Eghlimi, Ryan

Abstract Glioblastoma (GBM) and Alzheimer’s disease (AD) are devastating, age associated disorders with no cure. Epidemiologic data has established an inverse comorbidity between AD and cancer, including GBM, with up to a 50% reduction of cancer incidence in AD patients. While the underlying biology remains unclear, myeloid cells, which are central players in both neurodegeneration and tumor immunity, may serve as a mechanistic link. We hypothesize that myeloid cells activated by pre-existing AD pathology suppress tumor growth more effectively than homeostatic microglia. In this study, age-matched APPNL-G-F/MAPT double knock-in (henceforth “AD”) and wildtype (WT) mice received orthotopic xenografts of syngeneic GBM cell lines (GL261, SB28) (n=15/sex/genotype). We observed a significant survival difference, with AD mice living an average of 20% longer than WT mice (p<0.0001). In vivo luciferase imaging revealed impaired engraftment of tumor cells in AD mice compared to WT a week post-engraftment (p<0.05). At the microscopic level, tumors in AD mice were found to have nearly 2-fold the amount of tumor-infiltrating myeloid cells (p<0.01), with the proportion of anti-tumoral “activated” myeloid cells also being greater (p<0.05). Tumors in AD mice were found to have markedly higher immune infiltrate at the tumor rim and margin (p<0.001). We also observed striking astrogliosis (GFAP+) and microgliosis (P2RY12+) in both the tumor rim (p<0.05) and margin (p<0.01) in AD animals. This robust glial response was significantly higher in AD mice bearing tumor compared to both WT mice bearing tumor and sham AD mice. Lastly, we utilized a luciferase stain to visualize tumor cells that invaded beyond the bulk tumor. We found that both the quantity (p<0.01) and invasion distance of tumor cells (p<0.05) was lower in AD mice. We demonstrate that an AD-induced inflammatory microenvironment antagonizes glioma growth, providing a foundation for novel GBM treatment strategies that potentially exploit these immune signals.

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults, with few effective treatments. EGFR alterations, including expression of the truncated variant EGFRvIII, are among the most frequent genomic changes in these tumors. EGFRvIII is known to preferentially signal through STAT5 for oncogenic activation in GBM, yet targeting EGFRvIII has yielded limited clinical success to date. In this study, we employed patient-derived xenograft (PDX) models expressing EGFRvIII to determine the key points of therapeutic vulnerability within the EGFRvIII-STAT5 signaling axis in GBM. Our findings reveal that exogenous expression of paralogs STAT5A and STAT5B augments cell proliferation and that inhibition of STAT5 phosphorylation in vivo improves overall survival in combination with temozolomide (TMZ). STAT5 phosphorylation is independent of JAK1 and JAK2 signaling, instead requiring Src family kinase (SFK) activity. Saracatinib, an SFK inhibitor, attenuates phosphorylation of STAT5 and preferentially sensitizes EGFRvIII+ GBM cells to undergo apoptotic cell death relative to wild-type EGFR. Constitutively active STAT5A or STAT5B mitigates saracatinib sensitivity in EGFRvIII+ cells. In vivo, saracatinib treatment decreased survival in mice bearing EGFR WT tumors compared to the control, yet in EGFRvIII+ tumors, treatment with saracatinib in combination with TMZ preferentially improves survival.

Abstract Glioblastoma (GBM) and Alzheimer’s disease (AD) are two devastating central nervous system diagnoses with no known cure. Both diseases are associated with advanced age. The majority of GBM tumors form in the frontal and temporal lobes, two of the brain regions most impacted by amyloid and tau pathology in AD. Females have a higher risk of AD, while males have a higher risk of GBM. Several large case series have established epidemiological evidence that the two diseases are inversely correlated. Nevertheless, it remains unknown whether presence of subclinical amyloid or tau pathology antagonizes the establishment or progression of GBM. The present study sought to characterize GBM growth, progression, and immune response in the setting of concomitant AD pathology using the syngeneic murine GL261 tumor model. To further understand the tumor cell changes that occur in the presence or absence of AD pathology, we engineered GL261 with a dual-tagged vector consisting of RiboTag (to allow for full-length mRNA profiling) and TurboID (to allow for proteomic profiling). GL261 cells were injected intracranially into APPNL-G-F/NL-G-F - MAPThuman/human, APPNL-G-F/+ - MAPThuman/+, and wildtype C57BL/6J mice. Tumors in both wildtype and AD conditions contained ~50% intratumoral myeloid cells (Iba+, P2RY12-). Differential activation of intratumoral myeloid cells will be assessed with immunohistochemistry. Microgliosis (IBA1, P2RY12) and astrogliosis (GFAP) around the tumor in the setting of Alzheimer’s pathology versus normal conditions will be compared. Invasiveness, proliferation, DNA damage (pH2AX), and apoptosis (cleaved PARP, cleaved Caspase 3) will be compared. Tumor cell-specific changes unique to tumors in the AD microenvironment will be assessed transcriptionally with RiboTag profiling as well as proteomically with TurboID purifications. Our preliminary data suggests that the presence of concomitant AD pathology affects GBM growth and progression as well as the immune response to the tumor, and warrants further investigation. Citation Format: David Nascari, Ryan Eghlimi, Angad Beniwal, Drake Alton, John Fryer, Nhan L. Tran. Altered tumor microenvironment in animal model of concomitant GBM and Alzheimer's pathology [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 5562.

Abstract Glioblastoma (GBM) is a high grade glioma that accounts for the majority of primary malignant brain tumors. The standard of care for GBM consists of maximal surgical resection, concurrent radiation and temozolomide (TMZ), and adjuvant TMZ. However, this has only led to a modest increase in median patient survival. TROY (TNFRSF19) is an orphan member of the TNF receptor superfamily that is differentially upregulated in GBM with low expression in normal brain tissue. Previously, we have reported that TROY is overexpressed in GBM and signals to mediate cell invasion and therapeutic resistance. Propentofylline (PPF) is a well-studied synthetic methylxanthine derivative that has strong BBB penetrance. We have shown that PPF decreases TROY expression in gliomas, inhibits invasion, and sensitizes GBM to TMZ in vitro. Here we examined the efficacy of PPF in combination with TMZ using in vivo studies of intracranial models of GBM patient-derived xenograft (PDX) models in murine. We tested 3 models of GBM PDX cells with various TROY expression levels (GBM8-high TROY; GBM43-moderate TROY; GBM22-low/no TROY). Our intracranial PDX experiments showed that PPF treatment alone did not increase survival in all PDX cells. The combination of PPF and TMZ treatment did provide a survival benefit (p < 0.039) relative to TMZ alone (only in GBM8-high TROY and GBM43-moderate TROY) that was partially lost when treatment was discontinued. However, continuous PPF administration following a concomitant PPF and TMZ treatment course led to extended survival (p < 0.0015) compared to TMZ alone resulting in increased DNA damage (γH2AX) and apoptotic markers (Cleaved Caspase-3). Immunohistochemical analyses showed that TROY expression was decreased in tumors treated with PPF. In addition, PPF-treated tumors showed a differential myeloid population with well circumscribed tumors as compared to control-treated or TMZ-treated tumors. Thus, our data demonstrates that PPF may provide a pharmacological approach to targeting TROY concomitant with standard-of-care to increase patient survival. Citation Format: Ryan Eghlimi, David Nascari, Angad Beniwal, Drake Alton, Dustin Grief, Joseph Loftus, Melanie Haluska, David Coleman, Nhan Tran. Propentofylline enhances GBM sensitivity to temozolomide via targeting TROY signaling [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 5905.

Abstract Glioblastoma remains difficult to treat with immunotherapy due to the immunosuppressive tumor microenvironment and the ability of glioma cells to invade diffusely into normal brain parenchyma. While it is known that glioblastoma-associated myeloid cells (GAMs) comprise a large proportion of the tumor microenvironment and can suppress adaptive and innate immune responses through various mechanisms, detailed mapping of myeloid lineages in glioblastoma is still missing. Further characterization of pro-tumoral myeloid-lineages in glioblastoma is important for identifying myeloid-based drug targets that may synergize with T cell-based therapies and produce a more meaningful anti-tumor immune response. Towards this end, we examined numerous scRNAseq datasets derived from glioblastoma patients and identified expression of TNFRSF12A, the gene encoding the TNF- family receptor, Fn14, in a subset of GAMs enriched for expression of cytokines such as CCL3, CCL5, IL-6, and IL-8. This result is intriguing since TWEAK (TNFSF12), the ligand for Fn14, is abundantly expressed within numerous patient-derived glioblastoma cell lines, namely GBM6, GBM8, GBM22, and GBM39. To validate Fn14 protein expression in GAMs, we isolated murine bone-marrow derived macrophages and treated them with glioma-conditioned media, which induced Fn14 gene expression. We then conducted in vitro experiments utilizing the human microglia line HMC3, the murine microglia line SIM-A9, and primary murine bone-marrow-derived macrophages to assess the effects of Fn14 activation in myeloid cells. We found that treatment of Fn14+ myeloid cells with TWEAK ligand upregulates CCL3, CCL5, IL-6, and IL-8 expression. We tested the conditioned media generated from TWEAK-treated Fn14+ myeloid cell lines and found that it promotes glioblastoma invasion in a CCL5-dependent manner. This aligns with previous reports demonstrating the CCL5-CCR5 signaling in glioma cells promotes invasion through calcium-dependent matrix metalloproteinase 2. To assess the spatial distribution of this myeloid subpopulation, we performed RNAscope hybridization against AIF1, TMEM119, CCR5, CCL5, and Fn14 transcripts on sections of murine gliomas generated using the RCAS/tv-a model of human glioblastoma. We found an abundance of Fn14+ plus AIF1+ as well as Fn14+plus TMEM119+ myeloid cells throughout the tumor core and rim of murine tumors and identified CCL5 expression in pseudo-palisades bordering necrotic regions. Our results to date suggest that Fn14 expression in GAMs can promote the invasion of tumor cells into normal brain parenchyma by release of CCL5. Currently, we are utilizing orthotopic patient-derived xenograft models co-implanted with Fn14+/- myeloid cells to test the hypothesis that TWEAK-Fn14 signaling in GAMs provides strong pro-tumoral signals in the glioblastoma microenvironment. Citation Format: Angad Beniwal, Matthew Dufault, Pranjali Kanvinde, David Nascari, Ryan Eghlimi, Adarsha Malla, Dolores Hambardzumyan, Henrique Borges da Silva, Graeme Woodworth, Jeffrey Winkles, Nhan Tran. TWEAK-Fn14 signaling in glioblastoma-associated myeloid cells promotes tumor invasion via CCL5-CCR5 axis [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 6867.

Abstract Cellular heterogeneity within glioblastoma multiforme (GBM) is a key driver of resistance to standard-of-care therapeutic approaches such as DNA damage (temozolomide, TMZ), angiogenesis inhibition (Bevacizumab, BM), radiation necrosis, and surgical resection. As such, understanding and targeting heterogeneous tumor-associated populations is a critical aim of ongoing GBM research. Central nervous system (CNS) resident microglia constitute significant percentages of primary GBM samples, often up to 50% of bulk tumor. Additionally, glioblastoma-associated microglia have been implicated in intra-tumoral immunosuppression, and are thus a key barrier to successful implementation of immunotherapies that seek to enhance T-cell mediated anti-tumoral cytotoxicity. As such, insight into targetable pathways that upregulate immunosuppressive cytokines within GBM-associated microglia is necessary for overcoming immuno-therapeutic failure. Here, we identify expression of the cytokine TWEAK (TNFSF12) and its receptor Fn14 (TNFRSF12A) within the myeloid compartment of primary GBM samples via scRNA-seq. Furthermore, we demonstrate TWEAK-mediated reprogramming of GBM-associated microglia towards an immunosuppressive phenotype capable of inhibiting CD8+ T cell activation and proliferation. These findings unveil a potent, targetable axis of immunosuppression within GBM and are thus an exciting step towards enhancing efficacy of existing T-cell based anti-cancer strategies for GBM patients. Citation Format: Angad Beniwal, Ryan Eghlimi, Kenneth D. Alexander, Mylan R. Blomquist, Nhan Tran. Role of TWEAK-Fn14 axis in driving immunosuppression in glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6442.