SIGNAL TRANSDUCTION

LZTR1 is a regulator of RASubiquitination and signalingJohannes W. Bigenzahn1, Giovanna M. Collu2, Felix Kartnig1, Melanie Pieraks1,Gregory I. Vladimer1, Leonhard X. Heinz1, Vitaly Sedlyarov1, Fiorella Schischlik1,Astrid Fauster1,3, Manuele Rebsamen1, Katja Parapatics1, Vincent A. Blomen3,André C. Müller1, Georg E. Winter1, Robert Kralovics1,4, Thijn R. Brummelkamp1,3,5,6,Marek Mlodzik2, Giulio Superti-Furga1,7*

In genetic screens aimed at understanding drug resistance mechanisms in chronicmyeloid leukemia cells, inactivation of the cullin 3 adapter protein-encoding leucinezipper-like transcription regulator 1 (LZTR1) gene led to enhanced mitogen-activatedprotein kinase (MAPK) pathway activity and reduced sensitivity to tyrosine kinaseinhibitors. Knockdown of the Drosophila LZTR1 ortholog CG3711 resulted in a Ras-dependent gain-of-function phenotype. Endogenous human LZTR1 associates with themain RAS isoforms. Inactivation of LZTR1 led to decreased ubiquitination and enhancedplasma membrane localization of endogenous KRAS (V-Ki-ras2 Kirsten rat sarcomaviral oncogene homolog).We propose that LZTR1 acts as a conserved regulator of RASubiquitination and MAPK pathway activation. Because LZTR1 disease mutations failedto revert loss-of-function phenotypes, our findings provide a molecular rationale forLZTR1 involvement in a variety of inherited and acquired human disorders.

Chronic myeloid leukemia (CML) is char-acterized by the expression of the con-stitutively active oncogenic tyrosine kinasefusion BCR-ABL (1). The proliferation andsurvival of BCR-ABL+ CML cells depends

on the activation state of key cellular signalingnetworks, including themitogen-activated pro-tein kinase (MAPK) pathway (1). The devel-opment of the tyrosine kinase inhibitor (TKI)imatinib has provided a successful targetedtherapeutic, however limited by the developmentof resistance (2).Genetic screens in the near-haploid human

CML cell line KBM-7 allow unbiased identifica-tion of candidate genes that affect inhibitorresistance (3). We performed comprehensivehaploid genetic screening with six TKIs in clinicaluse or under evaluation (fig. S1A). Retroviral gene-trap mutagenized cells were exposed to the TKIsat concentrations corresponding to half maxi-mal inhibitory concentration (IC50) to IC70 dosage(fig. S1B), resistant cell populations were col-lected after selection, and genomic gene-trapinsertions were identified by means of deepsequencing. Each screen resulted in enrichment

of disruptive insertions in 5 to 18 different genes(Fig. 1A; figs. S1, C to G, and S2A; and table S1).We identified a recurrent set (≥4 screens) of sixgenes (NF1, WT1, PTPN1, PTPN12, LZTR1, andBAP1; “TOP6” set) (Fig. 1B)withoverrepresentationof disruptive genomic gene-trap integrations,strongly indicating a selective advantage upondrug treatment (fig. S2B).We used a lentiviral CRISPR/Cas9 multicolor

competition assay (MCA)–based coculture sys-tem to evaluate gene-mediated drug-resistanceeffects. SpCas9-expressing KBM-7 (KBM-7Cas9)cells were infected with lentiviral single-guideRNA (sgRNA) vectors that coexpress reporterfluorophores, which enabled the tracing of knock-out and control cell populations bymeans of flowcytometry in the same well (fig. S3A). MixedsgRen (targeting Renilla luciferase) control cellpopulations that express fluorescent proteins(GFP+ or mCherry+) did not show any prefer-ential outgrowth of resistant cells upon 14 daysof TKI treatment (Fig. 1C and fig. S3H). By con-trast, KBM-7Cas9 GFP+ cells harboring sgRNAsthat target the TOP6 genes showed a decreasedamount of cognate protein (fig. S3, B to G) anddemonstrated enhanced cell survival and out-growth in the presence of imatinib (Fig. 1C) andrebastinib (fig. S3H). Thus, we functionally val-idated the TOP6 genes as important for drugaction in BCR-ABL+ CML cells.Although NF1, PTPN1, and PTPN12 share the

ability to modulate MAPK pathway activation,and WT1 as well as BAP1 function through tran-scriptional regulation (fig. S2C), we could notdeduce any mechanistic explanation for therole of leucine zipper–like transcription regu-lator 1 (LZTR1) in enhanced CML cell survivalfrom the existing literature (4, 5). To excludecell line–specific effects, we confirmed that loss of

LZTR1 expression induced resistance to imatiniband rebastinib in other CML cell lines (fig. S4, Ato C). Although we identified significant LZTR1enrichment only in four of the genetic screens,KBM-7Cas9 sgLZTR1 cells exhibited various degreesof resistance against all tested BCR-ABL TKIs (fig.S3I).Weused aCRISPR/Cas9–based domain scan-ning strategy to test whether both N-terminalKelch domains and C-terminal Broad-Complex,Tramtrack, and Bric a brac (BTB) and partialBACKdomains are essential for thedrug-resistancephenotype (6–8). All protein domain-targetingsgRNAs showed efficient indel formation (fig. S3J)and induced resistant outgrowth of targeted cellpopulations exposed to rebastinib, indicatingthat the entire protein is functionally required(fig. S3K). To determine whether LZTR1 exertsits function only in a CML-specific context, weinfected FLT3-ITD+ acute myeloid leukemia(AML) MV4-11Cas9 cells with LZTR1, targetingsgRNAs (fig. S4D). FLT3 inhibitor treatment ledto an outgrowth of resistant cells, underlining amore general role for LZTR1 in the drug responseof hematopoietic cancers driven by differenttyrosine kinases (fig. S4, E to G).KBM-7Cas9 CML cells infected with distinct

sgRNAs targeting LZTR1 displayed enhancedphosphorylation of MAPK kinase 1 (MEK1) and-2 and extracellular signal–regulated kinase 1 (ERK1)and -2, which is indicative of augmented MAPKpathway activation (Fig. 2A). By contrast, globaltyrosine phosphorylation as well as phospho-rylation of AKT (at S473 and T308), the proteinkinase S6K1, ribosomal protein S6, and the directBCR-ABL substrate signal transducer and ac-tivator of transcription 5 (STAT5) remainedunchanged (fig. S4, J and K). Additional CML(K-562 and LAMA-84) and AML (MV4-11) celllines had similarly enhanced MAPK pathwayactivation under normal growth conditions aswell as, in the case of CML cells, upon increasingconcentration of imatinib treatment (Fig. 2A andfigs. S4, H and I, and S5, A to C). LZTR1 full-length cDNA complementation in K-562Cas9

sgLZTR1 cells reverted both enhanced MEK andERK phosphorylation as well as TKI resistance(Fig. 2, B and C). Treatment of K-562Cas9 sgLZTR1cells with the clinically approved inhibitor ofMEK1 and -2 trametinib reverted enhanced ac-tivation of ERK1 and -2 and pharmacologicallycounteracted the drug-resistance phenotype(Fig. 2, D and E). Furthermore, cDNA expressionof constitutively activeMek1D218, D222 (Mek1 DD)in K-562rtTA3 cells led to enhanced phosphoryla-tion of ERK1 and -2 and reduced imatinib sen-sitivity (fig. S6A, and B). Activation of the MAPKpathway could also be inferred by the geneexpression and transcription factor enrichmentsignature obtained with RNA sequencing experi-ments in KBM-7Cas9 sgLZTR1 compared withsgRen cells (fig. S5, D and E, and table S2).Altogether, the data established a causal rolefor enhanced MAPK pathway activation in theresistance of CML cells toward TKI therapy aselicited by loss of LZTR1 function.Because BTBdomain-containing proteins serve

as adaptor proteins for the cullin 3 (CUL3) E3

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1CeMM Research Center for Molecular Medicine of theAustrian Academy of Sciences, 1090 Vienna, Austria.2Department of Cell, Developmental, and RegenerativeBiology and Graduate School of Biomedical Sciences, IcahnSchool of Medicine at Mount Sinai, 1 Gustave L. Levy Place,New York, NY 10029, USA. 3Netherlands Cancer Institute,Plesmanlaan 121, 1066 CX, Amsterdam, Netherlands.4Department of Laboratory Medicine, Medical University ofVienna, 1090 Vienna, Austria. 5Oncode Institute, Division ofBiochemistry, Netherlands Cancer Institute, Plesmanlaan 121,1066 CX, Amsterdam, Netherlands. 6Cancer GenomicsCenter (CGC.nl), Plesmanlaan 121, 1066 CX, Amsterdam,Netherlands. 7Center for Physiology and Pharmacology,Medical University of Vienna, 1090 Vienna, Austria.*Corresponding author. Email: gsuperti@cemm.oeaw.ac.at

ubiquitin ligase complex, enabling specific sub-strate recognition and ubiquitination (9), wetested whether loss of CUL3 expression couldmimic the observed LZTR1 loss-of-function phe-notype. K-562Cas9 sgCUL3 cells demonstratedenhanced MAPK pathway activation and, incontrast to sgLZTR1 cells, increased phospho-rylation of AKT (fig. S4K). However, sgRNAs thattarget CUL3 had a pronounced antiproliferativeeffect both in KBM-7 Cas9 and K-562Cas9 cells (fig.S6, C and D), providing a potential explanationwhyCUL3wasnot detected in our genetic screens(fig. S2A).In contrast to the CML cell lines, depletion of

LZTR1 with CRISPR sgRNAs in HeLa, humanembryonic kidney (HEK)–293T, or HAP1 cellsdid not increaseMAPK pathway activation under

comparable culture conditions (fig. S7, A and C).However, after serum stimulation of cells culturedwithout serum,HEK293TCas9 sgLZTR1 cells showeda more pronounced activation of MEK and ERKthan in control cells (fig. S7B). Similarly, in HAP1cells, a non-hematopoietic derivative of theKBM-7 cell line (10), phorbol-12-myristat-13-acetat (PMA) treatment led to enhancedMAPKpathway activation in the absence of LZTR1compared with that in wild-type (WT) cells (fig.S7C). Whereas HAP1Cas9 sgCUL3 cells exhibitedincreased phosphorylation of ERK1, ERK2, andAKT (fig. S7F), loss of LZTR1 expression inHAP1Cas9 altered only the MAPK pathway asidentified by means of pathway array-based as-sessment of kinase activation (fig. S7, D and E,and table S3).

Genetic studies have identified LZTR1 muta-tions in glioblastoma (GBM) (11), schwannoma-tosis (SWNMT) (8), and Noonan syndrome (NS)(5), a developmental syndrome that is part of thelarger group of RASopathies characterized bymutations in components of the RAS-MAPKpathway (12). Identification of NF1 and LZTR1loss-of-function–induced MAPK pathway activa-tion in our haploid resistance screens combinedwith human LZTR1 mutations in NS indicatedthat LZTR1 might directly regulate guanosinetriphosphatases (GTPases) of the RAS family.Drosophila wing vein formation and eye de-

velopment serve as excellent in vivo readoutsfor RAS signaling (13–15). CG3711 encodes theDrosophila ortholog ofmammalianLZTR1, whichcontains a specific N-terminal domain (amino

Bigenzahn et al., Science 362, 1171–1177 (2018) 7 December 2018 2 of 7

Fig. 1. Haploidgenetic screens iden-tify gene knockoutsthat promote BCR-ABL inhibitorresistance. (A) Circosplot of the haploidgenetic screen inthe CML cell lineKBM-7 upon treatmentwith ponatinib.Each dot representsa mutagenized geneidentified in theresistant cell popula-tion, the dot sizecorresponds to thenumber of independentinsertions identifiedper gene, and thedistance from thecircos plot centerindicates the signifi-cance of enrichmentcompared withan unselected controldata set. Hits witha false discoveryrate (FDR)–adjustedP value lower than10−4 are labeled bygene name. (B) Bubbleplot depicting the“TOP6” set of genesidentified in four ormore of the six haploidscreens upon treat-ment with first-,second- and third-generation BCR-ABLinhibitors. Thesize of each bubblecorresponds to thenumber of independent insertions per gene, and the color gradient depictsthe FDR-adjusted P value of enrichment significance. (C) MCA-derivedfold change of cell populations after imatinib treatment of KBM-7Cas9

CML cells transduced with sgRNAs targeting the “TOP6” genes orsgRen.208 (targeting Renilla luciferase) as negative control. sgRNA-infected cell populations were mixed in a 1:1 ratio, treated with

increasing drug concentrations, and analyzed by means of flowcytometry after 14 days. Data are shown as mean value ± SDof at least two independent experiments (n ≥ 2) performedin duplicates. sgRNAs are labeled by gene name followed bythe genomic targeting sequence position numbered according tothe sequence position on the corresponding mRNA.

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acids 1 to 184) that is only found in Drosophila.This is followed by the highly conserved re-maining part of the protein (54% sequenceidentity) (fig. S8, A to C). Systemic depletion ofCG3711 with RNA interference (RNAi) by usingact5C-Gal4 yielded viable flies; however, the ma-jority of wings of these flies displayed wingvein defects characterized by extra veins andvein tissue (Fig. 3, A and B, and fig. S8D). Thisphenotype closely resembles a gain-of-function

increase of RAS-MAPK signaling (15) and couldbe rescued by a decrease in abundance of RASvia dRas (DrosophilaRas) heterozygosity (Fig. 3C).Drosophila R7 photoreceptor induction requiresRAS function (13, 14). We used a mild dominantnegative version, RasV12C40, which althoughlocked in the active guanosine 5′-triphosphate(GTP)–bound state does not activate MAPK sig-naling (16). RasV12C40 expression in the develop-ing eyes (via the sevenless/sev-Gal4 expression

system) led to a frequent loss of the R7 photo-receptor (~30% of ommatidia display R7 loss,and some also lost other R-cells) (fig. S8, E andH). Because RasV12C40 is constitutively active, italso causes defects in ommatidial rotation(16, 17), serving as internal control. When bothsev-Gal4–driven CG3711 RNAi and RasV12C40

were expressed, the loss of R7 phenotype wasalmost completely suppressed (fig. S8, F and H).The ommatidial rotation defects were enhanced

Bigenzahn et al., Science 362, 1171–1177 (2018) 7 December 2018 3 of 7

Fig. 2. Loss ofLZTR1 enhancesMAPK pathwayactivation.(A) Phosphorylationof MEK and ERKin KBM-7Cas9 andK-562Cas9 CML cellstransduced with theindicated sgRNAs.(B) Immunoblotanalysis of MEK andERK phosphorylationas well as LZTR1expression insgRen.208–expressing K-562Cas9

CML cells trans-duced with emptyvector, andsgLZTR1.466–expressing cellstransduced withempty vector orLZTR1-cDNA–containing murinestem cell virus(MSCV) retrovirus.Quantification of MEKand ERK phosphoryl-ation is shown next tothe correspondingimmunoblots.(C) Competitiveproliferation assay(MCA) of K-562Cas9

sgRen.208 cellstransduced withempty vector andsgLZTR1.466 cellstransduced withempty vector orLZTR1 cDNA aftertreatment withincreasing con-centrations ofimatinib for 14 days.(D) Phosphorylationof MEK and ERK inK-562Cas9 cells ex-pressing sgLZTR1.466and treated with in-creasing concentrations of trametinib for 3 hours. (E) Changes in cell popula-tions measured with MCA of K-562Cas9 CML cells expressing sgRen.208 orsgLZTR1.466 after 14 days of treatment with increasing concentrationsof imatinib alone or in combination with trametinib. Immunoblot results in (A), (B),

and (D) are representative of at least two independent biological experiments(n ≥ 2). MCA data in (C) and (E) are shown as mean value ± SD ofat least two independent experiments (n ≥ 2). Dimethyl sulfoxide (DMSO)treatment served as negative control.

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rather than suppressed because these involveMAPK-independent RAS signaling (fig. S8F)(16). Expression of CG3711 RNAi in the eye alonedid not induce phenotypic changes (fig. S8, Gand H). Thus, RAS is crucial for the phenotypesinduced by depletion of CG3711.To explore possible interactions of LZTR1 with

RAS, we used a BirA* fusion protein–based prox-imity biotinylation-dependent (BioID) proteomicapproach, which allows identification of weakinteraction partners. We expressed KRAS4A,KRAS4B, NRAS, andHRAS as N-terminal FLAG-BirA* fusion proteins in K-562rtTA3 CML cells.This led to enhanced MEK and ERK activation,demonstrating that the BirA* tag does not gen-erally interfere with RAS signaling (fig. S9A). Weidentified between 153 and 258 proteins in prox-imity of or interacting with human RAS proteins,among them several known interactors andpathway components (fig. S9, B to F, and tableS4). A common set of 123 proteins repeatedlypurified with all four RAS proteins, with geneontology analysis showing a significant enrich-ment in components associated with plasmamembrane and Golgi apparatus as well as theMAPK pathway (fig. S9, G and H). We identifiedLZTR1 among the most significant interactorsof all four RAS proteins (fig. S9, B to F). Inter-section of the 123 common interactors identifiedwith BioID, with a fluorescence-activated cellsorting (FACS)–based haploid genetic pathwayscreen for the identification of gene knockoutalleles that influence phosphorylation of ERK1and -2 (18), revealed LZTR1 as the only commonRAS interactor in our proteomic data with anegative regulatory functionwithin theRAS-MAPKpathway (fig. S10A).BioID proximity biotinylation-based and FLAG

tag–based coimmunoprecipitation experimentsconfirmed the interaction of all four RAS pro-teins with endogenous LZTR1 (fig. S10, B and C).Only the four main RAS isoforms interacted spe-cifically with LZTR1, whereas six related RASfamily GTPases showed no interaction (fig. S11A).Nine other canonical positive RAS-MAPK path-way regulators also failed to interact with endo-genous LZTR1 (fig. S11B). Immunoprecipitationswith extracts derived from LZTR1 knockout cellsconfirmed the specificity of the interaction (fig.S11C). The C-terminal posttranslational acyla-tions of KRAS proteins appeared to be requiredfor the interaction with LZTR1 (Fig. 3D and fig.S11D). Thus, proper membrane-associated lo-calizationmediatedby this regionmaybe requiredfor specific interaction with LZTR1.Stably expressed full-length hemagglutinin

(HA)–tagged LZTR1 protein showed a speckledand vesicular staining pattern inHeLa, HEK293T,andHAP1 cells, whereas all domainmutants hada homogenous cytoplasmic distribution (fig. S12,A to D). Full-length LZTR1 displayed an over-lapping localization within speckled and vesicularcompartments with transiently transfected V5-tagged CUL3 inHeLa cells (fig. S13A). The LZTR1-stained speckles further overlapped with theautophagosome marker LC3B fused to mCherry(fig. S13B), but we failed to detect LZTR1 lo-

calization with marker proteins of the Golgi,lysosome, peroxisome, or early and late endo-some compartments (fig. S13C). Isoform-specificantibodies that recognize endogenous humanRAS isoforms are not available (19). We there-fore endogenously tagged KRAS (fig. S14A) andconfirmed specificity of detection in immuno-fluorescence and immunoblotting by meansof genetic inactivation of the tagged genomicallele (fig. S14, B to D). KRAS localized to alarge number of small-punctate structures, likelymembrane-containing small vesicles (Fig. 3E andfig. S14D). Inactivation of endogenous LZTR1 ledto an increased RAS signal, particularly at theperiphery of cells, at regions of cell-to-cell contacts(Fig. 3E and fig. S14D). This phenotype ap-peared to be dependent on the CUL3 E3 ligasecomplex because genetic inactivation of CUL3led to similar phenotypic changes (Fig. 3E andfig. S14E).We also assessed interaction of endogenously

tagged KRAS with exogenous LZTR1 in HAP1cells (fig. S15A). Exogenous LZTR1 localized tofewer, larger, and more oblong structures thandid endogenous KRAS (fig. S15B). A fraction ofthe LZTR1-stained structures also containedKRAS. Treatment with the cullin neddylationinhibitor MLN4924 caused clustering of LZTR1-containing structures that appeared to surroundparticles strongly stained with KRAS (fig. S15B,MLN4924 panel). Caution is required for theinterpretation of these patterns because LZTR1was overexpressed, and KRAS was at endoge-nous concentration. However, the images couldrepresent a trapped proteostatic process thatinvolves the two proteins.We tested whether LZTR1 affected ubiquitina-

tion of RAS proteins by acting as a substrateadaptor for the CUL3 E3 ligase complex (11, 20).Ubiquitination of RAS proteins is known, butlittle is known of its consequences (21–25). Co-expression of HA-ubiquitin with either one ofthe four RAS isoforms alone only resulted in abasal state of ubiquitination, but addition ofMYC-tagged CUL3 and V5-tagged LZTR1 led toincreased ubiquitination of RAS proteins (fig.S16, A to D). LZTR1-deletion mutants that lackeither one or both C-terminal BTB domains orthe N-terminal Kelch domain failed to inducean equivalent degree of ubiquitination of KRAS4A(fig. S16A). KRAS4A required the presence of itsC-terminal hypervariable region (HVR) and thefarnesylated cysteine 186 for LZTR1-mediatedubiquitination (fig. S16E). LZTR1-induced ubi-quitination was unchanged in cells treated witha proteasome inhibitor but could be blocked bycullin neddylation inhibition, which is consistentwith the observed subcellular localization changes(fig. S16F). Specificity for LZTR1 in the processwas further underscored by the failure of twoprominent CUL3 adaptors, Kelch-like ECH-associated protein 1 (KEAP1) and Speckle-typePOZ protein (SPOP), to cause a comparable ubi-quitination (fig. S16G). We used HAP1 cells thatbear endogenously tagged KRAS and tandemubiquitin binding domain (TUBE) reagents tocapture endogenously ubiquitinated proteins and

evaluate the contribution of endogenous LZTR1(26). In the absence of any stimulation, immuno-precipitation with TUBE purified several proteinsthat reacted with pan-ubiquitin antibodies (Fig.3F, third from top, and fig. S16H). The same pre-cipitates contained proteins that react with pan-RAS antibodies (Fig. 3F, second from top). Last,FLAG antibodies detected a protein not visible inthe whole-cell extract (Fig. 3F, top, and fig. S16H).TUBE-mediated immunoprecipitation from cor-responding cells in which LZTR1 had been ge-netically inactivated did not contain amounts ofRAS detectable with these antibodies (Fig. 3F,top and second from top, and fig. S16H). Thesignal obtained with pan-ubiquitin antibodieswas unchanged (Fig. 3F, third from top, and fig.S16H). Thus, human LZTR1 appears to mediateubiquitination of endogenous human KRAS andpossibly other RAS proteins.If a main function of LZTR1 is to regulate RAS,

then loss of RAS function should compensate forloss of LZTR1 activity, as suggested in the fruitflyepistatic analysis, representing an ideal exper-imental setup to assess the disease-associatedLZTR1 mutations by their dependency on RASactivity. We confirmed that loss of LZTR1 func-tion enhanced RAS activity in K-562 cells (fig.S17A). We then performed CRISPR/Cas9–baseddouble-knockoutMCA experiments (fig. S17, B toD). Single sgLZTR1 and sgLZTR1-sgRen double-infected cells were resistant to imatinib comparedwith control sgRen cells (fig. S17, E and F). sgRNAsthat target KRAS abolished cellular outgrowth,whereas sgRNAs that target NRAS, HRAS, andRIT1 failed to do so (fig. S17, E and F). sgLZTR1-sgKRAS cells had reduced MEK phosphoryla-tion comparable with that of sgRen cells, whereassgNRAS and sgHRAS cells maintained enhancedMAPK pathway activation (fig. S17G). KRAS in-activation led to a strong antiproliferative phe-notype in KBM-7Cas9 cells and, to a lesser extent,K-562Cas9 cells (fig. S18, A and B), indicating thatthe selective requirement of KRAS in mediatingLZTR1-induced enhanced MAPK pathway acti-vation might represent a prerequisite of KRASfor BCR-ABL signaling in CML cells. We addi-tionally used a fibronectin-fold–based monobody,NS1, which bears low nanomolar affinity forthe dimerization interface of both KRAS andHRAS and is able to interfere with their signal-ing activities (27). Cells that stably express theNS1 monobody did not show increased activa-tion of the MAPK pathway or drug resistanceassociated with loss of LZTR1 function (fig. S18,C and D).More than 50 different mutations have been

mapped to the human LZTR1 gene in various dis-eases (fig. S19,AandB, and table S5) (5,8, 11,28–30).To establish a mechanistic link between mu-tations that affect human diseases and the bio-chemical processes described here, we focusedon LZTR1missense mutations identified in GBM(11), NS (5), and SWNMT (8) (fig. S20B).Mutation-bearing LZTR1 cDNAs were tested for their abilityto complement K-562 CML cells deficient inendogenous LZTR1 (fig. S20A). In contrast toWT LZTR1, the disease-associated mutations

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Fig. 3. LZTR1 modulates MAPK pathway activation through RAS regula-tion. (A) Morphology of adult wings from act5C-Gal4, UAS-wIR (act>wIR forshort), and act>CG3711IR #1 RNAi fly lines. Arrow, extra vein; arrowhead,partial extra vein. (B) Quantification of act>wIR and act>CG3711IR RNAi linesas percentage of wings with ectopic wing vein formation. P value forRNAi #1 and #3 in the wing is <0.0001 (****) and for #2 is 0.0255.(C) Quantification of act>wIR and act>CG3711IR RNAi lines alone or in aRase2F/+ background as percentage of wings with ectopic wing vein formation.For statistical assessment, partial extra vein and extra vein formation havebeen combined. P value for both RNAi line comparisons in the wing is

<0.0001 (****). (D) FLAG immunoprecipitates (IP) and whole-cell extracts(WCE) from K-562rtTA3 cells expressing FLAG-BirA*–tagged GFP or KRAS4AWT, DHVR, C180S, or C186S after 48 hours of doxycycline treatment wereimmunoblotted for the indicated proteins. (E) Confocal microscopy of HAP1WTcells and HAP1 cells with endogenously FLAG-tagged KRAS transducedwith sgRen.208, sgLZTR1.620, sgLZTR1.466, sgCUL3.852, or sgCUL3.1396 andstained with antibody to FLAG. Scale bars, 10 mm. (F) TUBE-based purificationsof ubiquitinated proteins and WCE from HAP1 WT and endogenouslyFLAG-tagged KRAS cells transduced with sgRen.208 or sgLZTR1.466 wereanalyzed by means of immunoblotting with the indicated antibodies.

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failed to reduceMAPKpathway activation, despitebeing expressed at a comparable or higher amount(Fig. 4, A to C). Similarly, all missense mutationsapart from theGBM-associated T288I andR810Wfailed to restore sensitivity to imatinib treatment(fig. S20, C to E). LZTR1 mutations within theKelch domain partially retained their localiza-tion pattern in comparison with the WT protein,whereas mutations in the BTB/BACK domainsmislocalized to the cytoplasm (fig. S21, A and B).In agreement, expression of LZTR1 Kelch domainmutations identified in GBM, NS, and SWNMTin K-562 WT cells had a resistance-mediating

effect inMCAassays in cells treatedwith imatinib,whereas BTB/BACK domain mutations did not(fig. S22, A to C). Furthermore, in contrast tothe LZTR1WT protein, the two mutants R198Gand G248R identified in GBM and NS failed toinduce a similar increase in ubiquitination onKRAS4A (fig. S22D). These findings provide func-tional evidence that human LZTR1 missensemutations identified in GBM, NS, and SWNMTrepresent loss-of-function toward ubiquitina-tion and inhibition of RAS activity.Together with the accompanying manuscript

on the role of LZTR1 in diseases driven by the

dysregulation of RAS ubiquitination and signal-ing (31), our work illustrates the importance ofan additional, underappreciated layer of RASregulation (Fig. 4D).

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Fig. 4. LZTR1 disease missense mutations fail to rescue the loss-of-function phenotype. (A to C) Immunoblotting for MEK and ERK phospho-rylation as well as LZTR1 expression of K-562Cas9 sgRen.208–expressing cellsretrovirally transduced with empty vector, and sgLZTR1.466-expressing cellstransduced with either empty vector, LZTR1 WT, or LZTR1 mutations identifiedin (A) GBM (blue), (B) NS (brown), or (C) SWNMT (orange).The LZTR1 G248R

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ACKNOWLEDGMENTS

We are grateful to all members of the Superti-Furga laboratoryfor help, discussions, and advice; E. Girardi and E. Salzer forcritically reading the manuscript; and P. Májek for feedback onthe bioinformatic analysis of BioID proteomic experiments. Wethank the Biomedical Sequencing Facility for advice on Illuminasequencing and the Core Facility Imaging of the MedicalUniversity of Vienna for technical support. Funding: This workwas supported by the Austrian Academy of Sciences, EuropeanResearch Council (ERC) grants (i-FIVE 250179 and Game ofGates 695214), and Austrian Science Fund grant (FWF SFBF4711) to G.S.-F; European Molecular Biology Organization long-term fellowships to M.R. (ALTF 1346-2011) and G.I.V. (ALTF1543-2012); an Austrian Science Fund grant (FWF SFB F4702)to R.K.; NIH grants R01 EY013256 and GM102811 to M.M.; andfunding from the CGC.nl, KWF grant NKI 2015-7609, and ERCStarting Grant (ERC-2012-StG 309634) to T.R.B. Authorcontributions: J.W.B. and G.S.-F. conceived the study based ondiscussions with T.R.B.; J.W.B., F.K., and M.P. performedresearch; and G.M.C. and M.M. designed and performedDrosophila experiments. L.X.H., G.I.V., A.F., M.R., andG.E.W. generated reagents and provided scientific insight. F.S.analyzed haploid genetic screening data and created circosplots and the graphical display of insertion sites. V.S. performedRNA sequencing and enrichment analysis and compiledcBioPortal mutation data. K.P. and A.C.M. performed proteomic

sample analysis. V.A.B. and T.R.B. provided reagents and gaveexperimental advice. R.K. supervised sequencing data analysisand gave experimental advice. J.W.B., G.M.C., F.K., G.I.V., L.X.H.,F.S., M.M., and G.S.-F. analyzed and interpreted the data. J.W.B.,M.M., and G.S.-F. wrote the paper. Competing interests: Theauthors declare no competing financial interest. T.R.B. is cofounderand SAB member of Haplogen GmbH and cofounder andmanaging director of Scenic Biotech. G.I.V. is cofounder andemployee of Allcyte GmbH. G.S.-F. is cofounder and shareholderof Allcyte GmbH and Haplogen GmbH. Data and materialsavailability: Deep sequencing datasets have been deposited inthe National Center for Biotechnology Information SequenceRead Archive under the accession no. SRP157890, and BioIDmass spectrometry proteomics data have been deposited to theProteomeXchange Consortium via the PRIDE partner repositoryunder the accession no. PXD011280. The KBM-7 cell line isavailable from T.R.B. under a materials transfer agreement withthe Netherlands Cancer Institute.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/362/6419/1171/suppl/DC1Materials and MethodsFigs. S1 to S22Tables S1 to S5References (32–59)

30 August 2017; resubmitted 26 June 2018Accepted 30 October 2018Published online 15 November 201810.1126/science.aap8210

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