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KEAP1-Mutant NSCLC: The Catastrophic Failure of a Cell-Protecting Hub

  • Stefano Scalera
    Affiliations
    SAFU Laboratory, Department of Research, Advanced Diagnostic, and Technological Innovation, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Regina Elena National Cancer Institute, Rome, Italy
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  • Marco Mazzotta
    Affiliations
    Department of Oncology and Hematology, Medical Oncology Unit, Central Hospital of Belcolle, Viterbo, Italy
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  • Clelia Cortile
    Affiliations
    SAFU Laboratory, Department of Research, Advanced Diagnostic, and Technological Innovation, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Regina Elena National Cancer Institute, Rome, Italy
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  • Eriseld Krasniqi
    Affiliations
    Division of Medical Oncology 2, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Regina Elena National Cancer Institute, Rome, Italy
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  • Ruggero De Maria
    Affiliations
    Fondazione Policlinico Universitario A. Gemelli Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Rome, Italy

    Institute of General Pathology, Università Cattolica del Sacro Cuore, Rome, Italy
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  • Federico Cappuzzo
    Affiliations
    Division of Medical Oncology 2, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Regina Elena National Cancer Institute, Rome, Italy
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  • Gennaro Ciliberto
    Affiliations
    Scientific Direction, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Regina Elena National Cancer Institute, Rome, Italy
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  • Marcello Maugeri-Saccà
    Correspondence
    Corresponding author. Address for correspondence: Marcello Maugeri-Saccà, MD, PhD, Clinical Trial Center, Biostatistics and Bioinformatics Division, Istituto di Ricovero e Cura a Carattere Scientifico Regina Elena National Cancer Institute, Via Elio Chianesi 53, 00144, Roma, Italy.
    Affiliations
    Division of Medical Oncology 2, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Regina Elena National Cancer Institute, Rome, Italy

    Clinical Trial Center, Biostatistics and Bioinformatics Unit, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Regina Elena National Cancer Institute, Rome, Italy
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Published:March 26, 2022DOI:https://doi.org/10.1016/j.jtho.2022.03.011

      Abstract

      Mutations in the KEAP1-NRF2 pathway are common in NSCLC, albeit with a prevalence of KEAP1 mutations in lung adenocarcinoma and an equal representation of KEAP1 and NFE2L2 (the gene encoding for NRF2) alterations in lung squamous cell carcinoma. The KEAP1-NRF2 axis is a crucial modulator of cellular homeostasis, enabling cells to tolerate oxidative and metabolic stresses, and xenobiotics. The complex cytoprotective response orchestrated by NRF2-mediated gene transcription embraces detoxification mechanisms, ferroptosis protection, and metabolic reprogramming. Given that the KEAP1-NRF2 pathway controls core cellular functions, it is not surprising that a number of clinical studies connected KEAP1 mutations to increased resistance to chemotherapy, radiotherapy, and targeted agents. More recently, an immune-cold tumor microenvironment was described as a typical feature of KEAP1-mutant lung adenocarcinoma. Consistently, a reduced efficacy of immunotherapy was reported in the KEAP1-mutant background. Nevertheless, the connection between KEAP1 and immune resistance seems more complex and dependent on coexisting genomic alterations. Given the clinical implications of deregulated KEAP1-NRF2 pathway in lung cancer, the development of pathway-directed anticancer treatments should be considered a priority in the domain of thoracic oncology.

      Keywords

      Introduction

      The KEAP1-NRF2 system represents the major defensive mechanism against oxidative and electrophilic stresses.
      • Hellyer J.A.
      • Padda S.K.
      • Diehn M.
      • Wakelee H.A.
      Clinical implications of KEAP1-NFE2L2 mutations in NSCLC.
      ,
      • Cuadrado A.
      • Rojo A.I.
      • Wells G.
      • et al.
      Therapeutic targeting of the NRF2 and KEAP1 partnership in chronic diseases.
      Preclinical and clinical studies in NSCLC revealed that loss-of-function (LOF) mutations in KEAP1 and gain-of-function mutations in NFE2L2 (the gene encoding for NRF2) confer resistance to chemotherapy, radiotherapy, and targeted agents.
      • Hellyer J.A.
      • Padda S.K.
      • Diehn M.
      • Wakelee H.A.
      Clinical implications of KEAP1-NFE2L2 mutations in NSCLC.
      More recently, KEAP1 mutations were connected to adverse survival outcomes in patients with advanced NSCLC treated with immunotherapy, particularly in the presence of specific co-occurring mutations.
      • Marinelli D.
      • Mazzotta M.
      • Scalera S.
      • et al.
      KEAP1-driven co-mutations in lung adenocarcinoma unresponsive to immunotherapy despite high tumor mutational burden.
      • Scalera S.
      • Mazzotta M.
      • Corleone G.
      • et al.
      KEAP1 and TP53 frame genomic, evolutionary, and immunologic subtypes of lung adenocarcinoma with different sensitivity to immunotherapy.
      • Ricciuti B.
      • Arbour K.C.
      • Lin J.J.
      • et al.
      Diminished efficacy of programmed death-(ligand)1 inhibition in STK11- and KEAP1-mutant lung adenocarcinoma is affected by KRAS mutation status.
      The increased appreciation of deregulated KEAP1-NRF2 axis in NSCLC is fueling the search of pharmacologic strategies for targeting aberrant pathway activation, and early phase clinical trials with compounds targeting metabolic vulnerabilities are ongoing. Here, we discuss the molecular functions of the KEAP1-NRF2 pathway, its role in lung tumorigenesis, evidence linking KEAP1 and NFE2L2 mutations to reduced efficacy of established anticancer treatments in patients with NSCLC, and the strategy proposed for targeting deregulated KEAP1-NRF2 activity.

      KEAP1-NRF2 Function

      In unstressed cells, the redox-sensitive KEAP1 protein binds NRF2 at DLG and ETGE degron motifs (conserved amino acid motifs) in the Neh2 domain, triggering NRF2 proteasomal degradation by means of the CUL3-RBX1 E3 ubiquitin ligase complex (Fig. 1A and B).
      • Hellyer J.A.
      • Padda S.K.
      • Diehn M.
      • Wakelee H.A.
      Clinical implications of KEAP1-NFE2L2 mutations in NSCLC.
      ,
      • Cuadrado A.
      • Rojo A.I.
      • Wells G.
      • et al.
      Therapeutic targeting of the NRF2 and KEAP1 partnership in chronic diseases.
      Figure thumbnail gr1
      Figure 1Schematic overview of the KEAP1-NRF2 pathway. (A) NRF2 and KEAP1 protein domains. (B) Activation and regulation of NRF2 in normal cells. In unstressed cells, KEAP1 binds NRF2 mediating the proteasome-dependent degradation of NRF2. ROS/nitrogen species and metabolic intermediates lead to conformational changes in KEAP1, resulting in impaired NRF2 targeting. In the nucleus, NRF2 modulates the transcription of target genes (genes containing antioxidant-responsive elements in their promoter), orchestrating a cytoprotective and genoprotective program. (C) Deregulated KEAP1-NRF2 pathway in NSCLC. In NSCLC, KEAP1 LOF mutations and NFE2L2 GOF mutations mediate an array of tumor-promoting functions, spanning from tumor progression (by means of oncogenic cooperation) and metabolic reprogramming to resistance to chemotherapy, radiotherapy, and targeted agents. An immune-cold microenvironment characterizes KEAP1-NFE2L2–mutant NSCLC, which may account for an increased ability to tolerate immune checkpoint inhibitors. CTR, C-terminal region; GOF, gain-of-function; LOF, loss-of-function; NTR, N-terminal region; ROS, reactive oxygen species; sMAF, small MAF.
      The accumulation of reactive oxygen and nitrogen species modifies sensor cysteines in KEAP1, leading to conformational changes in KEAP1 homodimers and impaired NRF2 ubiquitination.
      • Hellyer J.A.
      • Padda S.K.
      • Diehn M.
      • Wakelee H.A.
      Clinical implications of KEAP1-NFE2L2 mutations in NSCLC.
      ,
      • Cuadrado A.
      • Rojo A.I.
      • Wells G.
      • et al.
      Therapeutic targeting of the NRF2 and KEAP1 partnership in chronic diseases.
      Comparable effects are elicited by metabolic intermediates produced during glycolysis, tricarboxylic acid cycle, or lipid metabolism (Fig. 1B).
      • Adam J.
      • Hatipoglu E.
      • O’Flaherty L.
      • et al.
      Renal cyst formation in Fh1-deficient mice is independent of the Hif/Phd pathway: roles for fumarate in KEAP1 succination and Nrf2 signaling.
      • Bollong M.J.
      • Lee G.
      • Coukos J.S.
      • et al.
      A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signalling.
      • Kinch L.
      • Grishin N.V.
      • Brugarolas J.
      Succination of Keap1 and activation of Nrf2-dependent antioxidant pathways in FH-deficient papillary renal cell carcinoma type 2.
      • Mills E.L.
      • Ryan D.G.
      • Prag H.A.
      • et al.
      Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1.
      Beyond chemical and metabolic cues, oncogenic stimuli intersect the KEAP1-NRF2 axis (RAS/MAPK, p62).
      • DeNicola G.M.
      • Karreth F.A.
      • Humpton T.J.
      • et al.
      Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis.
      ,
      • Jain A.
      • Lamark T.
      • Sjøttem E.
      • et al.
      p62/SQSTM1 is a target gene for transcription factor NRF2 and creates a positive feedback loop by inducing antioxidant response element-driven gene transcription.
      When NRF2 is released from KEAP1 inhibition, it translocates to the nucleus, dimerizes with small MAF proteins, and induces the expression of target genes containing antioxidant response elements in their promoter.
      • Hellyer J.A.
      • Padda S.K.
      • Diehn M.
      • Wakelee H.A.
      Clinical implications of KEAP1-NFE2L2 mutations in NSCLC.
      ,
      • Cuadrado A.
      • Rojo A.I.
      • Wells G.
      • et al.
      Therapeutic targeting of the NRF2 and KEAP1 partnership in chronic diseases.
      The transcriptional program orchestrated by NRF2 aims at re-establishing the redox homeostasis and at protecting cells from xenobiotics (Fig. 1B).
      • Hellyer J.A.
      • Padda S.K.
      • Diehn M.
      • Wakelee H.A.
      Clinical implications of KEAP1-NFE2L2 mutations in NSCLC.
      ,
      • Cuadrado A.
      • Rojo A.I.
      • Wells G.
      • et al.
      Therapeutic targeting of the NRF2 and KEAP1 partnership in chronic diseases.
      Indeed, target genes encode for mediators of antioxidant detoxification, biotransformation enzymes, enzymes increasing the cellular reducing capability, and multidrug efflux pumps (Fig. 1B). A specific gene module is deputed to the prevention of ferroptosis, a nonapoptotic and iron-dependent cell death modality triggered by the accumulation of lipid reactive oxygen species.
      • Sun X.
      • Ou Z.
      • Chen R.
      • et al.
      Activation of the p62-Keap1-NRF2 pathway protects against ferroptosis in hepatocellular carcinoma cells.
      ,
      • Wohlhieter C.A.
      • Richards A.L.
      • Uddin F.
      • et al.
      Concurrent mutations in STK11 and KEAP1 promote ferroptosis protection and SCD1 dependence in lung cancer.
      Given that redox cycling mechanisms require NADPH and other substrates, the biological output of NRF2 activation also envisions the redirection of glucose, glycolytic intermediates, and glutamine toward anabolic pathways to fulfill this increased demand.
      • Mitsuishi Y.
      • Taguchi K.
      • Kawatani Y.
      • et al.
      Nrf2 redirects glucose and glutamine into anabolic pathways in metabolic reprogramming.
      • Sayin V.I.
      • LeBoeuf S.E.
      • Singh S.X.
      • et al.
      Activation of the NRF2 antioxidant program generates an imbalance in central carbon metabolism in cancer.
      • DeNicola G.M.
      • Chen P.H.
      • Mullarky E.
      • et al.
      NRF2 regulates serine biosynthesis in non-small cell lung cancer.
      As a result, NRF2 activation, elicited by redox, metabolic, or xenobiotic stressors, culminates in a profound reorganization of core cellular processes, coupling genoprotective and cytoprotective pathways to metabolic rewiring. Intersecting a number of processes lying at the centerpiece of cell fate decision, the KEAP1-NRF2 pathway was also connected with the DNA damage response machinery, the system balancing DNA damage repair, tolerance and apoptosis.
      • Deville S.S.
      • Luft S.
      • Kaufmann M.
      • Cordes N.
      Keap1 inhibition sensitizes head and neck squamous cell carcinoma cells to ionizing radiation via impaired non-homologous end joining and induced autophagy.
      ,
      • Goeman F.
      • De Nicola F.
      • Scalera S.
      • et al.
      Mutations in the KEAP1-NFE2L2 pathway define a molecular subset of rapidly progressing lung adenocarcinoma.
      A further branch of KEAP1-NRF2–regulated processes refers to immunomodulation.
      • Cuadrado A.
      • Rojo A.I.
      • Wells G.
      • et al.
      Therapeutic targeting of the NRF2 and KEAP1 partnership in chronic diseases.
      The immune-associated function of the pathway gained attention in tumors owing to the success of immune checkpoint inhibitors (ICIs) and the adverse survival outcomes of patients with NSCLC whose tumors harbored KEAP1 mutations.
      • Goeman F.
      • De Nicola F.
      • Scalera S.
      • et al.
      Mutations in the KEAP1-NFE2L2 pathway define a molecular subset of rapidly progressing lung adenocarcinoma.
      ,
      • Papillon-Cavanagh S.
      • Doshi P.
      • Dobrin R.
      • Szustakowski J.
      • Walsh A.M.
      STK11 and KEAP1 mutations as prognostic biomarkers in an observational real-world lung adenocarcinoma cohort.
      Evidence indicates that NRF2 interferes with the transcription of cytokines, chemokines, and the type I interferon-inducing cGAS/STING signaling.
      • Kitamura H.
      • Onodera Y.
      • Murakami S.
      • Suzuki T.
      • Motohashi H.
      IL-11 contribution to tumorigenesis in an NRF2 addiction cancer model.
      • Kobayashi E.H.
      • Suzuki T.
      • Funayama R.
      • et al.
      Nrf2 suppresses macrophage inflammatory response by blocking proinflammatory cytokine transcription.
      • Olagnier D.
      • Brandtoft A.M.
      • Gunderstofte C.
      • et al.
      Nrf2 negatively regulates STING indicating a link between antiviral sensing and metabolic reprogramming.
      Moreover, metabolites abnormally consumed or secreted after metabolic reprogramming modify the tumor microenvironment composition, in processes that may generate a hostile milieu for antitumor T-cell functions. Consistently, an immune-desert tumor microenvironment is emerging as a hallmark of KEAP1-mutant lung adenocarcinoma (LUAD).
      • Marinelli D.
      • Mazzotta M.
      • Scalera S.
      • et al.
      KEAP1-driven co-mutations in lung adenocarcinoma unresponsive to immunotherapy despite high tumor mutational burden.
      Likewise, low CD8+ tumor-infiltrating lymphocyte density, assessed by immunohistochemistry, was noticed in KEAP1-mutant lung squamous cell carcinoma (LUSC).
      • Jiang T.
      • Shi J.
      • Dong Z.
      • et al.
      Genomic landscape and its correlations with tumor mutational burden, PD-L1 expression, and immune cells infiltration in Chinese lung squamous cell carcinoma.

      Role of the KEAP1-NRF2 Pathway in Tumorigenesis

      Carcinogen-induced models and genetically engineered mouse models (GEMMs) have been instrumental in understanding KEAP1-NRF2 pathway function in neoplastic diseases. In particular, a “Janus-faced” role during carcinogenesis was proposed, which is, protumorigenic and antitumorigenic in a stage- and context-dependent manner. In normal cells, NRF2 activation ensures protection against cancer initiation by preventing cellular damage induced by chemicals and radiation (the canonical, protective role).
      • Rojo de la Vega M.
      • Chapman E.
      • Zhang D.D.
      NRF2 and the hallmarks of cancer.
      • Pillai R.
      • Hayashi M.
      • Zavitsanou A.M.
      • Papagiannakopoulos T.
      NRF2: KEAPing tumors protected.
      • Ramos-Gomez M.
      • Kwak M.K.
      • Dolan P.M.
      • et al.
      Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice.
      • Knatko E.V.
      • Ibbotson S.H.
      • Zhang Y.
      • et al.
      Nrf2 Activation protects against solar-simulated ultraviolet radiation in mice and humans.
      For instance, in a chemical carcinogenesis model, an increased tumor formation was observed in Nrf2−/− mice as compared with wild-type animals.
      • Ramos-Gomez M.
      • Kwak M.K.
      • Dolan P.M.
      • et al.
      Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice.
      Likewise, constitutive NRF2 activation protected mice from radiation-induced skin carcinogenesis.
      • Knatko E.V.
      • Ibbotson S.H.
      • Zhang Y.
      • et al.
      Nrf2 Activation protects against solar-simulated ultraviolet radiation in mice and humans.
      Conversely, in cancer cells, NRF2 activation promotes disease progression, metastatic dissemination, and resistance to cytotoxic agents (the “dark side” of NRF2; Fig. 1C).
      • Wang X.J.
      • Sun Z.
      • Villeneuve N.F.
      • et al.
      Nrf2 enhances resistance of cancer cells to chemotherapeutic drugs, the dark side of Nrf2.
      • Satoh H.
      • Moriguchi T.
      • Takai J.
      • Ebina M.
      • Yamamoto M.
      Nrf2 prevents initiation but accelerates progression through the Kras signaling pathway during lung carcinogenesis.
      • Wang H.
      • Liu X.
      • Long M.
      • et al.
      NRF2 activation by antioxidant antidiabetic agents accelerates tumor metastasis.
      • Lignitto L.
      • LeBoeuf S.E.
      • Homer H.
      • et al.
      Nrf2 activation promotes lung cancer metastasis by inhibiting the degradation of Bach1.
      • Binkley M.S.
      • Jeon Y.J.
      • Nesselbush M.
      • et al.
      KEAP1/NFE2L2 mutations predict lung cancer radiation resistance that can be targeted by glutaminase inhibition.
      In this setting, it was described that, after urethane exposure, Nrf2−/− mice developed a higher number of microscopic nodules than the Nrf2+/+ counterparts.
      • Satoh H.
      • Moriguchi T.
      • Takai J.
      • Ebina M.
      • Yamamoto M.
      Nrf2 prevents initiation but accelerates progression through the Kras signaling pathway during lung carcinogenesis.
      Nevertheless, on long-term exposure, lung tumors were more frequently observed in Nrf2+/+ mice and associated with Kras mutations.
      • Satoh H.
      • Moriguchi T.
      • Takai J.
      • Ebina M.
      • Yamamoto M.
      Nrf2 prevents initiation but accelerates progression through the Kras signaling pathway during lung carcinogenesis.
      Moreover, a tumor-promoting and oncogene-directed (e.g., KRAS, MYC) increased NRF2 activity was described in lung and pancreas tumorigenesis models,
      • DeNicola G.M.
      • Karreth F.A.
      • Humpton T.J.
      • et al.
      Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis.
      whereas Nrf2 loss hindered tumor initiation.
      • Hamada S.
      • Taguchi K.
      • Masamune A.
      • Yamamoto M.
      • Shimosegawa T.
      Nrf2 promotes mutant K-ras/p53-driven pancreatic carcinogenesis.
      ,
      • Chio I.I.C.
      • Jafarnejad S.M.
      • Ponz-Sarvise M.
      • et al.
      NRF2 promotes tumor maintenance by modulating mRNA translation in pancreatic cancer.
      Regarding KEAP1, its tumor-suppressive functions were clarified exploiting a CRISPR-Cas9–based approach in a GEMM of Kras-driven LUAD.
      • Romero R.
      • Sayin V.I.
      • Davidson S.M.
      • et al.
      Keap1 loss promotes Kras-driven lung cancer and results in dependence on glutaminolysis.
      Keap1 LOF resulted in higher tumor burden and faster tumor growth kinetics when compared with control animals. Likewise, combined inactivation of Keap1 and Pten promoted LUAD formation, suggesting the existence of an oncogenic cooperation between NRF2 and the PI3K/AKT pathway.
      • Best S.A.
      • De Souza D.P.
      • Kersbergen A.
      • et al.
      Synergy between the KEAP1/NRF2 and PI3K pathways drives non-small-cell lung cancer with an altered immune microenvironment.
      Last, combined loss of Keap1 and Trp53 resulted in the onset of tumors having the histologic and molecular features of LUSC.
      • Jeong Y.
      • Hoang N.T.
      • Lovejoy A.
      • et al.
      Role of KEAP1/NRF2 and TP53 mutations in lung squamous cell carcinoma development and radiation resistance.
      Recollecting the aforementioned evidence and considering that KEAP1-NRF2 alterations in NSCLC are significantly more common in smokers than in non-smokers, it is plausible that although chronic exposure to tobacco smoking induces a cytoprotective NRF2 activation, a switch toward tumor-enhancing functions occurs through oncogenic cooperation mechanisms. Thus, although available evidence indicates that KEAP1 and NFE2L2 alterations do not represent cancer-initiating events, their onset after a first mutational hit confers a fitness advantage by supporting tumor growth, dissemination, and therapeutic resistance.

      KEAP1 and NFE2L2 Mutations in NSCLC

      KEAP1 and NFE2L2 mutations occur in approximately 20% of LUAD and 25% to 30% of LUSC (available at https://genie.cbioportal.org). While in LUAD the majority of alterations are observed in KEAP1, a fairly equal representation of KEAP1 and NFE2L2 mutations is recorded in LUSC. In both settings, CUL3 alterations are uncommon (∼2%–3%). KEAP1 mutations have been detected, along with TP53, KRAS, and STK11 (also known as LKB1), in the normal airway epithelium in patients with early stage NSCLC, thus providing hints on the driver nature of these alterations.
      • Kadara H.
      • Sivakumar S.
      • Jakubek Y.
      • et al.
      Driver mutations in normal airway epithelium elucidate spatiotemporal resolution of lung cancer.
      Taking into account the key molecular function of KEAP1 and NFE2L2, it is not surprising the association between their mutations, smoking history, and mutual exclusivity with some actionable alterations (particularly EGFR).
      • Goeman F.
      • De Nicola F.
      • Scalera S.
      • et al.
      Mutations in the KEAP1-NFE2L2 pathway define a molecular subset of rapidly progressing lung adenocarcinoma.
      ,
      • Hellyer J.A.
      • Stehr H.
      • Das M.
      • et al.
      Impact of KEAP1/NFE2L2/CUL3 mutations on duration of response to EGFR tyrosine kinase inhibitors in EGFR mutated non-small cell lung cancer.
      Regardless of pathological subtype, KEAP1 and NFE2L2 alterations are mutually exclusive. Whether this mutual exclusivity is rooted in the detrimental effects of a double mutational hit on the same pathway or, rather, it reflects the existence of different disease entities remains an issue yet to be addressed.
      The mutational pattern of KEAP1 is consistent with its tumor-suppressive function. Indeed, pathogenic mutations are scattered throughout the whole gene length, and approximately one-third of them are stop-gain variants.
      • Hellyer J.A.
      • Padda S.K.
      • Diehn M.
      • Wakelee H.A.
      Clinical implications of KEAP1-NFE2L2 mutations in NSCLC.
      To some extent, KEAP1 displays similarities with TP53. For instance, some KEAP1 variants exhibited dominant-negative effects, which is, the encoded protein negatively interferes with the wild-type one.
      • Berger A.H.
      • Brooks A.N.
      • Wu X.
      • et al.
      High-throughput phenotyping of lung cancer somatic mutations.
      Furthermore, KEAP1 loss of heterozygosity was reported.
      • Singh A.
      • Misra V.
      • Thimmulappa R.K.
      • et al.
      Dysfunctional KEAP1-NRF2 interaction in non-small-cell lung cancer.
      Adding a further level of complexity, KEAP1 epigenetic silencing has been described.
      • Hanada N.
      • Takahata T.
      • Zhou Q.
      • et al.
      Methylation of the KEAP1 gene promoter region in human colorectal cancer.
      Conversely, the oncogenic nature of NFE2L2 is mirrored by hotspot mutations clustering at the Neh2 domain.
      • Hellyer J.A.
      • Padda S.K.
      • Diehn M.
      • Wakelee H.A.
      Clinical implications of KEAP1-NFE2L2 mutations in NSCLC.
      Given that NFE2L2 mutations mostly occur at KEAP1 binding sites (DLG and ETGE motifs), they hinder KEAP1-mediated NFR2 degradation, thus leading to the constitutive activation of NRF2-driven gene transcription.
      • Tong K.I.
      • Katoh Y.
      • Kusunoki H.
      • Itoh K.
      • Tanaka T.
      • Yamamoto M.
      Keap1 recruits Neh2 through binding to ETGE and DLG motifs: characterization of the two-site molecular recognition model.
      • Wu S.
      • Lu H.
      • Bai Y.
      Nrf2 in cancers: a double-edged sword.
      • Huppke P.
      • Weissbach S.
      • Church J.A.
      • et al.
      Activating de novo mutations in NFE2L2 encoding NRF2 cause a multisystem disorder.
      KEAP1 and NFE2L2 mutations have a distinct comutation repertoire. KEAP1 alterations often co-occur with STK11 and KRAS in LUAD, whereas NFE2L2 and TP53 mutations coexist in LUSC. The tendency toward KEAP1 and STK11 coalteration deserves particular mention. STK11 encodes a serine/threonine kinase (LKB1) acting upstream AMPK family members, which are involved in cellular energy regulation.
      • Shackelford D.B.
      • Shaw R.J.
      The LKB1-AMPK pathway: metabolism and growth control in tumour suppression.
      This suggests that KEAP1 and STK11 co-mutant LUAD configures a metabolically addicted phenotype. Moreover, a sharpened capability to tolerate ferroptosis was described in KEAP1 and STK11 double-mutant LUAD.
      • Wohlhieter C.A.
      • Richards A.L.
      • Uddin F.
      • et al.
      Concurrent mutations in STK11 and KEAP1 promote ferroptosis protection and SCD1 dependence in lung cancer.

      KEAP1/NFE2L2 and Immunotherapy

      The interest surrounding KEAP1 was fueled by pioneering molecular characterization studies shedding light on the recurrent nature of KEAP1 and NFE2L2 mutations in NSCLC, coupled with the deleterious effects of KEAP1 in NSCLC treated with chemotherapy and radiotherapy.
      • Goeman F.
      • De Nicola F.
      • Scalera S.
      • et al.
      Mutations in the KEAP1-NFE2L2 pathway define a molecular subset of rapidly progressing lung adenocarcinoma.
      ,
      • Binkley M.S.
      • Jeon Y.J.
      • Nesselbush M.
      • et al.
      KEAP1/NFE2L2 mutations predict lung cancer radiation resistance that can be targeted by glutaminase inhibition.
      ,
      Cancer Genome Atlas Research Network
      Comprehensive molecular profiling of lung adenocarcinoma.
      ,
      Cancer Genome Atlas Research Network
      Comprehensive genomic characterization of squamous cell lung cancers.
      Moreover, KEAP1 inactivation was associated with reduced sensitivity to EGFR-directed therapies (osimertinib) and agents targeting the RTK-RAS-MAPK pathway and ALK.
      • Foggetti G.
      • Li C.
      • Cai H.
      • et al.
      Genetic determinants of EGFR-driven lung cancer growth and therapeutic response in vivo.
      ,
      • Krall E.B.
      • Wang B.
      • Munoz D.M.
      • et al.
      KEAP1 loss modulates sensitivity to kinase targeted therapy in lung cancer.
      The advent of ICIs, a broader understanding of the pathway, and the increased use of sequencing technologies in clinical practice prompted a wave of novel studies striving to elucidate the relationship between KEAP1 and immunotherapy. The same holds true for its comutational background, relying on the concept of epistatic interactions.
      • Etxeberria I.
      • Teijeira A.
      • Montuenga L.M.
      • Berraondo P.
      • Melero I.
      Epistatic oncogenic interactions determine cancer susceptibility to immunotherapy.
      Coexisting mutations in KEAP1, STK11, SMARCA4, or PBRM1 have been noticed in a subset of ICI-treated LUAD patients with shorter survival outcomes when compared with single-mutant and wild-type cases.
      • Marinelli D.
      • Mazzotta M.
      • Scalera S.
      • et al.
      KEAP1-driven co-mutations in lung adenocarcinoma unresponsive to immunotherapy despite high tumor mutational burden.
      The subset of tumors with coexisting mutations had high tumor mutational burden, indicating the quality of alterations has greater predictive capability than the overall number of nonsynonymous mutations. Recently, two independent studies linked KEAP1 to TP53 in LUAD (immunotherapy-treated population on the first study, and early and advanced settings on the second one).
      • Scalera S.
      • Mazzotta M.
      • Corleone G.
      • et al.
      KEAP1 and TP53 frame genomic, evolutionary, and immunologic subtypes of lung adenocarcinoma with different sensitivity to immunotherapy.
      ,
      • Saleh M.M.
      • Scheffler M.
      • Merkelbach-Bruse S.
      • et al.
      Comprehensive analysis of TP53 and KEAP1 mutations and their impact on survival in localized- and advanced-stage NSCLC.
      The message conveyed was that KEAP1 and TP53 co-mutant LUAD shares clinical features of “pure” TP53-mutant tumors (which is, tumors carrying TP53 mutations in the absence of co-occurring KEAP1 mutations). Indeed, KEAP1 and TP53 double-mutant LUAD had intermediate prognosis, comparable with that of TP53-mutant tumors. Conversely, KEAP1 single-mutant LUAD had the shortest survival. In this context, the mutual exclusivity between TP53 and KRAS mutations suggests an enrichment for KRAS alterations in the KEAP1 single-mutant subgroup and raises the idea that including KRAS in this genomic predictor may further refine its prognostic and predictive capabilities.
      • Scalera S.
      • Mazzotta M.
      • Corleone G.
      • et al.
      KEAP1 and TP53 frame genomic, evolutionary, and immunologic subtypes of lung adenocarcinoma with different sensitivity to immunotherapy.
      ,
      • Skoulidis F.
      • Heymach J.V.
      Co-occurring genomic alterations in non-small-cell lung cancer biology and therapy.
      Next, the comparison between KEAP1 single-mutant and KEAP1 and TP53 double-mutant LUADs revealed distinct immunogenomic features and evolutionary trajectories, despite sharing an active super enhancer element sustaining the expression of ferroptosis-preventing genes (AKR).
      • Scalera S.
      • Mazzotta M.
      • Corleone G.
      • et al.
      KEAP1 and TP53 frame genomic, evolutionary, and immunologic subtypes of lung adenocarcinoma with different sensitivity to immunotherapy.
      Furthermore, it has been reported that KEAP1 and STK11 mutations negatively affected clinical outcomes in immunotherapy-treated patients with KRAS-mutant LUAD, but not in the KRAS wild-type setting.
      • Ricciuti B.
      • Arbour K.C.
      • Lin J.J.
      • et al.
      Diminished efficacy of programmed death-(ligand)1 inhibition in STK11- and KEAP1-mutant lung adenocarcinoma is affected by KRAS mutation status.
      This enforces the idea that an efficient subtyping can be achieved through the interrogation of multiple genomic markers, instead of a single feature.

      Therapeutic Targeting of KEAP1- or NFE2L2-Mutant NSCLC

      The deleterious impact of KEAP1 mutations on survival outcomes of patients with NSCLC is fueling an intense search of therapeutic strategies for targeting NRF2-addicted tumors. These efforts are mostly capitalizing on the concept of metabolic vulnerabilities, which is, the increased dependency on a given metabolic avenue stemming from NRF2-driven metabolic rewiring. For instance, NRF2-addicted tumors deplete intracellular glutamate pools, thus becoming dependent on extracellular glutamine. Thus, the inhibition of glutaminase, the enzyme that catalyzes the conversion of glutamine to glutamate, was proposed as a therapeutic strategy against NRF2-addicted NSCLC.
      • Sayin V.I.
      • LeBoeuf S.E.
      • Singh S.X.
      • et al.
      Activation of the NRF2 antioxidant program generates an imbalance in central carbon metabolism in cancer.
      On this basis, the glutaminase inhibitor telaglenastat (CB-839) is being evaluated in phase 2 trials, either in combination with chemoimmunotherapy or alone, in patients with advanced NSCLC whose tumors harbor KEAP1 or NFE2L2 mutations (KEAPSAKE and BeGIN trials; NCT04265534 and NCT03872427). Likewise, the dual mTORC1/2 inhibitor sapanisertib is being evaluated in the advanced setting, given that preclinical evidence suggested that NFE2L2 mutations induce mTOR pathway dependency (NCT02417701 and NCT04250545).
      • Shibata T.
      • Saito S.
      • Kokubu A.
      • Suzuki T.
      • Yamamoto M.
      • Hirohashi S.
      Global downstream pathway analysis reveals a dependence of oncogenic NF-E2-related factor 2 mutation on the mTOR growth signaling pathway.
      Finally, the use of GEMMs and CRISPR/Cas9 screens is shedding light on novel vulnerabilities. For instance, the endoplasmic reticulum-associated protein Slc33a1 was identified as a KEAP1 mutant-specific dependency.
      • Romero R.
      • Sánchez-Rivera F.J.
      • Westcott P.M.K.
      • et al.
      Keap1 mutation renders lung adenocarcinomas dependent on Slc33a1.

      Concluding Remarks

      After nearly two decades of success with tyrosine kinase inhibitors (and more recently ICIs), the advent of KEAP1 (and STK11) in the NSCLC mutational landscape is raising the “battlefield” to an entirely new and more complex level, dominated by pharmacologically orphan events shaping the natural history of the disease. We believe that future research should encompass three main domains. First, refining our current knowledge on oncogenic cooperation and lethal interactions. Preclinical models (e.g., GEMMs) and loss-of-function genetic screens hold the potential to significantly advance our understanding in KEAP1-NRF2 biology and uncover novel genotype-specific vulnerabilities. Second, mutational co-occurring models may represent a first generation of user-friendly genomic tools for routine clinical practice (e.g., KEAP1, STK11 and KRAS, KEAP1, and TP53). To some extent, this approach oversimplifies the complexity of genetic interactions. Consistently, we are investigating the clonal dynamics characterizing KEAP1-mutant LUAD with the aim of improving molecular subtyping. Finally, a first generation of ongoing clinical trials attempts to target KEAP1- and NFE2L2-mutant NSCLC. Although this mirrors an increased awareness on the biological and clinical relevance of the pathway, at the same time the proposed strategies mostly rely on an indirect targeting (metabolic dependencies). A direct inhibition of NRF2 should actively be pursued, as this strategy may more efficiently turn off the multiple oncogenic routes fueled by aberrant NRF2 activity. Overall, the new threat of driver tumor-suppressor genes with pleiotropic effects calls for global collaborations and academia-industry partnerships.

      CRediT Authorship Contribution Statement

      Stefano Scalera, Marco Mazzotta, Clelia Cortile, Eriseld Krasniqi, Marcello Maugeri-Saccà: Conceptualization, Writing - original draft.
      Ruggero De Maria, Federico Cappuzzo, Gennaro Ciliberto: Investigation, Writing - review & editing.
      Stefano Scalera, Marco Mazzotta, Clelia Cortile, Eriseld Krasniqi: Visualization.
      Marcello Maugeri-Saccà: Supervision, Wrote the final version of the manuscript.
      Stefano Scalera, Marco Mazzotta, Clelia Cortile, Eriseld Krasniqi, Ruggero De Maria, Federico Cappuzzo, Gennaro Ciliberto, Marcello Maugeri-Saccà: Drafting the manuscript, Read and approved the final version of the manuscript, Agree to be accountable for all aspects of the work.

      Acknowledgments

      Dr. Maugeri-Saccà is supported by the Italian Association for Cancer Research under MFAG 2019 (project identification 22940) and the Italian Ministry of Health (project identification GR-2016-02362025).

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