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Mutant RAS and the tumor microenvironment as dual therapeutic targets for advanced colorectal cancer

  • Jorien B.E. Janssen
    Affiliations
    Radboud University Medical Center, Radboud Institute for Health Sciences, Department of Medical Oncology, Nijmegen, the Netherlands
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  • Jan Paul Medema
    Affiliations
    Amsterdam UMC, University of Amsterdam, Laboratory for Experimental Oncology and Radiobiology, Center for Experimental Molecular Medicine, Cancer Center Amsterdam, Amsterdam, the Netherlands
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  • Elske C. Gootjes
    Affiliations
    Radboud University Medical Center, Radboud Institute for Health Sciences, Department of Medical Oncology, Nijmegen, the Netherlands
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  • Author Footnotes
    1 Shared last author.
    Daniele V.F. Tauriello
    Correspondence
    Corresponding authors at: Department of Medical Oncology, Radboud Institute for Health Sciences, Radboud University Medical Center, Geert Grooteplein Zuid 8, 6525GA Nijmegen, the Netherlands.
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    1 Shared last author.
    Affiliations
    Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, Department of Cell Biology, Nijmegen, the Netherlands
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    Henk M.W. Verheul
    Correspondence
    Corresponding authors at: Department of Medical Oncology, Radboud Institute for Health Sciences, Radboud University Medical Center, Geert Grooteplein Zuid 8, 6525GA Nijmegen, the Netherlands.
    Footnotes
    1 Shared last author.
    Affiliations
    Radboud University Medical Center, Radboud Institute for Health Sciences, Department of Medical Oncology, Nijmegen, the Netherlands
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  • Author Footnotes
    1 Shared last author.

      Highlights

      • RAS mutations occur in roughly half of the patients with colorectal cancer (CRC).
      • Oncogenic RAS induces a pro-metastatic immunosuppressive tumor microenvironment.
      • This suppressive environment prevents effective anti-tumor immune responses.
      • Effective treatments tailored to KRAS mutant metastatic CRC are needed.
      • Focus should be on combinations of immunotherapy with RAS-targeted treatments.

      Abstract

      RAS genes are the most frequently mutated oncogenes in cancer. These mutations occur in roughly half of the patients with colorectal cancer (CRC). RAS mutant tumors are resistant to therapy with anti-EGFR monoclonal antibodies. Therefore, patients with RAS mutant CRC currently have few effective therapy options. RAS mutations lead to constitutively active RAS GTPases, involved in multiple downstream signaling pathways. These alterations are associated with a tumor microenvironment (TME) that drives immune evasion and disease progression by mechanisms that remain incompletely understood. In this review, we focus on the available evidence in the literature explaining the potential effects of RAS mutations on the CRC microenvironment. Ongoing efforts to influence the TME by targeting mutant RAS and thereby sensitizing these tumors to immunotherapy will be discussed as well.

      Keywords

      Introduction

      The median overall survival (OS) for patients with metastatic CRC (mCRC) is estimated around 30 months [
      • Van Cutsem E.
      • Cervantes A.
      • Adam R.
      • Sobrero A.
      • Van Krieken J.H.
      • Aderka D.
      • et al.
      ESMO consensus guidelines for the management of patients with metastatic colorectal cancer.
      ]. Patients with RAS mutated CRC are confronted with fewer treatment options and overall worse prognosis. Although the prognostic value of KRAS mutations remains somewhat controversial, many larger cohorts point into the direction of KRAS mutations as a poor prognostic factor [
      • Meng M.
      • et al.
      The current understanding on the impact of KRAS on colorectal cancer.
      ,
      • Levin-Sparenberg E.
      • Bylsma L.C.
      • Lowe K.
      • Sangare L.
      • Fryzek J.P.
      • Alexander D.D.
      A Systematic Literature Review and Meta-Analysis Describing the Prevalence of KRAS, NRAS, and BRAF Gene Mutations in Metastatic Colorectal Cancer.
      ,

      Taieb J, et al. Prognostic Value of BRAF and KRAS Mutations in MSI and MSS Stage III Colon Cancer. JNCI: J Natl Cancer Instit 2016; 109(5).

      ,
      • Ottaiano A.
      • Normanno N.
      • Facchini S.
      • Cassata A.
      • Nappi A.
      • Romano C.
      • et al.
      Study of Ras Mutations' Prognostic Value in Metastatic Colorectal Cancer: STORIA Analysis.
      ]. The prognostic implications might even be mutation dependent. The high prevalence of RAS mutations in the CRC population causes an urgent need for new therapeutic strategies for this patient group. Mutations in KRAS, NRAS and HRAS occur in respectively 40–50%, 2–9%, and 1–2% of patients with mCRC [
      • Cox A.D.
      • et al.
      Drugging the undruggable RAS: Mission Possible?.
      ,
      • Kuhn N.
      • Klinger B.
      • Uhlitz F.
      • Sieber A.
      • Rivera M.
      • Klotz-Noack K.
      • et al.
      Mutation-specific effects of NRAS oncogenes in colorectal cancer cells.
      ,
      • Chang Y.-Y.
      • Lin P.-C.
      • Lin H.-H.
      • Lin J.-K.
      • Chen W.-S.
      • Jiang J.-K.
      • et al.
      Mutation spectra of RAS gene family in colorectal cancer.
      ]. KRAS mutations contribute to tumor formation by promoting tumor progression and hyperproliferation [
      • Haigis K.M.
      • Kendall K.R.
      • Wang Y.
      • Cheung A.
      • Haigis M.C.
      • Glickman J.N.
      • et al.
      Differential effects of oncogenic K-Ras and N-Ras on proliferation, differentiation and tumor progression in the colon.
      ], and tend to occur early during tumorigenesis [
      • Fearon E.R.
      • Vogelstein B.
      A genetic model for colorectal tumorigenesis.
      ]. NRAS mutations may be mainly responsible for suppression of stress-induced apoptosis [
      • Haigis K.M.
      • Kendall K.R.
      • Wang Y.
      • Cheung A.
      • Haigis M.C.
      • Glickman J.N.
      • et al.
      Differential effects of oncogenic K-Ras and N-Ras on proliferation, differentiation and tumor progression in the colon.
      ,

      Wang Y, et al. Mutant N-RAS protects colorectal cancer cells from stress-induced apoptosis and contributes to cancer development and progression. Cancer Discov 2013; 3(3): 294–307.

      ]. Less is known about the pro-tumorigenic function of the other RAS family members in CRC. In addition, the individual mutations and the tissue context are contributing factors that determine the phenotype during tumorigenesis [

      Zafra MP, et al. An In Vivo Kras Allelic Series Reveals Distinct Phenotypes of Common Oncogenic Variants. Cancer Discov 2020; 10(11): p. 1654–1671.

      ].
      RAS proteins activate multiple signaling pathways among which the mitogen-activated protein kinase (MAPK) and PI3K/AKT pathway [
      • Koveitypour Z.
      • et al.
      Signaling pathways involved in colorectal cancer progression.
      ]. Both pathways promote cell growth, differentiation and cell survival by up- or downregulation of multiple target genes. Interestingly, downstream signaling not only stimulates growth and survival of cancer cells, there is also accumulating evidence of crosstalk with cells of the surrounding tumor microenvironment (TME). This cellular, molecular and physical environment determines cancer growth and survival, and is shaped by cancer cells as well as by host factors. Indeed, through the TME, cancer cells can skew immunosurveillance to tolerance or even tumor support, contributing to resistance to cancer therapies [
      • Tauriello D.V.F.
      • Batlle E.
      Targeting the Microenvironment in Advanced Colorectal Cancer.
      ,
      • Sun Y.u.
      Tumor microenvironment and cancer therapy resistance.
      ]. In addition to cancer cell-centered therapies, there is a compelling rationale for therapeutic strategies with the potential to positively alter the TME towards a less suppressive state, potentially leading to increased efficacy of immunotherapy in MSS CRC [
      • Tauriello D.V.F.
      • Batlle E.
      Targeting the Microenvironment in Advanced Colorectal Cancer.
      ,
      • Janssen E.
      • Subtil B.
      • de la Jara Ortiz F.
      • Verheul H.M.W.
      • Tauriello D.V.F.
      Combinatorial Immunotherapies for Metastatic Colorectal Cancer.
      ]. Here, we describe the literature on the stromal effects of RAS mutations and give an overview of the current efforts to target mutant RAS signaling in CRC cells. As a consequence of the interplay between RAS mutations and the TME, we will discuss the potential of these treatments to modify the TME thereby opening avenues for immunotherapeutic strategies.

      RAS biology in CRC

      CRC prognosis is based on pathological and molecular parameters, including microsatellite (in)stability, tumor sidedness and TNM staging [
      • Popat S.
      • Hubner R.
      • Houlston R.S.
      Systematic review of microsatellite instability and colorectal cancer prognosis.
      ,
      • Dienstmann R.
      • Mason M.J.
      • Sinicrope F.A.
      • Phipps A.I.
      • Tejpar S.
      • Nesbakken A.
      • et al.
      Prediction of overall survival in stage II and III colon cancer beyond TNM system: a retrospective, pooled biomarker study.
      ,
      • Bahl A.
      • et al.
      Primary Tumor Location as a Prognostic and Predictive Marker in Metastatic Colorectal Cancer (mCRC).
      ]. Furthermore, CRC can be classified in four consensus molecular subtypes (CMS) based on gene-expression data. KRAS mutations are enriched in CMS3, but can be found in all subtypes, including the poor prognosis and TME-enriched CMS4 [
      • Guinney J.
      • Dienstmann R.
      • Wang X.
      • de Reyniès A.
      • Schlicker A.
      • Soneson C.
      • et al.
      The consensus molecular subtypes of colorectal cancer.
      ]. The majority of KRAS mutations are found in codons 12 and 13 of exon 2, with the most common amino acid substitutions being G12D, G12V, G12C and G13D [
      • Vaughn C.P.
      • ZoBell S.D.
      • Furtado L.V.
      • Baker C.L.
      • Samowitz W.S.
      Frequency of KRAS, BRAF, and NRAS mutations in colorectal cancer.
      ]. RAS proteins are either in an inactive guanosine diphosphate (GDP)-bound or in an active guanosine triphosphate (GTP)-bound conformation, depending on ligand binding to cell surface receptors such as the epidermal growth factor receptor (EGFR) [
      • Kamata T.
      • Feramisco J.R.
      Epidermal growth factor stimulates guanine nucleotide binding activity and phosphorylation of ras oncogene proteins.
      ,
      • Simanshu D.K.
      • Nissley D.V.
      • McCormick F.
      RAS Proteins and Their Regulators in Human Disease.
      ,
      • Vetter I.R.
      • Wittinghofer A.
      The guanine nucleotide-binding switch in three dimensions.
      ]. Signaling from this receptor tyrosine kinase prompts guanine nucleotide exchange factors (GEFs) to remove a GDP molecule, allowing it to be replaced by GTP [
      • Simanshu D.K.
      • Nissley D.V.
      • McCormick F.
      RAS Proteins and Their Regulators in Human Disease.
      ,
      • Boriack-Sjodin P.A.
      • Margarit S.M.
      • Bar-Sagi D.
      • Kuriyan J.
      The structural basis of the activation of Ras by Sos.
      ]. Independently regulated GTPase-activating proteins (GAPs) stimulate hydrolysis of GTP to GDP and thereby switch off the signal [
      • Simanshu D.K.
      • Nissley D.V.
      • McCormick F.
      RAS Proteins and Their Regulators in Human Disease.
      ,
      • Boguski M.S.
      • McCormick F.
      Proteins regulating Ras and its relatives.
      ]. Point mutations in RAS favor the formation of continuously GTP-bound, constitutively ‘active’ RAS, regardless of GAP activity or receptor stimulation [
      • Cox A.D.
      • et al.
      Drugging the undruggable RAS: Mission Possible?.
      ,
      • Gibbs J.B.
      • Sigal I.S.
      • Poe M.
      • Scolnick E.M.
      Intrinsic GTPase activity distinguishes normal and oncogenic ras p21 molecules.
      ].
      The latter explains why treatment with anti-EGFR monoclonal antibodies does not improve the outcome of patients with mutant KRAS mCRC [
      • 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.
      ,
      • 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.
      ]. Besides oncogenic KRAS, also NRAS and BRAF mutations render patients resistant to anti-EGFR therapy [
      • Pietrantonio F.
      • Petrelli F.
      • Coinu A.
      • Di Bartolomeo M.
      • Borgonovo K.
      • Maggi C.
      • et al.
      Predictive role of BRAF mutations in patients with advanced colorectal cancer receiving cetuximab and panitumumab: a meta-analysis.
      ,
      • Sorich M.J.
      • Wiese M.D.
      • Rowland A.
      • Kichenadasse G.
      • McKinnon R.A.
      • Karapetis C.S.
      Extended RAS mutations and anti-EGFR monoclonal antibody survival benefit in metastatic colorectal cancer: a meta-analysis of randomized, controlled trials.
      ]. Although HRAS mutations are the least prevalent in CRC, mutations in this gene will most likely contribute to anti-EGFR resistance through similar mechanisms. Testing of RAS and BRAF mutations has become standard-of-care to exclude patients for anti-EGFR therapy [
      • Van Cutsem E.
      • Cervantes A.
      • Adam R.
      • Sobrero A.
      • Van Krieken J.H.
      • Aderka D.
      • et al.
      ESMO consensus guidelines for the management of patients with metastatic colorectal cancer.
      ,
      • Lakatos G.
      • Köhne C.-H.
      • Bodoky G.
      Current therapy of advanced colorectal cancer according to RAS/RAF mutational status.
      ]. Moreover, acquired resistance of RAS wild-type mCRC during anti-EGFR therapy can be caused by the emergence of RAS-mutant cancer cells [
      • van Helden E.J.
      • et al.
      RAS and BRAF mutations in cell-free DNA are predictive for outcome of cetuximab monotherapy in patients with tissue-tested RAS wild-type advanced colorectal cancer.
      ]. However, a part of the patients with acquired resistance to anti-EGFR therapy cannot be linked to genetic drivers, leading to the hypothesis that the TME may play a role. Indeed, patient derived cancer-associated fibroblasts (CAFs) were shown to increase the secretion of EGF following anti-EGFR treatment (cetuximab), compensating for inhibition of the receptor [
      • Garvey C.M.
      • Lau R.
      • Sanchez A.
      • Sun R.X.
      • Fong E.J.
      • Doche M.E.
      • et al.
      Anti-EGFR Therapy Induces EGF Secretion by Cancer-Associated Fibroblasts to Confer Colorectal Cancer Chemoresistance.
      ]. Similar observations were done in another in vitro model with increased production of TGF-α and amphiregulin, two EGFR ligands, both were identified as factors contributing to cetuximab resistance [
      • Hobor S.
      • et al.
      TGFalpha and amphiregulin paracrine network promotes resistance to EGFR blockade in colorectal cancer cells.
      ]. Moreover, in another cohort of patients with non-genetic acquired resistance to anti-EGFR therapy, tumors were shown to have undergone stromal remodeling [
      • Woolston A.
      • et al.
      Genomic and Transcriptomic Determinants of Therapy Resistance and Immune Landscape Evolution during Anti-EGFR Treatment in Colorectal Cancer.
      ]. This involved a switch from CMS2 to CMS4, an increase in CAFs and evidence for immunomodulation, providing a rationale for added immunotherapy.
      Given the limited OS of patients with RAS mutant CRC, and their ineligibility to anti-EGFR treatment, options to block mutant RAS or downstream signaling pathways have been studied extensively. Until recently, this oncogene was considered to be undruggable. The high affinity of RAS proteins for GTP makes it extremely hard to design small molecule competitive direct RAS inhibitors [
      • John J.
      • Sohmen R.
      • Feuerstein J.
      • Linke R.
      • Wittinghofer A.
      • Goody R.S.
      Kinetics of interaction of nucleotides with nucleotide-free H-ras p21.
      ]. However, renewed efforts of direct inhibition have recently been made, and alternative targets downstream of mutant RAS are being explored as well. Whereas several monotherapies failed in the clinic, combinations of targeted therapies and immunotherapies may offer better results.

      Mutant RAS and the TME

      (Pre-) clinical research increasingly focusses on the TME surrounding cancer cells, comprising vascular cells, fibroblast, resident and infiltrating immune cells, and multiple signaling molecules [
      • Tauriello D.V.F.
      • Batlle E.
      Targeting the Microenvironment in Advanced Colorectal Cancer.
      ,
      • Janssen E.
      • Subtil B.
      • de la Jara Ortiz F.
      • Verheul H.M.W.
      • Tauriello D.V.F.
      Combinatorial Immunotherapies for Metastatic Colorectal Cancer.
      ,
      • Hanahan D.
      • Coussens L.
      Accessories to the crime: functions of cells recruited to the tumor microenvironment.
      ,
      • Quail D.F.
      • Joyce J.A.
      Microenvironmental regulation of tumor progression and metastasis.
      ,
      • Pietras K.
      • Östman A.
      Hallmarks of cancer: interactions with the tumor stroma.
      ]. Through crosstalk between multiple cell types in the TME, cancer cells exert multiple mechanisms to foster a favorable pro-inflammatory environment during tumorigenesis, and to evade immune destruction during subsequent stages. Indeed, in advanced CRC, immune suppression is associated with reduced inflammatory signaling. In fact, oncogenic KRAS may contribute to the formation of an anti-inflammatory, immunosuppressive TME [
      • Hamarsheh S.
      • Groß O.
      • Brummer T.
      • Zeiser R.
      Immune modulatory effects of oncogenic KRAS in cancer.
      ,

      Dias Carvalho P, et al. KRAS Oncogenic Signaling Extends beyond Cancer Cells to Orchestrate the Microenvironment. Cancer Res 2018; 78(1): 7–14.

      ]. Efficient inhibition of KRAS G12C in immunocompetent mice led to an influx of activated immune cells, resulting in a cytotoxic T lymphocyte (CTL)-mediated anti-tumor immune response [
      • 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.
      ]. The mechanisms by which oncogenic RAS affects the TME are only partially uncovered. Below, we will summarize the evidence across several relevant areas of cancer immunity (see Fig. 1). Despite the somewhat artificial piece-by-piece discussion, it is important to remember that cells within the TME communicate—either via direct contact or through the production of cytokines and chemokines—as a complex ecosystem [
      • Tauriello D.V.F.
      • Batlle E.
      Targeting the Microenvironment in Advanced Colorectal Cancer.
      ]. Mutant KRAS may furthermore influence the TME through oncogene-induced senescence. Senescent cells are in proliferative arrest and secrete multiple signaling molecules, which influence neighboring non-senescent cells and immune cells in the TME [
      • Serrano M.
      • Lin A.W.
      • McCurrach M.E.
      • Beach D.
      • Lowe S.W.
      Oncogenic ras Provokes Premature Cell Senescence Associated with Accumulation of p53 and p16INK4a.
      ,
      • Zhu H.
      • et al.
      Oncogene-induced senescence: From biology to therapy.
      ]. Oncogenic KRAS-induced senescence may be of therapeutic interest as well [
      • Zhu H.
      • et al.
      Oncogene-induced senescence: From biology to therapy.
      ], but is not further discussed in this review.
      Figure thumbnail gr1
      Fig. 1Summary of reported effects of mutant RAS on the TME in colorectal cancer. Accumulating evidence suggests that RAS mutations have a strong effect on the TME, mainly by attraction of or polarization of cells towards an immunosuppressive phenotype—TAMs, Treg cells and MDSCs; preventing an effective T cell response; and modulation of ECM and tumor vasculature. Abbreviations: CCL17, CC motif chemokine ligand 17; CIK cell, cytokine-induced killer cell; CSF2, colony stimulating factor 2; CXCL3, CXC motif chemokine ligand 3; CXCR2, CXC chemokine receptor 2; ECM, extracellular matrix; GM-CSF, granulocyte–macrophage colony-stimulating factor; IFN-γ, interferon gamma; IL-1β, interleukin-1 beta; IL-6, interleukin-6; IL-8, interleukin-8; IL-10, interleukin-10; IL-17, interleukin-17; IRF2, interferon regulatory factor 2; MDSC, myeloid derived suppressor cell; MHC-1, major histocompatibility complex-1; NET, neutrophil extracellular trap; TAM, tumor-associated macrophage; TGF—β, transforming growth factor beta; Th17, T helper 17 cell; TNFα, tumor necrosis factor alpha; Treg cell, regulatory T cell; VEGF, vascular endothelial growth factor.

      Cytokines

      Oncogenic RAS modulates the TME by influencing cytokine production—through activation of PI3K, MAPK and NF-κB signaling pathways—subsequently leading to immune cell polarization, recruitment to the TME, and altered activity. Signaling through MAPK/PI3K can induce the expression of IL-10, TGF-β, and GM-CSF. The former two have known immunosuppressive effects, such as the induction of regulatory T (Treg) cells and inhibition of effector T cell function and antigen presentation [
      • Cullis J.
      • Das S.
      • Bar-Sagi D.
      Kras and Tumor Immunity: Friend or Foe?.
      ,
      • Tauriello D.V.F.
      • Sancho E.
      • Batlle E.
      Overcoming TGFβ-mediated immune evasion in cancer.
      ]. GM-CSF is a hematopoietic and pro-inflammatory cytokine needed for proper differentiation of bone marrow-derived immune cells, which is also involved in the activation of signaling pathways related to cellular proliferation and survival. GM-CSF is expressed at higher levels in KRAS mutant than KRAS wild-type CRC patients or healthy controls [
      • Petanidis S.
      • et al.
      Differential expression of IL-17, 22 and 23 in the progression of colorectal cancer in patients with K-ras mutation: Ras signal inhibition and crosstalk with GM-CSF and IFN-gamma.
      ]. In fact, higher doses of this cytokine have been associated with immunosuppressive, rather than immunostimulatory effects [
      • Parmiani G.
      • et al.
      Opposite immune functions of GM-CSF administered as vaccine adjuvant in cancer patients.
      ].
      Several pro-inflammatory cytokines, such as TNF-α, IL-6, CXCL1, CXCL2, CXLC5 and CXCL8, are controlled by NF-κB. This transcription factor also regulates multiple key processes such as cell proliferation, angiogenesis and inflammation [
      • Cullis J.
      • Das S.
      • Bar-Sagi D.
      Kras and Tumor Immunity: Friend or Foe?.
      ,
      • Soleimani A.
      • et al.
      Role of the NF-kappaB signaling pathway in the pathogenesis of colorectal cancer.
      ]. In CRC cells, NF-κB p65 expression is significantly elevated in KRAS mutated vs KRAS wild-type samples. Conversely, both MEK-inhibitor treatment and knockdown of KRAS resulted in reduced transcriptional activity of NF-κB in SW620 cells [
      • Lin G.
      • et al.
      NF-kappaB activity is downregulated by KRAS knockdown in SW620 cells via the RAS-ERK-IkappaBalpha pathway.
      ]. In contrast to elevation of the above-mentioned cytokines, mRNA and protein levels of interferon (IFN)-γ—an important regulator of anti-cancer immune responses—are downregulated in KRAS mutant compared to wild-type CRCs and healthy controls. This is supported by in vitro data with a RAS inhibitor [
      • Petanidis S.
      • et al.
      Differential expression of IL-17, 22 and 23 in the progression of colorectal cancer in patients with K-ras mutation: Ras signal inhibition and crosstalk with GM-CSF and IFN-gamma.
      ].

      Dendritic cells and macrophages

      The TME often inhibits the activation of APC’s, such as dendritic cells (DCs). Their maturation and activation is required to initiate and sustain a profound T-cell-mediated anti-tumor immune response [
      • Subtil B.
      • et al.
      The Therapeutic Potential of Tackling Tumor-Induced Dendritic Cell Dysfunction in Colorectal Cancer.
      ]. Little is known about the relation between RAS mutations and DC function, but one study found an association between a polarization of DCs that is skewed to immature phenotypes and relapse in KRAS mutant CRC [
      • Kocián P.
      • et al.
      Tumor-infiltrating lymphocytes and dendritic cells in human colorectal cancer: Their relationship to KRAS mutational status and disease recurrence.
      ].
      Macrophages are plastic cells that can polarize in tissues into phenotypes that comprise both inflammatory (M1) and anti-inflammatory (M2) phenotypes. In established tumors, tumor-associated macrophages (TAMs; often described as M2-like) can promote cancer progression [
      • Hao N.B.
      • et al.
      Macrophages in tumor microenvironments and the progression of tumors.
      ]. Liu et al. describe how oncogenic KRAS mechanistically influences macrophage polarization to a TAM-like phenotype. In a cohort of 338 CRC patients, TAM density (defined as CD163+ and CD206+ cells) was significantly higher in KRAS mutant samples, which was associated with worse OS [
      • Liu H.
      • et al.
      Mutant KRAS triggers functional reprogramming of tumor-associated macrophages in colorectal cancer.
      ]. Furthermore, TAMs derived from KRAS mutant CRCs showed higher levels of immunosuppressive cytokines (IL-10, TGF-β) than TAMs from KRAS wild-type tumors. These changes were attributed to a combined effect of GM-CSF and lactate produced by HIF-1α signaling in the tumor cells. Lactate was described to abrogate the production of pro-inflammatory cytokines [
      • Liu H.
      • et al.
      Mutant KRAS triggers functional reprogramming of tumor-associated macrophages in colorectal cancer.
      ]. Moreover, this study showed that TAMs can aid in cetuximab resistance. In contrast, another study reported that high CD163+ (M2-like) macrophage counts are significantly correlated to a KRAS wild-type status in primary CRC [
      • Koelzer V.H.
      • et al.
      Phenotyping of tumor-associated macrophages in colorectal cancer: Impact on single cell invasion (tumor budding) and clinicopathological outcome.
      ]. Many aspects of the different macrophage polarization states and their effects in different stages of disease remain to be elucidated.

      Lymphocytes

      T cells play a crucial role in the adaptive immune response, and their presence is associated with a good prognosis in CRC [
      • Idos G.E.
      • et al.
      The Prognostic Implications of Tumor Infiltrating Lymphocytes in Colorectal Cancer: A Systematic Review and Meta-Analysis.
      ]. The recent successes in immune checkpoint blockade (ICB) therapies, which activate T cell effector functions, highlight the relevance and clinical potential of these immune cells [
      • Waldman A.D.
      • Fritz J.M.
      • Lenardo M.J.
      A guide to cancer immunotherapy: from T cell basic science to clinical practice.
      ]. However, poor responses to ICB in the majority of patients—especially in CRC—underline the complex biology that determines T cell activation, infiltration and cytotoxicity [
      • Huyghe N.
      • Baldin P.
      • Van den Eynde M.
      Immunotherapy with immune checkpoint inhibitors in colorectal cancer: what is the future beyond deficient mismatch-repair tumours?.
      ]. Indeed, poor prognosis CRCs are characterized by an immunosuppressive TME that has been linked to resistance to ICB therapy in preclinical models [
      • Tauriello D.V.F.
      • et al.
      TGFbeta drives immune evasion in genetically reconstituted colon cancer metastasis.
      ]. Interestingly, a relation between reduced T cell infiltration and KRAS mutations has been reported in mouse and in human tumors of colorectal origin [
      • Liao W.
      • et al.
      KRAS-IRF2 Axis Drives Immune Suppression and Immune Therapy Resistance in Colorectal Cancer.
      ,
      • Lal N.
      • et al.
      An immunogenomic stratification of colorectal cancer: Implications for development of targeted immunotherapy.
      ,
      • Lal N.
      • et al.
      KRAS Mutation and Consensus Molecular Subtypes 2 and 3 Are Independently Associated with Reduced Immune Infiltration and Reactivity in Colorectal Cancer.
      ]. Another study of stage III primary CRCs found reduced T cell infiltration to be related to the subgroup with KRAS G12D/V mutations, while such differences were not found for other oncogenic variants [
      • Park H.E.
      • et al.
      Tumor microenvironment-adjusted prognostic implications of the KRAS mutation subtype in patients with stage III colorectal cancer treated with adjuvant FOLFOX.
      ].
      In parallel to affecting T cell infiltration, oncogenic KRAS has been reported to inhibit T cell activation. Besides the above-mentioned effect on IFN-γ production, mutant KRAS can induce the formation of immunosuppressive Treg cells via the production of IL-10 and TGFβ1 [
      • Zdanov S.
      • et al.
      Mutant KRAS Conversion of Conventional T Cells into Regulatory T Cells.
      ] and thereby promote tumor progression. Silencing mutant KRAS with siRNA in tumor cells or neutralization of these cytokines reduced the number of Treg cells in CRC cell lines [
      • Zdanov S.
      • et al.
      Mutant KRAS Conversion of Conventional T Cells into Regulatory T Cells.
      ]. Effector T cells can be directly inhibited by immunosuppressive cytokines like IL-10 and TGFβ1 produced by malignant cells [
      • Cullis J.
      • Das S.
      • Bar-Sagi D.
      Kras and Tumor Immunity: Friend or Foe?.
      ,
      • Tauriello D.V.F.
      • Sancho E.
      • Batlle E.
      Overcoming TGFβ-mediated immune evasion in cancer.
      ,
      • Batlle E.
      • Massague J.
      Transforming Growth Factor-beta Signaling in Immunity and Cancer.
      ]. Furthermore, downregulation of pathways related to T cell function such as Th1/Th2 cell differentiation and T cell receptor signaling, was identified in gene expression profiles from KRAS mutant opposed to wild-type CRC patients. Estimation of immune cell populations based on these transcriptional data indicated fewer activated CD4 memory T cells and a significant increase of Treg cells in KRAS mutant samples [
      • Liu J.
      • et al.
      Immune landscape and prognostic immune-related genes in KRAS-mutant colorectal cancer patients.
      ]. However, conflicting results have also been reported, showing a decreased amount of Treg cells in a Kras mutant tumor mouse model [
      • Liao W.
      • et al.
      KRAS-IRF2 Axis Drives Immune Suppression and Immune Therapy Resistance in Colorectal Cancer.
      ]. The authors of this study hypothesize that KRAS mutant CRC cells block T cell infiltration in general, including Treg cells.
      Th17 cells are a subset of T helper lymphocytes with pro-inflammatory properties and the main producers of IL-17. Depending on their polarization state they can exert pro- and antitumor effects [
      • Asadzadeh Z.
      • et al.
      The paradox of Th17 cell functions in tumor immunity.
      ]. In patients with KRAS mutant CRC, IL-17 was significantly elevated in comparison with healthy controls and wild-type patients, and levels declined upon KRAS inhibition [
      • Petanidis S.
      • et al.
      Differential expression of IL-17, 22 and 23 in the progression of colorectal cancer in patients with K-ras mutation: Ras signal inhibition and crosstalk with GM-CSF and IFN-gamma.
      ]. IL-17 can promote tumor cell proliferation through the attraction of myeloid-derived suppressor cells (MDSCs) and induces the production of proinflammatory cytokines [
      • Asadzadeh Z.
      • et al.
      The paradox of Th17 cell functions in tumor immunity.
      ,
      • He D.
      • et al.
      IL-17 promotes tumor development through the induction of tumor promoting microenvironments at tumor sites and myeloid-derived suppressor cells.
      ].
      An additional mechanism by which KRAS mutations can inhibit T cell activation is downregulation of major histocompatibility complex (MHC) class I proteins, the molecules responsible for antigen presentation to CTLs [
      • El-Jawhari J.J.
      • et al.
      Blocking oncogenic RAS enhances tumour cell surface MHC class I expression but does not alter susceptibility to cytotoxic lymphocytes.
      ]. As expected, knockdown of Kras in another CRC mouse model decreased tumor development. The authors hypothesize that suppression of IL-18 and MHC-I downregulation might aid in the immune evasion caused by oncogenic RAS [
      • Smakman N.
      • et al.
      Dual effect of Kras(D12) knockdown on tumorigenesis: increased immune-mediated tumor clearance and abrogation of tumor malignancy.
      ]. Of note, MHC-I downregulation can activate natural killer (NK) cells, innate cytotoxic lymphocytes with key roles in immunosurveillance. However, the TME of most solid tumors effectively prevents NK cell cytotoxicity [
      • Melaiu O.
      • et al.
      Influence of the Tumor Microenvironment on NK Cell Function in Solid Tumors.
      ]. Related to NK cells—and sometimes characterized as NK-like T cells—cytokine-induced killer (CIK) cells are a cytotoxic cell product expanded from (patient) blood that may have a therapeutic potential. In 3D co-cultures with CIK cells, mutant KRAS was shown to contribute to increased CD155 surface expression, recapitulating observations in CRC patient samples. CD155 can decrease lymphocyte activity through interaction with TIGIT. Inhibiting CD155–TIGIT interactions with antibodies against CD155 (alone or combined with PD-1–PD-L1 inhibition) supported the ability of CIK cells to suppress tumor growth in vitro [
      • Nishi K.
      • et al.
      Mutant KRAS Promotes NKG2D(+) T Cell Infiltration and CD155 Dependent Immune Evasion.
      ], suggesting that oncogenic KRAS can inhibit cytotoxic activity of these cells. Taken together, mutant KRAS appears to prevent effective T cell responses.

      Neutrophils and myeloid-derived suppressor cells

      Neutrophils are part of the innate immune system, mainly described for their involvement in inflammation and defense against extracellular pathogens. Moreover, neutrophils are increasingly recognized for their role in the TME [
      • Uribe-Querol E.
      • Rosales C.
      Neutrophils in Cancer: Two Sides of the Same Coin.
      ], and were recently shown to suppress T cell responses in CRC through TGF-β activation [
      • Germann M.
      • et al.
      Neutrophils suppress tumor-infiltrating T cells in colon cancer via matrix metalloproteinase-mediated activation of TGFbeta.
      ]. Furthermore, they can drive cancer progression and metastasis by the formation of neutrophil extracellular traps (NETs). These net-like structures are ejected by dying neutrophils and consist of chromatin fibers and proteins that entrap extracellular pathogens [
      • Masucci M.T.
      • et al.
      The Emerging Role of Neutrophil Extracellular Traps (NETs) in Tumor Progression and Metastasis.
      ]. In an Apc/Kras G12D mouse model, oncogenic KRAS stimulated proliferation and malignant potential of cancer cells via IL-8-mediated neutrophil recruitment and NET formation, involving the transport of mutant KRAS protein via exosomes [
      • Shang A.
      • et al.
      Exosomal KRAS mutation promotes the formation of tumor-associated neutrophil extracellular traps and causes deterioration of colorectal cancer by inducing IL-8 expression.
      ]. In contrast, decreased neutrophil infiltration has also been reported in patients with KRAS mutant CRC based on transcriptomic analysis [
      • Lal N.
      • et al.
      KRAS Mutation and Consensus Molecular Subtypes 2 and 3 Are Independently Associated with Reduced Immune Infiltration and Reactivity in Colorectal Cancer.
      ].
      MDSCs are relatively immature immune cells that often expand in later stages of cancer and contribute to an immunosuppressive environment by inhibiting T cell proliferation and promoting Treg cell differentiation. Indeed, their presence is associated with disease progression and metastatic outgrowth [
      • Sieminska I.
      • Baran J.
      Myeloid-Derived Suppressor Cells in Colorectal Cancer.
      ]. In a Kras mutant mouse model it was shown that KRAS G12D attracts MDSCs to the TME by inhibiting the expression of interferon regulatory factor 2 (IRF2). This transcription factor negatively regulates CXCL3, which can attract CXCR2 chemokine receptor-expressing MDSCs, eventually leading to lower T cell infiltration [
      • Liao W.
      • et al.
      KRAS-IRF2 Axis Drives Immune Suppression and Immune Therapy Resistance in Colorectal Cancer.
      ]. Concurrently, the combination of the CXCR2 inhibitor SX-682 and anti-PD-1 treatment significantly prolonged survival in mice and was more effective than SX-682 monotherapy [
      • Liao W.
      • et al.
      KRAS-IRF2 Axis Drives Immune Suppression and Immune Therapy Resistance in Colorectal Cancer.
      ]. In line with observations of lower IRF2 expression levels in patients with KRAS mutant tumors, the combination of SX-682 and nivolumab is currently under clinical investigation (Table 1).
      Table 1Clinical trials with compounds targeting RAS or in a population of RAS mutant CRC or other solid tumors.
      ApproachTrial codePhaseDrugCombinationCancer typeStatus
      KRAS G12C inhibitorNCT03785249

      (KRYSTAL-1)
      I/IIAdagrasib

      (MRTX849)
      Pembrolizumab (NSCLC)

      Cetuximab (CRC)

      Afatinib (NSCLC)
      Solid tumors with KRAS G12C mutationRecruiting
      KRAS G12C inhibitor + SHP2 inhibitorNCT04330664

      (KRYSTAL-2)
      I/IIAdagrasibTNO155Solid tumors with KRAS G12C mutationActive, not recruiting
      KRAS G12C inhibitor + anti-EGFR antibodyNCT04793958

      (KRYSTAL-10)
      IIIAdagrasibCetuximabCRC with KRAS G12C mutationRecruiting
      KRAS G12C inhibitor + KRAS–SOS1 inhibitorNCT04975256IAdagrasibBI 1701963Solid tumors with KRAS G12C mutationActive, not recruiting
      KRAS G12C inhibitorNCT03600883

      (CodeBreak100)
      I/IISotorasib (AMG 510)Solid tumors with KRAS G12C mutationRecruiting
      KRAS G12C inhibitorNCT04185883

      (CodeBreak101)
      ISotorasibMEK inhibitor, PD1 inhibitor, SHP2 inhibitor

      EGFR inhibitor + chemo

      CDK 4/6 inhibitor

      mTOR inhibitor

      MEK + EGFR inhibitor
      Solid tumors with KRAS G12C mutationRecruiting
      KRAS G12C inhibitor + anti-EGFR antibodyNCT05198934

      (CodeBreak300)
      IIISotorasibPanitumumabCRC with KRAS G12C mutationRecruiting
      KRAS G12C inhibitorNCT04165031I/IILY3499446Abemaciclib

      Cetuximab

      Erlotinib

      docetaxel
      Solid tumors with KRAS G12C mutationTerminated (unexpected toxicity finding)
      KRAS G12C inhibitorNCT04006301IJNJ-74699157Solid tumors with KRAS G12C mutationCompleted
      KRAS G12C inhibitorNCT04449874IGDC-6036Atezolizumab

      Cetuximab

      Bevacizumab

      Erlotinib
      Solid tumors with KRAS G12C mutationRecruiting
      KRAS G12C inhibitorNCT04585035I/IID-1553Solid tumors with KRAS G12C mutationRecruiting
      KRAS G12C inhibitorNCT04699188I/IIJDQ443TNO 155

      Tislelizumab
      Solid tumors with KRAS G12C mutationRecruiting
      KRAS G12C inhibitorNCT04956640I/IILY3537982Abemaciclib

      Erlotinib

      Pembrolizumab

      Temuterkib

      LY3295668

      Cetuximab

      TNO155
      Solid tumors with KRAS G12C mutationRecruiting
      SHP2 inhibitorNCT04000529IbTNO155Spartalizumab

      Ribociclib
      Solid tumorsRecruiting
      SHP2 inhibitorNCT03518554IJAB-3068Solid tumorsRecruiting
      SHP2 inhibitorNCT03989115I/IIRMC-4630Cobimetinib (RAS/BRAF/NF1 mutation)Solid tumorsActive, not recruiting
      mRNA derived vaccine targeting mutant KRAS + ICBNCT03948763ImRNA-5671/V941Monotherapy or combination with pembrolizumabSolid tumorsActive, not recruiting
      KRAS peptide vaccine + ICBNCT04117087IPoly-ICLCNivolumab + IpilimumabKRAS mutant pancreatic cancer/CRCRecruiting
      Anti-PD-L1, MEKi, PARPiNCT03637491I/IIAvelumab, binimetinib, talazoparibDifferent combinationsRAS mutant solid tumorsTerminated (limited anti-tumor activity)
      MEKi + CDK4/6NCT03981614IIBinimetinibPalbociclibKRAS or NRAS mutant CRCActive, not recruiting
      Adoptive T cells with KRAS G12D/V engineered TCRNCT03745326

      NCT03190941
      I/IISolid tumorsRecruiting
      KRAS–SOS1 inhibitorNCT04111458IBI 1701963Monotherapy or with trametinibKRAS mutated solid tumorsActive not recruiting
      KRAS–SOS1 inhibitor + chemotherapyNCT04627142IBI1701963IrinotecanCRCTerminated (sponsor decision)
      CXCR1/2 inhibitor + ICBNCT04599140

      (STOPTRAFFIC-1)
      I/IISX-682NivolumabRAS mutated MSS CRCRecruiting
      PLK1 inhibitor + chemotherapyNCT03829410I/IIOnvansertibFOLFIRI-bevaCRCRecruiting
      MEKi + chemotherapyNCT02613650IBinimetinibFOLFIRICRCRecruiting
      MEKi + ICBNCT03271047I/IIBinimetinibNivolumab ± ipilimumabCRCCompleted
      MEKi + chemotherapyNCT03317119ITrametinibTrifluridine/ tipiracilRAS mutant CRCActive not recruiting
      Anti-EGFR + MEKiNCT03087071IITrametinib

      Panitumumab
      RAS/BRAF mutant CRCRecruiting
      CD137 agonist + anti-EGFR + chemotherapyNCT03290937I/IIUtolimumabCetuximab + irinotecanCRCActive not recruiting
      MDM2 inhibitor + MEK inhibitorNCT03714958IHDM201TrametinibRAS/RAF mutant CRCRecruiting
      Reovirus + chemotherapyNCT01274624IReolysinFOLFIRI-bevamCRCCompleted
      • Goel S.
      • et al.
      Elucidation of Pelareorep Pharmacodynamics in A Phase I Trial in Patients with KRAS-Mutated Colorectal Cancer.
      WEE1 inhibitor + chemotherapyNCT02906059IAdavosertib

      (AZD1775)
      IrinotecanRAS or BRAF mutated mCRCCompleted
      Pan-HER inhibitor + MEK inhibitorNCT03065387INeratinibTrametinibKRAS mutant solid tumor (a.o.)Recruiting
      c-MET inhibitor + MEK inhibitorNCT02510001ICrizotinibBinimetinibCRC with KRAS mutation or c-MET mutation/ amplificationCompleted
      RAF/MEK inhibitor + mTOR inhibitorNCT02407509ICH5126766EverolimusBRAF, KRAS, NRAS mutant solid tumorsRecruiting
      RAF/MEK inhibitor + FAK inhibitorNCT03875820ICH5126766DefactinibRAS mutant solid tumorsRecruiting
      Chemotherapy + anti PD-1NCT03519412IITemozolomide inductionPembrolizumabMMR-P RAS mutant CRC with MGMT hyper methylationRecruiting
      Anti-PD-L1 + Anti-CTLA4 + ChemotherapyNCT03202758I/IIDurvalumab

      Tremelimumab
      FOLFOXKRAS mutant CRCUnknown
      Anti PD-1 + chemotherapyNCT04194359IIISintilimabCAPOX-bevaKRAS mutant CRCRecruiting
      TRAIL receptor agonist + chemotherapyNCT03082209IEftozanermin (ABBV-321)FOLFIRI ± bevaKRAS mutant CRCCompleted
      ULK1/2 kinase inhibitor + MEK inhibitorNCT04892017IDCC-3116TrametinibRAS/RAF mutated solid tumorRecruiting
      XPO1 inhibitor + ICBNCT04854434IISelinexorPembrolizumabRAS mutated CRCRecruiting
      Abbreviations: KRAS; Kirsten rat sarcoma viral oncogene homolog; NSCLC, non-small cell lung cancer; CRC, colorectal cancer; SHP2, Src homology phosphotyrosyl phosphatase 2; EGFR, epidermal growth factor receptor; SOS1, Son of sevenless homolog 1; MEK, mitogen-activated protein kinase kinase; PD-1, programmed death-1; CDK4/6, cyclin-dependent kinases 4 and 6; mTOR, mammalian target of rapamycin; BRAF, v-raf murine sarcoma viral oncogene homolog B1; NF1, neurofibromin 1; ICB, immune checkpoint blockade; mRNA, messenger ribonucleic acid; anti-PD-L1, monoclonal antibody against programmed cell death-ligand 1; MEKi, inhibitor of mitogen-activated protein kinase kinase; PARPi, inhibitor of poly ADP ribose polymerase; TCR, T-cell receptor; CXCR1/2, C-X-C chemokine receptor 1/2; MSS, microsatellite stable; PLK-1, polo-like kinase 1, MDM2, mouse double minute 2 homolog, WEE1, WEE1 G2 checkpoint kinase; HER, human epidermal growth factor receptor; c-MET, tyrosine-protein kinase Met; NRAS, Neuroblastoma RAS viral oncogene homolog; FAK, focal adhesion kinase; MMR-P, mismatch repair proficient; MGMT, O6-alkylguanine DNA alkyltransferase; CTLA4, cytotoxic T-lymphocyte-associated protein 4; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; ULK1/2, unc-51-like autophagy activating kinase 1/2; XPO1, exportin-1.

      Fibroblasts and endothelial cells

      CAFs play a central role in extra cellular matrix (ECM) remodeling and can influence the TME through interaction with other cells via cytokines and growth factors. In pancreatic cancer, oncogenic KRAS is thought to activate fibroblasts via Hedgehog signaling [

      Dias Carvalho P, et al. KRAS Oncogenic Signaling Extends beyond Cancer Cells to Orchestrate the Microenvironment. Cancer Res 2018; 78(1): 7–14.

      ]. These activated CAFs, in turn, provide reciprocal AKT-mediated activation of tumor cells through IGF-1/IGF1R or GAS6/AXL signaling [
      • Tape C.J.
      • et al.
      Oncogenic KRAS Regulates Tumor Cell Signaling via Stromal Reciprocation.
      ]. A Kras-mutant MSS CRC mouse model also showed strong stromal activation, including CAF-specific TGF-β target genes that—similar to human CMS4 cancers—include ECM genes [
      • Tauriello D.V.F.
      • et al.
      TGFbeta drives immune evasion in genetically reconstituted colon cancer metastasis.
      ]. In contrast, a recent report links oncogenic KRAS to a significant downregulation of ECM/stroma-related genes, compared to wild-type counterparts, in locally advanced MSS rectal adenocarcinoma samples. IHC staining of rectal tumors showed decreased staining of POSTN (a regulator of collagen cross linking) in KRAS mutant samples [
      • Kim J.K.
      • et al.
      KRAS mutant rectal cancer cells interact with surrounding fibroblasts to deplete the extracellular matrix.
      ]. A genetically engineered mouse model confirmed lower expression of POSTN and FN1 proteins and ECM-related genes in Kras mutant compared to wild-type tumors [
      • Kim J.K.
      • et al.
      KRAS mutant rectal cancer cells interact with surrounding fibroblasts to deplete the extracellular matrix.
      ]. Perhaps this suggests a difference between rectal tumors and more proximal CRC in how mutant KRAS affects CAFs and stromal architecture.
      Endothelial cells cover the inner lining of blood- and lymphatic vessels. Tumors depend on blood vessels for growth and spreading. Angiogenesis, the formation of new vessels, is stimulated by tumor cells or by the TME—both by cellular signals and environmental factors such as local hypoxia, a consequence of vascular inadequacy. Newly formed tumor vessels are often immature and leaky, which may contribute to sustained hypoxia, as well as facilitate intravasation and dissemination [
      • Chen W.Z.
      • et al.
      Endothelial cells in colorectal cancer.
      ]. In CRC cell lines, KRAS mutations induce the production of pro-angiogenic proteins (e.g. VEGF, IL-8), while conditioned medium of mutant cells stimulates in vitro tube formation by endothelial cells [
      • Delle Monache S.
      • et al.
      Expression of pro-angiogenic factors as potential biomarkers in experimental models of colon cancer.
      ]. Induced HIF expression by PI3K and MAPK pathways was observed as a potential underlying mechanism. Knockdown of KRAS decreased levels of HIF-1α, bFGF and VEGF, suggesting that KRAS promotes angiogenesis in CRC [
      • Delle Monache S.
      • et al.
      Expression of pro-angiogenic factors as potential biomarkers in experimental models of colon cancer.
      ]. Additionally, KRAS can alter the expression of adhesion molecules on endothelial cells. In a liver metastasis mouse model with human KRAS mutant CRC cells, this was shown for BCAM and its ligand LAMA5, where the former was overexpressed in epithelial cells, and the latter was found on blood vessels in these tumors. Inhibition of BCAM or LAMA5 inhibited metastatic outgrowth in the mouse model by interfering with the adhesion of malignant cells to the endothelium [
      • Bartolini A.
      • et al.
      BCAM and LAMA5 Mediate the Recognition between Tumor Cells and the Endothelium in the Metastatic Spreading of KRAS-Mutant Colorectal Cancer.
      ].

      NRAS and HRAS mutations

      Besides the accumulating evidence for the TME-modulating effects of mutant KRAS, equivalent studies describing such roles for oncogenic NRAS and HRAS are limited. Although one can hypothesize that all RAS mutants exert similar effects through activation of a common set of pathways, differences in signaling by distinct mutant RAS isoforms may occur and can be context-dependent—i.e. depending on other signaling molecules and tissue specific factors [
      • Hood F.E.
      • et al.
      Isoform-specific Ras signaling is growth factor dependent.
      ]. One group introduced NRAS mutations (G12D and Q61K) into Caco-2 cells, showing elevated mRNA expression of IL-1, TNF-α, NF-κB and JAK/STAT signaling in cells expressing NRAS G12D [
      • Kuhn N.
      • Klinger B.
      • Uhlitz F.
      • Sieber A.
      • Rivera M.
      • Klotz-Noack K.
      • et al.
      Mutation-specific effects of NRAS oncogenes in colorectal cancer cells.
      ]. At the protein level, NRAS G12D led to strong elevation of IL-1A and IL-8, to higher levels than those observed in NRAS Q61K, linking this mutation to enhanced inflammation, immune infiltration, and tumor progression [
      • Kuhn N.
      • Klinger B.
      • Uhlitz F.
      • Sieber A.
      • Rivera M.
      • Klotz-Noack K.
      • et al.
      Mutation-specific effects of NRAS oncogenes in colorectal cancer cells.
      ].
      Since HRAS alterations are the least prevalent among RAS mutations in CRC, their influence on the TME has not been studied abundantly. However, expression of mutant variants of any of the three RAS isoforms led to increased squamous cell carcinoma antigen (SSCA) protein expression through MAPK pathway activation, including in CRC cells. This results in NF-kB signaling and subsequent cytokine production (IL-6, IL-8, CXCL-1, G-CSF and GM-CSF) in a mechanism that seems independent of oncogene-induced senescence [
      • Catanzaro J.M.
      • et al.
      Oncogenic Ras induces inflammatory cytokine production by upregulating the squamous cell carcinoma antigens SerpinB3/B4.
      ]. Overall, there are signs both of similar and of distinct effects on the TME amongst different RAS isotypes or within different mutations of the same isotype [
      • Hood F.E.
      • et al.
      Isoform-specific Ras signaling is growth factor dependent.
      ,
      • Horsch M.
      • et al.
      Overexpressed vs mutated Kras in murine fibroblasts: a molecular phenotyping study.
      ,
      • Hunter J.C.
      • et al.
      Biochemical and Structural Analysis of Common Cancer-Associated KRAS Mutations.
      ].

      BRAF mutations

      BRAF mutations, mainly BRAF V600E, occur in approximately 10% of CRC. These tumors have distinct clinical and pathological features. In contrast to KRAS mutations, they mainly occur in CMS 1 (known for immune infiltration and high tumor mutational burden) and are associated with microsatellite instability [
      • Guinney J.
      • Dienstmann R.
      • Wang X.
      • de Reyniès A.
      • Schlicker A.
      • Soneson C.
      • et al.
      The consensus molecular subtypes of colorectal cancer.
      ]. Still, CRC with a BRAF V600E mutation (BM) is a very heterogeneous group, which can be further divided in two subgroups based on transcriptomic data (BM1 and BM2). The former is characterized by KRAS/AKT/mTOR signaling and infiltration of immune cells while the latter shows differential expression of genes related to cell cycle regulation [
      • Barras D.
      • et al.
      BRAF V600E Mutant Colorectal Cancer Subtypes Based on Gene Expression.
      ]. The TME in these tumors is not as extensively studied as in KRAS mutant tumors. However in melanoma, BRAF mutations are evidently linked to an immunosuppressive environment [
      • Ilieva K.M.
      • et al.
      Effects of BRAF mutations and BRAF inhibition on immune responses to melanoma.
      ,
      • Frederick D.T.
      • et al.
      BRAF Inhibition Is Associated with Enhanced Melanoma Antigen Expression and a More Favorable Tumor Microenvironment in Patients with Metastatic Melanoma.
      ]. A comparison of BRAF mutant versus wild-type colorectal tumors using transcriptomics and IHC, showed more stromal cells, more immune cell infiltration, CD8+ T cell infiltration and higher expression levels of checkpoint molecule (PD-1, PD-L1, CTLA4 and LAG3) in BRAF mutant than in BRAF wild-type tumors [
      • Cen S.
      • et al.
      BRAF Mutation as a Potential Therapeutic Target for Checkpoint Inhibitors: A Comprehensive Analysis of Immune Microenvironment in BRAF Mutated Colon Cancer.
      ]. In vitro, BRAF mutations caused an increased production of IL-8 [
      • Conciatori F.
      • et al.
      BRAF status modulates Interelukin-8 expression through a CHOP-dependent mechanism in colorectal cancer.
      ]. A large study comparing the similarities and differences between TME of KRAS and BRAF mutant CRC is lacking. BRAF mutations and other MAPK pathway alterations will most likely exert similar immunosuppressive effects on cells in the TME via MAPK signaling. However, differences can be caused by the extent of pathway activation, signaling via alternative pathways and the other characteristics including BM subtype mentioned above. Combinations of BRAF/MEK inhibitors with ICB for pMMR BRAF V600E CRC are ongoing, with the rationale that this combination therapy can make these tumors more sensitive towards immunotherapy (NCT03668431; NCT04044430).

      Therapeutic avenues

      Targeting RAS

      Multiple approaches are employed to interfere with RAS expression or activation. Examples include therapeutically targeting of regulatory G-quadruplex (G4) structures or inhibition of RAS at the mRNA level (supplementary table 1). For proper plasma membrane localization and function, RAS is farnesylated, after which it requires PDEδ for intracellular transport. Therefore, inhibition of farnesylation or PDEδ seem attractive therapeutic approaches. Most of these strategies are either in a pre-clinical setting or lacked efficacy in clinical trials (supplementary table 1). SOS1, a GEF, and SHP-2, a tyrosine phosphatase that promotes RAS activation, are two modulators with potential clinical relevance [
      • Kessler D.
      • et al.
      Targeting Son of Sevenless 1: The pacemaker of KRAS.
      ,
      • Chou Y.T.
      • Bivona T.G.
      Inhibition of SHP2 as an approach to block RAS-driven cancers.
      ]. Inhibitors of SHP-2 and a drug that hinders the interaction between mutant KRAS and SOS1 (BI 1701963) are currently evaluated in clinical trials as monotherapy and in combination therapy (Table 1).
      A big step forward in the treatment of KRAS mutant cancers may be the discovery of small molecule inhibitors that directly target mutant KRAS G12C. These inhibitors bind a pocket next to the mutant cysteine forming a covalent bond with that residue—thus retaining the RAS protein in its GDP-bound state [
      • Ostrem J.M.
      • et al.
      K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions.
      ]. Adagrasib and sotorasib are two of the first examples of such G12C-specific, irreversible inhibitors to enter the clinic [
      • 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.
      • et al.
      The KRAS(G12C) Inhibitor MRTX849 Provides Insight toward Therapeutic Susceptibility of KRAS-Mutant Cancers in Mouse Models and Patients.
      ]. The results of the first clinical trials with these agents are promising, although response rates (RR) in CRC were much lower compared to NSCLC (supplementary table 2). Preliminary results from the KRYSTAL-1 trial with adagrasib report a 22% RR, stable disease in 64% and a mPFS of 5.6 months in patients with mCRC [
      • Weiss J.
      • et al.
      LBA6 KRYSTAL-1: Adagrasib (MRTX849) as monotherapy or combined with cetuximab (Cetux) in patients (Pts) with colorectal cancer (CRC) harboring a KRASG12C mutation.
      ]. The subgroup of patients with mCRC in the CodeBreak 100 trial treated with different doses of sotorasib revealed an objective response of 7%, disease control rate (DCR) of 74% and a mPFS of 4 months [
      • Hong D.S.
      • et al.
      KRAS(G12C) Inhibition with Sotorasib in Advanced Solid Tumors.
      ]. The follow-up phase II cohort with sotorasib (960 mg once daily) shows similar results compared to the phase I population with a response rate of 10%, DCR of 82% and a mPFS of 4 months in a cohort of 62 patients with mCRC [
      • Fakih M.G.
      • et al.
      Sotorasib for previously treated colorectal cancers with KRAS(G12C) mutation (CodeBreaK100): a prespecified analysis of a single-arm, phase 2 trial.
      ]. Several other KRAS G12C inhibitors have entered (pre)clinical evaluation (Table 1).
      Due to their modest activity as monotherapy and the anticipation of acquired resistance, research increasingly focusses on dissecting resistance mechanisms and combining G12C inhibitors with other therapeutic agents. Several potential mechanisms of acquired resistance were identified in patients treated with G12C inhibitors (supplementary table 2). Consequently, the addition of an inhibitor to an up- or downstream signaling factor was reported to overcome resistance to KRAS G12C inhibition in vitro and in vivo [
      • Ho C.S.L.
      • et al.
      HER2 mediates clinical resistance to the KRAS(G12C) inhibitor sotorasib, which is overcome by co-targeting SHP2.
      ,
      • Ryan M.B.
      • et al.
      Vertical Pathway Inhibition Overcomes Adaptive Feedback Resistance to KRAS(G12C) Inhibition.
      ]. In the clinic, analogous strategies are being tested, including combinations of KRAS G12C-directed agents with inhibitors of EGFR, SHP2, PI3K, CDK4/CDK6, AKT, MEK, and mTOR (Table 1). Auspiciously, promising preliminary data from the KRYSTAL-1 trial with cetuximab and adagrasib showing a RR of 43% and a DCR of 100% [
      • Weiss J.
      • et al.
      LBA6 KRYSTAL-1: Adagrasib (MRTX849) as monotherapy or combined with cetuximab (Cetux) in patients (Pts) with colorectal cancer (CRC) harboring a KRASG12C mutation.
      ], have made this the first combination to be investigated in a phase III trial in patients with mCRC (Table 1). First results from a similar combination of sotorasib with panitumumab revealed a 27% RR (of which 15% confirmed RR) and the combination advanced to a phase III setting for mCRC patients (Table 1) [
      • Fakih M.
      • et al.
      434P CodeBreaK 101 subprotocol H: Phase Ib study evaluating combination of sotorasib (Soto), a KRASG12C inhibitor, and panitumumab (PMab), an EGFR inhibitor, in advanced KRAS p. G12C-mutated colorectal cancer (CRC).
      ].
      The above-described approach targets a single specific mutation, which is only present in 3% of CRC patients. This limits widespread application of these drugs and combinations, leading to the search for less restrictive drugs. For example, direct inhibitors binding the more common G12D variant, by formation of a salt bridge with the aspartate residue, are in development (e.g. MRTX1133) [
      • Mao Z.
      • et al.
      KRAS(G12D) can be targeted by potent inhibitors via formation of salt bridge.
      ,
      • Wang X.
      • et al.
      Identification of MRTX1133, a Noncovalent, Potent, and Selective KRAS(G12D) Inhibitor.
      ]. Furthermore, KR12, a DNA-alkylating agent that binds with high affinity to the minor groove of specific KRAS codon 12 mutants (G12D and G12V), induces inhibition of cell growth in vivo and in vitro, by causing double-strand DNA breaks, cellular senescence and subsequently apoptosis [
      • Hiraoka K.
      • et al.
      Inhibition of KRAS codon 12 mutants using a novel DNA-alkylating pyrrole-imidazole polyamide conjugate.
      ]. Although this agent has not yet been tested in clinical trials, the fact that G12D/V mutations occur more frequently in CRC patients, renders this approach potentially beneficial for a broader CRC population. Among additional broad-spectrum direct KRAS inhibitors that are in pre-clinical development, BI 2852 targets the switch I/II pocket that is present in all three isoforms in the on and off state [
      • Kessler D.
      • et al.
      Drugging an undruggable pocket on KRAS.
      ].

      Targeting downstream signaling pathways

      Various treatments targeting single downstream signaling pathways of RAS, especially the RAS/RAF/MEK/ERK cascade, have shown little or no efficacy in CRC (supplementary table 3). Inhibition of individual downstream pathways (MAPK or PI3K) commonly evokes compensatory activation of the other, or of alternative signaling—indicating oncogenic redundancy. Improved efficacy is expected from inhibition of multiple downstream targets, or indeed pathways. However, this might be hampered by increased toxicity (supplementary table 3). Multiple combinations of MEK inhibitors with agents targeting the PI3K pathway have been tested—all with disappointing results [
      • Patelli G.
      • et al.
      Strategies to tackle RAS-mutated metastatic colorectal cancer.
      ].
      Additionally, MEK inhibitors are being tested in other combinations, such as with c-MET blockade, chemotherapy, ULK1/2 inhibitors and MDM2 inhibition (Table 1) [
      • Konopleva M.
      • et al.
      MDM2 inhibition: an important step forward in cancer therapy.
      ]. CH5126766 is a dual RAF/MEK kinase inhibitor currently being tested in intermittent schedules, as continuous dosing was restricted by toxicity. Objective responses were noted in 7/26 patients with a mutation in the RAS/RAF/MEK pathway, although none were observed in the mCRC group [
      • Guo C.
      • et al.
      Intermittent schedules of the oral RAF-MEK inhibitor CH5126766/VS-6766 in patients with RAS/RAF-mutant solid tumours and multiple myeloma: a single-centre, open-label, phase 1 dose-escalation and basket dose-expansion study.
      ]. Clinical trials with the combination of CH5126766 with either an mTOR inhibitor or a FAK inhibitor are ongoing (Table 1). The latter may have an added benefit besides causing a direct anti-tumor effect, as this is also a target in approaches aimed at the TME [
      • Murphy J.M.
      • et al.
      Targeting focal adhesion kinase in cancer cells and the tumor microenvironment.
      ]. Other combination strategies to improve the efficacy of inhibiting downstream effector pathways are currently under investigation. Synthetic lethal screens, aiming to search for targets essential in mutant opposed to wild-type KRAS, have identified multiple targets currently being tested in clinical trials [
      • Ku A.A.
      • et al.
      Integration of multiple biological contexts reveals principles of synthetic lethality that affect reproducibility.
      ,
      • Aguirre A.J.
      • Hahn W.C.
      Synthetic Lethal Vulnerabilities in KRAS-Mutant Cancers.
      ,
      • Grabocka E.
      • Commisso C.
      • Bar-Sagi D.
      Molecular pathways: targeting the dependence of mutant RAS cancers on the DNA damage response.
      ]. Examples include inhibitors of PLK1, PARP, WEE1 and CDK4/6 (Table 1).

      Immunotherapy combinations

      Given the many interactions between oncogenic RAS and the TME, combinatorial immunotherapy strategies with RAS-targeted treatments are increasingly being investigated. RAS mutations can give rise to immunogenic peptides that may be recognized by tumor-infiltrating lymphocytes (TILs). Indeed, KRAS G12D-responsive CTLs have been identified. In a case report, the ex vivo expansion and subsequent adoptive transfer of TILs able to recognize a KRAS-derived tumor-antigen resulted in a partial response [
      • Tran E.
      • et al.
      T-Cell Transfer Therapy Targeting Mutant KRAS in Cancer.
      ]. Peptide vaccines against KRAS G12D showed anti-tumor efficacy in a xenograft model [
      • Wan Y.
      • Recombinant K.R.A.S.
      • et al.
      G12D Protein Vaccines Elicit Significant Anti-Tumor Effects in Mouse CT26 Tumor Models.
      ]. Both KRAS peptide vaccines as well as mRNA-derived vaccines targeting mutant KRAS peptides are being tested in clinical trials either alone or in combination with ICB. In addition, T cell receptors (TCRs) recognizing mutant RAS may be exogenously expressed on T or NK cells before adoptive transfer into patients [
      • Chatani P.D.
      • Yang J.C.
      Mutated RAS: Targeting the “Untargetable” with T Cells.
      ,
      • Wang Q.J.
      • et al.
      Identification of T-cell Receptors Targeting KRAS-Mutated Human Tumors.
      ] (Table 1).
      Following promising preclinical findings with KRAS G12C-specific inhibitors [
      • 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.
      ,
      • Briere D.M.
      • et al.
      The KRAS(G12C) Inhibitor MRTX849 Reconditions the Tumor Immune Microenvironment and Sensitizes Tumors to Checkpoint Inhibitor Therapy.
      ], several combinations with ICB therapy are being evaluated in the clinic (Table 1). ICB therapy is also being utilized in many other combination strategies, including a combination with selinexor, a small molecule inhibitor of exportin-1 (XPO1). Inhibition of XPO1 results in the retention of tumor suppressor proteins in the nucleus and potentially leads to upregulation of PD-1/PD-L1 by inhibiting nuclear export of proteins involved in regulation of these checkpoint molecules, thereby sensitizing cells to ICB. In vivo, the combination showed a synergistic effect and the combination is currently being tested in a phase II trial in patients with KRAS mutant CRC (Table 1) [
      • Abdul Razak A.R.
      • et al.
      First-in-Class, First-in-Human Phase I Study of Selinexor, a Selective Inhibitor of Nuclear Export, in Patients With Advanced Solid Tumors.
      ,

      Elloul S, et al. Abstract 2219: Selinexor, a selective inhibitor of nuclear export (SINE) compound, shows synergistic anti-tumor activity when combined with PD-1 blockade in a mouse model of colon cancer. Cancer Res, 2016. 76(14 Supplement): p. 2219-2219.

      ].
      Besides an initial interest in statins as an alternative inhibitory mechanism of oncoprotein farnesylation, in vivo studies indicate they might be able to generate a broader immune response by inducing immunogenic cell death (ICD). Unlike the more common and orderly apoptosis, this type of cancer cell death can induce effective immune response through DC-mediated T cell priming. Combining statins with the ICD inducer oxaliplatin increased the sensitivity to ICB therapy and survival in a CT26 mouse model resistant to PD-1 blockade [
      • Nam G.H.
      • et al.
      Statin-mediated inhibition of RAS prenylation activates ER stress to enhance the immunogenicity of KRAS mutant cancer.
      ]. Due to the expanding knowledge of the TME and of the immune evasive roles of oncogenic RAS, immunotherapy combinations with RAS-targeted therapy may improve the treatment of patients with RAS mutant mCRC.
      As an alternative to focusing on T cells, NK cells might be used as immunotherapy treatment for KRAS mutant CRC. Unless inhibited by a suppressive TME, NK cells can have direct, unprimed cytotoxic effects against cancer cells. Moreover, they can be activated after binding of IgG1 monoclonal antibodies to Fc receptors located on the NK cell surface. Via this mechanism, cetuximab was shown to increase the anti-tumor efficacy of peripheral blood NK (PBNK) cells in multiple CRC cell lines regardless of KRAS status [
      • Veluchamy J.P.
      • et al.
      Combination of NK Cells and Cetuximab to Enhance Anti-Tumor Responses in RAS Mutant Metastatic Colorectal Cancer.
      ]. This synergistic effect was absent when umbilical cord blood (UCB)-derived NK cells were used [
      • Veluchamy J.P.
      • et al.
      In Vivo Efficacy of Umbilical Cord Blood Stem Cell-Derived NK Cells in the Treatment of Metastatic Colorectal Cancer.
      ]. The authors hypothesize that this difference is caused by the lower CD16 expression on UCB NK cells, which is important for antibody dependent cell-mediated cytotoxicity (ADCC). Nevertheless, the in vitro killing with UCB NK cells was equivalent to that of the combination of allogenic PBNK cells + cetuximab. Furthermore, in an in vivo model employing cetuximab resistant cell lines, UCB NK-cells showed significant antitumor efficacy [
      • Veluchamy J.P.
      • et al.
      Combination of NK Cells and Cetuximab to Enhance Anti-Tumor Responses in RAS Mutant Metastatic Colorectal Cancer.
      ,
      • Veluchamy J.P.
      • et al.
      In Vivo Efficacy of Umbilical Cord Blood Stem Cell-Derived NK Cells in the Treatment of Metastatic Colorectal Cancer.
      ]. These findings provide additional rationale for clinical testing of adoptive transfer of NK cells for the treatment of mCRC. One group engineered NK cells in such a way that they contain the anti-EGFR antibody cetuximab on their surface (NK92-CET cells). Treatment with these NK-cells in vitro and in vivo resulted in increased levels of perforin and IFN-γ, and yielded promising anti-tumor efficacy towards KRAS mutant CRC [
      • Wang X.
      • et al.
      Equipping Natural Killer Cells with Cetuximab through Metabolic Glycoengineering and Bioorthogonal Reaction for Targeted Treatment of KRAS Mutant Colorectal Cancer.
      ].
      Lastly, oncolytic viruses have the capacity to selectively infect and kill malignant cells, thereby inducing an immune response which potentially improves anti-tumor efficacy [
      • Hemminki O.
      • dos Santos J.M.
      • Hemminki A.
      Oncolytic viruses for cancer immunotherapy.
      ]. The combination of a reovirus with FOLFIRI plus bevacizumab was evaluated in KRAS mutant mCRC patients (Table 1). A partial response was observed in 3/6 patients with a median PFS of 66 weeks and OS of 25 months [
      • Goel S.
      • et al.
      Elucidation of Pelareorep Pharmacodynamics in A Phase I Trial in Patients with KRAS-Mutated Colorectal Cancer.
      ]. Multiple beneficial immune markers were observed, such as increased levels of GM-CSF and IFN-γ, and changes in immune cell populations including increased DCs and T lymphocytes [
      • Parakrama R.
      • et al.
      Immune characterization of metastatic colorectal cancer patients post reovirus administration.
      ].

      Concluding remarks

      Our knowledge of the various downstream effects of oncogenic RAS signaling is expanding: recent studies indicate that these mutations impact the TME in multiple key ways. Many of these stromal interactions indicate an even wider role for mutant RAS in driving cancer progression than was previously appreciated. Through interaction with cells in the surrounding microenvironment, oncogenic RAS can contribute to the formation of a pro-metastatic and immunosuppressive TME. Much of this knowledge is drawn from disparate research approaches—including cell lines, mouse models, and human tumor samples—and a lot remains to be discovered about these complex interactions between RAS mutant cancer cells and the TME. Many of the therapeutic agents discussed above are still in pre-clinical testing. However, a class of direct KRAS inhibitors that have recently entered clinical trials are anticipated to have promising efficacy, and to synergize with immunotherapies. A continued improvement of our understanding of the TME, supported by more sophisticated models, will likely facilitate new immunotherapeutic combinations tailored to KRAS mutant mCRC. Consequently, a new group of therapies may become available for a group of patients that currently lack efficacious targeted treatment options.

      CRediT authorship contribution statement

      Jorien B.E. Janssen: Writing – original draft, Visualization. Jan Paul Medema: Writing – review & editing. Elske C. Gootjes: Writing – review & editing. Daniele V.F. Tauriello: Conceptualization, Writing – original draft, Visualization. Henk M.W. Verheul: Conceptualization, Writing – original draft.

      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.

      Appendix A. Supplementary material

      The following are the Supplementary data to this article:

      References

        • Van Cutsem E.
        • Cervantes A.
        • Adam R.
        • Sobrero A.
        • Van Krieken J.H.
        • Aderka D.
        • et al.
        ESMO consensus guidelines for the management of patients with metastatic colorectal cancer.
        Ann Oncol. 2016; 27: 1386-1422
        • Meng M.
        • et al.
        The current understanding on the impact of KRAS on colorectal cancer.
        Biomed Pharmacother. 2021; 140111717
        • Levin-Sparenberg E.
        • Bylsma L.C.
        • Lowe K.
        • Sangare L.
        • Fryzek J.P.
        • Alexander D.D.
        A Systematic Literature Review and Meta-Analysis Describing the Prevalence of KRAS, NRAS, and BRAF Gene Mutations in Metastatic Colorectal Cancer.
        Gastroenterol Res. 2020; 13: 184-198
      1. Taieb J, et al. Prognostic Value of BRAF and KRAS Mutations in MSI and MSS Stage III Colon Cancer. JNCI: J Natl Cancer Instit 2016; 109(5).

        • Ottaiano A.
        • Normanno N.
        • Facchini S.
        • Cassata A.
        • Nappi A.
        • Romano C.
        • et al.
        Study of Ras Mutations' Prognostic Value in Metastatic Colorectal Cancer: STORIA Analysis.
        Cancers. 2020; 12: 1919https://doi.org/10.3390/cancers12071919
        • Cox A.D.
        • et al.
        Drugging the undruggable RAS: Mission Possible?.
        Nat Rev Drug Discovery. 2014; 13: 828-851
        • Kuhn N.
        • Klinger B.
        • Uhlitz F.
        • Sieber A.
        • Rivera M.
        • Klotz-Noack K.
        • et al.
        Mutation-specific effects of NRAS oncogenes in colorectal cancer cells.
        Adv Biol Regul. 2021; 79: 100778https://doi.org/10.1016/j.jbior.2020.100778
        • Chang Y.-Y.
        • Lin P.-C.
        • Lin H.-H.
        • Lin J.-K.
        • Chen W.-S.
        • Jiang J.-K.
        • et al.
        Mutation spectra of RAS gene family in colorectal cancer.
        Am J Surg. 2016; 212: 537-544.e3
        • Haigis K.M.
        • Kendall K.R.
        • Wang Y.
        • Cheung A.
        • Haigis M.C.
        • Glickman J.N.
        • et al.
        Differential effects of oncogenic K-Ras and N-Ras on proliferation, differentiation and tumor progression in the colon.
        Nat Genet. 2008; 40: 600-608
        • Fearon E.R.
        • Vogelstein B.
        A genetic model for colorectal tumorigenesis.
        Cell. 1990; 61: 759-767
      2. Wang Y, et al. Mutant N-RAS protects colorectal cancer cells from stress-induced apoptosis and contributes to cancer development and progression. Cancer Discov 2013; 3(3): 294–307.

      3. Zafra MP, et al. An In Vivo Kras Allelic Series Reveals Distinct Phenotypes of Common Oncogenic Variants. Cancer Discov 2020; 10(11): p. 1654–1671.

        • Koveitypour Z.
        • et al.
        Signaling pathways involved in colorectal cancer progression.
        Cell Biosci. 2019; 9: 97
        • Tauriello D.V.F.
        • Batlle E.
        Targeting the Microenvironment in Advanced Colorectal Cancer.
        Trends Cancer. 2016; 2: 495-504
        • Sun Y.u.
        Tumor microenvironment and cancer therapy resistance.
        Cancer Lett. 2016; 380: 205-215
        • Janssen E.
        • Subtil B.
        • de la Jara Ortiz F.
        • Verheul H.M.W.
        • Tauriello D.V.F.
        Combinatorial Immunotherapies for Metastatic Colorectal Cancer.
        Cancers (Basel). 2020; 12: 1875https://doi.org/10.3390/cancers12071875
        • Popat S.
        • Hubner R.
        • Houlston R.S.
        Systematic review of microsatellite instability and colorectal cancer prognosis.
        J Clin Oncol. 2005; 23: 609-618
        • Dienstmann R.
        • Mason M.J.
        • Sinicrope F.A.
        • Phipps A.I.
        • Tejpar S.
        • Nesbakken A.
        • et al.
        Prediction of overall survival in stage II and III colon cancer beyond TNM system: a retrospective, pooled biomarker study.
        Ann Oncol. 2017; 28: 1023-1031
        • Bahl A.
        • et al.
        Primary Tumor Location as a Prognostic and Predictive Marker in Metastatic Colorectal Cancer (mCRC).
        Front Oncol. 2020; 10: 964
        • Guinney J.
        • Dienstmann R.
        • Wang X.
        • de Reyniès A.
        • Schlicker A.
        • Soneson C.
        • et al.
        The consensus molecular subtypes of colorectal cancer.
        Nat Med. 2015; 21: 1350-1356
        • Vaughn C.P.
        • ZoBell S.D.
        • Furtado L.V.
        • Baker C.L.
        • Samowitz W.S.
        Frequency of KRAS, BRAF, and NRAS mutations in colorectal cancer.
        Genes Chromosom Cancer. 2011; 50: 307-312
        • Kamata T.
        • Feramisco J.R.
        Epidermal growth factor stimulates guanine nucleotide binding activity and phosphorylation of ras oncogene proteins.
        Nature. 1984; 310: 147-150
        • Simanshu D.K.
        • Nissley D.V.
        • McCormick F.
        RAS Proteins and Their Regulators in Human Disease.
        Cell. 2017; 170: 17-33
        • Vetter I.R.
        • Wittinghofer A.
        The guanine nucleotide-binding switch in three dimensions.
        Science. 2001; 294: 1299-1304
        • Boriack-Sjodin P.A.
        • Margarit S.M.
        • Bar-Sagi D.
        • Kuriyan J.
        The structural basis of the activation of Ras by Sos.
        Nature. 1998; 394: 337-343
        • Boguski M.S.
        • McCormick F.
        Proteins regulating Ras and its relatives.
        Nature. 1993; 366: 643-654
        • Gibbs J.B.
        • Sigal I.S.
        • Poe M.
        • Scolnick E.M.
        Intrinsic GTPase activity distinguishes normal and oncogenic ras p21 molecules.
        Proc Natl Acad Sci U S A. 1984; 81: 5704-5708
        • 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.
        N Engl J Med. 2008; 359: 1757-1765
        • 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.
        J Clin Oncol. 2008; 26: 1626-1634
        • Pietrantonio F.
        • Petrelli F.
        • Coinu A.
        • Di Bartolomeo M.
        • Borgonovo K.
        • Maggi C.
        • et al.
        Predictive role of BRAF mutations in patients with advanced colorectal cancer receiving cetuximab and panitumumab: a meta-analysis.
        Eur J Cancer. 2015; 51: 587-594
        • Sorich M.J.
        • Wiese M.D.
        • Rowland A.
        • Kichenadasse G.
        • McKinnon R.A.
        • Karapetis C.S.
        Extended RAS mutations and anti-EGFR monoclonal antibody survival benefit in metastatic colorectal cancer: a meta-analysis of randomized, controlled trials.
        Ann Oncol. 2015; 26: 13-21
        • Lakatos G.
        • Köhne C.-H.
        • Bodoky G.
        Current therapy of advanced colorectal cancer according to RAS/RAF mutational status.
        Cancer Metastasis Rev. 2020; 39: 1143-1157
        • van Helden E.J.
        • et al.
        RAS and BRAF mutations in cell-free DNA are predictive for outcome of cetuximab monotherapy in patients with tissue-tested RAS wild-type advanced colorectal cancer.
        Mol Oncol. 2019; 13: 2361-2374
        • Garvey C.M.
        • Lau R.
        • Sanchez A.
        • Sun R.X.
        • Fong E.J.
        • Doche M.E.
        • et al.
        Anti-EGFR Therapy Induces EGF Secretion by Cancer-Associated Fibroblasts to Confer Colorectal Cancer Chemoresistance.
        Cancers (Basel). 2020; 12: 1393https://doi.org/10.3390/cancers12061393
        • Hobor S.
        • et al.
        TGFalpha and amphiregulin paracrine network promotes resistance to EGFR blockade in colorectal cancer cells.
        Clin Cancer Res. 2014; 20: 6429-6438
        • Woolston A.
        • et al.
        Genomic and Transcriptomic Determinants of Therapy Resistance and Immune Landscape Evolution during Anti-EGFR Treatment in Colorectal Cancer.
        Cancer Cell. 2019; 36: 35-50.e9
        • John J.
        • Sohmen R.
        • Feuerstein J.
        • Linke R.
        • Wittinghofer A.
        • Goody R.S.
        Kinetics of interaction of nucleotides with nucleotide-free H-ras p21.
        Biochemistry. 1990; 29: 6058-6065
        • Hanahan D.
        • Coussens L.
        Accessories to the crime: functions of cells recruited to the tumor microenvironment.
        Cancer Cell. 2012; 21: 309-322
        • Quail D.F.
        • Joyce J.A.
        Microenvironmental regulation of tumor progression and metastasis.
        Nat Med. 2013; 19: 1423-1437
        • Pietras K.
        • Östman A.
        Hallmarks of cancer: interactions with the tumor stroma.
        Exp Cell Res. 2010; 316: 1324-1331
        • Hamarsheh S.
        • Groß O.
        • Brummer T.
        • Zeiser R.
        Immune modulatory effects of oncogenic KRAS in cancer.
        Nat Commun. 2020; 11https://doi.org/10.1038/s41467-020-19288-6
      4. Dias Carvalho P, et al. KRAS Oncogenic Signaling Extends beyond Cancer Cells to Orchestrate the Microenvironment. Cancer Res 2018; 78(1): 7–14.

        • 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.
        Nature. 2019; 575: 217-223
        • Serrano M.
        • Lin A.W.
        • McCurrach M.E.
        • Beach D.
        • Lowe S.W.
        Oncogenic ras Provokes Premature Cell Senescence Associated with Accumulation of p53 and p16INK4a.
        Cell. 1997; 88: 593-602
        • Zhu H.
        • et al.
        Oncogene-induced senescence: From biology to therapy.
        Mech Ageing Dev. 2020; 187111229
        • Cullis J.
        • Das S.
        • Bar-Sagi D.
        Kras and Tumor Immunity: Friend or Foe?.
        Cold Spring Harb Perspect Med. 2018; 8
        • Tauriello D.V.F.
        • Sancho E.
        • Batlle E.
        Overcoming TGFβ-mediated immune evasion in cancer.
        Nat Rev Cancer. 2022; 22: 25-44
        • Petanidis S.
        • et al.
        Differential expression of IL-17, 22 and 23 in the progression of colorectal cancer in patients with K-ras mutation: Ras signal inhibition and crosstalk with GM-CSF and IFN-gamma.
        PLoS ONE. 2013; 8e73616
        • Parmiani G.
        • et al.
        Opposite immune functions of GM-CSF administered as vaccine adjuvant in cancer patients.
        Ann Oncol. 2007; 18: 226-232
        • Soleimani A.
        • et al.
        Role of the NF-kappaB signaling pathway in the pathogenesis of colorectal cancer.
        Gene. 2020; 726144132
        • Lin G.
        • et al.
        NF-kappaB activity is downregulated by KRAS knockdown in SW620 cells via the RAS-ERK-IkappaBalpha pathway.
        Oncol Rep. 2012; 27: 1527-1534
        • Subtil B.
        • et al.
        The Therapeutic Potential of Tackling Tumor-Induced Dendritic Cell Dysfunction in Colorectal Cancer.
        Front Immunol. 2021; 12
        • Kocián P.
        • et al.
        Tumor-infiltrating lymphocytes and dendritic cells in human colorectal cancer: Their relationship to KRAS mutational status and disease recurrence.
        Hum Immunol. 2011; 72: 1022-1028
        • Hao N.B.
        • et al.
        Macrophages in tumor microenvironments and the progression of tumors.
        Clin Dev Immunol. 2012; 2012948098
        • Liu H.
        • et al.
        Mutant KRAS triggers functional reprogramming of tumor-associated macrophages in colorectal cancer.
        Signal Transduct Target Ther. 2021; 6: 144
        • Koelzer V.H.
        • et al.
        Phenotyping of tumor-associated macrophages in colorectal cancer: Impact on single cell invasion (tumor budding) and clinicopathological outcome.
        Oncoimmunology. 2016; 5e1106677
        • Idos G.E.
        • et al.
        The Prognostic Implications of Tumor Infiltrating Lymphocytes in Colorectal Cancer: A Systematic Review and Meta-Analysis.
        Sci Rep. 2020; 10: 3360
        • Waldman A.D.
        • Fritz J.M.
        • Lenardo M.J.
        A guide to cancer immunotherapy: from T cell basic science to clinical practice.
        Nat Rev Immunol. 2020; 20: 651-668
        • Huyghe N.
        • Baldin P.
        • Van den Eynde M.
        Immunotherapy with immune checkpoint inhibitors in colorectal cancer: what is the future beyond deficient mismatch-repair tumours?.
        Gastroenterol Rep (Oxf). 2020; 8: 11-24
        • Tauriello D.V.F.
        • et al.
        TGFbeta drives immune evasion in genetically reconstituted colon cancer metastasis.
        Nature. 2018; 554: 538-543
        • Liao W.
        • et al.
        KRAS-IRF2 Axis Drives Immune Suppression and Immune Therapy Resistance in Colorectal Cancer.
        Cancer Cell. 2019; 35: 559-572 e7
        • Lal N.
        • et al.
        An immunogenomic stratification of colorectal cancer: Implications for development of targeted immunotherapy.
        Oncoimmunology. 2015; 4e976052
        • Lal N.
        • et al.
        KRAS Mutation and Consensus Molecular Subtypes 2 and 3 Are Independently Associated with Reduced Immune Infiltration and Reactivity in Colorectal Cancer.
        Clin Cancer Res. 2018; 24: 224-233
        • Park H.E.
        • et al.
        Tumor microenvironment-adjusted prognostic implications of the KRAS mutation subtype in patients with stage III colorectal cancer treated with adjuvant FOLFOX.
        Sci Rep. 2021; 11: 14609
        • Zdanov S.
        • et al.
        Mutant KRAS Conversion of Conventional T Cells into Regulatory T Cells.
        Cancer Immunol Res. 2016; 4: 354-365
        • Batlle E.
        • Massague J.
        Transforming Growth Factor-beta Signaling in Immunity and Cancer.
        Immunity. 2019; 50: 924-940
        • Liu J.
        • et al.
        Immune landscape and prognostic immune-related genes in KRAS-mutant colorectal cancer patients.
        J Transl Med. 2021; 19: 27
        • Asadzadeh Z.
        • et al.
        The paradox of Th17 cell functions in tumor immunity.
        Cell Immunol. 2017; 322: 15-25
        • He D.
        • et al.
        IL-17 promotes tumor development through the induction of tumor promoting microenvironments at tumor sites and myeloid-derived suppressor cells.
        J Immunol. 2010; 184: 2281-2288
        • El-Jawhari J.J.
        • et al.
        Blocking oncogenic RAS enhances tumour cell surface MHC class I expression but does not alter susceptibility to cytotoxic lymphocytes.
        Mol Immunol. 2014; 58: 160-168
        • Smakman N.
        • et al.
        Dual effect of Kras(D12) knockdown on tumorigenesis: increased immune-mediated tumor clearance and abrogation of tumor malignancy.
        Oncogene. 2005; 24: 8338-8342
        • Melaiu O.
        • et al.
        Influence of the Tumor Microenvironment on NK Cell Function in Solid Tumors.
        Front Immunol. 2020; 10
        • Nishi K.
        • et al.
        Mutant KRAS Promotes NKG2D(+) T Cell Infiltration and CD155 Dependent Immune Evasion.
        Anticancer Res. 2020; 40: 4663-4674
        • Uribe-Querol E.
        • Rosales C.
        Neutrophils in Cancer: Two Sides of the Same Coin.
        J Immunol Res. 2015; 2015983698
        • Germann M.
        • et al.
        Neutrophils suppress tumor-infiltrating T cells in colon cancer via matrix metalloproteinase-mediated activation of TGFbeta.
        EMBO Mol Med. 2020; 12e10681
        • Masucci M.T.
        • et al.
        The Emerging Role of Neutrophil Extracellular Traps (NETs) in Tumor Progression and Metastasis.
        Front Immunol. 2020; 11: 1749
        • Shang A.
        • et al.
        Exosomal KRAS mutation promotes the formation of tumor-associated neutrophil extracellular traps and causes deterioration of colorectal cancer by inducing IL-8 expression.
        Cell Commun Signal. 2020; 18: 52
        • Sieminska I.
        • Baran J.
        Myeloid-Derived Suppressor Cells in Colorectal Cancer.
        Front Immunol. 2020; 11: 1526
        • Tape C.J.
        • et al.
        Oncogenic KRAS Regulates Tumor Cell Signaling via Stromal Reciprocation.
        Cell. 2016; 165: 910-920
        • Kim J.K.
        • et al.
        KRAS mutant rectal cancer cells interact with surrounding fibroblasts to deplete the extracellular matrix.
        Mol Oncol. 2021; 15: 2766-2781
        • Chen W.Z.
        • et al.
        Endothelial cells in colorectal cancer.
        World J Gastrointest Oncol. 2019; 11: 946-956
        • Delle Monache S.
        • et al.
        Expression of pro-angiogenic factors as potential biomarkers in experimental models of colon cancer.
        J Cancer Res Clin Oncol. 2020; 146: 1427-1440
        • Bartolini A.
        • et al.
        BCAM and LAMA5 Mediate the Recognition between Tumor Cells and the Endothelium in the Metastatic Spreading of KRAS-Mutant Colorectal Cancer.
        Clin Cancer Res. 2016; 22: 4923-4933
        • Hood F.E.
        • et al.
        Isoform-specific Ras signaling is growth factor dependent.
        Mol Biol Cell. 2019; 30: 1108-1117
        • Catanzaro J.M.
        • et al.
        Oncogenic Ras induces inflammatory cytokine production by upregulating the squamous cell carcinoma antigens SerpinB3/B4.
        Nat Commun. 2014; 5: 3729
        • Horsch M.
        • et al.
        Overexpressed vs mutated Kras in murine fibroblasts: a molecular phenotyping study.
        Br J Cancer. 2009; 100: 656-662
        • Hunter J.C.
        • et al.
        Biochemical and Structural Analysis of Common Cancer-Associated KRAS Mutations.
        Mol Cancer Res. 2015; 13: 1325-1335
        • Barras D.
        • et al.
        BRAF V600E Mutant Colorectal Cancer Subtypes Based on Gene Expression.
        Clin Cancer Res. 2017; 23: 104-115
        • Ilieva K.M.
        • et al.
        Effects of BRAF mutations and BRAF inhibition on immune responses to melanoma.
        Mol Cancer Ther. 2014; 13: 2769-2783
        • Frederick D.T.
        • et al.
        BRAF Inhibition Is Associated with Enhanced Melanoma Antigen Expression and a More Favorable Tumor Microenvironment in Patients with Metastatic Melanoma.
        Clin Cancer Res. 2013; 19: 1225-1231
        • Cen S.
        • et al.
        BRAF Mutation as a Potential Therapeutic Target for Checkpoint Inhibitors: A Comprehensive Analysis of Immune Microenvironment in BRAF Mutated Colon Cancer.
        Front Cell Dev Biol. 2021; 9
        • Conciatori F.
        • et al.
        BRAF status modulates Interelukin-8 expression through a CHOP-dependent mechanism in colorectal cancer.
        Commun Biol. 2020; 3: 546
        • Kessler D.
        • et al.
        Targeting Son of Sevenless 1: The pacemaker of KRAS.
        Curr Opin Chem Biol. 2021; 62: 109-118
        • Chou Y.T.
        • Bivona T.G.
        Inhibition of SHP2 as an approach to block RAS-driven cancers.
        Adv Cancer Res. 2022; 153: 205-236
        • Ostrem J.M.
        • et al.
        K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions.
        Nature. 2013; 503: 548-551
        • Hallin J.
        • et al.
        The KRAS(G12C) Inhibitor MRTX849 Provides Insight toward Therapeutic Susceptibility of KRAS-Mutant Cancers in Mouse Models and Patients.
        Cancer Discov. 2020; 10: 54-71
        • Weiss J.
        • et al.
        LBA6 KRYSTAL-1: Adagrasib (MRTX849) as monotherapy or combined with cetuximab (Cetux) in patients (Pts) with colorectal cancer (CRC) harboring a KRASG12C mutation.
        Ann Oncol. 2021; 32: S1294
        • Hong D.S.
        • et al.
        KRAS(G12C) Inhibition with Sotorasib in Advanced Solid Tumors.
        N Engl J Med. 2020; 383: 1207-1217
        • Fakih M.G.
        • et al.
        Sotorasib for previously treated colorectal cancers with KRAS(G12C) mutation (CodeBreaK100): a prespecified analysis of a single-arm, phase 2 trial.
        Lancet Oncol. 2022; 23: 115-124
        • Ho C.S.L.
        • et al.
        HER2 mediates clinical resistance to the KRAS(G12C) inhibitor sotorasib, which is overcome by co-targeting SHP2.
        Eur J Cancer. 2021; 159: 16-23
        • Ryan M.B.
        • et al.
        Vertical Pathway Inhibition Overcomes Adaptive Feedback Resistance to KRAS(G12C) Inhibition.
        Clin Cancer Res. 2020; 26: 1633-1643
        • Fakih M.
        • et al.
        434P CodeBreaK 101 subprotocol H: Phase Ib study evaluating combination of sotorasib (Soto), a KRASG12C inhibitor, and panitumumab (PMab), an EGFR inhibitor, in advanced KRAS p. G12C-mutated colorectal cancer (CRC).
        Ann Oncol. 2021; 32: S551
        • Mao Z.
        • et al.
        KRAS(G12D) can be targeted by potent inhibitors via formation of salt bridge.
        Cell Discovery. 2022; 8: 5
        • Wang X.
        • et al.
        Identification of MRTX1133, a Noncovalent, Potent, and Selective KRAS(G12D) Inhibitor.
        J Med Chem. 2022; 65: 3123-3133
        • Hiraoka K.
        • et al.
        Inhibition of KRAS codon 12 mutants using a novel DNA-alkylating pyrrole-imidazole polyamide conjugate.
        Nat Commun. 2015; 6: 6706
        • Kessler D.
        • et al.
        Drugging an undruggable pocket on KRAS.
        Proc Natl Acad Sci U S A. 2019; 116: 15823-15829
        • Patelli G.
        • et al.
        Strategies to tackle RAS-mutated metastatic colorectal cancer.
        ESMO Open. 2021; 6100156
        • Konopleva M.
        • et al.
        MDM2 inhibition: an important step forward in cancer therapy.
        Leukemia. 2020; 34: 2858-2874
        • Guo C.
        • et al.
        Intermittent schedules of the oral RAF-MEK inhibitor CH5126766/VS-6766 in patients with RAS/RAF-mutant solid tumours and multiple myeloma: a single-centre, open-label, phase 1 dose-escalation and basket dose-expansion study.
        Lancet Oncol. 2020; 21: 1478-1488
        • Murphy J.M.
        • et al.
        Targeting focal adhesion kinase in cancer cells and the tumor microenvironment.
        Exp Mol Med. 2020; 52: 877-886
        • Ku A.A.
        • et al.
        Integration of multiple biological contexts reveals principles of synthetic lethality that affect reproducibility.
        Nat Commun. 2020; 11: 2375
        • Aguirre A.J.
        • Hahn W.C.
        Synthetic Lethal Vulnerabilities in KRAS-Mutant Cancers.
        Cold Spring Harb Perspect Med. 2018; 8
        • Grabocka E.
        • Commisso C.
        • Bar-Sagi D.
        Molecular pathways: targeting the dependence of mutant RAS cancers on the DNA damage response.
        Clin Cancer Res. 2015; 21: 1243-1247
        • Tran E.
        • et al.
        T-Cell Transfer Therapy Targeting Mutant KRAS in Cancer.
        N Engl J Med. 2016; 375: 2255-2262
        • Wan Y.
        • Recombinant K.R.A.S.
        • et al.
        G12D Protein Vaccines Elicit Significant Anti-Tumor Effects in Mouse CT26 Tumor Models.
        Front Oncol. 2020; 10
        • Chatani P.D.
        • Yang J.C.
        Mutated RAS: Targeting the “Untargetable” with T Cells.
        Clin Cancer Res. 2020; 26: 537-544
        • Wang Q.J.
        • et al.
        Identification of T-cell Receptors Targeting KRAS-Mutated Human Tumors.
        Cancer Immunol Res. 2016; 4: 204-214
        • Briere D.M.
        • et al.
        The KRAS(G12C) Inhibitor MRTX849 Reconditions the Tumor Immune Microenvironment and Sensitizes Tumors to Checkpoint Inhibitor Therapy.
        Mol Cancer Ther. 2021; 20: 975-985
        • Abdul Razak A.R.
        • et al.
        First-in-Class, First-in-Human Phase I Study of Selinexor, a Selective Inhibitor of Nuclear Export, in Patients With Advanced Solid Tumors.
        J Clin Oncol: Off J Am Soc Clin Oncol. 2016; 34: 4142-4150
      5. Elloul S, et al. Abstract 2219: Selinexor, a selective inhibitor of nuclear export (SINE) compound, shows synergistic anti-tumor activity when combined with PD-1 blockade in a mouse model of colon cancer. Cancer Res, 2016. 76(14 Supplement): p. 2219-2219.

        • Nam G.H.
        • et al.
        Statin-mediated inhibition of RAS prenylation activates ER stress to enhance the immunogenicity of KRAS mutant cancer.
        J ImmunoTher Cancer. 2021; 9
        • Veluchamy J.P.
        • et al.
        Combination of NK Cells and Cetuximab to Enhance Anti-Tumor Responses in RAS Mutant Metastatic Colorectal Cancer.
        PLoS ONE. 2016; 11e0157830
        • Veluchamy J.P.
        • et al.
        In Vivo Efficacy of Umbilical Cord Blood Stem Cell-Derived NK Cells in the Treatment of Metastatic Colorectal Cancer.
        Front Immunol. 2017; 8: 87
        • Wang X.
        • et al.
        Equipping Natural Killer Cells with Cetuximab through Metabolic Glycoengineering and Bioorthogonal Reaction for Targeted Treatment of KRAS Mutant Colorectal Cancer.
        ACS Chem Biol. 2021; 16: 724-730
        • Hemminki O.
        • dos Santos J.M.
        • Hemminki A.
        Oncolytic viruses for cancer immunotherapy.
        J. Hematol. Oncol. 2020; 13: 84
        • Goel S.
        • et al.
        Elucidation of Pelareorep Pharmacodynamics in A Phase I Trial in Patients with KRAS-Mutated Colorectal Cancer.
        Mol Cancer Ther. 2020; 19: 1148-1156
        • Parakrama R.
        • et al.
        Immune characterization of metastatic colorectal cancer patients post reovirus administration.
        BMC Cancer. 2020; 20: 569