Molecular Therapy Oncology

Oncolytic viruses are most commonly administered via intratumoral injection; however, their clinical efficacy in achieving tumor eradication remains limited by several challenges, including insufficient penetration into all tumor cells and the inability to elicit robust systemic antitumor immune responses capable of eliminating metastatic microtumors. Here, we report an oncolytic adenovirus, OAd-2B6, with an engineered adenoviral E1 region for tumor selectivity and carrying the prodrug- activating enzyme cytochrome P450 2B6 (CYP2B6) to activate the anticancer prodrug cyclophosphamide (Cytoxan, CTX). OAd-2B6 alone induced dose-dependent tumor cell killing across multiple human tumor cell lines and exhibited strong synergistic antitumor effects when combined with CTX. Importantly, OAd-2B6-mediated local activation of CTX resulted in a potent bystander killing effect that eliminated tumor cells not directly infected by the virus. In a H1299 lung cancer xenograft nude mouse model, intratumoral injection of OAd-2B6 combined with CTX significantly inhibited tumor growth and even achieved complete tumor regression, with markedly superior efficacy compared with monotherapy. In immunocompetent mice bearing 4T1 breast cancer xenografts, OAd-2B6 alone inhibited tumor growth and was accompanied by upregulation of IFN-{gamma} and GzmB expression in the tumor-infiltrated T cells. CTX combination therapy further enhances this anti-tumor immune response, promoting the activation of T cells to suppress non-injected tumors at a distal site. Collectively, this study demonstrates that OAd-2B6 exerts potent antitumor effects through multiple mechanisms, including direct oncolysis, intratumoral prodrug activation leading to bystander killing, and enhancement of systemic antitumor immunity. These findings provide a promising strategy for improving the therapeutic efficacy of oncolytic therapy.

Epithelial ovarian cancer (EOC) is characterized by high relapse rates and the development of drug resistance, driven by adaptive DNA repair and survival pathways. Here, we develop a multimodal, chemically engineered miRNA therapeutic, MTX-5-FU-Gem-miR-15a, that integrates tumor-suppressive miR-15a activity with chemotherapeutic modifications and tumor-targeting capability. This modified miRNA exhibits potent nanomolar activity across diverse EOC models, including PARP inhibitor-resistant cells, without requiring delivery vehicles. Mechanistically, MTX-5-FU-Gem-miR-15a induces replication stress while suppressing G2/M checkpoint regulators (WEE1 and CHK1), resulting in genomic instability and apoptotic cell death. Transcriptomic and protein-level analyses revealed coordinated suppression of resistance-associated and oncogenic signaling pathways, alongside activation of DNA damage coupled with checkpoint abrogation and potential innate immune response. MTX-5-FU-Gem-miR-15a also demonstrates strong synergy with olaparib and robust antitumor efficacy in vivo. These findings establish a multimodal miRNA-based therapeutic strategy that targets replication stress and checkpoint dependency to overcome PARPi resistance in ovarian cancer.

Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor in adults, with median survival remaining approximately 12-15 months despite aggressive multimodal therapy. Therapeutic resistance and tumor recurrence are driven in part by limited drug penetration across the blood-brain barrier (BBB) and the persistence of brain cancer stem cells (BCSCs), highlighting the need for brain-penetrant therapeutic platforms capable of achieving sustained intratumoral delivery. Here, we developed a dendrimer-based nanotherapeutic by conjugating metformin to a fourth-generation hydroxyl-terminated polyamidoamine dendrimer (P4-MET) to enhance intracranial bioavailability and therapeutic efficacy in GBM. P4-MET exhibited favorable pharmacokinetic properties, including prolonged retention within the tumor microenvironment, and demonstrated enhanced cytotoxicity against GBM cell lines relative to free metformin (f-MET). Mechanistical studies with transcriptomic profiling by RNA sequencing revealed distinct treatment-associated molecular signatures, identifying BOLA2B as the most significantly differentially expressed gene between treatment groups. Specifically, BOLA2B expression was markedly elevated in f-MET-treated cells but not so following P4-MET treatment. Given the established association of BOLA2B with mTORC1 signaling and GPX4-mediated ferroptosis resistance, these findings suggest that P4-MET may, at least in part, enhance therapeutic efficacy by modulating ferroptosis-associated pathways. In orthotopic GBM models, combination treatment with P4-MET and radiotherapy (RT) significantly prolonged overall survival and increased tumor cell death compared with either monotherapy alone, consistent with a synergistic radiosensitizing effect. Importantly, P4-MET demonstrated minimal systemic toxicity, supporting its favorable therapeutic index and translational potential. Collectively, these findings establish P4-MET as a brain-penetrant nanomedicine platform that improves metformin delivery, modulates ferroptosis-related signaling networks, and potentiates radiotherapeutic response in GBM. This study highlights the potential of dendrimer-enabled metabolic nanotherapies to overcome therapeutic resistance in malignant brain tumors.

Cancer immunotherapies rely on tumor-specific T cells, which arise endogenously in most patients with cancer, but can be low frequency and poorly functional. Methods to specifically identify, expand, and manipulate tumor-specific T cells at the rare frequencies found in peripheral blood would enable new immunotherapeutic strategies. Here, we demonstrate an approach to virally transduce polyclonal tumor-reactive T cells across any MHC haplotype and in the absence of knowing the cognate antigen. By generating lentiviral vectors that selectively transduce cells expressing 4-1BB (CD137), a marker of T cell activation, we can transduce antigen-specific T cells with user-defined genetic cargoes that can selectively expand and track individual clonotypes via single-cell sequencing. Anti-4-1BB lentiviruses (4-1BB LVs) encoding therapeutic cargoes can also enhance antigen-specific T cells to extend survival in a xenograft model of human melanoma and transduce tumor-infiltrating T cells from patients with ovarian cancer. Overall, the 4-1BB LV platform targets antigen-specific T cells in a manner agnostic to both the antigen and presenting MHC, with potential applications in adoptive cell therapy manufacturing and TCR identification. One Sentence SummaryEngineered lentiviral vectors targeting 4-1BB selectively activate, expand, and transduce antigen-specific T cells with immunomodulatory cargo.

Multiple myeloma (MM) is a clonal plasma cell malignancy characterized by bone marrow infiltration, monoclonal immunoglobulin production, and microenvironmental dysregulation that leads to systemic organ damage. The advent of B-cell maturation antigen (BCMA)-directed chimeric antigen receptor (CAR) T-cell therapy has induced unprecedented responses and durability for patients with relapsed/refractory MM. These outcomes are rarely observed with prior salvage strategies, although relapse remains the predominant long-term challenge for most patients. The two currently approved BCMA CAR-T cell products use viral vectors to semi-randomly insert the CAR gene, which results in heterogeneous genomic composition and variability in efficacy, safety, and product consistency. To address these challenges, we integrated targeted CRISPR genome engineering with precise CAR transgene insertion at the T-cell receptor alpha constant (TRAC) locus, 1XX CAR signaling architecture to enhance potency and durability, and non-viral manufacturing with a single-stranded DNA repair template to improve efficiency and yield. This approach confers physiological CAR expression, reduces insertional mutagenesis, and improves persistence by mitigating tonic signaling and exhaustion. Our GMP manufacturing process consistently achieved high CAR integration (37.7-72.7%) and yields across all full-scale runs and met predefined release criteria for identity, purity, safety, and quality. In NSG mouse models of MM, the UCCT-BCMA-1 product exhibited exceptionally potent tumor control, CAR-T cell expansion 100-1000-fold greater than that of lentiviral constructs, and durable clearance of myeloma cells after multiple rechallenges. These findings establish a CRISPR-edited, fully non-viral manufacturing platform for next-generation 1XX-BCMA CAR-T therapies with enhanced persistence, safety, and efficacy. One Sentence SummaryCRISPR-engineered, TRAC-targeted 1XX-BCMA CAR-T therapy with improved safety, potency, and persistence in relapsed and refractory multiple myeloma.

Targeting membrane receptors underlies the success of antibody-drug conjugates (ADCs), yet single-receptor formats can be limited by heterogeneous expression, compensatory signaling, and variable internalization. Here we developed Multivalent Interchangeable Nanobody Degradation System (MINDS), a modular nanobody-Fc chassis that co-engages multiple membrane receptors, promotes their lysosomal co-depletion, and enables delivery of diverse intracellular payloads. As a proof of concept, we generated Tritazumab, a trispecific nanobody-Fc targeting three oncogenic receptors EGFR, cMET, and TfR1. Tritazumab incorporates a high-affinity, non-transferrin-competing anti-TfR1 nanobody that drives efficient uptake and lysosomal trafficking, enabling coordinated depletion of all three receptors. Across non-small cell lung cancer models, Tritazumab achieved rapid and sustained multi-receptor surface loss with picomolar degradation potency, reaching near-maximal depletion within approximately 1.5 hours. Conjugation of Tritazumab to MMAE preserved receptor binding and produced substantially greater antiproliferative activity and improved tumor selectivity relative to clinical ADCs in matched cell models, along with potent in vivo tumor growth inhibition and acceptable tolerability in a xenograft model. Extending the platform beyond cytotoxic payloads, a BRD4 molecular glue conjugate improved the selectivity window by > 100-fold and showed marked in vivo efficacy, while an EZH2-targeting PROTAC conjugate achieved an approximately 1,000-fold increase in intracellular degradation potency relative to the free PROTAC. These findings establish MINDS as a modular multispecific degrader-payload platform that integrates receptor co-depletion to enhance anticancer selectivity and efficacy.

Cancer-associated fibroblasts (CAFs) contribute to immune exclusion and therapy resistance in solid tumors, limiting the efficacy of chimeric antigen receptor (CAR) T cell and immune cell therapy. To overcome this, we developed a transcription factor (TF)-based strategy to reprogram prostate-derived CAFs (pCAFs) into normal fibroblast-like cells (NFs). We prioritized TFs enriched in quiescent stellate cells--Vitamin D receptor (VDR), Peroxisome Proliferator-Activated Receptor gamma (PPAR{gamma}), and p53--and selected VDR for proof-of-concept studies. Lentiviral VDR expression in pCAFs produced VDR-reprogrammed NFs (VDR-rpNFs) with reduced CAF markers, increased ATP, and suppressed TGF-{beta} and IL6, indicating phenotypic and metabolic reversion. In both in vitro 3D co-cultures and in vivo, VDR-rpNFs disrupted tumor architecture, enhanced CAR T cell infiltration, and reduced necrosis. PPAR{gamma}- and p53-rpNFs showed similar reprogramming effects. These results suggest TF-guided fibroblast reprogramming as a viable strategy to remodel the tumor microenvironment and improve CAR T cell efficacy in solid tumors.

Antibody-drug conjugates (ADCs) deliver cytotoxic payloads to tumors with antibody selectivity, yet all approved ADCs are administered by intravenous (IV) infusion despite a strong patient and clinical preference for subcutaneous (SC) delivery. SC administration would reduce treatment burden, but many ADC payloads are vesicants that cause tissue necrosis upon local release, a liability amplified, not mitigated, by the dispersion-enhancing excipients used for SC antibody formulations. We developed an injectable diacetyl-L-tartaric anhydride-functionalized chitosan hydrogel (TACT) that addresses this conflict by confining ADCs within a protective SC depot. TACT is compatible with clinically approved ADC formulations without drug-product modification and provides drug-to-antibody ratio (DAR)-dependent release kinetics that support a quantitative relationship with in vivo absorption timing. In direct comparison, recombinant human hyaluronidase (rHuPH20) co-formulated with vesicant ADCs caused severe tissue necrosis, whereas TACT prevented macroscopic injury while preserving antitumor efficacy comparable to intravenous dosing. TUNEL staining of injection sites showed that TACT attenuated peri-depot apoptotic injury 3-fold relative to T-DM1 alone and 2-fold relative to rHuPH20 co-formulation. In non-human primates, SC TACT achieved 78% relative bioavailability for total trastuzumab, reduced peak circulating T-DM1 catabolite (free DM1) exposure 7.6-fold compared to IV administration and produced only transient, self-resolving cutaneous reactions. These results identify depot-mediated confinement as a viable alternative to excipient-mediated dispersion for SC delivery of vesicant ADCs, demonstrated here for trastuzumab-based conjugates across two approved ADC drug products (T-DM1 and T-DXd, with non-cleavable MCC and cleavable peptide linkers), with supporting validation in a custom cleavable monomethyl auristatin E (MMAE) series. Additional validation with enfortumab vedotin (EV), a Nectin-4-targeting MMAE ADC, supported the applicability of this strategy beyond trastuzumab-based conjugates.

Current precision oncology--molecular targeted therapies and immunotherapies--relies on genomic or expressed biomarkers, yet most cancer patients remain ineligible for these treatments. Here, we establish enzyme activity as an actionable and orthogonal axis for precision cancer medicine. Strategic activity-based screening of mouse organs and human clinical specimens with a panel of enzyme-reactive fluorescence probes identified {beta}-galactosidase 1 (GLB1) and {beta}-hexosaminidases (HEX) as broadly elevated tumor-selective biomarkers. Leveraging these activities, we developed 7-ethyl-10-hydroxycamptothecin (SN38)-based GLB1-and HEX-reactive prodrugs. These prodrugs exhibited dramatically reduced systemic toxicities and improved therapeutic windows, compared to a clinically used SN38-based prodrug, irinotecan (CPT-11). Both prodrugs demonstrated activity-dependent therapeutic efficacy, affording a dramatic reduction of tumor volumes across multiple in vivo models, including a subcutaneous patient-derived xenograft (PDX) of lung squamous cell carcinoma that lacked genetic alterations targeted by current precision medicine. Furthermore, this strategy is broadly applicable across various cytotoxic payloads, establishing a generalizable platform for small-molecule precision medicines. Our results define an enzyme-targeting paradigm for precision oncology, in which fluorescence probes serve as companion diagnostic tools to guide development and selection of appropriately targeted prodrugs, which are expected to provide safer and more efficacious treatment options for cancer patients with elevated enzyme activities.

BackgroundResistance to 5-fluorouracil (5-FU)-based chemotherapy is a major clinical obstacle in colorectal cancer (CRC), highlighting the urgent need to overcome established resistance mechanisms. MicroRNA-based therapeutics have emerged as compelling candidates in this context, given their inherently pleiotropic mode of action; however, their clinical translation remains hindered by poor stability and suboptimal delivery. MethodsTo address these limitations, Gem-miR-15a, a unique gemcitabine-modified tumor-suppressor microRNA-15a was designed to synergistically integrate the tumor-suppressive activity of miR-15a with the chemotherapeutic potency of gemcitabine into a single molecular entity. Therapeutic efficacy of Gem-miR-15a was evaluated across a spectrum of preclinical models, including parental and drug-resistant CRC cell lines, 3D tumor spheroids, patient-derived organoids and in vivo metastatic models. Cell viability, apoptosis and cell cycle analyses were performed, along with RNA sequencing and protein validation. Statistical analyses were conducted using Students t-test or two-way ANOVA with mixed effects, and data were presented as mean {+/-} SD. ResultsGem-miR-15a exhibited potent anti-proliferative activity with IC50 values in the low nanomolar range, achieving [~]100-5000-fold greater potency relative to 5-FU and oxaliplatin. Importantly, it retained efficacy in both 5-FU- and oxaliplatin-resistant CRC models, effectively overcoming acquired chemoresistance. Mechanistically, Gem-miR-15a induced S-phase cell cycle arrest, eliminated the G2-phase cell population, and triggered apoptosis, accompanied by suppression of key oncogenic targets including WEE1, CHK1, YAP1 and BMI1. RNA-seq analysis further demonstrated modulation of pathways such as p53 signaling and reversal of resistance-associated gene expression, that were corroborated at the protein level. In vivo, Gem-miR-15a significantly reduced tumor growth at a dose [~]12-fold lower than gemcitabine, with no observable toxicity. ConclusionGem-miR-15a represents a potent, multi-targeted therapeutic strategy capable of overcoming chemoresistance in CRC. Its enhanced stability, effective delivery and robust efficacy across resistant models and a favorable safety profile highlight its strong potential for clinical translation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/720825v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@c20034org.highwire.dtl.DTLVardef@9b8478org.highwire.dtl.DTLVardef@161f1dorg.highwire.dtl.DTLVardef@54d826_HPS_FORMAT_FIGEXP M_FIG C_FIG

Lentiviral vectors enable efficient delivery of genetic cargoes for gene and cell therapies. With their [~]10-kb packaging limit, lentiviral vectors can encode multiple transcription units, supporting delivery of compact gene circuits. RNA-based devices offer highly compact control including ligand-responsive induction and closed-loop regulation. However, RNA devices such as ribozymes and splicing switches may interfere with vector production via activity on the single-stranded RNA genome. Here, we examine the impact of gene syntax and genetic parts to define design strategies for two-gene vectors encoding RNA devices. We find that titer decreases with genetic parts that interfere with transcription or processing of the viral transcript during production. Compared to initial vectors, our best-performing design boosts titer more than 30-fold, enabling fine-scale tuning of expression to optimize cell-fate conversion within a nonmonotonic landscape. Together, this work illuminates principles for constructing two-gene lentiviral vectors with both high titer and robust expression, enhancing efficacy for downstream applications.

Messenger RNA (mRNA) is canonically composed of ribonucleotides, with sporadic incorporation of deoxyribonucleotides into natural RNA transcripts being traditionally regarded as a rare, deleterious error arising from transcriptional infidelity. Here, we challenge this paradigm by demonstrating controlled partial substitution of ribonucleotides with deoxyribonucleotides during in vitro transcription (IVT) generates intact, stable and fully translationally competent IVT-mRNA. Unexpectedly, chimeric DNA-RNA backbone modification exhibits markedly enhanced IVT-mRNA translation several fold across multiple cell types and in vivo via diverse dosing routes relative to their ribonucleotide-based counterparts. 25% substitution of cytidine triphosphate with deoxycytidine triphosphate achieved best-performing translational output, surpassing the current gold-standard N1-methylpseudouridine (m1{Psi})-modified IVT-mRNA in a B16-OVA tumor vaccination model. These findings identify nucleotide class composition as a previously unrecognized parameter governing IVT-mRNA function and establish hybrid ribonucleotide-deoxyribonucleotide backbone engineering as a versatile strategy to expand the chemical space for next-generation mRNA therapeutics.

Hepatocellular carcinoma (HCC), a leading cause of cancer death, has a dynamic and heterogeneous tumor microenvironment (TME) that drives progression and therapeutic resistance. We previously elucidated that apoptosis antagonizing transcription factor (AATF) drives angiogenesis in HCC. However, its role in TME remains unexplored. We employed an orthotopic xenograft mouse model, implanting human HCC cells into the liver, and achieved liver-specific silencing via tail vein injection of AAV8 carrying mouse-specific siAATF or siControl. Histological, biochemical, and molecular analyses, combined with whole-genome transcriptomics mapped to mouse and human genomes, were used to study TME and tumor compartments separately. Silencing of AATF in the TME significantly reduced tumor growth compared with controls. Furthermore, AATF loss disrupted key processes in TME, including inflammation, immune response, angiogenesis, and extracellular matrix remodeling. Mechanistically, TGF-{beta} signaling was significantly suppressed in the TME, thereby affecting tumor cell cycle and metabolic activity, ultimately leading to tumor regression. The long noncoding RNA (lncRNA) analysis identified MIR100HG as a key downstream regulator of AATF in the TGF-{beta} signaling pathway. These findings expand the oncogenic role of AATF to include regulation of the TME via the AATF-MIR100HG-TGF-{beta} axis, highlighting its potential as a therapeutic target in HCC.

Chimeric antigen receptor (CAR) T-cell therapy has shown limited efficacy in solid tumors, in part due to variability in autologous T cells derived from heavily pretreated patients with advanced disease. To address these constraints, we developed an off-the-shelf allogeneic CAR-T platform using CRISPR-Cas9-mediated genome editing in T cells from healthy donors to enable targeted CAR insertion at the TRAC locus with concurrent disruption of B2M. Using adeno-associated virus (AAV) delivery, we designed CAR-T cells targeting glypican-2 (GPC2) and glypican-3 (GPC3), emerging antigens expressed in pediatric and adult solid tumors. Genome-edited allogeneic CAR-T cells exhibited potent, antigen-specific cytotoxicity across multiple tumor models. GPC2-directed allogeneic CAR-T cells demonstrated enhanced or comparable activity relative to conventional lentiviral CAR-T cells in neuroblastoma models and mediated tumor regression with prolonged survival in preclinical models. Notably, repeated dosing augmented antitumor efficacy without evidence of toxicity, supporting multi-dose regimens for solid tumors. Similarly, GPC3-targeted allogeneic CAR-T cells based on a single-domain antibody showed robust activity against hepatocellular carcinoma cells in vitro and in vivo. These findings establish a scalable, genome-engineered allogeneic CAR-T strategy with strong therapeutic potential and support the clinical development of off-the-shelf cell therapies for pediatric and adult solid tumors.

Poor aqueous solubility remains a major obstacle to the translational development of targeted anticancer compounds. VS1, a first-in-class inhibitor of the cholesterol-transfer protein STARD3, has emerged as a promising chemosensitizing agent in colorectal cancer (CRC), but its clinical applicability is limited by its poor water solubility. Here, we combine structural biology, nanotechnology, and functional pharmacology to establish STARD3 inhibition as a delivery-enabled strategy to potentiate fluoropyrimidine therapy. To define the molecular basis of STARD3 inhibition, we solved the crystal structure of VS1 bound to the STARD3 ligand-binding domain at 2.1 [A] resolution, revealing direct occupation of the sterol-binding cavity. Molecular dynamics simulations confirmed a stable binding mode and identified the {Omega}1 loop as a dynamic gate regulating ligand binding and dissociation. To overcome the formulation barrier of VS1, we engineered carrier-free, albumin-coated nanocrystals through sonication-assisted nanocrystallization followed by surfactant exchange with human serum albumin. The resulting rod-shaped nanocrystals displayed nanometric size, narrow size distribution, sustained release, and improved aqueous dispersibility, increasing the apparent solubility of VS1 by more than 14-fold while preserving its molecular integrity and crystallinity. Biologically, VS1 selectively potentiated 5-fluorouracil (5-FU) in CRC cells, with synergistic effects restricted to 5-FU-sensitive models and associated with enhanced reactive oxygen species accumulation. Albumin-coated formulation retained the chemosensitizing activity of the free compound. In HCT-116 xenografts, combined treatment with albumin-coated VS1 nanocrystals and 5-FU significantly reduced tumor growth, prolonged tumor doubling time, and increased intratumoral necrosis without exacerbating systemic toxicity. Together, these findings establish that albumin-coated nanocrystals can overcome the delivery limitations of an insoluble STARD3 inhibitor and provide a formulation-enabled strategy to enhance fluoropyrimidine therapy in colorectal cancer.

Immunoglobulins (e.g., IgGs) are critical effectors of the adaptive immune system that when overexpressed or dysregulated can result in autoimmune diseases. Thus, depletion of IgGs can be a promising therapeutic avenue. Here we developed genetically-encoded lysosome targeting chimeras (GELYTACs) that target circulating IgGs for clearance and degradation. The GELYTACs comprised two protein modules derived from insulin-like growth factor 2 (IGF2) and an IgG-binding nanobody, respectively, and mediated clearance of plasma IgG via the lysosomal trafficking receptor IGF2R. To achieve long-lasting IgG depletion, we encoded GELYTACs in an AAV gene therapy vector and established continuous expression in mice. We also developed conditional GELYACs that are activatable with disease-specific proteases or small molecule drugs. This work establishes GELYTACs as a possible therapeutic modality that is deliverable using genetic medicine approaches.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by a dense desmoplastic tumor microenvironment (TME) that limits drug penetration and promotes immune evasion. Effective treatment, therefore, requires simultaneous modulation of multiple signaling pathways. Here, we describe a directed polypharmacological strategy to identify dual modulators of c-MET and Smoothened (SMO), aiming to disrupt the protective stroma through SMO inhibition while directly suppressing tumor cell survival via c-MET targeting. An AI-guided virtual screening workflow combining the machine-learning platform PyRMD, trained on known c-MET and SMO ligands, with structure-based molecular docking was applied to a library of over 9 million compounds. This approach led to the identification of compound 21, an aminopyrimidine-benzamide-phenoxyquinoline derivative, as a dual c-MET/SMO inhibitor. Biochemical and cellular studies demonstrated that compound 21 selectively binds the SMO orthosteric site (pKi = 5.60), inhibits agonist-induced GLI (Glioma-associated oncogene) signaling (pIC50 = 5.50), and potently suppresses c-MET kinase activity (pIC50 = 6.94). Western blot analyses further revealed that compound 21 promotes ubiquitin-proteasome-mediated degradation of c-MET, eliminating receptor availability and limiting compensatory resistance signaling. In 3D heterotypic models comprising MIAPaCa2 pancreatic cancer cells and CAF154-hTERT fibroblasts, dual inhibition of SMO-mediated stromal support and c-MET-driven tumor progression resulted in greater cytotoxicity than the combination of the selective inhibitors Sonidegib and PHA-665752. Overall, compound 21 overcomes stromal-mediated resistance, enhances tumor cell death, and validates dual SMO/c-MET targeting as a promising single-agent therapeutic strategy for PDAC. One Sentence SummaryAn AI-identified dual SMO/c-MET inhibitor overcomes stromal resistance and degrades c-MET to suppress pancreatic cancer.

Oncolytic virotherapy is an emerging cancer therapy using genetically modified viruses. We previously reported engineering of measles virus with BNiP3, a proapoptotic gene for oncolytic purposes. The recombinant virus had shown promising results in breast cancer cells with a bias towards TNBC, an invasive and an aggressive subtype. Here, we investigated the mechanistic insights of anti-tumor effects induced by the recombinant virus. Initially, TNBC and non-TNBC tumor cell lines were compared bioinformatically using the available gene expression data through protein-protein interaction network using different topological properties. Four hub genes involved in tumor development and progression were identified to be the top genes in both the data sets. Of which, CTNNB1 gene encoding {beta}-catenin was found to be the significant one; as {beta}-catenin pathway is known to be a driver of tumor cell invasion and migration, the impact of the virus on this pathway was investigated in breast tumor cells. The results had demonstrated a notable decrease in {beta}-catenin expression and its downstream targets, cyclin D1, MMP7 reducing the migration potential of TNBC cells following virus infection. These findings suggest that the recombinant measles virus could be one of the effective treatment modalities to target invasive TNBC tumors. In vivo validation of engineered virus is ongoing to explore the therapeutic application of this virus. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/718830v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@19d321dorg.highwire.dtl.DTLVardef@1450ebeorg.highwire.dtl.DTLVardef@cb191eorg.highwire.dtl.DTLVardef@1de2d16_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIUse of recombinant measles virus with a pro-apoptotic gene, BNiP3 to target breast cancer cells C_LIO_LIIdentification of top regulatory genes in breast cancer development and progression C_LIO_LIReduction of {beta}-catenin expression encoded by CTNNB1 gene in TNBC cells following virus infection C_LIO_LIDownregulation of {beta}-catenin downstream targets in TNBC cells with virus infection C_LIO_LIInhibition of migratory potential of TNBC cells following infection C_LI

Chronic hepatitis B cure requires the inactivation and/or elimination of covalently closed circular DNA (cccDNA), together with silencing of integrated viral genomes and restoration of HBV-specific immune responses. The TLR1/2 agonist Pam3CSK4 has previously been identified as a potent direct anti-HBV agent in vitro. In the present study, we engineered a liver-targeting polymeric nanoparticle formulation of Pam3CSK4 to enhance its in vivo immunostimulatory and antiviral activity. We evaluated the antiviral efficacy of this novel nanoformulation carrying the TLR1/2 agonist (NP-Pam3CSK4) in monotherapy and started to investigate its mechanism of action through immunological correlates in an immune-competent AAV-HBV mouse model. AAV-HBV-infected mice received intravenous administrations of NP-Pam3CSK4 at doses of 5 or 20 g twice per treatment cycle over four cycles, followed by a 2-week follow-up period. Soluble Pam3CSK4 was administered at substantially higher doses (100 g). Serial blood samples were regularly collected to monitor virological and host immune parameters. At study completion, liver tissues were harvested for intrahepatic quantification of viral and immunological markers using immunoassays, quantitative PCR, and histological analyses. The most pronounced antiviral effects were observed in mice treated with NP-Pam3CSK4 formulations, which achieved greater viral suppression than free Pam3CSK4 despite markedly lower administered doses. Histological examination of liver biopsies from treated animals revealed prominent immune cell infiltration, including macrophages, monocytes, and T cells, organized in dense cluster-like structures. These findings support the induction of coordinated innate and adaptive immune responses contributing to HBV control and clearance. Collectively, our results demonstrate that nanoparticle-based delivery of TLR1/2 agonist represents a promising therapeutic strategy for chronic HBV infection and may improve the likelihood of achieving functional cure. Further mechanistic and translational studies (combination) are warranted to support clinical development.

Aberrant HGF-c-MET signaling is a major driver of hepatocellular carcinoma (HCC) progression and a clinically validated therapeutic axis, but current inhibitors predominantly target the intracellular kinase domain and remain vulnerable due to limited selectivity and resistance development. We therefore pursued an upstream strategy based on small molecules that target the extracellular HGF-c-MET interaction interface. We combined large-scale virtual screening of more than one million compounds from the ChemDiv and Enamine libraries with molecular dynamics (MD) simulations, steered MD, MM/GBSA profiling, and iterative lead optimization to identify candidate c-MET inhibitors targeting its extracellular (EC) domain. In HGF-stimulated HuH7 cells, selected compounds suppressed c-MET autophosphorylation, reduced cell viability, and inhibited long-term colony formation. Surface plasmon resonance (SPR) further confirmed direct binding of L083-1287 and 8008-3424 to the recombinant c-MET ectodomain. Mechanistic analyses identified previously unrecognized hotspot residues on the c-MET EC domain and a novel inhibitory network spanning multiple c-MET ectodomain interfaces. L083-0077 displayed the most consistent interaction pattern within this framework, including stabilization of key hotspot residues and preserved binding under acidic conditions relevant to the tumor microenvironment. Zebrafish xenograft assays with selected early hit compounds revealed compound-dependent developmental liabilities supporting the use of this model as an early in vivo prioritization step during lead optimization. These findings establish EC interface-directed c-MET inhibition as a promising therapeutic strategy in HCC and provide a mechanism-guided platform for the development of selective, upstream c-MET inhibitors with the potential to complement or overcome limitations of kinase-directed therapies.