Cancer Letters

Mcl1 is a major driver of therapeutic resistance across hematologic malignancies, but direct Mcl1 inhibition has been limited by on-target cardiotoxicity. Here, building on our development of an Mcl1-targeting autophagy-targeting chimera (AUTAC), we show that AUTAC-mediated degradation creates a tumor-selective therapeutic window that spares the heart. AUTAC induced robust cytotoxicity and Mcl1 degradation in multiple myeloma models, while showing minimal toxicity in cardiac cell lines, primary cardiomyocytes, and murine heart tissue. In vivo, AUTAC reduced tumor Mcl1 without measurably affecting cardiac Mcl1. Mechanistically, this selectivity was associated with lower expression of the p62/SQSTM1, TRAF6, and UBC13 machinery required for AUTAC activity in cardiac cells, together with lower intracellular AUTAC accumulation relative to tumor cells. AUTAC also enhanced the antitumor activity of carfilzomib and venetoclax, including in resistant models, without worsening cardiotoxicity or promoting cardiac Mcl1 loss. Compared with classical Mcl1 inhibitors, AUTAC caused markedly less cardiomyocyte death, mitochondrial depolarization, and apoptotic signaling. These findings identify AUTAC-mediated Mcl1 degradation as a cardiac-sparing strategy to target an otherwise clinically constrained vulnerability and support tumor-selective lysosomal degradation as a path to safer Mcl1-directed therapy.

Immune checkpoint inhibitors (ICIs) have transformed cancer therapy, but variable patient responses highlight the need to better regulators of immune sensitivity. Here, we identify the mitochondrial citrate carrier SLC25A1 as a determinant of anti-PD-L1 antibody therapy responsiveness through a dual regulation of type I interferon (IFN-I) signaling and of PD-L1 expression. SLC25A1 promotes a mitochondrial-to-nuclear retrograde signaling via cytosolic accumulation of mitochondrial DNA, activation of the cGAS-STAT1 axis, and establishment of a virus mimicry state that triggers the IFN-I response. This activation is enriched in cancer stem cell populations, consistent with the role for SLC25A1 in tumor stemness and therapy resistance. Moreover, SLC25A1 also regulates PD-L1 protein levels through a newly identified fumarate-Keap1-PD-L1 axis, whereby fumarate inhibits Keap1, leading to PD-L1 up-regulation. In vivo, tumors expressing high levels of SLC25A1 exhibit an inflammatory environment and increased sensitivity to PD-L1 blockade, but accelerated growth in the absence of anti-PD-L1 treatment. These findings position SLC25A1 as a novel regulator of mitochondrial-driven IFN-I signaling and PD-L1 stability, and suggest that SLC25A1 exploits PD-L1 to evade immune surveillance, while at the same time creating an intrinsic tumor vulnerability to checkpoint blockade. Thus, SLC25A1 may serve both as a biomarker of response and as a target to enhance the efficacy of immunotherapy.

Chimeric antigen receptor (CAR) T-cells have represented a groundbreaking advance in the control of hematological cancers. However, their efficacy in controlling solid tumors has been rather limited, highlighting the importance of new cell-based therapies strategies to curb the progression of solid cancers. Here, we generated functional macrophages from human umbilical cord blood derived CD34+ hematopoietic stem cells (HSCs) engineered to express CARs. Approximately 50% of the CAR-MacCD34 population expressed anti-HER2 CARs and maintained high viability throughout differentiation. Mass spectrometry (MS) and multiparametric flow cytometry analysis revealed upregulation of proteins associated with phagocytosis, matrix remodeling, and degradation, indicating enhanced tumor infiltration potential. In vitro, CAR-MacCD34 exhibited a significantly higher capacity to phagocytose HER2-positive tumor cells compared to untransduced MacCD34 cells. Additionally, CAR-MacCD34 cells that phagocytosed cancer cells showed increased nuclear translocation of NF-kB, suggesting CAR-mediated intracellular signaling. To assess functionality in a more physiologically relevant context, we used tumor spheroids embedded in a dense 3D collagen matrix. Confocal microscopy and live imaging revealed that CAR-MacCD34 cells exhibited superior infiltration of dense tumor spheroids compared to untransduced MacCD34 cells. Notably, we observed multiple instances of tumor cell phagocytosis by CAR-MacCD34 cells in this 3D model. In addition, we employed in vivo zebrafish larvae models of HER2-positive tumors. We noted that CAR-MacCD34 cells persisted for over 8 days post-injection and demonstrated significantly greater efficacy in controlling tumor growth compared to untransduced MacCD34 cells. Our findings introduce a novel CAR-macrophage therapeutic approach with promising clinical potential, leveraging a renewable and accessible cellular source. Optimizing CAR-MacCD34 functionality in combination with existing therapies may lead to durable and effective anti-tumor responses for patients with solid tumors.

Although KRAS G12C-specific inhibitors such as sotorasib have been approved by US FDA and currently used in clinic, treating non-G12C mutants and overcoming acquired resistance for these inhibitors remain critical challenges. Here, we introduce a reciprocal feedback blockade therapy combining the MEK inhibitor trametinib and the multi-tyrosine kinase inhibitor imatinib to overcome these limitations. Our study reveals their compensatory roles: trametinib suppresses MEK activity yet promotes tyrosine kinase signaling and angiogenesis, while imatinib, a pan-tyrosine kinase inhibitor unleashes the MEK/ERK pathway via phosphatase suppression. Combining these agents blocks the reciprocal survival signals, inducing robust cell death across diverse KRAS-mutant models. Mechanistically, this combination reprograms cellular metabolism, leading to autophagy-dependent lipid peroxidation accumulation and ferroptosis. This strategy was effective in sotorasib-resistant lung cancer cells and various mouse models, including pancreatic cancer patient-derived xenograft. Furthermore, a pilot clinical trial for KRAS-mutant pancreatic cancer yielded encouraging responses. Consequently, the trametinib-imatinib combination represents a promising, broad-spectrum therapeutic strategy to overcome the constraints of current KRAS-targeted therapies.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most challenging cancers to treat due to the dismal survival rate, poor post-treatment outcome and profound resistance to a wide range of therapies. With mutant KRAS being a key driver, small molecule inhibitors targeting KRAS or pan-RAS (KRASi) have demonstrated exciting preclinical and early clinical anti-tumor efficacy, and the pan-RAS(ON) inhibitor daraxonrasib (RMC-6236) recently achieved Phase 3 clinically meaningful improvements in patient survival compared to chemotherapy. But resistance to RAS/KRAS inhibitor inevitably develops, which limits and compromises the treatment outcome. In this study, we investigated the combination of stereotactic body radiation therapy (SBRT) and KRAS inhibition (MRTX1133 and daraxonrasib) in the treatment of preclinical PDAC models. We found that this combination strategy synergistically suppresses PDAC cell growth in vitro and enhances tumor control while minimizing local recurrence in orthotopically implanted KPC (LSL-KrasG12D/+;Trp53R172H/+;Pdx1-Cre) murine PDAC tumors in vivo. As radiation therapy (RT) induces ferroptosis in multiple cancer types and mutant KRAS promotes various anti-ferroptotic mechanisms, we tested the role of ferroptosis in promoting tumor-control efficacy. Intriguingly, the addition of a ferroptosis inhibitor, liproxstatin-1, to the combination therapy significantly abrogated the in vivo synergism between SBRT and KRAS inhibition, suggesting that treatment-induced ferroptosis at least partially drives the synergistic efficacy of this combination strategy. Our study indicates that this SBRT-KRASi combination has the potential to overcome treatment resistance and improve outcomes in PDAC patients. These data directly support the design of a planned multi-center Phase 2 clinical trial with this combination strategy in locally advanced PDAC.

SUMMARYChordoma, a rare malignant notochordal tumor of the skull base and spine, is typically resistant to chemotherapy and radiotherapy and exhibits aggressive local recurrence. Here we show that chordoma recurrence correlates with a coordinated upregulation of monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs), a low PFA/MUFA ratio and an adaptive, lipid peroxidation-resistant state that protects against DNA damage and cell death. Single-cell metabolic profiling identified a tumor subpopulation marked by a fatty acid biosynthesis-high state coupled to stemness. RT-tolerance was directly linked to elevated FASN and lipid droplet (LD) expansion, and MUFA-loading phenocopied RT-tolerance in chordoma cells. Mechanistically, LDs accumulated in response to RT via generation of ROS, and subsequent activation of ER-stress, SREBP1 and Fatty Acid Synthetase (FASN). DESI-MS showed that low-dose irradiation was sufficient to increase MUFAs early and build peroxidation resistant MUFA-LDs, whereas PUFA induction required a higher radiation dose. In a spatially defined manner in a patient-derived xenograft. Finally, in silico knockout and pharmacologic FASN blockade restored radiosensitivity and apoptosis in vitro and in vivo. Collectively, our result support a unifying model in which RT resistance in chordoma is shaped by an adaptive fatty acid metabolic program that buffers oxidative injury and increases survival of RT-resistant, stem-like tumor subpopulations. These findings further support FASN inhibition as a practical radiosensitization strategy for chordoma particulary where RT dose escalation is constrained by anatomy. KEYPOINTSO_LIRecurrent chordoma exhibits fatty acid-associated metabolic reprogramming. C_LIO_LIMUFA-associated lipid droplet accumulation is linked to radioresistance in chordoma cells. C_LIO_LITargeting FASN restores radiotherapy sensitivity of chordoma in vitro and in vivo. C_LI IMPORTANCE OF STUDYThis study underscores the clinical importance of targeting metabolic vulnerabilities to restore radiosensitivity in chordoma. By integrating transcriptomics, metabolomics, and in vitro and in vivo models, we identified adaptive fatty acid metabolic reprogramming as a central mechanism of RT resistance in chordoma. Recurrent tumors were characterized by coordinated enrichment of unsaturated fatty acids, especially monounsaturated fatty acids (MUFAs), together with a low PUFA/MUFA ratio and a lipid peroxidation-resistant state. Mechanistically, RT-tolerance chordoma cells exhibited a high-FASN state driven by activation of the ROS-ER stress-PERK/SREBP1/FASN axis, leading to intracellular lipid droplet expansion. Importantly, genetic and pharmacologic inhibition of FASN restored radiosensitivity and enhanced apoptosis in both in vitro and in vivo models, suggesting a translatable therapeutic strategy. Together, these findings link adaptive metabolic reprogramming to RT resistance and support new therapeutic approaches for chordoma management.

Regardless of the success of clinical surgery, disseminated tumor cells (DTCs) can persist in distant organs, with a fraction surviving chemotherapy, which can result in minimal residual disease (MRD), a relevant reservoir for metastatic relapse. Yet, the cellular states that enable the survival and outgrowth of MRD remain poorly defined. Here, using a patient-derived tongue cancer organoid (TCO) model, we recapitulated the key features of chemotherapy-tolerant DTCs by culturing TCOs under growth-factor deprivation and chemotherapeutic stress conditions that mimic the metastatic tissue environment. Clonal-level analyses revealed a distinct subset of cells that retained proliferative capacity without entering a therapy-induced cytostatic state (hereafter referred to as cycling persisters, CPs). CPs exhibited coordinated activation of the IFN signaling pathway, Xenobiotic metabolism, and inflammatory signaling pathways, defining a transcriptional and metabolic program that enables sustained proliferation of CPs under the poor conditions. Consistently, a cell population with similar features was identified in metastatic tissues from patients. Longitudinal clonal tracking demonstrated that early metastatic recurrence is more likely driven by CPs, suggesting a novel mechanism that differs from the prevailing view that relapse arises from reactivation of dormant non-CPs. Our findings highlight a critical therapeutic oversight in relapse prevention.

Irreversible electroporation (IRE) has shown promise for treating pancreatic ductal adenocarcinoma (PDAC), but whether IRE can induce an abscopal effect is not established. We demonstrated that the combination of IRE and anti-PD-1 antibody could trigger robust abscopal effects in preclinical models of metastatic PDAC. Data from multiple in vivo models, RNA-seq, scRNA-seq, and spatial immunofluorescence provide compelling evidence that IRE induced mitochondrial dysfunction and cellular stress, which triggered activation of the cGAS-STING pathway and subsequent systemic antitumor effects. IRE also led to inflammatory response characterized by tumor infiltration of myeloid cells and their polarization toward M1 state, turning immunologically "cold" tumors into "hot" tumors. Moreover, the presence of T cell/B cell clusters in tumors from mice treated with IRE plus PD-1 and the lack of antitumor efficacy in B cell knockout mice bearing orthotopic murine PDAC tumors indicate that B cells play an important role in IRE-mediated systemic antitumor immunity. SignificanceThis study shows that IRE plus a checkpoint inhibitor represents a promising therapeutic strategy for PDAC and supports advancing this treatment toward clinical translation. Our data also support potential combination strategies with immunomodulatory agents that can recruit and reprogram B cells to support T cell activation and cytotoxic effector functions.

Glypican-3 (GPC3) is an oncofetal protein widely being explored as a diagnostic and therapeutic target in hepatocellular carcinoma (HCC). Given that radiotherapy in the form of external beam and radioembolization are standard-of-care treatments for HCC, we aimed to determine whether there was any relationship between GPC3 and response to radiotherapy. Here, we demonstrate that GPC3 expression confers radioresistance in liver cancer through integrated in vitro, in vivo, and patient-level clinical analyses. Stable GPC3-knockout in liver cancer cell lines (HepG2, Hep3B, Huh7) and ectopic GPC3 expression in GPC3-negative liver cancer cells (SNU449), as well as in non-hepatic A431 cells, demonstrated that GPC3-mediated radioresistance is not restricted to hepatic lineage. Following irradiation, GPC3-deficient cells exhibited reduced proliferation, impaired clonogenic survival, persistent DNA damage, prolonged G2/M arrest, and increased apoptosis. Transcriptomic profiling demonstrated enrichment of cell-cycle and DNA damage response pathways in irradiated GPC3-deficient cells compared with GPC3-positive cells, and protein analyses confirmed sustained activation of the ATM/CHK2 axis. In vivo, GPC3 deletion markedly enhanced radiation-induced tumor growth delay in both HepG2 and A431 xenograft models. Consistent with these findings, high GPC3 expression was associated with inferior clinical outcomes in patients with HCC undergoing external-beam radiotherapy or radioembolization. Together, these findings identify GPC3 as a determinant of radioresistance in liver cancer and suggest its potential utility as a biomarker to guide radiotherapeutic strategies. Significance statementRadiotherapy is an important treatment option for HCC, but biomarkers that predict tumor response remain limited. GPC3 is highly expressed in most HCCs and is being investigated as an important biomarker for diagnosis and treatment of this disease, yet its relationship, if any, on radiosensitivity has not been previously reported. Here, we identify GPC3 as a modulator of radioresistance. GPC3 loss enhances radiosensitivity and is associated with persistent unresolved DNA damage, prolonged G2/M arrest, and sustained activation of the ATM/CHK2 pathway, resulting in delayed tumor growth after irradiation. In a clinical cohort of patients treated with radiotherapy, high GPC3 expression was associated with poorer overall survival. These findings suggest that GPC3 expressing tumors may necessitate either more dose-intense radiotherapy, radiobioligically ablative and/or combined with other modalities, or alternative therapeutic modalities to adequately treat HCC.

Gut microbiota metabolic remodeling is a pivotal determinant in irinotecan-induced enterotoxicity and epithelial damage, although the underlying mechanisms remain unclear. Herein, we discovered that Daikenchuto (DKT), a traditional Chinese prescription for intestinal disorders, alleviated irinotecan-induced enterotoxicity without compromising its anti-tumor efficacy by improving weight loss, diarrhea, intestinal inflammation, and barrier damage, and these effects were partially dependent on gut microbiota. DKT significantly restored microbial tryptophan metabolism in irinotecan-treated rats, which was characterized by the enrichment of Limosilactobacillus reuteri, and elevated levels of indole-3-ethanol (IE) and indole-3-propionic acid (IPA). Multi-omics analysis further revealed a positive correlation between L. reuteri and IE and IPA. Consistent with this, DKT promoted L. reuteri proliferation, leading to the conversion of tryptophan to IE and IPA, which improved epithelial barrier damage in the irinotecan-treated Caco-2 cells. In addition, DKT suppressed the growth of Loop 1 {beta}-glucuronidase ({beta}-GUS)-producing bacteria, such as Escherichia coli. Furthermore, the main constituents of DKT selectively inhibited Loop 1 {beta}-GUS activity independent of the gut microbiota, which reduced the intra-luminal level of 7-ethyl-10-hydroxycamptothecin, the toxic metabolite of irinotecan. Taken together, this study reveals a dual gut microbiota-driven mechanism by which DKT mitigates irinotecan-induced enterotoxicity, which provides a promising strategy for managing chemotherapy-related enterotoxicity.

Cancer-associated fibroblasts (CAFs) are now recognized as key regulators of tumor progression and therapeutic resistance, yet the cancer cells-fibroblasts crosstalk that ultimately promotes chemoresistance remain incompletely understood. Here, we show that direct physical contact between small cell lung cancer (SCLC) cells and lung fibroblasts induces early resistance to standard-of-care chemotherapeutic agents, etoposide and cisplatin. Using both 2D and 3D co-culture models, we demonstrate that early acquired therapy resistance entails cell-cell contact and cannot be recapitulated by conditioned media alone. Mechanistically, direct interaction promotes transcriptional reprogramming in cancer cells, including upregulation of YAP1 and epithelial-to-mesenchymal transcription factors (EMT-TFs), which partially mediate the resistant phenotype. A high-throughput drug screening identified idarubicin as a compound that retains efficacy despite fibroblast-mediated protection, suggesting it could bypass microenvironment-induced resistance early on. Together, our findings identify direct tumor-fibroblasts contact as an early driver of chemoresistance and highlight a potential therapeutic strategy targeting cell-cell interactions within the tumor microenvironment.

PurposeResidual ESCC after neoadjuvant CCRT reflects incomplete treatment response and carries a high risk of recurrence. We sought to define the spatial tumor microenvironmental features of CCRT-resistant residual ESCC -- using tumor regression grade (TRG) 2-3 tumors as a model of poor pathologic response -- with mechanistic refinement at single-cell resolution and outcome assessment in internal and external cohorts. Experimental DesignWe profiled post-CCRT ESCC tissue microarray (TMA) cores by Visium FFPE whole-transcriptome spatial transcriptomics and orthogonal Xenium single-cell-resolution in situ profiling on serial sections from the same TMA blocks. Spot-level cell-type composition was inferred by CellDART deconvolution against a published ESCC single-cell RNA sequencing (scRNAseq). Local malignant-cell-enriched regions were defined by CancerFinder. We additionally re-analyzed the scRNAseq dataset to characterize SPP1 and CXCL5 macrophage states and their ligand-receptor signaling using CellChat. Outcome associations were evaluated in the SNUH cohort and externally tested in the TCGA ESCC cohort. ResultsTRG 2-3 residual tumors exhibited effector immune-cell exclusion from malignant-cell-enriched regions, SPP1/CXCL5 macrophage accumulation, microvascular rarefaction, and proliferative-metabolic reprogramming. Single-cell re-analysis confirmed that CXCL5 macrophages constitute a transcriptional subset of the broader SPP1 macrophage population. CellChat showed that SPP1 macrophages dominantly signal through SPP1-CD44 and SPP1-integrin axes to stromal and epithelial targets, and additionally engage immunosuppressive NECTIN2-TIGIT, CD86-CTLA4, and LGALS9-HAVCR2 programs with CD8 T cells, providing a mechanistic context for local immune exclusion. Visium LIANA and Xenium distance-gradient profiling localized SPP1-associated signaling preferentially to tumor cells and CAFs. Higher SPP1 expression and lower endothelial abundance were associated with shorter disease-free survival in the SNUH cohort. Higher SPP1 expression was also associated with shorter disease-free survival in the independent TCGA ESCC cohort. ConclusionsCCRT-resistant residual ESCC is characterized by a spatially organized tumor microenvironmental niche centered on SPP1-associated macrophage programs and microvascular rarefaction. These spatially resolved findings identify candidate macrophage- and vasculature-targeted axes for overcoming treatment resistance. Translational RelevancePatients with esophageal squamous cell carcinoma (ESCC) who harbor residual disease after neoadjuvant chemoradiotherapy (CCRT) remain at high risk of recurrence, yet the spatial biology of this resistant residual state has been poorly defined. Here, we combine Visium whole-transcriptome and Xenium single-cell-resolution spatial transcriptomics with single-cell RNA-seq re-analysis and external TCGA context to define the tumor microenvironmental architecture of CCRT-resistant residual ESCC. The resistant niche is characterized by effector immune-cell exclusion from malignant-cell-enriched regions, accumulation of SPP1/CXCL5 macrophage programs that signal to tumor cells and cancer-associated fibroblasts through SPP1-CD44 and SPP1-integrin axes, microvascular rarefaction, and proliferative-metabolic reprogramming. These findings define a coordinated spatial tumor microenvironmental signature of CCRT-resistant residual ESCC and nominate macrophage-tumor/stromal interactions and vascular injury as biologic axes that may help guide future strategies to overcome treatment resistance.

Mono-ADP-ribosylation (MARylation) is emerging as an important regulator of anti-cancer immunity and immunosuppressive tumor microenvironment (TME). Our previous studies showed that PARP11, one of several enzymes that facilitate MARylation, regulates the activities of intratumoral cytotoxic T lymphocytes (CTLs) and regulatory T cells (Tregs). Here, we demonstrate that stimuli such as adenosine, epinephrine, or glucagon-like peptide-1 (GLP1) induced PARP11 in cancer cells. Upregulation of PARP11 in cancer cells led to PARP11-mediated MARylation, ubiquitination, and accelerated degradation of MHC-I through the autophagy-lysosomal pathway. Induction of PARP11 protected cancer cells from killing by specific CTLs and stimulated tumor growth and progression. Genetic ablation of PARP11 attenuated MHC-I MARylation, ubiquitination, and interaction with autophagy receptors. Pharmacologic inhibition of PARP11 in pancreatic ductal adenocarcinoma (PDAC) cells restored their MHC-I levels, sensitized them to killing by CTLs, inhibited tumor growth, and impeded their initial resistance to chemotherapy and their acquired resistance to targeted therapy with RAS inhibitors. Moreover, inhibition of PARP11 prevented hyperprogressive disease in a mouse melanoma model treated with immune checkpoint inhibitors (ICBs), suggesting that PARP11 is a major therapeutically actionable driver of immunosuppression in tumors. SYNOPSISInduction of PARP11 in the tumor microenvironment mediates immunosuppression. This study reports that PARP11-driven MARylation and ubiquitination of MHC-I in cancer cells drives immune evasion, tumor growth and resistance to therapies.

While a subpopulation termed cycling persisters (CPs) that is characterized by sustained proliferation, even under chemotherapy drug exposure, contributes more directly to tumor relapse, the molecular basis for the emergence of CPs has been unclear. Here, we used the human tongue cancer organoid (TCO) library to continuously track the in vitro fate of individual cancer cell clones during and after chemotherapy exposure using time-lapse imaging. Among the heterogeneous clones, we identified CPs that formed larger clusters than the others, which barely grew and remained small (non-CPs). Using differences in cell cluster size as an indicator, thousands of CP and non-CP clones were directly sampled from 3D matrix organoid cultures and were analyzed. Notably, tumor-intrinsic interferon (IFN) signaling and hypoxic pathways were inactivated, whereas the NR2F1-mediated cholesterol biosynthesis pathway was distinctly activated in CPs compared to non-CPs. Indeed, inhibiting cholesterol biosynthesis with simvastatin significantly suppressed the appearance of CP clones, showing that elevated cholesterol biosynthesis is essential for the emergence of CPs. These findings suggest that clonal-level variations in the intensity of these signaling pathways determine the fate of individual tumor cells exposed to chemotherapeutic agents, which may provide insights into cancer relapse mechanisms and identify potential molecular targets of CPs.

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.

Squamous cell carcinoma of the lung is a difficult-to-treat cancer with high prevalence in the US, and particularly in Kentucky. The goals of this work were to test if the EZH1/2 inhibitor valemetostat improves anti-PD1 responses in squamous cell lung cancer models, and to develop ex vivo models to test immunotherapy drug combinations. We found that valemetostat produced augmented anti-tumor responses to anti-PD1 therapy through up-regulation of tumor cell specific Major Histocompatibility Complex Class II (MHC Class II), and a shift towards activated CD8+ T cells. Neutrophils predominated in these tumors regardless of therapy, but examination of bone marrow revealed that valemetostat treated mice and mice that rejected tumors both had more mature neutrophils. Likewise, Ezh2 knock-out mice produced neutrophils that were more apoptotic, less migratory, and less able to produce extracellular nets, but had similar ability to kill bacteria as Ezh2-WT neutrophils. To test tumor responses to differing neutrophil populations, we engineered three-dimensional air-liquid interface cultures with tumoroids, lung mesenchymal cells, and T cells, with and without bone marrow containing neutrophils and myeloid progenitors from distinct donors. Bone marrow from tumor-naive or mice with actively growing untreated tumors boosted tumoroid growth, while bone marrow from tumor-rejected or mice with tumors treated with valemetostat was anti-tumor. MHC Class II blockade lowered the ability of bone marrow to boost tumor growth, and reduced the ability of valemetostat with anti-PD1 to reduce tumoroid growth. Patient samples revealed a strong negative correlation between EZH2 and MHC Class II, suggesting that targeting EZH2 activity could lead to marked increase in MHC Class II and improve treatment responses in lung squamous cell carcinomas.

IntroductionThe RAS(ON) multi-selective daraxonrasib (RMC-6236) inhibitor is effective in patients with NSCLC KRAS mutant cancers. Tolerance to daraxonrasib invariably develops by mechanisms that remain unclear. There is no known involvement of the M1C oncogenic protein in daraxonrasib resistance. MethodsNSCLC H358 KRAS(G12C), H2122 KRAS(G12C) and patient derived MGH1112 KRAS(G12C) cells with acquired daraxonrasib resistance were investigated for M1C dependence in studies of SHP2, STAT1/3 and NF-KB activation, clonogenicity, and self-renewal capacity. ResultsWe demonstrate that M1C is induced as a protective response in NSCLC KRAS(G12C) mutant cells treated with daraxonrasib. We report that M1C forms novel cell membrane-associated biomolecular condensates with the SHP2 protein tyrosine phosphatase in driving daraxonrasib resistance. M1C integrates SHP2 activation with induction of (i) oncostatin-m/gp130/STAT3 signaling, and (ii) the NF-{kappa}B-mediated epithelial-mesenchymal transition (EMT) pathway. The functional significance of this M1C-driven pathway is supported by the demonstration that targeting STAT3 and NF-{kappa}B reverses daraxonrasib resistance. Consistent with M1C dependence, we also show that targeting M1C is effective against daraxonrasib-resistant NSCLC KRAS mutant cell line and tumor models. In contrast, M1C drives sotorasib resistance by STAT1-mediated inflammatory signaling, demonstrating that M1C confers resistance to KRAS(G12C)-selective and RAS(ON) tri-complex inhibitors by noncongruent mechanisms. ConclusionsThese findings demonstrate that M1C is required for daraxonrasib tolerance and is a potential target for the treatment of patients with NSCLC KRAS(G12C) mutant tumors refractory to this agent.

Autophagy is a cellular degradation process that recycles dysfunctional components to maintain cellular homeostasis. Beyond this canonical role, autophagy-related proteins, such as the essential autophagy initiation protein ATG7, are increasingly recognized to have autophagy independent functions in diverse biological processes and disease contexts, including cancer progression and metastasis. However, the mechanisms underlying these autophagy independent functions remain unclear. Previously, we identified a short isoform, ATG7(2), that lacks canonical autophagy activity. To understand the unique role of ATG7(2), we analysed clinical data from publicly available databases and found that high ATG7(2) expression is associated with poor prognosis in pancreatic adenocarcinoma (PAAD). Using CRISPR/Cas9 in PAAD cells, we selectively knocked out the canonical isoform ATG7(1) or total ATG7. While total knock-out of ATG7 slowed proliferation and migration of PAAD cells, high levels of ATG7(2) were found to enhance both processes. In addition, RNA sequencing linked ATG7(2) with immune signalling, extracellular matrix organization and cell-cell interactions. Critically, ATG7(2) inhibition in a murine xenograft model substantially reduces tumour growth and overall progression in vivo, establishing functional relevance in a physiological tumour context. Together, these results suggest that ATG7(2) has a role in regulating immune signalling in PAAD cells, contributes to migration and proliferation in an autophagy-independent manner, and suggests ATG7(2) as a potential therapeutic target for the treatment of PAAD.

One-carbon (1C) metabolism is frequently upregulated in cancer to support anabolic growth and nucleotide biosynthesis. However, the contribution of mitochondrial 1C metabolism to cancer stemness and metastatic progression remains incompletely understood. Here, we identify methylenetetrahydrofolate dehydrogenase 1-like (MTHFD1L), a mitochondrial 1C metabolic enzyme, as a critical regulator of breast cancer stemness, tumor initiation, and lung metastasis. Genetic depletion of MTHFD1L in triple-negative breast cancer (TNBC) cell lines and patient-derived breast cancer models markedly impaired proliferation, sphere formation, tumorigenicity, and lung colonization. Metabolomic profiling revealed extensive metabolic rewiring following MTHFD1L loss, characterized by accumulation of the purine biosynthetic intermediates SAICAR and AICAR together with perturbations in glycolytic and pentose phosphate pathways. Importantly, suppression of lung metastasis was accompanied by reduced expression of the stemness-associated transcription factor SOX2 and decreased proliferative activity in metastatic lesions. Collectively, our findings establish MTHFD1L as a key metabolic dependency linking mitochondrial 1C metabolism to stem-like properties and metastatic progression in breast cancer, and highlight MTHFD1L as a promising therapeutic target in metastatic TNBC.

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.