Breast Cancer Cells Research Articles

Abstract 1312: FOXF2 expression triggered by endocrine therapy orchestrates therapeutic resistance through reorganization of chromatin architecture in breast cancer

Estrogen receptor-positive (ER+) breast cancer accounts for 80% breast cancer cases. The estrogen-ERα signaling pathway plays a pivotal role in driving the progression of ER+ breast cancer cells. Thus, endocrine therapy targeting the estrogen-ERα signaling pathway is a predominant treatment for ER+ breast cancer. Most patients with ER+ breast cancer require long-term treatment with endocrine agents. Although endocrine therapy effectively improves the survival of patients with ER+ breast cancer, bone metastasis frequently occurs in patients receiving long-term endocrine therapy due to acquired resistance. Elucidating the molecular mechanisms underlying endocrine-resistance and developing effective strategies to reverse resistance or treat resistant tumors are crucial unresolved issues. Enhancer reprogramming-mediated lineage-specific transcriptional rewiring systemically regulates the transdifferentiation and plasticity of cancer cells. Enhancers are DNA regulatory elements composed of clusters of transcription factor (TF) binding sites. Enhancer-bound TFs recruit chromatin-modifying enzymes to establish unique histone modifications in enhancer regions, subsequently recruiting cofactors to facilitate or repress the transcription of specific target genes by reorganizing chromatin architecture and altering DNA accessibility. Our previous studies found that forkhead box F2 (FOXF2), a mesenchymal TF, is barely expressed in ER+/luminal breast cancer (LumBC) cells but is commonly expressed in triple-negative breast cancer (TNBC)/basal-like breast cancer (BLBC) cells with mesenchymal characteristics. In the present study, we found that ERα represses FOXF2 transcription in ER+ breast cancer through H3K27me3 modification, therefore endocrine therapy triggers FOXF2 transcription by loss of H3K27me3. FOXF2 transactivation orchestrates endocrine-resistance and bone metastasis. Mechanically, FOXF2 globally activates enhancers of genes involved in epithelial-mesenchymal transition (EMT)/epithelial-osteogenic transition (EOT), as well as super-enhancers of NCOA3 (a coactivator of FOXF2) and SP1 (an upstream trans-activator of FOXF2) by recruiting SWI/SNF complex that mediates the reorganization of chromatin architecture. Targeting BRD4, an essential transcriptional coactivator of FOXF2, significantly inhibits FOXF2-orchestrated endocrine-resistance and bone metastases. Our findings uncover a mechanism underlying endocrine-resistance and provide a promising strategy for managing endocrine-resistant breast cancer. Citation Format: Rui Zhang, Wen-Jing Jiang, Shuai Zhao, Li-Juan Kang, Qing-Shan Wang, Yu-Mei Feng. FOXF2 expression triggered by endocrine therapy orchestrates therapeutic resistance through reorganization of chromatin architecture in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1312.

Abstract 6329: Edible Chinese mushroom-derived antioxidant, vialinin A, prevents breast cancer cell growth

Abstract Breast cancer ranks as the second most prevalent cancer worldwide, impacting millions annually, and stands as the leading cancer affecting women. Neoplasia occurs when free radicals, antioxidants, and reactive oxygen species increase in the cell, which can alter protein function and cause mutations and genomic instability. Current breast cancer treatments typically include chemotherapy and radiation. However, different breast cancer cell types have different antioxidative capacities, which allow them to resist these treatments, thereby reducing the efficacy of these expensive treatments. Several vitamins, antioxidants, and anti-inflammatory compounds have been tested for their effectiveness in preventing breast cancer. However, the role of vialinin A, an antioxidant and ubiquitin-specific peptidase inhibitor derived from edible Chinese mushrooms, is unknown. Therefore, in this study, we examined the chemopreventive effect of vialinin A in preventing breast cancer cell growth and migration. Human breast cancer cells (MCF-7) were treated with vialinin A in a time- and dose-dependent manner. Cell viability assay indicates that vialinin A prevents the growth of breast cancer cells in a dose-dependent manner. Further, vialinin also prevented the EGF-induced invasion of MCF-7 cells in a scratch assay. Vialinin A also prevented the EGF-induced trans-well migration of breast cancer cells. Vialinin A also regulated the growth factor-induced expression of various pro- and anti-apoptotic factors in breast cancer cells. Vialinin A also increased the activation of caspase-3 and cleavage of PARP in growth factor-treated breast cancer cells. Next, we plan to examine the chemopreventive efficacy of vialinin A in a nude mouse xenograft model. In conclusion, our results indicate that vialinin A could prevent breast cancer cell growth and invasion, suggesting its potential development as a chemopreventive drug. Citation Format: Malak Rafik, Afnan Khan, Kota V. Ramana. Edible Chinese mushroom-derived antioxidant, vialinin A, prevents breast cancer cell growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6329.

Abstract 6388: Exploiting CTPS1 dependency for the treatment of breast and ovarian cancer

Breast and ovarian cancer represent the most common and lethal cancers in women. Despite chemotherapy and targeted agents, recurrence is frequent and outcomes for patients with advanced disease are dismal, underscoring the need for novel therapeutic strategies. Using an unbiased approach with DepMap CRISPR screens across more than 1000 cancer cell lines, we identified six genes (CTPS1, RHOA, PRKRA, RAD9A, HUS1, and RAD1) that were essential for triple negative breast cancer (TNBC) cells, but not ERα+ breast cancer cells, that were also associated with poor outcomes. Validation of these effects by siRNA knockdown studies revealed that suppression of CTPS1 elicited the most potent inhibitory effects, characterized by S phase cell cycle arrest and apoptosis. Similar effects were also found in ovarian cancer cells which share many molecular features. CTPS1, along with CTPS2, is responsible for enzymatic conversion of UTP to CTP, an essential nucleoside triphosphate required for multiple cellular processes including DNA/RNA synthesis, phospholipid synthesis and glycosylation. CTPS1 was found to be highly expressed in tumor tissues, for both breast and ovarian cancer. RT-PCR assessment of CTPS1 expression in a panel of over 40 breast and ovarian cancer lines confirmed variable, but elevated expression relative to normal models. Notably, chemotherapy-resistant and PARP inhibitor (PARPi)-resistant models exhibited the highest levels of CTPS1 expression among all cells analyzed. In partnership with Step Pharma, which has developed a first-in-class highly selective CTPS1 inhibitor (STP938), we have demonstrated that STP938 elicits potent anti-proliferative effects at nM concentrations across many cell line and patient derived models, both in 2D and 3D culture systems. Furthermore, STP938 exhibited strong anti-cancer activity in chemotherapy- and PARPi-resistant ovarian cancer models, both as a monotherapy and in combination with standard-of-care treatments. RNA-seq analysis in cell models following STP938 treatment or CTPS1 knockdown/knockout showed significant dysregulation of DNA replication and cell cycle related genes, suggesting potential synergy with drugs targeting replication stress or DNA damage response pathways. Single-cell sequencing further revealed specific cell-cell interactions/cross talk/pathways that are disrupted by STP938 in vivo using ovarian cancer patient-derived xenograft. The first-in-human clinical trial for STP938 began in 2022 for relapsed/refractory B- and T-cell lymphomas (NCT05463263). In August 2024, a second phase I trial opened for advanced solid tumors, including an expansion cohort for CTPS2-null ovarian cancer (NCT06297525) at Mayo Clinic. These laboratory discoveries, and ongoing clinical studies, are expected to pave the way for a broader uptake of this novel therapeutic approach aimed at improving outcomes in patients with highly aggressive forms of these diseases. Citation Format: Xiyin Wang, Michael Emch, Lauren Voll, Rebecca Epp, Esther Rodman, Noa Odell, Nicole Pearson, Xiaonan Hou, Matthew Goetz, Scott Kaufmann, John Weroha, Philip Beer, John Hawse. Exploiting CTPS1 dependency for the treatment of breast and ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6388.

Abstract 5392: Breast cancer cells that preferentially metastasize to lung are more adaptable to varying glucose levels compared to those that metastasize to the liver

Abstract Metastasis is the primary cause of breast cancer-related deaths. We investigated the metabolic adaptations of glucose, a major energy substrate, in breast cancer cells that metastasize to lung versus liver to determine the metabolic adaptations that may support site-specific metastasis. Utilizing a breast cancer model that preferentially metastasizes to the lung (metM-WntLung cells; MLg) or liver (metM-WntLiver cells; MLr), we measured 14C-glucose uptake, 13C6-glucose flux into metabolic pathways, and viability under varying glucose concentrations. Results show that in 5 mM glucose (normal), MLg is more glycolytic with greater flux of 13C6-glucose-derived carbons into the glycolytic intermediates pyruvate and lactate in comparison to MLr. In contrast, MLr showed higher levels of 13C6-glucose-derived carbons in the tricarboxylic acid cycle metabolites including oxaloacetate and malate indicative of higher oxidative phosphorylation and higher pyruvate carboxylase (PC) activity. Interestingly, high glucose (25 mM) exposure reduced viability of MLr by 39%, suggesting MLg’s adaptability to varying glucose levels. Although glucose uptake similarly increased in all cell lines under high glucose, flux results showed greater oxidative phosphorylation and increased PC activity in MLg, while MLr shifted to a more glycolytic phenotype. These results were consistent with improved oxidative stress protection in high glucose in MLg, but not MLr. Further, MLg deprived of glucose were more viable (9%) compared to MLr, suggesting a potential alternate use of other substrates to overcome glucose deprivation. Inhibiting the rate-limiting enzyme for gluconeogenesis, phosphoenolpyruvate carboxykinase, reduced viability of MLg by 9% compared to MLr. Further, inhibiting the catabolism of glutamine, a gluconeogenic substrate, reduced viability of MLg by 13% compared to MLr, suggesting that MLg utilizes the contribution of glutamine and gluconeogenesis in conditions of low glucose. Overall, breast cancer cells with preferential metastasis to lung are more adaptable to varying glucose concentrations than breast cancer cells with preferential metastasis to liver by shifting metabolism from glycolysis to oxidative phosphorylation under high glucose conditions and utilizing glutamine and gluconeogenesis under low glucose conditions. Citation Format: Marjorie Anne A. Layosa, Mike Wendt, Stephen Hursting, Dorothy Teegarden. Breast cancer cells that preferentially metastasize to lung are more adaptable to varying glucose levels compared to those that metastasize to the liver [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5392.

Abstract 4157: Prodrug development of Cyclo-Creatine: Enhancing permeability and reducing toxicity for HER2+ breast cancer treatment

Abstract HER2+ breast cancer is an aggressive subtype of cancer that often develops resistance to the current first-line therapy, Trastuzumab. In our previous work, we demonstrated that HER2 signaling activates mitochondrial creatine kinase 1 (MtCK1), which enhances mitochondrial energy metabolism for rapid proliferation of breast cancer cells. However, pharmacological blockage of MtCK1 with a creatine analog, Cyclo-Creatine (CCr), requires millimolar concentration to inhibit breast cancer cell proliferation and is associated with brain toxicity, limiting its clinical application. This brain toxicity may result from the inhibition of the creatine transporter SLC6A8 by CCr, which decreases to transport creatine into brain. In this study, we took a prodrug development approach to identify and optimize protection groups for CCr, designed to bypass the SLC6A8 transporter, enhancing bioavailability and reducing toxicity. Liquid chromatography-mass spectrometry (LC-MS) was utilized to analyze drug uptake and creatine metabolites in cells treated with the prodrugs. Prodrug C, which is our lead prodrug candidate, demonstrated a seven-fold increase in cellular uptake and more than a hundred-fold improvement in cell-killing effects as compared to CCr in an MtCK1 specific manner in breast cancer cells. This approach offers a promising therapeutic strategy to inhibit HER2+ breast cancer cell by disrupting mitochondrial energy metabolism. Future investigations will focus on validating this prodrug’s pharmacokinetics (PK) and pharmacodynamics (PD) through further preclinical studies. Citation Format: Ya Li, Yetong Huang, Puja Dey, Sadae Hitosugi, Sarah A. Buhrow, Joel M. Reid, Taro Hitosugi. Prodrug development of Cyclo-Creatine: Enhancing permeability and reducing toxicity for HER2+ breast cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4157.

Abstract 5339: Single cell analysis enables tracking the evolution of resistance to CDK4/6 inhibitors in ER+ breast cancer

Abstract The combination of CDK4/6 inhibitors (CDK4/6i) with endocrine therapy prolongs survival of patients with ER+/HER2- metastatic breast cancer. Nearly all patients with advanced disease treated with this combination eventually progress, highlighting a need for therapeutic interventions that prevent the emergence of drug resistance. Drug tolerant persister (DTP) cells represent a reservoir of surviving cells from which drug-resistant clones eventually emerge. We hypothesized that targeting breast cancer cells in a DTP state that emerge under selective pressure from CDK4/6i would prevent the emergence of drug resistance. First, we aimed to delineate genomic and epigenomic features of DTPs under continuous CDK4/6i + antiestrogen therapy. We treated MCF7 and T47D ER+ breast cancer cells with palbociclib + estrogen deprivation long-term (4 weeks). Cells that survived long-term treatment exhibited characteristics of DTPs, such as reversible sensitivity to CDK4/6i + estrogen deprivation, reversible cell cycle arrest, and reversible transcriptional programs associated with the DTP state. To translate these findings to patients, we examined gene expression data from residual tumors after neoadjuvant treatment with palbociclib and the aromatase inhibitor anastrozole in the NeoPalAna trial and found that these tumors showed a similar enrichment of gene signatures associated with DTPs compared to tumors before treatment. To elucidate with DTPs give rise to drug resistant clones, we barcoded MCF7 and T47D cells with a lineage and RNA recovery (LARRY) barcode library with ∼315, 000 diverse barcodes, such that we can track them during long-term treatment. Barcoded cells were treated with palbociclib plus estrogen deprivation for 0 h, 72 h, and 4 weeks and then subjected to single-cell RNA-seq; 101 and 15 clones that were initially arrested in G0/G1 eventually expanded at 4 weeks, while 376 and 27 clones were eliminated during treatment of MCF7 and T47D cells, respectively. Comparing the expanding clones with the exhausted clones, we identified 50 marker genes for DTP cells in each cell line. We are currently using a CRISPR-Cas9 targeted knockout screen to identify which of these genes are causal to DTP survival. We will then compare results from these single cell approaches with RNA microarray analysis from residual tumors after neoadjuvant treatment in the NeoPalAna trial. In summary, a small subset of ER+ breast cancer cells survive long-term treatment with CDK4/6i plus estrogen deprivation and exhibit reversible transcriptomic features of DTPs. Single-cell RNA-seq analysis in barcoded breast cancer cells revealed genes significantly upregulated in expanding clones that show features of the DTP state. We proposed therapeutic targeting of these genes may reduce survival of drug-tolerant cells and prevent the emergence of drug resistance. Citation Format: Yuki Matsunaga, Emma Aleksandrovic, Hima M. Patel, Dhivya R. Sudhan, Khushi Ahuja, Dan Ye, Chang-Ching A. Lin, Lei Guo, Cynthia X. Ma, Jeon Lee, Siyuan Zhang, Carlos L. Arteaga, Ariella B. Hanker. Single cell analysis enables tracking the evolution of resistance to CDK4/6 inhibitors in ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5339.

Abstract 3801: Serine starvation inhibits SRSF protein expression and modulates RNA splicing in breast cancer cells

Breast cancer is the most common cancer in women worldwide, and luminal/estrogen receptor positive (ER+) breast cancer accounts for ∼50% of breast cancer deaths. Our lab has identified serine auxotrophy as a metabolic vulnerability of luminal/ER+ breast tumors due to low levels of the serine synthesis enzyme phosphoserine aminotransferase 1 (PSAT1). Serine is critically important for cancer cell growth due to its multiple downstream functions in protein, nucleotide, and lipid biosynthesis. There is considerable interest in starving serine auxotrophic tumors of serine for therapy, including an ongoing clinical trial in pancreatic cancer. While the metabolic effects of serine starvation have been extensively investigated, our goal is to better understand how serine starvation affects protein synthesis and expression. We found that serine starvation inhibits global translation, while inducing adaptive translation of ATF4, and causes ribosome stalling and fall-off at serine TCC codons. Analysis of changes in the proteome upon serine starvation in auxotrophic luminal breast cancer cells shows that serine-rich proteins are particularly sensitive to serine starvation; this includes the serine/arginine-rich splicing factor (SRSF) family that are important regulators of mRNA splicing. Accordingly, replicate multivariate analysis of transcript splicing (rMATS) on RNA-sequencing data suggests that serine starvation dramatically changes alternative mRNA splicing in multiple breast cancer cell lines, with many differential splicing events being conserved across cell lines. These results have led to our current hypothesis that serine starvation induces splicing changes due to reduced translation of serine-rich members of the SRSF family (such as SRSF6), which ultimately impacts DNA damage and cell survival. We believe that SRSF6 depletion and subsequent alteration of RNA splicing upon serine starvation may sensitize to DNA damaging agents (e.g., chemotherapy). Citation Format: Philippa Burns, Negar Tabatabaei, Laura Selfors, Aanshi Vashi, Debolanle O. Dahunsi, Patrick Murphy, Soroush Tahmasebi, Jonathan Coloff. Serine starvation inhibits SRSF protein expression and modulates RNA splicing in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3801.

Abstract 5241: Mapping spatial 3D genome landscape of breast cancer cells in different tumor immune microenvironments using onco-immune co-culture as a model

One of the leading causes of tumor cell heterogeneity is the complex nature of the tumor immune microenvironment involving crosstalk between different signaling pathways of cancer cells and immune cells. For cancer cells to grow into a primary tumor and then metastasize into secondary tumors, they must avoid or overcome the attack of different types of immune cells, including macrophages, natural killer (NK) cells, and T lymphocytes. For example, cancer cells can polarize macrophages from “tumor killing” to “tumor promoting” type using specific signaling molecules. Such tumor immune crosstalk is dependent on the clonal and sub-clonal heterogeneity in genomic, transcriptomic and protein signaling networks across different cancer cell sub-populations, immune cells and its surrounding microenvironment. Since structural aberrations affect gene dysregulation, the study of the 3D- genome organization in single cells can help in better understanding of disease progression and prognosis. The genome is organized and packaged in a highly structured manner inside the nucleus of mammalian cells and exhibits dynamic reorganization throughout disease progression. Understanding this 3D organization at the single-cell level with high spatial resolution is crucial for immune-oncology disease research such as investigating tumor-immune cell interactions. In this abstract, we present a novel jebFISHTM protocol on the PaintScapeTM platform that can be used to investigate immune cell modulated differences in global 3D genome organization among different localized and metastatic breast cancer cell lines at single cell, sub-population and population level. Using a genome wide panel of chromosomal targets on an onco-immune co-culture system involving different breast cancer cell lines and CD4+ T-cells or macrophages, we will show disruption in chromosome territory, radius of gyration, chromosomal instability between localized and metastatic breast cancer cells at single cell and sub-population level. We observed signatures of specific 3D genome structural alterations and interchromosomal interactions in cancer cells and/or immune cells, related to the effect of different types of immune response. We will show how the 3D architecture of key genomic loci and its associated regulatory elements belonging to major cancer signaling pathways exhibit specific signatures including differential folding, chromosomal instability, differential interchromosomal interaction and structural variation patterns at single cell, sub-chromosomal and sub-population level. We envision this study will improve our understanding on breast cancer tumor immune microenvironment and provide deeper insight into the underlying genomic heterogeneity of single cancer cells and sub-populations which might help to design better treatment options in the future. Citation Format: Jude Dunne, Huy Nguyen, Shyamtanu Chattoraj. Mapping spatial 3D genome landscape of breast cancer cells in different tumor immune microenvironments using onco-immune co-culture as a model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5241.

Abstract 1321: Glycosylation dependent metastatic seeding of circulating tumor cells in breast cancer

Abstract Chemotherapy has been standard of care for triple negative breast cancer for decades; however, how chemotherapy evasion enables metastasis remains elusive and is one of the biggest challenges impacting patient survival in cancer clinics. Circulating tumor cells (CTC) are pivotal component of longitudinal liquid biopsies. Most of the CTCs are detected as a single cell, whereas small proportion of CTCs are found in multicellular clusters which possess 20-100 times higher metastatic potential than single CTCs and corelates with unfavorable survival in breast cancer patients. Here we observed that chemotherapy evasion is linked to the aggregation of CTCs in breast cancer. Chemo-evasive linked aggregated CTCs show specific loss of ST6GAL1-catalyzed alpha2,6-sialylation. Deficiency of ST6GAL1 promotes CTC cluster formation for metastatic seeding and enables cellular quiescence to evade paclitaxel treatment in metastatic breast cancer. Glycoproteomic analysis identified protein substrates of ST6GAL1, such as adhesion and stemness markers PODXL, ICAM1, ECE1, ALCAM1, CD97, and CD44, contributing to CTC clustering (aggregation) and metastatic seeding. As proof-of-concept, neutralizing antibody against one of the most significant contributors, PODXL, inhibits CTC cluster formation and lung metastasis exacerbated by paclitaxel treatment for breast cancer. This study sheds light in the evaluation of chemotherapy response and developed potential therapy approach against chemotherapy resistance. Citation Format: Nurmaa Khund Dashzeveg. Glycosylation dependent metastatic seeding of circulating tumor cells in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1321.

Abstract 3007: The epigenetic regulators KDM5C and KAT6A influence AKT inhibitor response in ER positive breast cancer

Abstract Breast cancer is one of the most common cancers worldwide, and around 80% of breast cancers are oestrogen receptor positive (ER+ BC). For patients with ER+ advanced BC with one or more PIK3CA/AKT1/PTEN tumor alterations, capivasertib (AKT inhibitor) in combination with fulvestrant (selective oestrogen receptor (ER) degrader) is a recommended treatment. An innate PI3K/AKT pathway inhibitor resistance can occur in patients with ER+ BC, limiting their efficacy. Therefore, novel therapeutic strategies are needed to increase sensitivity to treatments in patients with ER+ BC. With this aim, from CRISPR-based sensitization screens in ER+ BC cells, we have found that the effectiveness of the AKT inhibitor capivasertib is improved by targeting the epigenetic regulators, KDM5C (lysine demethylase 5C) or KAT6A (lysine acetyltransferase 6A). Knockout (KO) of KDM5C or KAT6A strongly enhances the response to capivasertib monotherapy and its treatment in combination with fulvestrant in PIK3CA-mut and PTEN-null ER+ BC cells, as well as re-sensitizes capivasertib-resistant ER+ BC cells. Importantly, the effects of the genetic KO were recapitulated with a pan-KDM5 inhibitor and a selective KAT6A/B inhibitor in combination with capivasertib in ER+BC cells, suggesting that combination therapies, consisting of capivasertib with KDM5 or KAT6A inhibitors, could be beneficial in patients. Moreover, transcriptomic analysis and chromatin profiling techniques show that both KDM5C and KAT6A KO downregulate expression and alter chromatic accessibility of a subset of ER-target genes and this phenotype is enhanced in combination with capivasertib, suggesting that targeting KDM5C or KAT6A sensitizes cells to AKTi through a novel epigenetic regulation of ER-signaling. Also, this study reports for the first time a landscape of epigenetic differences upon capivasertib monotherapy treatment and in combination with fulvestrant in ER+BC cells, revealing changes in chromatin accessibility as a consequence of treatment with AKTi and the ER degrader. This suggests that epigenetic remodelling can lead to resistance to capivasertib and fulvestrant therapy in cancer cells. In summary, this findings identified and validated KDM5C and KAT6A as novel epigenetic targets, the inhibition of which enhances efficacy of AKTi in PI3K pathway mutated ER+ breast cancer. Inhibitors of KDM5C and KAT6A may be potential combination partners for capivasertib in ER+ breast cancer. Citation Format: Valentina Cutano, Shanade Dunn, Lorna Hopcroft, Eleanor M. Wigmore, Lambert Montava Garriga, Sungmi Park-Chouinard, Wu Qing, Lee Hyung Joo, Cath Eberlein, Michele Chirichella, Daniel Barrell, Toby Gurran, Omid Tavana, Neeraj Aryal, Jerome T. Mettetal, Huayang Liu, Ho Man Chan, Susan E. Critchlow, Ultan McDermott, Simon T. Barry. The epigenetic regulators KDM5C and KAT6A influence AKT inhibitor response in ER positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3007.

Abstract 3022: ODY-CDK, a brain-penetrant, selective CDK2 inhibitor that demonstrates antitumor activity in an intracranial CCNE1-amplified breast tumor model

Abstract Cyclin-dependent kinase 2 (CDK2) is a member of the serine/threonine family of protein kinases that acts in complex with cyclins E1/E2 and A1/A2 to drive entry into and progression through S/G2-phases of the cell cycle. Amplification of CCNE1 or overexpression of cyclin E1 protein leads to persistent CDK2-cyclin E1 activation and phosphorylation of the retinoblastoma protein (pRb), thereby promoting uncontrolled cell cycle progression. In addition, cancer cells with loss-of-function (LoF) mutations in the retinoblastoma gene (RB1) exhibit variable levels of dependence on CDK2-cyclin A2 activity for progression through S/G2 phases of the cell cycle and have shown vulnerability to CDK2 inhibition. Brain metastases are frequently observed in cancers that harbor CCNE1 or RB1 alterations, such as small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), and triple-negative breast cancer (TNBC). Clinical-stage CDK2 inhibitors are not brain penetrant, and a brain-penetrant CDK2 inhibitor could provide a targeted therapeutic option for these high need patients. Here, we report on a brain-penetrant, selective CDK2 inhibitor, ODY-CDK, for the treatment of cancers with CCNE1 or RB1 genetic alterations with brain metastases or at high risk of developing them. ODY-CDK potently inhibits CDK2 (Ki = 3.1 nM) and demonstrates 40 to 180-fold selectivity over CDK1/5/6/7/9 as measured by cellular NanoBRET assays. ODY-CDK inhibits pRb in CCNE1-amplified ovarian and breast cancer cells and demonstrates antiproliferative activity in CCNE1-amplified and RB1-null ovarian, lung and breast cancer cells. In a clinically relevant human bone marrow assay, ODY-CDK is predicted to have a low risk of neutropenia that enables safe combinations with other agents. In vivo, once daily oral administration of ODY-CDK to mice with subcutaneous CCNE1-amplified HCC1569 breast or RB1 LoF NCI-H526 SCLC xenografts was well tolerated and demonstrated antitumor activity comparable to the clinical-stage CDK2 inhibitor PF-07104091. ODY-CDK treatment provoked sustained pRb inhibition in HCC1569 tumors. Pharmacokinetic evaluation of ODY-CDK demonstrated brain penetration across species with equivalent free exposure observed between cerebrospinal fluid (CSF) and plasma in dog (CSF Kpuu 0.9). To evaluate the intracranial efficacy, CCNE1-amplified HCC1569 cells were implanted into the cranium of NOG mice and tumor growth was measured by MRI. Daily oral dosing of ODY-CDK resulted in tumor regression in 8 out of 11 mice, while PF-07104091 showed modest activity with regression in only a single mouse. These data demonstrate that ODY-CDK is a selective brain-penetrant CDK2 inhibitor with differentiating intracranial antitumor activity to existing clinical-stage CDK2 inhibitors and support the evaluation of ODY-CDK in patients with brain metastases that have a high unmet medical need. Citation Format: Joe Gozgit, Gerald Lelais, Ellen Vieux, Sam Zelman, Laura Stransky, Hyeseok Shim, Kim Aardalen, Casey Landry, Arun Thottumkara, Jin Choi, Chip Lesburg, Sebastian Schneider, Chaya Stern, Elizabeth McMillan, Hanbin Lu, Caroline Ho, Nigel Waters, John Shen, Natalie Dales, Shifeng Pan, Bob Abraham, Gary D. Glick, Elaine M. Pinheiro. ODY-CDK, a brain-penetrant, selective CDK2 inhibitor that demonstrates antitumor activity in an intracranial CCNE1-amplified breast tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3022.

Abstract 5117: Investigating the role of secreted plasminogen receptors in the establishment of the pre-metastatic breast cancer niche

Abstract Introduction: Breast cancer is the most common cancer in women, with 25% of patients presentingsecondary tumors at diagnosis. Metastasis, responsible for over 90% of cancer deaths, involves cancer cellsescaping the primary tumor to colonize distant organs. This process requires the preparation of target sites, known as premetastatic niches (PMNs). PMNs undergo structural remodeling facilitated by cancer cell-secreted factors, including the protease plasmin (Pm), which aids in cancer cell escape by degrading tissuebarriers. Cancer cells utilize plasminogen receptors (PgRs) to convert the blood protein plasminogen (Plg)into Pm. Our research has shown that conditioned media (CM) from breast cancer cells can activate Plg toPm, suggesting the presence of PgRs in these secretions. While cell surface PgRs are established in tumorprogression, the roles of secreted PgRs within the breast cancer secretome remains undefined. Additionally, the involvement of the Plg/Pm system in PMN modulation is unexplored. We have found that CM frommalignant BCCs has higher Pm-activating potential than from non-malignant BCCs. Protein purification ofCM identified several soluble PgR-active fractions, with phosphoglycerate kinase-1 (PGK-1) emerging asa key PgR candidate. Methods: CM was isolated from highly and weakly metastatic BCC lines (MDA MB 231 vs. MCF7 & 4T1vs. 67NR). Soluble proteins were separated by size- and charge-chromatography, and fractions wereassayed for PgR activity, measured through their ability to stimulate tPA-dependent Plg-to-Pm conversion.Active fractions were pooled and identified via mass spectrometry. Results: CM from MDA MB 231 and 4T1 cells exhibited higher PgR activity and distinct chromatographicprofiles compared to MCF7 and 67NR cells. Size-exclusion chromatography revealed multiple PgR activitypeaks. Mass spectrometry identified novel PgR candidates, including glycolytic enzymes and extracellularmatrix components, PGK-1, a glycolytic enzyme, was identified in a PgR activity peak, with recombinantPGK-1 showing significant in vitro Pm generation, confirming its PgR role. Conclusions: PGK-1 is secreted by breast cancer cells and has Pm-generating potential. Futureexperiments will validate PGK-1 and other proteins as PgRs and clarify their roles in metastasis. Citation Format: Gillian Okura. Investigating the role of secreted plasminogen receptors in the establishment of the pre-metastatic breast cancer niche [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5117.

Abstract 2656: Sec23b governs the migration of MDA-MB-231 breast cancer cells by regulating collagen I processing and secretion

Abstract Cancer metastasis is a complex, multi-step process that begins with the dissociation of primary cancer cells from their original site. These cells then migrate to distant areas, where they establish colonies and undergo uncontrolled proliferation at secondary locations. Thus, dysregulation of cell migration is one of the hallmarks of solid tumour metastasis. Given that the actin cytoskeleton plays crucial roles in normal cell migration, we developed a robust method that combines an amino acid sequence that binds to actin (LifeAct) with biotin-mediated proximal labelling (BioID) to identify actin-binding proteins. Specifically, to identify actin-associated protein complexes during the early stages of cells migrating into scratched areas, we conjugated LifeAct with the newest derivation of BioID (miniTurbo-LifeAct-BioID) and controlled its expression with doxycycline in MDA-MB-231 cells. After inducing cell migration by scratch wounding, we allowed cells to migrate into the scratched areas for 1.5 hours then pulse-labelled with biotin for 30 minutes. As controls, using the same system, we labelled actin-associated proteins in resting cells and in cells that had been treated with cytochalasin D (CD) to depolymerize actin filaments. Compared to the untreated cells, the CD-treated cells increased labelling of the G-actin binding proteins (e.g., profilin and JMY) while decreased labelling of the F-actin binding proteins (e.g., Coronin2A and ABLIM1/ABLIM2/ ABLIM3), validating the ability of miniTurbo-LifeAct-BioID to detect dynamic changes in cytoskeleton-associated protein complexes. By comparing miniTurbo-LifeAct-BioID labelled proteins in migrating versus stationary cells, eighteen proteins were significantly more biotin labelled and eight proteins were less biotin labelled. Unexpectedly, Sec23b, a component of the COPII secretion complex, was labelled to a greater extent in cells migrating into scratched areas. Depletion of Sec23b increased secretion of procollagen I but decreased deposition of collagen I filaments. Interestingly, the reduced migration of cells depleted of Sec23b was rescued by collagen I but not by fibronectin or gelatin. Based on our findings, metastatic breast cancer MDA-MB-231 cells appear to control the secretion and processing of collagen I to govern their extracellular matrix organization for efficient cell migration. Citation Format: E. Emily Joo, Audrey Astori, Jonathan St-Germain, Brian Raught, Michael F. Olson. Sec23b governs the migration of MDA-MB-231 breast cancer cells by regulating collagen I processing and secretion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2656.

Abstract 1418: Mapping the FOXA1 interactome in ER+ breast cancer cells using proximity labeling reveals a novel interaction with NR2C2

Abstract Approximately 70% of breast cancers are driven by the estrogen receptor alpha (ER), and despite the advent of endocrine therapy to block ER signaling pathways, a significant proportion of women develop resistance to these drugs. The pioneer factor FOXA1 has been shown to facilitate nearly all DNA-binding events of ER in response to estrogen in ER-positive breast cancer (ER+BC). Notably, up-regulation of FOXA1 is a hallmark of endocrine-resistant tumors. Furthermore, FOXA1 has been shown to alter the transcriptome in endocrine therapy resistance. FOXA1 interacting partners, such as ER, androgen receptor (AR), MLL3, YAP1 and GATA-3 mediate transcription activation of genes important for cancer progression. There have been previous studies showing interactors of FOXA1, but none have addressed a comprehensive list of interactors of FOXA1, especially in breast cancer. To address this challenge, we generated MCF-7 cell lines stably expressing the biotin ligase miniTurbo fused to the N- or C-terminus of FOXA1 to classify interactors of FOXA1 in ER+BC. Using proximity labeling (PL) coupled with LC-MS/MS, we aimed to comprehensively catalog interactors of FOXA1. We were able to identify expected proteins such as ER, AR, MLL3, YAP1, and GATA-3. Moreover, we have discovered 157 previously unpublished interactors of FOXA1. Importantly, poorer prognosis for relapse-free survival among ER+BC patients is associated with several of these novel interactors, including NR2C2, an orphan nuclear receptor previously shown to regulate various nuclear receptor signaling but whose roles in breast cancer remain unclear. NR2C2 ChIP-seq in MCF-7 cells under FOXA1-depleted conditions revealed NR2C2 binding loci dependent on FOXA1 levels and binding. By integrating proteomic and genomic approaches, we highlight new mechanisms of FOXA1-mediated transcriptional regulation, with promising potential for endocrine therapy resistance. Citation Format: Carla Sofia Rodriguez-Tirado, Rosemary Plagens, Shen Li, Christine Mills, Laure Herring, Hector Franco. Mapping the FOXA1 interactome in ER+ breast cancer cells using proximity labeling reveals a novel interaction with NR2C2. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1418.

Abstract 1865: Optimization of miRNA-6883 lipid nanoparticles for targeted CDK4/6 and HIF1a inhibition in colorectal and breast cancer cells

The design of delivery vehicles to protect microRNAs (miRNAs) from degradation, enable tissue-specific targeting, and avoid potential off-target effects is a central challenge for their therapeutic application. miRNA encapsulation by lipid nanoparticles (LNPs) has been used to extend in vivo half-life, and LNPs may also be conjugated to an antigen targeting moiety to guide delivery to tumors. In vitro studies have identified miR-6883 as a promising therapeutic agent which targets CDK4/6, induces apoptosis, and reduces HIF1α levels in colorectal and breast cancer. In the present study, we employ a partial factorial design of experiments (DOE) to determine the optimal molar ratios of LNP components for on-target tumor potency, and use the miRNA-LNPs to further explore the potential therapeutic application of miR-6883. Methods: LNPs were formulated with MC3 or Lipid 331, DOTAP, DSPC, cholesterol, and DMG-PEG2k. Aqueous and lipid phases were combined a 3:1 ratio, followed by rapid pipetting and overnight dialysis. Encapsulation and RNA concentration were determined by Ribogreen assay. Polydispersity index (PDI) and diameter were determined by dynamic light scattering. Cancer cell lines were reverse transfected with 50nM of LNP-encapsulated RNA (miR-6883 or control). Viability was determined via CellTiter-Glo assay and protein levels by western blot. Fluorescent imaging and flow cytometry were performed to evaluate uptake of fluorescently-labeled LNPs. Partial factorial DOE was used to survey a design space consisting of a 4-part mixture (ionizable lipid, phospholipid, cholesterol, and PEG molar ratios) and a binary categorical variable to include 5% cationic lipid (JMP software). Results: Initial dosing with fluorescently labeled LNPs showed a dose-dependent increase in fluorescence, at lipid doses as low as 1µg/mL. Following formation of 12 LNP formulations based on the DOE screening design, LNPs had a maximum RNA encapsulation of 94.32%, PDIs from 0.07-.279 and diameters from 98.85-240.3nm. Multiple formulations showed potent anticancer activity, with a maximal 93.52% reduction in viability observed in HCT116 after 72h of 50nM miR-6883 compared to an untreated control. Protein level modulation of CDK4 and CDK6 was observed in all LNPs tested, as well as a reduction of HIF1α after 4 days. Notably, multiple samples exhibited an increase in CDK6, warranting further investigation. Conclusions: Our DOE identified candidate miR-6883-LNP formulations for further optimization. Future work will assess the optimized miR-LNP therapeutic in murine models of colorectal and breast cancer. Currently, we are generating antibody-conjugated LNPs, with the aim of increasing potency and mitigating off target toxicity, and are continuing LNP optimization for in vivo studies. Citation Format: Connor Purcell, Kadhambari Rajendran, Wafik S. El-Deiry, Theresa M. Raimondo. Optimization of miRNA-6883 lipid nanoparticles for targeted CDK4/6 and HIF1a inhibition in colorectal and breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1865.

Abstract 5338: TY-1781, a potent and highly selective CDK2 inhibitor, demonstrates superior anti-tumor activity in CCNE1 high-expression solid tumor models

Abstract Background: The common prescription of Palbociclib and other approved CDK4/6 inhibitors in clinic have greatly benefitted breast cancer patients worldwide. The cell cycle regulation therapy however has been found that the long-term use of the CDK4/6 inhibition agents eventually causes primary or acquired resistance to the therapy, and patients eventually relapse from the treatment. Approximately 20% of patients treated with CDK4/6 inhibitors show initial treatment failure. Based on the finding that abnormal activation of CDK2/Cyclin E1 due to CCNE1 gene amplification is determined the key resistant mechanism to CDK4/6 inhibition. Amplification of CCNE gene is prevalent in multiple tumor types, particularly in high-grade ovarian, uterine, and gastric cancers. CDK2 activity is important for survival of CCNE aberrant cancer cells and is also associated with potential resistance to targeted therapies, such as CDK4/6 dual inhibitors. Therefore, CDK2 is a promising therapeutic target in cancer. However, due to the structural homology and functional conservation of CDK family members, as well as the toxic effects associated with the inhibition of other CDKs, the development of selective CDK2 inhibitor is challenging. Here, we present preclinical data of TY-1781, a novel CDK2 inhibitor for the treatment of CCNE1 high-expression tumors. Results: TY-1781 demonstrates excellent CDK2 kinase inhibitory activity (IC50 0.7 nM), and high selectivity against CDK1, CDK4, CDK6, CDK7, and CDK9 in a panel screening of CDK kinases. In vitro cell proliferation data shows that tumor cell lines bearing CCNE1 amplification, are highly sensitive to the TY-1781 treatment. TY-1781 inhibits cellular Rb phosphorylation in Cyclin E1 amplified OVCAR3 cells in a potent yet selective manner, but not in Cyclin E1 wild type TOV21G cells; TY-1781 blocks the G1/S transition in CCNE1 overexpression OVCAR3 cells; and the tumor inhibitory activity of TY-1781 is further underscored when combined with a CDK4/6 inhibitor (Palbociclib), as demonstrated in a cell colony formation assay with ER+/Her2- breast cancer cells. Consistent with the in vitro results, TY-1781 demonstrated a potent inhibitory effect in immune-defect mouse models of CCNE1-amplified cancer cells. When combined with a chemotherapy agent (gemcitabine) or PARP inhibitor, TY-1781 robustly suppressed tumor growth in the examined ovarian cancer models. Conclusion: Taken together, we identified a highly selective and potent CDK2 inhibitor through medicinal chemistry screening and rounds of refining. The data suggest that TY-1781 may be eligible for further clinical development for Cyclin E1 high-expression patients who relapse from previous standard of care treatments. Citation Format: Chengshan Niu, Meihua Li, Shaoqing Chen, Maolin Zheng, Mingtao Chen, Hui Xu, Kaige Ji, Yuanjie Li, Rui Wu, Shengli Dong, Yu Yu, Xinlong Yang, Chao Zhou, Apeng Liang, Wei Wu, Mingyu Jiang, Jun Li, Yusheng Wu. TY-1781, a potent and highly selective CDK2 inhibitor, demonstrates superior anti-tumor activity in CCNE1 high-expression solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5338.

Abstract 1393: Functional analysis of mitochondria-targeting Wnt-inhibitors on metabolically distinct breast cancer cell types

Abstract Mitochondria are essential organelles responsible for energy production and survival. In cancer cells, mitochondrial function is often altered through metabolic reprogramming, which enhances their growth, survival, and resistance to therapy. Recent findings suggest that bi-directional crosstalk between Wnt signaling and mitochondrial function plays a crucial role in tumorigenesis, as well as in the differentiation and self-renewal of stem cells. This study investigates the potential of targeting the interplay between Wnt signaling and mitochondrial function in breast cancer cells. We screened 71 agents known to alter Wnt signaling and assessed their impact on mitochondrial function and cell viability in BT474 and Hs578T breast cancer cell lines. The Mito Tox Index (MTI) obtained using the Agilent Seahorse XF MitoTox Assay was used as the parameter to evaluate the impact of test compounds on mitochondrial function. The screening identified Pyrvinium, Niclosamide, and Hexachlorophene as the most potent mitochondrial modulators. Pyrvinium and Niclosamide inhibited the mitochondrial electron transport chain, whereas Hexachlorophene acted as an uncoupler when the cells were exposed to the drugs for 24 hours. The mechanisms of mitochondrial perturbation induced by these drugs were further investigated using BT474 cells permeabilized with the Seahorse XF Plasma Membrane Permeabilizer (PMP), which selectively permeabilizes the cellular plasma membrane without affecting mitochondria integrity. This approach enabled detailed characterization of key components in mitochondrial function, including transporters, enzymes, and electron transport chain complexes. Our results indicated that Niclosamide inhibited complex I/II, either directly or indirectly, while Pyrvinium appeared to suppress mitochondrial function indirectly. Despite the different bioenergetic phenotypes of BT474 (highly oxidative) and Hs578T (highly glycolytic) cells, there was no significant difference in mitochondrial susceptibility to the three drugs. However, significant differential effects on ATP production rates were detected using the Agilent Seahorse XF Real-Time ATP Rate, BT474 cells exhibited a significant metabolic energy crisis in the presence of all three compounds, whereas Hs578T cells maintained stable total ATP production despite mitochondrial inhibition. This differential impact on ATP production rates corresponded directly to differences in cell viability, with BT474 cells showing higher susceptibility to these compounds. These findings suggest that the anti-cancer efficacy of mitochondrial modulators can vary depending on the metabolic phenotype of the target cell and should be considered in therapeutic design. Targeting mitochondrial function through Wnt signaling inhibitors presents a promising strategy for developing effective cancer therapies. Citation Format: Yoonseok Kam, Lisa Winer, Natalia Romero. Functional analysis of mitochondria-targeting Wnt-inhibitors on metabolically distinct breast cancer cell types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1393.

Abstract 1308: Protein profiling of breast cancer cell-derived extracellular vesicles in breast to bone metastasis

Abstract Breast cancer is the most common cancer among women worldwide, with bone being one of the most common sites for breast cancer metastasis. Despite improvements in systemic therapies for breast cancer, the mechanisms underlying breast cancer metastasis to bone remain incompletely understood thereby limiting the development of effective therapeutic options. Emerging evidence suggests that extracellular vesicles (EVs), nanosized particles secreted by cells including cancer cells, play a pivotal role in organ-specific metastasis. However, relatively little is known regarding the contribution of breast cancer cell-derived EVs to bone metastasis. To begin to investigate the potential role(s) of breast cancer cell-derived EVs in breast to bone metastasis, we first analyzed the protein profiles of EVs derived from parental MDA-MB-231 breast cancer cells (P-EVs) and from bone-seeking MDA-MB-231 breast cancer cells (B-EVs) using iTRAQ (isobaric Tags for Relative and Absolute Quantification) quantitative mass spectrometry. Among a total of 126 identified proteins, 13 were upregulated in B-EVs, 8 were upregulated in P-EVs and 105 were shared by both EVs. Gene Ontology (GO) enrichment analyses and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses identified three key biological processes of interest: osteoclastogenesis, EV-cell attachment and bone extracellular matrix-receptor interactions which may contribute to bone-tropism and pre-metastatic niche preparation. Importantly, confocal microscopy studies demonstrated that B-EVs are preferentially internalized by osteoclast precursor cells. This finding was further confirmed by quantitative flow cytometry which demonstrated significantly higher fluorescent signal intensities for B-EVs compared to P-EVs in these osteoclast precursor cells also suggesting enhanced uptake specificity. Moreover, we found that B-EVs can induce the differentiation of osteoclast precursors into multinucleated osteoclasts in long-term in vitro studies as confirmed by tartrate-resistant acid phosphatase staining, indicating that EVs derived from a bone-seeking subpopulation of breast cancer cells may promote osteoclastogenesis, a process critical for bone resorption. This, in turn, may contribute to the development of a pre-metastatic microenvironment for bone metastasis. Taken together, these studies begin to enhance our understanding of the role of breast cancer cell-derived EVs in breast to bone metastasis and may ultimately offer potential avenues for therapeutic intervention and early detection of bone metastasis. (This work was supported by the Breast Cancer Research Foundation, NIH R21 CA253051-01 and the Nile Albright Research Foundation. The authors acknowledge the gift of the MDA-MB-231 parental cell line and its bone-seeking variant from the Massagué Lab.) Citation Format: Guan Huang, Golnaz Morad, Marsha A. Moses. Protein profiling of breast cancer cell-derived extracellular vesicles in breast to bone metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1308.

Abstract 540: AVJ16 alleviates ABCB1-mediated doxorubicin chemoresistance via IGF2BP1 inhibition

Background: The efficacy of chemotherapy is often compromised by multidrug resistance (MDR), driven by the overexpression of ATP-binding Cassette Sub-family B Member 1 (ABCB1). While ABCB1 inhibitors are urgently needed, toxicity and pharmacokinetic challenges have limited clinical progress. Dysregulation of N6-Methyladenosine (m6A)—a key mRNA modification involved in gene regulation—is linked to chemoresistance, presenting a potential target to counteract ABCB1-mediated MDR. Aim: To examine m6A’s role in ABCB1-driven chemoresistance in uterine sarcoma and breast cancer and evaluate alternative ABCB1 inhibitors. Methods: RT-qPCR analysis identified Insulin-like Growth Factor 2 mRNA-binding protein 1 (IGF2BP1)—an m6A reader—as the most upregulated m6A regulator in MDR uterine sarcoma cells. Expression levels were validated in parental and MDR uterine sarcoma and breast cancer cells. GEO dataset of breast cancer patients treated with neoadjuvant chemotherapy was analyzed in silico to determine IGF2BP1-ABCB1 correlation. Functional assays were performed to evaluate IGF2BP1 knockdown and inhibition effects by AVJ16 (an IGF2BP1 inhibitor) on ABCB1 expression and doxorubicin resistance. Additionally, 3-deazaadenosine (DAA), an m6A inhibitor, was added to MDR cells with IGF2BP1 knockdown or AVJ16 treatment to confirm IGF2BP1’s role in m6A-dependent regulation of ABCB1 and chemoresistance. Results: IGF2BP1 and ABCB1 were significantly upregulated in MDR uterine sarcoma and breast cancer cells. In silico analysis confirmed a positive correlation between IGF2BP1 and ABCB1 expression, linking IGF2BP1 to ABCB1-driven chemoresistance clinically. IGF2BP1 knockdown in MDR uterine sarcoma cells reduced ABCB1 expression and doxorubicin resistance, evidenced by lower cell viability and colony formation. Moreover, AVJ16 significantly decreased doxorubicin’s half-maximal inhibitory concentration (IC50) in MDR uterine sarcoma by downregulating ABCB1 expression. DAA, combined with IGF2BP1 knockdown or AVJ16, markedly enhanced doxorubicin sensitivity, supporting IGF2BP1’s m6A-dependent role in MDR. Conclusion: IGF2BP1 is a novel m6A-dependent regulator of ABCB1-driven chemoresistance. These findings highlight AVJ16 as a promising therapeutic candidate for overcoming ABCB1-driven MDR cancers. Citation Format: Eveiyn Weng Yan Phoon, Kowit-Yu Chong. AVJ16 alleviates ABCB1-mediated doxorubicin chemoresistance via IGF2BP1 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 540.

Abstract 5627: Characterization of the Rac inhibitor CPV-337 in metastatic breast cancer

Rac and Cdc42 are homologous Rho GTPases that act as anticancer and antimetastatic targets regulating cancer progression and metastasis-associated cell functions such as cell survival, cell polarity, proliferation, migration, and invasion. Our lead compound MBQ-167, a dual Rac/Cdc42 inhibitor (IC50 of ∼100nM; GI50 of 130nM), is currently in Phase I clinical trials for advanced triple-negative breast cancer (NCT06075810). By screening a panel of MBQ-167 derivatives to identify molecules with a range of inhibitory mechanisms that address distinct cancer types, we identified CPV-337, with an IC50 of ∼50nM for inhibiting Rac activation, via pulldown assays to capture the GTP bound Rac, and a GI50 of ∼55nM for inhibiting the viability of metastatic breast cancer cells, as measured by MTT assays. However, CPV-337 does not affect the viability of human mammary epithelial cells (HMECs), demonstrating non-toxic effects in primary epithelial cells. Additionally, wound healing assays show that CPV-337 reduces breast cancer cell migration by ∼50% at 50nM. These data show that CPV-337 is ∼2.5X more effective than MBQ-167. We then compared the efficacy of CPV-337 and MBQ-167 in nude mice with orthotopic tumors established from green fluorescent protein-tagged metastatic HER2-positive breast cancer cells. Results demonstrate that CPV-337 administered at 5mg/kg Body Weight (BW) parallels MBQ-167 treatments of 10mg/kg Body Weight (BW) at 5X a week for 2 months. Compared to the vehicle, both CPV-337 and MBQ-167 reduced tumor growth by ∼90-95%, from quantification of the integrated density of fluorescent tumors for 62 days. Lung metastases were inhibited by ∼50% in CPV-337 treated mice, and both compounds inhibited liver and kidney metastases by ∼95%. At necropsy, blood was collected, and whole blood was analyzed by flow cytometry for the phosphorylated form of the Rac and Cdc42 downstream effector p21-activated kinase (p-PAK), using a fluorescent anti-p-PAKT423antibody, as a measure of Rac activation inhibition. CPV-337 inhibited PAK activation significantly by 37.4%, compared to vehicle. Interestingly, a significant increase in M1 anti-tumorigenic macrophages was observed in splenocytes of mice treated with CPV-337, implicating CPV-337 in the anticancer immune response. Further evaluation of CPV-337’s inhibitory mechanism, toxicity, bioavailability, and Rac/Cdc42 regulated processes in the tumor microenvironment are warranted to translate this promising anticancer compound to the clinic. Citation Format: Jessica Colon Gonzalez, Nilmary Grafals Ruiz, Anamaris Torres Sanchez, Stephanie Dorta Estremera, Cornelis Vlaar, Suranganie Dharmawardhane. Characterization of the Rac inhibitor CPV-337 in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5627.