bioRxiv Subject Collection: Cancer Biology

Induced Estrogen Receptor SUMOylation drives SERD activity

2026-6-26

The ligand dependent transcription factor estrogen receptor α (ERα) is a key driver of and important drug target in breast cancer. Patients with advanced disease are typically treated with selective ERα degraders (SERDs), whose therapeutic activity is commonly attributed to induced ERα protein degradation. Yet the exact mechanism and relevance of degradation for clinical efficacy remain unclear. We show that SERDs directly induce ERα SUMOylation, thereby triggering degradation via SUMO-targeted ubiquitin ligases (STUbLs). Inactivation of STUbLs prevents ERα degradation and counterintuitively further sensitizes breast cancer cells to SERDs, rather than conferring resistance. SERD efficacy is independent of ERα degradation, challenging the degradation-centric model of SERD action. Instead, SUMOylation recruits transcriptional co-repressors, turning SUMOylated ERα into a ‘dominant-negative’ repressor. Thus, SUMOylation rather than degradation is the direct consequence and driver of SERD activity. Our findings position SUMO-inducing drugs as a hitherto underappreciated yet clinically validated therapeutic modality with broad applicability.

Clear cell renal cell carcinoma consensus transcriptomic programs reveal converging trajectories towards aggressive disease

2026-6-26

Clear cell renal cell carcinoma (ccRCC) is characterized by a branching genomic trajectory in which early biallelic VHL inactivation splits into PBRM1 - and BAP1 -mutant lineages. However, driver mutations alone do not account for the molecular and phenotypic heterogeneity. To dissect this heterogeneity, we developed a non-negative matrix factorization (NMF)-based gene expression analytical framework for identifying recurrent transcriptional programs across factorization dimensionalities and across datasets. We applied it to three curated datasets (IMmotion151, n = 823; JAVELIN Renal 101, n = 726; TCGA, n = 614) to define 17 consensus transcriptomic programs (CTPs). Mapping CTPs onto single-cell RNAseq (scRNAseq) of human ccRCC tumors and patient-derived tumorgraft models linked these programs to their cellular sources, distinguishing RCC-intrinsic, tumor-cell-extrinsic, and mixed programs. RCC-intrinsic CTPs associated with canonical drivers, including VHL (R1), PBRM1 (R2), BAP1 (R4), PTEN / TSC1 (R3), TFE3/TFEB fusions (R5), NF2 (R6), and CDKN2A / TP53 (MP-Prolif). Additional CTPs captured tumor microenvironment (TME) composition (TME-Tcell, TME-Myelo, TME-Endo, TME-Stroma) and biological processes active across multiple cellular compartments, including proliferation, Y-chromosome–linked expression in male tumors, ciliary biology, and translation. Trajectory inference methods revealed PBRM1 -like and BAP1 -like branches that converged on a shared aggressive late transcriptomic stage (TS) associated with higher nuclear grade, additional driver alterations, myeloid/stromal infiltration, and poor clinical outcomes. Spatial transcriptomics (and multiregional sequencing) of paired conventional ccRCC and sarcomatoid regions linked TS advancement with morphological progression and clonal evolution. After adjusting for stage, grade, and BAP1 / PBRM1 status, TS remained independently prognostic. Furthermore, our data suggest that immune checkpoint inhibitor combinations are particularly beneficial for MP-Prolif and not R1 utilizing specimens. In summary, we present an atlas of recurring transcriptomic programs in RCC and an ontological framework bridging genotype, tumor-cell-intrinsic gene expression, and microenvironment remodeling, with implications for risk stratification and treatment selection in ccRCC.

Combined agonism and antagonism of canonical and non-canonical progesterone receptors in triple-negative breast cancer cells potentiates cytotoxicity enhanced by PI3K inhibition

Petrella, P., Chen, J., Cosgrove, B. — 2026-6-26

Confounding the treatment options available to patients with triple-negative breast cancer (TNBC) are not only its purported lack of hormone receptor and growth factor receptor targets (ER - / PR - / HER2 - ), but its enrichment in plastic and chemoresistant breast cancer stem cells (BCSCs). Although descriptions of non-canonical PR expression in TNBC are rife in the literature, only canonical PR is considered in the definition of TNBC and is used to determine therapeutic strategy, not least because the utility of non-canonical PR modulation in TNBC chemoresistance is largely unexplored and poorly understood. Here we document the expression of three non-canonical PRs and the canonical PR (PGR) phosphorylated at Ser345 (p-PGR S345) in a panel of TNBC and luminal breast cancer cell lines, and employ combined PR agonists and antagonists to investigate the influence of PR activity on TNBC cell viability and PI3K inhibitor cytotoxicity. To examine the contributions of non-canonical membrane-associated PRs mPRβ and PGRMC1, we tested the agonist Org OD 02-0, a synthetic progestin targeted to mPRs; the PGRMC1 antagonist Ag-205; and the antagonist SPA70 against the cytosolic/nuclear PXR, in the background of pan-PI3K inhibition with Buparlisib (BUP). We also reveal that combinations of agonists and antagonists targeted to canonical and non-canonical PRs robustly potentiate the cytotoxic effects of PI3K inhibition, and also exhibit significant cytotoxicity on their own. Using functional assays, flow cytometry, immunocytochemistry and protein expression analyses, we found that simultaneously perturbing PRs and inhibiting PI3K function resulted in significantly greater cell death than vehicle control or BUP alone, and reduced the proportion of ALDH1 + BCSCs in two TNBC cell lines. We conclude that four types of PR are tractable targets in TNBC which participate in cell viability and enhance chemotherapy-induced cytotoxicity, and should be re-evaluated in an evolving definition of this challenging disease.

A Paracrine Dietary Lipid Axis Constrains Antitumor Immunity in Liver Cancer

2026-6-26

Overnutrition-related liver dysfunction and cancer are increasingly prevalent and highly resistant to immunotherapy. While metabolic dysregulation is a hallmark of hepatocellular carcinoma (HCC), how nutrient overload impairs antitumor immunity remains unclear. Here, we show that short-term Western diet (WD) exposure drives near-complete loss of CD8⁺ T cell infiltration and antitumor function in HCC. We identify dietary linoleic acid (LA), the most abundant ω-6 fatty acid, as the dominant immunosuppressive driver. Cancer cell-restricted FADS2-mediated desaturation of LA to longer-chain ω-6 PUFAs drives their accumulation in the tumor interstitial fluid, suppressing infiltrating CD8⁺ T cells via lipid peroxidation. FADS2 inhibition restores CD8⁺ T cell function and sensitizes WD-driven HCC to PD-1-based immunotherapy. Further, the Parkinson’s disease-associated deglycase DJ-1 protects LA-handling proteins from methylglyoxal-mediated glycation, sustaining tumoral immunosuppressive PUFA production. Across multiple independent human MASLD-HCC cohorts, LA metabolic activity correlates with CD8⁺ T cell impairment, immune exclusion, and immunotherapy resistance. Overall, these studies identify a dietary lipid axis as a therapeutically actionable vulnerability in WD-associated HCC.

Targeting Cellular Pseudo-Senescence to Overcome PARP inhibitors Resistance in BRCA1 -Mutated Breast Cancer

2026-6-26

Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) are a mainstay therapy for homologous recombination (HR)-deficient cancers; however, resistance remains a major clinical challenge. Previously, through a genome wide CRISPR screen, we identified ZNF251 haploinsufficiency as a novel driver of PARPi resistance. In BRCA1 -mutant ( BRCA1 mut ) cells, ZNF251 deficiency led to HR hyperactivation, conferring PARPi resistance that could be reversed by RAD51 inhibition. In this study, we further show that ZNF251 deficiency induces replication stress and a pseudo-senescence state, in which cells exhibit molecular and phenotypic markers of senescence while retaining proliferative capacity. Because senomorphic and senolytic therapies can target senescent cells, we tested whether these approaches could overcome PARPi resistance in ZNF251 -deficient breast cancer cells. Critically, targeting this senescence-like state with either senomorphic agents, such as cytokine inhibitors, or senolytic agents, such as BCL-2 and BCL-XL inhibitors, overcame PARPi resistance ex vivo and in vivo . Importantly, pseudo-senescence was also observed in other PARPi-resistant contexts driven by HR hyperactivation, including 53BP1 - and Shieldin -mutant cells, suggesting that it may represent a broader mechanism underlying PARPi resistance in breast cancer. Furthermore, in two olaparib-resistant, BRCA -mutant triple-negative breast cancer organoid models, treatment with DT2216, a BCL-XL–targeting PROTAC, sensitized both models to olaparib. Together, our work defines a novel pathway linking HR hyperactivation, replication stress, and pseudo-senescence, and positions both senomorphic and senolytic therapies as promising strategies to overcome PARPi resistance in BRCA1 mut breast cancer. Highlights ZNF251 deficiency drives PARP inhibitor resistance through HR hyperactivation, replication stress, and pseudo-senescence in BRCA1 -mutant breast cancer. Senomorphic and senolytic therapies overcome PARP inhibitor resistance in vitro , in vivo , and in patient-derived organoid models. Pseudo-senescence represents a shared vulnerability of HR-hyperactivated PARPi-resistant cancers and can be therapeutically targeted.

An immunocompetent Merkel cell carcinoma model for preclinical studies

2026-6-26

Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine skin cancer that frequently carries integrated Merkel cell polyomavirus DNA and expresses oncogenic viral small T antigen (sTAg) and truncated large T antigen (tLTAg). We previously reported a mouse model of MCC with skin-targeted expression of sTAg, tLTAg, and the Merkel cell transcription factor ATOH1, combined with deletion of Trp53 . Here, we optimized this model to achieve 100% tumor penetrance with lymph node metastases, established four mouse MCC cell lines, and selected one line, mMCC2, for pilot preclinical trials. In immunocompetent C57BL/6J mice, mMCC2 cells reliably produce MCCs and lymph node metastases following subcutaneous or intradermal (orthotopic) injection, and liver and lung metastases after tail vein injection. Mouse MCC allografts resemble parental tumors histologically and express a full complement of MCC differentiation markers. Treatment of allografted mice with anti-PD-1 resulted in variable inhibition of tumor growth. In contrast, treatment with lysine-specific histone Wdemethylase 1 (LSD1) inhibitors, with or without anti-PD-1, led to consistently lower tumor volumes by 5.7-fold in both groups (P < 0.0001) and smaller or undetectable lymph node metastases. Growth-inhibited tumors in all groups showed a marked reduction in proliferating tumor cells and increased infiltration by F4/80+ macrophages and CD8+ T cells. These findings support a role for immune-cell recruitment in treatment response and underscore the importance of immunocompetent preclinical models, even in studies using targeted therapies. This unique virus-positive MCC allograft model, which produces local tumors as well as regional and distant metastases in immunocompetent hosts, provides a critical platform for preclinical evaluation of new therapeutic strategies and sets the stage for much-needed translational studies to inform future clinical trials.

Spatial Metabolic Modeling Reveals Zinc–Citrate Rewiring and Therapeutic Vulnerabilities in Prostate Cancer

Zargar, M., Malla, S., Raghunath, V., Saha, R., Chowdhury, R. — 2026-6-26

Prostate cancer exhibits a metabolic phenotype distinct from the Warburg paradigm, characterized by reprogramming of the zinc–citrate secretory axis that normally drives citrate accumulation and secretion in prostatic epithelium. How this metabolic rewiring is organized across the spatial architecture of tumors and linked to androgen receptor (AR) signaling remains poorly understood. We integrated spatial transcriptomics data from twelve regions spanning normal, inflamed, and Gleason-graded prostate tissues with genome-scale metabolic modeling to quantify metabolic activity across tissue states. The analysis identified the zinc–citrate–aconitase axis as a central feature of prostate cancer metabolic reprogramming, linking increased aconitase activity to enhanced citrate export, acetyl-CoA generation, and de novo lipogenesis. Metabolic similarity was more strongly associated with spatial proximity than with histological classification, suggesting field-level metabolic reprogramming beyond visible pathological boundaries. AR activity displayed grade-dependent associations with lipid metabolic pathways, indicating heterogeneous coupling between androgen signaling and metabolism across tumor regions. Consistent with the established biology of prostate cancer, predicted flux distributions did not support a classical Warburg phenotype. A flux-based target prioritization framework identified HMGCR, FASN, and SLC25A1 as candidate therapeutic targets, with independent support from TCGA-PRAD expression profiles and DepMap CRISPR essentiality data. Together, these findings provide a spatially resolved view of prostate cancer metabolism, establish the zinc–citrate axis as a dominant feature of metabolic organization, and identify candidate metabolic vulnerabilities for therapeutic intervention.

INHIBITION OF LIGAND-DEPENDENT BMP SIGNALING BLUNTS MELANOMA GROWTH

2026-6-26

Treatments for advanced melanoma have markedly improved, but a significant proportion of patients still receive little to no survival benefit with standard-of-care therapies due to resistance and relapse 1–5 . The discovery and development of novel targets and therapies are needed to continue to improve patient outcomes in advanced melanoma. The identification of ligand-dependent BMP signaling that inhibits differentiation and promotes survival of melanoma cells suggests it is a potential therapeutic target that could complement current therapies 6 . Expression of the BMP ligand GDF6 (a.k.a BMP13) is responsible for this activity, and its expression is correlated with poor outcomes for melanoma patients. Here, we describe a novel monoclonal antibody targeting GDF6 that causes melanoma cell differentiation and death and blunts tumor growth in vivo. Together, these results indicate BMP-directed therapy has significant potential as a novel therapy for patients with advanced melanoma.

The Neuro-Immune Axis in Glioma Spontaneous Remission: Evidence from Population-Level Latent Variable Modelling and Transcriptomic Profiling

Kapoor, A., Tiwari, A., Srivastava, S. — 2026-6-24

Gliomas are primary brain tumours that develop from neural stem or progenitor cells containing oncogenic alterations. Gliomas undergo remission with partial or complete disappearance of the disease, and in rare instances, spontaneous remission. Spontaneous remission happens without treatment or with inadequate medical intervention. This is a rare but well-documented phenomenon and has been observed across various tumour types, including gliomas, with varying frequency. While historically viewed as clinical anomalies, we hypothesize that SR may be driven by a measurable, latent neuro-immune axis specifically, autonomic vagal nerve modulation of the tumour microenvironment (TME) via the cholinergic anti-inflammatory pathway. Using a Gamma frailty Cox proportional hazards model on a SEER cohort of 6,939 glioma patients, we identified a latent biological variable (Z) that explains 27.1% of survival variance independent of age, grade, treatment, and tumour location. To determine the molecular basis of this latent survival advantage, we applied parallel frailty and Cox models to the TCGA Lower Grade Glioma and Glioblastoma (LGG+GBM) cohorts. Clinical validation confirmed expected hazards for age and grade, with the frailty model achieving high predictive accuracy (5-year AUC = 0.840; 10-year AUC = 0.841). Transcriptomic integration revealed that the alpha7 nicotinic acetylcholine receptor (CHRNA7) is highly protective, inversely correlating with biological frailty (r = -0.285, p < 0.0001). Conversely, pro-inflammatory cytokines (IL6) and M2 macrophage markers (CD163) positively correlated with frailty. Grade-stratified Cox regression and Kaplan-Meier analyses confirmed that CHRNA7 confers a significant survival advantage entirely independent of tumour grade. Single-cell RNA-sequencing data (Core GBmap) confirmed that CHRNA7 and TLR4 are expressed heavily on tumour-associated macrophages and microglia, rather than malignant cells. Our in-silico integration suggests that high vagal tone releases acetylcholine, binding to alpha7nAChR on TME macrophages. This triggers a signalling cascade that dampens the IL-6 production required for glioma proliferation, effectively halting tumour growth. Ongoing in vitro wet-lab experiments utilizing specific alpha7nAChR agonists (GTS-21) and physiological stress models aim to clinically validate this vagal-immune mechanism.

Dedifferentiation unlocks keratinocyte competence for metaplasia and tumorigenesis in the foregut

Drubbel, A., Pirard, S., BECK, B. — 2026-6-25

Summary How tissue injury shapes cell competence to undergo malignant transformation remains poorly understood. Esophageal metaplasia, a precancerous lesion driven by chronic acid reflux, can arise from the conversion of squamous progenitors into a columnar-like state, but the factors governing this plasticity remain unclear. Here we show that GATA4, overexpressed in esophageal metaplasia and adenocarcinoma, drives columnar metaplasia in squamous progenitors at the squamo-columnar junction but is insufficient, and even toxic, in keratinocytes outside this region. Using inducible transgenic mouse models, we find that reactivation of Hedgehog signaling expands the pool of progenitors permissive to GATA4-mediated reprogramming, driving gastric-like metaplasia even within the esophagus. Combined Hedgehog activation and GATA4 expression further induce adenosquamous-like neoplasms and stromal and immune remodeling reminiscent of the metaplastic microenvironment. Since Hedgehog signaling is reactivated by gastroesophageal reflux, chronic injury may generate a field of dedifferentiated progenitors poised for malignant progression upon oncogene acquisition. These findings demonstrate that a prior cell state transition, induced by environmental injury, can unlock oncogenic competence, establishing a mechanistic framework linking epithelial plasticity, developmental transcription factor reactivation, and lineage-specific cancer susceptibility with broad implications for precancerous metaplastic states.

A microscopy-based readout to assess tumour-specific viability in neuroblastoma co-cultures and short-term cultured patient samples

2026-6-25

High-risk neuroblastoma patients face poor survival despite intensive treatment. Drug testing using patient-derived models can support therapy prioritization for precision medicine and drug development. Models incorporating tumour microenvironmental components, such as co-cultures and short-term cultured patient samples containing substantial non-malignant cell fractions, could better recapitulate microenvironment-dependent drug responses. However, conventional viability assays measure the combined signal from all viable cells in a well and therefore cannot determine tumour-specific drug responses. Here, we establish a microscopy-based readout to quantify cell-type-specific viability in two complementary settings: neuroblastoma-PBMC co-cultures and freshly dissociated patient tumour samples. In the co-cultures, PBMCs were pre-labelled with a cell-tracking dye, and Calcein staining was used to independently quantify the viability of tumour cells and PBMCs in the same well. The Calcein-based viability readout correlated strongly with conventional CellTiter-Glo measurements and was compatible with automated high-throughput drug screening. The imaging workflow enabled identification of compounds with differential efficacy in co-culture versus monoculture and distinguished tumour-specific effects from PBMC toxicity. The microscopy-based viability readout was further adapted to short-term cultured patient samples. Neuroblastoma tumour cells were distinguished from the non-malignant cells using a combination of tumour-specific surface markers NCAM, L1CAM and B7H3. This enabled determination of tumour fractions and measurement of tumour-specific drug responses. Tumour fractions varied substantially between patient samples, highlighting the importance of tumour-specific viability measurements. Together, the microscopy-based viability readout for co-cultures and patient samples enables scalable assessment of tumour-specific drug responses.

Therapy-induced senescent-like cancer cells drive macrophage-mediated immunosuppression in cholangiocarcinoma

Li, B., Yang, J., Cai, M., Yee, S., Carlson, D., Smoot, R., Baker, D., Ilyas, S. — 2026-6-25

Cholangiocarcinoma (CCA) is a lethal biliary cancer in which chemoresistance is nearly universal, and its tumor immune microenvironment is dominated by immunosuppressive tumor-associated macrophages (TAMs) that exclude cytotoxic CD8 + T cells. How tumor cells sustain this immunosuppressive state during chemotherapy is undefined. Here we demonstrate that therapy-induced senescent-like (Sen-L) cancer cells accumulate after gemcitabine/cisplatin in human and murine CCA, are predominantly cancer cells, and predict shorter survival. Genetic elimination of Sen-L cancer cells reduces tumor burden, lowers TAM abundance, and restores intratumoral CD8 + T cells, establishing them as causal drivers. Growth differentiation factor 15 (GDF-15) is the dominant Sen-L-secreted factor and reprograms macrophages to suppress CD8 + T cells through the non-canonical receptor TGFBR2 and STAT6, and p16-restricted Gdf15 silencing phenocopies Sen-L elimination. Combined with chemotherapy, Sen-L elimination improves survival beyond chemotherapy alone. These findings establish Sen-L cancer cells and their GDF-15 output as causal, targetable drivers of macrophage-mediated immune evasion in CCA. SIGNIFICANCE STATEMENT Therapy-induced senescent-like cancer cells, not stromal cells, are the dominant senescent-like and immunosuppressive population in cholangiocarcinoma, and their elimination restores antitumor immunity. GDF-15 is their dominant secreted effector and engages a non-canonical macrophage receptor, TGFBR2, identifying a cancer-cell-to-macrophage axis and a Sen-L-elimination strategy to restore chemosensitivity. Figure Graphical abstract. Senescent-like CCA cells promote tumor immunosuppression through TAMs polarization by GDF-15

Multi-omics Profiling Reveals an NF-κB-Driven Anti-apoptotic Network Underlying Resistance to Oncolytic VSV in Prostate Cancer Cells

Abdelmageed, A., Dewhurst, S., Ferran, M. — 2026-6-25

The therapeutic efficacy of oncolytic viruses is often limited by the presence of tumor cells that resist virus-mediated killing. Here, we investigated the molecular mechanisms underlying resistance to Vesicular Stomatitis Virus (VSV) in PC3 cells, an aggressive metastatic prostate cancer (PrCa) cell line, using the VSV-sensitive LNCaP cell line as a comparator. RNA sequencing revealed that, relative to untreated cells, VSV-infected PC3 cells upregulated both pro-apoptotic genes, including BIM, PUMA, and NOXA, and anti-apoptotic and antiviral genes, including A20 and RIG-I. In addition, genes associated with antiviral and pro-survival pathways, including NFκB and PI3K-Akt signaling, were more highly expressed in PC3 cells than in LNCaP cells. At baseline, PC3 cells also exhibited elevated expression of multiple pro-survival genes, including BCL-xL, MCL1, and CK2, compared with LNCaP cells. Complementary proteomic analyses identified enhanced activation of NFκB, PI3K-Akt, and MSK1 signaling in VSV-infected PC3 cells relative to infected LNCaP cells. Furthermore, pharmacological inhibition of BCL-2 family proteins or NFκB signaling restored sensitivity to VSV-induced cell death in PC3 cells. Collectively, these findings identify NFκB-centered pro-survival signaling networks as key contributors to the resistant phenotype of PC3 cells and suggest that combining oncolytic virotherapy with targeted inhibitors may improve therapeutic efficacy in resistant prostate cancers.

Alternative splicing reshapes protein interaction networks through structured, directional rewiring across cancers

Zhong, J., Dannenfelser, R., Yao, V. — 2026-6-25

Alternative splicing generates extensive transcript diversity in cancer, but whether the resulting changes to protein-protein interaction (PPI) networks are structured across tumors or idiosyncratic to individual patients remains unresolved. Existing analyses catalog splicing events and their effects on individual interactions, but do not reconstruct patient-specific interaction networks at scale, leaving the systems-level architecture of splicing-driven rewiring uncharacterized. Here, focusing on exon skipping, the most prevalent form of alternative splicing in humans, we analyze 7,950 tumors across 28 cancer types to reconstruct patient-specific rewired PPI networks relative to matched normal tissues, distinguishing interaction gains from losses. We identify widespread and structured remodeling of protein connectivity, including a unique pattern of interaction gains in brain cancers, a conserved pan-cancer axis of interaction loss that converges on GTPase and Ras signaling, and a subset of genes that function as bidirectional switches, where rewiring direction varies across cancer types. At the patient level, tumors exhibiting extreme rewiring, especially pronounced interaction gains, are associated with significantly poorer survival, independent of tumor stage and mutational burden. These results delineate systems-level patterns through which alternative splicing reshapes protein interaction networks and contributes to tumor heterogeneity across cancers.

Pathway-centric multi-omics and functional precision medicine reveal shared drug vulnerabilities in heterogeneous adult Wilms tumor

2026-6-25

Wilms tumor, i.e., nephroblastoma, is rare in adults and lacks standardized treatment, complicating clinical decision-making. Within the functional precision medicine study (DEDUCER), we profiled two spatially distinct tumor regions (T1 and T2) of an adult Wilms tumor patient using integrated histopathology, whole-exome sequencing, FFPE transcriptomics, and ex vivo drug screening of short-term cultured patient-derived cancer cells (PDCs) with 528 compounds. Genomic profiling revealed a truncal ASXL1 frameshift and shared F7, UBA1, COL21A1, and ATM variants alongside region-specific alterations: a TP53 mutation and broad copy-number (CN) gains in T1, versus ARID1A and KMT2D stop-gains in copy-neutral T2. Transcriptomics of tumor areas identified convergent activation of the G2/M checkpoint, E2F targets, and mitotic spindle programs across regions, consistent with high proliferation and partially comparable biomarker signatures to those observed in an open-source pediatric Wilms tumor dataset (n = 130). Functional assays uncovered distinct and shared drug vulnerabilities: although ATM alterations were present in both tumors, T1 PDCs showed selective sensitivity to topoisomerase I and BCL-2 inhibition in the context of an additional T1-specific TP53 alteration, while broader single-agent sensitivity and stronger drug synergies were observed in T2. Pathway-centric data integration indicated that differential gene expression and copy-number gains, rather than single mutations alone, better predicted ex vivo drug responses, revealing actionable shared dependencies despite pronounced spatial heterogeneity and establishing a translational framework for individualized management in this rare disease. HIGHLIGHTS – In the adult Wilms tumor, multi-region genomics revealed a truncal ASXL1 frameshift together with F7, UBA1, COL21A1 and ATM mutations across two tumor regions (T1 and T2), as well as region-specific alterations: TP53 mutation and widespread copy-number gains in T1, versus ARID1A and KMT2D stop-gains in copy-neutral T2, illustrating spatial heterogeneity. – Transcriptomics showed convergent activation of E2F targets, G2/M checkpoint, and mitotic spindle programs in both regions, consistent with high proliferation and aligning with Wilms tumor signatures (TARGET dataset); these pathways were associated with higher ex vivo drug sensitivity scores. – Functional drug sensitivity testing of patient –derived cancer cells ex vivo uncovered distinct and shared vulnerabilities: Although both tumors shared an ATM mutation, T1-specific TP53 alteration and death-pathway/stress-response alterations may underlie selective sensitivity to topoisomerase I inhibitors and BCL-2 inhibition. – Clinically relevant combinations, including vincristine plus dactinomycin and doxorubicin plus dactinomycin, showed ex vivo synergy. These findings are consistent with the patient’s more than five-year relapse-free outcome following vincristine, doxorubicin, and dactinomycin treatment combined with surgery, supporting the translational relevance of the ex vivo drug testing approach. – Pathway-centric integration (copy-number gains and differential expression) predicted drug response better than single-gene biomarkers. Overall, pathway-level dependencies provide robust, actionable targets despite genomic and phenotypic heterogeneity in adult Wilms tumor.

Api5 Regulates Genomic Stability and Chemotherapy Resistance in Cancer

2026-6-25

Api5 is elevated in a number of cancers and is associated with many hallmarks of cancer, including resistance to apoptosis, immune escape, stemness, chemotherapy resistance, high proliferation, and cell-cycle dysregulation. In this study, we identified the DNA and chromatin-binding activities of Api5 in tumorigenic cells, as well as its association with genomic instability and chemotherapy resistance. Knockdown of Api5 resulted in reduced nuclear volume, DNA content, and chromosome number, and increased sensitivity to DNA damage. The survival of Api5-knockdown cells decreased following UV and cisplatin treatments due to the accumulation of damaged DNA and inefficient nucleotide excision repair. Interestingly, Api5 knockdown cells also exhibited low pChk1 levels following UV damage. Further, we confirmed the chemotherapy resistance phenotype in cancers with elevated Api5 levels, demonstrating that xenograft tumours with Api5 knockdown responded better to cisplatin, with significant tumour regression. Summary Apoptosis inhibitor 5 (Api5) contributes to chemotherapy resistance by conferring a survival advantage and promoting efficient DNA repair following genotoxic stress through regulation of Chk1 activation.

Efficacy of RMC-6236 (Daraxonrasib) and novel combination strategies targeting resistance in RAS pathway-driven neuroblastoma

Valencia-Sama, I., Kee, L., Weiss, A., Hayes, M., Ohh, M., Irwin, M. — 2026-6-25

Metastatic neuroblastoma (NB), the most common pediatric extra-cranial solid tumor, has a cure rate of <50%. DNA-sequencing studies have demonstrated rare recurrent driver mutations at diagnosis, with the most common alterations detected in ALK-RAS-MAPK pathway. Activating ALK and RAS-MAPK mutations are associated with inferior outcome and are increased at relapse, and thus, represent therapeutic vulnerabilities in NB. Previously, we identified combinations of RAS/MAPK inhibitors, including SHP2 and MEK, with efficacy in resistant MAPK-altered tumor cells, including those with the most common NB-associated RAS mutation NRAS-Q61K. However, toxicities of SHP2 inhibitors and promising results using compounds that directly target RAS suggest there may be superior strategies to target RAS/MAPK pathway in NB. Here, we have assessed the efficacy of RAS/MAPK inhibitors, including tovorafenib (pan-RAF), RMC-6236/daraxonrasib (pan-active-RAS) and avutometinib (RAF/MEK) in NB in vitro and in vivo using NB models harboring differing genomic status of RAS/MAPK pathway effectors. We demonstrate selective efficacy of RMC-6236 and avutometinib via RAS-MAPK pathway inhibition in NB cells and xenografts harboring RAS, NF1 or ALK alterations. Importantly, we demonstrate that presence of the NRAS-Q61K mutation confers drug sensitivity. Using newly generated and previously established NB cell models of acquired resistance to RMC-6236 or the ALK inhibitor lorlatinib, we identified targeted combinations, including RMC-6236 plus avutometinib, that demonstrate re-sensitization in resistant NB cell and xenograft models. Finally, transcriptomic studies of RMC-6236-resistant cells detected upregulation of RAS/MAPK signatures, as well as TNFα/NFκB and IL-6/JAK/STAT3 pathway enrichment, thus informing future combinations to enhance sensitivity to RAS inhibitors. STATEMENT OF SIGNIFICANCE Our work demonstrates that newly available RAS pathway inhibitors RMC-6236/daraxonrasib and avutometinib have efficacy in neuroblastoma tumors, which have frequent alterations in the RAS/MAPK pathway. These drugs with early efficacy results in adult RAS-driven tumors provide an important option for patients with relapsed neuroblastoma alone or in combination.

Focal radiotherapy improves CAR T cell therapy targeting prostate cancer

2026-6-24

Chimeric antigen receptor (CAR) T cell therapy has limited efficacy against solid tumors such as prostate cancer due to the immunosuppressive tumor microenvironment (TME). Combining CAR T cells with existing therapies that remodel the TME and promote endogenous immune responses, such as radiation therapy and chemotherapies, may strengthen antitumor responses. Here, we assessed the potency of combining focal radiotherapy (RT), cyclophosphamide (Cy) preconditioning, and prostate stem cell antigen (PSCA)-CAR T cells against syngeneic prostate cancer models. Focal RT alone increased T cell and dendritic cell infiltration and activation in the irradiated tumor. Furthermore, the combination of all three therapies was critical for enhanced antitumor responses and survival across multiple subcutaneous, bone-metastatic, and multifocal disease models. This combination, in the irradiated TME and tumor-draining lymph nodes (tdLN), led to greater antigen presentation by myeloid cells and endogenous T cell activation and cytotoxicity. Our study demonstrates the potency of combining focal RT with PSCA-CAR T cells, significantly improving therapeutic responses in the irradiated tumor and contributing to a more robust systemic immune response against metastatic burden in prostate cancer.

A glycoRNA switch for malignancy: SNORA73B activates TIAR-dependent oncogenic signaling in lung adenocarcinoma

2026-6-23

Although glycosylated small non-coding RNAs are emerging players in cancer, their functions in lung adenocarcinoma (LUAD) are largely unknown. We identify SNORA73B as a glycosylated small nucleolar RNA (glycol-snoRNA) that carries sialic acid-capped O-glycans in both normal lung epithelial and LUAD cells. SNORA73B is markedly elevated in LUAD, and its plasma levels distinguish early-stage LUAD from healthy controls with an area under the curve (AUC) of 0.7903. Subcellular fractionation reveals predominant nuclear localization. Functional assays demonstrate that SNORA73B depletion curbs LUAD cell proliferation, migration, and invasion, whereas its overexpression fosters these malignant phenotypes and accelerates tumor growth. Mechanistically, SNORA73B directly binds the T-cell-restricted intracellular antigen-related protein (TIAR), thereby enhancing TIAR protein abundance without affecting its mRNA levels. TIAR then recognizes the 3’-untranslated region (3’-UTR) of MYC mRNA to upregulate c-Myc, which subsequently augments AKT phosphorylation. Importantly, c-Myc knockdown largely rescues the oncogenic phenotypes and tumorigenesis induced by SNORA73B overexpression. Collectively, our data unveil a glycoRNA-dependent oncogenic axis SNORA73B-TIAR-c-Myc-AKT that drives LUAD progression. These findings position SNORA73B as a promising early diagnostic biomarker and a candidate therapeutic target in LUAD.

Deep Transfer Learning for Dormancy and Outbreaking State Classification in Metastatic Breast Tumor Cells: A Benchmark of Modern Deep Learning Models

Sharma, O., Weidenfeld, K., Barkan, D., Gal, O. — 2026-6-23

Breast cancer cells that disseminate to distant organs can remain dormant (non-proliferative) for years before reactivating and progressing into lethal metastatic disease. Understanding the transition between dormancy and reactivation is therefore critical for early intervention and treatment. In this study, we investigate a comprehensive range of deep learning (DL) architectures to classify dormant versus proliferative breast tumor cells within a 3-dimensional growth factor reduced basement membrane extract (3D BME) system that models tumor dormancy and outgrowth. To capture the underlying spatiotemporal dynamics, we evaluate both spatial and sequence-based learning approaches. We consider convolutional neural networks (EfficientNet, ResNet, DenseNet, MobileNet, VGG, AlexNet), segmentation-based models (U-Net, U-Net++, Attention U-Net, DeepLabV3, HRNet) and transformer-based architectures (Vision Transformer, Swin Transformer, SegFormer). We investigate transfer learning using both fixed and fine-tuned strategies. Experimental results show that classification performance is greatly enhanced through the integration of temporal information. EfficientNet-B7, EfficientNet-B6, DenseNet-169, and DenseNet201 are consistently better than competing architectures for all tested models. EfficientNet-B7 with the use of temporal sequences input reaches an accuracy of 98.86% with a ROC-AUC of 0.998. The results highlight the significance of spatio-temporal feature learning and the value of DL frameworks in automated classification of dormant versus proliferative breast cancer cells in physiologically relevant microenvironments.

M1C IS NECESSARY FOR DARAXONRASIB RESISTANCE OF NSCLC KRAS(G12C) MUTANT CELLS

2026-6-23

Introduction The 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. Methods NSCLC 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. Results We 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-κB-mediated epithelial-mesenchymal transition (EMT) pathway. The functional significance of this M1C-driven pathway is supported by the demonstration that targeting STAT3 and NF-κB 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. Conclusions These 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.

Tumor Microenvironment Modulates Lineage Plasticity in Lung Squamous Cell Carcinoma

2026-6-23

Lung squamous cell carcinoma (LUSC) is the second most common type of lung cancer, yet therapeutic options remain limited. A deeper understanding of its biology and molecular pathogenesis is essential for developing new treatment strategies. Here, we investigated the mechanisms of phenotypic plasticity in LUSC by comparing organoid-derived orthotopic lung models (ODOLs) and subcutaneous xenograft models (ODXs). ODXs showed greater tumor growth, squamous differentiation, and extracellular matrix (ECM) organization compared to ODOLs. Transcriptomic analyses revealed upregulation of multiple HIF1α and SOX2 target genes together with enhanced hypoxia signaling in ODXs. CRISPR/Cas9-mediated HIF1α -knockout ODXs showed reduced SOX2 expression, tumor growth, and ECM organization, whereas SOX2 -knockout ODXs reduced tumor growth without affecting HIF1α and ECM organization. These results indicate that HIF1α regulates squamous lineage maintenance through SOX2 and ECM remodeling. Spatial transcriptomics revealed enrichment of basal cell-like and squamous-differentiated tumor states in ODXs, whereas ODOLs displayed less differentiated phenotypes. These findings identify the tumor microenvironment as a critical determinant of lineage plasticity in LUSC and provide mechanistic insight into how hypoxia shapes tumor differentiation.

Rewiring of EGFR oncogenic program by opposing actions of membrane versus soluble CD109 in HNSCC

2026-6-23

The epidermal growth factor receptor (EGFR) expression is often dysregulated in head and neck squamous cell carcinoma (HNSCC), driving cancer cell proliferation, invasion, and metastasis through diverse pathways, thereby contributing to aggressive chemo- and radio-therapy resistance. A GPI-anchored protein, CD109 is upregulated in multiple cancers, including HNSCC. While membrane-anchored CD109 (mCD109) is pro-tumorigenic in SCC via EGFR/STAT3 activation, the role of protease-cleaved soluble CD109 (sCD109) is poorly understood. Our groundbreaking findings demonstrate that sCD109 antagonizes EGFR signaling by directly binding to the EGFR extracellular domain, preventing mCD109-EGFR stabilizing interactions on the cell surface, followed by inhibition of EGFR phosphorylation at Y1068 and downstream signaling cascades (AKT, MAPK, and STAT3) consequently suppressing cancer cell migration, invasion, 3D tumor spheroid formation and angiogenic tube formation. In addition, we found that sCD109 regulates EGFR fates by inhibiting nuclear localization of phosphorylated EGFR and promoting EGFR degradation. Additionally, sCD109 significantly reduces EGF-induced expression of cancer stem cell markers (CD44 and CD133) and embryonic stem cell markers (Nanog and Sox2), suggesting a suppressive role in cancer stemness. Taken together, these results underscore the opposing roles of mCD109 and sCD109: with sCD109 acting as an antagonist by inhibiting mCD109/EGFR-driven oncogenic signaling and phenotypes. Our current findings reveal a complex interplay among mCD109, sCD109, and EGFR, identifying a mechanism for targeting EGFR’s degradation in HNSCC, and lay the groundwork for future research on investigating sCD109’s modulatory role in preclinical models of HNSCC.

Tyrosine hydroxylase–mediated neuroimmune crosstalk regulates antitumor immunity in glioblastoma during oncolytic herpes virotherapy

2026-6-23

Neuroimmune crosstalk is increasingly recognized as a key regulator of tumor progression and therapeutic response, yet its role in central nervous system (CNS) tumors remains poorly understood. Here, we investigate tyrosine hydroxylase (TH)–mediated neuronal signaling in glioblastoma (GBM) and its impact on antitumor immunity and response to oncolytic virotherapy (OV). We show that TH⁺ cells are widely distributed within the GBM microenvironment, including neurons, astrocytes, and immune cells, and are enriched at the tumor margin. In addition, TH⁺ cells are present in the tumor-draining lymph nodes (TDLNs) of GBM, where they localize near lymphatic vessels and are associated with lymphangiogenesis. Notably, a subset of CD3⁺TH⁺ T cells is detected within lymphatic structures of TDLNs, suggesting immune-intrinsic catecholamine signaling. Single-cell RNA sequencing reveals that noradrenergic signaling, particularly via β2-adrenergic receptors (ADRB2), predominates in tumor-infiltrating myeloid cells and is dynamically regulated by therapy. Intratumoral administration of oncolytic herpes simplex virus (oHSV) upregulates ADRB2 expression in macrophages, whereas systemic chemo-immunotherapy induces distinct receptor modulation patterns in tumors and TDLNs. Functionally, pharmacologic β-adrenergic blockade significantly enhances the efficacy of oHSV therapy in orthotopic GBM and subcutaneous melanoma models, resulting in reduced tumor growth, increased tumor cell death, and enhanced CD8⁺ T cell infiltration. Similarly, direct inhibition of TH enzymatic activity suppresses tumor progression and further potentiates OV. Mechanistically, TH inhibition not only promotes tumor-infiltrated cytotoxic immune cells CD8, NK and γδ T cells, but also suppresses the activity of immunosuppressive myeloid cells, including transcriptional (Fos), and metabolism (Arg) modification in M2 macrophages and other immune cells. Collectively, these findings identify TH-mediated neuroimmune signaling as a critical regulator of tumor immunity in GBM and demonstrate that targeting catecholaminergic pathways or downstream neuroimmune crosstalk pathways can enhance the efficacy of OV. This study provides a rationale for integrating neural modulation into immunotherapeutic strategies for CNS malignancies.

Field–Flow–Front mapping of breast cancer evolutionary dynamics in situ

2026-6-23

Tumour progression reflects not only which clones arise but where, when and in what context they expand—dimensions genotype-centred reconstructions leave unresolved. Here we reconstruct breast cancer evolution in situ across 34 Visium HD, 16 Xenium, 32 MIBI-TOF and 10 CODEX samples, serial-section 3D reconstruction, single-cell and bulk transcriptomes, and genome-wide CRISPR dependency profiles. We partition tumours into 296 cancer microzones—stroma-bounded units within which expansion is reconstructed—and define in each a Cancer Progression Metric(CPM) coupling a transcriptomic clock to expansion geometry. Projected onto tissue, CPM yields a Field–Flow–Front model rendering progression as a continuous physical process and resolving subclonal architecture into spatially coherent domains rather than predefined branches. Unexpectedly, the most advanced fronts were not the most proliferative but low-dependency, slow-cycling populations with directional expansion, driven by an extracellular matrix programme whose evolutionary force exceeded inflammation by nearly an order of magnitude yet whose genes were the least cell-autonomous. Under chemotherapy it persisted while its clonal carriers reshuffled, marking a transferable, stroma-coupled front process—not a fixed clone—as the unit of advance. Distilled into an evolutionary advantage load, this front process predicted recurrence and survival across independent cohorts and improved on conventional staging, establishing a tissue-embedded paradigm for mapping tumour evolutionary dynamics, from local fronts to patient outcome.

COATS Identifies Copy-Number-Dependent Drivers and Enablers of Aneuploidy in Cancer

Wang, G., Dinh, K., Alfieri, F., Fani, S., Davoli, T. — 2026-6-23

Aneuploidy is a hallmark of most cancers but at the same time has been shown to decrease cellular fitness. Thus, to solve this conundrum, a current hypothesis in the field is that specific SCNAs may promote tolerance to the aneuploid state and/or promote additional chromosomal instability (CIN) and more aneuploidy. In other words, gains or losses of oncogenes (OGs) and tumor suppressor genes (TSGs) can, in turn, drive further CIN, promoting additional somatic alterations, or enhance aneuploid cell survival. Despite their importance, CN-dependent OGs and TSGs associated with aneuploidy remain largely unidentified. Here, we present a new method, Copy-number-dependent Oncogenes And Tumor Suppressors (COATS), to identify pan-cancer and cancer-specific CN-dependent OGs and TSGs associated with aneuploidy (Aneu-OGs and Aneu-TSGs). COATS integrates information theory and statistical tests to analyze gene expression, copy number, and aneuploidy, and incorporates timing analysis to distinguish early drivers of CIN from late tolerance enablers. Interestingly, using the CINner simulation framework, we show that aneuploidy drivers tend to occur earlier than aneuploidy enablers. Applying COATS to 33 TCGA cancer types, we identified 479 pan-cancer amplification-dependent Aneu-OGs and 141 deletion-dependent Aneu-TSGs. For validation, we used shRNA to knock down CCT5, a COATS-identified pan-cancer Aneu-OG predicted to promote aneuploidy tolerance, in isogenic aneuploid and near-diploid cells. Strikingly, CCT5 depletion was selectively toxic in aneuploid cells, supporting its classification as an aneuploidy tolerance enabler gene. Overall, our study defines a set of CN-dependent genes associated with aneuploidy and points to candidate therapeutic targets for chromosomally unstable cancers.

DLC1 loss drives multicellular streaming invasion by enforcing spatially coordinated Rho and β1 integrin signaling

2026-6-23

Efficient cancer cell invasion requires coordinated control of actomyosin contractility, extracellular matrix (ECM) engagement and remodeling, yet how these processes are integrated in complex, three-dimensional (3D) environments remains unclear. Here, we identify the tumor suppressor and RhoGAP protein DLC1 as molecular brake on multicellular streaming invasion in collagen-rich ECM. Using CRISPRoff-engineered breast cancer spheroids, DLC1 reconstitution models and patient-derived organoids embedded in collagen gels, we show that DLC1 downregulation promotes an efficient multicellular streaming phenotype. This invasion program requires matrix metalloproteinase activity, β1 integrin engagement and Rho–ROCK-dependent actomyosin contractility. Mechanistically, DLC1 downregulation stabilized the rear-polarization of RhoA activity and increased β1 integrin abundance, plasma membrane localization and activation. Separation-of-function mutants revealed that DLC1 restrains invasion through a dual mechanism: its RhoGAP activity limited actomyosin-driven streaming, whereas its LD-like talin-binding motif controlled β1 integrin enrichment at the plasma membrane. Together, our findings provide a molecular basis for the prominent role of DLC1 as a metastasis suppressor.

Epigallocatechin gallate and thermal cycling-stimulation synergistically promote apoptosis in A549 cells via endoplasmic reticulum stress-induced calcium ion dysregulation and oxidative stress

Hsu, F., Liu, H., Kung, Y., Lin, C., Chao, C. — 2026-6-23

Non-small cell lung cancer (NSCLC), as the predominant subtype of lung cancer, presents a considerable clinical challenge due to its high rates of recurrence and the significant adverse effects associated with conventional therapeutic modalities. In response to these challenges, this study explored the new combined anticancer effects of epigallocatechin gallate (EGCG) together with thermal cycling-stimulation (TCS). The findings demonstrated that the combination of EGCG and TCS synergistically decreased the viability of A549 and NCI-H460 NSCLC cells, while exhibiting minimal cytotoxic effects on IMR-90 normal lung fibroblasts. Further investigation revealed that EGCG mitigated the TCS-induced upregulation of heat shock proteins HSP70 and HSP105 and concurrently diminished the expression levels of proteasome subunits. This combined effect disrupted proteostasis, resulting in pronounced endoplasmic reticulum (ER) stress. Subsequently, a positive feedback mechanism was established between inositol 1,4,5-trisphosphate receptor (IP 3 R)-mediated ER Ca 2+ release and excessive reactive oxygen species (ROS) production, ultimately leading the cells to undergo mitochondrial apoptosis. This combined treatment reduces the necessary dosage of EGCG, thereby overcoming limitations related to its poor bioavailability and systemic toxicity, while also preventing the development of thermotolerance induced by TCS. Consequently, this method offers a new and potentially practical therapeutic strategy for treating NSCLC.

Tumor-Derived SPP1 Drives Immunosuppressive Macrophage Reprogramming in Gastric Peritoneal Carcinomatosis

2026-6-23

Peritoneal carcinomatosis is a major cause of death in gastric cancer, yet effective therapies remain limited. Tumor-derived soluble factors are increasingly recognized as key regulators of the peritumoral microenvironment. Here, we nominate osteopontin (SPP1) as a tumor-derived mediator that orchestrates macrophage-driven immunoregulation in gastric peritoneal carcinomatosis. Using integrated analyses of human clinical datasets and murine models, we demonstrate that tumor-secreted SPP1 promotes macrophage recruitment and induces tolerogenic IL-10 production. Clinically, SPP1 correlated with inferior overall survival and progression-free survival in gastric cancer. In syngeneic murine models of gastric peritoneal carcinomatosis, intracavitary pharmacologic inhibition of SPP1 restricted peritoneal dissemination, impaired macrophage infiltration and suppressed IL-10 production. Consistent with these findings, macrophage depletion phenocopied antitumor effects of SPP1 inhibition, resulting in decreased metastatic burden. Collectively, these findings define a mechanism of tumor-macrophage crosstalk that promotes peritoneal dissemination and provide a rationale for therapeutic targeting of SPP1 in gastric peritoneal carcinomatosis.

Bispecific antibody-drug conjugates targeting EGFR and LGR5 exert potent antitumor activity in colorectal cancer models

2026-6-23

Colorectal cancer (CRC) remains a significant contributor to cancer-associated deaths worldwide, indicating the need for new therapeutic targets and modalities. Antibody-drug conjugates (ADCs) have demonstrated remarkable potential for the treatment of various cancer types, although their efficacy as monotherapies is often limited by insufficient targeting of tumor heterogeneity, dose-limiting toxicities, and drug resistance. Accordingly, multi-targeting therapeutic strategies, such as bispecific ADCs (bsADCs), which simultaneously target two cancer-associated antigens or non-overlapping epitopes on the same antigen, may prove more effective at overcoming resistance and eliminating tumors compared to monospecific ADCs. In this work, we describe the development of EGFR:LGR5 bispecific antibodies (bsAbs) and bsADCs. EGFR:LGR5 bsAbs were shown to internalize to the lysosome to a greater extent than EGFR- and LGR5-targeting monoclonal antibodies (mAbs) and drive EGFR lysosomal degradation in an LGR5-mediated fashion. However, EGFR:LGR5 bsAbs exerted suboptimal cytotoxicity in CRC cell lines. We therefore engineered an EGFR:LGR5 bsADC that demonstrated 100- to 1000-fold enhanced efficacy over a previously developed LGR5-targeting monospecific ADC (8E11-CPT2) with an identical linker-payload in CRC cell lines of various genetic backgrounds and EGFR and LGR5 expression levels. EGFR:LGR5 bsADC potency was strongly correlated with cell line sensitivity to the CPT2 payload. EGFR:LGR5 bsADC induced tumor regression in select RAS MUT CRC xenograft models and demonstrated superior antitumor activity and prolonged survival benefit in all evaluated models versus EGFR mAb cetuximab (CTX), bsAb, and 8E11-CPT2. These findings strongly support the further development of EGFR and LGR5 dual-targeting approaches for CRC and other EGFR- and LGR5-expressing malignancies. One Sentence Summary EGFR:LGR5 bsADCs exert robust antitumor activity and outperform EGFR:LGR5 bsAb and LGR5 monospecific ADC in RAS WT and RAS MUT colorectal cancer models.