Kinase assay

Procedures for the preparation of KTC1101 are described in supplemental data. To investigate the PI3K inhibitory efficacy of KTC1101, its impact on the kinase activity of PI3K isoforms was assessed using the Adapta kinase assay as previously reported [39]. Initially, a dilution series of 2.5 µL KTC1101 or DMSO (control) was prepared and added to the respective wells of a 384-well plate. Subsequently, optimized 2.5 µL kinase solutions, including PIK3CA/PIK3R1, PIK3CB/PIK3R1, PIK3CG, and PIK3CD/PIK3R1, were introduced to each well. After adding the kinase solutions, 5 µL ATP and PIP2:PS lipid kinase substrate was introduced into each well and incubated for 1 h at room temperature. This step allows for the initiation and progression of the kinase reaction. Following the incubation, a detection solution (5 µL) was added to all the wells. This solution contained 30 mM EDTA to stop the kinase reaction, 6 nM Eu-labeled anti-ADP antibody, and a 3 × concentration of Alexa Fluor® 647 ADP tracer. The EDTA effectively terminates the kinase reaction, while the anti-ADP antibody and ADP tracer are crucial for the detection of ADP produced during the kinase reaction, which correlates with PI3K activity. The plate was then allowed to equilibrate for 30 min at room temperature, ensuring proper binding of the tracer and antibody to the generated ADP. Finally, the fluorescence of each well was measured using a Multi-Mode Microplate Reader (Spark, Tecan). To ascertain the selectivity of KTC1101 for PI3K isoforms and to rule out off-target effects, a kinase panel comprising 50 kinases was tested against KTC1101. This kinase profiling was conducted by Wuhan Heyan Biomedical Technology Co., Ltd.

Cell lines and culture

The B16 and PC3 cell lines were sourced from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The HSC2, HSC4, and CAL33 cell lines were obtained from the American Type Culture Collection (ATCC). S24 cell line was kindly provided by Gloria H. Su of Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center. The TMD8 cell line was generously provided by Dr. Xianhuo Wang of the Tianjin Medical University Cancer Institute and Hospital. Each cell line was maintained in media specified by their respective suppliers, ensuring optimal growth conditions.

Cell proliferation assay

Cell proliferation was measured by MTT assay as previously described [40]. The cells were seeded in a volume of 200 μL/well in 96-well plates. The next day, cells were treated with DMSO or a series of concentrations of KTC1101. After a 48-h incubation period, 20 μL of MTT (5 mg/mL) was added to each well. After further incubation for 4 h at 37 °C, the absorbance at 490 nm was measured using an iMark microplate reader (BIO-RAD, Hercules, CA, USA).

Cell cycle analysis

Cell cycle analysis was performed as previously described [41]. Cells were seeded into 6-well plates and treated with DMSO or a series of concentrations of KTC1101. After 48 h of incubation, cells were collected, suspended in PBS, and fixed in 75% ethanol at 4 °C overnight. Next, cells were washed and resuspended in PBS containing PI and RNase. The stained cells were acquired on a BD C6 Flow Cytometer using BD AccuriTM C6 Software (BD Biosciences).

qRT-PCR

qRT-PCR was performed as previously described [42]. RNA was isolated using Trizol and used to synthesize complementary DNA (cDNA) using a cDNA Synthesis Kit (GenStar), and RT-PCR was performed with aliquots of cDNA samples mixed with SYBR Green MasterMix (Applied Biosystems). Reactions were performed in triplicate. The fold differences in transcripts were calculated using the ΔΔCt method, and 18S rRNA was used as a control to normalize RNA expression (Supplementary Table 1 shows the primers used).

Western blots

Western blots were performed as previously described [43]. Cells were seeded into six-well plates and treated with DMSO or a series of concentrations of KTC1101. Cells were lysed, and total proteins were harvested. Equal amounts of protein (20–50 μg) were separated using 8% or 12% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Bio-Rad). After blocking in 5% nonfat dry milk, the membranes were incubated with appropriate primary antibodies overnight at 4 °C, washed, and incubated with respective HRP-conjugated secondary antibodies for 1 h at room temperature. The signals were detected using a ChemiDoc XRS + System (Bio-Rad) after exposure to chemiluminescence reagents (Bio-Rad), and β-actin served as the loading control.

Molecular docking study

For our molecular docking study, we utilized the CDOCKER module of Discovery Studio 3.5 software to predict the binding interactions between PI3K isoforms and KTC1101. This process comprised two primary steps: (1) Preparation of PI3Ks and KTC1101: The initial step involved preparing the PI3K proteins for docking. This was done using the Clean Protein module, which entailed adding hydrogens to the protein, removing any redundant conformations, adding missing residues, and setting the desired pH at 7. Additionally, water molecules within a 5 Å radius around the receptor were deleted to streamline the docking process. The active site of the protein, crucial for the docking simulation, was identified under the “from current selection” section of the receptor-ligand interactions module. The preparation of KTC1101 as the ligand involved assigning it to the Prepare Ligands module. (2) Molecular Docking: The prepared proteins and KTC1101 were then subjected to the docking process using the Dock Ligands (CDOCKER) protocol, providing detailed insights into the binding modes and interactions of KTC1101 with the PI3K isoforms.

Liver microsome metabolic stability assay

KTC1101 and positive control Midazolam were prepared as 10 mM stock solutions, diluted to 100 µM working solutions in acetonitrile. PBS containing 3 mM MgCl2 was prepared by diluting 200 mM MgCl2. Microsomes (20 mg/mL) were diluted to 0.56 mg/mL, and a 10 mM NADPH working solution was prepared. 5 µL of the compound working solution was added to 445 µL of microsome working solution in a 96-well plate, pre-incubated at 37°C for 5 min. 50 µL NADPH working solution was added to each well, mixed, and incubated at 37°C on a shaker for 120 min. Samples (40 µL) were collected at 0, 5, 15, 30, 45, 60, and 120 min, added to a 96-well plate, followed by 300 µL acetonitrile containing internal standard (phenacetin). After vortexing for 10 min, samples were centrifuged at 6000 rpm for 10 min. The supernatant (200 µL) was collected for analysis.

Plasma metabolic stability assay

KTC1101 and positive control Procaine were prepared as 10 mM stock solutions, diluted to 100 µM working solutions in acetonitrile. 495 µL of plasma was placed in a 96-well plate and pre-incubated at 37°C for 5 min. 5 µL of compound working solution was added, mixed, and incubated at 37°C on a shaker for 120 min. Samples (40 µL) were collected at 0, 15, 30, 60, and 120 min, added to a 96-well plate, followed by 300 µL acetonitrile containing internal standard. After vortexing for 10 min and centrifugation at 6000 rpm for 10 min, the supernatant (200 µL) was collected for analysis.

Pharmacokinetic study of oral administration of KTC1101

Chromatographic conditions employed a Waters BEH C18 column (2.1 mm × 50.0 mm, 1.7 µm) with a mobile phase comprising acetonitrile with 0.1% formic acid (A) and water with 0.1% formic acid (B), at a flow rate of 0.4 mL/min and a column temperature of 40°C. Gradient elution was used: 0–0.3 min, 20:80 → 20:80 (A:B); 0.3–2.5 min, 20:80 → 60:40 (A:B); 2.5–3.0 min, 60:40 → 100:0 (A:B); 3.0–3.01 min, 100:0 → 20:80 (A:B); 3.01–3.5 min, 20:80 → 20:80 (A:B). Mass spectrometry was performed using electrospray ionization (ESI) in positive ion mode, with a capillary voltage of 1 kV, a desolvation temperature of 450 °C, and a desolvation gas flow of 800 L/h. KTC1101 and internal standard phenacetin parameters were optimized for ionization and collision energies.

KTC1101 was prepared at 1 mg/mL concentration in acetonitrile, and further diluted to create a series of standard solutions. Blood samples were taken from BALB/c male mice (19 ± 2g) at various time points post-oral administration of KTC1101 (100 mg/kg). Samples were processed and analyzed using HPLC coupled with mass spectrometry. Data were calculated using Masslynx 4.1 and DAS 3.0 software.

Acute toxicity study

All mice care and experimental protocols were approved by the Ethical Committee of Tianjin Medical University (permit number: TMUaMEC 2023036). Preliminary testing was conducted on 6 groups of BALB/c mice (half male, half female, 18–22 g), fasted but not water-deprived for 12 h, and administered different doses of KTC1101 (200 to 6400 mg/kg). Post-administration, the animals were observed for 7 days for mortality, body weight changes, and other clinical signs. The LD50 was calculated based on the doses causing 0% and 100% mortality in animals. Subsequent testing was done using doses determined from the preliminary study. Each dose was administered to a single animal, with a 72-h interval between dosages. The administration continued until one of the following criteria was met: (a) three consecutive animals survived; (b) a transition from survival to death occurred in 5 out of 6 consecutive animals; (c) at least four animals were tested after the first transition, with an estimated LD50 range exceeding the critical value (2.5 times). The LD50 and its confidence interval were calculated, and the maximum likelihood method was used to determine the LD50 value using the AOT425StatPgm software.

Heterotopic nude mouse xenograft model

BALB/c nude, NSG, and C57BL/6J mice were obtained from Tianjin Medical University. The housing temperature was controlled, ranging from 20.5–24 °C, and humidity was monitored but not controlled ranging from 30–70%. The 12-h daily light cycle was from 06:00 to 18:00. To generate a murine subcutaneous tumor model, PC3 and HSC2 cells were subcutaneously injected into the right lateral flank of 4- to 5-week-old male BALB/c nude mice. TMD8, CAL33, and S24 cells were subcutaneously injected into the right lateral flank of 4- to 5-week-old male NSG mice. When tumors reached a volume of 300–500 mm3, they were excised, diced into 2 mm × 2 mm × 2 mm pieces, and implanted into the right flanks of BALB/c nude or NSG mice. Tumors were allowed to grow to a volume of 100 mm3, and then the animals were randomly divided into several groups. Each group of five mice was treated with either vehicle or KTC1101 (25, 50, or 100 mg/kg, PO). Tumor size was measured every 3 d or 4 d until the endpoint. The tumor volume was calculated using the following formula: length × width × height/2. At the end of the experimental period, mice were euthanized by overdosing on pentobarbital sodium, and the tumors were removed. Tumor tissue was formalin-fixed and paraffin-embedded for histological analysis.

Syngeneic mouse tumor model

To generate syngeneic mouse models, S24 and B16 cells were subcutaneously injected into the right lateral flank of 4- to 5-week-old male C57BL/6 mice. Tumors were allowed to grow to a volume of 100 mm3, and then the animals were randomly divided into several groups. For S24 tumor-bearing mice, each group of five mice was treated with either vehicle or KTC1101 (100 mg/kg, PO). Tumor size was measured every 3 d or 4 d until the endpoint. For B16 tumor-bearing mice, each group of five mice was treated with either vehicle, KTC1101 (50 or 100 mg/kg, PO), anti-PD-1 antibody (250 μg per mouse, clone RPM1-14, Bio X cell, IP), or KTC1101 and anti-PD-1 antibody. Tumor size was monitored every other day, and the tumor was harvested at indicated time points for analysis of tumor-infiltrating lymphocytes. The tumor volume was calculated using the following formula: length × width × height/2.

Drug concentration measurement in tumors

To evaluate the intratumoral concentration of KTC1101, the B16 murine cancer model was established by subcutaneously implanting 2.5 × 105 B16 cells into the right lateral flank of C57BL/6 mice. The mouse model was utilized for experiments when tumor volumes reached approximately 100 mm3. The B16 tumor-bearing C57BL/6 mice were divided into groups, with each group comprising three mice. They were treated with KTC1101 at doses of either 50 or 100 mg/kg (PO). The mice were sacrificed, and tumors were collected on days 1, 5, and 7 post-treatment. Approximately 50 mg of each tumor was homogenized in 1 mL of methanol, followed by centrifugation at 12,000 rpm at 4 °C for 10 min. The concentration of KTC1101 in the supernatant was quantified using LC–MS.

T cell suppression assay

Spleens from naive mice were isolated and ground through 70-μm filters to generate a single-cell suspension. After RBC lysis, single-cell suspensions were stained with CD3, CD8, CD4, CD25 or CD127 antibodies. Viable CD8+ T cells (CD3+CD8+), CD4+ T cells (CD3+CD4+), and Tregs (CD3+CD4+CD25+CD127low/−) from single-cell suspensions were sorted by FACS. Then T cells were labelled with 1 mM CFSE in pre-warmed PBS for 10 min at 37 °C. The CFSE-labelled T cells were seeded in 96-well plates pre-coated with 10 μg/mL CD3 and 1 μg/mL CD28 antibodies and cultured in 1640 media with 10% FBS, 1 mM Sodium Pyruvate (Thermo, 11,360,070), 50 μM β-ME and 100 IU/mL IL2, as well as different concentrations of KTC1101 or solvent control. After 72 h, cells were harvested and the CFSE signal was measured by flow cytometry. Cell growth rate was calculated by the percentage of live, proliferated cell number at each drug concentration vs. solvent control.

Flow cytometry

To obtain single-cell suspensions for analysis by flow cytometry, tumors were excised, minced, and dissociated in Deoxyribonuclease/collagenase/hyaluronidase buffer (0.1 mg/mL Deoxyribonuclease I, 0.2 mg/mL Collagenase IV and 0.2 mg/mL Hyaluronidase) for 60 min at 37 °C with agitation, and strained through a 70 μm strainer for downstream applications. Tissue was dissociated as described above to obtain single-cell suspensions. Cells were stained with antibodies for 30 min at 4 °C, and then fixed and intracellularly stained using Foxp3/ Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer’s instructions. Antibodies used in this study can be found in Supplementary Table 2. The stained cells were acquired on a BD C6 Flow Cytometer using BD AccuriTM C6 Software (BD Biosciences) and the data were processed using FlowJo software.

ELISA detection of secreted cytokines

We examined the secreted cytokines from B16 cells under both in vitro and in vivo conditions. For in vitro-cultured B16 cells, the cells were seeded into six-well plates and treated with either DMSO or a range of KTC1101 concentrations. After 48 h, the total cell count was determined, and the cell culture supernatant was collected for further analysis. For in vivo tumor tissues, the harvested tumor tissues were homogenized. Post-centrifugation, the supernatant was collected and used for protein quantification utilizing a BCA kit. The concentrations of murine CCL5 and CXCL10 in these supernatants were then quantified using specific ELISA kits, in accordance with the manufacturer's instructions.

H&E and immunohistochemical (IHC) staining

H&E and IHC staining were performed as previously described [44]. H&E staining was used to detect pathological changes in morphology. For histological analysis, formalin-fixed, paraffin-embedded tumors were sectioned, and slides were deparaffinized using xylene (Thermo Fisher). Endogenous peroxidases were quenched with 3% hydrogen peroxide in methanol. Staining was performed using antibodies against phosphorylated Akt (Ser473), phosphorylated S6 (Ser235/236), or Ki67. Counterstaining was performed with Mayer’s hematoxylin (Dako). Slides were observed under an Olympus CX21 microscope and scanned with a high-resolution digital slide scanner (Pannoramic 250, 3DHistech) to capture images.

Toxicity and safety profiling of KTC1101 in vivo

The B16 tumor-bearing C57BL/6 mice were divided into 4 groups randomly (n = 3 per group) and treated with vehicle, KTC1101 (100 mg/kg 3on/4off, PO), anti-PD-1 antibody (250 μg per mouse, IP), or a combination of KTC1101 and anti-PD-1 antibody. After 14 days of treatment, all mice were sacrificed. Blood samples were collected for analysis; one portion was centrifuged (3000 rpm, 4 °C) to obtain serum for biochemical parameter analysis, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA), and blood urea nitrogen (BUN). The other portion was used for routine blood examination. Major organs including the heart, liver, spleen, lung, and kidney were collected, sectioned, and stained with H&E for histopathological evaluation.

JFCR39 COMPARE analysis

JFCR39 drug discovery system [45, 46] was used to measure the growth inhibitory activities and identify the target of KTC1101. The JFCR39 panel consists of 39 cell lines seeded in 96-well plates. After 48-h treatment with KTC1101, cell growth was measured using the sulforhodamine B assay. The concentration of the compound that inhibited 50% of cell growth (GI50) was calculated from the dose–response curve. The deviation of log GI50 for each cell line from the average log GI50 across the JFCR39 panel was plotted as a fingerprint. MG-MID (mean of Log GI50 values) represents the average log GI50 for all 39 cell lines. The most sensitive and least sensitive cell lines were identified by calculating the difference in their log GI50 values (Range) and the difference between the most sensitive cell line and the average GI50 on the JFCR39 panel (Delta). The fingerprint of KTC1101 was compared with the fingerprints of reference compounds in the database. The Pearson correlation coefficient (r) was calculated (n = 39). Similarity to a reference compound suggests a similar mode of action or target for KTC1101.

Transcriptome data analysis

RNA-seq analysis was carried out as previously described [47]. B16 cells were treated with KTC1101 (0.04 and 0.2 μM) or DMSO for 48 h. C57BL/6J mice with subcutaneous tumors of B16 cells were treated with either a vehicle or 100 mg/kg KTC1101 for 3 days, followed by harvesting tumor tissues. Total RNA was isolated using TRIzol reagent. RNA concentrations were quantified using a NanoDrop Spectrophotometer (Thermo Fisher), and RNA integrity was assessed using the RNA Nano 6000 Assay Kit on a Bioanalyzer 2100 system (Agilent Technologies). Samples with RIN values of > 6.0 were used for experiments. A complementary DNA library was prepared, and sequencing was performed according to the Illumina standard protocol by Beijing Novel Bioinformatics Co., Ltd. (https://en.novogene.com/). Specifically, cDNA libraries were prepared using an Illumina NEBNext® UltraTM RNA Library Prep Kit. After cluster generation, the library preparations were sequenced on an Illumina NovaSeq 6000 platform, and 150 bp paired-end reads were generated. For the data analysis, raw data (raw reads) in fastq format were first processed through in-house Perl scripts. Clean reads were obtained by removing reads containing adapters, poly Ns, and low-quality reads from raw data. Reference genome and gene model annotation files were downloaded from the genome website directly. The index of the reference genome was built using Hisat2 v2.0.5, and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. Mapped reads of each sample were assembled using StringTie (v1.3.3b) in a reference-based approach. Feature Counts v1.5.0-p3 was used to quantify the read numbers mapped to each gene. Differential expression analysis was conducted using DESeq2 (v. 1.42.0). Differentially Expressed Genes (DEGs) were visualized as volcano plots using the ggplot2 package (v. 3.4.4). After normalizing FPKM values by Z score, DEGs were selected for hierarchical clustering, displayed as heatmaps, which were generated using the pheatmap package (v.1.0.12). The org.Hs.eg.db package (v.3.17.0) and the org.Mm.eg.db package (v.3.18.0) were used to convert official gene symbol IDs to Entrez IDs, after which DEGs were mapped to KEGG (Kyoto Encyclopedia of Genes and Genomes), GO (Gene Ontology), and Hallmark pathways using the clusterProfiler package (v.4.10.0). Gene Set Enrichment Analysis (GSEA) first calculated the fold change (FC) for each gene between subtypes, then genes were ordered in descending order based on FC values and visualized as volcano plots and ridgeline plots using ggplot2 (v. 3.4.4) and ggrepel (v. 0.9.4) packages. Gene Set Variation Analysis (GSVA) was performed using the GSVA package (v.1.50.0), and results were presented as bubble charts using the ggplot2 package (v. 3.4.4). All analyses were conducted in R v.4.3.1.

Connectivity map validation

The Connectivity Map (CMap) is a gene expression database developed by the Broad Institute, primarily used to uncover functional connections between small molecule compounds, gene perturbations, and disease states. CMap contains gene expression profiles resulting from various interventions, including small molecule treatments, gene overexpression, and gene knockouts, in different cell lines (PC3, A375, A549, HA1E, HCC515, HEPG2, HT29, MCF7, and VCAP). In this study, we utilized PC3 cells treated with 25 nM KTC1101 for 48 h. Following treatment, RNA-Seq was performed to identify the top 150 upregulated and downregulated differentially expressed genes (DEGs). These DEGs were uploaded to the Connectivity Map (CMap; https://clue.io/) database to analyze the similarity between DEGs and the expression profiles from small molecule drug treatments in the Compound database.

Tumor immune microenvironment analysis

Cell-type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT) was used to perform a deconvolution analysis of the gene expression matrix based on the principle of linear vector regression. The CIBERSORT algorithm, with a reference to 511 mouse gene signatures, categorized immune cell populations into seven groups and calculated composition scores: B cells including memory, naive, and plasma cells; CD8+ T cells including memory, naive, and activated cells; CD4+ T cells including memory, naive, follicular, Th1, Th2, and Th17 cells; DCs including activated and immature cells; the rest being mast cells, Tregs, and monocytes.

Statistical analysis

Data from three independent experiments are presented and expressed as the mean ± SD. An unpaired, 2-tailed Student’s t-test was used for 2-group comparisons. ANOVA with Bonferroni’s correction was used to compare multiple groups. A p-value of < 0.05 was considered statistically significant. Before statistical analysis, variations within each group and the assumptions of the tests were checked.