Neoplasia

Non-functioning gonadotroph (NFG) tumors are the most common type of non-functioning pituitary adenomas and can cause significant symptoms due to mass effect. However, the molecular programs underlying NFG tumor growth and their relationship to the normal anterior pituitary gland (APG) are poorly understood. To gain a deeper understanding of NFG tumor biology in the context of the normal APG, we performed single-cell/nucleus RNA-sequencing on 21 NFG tumors and 8 APG samples in human, generating the largest transcriptomic dataset of its kind. Single-cell/nucleus sequencing yielded 77,342, cells from APG samples and 152,649 cells from NFG tumor samples. Differential expression analysis within the APG identified novel marker genes of each neuroendocrine cell type and defined distinct transcriptional signatures of anterior and posterior pituitary stem cell populations. Comparison with tumor transcriptomes revealed that NFG tumor cells most closely resemble gonadotrophs, while also showing significant enrichment of thyrotroph and somatotroph markers. Notably, there was no significant overlap with stem cell markers, suggesting that NFG tumors most likely originate from differentiated gonadotrophs but can take on transcriptional characteristics of other neuroendocrine cell types. Pseudobulk profiling of NFG tumor cells demonstrated that tumor volume is significantly positively correlated with 83 genes including known oncogenes CAD, BRF2, and SOX12, along with 16 zinc finger transcription factors. Lastly, analysis of the tumor microenvironment revealed proportional increases in myeloid, endothelial, and mural cell populations in tumor samples compared to APG samples and highlighted cross talk between tumor and endothelial, mesenchymal, and immune populations via VEGF, PDGF, and MIF signaling pathways respectively. This study identifies novel transcriptomic signatures determining cell type identity in both APG neuroendocrine and NFG tumor cells. Our findings elucidate molecular programs driving NFG lineage infidelity and tumor growth, highlighting candidate predictors of patient outcomes and potential targets for therapeutic intervention.

Prostate cancer (PC) remains a significant health concern as the second leading cause of cancer-related death in American men. Androgen deprivation therapies represent the standard of care for patients with PC, regardless of disease stage. Gonadotropin-releasing hormone (GnRH) antagonists, such as Degarelix, are commonly used hormone therapies for PC and work by suppressing testosterone production. Previous studies indicate a prostate involution-regeneration cycle following surgical castration and testosterone addback. However, whether a similar response occurs following pharmacologic androgen deprivation with agents such as Degarelix remains poorly understood. In particular, the temporal dynamics of prostate involution and subsequent regeneration after chemical castration have not been systematically defined. Our study utilizes immunocompetent mouse models to evaluate the systemic and temporal effects of Degarelix treatment and determine its efficacy in suppressing PC tumor growth. We show that continuous Degarelix treatment significantly suppressed systemic testosterone levels in mice, whereas testosterone levels returned to physiological baseline following Degarelix withdrawal. Gross anatomical, H&E, and Trichrome analyses demonstrated that Degarelix induced castration resulted in prostate involution, characterized by reduced gland size and increased stromal remodeling. This phenotype was reversed after Degarelix withdrawal, highlighting the reversibility of pharmacological androgen deprivation. Immunofluorescence staining revealed changes in basal and luminal epithelial cell populations within the prostatic gland, indicating changes in prostate glandular architecture following Degarelix induced castration and subsequent regeneration. Next, we evaluated the impact of chemical castration on PC tumor growth. Although Degarelix initially suppressed tumor growth, this response was not sustained through the study endpoint, suggesting the emergence of acquired resistance to androgen deprivation. These findings establish this immunocompetent model as a physiologically relevant platform for dissecting the mechanisms that drive therapeutic resistance and tumor progression following androgen deprivation. Our findings highlight the effectiveness of chemical castration with Degarelix and establish a robust model to study both therapeutic responses and the emergence of castration resistance in PC.

Neuroblastoma is the most common extracranial solid tumor of childhood. Its clinical behavior ranges from spontaneous regression to lethal, treatment-refractory disease. Aberrant 2,6-sialylation contributes to aggressive phenotypes in many cancers, but the role of ST6Gal2, a neural-enriched 2,6-sialyltransferase, in neuroblastoma is largely unexplored. Here, we examine the clinical and functional significance of ST6Gal2 in neuroblastoma. In two independent public cohorts (SEQC, n=498; Kocak, n=649), high ST6GAL2 expression was associated with significantly worse overall and event-free survival. In the SEQC cohort, ST6GAL2 expression was higher in high-risk and MYCN-amplified tumors, varied across International Neuroblastoma Staging System stages, and correlated positively with a mesenchymal transcriptional signature (Spearman {rho}=0.181). The mesenchymal correlation was reproduced in the Kocak cohort ({rho}=0.204). Stable shRNA-mediated knockdown of ST6GAL2 in SK-N-AS and SK-N-BE(2) cells reduced proliferation and viability, impaired wound closure, and decreased migration and invasion. In preliminary experiments in SK-N-AS cells, ST6GAL2 knockdown reduced binding of Sambucus nigra agglutinin, consistent with a role for ST6Gal2 in 2,6-sialylation. Together, these findings link ST6Gal2 expression to aggressive clinical and transcriptional features and pro-tumorigenic phenotypes in neuroblastoma and nominate ST6Gal2-mediated sialylation as a candidate pathway for mechanistic study.

Neuroblastoma is a heterogeneous pediatric malignancy characterized by distinct genomic subgroups and cell-state plasticity between adrenergic (ADRN) and mesenchymal (MES) phenotypes. While small extracellular vesicles (sEVs) mediate pre-metastatic niche priming, the degree to which sEV proteomes reflect parental genomic and phenotypic identity remains unresolved. Here, we isolated sEVs from six neuroblastoma cell models (IMR-5/75, BE(2)-C, GI-ME-N, CLB-GA, LAN-6, and SK-N-FI) representing three genomic subgroups (MYCN-amplified, TERT-rearranged, and unaltered) using OptiPrep density gradient centrifugation followed by size exclusion chromatography and ultrafiltration. High-resolution mass spectrometry of sEV proteomes revealed that sEVs carry highly reproducible protein signatures that tightly correlate with the parental genomic subgroups. Analyzing paired cell/sEV samples, we demonstrate cell-to-EV concordance of lineage-specific markers: ADRN sEVs selectively enriched L1CAM and dopamine {beta}-hydroxylase (DBH), whereas MES sEVs enriched SERPINE1 and type III collagen (COL3A1). These findings indicate that neuroblastoma-derived sEVs systematically preserve both the genomic landscape and the lineage transdifferentiation states of their cells of origin. This study establishes a robust molecular foundation for utilizing sEV-based proteomics as a surrogate to track cellular plasticity and for studying sEV-mediated microenvironmental remodeling.

Glioblastoma (GBM) is characterized by near-universal recurrence and resistance to therapy. Although tumor-intrinsic mechanisms of radiation resistance have been extensively studied, how the extracellular matrix influences signaling in radiation-resistant GBM remains poorly understood. Here, we investigated discoidin domain receptor 1 (DDR1), a collagen-activated receptor tyrosine kinase, using matched radiation-sensitive parental and radiation-resistant patient-derived GBM models generated through serial in vivo irradiation. Within a three-dimensional collagen I matrix, radiation-resistant GBM cells exhibited distinct temporal patterns of Ki-67 positivity and ERK1/2 phosphorylation compared with parental cells. Despite increased DDR1 expression, radiation-resistant cells exhibited markedly reduced DDR1 phosphorylation and enhanced ectodomain shedding, characterized by accumulation of the membrane-associated C-terminal fragment and release of the N-terminal fragment. DDR1 depletion reduced ERK1/2 phosphorylation, supporting its contribution to ERK1/2 signaling under these conditions. ADAMTS1 was markedly upregulated in radiation-resistant cells, and its knockdown reduced DDR1 ectodomain shedding while increasing full-length DDR1 abundance, ERK1/2 and RB phosphorylation, cyclin D1 expression, and Ki-67 positivity. Together, these findings reveal a distinct DDR1 regulatory state in acquired radiation-resistant GBM, characterized by enhanced ADAMTS1-associated ectodomain shedding, reduced receptor phosphorylation, and attenuated proliferative signaling within a three-dimensional collagen I matrix.

The current treatment regimen for neuroblastoma involves immunotherapy, including a monoclonal antibody that recognizes disialoganglioside (GD2), expressed at high levels on neuroblastoma. GD2 is not present on most normal tissues except for nerves. Thus, anti-GD2 antibody treatment causes substantial, dose-limiting neuropathic pain. B7-H3 is overexpressed on multiple tumor types, including neuroblastoma, and is largely absent on nerves and other normal tissues. We designed a bispecific antibody (bsAb) that requires simultaneous binding of these two tumor antigens to achieve tight binding of tumor cells. Our preclinical research shows that, compared with a monospecific anti-GD2 antibody, the GD2xB7-H3 bsAb has improved tumor specificity, comparable antitumor efficacy, and reduced nerve binding and pain-associated toxicity. Since this bsAb does not bind to nerves, it may permit more tolerable and sustained treatment schedules than are currently feasible with monospecific anti-GD2 antibodies. In addition, its enhanced tumor specificity may support future development as a targeted delivery platform for antibody-drug conjugates or other payload-based therapies, potentially improving both efficacy and quality of life for patients with neuroblastoma.

Background: Glioblastoma (GBM) adapts to therapy through coordinated malignant-cell and microenvironmental responses, but the mechanisms linking treatment-associated inflammation to tumor-cell phenotypic plasticity remain poorly defined. Here, we investigated whether preoperative temozolomide exposure is associated with inflammatory-repair remodeling and a ROCK-linked therapeutic vulnerability in recurrent GBM using a window-of-opportunity clinical trial cohort. Methods: We integrated patient-resolved single-cell transcriptomics, spatial RNA profiling and multiplex protein imaging with experimental perturbations and orthotopic glioblastoma models. In the presurgical window-of-opportunity subgroup of NCT05236036, four patients with recurrent glioblastoma received additional preoperative temozolomide (TMZ; 150 mg/m2/day for 5 days) before resection and were compared with three recurrent comparators who did not receive additional preoperative TMZ. Results: Across seven tumors, malignant-cell inflammatory activity covaried with integrin-binding and wound-healing programs (median partial {rho} = 0.45 and 0.42, respectively). Tumors exposed to preoperative TMZ showed enrichment of inflammatory, chemokine, adhesion and wound-healing programs (GSEA q < 0.05), with the direction of enrichment preserved in all leave-one-patient-out analyses. In two tumors exposed to preoperative TMZ, CXCL12 localized to vascular territories, whereas chemokine and wound-repair programs increased near injury-reference regions. Across seven specimens analyzed by multiplex protein imaging, MYL9 abundance correlated with local CXCL12 ({rho} = 0.61), phosphorylated MYPT1 ({rho} = 0.48) and nuclear phosphorylated STAT3 ({rho} = 0.45), with positive associations in every specimen. Experimentally, TMZ increased CXCL8 and reactive-state markers and enhanced subsequent scratch closure, while CXCL8 and CXCL12 increased MLC2 phosphorylation. Fasudil attenuated TNF-NF-{kappa}B, inflammatory-response and IFN-{gamma}-response programs in TMZ-treated cells and reduced scratch closure and p-MLC2 in complementary assays. Across three orthotopic models, fasudil plus TMZ prolonged survival versus TMZ alone (model-stratified HR, 0.21; 95% CI, 0.07-0.61; P = 0.001). Conclusions: These findings link treatment-associated inflammatory-repair programs with cytoskeletal remodeling and support further translational evaluation of fasudil plus TMZ in glioblastoma.

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.

Advanced-stage high grade serous ovarian cancer (HGSC) initially responds to platinum-based chemotherapy; however, most HGSC tumors recur and develop chemotherapy-resistance, making recurrent ovarian cancer (OC) a fatal disease. Ovarian cancer stem cells (OCSCs), a subpopulation of cells capable of surviving treatment and repopulating tumors, contribute to disease relapse, and understanding the mechanisms(s) responsible for OCSC survival is critical for developing strategies to overcome acquired resistance and prevent tumor recurrence. However, whether platinum-induced OCSC enrichment results from the selection of pre-existing OCSCs or chemotherapy-induced cellular plasticity, and the molecular mechanisms underlying these processes, remain poorly understood. Here, we demonstrated that cisplatin treatment not only enriched for OCSCs but promoted the conversion of ALDH- cells to ALDH+ cells, revealing chemotherapy-induced plasticity as a potential mechanism for OCSC expansion. This conversion was associated with nuclear receptor signaling pathways, vitamin D receptor signaling (VDR/RXR) and retinoic acid receptor signaling (RAR/RXR). Importantly, activation of VDR/RXR signaling with vitamin D reduced stemness phenotypes and the cisplatin induced conversion to ALDH+ cells. In addition, vitamin D combined with cisplatin decreased tumor volume in vivo compared to either treatment alone. In contrast, cisplatin treatment increased RAR/RXR signaling, supporting an associative role for ligand-dependent nuclear receptor signaling in OCSC plasticity. Together, these findings reveal that platinum chemotherapy can actively promote the acquisition of stem-like properties and identifies VDR/RXR signaling as a potential regulatory mechanism of OCSC plasticity. Targeting this pathway with vitamin D may provide a therapeutic strategy to target OCSCs and improve responses to platinum-based chemotherapy.

Background. Progression of prostate cancer under androgen receptor (AR) pathway-targeted therapy spans a phenotypic spectrum, from tumors that sustain AR pathway output to tumors that become independent of AR signaling. Experimental models that occupy the intermediate portion of this spectrum, in which AR is still expressed but pathway output is reduced, are comparatively scarce. Methods. LNCaP cells expressing a triple-modality reporter (LNCaP-TR) were injected into nude mice by the intracardiac route, and a bone lesion that arose in the middle spine of one animal was recovered and propagated as LNCaP-TR-BoM. The subline and its parental line were compared by two-dimensional and three-dimensional culture assays, RNA-seq, qRT-PCR, and immunoblotting. AR pathway output was assessed using KLK3 expression under AR antagonist treatment. Public prostate cancer datasets provided clinical context. A CRISPR-Cas9 EPHA3-edited derivative was evaluated in aggregation and spheroid assays. Results. Relative to parental LNCaP-TR, LNCaP-TR-BoM formed more numerous and larger cell aggregates in monolayer culture and larger, less circular spheroids in both suspension and Matrigel three-dimensional culture. AR mRNA was modestly reduced and AR protein remained readily detectable, whereas KLK3 mRNA and protein were reduced disproportionately more than those of AR. Residual KLK3 expression remained suppressible by AR inhibitors enzalutamide and darolutamide. Transcriptome-based pseudotime placed LNCaP-TR-BoM between the parental line and the androgen-refractory LNCaP-RF variant. Subsequent differential gene expression (DGE) and pathway enrichment analysis revealed that LNCaP-TR-BoM exhibits transcriptomic rewiring concordant with that of clinical mCRPC, thus evidencing LNCaP-TR-BoM as a potentially clinically relevant model cell line. Finally, EPHA3 showed the largest fold change among the most significant differentially expressed genes in LNCaP-TR-BoM compared with the parental line. Therefore, EPHA3 was explored to demonstrate the utility of the LNCaP-TR-BoM cell line as a discovery system for genes that potentially drive the malignancy of bone-metastatic prostate cancer at the intermediate AR-attenuated progression stage. An EPHA3-silenced derivative of LNCaP-TR-BoM lacking detectable EPHA3 protein formed smaller aggregates and smaller, more circular spheroids, suggesting EPHA3 may play a role in driving the malignancy of AR-intermediate bone-metastatic prostate cancer. This finding provides support for the potential utility of the LNCaP-TR-BoM cell line as a discovery system for genes relevant to driving AR-intermediate bone-metastatic prostate cancer. Conclusions. LNCaP-TR-BoM is a bone lesion-derived prostate cancer cell line in which AR expression is retained but AR pathway output is attenuated, accompanied by an mCRPC-concordant transcriptomic shift and altered multicellular organization. It provides a paired discovery system for candidate genes such as EPHA3.

Objectives Low grade serous ovarian cancer (LGSOC) is characterized by frequent TP53 wild type status and limited responsiveness to conventional chemotherapy. This study investigated the cytotoxic effects of oxaliplatin combined with 5-fluorouracil on patient derived LGSOC cells. Results Clinically relevant concentrations of oxaliplatin and 5-fluorouracil had minimal effects on LGSOC cell viability but markedly reduced cellular proliferation and clonogenic recovery. Combined treatment induced pronounced accumulation of cells in the G0/G1 phase of the cell cycle and increased expression of p53 and p21, accompanied by reduced pRB phosphorylation, consistent with activation of the p53-p21-pRb pathway. Oxaliplatin plus 5-fluorouracil also increased senescence-associated beta-galactosidase activity, cellular enlargement and flattening, and reactive oxygen species production, supporting a senescence-like phenotype. However, after drug withdrawal, treated cells progressively regained proliferative capacity, indicating that the senescence phenotype was transient rather than fully irreversible. Together, these findings show that oxaliplatin plus 5-fluorouracil primarily arrest rather than kill LGSOC cells and induces a transient senescence-like phenotype and G0/G1-phase arrest by activating the p53-p21-pRb pathway.

Uterine leiomyomas, commonly known as fibroids, are benign tumors of the female reproductive tract. They are the most common tumor of the female reproductive system and the leading cause of hysterectomy in the United States. Progesterone, estrogen, and their receptors are important drivers for the development and growth of leiomyomas. How estrogen drives leiomyoma growth remains unclear. We have previously shown that simvastatin can inhibit leiomyoma growth by modulating intracellular calcium. This suggests that calcium signaling is important for leiomyoma growth. We therefore hypothesized that estrogen may drive leiomyoma growth by stimulating calcium release through the membrane estrogen receptor GPR30 (also known as GPER). We found that estrogen dose-dependently induced calcium release in leiomyoma cells. The GPR30-specific agonist G-1 also induced calcium release, indicating that GPR30 mediates estrogen-mediated calcium release. The GPR30 antagonist G-15 blocked estrogen-induced calcium release and significantly reduced leiomyoma proliferation. Our results demonstrate that GPR30 is a major driver of estrogen-dependent growth in leiomyoma cells by modulating intracellular calcium. These findings may have high therapeutic relevance to leiomyomas and other estrogen-dependent tumors.

ABSTRACT Background: High grade glioma (HGG), encompassing IDH-wildtype glioblastoma (CNS WHO grade 4), diffuse midline glioma (CNS WHO grade 4), oligodendroglioma (IDH-mutant and 1p/19q-codeleted) CNS WHO grade 3, together with IDH-mutant astrocytoma of CNS grade 3 and grade 4, represents the most lethal group of adult diffuse gliomas. Despite significant efforts, obtaining improvements in patient survival has been limited, in part, by difficulties in comprehensive ex vivo modeling of the complexities of HGG. Although organotypic slice cultures and other platforms have shown promise, existing models often fail to capture the intrinsic cellular heterogeneity, microenvironment, and architecture of HGG, restricting investigations into their fundamental biology as well as design of novel therapeutics. Methods: We developed a patient-derived HGG organotypic slice culture (GOSC) platform demonstrating long-term preservation of intrinsic tumor characteristics. Vibratome-sectioned slices were cultured in defined medium under conditions approximating those in situ: reduced oxygen (4%), low glucose (2.5 mM), and no added growth factors. The characteristics of GOSCs after 28 days in culture, including expression of immunotherapy targets, were compared to parental tumors using histological staining, immunofluorescence, multiplexed PhenoCycler (CODEX) imaging, bulk RNA-seq expression profiling, and CAR T cell killing assays. Results: GOSCs preserved the histopathology, cell heterogeneity, tumor microenvironment, and tissue architecture of their parental tumors throughout at least 28 days in culture. Importantly, GOSCs retained parental patterns of immunotherapy target expression, and CAR T cells co-cultured with GOSCs demonstrated target-specific killing consistent with parental tumors. Conclusions: This ex vivo GOSC platform offers long-term preservation of the cellular and structural properties of parental HGG tumors, thereby providing a robust tool for extended preclinical studies, and facilitating development and evaluation of advanced tumor-targeting strategies.

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.

Background: Relapsed and refractory osteosarcoma (OS) remains a major therapeutic challenge, with fewer than 20% of patients surviving beyond 3 years. Increasing evidence indicates that MYC amplification/overexpression is associated with inferior survival. Small molecule inhibitors and immunotherapies have limited single-agent efficacy in pediatric solid tumors. Using syngeneic cell lines derived from p53-driven and MYC-hyperactivated genetically engineered mouse models (GEMMs) of OS, we tested cabozantinib, a multi-tyrosine kinase inhibitor with immunomodulatory properties, with TIGIT immune checkpoint blockade and investigated mechanisms underlying therapeutic response and resistance. Methods: In vitro cabozantinib sensitivity was established in GEMM-derived cell lines. Mice bearing tibial tumors were randomized to vehicle control, cabozantinib, anti-TIGIT antibody, or combination therapy, and tumor growth and survival assessed after a 3-week treatment period. Temporal RNA sequencing was performed at early (8-15 days) and late (18-24 days) time points to characterize transcriptomic changes associated with efficacy. Results: MYC-hyperactivated cell lines were more resistant to cabozantinib in vitro than p53-driven lines (mean IC50 5.51 vs 0.65 mciroM, p=0.0016). In p53-driven orthotopic models, combination therapy significantly decreased tumor growth and improved survival compared to solvent and cabozantinib alone, while in MYC-hyperactivated models cabozantinib-containing regimens delayed tumor progression relative to control or anti-TIGIT monotherapy, however the addition of anti-TIGIT did not significantly improve survival over cabozantinib alone. Temporal transcriptomics revealed upregulated anti-tumor immune-response pathways and decreased M2 macrophages only with combination treatment in the p53-driven model. In contrast, combination-treated MYC-hyperactivated models demonstrated increased TNF signaling and elevated Cxcl5 and Ccr2 expression, indicative of increased myeloid cell recruitment, and upregulation of extracellular matrix (ECM) remodeling pathways suggest a therapy-induced stress adapted state that propagates treatment resistance over time. Conclusion: New therapies are needed for patients with relapse or refractory OS. By targeting tumor-intrinsic resistance mechanisms and modulating the tumor microenvironment using cabozantinib and anti-TIGIT therapy, improved tumor control and survival was achieved in p53-driven orthotopic OS models. MYC-hyperactivated models were able to overcome therapeutic pressure and employ myeloid recruitment and ECM remodeling programs to achieve treatment resistance. Targeting of these programs should be considered in future studies investigating therapeutic strategies in relapsed and refractory OS.

Background/Objectives: In high-grade serous ovarian cancer (HGSOC), the abilities of tumor cells to resist cytotoxic treatment and relapse are largely driven by ovarian cancer stem cells (CSCs). CSCs are a population of cells within a tumor that maintain stem-like qualities and have the capacity to produce metastasis and drive drug resistance. Based on the complexity of CSC signaling and emerging evidence of differential isoform usage in development, we hypothesize that CSCs possess a patient-specific, heterogeneous landscape which exploits isoform diversity to sustain stemness, metastasis and drug resistance. Methods: RNA-seq was generated from 3 patient-derived HGSOC (PD-HGSOC) to investigate stemness and differentiation status comparing standard culture and CSCs. The data was analyzed at both gene- and isoform-levels to identify patient-specific CSC features and define mechanisms of cancer-related stemness and plasticity. Results: CSCs exhibited higher stemness characteristics and identified differentially expressed gene sets across PD-HGSOCs. We noted that each patient had a distinct subset of genes regulating stemness phenotypes which suggested multiple mechanisms of CSC development and maintenance. Additionally, a large proportion of gene-level expression changes were driven by the differential expression of a single predominant isoform. Importantly, our isoform-level analysis enabled the identification of differential isoform usage that would not be detected with gene level expression analysis. Conclusions: CSC phenotypes such as chemoresistance, metastasis and self-renewal are conserved across patients, but arise from largely patient-specific gene and isoform programs. This CSC heterogeneity is reflective of the heterogeneity seen in patients and should be further evaluated in deriving precision-medicine approaches for HGSOC.

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.

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.

Glioblastoma (GBM) is an aggressive malignancy with limited therapeutic options and poor survival. Cyclin-dependent kinase 4 (CDK4) is overexpressed in GBM and promotes tumor progression through canonical cell-cycle regulation and extranuclear functions related to metabolism, invasion, and tumor microenvironment modulation. Current ATP-competitive CDK4/6 inhibitors show limited selectivity, potentially causing off-target effects and resistance. We developed a strategy to identify novel CDK4 inhibitors targeting the CDK4-Cyclin D1 protein-protein interaction, a key determinant of CDK4 activation. Virtual screening of approximately 950,000 compounds, combining molecular docking and molecular dynamics simulations, identified candidate molecules that were subsequently evaluated using proximity ligation assays, in vitro kinase activity assays, phosphokinase profiling, and functional analyses in patient-derived GBM cell lines and additional cancer models. Diophenic emerged as the lead candidate, demonstrating consistent antiproliferative activity across multiple GBM and epithelial cancer models. The compound selectively disrupted the CDK4-Cyclin D1 interaction while sparing the closely related CDK6 complex, and inhibited CDK4 kinase activity in vitro. Functional studies showed that diophenic induced substantial cell death in GBM cells, significantly reduced invasiveness, and exhibited distinct effects on cell migration compared with CDK4/6 inhibitors. Furthermore, phosphokinase profiling revealed that diophenic modulates a more restricted signaling network than ATP-competitive inhibitors, supporting greater functional selectivity and reduced off-target activity. Aiming the CDK4-Cyclin D1 interface represents a promising alternative to ATP-competitive CDK4/6 inhibition, which target a highly conserved site across the protein kinase superfamily. This approach provides a proof of concept for developing next-generation CDK4-selective therapeutics. Further studies should evaluate its translational potential in GBM.

Background: Giant cell tumor of bone (GCTB) is an intermediate osteolytic neoplasm characterized by a high rate of local recurrence. The tumor comprises two main cell populations: neoplastic stromal cells and osteoclast-like giant cells. Current adjuvant therapies primarily target osteoclast-like giant cells but have limited activity against the neoplastic stromal cells that drive tumor growth and recurrence. Here, we sought to identify actionable vulnerabilities in GCTB stromal cells by profiling druggable receptor tyrosine kinases (RTKs) and associated signaling pathways and evaluating their pharmacological inhibition in patient-derived in vitro models. Methods: RTK activation and downstream signaling were assessed using phospho-protein arrays and western blotting in therapy-naive and denosumab-treated GCTB patient samples and in patient-derived GCTB cell lines, representing clinically relevant models of the neoplastic component. Tyrosine kinase inhibitors (TKIs) targeting activated pathways were evaluated in patient-derived GCTB cell lines cultured under both two-dimensional (2D) and three-dimensional (3D) conditions. Effects on cell viability, proliferation, cell cycle distribution, and cell death were assessed using MTT and XTT assays, flow cytometry, live-cell imaging, and confocal microscopy. Results: Several activated RTKs, including EGFR, IGF-IR, InsR, PDGFR, and PDGFR{beta}, together with downstream signaling proteins MEK1/2 and ERK1/2, were identified as potential therapeutic targets. Among the inhibitors tested, trametinib, a MEK1/2 inhibitor, and axitinib, a multi-target inhibitor of VEGFR1-3, PDGFR{beta}, and c-Kit, demonstrated the strongest antiproliferative and cytotoxic effects, markedly reducing cell viability and proliferation while promoting cell death in vitro. Importantly, trametinib showed selective activity against GCTB stromal cells compared with healthy controls, suggesting a potential therapeutic window. Conclusions: This study identifies trametinib and axitinib as promising therapeutic candidates for targeting the neoplastic component of GCTB. Our findings provide a rationale for further preclinical and clinical evaluation of RTK-directed therapies to complement current treatments and specifically target the neoplastic GCTB stromal cells underlying tumor persistence and recurrence.