1. Introduction
Colorectal cancer (CRC) is a significant health concern as the third most frequently diagnosed and second most deadly cancer worldwide [
1]. In CRC, the mitogen-activated protein kinase (RAS/MAPK) pathway plays an integral role. Specifically, KRAS is one of the most widely mutated oncogenes in CRC, with almost 40% of CRC patients containing activating mutations in
KRAS. Specifically, the majority of these mutations occur at codons 12, 13, and 61 [
2]. Patients diagnosed with mutant
KRAS CRC have a poorer prognosis compared to those with wild-type
KRAS CRC, particularly in the context of metastatic disease [
3]. Among these mutations, the codon 12 mutation is the most prevalent, representing ~65% of
KRAS gene alterations. Furthermore, two of the most common subtypes in CRC are the G12D (glycine at position 12 to aspartate) and G12V (glycine at position 12 to valine) mutations [
4]. In addition, ~10% of CRC also contains
BRAF V600E mutations [
5]. V600E is a mutation of
BRAF, in which valine (V) is replaced by glutamic acid (E) at position 600.
KRAS proteins activate several signaling pathways through the direct binding of effector molecules to GTP-loaded RAS. CRAF was the first effector to be discovered. Subsequent biochemical analysis revealed that CRAF performs a crucial role in signal transduction from RAS proteins to MEK and ERK kinases, together with its associated family members ARAF and BRAF [
6]. Understanding the oncogenic mechanisms driven by KRAS is important, as it was considered an undruggable target. Presently, the FDA has approved two RAS inhibitors, Sotorasib and Adagrasib [
7,
8]. However, these inhibitors have several limitations, including their ineffectiveness in combating colorectal cancer and their lack of efficacy against other KRAS mutations [
6].
There are several ongoing studies to overcome the limitations of KRAS inhibitors. Using pan-RAF inhibitors is one of the most promising methods to circumvent the limitations of KRAS inhibitors. Pan-RAF inhibitors are designed to target A-Raf, B-Raf, and C-Raf, the three members of the RAF protein family [
9]. Consequently, pan-RAF targeting has become the primary treatment strategy for malignant tumors with non-G12C RAS mutations. For example, the pan-RAF inhibitor LY3009120 has demonstrated remarkable efficacy in CRC cells with different KRAS mutations, and the in vivo efficacy is also noteworthy [
10].
Nevertheless, patients who respond to these oncogene-targeted therapies usually develop resistances to treatment and recurrences of cancer [
11]. Substantial evidence indicates that resistance to RAF inhibition is caused by the formation of an immunosuppressive tumor microenvironment (TME) [
12,
13]. Accumulating evidence suggests that reversing the immunosuppressive TME is essential for enhancing the clinical efficacy of cancer therapy [
14]. It is well-established that tumor-associated macrophages (TAMs) and other myeloid cells are associated with the immunosuppressive TME. In response to tumor-derived CSF1, immunosuppressive M2 macrophages undergo active polarization and recruitment to the TME [
15,
16]. CSFR1 inhibition reduces TAM infiltrators that suppress T cells [
17,
18]. Thus, CSF1R inhibitors have emerged as an exciting partner for T-cell-enhancing immunotherapies. In particular, the attack of tumor cells by chemotherapy-induced stimulation of tumor-derived CSF1 release, followed by an increase in TAM infiltration, provides the tumor with additional growth and survival factors. A comparable process was described for anti-angiogenic therapy, which resulted in an increase in vascular endothelial growth factor (VEGF) production by TAMs. Therefore, the combination of RAF-targeted or anti-angiogenic treatments with CSF1R inhibitors holds the potential to significantly augment antitumor efficacy [
11,
14].
We hypothesized that attaining a superior antitumor effect can be attributed to two primary factors. First, the inhibition of the MAPK pathway in RAS mutant cells, facilitated by pan-RAF inhibition, may contribute to this effect. Second, the depletion of immune suppressor cells, specifically M2 macrophages and regulatory T cells (Tregs), can be caused by the inhibition of immunokinases such as CSF1R, which may further enhance the antitumor response. Our research findings support this hypothesis, and we observed that the inhibition of pan-RAF and CSF1R using SJ-C1044, a highly potent competitive inhibitor, resulted in a significant delay in tumor growth in both cell-based and mouse models harboring mutant KRAS.
2. Materials and Methods
2.1. Molecular Modeling Study of the BRAFV600E/ SJ-C1044 Complex
The X-ray crystal structure of the V600E-BRAF (PDB ID: 5C9C) was downloaded from the Protein Data Bank in PDB format. SJ-C1044 was drawn in ChemDraw (version 12, PerkinElmer; Waltham, MA, USA). For the molecular docking analysis of SJ-C1044 with the V600E-BRAF kinase domain (PDB ID: 5C9C), we utilized Discovery Studio® 2020 (BIOVIA; San Diego, CA, USA). The docking process involved a series of sequential steps, including Receptor–Ligand Interactions, Docking, Docking Optimization, and Dock Ligands. During the Dock Ligands step, the following parameter values were employed: Top Hits (10), Random Conformations (50), and Orientations to Refine (50). Subsequently, the obtained results were analyzed by comparing the CDOCKER Energy and CDOCKER Interaction Energy. The CDOCKER Energy represented the combined value of the ligand’s strain energy and the protein–ligand interaction energy. On the other hand, the CDOCKER Interaction Energy quantified the non-bonding interactions between the protein and the ligand, encompassing van der Waals energy and electrostatic interaction energy. Therefore, the CDOCKER Energy and CDOCKER Interaction Energy were assessed to evaluate the binding characteristics of the protein (V600E-BRAF)–ligand (SJ-C1044) complex.
2.2. Kinase Assay
Kinase profiling analysis was conducted by Eurofins (Poitiers, France). A total of 79 kinase assays were performed following standard protocols provided by Eurofins. Briefly, kinase activity was measured using the Eurofins’ KinaseProfiler radiometric assay for protein kinase assays and homogeneous time-resolved fluorescence assays for lipid and atypical kinase assays. For the radiometric assay, kinase profiling was performed by incubating a panel of kinases with the test compound SJ-C1044 at a concentration of 10 µM. The assay utilized radiolabeled phosphate as a substrate to measure kinase activity. For the homogeneous time-resolved fluorescence assay, the same panel of kinases was incubated with SJ-C1044 at the same concentration. However, in this assay, kinase activity was measured using a fluorescence-based method. For determination of the IC50 value, measurements were made using nine concentration points ranging from 0.001 µM to 10 µM of SJ-C1044.
2.3. Cell Lines and Cell Culture
HCT116, LS513, SW620, HUVECs, and HT29 cell lines were purchased from the Korean Cell Line Bank (Seoul, Korea). All cell lines were maintained in RPMI or DMEM (Gibco Inc., Billings, MT, USA) supplemented with 10% FBS (Gibco, USA) and penicillin–streptomycin in an incubator at 37 °C and 5% CO2. Bone marrow cells were isolated from the femur and tibia of mice using a single-pass bone marrow aspiration. Cells were cultured in RPMI medium supplemented with 10% FBS and 100 ng/mL CSF1 (Peprotech, Rocky Hill, NJ, USA) for 7 days to differentiate into bone-marrow-derived macrophages (BMDMs).
2.4. Cell-Based Kinase Assays
Kinase selectivity profiling in cells was carried out using the KiNativ assay developed by ActivX Biosciences (La Jolla, CA, USA). HCT 116 cells were utilized for the experiment. The cells were treated with SJ-C1044 at a concentration of 10 mmol/L for 2 h. Following the treatment, the cells were lysed, and the lysates were processed for further analysis. Probe labeling was performed using the lysates, and the labeled samples were subjected to LC/MS-MS (Liquid Chromatography/Mass Spectrometry) analysis [
19].
2.5. Cell Proliferation Assays
In a 96-well plate, HCT 116 cells were seeded and allowed to adhere for 24 h before treatment. An SJ-C1044 dilution series was prepared and added to the wells in duplicates or triplicates. The plates were incubated for 72 h to enable cell growth and proliferation. To assess cell viability, the CellTiter-Glo Luminescent Cell Viability Assay Reagent (Promega, Fitchburg, WI, USA) was added to each well. The assay measured ATP levels as an indicator of cell viability. All data points were normalized to the control samples treated with dimethyl sulfoxide when determining IC50 values. Statistical calculations and data analysis, including IC50 determination, were performed using GraphPad Prism software.
2.6. Protein Immunoblots
In 6-well plates, LS513 cells were seeded and allowed to adhere and proliferate for 24 h. Subsequently, the cells were treated with SJ-C1044 for 1 h. After treatment, the cells were harvested using RIPA lysis buffer to extract cellular proteins. The cell lysates were subjected to Western blot analysis. Antibodies specific to phosphorylated epidermal growth factor receptor (p-EGFR, Cell Signaling Technology, Danvers, MA, USA, #2234S), epidermal growth factor receptor (EGFR, Cell Signaling Technology #2232S), phosphorylated vascular endothelial growth factor receptor (p-VEGFR, Cell Signaling Technology #3770S), vascular endothelial growth factor receptor (VEGFR, Cell Signaling Technology #2479S), phosphorylated Akt (p-Akt, Cell Signaling Technology #9271S), Akt (Cell Signaling Technology #9272S), phosphorylated MEK (p-MEK, Cell Signaling Technology #9121S), MEK (Cell Signaling Technology #9122), phosphorylated ERK (p-ERK, Cell Signaling Technology #9101S), and ERK (Cell Signaling Technology #9102S) were used for protein detection. The antibodies were diluted 1:1000 for the specific target proteins. In addition, a β-actin antibody (Sigma-Aldrich, Seoul, Republic of Korea, #A5441) diluted 1:5000 was used as a loading control. Bone-marrow-derived macrophages (BMDMs) were plated and allowed to adhere and proliferate for 24 h. Next, the cells were treated with SJ-C1044 for 2 h. Subsequently, the cells were stimulated with recombinant human angiopoietin-2 (R&D Systems, Minneapolis, MN, USA, #623-AN) and CSF1 (Peprotech, #300-25) for 15 min. After stimulation, protein samples were obtained from the cells. The cell lysates were analyzed by Western blot using specific antibodies, such as p-CSF1R (Cell Signaling, #14591S), CSF1R (Cell Signaling, #67455S), p-TIE2 (Cell Signaling, #4221S), and TIE2 (Santa Cruz Biotechnology, Dallas, TX, USA, #sc-293414), to detect and analyze the respective proteins.
2.7. Tube Formation Assays
Cell culture plates were coated with Matrigel at a concentration of 7 mg/mL and incubated at 37 °C for 45 min to allow gel formation. Human umbilical vein endothelial cells (HUVECs) were harvested using trypsin and resuspended in endothelial cell growth medium. Following a 5 h incubation period, the formation of endothelial cell tubes was assessed using an inverted photomicroscope (Nikon, Tokyo, Japan). Microphotographs were taken, and the quantification of tube formation, specifically tube length, was performed. To quantify tube formation, measurements were obtained using ImageJ. The long axis of each tube or single cell, as well as groups of adjacent cells, was measured.
2.8. Pharmacokinetic Studies
In the male C57BL/6 mouse model (6–8 weeks old), SJ-C1044 was administered orally (po) at a dose of 40 mg/kg and intravenously (iv) at a dose of 10 mg/kg. Blood concentrations of SJ-C1044 were measured at specific time points, including 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, and 24 h post-administration. Blood samples were collected at each time point for analysis. Plasma samples were analyzed by liquid chromatography–mass spectrometry (LC-MS/MS).
2.9. Animals
Six-week-old female BALB/c nude mice or C57BL/6 mice were obtained from Orient Bio and housed in individual cages at a temperature of 22 ± 3 °C and 50 ± 20% humidity. The lights were turned on and off every 12 h. The mice were provided with sterilized bedding and feed, and their water supply was first-distilled water. All experiments were conducted according to the guidance of the Institutional Animal Care and Use Committee (IACUC) at Samjin Pharmaceutical Co. (Seoul, Republic of Korea), under Project Identification Code SJ-2016-004. and the IACUC at Hanyang University (Seoul, Republic of Korea).
2.10. In Vivo Efficacy
Human-derived colorectal cancer cell lines, LS513, HCT-116, and HT29, were subcutaneously transplanted into 6-week-old female BALB/c nude mice to establish tumor models. Additionally, the mouse cancer cell line MC38 was subcutaneously transplanted into 6-week-old female and C57BL/6 mice to induce tumor formation. Mice with an average tumor volume of ~100 mm3 were selected and randomly assigned to different treatment groups, including vehicle, low (20 mg/kg), medium (40 mg/kg), and high (80 mg/kg) concentration doses of SJP1601. The treatments were administered orally once daily for 2 weeks based on the growth rate observed in the vehicle group. Tumor volume was evaluated three times a week using the formula Tumor volume (TV) = (tumor short diameter 2 × tumor long diameter)/2. The body weight of the mice was measured three times a week to assess potential toxicity in vivo. The tumor growth inhibition rate (% TGI) was calculated using the formula %TGI = 100 × [1 − (TVfinal treated − TVinitial treated)/(TVfinal control − TVinitial control)].
2.11. Immunofluorescence
Immunofluorescence staining was performed on tumor tissues. First, tumor tissue fixation was performed using 4% paraformaldehyde (Sigma-Aldrich, #HT5011). Next, heat-induced epitope retrieval was performed by incubating the tissue in 10 mM sodium citrate for 10 min. Subsequently, permeabilization was achieved by treating the tissue with 0.2% Triton X-100 (Sigma-Aldrich, #X100) for 5 min. To block nonspecific binding, the tissue was incubated in 5% skim milk for 40 min. Primary antibodies, including FOXP3 (eBioscience, San Diego, CA, USA, #11-5773-82), CD8 (eBioscience #53-0081-82), CD31 (Abcam, Cambridge, UK, #ab28364), F480 (Abcam #ab6640), CD163 (Abcam #ab182422), and CD206 (Abcam #ab64693), were used. The secondary antibodies used were FITC-conjugated AffiniPure F(ab’)2 fragment goat anti-rat IgG (H + L) (Jackson ImmunoResearch, West Grove, PA, USA, #112-096-003) and Cy3-conjugated AffiniPure F(ab’)2 fragment goat anti-rabbit IgG (H + L) (Jackson ImmunoResearch #111-166-003). The stained tissue was mounted using ProLong™ Gold Antifade Mountant with DAPI (Invitrogen, Waltham, MA, USA) #14-5773-82). Fluorescent images of the samples were captured using the Agilent BioTek Cytation 1 device. The images were analyzed using the Gen5 program (Version3, BioTek, Winooski, VT, USA) to extract relevant data and perform quantitative analysis.
2.12. Material
In this study, SJ-C1044 was used at a purity level exceeding 99.79%, as confirmed by high-performance liquid chromatography (HPLC). SJ-C1044 was synthesized at Samjin Pharmaceutical Co. (Seoul, Republic of Korea). For the in vivo experiments, the vehicle formulation for SJ-C1044 was prepared by combining dimethylacetamide, cremophor phosphoric acid, and 21.5% hydroxypropyl-beta-cyclodextrin in a ratio of 1:3:11, respectively. LY3009120 was purchased from Selleckchem (Houston, TX, USA)
2.13. Statistical Analysis
Data were analyzed using GraphPad Prism (Version8, GraphPad; San Diego, CA, USA). A two-tailed Student’s t-test was used to establish statistical significance.
4. Discussion
The results presented in this study demonstrate the potential of SJ-C1044 as a novel pan-RAF inhibitor with promising therapeutic implications for cancer treatment. The discovery of SJ-C1044, its characterization, and the evaluation of its pharmacological properties provide valuable insights into its mechanism of action and potential clinical applications.
One of the key findings of this study is the potent inhibitory activity of SJ-C1044 against BRAF (V600E) as well as wild-type BRAF and CRAF. This broad-spectrum inhibitory activity is crucial considering the involvement of these RAF isoforms in tumors containing a RAS mutation. RAF inhibitors can be classified based on the conformation they stabilize in their target kinase. “αC-IN” inhibitors stabilize the αC-helix in the IN position (either αC-helix-IN/DFG-IN for type I or αC-helix-IN/DFG-OUT for type IIa), whereas “αC-OUT” inhibitors commonly stabilize the αC-helix in the OUT position, often occurring as αC-helix-OUT/DFG-IN for type IIb, which includes clinical RAF inhibitors such as vemurafenib and dabrafenib [
20]. Through molecular modeling, it was determined that SJ-C1044 establishes hydrogen bonds with GLU501, CYS532, and ASP594 of BRAF. This observation classifies SJ-C1044 as an αC-helix-IN/DFG-OUT (type IIa) BRAF inhibitor [
25]. Conversely, Vemurafenib exclusively forms a hydrogen bond with ASP594 of BRAF [
26]. Therefore, SJ-C1044 shares the common feature of hydrogen bonding with ASP594 of BRAF while additionally forming hydrogen bonds with GLU501 and CYS532 of BRAF, thus qualifying it as a type IIa BRAF inhibitor. The ability of SJ-C1044 to interact with BRAF (V600E) in a DFG-out and α-C helix-in configuration further highlights its specificity and potential to disrupt the aberrant MAPK signaling pathway as a type IIa RAF inhibitor. Recently, it was established that the mode of inhibitor binding plays an essential role in determining the occurrence of paradoxical activation [
27]. It is believed that Type IIb inhibitors, such as vemurafenib and dabrafenib, primarily bind to BRAF monomers and effectively inhibit BRAF V600E, which can activate signaling pathways in its monomeric state [
28]. However, the administration of type IIb inhibitors to KRAS mutant cancers results in the activation of the MAPK pathway within these tumors, eventually leading to the occurrence of paradoxical activation [
29]. Conversely, type IIa RAF inhibitors, which bind to a DFG-out and α-C-helix-in conformation, lack the propensity for inducing paradoxical activation in KRAS mutant cells [
30]. Indeed, treatment of KRAS-mutated colorectal cancer cells with SJ-C1044, a type IIa RAF inhibitor, resulted in the inhibition of MEK and ERK activity in a concentration-dependent manner, without any evidence of paradoxical activation (
Figure 2A). Moreover, in vivo animal efficacy studies conducted in xenograft models of mutant KRAS demonstrated that SJ-C1044 treatment led to a dose-dependent reduction in cancer tissue growth, without the development of drug resistance (
Figure 3). These findings highlight the complete inhibition of the MAPK pathway and the subsequent suppression of cancer cell growth and proliferation by SJ-C1044 in colorectal cancer cells harboring mutant KRAS, distinguishing it from Type IIb inhibitors that may exhibit reduced efficacy due to paradoxical activation.
From a therapeutic standpoint, the absence of effectiveness against ARAF may be either an advantage or a disadvantage. ARAF is highly expressed in the majority of human tissues, frequently associated with other RAF family members [
21]. A complete blockade of signaling through the RAS/RAF/MEK kinase cascade is associated with poor tolerance. Therefore, the absence of ARAF activity upon SJ-C1044 administration may present a more favorable therapeutic opportunity in comparison to other pan-RAF inhibitors. As a result, the expansion of this therapeutic window holds significant potential for improved treatment outcomes [
22]. Importantly, the selectivity profile of SJ-C1044 was thoroughly assessed using biochemical assays and the KiNativ system, which demonstrated minimal inhibition of ARAF and non-RAF kinases. This selectivity is crucial for minimizing off-target effects and potential toxicity.
Our results also highlight the potent inhibitory effects of SJ-C1044 on KRAS-activated MEK-ERK phosphorylation and cell proliferation. The selective cytotoxicity observed in KRAS-mutant and BRAF-mutant cancer cells further supports the potential therapeutic utility of SJ-C1044 in targeting specific oncogenic mutations. These findings provide a strong rationale for further exploration of SJ-C1044 as a targeted therapy for colorectal cancer and other malignancies harboring KRAS or BRAF mutations.
Notably, the inhibition of VEGFR2, TIE2, and CSF1R by SJ-C1044 suggests its potential as both an angiogenesis inhibitor and a modulator of macrophage function, which can have significant implications in the TME. The observed immunomodulatory properties of SJ-C1044 in the MC38 syngeneic mouse model provide valuable insights into its ability to modulate the tumor immune microenvironment. Additionally, the results of the immunofluorescence analysis revealed notable effects of SJ-C1044 on TAMs, further supporting its impact on the immune landscape within the tumor. The reduced intensity of F4/80+ immunostaining, a pan macrophage marker, in SJ-C1044-treated mice indicated the suppression of macrophage infiltration or activation within the tumor lesions. This effect was further supported by the decreased staining intensity of CD163 and CD206, markers associated with M2 macrophages known for their immunosuppressive functions. The depletion of M2 macrophages by SJ-C1044 treatment suggests a shift towards an anti-tumor immune response within the tumor microenvironment. In addition, SJ-C1044 treatment increased CD8+ T cell infiltration, indicating an enhanced anti-tumor immune response. Simultaneously, there is a reduction in the population of FOXP3+ cells, including Tregs responsible for immunosuppression. These findings suggest that SJ-C1044 promotes an immune cell composition within the tumor that favors an anti-tumor response and attenuates immunosuppressive mechanisms. The observed decrease in IL-10 levels, an immunosuppressive cytokine released by TAMs, further supports the immunomodulatory effects of SJ-C1044. By reducing IL-10 levels, SJ-C1044 counteracts the immunosuppressive tumor microenvironment, facilitating an immune response against the tumor. Manipulating intratumoral immunity holds significant promise as a strategic approach to overcome resistance and enhance the effectiveness of RAF inhibitors in the treatment of cancer patients [
11]. The findings from our study not only offer novel opportunities for concurrent inhibition but also emphasize the potential of synergistic targeting that encompasses both tumor cells and the immune system.
The favorable tolerability profile observed in the preclinical pharmacology studies, with no signs of toxicity or significant weight loss, is promising for the clinical development of SJ-C1044. However, further investigations are warranted to fully understand the safety profile and potential adverse effects of SJ-C1044 in clinical settings.
Collectively, the results of this study provide strong evidence for the therapeutic potential of SJ-C1044 in RAS or BRAF mutant colorectal cancers. By targeting multiple kinases, SJ-C1044 demonstrates its ability to reshape the tumor microenvironment and promote anti-tumor immune responses. These findings contribute to our understanding of the mechanisms underlying the anti-tumor activity of SJ-C1044 and provide a basis for further investigations and clinical developments of this multi-kinase inhibitor as an effective treatment option for RAS or BRAF mutant cancers.