Abstract Paediatric central nervous system (CNS) cancers represent a leading cause of cancer related mortality in children and are driven by distinct developmental and epigenetic mechanisms. However, systematic epigenetic characterisation across paediatric CNS cancer models remains limited. The Childhood Cancer Model Atlas (CCMA) was established as a comprehensive and globally accessible resource for paediatric cancer research, with a specific and strategic focus on CNS tumours. CCMA contains the largest and most diverse single-site collection of paediatric CNS tumour cell line models (n > 250), including high grade glioma, atypical teratoid rhabdoid tumour, ependymoma, medulloblastoma, and several rare CNS cancer types, alongside extensive molecular profiling, functional genomics, and drug response data. To deepen biological insight into these models, we initiated the development of CCMA-EPIC, a new epigenetic framework to characterise chromatin landscapes across CCMA cell lines. We employed Cut&Run, using six key histone modification markers that capture active promoters, enhancers, transcriptionally active regions, heterochromatin, and quiescent chromatin, to define chromatin states in four paediatric CNS tumour types and subtypes including ATRT (n = 6), H3K27M (n = 10), H3G34-altered (n = 8) and H3 wild-type high grade gliomas (n = 10). Integration of these markers enables systematic annotation of model specific chromatin regulatory states, revealing pronounced epigenetic heterogeneity across CNS tumour entities and highlighting lineage specific regulatory programs that are not apparent from genomic features alone. We further evaluated the functional relevance of CCMA-EPIC by integrating epigenetic features with machine learning models to predict CRISPR gene dependency and drug response profiles. Preliminary analyses show that inclusion of chromatin state features substantially improves prediction accuracy compared to models based solely on genomic and transcriptomic data. Collectively, our work reveals the critical role of the epigenome in defining genetic dependencies and drug sensitivities in paediatric CNS tumours and sets the stage for uncovering epigenetic biomarkers that may inform future precision medicine clinical trials.

Abstract Diffuse midline glioma, K27-altered (DMG) is a uniformly fatal cancer of the brainstem, thalamus, and spinal cord that usually arises in children. Next-generation sequencing in the last decade has revealed that 80% of patients with DMG harbour K27M mutations in histone variants H3.1 or H3.3, leading to global loss of H3K27me3 and a halted differentiation. Cells subsequently acquire oncogenic mutations resulting in tumour cells that transcriptionally resemble OPCs. DLX2 is a homeobox transcription factor necessary for GABAergic interneuron differentiation during healthy neurogenesis, which directly represses transcription factors necessary for OPC differentiation. We hypothesised that by pushing differentiation away from the halted OPC fate it would promote differentiation and lead to a reduction in tumourigenicity. RNA-sequencing data from 60 DMG cell lines revealed that inverse expression profiles of DLX2 and OLIG2 were maintained. Therefore, to test the plasticity of DMG cell fate two H3.1K27M and H3.3K27M cell lines underwent DLX2 overexpression, with the highest proportion of DLX2 expressing cells showing activation of DLX2 target neuronal genes by qPCR. Bulk RNAseq revealed activated neuronal programs, but that the cells maintained OPC and mesenchymal gene programs, suggesting incomplete transdifferentiation. Comparison of this data to an existing scRNAseq database of the fetal human brain showed subtype specific differences in transcriptional plasticity between H3.3 and H3.1 K27M cell lines. Thus, we show that transdifferentiation and transcriptional plasticity as it pertains to the existing developmental lineages present in DMG are dictated by histone subtype. Analysis of changes in chromatin structure and DNA methylation, as well as tumourigenesis in vitro and in vivo following DLX2 overexpression will be presented. These data show that mmanipulation of cell fate determination through altering a key transcriptional regulator in DMG provides a developmentally relevant model system for assessing the contribution differentiation and tumourigenicity of DMG.

Abstract Certain subgroups of paediatric-type diffuse high-grade gliomas (PDHGG) have highly infiltrative phenotypes spanning multiple anatomical sites and are commonly characterised by EGFR alterations. These include diffuse midline glioma, EGFR-altered (DMG-EGFR), overlapping with bithalamic glioma, and gliomatosis cerebri (GC). Although clinical studies with EGFR inhibitors have not shown substantial benefit, aberrant EGFR signalling may act as a driver of invasive growth in patient populations that could be selected for in future trials. Taking an unbiased approach to mapping EGFR alterations across PDHGG (n = 1487), we observed a mutation frequency of 8.2%, and amplification of 2.7% cases. These alterations were particularly enriched in the DMG-EGFR (32/52, 62%, enriched in exon7 and 20), and pedHGG-RTK2 subgroups (30/66, 45%), with amplifications restricted to pedHGG-RTK2B (5/18, 28%) and strong enrichment of mutations in pedHGG-RTK2A (21/48, 44%, enriched in exon7 and 15). We further established novel patient-derived models of EGFR-altered tumours for preclinical testing in vitro (n = 9) and in vivo (n = 4), with orthotopic PDXs implanted cortically recapitulating the GC infiltrative growth patterns by histology and MRI. Screening a panel of EGFR inhibitors in vitro, we observe the highest degree of single agent potency with afatinib and neratinib, particularly in a model with exon20 mutations. Screening these agents with drug-on screens against a ‘drugs and tools’ library of ∼900 compounds, we observed synergy with multiple chemotypes of FGFR inhibitors, pointing towards novel combinatorial therapies. Using in utero electroporation of day E12.5/13.5 embryos, addition of EGFR_A289V mutations accelerated tumour growth of both hemispheric TP53/PTEN/NF1 and midline H3.1K27M/EZHIP/TP53 mutant models, representing the first putative genetically engineered models of DMG-EGFR. By integrating molecular genetics, in vivo modelling, and pharmacological validation, this work suggests a role of EGFR-alterations in promoting diffuse infiltrative growth and co-inhibition of FGFR as an actionable vulnerability in EGFR-altered gliomas, addressing an urgent unmet need in paediatric neuro-oncology.

Abstract Synthetic lethality provides a powerful framework for cancer therapy by targeting gene interactions that are selectively essential in tumor cells, but are non-essential in normal tissues.. This approach is particularly attractive in malignancies driven by oncogenes considered “undruggable”. Using a machine-learning multi-omics approach we identify UBE2Z/USE1, a key ubiquitin-conjugating enzyme of non-canonical E1 UBA6-charged ubiquitination cascade, as a synthetic lethal partner of oncogenic β-catenin. UBE2Z knockout markedly impairs nuclear β-catenin accumulation, suppresses Wnt target gene expression, and induces differentiation in cell lines, tumouroids and in vivo models. Strikingly, UBE2Z is exclusively necessary for oncogenic β-catenin activity and completely dispensable for physiological Wnt/β-catenin signaling, highlighting its tumor-specific role. Genome-wide CRISPR rescue screens identified E-Cadherin as a critical intermediate of UBE2Z activity. Integrative transcriptomic and proteomics analyses suggest that UBE2Z supports oncogenic β-catenin transcriptional activity by promoting the degradation of intracellular E-cadherin via UBRfamily N-end rule E3 ligases. Manipulating UBE2Z’s ubiquitin-conjugating activity recapitulates the potent inhibitory effect of E-cadherin overexpression on oncogenic β-catenin activity, underscoring its translational significance. These findings reveal the UBA6-UBE2Z non-canonical ubiquitination cascade as a druggable vulnerability in β-catenin-addicted cancers and underscore synthetic lethality as a rational strategy for targeting β-catenin-driven tumorigenesis. Citation Format: Chunhua Wan, Hugh Gao, Claire Sun, Ron Firestein. Integrative computational and functional genomic approach reveals UBE2Z-UBR2-Ecadherinaxis as a beta-catenin-specific vulnerability in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 306.

Abstract H3K27M diffuse midline gliomas (DMGs) are devastating paediatric brain tumours for which radiotherapy (RT) remains the primary treatment modality. Although responses are transient, re-irradiation can provide modest benefit, underscoring the continued clinical reliance on RT. As most recurrent DMGs arise after prior RT, they frequently exhibit acquired radioresistance, raising questions about how radiation-adapted tumour cells modulate immune-relevant pathways. Beyond cytotoxicity, RT can induce immunostimulatory effects, including upregulation of HLA class I (HLA-I) expression and antigen presentation. However, how radiation reshapes the HLA-I immunopeptidome in H3K27M DMGs remains largely unexplored. In vitro RT responses were assessed in three paediatric H3.3K27M DMG cell lines (SF7761, SU-DIPG19, SU-DIPG27), comprising one radiation-naïve line and two autopsy-derived, previously irradiated. Cells were irradiated at 5 Gy or 10 Gy and harvested 72 hours post-treatment. HLA-I surface expression was measured by flow cytometry. Triplicate pellets were processed for HLA-I immunopeptidomics and global proteomics. IFN-γ–treated cells served as an internal control. Radiation induced a 1.5–1.8-fold increase in HLA-I surface expression across all lines. Across conditions, 10,000–35,000 HLA-I peptides were detected, with >45% shared, indicating a substantial core immunopeptidome that persists despite treatment. Both shared peptides with increased intensity post-radiation and radiation-exclusive peptides were enriched for source proteins involved in DNA repair, cell-cycle regulation, and oxidative stress responses, demonstrating that the immunopeptidome of irradiated cells reflects key features of the radiation-induced stress state. For example, in SF7761, radiation-responsive proteins such as TIGAR, TP53I3 and CDKN1A showed concordant increases in both peptide presentation and protein abundance, illustrating that radiation imprints a stress-associated antigenic signature detectable across the proteome and immunopeptidome. Overall, global proteomic profiling (>10,000 proteins) showed that irradiated cells formed distinct clusters separate from untreated and IFN-γ–treated groups, reflecting cell-line–specific features such as TP53 status and prior RT exposure that shape radiation-responsive antigen presentation.

Abstract Diffuse midline glioma (DMG) remains among the most lethal paediatric malignancies, characterised by the H3K27M onco-histone mutation and profound resistance to current therapies. Despite remarkable progress in cancer immunotherapy, the antigenic determinants capable of eliciting effective T-cell responses in DMG remain undefined. Here, we delineate the first comprehensive map of H3K27M-derived and H3K27M-induced antigens, spanning canonical and noncanonical sources, and demonstrate their therapeutic tractability across multiple HLA contexts. Using isogenic H3K27M mutant–wild-type DMG cell line pairs, we demonstrated that the onco-histone fundamentally reshapes the HLA class I ligandome. Through an integrated multi-omics and functional pipeline combining deep immunopeptidomics, predictive modelling, and T-cell functional assays we identified and validated five naturally presented, immunogenic H3.3K27M neoepitopes restricted by three distinct HLA supertypes. These neoantigens were confirmed in patient tumours by PRM-targeted mass spectrometry and elicited potent cytotoxicity when targeted by cloned H3K27M-specific TCRs in co-culture assays. To chart the broader antigenic landscape, we profiled 22 patient-derived DMG lines and 19 primary tumours, integrating fractionated data-dependent and data-independent acquisition immunopeptidomics. By filtering tumour ligandomes against benign brain and reference HLA datasets and using a custom proteogenomic database incorporating Ribo-seq-defined noncanonical ORFs, we uncovered a rich layer of cryptic antigens arising from H3K27M-driven chromatin dysregulation. Remarkably, 18% of these tumour-exclusive ligands were shared across samples, indicating recurrent, non-mutational vulnerabilities exploitable by “off-the-shelf” immunotherapies. Functional testing confirmed that selected dark-proteome-derived peptides robustly activated T cells in an HLA-restricted manner. Together, this work defines the first integrated antigenic atlas of DMG, encompassing both mutation-derived and epigenetically induced antigens. It establishes a direct mechanistic link between H3K27M-mediated chromatin remodelling and tumour-specific antigen presentation providing an actionable foundation for next-generation TCR-T and vaccine therapies in a cancer long deemed immunologically silent.

Abstract Paediatric cancers are the leading cause of disease-related mortality in children, with approximately 400,000 diagnoses annually worldwide. Central nervous system (CNS) tumours are the most common childhood solid malignancies and account for the highest proportion of cancer-related deaths. Cancer immunotherapy offers a potentially less toxic treatment option for paediatric patients, with tumour-derived neoepitopes presented by human leukocyte antigen (HLA) class I molecules emerging as promising targets. However, HLA downregulation and poor antigen presentation limit anti-tumour immune responses in paediatric CNS tumours. A detailed understanding of antigen processing and presentation is critical for advancing next-generation precision immunotherapies in paediatric oncology. Using the Childhood Cancer Model Atlas (CCMA), we curated a catalogue of class I HLA types and neoepitopes across 287 cell line models, with a focus on CNS tumours (177 models). HLA-A, B, and C types were inferred at four-digit resolution from sequencing data and curated to assess allele diversity, balance, and expression. Interestingly, we observed homozygosity across all three HLA class I loci in several models. HLA allele-specific expression loss was also commonly detected across paediatric cancer types, consistent with tumour immune evasion. To further characterise the immunogenic potential of CCMA models, neoepitopes were predicted by integrating somatic variants with matched HLA types and prioritised using binding affinity and elution likelihood. From this refined set of high-confidence candidates, we identified neoepitopes derived from recurrent driver mutations and observed overlap with experimentally validated epitopes from the Immune Epitope Database. Collectively, our analysis reveals significant HLA allele-specific downregulation in CNS tumours and other paediatric cancers. Integration of HLA genotype, expression, and the predicted neoepitope repertoire provides a foundation for neoantigen-based immunotherapy development. We believe the CCMA dataset will serve as a valuable public resource and enable a broader range of research for the treatment of paediatric cancer.

Abstract Background: Atypical teratoid rhabdoid tumor (ATRT) is an aggressive central nervous system tumor that mostly affects infants and children under the age of 3. While there have been some improvements in clinical outcomes with multimodal therapy, there remains significant morbidity and toxicities associated with intensive therapy. Therefore, there is a dire need for less toxic and improved therapies for children with ATRT. The identification of cancer dependencies can be utilized to determine novel therapeutic approaches. We identified exportin-1 (XPO1), a nuclear export protein that transports cargo proteins from the nucleus to the cytoplasm, as a novel dependency in ATRT. In other cancers, elevated XPO1 expression has been associated with poor prognosis. Methods: We utilized an integrative approach harnessing in vitro ATRT models, functional genomics, drug assays, flow cytometry, transcriptomics, and in vivo intracranial xenograft models to systematically test the hypothesis that XPO1 is a therapeutic vulnerability in ATRT. Results: RNA-sequencing data across pediatric brain tumor cell lines reveals that XPO1 is highly expressed in ATRT cells. We utilized CRIPSR-Cas9 to knockout XPO1 expression in a panel of patient-derived ATRT cells and found significant defects in cell viability and proliferation. Pharmacologic inhibition of XPO1 using six different selective inhibitors of nuclear export (SINEs) in multiple ATRT cell lines showed sub-10 nM IC50 values. We found that XPO1 inhibition led to on target degradation of XPO1 protein levels. In ATRT cells treated with an XPO1 inhibitor selinexor, Annexin V flow cytometry showed increased apoptosis relative to DMSO. In addition, ATRT cells treated with selinexor demonstrated G1 cell cycle arrest. Western blotting revealed a significant increase in cleaved caspase-3 levels and activation of TP53 with pharmacologic XPO1 inhibition. Transcriptomic analysis of ATRT cells with genetic and pharmacologic inhibition of XPO1 showed significant upregulation of apoptosis and TP53 signaling pathways, with concurrent depletion of cell cycle gene sets. Lastly, using in vivo intracranial xenograft models, the combination of selinexor with radiation and cyclophosphamide led to a reduction of tumor size and significant increase in animal survival. Conclusion: We demonstrate that XPO1 is a dependency in ATRT, and targeting XPO1 in combination with cytotoxic chemotherapy shows high translational potential. Citation Format: Tessa O. House, Irina Alimova, Shawna Larsen, Gillian Murdock, Angela Pierce, Breauna Brunt, Stefania Tocci, Sofia Krykunenko, Marissa Coppola, Anat Erdreich-Epstein, Ron Firestein, Natalie Serkova, Rajeev Vibhakar, Jessica W. Tsai. XPO1 as a therapeutic vulnerability in atypical teratoid rhabdoid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3487.

Abstract Background Atypical teratoid rhabdoid tumor (ATRT) is an aggressive central nervous system tumor that mostly affects children under age 3. Current intensive, multimodal therapy results in profound treatment-related toxicities. Thus, there is a dire need for less toxic and improved therapies for children with ATRT. The identification of cancer dependencies can be utilized to determine novel therapeutic approaches. We identified exportin-1 (XPO1), a nuclear export protein that transports cargo proteins from the nucleus to the cytoplasm, as a novel dependency in ATRT. Methods We harnessed an integrative approach utilizing in vitro ATRT models, drug assays, functional genomics, flow cytometry, transcriptomics, and in vivo intracranial xenograft models to systematically test the hypothesis that XPO1 is a therapeutic vulnerability in ATRT. Results RNA-sequencing data across pediatric brain tumor cell lines revealed that XPO1 is highly expressed in ATRT. We utilized CRISPR-Cas9 to knockout XPO1 expression in a panel of patient-derived ATRT cells and found significant defects in cell viability and neurosphere formation. Pharmacologic inhibition of XPO1 using six different selective inhibitors of nuclear export (SINEs) across multiple ATRT cell lines showed sub-10 nM IC50 values. XPO1 inhibition led to on target degradation of XPO1 protein levels. Moreover, ATRT cells treated with selinexor showed increased apoptosis by Annexin V flow cytometry and also demonstrated G1 cell cycle arrest. Transcriptomic analysis of ATRT cells with genetic and pharmacologic inhibition of XPO1 showed significant depletion of cell cycle gene sets, with concurrent upregulation of apoptosis and TP53 signaling pathways. Lastly, using in vivo intracranial xenograft models, the combination of selinexor, radiation, and cyclophosphamide led to a reduction of ATRT tumor size and significant increase in animal survival. Conclusion We demonstrate that XPO1 is a therapeutic vulnerability in ATRT, and targeting XPO1 in combination with cytotoxic chemotherapy demonstrates high translational potential.

Aberrant epigenetic regulation is a hallmark of diffuse midline glioma (DMG), an incurable pediatric brain tumor. The H3K27M driver histone mutation leads to transcriptional dysregulation, indicating that targeting the epigenome and transcription may be key therapeutic strategies against this highly aggressive cancer. One such target is the facilitates chromatin transcription (FACT) histone chaperone. We found FACT to be enriched at developmental gene promoters, coinciding with open chromatin and binding motifs of core DMG regulatory transcription factors. Furthermore, FACT co-occurred with the bromodomain and extraterminal domain (BET) protein BRD4 at promoters and enhancers, suggesting functional cooperation between FACT and BRD4 in DMG. In vitro, a combinatorial therapeutic approach using the FACT inhibitor CBL0137, coupled with BET inhibition, revealed potent and synergistic cytotoxicity across a range of DMG cultures. These results were recapitulated in vivo, extending survival in three independent orthotopic patient-derived xenograft models of DMG. Mechanistically, we show that CBL0137 treatment decreased chromatin accessibility and combined with BET inhibition to cause broad transcriptional collapse; silencing of several key oncogenes including MYC, PDGFRA, MDM4, and SOX2; and alterations to the splicing landscape. This combination also elicited immune-related effects, including activation of the interferon response and antigen presentation mechanisms in DMG cells and induction of an activated state in macrophages and T cells, as demonstrated in an immunocompetent setting with spatial transcriptomics. Together, our data highlight the therapeutic promise of simultaneously targeting FACT and BET proteins in DMG, offering a dual tumor-intrinsic and immune-mediated strategy for combating this devastating pediatric brain tumor.

Diffuse midline glioma (DMG) is a terminal paediatric brain cancer which has no current cure, leading to an urgent demand to re-evaluate therapeutic strategies. DMG is an ‘epigenetically driven’ cancer, as most patients harbor the epigenetic mutation H3K27M. In 2020, a novel DMG subtype was discovered for cases lacking H3K27M, which instead aberrantly expressed the Enhancer of zeste inhibitory protein (EZHIP). Interestingly, EZHIP is a ‘mimic’ of H3K27M due to sequence similarity. This project aims to study the novel EZHIP-DMG subtype by establishing the epigenetic profile of these tumours for comparison with other DMG subtypes, and explore the EZHIP-specific epigenetic effects to predict the best therapeutic strategy. To compare epigenetic profiles of DMG subtypes, patient-derived DMG cell lines expressing EZHIP or H3K27M, H3-wildtype (without EZHIP) and normal controls were used. Epigenomic profiling was performed in cell lines for active and repressive chromatin marks by ChIP-sequencing (H3K27ac, H3K4me3, H3K4me1, H2A.Zac, H3K36me3, H3K27me3 and H3K9me3) and for chromatin accessibility by ATAC-sequencing. Integration of transcriptomic signatures was done using RNA-sequencing. Overall, EZHIP-DMG cell lines are epigenetically more similar to H3.3K27M-DMG than H3-wildtype DMG cell lines. This was confirmed by principal component analysis for chromatin accessibility, histone modifications and gene expression. However, specific epigenetic patterns of EZHIP-DMG were also identified. Particularly, for H3K27ac and H2A.Zac histone modifications, which putatively mark active enhancers. Further analysis of the top variant H3K27ac-enhancers by hierarchical clustering revealed unique enhancers in EZHIP-DMG, reasoning the differences observed between subtypes. This study is the first to confirm that EZHIP has epigenetic similarities with H3K27M in DMG beyond H3K27me3 loss, but also, EZHIP-DMG may have unique signatures at enhancers that could influence therapeutic response. Further work into identifying EZHIP-DMG specific targets in addition to targeting common epigenetic vulnerabilities for H3K27M/EZHIP in DMG will be a promising therapeutic strategy.

Pediatric high-grade gliomas (pHGGs) are the most aggressive brain tumors in children, necessitating innovative therapies to improve outcomes. Unlike adult gliomas, recent research reveals that childhood gliomas have distinct biological features, requiring specific treatment strategies. Here, we focused on deciphering unique genetic dependencies specific to childhood gliomas. Using a pooled CRISPR/Cas9 knockout screening approach on 65 pediatric and 10 adult high-grade glioma (HGG) cell lines, myeloid cell leukemia 1 ( MCL1 ) emerged as a key antiapoptotic gene essential in pediatric but not adult gliomas. We demonstrated that MCL1 is targetable using current small molecule inhibitors, and its inhibition leads to potent anticancer activity across pediatric HGG cell lines irrespective of genotype. Employing predictive modeling approaches on a large set of childhood cancer cell lines with multiomics data features, we identified a potentially previously unreported cluster of CpG sites in the antiapoptotic BCL-xL/BCL2L1 gene, which predicted MCL1 inhibitor response. We extended these data across multiple pediatric tumor types, showing that BCL2L1 methylation is a broad predictor of MCL1 dependency in vitro and in vivo. Overall, our multidimensional, integrated genomic approach identified MCL1 as a promising therapeutic target in several BCL2L1-methylated pediatric cancers, offering a translational strategy to identify patients most likely to benefit from MCL1 inhibitor therapy.

Cancer immunotherapy has revolutionized treatment by leveraging the immune system to recognize and destroy tumor cells, offering a promising, less toxic option for pediatric patients. A key component of this response is antigen presentation, which depends on accurate human leukocyte antigen (HLA) typing and expression. However, immune-focused resources for pediatric cancers remain limited. In this study, we present a comprehensive immunogenomic resource covering 231 cancer cell lines and 56 tumor-associated fibroblast cell lines from the Childhood Cancer Model Atlas (CCMA). We inferred high-resolution HLA types, predicted neoantigens arising from somatic single nucleotide variants, gene fusions, and splicing isoforms across multiple tumor types, and quantified HLA expression levels. We also explored immune escape mechanisms, including loss of heterozygosity and allele-specific expression loss of HLA genes. This publicly accessible dataset provides critical insight into the immune landscape of pediatric cancers and serves as a foundational tool for immunotherapy development.

The discovery that ACVR1-mutations arise in ~25% of DMG H3K27-altered patients has led assessment of both the dependency of ACVR1-mutant cells on ALK2 and the effectiveness of ALK2 inhibitors (ALK2i), however as single-agents these are yet to translate into the clinic. Here, we perform high-throughput screens to identify therapeutic ALK2i combination partners and characterise pre-clinical ALK2 inhibitors of different chemotypes using a multi-omic approach. Combinatorial CRISPR screens identified multiple ALK2i sensitising hit genes which encode for key enzymes in the cholesterol synthesis pathway (EBP/DHCR24/LSS). Drug-combination screens identified clinically well-tolerated statins, including simvastatin/lovastatin which target the rate-limiting cholesterol synthesis enzyme HMG-CoA reductase, and estrogen receptor inhibitors, including Tamoxifen, which exhibits off-target effects on EBP, as sensitisers to ALK2i. Global transcriptomics, proteomics and metabolomics revealed a novel link between ALK2 signalling and cholesterol homeostasis, with ALK2i treatment significantly decreasing the expression cholesterol biosynthesis genes/proteins (SREBF2/HMGCR/EBP) while increasing those associated with cholesterol transport (ABCA1/MYLIP). Metabolomic analysis confirmed these changes were associated with a significant decrease in cholesterol and an increase in the precursor desmosterol. Validation of screening hits revealed strong synergy between ALK2i and cholesterol biosynthesis inhibitors targeting different nodes of the pathway. This was also observed in vivo, the combination treatment significantly increased the median survival compared to vehicle but not to single-agents. Forced differentiation of DMG cells to an astrocyte-like cell state increased cholesterol production and led to a significant decrease in M4K2009 sensitivity and synergy with statins, which was phenocopied when DMG cells were co-cultured with normal astrocytes. This was overcome in vitro, ex vivo and in vivo using a triple combination including a LXR agonist (LXR623) to promote cholesterol export. We identify a previously unappreciated link between ALK2 signalling and cholesterol homeostasis which may be exploited clinically by combining ALK2i with routinely-used statins and BBB-penetrant LXR agonists.

Uterine fibroids (UFs) are the most common non-cutaneous tumors in women worldwide. UFs arise from genetic alterations in myometrial stem cells (MM SCs) that trigger their transformation into tumor initiating cells (UF SCs). Mutations in the RNA polymerase II Mediator subunit MED12 are dominant drivers of UFs, accounting for 70% of these clinically significant lesions. Biochemically, UF driver mutations in MED12 disrupt CDK8/19 kinase activity in Mediator, but how Mediator kinase disruption triggers MM SC transformation remains unknown. Here, we show that pharmacologic inhibition of CDK8/19 in MM SCs removes a barrier to myogenic differentiation down an altered pathway characterized by molecular phenotypes characteristic of UFs, including oncogenic growth and extracellular matrix (ECM) production. These perturbations appear to be induced by transcriptomic changes, arising in part through epigenomic alteration and super-enhancer reprogramming, that broadly recapitulate those found in MED12-mutant UFs. Altogether these findings provide new insights concerning the biological role of CDK8/19 in MM SC biology and UF formation.

Aberrant epigenetic regulation is a hallmark of Diffuse Midline Glioma (DMG), an incurable pediatric brain tumor. The H3K27M driver histone mutation leads to transcriptional dysregulation, indicating that targeting the epigenome and transcription may be key therapeutic strategies against this highly aggressive cancer. One such target is the Facilitates Chromatin Transcription (FACT) histone chaperone. Using Cleavage Under Targets and Release Using Nuclease (CUT&RUN), we found FACT to be enriched at developmental gene promoters, coinciding with regions of open chromatin and binding motifs of core DMG regulatory transcription factors. Furthermore, FACT interacted and co-localized with the Bromodomain and Extra-Terminal Domain (BET) protein BRD4 at promoters and enhancers, suggesting functional cooperation between FACT and BRD4 in DMG. In vitro, a combinatorial therapeutic approach using the FACT inhibitor CBL0137, coupled with BET inhibition revealed potent and synergistic cytotoxicity across a range of DMG cultures. These results were recapitulated in vivo, significantly extending survival in three independent orthotopic PDX models of DMG. Using ATAC-seq and nascent RNA-seq, we show that CBL0137 treatment decreased chromatin accessibility, synergizing with BET inhibition to cause broad transcriptional collapse. This included silencing of several key oncogenes including MYC, PDGFRA, MDM4 and SOX2, as well as causing alterations to the splicing landscape. Notably, this combination also elicited immune-related effects, including activation of the interferon response and antigen presentation in DMG cells and induction of an activated state in macrophages and T cells, as demonstrated in an immunocompetent setting using Xenium spatial profiling with a 5000 gene panel. Altogether, our data highlights the therapeutic promise of simultaneously targeting FACT and BET proteins in DMG, offering a dual tumor-intrinsic and immune-mediated strategy for combating this devastating pediatric brain tumor.

Colorectal cancers (CRCs) present across a range of differentiation grades, which impact patient outcome and management; however, the molecular features and drivers of differentiation status are not fully understood. To address this, 84 commonly used human CRC cell lines were grown as xenografts in mice, revealing models of low-grade (LG) and high-grade (HG) CRC. Transcriptional profiling revealed coordinate downregulation of multiple transcription factors involved in intestinal development and differentiation, markers of colonic lineage-specific differentiation, and effectors of normal functions of the colonic epithelium in HG tumours. Mechanistically, multiple genes suppressed in HG tumours harboured promoter methylation, indicative of stable epigenetic silencing. Furthermore, markers of LGR5+ colon stem cells were suppressed in HG tumours, while markers of cell proliferation, fetal-like intestinal stem cells, and non-canonical cell types including mesenchymal cells were increased. These changes manifested in HG cell line displaying increased proliferation, migration and metastatic capacity. Importantly, CRC cell line-derived transcriptional profiles of differentiation grade were reflected in LG and HG patient-derived tumour organoids and primary CRCs, revealing cell lines accurately model differentiation grade. The models and tumour differentiation-related properties identified herein may inform new approaches for tailored CRC treatments based on tumour grade.

Diffuse intrinsic pontine glioma (DIPG) is a devastating brainstem cancer in children, with a median survival of under one year and limited treatment options. Over 80% of DIPGs possess a H3K27M mutation. To identify metabolic vulnerabilities linked to this mutation, we utilized a multi-omics approach in H3K27M-expressing cells, patient-derived cell lines, and mouse models. We show that by reprogramming chromatin landscape the mutation aberrantly induces NFI transcriptional activity, leading to misregulated purine metabolism. The mutation amplifies purine biosynthesis and degradation via the enzymes ATIC and PNP, respectively. Unregulated purine degradation relieves the negative feedback of purines on their own synthesis allowing continuous synthesis, use and degradation making DIPGs reliant on purine biosynthesis. Targeting ATIC reduced tumor progression and improved survival in mice. We propose ATIC as a potential novel target in DIPG.

Simple Summary Diffuse midline gliomas (DMGs) are aggressive childhood brain tumours with no effective treatments. Over 80% of cases carry the histone H3K27M mutation, which alters chromatin structure and gene regulation and induce tumours growth. Immunotherapy, which uses the body’s immune system to fight cancer, relies on tumour- specific antigen molecules that immune cells can recognise. However, because DMGs have few mutations, finding suitable antigens for therapeutic purposes has been challenging. In this study, we investigated how the H3K27M mutation affects tumour antigen presentation in DMG. Using patient-derived DMG models, we found that H3K27M alters the landscape of antigens displayed on tumour cell surface, creating unique immune targets. We identified six immunogenic peptides, that triggered strong T cell responses. These antigens were absent when H3K27M was removed, confirming their link to the mutation. Our findings provide a blueprint for developing T cell-based immunotherapies for DMG, offering new hope for targeted treatments against this devastating disease. Background: Diffuse midline gliomas (DMGs) are among the most aggressive paediatric brain tumours, with the pathognomonic H3K27M mutation present in over 80% of cases. This mutation drives epigenetic dysregulation and transcriptional reprogramming, yet its impact on the tumour antigenic landscape remains poorly understood. Given the low mutational burden of DMG, an expanded search beyond neoantigens to include epigenetically dysregulated tumour-associated antigens (TAAs) is critical for advancing antigen-specific immunotherapies. Methods: To assess how H3K27M influences antigenic landscape of DMG, we performed a comprehensive immunopeptidomic analysis using patient-derived DMG cell line models (SU-DIPG13 and BT245) that harbour the H3K27M mutation and their CRISPR-edited H3K27M-knockout (KO) counterparts. High-resolution mass spectrometry and bioinformatics were employed to define H3K27M-driven changes in the immunopeptidome. Functional T cell assays using HLA-matched healthy donor PBMCs were conducted to evaluate the immunogenicity of H3K27M-associated peptides. Results: Our findings reveal that the H3K27M mutation reshapes the tumour antigenic landscape in a model-specific manner. While H3K27M knockout increased HLA-I expression in SU-DIPG13 but not BT245, immunopeptidomic profiling uncovered distinct shifts in the presentation of tumour-associated peptides, independent of direct effects on antigen processing machinery. Among these, we identified six immunogenic peptides, derived from SLITRK2, PRAME, XKR5, and CBX2, that elicited CD8⁺ T cell responses in in vitro functional assays. Notably, PRAME, a well-characterised cancer-testis antigen was confirmed as an H3K27M-associated immunogenic target, reinforcing its therapeutic relevance. Peptides identified exclusively in H3K27M+ cells were absent in KO models, demonstrating a direct link between H3K27M-driven transcriptional dysregulation and tumour antigenicity. Conclusions: This study provides the first systematic assessment of how H3K27M reshapes the antigenic landscape in DMG, uncovering novel, immunogenic tumour- associated peptides that could serve as targets for precision immunotherapy. By demonstrating that H3K27M mutation drives context-dependent antigen presentation, our findings establish a foundation for T cell-based therapies targeting H3K27M-associated antigens. These insights pave the way for next-generation personalised immunotherapies for this otherwise treatment-refractory disease. Key Points H3K27M mutation induces expression of tumour-associated antigens in DMG H3K27M alters the DMG immunopeptidome without uniformly changing HLA-I levels PRAME- and CBX2-derived peptides are immunogenic and targetable by CD8⁺ T cells Importance of Study Diffuse midline gliomas (DMGs) are universally fatal paediatric brain tumours with limited treatment options and poor immune visibility. While the H3K27M mutation is a defining hallmark, its impact on tumour immunogenicity remains unclear. This study presents the first comprehensive to explore the effect of H3K27M-mution on the DMG immunopeptidome, revealing six immunogenic peptides derived from epigenetically dysregulated tumour- associated antigens, including SLITRK2, PRAME, XKR5, and CBX2. These antigens elicited CD8⁺ T cell responses, establishing a direct link between H3K27M-driven transcriptional dysregulation and tumour antigenicity. By leveraging these altered antigens, we highlight actionable vulnerabilities for T cell-based immunotherapy. ### Competing Interest Statement The authors have declared no competing interest.