Therapy For Triple-negative Breast Cancer Research Articles

Advancements and challenges in triple-negative breast cancer: a comprehensive review of therapeutic and diagnostic strategies

Triple-negative breast cancer (TNBC) poses significant challenges in oncology due to its aggressive nature, limited treatment options, and poorer prognosis compared to other breast cancer subtypes. This comprehensive review examines the therapeutic and diagnostic landscape of TNBC, highlighting current strategies, emerging therapies, and future directions. Targeted therapies, including PARP inhibitors, immune checkpoint inhibitors, and EGFR inhibitors, hold promise for personalized treatment approaches. Challenges in identifying novel targets, exploring combination therapies, and developing predictive biomarkers must be addressed to optimize targeted therapy in TNBC. Immunotherapy represents a transformative approach in TNBC treatment, yet challenges in biomarker identification, combination strategies, and overcoming resistance persist. Precision medicine approaches offer opportunities for tailored treatment based on tumor biology, but integration of multi-omics data and clinical implementation present challenges requiring innovative solutions. Despite these challenges, ongoing research efforts and collaborative initiatives offer hope for improving outcomes and advancing treatment strategies in TNBC. By addressing the complexities of TNBC biology and developing effective therapeutic approaches, personalized treatments can be realized, ultimately enhancing the lives of TNBC patients. Continued research, clinical trials, and interdisciplinary collaborations are essential for realizing this vision and making meaningful progress in TNBC management.

Recent Developments in Combination Immunotherapy with Other Therapies and Nanoparticle-Based Therapy for Triple-Negative Breast Cancer (TNBC)

Recent Developments in Combination Immunotherapy with Other Therapies and Nanoparticle-Based Therapy for Triple-Negative Breast Cancer (TNBC)

Oncolytic Tanapoxvirus Recombinants Expressing Flagellin C or Mouse Interleukin-2 Are Capable of Regressing Human Triple-Negative Breast Cancer Xenografts in Immuno-Competent BALB/c Nude Mice.

Triple-negative breast cancer (TNBC) in humans is the most aggressive and deadly form of BC. Although TNBCs are about 15 percent of the total number of BC cases, they are associated with the highest mortalities. Current treatment options are limited, and most modalities are toxic and have not increased the 5-year survival rates of TNBC. Many oncolytic viruses are emerging as potential therapies for TNBC. In this study, two Tanapoxvirus (TPV) recombinants, one expressing FliC and the other expressing mouse interleukin-2 (mIL-2), were assessed for their efficacy in an immuno-competent xenograft mouse model. MDA-MB-231 tumors were planted in BALB/c nude mice, treated, made immuno-competent via adoptive transfer of splenocytes from healthy BALB/c donors, and then monitored for 40 days. TPV/Δ2L/66R/FliC and TPV/Δ66R/mIL-2 demonstrated significant tumor reduction (p = 0.01602 and p = 0.03890, respectively) compared to the reconstituted control (RC), whereas wtTPV did not. Pathological analyses of treated tumors revealed cells consistent with lymphocyte and plasma cell morphology in reconstituted mice treated with TPV recombinants. Anti-viral plaque reduction assays conducted using harvested serum from treated animals indicated the presence of anti-TPV antibodies in mice reconstituted and treated with TPV that were missing from immune-deficient nude mice, including those exposed to TPV and of statistically equivalent serum concentrations to normal BALB/c mice immunized against TPV. The results suggest immuno-deficient BALB/c nude mice can become immuno-competent via adoptive transfer of splenocytes from genetically identical donors and allow for testing of tumor xenografts in a competent model system. The TPV recombinants tested should be further studied for the potential treatment of human TNBC.

Ternary heterostructure-driven photoinduced electron-hole separation enhanced oxidative stress for triple-negative breast cancer therapy

Zinc oxide nanoparticles (ZnO NPs) stand as among the most significant metal oxide nanoparticles in trigger the formation of reactive oxygen species (ROS) and induce apoptosis. Nevertheless, the utilization of ZnO NPs has been limited by the shallowness of short-wavelength light and the constrained production of ROS. To overcome these limitations, a strategy involves achieving a red shift towards the near-infrared (NIR) light spectrum, promoting the separation and restraining the recombination of electron-hole (e−-h+) pairs. Herein, the hybrid plasmonic system Au@ZnO (AZ) with graphene quantum dots (GQDs) doping (AZG) nano heterostructures is rationally designed for optimal NIR-driven cancer treatment. Significantly, a multifold increase in ROS generation can be achieved through the following creative initiatives: (i) plasmonic Au nanorods expands the photocatalytic capabilities of AZG into the NIR domain, offering a foundation for NIR-induced ROS generation for clinical utilization; (ii) elaborate design of mesoporous core-shell AZ structures facilitates the redistribution of electron-hole pairs; (iii) the incorporation GQDs in mesoporous structure could efficiently restrain the recombination of the e−-h+ pairs; (iv) Modification of hyaluronic acid (HA) can enhance CD44 receptor mediated targeted triple-negative breast cancer (TNBC). In addition, the introduced Au NRs present as catalysts for enhancing photothermal therapy (PTT), effectively inducing apoptosis in tumor cells. The resulting HA-modified AZG (AZGH) exhibits efficient hot electron injection and e−-h+ separation, affording unparalleled convenience for ROS production and enabling NIR-induced PDT for the cancer treanment. As a result, our well-designed mesoporous core-shell AZGH hybrid as photosensitizers can exhibit excellent PDT efficacy.

Mitomycin C in the treatment of early triple-negative locally advanced BRCA-associated breast cancer

Rationale. BRCA1 associated triple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer. At the same time, carcinomas that develop in carriers of BRCA1 mutations are characterized by extremely high sensitivity to DNA-damaging chemotherapy. Mitomycin C alone or in combination with platinum agents has already demonstrated promising results in the treatment of BRCA-associated ovarian cancer (OC) and metastatic breast cancer. In this article, we present the results of a retrospective study aimed at comparing standard neoadjuvant chemotherapy regimens (NACT) with mitomycin-based regimens for primary locally advanced BRCA1-associated TNBC.The aim of the study is to determine the effectiveness of the combination of mitomycin and platinum compounds during neoadjuvant therapy in patients with primary locally advanced BRCA1 – associated TNBC.Materials and methods. The study included 89 patients diagnosed with primary locally advanced BRCA1-associated TNBC. Patients were divided into three groups depending on the therapy: 1) 4 cycles of anthracycline and cyclophosphamide followed by 12 weekly injections of paclitaxel (n = 48) (AC + T), 2) 4 cycles of anthracycline and cyclophosphamide followed by 12 weekly injections of paclitaxel and carboplatin (n = 27) (AC + TCbP), 3) mitomycin C plus platinum followed by 12 weekly injections of paclitaxel (n = 14) (MR + T). Pathological complete response (pCR) rates were compared.Results. The pCR rate in the MP+T group was 10/14 (71%). In patients with BRCA1-associated breast cancer who received AC + T and AC + TCbP regimens as NACT, the pCR rate was 17/48 (35%) and 19/27 (70%), respectively. The difference in pCR rate between mitomycin-containing therapy and the standard AC + T regimen was statistically significant (p = 0.03); the frequency of regressions was comparable to the frequency in the AC + TCbP group. During the 20-month follow-up period, no relapses were observed in the MR + T group. Relapses were more frequent in the AC + T group compared with the AC + TCbP group (16/48 (33%) vs 1/27 (4%), p = 0.003, Fisher’s exact test). The toxicity profile of the mitomycin-containing regimen included hematologic adverse events, the most common of which were anemia and leukopenia. Compared to standard regimens, nausea was significantly less pronounced. No patients reported alopecia with this regimen.Conclusions. The addition of mitomycin C to neoadjuvant therapy for BRCA1-associated TNBC may be a promising treatment option for this category of patients and merits further study.

Emergence of Quantum Dots as Innovative Tools for Early Diagnosis and Advanced Treatment of Breast Cancer.

Quantum dots (QDs) semiconducting nanomaterials, have garnered attention due to their distinctive properties, including small size, high luminescence, and biocompatibility. In the context of triple-negative breast cancer (TNBC), notorious for its resistance to conventional treatments, QDs exhibit promising potential for enhancing diagnostic imaging and providing targeted therapies. This review underscores recent advancements in the utilization of QDs in imaging techniques, such as fluorescence tomography and magnetic resonance imaging, aiming at the early and precise detection of tumors. Emphasis is placed on the significance of QD design, synthesis and functionalization processes as well as their use in innovative strategies for targeted drug delivery, capitalizing on their ability to selectively deliver therapeutic agents to cancer cells. As the research in this field advances rapidly, this review covers a classification of QDs according to their composition, the characterization techniques than can be used to determine their properties and, subsequently, emphasizes recent findings in the field of TNBC-targeting, highlighting the imperative need to address challenges, like potential toxicity or methodologies standardization. Collectively, the findings explored thus far suggest that QDs could pave the way for early diagnosis and effective therapy of TNBC, representing a significant stride toward precise and personalized strategies in treating TNBC.

In silico identification of a novel Cdc2‐like kinase 2 (CLK2) inhibitor in triple negative breast cancer

AbstractDysregulation of RNA splicing processes is intricately linked to tumorigenesis in various cancers, especially breast cancer. Cdc2‐like kinase 2 (CLK2), an oncogenic RNA‐splicing kinase pivotal in breast cancer, plays a significant role, particularly in the context of triple‐negative breast cancer (TNBC), a subtype marked by substantial medical challenges due to its low survival rates. In this study, we employed a structure‐based virtual screening (SBVS) method to identify potential CLK2 inhibitors with novel chemical structures for treating TNBC. Compound 670551 emerged as a novel CLK2 inhibitor with a 50% inhibitory concentration (IC50) value of 619.7 nM. Importantly, Compound 670551 exhibited high selectivity for CLK2 over other protein kinases. Functionally, this compound significantly reduced the survival and proliferation of TNBC cells. Results from a cell‐based assay demonstrated that this inhibitor led to a decrease in RNA splicing proteins, such as SRSF4 and SRSF6, resulting in cell apoptosis. In summary, we identified a novel CLK2 inhibitor as a promising potential treatment for TNBC therapy.

New Vaccine Therapy for Triple-Negative Breast Cancer

Abstract Purpose of the review The objective of this review is to provide an analysis of early-phase clinical trials investigating vaccine therapies for triple-negative breast cancer (TNBC). Specifically, the focus is on ongoing trials that are actively recruiting or in progress, while excluding vaccines that target neoantigens or those that have already completed trials. Recent findings Over the past decade, notable transformations have occurred in the strategy of breast cancer vaccine design. Traditional approaches to identifying tumor antigens, such as SEREX, have been replaced with modern techniques, such as RNA sequencing, HLA typing, and immunoinformatics. These new methods enable the identification and characterization of tumor antigens. Notably, current clinical investigations into tumor targets extend beyond mutated self-proteins or proteins that are overexpressed following neoplastic transformation. Clinical researchers are currently examining protein targets associated with cancer stem cells or non-malignant immune regulatory cell types within the tumor microenvironment. However, the application of up-to-date antigen delivery methods for certain types of breast cancer vaccine therapies still lags behind. Another significant transformation in comparison to previous breast cancer vaccine therapies is the emphasis on stimulating robust T-cell responses against breast cancer cells, independent of any B-cell response directed at the tumor. Summary In conclusion, we critically assessed the tumor antigens targeted by vaccine immunotherapies in these new clinical trials, the delivery methods used for these antigens, and conclude by discussing potential future directions for the development of new TNBC vaccine therapies.

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.

Abstract PO1-14-07: Programme of mast cell subsets to potentiate breast cancer immunotherapy: from bed to bench to bed (the phase 2 platform RENAISSANCE trial)

Abstract Background: Immune checkpoint inhibitors (ICIs) have heralded a new era in breast cancer treatment; however, response rates remain limited, making precision immune-oncology a major unmet need. In addition to T cells, effective immune responses to ICIs rely on coordinated interactions between innate and adaptive immune cells. Mast cells are evolutionarily conserved, tissue-resident cells of importance to human health. Specific subsets of mast cells might be endowed with opposite roles in cancer treatment, yet the extent of mast cell heterogeneity and its clinical merit in immunotherapy remain undefined. Objective: We sought to comprehensively characterize mast cells in breast cancer, investigate their association with immunotherapy response with in-depth mechanistic insights, and identify actionable strategies to modulate mast cell functional states, thereby optimizing immunotherapy efficacy. Methods: We employed single-cell profiling on longitudinal breast cancer samples from three independent clinical trials (NCT04613674, NCT03197389 and GSE169246) to delineate mast cell heterogeneity in anti-PD-(L)1 therapy. By integrating multi-omic analyses, tissue characterization, preclinical experiments, transgenic mice, and high-throughput drug screening, we outlined the molecular features, underlying mechanisms, and clinical relevance of distinct mast cells to elicit ICI-responsive microenvironments. Subsequently, we launched RENAISSANCE (NCT05076682), a proof-of-concept, Bayesian adaptive, phase 2 platform trial, to evaluate the efficacy and safety of combining mast cell therapeutics with anti-PD-1 backbone therapy in metastatic triple-negative breast cancer (TNBC) patients who progressed after immunotherapy. The primary endpoint was the objective response rate (ORR) assessed using RECIST v1.1 criteria. Results: We identified a distinct population of mast cells termed antigen-presenting mast cells (APMCs), constituting approximately 30% of intratumoral mast cells and correlating with improved clinical benefit of anti-PD-(L)1 therapy in TNBC. APMCs displayed MHC-II and costimulatory molecules, and indicated the presence of tumor-reactive T cells and tertiary lymphoid structures. Using three immunocompetent mouse models, we confirmed the immunomodulatory capacity of APMCs in immunotherapy. Mechanistically, by employing Cpa3CreERT2Cd74fl/fl mice, we demonstrated that APMCs potentiate anti-PD-1 efficacy and antitumor T cell immunity through their antigen-presentation machinery. Interestingly, we identified cromolyn, an FDA-approved drug for allergy, as a potential therapeutic agent that elicited APMC-dependent CD8+ T cell cytotoxicity to synergize with anti-PD-1 therapy. Between February 2022 and March 2023, 10 patients with immunotherapy-refractory metastatic TNBC were enrolled to receive cromolyn plus camrelizumab backbone treatment. Given Bayesian predictive probability, this arm was “graduated” due to meeting the pre-specified efficacy boundary, with an ORR of 40.0% (4/10). The treatment was well tolerated with similar safety profiles of relevant drugs. Conclusions: Our findings provide crucial insights into the impact of mast cell heterogeneity on the clinical response to ICIs at a single-cell level, and pave the way for APMC-directed therapeutic interventions in cancer treatment. To our knowledge, this is the first prospective study in breast cancer of cromolyn plus anti-PD-1 backbone regimen after anti-PD-(L)1 immunotherapy failure, demonstrating significant antitumor activity and commendable tolerability. Consequently, we suggest a phase 3 randomized study to consolidate this finding, which might be an effective treatment in patients for whom there are few effective treatment options. Citation Format: Song-Yang Wu, Xi Jin, Yin Liu, Wen-Jia Zuo, Li Chen, Xiyu Liu, Lei Fan, Zhong-Hua Wang, Yan-Fei Liu, Yi-Zhou Jiang, Zhi-Ming Shao. Programme of mast cell subsets to potentiate breast cancer immunotherapy: from bed to bench to bed (the phase 2 platform RENAISSANCE trial) [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 PO1-14-07.

Abstract PO4-03-13: High B7-H3 expression with low PD-L1 expression identifies armored-cold tumors suggesting potential anti-B7-H3 therapy in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is generally regarded as the most aggressive subtype among breast cancer. B7 homolog 3 protein (B7-H3), also known as CD276, is an emerging immune checkpoint molecule, which plays a dual role in the immune system. Multiple anti-B7-H3 therapies has been raised, such as DS-7300, MGA271, MGD009, and B7-H3 chimeric antigen receptor T-cell immunotherapy (CAR-T). However, the expression of B7-H3 in TNBC and its value as a biomarker identifying therapeutic options has not been elucidated Methods: A total of 165 samples (83 non-TNBC and 82 TNBC) were included in this study, and 6 samples included paired para-tumor samples. In addition, 30 TNBC patients receiving standardized neoadjuvant chemotherapy (NAT) were recruited. After 8 cycles of NAT, the response to NAT was assessed by RECIST1.1 criterion before surgical operation. Moreover, multiple public cohorts of breast cancer and TNBC were obtained, including the TCGA dataset, the METABRIC dataset, the GSE176307 (durvalumab-based therapy) dataset, the PRJNA558949 (durvalumab-based therapy) dataset, the GSE194040 (paclitaxel-based therapy) dataset, and the GSE34138 (anthracycline-based therapy) dataset. Based on multiple in-house and public cohorts, we investigated the expression features of B7-H3 in breast cancer and checked the anti-tumor effect of the B7-H3 monoclonal antibody in mouse model. We also developed a novel classifier combined B7-H3 and PD-L1 expression in TNBC. Results: B7-H3 was highly expressed in tumor tissues in the in-house and the TCGA cohorts. B7-H3 expression did not differ significantly between non-TNBC and TNBC samples in the in-house and the TCGA cohorts, excepting for the METABIRC cohort. There was no notable difference in CD8+ cells infiltration between B7-H3 low and high expressed non-TNBC samples, but CD8+ cells was notably higher in the B7-H3 low expressed TNBC samples. The similar findings were also observed in the TCGA and the METABIRC cohorts. In addition, enrichment analysis of transcriptome data and analysis of in-house samples showed that B7-H3 was positively related to most collagens. Moreover, anti-B7-H3 therapy significantly inhibited tumor growth in mouse TNBC model, notably increased immune cells infiltration, and also reduced collagen deposition within the tumor. Given the notable value of PD-L1 in predicting immunotherapy responses, we also combined B7-H3 and PD-L1 expression to establish a novel subtyping strategy. TNBC patients with B7-H3highPDL1low feature exhibited the lowest anti-tumor immune infiltration and higher collagen deposition level. In the durvalumab-dependent cohort, paclitaxel-dependent cohort, anthracycline-dependent cohort, and our recruited NAT cohort, the B7-H3highPDL1low subgroup exhibited the lowest therapeutic response. Conclusions: Overall, this research provides a novel subtyping strategy based on the combination of B7-H3/PD-L1 expression that could potentially be applied to predict the therapeutic responses in TNBC, and suggests a potential biomarker-guided anti-B7-H3 therapy. Based on the classifier, we can select potential beneficiaries to deliver personalized medical services. Citation Format: Jie Mei, Yun Cai, Ziyi Fu, Yongmei Yin. High B7-H3 expression with low PD-L1 expression identifies armored-cold tumors suggesting potential anti-B7-H3 therapy 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 PO4-03-13.

Abstract PO2-16-01: Molecular correlates of drug response to guide therapy in triple-negative breast cancer

Abstract TNBC accounts for 10-20% of total breast cancer cases in the US and is more aggressive, higher grade, and has a poorer prognosis than other forms of breast cancer. TNBC is more likely to metastasize within 3-5 years than other forms of breast cancer and has a median survival of 17.6-21.3 months after metastasis. Advances in immunotherapy are promising, but recent trial results of immunotherapy-chemo combination have resulted in a 3-year overall survival rate under 36% in a PD-L1 based selected cohort. Thus, there is an unmet need for innovations that lead to new treatment options and improve outcomes for patients with TNBC in a personalized manner beyond only one or a few markers. In our recently published study (PMID: 31655920), we tested the dose response for a panel of 78 investigational drugs and plotted this readout against molecular characterization to identify statistically significant DNA, RNA, and protein predictors of drug efficacy in TNBC. We have expanded on this published study by identifying correlates from 3D cell culture. According to the literature, 3D culture systems are more representative of in vivo activity. We have increased our sample number (now 27 cell lines and 8 ex vivo PDXs) and expanded our drug library to 387 compounds, selected for their diverse mechanisms and promise in other cancers. Following screening, we prioritized 35 compounds based on their elevated activity and variance of response. From these 35 prioritized compounds, we identified molecular correlates of drug response for 27 compounds (77% of prioritized compounds yielding molecular correlates compared to 50% in our previously published study). Focusing on a known active drug in TNBC that is approved for patients with metastatic TNBC, we identified multi-omic (protein expression/activity and RNA expression) molecular correlates of response to a chemotherapeutic- SN-38, the payload of Sacituzumab and the active metabolite of irinotecan. We utilized a sub-panel of RNA correlates to characterize treatment-naive PDXs and predict response to SN-38. We then tested SN-38 response ex vivo for 5 PDXs to show that we could accurately identify sensitive/resistant models to SN-38 therapy. From significant protein correlates, we identified that the autophagy and AKT pathways were elevated in models with poor response to SN-38. Co-targeting the autophagy or AKT pathway with SN-38 was an effective strategy to identify rational, synergistic therapeutic combinations at therapeutically relevant levels (effective dose (ED) 50 and ED75). We evaluated irinotecan in vivo using a cell line with sensitivity in our 3D assay, demonstrating strong activity in vivo to validate that our screening assay accurately predicts in vivo response. We have further applied this methodology to targeted inhibitors with potential activity in TNBC. We have identified an ATR inhibitor, berzosertib, that targets the DNA damage response (DDR) pathway and correlates with protein expression and activity of Aurora kinases (AKs). Furthermore, combining the pan-AK inhibitor danusertib with berzosertib results in synergistic killing of TNBC cells. As a future direction, we are evaluating this combination in vivo utilizing an ethnically diverse patient-derived xenograft library grown in mice to evaluate this novel combination in a mock clinical trial and to identify molecular correlates of response and resistance that could be used to guide future human trials. Citation Format: Nathan Merrill, Nathalie Vandecan, Athena Apfel, Habib Serhan, Peter Ulintz, Liwei Bao, Xu Cheng, Aki Morikawa, Sofia Merajver, Matthew Soellner. Molecular correlates of drug response to guide therapy 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 PO2-16-01.

Abstract PO2-13-07: SMARCA2 compensation of SMARCA4 loss mediates adaptive resistance to neoadjuvant systemic therapy in triple-negative breast cancer: paired transcriptomic analysis of pre- and post-treatment samples from NeoSTOP and NeoPACT

Abstract Objectives/Rationale: Triple-negative breast cancer (TNBC) patients with residual disease (RD) after neoadjuvant systemic therapy (NAST) are at high risk for disease recurrence and death. While multiple prognostic biomarkers associated with response to NAST have been identified, adaptive resistance mechanisms to NAST remain poorly understood. Identifying and characterizing adaptive resistance mechanisms can illuminate novel therapeutic approaches and improve outcomes for TNBC patients with RD. Methods: Total RNA was isolated from pre-treatment and paired surgical specimens (for patients with RD) from TNBC patients treated with chemotherapy on the NeoSTOP (NCT02413320) or chemoimmunotherapy on the NeoPACT (NCT03639948) neoadjuvant trials and was subjected to RNA exome sequencing. Comparative marker selection analysis was performed by computing the two-sided paired samples t-test for each gene followed by pre-ranked gene set enrichment analysis (GSEA) amongst 23,797 annotated gene sets. Gene sets with false-discovery rate (FDR) corrected P values < 0.001 were determined to be significant. Results: Pre-treatment sequencing data were available for N=200 patients and the overall pathologic complete response (pCR) rate was 56.5%. Paired pre- and post-treatment sequencing data were available for N=58 patients with RD (N=27 from NeoSTOP and N=31 from NeoPACT). 165/23,797 gene sets were significantly enriched in post-treatment compared to pre-treatment samples. SHEN_SMARCA2_TARGETS_UP (M29) was among the top five significantly enriched gene set and was selected for further analysis based on the known role of the SWI/SNF complex in cell survival and the availability of clinically viable SMARCA2 degraders. Pre-treatment SMARCA4 expression was associated with pCR, with lower median expression noted in patients with RD compared to patients who achieved pCR (P=0.04). Pre-treatment SMARCA2 expression was not associated with pCR (P=0.48). On paired sample analysis there was pronounced downregulation of SMARCA4 in post-treatment compared to pre-treatment samples in all patients (mean Z=-1.46, P< 0.0001), in patients treated with chemotherapy (mean Z=-1.65, P< 0.0001), and in patients treated with chemoimmunotherapy (mean Z=-1.28, P< 0.0001). Although there was no difference in SMARCA2 expression between paired pre- and post-treatment samples (mean Z=0.03, P=0.83), there was enrichment of SMARCA2 target expression on GSEA analysis (NES=2.63, FDR q< 0.00001) indicating that SMARCA2 activity is compensating for therapy-induced SMARCA4 loss. Conclusions: We identified 165 gene sets that were significantly enriched in paired post-treatment compared to pre-treatment TNBC samples. Altered activity of the SMARCA2 subunit of the SWI/SNF chromatin remodeling complex was identified as one of the top hits. SMARCA2 and SMARCA4 are mutually exclusive subunits of SWI/SNF, and inactivation of both SMARCA2 and SMARCA4 is synthetic lethal. We observed an association of lower pre-treatment SMARCA4 expression with resistance to NAST and pronounced therapy-induced downregulation of SMARCA4 and induction of SMARCA2 target genes in post-treatment compared to pre-treatment samples, suggesting that SMARCA2 compensates for adaptive SMARCA4 loss. SMARCA2 degraders are in early-phase clinical trials and are known to induce synthetic lethality in SMARCA4-deficient cells. Our results identify adaptive loss of SMARCA4 and synthetic lethal targeting of compensatory SMARCA2 as an attractive therapeutic target in TNBC patients with RD after neoadjuvant chemotherapy or chemoimmunotherapy. Citation Format: Shane Stecklein, Rachel Yoder, Joshua Staley, Zachary Schmitt, Anne O'Dea, Lauren Nye, Deepti Satelli, Gregory Crane, Rashna Madan, Maura O'Neil, Andrew Godwin, Harsh Pathak, Qamar Khan, Joyce O'Shaughnessy, Priyanka Sharma. SMARCA2 compensation of SMARCA4 loss mediates adaptive resistance to neoadjuvant systemic therapy in triple-negative breast cancer: paired transcriptomic analysis of pre- and post-treatment samples from NeoSTOP and NeoPACT [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 PO2-13-07.

Abstract PO1-17-02: Real-world outcomes of systemic therapy for advanced triple-negative breast cancer: a tertiary centre experience

Abstract Background Despite recent therapeutic developments for advanced triple-negative breast cancer (aTNBC), outcomes are still suboptimal for a significant proportion of patients. Clinical trials focus on a single line of therapy and do not examine outcomes across the whole treatment pathway involving multiple lines of therapy and may not be representative of outcomes in the real world. We evaluated the efficacy of sequential lines of systemic therapies (SACT) in patients with aTNBC treated at our Institution. Methods We conducted a retrospective analysis of patients with aTNBC who commenced SACT at our Institution between 1/12/2011 and 31/12/2020. Patients’ demographics and tumour characteristics were recorded, along with SACT regime used and treatment outcomes. Median overall survival (mOS) was calculated, as was overall response rate (ORR), median progression survival (mPFS) for each line of therapy. Results In total, 239 patients were eligible for inclusion, with a median age of 55 years at diagnosis of aTNBC (range 26-91). Of these, 65 (27.2%) were ≥65. Two hundred and eleven patients (88.3%) had ECOG performance status (PS) 0 or 1 at first-line SACT initiation. Thirty-two (13.4%) had de-novo aTNBC. Of those with recurrent disease (N=207), 110 (53.1%) received neoadjuvant treatment in the early disease setting. Of these, only 8 (7.3%) achieved pathological complete response. In the recurrent disease cohort, 75 (36.2%) had a disease-free interval (DFI) ≤12 months. There was visceral involvement in 144 (60.2%) patients and 10 (4.2%) had bone only disease. The most common histology was invasive ductal (n=142, 59.4%) at metastatic biopsy and 30 (12.6%) were germline BRCA 1/2 mutation carriers. Number of SACT lines ranged from 1 to 8, with most patients (n=140, 58.6%) receiving 1 to 2 lines of treatment only; with 99 (41.4%) receiving ≥3 lines, 60 (25.1%) ≥4 lines and 36 (15.1%) ≥5 lines. Fluoropyrimidines was most commonly used drug class in 1st and in 2nd lines (40.6% and 32.7%, respectively). Eribulin was the most common choice in 3rd line and 4th lines (37.4% and 30%, respectively). Check-point inhibitor-based therapy was used in 6.3% of patients in 1st line, 2.5% in 2nd line and 3.0% in 3rd line. Overall, 25.5% (n=61) of patients were enrolled onto clinical trials. Clinical trial enrolment was most common in third-line (12.1%, n=12). ORR and mPFS were 42.2% (95% CI 35.7-49) and 3.7 months (95% CI 3.0-5.0) respectively in 1st line, 38.5% (95% CI 30.8-46.6) and 3.5 months (95% CI 2.8-4.0) in 2nd line, 30.2% (95% CI 21.3-40.4) and 2.5 months (95% CI 2.1-3.0) in 3rd line, 23.7% (95% CI 13.6-36.6) and 2.1 months (95% CI 2.0-2.8) in 4th line Patients with a DFI >12 months had a longer mPFS compared to patient with DFI ≤12 months at 5.4 (95% CI 3.7– 6.4) and 2.75 (95% CI 2.2– 3.6) months, respectively (P=0.009). At the censor date 18 patients (7.5%) were still alive and mOS was 11.8 months. Conclusions Our real-world data shows that over half of patients only receive one or two lines of systemic therapy for aTNBC, emphasising the importance of therapy choice in the early line setting for aTNBC. These data are important to inform decision-making, discussions with patients and considerations of clinical trials. Table 1. ORR and PFS to First, second, third and fourth-line systemic treatment. Citation Format: Shuai Zhang, Matilde Coriano, Colm Mac Eochagain, Dorothy Yang, Daniel Yiu, Alistair Ring, Nicolo M.L. Battisti. Real-world outcomes of systemic therapy for advanced triple-negative breast cancer: a tertiary centre experience [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 PO1-17-02.

Abstract PO3-06-13: A novel stable 6-aryl-2-benzoyl-pyridine colchicine-binding site inhibitor targeting microtubules (60c) is effective against taxane-resistant, metastatic breast cancer

Abstract Background: Improving survival for patients diagnosed with metastatic disease and treating spontaneous chemoresistance remain major clinical challenges in treating breast cancer. Triple negative breast cancer (TNBC), characterized by a lack of therapeutically targetable receptors (ER/PR/HER2), is an aggressive subtype of breast cancer. TNBC therapy relies on a combination of systemically administered chemotherapies, including microtubule-targeting agents (MTAs) like paclitaxel (taxane class) or eribulin (vinca class); however, there are currently no FDA approved MTAs that bind to the colchicine-binding site. Approximately 70% of patients who initially respond to paclitaxel, a first-line MTA, will develop taxane resistance (TxR) within 6 months. We have previously reported that an orally bioavailable colchicine-binding site inhibitor (CBSI), sabizabulin (formerly known as Veru-111), not only inhibits TNBC tumor progression but also treats pre-established metastatic disease in a taxane-refractory PDX model, HCI-10-Luc2. Sabizabulin is currently in phase II/III trials for advanced prostate cancer. To further improve potency and metabolic stability, we created next-generation derivatives of the sabizabulin scaffold, including 60c. Results: 60c shows improved potency compared to sabizabulin against taxane-sensitive and TxR TNBC models, inhibiting proliferation and clonogenicity at low nanomolar concentrations, and inducing apoptosis in a dose-dependent manner. In vivo, 60c therapy significantly suppressed primary tumor growth and lung metastasis in an orthotopic MDA-MB-231-TxR xenograft model without gross toxicity, and with equivalent efficacy to combrestatin A4 phosphate (CA-4P), another CBSI in clinical trials. In the taxane-refractory HCI-10-Luc2 PDX model, 60c significantly suppressed both the growth of the primary tumor (by ~3.5 fold) and the expansion of pre-established axillary metastases (by ~26 fold) whereas paclitaxel had no effect. Analysis of total photon flux of organs excised at study endpoint showed that 60c had distinct anti-metastatic tropism as compared to sabizabulin. Whereas 60c completely suppressed metastases to the spleen (0/9 mice had signal), and significantly reduced metastatic burden in the leg bones (only 3/9 mice had signal) and in the kidney (only 2/9 mice had signal), 60c had no statistically significant effect on liver metastases. In contrast, we had previously reported that sabizabulin strongly repressed liver metastases in the same PDX model (0 mice with detectable liver metastases), with less potent effects on bone. Unlike 60c, sabizabulin showed no improvement relative to the vehicle control for spleen and kidney metastases. Together, our data positions 60c as a promising candidate for TNBC therapy, particularly for patients with TxR disease. Our results also suggest that different CBSI scaffolds may preferentially inhibit metastasis to specific organs, which requires further investigation. Citation Format: Damilola Oluwalana, Kelli Hartman, Raisa Krutilina, Hao Chen, Hilaire Playa, Shanshan Deng, Deanna Parke, Duane Miller, Tiffany Seagroves, Wei Li. A novel stable 6-aryl-2-benzoyl-pyridine colchicine-binding site inhibitor targeting microtubules (60c) is effective against taxane-resistant, 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 PO3-06-13.

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 PO2-15-07: HER2DX genomic assay in triple-negative breast cancer (TNBC) patients treated with 12-weeks of neoadjuvant chemotherapy: a correlative analysis from WSG-ADAPT-TN phase II trial

Abstract HER2DX genomic assay in triple-negative breast cancer (TNBC) patients treated with 12-weeks of neoadjuvant chemotherapy: a correlative analysis from WSG-ADAPT-TN phase II trial Background: Biomarkers for de-escalation and escalation of systemic therapy in early-stage TNBC are needed. HER2DX is a standardized prognostic (risk-score) and predictive (pathological complete response [pCR]-score) assay based on clinical and gene expression-based data. Here we aimed to test the value of HER2DX assay in early TNBC. Methods: Standardized HER2DX was evaluated centrally using RNA from baseline FFPE tumor biopsies from the WSG-ADAPT-TN study (NCT01815242), a multicenter phase II trial that randomized 336 patients with stage I–III early TNBC to 12-weeks of nab-paclitaxel 125 mg/m2 plus gemcitabine 1000 mg/m2 d1,8 every 3 weeks (arm A) versus nab-paclitaxel 125 mg/m2 plus carboplatin AUC2 day 1,8 every 3 weeks (arm B). The primary aim was to test the ability of HER2DX pCR and risk-score models to predict pCR (ypT0/isN0) and survival endpoints, respectively, such as invasive disease-free survival (iDFS), distant disease-free survival (DDFS) and overall survival (OS). Secondary objectives were to assess the association of the i) HER2DX immune signature score and ii) percentage (%) of stromal tumor infiltrating lymphocytes (sTILs) with efficacy endpoints. Uni- and multi-variable logistic regression and Cox models tested the association of each variable with each endpoint. Results: HER2DX was evaluated in 126 (37.5%) baseline pre-treatment tumors. Mean age was 52.1 (range 26-76). cT1 represented 40.5% of cases and 72.2% were cN0. Overall, pCR rate was 34.1% (95%CI 26.1 – 43.2) and median follow-up was 5.0 years. Median sTILs at baseline was 31.6% (range 0-90) and moderately correlated with the IGG signature (coefficient=0.57). The % of HER2DX high-, medium- and low-pCR groups was 41.3%, 47.6% and 11.1%, respectively. HER2DX pCR score (as a continuous variable) was significantly associated with pCR in univariate (odds ratio [OR per 10-unit increase]=1.31, 1.06-1.64, p=0.016) and after adjusting by treatment arm (OR=1.31, 1.06-1.65, p=0.015). Overall, the pCR rates in HER2DX pCR-high, -medium and -low groups were 46.2%, 26.7% and 21.4%, respectively (high vs medium/low OR=2.48, 1.17-5.34, p=0.018). In arm B (n=51), the pCR rates in HER2DX pCR-high, -medium and -low groups were 47.8%, 25.0% and 0.0% respectively (high vs medium/low OR=3.36, 1.02-12.0, p=0.051). In terms of the risk-score, the % of HER2DX low-risk and high-risk groups was 60.3% and 39.7%, respectively. HER2DX risk score as a continuous variable was significantly associated with iDFS, DDFS and OS (p< 0.001 in all univariate analysis), and after adjusting by variables such as arm and pCR status (all p< 0.05). sTILs were not associated with any survival endpoint (all p >0.35). The HER2DX immune signature was significantly associated with iDFS (p=0.012), DDFS (p=0.022) and OS (p=0.012). Conclusion: The HER2DX genomic test in TNBC provides valuable insights into the response and survival following neoadjuvant taxane-based chemotherapy in the absence of immunotherapy. The development of a tailored genomic assay for TNBC is currently in progress. Citation Format: Oleg Gluz, Fara Brasó-Maristany, Ulrike Nitz, Laia Paré, Matthias Christgen, Guillermo Villacampa, Benedetta Conte, Sherko Kuemmel, Ronald Kates, Cornelia Kolberg-Liedtke, Eva-Maria Grischke, Helmut Forstbauer, Michael Braun, Mathias Warm, John Hackmann, Christoph Uleer, Bahriye Aktas, Claudia Schumacher, Ana Vivancos, Rachel Wuerstlein, Joel S. Parker, Monika Graeser, Charles M. Perou, Christine zu Eulenburg, Patricia Villagrasa, Hans-Heinrich Kreipe, Aleix Prat, Nadia Harbeck. HER2DX genomic assay in triple-negative breast cancer (TNBC) patients treated with 12-weeks of neoadjuvant chemotherapy: a correlative analysis from WSG-ADAPT-TN phase II trial [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 PO2-15-07.

Abstract PO2-15-08: Exploring Essential Immune Genes Linked to Pathological Complete Response and Survival in Early-Stage Triple-Negative Breast Cancer (TNBC) after Chemotherapy Treatment

Abstract Background: De-escalation and escalation of systemic therapy in early-stage TNBC will require the implementation of biomarkers to guide therapy. To date, high expression of a variety of immune genes has been associated with higher pathological complete response (pCR) rates following neoadjuvant chemotherapy or with improved survival, or both, in early-stage TNBC. However, it is currently unknown which are the key genes influencing survival (i.e., the Core Immune Genes [CIG]) and if combinations of these genes can improve their association with both clinical endpoints. Methods: The expression of up to 185 genes was evaluated on pre-treatment tumor biopsies from 3 independent publicly available datasets of early-stage TNBC: CALGB40603 (n=388) (NCT00861705), BrighTNess (n=482) (NCT02032277), and SCAN-B (n=498). Patients in CALGB40603 and BrighTNess were treated with neoadjuvant chemotherapy-based therapy and had response data. Patients in CALGB40603 and SCAN-B had survival outcome data (event-free survival [EFS] or relapse-free interval [RFi]). Each individual gene was associated with each clinical endpoint within each dataset using Cox model and logistic regression analyses, adjusting for treatment arm. Genes found in common across datasets were selected as CIG, except for PDCD1, which was not available in BrighTNess. Using the CIGs, we evaluated whether combinations of 2 genes enhance their association with each clinical endpoint using different methods: adding, subtracting, multiplying, and ratio. Likelihood ratio testing was used to evaluate the added contribution of each gene in 2 combined cohorts evaluating pCR (i.e., CALGB40603 and BrighTNess) and survival (i.e., CALGB40603 and SCAN-B). Results: 10 CIGs related to B-cells and T-cells (i.e., IRF4, LAX1, POU2AF1, CD274, CD79A, TNFRSF17, PIM2, PDCD1, CXCR6, and SLAMF1) were associated with higher pCR rates and improved survival outcome across datasets. Of note, 5 (50%) genes are known components of the 14-gene immunoglobulin/B-cell (IGG) signature. The correlation coefficient among the 10 CIG genes was 0.63 (moderate). In general, the sum of 2 CIGs provided more prognostic information than a single gene (p< 0.001). Specifically, the sum of 2 CIGs provides more prognostic information than a single gene in 30 of 45 (66.6%) cases. Two-gene subtraction, multiplication or ratio decreased the amount of prognostic information (p< 0.001). The top 10 2-gene CIGs associated with prognosis (p< 0.001) were CD274_TNFRSF17, IRF4_CD274, PDCD1_TNFRSF17, CD79A_CD274, CD274_POU2AF1, CXCR6_TNFRSF17, LAX1_TNFRSF17, SLAMF1_TNFRSF17, CD274_LAX1 and CD79A_CXCR6. In general, the sum of 2 CIGs provided more information about pCR than a single gene (p< 0.001). Specifically, the sum of 2 CIGs provided numerically more information about pCR than 1 gene in 17 of 36 (47.2%) cases. Two-gene subtraction, multiplication or ratio decreased the amount of information about pCR (p< 0.001). The top 10 2-gene CIGs associated with pCR were CD274_PIM2, CD274_POU2AF1, CXCR6_PIM2, CD274_CXCR6, IRF4_CD274, CXCR6_POU2AF1, CD274_LAX1, IRF4_CXCR6, CXCR6_LAX1, and POU2AF1_SLAMF1. Conclusion: The expression of CIGs associated with B-cell and T-cell adaptive immune response is closely linked to pCR and survival outcomes following (neo)adjuvant chemotherapy. When combining the expression of 2 specific CIGs through addition, we found that this method provides more informative insights into pCR and survival outcomes compared to other combination methods. This approach enhances the predictive power of CIGs, enabling a better understanding of treatment response and patient prognosis, ultimately contributing to improved clinical decision-making and patient outcomes. Citation Format: Fara Brasó-Maristany, Laia Paré, Benedetta Conte, Laura Angelats, Ana Vivancos, Joel S Parker, Charles M. Perou, Patricia Villagrasa, Aleix Prat. Exploring Essential Immune Genes Linked to Pathological Complete Response and Survival in Early-Stage Triple-Negative Breast Cancer (TNBC) after Chemotherapy Treatment [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 PO2-15-08.

Abstract PO3-26-10: Small molecule inhibitor of PELP1 exhibit cytotoxic effects on breast cancer cells by blocking ribosome biogenesis

Abstract Introduction: Breast cancer (BC) is the second-leading cause of cancer-related deaths in women. Despite responsive to endocrine therapy, a sizeable portion of estrogen receptor positive (ER+) BC goes on to develop therapy-resistant breast cancer (TR-BC). Furthermore, triple-negative breast cancer (TNBC) has a more aggressive clinical course with fewer targeted therapeutic options. The development of more effective therapies for TR-BC or TNBC represent an unmet need. Excessive ribosome biogenesis has recently been linked to BC progression, and resistance to therapy. Oncogene PELP1, which is required for ribosome biogenesis, is frequently dysregulated in BC and serves as a prognostic factor for poor BC survival and therapy resistance. We recently identified SMIP34 as a PELP1 small molecule inhibitor of its kind and it is effective against both TR-BC and TNBC. The objective of this study is to determine the mechanisms by which SMIP34 exhibit cytotoxic effects on BC cells. Methods: We have used multiple ER+, therapy resistant and TNBC models in cell viability, and apoptosis assays. Modelling studies were done based on Cryo-EM structure of the WDR18/PELP1 Rix1 complex (PDB code 7UWF). Biochemical assays include immune precipitation, reporter assays, GST pulldown, and Western Blotting. SMIP34 effect on protein synthesis was measured by using Western blot of puromycin treated total lysates for puromycinylated protein content. SMIP34 effect on protein synthesis assessed using Cayman’s Protein Synthesis Assay Kit. Ribosome biogenesis in SMIP34 treated cells was monitored using confocal microscopy using rpS6 antibody. Results: In in vitro cell based assays, SMIP34 was highly effective in reducing the cell viability, reducing colony formation and inducing apoptosis of ER+, endocrine therapy resistant, and TNBC model cells. PELP1 homodimer has recently been predicated to act as the core of the WDR18/PELP1 assembly. Our biochemical assay results suggest that SMIP34 binds to the same region involved in the PELP1 homodimerization and disrupts the formation of PELP1 homodimer and subsequently blocks the formation of WDR18/PELP1 Rix1 complex. Modeling studies predicted that altering the conformation of PELP1 aa 696-720 loop by SMIP34 binding may influence the conformation of the following C-terminal region, which may disturb the interaction of other PELP1 partners such as SENP3 and TEX10. Accordingly, the results from immunoprecipitation experiments demonstrated decreased association of Rix1 complex proteins WDR18, TEX10, and SENP3 with PELP1 under conditions of SMIP34 treatment. Further, Western blotting analysis confirmed that SMIP34 treatment decreased levels of PELP1 and its complex proteins, such as WDR18, TEX10, LAS1L, and SENP3. SMIP34 significantly reduced ribosomal biogenesis in a dose dependent manner. Additionally, puromycin labelling experiments confirmed a dose dependent decrease in new protein synthesis upon SMIP34 administration. Data from the DepMap database analysis revealed that PELP1 is required for cancer cell survival. Additionally, analysis of TCGA breast cancer datasets revealed that PELP1 expression is positively correlated with WDR18, TEX10, LAS1L, and SENP3. In in vivo xenograft assays and ex vivo explant assays using tumor tissue, SMIP34 strongly inhibited the growth of ER+, endocrine treatment resistant, and TNBC model cells. Conclusion: These findings provide solid evidence that the Rix1 complex, which is required for ribosomes production, is disturbed by SMIP34 binding to PELP1. SMIP34 represents a new therapeutic for the treatment of both ER+ and TNBC breast cancer. Supported by VA grant 1 I01 BX004545-01A1. Citation Format: Xue Yang, Khaled Mohamed Nassar, Uday Pratap, Rahul Gopalam, Behnam Ebrahimi, Durga Meenakshi Panneerdoss, Xiaonan Li, Yaxia Yuan, Daohong Zhou, Gangadhara R. Sareddy, Rajeshwar R. Tekmal, Manjeet Rao, Suryavathi Viswanadhapalli, Ratna Vadlamudi. Small molecule inhibitor of PELP1 exhibit cytotoxic effects on breast cancer cells by blocking ribosome biogenesis [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 PO3-26-10.

Abstract PS12-06: A phase 2 study of abemaciclib monotherapy for patients with retinoblastoma-positive (Rb+), triple-negative metastatic breast cancer

Abstract Background: Cyclin-dependent kinase (CDK) 4/6 inhibitors can significantly extend survival when given in combination with endocrine therapy as treatment for hormone receptor-positive metastatic breast cancer. Preclinical studies suggest CDK4/6 inhibitor monotherapy might also be effective in a subset of triple-negative breast cancers (TNBCs), including those that express a functional retinoblastoma (RB) protein and/or those of the Luminal Androgen Receptor (LAR) subtype. Currently, the clinical activity of CDK4/6 inhibitor therapy in TNBC has not been reported. Methods: We conducted a single-arm phase II study of abemaciclib monotherapy in patients with locally advanced or metastatic TNBC. Key eligibility criteria included: (i) Measurable disease by RECIST 1.1; (ii) Between 1-3 prior lines of systemic therapy for advanced TNBC; (iii) RB-positive tumor (defined as ≥ 50% of tumor cells staining positive for RB by immunohistochemistry [archival or fresh sample] on central testing); (iv) ECOG PS 0/1. Patients were treated with abemaciclib 200 mg orally (28-day cycles) twice daily until disease progression, unacceptable toxicity, withdrawal of consent, or death. Tumor biopsies were mandatory at baseline and C2D1 if tumor tissue was safely accessible. The primary outcome was objective response rate (ORR). Key secondary endpoints included progression-free survival (PFS), overall survival (OS), clinical benefit rate (CBR: CR + PR + SD ≥ 24 weeks), and safety and tolerability. The study had a two-stage design. Thirteen patients were enrolled in the first stage, with the plan to enrol a further 25 patients if at least one objective response was observed. Results: One unconfirmed partial response was observed in stage 1, and a total of 27 patients were enrolled before the trial was closed early due to slow accrual. The median age was 61 years and patients had received a median of 2 prior lines of systemic therapy for metastatic disease. Twelve patients had received prior immunotherapy. After a median follow-up of 28.5 months, the confirmed ORR was 0% and the CBR was 15%, with 4 of 27 patients experiencing stable disease for ≥ 24 weeks. The median PFS was 1.94 months (95% CI: 1.8 – 11.5 months), and the median OS was 8.44 months (95% CI: 4.6 – 15.6 months). There was no significant difference in PFS or OS between patients with PD-L1-positive versus PD-L1-negative disease, or Androgen Receptor (AR) positive versus negative tumors by immunohistochemistry. The most common adverse events of grade 2 or higher were diarrhea (41%), neutropenia (41%), anemia (30%), and nausea (30%). RNA-sequencing of baseline biopsies has been performed to identify biomarkers associated with clinical benefit, and results will be presented at the meeting. Conclusions: Abemaciclib monotherapy did not show clinical activity in patients with Rb+ metastatic TNBC. This finding suggests that future trials of CDK4/6 inhibition as monotherapy in TNBC are not warranted. Citation Format: Shom Goel, Bojana Jovanović, Xiangying Chu, Melissa Hughes, Ayesha Mohammed-Abreu, Julie Kasparian, Timothy Erick, Molly DiLullo, Eileen Wrabel, Rinath Jeselsohn, Nabihah Tayob, Stuart Schnitt, Nancy Lin, Sara Tolaney. A phase 2 study of abemaciclib monotherapy for patients with retinoblastoma-positive (Rb+), triple-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 PS12-06.