DNA hypomethylation silences anti-tumor immune genes in early prostate cancer and CTCs.

Cancer is characterized by hypomethylation-associated silencing of large chromatin domains, whose contribution to tumorigenesis is uncertain. Through high-resolution genome-wide single-cell DNA methylation sequencing, we identify 40 core domains that are uniformly hypomethylated from the earliest detectable stages of prostate malignancy through metastatic circulating tumor cells (CTCs). Nested among these repressive domains are smaller loci with preserved methylation that escape silencing and are enriched for cell proliferation genes. Transcriptionally silenced genes within the core hypomethylated domains are enriched for immune-related genes; prominent among these is a single gene cluster harboring all five CD1 genes that present lipid antigens to NKT cells and four IFI16-related interferon-inducible genes implicated in innate immunity. The re-expression of CD1 or IFI16 murine orthologs in immuno-competent mice abrogates tumorigenesis, accompanied by the activation of anti-tumor immunity. Thus, early epigenetic changes may shape tumorigenesis, targeting co-located genes within defined chromosomal loci. Hypomethylation domains are detectable in blood specimens enriched for CTCs.

Visualizing Engrafted Human Cancer and Therapy Responses in Immunodeficient Zebrafish.

Xenograft cell transplantation into immunodeficient mice has become the gold standard for assessing pre-clinical efficacy of cancer drugs, yet direct visualization of single-cell phenotypes is difficult. Here, we report an optically-clear prkdc-/-, il2rga-/- zebrafish that lacks adaptive and natural killer immune cells, can engraft a wide array of human cancers at 37°C, and permits the dynamic visualization of single engrafted cells. For example, photoconversion cell-lineage tracing identified migratory and proliferative cell states in human rhabdomyosarcoma, a pediatric cancer of muscle. Additional experiments identified the preclinical efficacy of combination olaparib PARP inhibitor and temozolomide DNA-damaging agent as an effective therapy for rhabdomyosarcoma and visualized therapeutic responses using a four-color FUCCI cell-cycle fluorescent reporter. These experiments identified that combination treatment arrested rhabdomyosarcoma cells in the G2 cell cycle prior to induction of apoptosis. Finally, patient-derived xenografts could be engrafted into our model, opening new avenues for developing personalized therapeutic approaches in the future.

Stromal Microenvironment Shapes the Intratumoral Architecture of Pancreatic Cancer.

Single-cell technologies have described heterogeneity across tissues, but the spatial distribution and forces that drive single-cell phenotypes have not been well defined. Combining single-cell RNA and protein analytics in studying the role of stromal cancer-associated fibroblasts (CAFs) in modulating heterogeneity in pancreatic cancer (pancreatic ductal adenocarcinoma [PDAC]) model systems, we have identified significant single-cell population shifts toward invasive epithelial-to-mesenchymal transition (EMT) and proliferative (PRO) phenotypes linked with mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling. Using high-content digital imaging of RNA in situ hybridization in 195 PDAC tumors, we quantified these EMT and PRO subpopulations in 319,626 individual cancer cells that can be classified within the context of distinct tumor gland "units." Tumor gland typing provided an additional layer of intratumoral heterogeneity that was associated with differences in stromal abundance and clinical outcomes. This demonstrates the impact of the stroma in shaping tumor architecture by altering inherent patterns of tumor glands in human PDAC.

A Landscape of Pharmacogenomic Interactions in Cancer.

Systematic studies of cancer genomes have provided unprecedented insights into the molecular nature of cancer. Using this information to guide the development and application of therapies in the clinic is challenging. Here, we report how cancer-driven alterations identified in 11,289 tumors from 29 tissues (integrating somatic mutations, copy number alterations, DNA methylation, and gene expression) can be mapped onto 1,001 molecularly annotated human cancer cell lines and correlated with sensitivity to 265 drugs. We find that cell lines faithfully recapitulate oncogenic alterations identified in tumors, find that many of these associate with drug sensitivity/resistance, and highlight the importance of tissue lineage in mediating drug response. Logic-based modeling uncovers combinations of alterations that sensitize to drugs, while machine learning demonstrates the relative importance of different data types in predicting drug response. Our analysis and datasets are rich resources to link genotypes with cellular phenotypes and to identify therapeutic options for selected cancer sub-populations.

Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis.

Circulating tumor cell clusters (CTC clusters) are present in the blood of patients with cancer but their contribution to metastasis is not well defined. Using mouse models with tagged mammary tumors, we demonstrate that CTC clusters arise from oligoclonal tumor cell groupings and not from intravascular aggregation events. Although rare in the circulation compared with single CTCs, CTC clusters have 23- to 50-fold increased metastatic potential. In patients with breast cancer, single-cell resolution RNA sequencing of CTC clusters and single CTCs, matched within individual blood samples, identifies the cell junction component plakoglobin as highly differentially expressed. In mouse models, knockdown of plakoglobin abrogates CTC cluster formation and suppresses lung metastases. In breast cancer patients, both abundance of CTC clusters and high tumor plakoglobin levels denote adverse outcomes. Thus, CTC clusters are derived from multicellular groupings of primary tumor cells held together through plakoglobin-dependent intercellular adhesion, and though rare, they greatly contribute to the metastatic spread of cancer.

NR4A1 regulates expression of immediate early genes, suppressing replication stress in cancer.

Deregulation of oncogenic signals in cancer triggers replication stress. Immediate early genes (IEGs) are rapidly and transiently expressed following stressful signals, contributing to an integrated response. Here, we find that the orphan nuclear receptor NR4A1 localizes across the gene body and 3' UTR of IEGs, where it inhibits transcriptional elongation by RNA Pol II, generating R-loops and accessible chromatin domains. Acute replication stress causes immediate dissociation of NR4A1 and a burst of transcriptionally poised IEG expression. Ectopic expression of NR4A1 enhances tumorigenesis by breast cancer cells, while its deletion leads to massive chromosomal instability and proliferative failure, driven by deregulated expression of its IEG target, FOS. Approximately half of breast and other primary cancers exhibit accessible chromatin domains at IEG gene bodies, consistent with this stress-regulatory pathway. Cancers that have retained this mechanism in adapting to oncogenic replication stress may be dependent on NR4A1 for their proliferation.

TAK1 inhibition promotes apoptosis in KRAS-dependent colon cancers.

Colon cancers frequently harbor KRAS mutations, yet only a subset of KRAS mutant colon cancer cell lines are dependent upon KRAS signaling for survival. In a screen for kinases that promote survival of KRAS-dependent colon cancer cells, we found that the TAK1 kinase (MAP3K7) is required for tumor cell viability. The induction of apoptosis by RNAi-mediated depletion or pharmacologic inhibition of TAK1 is linked to its suppression of hyperactivated Wnt signaling, evident in both endogenous and genetically reconstituted cells. In APC mutant/KRAS-dependent cells, KRAS stimulates BMP-7 secretion and BMP signaling, leading to TAK1 activation and enhancement of Wnt-dependent transcription. An in vitro-derived "TAK1 dependency signature" is enriched in primary human colon cancers with mutations in both APC and KRAS, suggesting potential clinical utility in stratifying patient populations. Together, these findings identify TAK1 inhibition as a potential therapeutic strategy for a treatment-refractory subset of colon cancers exhibiting aberrant KRAS and Wnt pathway activation.

RNA transcripts stimulate homologous recombination by forming DR-loops.

Homologous recombination (HR) repairs DNA double-strand breaks (DSBs) in the S and G2 phases of the cell cycle1-3. Several HR proteins are preferentially recruited to DSBs at transcriptionally active loci4-10, but how transcription promotes HR is poorly understood. Here we develop an assay to assess the effect of local transcription on HR. Using this assay, we find that transcription stimulates HR to a substantial extent. Tethering RNA transcripts to the vicinity of DSBs recapitulates the effects of local transcription, which suggests that transcription enhances HR through RNA transcripts. Tethered RNA transcripts stimulate HR in a sequence- and orientation-dependent manner, indicating that they function by forming DNA-RNA hybrids. In contrast to most HR proteins, RAD51-associated protein 1 (RAD51AP1) only promotes HR when local transcription is active. RAD51AP1 drives the formation of R-loops in vitro and is required for tethered RNAs to stimulate HR in cells. Notably, RAD51AP1 is necessary for the DSB-induced formation of DNA-RNA hybrids in donor DNA, linking R-loops to D-loops. In vitro, RAD51AP1-generated R-loops enhance the RAD51-mediated formation of D-loops locally and give rise to intermediates that we term 'DR-loops', which contain both DNA-DNA and DNA-RNA hybrids and favour RAD51 function. Thus, at DSBs in transcribed regions, RAD51AP1 promotes the invasion of RNA transcripts into donor DNA, and stimulates HR through the formation of DR-loops.

The evolving war on cancer.

Building on years of basic scientific discovery, recent advances in the fields of cancer genetics and medicinal chemistry are now converging to revolutionize the treatment of cancer. Starting with serendipitous observations in rare subsets of cancer, a paradigm shift in clinical research is poised to ensure that new molecular insights are rapidly applied to shape emerging cancer therapies. Could this mark a turning point in the "War on Cancer"?

Integrating genomic features for non-invasive early lung cancer detection.

Radiologic screening of high-risk adults reduces lung-cancer-related mortality1,2; however, a small minority of eligible individuals undergo such screening in the United States3,4. The availability of blood-based tests could increase screening uptake. Here we introduce improvements to cancer personalized profiling by deep sequencing (CAPP-Seq)5, a method for the analysis of circulating tumour DNA (ctDNA), to better facilitate screening applications. We show that, although levels are very low in early-stage lung cancers, ctDNA is present prior to treatment in most patients and its presence is strongly prognostic. We also find that the majority of somatic mutations in the cell-free DNA (cfDNA) of patients with lung cancer and of risk-matched controls reflect clonal haematopoiesis and are non-recurrent. Compared with tumour-derived mutations, clonal haematopoiesis mutations occur on longer cfDNA fragments and lack mutational signatures that are associated with tobacco smoking. Integrating these findings with other molecular features, we develop and prospectively validate a machine-learning method termed 'lung cancer likelihood in plasma' (Lung-CLiP), which can robustly discriminate early-stage lung cancer patients from risk-matched controls. This approach achieves performance similar to that of tumour-informed ctDNA detection and enables tuning of assay specificity in order to facilitate distinct clinical applications. Our findings establish the potential of cfDNA for lung cancer screening and highlight the importance of risk-matching cases and controls in cfDNA-based screening studies.

Targeting breast and pancreatic cancer metastasis using a dual-cadherin antibody.

The successful application of antibody-based therapeutics in either primary or metastatic cancer depends upon the selection of rare cell surface epitopes that distinguish cancer cells from surrounding normal epithelial cells. By contrast, as circulating tumor cells (CTCs) transit through the bloodstream, they are surrounded by hematopoietic cells with dramatically distinct cell surface proteins, greatly expanding the number of targetable epitopes. Here, we show that an antibody (23C6) against cadherin proteins effectively suppresses blood-borne metastasis in mouse isogenic and xenograft models of triple negative breast and pancreatic cancers. The 23C6 antibody is remarkable in that it recognizes both the epithelial E-cadherin (CDH1) and mesenchymal OB-cadherin (CDH11), thus overcoming considerable heterogeneity across tumor cells. Despite its efficacy against single cells in circulation, the antibody does not suppress primary tumor formation, nor does it elicit detectable toxicity in normal epithelial organs, where cadherins may be engaged within intercellular junctions and hence inaccessible for antibody binding. Antibody-mediated suppression of metastasis is comparable in matched immunocompetent and immunodeficient mouse models. Together, these studies raise the possibility of antibody targeting CTCs within the vasculature, thereby suppressing blood-borne metastasis.

A conduit to metastasis: circulating tumor cell biology.

Advances in the enrichment and analysis of rare cells from the bloodstream have allowed for detection and characterization of circulating tumor cells (CTCs) from patients with cancer. The analysis of CTCs has provided significant insight into the metastatic process. Studies on the biology of CTCs have begun to elucidate the molecular mechanisms of CTC generation, intravasation, survival, interactions with components of the blood, extravasation, and colonization of distant organs. Additionally, the study of CTCs has exposed dramatic intrapatient and interpatient heterogeneity and their evolution over time. In this review, we focus on the current knowledge of CTC biology and the potential clinical implications.

ATR inhibition disrupts rewired homologous recombination and fork protection pathways in PARP inhibitor-resistant BRCA-deficient cancer cells.

Poly-(ADP-ribose) polymerase (PARP) inhibitors (PARPis) selectively kill BRCA1/2-deficient cells, but their efficacy in BRCA-deficient patients is limited by drug resistance. Here, we used derived cell lines and cells from patients to investigate how to overcome PARPi resistance. We found that the functions of BRCA1 in homologous recombination (HR) and replication fork protection are sequentially bypassed during the acquisition of PARPi resistance. Despite the lack of BRCA1, PARPi-resistant cells regain RAD51 loading to DNA double-stranded breaks (DSBs) and stalled replication forks, enabling two distinct mechanisms of PARPi resistance. Compared with BRCA1-proficient cells, PARPi-resistant BRCA1-deficient cells are increasingly dependent on ATR for survival. ATR inhibitors (ATRis) disrupt BRCA1-independent RAD51 loading to DSBs and stalled forks in PARPi-resistant BRCA1-deficient cells, overcoming both resistance mechanisms. In tumor cells derived from patients, ATRis also overcome the bypass of BRCA1/2 in fork protection. Thus, ATR inhibition is a unique strategy to overcome the PARPi resistance of BRCA-deficient cancers.

Modeling the novel SERD elacestrant in cultured fulvestrant-refractory HR-positive breast circulating tumor cells.

PURPOSE: Metastatic hormone receptor-positive (HR+) breast cancer initially responds to serial courses of endocrine therapy, but ultimately becomes refractory. Elacestrant, a new generation FDA-approved oral selective estrogen receptor degrader (SERD) and antagonist, has demonstrated efficacy in a subset of women with advanced HR+breast cancer, but there are few patient-derived models to characterize its effect in advanced cancers with diverse treatment histories and acquired mutations. METHODS: We analyzed clinical outcomes with elacestrant, compared with endocrine therapy, among women who had previously been treated with a fulvestrant-containing regimen from the recent phase 3 EMERALD Study. We further modeled sensitivity to elacestrant, compared with the currently approved SERD, fulvestrant in patient-derived xenograft (PDX) models and cultured circulating tumor cells (CTCs). RESULTS: Analysis of the subset of breast cancer patients enrolled in the EMERALD study who had previously received a fulvestrant-containing regimen indicates that they had better progression-free survival with elacestrant than with standard-of-care endocrine therapy, a finding that was independent estrogen receptor (ESR1) gene mutations. We modeled elacestrant responsiveness using patient-derived xenograft (PDX) models and in ex vivo cultured CTCs derived from patients with HR+breast cancer extensively treated with multiple endocrine therapies, including fulvestrant. Both CTCs and PDX models are refractory to fulvestrant but sensitive to elacestrant, independent of mutations in ESR1 and Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha (PIK3CA) genes. CONCLUSION: Elacestrant retains efficacy in breast cancer cells that have acquired resistance to currently available ER targeting therapies. Elacestrant may be an option for patients with HR+/HER2- breast cancer whose disease progressed on fulvestrant in the metastatic setting. TRANSLATIONAL RELEVANCE: Serial endocrine therapy is the mainstay of management for metastatic HR+breast cancer, but acquisition of drug resistance highlights the need for better therapies. Elacestrant is a recently FDA-approved novel oral selective estrogen receptor degrader (SERD), with demonstrated efficacy in the EMERALD phase 3 clinical trial of refractory HR+breast cancer. Subgroup analysis of the EMERALD clinical trial identifies clinical benefit with elacestrant in patients who had received prior fulvestrant independent of the mutational status of the ESR1 gene, supporting its potential utility in treating refractory HR+breast cancer. Here, we use pre-clinical models, including ex vivo cultures of circulating tumor cells and patient-derived xenografts, to demonstrate the efficacy of elacestrant in breast cancer cells with acquired resistance to fulvestrant.

Dependency of a therapy-resistant state of cancer cells on a lipid peroxidase pathway.

Plasticity of the cell state has been proposed to drive resistance to multiple classes of cancer therapies, thereby limiting their effectiveness. A high-mesenchymal cell state observed in human tumours and cancer cell lines has been associated with resistance to multiple treatment modalities across diverse cancer lineages, but the mechanistic underpinning for this state has remained incompletely understood. Here we molecularly characterize this therapy-resistant high-mesenchymal cell state in human cancer cell lines and organoids and show that it depends on a druggable lipid-peroxidase pathway that protects against ferroptosis, a non-apoptotic form of cell death induced by the build-up of toxic lipid peroxides. We show that this cell state is characterized by activity of enzymes that promote the synthesis of polyunsaturated lipids. These lipids are the substrates for lipid peroxidation by lipoxygenase enzymes. This lipid metabolism creates a dependency on pathways converging on the phospholipid glutathione peroxidase (GPX4), a selenocysteine-containing enzyme that dissipates lipid peroxides and thereby prevents the iron-mediated reactions of peroxides that induce ferroptotic cell death. Dependency on GPX4 was found to exist across diverse therapy-resistant states characterized by high expression of ZEB1, including epithelial-mesenchymal transition in epithelial-derived carcinomas, TGFß-mediated therapy-resistance in melanoma, treatment-induced neuroendocrine transdifferentiation in prostate cancer, and sarcomas, which are fixed in a mesenchymal state owing to their cells of origin. We identify vulnerability to ferroptic cell death induced by inhibition of a lipid peroxidase pathway as a feature of therapy-resistant cancer cells across diverse mesenchymal cell-state contexts.

Ultrahigh-throughput magnetic sorting of large blood volumes for epitope-agnostic isolation of circulating tumor cells.

Circulating tumor cell (CTC)-based liquid biopsies provide unique opportunities for cancer diagnostics, treatment selection, and response monitoring, but even with advanced microfluidic technologies for rare cell detection the very low number of CTCs in standard 10-mL peripheral blood samples limits their clinical utility. Clinical leukapheresis can concentrate mononuclear cells from almost the entire blood volume, but such large numbers and concentrations of cells are incompatible with current rare cell enrichment technologies. Here, we describe an ultrahigh-throughput microfluidic chip, LPCTC-iChip, that rapidly sorts through an entire leukapheresis product of over 6 billion nucleated cells, increasing CTC isolation capacity by two orders of magnitude (86% recovery with 105 enrichment). Using soft iron-filled channels to act as magnetic microlenses, we intensify the field gradient within sorting channels. Increasing magnetic fields applied to inertially focused streams of cells effectively deplete massive numbers of magnetically labeled leukocytes within microfluidic channels. The negative depletion of antibody-tagged leukocytes enables isolation of potentially viable CTCs without bias for expression of specific tumor epitopes, making this platform applicable to all solid tumors. Thus, the initial enrichment by routine leukapheresis of mononuclear cells from very large blood volumes, followed by rapid flow, high-gradient magnetic sorting of untagged CTCs, provides a technology for noninvasive isolation of cancer cells in sufficient numbers for multiple clinical and experimental applications.

HER2 expression identifies dynamic functional states within circulating breast cancer cells.

Circulating tumour cells in women with advanced oestrogen-receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancer acquire a HER2-positive subpopulation after multiple courses of therapy. In contrast to HER2-amplified primary breast cancer, which is highly sensitive to HER2-targeted therapy, the clinical significance of acquired HER2 heterogeneity during the evolution of metastatic breast cancer is unknown. Here we analyse circulating tumour cells from 19 women with ER+/HER2- primary tumours, 84% of whom had acquired circulating tumour cells expressing HER2. Cultured circulating tumour cells maintain discrete HER2+ and HER2- subpopulations: HER2+ circulating tumour cells are more proliferative but not addicted to HER2, consistent with activation of multiple signalling pathways; HER2- circulating tumour cells show activation of Notch and DNA damage pathways, exhibiting resistance to cytotoxic chemotherapy, but sensitivity to Notch inhibition. HER2+ and HER2- circulating tumour cells interconvert spontaneously, with cells of one phenotype producing daughters of the opposite within four cell doublings. Although HER2+ and HER2- circulating tumour cells have comparable tumour initiating potential, differential proliferation favours the HER2+ state, while oxidative stress or cytotoxic chemotherapy enhances transition to the HER2- phenotype. Simultaneous treatment with paclitaxel and Notch inhibitors achieves sustained suppression of tumorigenesis in orthotopic circulating tumour cell-derived tumour models. Together, these results point to distinct yet interconverting phenotypes within patient-derived circulating tumour cells, contributing to progression of breast cancer and acquisition of drug resistance.

COX-2 mediates tumor-stromal prolactin signaling to initiate tumorigenesis.

Tumor-stromal communication within the microenvironment contributes to initiation of metastasis and may present a therapeutic opportunity. Using serial single-cell RNA sequencing in an orthotopic mouse prostate cancer model, we find up-regulation of prolactin receptor as cancer cells that have disseminated to the lungs expand into micrometastases. Secretion of the ligand prolactin by adjacent lung stromal cells is induced by tumor cell production of the COX-2 synthetic product prostaglandin E2 (PGE2). PGE2 treatment of fibroblasts activates the orphan nuclear receptor NR4A (Nur77), with prolactin as a major transcriptional target for the NR4A-retinoid X receptor (RXR) heterodimer. Ectopic expression of prolactin receptor in mouse cancer cells enhances micrometastasis, while treatment with the COX-2 inhibitor celecoxib abrogates prolactin secretion by fibroblasts and reduces tumor initiation. Across multiple human cancers, COX-2, prolactin, and prolactin receptor show consistent differential expression in tumor and stromal compartments. Such paracrine cross-talk may thus contribute to the documented efficacy of COX-2 inhibitors in cancer suppression.

Epithelial to mesenchymal plasticity and differential response to therapies in pancreatic ductal adenocarcinoma.

Transcriptional profiling has defined pancreatic ductal adenocarcinoma (PDAC) into distinct subtypes with the majority being classical epithelial (E) or quasi-mesenchymal (QM). Despite clear differences in clinical behavior, growing evidence indicates these subtypes exist on a continuum with features of both subtypes present and suggestive of interconverting cell states. Here, we investigated the impact of different therapies being evaluated in PDAC on the phenotypic spectrum of the E/QM state. We demonstrate using RNA-sequencing and RNA-in situ hybridization (RNA-ISH) that FOLFIRINOX combination chemotherapy induces a common shift of both E and QM PDAC toward a more QM state in cell lines and patient tumors. In contrast, Vitamin D, another drug under clinical investigation in PDAC, induces distinct transcriptional responses in each PDAC subtype, with augmentation of the baseline E and QM state. Importantly, this translates to functional changes that increase metastatic propensity in QM PDAC, but decrease dissemination in E PDAC in vivo models. These data exemplify the importance of both the initial E/QM subtype and the plasticity of E/QM states in PDAC in influencing response to therapy, which highlights their relevance in guiding clinical trials.

Evaluation of endocrine resistance using ESR1 genotyping of circulating tumor cells and plasma DNA.

PURPOSE: Therapeutic efficacy of hormonal therapies to target estrogen receptor (ER)-positive breast cancer is limited by the acquisition of ligand-independent ESR1 mutations, which confer treatment resistance to aromatase inhibitors (AIs). Monitoring for the emergence of such mutations may enable individualized therapy. We thus assessed CTC- and ctDNA-based detection of ESR1 mutations with the aim of evaluating non-invasive approaches for the determination of endocrine resistance. PATIENTS AND METHODS: In a prospective cohort of 55 women with hormone receptor-positive metastatic breast cancer, we isolated circulating tumor cells (CTCs) and developed a high-sensitivity method for the detection of ESR1 mutations in these CTCs. In patients with sufficient plasma for the simultaneous extraction of circulating tumor DNA (ctDNA), we performed a parallel analysis of ESR1 mutations using multiplex droplet digital PCR (ddPCR) and examined the agreement between these two platforms. Finally, we isolated single CTCs from a subset of these patients and reviewed RNA expression to explore alternate methods of evaluating endocrine responsiveness. RESULTS: High-sensitivity ESR1 sequencing from CTCs revealed mono- and oligoclonal mutations in 22% of patients. These were concordant with plasma DNA sequencing in 95% of cases. Emergence of ESR1 mutations was correlated both with time to metastatic relapse and duration of AI therapy following such recurrence. The Presence of an ESR1 mutation, compared to ESR1 wild type, was associated with markedly shorter Progression-Free Survival on AI-based therapies (p = 0.0006), but unaltered to other non-AI-based therapies (p = 0.73). Compared with ESR1 mutant cases, AI-resistant CTCs with wild-type ESR1 showed an elevated ER-coactivator RNA signature, consistent with their predicted response to second-line hormonal therapies. CONCLUSION: Blood-based serial monitoring may guide the selection of precision therapeutics for women with AI-resistant ER-positive breast cancer.