Epigenetic editing and epi-drugs: a combination strategy to simultaneously target KDM4 as a novel anticancer approach

Abstract

KDM4-A/B/C, preferentially demethylating di- and tri-methylated lysine 9 on histone H3, are overexpressed in cancers and considered interesting therapeutic targets. Consequently, KDM4 inhibitors have been developed to block their enzymatic activity. However, the potential lack of specificity of such small molecules (epi-drugs) may contribute to dose-limiting toxicities. In the pursuit of more specific interventions, epigenetic editing (epi-editing) has emerged as a powerful tool to modulate gene expression by modifying the epigenetic profile of specific genomic locations. The recently developed CRISPRoff (dCas9 fused to DNMT3A/3L and KRAB), guided by sgRNAs, is successfully used for gene repression by introducing methylation of DNA and (indirectly) of histones at the targeted genomic region. We propose that combining epi-editing (here to prevent the expression of KDM4) with epi-drugs (to inhibit the KDM4 protein activity) may represent a novel path for synergistic anticancer effects through simultaneous inhibition of gene expression and protein activity. Upon validating the downregulation of KDM4A in HEK293T cells through epi-editing, we demonstrated its repression in colon, breast and hepatocellular carcinomas which was effective in preventing (breast, MCF7) or inhibiting (colon, HCT116) cancer cell growth. Anticancer effect was also confirmed for these cell lines using the KDM4 inhibitor QC6352. In parallel, our studies demonstrate a previously unnoticed increase in the expression of KDM4-A/B/C genes following the inhibition of protein activity using the pan-KDM4 inhibitors QC6352 and JIB-04. Importantly, this induction of gene expression was fully prevented or even further inhibited by epi-editing. Then, we assessed the efficacy of our dual-targeted silencing approach in cancer cells and demonstrated that the inhibition in cancer cell growth by epi-drug or epigenetic editing could be further improved by combining the treatments. Building upon these findings, we introduce a novel, potentially synergistic, therapeutic strategy that combines epi-drug administration with epi-editing. This innovative approach aims to reduce drug toxicity and the potential development of resistance by preventing drug-induced upregulation of target enzyme expression, thereby further increasing anticancer effects.

Keywords: CRISPR/dCas9; Epi-drugs; Epigenetic editing; JIB-04; KDM4; QC6352.

Fig. 1

(A)KDM4A gene expression reduction by epi-editing using CRISPRoff in HEK293T, HCT116 and MCF7; (B) Proliferation assay in HCT116 and MCF7 cells upon downregulating KDM4A. The dark gray lines show the untransfected cells (symbol: circle), in solid black lines the cells transfected with CRISPRoff and sgRNA-KDM4A (symbol: square), the dashed lines show the cells transfected only with sgRNA-KDM4A (symbol: triangle), and in light gray the cells only with CRISPRoff (symbol: diamond)

Fig. 2

(A) KDM4A expression evaluation in HEK239T, HCT116, MCF7, HepG2 and MDA-MB-231 cells treated with KDM4 inhibitor, QC6352, for 24 h at 5 µM, or with DMSO (ctr); (B) KDM4A/B/C expression in HCT116, MCF7, HEK239T and HepG2 cells after 24 h of treatment with the pan-KDM inhibitors JIB-04 and KDM6 inhibitor GSK-J4 at 10 µM (data represent the mean of three independent experiments)

Fig. 3

KDM4A expression after QC6352 (1 µM) treatment and/or epi-editing using CRISPRoff + sgRNA-KDM4A in (A) HEK293T, (B) MCF7, (C) HCT116 and (D) HepG2 cells. All samples were normalized to cells transfected only with CRISPRoff. The first samples of each graph show the negative control (mock): cells transfected with MLM3636, without inserted target sequence

Fig. 4

Proliferation assay in (A-B) HCT116 and (D) MCF7 cells with/without sgRNA-KDM4A (sgRNA) treated with QC6352 at (A) 0.05 µM, (B) 0.25 µM and (D) 1 µM or DMSO (ctr); MTS assay in (C) HCT116 and (E) MCF7 cells with/without sgRNA-KDM4A treated with QC6352 at (C) 0.25 µM and (E) 1 µM or DMSO (ctr). The dark gray bars show the untransfected cells (symbol: circle), in white bars the cells only with CRISPRoff (symbol: diamond), in black the cells transfected with CRISPRoff + sgRNA-KDM4A (symbol: square). The solid bars represent the cells only transfected with epi-editing, and the dashed bars, the cells treated with QC6352 after transfection

Fig. 5

KDM4C gene expression reduction by epi-editing in (A) HEK293T and (B) sorted MCF7 cells 3 days after CRISPRoff and sgRNA-KDM4C transfection; (C) KDM4C gene evaluation in sorted HCT116 cells after 3 and 21 days after transfection; (D) KDM4C expression in HEK293T cells after JIB-04 (5 µM) treatment and CRISPRoff transfection