Citations

Abstract Estrogen receptor alpha (ERα)-positive breast cancer accounts for up to 80% of cases. Despite advances in CDK4/6 inhibitors (CDK4/6i) and endocrine therapies, recurrence and late relapse remain significant challenges. Recurrence often arises from disseminated cancer cells that persist despite 5-10 years of systemic adjuvant treatment. While CDK4/6i and endocrine therapies induce prolonged growth arrest resembling senescence, they do not significantly kill cancer cells. Senescent/pseudo-senescent cancer cells are implicated in aggressive variant formation, recurrence, and metastasis. We explored eliminating breast cancer cells via senolysis while they are in therapy-induced senescent state to prevent their potential to escape, repopulate, or cause long-term adverse effects. Live-cell imaging of MCF7 and T47D cells treated with abemaciclib and endocrine therapies (endoxifen or estrogen deprivation to mimic aromatase inhibition) at clinically relevant concentrations for eight days demonstrated prolonged growth cessation and G1 cell cycle arrest even two weeks post-drug withdrawal. These cells displayed a senescence-like gene expression profile, with increased CDKN2B (p15), CDKN1A (p21), BCL2, reduced KI67, and increased TGFβ expression, a known Senescence-Associated Secretory Phenotype (SASP) factor linked to deep senescence. These results confirm effective senescence induction by various CDK4/6i and endocrine therapy combinations. Further analysis using established senescence markers (e.g., nuclear size, telomere-associated foci, satellite distension, and loss of nuclear HMGB1 and LaminB1) revealed unique senescence phenotypes across therapy combinations. Cell viability assays and live imaging of Caspase 3/7 activation demonstrated differential vulnerabilities of senescent/senescent-like ERα+ cancer cells (MCF7, T47D, UCD4, UCD12) to 13 senolytics (1 μM), including natural flavonoids and inhibitors targeting pro-survival factors, tyrosine kinases, and the Notch pathway. For example, MCF7 and UCD4 cells responded to MCL1i and RSL3, T47D to BCLXLi and RSL3, and UCD12 to BCLXLi and Navitoclax. Notably, senolytic cocktails (e.g., RSL3, GNF7, Venetoclax, Dasatinib) at reduced concentrations (0.25 μM each) effectively eliminated senescent/senescent-like ERα+ cancer cells across all cancer cell lines, achieving 100% elimination in T47D cells. Collectively, these findings demonstrate that despite variability in therapy-induced senescence, senolytic combinations effectively target senescent ERα+ cancer cells. This provides preliminary evidence for the potential integration of this strategy into standard breast cancer therapy with a goal to improve recurrence-free survival and increase the cure rates for ERα+ breast cancer patients. In vivo studies are underway to evaluate this strategy in preclinical models. Citation Format: Lena Batoon, Angelo J. Guilatco, Anna Detry, Xiyin Wang, Megan M. Weivoda, John R. Hawse. CDK4/6 inhibitors and endocrine therapies induce senescence with drug- and cell line-specific vulnerabilities in ERα+ 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 519.

Abstract Purpose: Black women are disproportionately affected by aggressive breast cancers compared to white women, suggesting underlying biological differences in normal breast tissue. This study aims to identify molecular disparities in normal breast tissue between premenopausal Black and white women that may contribute to increased breast cancer risk. Experimental Procedures: Visium spatial transcriptomic analysis was performed on a pilot cohort of normal breast tissue samples from premenopausal Black and white women. Tissue sections were spatially profiled, and capture spots were classified based on predominant cell types: epithelial, stromal, or endothelial. Differential gene expression analysis between racial groups was conducted for each cell type classification. Multiplex immunofluorescence (mIF) was used to validate selected markers in the pilot cohort. Meta-analysis compared differentially expressed genes with published gene sets associated with breast cancer subtypes. Results: Significant racial differences in gene expression profiles were identified in both epithelial and stromal spots. In epithelial spots, genes upregulated in Black women significantly overlapped with over 20 published gene sets associated with estrogen receptor-negative (ER−) breast cancer, including key genes involved in Wnt signaling inhibition (e.g., WIF1) and basal epithelial markers (e.g., KRT14, KRT15). This suggests alterations in pathways linked to more aggressive breast cancer phenotypes. In stromal spots, differentially expressed genes related to extracellular matrix remodeling and fibroblast activation (e.g., PI16, DPT, EFEMP1) were observed, indicating potential differences in stromal-epithelial interactions. Ongoing deconvolution and spatial analyses are being performed to further elucidate these findings. Preliminary mIF validation confirmed increased expression of PI16 in samples from Black women. Validation of additional markers is underway in a larger cohort of premenopausal Black and white women. Conclusions: Spatial transcriptomic profiling of normal breast tissue reveals distinct molecular differences between Black and white women, particularly in pathways associated with aggressive breast cancer subtypes and stromal interactions. These findings suggest that inherent variations in the normal breast microenvironment may contribute to the increased risk and aggressiveness of breast cancer in Black women. Further validation and analysis may identify potential biomarkers for risk assessment and inform targeted prevention strategies. Citation Format: Camila Pacheco Erak, Nicole Cruz-Reyes, Pria Westerman, Joshua Ogony, Jean R. Clemenceau, Tae Hyun Hwang, Sarah McLaughlin, Derek Radisky. Spatial transcriptomic profiling of normal breast tissue reveals potential mechanisms underlying increased breast cancer risk in Black women [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 765.

Abstract VSV-IFNβ-NIS (VV1) is a Vesicular Stomatitis Virus (VSV) that encodes the interferon beta (IFNβ) and the iodide symporter (NIS) gene. The IFNβ gene confers tumor selectivity and enhances host antitumor immunity while the NIS gene acts as an imaging biomarker, allowing noninvasive imaging of viral infection sites using PET/SPECT. Multiple myeloma (MM) is a cancer of plasma cells and poses a challenge for treatment due to its biological heterogeneity. As MM usually relapses and is refractory, current therapies face the issue of drug resistance and patients may have to turn to novel therapies. Therefore, we set out to understand the activity of VSV which could be a new approach for patients with relapsed refractory MM (RRMM). Our study utilized two unique bortezomib resistant Vk*MYC transplantable models, Vk12598 and Vk12653, with distinct genomic features. Upon re-transplantation of splenocytes from these mice, flow cytometry revealed that Vk12598 and Vk12653 mice had high MM populations in the spleen and bone marrow. To assess VV1 efficacy in tumor bearing mice, we performed a VV1 dose escalation study, showing efficacy in delaying MM development and prolonging survival in both mice models, with Vk12598 being the more responsive model. Later studies also showed that VV1 induces tumor remission in the Vk12598 model when treatment was initiated at low tumor burden (gamma/albumin ratio ∼0.2). IFNβ levels increased significantly post 24-72h of VV1 treatment, indicating targeted infection and replication of the virus in MM cells. However, high doses of VV1 led to toxicity, as observed from early mortality and weight loss. To determine the sites of MM metastasis and VV1 infection, timepoint harvests were carried out. Hematoxylin and eosin and immunostainings showed the onset of MM of Vk*MYC in the bone marrow and spleen first, then later in the liver. Immunofluorescent staining confirmed that the higher the tumor burden observed, the more VV1 was present in the tissues. To enhance VV1 efficacy, we performed in vivo combination therapies of VV1 with anti-PD-1, an inhibitor of PD-1 which is overexpressed on MM patient T cells; and bortezomib, which inhibits NF-κB which in turn prevents IFN pathway protein transcription, potentially enhancing the replication of VV1 virus. In Vk12598 mice, VV1 combined with both drugs induced tumor regression and doubled the median survival time compared to treatment with VV1 only or VV1 coupled with either of the drugs individually. Addition of VV1 to either anti-PD-1 or bortezomib alone also increased survival time significantly. Studies are still ongoing to understand the mechanisms of VSV and its combination treatments in Vk*MYC models. This translational study will allow us to gain the knowledge needed to refine VSV treatments and provide novel therapy combinations to improve MM patient survival. Citation Format: Yi Ying Ma, Lianwen Zhang, Rebecca A. Nace, Marta Chesi, Stephen J. Russell, Kah Whye Peng. Harnessing oncolytic virus VSV and combination therapy to advance treatments for relapsed refractory multiple myeloma using Vk*MYC 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 940.

Abstract Background: Postpartum breast cancer (PPBC) is associated with increased morbidity and mortality among women within 5 to 10 years following childbirth. The postpartum breast undergoes involution, involving tissue remodeling and immune cell infiltration, potentially creating a pro-tumorigenic microenvironment. The molecular mechanisms underlying this elevated risk remain poorly understood. In this study, we compared normal breast tissue samples and human mammary epithelial cells (HMECs) from postpartum women to those from age- and BMI-matched nulliparous women to elucidate potential molecular contributors to PPBC risk. Methods: Transcriptomic profiling was performed on RNA extracted from FFPE normal breast tissues using the NanoString IO 360 and BC 360 platforms to examine gene expression in nulliparous and postpartum women under 45 years of age. HMECs were isolated from organoids derived from breast tissue samples and used for next-generation sequencing and knockdown experiments targeting STAT1 and GBP1. Gene set enrichment analysis (GSEA) was applied to NanoString and RNA-seq datasets to identify differentially enriched genes between postpartum and nulliparous women. Results: Transcriptomic analysis revealed significant upregulation of interferon-stimulated genes, including STAT1 and GBP1, in postpartum breast tissues, consistent with increased immune responses during involution. NanoString data showed that markers of cell proliferation, such as Ki67, STAT1, and GBP1, were significantly higher in samples from parous compared to nulliparous women (p < 0.05). GSEA revealed that cell cycle-promoting genes (GBP1, cyclins, CDKs) were upregulated, while cyclin-dependent kinase inhibitors (CDKNs) were downregulated in postpartum breast tissues. Immunohistochemistry confirmed significantly higher expression of Ki67, STAT1, and GBP1 in postpartum versus nulliparous breast tissues (p < 0.0001). Silencing STAT1 and GBP1 in HMECs resulted in decreased cell proliferation, G1 cell cycle arrest, and increased expression of CDKN1A and CDKN1C. Conclusion: These findings suggest that STAT1 and GBP1 promote proliferation in postpartum breast epithelial cells by downregulating cyclin-dependent kinase inhibitors, potentially contributing to the increased risk of PPBC. Targeting these pathways may offer novel strategies for mitigating postpartum breast cancer risk. Citation Format: Joshua Ogony, Melody Stallings-Mann, Sarah McLaughlin, Mark Sherman, Derek C. Radisky. STAT1 and GBP1 promote proliferation in postpartum breast epithelium by downregulating cyclin-dependent kinase inhibitors: implications for increased postpartum breast cancer risk [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 187.

Abstract Background: Targeted agents, such as nanobodies, can be covalently linked to radionuclide chelators for theranostics. Nanobodies are single-domain immunoglobulin variable regions with a short blood half-lives and high specificity. Calreticulin (CALR) is endoplasmic reticular (ER) protein that translocates to the cell surface upon induction with certain agents, namely doxorubicin. We hypothesized surface CALR on tumors could be targeted using a novel nanobody after induction with doxorubicin. Methods: Studies were conducted using in vitro and in vivo models of pancreatic ductal adenocarcinoma (PDAC). Surface CALR expression was determined by western blot on fractionated lysates, flow cytometry, or immunofluorescence (IF). Doxorubicin was administered by intraperitoneal injection to mouse models of PDAC. The anti-CALR nanobody was developed via the panning of a synthetic nanobody phage display library. The top CALR-binding nanobody was confirmed by ELISA, immunohistochemistry (IHC), and flow cytometry. For in vivo studies, the nanobody was conjugated with the chelator NOTA and radiolabeled with 64Cu. PET images were acquired 1-hour after injection. Significance was determined using an independent t-test and p values < 0.05 were considered statistically significant. Results: PDAC cells treated with doxorubicin showed peak surface CALR expression between 24 and 48 hours. Flow cytometry revealed that 61.7% of live PDAC cultured cells treated for 24 hours expressed surface CALR, compared to 2.2% in controls (p < 0.001). In a subcutaneous syngeneic PDAC mouse model, two doses of doxorubicin 48 hours apart significantly increased surface CALR, with 30.4% of live-cells expressing CALR in treated tumors versus 1.5% in controls (p < 0.001). Similarly, doxorubicin-treated tumors from a PDAC patient derived xenograft (PDX) model had a 6.7-fold increase in live-cells expressing surface CALR compared to controls (p < 0.01). IF confirmed a 1.4-fold increase in CALR expression in doxorubicin-treated tumors from the subcutaneous PDAC mouse model (p < 0.01) and a 1.7-fold increase in the PDAC PDX model (p < 0.01). Our anti-CALR nanobody has an apparent 37 nM affinity for recombinant CALR and successfully detected surface CALR by IHC and flow cytometry. In vivo PET imaging showed significant increased uptake of the 64Cu-NOTA-nanobody in tumors from doxorubicin-treated mice over controls, with no significant changes in heart, kidney, or liver accumulation by PET quantification and flow cytometry (p < 0.05). Conclusions: Systemic doxorubicin treatment induces CALR surface translocation in tumors that can be successfully targeted with a radiolabeled nanobody for PET imaging. Targeting chemotherapy-induced surface CALR using our radiolabeled nanobody could provide a theragnostic strategy for the detection and treatment of PDAC. Citation Format: Rachael Guenter, Yuvasri Golivi, Lucinda Hall, Amro Abdelrahman, Chloe La Prairie, Caleb Miller, Tejeshwar C. Rao, Danielle M. Carlson, Mark J. Truty, Benjamin Larimer, J. Bart Rose. Detection of tumor cell surface calreticulin after doxorubicin treatment using a novel radiolabeled nanobody [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 574.