Abstract P3-05-03: KMT2D as a novel therapeutic target for HER2+ breast cancers
Authors: Ma Emily , Zlobin Andrei , Wyatt Debra , Ng Jeffrey , Dingwall Andrew , Osipo Clodia 
Published: March 2022

Background: Breast cancer is the second leading cause of cancer-related deaths among women in the US. Major factors that contribute to breast cancer carcinogenesis include disruptions in both genetic and epigenetic processes. Histone modifications and DNA-methylation are common epigenetic changes that have been implicated in several breast cancers, including an aggressive subset known as human epidermal growth factor receptor 2 (HER2+) breast cancers. One prognostic marker for poor survival in women with HER2+ breast cancer is high RNA expression of the critical epigenetic regulator, Histone-lysine methyltransferase 2D (KMT2D). KMT2D belongs to a family of histone modifying proteins which plays an essential role in regulating developmental processes. Specifically, KMT2D is a histone methyltransferase that monomethylates histone 3 lysine 4 at enhancers throughout the genome and is required for transcriptional activation. Interestingly, in HER2+ cells (BT474), KMT2D mRNA was found to be significantly increased in stem-like tumor initiating cells (TICs) compared to bulk cells. BT474 cells that have acquired resistance to trastuzumab (BTR) from prolonged treatment express higher levels of KMT2D than those of the isogenic parental sensitive cells (BTS). Furthermore, siRNA-specific knockdown of KMT2D significantly decreased bulk cell proliferation in both BTS and BTR cells, and significantly inhibited TIC survival in BTS cells. Thus, we hypothesized that KMT2D is a critical epigenetic regulator that contributes to resistance by promoting bulk cell proliferation and TIC survival. Methods: RNA-sequencing was performed in BTS and BTR cells-expressing or depleted for KMT2D. Differentially regulated genes were identified and pathway analysis was performed. Chromatin immunoprecipitation (ChIP) PCR and sequencing were performed to identify novel KMT2D-bound regions in both BTS and BTR cells. SiRNA induced silencing was also utilized to study various gene functions in BT474 cells. Results: We identified and confirmed by RT-qPCR several differentially expressed genes that were positively and negatively regulated by KMT2D, suggesting they may be critical direct targets of KMT2D. One gene, ITGB6, encodes for integrin subunit beta 6 and was found to be decreased upon KMT2D depletion. ITGB6 regulates multiple processes which contribute to cancer progression and metastases, including cellular proliferation and epithelial-mesenchymal transition. Similar to KMT2D knockdown, ITGB6 knockdown resulted in decreased BTS and BTR bulk cell proliferation and a reduction in TICs. Interestingly, KMT2D enrichment at an ITGB6 enhancer region was lower in resistant cells compared to sensitive cells. Future studies will include knockdown of KMT2D with simultaneous rescue of wild type or a mutant of ITGB6 to determine if KMT2D-mediated increase in ITGB6 is required for HER2+ cell growth and/or TIC survival. Furthermore, our data also suggests an anti-apoptotic and cell-cell adhesion role for KMT2D, which may in part be due to its direct effects on ITGB6. Additional future studies will investigate the mechanism of how KMT2D regulates its target genes through ChIP-sequencing analysis as well as studies to evaluate KMT2D’s role in vivo. Conclusions: Our results suggest a critical role for KMT2D in HER2+ breast cancer progression, and may yield novel therapeutic targets and pathways for the treatment of HER2+ breast cancers.

Citation Format: Emily Ma, Andrei Zlobin, Debra Wyatt, Jeffrey Ng, Andrew Dingwall, Clodia Osipo. KMT2D as a novel therapeutic target for HER2+ breast cancers [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-05-03.