Molecular Therapy Oncology

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.

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.

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.

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.

Canine hemangiosarcoma (HSA) is an aggressive endothelial malignancy with limited therapeutic options, and its progression is closely associated with vascular architecture, stromal remodeling, and inflammatory cell recruitment. Sulfoquinovosylacylpropanediol (SQAP) is a sulfoquinovosyl lipid radiosensitizer reported to affect angiogenic and tumor-microenvironmental pathways, but its effects in canine HSA are unknown. Here, we evaluated SQAP in canine HSA cell lines and patient-derived xenograft (PDX) models. SQAP showed minimal direct cytotoxicity against HSA cell lines in vitro, whereas it significantly suppressed tumor growth in three canine HSA PDX models. Transcriptome analysis of SQAP-treated HSA PDX tumors detected more SQAP-responsive genes in mouse host-derived cells than in canine tumor cells. Gene-set enrichment analysis of the mouse host-derived fraction showed positive enrichment of angiogenesis, hypoxia, and stromal remodeling-related gene sets after SQAP treatment. Subsequent tissue analysis showed that SQAP reduced host-derived CD31-positive vascular area and increased -smooth muscle actin coverage of remaining vessels in two of the three PDX models, while altering macrophage-associated marker profiles in a model-dependent manner. These findings indicate that SQAP suppresses canine HSA PDX growth primarily through vascular and macrophage-associated remodeling of the tumor microenvironment rather than direct tumor-cell cytotoxicity.

Harnessing the immunomodulatory capacity of commensal bacteria is an emerging avenue in cancer therapy. Bacterial extracellular vesicles (BEVs) provide a non-replicating, nanoscale alternative to live microbes with the potential for safer systemic delivery. Here, we investigated BEVs from a novel Gram-positive strain of Bifidobacterium pseudocatenulatum (Bif-210). Intravenous administration of Bif-210 BEVs reduced B16-F10 melanoma growth in C57BL/6J mice. Mechanistically, BEVs increased tumour-infiltrating Ly6G+ granulocytes in vivo, increased CD11b+Ly6G+ and ICAM-1+Ly6G+ bone marrow populations, and induced production of the neutrophil-attracting chemokines KC/CXCL1 (mouse) and IL-8 (human). Although Ly6G+ depletion independently inhibited tumour growth, it did not combine additively with BEVs, supporting a model in which Bif-210 BEVs alter Ly6G+ granulocyte function rather than simply expanding a conventional pro-tumour granulocyte pool. BEVs activated TLR2, did not activate TLR4, and upregulated TLR2 on Ly6G+ cells, while proxy assays provided no evidence of NETosis-associated activation. Repeated intravenous BEV administration produced no overt toxicity by tissue histology, body temperature, or body weight monitoring. These findings position B. pseudocatenulatum BEVs as a promising systemic immunotherapy that recruits and re-educates granulocytes via a TLR2-centred pathway to restrain melanoma progression. HIGHLIGHTSO_LIIntravenous Bif-210 BEVs reduce established B16-F10 melanoma growth in mice. C_LIO_LIBif-210 BEVs selectively increase tumour-associated Ly6G+ granulocytes. C_LIO_LIBEV treatment and Ly6G depletion are non-additive, linking BEV activity to granulocyte biology. C_LIO_LIBif-210 BEVs expand Ly6G+ bone marrow populations and induce granulocyte-recruiting chemokines. C_LIO_LIBif-210 BEVs engage TLR2 and enhance granulocyte fitness without NETosis-associated activation. C_LI

Although anthracyclines such as epirubicin are potent DNA-damaging agents, their application in glioblastoma (GBM) is limited by poor intracellular penetration, lack of durable responses and rapid emergence of adaptive tumor phenotypes. Here, we demonstrate that nanoarchaeosome-mediated delivery of epirubicin (NanoEpi) enables functional reprogramming of GBM survival under therapeutic stress. Nanoarchaeosomes composed of archaeal ether lipids exhibited high encapsulation efficiency ([~]96%) and nanoscale stability. Although both Epi and NanoEpi showed similar bulk uptake, both in established (U251-MG) and patient-derived (Gli5) glioblastoma models, NanoEpi induced significantly greater cytotoxicity than free epirubicin, indicating enhanced intracellular drug engagement. NanoEpi induced enhanced DNA damage, elevated reactive oxygen species, and mitochondrial depolarisation, leading to cytoskeletal collapse. In 3D gliomasphere systems, NanoEpi showed improved penetration and sustained retention, resulting in suppression of core viability and invasion. This correlates with its increased uptake by the lysosomes. Notably, even a transient exposure led to depletion of sphere-initiating capacity and complete loss of clonogenic recovery, indicating targeting of the stem-like compartment. This was accompanied by attenuation of MMP-2/9 activity and reduced angiogenic signalling in a chorioallantoic membrane model. These findings establish nanoarchaeosomes as a robust lysosome-directed delivery platform that extends beyond passive drug transport to sustain intracellular stress and suppress invasive adaptation and limit recurrence in GBM.

Pancreatic ductal adenocarcinoma (PDAC) remains a lethal malignancy with limited therapeutic options, underscoring the need for innovative treatments. Chimeric antigen receptor (CAR) therapy has transformed hematologic malignancies but faces key challenges in solid tumors, particularly on-target/off-tumor toxicity and antigen heterogeneity. Adapter CAR (AdCAR) platforms offer enhanced control by decoupling antigen recognition from CAR activation, enabling controllable, reversible, and multi-antigen targeting. Recent studies suggest AdCARs can function as an AND-gate using combinations of adapter molecules at controlled surface densities. This defines activation thresholds, termed the Surface Activation Matrix, that restricts full activation to tumor cells overexpressing the target antigen combination, thereby reducing off-tumor toxicity. In this study, we evaluated its applicability to PDAC using adapters targeting CD318, TSPAN8 and CD66c. We systematically evaluated single and combinatorial adapter dosing in co-culture assays with AsPC1 cells, in a donor-dependent manner. Low concentrations of individual adapters were non-cytotoxic, whereas combining them at identical sub-threshold doses restored potent tumor killing, demonstrating that AdCAR activation depends on cumulative adapter density rather than total amount. However, the activation threshold required for AND-gate cytotoxicity varied between donors, highlighting the need for patient-specific titration to achieve selective tumor killing. These findings validate that AdCAR T cell activity in PDAC can be finely tuned through adapter concentration and combinatorial targeting, enabling selective tumor recognition while minimizing on-target/off-tumor toxicity. This flexible, safety-oriented strategy supports targeting heterogeneous PDAC tumors, though donor-dependent variability remains a critical consideration for clinical implementation.

Glioblastoma (GBM) remains one of the most lethal primary brain tumors, in part because the blood-brain barrier (BBB), restricts delivery of most systemically administered chemotherapeutics. Although focused ultrasound (fUS) can transiently increase BBB permeability, therapeutic efficacy remains limited by reliance on systemic drug exposure and heterogeneous intratumoral distribution. Here, we report a pressure-gated ultrasound-triggered drug delivery strategy that enables localized intravascular release of chemotherapy at the site of sonication. Freebase doxorubicin and afatinib were encapsulated within ultrasound-sensitive mPEG-PDLLA/PFOB microdroplets and administered systemically to N-TVA::Ink4a/Arflox/lox;Ptenlox/lox mice bearing genetically engineered glioblastomas. Animals received repeated transcranial focused ultrasound over a 30-day treatment period. Ultrasound-triggered release of the dual-drug formulation significantly extended survival compared with untreated controls, with median survival increased by over two weeks - approximately a 30% improvement. Furthermore, this survival improvement was reflected in histological analysis, showing decreased tumor burden and severity. These improvements were not found in any control groups, demonstrating that spatially and temporally controlled intravascular drug release can substantially improve therapeutic efficacy in an aggressive immunocompetent glioblastoma model. These findings support pressure-gated ultrasound-triggered chemotherapy as a promising activation-based strategy for overcoming BBB-associated delivery limitations and improving outcomes in malignant brain tumors. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/735435v2_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1767043org.highwire.dtl.DTLVardef@c46048org.highwire.dtl.DTLVardef@8d3b44org.highwire.dtl.DTLVardef@2df0b8_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPressure-gated focused ultrasound enables localized release of doxorubicin and afatinib in glioblastoma. C_LIO_LIUltrasound-triggered chemotherapy significantly extends survival in a genetically engineered immunocompetent GBM model. C_LIO_LILocal activation outperforms systemic administration of identical drug combinations. C_LIO_LIThis strategy shifts focused ultrasound therapy from general BBB opening to spatially controlled drug activation. C_LI

Homologous recombination deficiency (HRD) is an actionable vulnerability found in a substantial fraction of human cancers, yet current HRD-directed therapies are limited by toxicity, incomplete responses, and acquired resistance. Many DNA-damaging agents were developed before DNA repair biomarkers were available, suggesting that abandoned agents may harbor previously unrecognized genotype-selective activity. Here, through a focused screen of DNA-damaging agents in isogenic homologous recombination-proficient and -deficient models, we identify CB1954, a decades-old nitrobenzamide aziridine prodrug, as highly selective for BRCA2-deficient tumor cells. CB1954 forms DNA interstrand crosslinks independent of HR status, but selectively induces DNA-damage signaling, apoptosis, and loss of clonogenic survival in HR-deficient cells. Targeted DDR CRISPR screening and isogenic validation define a distinct repair dependency for the Fanconi anemia and homologous recombination pathways, with limited dependence on mismatch repair or nucleotide excision repair. Genetic and pharmacologic perturbation of NQO2, the bioactivating enzyme for CB1954, reveals a bifurcated mechanism in which NQO2-dependent activation selectively contributes to HRD cytotoxicity, while aziridine-dependent lesions likely account for residual activity in HR-proficient cells. CB1954 exhibits favorable preclinical pharmacokinetic properties and genotype-dependent antitumor activity in BRCA2-deficient xenografts. These findings reposition CB1954 as a historically overlooked HRD-selective agent and demonstrate that biomarker-guided profiling of DNA-damaging agents can uncover new opportunities for precision oncology.

Background: Medulloblastoma (MB) is the most common malignant brain tumour in children, and aggressive subgroups are frequently driven by the oncoproteins MYC or MYCN. Direct therapeutic targeting of MYC/MYCN has been challenging because of their intrinsically disordered protein structures. The aim of this study was to determine whether novel SE486-11 analogues (UNSW-SCs) can therapeutically target MYC/MYCN-driven MB. Methods: The anticancer activity of UNSW-SCs was assessed in MB cell lines with differential MYC/MYCN expression. Target engagement was evaluated using surface plasmon resonance and drug affinity responsive target stability assays. Blood-brain barrier penetration, MYC/MYCN protein degradation, cell cycle effects, apoptosis, DNA damage, and synergy with histone deacetylase (HDAC) inhibitors were examined. Therapeutic efficacy was evaluated in murine models of MYC- and MYCN-driven human MB. Results: UNSW-SCs showed potent anticancer activity, with preferential selectivity toward MB cells expressing high MYC/MYCN levels and IC50 values ranging from 0.22 to 1.18 M. The lead molecule, UNSW-SC-22, directly bound MYC, crossed the blood-brain barrier, and achieved a brain-to-plasma ratio of 1.44 at peak concentrations. UNSW-SC-22 induced MYC/MYCN-dependent cytotoxicity associated with enhanced proteasomal degradation, cell cycle arrest, apoptosis, and DNA damage. Combined treatment with HDAC inhibitors further reduced MYC/MYCN protein levels, increased DNA damage, and enhanced apoptosis. In vivo, UNSW-SC-22, either alone or with entinostat, significantly suppressed intracranial tumour growth and prolonged survival. Conclusions: UNSW-SC-22 is a brain-penetrant MYC/MYCN-targeting molecule with potent preclinical activity in MYC/MYCN-driven MB, supporting its development as a monotherapy or combination strategy with HDAC inhibition.

Osteosarcoma (OS) and Ewing sarcoma (EWS) are the most common malignant bone tumors in children and adolescents, with survival rates around 25% in metastatic disease and few advances in treatment in decades. High DDX3 expression has been reported across various sarcoma subtypes. Depending on the context, DDX3 appears to have opposing roles in regulating the tumor immune microenvironment. Within macrophages, DDX3 promotes inflammatory cytokine expression and supports immune cell function. In contrast, in tumor cells DDX3 suppresses a pro-inflammatory state by unwinding dsRNAs, preventing a Type I interferon response. We show that inhibiting DDX3 with RK-33 leads to dsRNA accumulation, inducing a Type I interferon response and broader inflammatory gene expression changes across multiple sarcoma models, shifting macrophage polarization toward a pro-inflammatory M1-like phenotype. To evaluate whether this innate immune microenvironmental remodeling could translate into clinical benefit, we assessed the therapeutic efficacy of RK-33 alone or in combination with mifamurtide, an immunostimulant, in immune competent mouse models of osteosarcoma, with metastatic burden as the primary outcome. We found that in the absence of MYC over-expression, the combination treatment significantly reduced metastatic spread. These findings support targeting DDX3 as a novel innate immune based therapeutic strategy and highlight that the tumors molecular landscape critically influences therapeutic responsiveness.

Pancreatic cancer remains a highly lethal malignancy with limited therapeutic options. Chimeric antigen receptor (CAR) therapy has revolutionized the treatment of hematological cancers but still faces major limitations in solid tumors, particularly due to the scarcity of tumor-specific targets. Cutaneous lymphocyte antigen (CLA) recently emerged as a promising PDAC target due to its high tumor expression and limited presence in healthy tissues. However, previously reported CLA-directed CAR constructs lacked antitumor functionality. Here, we investigated multiple strategies to generate functional CLA-targeting CAR approaches. We first hypothesized that impaired activity resulted from fratricide caused by CLA expression on activated T cells. CLA knockout was successfully achieved through deletion of fucosyltransferase-7, but not by knockout of the major CLA carrier backbones CD162, CD44 or CD43, suggesting additional CLA carriers or compensatory regulation. As CLA knockout alone did not restore CAR-mediated killing, we explored whether insufficient binding affinity limited CAR activity. Affinity maturation was performed in silico and in vitro using yeast surface display, identifying 39 candidate mutations, although none restored cytotoxicity. We finally switched to an AdCAR strategy using anti-biotin CAR T cells combined with biotinylated anti-CLA scFv-Fc adapters. This approach enabled efficient, concentration-dependent cytotoxicity with both CLA-targeting binders. Additionally, we identified a dynamic, cell density-dependent regulation of CLA expression. Finally, glycan profiling of CLA binders further revealed broader-than-expected glycan interactions, suggesting a potentially wider definition of the CLA family. Overall, our findings establish CLA as a functional PDAC immunotherapy target while revealing unexpected complexity in its regulation and molecular presentation.

Chemotherapy resistance remains a critical barrier in treating osteosarcoma. Hypoxia-activated prodrugs (HAPs) target oxygen-deprived tumor regions that evade conventional chemotherapy. Here, we applied integrated spatial multimodal mass spectrometry imaging of metabolites (DESI-MSI, MALDI-MSI), targeted proteomics (IMC), and metallomics (LA-ICP-MSI) to naive and newly developed doxorubicin-resistant osteosarcoma spheroids treated with a novel HAP tirapazamine analogue, TPZ-A-X, and doxorubicin. Combination treatment selectively downregulated GLUT1 and suppressed pro-survival pAkt in doxorubicin-resistant spheroids whilst inducing comparable DNA damage ({gamma}H2AX) across both phenotypes. Metabolomics imaging identified ferroptosis pathway suppression in doxorubicin resistance, which combination treatment reversed, whilst simultaneously depleting glycolytic fuels. Integrative protein-metabolite correlation analysis uncovered functional couplings between glucose transport and CoA-dependent metabolism and spatially revealed anabolic signaling at spheroid peripheries. Combination treatment induced endogenous copper, zinc and magnesium depletion, independent of ATP/ADP collapse reflecting an adaptive survival remodeling of the metalloproteome. HAP/chemotherapy combinations exploit metabolic vulnerabilities via coordinated disruption of ferroptosis suppression, glycolytic dependence, and survival pathways underlying apoptotic resistance. These findings demonstrate a framework for informing mechanistic reasoning and combination strategy design in chemotherapy-resistant tumors.

Background: Juvenile nasopharyngeal angiofibroma (JNA) is a rare locally aggressive vascular sinonasal tumor that primarily affects adolescent males. Despite advances in endoscopic surgery and preoperative embolization, JNA can be associated with major operative bleeding risk and clinically meaningful recurrence, while non-surgical treatment options remain limited. Methods: To define the cellular programs underlying JNA vascularity, we performed single-cell RNA sequencing of JNA tumors (n=2), tumor-adjacent mucosa, and control sinonasal tissue. We analyzed cell composition, differential gene expression, pathway enrichment, and cell-cell communication, followed by Drug2cell-based mapping of transcriptional states to candidate therapeutic targets. Results: JNA contained an expanded fibrovascular compartment composed of endothelial cells, fibroblasts, pericytes, vascular smooth muscle cells, and neural crest-like cells. Neural crest-like cells were enriched in JNA but showed relatively limited transcriptional differences from tumor-adjacent tissue. By contrast, endothelial cells demonstrated the strongest disease-associated remodeling, with enrichment of angiogenesis, extracellular matrix organization, hypoxia response, and cell migration pathways. Endothelial cells also showed downregulation of adaptive immune signaling pathways, suggesting reduced immune engagement within the tumor microenvironment. Intercellular communication analyses revealed dense endothelial-stromal signaling across the JNA fibrovascular network. Drug2cell analysis nominated VEGF/VEGFR signaling as a candidate therapeutic vulnerability, with VEGFR-targeting agents predicted to act primarily on vascular and lymphatic endothelial populations. Conclusions: JNA is organized around an angiogenesis-dominant fibrovascular program driven by endothelial-centered signaling. These data support further investigation of VEGF/VEGFR-directed therapy as a potential adjunctive strategy for patients with recurrent, unresectable, or surgically high-risk JNA.

Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related mortality worldwide despite recent therapeutic advances, driven in part by its marked etiological and molecular heterogeneity and the lack of broadly effective therapeutic targets. Identifying conserved tumor dependencies shared across distinct etiological backgrounds may provide new opportunities for targeted therapy. Here, we developed an integrative computational framework to systematically integrate transcriptomic, functional genomics, and clinical datasets for the identification and prioritization of candidate tumor dependency genes in liver cancer. We reanalyzed transcriptomic data from murine models of liver cancer driven by genotoxic (DEN), oncogenic (c-Myc), and inflammatory (lymphotoxin) stimuli, identifying more than 380 genes consistently upregulated across all tumor models. Functional enrichment analysis revealed a strong overrepresentation of cell cycle-related pathways and liver cancer signatures. Integration with DepMap dependency datasets identified 26 genes with strong dependency scores. Candidate genes were further prioritized by comparing their expression across models of liver regeneration, chronic liver injury, and liver cancer. Analysis of the TCGA-LIHC cohort confirmed significant overexpression of all 26 genes in human HCC, with high expression associated with poor patient survival. Together, these findings establish an integrative framework for identifying conserved tumor dependencies, providing a prioritized set of proliferation-associated genes for functional evaluation as therapeutic targets in HCC.

177Lu-PSMA-617 (PluvictoTM, Lu-177 RLT) is an FDA-approved targeted radioligand therapy (RLT) for metastatic castration-resistant prostate cancer (mCRPC), but its durability of response to this singular approach poses a challenge to the field. Chimeric antigen receptor (CAR) T cell therapy has revolutionized clinical practice for hematological malignancies, but its clinical development for solid tumors, including mCRPC, has been encumbered by antigen heterogeneity and the immunosuppressive tumor microenvironment (TME). Here, we evaluate the therapeutic combination of Lu-177 RLT and PSCA-CAR T cells to overcome these barriers. In human xenograft and mouse syngeneic prostate cancer models with homogeneous or heterogeneous antigen expression, the sequential administration of Lu-177 RLT, cyclophosphamide (Cy), and PSCA-CAR T cells improves tumor control and prolongs survival compared to monotherapies. Mechanistically, Lu-177 RLT alone or with Cy remodels the TME by promoting pro-inflammatory myeloid responses and activating endogenous T cells, while enhancing CAR T cell activation and effector function. We additionally evaluated 225Ac-PSMA-617 RLT as an emerging approach in combination with CAR T cells and observed anti-tumor responses, supporting its potential as an alternative RLT partner. These findings support RLT as an immune priming strategy to enhance CAR T cell therapy and provide a rationale for clinical translation of this combination in mCRPC. One Sentence SummaryCombining 177Lu-PSMA-617 radioligand therapy with PSCA-CAR T cells improves tumor control and survival in prostate cancer models by overcoming the antigen heterogeneity and reshaping the immunosuppressive tumor microenvironment.

Scaffold integrity is essential for the activity of proteins that function through protein-protein interactions rather than catalytic output. RAF1 exemplifies this duality: although it is a bona fide kinase and a core component of the MAPK cascade, its tumor-promoting role is largely kinase-independent, relying instead on scaffold-mediated suppression of apoptosis. Genetic Raf1 ablation in KRAS-driven lung adenocarcinoma mouse models induces tumor regression without systemic toxicity, making it an attractive candidate for targeted protein degradation. Chemogenetic systems like the dTAG platform are widely used for preclinical target validation. Here, we generated a dTAG-RAF1 mouse model and showed that pharmacological degradation is efficient and systemically well tolerated, but fails to reproduce the tumor regression observed upon genetic Raf1 ablation. Mechanistically, the N-terminal FKBP12F36V tag (dTAG) perturbs the RAF1 interactome, including scaffold associations with apoptotic regulators, thereby blunting the phenotypic consequences of its degradation. These results establish scaffold integrity as a determinant of chemogenetic system fidelity and argue that degradation tools must be validated at the functional level, not only for target elimination, before assessing their therapeutic relevance.

Melanoma is an aggressive malignancy that rapidly adapts to therapy. While chemotherapy resistance has traditionally been attributed to tumour-intrinsic mechanisms, growing evidence implicates the tumour microenvironment in shaping drug tolerance. However, few in vitro models capture the stromal complexity needed to study this interaction. We developed two multicellular melanoma spheroid models of increasing stromal complexity: a baseline model of melanoma, endothelial, and macrophage cells (BEM), and a fibroblast-containing counterpart (BEMF), and compared their transcriptional response to doxorubicin. Fibroblast inclusion increased the doxorubicin concentration required to achieve comparable growth inhibition. While untreated BEMF spheroids exhibited only modest baseline transcriptional differences, they showed a profoundly reshaped transcriptional response after doxorubicin exposure, displaying broader and higher-magnitude changes. These responses were characterized by suppression of proliferative and cell-cycle programmes, together with activation of inflammatory, immune-associated, metabolic, and stress-adaptive pathways. Higher-resolution pathway analyses further revealed coordinated attenuation of mitotic progression, checkpoint regulation, homologous recombination repair, and Rho GTPase signalling, consistent with a shift toward stress-adaptive and phenotypically plastic states, rather than classical resistance mechanisms. Transcriptome-derived transcription factor activity inference supported this regulatory rewiring. Integration with curated resistance-associated genes and external transcriptomic datasets demonstrated strong conservation of core transcriptional features across heterogeneous experimental systems, including consistent suppression of proliferation-associated genes and induction of inflammatory signalling programmes. Together, these findings indicate that fibroblasts redirect chemotherapy responses toward a stress-adaptive, persister-like phenotype and establish fibroblast-containing 3D melanoma spheroids as a physiologically relevant platform for studying tumour microenvironment-mediated chemotherapy tolerance and stromal-tumour interactions.

Androgen receptor (AR) antagonists, such as enzalutamide, suppress prostate cancer (PCa) cells to achieve temporary therapeutic effects. In addition to tumor cell-autonomous suppressive function, AR antagonists can also potentially exert anti-tumor effects via mitigating cytotoxic T cells exhaustion. However, strategies for effectively harnessing enzalutamides immunotherapeutic effects remain elusive. In studying a recently described glutamine antagonist prodrug (DRP-104) in PCa models, we found that despite the initial response, tumors ultimately became resistant. Intriguingly, compared to the untreated (DRP-104 treatment-naive) tumors, the resistant tumors became highly susceptible to enzalutamide in vivo. Additionally, treating tumors with DRP-104 and enzalutamide simultaneously also yielded superior therapeutic efficacy. We demonstrated that DRP-104 therapy promoted the infiltration of CD8+ T cells as well as regulatory T cells (Treg) in responsive tumors, and the tumor-infiltrating Treg were mostly depleted upon enzalutamide treatments. Mechanistically, we showed that Treg differentiation from mouse CD4+ T cells was attenuated by enzalutamide. We further demonstrated that Treg induction was accompanied by the interaction between AR and aryl hydrocarbon receptor (AhR), the nuclear receptor indispensable for Treg differentiation, in the nuclei of CD4+ T cells, and this interaction was diminished by enzalutamide. In further support of AR signaling in Treg biogenesis, analysis of available gene expression datasets found that AR expression was elevated in Treg when compared to CD4+ T cells in human peripheral blood mononuclear cells (PBMCs). In addition, it was positively correlated with Treg module scores in several human cancer types. Finally, using an anti-GPC3 (Glypican 3) vaccination model, we demonstrated that CD4+ T cells subjected to Treg induction in the presence of enzalutamide were less effective in protecting GPC3-expressing tumor cells from CD8+ T cells cytotoxic killing. Collectively, these results suggest that AR promotes Treg s differentiation and/or immunosuppressive functions, and nominate enzalutamide as a Treg-mitigating agent for potentiating immunotherapies. Our results also demonstrate that an otherwise unintended, Treg-promoting property of DRP-104 can be leveraged to unleash the immune-regulatory function of enzalutamide for the treatment of PCa.