Molecular Cancer Research

Long noncoding RNAs (lncRNAs) are important regulators of gene expression and are frequently dysregulated in cancer. The mitotically associated lncRNA MANCR is highly expressed in aggressive cancers and contributes to genomic instability in triple-negative breast cancer (TNBC), but the molecular mechanisms underlying its activity remain poorly defined. Here we integrate computational and experimental approaches to examine the structure and regulatory interactions of MANCR isoforms. Analysis of transcriptomic datasets revealed tumor-type-specific expression patterns for seven MANCR isoforms in breast cancer cell lines. Computational prediction of RNA secondary structures identified conserved structural features across isoforms, suggesting potential functional specialization. We identify p53 as a MANCR-interacting protein through computational docking and RNA immunoprecipitation sequencing (RIP-seq) and demonstrate that MANCR depletion reduces p53-dependent transcriptional activity. Chromatin isolation by RNA purification sequencing (ChIRP-seq) revealed 1, 250 genomic regions associated with MANCR, including enrichment of p53 consensus motifs and GC-rich sequence elements. Motif analysis further identified candidate sequence features associated with MANCR-occupied chromatin regions. Computational prediction of RNA-miRNA interactions identified multiple potential miRNA binding sites across MANCR isoforms, including miR-6756-5p, which targets the androgen receptor (AR). Consistent with this prediction, AR expression decreased following MANCR knockdown in TNBC cells. Together, these results suggest that MANCR isoforms may contribute to transcriptional regulation in TNBC through interactions with chromatin, p53 signaling pathways, and potential miRNA regulatory networks. One Sentence SummaryMitotically-associated lncRNA (MANCR) is prevalent in aggressive cancers interacting with DNA, P53, and miRNAs, to mediate multiple levels of epigenetic transcriptional control in triple negative breast cancer.

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.

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.

Keratin-positive giant cell-rich tumor (KPGCT) is a newly described bone and soft tissue tumor. The tumor is characterized by scattered keratin-positive cells and the presence of HMGA2::NCOR2 fusions. It is not known if the HMGA2::NCOR2 fusion is located in the keratin-positive cells, and little is known about how KPGCT develops. KPGCT shares some histologic features with tenosynovial giant cell tumor (TGCT), a soft tissue tumor with CSF1 rearrangements. Single-nuclei RNA sequencing (snRNA-seq) and Xenium spatial transcriptomics were used to elucidate the mechanisms driving KPGCT and compare KPGCT to TGCT. We show that the neoplastic cells in KPGCT constitute only a minority of cells in the tumor, and that they co-express keratin, HMGA2 and CSF1. The neoplastic cells in KPGCT express no synovial markers, confirming KPGCT as a distinct entity, separate from TGCT. The bulk of the tumor consists of CSF1R-expressing macrophages and osteoclast-like giant cells, suggesting an important role for CSF1-CSF1R signaling. In addition, we find that the cells with the HMGA2 translocation show activation of the hippo signaling pathway, which is known to regulate CSF1 expression. We show that the CSF1-CSF1R axis, possibly regulated through the hippo signaling pathway, plays an important role in KPGCT. This axis likely stimulates the migration and proliferation of macrophages, which form the majority of cells in the tumor, as well as their differentiation into osteoclasts-like giant cells. These results provide a rationale for the use of CSF1R inhibitors, which have already shown efficacy in TGCT, as a therapy for KPGCT. SignificanceKeratin-positive giant cell-rich tumor (KPGCT) is a rare, newly described soft tissue and bone tumor. By examining this tumour on a single-cell level, we confirm the identity of the neoplastic cells on a molecular level, showing these form a minority of cells in the tumor. We show that activation of the hippo pathway in the neoplastic cells is a likely driver of tumorigenesis. Additionally, we show the neoplastic cells produce large amounts of CSF1, attracting the macrophages that form the majority of cells in the tumor. This finding gives supporting evidence for anecdotal reports of response to CSF1 inhibitor therapy. Finally, we identify key differences between KPGCT and tenosynovial giant cell tumor, a tumor that shares histological features with KPGCT.

Macrophages/osteoclasts are highly fusogenic cells that interact closely with bone-metastatic breast cancer cells. These cancer cells adapt to bone microenvironments by undergoing osteomimicry, acquiring bone-like phenotypes. Exploration using human breast cancer-bone metastases dataset revealed that a small population of epithelial breast cancer cells express osteoclast-like and osteomimicry genes at the single-cell level. Cell fusion and cell-in-cell (CIC) processes are two uncommon yet prognostically significant mechanisms in cancer. We showed that co-culture between murine breast cancer cells and osteoclasts yielded a unique osteoclast phenotype through dynamic cell-in-cell (CIC) interactions and fusion-like behaviours between pre-osteoclasts/mature osteoclasts and breast tumor cells, resulting in osteoclast-tumor hybrid-like cells. These tumor cell interactions characterized by membrane retention and nuclear adjacency to host nuclei were consistently observed throughout osteoclast differentiation. Single-cell sequencing analysis and interpretative assays on hybrid-like cells revealed altered extracellular matrix (ECM) modification processes, immunoregulatory, and cancer-associated pathways compared to unfused osteoclasts. Tumor cells co-cultured with osteoclasts expressed hematopoietic and osteoclast-lineage factors more strongly than tumor cells cultured alone with their effects amplified under direct cell-cell contact. The presence of these hybrid-like cells was validated in human breast cancer-bone metastases. We propose that disseminated bone-tropic breast cancer cells were stimulated by osteoclasts to undergo a non-canonical, dynamic osteoclast differentiation and CIC formation to form hybrid-like cells that may facilitate bone metastatic lesions.

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 PBAF is one of three biochemically distinct BAF chromatin remodelers in humans. We previously proposed the role of ARID2, a PBAF component, as a bonafide tumor suppressor in colorectal cancer (CRC). Here, we validated loss of tumor suppression under conditions of ARID2 deficiency emanating from a marked reduction in PBAF complex assembly resulting from destabilization of PBAF-specific components BRD7, PHF10, and PBRM1. Transcriptome profiling of ARID2 deficient CRC cells revealed perturbation of disease processes, including CRC and neurodegenerative disorders, as well as CRC relevant pathways including Wnt/{beta}-catenin signalling, but transcript levels of PBAF-specific components remained unchanged, confirmed by RT-qPCR and TCGA data analysis. Our study establishes ARID2 as a critical stabilizer of the PBAF complex of relevance to CRC.

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.

La-related protein 1 (LARP1) is an RNA-binding protein that post-transcriptionally regulates mRNA with potential oncogenic role in multiple cancers; however, its function in endometrial cancer remains unknown. An analysis of the TCGA endometrial cancer cohort showed that overexpression of LARP1 is associated with shorter overall survival (OS) and progression-free interval (PFI) as indicated by Kaplan-Meier analysis. Functional in vitro studies revealed that LARP1 knockdown by two different siRNAs markedly suppressed cell viability and triggered apoptosis, as confirmed by increased protein levels of cleaved PARP1 and cleaved caspase-3. Mechanistically, LARP1 knockdown remarkably reduced E2F1 protein levels as confirmed by immunofluorescence and Western blotting. Clinically, co-overexpression of LARP1 and E2F1 further decreased OS and PFI, suggesting a co-operative oncogenic axis. Importantly, LARP1 knockdown enhanced the sensitivity of ISHI and HEC-1A endometrial cancer cell lines to carboplatin treatment. These findings suggest that LARP1 promotes endometrial cancer survival and resistance to chemotherapy, at least in part, through the regulation of E2F1 and suppression of apoptosis. Targeting LARP1 could represent a promising therapeutic strategy to suppress tumor growth and enhance sensitivity to platinum-based chemotherapy.

BackgroundLong non-coding RNAs (lncRNAs) have emerged as key regulators of tumor biology, however, thus far none have translated to cancer therapies. The lncRNA MALAT1 is overexpressed in more than 20 cancers, including breast cancer and has been shown to function via various mechanisms in a context-dependent manner, in 2D cell lines and mouse models. However, its functional role and therapeutic potential have not been evaluated in clinically relevant patient-derived models. MethodsWe investigated the therapeutic potential of a MALAT1-targeting antisense oligonucleotide (ASO) for breast cancer, using clinically relevant 3D human patient-derived organoids (PDOs) and PDO-xenograft (PDO-X) models. We systematically evaluated the efficiency of MALAT1-targeting ASOs using a biobank of 28 PDO models. Using three independent PDO-X models of triple negative breast cancer (TNBC), we targeted MALAT1 in vivo to study its impact on transcription, alternative splicing, stromal remodeling and metastasis. ResultsAcross PDO-X models, MALAT1 depletion reproducibly drove widespread alternative splicing changes across all event types, particularly intron retention events, accompanied by modest gene expression alterations. Differentially spliced transcripts were enriched for targets of shared cancer-associated transcription factors, and MALAT1 knockdown altered the relative abundance of previously unannotated splicing isoforms. Beyond tumor-intrinsic effects, tumor-specific MALAT1 depletion induced a consistent reduction in macrophage-associated gene signatures and reduced lung metastatic burden. ConclusionsOur data define MALAT1s multifaceted role in TNBC, coordinating alternative splicing, transcriptional fine-tuning, tumor-stroma crosstalk, and metastatic progression. Our study provides strong preclinical evidence supporting MALAT1-targeted ASO therapy and establishes PDO-X models as a clinically relevant platform for functional interrogation of TNBC therapies.

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.

Cancer metabolism rewiring is one of the hallmarks of cancer, enabling cancer cell survival in a nutrient deprived microenvironment. Key to this is nutrient scavenging where cancer cells rely on extracellular proteins, including extracellular matrix (ECM) components, to sustain their proliferation. ECM uptake is mediated by 2{beta}1 integrin, however it is not clear how this process is controlled by nutrient availability. Here we demonstrated that amino acid starvation promoted ECM internalisation, by inducing the expression of 2 integrin. Mechanistically, starvation-driven RAS/MAPK pathway activation in cells harbouring oncogenic RAS mutations and mTOR inhibition increased 2 integrin, while the GCN2-depedent integrated stress response was not required. Functionally, elevated 2 integrin levels promoted cell adhesion and migration in nutrient starved cells. Finally, 2 integrin was found upregulated in pancreatic tumours and correlated with poor prognosis in pancreatic adenocarcinoma patients. Together, these data indicate that the nutrient- starved pancreatic cancer microenvironment synergises with KRAS mutation to drive pancreatic cancer aggressiveness.

Cancer testis antigens are widely expressed in human malignancies. Melanoma-Associated Antigens (MAGE) A3 and A6 have been proposed to modulate protein turnover and metabolism in cancer cells. However, the substrate specificity of MAGE-A3/6 and the impact on cancer cell behavior remain poorly understood. Although previous research has identified binding partners, a molecularly validated target for MAGE-A3/6-mediated proteasomal degradation has not been described. In this study, we redefine the substrate specificity of MAGE-A3/6 and present a mechanistic framework for substrate binding, polyubiquitination, and subsequent degradation. We identify BPTF-Associated Protein of 18kDa (BAP18) as a bona fide novel substrate of MAGE-A3/6 and demonstrate its direct regulation via a molecularly defined substrate-degron-E3-adaptor interaction. The degradation of BAP18 by MAGE-A3/6 underlies phenotypic alterations in cancer cells, such as enhanced migratory capacity. This previously unrecognized molecular link is observed in both cancer cell lines and human cancer tissues, supporting a role as a fundamental oncogenic process. The discovery of a molecularly defined interaction between MAGE-A3/6 and their substrate enables systematic investigation into oncogenic protein degradation in human cancers and may inform future therapeutic strategies that leverage the molecular function of aberrantly reexpressed germline proteins in cancer.

Colonic adenocarcinoma (COAD) is a major cause of cancer-related mortality worldwide. Various tumors are linked to metastasis-associated in colon cancer 1 (MACC1). This study aimed to analyze public datasets to examine MACC1 expression, signaling pathways, copy number variations, and associations with immune cell subsets in COAD employing bioinformatics. MACC1 expression was elevated in COAD, especially in Wnt signaling and chromatin modifier pathways. Analysis of somatic copy number alterations in The Cancer Genome Atlas-COAD dataset revealed a link between MACC1 and DNA damage repair. MACC1 also showed a negative correlation with genes involved in immune cell infiltration in patients with COAD, including cluster of differentiation (CD)8+ T cells, activated dendritic cells, CD8 T cells, and cytotoxic cells. Collectively, these findings suggest MACC1 as a potential prognostic biomarker and therapeutic target for COAD.

B7-H3 (CD276) is an immune checkpoint molecule frequently overexpressed in hepatocellular carcinoma (HCC) and represents a promising therapeutic target. However, its roles in tumor cell adhesion, metastatic behavior and immune evasion--particularly in interactions with natural killer (NK) cells--remain incompletely understood. In the present study, B7-H3 was depleted using small interfering RNA and CRISPR/Cas9 in epithelial (Huh7 and HepG2) and mesenchymal (SNU449) HCC cell lines. Tumor cell survival, apoptosis, adhesion, migration and invasion were evaluated using functional assays. Expression of adhesion molecules and immune checkpoint proteins was assessed by flow cytometry and western blotting. Oncogenic signaling pathways were analyzed by examining phosphorylation of Akt, ERK, FAK and STAT3. NK cell-mediated cytotoxicity was assessed using primary human NK cells. B7-H3 depletion reduced clonogenic survival and increased apoptosis in mesenchymal HCC cells under anchorage-independent conditions. Loss of B7-H3 impaired cell adhesion, migration and invasion, accompanied by downregulation of PTGFRN, E-cadherin, integrin 3 and integrin V, and reduced cell-to-cell aggregation under anchorage-independent conditions. B7-H3 depletion also decreased surface expression of PD-L1, PD-L2 and CD47. Notably, B7-H3-deficient cells exhibited enhanced susceptibility to primary NK cell-mediated cytotoxicity. Mechanistically, B7-H3 promoted tumorigenic signaling through Akt/S6, MVP/ERK and FAK/Src pathways in epithelial cells, and through FAK/Src and JAK2/STAT3 pathways in mesenchymal cells. Together, these findings reveal previously unrecognized roles for B7-H3 in coordinating adhesion and NK immune evasion in HCC, and support its therapeutic targeting for next-generation immunotherapies.

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

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.

Ewing sarcoma (EwS) is an aggressive, human-exclusive tumor typically driven by the EWS::FLI1 fusion protein. To assess whether the neomorphic functions of EWS::FLI1 are fundamentally dependent on evolutionarily recent cofactors such as ETS transcription factors (ETS-TFs), Plycomb group (PcG) proteins, CBP/p300, or specific subunits of the BAF complex, we expressed EWS::FLI1 in the model organism Saccharomyces cerevisiae. This minimal system was chosen because several key EWS::FLI 's cofactors possess greatly reduced sequence homology (e.g., BAF) or are lacking altogether (e.g., ETS-TFs, PcG, or CBP/p300). We used co-IP/MS to map the yeast interactome, Chip-Seq to identify gDNA binding sequences, RNA-Seq for global gene expression, and engineered reporters to test conversion of (GGAA) tandem repeats (GGAASat) into neoenhancers. We found that the yeast EWS::FLI1 interactome was more limited and qualitatively distinct from its human counterpart, sharing core machinery (e.g. RNA Polymerase II, FACT) but lacking the BAF/SWI-SNF and spliceosome complexes, and showing strong enrichment for the SAGA chromatin remodeling complex. We also found that EWS::FLI1 binds to hundreds of sites in the yeast genome with a clear preference for putative ETS-TF consensus sequences and (CA) dinucleotide repeats. Yet, EWS::FLI1 expressing cells presented only minimal transcriptional dysregulation, a stark contrast to the extensive changes observed in humans and Drosophila cells. Finally, we found that EWS::FLI1 successfully converted silent GGAASat sequences into active enhancers in yeast. This remarkable result occurs despite the absence of homologs for key human activators, such as CBP/p300, strongly suggesting that EWS::FLI1 can mobilize functionally related, non-homologous pathways to establish neoenhancers at GGAASat sites. Altogether, our results indicate that EWS::FLI1's core ability to drive GGAASat-dependent gene expression is a conserved, ancient property, while GGAASat-independent extensive transcriptome reprogramming is dependent on co-factors and pathways specific to animal cells.

Diffuse large B-cell lymphoma (DLBCL), the most common aggressive lymphoma, encompasses histologically similar but genetically distinct cancers. Recent genetic studies have defined at least six molecular subtypes, yet none account for Epstein-Barr virus (EBV), despite 5-15% of DLBCLs being EBV-associated. By reanalyzing published whole-exome and RNA-sequencing data from 481 tumors, we identified 19 EBV-positive cases. These were significantly enriched in the BN2 subtype (6/19), while most (11/19) remained unclassified. In BN2 tumors, several subtype-defining mutations were reduced in frequency among EBV-positive cases, supporting the hypothesis that EBV oncogenes substitute for specific cellular alterations and may confound DLBCL classification algorithms. Extending our analysis to cell lines, we found that the widely used Val cell line harbors the B95-8 laboratory EBV strain; other EBV-positive lines appeared authentic but modeled only non-BN2 subtypes and expressed an atypical viral latency III program, whereas some DLBCL tumors expressed the atypical latency III program and others latency I or II. Together, these findings demonstrate that EBV-positive DLBCL, like DLBCL itself, is not a single disease, and that current in vitro models only partially capture its biological heterogeneity. Key pointsO_LIEBV-positive DLBCL is not a single disease and EBV status can impact genetic-based classifications. C_LIO_LICurrent EBV-positive DLBCL cell lines do not adequately capture tumor complexity; we determined that Val is a problematic cell line. C_LI

PRMT5 is a type II arginine methyltransferase that forms an active complex with methylosome protein WDR77 (MEP50) to catalyze the symmetric dimethylation (SDMA) of arginine residues in proteins that regulate biological roles including apoptosis, DNA damage response and RNA processing. Some of the best characterized PRMT5 substrates are the small nuclear ribonucleoproteins SNRPB, SNRPD1 and SNRPD3, which are critical for spliceosome assembly and RNA splicing fidelity. MTAP-deleted cancers exhibit increased sensitivity to PRMT5 inhibition due to elevated levels of methylthioadenosine (MTA), a natural inhibitor of PRMT5. This vulnerability is exploited by MTA-cooperative PRMT5 inhibitors, exemplified by TNG908 and TNG462 which selectively target PRMT5 in MTAP-deleted cells while sparing MTAP-wildtype (WT) cells. Consistent with this mechanism, treatment with TNG908 in preclinical studies induces widespread splicing alterations in MTAP-deleted cancer models, with minimal effects in MTAP-WT cells. These splicing changes are consistent across diverse MTAP-deleted tumor types, including glioblastoma, pancreatic, and non-small cell lung cancer, indicating a histology-agnostic response to PRMT5 inhibition. Moreover, treatment of MTAP-WT cells with exogenous MTA mimics the splicing alterations observed with PRMT5 inhibition, as does pharmacologic inhibition of MTAP further supporting a mechanistic link between MTA accumulation, PRMT5 modulation, and aberrant splicing. Given that MTAP deletions occur in approximately 10-15% of human cancers, the identification of a robust RNA splicing signature offers a valuable pharmacodynamic biomarker for monitoring the activity of PRMT5 inhibitors. This splicing-based readout may also serve as a predictive biomarker of therapeutic response, offering greater specificity than global SDMA levels. Collectively these data suggest that a PRMT5-dependent RNA splicing signature can monitor the pharmacodynamic activity of MTA-cooperative PRMT5 inhibitors in MTAP-deleted cells.