Similar but different: Oncogenic MAPK pathway mutations in colorectal cancer

In metastatic colorectal cancer (mCRC), resistance to EGFR-targeted therapy is associated with MAPK pathway activating mutations. As a result, mCRC patients with such mutations, like oncogenic RAS, are currently excluded from anti-EGFR targeted therapy. However, an increasing amount of evidence supports the notion that not all oncogenic RAS proteins are equal, but impose isoform- and mutation-specific phenotypes in a context-dependent manner. Diverging phenotypes are likely to exist for most types of activating mutations. Unfortunately, these concepts are challenging to support with clinical evidence, as ideal stratification of patient cohorts per mutation type would severely dilute the desired patient numbers to draw significant conclusions.
In this thesis the similarities and discrepancies between various oncogenic MAPK pathway mutations that are prevalent in CRC progression and therapy resistance have been investigated with the aim to improve future patient stratification for treatment regimens. Patient-derived organoids from CRC samples were used that maintain the histopathological features of the native tumor. Subsequently, patient-derived CRC organoids were genetically modified using CRISPR/Cas9 technology to introduce various oncogenic mutations.
Chapter 2 provides a guide to create state-of-the-arty genetic model systems using the CRISPR/Cas9 technology for homologous recombination. Strategies to enhance current genome editing efficiencies in various model systems are described, allowing us to study oncogene-induced mutation effects at endogenous expression levels.
In chapter 3, CRISPR-mediated knock-out strategies were used to reveal that of all RASGAPs only the loss of NF1 promotes EGFR signaling independence. In comparison to oncogenic KRAS, we show that the degree of EGFR independence is less robust in NF1-deficient organoids. Furthermore, we observed that enhanced growth of NF1-deficient organoids mainly manifests itself in growth condition supplemented with EGF, demonstrating that the loss of NF1 acts as an amplifier of RAS-MAPK signaling.
In chapter 4, 5' gene fusion partners of BRAF were characterized that replace its autoinhibitory domain, thereby creating oncogenic BRAF variants. In particular, potential 5' gene fusion partners of BRAF influence BRAF localization and its efficacy to activate downstream signaling. While all BRAF fusions exhibit resistance to EGFR inhibition, they revealed differential responses to targeted therapy of downstream MAPK pathway components. Patients with mCRC expressing BRAF fusions are relatively rare and no specific therapeutic strategy is present. Our data indicate that these mutant tumors should be excluded from anti-EGFR targeted therapy.
In chapter 5, codon- and isoform-specific mutations in RAS and BRAF in mCRC progression and therapy resistance were investigated. Using CRISPR-mediated homologous recombination a panel of isogenic patient-derived organoid lines were created to study the similarities and discrepancies between various oncogenic mutations in the MAPK pathway. We show that different oncogenic MAPK pathway mutations impose distinct phenotypes in a context-dependent manner, in accordance with observations in mCRC patients. Furthermore, we show that RAS and BRAF mutant organoids are still dependent on upstream EGFR signaling, as tumor growth and corresponding levels of MEK-ERK activation decreased upon pan-HER inhibition.
Finally, the main findings of this thesis are summarized in chapter 6 and the novel insights on oncogenic RAS and BRAF signaling in CRC biology are discussed.