bluuebunny/crossref_metadata_2025 huggingface dataset:
MODL-42. ESTABLISHMENT AND CHARACTERIZATION OF A NOVEL MOUSE SYNGENEIC GLIOBLASTOMA CELL LINE
Authors: Ye Juan , Wang Herui , Yang Chunzhang , Zhuang Zhengping
Published: November 2022

Glioblastoma (GBM) is the most common and the deadliest type of brain tumor affecting the adult central nervous system, carrying a median survival of 14 months. In vivo syngeneic animal models of GBM that recapitulate human GBM are urgently needed to decipher glioma biology and develop novel therapeutics, including immunotherapy. Here, we generated a syngeneic mouse glioblastoma stem cell line (RCAS-TVA-GSC01) through repeated in-vitro passages of stem-like tumor cells isolated from the RCAS-TVA glioma mouse model, which overexpresses PDGFA and carries a homozygous deletion of Ink4a-Arf locus. We characterized this new GSC line both in vitro and in vivo. This cell line expresses stemness markers CD133 and Nestin, and is able to differentiate into astrocyte, neuron, and oligodendrocyte-like cells in 2% FBS differentiating medium with markers GFAP, β-III tubulin, and O4, respectively. Transcriptomic and genomic profiling of RCAS-TVA-GSC01 cells revealed molecular heterogeneity comparable to human glioblastoma. When intracranially transplanted into wild-type C57BL/6 mice, this cell line formed high-grade gliomas with a median survival time of 56.5 days. The differentiation potential and molecular heterogeneity make RCAS-TVA GSC01 line a unique and useful tool for studying multiple aspects of glioblastoma pathogenesis and a promising platform for testing new GBM therapeutics study, especially for immunotherapy that requires a syngeneic mouse model.
STEM-04. CONNEXIN 43 DRIVES GLIOBLASTOMA CANCER STEM CELL PHENOTYPES THROUGH A WNK1 SIGNALING AXIS
Authors: Mulkearns-Hubert Erin , Hajdari Nicole , Lathia Justin
Published: November 2022

Tumors represent a highly dynamic system that relies on coordinated signaling to drive growth and adapt to selective pressures, including those induced by anti-cancer therapies. Given the need for tumors to engage in rapid and coordinated cell-cell communication, mechanisms including gap junction intracellular communication would be essential. Surprisingly, the connexin proteins that make up gap junctions have traditionally been considered tumor suppressors. This hypothesis was based on the loss of GJIC in cancer cells and, frequently, lower connexin expression in tumor cells compared to non-neoplastic tissue, including in glioblastoma (GBM), where connexin 43 (Cx43) was shown to be decreased. However, recent data suggest that this tumor-suppressive effect is context dependent and that connexins can function in a tumor-promoting role. We previously found that GBM cancer stem cells (CSCs) rely on gap junctional intercellular communication through connexin 46 (Cx46) for survival. We now identify a subset of GBM patient-derived xenograft (PDX) CSC models that also rely on Cx43 in addition to Cx46. This dependence on Cx43 is independent of Cx43 expression level. Knockdown of Cx43 using shRNA in these PDX models compromises their survival and self-renewal while inducing apoptosis. We leveraged a protein kinase phospho array to identify signaling changes associated with Cx43 knockdown and observed reductions in phosphorylation of WNK lysine-deficient protein kinase 1 (WNK1) in addition to ERK1/2, AKT, and p38MAPK. Immunoblotting validated that WNK1 phosphorylation is lost in the absence of Cx43. Treatment with a WNK1 inhibitor was toxic to CSCs. Current work is focused on interrogating the link among Cx43, WNK1 phosphorylation, and downstream signaling events and the therapeutic window of WNK1 inhibition. This work identifies a potential new signaling axis downstream of Cx43 in glioblastoma CSCs and further strengthens the hypothesis that connexins may act as both tumor suppressors and tumor drivers depending on context.
STEM-09. NEURAL STEM CELLS OF THE SUBVENTRICULAR ZONE EVOLVE INTO TUMORS AROUND THE RESECTION CAVITY AFTER SURGICAL RESECTION OF GLIOBLASTOMA VIA THE CXCL12/CXCR4 AXIS
Authors: Lee Joo Ho , Li Xue , Kim Hyun Jung , Lee Jeong Ho , Kang Seok-Gu , Wu Hong-Gyun
Published: November 2022

Neural stem cells (NSCs) harboring cancer-driving mutations in the subventricular zone (SVZ) have recently been reported as the cells of origin for human GBM. However, the pathological role of residual tumor-initiating NSCs (tNSCs) in the SVZ is undetermined for recurrent tumors after surgical removal of the primary tumor. After the primary resection of GBM, most recurrences still appear as local tumors around the resection cavity (RC). Accordingly, after surgical removal, we hypothesized that the tNSCs in SVZ might be the source for tumor construction at RC as local recurrence. Here, we examined the role of tNSCs in developing local tumors around RC using a mouse model and human tissues. First, cancer-driving mutations, including Trp53, Pten, and Egfr mutations, were introduced to NSCs in SVZ through in vivo electroporation, and the RC was generated in the remote cortex through surgical resection. We found that the NSCs harboring driver mutations migrated specifically to the RC through the aberrant growth of oligodendrocyte-precursor cells (OPCs). Furthermore, the SVZ-originated cells constituted the GBM around the RC at four weeks post-resection in 64% of mouse models. Second, the CXCR4/CXCL12 axis was upregulated through migration from the SVZ and tumor formation at the RC compared with primary tumors without the RC. Blockage of the CXCR4/CXCL12 axis decreased the number of immigrating OPC lineage and switched the differentiation of tNSC to astrocyte lineage. Furthermore, it improved survival rates in vivo. Third, we validated the independent clonal evolution of recurrent tumor from SVZ and the upregulation of CXCR4 axis in the recurrent tumor in a human patient. Taken together, our results reveal that the residual tNCSs harboring cancer-driving mutations in the SVZ can re-construct tumors after surgical resection of GBM. Blocking CXCL12/CXCR4 might be a potential treatment strategy for preventing recurrences after resection.
STEM-13. GLIOMA STEM CELLS MIMIC REGULATORY T CELL FUNCTION TO SUPPRESS THE IMMUNE RESPONSE AND PROMOTE TUMOR PROPAGATION
Authors: Johnson Amanda , Korleski Jack , Sall Sophie , Lopez-Bertoni Hernando , Laterra John
Published: November 2022

The advent of immunotherapy has transformed our approach to cancer treatment; however, it is virtually ineffective in glioblastoma (GBM), the most common & malignant brain cancer in adults, due in part to the inherently immunosuppressive tumor microenvironment (TME). Recent research has identified GBM cancer stem cells (GSCs) as key modulators of the TME, mainly through activation and recruitment of pro-tumor immune cells (e.g., TAMs, regulatory T-cells (Tregs), MDSCs). We identified a novel immunosuppressive phenotype whereby GSCs mimic Treg function via TGF-b type II receptor (TGFBR2) signaling to suppress the immune response with the potential to promote tumor propagation and resist immunotherapy. Preliminary experiments revealed that TGFBR2 drives GBM cells to a CD44-high, mesenchymal-like GSC state. Transgenic TGFBR2 was found to induce a Treg-like transcriptional profile defined by expression of canonical Treg effector genes (e.g., CD274, ENTPD1, NT5E, LGALS1, TGFB1, PDCD1LG2) in human GBM neurospheres. Single-cell RNA-Seq and bioinformatics analyses confirmed the presence of this TGFBR2-mediated transcriptional signature in both patient-derived GSCs and patient tumors. Notably, in silico deconvolution of bulk GBM sequencing predicts that high expression of a TGFBR2-induced transcriptome associates with an immunosuppressive TME characterized by high infiltration of pro-tumor immune cells (e.g., TAMs, neutrophils). To target this Treg-like, immunosuppressive GSC phenotype, we utilized ITD-1, a small molecule inhibitor of TGFBR2. In vitro ITD-1 treatment reduced GSC proliferation capacity and downregulated expression of CD44 and Treg effectors. In T-cell co-cultures, pre-treating GSCs with ITD-1 increased T-cell viability and T-cell-mediated tumor cell killing. Administration of ITD-1 in tumor-bearing, immunocompetent mice reduced tumor volume and increased anti-tumor immune cell infiltration relative to vehicle controls. In all, ITD-1-mediated TGFBR2 inhibition attenuates a unique, Treg-like immunosuppressive and potentially tumor-protective phenotype in GSCs. Future studies will combine ITD-1 and checkpoint inhibitor therapy in an effort to increase the efficacy of immunotherapy in GBM.
STEM-15. THERAPY-INDUCED CHANGES BY BRAF AND MEK INHIBITORS IN BRAF V600E-MUTATED GLIOMA MODELS PROVIDE POTENTIAL NOVEL THERAPEUTIC OPPORTUNITIES
Authors: Park Jongwhi , Lancero Hope , Nasajpour Emon , Garcia Cesar , Prolo Laura , Grant Gerald , Petritsch Claudia
Published: November 2022

Combinations of the MEK inhibitor trametinib, and BRAF inhibitor dabrafenib (BRAFi+MEKi) show rapid and sustained responses in patients with BRAF V600E-mutated low-grade glioma, but tumor rebound after treatment discontinuation is frequent. Moreover, a lack of response is common in patient with high-grade glioma raising the need for further research into BRAFi+MEKi effects on tumors. We showed previously that BRAF V600E-mutated glioma cells positive for CD133 (Prominin-1), a marker of brain tumor stem cells, show decreased sensitivity to BRAFi, indicative of their role in promoting therapy resistance. BRAF V600E-mutated murine and patient-derived glioma cell lines (STN-10049, SU-aGBM5) were generated and together with established BRAF V600E-mutated cell lines (DBTRG, AM38) were analyzed for changes in gene expression in response to 48 hrs treatment with BRAFi dabrafenib and MEKi trametinib. Cells were analyzed by RNA-seq and gene enrichment analyses while cell culture supernatant was analyzed for cytokine production using an ELISA. Syngeneic, orthotopic BRAF V600E-mutated tumor allograft-bearing mice were treated with BRAFi+MEKi, with therapeutic antibodies against immune checkpoint molecules (anti-PD-L1 and anti-CTLA-4) and with combination of all four agents, and tumors were analyzed by mass cytometry and immunofluorescence for stem and T cell markers. BRAFi+MEKi treatment induced an interferon gamma (IFNg) response gene signature in BRAF V600E-mutated glioma cells and increased HLA gene expression. The frequency of tumor-infiltrating CD4+ CD8+ T cells in syngeneic BRAF V600E-mutated tumor allografts increased with BRAFi+MEKi treatment. Combining BRAFi+MEKi with anti-PD-L1 and anti-CTLA-4 treatment decreased CD133+ cells more effectively than either therapy alone, and resulted in a T cell-dependent survival benefit of mice with orthotopic BRAF V600E-mutated high-grade glioma. Combination of BRAFi+MEKi with immune checkpoint inhibition should be further explored as a viable option to prevent tumor rebound and therapy resistance in patients with BRAF V600E-mutated glioma.
STEM-17. IDENTIFICATION OF LOMERIZINE, A PROPHYLACTIC DRUG FOR MIGRAINES, AS A POTENTIAL ANTI-GLIOBLASTOMA DRUG
Authors: Ichinose Toshiya , Tamai Sho , Sabit Hemragul , Tanaka Shingo , Kinoshita Masashi , Nakada Mitsutoshi
Published: November 2022

BACKGROUND

Glioblastoma (GBM) is one of the most malignant primary brain tumors. Despite development of treatment, prognosis of GBM is still poor. Here, we identified lomerizine, a prophylactic drug for migraine, as a potential anti-GBM drug from thousands of existing compounds. We investigated its therapeutic effects both in vitro and in vivo with the purpose of drug repositioning. Materials and method: Human patient-derived glioma stem cell line (KGS01, KGS10, KGS15), their differentiated cells, and GBM cell lines (A172, SNB19, T98, U87) were used. Proliferation assay, migration assay, invasion assay and sphere-forming assay were performed. We analyzed various signaling pathways altered by lomerizine by Western blotting. In addition, induction of apoptosis was evaluated by immunofluorescence and Annexin V assay. Anti-GBM effects of lomerizine in vivo with a mouse brain tumor model was validated Result: Lomerizine inhibited proliferation, migration, and invasion dose-dependently in all cell lines, especially in GSCs. Sphere formation was inhibited by lomerizine in all GSCs. STAT3, which is involved in tumorigenesis, was dephosphorylated after the treatment with lomerizine in a dose-dependent manner in all cell lines. Furthermore, lomerizine induced apoptosis in both immunofluorescence and Annexin V assay. In vivo experiments showed a significant tumor suppression and prolongation of overall survival in the group of mice treated with lomerizine.

CONCLUSION

Lomerizine has anti-GBM effects.
STEM-25. IN VIVO FUNCTIONAL GENOMICS IDENTIFIES ESSENTIALITY OF POTASSIUM HOMEOSTASIS IN MEDULLOBLASTOMA
Authors: Fan Jerry , Skowron Patryk , Erickson Anders , Wang Xian , Wang Xin , Chen Xin , Sun Yu , Wanggou Siyi , Li Xuejun , Taylor Michael , Huang Xi
Published: November 2022

Distinguishing cancer maintenance genes—i.e. genes essential and specific to tumor survival—from initiation, progression, and passenger genes is critical for developing effective cancer therapeutics. We engineered the Lazy Piggy genetic screening system, which uses a Sleeping Beauty / piggyBac hybrid transposon to dysregulate and later restore gene expression. In vivo spatiotemporal control of Lazy Piggy transposon insertion and remobilization depletes insertions that are non-essential for tumor survival while enriching for maintenance driver insertions. Using this functional genomic approach, we identify potassium channels as a regulator for medulloblastoma, the most common pediatric brain malignancy. Loss of potassium channel Kcnb2 diminishes the pool of medulloblastoma-propagating cells by hampering their replication potential, thereby prolonging the survival of tumor-bearing mice. Mechanistically, Kcnb2 governs potassium homeostasis to regulate plasma membrane tension-gated EGFR signaling, which drives the proliferative expansion of medulloblastoma-propagating cells. Our in vivo functional genomics reveals potassium homeostasis as a tumor maintenance essentiality, establishes a link between potassium channel activity and plasma membrane tension, and defines a mechanism by which ion homeostasis integrates biomechanical and biochemical signaling to promote medulloblastoma aggression.
CCRG-01. KAT5 ACTIVITY REGULATES G0-LIKE STATES IN HUMAN GLIOMAS
Authors: Mihalas Anca , Arora Sonali , O'Connor Samantha , Feldman Heather , Patel Anoop , Paddison Patrick
Published: November 2022

Glioblastoma, the most aggressive type of primary brain cancer, benefits little from standard of care therapy. Over the past few decades not much advancement has been made in improving the recurrence-free survival after treatment, with a median survival period of 15 month from diagnostic. G0-like states are thought to act a reservoir for tumor recurrence after treatment in glioblastoma. Targeting G0 states in glioblastoma poses an attractive therapeutic approach, however, little progress has been made in the field, likely due to poorly understood mechanisms that regulate G0 ingress and egress. To find regulators of G0 states, we performed a genome-wide CRISPR-Cas9 screen of patient-derived glioblastoma stem cells. We identified KAT5, a histone acetyltransferase coding gene which is the catalytic subunit of the histone acetyltransferase complex NuA4, as an important candidate for regulating G0 ingress and egress. We found that in primary gliomas, KAT5-low cells display G0-like properties, while overall KAT5 activity increases from low to high grade tumors. To study G0 states in glioblastoma, we have engineered an inducible KAT5 system that functions as a fully tunable model system, where we can control expression of KAT5 and, therefore, effectively control glioma cell entry and exit from G0-like states. We provide evidence that G0-like states are characterized by hypoacetylated histones and low protein synthesis rates, which remarkably induces shifts in cell state reminiscent of dedifferentiation and acquisition of stem-like behavior. Our work demonstrates that regulation of G0 like states may be coupled to the generation of tumor heterogeneity and, more importantly, suggests a potential strategy whereby inhibiting KAT5 activity could effectively "down grade" GBM tumors by lengthening residence time in G0-like states, significantly increasing survival times. For these studies we will present a variety of techniques, including single cell RNA-seq to characterize cellular subpopulations in tumors and GBM stem-like cell cultures.
CSIG-03. EGFR LIGAND SHIFTS THE ROLE OF EGFR FROM ONCOGENE TO TUMOR SUPPRESSOR IN EGFR AMPLIFIED GLIOBLASTOMA
Authors: Guo Gao , Gong Ke , Beckley Nicole , Zhang Yue , Yang Xiaoyao , Nayab Arifa , Habib Amyn
Published: November 2022

Glioblastoma (GBM) is a devastating cancer largely because of its highly invasive nature. Although pathways that promote GBM invasion have been identified, no pathway that actively suppresses GBM invasion is known and there is no treatment that specifically blocks GBM invasion. EGFR is a prime oncogene and is frequently amplified and mutated in GBM. However multiple clinical trials of EGFR inhibition in GBM have failed. Here, we demonstrate that EGFR ligands activate a tumor suppressor for the EGFR in EGFR-amplified GBMs. Analysis of TCGA data reveals that GBM patients with EGFR amplification and a low level of EGFR ligands have a worse prognosis compared to those with EGFR amplification and a high level of EGFR ligands. We identify the cytoskeletal BAR domain protein BIN3 as a critical suppressor of invasion upregulated by EGFR ligand in EGFR amplified GBMs. BIN3 inhibits a DOCK-7 Rho GTPase pathway, resulting in small hyper-proliferating noninvasive tumors and a better prognosis, while constitutive EGFR wild type or EGFRvIII signaling promotes invasion via activation of a TAB1-TAK1-NF-κB-EMP1 pathway, resulting in large tumors and worse survival in orthotopic models. Consistent with its role as a negative regulator of invasion, BIN3 is deleted in 10% of GBMs and increased expression of BIN3 confers a better prognosis. Thus, increased EGFR ligand shifts the role of EGFR from oncogene to tumor suppressor in EGFR amplified GBMs by suppressing invasion. We identify tofacitinib, a BBB penetrant drug as an upregulator of BIN3 and show that tofacitinib suppresses invasion and prolongs survival in orthotopic models. Our study identifies a new tumor suppressive function of EGFR in GBM and we propose the use of tofacitinib to directly target invasion in EGFR-amplified and EGFR ligand-poor GBMs.
CSIG-05. A GABP DOMINANT NEGATIVE APPROACH TO THE REVERSAL OF TUMOR IMMORTALITY
Authors: Stevers Nicholas , Collins Sara , Kasahara Noriyuki , Costello Joseph F
Published: November 2022

Immortality is a fundamental hallmark of human cancer cells and therapeutic reversal is of great interest. Telomerase Reverse Transcriptase promoter (TERTp) mutations reactivate TERT expression, the rate limiting step in telomerase activity and cellular immortality. TERTp mutations are the most common non-coding mutation across all cancer types, including glioblastoma (GBM), oligodendroglioma, medulloblastoma, and high-grade meningioma. While prior telomerase therapies lack tumor selectivity and are poorly tolerated, TERTp mutations and their regulation offer a unique opportunity for tumor specific reversal of cellular immortality. The mutations generate a de novo E26 Transformation Specific (ETS) binding motif that recruits the GA-Binding Protein (GABP) multimeric complex to reactivate TERT expression. Initially we found a GABP tetramer reactivates the mutant TERTp, suggesting targeting only the tetramer may induce telomere shortening and tumor cell death. However, through knocking out this tetramer in cell cultures, we found that the GABP dimer is upregulated and maintains TERT expression. In the absence of both the dimer and one tetramer isoform, many tumor cells senesce or undergo cell death. However, some tumor cells escape this fate via upregulation of a second GABP tetrameric paralogue that maintains TERT expression. Therefore, to block all GABP complexes with a single approach, we devised a transactivation domain null, putative dominant negative GABP subunit that ultimately could be delivered to patients via a replicating retroviral system. Introduction of the construct into TERTp mutant glioma cells significantly reduced TERT expression while not altering expression in TERTp wildtype GBM cells, suggesting tumor selectivity. TERT expression was also reduced in a second TERTp mutant tumor type. We are currently investigating the long-term effects of the dominant negative GABP on telomere length and tumor immortality. Dominant negative GABP is a promising candidate for tumor selective reversal of cellular immortality by inhibiting mutant TERTp activation by all GABP multimeric forms.
CSIG-06. RETROSPECTIVE REVIEW OF GLIOMA PATIENTS WITH CONFIRMED MET AMPLIFICATION OR FUSION
Authors: Tadipatri Ramya , Ekhator Chukwuyem , Miller Adela , Grewal Jai , Ahluwalia Manmeet , Fonkem Ekokobe
Published: November 2022

BACKGROUND

The MET pathway is a key oncogenic pathway in gliomas and is involved in tumor survival, angiogenesis, and invasion. MET alterations have been increasingly identified as a potential therapeutic target.

METHODS

Our objective was to further characterize the natural history of gliomas which harbor MET alterations and study outcomes as determined by progression free survival (PFS) and overall survival (OS).

RESULTS

We identified 22 glioma patients seen at our institution between 2015 and 2021 who had a MET alteration confirmed through comprehensive molecular profiling. There were 18 grade 4 astrocytomas/glioblastomas, 3 grade 3 gliomas (2 astrocytomas, 1 oligodendroglioma), and 1 grade 2 glioma (oligodendroglioma). MET amplification was observed in 13 patients and fusion in 16 patients , with 7 patients carrying both amplification and fusion alterations. MET fusion partners included PTPRZ1 (8 patients), CAPZA2 (6 patients), and ST7 (3 patients). Median OS was 12.8 months, and median PFS was 8.47 months. IDH1/2 mutation was associated with significantly improved survival, with an estimated OS of 146 months for IDH1/2 mutant tumors and 17.9 months for IDH1/2 wildtype tumors (p< 0.001), MGMT methylation status had no significant survival impact. No significant survival differences were observed between the fusion only, amplification only, and combined fusion and amplification groups. No therapy was predictive of improved outcomes.

CONCLUSION

MET amplification and fusion alterations were observed in both low and high grade gliomas, suggesting that they may be involved in early tumorigenesis. Survival outcomes were poor compared with historical data, with no differences observed between treatment groups, suggesting that current therapeutic modalities may be inadequate. This study indicates that MET may be an appropriate therapeutic target, laying the groundwork for clinical trials investigating MET-targeted agents.
CSIG-10. PHARMACO-PHOSPHO-PROTEO-GENOMICS OF PEDIATRIC HIGH-GRADE GLIOMAS – A PILOT STUDY
Authors: Findlay Izac , Staudt Dilana , Kearney Padraic , McEwen Holly , Duchatel Ryan , Jackson Evangeline , Beitaki Tyrone , Smith Nathan , Vitanza Nicholas , Firestein Ron , Cain Jason , Mueller Sabine , Pasquier Eddy , Koschmann Carl , Hulleman Esther , Nazarian Javad , Hansen Mitchell , Alvaro Frank , Davis Melissa , Waszak Sebastian , Dun Matthew
Published: November 2022

Pediatric high-grade gliomas (pHGG) are the leading cause of cancer-related death in children and young adults. Current treatment strategies are centered on maximal safe resection, followed by radiotherapy, and interrogation of the tumor genome to identify targetable mutations. Unfortunately, we are yet to see an improvement in patient outcomes with a median overall survival remaining 15-months. To improve patient outcomes, we have begun to characterize the genome, proteome, and phosphoproteome of 168 pHGGs to better understand the functional consequences of their somatic alterations as well as their influence of the tumor microenvironment. Employing a novel ‘pharmaco-phospho-proteo-genomics’ pipeline, we have analyzed pHGG cell lines and tumor tissue specimens at diagnosis, relapse (partial resection congenital glioblastoma), and autopsy. Genomic profiling was conducted utilizing the 523-gene TruSight Oncology 500 (TSO500) next-generation sequencing panel. Simultaneously, tumor proteomes and phosphoproteomes were characterized using our high-throughput global phospho-proteomic profiling technique termed pHASED (phospho Heavy-labelled-spiketide FAIMS StEpped-CV DDA). High-fidelity tumor proteomic and phospho-proteomic data were identified and compared to normal control brain samples. Across 40 pHGG samples, we identified 290 unique somatic alterations with a high predicted impact severity and quantified 7,345 unique proteins and 3,327 phosphoproteins. Gene panel sequencing of a critical pediatric glioblastoma patient sample identified 18 somatic alterations, eight of which had a high predicted impact severity, however, none were targetable. Conversely, phosphoproteomic profiling identified enriched MAPK and PRKCB signaling, relative to normal brain tissues, thereby encouraging the use of the TGA/FDA approved therapies trametinib (MAPKs) and enzastaurin (PRKCB). In vitro investigations confirmed the utility of these treatment approaches and in vivo patient derives xenograft mouse models for this sample are under investigation. This pilot study provides critical data to support the benefit of interrogating the genome, proteome, and phospho-proteome of these devastating tumours to aid in the selection/development of effective treatment strategies.
CSIG-15. DEFINING THE ROLE OF HISTONE H3 K27 METHYLATION AND H3.3 S31 PHOSPHORYLATION IN H3.3K27M MUTANT DIFFUSE MIDLINE GLIOMAS
Authors: Hakkim Faruck , Day Charles , Grigore Florina , Langfald Alyssa , Entin Igor , Hincliffe Edward , Robinson James
Published: November 2022

Diffuse midline gliomas (DMG) are aggressive high-grade gliomas that characteristically occurs in children. Heterozygous K27M mutation occurs in histone H3.3 as early events in DMG and are sufficient to drive a global reduction of H3K27 methylation (H3K27me) on all histone H3 variants. Histone H3K27me is responsible for epigenetic gene repression, and loss of K27me is associated with epigenetic dysregulation. However, histone H3.3 has a unique Chk1 phosphorylation site, Serine 31, critical for mitotic checkpoint regulation. Masking of S31 phosphorylation results in the abrogation of cell cycle checkpoints that monitor the alignment and segregation of chromosomes. DMG cells have decreased levels of mitotic S31P and are chromosomally unstable and aneuploid. Genomic-editing of H3.3 to revert the K27M mutation restores mitotic S31P and K27me and significantly reduces the rate of chromosome instability (CIN). Expression of H3.3 K27M or a non-phosphorylatable S31A mutant in normal, diploid cells results in chromosome missegregation and cell cycle checkpoint defects; and cells fail to undergo G1 cycle arrest leading to aneuploidy. H3.3 K27M and S31A inhibit p53 accumulation. In a mouse model of DMG, H3.3K27M and H3.3S31A promoted the development of high-grade gliomas, whereas H3.3WT controls did not. H3.3S31A is WT for K27me, demonstrating that loss of S31P is oncogenic. It remains unclear if it is the H3.3 K27M mutations or the associated loss of H3K27me3 that drives the reduction of S31P. The PRC2 complex is responsible for H3K27me, catalyzed by methyltransferases subunits EZH1 and EZH2. We used genome editing to knockout EZH1 and EZH2 in DMG cells and untransformed human diploid cell lines. Deletion of EZH2 causes a loss in K27 tri-methylation, and EZH1/2 knockouts causes a global loss of K27me. Ongoing studies examine how the loss of K27me affects gene expression, proliferation, H3.3S31P, chromosome missegregation, and aneuploid.
CSIG-19. THE PI3K/AKT/MTOR SIGNALING CASCADE MAY CONTRIBUTE TO SEX DIFFERENCES IN GLIOBLASTOMA
Authors: Sponagel Jasmin , Johnston Hannah , Gass Hannah , Nunez Elena , Ippolito Joseph , Rubin Joshua
Published: November 2022

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. It is more prevalent in males and female patients exhibit a survival advantage. Understanding the molecular mechanisms that underlie those sex differences could support novel treatment strategies. The PI3K/Akt/mTOR signaling cascade plays a crucial role in GBM development and progression as it is involved in the regulation of cellular growth, survival, nutrient sensing, and metabolic activity. To investigate whether the PI3K/Akt/mTOR signaling cascade may differ in male and female GBM patients, we first assessed survival data of male and female GBM patients using the TCGA RPPA phosphoproteome data set and found that changes in protein phosphorylation of components of the PI3K/Akt/mTOR pathway, including mTOR phosphorylation, worsen the outcome of male but not female GBM patients in a dose-dependent fashion. This was supported by in vitro experiments of murine transformed astrocytes where pathway stimulation via treatment with insulin, IGF-1, or EGF significantly increased pathway activity in male but not female cells. Furthermore, pathway inhibition via serum deprivation resulted in a significant decrease in pathway activity in male but not female cells, indicating that male transformed astrocytes exhibit higher sensitivity to inhibitory conditions (serum deprivation) and stimulatory conditions (insulin, IGF-1, or EGF treatment). Together, these data suggest that (i) the PI3K/Akt/mTOR pathway activity may affect male and female GBM outcome differently, and (ii) there are sex differences in the regulation of the PI3K/Akt/mTOR signaling cascade in GBM which may contribute to the sex disparity in GBM. Our data add to the growing body of literature regarding the presence of sex differences in PI3K/Akt/mTOR signaling in health and disease and provide important insight for the development of translatable approaches to treatment for male and female GBM patients.
CSIG-27. MODULATION OF GPR133 (ADGRD1) SIGNALING BY ITS INTRACELLULAR INTERACTION PARTNER EXTENDED SYNAPTOTAGMIN 1 (ESYT1)
Authors: Stephan Gabriele , Erdjument-Bromage Hediye , Liu Wenke , Frenster Joshua , Neubert Tomas , Fenyo David , Placantonakis Dimitris
Published: November 2022

GPR133 (ADGRD1), a member of the adhesion GPCR (aGPCR) family, has been recently implicated in the pathogenesis of glioblastoma. Like other aGPCRs, GPR133 is characterized by a large N-terminus, which possesses a conserved GPCR autoproteolysis-inducing (GAIN) domain catalyzing cleavage at a GPCR proteolysis site (GPS), resulting in a C-terminal fragment (CTF) and an N-terminal fragment (NTF). We showed that dissociation of the cleaved NTF and CTF at the plasma membrane correlates with increased GPR133 signaling, which is mediated by coupling to Gs and increase in cytosolic cAMP. Since GPR133 is absent in normal brain cells and de novo expressed in GBM, it represents a potential target for GBM treatment. However, little is known about the receptor’s protein interactome and its effects on receptor function and signaling. To identify intracellular interactors of GPR133, we used a proximity biotinylation/mass spectrometry approach in HEK293T cells expressing the wild-type or a signaling-incompetent mutant GPR133. We identified Extended Synaptotagmin 1 (ESYT1), a protein responsible for Ca2+-dependent formation of endoplasmic reticulum-plasma membrane bridges, as the strongest interaction partner for both versions of the receptor. Co-immunoprecipitation confirmed robust binding of ESYT1 to GPR133. Knockdown/knockout of ESYT1 increased GPR133 signaling, while overexpression of ESYT1 had the opposite effect. This effect was not mediated by changes in GPR133 expression or plasma membrane trafficking. We are currently performing additional studies to elucidate the mechanism whereby ESYT1 modulates GPR133 signaling. Collectively, these data suggest that GPR133 signaling is modulated through a novel cytosolic, signaling-independent interaction with ESYT1.
CSIG-31. T-TYPE CALCIUM CHANNEL BLOCKERS INDUCE METABOLIC ALTERATIONS AND APOPTOSIS IN MEDULLOBLASTOMA CELLS
Authors: Sedeeq Mohammed , Maklad Ahmed , Feng Zikai , Dutta Taush , Wilson Richard , Guven Nuri , Azimi Iman
Published: November 2022

Medulloblastoma (MB) is the most common malignant paediatric brain tumour. In our previous studies, in a screen of a panel of calcium channel modulators in our novel high-throughput 3D assay, we identified T-type (CaV3) channel inhibitors, mibefradil and NNC-55-0396 (NNC) as selective inhibitors of MB cell growth. Mibefradil was originally approved for the treatment of hypertension and angina pectoris, and recently successfully completed a Phase-I trial for recurrent high-grade glioma. NNC is an analogue of mibefradil with multiple advantages compared to mibefradil that makes it attractive for potential future clinical trials. T-type channels have a unique low voltage-dependent activation/inactivation, and many studies suggest that they have a direct regulatory role in controlling Ca2+ signalling in non-excitable tissues, including cancers. In this study, we characterized the effect of mibefradil and NNC on MB cells and elucidated their mechanism of action. This study demonstrates that the induction of toxicity in MB cells is selective to T-type but not to L-type Ca2+ channel inhibitors. Addition of CaV3 inhibitors to vincristine sensitised MB cells to this MB chemotherapeutic agent, suggesting an additive effect. Furthermore, CaV3 inhibitors induced cell death in MB cells via apoptosis. Supported by proteomics data and cellular assays, apoptotic cell death was associated with reduced mitochondrial membrane potential and reduced ATP levels, which suggests that both compounds alter the metabolism of MB cells. We also identified that CACNA1H (encoding the CaV3.2 protein) is significantly upregulated in aggressive Groups 3 and 4 MB patient samples, and its higher expression is associated with higher metastasis and lower patient survival rates. This study offers new insights into the action of mibefradil and NNC and will pave the way to test these molecules or their analogues in pre-clinical MB models alone and in combination with vincristine to assess their suitability as a potential MB therapy.
CSIG-35. TARGETING HYPOXIC CANCER CELLS IN MEDULLOBLASTOMA
Authors: Kuranaga Yuki , Van Meir Erwin
Published: November 2022

New treatment approaches are urgently needed for children with medulloblastoma (MB) to augment survival and reduce the long-term side effects from the treatment. Malignant tumors often display hypoxic regions where hypoxia-induced transcription factors (HIFs) increase the expression of genes that promote tumor growth (e.g., metabolic adaptation and invasion/metastasis). Based on the known roles of HIF in many solid tumors, we hypothesized that Hypoxia/HIFs contribute to MB cell growth and targeting HIF proteins is a viable strategy for treating MB. To test our hypothesis, first, we exposed MB cell lines (ONS-76; Shh and D556; Group 3) to 1% hypoxia in cell culture. We found increased expression of both HIF1a and HIF2a proteins and activation of a hypoxia-response element driven luciferase reporter assay. Treatment of the cells with 64B, an arylsulfonamide HIF inhibitor abrogated reporter activity and reduced expression of hypoxia-induced genes (GLUT-1, VEGF). Next, we used CRISPR/Cas9 to knockout singly or in combination HIF1a/HIF2a genes. The inhibition of HIF reporter activity by 64B was partially canceled in single HIF-KO cells and completely canceled in double HIFs-KO cells, showing both isoforms were active. Second, to investigate HIFs can be therapeutic targets, we established orthotopic human MB xenografts in mice. We first examined the tumor for the presence of hypoxic areas and used pimonidazole (Hypoxyprobe) injections followed by immunohistochemistry and found hypoxic areas throughout the tumors where tumor cells expressed HIF1a and/or HIF2a. Evaluation of human MB samples for HIF1/2 expression is also ongoing. Next, we plan on injecting mice with cells devoid of HIFs genes and examine their growth. We will also treat the mice with 64B to test whether it has anti-tumor effects and extends survival. In conclusion, these results indicate that HIF activation is part of the pathobiology of MB and HIF proteins may be a target for MB treatment.
CSIG-41. SENSITIZING CANCER CELLS TO TUMOR TREATING FIELDS (TTFIELDS) BY INHIBITION OF PI3K
Authors: Klein-Goldberg Anat , Voloshin Tali , Zemer-Tov Efrat , Paz Rom , Koren Lilach , Wainer-Katsir Kerem , Volodin Alexandra , Koltun Bella , Brant Boris , Barsheshet Yiftah , Kan Tal , Cohen Gadi , David Cfir , Khalil Tharwat Haj , Haber Adi , Giladi Moshe , Weinberg Uri , Palti Yoram
Published: November 2022

Tumor Treating Fields (TTFields) are alternating electric fields, which disrupt cellular process critical for cancer cell survival and tumor progression. TTFields therapy is approved for the treatment of glioblastoma (GBM) and unresectable malignant pleural mesothelioma, and is being tested in clinical studies for the treatment of other solid tumors, including ovarian cancer, non-small cell lung carcinoma (NSCLC), and hepatocellular carcinoma (HCC). The current study aimed to detect potential mechanisms that may reduce cellular sensitivity to TTFields, and target these pathways in order to re-sensitize the cells to TTFields. Cancer cells (Ovarian A2780, GBM U-87 MG, and NSCLC H1299) that display reduced sensitivity to TTFields were generated by continuous long-term TTFields application (7 or 13 days, depending on the cell line). A Luminex multiplex assay revealed activation of the PI3K/AKT/mTOR signaling pathway in these cells, with significant increases in phosphorylation levels of AKT and RPS6. This elevation was also observed by immunohistochemistry in tumor sections from N1S1 HCC tumor-bearing rats treated with TTFields relative to sham. Treatment of cells with PI3K inhibitors re-sensitized them to TTFields and downregulated the phosphorylation of AKT. Concomitant application of TTFields with the PI3K inhibitor alpelisib in mice orthotopically implanted with MOSE-L firefly luciferase (FFL) ovarian cancer cells resulted in enhanced efficacy, as determined by In Vivo Imaging System (IVIS) measurements of tumor volume. Overall, this study demonstrated that the PI3K/AKT/mTOR signaling pathway is involved in reduced cancer cell sensitivity to long-term application of TTFields, and that re-sensitization may be achieved with relevant inhibitors. The results provide a rationale for further examining the potential benefit of TTFields concomitant with PI3K inhibitors.
RADT-02. IMPACT OF SINGLE AND DUAL IMMUNE CHECKPOINT BLOCKADE ON RISK OF RADIATION NECROSIS AND LOCAL CONTROL AMONG PATIENTS WITH BRAIN METASTASES TREATED WITH STEREOTACTIC RADIOSURGERY
Authors: Vaios Eugene , Shenker Rachel , Hendrickson Peter , D'Anna Rachel , Niedzwiecki Donna , Carpenter David , Floyd Warren , Winter Sebastian , Dietrich Jorg , Salama April , Clarke Jeffrey , Allen Karen , Mullikin Trey , Floyd Scott , Kirkpatrick John , Reitman Zachary
Published: November 2022

PURPOSE

Single and dual immune checkpoint inhibition (ICPI) are common treatment options for patients, particularly with melanoma and non-small cell lung cancer (NSCLC). While data suggest a cancer control benefit of combining stereotactic radiosurgery (SRS) and ICPI, we hypothesized that concurrent dual ICPI and SRS increases risk for radiation necrosis (RN).

METHODS

We retrospectively reviewed patients with metastatic melanoma or NSCLC treated with SRS for intact brain metastases from 2014-2020. Patients were stratified by receipt of dual ICPI, single ICPI, and SRS alone. Concurrent ICPI was defined as treatment within 30 days of SRS. RN and local control (LC) were biopsy confirmed or determined radiographically and longitudinally, in combination with clinical assessment and steroid use. Kaplan-Meier estimates were used to compare rates of RN and LC between cohorts.

RESULTS

673 brain lesions from 93 patients met inclusion criteria [median (Q1, Q3): 5.0 (2.0-10.0) lesions per patient]. Median follow-up was 8.1 months (95% CI: 7.3-8.7). Histologies included melanoma (53.5%), adenocarcinoma NSCLC (27.3%), squamous cell NSCLC (6.1%), and NSCLC NOS (6.1%). 88 lesions from 25 patients (27%) developed RN and 11 (13%) were biopsy-proven. ICPI use was enriched among lesions that developed RN (85.2%) versus those that did not (19.8%). RN was associated with concurrent ICPI (p< 0.001). Freedom from RN at 6 months was 80% for dual ICPI, 82% for single ICPI, and 97% for SRS alone; 12-month rates were 78% in each ICPI cohort and 95% with SRS alone (p=0.0002). LC differed among dual (97.5%), single (88.7%), and no ICPI (79.7%, p < 0.001). There was a trend toward improved LC with RN (96.6% vs 91.8%; p=0.087).

CONCLUSIONS

In a large cohort of brain metastases, we observed increased risk of RN and improved LC with SRS plus concurrent dual or single ICPI. Awareness of these associations is critical for patient management.
RADT-09. PREOPERATIVE STEREOTACTIC RADIOSURGERY FOR LARGE CEREBRAL METASTASES – A PROSPECTIVE CLINICAL TRIAL SHOWING EFFICACY AND DECREASED TIME TO RESUMPTION OF IMMUNOTHERAPY
Authors: Mallela Arka , Iheagwara Uzoma , Fogg David , Anthony Austin Walker , Zhang Xiaoran , Gersey Zachary , Abou-Al-Shaar Hussam , Xu Emily , Zinn Pascal , Burton Steve , Quinn Annette , Clump David , Siddiqui Zaid , Amankulor Nduka
Published: November 2022

INTRODUCTION

Preoperative stereotactic radiosurgery (SRS) is an emerging therapeutic strategy for treatment of brain metastasis requiring surgical resection. Prior reports have been highly-controlled, excluding very large brain metastases. Here, we report a prospectively-collected preoperative series reflective of a real-world neurosurgical patient cohort, with a particular focus on resumption of immunotherapy.

METHODS

Our series included Nf27 prospective preoperative compared to concurrently collected Nf37 postoperative patients. Demographic, SRS volumes/dose, pre/postoperative immunotherapy, and clinical outcomes were collected. Subsequent univariate comparisons between groups were performed.

RESULTS

Pre/postoperative tumor volumes (19.1 [range: 2.4-65.1] vs. 12.5 [3.6-80.3]cc, p=0.20); local tumor control at 1 year (77.6% vs. 71.4%, p=0.616); overall survival (1.0 [0.8-2.2] vs. 1.3 [0.7-NR] years, p=0.8) were comparable between groups. Rates of LMD (3.7% vs. 8.0%, p=0.39) and subsequent WBRT (8.0% vs. 16.2%, p=0.46) were not statistically different. OR time (168 vs. 173 min, p=0.64), estimated blood loss (133 vs. 180 mL, p=0.31), and wound infections (1 vs. 1, p=1.00) were not significantly different.An ostensible advantage to preoperative SRS is that steroids may be weaned more quickly to allow resumption of immunotherapy. Median steroid taper length was significantly shorter (8 vs. 21.5 days, p=0.007), and median daily Dexamethasone dose at 1 week post-op was significantly lower in the preoperative group (1 vs. 7mg d, p=0.007). All preoperative patients eligible for resumption of immunotherapy (Nf5) resumed treatment within 6 weeks. Conversely, only 2/6 in postoperative patients resumed treatment within 6 weeks, (p=0.07), while 4 failed to resume immunotherapy altogether. In patients initiating immunotherapy after OR/SRS, time to initiation was non-significantly lower in the preoperative group (52[38-84] vs.163[108-208] days, p=0.06).

CONCLUSIONS

Preoperative SRS is safe and provides comparable local tumor control rate even in large tumors ( >20cc). In patients requiring resumption of immunotherapy, preoperative SRS may provide a faster pathway towards resumption of systemic therapy.
RADT-15. 18F-DOPA PET/CT SURVEILLANCE FOR GLIOBLASTOMA: A RADIOMIC MODEL FROM A PROSPECTIVE PHASE II CLINICAL TRIAL PREDICTING SURVIVAL IN IDH-WILDTYPE, MGMT-UNMETHYLATED PATIENTS
Authors: Qian Jing , Pafundi Deanna , Breen William , Brown Paul , Hunt Christopher , Jacobson Mark , Johnson Derek , Kaufmann Timothy , Kemp Bradley , Kizilbash Sani , Lowe Val , Ruff Michael , Sarkaria Jann , Uhm Joon , Chan Tseung Hok Seum Wan , Yan Elizabeth , Zhang Yan , Laack Nadia , Brinkmann Debra
Published: November 2022

BACKGROUND

Interpretation of serial magnetic resonance imaging (MRI) for glioblastoma following radiation therapy (RT) is complicated by difficulty differentiating tumor from treatment-related changes, even using updated RANO criteria. The incorporation of novel imaging such as 3,4-dihydroxy-6-[18F]-fluoro-L-phenylalanine (18F-DOPA) PET/CT to post-treatment serial imaging may improve prognostication and better facilitate future treatment decisions.

METHODS

The secondary analysis of a recent phase II prospective clinical trial of 18F-DOPA PET/CT-directed dose-escalated RT included patients with IDH-wildtype, MGMT-unmethylated glioblastoma who underwent post-treatment serial 18F-DOPA PET/CT surveillance. Quantitative features were extracted from pre-RT and post-RT serial PET/CT images, and robust prognostic features were selected using an in-house workflow. Both an automated machine learning (ML) algorithm and an interpretable ML algorithm were utilized to correlate surveillance PET image features with subsequent survival of greater than 12 months versus less than 12 months from the surveillance timepoint. Changes from pre-RT to post-RT PET/CT (delta model) were also assessed for association with post-RT survival and validated with a separate cohort.

RESULTS

Thirty-five patients with IDH-wildtype, MGMT-unmethylated glioblastoma who had at least one available (range: 1-14) post-treatment 18F-DOPA PET/CT were included. Twenty-four were used for model training, while 11 were used for validation. Ultimately, a five-feature post-RT model utilizing two shape, two texture, and one first-order radiomic feature was selected. For the delta model, five texture, two shape, and one first order radiomic feature were selected. The models show 90% accuracy in predicting survival < 12 months post-surveillance on the training set, and 68%-73% accuracy (AUC 0.64-0.73) for the validation cohort. Delta features were significantly associated with overall survival (p < 0.05).

CONCLUSIONS

Post-RT serial 18F-DOPA PET/CT imaging can help distinguish true tumor progression in patients with glioblastoma using a radiomics model. Tumor response evaluated for changes from pre-RT to post-RT 18F-DOPA PET/CT also predicted subsequent overall survival.