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KRAS: From undruggable to a druggable Cancer Target

      Highlights

      • RAS is the most frequently mutated oncogene in human cancers, accounting for approximately 30% of mutations in all human cancers.
      • Despite playing a distinct role in tumorigenesis, various attempts to inhibit K-RAS directly in the past were unsuccessful.
      • Additionally, inhibiting downstream Kras signaling through approaches such as inhibiting RAF, MEK and ERK have been unsuccessful.
      • Recently, a binding pocket (S-IIP) has been identified in K-RAS G12C that can be targeted by covalent inhibitors.
      • The K-RAS G12C mutation is present in about 13% of lung adenocarcinoma and 3% of colorectal cancer cases. Several inhibitors of this specific mutation have been developed, with initial evidence of impressive clinical activity.
      • Other approaches including, SHP2, SOS1 and eIF4 inhibition, are being evaluated to abrogate tumor growth in K-RAS mutant cells.

      Abstract

      RAS is the most frequently mutated oncogene in human cancers, with mutations in about 30% of all cancers. RAS exists in three different isoforms (K-RAS, H-RAS and N-RAS) with high sequence homology. K-RAS is the most commonly mutated RAS isoform. The Ras protein is a membrane bound protein with inherent GTPase activity and is activated by numerous extracellular stimuli, cycling between an inactive (GDP-bound) and active (GTP-bound) form. When bound to GTP, it is switched “on” and activates intracellular signaling pathways, critical for cell proliferation and angiogenesis. Mutated RAS is constitutively activated and persistently turned “on” thereby enhancing downstream signaling and leading to tumorigenesis. Various attempts to inhibit Kras in the past were unsuccessful. Recently, several small molecules (AMG510, MRTX849, JNJ-74699157, and LY3499446) have been developed to specifically target K-RAS G12C. Additionally, various other approaches including, SHP2, SOS1 and eIF4 inhibition, have been utilized to abrogate tumor growth in K-RAS mutant cells, resulting in a renewed interest in this pathway. In this review article, we provide an overview on the role of K-RAS in tumorigenesis, past approaches to inhibiting Kras, and current and future prospects for targeting Kras.

      Keywords

      Introduction

      Ras is a membrane-bound protein that possesses inherent GTPase (guanosine triphosphatases) activity and is expressed in all humans [
      • Barbacid M.
      ras genes.
      ]. When activated, it can “switch on” downstream pathways, which ultimately turn on genes that are involved in various physiological processes, including cell growth, differentiation, and survival. It was initially identified in the 1960s by Harvey and Kirsten as a retroviral oncogene when sarcomas were induced in rodents from a murine leukemogenic virus preparation; hence it’s named- Kirsten rat sarcoma 2 viral oncogene homolog [
      • Harvey J.J.
      An unidentified virus which causes the rapid production of tumours in mice.
      ,
      • Kirsten W.H.
      • Mayer L.A.
      Morphologic responses to a murine erythroblastosis virus.
      ]. In the early 1980s, a mutated K-RAS oncogene was identified in a tumor biopsy of a 66-year-old male with squamous cell lung carcinoma [
      • Santos E.
      • Martin-Zanca D.
      • Reddy E.P.
      • Pierotti M.A.
      • Della Porta G.
      • Barbacid M.
      Malignant activation of a K-ras oncogene in lung carcinoma but not in normal tissue of the same patient.
      ]. This mutation was not identified in patient’s white cells and in normal bronchial and parenchymal tissue, demonstrating the significance of somatic mutations in tumorigenesis. Subsequently, it was found that somatic K-RAS mutations are present in approximately 30% of all human cancers, commonly in lung, pancreas, colorectal, and cholangiocarcinoma [
      • Fernandez-Medarde A.
      • Santos E.
      Ras in cancer and developmental diseases.
      ,
      • Friday B.B.
      • Adjei A.A.
      K-ras as a target for cancer therapy.
      ,
      • Wang J.Y.
      • Lian S.T.
      • Chen Y.F.
      • Yang Y.C.
      • Chen L.T.
      • Lee K.T.
      • et al.
      Unique K-ras mutational pattern in pancreatic adenocarcinoma from Taiwanese patients.
      ,
      • Shibata D.
      • Almoguera C.
      • Forrester K.
      • Dunitz J.
      • Martin S.E.
      • Cosgrove M.M.
      • et al.
      Detection of c-K-ras mutations in fine needle aspirates from human pancreatic adenocarcinomas.
      ,
      • Pao W.
      • Girard N.
      New driver mutations in non-small-cell lung cancer.
      ,
      • Mascaux C.
      • Iannino N.
      • Martin B.
      • Paesmans M.
      • Berghmans T.
      • Dusart M.
      • et al.
      The role of RAS oncogene in survival of patients with lung cancer: a systematic review of the literature with meta-analysis.
      ,
      • Peeters M.
      • Kafatos G.
      • Taylor A.
      • Gastanaga V.M.
      • Oliner K.S.
      • Hechmati G.
      • Terwey J.-H.
      • van Krieken J.H.
      Prevalence of RAS mutations and individual variation patterns among patients with metastatic colorectal cancer: a pooled analysis of randomised controlled trials.
      ,
      • Munoz-Maldonado C.
      • Zimmer Y.
      • Medova M.
      A comparative analysis of individual RAS mutations in cancer biology.
      ]. In this review, we discuss Kras signaling, it’s role in tumorigenesis and why this target has been considered “undruggable” historically. We also outline some strategies for targeting K-RAS mutant cancers by discussing promising new agents against Kras, including specific G12C inhibitors, SHP2 inhibitors, SOS1 inhibitors, and conclude by summarizing ongoing trials.

      Ras family members

      Ras structure and function

      There are two copies of K-RAS, namely K-RAS1 and K-RAS2[
      • Jancik S.
      • Drabek J.
      • Radzioch D.
      • Hajduch M.
      Clinical relevance of KRAS in human cancers.
      ]. K-RAS1 and K-RAS2 are located on chromosome 6p11-12 and 12p11.1-12.1 respectively [
      • McBride O.W.
      • Swan D.C.
      • Tronick S.R.
      • Gol R.
      • Klimanis D.
      • Moore D.E.
      • et al.
      Regional chromosomal localization of N-ras, K-ras-1, K-ras-2 and myb oncogenes in human cells.
      ,
      • Popescu N.C.
      • Amsbaugh S.C.
      • DiPaolo J.A.
      • Tronick S.R.
      • Aaronson S.A.
      • Swan D.C.
      Chromosomal localization of three human ras genes by in situ molecular hybridization.
      ]. K-RAS1 is a pseudo-gene [
      • Jancik S.
      • Drabek J.
      • Radzioch D.
      • Hajduch M.
      Clinical relevance of KRAS in human cancers.
      ]. Activating K-RAS2 mutations have been identified in various human cancers. K-RAS2 is simply referred to as K-RAS. The K-RAS gene consists of 6 exons spread over 35 kb of genomic DNA [
      • Sameer A.S.
      Colorectal cancer: molecular mutations and polymorphisms.
      ]. The structure of the K-RAS gene is depicted in Fig. 1. K-RAS is alternatively spliced to form K-RAS 4A and K-RAS 4B. The term K-RAS is generally used to indicate K-RAS 4B [
      • Chen Z.
      • Otto J.C.
      • Bergo M.O.
      • Young S.G.
      • Casey P.J.
      The C-terminal polylysine region and methylation of K-Ras are critical for the interaction between K-Ras and microtubules.
      ]. The Ras protein includes three closely related 21-kDa isoforms, H (Harvey rat sarcoma virus oncogene), N (human neuroblastoma) and K-ras (Kirsten rat sarcoma virus oncogene) [
      • Barbacid M.
      ras genes.
      ]. Ras has three major domains: the G-domain, the C-terminal and the C-terminal CAAX box [
      • Vogler O.
      • Barcelo J.M.
      • Ribas C.
      • Escriba P.V.
      Membrane interactions of G proteins and other related proteins.
      ,
      • Lemmon M.A.
      • Schlessinger J.
      Regulation of signal transduction and signal diversity by receptor oligomerization.
      ]. The G-domain, containing switch I and switch II loops, is a highly conserved domain and is responsible for GDP-GTP exchange [
      • Friday B.B.
      • Adjei A.A.
      K-ras as a target for cancer therapy.
      ,
      • Vogler O.
      • Barcelo J.M.
      • Ribas C.
      • Escriba P.V.
      Membrane interactions of G proteins and other related proteins.
      ]. The C-terminal including the CAAX box demonstrates a significant variation between RAS family members, and is required for post-translational modification [
      • Friday B.B.
      • Adjei A.A.
      K-ras as a target for cancer therapy.
      ,
      • Gao J.
      • Liao J.
      • Yang G.Y.
      CAAX-box protein, prenylation process and carcinogenesis.
      ]. Ras proteins bind with GDP (guanosine diphosphate) and GTP (guanosine triphosphate) with great affinity [
      • Lowy D.R.
      • Willumsen B.M.
      Function and regulation of ras.
      ]. They act as “molecular switches” and cycle between the GDP-bound (inactive) and GTP-bound (active) forms. In the active state, they transmit signals from the cell membrane to the nucleus, leading to activation of transcription factors which lead to the regulation of cell growth and differentiation (Fig. 2) [
      • Adjei A.A.
      Blocking oncogenic Ras signaling for cancer therapy.
      ].
      Figure thumbnail gr1
      Fig. 1Structure of K-RAS gene with associated mutations and their protein domains. Reproduced with permission from Ramakrishnan V et al. Effects of KRAS Gene Mutations in Gynecological Malignancies. Investigations in Gynecology Research & Womens Health.
      Figure thumbnail gr2
      Fig. 2Simplified scheme of Mitogen Activation Protein Kinase activation and signaling cascade.

      Ras signaling

      Ras signaling begins when a ligand binds to an upstream receptor, such as a tyrosine kinase receptor. Almost all of the receptor tyrosine kinases are monomers [
      • Schlessinger J.
      Cell signaling by receptor tyrosine kinases.
      ]. A well-known pathway involves the interaction of epidermal growth factor (EGF) to its receptor (EGFR) [
      • Liebmann C.
      Regulation of MAP kinase activity by peptide receptor signalling pathway: paradigms of multiplicity.
      ]. Binding of EGF to EGFR induces dimerization of the receptor, followed by auto-phosphorylation [
      • Lemmon M.A.
      • Schlessinger J.
      Regulation of signal transduction and signal diversity by receptor oligomerization.
      ,
      • Heldin C.H.
      Dimerization of cell surface receptors in signal transduction.
      ]. The phosphorylated receptor binds to an adaptor protein Grb2 (Growth factor receptor-bound protein 2). This complex recruits son of sevenless (SOS) to the plasma membrane [
      • Schlessinger J.
      Cell signaling by receptor tyrosine kinases.
      ]. Once recruited to the plasma membrane, SOS is capable of displacing GDP from Ras, allowing Ras-GTP interaction. Ras can also regulate SOS activity suggesting that the pathway could be bidirectional [
      • Iversen L.
      • Tu H.L.
      • Lin W.C.
      • Christensen S.M.
      • Abel S.M.
      • Iwig J.
      • et al.
      Molecular kinetics. Ras activation by SOS: allosteric regulation by altered fluctuation dynamics.
      ,
      • Margarit S.M.
      • Sondermann H.
      • Hall B.E.
      • Nagar B.
      • Hoelz A.
      • Pirruccello M.
      • et al.
      Structural evidence for feedback activation by Ras.GTP of the Ras-specific nucleotide exchange factor SOS.
      ,
      • Sondermann H.
      • Soisson S.M.
      • Boykevisch S.
      • Yang S.S.
      • Bar-Sagi D.
      • Kuriyan J.
      Structural analysis of autoinhibition in the Ras activator Son of sevenless.
      ]. The binding of GTP to Ras induces changes in switch I and switch II loops of the G-domain, thereby activating Ras.
      Hydrolysis of GTP to GDP inactivates Ras. Ras inherently has low GTPase activity. The intrinsic GTPase activity is stimulated further by GTPase Activating Proteins (GAPs) such as p120-GAP and NF1 (neurofibromin) [
      • Campbell P.M.
      • Der C.J.
      Oncogenic Ras and its role in tumor cell invasion and metastasis.
      ,
      • Drugan J.K.
      • Rogers-Graham K.
      • Gilmer T.
      • Campbell S.
      • Clark G.J.
      The Ras/p120 GTPase-activating protein (GAP) interaction is regulated by the p120 GAP pleckstrin homology domain.
      ,
      • Pamonsinlapatham P.
      • Hadj-Slimane R.
      • Lepelletier Y.
      • Allain B.
      • Toccafondi M.
      • Garbay C.
      • et al.
      p120-Ras GTPase activating protein (RasGAP): a multi-interacting protein in downstream signaling.
      ]. This keeps Ras in the inactive form and prevents its persistent activation. In addition to p120 and NF1, numerous other Ras GTPases have been identified [
      • Maekawa M.
      • Li S.
      • Iwamatsu A.
      • Morishita T.
      • Yokota K.
      • Imai Y.
      • et al.
      A novel mammalian Ras GTPase-activating protein which has phospholipid-binding and Btk homology regions.
      ,

      Jin H, Wang X, Ying J, Wong AH, Cui Y, Srivastava G, et al. Epigenetic silencing of a Ca(2+)-regulated Ras GTPase-activating protein RASAL defines a new mechanism of Ras activation in human cancers. In: Proceedings of the national academy of sciences of the United States of America. 2007;104(30):12353–8.

      ,
      • Kim J.H.
      • Liao D.
      • Lau L.F.
      • Huganir R.L.
      SynGAP: a synaptic RasGAP that associates with the PSD-95/SAP90 protein family.
      ]. GAP represents a notable class of tumor suppressor genes. Normally, Ras signaling is transient. Mechanistically, inactivation of the Ras GAPs will persistently activate Ras and its effectors leading to malignant transformation. The most extensively studied tumor suppressor gene is NF1-GAP. Germline mutation of the NF1 gene predisposes to variety of tumors, including gliomas, neurofibromas, pheochromocytoema and leukemia [
      • Ledbetter D.H.
      • Rich D.C.
      • O'Connell P.
      • Leppert M.
      • Carey J.C.
      Precise localization of NF1 to 17q11.2 by balanced translocation.
      ,
      • Tucker T.
      • Riccardi V.M.
      • Sutcliffe M.
      • Vielkind J.
      • Wechsler J.
      • Wolkenstein P.
      • et al.
      Different patterns of mast cells distinguish diffuse from encapsulated neurofibromas in patients with neurofibromatosis 1.
      ,
      • Sorensen S.A.
      • Mulvihill J.J.
      • Nielsen A.
      Long-term follow-up of von Recklinghausen neurofibromatosis. Survival and malignant neoplasms.
      ]. Additionally, recent studies have demonstrated a high frequency of somatic NF1 mutations in a variety of sporadic tumors, including lung adenocarcinoma, leukemia, ovarian, multiple myeloma, glioblastoma and melanoma [
      • Ding L.
      • Getz G.
      • Wheeler D.A.
      • Mardis E.R.
      • McLellan M.D.
      • Cibulskis K.
      • et al.
      Somatic mutations affect key pathways in lung adenocarcinoma.
      ,
      • Boudry-Labis E.
      • Roche-Lestienne C.
      • Nibourel O.
      • Boissel N.
      • Terre C.
      • Perot C.
      • et al.
      Neurofibromatosis-1 gene deletions and mutations in de novo adult acute myeloid leukemia.
      ,
      • Philpott C.
      • Tovell H.
      • Frayling I.M.
      • Cooper D.N.
      • Upadhyaya M.
      The NF1 somatic mutational landscape in sporadic human cancers.
      ].
      There are a number of effector molecules that an activated Ras can act upon including Raf, PI3K [
      • Friday B.B.
      • Adjei A.A.
      K-ras as a target for cancer therapy.
      ]. The Raf family is the best characterized Ras effector and the one with the strongest role in human cancer. Raf (Rapidly Accelerated Fibrosarcoma) protein is a serine/threonine kinase initially isolated in avian retrovirus and murine sarcoma virus [
      • Zebisch A.
      • Troppmair J.
      Back to the roots: the remarkable RAF oncogene story.
      ]. It consists of three subtypes, A-raf, B-raf and Raf-1 (C-raf). Binding of GTP to Ras promotes recruitment of Raf to the cell membrane, dimerization of Raf and phosphorylation. Additionally, many factors that are not completely understood are involved in the proper activation of Raf [
      • Fernandez-Medarde A.
      • Santos E.
      Ras in cancer and developmental diseases.
      ,
      • Stokoe D.
      • Macdonald S.G.
      • Cadwallader K.
      • Symons M.
      • Hancock J.F.
      Activation of Raf as a result of recruitment to the plasma membrane.
      ]. Activated Raf phosphorylates MEK (Mitogen Activated Protein Kinase), which in turn, phosphorylates ERK (extracellular-signal-regulated kinase). B-RAF is frequently mutated in human cancers, including melanoma, thyroid malignancy and hairy cell leukemia [
      • Long G.V.
      • Menzies A.M.
      • Nagrial A.M.
      • Haydu L.E.
      • Hamilton A.L.
      • Mann G.J.
      • et al.
      Prognostic and clinicopathologic associations of oncogenic BRAF in metastatic melanoma.
      ,
      • Kimura E.T.
      • Nikiforova M.N.
      • Zhu Z.
      • Knauf J.A.
      • Nikiforov Y.E.
      • Fagin J.A.
      High prevalence of BRAF mutations in thyroid cancer: genetic evidence for constitutive activation of the RET/PTC-RAS-BRAF signaling pathway in papillary thyroid carcinoma.
      ,
      • Tiacci E.
      • Trifonov V.
      • Schiavoni G.
      • Holmes A.
      • Kern W.
      • Martelli M.P.
      • et al.
      BRAF mutations in hairy-cell leukemia.
      ]. When compared with A-RAF and C-RAF, B-RAF has a higher basal kinase activity and is easily activated by RAS [
      • Wellbrock C.
      • Karasarides M.
      • Marais R.
      The RAF proteins take centre stage.
      ,
      • Emuss V.
      • Garnett M.
      • Mason C.
      • Marais R.
      Mutations of C-RAF are rare in human cancer because C-RAF has a low basal kinase activity compared with B-RAF.
      ].
      The second best characterized Ras effector is Phosphoinositide 3′-kinase (PI3-K), which is activated by numerous mechanisms. One of the mechanisms involves binding of extracellular growth factor to its receptor tyrosine kinase, leading to dimerization of the receptor monomer followed by auto-phosphorylation. Insulin Receptor Substrate-1 (IRS-1) then binds to the catalytic site of the phosphorylated dimer. Once bound to the dimer, IRS-1 serves as a binding and an activation site for PI3-K. A totally different mechanism of PI3-K activation involves direct binding of PI3-K with GTP-bound Ras. The activated PI3-K then migrates to the inner aspect of cell membrane leading to phosphorylation of phosphatidylinositol bisphosphate (PIP2) to phosphatidylinositol (3,4,5) trisphosphate (PIP3), which then activates a protein kinase AKT [
      • Castellano E.
      • Downward J.
      RAS interaction with PI3K: more than just another effector pathway.
      ]. This ultimately activates mTOR. mTOR then activates the translation factor S6K. By binding to the larger ribosomal subunit, S6K induces translation of mRNA into protein. All the essential steps of the RAS-RAF and PI3-K signaling pathway are illustrated in Fig. 2.

      K-RAS mutations in human tumors

      K-RAS mutation subtypes

      Various mutant forms of K-RAS are now recognized and are divided into three broad categories based on the mutated codon: G12 (mutation at codon 12), G13 (mutation at codon 13), and Q61 (mutation at codon 61).
      The prevalence of K-RAS mutations in non-small cell lung cancer (NSCLC) is about 30% in adenocarcinoma and 5% in squamous cell carcinoma [
      • Roberts P.J.
      • Stinchcombe T.E.
      • Der C.J.
      • Socinski M.A.
      Personalized medicine in non-small-cell lung cancer: is KRAS a useful marker in selecting patients for epidermal growth factor receptor-targeted therapy?.
      ]. About 97% of K-RAS mutations in NSCLC occurs at exons 2 and 3 (G12, G13, and Q61) [
      • Wood K.
      • Hensing T.
      • Malik R.
      • Salgia R.
      Prognostic and predictive value in KRAS in non-small-cell lung cancer: a review.
      ]. Also, they usually do not exist concomitantly with other sensitizing mutations, such as EGFR, B-RAF and ALK rearrangement [
      • Gainor J.F.
      • Varghese A.M.
      • Ou S.H.
      • Kabraji S.
      • Awad M.M.
      • Katayama R.
      • et al.
      ALK rearrangements are mutually exclusive with mutations in EGFR or KRAS: an analysis of 1,683 patients with non-small cell lung cancer.
      ]. G12C is the most common mutation subtype, accounting for about 40% of all K-RAS mutations followed by G12V [
      • 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.
      ,
      • Karachaliou N.
      • Mayo C.
      • Costa C.
      • Magri I.
      • Gimenez-Capitan A.
      • Molina-Vila M.A.
      • et al.
      KRAS mutations in lung cancer.
      ,
      • Biernacka A.
      • Tsongalis P.D.
      • Peterson J.D.
      • de Abreu F.B.
      • Black C.C.
      • Gutmann E.J.
      • et al.
      The potential utility of re-mining results of somatic mutation testing: KRAS status in lung adenocarcinoma.
      ].
      In colorectal cancer, K-RAS mutations occur in about 30–50% of cases [
      • Peeters M.
      • Kafatos G.
      • Taylor A.
      • Gastanaga V.M.
      • Oliner K.S.
      • Hechmati G.
      • Terwey J.-H.
      • van Krieken J.H.
      Prevalence of RAS mutations and individual variation patterns among patients with metastatic colorectal cancer: a pooled analysis of randomised controlled trials.
      ,
      • Kafatos G.
      • Niepel D.
      • Lowe K.
      • Jenkins-Anderson S.
      • Westhead H.
      • Garawin T.
      • et al.
      RAS mutation prevalence among patients with metastatic colorectal cancer: a meta-analysis of real-world data.
      ,
      • Watanabe T.
      • Yoshino T.
      • Uetake H.
      • Yamazaki K.
      • Ishiguro M.
      • Kurokawa T.
      • et al.
      KRAS mutational status in Japanese patients with colorectal cancer: results from a nationwide, multicenter, cross-sectional study.
      ,
      • Andreyev H.J.
      • Norman A.R.
      • Cunningham D.
      • Oates J.R.
      • Clarke P.A.
      Kirsten ras mutations in patients with colorectal cancer: the multicenter “RASCAL” study.
      ]. G12D and G12V are the two most common mutation subtypes [
      • Hayama T.
      • Hashiguchi Y.
      • Okamoto K.
      • Okada Y.
      • Ono K.
      • Shimada R.
      • et al.
      G12V and G12C mutations in the gene KRAS are associated with a poorer prognosis in primary colorectal cancer.
      ,
      • Hershkovitz D.
      • Simon E.
      • Bick T.
      • Prinz E.
      • Noy S.
      • Sabo E.
      • et al.
      Adenoma and carcinoma components in colonic tumors show discordance for KRAS mutation.
      ]. Additionally, K-RAS mutations have also been identified in colorectal adenoma [
      • Vogelstein B.
      • Fearon E.R.
      • Hamilton S.R.
      • Kern S.E.
      • Preisinger A.C.
      • Leppert M.
      • et al.
      Genetic alterations during colorectal-tumor development.
      ]. The prevalence of K-RAS mutations in pancreatic carcinoma is the highest with various studies showing the prevalence rate well above 80% with G12D being the most common subtype [
      • Jones S.
      • Zhang X.
      • Parsons D.W.
      • Lin J.C.
      • Leary R.J.
      • Angenendt P.
      • et al.
      Core signaling pathways in human pancreatic cancers revealed by global genomic analyses.
      ,
      • Hruban R.H.
      • van Mansfeld A.D.
      • Offerhaus G.J.
      • van Weering D.H.
      • Allison D.C.
      • Goodman S.N.
      • et al.
      K-ras oncogene activation in adenocarcinoma of the human pancreas. A study of 82 carcinomas using a combination of mutant-enriched polymerase chain reaction analysis and allele-specific oligonucleotide hybridization.
      ]. In cholangiocarcinoma, the prevalence of K-RAS mutation varies from 10% to 15% for intrahepatic cholangiocarcinoma and from 45% to 54% for extrahepatic cholangiocarcinoma [
      • Hezel A.F.
      • Deshpande V.
      • Zhu A.X.
      Genetics of biliary tract cancers and emerging targeted therapies.
      ]. K-RAS mutations are also found in various hematological malignancies (multiple myeloma, acute myeloid leukemia, and diffuse large B-cell lymphoma), other gastrointestinal malignancies (esophageal adenocarcinoma, gastric cancer), uterine carcinoma, and cervical adenocarcinoma [
      • Walker B.A.
      • Boyle E.M.
      • Wardell C.P.
      • Murison A.
      • Begum D.B.
      • Dahir N.M.
      • et al.
      Mutational spectrum, copy number changes, and outcome: results of a sequencing study of patients with newly diagnosed myeloma.
      ,
      • Bezieau S.
      • Devilder M.C.
      • Avet-Loiseau H.
      • Mellerin M.P.
      • Puthier D.
      • Pennarun E.
      • et al.
      High incidence of N and K-Ras activating mutations in multiple myeloma and primary plasma cell leukemia at diagnosis.
      ,
      • Cox A.D.
      • Fesik S.W.
      • Kimmelman A.C.
      • Luo J.
      • Der C.J.
      Drugging the undruggable RAS: mission possible?.
      ,
      • Peng N.
      • Zhao X.
      Comparison of K-ras mutations in lung, colorectal and gastric cancer.
      ,
      • Ayatollahi H.
      • Tavassoli A.
      • Jafarian A.H.
      • Alavi A.
      • Shakeri S.
      • Shams S.F.
      • et al.
      KRAS codon 12 and 13 mutations in gastric cancer in the Northeast Iran.
      ,
      • Arber N.
      • Shapira I.
      • Ratan J.
      • Stern B.
      • Hibshoosh H.
      • Moshkowitz M.
      • et al.
      Activation of c-K-ras mutations in human gastrointestinal tumors.
      ,
      • Moul J.W.
      • Theune S.M.
      • Chang E.H.
      Detection of RAS mutations in archival testicular germ cell tumors by polymerase chain reaction and oligonucleotide hybridization.
      ,
      • Hacioglu B.M.
      • Kodaz H.
      • Erdogan B.
      • Cinkaya A.
      • Tastekin E.
      • Hacibekiroglu I.
      • et al.
      K-RAS and N-RAS mutations in testicular germ cell tumors.
      ,
      • Ridanpaa M.
      • Lothe R.A.
      • Onfelt A.
      • Fossa S.
      • Borresen A.L.
      • Husgafvel-Pursiainen K.
      K-ras oncogene codon 12 point mutations in testicular cancer.
      ,
      • Jiang W.
      • Xiang L.
      • Pei X.
      • He T.
      • Shen X.
      • Wu X.
      • et al.
      Mutational analysis of KRAS and its clinical implications in cervical cancer patients.
      ,
      • Wright A.A.
      • Howitt B.E.
      • Myers A.P.
      • Dahlberg S.E.
      • Palescandolo E.
      • Van Hummelen P.
      • et al.
      Oncogenic mutations in cervical cancer: genomic differences between adenocarcinomas and squamous cell carcinomas of the cervix.
      ,
      • Nagel P.D.
      • Feld F.M.
      • Weissinger S.E.
      • Stenzinger A.
      • Moller P.
      • Lennerz J.K.
      Absence of BRAF and KRAS hotspot mutations in primary mediastinal B-cell lymphoma.
      ,
      • Liu Q.W.
      • Fu J.H.
      • Luo K.J.
      • Yang H.X.
      • Wang J.Y.
      • Hu Y.
      • et al.
      Identification of EGFR and KRAS mutations in Chinese patients with esophageal squamous cell carcinoma.
      ,
      • Lorenzen S.
      • Schuster T.
      • Porschen R.
      • Al-Batran S.E.
      • Hofheinz R.
      • Thuss-Patience P.
      • et al.
      Cetuximab plus cisplatin-5-fluorouracil versus cisplatin-5-fluorouracil alone in first-line metastatic squamous cell carcinoma of the esophagus: a randomized phase II study of the Arbeitsgemeinschaft Internistische Onkologie.
      ,
      • Essakly A.
      • Loeser H.
      • Kraemer M.
      • Alakus H.
      • Chon S.H.
      • Zander T.
      • et al.
      PIK3CA and KRAS amplification in esophageal adenocarcinoma and their impact on the inflammatory tumor microenvironment and prognosis.
      ,
      • Hollestelle A.
      • Elstrodt F.
      • Nagel J.H.
      • Kallemeijn W.W.
      • Schutte M.
      Phosphatidylinositol-3-OH kinase or RAS pathway mutations in human breast cancer cell lines.
      ,
      • Pereira C.B.
      • Leal M.F.
      • de Souza C.R.
      • Montenegro R.C.
      • Rey J.A.
      • Carvalho A.A.
      • et al.
      Prognostic and predictive significance of MYC and KRAS alterations in breast cancer from women treated with neoadjuvant chemotherapy.
      ,
      • Ahmad E.I.
      • Gawish H.H.
      • Al Azizi N.M.
      • Elhefni A.M.
      The prognostic impact of K-RAS mutations in adult acute myeloid leukemia patients treated with high-dose cytarabine.
      ,
      • Stirewalt D.L.
      • Kopecky K.J.
      • Meshinchi S.
      • Appelbaum F.R.
      • Slovak M.L.
      • Willman C.L.
      • et al.
      FLT3, RAS, and TP53 mutations in elderly patients with acute myeloid leukemia.
      ,
      • Neubauer A.
      • Dodge R.K.
      • George S.L.
      • Davey F.R.
      • Silver R.T.
      • Schiffer C.A.
      • et al.
      Prognostic importance of mutations in the ras proto-oncogenes in de novo acute myeloid leukemia.
      ,
      • Vendramini E.
      • Bomben R.
      • Pozzo F.
      • Benedetti D.
      • Bittolo T.
      • Rossi F.M.
      • et al.
      KRAS, NRAS, and BRAF mutations are highly enriched in trisomy 12 chronic lymphocytic leukemia and are associated with shorter treatment-free survival.
      ,
      • Gimenez N.
      • Martinez-Trillos A.
      • Montraveta A.
      • Lopez-Guerra M.
      • Rosich L.
      • Nadeu F.
      • et al.
      Mutations in the RAS-BRAF-MAPK-ERK pathway define a specific subgroup of patients with adverse clinical features and provide new therapeutic options in chronic lymphocytic leukemia.
      ,
      • Ouerhani S.
      • Bougatef K.
      • Soltani I.
      • Elgaaied A.B.
      • Abbes S.
      • Menif S.
      The prevalence and prognostic significance of KRAS mutation in bladder cancer, chronic myeloid leukemia and colorectal cancer.
      ,
      • Kodaz H.K.O.
      • Hacioglu M.B.
      • et al.
      Frequency of RAS mutations (KRAS, NRAS, HRAS) in human solid cancer.
      ,
      • Lax S.F.
      • Kendall B.
      • Tashiro H.
      • Slebos R.J.
      • Hedrick L.
      The frequency of p53, K-ras mutations, and microsatellite instability differs in uterine endometrioid and serous carcinoma: evidence of distinct molecular genetic pathways.
      ]. The frequency of K-RAS mutations in various tumor types is summarized in Table 1.
      Table 1Prevalence of K-RAS mutations in human cancers.
      Tumor TypeFrequency of K-RAS mutation
      Pancreatic adenocarcinoma (61,62)≥ 80%
      Colorectal cancer (11,55–57)30–50%
      Non-small cell lung cancer (49)
      Adenocarcinoma30%
      Squamous cell carcinoma5%
      Cholangiocarcinoma (63)
      Extrahepatic45–54%
      Intrahepatic10–15%
      Multiple myeloma (64,65)21–33%
      Uterine endometrial (66, 88)
      Endometrioid21–26%
      Serous2%
      Gastric cancer (67–69)3–13%
      Testicular cancer (70–72)9–16%
      Cervical adenocarcinoma (73,74)7–18%
      Diffuse Large B-cell lymphoma (75)1.6%
      Esophageal (66, 76–78)
      Adenocarcinoma3.8–17%
      Squamous cell carcinoma0–12%
      Breast Cancer (79,80)8–13%
      Acute Myeloid Leukemia (81–83)5–23%
      Chronic Lymphocytic Leukemia (84,85)0.7–7%
      Bladder Cancer (86)4%
      Cutaneous malignant melanoma (87)<1%

      Predictive and prognostic value of K-RAS mutations

      Prognostic value

      The prognostic value of K-RAS mutations in various tumor types remains unclear. In NSCLC, K-RAS mutant NSCLC patients were considered to have a worse prognosis [
      • Slebos R.J.
      • Kibbelaar R.E.
      • Dalesio O.
      • Kooistra A.
      • Stam J.
      • Meijer C.J.
      • et al.
      K-ras oncogene activation as a prognostic marker in adenocarcinoma of the lung.
      ]. However, various studies have demonstrated conflicting results [
      • Mascaux C.
      • Iannino N.
      • Martin B.
      • Paesmans M.
      • Berghmans T.
      • Dusart M.
      • et al.
      The role of RAS oncogene in survival of patients with lung cancer: a systematic review of the literature with meta-analysis.
      ,
      • Shepherd F.A.
      • Domerg C.
      • Hainaut P.
      • Janne P.A.
      • Pignon J.P.
      • Graziano S.
      • et al.
      Pooled analysis of the prognostic and predictive effects of KRAS mutation status and KRAS mutation subtype in early-stage resected non-small-cell lung cancer in four trials of adjuvant chemotherapy.
      ,
      • Zer A.
      • Ding K.
      • Lee S.M.
      • Goss G.D.
      • Seymour L.
      • Ellis P.M.
      • et al.
      Pooled analysis of the prognostic and predictive value of KRAS mutation status and mutation subtype in patients with non-small cell lung cancer treated with epidermal growth factor receptor tyrosine kinase inhibitors.
      ,
      • Pan W.
      • Yang Y.
      • Zhu H.
      • Zhang Y.
      • Zhou R.
      • Sun X.
      KRAS mutation is a weak, but valid predictor for poor prognosis and treatment outcomes in NSCLC: a meta-analysis of 41 studies.
      ]. Mascaux and colleagues performed a systematic review of 5216 stage I-IV patients in forty-three studies from 1990 to 2003 [
      • Mascaux C.
      • Iannino N.
      • Martin B.
      • Paesmans M.
      • Berghmans T.
      • Dusart M.
      • et al.
      The role of RAS oncogene in survival of patients with lung cancer: a systematic review of the literature with meta-analysis.
      ]. The study demonstrated a worse survival outcome in patients with K-RAS mutations or p21 expression compared to those without these aberrations (HR, 1.35 [95% CI 1.16–1.56]). Moreover, the study revealed no significant impact of K-RAS mutations on survival for squamous histology and for the stage I and stage I-III cohorts. On the contrary, a pooled analysis utilizing the Lung Adjuvant Cisplatin Evaluation (LACE) database of 3,533 patients with stage I-III disease, demonstrated no difference in overall survival in patients with K-RAS mutant versus K-RAS wild-type NSCLC [
      • Shepherd F.A.
      • Domerg C.
      • Hainaut P.
      • Janne P.A.
      • Pignon J.P.
      • Graziano S.
      • et al.
      Pooled analysis of the prognostic and predictive effects of KRAS mutation status and KRAS mutation subtype in early-stage resected non-small-cell lung cancer in four trials of adjuvant chemotherapy.
      ]. A more recent study by Pan and colleagues utilizing 41 studies from 2005 to 2015 with 13,103 patients, showed worse overall survival (HR, 1.56 [95% CI 1.39–1.76]) and disease free survival (HR, 1.57 [95% CI 1.17–2.09]) with K-RAS mutation in patients with early-stage resected NSCLC [
      • Pan W.
      • Yang Y.
      • Zhu H.
      • Zhang Y.
      • Zhou R.
      • Sun X.
      KRAS mutation is a weak, but valid predictor for poor prognosis and treatment outcomes in NSCLC: a meta-analysis of 41 studies.
      ].
      In colon cancer, K-RAS mutations may confer poor prognosis but the data are not consistent for localized disease. While many studies demonstrated a negative impact of K-RAS mutations on survival [
      • Cerottini J.P.
      • Caplin S.
      • Saraga E.
      • Givel J.C.
      • Benhattar J.
      The type of K-ras mutation determines prognosis in colorectal cancer.
      ,
      • Samowitz W.S.
      • Curtin K.
      • Schaffer D.
      • Robertson M.
      • Leppert M.
      • Slattery M.L.
      Relationship of Ki-ras mutations in colon cancers to tumor location, stage, and survival: a population-based study.
      ,
      • Yoon H.H.
      • Tougeron D.
      • Shi Q.
      • Alberts S.R.
      • Mahoney M.R.
      • Nelson G.D.
      • et al.
      KRAS codon 12 and 13 mutations in relation to disease-free survival in BRAF-wild-type stage III colon cancers from an adjuvant chemotherapy trial (N0147 alliance).
      ,
      • Modest D.P.
      • Ricard I.
      • Heinemann V.
      • Hegewisch-Becker S.
      • Schmiegel W.
      • Porschen R.
      • et al.
      Outcome according to KRAS-, NRAS- and BRAF-mutation as well as KRAS mutation variants: pooled analysis of five randomized trials in metastatic colorectal cancer by the AIO colorectal cancer study group.
      ,
      • Taieb J.
      • Zaanan A.
      • Le Malicot K.
      • Julie C.
      • Blons H.
      • Mineur L.
      • et al.
      Prognostic effect of BRAF and KRAS mutations in patients with stage III colon cancer treated with leucovorin, fluorouracil, and oxaliplatin with or without cetuximab: a post hoc analysis of the PETACC-8 trial.
      ], including those with localized disease [
      • Cerottini J.P.
      • Caplin S.
      • Saraga E.
      • Givel J.C.
      • Benhattar J.
      The type of K-ras mutation determines prognosis in colorectal cancer.
      ,
      • Yoon H.H.
      • Tougeron D.
      • Shi Q.
      • Alberts S.R.
      • Mahoney M.R.
      • Nelson G.D.
      • et al.
      KRAS codon 12 and 13 mutations in relation to disease-free survival in BRAF-wild-type stage III colon cancers from an adjuvant chemotherapy trial (N0147 alliance).
      ,
      • Taieb J.
      • Zaanan A.
      • Le Malicot K.
      • Julie C.
      • Blons H.
      • Mineur L.
      • et al.
      Prognostic effect of BRAF and KRAS mutations in patients with stage III colon cancer treated with leucovorin, fluorouracil, and oxaliplatin with or without cetuximab: a post hoc analysis of the PETACC-8 trial.
      ], Roth and colleagues demonstrated that K-RAS mutations did not affect survival in stage II or III colon cancer [
      • Roth A.D.
      • Tejpar S.
      • Delorenzi M.
      • Yan P.
      • Fiocca R.
      • Klingbiel D.
      • et al.
      Prognostic role of KRAS and BRAF in stage II and III resected colon cancer: results of the translational study on the PETACC-3, EORTC 40993, SAKK 60–00 trial.
      ]. Furthermore, the RASCAL-II study demonstrated that out of the 12 possible mutations on codon 12 or 13 of the K-RAS gene, only one mutation on codon 12, G12V (glycine to valine), was associated with inferior survival [
      • Andreyev H.J.
      • Norman A.R.
      • Cunningham D.
      • Oates J.
      • Dix B.R.
      • Iacopetta B.J.
      • et al.
      Kirsten ras mutations in patients with colorectal cancer: the 'RASCAL II' study.
      ].
      In pancreatic cancer, studies have demonstrated conflicting results on the prognostic value of K-RAS mutations on survival [
      • Haas M.
      • Ormanns S.
      • Baechmann S.
      • Remold A.
      • Kruger S.
      • Westphalen C.B.
      • et al.
      Extended RAS analysis and correlation with overall survival in advanced pancreatic cancer.
      ,
      • Shin S.H.
      • Kim S.C.
      • Hong S.M.
      • Kim Y.H.
      • Song K.B.
      • Park K.M.
      • et al.
      Genetic alterations of K-ras, p53, c-erbB-2, and DPC4 in pancreatic ductal adenocarcinoma and their correlation with patient survival.
      ,
      • Windon A.L.
      • Loaiza-Bonilla A.
      • Jensen C.E.
      • Randall M.
      • Morrissette J.J.D.
      • Shroff S.G.
      A KRAS wild type mutational status confers a survival advantage in pancreatic ductal adenocarcinoma.
      ]. A study conducted by Bournet and colleagues involving 219 patients with locally advanced or metastatic pancreatic adenocarcinoma, demonstrated no difference in survival between K-RAS mutant and K-RAS wild-type tumors [
      • Bournet B.
      • Muscari F.
      • Buscail C.
      • Assenat E.
      • Barthet M.
      • Hammel P.
      • et al.
      KRAS G12D mutation subtype is a prognostic factor for advanced pancreatic adenocarcinoma.
      ]. The study however showed that the G12D (glycine to aspartic acid) mutation had worse prognosis when compared with other mutation subtypes and K-RAS wild type. Additionally, coexistence of CDKN2 aberrations and K-RAS mutation appeared to confer the worst prognosis [
      • Rachakonda P.S.
      • Bauer A.S.
      • Xie H.
      • Campa D.
      • Rizzato C.
      • Canzian F.
      • et al.
      Somatic mutations in exocrine pancreatic tumors: association with patient survival.
      ].
      In summary, the prognosis conferred by K-RAS mutations may differ based on the specific mutation and tumor type. This may, in part, explain the conflicting results from different studies. More studies examining mutation subtypes are needed to assess the real prognostic value of K-RAS mutations in tumors.

      Predictive value

      The predictive value of K-RAS mutations in NSCLC has been evaluated in multiple trials [
      • Rodenhuis S.
      • Boerrigter L.
      • Top B.
      • Slebos R.J.
      • Mooi W.J.
      • van't Veer L.
      • et al.
      Mutational activation of the K-ras oncogene and the effect of chemotherapy in advanced adenocarcinoma of the lung: a prospective study..
      ,
      • Herbst R.S.
      • Prager D.
      • Hermann R.
      • Fehrenbacher L.
      • Johnson B.E.
      • Sandler A.
      • et al.
      TRIBUTE: a phase III trial of erlotinib hydrochloride (OSI-774) combined with carboplatin and paclitaxel chemotherapy in advanced non-small-cell lung cancer.
      ]. These trials have demonstrated similar response rates between the K-RAS mutant and K-RAS wild-type NSCLC. However, data suggest that K-RAS mutations may be a negative predictor of response to EGFR tyrosine kinase inhibitors in the minority of patients with concomitant K-RAS and sensitizing EGFR mutations [
      • Zhu C.Q.
      • da Cunha Santos G
      • Ding K.
      • Sakurada A.
      • Cutz J.C.
      • Liu N.
      • et al.
      Role of KRAS and EGFR as biomarkers of response to erlotinib in National Cancer Institute of Canada Clinical Trials Group Study BR.21..
      ,
      • Douillard J.Y.
      • Shepherd F.A.
      • Hirsh V.
      • Mok T.
      • Socinski M.A.
      • Gervais R.
      • et al.
      Molecular predictors of outcome with gefitinib and docetaxel in previously treated non-small-cell lung cancer: data from the randomized phase III INTEREST trial.
      ,
      • Schneider C.P.
      • Heigener D.
      • Schott-von-Romer K.
      • Gutz S.
      • Laack E.
      • Digel W.
      • et al.
      Epidermal growth factor receptor-related tumor markers and clinical outcomes with erlotinib in non-small cell lung cancer: an analysis of patients from german centers in the TRUST study.
      ]. The situation with immune checkpoint inhibitors is more complex. While there are conflicting individual studies on the outcome of K-RAS mutant NSCLC patients treated with PD-1/PD-L1 inhibitors, a recent meta-analysis of three prospective studies (CheckMate 057, POPLAR and OAK trial), demonstrated that patients with K-RAS mutant NSCLC had a superior survival compared to K-RAS wild type patients [
      • 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.
      ]. However, subset analyses suggest that patients with concomitant STK11/LKB1 gene alterations may be less responsive to PD-1/PD-L1 inhibitors [
      • 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.
      ].
      For colorectal cancer, K-RAS mutations are a major predictor of lack of response to therapy with monoclonal antibodies targeting EGFR (panitumumab, cetuximab) [
      • Amado R.G.
      • Wolf M.
      • Peeters M.
      • Van Cutsem E.
      • Siena S.
      • Freeman D.J.
      • et al.
      Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer.
      ,
      • De Roock W.
      • Piessevaux H.
      • De Schutter J.
      • Janssens M.
      • De Hertogh G.
      • Personeni N.
      • et al.
      KRAS wild-type state predicts survival and is associated to early radiological response in metastatic colorectal cancer treated with cetuximab.
      ,
      • Karapetis C.S.
      • Khambata-Ford S.
      • Jonker D.J.
      • O'Callaghan C.J.
      • Tu D.
      • Tebbutt N.C.
      • et al.
      K-ras mutations and benefit from cetuximab in advanced colorectal cancer.
      ,
      • Douillard J.Y.
      • Oliner K.S.
      • Siena S.
      • Tabernero J.
      • Burkes R.
      • Barugel M.
      • et al.
      Panitumumab-FOLFOX4 treatment and RAS mutations in colorectal cancer.
      ]. In addition, patients with K-RAS G13D mutations have an inferior response to chemotherapy as compared to those with other K-RAS mutation subtypes or those with K-RAS wild-type tumors [
      • Tejpar S.
      • Celik I.
      • Schlichting M.
      • Sartorius U.
      • Bokemeyer C.
      • Van Cutsem E.
      Association of KRAS G13D tumor mutations with outcome in patients with metastatic colorectal cancer treated with first-line chemotherapy with or without cetuximab.
      ,
      • De Roock W.
      • Jonker D.J.
      • Di Nicolantonio F.
      • Sartore-Bianchi A.
      • Tu D.
      • Siena S.
      • et al.
      Association of KRAS p.G13D mutation with outcome in patients with chemotherapy-refractory metastatic colorectal cancer treated with cetuximab.
      ]. There is a lack of data on the predictive value of K-RAS mutations on response to therapy as majority of pancreatic cancers harbor K-RAS mutations.

      Rationale for targeting Kras in cancer therapy

      Three factors support the hypothesis that Kras is a valid therapeutic target. First, K-RAS plays a distinct role in tumorigenesis. A study by Janseen and colleagues, in their transgenic mouse model, demonstrated that the transfection of oncogenic K-RAS V12G in epithelial cells of the large and small intestine led to the development of intestinal lesions including, invasive adenocarcinomas indicating a clear role of K-RAS in tumorigenesis [
      • Janssen K.P.
      • el-Marjou F.
      • Pinto D.
      • Sastre X.
      • Rouillard D.
      • Fouquet C.
      • et al.
      Targeted expression of oncogenic K-ras in intestinal epithelium causes spontaneous tumorigenesis in mice.
      ]. Additionally, various mouse models have demonstrated the formation of frank tumors with the activation of oncogenic K-RAS [
      • Caulin C.
      • Nguyen T.
      • Longley M.A.
      • Zhou Z.
      • Wang X.J.
      • Roop D.R.
      Inducible activation of oncogenic K-ras results in tumor formation in the oral cavity.
      ,
      • Vitale-Cross L.
      • Amornphimoltham P.
      • Fisher G.
      • Molinolo A.A.
      • Gutkind J.S.
      Conditional expression of K-ras in an epithelial compartment that includes the stem cells is sufficient to promote squamous cell carcinogenesis.
      ]. Second, K-RAS mutant cancer cells are K-RAS dependent. Preclinical abrogation of mutant K-RAS inhibits tumor growth. A study by Collins and colleagues in a mouse model demonstrated that both primary and metastatic pancreatic adenocarcinoma lesions rely on constant Kras activity [
      • Collins M.A.
      • Brisset J.C.
      • Zhang Y.
      • Bednar F.
      • Pierre J.
      • Heist K.A.
      • et al.
      Metastatic pancreatic cancer is dependent on oncogenic Kras in mice.
      ]. This notion is further supported by preclinical studies in different tumor types [
      • Fisher G.H.
      • Wellen S.L.
      • Klimstra D.
      • Lenczowski J.M.
      • Tichelaar J.W.
      • Lizak M.J.
      • et al.
      Induction and apoptotic regression of lung adenocarcinomas by regulation of a K-Ras transgene in the presence and absence of tumor suppressor genes.
      ,
      • Kwong L.N.
      • Costello J.C.
      • Liu H.
      • Jiang S.
      • Helms T.L.
      • Langsdorf A.E.
      • et al.
      Oncogenic NRAS signaling differentially regulates survival and proliferation in melanoma.
      ]. Third, K-RAS mutant cancers represent about 30% of all human cancers as previously mentioned. Taken together, these factors make a compelling argument for targeting Kras for cancer therapy.

      Historical approaches to Kras targeting for cancer therapy

      The most successful approach to inhibiting oncogenic kinases has been the development of inhibitors that compete with ATP binding to the kinase domain. Kras utilizes GTP rather than ATP as a phosphate donor for signaling. Because of the tight binding of GTP (a thousand-fold tighter than ATP) to Kras, this approach has not been feasible, based on current technology.
      Thus, a number of approaches have been utilized, as outlined below

      Direct inhibition of Kras

      Direct inhibition of Kras has been a goal of cancer drug development for several decades. SCH-53239 was the first small non-nucleotide molecule which was designed to prevent GDP to GTP nucleotide transition by binding with Ras protein and thereby preventing Ras activation [
      • Taveras A.G.
      • Remiszewski S.W.
      • Doll R.J.
      • Cesarz D.
      • Huang E.C.
      • Kirschmeier P.
      • et al.
      Ras oncoprotein inhibitors: the discovery of potent, ras nucleotide exchange inhibitors and the structural determination of a drug-protein complex.
      ]. Additionally, a water-soluble analog SCH-54292, which was able to bind to the switch II region of the Ras molecule, was developed [
      • Ganguly A.K.
      • Wang Y.S.
      • Pramanik B.N.
      • Doll R.J.
      • Snow M.E.
      • Taveras A.G.
      • et al.
      Interaction of a novel GDP exchange inhibitor with the Ras protein.
      ]. However, the development of these compounds was dropped because of lack of potency.

      Inhibition of RAS protein expression

      Antisense oligonucleotides

      Antisense oligonucleotides bind to their complimentary mRNAs at a specific strand and thereby inhibit mRNA translation and ultimately the protein synthesis [
      • Jansen B.
      • Zangemeister-Wittke U.
      Antisense therapy for cancer–the time of truth.
      ]. With this approach, a study by Gray and colleagues demonstrated a 90% reduction in Ras protein expression after targeting the 5′-flanking region of H-RAS in NIH-3T3 cells transformed by the H-RAS oncogene [
      • Gray G.D.
      • Hernandez O.M.
      • Hebel D.
      • Root M.
      • Pow-Sang J.M.
      • Wickstrom E.
      Antisense DNA inhibition of tumor growth induced by c-Ha-ras oncogene in nude mice.
      ]. ISIS 2503 is an oligonucleotide that targets the 5′-untranslated region of human H-RAS mRNA and thereby reduces mRNA expression [
      • Adjei A.A.
      • Dy G.K.
      • Erlichman C.
      • Reid J.M.
      • Sloan J.A.
      • Pitot H.C.
      • et al.
      A phase I trial of ISIS 2503, an antisense inhibitor of H-ras, in combination with gemcitabine in patients with advanced cancer.
      ]. Cunningham et al conducted a phase I study utilizing this compound in 23 patients with solid tumors [
      • Cunningham C.C.
      • Holmlund J.T.
      • Geary R.S.
      • Kwoh T.J.
      • Dorr A.
      • Johnston J.F.
      • et al.
      A Phase I trial of H-ras antisense oligonucleotide ISIS 2503 administered as a continuous intravenous infusion in patients with advanced carcinoma.
      ]. The compound was well tolerated. None of the patients achieved an objective response and only four patients (17%) had disease stability for 2 months or more. To evaluate the clinical activity of single agent ISIS 2503, a phase II study was conducted in sixteen patients with refractory colorectal cancer [
      • Marshall J.L.
      • Eisenberg S.G.
      • Johnson M.D.
      • Hanfelt J.
      • Dorr F.A.
      • El-Ashry D.
      • et al.
      A phase II trial of ISIS 3521 in patients with metastatic colorectal cancer.
      ]. None of the patients achieved an objective response. Only one patient achieved disease stability after two cycles of treatment. Additionally, phase I and II studies were also conducted with this compound in combination with gemcitabine. In a phase I study of 27 patients with advanced cancer, the combination of ISIS 2503 and gemcitabine was well tolerated; partial response was noted in a single patient and disease stability in 5 patients [
      • Adjei A.A.
      • Dy G.K.
      • Erlichman C.
      • Reid J.M.
      • Sloan J.A.
      • Pitot H.C.
      • et al.
      A phase I trial of ISIS 2503, an antisense inhibitor of H-ras, in combination with gemcitabine in patients with advanced cancer.
      ]. This combination was further evaluated in a phase II study in 48 patients with unresectable or metastatic pancreatic adenocarcinoma [
      • Alberts S.R.
      • Schroeder M.
      • Erlichman C.
      • Steen P.D.
      • Foster N.R.
      • Moore Jr, D.F.
      • et al.
      Gemcitabine and ISIS-2503 for patients with locally advanced or metastatic pancreatic adenocarcinoma: a North Central Cancer Treatment Group phase II trial.
      ]. At a median follow-up of 12.6 months, the study reported a 6-month survival rate of 57.5%, median survival of 6.6 months and a response rate of 10.4%. Further development of this agent was discontinued because of minimal efficacy.

      Inhibition of K-RAS processing

      Farnesyltransferase inhibitors

      Prenylation is a post-translational addition of either a farnesyl (farnesylation) or geranylgeranyl (geranylgeranylation) moiety to the carboxyl terminus of Ras proteins that help in membrane localization. This is a rate limiting step in the post-translational modification of Kras [
      • Haluska P.
      • Dy G.K.
      • Adjei A.A.
      Farnesyl transferase inhibitors as anticancer agents.
      ]. Farnesyltransferase inhibitors were expected to block Ras farnesylation, thus preventing membrane localization and inhibiting Ras-mediated cellular proliferation. Various trials, utilizing a variety of farnesyltransferase inhibitors, including tipifarnib (R115777), and lonafarnib (SCH 66336) were conducted in different tumor types [
      • Adjei A.A.
      • Mauer A.
      • Bruzek L.
      • Marks R.S.
      • Hillman S.
      • Geyer S.
      • et al.
      Phase II study of the farnesyl transferase inhibitor R115777 in patients with advanced non-small-cell lung cancer.
      ,
      • Sharma S.
      • Kemeny N.
      • Kelsen D.P.
      • Ilson D.
      • O'Reilly E.
      • Zaknoen S.
      • et al.
      A phase II trial of farnesyl protein transferase inhibitor SCH 66336, given by twice-daily oral administration, in patients with metastatic colorectal cancer refractory to 5-fluorouracil and irinotecan.
      ,
      • Winquist E.
      • Moore M.J.
      • Chi K.N.
      • Ernst D.S.
      • Hirte H.
      • North S.
      • et al.
      A multinomial Phase II study of lonafarnib (SCH 66336) in patients with refractory urothelial cancer.
      ,
      • Gajewski T.F.
      • Salama A.K.S.
      • Niedzwiecki D.
      • Johnson J.
      • Linette G.
      • Bucher C.
      • et al.
      Phase II study of the farnesyltransferase inhibitor R115777 in advanced melanoma (CALGB 500104).
      ]. A phase II trial of tipifarnib was conducted by Adjei et al in forty-four patients with stage IIIB or stage IV NSCLC [
      • Adjei A.A.
      • Mauer A.
      • Bruzek L.
      • Marks R.S.
      • Hillman S.
      • Geyer S.
      • et al.
      Phase II study of the farnesyl transferase inhibitor R115777 in patients with advanced non-small-cell lung cancer.
      ]. There were no objective responses, the disease stability rate was 16%, median time to progression was 2.7 months and median OS was 7.7 months. Additionally, various phase II studies of single agent tipifarnib failed to show any objective responses in various solid tumors [
      • Gajewski T.F.
      • Salama A.K.S.
      • Niedzwiecki D.
      • Johnson J.
      • Linette G.
      • Bucher C.
      • et al.
      Phase II study of the farnesyltransferase inhibitor R115777 in advanced melanoma (CALGB 500104).
      ,
      • Cohen S.J.
      • Ho L.
      • Ranganathan S.
      • Abbruzzese J.L.
      • Alpaugh R.K.
      • Beard M.
      • et al.
      Phase II and pharmacodynamic study of the farnesyltransferase inhibitor R115777 as initial therapy in patients with metastatic pancreatic adenocarcinoma.
      ]. Also, a phase III study of tipifarnib failed to improve survival over best supportive care in advanced colorectal cancer [
      • Rao S.
      • Cunningham D.
      • de Gramont A.
      • Scheithauer W.
      • Smakal M.
      • Humblet Y.
      • et al.
      Phase III double-blind placebo-controlled study of farnesyl transferase inhibitor R115777 in patients with refractory advanced colorectal cancer.
      ]. A phase III study of tipifarnib in patients with advanced pancreatic adenocarcinoma randomized 688 patients to receive gemcitabine and tipifarnib or gemcitabine and placebo [
      • Van Cutsem E.
      • van de Velde H.
      • Karasek P.
      • Oettle H.
      • Vervenne W.L.
      • Szawlowski A.
      • et al.
      Phase III trial of gemcitabine plus tipifarnib compared with gemcitabine plus placebo in advanced pancreatic cancer.
      ]. The study demonstrated no survival benefit with the addition of tipifarnib (median OS was 193 days and 182 days in the combination arm and gemcitabine arm respectively). In a phase III Intergroup study, 144 AML patients in remission were randomized to either receive tipifarnib or observation [
      • Luger S.X.V.
      • Paietta E.
      • et al.
      Tipifarnib as maintenance therapy in acute myeloid leukemia (AML) improves survival in a subgroup of patients with high risk disease. Results of the phase III intergroup trial E2902.
      ]. The study demonstrated no improvement in DFS with tipifarnib maintenance therapy.
      Likewise, phase II trials of lonafarnib conducted in metastatic colorectal cancer and urothelial cancer showed no objective responses [
      • Sharma S.
      • Kemeny N.
      • Kelsen D.P.
      • Ilson D.
      • O'Reilly E.
      • Zaknoen S.
      • et al.
      A phase II trial of farnesyl protein transferase inhibitor SCH 66336, given by twice-daily oral administration, in patients with metastatic colorectal cancer refractory to 5-fluorouracil and irinotecan.
      ,
      • Winquist E.
      • Moore M.J.
      • Chi K.N.
      • Ernst D.S.
      • Hirte H.
      • North S.
      • et al.
      A multinomial Phase II study of lonafarnib (SCH 66336) in patients with refractory urothelial cancer.
      ]. Additionally, a combination of lonafarnib and paclitaxel in a phase II trial of 33 patients with advanced NSCLC demonstrated a partial response of 10% and a disease stability rate of 38% [
      • Kim E.S.
      • Kies M.S.
      • Fossella F.V.
      • Glisson B.S.
      • Zaknoen S.
      • Statkevich P.
      • et al.
      Phase II study of the farnesyltransferase inhibitor lonafarnib with paclitaxel in patients with taxane-refractory/resistant nonsmall cell lung carcinoma.
      ]. Based on the results of this trial, a phase III trial of lonafarnib in combination with carboplatin-paclitaxel versus carboplatin-paclitaxel and placebo was initiated in patients with NSCLC (NCT00050336). But the study was terminated because of inadequate activity at interim analysis. These studies failed to demonstrate any benefit likely because Kras can be alternatively prenylated through geranylgeranylation [
      • Whyte D.B.
      • Kirschmeier P.
      • Hockenberry T.N.
      • Nunez-Oliva I.
      • James L.
      • Catino J.J.
      • et al.
      K- and N-Ras are geranylgeranylated in cells treated with farnesyl protein transferase inhibitors.
      ].
      Hras on the other hand is dependent solely on farnesylation for post translational modification, and the farnesyltransferase inhibitors will be expected to show activity. In support of this hypothesis, tipifarnib has recently being shown to have activity in patients with H-RAS mutant head and neck cancer, in a phase II study which demonstrated an overall response rate (ORR) of 56% and a median duration of response of 8.3 months [

      Ho A BI, Haddad R, et al. Preliminary results from a phase 2 trial of tipifarnib in Head and Neck Squamous Cell Cercinomas (HSCCs) with HRAS mutations. In: Presented at AACR-NCI-EORTC international conference on molecular targets and cancer; October 26-30, 2019; Boston, MA. Abstract 384.

      ].

      Geranylgeranyltransferase inhibitors

      Based on data indicating that Kras may be prenylated through geranylgeranylation, geranylgeranyltransferase inhibitors were evaluated in the clinic. GGTI-2418, a geranylgeranyl transferase inhibitor, was utilized in a phase I study, in 14 patients with advanced solid tumors [
      • Karasic T.B.
      • Chiorean E.G.
      • Sebti S.M.
      • O'Dwyer P.J.
      A phase I study of GGTI-2418 (geranylgeranyl transferase I inhibitor) in patients with advanced solid tumors.
      ]. The drug was well tolerated and no dose limiting toxicity was noted. However, no objective response was noted and the development of this class of agents was abandoned because of lack of efficacy.

      Targeting downstream RAS effectors

      RAF kinase inhibitors

      Because of the multiple unsuccessful attempts at direct inhibition, subsequent approaches focused on the inhibition of downstream signaling of Kras.
      Raf is the first protein that is phosphorylated by activated Ras in the mitogen-activated protein kinase pathway. Sorafenib (BAY 43-9006) was the first compound initially developed to specifically target Raf. It is currently approved for numerous cancers including hepatocellular carcinoma, gastrointestinal stromal tumor, renal cell carcinoma and thyroid cancer [
      • Llovet J.M.
      • Ricci S.
      • Mazzaferro V.
      • Hilgard P.
      • Gane E.
      • Blanc J.F.
      • et al.
      Sorafenib in advanced hepatocellular carcinoma.
      ,
      • Escudier B.
      • Eisen T.
      • Stadler W.M.
      • Szczylik C.
      • Oudard S.
      • Siebels M.
      • et al.
      Sorafenib in advanced clear-cell renal-cell carcinoma.
      ,
      • Brose M.S.
      • Nutting C.M.
      • Jarzab B.
      • Elisei R.
      • Siena S.
      • Bastholt L.
      • et al.
      Sorafenib in radioactive iodine-refractory, locally advanced or metastatic differentiated thyroid cancer: a randomised, double-blind, phase 3 trial.
      ]. It is, however, not a specific or potent Raf kinase inhibitor and its antitumor activity is due to inhibition of several other receptor tyrosine kinases [
      • Wilhelm S.M.
      • Carter C.
      • Tang L.
      • Wilkie D.
      • McNabola A.
      • Rong H.
      • et al.
      BAY 43–9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis.
      ]. For instance, in pancreatic cancer, where the prevalence of K-RAS mutation is high, sorafenib when used in combination with chemotherapy did not demonstrate any significant clinical activity [
      • Cascinu S.
      • Berardi R.
      • Sobrero A.
      • Bidoli P.
      • Labianca R.
      • Siena S.
      • et al.
      Sorafenib does not improve efficacy of chemotherapy in advanced pancreatic cancer: a GISCAD randomized phase II study.
      ,
      • Kindler H.L.
      • Wroblewski K.
      • Wallace J.A.
      • Hall M.J.
      • Locker G.
      • Nattam S.
      • et al.
      Gemcitabine plus sorafenib in patients with advanced pancreatic cancer: a phase II trial of the University of Chicago Phase II Consortium.
      ]. Subsequently, various potent inhibitors of B-Raf (dabrafenib, vemurafenib and encorafenib) were introduced and are now approved for numerous tumor types with a B-RAF mutation (particularly B-RAF V600E), including melanoma, NSCLC, anaplastic thyroid and colon cancer [
      • Hauschild A.
      • Grob J.J.
      • Demidov L.V.
      • Jouary T.
      • Gutzmer R.
      • Millward M.
      • et al.
      Dabrafenib in BRAF-mutated metastatic melanoma: a multicentre, open-label, phase 3 randomised controlled trial.
      ,
      • Subbiah V.
      • Kreitman R.J.
      • Wainberg Z.A.
      • Cho J.Y.
      • Schellens J.H.M.
      • Soria J.C.
      • et al.
      Dabrafenib and trametinib treatment in patients with locally advanced or metastatic BRAF V600-mutant anaplastic thyroid cancer.
      ,
      • Hyman D.M.
      • Puzanov I.
      • Subbiah V.
      • Faris J.E.
      • Chau I.
      • Blay J.Y.
      • et al.
      Vemurafenib in multiple nonmelanoma cancers with BRAF V600 mutations.
      ]. Mechanistically, these agents should be effective in K-RAS mutant cancers as Raf is downstream of Ras, and these agents are specific inhibitors of B-Raf unlike sorafenib. However, B-raf inhibition paradoxically activates ERK signaling in wild-type B-RAF cells [
      • Poulikakos P.I.
      • Zhang C.
      • Bollag G.
      • Shokat K.M.
      • Rosen N.
      RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF.
      ]. In K-RAS mutant cells, B-raf inhibition activates upstream proteins leading to ERK activation through an alternative pathway. One of the mechanisms by which this happens is through C-Raf activation [
      • Heidorn S.J.
      • Milagre C.
      • Whittaker S.
      • Nourry A.
      • Niculescu-Duvas I.
      • Dhomen N.
      • et al.
      Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
      ]. In support of these data, the C-Raf activation cascade has been noted in B-RAF wild type cells and not in B-RAF mutant cells [
      • Hatzivassiliou G.
      • Song K.
      • Yen I.
      • Brandhuber B.J.
      • Anderson D.J.
      • Alvarado R.
      • et al.
      RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth.
      ]. Currently, there are no active clinical trials utilizing single-agent B-RAF inhibitors in K-RAS mutant solid tumors.

      MEK inhibitors

      MEK inhibitors when used in conjunction with B-RAF inhibitors are superior to B-RAF inhibitors alone in B-RAF mutant cancers. Currently, three MEK inhibitors, trametinib, cobimetinib and binimetinib, are approved in combination with B-RAF inhibitors for patients with B-RAF mutant melanoma, NSCLC and colon cancer (along with cetuximab) [
      • Dummer R.
      • Ascierto P.A.
      • Gogas H.J.
      • Arance A.
      • Mandala M.
      • Liszkay G.
      • et al.
      Encorafenib plus binimetinib versus vemurafenib or encorafenib in patients with BRAF-mutant melanoma (COLUMBUS): a multicentre, open-label, randomised phase 3 trial.
      ,
      • Van Cutsem E.
      • Huijberts S.
      • Grothey A.
      • Yaeger R.
      • Cuyle P.J.
      • Elez E.
      • et al.
      Binimetinib, encorafenib, and cetuximab triplet therapy for patients with BRAF V600E-mutant metastatic colorectal cancer: safety lead-in results from the phase III BEACON colorectal cancer study.
      ,
      • Planchard D.
      • Besse B.
      • Groen H.J.M.
      • Souquet P.J.
      • Quoix E.
      • Baik C.S.
      • et al.
      Dabrafenib plus trametinib in patients with previously treated BRAF(V600E)-mutant metastatic non-small cell lung cancer: an open-label, multicentre phase 2 trial.
      ].
      Single agent MEK inhibition has shown disappointing results in various tumor types. A phase II study, of an oral MEK inhibitor (CI-1040), demonstrated minimal efficacy with no complete and partial responses among 67 patients with NSCLC, breast, colon and pancreatic cancer [
      • Rinehart J.
      • Adjei A.A.
      • Lorusso P.M.
      • Waterhouse D.
      • Hecht J.R.
      • Natale R.B.
      • et al.
      Multicenter phase II study of the oral MEK inhibitor, CI-1040, in patients with advanced non-small-cell lung, breast, colon, and pancreatic cancer.
      ]. In another phase II trial, 84 patients with NSCLC, who had received one or two prior lines of treatment, were randomized to either receive pemetrexed or selumetinib (AZD6244, a MEK inhibitor) [
      • Hainsworth J.D.
      • Cebotaru C.L.
      • Kanarev V.
      • Ciuleanu T.E.
      • Damyanov D.
      • Stella P.
      • et al.
      A phase II, open-label, randomized study to assess the efficacy and safety of AZD6244 (ARRY-142886) versus pemetrexed in patients with non-small cell lung cancer who have failed one or two prior chemotherapeutic regimens.
      ]. The study demonstrated no significant difference in progression-free survival between the two arms.
      Inhibition of MEK induces PI3K activation leading to activation of EGFR [
      • Mirzoeva O.K.
      • Collisson E.A.
      • Schaefer P.M.
      • Hann B.
      • Hom Y.K.
      • Ko A.H.
      • et al.
      Subtype-specific MEK-PI3 kinase feedback as a therapeutic target in pancreatic adenocarcinoma.
      ]. Based on these data, various trials have utilized MEK inhibitors in combination with EGFR inhibitors. Phase II studies of selumetinib combined with erlotinib failed to show activity in previously treated NSCLC [
      • Carter C.A.
      • Rajan A.
      • Keen C.
      • Szabo E.
      • Khozin S.
      • Thomas A.
      • et al.
      Selumetinib with and without erlotinib in KRAS mutant and KRAS wild-type advanced nonsmall-cell lung cancer.
      ] and pancreatic carcinoma [
      • Ko A.H.
      • Bekaii-Saab T.
      • Van Ziffle J.
      • Mirzoeva O.M.
      • Joseph N.M.
      • Talasaz A.
      • et al.
      A Multicenter, open-label phase II clinical trial of combined MEK plus EGFR inhibition for chemotherapy-refractory advanced pancreatic adenocarcinoma.
      ].
      The combination of MEK inhibitors and chemotherapy has also been evaluated for K-RAS mutant NSCLC. A number of phase II studies were performed [
      • Gandara D.R.
      • Leighl N.
      • Delord J.P.
      • Barlesi F.
      • Bennouna J.
      • Zalcman G.
      • et al.
      A phase 1/1b study evaluating trametinib plus docetaxel or pemetrexed in patients with advanced non-small cell lung cancer.
      ,
      • Kawaguchi K.
      • Igarashi K.
      • Miyake K.
      • Lwin T.M.
      • Miyake M.
      • Kiyuna T.
      • et al.
      MEK inhibitor trametinib in combination with gemcitabine regresses a patient-derived orthotopic xenograft (PDOX) pancreatic cancer nude mouse model.
      ,
      • Kawaguchi K.
      • Igarashi K.
      • Murakami T.
      • Kiyuna T.
      • Lwin T.M.
      • Hwang H.K.
      • et al.
      MEK inhibitors cobimetinib and trametinib, regressed a gemcitabine-resistant pancreatic-cancer patient-derived orthotopic xenograft (PDOX).
      ,
      • Infante J.R.
      • Somer B.G.
      • Park J.O.
      • Li C.P.
      • Scheulen M.E.
      • Kasubhai S.M.
      • et al.
      A randomised, double-blind, placebo-controlled trial of trametinib, an oral MEK inhibitor, in combination with gemcitabine for patients with untreated metastatic adenocarcinoma of the pancreas.
      ] culminating in the phase III SELECT-1 trial which randomized patients with K-RAS mutant advanced NSCLC with disease progression after first-line treatment to either selumetinib plus docetaxel or placebo plus docetaxel [
      • Janne P.A.
      • van den Heuvel M.M.
      • Barlesi F.
      • Cobo M.
      • Mazieres J.
      • Crino L.
      • et al.
      Selumetinib plus docetaxel compared with docetaxel alone and progression-free survival in patients with KRAS-mutant advanced non-small cell lung cancer: the SELECT-1 randomized clinical trial.
      ]. Median PFS was 3.9 months with selumetinib plus docetaxel therapy versus 2.8 months in placebo plus docetaxel (HR, 0.93 [95% CI 0.77–1.12]; P = 0.44). In summary, MEK inhibition either alone or in combination is not an effective therapy for K-RAS mutant cancers.

      ERK inhibitors

      Since ERK is the final downstream kinase of the MAP Kinase pathway, it has been hypothesized that ERK inhibition may be effective in K-RAS mutant tumors. This hypothesis is supported by preclinical data [
      • Bhagwat S.V.
      • McMillen W.T.
      • Cai S.
      • Zhao B.
      • Whitesell M.
      • Shen W.
      • et al.
      ERK inhibitor LY3214996 targets ERK pathway-driven cancers: a therapeutic approach toward precision medicine.
      ]. Various ERK inhibitors, including, LY3214496, BVD-523, MK-8353 and KO-947, are in early phase of clinical development either alone or in combination (NCT02857270, NCT-01781429, NCT02972034, NCT03745989, NCT03051035). The dose escalation portion of a phase I trial of LY3214496 in patients with K-RAS, N-RAS or B-RAF mutant advanced or metastatic cancer has been reported [

      Pant S, Bendell JC, Sullivan RJ, Shapiro G, Millward M, Mi G, et al. A phase I dose escalation (DE) study of ERK inhibitor, LY3214996, in advanced (adv) cancer (CA) patients (pts). 2019;37(15_suppl):3001.

      ]. No concerning toxicities were noted. The study is now in the second phase, where LY3214496 is utilized either alone or in combination with abemaciclib or nab-paclitaxel plus gemcitabine in various tumor types (NCT02857270).

      CDK4/6 inhibitors

      Abemaciclib is a cyclin-dependent kinase (CDK 4/6) inhibitor currently approved in combination with hormonal therapy for patients with advanced or metastatic hormone receptor positive and HER- negative breast cancer. In a mouse tumor model that recapitulates human NSCLC, Puyol and colleagues demonstrated that CDK4 inhibition can induce selective death of K-RAS mutant cancer cells [
      • Puyol M.
      • Martin A.
      • Dubus P.
      • Mulero F.
      • Pizcueta P.
      • Khan G.
      • et al.
      A synthetic lethal interaction between K-Ras oncogenes and Cdk4 unveils a therapeutic strategy for non-small cell lung carcinoma.
      ]. In a phase III open-label trial, patients with stage IV, K-RAS mutant NSCLC after having disease progression on platinum-doublet were randomized to abemaciclib or erlotinib. ORR was 8.9% in the abemaciclib arm versus 2.7% in the erlotinib arm (P = 0.01) [
      • Goldman J.W.
      • Mazieres J.
      • Barlesi F.
      • Koczywas M.
      • Dragnev K.H.
      • Göksel T.
      • et al.
      A randomized phase 3 study of abemaciclib versus erlotinib in previously treated patients with stage IV NSCLC with KRAS mutation.
      ]. Likewise, median PFS was 3.6 months with abemaciclib versus 1.9 months with erlotinib (HR, 0.58; 95% CI 0.47–0.72). However, despite having a better response rate and PFS, abemaciclib did not improve overall survival (median OS with abemaciclib was 7.4 months versus 7.8 months with erlotinib).
      The results of the above mentioned studies are summarized in Table 2 [
      • Cascinu S.
      • Berardi R.
      • Sobrero A.
      • Bidoli P.
      • Labianca R.
      • Siena S.
      • et al.
      Sorafenib does not improve efficacy of chemotherapy in advanced pancreatic cancer: a GISCAD randomized phase II study.
      ,
      • Kindler H.L.
      • Wroblewski K.
      • Wallace J.A.
      • Hall M.J.
      • Locker G.
      • Nattam S.
      • et al.
      Gemcitabine plus sorafenib in patients with advanced pancreatic cancer: a phase II trial of the University of Chicago Phase II Consortium.
      ,
      • Rinehart J.
      • Adjei A.A.
      • Lorusso P.M.
      • Waterhouse D.
      • Hecht J.R.
      • Natale R.B.
      • et al.
      Multicenter phase II study of the oral MEK inhibitor, CI-1040, in patients with advanced non-small-cell lung, breast, colon, and pancreatic cancer.
      ,
      • Hainsworth J.D.
      • Cebotaru C.L.
      • Kanarev V.
      • Ciuleanu T.E.
      • Damyanov D.
      • Stella P.
      • et al.
      A phase II, open-label, randomized study to assess the efficacy and safety of AZD6244 (ARRY-142886) versus pemetrexed in patients with non-small cell lung cancer who have failed one or two prior chemotherapeutic regimens.
      ,
      • Carter C.A.
      • Rajan A.
      • Keen C.
      • Szabo E.
      • Khozin S.
      • Thomas A.
      • et al.
      Selumetinib with and without erlotinib in KRAS mutant and KRAS wild-type advanced nonsmall-cell lung cancer.
      ,
      • Ko A.H.
      • Bekaii-Saab T.
      • Van Ziffle J.
      • Mirzoeva O.M.
      • Joseph N.M.
      • Talasaz A.
      • et al.
      A Multicenter, open-label phase II clinical trial of combined MEK plus EGFR inhibition for chemotherapy-refractory advanced pancreatic adenocarcinoma.
      ,
      • Infante J.R.
      • Somer B.G.
      • Park J.O.
      • Li C.P.
      • Scheulen M.E.
      • Kasubhai S.M.
      • et al.
      A randomised, double-blind, placebo-controlled trial of trametinib, an oral MEK inhibitor, in combination with gemcitabine for patients with untreated metastatic adenocarcinoma of the pancreas.
      ,
      • Janne P.A.
      • van den Heuvel M.M.
      • Barlesi F.
      • Cobo M.
      • Mazieres J.
      • Crino L.
      • et al.
      Selumetinib plus docetaxel compared with docetaxel alone and progression-free survival in patients with KRAS-mutant advanced non-small cell lung cancer: the SELECT-1 randomized clinical trial.
      ,
      • Goldman J.W.
      • Mazieres J.
      • Barlesi F.
      • Koczywas M.
      • Dragnev K.H.
      • Göksel T.
      • et al.
      A randomized phase 3 study of abemaciclib versus erlotinib in previously treated patients with stage IV NSCLC with KRAS mutation.
      ,
      • Blumenschein Jr., G.R.
      • Smit E.F.
      • Planchard D.
      • Kim D.W.
      • Cadranel J.
      • De Pas T.
      • et al.
      A randomized phase II study of the MEK1/MEK2 inhibitor trametinib (GSK1120212) compared with docetaxel in KRAS-mutant advanced non-small-cell lung cancer (NSCLC)†.
      ]. In addition, various ongoing trials that have targeted downstream Kras effectors or utilized indirect Kras inhibition approaches are summarized in table 3.
      Table 2Completed phase II or phase III studies of downstream Kras signaling inhibition in tumors with high prevalence of K-RAS mutations.
      StudyTumor typeStudy design/PhaseSample SizeInterventionORR (%)Median PFS (months)Median OS (months)
      GISCAD study Cascinu et al (151)Pancreatic adenocarcinomaOpen-label

      Randomized

      Phase II
      114Gemcitabine/cisplatin plus sorafenib versus Gemcitabine/cisplatin3.4 vs. 3.64.3 vs. 4.5

      7.5 vs. 8.3
      Kindler et al (152)Pancreatic adenocarcinomaOpen-label

      Single arm

      Phase II
      17Gemcitabine plus sorafenib03.24.0
      Rinehart et al (162)CRC, NSCLC, breast, pancreatic cancerOpen-label

      Single arm

      Phase II
      67CI-104004.4*NRe
      Hainsworth et al (163)NSCLCOpen-label

      Randomized

      Phase II
      84Selumetinib (AZD6244) versus pemetrexed5 vs. 52.2 vs. 3.0NRe
      Carter et al (165)NSCLCRandomized

      Phase II
      79K-RAS wild-type: erlotinib versus erlotinib plus selumetinib5 vs. 122.4 vs. 2.16.3 vs. 12.9
      K-RAS mutant: selumetinib versus selumetinib plus erlotinib0 vs. 104.0 vs. 2.310.5 vs. 21.8
      Blumenschein et al (176)NSCLC

      K-RAS, NRAS, BRAF, or MEK1 mutant
      Open-label

      Randomized

      Phase II
      134Trametinib versus docetaxel12 vs. 122.8 vs. 2.68 vs. NR
      SELECT-1

      Janne et al (171)
      NSCLC

      K-RAS mutant
      Randomized

      Double Blind

      Phase III
      510Selumetinib plus docetaxel versus placebo plus docetaxel20.1 vs. 13.73.9 vs. 2.88.7 vs. 7.9
      Ko et al (166)PDACNon-randomized

      Single arm

      Phase II
      46Selumetinib plus erlotinib01.97.3
      Infante et al (170)Pancreatic adenocarcinomaDouble-blind

      Randomized

      Phase II
      160Trametinib plus gemcitabine versus placebo plus gemcitabine22 vs. 183.8 vs. 3.58.4 vs. 6.7
      JUNIPER

      Goldman et al (175)
      NSCLC

      K-RAS mutant
      Open-label

      Randomized

      Phase III
      453Abemaciclib versus erlotinib8.9 vs. 2.73.6 vs. 1.97.4 vs. 7.8
      ORR, Objective Response Rate; PFS, Progression Free Survival; OS, Overall Survival; CRC, colorectal carcinoma; NSCLC, non-small cell lung cancer; NRe, not reported; * median duration of stable disease; NR, Not Reached; PDAC, pancreatic ductal adenocarcinoma.
      Table 3Ongoing trials targeting Kras downstream signaling in K-RAS mutant solid tumors.
      Site of actionStudy/Clinical Trial IdentifierTumor TypeMutation profilePhase/Study DesignInterventionRecruitment statusEstimated Enrollment
      PI3KNCT04073680Solid tumorPIK3CA or K-RASIb/II

      Open-label
      Serabelisib and CanagliflozinNot yet recruiting60
      RAFNCT02974725NSCLC, melanomaK-RAS or BRAF (NSCLC), N-RAS (melanoma)Ib

      Open-label
      LXH254 with LTT462 or trametinib or ribociclibRecruiting195
      RAFNCT04249843Solid tumorB-RAF, K-RAS, N-RASIa/Ib

      Open-label
      BGB-3245Recruiting69
      RAF

      MEK
      NCT03681483NSCLCK-RASI

      Open-label
      RO5126766Recruiting31
      MEKNCT03704688NSCLCK-RASI

      Open-label
      Trametinib and ponatinibRecruiting37
      MEKNCT04132505Pancreatic adenocarcinomaK-RASI

      Open-label
      Binimetinib and hydroxychloroquineRecruiting39
      MEKNCT03299088NSCLCK-RASIb

      Open-label
      Trametinib and pembrolizumabRecruiting42
      MEKNCT02613650ColorectalH-RAS, N-RAS or K-RASIb

      Open-label
      MEK162 and mFOLFIRIRecruiting30
      MEKNCT03981614ColorectalK-RAS or N-RASII

      Randomized

      Open-label
      Binimetinib plus palbociclib versus TAS-102 (trifluridine and tipiracil)Recruiting112
      MEKSELECT-1 NCT01933932NSCLCK-RASIII

      Randomized

      Double-blind
      Selumetinib plus docetaxel versus docetaxel plus placeboActive, not recruiting510
      MEKM14LTK

      NCT02230553
      NSCLCK-RAS mutant and PIK3CA wild-typeI/II

      Open-label
      Trametinib and lapatinibRecruiting30
      MEKNCT03990077NSCLCK-RASI

      Open-label
      HL-085 and docetaxelNot yet recruiting27
      MEKM14AFS

      NCT02450656
      NSCLCK-RAS mutant and PIK3CA wild-typeI/II

      Randomized

      Open-label
      Selumetinib plus afatinib versus docetaxelRecruiting320
      MEKMEKiAUTO

      NCT04214418
      Pancreatic adenocarcinoma

      colorectal
      K-RASI/II

      Open-label
      Cobimetinib, hydroxychloroquine and atezolizumabNot yet recruiting175
      MEKNCT03170206NSCLCK-RASI/II

      Open-label
      Binimetinib and palbociclibRecruiting72
      MEKNCT02079740Solid tumorK-RAS or N-RASIb/II

      Open-label
      Trametinib and navitoclaxRecruiting130
      MEKNCT03637491Solid tumorK-RAS or N-RASIb/II

      Open-label

      Randomized
      Binimetinib and avelumab or binimetinib, avelumab and talazoparib or binimetinib and talazoparibRecruiting122
      ERKNCT02857270Melanoma, NSCLC, colorectal, pancreatic adenocarcinomaBRAF or RAS NSCLC, BRAF or NRAS melanoma, BRAF colorectalI

      Open-label
      LY3214996 alone and in combination with abemaciclib, nab-paclitaxel and gemcitabine or cetuximab and encorafenibRecruiting272
      ERKNCT04145297GI malignancyKRAS, NRAS, HRAS, BRAF non-V600, MEK, ERKI

      Open-label
      Ulixertinib (BVD-523) and hydroxychloroquineRecruiting12
      ERKNCT03051035Solid tumorBRAF, KRAS, NRAS or HRAS in non-squamous histologyI

      Open-label
      KO-947 aloneActive but not recruiting100
      ERKNCT03415126Solid tumorBRAF mutant melanoma, N-RAS or H-RAS mutant solid tumors, K-RAS mutant CRC, K-RAS mutant NSCLCI

      Open-label
      ASN007Active but not recruiting49
      mTORNCT03520842Non-squamous NSCLCK-RASII

      Open-label
      Regorafenib and methotrexateRecruiting18
      NSCLC, non-small cell lung cancer.

      Covalent Kras G12C inhibitors

      Kras has been considered “undruggable” despite decades of extensive attempts to develop an effective anti-Ras therapy, as described above. Recent studies have identified small molecules that can selectively target and inactivate the K-RAS G12C mutant variant [
      • 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.
      ,
      • 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.
      ,
      • Lito P.
      • Solomon M.
      • Li L.S.
      • Hansen R.
      • Rosen N.
      Allele-specific inhibitors inactivate mutant KRAS G12C by a trapping mechanism.
      ,
      • Janes M.R.
      • Zhang J.
      • Li L.S.
      • Hansen R.
      • Peters U.
      • Guo X.
      • et al.
      Targeting KRAS mutant cancers with a covalent G12C-specific inhibitor.
      ,
      • Canon J.
      • Rex K.
      • Saiki A.Y.
      • Mohr C.
      • Cooke K.
      • Bagal D.
      • et al.
      The clinical KRAS(G12C) inhibitor AMG 510 drives anti-tumour immunity.
      ,
      • Hallin J.
      • Engstrom L.D.
      • Hargis L.
      • Calinisan A.
      • Aranda R.
      • Briere D.M.
      • et al.
      The KRAS(G12C) inhibitor MRTX849 provides insight toward therapeutic susceptibility of KRAS-mutant cancers in mouse models and patients.
      ]. K-RAS G12C results from a missense mutation (glycine-to-cysteine substitution) at codon 12. This leads to impairment of GAP mediated hydrolysis of GTP to GDP, thereby locking the Kras protein in a hyperexcitable state.
      K-RAS G12C is the most common mutant variant in NSCLC accounting for about 40% of all K-RAS mutant tumors and about 13% of all lung adenocarcinoma [
      • 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.
      ,
      • Biernacka A.
      • Tsongalis P.D.
      • Peterson J.D.
      • de Abreu F.B.
      • Black C.C.
      • Gutmann E.J.
      • et al.
      The potential utility of re-mining results of somatic mutation testing: KRAS status in lung adenocarcinoma.
      ,
      • Bar-Sagi D.
      • Knelson E.H.
      • Sequist L.V.
      A bright future for KRAS inhibitors.
      ]. Additionally, it is present in about 3% of colorectal cancer cases and a small subset of patients with pancreatic, endometrial and urothelial cancers [
      • Cox A.D.
      • Fesik S.W.
      • Kimmelman A.C.
      • Luo J.
      • Der C.J.
      Drugging the undruggable RAS: mission possible?.
      ,
      • Neumann J.
      • Zeindl-Eberhart E.
      • Kirchner T.
      • Jung A.
      Frequency and type of KRAS mutations in routine diagnostic analysis of metastatic colorectal cancer.
      ]. The frequency of different K-RAS mutations in various tumor types is shown in Fig. 3 [
      • Miller M.S.
      • Miller L.D.
      RAS mutations and oncogenesis: not all RAS mutations are created equally.
      ].
      Figure thumbnail gr3
      Fig. 3K-RAS mutation frequency in different tumor types.
      Ostrem and colleagues developed a series of compounds that could target the mutant Kras G12C protein by covalently binding to the mutant cysteine residue [
      • 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.
      ]. With this approach, they were able to selectively target the mutant cells and spare the normal ones. Additionally, they found that these inhibitors were binding to a new allosteric binding pocket, the switch-II pocket (S-IIP). This pocket extends from the mutant cysteine residue into a pocket comprising mainly of the switch II region. By targeting this specific pocket, these compounds displace glycine 60 towards the switch I region leading to conformational disruption of GTP bound Ras and thereby preventing further downstream signaling. However, the initial lead compound developed by Ostrem and colleagues (compound 12) had poor pharmacologic properties [
      • 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.
      ,
      • Lito P.
      • Solomon M.
      • Li L.S.
      • Hansen R.
      • Rosen N.
      Allele-specific inhibitors inactivate mutant KRAS G12C by a trapping mechanism.
      ].
      Consequently, ARS853, which had more than 600-fold improved engagement with Kras G12C over compound 12, was discovered [
      • 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.
      ,
      • Lito P.
      • Solomon M.
      • Li L.S.
      • Hansen R.
      • Rosen N.
      Allele-specific inhibitors inactivate mutant KRAS G12C by a trapping mechanism.
      ]. Two studies, by Lito et al and Patricelli et al, demonstrated reduction in GTP-bound Kras levels in K-RAS G12C mutant cancer cell lines after treatment with ARS853 [
      • 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.
      ,
      • Lito P.
      • Solomon M.
      • Li L.S.
      • Hansen R.
      • Rosen N.
      Allele-specific inhibitors inactivate mutant KRAS G12C by a trapping mechanism.
      ]. Additionally, they also showed that ARS833 bound preferentially to the GDP-bound state of Kras G12C. Since this compound was selectively inhibiting GDP-bound Kras, there was a significant concern about its efficacy in vivo. Consequently, Janes and colleagues identified a compound, ARS-1260, which selectively targets the switch II pocket and also inhibits Kras in the GTP-bound state [
      • Janes M.R.
      • Zhang J.
      • Li L.S.
      • Hansen R.
      • Peters U.
      • Guo X.
      • et al.
      Targeting KRAS mutant cancers with a covalent G12C-specific inhibitor.
      ]. They demonstrated that ARS-1260 covalently inhibits Kras G12C activity in vitro, and exhibited antitumor activity in subcutaneous xenograft models bearing K-RAS G12C but not G12V mutations.
      Subsequently, Canon and colleagues demonstrated the activity of AMG 510 in K-RAS G12C mouse xenografts [
      • Canon J.
      • Rex K.
      • Saiki A.Y.
      • Mohr C.
      • Cooke K.
      • Bagal D.
      • et al.
      The clinical KRAS(G12C) inhibitor AMG 510 drives anti-tumour immunity.
      ]. Similar preclinical studies of another Kras G12C inhibitor, MRTX849 have been published [
      • Hallin J.
      • Engstrom L.D.
      • Hargis L.
      • Calinisan A.
      • Aranda R.
      • Briere D.M.
      • et al.
      The KRAS(G12C) inhibitor MRTX849 provides insight toward therapeutic susceptibility of KRAS-mutant cancers in mouse models and patients.
      ].
      A phase I study of AMG 510 was presented at the European Society for Medical Oncology (ESMO) annual meeting and at the World Lung Cancer Conference in 2019, by Govindan and colleagues [

      Govindan RFM, Price T, et al. Phase 1 study of safety, tolerability, PK, and efficacy of AMG 510, a novel KRAS G12C inhibitor, evaluated in NSCLC. In: Presented at the IASLC 2019 world lung cancer conference; September 7–10, 2019; Barcelona, Spain.

      ]. A total of 76 patients with K-RAS G12C mutant solid tumors were enrolled in the study. There was no dose limiting toxicity. Most of the patients (34.2%) had grade 1 or grade 2 treatment-related adverse events. 6 patients had grade 3 adverse events, which included anemia and diarrhea. The recommended phase II dose was 960 mg once daily. Among NSCLC cohort (n = 23), the ORR was 48% and disease control rate (DCR) was 96%. In the colorectal cancer cohort (n = 29), the ORR was 3% and the DCR was 79%. There are two other phase I trials (NCT03600883, NCT04185883), which are actively recruiting patients with K-RAS G12C mutant solid malignancies. These trials will also assess the safety and feasibility of various therapeutic agents in combination with AMG510, including a PD-1 inhibitor, MEK inhibitor, a SHP2 allosteric inhibitor, and a pan-ErbB tyrosine kinase inhibitor. Additionally, a phase III trial is scheduled to start accrual this summer in patients with previously treated locally advanced and unresectable or metastatic K-RAS G12C mutant NSCLC with randomization to AMG510 or docetaxel (NTC04303780). The clinical activity of AMG 510 in colorectal cancer is minimal to modest compared to the activity in NSCLC, suggesting that signaling networks in colorectal cancer are different from NSCLC. As an example, Braf inhibition in BRAF V600E mutation is much more effective in NSCLC compared to colorectal cancer, where bypass signaling in EGFR abrogates the effect of Braf inhibition. Thus, concurrent inhibition of EGFR is needed to achieve impressive responses after Braf inhibition. This same mechanism seems to be present in K-RAS G12C mutant colorectal cancer, as demonstrated by Amodio and colleagues [
      • Amodio V.
      • Yaeger R.
      • Arcella P.
      • Cancelliere C.
      • Lamba S.
      • Lorenzato A.
      • et al.
      EGFR blockade reverts resistance to KRAS G12C inhibition in colorectal cancer.
      ].
      Studies with another Kras G12C inhibitor are also ongoing. A phase 1/2 multiple expansion study of MRTX849 is currently accruing patients (NCT03785249). In this trial, patients with advanced, unresectable or metastatic solid tumors with a K-RAS G12C mutation will be enrolled to access the safety, pharmacokinetics, tolerability and clinical activity of MRTX849. This trial will also evaluate the safety of the combination of MRTX849 with other therapeutic agents, including, a PD-1 inhibitor in patients with NSCLC and cetuximab in patients with colorectal cancer. Another phase 1/2 study will be opening in the near future utilizing a combination of MRTX849 and TNO155 in patients with KRAS G12C mutant cancer (NCT04330664). TNO155 is a SHP2 inhibitor and will be discussed in detail below. Two other K-RAS G12C inhibitors, ARS-3248/JNJ-74699157, and LY3499446 are under investigation (NCT04006301, NCT04165031). Table 4 summarizes all the active trials in K-RAS mutant solid tumors which utilize novel direct inhibitors of Kras.
      Table 4Ongoing clinical trials of novel Kras inhibitors in K-RAS mutant solid tumors.
      Site of actionStudy/Clinical Trial IdentifierTumor TypeMutation profilePhase/Study DesignInterventionRecruitment statusEstimated Enrollment
      K-RAS G12CCodeBreak 101

      NCT04185883
      Solid tumorK-RAS

      G12C
      Ib

      Open-label
      AMG 510 alone and in combination with

      MEK, PD-1, SHP2 or ErbB inhibitor
      Recruiting250
      K-RAS G12CCodeBreak

      200

      NCT04303780
      NSCLCK-RAS G12CIII

      Randomized

      Open-label
      AMG 510 plus Docetaxel versus DocetaxelNot yet recruiting650
      K-RAS G12CCodeBreak 100

      NCT04303780
      Solid tumorK-RAS G12CI/II

      Open-label
      AMG 510 alone and in combination with PD-1 inhibitorRecruiting533
      K-RAS

      G12C
      NCT03785249Solid tumorK-RAS G12CI/II

      Open-label
      MRTX849 aloneRecruiting200
      K-RAS G12CNCT04330664Solid tumorK-RAS G12CI/II

      Open-label
      MRTX849 in combination with TNO155Recruiting148
      K-RAS G12CNCT04165031Solid tumorK-RAS

      G12C
      I/II

      Open-label
      LY3499446 alone or in combination with abemaciclib, cetuximab, erlotinib, docetaxelRecruiting230
      K-RAS G12CNCT04006301Solid TumorK-RAS

      G12C
      I

      Open-label
      JNJ-74699157 aloneRecruiting140
      SOS1NCT04111458Solid tumorK-RASI

      Open-label
      BI 1,701,963 alone and in combination with trametinibRecruiting140
      SHP2NCT03634982Solid tumorRTK, K-RAS G12, BRAF class 3, NF1 LOFI

      Open-label
      RMC-4630 aloneRecruiting240
      SHP2NCT03989115Solid tumorK-RAS, BRAF class 3, NF1 LOFIb/II

      Open-label
      RMC-4630 in combination with cobimetinibRecruiting144
      SHP2NCT03114319Solid tumorEGFR NSCLC, K-RAS G12C NSCLC, CRC, Esophageal SCC, HN SCC, RAS/RAF wild-type other solid tumorI

      Open-label
      TNO155 aloneRecruiting135
      SHP2NCT04000529NSCLC, CRC, HNSCCwild-type (dose escalation);

      K-RAS G12C NSCLC, K-RAS wild-type NSCLC, K-RAS codon 12, 13, or 61 for CRC (dose escalation)
      Ib

      Open-label
      TNO155 in combination with spartalizumab or ribociclibRecruiting126
      mRNA based vaccineNCT03948763NSCLC, CRC, pancreatic adenocarcinomaK-RAS G12D, G12V, G13D, G12CI

      Open-label
      mRNA-5671/V941 alone and in combination with pembrolizumabRecruiting100
      eIF4NCT04092673Solid tumorHER2, ERBB3, FGFR1, FGFR2, K-RASI/II

      Open-label
      eFT226 (Zotatifin) aloneRecruiting45
      CRC, colorectal carcinoma; NSCLC, non-small cell lung cancer.

      PAN K-RAS inhibitors

      SOS1 inhibitors

      BI-3406 is an orally bioavailable drug designed to inhibit the son of sevenless 1 (SOS1) protein. Hofmann and colleagues have demonstrated that this Boehringer-Ingelheim drug only inhibits SOS1, and not SOS2 [

      Hofmann MHGM, Ramharter J, et al. Discovery of BI-3406: a potent and selective SOS1:: KRAS inhibitor opens a new approach for treating KRAS-driven tumors. In: Presented at AACR-NCI-EORTC international conference on molecular targets and cancer therapeutics; October 26-30, 2019; Boston, MA. Abstract PL06-01.

      ]. They further demonstrated that in K-RAS-mutant cancer, including G12 and G13. By inhibiting SOS1, BI-3406 reduced GTP-KRAS levels thereby restricting tumor cell proliferation. BI 1701963, which is a BI-3406 analog, is in phase I trials, either alone or in combination with Trametinib in patients with K-RAS mutant solid tumors (NCT04111458).

      SHP2 inhibitors

      SHP2, a protein tyrosine phosphatase (PTPN11), relays stimulatory signals from various membrane receptor tyrosine kinases to the MAPK kinase signaling pathway [
      • Dance M.
      • Montagner A.
      • Salles J.P.
      • Yart A.
      • Raynal P.
      The molecular functions of Shp2 in the Ras/Mitogen-activated protein kinase (ERK1/2) pathway.
      ]. Chen and colleagues initially developed SHP099, a selective and orally bioavailable allosteric inhibitor of SHP2, and demonstrated its antitumor activity in receptor tyrosine kinase-driven cancers in patient derived tumor xenograft models [
      • Chen Y.N.
      • LaMarche M.J.
      • Chan H.M.
      • Fekkes P.
      • Garcia-Fortanet J.
      • Acker M.G.
      • et al.
      Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases.
      ]. Later, Mainardi and colleagues demonstrated an importance of SHP2 inhibition in controlling K-RAS mutant tumor growth by MEK inhibition [
      • Mainardi S.
      • Mulero-Sanchez A.
      • Prahallad A.
      • Germano G.
      • Bosma A.
      • Krimpenfort P.
      • et al.
      SHP2 is required for growth of KRAS-mutant non-small-cell lung cancer in vivo.
      ]. They demonstrated that MEK inhibition can reduce phosphorylated ERK in cell lines of three tumor types (NSCLC, pancreatic cancer and colon cancer). Furthermore, they found that ERK levels slowly started rising along with a rise in SHP2 levels suggesting the activation of a feedback loop involving receptor tyrosine kinase. Additionally, when they simultaneously blocked SHP2 and MEK, they found a strong synergy between a SHP2 inhibitor and a MEK inhibitor in all three cells lines, and the strongest effect was observed in NSCLC cell lines. In addition, they demonstrated that the PTPN11-knockout cells demonstrated lower baseline RAS-GTP levels and had an increased sensitivity to MEK inhibitor. Based on these preclinical data, it is reasonable to utilize SHP inhibitor in K-RAS mutant tumor. There are two SHP 2 inhibitors, namely, RMC 4630 and TNO155, in early phase of development, (NCT03634982, NCT03989115, NCT04000529, NCT03114319, NCT04330664).

      Transcription regulator elF4 inhibitors

      Protein synthesis is catalyzed by eukaryotic translational initiation factor 4 (eIF4) which is responsible for recruitment of the 5′-untranslated segment of the mRNA to the ribosomal subunit [
      • Hernandez G.
      • Vazquez-Pianzola P.
      Functional diversity of the eukaryotic translation initiation factors belonging to eIF4 families.
      ]. eIF4A, a component of eIF4 complex, is an ATPase/RNA helicases and its specific role in this process is mRNA unwinding to facilitate ribosome binding [
      • Hernandez G.
      • Vazquez-Pianzola P.
      Functional diversity of the eukaryotic translation initiation factors belonging to eIF4 families.
      ]. Since this protein complex is an essential component of the translation initiation of multiple oncogenic pathways, including K-RAS, targeting this protein in K-RAS mutant cancer cases can potentially control tumor growth and proliferation. eFT226 is a first in class selective inhibitor of eIF4A. Thompson and colleagues have demonstrated antitumor activity of this compound in a preclinical study in B-cell lymphoma where there is a PI3K/AKT/mTOR pathway aberrancy [

      Thompson PAEB, Young NP, et al. eFT226, a potent and selective inhibitor of eIF4A, is efficacious in preclinical models of lymphoma. In: Presented at AACR Annual Meeting 2019; March 29-April 3, 2019; Atlant, GA. Abstract 2698.

      ]. Additionally, Thompson and colleagues have demonstrated in vivo tumor growth inhibition in solid tumor xenograft models with FGFR1/2 or HER2 amplifications, including NSCLC, breast and colorectal cancers [

      Thompson PAYN, Stumpf CR, et al. eFT226, a first in class inhibitor of eIF4A1, targets FEGFR1/2 and HER2 driven cancers. In: Presented at AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; October 26–30, 2019; Boston, MA. Abstract B133.

      ]. A phase I trial of eFT 226 (zotatifin) is currently recruiting patients with HER2, ERBB3, FGFR1, FGFR2 and K-RAS mutant solid tumors (NCT 04092673). Various drugs targeting at different levels on Kras pathway is depicted in Fig. 4.
      Figure thumbnail gr4
      Fig. 4Various drugs targeting at different levels on Kras pathway.

      Other agents in late preclinical development

      Recently, mRNA-based vaccination is being utilized to investigate specific immune responses against cancer cells. Mutanome is a distinct set of somatic mutations unique to an individual’s tumor. As majority of these mutations are unique to each individual, Sahin et al investigated the concept of individualized mutanome vaccines by implementing an RNA-based neo-epitope approach in patients with stage III or IV melanoma [
      • Sahin U.
      • Derhovanessian E.
      • Miller M.
      • Kloke B.P.
      • Simon P.
      • Löwer M.
      • et al.
      Personalized RNA mutanome vaccines mobilize poly-specific therapeutic immunity against cancer.
      ]. After identifying non-synonymous mutations in 13 patients, an RNA vaccine was engineered encoding 10 selected mutations per patient, which was then injected intranodally. Following vaccination, all patients developed T-cell responses and two of the five patients with metastatic disease achieved an objective response. This study unlocked a novel path for a more personalized treatment and has drawn significant attention. A phase I trial of mRNA-5671/V941 (that encodes antigen for K-RAS G12D, G12V, G12C and G13D) as monotherapy and in combination with pembrolizumab in patients with solid tumors with four prevalent K-RAS mutations is currently underway (NCT03948763). The mRNA-5671/V941 vaccine is intended to target majority of the K-RAS mutations that occur in solid tumors.
      Additionally, a novel short inhibitory peptide, KRpep-2d, is recently identified using a T7 phage display technique. KRpep-2d is a 19-mer cyclic peptide which is able to non-covalently and selectively inhibit Kras G12D activity with high potency [
      • Sakamoto K.
      • Kamada Y.
      • Sameshima T.
      • Yaguchi M.
      • Niida A.
      • Sasaki S.
      • et al.
      K-Ras(G12D)-selective inhibitory peptides generated by random peptide T7 phage display technology.
      ,
      • Niida A.
      • Sasaki S.
      • Yonemori K.
      • Sameshima T.
      • Yaguchi M.
      • Asami T.
      • et al.
      Investigation of the structural requirements of K-Ras(G12D) selective inhibitory peptide KRpep-2d using alanine scans and cysteine bridging.
      ]. It acts as an allosteric inhibitor by binding near the switch II pocket [
      • Sogabe S.
      • Kamada Y.
      • Miwa M.
      • Niida A.
      • Sameshima T.
      • Kamaura M.
      • et al.
      Crystal structure of a human K-ras G12D mutant in complex with GDP and the cyclic inhibitory peptide KRpep-2d.
      ]. This molecule is still in its infancy but is likely to enter clinical trial in near future for K-RAS G12D mutant tumors.

      Conclusion

      While K-RAS has been seen as an attractive target for cancer therapy, all approaches taken to inhibit K-RAS either directly or indirectly through inhibiting post translational modification or downstream signaling to date have been ineffective. Advances in genomics and molecular biology, however, have for the first time suggested that direct inhibition of K-RAS may be possible. An identification of a targetable binding pocket (S-IIP) in K-RAS G12C recently resulted in a renewed interest in targeting K-RAS via G12C inhibition. The covalent Kras G12C inhibitors have provided the first clinical evidence of the ability to inhibit a class of K-RAS mutant tumors. This initial success has rekindled interest in Kras inhibition, with a number of other approaches including SHP2, SOS1 and eIF4 inhibition being tested in the clinic. These newer approaches, if successful would abrogate the activity of all mutant isoforms of K-RAS. Thus, we are for the first time at the cusp of successfully drugging this hitherto undruggable target.

      Declaration of Competing Interest

      The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

      References

        • Barbacid M.
        ras genes.
        Annu Rev Biochem. 1987; 56: 779-827
        • Harvey J.J.
        An unidentified virus which causes the rapid production of tumours in mice.
        Nature. 1964; 204: 1104-1105
        • Kirsten W.H.
        • Mayer L.A.
        Morphologic responses to a murine erythroblastosis virus.
        J Natl Cancer Inst. 1967; 39: 311-335
        • Santos E.
        • Martin-Zanca D.
        • Reddy E.P.
        • Pierotti M.A.
        • Della Porta G.
        • Barbacid M.
        Malignant activation of a K-ras oncogene in lung carcinoma but not in normal tissue of the same patient.
        Science. 1984; 223: 661-664
        • Fernandez-Medarde A.
        • Santos E.
        Ras in cancer and developmental diseases.
        Genes Cancer. 2011; 2: 344-358
        • Friday B.B.
        • Adjei A.A.
        K-ras as a target for cancer therapy.
        BBA. 2005; 1756: 127-144
        • Wang J.Y.
        • Lian S.T.
        • Chen Y.F.
        • Yang Y.C.
        • Chen L.T.
        • Lee K.T.
        • et al.
        Unique K-ras mutational pattern in pancreatic adenocarcinoma from Taiwanese patients.
        Cancer Lett. 2002; 180: 153-158
        • Shibata D.
        • Almoguera C.
        • Forrester K.
        • Dunitz J.
        • Martin S.E.
        • Cosgrove M.M.
        • et al.
        Detection of c-K-ras mutations in fine needle aspirates from human pancreatic adenocarcinomas.
        Cancer Res. 1990; 50: 1279-1283
        • Pao W.
        • Girard N.
        New driver mutations in non-small-cell lung cancer.
        Lancet Oncol. 2011; 12: 175-180
        • Mascaux C.
        • Iannino N.
        • Martin B.
        • Paesmans M.
        • Berghmans T.
        • Dusart M.
        • et al.
        The role of RAS oncogene in survival of patients with lung cancer: a systematic review of the literature with meta-analysis.
        Br J Cancer. 2005; 92: 131-139
        • Peeters M.
        • Kafatos G.
        • Taylor A.
        • Gastanaga V.M.
        • Oliner K.S.
        • Hechmati G.
        • Terwey J.-H.
        • van Krieken J.H.
        Prevalence of RAS mutations and individual variation patterns among patients with metastatic colorectal cancer: a pooled analysis of randomised controlled trials.
        Eur J Cancer. 2015; 51: 1704-1713https://doi.org/10.1016/j.ejca.2015.05.017
        • Munoz-Maldonado C.
        • Zimmer Y.
        • Medova M.
        A comparative analysis of individual RAS mutations in cancer biology.
        Front Oncol. 2019; 9: 1088
        • Jancik S.
        • Drabek J.
        • Radzioch D.
        • Hajduch M.
        Clinical relevance of KRAS in human cancers.
        J Biomed Biotechnol. 2010; 2010: 150960
        • McBride O.W.
        • Swan D.C.
        • Tronick S.R.
        • Gol R.
        • Klimanis D.
        • Moore D.E.
        • et al.
        Regional chromosomal localization of N-ras, K-ras-1, K-ras-2 and myb oncogenes in human cells.
        Nucleic Acids Res. 1983; 11: 8221-8236
        • Popescu N.C.
        • Amsbaugh S.C.
        • DiPaolo J.A.
        • Tronick S.R.
        • Aaronson S.A.
        • Swan D.C.
        Chromosomal localization of three human ras genes by in situ molecular hybridization.
        Somat Cell Mol Genet. 1985; 11: 149-155
        • Sameer A.S.
        Colorectal cancer: molecular mutations and polymorphisms.
        Front Oncol. 2013; 3: 114
        • Chen Z.
        • Otto J.C.
        • Bergo M.O.
        • Young S.G.
        • Casey P.J.
        The C-terminal polylysine region and methylation of K-Ras are critical for the interaction between K-Ras and microtubules.
        J Biol Chem. 2000; 275: 41251-41257
        • Vogler O.
        • Barcelo J.M.
        • Ribas C.
        • Escriba P.V.
        Membrane interactions of G proteins and other related proteins.
        BBA. 2008; 1778: 1640-1652
        • Lemmon M.A.
        • Schlessinger J.
        Regulation of signal transduction and signal diversity by receptor oligomerization.
        Trends Biochem Sci. 1994; 19: 459-463
        • Gao J.
        • Liao J.
        • Yang G.Y.
        CAAX-box protein, prenylation process and carcinogenesis.
        Am J Transl Res. 2009; 1: 312-325
        • Lowy D.R.
        • Willumsen B.M.
        Function and regulation of ras.
        Annu Rev Biochem. 1993; 62: 851-891
        • Adjei A.A.
        Blocking oncogenic Ras signaling for cancer therapy.
        J Natl Cancer Inst. 2001; 93: 1062-1074
        • Schlessinger J.
        Cell signaling by receptor tyrosine kinases.
        Cell. 2000; 103: 211-225
        • Liebmann C.
        Regulation of MAP kinase activity by peptide receptor signalling pathway: paradigms of multiplicity.
        Cell Signal. 2001; 13: 777-785
        • Heldin C.H.
        Dimerization of cell surface receptors in signal transduction.
        Cell. 1995; 80: 213-223
        • Iversen L.
        • Tu H.L.
        • Lin W.C.
        • Christensen S.M.
        • Abel S.M.
        • Iwig J.
        • et al.
        Molecular kinetics. Ras activation by SOS: allosteric regulation by altered fluctuation dynamics.
        Science. 2014; 345: 50-54
        • Margarit S.M.
        • Sondermann H.
        • Hall B.E.
        • Nagar B.
        • Hoelz A.
        • Pirruccello M.
        • et al.
        Structural evidence for feedback activation by Ras.GTP of the Ras-specific nucleotide exchange factor SOS.
        Cell. 2003; 112: 685-695
        • Sondermann H.
        • Soisson S.M.
        • Boykevisch S.
        • Yang S.S.
        • Bar-Sagi D.
        • Kuriyan J.
        Structural analysis of autoinhibition in the Ras activator Son of sevenless.
        Cell. 2004; 119: 393-405
        • Campbell P.M.
        • Der C.J.
        Oncogenic Ras and its role in tumor cell invasion and metastasis.
        Semin Cancer Biol. 2004; 14: 105-114
        • Drugan J.K.
        • Rogers-Graham K.
        • Gilmer T.
        • Campbell S.
        • Clark G.J.
        The Ras/p120 GTPase-activating protein (GAP) interaction is regulated by the p120 GAP pleckstrin homology domain.
        Jo Boil Chem. 2000; 275: 35021-35027
        • Pamonsinlapatham P.
        • Hadj-Slimane R.
        • Lepelletier Y.
        • Allain B.
        • Toccafondi M.
        • Garbay C.
        • et al.
        p120-Ras GTPase activating protein (RasGAP): a multi-interacting protein in downstream signaling.
        Biochimie. 2009; 91: 320-328
        • Maekawa M.
        • Li S.
        • Iwamatsu A.
        • Morishita T.
        • Yokota K.
        • Imai Y.
        • et al.
        A novel mammalian Ras GTPase-activating protein which has phospholipid-binding and Btk homology regions.
        Mol Cell Biol. 1994; 14: 6879-6885
      1. Jin H, Wang X, Ying J, Wong AH, Cui Y, Srivastava G, et al. Epigenetic silencing of a Ca(2+)-regulated Ras GTPase-activating protein RASAL defines a new mechanism of Ras activation in human cancers. In: Proceedings of the national academy of sciences of the United States of America. 2007;104(30):12353–8.

        • Kim J.H.
        • Liao D.
        • Lau L.F.
        • Huganir R.L.
        SynGAP: a synaptic RasGAP that associates with the PSD-95/SAP90 protein family.
        Neuron. 1998; 20: 683-691
        • Ledbetter D.H.
        • Rich D.C.
        • O'Connell P.
        • Leppert M.
        • Carey J.C.
        Precise localization of NF1 to 17q11.2 by balanced translocation.
        Am J Hum Genet. 1989; 44: 20-24
        • Tucker T.
        • Riccardi V.M.
        • Sutcliffe M.
        • Vielkind J.
        • Wechsler J.
        • Wolkenstein P.
        • et al.
        Different patterns of mast cells distinguish diffuse from encapsulated neurofibromas in patients with neurofibromatosis 1.
        J Histochem Cytochem: Off J Histochem Soc. 2011; 59: 584-590
        • Sorensen S.A.
        • Mulvihill J.J.
        • Nielsen A.
        Long-term follow-up of von Recklinghausen neurofibromatosis. Survival and malignant neoplasms.
        New Engl J Med. 1986; 314: 1010-1015
        • Ding L.
        • Getz G.
        • Wheeler D.A.
        • Mardis E.R.
        • McLellan M.D.
        • Cibulskis K.
        • et al.
        Somatic mutations affect key pathways in lung adenocarcinoma.
        Nature. 2008; 455: 1069-1075
        • Boudry-Labis E.
        • Roche-Lestienne C.
        • Nibourel O.
        • Boissel N.
        • Terre C.
        • Perot C.
        • et al.
        Neurofibromatosis-1 gene deletions and mutations in de novo adult acute myeloid leukemia.
        Am J Hematol. 2013; 88: 306-311
        • Philpott C.
        • Tovell H.
        • Frayling I.M.
        • Cooper D.N.
        • Upadhyaya M.
        The NF1 somatic mutational landscape in sporadic human cancers.
        Human Genomics. 2017; 11: 13
        • Zebisch A.
        • Troppmair J.
        Back to the roots: the remarkable RAF oncogene story.
        Cell Mole Life Sci: CMLS. 2006; 63: 1314-1330
        • Stokoe D.
        • Macdonald S.G.
        • Cadwallader K.
        • Symons M.
        • Hancock J.F.
        Activation of Raf as a result of recruitment to the plasma membrane.
        Sci (New York, NY). 1994; 264: 1463-1467
        • Long G.V.
        • Menzies A.M.
        • Nagrial A.M.
        • Haydu L.E.
        • Hamilton A.L.
        • Mann G.J.
        • et al.
        Prognostic and clinicopathologic associations of oncogenic BRAF in metastatic melanoma.
        J Clin Oncol: Off J Am Soc Clin Oncol. 2011; 29: 1239-1246
        • Kimura E.T.
        • Nikiforova M.N.
        • Zhu Z.
        • Knauf J.A.
        • Nikiforov Y.E.
        • Fagin J.A.
        High prevalence of BRAF mutations in thyroid cancer: genetic evidence for constitutive activation of the RET/PTC-RAS-BRAF signaling pathway in papillary thyroid carcinoma.
        Cancer Res. 2003; 63: 1454-1457
        • Tiacci E.
        • Trifonov V.
        • Schiavoni G.
        • Holmes A.
        • Kern W.
        • Martelli M.P.
        • et al.
        BRAF mutations in hairy-cell leukemia.
        New Engl J Med. 2011; 364: 2305-2315
        • Wellbrock C.
        • Karasarides M.
        • Marais R.
        The RAF proteins take centre stage.
        Nat Rev Mol Cell Biol. 2004; 5: 875-885
        • Emuss V.
        • Garnett M.
        • Mason C.
        • Marais R.
        Mutations of C-RAF are rare in human cancer because C-RAF has a low basal kinase activity compared with B-RAF.
        Cancer Res. 2005; 65: 9719-9726
        • Castellano E.
        • Downward J.
        RAS interaction with PI3K: more than just another effector pathway.
        Genes Cancer. 2011; 2: 261-274
        • Roberts P.J.
        • Stinchcombe T.E.
        • Der C.J.
        • Socinski M.A.
        Personalized medicine in non-small-cell lung cancer: is KRAS a useful marker in selecting patients for epidermal growth factor receptor-targeted therapy?.
        J Clin Oncol: Off J Am Soc Clin Oncol. 2010; 28: 4769-4777
        • Wood K.
        • Hensing T.
        • Malik R.
        • Salgia R.
        Prognostic and predictive value in KRAS in non-small-cell lung cancer: a review.
        JAMA Oncol. 2016; 2: 805-812
        • Gainor J.F.
        • Varghese A.M.
        • Ou S.H.
        • Kabraji S.
        • Awad M.M.
        • Katayama R.
        • et al.
        ALK rearrangements are mutually exclusive with mutations in EGFR or KRAS: an analysis of 1,683 patients with non-small cell lung cancer.
        Clin Cancer Res: Off J Am Assoc Cancer Res. 2013; 19: 4273-4281
        • 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.
        J Thoracic Oncol: Offic Publ Int Assoc Study Lung Cancer. 2019; 14: 606-616
        • Karachaliou N.
        • Mayo C.
        • Costa C.
        • Magri I.
        • Gimenez-Capitan A.
        • Molina-Vila M.A.
        • et al.
        KRAS mutations in lung cancer.
        Clin Lung Cancer. 2013; 14: 205-214
        • Biernacka A.
        • Tsongalis P.D.
        • Peterson J.D.
        • de Abreu F.B.
        • Black C.C.
        • Gutmann E.J.
        • et al.
        The potential utility of re-mining results of somatic mutation testing: KRAS status in lung adenocarcinoma.
        Cancer Genet. 2016; 209: 195-198
        • Kafatos G.
        • Niepel D.
        • Lowe K.
        • Jenkins-Anderson S.
        • Westhead H.
        • Garawin T.
        • et al.
        RAS mutation prevalence among patients with metastatic colorectal cancer: a meta-analysis of real-world data.
        Biomarkers Med. 2017; 11: 751-760
        • Watanabe T.
        • Yoshino T.
        • Uetake H.
        • Yamazaki K.
        • Ishiguro M.
        • Kurokawa T.
        • et al.
        KRAS mutational status in Japanese patients with colorectal cancer: results from a nationwide, multicenter, cross-sectional study.
        Jpn J Clin Oncol. 2013; 43: 706-712
        • Andreyev H.J.
        • Norman A.R.
        • Cunningham D.
        • Oates J.R.
        • Clarke P.A.
        Kirsten ras mutations in patients with colorectal cancer: the multicenter “RASCAL” study.
        J Natl Cancer Inst. 1998; 90: 675-684
        • Hayama T.
        • Hashiguchi Y.
        • Okamoto K.
        • Okada Y.
        • Ono K.
        • Shimada R.
        • et al.
        G12V and G12C mutations in the gene KRAS are associated with a poorer prognosis in primary colorectal cancer.
        Int J Colorectal Dis. 2019; 34: 1491-1496
        • Hershkovitz D.
        • Simon E.
        • Bick T.
        • Prinz E.
        • Noy S.
        • Sabo E.
        • et al.
        Adenoma and carcinoma components in colonic tumors show discordance for KRAS mutation.
        Hum Pathol. 2014; 45: 1866-1871
        • Vogelstein B.
        • Fearon E.R.
        • Hamilton S.R.
        • Kern S.E.
        • Preisinger A.C.
        • Leppert M.
        • et al.
        Genetic alterations during colorectal-tumor development.
        New Engl J Med. 1988; 319: 525-532
        • Jones S.
        • Zhang X.
        • Parsons D.W.
        • Lin J.C.
        • Leary R.J.
        • Angenendt P.
        • et al.
        Core signaling pathways in human pancreatic cancers revealed by global genomic analyses.
        Science (New York, NY). 2008; 321: 1801-1806
        • Hruban R.H.
        • van Mansfeld A.D.
        • Offerhaus G.J.
        • van Weering D.H.
        • Allison D.C.
        • Goodman S.N.
        • et al.
        K-ras oncogene activation in adenocarcinoma of the human pancreas. A study of 82 carcinomas using a combination of mutant-enriched polymerase chain reaction analysis and allele-specific oligonucleotide hybridization.
        Am J Pathol. 1993; 143: 545-554
        • Hezel A.F.
        • Deshpande V.
        • Zhu A.X.
        Genetics of biliary tract cancers and emerging targeted therapies.
        J Clin Oncol: Off J Am Soc Clin Oncol. 2010; 28: 3531-3540
        • Walker B.A.
        • Boyle E.M.
        • Wardell C.P.
        • Murison A.
        • Begum D.B.
        • Dahir N.M.
        • et al.
        Mutational spectrum, copy number changes, and outcome: results of a sequencing study of patients with newly diagnosed myeloma.
        J Clin Oncol: Off J Am Soc Clin Oncol. 2015; 33: 3911-3920
        • Bezieau S.
        • Devilder M.C.
        • Avet-Loiseau H.
        • Mellerin M.P.
        • Puthier D.
        • Pennarun E.
        • et al.
        High incidence of N and K-Ras activating mutations in multiple myeloma and primary plasma cell leukemia at diagnosis.
        Hum Mutat. 2001; 18: 212-224
        • Cox A.D.
        • Fesik S.W.
        • Kimmelman A.C.
        • Luo J.
        • Der C.J.
        Drugging the undruggable RAS: mission possible?.
        Nat Rev Drug Discov. 2014; 13: 828-851
        • Peng N.
        • Zhao X.
        Comparison of K-ras mutations in lung, colorectal and gastric cancer.
        Oncol Lett. 2014; 8: 561-565
        • Ayatollahi H.
        • Tavassoli A.
        • Jafarian A.H.
        • Alavi A.
        • Shakeri S.
        • Shams S.F.
        • et al.
        KRAS codon 12 and 13 mutations in gastric cancer in the Northeast Iran.
        Iran J Pathol. 2018; 13: 167-172
        • Arber N.
        • Shapira I.
        • Ratan J.
        • Stern B.
        • Hibshoosh H.
        • Moshkowitz M.
        • et al.
        Activation of c-K-ras mutations in human gastrointestinal tumors.
        Gastroenterology. 2000; 118: 1045-1050
        • Moul J.W.
        • Theune S.M.
        • Chang E.H.
        Detection of RAS mutations in archival testicular germ cell tumors by polymerase chain reaction and oligonucleotide hybridization.
        Genes Chromosom Cancer. 1992; 5: 109-118
        • Hacioglu B.M.
        • Kodaz H.
        • Erdogan B.
        • Cinkaya A.
        • Tastekin E.
        • Hacibekiroglu I.
        • et al.
        K-RAS and N-RAS mutations in testicular germ cell tumors.
        Bosnian J Med Sci. 2017; 17: 159-163
        • Ridanpaa M.
        • Lothe R.A.
        • Onfelt A.
        • Fossa S.
        • Borresen A.L.
        • Husgafvel-Pursiainen K.
        K-ras oncogene codon 12 point mutations in testicular cancer.
        Environ Health Perspect. 1993; 101: 185-187
        • Jiang W.
        • Xiang L.
        • Pei X.
        • He T.
        • Shen X.
        • Wu X.
        • et al.
        Mutational analysis of KRAS and its clinical implications in cervical cancer patients.
        J Gynecol Oncol. 2018; 29: e4
        • Wright A.A.
        • Howitt B.E.
        • Myers A.P.
        • Dahlberg S.E.
        • Palescandolo E.
        • Van Hummelen P.
        • et al.
        Oncogenic mutations in cervical cancer: genomic differences between adenocarcinomas and squamous cell carcinomas of the cervix.
        Cancer. 2013; 119: 3776-3783
        • Nagel P.D.
        • Feld F.M.
        • Weissinger S.E.
        • Stenzinger A.
        • Moller P.
        • Lennerz J.K.
        Absence of BRAF and KRAS hotspot mutations in primary mediastinal B-cell lymphoma.
        Leukemia Lymphoma. 2014; 55: 2389-2390
        • Liu Q.W.
        • Fu J.H.
        • Luo K.J.
        • Yang H.X.
        • Wang J.Y.
        • Hu Y.
        • et al.
        Identification of EGFR and KRAS mutations in Chinese patients with esophageal squamous cell carcinoma.
        Dis Esophagus: Off J Int Soc Dis Esophagus. 2011; 24: 374-380
        • Lorenzen S.
        • Schuster T.
        • Porschen R.
        • Al-Batran S.E.
        • Hofheinz R.
        • Thuss-Patience P.
        • et al.
        Cetuximab plus cisplatin-5-fluorouracil versus cisplatin-5-fluorouracil alone in first-line metastatic squamous cell carcinoma of the esophagus: a randomized phase II study of the Arbeitsgemeinschaft Internistische Onkologie.
        Ann Oncol: Off J Eur Soc Med Oncol. 2009; 20: 1667-1673
        • Essakly A.
        • Loeser H.
        • Kraemer M.
        • Alakus H.
        • Chon S.H.
        • Zander T.
        • et al.
        PIK3CA and KRAS amplification in esophageal adenocarcinoma and their impact on the inflammatory tumor microenvironment and prognosis.
        Transl Oncol. 2020; 13: 157-164
        • Hollestelle A.
        • Elstrodt F.
        • Nagel J.H.
        • Kallemeijn W.W.
        • Schutte M.
        Phosphatidylinositol-3-OH kinase or RAS pathway mutations in human breast cancer cell lines.
        Mole Cancer Res: MCR. 2007; 5: 195-201
        • Pereira C.B.
        • Leal M.F.
        • de Souza C.R.
        • Montenegro R.C.
        • Rey J.A.
        • Carvalho A.A.
        • et al.
        Prognostic and predictive significance of MYC and KRAS alterations in breast cancer from women treated with neoadjuvant chemotherapy.
        PLoS ONE. 2013; 8: e60576
        • Ahmad E.I.
        • Gawish H.H.
        • Al Azizi N.M.
        • Elhefni A.M.
        The prognostic impact of K-RAS mutations in adult acute myeloid leukemia patients treated with high-dose cytarabine.
        OncoTargets Therapy. 2011; 4: 115-121
        • Stirewalt D.L.
        • Kopecky K.J.
        • Meshinchi S.
        • Appelbaum F.R.
        • Slovak M.L.
        • Willman C.L.
        • et al.
        FLT3, RAS, and TP53 mutations in elderly patients with acute myeloid leukemia.
        Blood. 2001; 97: 3589-3595
        • Neubauer A.
        • Dodge R.K.
        • George S.L.
        • Davey F.R.
        • Silver R.T.
        • Schiffer C.A.
        • et al.
        Prognostic importance of mutations in the ras proto-oncogenes in de novo acute myeloid leukemia.
        Blood. 1994; 83: 1603-1611
        • Vendramini E.
        • Bomben R.
        • Pozzo F.
        • Benedetti D.
        • Bittolo T.
        • Rossi F.M.
        • et al.
        KRAS, NRAS, and BRAF mutations are highly enriched in trisomy 12 chronic lymphocytic leukemia and are associated with shorter treatment-free survival.
        Leukemia. 2019; 33: 2111-2115
        • Gimenez N.
        • Martinez-Trillos A.
        • Montraveta A.
        • Lopez-Guerra M.
        • Rosich L.
        • Nadeu F.
        • et al.
        Mutations in the RAS-BRAF-MAPK-ERK pathway define a specific subgroup of patients with adverse clinical features and provide new therapeutic options in chronic lymphocytic leukemia.
        Haematologica. 2019; 104: 576-586
        • Ouerhani S.
        • Bougatef K.
        • Soltani I.
        • Elgaaied A.B.
        • Abbes S.
        • Menif S.
        The prevalence and prognostic significance of KRAS mutation in bladder cancer, chronic myeloid leukemia and colorectal cancer.
        Mol Biol Rep. 2013; 40: 4109-4114
        • Kodaz H.K.O.
        • Hacioglu M.B.
        • et al.
        Frequency of RAS mutations (KRAS, NRAS, HRAS) in human solid cancer.
        EJMO. 2017; 1: 1-7
        • Lax S.F.
        • Kendall B.
        • Tashiro H.
        • Slebos R.J.
        • Hedrick L.
        The frequency of p53, K-ras mutations, and microsatellite instability differs in uterine endometrioid and serous carcinoma: evidence of distinct molecular genetic pathways.
        Cancer. 2000; 88: 814-824
        • Slebos R.J.
        • Kibbelaar R.E.
        • Dalesio O.
        • Kooistra A.
        • Stam J.
        • Meijer C.J.
        • et al.
        K-ras oncogene activation as a prognostic marker in adenocarcinoma of the lung.
        New Engl J Med. 1990; 323: 561-565
        • Shepherd F.A.
        • Domerg C.
        • Hainaut P.
        • Janne P.A.
        • Pignon J.P.
        • Graziano S.
        • et al.
        Pooled analysis of the prognostic and predictive effects of KRAS mutation status and KRAS mutation subtype in early-stage resected non-small-cell lung cancer in four trials of adjuvant chemotherapy.
        J Clin Oncol: Off J Am Soc Clin Oncol. 2013; 31: 2173-2181
        • Zer A.
        • Ding K.
        • Lee S.M.
        • Goss G.D.
        • Seymour L.
        • Ellis P.M.
        • et al.
        Pooled analysis of the prognostic and predictive value of KRAS mutation status and mutation subtype in patients with non-small cell lung cancer treated with epidermal growth factor receptor tyrosine kinase inhibitors.
        J Thoracic Oncol: Offic Publ Int Assoc Study Lung Cancer. 2016; 11: 312-323
        • Pan W.
        • Yang Y.
        • Zhu H.
        • Zhang Y.
        • Zhou R.
        • Sun X.
        KRAS mutation is a weak, but valid predictor for poor prognosis and treatment outcomes in NSCLC: a meta-analysis of 41 studies.
        Oncotarget. 2016; 7: 8373-8388
        • Cerottini J.P.
        • Caplin S.
        • Saraga E.
        • Givel J.C.
        • Benhattar J.
        The type of K-ras mutation determines prognosis in colorectal cancer.
        Am J Surg. 1998; 175: 198-202
        • Samowitz W.S.
        • Curtin K.
        • Schaffer D.
        • Robertson M.
        • Leppert M.
        • Slattery M.L.
        Relationship of Ki-ras mutations in colon cancers to tumor location, stage, and survival: a population-based study.
        Cancer Epidemiol Biomarkers Prevent: Publ Am Assoc Cancer Res Cosponsored Am Soc Prevent Oncol. 2000; 9: 1193-1197
        • Yoon H.H.
        • Tougeron D.
        • Shi Q.
        • Alberts S.R.
        • Mahoney M.R.
        • Nelson G.D.
        • et al.
        KRAS codon 12 and 13 mutations in relation to disease-free survival in BRAF-wild-type stage III colon cancers from an adjuvant chemotherapy trial (N0147 alliance).
        Clin Cancer Res: Off J Am Assoc Cancer Res. 2014; 20: 3033-3043
        • Modest D.P.
        • Ricard I.
        • Heinemann V.
        • Hegewisch-Becker S.
        • Schmiegel W.
        • Porschen R.
        • et al.
        Outcome according to KRAS-, NRAS- and BRAF-mutation as well as KRAS mutation variants: pooled analysis of five randomized trials in metastatic colorectal cancer by the AIO colorectal cancer study group.
        Ann Oncol: Off J Eur Soc Med Oncol. 2016; 27: 1746-1753
        • Taieb J.
        • Zaanan A.
        • Le Malicot K.
        • Julie C.
        • Blons H.
        • Mineur L.
        • et al.
        Prognostic effect of BRAF and KRAS mutations in patients with stage III colon cancer treated with leucovorin, fluorouracil, and oxaliplatin with or without cetuximab: a post hoc analysis of the PETACC-8 trial.
        JAMA Oncol. 2016; 2: 643-653
        • Roth A.D.
        • Tejpar S.
        • Delorenzi M.
        • Yan P.
        • Fiocca R.
        • Klingbiel D.
        • et al.
        Prognostic role of KRAS and BRAF in stage II and III resected colon cancer: results of the translational study on the PETACC-3, EORTC 40993, SAKK 60–00 trial.
        J Clin Oncol: Off J Am Soc Clin Oncol. 2010; 28: 466-474
        • Andreyev H.J.
        • Norman A.R.
        • Cunningham D.
        • Oates J.
        • Dix B.R.
        • Iacopetta B.J.
        • et al.
        Kirsten ras mutations in patients with colorectal cancer: the 'RASCAL II' study.
        Br J Cancer. 2001; 85: 692-696
        • Haas M.
        • Ormanns S.
        • Baechmann S.
        • Remold A.
        • Kruger S.
        • Westphalen C.B.
        • et al.
        Extended RAS analysis and correlation with overall survival in advanced pancreatic cancer.
        Br J Cancer. 2017; 116: 1462-1469
        • Shin S.H.
        • Kim S.C.
        • Hong S.M.
        • Kim Y.H.
        • Song K.B.
        • Park K.M.
        • et al.
        Genetic alterations of K-ras, p53, c-erbB-2, and DPC4 in pancreatic ductal adenocarcinoma and their correlation with patient survival.
        Pancreas. 2013; 42: 216-222
        • Windon A.L.
        • Loaiza-Bonilla A.
        • Jensen C.E.
        • Randall M.
        • Morrissette J.J.D.
        • Shroff S.G.
        A KRAS wild type mutational status confers a survival advantage in pancreatic ductal adenocarcinoma.
        J Gastrointestinal Oncol. 2018; 9: 1-10
        • Bournet B.
        • Muscari F.
        • Buscail C.
        • Assenat E.
        • Barthet M.
        • Hammel P.
        • et al.
        KRAS G12D mutation subtype is a prognostic factor for advanced pancreatic adenocarcinoma.
        Clin Translat Gastroenterol. 2016; 7: e157
        • Rachakonda P.S.
        • Bauer A.S.
        • Xie H.
        • Campa D.
        • Rizzato C.
        • Canzian F.
        • et al.
        Somatic mutations in exocrine pancreatic tumors: association with patient survival.
        PLoS ONE. 2013; 8: e60870
        • Rodenhuis S.
        • Boerrigter L.
        • Top B.
        • Slebos R.J.
        • Mooi W.J.
        • van't Veer L.
        • et al.
        Mutational activation of the K-ras oncogene and the effect of chemotherapy in advanced adenocarcinoma of the lung: a prospective study..
        J Clin Oncol: Off J Am Soc Clin Oncol. 1997; 15: 285-291
        • Herbst R.S.
        • Prager D.
        • Hermann R.
        • Fehrenbacher L.
        • Johnson B.E.
        • Sandler A.
        • et al.
        TRIBUTE: a phase III trial of erlotinib hydrochloride (OSI-774) combined with carboplatin and paclitaxel chemotherapy in advanced non-small-cell lung cancer.
        J Clin Oncol: Off J Am Soc Clin Oncol. 2005; 23: 5892-5899
        • Zhu C.Q.
        • da Cunha Santos G
        • Ding K.
        • Sakurada A.
        • Cutz J.C.
        • Liu N.
        • et al.
        Role of KRAS and EGFR as biomarkers of response to erlotinib in National Cancer Institute of Canada Clinical Trials Group Study BR.21..
        J Clin Oncol: Off J Am Soc Clin Oncol. 2008; 26: 4268-4275
        • Douillard J.Y.
        • Shepherd F.A.
        • Hirsh V.
        • Mok T.
        • Socinski M.A.
        • Gervais R.
        • et al.
        Molecular predictors of outcome with gefitinib and docetaxel in previously treated non-small-cell lung cancer: data from the randomized phase III INTEREST trial.
        J Clin Oncol: Off J Am Soc Clin Oncol. 2010; 28: 744-752
        • Schneider C.P.
        • Heigener D.
        • Schott-von-Romer K.
        • Gutz S.
        • Laack E.
        • Digel W.
        • et al.
        Epidermal growth factor receptor-related tumor markers and clinical outcomes with erlotinib in non-small cell lung cancer: an analysis of patients from german centers in the TRUST study.
        J Thoracic Oncol: Offic Publ Int Assoc Study Lung Cancer. 2008; 3: 1446-1453