SB202190 Predicts BRAF-Activating Mutations in Primary Colorectal Cancer Organoids via Erk1-2 Modulation

Abstract

The p38 inhibitor SB202190 is a necessary component of the medium used for normal colorectal mucosa cultures. Sato et al. suggested that the primary activity of SB202190 may be EGFR signaling stabilization, causing an increased phosphorylation of Erk1-2 sustaining organoid proliferation. However, the growth of some colorectal cancer (CRC)-derived organoid cultures is inhibited by this molecule via an unknown mechanism. We biochemically investigated SB202190 activity on a collection of 25 primary human CRC organoids, evaluating EGFR, Akt and Erk1-2 activation using Western blot. We found that Erk1-2 phosphorylation was induced by SB202190 in 20 organoid cultures and inhibited in 5 organoid cultures. A next-generation sequencing (NGS) analysis revealed that the inhibition of p-Erk1-2 signaling corresponded to the cultures with BRAF mutations (with four different hits, one being undescribed), while p-Erk1-2 induction was apparently unrelated to other mutations involving the EGFR pathway (Her2, KRAS and NRAS). We found that SB202190 mirrored the biochemical activity of the BRAF inhibitor Dabrafenib, known to induce the paradoxical activation of p-Erk1-2 signaling in BRAF wild-type cells. SB202190 was a more effective inhibitor of BRAF-mutated organoid growth in the long term than the specific BRAF inhibitors Dabrafenib and PLX8394. Overall, SB202190 can predict BRAF-activating mutations in patient-derived organoids, as well as allowing for the identification of new BRAF variants, preceding and enforcing NGS data.

Keywords: BRAF; Erk1-2; SB202190; colorectal cancer; organoid.

Figure 1

Villin1 (Vil1, enterocyte), Mucin2 (MUC2, goblet cell) and chromogranin-A (CGA, neuroendocrine cell) marker expressions in some representative organoids.

Figure 2

The biochemical signature of SB202190 (SB) on key transducers of the EGFR pathway identifies BRAF-specific modulations of p-Erk1-2. (a) An example of the WB analysis of four samples; −control of basal activation (48 h old medium), + positive control (new culture medium incubated for 2 h), SB (new culture medium incubated for 2 h with 10 µM SB202190). Green rectangles highlight p-Erk1-2 modulation in BRAF wild-type organoids; the red rectangle indicates p-Erk1-2 in a BRAF-mutated organoid (OMCR18-025TK BRAF V600E). Orange rectangles highlight the inhibition of Akt phosphorylation. (b) Analysis of the fold increase in phosphorylation of EGFR, Akt and Erk1-2 in BRAF wild-type (wt) or -mutant (mut) organoids. Single cohorts (dots) and merged results (boxes) are reported. The green arrow indicates the powerful upregulation of p-Erk1-2 signaling induced by SB202190 in BRAF wt organoids as compared to its downregulation in BRAF-mutated ones (red arrow). The orange arrows indicate the inhibition of Akt phosphorylation by SB202190 in all samples. (c) Same conditions reported in b, comparing NRAS or KRAS (N-KRAS) wild-type and mutated organoids. Statistics: * p ≤ 0.05; *** p ≤ 0.001; n = 25.

Figure 3

SB202190 mimics the biochemical activity of the BRAF inhibitor Dabrafenib on p-Erk1-2 modulation, unaffected by EGFR activation. (a) An example of the signaling of a BRAF wild-type organoid (OMCR18-035TK). The green rectangles highlight increased p-Erk1-2 activity induced by SB202190 and Dabrafenib, both in the presence (left) or absence (right) of EGF. (b) An example of the signaling of a BRAF mutated organoid (OMCR16-005TK, BRAF V600E). The red rectangles highlight the inhibition of p-Erk1-2 activity mediated by SB202190 and Dabrafenib, both in the presence (left) or absence (right) of EGF. Organoids were treated for 2 h in new culture medium with (C+) or without EGF (-EGF); 48 h old medium was used as control of basal activation (C−). SB202190 (SB) 10 µM, Dabrafenib (DBF) 1 µM, PLX8394 (PLX) 1 µM, BIRB796 (BIRB) 0.2 µM. (c) The effect of EGF (25 ng/mL) on SB202190- or Dabrafenib-treated organoids is represented for p-EGFR, p-Akt and p-Erk1-2, according to BRAF wild-type or -mutant status. No condition achieved statistically significant difference. The green arrows indicate the powerful upregulation of p-Erk1-2 signaling induced by SB202190 and Dabrafenib in BRAF wt organoids as compared to its downregulation in the BRAF-mutated ones (red arrows). (d) Comparison of the effects of SB, DBF and PLX on p-EGFR, p-Akt and p-Erk1-2, in BRAF wild-type or -mutant organoids. (e) Effects of PLX+SB or PLX+DBF co-treatment in BRAF wild-type organoids. Statistics: * p ≤ 0.05 (wt versus mutant BRAF), n = 12.

Figure 4

Modulation of BRAF-mutated organoid growth by SB202190 (10 µM), Dabrafenib (1 µM) and PLX8394 (1 µM). Left column: interpolation of the growth curves live-monitored for 9 days (12 h steps); all data are normalized against point 0 values to reduce variations in plating efficiency among different organoids. Each experimental point represents the mean of 4–5 replicates (geltrex domes) in three tests. Mutations: OMCR13-011TK BRAF K601E; OMCR16-005TK and OMCR18-025TK BRAF V600E; OMCR18-059TK BRAF Val471dup; OMCR19-011TK BRAF G469V. The statistics of each experimental point vs. controls (CTRL) are detailed in TabS3. Center column: example images from the last experimental point; the organoids monitored in a single geltrex dome are shown. Right column: the cytotoxic effects of drugs were assessed using live fluorescent marking and normalized against total cells (in the same tests shown in the left column). Statistics vs. controls (CTRL): ** p ≤ 0.01; *** p ≤ 0.001.

Figure 5

Organoids with wild-type EGFR signaling; modulation of growth by SB202190 (10 µM), Dabrafenib (1 µM) and PLX8394 (1 µM). Left column: interpolation of the growth curves live-monitored for 9 days (12 h steps); all data are normalized against point 0 values to reduce variations in plating efficiency among different organoids. Each experimental point represents the mean of 4–5 replicates (geltrex domes) in three tests. Mutations: OMCR18-035TK (frame-shift mutation of TP53 with undefined effect); OMCR18-060TK (no mutation in our NGS panel); and OMCR19-010TK (double PTEN truncation, undescribed ALK mutation, TP53 R181H). The statistics of each experimental point vs. controls (CTRL) are detailed in TabS3. Center column: example images from the last experimental point; the organoids monitored in a single geltrex dome are shown. Right column: the cytotoxic effects of drugs were assessed using live fluorescent marking and normalized against total cells (in the same tests shown in the left column). Statistics vs. controls (CTRL): * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.