British Journal of Cancer

Background and AimsA major goal of personalised liver oncology is the ability to make targeted predictions about cancer-specific toxicity, however there are limited methods available. To address this, we validated the performance of our bioinformatics framework, TARGET-SL, through ex vivo drug screening. MethodsUsing TARGET-SL we predicted gain of function (GOF), loss of function (LOF) and synthetic lethal (SL) genetic events, and corresponding drug candidates. We validated drug predictions across hepatocellular carcinoma (HCC) cell lines, and a cohort of HCC and cholangiocarcinoma (CCA) patient-derived organoids (PDOs). ResultsFor HCC cells and PDOs we found 37.5% and 25% of the respective selected compounds induced unique target-specific growth inhibition based on genetic biomarkers, suggesting novel biomarker-driven drug sensitivities. ConclusionsOur analyses demonstrate TARGET-SLs potential to enhance personalized drug screening for liver cancer, by focusing on genetically informed targets. This will reduce experimental costs and accelerate the pace of therapeutic discovery. Impact and ImplicationsPrimary liver cancer (PLC) is a cancer with poor prognosis, and current therapies increase survival only for a minority of patients. Through the application of TARGET-SL we can predict, for each patient, the essential genes and corresponding small molecule inhibitors. These data support further investigation in larger patient cohorts and offer the possibility to specify new small molecule inhibitors and to repurpose current drugs for PLC treatment. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/725819v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@10cb252org.highwire.dtl.DTLVardef@8f3049org.highwire.dtl.DTLVardef@ab4467org.highwire.dtl.DTLVardef@17f9d3_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LITARGET-SL can predict gene and drug sensitivities for cell lines and patient-derived organoids C_LIO_LIThis may reduce drug screening costs and accelerate the pace of therapeutic discovery. C_LIO_LITARGET-SL may assist in the repurposing of current drugs and their rapid translation for primary liver cancer C_LIO_LITARGET-SL is tumour-type agnostic, and therefore may have application in other cancers with poor prognosis C_LI

Background and aimsTherapeutic outcomes for advanced hepatocellular carcinoma remain inadequate, despite recent advances using immunotherapy. Long-term effectiveness of systemic therapies, including second-line multi-tyrosine kinase inhibitor sorafenib, is limited by resistance mechanisms and adverse effects. Upregulated deubiquitinase UCH-L1 is frequently correlated with poor prognosis in cancers. Here, we investigated the therapeutic potential of combining pharmacological UCH-L1-inhibition with sorafenib in HCC. MethodsUCH-L1 expression was analysed in TCGA-LIHC data and patient-derived HCC tissues. Sorafenib and LDN57444 effects were evaluated in vitro in cytotoxicity and invasion assays. Gene and protein expression were examined by RT-qPCR, Western blotting and immunohistochemistry. In vivo efficacy of drug synergy was assessed in an orthotopic xenograft mouse HCC model. ResultsIn silico data-analysis revealed significantly higher UCH-L1 levels in patient HCC tumours versus non-tumour, associated with reduced overall survival. Low-dose sorafenib upregulated UCH-L1 in HCC cell line Hep3B. Paradoxically, this also promoted invasiveness and sustained MEK1/2-ERK1/2-pathway activation. Combining low-dose sorafenib with LDN57444 produced strong synergistic cytotoxicity in vitro, reverted MAPK-activation and suppressed invasion. Consistently, at low sorafenib dose co-treatment with LDN57444 completely inhibited tumour growth of Hep3B xenografts and enhanced sorafenib efficacy. ConclusionLDN57444 sensitises HCC cells to low-dose sorafenib by reverting drug-induced pro-oncogenic signalling and thereby strongly synergises with sorafenib to enhance anti-tumour efficacy in a HCC mouse model. This presents UCH-L1 as a player in treatment-induced adaptive response and supports further exploring UCH-L1-targeting in combination with sorafenib as therapeutic avenue for advanced HCC. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/725527v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@176dc91org.highwire.dtl.DTLVardef@8acae8org.highwire.dtl.DTLVardef@f71bborg.highwire.dtl.DTLVardef@1f3c5aa_HPS_FORMAT_FIGEXP M_FIG C_FIG Lay summaryThis study explores a new treatment approach for hepatocellular carcinoma (HCC) by combining two drugs: LDN57444, which blocks the enzyme UCH-L1, and sorafenib, a FDA-approved multi-tyrosine kinase inhibitor. We evaluated the effect of this drug combination in vitro using a HCC cell line and in an mouse HCC-model. The drug combination displayed strong, synergy in lowering HCC cell viability, and greatly reduced invasiveness and in vivo tumour growth. LDN57444 sensitised HCC cells to low doses of sorafenib by preventing UCH-L1-mediated activation of pro-oncogenic signalling. These findings highlight the potential of this new drug combination for treating advanced HCC thereby potentially reducing side-effects and countering drug resistance. Impact and implicationsOur preclinical research introduces a novel combination strategy against advanced HCC that holds potential to improve existing therapies, particularly the second-line multi-tyrosine kinase inhibitor sorafenib. The proposed combination of sorafenib with an inhibitor of the deubiquitinase UCH-L1 not only enhances sorafenib efficacy but present promise to also counter resistance mechanisms. Moreover, because effective responses are achieved at lower drug doses, this may in addition reduce therapy-associated adverse effects further increasing potential impact. While sorafenib is FDA-approved, the UCH-L1 inhibitor LDN57444 needs further (clinical) development to bring our promising findings to full translational potential for HCC patients and physicians.

Background and ObjectivesIntravesical gemcitabine/docetaxel (Gem/Doce) is an effective therapy for Bacillus Calmette- Guerin (BCG)-unresponsive non-muscle-invasive bladder cancer (NMIBC), achieving 50% complete responses at 2 years. However, the genomic determinants underlying response and resistance to Gem/Doce remain poorly defined. Our objective was to define the mutational landscape of BCG-unresponsive NMIBC and nominate genomic features associated with response or resistance Gem/Doce. MethodsPatients with BCG-unresponsive NMIBC treated with Gem/Doce were classified as responders (recurrence-free survival [RFS] >12 months) or non-responders (RFS <12 months). Whole-exome sequencing was performed on tumors prior to Gem/Doce treatment (n=23). Single nucleotide variants were identified and annotated using a Cancer Genome Analysis pipeline. Copy number alterations were inferred with ABSOLUTE, and clonal architecture was reconstructed using PhylogicNDT. Key Findings and LimitationsResponders demonstrated significantly prolonged time to high-grade recurrence (3.5 vs 42 months, p<0.001) and cystectomy compared with non-responders (9.5 months vs not reached; p<0.001). Non-responders exhibited higher tumor mutational burden (13.66 vs 8.71; p=0.02) and more frequent whole-genome doubling (2/2 non-responders vs 0/1 responders; p=0.33). Phylogenetic analyses revealed clonal BAP1 and subclonal BRCA2 mutations in responders, whereas non-responders harbored clonal FGFR3 mutations. Limitations include small sample size and retrospective design. Conclusions and Clinical ImplicationsDistinct genomic features underlie differential response to Gem/Doce in BCG-unresponsive NMIBC. In responders, alterations in DNA repair pathways (e.g., BRCA2) may sensitize tumors to chemotherapy, while non-responders with FGFR3 mutations may benefit from alternative targeted strategies. These findings warrant validation in larger cohorts and support the development of biomarker-driven clinical trials. Patient summaryIn this report we analyzed bladder tumors and found that some tumors respond well to treatment because they have defects in repairing DNA, making them more vulnerable to chemotherapy. In contrast, tumors that do not respond to chemotherapy harbor different genetic changes that help them survive and grow. These findings may help physicians choose more effective and personalized treatments in the future.

Objective To determine if it is possible to assess individual patient risk of the development of colorectal cancer (CRC) in people in high-risk groups due to their family history. Design/Method Retrospective observational study of prospectively collected data from consecutive patients referred for a colonoscopy. 2,478 consecutive patients were referred to a single colorectal surgical practice in Sydney, Australia between 1977 and 2018 for a colonoscopy because of a family history of CRC. Of these, 1,963 have been followed for more than 10 years and are the subject of this paper. Histopathological findings categorised as normal (N), non-advanced adenoma (NAA) or advanced neoplasia (AN) with AN proven to be the precursor to CRC. Intervention Colonoscopic screening on the basis of contemporary practice to 2006 and subsequently according to Australian National Health and Medical Research Council guidelines. Results Participants with normal or low-risk findings in the first decade remain at lower risk of CRC for 30 years from the commencement of screening. Conclusion It is possible to stratify individual patients in a high relative risk cohort into those with high or low personal risk of CRC based on colonoscopic findings in the first 10 years of surveillance. Those with no AN in the first ten years have a lower 30-year risk of developing AN than the general community. This offers the possibility of structuring surveillance programs around individual risk rather than group risk, lessening the need for multiple surveillance colonoscopies in the majority of such patients and improving the cost effectiveness of CRC screening at the population level.

Importance: Enfortumab vedotin (EV) is an antibody-drug conjugate approved for the treatment of locally advanced or metastatic urothelial cancer (la/mUC). Cutaneous adverse events (cAEs) are common during EV therapy, with prior studies suggesting an association between EV-related cAEs and improved survival; however, there is insufficient data to delineate the survival benefit of EV-induced cAEs from those associated with concurrent immune checkpoint inhibitors (ICIs). Objective: This study aims to evaluate the association of EV-induced cAEs and survival, and to characterize the timing and morphology of EV-induced cAEs. Design: We conducted a multi-institutional retrospective study of patients with la/mUC treated with EV between 2020 and 2025. Setting: Multicenter academic referral center. Participants: A total of 449 EV-treated patients were included. Patient characteristics were extracted manually, and likelihood scoring was used to attribute cAEs to either EV or other etiologies. Exposure: EV treatment. Main Outcomes and Measures: We estimated progression-free (PFS) and overall (OS) survival using Kaplan-Meier method. Multivariable time-varying and landmark Cox regression models were used to evaluate associations between EV-induced cAE and survival. Sensitivity analyses were performed at landmarks from 15 to 105 days. Results: Of 449 patients, 206 (45.9%) developed a cAE; 39 (18.9%) were high-grade and 127 (61.7%) were attributed to EV. The most common cAEs were pruritus (41.3%), unspecified and desquamating dermatitis (37.3%), and morbilliform dermatitis (27.7%). Across all treatment groups, survival was longer in patients with EV-induced cAEs. Developing an EV-induced cAE was protective across all examined landmark times, with hazard ratio (HR) 0.60 (95% CI: 0.43-0.82, p<0.001) for PFS and HR 0.46 (95% CI: 0.31-0.67, p<0.001) for OS at primary landmark time of 30 days. Early-onset EV-induced cAEs were protective at all landmark times and high-grade EV-induced cAEs were not associated with worse survival. Conclusions and Relevance: EV-induced cAEs were independently associated with improved PFS and OS in patients with la/mUC, even after accounting for immortal time bias and ICI exposure. Distinguishing EV-induced cAEs from other etiologies in timeline and morphology may help guide oncology and dermatology management.

Therapeutic resistance remains a major challenge in advanced and recurrent endometrial cancer (EC). Aberrant NOTCH signaling has been associated with aggressive tumor behavior and therapeutic resistance across multiple malignancies, yet its therapeutic significance in EC remains incompletely defined. We investigated whether auranofin (AuR), a noncanonical modulator of NOTCH signaling through the transcriptional effector RBPJ, alters platinum responsiveness in EC models. Elevated NOTCH3 copy-number was associated with poorer overall survival in the TCGA-UCEC cohort. AuR treatment reduced RBPJ occupancy at canonical NOTCH target loci, including HES1 and HES4, across multiple EC models. Stable NOTCH3 depletion altered AuR responsiveness in a context-dependent manner while significantly enhancing cisplatin (CDDP) sensitivity in AN3CA cells. Pharmacologic AuR treatment similarly potentiated CDDP response in AN3CA cells and AN3CA xenografts, resulting in reduced tumor burden and prolonged endpoint-free survival following combination treatment. In contrast, ARK-1 xenografts demonstrated limited additional benefit from combined AuR plus CDDP therapy despite detectable suppression of RBPJ occupancy. Together, these findings identify context-dependent NOTCH-associated therapeutic vulnerabilities in EC and support further development of biomarker-guided AuR-based platinum-sensitization strategies. Statement of significanceAuranofin suppresses RBPJ-associated transcriptional activity and enhances cisplatin response in biologically distinct subsets of endometrial cancer.

Background: Circulating tumour DNA (ctDNA) liquid biopsy is now established across oncology for early cancer detection, minimal residual disease surveillance, and treatment monitoring. Detection thresholds for all current ctDNA assays are derived empirically through receiver operating characteristic analysis on training cohorts - a statistically valid but theoretically uninformed approach that does not specify the minimum detectable tumour fraction given assay technical characteristics, nor identify when increasing sequencing depth ceases to provide additional clinical information. Methods: We model ctDNA detection as a binary hypothesis testing problem with Binomial-distributed mutant allele counts against a sequencing error noise floor. The Neyman-Pearson lemma is applied to derive the uniformly most powerful detector and the minimum detectable tumour fraction in closed form. The sequencing assay is modelled as a binary symmetric channel and Shannon channel capacity is calculated. Empirical validation uses n=61 data points extracted from five published peer-reviewed analytical validation studies across five independent institutions in the US and EU (2018 - 2025): Yu et al. 2022, Stetson et al. 2018, Frydendahl et al. 2023, Northcott et al. 2024, and Cheng et al. 2025. Results: The minimum detectable tumour fraction is derived in closed form as f_min approximately equal to (z_alpha + z_beta) multiplied by the square root of (epsilon divided by N), where N is sequencing depth, epsilon is the platform error rate, and z_alpha, z_beta are standard normal quantiles at the specified false positive and false negative rates. Shannon channel capacity is C = 1 minus H(epsilon) bits per read, where H(epsilon) is binary entropy. Empirical validation yields 84.3% agreement for single-locus assays. Discordance for multi-locus tumour-informed assays (NeXT Personal, duplex WGS) is consistent with the single-locus model scope and identifies the principal theoretical extension required. Conclusions: This framework provides the first formal Neyman-Pearson optimality proof for ctDNA detection, a closed-form detection limit, and a platform-independent efficiency metric for NHS and regulatory standardisation. Keywords: circulating tumour DNA; liquid biopsy; Neyman-Pearson detection; Shannon channel capacity; sequencing depth; limit of detection; minimal residual disease; signal detection theory

BackgroundTumor-associated macrophages (TAMs) are key drivers of the immunosuppressive tumor microenvironment (TME), supporting tumor progression through diverse functions. However, mechanistic studies of TAM polarization remain limited by the lack of physiologically relevant human model systems that capture stromal-immune interactions and macrophage heterogeneity. MethodsWe established advanced human co-culture systems that integrate healthy donor-derived macrophages with patient-derived organoids and tumoroids (PDOs and PDTs), as well as matched normal fibroblasts (NFs) and cancer-associated fibroblasts (CAFs). These multicellular models enabled the investigation of interactions among stromal, epithelial, and immune cells within tumor and adjacent normal tissue environments. ResultsThe co-culture systems recapitulated distinct macrophage states associated with tumor and adjacent normal environments and identified fibroblasts as major regulators of macrophage phenotypes. CAFs promoted macrophage metabolic remodeling characterized by altered lipid handling and enrichment of TAM-like signatures. Mechanistically, we identified thrombospondin 1 (TSP1) as a CAF-secreted factor linked to metabolic priming. Recombinant TSP1 induced transient lipid accumulation followed by mitochondrial remodeling. In tumor co-culture conditions, CD36 inhibition reduced lipid accumulation in macrophages, supporting a role for TSP1-linked lipid crosstalk in stromal-immune interactions. ConclusionOur study establishes advanced patient-derived co-culture models as a platform to investigate human TAM biology and stromal-immune interactions in CRC. Using these systems, we identify a fibroblast-associated TSP1-lipid axis linked to macrophage metabolic remodeling and TAM-like polarization, highlighting stromal metabolic communication as a potential targetable feature of the CRC microenvironment. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=176 HEIGHT=200 SRC="FIGDIR/small/728363v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@16d60aborg.highwire.dtl.DTLVardef@1e593e7org.highwire.dtl.DTLVardef@15124b0org.highwire.dtl.DTLVardef@3fca92_HPS_FORMAT_FIGEXP M_FIG C_FIG

PurposeBladder cancer (BLCA) is a heterogeneous tumor type. Only one third of muscle-invasive (MIBC) patients respond to immune checkpoint inhibitors (ICIs). Reliable resistance markers are needed to guide clinical decisions. We investigated the nerve growth factor receptor (NGFR) in BLCA and analyzed its correlation with disease progression and response to immunotherapy. Experimental DesignWe analyzed NGFR expression in BLCA cell lines, organoids, mouse models and patient samples. The cohorts used were The Cancer Genome Atlas (TCGA), enriched in muscle-invasive bladder cancer (MIBC) (n=407); IMvigor210, representing MIBC patients treated with ICIs (n=348); and UROMOL2, as a non-muscle-invasive bladder cancer (NMIBC)-specific cohort (n=535). IMvigor010 was also included (n=728). Patients were stratified by NGFR expression quartiles. We analyzed survival and tumor subtypes and performed stromal deconvolution and functional profiling. We assessed stemness- and invasion-related features in SCaBER cells. ResultsNGFR marks a basal tumor cell subcluster and is independently associated with poor prognosis in TCGA and IMvigor210. NGFR-high tumors show stromal content enriched in cancer-associated fibroblasts, lower neoantigen burden, higher CD8+ T effector signature together with an immune-excluded phenotype, and a CAF-specific TGF{beta} signature. In the immunotherapy-treated cohort, high NGFR expression was also associated with poorer outcome. Functionally, NGFR appears to promote a stem-like/pro-invasive program in BLCA cells. ConclusionsNGFR identifies a basal-like BLCA subpopulation linked to poor survival, while its association with immunotherapy response requires further validation. In addition, our in vitro analyses support a role of NGFR in stem-like and invasive traits, highlighting its relevance as a biomarker in BLCA.

Aims Active surveillance (AS) allows selected men with localized prostate cancer to defer curative therapy and reduce treatment morbidity. Conversion from AS to treatment is commonly triggered by Gleason grade group (GGG) upgrading on confirmatory biopsy. We developed and validated a digital pathology artificial intelligence (DPAI) biomarker to predict GGG upgrading in AS-eligible patients. Materials & Methods The DPAI model was trained using histopathology image features from diagnostic biopsies of 998 patients and validated in an independent cohort of 296 patients meeting criteria for AS. Logistic regression estimated the probability of confirmatory-biopsy GGG increase, and feature selection identified the most predictive variables. Results AI-GUR (Artificial Intelligence-Gleason Upgrade Risk) predicted GGG reclassification at confirmatory biopsy (OR 1.60; p=0.0003), and provided information beyond conventional stratification (risk group, CAPRA) and cribriform morphology (all p<0.01). Predicted risks were similar across time from diagnosis (~10-15% to ~85% at 1, 1.5, or 2 years; p for time=0.50), consistent with initial biopsy mischaracterization rather than time-dependent progression. Conclusions AI-GUR provides individualized estimates of confirmatory-biopsy GGG upgrading for AS candidates. Using DPAI may improve shared decision-making by complementing standard clinicopathologic tools and molecular testing using the same biopsy specimen, while informing the likelihood of grade upgrade at confirmation.

High grade serous ovarian cancer patients initially respond to platinum-based chemotherapy, but usually relapse within two years and ultimately develop therapy resistance. Management of response and effective clinical decisions are currently based on unspecific biomarkers and limited imaging techniques, illustrating the clear clinical need for reliable predictors of response. In this work, we evaluated the performance of patient-derived organoids generated from ascitic fluid and functionally tested in parallel to the patients clinical course, in the prediction of treatment response, and guiding clinical decision-making in a patient-specific manner. Ascites derived organoids reliably recapitulated the histological and molecular features of a paradigmatic HGSOC patient with an apparent dissociated response, and demonstrated chemoresistance months before laparoscopy confirmed persistent inoperable disease with poor pathological response. Drug screening identified alternative therapeutic options, while multi-omics provided additional insights into the tumor-specific biological features, to assist in the personalized clinical management in ovarian cancer.

Background Emerging evidence suggests irreversible electroporation (IRE) with standard-of-care (SOC) chemo-therapy may improve survival in patients with Stage 3 pancreatic ductal adenocarcinoma (PDAC) when compared to SOC alone. This study evaluates the overall survival (OS) and progression-free survival (PFS) of Stage 3 PDAC patients treated with SOC plus IRE with the NanoKnife System versus SOC alone. Methods This prospective, multicenter study included two cohorts from the DIRECT registry: an IRE cohort from sites offering IRE as part of clinical care, and a comparator SOC cohort of prospectively enrolled and contemporaneous retrospective patients. Enrollment spanned 08/05/2019 to 02/05/2023, with follow-up through at least 24 months, death, or loss to follow-up. Included were 137 patients (99 IRE; 38 SOC), aged [&ge;]18 years with Stage 3 PDAC and no progression after three months of SOC therapy. Results Median (interquartile range) time from diagnosis to enrollment was 8 (6-10) months for IRE and 4 (3-6) for SOC (p<0.0001). Median OS and PSF from enrollment were 18 (95% confidence interval [CI]: 15-24) months and 9 (95% CI: 7-12) months for IRE, and 10 (95% CI: 8-14) months and 6 (5-8) months for SOC, respectively (p<0.0001 and p=0.009). Adverse events occurred in 80% (79/99) of IRE patients and 95% (36/38) of SOC patients; 29% (29/99) of the IRE cohort experiencing an IRE-related adverse event. Conclusions IRE was associated with improved OS versus SOC alone and may be an effective consolidative treatment for Stage 3 PDAC after three months of induction chemotherapy.

Multimorbidity, the co-existence of two or more long-term conditions, is up to three times more prevalent among people with cancer than in the general population and is associated with poorer survival, particularly for cancers with a more favourable prognosis such as colorectal cancer. In Scotland, multimorbidity is the norm among older adults, emerges earlier in socioeconomically deprived populations, and may contribute to comparatively low cancer survival rates. Despite this, the influence of multimorbidity on the colorectal cancer pathway remains poorly understood. We conducted a Scottish data-linkage study of adults diagnosed with colorectal cancer between 2010 and 2014, linking the Scottish Cancer Registry to national prescribing, hospital admissions, death registration, and bowel screening datasets. Prescribing data were used to derive overall and system-specific comorbidity measures as a proxy for multimorbidity and active disease burden. Associations with stage at diagnosis, treatment, survival, and screening uptake were examined using logistic regression and Cox proportional hazards models adjusted for demographic and clinical covariates. Among 19,043 patients, 87% had at least one prescribing-based comorbidity, most commonly cardiovascular, nervous system, and gastrointestinal conditions. Overall comorbidity burden was not associated with stage at diagnosis, although laxative-related prescribing was associated with later-stage disease. Increasing comorbidity burden reduced the likelihood of receiving any treatment and surgery, while associations varied across system-specific comorbidities. Higher comorbidity burden was also associated with increased all-cause and colorectal cancer-specific mortality, particularly among patients with respiratory, nervous system, and haematological/nutritional conditions. Screening uptake was not associated with overall comorbidity burden but did differ by system-specific comorbidity. Prescribing-based multimorbidity was highly prevalent and strongly associated with treatment patterns and mortality among patients with colorectal cancer. System-specific multimorbidity measures provided greater discrimination than overall morbidity counts, highlighting the importance of considering distinct multimorbidity profiles when assessing cancer pathways and designing targeted interventions for optimising treatment and survival. Keywords (primary health care, general practice, multimorbidity, comorbidity, colorectal cancer, early diagnosis, cancer treatment, survival)

Uveal melanoma (UM) is the most common form of intraocular cancer in adults and has a median survival rate of [~]1 year after metastasis occurs. Metastatic UM is largely refractory to treatment and there are no effective pharmacological therapies, resulting in poor overall survival. Activating mutations in GNAQ and GNA11 proteins (GNAQ/11) are the oncogenic initiators in >90% UM cases. While there are no targeted therapies yet identified for the GNAQ/11 oncoproteins, a natural compound called FR900359 (FR) is a selective inhibitor for both oncogenic and wild type GNAQ/11. We performed a functional genomics screen to identify drivers of FR resistance in two UM cell lines (92.1 and MEL202). The screen identified eleven genes as candidate FR resistance drivers in both cell lines. Over-expression of five of these genes (ABCB1, PLCB4, GRM1, PLCE1, PDGFRB) was predicted to provide resistance to FR treatment. Enforced expression of ABCB1 or PLCB4 did not provide immediate resistance to FR, although over-expression of either transgene led to the emergence of resistant colonies at a much higher rate than occurs spontaneously in parental cells. We show that a relatively small fraction of UM cells can tolerate the initial over-expression of PLCB4 and ABCB1, but FR treatment leads to expansion of this cell population. Expression of an ABCB1-tGFP fusion protein was used to isolate drug naive UM cells. We show that these cells are uniformly resistant to FR, unlike the bulk tumor cell population. Finally, additional experiment of the drug naive ABCB1-tGFP+ UM cells led to the observation that these cells exhibit a significantly lower rate of protein translation, like BAP1-deficient UM cells. These findings suggest that resistance to targeted GNAQ/11 inhibitors is dictated by interaction between acquired genetic alterations and epigenetic states within heterogenous UM cell populations.

BackgroundIn RAS-mutant tumors, ERK phosphorylates the mitochondrial fission GTPase DRP1 to promote mitochondrial fission. DRP1 activity is tumor-promoting in pancreatic and other RAS-driven cancers, but its role in therapeutic resistance is unknown. MethodsWe developed a panel of patient-derived pancreatic cancer cell lines resistant to the MEK inhibitor trametinib. We used immunofluorescence imaging, in vitro growth assays and orthotopic xenografts to determine the role of DRP1 in trametinib resistance. ResultsWe find that trametinib-resistant cells exhibit increased expression and phosphorylation of DRP1 compared to sensitive counterparts. Quantitative analysis of mitochondrial structure reveals that mitochondria in resistant cells are morphologically distinct and relatively smaller than sensitive cells treated with trametinib. Genetic and pharmacological inhibition of both c-Myc and CDK6 are sufficient to block DRP1 phosphorylation in resistant cells, suggesting that activation of a c-Myc-CDK6 signaling axis drives reactivation of mitochondrial fission in the absence of MAPK signaling. Importantly, deletion of DRP1 leads to either growth inhibition or re-sensitization to trametinib in resistant lines. ConclusionThese findings suggest DRP1 contributes to drug resistance, and that inhibition of mitochondrial fission might be a promising therapeutic strategy to combat resistance to MAPK and RAS inhibitors.

BackgroundmTOR signaling promotes cell growth and anabolic processes in all eukaryotes. Hyperactivation of mTOR signaling is associated with various cancers along with hepatocellular carcinoma (HCC). HCC is a highly lethal malignancy with multiple aetiologies such as viral infection, alcohol abuse, and metabolic dysfunction. Therapeutic options for HCC remain limited due to an incomplete understanding of oncogenic drivers and poorly characterized mechanisms of disease progression. MethodsVarious regimens of DEN and CCl4 carcinogen dosage were investigated on C57BL/6J mice to induce HCC. The histological analysis for fibrosis and serum markers for liver function were performed. Molecular analyses of oncogenic drivers were performed in the HCC tissues obtained from mice and HCC patients. The impact of inhibition of mTOR signaling was assessed on HCC progression. ResultsWe established a rapid DEN+CCl4 induced (DCI) HCC model in C57BL/6 mice to study disease progression longitudinally. The molecular analysis revealed upregulation of MAPK and downregulation of mTORC1-S6K-S6 signaling in HCC. However, other branches of mTOR such as mTORC1-ULK1, mTORC1-4EBP1, and mTORC2-PKC were upregulated due to the increased expression. Similar observations were found in tissues derived from HCC patients. Furthermore, inhibition of mTORC1 alone by Rapamycin did not reduce HCC progression but Torin 1 mediated inhibition of both mTORC1 and mTORC2 significantly reduced HCC progression. ConclusionsWe propose this biased mTOR signaling modulates mTOR activity towards specific downstream processes that are crucial for cancer cell growth and targeting both the mTOR complexes has better therapeutic potential in HCC. Impact and ImplicationsThis study provides a rapid pre-clinical model for understanding HCC progression and to explore various intervention strategies. The study reports a novel phenomenon of biased mTOR signaling where deregulation of downstream substrate levels modulates the mTOR activity towards the specific branches, the master regulator of cell growth and metabolism. Furthermore, the study suggests that the clinical investigations exploring the rapalogs against HCC should be cautiously considered depending on the aetiology and signaling status of HCC. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/727188v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@58834forg.highwire.dtl.DTLVardef@114f43corg.highwire.dtl.DTLVardef@aea643org.highwire.dtl.DTLVardef@2596ce_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDEN and CCl4 treatment generated a well-established HCC within 4 months. C_LIO_LILiver fibrosis and serum markers correlated with HCC progression. C_LIO_LIUpregulation of mTOR pathway substrates create biased mTOR signaling. C_LIO_LIDual inhibition of mTORC1 and mTORC2 reduced HCC progression significantly. C_LI

Background Endometrial cancer exhibits marked molecular and immune heterogeneity that is only partially explained by established genomic biomarkers. We investigated whether T cell receptor (TCR) repertoire architecture captures complementary dimensions of antitumor immunity beyond conventional molecular classification. Methods Paired tumor and peripheral blood samples from eight patients with molecularly characterized endometrial cancer underwent TCR repertoire profiling. Diversity, clonality, and tumor blood overlap metrics were integrated with genomic variables, including tumor mutational burden (TMB), genomic instability metric (GIM), and POLE status. Principal component analysis and correlation analyses were used to identify major dimensions of repertoire organization. Composite Immune Focusing and Immune Sharing Scores were derived to summarize dominant repertoire patterns. Results The first two principal components explained 70.1% of total repertoire variance and revealed substantial heterogeneity independent of histological subtype. TMB was strongly associated with reduced repertoire diversity and increased clonal dominance, resulting in a robust association with the Immune Focusing Score ({rho} = 0.88, p = 0.004). POLE mutated tumors occupied the extreme end of this focusing continuum. In contrast, genomic instability was associated with increased tumor blood repertoire overlap and preserved diversity, reflected by a strong correlation between GIM and the Immune Sharing Score ({rho} = 0.76, p = 0.027). The two immune scores showed minimal correlation with each other ({rho} = -0.24, p = 0.57), indicating that they capture largely independent aspects of immune organization. Conclusion Integrative analysis of TCR repertoire architecture and tumor genomics identifies distinct immunogenomic states in endometrial cancer that are not fully captured by conventional molecular classification. If validated in larger cohorts, immune focusing and immune sharing metrics may provide complementary biomarkers for patient stratification and immunotherapy-oriented precision oncology

Importance Prognostic tools beyond staging are needed to guide treatment and counseling in head and neck squamous cell carcinoma (HNSCC). Objective To develop and externally validate a machine learning model predicting survival in advanced HNSCC using routinely collected clinical and biomarker data. Design, Setting, and Participants Retrospective, multi-institutional cohort study including 2,385 patients with stage III-IV HNSCC diagnosed from 2012-2022 in the University of California Health Data Warehouse (UCHDW). Patients were randomly split into training (n = 1,908) and test (n = 477) sets. Partial external validation used 7,749 patients from the Surveillance, Epidemiology, and End Results (SEER) registry (2010-2020). Exposures Demographic, tumor, treatment, comorbidity, and biomarker variables recorded at or before diagnosis. Main Outcomes and Measures The primary outcome was all-cause mortality within 70 months. Cox proportional hazards models included all predictors. Discrimination was assessed with Harrell's concordance index (C-index), calibration with predicted vs observed survival, and stratification with Kaplan-Meier curves. A Random Survival Forest (RSF) was trained for benchmarking and interpretability using Shapley Additive exPlanations (SHAP). Results Among 2,385 patients in UCHDW (median age, 63 years; 29.0% mortality), the Cox model achieved a C-index of 0.735 in the internal test set. Risk quartiles showed clear separation on Kaplan-Meier curves (log-rank p < 0.0001). In the SEER cohort (n = 7,749), where only demographic, staging, subsite, and treatment variables were available, the reduced Cox model achieved a C-index of 0.688, with calibration showing modest underestimation of survival in high-risk groups. Age, T stage, Charlson Comorbidity Index, neutrophil-to-lymphocyte ratio, and platelet count were among the strongest predictors, while surgery was associated with improved survival. The RSF achieved a C-index of 0.758 internally, with SHAP highlighting nonlinear effects of albumin, BMI, and inflammatory markers. Conclusions and Relevance A machine learning model using routine clinical and biomarker data demonstrated good prognostic performance in advanced HNSCC, with partial external validation. Such approaches may support individualized survival estimates, risk stratification, and treatment discussions, but broader validation is required before clinical adoption.

Background & AimsColorectal cancer liver metastasis is the leading cause of mortality in affected patients, with liver sinusoidal endothelial cells playing a pivotal role in metastatic niche formation. Proprotein convertase subtilisin/kexin type 9 has emerged as a regulator of tumor biology, but its function in the hepatic microenvironment remains poorly defined. This study aimed to characterize the role and subcellular localization of PCSK9 in liver sinusoidal endothelial cells and to evaluate the therapeutic potential of its endothelial-specific inhibition in colorectal liver metastasis. MethodsIn vitro studies were performed using human and murine liver sinusoidal endothelial cells stimulated with conditioned media from metastatic colorectal cancer cells and cancer stem cells. Subcellular localization was assessed by immunofluorescence, immunogold electron microscopy, and biochemical fractionation. Protein interactions were investigated using co-immunoprecipitation and proteomic analyses. For in vivo validation, a murine model of colorectal liver metastasis was generated by intrasplenic injection of tumor cells, followed by systemic administration of chondroitin sulfate-targeted nanoparticles delivering PCSK9 siRNA every 5 days for 18 days. ResultsPCSK9 was consistently expressed in liver sinusoidal endothelial cells and displayed a predominant nuclear localization, which increased upon tumor-induced activation. Proteomic integration identified multiple candidate interacting proteins involved in metabolic and tumor-related pathways. Targeted nanoparticle-mediated delivery achieved efficient PCSK9 silencing in vitro. In vivo, endothelial-specific PCSK9 inhibition significantly reduced liver metastatic tumor burden compared with control groups, whereas free siRNA showed no significant effect. ConclusionsPCSK9 exhibits a novel nuclear localization in liver sinusoidal endothelial cells and potentially interacts with proteins implicated in tumor mediated pathways. Selective inhibition of endothelial PCSK9 using targeted nanoparticles significantly reduces colorectal liver metastasis, highlighting a novel therapeutic strategy focused on the hepatic microenvironment. Impact and ImplicationsThis study provides mechanistic insight into how PCSK9 contributes to colorectal liver metastasis by identifying its novel nuclear localization and potential function in liver sinusoidal endothelial cells. These findings are important for researchers and clinicians seeking to understand microenvironment-driven metastasis and resistance to current therapies. The demonstration that endothelial-specific targeting of PCSK9 reduces metastatic burden suggests a new avenue for therapeutic development beyond systemic inhibition. Such strategies could be translated into precision nanomedicine approaches to improve outcomes in patients with metastatic colorectal cancer while minimizing off-target effects.

BackgroundCDH1 (E-cadherin) is a key epithelial adhesion molecule traditionally associated with tumor suppression and epithelial-mesenchymal transition (EMT). However, its roles across cancers remain incompletely understood, particularly within multilayer regulatory contexts involving genomic, epigenetic, transcriptional, and immune mechanisms. MethodsCDH1 expression, survival associations, EMT-correlated gene profiles (VIM, SNAI1, ZEB1), immune infiltration patterns, immune checkpoint correlations (PDCD1, CD274, CTLA4), promoter methylation, and genomic alterations were assessed across five epithelial cancers, breast invasive carcinoma (BRCA), colon adenocarcinoma (COAD), lung adenocarcinoma (LUAD), ovarian cancer (OV), and stomach adenocarcinoma (STAD). Cross-platform validation was performed using TCGA/GDC datasets, GEPIA2, UALCAN, TIMER, KM Plotter, cBioPortal, and g:Profiler. ResultsCDH1 was overexpressed but showed variable prognostic significance; higher expression predicted better survival in COAD, LUAD and STAD, worse survival in BRCA and had no impact in OV. Classic inverse relationships between CDH1 and VIM or ZEB1 were evident only in STAD, and SNAI1 showed no consistent association. Immune infiltration patterns were tumor-specific, ranging from cytotoxic T-cell dominance in LUAD to macrophage-rich profiles in OV; immune checkpoint correlations were similarly context-dependent. Co-expressed genes were enriched for endomembrane transport rather than adhesion pathways. Promoter methylation patterns varied by cancer, whereas genomic alterations of CDH1 were rare. ConclusionsCDH1 does not function as a universal epithelial or EMT marker across epithelial cancers. Instead, its associations with EMT, immune contexture, methylation, and prognosis are context-dependent, supporting a model of CDH1 as a heterogeneous regulator of epithelial plasticity. These findings challenge single-function interpretations and support cancer-specific CDH1 evaluation in translational research.