Antitumorigenic effect of combination treatment with BRAF inhibitor and cisplatin in colorectal cancer in vitro and in vivo

Antitumorigenic effect of combination treatment with BRAF inhibitor and cisplatin in colorectal cancer in vitro and in vivo | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Antitumorigenic effect of combination treatment with BRAF inhibitor and cisplatin in colorectal cancer in vitro and in vivo Kassandra Koumaki, Salomi Skarmalioraki, Vivian Kosmidou, Lida Krikoni, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4109451/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose In colorectal cancer (CRC), BRAF inhibitor (BRAFi) monotherapy appears ineffective, while cisplatin treatment is associated with adverse effects, drug resistance and reduced efficacy. Herein, we seek to explore a combinatorial approach to increase the likelihood of effectively killing colorectal cancer cells. Methods We examined the combined effect of BRAFi (PLX4720, Vemurafenib, Dabrafenib, Encorafenib) and cisplatin treatment in BRAFV600E-mutated (RKO, HT29, Colo-205) and BRAFwt (Caco-2) cell lines, as well as in mouse xenografts of RKO cells. Results Following cisplatin-only treatment, all cell lines showed accumulation within subG1 (apoptotic cells) and G2/M phases, as well as phosphorylation of ERK1/2 and H2AX. Following BRAFi-only treatment, BRAFV600E-mutated cells showed accumulation within G0/G1 phase, reduced distribution in the S and G2/M phases, inhibition of ERK1/2 phosphorylation and increased phosphorylation of H2AX. BRAFi had no effect on BRAFwt Caco-2 cell line. Combined BRAFi and cisplatin treatment synergistically decreased RKO cells viability, reduced phosphorylation of ERK1/2 and increased phosphorylation of H2AX. Importantly, in mouse xenografts of RKO cells, combined PLX4720 and cisplatin treatment showed superior therapeutic potential than each monotherapy ( P < 0.001). Conclusion In in vitro and in vivo preclinical models, BRAFi and cisplatin combined treatment has shown an improved antitumor effect, rendering it a potential anticancer treatment strategy for BRAF-mutant colon cancer patients. colorectal cancer BRAF inhibitor cisplatin combination therapy BRAFV600E-mutated cells mouse xenografts Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 Introduction Colorectal cancer (CRC) is the third most common cancer worldwide [ 1 , 2 ]. Approximately 65% of CRC cases are sporadic and believed to be associated with environmental factors [ 3 ]. The remaining cases exhibit inheritance, of which approximately 3% exhibit highly penetrant inheritance, whereas the remaining less penetrant [ 3 , 4 ]. Despite availability of early detection methods, it has been reported that up to 25% of patients with CRC have already metastases at initial diagnosis and nearly twice as many will develop metastases in time, resulting in low survival rates [ 5 ]. CRC incidence and mortality rates differ among males compared to females. In particular, CRC is the third most frequent cancer and the fourth most common cause of cancer death in men, while among females CRC is the second most frequent cancer and the third most common cause of cancer death [ 6 ]. CRC comprises a complex and heterogeneous disease, resulting from genomic, proteomic, epigenetic and other changes. Surgical resection of the tumor, chemotherapy and radiotherapy are the key treatment options for CRC patients. However, as previously stated, up to a quarter of CRC patients are diagnosed at a late stage with metastases, limiting effective surgical control, leading to poor prognosis and increased mortality rates [ 7 ]. Although cisplatin remains the gold standard for solid tumors, in CRC, cisplatin treatment is correlated with adverse effects, drug resistance and reduced efficacy. Therefore, improving treatment options, especially for metastatic CRC patients, is crucial, in order to prolong survival. The RAS/RAF/MEK/extracellular signal-regulated kinase (ERK) mechanism, also known as the mitogen-activated protein kinase (MAPK) pathway, regulates cell proliferation, differentiation and survival. Activating mutations in the genes involved in this pathway, including BRAF, transfer signals from the cell membrane to the nucleus through downstream components, resulting in deregulation of key cellular activities and tumor development by uncontrolled cell proliferation and survival [ 8 ]. BRAF mutations have been reported in 5–12% of patients with metastatic CRC and affect treatment and prognosis of these patients [ 9 , 10 ]. The most frequent mutation in BRAF, accounting for more than 95% of BRAF mutations, is a single substitution at nucleotide 1799, replacing valine with glutamic acid (V600E mutation). BRAF mutations have been correlated with a poor prognosis, with a median survival of less than 12 months. Treatment strategies for patients with BRAF-mutant metastatic CRC are inadequate, therefore efficient anticancer treatment is urgently needed. During the past few years, targeting mutated ΒRAF proteins with selective inhibitors (BRAFi) has produced remarkable antitumor activity. Indeed, Vemurafenib, Dabrafenib and Encorafenib have been approved by the United States Food and Drug Administration (FDA) for the treatment of BRAF-mutant metastatic melanoma. Vemurafenib has been shown to inhibit the kinase activity of BRAFV600E mutation in patients with metastatic melanoma and was the first potent and selective tyrosine kinase inhibitor to inactivate the MAPK pathway, demonstrating antitumor activity [ 11 , 12 ]. Dabrafenib was the second kinase inhibitor approved by the FDA for the treatment of patients with metastatic melanoma harboring the BRAFV600E mutation [ 11 ]. It is also used, in combination with Trametinib (MEK inhibitor), for the treatment of patients with BRAFV600E-mutant metastatic non-small cell lung cancer (NSCLC) or anaplastic thyroid cancer (ATC) [ 13 , 14 ]. Encorafenib is a potent BRAF inhibitor that has been shown to decrease ERK phosphorylation and inhibit proliferation in BRAFV600E-mutant melanoma, which accounts for 50% of all melanomas. It also induces senescence, rather than apoptosis, by down-regulating Cyclin D1 and arresting the cell cycle in the G1 phase [ 15 , 16 ]. BRAF inhibitors have been reported to show improved rates of rapid response in patients with BRAF-mutant metastatic melanoma, ranging from 48% in a phase III study assessing Vemurafenib efficacy to 59% in a phase II study of the clinical activity of Dabrafenib [ 12 , 17 ]. Moreover, the phase II COLUMBUS trial showed superior efficacy data of Encorafenib over Vemurafenib monotherapy [ 18 , 19 ]. However, BRAFi monotherapy does not usually have an enduring anticancer effect, since patients have been reported to progress to more advanced stages 6 to 7 months after the beginning of treatment. This acquired resistance to BRAF inhibitor therapy has been attributed to MAPK pathway reactivation and the reported mechanisms include activating mutations in NRAS, KRAS, MEK1/2 and AKT1, BRAF amplification and splicing and CDKN2A loss, along with PI3K-PTEN-AKT pathway mutations overlapping with the MAPK pathway [ 20 – 22 ]. Thus, further research is urgently needed to find new therapeutic options, in order to increase treatment efficacy and prevent the development of BRAFi-associated resistance. Rational combinatorial treatment protocols of BRAF inhibitors have offered high potential against resistant tumors. The currently investigated strategies include among other, co-treatment protocols with inhibitors of EGFR, MEK, or PI3K/AKT pathway, or immunotherapies. The best results so far have been observed by combining Vemurafenib with the selective MEK inhibitor Cobimetinib, a combination approved by the FDA in 2015 for the treatment of patients with BRAF-mutant metastatic or unresectable melanoma [ 23 ]. Combination of Dabrafenib with Trametinib was also approved by the FDA for BRAFV600E-mutant anaplastic thyroid cancer in 2018 [ 24 ] and for BRAFV600E-mutant non-small cell lung cancer (NSCLC), where a major favorable effect has been demonstrated [ 25 , 26 ]. The Encorafenib-Cetuximab (EGFR targeting antibody) combination was approved in April 2020 for the previously treated BRAFV600E metastatic CRC patients, after it was shown to significantly improve their overall and progression-free survival during the trial. The doublet therapy was selected being to be equally effective to the triplet protocol, but with slightly less (MEKi-related) adverse effects [ 27 , 28 ]. The present study examined whether combined treatment of BRAFi and cisplatin is more effective than each drug alone in human colon cancer cell lines both in vitro and in vivo . For this purpose, the effects of BRAFi and/or cisplatin treatment on the cell viability, the cell cycle, as well as the phosphorylation of critical molecular components of the MAPK pathway and the DNA damage response (DDR) network were investigated in BRAFV600E mutated and BRAFwt cell lines. The antitumor activity of the combined treatment of BRAFi and cisplatin was also studied in vivo , using a mouse xenografts model. 2 Materials and Methods 2.1 Cell lines Human colorectal adenocarcinoma cell lines (Colo-205, HT29, RKO and Caco-2) were obtained from the American Type Culture Collection (ATCC). All cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% Fetal Bovine Serum (FBS), 1% nonessential amino acids and 1% penicillin/streptomycin (all from Thermo Fisher Scientific) at 37°C, 5% CO 2 . 2.2 Inhibitors The BRAF inhibitors PLX4720 (#S1152) and Vemurafenib (#S1267) were purchased from Selleckchem, while Encorafenib (#HY-15605) and Dabrafenib (#HY-14660) from MedChemExpress. Human recombinant SuperKiller cc-TRAIL (Alexis, ALX-522-020) was used as a control of apoptotic cell death. 2.3 Western blotting Whole cell protein lysates were extracted with lysis buffer containing protease inhibitors separated in sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Amersham, UK) as described previously [ 29 ]. The antibodies used were directed against pERK1/2 (Santa Cruz Biotechnology, sc-7383), γH2ΑΧ (Cell Signaling Technology, #9718T), and GAPDH (Santa Cruz Biotechnology, sc-47724). The secondary antibodies used were mouse anti-rabbit IgG-HRP (Santa Cruz Biotechnology, sc-2357) and goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, sc-2005). The antibody signal was enhanced with chemiluminescence and captured on X-ray film Super RX-N (Fujifilm Tokyo, Japan). Values were measured using Studio Lite software (LI-COR Biotechnology, Lincoln, NE, USA) and levels were normalized against the housekeeping GAPDH protein. The blots presented are representative of three independently repeated experiments. 2.4 Flow cytometry Cells were cultured and treated in 6-well plates. Upon the selected time-point, they were detached and fixed/permeabilized with ice-cold ethanol overnight. DNA was marked with propidium iodide (PI; BD Biosciences; #556463) for 1h at room temperature at a concentration of 50µg/ml. RNA binding was avoided with the use of RNase A, 10µg/ml (Thermo Fisher Scientific, #EN0531). Cell cycle was analyzed using a BD FACSAria II flow cytometer and the BD FACSDiva v8.0 software (BD Biosciences). 2.5 Cell viability assay Cell viability was estimated with the Sulforhodamine B (SRB; Sigma-Aldrich, S1402) assay [ 30 ]. Cells were seeded for 24h into 96-well microtiter plates. After completion of the treatment, fixation was performed with 10% trichloroacetic acid (TCA; Sigma-Aldrich, #T6399) and staining with 0.4% SRB in 1% acetic acid. Absorbance was measured using a TECAN microplate reader (TECAN, Mannedorf, Switzerland) and cell viability was estimated. 2.6 In vivo studies A total of 1x10 6 RKO cells diluted in PBS were injected subcutaneously into the left and right flanks of 6-week-old female SCID mice. When the tumors became palpable, reaching an appropriate volume of 21-33mm 3 (Day 15), the mice were randomly assigned to 5 groups (4 mice per group). The first group was used as a negative control (untreated) group. The second group was injected with DMSO (5% in distilled H 2 O). The third group was treated intratumorally with a combination of 5mg/kg cisplatin plus 10mg/kg PLX4720 in 5% DMSO (100µg cisplatin plus 200µg PLX4720/mouse every five days). The fourth group was treated intratumorally with 5mg/kg cisplatin alone (100µg/mouse every five days). The fifth group was treated intratumorally with 10mg/kg PLX4720 (200µg/mouse every five days). The mice received 5 treatment doses in a total period of 25 days. During this period, tumor sizes were measured every 5 days using caliper and tumor volumes were calculated using the formula V = (height x width x length) / 2. The Standard Deviation (SD) was used for error bar generation between all tumors of the same group of animals. Statistical analysis of the data was performed using two-way ANOVA Bonferroni’s multiple comparisons test in Graphpad Prism 9.0. At the end of the observation period, the mice were sacrificed due to tumor burden. Tumors were subsequently excised and photographed. 2.7 Apoptosis assay Cells were treated with 0-100µg/ml cisplatin for 3h, followed by 48h post-incubation time in drug-free medium. The Cell Death Detection ELISAPLUS kit (Roche Diagnostics, Switzerland, #11544675001) was used to determine apoptosis as previously described [ 31 ]. 2.8 Statistical Analysis Continuous variables were compared among groups with Student's t-test, or Mann-Whitney U test when normal distribution did not apply, whereas paired comparisons were performed by paired t-test or Wilcoxon's test. Correlations were examined with Spearman's rank test. All statistical analyses were performed with SPSS v.24.0. Results were considered significant when P < 0.05. 3 Results 3.1 Cisplatin-only treatment of colon cancer cell lines First, we analyzed the cytotoxic effects of cisplatin on a number of human colorectal adenocarcinoma cells, such as the BRAFV600E bearing RKO, HT29 and Colo-205 cell lines, as well as the BRAFwt colon adenocarcinoma Caco-2 cells (Fig. 1 A-G). For all cell lines tested, the decrease in cell viability was time- and concentration-dependent. All BRAFV600E bearing cell lines were resistant to cisplatin 24h after treatment with concentrations up to 10µg/ml. In RKO and HT29 cells, cisplatin doses higher than 25µg/ml led to increased cytotoxicity 24h after treatment, while Colo-205 cells seemed more resistant, since reduction of cell viability was observed following treatment with drug doses higher than 50µg/ml (Fig. 1 A-C). Interestingly, the BRAFwt Caco-2 cell line was more resistant than HT29 cells at 72h, 96h and 120h time-points (Fig. 1 D-F) and more resistant than RKO cells 72h following treatment with various doses of cisplatin (Fig. 1 G). Next, we studied the progression of the cell cycle following treatment with cisplatin (Fig. 1 H). In all cell lines tested, 48h following treatment with 5µg/ml cisplatin, increase in the percentages of cells at the G2/M phase, combined with increase in the percentages of cells at the subG1 phase (apoptotic cells) were observed. Driven by the reduction of the cell viability and the increase of the SubG1 phase upon treatment with cisplatin, we hypothesized that apoptotic pathway is possibly triggered. Therefore, the apoptosis rates of all colorectal cell lines were evaluated 48h after treatment with cisplatin treatment using an ELISA assay. The lowest concentrations of cisplatin needed for induction of apoptosis were 17.5 ± 5.2µg/ml for Colo-205 cells, 15.8 ± 4.9µg/ml for Caco-2 cells, 12.5 ± 6.1µg/ml for RKO cells and 9.2 ± 4.9µg/ml for HT29 cells indicating that the HT29 cell line showed the highest cisplatin-induced apoptosis rates (all P < 0.05; Fig. 1 I). The effect of cisplatin on the phosphorylation of critical molecular components of the MAPK and DDR pathways, namely ERK1/2 and H2AX, respectively, was also evaluated 0, 24h and 48h after treatment with 5, 10, 25, 50 or 100µg/ml cisplatin. In all cell lines analyzed, we found that at the 48h time-point, cisplatin induced phosphorylation of ERK1/2 (Fig. S1 A-C) and H2AX (Fig. S1 D-F). 3.2 BRAFi-only treatment of colon cancer cell lines The effects of various BRAF inhibitors, on the same colon cancer cell lines were also analyzed. At first, the viability of the BRAF mutant cell lines was evaluated 48h and 72h following treatment with a BRAFi. After treatment of BRAFV600E-mutated cell lines with the Vemurafenib analog PLX4720, a dose-dependent decrease in the viability was observed in both RKO and HT29 cells; Colo-205 was the most sensitive and RKO the most resistant cell line (Fig. 2 A-C). As expected, treatment with PLX4720 had no effect on the viability of BRAFwt Caco-2 cells (Fig. 2 D). Moreover, 48h after treatment all BRAF mutant cell lines exhibited similar sensitivity to Dabrafenib or Encorafenib (Fig. 2 E). The apoptosis rates of the BRAFV600E-mutated cell lines were also evaluated 48h after treatment with PLX4720 using an ELISA assay. The lowest concentrations of PLX4720 needed for induction of apoptosis were 10.8 ± 5.8µM for RKO cells, 4.5 ± 3.3µM for HT29 cells and 0.7 ± 0.3µM for Colo-205 cells, indicating that Colo-205 cells showed the highest apoptosis rates (all P < 0.001; Fig. 2 F). The progression of the cell cycle was also investigated following treatment with PLX4720 and Encorafenib. Both these BRAF inhibitors showed similar effects in all cell lines tested. That is, 48h after treatment, increases were found in the percentages of cells at G0/G1 phase, combined with reduction in their percentage at S and G2/M phases; no increase in the percentages of cells at the subG1 phase was observed (Figs. 2 G, H and Figs. S2-S4). The effect of PLX4720, Vemurafenib, Dabrafenib and Encorafenib on ERK1/2 and H2AX phosphorylation was also evaluated by Western blot analysis (Fig. 2 I, J). It was found that 48h after treatment, each one of these BRAFi inhibited phosphorylation of ERK1/2 and increased phosphorylation of H2AX. 3.3 The effect of cisplatin and BRAFi combined treatment on CRC cell lines In order to examine the combined effect of BRAFi and cisplatin on CRC cell lines, we tested five different treatment schedules. In the 1st treatment schedule, cells were exposed for 3h to 1µM PLX4720, followed by 3h treatment with 5µg/ml cisplatin and 72h post-incubation in drug-free medium. Firstly, the effect of this combined treatment on the phosphorylation of ERK1/2 and H2AX was evaluated (Fig. 3 A-C). In all cell lines analyzed, compared with non-treated cells, the combined treatment inhibited the phosphorylation of the ERK1/2 kinase and increased the phosphorylation of H2AX at the 24h time-point. Moreover, the combined treatment increased the percentage of all cells analyzed at the subG1 (apoptotic cells) and G0/G1 phases and decreased the percentage at the G2/M phase (Fig. 3 D-F). Using the SRB viability assay, RKO and HT29 cells exhibited synergistic effects 48h after combined treatment, while Colo-205 at the 72h time-point (Fig. 3 G-I). In the 2nd treatment schedule, cells were exposed to 5µg/ml cisplatin for 3h, followed by 3h treatment with 1µM PLX4720 and 72h post-incubation in drug-free medium, while in the 3rd treatment schedule, the simultaneous exposure to both 5µg/ml cisplatin and 1µM PLX4720 for 3h, followed by 72h post-incubation in drug-free medium was evaluated (Fig. 3 G-I). Both 2nd and 3rd treatment schedules showed similar viability results to those obtained following the 1st treatment schedule mentioned above. In the 4th treatment schedule, cells were exposed for 24h to 0.5µM or 1µM PLX4720, followed by 3h treatment with 5µg/ml cisplatin in the presence of the inhibitor and 72h post-incubation in drug-free medium. When the 4th treatment schedule was validated experimentally, no synergistic effect was found (Fig. S5). Finally, in the 5th treatment schedule the simultaneous exposure to 5µg/ml cisplatin and 1µM PLX4720 or 1µM Dabrafenib or 1µM Encorafenib for 48h was evaluated. In RKO cells, all BRAF-containing combinations exhibited synergistic effects, as shown by the SRB viability assay (Fig. 4 A). In Colo-205 cells, PLX4720- and Dabrafenib-containing treatments showed synergistic effect. No synergy on cell viability was found following treatment of Colo-205 cells with the Encorafenib-containing schedule and after exposure of HT29 cells with any of the combinations used. The effect of the 5th treatment schedule on the phosphorylation of H2AX and ERK1/2 was also evaluated (Fig. 4 B). In all BRAFi-containing treatments, phosphorylation of H2AX was observed, combined with reduction in the phosphorylation of ERK1/2. 3.4 Combinatorial treatment with cisplatin and BRAFi results in strong anti-tumor effects in vivo To shed light on the potential anti-tumorigenic effects of combined BRAFi and cisplatin treatment in vivo , xenografts of RKO cells were subcutaneously implanted into both flanks of severe combined immune-deficient (SCID) mice. When the tumors reached appropriate sizes, the mice were divided into five groups: control (untreated), DMSO-treated, cisplatin plus PLX4720-treated, cisplatin alone-treated and PLX4720 alone-treated. Tumor growth was monitored for 20 days after their formation, which was considered the reference point (Day 15) of the experiment. The combined treatment with 5mg/kg cisplatin and 10mg/kg PLX4720 caused a remarkable attenuation of colon cancer progression in mice bearing RKO xenografts, which was significantly higher than each separate monotherapy ( P < 0.001; Fig. 5 A, B). After comparison of the excised tumor volume of the five experimental groups, cisplatin or PLX4720 monotherapy were found to reduce tumor sizes efficiently by 2.6 and 1.7 folds, respectively, as compared to untreated mice. Importantly, combined treatment with cisplatin plus PLX4720 resulted in about 3.3-fold tumor size reduction, as compared to the control untreated mice by the end of the experiment (Fig. 5 C, D). Those results clearly indicate that the combination of a BRAF inhibitor and cisplatin holds superior therapeutic potential than each monotherapy in vivo . 4. Discussion Certain combination treatment regimens produce better response than the individual drugs alone [ 32 ]. Herein, we examined the combined effect of BRAFi with cisplatin in colorectal cancer cell lines and a mouse xenograft model. Firstly, the effect of cisplatin on CRC cell lines was analyzed. We found that following treatment of BRAF mutant cells with cisplatin, reduction of cell viability, G2/M phase arrest and phosphorylation of H2AX were observed. Cisplatin is a genotoxic drug inducing the formation of single-nucleotide damage of guanine (monoadducts), intrastrand and interstrand cross-links [ 33 ]. Monoadducts and intrastrand cross-links are repaired by the nucleotide excision repair (NER) mechanism [ 34 ], while the removal of interstrand cross-links requires the activation of several DNA repair pathways, such as homologous recombination, NER and translesion synthesis [ 35 – 37 ]. Of note, DNA double-strand breaks (DSBs) are produced as intermediates in the interstrand cross-links repair process [ 38 ]. Following the detection of DSBs, cells induce the phosphorylation of histone H2AX on serine 139 (γH2AX), by the apical signaling kinases ataxia telangiectasia mutated (ATM) and ATM-Rad3-related (ATR) [ 39 ]. Since the induction of γH2AX is an early event in the activation of the DNA damage response network, it is generally utilized as a marker of DSBs. Previous studies have also demonstrated that cisplatin impairs the synthesis of the DNA [ 40 , 41 ], induces S-phase slowdown [ 42 ] and triggers a G2/M arrest through the inhibition of the CDK1/cyclin B activity [ 42 , 43 ]. In addition, we found that following treatment of BRAF mutant cells with cisplatin, phosphorylation of the ERK1/2 kinase was observed. In accordance with our results, previous studies have shown that cisplatin treatment of HeLa cells caused a dose- and time-dependent phosphorylation, and therefore activation, of ERK, and that activation of this kinase is important for the induction of the apoptosis pathway via the cytochrome c release from mitochondria [ 44 ]. In contrast, Wei et al. [ 45 ] have shown that ERK signaling inhibition enhanced the susceptibility of ovarian cancer cells to cisplatin. Another report has shown that various genotoxic insults, such as adriamycin, ultraviolet irradiation, etoposide and ionizing radiation induced activation of ERK1/2 in the MEF and IMR90 primary cells, the NIH3T3 immortalized cells and the transformed MCF-7 cells, thus leading to cell cycle arrest or induction of apoptosis, depending on the drug concentration used [ 46 ]. Next, the effect of BRAF inhibition on CRC cells was analyzed. We found that treatment of BRAF mutant cells with a BRAFi resulted in decreased cell viability and reduction of ERK activation/phosphorylation. As expected, the inhibition of BRAF had no effect on cell viability of BRAFwt cells. It is known that the pathway that leads to the activation of the two isoforms of ERK (ERK1 and ERK2) is initiated after ligand binding to a plasma membrane receptor tyrosine kinase and the activation of the GTP-binding protein Ras. Then, Ras activates the MAP3K kinase Raf, followed by the activation of the mitogen-activated protein kinase kinase (MEK or MAP2K), which in turn phosphorylates threonine and tyrosine residues in the Thr-Glu-Tyr (TEY) sequence of ERK1/2 [ 47 ]. In several cancer types, such as melanoma, hairy cell leukemia, colon carcer and papillary thyroid carcinoma, a mutation in codon 600 of exon 15 (V600E) has been reported. This mutation has been implicated in various mechanisms of cancer progression, such as stimulation of the MEK/ERK pathway, prevention of immune response, avoidance of apoptosis and senescence, angiogenesis, tissue invasion and metastasis [ 48 ]. Interestingly, the inhibition of the BRAF kinase results in decreased pERK activity and the reduction of cell proliferation, indicating that the decreased pERK activity can be used as a pharmacodynamic biomarker of the BRAF inhibition. Moreover, we found that treatment of BRAF mutant cells with a BRAFi resulted in perturbation of cell cycle progression, inducing a G0/G1 arrest. An accumulating body of evidence suggests that the ERK kinases are involved in the cell cycle progression from G1 to S phase, which occurs immediately after growth factor stimulation. Indeed, previous studies have shown that ERK inhibition by a MEK inhibitor that was given even immediately prior to the beginning of the S phase, blocked cell cycle entry into the S phase [ 49 ]. That is, after growth factor stimulation, the activation of ERK causes the phosphorylation, and thus activation of the ETS transcription factor Elk-1 and results in the upregulation of immediate-early genes, such as the proto-oncogene c-fos, the expression of which is involved in the induction of delayed-early genes, such as cyclin D [ 50 – 52 ]. Then, the Cyclin D/Cdk4 complex initiates the phosphorylation of retinoblastoma, a protein known to activate the E2F family of transcription factors, and regulates the expression of several target genes, such as cyclin E. Next, the complex of cyclin E with Cdk2 further phosphorylates the retinoblastoma protein, and activates the E2F transcription factors. These sequential events regulate the synthesis of various proteins that are involved in the entry of the cells in the S phase [ 53 ]. Previous studies have revealed a set of genes whose expression levels were rapidly reduced after ERK inactivation. Interestingly, several of these genes were found to possess antiproliferative properties, i.e. they have the ability to suppress the entry of the cells into the S phase. Together, these data suggest that the BRAF inhibitor-induced ERK inactivation blocks S phase entry [ 49 , 54 ]. Also, in accordance with our previous study [ 29 ], herein we found that following treatment of BRAF mutant cells with a BRAFi, phosphorylation of H2AX was observed. In fact, evidence has accumulated that histone H2AX phosphorylated on Ser-139 (γH2AX), in addition to be a critical component of the DSB repair mechanism, is also implicated in many other biological processes. For example, Fragkos et al. [ 55 ] have shown that, in the absence of DNA damage, phosphorylation of H2AX is an early sign of replication stalling, inhibiting cell cycle progression from the G1 to the S phase. Interestingly, they found that after inhibition of the replication machinery, γH2AX is needed for increasing the levels of p21, thus resulting in checkpoint activation and cell cycle arrest. These results suggest that the phosphorylation of H2AX on Ser-139 that was observed in the present study correlated with the G0/G1 arrest after treatment of BRAF mutant cells with a BRAFi. Although the exact mechanism of the interaction between γH2AX and the p53/p21 pathway are still unknown, it is possible that H2AX phosphorylation affects the interaction of PCNA with chromatin, allowing the stable binding of PCNA to p21 and blocking its ubiquitination [ 56 ]. Importantly, the antitumor activity of the combination treatment with cisplatin and PLX4720 was further validated in vivo in mouse xenografts of RKO cells, with the combinatorial treatment showing superior therapeutic potential than each drug alone. Combinatorial treatments of PLX4720 and other therapeutic compounds have proven efficient in growth suppression of distinct tumor types [ 57 , 58 ]. Especially in the case of colorectal cancer, in which Vemurafenib monotherapy is of no appreciable value for patients [ 59 ], combination of the RAS/MAPK pathway inhibition and chemotherapy could potentially ameliorate the observed resistance mechanisms that contribute to reactivation of cancer cell proliferation. Finally, drug combinations can achieve higher therapeutic responses in lower individual doses, thus avoiding the complications of off-target toxicities induced by high drug concentrations. Herein, the in vivo confirmation of the remarkable in vitro results paves the way for the exploitation of similar therapeutic combinations in multiple preclinical and potentially, clinical settings. However, more in vivo studies regarding pharmacokinetics and other pharmacological parameters are required in order to accurately characterize the therapeutic value of the proposed regime. Taken together, our study demonstrates that the combined treatment with BRAFi and cisplatin is more effective than single-drug treatment in preclinical models in vitro and in vivo (Fig. 6 ), suggesting that these data, once further validated at the preclinical level, can be exploited for the design of new therapies for the treatment of CRC. Declarations Supplementary Materials Fig. S1 The effect of cisplatin on MAPK and DDR pathways; Fig. S2 Cell cycle phase distribution of RKO cells treated with Encorafenib; Fig. S3 Cell cycle phase distribution of HT29 cells treated with Encorafenib; Fig. S4 Cell cycle phase distribution of Colo-205 cells treated with Encorafenib, Fig. S5 The effect of the combined treatment with BRAFi and cisplatin on human CRC cells. Author Contributions: Conceptualization, V.Z., A.P. and V.L.S.; Data curation, V.Z., A.P. and V.L.S.; Formal analysis, K.K., V.K., M.G., V.Z., A.P. and V.L.S.; Funding acquisition, V.Z., A.P. and V.L.S.; Investigation, K.K., S.S., V.K., L.K. and M.G.; Project administration, A.P. and V.L.S.; Resources, V.Z., A.P. and V.L.S.; Supervision, A.P. and V.L.S.; Visualization, A.P. and V.L.S.; Writing – original draft, A.P. and V.L.S.; Writing – review & editing, all authors. Funding: This work was supported by the project “STHENOS-b: Targeted therapeutic approaches against degenerative diseases with special focus on cancer and ageing-optimisation of the targeted bioactive molecules” (MIS 5002398) which is implemented under the Action “Action for the Strategic Development on the Research and Technological Sector”, funded by the Operational Programme “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014–2020) and co-financed by Greece and the European Union (European Regional Development Fund). Institutional Review Board Statement: The study was carried out in accordance with the EU Directive 2010/63/EU for animal experiments and was approved by the Ethics Committee of the National Hellenic Research Foundation (approval No. 431956). Data Availability Statement: Data are available upon reasonable request. Conflicts of Interest: The authors declare no conflict of interest. References L.A. Torre, R.L. Siegel, E.M. Ward, A. Jemal, Global cancer incidence and mortality rates and trends-an update, Cancer Epidemiol. Biomarkers Prev. 25 , 16–27 (2016). https://doi.org/10.1158/1055-9965.EPI-15-0578 J. Ferlay, I. Soerjomataram, R. Dikshit, S. Eser, C. Mathers, M. Rebelo, D.M. Parkin, D. Forman, F. Bray, Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. Int. J. Cancer. 136 , E359–E386 (2015). https://doi.org/10.1093/annonc/mdv621 R. Burt, Inheritance of colorectal cancer, Drug Discov. Today Dis. Mech. 4 , 293–300 (2007). https://doi.org/10.1016/j.ddmec.2008.05.004 S.E. Hendon, J.A. DiPalma, U.S. practices for colon cancer screening. Keio J. Med. 54 , 179–183 (2005). https://doi.org/10.2302/kjm.54.179 M.A. Gómez-España, J. Gallego, E. González-Flores, J. Maurel, D. Páez, J. Sastre, J. Aparicio, M. Benavides, J. Feliu, R. Vera, SEOM clinical guidelines for diagnosis and treatment of metastatic colorectal cancer (2018). Clin. Transl Oncol. 21 , 46–54 (2019). https://doi.org/10.1007/s12094-018-02002-w F. Bray, J. Ferlay, I. Soerjomataram, R.L. Siegel, L.A. Torre, A. Jemal, Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 68 , 394–424 (2018). https://doi.org/10.3322/caac.21492 Y.H. Xie, Y.X. Chen, J.Y. Fang, Comprehensive review of targeted therapy for colorectal cancer. Sig Transduct. Target. Ther. 5 , 22 (2020). https://doi.org/10.1038/s41392-020-0116-z P.A. Ascierto, J.M. Kirkwood, J.J. Grob, E. Simeone, A.M. Grimaldi, M. Maio, G. Palmieri, A. Testori, F.M. Marincola, N. Mozzillo, The role of BRAF V600 mutation in melanoma. J. Transl Med. 10 , 85 (2012). https://doi.org/10.1186/1479-5876-10-85 W. De Roock, B. Claes, D. Bernasconi, J. De Schutter, B. Biesmans, G. Fountzilas, K.T. Kalogeras, V. Kotoula, D. Papamichael, P. Laurent-Puig, F. Penault-Llorca, P. Rougier, B. Vincenzi, D. Santini, G. Tonini, F. Cappuzzo, M. Frattini, F. Molinari, P. Saletti, S. De Dosso, M. Martini, A. Bardelli, S. Siena, A. Sartore-Bianchi, J. Tabernero, T. Macarulla, F. Di Fiore, A.O. Gangloff, F. Ciardiello, P. Pfeiffer, C. Qvortrup, T.P. Hansen, E. Van Cutsem, H. Piessevaux, D. Lambrechts, M. Delorenzi, S. Tejpar, Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis. Lancet Oncol. 11 , 753–762 (2010). https://doi.org/10.1016/S1470-2045(10)70130-3 H. Sorbye, A. Dragomir, M. Sundström, P. Pfeiffer, U. Thunberg, M. Bergfors, K. Aasebø, G.E. Eide, F. Ponten, C. Qvortrup, B. Glimelius, High BRAF mutation frequency and marked survival differences in subgroups according to KRAS/BRAF mutation status and tumor tissue availability in a prospective population-based metastatic colorectal cancer cohort. PLoS One. 10 , e0131046 (2015). https://doi.org/10.1371/journal.pone.0131046 L.A. Dossett, R.R. Kudchadkar, J.S. Zager, BRAF and MEK inhibition in melanoma, Expert Opin. Drug Saf. 14 , 559–570 (2015). https://doi.org/10.1517/14740338.2015.1011618 P.B. Chapman, A. Hauschild, C. Robert, J.B. Haanen, P. Ascierto, J. Larkin, R. Dummer, C. Garbe, A. Testori, M. Maio, D. Hogg, P. Lorigan, C. Lebbe, T. Jouary, D. Schadendorf, A. Ribas, S.J. O'Day, J.A. Sosman, J.M. Kirkwood, A.M. Eggermont, B. Dreno, K. Nolop, J. Li, B. Nelson, J. Hou, R.J. Lee, K.T. Flaherty, G.A. McArthur, BRIM-3 Study Group, Improved survival with vemurafenib in melanoma with BRAF V600E mutation, N. Engl. J. Med. 364 , 2507–2516 (2011). https://doi.org/10.1056/NEJMoa1103782 D. Planchard, E.F. Smit, H.J.M. Groen, J. Mazieres, B. Besse, Å. Helland, V. Giannone, A.M. Jr. D'Amelio, P. Zhang, B. Mookerjee, B.E. Johnson, Dabrafenib plus trametinib in patients with previously untreated BRAF V600E-mutant metastatic non-small-cell lung cancer: an open-label, phase 2 trial. Lancet Oncol. 18 , 1307–1316 (2017). https://doi.org/10.1016/S1470-2045(17)30679-4 V. Subbiah, R.J. Kreitman, Z.A. Wainberg, J.Y. Cho, J.H.M. Schellens, J.C. Soria, P.Y. Wen, C. Zielinski, M.E. Cabanillas, G. Urbanowitz, B. Mookerjee, D. Wang, F. Rangwala, B. Keam, Dabrafenib and Trametinib Treatment in Patients With Locally Advanced or Metastatic BRAF V600–Mutant Anaplastic Thyroid Cancer. J. Clin. Oncol. 36 , 7–13 (2018). https://doi.org/10.1200/JCO.2017.73.6785 Z. Li, K. Jiang, X. Zhu, G. Lin, F. Song, Y. Zhao, Y. Piao, J. Liu, W. Cheng, X. Bi, P. Gong, Z. Song, S. Meng, Encorafenib (LGX818), a potent BRAF inhibitor, induces senescence accompanied by autophagy in BRAF V600E melanoma cells. Cancer Lett. 370 , 332–344 (2016). https://doi.org/10.1016/j.canlet.2015.11.015 E. Hodis, I.R. Watson, G.V. Kryukov, S.T. Arold, M. Imielinski, J.P. Theurillat, E. Nickerson, D. Auclair, L. Li, C. Place, D.A.H. Dicara Ramos, M.S. Lawrence, K. Cibulskis, A. Sivachenko, D. Voet, G. Saksena, N. Stransky, R.C. Onofrio, W. Winckler, K. Ardlie, N. Wagle, J. Wargo, K. Chong, D.L. Morton, K. Stemke-Hale, G. Chen, M. Noble, M. Meyerson, J.E. Ladbury, M.A. Davies, J.E. Gershenwald, S.N. Wagner, D.S. Hoon, D. Schadendorf, E.S. Lander, S.B. Gabriel, G. Getz, L.A. Garraway, L. Chin, A landscape of driver mutations in melanoma. Cell. 150 , 251–263 (2012). https://doi.org/10.1016/j.cell.2012.06.024 P.A. Ascierto, D. Minor, A. Ribas, C. Lebbe, A. O'Hagan, N. Arya, M. Guckert, D. Schadendorf, R.F. Kefford, J.J. Grob, O. Hamid, R. Amaravadi, E. Simeone, T. Wilhelm, K.B. Kim, G.V. Long, A.M. Martin, J. Mazumdar, V.L. Goodman, U. Trefzer, Phase II trial (BREAK-2) of the BRAF inhibitor dabrafenib (GSK2118436) in patients with metastatic melanoma. J. Clin. Oncol. 31 , 3205–3211 (2013). https://doi.org/10.1200/JCO.2013.49.8691 P. Koelblinger, O. Thuerigen, R. Dummera, Development of encorafenib for BRAF-mutated advanced melanoma. Curr. Opin. Oncol. 30 , 125–133 (2018). https://doi.org/10.1097/CCO.0000000000000426 R. Dummer, K. Flaherty, C. Robert, A.M. Arance, J.W. de Groot, C. Garbe, H. Gogas, R. Gutzmer, I. Krajsová, G. Liszkay, C. Loquai, M. Mandalà, D. Schadendorf, N. Yamazaki, M.D. Pickard, F. Zohren, M.L. Edwards, P.A. Ascierto, Five-year overall survival (OS) in COLUMBUS: A randomized phase 3 trial of encorafenib plus binimetinib versus vemurafenib or encorafenib in patients (pts) with BRAF V600-mutant melanoma. J. Clin. Oncol. 39 (15suppl), 9507–9507 (2021). https://doi.org/10.1200/JCO.2021.39.15_suppl.9507 H. Shi, W. Hugo, X. Kong, A. Hong, R.C. Koya, G. Moriceau, T. Chodon, R. Guo, D.B. Johnson, K.B. Dahlman, M.C. Kelley, R.F. Kefford, B. Chmielowski, J.A. Glaspy, J.A. Sosman, N. van Baren, G.V. Long, A. Ribas, R.S. Lo, Acquired resistance and clonal evolution in melanoma during BRAF inhibitor therapy. Cancer Discov. 4 , 80–93 (2014). https://doi.org/10.1158/2159-8290.CD-13-0642 H. Rizos, A.M. Menzies, G.M. Pupo, M.S. Carlino, C. Fung, J. Hyman, L.E. Haydu, B. Mijatov, T.M. Becker, S.C. Boyd, J. Howle, R. Saw, J.F. Thompson, R.F. Kefford, R.A. Scolyer, G.V. Long, BRAF inhibitor resistance mechanisms in metastatic melanoma: spectrum and clinical impact, Clin. Cancer Res. 20 , 1965–1977 (2014). https://doi.org/10.1158/1078-0432.CCR-13-3122 N. Wagle, C. Emery, M.F. Berger, M.J. Davis, A. Sawyer, P. Pochanard, S.M. Kehoe, C.M. Johannessen, L.E. Macconaill, W.C. Hahn, M. Meyerson, L.A. Garraway, Dissecting therapeutic resistance to RAF inhibition in melanoma by tumor genomic profiling. J. Clin. Oncol. 29 , 3085–3096 (2011). https://doi.org/10.1200/JCO.2010.33.2312 A. Kim, M.S. Cohen, The discovery of vemurafenib for the treatment of BRAF-mutated metastatic melanoma, Expert Opin. Drug Discov. 11 , 907–916 (2016). https://doi.org/10.1080/17460441.2016.1201057 A. Puszkiel, G. Noé, A. Bellesoeur, N. Kramkimel, M.N. Paludetto, A. Thomas-Schoemann, M. Vidal, F. Goldwasser, E. Chatelut, B. Blanchet, Clinical Pharmacokinetics and Pharmacodynamics of Dabrafenib. Clin. Pharmacokinet. 58 , 451–467 (2019). https://doi.org/10.1007/s40262-018-0703-0 A. Khunger, M. Khunger, V. Velcheti, Dabrafenib in combination with trametinib in the treatment of patients with BRAF V600-positive advanced or metastatic non-small cell lung cancer: clinical evidence and experience, Ther. Adv. Respir Dis. 12 , 1753466618767611 (2018). https://doi.org/10.1177/1753466618767611 D. Planchard, B. Besse, H.J.M. Groen, S.M.S. Hashemi, J. Mazieres, T.M. Kim, E. Quoix, P.J. Souquet, F. Barlesi, C. Baik, L.C. Villaruz, R.J. Kelly, S. Zhang, M. Tan, E. Gasal, L. Santarpia, B.E. Johnson, Phase 2 Study of Dabrafenib Plus Trametinib in Patients With BRAF V600E-Mutant Metastatic NSCLC: Updated 5-Year Survival Rates and Genomic Analysis. J. Thorac. Oncol. 17 , 103–115 (2022). https://doi.org/10.1016/j.jtho.2021.08.011 I.N. Okten, S. Ismail, B.M. Withycombe, Z. Eroglu, Preclinical discovery and clinical development of encorafenib for the treatment of melanoma, Expert Opin. Drug Discov. 15 , 1373–1380 (2020). https://doi.org/10.1080/17460441.2020.1795124 J. Tabernero, A. Grothey, E. Van Cutsem, R. Yaeger, H. Wasan, T. Yoshino, J. Desai, F. Ciardiello, F. Loupakis, Y.S. Hong, N. Steeghs, T.K. Guren, H.T. Arkenau, P. Garcia-Alfonso, E. Elez, A. Gollerkeri, K. Maharry, J. Christy-Bittel, S. Kopetz, Encorafenib Plus Cetuximab as a New Standard of Care for Previously Treated BRAF V600E–Mutant Metastatic Colorectal Cancer: Updated Survival Results and Subgroup Analyses from the BEACON Study. J. Clin. Oncol. 39 , 273–284 (2021). https://doi.org/10.1200/JCO.20.02088 K. Koumaki, G. Kontogianni, V. Kosmidou, F. Pahitsa, E. Kritsi, M. Zervou, A. Chatziioannou, V.L. Souliotis, O. Papadodima, A. Pintzas, BRAF paradox breakers PLX8394, PLX7904 are more effective against BRAFV600Ε CRC cells compared with the BRAF inhibitor PLX4720 and shown by detailed pathway analysis. Biochim. Biophys. Acta Mol. Basis Dis. 1867 , 166061 (2021). https://doi.org/10.1002/cmdc.202300322 V. Vichai, K. Kirtikara, Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat. Protoc. 1 , 1112–1116 (2006). https://doi.org/10.1038/nprot.2006.179 A. Psyrri, M. Gkotzamanidou, G. Papaxoinis, L. Krikoni, P. Economopoulou, I. Kotsantis, M. Anastasiou, V.L. Souliotis, The DNA damage response network in the treatment of head and neck squamous cell carcinoma. ESMO Open. 6 , 100075 (2021). https://doi.org/10.1016/j.esmoop.2021.100075 P. Jaaks, E.A. Coker, D.J. Vis, O. Edwards, E.F. Carpenter, S.M. Leto, L. Dwane, F. Sassi, H. Lightfoot, S. Barthorpe, D. van der Meer, W. Yang, A. Beck, T. Mironenko, C. Hall, J. Hall, I. Mali, L. Richardson, C. Tolley, J. Morris, F. Thomas, E. Lleshi, N. Aben, C.H. Benes, A. Bertotti, L. Trusolino, L. Wessels, M.J. Garnett, Effective drug combinations in breast, colon and pancreatic cancer cells. Nature. 603 , 166–173 (2022). https://doi.org/10.1038/s41586-022-04437-2 A.M. Fichtinger-Schepman, J.L. van der Veer, J.H. den Hartog, P.H. Lohman, J. Reedijk, Adducts of the antitumor drug cis-diamminedichloroplatinum(II) with DNA: formation, identification, and quantitation. Biochemistry. 24 , 707–713 (1985). https://doi.org/10.1021/bi00324a025 M. Kartalou, J.M. Essigmann, Mechanisms of resistance to cisplatin. Mutat. Res. 478 , 23–43 (2001). https://doi.org/10.1016/s0027-5107(01)00141-5 M.L. Dronkert, R. Kanaar, Repair of DNA interstrand cross-links. Mutat. Res. 486 , 217–247 (2001). https://doi.org/10.1016/s0921-8777(01)00092-1 A.J. Deans, S.C. West, DNA interstrand crosslink repair and cancer. Nat. Rev. Cancer. 11 , 467–480 (2011). https://doi.org/10.1038/nrc3088 L.H. Thompson, J.M. Hinz, Cellular and molecular consequences of defective Fanconi anemia proteins in replication-coupled DNA repair: mechanistic insights. Mutat. Res. 668 , 54–72 (2009). https://doi.org/10.1016/j.mrfmmm.2009.02.003 S. Mogi, D.H. Oh, Gamma-H2AX formation in response to interstrand crosslinks requires XPF in human cells. DNA Repair. (Amst). 5 , 731–740 (2006). https://doi.org/10.1016/j.dnarep.2006.03.009 L.J. Kuo, L.X. Yang, Gamma-H2AX-a novel biomarker for DNA double-strand breaks, In Vivo 22, 305–309 (2008). PMID: 18610740 B. Salles, J.L. Butour, C. Lesca, Macquet, cis-Pt(NH3)2Cl2 and trans-Pt(NH3)2Cl2 inhibit DNA synthesis in cultured L1210 leukemia cells. Biochem. Biophys. Res. Commun. 112 , 555–563 (1983). https://doi.org/10.1016/0006-291x(83)91500-0 C.M. Sorenson, A. Eastman, Influence of cis-diammine-dichloro-platinum (II) on DNA synthesis and cell cycle progression in excision repair proficient and deficient Chinese hamster ovary cells, Cancer Res. 48, 6703–6707 (1988). PMID: 3180081 M.G. Ormerod, R.M. Orr, J.H. Peacock, The role of apoptosis in cell killing by ciplatin: a flow cytometric study. Br. J. Cancer. 69 , 93–100 (1994). https://doi.org/10.1038/bjc.1994.14 K. Nishio, Y. Fujiwara, Y. Miyahara, Y. Takeda, T. Ohira, N. Kubota, S. Ohta, Y. Funayama, H. Ogasawara, M. Ohata, cis-Diammine-dichloro-platinum (II) inhibits p34cdc2 protein kinase in human lung-cancer cells. Int. J. Cancer. 55 , 616–622 (1993). https://doi.org/10.1002/ijc.2910550417 X. Wang, J.L. Martindale, N.J. Holbrook, Requirement for ERK activation in cisplatin-induced apoptosis. Biol. Chem. 275 , 39435–39443 (2000). https://doi.org/10.1074/jbc.M004583200 S.Q. Wei, L.H. Sui, J.H. Zheng, G.M. Zhang, Y.L. Kao, Role of ERK1/2 kinase in cisplatin-induced apoptosis in human ovarian carcinoma cells, Chin. Med. Sci. J. 19, 125–129 (2004). PMID: 15250250 D. Tang, D. Wu, A. Hirao, J.M. Lahti, L. Liu, B. Mazza., V.J. Kidd, T.W. Mak, A.J. Ingram, ERK activation mediates cell cycle arrest and apoptosis after DNA damage independently of p53. J. Biol. Chem. 277 , 12710–12717 (2002). https://doi.org/10.1074/jbc.M111598200 T. Knight, J.A.E. Irving, Ras/Raf/MEK/ERK Pathway activation in childhood acute lymphoblastic leukemia and its therapeutic targeting. Front. Oncol. 4 , 160 (2014). https://doi.org/10.3389/fonc.2014.00160 G. Maurer, B. Tarkowski, M. Baccarini, Raf kinases in cancer-roles and therapeutic opportunities. Oncogene. 30 , 3477–3488 (2011). https://doi.org/10.1038/onc.2011.160 T. Yamamoto, M. Ebisuya, F. Ashida, K. Okamoto, S. Yonehara, E. Nishida, Continuous ERK activation downregulates antiproliferative genes throughout G1 phase to allow cell-cycle progression. Curr. Biol. 16 , 1171–1182 (2006). https://doi.org/10.1016/j.cub.2006.04.044 J.R. Brown, E. Nigh, R.J. Lee, H. Ye, M.A. Thompson, F. Saudou, R.G. Pestell, M.E. Greenberg, Fos family members induce cell cycle entry by activating cyclin D1, Mol. Cell. Biol. 18 , 5609–5619 (1998). https://doi.org/10.1128/MCB.18.9.5609 E. Kerkhoff, U.R. Rapp, Cell cycle targets of Ras/Raf signalling. Oncogene. 17 , 1457–1462 (1998). https://doi.org/10.1038/sj.onc.1202185 K. Roovers, R.K. Assoian, Integrating the MAP kinase signal into the G1 phase cell cycle machinery. Bioessays. 22 , 818–826 (2000). https://doi.org/10.1002/1521-1878(200009)22:93.0.CO;2-6 C.J. Sherr, J.M. Roberts, CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev. 13 , 1501–1512 (1999). https://doi.org/10.1101/gad.13.12.1501 P. Salerno, V. De Falco, A. Tamburrino, T.C. Nappi, G. Vecchio, R.E. Schweppe, G. Bollag, M. Santoro, G. Salvatore, Cytostatic activity of adenosine triphosphate-competitive kinase inhibitors in BRAF mutant thyroid carcinoma cells. J. Clin. Endocrinol. Metab. 95 , 450–455 (2010). https://doi.org/10.1210/jc.2009-0373 M. Fragkos, J. Jurvansuu, P. Beard, H2AX is required for cell cycle arrest via the p53/p21 pathway. Mol. Cell. Biol. 29 , 2828–2840 (2009). https://doi.org/10.1128/MCB.01830-08 M. Bendjennat, J. Boulaire, T. Jascur, H. Brickner, V. Barbier, A. Sarasin, A. Fotedar, R. Fotedar, UV irradiation triggers ubiquitin-dependent degradation of p21(WAF1) to promote DNA repair. Cell. 114 , 599–610 (2003). https://doi.org/10.1016/j.cell.2003.08.001 M. Mao, F. Tian, J.M. Mariadason, C.C. Tsao, R.J. Lemos, F. Dayyani, Y.N. Gopal, Z.Q.I.I. Wistuba, X.M. Tang, W.G. Bornman, G. Bollag, G.B. Mills, G. Powis, J. Desai, G.E. Gallick, M.A. Davies, S. Kopetz, Resistance to BRAF inhibition in BRAF-mutant colon cancer can be overcome with PI3K inhibition or demethylating agents, Clin. Cancer Res. 19 , 657–667 (2013). https://doi.org/10.1158/1078-0432.CCR-11-1446 E. Jiménez-Mora, B. Gallego, S. Díaz-Gago, M. Lasa, P. Baquero, A. Chiloeches, (V600E)BRAF Inhibition Induces Cytoprotective Autophagy through AMPK in Thyroid Cancer Cells. Int. J. Mol. Sci. 22 , 6033 (2021). https://doi.org/10.3390/ijms22116033 S. Kopetz, J. Desai, E. Chan, J.R. Hecht, P.J. O' Dwyer, D. Maru, V. Morris, F. Janku, A. Dasari, W. Chung, J.P. Issa, P. Gibbs, B. James, G. Powis, K.B. Nolop, S. Bhattacharya, Saltz, Phase II Pilot Study of Vemurafenib in Patients with Metastatic BRAF-Mutated Colorectal Cancer. J. Clin. Oncol. 33 , 4032–4038 (2015). https://doi.org/10.1200/JCO.2015.63.2497 Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterials.doc Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4109451","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":280917572,"identity":"e0bac412-2300-4d42-9d47-d9839f6c04c3","order_by":0,"name":"Kassandra Koumaki","email":"","orcid":"","institution":"Institute of Chemical Biology, National Hellenic Research Foundation","correspondingAuthor":false,"prefix":"","firstName":"Kassandra","middleName":"","lastName":"Koumaki","suffix":""},{"id":280917574,"identity":"3351f829-3a05-4e3e-ac17-66ad770a898c","order_by":1,"name":"Salomi Skarmalioraki","email":"","orcid":"","institution":"Institute of Chemical Biology, National Hellenic Research Foundation","correspondingAuthor":false,"prefix":"","firstName":"Salomi","middleName":"","lastName":"Skarmalioraki","suffix":""},{"id":280917576,"identity":"f0045029-5f5f-47df-83de-a4acb2d762fe","order_by":2,"name":"Vivian Kosmidou","email":"","orcid":"","institution":"Institute of Chemical Biology, National Hellenic Research Foundation","correspondingAuthor":false,"prefix":"","firstName":"Vivian","middleName":"","lastName":"Kosmidou","suffix":""},{"id":280917577,"identity":"4b4ae479-74f9-42bb-abed-1869e6b8ed2c","order_by":3,"name":"Lida Krikoni","email":"","orcid":"","institution":"Institute of Chemical Biology, National Hellenic Research Foundation","correspondingAuthor":false,"prefix":"","firstName":"Lida","middleName":"","lastName":"Krikoni","suffix":""},{"id":280917578,"identity":"1c861c81-cad7-4ec4-8cd5-c46f62d92c9e","order_by":4,"name":"Maria Goulielmaki","email":"","orcid":"","institution":"Institute of Chemical Biology, National Hellenic Research Foundation","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"","lastName":"Goulielmaki","suffix":""},{"id":280917581,"identity":"611f39a6-ad70-425a-b096-3e9f0e047239","order_by":5,"name":"Vassilis Zoumpourlis","email":"","orcid":"","institution":"Institute of Chemical Biology, National Hellenic Research Foundation","correspondingAuthor":false,"prefix":"","firstName":"Vassilis","middleName":"","lastName":"Zoumpourlis","suffix":""},{"id":280917582,"identity":"a065a4f9-1465-44e0-b414-6d1755bdd80a","order_by":6,"name":"Alexander Pintzas","email":"","orcid":"","institution":"Institute of Chemical Biology, National Hellenic Research Foundation","correspondingAuthor":false,"prefix":"","firstName":"Alexander","middleName":"","lastName":"Pintzas","suffix":""},{"id":280917583,"identity":"85ee1369-9b56-4812-b431-025e2a0da165","order_by":7,"name":"Vassilis L. Souliotis","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAkElEQVRIiWNgGAWjYDACdgbGBwwGJGlhZmA2IFkLmwRJGhgMDrM/q/hQYMMgH32AaC08ZjdnGKQxGJ5LIFKLZDMP220eg8MMhj3EOkyymf1Z8R+D/yRo4WdmMGNmMDjAIM9DvBYeY8keg2QeA6K1sLG3P/zw44+dnDzRDoMBHqDbSAXyDSRrGQWjYBSMgpECAMbbHcRWNGABAAAAAElFTkSuQmCC","orcid":"","institution":"Institute of Chemical Biology, National Hellenic Research Foundation","correspondingAuthor":true,"prefix":"","firstName":"Vassilis","middleName":"L.","lastName":"Souliotis","suffix":""}],"badges":[],"createdAt":"2024-03-15 16:59:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4109451/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4109451/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53184594,"identity":"ca9a8aa9-e463-4bb8-a7c6-929cd3a51062","added_by":"auto","created_at":"2024-03-21 16:06:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1087321,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of cisplatin-only treatment on CRC cells. Cytotoxicity and cell proliferation using the SRB assay at 0, 24h and 48h after treatment of RKO (A), HT29 (B) and Colo-205 cells (C) with various doses of cisplatin (0-100μg/ml) for 3h. Cytotoxicity and cell proliferation was also measured in HT29 and Caco-2 cells at 72h (D), 96h (E) and 120h (F) following treatment with PLX4720 (1μM), cisplatin (5μg/ml) or co-treatment. (G) Cytotoxicity and cell proliferation was measured in RKO and Caco-2 cells after treatment with PLX4720 (1μM), various doses of cisplatin, or co-treatment. (H) Cell cycle phase distribution for RKO, HT29 and Colo-205 cells treated with cisplatin. (I) Bar charts showing distribution of the lowest concentrations of cisplatin required for the induction of apoptosis 48h after cisplatin treatment. Error bars represent SD. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4109451/v1/3cd961f3b4ab3658f5bdb720.png"},{"id":53184589,"identity":"ebb6d089-abfe-4e4d-9d96-5a14ebff28ad","added_by":"auto","created_at":"2024-03-21 16:06:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1161158,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of BRAFi-only treatment on human CRC cells. Cytotoxicity and cell proliferation using the SRB assay 48h after treatment of RKO (A), HT29 (B), Colo-205 cells (C) and Caco-2 (D) with various doses of PLX4720. In (E) cytotoxicity and cell proliferation of the same cell lines, 48h following treatment with 1μM and 5μM of PLX4720, Dabrafenib or Encorafenib. (F) Bar charts showing distribution of the lowest concentrations of cisplatin required for the induction of apoptosis, 48h after cisplatin treatment. Error bars represent SD. ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001. Cell cycle phase distribution of the same cell lines treated with PLX4720 (G) or Encorafenib (H) is also presented. Western blot analysis for the phosphorylation of ERK1/2 and γH2AX in RKO (I), Colo-205 and HT29 (J) cells, following treatment with PLX4720, Vemurafenib, Dabrafenib or Encorafenib. Averaged densitometry data normalized to the control (GAPDH) at the same time points are also shown.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4109451/v1/cb2deb47913c964935b103b5.png"},{"id":53184592,"identity":"88d4392b-52b1-493a-89a3-8d5f435e38a0","added_by":"auto","created_at":"2024-03-21 16:06:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1054930,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of the combined treatment with BRAFi and cisplatin on CRC cells. Western blot analysis for the phosphorylation of ERK1/2 and γH2AX (A-C), cell cycle phase distribution (D-F), as well as cytotoxicity and cell proliferation (G-I) for RKO (A, D, G), HT29 (B, E, H) and Colo-205 (C, F, I) cell lines following combined treatment with BRAFi and cisplatin. Averaged densitometry data normalized to the control (GAPDH) at the same time points are also shown.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4109451/v1/c8b29a38467bdc48a1e44538.png"},{"id":53184605,"identity":"eec30af6-5981-4768-b68a-3230ee219614","added_by":"auto","created_at":"2024-03-21 16:06:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":490499,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of simultaneous exposure to cisplatin and BRAFi. (A) Cytotoxicity and cell proliferation using the SRB assay after simultaneous exposure to 5μg/ml cisplatin and 1μM PLX4720 or 1μM Dabrafenib or 1μM Encorafenib for 48h. (B) Western blot analysis for the phosphorylation of ERK1/2 and γH2AX following simultaneous treatment with BRAFi and cisplatin. Averaged densitometry data normalized to the control (GAPDH) at the same time points are also shown.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4109451/v1/19196e9d334af3091ff47c5d.png"},{"id":53186013,"identity":"55035f1b-101d-4199-b1ce-88d13655c027","added_by":"auto","created_at":"2024-03-21 16:14:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":973101,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of the combined treatment with BRAFi and cisplatin on colon tumors \u003cem\u003ein vivo\u003c/em\u003e. (A) Growth rates of RKO tumor xenografts in SCID mice. Twenty mice were equally divided into the following five groups based on the applied treatment: control (untreated), DMSO, combination of 5mg/kg cisplatin and 10mg/kg PLX4720, 5mg/kg cisplatin alone, 10mg/kg PLX4720 alone. Chart lines represent tumor growth within a total period of 20 days after the first administration of the compounds to the developed tumors (Day 15). Standard deviation (SD) was used for error bar generation between all tumors of the same group (bars indicate standard deviation of tumor volumes, \u003cem\u003en\u003c/em\u003e=8 sites injected). (B) Statistical analysis was performed using two-way ANOVA, Bonferroni’s multiple comparisons test. Statistical significance represents the comparison of each indicated sample with the control. ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001, ns: non-significant. (C) Size values of the tumors at the experiment onset and during the course of the administrations. (D) Representative image of the tumors excised from each group of mice at the end of the \u003cem\u003ein vivo \u003c/em\u003eexperiment (Day 35).\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4109451/v1/26cd8c0fea678798b54db086.png"},{"id":53184600,"identity":"184d8c26-7016-4cf8-be7c-561466be8ec3","added_by":"auto","created_at":"2024-03-21 16:06:13","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":464360,"visible":true,"origin":"","legend":"\u003cp\u003eIn preclinical models, combined BRAFi and cisplatin treatment, decreased phosphorylation of ERK, increased phosphorylation of H2AX, induced G2/M arrest and decreased cancer cells viability. Norably, in a mouse xenografts model, combination treatment demonstrated greater therapeutic effects compared to monotherapies.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-4109451/v1/ec9ae84969074d1fa0d22a08.png"},{"id":53200140,"identity":"afb714e5-b426-49c9-aa91-963ee57f9b76","added_by":"auto","created_at":"2024-03-21 19:22:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2283543,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4109451/v1/b31770f7-5696-4cf3-ba16-39b100134e41.pdf"},{"id":53184591,"identity":"3d6e3b49-5f35-4c90-9bd9-4c9d6d20fa65","added_by":"auto","created_at":"2024-03-21 16:06:08","extension":"doc","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":1546240,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.doc","url":"https://assets-eu.researchsquare.com/files/rs-4109451/v1/2cd34667070786835719d989.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"Antitumorigenic effect of combination treatment with BRAF inhibitor and cisplatin in colorectal cancer in vitro and in vivo","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eColorectal cancer (CRC) is the third most common cancer worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Approximately 65% of CRC cases are sporadic and believed to be associated with environmental factors [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The remaining cases exhibit inheritance, of which approximately 3% exhibit highly penetrant inheritance, whereas the remaining less penetrant [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Despite availability of early detection methods, it has been reported that up to 25% of patients with CRC have already metastases at initial diagnosis and nearly twice as many will develop metastases in time, resulting in low survival rates [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. CRC incidence and mortality rates differ among males compared to females. In particular, CRC is the third most frequent cancer and the fourth most common cause of cancer death in men, while among females CRC is the second most frequent cancer and the third most common cause of cancer death [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCRC comprises a complex and heterogeneous disease, resulting from genomic, proteomic, epigenetic and other changes. Surgical resection of the tumor, chemotherapy and radiotherapy are the key treatment options for CRC patients. However, as previously stated, up to a quarter of CRC patients are diagnosed at a late stage with metastases, limiting effective surgical control, leading to poor prognosis and increased mortality rates [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Although cisplatin remains the gold standard for solid tumors, in CRC, cisplatin treatment is correlated with adverse effects, drug resistance and reduced efficacy. Therefore, improving treatment options, especially for metastatic CRC patients, is crucial, in order to prolong survival.\u003c/p\u003e \u003cp\u003eThe RAS/RAF/MEK/extracellular signal-regulated kinase (ERK) mechanism, also known as the mitogen-activated protein kinase (MAPK) pathway, regulates cell proliferation, differentiation and survival. Activating mutations in the genes involved in this pathway, including BRAF, transfer signals from the cell membrane to the nucleus through downstream components, resulting in deregulation of key cellular activities and tumor development by uncontrolled cell proliferation and survival [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. BRAF mutations have been reported in 5\u0026ndash;12% of patients with metastatic CRC and affect treatment and prognosis of these patients [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The most frequent mutation in BRAF, accounting for more than 95% of BRAF mutations, is a single substitution at nucleotide 1799, replacing valine with glutamic acid (V600E mutation). BRAF mutations have been correlated with a poor prognosis, with a median survival of less than 12 months. Treatment strategies for patients with BRAF-mutant metastatic CRC are inadequate, therefore efficient anticancer treatment is urgently needed.\u003c/p\u003e \u003cp\u003eDuring the past few years, targeting mutated ΒRAF proteins with selective inhibitors (BRAFi) has produced remarkable antitumor activity. Indeed, Vemurafenib, Dabrafenib and Encorafenib have been approved by the United States Food and Drug Administration (FDA) for the treatment of BRAF-mutant metastatic melanoma. Vemurafenib has been shown to inhibit the kinase activity of BRAFV600E mutation in patients with metastatic melanoma and was the first potent and selective tyrosine kinase inhibitor to inactivate the MAPK pathway, demonstrating antitumor activity [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Dabrafenib was the second kinase inhibitor approved by the FDA for the treatment of patients with metastatic melanoma harboring the BRAFV600E mutation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. It is also used, in combination with Trametinib (MEK inhibitor), for the treatment of patients with BRAFV600E-mutant metastatic non-small cell lung cancer (NSCLC) or anaplastic thyroid cancer (ATC) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Encorafenib is a potent BRAF inhibitor that has been shown to decrease ERK phosphorylation and inhibit proliferation in BRAFV600E-mutant melanoma, which accounts for 50% of all melanomas. It also induces senescence, rather than apoptosis, by down-regulating Cyclin D1 and arresting the cell cycle in the G1 phase [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBRAF inhibitors have been reported to show improved rates of rapid response in patients with BRAF-mutant metastatic melanoma, ranging from 48% in a phase III study assessing Vemurafenib efficacy to 59% in a phase II study of the clinical activity of Dabrafenib [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Moreover, the phase II COLUMBUS trial showed superior efficacy data of Encorafenib over Vemurafenib monotherapy [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, BRAFi monotherapy does not usually have an enduring anticancer effect, since patients have been reported to progress to more advanced stages 6 to 7 months after the beginning of treatment. This acquired resistance to BRAF inhibitor therapy has been attributed to MAPK pathway reactivation and the reported mechanisms include activating mutations in NRAS, KRAS, MEK1/2 and AKT1, BRAF amplification and splicing and CDKN2A loss, along with PI3K-PTEN-AKT pathway mutations overlapping with the MAPK pathway [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Thus, further research is urgently needed to find new therapeutic options, in order to increase treatment efficacy and prevent the development of BRAFi-associated resistance. Rational combinatorial treatment protocols of BRAF inhibitors have offered high potential against resistant tumors. The currently investigated strategies include among other, co-treatment protocols with inhibitors of EGFR, MEK, or PI3K/AKT pathway, or immunotherapies. The best results so far have been observed by combining Vemurafenib with the selective MEK inhibitor Cobimetinib, a combination approved by the FDA in 2015 for the treatment of patients with BRAF-mutant metastatic or unresectable melanoma [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Combination of Dabrafenib with Trametinib was also approved by the FDA for BRAFV600E-mutant anaplastic thyroid cancer in 2018 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and for BRAFV600E-mutant non-small cell lung cancer (NSCLC), where a major favorable effect has been demonstrated [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The Encorafenib-Cetuximab (EGFR targeting antibody) combination was approved in April 2020 for the previously treated BRAFV600E metastatic CRC patients, after it was shown to significantly improve their overall and progression-free survival during the trial. The doublet therapy was selected being to be equally effective to the triplet protocol, but with slightly less (MEKi-related) adverse effects [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe present study examined whether combined treatment of BRAFi and cisplatin is more effective than each drug alone in human colon cancer cell lines both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. For this purpose, the effects of BRAFi and/or cisplatin treatment on the cell viability, the cell cycle, as well as the phosphorylation of critical molecular components of the MAPK pathway and the DNA damage response (DDR) network were investigated in BRAFV600E mutated and BRAFwt cell lines. The antitumor activity of the combined treatment of BRAFi and cisplatin was also studied \u003cem\u003ein vivo\u003c/em\u003e, using a mouse xenografts model.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cell lines\u003c/h2\u003e \u003cp\u003eHuman colorectal adenocarcinoma cell lines (Colo-205, HT29, RKO and Caco-2) were obtained from the American Type Culture Collection (ATCC). All cell lines were maintained in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM), supplemented with 10% Fetal Bovine Serum (FBS), 1% nonessential amino acids and 1% penicillin/streptomycin (all from Thermo Fisher Scientific) at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Inhibitors\u003c/h2\u003e \u003cp\u003eThe BRAF inhibitors PLX4720 (#S1152) and Vemurafenib (#S1267) were purchased from Selleckchem, while Encorafenib (#HY-15605) and Dabrafenib (#HY-14660) from MedChemExpress. Human recombinant SuperKiller cc-TRAIL (Alexis, ALX-522-020) was used as a control of apoptotic cell death.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Western blotting\u003c/h2\u003e \u003cp\u003eWhole cell protein lysates were extracted with lysis buffer containing protease inhibitors separated in sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Amersham, UK) as described previously [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The antibodies used were directed against pERK1/2 (Santa Cruz Biotechnology, sc-7383), γH2ΑΧ (Cell Signaling Technology, #9718T), and GAPDH (Santa Cruz Biotechnology, sc-47724). The secondary antibodies used were mouse anti-rabbit IgG-HRP (Santa Cruz Biotechnology, sc-2357) and goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, sc-2005). The antibody signal was enhanced with chemiluminescence and captured on X-ray film Super RX-N (Fujifilm Tokyo, Japan). Values were measured using Studio Lite software (LI-COR Biotechnology, Lincoln, NE, USA) and levels were normalized against the housekeeping GAPDH protein. The blots presented are representative of three independently repeated experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Flow cytometry\u003c/h2\u003e \u003cp\u003eCells were cultured and treated in 6-well plates. Upon the selected time-point, they were detached and fixed/permeabilized with ice-cold ethanol overnight. DNA was marked with propidium iodide (PI; BD Biosciences; #556463) for 1h at room temperature at a concentration of 50\u0026micro;g/ml. RNA binding was avoided with the use of RNase A, 10\u0026micro;g/ml (Thermo Fisher Scientific, #EN0531). Cell cycle was analyzed using a BD FACSAria II flow cytometer and the BD FACSDiva v8.0 software (BD Biosciences).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Cell viability assay\u003c/h2\u003e \u003cp\u003eCell viability was estimated with the Sulforhodamine B (SRB; Sigma-Aldrich, S1402) assay [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Cells were seeded for 24h into 96-well microtiter plates. After completion of the treatment, fixation was performed with 10% trichloroacetic acid (TCA; Sigma-Aldrich, #T6399) and staining with 0.4% SRB in 1% acetic acid. Absorbance was measured using a TECAN microplate reader (TECAN, Mannedorf, Switzerland) and cell viability was estimated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 In vivo studies\u003c/h2\u003e \u003cp\u003eA total of 1x10\u003csup\u003e6\u003c/sup\u003e RKO cells diluted in PBS were injected subcutaneously into the left and right flanks of 6-week-old female SCID mice. When the tumors became palpable, reaching an appropriate volume of 21-33mm\u003csup\u003e3\u003c/sup\u003e (Day 15), the mice were randomly assigned to 5 groups (4 mice per group). The first group was used as a negative control (untreated) group. The second group was injected with DMSO (5% in distilled H\u003csub\u003e2\u003c/sub\u003eO). The third group was treated intratumorally with a combination of 5mg/kg cisplatin plus 10mg/kg PLX4720 in 5% DMSO (100\u0026micro;g cisplatin plus 200\u0026micro;g PLX4720/mouse every five days). The fourth group was treated intratumorally with 5mg/kg cisplatin alone (100\u0026micro;g/mouse every five days). The fifth group was treated intratumorally with 10mg/kg PLX4720 (200\u0026micro;g/mouse every five days). The mice received 5 treatment doses in a total period of 25 days. During this period, tumor sizes were measured every 5 days using caliper and tumor volumes were calculated using the formula V = (height x width x length) / 2. The Standard Deviation (SD) was used for error bar generation between all tumors of the same group of animals. Statistical analysis of the data was performed using two-way ANOVA Bonferroni\u0026rsquo;s multiple comparisons test in Graphpad Prism 9.0. At the end of the observation period, the mice were sacrificed due to tumor burden. Tumors were subsequently excised and photographed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Apoptosis assay\u003c/h2\u003e \u003cp\u003eCells were treated with 0-100\u0026micro;g/ml cisplatin for 3h, followed by 48h post-incubation time in drug-free medium. The Cell Death Detection ELISAPLUS kit (Roche Diagnostics, Switzerland, #11544675001) was used to determine apoptosis as previously described [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical Analysis\u003c/h2\u003e \u003cp\u003eContinuous variables were compared among groups with Student's t-test, or Mann-Whitney U test when normal distribution did not apply, whereas paired comparisons were performed by paired t-test or Wilcoxon's test. Correlations were examined with Spearman's rank test. All statistical analyses were performed with SPSS v.24.0. Results were considered significant when \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Cisplatin-only treatment of colon cancer cell lines\u003c/h2\u003e \u003cp\u003eFirst, we analyzed the cytotoxic effects of cisplatin on a number of human colorectal adenocarcinoma cells, such as the BRAFV600E bearing RKO, HT29 and Colo-205 cell lines, as well as the BRAFwt colon adenocarcinoma Caco-2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-G). For all cell lines tested, the decrease in cell viability was time- and concentration-dependent. All BRAFV600E bearing cell lines were resistant to cisplatin 24h after treatment with concentrations up to 10\u0026micro;g/ml. In RKO and HT29 cells, cisplatin doses higher than 25\u0026micro;g/ml led to increased cytotoxicity 24h after treatment, while Colo-205 cells seemed more resistant, since reduction of cell viability was observed following treatment with drug doses higher than 50\u0026micro;g/ml (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C). Interestingly, the BRAFwt Caco-2 cell line was more resistant than HT29 cells at 72h, 96h and 120h time-points (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-F) and more resistant than RKO cells 72h following treatment with various doses of cisplatin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we studied the progression of the cell cycle following treatment with cisplatin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). In all cell lines tested, 48h following treatment with 5\u0026micro;g/ml cisplatin, increase in the percentages of cells at the G2/M phase, combined with increase in the percentages of cells at the subG1 phase (apoptotic cells) were observed.\u003c/p\u003e \u003cp\u003eDriven by the reduction of the cell viability and the increase of the SubG1 phase upon treatment with cisplatin, we hypothesized that apoptotic pathway is possibly triggered. Therefore, the apoptosis rates of all colorectal cell lines were evaluated 48h after treatment with cisplatin treatment using an ELISA assay. The lowest concentrations of cisplatin needed for induction of apoptosis were 17.5\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2\u0026micro;g/ml for Colo-205 cells, 15.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9\u0026micro;g/ml for Caco-2 cells, 12.5\u0026thinsp;\u0026plusmn;\u0026thinsp;6.1\u0026micro;g/ml for RKO cells and 9.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9\u0026micro;g/ml for HT29 cells indicating that the HT29 cell line showed the highest cisplatin-induced apoptosis rates (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003eThe effect of cisplatin on the phosphorylation of critical molecular components of the MAPK and DDR pathways, namely ERK1/2 and H2AX, respectively, was also evaluated 0, 24h and 48h after treatment with 5, 10, 25, 50 or 100\u0026micro;g/ml cisplatin. In all cell lines analyzed, we found that at the 48h time-point, cisplatin induced phosphorylation of ERK1/2 (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-C) and H2AX (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD-F).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 BRAFi-only treatment of colon cancer cell lines\u003c/h2\u003e \u003cp\u003eThe effects of various BRAF inhibitors, on the same colon cancer cell lines were also analyzed. At first, the viability of the BRAF mutant cell lines was evaluated 48h and 72h following treatment with a BRAFi. After treatment of BRAFV600E-mutated cell lines with the Vemurafenib analog PLX4720, a dose-dependent decrease in the viability was observed in both RKO and HT29 cells; Colo-205 was the most sensitive and RKO the most resistant cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C). As expected, treatment with PLX4720 had no effect on the viability of BRAFwt Caco-2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Moreover, 48h after treatment all BRAF mutant cell lines exhibited similar sensitivity to Dabrafenib or Encorafenib (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe apoptosis rates of the BRAFV600E-mutated cell lines were also evaluated 48h after treatment with PLX4720 using an ELISA assay. The lowest concentrations of PLX4720 needed for induction of apoptosis were 10.8\u0026thinsp;\u0026plusmn;\u0026thinsp;5.8\u0026micro;M for RKO cells, 4.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3\u0026micro;M for HT29 cells and 0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u0026micro;M for Colo-205 cells, indicating that Colo-205 cells showed the highest apoptosis rates (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eThe progression of the cell cycle was also investigated following treatment with PLX4720 and Encorafenib. Both these BRAF inhibitors showed similar effects in all cell lines tested. That is, 48h after treatment, increases were found in the percentages of cells at G0/G1 phase, combined with reduction in their percentage at S and G2/M phases; no increase in the percentages of cells at the subG1 phase was observed (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, H and Figs. S2-S4). The effect of PLX4720, Vemurafenib, Dabrafenib and Encorafenib on ERK1/2 and H2AX phosphorylation was also evaluated by Western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI, J). It was found that 48h after treatment, each one of these BRAFi inhibited phosphorylation of ERK1/2 and increased phosphorylation of H2AX.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 The effect of cisplatin and BRAFi combined treatment on CRC cell lines\u003c/h2\u003e \u003cp\u003eIn order to examine the combined effect of BRAFi and cisplatin on CRC cell lines, we tested five different treatment schedules. In the 1st treatment schedule, cells were exposed for 3h to 1\u0026micro;M PLX4720, followed by 3h treatment with 5\u0026micro;g/ml cisplatin and 72h post-incubation in drug-free medium. Firstly, the effect of this combined treatment on the phosphorylation of ERK1/2 and H2AX was evaluated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C). In all cell lines analyzed, compared with non-treated cells, the combined treatment inhibited the phosphorylation of the ERK1/2 kinase and increased the phosphorylation of H2AX at the 24h time-point. Moreover, the combined treatment increased the percentage of all cells analyzed at the subG1 (apoptotic cells) and G0/G1 phases and decreased the percentage at the G2/M phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-F). Using the SRB viability assay, RKO and HT29 cells exhibited synergistic effects 48h after combined treatment, while Colo-205 at the 72h time-point (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-I).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the 2nd treatment schedule, cells were exposed to 5\u0026micro;g/ml cisplatin for 3h, followed by 3h treatment with 1\u0026micro;M PLX4720 and 72h post-incubation in drug-free medium, while in the 3rd treatment schedule, the simultaneous exposure to both 5\u0026micro;g/ml cisplatin and 1\u0026micro;M PLX4720 for 3h, followed by 72h post-incubation in drug-free medium was evaluated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-I). Both 2nd and 3rd treatment schedules showed similar viability results to those obtained following the 1st treatment schedule mentioned above.\u003c/p\u003e \u003cp\u003eIn the 4th treatment schedule, cells were exposed for 24h to 0.5\u0026micro;M or 1\u0026micro;M PLX4720, followed by 3h treatment with 5\u0026micro;g/ml cisplatin in the presence of the inhibitor and 72h post-incubation in drug-free medium. When the 4th treatment schedule was validated experimentally, no synergistic effect was found (Fig. S5).\u003c/p\u003e \u003cp\u003eFinally, in the 5th treatment schedule the simultaneous exposure to 5\u0026micro;g/ml cisplatin and 1\u0026micro;M PLX4720 or 1\u0026micro;M Dabrafenib or 1\u0026micro;M Encorafenib for 48h was evaluated. In RKO cells, all BRAF-containing combinations exhibited synergistic effects, as shown by the SRB viability assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In Colo-205 cells, PLX4720- and Dabrafenib-containing treatments showed synergistic effect. No synergy on cell viability was found following treatment of Colo-205 cells with the Encorafenib-containing schedule and after exposure of HT29 cells with any of the combinations used. The effect of the 5th treatment schedule on the phosphorylation of H2AX and ERK1/2 was also evaluated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In all BRAFi-containing treatments, phosphorylation of H2AX was observed, combined with reduction in the phosphorylation of ERK1/2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Combinatorial treatment with cisplatin and BRAFi results in strong anti-tumor effects in vivo\u003c/h2\u003e \u003cp\u003eTo shed light on the potential anti-tumorigenic effects of combined BRAFi and cisplatin treatment \u003cem\u003ein vivo\u003c/em\u003e, xenografts of RKO cells were subcutaneously implanted into both flanks of severe combined immune-deficient (SCID) mice. When the tumors reached appropriate sizes, the mice were divided into five groups: control (untreated), DMSO-treated, cisplatin plus PLX4720-treated, cisplatin alone-treated and PLX4720 alone-treated. Tumor growth was monitored for 20 days after their formation, which was considered the reference point (Day 15) of the experiment. The combined treatment with 5mg/kg cisplatin and 10mg/kg PLX4720 caused a remarkable attenuation of colon cancer progression in mice bearing RKO xenografts, which was significantly higher than each separate monotherapy (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). After comparison of the excised tumor volume of the five experimental groups, cisplatin or PLX4720 monotherapy were found to reduce tumor sizes efficiently by 2.6 and 1.7 folds, respectively, as compared to untreated mice. Importantly, combined treatment with cisplatin plus PLX4720 resulted in about 3.3-fold tumor size reduction, as compared to the control untreated mice by the end of the experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). Those results clearly indicate that the combination of a BRAF inhibitor and cisplatin holds superior therapeutic potential than each monotherapy \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eCertain combination treatment regimens produce better response than the individual drugs alone [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Herein, we examined the combined effect of BRAFi with cisplatin in colorectal cancer cell lines and a mouse xenograft model. Firstly, the effect of cisplatin on CRC cell lines was analyzed. We found that following treatment of BRAF mutant cells with cisplatin, reduction of cell viability, G2/M phase arrest and phosphorylation of H2AX were observed. Cisplatin is a genotoxic drug inducing the formation of single-nucleotide damage of guanine (monoadducts), intrastrand and interstrand cross-links [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Monoadducts and intrastrand cross-links are repaired by the nucleotide excision repair (NER) mechanism [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], while the removal of interstrand cross-links requires the activation of several DNA repair pathways, such as homologous recombination, NER and translesion synthesis [\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Of note, DNA double-strand breaks (DSBs) are produced as intermediates in the interstrand cross-links repair process [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Following the detection of DSBs, cells induce the phosphorylation of histone H2AX on serine 139 (γH2AX), by the apical signaling kinases ataxia telangiectasia mutated (ATM) and ATM-Rad3-related (ATR) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Since the induction of γH2AX is an early event in the activation of the DNA damage response network, it is generally utilized as a marker of DSBs. Previous studies have also demonstrated that cisplatin impairs the synthesis of the DNA [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], induces S-phase slowdown [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] and triggers a G2/M arrest through the inhibition of the CDK1/cyclin B activity [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition, we found that following treatment of BRAF mutant cells with cisplatin, phosphorylation of the ERK1/2 kinase was observed. In accordance with our results, previous studies have shown that cisplatin treatment of HeLa cells caused a dose- and time-dependent phosphorylation, and therefore activation, of ERK, and that activation of this kinase is important for the induction of the apoptosis pathway via the cytochrome c release from mitochondria [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In contrast, Wei et al. [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] have shown that ERK signaling inhibition enhanced the susceptibility of ovarian cancer cells to cisplatin. Another report has shown that various genotoxic insults, such as adriamycin, ultraviolet irradiation, etoposide and ionizing radiation induced activation of ERK1/2 in the MEF and IMR90 primary cells, the NIH3T3 immortalized cells and the transformed MCF-7 cells, thus leading to cell cycle arrest or induction of apoptosis, depending on the drug concentration used [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNext, the effect of BRAF inhibition on CRC cells was analyzed. We found that treatment of BRAF mutant cells with a BRAFi resulted in decreased cell viability and reduction of ERK activation/phosphorylation. As expected, the inhibition of BRAF had no effect on cell viability of BRAFwt cells. It is known that the pathway that leads to the activation of the two isoforms of ERK (ERK1 and ERK2) is initiated after ligand binding to a plasma membrane receptor tyrosine kinase and the activation of the GTP-binding protein Ras. Then, Ras activates the MAP3K kinase Raf, followed by the activation of the mitogen-activated protein kinase kinase (MEK or MAP2K), which in turn phosphorylates threonine and tyrosine residues in the Thr-Glu-Tyr (TEY) sequence of ERK1/2 [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In several cancer types, such as melanoma, hairy cell leukemia, colon carcer and papillary thyroid carcinoma, a mutation in codon 600 of exon 15 (V600E) has been reported. This mutation has been implicated in various mechanisms of cancer progression, such as stimulation of the MEK/ERK pathway, prevention of immune response, avoidance of apoptosis and senescence, angiogenesis, tissue invasion and metastasis [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Interestingly, the inhibition of the BRAF kinase results in decreased pERK activity and the reduction of cell proliferation, indicating that the decreased pERK activity can be used as a pharmacodynamic biomarker of the BRAF inhibition.\u003c/p\u003e \u003cp\u003eMoreover, we found that treatment of BRAF mutant cells with a BRAFi resulted in perturbation of cell cycle progression, inducing a G0/G1 arrest. An accumulating body of evidence suggests that the ERK kinases are involved in the cell cycle progression from G1 to S phase, which occurs immediately after growth factor stimulation. Indeed, previous studies have shown that ERK inhibition by a MEK inhibitor that was given even immediately prior to the beginning of the S phase, blocked cell cycle entry into the S phase [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. That is, after growth factor stimulation, the activation of ERK causes the phosphorylation, and thus activation of the ETS transcription factor Elk-1 and results in the upregulation of immediate-early genes, such as the proto-oncogene c-fos, the expression of which is involved in the induction of delayed-early genes, such as cyclin D [\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Then, the Cyclin D/Cdk4 complex initiates the phosphorylation of retinoblastoma, a protein known to activate the E2F family of transcription factors, and regulates the expression of several target genes, such as cyclin E. Next, the complex of cyclin E with Cdk2 further phosphorylates the retinoblastoma protein, and activates the E2F transcription factors. These sequential events regulate the synthesis of various proteins that are involved in the entry of the cells in the S phase [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Previous studies have revealed a set of genes whose expression levels were rapidly reduced after ERK inactivation. Interestingly, several of these genes were found to possess antiproliferative properties, i.e. they have the ability to suppress the entry of the cells into the S phase. Together, these data suggest that the BRAF inhibitor-induced ERK inactivation blocks S phase entry [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlso, in accordance with our previous study [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], herein we found that following treatment of BRAF mutant cells with a BRAFi, phosphorylation of H2AX was observed. In fact, evidence has accumulated that histone H2AX phosphorylated on Ser-139 (γH2AX), in addition to be a critical component of the DSB repair mechanism, is also implicated in many other biological processes. For example, Fragkos et al. [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] have shown that, in the absence of DNA damage, phosphorylation of H2AX is an early sign of replication stalling, inhibiting cell cycle progression from the G1 to the S phase. Interestingly, they found that after inhibition of the replication machinery, γH2AX is needed for increasing the levels of p21, thus resulting in checkpoint activation and cell cycle arrest. These results suggest that the phosphorylation of H2AX on Ser-139 that was observed in the present study correlated with the G0/G1 arrest after treatment of BRAF mutant cells with a BRAFi. Although the exact mechanism of the interaction between γH2AX and the p53/p21 pathway are still unknown, it is possible that H2AX phosphorylation affects the interaction of PCNA with chromatin, allowing the stable binding of PCNA to p21 and blocking its ubiquitination [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eImportantly, the antitumor activity of the combination treatment with cisplatin and PLX4720 was further validated \u003cem\u003ein vivo\u003c/em\u003e in mouse xenografts of RKO cells, with the combinatorial treatment showing superior therapeutic potential than each drug alone. Combinatorial treatments of PLX4720 and other therapeutic compounds have proven efficient in growth suppression of distinct tumor types [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Especially in the case of colorectal cancer, in which Vemurafenib monotherapy is of no appreciable value for patients [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], combination of the RAS/MAPK pathway inhibition and chemotherapy could potentially ameliorate the observed resistance mechanisms that contribute to reactivation of cancer cell proliferation. Finally, drug combinations can achieve higher therapeutic responses in lower individual doses, thus avoiding the complications of off-target toxicities induced by high drug concentrations. Herein, the \u003cem\u003ein vivo\u003c/em\u003e confirmation of the remarkable \u003cem\u003ein vitro\u003c/em\u003e results paves the way for the exploitation of similar therapeutic combinations in multiple preclinical and potentially, clinical settings. However, more \u003cem\u003ein vivo\u003c/em\u003e studies regarding pharmacokinetics and other pharmacological parameters are required in order to accurately characterize the therapeutic value of the proposed regime.\u003c/p\u003e \u003cp\u003eTaken together, our study demonstrates that the combined treatment with BRAFi and cisplatin is more effective than single-drug treatment in preclinical models \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), suggesting that these data, once further validated at the preclinical level, can be exploited for the design of new therapies for the treatment of CRC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Materials\u0026nbsp;\u003c/strong\u003eFig. S1 The effect of cisplatin on MAPK and DDR pathways;\u0026nbsp;Fig. S2 Cell cycle phase distribution of RKO cells treated with Encorafenib;\u0026nbsp;Fig. S3 Cell cycle phase distribution of HT29 cells treated with Encorafenib;\u0026nbsp;Fig. S4 Cell cycle phase distribution of Colo-205 cells treated with Encorafenib,\u0026nbsp;Fig. S5 The effect of the combined treatment with BRAFi and cisplatin on human CRC cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e Conceptualization, V.Z., A.P. and V.L.S.; Data curation, V.Z., A.P. and V.L.S.; Formal analysis, K.K., V.K., M.G., V.Z., A.P. and V.L.S.; Funding acquisition, V.Z., A.P. and V.L.S.; Investigation, K.K., S.S., V.K., L.K. and M.G.; Project administration, A.P. and V.L.S.; Resources, V.Z., A.P. and V.L.S.; Supervision, A.P. and V.L.S.; Visualization, A.P. and V.L.S.; Writing \u0026ndash; original draft, A.P. and V.L.S.; Writing \u0026ndash; review \u0026amp; editing, all authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was supported by the project \u0026ldquo;STHENOS-b: Targeted therapeutic approaches against degenerative diseases with special focus on cancer and ageing-optimisation of the targeted bioactive molecules\u0026rdquo; (MIS 5002398) which is implemented under the Action \u0026ldquo;Action for the Strategic Development on the Research and Technological Sector\u0026rdquo;, funded by the Operational Programme \u0026ldquo;Competitiveness, Entrepreneurship and Innovation\u0026rdquo; (NSRF 2014\u0026ndash;2020) and co-financed by Greece and the European Union (European Regional Development Fund).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u0026nbsp;\u003c/strong\u003eThe study was carried out in accordance with the EU Directive 2010/63/EU for animal experiments and was approved by the Ethics Committee of the National Hellenic Research Foundation (approval No. 431956).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003eData are available upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e The authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eL.A. Torre, R.L. Siegel, E.M. Ward, A. Jemal, Global cancer incidence and mortality rates and trends-an update, Cancer Epidemiol. Biomarkers Prev. \u003cb\u003e25\u003c/b\u003e, 16\u0026ndash;27 (2016). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1158/1055-9965.EPI-15-0578\u003c/span\u003e\u003cspan address=\"10.1158/1055-9965.EPI-15-0578\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJ. Ferlay, I. Soerjomataram, R. Dikshit, S. Eser, C. Mathers, M. Rebelo, D.M. Parkin, D. Forman, F. Bray, Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. Int. J. Cancer. \u003cb\u003e136\u003c/b\u003e, E359\u0026ndash;E386 (2015). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/annonc/mdv621\u003c/span\u003e\u003cspan address=\"10.1093/annonc/mdv621\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR. Burt, Inheritance of colorectal cancer, Drug Discov. Today Dis. Mech. \u003cb\u003e4\u003c/b\u003e, 293\u0026ndash;300 (2007). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ddmec.2008.05.004\u003c/span\u003e\u003cspan address=\"10.1016/j.ddmec.2008.05.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS.E. Hendon, J.A. DiPalma, U.S. practices for colon cancer screening. Keio J. Med. \u003cb\u003e54\u003c/b\u003e, 179\u0026ndash;183 (2005). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2302/kjm.54.179\u003c/span\u003e\u003cspan address=\"10.2302/kjm.54.179\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM.A. G\u0026oacute;mez-Espa\u0026ntilde;a, J. Gallego, E. Gonz\u0026aacute;lez-Flores, J. Maurel, D. P\u0026aacute;ez, J. Sastre, J. Aparicio, M. Benavides, J. Feliu, R. Vera, SEOM clinical guidelines for diagnosis and treatment of metastatic colorectal cancer (2018). Clin. Transl Oncol. \u003cb\u003e21\u003c/b\u003e, 46\u0026ndash;54 (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12094-018-02002-w\u003c/span\u003e\u003cspan address=\"10.1007/s12094-018-02002-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eF. Bray, J. Ferlay, I. Soerjomataram, R.L. Siegel, L.A. Torre, A. Jemal, Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. \u003cb\u003e68\u003c/b\u003e, 394\u0026ndash;424 (2018). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3322/caac.21492\u003c/span\u003e\u003cspan address=\"10.3322/caac.21492\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eY.H. Xie, Y.X. Chen, J.Y. Fang, Comprehensive review of targeted therapy for colorectal cancer. Sig Transduct. Target. Ther. \u003cb\u003e5\u003c/b\u003e, 22 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41392-020-0116-z\u003c/span\u003e\u003cspan address=\"10.1038/s41392-020-0116-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eP.A. Ascierto, J.M. Kirkwood, J.J. Grob, E. Simeone, A.M. Grimaldi, M. Maio, G. Palmieri, A. Testori, F.M. Marincola, N. Mozzillo, The role of BRAF V600 mutation in melanoma. J. Transl Med. \u003cb\u003e10\u003c/b\u003e, 85 (2012). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/1479-5876-10-85\u003c/span\u003e\u003cspan address=\"10.1186/1479-5876-10-85\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eW. De Roock, B. Claes, D. Bernasconi, J. De Schutter, B. Biesmans, G. Fountzilas, K.T. Kalogeras, V. Kotoula, D. Papamichael, P. Laurent-Puig, F. Penault-Llorca, P. Rougier, B. Vincenzi, D. Santini, G. Tonini, F. Cappuzzo, M. Frattini, F. Molinari, P. Saletti, S. De Dosso, M. Martini, A. Bardelli, S. Siena, A. Sartore-Bianchi, J. Tabernero, T. Macarulla, F. Di Fiore, A.O. Gangloff, F. Ciardiello, P. Pfeiffer, C. Qvortrup, T.P. Hansen, E. Van Cutsem, H. Piessevaux, D. Lambrechts, M. Delorenzi, S. Tejpar, Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis. Lancet Oncol. \u003cb\u003e11\u003c/b\u003e, 753\u0026ndash;762 (2010). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S1470-2045(10)70130-3\u003c/span\u003e\u003cspan address=\"10.1016/S1470-2045(10)70130-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eH. Sorbye, A. Dragomir, M. Sundstr\u0026ouml;m, P. Pfeiffer, U. Thunberg, M. Bergfors, K. Aaseb\u0026oslash;, G.E. Eide, F. Ponten, C. Qvortrup, B. Glimelius, High BRAF mutation frequency and marked survival differences in subgroups according to KRAS/BRAF mutation status and tumor tissue availability in a prospective population-based metastatic colorectal cancer cohort. PLoS One. \u003cb\u003e10\u003c/b\u003e, e0131046 (2015). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0131046\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0131046\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eL.A. Dossett, R.R. Kudchadkar, J.S. Zager, BRAF and MEK inhibition in melanoma, Expert Opin. Drug Saf. \u003cb\u003e14\u003c/b\u003e, 559\u0026ndash;570 (2015). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1517/14740338.2015.1011618\u003c/span\u003e\u003cspan address=\"10.1517/14740338.2015.1011618\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eP.B. Chapman, A. Hauschild, C. Robert, J.B. Haanen, P. Ascierto, J. Larkin, R. Dummer, C. Garbe, A. Testori, M. Maio, D. Hogg, P. Lorigan, C. Lebbe, T. Jouary, D. Schadendorf, A. Ribas, S.J. O'Day, J.A. Sosman, J.M. Kirkwood, A.M. Eggermont, B. Dreno, K. Nolop, J. Li, B. Nelson, J. Hou, R.J. Lee, K.T. Flaherty, G.A. McArthur, BRIM-3 Study Group, Improved survival with vemurafenib in melanoma with BRAF V600E mutation, N. Engl. J. Med. \u003cb\u003e364\u003c/b\u003e, 2507\u0026ndash;2516 (2011). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1056/NEJMoa1103782\u003c/span\u003e\u003cspan address=\"10.1056/NEJMoa1103782\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD. Planchard, E.F. Smit, H.J.M. Groen, J. Mazieres, B. Besse, \u0026Aring;. Helland, V. Giannone, A.M. Jr. D'Amelio, P. Zhang, B. Mookerjee, B.E. Johnson, Dabrafenib plus trametinib in patients with previously untreated BRAF V600E-mutant metastatic non-small-cell lung cancer: an open-label, phase 2 trial. Lancet Oncol. \u003cb\u003e18\u003c/b\u003e, 1307\u0026ndash;1316 (2017). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S1470-2045(17)30679-4\u003c/span\u003e\u003cspan address=\"10.1016/S1470-2045(17)30679-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eV. Subbiah, R.J. Kreitman, Z.A. Wainberg, J.Y. Cho, J.H.M. Schellens, J.C. Soria, P.Y. Wen, C. Zielinski, M.E. Cabanillas, G. Urbanowitz, B. Mookerjee, D. Wang, F. Rangwala, B. Keam, Dabrafenib and Trametinib Treatment in Patients With Locally Advanced or Metastatic BRAF V600\u0026ndash;Mutant Anaplastic Thyroid Cancer. J. Clin. Oncol. \u003cb\u003e36\u003c/b\u003e, 7\u0026ndash;13 (2018). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1200/JCO.2017.73.6785\u003c/span\u003e\u003cspan address=\"10.1200/JCO.2017.73.6785\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZ. Li, K. Jiang, X. Zhu, G. Lin, F. Song, Y. Zhao, Y. Piao, J. Liu, W. Cheng, X. Bi, P. Gong, Z. Song, S. Meng, Encorafenib (LGX818), a potent BRAF inhibitor, induces senescence accompanied by autophagy in BRAF V600E melanoma cells. Cancer Lett. \u003cb\u003e370\u003c/b\u003e, 332\u0026ndash;344 (2016). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.canlet.2015.11.015\u003c/span\u003e\u003cspan address=\"10.1016/j.canlet.2015.11.015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eE. Hodis, I.R. Watson, G.V. Kryukov, S.T. Arold, M. Imielinski, J.P. Theurillat, E. Nickerson, D. Auclair, L. Li, C. Place, D.A.H. Dicara Ramos, M.S. Lawrence, K. Cibulskis, A. Sivachenko, D. Voet, G. Saksena, N. Stransky, R.C. Onofrio, W. Winckler, K. Ardlie, N. Wagle, J. Wargo, K. Chong, D.L. Morton, K. Stemke-Hale, G. Chen, M. Noble, M. Meyerson, J.E. Ladbury, M.A. Davies, J.E. Gershenwald, S.N. Wagner, D.S. Hoon, D. Schadendorf, E.S. Lander, S.B. Gabriel, G. Getz, L.A. Garraway, L. Chin, A landscape of driver mutations in melanoma. Cell. \u003cb\u003e150\u003c/b\u003e, 251\u0026ndash;263 (2012). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cell.2012.06.024\u003c/span\u003e\u003cspan address=\"10.1016/j.cell.2012.06.024\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eP.A. Ascierto, D. Minor, A. Ribas, C. Lebbe, A. O'Hagan, N. Arya, M. Guckert, D. Schadendorf, R.F. Kefford, J.J. Grob, O. Hamid, R. Amaravadi, E. Simeone, T. Wilhelm, K.B. Kim, G.V. Long, A.M. Martin, J. Mazumdar, V.L. Goodman, U. Trefzer, Phase II trial (BREAK-2) of the BRAF inhibitor dabrafenib (GSK2118436) in patients with metastatic melanoma. J. Clin. Oncol. \u003cb\u003e31\u003c/b\u003e, 3205\u0026ndash;3211 (2013). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1200/JCO.2013.49.8691\u003c/span\u003e\u003cspan address=\"10.1200/JCO.2013.49.8691\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eP. Koelblinger, O. Thuerigen, R. Dummera, Development of encorafenib for BRAF-mutated advanced melanoma. Curr. Opin. Oncol. \u003cb\u003e30\u003c/b\u003e, 125\u0026ndash;133 (2018). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1097/CCO.0000000000000426\u003c/span\u003e\u003cspan address=\"10.1097/CCO.0000000000000426\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR. Dummer, K. Flaherty, C. Robert, A.M. Arance, J.W. de Groot, C. Garbe, H. Gogas, R. Gutzmer, I. Krajsov\u0026aacute;, G. Liszkay, C. Loquai, M. Mandal\u0026agrave;, D. Schadendorf, N. Yamazaki, M.D. Pickard, F. Zohren, M.L. Edwards, P.A. Ascierto, Five-year overall survival (OS) in COLUMBUS: A randomized phase 3 trial of encorafenib plus binimetinib versus vemurafenib or encorafenib in patients (pts) with BRAF V600-mutant melanoma. J. Clin. Oncol. \u003cb\u003e39\u003c/b\u003e(15suppl), 9507\u0026ndash;9507 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1200/JCO.2021.39.15_suppl.9507\u003c/span\u003e\u003cspan address=\"10.1200/JCO.2021.39.15_suppl.9507\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eH. Shi, W. Hugo, X. Kong, A. Hong, R.C. Koya, G. Moriceau, T. Chodon, R. Guo, D.B. Johnson, K.B. Dahlman, M.C. Kelley, R.F. Kefford, B. Chmielowski, J.A. Glaspy, J.A. Sosman, N. van Baren, G.V. Long, A. Ribas, R.S. Lo, Acquired resistance and clonal evolution in melanoma during BRAF inhibitor therapy. Cancer Discov. \u003cb\u003e4\u003c/b\u003e, 80\u0026ndash;93 (2014). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1158/2159-8290.CD-13-0642\u003c/span\u003e\u003cspan address=\"10.1158/2159-8290.CD-13-0642\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eH. Rizos, A.M. Menzies, G.M. Pupo, M.S. Carlino, C. Fung, J. Hyman, L.E. Haydu, B. Mijatov, T.M. Becker, S.C. Boyd, J. Howle, R. Saw, J.F. Thompson, R.F. Kefford, R.A. Scolyer, G.V. Long, BRAF inhibitor resistance mechanisms in metastatic melanoma: spectrum and clinical impact, Clin. Cancer Res. \u003cb\u003e20\u003c/b\u003e, 1965\u0026ndash;1977 (2014). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1158/1078-0432.CCR-13-3122\u003c/span\u003e\u003cspan address=\"10.1158/1078-0432.CCR-13-3122\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eN. Wagle, C. Emery, M.F. Berger, M.J. Davis, A. Sawyer, P. Pochanard, S.M. Kehoe, C.M. Johannessen, L.E. Macconaill, W.C. Hahn, M. Meyerson, L.A. Garraway, Dissecting therapeutic resistance to RAF inhibition in melanoma by tumor genomic profiling. J. Clin. Oncol. \u003cb\u003e29\u003c/b\u003e, 3085\u0026ndash;3096 (2011). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1200/JCO.2010.33.2312\u003c/span\u003e\u003cspan address=\"10.1200/JCO.2010.33.2312\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eA. Kim, M.S. Cohen, The discovery of vemurafenib for the treatment of BRAF-mutated metastatic melanoma, Expert Opin. Drug Discov. \u003cb\u003e11\u003c/b\u003e, 907\u0026ndash;916 (2016). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/17460441.2016.1201057\u003c/span\u003e\u003cspan address=\"10.1080/17460441.2016.1201057\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eA. Puszkiel, G. No\u0026eacute;, A. Bellesoeur, N. Kramkimel, M.N. Paludetto, A. Thomas-Schoemann, M. Vidal, F. Goldwasser, E. Chatelut, B. Blanchet, Clinical Pharmacokinetics and Pharmacodynamics of Dabrafenib. Clin. Pharmacokinet. \u003cb\u003e58\u003c/b\u003e, 451\u0026ndash;467 (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s40262-018-0703-0\u003c/span\u003e\u003cspan address=\"10.1007/s40262-018-0703-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eA. Khunger, M. Khunger, V. Velcheti, Dabrafenib in combination with trametinib in the treatment of patients with BRAF V600-positive advanced or metastatic non-small cell lung cancer: clinical evidence and experience, Ther. Adv. Respir Dis. \u003cb\u003e12\u003c/b\u003e, 1753466618767611 (2018). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1177/1753466618767611\u003c/span\u003e\u003cspan address=\"10.1177/1753466618767611\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD. Planchard, B. Besse, H.J.M. Groen, S.M.S. Hashemi, J. Mazieres, T.M. Kim, E. Quoix, P.J. Souquet, F. Barlesi, C. Baik, L.C. Villaruz, R.J. Kelly, S. Zhang, M. Tan, E. Gasal, L. Santarpia, B.E. Johnson, Phase 2 Study of Dabrafenib Plus Trametinib in Patients With BRAF V600E-Mutant Metastatic NSCLC: Updated 5-Year Survival Rates and Genomic Analysis. J. Thorac. Oncol. \u003cb\u003e17\u003c/b\u003e, 103\u0026ndash;115 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jtho.2021.08.011\u003c/span\u003e\u003cspan address=\"10.1016/j.jtho.2021.08.011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eI.N. Okten, S. Ismail, B.M. Withycombe, Z. Eroglu, Preclinical discovery and clinical development of encorafenib for the treatment of melanoma, Expert Opin. Drug Discov. \u003cb\u003e15\u003c/b\u003e, 1373\u0026ndash;1380 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/17460441.2020.1795124\u003c/span\u003e\u003cspan address=\"10.1080/17460441.2020.1795124\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJ. Tabernero, A. Grothey, E. Van Cutsem, R. Yaeger, H. Wasan, T. Yoshino, J. Desai, F. Ciardiello, F. Loupakis, Y.S. Hong, N. Steeghs, T.K. Guren, H.T. Arkenau, P. Garcia-Alfonso, E. Elez, A. Gollerkeri, K. Maharry, J. Christy-Bittel, S. Kopetz, Encorafenib Plus Cetuximab as a New Standard of Care for Previously Treated BRAF V600E\u0026ndash;Mutant Metastatic Colorectal Cancer: Updated Survival Results and Subgroup Analyses from the BEACON Study. J. Clin. Oncol. \u003cb\u003e39\u003c/b\u003e, 273\u0026ndash;284 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1200/JCO.20.02088\u003c/span\u003e\u003cspan address=\"10.1200/JCO.20.02088\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eK. Koumaki, G. Kontogianni, V. Kosmidou, F. Pahitsa, E. Kritsi, M. Zervou, A. Chatziioannou, V.L. Souliotis, O. Papadodima, A. Pintzas, BRAF paradox breakers PLX8394, PLX7904 are more effective against BRAFV600Ε CRC cells compared with the BRAF inhibitor PLX4720 and shown by detailed pathway analysis. Biochim. Biophys. Acta Mol. Basis Dis. \u003cb\u003e1867\u003c/b\u003e, 166061 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/cmdc.202300322\u003c/span\u003e\u003cspan address=\"10.1002/cmdc.202300322\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eV. Vichai, K. Kirtikara, Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat. Protoc. \u003cb\u003e1\u003c/b\u003e, 1112\u0026ndash;1116 (2006). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nprot.2006.179\u003c/span\u003e\u003cspan address=\"10.1038/nprot.2006.179\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eA. Psyrri, M. Gkotzamanidou, G. Papaxoinis, L. Krikoni, P. Economopoulou, I. Kotsantis, M. Anastasiou, V.L. Souliotis, The DNA damage response network in the treatment of head and neck squamous cell carcinoma. ESMO Open. \u003cb\u003e6\u003c/b\u003e, 100075 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.esmoop.2021.100075\u003c/span\u003e\u003cspan address=\"10.1016/j.esmoop.2021.100075\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eP. Jaaks, E.A. Coker, D.J. Vis, O. Edwards, E.F. Carpenter, S.M. Leto, L. Dwane, F. Sassi, H. Lightfoot, S. Barthorpe, D. van der Meer, W. Yang, A. Beck, T. Mironenko, C. Hall, J. Hall, I. Mali, L. Richardson, C. Tolley, J. Morris, F. Thomas, E. Lleshi, N. Aben, C.H. Benes, A. Bertotti, L. Trusolino, L. Wessels, M.J. Garnett, Effective drug combinations in breast, colon and pancreatic cancer cells. Nature. \u003cb\u003e603\u003c/b\u003e, 166\u0026ndash;173 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41586-022-04437-2\u003c/span\u003e\u003cspan address=\"10.1038/s41586-022-04437-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eA.M. Fichtinger-Schepman, J.L. van der Veer, J.H. den Hartog, P.H. Lohman, J. Reedijk, Adducts of the antitumor drug cis-diamminedichloroplatinum(II) with DNA: formation, identification, and quantitation. Biochemistry. \u003cb\u003e24\u003c/b\u003e, 707\u0026ndash;713 (1985). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/bi00324a025\u003c/span\u003e\u003cspan address=\"10.1021/bi00324a025\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM. Kartalou, J.M. Essigmann, Mechanisms of resistance to cisplatin. Mutat. Res. \u003cb\u003e478\u003c/b\u003e, 23\u0026ndash;43 (2001). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/s0027-5107(01)00141-5\u003c/span\u003e\u003cspan address=\"10.1016/s0027-5107(01)00141-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM.L. Dronkert, R. Kanaar, Repair of DNA interstrand cross-links. Mutat. Res. \u003cb\u003e486\u003c/b\u003e, 217\u0026ndash;247 (2001). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/s0921-8777(01)00092-1\u003c/span\u003e\u003cspan address=\"10.1016/s0921-8777(01)00092-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eA.J. Deans, S.C. West, DNA interstrand crosslink repair and cancer. Nat. Rev. Cancer. \u003cb\u003e11\u003c/b\u003e, 467\u0026ndash;480 (2011). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nrc3088\u003c/span\u003e\u003cspan address=\"10.1038/nrc3088\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eL.H. Thompson, J.M. Hinz, Cellular and molecular consequences of defective Fanconi anemia proteins in replication-coupled DNA repair: mechanistic insights. Mutat. Res. \u003cb\u003e668\u003c/b\u003e, 54\u0026ndash;72 (2009). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.mrfmmm.2009.02.003\u003c/span\u003e\u003cspan address=\"10.1016/j.mrfmmm.2009.02.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS. Mogi, D.H. Oh, Gamma-H2AX formation in response to interstrand crosslinks requires XPF in human cells. DNA Repair. (Amst). \u003cb\u003e5\u003c/b\u003e, 731\u0026ndash;740 (2006). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.dnarep.2006.03.009\u003c/span\u003e\u003cspan address=\"10.1016/j.dnarep.2006.03.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eL.J. Kuo, L.X. Yang, Gamma-H2AX-a novel biomarker for DNA double-strand breaks, In Vivo 22, 305\u0026ndash;309 (2008). PMID: 18610740\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eB. Salles, J.L. Butour, C. Lesca, Macquet, cis-Pt(NH3)2Cl2 and trans-Pt(NH3)2Cl2 inhibit DNA synthesis in cultured L1210 leukemia cells. Biochem. Biophys. Res. Commun. \u003cb\u003e112\u003c/b\u003e, 555\u0026ndash;563 (1983). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0006-291x(83)91500-0\u003c/span\u003e\u003cspan address=\"10.1016/0006-291x(83)91500-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eC.M. Sorenson, A. Eastman, Influence of cis-diammine-dichloro-platinum (II) on DNA synthesis and cell cycle progression in excision repair proficient and deficient Chinese hamster ovary cells, Cancer Res. 48, 6703\u0026ndash;6707 (1988). PMID: 3180081\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM.G. Ormerod, R.M. Orr, J.H. Peacock, The role of apoptosis in cell killing by ciplatin: a flow cytometric study. Br. J. Cancer. \u003cb\u003e69\u003c/b\u003e, 93\u0026ndash;100 (1994). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/bjc.1994.14\u003c/span\u003e\u003cspan address=\"10.1038/bjc.1994.14\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eK. Nishio, Y. Fujiwara, Y. Miyahara, Y. Takeda, T. Ohira, N. Kubota, S. Ohta, Y. Funayama, H. Ogasawara, M. Ohata, cis-Diammine-dichloro-platinum (II) inhibits p34cdc2 protein kinase in human lung-cancer cells. Int. J. Cancer. \u003cb\u003e55\u003c/b\u003e, 616\u0026ndash;622 (1993). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/ijc.2910550417\u003c/span\u003e\u003cspan address=\"10.1002/ijc.2910550417\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eX. Wang, J.L. Martindale, N.J. Holbrook, Requirement for ERK activation in cisplatin-induced apoptosis. Biol. Chem. \u003cb\u003e275\u003c/b\u003e, 39435\u0026ndash;39443 (2000). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1074/jbc.M004583200\u003c/span\u003e\u003cspan address=\"10.1074/jbc.M004583200\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS.Q. Wei, L.H. Sui, J.H. Zheng, G.M. Zhang, Y.L. Kao, Role of ERK1/2 kinase in cisplatin-induced apoptosis in human ovarian carcinoma cells, Chin. Med. Sci. J. 19, 125\u0026ndash;129 (2004). PMID: 15250250\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD. Tang, D. Wu, A. Hirao, J.M. Lahti, L. Liu, B. Mazza., V.J. Kidd, T.W. Mak, A.J. Ingram, ERK activation mediates cell cycle arrest and apoptosis after DNA damage independently of p53. J. Biol. Chem. \u003cb\u003e277\u003c/b\u003e, 12710\u0026ndash;12717 (2002). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1074/jbc.M111598200\u003c/span\u003e\u003cspan address=\"10.1074/jbc.M111598200\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eT. Knight, J.A.E. Irving, Ras/Raf/MEK/ERK Pathway activation in childhood acute lymphoblastic leukemia and its therapeutic targeting. Front. Oncol. \u003cb\u003e4\u003c/b\u003e, 160 (2014). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fonc.2014.00160\u003c/span\u003e\u003cspan address=\"10.3389/fonc.2014.00160\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eG. Maurer, B. Tarkowski, M. Baccarini, Raf kinases in cancer-roles and therapeutic opportunities. Oncogene. \u003cb\u003e30\u003c/b\u003e, 3477\u0026ndash;3488 (2011). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/onc.2011.160\u003c/span\u003e\u003cspan address=\"10.1038/onc.2011.160\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eT. Yamamoto, M. Ebisuya, F. Ashida, K. Okamoto, S. Yonehara, E. Nishida, Continuous ERK activation downregulates antiproliferative genes throughout G1 phase to allow cell-cycle progression. Curr. Biol. \u003cb\u003e16\u003c/b\u003e, 1171\u0026ndash;1182 (2006). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cub.2006.04.044\u003c/span\u003e\u003cspan address=\"10.1016/j.cub.2006.04.044\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJ.R. Brown, E. Nigh, R.J. Lee, H. Ye, M.A. Thompson, F. Saudou, R.G. Pestell, M.E. Greenberg, Fos family members induce cell cycle entry by activating cyclin D1, Mol. Cell. Biol. \u003cb\u003e18\u003c/b\u003e, 5609\u0026ndash;5619 (1998). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/MCB.18.9.5609\u003c/span\u003e\u003cspan address=\"10.1128/MCB.18.9.5609\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eE. Kerkhoff, U.R. Rapp, Cell cycle targets of Ras/Raf signalling. Oncogene. \u003cb\u003e17\u003c/b\u003e, 1457\u0026ndash;1462 (1998). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/sj.onc.1202185\u003c/span\u003e\u003cspan address=\"10.1038/sj.onc.1202185\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eK. Roovers, R.K. Assoian, Integrating the MAP kinase signal into the G1 phase cell cycle machinery. Bioessays. \u003cb\u003e22\u003c/b\u003e, 818\u0026ndash;826 (2000). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/1521-1878(200009)22:9\u0026lt;818::AID-BIES7\u0026gt;3.0.CO;2-6\u003c/span\u003e\u003cspan address=\"10.1002/1521-1878(200009)22:9%3C818::AID-BIES7%3E3.0.CO;2-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eC.J. Sherr, J.M. Roberts, CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev. \u003cb\u003e13\u003c/b\u003e, 1501\u0026ndash;1512 (1999). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1101/gad.13.12.1501\u003c/span\u003e\u003cspan address=\"10.1101/gad.13.12.1501\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eP. Salerno, V. De Falco, A. Tamburrino, T.C. Nappi, G. Vecchio, R.E. Schweppe, G. Bollag, M. Santoro, G. Salvatore, Cytostatic activity of adenosine triphosphate-competitive kinase inhibitors in BRAF mutant thyroid carcinoma cells. J. Clin. Endocrinol. Metab. \u003cb\u003e95\u003c/b\u003e, 450\u0026ndash;455 (2010). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1210/jc.2009-0373\u003c/span\u003e\u003cspan address=\"10.1210/jc.2009-0373\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM. Fragkos, J. Jurvansuu, P. Beard, H2AX is required for cell cycle arrest via the p53/p21 pathway. Mol. Cell. Biol. \u003cb\u003e29\u003c/b\u003e, 2828\u0026ndash;2840 (2009). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/MCB.01830-08\u003c/span\u003e\u003cspan address=\"10.1128/MCB.01830-08\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM. Bendjennat, J. Boulaire, T. Jascur, H. Brickner, V. Barbier, A. Sarasin, A. Fotedar, R. Fotedar, UV irradiation triggers ubiquitin-dependent degradation of p21(WAF1) to promote DNA repair. Cell. \u003cb\u003e114\u003c/b\u003e, 599\u0026ndash;610 (2003). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cell.2003.08.001\u003c/span\u003e\u003cspan address=\"10.1016/j.cell.2003.08.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM. Mao, F. Tian, J.M. Mariadason, C.C. Tsao, R.J. Lemos, F. Dayyani, Y.N. Gopal, Z.Q.I.I. Wistuba, X.M. Tang, W.G. Bornman, G. Bollag, G.B. Mills, G. Powis, J. Desai, G.E. Gallick, M.A. Davies, S. Kopetz, Resistance to BRAF inhibition in BRAF-mutant colon cancer can be overcome with PI3K inhibition or demethylating agents, Clin. Cancer Res. \u003cb\u003e19\u003c/b\u003e, 657\u0026ndash;667 (2013). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1158/1078-0432.CCR-11-1446\u003c/span\u003e\u003cspan address=\"10.1158/1078-0432.CCR-11-1446\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eE. Jim\u0026eacute;nez-Mora, B. Gallego, S. D\u0026iacute;az-Gago, M. Lasa, P. Baquero, A. Chiloeches, (V600E)BRAF Inhibition Induces Cytoprotective Autophagy through AMPK in Thyroid Cancer Cells. Int. J. Mol. Sci. \u003cb\u003e22\u003c/b\u003e, 6033 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms22116033\u003c/span\u003e\u003cspan address=\"10.3390/ijms22116033\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS. Kopetz, J. Desai, E. Chan, J.R. Hecht, P.J. O' Dwyer, D. Maru, V. Morris, F. Janku, A. Dasari, W. Chung, J.P. Issa, P. Gibbs, B. James, G. Powis, K.B. Nolop, S. Bhattacharya, Saltz, Phase II Pilot Study of Vemurafenib in Patients with Metastatic BRAF-Mutated Colorectal Cancer. J. Clin. Oncol. \u003cb\u003e33\u003c/b\u003e, 4032\u0026ndash;4038 (2015). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1200/JCO.2015.63.2497\u003c/span\u003e\u003cspan address=\"10.1200/JCO.2015.63.2497\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"colorectal cancer, BRAF inhibitor, cisplatin, combination therapy, BRAFV600E-mutated cells, mouse xenografts","lastPublishedDoi":"10.21203/rs.3.rs-4109451/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4109451/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eIn colorectal cancer (CRC), BRAF inhibitor (BRAFi) monotherapy appears ineffective, while cisplatin treatment is associated with adverse effects, drug resistance and reduced efficacy. Herein, we seek to explore a combinatorial approach to increase the likelihood of effectively killing colorectal cancer cells.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe examined the combined effect of BRAFi (PLX4720, Vemurafenib, Dabrafenib, Encorafenib) and cisplatin treatment in BRAFV600E-mutated (RKO, HT29, Colo-205) and BRAFwt (Caco-2) cell lines, as well as in mouse xenografts of RKO cells.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eFollowing cisplatin-only treatment, all cell lines showed accumulation within subG1 (apoptotic cells) and G2/M phases, as well as phosphorylation of ERK1/2 and H2AX. Following BRAFi-only treatment, BRAFV600E-mutated cells showed accumulation within G0/G1 phase, reduced distribution in the S and G2/M phases, inhibition of ERK1/2 phosphorylation and increased phosphorylation of H2AX. BRAFi had no effect on BRAFwt Caco-2 cell line. Combined BRAFi and cisplatin treatment synergistically decreased RKO cells viability, reduced phosphorylation of ERK1/2 and increased phosphorylation of H2AX. Importantly, in mouse xenografts of RKO cells, combined PLX4720 and cisplatin treatment showed superior therapeutic potential than each monotherapy (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eIn \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e preclinical models, BRAFi and cisplatin combined treatment has shown an improved antitumor effect, rendering it a potential anticancer treatment strategy for BRAF-mutant colon cancer patients.\u003c/p\u003e","manuscriptTitle":"Antitumorigenic effect of combination treatment with BRAF inhibitor and cisplatin in colorectal cancer in vitro and in vivo","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-21 16:05:44","doi":"10.21203/rs.3.rs-4109451/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cc5fc15f-de5b-411d-b884-c21e6ad2dfaa","owner":[],"postedDate":"March 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-21T19:14:17+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-21 16:05:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4109451","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4109451","identity":"rs-4109451","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}