Bulk RNA-seq Research Articles

Abstract IA011: Heterogeneity of cancer-derived extracellular vesicles

Abstract Extracellular vesicles (EVs) are heterogenous in size, biogenesis, cargo and function. Beside small EVs, aggressive tumor cells release a population of particularly large EVs, namely large oncosomes (LO). This study provides the first resource of label-free quantitative proteomics of LO and small EVs obtained from distinct cancer cell types (prostate, breast, and glioma). This dataset was integrated with a SWATH Proteomic assay on LO, rigorously isolated from the plasma of patients with metastatic prostate cancer (PC). Proteins enriched in LO, which were identified also at the RNA level, and found in plasma LO significantly correlated with PC progression. Single EV RNA-Seq of the PC cell-derived LO confirmed some of the main findings from the bulk RNA-Seq, providing first evidence that single cell technologies can be successfully applied to EVs. This multiomics resource of cancer EVs can be leveraged for developing a multi-analyte approach for liquid biopsy. Citation Format: Dolores Di Vizio. Heterogeneity of cancer-derived extracellular vesicles [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr IA011.

Abstract 4120623: Sphingosine Kinase 1 Is Integral For Elastin Deficiency-induced Arterial Hypermuscularization

Introduction: Defective elastic lamellae and smooth muscle cell (SMC) accumulation are characteristics of diverse obstructive arterial diseases (e.g., atherosclerosis, pulmonary hypertension, and supravalvular aortic stenosis [SVAS]) as well as physiological closure of the ductus arteriosus (DA). Mechanistic links between defective elastin and SMC proliferation are not well elucidated. Methods: Immunostaining for proliferation marker Ki67 was performed on wild-type (WT) and Eln (-/-) mouse aortas at embryonic day (E) 13.5 and E15.5 because elastin (ELN) is expressed in the mouse aorta from E14. Bulk RNA-seq was conducted on mouse aortic SMCs isolated from WT or Eln (-/-) embryos at E15.5. As sphingosine kinase 1 (SPHK1) was found as a highly promising candidate, its expression was evaluated in human SVAS patient aortas and in mouse Eln (-/-) aortas and WT DA. S1P receptor 1 (S1PR1) activity was assessed in aortic SMCs from S1pr1 (knock-in/knock-in) , H2B-GFP mice. Genetic deletion of Sphk1 in SMCs was performed on the elastin mutant background. Pharmacological SPHK1 inhibition was evaluated on both elastin mutants and WT embryos. Regulatory mechanisms of SPHK1 were assessed by mRNA stability assay and TRANSFAC database analysis. Results: SMC hyperproliferation was first observed in Eln (-/-) aorta at E15.5, prior to morphological differences. Bulk RNA-seq revealed that Sphk1 is the most upregulated transcript in Eln (-/-) aortic SMCs at E15.5. Reduced ELN increases SPHK1 levels in SMCs of human patient aortas and mouse aorta and DA. S1PR1 expression and activity are increased by elastin insufficiency. SMC Sphk1 deletion attenuates SMC proliferation and muscularization in the elastin-defective aorta, leading to extended viability of Eln (-/-) mice. Similarly, pharmacological SPHK1 inhibition ameliorates elastin aortopathy but leads to patent DA in WT mice. mRNA stability assay indicated Sphk1 is upregulated by enhanced transcription. TRANSFAC and bulk RNA-seq data suggested that transcription factor early growth response 1 (Egr1) induces Sphk1 transcription. Indeed, EGR1 was upregulated in elastin mutant aortas and DA. Conclusions: Elastin deficiency upregulates SPHK1 transcription, leading to SMC proliferation and hypermuscularization in elastin aortopathy as well as during physiological DA closure. Inhibiting SPHK1 is a promising therapeutic strategy for elastin aortopathy and for select congenital heart diseases in which patent DA maintains circulation.

Upregulation of multiple key molecules is correlated with poor prognosis and immune infiltrates in hepatocellular carcinoma by bulk and single-cell RNA-seq

Upregulation of multiple key molecules is correlated with poor prognosis and immune infiltrates in hepatocellular carcinoma by bulk and single-cell RNA-seq

Applications of single-cell RNA sequencing in rheumatoid arthritis

Single cell RNA sequencing (scRNA-seq) is a relatively new technology that provides an unprecedented, detailed view of cellular heterogeneity and function by delineating the transcriptomic difference among individual cells. This will allow for mapping of cell-type-specific signaling during physiological and pathological processes, to build highly specific models of cellular signaling networks between the many discrete clusters that are present. This technology therefore provides a powerful approach to dissecting the cellular and molecular mechanisms that contribute to autoimmune diseases, including rheumatoid arthritis (RA). scRNA-seq can offer valuable insights into RA unique cellular states and transitions, potentially enabling development of novel drug targets. However, some challenges that still limit its mainstream utilization and include higher costs, a lower sensitivity for low-abundance transcripts, and a relatively complex data analysis workflow relative to bulk or traditional RNA-seq. This minireview explores the emerging application of scRNA-seq in RA research, highlighting its role in producing important insights that can help pave the way for innovative and more effective therapeutic strategies.

Abstract 4141486: Site-Specific Transcriptomic Patterns and Cell Types Associated with Hypertension

Introduction: The biology of early human hypertensive heart disease (HHD) is largely unexplored due to limited cardiac tissue access. Site-specific transcriptomics of human cardiac tissue before development of overt HHD offers a unique vantage to understand the biology of HHD in human cardiac tissue. Hypothesis: We hypothesize that transcriptomic signatures and cell subtype proportions differ in human heart tissue from HTN donors compared to normotensive (NTN) donors in a site-specific manner. Methods: We performed bulk RNA-seq on rRNA-depleted libraries from left (LV) and right (RV) mid-ventricular-wall tissue (average depth 114 million reads/sample) collected postmortem from HTN (n=3, 67% Black/Latinx) and NTN (n=2, 50% Black/Latinx) donors. We performed single-nucleus RNA-seq (snRNA-Seq) on cardiac tissue from the same donors (average of 4,062 LV nuclei). Differential gene expression, lineage identification, and Gene Set Enrichment Analysis (GSEA) were performed with publicly available software. Results: All donors were female with matched postmortem intervals (time from death to tissue preservation; HTN 19±9 hours, NTN 18±6 hours). HTN donors were older (65±7 years) than NTN donors (36±16 years). A total of 416 genes were associated with HTN (Wald test, FDR q <0.05) across both ventricles (correcting for site). When tested separately, 72 (LV) or 83 (RV) genes were associated with HTN (FDR q <0.05), with 13 common genes, indicating site-specific differences. GSEA of genes ranked by Wald statistic computed across both ventricles showed that transcripts involved in single-stranded DNA helicase activity, branched-chain amino acid catabolism, and NAD-dependent deacetylase activity were coordinately associated with HTN (FDR q <0.05). snRNA-seq revealed 9 cell types in these samples, including immune cells and chamber-specific cardiomyocytes (CMs) with higher proportions of LV CMs and lymphocytes associated with HTN compared to NTN. MYH6 and MYL7 expression was unchanged with respect to HTN by RNA-seq but lower in LV HTN CMs compared to NTN by snRNA-seq, suggesting dysregulation of contractile programming in HTN LV CMs. Conclusion: HTN is associated with altered cardiomyocyte/lymphocyte ratios, and site-specific differential expression of cardiac contractile elements. Further study is required to validate these findings with additional samples and elucidate mechanisms driving transcriptional program differences in the hypertensive human heart.

EPCO-57. INTEGRATED ANALYSIS OF BULK AND SINGLE-CELL RNA SEQUENCING DATA OF PRIMARY AND METASTATIC PEDIATRIC MEDULLOBLASTOMA

Abstract Medulloblastoma (MB) is a highly aggressive and the most common brain tumor in childhood. MB presents a high intertumoral heterogeneity, with at least four molecular subgroups identified (SHH, WNT, Group 3, and Group 4). Metastatic MB, or Leptomeningeal Disease (LMD), is predominantly found in the MB Group 3 type. Although LMD represents a main clinical challenge, its molecular mechanisms remain poorly characterized. Recent research has shown that primary and MB metastasis diverge dramatically. Our work has focused on establishing therapy naïve Group 3 Patient-Derived Xenografts models of primary and metastatic Medulloblastoma to conduct transcriptomic profiling at the bulk and single-nuclei RNAseq levels to identify genetic drivers/pathways that sustain leptomeningeal disease compartment. Our results show various signaling pathways enriched across LMD models, such as MYC targets, unfolded protein response, and fatty acid metabolism. Using single-sample GSEA analysis (ssGSEA) and deconvolution approaches, we have also identified that our PDXes models retain neoplastic subpopulations previously identified in MB single-cell sequencing studies. Similarly, we have identified slight differences in cell subpopulation proportions between primary and leptomeningeal compartments. Our single-nuclei studies have confirmed these results and differentially expressed genes previously found in bulk RNAseq analyses. These results suggest the presence of cell populations enriched in the metastatic compartment with an aberrant transcription phenotype and adaptations in fatty acid metabolism to survive the leptomeningeal space. In conclusion, our work supports the notion that primary and LMD retain subpopulation clusters present in MB Group 3 tumors with different proportions in the metastatic compartment shown by bulkRNAseq deconvolution and single nuclei RNA analysis. We also show that primary and LMD are transcriptionally different, with various enriched pathways in the metastatic compartment and shared among LMD Group 3 PDX models. Through these approaches, we aim to elucidate the genetic dependencies of metastatic Medulloblastoma that will help for targeted therapies.

IMMU-48. CRACKING THE ROLE OF ONCOMETABOLITES IN VIROIMMUNOTHERAPY FOR PEDIATRIC MALIGNANT BRAIN TUMORS

Abstract Diffuse midline gliomas (DMGs) are among the most frequent and lethal pediatric tumors. The standard of care for DMGs is palliative, resulting in a median survival of 12 months. Viroimmunotherapy is an emerging alternative treatment for these tumors. Our group developed a platform of oncolytic adenoviruses that was translated to the clinic. Specifically, Delta-24-RGD was recently tested in a Phase I clinical trial for patients with newly diagnosed DMG (NCT03178032) with encouraging results. To further stimulate the immune arm of this therapy, we developed Delta-24-RGDOX, expressing the T-cell activator OX40L (NCT03714334, NCT04714983). Using bulk RNAseq, we showed that treatment with Delta-24-RGDOX upregulated the immunosuppressive immunometabolic IDO-AhR pathway. Of importance, treatment with Th1-related cytokine IFNg resulted in the upregulation of IDO1 expression in a panel of human and murine DMG cells. In agreement with these data, we showed synergy between the oncolytic virus and several IDO1-AhR inhibitors in tumor and immune cell co-cultures, as indicated by T-cell activation and proliferation. To further dissect the reshaping of the TME in clinically relevant DMG models treated with Delta-24-RGD or Delta-24-RGDOX, we performed scRNA seq analysis. We showed a pronounced T-cell infiltration in virus-treated animals versus control-treated mice, further confirmed by flow cytometry. Additionally, Th1 and CD8 effector memory subsets were preferentially enriched in the Delta-24-RGDOX group, indicating an enhanced proinflammatory shift. Importantly, we detected, for the first time, upregulation of IDO-AhR downstream effectors in the myeloid compartment in the virus-treated groups. Interestingly, we detected IL4I1 in myeloid cells, a recently discovered checkpoint upstream of AhR never described in the context of virotherapy. Preliminary data using a combination of Delta-24-RGDOX and AhR inhibitors prolongs the survival of glioma-bearing mice. Collectively, our in vivo and in vitro data support the rationale to propel a future clinical trial combining Delta-24-RGDOX with AhR inhibitors for DMG.

Single-cell landscape identified SERPINB9 as a key player contributing to stemness and metastasis in non-seminomas

AbstractEmbryonal carcinoma (EC), characterized by a high degree of stemness similar to that of embryonic stem cells, is the most malignant subtype within non-seminomatous testicular germ cell tumors (TGCTs). However, the mechanisms underlying its malignancy remain unknown. In this study, we employed single-cell RNA sequencing to analyze four non-seminoma samples. Our differential expression analysis revealed high expression of SERPINB9 in metastatic EC cells. We conducted in vitro experiments to further investigate SERPINB9’s role in the progression of EC. Functionally, the knockdown of SERPINB9 in NCCIT and NTERA-2 leads to a diminished migratory capability and decreased cis-platin resistance, as demonstrated by Transwell migration assay and drug sensitivity assay. Moreover, embryoid bodies showed reduced size and lower OCT4 expression, alongside heightened expression of differentiation markers AFP, ACTA2, and CD57 in shSERPINB9 cells. In vivo, the role of SERPINB9 in maintaining cancer stemness was validated by the limiting dilution assay. Mechanistically, Bulk RNA-seq further showed downregulation of ERK1/2 signaling and WNT signaling pathways with concomitant upregulation of differentiation pathways subsequent to SERPINB9 knockdown. Additionally, the analysis indicated increased levels of cytokines linked to tertiary lymphoid structures (TLS), such as IL6, IL11, IL15, CCL2, CCL5, and CXCL13 in shSERPINB9 cells, which were further validated by ELISA. Our research indicates that SERPINB9 plays a key role in driving tumor progression by enhancing tumor stemness and suppressing TLS. This study stands as the first to elucidate the molecular signature of non-seminomas at a single-cell level, presenting a wealth of promising targets with substantial potential for informing the development of future therapeutic interventions.

EPCO-43. DECONVOLUTION OF GLIOBLASTOMA RNA-SEQ REVEALS IMMUNE SUBPOPULATIONS PREDICTING THERAPEUTIC RESPONSE

Abstract Bulk RNA-seq is now among the standard of care for patients with newly diagnosed glioblastoma (GBM), but whether this data can predict therapeutic response is unclear. In silico deconvolution of bulk RNA-seq data allows for quantification of cell types comprising the tumor and its microenvironment. Our study tests whether cell type proportions predict therapeutic response in patients with GBM and high-grade glioma (HGG) enrolled in trials of investigational agents. 127 patients with GBM/HGG underwent post-resection/biopsy whole exome RNA-seq and reads were post-processed using standard computational pipelines. In silico deconvolution was performed using GBMDeconvoluteR, which generates cellular proportions comprising the RNA-seq reads. Multivariate Cox regression models were used to examine each cell proportion as a predictor of progression-free (PFS) and overall (OS) survival. Statistical significance was ascertained using Bonferroni correction for multiple comparisons. Among 54 patients treated with a PD-L1 inhibitor (nivolumab) and VEGF inhibitor (bevacizumab), the proportions of NK cells (p=0.0024), radial glial cells (p=0.0010), and mural cells (p=0.0025), was associated with increased PFS, while proportion of endothelial cells (p=0.0008) was associated with decreased PFS. Astrocyte (p=0.0022), macrophage (p=0.0015), NK cell (p<0.0001), and mural cell (p=0.0004) proportions were associated with longer OS, while endothelial cell proportion (p=0.0001) was associated with shorter OS. Among 21 patients treated with a tyrosine kinase inhibitor (ibrutinib) combined with radiation and temozolomide (RT/TMZ), cell proportions assessed at the time of diagnosis did not predict PFS or OS. In 52 patients with HGG treated with a JAK inhibitor (ruxolitinib) with RT/TMZ, only mural cell proportion was associated with a longer OS (p=0.0017). Our results highlight how bulk RNA-seq data can be used to predict therapeutic response. In GBM patients treated with immunotherapies, the proportion of NK cells may predict response. Further work will examine which gene programs are predictive of therapeutic response.

Small and Long Non-Coding RNA Analysis for Human Trophoblast-Derived Extracellular Vesicles and Their Effect on the Transcriptome Profile of Human Neural Progenitor Cells

In mice, the fetal brain is dependent upon the placenta for factors that guide its early development. This linkage between the two organs has given rise to the term, the placenta–brain axis. A similar interrelationship between the two organs may exist in humans. We hypothesize that extracellular vesicles (EVs) released from placental trophoblast (TB) cells transport small RNA and other informational biomolecules from the placenta to the brain where their contents have pleiotropic effects. Here, EVs were isolated from the medium in which human trophoblasts (TBs) had been differentiated in vitro from induced pluripotent stem cells (iPSC) and from cultured iPSC themselves, and their small RNA content analyzed by bulk RNA-seq. EVs derived from human TB cells possess unique profiles of miRs, including hsa-miR-0149-3p, hsa-302a-5p, and many long non-coding RNAs (lncRNAs) relative to EVs isolated from parental iPSC. These miRs and their mRNA targets are enriched in neural tissue. Human neural progenitor cells (NPCs), generated from the same iPSC, were exposed to EVs from either TB or iPSC controls. Both sets of EVs were readily internalized. EVs from TB cells upregulate several transcripts in NPCs associated with forebrain formation and neurogenesis; those from control iPSC upregulated a transcriptional phenotype that resembled glial cells more closely than neurons. These results shed light on the possible workings of the placenta–brain axis. Understanding how the contents of small RNA within TB-derived EVs affect NPCs might yield new insights, possible biomarkers, and potential treatment strategies for neurobehavioral disorders that originate in utero, such as autism spectrum disorders (ASDs).

TMIC-56. LONGITUDINAL SINGLE CELL RNA SEQUENCING UNVEILS PHENOTYPIC CO-OPTION AND LINEAGE TRAITS OF MESENCHYMAL GLIOBLASTOMA AND GLIOMA-ASSOCIATED FIBROBLASTS

Abstract BACKGROUND Adult gliomas exhibit a high level of heterogeneity presenting a substantial challenge hampering development of targeted therapies. Bulk RNAseq studies have identified a mesenchymal gene expression signature across the glioma continuum, based on which a mesenchymal subtype of glioblastoma (MES-GBM) has been described. However, the mesenchymal phenotype of gliomas remains inconsistently defined and the origin of the mesenchymal signature has been debated). Single cell transcriptomic profiling has allowed resolution of gene expression profiles of tumor and non-tumor cells, leading to recent identification of cancer-associated fibroblasts in gliomas and improved understanding of the mesenchymal gene expression signature. METHODS We performed longitudinal scRNAseq analysis of organotypic human glioma slices from 8 patients cultured ex vivo for up to 3 weeks. RESULTS We found 2 of the 6 IDHwt glioblastomas exhibit the mesenchymal phenotype based on Consensus Methylation Profiling (NIH) and scRNAseq analysis, annotated as Mes-1 and Mes-2. The gene expression profiles of mesenchymal glioblastomas show limited concordance with that of either the proneural or classical glioblastoma signatures or of cell types in physiological human brain, with Mes-2 deviating more extensively from non-mesenchymal gliomas. Instead, single cell transcriptomics suggests a hyper-secretory phenotype and overlaps broadly with that of cancer-associated fibroblasts (CAFs) and both express FGF2, other growth factors, and cytokines. Notably, Mes-2 cells express minimal to no levels of genes associated with brain cell lineages, such as PTPRZ1 and SCG3, which are abundantly expressed in other glioma subtypes. CONCLUSIONS Taken together, our data reveals extended depth of glioma heterogeneity that distinguishes MES-GBM from other glioma subtypes and suggests a phenotypic and lineage linkage between MES-GBM and glioma-associated fibroblasts. Thus, new diagnostic and treatment strategies leveraging auto- and paracrine mechanisms underpinning tumor and non-tumor interactions including disruption of the FGF2-FGFR1 circuitry may apply specifically to MES-GBM.

BIOM-38. OPPOSING EFFECTS OF LINKED CX3CR1 GERMLINE VARIANTS ON INCIDENCE, SURVIVAL, AND IMMUNE ACTIVATION IN GLIOMA

Abstract Linkage-disequilibrium connects CX3CR1 V249I and T280M single-nucleotide polymorphisms; V249I occurs as a single-variant (SV) and T280M exclusively as a double-variant (DV) concurrent with V249I. The effect of these combined haplotypes remains unexplored in glioma. We identified CX3CR1 haplotypes in internal LGG and TCGA GBM cohorts and analyzed median overall survival (mOS). The proportion of each haplotype in glioma and normal patients was assessed via meta-analysis. Bulk RNAseq analysis was conducted in TCGA and internal GBM cohorts and scRNAseq in internal IDH mutant gliomas. In LGG, SV improved mOS (n=14, 21.03yrs) by twofold compared to WT (n=45, 9.41yrs) and DV (n=31, 10.49yrs; p=0.037). In TCGA GBM, SV (n=69, 15.65mo) improved mOS over DV (n=106, mOS 11.74mo; p=0.044). In combined meta-analysis, SV patients had increased risk for glioma development (OR 1.11, 1.03-1.19 p = 0.006) and the WT genotype was protective (OR 0.95, p=0.048). Collagen genes associated with increased immunosuppression (COL6A3: log2FC=2.33, p-adj=0.004; COL6A2: log2FC=1.55, p-adj=.059) were reduced in SV. MME, an endopeptidase associated with radiation resistance, was downregulated in SV (log2FC=3.29, p-adj=2.53e-6). MN1, associated with improved glioma survival, was upregulated in SV (log2FC=-1.76, p-adj=0.001). CCL18, an immunosuppressive chemokine associated with M2 macrophage polarization was reduced in SV (log2FC=2.36, p-adj=0.014). Thus, concomitant with its improved survival, SV is biologically distinct from DV and WT. Using scRNAseq, myeloid-derived cells (microglia/macrophages; 7758 cells) were re-clustered, identifying fourteen clusters. Cells clustered by CX3CR1 status, with 58.2% of SV cells in cluster five and 84.9% of DV cells in clusters six or seven. Cluster five over-expressed IL1B (average-log2FC 3.16), a tumor-suppressive M1 macrophage marker. Clusters six and seven were characterized by IFIT genes (IFIT3 average-log2FC 2.72 and 2.16 respectively), which corresponds to glioma associated microglia from recent scRNAseq microglial atlases. We demonstrate that CX3CR1 SV has improved survival and reduced immunosuppression over wildtype and this is lost with in the linked CX3CR1 DV genotype.

CNSC-36. REGULATION OF CELL FATE IN PAEDIATRIC HIGH-GRADE GLIOMA BY THE HOMEOBOX TRANSCRIPTION FACTOR DLX2

Abstract Paediatric high-grade gliomas (pHGG) are nearly uniformly fatal cancers that arise predominantly in children and adolescents and encompass Diffuse Midline Glioma (DMG) and Diffuse Hemispheric Glioma (DHG). Most patients harbour mutations in histone variants H3.1 or H3.3, resulting in global epigenetic dysregulation and a stalled oligodendroglial progenitor (OPC) or GABAergic interneuron progenitor (IP) fate for DMG and DHG, respectively. DLX2, a homeobox transcription factor, represses OPC fate and promotes IP cell fate and migration during early neurogenesis. Our lab has shown that Dlx2 binds to and regulates the promoters of Gad1/Gad2, as well as early (Olig2, Nkx2.2) and late (Myt1, Plp1) genes required for OPC differentiation in vivo. Co-expression of Dlx2 with these target sequences reduces reporter gene expression in vitro and Dlx1/2 double knockout mice exhibit increased expression of these OPC markers in vivo. Transient overexpression of a Dlx2-GFP construct in murine DMG cells led to significant IP marker increase (Gad1/2), decrease of OPC markers (Olig2, Nkx2.2) and global restoration of H3K27me3. Additionally, decreased proliferation, colony growth, invasion and migration were observed in vitro. To translate these findings in human pHGG, analysis of a bulk RNA-seq database of 62 patient-derived pHGG cell lines revealed an inverse relationship between DLX2 and OLIG2. DLX2 was knocked out of DLX2high/OLIG2low DHG cell lines using CRISPR-Cas9, and overexpressed in DLX2low/OLIG2high DMG cell lines using a DLX2-mCherry overexpression construct. Evaluation of cell fate markers and epigenetic marks will be presented. Changes in migration, invasion, and proliferation in vitro will also be presented, in addition to changes in tumour growth using a neonatal xenograft murine model in vivo. Manipulation of DLX2 in pHGG provides a model for investigating the link between the factors that control cell fate decisions in the developing brain and the aberrant differentiation state in pHGG.

CTIM-28. NEOADJUVANT ANTI-PD1 IMMUNOTHERAPY FOR SURGICALLY ACCESSIBLE RECURRENT GLIOBLASTOMA: CLINICAL AND MOLECULAR OUTCOMES OF A STAGE 2 SINGLE-ARM EXPANSION COHORT

Abstract Glioblastoma is immunologically “cold” and resistant to single-agent immune-checkpoint inhibitors (ICI). Our previous study of neoadjuvant pembrolizumab in surgically-accessible recurrent glioblastoma identified a molecular signature of response to ICI and suggested that neoadjuvant pembrolizumab may improve survival. To increase the power of this observation, we performed bulk-RNA seq on resected tumor tissue from an additional 25 patients with recurrent glioblastoma treated with neoadjuvant pembrolizumab. Neoadjuvant pembrolizumab was associated with suppression of cell cycle/cancer proliferation genes and upregulation of T-cell/interferon-related gene expression. This signature was unique to patients treated with neoadjuvant pembrolizumab and was an independent positive risk factor for survival. PF6 for the stage 2 expansion cohort was 0%, while overall survival and progression-free survival were similar to historical controls at 6.8 months and 2.5 months, respectively. Our results demonstrate a clear pharmacodynamic effect of anti-PD1 therapy in glioblastoma and identify pathways that may mediate resistance. However, we did not observe a survival benefit to neoadjuvant pembrolizumab in recurrent glioblastoma in our expansion cohort. Our new data suggests some patients may exhibit innate resistance to pre-surgical ICI and require other concomitant therapies to sensitize effectively.

IMMU-35. TARGETING THE AHR IMMUNOMETABOLIC HUB DURING VIROIMMUNOTHERAPY OF GLIOMAS

Abstract Glioblastoma (GBM) poses a significant therapeutic challenge in oncology. Recent clinical trials have shown the feasibility of virotherapy to treat patients with GBM. Our laboratory developed an oncolytic adenovirus termed Delta-24-RGD that has been tested in clinical trials for recurrent GBM patients with encouraging results (NCT00805376, NCT02798406). These clinical studies, together with preclinical data, showed that the efficacy of Delta-24-RGD was due to direct tumor cell oncolysis and indirect activation of anti-tumor immune response. To further leverage this immune component, our group generated Delta-24-RGDOX, an armed version of Delta-24-RGD expressing the T-cell activator OX40-L, which exhibits a superior anti-cancer effect. In this project, we aim to further boost the effectiveness of Delta-24-RGDOX by targeting factors that maintain the immunosuppressive characteristic of gliomas. Specifically, we identified the environmental sensor and transcription factor Aryl Hydrocarbon Receptor (AhR) as one of the immunosuppressive factors. We show that AhR is overexpressed in GBM surgical specimens compared to healthy brain tissue, and high AhR expression correlates with poorer survival in glioma patients. Immunofluorescence staining of glioma cells treated with Delta-24-RGDOX demonstrated that AhR translocates to the nucleus, and qPCR showed that translocation resulted in the enhanced transcription of AhR-responsive elements. Additionally, bulk RNA-seq reveals that in vivo glioma models treated with Delta-24-RGDOX exhibit activation of the AhR pathway network. This unexpected and paradoxical activation may be responsible for immune suppression mechanisms, which might hinder the effect of Delta-24-RGDOX. Based on these data, we combined two innovative strategies, the oncolytic virus Delta-24-RGDOX and AhR inhibitors. Survival analysis in immunocompetent models of gliomas treated with this combined therapy showed the greatest therapeutic benefit compared to monotherapy controls. These findings highlight the critical role of AhR activation triggered by Delta-24-RGDOX and position AhR as a potential therapeutic target for enhancing the efficacy of oncolytic adenovirus therapy in GBM.

EPCO-53. CHARACTERIZING THE FUNCTIONAL IMPACT OF GERMLINE-INHERITED RISK ALLELES IN GLIOMA EVOLUTION

Abstract Efforts to understand etiology of glioma led researchers to investigate associated genetic factors. Genome Wide Association Studies (GWAS) conducted to that end, uncovered an association of various single nucleotide polymorphisms (SNP) among hallmark oncogenes and tumor suppressors as risk variants in glioma incidence. Here, we perform in vitro modeling of four risk alleles/SNPs((rs723527 (EGFR), rs55705857 (MYC), rs7572263 (IDH1), rs78378222 (TP53)) mapped to non-coding regulatory regions of the genome, to assess their causal contribution to glioma evolution. To model the aforementioned risk alleles in an in-vitro system, we used a CRISPR-knock in strategy in human induced pluripotent stem cells (hiPSCs) and verified by Sanger sequencing. To study the effects of the risk alleles on brain cell types, we generated a cerebral organoid model from the edited and control lines and subjected them to downstream morphological and transcriptomic analyses. While we did not see morphological changes in the histological sections relative to controls at the early 14-day timepoint, we observed increased differentiation in both control and risk cohorts, along with an absence of neural rosettes, at the late 90-day timepoint. Bulk RNA-seq of the organoids at 14-days resulted in positive enrichment of oncogenic signaling pathways and negative enrichment of DNA repair pathways in the risk allele cohorts. Furthermore, single cell RNA seq of 14 and 90-day samples revealed a sharp decrease in neural stem cell clusters in both IDH and TP53 cohorts, while control organoids demonstrated robust production of immature neurons at 90 days. Overall, we observed a significant increase in differentiation into mature cell clusters in the control, relative to the risk allele organoids. Ongoing efforts are aimed at understanding this apparent differentiation block in the IDH and TP53 compartments, relative to control. Further analysis will lead to addressing key understudied areas of glioma etiology and associated mechanisms of risk incidence.

TMIC-46. TRANSCRIPTIONAL HETEROGENEITY AND MECHANISTIC PATHWAYS OF RECURRENT GLIOBLASTOMA: INSIGHTS FROM A PRE-CLINICAL RECURRENT TUMOR MODEL

Abstract Despite aggressive therapy combining surgical resection, radiation and chemotherapy, glioblastoma (GBM) almost always recurs. Currently, there is no standard treatment for recurrent GBM, and patient inclusion in clinical trials is based on the genetic and molecular profile of the primary tumor even though the effects of chemoradiation on the transcriptional subtype in matched primary and recurrent GBM remain largely understudied. Paralleling the GLASS consortium, we sought to perform a longitudinal assessment of GBM recurrence after therapy based on molecular subtype with the hypothesis that recurrent tumors exhibit a distinct transcriptional landscape different from treatment-naive tumors. To address this, we utilized primary tumor-derived glioma stem cells (GSCs) representing classical and proneural subtypes. We then undertook in vivo intracranial implantation studies and performed standard concomitant treatment that mimics the Stupp protocol. Upon bulk RNAseq data analyses of untreated (n=21) and recurrent tumors (n=17) we identified that while the molecular subtype of recurrent tumors does not change post therapy, their transcriptional profile is more heterogenous compared to untreated tumors for both subtypes. Hierarchical cluster analyses revealed distinct sub-clusters of recurrent tumors. Common mechanisms of recurrence include upregulation of angiogenesis pathways and PI3k/Akt/mTOR signaling pathways. Interestingly, recurrent tumors exhibit upregulation of genes involved in cellular metabolism and membrane transport/ potential. The source of heterogeneity in recurrent tumors in both subtypes appears to be due to the diversity in expression of gene sets involved in hallmark of cancer pathways such as cell cycle, Notch and Wnt signaling pathways and heterogeneity in oncogenic signatures and cell states resembling fetal or neural cells. Further research into the recurrent tumor microenvironment by spatial multi-omics approaches using this pre-clinical recurrent GBM model will elucidate the linkage between cellular as well as molecular heterogeneity and enable identification of targets that can help drive therapeutic decisions for recurrent GBM patients.

EPCO-20. MUTANT IDH INHIBITORS INDUCE LINEAGE DIFFERENTIATION IN IDH-MUTANT OLIGODENDROGLIOMA

Abstract IDH-mutant gliomas exhibit variable responses to mutant-IDH inhibitor (IDHi) therapy; yet, the molecular mechanisms underlying these responses remain poorly understood. Here, we investigate the cellular underpinnings of response by leveraging single cell/nuclei RNA-sequencing of on-treatment IDH-mutant oligodendroglioma tumor samples resected from three patients who benefited clinically from treatment. We integrate these findings with single cell and bulk RNA-seq data from independent cohorts and experimental models. We find that IDHi treatment promotes robust differentiation towards the astrocytic lineage, accompanied by stem-like cell depletion and reduced proliferation. Notably, NOTCH1 mutations are associated with impaired astrocytic differentiation, potentially negatively impacting response to IDHi. This study unveils the differentiating effects of IDHi on oligodendroglioma cellular hierarchies and identifies a potential genetic marker for optimizing patient selection. Paper was recently published in Cancer Cell (10.1016/j.ccell.2024.03.008).

CTIM-36. IN-SITU VACCINATION OF BIOPSY-ONLY GBM TUMORS BY TUMOR TREATING FIELDS PLUS ANTI-PD-1 IMMUNOTHERAPY RESULTS IN ROBUST ANTIGEN-SPECIFIC T CELL SELECTION AND EXPANSION, HIGH RESPONSE RATE, AND SIGNIFICANTLY EXTENDED SURVIVAL

Abstract We have recently described a complete in-situ immunizing protocol against GBM using Tumor Treating Fields (TTFields) to activate the DNA sensor-dependent inflammasomes and immunogenic cell death. Here, we report successful in-situ vaccination of patients with newly diagnosed GBM, especially in those with bulky, biopsy-only tumors as compared to those with maximally resected disease, using TTFields together with pembrolizumab, an anti-PD-1 immunotherapy. Patients with biopsy-only tumors displayed a marked improvement in both progression-free survival (27.2m vs 9.6m; HR 0.37, 95%CI 0.16-0.85; P=0.014), overall survival (31.6m vs 18.8m; HR 0.4, 95%CI 0.17-0.92; P=0.023), and response rates (66.6% vs 25%) when compared to those with maximally resected tumors. Using a combination of bulk RNA-seq of enriched T cells and single-cell RNA-seq and TCR clonotyping of tumors and PBMC, we tracked clonal evolution of T cells and confirmed that TTFields’ adaptive immune activation followed a type 1 interferon trajectory (T1IFN) specifically through the DC compartment in patients with biopsy-only tumor. From DC, the T1IFN pathway engaged the adaptive immune system, highly correlated with T cell activation (Pearson r=0.81; P=0.000027), and strongly predicted for extended survival (HR 0.3; P=0.034). Importantly, we discovered the adaptive replacement of expanded T cell clones induced by TTFields and enhanced by pembrolizumab predicted for prolonged survival (P=0.031), leading to sustained T cell activation (Pearson r=0.72; P=0.0018) and robust generation of central memory CD4+ and CD8+ T cells, especially in responders with biopsy-only tumors. In recurrent tumors, the PD-1/PD-L1 axis was suppressed, while concurrent upregulation of alternative immune checkpoints was observed in the periphery and TME. This was coupled with ineffective compensatory T cell activation, suggesting potential mechanisms of resistance. These findings highlight the potent in-situ vaccination effects of TTFields and support its co-application with immune checkpoint inhibitors as a promising strategy for GBM, especially in patients with substantial residual tumor.

CTIM-18. HIGH LEVEL OF MUTATIONAL LOAD AND DIVERSITY OF TUMOR INFILTRATING T LYMPHOCYTES ARE FAVORABLE PROGNOSTIC MARKERS FOR CANCER SURVIVAL IN CHILDREN WITH RECURRENT MEDULLOBLASTOMA DURING ADOPTIVE CELLULAR THERAPY

Abstract BACKGROUND Adoptive cellular therapy (ACT) has shown remarkable efficacy in some patients with solid tumors, resulting in durable complete responses. A key mechanism behind this success is the targeting of neoantigens by T cells. There is culminating evidence that tumor-infiltrating lymphocytes (TILs) have a major effect on the clinical attributes of human cancer and can influence the tumor response to various therapy regimens. We have been sequencing RNA isolated from patient’s tumors to evaluate their mutational burden and identify tumor-specific neoantigens. Additionally, we have identified the entire repertoire of tumor-infiltrating lymphocytes in patients with recurrent medulloblastoma. METHODS Total tumor RNA was extracted from resected brain tumor specimens and after we applied in-house bulk RNA-Seq and TCR-Seq techniques using rapid amplification of cDNA ends (RACE) technology with the addition of a common adapter at the 5’ end. RESULTS & CONCLUSIONS Analysis of the 22 tumor tissues revealed that the frequency of non-synonymous mutations varied dramatically more than 10-fold among patients with recurrent medulloblastoma undergoing adoptive cellular therapy (Re-MATCH protocol, FDA IND BB-14058). Patients with shorter overall survival exhibited non-synonymous mutation frequencies between 0.113 and 0.335 mutations per megabase (Mb). Children with prolonged overall survival had higher mutation frequencies, ranging from 0.6 to 1.1 mutations per Mb. These findings highlight the potential of using mutational burden as a biomarker for patient selection and treatment customization, ultimately aiming to improve outcomes in ACT for recurrent medulloblastoma. Up to 2000 unique TCR clones were found among TIL populations in patients who responded well to ACT. Patients with poor outcomes had significantly lower TCR diversity, with only 10-40 unique TCR clones among TILs. The correlation between TCR diversity among TILs and clinical outcomes in pediatric brain tumors underscores the importance of a diverse T cell repertoire for the success of ACT.