PII-045 - DNA METHYLTRANSFERASE INHIBITORS REDUCE TRIPLE NEGATIVE BREAST CANCER CELL GROWTH THROUGH ENHANCED IMMUNE RESPONSE BY DOWNREGULATION OF ZNF101

X. Wang¹, H. Gao², J. Yu³, M. Wang¹, D. Liu¹, R. Weinshilboum¹, L. Wang¹; ¹Mayo Clinic, Rochester, MN, USA, ²Yale University, CT, United States, ³Mayo clinic, MN, United states.

Background: Triple Negative Breast Cancer (TNBC) is the most aggressive subtype of breast cancer (BC) and has a relative lack of treatment options. Our previous studies showed that a subgroup of TNBC tumors with high expression of DNA methylation transferases 3A and 3B (DNMT3A and DNMT3B) responded to DNMT inhibitor (DNMTi) treatment, and that DNMTi synergized with immune check point inhibitors in mouse models. However, the therapeutic mechanisms of DNMTi in TNBC, particularly the interaction with immunotherapy remain unclear.

Methods: To investigate molecular mechanism(s) of DNMTi treatment in TNBC, two TNBC cell lines (BT549 and MDA-MB-231) were treated with vehicle or two DNMTis (Decitabine and 5-aza-4’-thio-2’-deoxycytidine), respectively. After DNMTi treatment, a series of next-gen sequencing-based assays, including RNA-seq, reduced representation bisulfite sequencing (RRBS), Chromatin immunoprecipitation (ChIP) assay and assay for transposase-accessible chromatin with sequencing (ATAC-seq), were performed to understand transcriptomic and epigenomic responses to DNMTi in TNBC. Gene(s) identified by those sequencing data were functional validated by molecular assays such as qPCR, Western blot, CyQUANT assay and immunoprecipitation.

Results: RNA-seq identified highly enriched upregulated immune-related pathways such as interferon (IFN) signaling pathway, consistent between the two TNBC cell lines after two DNMTi treatments. By integrating those transcriptomic and epigenomic data, ZNF101 was identified as the one of the most altered genes within regions showing chromatin status changes. An unique cluster of genes in IFNα/β pathways was found to be associated with ZNF101. Ectopic expression of ZNF101 increases TNBC cellular resistance to DNMTi treatment. Furthermore, ZNF101 was found to bind with interferon regulatory factor 3 (IRF3) and to negatively regulate IRF3 function, thus influencing the expression of IFN/ISGs.

Conclusion: Our data suggest that the downregulation of ZNF101 by DNMTi activates the IFN signaling pathway which is involved in DNMTi-induced immune responses and apoptosis in TNBC cells. These results support the future study of the application of DNMTi in the treatment of TNBC.