Triple-negative Breast Cancer Cell Lines Research Articles

Cellular Responses Induced by NCT-503 Treatment on Triple-Negative Breast Cancer Cell Lines: A Proteomics Approach

Cellular Responses Induced by NCT-503 Treatment on Triple-Negative Breast Cancer Cell Lines: A Proteomics Approach

Autologous patient-derived exhausted nano T-cells exploit tumor immune evasion to engage an effective cancer therapy

BackgroundActive targeting by surface-modified nanoplatforms enables a more precise and elevated accumulation of nanoparticles within the tumor, thereby enhancing drug delivery and efficacy for a successful cancer treatment. However, surface functionalization involves complex procedures that increase costs and timelines, presenting challenges for clinical implementation. Biomimetic nanoparticles (BNPs) have emerged as unique drug delivery platforms that overcome the limitations of actively targeted nanoparticles. Nevertheless, BNPs coated with unmodified cells show reduced functionalities such as specific tumor targeting, decreasing the therapeutic efficacy. Those challenges can be overcome by engineering non-patient-derived cells for BNP coating, but these are complex and cost-effective approaches that hinder their wider clinical application. Here we present an immune-driven strategy to improve nanotherapeutic delivery to tumors. Our unique perspective harnesses T-cell exhaustion and tumor immune evasion to develop a groundbreaking new class of BNPs crafted from exhausted T-cells (NExT) of triple-negative breast cancer (TNBC) patients by specific culture methods without sophisticated engineering.MethodsNExT were generated by coating PLGA (poly(lactic-co-glycolic acid)) nanoparticles with TNBC-derived T-cells exhausted in vitro by acute activation. Physicochemical characterization of NExT was made by dynamic light scattering, electrophoretic light scattering and transmission electron microscopy, and preservation and orientation of immune checkpoint receptors by flow cytometry. The efficacy of chemotherapy-loaded NExT was assessed in TNBC cell lines in vitro. In vivo toxicity was made in CD1 mice. Biodistribution and therapeutic activity of NExT were determined in cell-line- and autologous patient-derived xenografts in immunodeficient mice.ResultsWe report a cost-effective approach with a good performance that provides NExT naturally endowed with immune checkpoint receptors (PD1, LAG3, TIM3), augmenting specific tumor targeting by engaging cognate ligands, enhancing the therapeutic efficacy of chemotherapy, and disrupting the PD1/PDL1 axis in an immunotherapy-like way. Autologous patient-derived NExT revealed exceptional intratumor accumulation, heightened chemotherapeutic index and efficiency, and targeted the tumor stroma in a PDL1+ patient-derived xenograft model of triple-negative breast cancer.ConclusionsThese advantages underline the potential of autologous patient-derived NExT to revolutionize tailored adoptive cancer nanotherapy and chemoimmunotherapy, which endorses their widespread clinical application of autologous patient-derived NExT.

Exogenous Metabolic Modulators Improve Response to Carboplatin in Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) lacks targeted therapies, leaving cytotoxic chemotherapy as the current standard treatment. However, chemotherapy resistance remains a major clinical challenge. Increased insulin-like growth factor 1 signaling can potently blunt chemotherapy response, and lysosomal processes including the nutrient scavenging pathway autophagy can enable cancer cells to evade chemotherapy-mediated cell death. Thus, we tested whether inhibition of insulin receptor/insulin-like growth factor 1 receptor with the drug BMS-754807 and/or lysosomal disruption with hydroxychloroquine (HCQ) could sensitize TNBC cells to the chemotherapy drug carboplatin. Using in vitro studies in multiple TNBC cell lines, in concert with in vivo studies employing a murine syngeneic orthotopic transplant model of TNBC, we show that BMS-754807 and HCQ each sensitized TNBC cells and tumors to carboplatin and reveal that exogenous metabolic modulators may work synergistically with carboplatin as indicated by Bliss analysis. Additionally, we demonstrate the lack of overt in vivo toxicity with our combination regimens and, therefore, propose that metabolic targeting of TNBC may be a safe and effective strategy to increase sensitivity to chemotherapy. Thus, we conclude that the use of exogenous metabolic modulators, such as BMS-754807 or HCQ, in combination with chemotherapy warrants additional study as a strategy to improve therapeutic responses in women with TNBC.

MerTK Drives Proliferation and Metastatic Potential in Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is characterized by the absence of the estrogen receptor, progesterone receptor, and receptor tyrosine kinase HER2 expression. Due to the limited number of FDA-approved targeted therapies for TNBC, there is an ongoing need to understand the molecular underpinnings of TNBC for the development of novel combinatorial treatment strategies. This study evaluated the role of the MerTK receptor tyrosine kinase on proliferation and invasion/metastatic potential in TNBC. Immunohistochemical analysis demonstrated MerTK expression in 58% of patient-derived TNBC xenografts. The stable overexpression of MerTK in human TNBC cell lines induced an increase in proliferation rates, robust in vivo tumor growth, heightened migration/invasion potential, and enhanced lung metastases. NanoString nCounter analysis of MerTK-overexpressing SUM102 cells (SUM102-MerTK) revealed upregulation of several signaling pathways, which ultimately drive cell cycle progression, reduce apoptosis, and enhance cell survival. Proteomic profiling indicated increased endoglin (ENG) production in SUM102-MerTK clones, suggesting that MerTK creates a conducive environment for increased proliferative and metastatic activity via elevated ENG expression. To determine ENG’s role in increasing proliferation and/or metastatic potential, we knocked out ENG in a SUM102-MerTK clone with CRISPR technology. Although this ENG knockout clone exhibited similar in vivo growth to the parental SUM102-MerTK clone, lung metastasis numbers were significantly decreased ~4-fold, indicating that MerTK enhances invasion and metastasis through ENG. Our data suggest that MerTK regulates a unique proliferative signature in TNBC, promoting robust tumor growth and increased metastatic potential through ENG upregulation. Targeting MerTK and ENG simultaneously may provide a novel therapeutic approach for TNBC patients.

Peposertib, a DNA-PK Inhibitor, Enhances the Anti-Tumor Efficacy of Topoisomerase II Inhibitors in Triple-Negative Breast Cancer Models

Triple-negative breast cancer (TNBC) remains the most lethal subtype of breast cancer, characterized by poor response rates to current chemotherapies and a lack of additional effective treatment options. While approximately 30% of patients respond well to anthracycline- and taxane-based standard-of-care chemotherapy regimens, the majority of patients experience limited improvements in clinical outcomes, highlighting the critical need for strategies to enhance the effectiveness of anthracycline/taxane-based chemotherapy in TNBC. In this study, we report on the potential of a DNA-PK inhibitor, peposertib, to improve the effectiveness of topoisomerase II (TOPO II) inhibitors, particularly anthracyclines, in TNBC. Our in vitro studies demonstrate the synergistic antiproliferative activity of peposertib in combination with doxorubicin, epirubicin and etoposide in multiple TNBC cell lines. Downstream analysis revealed the induction of ATM-dependent compensatory signaling and p53 pathway activation under combination treatment. These in vitro findings were substantiated by pronounced anti-tumor effects observed in mice bearing subcutaneously implanted tumors. We established a well-tolerated preclinical treatment regimen combining peposertib with pegylated liposomal doxorubicin (PLD) and demonstrated strong anti-tumor efficacy in cell-line-derived and patient-derived TNBC xenograft models in vivo. Taken together, our findings provide evidence that co-treatment with peposertib has the potential to enhance the efficacy of anthracycline/TOPO II-based chemotherapies, and it provides a promising strategy to improve treatment outcomes for TNBC patients.

Targeted blocking of EGFR and GLUT1 by compound H reveals a new strategy for treatment of triple-negative breast cancer and nasopharyngeal carcinoma

BackgroundCytoplasmic epidermal growth factor receptor (EGFR) is overexpressed in both nasopharyngeal carcinoma (NPC) and triple-negative breast cancer (TNBC), while clinical outcome and prognosis vary greatly among patients treated with gefitinib, and all patients eventually develop resistance to this agent. Therefore, we propose a new concept for synthesizing multitarget compounds and reveal new therapeutic strategies for NPC and TNBC expressing EGFR. MethodsCompound H was synthesized in our previous study. Molecular docking, and cell thermal shift assays (CETSAs) and drug affinity responsive target stability(DARTS) were used to confirm the binding of compound H to EGFR and GLUT1. Methylthiazolyldiphenyl-tetrazolium bromide(MTT), annexin V-PE assays, mitochondrial membrane potential (MMP) assays, and animal models were used to evaluate the inhibitory effect of compound H on TNBC cell lines. Energy metabolism tests, Western blotting, and immunofluorescence staining were performed to evaluate the synergistic effects on EGFR- and glucose transporter type 1(GLUT1)-mediated energy metabolism. ResultsCompound H can simultaneously act on the EGFR tyrosine kinase ATP-binding site and inhibit GLUT1-mediated energy metabolism, resulting in reductions in ATP, MMP, intra-cellular lactic acid, and EGFR nuclear transfer. The anti-tumor activity of compound H is significantly superior to the combination of GLUT1 inhibitor BAY876 and EGFR inhibitor gefitinib. Compound H has remarkable anti-proliferative effects on TNBC MDA-MB231 cells, and importantly, no obvious toxicity effects of compound H were found in vivo. ConclusionsSynergistic effects of inhibition of EGFR- and GLUT1-mediated energy metabolism by compound H may present a new strategy for the treatment of TNBC and NPC.

Exploring the regulatory role of FBXL19-AS1 in triple-negative breast cancer through the miR-378a-3p/OTUB2 axis.

The regulatory potential of long noncoding RNA (lncRNA) FBXL19-AS1 has been highlighted in various cancers, but its effect on triple-negative breast cancer (TNBC) remains unclear. Here, we aimed to elucidate the role of FBXL19-AS1 in TNBC and its underlying mechanism. RT-qPCR was employed to detect the expressions of FBXL19-AS1 and miR-378a-3p in tissues and cells. Immunohistochemical staining and western blot were utilized to detect the expression levels of proteins. Cell activities were detected using flow cytometry, CCK-8, and transwell assay. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were deployed to investigate interactions of different molecules. Protein-protein interaction (PPI) network, gene ontology (GO), and Kyoto encyclopedia of genes and genomes (KEGG) pathways were used to analyze the downstream pathway. In vivo xenograft model was conducted to detect the effect of FBXL19-AS1 on tumor growth. FBXL19-AS1 was overexpressed in TNBC tissues and cell lines compared with counterparts. FBXL19-AS1 knockdown suppressed TNBC cell activities, whereas its overexpression exhibited the opposite effect. Mechanistically, FBXL19-AS1 was found to interact with miR-378a-3p. Further analysis revealed that miR-378a-3p exerted tumor-suppressive effects in TNBC cells. Additionally, miR-378a-3p targeted and downregulated the expression of ubiquitin aldehyde binding 2 (OTUB2), a deubiquitinase associated with TNBC progression. In vivo experiments substantiated the inhibitory effects of FBXL19-AS1 knockdown on TNBC tumorigenesis, and a miR-378a-3p inhibitor partially rescued these effects. The downstream pathway of the miR-378a-3p/OTUB2 axis was explored, revealing connections with proteins involved in modifying other proteins, removing ubiquitin molecules, and influencing signaling pathways, including the Hippo signaling pathway. Western blot analysis confirmed changes in YAP and TAZ expression levels, indicating a potential regulatory network. In summary, FBXL19-AS1 promotes exacerbation in TNBC by suppressing miR-378a-3p, leading to increased OTUB2 expression. The downstream mechanism may be related to the Hippo signaling pathway. These findings propose potential therapeutic targets for TNBC treatment.

Abstract PO4-28-07: SLC26A9 promotes triple-negative breast cancer progression by regulating lipid metabolism

Abstract Background: The disturbance of lipid metabolism in the microenvironment plays an important role in the occurrence and development of triple-negative breast cancer(TNBC). SLC26A9, a member of the SLC26A family of anion transporters, is involved in breast oncogenesis. However, the effect of SLC26A9 in triple-negative breast cancer on lipid metabolism in the tumor microenvironment remains unclear. Methods: Bioinformatics, tissue microarrays and TNBC cell lines were used to detect the expression of SLC26A9 and its clinical significance. By altering the expression of SLC26A9 gene in TNBC cells, the effects of SLC26A9 on the regulation of lipid metabolism, cell biological behavior and its related molecular mechanisms in the TNBC microenvironment were explored using adoptive staining techniques, flow cytometric analysis and animal models. Results: We found by tissue microarray (TNBC=105 cases, Nor=67 cases) that the expression of SLC26A9 was significantly up-regulated in BC compared to paracancerous tissues, where SLC26A9 expression was higher in TNBC, and the same results were obtained in BC cell lines. Secondly, we found that SCL26A9 was positively correlated with lipid metabolism status in TNBC microenvironment and possessed a close association with P53 mutation by analyzing GEO and TCGA databases. Therefore, we used two cell lines (MDA-MB-231, MDA-MB-468) in which SCL26A9 was highly expressed in TNBC cell lines. It was demonstrated by oil red O staining and lipid fluorescence staining that knockdown/silencing of SLC26A9 diminished the ability of MDA-MB-231 and MDA-MB-468 cells to uptake and utilize lipids, especially for metabolites such as neutral lipids: triglycerides, lipid droplets, and steroidal substances. Then we demonstrated by using fatty acid flow cytometric analysis that knockdown/silencing of SLC26A9 significantly reduced the utilization and production of fatty acids in MDA-MB-231 and MDA-MB-468 cells, which are the key metabolic indicators of cancer cells as well as important energy-providing substances for cancer progression. Therefore, we further verified functionally that cell proliferation, migration, invasion and anti-apoptosis were significantly inhibited in in vitro after knockdown/silencing of SLC26A9, and in vivo tumorigenesis was significantly inhibited in BALB/c nude mice in in vivo. In addition, the expression of markers of lipid metabolic activity (FASN, ACLY) was reduced after knockdown/silencing of SLC26A9 in MDA-MB-231 cells by Western Blot, and the reduced expression of FASN and ACLY mainly inhibited the ab initio synthesis, uptake and utilization of fatty acids in TNBC cells, which in turn regulated the proliferation and invasion of TNBC. Mechanistically, SLC26A9 silencing/knockdown inhibited the synthesis, uptake, and utilization of multiple metabolites of lipids in TNBC, which combined with the TCGA database results revealed the activation of the key signaling pathway, the JAK-STAT3 signaling pathway, to promote the progression of TNBC. Conclusion(s): SLC26A9 is involved in the disturbance of lipid metabolism in the TNBC microenvironment and regulates the progression of TNBC. The search for new SLC26A9 inhibitors is expected to provide a new direction for the treatment of TNBC, but the exact molecular mechanisms need to be further explored. SLC26A9 upregulation in TNBC leads to a poor prognosis by referencing lipid metabolism in the TNBC microenvironment Wench. SLC26A9 is involved in lipid metabolism disorders and cell biological functions in the TNBC microenvironment in vivo. SLC26A9 is involved in disordered lipid metabolism in the TNBC microenvironment. Citation Format: Yingming Zhou, Hu Wang, Zhengxing Zhou, Xuemei Liu, Biguang Tuo, Taolang Li. SLC26A9 promotes triple-negative breast cancer progression by regulating lipid metabolism [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-28-07.

Abstract PO2-14-10: A selective MCL-1 inhibitor AZD5991 showed tumor regression in a triple-negative inflammatory breast cancer xenograft model

Abstract Background Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancer diagnoses but up to 30% of breast cancer-related deaths. Similarly, Inflammatory breast cancer (IBC) accounts for 2-4% of breast cancer diagnosis but causes 8%–10% of all breast cancer-related deaths. The myeloid cell leukemia-1 (MCL-1) is a member of the BCL-2 family of proteins, which is highly amplified in numerous human cancers and associated with cell immortalization, transformation, and chemoresistance. In fact, MCL-1 is amplified in 55% of TNBC after preoperative chemotherapy. Amongst the MCL-1 inhibitors that have been developed, AZD5991 showed high selectivity and affinity for MCL-1. In myeloma and acute myeloid leukemia cells AZD5991 induces apoptosis and showed anti-tumor effects in in vivo models. In the present study, we evaluated the biological role of the MCL-1 inhibitor AZD5991 in IBC and TNBC in vitro and in vivo models. Materials and methods The expression of the MCL-1 gene was analyzed in the World Consortium IBC patient dataset. Next, we screened a panel of TNBC and IBC cell lines for MCL-1 expression by western blot analysis. The effect of AZD5991 on TNBC and IBC cells was analyzed via cell titer-blue proliferation, clonogenic assays and soft agar assay. To further validate these findings, we used siRNA to knockdown MCL-1 and performed cell proliferation, clonogenic and migration assays. To identify signaling pathways involved in MCL-1 Inhibitor’s effects we performed a proteome profiler human apoptosis array in TNBC cells in the presence of AZD5991. The anti-tumor effect of the MCL-1 inhibitor was evaluated using a SUM-149 (TN-IBC) orthotopic xenograft mouse model. The mice were treated with AZD5991 at doses of 15 and 30mg/kg once weekly via tail vein injection for a duration of 4 weeks. Results The mean expression level of MCL-1 was higher in patients with TNBC than in those with non-TNBC. Further, compared to the patients with ER positive, HER-2 negative tumors those with TNBC showed an increased in the mean expression of MCL-1 gene (p value= 0.05). We screened a panel of TNBC and IBC cell lines in which most of the cell lines showed high levels of MCL-1. The cell proliferation assay showed IC50 ranging from 0.27 – 19.6 μM. The SUM149, SUM190, FCIBC02, MDA-IBC3 and MDA-MB-468 cells showed high sensitivity and showed significant decrease in the proliferation rate while BCX010, KPL4, MDA-MB-231, 4T1.2 and E0771 cells as well as the normal breast cells MCF10A exhibited resistance with no significant decrease in proliferation. The clonogenic and soft agar assays with these sensitive and resistant cell lines showed similar results. Further, these findings were validated via genetic knockdown of MCL-1 which resulted in a significant decrease in cell proliferation, colony formation and migration capability of the TNBC cells. To identify signaling pathways involved in MCL-1 Inhibitor’s phenotypic changes, we performed a proteome profiler human apoptosis array which showed a decreased in BCLx expression, and an increase in expression of BAX and phospho-Rad17 proteins in the AZD5991 treated TN-IBC cells. Finally, in a SUM-149 (TN-IBC) orthotopic xenograft mouse model, mice injected with 30mg/kg showed a significant tumor regression compared to the vehicle-treated mice (vehicle vs 30 mg/kg, p value=0.0006). Conclusions and Future directions Our data demonstrates that AZD5991 inhibits tumorigenesis in an inflammatory breast cancer xenograft model. We are currently validating several significant targets to understand the biological mechanisms related to our candidate drug. Our long-term goal is to develop MCL-1 targeted therapy in combination with standard-of-care treatment for inflammatory and triple-negative breast cancers. Citation Format: Mohd Mughees, Moises Tacam, Alex Tan, Michael White, Bisrat Debeb, Emilly Villodre, Xiaoding Hu, Debu Tripathy, Wendy Woodward, Rachel Layman, Geoffrey Bartholomeusz, Chandra Bartholomeusz. A selective MCL-1 inhibitor AZD5991 showed tumor regression in a triple-negative inflammatory breast cancer xenograft model [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-14-10.

Abstract PO3-24-02: Molecular characterization and therapeutic approach for VISTA+ triple-negative breast cancers

Abstract Introduction: Current FDA-approved immunotherapeutics for triple-negative breast cancer are only available for a subset of patients, highlighting the need to develop improved immune-targeting strategies. VISTA (V-domain Ig suppressor of T cell activation) is a single-pass transmembrane immune checkpoint receptor that is an emerging clinical target for cancer immunotherapy. It is expressed widely on lymphoid and myeloid cells in healthy individuals, is commonly expressed on cells of the tumor microenvironment and can be abnormally upregulated on tumor cells in a wide variety of tumors. Although VISTA confers quiescence to naïve T cells, molecular mechanisms responsible for this effect remain unclear. Also, the effects of VISTA expression on tumor cells remains poorly defined. Methods: Multi-omic profiling was performed in human breast cancer cells lines to determine immune regulatory genes controlled by growth factor stimulation. Human triple-negative breast cancer tumor specimens (N=248) were examined by immunohistochemistry for expression of VISTA, PDL1, PD1 and FoxP3. VISTA expression was engineered into human and mouse triple-negative breast cancer cell lines and growth metrics were characterized in vitro and in immunocompetent (syngeneic) and immunodeficient (xenograft) mouse tumor models. Membrane receptor trafficking and localization of EGFR was performed in human TNBC cells lines expressing VISTA and VISTA mutants. Proximity biotinylation identified protein partners that interacted with VISTA’s intracellular domain. Deletion mutagenesis was performed to identify motifs required for biochemical interactions between VISTA and clathrin adaptor proteins. Clathrin adapter localization was examined in WT and VISTA knock-out cell lines. Cytokine response, clathrin adaptor localization and tumor growth kinetics were measured in TNBC cell lines expressing VISTA or VISTA mutants. Results: A class of human triple-negative breast cancers were identified with high VISTA expression, low tumor infiltrating lymphocytes and low proliferative index (8-20% of all TNBCs). Tumor models demonstrated that enforced expression of VISTA in tumor cells caused growth suppression, even in the absence of an immune system. This effect was mapped to the cytoplasmic domain of VISTA, which lacks defined signaling domains. Through biochemical approaches, the clathrin-adaptor molecules NUMB and GULP1 were found in complex with a VISTA intracellular NPGF motif. This motif sequestered NUMB at early endosomes, resulting in diminished NUMB recycling for EGFR endocytosis, thereby causing growth suppression. VISTA impaired NUMB recycling by blocking Rab11 effector complex formation. VISTA NPGF mutations did not disrupt cytokine secretion in mixed lymphocyte assays. VISTA-targeted antibodies did not disrupt NUMB sequestration. VISTA+ tumors were selectively sensitive to VISTA-blocking antibodies. Conclusions: VISTA+ TNBC have distinct features including slower proliferative index and increased sensitivity to VISTA-blocking antibodies. This work identifies mechanisms by which VISTA enforces a quiescent cellular state by sequestering clathrin adapter proteins away from sites of endocytosis in cancer cells. This is the first description of intracellular molecular mechanisms controlled by VISTA’s intracellular tail. It may inform features of the VISTA-dependent immune checkpoint that could be exploited to improve anti-tumor immunity. Citation Format: Yan Zhao, Fatima Charles, Andrew Lipchik, Yan Peng, James Ford, Melinda Telli, Song Zhang, Allison Kurian, Michael Snyder, Joshua Gruber. Molecular characterization and therapeutic approach for VISTA+ triple-negative breast cancers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-02.

Abstract PO3-25-10: Selective and Effective Targeting of Triple-Negative Breast Cancer by CMPD1: Inhibition of MAP kinase-activated protein kinase 2 and Microtubule Dynamics

Abstract Background: Microtubule-targeting agents (MTAs) have been widely utilized in cancer treatment, but the development of novel MTAs has faced limitations. CMPD1, a highly selective inhibitor of MAP kinase-activated protein kinase 2 (MK2) with dual functions as a microtubule inhibitor, shows promise as a targeted therapy for triple-negative breast cancer (TNBC). This study aimed to investigate the effects of CMPD1, targeting MK2 and acting as a microtubule inhibitor, on cell cycle progression, microtubule dynamics, and TNBC-related processes. Methods: CMPD1 was evaluated in non-transformed retinal pigment epithelial cells (RPE1) and TNBC cell lines, including MDA-MB-231 and MDA-MB-468 cells. Time-lapse microscopy and immunofluorescence staining were employed to observe the effects of CMPD1 on mitotic progression, cellular morphology, activation of the spindle assembly checkpoint (SAC), and microtubule dynamics. Anchorage-independent growth assays and in vivo studies using a TNBC xenograft model were performed to assess the impact of CMPD1 on colony formation, tumor growth, and metastasis. Results: CMPD1 treatment induced prometaphase arrest and mitotic slippage in both non-transformed and TNBC cells. This arrest was associated with SAC activation, evidenced by increased levels of Mad1 at kinetochores. CMPD1 exhibited robust microtubule depolymerization, primarily targeting microtubule plus-ends and inhibiting microtubule polymerization in vitro. Furthermore, CMPD1 disrupted the organization of the microtubule network in interphase cells and perturbed the mitotic spindle during mitosis. Additionally, CMPD1 treatment resulted in the formation of cytoplasmic protrusions, membrane deformations, and dynamic movement of clustered mitotic chromosomes. These morphological alterations were indicative of the impact of CMPD1 on cellular structures and processes during mitosis. Notably, CMPD1 demonstrated significant efficacy in inhibiting anchorage-independent growth in TNBC cells, highlighting its potential as an anti-TNBC agent. In the TNBC xenograft model, CMPD1 exhibited superior anti-tumor activity compared to the standard chemotherapeutic agent Taxol. Tumor growth was markedly suppressed, and the formation of metastatic lesions was significantly reduced upon CMPD1 treatment. Furthermore, CMPD1 treatment effectively inhibited TNBC cell migration and invasion, likely through its modulation of microtubule dynamics. The disruption of microtubule dynamics by CMPD1 impaired the ability of TNBC cells to migrate and invade surrounding tissues, which are crucial processes in cancer metastasis. Importantly, CMPD1 showed high selectivity for TNBC cells, exerting minimal cytotoxic effects on normal cells, and could be efficiently washed out. This selectivity and washout ability enhance its potential as a targeted therapeutic agent for TNBC treatment. Conclusions: CMPD1, a highly selective MK2 inhibitor and microtubule inhibitor, exerts profound effects on cell cycle progression, microtubule dynamics, and TNBC-related processes. It induces prometaphase arrest, mitotic slippage, and microtubule depolymerization, resulting in altered cellular morphology. CMPD1 displays high selectivity for TNBC cells, while sparing normal cells and allowing efficient washout, making it a promising therapeutic agent for TNBC treatment. Its inhibition of anchorage-independent growth, suppression of tumor growth in vivo, and ability to impair TNBC cell migration and invasion further underscore its potential as a targeted anti-TNBC therapy. Citation Format: Mamoru Takada, Yu-Chia Chen, Yu Muhan, Hideyuki Yamada, Junta Sakakibara, Hiroshi Fujimoto, Takeshi Nagashima, Masayuki Otsuka, Motoki Takaku, Aussie Suzuki. Selective and Effective Targeting of Triple-Negative Breast Cancer by CMPD1: Inhibition of MAP kinase-activated protein kinase 2 and Microtubule Dynamics [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-25-10.

Abstract PO5-05-02: Discovery of BTX-9341, a bifunctional degrader of CDK4 and CDK6 for HR+/HER2- breast cancer

Abstract CDK4/6 inhibitors (CDK4/6i) such as palbociclib, abemaciclib and ribociclib are used to treat HR+/HER2- breast cancer, but patients can develop resistance via many mechanisms, several of which converge on the upregulation of cyclin D-CDK4/6 signaling node. This has been shown to limit the effectiveness of CDK4/6i in ER+ breast cancer with up to 20% patients exhibiting innate resistance and up to 70% patients developing acquired resistance after 3 years on therapy (Scheidemann, 2021). To address acquired resistance, we sought a degrader approach. We utilized our PRODEGY platform of Cereblon (CRBN) binders to synthesize CRBN mediated CDK4/6 bifunctional degraders and identified BTX-9341 as a development candidate due to its potency in vitro, in CDK4/6i resistant models and its effectiveness at inhibiting tumor growth in vivo. Degradation of CDK4/6 by immunoblot analysis of the triple negative breast cancer (TNBC) cell line, MDA-MB-231, treated with BTX-9341 for 6 hours showed up to 85% degradation of CDK4 and CDK6 with DC50s of < 1nM. Similar degradation depth and DC50 values were seen in HR+ breast cancer cells including T47D, MCF7 and ZR-751. CDK4/6 phosphorylates the protein RB which releases the transcription factor E2F, inducing the expression of genes which promote cell cycle progression. To determine the effect of CDK4/6 degradation on downstream signaling, we examined RB phosphorylation by in-cell western. Twenty-four hours treatment with BTX-9341 blocked RB phosphorylation robustly, with phospho-RB IC50s at < 30nM in HR+ breast cancer cells as well as MDA-MB-231 cells. Cell cycle analysis by staining with propidium iodide after 24 hours of treatment with BTX-9341 caused G0/G1 cell cycle arrest at concentrations as low as 10nM. We used a 2D colony formation assay (CFA) as a readout for inhibition of proliferation by cell cycle arrest. BTX-9341 showed potent inhibition of cell proliferation with CFA IC50s of < 50nM in TNBC cell lines and < 20nM in HR+ cell lines. We demonstrated that BTX-9341 was significantly more potent in vitro than the CDK4/6i, which had CFA values between 50 and 700nM in HR+ cell lines and between 100 and 1000nM in TNBC. This increased activity was due to CRBN mediated target degradation, as demonstrated by a shift in CFA IC50 values in a CRBN knockout cell line towards the values seen with the inhibitors. In palbociclib-resistant HR+/HER2- cell lines models which exhibit high CDK6 expression, BTX-9341 maintained a low CFA IC50 (< 150nM) while other CDK4/6 inhibitors displayed micromolar CFA IC50s. BTX-9341 displays excellent pharmacokinetic properties and oral bioavailability, which allowed for oral dosing in xenograft studies. MCF7 xenograft results with BTX-9341 showed dose-dependent tumor growth inhibition, tumor regression at higher doses and overall greater potency compared to the clinical CDK4/6i. BTX-9341 shows good exposure in the brain and exhibits tumor growth inhibition in an intracranial MCF-7 xenograft model, indicating that BTX-9341 could inhibit the growth of brain metastases. These results show that BTX-9341 displays excellent single agent activity in vitro and in vivo particularly in comparison to clinically approved CDK4/6i and that this activity is maintained in CDK4/6i resistant models. This indicates that a degrader approach to targeting this pathway may be more effective than current therapies, and that using this modality in a post CDK4/6i setting may be more effective than switching CDK4/6 inhibitors. Reference: Scheidemann, Erin R, and Ayesha N Shajahan-Haq. “Resistance to CDK4/6 Inhibitors in Estrogen Receptor-Positive Breast Cancer.” International journal of molecular sciences vol. 22,22 12292. 14 Nov. 2021, doi:10.3390/ijms222212292 Citation Format: Hannah Majeski, Akinori Okano, Kirti Chahal, Angela Pasis, Casey Carlson, Arvind Shakya, Qiao Liu, Shenlin Huang, Aparajita Hoskote Chourasia, Leah Fung. Discovery of BTX-9341, a bifunctional degrader of CDK4 and CDK6 for HR+/HER2- breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-05-02.

Abstract PO1-13-11: ZNF689 promotes homologous recombination via NBS1 ubiquitination and its loss confers sensitivity to PARP inhibition in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) poses significant clinical challenges due to the lack of targeted therapies and poor prognosis. Homologous recombination (HR) deficiency is a defining characteristic of TNBC and renders tumors susceptible to Poly (ADP-ribose) polymerase (PARP) inhibitors. However, not all TNBC patients respond to PARP inhibitors, highlighting the need to identify novel biomarkers to predict therapeutic response and improve treatment strategies. Methods: In this study, we investigated the role of zinc finger protein 689 (ZNF689) in DNA damage response (DDR) and its impact on PARP inhibitor sensitivity in TNBC. Using a comprehensive set of molecular and cellular techniques, including immunoprecipitation, in vitro and in vivo ubiquitination assays, protein expression analysis, and functional assays using TNBC cell lines, patient-derived organoids (PDOs), patient-derived xenograft (PDX) models, and cell line xenograft models, we elucidated the mechanistic role of ZNF689 in HR and PARP inhibitor response. Results: Our findings revealed ZNF689 as a novel player in DDR. ZNF689 is activated by ATM-mediated phosphorylation and recruited to DNA damage sites in an ATM-dependent manner. At the DNA double-strand break sites, ZNF689 collaborates directly with NBS1 to facilitate K63-linked ubiquitination of NBS1 via the E3 ligase SKP2. Furthermore, ZNF689-mediated NBS1 ubiquitination stabilizes the MRE11-RAD50-NBS1 (MRN) complex and feedbacks activation of ATM, therefore accelerating HR repair. Loss of ZNF689 results in HR deficiency, leading to increased sensitivity of TNBC cells to PARP inhibitors in both in vitro and in vivo models. Additionally, the combination of PARP inhibitors and paclitaxel demonstrated potential for improved efficacy in treating ZNF689-deficient TNBC. In our clinical analysis, we observed a positive correlation between ZNF689 expression and the levels of NBS1 and phosphorylated ATM in TNBC patient samples. Importantly, low ZNF689 expression was associated with a favorable response to PARP inhibitors in TNBC patients. Conclusions: Our study highlights the significant role of ZNF689 in HR and its impact on the response to PARP inhibitors in TNBC. These findings suggest ZNF689 as a promising therapeutic target for enhancing the effectiveness of PARP inhibitors in TNBC patients. Working Modle Citation Format: Li-Ping Ge, Zi-Yu Wang, Xi Jin, Yi-Zhou Jiang, Zhi-Ming Shao. ZNF689 promotes homologous recombination via NBS1 ubiquitination and its loss confers sensitivity to PARP inhibition in triple-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-13-11.

Abstract PO2-18-08: Identification of novel genetic/therapeutic vulnerabilities in breast cancer

Abstract Background: Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death among women worldwide. Triple negative breast cancer (TNBC) is an aggressive subtype that is defined by the lack of expression of estrogen and progesterone receptors in the absence of HER2 amplification or overexpression. TNBC represents 15-20% of all breast cancers and occurs more frequently in young premenopausal women. TNBC often ends with a poor clinical outcome due to high histological grade, rapid recurrence in many patients combined with few treatment options. These realities highlight the urgent need to identify novel therapeutics vulnerabilities that can be leveraged to improve TNBC patient outcomes. Methods: We utilized the publicly available DepMap and Kaplan Meier datasets to identify essential genes that were unique to TNBC cells and whose expression levels were associated with worse patient outcomes. siRNA based approaches were used to confirm the relevance of top hits in mediating cancer cell phenotypes including viability, cell-cycle profiles and induction of apoptosis among others. A novel drug currently being developed was identified for the top hit in these studies and was subsequently employed in multiple in vitro approaches to characterize the mechanisms of action in TNBC as well as initial efficacy studies using clinically relevant ex vivo patient derived models. Results: Using the DepMap database, we interrogated genome-wide sgRNA knockout screens conducted in over 40 breast cancer cell lines to identify specific genes whose expression was required for cell viability in TNBC cells, but not other breast cancer cell lines. This endeavor identified 6 genes (CTPS1, HUS1, PRKRA, RAD1, RAD9A and RHOA) that were preferentially essential in TNBC cells. Further profiling of these genes revealed that CTPS1 expression is higher in cancer relative to normal tissue and that its expression is further upregulated in chemotherapy- and PARPi-resistant models. Knockdown of CTPS1 in TNBC cells was shown to decrease cell proliferation and viability with no effects observed in estrogen receptor+ cell lines. Using a first-in-class, highly selective and orally bioavailable CTPS1 inhibitor (STP938) developed by Step-Pharma, we identified IC50s in the low nM range for many TNBC cell lines, including cell models that are resistant to existing standard-of-care therapies. Inhibition of CTPS1 was shown to arrest cells in S phase. Conclusions: Our studies have identified CTPS1 as an essential gene in TNBC. CTPS1 encodes an enzyme responsible for the catalytic conversion of UTP (uridine triphosphate) to CTP (cytidine triphosphate), an essential step in the biosynthesis of nucleic acids. These findings are timely in light of the recent and ongoing development of the first-in-class CTPS1 inhibitor, STP938, for which first-in-human studies are underway for refractory B- and T-cell lymphomas. Based on our initial studies using this drug, we provide pre-clinical evidence of its efficacy in multiple models of primary and advanced TNBC. Efforts are currently underway to move this work toward a clinical trial for TNBC patients. Citation Format: Xiyin Wang, Michael Emch, Lauren Voll, Rebecca Russell, Esther Rodman, Philip Beer, John Hawse. Identification of novel genetic/therapeutic vulnerabilities in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-18-08.

Abstract PO1-24-08: Targeting of HER3 potentiates the antitumor activity of paclitaxel against triple negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) is characterized by the absence of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) overexpression. This aggressive form of breast cancer carries a higher risk of recurrence compared to other subtypes, presenting a significant clinical challenge. Although chemotherapy is commonly used as the primary treatment for a substantial number of TNBC patients, it is often accompanied by drug resistance and frequent tumor recurrence. Urgent efforts are needed to identify novel molecular targets specific to TNBC and develop effective therapies to combat this aggressive disease. The ultimate objective is to discover treatments capable of overcoming drug resistance and improving the overall survival of patients with TNBC. Methods: Immunohistochemistry was performed to examine the expression of HER3 in TNBC samples. Western blots were used to assess protein expression and activation. Cell proliferation and viability were determined by cell growth (MTS) assays. TCGA databases were analyzed to correlate HER3 mRNA expression with the clinical outcomes of TNBC patients. Specific shRNA was used to knockdown HER3 expression. LIVE/DEAD Cell Imaging was to detect live and dead cells. Orthotopic tumor models were established in nude mice to determine the capability of TNBC cells forming tumors and to test if our newly developed anti-HER3 monoclonal antibody (mAb) 4A7 could potentiate the antitumor activity of paclitaxel in vivo. Results: The chemo-resistant subline HCC1806-TR was obtained by subjecting TNBC HCC1806 cells to prolonged treatment with paclitaxel. In comparison to the parental cell line HCC1806-P, HCC1806-TR exhibited enhanced HER3 expression and activation. It is noteworthy that approximately half of the tested TNBC specimens and cell lines displayed elevated expression of HER3. Analyzing TCGA databases revealed that TNBC patients with high levels of HER3 mRNA expression in their tumors had significantly poorer overall survival (OS) and relapse-free survival (RFS) compared to those with low HER3 expression. Specifically knockdown of HER3 markedly inhibited TNBC cell proliferation and mammosphere formation in vitro and tumor growth in vivo. Blockade of HER3 signaling with our mAb (4A7) in combination with paclitaxel exerted significant antitumor activity against TNBC in vitro and in vivo. Conclusions: Our data demonstrate that increased HER3 is an effective therapeutic target for TNBC and our anti-HER3 mAb (4A7) may enhance the efficacy of paclitaxel in TNBC. Keywords: HER3, Anbibody, Chemotherapy, Triple Negative Breast Cancer Citation Format: Hui Lyu, Sanbao Ruan, Congcong Tan, Ann Thor, Bolin Liu. Targeting of HER3 potentiates the antitumor activity of paclitaxel against triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-24-08.

Abstract PO4-14-01: Synergistic Activity of CDK8/19 Inhibitor RVU120 and MEK Inhibitors in Hormone-Negative Breast Cancer: Implications for Targeted Therapy

Abstract Breast cancer (BC) is a complex disease with distinct subtypes, including triple-negative breast cancer (TNBC), which lacks targeted therapies. CDK8 and CDK19, components of the mediator complex involved in transcriptional regulation, have emerged as potential therapeutic targets in BC. Transcriptomic analysis revealed that elevated CDK8 expression was associated with poor prognosis across all BC subtypes. Furthermore, previous studies demonstrated that sensitivity to CDK8/CDK19 inhibition correlated with high STAT3 phosphorylation and enrichment of TNF/NF-kB and STAT target genes in responder cells. In this study, we evaluated the responsiveness of TNBC cell lines to RVU120, a highly selective and potent CDK8/CDK19 inhibitor currently undergoing clinical trials for acute myeloid leukemia (AML), high-risk myelodysplastic syndrome (HR-MDS), and solid tumors. Notably, the sensitivity of TNBC cell lines to RVU120 varied depending on the culture conditions, with the presence of epidermal growth factor (EGF) identified as a major determinant of response variability. Considering the role of EGFR in activating downstream signaling pathways, including MAPK and STAT, and previous observations of synergy between small molecule CDK8 inhibitors and MEK inhibitors in neuroblastoma, we investigated MEK inhibitors as potential combination partners in hormone-negative BC. Among the 15 tested BC cell lines, a synergy between CDK8/CDK19 inhibitors and MEK inhibitors was observed in 5 cell lines, antagonism in another 5 cell lines, and no synergy in the remaining 5 cell lines, based on the analysis with Combenefit software (Loewe additivity model). Importantly, cell lines where synergy of the combination therapy has been observed, exhibited EGFR amplification and markers of activated RAS pathway. This pattern of response was consistent across different MEK and MEK/RAF inhibitors, including selumetinib, trametinib, and avutometinib. These findings highlight the synergistic potential of combining RVU120 with MEK inhibitors in hormone-negative BC, particularly in TNBC with EGFR amplification and an active RAS pathway. Ongoing studies involving RNAseq analysis and organoid screening aim to further elucidate the underlying mechanisms and confirm the efficacy of this combination therapy. Furthermore, in vivo efficacy experiments are necessary to validate in vitro activity of both compounds. Successful development of this therapeutic concept relies on the validation and implementation of selected partner drugs targeting RAS pathway, as well as the identification of suitable biomarkers such as EGFRamp or RAS-score. Promising findings of this study provide a rationale for exploring the combination of CDK8/CDK19 inhibitor RVU120 and MEK inhibitors as a potential targeted therapy strategy for hormone-negative BC. Citation Format: Urszula Pakulska, Adrianna Moszynska, Justyna Martyka, Jerzy Woznicki, Elzbieta Adamczyk, Marta Obacz, Kinga Keska Izworska, Katarzyna Wiklik, Agata Stachowicz, Hendrik Nogai, Milena Mazan, Tomasz Rzymski. Synergistic Activity of CDK8/19 Inhibitor RVU120 and MEK Inhibitors in Hormone-Negative Breast Cancer: Implications for Targeted Therapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-14-01.

Abstract PO5-06-07: Unravelling the role of PKC-eta in modulating the Hippo Pathway: a novel therapeutic strategy for triple-negative breast cancer metastasis

Abstract Breast cancer (BC) is the leading cause of cancer-related deaths in women, with metastasis being the primary reason for BC mortality. Triple-Negative Breast Cancer (TNBC) is aggressive and highly metastatic, having fewer treatment options than other types of breast cancer. Protein kinase C eta (PKCη), an anti-apoptotic kinase of the novel PKC subfamily, is associated with poor prognosis in invasive BC patients. In this study, we show the role of PKCη in promoting cell migration and invasion, and we demonstrate that its expression is required to maintain the mesenchymal state during EMT. Furthermore, we show that PKCη activates YAP/TAZ, tumor aggressiveness, and metastasis using in vitro and in vivo models. PKCη promotes metastasis in TNBC cells, showing that the PKCƞ-YAP signaling axis mediates this. Data analysis of BC patients with a high metastatic invasive propensity and low survival BC revealed a positive correlation between the elevated expression levels of PKCη and the expression of YAP. Knockout of PKCη (PKCηKO) in the TNBC cells 4T1 and MDA-MB-231 markedly inhibited their invasion and migration capability. We further show that PKCη enhances cell survival and anoikis prevention ability, which have crucial roles in metastasis formation, allowing cancer cells to circulate and home to distant sites in the body. Further substantiated by in vivo experiments with 4T1 xenografts in NSG mice, demonstrating that 4T1 PKCηKO cells depicted reduced primary tumor size and a significant decrease in size and number of tumor metastases in the lungs, with fewer tumor nodules. MDA-MB-231 PKCηKO xenografts also expressed reduced metastasis in the distant brain, lung, and liver organs. Mechanistically, we show that PKCη regulates epithelial-to-mesenchymal transition (EMT) markers as knockout of PKCη in TNBC cell lines increased expression of E-cadherin, EpCAM, and slug, and decreased expression of vimentin and ZEB1. Further profiling of the Hippo-YAP axis showed that PKCη is a negative regulator of the Hippo pathway that leads to YAP stabilization and is associated with phosphorylation at Ser128, which allows YAP to translocate to the nucleus and contributes to metastasis of TNBC cells. Moreover, our data support direct interaction between PKCη and YAP1 in TNBC cells. Finally, we show that treatment of TNBC cells with uPEP2 (a uORF-encoded peptide of the PKCη transcript) downregulates the PKCη expression, activates the Hippo signaling pathway and promotes YAP degradation. Our findings highlight the importance of PKCη in TNBC metastasis and provide a new avenue for therapeutic intervention in this aggressive and lethal disease. In conclusion, our studies identify a novel role of PKCη as a negative regulator of the Hippo pathway and reveal its function in promoting EMT and metastasis by modulating YAP/TAZ activity in TNBC. Results suggest that PKCη may represent a therapeutic target for this highly lethal and metastatic TNBC. Citation Format: Liju Vijaya Steltar Belsamma, Amitha Muraleedharan, Divya Ram Jayaram, Kamran Waidha, Sankar Jagadeeshan, Rose Sinay, Ekaterina Eremenko, Omer Berner, Moshe Elkabets, Etta Livneh. Unravelling the role of PKC-eta in modulating the Hippo Pathway: a novel therapeutic strategy for triple-negative breast cancer metastasis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-06-07.

Abstract PO1-29-01: Single agent and combinatorial efficacy with imipridone ONC206 via inhibition of mitochondrial function in preclinical models of chemorefractory triple negative breast cancer

Abstract Breast cancer remains a major global health concern, with triple-negative breast cancer (TNBC) being one of the most aggressive subtypes associated with limited targeted therapies. Chemotherapy resistance in TNBC patients poses a significant clinical challenge and is associated with poor progression-free and overall survival. Thus, exploring novel approaches to improve chemotherapeutic efficacy is of paramount importance. We previously demonstrated a role for mitochondrial fusion in supporting oxidative phosphorylation (oxphos) activity and cell survival in residual TNBC (PMID: 30996079 and 36813854) cells refractory to chemotherapy treatments. Based on these observations, we sought to inhibit mitochondrial function to improve chemotherapeutic efficacy. We used ONC206, a novel agonist of mitochondrial serine protease, ClpP (Caseinolytic Mitochondrial Matrix Peptidase Proteolytic Subunit) to induce mitochondrial proteotoxic stress. ONC201, a parent molecule of ONC206, has shown efficacy in TNBC as a single agent or when combined with a MEK inhibitor in vitro (PMID: 34680527). We demonstrate treatment of TNBC cell lines with ONC206 results in reduced mitochondrial fusion and OXPHOS. Furthermore, ONC206 co-treatment enhanced chemo-sensitivity in vitro. Based on these promising findings, we tested this in orthotopic patient-derived xenograft (PDX) mouse preclinical trials. Three PDX models of TNBC were tested, BCM-2665, BCM-5471, and BCM-0002. ONC206 as a single agent (twice weekly for 4 weeks, oral gavage, 100mg/kg) significantly decreased tumor growth in all three PDX models. Remarkably, treatment ONC206 achieved nearly complete tumor regression as a single agent or when combined with carboplatin in BCM-2665. Regressed tumors did re-grow, but this was significantly delayed when combined with carboplatin. Treatment with ONC206 in BCM-5471 resulted in a modest but significant reduction in tumor growth rate as a single agent. However, ONC206 did not improve carboplatin (weekly, i.p., 50mg/kg) nor docetaxel (weekly, i.p., 20mg/kg) efficacy in this model. ONC206 treatment of BCM-0002 resulted in prolonged tumor stasis and a slight enhancement of docetaxel and carboplatin efficacy. Interestingly, the efficacy of ONC206 in these PDX models correlates with their relative mRNA expression of ClpP. The mice demonstrated tolerance to ONC206 as a single agent or when combined with chemotherapy during the course of treatment. Our study underscores the potentially crucial role of mitochondria in driving chemotherapy resistance in TNBC and provides novel insights into mitochondria-targeted therapeutic approaches. We report a novel targeting option for TNBC and anticipate that these findings will stimulate further research into innovative combination therapies for TNBC patients, and may lead us to a viable inhibitor. Citation Format: Lily Baek, Lacey Dobrolecki, Stefanie Faucher, Christina Sallas, Nia Griffith, Varun Prabhu, Bora Lim, Michael Lewis, Gloria Echeverria. Single agent and combinatorial efficacy with imipridone ONC206 via inhibition of mitochondrial function in preclinical models of chemorefractory triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-29-01.

Abstract PO4-19-11: A Multicenter, Phase Ib/II Study of Abemaciclib in Combination with Bicalutamide for Androgen Receptor-positive, HER2-negative Metastatic Breast Cancer

Abstract Background The androgen receptor (AR) is expressed in approximately 30% of breast cancers. Phase I and II trials of bicalutamide in HR-negative and enzalutamide in both triple negative breast cancer (TNBC) and hormone receptor (HR) positive populations have shown safety and favorable clinical benefit rates (CBR). Additionally, cell cycle (CC) inhibitors, such as abemaciclib, have demonstrated improved progression free survival (PFS) for patients with HR-positive metastatic breast cancer (MBC) in combination with ET in the first line and subsequent lines of therapy. A preclinical study showed that anti-androgen therapies enhanced CKD4/6 induced cytostatic effect in AR-positive TNBC cell lines. We performed a retrospective, institutional study evaluating metastatic HR+ tumor samples with immunohistochemistry (IHC) from patients treated with CDK4/6 inhibitor, palbociclib. We found a statistically significant increase in event free survival (EFS) for those that had any expression of AR (>0%, median EFS 19 months vs 5 months, HR 0.26, p=0.01). Additionally, AR expression was associated with intact phosphorylated retinoblastoma (Rb) expression (100%, P=0.02), which may represent a mechanism by which CC inhibition is effective in AR+ MBC. Trial Design NCT 05095207 is an ongoing, Phase Ib/II, open label, multi-center study. The primary objective for the phase I portion is to determine the recommended phase II dose (RP2D) of abemaciclib in combination with bicalutamide. The primary objective for the phase II portion is to determine the disease control rate (DCR) at 6-month treatment with abemaciclib and bicalutamide. Patients with HER2-negative MBC who are AR+ (IHC >1%) are eligible. HR-positive participants must have had one prior line of ET in the metastatic setting and no more than 2 prior lines of cytotoxic chemotherapy. HR-negative participants may have had up to 4 prior lines of cytotoxic chemotherapy. In the Phase I portion, up to 12 patients are given abemaciclib 100 mg oral, twice daily on days 1 to 28 and bicalutamide 150 mg oral, daily on days 1 to 28. If there is no toxicity after cycle 1 for the first 3 patients, the dose of abemaciclib will increase to 150 mg twice daily. However, if there is a dose limiting toxicity (DLT) within the first cycle (28 days) of therapy, the dose will decrease to 50 mg twice daily. In the Phase II portion, 51 patients will receive abemaciclib at the RP2D twice daily in addition to bicalutamide 150 mg daily on days 1 to 28. The estimated study duration is 36 months. The drugs will be administered until disease progression or unacceptable toxicity. The primary end points include the DLT and the RP2D for abemaciclib administered with bicalutamide and the DCR at 6 months. The secondary end points include the frequency of adverse effects, the DCR stratified by HR status, and PFS. Exploratory endpoints are to determine whether Ki67, cyclin D1, p16, and phosphorylated Rb expression at baseline are predictors of response to abemaciclib and to determine potential mechanisms for resistance to treatment. For Phase Ib, we will utilize the Bayesian optimal interval (BOIN) design, the RP2D will be selected as the dose closest to the target toxicity rate of 0.3. The observed DLT rate at the current dose is ≤ 0.236, the next cohort will be treated at the next higher dose level, if it is ≥ 0.359 the next cohort will be treated with the next lower dose level. For Phase II, we will assess the DCR using a Simon 2 stage optimal design. If among the 46 evaluable patients, 12 or fewer achieve disease control, the treatment will be rejected, however if 13 or more have disease control, the treatment will be considered worthy for further study. We define inactivity as DCR of 0.20 and activity as DCR of 0.35. To date, the study has recruited 21 participants, the goal accrual is 60 patients. Citation Format: Nicole Casasanta, Chrystal Landry, Theresa Shao, Paula Klein, Julie Fasano, Aarti Bhardwaj, Natalie Berger, Erin Moshier, Gargi Atul Joshi, Rita Vaccaro, Joseph Sparano, Amy Tiersten. A Multicenter, Phase Ib/II Study of Abemaciclib in Combination with Bicalutamide for Androgen Receptor-positive, HER2-negative Metastatic Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-19-11.

Abstract PO1-06-11: Exosomal HMGB1 induce PD-L1+-tumor associated macrophages through glycometabolic reprogramming to promote lung-tropic metastasis of triple negative breast cancer

Abstract Background: Lung metastasis occurs in 30% of triple negative breast cancer (TNBC), the detailed molecular mechanism remains largely unexplored. Accumulating evidence suggests that PD-L1+-tumor associated macrophages (PD-L1+-TAMs) are closely related to immune suppression, invasion, and metastasis. Tumor-derived exosomes have been reported to remodel tumor microenvironment and accelerate metastasis via packing and delivering a variety of biologically active molecules. Our previous studies found that exosomes from breast cancer cells polarized macrophages toward TAMs within the primary tumor nest. The aim of this study was to reveal whether TNBC-derived exosomes were able to polarize lung macrophages towards PD-L1+-TAMs and generate a pre-metastatic immunosuppressive niche, therefore discovering the underlying mechanism of lung-tropic metastasis. Methods: Exosomes isolated from EO771 TNBC cell line (EO771/exo) and HC11 normal breast epithelial cell line (HC11/exo) were characterized. Uptake of exosomes by macrophages was visualized by confocal microscopy. Mass spectrometry was applied to identify the significantly highly expressed HMGB1 protein in EO771/exo, and ligands of HMGB1 on lung macrophages were detected by co-immunoprecipitation. Functions of EO771/exo and HMGB1 in inducing PD-L1+-TAMs and generating a pre-metastatic immunosuppressive niche were confirmed by both in vitro and in vivo studies. Roles of EO771/exo and HMGB1 on glycolysis and lactate production were evaluated by metabolism assays. Associations between PD-L1+-TAMs and CD3+CD8+ T lymphocytes within TNBC axillary lymph nodes and lung metastasis specimens were analyzed by immunofluorescence and immunohistochemistry. Results: Confocal microscopy showed constant internalization and absorption of EO771/exo by macrophages. EO771/exo could polarize macrophages toward PD-L1+-TAMs and generate a pre-metastatic immunosuppressive niche, with respect to HC11/exo. Mass spectrometry, clinical samples, and bioinformatics analysis revealed that HMGB1 was highly expressed in EO771/exo and was involved in cellular communication and metabolism processes. Co-immunoprecipitation and 3-dimensional modeling indicated a strong binding of HMGB1 with Tim-3 ligand on lung macrophages. Macrophages treated with EO771/exo displayed an enhanced glycolysis and lactate production. Flow cytometry demonstrated that green fluorescent protein (GFP)-EO771 tumor-bearing mice had more lung micro-metastases than brain and liver. Immunofluorescence and laser confocal microscopy confirmed that EO771/exo, with respect to HC11/exo, were able to increase lung micro-metastases and escalate PD-L1 expression on lung macrophages. Moreover, elevated PD-L1+-TAMs and reduced CD3+CD8+ T lymphocytes were found in TNBC positive axillary lymph nodes and lung metastasis specimens than TNBC negative axillary lymph nodes. Conclusions: Exosomal HMGB1 from TNBC could target Tim-3 ligand on lung macrophages and accelerate glycolysis and lactate production to induce PD-L1+-TAMs, thereby suppressing CD3+CD8+ T lymphocytes immunity and generating a pre-metastasis immune-suppressive niche. Our research would uncover a novel mechanism of lung-tropic metastasis and provide a new therapeutic approach for TNBC treatment. Keywords: breast cancer; lung metastasis; exosomes; tumor-associated macrophages; metabolic reprogramming Citation Format: Wei-Xian Chen, Jia-Hao Wu, Tian-Cheng Cheng, Sujin Yang. Exosomal HMGB1 induce PD-L1+-tumor associated macrophages through glycometabolic reprogramming to promote lung-tropic metastasis of triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-06-11.