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Supplementary Table 3. Z-scores of significantly differential expressed genes (provided separately due to size)

Supplementary Table 2. Differential Gene Expression Analysis on BRAF (fusion) gene expressing organoid lines (provided separately due to size)

Fusion genes can be oncogenic drivers in a variety of cancer types and represent potential targets for targeted therapy. The BRAF gene is frequently involved in oncogenic gene fusions, with fusion frequencies of 0.2%–3% throughout different cancers. However, BRAF fusions rarely occur in the same gene configuration, potentially challenging personalized therapy design. In particular, the impact of the wide variety of fusion partners on the oncogenic role of BRAF during tumor growth and drug response is unknown. Here, we used patient-derived colorectal cancer organoids to functionally characterize and cross-compare BRAF fusions containing various partner genes (AGAP3, DLG1, and TRIM24) with respect to cellular behavior, downstream signaling activation, and response to targeted therapies. We demonstrate that 5′ fusion partners mainly promote canonical oncogenic BRAF activity by replacing the auto-inhibitory N-terminal region. In addition, the 5′ partner of BRAF fusions influences their subcellular localization and intracellular signaling capacity, revealing distinct subsets of affected signaling pathways and altered gene expression. Presence of the different BRAF fusions resulted in varying sensitivities to combinatorial inhibition of MEK and the EGF receptor family. However, all BRAF fusions conveyed resistance to targeted monotherapy against the EGF receptor family, suggesting that BRAF fusions should be screened alongside other MAPK pathway alterations to identify patients with metastatic colorectal cancer to exclude from anti-EGFR–targeted treatment.Implications:Although intracellular signaling and sensitivity to targeted therapies of BRAF fusion genes are influenced by their 5′ fusion partner, we show that all investigated BRAF fusions confer resistance to clinically relevant EGFR inhibition.

Supplementary Figure Legends

Supplementary Table 1. KSEA kinase scores and kinase-substrate links identified in phosphoproteomic screen (provided separately due to size)

Supplementary Table 4. List of BRAF (fusion) gene and GFP-specific primers. Primer used for breakpoint PCR, HA-tag spanning PCR and for the gateway cloning

S1. Impact of BRAF (fusion) gene expression on phosphorylation of AKT. S2. BRAF (fusion) gene mRNA and protein expression levels. S3. Phosphoproteomics screen in HEK293 cells reveals that BRAF fusions and BRAFV600E activate similar signaling pathways. S4. BRAF (fusion) gene expression and MAPK pathway activation in HEK293 cells. S5. BRAF (fusion) protein localization in HEK293 cells. S6. Establishment of optimal duration of dox induction for BRAF (fusion) protein expression. S7. Validation of phospho-proteins and transcriptomic analysis of BRAF (fusion) gene expressing P18T CRC organoid lines. S8. Expression of most differentially expressed genes in DLG1-BRAF expressing P18T organoids and CRC patients. S9. BRAF (fusion) genes confer resistance to EGFR inhibition. S10. Impact of afatinib exposure on pAKT levels in BRAF (fusion) gene expressing CRC organoids. S11. Differential sensitivities of BRAF (fusion) genes to combinatorial targeting of EGFR and MEK.

Supplementary Materials and Methods

Fusion genes can be oncogenic drivers in a variety of cancer types and represent potential targets for targeted therapy. The BRAF gene is frequently involved in oncogenic gene fusions, with fusion frequencies of 0.2%–3% throughout different cancers. However, BRAF fusions rarely occur in the same gene configuration, potentially challenging personalized therapy design. In particular, the impact of the wide variety of fusion partners on the oncogenic role of BRAF during tumor growth and drug response is unknown. Here, we used patient-derived colorectal cancer organoids to functionally characterize and cross-compare BRAF fusions containing various partner genes (AGAP3, DLG1, and TRIM24) with respect to cellular behavior, downstream signaling activation, and response to targeted therapies. We demonstrate that 5′ fusion partners mainly promote canonical oncogenic BRAF activity by replacing the auto-inhibitory N-terminal region. In addition, the 5′ partner of BRAF fusions influences their subcellular localization and intracellular signaling capacity, revealing distinct subsets of affected signaling pathways and altered gene expression. Presence of the different BRAF fusions resulted in varying sensitivities to combinatorial inhibition of MEK and the EGF receptor family. However, all BRAF fusions conveyed resistance to targeted monotherapy against the EGF receptor family, suggesting that BRAF fusions should be screened alongside other MAPK pathway alterations to identify patients with metastatic colorectal cancer to exclude from anti-EGFR–targeted treatment. Implications: Although intracellular signaling and sensitivity to targeted therapies of BRAF fusion genes are influenced by their 5′ fusion partner, we show that all investigated BRAF fusions confer resistance to clinically relevant EGFR inhibition.

Contains fulltext : 215760.pdf (Publisher’s version ) (Open Access) Anti-EGFR therapy is used to treat metastatic colorectal cancer (CRC) patients, for which initial response rates of 10-20% have been achieved. Although the presence of HER2 amplifications and oncogenic mutations in KRAS, NRAS, and BRAF are associated with EGFR-targeted therapy resistance, for a large population of CRC patients the underlying mechanism of RAS-MEK-ERK hyperactivation is not clear. Loss-of-function mutations in RASGAPs are often speculated in literature to promote CRC growth as being negative regulators of RAS, but direct experimental evidence is lacking. We generated a CRISPR-mediated knock out panel of all RASGAPs in patient-derived CRC organoids and found that only loss of NF1, but no other RASGAPs e.g. RASA1, results in enhanced RAS-ERK signal amplification and improved tolerance towards limited EGF stimulation. Our data suggests that NF1-deficient CRCs are likely not responsive to anti-EGFR monotherapy and can potentially function as a biomarker for CRC progression.

Direct targeting of the downstream mitogen-activated protein kinase (MAPK) pathway to suppress extracellular-regulated kinase (ERK) activation in KRAS and BRAF mutant colorectal cancer (CRC) has proven clinically unsuccessful, but promising results have been obtained with combination therapies including epidermal growth factor receptor (EGFR) inhibition. To elucidate the interplay between EGF signalling and ERK activation in tumours, we used patient-derived organoids (PDOs) from KRAS and BRAF mutant CRCs. PDOs resemble in vivo tumours, model treatment response and are compatible with live-cell microscopy. We established real-time, quantitative drug response assessment in PDOs with single-cell resolution, using our improved fluorescence resonance energy transfer (FRET)-based ERK biosensor EKAREN5. We show that oncogene-driven signalling is strikingly limited without EGFR activity and insufficient to sustain full proliferative potential. In PDOs and in vivo, upstream EGFR activity rigorously amplifies signal transduction efficiency in KRAS or BRAF mutant MAPK pathways. Our data provide a mechanistic understanding of the effectivity of EGFR inhibitors within combination therapies against KRAS and BRAF mutant CRC. Ponsioen et al. use a FRET‐based ERK biosensor EKAREN5 in patient‐derived organoids to show that EGFR activity amplifies signal transduction efficiency in KRAS or BRAF mutant MAPK pathways.

Model systems with defined genetic modifications are powerful tools for basic research and translational disease modelling. Fortunately, generating state-of-the-art genetic model systems is becoming more accessible to non-geneticists due to advances in genome editing technologies. As a consequence, solely relying on (transient) overexpression of (mutant) effector proteins is no longer recommended since scientific standards increasingly demand genetic modification of endogenous loci. In this review, we provide up-to-date guidelines with respect to homology-directed repair (HDR)-mediated editing of mammalian model systems, aimed at assisting researchers in designing an efficient genome editing strategy.

Colorectal cancers (CRCs) with oncogenic mutations in RAS and BRAF are associated with anti-EGFR therapy resistance. Consequently, all RAS mutant CRC patients are being excluded from this therapy. However, heterogeneity in drug response has been reported between RAS mutant CRC patients. It is poorly understood to what extent such differences are derived from different genetic backgrounds or intrinsic differences between the various RAS pathway mutations. Therefore, using CRISPR technology we generated an isogenic panel of patient-derived CRC organoids with various RAS pathway mutations (i.e. KRASG12D, BRAFV600E, KRASG13D and NRASG12D). All RAS pathway mutants promote ERK activation and tumor growth. However, KRASG12D and BRAFV600E mutations in particular conferred robust resistance to anti-EGFR therapy, both in vitro and in vivo. Moreover, untreated KRASG13D mutants showed fastest growth in mice but remained sensitive to anti-EGFR therapy. Together, introducing mutation-specific oncogene signaling in CRC organoids resembles clinical phenotypes and improves understanding of genotype-phenotype correlations.

RAS and BRAF proteins are frequently mutated in colorectal cancer (CRC) and have been associated with therapy resistance in metastatic CRC patients. RAS isoforms are considered to act as redundant entities in physiological and pathological settings. However, there is compelling evidence that mutant variants of RAS and BRAF have different oncogenic potentials and therapeutic outcomes. In this review we describe similarities and differences between various RAS and BRAF oncogenes in CRC development, histology, and therapy resistance. In addition, we discuss the potential of patient-derived tumor organoids for personalized therapy, as well as CRC modeling using genome editing in preclinical model systems to study similarities and discrepancies between the effects of oncogenic MAPK pathway mutations on tumor growth and drug response.

Fusion genes can be oncogenic drivers in a variety of cancer types and represent potential targets for targeted therapy. The

The CRISPR/Cas9 system is a highly effective tool for genome editing. Key to robust genome editing is the efficient delivery of the CRISPR/Cas9 machinery. Viral delivery systems are efficient vehicles for the transduction of foreign genes but commonly used viral vectors suffer from a limited capacity in the genetic information they can carry. Baculovirus however is capable of carrying large exogenous DNA fragments. Here we investigate the use of baculoviral vectors as a delivery vehicle for CRISPR/Cas9 based genome-editing tools. We demonstrate transduction of a panel of cell lines with Cas9 and an sgRNA sequence, which results in efficient knockout of all four targeted subunits of the chromosomal passenger complex (CPC). We further show that introduction of a homology directed repair template into the same CRISPR/Cas9 baculovirus facilitates introduction of specific point mutations and endogenous gene tags. Tagging of the CPC recruitment factor Haspin with the fluorescent reporter YFP allowed us to study its native localization as well as recruitment to the cohesin subunit Pds5B.