Targeting RAF Isoforms and Tumor Microenvironments in RAS or BRAF Mutant Colorectal Cancers with SJ-C1044 for Anti-Tumor Activity

Colorectal cancer (CRC) is a significant global health issue characterized by a high prevalence of KRAS gene mutations. The RAS/MAPK pathway, involving KRAS, plays a crucial role in CRC progression. Although some RAS inhibitors have been approved, their efficacy in CRC is limited. To overcome these limitations, pan-RAF inhibitors targeting A-Raf, B-Raf, and C-Raf have emerged as promising therapeutic strategies. However, resistance to RAF inhibition and the presence of an immunosuppressive tumor microenvironment (TME) pose additional obstacles to effective therapy. Here, we evaluated the potential of a novel pan-RAF inhibitor, SJ-C1044, for targeting mutant KRAS-mediated signaling and inhibiting CRC cell proliferation. Notably, SJ-C1044 also exhibited inhibitory effects on immunokinases, specifically, CSF1R, VEGFR2, and TIE2, which play crucial roles in immune suppression. SJ-C1044 demonstrated potent antitumor activity in xenograft models of CRC harboring KRAS or BRAF mutations. Importantly, treatment with SJ-C1044 resulted in increased infiltration of T cells and reduced presence of tumor-associated macrophages and regulatory T cells within the TME. Thus, SJ-C1044 shows immunomodulatory potential and the ability to enhance antitumor responses. The study underscores the therapeutic potential of SJ-C1044 as a novel pan-RAF inhibitor capable of targeting oncogenic signaling pathways and overcoming immune suppression in CRC.


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 the CDOCKER Energy and CDOCKER Interaction Energy were assessed to evaluate the binding characteristics of the protein (V600E-BRAF)-ligand (SJ-C1044) complex.

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 IC 50 value, measurements were made using nine concentration points ranging from 0.001 µM to 10 µM of SJ-C1044.

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% CO 2 . 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).

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].

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 IC 50 values. Statistical calculations and data analysis, including IC 50 determination, were performed using GraphPad Prism software.

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.

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).

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).

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 mm 3 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)].

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)

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.

Discovery of Pan-RAF Inhibitor SJ-C1044
SJ-C1044, with the chemical formula N-(5-(3-(9H-purin-6-yl)pyridin-2-ylamino)-2fluorophenyl)-3,5-bis(trifluoromethyl)benzamide ( Figure 1A), is a synthetic BRAF inhibitor designed to bind to the ATP-binding site of the BRAF(V600E) protein. Figure 1B represents the docking model for SJ-C1044 with the released X-ray crystal structure of BRAF(V600E) (PDB 5C9C). By evaluating the binding energies obtained from the docking analysis, we discerned the presence of three key residues within the BRAF protein that engaged in hydrogen bonding interactions with SJ-C1044 (Supplementary Figure S1). The purine ring of SJ-C1044 interacted with the amide backbone of CYS532 of the hinge region. Two hydrogen bonds existed between the amide oxygen and NH of SJ-C1044 and ASP594 and GLU501, respectively. These hydrogen bonding interactions supported SJ-C1044 interactions with BRAF in a DFG-out and α-C helix-in configuration [20].

SJ-C1044 Is a Pan-RAF Inhibitor of RAF Isoforms and Immunokinases
The biochemical activity of SJ-C1044 was characterized using recombinant proteins. In kinase assays, SJ-C1044 inhibited wild-type BRAF, wild-type CRAF, and BRAF(V600E) with IC 50 values of 331, 257, and 187 nM, respectively ( Table 1). The lack of specificity for RAF compared to other kinases was an issue that potentially restricted the clinical effectiveness of type II RAF inhibitors [10,[21][22][23]. We investigated the selectivity of SJ-C1044 using two alternate screening methods. SJ-C1044 had IC 50 values > 10 µM against 73 of 76 non-RAF kinases in biochemical assays with purified proteins. The only kinases inhibited by more than 50% were VEGFR2 (Vascular Endothelial Growth Factor Receptor 2), TIE2 (TEK Receptor Tyrosine Kinase), and CSF1R (Colony Stimulating Factor-1 Receptor) ( Table 1,  Supplementary Table S1). With IC 50 values of 100, 23, and 235 nM, respectively, SJ-C1044 inhibited VEGFR2, TIE2, and CSF1R. Consistent with the results of the kinase assay, our molecular docking studies confirmed the binding of SJ-C1044 to both VEGFR2 and CSF1R (Supplementary Figures S2 and S3). Next, we analyzed SJ-C1044 using the KiNativ system, in which kinase selectivity in cells was determined by competing with an ATP-competitive covalent probe, followed by mass spectrometry analysis [24]. To evaluate the universal selectivity of SJ-C1044, we analyzed it in HCT 116 cell line lysates. LC/MS-MS analysis was employed to analyze and identify the kinase targets within the lysates, providing information on the selectivity of SJ-C1044 towards specific kinases in the cellular context. The profiling was conducted at concentrations of 10, 1, 0.1, and 0.01 µM. In HCT 116 cells incubated for 2 h with 10 µM SJ-C1044, the only kinases where probe binding was inhibited >80% were BRAF (86%) and CRAF (83%), having IC 50 values of 1.3 and 1.4 µM, respectively (Table 2). Notably, the analysis did not include VEGFR2, TIE2, or CSF1R, as these kinases were not expressed in the HCT116 cell line.
Curr. Issues Mol. Biol. 2023, 3, FOR PEER REVIEW 6 analysis, we discerned the presence of three key residues within the BRAF protein that engaged in hydrogen bonding interactions with SJ-C1044 (Supplementary Figure S1). The purine ring of SJ-C1044 interacted with the amide backbone of CYS532 of the hinge region. Two hydrogen bonds existed between the amide oxygen and NH of SJ-C1044 and ASP594 and GLU501, respectively. These hydrogen bonding interactions supported SJ-C1044 interactions with BRAF in a DFG-out and α-C helix-in configuration [20].

SJ-C1044 Is a Pan-RAF Inhibitor of RAF Isoforms and Immunokinases
The biochemical activity of SJ-C1044 was characterized using recombinant proteins. In kinase assays, SJ-C1044 inhibited wild-type BRAF, wild-type CRAF, and BRAF(V600E) with IC50 values of 331, 257, and 187 nM, respectively ( Table 1). The lack of specificity for RAF compared to other kinases was an issue that potentially restricted the clinical effectiveness of type II RAF inhibitors [10,[21][22][23]. We investigated the selectivity of SJ-C1044 using two alternate screening methods. SJ-C1044 had IC50 values > 10 µM against 73 of 76 non-RAF kinases in biochemical assays with purified proteins. The only kinases inhibited by more than 50% were VEGFR2 (Vascular Endothelial Growth Factor Receptor 2), TIE2 (TEK Receptor Tyrosine Kinase), and CSF1R (Colony Stimulating Factor-1 Receptor) (Table 1, Supplementary Table S1). With IC50 values of 100, 23, and 235 nM, respectively, SJ-C1044 inhibited VEGFR2, TIE2, and CSF1R. Consistent with the results of the kinase assay, our molecular docking studies confirmed the binding of SJ-C1044 to both VEGFR2 and CSF1R ( Supplementary Figures S2 and S3). Next, we analyzed SJ-C1044 using the KiNativ system, in which kinase selectivity in cells was determined by competing with an ATPcompetitive covalent probe, followed by mass spectrometry analysis [24]. To evaluate the universal selectivity of SJ-C1044, we analyzed it in HCT 116 cell line lysates. LC/MS-MS analysis was employed to analyze and identify the kinase targets within the lysates, providing information on the selectivity of SJ-C1044 towards specific kinases in the cellular context. The profiling was conducted at concentrations of 10, 1, 0.1, and 0.01 µM. In HCT 116 cells incubated for 2 h with 10 µM SJ-C1044, the only kinases where probe binding was inhibited >80% were BRAF (86%) and CRAF (83%), having IC50 values of 1.3 and 1.4 µM, respectively (Table 2). Notably, the analysis did not include VEGFR2, TIE2, or CSF1R, as these kinases were not expressed in the HCT116 cell line.

SJ-C1044 Potently Inhibits KRAS-Activated MEK-ERK Phosphorylation and Cell Proliferation
The effect of SJ-C1044 on oncogenic KRAS-mediated MEK and ERK phosphorylation was investigated in LS513, a KRAS(G12D)-mutant colorectal cancer cell line (Figure 2A). SJ-C1044 inhibited MEK and ERK phosphorylation potently, which was consistent with the biochemical characterization data. There was no detectable difference in AKT phosphorylation. Interestingly, VEGFR2 was also expressed in LS513, and SJ-C1044 inhibited VEGFR2 activity at the cellular level. We found that SJ-C1044 effectively inhibited the receptor kinase activity of VEGFR2 in colorectal cancer cell lines. These findings highlighted the ability of SJ-C1044 to modulate VEGFR2 activity in the context of colorectal cancer. To test its potency against KRAS and BRAF mutant colorectal cancer cells, we treated three KRAS-mutant cancer cell lines (HCT116, LS513, and SW620) or a BRAF-mutant cancer cell line (HT29) with SJ-C1044 in a cell growth assay. Our results showed that SJ-C1044 impaired the growth of all KRAS and BRAF mutant cancer cells, with IC 50 values ranging from 160 to 628 nM (Table 3). These results confirmed that SJ-C1044 induced cytotoxicity in KRAS-mutated cancer cells in vitro.

SJ-C1044 Potently Inhibits KRAS-Activated MEK-ERK Phosphorylation and Cell Proliferation
The effect of SJ-C1044 on oncogenic KRAS-mediated MEK and ERK phosphorylation was investigated in LS513, a KRAS(G12D)-mutant colorectal cancer cell line (Figure 2A). SJ-C1044 inhibited MEK and ERK phosphorylation potently, which was consistent with the biochemical characterization data. There was no detectable difference in AKT phosphorylation. Interestingly, VEGFR2 was also expressed in LS513, and SJ-C1044 inhibited VEGFR2 activity at the cellular level. We found that SJ-C1044 effectively inhibited the receptor kinase activity of VEGFR2 in colorectal cancer cell lines. These findings highlighted the ability of SJ-C1044 to modulate VEGFR2 activity in the context of colorectal cancer. To test its potency against KRAS and BRAF mutant colorectal cancer cells, we treated three KRAS-mutant cancer cell lines (HCT116, LS513, and SW620) or a BRAF-mutant cancer cell line (HT29) with SJ-C1044 in a cell growth assay. Our results showed that SJ-C1044 impaired the growth of all KRAS and BRAF mutant cancer cells, with IC50 values ranging from 160 to 628 nM (Table 3). These results confirmed that SJ-C1044 induced cytotoxicity in KRAS-mutated cancer cells in vitro.

Potent Inhibition of TAM Signaling and Angiogenesis by SJ-C1044
SJ-C1044, a potent inhibitor of CSF1R and TIE2, displayed significant inhibitory effects on the ligand-dependent phosphorylation of CSF1R and TIE2 in BMDMs ( Figure 2B). Furthermore, SJ-C1044 demonstrated strong activity against VEGF-stimulated endothelial tube formation, indicative of its potent anti-angiogenic properties through inhibition of VEGFR2 ( Figure 2C,D). SJ-C1044 inhibited the formation of the tube to a greater degree compared to LY3009120. These findings highlight the potential of SJ-C1044 as a multitargeting therapeutic agent with the ability to inhibit angiogenesis in endothelial cells and disrupt crucial cellular signaling pathways in TAMs.

Pharmacokinetic Studies of SJ-C1044
The pharmacokinetics of SJ-C1044 were evaluated in C57BL/6 mice. The study involved oral (po) and intravenous (iv) administration of SJ-C1044 at doses of 40 mg/kg and 5 mg/kg, respectively. Blood samples were collected at various time intervals up to 24 h post-administration to measure the concentrations of SJ-C1044. Following po administration, the mean peak plasma concentration (C max ) reached 14,885 ng/mL, with a mean time to C max (T max ) of 4.3 h. In the case of iv administration, the corresponding values were 11,408 ng/mL for C max and 1.6 h for T max (Table 4). Plasma concentration/time curves obtained from an acute dose pharmacokinetic study in mice revealed that SJ-C1044 exhibited sustained plasma concentrations at or above 8000 ng/mL (14 µM) for 16 h per day when administered at a dose of 40 mg/kg once daily. The oral bioavailability of SJ-C1044 was determined to be 23%. These findings suggested that SJ-C1044 achieved and maintained therapeutically relevant plasma concentrations, supporting its potential efficacy as a treatment option ( Figure 3A).

SJ-C1044 Demonstrates Antitumor Activity in KRAS or BRAF Mutant Xenograft Models of Colorectal Cancer
In the KRAS G12D LS513 colorectal cancer model, SJ-C1044 was administered orally at 20, 40, or 80 mg/kg, once a day (QD), and significant tumor growth suppression was observed in a dose-dependent manner ( Figure 3B). The tumor growth inhibition (TGI) at the end of the 17th day was 50%, 63%, and 66% (p < 0.05) for the 20, 40, or 80 mg/kg dose group administered SJ-C1044, respectively (Table 5). Similarly, in the KRAS G13D HCT116 colorectal tumor model, SJ-C1044 administered at 20, 40, or 80 mg/kg QD showed dose-dependent tumor growth inhibition. In the HCT116 colorectal tumor model, treatment with SJ-C1044 for 16 days produced a significant growth inhibitory effect ranging from 72% to 107% (Table 5). The in vivo efficacy of SJ-C1044 was examined in xenograft

SJ-C1044 Demonstrates Antitumor Activity in KRAS or BRAF Mutant Xenograft Models of Colorectal Cancer
In the KRAS G12D LS513 colorectal cancer model, SJ-C1044 was administered orally at 20, 40, or 80 mg/kg, once a day (QD), and significant tumor growth suppression was observed in a dose-dependent manner ( Figure 3B). The tumor growth inhibition (TGI) at the end of the 17th day was 50%, 63%, and 66% (p < 0.05) for the 20, 40, or 80 mg/kg dose group administered SJ-C1044, respectively (Table 5). Similarly, in the KRAS G13D HCT116 colorectal tumor model, SJ-C1044 administered at 20, 40, or 80 mg/kg QD showed dosedependent tumor growth inhibition. In the HCT116 colorectal tumor model, treatment with SJ-C1044 for 16 days produced a significant growth inhibitory effect ranging from 72% to 107% (Table 5). The in vivo efficacy of SJ-C1044 was examined in xenograft models derived from HT29 colorectal cancer cell lines carrying the BRAFV600E mutation ( Figure 3C). A clear dose response was observed in this model. Treatment of HT29 tumor xenografts with SJ-C1044 resulted in TGI with a range of 58% to 117% ( Table 5). The results of these in vivo efficacy analyses suggested that SJ-C1044 inhibited the growth of tumors with KRAS or BRAF mutations.

SJ-C1044 Increased T-Cell Infiltration While Reducing the Proportion of TAMs
To investigate the immunomodulatory properties of SJ-C1044, the MC38 syngeneic mouse model was used. Due to their intact immune systems, syngeneic mice are particularly useful for immunotherapeutic analyses. Compared to the control, SJ-C1044 completely inhibited MC38 tumor growth in vivo (Figure 4). In the immunofluorescence analysis of the tumor tissue, we observed that CD31 staining, an endothelial cell (EC) marker to detect angiogenesis intensity, was reduced in mice treated with SJ-C1044, indicating an inhibitory effect on angiogenesis ( Figure 5A, upper panel). Additionally, F4/80+ immunostaining, which served as a pan macrophage marker, exhibited high intensity within the lesions of the control group. However, in mice treated with SJ-C1044, the F4/80+ staining intensity was notably reduced, suggesting the suppression of macrophage infiltration or activation ( Figure 5A, upper panel). Furthermore, the mice treated with SJ-C1044 showed a significant decrease in CD163+ or CD206+ staining, which are markers for M2 macrophages ( Figure 5A, middle panel). These results provided evidence of the effective depletion of M2 macrophages by SJ-C1044 treatment. Furthermore, treatment with SJ-C1044 caused an increase in the infiltration of CD8+ T cells ( Figure 5A, bottom panel) while simultaneously reducing the population of FOXP3+ cells, which included immunosuppressive cells such as Tregs within the tumors. Interestingly, treatment with SJ-C1044 resulted in a notable decrease in IL-10 levels, which is an immunosuppressive cytokine released by TAMs ( Figure 5B). These findings collectively demonstrated the potential of SJ-C1044 to modulate immune cell activity in vivo. The tolerability of SJ-C1044 was thoroughly assessed in all preclinical pharmacology studies. A single oral dose toxicity study in Sprague Dawley rats at 500 mg/kg showed no respiratory effects or signs of toxicity. Similarly, a 2-week repeated oral DRF (Dose Range Finding) study in Sprague Dawley rats at 500 mg/kg revealed no indications of toxicity. In ICR mice, a single oral dose of SJ-C1044 at 500 mg/kg did not result in any abnormal changes in the central nervous system. In Beagle Dogs, a 2-week repeated oral DRF toxicity study demonstrated no toxicity at 250 mg/kg, although decreased body weight was observed at 500 mg/kg. Based on these findings, further 4-week repeated oral toxicity studies will be conducted at various dose levels to assess safety. Overall, these preclinical studies indicated a favorable tolerability profile of SJ-C1044 within the tested dose ranges in the respective animal models. In the mouse xenograft experiments described in this paper, no signs of toxicity or significant weight loss were observed when SJ-C1044 was administered.
Similarly, a 2-week repeated oral DRF (Dose Range Finding) study in Sprague Dawley rats at 500 mg/kg revealed no indications of toxicity. In ICR mice, a single oral dose of SJ-C1044 at 500 mg/kg did not result in any abnormal changes in the central nervous system. In Beagle Dogs, a 2-week repeated oral DRF toxicity study demonstrated no toxicity at 250 mg/kg, although decreased body weight was observed at 500 mg/kg. Based on these findings, further 4-week repeated oral toxicity studies will be conducted at various dose levels to assess safety. Overall, these preclinical studies indicated a favorable tolerability profile of SJ-C1044 within the tested dose ranges in the respective animal models. In the mouse xenograft experiments described in this paper, no signs of toxicity or significant weight loss were observed when SJ-C1044 was administered.

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

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 dosedependent 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.

Data Availability Statement:
The data that support the findings of this study are available on request from the corresponding author Soon Kil Ahn.