Oncotarget

Acute radiation syndrome remains a major consequence of high-dose ionizing radiation exposure, and effective pharmacological countermeasures are limited. Nitro-fatty acids possess anti-inflammatory and cytoprotective properties, suggesting potential effectiveness in radiation injury. Here, we investigated the radioprotective effects of 10-nitro-oleic acid (10-NO2OA) in mouse models of total body irradiation (TBI). C57BL/6 mice were exposed to either sublethal (4 Gy) or LD50-level (7.5 Gy) gamma irradiation and treated with 10-NO2OA according to pre-irradiation, post-irradiation, or combined treatment regimens. Survival, hematological responses, bone marrow cells (BMCs)/colony-forming units (CFU), plasma granulocyte colony-stimulating factor (G-CSF), NRF2-responsive signaling, and intestinal responses were evaluated. Administration of 10-NO2OA before and after irradiation significantly improved 30-day survival following 7.5 Gy TBI (57% vs. 20% in vehicle-treated controls). In contrast, post-irradiation treatment alone was ineffective, whereas pre-irradiation treatment alone produced a non-significant trend toward improved survival. The protective effect was absent in BALB/c mice, indicating strain dependence. Although 10-NO2OA did not prevent long-term radiation-induced cytopenia, it increased neutrophil and platelet counts, elevated plasma G-CSF levels shortly after irradiation, and transiently enhanced BMC counts. In irradiated BMCs, 10-NO2OA activated NRF2-responsive gene expression in vitro and in vivo. Exploratory intestinal analyses revealed improved macroscopic appearance and partial restoration of irradiation-suppressed cytokine and growth factor production. In conclusion, 10-NO2OA improves survival following LD50-level gamma irradiation and is associated with modulation of hematopoietic, cytoprotective, and intestinal responses. These findings identify 10-NO2OA as a promising candidate radioprotective agent and support a multifactorial mechanism of action.

Ovarian cancer stands out as one of the most lethal gynecological cancers among women in the United States. While patients with ovarian cancer often show positive responses to treatment, it is common for the cancer to reappear and develop resistance to treatment, so it is important to explore and create novel combinations to increase patient survival rates. ATRi (Ataxia telangiectasia and Rad3-related inhibitors) and ONC201 monotherapies have shown efficacy in ovarian cancer in preclinical models. We hypothesized that combining ATRi with ONC201 would enhance this efficacy. ONC201 induces apoptosis through the TRAIL-pathway following activation of the integrated stress response and inactivation of Akt which is upregulated in ovarian cancer. Six human ovarian cancer cell lines were treated alone and with the novel drug combination of ceralasertib (ATRi) and ONC201. We found an IC50 range of 0.88-20.16 microM for ONC201 and IC50 range of 0.55-54.75 microM for ceralasertib. The combination of ONC201 plus ceralasertib resulted in PARP cleavage consistent with apoptosis along with reduction in Bcl-XL, Xiap and p-Akt expression after 72 hours of treatment. Cytokine profiling showed a decrease in markers of inflammation and tumor progression. Our data provide evidence of synergy from the combination of ATRi ceralasertib and ONC201 against Ovarian cancer cells that merits further investigation including clinical translation.

Abstract Background Liposarcoma is a rare cancer, comprising four major histological subtypes: well-differentiated, dedifferentiated, myxoid and pleomorphic liposarcoma. Therapeutic options remain limited for advanced disease, and additional biomarkers that may support biological characterization, diagnosis, or risk stratification are needed. Methods Bulk RNA sequencing (RNA-seq) was performed on paired tumour and adjacent non-tumour tissues from an analysable subset of 16 patients (22 tumour specimens) within a clinicopathological cohort of 21 patients. Differential gene expression analysis, principal component analysis, gene set enrichment (GSEA) analysis and single-sample GSEA (ssGSEA) were used to define subtype-specific transcriptional programs and imprinted-gene dysregulation. PEG10 expression was validated by RT-qPCR and RNA fluorescence in situ hybridization (FISH), and its cellular localization was explored by combined FISH and immunofluorescence for P53, vimentin, Ki67, and CD34. PEG10 expression in liposarcoma was further investigated in an external single-cell RNA-sequencing dataset of dedifferentiated liposarcoma. An exploratory survival analysis was performed in the pan-sarcoma TCGA-SARC cohort. Results Transcriptomic profiling identified distinct subtype-specific gene expression patterns and broad dysregulation of imprinted genes in liposarcoma compared with adjacent non-tumour tissue. Among all the imprinted genes, PEG10 showed a marked tumour-to-normal expression difference and good discriminatory performance between tumour and normal tissues in the discovery cohort (AUC=0.867). In the TCGA sarcoma cohort high PEG10 expression was associated with shorter overall survival. Immuno-FISH and single-cell analyses revealed that PEG10 was predominantly detected in tumour and proliferating cell populations. Importantly, PEG10 expression was higher in high-grade tumours. In bulk RNA-seq data, PEG10 expression correlated with hypoxia, extracellular matrix remodelling and invasion ssGSEA signatures. In the external single-cell RNA-seq dataset PEG10high tumours showed a reduced immune cell infiltration. Conclusions This study provides an integrated transcriptomic characterisation of liposarcoma and identifies PEG10 as a candidate tumour-associated biomarker linked to aggressive disease features. The diagnostic and prognostic value of PEG10 requires validation in larger, independent, subtype-balanced liposarcoma cohorts, and its potential therapeutic relevance requires direct functional investigation.

Background: This systematic review and meta-analysis evaluated the prognostic value of Hippo signaling pathway biomarkers in oral squamous cell carcinoma. Methods: Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, electronic database searches identified 17 eligible studies involving 1,194 patients. Associations between the immunohistochemical expression of Hippo pathway effectors, primarily YAP and TAZ, and clinicopathological characteristics were assessed. Meta-analyses using random-effects models estimated pooled hazard ratios for overall survival. Results: Predominantly nuclear expression of YAP and TAZ was consistently associated with adverse clinicopathological features, including advanced clinical stage, lymph node metastasis, and poorer histological differentiation. Meta-analysis demonstrated that YAP overexpression was associated with worse overall survival (hazard ratio, 1.918; 95% confidence interval, 1.036 to 3.549; p = 0.038). TAZ overexpression was similarly associated with poorer overall survival (hazard ratio, 2.845; 95% confidence interval, 1.682 to 4.811; p < 0.001). Although substantial between-study heterogeneity was observed, the direction of the prognostic association remained consistent across studies. Conclusions: Increased nuclear expression of YAP and TAZ is consistently associated with more aggressive disease and poorer overall survival in oral squamous cell carcinoma. These findings support the prognostic value of Hippo pathway biomarkers, although methodological heterogeneity warrants cautious interpretation.

Purpose: Uveal melanoma (UM) is a rare but aggressive intraocular malignancy originating in the uvea (choroid, iris, and ciliary body). Despite primary treatment, 50% of UM patients develop metastases to the liver, lungs, bones, and skin. Topotecan, a topoisomerase I inhibitor, and SP141, an MDM2 inhibitor, have been studied in cancers, including retinoblastoma. This study explores a combined approach with these drugs in the 92-1 UM cell line. Methods: Primary choroidal melanoma-derived 92-1 cells were treated with topotecan and SP141 individually to determine IC50 values. Cell viability and proliferation were assessed using the MTT assay and the xCELLigence real-time monitoring system. The synergistic effects were analyzed quantitatively using the Loewe Additivity and Highest Single-Agent (HSA) models. Flow cytometry was employed to analyze cell cycle distribution, while cell death and apoptosis were monitored via high-throughput, real-time quantitative analysis using the IncuCyte live-cell kinetic imaging system. Results: Both Topotecan and SP141 induced significant cell death in 92-1 cells. xCELLigence showed dose- and time-dependent inhibition of proliferation. Combining sub-IC50 concentrations of topotecan and SP141 exhibited significant synergistic effects, markedly reducing growth. Cell cycle analysis revealed that topotecan causes a dose-dependent biphasic arrest in G2 and S phases, while SP141 causes G2/M arrest, leading to cell death. Cytotoxicity assays corroborated increased cell death following combination therapy, and the Caspase 3/7 assay indicated that apoptosis is the mechanism underlying cell death. Conclusions: The multi-targeted approach combining topotecan and SP141 drugs demonstrated synergistic therapeutic effects in 92-1 cells, suggesting a potential treatment strategy for managing UM.

Kaposi's sarcoma (KS) is an angioproliferative tumor associated with infection by human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV). Among the viral genes involved in KS pathogenesis, the viral G protein-coupled receptor (vGPCR) plays a key role in oncogenic transformation and angiogenesis, and its sustained activity contributes to tumor development and maintenance. Quercetin (QUE), a naturally occurring flavonoid with well-recognized anticancer properties, has been shown to reduce cell viability and proliferation and to induce apoptosis in vGPCR-transformed endothelial cells. This study further investigated the mechanisms underlying the antineoplastic effects of QUE in vGPCR cells, an in vitro model of Kaposi's sarcoma. Initially, QUE promoted the activation of MAPK signaling pathways in vGPCR cells, increasing the phosphorylation levels of ERK1/2, JNK1/2, and p38, without affecting AKT phosphorylation. In parallel, QUE increased intracellular oxidant levels, followed by lipid peroxidation and DNA damage, supporting a link between oxidative imbalance and genotoxicity. However, increased total glutathione content, and unaltered Gpx4 mRNA expression, suggest the induction of oxidative stress and compensatory antioxidant responses. Furthermore, increased intracellular oxidant levels were associated with MAPK signaling activation, particularly JNK1/2, whereas ERK1/2 and p38 activation may reflect a compensatory response that is insufficient to overcome QUE-induced cytotoxicity. Taken together, these findings indicate that QUE exerts antiproliferative effects in vGPCR cells through a mechanism involving oxidative stress, MAPK activation, and DNA damage, supporting its potential relevance in the study of therapeutic strategies for virus-associated malignancies.

Background: Brain-penetrant histone deacetylase inhibitors (HDACi) are promising radiosensitizing agents for glioblastoma (GBM). HDAC inhibitors can impair DNA damage response (DDR) pathways and promote persistent DNA damage. Here, we evaluated whether combined inhibition of HDAC and the DNA repair kinase, ataxia-telangiectasia mutated (ATM), could synergize with radiation (IR) and improve therapeutic efficacy in GBM. Methods: In vitro dose-response assays were performed in patient-derived xenograft (PDX)-derived glioma stem cells (GSCs) with both inhibitors of HDAC (quisinostat, QST) and ATM (AZD1390) with or without IR treatment to assess synergy. Pharmacodynamic responses were evaluated by immunoblotting for histone acetylation, DDR signaling, and DNA damage markers. Transcriptomic analyses were performed to evaluate treatment-associated changes in DNA repair pathways. The combination of QST, AZD1390, and IR was further evaluated in an orthotopic GBM survival study. Results: Combined treatment with QST and AZD1390 produced synergistic radiosensitization in multiple GSC models in vitro. Furthermore, QST combined with IR suppressed transcription of homologous recombination (HR) pathway genes in GSCs, impairing double-strand break repair capacity through an epigenetic mechanism that does not directly inhibit ATM kinase activity. Pharmacodynamic analyses confirmed that QST increased histone acetylation while AZD1390 suppressed ATM signaling, together impairing complementary mechanisms to increase radiosensitivity of GSCs. In an orthotopic GBM model, QST combined with IR significantly prolonged survival compared with IR alone. However, the addition of AZD1390 did not provide a significant survival benefit beyond QST+IR, and AZD1390 combined with IR did not significantly improve survival relative to IR alone. Conclusions: Combined HDAC and ATM inhibition with QST and AZD1390 enhances radiosensitivity in patient-derived GBM cells, mechanistically underpinned by QST-mediated increase in DNA damage and dual disruption of DDR through complementary, non-overlapping mechanisms. However, this in vitro synergy did not confer additional survival benefit beyond QST+IR in an orthotopic GBM model under the dosing conditions tested, suggesting pharmacological limitations with the tumor microenvironment or suboptimal dosing schedules may have constrained in vivo efficacy.

Purpose: There is an unmet need in the treatment of triple-negative breast cancer (TNBC), as recurrence rates remain high; thus, novel treatment strategies are warranted to improve outcomes for patients. Smac mimetics (SMs) are small molecules that inhibit the inhibitors of apoptosis. In this study, we investigated the properties of the SM Xevinapant (AT406) as a radiosensitizer in TNBC. Methods: TNBC cell lines were treated with Xevinapant (AT406) in the presence or absence of ionizing RT and cell viability and apoptosis measured in vitro. Clonogenic assays were performed to confirm radiosensitization. We also assessed xevinapant radiosensitization in the presence of the Tumor Necrosis Factor (TNF) cytokine, which has been shown to enhance SM-induced apoptosis. Radiosensitization mechanisms were further validated by western blotting, flow cytometry and cell cycle analysis. Results: We show that Xevinapant increased cell death in a panel of irradiated TNBC cell lines. In clonogenic assays, Xevinapant sensitized MDA-MB-231 cells to RT-induced cell death, and this was significantly enhanced in the presence of TNF, with a dose-enhancing factor (DEF) of 1.57. We also show that Xevinapant radiosensitization is mediated by TNF-dependent apoptosis. Conclusions: Xevinapant is a radiosensitizer that enhances RT-induced cell death in TNBC cells, and TNF is central to this process through activation of apoptosis. These results are promising for combining RT with Xevinapant to improve local control in TNBC.

Cisplatin remains as standard chemotherapy for patients with HNSCC, but rapid development of drug resistance has limited patient benefit. The p53 tumor suppressor plays a central role in the cellular response to DNA damage in cancer, triggering apoptosis to prevent propagation of damaged cells in tumor development. TP53 gene mutations occur in 65-86% of HNSCC. Small molecule PG3 induces the integrated stress response (ISR), leading to apoptosis via the HRI-eIF2-ATF4-PUMA axis. We hypothesized that a combination of PG3 plus cisplatin could increase apoptosis in TP53-mutated HNSCC cells through enhanced induction of the ISR and ATF4. PG3 synergized with cisplatin to inhibit cell viability, leading to potent apoptosis in TP53-deficient cells. The effect was regulated through the HRI-ATF4-NOXA pathway. Furthermore, we identified that cisplatin activates HRI and leads to the degradation of CReP (constitutive repressor of eIF2 phosphorylation) via E3 ligase {beta}-TrCP, contributing to the induction of the ISR. We noted decreased ATF4 levels after treatment with cisplatin, CPT, or PG3 and cisplatin. Thus, combined therapy of PG3 plus cisplatin likely results in adaptation and acquired resistance via degradation of ATF4. We targeted the degradation mechanism of ATF4 by inhibiting {beta}-TrCP1, CK1{delta}, or CK2, respectively. Each approach successfully blocked ATF4 degradation induced by cisplatin or PG3 plus cisplatin and enhanced apoptosis. Our results provide a rational strategy for triple treatments, involving an ISR inducer, a DNA damaging drug, and a {beta}-TrCP inhibitor/CK1{delta} inhibitor/CK2 inhibitor, to achieve potent and prolonged anti-tumor effects and overcome chemoresistance in HNSCC.

Background: Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine neoplasms originating from chromaffin cells that express somatostatin receptors (SSTRs) and may therefore be susceptible to somatostatin analogue therapy. Despite this biological rationale, prospective evidence supporting the antiproliferative activity of somatostatin analogues in PPGL is limited. We conducted a multicenter phase 2 clinical trial to assess the efficacy and safety of lanreotide in patients with advanced or metastatic PPGL. [LAMPARA, NCT03946527] Methods: Patients with advanced or metastatic PPGL and evidence of recent disease progression received lanreotide depot/autogel 120 mg subcutaneously every 4 weeks. Treatment was planned for 52 weeks with an option to continue for an additional 52 weeks. Endpoints included overall survival (OS), progression-free survival (PFS), and response according to RECIST. Serum chromogranin A (CgA) was evaluated as an exploratory biomarker. A tumor growth rate analysis was planned to allow comparison with data from the CLARINET trial in gastroenteropancreatic NETs. Results: Eighteen patients (median age 42 years; range 28-77) across three centers participated; 78% carried SDHx mutations. Lanreotide was well tolerated with predominantly grade 1-2 adverse events and no treatment discontinuations due to toxicity. The most common treatment-emergent adverse events were diarrhea (67%), injection-site reaction (44%), constipation (39%), fatigue (39%), and abdominal pain (39%). Serious adverse events were reported in four patients and were assessed as not related or unlikely related to lanreotide. All 18 patients had at least one recorded post-baseline tumor assessment. Two partial responses were recorded during extended follow-up. At approximately 1 year, disease stabilization was observed in 11 of 13 patients (85%) with an available assessment, while two patients (15%) had progressive disease. Biomarker analysis showed substantial variability in serum CgA values. Tumor growth rate analysis revealed a median growth rate of 0.00124/day (tumor doubling time 559 days), comparable in order of magnitude to the rate of 0.00046/day observed in 83 lanreotide-treated patients in CLARINET, and consistent with a median PFS [&ge;]2 years. Conclusions: Lanreotide exhibits promising antiproliferative activity in PPGL, with a favorable safety profile and extended periods of disease stabilization. These findings support the consideration of SSAs as an option in managing SSTR-positive PPGLs, delaying more aggressive treatments until disease progression mandates escalation.

Blood-based metabolomic profiling has been widely investigated for breast cancer (BC) detection; however, clinical implementation remains limited due to variability in sample handling, analytical reproducibility, and overfitting during statistical analysis. We established a plasma GC/MS metabolomics workflow for discriminating BC from healthy controls (HC) using conventional machine-learning algorithms. Plasma samples (n = 360; BC = 180, HC = 180) were collected prospectively under standardized preanalytical conditions before surgery and the initiation of systematic anticancer therapy and analyzed using a quantitative GC/MS platform with automated derivatization. Feature selection and model development were conducted using three machine-learning (ML) algorithms (Lasso logistic regression (LR), random forest classifier (RFC), and support vector machine (SVM)). A total of 45 metabolite candidate biomarkers were identified, and the optimal number of metabolite features for each algorithm was estimated by a recursive feature elimination (RFE)-based strategy. The best-performing models achieved area under the ROC curve values (AUC) of 0.910 (LR), 0.893 (RFC), and 0.843 (SVM). We selected prioritizing candidate biomarkers consistently expressed across the multi-algorithm pipeline. A bagging ensemble model improved stability (AUC = 0.911) and reduced false-positive predictions in the independent HC dataset. In addition, model stability with respect to false-positive predictions was assessed using an independent HC cohort (n = 15) that was collected at a separate institution. These results indicate that a plasma metabolomics workflow combined with conventional multi-algorithm ML, algorithm-specific feature selection, and independent assessment provides stable discrimination between BC and HC in a moderately sized cohort.

Background: Chimeric antigen receptor (CAR) T cell therapy has had clinical success in hematologic malignancies, but limited efficacy is seen in solid tumors. In this study, we investigated whether systemic CAR T cell therapy could be enhanced in metastatic models of neuroblastoma when combined with radiopharmaceutical therapy (RPT). Methods: Non-irradiated or irradiated tumor cells were co-cultured with CAR T cells (1:1) in vitro and supernatant media was subsequently collected for cytokines analyses. CAR T cell phenotypes were characterized by flow cytometry including checkpoint marker expression. Xenograft models of metastatic neuroblastoma were generated in NOD-Rag1nullIL2rgnull (NRG) mice. Tumor-bearing mice received 1.8 Gy of radiation delivered by 177Lu-NM600 RPT five days after tumor implantation. Nine days after RPT, CAR T cells were administered intravenously. To evaluate tumor burden, mice were imaged weekly for 4 weeks. Results: In models of metastatic neuroblastoma, 177Lu-NM600 RPT significantly increased overall survival when combined with CAR T cell therapy in vivo. Pre-treatment of tumor cells with 177Lu also significantly increased CAR T cell cytotoxicity while decreasing production of IL-4 and IL-10 in vitro. Co-culture of CAR T cells with irradiated tumors led to increases in PD-1+TIM3+LAG3+ T cells, suggesting that further combination with immune checkpoint inhibitors may enhance clinical efficacy. Conclusions: Our findings demonstrate that low-dose RPT can potentiate the anti-tumor efficacy of CAR T cells in metastatic neuroblastoma. To our knowledge, this is the first report of dosimetry-based RPT being combined with CAR T cells in a metastatic solid tumor setting. These findings underscore the potential of combining RPT and CAR T cells to overcome the unique challenges of solid tumors, particularly when treating metastatic disease.

Germ cell tumors (GCTs) are highly sensitized to cell death in response to DNA damaging agents, a property that underlies the success of current chemotherapeutic regimens. To address the molecular basis for this, known as apoptotic priming, we evaluated how different BCL2 family members modulate the heightened sensitivity of GCTs to therapy. Our analysis of human GCTs finds consistently high expression of the pro-survival factors MCL1 and BCL2L1 (BCLX) in a cohort of primary tumors and in their embryonic precursor cells, frequently accompanied by copy number gains of these loci and reciprocal losses of their pro-apoptotic interaction partners and inhibitors, PMAIP1 (NOXA) and BAD. We find that co-inhibition of MCL1 and BCLX using selective BH3 mimetics results in a potent synthetic lethality in multiple GCT embryonal carcinoma cell lines. When these cell lines were cultured with the DNA damaging agents cisplatin or etoposide, inhibition of MCL1 or BCLX potentiated their apoptotic effect in undifferentiated embryonal carcinoma cell lines, but not in retinoic acid-differentiated cells. The inhibition of MCL1 also heightened cisplatin sensitivity in p53-deficient or -mutant cell lines, which is associated with resistance to therapy. Employing an in ovo human xenograft model, we validate that the combination of cisplatin and MCL1 inhibition enhanced the therapeutic response by eliminating tumor cells. Our findings identify MCL1 and BCLX as critical factors to maintain GCT viability and as putative therapeutic targets to further augment GCT responsiveness to DNA damaging agents.

Pannexin 1 (PANX1) is a channel-forming glycoprotein that has been implicated in regulating melanoma cell properties such as growth, migration, and invasion. Here we report that the CRISPR-Cas9 mediated ablation of PANX1 or channel inhibition with probenecid shifted A375-P cell cycle distribution towards G2/M, while channel inhibition with spironolactone stalled cell cycle progression at the G1/S checkpoint regardless of PANX1 expression. Bulk RNA sequencing revealed cell cycle related gene expression was downregulated with PANX1 targeting strategies and this was exacerbated when PANX1 deficient cells were treated with spironolactone. When grown as spheroids, we found the deletion and inhibition of PANX1 disrupted spheroid compaction, with spheroids displaying poorly defined borders and reduced circularity. The deletion of PANX1 increased spheroid fragmentation, and this phenotype was intensified when spheroids devoid of PANX1 were treated with spironolactone, highlighting a potential synthetic lethal effect. Using liquid chromatography mass spectrometry, we sought to determine whether PANX1 had binding partners that may act to stabilize spheroid integrity and found that PANX1 co-immunoprecipitated various cell adhesion molecules such as {beta}-actin and F-actin-capping protein subunit -1, suggesting PANX1 and its interacting partners may stabilize cell-cell contacts. When control and PANX1 ablated cells were grown in chick chorioallantoic membrane xenografts to assess their tumour growth characteristics, we discovered that the deletion of PANX1 reduced tumour surface area and viability. Our findings indicate that PANX1 regulates cell cycle progression and the structural architecture of A375-P melanoma tumours, highlighting the therapeutic utility of targeting PANX1 in melanoma.

Purpose: How sarcoma subtypes engage telomerase versus alternative lengthening of telomeres (ALT), and with what consequence, is incompletely defined. Methods: We analyzed 11,792 patients with sarcoma sequenced across 19 centers in AACR Project GENIE v20.0. Telomerase activation was defined as TERT promoter mutation, amplification, or rearrangement, and ATRX/DAXX inactivation served as the ALT proxy, each assessed only where the assay interrogated the locus. Findings were tested for TERT expression in The Cancer Genome Atlas (TCGA), for overall survival (OS) in MSK-IMPACT 50K, and for replication in an independent Foundation Medicine cohort. Mendelian randomization (MR) in FinnGen tested germline telomere length. Results: Telomerase activation (8.8%) and ATRX/DAXX loss (8.0%) were mutually exclusive across subtypes (odds ratio [OR], 0.52; 95% CI, 0.29 to 0.92) and segregated by histology. In MDM2-amplified dedifferentiated liposarcoma (DDLPS), telomerase was activated through TERT amplification (17.2%) or rearrangement (2.7%), never promoter mutation (0/261), and never with ATRX/DAXX loss (0/49; P = .011). TERT-amplified tumors expressed TERT (TCGA; P = .002), and amplification was largely focal. Telomere maintenance alterations were present in 28.0% of DDLPS versus 8.7% of well-differentiated liposarcoma (P < .001) and predicted shorter OS in DDLPS (hazard ratio, 2.04; 95% CI, 1.23 to 3.39; P = .005). ATRX/DAXX loss was more frequent in uterine than extrauterine leiomyosarcoma in GENIE (adjusted OR, 1.96) and in the independent cohort (OR, 1.95; both P < .001). Genetically longer telomeres were associated with liposarcoma (OR per SD, 4.10; 95% CI, 2.15 to 7.81). Conclusion: Sarcoma telomere maintenance is histology-specific and mutually exclusive. In liposarcoma it is acquired at dedifferentiation through focal TERT amplification or ALT and identifies DDLPS with inferior survival.

Chemobrain, characterized by cognitive dysfunction, has emerged as a significant health concern following cancer treatment. Doxorubicin, an FDA-approved chemotherapeutic agent, is widely used to treat various cancers. However, its adverse effects raise concerns regarding toxicity in non-cancerous cell types. Microglia, the resident immune cells of the brain, play a central role in maintaining neural homeostasis. Despite this, the mechanisms by which microglia become dysfunctional following doxorubicin exposure remain poorly understood. Understanding the transcriptomic alterations induced by doxorubicin in microglia could provide critical insights into mechanisms underlying its neurotoxic effects. In this study, the transcriptomic response of human microglia to doxorubicin using mRNA sequencing combined with bioinformatic analysis was investigated. Exposure to doxorubicin significantly altered microglial morphology, promoting a shift toward a process-bearing phenotype in human HMC3 cells. This phenotypic change was accompanied by elevated levels of DNA damage and was associated with the induction of cellular senescence. At the gene regulation level, the Illumina-based transcriptomic analysis provided 25,834 gene expression profiling in which 5,669 were differentially expressed. The future analyses revealed that 3,431 (upregulated) and 2,238 (downregulated) were differentially expressed under the treatment of doxorubicin. Genes associated with the p53 signaling pathway and inflammatory responses were upregulated, whereas genes involved in translation, DNA replication, and cell-cycle progression were downregulated. This transcriptional dysregulation was accompanied by differential expression of key transcription factors. This study provides transcriptomic evidence elucidating the genotoxic response to doxorubicin in microglial cells, offering mechanistic insights into chemobrain and highlighting key molecular pathways as potential targets for therapeutic intervention.

Background. Hematoporphyrin derivative (HpD, Hiporfin) is used for photodynamic therapy (PDT) of solid tumors but had never been evaluated for fluorescence guided resection (FGR) in glioma. We assessed Hiporfin fluorescence accuracy against histopathology in recurrent glioblastoma (rGBM) and the feasibility of adding cavity PDT to FGR. Methods. In this prospective single arm series, adults with rGBM received a single preoperative intravenous Hiporfin dose of 5 mg/kg before FGR under blue light excitation and 630 nm cavity PDT. Intraoperative fluorescence assessment was the index test and histopathology the reference standard. Specimen level diagnostic accuracy was pooled with bivariate random-effects modeling. Results. Of 63 specimens, 35 were true positive, 7 false positive, 8 false negative and 13 true negative. Pooled sensitivity was 0.751 (95% confidence interval 0.600 to 0.858) and specificity 0.606 (0.407 to 0.775); the crude positive predictive value was 0.833 (0.694 to 0.917) and the negative predictive value 0.619 (0.409 to 0.792). All seven false positive specimens were peritumoral tissue and the eight false negatives were non fluorescent tumor. FGR combined with cavity PDT was completed in all six patients without new neurological deficits. Conclusions. Hiporfin fluorescence identified rGBM with moderate accuracy: fluorescent regions were likely tumor, while non fluorescent tissue often still contained tumor. The main source of error was peritumoral tissue of the treated cavity, and not the drug or the optics. Hiporfin is a new fluorescence agent for glioma. One drug provides both fluorescence guidance during resection and immediate cavity PDT.

Ferroptosis has attracted significant attention for its potential to treat drug resistant tumors, along with its role in neurodegeneration and ischemia. Several ferroptosis targets were identified in the past decade, yet it is also highly affected by cellular metabolism and epithelial or mesenchymal phenotypes which limits the utility of these targets across diverse cell and tissue types. To address this, we utilized chemo-resistant cancer cell lines with different metastatic potential and tissue origins and discovered a common role of cytoplasmic ion changes during ferroptosis. We observed significant changes in intracellular potassium, sodium and chloride levels upon induction of ferroptosis. Therefore, we screened several ion channel inhibitors and activators (i.e. modulators) and identified Kv7.2 and CFTR modulators influence ferroptosis execution despite the fact that these cell lines do not express these proteins. We demonstrated that clinically approved modulators of CFTR and Kv7.2 can inhibit ferroptosis which indicates that these inhibitors could be repurposed as treatments in ferroptosis-associated diseases such as neurodegeneration and kidney diseases independent of ion channel expression. These modulators also influenced potassium and chloride levels in different contexts indicating off-target effects may also lead to changes in ion levels and may serve as a ferroptosis target. Further, we demonstrated that sustained imbalance of sodium, potassium and chloride ions with ferroptosis inducer salinomycin and digoxin, a Na+/K+ ATPase inhibitor, enhanced ferroptosis. The combination of salinomycin and digoxin successfully enhanced ferroptotic cell death across both cell lines with different metabolic signatures in vitro, offering a novel ferroptosis target. This combination significantly reduced tumor growth in vivo compared with the single agent alone, offering a new therapeutic modality against drug resistant tumors.

Background: Succinate dehydrogenase (SDH) is a heterotetrameric enzyme in both the tricarboxylic acid (TCA) cycle and the electron transport chain. SDH-deficiency has been shown to drive the oncogenesis of cancer types including gastrointestinal stromal tumors (GIST), paraganglioma/pheochromocytoma, and clear cell renal cell carcinoma. Previous work on SDH loss has shown increases in succinate, decreases in fumarate, as well as increase in the succinate to -ketoglutarate ratio, and correlated these changes to metabolite signaling and epigenetic dysregulation. Alterations to other TCA intermediates, including succinyl-CoA, the acyl-donor for succinylation, and the resulting cellular effects has not been fully explored. Methods: We used liquid chromatography high resolution mass spectrometry to quantify TCA cycle metabolites across the NCI-60 panel of cell lines, three SDH-competent and SDH-deficient model systems, and human tumor samples. We measured histone post-translational modifications in SHD-deficient and SDH-competent models and examined the correlation and potential pharmacological targeting of described desuccinylase enzymes by protein and cell assays. Results: We found an increase in succinyl-CoA in multiple SDH-deficient models, but no correlation between succinate to fumarate ratio and succinyl-CoA concentrations in the NCI-60. Interestingly, we found that while there was an increase in succinyl-CoA in the SDH-deficient cells, there was no corresponding increase in histone succinylation. We also observed a decrease in lysine malonylation and glutarylation, two other anionic lysine modifications. Cell viability and clonogenic assays revealed very weak or no selective effect of desuccinylase targeting in SDH-deficient models. Similarly, using SDH-deficient versus SDH-competent GIST patient samples, we examined both protein expression and metabolite abundance at the bulk tumor level and found no significant increase in succinyl-CoA or desuccinylase expression, despite the increase in succinate to fumarate ratio. Conclusions: We found that SDH-deficiency led to altered central carbon metabolism including increases to succinate and succinyl-CoA. Despite data that suggested the upregulation of desuccinylases may mediate a disconnect between succinyl-CoA concentration and histone succinylation, we found weak or no reductions in cell viability in response to pharmacological inhibition of desuccinylases. Additionally, no correlation of SDH loss with desuccinylase expression and succinyl-CoA in bulk tumor samples was observed.

Chimeric antigen receptor (CAR) T cells are patient-derived T cells engineered to recognize specific antigens on malignant cells, representing a major breakthrough in personalized cancer immunotherapy. While CAR-T cell therapy is an established treatment for relapsed or refractory hematologic malignancies, understanding the rationale and methodology behind its development is essential for training the next generation of oncology researchers. Here, we describe a novel, hands-on small-enrollment laboratory course designed to teach upper-level undergraduate and graduate students about immunotherapies and CAR-T cell technology by paralleling a typical bench-scale development process. Students isolated T cells from porcine peripheral blood mononuclear cells (PBMCs) via immunomagnetic selection, followed by flow cytometry to assess purity, viability, and yield. Students then used pre-engineered B7H3 CAR-T cells to perform co-culture assays with U87 glioblastoma cells to evaluate tumor cell killing efficacy. Cytotoxicity was qualitatively evaluated using immunofluorescence microscopy, while IFN-gamma release was quantified using Enzyme-linked Immunosorbent Assay (ELISA). Student performance was evaluated through electronic laboratory notebooks, discussion questions, a capstone project, and student-generated data. Our results demonstrate students high academic performance across assignments and the successful execution of complex laboratory workflows. Collectively, these findings establish the feasibility and implementation of a research-integrated curriculum providing foundational exposure to immuno-oncology research tools and CAR-T cell production techniques.