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. 2024 Jul 9;22(1):646.
doi: 10.1186/s12967-024-05467-2.

Novel drug resistance mechanisms and drug targets in BRAF-mutated peritoneal metastasis from colorectal cancer

Christin Lund-Andersen #  1 , Annette Torgunrud #  2 , Chakravarthi Kanduri #  3 , Vegar J Dagenborg #  4 , Ida S Frøysnes  2 , Mette M Larsen  4   5 , Ben Davidson  6   5 , Stein G Larsen  4 , Kjersti Flatmark  2   4   5
Affiliations

Novel drug resistance mechanisms and drug targets in BRAF-mutated peritoneal metastasis from colorectal cancer

Christin Lund-Andersen et al. J Transl Med. .

Abstract

Background: Patients with peritoneal metastasis from colorectal cancer (PM-CRC) have inferior prognosis and respond particularly poorly to chemotherapy. This study aims to identify the molecular explanation for the observed clinical behavior and suggest novel treatment strategies in PM-CRC.

Methods: Tumor samples (230) from a Norwegian national cohort undergoing surgery and hyperthermic intraperitoneal chemotherapy (HIPEC) with mitomycin C (MMC) for PM-CRC were subjected to targeted DNA sequencing, and associations with clinical data were analyzed. mRNA sequencing was conducted on a subset of 30 samples to compare gene expression in tumors harboring BRAF or KRAS mutations and wild-type tumors.

Results: BRAF mutations were detected in 27% of the patients, and the BRAF-mutated subgroup had inferior overall survival compared to wild-type cases (median 16 vs 36 months, respectively, p < 0.001). BRAF mutations were associated with RNF43/RSPO aberrations and low expression of negative Wnt regulators (ligand-dependent Wnt activation). Furthermore, BRAF mutations were associated with gene expression changes in transport solute carrier proteins (specifically SLC7A6) and drug metabolism enzymes (CES1 and CYP3A4) that could influence the efficacy of MMC and irinotecan, respectively. BRAF-mutated tumors additionally exhibited increased expression of members of the novel butyrophilin subfamily of immune checkpoint molecules (BTN1A1 and BTNL9).

Conclusions: BRAF mutations were frequently detected and were associated with particularly poor survival in this cohort, possibly related to ligand-dependent Wnt activation and altered drug transport and metabolism that could confer resistance to MMC and irinotecan. Drugs that target ligand-dependent Wnt activation or the BTN immune checkpoints could represent two novel therapy approaches.

Keywords: Colorectal cancer; Drug resistance; Peritoneal metastasis; Therapeutic targets.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
DNA aberrations in PM-CRC. A Genes mutated in PM-CRC patients (n = 230) detected by targeted DNA sequencing: unsupervised clustering of mutational profiles, blue marker: gene mutation, grey marker: data not available, red and green outline: co-occurring or mutually exclusive mutations before multiple testing, *padj < 0.1, **padj < 0.01, ***padj < 0.001, ****padj < 0.0001. BRAF mutations frequently co-occur with RNF43, NOTCH1 and NF1 mutations and are mutually exclusive with KRAS and APC mutations. B gene mutation frequency of genes mutated in more than five cases; colors indicate different mutation types. BRAF mutations are surprisingly frequent, present in 27% of the patients. C and D Copy number gains found in PM-CRC patients (n = 137). Colors indicate co-occurrence with mutations in BRAF, KRAS or wild-type (WT). E and F Fusion genes found in PM-CRC patients (n = 128) by targeted RNA sequencing, colors indicate co-occurrence with mutations in BRAF, KRAS or wild-type (WT). BRAF mutations frequently co-occur with R-Spondin (RSPO) fusions (p = 0.02), while KRAS mutations co-occur with PIK3CA fusions (p = 0.04)
Fig. 2
Fig. 2
Associations between mutations and long-term outcome. A Significant findings from univariable analyses of overall survival (OS) for the total cohort (n = 230); mutated BRAF, SMAD4 and MSH6 compared to wild-type (WT) (upper panel) and for cohort subgroups; mutated BRAF (CC = 0), SMAD4 (CC ≥ 1) and MSH6 (CC = 0) compared to WT (lower panel). B Progression-free survival (PFS, n = 164). C Mutated genes associated with primary tumor location, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. BRAF and RNF43 mutations are associated with right sided primary CRC
Fig. 3
Fig. 3
Differential gene expression and gene set enrichment analyses. A Heatmap of significant differential expressed genes (DEGs, p < 0.1) for individual patient samples comparing cases with mutated BRAF to mutated KRAS or WT, blue color: low expression, red color: high expression. Normalized counts from DESeq2 are used for visualization. Rows are centred and scaled. B Volcano plots showing DEGs significantly associated with BRAF mutation compared to WT (left panel) and BRAF mutation compared to KRAS mutation (right panel). C Gene set enrichment analysis showing affected signaling pathways and the DEGs involved, red markers: DEGs upregulated in BRAF mutated cases, blue markers: DEGs downregulated in BRAF mutated cases. Drug metabolism and immune signaling pathways are enriched in BRAF mutated cases, while SLC-mediated transport and Wnt signaling pathways are diminished
Fig. 4
Fig. 4
Differential expression of genes involved in drug metabolism, transport, immune signaling and Wnt signaling. Heatmaps with relative average gene expression levels within each group (BRAF, KRAS, WT) and boxplots of selected genes (indicating median, 25 and 75 percentile): A and B Altered expression of drug metabolism genes e.g. CYP3A4 and CES1 known to metabolize irinotecan, and reduced expression of SLC genes (e.g. SLC7A6) involved in transport of mitomycin C in BRAF mutated cases. C and D Increased expression of interferon stimulated genes (ISG, compared to KRAS mutated only) and BTN checkpoint molecules in BRAF mutated cases. E and F Similar expression of genes involved in the Wnt activation signature (Hallmark M5895) between the groups, however reduced expression of negative Wnt regulators in BRAF mutated cases, associated with ligand-dependent Wnt signaling. *padj < 0.1, **padj < 0.01, ***padj < 0.001, ****padj < 0.0001. Mean normalized read counts (DESeq2) in log2 scale were centred and scaled for visualization in heatmaps (A, C, E)
Fig. 5
Fig. 5
Consensus molecular subtype (CMS) classification. A Distribution of CMS subtypes in PM-CRC (n = 30). CMS1 and CMS4 are enriched compared to pCRC. B Distribution of CMS subtypes in PM-CRC mutational subgroups (KRAS, BRAF, WT). BRAF mutated cases are enriched with CMS1
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