Neoplasia

Temozolomide (TMZ) resistance remains a major obstacle in glioblastoma (GBM) therapy, yet the metabolic adaptations underlying this phenotype are incompletely understood. Here, we performed integrative lipidomic, ultrastructural, and pathway analyses to define lipid metabolic reprogramming associated with TMZ resistance and failure of statin-mediated sensitization. Targeted LC-MS lipidomics quantified 322 lipid species across 25 lipid classes in TMZ-sensitive and TMZ-resistant U251 cells under basal conditions and following TMZ, simvastatin, or combination treatment. Multivariate analyses (PCA, PLS-DA, and volcano plots) revealed a robust and treatment-resilient lipidomic signature in resistant cells characterized by enrichment of lysophospholipids, sphingolipids, and cholesteryl esters, alongside depletion of glycerolipid and phospholipid pools. Complementary univariate analysis confirmed these changes at the species level, demonstrating consistent elevation of lysophosphatidylcholine/ethanolamine, glycosphingolipid subclasses, and cholesteryl esters, together with reductions in phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and diacylglycerol intermediates across multiple treatment conditions. In contrast, sensitive cells displayed dynamic lipid remodeling, including phosphatidylinositol and phosphatidylethanolamine enrichment associated with autophagic membrane expansion. KEGG pathway analysis linked the resistant phenotype to Rap1, PI3K-Akt, and phospholipase D signaling networks regulating vesicle trafficking and membrane homeostasis. Transmission electron microscopy confirmed a vesicle-rich intracellular architecture consistent with persistent autophagy flux blockade in resistant cells. Collectively, these findings define a stable lipid metabolic program characterized by lysophospholipid expansion and cholesteryl ester accumulation that supports membrane integrity and therapeutic resistance. Targeting lipid buffering and cholesterol storage pathways may represent a promising strategy to overcome chemoresistance in glioblastoma. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=134 HEIGHT=200 SRC="FIGDIR/small/712341v1_ufig1.gif" ALT="Figure 1"> View larger version (78K): org.highwire.dtl.DTLVardef@178acd7org.highwire.dtl.DTLVardef@19b6a79org.highwire.dtl.DTLVardef@6b3904org.highwire.dtl.DTLVardef@16c3d01_HPS_FORMAT_FIGEXP M_FIG C_FIG Lipidomic and autophagy differences between non-resistant (NR) and temozolomide-resistant (R) glioblastoma cells. NR cells show dynamic lipid remodeling and treatment-dependent autophagy responses, whereas R cells maintain blocked autophagy flux and persistent enrichment of LPC, SM, and cholesteryl esters across treatments.

High-risk neuroblastoma (NB) is a pediatric malignancy associated with metastases and an immunosuppressive tumor microenvironment. Standard-of-care treatments like chemotherapy are often ineffective, which motivates the investigation of adjuvant approaches. Histotripsy is a noninvasive focused ultrasound therapy that ablates tissue through the mechanical action of bubble clouds. In addition to disruption of the targeted tumor, non-targeted lesions exhibit growth delay after the histotripsy procedure. The primary hypothesis of this study was histotripsy-induced shifts in the tumor microenvironment will improve the response of metastatic NB to chemotherapy. Female A/J mice flanks were inoculated bilaterally with 1x10 Neuro-2a cells. Histotripsy was applied to one tumor (200-500 mm3), with or without concurrent administration of liposomal doxorubicin (LDOX). The contralateral tumor served as a model of non-targeted distal metastases. Following treatment, tumors were monitored indefinitely for growth, or assessed after 5-7 days with flow cytometry, single-cell RNA sequencing, and immunohistochemistry. Histotripsy alone delayed the growth of treated and contralateral tumors relative to controls (p = 0.01 and p < 0.0001, respectively) and increased CD8 T and CD11b+ cells (p < 0.05 for both comparisons). Further, NB cells in targeted and contralateral tumors exhibited a decrease in Myc expression and cell-cycle activity, and upregulation of interferon and apoptosis pathways. Histotripsy combined with LDOX had the longest delay in tumor growth (p < 0.01) and greatest expression of CD8 and MOMA staining. These findings indicate that histotripsy induces a systemic antitumor immune response that potentiates chemotherapy efficacy in this model of metastatic NB. SignificanceMechanical ablation with histotripsy drives systemic antitumor immunity, reshapes the tumor microenvironment, and enhances chemotherapy efficacy in a syngeneic model of metastatic, high-risk neuroblastoma.

tRNA-derived fragments (tRFs) are relatively recently discovered class of small RNAs implicated in gene-regulatory processes in diverse biological contexts but there have been very few reports of a clear phenotypic role of these small RNAs in cancer progression. By analyzing small RNA-seq data from The Cancer Genome Atlas (TCGA), we found that high expression of three 3' tRFs (tRF-3a), tRF-3009a, tRF-3021a or tRF-3030a, is significantly associated with poor overall survival in low-grade glioma (LGG). In glioblastoma cells, tRF-3009a, tRF-3021a and tRF-3030a enhance cell invasion and migration but tRF-3021a was uniquely required for cell proliferation and suppression of apoptosis. Interestingly, tRF-3021a knockdown decreases global protein synthesis prior to and independent of apoptosis. These data indicate that tRF-3021a supports glioma cell survival and particularly protein synthesis while promoting cellular invasion and migration. Given its association with poor outcome in LGG patients, tRF-3021a represents a promising biomarker and potential therapeutic target in gliomas and these results provide a foundation for future studies to define its molecular interactors and downstream pathways controlling protein synthesis and apoptosis in cancer cells. ImplicationtRF-3021a promotes malignant glioma phenotypes, sustains global protein synthesis and prevents spontaneous apoptosis, motivating efforts to evaluate it as a biomarker and therapeutic target.

Boys experience an overall increased incidence of several childhood cancers, including medulloblastoma, a clinically heterogeneous cerebellar tumor. In subtypes of Group 3 and Group 4 medulloblastoma, males are three times more prevalent than females. As medulloblastoma is suspected to initiate during fetal development, we hypothesized that this sex bias reflects a combination of prenatal, sex-specific developmental processes and somatic alterations. To test these hypotheses, we compiled a large multi-omics dataset from children with medulloblastoma, which revealed sex-specific alterations, including frequent loss of the inactive X chromosome in females with Group 4. Generation of a sex-matched single-cell transcriptome atlas of the developing murine cerebellum enabled investigation of putative developmental factors underlying sex bias. Progenitors giving rise to Group 3/4 subgroups were more abundant, more proliferative, and harbored more open chromatin for recruitment of LMX1A and OTX2, master transcription factors defining Group 3/4 identity. Advanced genetically engineered mouse models and human cerebellar organoids were leveraged to determine whether sexual dimorphism arises from intrinsic or extrinsic factors. These models showed that the XY genotype contributed to the phenotype, but the predominant effect was driven by presence of the male gonadal hormone testosterone. Our findings provide a sex-specific genetic and neurodevelopmental explanation for male bias in an aggressive pediatric brain tumor. Outcomes from this study may inform novel treatment strategies delivered according to sex and are likely to be broadly applicable to other sex-biased malignancies arising in early life. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/714163v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@3a06faorg.highwire.dtl.DTLVardef@1a01bb7org.highwire.dtl.DTLVardef@7bc9c2org.highwire.dtl.DTLVardef@fb206d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Relapsed or refractory (R/R) high-risk (HR) NB tumors continue to exhibit poor outcomes despite intensive and protractive multimodal therapy. Activating mutations in the RAS- mitogen-activated protein kinase (MAPK) pathway are frequently observed in R/R HRNB. The early promise of ALK inhibitors to treat ALK-mutant NB underscores the ability of appropriate targeted therapies to improve outcomes for HRNB patients. While MAPK pathway activation is prominent in HRNB, FDA-approved MEK inhibitors and KRAS G12C inhibitors have failed to demonstrate significant preclinical single-agent activity. Daraxonrasib (RMC-6236), a potent and selective RAS(ON) inhibitor, has demonstrated activity in both preclinical models and early phase clinical trials of RAS-mutant adult cancers. A subset of R/R HRNB tumors is noteworthy for containing diverse RAS-mutations, providing rationale for RMC-6236 investigation. In this study, we evaluated the therapeutic efficacy and oncogenic signaling modulation of RMC-6236 across NB models harboring RAS pathway activation. RMC-6236 as a single-agent treatment led to a significant decrease in cell viability, suppression of downstream MAPK signaling, upregulation of the MAPK pathway effector protein BIM, and increased cell death in RAS-mutant NB models as well as in NF1-mutant NB models. In vivo studies evidenced that RMC-6236 had on-target activity that significantly reduced tumor growth and extended survival in RAS-mutant HRNB mouse models. Furthermore, RMC-6236-induced both BCL-2 and BIM upregulation and enhancement of BIM:BCL-2 complexes in RAS-mutant NB. As such, the BCL-2 inhibitor venetoclax further enhanced RMC-6236-mediated killing by disrupting RMC-6236 enhanced BIM:BCL-2 complexes. These findings demonstrate that RMC-6236 is a rationale targeted therapy for RAS-mutant NB, a subset of NB that is progressively understood as conferring particularly poor outcomes. RMC-6236 is a clinically relevant drug that can successfully target the MAPK pathway in these cancers. This study supports expanded clinical testing of this novel therapy to this important subset of neuroblastoma.

Lynch syndrome, historically known as hereditary nonpolyposis colorectal cancer, is caused by germline mutations in the DNA mismatch repair (MMR) genes, MLH1, MSH2 (EPCAM), MSH6, and PMS2. While the genetic changes associated with Lynch Syndrome have previously been characterized, there have not been studies of the associated proteomic alterations, in part because of the limited availability of primary samples and the absence of in vitro model systems. In this study, the first large-scale tissue proteomic assessment of Lynch Syndrome samples as well as three other subtypes of colorectal cancer was completed with specimens from the Ohio Colorectal Cancer Prevention Initiative. The cohort contained three groups of microsatellite unstable (MSI-high) CRC patients (Lynch syndrome, double somatic MMR mutation, and MLH1 hypermethylation) and a group of microsatellite stable (MSS) CRC patients. A total of 122 tumor and complimentary normal mucosa samples from 61 patients were evaluated using label-free bottom-up proteomic analysis. Hierarchical clustering analysis of the global proteome showed that the MSS group was significantly different than the three MSI-high groups. Of the 1,084 proteins found to be dysregulated across all four colorectal cancer subtypes, there were age at diagnosis associated shifts in proteins correlated with tumor proliferation and immune regulation for the Lynch syndrome and Double Somatic samples. The proteins TPD52, GMDS, and DSP showed increased protein abundance correlated with older age at diagnosis. In addition, the Lynch syndrome samples showed substantial sex-based differences in immune and inflammatory pathways, for example, downregulation of ZG16, DIS3, and WDR43. This study fills a critical gap as the first proteomic characterization of Lynch syndrome samples to date. Data are available via ProteomeXchange with identifier PXD073693. TeaserThis is the first study of the global proteomic differences between Lynch Syndrome and other forms of colorectal cancer.

PurposeEarly noninvasive assessments of treatment response are desperately needed to improve outcomes in glioblastoma (GBM). Molecular imaging techniques that measure glycolytic metabolism are being increasingly studied, but limitations such as variable substrate delivery present significant barriers to clinical interpretation. To develop more robust translational imaging biomarkers, we propose utilizing the interrogation of oxidative stress, a critical component of tumor metabolism for which no method of clinical measurement currently exists. This study investigates the simultaneous measure of oxidative stress and glycolytic flux using co-hyperpolarized [1-13C] dehydroascorbate and [1-13C] pyruvate (HP DHA/PA) as a predictor of treatment response in GBM. Experimental DesignTo establish a model that exhibits known metabolic responses to oxidative stress, we characterize radiation induced metabolic reprogramming in four human GBM lines (U87, U251, A172, T98) in vitro. We extend this in vivo and establish radiosensitive and radioresistant orthotopic xenograft models to investigate HP DHA/PA magnetic resonance imaging as a predictor of treatment response. ResultsIn vitro analyses revealed that radiation upregulates the pentose phosphate pathway and response is augmented by glutathione depletion. In vivo metabolomic profiling identified preferential nucleotide metabolism pathways in each tumor type. HP DHA/PA imaging revealed that DHA perfusion was not impacted by blood-brain-barrier integrity and detected reductions in DHA-to-vitamin C and pyruvate-to-lactate conversion in treatment-sensitive tumors, reflecting diminished reductive capacity following radiation. ConclusionsThese findings demonstrate successful prediction of radiosensitivity in GBM utilizing measurement of oxidative stress and establish HP DHA/PA imaging as an innovative method to address existing clinical limitations in treatment response assessment.

BackgroundYoung age is an independent risk factor for the development of breast cancer brain metastases (BM). Prior work showed that 17{beta}-estradiol (E2), the predominant premenopausal hormone, promotes BM of tumors intrinsically unresponsive to E2, in part through modulating estrogen receptor-alpha expressing (ER) glial cells. However, how E2 reshapes the brain tumor microenvironment (TME), particularly microglia-mediated immunity, and its impact to BM progression remains unclear. MethodsscRNA sequencing and multiparametric flow cytometry were used to define the impact of E2 and E2-suppression on brain immune-cell populations across different stages of BM progression using spontaneous and experimental models of BM. Depletion of microglia and T cell co-cultures were used to study microglias role in E2-induced BM. The effects of E2-suppression alone or in combination with whole brain radiotherapy were tested in preclinical models mimicking late-stage BM. ResultsE2 repressed immune surveillance and immune activation programs in microglia from early to late stages of brain metastatic progression, suppressing recruitment of effector immune cells to BM. Estrogen suppression, in turn reactivated anti-tumoral signaling in microglia and increased recruitment of effector immune cells to the brain. Microglia from E2-stimulated BM-bearing mice showed decreased ability to induce interferon cytotoxic function and expansion of activated T cells. Conversely, E2-suppression reactivated an effective anti-tumoral response and synergized with RT to significantly decrease BM progression. ConclusionThese findings reveal a previously unrecognized mechanism by which E2 accelerates BC-BM progression through microglial immunosuppression and support evaluation of endocrine therapies as adjunct treatments for ER- brain metastases. Importance of the StudyStandard of care for BM includes stereotactic radiosurgery (SRS) alone or in combination with surgery, systemic chemotherapy or targeted therapies. Our studies show that ovariectomy (which eliminates ovarian E2) and aromatase inhibitors (AIs, which eliminate peripheral E2 synthesis) reduce progression of BM when used in combination with WBRT and in immuno-competent models. We demonstrate E2 promotes an immunosuppressive brain microenvironment from early stages of metastatic progression, in part through modulation of myeloid cells and repression of recruitment of effector T cells to the brain. Thus, these studies suggest that FDA-approved E2-depletion therapies (aromatase inhibitors and selective-estrogen modulators) could be used in combination with brain irradiation to decrease BM progression.

Diffuse hemispheric gliomas (DHGs) are highly aggressive and infiltrative CNS tumors that are refringent to treatment, and with a 5-year overall survival of around 20%. A fraction of DHGs is driven by mutations in the histones H3.1 and H3.3. In this study, we demonstrate that the expression of histone H3.3 glycine 34 to arginine mutations (H3.3-G34R) result in the epigenetic and transcriptional activation of the NF-{kappa}B signaling pathway in DHG. To target this vulnerability, we designed high density lipoprotein (HDL) nanoparticles loaded with unmethylated CpG dinucleotides, which mimic the immune stimulatory activity of bacterial DNA. CpG are recognized by Toll-like receptor 9 (TLR9), activating the NF-{kappa}B signaling. The CpG-mediated NF-{kappa}B activation results in the release of immuno-stimulating cytokines that promote an antitumoral response. As we previously established that G34-mutant DHGs are characterized by DNA repair impairment, we combined CpG dinucleotides with a PARP (poly (ADP-ribose) polymerase) inhibitor, olaparib, in the HDL nanoparticles.

Brain cancers hijack biological systems involved in neural development and synaptic plasticity. Medulloblastoma (MB), the most common malignant brain tumor in children, is thought to arise from disruptions in neurodevelopmental programs. Glutamatergic transmission mediated by -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs) has been implicated in synaptic communication between adult brain tumors and surrounding neurons; however, the possible role of AMPARs in MB remains largely unexplored. Here, we analyzed the expression of genes encoding AMPAR subunits, GRIA1-4, in datasets of MB tumors, revealing distinct expression patterns and subgroup-specific associations with overall survival (OS) across molecular subgroups and histological variants. Expression of GRIA1, GRIA3, and GRIA4 was significantly lower in MB in comparison with normal cerebellar tissue. Higher GRIA1, GRIA2, and GRIA4 transcription was associated with more favorable patient outcomes in specific MB subgroups. In contrast, high expression of GRIA3 in SHH, or of either GRIA3 or GRIA4 in Group 3 MB, was associated with worse prognosis. Particularly robust but opposing associations with patient survival were found for GRIA3 and GRIA4 in SHH MB. Analysis of GRIA mRNA levels in MB cell lines representing different molecular subgroups, using data from The Human Protein Atlas, showed partial concordance with expression patterns observed in tumors. Together, these findings suggest that GRIA genes and their corresponding AMPAR subunits may have subgroup-specific prognostic relevance in MB and merit further investigation.

BackgroundMore than 90% of advanced ovarian cancer patients develop malignant ascites, which describes a buildup of fluid in the peritoneal cavity caused by increased vascular permeability and obstructed lymphatic drainage. Malignant ascites contains spheroidal tumor cell clusters that contain stromal cells, cancer-associated fibroblasts, and blood cells. These spheroids promote peritoneal metastasis and treatment resistance, yet the phenotypic and proteomic changes of cells caused by the ascites environment remain poorly understood, as does its influence on ex vivo responses to chemotherapeutics in personalized medicine approaches. MethodsUsing mass spectrometry, we compared the proteome profiles of cell-free ascites to serum from ovarian cancer patients. We then analyzed the proteomes of immortalized cancer cells grown as monolayers or spheroids in either malignant ascites or standard cell culture medium. The effects of this fluid on the phenotype, molecular composition, and ex vivo chemotherapy responses of cancer cells were also investigated. ResultsProteome analysis revealed that cell-free ascites had higher levels of extracellular, secreted, and membrane proteins compared to serum. Ascites enhanced cell viability and spheroid formation in immortalized ovarian cancer cell lines more effectively than standard cell culture medium. Despite this altered baseline viability, growth of spheroids in ascites versus cell culture medium did not hinder chemotherapy response assessments, indicating the appropriateness of standard cell culture medium in ex vivo applications. The observed phenotypic changes of cells grown in ascites could not be recapitulated by adding chemokines or periostin to the cell culture medium, suggesting that additional factors are required. Notably, elevated levels of transglutaminase 2 were identified in SKOV-3 cells grown in ascites, indicating that ascites directly influences protein expression in cancer cells.

Engineering macrophages with chimeric antigen receptors is emerging as a promising cancer therapeutic. Chimeric antigen receptor-expressing macrophages (CAR-Ms) engineered to recognize tumor-specific antigens have been shown to inhibit tumor growth and activate adaptive immune responses, leading to robust tumor control in animal studies. Based on this work, clinical trials have been initiated. While the trials have shown promise, challenges remain. The dynamic interactions between CAR-Ms and cancer cells and the exact mechanisms driving anti-tumor effects remain poorly defined. Defining the dynamic interactions between CAR-Ms and cancer cells will provide critical insights for optimizing future CAR-M design and improving therapeutic efficacy. We sought to directly visualize CAR-M interactions with glioblastoma cells at high-resolution and in real-time using CAR-Ms engineered to recognize Neural-Glial Antigen 2 (NG2), an antigen expressed on glioblastoma cells. Using patient-derived glioblastoma cells, we formed glioblastoma spheroids and embedded them in a 3D matrix together with CAR-Ms. Using time-lapse microscopy, as expected, we found that NG2-targeting CAR-Ms engulfed glioblastoma cells. However, excitingly, we found that NG2-targeting CAR-Ms blocked >85% of glioblastoma cell invasion in 3D. This inhibition of glioblastoma invasion was not due to a significant change in CAR-M polarization states. Together, these data suggest that NG2-targeting CAR-Ms both engulf glioblastoma cells and block glioblastoma invasive behavior.

Brain metastases are a common and often fatal complication of certain cancer types, such as triple-negative breast cancer. However, the molecular pathways driving brain metastasis formation, including the migration of cancer cells from the bloodstream to the brain parenchyma across the blood-brain barrier, are not yet fully defined. Therefore, using highly relevant mouse and human model systems, the mechanisms by which triple-negative breast cancer cells and their released extracellular vesicles modulate the blood-brain barrier-forming endothelium to increase its permissiveness to tumour cell entry into the brain are investigated. It is observed that extracellular vesicles derived from tumour cells are taken up by cerebral endothelial cells, where they induce miR-146a-5p- and TGF-{beta}1-mediated downregulation of PAQR5/mPR{gamma}, a membrane progesterone receptor. This, in turn, leads to disruption of interendothelial tight junctions, particularly through repression of claudin-5 expression, a critical protein for maintaining barrier function. Altogether this identifies a novel mechanism by which triple-negative breast cancer-derived extracellular vesicles compromise blood-brain barrier integrity, thereby facilitating transendothelial migration of cancer cells and promoting brain metastasis development. Moreover, this study is the first to highlight the role of membrane progesterone receptors in regulating the blood-brain barrier. Table of contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/701753v2_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@15252aeorg.highwire.dtl.DTLVardef@1b23beforg.highwire.dtl.DTLVardef@7cd517org.highwire.dtl.DTLVardef@189db4e_HPS_FORMAT_FIGEXP M_FIG Extracellular vesicles from triple-negative breast cancer cells induce miR-146a-5p- and TGF-1-mediated downregulation of PAQR5/mPR{gamma}, a membrane progesterone receptor, in blood-brain barrier-forming endothelial cells. This results in disruption of interendothelial tight junctions, thereby promoting enhanced migration of cancer cells into the brain. This mechanism highlights the role of membrane progesterone receptors in regulating the blood-brain barrier. C_FIG

Tumor-associated macrophages (TAMs) are key mediators of tumor immunosuppression, yet the factors governing their polarization remain poorly understood, especially in highly immunosuppressive cancers, including cancers affecting the central nervous system. This study investigates the molecular pathways underlying TAM polarization in glioblastoma, one of the most immunosuppressive cancer types. Using a multi-omics approach integrating spatial proteomics, RNA-sequencing, and proteomic profiling of tumor cells and macrophages, we demonstrate that circulating monocytes polarize toward an immunosuppressive state when they exit tumor blood vessels, in response to glioblastoma cell-secreted cytokines. In situ and in vitro data shows that macrophage polarization is regulated via the cAMP-CREB signaling-transcription axis.

Glioblastoma (GBM) is an aggressive, high-grade glioma with a near-universally fatal prognosis. Therapeutic failure is often attributed to the highly selective blood brain barrier (BBB), the diffuse infiltrative nature of the tumor, and the marked intratumoral heterogeneity of GBM. Although antibody drug conjugates ADCs have shown promise for high grade gliomas such as GBM, efficacy is limited by ADC size. Aptamers--short, synthetic, single-stranded DNA or RNA molecules--can be [~]6-fold lower in molecular weight than IgG antibodies and have the potential to cross the intact BBB. Unlike other nucleic acid-based therapies, aptamer function arises from three-dimensional shape rather than genetic coding. Here we aim to replace the targeting component of the ADC paradigm with a DNA aptamer, thus creating an aptamer-drug conjugate (ApDC). We employed in vivo SELEX using an orthotopic patient-derived xenograft (PDX) GBM mouse model and a vast ([~]100 trillion 80-mer sequences) ApDC library. We report the results from this first in vivo ApDC selection of its kind. We characterize target tissue binding ex vivo, cell association, biodistribution, and pharmacokinetics from this selection. This study exemplifies an unbiased approach to a problem that rational design has yet to overcome, offering a new direction for GBM therapeutic development.

Atypical teratoid rhabdoid tumor (ATRT) is a malignant brain tumor of children that has an overall survival of less than 40 percent even with aggressive therapy. We identified upregulation of the mitogen activated protein (MAP) kinase pathway in ATRT. The novel, brain-penetrant MEK inhibitor mirdametinib inhibited the growth of ATRT cell lines in culture at nanomolar concentrations. Mirdametinib suppressed proliferation as measured by BrdU incorporation and induced apoptosis as measured by cPARP and Annexin V staining. Monotherapy with mirdametinib extended the life of mice bearing orthotopic xenografts. Combination therapy with the brain-penetrant cyclin dependent kinase 4/6 inhibitor abemaciclib further suppressed growth and BrdU incorporation in ATRT cell lines representing all molecular subgroups. Mirdametinib and abemaciclib combined to extend survival of mice bearing orthotopic ATRT xenografts. In conclusion, mirdametinib has single agent activity against ATRT and combines with abemaciclib to decrease proliferation and extend survival in orthotopic xenograft models of ATRT.

BackgroundGlioblastoma (GBM) is an aggressive form of primary brain cancer. Recent efforts to characterize GBM using single-cell or spatially-resolved transcriptomics have revealed a tremendous intra-tumoral heterogeneity between malignant cells and between different tumor areas. However, most efforts have focused on malignant cells, and the spatial and cellular heterogeneity of the tumor microenvironment (TME) remains poorly understood. Moreover, it is unclear how TME compositions and organizations influence clinical outcomes for patients. ResultsIntegrating spatial transcriptomics, single-cell RNA-seq and histology on 25 tumors, cellular composition of the TME was estimated on over 46,000 55-m wide spots. Spatial associations were revealed between mesenchymal-like cancer cells and monocyte-derived macrophages. Spots were clustered into six unique classes of TME, exhibiting differential composition of malignant and immune cells, and distinct activation of biological pathways. Spatial transcriptomics-informed deconvolution of large-scale bulk RNA-seq datasets revealed that the niche composition of tumors associated significantly with patient survival and response to immunotherapy. Mesenchymal-like, monocyte-derived macrophages-rich and hypoxic niche N1 associated with lower overall survival while oligodendrogial progenitor-like and microglia-derived macrophages-enriched niche N5 is associated with longer patients survival. Analysis of data from patients treated with immunotherapy showed that niches N1 and mixed mesenchymal-like and astrocyte-like niche N3 associated with response to PD-1 inhibitors. ConclusionsOur results show that GBM exhibits a strong spatial heterogeneity of TMEs, with distinct categories of niche. The niche composition of tumors associated with survival and immunotherapy response. Our results suggest incorporation of TME niches as biomarkers for risk stratification and therapeutic decisions for patients.

Ionizing radiation can be an effective therapy for prostate cancer. Unfortunately, however, more aggressive prostate cancers such as neuroendocrine prostate cancer (NEPC) are often radiation resistant, which contributes to their high degree of morbidity and mortality. In this study, we used an unbiased approach to identify novel mechanisms that contribute to resistance to radiation and that are associated with neuroendocrine differentiation. Specifically, we compared the expression of cell surface proteins by mass spectrometry in prostate cancer cell lines that had been either untreated or treated with radiation to induce resistance, a process that also promotes neuroendocrine differentiation. Among the proteins identified by this screen, we focused on folate receptor (FR) because of its known biological functions and the fact that it is a validated therapeutic target. Our data reveal that FR has a causal role in enabling prostate cancer cells to resist radiation. Importantly, we also demonstrate that the expression of FR is regulated by HIF-1, which also has a causal role in radiation resistance and neuroendocrine differentiation. Given that the ability of cells to resist damage and death in response to ionizing radiation depends largely on their ability to buffer the substantial increase in reactive oxygen species (ROS) that is generated by radiation, we also demonstrate that the folate-FR axis promotes radiation resistance by sustaining intracellular glutathione levels that buffer this increase in ROS. In summary, the data reported here highlight a novel role for FR in resistance to ionizing radiation that is intimately associated with the hypoxic microenvironment of NEPC and the ability of the folate-FRa axis to maintain redox homeostasis.

BackgroundNeuroendocrine Prostate Cancer (NEPC) is an incurable malignancy, originating from the trans-differentiation of prostate adenocarcinoma (PRAD). Compared to PRAD, NEPC shows over-activation of Polycomb Repressive complex-1(PRC1) and-2 (PRC2), which are multiprotein epigenetic writers that drive cancer progression via tumour suppressor gene silencing. Tazemetostat is a PRC2 inhibitor approved for the treatment of sarcomas and lymphomas. ORIC-944 is a novel EED (Embryonic Ectoderm Development) inhibitor, which is being tested in clinical trials. EED is an attractive target as it functions as a key component of both PRC1 and PRC2. Objective and MethodsWe compared the anticancer effects of tazemetostat and ORIC-944 in NEPC and PRAD cells. Cells were exposed to various concentrations of the two compounds to measure effects on cell viability (IC50) and apoptosis (flow cytometry). PRC2 inhibition was confirmed by measuring histone H3 Lys 27 trimethylation (H3K27me3) via ELISA and Western Blot. RNA Sequencing and pathway analysis was conducted to study modes of actions of tazemetostat vs ORIC-944. ResultsUnlike tazemetostat, ORIC-944 causes dose-dependent growth inhibition in both NEPC and PRAD cells. In this context, EED targeting achieves IC50 values that are comparable to those of compounds used for the clinical treatment of advanced prostate cancer. Moreover, ORIC-944 (but not tazemetostat) causes significant apoptosis in NEPC cells. Both tazemetostat and ORIC-944 reduce H3K27me3. Mechanistically, both compounds reactivate the expression of known PRC2 targets, such as genes that control neural differentiation. However, the EED inhibitor also reactivates PRC1 targets, including pro-apoptotic and anti-proliferating genes (e.g. metallothionines). This evidence suggests that EED inhibition is a promising therapeutic strategy for NEPC.

BackgroundGlioblastoma (GB) is a highly aggressive brain tumor with a median survival of approximately 14 months, primarily due to its ability to infiltrate healthy brain tissue both as single cells and in collectives. A deeper understanding of GB cell motility, both individual and collective, is crucial for developing patient-specific therapies. We aimed to characterize migration in patient-derived GB cells using advanced modeling to identify stratification markers and therapeutic vulnerabilities. MethodsWe developed Single-Cell Behavior Live Imaging (ScBLI), an approach integrating live imaging with computational analysis, applied to 30 GB primary cell cultures. Trajectories and morphological features were tracked and analyzed. Diffusion Entropy Analysis (DEA) was applied to classify trajectories based on the Delta Scaling parameter ({delta} scaling). We evaluated functional responses correlating all findings with clinical outcomes and transcriptomic profiles. ResultsWe analyzed 4,279 cell trajectories. Based on {delta} scaling (range 0.28-0.837), we defined three distinct motility groups: Low (L, {delta} scaling [&le;]0.5), Medium (M, 0.5 < {delta} scaling [&le;] 0.7), and High (H, {delta} scaling >0.7). Functional assays demonstrated that Group H cells are more performant in both positive and negative chemotaxis. Clinically, the three groups showed a clear linear progression with patient survival: High {delta} scaling correlated with the shortest survival (poorer prognosis), while Low {delta} correlated with the longest survival, suggesting that structured motility drives invasiveness. Integrative multi-omic analysis, encompassing both exome and transcriptome profiling, demonstrated that these groups are defined by distinct molecular landscapes rather than poor behavioral traits. Moreover, exome data revealed that Group H is significantly enriched in PTEN alterations (75% vs. 8% in Group L), with PTEN gain-of-function (GoF) mutations exclusively restricted to this group (100% vs 0% in Group L). Notably, within our extended cohort (n=51) currently characterized by whole-exome sequencing, we observed that specific PTEN GoF mutations were associated with a significantly shorter survival compared to PTEN wild-type cases (median OS 6.4 vs 16.6 months; p=0.02), which typically harbor the canonical loss of chromosome 10q. A similar clinical trend was observed when comparing directly GoF carriers to patients with truncating (Ter) alterations (median OS 6.4 vs 14.3 months; p=0.09). Conversely, no survival difference was found between truncating (Ter) mutations and wild-type cases. ConclusionOur findings demonstrate for the first time that migratory efficiency, quantified through DEA, represents a powerful predictor of glioblastoma aggressiveness. Tumor cells adopting highly efficient exploration strategies are strongly associated with poor clinical outcomes and are characterized by distinct molecular signatures, notably PTEN gain-of-function alterations. Statement of significanceOur multi-scale computational framework elucidates emergent behavioral phenotypes as pivotal drivers of glioblastoma progression. By demonstrating a correlation between enhanced migratory efficiency, PTEN gain-of-function, and significantly reduced overall survival, we establish a foundational paradigm for deciphering the emergent complexity governing tumor invasiveness.