Citations

ABSTRACT Intronic GGGGCC hexanucleotide repeat expansions in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Despite its intronic location, this repeat avidly supports synthesis of pathogenic dipeptide repeat (DPR) proteins via repeat-associated non-AUG (RAN) translation. However, the template RNA species that undergoes RAN translation endogenously remains unclear. Using long-read based 5’ RNA ligase-mediated rapid amplification of cDNA ends (5’ Repeat-RLM-RACE), we identified novel C9orf72 transcripts initiating within intron 1 in a C9BAC mouse model, patient-derived iNeurons, and iNeuron-derived polysomes. These cryptic m 7 G-capped mRNAs are at least partially polyadenylated and are more abundant than transcripts derived from intron retention or circular intron lariats. In RAN translation reporter assays, novel intronic template transcripts – even those with short (32 nucleotide) leaders – exhibited robust expression compared to exon-intron and repeat-containing lariat reporters. To assess endogenous lariat repeat RNA contributions to RAN translation, we enhanced endogenous lariat stability by knocking down the lariat debranching enzyme Dbr1. However, this modulation did not impact DPR production in patient-derived iNeurons. These findings identify cryptic, linear, m 7 G-capped intronic-initiating C9orf72 mRNAs as an endogenous template for RAN translation and DPR production, with implications for disease pathogenesis and therapeutic development. SIGNIFICANCE STATEMENT An intronic GGGGCC repeat expansion in C9orf72 supports an unusual translational initiation process known as repeat-associated non-AUG (RAN) translation to produce toxic dipeptide repeat (DPR) proteins that contribute to neurodegeneration in ALS and FTD. How an intronic repeat RNA engages with ribosomes to support such translation is unclear. Here we identify a series of novel mRNA transcripts that initiate within the repeat-containing intron to create linear m 7 G-capped templates for RAN translation from GGGGCC repeats. These cryptic mRNAs are present in patient iNeurons, engage with ribosomes, and robustly support RAN translation. This finding has important implications for both our understanding of the mechanism by which RAN translation occurs and on therapeutic development in this currently untreatable class of neurodegenerative disorders.

Abstract O.A. Shariq: None. K.E. Lines: None. K.A. English: None. I. Rioja: Employee; Self; GlaxoSmithKline. A. Selberherr: None. H. Ferry: None. A.L. Oberg: None. R. Prinjha: Employee; Self; GlaxoSmithKline. D. Richards: None. R.V. Thakker: None. Background: Effective treatments for metastatic pancreatic neuroendocrine tumors (PNETs) are scarce. We previously showed that inhibiting bromodomain and extra-terminal (BET) epigenetic reader proteins via non-selective targeting of their two bromodomains (BD1 and BD2) reduces proliferation in preclinical PNET models. However, the clinical relevance of BET proteins in PNETs and functional differences between BD1/BD2 are unclear. Here, we characterised BET expression in human PNETs and performed efficacy and mechanistic studies using next-generation BD1- (BD1i) and BD2-selective (BD2i) BET inhibitors. Methods: BET expression was evaluated by immunohistochemistry in functioning and non-functioning human PNETs (n=52) vs normal adjacent islets (n=18). RNA sequencing (RNA-seq) and gene set enrichment analyses (GSEA) were performed in an independent PNET cohort (n=46) to identify biological pathways upregulated in metastatic vs non-metastatic tumors. Effects of BD1i and BD2i on viability, apoptosis, and cell cycle progression were assessed in three PNET cell lines (BON1, QGP1, MIN6). Early (6h) and late (48h) transcriptional responses to BET inhibition were examined in vitro using nascent RNA-seq (SLAM-seq) and standard RNA-seq, respectively. Epigenomic responses were explored using chromatin immunoprecipitation sequencing (ChIP-seq) for the histone mark H3K27ac. Effects of BD1i and BD2i on viability were evaluated in ex vivo neoplastic pancreatic islets derived from Men1L/L/RIP2-Cre mice. Results: BET proteins (BRD2, BRD3 and BRD4) were overexpressed by 1.5-2.5-fold in human PNETs vs islets (p<0.01). GSEA showed enrichment of BET-regulated gene sets in metastatic tumors (normalized enrichment score 1.31, adjusted p=0.04). BD2i marginally affected cell viability (IC50 55-90 µM), apoptosis, and cell cycle profile, while BD1i had a moderate impact on all phenotypes (IC50 1-1.5 µM). Synergistic effects were observed when BD1i+BD2i were combined (IC50 0.2-0.5 µM). RNA-seq and SLAM-seq revealed transcriptional changes that were minimal with BD2i (n=73 and n=10 differentially expressed genes, respectively), modest with BD1i (n=354 and n=60 genes), and greatest with BD1i+BD2i combined (n=1739 and n=375 genes). ChIP-seq showed a reduction in H3K27ac enrichment at promoters and super-enhancers of genes involved in BET protein-regulated pathways (e.g. cell cycle progression), with the greatest decrease observed with BD1i+BD2i combined. In ex vivo cultured neoplastic islets, viability was reduced by <30% with BD2i (p<0.005), ∼40% with BD1i (p<0.0001), and ∼60% with BD1i+BD2i (p<0.0001). Conclusions: Our studies confirm BET proteins as clinically-relevant targets in human PNETs and show that dual (BD1+BD2) inhibition has synergistic efficacy. These findings have important implications for the clinical application of next-generation epigenetic modulators. Sunday, June 2, 2024

OBJECTIVE Despite the known negative physiological impact of repeated mild head trauma events, their multiplicative impact on long-term seizure risk remains unclear. The objective of this study was to evaluate how multiple mild traumatic brain injuries (mTBIs) impact long-term seizure risk by testing 3 distinct machine learning approaches. Baseline and injury-specific characteristics were incorporated to enhance prognostication of individual seizure risk. METHODS Children with at least 1 mTBI event without prior evidence of seizure or antiepileptic drug treatment, from 2003 to 2021, were identified from a nationally sourced administrative claims database. The primary outcome of interest was a seizure event after mTBI, defined by qualifying principal diagnosis codes. Time-varying multivariable Cox regression was used to assess the impact of repeated mTBI. RESULTS A total of 156,118 children (mean age 11.7 ± 4.7 years) were included, with a median follow-up duration of 22.6 months (IQR 9.2–45.4 months). Among patients who experienced seizure after mTBI, the median time to seizure was 306 days. Seizures among those with radiographic findings and/or loss of consciousness occurred earlier (median time to seizure 112.5 days [imaging findings only, IQR 5–526.25 days], 80 days [loss of consciousness only, IQR 7–652 days], 22 days [both, IQR 5–192 days]). Both mTBI without and with short-term loss of consciousness resulted in increasing seizure risk with repeated trauma (HR 1.196, 95% CI 1.082–1.322; HR 2.025, 95% CI 1.828–2.244; respectively). The random survival forest approach achieved fixed-time areas under the receiver operating characteristic curve of 0.780 and 0.777 at 30 and 90 days after mTBI, and children predicted at high risk by the final model experienced a significantly higher burden of early seizure after mTBI (46.7% within the first 30 days vs 17.7% and 19.9% of children at low and medium risk). A simplified model using the top 12 contributing features achieved 95% of the full model’s performance in the validation set. CONCLUSIONS A novel machine learning model was developed and validated for personalized prediction of long-term seizure risk following multiple mTBIs. Model performance remained robust with a limited feature set, suggesting the feasibility of real-time incorporation into clinical workflows for individualized prognostication following each repeat mTBI event. In children predicted to be at high risk, early intervention should be considered.

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths in the U.S. mainly because of late-stage diagnosis. Heritable genetic information can improve risk stratification for individuals with moderate to high-risk of PDAC for targeted screening and early detection. We aimed to develop an enhanced polygenic risk score (PRS) for PDAC using a Bayesian approach, LDpred2, that reweights risk estimates attributed to millions of polymorphisms (SNPs) estimated from genome-wide association studies (GWASs). We then compared the performance of LDpred2 with the commonly used genetic risk score (GRS) approach that is based only on genome-wide significant SNPs. Methods: GWAS summary statistics were obtained from the Pancreatic Cancer Cohort Consortium and the Pancreatic Cancer Case Control Consortium for 9040 cases and 12496 controls and were used to compute weights for a GRS (17 SNPs) using a p-value threshold of 5e-8 and weights for an enhanced PRS (1.3 million SNPs) using LDpred2. These weights were then used to compute risk scores in an independent validation sample of 2616 cases and 45407 controls from the Mayo Clinic. We used logistic regression to test for associations of the GRS and PRS with PDAC risk, adjusting for age, sex, and the first 10 principal components of ancestry. Results: The validation sample was of mostly European ancestries (>95%). Cases were slightly older than controls (median age: 66 yrs. vs. 63 yrs.) and had a higher frequency of men than controls (57% vs. 42%). Comparisons between the GRS and the PRS are shown in Table 1. DeLong’s test comparing the AUCs between the two genetic scores showed the PRS significantly outperformed the GRS (AUC = 0.68 vs. 0.67; p = 7.6e-7). Conclusions: The LDpred2 PRS model that used a larger set of SNPs significantly improved prediction of PDAC. Comparing the top percentile of the PRS versus the rest showed that the PRS is a clinically significant risk score (OR > 3) for PDAC and could inform clinical decision making and genetic counseling of individuals identified as having a high risk of PDAC. Citation Format: Samuel O. Antwi, Brandon J. Coombes, Nicholas B. Larson, Erin E. Carlson, Kari G. Rabe, Hunter J. Atkinson, Shounak Majumder, Jun Zhong, David McKean, Pancreatic Cancer Cohort & Case Control Consortia (PanScan and PanC4), Brian M. Wolpin, Rachael Z. Stolzenberg-Solomon, Alison P. Klein, Laufey T. Amundadottir, Ann L. Oberg. Enhanced polygenic risk score for pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB331.

AbstractINTRODUCTIONHigher male prevalence in sporadic behavioral variant frontotemporal dementia (bvFTD) has been reported. We hypothesized differences in phenotypes between genetic and sporadic bvFTD females resulting in underdiagnosis of sporadic bvFTD females.METHODSWe included genetic and sporadic bvFTD patients from two multicenter cohorts. We compared behavioral and cognitive symptoms, and gray matter volumes, between genetic and sporadic cases in each sex.RESULTSFemales with sporadic bvFTD showed worse compulsive behavior (p = 0.026) and language impairments (p = 0.024) compared to females with genetic bvFTD (n = 152). Genetic bvFTD females had smaller gray matter volumes than sporadic bvFTD females, particularly in the parietal lobe.DISCUSSIONFemales with sporadic bvFTD exhibit a distinct clinical phenotype compared to females with genetic bvFTD. This difference may explain the discrepancy in prevalence between genetic and sporadic cases, as some females without genetic mutations may be misdiagnosed due to atypical bvFTD symptom presentation.Highlights Sex ratio is equal in genetic behavioral variant of frontotemporal dementia (bvFTD), whereas more males are present in sporadic bvFTD. Distinct neuropsychiatric phenotypes exist between sporadic and genetic bvFTD in females. Phenotype might explain the sex ratio difference between sporadic and genetic cases.

PURPOSE Sertoli-Leydig cell tumors (SLCTs) are rare sex cord-stromal tumors, representing <0.5% of all ovarian tumors. We analyze the role of germline DICER1 status in outcomes of ovarian SLCT. METHODS Patients with SLCT were enrolled in the International Pleuropulmonary Blastoma/ DICER1 Registry and/or the International Ovarian and Testicular Stromal Tumor Registry. Medical records were systematically abstracted, and those with known germline DICER1 status were selected for analysis. RESULTS Of 162 patients with SLCT, 60% had a germline DICER1 pathogenic or likely pathogenic (P/LP) variant. The adjusted 3-year recurrence-free survival (RFS) was 87.2% (95% CI, 79.4 to 95.8) for patients with a germline DICER1 P/LP variant compared with 78.1% (95% CI, 66.4 to 91.9) for those without a germline DICER1 P/LP variant ( P = .043). The adjusted 3-year and 5-year overall survival (OS) was 93.9% (95% CI, 87.3 to 100.0) for those with a germline DICER1 P/LP variant compared with the 3-year OS of 91.3% (95% CI, 83.4 to 100.0) and the 5-year OS of 78.2% (95% CI, 63.8 to 95.9) for those without a germline DICER1 P/LP variant ( P = .021). Among patients with a germline DICER1 P/LP variant, the risk of a subsequent, nonrecurrent event was 36.2% (95% CI, 21.4 to 48.1) within 10 years. Previous/concurrent and subsequent neoplasms were rare among those without a germline DICER1 P/LP variant. CONCLUSION This cohort study of patients with SLCT demonstrated that those with germline DICER1 P/LP variants had superior RFS and OS even when adjusting for other prognostic factors. Beyond prognostic implications of a germline DICER1 P/LP variant, germline testing helps identify patients at risk of subsequent neoplasms, including metachronous SLCT.

AbstractBackgroundOvarian juvenile granulosa cell tumors (juvGCT) are rare sex cord‐stromal tumors that occur primarily in children and adolescents. This study summarizes the clinical presentation and outcomes of patients with juvGCT.MethodsPatients were enrolled in the International Ovarian and Testicular Stromal Tumor and/or International Pleuropulmonary Blastoma/DICER1 Registries. Available medical records were abstracted, and pathology was centrally reviewed. Surgical staging was classified using the 2014 International Federation of Gynecology and Obstetrics (FIGO) criteria.ResultsIn total, 70 patients with juvGCT enrolled and were diagnosed between 2001 and 2024; most patients (81%, 57 of 70) presented with FIGO stage I disease. Adjuvant chemotherapy was given in 30% (21 of 70); all regimens were platinum‐based. Three‐year event‐free survival among patients with stage IA tumors was 80.2% (95% confidence interval [CI], 62.4%–100.0%), IC1 was 87.4 (95% CI, 72.4%–100.0%), IC2‐IC3 was 63.6% (95% CI, 40.7%–99.5%), and II‐IV was 48% (95% CI, 24.6%–93.8%). Of the patients with recurrent juvGCT with known mitotic index (MI), all had MI greater than 19 mitoses per 10 high power fields (HPF) at diagnosis.ConclusionOutcomes were worse for patients with FIGO stage ≥IC2 disease and for tumors with >19 mitoses per 10 HPF. Given the prognostic significance of MI, the authors strongly recommend the assessment of MI for all juvGCTs. More information about tumor biology is critical to identify which patients may benefit from adjuvant chemotherapy and to facilitate the development of novel therapies.

PURPOSE Chemotherapy-induced peripheral neuropathy is a potentially debilitating adverse effect of cancer therapy that can lead to delay, reduction, or discontinuation of cancer treatment. Population-based data on peripheral neuropathy incidence among young cancer survivors are lacking. METHODS Using a linkage between Medicaid, the California Cancer Registry, and hospitalization and emergency department data, we identified 6,028 adolescents and young adults (15-39 years) with Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), breast cancer, colorectal cancer, or testicular cancer and 418 children (<15 years) with HL or NHL during 2005-2017. We determined the cumulative incidence of peripheral neuropathy and its association with neurotoxic chemotherapy identified from Medicaid claims, using multivariable Cox proportional hazards regression models. RESULTS Of 6,446 patients, 1,007 were diagnosed with peripheral neuropathy. Across each cancer type, incidence was higher among patients receiving neurotoxic drugs. For example, compared with non-neurotoxic agents, 5-year cumulative incidence was higher with oxaliplatin for colorectal cancer (24.0% v 6.2%) and paclitaxel for breast cancer (22.6% v 5.1%). In multivariable analysis, the agents most strongly associated with peripheral neuropathy were brentuximab (±other neurotoxic drugs) for HL (hazard ratio [HR], 9.53 [95% CI, 5.95 to 15.26]); brentuximab (±vinca alkaloids; HR, 7.00 [95% CI, 4.13 to 11.87]) for NHL, paclitaxel for breast cancer (HR, 4.03 [95% CI, 3.05 to 5.31]); oxaliplatin for colorectal cancer (HR, 3.46 [95% CI, 2.23 to 5.36]); and cisplatin and etoposide for testicular cancer (HR, 2.06 [95% CI, 1.37 to 3.11]). CONCLUSION The high incidence of peripheral neuropathy highlights the need for frequent monitoring, new supportive care approaches, and development of novel therapeutic agents to minimize toxicity while maintaining treatment efficacy.

Background: Tumor immune microenvironment (TIME) organization fundamentally shapes cancer progression and immunotherapy outcomes. Traditional 2D spatial molecular approaches inadequately capture the complex three-dimensional architecture and cellular interactions within TIME. We developed an AI-driven platform integrating multiple spatial molecular datasets, enabling comprehensive 3D visualization for therapeutic discovery and patient selection. Methods: Our platform combines holotomography imaging, providing 3D refractive index maps, with diverse spatial molecular data: in situ molecular imaging, spatial proteomics, multiplex immunohistochemistry, and spatial transcriptomics. AI algorithms align datasets within a unified 3D coordinate system, while Poisson implicit surface reconstruction generates subcellular-resolution models. Platform validation used clinical samples from immunotherapy-treated patients. Results: The platform integrated multimodal spatial data into high-resolution 3D TIME representations, revealing novel relationships between protein expression, transcriptional patterns, and cellular architecture. 3D surface protein analysis with subcellular morphology identified patients with target protein expression outside tight junctions, indicating higher risk for adverse effects from antibody-drug conjugates and cellular therapies. This insight, unavailable through 2D analysis, enabled precise patient selection. Conclusions: This platform provides detailed subcellular-resolution TIME architecture analysis, offering actionable insights for therapeutic targeting, biomarker discovery, and patient selection. AI-driven 3D spatial multimodal TIME analysis advances precision medicine across multiple therapeutic approaches, including immunotherapy, targeted therapy, and drug delivery optimization. AI Disclosure: Original abstract content was written by the authors. Claude was used for editorial assistance and formatting without altering the scientific content or context of the work. Citation Format: Inyeop Jang, Yoonho Choi, Minji Kim, Jeonghwan Park, Jiyeon Sung, Byungsoo Ann, Clemenceau R. Jean, TaeHyun Hwang. AI-enabled 3D multimodal integration platform reveals subcellular architecture of tumor immune microenvironment for therapeutic target and biomarker discovery and patient selection [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 3648.

Temozolomide (TMZ) is the main chemotherapy for GBM, but resistance develops in most patients, highlighting the need for sensitizing strategies. PARP inhibitors (PARPi), which disrupt DNA repair, are being investigated as TMZ-sensitizing agents. Here we evaluated the efficacy and explored molecular mechanisms of pamiparib-mediated TMZ sensitization. In vitro, low concentrations of pamiparib (0.1-1.0 μM) synergized with TMZ and enhanced DNA damage, particularly during the S- and G2/M phases. In vivo, pamiparib monotherapy (0.75 or 3 mg/kg, twice daily) was ineffective, however, TMZ afforded survival benefit in orthotopic patient-derived xenograft (PDX) models (GBM12, GBM22, and GBM84) of MGMT-methylated primary GBM. Pamiparib combined with TMZ (25 or 50 mg/kg daily) prolonged survival in GBM12 [median survival 272 vs. 114 days with TMZ (P=0.02), 30 days with placebo (P&lt;0.001)] and GBM22 (median survival 88 vs. 60 days with TMZ (P=0.05), 26 days with placebo, (P&lt;0.001)] but not in GBM84 [median survival 355 vs. 404 days with TMZ (P=0.44), 37 days with placebo (P&lt;0.001)]. These findings align with prior studies indicating that PARPi can potentiate TMZ efficacy in a subset of primary GBM models sensitive to TMZ. TMZ induces cytotoxic DNA adducts, N7MeG, N3MeA, and O6MeG. While N7MeG and N3MeA are repaired by base-excision repair (BER), MGMT removes O6MeG. Suppressed MGMT expression, due to promoter methylation, drives TMZ sensitivity in ∼40% of primary GBM cases. In MGMT-methylated tumors, O6MeG-thymine mispairs trigger futile mismatch repair, causing replication stress. Although BER is PARP-dependent, BER inhibition did not replicate the TMZ-sensitizing effects of pamiparib. Beyond DNA repair, PARP regulates replication stress by recruiting translocases like SMARCAL1 and controlling endonucleases such as MRE11 and EXO1, protecting stressed forks and nascent DNA. We targeted these components using siRNA and small-molecule inhibitors to elucidate their roles in TMZ sensitization. Unexpectedly, silencing SMARCAL1 or MRE11, or inhibiting MRE11 with mirin, did not affect pamiparib-mediated sensitization in U251 cells. While EXO1 silencing attenuated DNA damage signaling, it had minimal impact on TMZ sensitivity or pamiparib-mediated sensitization. These results suggest that fork protection mechanisms may be epistatic to PARP but redundant for TMZ sensitivity. Alternatively, replication stress may be mitigated by lesion bypass via translesion synthesis (TLS), where replicative polymerases are replaced with low-fidelity polymerases. Consistent with this, silencing RAD18, involved in TLS, enhanced sensitivity to both TMZ and pamiparib, with the combination being more effective than either drug alone. In conclusion, pamiparib enhances TMZ efficacy in a subset of primary GBM. While further in vivo validation is needed, TLS deficiency may be a key determinant of pamiparib-mediated sensitization. Citation Format: Shiv K. Gupta, Ann C. Mladek, Sonia Jain, Katrina K. Bakken, Zeng HU, Brett L. Carlson, Danielle M. Burgenske, Jann N. Sarkaria. Pamiparib-induced replication stress augments the efficacy of temozolomide in GBM PDX models [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 2910.