Targeting the dysregulated BRaf-MEK-ERK pathway in cancer has increasingly emerged in clinical trial design. Despite clinical responses in specific cancers using inhibitors targeting BRaf and MEK, resistance develops often involving non-genomic adaptive bypass mechanisms. Inhibition of MEK1/2 by trametinib in triple negative breast cancer (TNBC) patients induced dramatic transcriptional responses, including upregulation of receptor tyrosine kinases (RTKs) comparing tumor samples before and after one week of treatment. In preclinical models MEK inhibition induced genome-wide enhancer formation involving the seeding of BRD4, MED1, H3K27 acetylation and p300 that drives transcriptional adaptation. Inhibition of P-TEFb associated proteins BRD4 and CBP/p300 arrested enhancer seeding and RTK upregulation. BRD4 bromodomain inhibitors overcame trametinib resistance, producing sustained growth inhibition in cells, xenografts and syngeneic mouse TNBC models. Pharmacological targeting of P-TEFb members in conjunction with MEK inhibition by trametinib is an effective strategy to durably inhibit epigenomic remodeling required for adaptive resistance.

Screening of an inhibitor library targeting kinases and epigenetic regulators identified several molecules having antiproliferative synergy with extraterminal domain (BET) bromodomain (BD) inhibitors (JQ1, OTX015) in triplenegative breast cancer (TNBC). GSK2801, an inhibitor of BAZ2A/B BDs, of the imitation switch chromatin remodeling complexes, and BRD9, of the SWI/SNF complex, demonstrated synergy independent of BRD4 control of P-TEFbmediated pause-release of RNA polymerase II. GSK2801 or RNAi knockdown of BAZ2A/B with JQ1 selectively displaced BRD2 at promoters/enhancers of ETS-regulated genes. Additional displacement of BRD2 from rDNA in the nucleolus coincided with decreased 45S rRNA, revealing a function of BRD2 in regulating RNA polymerase I transcription. In 2D cultures, enhanced displacement of BRD2 from chromatin by combination drug treatment induced senescence. In spheroid cultures, combination treatment induced cleaved caspase-3 and cleaved PARP characteristic of apoptosis in tumor cells. Thus, GSK2801 blocks BRD2-driven transcription in combination with BET inhibitor and induces apoptosis of TNBC. Implications: Synergistic inhibition of BDs encoded in BAZ2A/B, BRD9, and BET proteins induces apoptosis of TNBC by a combinatorial suppression of ribosomal DNA transcription and ETS-regulated genes.

Daniel R. Goulet: Discrete Adaptive Responses to MEK Inhibitor in Subpopulations of Triple Negative Breast Cancer (Under the direction of Gary Johnson) Triple-negative breast cancer (TNBC) is characterized by genomic heterogeneity and high risk of relapse. Recent studies have demonstrated that breast cancers can contain a spectrum of epithelial and mesenchymal phenotypes. We identified epithelial (POS) and mesenchymal (NEG) subpopulations in a triple negative breast cancer cell line that have similar coding sequences, but unique enrichment of the epigenetic activators H3K27ac and BRD4. We show that localization of these epigenetic activators correlates with the expression of epithelial transcription factors TP63 and ELF3 in epithelial cells, and ZEB1 and TWIST1 in mesenchymal cells. Currently, there are no targeted therapies available for the treatment of triple negative breast cancer. However, overexpression of EGFR, KRAS, or BRAF occurs in approximately 30% of TNBC, suggesting that inhibiting the MAPK signaling pathway may be an effective therapy for TNBC. Acute treatment with the MEK inhibitor trametinib induced distinct transcriptional responses in epithelial and mesenchymal subpopulations. ChIP-Seq analysis of H3K27ac and BRD4 binding revealed epigenetic remodeling in response to trametinib treatment, especially at binding sites for AP-1 in NEG cells and epithelial-specific ETS transcription factors in POS cells. Chronic treatment with trametinib induced the emergence of trametinib resistant POS cells, but not NEG cells. In trametinib resistant POS cells, we observe increased expression of the Gi-coupled chemokine receptor CXCR7, and its cognate ligand adrenomedullin, as well as increased expression of KRAS. ChIP-Seq analysis revealed enrichment of H3K27ac and BRD4 at the CXCR promoter and enhancer, indicating epigenetic remodeling induces transcriptional activation. siRNA knockdown of CXCR7 and KRAS demonstrate these genes are essential for the proliferation of trametinib resistant cells, and reveals that epigenetic remodeling at the CXCR7 locus may be a mechanism for drug resistance. CCND1 Cyclin D1 CD49f Integrin alpha 6 CDH1 E-Cadherin CDK4 Cyclin-dependent kinase 4 CDK6 Cyclin-dependent kinase 6 CDKN2A Cyclin dependent kinase inhibitor 2A cDNA Complementary DNA CHD1 Chromodomain helicase binding protein 1 ChIP-Seq Chromatin Immunoprecipitation Sequencing xiv cm Centimeter CML Chronic Myelogenous Leukemia CpG Cytosine-Guanine dinucleotide CRISPR Clustered Regularly Interspaced Short Palindromic Repeats CTCF CCCTC-binding factor CTD C-terminal domain CXCR7 Chemokine receptor 7 DCLK1 Doublecortin-like kinase 1

Inhibition of the HER2/ERBB2 receptor is a keystone to treating HER2-positive malignancies, particularly breast cancer, but a significant fraction of HER2-positive (HER2+) breast cancers recur or fail to respond. Anti-HER2 monoclonal antibodies, like trastuzumab or pertuzumab, and ATP active site inhibitors like lapatinib, commonly lack durability because of adaptive changes in the tumor leading to resistance. HER2+ cell line responses to inhibition with lapatinib were analyzed by RNAseq and ChIPseq to characterize transcriptional and epigenetic changes. Motif analysis of lapatinib-responsive genomic regions implicated the pioneer transcription factor FOXA1 as a mediator of adaptive responses. Lapatinib in combination with FOXA1 depletion led to dysregulation of enhancers, impaired adaptive upregulation of HER3, and decreased proliferation. HER2-directed therapy using clinically relevant drugs (trastuzumab with or without lapatinib or pertuzumab) in a 7-day clinical trial designed to examine early pharmacodynamic response to antibody-based anti-HER2 therapy showed reduced FOXA1 expression was coincident with decreased HER2 and HER3 levels, decreased proliferation gene signatures, and increased immune gene signatures. This highlights the importance of the immune response to anti-HER2 antibodies and suggests that inhibiting FOXA1-mediated adaptive responses in combination with HER2 targeting is a potential therapeutic strategy.