Diverse BRAF Gene Fusions Confer Resistance to EGFR-Targeted Therapy Via Differential Modulation of BRAF Activity Christina Stangl1,2, Jasmin B

Published OnlineFirst January 7, 2020; DOI: 10.1158/1541-7786.MCR-19-0529

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Diverse BRAF Gene Fusions Confer Resistance to EGFR-Targeted Therapy via Differential Modulation of BRAF Activity Christina Stangl1,2, Jasmin B. Post3, Markus J. van Roosmalen1,4, Nizar Hami3, Ingrid Verlaan-Klink3, Harmjan R. Vos3, Robert M. van Es3, Marco J. Koudijs1,5, Emile E. Voest2, Hugo J.G. Snippert3,and W.P. Kloosterman1,6,7

ABSTRACT ◥ Fusion genes can be oncogenic drivers in a variety of cancer types influences their subcellular localization and intracellular signaling and represent potential targets for targeted therapy. The BRAF gene capacity, revealing distinct subsets of affected signaling pathways is frequently involved in oncogenic gene fusions, with fusion and altered gene expression. Presence of the different BRAF frequencies of 0.2%–3% throughout different cancers. However, fusions resulted in varying sensitivities to combinatorial inhibi- BRAF fusions rarely occur in the same gene configuration, poten- tion of MEK and the EGF receptor family. However, all BRAF tially challenging personalized therapy design. In particular, the fusions conveyed resistance to targeted monotherapy against the impact of the wide variety of fusion partners on the oncogenic role of EGF receptor family, suggesting that BRAF fusions should be BRAF during tumor growth and drug response is unknown. Here, screened alongside other MAPK pathway alterations to identify we used patient-derived colorectal cancer organoids to functionally patients with metastatic colorectal cancer to exclude from anti- characterize and cross-compare BRAF fusions containing various EGFR–targeted treatment. partner genes (AGAP3, DLG1, and TRIM24) with respect to cellular behavior, downstream signaling activation, and response to targeted Implications: Although intracellular signaling and sensitivity to therapies. We demonstrate that 50 fusion partners mainly promote targeted therapies of BRAF fusion genes are influenced by their 50 canonical oncogenic BRAF activity by replacing the auto-inhibitory fusion partner, we show that all investigated BRAF fusions confer N-terminal region. In addition, the 50 partner of BRAF fusions resistance to clinically relevant EGFR inhibition.

Introduction tively, fusions may facilitate the connection of a strong promoter to the coding sequence of a second gene, causing high expression of a protein Cancer genomes are often subject to genomic instability, which can such as kinases (1, 2). result in various genomic rearrangements, including translocations (1). Recent advances in sequencing technologies and bioinformatic Some genomic rearrangements can lead to oncogenic transformation, solutions have enabled the straightforward identification of novel in particular when tumor suppressor genes are being disrupted or fusion genes (2, 3). This has revealed that their frequency of oncogenic fusion genes are created (2). Fusion genes are chimeric occurrence varies between many cancer types, ranging from high genes resulting in proteins with altered or novel functions. Alterna- frequencies in breast cancer (14.7%) to low frequencies in uveal melanoma (0.16%; ref. 2), but also highlighted the vast diversity of > fi 1Department of Genetics, Center for Molecular Medicine, University Medical fusion partners ( 10,800 unique fusion con gurations, Quiver Center Utrecht and Utrecht University, Utrecht, the Netherlands. 2Division of Fusion Database). Molecular Oncology, Netherlands Cancer Institute, and Oncode Institute, Oncogenic fusion proteins frequently interfere with signaling 3 Amsterdam, the Netherlands. Molecular Cancer Research, Center for Molecular pathways that regulate cellular differentiation and proliferation and Medicine, and Oncode Institute, University Medical Center Utrecht and Utrecht 4 therefore are excellent targets for personalized cancer therapy. University, Utrecht, the Netherlands. Princess Maxima Center for Pediatric BRAF Oncology and Oncode Institute, Utrecht, the Netherlands. 5Center for Person- Fusions that involve the oncogene are mutually exclusive alized Cancer Treatment, University Medical Center Utrecht, Utrecht, the Nether- with other oncogenic mutations in the MAPK pathway (e.g., V600E lands. 6Cyclomics, Utrecht, the Netherlands. 7Frame Cancer Therapeutics, BRAF ), suggesting that BRAF fusion genes promote constitu- Amsterdam, the Netherlands. tive MAPK signaling (4). Indeed, previous studies have shown that Note: Supplementary data for this article are available at Molecular Cancer loss of the auto-inhibitory N-terminal domain of BRAF promotes Research Online (http://mcr.aacrjournals.org/). BRAF kinase activity independent of upstream RAS signaling C. Stangl and J.B. Post contributed equally to the article. activity resulting in enhanced downstream MAPK signaling (5, 6). Furthermore, specific BRAF fusion genes have been implicated in H.J.G. Snippert and W.P. Kloosterman are co-senior authors for this article. acquired resistance to targeted therapies (7). For example, the Corresponding Authors: W.P. Kloosterman, Universiteitsweg 100, Utrecht AGAP3-BRAF fusion has been reported to induce resistance to the 3584 CG, the Netherlands. Phone: 318-8755-0406; E-mail: BRAF inhibitor vemurafenib in a patient with BRAFV600E-mutated [email protected]; and Emile E. Voest, Netherlands Cancer Insti- tute, Plesmanlaan, Amsterdam, the Netherlands. E-mail: [email protected] melanoma that was previously responsive to treatment (7). Hence, BRAF fusion genes may represent an important and understudied Mol Cancer Res 2020;XX:XX–XX mode to activate oncogenic MAPK signaling. doi: 10.1158/1541-7786.MCR-19-0529 BRAF fusion genes have been identified in multiple cancers with 0 2020 American Association for Cancer Research. a wide variety of 5 fusion partners (>60 different partners published,

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some of which have only been described once; refs. 4, 8). The AB_2107445), beta-catenin (RRID:AB_397555), pERK (RRID: large variety of BRAF fusion partners complicates straightforward AB_331646), ERK (RRID:AB_390779), pMEK (RRID:AB_331648), discrimination between oncogenic effects that are shared versus MEK (RRID:AB_823567), pRAF1 (AB_2492224), RAF1 (RRID: actions that are influenced or even determined by the unique fusion AB_390808), pAKT (RRID: AB_2315049), and AKT (RRID: partner. Therefore, in contrast to common BRAF hotspot mutations, AB_1147620). this high diversity of fusion gene configurations impedes the clinical interpretation of BRAF fusion genes in relation to their oncogenic Phenotypic drug screen potential and treatment responses. Phenotypic drug screen was performed as described previously (14). To explore cellular and molecular effects of different BRAF In brief, 5-day-old organoids were cultured with medium containing fusion configurations that have recently been identified in colorectal either 1 mg/mL doxycycline or ddH2O, together with 1 mmol/L cancers (e.g., AGAP3-BRAF, DLG1-BRAF,andTRIM24-BRAF; of afatinib or DMSO. After 7 days, cell viability was visualized ref. 4), we employed patient-derived colorectal cancer organoids by microscopy with Hoechst 33342 (Life Technologies) and (CRC PDO) that are representative patient models (9). We here 1.5 mmol/L DRAQ7 (Cell Signaling Technology catalog no. 7406) show that expression of different BRAF fusions in CRC PDOs staining. For calculating organoid viability and size, organoids were renders overall resistance to targeted inhibition of the MAPK scored by morphology and analyzed by automated brightfield mor- V600E pathway, similar as the common BRAF mutation. However, phometry using OrganoSeg (15). notable differences in signaling activity and sensitivity toward MAPK-targeting drugs were observed between the different BRAF Immunofluorescence V600E oncogenes, for example, BRAF fusions and BRAF . On the basis For immunofluorescence, organoids and HEK293 cells were probed of quantitative phosphoproteomics and RNA sequencing, we show with an HA-antibody (RRID:AB_390929). Hoechst 33342 was added that 5fusion partners mainly promote canonical oncogenic BRAF together with secondary antibodies to stain for DNA. Images were activity by replacing the auto-inhibitory N-terminal region, but also captured with a Leica SP8X microscope using a 40 objective. impose unique features that can influence, among others, strength Postacquisition analyses of phenotypes were performed manually of downstream pathway activation. using ImageJ.

Drug screen and viability assessment Materials and Methods The 4-day drug screen was performed as described previously (14). BRAF (fusion) gene cloning Organoids were treated with afatinib, selumetinib, encorafenib, navi- BRAF (fusion) genes were cloned from patient RNA (4) into the toclax (Selleck Chemicals), SCH772984 (MedChem Express), and pInducer20 vector (10). TRIM24-BRAF was ordered from Twist dabrafenib (Bio-Connect). Organoid size was measured by integrating Bioscience. Gateway cloning (Invitrogen) was performed with BRAF Hoechst signal and contrast using Columbus Cellular Imaging and (fusion)-specific primers (Supplementary Table S1) according to the Analyses (Perkin Elmer). Multiple identical drug combinations were manufacturer's protocol and correct insertion of fusion gene into the averaged. Dose–response curves were generated using GraphPad pInducer20 was verified by Sanger sequencing. software by performing nonlinear regression (curve fit), assuming a standard Hill equation [chosen method: log(inhibitor) vs. Response, CRC PDO and HEK293 culture and maintenance constrain top ¼ 100]. The P18T patient-derived organoids were previously established and characterized (11, 12). P18T colorectal cancer organoids and RNA-sequencing HEK293 cells (ATCC, ordered April 4, 2018 and thereafter not Paired-end RNA-sequencing was performed by Macrogen authenticated) were cultured as described previously (11, 13). For (Korea) on the NovaSeq platform (2 100 bp, 60M reads per selection of cells stably expressing BRAF (fusion) genes, cells were sample). Data were processed with our in-house RNA analysis grown in culture medium containing 400 mg/mL G418 (Santa Cruz pipeline (https://github.com/UMCUGenetics/RNASeq, v.2.4.0, Biotechnology). P18T organoid line was validated by SNP array and default settings). Principal component analysis, Euclidean dis- confirmed Mycoplasma negative with the Mycoplasma PCR ELISA Kit tance-based clustering, and differential expression calculations were (Roche; last test was performed September 12, 2019) and organoids performed with the DESeq2 package (16). Geneset overrepresen- were kept in culture for 10 passages until final experiments were tation analysis (ORA) was performed on WebGestalt (17). RNA performed. HEK293 were kept in culture for 16 passages until final sequencing data were deposited to EGA with the dataset identifier experiments were performed. EGAS00001003558.

Lentiviral organoid and HEK293 transduction Mass spectrometry Each BRAF (fusion) gene construct was stably integrated into the For SILAC labeling, HEK293 cells were cultured in high-glucose genome of patient-derived P18T organoids or HEK293 cells utilizing DMEM (Thermo Fisher Scientific) lacking lysine and arginine sup- lentiviral transduction resulting in polyclonal BRAF (fusion) gene- plemented with Lys-0/Arg-0 or Lys-8/Arg-10 (Silantes). Mass spec- expressing lines. Virus was produced with HEK293T cells and after trometry was performed by the Proteomics Facility (UMC Utrecht). 3 days virus was sterile filtered (45 mm) and concentrated, and target Raw files were analyzed with the MaxQuant software version cells were infected. 1.6.1.0 (18). Phosphorylation sites were analyzed in Perseus software (Version 1.5) using MaxQuant normalized H/L ratios. Data were Western blot assay deposited in the ProteomeXchange Consortium via the PRIDE partner Western blotting was performed as described before (14). Mem- repository with the dataset identifier PXD013461. branes were probed with antibodies directed against HA (RRID: A detailed description of Materials and Methods is described in the AB_631618), Vinculin (RRID:AB_477629), GAPDH (RRID: Supplementary Materials and Methods.

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Results cline, no BRAF (fusion) mRNA or protein was detected, demonstrat- Establishment of inducible BRAF fusion gene expression in ing tight control of gene expression by tetracycline-responsive colorectal cancer organoids promoters. In a recent RNA-sequencing screen of primary colon tumors, we identified BRAF fusion genes containing different 50 partner genes (4). 50 partner choice affects subcellular localization and To characterize and cross-compare the influence of the 50 partner intracellular signaling capacity of BRAF fusion proteins between different BRAF fusions, we used CRC PDOs (P18T line) with a Whereas various studies have shown that BRAF fusions promote mutational background characteristic for BRAF fusion–positive colo- MAPK pathway activation by effectuating loss of the auto-inhibitory rectal cancer tumors, that is, non-functional APC and TP53, and KRAS N-terminal domain of BRAF (4, 19), specific effects imposed by the 50 wild-type (4, 11). We employed this platform (Fig. 1A) to stably partner on the fusion protein have only been studied to a limited integrate a panel of BRAF fusion genes (AGAP3-BRAF, DLG1-BRAF, extent (20). Indeed, the 5 fusion partners of BRAF, for example, WT and TRIM24-BRAF) as well as a wild-type BRAF (BRAF ), a trun- AGAP3 (21), DLG1 (22), or TRIM24 (23) manifest distinct biological Kinase cated BRAF gene containing only the kinase domain (BRAF ), and functions in their original conformation and a potential carry-over V600E a BRAF gene with the canonical V600E mutation (BRAF ; Fig. 1B). toward the fusion is likely. BRAF (fusion) gene expression was under the control of doxycycline- First we characterized the effects of fusion gene expression during inducible Tet-On activity to ensure controlled and selective construct normal culture conditions on MAPK pathway activation, which is expression (Fig. 1C). implicated in cellular growth and proliferation (24). Normal culture We confirmed selective mRNA expression of all BRAF variants conditions of CRC PDOs include EGF that induces baseline levels of upon doxycycline administration (Fig. 2A). Furthermore, the presence MAPK pathway activity. Only the expression of DLG1-BRAF resulted of the various BRAF proteins was visualized by immunoblot staining in enhanced pERK levels, while the other BRAF fusions, as well as the against the C-terminal HA-tag (Fig. 2B). In the absence of doxycy- well-known oncogenic mutant BRAFV600E did not deviate from

Figure 1. Overview of experimental set-up for characterization of BRAF (fusion) genes. A, Schematic overview of the fusion gene expression platform and workflow. Fusion genes are identified by sequencing and fusion breakpoints are validated by PCR and sequencing. (Fusion) gene constructs are cloned into the inducible expression vector pInducer20. The pInducer20 constructs are stably integrated into colorectal cancer organoids by means of lentiviral transduction. Thereafter, BRAF (fusion) genes are characterized through analysis of localization, MAPK pathway activation, phosphoproteomics, RNA-sequencing (RNA-seq), and drug screenings. B, Schematic overview of the investigated BRAF fusion genes. BRAF fusions and their respective 50 partners (AGAP3, DLG1,andTRIM24) and BRAF variants (BRAF-WT, BRAF-Kinase,andBRAF-V600E) are depicted with their retained functional domains. PH, Pleckstrin homology; L27, L27 protein interaction module; RING, zinc finger domain ring type; BBOX1, B-box-type zinc finger domain; RBD, Ras-binding domain; CRD, cysteine-rich domain; S/Th kinase domain, serine/threonine kinase domain. C, Depiction of the inducible pInducer20 vector cassette. cDNA of a BRAF (fusion) transcript (flanked by attR1/R2 sites) with an HA-tag linked to the C-terminus. The expression of the BRAF (fusion) transcript is under the control of a tetracycline-responsive element (TRE) which is activated upon interaction with doxycycline- bound reverse tetracycline-controlled transactivator 3 (rtTA3). The rtTA3 expression is under the control of a constitutively active Ubc promoter but can only interact with the TRE upon doxycycline binding.

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Figure 2. BRAF (fusion) gene expression, MAPK pathway activation, and localization in P18T colorectal cancer (CRC) organoids. A, Breakpoint-PCR verifying the expression of the BRAF (fusion) transcripts in P18T colorectal cancer organoids upon doxycycline administration. Respective breakpoint primers are used on uninduced (), induced (þ;1mg/mL doxycycline for 24 hours), positive (respective pInducer20-plasmid; P), and negative control (water; N). B, Immunoblotting for HA-tagged BRAF (fusion) proteins verifying the protein expression in P18T colorectal cancer organoids at the expected height and upon doxycycline administration (1 mg/mL for 24 hours). Correct BRAF (fusion) protein bands are highlighted with an asterisk. Vinculin was used as loading control. C, Organoids expressing BRAF genes were not induced () or induced (þ) with doxycycline (1 mg/mL) for 24 hours and immunoblotted for pERK and tERK. GAPDH was used as loading control. D, P18T colorectal cancer organoids expressing BRAF constructs were stained for the HA-tag to visualize the protein localization (HA-tag ¼ green) and the nucleus (Hoechst ¼ blue). White arrows are pointing to the protein localization at the plasma membrane.

baseline pERK levels under normal culture conditions (Fig. 2C). In We hypothesized that the 50 fusion partners can redirect the agreement with previous literature (25), none of the BRAF (fusion) subcellular localization of the BRAF fusion proteins, thereby affecting proteins had an effect on AKT activity (Supplementary Fig. S1). the efficiency by which the BRAF kinase domain can activate down- Importantly, BRAF (fusion) protein levels vary between organoid stream MEK. Therefore, we visualized the intracellular localization of lines (Fig. 2B), potentially influencing observed phenotypes. The the BRAF variants by using immunofluorescent staining against the C- expression levels of the BRAF fusions do not correlate with the levels terminal HA-tag. Indeed, we observed differences in the cellular of ERK phosphorylation, which indicates that there is a specific localization of the different BRAF fusions (Fig. 2D). The BRAFWT, influence of the 50 fusion partner on the downstream MAPK pathway BRAFKinase, and BRAFV600E proteins exhibited, as expected, a diffuse activation (Supplementary Fig. S2). localization pattern throughout the cytoplasm (26). Whereas a similar

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localization pattern was observed for the AGAP3-BRAF and TRIM24- In general, we observed that expression of BRAF fusions, BRAF fusion proteins, DLG1-BRAF fusions were primarily localized BRAFV600E, and BRAFKinase mainly induced an increase in protein to the plasma membrane. This is most likely due to the retained L27 phosphorylation in 4 hours (Fig. 3A, Table 1, Supplementary Figs. S3B domain of DLG1 in the DLG1-BRAF fusion, which triggers native and S6D). BRAFWT expression only resulted in one significantly DLG1 localization likewise to the apical plasma membrane (27). downregulated phosphosite belonging to the BRAF protein itself, indicative of a negative feedback response induced by BRAF over- Phosphoproteomics reveals that BRAF fusions and BRAFV600E expression (Fig. 3A). In contrast to BRAFV600E and BRAF fusions, the induce similar signaling pathways in HEK293 cells presence of BRAFKinase resulted in a rather low number of upregulated On the basis of the observed differences between the BRAF fusions phosphosites (Table 1, Supplementary Fig. S3B). with respect to ERK activation, we aimed to characterize the type and To explore kinase activities responsible for the deregulated targets extent of unique intracellular signaling pathways that are specifically within the phosphoproteomics data, we used kinase-substrate enrich- activated by a BRAF fusion, in comparison with BRAFV600E and ment analysis (KSEA; ref. 30). With the exception of GFP, we noticed BRAFWT. that all BRAF variants showed a significant activation of kinases To identify all downstream phosphosites that are regulated by a involved in the MAPK signaling pathway, including MEK1/2, BRAF fusion, we performed an unbiased SILAC-based phospho- ERK1/2, and Raf (Fig. 3B, Supplementary Fig. S3C, Supplementary proteomics screen (Supplementary Fig. S3A). Because organoids are Table S2). In addition, BRAF fusions and BRAFV600E also showed not a suitable platform to scale to sufficient protein quantities significant activation of kinases involved in cell-cycle progression and required for phosphoproteomic screens, and organoid growth DNA damage response, such as CDK1, CDK2, Aurora kinase B, and kinetics may potentially be affected in SILAC medium, we opted CHEK1 (Fig. 3B, Supplementary Table S2). to use HEK293 cells instead. HEK293 cells are a well-characterized Next, we explored phosphotargets that are common to all BRAF cell system with an unperturbed MAPK pathway (28), a major fusions, versus unique targets per BRAF variant. We identified 289 prerequisite to not mask potential BRAF (fusion)-induced effects on phosphosites and 298 proteins that are shared as substrate (direct or MAPK signaling, and are compatible with growth in both types of indirect) by all BRAF fusions, BRAFV600E,andBRAFKinase (Sup- SILAC media, which is not trivial for most well-known cancer cell plementary Fig. S3D). As indicated already, most of the overlapping lines. Like our organoid lines, we confirmed inducible BRAF fusion targets include members of the MAPK signaling pathway, such as gene expression by mRNA RT-PCR and Western blot analysis ERK1/2 (MAPK3/1), BRAF, and MEK2 (MAP2K2). Moreover, (Supplementary Fig. S4A and S4B). In contrast to P18T organoids, these phosphotargets turned out to be most strongly upregulated weak BRAF fusion gene expression was detected in the uninduced (Fig. 3A, Supplementary Fig. S3B). Besides shared downstream state (doxycycline) of HEK293 cells (Supplementary Figs. S4A targets, we found that each BRAF fusion also induced the phos- and S4B and S5), presumably because of supplemented FBS in phorylation of a unique set of substrates (Fig. 3A, Supplementary HEK293 culture medium (29). Importantly, however, except for Fig. S3B). Intriguingly, however, the degree by which these unique WT GFP- and BRAF -expressing cells, a strong increase in pERK phosphosites are phosphorylated was generally lower when com- levels was detected in HEK293 cells upon expression of all onco- pared with the fold change (FC) of shared phosphosites (Supple- genic BRAF variants (Supplementary Fig. S4C). Whereas a signif- mentary Fig. S3B). icant increase in ERK phosphorylation during unperturbed culture Next, when analyzing which pathways are represented by the set of conditions was only observed upon DLG1-BRAF expression in our downstream phosphotargets [ORA (WebGestalt); ref. 17], we noticed CRC PDOs (Fig. 2C),thiswasnotthecaseinHEK293cells. a high degree of overlap between the different BRAF fusion and V600E Presumably, this discrepancy may be the result of technical differ- BRAF -expressing cells (Fig. 3C). In particular, the pathways ences between the 2D and 3D models, such as different integration mainly converged on MAPK signaling pathway regulation and cell- and expression efficiencies of the fusion proteins, or may be cycle progression (Fig. 3C and D). attributed to the inherent biological differences between the two Together, from a protein intrinsic point of view, these data indicate model systems. In support of the latter, the distinctive localization that the different BRAF fusions have largely similar substrates as patterns of BRAF fusion proteins observed in organoids (Fig. 2D) BRAFV600E and activate similar signaling pathways, mainly involving was absent in unpolarized HEK293 cells (Supplementary Fig. S5; MAPK pathway activation and cell-cycle progression. To complement ref. 24). Moreover, the MAPK signaling pathway is already active in the phosphoproteomic analysis and to improve our understanding of organoids prior to induction of the fusion variants, while largely the global impact of BRAF (fusion) genes, we performed Western blot inactive in HEK293 cells, potentially masking their effects during analysis and RNA-sequencing (Materials and Methods) on CRC normal growth conditions. As a result, we now reveal phosphor- PDOs. Western blot analysis showed that doxycycline-induced expres- ylation targets of the different oncogenic BRAF variants as a result sion of BRAFV600E, AGAP3-BRAF, and DLG1-BRAF in CRC PDOs of intrinsic capacity, rather than induced by differences in subcel- indeed resulted in increased MEK-ERK phosphorylation and con- lular localization. firmed varying degrees of MAPK pathway activation between onco- To identify targets directly downstream of BRAF (fusion) protein genic BRAF variants (Supplementary Fig. S7A). On the basis of gene signaling by phosphoproteomic analysis, we measured the earliest expression analysis (Materials and Methods) of organoids expressing timepoint of BRAF (fusion) protein expression upon doxycycline- fusion genes, we indirectly confirmed the activation of MAPK signal- mediated induction. Time-course measurements of HA-tagged pro- ing by BRAF fusions, specifically for the DLG1-BRAF fusion. The tein expression and ERK phosphorylation in the GFP- and DLG1- DLG1-BRAF fusion induced a large amount of differentially expressed BRAF–expressing cell lines identified 4 hours as the minimal duration genes in CRC PDOs that were mainly involved in the cell cycle and of doxycycline exposure for protein expression and robust down- signaling by Rho GTPases (Supplementary Fig. S7B–S7D; Supple- stream MAPK pathway activation (Supplementary Fig. S6A and S6B). mentary Tables S3 and S4; refs. 31, 32). Indeed, MAPK pathway Same kinetics was confirmed in the remaining BRAF fusion cell lines activation promotes cell proliferation (33) and migration (33, 34), (Supplementary Fig. S6C). which corresponds to the strong effects of DLG1-BRAF on ERK

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Figure 3. Phosphoproteomics screen in HEK293 cells reveals that BRAF fusions and BRAFV600E activate similar signaling pathways. A, Heatmap of ratio changes of signiﬁcantly

affected (1.5 < log2 FC > 1.5, P < 0.05) phosphosites induced upon BRAF (fusion) gene expression in HEK293 cells. Phosphosites in the MAPK signaling pathway that are shared between BRAF fusion and BRAFV600E-expressing cells are indicated at the top left. B KSEA results showing kinase activity scores of BRAF fusion and BRAFV600E-expressing HEK293 cells. C, ORA of signiﬁcantly enriched pathways (heatmap of P values) upon BRAF (fusion)geneandBRAFV600E expression in HEK293 cells. Heatmap shows that the majority of highly enriched pathways are shared among oncogenic BRAFV600E and fusion variants (most signiﬁcant common pathways are indicated in top left). D, Venn diagram depicting the overlap of overrepresented pathways between oncogenic BRAF variants (fusion genes and BRAFV600E)in HEK293 cells.

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Table 1. Summary of phosphoproteomics screen in BRAF (fusion)-expressing HEK293 cells.

Phosphosites/ Signiﬁcant Upregulated Downregulated proteins phosphosites/ phosphosites/ phosphosites/ (detected in fw proteins proteins proteins

and rv experiments) (Log2 1.5 FC) (Log2 1.5 FC) (Log2 1.5 FC)

AGAP3-BRAF 5765/2456 100/87 100/87 0/0 DLG1-BRAF 6180/2526 65/61 65/61 0/0 TRIM24-BRAF 6914/2801 150/135 145/131 5/5 BRAFV600E 7159/2843 202/178 202/177 1/1 BRAFKinase 4390/2001 8/7 7/6 1/1 BRAFWT 6626/2656 6/1 0/0 6/1 GFP 6740/2705 0/0 0/0 0/0

Abbreviations: fw, forward; rv, reverse. activation. Furthermore, we confirmed that the expression of genes the MAPK pathway seem already activated to significant levels implicated in cell-cycle regulation, such as CHEK1, CHEK2, and during normal growth conditions (Fig. 2C). In addition, no influ- CCNB2 was similarly regulated in organoids expressing DLG1-BRAF ence of BRAF fusions on pAKT levels was observed (Supplementary and in a tumor sample positive for the DLG1-BRAF fusion, corrob- Fig. S10). Intriguingly, although BRAFV600E-andBRAFKinase- orating the validity of these findings (Materials and Methods, Sup- expressing organoids were able to sustain ERK phosphorylation, plementary Fig. S8A and S8B). the degree of ERK phosphorylation was affected by EGFR inhibition. This is concordant with two recent studies, which show that BRAF fusion genes confer resistance to targeted EGFR KRAS-mutant lung cancer cells still require upstream receptor inhibition signaling for tumor growth and survival (35, 38). Dependency on Besides KRAS, oncogenic mutations in BRAF (e.g., V600E) have continuous upstream signaling input at the receptor level, however, been associated with resistance to anti-EGFR–targeted therapy in was not observed in organoids expressing the BRAF fusion genes. metastatic colorectal cancer (mCRC; refs. 35, 36). BRAF fusions Furthermore, the DLG1-BRAF fusion consistently showed stronger may also influence the sensitivity of tumor cells to anti-EGFR– ERK phosphorylation, both at unperturbed (Fig. 2C)andafatinib- targeted therapy due to constitutive activation of downstream treated conditions (Fig. 4E), pointing toward an enhanced capacity MAPK signaling. Therefore, we challenged the CRC PDOs with to activate the downstream MAPK pathway, possibly due to its a pan-HER inhibitor (afatinib) for 7 days and scored viability by localization at the plasma membrane. microscopy (Fig. 4A). Consistent with previous patient-derived WT colorectal cancer studies (13), most of the GFP- and BRAF - BRAF fusions elicit differential sensitivities to combinatorial expressing organoids died upon EGFR inhibition, while the major- targeting of EGFR and MEK V600E ity of BRAF -mutant organoids showed resistance to similar To identify drugs that could be used for the treatment of patients treatment (Fig. 4B; Supplementary Fig. S9A). In addition, we with mCRC with BRAF fusion genes, we tested a panel of MAPK observed that expression of all BRAF fusion variants provided pathway–targeting agents in a drug screen (Fig. 5A). resistance to afatinib (Fig. 4C and D; Supplementary Fig. S9A). Previously, a synergistic effect was observed with dual inhibition of Kinase Interestingly, BRAF organoids exhibited an intermediate phe- the MAPK pathway on BRAF- and RAS-mutant colorectal cancer notype to anti-EGFR–targeted treatment compared with the BRAF cells (13, 39). Therefore, we investigated whether a similar effect could fusion organoids, showing few large surviving organoids together be achieved with combinatorial targeting of the MAPK pathway. WT with significant cell death (Fig. 4B and D; Supplementary Fig. S9A Organoids expressing BRAF or GFP were highly sensitive to afatinib Kinase V600E and S9B). As BRAF expression is comparable with expression (pan-HERi) treatment in this drug screen assay, while BRAF , Kinase of BRAF fusions (Fig. 2B), these data suggest that a protein domain BRAF , and BRAF fusion–expressing organoids showed resis- V600E at the N-terminal side of the BRAF kinase domain is important to tance, with BRAF -, DLG1-BRAF-, and TRIM-BRAF–mutant maximize BRAF activity. organoids showing resistance at even high concentrations (Fig. 5B, Previous studies have shown that constitutive MAPK pathway Supplementary Fig. S11A). MEK inhibition (selumetinib) had a similar WT V600E Kinase activation plays an essential role in anti-EGFR therapy resistance in inhibitory effect on BRAF , BRAF , BRAF , and BRAF mCRC (13, 37). To validate that BRAF fusion genes can promote fusion–expressing organoids, which was slightly lower compared with MAPK pathway signaling independent of external EGF stimulation, its inhibitory effect on the control GFP-expressing line (Fig. 5C, we investigated ERK and AKT activity upon afatinib treatment. In Supplementary Fig. S11A). Combining selumetinib with afatinib Kinase accordance to the observed phenotypes, the afatinib-sensitive lines resulted in an additive effect on BRAF - and AGAP3-BRAF– WT (GFP and BRAF ) failed to phosphorylate ERK upon EGFR expressing organoids that already exhibited higher sensitivity to V600E inhibition while afatinib-resistant lines (BRAF fusions, BRAF , afatinib compared with organoids with the other BRAF fusions Kinase and BRAF )wereabletosustainERKphosphorylation (Fig. 5D; Supplementary Fig. S11A). We observed only a minor (Fig. 4E). Along similar lines, we confirmed sustained phosphor- additive effect of this combinatorial treatment on highly afatinib- V600E V600E ylation by BRAF fusions and BRAF of additional targets resistant DLG1-BRAF and TRIM-BRAF fusions, as well as BRAF identified with the phosphoproteomics screen, like phosphorylated mutants, compared with afatinib or selumetinib alone. We conclude MEK (pMEK) and pCDK2 (Fig. 4F). Intriguingly, this was espe- that, unlike mutant KRAS, the combination of pan-HER and MEK cially apparent during EGFR inhibition as common targets of inhibition only has an additive effect in some, that is, AGAP3-BRAF

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Figure 4. BRAF (fusion) genes confer resistance to EGFR inhibition. A, Schematic overview of the phenotypic screening method to measure organoid viability after afatinib treatment. B, P18T colorectal cancer organoids with [þdoxycycline (dox, 1 mg/mL)] or without (doxycycline) induced expression of GFP, BRAFWT, BRAFKinase,orBRAFV600E were treated with DMSO or afatinib (afa, 1 mmol/L) for 7 days and stained for nuclei (Hoechst ¼ blue) and dead cells (DRAQ7 ¼ red). White squares represent zoomed-in areas. C, P18T colorectal cancer organoids with [þdoxycycline (1 mg/mL)] or without (doxycycline) induced expression of AGAP3-BRAF, DLG1-BRAF,or TRIM24-BRAF fusion genes were treated with DMSO or afatinib (1 mmol/L) for 7 days and stained for nuclei (Hoechst ¼ blue) and dead cells (DRAQ7 ¼ red). White squares represent zoomed-in areas. D, Bar graph depicts the percent- age of viable organoids after 7 days of DMSO or afatinib (1 mmol/L) treatment in the presence or absence of doxycycline (1 mg/mL; from two independent experiments). E, BRAF (fusion) protein– expressing organoids were treated with DMSO ()or1mmol/L afatinib (þ)for 24 hours and immunoblotted for pERK and tERK. Vinculin was used as loading control. F, Same as in E, immunoblotted for pMEK and CDK2 (pCDK2), and total MEK (tMEK) and CDK2 (tCDK2). GAPDH was used as a loading control. A, AGAP3- BRAF; D, DLG1-BRAF; T, TRIM24-BRAF; G, GFP; W, BRAF-WT; K, BRAF-Kinase; and V, BRAF-V600E.

Kinase and BRAF , but not all oncogenic BRAF-expressing colorectal been reported previously (39), organoid growth was barely affected by cancer organoids. the abrogation of BRAF kinase activity by encorafenib or dabrafenib, Next, we tested whether combining a MEK inhibitor with an ERK independent of whether oncogenic BRAF variants were present or not inhibitor has additive or synergistic effect. All oncogenic BRAF (Fig. 5G and I, Supplementary Fig. S11A). Surprisingly, combinatorial variants showed approximately similar sensitivity to the speciﬁc targeting of the MAPK pathway that includes BRAF inhibition, either ERK1/2 inhibitor SCH772984 (40), which was comparable with the combined with pan-HER or with MEK inhibitors, did not reveal control GFP-expressing organoids (Fig. 5E, Supplementary signiﬁcant improvements over pan-HER or MEK inhibition alone Fig. S11A). Combinatorial targeting of MEK (selumetinib) and ERK (Fig. 5H and J, Supplementary Fig. S11A). (SCH772984) improved overall sensitivity, without major differences Together, screening drug sensitivities across multiple oncogenic between the lines (Fig. 5F, Supplementary Fig. S11A). BRAF lines revealed intertumoral differences against the pan-HER Finally, organoids were exposed to the selective, ATP-competitive inhibitor afatinib. AGAP3-BRAF–expressing organoids behaved over- WT Kinase BRAF inhibitor encorafenib or dabrafenib (41). Similar to what has all similarly as drug sensitive BRAF and the BRAF mutants to

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Figure 5. Differential sensitivities of BRAF (fusion) genes to targeted BRAF and ERK inhibition. A, Schematic overview of the drug screening method. In short, a 4-day drug screen is started on 5 days old organoids in which fusion genes are expressed 24 hours prior to the start of the screen. Heatmap of drug response (growth) to afatinib (B), selumetinib (C), afatinib plus selumetinib (D), SCH772984 (E), SCH772984 plus selumetinib (F), encorafenib (G), encorafenib plus selumetinib (H), dabrafenib (I), and dabrafenib plus afatinib (J). Asterisks indicate analysis artefacts due to organoid swelling at high concentrations of encorafenib (Supplementary Fig. S11B).

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combined EGFR and MEK inhibition, while DLG1- and TRIM24- culture conditions. The expression of the DLG1-BRAF fusion V600E BRAF fusions approximated resistance phenotypes of the BRAF uniquely affected a subset of genes, which mainly converged on mutant. In contrast, ERK or BRAF inhibition, alone or in combination, the cell cycle. Similarly, cell-cycle–related genes were deregulated in gave similar responses between all the lines, irrespective of oncogenic apatientwith DLG1-BRAF fusion–positive colorectal cancer, sub- WT BRAF variants, BRAF , or normal control (GFP). stantiating this observation. Together, this data show that BRAF fusion genes commonly impact gene sets involved in cell proliferation and migration and that specific BRAF fusions can affect Discussion unique and distinct sets of genes. BRAF fusions are recurrent events throughout various cancer In clinical practice, patients with mCRC are treated with anti- types and form oncogenic drivers (8). Thus far, few studies have EGFR–targeting mAbs, given that the patient does not harbor onco- addressed the effect of BRAF fusion expression on intracellular genic KRAS or NRAS mutations (47, 48). In addition, oncogenic signaling and cellular processes (19, 42). Moreover, while some mutations in BRAF (e.g., V600E) are also associated with anti- results have been obtained for ALK and BRAF fusions (20, 43), the EGFR therapy resistance in colorectal cancer (49). Here, we observed possible effects induced by specific50 fusion partners of BRAF have that all BRAF fusion genes tested were able to confer resistance to not been thoroughly investigated in a clinically relevant model targeted inhibition of EGFR with the small-molecule inhibitor afatinib. system. It is generally assumed that the loss of the N-terminal Our data highlight that BRAF fusions activate the MAPK pathway to V600E domain is responsible for enhanced oncogenic BRAF activity (19). equal or even more pronounced levels than oncogenic BRAF , Whereasthisisconsistentwithourfindings showing enhanced both at unperturbed and afatinib-treated growth conditions. Further- MAPK pathway activation upon BRAF fusion gene expression, we more, sustained activity of the downstream MAPK signaling pathway systematically investigated the influence of 50 partner genes on in the presence of afatinib is in concordance with resistance mechan- BRAF activity. isms observed in patients that are insensitive to EGFR-targeting As opposed to previous studies describing fusion gene charac- agents (50). These findings emphasize the clinical relevance of this terization in cell lines, we here used a patient-derived organoid study that suggest to include BRAF fusions to genetic screening model that is the closest representative of human colorectal tumors programs for patients with colorectal cancer to assist personalized that is compatible with biochemical analysis of signaling pathway therapy design. alterations by BRAF fusions. Using the organoid system, we In conclusion, we show in a patient relevant model system that 50 observed that subcellular localization of the BRAF fusion protein, fusion partners can impose a unique influence on the oncogenic effects as well as the level of MAPK pathway activation were affected by the of BRAF, among others by redirecting its subcellular localization. 5'partner. A distinct localization of DLG1-BRAF proteins was Nevertheless, all BRAF fusion genes showed insensitivity toward detected at the plasma membrane, possibly responsible for the targeted inhibition of EGFR family members. Therefore, we provide enhanced activation of the MAPK pathway in unperturbed as well a strong incentive to include BRAF fusion genes to genetic screening as in drug-treated conditions as compared with AGAP3-BRAF, programs for patients with colorectal cancer amenable for anti-EGFR TRIM24-BRAF, and the canonical BRAFV600E mutation. Wild-type therapy. BRAF is usually expressed throughout the cytoplasm and gets recruited to the plasma membrane through activated Ras (44). Disclosure of Potential Conflicts of Interest Previous studies showed that localization of the BRAF protein to No potential conflicts of interest were disclosed. the plasma membrane potentiates BRAF signaling through prox- imity to downstream effectors (45). Mediated by its L27 domain, Authors’ Contributions DLG1 is known to localize to junctions at the plasma mem- Conception and design: C. Stangl, J.B. Post, M.J. Koudijs, E.E. Voest, H.J.G. Snippert, brane (27). Exactly this domain is retained in the DLG1-BRAF W.P. Kloosterman fusion and is likely to promote its plasma membrane localization Development of methodology: C. Stangl, J.B. Post, E.E. Voest, W.P. Kloosterman independent of active RAS. Redirected subcellular localization Acquisition of data (provided animals, acquired and managed patients, provided fl facilities, etc.): C. Stangl, H.R. Vos, R.M. van Es showcases how the 5 fusion partners can in uence functionality Analysis and interpretation of data (e.g., statistical analysis, biostatistics, of oncogenic BRAF. In concordance with previous studies, the computational analysis): C. Stangl, J.B. Post, M.J. van Roosmalen, N. Hami, unique localization of DLG1-BRAF at the plasma membrane was H.R. Vos, E.E. Voest, H.J.G. Snippert lost in unpolarized 2D HEK293 cells and underscores the advantage Writing, review, and/or revision of the manuscript: C. Stangl, J.B. Post, E.E. Voest, of using 3D tumor organoid models for assessing functional effects H.J.G. Snippert, W.P. Kloosterman of oncogenes (46). Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C. Stangl We performed an unbiased phosphoproteomic screen to identify Study supervision: E.E. Voest, W.P. Kloosterman proteins that are differentially phosphorylated by each of the Other (performed viral transductions): I. Verlaan-Klink different BRAF (fusion) genes. We observed that BRAF fusion signaling mainly converges on the same signaling targets and Acknowledgments V600E pathways (e.g., MAPK) as the oncogenic BRAF mutant, with This work was funded by the Oncode Institute, which was partly financed by the very few BRAF fusion–specific targets. On the other hand, whole- Dutch Cancer Society, and was funded by the gravitation program CancerGenomiCs.nl transcriptome expression analysis identified shared as well as from the Netherlands Organization for Scientific Research, by grants from the Dutch fusion-specific effects. Intriguingly, most of the deregulated genes Cancer Society [Koningin Wilhelmina Fonds (KWF), UU 2013-6070 and UU 2012- that all BRAF fusions have in common are presumably beyond the 5710], by a SU2C-DCS International Translational Cancer Research Dream Team Grant (SU2C-AACR-DT1415), and by a ERC starting grant (to H.J.G. Snippert), and traditional effects of MAPK pathway activity. ERK activity levels a Dutch Cancer Society grant. Stand Up To Cancer is a division of the Entertainment (with the exception of enhanced levels in DLG1-BRAF) were similar Industry Foundation. Research grants were administered by the American Associ- between all lines, which may be attributed to the already default ation for Cancer Research, the Scientific Partner of SU2C. The authors thank KWF for active MAPK signaling pathway due to EGF presence at normal funding this project. Furthermore, we thank all the members of the Kloosterman,

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Voest, Snippert, and Bos laboratories for fruitful discussions and support. We thank The costs of publication of this article were defrayed in part by the payment of page the Proteomics facility (UMCU) and Macrogen (Korea) for their help with the charges. This article must therefore be hereby marked advertisement in accordance Phosphoproteomics and RNA sequencing experiments. We thank Glen Monroe and with 18 U.S.C. Section 1734 solely to indicate this fact. Hans Bos for critical reading of the article and Francis Blokzijl for providing a script for the RNA sequencing analysis. We thank Robert Coebergh, Jan Ijzermans, Anieta Received May 17, 2019; revised November 13, 2019; accepted January 2, 2020; Sieuwerts, and John Martens for collaborations that led up to this work. published ﬁrst January 7, 2020.

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Diverse BRAF Gene Fusions Confer Resistance to EGFR-Targeted Therapy via Differential Modulation of BRAF Activity

Christina Stangl, Jasmin B. Post, Markus J. van Roosmalen, et al.

Mol Cancer Res Published OnlineFirst January 7, 2020.

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