Cancer Research

TP53 mutations occur in 80-90% of triple-negative breast cancers (TNBCs) and drive genomic instability and metastatic progression. Poly (ADP-ribose) polymerase (PARP) is critical for DNA repair and replication fork stability. How oncogenic signaling influences PARP function to sustain proliferation during replication stress remains unclear. Mutant p53 (mtp53) R273H associates tightly with chromatin, forms complexes with PARP, and enhances PARP recruitment to replication forks [1-3]. The C-terminal region of mtp53 mediates mtp53-PARP and mtp53-Poly (ADP-ribose) (PAR) interactions that facilitate S phase progression [4, 5]. The PARP inhibitor talazoparib (TAL) combined with the alkylating agent temozolomide (TMZ) produces synergistic cytotoxicity selectively in mtp53, but not wild-type p53 (wtp53), breast cancer cells and organoids. Herein we evaluated the mechanism of mtp53-associated cell death and tested if this could translate to a preclinical xenograft model. We found that TMZ+TAL treatment induced elevated cleaved PARP and {gamma}H2AX and reduced the metastasis-promoting oncoprotein MDMX. In orthotopic xenografts expressing mtp53 R273H, but not wtp53, combination therapy significantly decreased circulating tumor cells (CTCs) and lung metastases. Transcriptomic profiling of tumors from combination treated animals demonstrated downregulation of MDMX, VEGF, and NF-{kappa}B, consistent with the observed suppression of CTCs and lung metastasis, and increased {gamma}H2AX, indicative of replication stress in mtp53 xenografts. Inhibition of metastasis was also observed in mtp53 R273H WHIM25 and p53-undetectable WHIM6 TNBC patient-derived xenografts (PDX). The mtp53 C-terminal domain (347-393) demonstrated a critical tumor promoting function, as CRISPR-mediated deletion impaired replication fork progression, tumor growth, and metastatic dissemination. DNA fiber combing showed that expression of full-length mtp53 R273H, but not C-terminal deleted {Delta}347-393, supported sustained single-stranded DNA gaps (ssGAPs) following Poly (ADP-ribose) glycohydrolase (PARG) inhibition. These findings support that mtp53 uses C-terminal amino acids to exploit PARP to enable replication stress adaptation and that mtp53 is a predictive biomarker for combined PARP inhibitor and DNA damaging therapies targeting TNBC. Significance statementTP53 mutations are the most common genetic alterations in TNBC and a major driver of replication stress and metastasis. This study shows that missense mutant p53 uses C-terminal amino acids to reprogram PARP activity to maintain tumor cell survival under replication stress. We demonstrate that p53 status governs the response to combined PARP inhibitor (PARPi) and DNA-damaging chemotherapy, establishing an additional molecular basis beyond BRCA1 mutations for treating TNBC with PARPi therapy. These findings reveal a previously unrecognized mechanism by which the mutant p53-PARP axis enables replication stress tolerance and drives cancer metastasis. We show mutation of p53 in TNBC provides an additional biomarker-guided framework to improve PARPi therapeutic outcomes.

Soft tissue leiomyosarcoma (STLMS) is an aggressive malignancy for which robust molecular subclassification and mechanism-based therapeutic strategies still remain limited. We performed integrative proteogenomic analyses of primary and metastatic STLMS to define subtype-associated molecular programs. Joint analysis of the proteome and phosphoproteome identified 3 biologically distinct subtypes. P1 was characterized by relative genomic stability, low proliferative activity, and enrichment of FGFR2- and PDK-associated signaling. In contrast, P2 and P3 showed greater chromosomal instability and more aggressive clinical behavior, but with distinct molecular features. Notably, P2 was associated with inflammatory and RTK-RAS pathway programs, activation of CDK-AURKA/B-mTOR-ERK kinase networks, IGF1R/PDGFRA alterations, and the poorest outcomes. On the other hand, P3 showed strong cell cycle and DNA repair programs, elevated NCOR1 expression, and increased expression of nonhomologous end joining components, including PARP1. Homologous recombination deficiency analyses distinguished HRD-low P1 from HRD-high P2/P3, and paired analyses suggested increased HRD-related features in metastatic lesions within P3. Immune profiling identified an immune-hot yet potentially suppressive state in P2, marked by higher LGALS9 expression and M2-like macrophage infiltration. To support clinical translation, we developed a tissue microarray-based immunohistochemical classifier that enabled surrogate assignment of proteome-defined subtypes in an independent cohort and showed recurrence-free survival differences across inferred subtypes. These findings together establish a proteogenomic framework for STLMS heterogeneity and nominate subtype-associated biological vulnerabilities for future translational and clinical investigation.

AbstractRecurrent loss-of-function mutations in RUNX1 occur in estrogen receptor-positive (ER+) breast cancers, yet how RUNX1-loss contributes to breast tumorigenesis remains unclear. Here we used genetically engineered mouse models with luminal mammary epithelial cell (MEC)-restricted gene disruption to investigate its role in breast cancer initiation. Loss of RUNX1 alone, or together with RB1, was insufficient to drive tumor formation. In contrast, combined loss of RUNX1 and p53 induced mammary tumors with full penetrance. These tumors contained ER+ cancer cells and exhibited extensive T cell and macrophage infiltration, indicative of an immune hot microenvironment. Mechanistically, RUNX1-deficiency activated interferon signaling in luminal MECs, associated with derepression of RUNX1 target STAT1 and enhanced inflammatory responses. Consistent with these findings, human ER+ breast cancers with low RUNX1 expression displayed elevated immune signatures and poorer patient survival. Together, our results identify RUNX1-loss as a driver of an immune-active subtype of ER+ breast cancer.

Secretory breast carcinoma (SBC) is typically indolent, yet mechanisms underlying aggressiveness and therapeutic resistance to tropomyosin receptor kinase inhibitors (TRKi) remain unclear. Autopsy-based longitudinal multi-organ high-dimensional profiling of metastatic TRKi-resistant SBC demonstrated histopathological heterogeneity, including secretory and squamous components, arising from a shared clonal origin. Integrated genomic and transcriptomic analyses revealed hierarchical transcriptional rewiring consistent with a lineage-plastic state, suggesting a potential link to tumor aggressiveness and therapeutic resistance.

Well-differentiated and dedifferentiated liposarcoma (WDLPS and DDLPS) exhibit markedly different clinical behaviors, with DDLPS showing greater aggressiveness, higher recurrence and metastasis rates, and worse outcomes. Using single-nucleus multiome sequencing, epigenomic profiling, and spatial transcriptomics, we characterized cellular and epigenetic heterogeneity between these subtypes at single-cell and spatial resolution. We found distinct phenotypic states reflecting altered lineage differentiation and plasticity: DDLPS is dominated by early-differentiated progenitor-like cells, sclerotic WDLPS displays broader mesenchymal lineage plasticity, and adipocytic WDLPS contains abundant committed adipocytes. The DDLPS immune microenvironment was dominated by immunosuppressive macrophages, whereas WDLPS harbored more T cells and inflammatory macrophages. Notably, sclerotic WDLPS displayed intermediate cellular and molecular features, suggesting it may represent a distinct WDLPS subtype. Importantly, we identified novel gene regulatory circuits underlying each state, including FABP4/PPARG programs in adipocytic WDLPS, GLI2/TCF7L2/RBPJ/KLF7 programs in sclerotic WDLPS, and KLF7/FOSL2/SP3/GLI2/RBPJ programs in DDLPS. H3K27ac-marked enhancers were enriched near adipocytic marker genes in WDLPS and mesenchymal markers in DDLPS. Together, these findings reveal the cellular heterogeneity of tumor and immune compartments across liposarcoma subtypes and identify regulatory programs driving their differentiation states. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/713651v1_ufig1.gif" ALT="Figure 1"> View larger version (73K): org.highwire.dtl.DTLVardef@1c84ee1org.highwire.dtl.DTLVardef@1b2ad42org.highwire.dtl.DTLVardef@18ce5a6org.highwire.dtl.DTLVardef@138f615_HPS_FORMAT_FIGEXP M_FIG C_FIG

Well- and dedifferentiated liposarcomas (WDLPS and DDLPS) are characterized by extensive copy- number alterations rather than recurrent gene-inactivating mutations, obscuring the molecular mechanisms that drive disease progression and, critically, the transition from well-differentiated to the more aggressive dedifferentiated tumor states. Despite marked differences in clinical behavior and prognosis, the regulatory events underlying adipocytic lineage destabilization in DDLPS remain poorly understood. Here, we establish an in vivo model of retroperitoneal liposarcoma in Xenopus tropicalis through early embryonic mosaic perturbation of p53 and Rb pathway components. Combined disruption reproducibly induced retroperitoneal WDLPS development, demonstrating that pathway-level deregulation of the MDM2-p53 and CDK4-Rb axes is sufficient to initiate liposarcoma development in vivo. Strikingly, additional perturbation of transcriptional co-activator ep300 in this context resulted in increased tumor dedifferentiation, yielding lesions composed of spatially coexisting well- and dedifferentiated adipocytic states. In contrast, direct targeted disruption of downstream adipogenic regulators recurrently lost in human DDLPS, including cebpa, g0s2, and dgat2, failed to induce dedifferentiation in the same genetic context in vivo. These findings indicate that dedifferentiation cannot be explained by loss of downstream adipocytic effectors alone but instead reflects destabilization of higher-order regulatory programs governing adipocytic identity. Together, these results establish an in vivo model that closely reflects the clinical situation on a pathway level and provides initial mechanistic insight into how adipocytic differentiation may become destabilized during disease progression. This framework offers a foundation for future studies leveraging higher-order and multi-omic approaches to dissect the molecular processes underlying the WDLPS-to-DDLPS transition.

Aberrant neurotransmitter signaling and transcriptional dysregulation are hallmarks of gliomagenesis and represent potential therapeutic targets. Monoamine neurotransmitters such as dopamine and serotonin primarily activate GPCRs but can also function epigenetically as histone H3 modifications. Here, we uncover mechanisms of crosstalk between monoamine neurotransmitter signaling, H3 dopaminylation, and RNA polymerase II (Pol2) transcription in diffuse midline glioma (DMG). We find that co-treatment with Pol2-targeting CDK9 inhibitors (CDK9i) and FDA-approved neuropsychiatric drugs, including selective serotonin reuptake inhibitors (SSRIs), synergistically reduces DMG growth. Mechanistically, CDK9i+SSRI treatment alters H3 dopaminylation patterns and represses synaptic and neurodevelopmental gene transcription associated with CDK9i resistance. Further phospho-proteomic analyses show that CDK9i monotherapy activates pro-survival CaMKII signaling, which can be suppressed by co-treatment with neuromodulatory drugs. These studies establish roles for H3 dopaminylation and neurotransmitter signaling in DMG gene regulation and response to CDK9i, suggesting that monoamine neurotransmitter pathways may be exploited as a therapeutic strategy for DMG.

BackgroundGallium (Ga) is a promising anti-tumor agent; however, its precise molecular targets in osteosarcoma remain debated. While current paradigms largely attribute its toxicity to reactive oxygen species (ROS) and ferroptosis, understanding its true mechanism is essential for overcoming therapeutic resistance. This highlights the need for interdisciplinary approaches, such as metabolomics, to unveil novel vulnerabilities in cancer metabolism. MethodsWe employed an interdisciplinary strategy utilizing high-resolution liquid chromatography-mass spectrometry (LC-MS) metabolomics and 13C2-glutamine stable isotope tracing in osteosarcoma cells to elucidate the cytotoxic mechanisms of gallium nitrate. Scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDS) was utilized for elemental mapping, and in silico modeling was applied to evaluated metal binding dynamics. Furthermore, synergistic effects were tested by combining gallium with the DNA-damaging agent cisplatin. ResultsOur metabolic profiling revealed a profound bifurcation characterized by the systemic depletion of glycolysis and pentose phosphate pathway intermediates, coupled with a novel ribonucleotide accumulation bottleneck. The observed distinct signature strongly implicated ribonucleotide reductase (RNR) as the primary enzymatic target. In silico modeling and SEM-EDS visually and thermodynamically confirmedthat gallium acts as a structural decoy for iron within the RNR active site. The co-localization induces functional iron starvation rather than canonical ferroptosis. Furthermore, isotope tracing confirmed that elevated ROS is a consequence of overall metabolic failure, not the primary driver of cell death. Crucially, gallium functioned as a metabolic DNA repair inhibitor, synergizing potently with cisplatin to prevent the repair of platinum-induced DNA lesions. ConclusionsGallium selectively sensitizes highly proliferative sarcoma cells by disrupting RNR-mediated DNA precursor synthesis, while sparing normal osteoblasts. Leveraging metabolomics to uncover this state of functional iron starvation provides a rational, interdisciplinary framework for developing gallium-based combination therapies designed to break platinum resistance in clinical oncology.

Despite the availability of several FDA-approved therapies, metastatic melanoma remains a significant clinical challenge, particularly for patients with brain metastases, which frequently represent the site of treatment failure and a major cause of melanoma-related mortality. Melanoma exhibits a strong propensity to metastasize to the brain, yet the molecular mechanisms driving this lethal progression remain incompletely understood, limiting the development of effective treatment options. Building on our prior discovery that focal adhesion kinase (FAK) is a key mediator of AKT1-driven brain metastasis, we sought to validate the role of FAK in melanoma progression and metastatic dissemination. Using complementary autochthonous and syngeneic mouse models of BRAF-mutant melanoma, we evaluated the impact of FAK expression on overall survival, primary tumor growth, and metastasis. Through the generation of targeted FAK mutants, we distinguished kinase-dependent from kinase-independent functions and demonstrate that FAK promotes melanoma metastasis in a kinase-dependent manner. Furthermore, we establish that FAK functions downstream of PTEN to drive metastatic progression. Collectively, these findings support the therapeutic potential of FAK inhibition, either alone or in combination with existing treatments, to more effectively combat metastatic melanoma and inform the development of emerging FAK-targeted therapies.

We generated interferon signaling reporters in human and mouse melanoma cells and observed heterogeneity in interferon responses among cells in the same tumors. This was marked by inflamed regions within primary tumors that contained increased numbers of interferon-expressing macrophages/monocytes and elevated type I interferon signaling in melanoma cells. Melanoma cells that expressed GFP-Interferon stimulated gene 15 (ISG15) or GFP-Interferon-induced protein with tetraticopeptide repeats 3 (IFIT3) fusion reporters exhibited a profoundly increased ability to form metastatic tumors as compared to GFP-ISG15- melanoma cells from the same tumors, particularly when transplanted into immunocompetent mice. The increased metastatic potential of GFP-ISG15+ cells was driven partly by increased CD47 expression, which protected metastasizing cells from phagocytosis by macrophages. Macrophages are thus a double-edged sword, inhibiting the development of metastatic disease by phagocytosing disseminated melanoma cells, but promoting the emergence of a hyper-metastatic subpopulation of cells in inflamed regions of primary tumors as a consequence of sustained interferon production. One sentence summarySustained interferon exposure within inflamed regions of primary tumors dramatically increases the metastatic potential of a subpopulation of melanoma cells, partly by promoting CD47 expression, which protects against phagocytosis by macrophages.

Pancreatic ductal adenocarcinoma (PDAC) remains a lethal malignancy, with therapeutic resistance influenced by a dense desmoplastic stroma dominated by cancer-associated fibroblasts (CAF). Using single-cell RNA-sequencing and gene regulatory network modeling of 42 PDAC tumors, we identified a CAF subpopulation characterized by elevated NFATC2 expression that is enriched in patients with improved therapeutic response and survival. NFATC2+ CAFs exhibited tumor-suppressive features, including enhanced apoptotic signaling and suppression of ERBB pathway activity. Co-culture experiments demonstrated that NFATC2+ CAFs restrain pancreatic cancer cell growth and enhance chemotherapy-induced apoptosis, increasing sensitivity to standard-of-care chemotherapy regimens and synergizing with ERBB-targeted therapies. The favorable effect of NFATC2+ CAFs on chemotherapy response was validated in two other PDAC cohorts and in rectal cancer. Together, these findings identify NFATC2+ CAFs as a therapy-conditioned stromal state linked to improved treatment response and uncover a context-dependent vulnerability within the tumor microenvironment that may be exploited to rationally optimize combination therapies.

3-mercaptopyruvate sulfurtransferase (3-MST) is a mammalian enzyme that contributes to hydrogen sulfide and reactive sulfur species generation. Here we show that 3-MST is markedly upregulated in colorectal cancer stem cells (CSCs) and functions as a critical metabolic support mechanism for this therapy-resistant tumor cell population. CSCs exhibit low proliferation rate, high membrane rigidity and a metabolically restrained phenotype characterized by low oxidative phosphorylation rate, combined with a reduced rate of glycolysis. Genetic or pharmacological inhibition of 3-MST further suppresses cellular bioenergetics in CSCs, and this bioenergetic collapse impairs CSC proliferation, spheroid formation, migration and promotes cell death and attenuates tumor growth. Integrated transcriptomic, proteomic, metabolomic, and lipidomic analyses reveal extensive metabolic remodeling of the CSCs following 3-MST inhibition, including disruption of the glycolysis-TCA axis and marked remodeling of membrane lipid composition, including enrichment of ceramides and sphingolipids and increased incorporation of polyunsaturated phospholipids, resulting in increased membrane fluidity. 3-MST inhibition induced an activation of integrated stress pathways, proteotoxic stress responses and inflammatory signaling, linking the metabolic failure of CSCs to the induction of mixed-mode cell death. These findings identify 3-MST as a metabolic vulnerability in colorectal CSCs. Targeting this enzyme may be a translatable strategy to eliminate therapy-resistant tumor stem cell populations.

BackgroundNon-invasive electromagnetic field (EMF)-based therapies offer a potential route to modulate local tumor-immune interactions but their mechanistic basis remains poorly defined. MethodsWe evaluated Asha therapy, a proprietary low-intensity (50khz, 2 mT, 25% duty cycle) alternating magnetic-field treatment in preclinical breast cancer models. Cellular responses in human triple negative breast cancer cell lines (MDA-MB-231 and MDA-MB-468) were evaluated using bulk RNA sequencing, quantitative proteomics, flow cytometry, and cytokine analysis and proteomics analysis. Tumor microenvironment responses in mouse 4T1 breast cancer model was characterized using single-cell CITE-seq analysis. Functional efficacy was assessed in vivo using the murine 4T1 triple-negative breast cancer model, both as monotherapy and in combination with anti-PD1 checkpoint blockade. Clinical relevance was assessed by deriving a 19-gene neutrophil activation signature from Asha-induced transcriptional changes and projecting it onto two independent TNBC patient cohorts (METABRIC n=338, SCAN-B n=874) for survival analysis. ResultsAsha therapy induced endoplasmic reticulum (ER) stress and activated an adaptive unfolded-protein response in tumor cells, triggering robust NF-{kappa}B and interferon signaling and time-dependent secretion of inflammatory cytokines. In vivo, these tumor-intrinsic changes propagated to the tumor microenvironment (TME), reprogramming fibroblasts from contractile states to immune-recruiting, interferon-responsive phenotypes and enriching for interferon-stimulated, metabolically active neutrophils and macrophages. These coordinated innate immune changes occurred without overt cytotoxicity and were associated with significant reductions in metastasis and improved survival. Combination with anti-PD1 therapy markedly enhanced efficacy, reducing lung metastasis and mortality by 88% compared with control. The neutrophil activation signature derived from Asha-treated tumors was associated with improved overall survival in both METABRIC (log-rank p=0.036) and SCAN-B (p=0.048) TNBC cohorts by Kaplan-Meier analysis, with pooled multivariable Cox regression confirming significant survival benefit (HR=0.75, 95% CI 0.59-0.94, p=0.01). ConclusionsAsha therapy triggers a controlled ER stress response in tumor cells that drives interferon-mediated cytokine release and immune reprogramming of the TME, resulting in anti-metastatic and survival benefits. These findings identify electromagnetic-field exposure as a potential non-pharmacologic strategy to activate innate immunity and sensitize tumors to checkpoint blockade, supporting further clinical development of EMF-based immunotherapy.

The central nervous system (CNS) is a common site of metastatic spread for both non-small cell and small cell lung cancer, yet the therapeutic strategies to prevent and decrease lung cancer brain metastases remain limited. Tyrosine kinase inhibitors have shown promising results in increasing the overall response in brain metastases, owing to their brain penetrance and increased effectiveness; however, their use is limited to the small group of tumors carrying specific oncogenic drivers. Among these, inhibitors with activity against neurotrophic tyrosine receptor kinases (NTRKs) are showing promising effects in reducing CNS metastases in cancers driven by gene rearrangements of these drugs targets. However, wild-type NTRKs are susceptible to activation by their canonical ligands, which are expressed throughout the brain metastatic niche and can, in a paracrine manner, activate NTRK function in cancer cells. Here we show that NTRKs are expressed in primary tumors, brain metastases, and lung cancer cells with various driver mutations expressing wild-type NTRK2 (WT-TrkB). We demonstrate that WT-TrkB activates downstream signaling and proliferation in response to exogenous BDNF and conditioned media from reactive astrocytes known to secrete BDNF in the brain niche. Importantly, the FDA-approved NTRK inhibitor entrectinib blocked BDNF and astrocyte-induced survival pathways in multiple lung cancer cell lines, decreased their proliferation in vitro, and effectively prevented brain metastatic colonization and progression in vivo without significant effects on extracranial disease. Thus, these studies suggest that brain-dependent activation of NTRK is critical for brain metastases of WT-NTRK+ lung cancers, and therefore, NTRK inhibitors can be used to target non-fusion NTRK function to prevent or decrease brain metastases. SIGNIFICANCEThese studies demonstrate that NTRK wild-type receptors are important drivers of brain metastatic colonization and progression in different subtypes of lung cancer, independent of their driver alterations. Thus, they provide rationale to expand the use of FDA-approved NTRK inhibitors with brain penetrance for the prevention of CNS metastases.

Macrophages are pivotal mediators of wound healing, yet the cellular programs they employ can be hijacked by cancers to drive tumorigenesis. Although similar macrophage programs support both physiological tissue regeneration and pathological cell growth, the molecular and functional difference between wound-associated macrophages (WAMs) and tumor-associated macrophages (TAMs) remain poorly defined. Here, we perform comparative single-cell RNA sequencing to delineate the dynamic cell states of macrophages during skin wound healing and the progression of cutaneous squamous cell carcinoma. Our analyses reveal that aberrantly regulated lipid metabolism is a distinct feature of TAMs. Critically, our genetic manipulations allow us to identify SOX2High tumor-initiating stem cells as key orchestrators that modulate the lipid metabolism of TAMs and shape their cell states. These findings suggest that disrupting the metabolic crosstalk between tumor-initiating stem cells and TAMs represents a promising strategy to normalize myeloid cell function and enhance cancer immunotherapy efficacy.

BackgroundNeoadjuvant chemoimmunotherapy (nCIT) has become a standard treatment for locally advanced resectable non-small cell lung cancer (NSCLC), yet the spatial biology underlying treatment resistance remains poorly understood. We used spatial transcriptomics to define the microenvironmental architecture of residual cancers in patients who did not achieve major pathologic response (non-MPR) compared with those who did (MPR). MethodsSpatial transcriptomics was performed on 10 formalin-fixed paraffin-embedded (FFPE) tumor blocks (5 MPR, 5 non-MPR) obtained from 8 patients treated with nCIT. A deep learning algorithm was applied to detect viable residual cancer spots from treatment-induced fibrosis and necrosis. Spatial deconvolution, distance modeling, ligand-receptor analysis, and functional pathway scoring were integrated to characterize niche-specific programs. ResultsMPR cancer core displayed an immune-permissive remodeling environment with deep infiltration of cytotoxic CD8+ T cells, mature dendritic cells (LAMP3+, CCR7+), and active efferocytosis signaling (APOE-TREM2), alongside robust MHC class II expression. Non-MPR cancer core, by contrast, exhibited spatial immune exclusion: a dense fibroblast barrier reinforced by TIMP1-CD63 signaling and Treg-enriched boundaries physically restricted effector T cell access to the cancer core. Residual cancer cells in non-MPR samples maintained active cell cycling and independently upregulated cytochrome P450-mediated drug detoxification and DNA damage response pathways without inducing MHC class II expression -- effectively decoupling intrinsic survival from immune recognition. The non-MPR core also showed a hyper-metabolic profile, including elevated glutathione metabolism consistent with antioxidant buffering against chemotherapy-induced oxidative stress. TROP2 was broadly expressed across the non-MPR cancer core and co-localized with DNA damage response and nuclear factor erythroid 2-related factor 2 resistance signatures. ConclusionsResidual cancer cores in non-MPR tumors appear to represent evolved resistant niches sustained by structural immune exclusion, metabolic rewiring, and DNA repair proficiency. These findings highlight the spatial co-localization of epithelial anchors, such as TROP2, with intrinsic resistance pathways, providing a structural rationale for developing novel precision therapeutic strategies to bypass stromal barriers and overcome the cancer cores intrinsic repair capacity.

Osteosarcoma is a malignant bone tumor with a high risk of metastatic relapse and poor outcomes due to primary and acquired chemoresistance. This highlights the medical need to develop effective targeted approaches to overcome chemoresistance. Recent studies have revealed the roles of metabolic reprogramming and mitochondria-nucleus crosstalk in osteosarcoma progression, indicating the potential of these cellular processes as therapeutic targets. The complex formed by mitochondrial apoptosis-inducing factor (AIF) and coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4) orchestrates the import and oxidative folding of cysteine-rich, nuclear-encoded proteins, thereby regulating key mitochondrial functions and metabolism. Here, we identified mitoxantrone as an inhibitor of the AIF/CHCHD4 mitochondrial import machinery and revealed a new mitoxantrone-induced metabolic vulnerability in some osteosarcoma cell line models, characterized by intracellular glutamine accumulation and an increase in nucleotide synthesis. As a result, synergy was found between mitoxantrone and the glutaminase inhibitor telaglenastat in both in vitro and in vivo osteosarcoma models. Collectively, our findings position the AIF/CHCHD4 complex as a druggable therapeutic target and provide a combination strategy for mitoxantrone/telaglenastat treatment to overcome metabolic adaptations and chemoresistance in osteosarcoma. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/716303v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1229e58org.highwire.dtl.DTLVardef@1c9af45org.highwire.dtl.DTLVardef@120d2borg.highwire.dtl.DTLVardef@11e8216_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

Y-box binding protein 1 (YB-1; YBX1) is a multifunctional DNA- and RNA-binding protein involved in cell cycle regulation, DNA repair, stress adaptation, and therapy resistance. Elevated YBX1 mRNA expression is associated with aggressive disease across multiple cancers, yet its pan-cancer genomic and clinical correlates remain unclear. Here, we performed a comprehensive pan-cancer analysis across 53 datasets spanning 33 tumour types, integrating RNA expression, somatic mutations, copy number, hypoxia, and clinical outcomes. YBX1 was rarely mutated or amplified, indicating that oncogenic relevance is primarily driven by its expression. Tumours with high YBX1 mRNA exhibited a conserved transcriptional program enriched for cell cycle, DNA repair, and chromatin regulation pathways, and were preferentially mutated in genes involved in maintaining genomic stability, including TP53. These tumours were associated with increased mutation burden, fraction of genome altered, homologous recombination deficiency, and elevated hypoxia. Clinically, high YBX1 mRNA associated with advanced stage, higher grade, shorter progression-free survival, and reduced overall survival. Collectively, high YBX1 mRNA expression defines a conserved, genomically unstable, and clinically aggressive tumour state across multiple cancer types.

The normal adult male breast has not been characterized at single-cell resolution, leaving the cellular basis of male breast cancer (MBC) biology undefined. Here we present an integrated single-cell RNA sequencing atlas of the adult human breast comprising 174,471 cells from 17 donors (3 male, 14 female), including 18,117 male-derived cells. This revealed that the male breast retains all three epithelial populations, basal (BC), luminal progenitor (LP), and luminal committed cells (LC), but with an increase in LC at the expense of BC and LP across all three male donors. Male LC were distinguished from female by elevated ESR1 and PGR mRNA, enrichment of RNA processing and ribosome biogenesis programs, reduced inflammatory cytokine and growth factor signaling, elevated estradiol gene set enrichment scores, and higher inferred activity of developmental patterning transcription factors. This pattern was observed across differential expression, gene ontology, ligand profiling, and regulon-based analyses, and was not restricted to sex chromosome-linked gene expression. This is consistent with the near-universal estrogen receptor (ER) positivity that characterizes MBC clinically. This atlas provides the first cellular and transcriptional reference for the normal male breast and a resource for investigating sex differences in mammary biology, germline susceptibility variant interpretation, and modeling breast malignancies.