Investigating the Role of ABL Kinases in Epigenetic Reprogramming in Metastatic Triple-Negative Breast Cancer

Abstract

Triple-Negative Breast Cancer (TNBC) is a highly aggressive breast cancer type with a high propensity to metastasize to distant sites. Despite advances in the treatment of other breast cancer types, mortality in patients with TNBC remains high, highlighting the clinical need for improved molecular characterization of the factors that contribute to TNBC metastasis. TNBC metastasis is a highly dynamic multistep process that involves both genetic and epigenetic networks. Prior work established a role of the Abelson (ABL) family of tyrosine kinases in promoting TNBC metastasis by engaging with specific transcriptional programs, and treatment with ABL-specific inhibitors induced a profound decrease in TNBC bone metastasis in pre-clinical mouse models. Accumulating reports have shown that specific chromatin landscapes are linked to metastatic relapse. Notably, approximately 50% of all human cancers have mutations in chromatin modifying proteins. However, single agent treatment with drugs targeting epigenetic modulators have not yet been successful, owing in part to TNBC tumor cell plasticity and therapy-resistance. Thus, new therapeutic strategies are needed to treat metastatic TNBC. Here we show for the first time that treatment of TNBC with an ABL allosteric inhibitor increased accessibility at gene loci that correspond to transcription factor binding sites at promoter elements, and unexpectedly, identified intronic loci with increased accessibility after ABL inhibitor treatment. These data suggested that ABL kinase activity may promote metastatic spread not only through transcription factor engagement, but also by regulation of chromatin remodeling. Using an unbiased CRISPR-loss-of-function screen, we have identified epigenetic modulators as sensitizers to ABL allosteric inhibition in TNBC cells and uncovered a role for ABL kinases in the regulation of Enhancer of Zeste Homolog 2 (EZH2), the key catalytic element of the Polycomb Repressive Complex 2 (PRC2). Enhanced EZH2 expression is correlated with breast cancer metastasis. EZH2 is a histone methyl-transferase that functions as the catalytic subunit of PRC2, which functions to silence transcription by tri-methylation of lysine on histone H3 (H3K27me3). Emerging data have shown that EZH2 promotes tumorigenesis independently of the PRC2 complex through non-canonical interactions with transcription factors and other targets. Here we report that ABL kinases modulate the activity of EZH2 in metastatic TNBC cells by increasing the canonical activity of EZH2 targeting H3K27me3, while also targeting non-canonical EZH2 signaling. Unexpectedly, we found that ABL inhibition enhances EZH2 binding to non-canonical targets linked to the accumulation of phosphorylated EZH2 (p-EZH2) on threonine 487 (T487), a post translational modification of EZH2 that promotes a shift in binding activity to non-canonical targets. ABL inactivation promotes p-EZH2-T487 accumulation through the regulation of a Focal Adhesion Kinase (FAK)-Cyclin Dependent Kinase (CDK1) signaling axis. Notably, combination treatment with allosteric ABL inhibitors and EZH2 inhibitors elicits a synergistic decrease in TNBC cell survival in vitro and impairs metastasis and extends survival of tumor-bearing mice treated with the combination therapy. Together these data suggest that co-inhibition of ABL and EZH2 might be exploited for the treatment of metastatic TNBC patients.