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

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.

Patient-Derived Ovarian Cancer Organoids Mimic Clinical Response and Exhibit Heterogeneous Inter- and Intrapatient Drug Responses.

There remains an unmet need for preclinical models to enable personalized therapy for ovarian cancer (OC) patients. Here we evaluate the capacity of patient-derived organoids (PDOs) to predict clinical drug response and functional consequences of tumor heterogeneity. We included 36 whole-genome-characterized PDOs from 23 OC patients with known clinical histories. OC PDOs maintain the genomic features of the original tumor lesion and recapitulate patient response to neoadjuvant carboplatin/paclitaxel combination treatment. PDOs display inter- and intrapatient drug response heterogeneity to chemotherapy and targeted drugs, which can be partially explained by genetic aberrations. PDO drug screening identifies high responsiveness to at least one drug for 88% of patients. PDOs are valuable preclinical models that can provide insights into drug response for individual patients with OC, complementary to genetic testing. Generating PDOs of multiple tumor locations can improve clinical decision making and increase our knowledge of genetic and drug response heterogeneity.

CRISPR-induced RASGAP deficiencies in colorectal cancer organoids reveal that only loss of NF1 promotes resistance to EGFR inhibition.

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.

Ongoing chromosomal instability and karyotype evolution in human colorectal cancer organoids.

Chromosome segregation errors cause aneuploidy and genomic heterogeneity, which are hallmarks of cancer in humans. A persistent high frequency of these errors (chromosomal instability (CIN)) is predicted to profoundly impact tumor evolution and therapy response. It is unknown, however, how prevalent CIN is in human tumors. Using three-dimensional live-cell imaging of patient-derived tumor organoids (tumor PDOs), we show that CIN is widespread in colorectal carcinomas regardless of background genetic alterations, including microsatellite instability. Cell-fate tracking showed that, although mitotic errors are frequently followed by cell death, some tumor PDOs are largely insensitive to mitotic errors. Single-cell karyotype sequencing confirmed heterogeneity of copy number alterations in tumor PDOs and showed that monoclonal lines evolved novel karyotypes over time in vitro. We conclude that ongoing CIN is common in colorectal cancer organoids, and propose that CIN levels and the tolerance for mitotic errors shape aneuploidy landscapes and karyotype heterogeneity.

An organoid platform for ovarian cancer captures intra- and interpatient heterogeneity.

Ovarian cancer (OC) is a heterogeneous disease usually diagnosed at a late stage. Experimental in vitro models that faithfully capture the hallmarks and tumor heterogeneity of OC are limited and hard to establish. We present a protocol that enables efficient derivation and long-term expansion of OC organoids. Utilizing this protocol, we have established 56 organoid lines from 32 patients, representing all main subtypes of OC. OC organoids recapitulate histological and genomic features of the pertinent lesion from which they were derived, illustrating intra- and interpatient heterogeneity, and can be genetically modified. We show that OC organoids can be used for drug-screening assays and capture different tumor subtype responses to the gold standard platinum-based chemotherapy, including acquisition of chemoresistance in recurrent disease. Finally, OC organoids can be xenografted, enabling in vivo drug-sensitivity assays. Taken together, this demonstrates their potential application for research and personalized medicine.