Background: We previously published a literature based pipeline for sepsis gene prioritization (PS3 and candidate genes) using an LLM enabled retrieval and judging framework. Here, we extend that work to ask whether these prioritized genes show independent clinical validity and whether the same strategy generalizes to a "drug/obesity/infection" setting. Methods: Using the original LLM guided workflow, we evaluated PS3 and the Candidate set in two new settings. First, we tested 28 day mortality prediction in the independent VANISH sepsis trial, benchmarking PS3 and Candidate against two established immune signatures the Severe or Mild (SoM) signature and Immune Health Metric (IHM) under a uniform logistic regression framework with clinical covariates. Second, we applied the same genome wide screening and tiered judging pipeline to GLP 1/obesity/infection biology centered on semaglutide, comparing Tier 1 and Tier 2 gene sets to STEP trial serum proteomics at gene and Hallmark pathway levels. In parallel, we fine tuned an open weight GPT OSS 20B model on curated sepsis justifications to obtain a domain aware LLM as judge, and compared its scoring behavior with the base model on semaglutide Tier 2 genes. Results: In the full VANISH cohort, PS3 and the Candidate set showed moderate discrimination, whereas SoM remained the strongest single predictor of 28 day mortality. In the Critical/High APACHE II subgroup, PS3 achieved ROC and precision recall performance comparable to, or slightly better than, SoM despite its smaller, knowledge derived composition, indicating that literature prioritized genes capture mortality relevant immune dysregulation under severe illness. In the semaglutide case study, gene level overlap between LLM prioritized genes and differentially abundant serum proteins was modest, but Tier 1 genes recapitulated the main semaglutide responsive metabolic programs from STEP and highlighted additional immune metabolic pathways relevant to infection, with discordances largely explained by serum proteome coverage. The fine tuned judge remained moderately concordant with the base GPT OSS across mechanistic themes, preserving overall ranking while inducing systematic, biologically interpretable shifts in immune and infection related scores. Conclusions : An LLM guided, literature based gene prioritization framework yields compact gene sets that show independent sepsis mortality signal and pathway level concordance in a semaglutide/obesity/infection setting, while a sepsis aware LLM as judge provides domain specific refinements without overturning core rankings. Together, these findings support knowledge grounded, LLM derived gene sets and judges as interpretable components for probing immune dysregulation across diseases and therapies. ### Competing Interest Statement The authors have declared no competing interest.

Molecular mechanisms underlying sex-specific differences in cancer incidence and therapy responses are under intense investigation. Here, we report sex-biased functions of Yap1 in multiple cancer types in human and mouse. Through integrated multi-omics analyses, we demonstrate that Yap1 deletion significantly extends survival in male but not female Sonic Hedgehog (SHH) medulloblastomas (MB) models. While Yap1 is required to maintain stem-like cells in both sexes, Yap1 plays a more critical role in immune evasion in males. Mechanistically, YAP1 is essential for activating Cd276/B7-H3 expression to mediate CD8+ T cell suppression in males. Consistently, CD276 inhibition extends survival in male but not female SHH MB. Moreover, in vivo targets of YAP1 stratify survival in male but not female patients with medulloblastoma, glioblastoma, mesothelioma, and lung cancer. This study provides evidence for sex-biased functions of Yap1 and CD276 in MB immune suppression and highlights the importance of biological sex in cancer:immune interactions.

The molecular mechanisms driving sex disparities in cancer prevalence, progression, and treatment outcomes represent a crucial yet understudied area with significant implications for cancer therapy. Medulloblastoma (MB), exhibits a pronounced sex bias in incidence and survival rates, with males experiencing higher incidence and poorer prognoses compared to females. This study focused on the role of Yap1 in Sonic Hedgehog medulloblastomas (SHH MBs), which revealed an unanticipated sex-biased role of Yap1 in MB immune evasion. Hippo/Yap pathway is one of the top 10 most dysregulated pathways in human cancer. Our in vivo studies uncovered that Yap1 deletion in a spontaneous SHH MB model significantly extended survival, providing genetic evidence that Yap1 is a critical oncogene in MB formation. Notably, Yap1 deletion/inhibition benefitted males, but not females, revealing its sex-biased function for the first time. Integrated multi-omics analyses showed Yap1 is essential for maintaining MB stem/progenitor cells by activating stemness genes (Sox2) and repressing differentiation genes (Neurod1, Zic1/2) in both sexes. However, Yap1 plays a more pivotal role in immune evasion in males, particularly by regulating the immune checkpoint molecule; Cd276/B7-H3. In males, either CD276 blockade or Yap1 deletion reverses T cell suppression. In contrast, in females, CD276 blockade or Yap1 deletion is not sufficient to overcome T cell suppression, and it requires both interventions to activate T cells. Furthermore, our newly discovered transcriptional target gene signatures of YAP1 predict survival in male but not female patients across multiple human cancers, indicating a highly conserved mechanism across species and cancer types. This study highlights sex-specific differences in tumor:immune interactions downstream of YAP1/CD276. In addition, it reveals a novel sex-biased role of YAP1 transcriptional programs, providing molecular entry points for future research on sex differences in immune response. These results underscore the importance of considering sex as a major variable in mechanistic and therapeutic studies involving Yap1 and CD276, with broader implications for targeted cancer therapies. Nourhan Abdelfattah, Sivaraman Natarajan, Han Nhat Tran, Thomas Wong, Jose Maldonado, Rachael McMinimy, Hannah Borland, Shu-hsia Chen, Fernando Camargo, James Olson, Joshy George, Kyuson Yun. Yap1 and CD276/B7-H3: key players in Sex-biased SHH medulloblastoma immune evasion [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 6446.

BACKGROUND:Ovarian high-grade serous carcinomas (HGSC) comprise four distinct molecular subtypes based on mRNA expression patterns, with differential survival. Understanding risk factor associations is important to elucidate the etiology of HGSC. We investigated associations between different epidemiologic risk factors and HGSC molecular subtypes. METHODS:We pooled data from 11 case-control studies with epidemiologic and tumor gene expression data from custom NanoString CodeSets developed through a collaboration within the Ovarian Tumor Tissue Analysis consortium. The PrOTYPE-validated NanoString-based 55-gene classifier was used to assign HGSC gene expression subtypes. We examined associations between epidemiologic factors and HGSC subtypes in 2,070 cases and 16,633 controls using multivariable-adjusted polytomous regression models. RESULTS:Among the 2,070 HGSC cases, 556 (27%) were classified as C1.MES, 340 (16%) as C5.PRO, 538 (26%) as C2.IMM, and 636 (31%) as C4.DIF. The key factors, including oral contraceptive use, parity, breastfeeding, and family history of ovarian cancer, were similarly associated with all subtypes. Heterogeneity was observed for several factors. Former smoking [OR = 1.25; 95% confidence interval (CI) = 1.03, 1.51] and genital powder use (OR = 1.42; 95% CI = 1.08, 1.86) were uniquely associated with C2.IMM. History of endometriosis was associated with C5.PRO (OR = 1.46; 95% CI = 0.98, 2.16) and C4.DIF (OR = 1.27; 95% CI = 0.94, 1.71) only. Family history of breast cancer (OR = 1.44; 95% CI = 1.16, 1.78) and current smoking (OR = 1.40; 95% CI = 1.11, 1.76) were associated with C4.DIF only. CONCLUSIONS:This study observed heterogeneous associations of epidemiologic and modifiable factors with HGSC molecular subtypes. IMPACT:The different patterns of associations may provide key information about the etiology of the four subtypes.

This table shows the associations between epidemiologic factors and HGSC subtypes restricted to HGSC cases with probability of subtype assignment >80%.

Background/Objectives: Osteosarcoma is the most common malignant bone tumor in children, characterized by a high degree of genomic instability, resulting in copy number alterations and genomic rearrangements without disease-defining recurrent mutations. Clinical trials based on molecular characterization have failed to find new effective therapies or improve outcomes over the last 40 years. Methods: To better understand the immune microenvironment of osteosarcoma, we performed single-cell RNA sequencing on six tumor biopsy samples, combined with a previously published cohort of six samples. Additional osteosarcoma samples were profiled using spatial transcriptomics for the validation of discovered subtypes and to add spatial context. Results: Analysis revealed immunosuppressive cells, including myeloid-derived suppressor cells (MDSCs), regulatory and exhausted T cells, and LAMP3+ dendritic cells. Conclusions: Using cell-cell communication modeling, we identified robust interactions between MDSCs and other cells, leading to NF-κB upregulation and an immunosuppressive microenvironment, as well as interactions involving regulatory T cells and osteosarcoma cells that promoted tumor progression and a proangiogenic niche.

Background Molecular analysis of advanced tumors can increase tumor heterogeneity and selection bias. We developed a robust prognostic signature for gastric cancer by comparing RNA expression between very rare early gastric cancers invading only mucosal layer (mEGCs) with lymph node metastasis (Npos) and those without metastasis (Nneg). Methods Out of 1003 mEGCs, all Npos were matched to Nneg using propensity scores. Machine learning approach comparing Npos and Nneg was used to develop prognostic signature. The function and robustness of prognostic signature was validated using cell lines and external datasets. Results Extensive machine learning with cross-validation identified the prognostic classifier consisting of four overexpressed genes (HDAC5, NPM1, DTX3, and PPP3R1) and two downregulated genes (MED12 and TP53), and enabled us to develop the risk score predicting poor prognosis. Cell lines engineered to high-risk score showed increased invasion, migration, and resistance to 5-FU and Oxaliplatin but maintained sensitivity to an HDAC inhibitor. Mouse models after tail vein injection of cell lines with high-risk score revealed increased metastasis. In three external cohorts, our risk score was identified as the independent prognostic factor for overall and recurrence-free survival. Conclusion The risk score from the 6-gene classifier can successfully predict the prognosis of gastric cancer.

The molecular pathogenesis of diabetes is multifactorial, involving genetic predisposition and environmental factors that are not yet fully understood. However, pancreatic β-cell failure remains among the primary reasons underlying the progression of type-2 diabetes (T2D) making targeting β-cell dysfunction an attractive pathway for diabetes treatment. To identify genetic contributors to β-cell dysfunction, we investigated single-cell gene expression changes in β-cells from healthy (C57BL/6J) and diabetic (NZO/HlLtJ) mice fed with normal or high-fat, high-sugar diet (HFHS). Our study presents an innovative integration of the causal network perturbation assessment (ssNPA) framework with meta-cell transcriptome analysis to explore the genetic underpinnings of type-2 diabetes (T2D). By generating a reference causal network and in silico perturbation, we identified novel genes implicated in T2D and validated our candidates using the Knockout Mouse Phenotyping (KOMP) Project database.

Abstract Unveiling the molecular mechanisms underlying sex-specific differences in cancer initiation, progression and treatment outcomes will provide novel insights that will advance cancer research and clinical care. This study highlights an unexpected, sex-biased role of the oncogene Yap1 in SHH medulloblastoma (MB). We discovered that Yap1 deletion in SmoM2-driven SHH MB significantly prolongs survival in male but not female mice. Using an integrated multi-omics approach, we show that YAP1 promotes cancer stem cell maintenance through concurrent transcriptional activation of stemness genes, such as Sox2, and repression of differentiation genes such as NeuroD1 and Zic1/2. Interestingly, while Yap1 is essential for maintaining cancer stem cells in both sexes, it plays a more critical role in immune evasion in males. Specifically, YAP1 regulates the expression of an immune checkpoint molecule Cd276 to suppress T cell function. Blocking CD276 or deleting Yap1 is sufficient to significantly reverse T cell suppression in males but not females. Furthermore, YAP1 direct targets of transcriptional regulation, including CD276, predicts survival in male but not female patients across multiple human cancers, suggesting that our findings have broader implications beyond medulloblastomas and is conserved across species. This study provides compelling evidence for male-biased susceptibility to Yap1 inhibition and uncovers the YAP1-CD276 axis as a sexually diverse pathway in T cell suppression in tumors. Citation Format: Nourhan Abdelfattah, Sivaraman Natarajan, Han Nhat Tran, Jose Maldonado, Rachael McMinimy, Hannah Borland, Shu-hsia Chen, Fernando Camargo, James Olson, Joshy George, Kyuson Yun. Sex-specific differences in Yap1 and Cd276 function and expression regulate medulloblastoma progression and immune evasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3037.

Abstract The molecular mechanism governing sex disparities in cancer prevalence, progression, and treatment outcomes represents an important yet understudied area of research with significant implications for cancer treatment. Medulloblastoma (MB), the most prevalent pediatric brain malignancy, exhibits a notable sex bias in incidence and survival rates. Males have a higher incidence rate across all age groups and have worse prognoses than their females for age groups over three years old. Here, we report an unanticipated sex-biased role of the oncogene Yap1 in SHH medulloblastoma. Our results reveal that Yap1 deletion significantly prolongs survival in male, but not female mice with SmoM2-driven SHH MB. Employing an integrated multi-omics approach, we demonstrate that YAP1 is required to maintain cancer stem cells simultaneously activating stemness genes (such as Sox2) and repressing differentiation genes (such as Neurod1 and Zic1/2) in both sexes. In contrast, Yap1 plays a more critical role in immune evasion in males than in females. Specifically, YAP1 regulates the expression of the immune checkpoint molecule Cd276/B7-H3, which suppresses cytotoxic T cell function. In males, CD276 blockade or Yap1 deletion in MB cells is enough to reverse T cell suppression effectively. In females, neither CD276 blockade nor Yap1 deletion alone in MB cells can reverse T cell suppression. Furthermore, YAP1’s direct transcriptional target gene signatures, including CD276, predict survival across multiple human cancers in male patients. These findings suggest broader implications of our findings beyond medulloblastoma and indicate a highly conserved mechanism across species. Our study provides compelling evidence for the male-biased efficacy of Yap1 or CD276 inhibitors and presents novel insights into sex-specific mechanisms of cancer immune evasion.

Parvalbumin-positive interneurons (PV-INs) regulate neuronal and circuit activity, and their dysfunction is observed across neurological conditions, including traumatic brain injury (TBI), epilepsy, Alzheimer’s disease, and schizophrenia. PV-INs are particularly vulnerable to cell loss, potentially due to their increased metabolic demands arising from their uniquely high level of electrical activity, which render them susceptible to metabolic pressure. Here, we use single-nucleus RNA-sequencing (snRNAseq) data from a rodent model of TBI, as well as human TBI data, and demonstrate PV-INs have unique metabolic specializations that are lost after injury and can be rescued by in vivo treatment with the glycolytic inhibitor, 2-deoxyglucose. We generated a novel PV-IN transcriptional identity module comprised primarily of genes encoding specialized ion channels, metabolic enzymes, and synaptic machinery, that identifies heterogenous subsets of injury-associated PV-INs with loss of PV-IN transcriptional identity. We show that changes in metabolic specialization are coupled to changes in transcriptional identity in PV-INs and implicate the PV-IN-enriched transcriptional co-activator, Ppargc1a , as a key driver of PV-IN transcriptional metabolic dysfunction. We also identify a family of long non-coding RNAs enriched in this subset of transcriptionally dysfunctional PV-INs that negatively correlates with PV-IN metabolic specialization. Lastly, we utilize these tools to interrogate a published human TBI snRNAseq data set and find nearly identical changes, underscoring the importance of PV-IN metabolic dysfunction in the pathology of TBI. ### Competing Interest Statement The authors have declared no competing interest.

Immature oocytes enclosed in primordial follicles stored in female ovaries are under constant threat of DNA damage induced by endogenous and exogenous factors. Checkpoint kinase 2 (CHEK2) is a key mediator of the DNA damage response in all cells. Genetic studies have shown that CHEK2 and its downstream targets, p53 and TAp63, regulate primordial follicle elimination in response to DNA damage, however the mechanism leading to their demise is still poorly characterized. Single-cell and bulk RNA sequencing were used to determine the DNA damage response in wildtype and Chek2-deficient ovaries. A low but oocyte-lethal dose of ionizing radiation induces a DNA damage response in ovarian cells that is solely dependent on CHEK2. DNA damage activates multiple ovarian response pathways related to apoptosis, p53, interferon signaling, inflammation, cell adhesion, and intercellular communication. These pathways are differentially employed by different ovarian cell types, with oocytes disproportionately affected by radiation. Novel genes and pathways are induced by radiation specifically in oocytes, shedding light on their sensitivity to DNA damage, and implicating a coordinated response between oocytes and pre-granulosa cells within the follicle. These findings provide a foundation for future studies on the specific mechanisms regulating oocyte survival in the context of aging, as well as therapeutic and environmental genotoxic exposures.

<p>Supplementary Table 5 showing IPA prediction of upstream regulators for up-regulated genes in S100A4 knockdown cells</p>

<p>Supplementary figure legends for Supplementary Figures 1-3. In addition, supplementary Methods with detailed protocols and sequence information.</p>

Supplementary Methods - PDF file 70K, CDK2 sequencing methods and primer sequences

Supplementary Figures S1-S9 - PDF file 327K, Supplementary Figure S1: A) Baseline expression of CCNE1 and CDK2 in OVCAR-3 (CCNE1 amplified) and OVCAR-4 (CCNE1 gained) cell lines compared to SK-OV-3 (CCNE1 unamplified). *detects both isoforms of CDK2. B) CCNE1 and CDK2 gene expression by RT-PCR after gene knockdown normalized to no siRNA control cells within each cell line. Representative data shown. *detects both isoforms of CDK2. C) Cell viability normalized to no siRNA control cells after transfection with siRNAs against CCNE1 or CDK2. Statistical significance calculated by comparison to non-silencing (NS) siRNA in the same cell line using data from three independent MTS assays performed in triplicate. Average normalized absorbance at 490 nm and SEM plotted (n = 3), T-test *p-value <0.05, ***p-value <0.01 and ***p-value <0.001; Supplementary Figure S2: A) Relative abundance of shRNAs targeting CCNE1 and CDK2 in shRNA screen of 102 tumor cell lines and B) average normalized abundance of CCNE1 shRNA3 in tumor cell lines stratified by CCNE1 copy number status; Supplementary Figure S3: A) PHA-533533 and B) dinaciclib 72 hour MTS proliferation assay dose-response and average IC50 values. Curves show average absorbance normalized to untreated control cells. Error bars are SEM (n=3). T-test **p-value <0.01, ***p-value <0.001; Supplementary Figure S4: A) Clonogenic survival dose response to PHA-533533 in OVCAR-3 parental and R1 cell line. Mean of replicates and SEM from one representative experiment shown. B) MTS dose response to dinaciclib in OVCAR-3 parental and dinacicilib resistant (RD1) cell line. Mean of replicates and SEM shown. C) IC50 values for Dinaciclib-RD1 over time. Cells were cultured in the presence (solid line) or absence of dinaciclib (dashed line). The IC50 value for parental cells is indicated by the dotted line. D) Proliferation rate of parental and two resistant cell lines (533533-R1 and -R6); Supplementary Figure S5: A) Cycle profile of PI stained cells analyzed by flow cytometry after 48 hr treatment with PHA-533533; Supplementary Figure S6: A) Baseline protein expression in proliferating parental and PHA-533533 resistant cell lines (R1, R3, R5, R6, R7) by western blot. B) Gene expression of targets of the PHA-533533 small molecule inhibitor in parental compared to resistant cell lines (n = 4) after drug selection (selected) and subsequent passage in media (stable); Supplementary Figure S7: A) Cell cycle profile of PI stained cells analyzed by flow cytometry. PHA-53353 resistant cell lines (R3, R5 and R7) and B) dinaciclib resistant line (RD1) are comprised of a hyperpentaploid cell population. G1 and G2 peaks (BG1 and BG2) are labeled; Supplementary Figure S8: A) Proportion of hypotriploid and hyperpentaploid cells in the parental, 533533-R1 and -R6 resistant cell lines estimated by counting 50 metaphase karyotypes. Additional representative hypotriploid and hyperpentaploid karyotypes of B) OVCAR-3 parental, C) 533533-R1 (hyperpentaploid only) and D) 533533-R6 cells; Supplementary Figure S9: A) DNA ploidy estimated by ABSOLUTE in TCGA ovarian primary tumors stratified by CCNE1 log2 copy number ratio; >0.3 for gains and >0.8 for amplifications. Fraction of genome lost (log2 copy number ratio < -0.3) in ovarian tumor samples by B) number of predicted whole genome doublings and C) CCNE1 copy number status. T-test ***p-value < 0.001

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