Targeting of the G9a, DNMT1 and UHRF1 epigenetic complex as an effective strategy against pancreatic ductal adenocarcinoma

Abstract

Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with limited treatment options and a poor prognosis. The critical role of epigenetic alterations such as changes in DNA methylation, histones modifications, and chromatin remodeling, in pancreatic tumors progression is becoming increasingly recognized. Moreover, in PDAC these aberrant epigenetic mechanisms can also limit therapy efficacy. This study aimed to investigate the expression and prognostic significance of a key epigenetic complex encompassing DNA methyltransferase-1 (DNMT1), the histone methyltransferase G9a, and the scaffold protein UHRF1 in PDAC. We also evaluated the therapeutic potential of an innovative inhibitor targeting these epigenetic effectors.

Methods: Immunohistochemical analysis of DNMT1, G9a, and UHRF1 expression was conducted in human PDAC tissue samples. Staining was semi-quantitatively scored, and overexpression was defined as moderate to strong positivity. The prognostic impact was assessed by correlating protein expression with patient survival. The antitumoral effects of the dual DNMT1-G9a inhibitor CM272 were tested in PDAC cell lines, followed by transcriptomic analyses to identify underlying mechanisms. The in vivo antitumoral efficacy of CM272 was evaluated in PDAC xenograft and syngeneic mouse models, both alone and in combination with anti-PD1 immunotherapy.

Results: DNMT1, G9a, and UHRF1 were significantly overexpressed in PDAC cells and stroma compared to normal pancreatic tissues. Simultaneous overexpression of the three proteins was associated with significantly reduced survival in resected PDAC patients. CM272 exhibited potent antiproliferative activity in PDAC cell lines, inducing apoptosis and altering key metabolic and cell cycle-related genes. CM272 also enhanced chemotherapy sensitivity and significantly inhibited tumor growth in vivo without detectable toxicity. Combination of CM272 with anti-PD1 therapy further improved antitumor responses and immune cell infiltration, particularly CD4 + and CD8 + T cells.

Conclusions: The combined overexpression of DNMT1, G9a, and UHRF1 in PDAC is a strong predictor of poor prognosis. CM272, by targeting this epigenetic complex, shows promising therapeutic potential by inducing apoptosis, reprogramming metabolic pathways, and enhancing immune responses. The combination of CM272 with immunotherapy offers a novel, effective treatment strategy for PDAC.

Keywords: Apoptosis; CM272; Chemotherapy sensitivity; DNMT1; Epigenetic mechanisms; G9a; Immunotherapy; Metabolic reprogramming; Pancreatic ductal adenocarcinoma; UHRF1.

Fig. 1

Expression of G9a, DNMT1, and UHRF1 mRNA in human PDAC. A Scoring of G9a, DNMT1 and UHRF1 expression in pancreatic tissues from PDAC patients. Each image shows the representative IHC staining of the three proteins according to each score assigned. B Differential G9a, DNMT1 and UHRF1 expression (3 +) versus (2 + /1 + /0) in tumor tissue and normal pancreatic ducts. C DFS and (D) OS in PDAC patients according a combined high expression (3 +) or low expression (2 + /1 + /0) of DNMT1/G9a/UHRF1. *p < 0.05

Fig. 2

Expression and simultaneous targeting of G9a, DNMT1 and UHRF1 in human PDAC cell lines. A G9a, DNMT1 and UHRF1 mRNA levels in PDAC cell lines according to their classification as classical (n = 9) or quasimesenchymal, QM (basal) (n = 36) categories extracted from Collisson dataset. B Immunoprecipitation of endogenous G9a in MIA PaCa-2 cells. Immunoprecipitates were probed with anti-G9a, anti-DNMT1 and anti-UHRF1 antibodies. Pre-immune IgG immunoprecipitates and Inputs are shown as controls. C Combination study of the growth inhibitory effects of G9a (UNC0642, UNC) and DNMT1 (AZA) inhibitors in MIA PaCa-2 and PANC-1 cells. D GI₅₀ values for CM272 in the indicated human and mouse PDAC cell lines. E Effect of CM272 treatment (72 h) on the levels of H3K9me2 in MIA PaCa-2 and PANC-1 cells. H3 total (H3T) levels and isolated histones (Ponceau) are also shown. F Effect of CM272 treatment (72 h) on the global levels of DNA CpG methylation in MIA PaCa-2 and PANC-1 cells. G Colony formation assays in MIA PaCa-2 and PANC-1 cells treated with CM272 at half of their respective GI₅₀. *p < 0.05; **p < 0.01

Fig. 3

Mechanisms of CM272 antitumoral activity in PDAC cells. A Volcano plot of the genes differentially expressed in MIA PaCa-2 (left panel) and PANC-1 (right panel) cells treated with CM272 (GI₅₀, 72 h). Selected functionally relevant genes are indicated. B Most relevant GO functional categories of genes undergoing changes in expression identified by RNA-sequencing in MIA PaCa-2 cells treated with CM272 (GI₅₀, 72 h). C GSEA analysis of specific categories including heatmaps with a ranked list of genes modulated by CM272 from the RNA-seq data. D Apoptosis vs Viability determination in PDAC cells treated with vehicle (Control) or CM272 at their GI₅₀ during 72 h. E Representative western blots of the indicated proteins in Control and CM-272-treated MIA PaCa-2 and PANC-1 cells. Blots were probed for α-TUBULIN to show equivalent loading. **p < 0.01; ***p < 0.001

Fig. 4

CM272 synergizes with antitumor drugs in the inhibition of PDAC cells growth. A Combination study of the growth inhibitory effects of CM272 and gemcitabine (GEM), cisplatin (CDDP) and FOLFIRINOX (FOLFX) in MIA PaCa-2 cells. B Combination study of the growth inhibitory effects of CM272 and lovastatin (LVT) in MIA PaCa-2 cells. *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 5

The simultaneous inhibition of the epigenetic complex G9a/DNMT1/UHRF1 activates the expression of immune response-related genes in PDAC cells. A qPCR analysis of CCL5, HLA-A, HLA-B and HLA-C expression in MIA PaCa-2 cells pretreated with CM-272 for 24 h and then induced with IFNγ (100U/mL) for another 24 h. B qPCR analysis of CCL5, HLA-A, HLA-B and HLA-C expression in MIA PaCa-2 cells transfected with G9a, DNMT1 or UHRF1-specific siRNAs, specific siRNAs combinations (siG9a + siDNMT1 and siG9a + SiDNMT1 + siUHRF1) and control siRNAs (siC) for 48 h. C qPCR analysis of CCL5, HLA-A, HLA-B and HLA-C expression in MIA PaCa-2 cells treated with UNC, AZA, and the combination (UNC + AZA) at their GI₅₀ during 72 h. *p < 0.05; **p < 0.01; ***p < 0.001; ns: not significant

Fig. 6

In vivo antitumoral effects of CM272. A Diagram of the experimental protocol. Effect of CM272 treatment on the growth of PDAC PDX mice. B Representative images of the immunohistochemical analysis of Ki67 protein in mouse PDX tumor samples and quantification. C Diagram of the experimental protocol. Representative tumor images of tumors in the PDAC orthotopic model and tumor size in Vehicle and CM272-treated groups. D Representative images of the immunohistochemical analysis of CD4 and CD8 proteins in the orthotopic tumor tissue samples and their quantification. *p < 0.05; **p < 0.01; ***p < 0.001; ns: not significant

Fig. 7

In vivo antitumoral effects of CM272 and its combination with immunotherapies. A Diagram of the experimental protocol. Representative tumor images and tumor weights of the subcutaneous PDAC model in which mice were treated with vehicle, CM272, anti-PD-1 and the combination of CM272 and anti-PD-1. B qPCR analysis of the expression of Cxcl10 and Cdk1a genes in in tumor tissues from mice treated with vehicle, CM272, α-PD1 and the combination of CM272 and α-PD1. C Representative images showing the immunohistochemical detection of CD4, CD8 and CDKN1A and their quantification in tumors from mice treated with vehicle, CM272, α-PD1 and the combination of CM272 and α-PD-1. *p < 0.05; **p < 0.01; ***p < 0.001; ns: not significant

Fig. 8

Overview of the main conclusions of the study. The combined overexpression of DNMT1, G9a, and UHRF1 in PDAC is a strong predictor of poor prognosis. CM272, by targeting this epigenetic complex, shows promising therapeutic potential by inducing apoptosis, reprogramming metabolic pathways, and enhancing immune responses. The combination of CM272 with immunotherapy offers a novel, effective treatment strategy for PDAC