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Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer. Among TNBC subtypes, the luminal androgen receptor (LAR) subtype expresses high levels of androgen receptor (AR), tends to be less proliferative, and generally responds poorly to neoadjuvant therapy. Previous studies have shown that AR inhibition suppressed proliferation of AR+ or LAR subtype TNBC cells and tumor growth in xenograft models. Thus, AR is a promising therapeutic target for this TNBC subtype. Here, we identified kinase targets for enhancing the antitumor efficacy of the AR inhibitor enzalutamide in preclinical models and investigated the underlying molecular mechanisms. Methods: We performed a nonbiased high-throughput kinome siRNA screening (targeting 709 genes) to identify a synergistic partner for enhancing the antitumor efficacy of the AR inhibitor enzalutamide in AR+ LAR TNBC. The growth inhibitory effects of enzalutamide alone or combined with kinase inhibitors were determined using the sulforhodamine B staining assay and clonogenic assay. The effect of treatments on the expression of target proteins of interest was examined by Western blot analysis. Results: Enzalutamide was not effective as a single agent to inhibit the proliferation of AR+ TNBC cells (IC50 >15 µM in MDA-MB-453, CAL-51, CAL-148, MFM223, HCC2185, SUM149, SUM159, and SUM185 cells). Nonbiased high-throughput kinome siRNA screening identified PI3K/AKT/mTOR, cell cycle, and JNK pathways as potential canonical targets for combination with enzalutamide to enhance its antitumor efficacy in AR+ LAR TNBC. Among these pathways, inhibition of cell cycle progression using the CDK7 inhibitor UD-017 showed the most synergistic anti-proliferation effect with enzalutamide in AR+ LAR MDA-MB-453 (50.61% reduction in proliferation compared with enzalutamide alone, P < 0.001; and 48.98% reduction in proliferation compared with UD-017 alone, P < 0.001) and SUM185 (40.41% reduction in proliferation compared with enzalutamide alone, P < 0.05; and 42.76% reduction in proliferation compared with UD-017 alone, P < 0.05) TNBC cells. Furthermore, CDK7 knockdown using siRNA significantly enhanced the sensitivity of MDA-MB-453 (60.98%, P < 0.01) and SUM185 (30.99%, P < 0.01) cells to enzalutamide, and AR knockdown significantly enhanced the sensitivity of MDA-MB-453 (32.64%, P < 0.01) and SUM185 (43.62%, P < 0.01) cells to UD-017, in both cases with a sensitivity similar to that of enzalutamide plus UD-017. This result suggests that the synergy of enzalutamide and UD-017 results from specific targeting of AR and CDK7. Downstream target analysis revealed that the combination of enzalutamide and UD-017 dramatically reduced the expression of c-MYC. c-MYC knockdown using siRNA dramatically suppressed colony formation in both MDA-MB-453 and SUM185 cells at a degree similar to that of enzalutamide plus UD-017, whereas c-MYC overexpression reversed the synergistic effect of the combination treatment. This result suggests that UD-017 synergizes with enzalutamide through inhibition of c-MYC–mediated tumorigenesis. Conclusion: Our results suggest that enzalutamide synergizes with UD-017 by inhibiting c-MYC–mediated oncogenic activity. These in vitro data warrant future in vivo studies of the antitumor synergy of enzalutamide plus UD-017 in AR+ LAR TNBC models. Citation Format: Xuemei Xie, Marwa Manai, Jon A. Fuson, Troy Pearson, Dileep R. Rampa, Debu Tripathy, Jangsoon Lee, Naoto T. Ueno. Targeting CDK7 enhances the antitumor efficacy of enzalutamide in androgen receptor-positive triple-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-01.

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.

Supplementary Data

Background: Inflammatory breast cancer (IBC) is the most lethal and aggressive form of breast cancer, yet no targeted therapy has been approved specifically for this disease. There is a critical need for innovative treatment approaches for patients with IBC. There is a paucity of data on immune checkpoint inhibitors (ICIs) in IBC, yet a strong biological rationale exists to lay the groundwork for testing the efficacy of ICIs in IBC. We have previously shown that an anti–epidermal growth factor receptor (EGFR) antibody, panitumumab (sponsored by Amgen), combined with Abraxane (sponsored by Bristol Myers Squibb), and carboplatin followed by 5-fluorouracil, epirubicin, and cyclophosphamide achieves a very high pathologic complete response (42%) in patients with triple-negative receptor status (TN-IBC) (NCT01036087). We hypothesize that inducing a broad shift in the IBC tumor microenvironment (TME), a critical driver of the IBC clinical phenotype and metastasis, from an immunosuppressive to an immunoreactive phenotype can enhance the efficacy of ICIs. Here we report the impact of targeting EGFR on ICI effectiveness by modulating the immunosuppressive TME in TN-IBC. Methods: We examined the effects of panitumumab on components of the immune TME in IBC patient tissues collected before and after panitumumab treatment. Furthermore, we analyzed panitumumab effects in an IBC SUM149 humanized mouse model (SUM149-hu-NSG-SGM3) by using multiplex immunofluorescence staining (mIF) of single-cell RNA-sequencing (scRNA-seq) and multicolor flow cytometry. We examined the changes in chemokines in humanized mouse tissue after panitumumab treatment using a cytokine antibody array and enzyme-linked immunosorbent assay (ELISA). We also studied the mechanism of EGFR regulation of immunosuppressive chemokine expression via transcription factor EGR1 by using reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and chromatin immunoprecipitation (ChIP) assays. In addition, we tested the efficacy of panitumumab combined with anti-PD-L1 antibody (Bio X Cell, clone 29E.2A3) in reducing IBC tumor growth in a humanized IBC SUM149 mouse model. Results: IBC patients who achieved pathologic complete response after receiving panitumumab/neoadjuvant chemotherapy had increased levels of CD8+ T-cells and decreased levels of M2 macrophages and T-reg cells. In the humanized mouse model, panitumumab treatment reduced IBC tumor growth and increased immune response that favors an antitumor effect, as shown by scRNA-seq, flow cytometry, and mIF analyses. In addition, we observed that panitumumab treatment increased the expression of chemokines that function as a chemoattractant for CD8+ T-cells, including CXCL10, CCL4, and CXCL9. We also observed that panitumumab treatment reduced EGR1 expression, leading to the downregulation of chemokines involved in the recruitment of immunosuppressive M2 macrophages and T-reg cells, including CCL2, CCL20 CXCL5, and IL-8. Finally, panitumumab enhanced the reduction in tumor growth by anti-PD-L1 antibody treatment; furthermore, the combination of panitumumab and anti-PD-L1 antibody increased the presence of CD8+ T-cells and reduced the presence of T-reg cells and M2 macrophages more effectively than either treatment alone. Conclusion: Panitumumab treatment converts the immunosuppressive IBC TME to an immunoreactive status by modulating the global expression of chemokines by downregulating the EGR1 transcription factor. This modulation of the TME by panitumumab improves the inhibition of IBC tumor growth by an ICI. Our study is the first to demonstrate that targeting the EGFR/EGR1 axis remodels the IBC TME by regulating chemokine expression, thus boosting the antitumor immune response in IBC and response to ICI. Citation Format: Xiaoping Wang, Takashi Semba, Ganiraju C. Manyam, Jing Wang, Shan Shao, Francois Bertucci, Pascal Finetti, Savitri Krishnamurthy, Lan ThiHanh Phi, Troy Pearson, Jared K. Burks, Evan N. Cohen, James M. Reuben, Fei Yang, Hu Min, Nicholas Navin, Toshiaki Iwase, Yichao Shen, Xiang Zhang, Debu Tripathy, Naoto T. Ueno. Remodeling the inflammatory breast cancer tumor microenvironment to enhance immunotherapy: Novel therapeutic development [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 PD10-08.

Background: LMP7 (beta5i/PSMB8) is a proteolytic subunit of the immunoproteasome that is predominantly expressed in leukocytes and inflamed tissues. The immunoproteasome degrades ubiquitinated proteins for maintenance of cell homeostasis and generation of peptides for MHC class I presentation. LMP7 has been implicated in multiple myeloma, autoimmune and inflammatory diseases, and inflammation-related cancers. Here, we evaluated the effect of M3258, an orally bioavailable, highly-selective, and reversible inhibitor of LMP7, on the tumor microenvironment (TME) using in vitro and in vivo models of triple-negative breast cancer (TNBC) and inflammatory breast cancer (IBC). Methods: The proteolytic activity of M3258 was determined using the fluorogenic LMP7-specific peptidic substrate (Ac-ANW)2R110. Its anti-proliferation activity was determined using the CellTiter-Blue cell viability and sulforhodamine B staining assays. Expression of immunoproteasome subunits under different conditions was examined by Western blotting. All in vitro assessments were done with or without IFNγ, which is known to increase immunoproteasome expression. The in vivo anti-tumor efficacy of M3258 was evaluated using a SUM-149 PT TN-IBC xenograft mouse model implanted in humanized mice. Single-cell RNA sequencing (scRNA-seq) was used in this same study to examine the impact of M3258 on the TME. Results: M3258 inhibited LMP7 activity (IC50 = 6.5-212.8 nM) and had no effects on LMP7 protein expression in a panel of TNBC cells (DU4475, HCC1187), IBC cells (SUM190, IBC3, KPL4), and TN-IBC cells (SUM-149 PT, BCX-010, FC-IBC02). M3258 inhibited proliferation of TNBC and IBC cell lines in vitro (IC50 = 1-20 µM). In keeping with previous reports, IFNγ treatment strongly induced LMP7 expression and enhanced its proteolytic activity in these cell lines in vitro and increased M3258 sensitivity (2- to 5-fold). In the humanized SUM-149 PT xenograft mouse model, M3258 orally administered at 10 mg/kg significantly inhibited tumor growth versus vehicle (31.4%, P < 0.01) and was tolerated. ScRNA-seq analysis of the terminal tumor samples from this study revealed that M3258 reduced the abundance of CD4+ and CD8+ T cells, Tregs, M1 and M2 macrophages, and dendritic cells. Gene expression profiling indicated a particularly pronounced inhibitory effect of M3258 on M1 and M2 macrophages. Furthermore, M3258 treatment activated CD8+ T cells and suppressed expression of genes involved in inflammatory response, IFNα signaling, IFNγ signaling, and TNFα signaling in immune cells. In light of the scRNA-seq findings, we assessed the impact of M1 and M2 macrophages on the invasiveness of TN-IBC cells in vitro. Co-cultures with M1 or M2 macrophages enhanced invasiveness of SUM-149 PT cells (4.6-fold by M1, P < 0.001; 6.3-fold by M2, P < 0.01) and BCX-010 cells (2.1-fold by M1, P < 0.05; 4.0-fold by M2, P < 0.01). Since M2 macrophages showed a superior effect than M1 macrophages on TN-IBC cell invasiveness, we further assessed M3258’s ability to inhibit such activity. M3258 at 500 nM significantly inhibited M2 macrophage-induced invasion of SUM-149 PT cells (29.6%, P < 0.05) and BCX-010 (49.5%, P < 0.01). Conclusion: Using the exquisitely selective LMP7 inhibitor M3258, we were able to demonstrate for the first time that LMP7 plays an important role in the inflammatory microenvironment, proliferation and invasiveness of TNBC and IBC cells, in particular by modulating the pathogenic role of M2 macrophages. These data warrant future in vivo studies into the antitumor efficacy of M3258 when used with immunotherapy agents. Citation Format: Xuemei Xie, Jangsoon Lee, Ganiraju C Manyam, Troy Pearson, Gina Walter-Bausch, Manja Friese-Hamim, Samantha M Goodstal, Debu Tripathy, Wang Jing, Michael Sanderson, Naoto T Ueno. LMP7-specific inhibitor M3258 modulates the tumor microenvironment of aggressive breast cancer [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 P5-08-04.

Background: Neratinib is a potent, irreversible pan-HER inhibitor that inhibits the ErbB family members EGFR, HER2, and HER4 and downstream signal transduction of these receptors. Triple-negative breast cancer (TNBC) is a heterogeneous disease that lacks druggable levels of receptors for estrogen, progesterone and HER2 and therefore challenging to treat. There is evidence that some cases of TNBC have activated signaling pathways mediated by ErbB family members that may contribute to aggressive behavior. The purpose of this preclinical study was to identify and validate kinases whose targeting may enhance the antitumor activity of neratinib in TNBC cell lines. Methods: In vitro proliferation assays were used to evaluate the efficacy of neratinib in TNBC cell lines. Baseline and post-neratinib-treatment expression of EGFR and phosphorylated EGFR (phospho-EGFR) were assessed via Western blot analysis in 18 TNBC cell lines. Reverse-phase protein array (RPPA) was used to profile and validate the signaling networks induced by neratinib. To identify potential targets or pathways that may synergize with neratinib treatment, we performed high-throughput RNA interference (HT RNAi) screening using a 709-kinome library. CellTiter-Blue, sulforhodamine B, and soft-agar assays were performed to evaluate the antiproliferative effect of neratinib alone and with target inhibitor. Mammary fat pad xenograft models were used to evaluate the efficacy of neratinib alone or with inhibitor in vivo. Results: In vitro proliferation assays showed that the half-maximal inhibitory concentration (IC50) of neratinib in tested TNBC cell lines ranged from 0.16 µM to 1.25 µM. RPPA and Western blot analyses revealed that the efficacy of neratinib correlated with phospho-EGFR expression levels across the TNBC cell lines tested (R2 = 0.3245). Among the tested TNBC cell lines, SUM149 cells (PIK3CA wild-type) were selected for high throughput RNAi screening because this cell line has high EGFR expression and is moderately sensitive to neratinib (IC50 = 0.35 µM). We identified the 40 most relevant kinase targets by the sensitivity index analysis, and further pathway analysis identified PI3K/AKT/mTOR (drug: everolimus) and MAPK (drug: trametinib) as major canonical pathways whose targeting enhanced the cytotoxic effect of neratinib. Everolimus (mTOR inhibitor) produced a strong antiproliferative effect when combined with neratinib in most tested TNBC cell lines (12 of 15 cell lines; combination index [CI] values, 0.1-0.5) and was more effective in PIK3CA-mutated compared to wildtype cell lines. Trametinib (MEK inhibitor) showed a moderate antiproliferative effect (effective in 10 of 15 cell lines; CI values, 0.2-0.9). Synergistic antitumor effects of neratinib combined with everolimus or with trametinib were also observed in anchorage-independent growth conditions (P < 0.05). In vivo experiments demonstrated that neratinib plus everolimus and neratinib plus trametinib combinations inhibited tumor growth in the SUM149 xenograft model for than single drug (neratinib, 42.3% growth inhibition; everolimus, 29.7%; trametinib, 47.1%; neratinib plus everolimus, 69.7%; neratinib plus trametinib, 77.7%; P < 0.0001). Conclusion: Combining neratinib with everolimus or with trametinib enhanced the antitumor effects of these drugs in TNBC regardless of PIK3CA mutation status, and clinical investigations evaluating these combination regimens for the treatment of TNBC are warranted. Citation Format: Jangsoon Lee, Troy Pearson, Huey Liu, Jon A. Fuson, Toshiaki Iwase, Irmina Diala, Alshad S. Lalani, Lisa D. Eli, Debu Tripathy, Bora Lim, Naoto T. Ueno. Identification of novel molecules that enhance neratinib efficacy in triple-negative breast cancer by high-throughput RNA interference [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-07.

Adipose stromal cells (ASCs) recruited by tumors contribute to the population of cancer-associated fibroblasts. ASCs have been reported to induce tumor growth and chemotherapy resistance. The effect of ASCs on metastasis has not been explored. Here, we investigated the role of ASCs in cancer aggressiveness and tested them as a therapy target. We show that ASCs promote the epithelial-mesenchymal transition and invasiveness of triple-negative breast cancer cells. In human cell lines derived from various types of breast tumors, ASCs suppressed cytotoxicity of cisplatin and paclitaxel. D-CAN, a proapoptotic peptide targeting ASC, suppressed spontaneous breast cancer lung metastases in a mouse allograft model when combined with cisplatin. Moreover, in a human breast cancer xenograft model, treatment with D-CAN alone was sufficient to suppress lung metastases. This study improves our understanding of how tumor stromal cells recruited from fat tissue stimulate carcinoma progression to chemotherapy resistance/metastasis and outlines a new approach to combination cancer treatment., Graphical Abstract, This study improves our understanding of how tumor stromal cells recruited from fat tissue stimulate carcinoma progression to chemotherapy resistance and metastasis. A combination of chemotherapy and experimental pharmacological adipose stromal cell ablation reduced the metastatic burden in mouse models, hence establishing a new approach to cancer treatment.

BACKGROUND: Eribulin mesylate, a non-taxane microtubule-dynamics inhibitor, is an effective chemotherapy drug for patients with metastatic breast cancer who have previously received at least two chemotherapeutic regimens. We are seeking to enhance the anti-tumor activity of eribulin in metastatic breast cancer cell lines. The purpose of this study was to identify kinase targets that markedly enhance the efficacy of eribulin using high-throughput nonbiased synthetic lethal RNA interference (RNAi) screening and to validate these targets in combination with eribulin using triple-negative breast cancer (TNBC) and inflammatory breast cancer (IBC) cell lines which are heterogeneous diseases and characterized by their high metastatic potential and poor prognosis. METHODS: High-throughput RNAi screening (709-human-kinome library) was performed to identify target kinases. The Bliss scoring method was used to identify potential targets from the screening (using a Bliss score cut-off of 0.15), and protein-protein interactome analysis (STRING V.11) and Ingenuity Pathway Analysis (IPA) were used to identify therapeutic target pathways. To validate the in vitro efficacy of selected targets, we performed CellTiter-Blue or sulforhodamine B cell proliferation and anchorage-independent colony-formation assays in 18 TNBC and 6 IBC cell lines treated with eribulin with or without inhibitors of the discovered targets. RESULTS: In vitro proliferation data demonstrated that single-agent eribulin had a nanomolar range of half-maximal inhibitory concentrations (0.2 - 4.0 nM) in most of the tested cell lines. RNAi screening identified 130 relevant kinase targets. IPA and STRING analysis revealed PI3K/AKT/mTOR, MAPK, JNK, and PAK1 as major canonical pathways that could be targeted to enhance the cytotoxic effect of eribulin. In in vitro cell line validation, PAK1 inhibitor NVS-PAK1-1 and mTOR inhibitor everolimus showed strong combinational anti-tumor effect (combination index values = 0.1 - 0.9), and JNK inhibitor JNK-In-8 showed moderate combinational anti-tumor effect (combination index values = 0.5 - 0.9) in combination with eribulin. Inhibitors of PI3K (copanlisib) and MEK (trametinib) showed combinational anti-tumor effect in some cell lines. CONCLUSION: We identified that PAK1, mTOR, and JNK inhibitors enhanced the anti-proliferation effects of eribulin in TNBC and IBC cell lines. Our data inform new treatment strategies with eribulin for TNBC and IBC that could lead to clinical trials of innovative combination therapy based on eribulin. Citation Format: Jangsoon Lee, Jon A Fuson, Huey Liu, Troy Pearson, Debu Tripathy, Bora Lim, Naoto T Ueno. Identification of molecules that enhance the efficacy of eribulin in TNBC and IBC cell lines [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-16.

Background: Triple negative breast cancer (TNBC) has a relative paucity of effective targeted therapies compared to other molecular subtypes, and with poor prognosis. Thus, new targeted therapeutics are needed. ONC201 is a first-in-class small molecule that induces caspase-mediated apoptosis in cancers. It recently showed efficacy in pediatric glioma, harboring the potential to be translated in advanced breast cancer. Here we evaluated the efficacy of ONC201 in TNBC cell lines, investigated proteomic/genomic markers that predict efficacy, and performed a synthetic lethal RNAi screening to identify rational combinational targets. Method: The CellTiter-Blue (CTB) cell viability or sulforhodamine B assay was used to measure the range of half-maximal inhibitory concentrations (IC50) of ONC201 in TNBC cell lines. 2D/3D high-throughput RNAi kinome screening was performed using ONC201-sensitive CAL51, resistant HCC70 cells. The top genes noted to augment ONC201 sensitivity by siRNA were analyzed using both protein-protein interactome analysis (STRING V.11) and Ingenuity Pathway Analysis (IPA) to identify contributing canonical pathways. Combinational anti-proliferative activity of ONC201 and target inhibitors were evaluated by CTB cell viability assay and quantified by CalcuSyn software. Identified potential partners were subsequently tested using 3D ex vivo spheroid assay, tumor xenograft animal model to measure tumor growth inhibition (TGI). Five NOD/SCID mice were treated per each group. Mice were treated with 50mg/kg twice daily dosing of ONC201, and/or 25mg /kg of palbociclib after tumor grew to 100mm3. Reverse phase protein array (RPPA) of both ONC201 treated and non-treated TNBC cell lysates was performed to identify pathways affecting the sensitivity to ONC201 by comparing the effect of protein level changes (individual and set) using 3-way ANOVA, R program. Results: Twenty TNBC cell lines exhibited varying IC50 values (2 μM to 40 μM). No correlation between Vanderbilt subtypes and IC50 was observed. RNAi kinome screening identified 65 overlapping target kinases. Pathway analyses revealed PI3K/Akt, MEK/ERK, CDK 2/4/6 as potential combination therapeutic partners. In the in vitro study, the MEK inhibitor trametinib and CDK 4/6 inhibitor palbociclib consistently showed synergistic TGI with ONC201 (combination index values [CI] = 0.1 - 0.7). AKT inhibitor MK-2206 showed moderate combinational TGI (CI = 0.5 - 0.9). PI3K inhibitors PF04691052, buparlisib, and dactolisib showed cell type-specific combinational TGI. RPPA analysis did not reveal direct apoptosis-regulators mediators of sensitivity to ONC201, yet identified 7 potential predictive markers (fibronectin, pHER2, PAR, PLK1, pRb, SOD2, and EMA) of resistance/sensitivity. It also provided the rationale to study CDK 4/6 inhibition as a combination partner. Ex vivo study of ONC201/palbociclib (CDK 4/6 inhibitor) treatment inhibited of the colony formation in CAL51 cells. Interestingly, ONC201/palbociclib did not induce synergy in HCC70 cells, while the ONC-201/trametinib pair was synergistic in both cell lines. CAL51 tumor xenograft in vivo study confirmed the synergy of ONC201/palbociclib. Conclusion: MEK inhibitor trametinib and CDK4/6 inhibitor palbociclib demonstrated synergy with ONC201 yet palbociclib only in ONC201 sensitive CAL51. Detailed mechanisms of synergy are being investigated that can further guide clinical translation. Citation Format: Bora Lim, Christine Peterson, Elin Cho, Alexander Davis, Troy Pearson, Huey Liu, Minha Hwang, Debu Tripathy, Naoto T Ueno, Jangsoon Lee. ONC201 synergistically induces cell death in triple negative breast cancer with CDK 4/6 and MEK inhibition [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-14.

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 a heterogeneous group of estrogen, progesterone, and HER2-negative breast cancers with poor clinical outcomes. The imipridone ONC201 is a G-protein-coupled dopamine receptor D2 modulator and an allosteric agonist of the mitochondrial protease caseinolytic protease P(ClpP), which induces apoptosis. Here, we aimed to develop a novel ONC201-based combination therapy targeting TNBC. We performed a reverse-phase protein array analysis of ONC201-treated/-untreated and -sensitive/-resistant cell lines to identify potential predictive biomarkers. A principal component analysis using measured protein expression levels, the apoptosis score (AS), and heatmaps of all the measured protein and AS-related protein expression levels did not show a clear correlation between the expression levels of a specific protein and ONC201 efficacy. Three-dimensional RNA interference kinome-wide library screening revealed the MAPK and PI3K/Akt pathways as potential synergistic therapeutic partners. The combination with the MEK inhibitor trametinib successfully inhibited the growth of both ONC201-sensitive/-resistant TNBC cell lines. The baseline ClpP level correlated with the efficacy of single-agent ONC201. Single and combination therapy increased caspase 3/7 activity. The predictive biomarkers and a detailed mechanism of synergy beyond an induction of caspase activation should be tested for translation into future studies.

Background: Inflammatory breast cancer (IBC) is the most lethal and aggressive form of breast cancer and there are no approved targeted therapies specifically for IBC. Targeting the epidermal growth factor receptor (EGFR) pathway is a promising therapeutic target for patients with triple-negative IBC (TN-IBC) with a reported pathological complete response rate of 42% (JAMA Oncology, 2018). The tumor microenvironment (TME) is a critical contributor to the aggressiveness of IBC. Delineating cross-talk between EGFR-targeted therapies and TME components, which define IBC, could inform more efficient combination regimens and novel clinical trial designs for IBC. However, such studies have not been conducted due to the lack of a syngeneic IBC mouse model. Here we report the establishment of an IBC immunocompetent mouse model and the effects of panitumumab (PmAb) on IBC tumor growth and the TME. Methods: TN-IBC cell lines, SUM149 or FC-IBC-02, were mixed with 50% Matrigel and inoculated into mammary fat pads of hu-NSG-SGM3 mice engrafted with hematopoietic stem cells (The Jackson Laboratory). SUM149 tumor growth in hu-NSG-SGM3 mice treated with either IgG2 (isotype control, 4 mg/kg) or PmAb (1 mg/kg and 4 mg/kg) was measured. The percentages of TME components, including human CD4+ T, CD8+ T, regulatory T (Tregs), and natural killer (NK) cells, and M1 or M2 macrophages, in the peripheral blood and tumor tissues treated with IgG2 and PmAb for 7 weeks were measured by flow cytometry. Results: Hu-NSG-SGM3 mice supported the growth of TN-IBC SUM149 and FC-IBC-02 xenografts. These humanized mouse models were named SUM149-huSGM3 and FC-IBC-02-huSGM3, respectively. Analysis of the blood cells showed that SUM149-huSGM3 mice display human CD4+ T, CD8+ T, Tregs, M1 and M2 macrophages. T cell infiltration and M1 and M2 macrophages were also detected in SUM149-huSGM3 tumors. NK cells were not detected in both peripheral blood and tumors. PmAb treated SUM149-huSGM3 mice had significantly reduced SUM149 tumor growth, compared with mice that received the IgG2 control. PmAb treatment increased the percentage of CD8+ T cells and reduced the percentage of Tregs in peripheral blood. A similar analysis of tumor infiltrating lymphocytes isolated from each group showed an increase in percent CD8+ T cells in mice treated with PmAb. There were no significant changes of M1 or M2 macrophages following PmAb treatment. These results suggest that the increase in percentage of CD8+ T cells in peripheral blood and IBC tumors, and the decrease in percentage of Tregs in peripheral blood may contribute to the therapeutic efficacy of PmAb. Conclusion: We established the first immunocompetent mouse model to study the TME and immune response in IBC, which provides the premise for conducting a diversity of novel preclinical therapeutic studies. The mechanism of how immune responses of TN-IBC xenografts mediates the therapeutic efficacy of PmAb in IBC tumors needs to be further investigated. Our study also suggests that combination therapy with immune checkpoint inhibitors may potentiate the efficacy of anti-EGFR therapy in IBC. The therapeutic efficacy of PmAb and anti-PD-L1 combination in SUM149 humanized mice is in progress. Citation Format: Wang X, Shao S, Pearson T, Cheng Y, Reuben JM, Tripathy D, Ueno NT. Immune modulation with humanized anti-EGFR antibody panitumumab in an immunocompetent mouse model for inflammatory breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-17-06.

Human epidermal growth factor receptor (EGFR) 2 (HER2) is overexpressed/amplified in about 25% of all breast cancers, and EGFR is overexpressed in up to 76% and amplified in up to 24% of triple-negative breast cancers (TNBC). Here, we aimed to identify inhibitors that may enhance the anti-tumor activity of neratinib for HER2+ breast cancer and TNBC. By conducting a non-biased high-throughput RNA interference screening, we identified PI3K/AKT/mTOR and MAPK as two potential inhibitory synergistic canonical pathways. We confirmed that everolimus (mTOR inhibitor) and trametinib (MEK inhibitor) enhances combinatorial anti-proliferative effects with neratinib under anchorage-independent growth conditions (p &lt, 0.05). Compared to single agent neratinib, the combination therapies significantly enhanced tumor growth inhibition in both SUM190 HER2+ breast cancer (neratinib plus everolimus, 77%, neratinib plus trametinib, 77%, p &lt, 0.0001) and SUM149 TNBC (neratinib plus everolimus, 71%, neratinib plus trametinib, 81%, 0.0001) xenograft models. Compared to single-agent neratinib, everolimus, or trametinib, both everolimus plus neratinib and trametinib plus neratinib significantly suppressed proliferation marker Ki67 and enhanced antitumor efficacy by activating the apoptosis pathway shown by increased Bim and cleaved-PARP expression. Taken together, our data justify new neratinib-based combinations for both HER2+ breast cancer and TNBC.

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.

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.

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 dose-dependent 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. CONCLUSION: These results indicate that E6201 exhibits anti-tumor efficacy against TNBC in vitro and antimetastasis efficacy against TNBC in vivo. These results provide a rationale for further clinical development of E6201 as a MAPK-pathway-targeted therapy for TNBC.

Background: Apoptosis resistance is a hallmark of cancer, and apoptosis regulators have been targeted for cancer treatment. A class of such regulators is inhibitor of apoptosis proteins (IAPs), which suppress caspase activity by inducing degradation of active caspases or blocking interaction of caspases with their substrates. Dysregulated IAP expression has been reported in many cancers, including breast cancer, and is involved in chemoresistance. IAPs have therefore become attractive targets for cancer treatment. Birinapant (TL32711), a biindole-based bivalent mimetic of second mitochondria-derived activator of caspase (SMAC, an endogenous antagonist of IAPs), has been shown to effectively activate apoptotic signaling by targeting IAPs and has potential application for treatment of multiple cancers. In addition, birinapant showed synergistic anti-tumor effects with several widely used chemotherapeutic agents in various cancers. Here, we assessed whether birinapant synergizes with commonly used anti-cancer drugs, including entinostat (class I histone deacetylase inhibitor), cisplatin, paclitaxel, voxtalisib (PI3K inhibitor), dasatinib (Src inhibitor), everolimus (mTOR inhibitor), and gemcitabine, in triple-negative breast cancer (TNBC). Among these drugs, gemcitabine strongly synergized with birinapant (combination index [CI] Results: Birinapant as a single agent had varying anti-proliferation effects in TNBC cells (IC50 = 0.21 to >20 µM). It had no synergistic effects with entinostat, cisplatin, paclitaxel, voxtalisib, dasatinib, or everolimus but strongly synergized with gemcitabine (CI Conclusion: Our findings demonstrate the therapeutic potential of birinapant for enhancing the anti-tumor efficacy of gemcitabine in TNBC by targeting the IAP family of proteins. Citation Format: Xuemei Xie, Jangsoon Lee, Troy Pearson, Alexander Y. Lu, Debu Tripathy, Gayathri R. Devi, Chandra Bartholomeusz, Naoto T. Ueno. Birinapant enhances gemcitabine's anti-tumor efficacy in triple-negative breast cancer by inducing intrinsic pathway-dependent apoptosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 548.

Inflammatory breast cancer (IBC) is the most lethal and aggressive form of breast cancer, yet no approved targeted therapies specifically for IBC have been approved. The epidermal growth factor receptor (EGFR) pathway is a promising therapeutic target for patients with triple-negative IBC, with a reported pathological complete response rate of 42%. The tumor microenvironment (TME) is a critical contributor to the aggressiveness of IBC. Therefore, delineating the cross-talk between EGFR-targeted therapy and TME components could lead to more efficient combination regimens and novel clinical trial designs for IBC. Because an immunocompetent IBC animal model is not available for our study, we established an IBC SUM149 immunocompetent mouse model (SUM149-hu-NSG-SGM3) for such studies. Here we report the effects of panitumumab (PmAb), a humanized monoclonal anti-EGFR antibody, on tumor growth and on the TME in the SUM149-hu-NSG-SMG3 mouse model. Methods: SUM149 cells were mixed with 50% Matrigel and inoculated into mammary fat pads of hu-NSG-SGM3 mice engrafted with human hematopoietic stem cells. SUM149 tumor growth in mice treated with either isotype IgG2 or PmAb was measured. We used flow cytometry to measure the percentages of human immune cells in the peripheral blood and tumor tissues from mice treated with IgG2 and PmAb. The effects of PmAb treatment on the expression and distribution of TME components in tumor tissues was examined by multiplex immunohistochemical staining. Cytokine expression in tumor tissues from IgG2- and PmAb-treated mice was measured by cytokine antibody array, and the changes in cytokines expression were validated with RT-PCR. Results: PmAb treatment, compared with IgG2 treatment, significantly reduced SUM149 tumor growth. Flow cytometry analysis showed infiltration of lymphocytes in SUM149 tumors with an observed increase in the level of CD8+ T cells and a reduced level of Tregs in both peripheral blood and tumor tissues from PmAb-treated mice compared with IgG2-treated mice. Cytokine array and RT-PCR showed that PmAb treatment increased expression of cytokines that function as a chemoattractant for T cells, natural killer cells, and monocytes, including CXCL10, CCL5, CCL4, and CXCL9. In contrast, PmAb treatment reduced expression of cytokines involved in antitumor immune suppression, including osteoprotegerin (OPG), CCL2, CXCL5, and CCL20. Conclusion: PmAb treatment reduced IBC tumor growth by increasing cytotoxic T cells and reducing immunosuppressive Treg cells. Our data also suggest that the combination of PmAb with an immune checkpoint inhibitor may potentiate the efficacy of anti-EGFR therapy in IBC. A study of the therapeutic efficacy of the PmAb and anti-PD-L1 combination in the SUM149-hu-NSG-SMG3 mouse model is in progress. Citation Format: Xiaoping Wang, Shan Shao, Takashi Semba, Troy Pearson, James M. Re, Debu Tripathy, Naoto Ueno. Humanized anti-EGFR antibody panitumumab inhibits tumor growth of inflammatory breast cancer by inducing antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4492.

Unfortunately in the original publication of the article, the author’s funding support has been mentioned incorrectly. The correct funding statement should read as “This work was supported by the Morgan Welch Inflammatory Breast Cancer Research Program, the State of Texas Rare and Aggressive Breast Cancer Research Program, MD Anderson’s Cancer Center Support Grant (P30CA016672, used the Characterized Cell Line Core Facility and Flow Cytometry and Cellular Imaging Facility), and Spirita Oncology, LLC.” The first affiliations was incorrect in the original article. The correct information is given below.

Background: Triple-negative breast cancer (TNBC) lacks the receptor targets ER, PR, and HER2 and thus it does not respond to receptor-targeted treatments such as hormonal therapy and trastuzumab, leaving chemotherapy as the mainstay of treatment. TNBC also has higher recurrence, metastasis, and mortality rates than other receptor subtypes. The MAPK (RAS/MEK/ERK) pathway plays a crucial role in cancer cell survival, invasion, and metastasis and critically contributes to aggressive features of TNBC, as demonstrated by our novel therapeutic synergy identification screening of RNA interference. Two BRAF inhibitors (vemurafenib and dabrafenib) and two MEK inhibitors (trametinib and cobimetinib) have received U.S. Food and Drug Administration approval for treatment of melanoma, clinically validating the potential of MAPK pathway inhibition to meaningfully benefit patients with TNBC. However, the rapidly emerging resistance of TNBC to current standard treatments and disease progression remain clinical challenges for these pathway-targeted agents. E6201 is a novel MEK1 inhibitor being evaluated in phase 1 clinical trials in patients with advanced solid tumors (NCT00794781) and with melanoma metastasized to the central nervous system (NCT03332589). In the preclinical study described herein, we determined the in vitro anti-tumor efficacy and in vivo anti-metastatic efficacy of E6201 in TNBC to prepare for further development of E6201 in the clinic. Methods: The anti-tumor effects of E6201 were examined using cell proliferation and anchorage-independent colony-formation assays. To evaluate the anti-metastatic activity of E6201 in vitro and in vivo, a migration/invasion assay and an experimental/spontaneous metastasis assay were performed, respectively. Results: In vitro cell proliferation data demonstrated that E6201 inhibited growth more effectively (half maximal inhibitory concentration [IC50] range: 0.05-5 µM) than did other MEK inhibitors (selumetinib, pimasertib, and trametinib). E6201 inhibited TNBC cell-colony formation in a dose-dependent manner (P = 0.0001). Also, E6201 suppressed the migration and invasion of MDA-MB-231 (IC50: 1 µM) and SUM149 (IC50: 0.25 µM) cells in a dose-dependent manner (P = 0.001). In vivo experimental and spontaneous metastasis assays demonstrated that E6201 inhibited lung metastases of the SUM149 and MDA-MB-231-LM2 cell lines (P = 0.0001). Immunohistochemical staining demonstrated that E6201 decreased the metastatic burden in the lung (P = 0.011) and decreased phosphorylated ERK expression in a dose-dependent manner. Conclusion: Taken together, these data demonstrate that E6201 is an effective inhibitor targeting the MAPK pathway in TNBC, showing anti-tumor efficacy in vitro and anti-metastatic efficacy in vivo. Our data provide a rationale for further clinical development of E6201 as MEK-targeted therapy for treatment of TNBC. Citation Format: Jangsoon Lee, Bora LIM, Kuicheon Choi, Troy Pearson, Linda Paradiso, Thomas Myers, Debu Tripathy, Naoto T. Ueno. E6201, a novel MEK1 inhibitor, suppresses the metastatic capability of triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5780.

Background: Aberrant expression of members of the ErbB family of receptor tyrosine kinases and its downstream pathways is known to play pivotal roles in breast cancer malignancy. HER2-positive breast cancer has an aggressive nature that is driven by HER2 gene amplification. Inflammatory breast cancer is a rare but the most aggressive clinical subtype of breast cancer. Triple-negative breast cancer is an aggressive receptor subtype of breast cancer. More than 30% of cases of each of these groups express EGFR. Emerging therapeutic resistance continues to be a challenge in recurrent and/or metastatic disease. Neratinib is a potent irreversible EGFR, HER2, and HER4 tyrosine kinase inhibitor that blocks signal transduction generated via these receptors. In this study, we sought to identify the optimal targets in different subtypes of breast cancer and synergistic partners to maximize the antitumor effect of neratinib against breast cancer cells in vitro and in vivo. Methods: Expression of EGFR and HER2 was assessed via Western blot in 31 breast cancer cell lines, including HER2-targeted drug-resistant cell lines (14 triple-negative breast cancer and 10 HER2-positive). The antiproliferative effects of neratinib were measured using cell proliferation and anchorage-independent colony-formation (soft agar) assays. A reverse-phase protein array was used to profile and validate the signaling networks induced by neratinib-based treatment. To identify the synergistic targets, RNA interference screening of a 779-kinome library was performed under multicellular tumor spheroid culture conditions. Results: In vitro proliferation data demonstrated that neratinib had a nanomolar range of half-maximal inhibitory concentrations in most of the tested cell lines, including T-DM1-resistant HER-2 breast cancer cell lines. Neratinib inhibited colony formation in a dose-dependent manner (P = 0.0001). Reverse-phase protein array data revealed that the efficacy of neratinib correlated with phosphorylated EGFR (R2 = 0.6055) or phosphorylated HER2 (R2 = 0.6734) expression level. Statistical analysis identified the 25 most relevant targets in the RNA interference screening, which we will validate for combinational antitumor effects with neratinib in in vitro and in vivo studies. Conclusion: We determined the efficacy of neratinib and identified target molecules to enhance neratinib efficacy in different breast cancer subtype cell lines with ErbB alterations. Our data inform new combined treatment strategies with neratinib for breast cancer that will lead to innovative designs using optimized neratinib combinations in clinical trials. Citation Format: Bora Lim, Jangsoon Lee, Troy Pearson, Liu Huey, Minha Hwang, Kuicheon Choi, Francesca Avogadri-Connors, Richard E. Cutler, Debu Tripathy, Naoto T. Ueno. Identification of molecular therapeutic targets that enhance the antitumor activity of neratinib in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 825.

Background: Triple negative breast cancer (TNBC) lacks effective targeted therapy, leaving chemotherapy as the only therapeutic option. Metastatic TNBCs eventually develop resistance to chemotherapy via several mechanisms including apoptosis evasion, hence representing an urgent unmet need for novel therapies. ONC201 is a first-in-class imipridone that induces caspase-mediated apoptosis in multiple cancers including TNBC, selectively antagonizing dopamine D2-like receptors (DRD2) resulting in inhibition of Ras signaling and cell death. Selective antagonism of D2-like receptors has been shown to induce cell death in cancer cells via the same signaling pathways engaged by ONC201. To translate these early findings for breast cancers patients, more studies to prove ONC201’s activity and mechanism of action in breast cancers are needed. In this study, we tested the efficacy of ONC201 in TNBC cell lines, and further confirmed the mechanism of action of ONC201 by studying the association between DRD2/DRD5 and ONC201 efficacy. Additionally, we performed siRNA kinome screening to identify combinatorial opportunities to further enhance ONC201 efficacy in TNBC. Method: We determined the efficacy of ONC201 in 20 TNBC cell lines by determining the IC50. We then selected one cell line with low IC50 (CAL51) and high IC50 (HCC70) for siRNA synthetic lethality screening via 2D and 3D culture systems. Additionally, we correlated the IC50 with the mRNA expression of DRD2 measured by Affymetrix microarray on each TNBC cell lines with IC50 of the ONC201. Result: IC50 ranged from 2.09 to 40μM, with median IC50 of 10μM. No correlation between Vanderbilt TNBC molecular subtype and IC50. 2D/3D siRNA kinome screening and Ingenuity pathway analysis showed PIK3, MAPK, and Ephrin receptor pathways as top three target candidates correlating with tumor growth inhibition efficacy of ONC201. The long variant of DRD2 mRNA expression and ONC201 IC50 showed a correlation. There was a negative correlation between mRNA expression of DRD5 and IC50 (R2=0.3601, P=0.018). Conclusion: Our preclinical results suggest that ONC201 is active is TNBC cells within previously proven safe therapeutic dose and provides potential combinatorial opportunities with PI3K/MAPK targeted drugs. These results highlight the importance of targeting the dopamine receptor pathway for ONC201 efficacy and supports DRD5 as a potential predictive marker. Targeting DRD2 and apoptosis induction could be one of therapeutic strategy in triple negative breast cancers. Ongoing work involves further in vitro and in vivo studies to determine the best synergistic therapeutic partner and conducting a phase II single agent trial to study the pharmacodynamics changes induced by ONC201 in TNBC. Citation Format: Bora Lim, Jangsoon Lee, Huey Liu, Troy Pearson, Larry Coffer, Debu Tripathy, Naoto T. Ueno. Preclinical evaluation of imipridone ONC201 in triple negative breast cancer identifies predictive biomarkers and combinatorial opportunities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4294. doi:10.1158/1538-7445.AM2017-4294