CDK4/6 inhibition to resensitize BRAF/EGFR inhibitor in patient-derived BRAF/PTEN-mutant colon cancer cells

Introduction

Approximately 5% to 9% of cases of colorectal cancers (CRCs) exhibit a distinct mutation within v-raf murine sarcoma viral oncogene homolog B1 (BRAF) gene known as V600E (1,2). BRAF V600E mutated CRCs have a poor prognosis, resulting in the risk of mortality increasing by more than two times that of the wild-type BRAF CRCs (3). Activation of the protein product of the non-mutated BRAF gene occurs downstream of the activated Kirsten rat sarcoma viral oncogene homolog (KRAS) protein in the epidermal growth factor receptor (EGFR) pathway. It is hypothesized that the mutated BRAF protein product exhibits constitutively activity (4-6), potentially circumventing the inhibition of EGFR by cetuximab or panitumumab.

Although melanomas harboring BRAF V600E mutation are highly sensitive to BRAF inhibitors as single agents, previous clinical trials with BRAF inhibitors in combination with EGFR or mitogen-activated protein kinase kinase (MEK) inhibitors in CRCs showed only a modest overall response rate (ORR) and disappointing survival results (7). The BEACON trial reported that the double combination of encorafenib-cetuximab and triple combination of encorafenib-cetuximab-binimetinib significantly improved ORR to over 20% and median overall survival of around 8–9 months compared to standard chemotherapy (cetuximab plus irinotecan or folinic acid/fluorouracil/irinotecan; FOLFIRI) (8,9). Based on these results, encorafenib plus cetuximab has become a new standard care regimen for previously treated patients with BRAF V600E mutant CRC. However, the median progression-free survival (PFS) was reported to be 4.3 months and several studies are underway to overcome the acquired resistance caused by the reactivation of the mitogen-activated protein kinase (MAPK) pathway (10-13).

Cyclin-dependent kinase-4 and 6 (CDK4/6) play key roles in cell proliferation. Dysregulation of cell cycle machinery and activation of CDK results in uncontrolled cellular proliferation in cancer (14,15). Ribociclib is an orally bioavailable and highly selective CDK4/6 targeting agent that exhibits an IC₅₀ value in the low nanomolar range and is often used in combination with endocrine therapy in breast cancer (16). In BRAF V600E mutant melanoma, a preclinical study showed that BRAF inhibitor (vemurafenib) resistant tumors via MAPK reactivation showed cyclin D1 elevation but the inhibition of CDK4/6 by LY2835219 caused tumor growth regression (17).

We established patient-derived cells (PDCs) from a patient with BRAF V600E mutant CRC whose tumors rapidly progressed after encorafenib and cetuximab treatment. The aim of the study is to investigate the resistance mechanisms in BRAF-mutant CRC patient refractory to BRAF/EGFR targeted therapy and to explore therapeutic options to overcome intrinsic resistance of BRAF and EGFR inhibitors. We investigated the therapeutic impact of ribociclib and the combination of encorafenib plus cetuximab in BRAF-mutant CRC. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-24-20/rc).

Methods

Tumor DNA extraction and TruSight oncology 500 (TSO500) assays

Before the assay, genomic DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tumor tissues using an AllPrep DNA/RNA FFPE Kit (Qiagen, Venlo, The Netherlands). The concentration of DNA was measured by a Qubit dsDNA HS assay (Thermo Fisher Scientific, Waltham, USA), then 40 ng of DNA were sheared using a Covaris E220 Focused-ultrasonicator (Woburn, USA) and eight microTUBE-50 Strip AFA Fiber V2 following the manufacturer’s instructions. DNA libraries were generated with a TSO 500 kit (Illumina, Madison, USA), according to the manufacturer’s protocol. Data exported from the TSO 500 pipeline were analyzed using the Ensembl Variant Effect Predictor Annotation Engine with information from databases to identify genomic changes, such as copy number variants, insertions/deletions, gene infusions, and single-nucleotide variants.

PDC line culture

Peritoneal ascites from the patient were collected for therapeutic purposes after obtaining informed consent documents, and all procedures were performed according to the Declaration of Helsinki (as revised in 2013). The study was approved by the Institutional Review Board committee of Samsung Medical Center (SMC), Seoul, Republic of Korea, on the use of human samples for experimental studies (No. 2021-09-052).

Approximately 1-L effusions were used to prepare PDCs as described previously with minor modifications (18,19). Of the collected cells, 2×10⁷ cells were seeded onto a 150-mm culture plate (Corning Costar, New York, USA) and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% antibiotic-antimycotic solution (Gibco BRL, UK), 0.5 µg/mL hydrocortisone (Sigma Aldrich, St. Louis, USA), 5 µg/mL insulin (PeproTech, Waltham, USA), and 5 ng/mL epidermal growth factor (PeproTech). The cells were maintained at 37 ℃ in a humidified 5% CO₂ incubator. Adherent PDCs were washed with Dulbecco’s phosphate-buffered saline (Welgene, South Korea) after reaching 80–90% confluence and incubated with TrypLE Express (Gibco BRL) to detach the cells. Then, the cells were harvested and cryopreserved for further experiments using Cell Banker 1 (Zenogen Pharma, Tokyo, Japan).

Pharmacological inhibitors

Encorafenib (LGX818), cetuximab (C225), and ribociclib (LEE011) were all purchased from Selleck Chemicals LLC (Huston, USA) and prepared for use following the provider’s instructions.

Proliferation and cytotoxicity assay

Relative cell number was evaluated by using a CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, USA) following the manufacturer’s instructions.

For the proliferation assay, 3×10³ cells/well were seeded in 96-well plates (Corning Costar) 24 h before the inhibitor treatment. After 72 h exposure to the serially diluted reagents, each well was incubated with the assay solution for 2 h at 37 ℃ in 5% CO₂. A microplate reader (X-Mark Microplate; Bio-Rad Laboratories, Hercules, USA) was used to determine the absorbance at 490 nm. PDCs with vehicle media served as a control, and wells with media alone as a blank. Relative live cell percentage was calculated as follows: (absorbance of interest − blank absorbance)/(PDC alone absorbance − blank absorbance) ×100.

For the cytotoxicity assay, 6×10³ cells/well were used and the cells were incubated with the cytotoxic reagents for 24 h. The rest of the procedure is the same as the proliferation assay above. All assays were performed in triplicate in 96-well plates.

Assessment of Ki-67 expression with flow cytometry

The cells were incubated with the inhibitors for 24 h and harvested for flow cytometry analysis. The Zombie NIR Fixable Viability Kit (BioLegend, San Diego, USA) was used to discriminate live cells from dead cells. Intracellular Ki-67 staining (BD Horizon BV480 Mouse Anti-Ki-67 antibody; BD Biosciences) was performed after fixation and permeabilization (eBioscience, Inc., San Diego, USA). Cytek Aurora flow cytometer (Cytek Biosciences, Fremont, USA) was used to acquire and analyze immunofluorescence data.

Drug combination index (CI)

Drug combination effects on cell viability from the cytotoxic assay were assessed by data-analytic software CompuSyn 3.0.1 (ComboSyn Pharmaceutics Inc., Paramus, New Jersey, USA). The CI values were calculated according to the publisher’s instructions with the relative cell viability values. Combination effects were considered synergistic (CI <0.9), additive (CI =0.9–1.1), or antagonistic (CI >1.1).

Statistical analyses

All statistical analyses were carried out using GraphPad Prism (GraphPad software). The results are presented as the mean ± standard error of the mean (SEM) and differences were assessed using one-way analysis of variance (ANOVA) or paired t-test as indicated in figure legends. A P value <0.05 was considered statistically significant.

Results

Patient

A 41-year-old woman initially presented with mechanical bowel obstruction due to sigmoid colon cancer with multiple liver and lymph node metastases. She underwent colonic stent insertion; however, colon perforation occurred after stent insertion, and she underwent radical sigmoid colectomy. Pathology revealed adenocarcinoma, moderately differentiated with focal micropapillary features, and the next-generation sequencing (NGS) TruSight results confirmed BRAF V600E mutation with microsatellite stable (MSS) tumor. Targeted sequencing also revealed concomitant phosphatase and tensin homolog (PTEN), FANCA, and PALB2 aberrations. Although she started palliative first-line chemotherapy with FOLFOX (oxaliplatin, leucovorin, and 5-fluorouracil) plus bevacizumab, liver metastases and retroperitoneal metastatic lymph nodes progressed after 4 cycles of FOLFOX plus bevacizumab. She was treated with encorafenib and cetuximab as second-line therapy and after 2 cycles she had rapid disease progression with newly developed peritoneal seeding, ascites, and multiple lung metastases (Figure 1). At this time, paracentesis was performed and after ascite cytology confirmed metastatic adenocarcinoma, we generated PDCs from the patient.

Figure 1 Abdomen-pelvis and chest CT scan evaluation under encorafenib with cetuximab treatment. Compared to a baseline CT scan, after 4 cycles of encorafenib plus cetuximab: yellow circles indicate the increased size of metastatic lymphadenopathy in the Lt. supraclavicular lymph node. The yellow arrow indicates obstructive ileus in the lower abdomen due to peritoneal seeding. Red arrows indicate increased size of osteolytic lesion of L1 destructing bony cortex. The red circle indicates newly developed small lung metastasis. CT, computed tomography.

Addition of ribociclib, a CDK4/6 inhibitor, to encorafenib-cetuximab combination therapy attenuates the proliferation of BRAF V600E mutant CRC PDCs

To investigate why the patient did not respond to the standard therapy with encorafenib plus cetuximab, we used a PDC model. We collected nucleated cells from the ascites of the patient and produced PDCs. First, we examined the cytotoxic effect of the encorafenib-cetuximab combination on PDCs and found that even at a high concentration of cetuximab (up to 500 µg/mL), the cells were not responsive (Figure S1). The reported IC₅₀ value of cetuximab for known colon and rectum adenocarcinoma cell lines ranges from 183.7 ng/mL to 377.6 µg/mL (20).

According to a result obtained from a TruSight Oncology 500 assay, the patient has an R173C substitution loss-of-phosphatase function mutation on PTEN (21). Concomitant occurrence of BRAF mutation and PTEN loss is considered a predictive marker for poor prognosis of cetuximab treatment (22-24). Thus, we checked whether a CDK4/6 inhibitor, ribociclib, could be used to overcome cetuximab-mediated resistance. In a proliferation assay, encorafenib has no synergistic effect when combined with cetuximab (Figure 2A). However, the addition of ribociclib to the dual combination of cetuximab plus encorafenib (CE) attenuated cell proliferation more efficiently than CE alone. The anti-proliferative impact of the triple combination of cetuximab, encorafenib, and ribociclib (CER) was confirmed by flow cytometry analysis using Ki-67 expression, a widely used marker for cell proliferation (Figure 2B). As with the proliferation assay (Figure 2A), CER triple therapy resulted in lower expression of Ki-67 than CE or the dual combination of cetuximab plus ribociclib, even though there was no significant difference in Ki-67 expression between encorafenib plus ribociclib and CER-treated cells.

Figure 2 Effects of the triple combination of encorafenib, cetuximab plus ribociclib on the proliferation of BRAF V600E-mutant CRC PDCs. (A) BRAF V600E-mutant CRC PDCs were treated with indicated drugs for 72 h. Then, the number of viable cells was determined using a CellTiter 96 Aqueous One Solution Cell Proliferation Assay (MTS) and the relative viable cell number was calculated as a percentage of the untreated control. (B) Proliferation marker Ki-67-expressing PDCs were assessed by flow cytometry analysis after 24 h incubation with indicated drugs. At least 3 independent experiments were performed. Statistical differences were determined using one-way ANOVA with Tukey-adjusted post hoc tests for multiple comparisons (A) or paired t-test (B). *, P<0.05; **, P<0.01. CRC, colorectal cancer; PDCs, patient-derived cells; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; ANOVA, analysis of variance.

Triple treatment of encorafenib, cetuximab, and ribociclib has combined cytotoxic effects on BRAF V600E mutant CRC PDCs

Next, we tested the combined effect of the triple treatment on cytotoxic ability (Figure 3A). The relative viability was determined by using an MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. The MTS assay showed that combined treatment with CER exerted more cytotoxicity than CE treatment and was consistent with the result from the proliferation assay (Figure 2A).

Figure 3 Cytotoxic effects of the triple combination of encorafenib, cetuximab plus ribociclib on BRAF V600E-mutant CRC PDCs. (A) BRAF V600E-mutant CRC PDCs were treated with indicated drugs for 24 h. Then, the number of viable cells was determined using CellTiter 96^® Aqueous One Solution Cell Proliferation Assay (MTS) and viability was calculated as a percent of the untreated control. At least 3 independent experiments were performed. Statistical differences were determined using one-way ANOVA with Tukey-adjusted post hoc tests for multiple comparisons (A) or paired t-test (B). (B) Cell viability data further proceeded with the CompuSyn 3.0.1 software, where the CI values were calculated and FA-CI plots were drawn for combination effects were defined according to the CI values as follows: synergistic (CI <0.9), additive (CI =0.9–1.1), or antagonistic (CI >1.1). At least 3 independent experiments were performed. ***, P<0.001. CI, combination index; FA, fractions of cells affected; CRC, colorectal cancer; PDCs, patient-derived cells; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; ANOVA, analysis of variance.

With the figures obtained from the cytotoxicity assay (Figure 3A), we inspected the combinational effects of three drugs by determining the CI that was proposed by Chou (25). As a result, synergism of the triple combination was confirmed throughout several fractional inhibition (Fa) points (Fa =0.15, 0.25, 0.5, 0.75, and 0.97, CI: 0.07969) (Figure 3B).

Collectively, we found that the CDK4/6 inhibitor, ribociclib, granted an additive impact to the encorafenib-cetuximab dual combination by reversing the dysregulated cell cycle in a patient with CRC who had a loss-of-function mutation in PTEN in addition to a BRAF V600E mutation.

Discussion

Several clinical trials in colon cancer have demonstrated that EGFR inhibition lacks clinical benefit when either KRAS or BRAF is mutated downstream of EGFR (26,27). Therefore, the NCCN only recommends anti-EGFR monoclonal antibodies for CRC patients with wild-type KRAS, NRAS, or BRAF (28). However, BRAF activation in metastatic CRC is more intricate and heterogeneous than that in melanoma. Therefore, in BRAF-mutant CRC, BRAF inhibitor monotherapy did not show a clinically meaningful anti-tumor activity because of the negative feedback of EGFR pathway activation. Therefore, dual inhibition by anti-EGFR and anti-BRAF is rational and has demonstrated clinical benefit for patients with BRAF-mutant CRC.

Our patient was treated with second-line encorafenib plus cetuximab but did not respond and the disease progressed rapidly. A concomitant loss-of-function mutation in PTEN occurred in the tumor of the patient and we found that encorafenib plus cetuximab had no synergistic effect compared with encorafenib alone in the PDCs. Previously, the role of PIK3CA/PTEN signaling has been examined and PIK3CA-activating mutations and/or PTEN loss seem to be involved in the resistance to cetuximab treatment (24,29). PTEN is a negative modifier of the PI3K/AKT pathway and is detected in 20–40% of metastatic colorectal cancer (mCRC) cases, the loss of PTEN results in tumor growth by activating PI3K/AKT (30). Several studies report that PTEN deregulation contributes to the response by regulating cyclin D expression and, therefore, accelerating the cyclin D/CDK-4/6-mediated cell cycle pathway (31,32).

Preclinical studies using a PTEN-deficient mouse model have shown that the CDK4/6 inhibitor palbociclib reduced tumor cell proliferation and disrupted the tumorigenic process in endometrial cancer (33). In glioblastoma (GBM) cell lines, wild-type PTEN were more sensitive to the CDK4/6 inhibitor than PTEN-deficient cell lines, indicating that PTEN enhances the sensitivity to palbociclib (34).

Of note, in our PDC model, the addition of ribociclib to encorafenib plus cetuximab attenuated cell proliferation more efficiently and expressed lower Ki-67 than encorafenib plus cetuximab treatment. A cytotoxicity assay also showed a significant synergistic effect from the triple combination treatment.

In BRAF-mutant CRCs, MAPK pathway reactivation via an alternative pathway is the most well-known resistance mechanism of BRAF and EGFR inhibition. A study using a matched biopsy before and at the time of progression showed the emerging amplification of wild-type RAS as a recurrent mechanism of resistance (13). A serial circulating tumor DNA study also demonstrated MAPK activation (NRAS/KRAS mutations or amplifications) during dual BRAF/EGFR inhibition treatment (11,12). The clonal expansion of MET gene amplification during panitumumab-vemurafenib treatment and MET mutation during encorafenib-cetuximab has been introduced (10,35). The MAPK pathway positively regulates the cell cycle by transcriptionally controlling cyclin D1 expression through ERK (36). Recently, Nassar et al. showed that the combination of CDK4/6 and MEK inhibitors overcomes acquired resistance to BRAF/MEK inhibitors in a BRAF-V600E-mutant melanoma patient tumor (37), and suggested that loss of CDKN2A represents a biomarker of response to the combination.

The primary resistance to BRAF inhibitors in BRAF-mutated CRC is reported to be as high as 25% (8,38), and relatively few studies have been conducted on the primary resistance of BRAF inhibitors in BRAF-mutated metastatic CRC. The NGS results from our patient showed a low tumor mutation burden (TMB) of 7.8 Muts/Mb and MSS. However, a small case-control study showed a higher TMB (≥20 Muts/Mb) was associated with limited benefit from BRAF/EGFR blockade in patients with MSS and BRAF-mutated metastatic CRC, and PTEN loss and genetic alterations in the PIK3CA/MTOR pathway were not significantly different between a sensitive and resistance cohort (39).

A limitation of this study is that it is difficult to generalize from a single case. We attempted to conduct a cell line study to further confirm the findings of our study; however, we were unable to obtain a cell line with BRAF/PTEN loss of function (LoF) co-alteration, so we could not proceed. The heterogeneity and molecular diversity of BRAF-mutant CRC can easily promote drug resistance and are associated with adverse outcomes. Further research is warranted to determine whether the inhibition of the cell cycle and MAPK pathway may represent a promising strategy for patients with metastatic CRC who are refractory to BRAF/EGFR inhibitor therapy.

Conclusions

In conclusion, the present study showed that a CDK 4/6 inhibitor (ribociclib) in combination with encorafenib plus cetuximab might have anti-tumor activity in a patient with V600E mutant CRC with concomitant PTEN loss-of-function mutation.

Acknowledgments

Funding: The study was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIT) (No. RS-2023-00222838).

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-24-20/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-24-20/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-24-20/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-24-20/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the Institutional Review Board committee of Samsung Medical Center (SMC), Seoul, Republic of Korea, on the use of human samples for experimental studies (No. 2021-09-052). Written informed consent was obtained from study participant prior to enrollment.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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