Targeting the rRNA m6A methyltransferase ZCCHC4 in hepatocellular carcinoma with small-molecule inhibitors

• Applicant: Dr. Ines Helen Kaltheuner
• Subject Area: Biochemistry ; Hematology, Oncology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 563607013

RNA methylation, particularly N6-methyladenosine (m6A), is a key regulator in gene expression and various cellular processes. In human, m6A modification is most common in mRNA, which has been the major focus of research. However, most of the global m6A transcriptome content is contributed by rRNA m6A on the small and large ribosomal subunits. Interestingly, human rRNA contains only two m6A modifications, one in 28S rRNA at position A4220, catalyzed by ZCCHC4, and the second one in 18S rRNA at position A1832, mediated by METTL5. In contrast to METTL5, loss of ZCCHC4 impacts global translation in human cells. Furthermore, an increasing amount of data demonstrates that ZCCHC4 is overexpressed in multiple human cancer tissues, such as hepatocellular carcinoma, colorectal cancer, and glioblastoma, where it contributes to tumorigenesis, chemoresistance, and poor prognosis, highlighting the potential of ZCCHC4 as a novel therapeutic target. Strikingly, METTL3, the mRNA m6A enzyme, is the only RNA m6A methyltransferase with any reported inhibitors to date, one of which has progressed to clinical phase-1 trials. To further investigate the therapeutic potential of ZCCHC4, I aim to develop a small-molecule inhibitor of ZCCHC4. A biochemical activity assay (for IC50 determination), a thermal shift assay, surface plasmon resonance (for KD determination), a mass spectrometry-based cell assay, and co-crystallization of ZCCHC4 with potential inhibitors are already in place, providing the ideal set-up for the screening and validation of potential inhibitors. Initial screening will focus on targeted libraries from structurally related DNA methyltransferases, which share both, a similar cofactor and nucleotide binding site with ZCCHC4. After validation and lead compound optimization, inhibitors will be tested for their inhibitory potency and selectivity and if necessary, covalent inhibitors or proteolysis-targeting chimeras (PROTACs) will be developed, which will ultimately result in potent and selective ZCCHC4 inhibitors. Second, I will explore the therapeutic potential of ZCCHC4 inhibitors and potential beneficial effects on chemotherapeutic resistance in a hepatocellular carcinoma cell line, as well as other cancer cell lines. This will help to identify cancer types that could benefit from therapeutically targeting ZCCHC4. This project represents a unique opportunity, not only to develop the first in class rRNA m6A inhibitor, but is also timely considering that an increasing amount of data highlights the oncogenic relevance of RNA m6A enzymes, such as ZCCHC4, suggesting that inhibitors for these family members will soon become the focus of therapeutic research. By combining inhibitor development with functional analysis in cancer cells, this project aims to validate ZCCHC4 as a promising therapeutic target in cancer treatment and to further contribute on the understudied mechanisms of epitranscriptomic regulation in cancer.

• DFG Programme: WBP Fellowship
• International Connection: United Kingdom
• Host: Professor Dr. Yang Shi