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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.

Redox signaling is controlled by the reversible oxidation of cysteine thiols, a post-translational modification triggered by H2O2 acting as a second messenger. However, H2O2 actually reacts poorly with most cysteine thiols and it is not clear how H2O2 discriminates between cysteines to trigger appropriate signaling cascades in the presence of dedicated H2O2 scavengers like peroxiredoxins (PRDXs). It was recently suggested that peroxiredoxins act as peroxidases and facilitate H2O2-dependent oxidation of redox-regulated proteins via disulfide exchange reactions. It is unknown how the peroxiredoxin-based relay model achieves the selective substrate targeting required for adequate cellular signaling. Using a systematic mass-spectrometry-based approach to identify cysteine-dependent interactors of the five human 2-Cys peroxiredoxins, we show that all five human 2-Cys peroxiredoxins can form disulfide-dependent heterodimers with a large set of proteins. Each isoform displays a preference for a subset of disulfide-dependent binding partners, and we explore isoform-specific properties that might underlie this preference. We provide evidence that peroxiredoxin-based redox relays can proceed via two distinct molecular mechanisms. Altogether, our results support the theory that peroxiredoxins could play a role in providing not only reactivity but also selectivity in the transduction of peroxide signals to generate complex cellular signaling responses.

Cholestasis‐induced accumulation of bile acids in the liver leads to farnesoid X receptor (FXR)‐mediated transcriptional down‐regulation of the bile acid importer Na+‐taurocholate cotransporting protein (NTCP) and to induction of endoplasmic reticulum (ER) stress. However, whether ER stress affects bile acid uptake is largely unknown. Here, we investigated the role of ER stress on the regulation and function of the bile acid transporter NTCP. ER stress was induced using thapsigargin or subtilase cytotoxin in human osteosarcoma (U2OS) and human hepatocellular carcinoma (HepG2) cells stably expressing NTCP. Cellular bile acid uptake was determined using radiolabeled taurocholate (TCA). NTCP plasma membrane expression was determined by cell surface biotinylation. Mice received a single injection of thapsigargin, and effects of ER stress on NTCP messenger RNA (mRNA) and protein were measured by reverse‐transcription polymerase chain reaction (RT‐PCR) and western blot analysis. Effects of cholestasis on NTCP and ER stress were assessed in response to 3, 5‐diethoxycarbonyl‐1, 4‐dihydrocollidine (DDC) feeding or bile duct ligation in FXR–/– mice after 7 or 3 days, respectively. Novel NTCP‐interacting proteins were identified by mass spectrometry (MS), interaction verified, and assessed by co‐immunoprecipitation and TCA uptake for functional relevance in relation to ER stress. ER stress induction strongly reduced NTCP protein expression, plasma membrane abundance, and NTCP‐mediated bile acid uptake. This was not controlled by FXR or through a single unfolded protein response (UPR) pathway but mainly depended on the interaction of NTCP with calnexin, an ER chaperone. In mice, expression of both NTCP and calnexin was reduced by thapsigargin or cholestasis‐induced ER stress. Calnexin down‐regulation in vitro recapitulated the effect of ER stress on NTCP. Conclusion: ER stress‐induced down‐regulation of calnexin provides an additional mechanism to dampen NTCP‐mediated bile acid uptake and protect hepatocytes against bile acid overload during cholestasis.

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

Mutations in the RAS genes are identified in a variety of clinical settings, ranging from somatic mutations in oncology to germline mutations in developmental disorders, also known as ‘RASopathies’, and vascular malformations/overgrowth syndromes. Generally single amino acid substitutions are identified, that result in an increase of the GTP bound fraction of the RAS proteins causing constitutive signalling. Here, a series of 7 in-frame insertions and duplications in HRAS (n = 5) and KRAS (n = 2) is presented, resulting in the insertion of 7–10 amino acids residues in the switch II region. These variants were identified in routine diagnostic screening of 299 samples for somatic mutations in vascular malformations/overgrowth syndromes (n = 6) and in germline analyses for RASopathies (n = 1). Biophysical characterization shows the inability of Guanine Nucleotide Exchange Factors to induce GTP loading and reduced intrinsic and GAP-stimulated GTP hydrolysis. As a consequence of these opposing effects, increased RAS signalling is detected in a cellular model system. Therefore these in-frame insertions represent a new class of weakly activating clinically relevant RAS variants.

The RNA polymerase II complex (pol II) is responsible for transcription of all similar to 21,000 human protein-encoding genes. Here, we describe sixteen individuals harboring de novo heterozygous variants in POLR2A, encoding RPB1, the largest subunit of pol II. An iterative approach combining structural evaluation and mass spectrometry analyses, the use of S. cerevisiae as a model system, and the assessment of cell viability in HeLa cells allowed us to classify eleven variants as probably disease-causing and four variants as possibly disease-causing. The significance of one variant remains unresolved. By quantification of phenotypic severity, we could distinguish mild and severe phenotypic consequences of the disease-causing variants. Missense variants expected to exert only mild structural effects led to a malfunctioning pol II enzyme, thereby inducing a dominant-negative effect on gene transcription. Intriguingly, individuals carrying these variants presented with a severe phenotype dominated by profound infantile-onset hypotonia and developmental delay. Conversely, individuals carrying variants expected to result in complete loss of function, thus reduced levels of functional pol II from the normal allele, exhibited the mildest phenotypes. We conclude that subtle variants that are central in functionally important domains of POLR2A cause a neurodevelopmental syndrome characterized by profound infantile-onset hypotonia and developmental delay through a dominant-negative effect on pol-II-mediated transcription of DNA.

Covalent modifications of histones have an established role as chromatin effectors, as they control processes such as DNA replication and transcription, and repair or regulate nucleosomal structure. Loss of modifications on histone N tails, whether due to mutations in genes belonging to histone-modifying complexes or mutations directly affecting the histone tails, causes developmental disorders or has a role in tumorigenesis. More recently, modifications affecting the globular histone core have been uncovered as being crucial for DNA repair, pluripotency and oncogenesis. Here we report monoallelic missense mutations affecting lysine 91 in the histone H4 core (H4K91) in three individuals with a syndrome of growth delay, microcephaly and intellectual disability. Expression of the histone H4 mutants in zebrafish embryos recapitulates the developmental anomalies seen in the patients. We show that the histone H4 alterations cause genomic instability, resulting in increased apoptosis and cell cycle progression anomalies during early development. Mechanistically, our findings indicate an important role for the ubiquitination of H4K91 in genomic stability during embryonic development.