk.

Targeting mTOR

mTOR is a serine/threonine kinase downstream from PI3K in the PI3K/AKT/mTOR pathway. Preclinical data demonstrate that mTOR inhibition blocks tumor growth in mouse models of KRAS-mutant lung adenocarcinoma [72]. Several mTOR inhibitors (e.g., everolimus, ridaforolimus) have been investigated in NSCLC treatment, with promising results [73]. Ridaforolimus is a rapamycin analog and selectively targets mTOR [74]. In a phase II clinical trial, 70 patients with KRAS-mutant NSCLC received 8 weeks of ridaforolimus. After 8 weeks, 28 patients who were deemed to have stable disease were randomly assigned to receive placebo or further ridaforolimus. PFS was significantly higher with ridaforolimus (4 months vs. 2 months; HR, 0.36; p = .013) and there was a trend for better OS (18 months vs. 5 months; HR, 0.46; p = .09) [75]. Thus, mTOR is a promising target for the treatment of KRAS-mutant NSCLC.

Targeting BRAF

BRAF is another serine/threonine kinase that directly interacts with GTP-bound KRAS and activates the BRAF/MEK/ERK/MAPK pathway [76]. BRAF activation is required in KRAS-mediated transformation and tumorigenesis [77]. BRAF mutations are found in 1% to 4% of NSCLC, with V600E mutations composing 50% [78]. BRAF inhibitors dabrafenib and vemurafenib, specific to BRAF V600E mutant tumors, were first investigated in patients with melanoma, in whom V600E accounts for more than 80% of BRAF mutations [79, 80]. In NSCLC, dabrafenib has demonstrated activity in advanced NSCLC with V600E BRAF mutation, with an estimated response rate of 40%; phase II expansion is ongoing (NCT01336634) [81]. At present, there are no data regarding the activity of these agents against non-V600E BRAF mutant tumors nor on the sensitivity of KRAS-mutant NSCLC. Sorafenib is a multikinase inhibitor that can also target BRAF independently of BRAF mutational status [82]. Studies looking at KRAS mutations as a predictor of sensitivity to sorafenib in NSCLC have yielded differing results. Although one early phase study suggested activity of sorafenib [83], others have not, including the phase III MISSION study of sorafenib vs. placebo [84–86]. Sorafenib failed to significantly improve efficacy in any subset of lung adenocarcinoma, including KRAS mutant adenocarcinoma [86].

In NSCLC, dabrafenib has demonstrated activity in advanced NSCLC with V600E BRAF mutation, with an estimated response rate of 40%; phase II expansion is ongoing. At present, there are no data regarding the activity of these agents against non-V600E BRAF mutant tumors nor on the sensitivity of KRAS-mutant NSCLC.

Targeting MEK

The MEK protein kinases are downstream of KRAS and BRAF in the RAS/RAF/MEK/ERK pathway, also known as the MAP kinase cascade. Phosphorylated BRAF activates the serine/threonine kinase MEK, which, in turn, activates the serine/threonine kinase ERK, resulting in activation of transcriptional factors promoting cell cycle progression and cell proliferation [87]. Several small molecules have been developed to selectively inhibit MEK. Table 2 summarizes MEK inhibitors in development in KRAS-mutant NSCLC. Preclinical studies have shown that BRAF and KRAS mutations confer sensitivity to these molecules [97]. However, despite preclinical activity, the clinical outcomes with these agents were not as promising, including CI-1040, RO5126766, and PD-0325901 [88–92, 98, 99]. More recently, other MEK-1 and MEK-2 inhibitors have been developed, including selumetinib and trametinib, and studied in patients with KRAS-mutant NSCLC.

Table 2.

MEK inhibitors investigated in non-small cell lung cancer for the treatment of patients with KRAS mutation

Preclinical studies of selumetinib show growth inhibition in BRAF- and KRAS-mutated cell lines [93]. Furthermore, in NSCLC murine models, selumetinib induces tumor shrinkage [94]. Phase I clinical trials of selumetinib in solid tumors defined the suitable dose as 100 mg orally twice daily [95]. Several phase II studies were then conducted in NSCLC, including a trial of selumetinib alone in comparison with pemetrexed in second- or third-line treatment of unselected patients with NSCLC. Selumetinib showed some clinical activity but offered no survival advantage compared with pemetrexed in this unselected population, suggesting further need for study in BRAF- or KRAS-mutant NSCLC [96]. The most commonly reported adverse events with selumetinib were acneiform rash, diarrhea, nausea, and vomiting. Based on these data and preclinical models, selumetinib was combined with docetaxel versus docetaxel alone in KRAS-mutant NSCLC. The addition of selumetinib in this phase II study significantly improved response rate (37% vs. 0%; p < .0001) and PFS (5.3 vs. 2.1 months; p = .014), but did not significantly improve OS (9.4 months vs. 5.2 months with docetaxel alone; p = .21) [100]. The most common side effects were febrile neutropenia and weakness (18% and 9%, respectively, in the selumetinib arm vs. 0% in the placebo arm). Based on this exciting signal of activity, a phase III trial was initiated in patients with KRAS-mutant NSCLC, comparing docetaxel plus selumetinib to chemotherapy alone, but did not significantly improve RR, PFS, or OS [101]. A subgroup analysis of KRAS-mutation subtypes from the phase II selumetinib/docetaxel trial suggests that those with G12V and, to a lesser extent, G12C mutations may have higher response rates and PFS with selumetinib than those with other mutation subtypes [102]. Those with G12C mutations also had a nonsignificant trend toward better survival. Although this was not statistically significant, this report suggests that patients with G12V and G12C mutations may derive preferential benefit from MEK inhibition, a hypothesis that should be tested in future trials. Selumetinib has also been studied in combination with erlotinib in both KRAS-mutant and wild-type NSCLC but failed to show improved outcomes compared with erlotinib alone, and increased toxicity was reported [103]. A recent preclinical study has shown that selumetinib might reverse resistance to bevacizumab and merits further study [104].

More recently, another MEK inhibitor, trametinib, has been studied in NSCLC. Trametinib induces cell-growth inhibition in KRAS-mutant cell lines and xenograft models [105], and was first studied as a single agent in phase I trials in which 2 mg once daily was established as the recommended dose for further study [106]. The most commonly reported adverse events with trametinib were febrile neutropenia, transaminitis, and uveitis. It has been combined with multiple agents in phase Ib studies, including gemcitabine [107], pemetrexed [108], and docetaxel [109]. All of these combinations demonstrated tolerability and clinical activity warranting further study. Trametinib has also been compared with docetaxel in a randomized phase II study in patients with KRAS-mutant advanced NSCLCs. Although single-agent trametinib did not improve PFS, it demonstrated similar activity to docetaxel, including response rate and PFS [110].

Dual Inhibition of PI3K/AKT/mTOR and BRAF/MEK/ERK Pathways

Because KRAS activates PI3K/AKT/mTOR and BRAF/MEK/ERK pathways, inhibition of both pathways may be required to fully block KRAS signaling. Moreover, a single-agent pathway inhibitor can release negative feedback loops in the alternative signaling pathway [111]. In preclinical studies, dual inhibition of both pathways has led to synergistic tumor shrinkage [112]. Several phase I trials of MEK and either PI3K or PI3K plus mTOR inhibitors have been conducted. These combinations appear well tolerated with early signs of anticancer activity [113–116]. These results warrant further investigation of this strategy in phase II trials.

Targeting MET

MET, a transmembrane tyrosine kinase receptor involved in invasion, proliferation, angiogenesis, and metastasis, can also activate the KRAS pathway [117]. MET amplifications are found in approximately 4% of lung adenocarcinomas [118] and have been associated with acquired resistance to EGFR-TKIs via constitutive activation of the KRAS/PI3K/AKT/mTOR pathway despite EGFR inhibition [119]. MET has been studied as target in all NSCLC, and it has also been studied as an activator of KRAS signaling. The best studied MET inhibitors in lung cancer include tivantinib, a triphosphate-competitive small-molecule MET inhibitor, and onartuzumab, a MET-receptor monoclonal antibody. Tivantinib combined with erlotinib did not improve outcomes in unselected advanced NSCLC, but results suggested a significant PFS improvement in patients with KRAS-mutant NSCLC (HR, 0.18; 95% CI, 0.05-0.70; p < .01, interaction p = .006) [120]. Although the phase III MARQUEE study was stopped prematurely for lack of efficacy, the preliminary analysis in molecular subgroups seems to confirm these results [121]. Onartuzumab has also been combined with erlotinib, and an initial phase II study suggested better outcomes in patients with MET-protein expressing (MET-positive) NSCLC, although the phase III METLung study in this enriched population has been halted for futility [122]. No response was observed in patients with KRAS-mutant NSCLC [123].

Targeting HSP90

Heat shock proteins (HSPs) are molecular chaperones involved in the posttranslational folding and synthesis of proteins [124]. Preclinical data from KRAS-mutant NSCLC cell lines and mouse models suggest sensitivity to HSP90 inhibition [125]. HSP90 inhibitors disrupt proper functioning of oncogenic driver proteins, including mutant EGFR, BRAF, HER2, and EML4-ALK proteins [126]. Combinations of ganetespib, an HSP90 inhibitor, with MEK or PI3K/mTOR inhibitors increase cytotoxic activity against KRAS-mutant cells [127]. Ganetespib was recently studied as monotherapy in previously treated patients with NSCLC in a phase II clinical trial. Among 17 patients with KRAS mutation, 47% had tumor shrinkage, although no objective response was observed, and the 16-week PFS rate was 5.9% [128]. Although single-agent HSP90 inhibitors may have some therapeutic activity, they are clearly more active in other genomic subgroups such as EGFR-mutant or ALK-rearranged lung cancer. Combination studies with chemotherapy are ongoing, although they do not specifically enrich for KRAS mutations [129]. In a phase II trial of ganetespib and docetaxel, the combination failed to improve PFS or OS in patients with KRAS-mutant lung cancer [130]. Another trial of retaspimycin in combination with docetaxel failed to improve overall survival compared with docetaxel/placebo (NCT01362400) [131]. To date, HSP90 inhibitors have not yielded improved outcomes for patients with KRAS-mutant NSCLC as monotherapy or in combination. However, based on preclinical data, combination therapy of ganetespib and PI3K/MTOR inhibitors is under clinical evaluation, including in KRAS-mutant tumors [132].

Other Targets

RNA interference technology has been used to screen genes interacting with RAS [133], leading to the identification of key regulators of the KRAS pathway and in KRAS-driven cancer. Many, including epiregulin [134] WT1 [135], GATA2 [136], or molecules involved in the nuclear factor (NF)-κB pathway [137], are appealing targets, and specific inhibitors of these molecules are being investigated in preclinical models. Bortezomib, an NF-κB proteasome inhibitor, was investigated in a phase II study of 16 patients with KRAS G12D-mutant lung adenocarcinoma. A median survival of 13 months was reported [138], with the potential for further study.

Focal adhesion kinase (FAK), involved in cell adhesion to the extracellular matrix, has also been explored as a target. The KRAS-RHOA-FAK pathway plays a major role in tumorigenesis in lung cancers with inactivated TP53 or deficient CDKN2A [126]. A phase II study evaluating VS-6063 (defactinib), a FAK inhibitor, in KRAS-mutant lung cancer (NCT01951690) yielded a low response rate (1 of 44 patients) but a 12-week PFS rate of 36%, which the investigators deemed promising [139].

Researchers have also found an association between KRAS mutation and mesothelin expression. In 93 patients with advanced lung adenocarcinoma, high mesothelin expression conferred a poorer prognosis (p < .0001) [140]. With the ongoing development of mesothelin-directed agents, there may be an opportunity to explore this in KRAS-mutant lung cancer.

Given the frequency of smoking in patients with KRAS-mutant lung cancer, this subset may have higher nonsynonymous mutation load, neo-antigen burden, and potentially greater response to immune checkpoint inhibitors. To date, however, response rates appear similar among patients with and without KRAS mutant tumors.

Although not directed at a specific pathway, immunotherapy has become part of standard lung cancer treatment and a rapidly evolving area of clinical investigation. Given the frequency of smoking in patients with KRAS-mutant lung cancer, this subset may have higher nonsynonymous mutation load, neo-antigen burden, and potentially greater response to immune checkpoint inhibitors [141, 142]. To date, however, response rates appear similar among patients with and without KRAS mutant tumors [143–145]. For example, the response rate with pembrolizumab was 17.2% in patients with KRAS-mutant lung cancer and 16.4% in those with KRAS-WT lung cancer, although strong PDL-1 expression did correlate with higher response (30.8% in KRAS-mutant lung cancer; 29.4% in KRAS-WT lung cancer) [143]. To date, although clinical studies support an association of benefit from PD-1 axis inhibition, smoking status, and transversion mutation load, KRAS mutations have not emerged as an independent predictor of benefit from PD-1 inhibition [141, 142]. Indeed, studies suggest heterogeneity among KRAS-mutant lung cancer samples and PDL-1 and PD-1 expression [146, 147]. With respect to other immune agents, reovirus type 3 Dearing (Reolysin; Oncolytics Biotech, Calgary, AB, Canada, http://www.oncolyticsbiotech.com/) has been explored in combination with paclitaxel and carboplatin in patients with KRAS-mutant lung cancer, with a response rate of 31%, which may be pursued further [148].

Conclusion

The identification of driver oncogenic mutations and the development of biomarker-guided targeted agents have improved outcomes of patients with NSCLC and revolutionized NSCLC therapeutic strategies. Given the high incidence of KRAS mutations in NSCLC and lack of a current approved targeted therapy in this genomic subgroup, KRAS remains an important and appealing therapeutic target. Attempts to target KRAS directly or its regulators, or to alter KRAS membrane localization have yielded disappointing results to date. However, novel molecules are in development that may change this. Indirect targeting of KRAS, via inhibition of downstream effector molecules, appears more promising, and dual inhibition of PI3K/AKT/mTOR and BRAF/MEK/ERK pathways may further advance our attempts to target KRAS-mutant NSCLC. Given that the prognostic and predictive roles of KRAS remain unclear, initial KRAS testing for use in treatment selection remains confined to clinical research [149, 150]. However, with the emergence of promising new agents, KRAS testing may be integrated into routine care of patients with NSCLC in the near future.

Author Contributions

Conception/Design: Pascale Tomasini, Natasha B. Leighl

Collection and/or assembly of data: Pascale Tomasini, Preet Walia, Natasha B. Leighl

Data analysis and interpretation: Pascale Tomasini, Preet Walia, Natasha B. Leighl

Manuscript writing: Pascale Tomasini, Preet Walia, Catherine Labbe, Kevin Jao, Natasha B. Leighl

Final approval of manuscript: Pascale Tomasini, Preet Walia, Catherine Labbe, Kevin Jao, Natasha B. Leighl

Disclosures

The authors indicated no financial relationships.