Cancer Research Communications

Genetic uniqueness of the tumor microenvironment significantly influences cancer growth, survival, and response to therapy, independent of the cancer cells intrinsic properties or the adaptive immune system. Using genetically distinct Rag1-/- mouse models, this study shows that different strains exhibit varied tumor growth kinetics and survival outcomes when xenografted with identical leukemic and solid tumor cell lines. This study further highlights the critical role of the myeloid immune compartment and shows that disrupting both lymphoid and myeloid systems alters cancer progression. These results also reveal that the tumor microenvironment can permanently alter cancer cell phenotypes and significantly affect chemotherapy efficacy, as seen with Cisplatins varying effects across strains. These findings underscore the importance of considering genetic background in preclinical cancer models, suggesting that reliance upon a single mouse strain may lead to incomplete conclusions about cancer biology and treatment efficacy. SUMMARY STATEMENTPre-clinical xenograft mammalian models are used to study human diseases. Here we report that the genetic uniqueness of the tumor microenvironment, independent of the immune system, can determine the fate of cancer progression, survival, and therapy response.

PurposeSWI/SNF (BAF) chromatin remodeling complex alterations are common in urothelial carcinoma, yet no biomarker-directed therapeutic strategies have been established for this population. We investigated whether BAF alterations delineate a biologically distinct, therapeutically actionable urothelial carcinoma subtype. Experimental DesignWe performed integrative genomic and transcriptomic analyses of 792 urothelial carcinoma tumors from the Oncology Research Information Exchange Network (ORIEN) and validated findings in the TCGA-BLCA cohort. Mechanistic studies incorporated RNA sequencing and ATAC-seq following histone deacetylase (HDAC) inhibition. Functional dependencies were assessed using patient-derived xenograft organoids and cell line models. Clinical relevance was explored in a biomarker-enriched investigator-initiated trial. ResultsApproximately half of urothelial carcinoma tumors exhibited BAF alterations, defining a previously unrecognized chromatin-altered molecular subtype characterized by activation of proliferative programs, loss of lineage identity, and altered metabolic signaling. This subtype was enriched for transcriptomic programs associated with HDAC inhibitor sensitivity and depleted of HDAC inhibitor resistance signatures. Mechanistically, HDAC inhibition induced widespread chromatin remodeling with reduced accessibility at AP-1 and TEAD-associated regions, and downregulation of E2F- and MYC-driven transcriptional networks. Functional studies confirmed enhanced HDAC inhibition sensitivity in ARID1A-mutated cell lines and a patient-derived organoid model. Early clinical observations demonstrated a durable responder treated with HDAC inhibitors and immunotherapy. ConclusionsBAF alterations define a chromatin-dependent tumor state in urothelial carcinoma that is selectively vulnerable to HDAC inhibition. Integrating genomic, epigenomic, functional, and early clinical evidence, these findings provide a rationale for biomarker-enriched clinical trials and HDAC inhibitor-based combination strategies in urothelial carcinoma.

Endometrial cancer (EC), the most common gynecologic malignancy in the USA, has seen limited improvement in patient outcomes over recent decades, underscoring the need for relevant preclinical models. To address EC heterogeneity, we established an integrated platform of patient-derived xenografts (PDXs) and matched patient-derived primary cancer cells (PDCs) for disease modeling and systematic drug sensitivity testing. Fresh tumor specimens (n=103) were collected from EC patients to generate PDXs in immunodeficient mice and corresponding PDCs. Fifty-three PDX models were successfully established (52% engraftment rate), with higher success observed in high-grade, recurrent, metastatic tumors (70%), compared with their low-grade counterparts (56%). Histopathologic and immunohistochemical analyses confirmed that PDX tumors faithfully preserved morphology, hormone receptor status, and intertumoral heterogeneity across multiple passages. Using 13 PDC models, we performed an unbiased screening of 179 FDA-approved oncology drugs, revealing marked intertumoral variability in drug response. Almost all PDC models exhibited limited sensitivity to NCCN-recommended therapies, highlighting the need for alternative treatment strategies. In contrast, multiple FDA-approved agents including epigenetic modulators, dual PI3-kinase/HDAC inhibitors, topoisomerase II inhibitors, and proteasome inhibitors demonstrated potent antitumor activity. Importantly, a low-dose combination of the DNA methyltransferase inhibitor 5-azacytidine and the histone deacetylase inhibitor romidepsin significantly suppressed tumor growth across six independent PDX models. Together, these findings establish a comprehensive PDX and PDC platform as a robust translational resource. By capturing the histopathologic and molecular diversity of EC and identifying clinically actionable therapeutic advantages, including an epigenetic combination regimen, this study offers a translational resource for preclinical drug evaluation.

Treatment-emergent neuroendocrine prostate cancer (NEPC) is an aggressive, therapy-resistant disease arising in up to 20% of castration resistant prostate cancers, yet robust biologically relevant preclinical models remain scarce. Here, we describe a technical blueprint for establishing an integrated platform of patient-derived models from visceral and bone metastases collected through a prostate cancer rapid autopsy program (PC RAP). We report the establishment and characterization of patient-derived xenograft (PDX) models from liver metastasis tissue, liver and bone metastasis-derived organoid lines (PDOs), and corresponding patient-derived organoid xenograft (PDOX) models. In addition, we established, to our knowledge, the first mesenchymal stem cell (MSC) cultures derived from neuroendocrine prostate cancer (NEPC) bone metastases. The PDOs preserved intratumoral heterogeneity, displaying both CRPC-NE and CRPC-adenocarcinoma features. These organoids retained neuroendocrine identity across multiple passages, with transcriptomic profiles concordant with the original patient tissue and matched PDX models generated at our institution and at the National Cancer Institute (NCI Patient-Derived Models Repository). To model the bone metastatic microenvironment, we generated novel organoid-based New Approach Methodologies (NAMs) by co-culturing PDOs with iPSC-derived bone marrow organoids, establishing a physiologically relevant vascularized organotypic model of PC bone metastasis. To extend our studies in vivo, we established preclinical models using the liver and bone metastasis-derived organoid models. The PDOX models were tumorigenic and developed spontaneous lymph node metastases, providing clinically relevant models for investigating lethal NEPC biology. Together, these complementary patient-derived models provide a robust and versatile platform for investigating NEPC biology, metastatic progression, and evaluating new therapeutic strategies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=184 SRC="FIGDIR/small/740121v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@a4b747org.highwire.dtl.DTLVardef@1fcb778org.highwire.dtl.DTLVardef@7167e6org.highwire.dtl.DTLVardef@15c5b6d_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LINovel preclinical models of visceral and bone metastases established from a prostate cancer rapid autopsy program. C_LIO_LIThis study is the first to establish mesenchymal stem cell cultures from NEPC bone metastases. C_LIO_LIPDOs preserve heterogeneity, showing both CRPC-NE and CRPC-Adeno features, with transcriptomic profiles concordant with originator tissue and PDX models. C_LIO_LIPC RAP-derived organoids are tumorigenic in vivo and generate spontaneous lymph node metastases. C_LI

Survival analysis in head and neck squamous cell carcinoma (HNSCC) is traditionally performed using Cox proportional hazards models, alongside some exploration into black-box machine learning methods. The Super Learner (SL) algorithm addresses this model selection dilemma by combining diverse candidate algorithms into a weighted ensemble to perform comparably to the best candidate method. This study evaluates the performance of SL in HNSCC. Proteomic features as well as clinical covariates from 96 CPTAC HNSCC samples were modeled with three candidate algorithms (Cox LASSO, Cox Ridge, and Random Survival Forest) as well as the ensemble SL method. Models were optimized via Unos time-dependent Concordance Index (C-index) and tested at 1- and 3-year time horizons using 2000 bootstrap resamples. The Cox Ridge regression model achieved the highest predictive accuracy among the four total methods. However, the SL demonstrated stable performance over both time horizons (1-year C-index: 0.985; 3-year C-index: 0.960). Variable importance analysis of the Cox Ridge model successfully identified malignant proteins (ATR, MAML1, MIEN1) alongside novel potential prognostic indicators (ZNF800, KERA). This analysis emphasizes the statistical necessity for larger cohorts for ensemble learning, while providing a benchmark of proteomic indicators in HNSCC.

Epithelioid sarcoma (EpS) is an ultra-rare, aggressive soft tissue sarcoma (STS) driven by INI1 loss and consequent hyperactivation of the chromatin-modifying enzyme EZH2. Although the EZH2 inhibitor tazemetostat has shown clinical activity, responses remain limited, highlighting the need for improved treatment strategies. Here, using two in-house generated patient-derived xenograft models and matched cell lines derived from INI-1 deficient EpS, we investigated EZH2 inhibition in combination with doxorubicin, the first-line standard for advanced STSs, identifying distinct patterns of response and resistance. Integrated transcriptomic and functional analyses revealed that response to EZH2 inhibition-based therapy was associated with chromatin remodeling, characterized by increased H3K27 acetylation and downregulation of histone deacetylase (HDAC)-related transcriptional programs. Conversely, the intrinsically resistant model failed to undergo this epigenetic transition despite EZH2 inhibition. Pharmacological HDAC inhibition restored H3K27 acetylation, promoted apoptosis, and enhanced the activity of EZH2 inhibition-based therapy. These findings identify failure to accumulate H3K27 acetylation as a hallmark of resistance to EZH2 inhibition-based treatment, and show that pharmacological HDAC inhibition can restore this chromatin transition and re-sensitize resistant tumors, providing a rationale for combined epigenetic targeting strategies in INI1-deficient malignancies. Translational relevanceProspective trials are challenging in rare tumors such as epithelioid sarcoma (EpS), limiting the level of evidence for existing therapies and the development of new agents. This is particularly relevant for EpS, where drug regimens are those used for all soft tissue sarcomas (STSs), and the specific mechanisms of drug response remain poorly understood. This preclinical study of tazemetostat in combination with doxorubicin shows differential outcomes in two INI1-deficient proximal-type EpS models, providing evidence of the heterogeneity that exists even within the same tumor subtype and fostering the need to better understand the molecular mechanisms driving drug sensitivity/resistance in this disease. In addition, we demonstrated the potential to treat EpS models through modulation of epigenetic mechanisms, showing that HDAC inhibition may restore sensitivity to EZH2-targeted therapy. These findings support the rationale for developing combination strategies incorporating epigenetic modulators and provide a preclinical framework for overcoming resistance to current therapies in EpS.

Traditional animal models are often inbred and genetically uniform. This makes them powerful for controlled experiments, but it limits how well they represent the patient-to-patient variation seen in real-world disease. Comparative oncology seeks to address this gap by studying naturally occurring cancers in outbred companion animals, especially dogs. Canine medicine offers two important advantages: first, prospective trials can often be completed faster than in humans and second, dogs are already part of the translational pipeline through pharmacokinetic and toxicology studies. Here, we assessed the transcriptional fidelity of human and canine invasive urothelial carcinoma in primary tumors and patient-derived organoids. We then used single-cell and spatial data to resolve the underlying cellular organization. Despite strong species and platform differences, human and canine tumors preserved the same major luminal-basal structure and a similar tumor microenvironment. The two species reached this shared biology through different recurrent mutations. These included FGFR3 alterations in humans and BRAF alterations in dogs, which converged on overlapping pathways and a luminal phenotype. Human and canine organoids also underwent a similar shift in culture. Both became more proliferative and metabolic while losing inflammatory programs. Thus, organoids preserved important tumor biology while introducing predictable platform effects. Single-cell and spatial analyses showed that the luminal-basal axis reflects a gradient of cell states organized around the tumor-stroma boundary, rather than two discrete tumor types. This helps explain why bulk RNA-sequencing subtypes are reproducible but coarse. Together, these findings define where canine and human bladder cancer agree, where they differ, and how dogs can support parallel therapeutic and diagnostic development.

Cisplatin resistance remains a major barrier in head and neck squamous cell carcinoma (HNSCC) treatment. ATP-binding cassette (ABC) transporters contribute to chemoresistance by limiting intracellular drug accumulation. Bitter taste receptor 10 (T2R10) has been implicated in ABC transporter regulation, but its role in HNSCC remains undefined. HNSCC cell lines were treated with T2R10-agonist caffeine (100 or 200 M), cisplatin, or a combination, and viability was assessed by crystal violet assay. T2R10 promoter activity and expression following caffeine exposure were evaluated using a promoter-driven mCherry reporter and RT-qPCR. ABC transporter expression was measured after caffeine treatment and T2R10 gene (TAS2R10) knockdown or overexpression. Associations between tumor TAS2R10 expression and survival were assessed using TCGA data through GEPIA2. Caffeine enhanced the cisplatin-associated reduction in viability in a cell line- and concentration-dependent manner, with the strongest effect seen in UM-SCC47. A significant effect was observed in FaDu at 200 M of caffeine, and minimal response in RPMI 2650. RPMI 2650 cells and FaDu cells exhibited lower baseline TAS2R10 expression and RPMI 2650 cells did not demonstrate enhanced cisplatin sensitivity following caffeine treatment. Caffeine treatment increased TAS2R10 promoter activity and expression and was associated with decreased ABCG2 expression. TAS2R10 knockdown increased ABCG2 and ABCF1 expression, whereas TAS2R10 overexpression reduced ABCG2 and ABCC1 expression. High tumor TAS2R10 expression was associated with improved disease-free survival (log-rank p=0.0071; HR=0.61) but not overall survival. Caffeine enhances cisplatin sensitivity in selected HNSCC models. Caffeine exposure is associated with increased TAS2R10 expression and reduced expression of chemoresistance-associated transporters, particularly ABCG2.

IntroductionNectin-4 targeting antibody-drug conjugate (ADC) enfortumab vedotin (EV), in combination with pembrolizumab, is the first-line treatment for patients with locally advanced or metastatic urothelial carcinoma (UC). Optimal treatment strategies for patients who are non-responders or progress on EV with pembrolizumab remain an unmet clinical need. We sought to characterize ADC and immunotherapy (IO)-associated target expression profiles to identify candidate therapeutic vulnerabilities beyond EV. MethodsWe conducted a literature review to identify ADC and IO targets with approved or investigational relevance in UC. Unsupervised hierarchical clustering was used to identify clusters of target gene expression in RNA-seq data. Transcriptomic clustering analyses were performed in 434 patients from The Cancer Genome Atlas Bladder Urothelial Carcinoma cohort (TCGA-BLCA) and validated in an independent cohort of 478 patients from the Oncology Research Information Exchange Network (ORIEN) consortium. Proteomic interrogation of these targets was performed using mass spectrometry data from additional cohort of 116 patients. Differential gene expression analyses evaluated associations between target expression patterns, histologic variants, and consensus molecular subtypes of muscle-invasive bladder cancer (CMIBC). ResultsWe identified 13 ADC and 10 IO-associated targets with translational relevance in UC. Transcriptomic analyses revealed three reproducible clusters of overexpressed target genes across independent cohorts: 1) a luminal/epithelial-associated cluster enriched for VTCN1, SLITRK6, FGFR3, NECTIN4, TACSTD2, ERBB2, and ERBB3; 2) an immune target predominant cluster enriched for BTLA, LAG3, PDCD1, TIGIT, CTLA4, TNFRSF9, TNFRSF18, TNFRSF4; and 3) a basal/neuroendocrine-associated cluster characterized by CD274, F3, NT5E, EGFR, MET and DLL3. Similar clusters were largely conserved at the proteomic level. Adenocarcinomas overexpressed ERBB3 compared to neuroendocrine and squamous cell carcinomas. Pure squamous cell carcinomas overexpressed TACSTD2 compared to adenocarcinomas. In CMIBC subtypes, basal/squamous tumors expressed higher levels of CD274, EGFR, F3, LAG3, NT5E, and TNFRSF18, whereas luminal tumors demonstrated higher ERBB2 and ERBB3 expression. Neuroendocrine-like tumors showed higher DLL3 expression compared to all other subtypes. Tumors with low expression of NECTIN4, TACSTD2, and FGFR3 were enriched for alternative targets including DLL3, CD274, and CD276. Our findings provide a framework for hypothesis-driven therapeutic prioritization in advanced UC. Conclusions: UC is characterized by reproducible, biologically distinct patterns of ADC and IO target expressions. The degree of expression of NECTIN4 was positively associated with TACSTD2, FGFR3 and inversely associated with DLL3, CD276, and CD274, supporting alternative biologically informed treatment strategies besides EV . Histologic variants and molecular subtypes of UC also display distinct patterns of target expression. This study provides the first integrated transcriptomic framework linking ADC and IO target co-expression patterns for hypothesis-driven therapeutic prioritization. These findings provide a basis for rational ADC and immunotherapy development in advanced UC and support prospective proteomic validation in treatment stratified cohorts. Statement of Translational RelevanceEnfortumab vedotin plus pembrolizumab has redefined first-line therapy for advanced urothelial carcinoma, yet treatment selection following resistance or progression remains undefined. In this study, we integrate transcriptomic and proteomic analyses across independent cohorts to define reproducible patterns of antibody-drug conjugate (ADC) and immunotherapy target co-expression in urothelial carcinoma. We identify biologically distinct target-expression patterns that are associated with histologic and molecular subtypes and demonstrate coordinated and, in some cases, mutually exclusive relationships among therapeutically actionable targets. These findings have direct translational implications. First, they provide biologic rationale for rational sequencing and combination strategies based on co-expressed targets in NECTIN4-enriched tumors. Second, they identify alternative therapeutic vulnerabilities, including DLL3- and CD274-associated pathways, in tumors with low NECTIN4 expression, a population potentially enriched for resistance to EV-based therapy. Finally, this framework establishes a foundation for biomarker-driven clinical trials in urothelial carcinoma and supports the development of precision therapeutic approaches beyond current standards.

Tumor cellular composition--including malignant cell states, immune populations, and stromal populations--is increasingly recognized as a determinant of therapeutic response and resistance to anti-cancer agents, yet comprehensive cellular profiling remains largely confined to research settings. Here, we present a clinically compatible sample-to-report workflow for tumor composition profiling from routine formalin-fixed paraffin-embedded (FFPE) clinical specimens. By combining low-input single-nucleus RNA sequencing with foundation model- based automated cell annotation, this workflow enables prospective sample-by-sample analysis without dedicated research material or cohort-based processing. Across 116 clinical specimens representing six cancer types, we generated reproducible measurements of cellular composition and cell-type-specific gene expression, demonstrated high technical reproducibility, and showed concordance with pathological assessment of immune infiltration. The workflow was similarly applicable to archival FFPE material and ultra-low-input biopsy specimens. Together, these findings establish a practical framework for routine single-cell profiling from standard pathology specimens and open the perspective of prospective evaluation of cellular composition as a clinical biomarker in precision oncology.

Radiation therapy is a cornerstone of breast cancer treatment and reduces recurrence overall. However, patients with triple negative breast cancer (TNBC) continue to experience recurrence at higher rates than patients with other subtypes, especially when immunocompromised. While CD8 T-cells are known to mitigate recurrence, the role of CD4+ T-cell subsets in shaping the irradiated microenvironment remains unclear. We show that irradiated mammary tissue from mice accumulates CD4+ T-cells and exhibits a TGF{beta}-enriched cytokine milieu coincident with macrophage infiltration. We demonstrate that Th2-polarized CD4+ T-cells promote invasion of TNBC cells and macrophages through secretion of TGF{beta}. Neutralization of TGF{beta} significantly reduces this invasive phenotype. Mechanistically, Th2-conditioned media induces Tgfb1 expression in both TNBC cells and macrophages, establishing a TGF{beta}-dependent feed-forward amplification loop. In TNBC cells, Th2-derived TGF{beta} activates non-canonical signaling characterized by increased p38 MAPK and NF-{kappa}B phosphorylation, linking cytokine exposure to pro-invasive behavior. Together, these findings identify Th2-derived TGF{beta} as a driver of pro-invasive tumor reprogramming and suggest that interruption of Th2-TGF{beta} signaling may prevent recurrence following therapy.

Patient-derived organoids (PDOs) have transformed translational cancer research, allowing tractable models that better represent clinical features than traditional immortalized cell lines. Here we describe two PDOs with differential responses to carboplatin derived from sequential ascites fluid collections from a patient with high-grade mullerian carcinoma, that could not be further subclassified on the omental biopsy. Uterine origin was clinically excluded by pelvic imaging/CT scan of the uterus and absence of vaginal bleeding. Successful derivation from independent collections enabled comparison of intra-patient heterogeneity across sequential ascites samples and demonstrates that PDO efficiency rate is at least partly patient-specific or tumor-dependent. We performed long-read whole genome sequencing on the two PDOs, OC104 and OC109, to better characterize the structural variant landscape while also obtaining information on single nucleotide variants and DNA methylation. In addition to confirming single nucleotide variants noted in clinical sequencing (TP53, KRAS, SPOP, PPP2R1A, KMT2D), we identified additional variants in TSC2, NCOR2, and CTNNA2 that are predicted to be likely pathogenic. The spectrum of mutations, particularly the coincident KRAS and TP53, highlighted unexpected overlap with ovarian mucinous carcinoma. We also identified larger insertions and deletions that result in non-synonymous variants in MUC5AC, TPRX1, and BMX, as well as four translocation events, including two that could not have been resolved with short-read sequencing. Differentially methylated promoters between the two PDOs include 201 oncogenes and tumor suppressor genes, with HNF1A, MSI2, and SETBP1 having methylation directions consistent with these genes' roles in platinum response differences observed between the PDOs. Notably, the clonal nature of PDOs produced from two samples taken one week apart is important for the field to appreciate, particularly since they have clonal differences in platinum response. The temporal differences in clonality may indicate a limitation of low volume sampling, however may provide opportunity to longitudinally predict clinical outcomes. We also demonstrate the ability of long-read sequencing to add detail into the genomics and epigenetics of ovarian cancer.

Decades of research have shown that tumor hypoxia is associated with resistance to anti-cancer treatments. Analysis of TCGA gene expression profiles indicates that NSCLC is among the most hypoxic of cancers despite the high levels of oxygen in the surrounding lung tissue. Several groups have shown that extrinsic factors such as poorly formed tumor vascular contributes to tumor hypoxia. Here, we have investigated the possibility that genetic abnormalities within the tumor also contribute to the development of hypoxia. Our analysis of NSCLC patient datasets in the Cancer Genome Atlas (TCGA) PanCancer and ORIEN datasets revealed a strong correlation between tumor hypoxia and amplification of chromosome 3q which is found in up to 40% of NSCLC. Several oncogenic driver genes have been identified in 3q, and we identified a passenger gene encoding mitochondrial complex I subunit NDUFB5 at 3q26.33. To provide experimental evidence that NDUFB5 amplification can drive tumor hypoxia, we have used CRISPR activation technology to generate murine cells overexpressing the endogenous NDUFB5 gene. We found that cells overexpressing NDUFB5 have elevated rates of oxygen consumption, and tumors grown from these cells have increased amounts of hypoxia with associated treatment resistance. Here, we investigate the impact of manipulating NDUFB5 gene expression on mitochondrial complex I activity and experimentally validate the clinical observations that NDUFB5 overexpression leads to increased levels of intratumoral hypoxia and increased resistance to radiation therapy and immunotherapy.

Neuroblastoma is the most common extracranial solid tumor in children, presenting remarkable clinical heterogeneity with survival outcomes ranging from spontaneous regression to aggressive progression. MYCN oncogene amplification and age at diagnosis are established prog-nostic factors that are typically treated as independent covariates in risk stratification, yet their joint influence on the tumor immune microenvironment remains poorly understood. Here we show that stratifying patients by both variables simultaneously reveals six reproducible immune subtypes with distinct transcriptional programs and prognostic significance. Consensus clustering of immunomodulatory gene expression profiles from 149 patients in the TARGET-NBL cohort identified subtypes whose survival trajectories differ significantly within clinical strata defined by MYCN status and age at diagnosis. A linear Support Vector Machine classifier trained on these subtypes, using immunomodulatory gene expression combined with MYCN amplification status and age at diagnosis as predictive features, achieved 97.2% accuracy and a Cohens Kappa of 0.963 under 10-fold cross-validation, and generalized to an independent cohort of 493 patients (GSE62564). Kaplan-Meier analysis revealed significant survival differences across subtypes in both cohorts (TARGET-NBL: log-rank p = 0.0018; GSE62564: log-rank p < 0.0001). Single-sample gene set enrichment analysis identified differential activation of proliferative and immune response pathways across subtypes, consistent between both cohorts. These findings suggest that integrating MYCN amplification status and age at diagnosis as joint determinants of immune organization may reveal prognostic heterogeneity that is not fully captured when these factors are considered independently.

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

With the advent of genome sequencing and its widespread use in the clinic, there is a great need to identify mutational biomarkers that predict therapeutic responses. DNA polymerase Beta (Pol{beta}) and the base excision repair (BER) pathway have been previously implicated as modulators of response to platinum-based chemotherapies and are mutated in as high as 30% of cancers. Here, we show in a triple-negative breast cancer (TNBC) model that two classes of mutations in Pol{beta}, reduced catalytic activity (E295K and D256A mutation) and reduced fidelity (I260M), are sufficient to drive cisplatin and carboplatin-specific sensitivity. Cellular response to oxaliplatin in these Pol{beta} mutant models is minimal relative to cisplatin and carboplatin. Additionally, we show that sensitivity is associated with reduced repair of both platinum-induced DNA intrastrand adducts and interstrand crosslinks (ICLs). Downregulation of the upstream BER factor uracil DNA glycosylase (UNG) reverses drug sensitivity consistent with these Pol{beta} mutations negatively impacting ICL DNA repair to drive drug sensitivity. In addition, intrastrand adduct repair readout indicates these lesions also play a role in the sensitivity observed in Pol{beta} mutant models. In vivo studies demonstrate a significant effect on tumor growth delay with cisplatin treatment in tumor xenografts harboring Pol{beta} mutations. These results support the potential for using Pol{beta} mutations as predictive biomarkers for cisplatin and carboplatin therapies in the clinical setting.

Head and neck squamous cell carcinoma (HNSCC) remain a major clinical challenge due to its high heterogeneity and limited therapeutic response, resulting in a 5-year overall survival rate of only [~]50%. Identifying molecular pathways that drive tumor progression while suppressing anti-tumor immunity is therefore critical for developing more effective therapies. NAT10 (N-acetyltransferase 10) is the only known enzyme responsible for catalyzing the RNA modification N4-acetylcytidine (ac4C) on rRNA, tRNA, and mRNA, and has been implicated in tumor progression in several cancers. In this study, we identify NAT10 as a key suppressor of tumor-intrinsic immune signaling in HNSCC. NAT10 expression was significantly elevated in tumor tissues and HNSCC cell lines and was associated with poor overall survival. Moreover, high-risk HPV, a major etiological factor in HNSCC, upregulated NAT10 protein expression through the viral oncoproteins E6 and E7. Functional inhibition of NAT10, either by genetic depletion or the small-molecule inhibitor Remodelin, activated tumor-intrinsic innate immune responses, as evidenced by increased IRF3 phosphorylation and induction of type I/II interferons and interferon-stimulated genes. Depletion of NAT10 was able to suppress tumorigenic phenotypes, including cell proliferation, migration, and colony formation in HNSCC cells. Importantly, activation of the STING signaling pathway using agonist cyclic di-GMP further amplified immune activation in NAT10-inhibited cancer cells. Together, our findings establish NAT10 as a previously unrecognized negative regulator of tumor-intrinsic immunity in HNSCC and support NAT10 targeting, particularly in combination with STING agonists, as a promising immunotherapeutic strategy.

Multiple myeloma (MM) is a plasma cell malignancy that frequently harbors activating mutations in NRAS and KRAS oncogenes. Previous clinical trials targeting the Ras/MAPK oncogenic pathway with MEK inhibitors (MEKi) were met with limited efficacy, and newer generation of Ras inhibitors (RASi) have not been specifically evaluated in MM patients. To investigate the vulnerabilities of Ras-mutant MM to targeted therapies, we examined the sensitivity of a panel of human MM cell lines to the RASi RMC-6236 (daraxonrasib) and the MEKi trametinib. Although Ras-mutant MM cells are responsive to oncogenic Ras signaling and are sensitive to RAS inhibition, their sensitivity to MEK inhibition is heterogeneous. Mechanistic studies revealed that c-Myc protein is destabilized by MEK inhibition only in MEKi-sensitive MM cells but not in MEKi-resistant cells, and pharmacological and genetic stabilization of c-Myc is sufficient to confer MEKi resistance. In contrast, Ras inhibition reduced c-Myc protein across all MM cell lines tested, regardless of their dependency on the MAPK pathway, and c-Myc expression was insufficient to promote RASi resistance. Together, these findings demonstrate that c-Myc protein stability differentiates the response of Ras-mutant MM cells to Ras and MEK inhibition, and suggest that direct targeting of the Ras oncoprotein, rather than its downstream MAPK pathway, may present a more effective strategy.

CD47 is a ubiquitously expressed transmembrane protein that functions as a negative immune checkpoint, marking host cells as "self" by delivering an inhibitory "dont-eat-me" signal to phagocytes. Cancer cells co-opt this mechanism, upregulating CD47 to evade immunosurveillance and phagocytosis by innate immune cells, a pattern observed across solid tumours and haematological malignancies. CD47 overexpression correlates with poor prognosis across most cancer types, including therapy-resistant disease. Despite extensive efforts to develop CD47-targeted therapies, the downstream biological consequences of aberrant CD47 expression within tumour cells remain poorly characterised beyond its established anti-phagocytic role. This study investigated non-immunological, pro-tumorigenic functions of CD47 to define the cellular effects, beyond immune evasion, that CD47-targeted therapy might disrupt. We found that CD47 exerts cancer type-specific effects: in DLBCL, CD47 loss impaired mitochondrial metabolism and sensitised cells to R-CHOP standard-of-care chemoimmunotherapy, whereas in triple-negative breast cancer (TNBC), CD47 knockdown delayed cell cycle progression, enhanced migration, and conferred resistance to specific chemotherapeutic agents. These findings indicate that CD47 has multifaceted, context-dependent roles in tumour biology that extend beyond immune checkpoint signalling. Clinically, this suggests CD47-targeted therapies may produce cancer type-specific off-target effects on tumour metabolism, proliferation, and drug sensitivity, which are considerations that should inform their rational combination with existing targeted therapies.

Radiotherapy induces cytotoxic DNA damage, but activation of DNA repair pathways and cell-cycle checkpoints can limit therapeutic efficacy. Here, we developed a high-throughput, flow cytometry-based screening platform to identify compounds that inhibit radiation-induced DNA repair and checkpoint activation. Reh leukemia and A549 lung cancer cells were irradiated and screened against up to 700 bioactive compounds, with DNA damage persistence quantified by {gamma}H2AX levels across independent screens. Cell barcoding using Pacific Blue staining was incorporated to enable highly accurate quantification of {gamma}H2AX across treatment conditions. The platform yielded robust and reproducible results and supported multiparametric analysis, including assessment of G2 checkpoint activation by phospho-histone H3. Largely overlapping candidate radiosensitizers were identified in both cell lines, including the multi-kinase inhibitor 5-iodotubercidin and the PI3K/mTOR inhibitor omipalisib. Validation studies in lung cancer and glioblastoma models confirmed screen performance. Mechanistically, omipalisib reduced phosphorylation of the non-homologous end-joining protein DNA-PK, consistent with impaired double-strand break repair. Both compounds enhanced radiosensitivity in clonogenic survival assays. Notably, 5-iodotubercidin increased radiosensitivity in glioblastoma cells despite previous reports of radioprotective effects in normal brain tissue. Together, these findings establish a robust barcoded screening approach for identifying radiosensitizers that target DNA damage repair and checkpoint responses.