Efficacy of DNA methyltransferase inhibition in combination with immune-checkpoint/PARP-inhibitor in colorectal cancer preclinical model

Background. Epigenetic alterations in the form of DNA methylation changes in genes have been associated with colorectal cancer (CRC) progression. DNA methyltransferase (DNMT) enzyme plays central role in epigenetic regulation. In this study, we evaluated the therapeutic potential of epigenetic modulation using DNMT inhibitor 5-AZA-2ʹ-deoxycytidine (5AZA) in combination with immune-checkpoint inhibitor (ICI) anti-PD1 or PARP inhibitor BMN673 in syngeneic CRC mouse model. Methods. 8 weeks old C57BL/6 J female mice were subcutaneously injected with 10 6 MC38 cells in the right flank. Mice (n=10 per group) were randomized into following treatment groups: control, 5AZA 0.5 mg/kg, anti-PD1 100µg/mouse, BMN673 0.3 mg/kg, combinations 5AZA+anti-PD1, and 5AZA+BMN673. Upon endpoint, mice were euthanized, tumors and plasma were collected. Plasma cytokine levels were subsequently measured using V-PLEX Proinflammatory Panel 1 kit (MSD). Isolated DNA was subjected to reduced representation bisulfite sequencing, followed by analysis of statistically significant (adjusted p <0.05) differentially methylated regions (DMRs) and their associated genes using metilene and Ingenuity Pathway Analysis (IPA). RNA sequencing was performed using Novaseq (Illumina) and significant differentially expressed genes (DEGs) (adjusted p <0.05) were analyzed using DESeq2. Results. We showed that combining 5AZA with anti-PD1 significantly reduced ( P <0.0001) tumor volume compared to control and with either 5AZA or anti-PD1 alone. Similarly, combination of 5AZA and BMN673 significantly reduced ( P <0.0001) the tumor volume compared to control and BMN673 alone. Methylation profiling revealed 940 and 299 DMRs in the combination 5AZA+anti-PD1 and 5AZA+BMN673, respectively when compared to single agent treated groups. IPA analysis revealed upregulation of RHO GTPase cycle and downregulation of adipogenesis in all 5AZA-treated groups. Furthermore, proinflammatory cytokines were differentially expressed among treatment groups, with cytokines IL-1beta and TNF-alpha detected at higher concentration in BMN673 treated groups and higher levels of IL-10 and IL-2 in 5AZA treated groups. Transcriptomics profiling showed 203 and 135 upregulated, and 126 and 237 downregulated DEGs in 5AZA+anti-PD1 and 5AZA+BMN673, respectively. Notably, both combination groups showed significant downregulation of Hba and Hbb genes which encode the hemoglobin subunits, suggesting increased reactive oxygen species and associated apoptosis in tumors as a countereffect to the observed upregulation of Tuba3a , Tuba3b and Trap1a genes all associated with hypoxia in both combination groups. Conclusion. Our findings suggest that administration of DNMT inhibitors in combination with ICI or PARP inhibitors could be a promising strategy to substantially improve the treatment and survival outcomes in CRC.