Purpose-Triple-negative breast cancer (TNBC) lacks the receptor targets estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2, and thus it does not respond to receptor-targeted treatments. TNBC has higher recurrence, metastasis, and mortality rates than other subtypes of breast cancer. Mounting data suggest that the MAPK (also known as RAS-RAF-MEK-ERK) pathway is an important therapeutic target in TNBC.Methods-To evaluate anti-tumor and anti-metastasis efficacy of E6201, we used cell proliferation assay, soft agar assay, cell cycle assay, Annexin V staining assay, immunoblotting analysis, immunohistochemistry, migration assay, invasion assay, mammary fat pad xenograft, and experimental and spontaneous metastasis xenograft models. We also evaluated the anti-tumor efficacy of E6201 plus CDK4/6 inhibitor, mTOR inhibitor, or ATR inhibitor.Results-E6201 inhibited TNBC cell colony formation, migration, and invasion in a dosedependent manner. E6201 induced G1 cell cycle arrest and apoptosis. E6201 inhibited TNBC xenograft growth and inhibited TNBC lung metastasis and improved mouse survival in experimental metastasis and spontaneous metastasis assays. Immunohistochemical staining demonstrated that E6201 decreased the metastatic burden in the lung and decreased phosphorylated ERK expression in a dose-dependent manner. Combination of E6201 with CDK4/6 inhibitor or mTOR inhibitor enhanced E6201's in vitro anti-tumor efficacy.

The original algorithm that classified triple-negative breast cancer (TNBC) into six subtypes has recently been revised. The revised algorithm (TNBCtype-IM) classifies TNBC into five subtypes and a modifier based on immunological (IM) signatures. The molecular signature may differ between cancer cells in vitro and their respective tumor xenografts. We identified cell lines with concordant molecular subtypes regardless of classification algorithm or analysis of cells in vitro or in vivo, to establish a panel of clinically relevant molecularly stable TNBC models for translational research. Gene expression data were used to classify TNBC cell lines using the original and the revised algorithms. Tumor xenografts were established from 17 cell lines and subjected to gene expression profiling with the original 2188-gene algorithm TNBCtype and the revised 101-gene algorithm TNBCtype-IM. A total of six cell lines (SUM149PT (BL2), HCC1806 (BL2), SUM149PT (BL2), BT549 (M), MDA-MB-453 (LAR), and HCC2157 (BL1)) maintained their subtype classification between in vitro and tumor xenograft analyses across both algorithms. For TNBC molecular classification-guided translational research, we recommend using these TNBC cell lines with stable molecular subtypes.

Inflammatory breast cancer (IBC), the most aggressive breast cancer subtype, is driven by an immunosuppressive tumor microenvironment (TME). Current treatments for IBC have limited efficacy. In a clinical trial (NCT01036087), an anti-EGFR antibody combined with neoadjuvant chemotherapy produced the highest pathological complete response rate ever reported in patients with IBC having triple-negative receptor status. We determined the molecular and immunological mechanisms behind this superior clinical outcome. Using novel humanized IBC mouse models, we discovered that EGFR-targeted therapy remodels the IBC TME by increasing cytotoxic T cells and reducing immunosuppressive regulatory T cells and M2 macrophages. These changes were due to diminishing immunosuppressive chemokine expression regulated by transcription factor EGR1. We also showed that induction of an immunoactive IBC TME by an anti-EGFR antibody improved the antitumor efficacy of an anti–PD-L1 antibody. Our findings lay the foundation for clinical trials evaluating EGFR-targeted therapy combined with immune checkpoint inhibitors in patients with cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subgroup of breast cancer, and patients with TNBC have few therapeutic options. Apoptosis resistance is a hallmark of human cancer, and apoptosis regulators have been targeted for drug development for cancer treatment. One class of apoptosis regulators is the inhibitors of apoptosis proteins (IAPs). Dysregulated IAP expression has been reported in many cancers, including breast cancer, and has been shown to be responsible for resistance to chemotherapy. Therefore, IAPs have become attractive molecular targets for cancer treatment. Here, we first investigated the antitumor efficacy of birinapant (TL32711), a biindole-based bivalent mimetic of second mitochondria-derived activator of caspases (SMACs), in TNBC. We found that birinapant as a single agent has differential antiproliferation effects in TNBC cells. We next assessed whether birinapant has a synergistic effect with commonly used anticancer drugs, including entinostat (class I histone deacetylase inhibitor), cisplatin, paclitaxel, voxtalisib (PI3K inhibitor), dasatinib (Src inhibitor), erlotinib (EGFR inhibitor), and gemcitabine, in TNBC. Among these tested drugs, gemcitabine showed a strong synergistic effect with birinapant. Birinapant significantly enhanced the antitumor activity of gemcitabine in TNBC both in vitro and in xenograft mouse models through activation of the intrinsic apoptosis pathway via degradation of cIAP2 and XIAP, leading to apoptotic cell death. Our findings demonstrate the therapeutic potential of birinapant to enhance the antitumor efficacy of gemcitabine in TNBC by targeting the IAP family of proteins.

BACKGROUND: CDK4/6 regulates the G1-S phase transition by phosphorylating the retinoblastoma protein (Rb). Given their potent clinical efficacy, CDK4/6 inhibitors used in combination with hormone receptor (HR) blockade (with an aromatase inhibitor or fulvestrant) are emerging as the standard of care for patients with metastatic HR-positive breast cancers. The CDK4/6 inhibitors palbociclib and ribociclib are FDA-approved for use in HR-positive breast cancer patients, and abemaciclib is currently in phase III trials. We observed that approximately 74% (25/34) of breast cancer cell lines had high phosphorylated Rb (phospho-Rb) expression levels and that triple-negative breast cancer (TNBC) cell lines often expressed phospho-Rb, suggesting that targeting phospho-Rb via CDK4/6 inhibition may be effective against TNBC. The histone deacetylase (HDAC) inhibitors increase p21Cip1 levels, promoting proteasomal degradation of cyclin B1 and resulting in G2/M arrest. Entinostat is an oral, class 1, selective HDAC inhibitor currently in phase III testing in HR-positive breast cancer. Preclinical and clinical data demonstrate that entinostat, in combination with HR blockade, has anticancer activity. Our group recently reported that entinostat combined with other anticancer drugs induced apoptosis via induction of proapoptotic proteins such as Noxa and Bim in breast cancer cell lines. Based on these findings, we hypothesized that entinostat-induced apoptosis and palbociclib-induced cell cycle arrest synergize to produce enhanced antitumor effects in estrogen receptor (ER)-positive breast cancer and TNBC cell lines with high phospho-Rb expression levels. METHODS: We assessed the combination antitumor effects and their mechanisms via CellTiter Blue and sulforhodamine B assays, flow cytometry, apoptosis (caspase 3/7) assays, anchorage-independent growth assays, Western blotting, reverse phase protein array (RPPA), and mammary fat pad xenograft mouse models. RESULTS: RPPA data showed that ER-positive and TNBC cell lines more often expressed phospho-Rb than did other breast cancer cell subtypes (7/10 and 8/17 cell lines, respectively). We found that the combination of entinostat and palbociclib synergistically inhibited tumor cell proliferation (combinational index less than 1.0), reduced in vitro colony formation (P < 0.05), inhibited in vivo tumor growth in ER-positive MCF-7 breast cancer cells (P < 0.05), and inhibited tumor growth in TNBC xenograft mouse models (MDA-MB-231) more effectively than did either drug alone. CONCLUSION: Taken together, our data provide evidence that combining entinostat with palbociclib enhances the antitumor effects of these drugs. Along with our continued effort to determine predictive biomarkers, our findings justify conducting a clinical trial of combination treatment with entinostat and palbociclib in patients with ER-positive breast cancer or TNBC. Citation Format: Lee J, Lim B, Pearson T, Tripathy D, Ordentlich P, Ueno NT. The synergistic antitumor activity of entinostat (MS-275) in combination with palbociclib (PD 0332991) in estrogen receptor-positive and triple-negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-21-15.