Human PDAC specimen collection

This study was conducted on a first cohort of 42 patients diagnosed with PDAC and treated at the University Hospital of Modena, Italy, and a second cohort of 49 patients diagnosed and treated at Hospital Universitario de Navarra, Spain, from January 2000 to February 2019.

Patients with histologically proven PDAC, diagnosed at early/surgical stage without previous neoadyuvant therapy, with available formalin-fixed, paraffin-embedded (FFPE) histological samples were eligible for our analyses. Study population included patients either with ab initio surgically resected tumor. Patients who had received systemic therapies or radiotherapy prior to histological sample collection were excluded. Written informed consent was provided by all patients, and the study protocol was approved by the medical ethics committee of each hospital. University Hospital of Modena (Comitato Etico dell'Area Vasta Emilia Nord- Protocol Number: 0013043/19—Pratica 299/2019/OSS/AOUMO) and Clinical Research Ethical Committee of Navarra (Project 2015/120). All patients provided written informed consent.

Cell culture and treatments

Human PDAC cell lines (MIA PaCa-2, PANC-1, Panc8902) and murine PDAC cell lines (DT6606 and PAN02) were maintained in Dulbecco's Modified Eagle Medium (DMEM) that was enriched with 10% heat-inactivated Fetal Bovine Serum (FBS), penicillin (100 U/mL), and streptomycin (100 mg/mL). Another set of human PDAC cell lines (ASPC-1, NP18, and HPAF-II) and murine PDAC cell lines (511,950, PDAC80, PM12167) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium, also supplemented with 10% heat-inactivated FBS, penicillin (100 U/mL), and streptomycin (100 mg/mL), all from Gibco-Life Technology (Waltham, MA, USA). The human Pancreatic Duct Epithelial Cell Line (H6c7) was cultured in Keratinocyte SFM, + EGF + bovine pituitary extract (Invitrogen) supplemented with 1 × antibiotic‐antimycotic (Gibco-Life Technology).

PDAC cancer cell lines (MIA PaCa-2, PANC-1, AsPC-1, HPAF-II) were acquired from the American Type Culture Collection (ATCC). NP18 and DT6606 were provided by Dr. Ruben Hernandez, PAN02 cells were provided by Dr Pedro Berraondo and Panc8902, 511,950, PDAC80, PM12167 and H6c7 cells were provided by Dr. Silvestre Vicent, all three researchers from CIMA-University of Navarra, Pamplona, Spain.

Treatment times and dosages in the experiments are specified throughout the manuscript, with controls receiving equivalent concentrations of dimethyl sulfoxide (DMSO) (always < 0.1% of the final volume). For cell viability assays, 2,000–4,000 cells were plated into each well of a 96-well plate with the appropriate culture media. After 24 h, the media/drug was added and cells were incubated for an additional 72 h. Following this period, 20 µL of CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA) was introduced. After three hours, absorbance at 490 nm was measured using a plate reader. The drug concentration required to inhibit cell growth by 50% compared to the untreated control (GI50) was determined through curve fitting using GraphPad Prism-v10.2.0 software as previously described [23]. The combination index (CI) for UNC0642 and AZA (5-azacytidine) was calculated as detailed [25]. For apoptosis detection, cells were plated and treated following the same indications as for viability determinations, and Caspase-Glo® 3/7 Assay (Promega, Madison, WI, USA) was employed according to manufacture´s instructions.

Chemotherapeutic agents’ preparation: Irinotecan, 5-FU, oxaliplatin, leucovorin and cisplatin were obtained from Selleckchem (Houston, TX, USA). Gemcitabine, UNC0642 and AZA were from Sigma-Aldrich (St. Louis, MO, USA). The drugs were dissolved in DMSO or nuclease-free water and stored at − 80˚C. Drugs constituting FOLFIRINOX were combined in the following molar ratios analogous to those used in patients: 1.00 irinotecan, 80.95 5-FU, 0.80 oxaliplatin, 1.07 leucovorin. CM272 was produced by WuXi AppTech (Shanghai, China).

Histones extraction

Histones were isolated as described [23]. In brief, cells were lysed using a buffer composed of 10 mM Tris–HCl (pH 7.4), 10 mM NaCl, and 3 mM MgCl2. After centrifugation at 2,500 rpm for 10 min at 4 °C, the supernatant was discarded, and the pellets were re-lysed in the same buffer with the addition of 0.5% NP40 on ice for 10 min with gentle agitation. The nuclei were then pelleted by centrifugation at 2,500 rpm for 10 min at 4 °C and resuspended in a solution of 5 mM MgCl2 and 0.8 M HCl. This suspension was incubated on ice for 30 min to facilitate histone extraction. Following incubation, the samples were centrifuged at 14,000 rpm for 10 min at 4 °C to remove debris, and the supernatants were transferred to a clean tube. To precipitate the histones, 50% trichloroacetic acid was added. The resulting pellets were washed with acetone, air-dried, and then resuspended in a solution of 100 mM Tris–HCl (pH 7.5), 1 mM EDTA, and 1% sodium dodecyl sulfate (SDS). The concentration of histones in the extract was determined using the BCA assay (Pierce Technologies, Rockford, IL) according to the manufacturer's instructions.

Immunoprecipitation

For immunoprecipitation (IP) experiments, cells were lysed using an IP buffer containing protease and phosphatase inhibitors (20 mM Tris–HCl at pH 8, 137 mM NaCl, 1% NP-40, and 2 mM EDTA) at 4 °C for 30 min with continuous rotation. The cell lysates were clarified by centrifuging at 12,000 rpm for 20 min at 4 °C. Afterward, protein concentration was measured, and 800 µg of protein were pre-cleared by incubating with 25 µl of Dynabeads G (Invitrogen) for 2 h with constant rotation at 4 °C. Simultaneously, 5 µg of the primary antibody and an equivalent amount of control IgG were incubated with 20 µl of Dynabeads G for 2 h at room temperature with continuous rotation. The antibody-bound Dynabeads were washed three times with citrate phosphate buffer (pH 5) containing 0.01% Tween-20 and then incubated overnight at 4 °C with the pre-cleared protein samples under constant rotation. The next day, the samples were washed three times using PBS (Gibco-Life Technology) with protease and phosphatase inhibitors, then boiled in Laemmli buffer at 95 °C for 5 min. The beads were separated using a magnetic rack, and the supernatant was collected for Western blot analysis. Antibodies used for IP were: G9a (435,200, Invitrogen) and IgG mouse (SC2025, Santa Cruz Biotechnology, Dallas, TX, USA).

Immunoblot analyses

Cells were lysed in RIPA buffer as described previously [28]. Histone extracts, as well as IP products and homogenates from cells, were subjected to immunoblot (Western blot) analysis as reported [23]. Total protein was separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and electrotransferred onto PVDF membranes (Millipore, Burlington, MA, USA). The following primary antibodies were used for protein detection: anti-H3K9me2 (1:1000, 07–212, Millipore), anti-Total H3 (1:2000, 05–928, Millipore), anti-ATF3 (1:1000, sc-188, Santa Cruz Biotechnology), anti-CDK1A (1:1000, ab188224, Abcam, Cambridge, UK), anti-EGFR (1:1000, 06–847, Millipore), anti-alpha-TUBULIN (1:1000, 2144, Cell Signaling, Danvers, MA, USA), anti-G9a (1:1000, 33,065, Cell Signaling), anti-DNMT1 (1:1000 5032, Cell Signaling) and anti-UHRF1 (1:500, ab57083, Abcam). Secondary HRP-conjugated goat anti-mouse IgG (1:5000, 68,860, Cell Signaling) or anti-rabbit IgG (1:5000, 2729, Cell Signaling) were also used. Target antigens were visualized using SuperSignal™ West Pico PLUS chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA, USA). Images were scanned with a ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA).

5meC Immunofluorescence

For immunofluorescence staining, cells were grown on coverslips and treated with either vehicle or CM272 for 72 h. They were then fixed using ice-cold methanol for 15 min at room temperature and subsequently washed twice with PBS. To quench, cells were exposed to 50 mM NH4Cl in PBS for 10 min. Following three washes with PBS, cells were permeabilized with 0.2% Triton X-100 for 5 min at 4 °C, and DNA was denatured using 4 M HCl for 15 min, followed by treatment with 100 mM Tris–HCl (pH 8.5) for 10 min. After washing, coverslips were blocked with Superblocking buffer (Thermo Fisher Scientific) for 1 h at room temperature. They were then incubated overnight at 4 °C with anti-5-methylCytosine antibody (Eurogentec, Seraing, Belgium, BI-MECY 0100), diluted in 1% BSA in PBS. After washing, cells were treated with fluorophore-conjugated secondary antibodies in 1% BSA in PBS for 1 h at room temperature, washed again, and then stained with Vectashield (Vector Laboratories, Burlingame, CA, USA) containing DAPI. Images were captured using a Zeiss Axio Imager.M1 microscope (Zeiss, Oberkochen, Germany).

Colony formation assays

Colony formation assays were conducted using MIA PaCa-2, PANC-1, AsPC-1, and NP18 cells following the protocol outlined previously [23]. In brief, 3,000 cells were plated in complete medium in six-well plates and treated with CM-272 the following day. The medium was replaced every two days, and cultures were maintained until observable differences between treatment conditions emerged (approximately 4 weeks). After the incubation period, plates were washed with PBS, fixed with 4% formaldehyde (Sigma) in PBS for 10 min, and stained with crystal violet. Representative images were captured.

siRNAs

Human-specific siRNAs targeting G9a, DNMT1, and UHRF1, along with control siRNA (siC), were sourced from Santa Cruz Biotechnology (Santa Cruz, CA). Transfections were carried out using 75 nM of each siRNA with Lipofectamine RNAiMAX reagent (Invitrogen, Grand Island, NY, USA), following the protocol detailed previously [28] and according to the manufacturer's guidelines. When multiple siRNAs were used simultaneously, each specific siRNA was applied at 32.5 nM for combinations of two siRNAs or 25 nM for combinations of three siRNAs. Cells were collected 48 h post-silencing, and gene expression was assessed by qPCR following the transfections.

RNA isolation and quantitative real-time RT-qPCR

Total RNA from cell lines was extracted using the automated Maxwell system from Promega (Madison, WI, USA). For reverse transcription, RNA samples were initially heated at 90 °C for 1 min to denature, followed by incubation at 37 °C for 1 h. The reverse transcription mix contained 50 mM Tris–HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 ng/μL of random primers, 0.5 mM of each deoxyribonucleic triphosphate (dNTP), 5 mM dithiothreitol (DTT), 1.2 U/μL RNase inhibitors (RNase Out), and 6 U/μL M-MLV reverse transcriptase enzyme. All reagents were sourced from Invitrogen (Carlsbad, CA, USA), except for dNTPs, which were obtained from Roche Diagnostics (Mannheim, Germany). Quantitative reverse transcription PCR (qRT-PCR) was conducted as described, with gene expression levels normalized to the housekeeping gene H3F3A as previously outlined [23]. Primer sequences are available upon request.

RNA sequencing (RNAseq)

RNA was assessed for quantity and quality using the Qubit HS RNA Assay Kit (Thermo Fisher Scientific) and the 4200 Tapestation with High Sensitivity RNA ScreenTape (Agilent Technologies, Santa Clara, CA, USA). All RNA samples met high-quality standards, with RIN values above 8. Library preparation was carried out with the Illumina Stranded mRNA Prep Ligation kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Sequencing libraries were prepared from 100 ng of total RNA. The process involved selecting and purifying poly(A)-containing RNA molecules with magnetic beads coated with poly(T) oligos. These poly(A)-RNAs were then fragmented and reverse transcribed into the first cDNA strand using random primers. The second cDNA strand was synthesized with dUTP to maintain strand specificity. The resulting cDNA fragments were purified with AMPure XP beads (Beckman Coulter, Brea, CA, USA), adenylated at the 3′ ends, and ligated with uniquely indexed sequencing adapters. After purification, the fragments were PCR amplified to generate the final libraries. The quality and quantity of these libraries were confirmed using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and the 4200 Tapestation with High Sensitivity D1000 ScreenTape (Agilent Technologies). The libraries were sequenced on a NextSeq2000 sequencer (Illumina), yielding 30–40 million paired-end reads per sample. The data were demultiplexed using Cutadapt. RNAseq was performed at the Genomics Unit of the Center for Applied Medical Research (CIMA), Universidad de Navarra, Pamplona, Spain.

Immunohistochemical analyses

Three-micrometer-thick sections were prepared from formalin-fixed paraffin-embedded pancreatic tissues. The sections were deparaffinized using xylene, dehydrated with ethanol, and treated with 3% hydrogen peroxide to inhibit endogenous peroxidase activity. Antigen retrieval was achieved by heating the sections in a 10 mM Tris–EDTA buffer at pH 9 before incubating with primary antibodies for G9a (1:200, ab185050, Abcam), DNMT1 (1:100, ab188453, Abcam), UHRF1 (1:100, ab194236, Abcam), CD8 (1:100, 98941 T, Cell Signalling), CD4 (1:100, ab183685, Abcam), CDKN1A (1:100, ab188224, Abcam), and anti-H3K9me2 (1:100, 07–212, Millipore). For detection, an HRP-conjugated Envision secondary antibody (K4003, Dako, Santa Clara, CA, USA) was used, followed by DAB reagent (K3468, Dako). Signal quantification was performed using QuPath software v0.3.217. The tissue sections were counterstained with Hematoxylin (Sigma-Aldrich) and dehydrated. Negative controls were included by omitting the primary antibodies.

In vivo experiments

For the patient-derived xenograft (PDX) model, experiments performed at Vall d'Hebron Institute of Oncology (Barcelona, Spain) were approved by the institution's Ethical Committee and the Catalan Regional Government. PDX92 was created by implanting 3–4 mm tumor fragments from a metastatic liver biopsy of a PDAC patient into the flanks of 6-week-old female NOD.CB-17-Prkdc scid/Rj mice (Janvier Labs, Saint-Berthevin, France, RRID:MGI:3,760,616). When tumors reached 100–150 mm3, mice were divided into two groups for daily intraperitoneal treatment (i.p.) with either vehicle (PBS) or CM272 (5 mg/kg) for 28 days. Tumor growth and animal weight were monitored bi-weekly. Mice were euthanized when tumors reached 1–1.5 cm3 or if significant weight loss occurred. Mice were kept in filtered cages with a 12-h light/dark cycle and had ad libitum access to food and water.

For the orthotopic PDAC model, 8-week-old male C57/BL6 mice (Jackson Laboratories, Bar Harbor, ME, USA) were housed four per cage with standard chow and water at 22 °C on a 12-h light/dark cycle. A 1.5-cm midline laparotomy was performed under anesthesia, and DT6606 cells (5 × 105 cells) were injected into the pancreas. The abdominal wall was closed with 5–0 Vicryl suture (Ethicon, Raritan, NJ, USA). Mice were randomly assigned to two different treatment groups: (1) saline, (2) CM272 (5 mg/kg, i.p., 5 times/week). After 4 weeks, mice were euthanized, and tumors were excised, weighed, and processed for histology and immunohistochemistry.

In a combination treatment model, 1 × 106 PAN02 cells were injected subcutaneously into the right flank of 8-week-old male C57/BL6 mice. When tumors reached ~ 100 mm3, mice (n = 6) were divided into four treatment groups: (1) saline; (2) CM272 (5 mg/kg, i.p., 5 times/week); (3) anti-PD1 (10 mg/kg, i.p., twice a week); and (4) combination (CM272 and anti-PD1, using the same dosis and times). After 4 weeks, mice were euthanized, tumors excised, weighed, and analyzed for histology and immunohistochemistry. These procedures were also approved by the University of Navarra's Animal Care Committee (ethical committee approval #R-CP001-15GN).

Biochemical parameters

Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lipase (LIPC), and amylase (AMYL) were measured using a C311 Cobas Analyzer (Roche Diagnostics GmbH, Mannheim, Germany) following manufacturer’s instructions.

Statistical analyses

Association between IHC expression of DNMT1, G9a and UHRF1 was tested using Fisher exact test. Disease-free survival (DFS), overall survival (OS) and their two-sided 95% CI were estimated by the Kaplan–Meier method and curves were compared by the log-rank test (at a significance level of 5%). Estimated HRs and their two-sided 95% CI were calculated using the Cox-proportional hazard model. Other statistical analyses were conducted using GraphPad Prism 10.2.0 software. Comparisons between two groups were made using either the paired two-tailed Student’s t-test or the Kruskal–Wallis ANOVA test, depending on the data distribution. Survival rates were evaluated with Kaplan–Meier curves, and differences were tested using the log-rank test. All p-values were two-tailed, and significance was defined as p < 0.05.