2-Pyridin-4-yl-methylene-beta-boswellic Acid—A Potential Candidate for Targeting O⁶-Methylguanine-DNA Methyltransferase Epi-transcriptional Reprogramming in KRAS G13D—Microsatellite Stable, G12V—Microsatellite Instable Mutant Colon Cancer

Abstract

PMBA (2-Pyridin-4-yl-methylene-beta-boswellic acid), screened from among the 21 novel series of semisynthetic analogues of β-boswellic acid, is being presented as a lead compound for integrative management of KRAS mutant colorectal cancer (CRC), upon testing and analysis for its anticancerous activity on a panel of NCI-60 cancer cell lines and in vivo models of the disease. PMBA (1.7–29 μM) exhibited potent proliferation inhibition on the cell lines and showed sensitivity in microsatellite instability and microsatellite stable (GSE39582 and GSE92921) subsets of KRAS gene (Kirsten rat sarcoma viral oncogene homolog)-mutated colon cell lines, as revealed via flow cytometry analysis. A considerable decrease in mitogen-activated protein kinase pathway downstream effectors was observed in the treated cell lines via the western blot and STRING (Search tool for the retrieval of interacting genes/proteins) analysis. PMBA was further found to target KRAS at its guanosine diphosphate site. Treatment of the cell lines with PMBA showed significant reduction in MGMT promoter methylation but restored MGMT (O⁶-methylguanine-DNA methyltransferase) messenger ribonucleic acid expression via significant demethylation of the hypermethylated CpG (Cytosine phosphate guanine) sites in the MGMT promoter. A significant decrease in dimethylated H3K9 (Dimethylation of lysine 9 on histone 3) levels in the MGMT promoter in DNA hypo- and hypermethylated HCT-116^G13D and SW-620^G12V cells was observed after treatment. In the MNU (N-methyl-N-nitrosourea)-induced CRC in vivo model, PMBA instillation restricted and repressed polyp formation, suppressed tumor proliferation marker Ki67 (Marker of proliferation), ablated KRAS-associated cytokine signaling, and decreased mortality. Clinical trial data for the parent molecule revealed its effectiveness against the disease, oral bioavailability, and system tolerance. Comprehensively, PMBA represents a new class of KRAS inhibitors having a therapeutic window in the scope of a drug candidate. The findings suggest that the PMBA analogue could inhibit the growth of human CRC in vivo through downregulation of cancer-associated biomarkers as well as reactivate expression of the MGMT gene associated with increased H3K9 acetylation and H3K4 methylation with facilitated transcriptional activation, which might be important in silencing of genes associated with upregulation in the activity of KRAS.

The health care system, worldwide, has achieved a commendable feat except in cancer therapy wherein mortality, till date, has not dwindled to the desired rate. Among the various types of the disease, colorectal cancer (CRC), with a prevalence of 10.2% and almost the same mortality, stands out at the third position, just after lung cancer and breast cancer (WHO, 2018).¹ The worrying scenario is the exacerbating trend of the disease, which is expected to increase by 60% in the next 15 years, affecting 13 million people by 2030.² CRC has been shown to develop from a stem cell or a stem cell-like cell residing at the base of the colon crypts due to the standalone or combinatorial effect of the following: (i) mutations (in oncogenes and tumor suppressor genes); (ii) genomic instability; (iii) epigenetic alterations; and (iv) aberrations in signaling pathways such as WNT (Wingless-related integration site), MAPK/PI3K (Mitogen-activated protein kinase/Phosphatidyl inositol 3-kinase), TGF-β (Transforming growth factor beta), and TP53 (Tumor protein P53), which arise mostly due to mutations in different genes such as c-MYC (Cellular myelocytomatosis oncogene), BRAF (Rapidly accelerated fibrosarcoma B-type), PIK3CA (Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha), PTEN (Phosphatase and tension homolog deleted on chromosome 10), SMAD2 (Suppressor of mothers against decapentaplegic 2), SMAD4 (Suppressor of mothers against decapentaplegic 4), and RAS (Rat sarcoma virus). Association of genomic instability with microsatellite instability (MSI) and p53 mutation, CpG island methylator phenotype (CIMP), methylation of MLH1 (MutL homolog 1), MGMT (O⁶-methylguanine-DNA methyltransferase), WRN (Werner syndrome ATP-dependent helicase), prognostic biomarkers such as LINE-1 (Long interspersed nuclear elements) hypomethylation, and chromosomal instability (CIN) represent up to 80–85% of the root of all CRC cases.³ Mutant KRAS has been found to constitute about 35–45% of all CRCs. Its 12th and 13th codons, with 80 and 15% prevalence values, respectively, account for about 95% of all mutation genre and are considered as dyad hotspots, while mutations in other codons such as 61, 146, and 154 occur to a lesser degree (5%).^4,5 RASCAL (Kirsten ras in-colorectal-cancer collaborative group) analysis has revealed distinct mutation in the KRAS gene at codon 12 in 8.6% of all patients with CRC and a further increase in recurrence or death by 30%. Different mutations pertaining to KRAS such as that of guanine (G) to thymidine (T) escalates the risk of death by 44%, while the mutation of glycine to valine at the 12th codon has been found in about 10% of the patients and is considered as an independent risk factor for recurrence and death.⁵ The catalogue of somatic mutations in cancer database (COSMIC) has associated more than 5000 mutations in the KRAS gene with CRC.⁶ Thus, KRAS mutations not only are associated with CRCs but also present an increased risk of relapse and death also. Various platforms have been developed relating to phenotype screening, a personalized approach to improved detection and mortality statistics. An array of different combinations of cytotoxic and biologic drugs have been studied and used in the treatment of metastatic CRCs. Clinical trials have been acquired by integrating different cytotoxic drugs, such as a phase I study of REOLYSIN in combination with FOLFIRI (Folinic acid, Flluorouracil, and Irinotecan) and Bevacizumab in FOLFIRI naive patients and a phase 2a study of BAX69 (Imalumab) and 5-FU (Flluorouracil)/Leucovorin or anitumumab (NCT01274624). In spite of these advances, numerous patients with progressed and metastatic tumors capitulate to the disease due to plasticity of the phenotype caused by epigeneticity. Considering the fact that so many KRAS mutations are associated with the development of CRC, development of a specific and selective therapy against the mutant KRAS-induced CRC is an urgent need of the hour. The treatment of the disease is further compounded by the development of resistance to almost all the chemotherapeutic drugs screened so far. To ameliorate these complications, the current research focuses on the integrative approach by studying the natural compounds that could provide effective treatment with fewer undesirable effects. In this context, we report the effect of a lead compound, PMBA (2-Pyridin-4-yl-methylene-beta-boswellic acid) (Figure S1), screened from among the 21 novel series of semi-synthetic analogues of β-boswellic acid that we had developed previously, on the treatment of CRC. The novel analogue showed several anticancerous activities such as inhibition of colony formation in HCT-116 (KRAS^G13D) and induction of apoptosis.⁷ There was no testimony on its KRAS potential and effect on the downstream signaling cascade. Here, we report, for the first time, the repression of KRAS-mutated CRC in both in vitro and in vivo models by PMBA involving the RAS/RAF/MEK/ERK (Rat sarcoma virus/Rapidly accelerated fibrosarcoma/Mitogen-activated protein kinase/Extracellular signal-regulated kinase) signaling cascade with prioritization using genomic features of KRAS mutant cancer cells, which is a step toward personalized medicine.

Methodologies

Cell Culture

The panel of NCI-60 human cancer cell lines was sourced from the NCI-Frederick cancer DCTD. FR-2, HEK-293, and HGF normal cell lines were sourced from ECACC (European collection of cell cultures) and ATCC (American type culture collection). Cell line authentication was performed through information provided by the cell line repository. The lines were maintained and cultured under standard culture conditions, as prescribed by the provider, and were utilized before passage 20, preferably between 50% and 75% confluency.

Sulforhodamine-B Assay

Cells were seeded into 96-well microtiter plates at densities ranging from 5000 to 10,000 cells/100 μL/well, depending on the doubling time of the individual cell line. After 24 h, PMBA was added to the cultures (at a seven-point dose scale using a 2-fold dilution), and further incubation was performed for 24, 48, and 72 h to examine the inhibition using an SRB (Sulforhodamine B) dye assay. Paclitaxel (Sigma-Aldrich) was used as a positive control, and the assay was performed in biological quadruplicates according to the previously mentioned protocol.⁸

Bromodeoxyuridine Proliferation Assay

2 × 10⁴ cells were cultured in 96-well plates and treated separately with the following: (i) PMBA (1.7 μM); (ii) 5-FU (2.2 μM); and (iii) PMBA + 5-FU (1.7 + 14.7 and 2.2 + 78 μM, respectively) for 48 h. Proliferation indices for HCT-116^G13D and SW-620^G12V were analyzed using the colorimetric bromodeoxyuridine (BrdU) kit (BioVision, k306) according to the manufacturer’s instructions.

KRAS Expression Validation through Mass Spectrometry

All MS (Mass spectrometry) experiments were carried out on an Agilent 6540 high-resolution MS system equipped with an Agilent 1260 series. The protein mixture was directly injected into the electrospray ionization source and eluted through a union using a 0.5 mL min^–1 flow with an isocratic system (70% of B and 30% of A; eluent A being 0.1% formic acid in water and eluent B being acetonitrile). The MS acquisition parameters were as follows: 4 kV capillary voltage, 300 °C gas temperature, 12 L min^–1 drying gas flow, and 35 psi nebulizer pressure. The scan source parameters for the skimmer, fragmenter, and octopole RF peaks were at 65, 175, and 700 V, respectively. The data were acquired in both profile and centroid modes with a mass scan range of 100–3000 m/z. The mass spectra were deconvoluted using Agilent Mass hunter software at maximum entropy, with a 10,000–170,000 mass range and a mass step of 1 Da.⁹

In Vitro RAS Activation Assay

Levels of active GTP (Guanosine-5′-triphosphate)-loaded RAS were determined via a GST-RAF1-RBD (Glutathione-S-transferase-rapidly accelerated fibrosarcoma-ras binding domain) pull-down assay (Merck Millipore, 17-218). In brief, GST-RAF1-RBD fusion proteins were incubated with glutathione agarose beads, and cell lysates were collected in lysis buffer, as recommended by the manufacturer. Bound proteins were eluted with SDS (Sodium dodecyl sulfate) sample buffer and analyzed via the western blot for RAS protein levels, according to the manufacturer’s instructions.

In Silico Analysis of PMBA Binding for KRAS Protein

The crystal structures of KRAS and its mutant forms were selected from the Protein Data Bank (PDB) database (PDB entries: 4TQA, 4TQ9, 4OBE, and 5VPI) on the basis of resolution, missing residues, and ligands. The protein structures were prepared for docking, using the protein preparation wizard in Maestro software provided by Schrödinger. Sequential steps involving preprocessing, optimization, and minimization of crystal structures were performed. Afterward, molecular docking was performed by generating a grid using default settings in the receptor grid generation panel of Glide.

Molecular Dynamics Simulations

The simulations were performed for a period of 50 ns by adopting sequential steps of the conventional MD simulation process. It comprised three steps, namely, system builder, minimization, and molecular dynamics simulations using Desmond. Initially, the protein ligand complexes were immersed in an orthorhombic box containing single point charge (SPC) water molecules. Counter ions and 0.15 M NaCl were added to neutralize the system. The prepared system was minimized using the steepest descent and LBGFS (Llyod–Broyden–Fletcher–Goldfarb–Shanno) method with a convergence threshold of 2.0 kcal/mol and 1000 iterations to remove steric clashes. The molecular dynamics protocol devised by the D. E. Shaw group was used for running simulations. Temperature and pressure were controlled using the Nose Hoover chain method and Martyna–Tobias–Klein method, respectively.

Global Methylation Assay

HCT-116^G13D and SW-620^G12V cells were assayed for global methylation using an MDQ1 imprint methylated DNA quantification kit (Sigma-Aldrich, Germany). Quantification was performed by calculating the number of methylated cytosine in the sample (5mC) relative to global cytidine (5mC + dC) residues in the previously methylated positive control.

Immunoblotting

HCT-116^G13D and SW-620^G12V cells were treated with PMBA, 5-FU, or both at 1.7/2.2, 14.7/78 μM for 48 h in the presence and absence of EGF (Epidermal growth factor) (10 ng/mL). The treated cells were lysed in RIPA (Radioimmunoprecipitation assay) lysis buffer (Sigma, R0278), supplemented with a protease inhibitor mixture (Promega, G6521) and a phosphatase inhibitor mix (Thermo, 78420). Lysates were centrifuged at 15,000 rpm for 15 min, and protein concentration was determined using a Coomassie protein assay (Thermo Scientific, 1856209). Equal amounts of protein were subjected to SDS–polyacrylamide gel electrophoresis analysis and transferred to the polyvinylidene fluoride membrane (Millipore, IPVH00010). After blocking with 5% non-fat milk–TBS or 3% BSA–TBS (for phosphorylated antibodies) in blocking buffer, membranes were incubated overnight at 4 °C with the specific primary antibodies [P21 (CST #2947), P27 (CST #3686), cyclin E1 (CST #20808), cyclin A2 (CST #4656), CDK2 (CST #2546), CDC25A (CST #3652), PRB-795 (CST #9301), ATM-Ser1981 (CST #13050), p-CDK2-Thr160 (CST #2561), p-CHK1-Ser345 (CST #2348), CDK4 (CST #12790), EGFR (CST #4267), EREG (CST #12048), AKT (CST #4685), Erk1/2 (CST #4695), E2F1 (CST #3742), STAT3 (CST #4904), p-CHK2-Thr68 (CST #2661), p-P53-Ser15 (CST #9284), p-AKT-Ser473 (CST #4060), p-ERK1/2-Thr/Tyr204 (CST #4370), p-STAT-3-Tyr705 (CST #9145), p-MEK1/2-Ser217/221 (CST #9154), and p-P90RSK-Ser380 (CST #11989)] and subsequently incubated with secondary antibodies [anti-mouse IgG (CST #7076) and anti-rabbit IgG (CST #7074)]. After incubation, the membranes were treated with a chemiluminescent HRP substrate and exposed to X-ray film, as per the already established protocol.⁷

Cell Cycle Analysis

HCT-116^G13D and SW-620^G12V cells treated with PMBA, 5-FU, or both at (i) 1.4, 14.7, 1.7 + 14.7 μM and (ii) 2.2, 78, 2.2 + 78 μM, respectively, for 48 h in the presence of EGF (10 ng/mL) were analyzed for the cell cycle stage via flow cytometry, as per the already established protocol.¹⁰

Methylation Profiling Assay

Genomic DNA from HCT-116^G13D and SW-620^G12V cell lines was separately extracted using a Wizard Genomic DNA isolation kit (Promega, A1120) according to the manufacturer’s instructions. 2 μg of the genomic DNA was bisulfite-modified using a MethylEdge Bisulfite Conversion System (Promega, N1301). Methylation-specific polymerase chain reaction (MSP) was performed using the specific primers from a CpG WIZ amplification kit (Merck, S7803) for the fragment located in the promoter region of the MGMT gene (Entrez Gene Number: NM_002412.2, Genbank accession no: U95038). PCR products were separated via electrophoresis on 2% agarose gels and photographed using GeneSnap from SynGene. Methylation status was measured as the ratio of the signal from a methylated probe relative to both methylated and unmethylated probe signals.

Chromatin Immunoprecipitation Assay

Chromatin immunoprecipitation assay (ChIP) experiments for HT-29^WT, HCT-116^G13D, and SW-620^G12V cell lines were carried out using a SimpleChIP plus enzymatic chromatin IP kit (Cell Signaling Technology). Briefly, chromatin from fixed cells was fragmented to a size range of 150–900 bases with a Sartorius LABSONIC M sonicator. Solubilized chromatin was immunoprecipitated with antibodies against H3K9me2 (CST #4658), H3K4me2 (CST #9725), H3K9ac (CST #9649), and histone-H3 (CST #4620). Antibody–chromatin complexes were pulled-down using protein G-agarose contained salmon sperm DNA, washed, and then eluted. After cross-link reversal and Proteinase K treatment, immunoprecipitated DNA was quantified using qRT-PCR (Quantitative reverse transcription polymerase chain reaction) according to the already established protocol.

Histone Extraction

HCT-116^G13D and SW-620^G12V cell lines, treated as described above, were lysed in RIPA buffer containing SDS. After incubating in ice for 10 min, the cell pellets were washed with Tris (1 M) ethylenediamine tetraacetic acid buffer (pH 8), centrifuged at 10,000×g for 5 min at 4 °C, and then resuspended in 0.4 N H₂SO₄. After overnight incubation at 4 °C, the supernatant was collected via centrifugation at 14,000g for 15 min, mixed with cold acetone, and kept at −20 °C overnight. Histone pellets were collected at 14,000g for 15 min, washed with 0.5 mL of acetone, and dried under the hood for 30 min. The dried pellets were suspended in water and estimated using a Bradford assay.

RT-PCR Analysis of Immunoprecipitated DNA

RT-PCR reactions were carried out using 2 μL of immunoprecipitated DNA, a negative control, and a DNA input control. Three pairs of primers (GenBank: NT_008818.15), based on a previous study and located in the MGMT promoter region, were used for ChIP–PCR (Tables S12 and S13). The levels of histone modifications in each immunoprecipitation process were measured by quantifying immunoprecipitated DNA versus the DNA input control through qRT-PCR.¹¹

Animals

Wistar rats (100–110 g) were procured and maintained at the institutional facility with approval from the Institutional Animals Ethics Committee (IAEC [85/2/16]) of CSIR-Indian Institute of Integrative Medicine, Jammu, India. All methods were performed in accordance with the relevant guidelines and regulations published by the Institutional Animals Ethics Committee and ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. The animals were reared under standard laboratory conditions, that is, a temperature of 25 ± 2 °C, a relative humidity of 65–75%, and a photoperiod of 12:12 h (Light/Dark), and were provided with pellets and water ad libitum.

Test Inhibitors

PMBA was previously prepared in the laboratory as a suspension in normal saline (NS) at various concentrations and checked for its purity and integrity.⁷ 5-FU, purchased from Sigma-Aldrich, was prepared in NS. PMBA, 5-FU, PMBA + 5-FU, and vehicle control dosing solutions were prepared as and when needed.

MNU-Induced Rat CRC Model

The tumor model was developed as described previously.^12,13 The experiments were conducted in accordance with Institutional Animal Ethics Committee (IAEC) approval no. 85/2/16 of CSIR-Indian Institute of Integrative Medicine, Jammu, India. The orthotopic carcinogen-induced rat colorectal model was used to evaluate the anticancer efficacy of PMBA, 5-FU, and both. Healthy 5–8 week old wistar male rats (BW 125–135 g) at six animals per group were taken for the study and given intrarectal instillation of 0.5 mL of freshly prepared 0.4% aqueous solution of MNU three times weekly for 4 weeks for development of colorectal tumors. Treatment was started 22 weeks after MNU administration when colorectal tumor development was confirmed. The control group received 0.9% NS, the positive control group received 5-FU at a concentration of 25 mg/kg BW, while the test groups received PMBA (50 mg/kg) and PMBA + 5-FU (50 + 25 mg/kg) formulations at alternate days for 4 weeks. The experiment was terminated at the 25th week, and animals were sacrificed using the cervical dislocation method. The unexpurgated large bowls of the animals were dissected cautiously and observed for the shape, size, and location of the tumor.

Cytokine Expression Estimation

IL-2 (Interleukin-2), IL-8 (Interleukin-8), IFN-γ (Interferon gamma), TNF-α (Tumor necrosis factor alpha), IL-6 (Interleukin-6), TGF-β (Transforming growth factor beta), and IL-1β (Interleukin-1 beta) expression was measured in serum isolated from the blood of the orthotropic carcinogenic induced animal model. ELISA (Enzyme-linked immunoassay) was performed as per the protocol of the commercially available kits (R&D Systems).

Histology and Immunohistochemistry

Colon sections were isolated and rinsed in ice-cold PBS, fixed in 10% neutral buffered formalin overnight at 4 °C, dehydrated, and embedded in paraffin. Sections (5 mm) were stained with hematoxylin and eosin (H&E), deparaffinized, rehydrated, and then treated for antigen retrieval. After blocking in protein block serum-free solution, tissues were incubated with the primary antibody overnight at 4 °C, followed by incubation with the Alexa-conjugated secondary antibody (KRAS #53270, caspase 3 #9661, Ki67 #9129) and examined using a confocal laser scanning microscope (Olympus fluoview fv1000).

In Vivo Acute Toxicity Study

Healthy 5–8 week old male Wistar rats weighing 125–130 g were randomly assigned to treatment and control groups, with each group having five animals. Dose formulation of the test compound was prepared in 2% gum acacia suspension. PMBA, 5-FU, or both were administered to the fasted (10 h) animals via oral gavage. Control animals received only the vehicle for dose preparation. All the animals were observed daily for clinical signs of toxicity, mortality, and food and water consumption. Body weights of each animal were measured before the start of the experiment and then on a weekly basis. After 14 days, blood samples were collected from both the groups of overnight fasted animals for hematological and biochemical analysis and sacrificed after 14 days of dosing. Hematological parameters including total red blood cell (RBC) count, hemoglobin (Hb) concentration, hematocrit, mean corpuscular volume, mean corpuscular Hb concentration , platelet count, total white blood cell (WBC) count, and differential WBC counts such as neutrophil, lymphocyte, eosinophil, monocyte, and basophil were examined using automated hematology analyzers (XT1800i, Sysmex, USA). Serum biochemistry parameters, namely, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), creatinine, glucose, total protein, urea, total bilirubin, cholesterol, triglycerides, and uric acid, were determined using an automated clinical chemistry analyzer (EM360, Erba Mannheim group, Germany). Statistical analysis was performed between the treatment and control groups using Student’s t-test where the P-value of less than 0.05 was considered significant. The present study was performed according to the “guideline for the acute oral toxicity study in rodents” provided by the Organization for Economic Co-operation and Development.¹⁴

Quantification and Statistical Analysis

The ratio results were expressed as mean ± standard error mean (SEM). Significance between controls and treated samples was calculated using Student’s t-tests. Significance between controls in different cell lines was calculated using one-way and two-way ANOVA (Analysis of variance) and t-tests. Statistical calculations were performed using GraphPAD prism version 5.0 (Chicago, USA). All analyses used P < 0.05, P < 0.01, P < 0.001, and P < 0.0001 for determining significance, marked as *, **, ***, and ****, respectively. For two-group comparisons with normally distributed data, two-sided t-tests were used. In boxplots, the line and box represent, respectively, the median value ±25th and 75th percentiles or the interquartile range.

Software and Computational Methods

Comprehensive comparative data hosted in the Gene Expression Omnibus (GEO) by NCBI under accession number GSE108464. Molecular docking and modeling were performed using Glide version V 11.4 (Schrodinger). The chemical structure was drawn using Chembio Draw Ultra version 10.0 (PerkinElmer). All statistical analyses, potency determinations, and inhibition curves were produced using Prism 5.0 (GraphPad Software) and GraphPad Instat3 version 3.05. All Immunohistochemistry images were quantified using ImageJ (Version 1.48).

Results

PMBA Inhibits Cancer Cell Line Proliferation

The cell-based colorimetric SRB assay was conducted on the authenticated tumor cell lines with diverse cell lineage annotated gene mutations as extracted information from the Sanger institute COSMIC database, namely, A-431, MDAMB-468, KM-12, HCT-116, SW-620, COLO-205, HCC2998, A-498, 786-O, ACHN, SF-268, SF-295, A-549, EKVX, HOP-92, MDA MB-435, LOXIMVI, SK MEL-2, PC-3, DU-145, OVCAR-5, MCF-7, and T47D (NCI-DCTD, USA). The half maximal inhibitory concentration (IC₅₀) values were interpolated from a seven-point scale (2-fold dilution) dose–response curve using nonlinear regression. Treatment with PMBA resulted in IC₅₀ values in the range of 1.7–30 μM on the cancer cell line panel (Figure 1A and Table S1). At 48 h, PMBA in the concentration range of 1.7–29 μM exhibited potent inhibitory activity on various colon cancer cell lines (Figure 1B,C and Table S2). Using the average of the tumor cell IC₅₀ values, PMBA was found to be more potent on the tumor cell lines than on the normal epithelial and fibroblast cells as inferred from the selectivity index (Table S3).

Figure 1.

Cellular activity and selectivity of PMBA. (A) A total of 24 cancer cell lines were profiled for sensitivity to PMBA in cell inhibition studies as illustrated. Half maximal inhibitory concentration (IC₅₀, μM) values were determined using a seven-parameter fit via nonlinear regression analysis. (B) KRAS mutant (red) and WT colon cancer cell lines (green) were profiled against the selective inhibition by PMBA. Error bars represent the mean of three measurements ± SEM of mean. (C) Dose- and time-dependent response of PMBA and 5-FU on cell inhibition in HCT-116 and SW-620 (KRAS^G13D, G12V) mutant cell lines. The mean differences are plotted as a line gradient colored graph. Mean (n = 3) ± SEM. (D) KRAS mutant HCT-116^G13D and SW-620^G12V cells were cultured with EGF (10 ng/mL) for 24 h and subsequently treated for an additional 48 h with PMBA, 5-FU, and the combination, in which the level of active KRAS (GTP-KRAS) was determined using a KRAS activation assay. Western blotting subsequently monitored KRAS-GTP via pull-down signaling, showing dose response of PMBA, 5-FU, or both. (n = 3, ns; **P < 0.01; and ***P < 0.001).

PMBA Selectively Restricts KRAS-Mutant CRC Cell Proliferation

The COSMIC data have been accumulated to determine the functional importance of PMBA in KRAS mutant CRC cell lines as compared to that in KRAS wild-type cancer cell lines (Figure 1B). PMBA suppressed the cellular growth (*P ≤ 0.5) in 3 KRAS mutant CRC cell lines (HCT-116, HCT-15, HCC 2998, and SW-620), while it had a lower effect in three KRAS wild-type CRC cell lines (COLO205, HT-29, and KM-12) (Figure 1B and Table S2), suggesting that PMBA selectively targets KRAS mutant CRC cells and preferentially inhibits the growth of EGFR- or ErbB-2-overexpressing cell lines. The IC₅₀ values for KRAS WT cell lines were 23.18, 25.89, and 27.43 μM, and were found to be ∼10–16 fold more than the IC₅₀ values for the KRAS mutant colon cancer cell lines for 48 h treatment period, while the corresponding IC₅₀ values for 5-FU treatment (14 and 78 μM) were found to be 8.6- and 35.4-fold higher, respectively (Figure 1C). The selective effect of PMBA was further validated using an active RAS pull-down assay (Figure 1D) and MS, in which the acquired spectrum designated a well-resolved charge state series corresponding to KRAS (G13D and G12V) (Figure S2).

PMBA Inhibits KRAS Mutant CRC via Apoptosis and Cell Cycle Arrest

Following 48 h of PMBA treatment, a considerable increase in the percentage of HCT-116^G13D (at a PMBA concentration of 1.7 μM) and SW-620^G12V (at a PMBA concentration of 2.2 μM) cells arrested in the S phase was observed (Figure 2A), whereas KRAS wild-type HT-29 cells (at a PMBA concentration of 25.8 μM) were arrested in the G1 phase. A decrease in the S phase population of the cell cycle by 56% in HCT-116^G13D and 61% in SW-620^G12V cells was observed as compared to 75% in the combination treatment of PMBA + 5-FU (1.7 + 14.7 and 2.2 + 78 μM, respectively), correlating with the antiproliferative effects observed in these cells, in response to the downregulation of key S phase cell cycle regulators, namely, CDK2 and cyclin A/E validated through western blotting (Figure 2C). Hence, PMBA halted cell proliferation in KRAS mutant CRC by inducing apoptosis and cell cycle arrest (Table S5). Using the diverse CRC cell line panel, a clear relationship between PMBA sensitivity to MSI and microsatellite stable (MSS) (GSE39582 and GSE92921) subsets of KRAS mutated colon cell lines reflected an objective categorization of the response (Figure 2D and Table S6).

Figure 2.

PMBA inhibits KRAS mutant CRC cell growth and proliferation. (A) Quantitative flow cytometric analysis of cell cycle distribution in HCT-116^G13D and SW-620^G12V cells treated with DMSO, 5-FU (14.7, 78 μmol/L), PMBA (1.7, 2.2 μmol/L), or the combination, showing increased S phase arrest in contrast to KRAS^WT arrested in the G1 phase. n = 2, ns,*P < 0.05; and **P < 0 0.01. (B) Quantitative proliferation analysis (BrdU-fluorescein isothiocyanate) is shown for both cell lines. Cells were resynchronized via serum starvation for 48 h, released in 10% FBS medium in the presence of PMBA, 5-FU, or the combination, and incubated with BrdU for 4 h with an effective decrease in the proliferation rate of both the mutant cell lines. n = 3, Tukey’s post-test (ANOVA) *P < 0.05; **P < 0.01; and ***P < 0.001. (C) Representative western blot analysis showing the intracellular signaling altered by PMBA, 5-FU, or both in HCT-116^G13D and SW-620^G12V cells, as shown by active phosphorylated checkpoint kinases and cell cycle proteins ATM (Ser1981), p21, p27, CDK4, CDK6, CDK2, p-CDK2 (Thr160), p-CHK1 (Ser345), p-CHK2 (Thr68), cyclin A2 and E1, p-P53 (Ser15), pRB (795), CDC25A, and p-CDC2 with β-actin as a loading control. (D) Mechanism of defect in DNA repair and/or replication (MSS) ultimately leads to alteration in the chromosome alteration profile (MSI), resulting in genomic instability.

PMBA Administration Reduces KRAS Cancer Cell Population Expansion

HT-29^WT, HCT-116^G13D, and SW-620^G12V cells were treated with PMBA (25.8, 1.7, and 2.2 μM), 5-FU (82.4, 14.7, and 78 μM) and both PMBA + 5-FU (25.8 + 82.4, 1.7 + 14.7, and 2.2 + 78 μM) at indicated concentrations for 48 h. The expansion in growth curves was seen in control and treated cells after BrdU (BioVision) addition and detected at an absorbance of 450 nm. After the treatment, a statistically (***P ≤ 0.001) significant dose-responsive decrease in the number of HCT-116^G13D cells and SW-620^G12V cells was observed, as compared to HT-29^WT cells (**P ≤ 0.01), where it was more distinct with increasing rounds of replication (data are represented as population doublings with time, and statistical evaluation was conducted on the total cell number). The reduction in the proliferation rate was followed by a successive inclination as the concentration peaked up over the cell lines (Figure 2B).

PMBA Downregulates KRAS-Induced MAPK/ERK Signaling

To further explore the dependency of MEK and AKT events in KRAS^G13D, G12V cell-autonomous signaling, KRASWT-HT-29 and KRASG13D, G12V-HCT-116, and SW-620 colon cancer cells, treated with PMBA (25.8, 1.7, and 2.2 μM), 5-FU (82.4, 14.7, and 78 μM), and both PMBA and 5-FU (25.8 + 82.4, 1.7 + 14.7, and 2.2 + 78 μM) for 48 h showed decreased expression of various downstream signaling effectors of the MAPK pathway (Figure 3A). PMBA, thereby, acts to control the differential phospho-proteome of KRAS^G13D, G12V. Collectively, these observations revealed that cell-autonomous KRAS modulates a disparate section of the tumor cell phospho-proteome via MEK-ERK to regulate the AKT expression, which was further confirmed through the STRING protein–protein interaction database having a confidence score range between 0.4 and 0.9 (Figure 3B). Getting the highest possible confidence score with EGFR (0.99) for PMBA, 5-FU, and both PMBA + 5-FU, the dose response was obtained using 4 ng of EGFR to determine the potency of the inhibitors (IC₅₀) on the EGFR kinase domain (n = 3, ***P < 0.001) (Table S6).

Figure 3.

Inhibition of mutant KRAS G13D, G12V in active cells. (A) Western blot analysis showing the downstream effectors altered by PMBA, 5-FU, or both in HCT-116^G13D and SW-620^G12V, as shown by active phosphorylated checkpoint kinases and cell cycle proteins, p-AKT (Ser473), and activated protein kinase cascades p-ERK1/2 (Thr202/Tyr204) and p-STAT-3 (Tyr705), p-MEK (Ser217), and p-p90RSK (Ser380). β-Actin was used as a loading control. (B) Protein–protein interaction database STRING was utilized to search the KRAS protein interactions in the metabolic map in the KEGG database. Proteins differentially expressed in HCT-116^G13D and SW-620^G12V are highly interactive. (C,D) In silico interpretation displayed interaction analysis of PMBA with various amino acid residues in KRAS G12V and G13D. (E) Binding mode of PMBA and KRAS^G13D,G12V from the co-crystal structure.

PMBA Specifically Targets KRAS^G13D, G12V at Its Guanosine Diphosphate Site

Molecular docking studies were performed for KRAS native and mutant forms at the GDP/GTP site. PMBA shares precise binding affinity scores for native as well as mutant forms at the GDP site. The binding affinity scores for the GTP site were comparatively lower than those at the GDP site (Tables S7-1 and S7-2).

Interaction Analysis of PMBA with KRAS Structural Transitions for the GDP Site

Major interaction formed in native KRAS was found to be due to direct hydrogen bonding and ionic interactions, while PMBA majorly interacted to the native KRAS enzyme through hydrophobic contacts and water-mediated hydrogen bonding. Comparatively, a large number of contacts contribute to a stable trajectory for PMBA and the native (4OBE and 5VPI) KRAS enzyme. Phe 28 (phenylalanine 28), Pro 34 (proline 34), and Lys 147 (lysine 147) were found to be important interacting residues (Figures S2 and S5). The pyridyl ring of PMBA showed Π–Π stacking with Phe 28, hydrogen bonding with Asn116 (asparagine 116), and Π-cation bonding with Lys 117 in the G12V KRAS mutant (Figure 3C). The pyridyl ring was oriented inside the KRAS G12V mutant, while in the case of the G13D mutant, it is oriented outside the protein. The critical residues such as Phe 28, Asn 116, Lys 117, and 147 showed interactions in the G12V mutant unlike those in the G13D mutant (Figure 3D). This renders selectivity to PMBA while gaining an extra edge in inhibition to G12V against the G13D KRAS mutant. The mode of PMBA binding in the switch I region of KRAS protein (PDB ID: 4OBE) is shown in Figure 3E.

For the GTP Site

The amino acid residues involved in native KRAS protein binding to PMBA were Lys 16, Ser 17, Phe 28, Tyr 32, Asp 33, and Tyr 64 (Figure S5). In the G12V mutant form, loss of contacts with key amino acid residues at the GTP binding site was observed (Figure S6). The frequency of contacts with key amino acid residues in the native protein was hydrophobic,within significant water bridged bonding observed at residues such as Gly 25 (Glycine 25), Asn 26 (Asparagine 26), and His 27 (Histidine 27). However, in the G13D mutant form, key interactions were retained, and significant hydrophobic interactions were observed with binding acid residues (Figure S7). The orientation of the pyridyl ring at the GTP site of the native KRAS protein had Π–Π stacking interactions with Phe 28 (Phenylalanine 28) and Tyr 32 (Tyrosine 32), while no such interactions were observed in mutant forms. Suggestive from docking and molecular dynamics simulations, PMBA interacts through hydrophobic contacts with the KRAS protein.

Molecular Dynamics Simulations

The root mean square deviation (RMSD) of all complexes converged to a stable trajectory, suggesting that a 50 ns time period was enough for observing conformational changes in KRAS native and mutant forms. The RMSDs of the complexes have been presented in Figures S2–S7 and are in the range between 1.5 and 3 Å. Root mean square fluctuation for side chains of all complexes was found to be in range, that is, terminal residues were found to be more flexible than deeply buried residues. Superimposed conformations of protein ligand complexes for G13D as well as G12V mutant forms have been depicted for initial and final frames of MD simulations (Figure S8). For the G13D mutant, the RMSD for superimposed conformations was found to be 3.06 Å, and for G12V, it was found to be 1.89 Å.

Correlation of KRAS with MGMT Promoter Methylation in Transcriptional Silencing

A comprehensive methylome analysis of a CIMP in colon adenocarcinoma harboring an MGMT mutation in KRAS mutant cell line HCT-116^G13D provided by the UCSC (University of California Santa Cruz) (http://genome.ucsc.edu/) was performed (Figure 4A). The methylation status of CRC subtypes—MSS (SW-620) and MSI (HCT-116)—in KRAS mutant colon cancer has been analyzed (Figure 4B). MSP analysis of DNA methylation at the promoter region of MGMT revealed hypermethylation of its both alleles in SW-620^G12V cells, while only one allele was methylated in HCT-116^G13D cells (partially methylated) (Figure 4C). The MSP assay exhibited distinct variability in CpG methylation of the MGMT promoter between SW-620^G12V, a methylated band (lanes indicated by M), and HCT-116^G13D cells, a partially methylated band (lanes indicated by U). SW-620^G12V and HCT-116^G13D cells upon treatment with PMBA (1.7 and 2.2 μM), 5-FU (14.7 and 78 μM), and PMBA + 5-FU (1.7 + 14.7 and 2.2 + 78 μM) showed significant reduction in MGMT promoter methylation (Figure 4C) and restored MGMT mRNA expression via significant demethylation of the hypermethylated CpG sites in the MGMT promoter. 5-FU alone had no significant effect on DNA methylation in the KRAS mutant cell lines, while treatment with both agents has an additional impact on DNA demethylation.

Figure 4.

Focal methylation corresponding to distinct epigenomic signatures. (A) Epigenetic landscape of region 21 (10q21.3-q22.1) in HCT-116^G13D cells. The UCSC Genome Browser plot of downregulated MGMT genes reveals that the methylation pattern (Regions of focal DNA hypermethylation and long-range hypomethylation in colorectal cancer coincide with nuclear lamina-associated domains, Berman et al. 2011) around colon cancer long-range epigenetic silencing often coincide with a combination of promoter or enhancer histone modifications (H3K4 methylation), DNase I hypersensitivity, and transcription-factor binding. In the enhancer and promoter tracks, each color represents an individual ENCODE cell line, and all cell lines were combined in the DNase H and transcription factor tracks. Genome browser representations of H3K4me1, H3K27ac, and H3K4me3 enriched the profile in HESC and HUVEC cell lines, where methylation prone elements correspond to known promoters from start sites and as regions with simultaneous enrichment of histone modification of H3K4me1 and H3K27ac. This region is already silent in HCT-116^G13D cells but is remodeled up on treatment with PMBA and both PMBA + 5-FU with a loss of H3K27me3 and a gain in DNA methylation. Gene expression changes for genes within MGMT region 21 in colon cancer and adjacent normal tissue. (B) Reduced expression of consecutive genes in HCT-116^G13D across MGMT region 21 from experiment data (detection range: [0–0.6]) (session name: hg19_chr10_Human). (C) Global DNA methylation level in the DNA pattern in HCT-116^G13D and SW-620^G12V relative to the methylated control. Bisulfite converted DNA from HCT-116^G13D and SW-620^G12V cells treated with PMBA, 5-FU, or both for 48 h were found to be lower in PMBA and combinational treatment, as quantitatively determined using a methylated DNA quantification assay. Box plot: median, 25th and 75th percentiles, whiskers extend to the minimum and maximum values; n = 3 for each cell line; ***P < 0.001. (D) MSP detected MGMT (GeneBank accession no. NM_002412.2) methylation status before and after the addition of PMBA, 5-FU, or both in different concentrations. A PCR product in lanes U illustrates the methylation status of all the loci analyzed in an MSI cell line (HCT-116^G13D), whereas the left panel shows the methylation status of the representative MSS cell line (SW-620^G12V). Abbreviations: NTC, normal treated control (containing water as the template); U, a lane for the unmethylated MSP product; and M, a lane for the methylated MSP product. The presence of 92-bp and 80-bp PCR products indicates the unmethylated and methylated final products. (E) Real-time RT-PCR analysis of MGMT mRNA expression before and after treatment of HT-29 ^WT, HCT-116^G13D, and SW-620^G12V cells with PMBA, 5-FU, or both. MGMT mRNA expression levels in each sample were normalized to GAPDH expression. Distribution of MGMT hypermethylation-associated inactivation in colorectal tumorigenesis as a function of the KRAS mutational spectrum. Results are shown as mean (n = 3) ± SEM. *P < 0.05; ***P < 0.001; and ns.

Status of MGMT Methylation and the mRNA Level in KRAS^WT,MUT (Wildtype and Mutant) Cell Lines

SW-620^G12V and HCT-116^G13D cells with low levels of MGMT mRNA expression were shown to have a high percentage of CpG methylation in the promoter, whereas HT-29^WT cells with high levels of MGMT expression had a low percentage of CpG methylation (Figure 4D).^15,16 Most colon cancer cells manifested a close inverse interaction between the expression level of MGMT and CpG methylation in the promoter. The results suggest that CpG sites in the MGMT promoter have an important role in CpG methylation-related gene silencing of MGMT, as detected by qRT-PCR and consistent with data (GSE5720) suggesting that about 30% of colon cancer cells are devoid of MGMT expression despite cancer-determined aberrant DNA methylation of the MGMT promoter.¹⁷

PMBA Restored MGMT Expression in KRAS Mutant Colon Cancer Cell Lines

To examine whether DNA demethylation or histone hyperacetylation could restore MGMT silencing, cells were treated with PMBA (1.7 and 2.2 μM), 5-FU (14.7 and 78 μM), and both PMBA + 5-FU (1.7 + 14.7 and 2.2 + 78 μM), respectively. In MGMT-negative SW-620^G12V and HCT-116^G13D cells, treatment with either PMBA or integrated treatment with both PMBA + 5-FU (1.7 + 14.7 and 2.2 + 78 μM) restored MGMT expression to a significantly considerable extent compared to that of treatment with 5-FU alone. In MGMT-positive HT-29^WT cells, treatment with PMBA (25.8 μM) and 5-FU (82.4 μM) or in combination PMBA + 5-FU (25.8 + 82.4 μM) had no significant effect on the expression of MGMT (Figure 4D).

Histone Modification under Different DNA Methylation States in the MGMT Promoter

The ChIP assay accompanied with qPCR for H3K9 dimethylation of the promoter regions in HT-29^WT cells (where MGMT was expressed) was found to be nominal compared to the levels (10–15-fold higher) in MGMT-negative HCT-116^G13D and SW-620^G12V colon cancer cell lines (Figure 5A). Di-methylation of H3K4 at the MGMT promoter region was remarkably higher in HT-29^WT cells (MGMT-positive) than in HCT-116^G13D and SW-620^G12V cells (MGMT-negative). H3K9 acetylation was similar to that of H3K4 dimethylation levels (Figure 5A–C).¹⁸

Figure 5.

ChIP assay demonstrating the relative enrichment of H3K4/H3K9me2 on the MGMT promoter in KRAS^G13D/G12V colon cancer cells. (A–C) A ChIP experiment using HCT-116^G13D and SW-620^G12V cells is shown. MGMT promoter regions in HCT-116^G13D and SW-620^G12V cells showed a high degree of H3K9me2 and a low degree of H3K9ac with gene inactivation by contrast; HT-29^WT showed a low degree of H3K9me2 and a high degree of H3K9ac and H3K4me2 in MGMT. Synergistic restoration of gene expression of H3K9ac and H3K4me2 by PMBA and PMBA + 5-FU-modulated MGMT expression. Ratios of precipitated DNA over input DNA were calculated as a relative precipitated fold shown below each sample. Antibodies against H3K9me2, H3K9ac, and H3K4me2 or no antibody was used. A fixed portion of the total input (0.2%) was also examined using qRT-PCR (not shown). Ratios of K9 methylation density over K9 acetylation density (K9 Me/Ac) were calculated for each cell line and are shown on the y-axis. Error bars represent SD (n = 2), Bonferroni test (ANOVA): *P < 0.05; **P < 0.01; and ***P < 0.001. (D) The linkage hierarchical clustering corresponds to the expression results showing increased methylated MGMT in KRAS mutant cell lines. (E) Representative western blots of acid-extracted proteins from whole cell lysates of HCT-116^G13D and SW-620^G12V cells before and after treatment with PMBA, 5-FU, or both, showing global levels of H3K9/H3K4me2 and HEK9ac. Relative average protein normalized to Histone H3 served as a loading control. Mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001; and ns. (F) Mechanistic illustration of S phase-specific downregulation of antimutagenic human DNA repair protein MGMT through its serendipitous epigenetic interaction with KRAS mutated colon cancer.

PMBA Alters Histone Modifications in MGMT Promoter CpG Islands

The levels of H3K9/H3K4 dimethylation and H3K9 acetylation in MGMT promoter CpG islands in untreated tumor cells were evaluated via ChIP and compared with those treated with PMBA (1.7/2.2 μmol/L), 5-FU (14.7/78 μmol/L), and PMBA + 5-FU (1.7 + 14.7 and 2.2 + 78 μM). Dimethylated H3K9 levels in the MGMT promoter in DNA hypo- and hypermethylated HCT-116^G13D and SW-620^G12V cells decreased significantly after treatment with PMBA and PMBA + 5-FU. In HCT-116^G13D cells, where the promoter showed a high expression level of both dimethylated H3K4 and acetylated H3K9, PMBA restored H3K4 dimethylation and H3K9 acetylation. The efficacy of the combined treatment with PMBA and 5-FU on H3K9 and H3K4 dimethylation and H3K9 acetylation in HCT-116^G13D and SW-620^G12V cells was significantly greater than those of PMBA and 5-FU. Conversely, the outcome of the combined treatment on H3K9/H3K4 dimethylation and H3K9 acetylation in HT-29^WT cells was not significantly altered from that of the treatment with 5-FU. 5-FU independently had no significant effect on H3K9 and H3K4 dimethylation and H3K9 acetylation levels irrespective of the DNA methylation influence (Figure 5A–C). The linkage hierarchical clustering corresponding to the expression results has increased methylated MGMT expression in KRAS cell lines bearing mutations (Figure 5D). The detailed mechanistic insights of hypermethylated DNA repair protein MGMT in KRAS mutant colon cancer through epigenetic silencing are shown in Figure 5F.

Effect of PMBA on the Global Histone-Methyl Level

The histone extracted from HCT-116^G13D and SW-620^G12V cells were investigated via western blotting. Immunoblots probed with dimethylated H3K9 and H3K4 and acetylated H3K9 antibodies showed that treatment with PMBA (1.7 and 2.2 μM), 5-FU (14.7 and 78 μM), and PMBA + 5-FU (1.7 + 14.7 and 2.2 + 78 μM) had no significant effect on the global levels of dimethyl-H3K9 and dimethyl-H3K4 (Figure 5E). In contrast, treatment with PMBA or with 5-FU significantly amplified the overall level of acetyl-H3K9.

PMBA Displays Potent In Vivo Antitumor Activity

By instillation of MNU, a carcinogen, an orthotopic colon cancer rat model was developed in which the overall growth of the tumor was reduced, followed by a prolonged decrease in the tumor volume in comparison to that of the control group. Consistent to the treatment with PMBA (50 mg), 5-FU (25 mg) and the combined PMBA + 5-FU (50 + 25 mg) therapy also resulted in statistically significant tumor regression in the established model (***P < 0.001 vs vehicle, t-test) (Figure 6A). No over drug-related toxicity was observed, though with minimal disease progression in the responding rat even at the 25th week. A statistically significant decrease in mortality was observed in the combination therapy (only 1 death) and PMBA therapy (2 deaths), compared to the control group (4 deaths), as depicted in the Kaplan–Meier survival graph (Figure 6A). Moreover, there was a statistically significant increase in the animal survival rate (Table S8). After cervical dislocation of animals, the colorectal tumor was subjected to histopathological and immunohistochemistry studies.

Figure 6.

Therapeutic efficacy of PMBA in MNU-induced rat colon carcinoma. (A) Upper panel showing representative pictures of colon tumors developed in rats treated with MNU (0.4%). Graphs show the number of polyps and the scoring of polyps developed treated with both MNU and either PMBA (50 mg/kg), 5-FU (25 mg/kg), or both. Orally administered PMBA reduces colonic tumorigenesis in male Wistar rats as changes in the tumor burden of KRAS MUT/WT colon cancer treated with vehicle, PMBA, 5-FU, or the combination were imaged at the 25th week following the PMBA, 5-FU, or PMBA/5-FU therapy (Kaplan–Meier survival curve). **P < 0.001, t-test. Data indicate mean ± SEM of three independent experiments, in which at least six mice per group were considered. Differences were calculated using the two-tailed Student’s t-test. (B) Micrographs of the gross anatomy of the colonic sections through H&E-stained tissue specimens were obtained at the end of MNU induction, PMBA, 5-FU, or both, as determined by a pathologist in a blinded manner. (C) Representative immunohistochemical images probed with indicated antibodies show percentage changes in expression levels for KRAS and Ki67 positive cells in colonic sections taken from MNU-induced treated tumors from the vehicle, 5-FU, PMBA, or PMBA + 5-FU sacrificed at the 25th week and was quantified by using ImageJ. Mean ± SEM (n = 3); *P < 0.05; **P < 0.01; ***P < 0.001 t-test; and ns vs the vehicle-treated group. (D) The mRNA expression level of MGMT shows indicated protein changes in tumor tissues after treatment with 5-FU, PMBA, or both in MNU-induced rats, analyzed using RT-PCR. Mean ± SEM of triplicate samples shown. (E) Immunoblots of KRAS, Ki67, and caspase-3 expression levels in tumors from the vehicle or two different PMBA treatments. Mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001 t-test; and ns. (F) Analysis of cytokine production in the orthotopic carcinogenic mouse, in which the representative box plot shows the fraction of IFN-γ, IL-1β, IL-8, TGF-β, TNF-α, and IL-6 in the serum of the MNU-induced colorectal rat model. The serum is massively infiltrated with cytokines-TNF-α, IL-6, IL-8, and IL-1β in the vehicle analyzed using an ELISA, whereas significant reduction with PMBA, 5-FU, or combination-derived supernatants was seen, and data are expressed as picogram per milliliter. Data are expressed as mean ± SEM, and differences were calculated using the two-tailed Student’s t-test.

Development of Polyps by MNU

The developed colon polyps induced by MNU was significantly higher in the control group. However, the mean number of polyps per animal was reduced insignificantly in treated groups compared to that in the control group. The rats had 11–14 polyps in the control group (Figure 6A). The data demonstrated that the PMBA therapy inhibited the formation of polyps to a large extent. Each of the tumor was localized in the distal large colon and was a polypoid lesion. Histologically, the tumor was signet ring cell carcinoma and appeared as well-differentiated adenocarcinoma. The tumor was small, with minimal extension and no metastasis into the lymph nodes or other organs. There were no additional pathological findings in the gastrointestinal tract and other organs such as the liver, kidney, and urinary bladder.

Histopathological Analysis

Paraffin-embedded colon sections (5 mm thick) were stained with H&E and studied in an imperceptive fashion for tumor grades, epithelial damage, and inflammation using the scoring process described. Following the WHO definition, the lesion was classified according to the histological grade as carcinoma, high-grade dysplasia, and low-grade dysplasia. In this study, carcinoma showed in situ cellular and structural atypia and consecutive arrangement or a cribriform pattern. Therefore, the diagnostic grade of a tumor was based on the most severely dysplastic area. Almost all of the tumors were polypoid lesions and were distributed evenly within the large intestine in the MNU-treated Wistar rats, but in control rats, no visible colon tumor was observed (Figure 6B).

Immunohistochemistry

Colon sections were stained with the Ki67 and KRAS antibodies, and expression levels were quantified through ImageJ. The expression of both Ki67 and KRAS probed with secondary antibodies decreased significantly, as analyzed using confocal microscopy (Figure 6C). In this KRAS mimetic syngeneic tumor model, the mRNA expression level of MGMT was significantly increased when treated with PMBA (50 mg) and PMBA + 5-FU (50 + 25 mg) (***P ≤ 0.001) in MNU-induced rats (Figure 6D). In parallel, western blotting analysis revealed a decrease in proliferating cells stained with Ki67 in MNU-treated mice and the expression of caspase-3, which demonstrates that the cells died of apoptosis and not by necroptosis (Figure 6E).

Effect of PMBA Treatment on Immunomodulatory Cytokine Expression

KRAS mutation causes tumor cells to support heterocellular communication via increased secretion of soluble growth factors and pro-inflammatory cytokines. PMBA (1.7 and 2.2 μM), 5-FU (14.7 and 78 μM), and PMBA + 5-FU (1.7 + 14.7 and 2.2 + 78 μM) treatment (in vitro) suppressed the MAPK pathway activation in KRAS-dependent/TGF-β mutant HCT-116^G13D and TGF-β resistant SW-620^G12V cells, with decreased pERK1/2 and pSTAT3 levels compared to that of the control. To determine whether PMBA disrupts the autocrine cytokine circuit in vivo; the in vivo assay of pro-inflammatory marker activity was conducted. PMBA (50 mg), 5-FU (25 mg), and PMBA + 5-FU (50 + 25 mg) treatment proficiently elevated IFN-γ and IL-2 levels (***P ≤ 0.001 and **P ≤ 0.01) as compared to that with the vehicle (Figure 6F). It appears that the loss of TGF-β signaling and oncogenic KRAS cooperate to generate a permissive state for metastatic behavior. As a consequence, levels of IL-6, IL-8, TGF-β, and TNF-α were all preferentially reduced in PMBA- and PMBA + 5-FU-treated animals (Figure 6F) (Table S9). Thus, the response to PMBA therapy correlated with effective disruption of this cytokine circuit in vivo, resulting in high levels of IL-1β-activated NF-κB, which results in chemoresistance in colon cancer cells. The increased IL-1β production, as a consequence of genetic polymorphism, reduces survival in colon cancer patients.^19,20

Effect of PMBA on Hematological and Biochemical Parameters

Blood was collected from Wistar rats in which an acute toxicity study at 25, 50, and 50 + 25 mg/kg for PMBA, 5-FU, and PMBA + 5-FU, respectively, was elucidated in the specified ways that (a) there was no treatment-related mortality in the animals treated with the compound during the study period; (b) feed and water consumptions had similar natures for treated and control groups; and (c) all the parameters for hematological and biochemical consideration were significantly within the range, and no adverse effects was seen in the treated group in comparison to the control group, in which the Hb level, RBC, and platelet count of PMBA and PMBA + 5-FU groups were significantly increased. On the contrary, the treated groups showed reduced serum TG, ALP, AST, and ALT levels compared to untreated control groups (Tables S10 and S11).

Discussion

The comprehensive investigation of KRAS^G13D,G12V mutant CRC under both in vitro and in vivo conditions revealed that PMBA, derived from β-boswellic acid, could be an effective therapeutic agent against the disease.²¹ The parent molecule, under the various clinical trials conducted so far (https://clinicaltrials.gov/ct2/show/NCT03149081, https://clinicaltrials.gov/ct2/show/NCT00243022, and https://clinicaltrials.gov/ct2/show/NCT02977936), proved to be significantly effective against the disease, orally bioavailable (in vivo), and well-tolerated. The distinct therapeutic potential of the molecule as a single therapeutic agent was well-established in diverse panel of NCI-60 cancer cell lines as well as from inhibition studies in three colon cancer cell lines carrying the KRAS wild-type and mutant profiles and in extended in vivo studies. In parallel, for validation of KRAS as a target protein, in silico studies were carried out for KRAS G13D and G12V mutant forms, in which PMBA shared precise affinity scores for native as well as mutant forms. The switching of PMBA at two different mutations (G13D and G12V) demonstrated that in the KRAS G13D (PDB ID 4TQA) aspartate side chain, the rate of SOS-independent nucleotide exchange is faster than in WT KRAS due to a change in the electrostatic environment around the nucleotide and increased repulsion between the negatively charged carboxylic group of D13 and the α-phosphate group. In contrast, KRAS G12V insertion of valine, a nonpolar amino acid, directly above the γ-phosphate of GTP may alter the charge distribution or cause some reordering of the solvent, which accounts for the slow hydrolysis rate, while cysteine, a polar amino acid, may improve the local environment and, therefore, show less of an overall impact on the intrinsic hydrolysis rate. In the crystal structure of KRAS G12V bound to PMBA (PDB ID 4TQ9), one conformation of the lysine and asparagine side chain shows the nitrogen atom involved in the hydrogen bonding network with Y32 coordinated with the water molecule adjacent to the phosphate. This additional coordination may account for its near WT rate of intrinsic hydrolysis. Another therapeutic consideration that emerged from the results that boosted compound arrangement with KRAS^G13D,G12V through coherent targeted combination inhibited upstream RTK (e.g., EGFR), which facilitated the approachability of the GDP-bound state of KRAS^G13D,G12V by displacing the cellular KRAS bound GDP/GTP ratio in support of KRAS G13D, G12V-GDP, as manifested in vitro with PMBA.²² Besides the on-target selectivity of KRAS^G13D,G12V inhibitors for the mutant allele, PMBA was well-tolerated in mice and showed fewer side effects and higher sustainability in vivo. The methylome and transcriptome analysis of KRAS mutant cells revealed decreased MGMT expression in the colon tumors (TCGA), which correlated with pCHK2 effects with optimal activation of ATM of RAS/MAPK pathways, in accordance with the findings.²³⁻²⁶ The Geo omnibus data of the KRAS WT, G13D, and G12V mutant patients, for whom we had DNA methylation data with genomic alterations in the tumor, provided markers for lineage tracing and were classified into several genetic sub-lineages (GSE92921, GSE39582, and GSE5720). For MSI, we explored the mutations having CIMP (+) (CpG island methylator phenotype), CIN (−) (Chromosomal instability), dMMR (Deficient mismatch repair), KRASG13D, BRAFwt, and the TP53mut profile, whereas for MSS, all patients showed CIMP (−), CIN (+), pMMR (Proficient mismatch repair), KRASG12V, BRAFwt, and the TP53mut profile (supplementary E1-2). Tumor lineage hypomethylated regions were significantly enriched for long terminal repeats, long interspersed nuclear element 1 (LINE-1), and heterochromatin regions (H3K9me3). In comparison, tumor lineage hypermethylated regions were enriched in CpG islands (CGIs), H3K4me3, and open chromatin.²⁷ The alterations in H3K9me2 and H3K4me2 linked with MGMT silencing were found to be region-specified, other than global, as further investigated through a histone modification study. PMBA was found to be capable of re-establishing the expression of the MGMT gene.

This work contributed the first in vivo evidence in which a PMBA-targeted approach was proposed to be a promising therapeutic strategy for patients with KRAS G13D, G12V mutant cancers. We envisaged an approach that maximizes KRAS target tenancy (in vivo), a key attribute to employ while going forward into clinical development for this class of compounds. With this challenge, we investigated the influence of KRAS on cytokine signaling in an MNU-induced colon cancer model that stimulated the secretion of pro-inflammatory cytokines such as IL-6, IL-8, IL-1β, and TNF-α, identified as the main promoters for Treg induction of differentiation, as cancer cells having KRAS mutations have been described to utilize an immune-suppression phenotype across various molecular mechanisms.²⁷ Downstream targets of TGF-β signaling are key cell cycle checkpoint genes including CDKN1A (p21) (Cyclin dependent kinase inhibitor 1A), CDKN1B (p27) (Cyclin dependent kinase inhibitor 1B), and CDKN2B (p15) (Cyclin dependent kinase 4 inhibitor B), thus generating the expression of regulatory genes needed for entry into the S phase of the cell cycle. Thus, it is reported that the signaling pathway constituting to neither the activation of KRAS and MAPK-ERK nor the inactivation of TGF-β alone has the sufficient capacity to initiate and promote tumor formation. However, the concurrence of both KRAS mutation and TGF-β deletion stimulates the formation of adenocarcinomas in the intestine.²⁸ These results suggested that the tumor suppressor role of TGF-β acting in the intestines is obvious due to other deregulated signaling pathways, such as the Wnt-APC-β-catenin pathway. Furthermore, it is also seen that the tumor-promoting action is auxiliary to autocrine and paracrine effects mediated through the EGFR pathway. Epiregulin expression is also increased in tumor cells of advanced adenomas and may be one of the mechanisms through which the TGF-β loss mediates the malignant transformation of colon adenoma.²⁹⁻³¹ Collectively, the in vivo studies confirmed that PMBA is a broadly efficacious agent throughout the CRC model and provided further evidence that a significant portion of patients with KRAS G13D, G12V mutations get assistance from KRAS^G13D,G12V-directed therapies. Comprehensively, PMBA represents a new class of KRAS inhibitors having a therapeutic window in the scope of a drug candidate. Collectively, these results suggest that using PMBA could provide an effective strategy for targeting colon adenocarcinomas harboring KRAS mutations with an induced immune escape phenotype.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.1c00228.

• Chemical structure, liquid chromatography (LC)/MS-based detection, in silico analysis, calculation of IC₅₀ through the SRB assay on various cancer cell lines and noncancerous cell lines, GEODATA expression analysis, EGFR kinase assay, binding affinity score, cytokine level in MNU-induced wistar rat, biochemical and hematology parameters, and primer set (PDF)

Author Contributions

Conceptualization and methodology by A.Q., A.K., B.A.S., and S.K.S. Data curation by A.Q. and S.S. Visualization and investigation of mass spectrometric analysis carried out by A.Q. and M.K. Software and validation by A.Q., R.M., and S.S. Writing, reviewing, and editing carried out by A.Q., S.M.S., and S.K.S. In vivo experiments executed by A.Q. and J.S. PK–PD study carried out by U.N. and A.M. All authors read and approved the final manuscript.

Supported by the CSIR Project-BSC0205.

The authors declare no competing financial interest.

Notes

The in vivo study has been carried out with the approval from the IAEC (85/2/16) of CSIR-Indian Institute of Integrative Medicine, Jammu, India. All methods were performed in accordance with the relevant guidelines and regulations by the Institutional Animals Ethics Committee. All data generated or analyzed during this study are included in this published article and its Supporting Information files. Genomic data for the patients used are available from the GEO repository in the GSE39582, GSE5720, and GSE92921 data sets.