GSE202065
Targeting the ADPKD methylome using nanoparticle-mediated combination therapy
DNA methylation aberrancies are found in autosomal dominant polycystic kidney disease (ADPKD) which suggest the methylome to be a promising therapeutic target. However, the impact of combining DNA methylation inhibitors (DNMTi) and ADPKD drugs in treating ADPKD and on disease-associated methylation patterns has not been fully explored. To test this, ADPKD drugs, metformin and tolvaptan (MT), were delivered in combination with DNMTi 5-aza-2'-deoxycytidine (Aza) to 2D or 3D cystic Pkd1 heterozygous renal epithelial cells (PKD1-Het cells) as free drugs or within nanoparticles to enable direct delivery for future in vivo applications. We found Aza synergizes with MT to reduce cell viability and cystic growth. Reduced representation bisulfite sequencing (RRBS) was performed across 4 groups: PBS, Free-Aza (Aza), Free-Aza+MT (F-MTAza), and Nanoparticle-Aza+MT (NP-MTAza). Global methylation patterns showed that while Aza alone induces a unimodal intermediate methylation landscape, Aza+MT recovers the bimodality reminiscent of somatic methylomes. Importantly, site-specific methylation changes associated with F-MTAza and NP-MTAza were largely conserved including hypomethylation at ADPKD-associated genes. Notably, we report hypomethylation of cancer-associated genes implicated in ADPKD pathogenesis as well as new target genes that may provide additional therapeutic effects. Overall, this study motivates future work to further elucidate the regulatory mechanisms of observed drug synergy and apply these combination therapies in vivo. Overall design: Reduced-Representation Bisulfite Sequencing (RRBS) on Pkd1 heterozygous renal epithelial cells (PKD1-Het cells) treated for 24h with a PBS control (PBS, n=3), 50 µM Aza (Aza, n=3), and a drug combination consisting of 50 µM Aza, 300 µM metformin, and 10 µM tolvaptan delivered as free molecules (F-MTAza, n=3), or the aforementioned drug combination delivered in micelle formulation (NP-MTAza, n=3).