Biology subjects

Zheng, L.

Publications and source records attributed to Zheng, L..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Deep learning-guided design of hydrolases for crystalline PET depolymerization

Poly(ethylene terephthalate) (PET), a ubiquitous polyester used in packaging and textiles, persists in the environment due to its high crystallinity and stability, contributing substantially to global plastic pollution. Enzymatic depolymerization by PET hydrolase (PETase) offers a chemically precise and environmentally sustainable route to convert PET into its monomeric building blocks, enabling recycling. However, practical implementation remains hindered by the rigid, crystalline architecture of PET, which restricts enzyme access and necessitates energy-intensive pretreatment to enable efficient depolymerization. In addition, most PETases achieve only partial depolymerization with low terephthalic acid (TPA) yields and accumulate inhibitory intermediates, while limited thermostability and slow surface kinetics further restrict efficiency. To overcome these barriers, we report VenusPETase, an engineered variant of KbPETase designed using PET-Flow, a state-of-the-art computational framework for PETase engineering. Compared with KbPETase, VenusPETase exhibits a 3.4-fold increase in hydrolytic activity, up to a 27-fold improvement after heat treatment of protein, and a 12 {degrees}C enhancement in thermostability. VenusPETase exhibits rapid degradation across a wide crystallinity range (8%-50%) at 50 {degrees}C, effectively spanning the entire spectrum of commercial PET products. Moreover, VenusPETase outperformed nine high-performance PETases under their respective optimal conditions and degraded untreated PET substrates across 8%-50% crystallinity, producing TPA as over 95% of the released products with minimal intermediate accumulation. X-ray crystallography and molecular dynamics simulations suggest that dynamic modulation, elevated surface electrostatic potential enhance the interaction of VenusPETase with crystalline PET, thereby lowering the hydrolytic energy barrier and improving catalytic performance. We also demonstrate that untreated, postconsumer-PET from nine different products can all be degraded by VenusPETase. The recovered monomers can be directly repolymerized into virgin-quality PET, demonstrating a closed-loop enzymatic recycling process. In a 100 L bioreactor, VenusPETase completely depolymerizes post-consumer crystalline PET (28% crystallinity) within 24 h under 50 {degrees}C. These results establish VenusPETase as a robust biocatalyst that enables efficient, closed-loop recycling of crystalline PET under mild conditions.

biochemistry↗

bioRxiv2026

The in vivo inhibitory function of the MHC-I alpha3 domain/CD8alpha interaction

The interaction between the Major Histocompatibility Complex Class I (MHC-I) 3 domain and CD8 has classically been viewed as a positive coreceptor interaction that stabilizes TCR signaling during antigen recognition. However, its physiological function in mature peripheral CD8+ T cells in vivo remains incompletely understood. Here, we identify the MHC-I 3 domain-CD8 interaction as a previously unrecognized inhibitory pathway that tonically restrains peripheral CD8+ T-cell activation and maintains T-cell tolerance in vivo. Antibody-mediated disruption of the MHC-I 3 domain-CD8 interaction induced spontaneous activation of peripheral CD8+ T cells without impairing their survival, lowered the threshold for antigen-induced activation, and enhanced responsiveness to cognate peptide stimulation. In peptide-induced OT-I T-cell anergy models, blockade of either H-2Db or H-2Kb 3 domain interactions with CD8 prevented the induction of anergy and restored responsiveness of previously anergic T cells. Notably, blockade of the H-2Kb 3 domain enhanced OT-I responses despite simultaneously disrupting the classical positive coreceptor interaction within the TCR-peptide-MHC complex, indicating that tonic inhibitory signaling mediated by the MHC-I 3 domain predominates under these conditions. Together, these findings redefine the classical MHC-I-CD8 interaction as a bidirectional pathway that not only supports antigen recognition but also imposes tonic inhibitory control over peripheral CD8+ T cells. These results identify the MHC-I 3 domain-CD8 axis as a potential target for reversing T-cell tolerance and enhancing antitumor or antiviral immunity.

immunology↗

bioRxiv2026

Simultaneous brain-wide single-cell recording resolves spatiotemporal memory architecture

Many fundamental mammalian brain functions emerge from the coordinated activity of cells distributed across large, brain-wide networks. To understand these processes in healthy and diseased states, ideally one would simultaneously measure and analyze single-cell activity at the brain-wide scale, an enduring challenge for live-measurement approaches that often face an inherent tradeoff between spatial resolution and scale. Here, we present GLOBE (sinGle-cell spatiotemporaL recOrding Brain-widE), a technology for brain-wide single-cell recording of cellular activity in vivo with spatiotemporal resolution, physiological sensitivity, and parallelization-accelerated readout. GLOBE leverages genetically encoded intracellular protein tape recorders and a high-throughput computational platform for integrated image and signal analysis. GLOBE records analog signal amplitudes across a continuous time axis, requires only standard light microscopy for in situ readout, and is compatible with expansion microscopy and RNA readouts. We applied GLOBE to simultaneously record transcriptional activity of the immediate early gene Fos in up to 219,703 neurons simultaneously across a single mouse brain over 5.5 continuous days, with a timestamp precision of 3.1-6.7 hours (median absolute error), a local recording density of 69-90% of neurons per imaging field of view, and a post-mortem imaging readout speed of 2.9 seconds per neuron on average. GLOBE resolves the brain-wide spatiotemporal structure of single-cell activity, revealing that Fos transcriptional dynamics associated with fear learning and memory retrieval are distributed across the brain with region-specific temporal heterogeneity, and that the variance of this structure scales down as the number of sampled cells increases. We envision GLOBE to have broad applications for dissecting and decoding physiological and pathological processes at the brain-wide scale.

neuroscience↗

bioRxiv2026

Large-domain histology-based diffusion MRI simulation via independent local simulations

Diffusion MRI simulations based on realistic tissue microstructure provide a means to validate biophysical models and optimize acquisition protocols, but their computational cost restricts most studies to domains far smaller than a clinical voxel. The objective of this study was to develop an automated and scalable framework that converts whole-slide histology into diffusion MRI simulations at clinically relevant spatial scales while remaining feasible on standard workstation hardware. We present an end-to-end pipeline integrating two-dimensional whole-slide cell segmentation, mesh generation, and finite element Bloch-Torrey simulation. To enable simulations at large spatial scales without prohibitive memory growth, we introduce a subdomain tiling strategy in which the tissue domain is partitioned into extended subdomains simulated independently under no-flux boundary conditions. Signals are aggregated only from the central regions of each subdomain to minimize boundary artifacts. For an 800 {micro}m x 800 {micro}m histology-based domain, the aggregated signal differed by 0.07% from the corresponding full-domain finite element simulation while reducing wall-clock time from several days to hours and maintaining bounded memory usage independent of global domain size. When applied to a 2016 {micro}m x 2016 {micro}m heterogeneous region approximating the in-plane dimensions of a clinical voxel, the apparent diffusion coefficient obtained from the full domain differed from values computed in smaller dense and sparse subregions, demonstrating the influence of structural heterogeneity at clinically relevant scales on derived diffusion metrics. The proposed framework establishes an automated and memory-stable approach for generating diffusion MRI simulations directly from routine histology.

biophysics↗

bioRxiv2026

Molecular Structure and DNA Binding Mode of Unsymmetric Cyanine Dyes RiboGreen and OliGreen

The binding of fluorescent dyes to nucleic acids and their fluorogenic properties are indispensable tools for nucleic acid detection, quantification, and imaging, yet the molecular structures of several widely used commercial dyes have remained unknown. Here, we de novo determined the molecular structures of RiboGreen and OliGreen and confirmed the previously proposed structure of PicoGreen using high-field NMR spectroscopy and ultra-high resolution mass spectrometry. All three dyes were identified as unsymmetric cyanine dyes, where benzazolium and 4-quinolinium moieties are linked by a monomethine bridge. Complete 1H and 13C resonance assignments enabled us to expand the existing chemical shift reference set for this important class of dyes. Photophysical characterization with standardized single- and double-stranded DNA and RNA targets indicated that all dyes performed similarly upon binding despite being marketed towards different nucleic acid types. NMR spectroscopy and long-timescale molecular dynamics simulations showed that RiboGreen interacts with double-stranded DNA predominantly by two binding modes, electrostatic interactions with the phosphodiester backbone and {pi}-{pi} stacking with accessible nucleobases of the DNA molecule. These results establish the molecular structures of three widely used commercial dyes and provide a structural and mechanistic framework for understanding the fluorogenic properties of this class of dyes. HighlightsO_LIDetermination of the molecular structures of nucleic acid dyes RiboGreen, OliGreen, and PicoGreen C_LIO_LINMR spectroscopic characterization of all three dyes. C_LIO_LINMR and MD data indicate binding to be dominated by electrostatic and {pi}-{pi} stacking interactions C_LI

molecular biology↗

bioRxiv2026

PARP1-mediated 5' flap dynamics facilitate Okazaki fragment maturation

Abstract/SummaryPrecise regulation of enzyme recruitment during Okazaki fragment maturation (OFM) is essential for faithful and efficient lagging-strand DNA synthesis. Emerging evidence suggests that PARP1 contributes to OFM yet its specific functions remain unclear. Here, we define context-dependent functions of PARP1 during OFM. Under physiological conditions, PARP1 co-localizes with PCNA in early S phase and restrains Pol {delta}-PCNA- mediated strand-displacement DNA synthesis, thereby preventing the formation of long 5' flaps, which is refractory to FEN1 cleavage. On the other hand, in LIG1-deficient cells, in which DNA nicks and unexpectedly long 5' flaps accumulate, PARP1 promotes the recruitment of LIG3 to catalyze OF ligation and DNA2 to facilitate long 5' flap processing. Collectively, our findings uncover previously unrecognized roles of PARP1 in regulating 5' flap dynamics to ensure efficient OFM and cell viability. HighlightsO_LIPARP1 plays context-dependent regulatory functions in Okazaki fragment maturation (OFM). C_LIO_LIPARP1 controls strand displacement DNA synthesis by the PCNA-Pol{delta} complex to dictate generation of short over long RNA-DNA flaps during canonic OFM. C_LIO_LIPARP1 senses unligated Okazaki fragments in DNA Ligase 1 deficient cells and suppresses unwanted conversion of DNA nicks into 5 flaps. C_LIO_LIProcessing of unligated nicks or flaps by DNA ligase 3 or DNA2, respectively in LIG1 deficient cells depends on PARP1. C_LIO_LIPARP1 inhibitors induce synthetic lethality with DNA ligase 1 or DNA2 inhibition. C_LI

molecular biology↗

bioRxiv2026

Pyruvate-driven hydrogen production promotes polyphenol bioconversion by gut bacteria

Gut microbial biotransformation of poorly absorbable polyphenols into bioactive, bioavailable metabolites is increasingly recognized as a key mechanism underlying their health benefits of polyphenols. Microbial ellagic acid (EA)-to-urolithin conversion represents a typical example, but the environmental factors that facilitate such metabolism remain underexplored. We discovered that urolithin production by a gut commensal bacterium, Gordonibacter urolithinfaciens (G. uro), is metabolically repressed by arginine. To overcome such limitations, we developed PhenolBoost Medium (PBM) that induces a metabolic shift by suppressing the arginine deiminase pathway while activating pyruvate metabolism and hydrogen production in G. uro, thereby driving urolithin dehydroxylation. Transcriptomic profiling and 13C-isotopic tracing analysis revealed that pyruvate metabolism in PBM upregulates hydrogenase expression, facilitating the dehydroxylation of EA. PBM also promoted the complete conversion of EA to urolithin A in G. uro-Enterocloster bolteae co-culture, and other polyphenol biotransformations. In addition, co-culturing G. uro with hydrogen-producing Bacteroides species significantly increased urolithin production. Furthermore, an arginine-limited, pyruvate-enriched dietary regimen proved effective in vivo, resulting in significantly higher urolithin production and bioavailability in a mouse model. Our findings reveal the critical role of hydrogen in facilitating polyphenol dehydroxylation, and offer a viable nutritional strategy for boosting microbial production of beneficial metabolites from polyphenols.

microbiology↗

bioRxiv2026

Ketogenic diet synergistic reprogramming of both host and microbiome promotes tissue regeneration

Nutrition influences host physiological processes, yet how diets reshape host physiology, microbial functions, or host-microbe interactions to promote regeneration remains poorly explored. Here, we show that a ketogenic diet (KD), enriched in fats and low in carbohydrates, reprograms both skin microbial and immune functions to promote tissue repair. KD enhances IL-17A activity in {gamma}{delta} T cells and mucosal-associated invariant T (MAIT) cells, accelerating tissue repair, while KD-induced skin lipidomic alterations enhance both the abundance and metabolic output of Staphylococcus epidermidis. Metatranscriptomic and lipidomic analyses revealed increased riboflavin biosynthesis and sphingomyelinase (Sph)-dependent ceramide production in S. epidermidis under KD conditions. Genetic depletion of microbial ribD, a key enzyme for riboflavin biosynthesis, or of sph compromised the ability of the bacteria to promote tissue repair. Thus, host nutritional status drives tissue regeneration by synergistically rewiring host and microbial functions, providing new insights into how diet can be harnessed to regulate host physiology.

immunology↗

bioRxiv2026

Towards a Cytometry Foundation Model: Interpretable Sample-level Predictive Modelling via Pretrained Transformers

Foundation models have transformed scientific data modelling across domains, yet flow cytometry has lacked one. Despite the abundance of high-dimensional cellular data, automated analysis remains bottlenecked by marker variability: prior studies are typically confined to fixed marker panels and homogeneous data, limiting scalability and generalisation due to architectural constraints. We present the Generalised Pretrained Cytometry Transformer (GPCT), an interpretable framework designed to learn from heterogeneous marker panels for sample-level predictive modelling. Through a novel cytometry-specific pretraining regime, GPCT learns transferable cellular representations that achieve high classification accuracy across diverse datasets. Notably, pretraining significantly boosts performance on data-scarce downstream tasks, marking a pivotal step towards a cytometry foundation model. Furthermore, GPCT maintains interpretability and identifies the specific cell subsets most influential to its predictions. This enables direct biological validation of learned patterns and provides a data-driven basis for refining traditional gating strategies.

bioinformatics↗

bioRxiv2026

APEX1 Protects Cardiomyocytes Against Oxidative Stress by Regulating p53 Protein Stability

Withdrawal statementThe authors have withdrawn this manuscript because the preprint was uploaded to bioRxiv before all co-authors had been fully informed and had provided their explicit consent to make the manuscript publicly available as a preprint. Following internal discussions, the authors have reached a consensus that they do not wish this version to be publicly available. We therefore kindly request that this preprint be withdrawn on the bioRxiv platform. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

molecular biology↗

bioRxiv2026

Introducing a fusogenicity metric for lipid nanoparticle formulation

Lipid nanoparticles (LNPs) are the most successful drug delivery carrier to date, but optimizing lipid formulations to improve membrane fusion capabilities for effective drug release has been challenging due to lack of a quantitative measure for fusogenicity. Here we introduce a new framework based on small angle X-ray scattering to experimentally measure [Formula] for lipids used in LNP formulations such as glycerol monooleate (GMO) and ionizable lipids (SM-102 and ALC-0315). Q intrinsically captures spontaneous curvature (J0), which is traditionally used to assess fusogenicity. The change of cubic lattice parameters with temperature was measured for GMO-containing lipid mixtures, and the Q extracted quantitatively correlated with LNP fusogenicity power validated by fluorescence-based fusion assays and cryogenic electron microscopy. Fusogenicity of SM-102 and ALC-0315 was quantified by adding them to host membranes and assessing change in Q. This framework provides researchers with the ability to optimize the fusogenicity of LNP formulations for potent drug release and enhances understanding of parameters governing fusion in all biomembranes.

biophysics↗

bioRxiv2026

Plasma regulates homeostatic pulmonary endothelial signaling to mitigate vascular leak following polytrauma and hemorrhagic shock

IntroductionIn hemorrhagic shock, plasma resuscitation preserves vascular integrity and protects against trauma-induced coagulopathy and organ injury. Despite demonstrated clinical benefit, the endothelial mechanisms underlying plasma resuscitation remain incompletely defined. This study investigated endothelial-specific responses to plasma resuscitation to identify targetable pathways that promote vascular repair after traumatic injury. MethodsA murine model of severe polytrauma-hemorrhagic shock (PT/HS) with demonstrable vascular endotheliopathy by 24 hours was used to compare pulmonary vascular endothelial cell (EC) responses to resuscitation with lactated Ringers (LR) relative to fresh frozen plasma (FFP). Whole blood was collected for inflammatory biomarker analysis, and pulmonary vascular leak was quantified by dextran extravasation. Pulmonary EC glycocalyx (eGC) structure was assessed by transmission electron microscopy and immunofluorescence. Spatial transcriptomic profiling of pulmonary ECs was performed using a GeoMx Digital Spatial Profiler. Key transcriptomic findings related to mitochondrial biogenesis were validated by immunostaining and by treating primary human lung EC with FFP or LR. ResultsAt 24 hours after injury, FFP reduced systemic inflammatory cytokines, pulmonary innate immune cell infiltration, and PT/HS-induced vascular leak compared to LR. Plasma levels of syndecan-1, syndecan-4, and hyaluronan were decreased, consistent with enhanced pulmonary eGC expression. Although few differences in eGC-related genes were detected, pathway analysis revealed enrichment of cellular bioenergetics and metabolic recovery pathways in ECs after FFP, whereas LR was associated with oxidative stress and inflammatory signaling. FFP enhanced mitochondrial content in pulmonary EC after PT/HS and in treated human EC compared to LR-treated controls. ConclusionsFFP resuscitation after PT/HS reduces systemic inflammation and preserves pulmonary vascular barrier function, potentially through promotion of mitochondrial signaling, metabolic recovery, and endothelial stress regulation.

cell biology↗

bioRxiv2026

Thermal Nano-Engineering of Ginger Extracellular Vesicles for Targeted Oral Therapy of Colitis

Plant-derived extracellular vesicles (PEVs) are promising candidates for oral drug delivery, yet their clinical translation is hindered by limited targeting precision and inconsistent systemic absorption. While surface engineering can enhance tissue accumulation, strategies that preserve biocompatibility and enable scalable production remain limited. Here, we introduce a simple thermal processing approach, boiling, to structurally reconfigure ginger EVs into functionally enhanced, thermally reassembled nanoparticles (B-GEVs). The surface architecture of B-GEVs is enriched with key vesicle trafficking regulators, including V-type proton ATPase subunit G, ARF1, and {beta}-adaptin-like protein. This specific composition drives their tissue-specific accumulation in the intestine and liver and potentiates clathrin-dependent cellular uptake in intestinal cells by 8.57-fold. Beyond superior intrinsic anti-inflammatory activity through NLRP3 inflammasome suppression, B-GEVs function as an efficient oral delivery platform. When loaded with TNF- siRNA, they enable a synergistic therapy that simultaneously modulates upstream inflammation and silences key downstream mediators, showing potent efficacy in colitis. Our findings position boiling as a natural strategy for enhancing the bioactivity and targeted oral delivery potential of ginger-derived EVs.

bioengineering↗

bioRxiv2026

The BELL-type homeobox transcription factor RLC3/OsBLH4 controls leaf rolling and drought tolerance via KNOX-BELL-lignin regulatory network in rice

O_LIModerate leaf rolling in rice is crucial for plant architecture and stress adaptation, but its molecular regulation remains unclear. We investigated the role of RLC3/OsBLH4, a BELL-type homeobox transcription factor, in controlling leaf rolling and drought tolerance, addressing gaps in lignin biosynthesis and cell wall development mechanisms. C_LIO_LIWe used gene map-based cloning (rlc3-1, rlc3-2), CRISPR/Cas9 knockout lines (rlc3-ko#11, rlc3-ko#12), and allelic complementation to validate RLC3s function. Additionally, we employed biochemical assays, gene expression analysis, and protein interaction studies to explore its regulatory network. C_LIO_LIRLC3 mutations impaired lignin biosynthesis and secondary cell wall formation, reducing bulliform cells area and causing midrib defects. These structural abnormalities accelerated water loss, leading to excessive leaf rolling and compromised drought tolerance. Mechanistically, RLC3 directly activates lignin synthesis genes (OsPAL5, OsCOMT5, OsCCR4, OsCAld5H1) and interacts with KNOX transcription factors (OSH1, OSH45, OSH71) to form a KNOX-BELL complex, further regulating lignin content and cell wall development. C_LIO_LIRLC3 orchestrates lignin deposition and secondary cell wall development to control leaf rolling, water transport, and drought tolerance. This study reveals a novel KNOX-BELL-lignin regulatory module governing leaf morphology and stress adaptation, offering targets for crop improvement under drought conditions. C_LI

plant biology↗

bioRxiv2026

m6A-dependent microRNA binding to chromatin-associated RNA for transcriptional activation

For decades, microRNAs (miRNAs) have been canonically viewed as post-transcriptional repressors. We discovered extensive binding of microRNAs to chromatin-associated RNAs (caRNAs) and uncovered an N6-methyladenosine (m6A)-dependent transcriptional activation mechanism of microRNAs. We show that m6A-binding proteins FXR1/2 anchor AGO1/2 at m6A-marked caRNAs, where AAGUGC-seed microRNAs function as guide RNAs to direct AGO positioning. This dual anchoring stabilizes the AGO-microRNA/FXR-m6A complex at specific loci, which in turn recruits the ATP-dependent chromatin remodeler SMARCA4 (BRG1) to promote local chromatin opening and TET1 for DNA demethylation, respectively. Together, these coordinated activities establish a transcriptionally permissive chromatin environment, enhancing accessibility and transcription across hundreds of genes in diverse cell types. Beyond the AAGUGC-seed family, additional microRNAs and siRNAs also enhance transcription, suggesting that caRNA binding and transcriptional activation may represent a broader property of small RNAs.

molecular biology↗

bioRxiv2026

Immunoregulatory gene GIMAP6 suppresses lethal atherosclerotic vasculopathy and ischemic heart failure

Controlling hyperlipidemia has reduced but not eliminated atherosclerotic cardiovascular disease as a predominant cause of human mortality. Here, we report that loss of the immunoregulatory gene GTPase of immunity-associated protein 6 (GIMAP6), causes an inflammatory vasculopathy and accelerated atherosclerosis in the absence of hyperlipidemia. These pathologic changes in turn result in progressive cardiac ischemia, myocardial infarction, and heart failure, culminating in early death. In humans, rare deleterious GIMAP6 variants are associated with premature severe cardiovascular disease. These findings reveal GIMAP6 to play an important protective role against atherosclerotic cardiovascular disease whose identification offers opportunities for improved risk management and a target for new therapies.

immunology↗

bioRxiv2026

Microsurgical Isolation and Molecular Characterization of the Mouse Left Internal Mammary Artery: Insights into Natural Resistance to Atherosclerosis

Atherosclerosis develops unevenly across the vascular tree, yet the molecular basis for this regional susceptibility remains poorly defined. The left internal mammary artery (LIMA), the most durable conduit for coronary artery bypass (CABG) surgery, is uniquely resistant to atherosclerosis in humans; however, it has never been isolated or studied in mouse models, limiting mechanistic insight into atheroprotective pathways. Here, we establish the first method to identify and isolate the murine LIMA and generate the first transcriptomic atlas of this artery. Cross-species analyses of human and mouse LIMA reveal a conserved protective signature that distinguishes the LIMA from atheroprone vessels and uncover fundamental molecular differences that govern vascular resilience. This study presents a transformative experimental platform for dissecting the determinants of atheroprotection and identifying molecular targets to improve CABG graft performance and long-term cardiovascular outcomes.

physiology↗

bioRxiv2026

The combination of BCL-xL PROTAC and mTOR inhibitor sensitizes pancreatic ductal adenocarcinoma to KRASG12D inhibitor treatment by enhancing apoptosis induction

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with a five-year survival rate of approximately 13%, partly because of limited treatment options and resistance to therapies. Although the recently discovered KRAS G12D inhibitor MRTX1133 has shown promise for PDAC treatment in preclinical studies, its clinical efficacy as a single agent is expected to be limited, as is the case with KRAS G12C inhibitors. Therefore, in this study, we evaluated potential combination strategies to enhance the therapeutic effect of MRTX1133. We rationally combined MRTX1133 with the BCL-xL proteolysis-targeting chimera (PROTAC) DT2216 and the mTOR inhibitor everolimus, which significantly potentiated the anti-tumor activity of MRTX1133 in multiple G12D-mutated PDAC cells in vitro by enhancing apoptosis induction. Mechanistically, MRTX1133 treatment increased BIM and decreased NOXA levels, while the combination of DT2216/everolimus was shown to simultaneously enhance BIM release and stabilize NOXA. In vivo, DT2216/everolimus combination significantly potentiated the anti-tumor activity of MRTX1133 in AsPC1 PDAC xenograft model. Furthermore, the triple combination of MRTX1133, DT2216, and everolimus effectively overcame acquired resistance to MRTX1133 in AsPC1 cells in vitro and in xenograft model. Collectively, our findings suggest that the single-agent efficacy of MRTX1133 is limited by apoptosis inhibition, and that its combination with DT2216/everolimus can enhance apoptosis and potentially overcome resistance in KRAS G12D-mutated PDAC. SIGNIFICANCEKRAS inhibitors, including KRAS G12D inhibitor MRTX1133, are promising therapeutics against KRAS-mutated pancreatic ductal adenocarcinoma (PDAC), but drug resistance limits their efficacy. Our study reveals that robust induction of apoptosis using a combination of BCL-xL PROTAC degrader and an mTOR inhibitor, significantly enhances MRTX1133 efficacy in PDAC models without increasing toxicity to normal tissues.

cancer biology↗