Eukaryotic transcription is a highly dynamic and adaptable process that underpins the gene expression programs regulating development, cellular identity, and responses to extracellular signals. Gene-specific regulation of transcription across different cell types and environmental conditions is therefore fundamental to both normal physiology and disease. This specificity is shaped by phosphorylation of the RNA polymerase II (RNAPII) C-terminal domain (CTD) and its associated transcription factors. While cyclin-dependent kinases (CDKs) have long been recognized as the regulators of RNAPII activity, emerging evidence points to a broader, more diverse network of transcriptional kinases. Here, we highlight non-canonical transcriptional kinases that could operate alongside and beyond CDKs to modulate transcription by RNAPII. We discuss how these kinases could introduce context-specific CTD modifications that enable transcriptional plasticity, facilitate rapid loci-specific activation, and integrate signaling and stress-responsive pathways, ultimately adding a layer of regulatory complexity with profound implications for dynamic transcriptional regulation in development, homeostasis, and disease.

Many epigenetic regulatory factors are targets of the somatic mutations found in patient tumours. Amongst the family of epigenetic regulatory complexes known as Complex of Proteins Associated with Set1 (COMPASS), the enhancer regulators histone-lysine N-methyltransferase 2C (KMT2C)-COMPASS and KMT2D-COMPASS are particularly critical for differentiation and cell fate specification. Their catalytic subunits, including the histone H3 lysine 4 (H3K4) monomethyltransferases KMT2C (also known as MLL3) and KMT2D (also known as MLL4) and the H3K27-specific demethylase lysine-specific demethylase 6A (KDM6A; also known as UTX), are encoded by some of the most frequently mutated genes across human cancers, particularly epithelial cancers. The multifaceted roles of KMT2C-COMPASS and KMT2D-COMPASS, the variety of KMT2C, KMT2D and KDM6A mutations found across all cancer types, and the tissue-specific impacts of compromised enhancer regulatory function have posed challenges for direct therapeutic targeting. However, KMT2C-COMPASS and KMT2D-COMPASS mutations also create tumour-specific and potentially targetable vulnerabilities. In this Review, we discuss the functional roles of KMT2C-COMPASS and KMT2D-COMPASS and the impact of their mutations on cancer progression. We outline potential therapeutic strategies to exploit vulnerabilities in cancer cells with altered KMT2C-COMPASS or KMT2D-COMPASS activity, including aberrant epigenetic regulatory complex activity, metabolic rewiring, defects in cell-cycle control and DNA repair, and increased immunogenicity.

Regulation of RNA polymerase II (Pol II) transcription is closely associated with cell proliferation. However, it remains unclear how the Pol II transcription program is rewired in cancer to promote uncontrolled growth. Here, we find that expression of NELFCD, a known negative transcription elongation factor, is upregulated in colorectal tumors. Auxin-dependent protein degradation of NELF-C in combination with nascent transcript sequencing demonstrates a direct role of NELF-C on Pol II transcription in this cancer. Strikingly, we demonstrate that the acute loss of NELF-C protein globally redistributes termination factors and perturbs Pol II transcription termination. These changes drive pervasive Pol II transcription into DNA replication zones, leading to transcription-replication conflict that may block the cell cycle in G1 or early S phase. Our findings reveal a previously unrecognized role of NELF in transcription termination and highlight NELF as a potential therapeutic target in colorectal cancer.

COMPASS (Complex of proteins associated with Set1) are highly conserved chromatin regulatory complexes responsible for all methylation marks on histone H3 lysine 4 (H3K4). The COMPASS protein SET1A is upregulated in metastatic breast cancer, and its H3K4 methyltransferase activity promotes metastasis in a palmitic acid diet setting. We identify and characterize FCD-11, a first-in-class small molecule inhibitor of COMPASS activity designed to disrupt the termolecular interface between COMPASS SET domains, ASH2L, and RBBP5. FCD-11 significantly inhibited the H3K4me3 methyltransferase activity of SET1A/COMPASS and MLL1/COMPASS in vitro. ChIP-seq and CETSA indicated that FCD-11 selectively inhibits SET1A/COMPASS activity in mouse embryonic stem cells and breast cancer cell lines. FCD-11 significantly reduced tumor size and extended survival in mouse models of breast cancer. Our findings establish FCD-11 as a potent, COMPASS-specific inhibitor lead compound with preclinical efficacy in breast cancer models and therapeutic potential for cancers with abnormal dependence on SET1A/COMPASS activity.

L-2-Hydroxyglutarate (L-2-HG) is a low-abundance metabolite in mammals because the mitochondrial enzyme L-2-HG dehydrogenase (L2HGDH) oxidizes L-2-HG to 2-oxoglutarate (2-OG) to prevent its accumulation1. In humans, a lack of L2HGDH activity leads to L-2-HG accumulation and causes L-2-hydroxyglutaric aciduria2. Thus, L-2-HG is often classified as a toxic metabolite2-5. However, whether L-2-HG has any physiological function is unclear. Here we investigate whether L-2-HG qualifies as a physiological signalling metabolite by testing three criteria: regulated levels, defined molecular targets and a measurable physiological function. We report that an increase in mitochondrial NADH/NAD+ ratio drives malate dehydrogenase 2 (MDH2) to reduce 2-OG into L-2-HG. Moreover, L2HGDH oxidizes L-2-HG back to 2-OG in the mitochondrial matrix without requiring a functional electron transport chain. Through proteome integral solubility alteration assays, we show that the KDM4 family of H3K9 demethylases are L-2-HG-responsive targets. L-2-HG represses the nascent transcription of specific genes in mouse embryonic stem cells and increases H3K9me3 (a repressive histone mark) at these loci. In vivo, early embryonic L2HGDH overexpression in mice systemically reduces L-2-HG levels, impairs postnatal growth, causes mortality and produces selective functional and histological renal vulnerabilities. In postnatal kidneys, this reduction in L-2-HG causes H3K9me3 loss at L1MdTf retrotransposons and their derepression, which coincides with the activation of the integrated stress response and inflammation pathways. Our findings establish mitochondrial L-2-HG as a physiological signalling metabolite and indicate that metabolites previously regarded as toxic may also have crucial physiological functions.

Abstract Lineage-restricted transcriptional programs establish cell identity and can create selective dependencies in cancer. Here, we identify POU2AF2, encoding the transcriptional co-activator OCA-T1, as a critical lineage-specific dependency in a subset of diffuse large B-cell lymphoma (DLBCL). Pan-cancer dependency analyses and patient cohorts reveal elevated POU2AF2 expression in genetically aggressive DLBCL, where its depletion markedly suppresses tumor growth in vitro and in vivo . Mechanistically, POU2AF2 cooperates with the B-cell lineage– defining transcription factor POU2F2 (OCT2) to activate lymphocyte activation gene programs through direct chromatin engagement, thereby sustaining malignant transcriptional networks. We further identified a key epigenetic regulatory axis composed of the lineage-specific transcription factor TCF3 and the histone methyltransferase SET1A-COMPASS that drives POU2AF2 expression downstream of B-cell receptor signaling. Single-cell transcriptomic analysis reveals that POU2AF2 marks and sustains an innate-like B1 B-cell population in vivo , a candidate cell of origin for lymphoma. Together, these findings define a lineage-restricted POU2AF2/POU2F2 transcriptional module, controlled by a TCF3/SET1A epigenetic network, that sustains both innate-like B-cell identity and malignant fitness in DLBCL. Our study uncovers a previously unrecognized lineage-specific transcriptional dependency and highlights POU2AF2 and its associated regulatory circuitry as potential therapeutic targets in aggressive B-cell malignancies.

Transcription by RNA polymerase II (RNAPII), which is essential for protein-coding gene expression and cellular function, is increasingly understood to become dysregulated with aging. Here, we use a multimodal approach to comprehensively characterize age-dependent changes in RNAPII-mediated transcription in both mouse and human tissues. Short-read total RNA sequencing (RNA-seq) to profile nascent transcription reveals a global reduction in overall transcriptional activity/frequency in aged tissues, without apparent change in elongation rates. Transcriptomic analysis reveals a shift toward preferential expression of short genes in aged tissues, with notable upregulation of short stress-response genes and downregulation of long neurodevelopmental genes in the aged mouse brain. These results are recapitulated by analysis of total RNA-seq data from human tissues. Leveraging long-read RNA-seq, we determine that the representation of aberrant mono-exonic and intron-retention splice isoforms is increased in the aged mouse brain. Finally, we characterize the composition of RNAPII transcriptional machinery, finding that interactions between RNAPII and the Mediator complex are decreased in the chromatin of aged mouse liver and brain. Collectively, these analyses provide insight for future aging studies and reveal potential transcriptional control targets for anti-aging drug development.

The P-TEFb transcriptional kinase complex regulates the pause release checkpoint step in transcription by RNA polymerase II (RNAPII). We sought to identify hypoxia-specific interactions that could direct P-TEFb activity to hypoxia-responsive genes. Using a biochemical purification approach, we discovered a hypoxia-specific, chromatin-associated interaction between the P-TEFb subunit cyclin T1 (CCNT1), nuclear localized mitochondrial chaperone Tim8-Tim13 complexes, and the hypoxia-inducible, DNA binding transcription factor BHLHE40. This interaction is confirmed across multiple human cell lines. Tim8-Tim13 complex disruption and BHLHE40 silencing both impair the transcriptional response to acute hypoxia. HIF is not involved in the CCNT1/BHLHE40/Tim8-Tim13 interaction, and neither genetic HIF-1β knockout nor pharmacological HIF-2α inhibition (belzutifan) eliminates BHLHE40 expression. Finally, BHLHE40 depletion compromises the proliferation of 786-O clear cell renal carcinoma cells, which constitutively express HIF-2α and hypoxia-responsive genes. Together, these findings reveal a partially HIF-independent regulatory axis, in which Tim8-Tim13 complexes and BHLHE40 modulate P-TEFb activity in the transcriptional response to hypoxia.

Metabolic dysfunction-associated fatty liver disease (MASLD) has emerged as a major global health concern, with prevalence rising alarmingly in the pediatric population. Maternal nutrition and maternal body mass index (BMI) influence the propensity of the offspring to develop MASLD during childhood and adulthood, but the mechanisms underlying this maternal inheritance are unknown. Here, we have explored the developmental origins of MASLD, focusing on the effect of maternal nutrition and its epigenetic consequences in offspring. Using a mouse model, we demonstrate that maternal consumption of a Palmitic Acid-enriched High-Fat and high-sugar Diet (PA-HFD) during pregnancy and lactation induces stable epigenetic modifications, particularly related to H3K4me3 deposition, in the liver of male offspring through the action of the histone methyltransferase SET1a/COMPASS. This imprinting establishes a persistent transcriptional reprogramming, notably an enrichment in genes governing fatty acid β-oxidation, generating a fasted hepatocyte-like phenotype. Integrated transcriptomic, proteomic and metabolomic analyses reveal that upon re-exposure to a HFD in adulthood, this epigenetically primed hepatic state drives exaggerated metabolic responses, including lipidome reprogramming, leading to exacerbated hepatic steatosis, peroxisomal hyperactivation, oxidative stress, toxic lipid accumulation, and impaired glucose tolerance. Importantly, these gene expression and lipidome changes predict the severity of metabolic liver disease later in life, and can be recapitulated in human pluripotent stem cell-derived hepatocytes when Plamitic Acid is exposed in developmental stage. Moreover, genetic or pharmacological inhibition of SETD1A/COMPASS eliminates the priming marks, restores hepatic transcriptomic programs, and reduces circulating insulin levels and hepatic steatosis in vivo. Together, our findings identify a mechanistic epigenetic link between maternal nutrition and lifelong MASLD susceptibility and uncover a therapeutic strategy to erase the intergenerational transmission of MASLD predisposition.

Epigenetic status-altering mutations in chromatin-modifying enzymes are a feature of human diseases, including many cancers. We investigated cellular dependencies, or vulnerabilities, that arise when enhancer function is compromised by loss of the frequently mutated COMPASS family members MLL3 (KMT2C) and MLL4 (KMT2D). We performed a CRISPR dropout screen that revealed a targetable metabolic dependency arising from epigenetic factor deficiency, identifying potential therapies for cancers with epigenetic alterations secondary to MLL3/4-COMPASS dysfunction. We identified strong evidence that there is an internal balance between promoter and enhancer usage dictated by MLL1- versus MLL4-COMPASS, and this equilibrium is subject to disruption during cancer. Using an autochthonous carcinogen model of bladder cancer, we demonstrate that truncated, cytoplasmic MLL4 predicts response to targeted metabolic inhibition therapy for bladder cancer and could be developed as a biomarker for KMT2D-mutated cancers. We also highlight the broader potential for prognosis, patient stratification and treatment decision-making based on KMT2D mutation status in MLL4 truncation-relevant diseases, including human cancers and Kabuki Syndrome. Due to the high prevalence of loss-of-function mutations affecting MLL4 or its COMPASS partner UTX (KDM6A) in bladder cancer, we hypothesized that KMT2D mutation status may predict responsiveness to purine-targeting therapy, including methotrexate and pemetrexed. Our study found that KMT2D mutant bladder cancer cells were selectively dependent on TYMS and showed heightened sensitivity to pemetrexed, a drug that inhibits TYMS, DHFR, and GART. This suggests that targeting multiple enzymes within the one-carbon metabolism and de novo purine synthesis pathway could be promising for the treatment of MLL4/UTX-COMPASS mutant bladder cancer. Based on our previous and current findings, a clinical trial using pemetrexed in MLL4/UTX-COMPASS mutant solid tumors is being launched at Northwestern Medicine. Zibo Zhao, Sarah Gold, Yuki Aoi, Khyati A. Meghani, Luke St John, Rukkia Liaqat, Carolyn Moloney, Yanni Yu, Jun Qian, Issam Ben-Sahra, Rintaro Hashizume, Devalingam Mahalingam, Joshua Meeks, Ali Shilatifard. MLL4/COMPASS dysfunction in cancer and treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4103.

The stability of RNA polymerase II (Pol II) is tightly regulated during transcriptional elongation for proper control of gene expression. Our recent studies revealed that promoter-proximal Pol II is destabilized via the ubiquitin E3 ligase cullin 3 (CUL3) upon loss of transcription elongation factor SPT5. Here, we investigate how CUL3 recognizes chromatin-bound Pol II as a substrate. Using an unbiased proteomic screening approach, we identify armadillo repeat-containing 5 (ARMC5) as a CUL3 adaptor required for VCP/p97-dependent degradation of SPT5-depleted, chromatin-bound Pol II. Genome-wide analyses indicate that ARMC5 targets promoter-proximal Pol II in a BTB domain-dependent manner. Further biochemical analysis demonstrates that interaction between ARMC5 and Pol II requires the transcriptional cyclin-dependent kinase 9 (CDK9), supporting a phospho-dependent degradation model. We propose that defective, promoter-proximal Pol II that lacks SPT5 is rapidly eliminated from chromatin in a noncanonical early termination pathway that requires CDK9-dependent interaction with the CUL3-ARMC5 ubiquitin ligase complex.

We previously established distinct roles for the transcriptional elongation factors PAF1, negative elongation factor (NELF), SPT4/5, and SPT6 using auxin-inducible degron systems in human cell lines. Here, we integrate long-and short-read RNA-seq data from these degron lines to quantify transcript isoform usage at single-molecule resolution, identifying elongation factor-specific RNA processing regulons, including a cellular senescence-enriched regulon impacted by NELF and SPT6. Long-term NELF or SPT6 depletion causes reversible growth arrest following early upregulation of senescence-associated genes. Our genetic suppressor screens implicate the elongation factor Elongin A (ELOA) in these effects. ELOA knockout suppresses the progression of RNA polymerase II (RNAPII) past transcription end sites (TESs) at NELF depletion-induced genes. Acute depletion of TES-proximal ELOA causes a loss of RNAPII processivity at the 3 ' end of genes. ELOA loss also confers a growth advantage to aging primary human fibroblasts. These findings establish NELF/ELOA-dependent mechanisms regulating transcriptional elongation and RNA processing and link them to senescence and aging.

The mechanisms by which the expression of pluripotency and Polycomb networks are harmonized to allow the transition from pluripotency to a differentiated state have not been fully elucidated. Integrator complex regulates transcription pause release and RNA processing in metazoans. We show that Integrator is required for stemness and plays a critical role as early as day 2 in embryonic development. While the catalytic endonuclease activity enhances cellular reprogramming, Integrator recruits RNA polymerase II (RNAPII) to promoters and super enhancers of pluripotency and Polycomb genes. Integrator coordinates expression of pluripotency and Polycomb networks by fostering the association of RNAPII and basal transcription factors. We pinpoint a critical role for TATA-binding protein-associated factors (TAFs) in Integrator entry into the preinitiation complex. Taken together, beyond its role in RNAPII pause release, Integrator recruitment of RNAPII ensures an orderly cellular differentiation during development.

Monocyte-derived alveolar macrophages drive lung injury and fibrosis in murine models and are associated with pulmonary fibrosis in humans. Monocyte-derived alveolar macrophages have been suggested to develop a phenotype that promotes lung repair as injury resolves. We compared single-cell and cytokine profiling of the alveolar space in a cohort of 35 patients with post-acute sequelae of COVID-19 who had persistent respiratory symptoms and abnormalities on a computed tomography scan of the chest that subsequently improved or progressed. The abundance of monocyte-derived alveolar macrophages, their gene expression programs, and the level of the monocyte chemokine CCL2 in bronchoalveolar lavage fluid positively associated with the severity of radiographic fibrosis. Monocyte-derived alveolar macrophages from patients with resolving or progressive fibrosis expressed the same set of profibrotic genes. Our findings argue against a distinct reparative phenotype in monocyte-derived alveolar macrophages, highlighting their utility as a biomarker of failed lung repair and a potential target for therapy. Misharin, Sala and colleagues show that in patients with lung fibrosis after COVID-19, monocyte-derived alveolar macrophages activate an inflammatory and fibrotic program that was similar in patients with either resolving or progressing fibrosis.

TPS4621 Background: MLL4 (encoded by KMT2D) and UTX (encoded by KDM6A) are protein components of the epigenetic chromatin modifier complex COMPASS. MLL4 alterations are found in ~10% of all cancers including ~29% of bladder cancer (BLCA). UTX alterations are found in up to ~5% of all cancers including ~29% of BLCA. These alterations have not been previously therapeutically targeted as a precision oncology strategy in humans despite their frequency. We recently published the results of a CRISPR/Cas9 knockout screen in cells lacking MLL4/UTX-COMPASS function, which revealed synthetic lethality upon loss of genes that encode enzymes involved in de novo nucleotide synthesis (dnNS) [Zhao et al. J Clin Invest. 2023; Zhao et al. PNAS. 2023]. We also reported that MLL4 truncation mutations confer an inhibitor-targetable dependence on dnNS in colorectal cancer (CRC) and BLCA. We demonstrated sensitivity to lometrexol, which targets the enzyme GART (glycinamide ribonucleotide formyltransferase), in animal models of CRC and BLCA with MLL4 truncation. Our preclinical results clearly indicated the potential for dnNS inhibition as a targeted therapy for patients stratified by MLL4 or UTX status. Pemetrexed was identified as a more clinically relevant purine synthesis inhibitor for further development due to its well-established safety profile and prior use in BLCA. Methods: We have initiated an investigator-initiated, open-label phase II basket clinical trial at Northwestern University (NCT06630416). Patients with advanced, treatment-refractory tumors with MLL4 (KMT2D) or UTX (KDM6A) mutations (as identified by next generation sequencing) are enrolled in 2 cohorts: a) BLCA and b) other solid tumors. Other key inclusion criteria include ECOG performance status 0-2 and adequate organ function. Prior pemetrexed use is a key exclusion criterion. Patients are treated with pemetrexed 500mg/m2 IV Q 3 weeks. We intend to enroll up to 64 patients to allow for 58 evaluable patients (29 in each cohort) to achieve the null hypothesis. We will use a Simon 2-stage design, with 10 patients enrolled in each cohort in the first stage. The null hypothesis is that the true response rate is 0.1, and the alternative hypothesis is that the true response rate is 0.3. If there are 5 or more responses among these 29 patients, we reject the null hypothesis and claim that the treatment is promising. The design controls the type I error rate at 0.05 and yields a power of 0.8. This clinical trial has accrued 1 patient as of January 28th, 2025. Correlative studies will be carried out alongside the study to assess for mechanisms of resistance to pemetrexed. Molecular analysis of ctDNA will be performed on plasma for both arms and for plasma and urine for cohort A (bladder cohort) at pre-determined time points during treatment. Clinical trial information: NCT06630416 .

Cleverly exploiting the natural genetic variation among cells from unrelated individuals enables high throughput, highly parallel droplet single-cell RNA sequencing. We sought to lift this principle and bring analogous improvements to the setting of single-molecule long read sequencing. Toward this end, we present an error-robust method for matching individual long genomic reads to their genetic background based on a Naive Bayes Classifier. As a case study, we apply this to explore allele-specific splicing variation in the colorectal adenocarcinoma cell line DLD1 using Iso-Seq long-read RNA sequencing. Surprisingly, we uncover many allele differentially-expressed isoforms even across genes with no allele-specific expression bias in aggregate. Further, we investigate RNA-binding proteins and functional pathways potentially involved in mediating this allele-specific isoform expression pattern. In future work, our method will be applied to pooled single-molecule epigenomic screens of cancer cell line responses to treatment. Deniz Guney Olgun, Simai Wang, Saeid Parast, Ali Shilatifard, Hani Goodarzi, Vijay Ramani. Barcode-free demultiplexing of HiFi reads using natural genetic variation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5035.

Diffuse midline glioma (DMG) K27-altered is one of the most aggressive pediatric cancers. The identification of effective therapies has been extremely challenging. Over 250 clinical trials involving different combinations of chemotherapy and radiation have been unsuccessful in DMG patients, highlighting the need to develop new therapeutic strategies based on molecular characteristics to improve treatment outcomes. We recently showed that targeting BET domain protein 4 (BRD4) activity using small-molecule inhibitors results in delayed tumor progression and extended survival of mice bearing K27M DMG patient-derived xenografts (PDX). However, tumors that initially respond to small-molecule inhibitors, such as those targeting BRD4 activity, eventually show resistance to monotherapy treatments. To find new therapeutic targets and discover novel combinatorial approaches to prevent or delay acquired resistance to BRD4 monotherapy, we performed an unbiased genome-wide CRISPR/Cas9-based genetic screening of patient-derived DMG cells. We found that DMG cell maintenance depends on a set of downstream effectors including POLR2I, which encodes a subunit of RNA Polymerase II (Pol II), in addition to cyclin-dependent kinase 9 (CDK9), a key component of the super elongation complex involved in transcriptional elongation. We observed that targeting POLR2I and CDK9 activity using short-hairpin RNA knockdown and small-molecule inhibitors blocks Pol II transcriptional elongation and inhibits the growth of DMG in vitro and in vivo. We hypothesized that dual inhibition of BRD4 and CDK9 activity will further suppress gene transcription at levels of both transcriptional initiation and elongation and will either delay or prevent DMG from acquiring resistance to monotherapies. DMG cells treated with combined BRD4 and CDK9 inhibitors showed time-dependent growth inhibition and increased apoptosis, outperforming each monotherapy. Combination therapy in mice bearing DMG PDX also inhibited tumor growth and extended survival. This study has shown promising results demonstrating that this combined therapy is a potentially effective strategy for treating DMG.

Background:Diffuse midline gliomas, H3 K27-altered (DMG), are rare CNS WHO grade 4 tumors characterized by the global loss of K27me3 on histone H3. DMGs cannot be safely resected and are associated with poor outcomes in children, though relatively few DMGs have undergone extensive molecular and clinical profiling. Herein, we describe the clinical and molecular profiles of 36 pediatric DMGs as they relate to patient outcomes. Methods:Pediatric patients (<18 y/o) between 2015 and 2024 with biopsy-proven DMGs and next-generation sequencing (NGS) panels of 86 hotspot genes were reviewed for clinicopathologic characteristics and survival outcomes. Gene Ontology (GO) enrichment analysis was performed. Progression-free survival (PFS) and overall survival (OS) were calculated according to the Kaplan-Meier method. Multivariate Cox regression analysis was performed. Results:Thirty-six patients were included (median age = 9 y/o). Patients <10 y/o at diagnosis (n = 24) progressed significantly earlier and experienced significantly greater mortality than patients ≥10 y/o at diagnosis (n = 11); PFS and OS at 12 months were 9.0% and 26% for <10 y/o and 36% and 64% for ≥10 y/o (PFS: P ≤ .03, OS: P < .03). NGS findings revealed PIK3CA mutations occurred only in patients <10 y/o (10/25), and GO analysis revealed patients <10 y/o were significantly more enriched for PI3K/AKT signaling pathway alterations than patients ≥10 y/o (P ≤ .03). Conclusion:We present findings that suggest "adolescent" DMGs carry more favorable prognosis and are molecularly distinct from earlier onset pediatric DMGs. These findings have implications in the design and interpretation of clinical trials, in addition to informing clinical practice.

The testis-specific BET protein BRDT structurally resembles the ubiquitous BRD4 and is misexpressed in cancer, and we show that BRDT misexpression may affect lung cancer progression. BRDT knockdown in lung cancer cells slowed tumor growth and prolonged survival in a xenograft model. Comparative characterization of PTEFb complex participation and chromatin binding indicates BRD4-redundant and BRD4-distinct BRDT functions. Unlike dual depletion, individual BRD4 or BRDT knockdown did not impair transcriptional responses to hypoxia in BRDT-expressing cells, consistent with redundant function. However, BRD4 depletion/BRDT complementation revealed that BRDT can also release paused RNA polymerase II independently of its bromodomains as we previously demonstrated not to be required for Pol II pause/release function of BRD4, underscoring the functional importance of the C-terminal domains in both BRD4 and BRDT and their potential as therapeutic targets in solid tumors. Based on this study, future investigations should explore BRD4-distinct BRDT functions and BRDT misexpression driving cancer pathogenesis.