Abstract A020: Histone modification by Kmt2c and Kmt2d regulate metastasis in triple-negative breast cancer in vivo

Abstract

Abstract Breast cancer is the most common cancer type worldwide accounting for more than 2 million newly diagnosed cases in 2020. Approximately 20% of all breast tumors in the USA is the highly metastatic subtype triple-negative breast cancer (TNBC). Despite recent development of new therapies, disease-free survival rates of TNBC patients are still relatively low. In this study we describe the role of histone modifier lysine methyltransferase 2C and 2D (KMT2C and KMT2D) in metastasis. KMT2C and KMT2D are among the most commonly mutated genes across all cancer types including TNBC. To examine the functional relevance of Kmt2c and Kmt2d mutations in a murine TNBC mouse model, we mutated Kmt2c and Kmt2d using CRISPR/Cas9. In order to minimize confounder effects of constitutive CRISPR/Cas9 expression we utilized a unique strategy of consecutive transient transfections. Mutation of Kmt2c or Kmt2d did not impact tumor growth in vivo, however, Kmt2c and Kmt2d mutant tumors were highly metastatic to multiple organs while no metastases were found in mice with wildtype tumors. To unravel the underlying mechanisms, we first performed histone mass spectrometry. As expected, direct histone modification target of Kmt2cd and Kmt2d, H3K4me1 levels were reduced in mutant cells. Interestingly, we also identified decreased levels of H3K27me3 and increased levels of H3K27ac. To further delineate the impact of these changes we performed ChIP-seq for these three histone modifications. In accordance with the histone mass spectrometry data peak intensities for H3K4me1, H3K27me3, and H3K27ac were increased or decreased, respectively, in mutant cells. Additionally, to examine effects of these changes on chromatin remodeling we performed ATAC sequencing. We identified differencent chromatin structures between wildtype and mutant cells, validating the impact of Kmt2c and Kmt2d perturbation on histone modification and chromatin remodeling. Lastly, we performed RNA-seq and PRO-seq analyses to identify genes and pathways that could explain the increased metastatic capacity of the mutant cells. Gene set enrichment analyses revealed differential regulation of pro-inflammatory pathways in Kmt2c mutant cells correlating with increased number of T-cells in Kmt2c mutant tumors. Kmt2d mutant cells showed alterations in metabolic pathways which we also identified by unbiased metabolomics showing significant changes. This study revealed that mutations in Kmt2c and Kmt2d are sufficient to induce distant metastases and we deciphered underlying mechanism that could be explored as novel strategies to treat or prevent metastasis. Citation Format: Marco Seehawer, Zheqi Li, Jun Nishida, Paloma Cejas, Andrew Reiter, Seth Goldman, Pierre Foidart, Ernesto Rojas, Malvina Papanastasiou, Karen Adelman, Henry Long, Kornelia Polyak. Histone modification by Kmt2c and Kmt2d regulate metastasis in triple-negative breast cancer in vivo. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A020.