Abstract Activating HER2 mutations are significantly enriched in both primary and metastatic invasive lobular breast cancer (ILC), with large public datasets of primary breast tumors linking them to a worse prognosis in ILC. Despite their oncogenic role, no FDA-approved therapies currently target HER2-mutant breast cancers. While the HER2-directed antibody-drug conjugate (ADC) trastuzumab deruxtecan (T-DXd) has shown efficacy in HER2-mutant non-small cell lung cancer, its activity in HER2-mutant ILC remains unknown. Using the Caris real-world database, one of the largest cohorts with survival data in advanced breast cancers, we confirmed that HER2 mutations are more prevalent in advanced ILC than in invasive breast cancer of no special type (NST) tumors, are associated with worse survival in both histologies, yet predict improved response to T-DXd across subtypes, highlighting the need for mutation-directed, histology-informed therapies. Using endogenous HER2-mutant ILC cell lines (UACC3133-S310F, BCK4-L755S) and CRISPR-engineered isogenic ILC models with clinically relevant HER2 mutations (S310F, V777L), we found these mutations drive HER2/HER3 hyperactivation and downstream signaling, conferring increased sensitivity to HER2 tyrosine kinase inhibitors (TKIs) and T-DXd. Mechanistically, HER2 mutants showed enhanced receptor ubiquitination, internalization, and lysosomal degradation upon T-DXd treatment, explaining the observed drug sensitivity. While combining T-DXd with neratinib or the HSP90 inhibitor ganetespib yielded synergistic effects in long-term growth assays, accompanied by increased HER2 ubiquitination, the concurrent hyperactivation of HER3 in HER2-mutant cells suggested that co-targeting HER3 could provide an effective alternative strategy. Accordingly, HER2-mutant ILC exhibited enhanced sensitivity to the HER3-directed ADC patritumab deruxtecan (P-DXd) or LJM716, a HER3-targeting antibody. We further uncovered a previously unrecognized mechanism of P-DXd beyond HER3 ligand blockade and payload delivery: P-DXd promotes HER2/HER3 association, increases HER2 ubiquitination, and enhances T-DXd internalization, resulting in potent synergy with T-DXd. Mechanistically, we identified HER3 extracellular domains I and II as essential for P-DXd binding and for mediating P-DXd-induced HER2/HER3 association, establishing a structural basis for this activity. In vivo , both T-DXd and P-DXd suppressed UACC3133 and BCK4 xenograft growth, with combination therapy trending toward greater efficacy and prevented regrowth of tumors. Extending these findings beyond HER2-mutant ILC, combination treatment with T-DXd and P-DXd demonstrated synergistic activity across multiple breast cancer models, including (i) HER2-amplified NST patient-derived organoids (PDOs) harboring hotspot HER2 mutations, (ii) HER2-wild-type NST PDOs with clinically intrinsic or acquired T-DXd resistance, and (iii) isogenic HER2-mutant ILC PDOs with experimentally induced resistance after prolonged T-DXd exposure. Collectively, these findings support HER2 as an actionable target in HER2-mutant ILC and position T-DXd-based regimens, particularly in combination with HER3 inhibition, as a promising therapeutic strategy for this underserved patient population.

Chemotherapy remains the primary treatment for ovarian cancer (OvCa), and chemoresistance drives patient mortality. Cellular quiescence, reversible exit from the cell cycle, increases chemotherapy resistance as chemotherapies primarily target rapidly proliferating cells. Here, we report that CHD4 and MBD3, components of the nucleosome remodeling and deacetylase (NuRD) complex, are downregulated in quiescent OvCa cells (qOvCa). We find that either CHD4 or MBD3 knockdown or histone deacetylase inhibitors (HDACi), induce quiescence in OvCa cells. RNA-Seq and ATAC-seq analysis of HDACi-treated cells confirmed expression changes consistent with induction of quiescence. Additionally, HDACi-treated cells revealed downregulation of the RHO/RAC pathways. Suggesting downregulation of the RAC pathway could play a role in HDACi-mediated quiescence, RAC inhibitors (RACi) similarly induced quiescence. Both HDACi and RACi resulted in nuclear-to-cytoplasmic shifting of the pro-proliferative transcription factor MRTFA. which has been linked to quiescence. Further analysis of HDACi qOvCa indicated multiple alterations in proteostasis, including increased proteasome activity and autophagy. We find qOvCa cells are dependent on these pathways for survival, such that there is profound synergistic OvCa cell death with HDACi and proteasome- or autophagy-inhibitor combination therapy. Combined, this work supports HDACi as pharmacologic means to induce a quiescent state in OvCa cells and sensitize them to proteostasis-targeting drugs.

Chemoresistance is a major cause of cancer deaths. One understudied mechanism of chemoresistance is quiescence. We used single-cell culture to identify and isolate patient-derived proliferating and quiescent ovarian cancer cells (qOvCa). RNA-seq analysis indicated that hundreds of genes that are differentially expressed in qOvCa cells are transcriptional targets of the Myocardin-Related Transcription Factor-A/Serum Response Factor (MRTFA/SRF) pathway, and both genetic disruption and pharmacologic inhibition of MRTFA/SRF interaction (with the inhibitor CCG257081) induced quiescence across multiple cancer types. MRTFA/SRF inhibition-mediated quiescence is p27/Kip1 dependent and associated with a downregulation of cell cycle regulators, NCL, MYH9, and alterations in the proteasome. We show that the MRTFA/SRF axis plays a dual role in chemotherapy resistance, with both pathway inhibition and activation contributing to chemotherapy resistance in vitro and in patient samples. CCG081 treatment results in a proteasome-dependent downregulation of the stem-cell marker CD133. Suggesting a critical role for the proteasome in quiescent cells, CCG081 therapy sensitized OvCa cells to proteasome inhibitors. In vivo, we found that CCG257081 therapy could be used to induce tumor growth-arrest and delay disease growth to improve overall survival. Moreover, we found that dual therapy with CCG081 and proteasome inhibition further improved outcomes, leading to undetectable tumors in ∼20% of mice. Together, these data suggest that the MRTFA/SRF pathway is a critical regulator of quiescence in cancer and a potential therapeutic target.

Accurately inferring copy number variation (CNV) from scRNA-seq data is critical for identifying malignant cells, reconstructing tumor subclonal architecture, and uncovering the genomic drivers that dictate cancer cell biology. However, the performance of existing tools varies significantly, and current benchmarks lack the breadth of datasets and methods necessary to provide definitive guidance. We present a comprehensive benchmark of 12 CNV inference methods across 28 real datasets (>100,000 cells) and diverse synthetic datasets. By evaluating methods based on malignant cell classification accuracy, CNV inference accuracy, scalability, and robustness, we establish a definitive practitioner's guideline: allele-aware methods like Numbat excel when high-quality allelic inference can be achieved, whereas expression-centric tools such as Clonalscope, CopyKAT, inferCNV, and SCEVAN remain reliable when raw sequencing data are unavailable. Our study provides both a practical decision-making framework for researchers and a public repository of standardized CNV profiles to catalyze further methodological innovation.

Estrogen receptor-positive (ER+) breast cancer is the most common subtype of breast cancer and is an age-related disease. How systemic and tumor microenvironment hormone signaling intersects with chronic inflammation and how this shapes aging-associated tumor biology remain incompletely understood. Here, using an aged rat model of ER+ tumors, we identify age-associated differences in tumor development and immune features. In parallel, analysis of samples from patients with ER+/HER2- breast cancer and age-matched controls demonstrated estrone-predominant systemic estrogen patterns and increased tumor HSD17B7 expression in older patients, consistent with enhanced local estrone-to-estradiol conversion. In patient-derived organoids from older women, pharmacologic inhibition of HSD17B7 reduced estrogen conversion and proliferation-associated transcriptional programs. Tumors from older patients also exhibited chemokine enrichment, including CCL2, associated with immunosuppressive macrophage features. Targeting both estrogen signaling and chemokine pathways attenuated macrophage polarization ex vivo. These findings support a model linking age-associated hormonal and inflammatory changes to features of a tumor-permissive microenvironment in ER+ breast cancer.

Immune escape during the ductal carcinoma in situ (DCIS)-to-invasive breast cancer (IBC) transition shapes tumor evolution. Through transcriptomic mapping of the immune landscapes of normal breast, DCIS, and IBC from large patient cohorts, we identified T and myeloid cells as the primary distinguishing features between DCIS and IBC. We discovered cycling regulatory T cells (cycTreg) as an orchestrator of immunosuppression in IBC. cycTreg frequency predicts cytotoxic CD8+, TCR diversity, disease-specific survival in IBC, and recurrence in DCIS. In a rat model of breast cancer, we demonstrated that cycTreg act as precursors to mature Treg and are inducible by tumor-localized type 2 dendritic cells. Profiling of tumors subjected to αOX40 and αPD-L1 therapies revealed an IL-33-mediated fibroblast-cycTreg signaling loop, the disruption of which enhances intratumoral antigen-experienced CD8+ effectors and systemic immunosurveillance. Our study defines cycTreg as critical inducers of immune escape and promising immuno-oncology targets in breast cancer.

Invasive lobular cancer (ILC) is the most common special breast cancer subtype, accounting for 10-15% of all cases. The pathognomonic feature of ILC is loss of E-cadherin (encoded by CDH1), leading to discohesive single-file growth. Although ILCs show better prognostic factors than 'no special type' (NST) breast cancer, patients with ILC have worse long-term outcomes. We identified and validated patient-derived xenograft (PDX) models of ILC from 122 breast cancer PDX models based on truncating CDH1 mutations and/or low CDH1 mRNA expression. Eight PDX models were selected for validation using immunohistochemistry for E-cadherin, p120, estrogen receptor, progesterone receptor and HER2. Seven models were confirmed as ILC, and one showed mixed NST-ILC features. Confirmed ILC PDX models showed enrichment of truncating CDH1 mutations, significantly lower CDH1 mRNA expression and predominantly luminal subtypes compared to NST models, consistent with human ILC characteristics. Commonly altered genes included PIK3CA (57%), CDH1 (57%) and TP53 (57%). These validated ILC PDX models provide valuable tools to advance understanding of ILC biology and support development of targeted therapies.

Invasive lobular breast carcinoma (ILC), the most common special histological subtype of breast cancer, is characterized by nearly universal expression of estrogen receptor alpha (ER) and unique sites of metastases, neither of which is fully recapitulated by genetically engineered mouse models. Using reporter-labeled ILC mouse xenografts, herein we used mammary fat pad, tail vein and intracardiac orthotopic growth to analyze spontaneous and experimental metastasis and gene expression. We observed ER-positive primary tumors with single-file histology and collagen deposition, and spontaneous metastasis from the mammary fat pad to bones, ovaries, and brain including the leptomeninges, thereby closely mirroring the growth and metastatic spread of human ILC. Brain metastases showed strong ER staining, confirmed by sequencing analyses which identified estrogen signaling as top activated pathway, and the lesions exhibited robust response to endocrine therapy. In summary, we report endocrine responsive mammary fat pad, tail vein and intracardiac xenografts that faithfully demonstrate unique ILC features and can serve as invaluable pre-clinical translational platforms for validating candidate ILC genetic drivers and testing novel therapeutics.

Whole-genome doubling (WGD) is a common yet poorly understood event associated with poor clinical outcomes. Here, we characterize mechanisms by which WGD drives tumor evolution, utilizing mouse mammary tumor models of WGD established through cell fusion. We find that WGD increases transcriptomic and epigenetic heterogeneity and identify the YM155 BIRC5 inhibitor as a compound specifically suppressing WGD+ tumors. WGD triggers immune evasion by escaping CD8+ T cell responses, rendering WGD+ tumors more sensitive to anti-PD-L1. Through single-cell profiling, we discover that WGD+ cancer cells exhibit reduced antigen presentation and response to IFNγ, attributed to the epigenetic silencing of MHCI transcriptional regulators via elevated histone H3 lysine 27 trimethylation. Further investigations reveal decreased KDM6 activity and increased succinate levels in WGD+ tumors. PRC2 inhibition preferentially suppresses WGD+ tumor growth, enhances antigen presentation, and CD8+ T cell infiltration. Our results underscore metabolic and epigenetic alterations as critical drivers of WGD-associated immune escape.

Despite advances in precision oncology, effective personalized treatments are still lacking for most patients with cancer1. The Cancer Dependency Map (DepMap) accelerates this field by systematically identifying cancer vulnerabilities in diverse preclinical models. Data from over 1,300 cell lines have led to the discovery of new therapeutic strategies across multiple tumour types2. However, mapping cancer vulnerabilities using traditional cell lines has limitations, including insufficient cancer subtype representation and the impact of culture conditions on perturbation responses. Here we perform 147 genome-scale CRISPR screens and multi-omic characterizations of next-generation (NextGen) cancer models (organoids and spheroids) across 10 cancer types. This strategy enables the expansion of DepMap to cover new genomic and molecular subtypes and to identify new biomarker-associated vulnerabilities. These new models also preserve transcriptional programs that are silenced in traditional cell lines and facilitate the discovery of specific gene dependencies associated with these programs. Comparisons of traditional and NextGen cancer models enable further identification of distinct effects of growth format and culture medium on gene essentiality. The integrated dataset combines data from both model types to offer a valuable, expansive resource for exploring cancer vulnerabilities and is accessible via the DepMap portal.

Resistance to human epidermal growth factor receptor 2 (HER2)-targeted therapies and immuno-oncology agents poses a major challenge in treating HER2-positive breast cancer. Here we demonstrate that p95HER2, a truncated form of HER2, drives immune evasion in HER2-positive female breast cancer, enhancing tumor growth and conferring therapy resistance. This stems from the unique ability of p95HER2 to promote cancer cell-intrinsic programmed death ligand 1 expression and secretion of immunosuppressive mediators including interleukin 6. In preclinical models, this impairs the efficacy of trastuzumab deruxtecan, a HER2-directed antibody–drug conjugate (ADC) that relies on immunogenic responses to cell death for full efficacy. Importantly, we find that neratinib potently directs proteasomal degradation of p95HER2, relieving its immunosuppressive effects, and provide proof of concept that neratinib and/or agents targeting p95HER2 downstream mediators can restore antitumor immunity and trastuzumab deruxtecan efficacy. This study reveals a p95HER2-specific therapy resistance mechanism in HER2-positive female breast cancer and highlights the potential value of targeting p95HER2 to improve outcomes with ADCs or immuno-oncology agents. Hu et al. discovered that the truncated form of human epidermal growth factor receptor 2 (HER2), called p95HER2, drives tumor progression and resistance to the antibody–drug conjugate trastuzumab deruxtecan in HER2+ breast cancer. Blocking p95HER2 restores antitumor immunity.

BRM014 represses cell proliferation and DEX-induced chemoresistance.

Invasive lobular carcinoma (ILC) is responsible for 10%-15% of all invasive breast cancers and is associated with worse long-term outcomes compared to the most common no special type (NST). Despite known differences between ILC and NST, the treatment for ILC is the same as stage matched NST. Therefore, identifying the biological molecules that are specific for ILC and can be therapeutically targeted is an urgent need. In this context, we recently identified the transcription factor AP2b (TFAP2B) as one of the most hypomethylated genes in ILC compared with NST and normal mammary tissues. Consistently, differential gene expression analysis in TCGA, METABRIC and SCAN-B datasets confirmed the upregulation of TFAP2B expression in ILC. Methylation of CG island in the first exon possesses the highest correlation with mRNA expression levels. Here, we examined the role of this transcription factor in ILC using various models including cell lines, patient derived organoids (PDOs) and tumor samples. Immunoblotting and RT-qPCR confirmed the specific upregulation of TFAP2B in ILC compared with NST cell lines and PDOs. Immunofluorescence showed a clear nuclear localization of TFAP2B and immunohistochemistry revealed a strong and uniform expression in tissues isolated from ILC patients. TFAP2B knockdown (using siRNA and inducible shRNA) significantly reduces the growth of ILC cell lines and PDOs in anchorage dependent and independent conditions as well as colony formation assay. The actions of TFAP2B seem to be mediated, at least in part, through cell cycle as knockdown of this factor decreases and increases the percentage of cells in S and G0/G1 phase, respectively. CDH1 knockout or inducible expression in NST or ILC cell lines failed to affect TFAP2B levels indicating that changes in E-cadherin levels, in the transformed cells, are not sufficient to alter TFAP2B expression. Preliminary data from CUT and RUN sequencing indicate possible binding sites for TFAP2B in growth factors and cell cycle related genes. Collectively, the data support the specific enhancement of TFAP2B in ILC and its importance for cell proliferation. Future studies will be conducted to uncover the interaction partners and pathways regulated by TFAP2B. This could serve as a basis for the development of novel therapeutic approaches that can be tailored specifically to patients with ILC tumors. Abdalla M. Wedn, Hunter Waltermire, Haley Arbore, Daniel Brown, Osama Shah, Fangyuan Chen, Sanghoon Lee, Jagmohan Hooda, Adrian Lee, Steffi Oesterreich. Hypomethylation and overexpression of transcription factor AP2B promote growth in ILC via cell cycle modulation [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 4044.

Breast cancer affects 1/8 of women throughout their lifetimes, with over 90% of cancer deaths being caused by metastasis. However, metastasis poses unique challenges to research, as complex changes in the microenvironment in different metastatic sites and difficulty obtaining tissue for study hinder the ability to examine in depth the changes that occur during metastasis. Rapid autopsy programs thus fill a unique need in advancing metastasis research. Here, we describe our protocol and processes for establishing and improving the US-based Hope for OTHERS (Our Tissue Helping Enhance Research and Science) program for organ donation in metastatic breast cancer. As of August 2024, we consented 114 patients and performed 37 autopsies, from which we collected 551 unique metastatic frozen tumor samples, 1244 FFPE blocks, 90 longitudinal liquid biopsy samples and developed 14 patient-derived organoid and 8 patient-derived xenograft models. We report in-depth clinical and histopathological information and discuss extensive new research and novel findings in patient outcomes, metastatic phylogeny, and factors in successful living model development. Our results reveal key logistical and protocol improvements that are uniquely beneficial to certain programs based on identifiable features, such as working closely with patient advocates, methods to rescue RNA quality in cases where tissue quality may degrade due to time delays, as well as guidelines and future expansions of our program.

Overall data set and experiments included in the EstroGene database

BRG1, but not BRM, is overexpressed in TNBC.

High confident E2-regulated genes and BETA score

Cytotoxic chemotherapy remains the primary treatment for ovarian cancer (OvCa). Development of chemoresistance typically results in patient death within two years. As such, understanding chemoresistance is critical. One underexplored mechanism of chemotherapy resistance is quiescence. Quiescent cells, which have reversibly exited the cell cycle, are refractory to most chemotherapies which primarily target rapidly proliferating cells. Here, we report that CHD4 and MBD3, components of the nucleosome remodeling and deacetylase (NuRD) complex, are downregulated in quiescent OvCa cells (qOvCa). Indicating a direct role for NuRD complex downregulation in the induction of quiescence, either CHD4 or MBD3 knockdown or histone deacetylase inhibitors (HDACi), such as vorinostat, induce quiescence in OvCa cells. RNA-Seq analysis of HDACi-treated cells confirmed expression changes consistent with induction of quiescence. We also find that both primary qOvCa and vorinostat-induced qOvCa demonstrate altered proteostasis, including increased proteasome activity and autophagy, and combination therapy of HDACi and proteasome inhibitors or autophagy inhibitors demonstrated profound synergistic death of OvCa cells. Finally, we overlapped RNA-Seq signatures from quiescent ovarian cancer cells with genes essential for quiescence in yeast to identify a "quiescent cell core signature." This core quiescent cell signature appeared to be conserved across multiple cancer types, suggesting new therapeutic targets.

LISA regulator predictions of all signatures

E2-induced DEGs from inter-dataset concordance analysis