Turing patterns are a cornerstone of biological self-organization, yet their emergence typically requires finely tuned parameters occupying narrow regions of high-dimensional space. This poses a fundamental challenge: how can evolving biological systems reliably find and exploit such rare conditions? In this work, we propose that common biochemical limit cycles, such as those arising from genetic feedback loops, can act as natural explorers of Turing space. By coupling a reaction-diffusion system to an orbit that modulates some of its parameters, we show that the system can dynamically sweep through Turing-permissive regimes and generate transient spatial patterns. We use an entropy-based measure in Fourier space to quantify pattern formation and demonstrate how cycles enhance the detectability and robustness of Turing islands. We further explore how coupling to positional gradients increases reproducibility, suggesting a route from oscillatory dynamics to stable developmental programs. Our results highlight a powerful mechanism by which nature might bootstrap complex spatial structure from simple temporal motifs.
Latent prostate cancers discovered at autopsy may represent the earliest, subclinical stage of the disease. This study aimed to construct three-dimensional (3D) spatial distribution maps of these tumors to infer their likely origin and growth patterns. A total of 161 tumor lesions from 230 cadaver donors were stratified by volume (V1: 0-0.05 ml; V2: 0.05-0.1 ml; V3: 0.1-0.5 ml; V4: > 0.5 ml) and registered to a standardized MRI-based reference prostate model using Advanced Normalization Tools. Two-dimensional frequency maps were generated per axial layer and stacked into a 3D map. The smallest tumors (V1) originated almost exclusively in the peripheral zone (83.5%), concentrated in the apical two-thirds of the gland (95.9%), with a uniform distribution across the anterior-posterior and left-right axes. As tumor volume increased (V2 to V4), three distinct growth patterns emerged: (1) increased invasion into the transition zone; (2) horizontal convergence into three "hotspots"-the anterior midline and bilateral posterolateral regions; and (3) vertical concentration within layers III to IX. A compact "susceptible region" occupying only 9.8% of the gland volume harbored 88.2% of the largest (V4) tumors. Latent prostate cancers appear to originate predominantly within the apical-to-mid peripheral zone with an initially uniform spatial distribution. Their growth is anisotropic, favoring the anterior midline and bilateral posterolateral zones while concentrating vertically within the central 20-40% of the prostatic height. These spatial patterns point to target areas that may improve biopsy strategies and focal therapy planning.
Viral transcriptional regulators (vTRs) reprogram host cell transcriptional and epigenetic networks to promote infection, persistence, and oncogenic transformation. Despite their broad essentiality to viral pathogenesis, thousands of putative vTRs remain functionally uncharacterized. Here we apply PROD-ATAC, a pooled single-cell epigenomic screening platform, to map chromatin accessibility dysregulation induced by over 100 vTRs in a single assay. Our screen identified dozens of vTRs which directly and indirectly remodel chromatin, including variants with no previously reported epigenetic activity. Comparative analyses revealed that unrelated vTRs frequently opened chromatin regions associated with the same host transcription factor networks, including the NF-κB, AP-1, p53, and Sp1/KLF families. Integration of chromatin accessibility and transcriptomic datasets highlighted that epigenetic perturbations often coincide with downstream gene expression changes. By adapting PROD-ATAC to incorporate small-molecule perturbations, we demonstrate that targeted inhibition of host features (including the MAPK signaling cascade) can illuminate host dependencies for vTR-induced epigenomic dysregulation. These results establish a scalable framework for discovering virus-host chromatin interactions and suggest that epigenetic manipulation of host regulatory networks is a widespread and conserved function of many diverse vTRs.
Regulated necrosis, a type of programmed cell death, is a major factor in cancer development. The function of regulated necrosis-related genes (RNRGs) in pancreatic cancer (PC) remains unclear. This study aimed to determine the heterogeneity associated with RNRGs in PC. Single-cell RNA-seq was downloaded from the Gene Expression Omnibus database. Transcriptome profiling, somatic mutations, somatic copy-number alterations, and the clinical data of PC samples were downloaded from The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) databases. At single-cell resolution, we discovered that the regulated necrosis status of non-malignant cells, rather than that of malignant cells, was a significant contributor. Notably, immune cells with immunosuppressive functions displayed higher regulated necrotic activity among distinct immune cells and clusters with discernible regulated necrosis traits. PCs were divided into two prognostic groups using signatures associated with regulated necrosis. Cluster 1 showed low regulated necrosis activity and a higher survival rate, whereas cluster 2 showed significant regulated necrosis activity and a lower survival rate. The clustering of TCGA cohorts mirrored the variety of intertumoral regulated necrosis, while ICGC cohorts further confirmed the intricacy. Finally, we discovered that the candidate gene for regulated necrotic activity in PC may be A2ML1. Using a classifier that integrate single-cell and bulk RNA-seq, we identified the heterogeneity of RNRGs in PC. These results may deepen our understanding of RNRGs in PC, offer new perspectives for physicians to forecast prognoses, and help create more efficient and customized therapeutic approaches in the future.
The pigmented rice types, i.e., black, red, and purple rice, are highly nutritious substitutes for the ordinary white rice, providing compounds such as anthocyanins, flavonoids, and proanthocyanidins. These bioactive compounds confer antioxidant, anti-inflammatory, anti-cancer, anti-diabetic, and cardiovascular-protective effects, while also assisting in weight management, gut health, and metabolism. The genetic basis of pigmentation includes regulatory genes like OsC1, OsDFR, and OsMYB, which comprise the MYB-bHLH-WD40 complex regulating anthocyanin biosynthesis. Anthocyanin pathways interact with flavonoid and proanthocyanidin synthesis, which is essential for colouration and stress adaptation. The environmental factors, such as temperature, pH, and storage conditions, cause degradation of anthocyanins. Acylation and encapsulation are techniques used in preserving anthocyanins better for industrial use. However, degradation of anthocyanins remains unexplored. Emerging evidence shows that degradation is not only chemical but also enzymatically regulated, mediated by polyphenol oxidases, peroxidases, and β-glucosidases, which accelerate oxidation and hydrolysis reactions and contribute to organ-specific pigment loss. Studies confirm first-order kinetics of anthocyanin breakdown under heat, alkaline pH, and oxygen, while stability improves with encapsulation, co-pigmentation, and gamma irradiation. This review explores the pigmentation of rice (Oryza sativa L.), focusing on its respective health benefits, genetics, synthesis, and degradation. Additionally, much remains to be discovered about its genetic and molecular basis. The regulation of vacuolar enzymes and degradation-related genes in rice tissues is still poorly understood, representing a major knowledge gap compared with biosynthesis.
Plant viruses inflict annual economic losses exceeding $30 billion and pose a significant threat to global food security. Discovering reliable antiviral targets is therefore essential to developing effective agents that can substantially reduce these agricultural losses. Despite ongoing efforts, the rapid discovery of such antiviral targets remains a significant challenge. This study employs an accelerated framework that integrates a machine learning-driven (MLD) approach with bioinformatics and in silico chemical screening to rapidly predict essential plant viral genes and validate novel antiviral targets. A MLD web-based prediction tool, Vgep, was developed to predict the viral essential gene. Subsequent phylogenetic, structural, and target-likeness analyses assessed the targetability of the essential protein it encodes. Finally, in silico virtual screening, biological activity evaluation, virus morphology observation, gene expression analysis, and molecular simulations were employed to identify a chemical probe for evaluating the druggability of the essential protein. Using the created Vgep tool, we predicted viral essential genes and identified the viral helicase as a key target in tobamoviruses. Viral helicase reveals high conservation and similarity to benchmark antiviral targets. The chemical probe Amidoca, identified through virtual screening exhibits strong binding affinity (Kd = 4.59 µM) to tobacco mosaic virus helicase. In antiviral bioassays, Amidoca outperformed Ribavirin, exhibiting EC50 values of 155.85 mg/L (inactive), 244.32 mg/L (curative), and 344.59 mg/L (protective), thereby confirming the druggability of the tobamovirus helicase. Mechanistic studies revealed that Amidoca may competitively bind at the helicase's NTP-binding site. This interaction may interfere with the energy release required for unwinding viral dsRNA and lead to a dramatic reduction in helicase accumulation by nearly 95%. This work establishes a systematic framework for the rapid discovery and validation of next-generation targets in plant virus therapies, highlighting RNA helicase as a promising antiviral candidate and advancing antiviral agent development efforts.
Here, we review the history, advancements, and broad utility of the NTR/prodrug system, and suggest future strategies for developing versatile ablation models. As a chemogenetic tool, the nitroreductase (NTR)/prodrug system enables precise spatiotemporal control over cell ablation. The technology leverages bacterial NTR enzymes (e.g. nfsB) to convert inert prodrugs into cytotoxic agents, thereby allowing researchers to induce targeted cell death. Although the NTR/prodrug approach was first implemented in transgenic mice, it was subsequently adapted to zebrafish, where it has been extensively optimized and applied. Consequently, zebrafish remain the primary focus of this review. Nevertheless, the utility of the NTR/prodrug system has expanded to other important model organisms, including Drosophila, Nematostella, Xenopus, medaka, and rats, enabling detailed studies of tissue damage and regeneration. This review highlights how the NTR system has been deployed to model a spectrum of human diseases, including Parkinson's disease, retinal degeneration, demyelinating disorders, and kidney disease. These models provide valuable platforms to study pathogenesis in vivo. Furthermore, the precise and controllable nature of NTR ablation makes it an ideal tool for high-throughput chemical and genetic screens aimed at discovering pro-regenerative and protective compounds. The development of NTR2.0, an enzyme variant with over 100-fold greater activity, along with more potent prodrugs such as ronidazole (RNZ), has dramatically broadened experimental possibilities. These improvements permit chronic ablation and long-term disease modeling at well-tolerated drug concentrations. Here, we present some key considerations, including transgenic design for optimal cell-type specificity, calibrating expression levels for desired ablation kinetics, and suitable controls to allow interpretation. These best practices will allow the researcher to develop a precise, reproducible, and versatile platform for either modeling human disease or dissecting regenerative mechanisms.
Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia/lymphoma (ATLL), but the full scope of its oncogenic mechanisms remains elusive. While the viral oncoprotein Tax drives early oncogenesis, its expression is frequently silenced in later stages of ATLL, whereas the HTLV-1 basic leucine zipper factor (HBZ) is constitutively expressed throughout infection. This shifting viral expression profile underscores the critical need to define HBZ-specific oncogenic mechanisms. Here, we identify miR-155 as a key oncogenic driver in ATLL and reveal a novel, HBZ-dependent post-transcriptional regulatory axis that sustains its overexpression. We confirm Tax-mediated transcriptional activation of miR-155 but further demonstrate that HBZ enhances miR-155 maturation by elevating Dicer expression and promoting its processing of pre-miR-155. The elevated miR-155 promotes tumorigenesis through targeting PTEN and activating PI3K-Akt pathway. Functional studies demonstrate that miR-155 overexpression drives ATLL progression by enhancing proliferation and inhibiting apoptosis, while its inhibition reverses these malignant phenotypes. Xenograft models confirm that miR-155 blockade significantly reduces tumor growth. Our results uncover a cooperative mechanism by which HTLV-1 Tax and HBZ jointly drive miR-155 overexpression to promote leukemogenesis and identify miR-155 as a potential therapeutic target in ATLL. Adult T-cell leukemia/lymphoma (ATLL) is an aggressive cancer caused by HTLV-1 with limited treatment options. We show that HTLV-1 hijacks the host microRNA miR-155 to drive tumor growth. While the viral protein Tax activates miR-155 transcription, we discovered that HBZ-constitutively expressed even when Tax is silenced-sustains miR-155 expression by upregulating Dicer and enhancing miR-155 processing. Elevated miR-155 then suppresses PTEN and activates the PI3K-Akt pathway, promoting cancer cell proliferation. Importantly, blocking miR-155 significantly reduces tumor growth in mouse models, identifying miR-155 as a promising therapeutic target for ATLL.
Diterpenoids are important natural secondary metabolites characterized by a 20-carbon backbone and classified into acyclic, monocyclic, bicyclic, tricyclic, tetracyclic, and macrocyclic types based on their ring structures. Abundant in medicinal plants, these compounds function in defense against pathogens and herbivores and exhibit diverse pharmacological activities, including anticancer, anti-inflammatory, cardiovascular-protective, antidiabetic, and antimicrobial effects. The biosynthetic precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) is primarily generated through the methylerythritol 4-phosphate (MEP) pathway in plants. The carbon skeleton of diterpenoids is then constructed by diterpene synthases (diTPSs) and undergoes diverse modifications catalyzed by cytochrome P450 monooxygenases (CYP450s), alcohol dehydrogenases (ADHs), and UDP-dependent glycosyltransferases (UGTs), while transcription factors orchestrate pathway gene expression. Advances in synthetic biology and metabolic engineering have enabled heterologous production of diterpenoids in genetically tractable hosts, with yields enhanced through promoter optimization, targeted mutagenesis, and fermentation optimization. This review systematically summarizes the structure, distribution, biological activities, and biosynthetic pathways of diterpenoids, and discusses future directions including structural modification for enhanced bioactivity, pathway elucidation via omics analysis, modular biosynthesis for overproduction, and machine learning applications in pharmacology and enzyme engineering. These efforts provide a foundation for discovering novel bioactive diterpenoids, elucidating complete biosynthetic pathways, and enabling sustainable production through biotechnological breeding and synthetic biology.
Scientific models are widely used across the natural sciences as an interface between scientific theories and empirical data [1]. Such models play a key role, for example, in the study of human and animal learning, where they express algorithmic hypotheses and relate them to psychology and neuroscience data [2, 3]. These models are traditionally handcrafted by expert researchers based on existing theory or new insights. Such handcrafted models, however, are now known to fall short of capturing the full richness of behavior, even in their narrow domains [4-7]. An alternative data-driven approach has emerged, seeking to discover new insights by fitting and interpreting flexible models [8-11]. However, these tools require substantial human effort to derive insight from data, and it has been unclear how to discover new ideas from data efficiently. Here, we present DataDIVER, a general approach for automatically discovering computational models from data, and demonstrate that these models surface novel mechanistic insights into human and animal learning. Our approach delivers models that take the form of short computer programs, which are optimized both to fit data well and to be simple. These programs explicitly connect with existing theoretical frameworks and are readily understandable by human scientists. They can also be used to make novel predictions, some of which we show are borne out in re-analysis of existing data. General-purpose tools for surfacing new ideas from data, especially in combination with the large datasets that are increasingly available in many fields, stand to dramatically accelerate scientific discovery.
Acetobacter indonesiensis UNPADCC 01-5 is a recently discovered acetic acid bacterium isolated from fermented food, oncom merah. With its current lack of polyphasic approach in classification, this study aims to provide the first morphological, physiological, biochemical and molecular analyses. These analyses are achieved by morphological and physiological characterization, Kirby-Bauer antibiotic susceptibility, biochemical profiling, gas chromatography-mass spectrometry and whole-genome sequencing (WGS). The results found that A. indonesiensis UNPADCC 01-5, a non-motile cell, is a gram-negative, short rod-shaped cell that formed cream-white colonies with 0.3-1.3 in diameter. It exhibited environmental resilience, including acid-tolerant, halotolerant and thermotolerant. Against antibiotics, it showed resistance towards chloramphenicol. Biochemical tests revealed its ability to ferment glucose, rhamnose, melibiose and arabinose, as well as to produce bioactive metabolites such as acetoin and acetic acid. WGS identified potential gene expressions associated with acetic acid production, stress tolerance, nitrogen fixation, hydrocarbon degradation and heavy-metal resistance. WGS analysis revealed no detectable genes associated with human virulence factors or pathogenic secretion systems. The results suggested that A. indonesiensis UNPADCC 01-5 from oncom merah possessed adaptive traits which, with future assessments and industrial-scale tests, hold potential as a functional/adjunct culture for acidic fermentations and a biofunctional agent.
Developmental biology seeks to understand how multicellular organization emerges from cell-cell interactions. Advances in stem cell and synthetic biology now enable researchers to rebuild developmental processes outside the embryo, with varying degrees of resemblance to natural systems. While some reconstituted systems reveal how development occurs, others uncover what is possible. This perspective examines how such bottom-up approaches have elucidated general principles and causal mechanisms of multicellular organization. We argue that synthetic systems, though simplified, provide powerful platforms to test the limits of developmental potential, disentangle causal relationships, and inform predictive models. With rapid advances in genomic engineering, imaging, and computational modeling, leveraging these engineered systems to discover what is possible holds transformative promise for understanding what is happening in nature.
Natural products (NPs) remain vital to drug discovery, yet the identification of novel bioactive NPs is frequently hampered by the rediscovery of known compounds in traditional screening and the "bioactivity gap" in genome mining. Self-resistance-guided genome mining has emerged as a transformative strategy to address these challenges, leveraging co-localized resistance genes within biosynthetic gene clusters (BGCs) to predict NPs' molecular targets. This review summarizes recent progress in discovering novel NPs that target essential cellular processes, including protein synthesis, protein degradation, DNA integrity, and primary metabolism. We further highlight key technologies and strategies designed to accelerate this discovery workflow and discuss the limitations and opportunities of self-resistance-guided genome mining for the systematic discovery of precision therapeutics in the genomic era.
Mesenteric neurofibromatosis, a rare complication of neurofibromatosis type 1 (NF-1), comprises benign plexiform neurofibromas with recognized potential for malignant transformation. These lesions are frequently asymptomatic and diagnostically elusive, mimicking more common gastrointestinal pathologies. We report the incidental intraoperative discovery of mesenteric neurofibromatosis during surgery for acute appendicitis in an NF-1 patient, a presentation scarcely documented previously. This case highlights the occult nature of gastrointestinal involvement in NF-1 and underscores the critical need for systematic imaging surveillance to enable early detection and intervention. A 17-year-old female presented with a one-week history of right lower quadrant pain. An abdominal CT scan was consistent with acute appendicitis. Physical examination revealed numerous café-au-lait macules, and a significant family history confirmed a concurrent diagnosis of neurofibromatosis type 1 (NF-1). During a scheduled laparoscopic appendectomy, diffuse nodular lesions were incidentally discovered throughout the small bowel mesentery. Histopathological analysis of a biopsied lesion confirmed mesenteric neurofibromatosis. The patient had an uncomplicated postoperative recovery and remained asymptomatic at three-month follow-up. These findings underscore the necessity for imaging-based screening to detect gastrointestinal lesions early in patients with neurofibromatosis type 1 (NF-1). Heightened awareness of the potential presence of mesenteric neurofibromatosis, a rare manifestation of NF-1, is particularly warranted in the surgical setting when abdominal procedures are indicated for these patients.
Breastfeeding counseling is an effective public health intervention. However, there is a scarcity of studies detailing protocols, especially those applied during the dyad's stay in the Rooming-In Unit. Thus, this research aimed to describe a breastfeeding counseling protocol during the dyad's hospitalization in Rooming-in. A methodological development study was carried out from June to September 2022 in four stages: discovering from a bibliographical survey; defining the target audience, contexts, and answers to the research question; developing from the construction of the instrument; and delivering the final product after content validation by eight experts. The intervention components protocol includes items necessary for execution; intervention script; breastfeeding assessment instrument; adverse situations, solutions, and initial difficulties in breastfeeding; and counseling approach in the face of situations. For each item of the protocol, a Likert scale was applied, and the Content Validity Index was calculated to verify the agreement between the experts. Items with agreement above 80% were considered valid. Content validation was performed in a single evaluation round. The final version of the protocol consisted of 11 sections. It describes in detail the necessary conditions, expected and unexpected procedures, and how to intervene using the counseling approach in the initial difficulties presented during the hospitalization of the dyad in Rooming-in.
Hypoxic-ischemic brain damage (HIBD) is a major contributor to neurological disabilities. Our previous studies have demonstrated that DL-3-n-butylphthalide (NBP) confers protective effects against HIBD and mitigates blood-brain barrier (BBB) impairment, but the underlying mechanism remains unclear. This study aimed to investigate the mechanism by which NBP protects against ferroptosis-induced BBB damage. Our results showed that NBP treatment significantly reduced infarct volume (by 24.09%), alleviated cerebral edema (brain water content decreased by 1.651%), attenuated BBB disruption, suppressed neuroinflammation, promoted neuronal repair, and improved functional recovery. Using RNA sequencing, molecular docking, and surface plasmon resonance (SPR) assays, we discovered for the first time that NBP directly binds to membrane-bound SLC7A11 with a KD value of 222.0 µM, thereby activating the SLC7A11/GSH/GPX4 pathway and inhibiting ferroptosis in cerebrovascular endothelial cells. Different from previously reported anti-apoptotic and anti-inflammatory mechanisms of NBP, the present study reveals a novel mechanism by which NBP protects the BBB through inhibiting endothelial ferroptosis. The protective effect of NBP was further validated by knocking down SLC7A11 with specific siRNA, confirming its dependence on SLC7A11. Ultimately, NBP ameliorated BBB damage both in vitro and in vivo. In summary, NBP attenuates BBB disruption by targeting the SLC7A11/GSH/GPX4 pathway and inhibiting endothelial ferroptosis, thereby reducing brain injury and promoting neural recovery.
Individual microbes often respond differently to the same environment, yet the magnitude of such niche variation inherent to individuals remains unresolved and is anticipated to differ substantially from community-level average responses. We conducted metagenomic binning on monthly time-series soil samples from three sites across seasonal cycles. By considering 440,571 genes as dimensions of the fundamental individualised niche (FIN), we traced FIN trajectories of archaea and bacteria during warming, cooling, and turning periods. We found that neither mean temperature nor temperature difference had a significant effect on FIN breadth or overlap. Instead, we discovered a temporally constant, stepwise gradient of niche differentiation across taxonomic categories. At the interdomain level (Archaea vs. Bacteria), niche overlap is approximately 25%, rising to ~40% at the interphylum level and ~60% at the interorder level. This discontinuous gradient likely marks the limit boundaries of niche variation, is closely linked to functional synergy within FINs, and provides a preliminary comparable ecological carrying capacity for each niche step, particularly regarding the interdomain balance.
Cardiac contractility requires precise regulation. A recently discovered form of regulation of cardiac contractility involves a β-cardiac myosin off-state, the Interacting-Heads Motif (CarIHM). Despite its central role in cardiac physiology and disease, CarIHM structural dynamics remain poorly understood. Here, we integrate near-atomic resolution cryo-EM with all-atom molecular dynamics to characterize CarIHM in solution and in the context of the filament. We describe its conformational ensembles maintained by dynamic interfaces, and the stabilizing effect of the dilated cardiomyopathy mutation E525K, which limits S2 coiled-coil flexibility and impairs myosin activation. Intrinsically disordered regions of CarIHM and MyBP-C further modulate these dynamics. Our findings provide evidence for how CarIHM ensembles balance off/on states and anchor myosin heads in distinct thick filament environments. This integrated structural and dynamic approach enhances the understanding of thick filament regulation and facilitates predictions of the effects of genetic variants in inherited cardiomyopathies.
Extrachromosomal circular DNAs (eccDNAs) are well-established drivers of tumorigenesis, yet their landscape and functional significance in human heart failure (HF) remain largely unknown. Using Tn5 transposase-based sequencing, we comprehensively profiled plasma eccDNAs from healthy controls and HF patients with myocardial ischemia (MI-HF) or non-myocardial ischemia (NMI-HF). Bioinformatics was employed to probe their chromosomal origins, genomic features, and potential roles in the development of HF. Circular structures of candidate eccDNAs were validated by inward and outward PCR followed by Sanger sequencing. Their clinical prognostic value was assessed by Kaplan-Meier and Cox regression analyses in a patient cohort. Most plasma eccDNAs were shorter than 1 kb and originated from all chromosomes, with selective enrichment from specific genomic regions including 5' UTRs, CpG islands, and Alu elements. Characteristic nucleotide repeats were identified at eccDNA junction sites. The circular structure of eccDNAs was confirmed. Furthermore, we discovered that the eccDNA ENPP1circle exon 25 was specifically detected in MI-HF patients. Its presence was significantly associated with a higher incidence of major adverse cardiac events (MACEs), and it served as an independent prognostic biomarker in multivariate analysis. This study delineates the first detailed landscape of plasma eccDNAs in HF and reveals their potential as noninvasive biomarkers for risk stratification. Our findings lay a crucial foundation for future research into eccDNA biology and their translational applications in cardiovascular disease.
While Trastuzumab emtansine (T-DM1) and other HER2-targeting antibody-drug conjugates (ADCs) are used to treat cancer patients with HER2-amplified tumors, there is a need to improve the efficacy through the understanding of their mechanism of uptake into cells. Flotillin-2 (FLOT2) regulates the internalization of epidermal growth factor receptor (EGFR), leading us to investigate FLOT2 effects on HER2 internalization. Higher FLOT2 expression in nine HER2 amplified cell lines correlated with a higher T-DM1 IC50 in vitro , and breast cancer patients with high FLOT2 expression had worse survival when receiving either T-DXd (16.2 months (m) vs 18.3 m, p=0.04) or T-DM1 (38.0 m vs 41.3 m, p=0.1) in real-world Caris Life Sciences data. FLOT2 interacts with HER2 and positively regulates HER2 activation and downstream signaling, while FLOT2 knockdown reduces the viability of HER2 amplified cancer cells. FLOT2 knockdown results in increased HER2 internalization upon binding of T-DM1, mediated by ubiquitination by the Cbl ubiquitin ligases. We investigated the effects of various small molecules and discovered that zoledronic acid binds to FLOT2 and disrupts the HER2/FLOT2 interaction, which enhances T-DM1 internalization and cytotoxicity. In conclusion, FLOT2 regulates the internalization and cytotoxicity of T-DM1 mediated by Cbl-dependent ubiquitination of HER2. Zoledronic acid disrupts the HER2/FLOT2 interaction, therefore increasing the internalization and cytotoxicity of T-DM1, providing proof of principle that a small molecule inhibitor of the HER2/FLOT2 interaction can enhance the activity of the HER2-targeting ADC.