bluuebunny/crossref_metadata_2025 huggingface dataset:
CNSC-36. VRK1 IS A PARALOG SYNTHETIC LETHAL TARGET IN VRK2-METHYLATED GLIOBLASTOMA
Authors: Shields Julie , Meier Samuel , Bandi Madhavi , Ferdinez Maria Dam , Engel Justin , Mulkearns-Hubert Erin , Hajdari Nicole , Shen Binzhang , Hubert Christopher , Mitchell Kelly , Zhang Wenhai , Zhao Shan-chuan , Bejnood Alborz , Zhang Minjie , Sjin Robert Tjin Tham , Wilker Erik , Lathia Justin , Andersen Jannik , Chen Yingnan , Li Fang , Weber Barbara , Huang Alan , Emmanuel Natasha
Published: November 2022

Synthetic lethality — a genetic interaction that results in cell death when two genetic deficiencies co-occur but not when either deficiency occurs alone — can be co-opted for cancer therapeutics. A pair of paralog genes is among the most straightforward synthetic lethal interaction by virtue of their redundant functions. Here we demonstrate a paralog-based synthetic lethality by targeting Vaccinia-Related Kinase 1 (VRK1) in Vaccinia-Related Kinase 2 (VRK2)-methylated glioblastoma (GBM). VRK2 is silenced by promoter methylation in approximately two-thirds of GBM, an aggressive cancer with few available targeted therapies. Genetic knockdown of VRK1 in VRK2-methylated GBM cell lines and patient-derived models was lethal and resulted in decreased activity of the downstream substrate Barrier to Autointegration Factor (BAF), a regulator of post-mitotic nuclear envelope formation. VRK1 knockdown, and thus reduced BAF activity, caused nuclear lobulation, blebbing and micronucleation, which subsequently resulted in G2/M arrest and DNA damage. The VRK1-VRK2 synthetic lethal interaction was dependent on VRK1 kinase activity and was rescued by ectopic VRK2 expression. Knockdown of VRK1 led to robust tumor growth inhibition in VRK2-methylated GBM xenografts. These results indicate that inhibiting VRK1 kinase activity could be a viable therapeutic strategy in VRK2-methylated GBM.
MODL-41. ORGANOTYPIC BRAIN SLICE CULTURE PLATFORM AS A NOVEL PRE-CLINICAL MODEL FOR PATIENT DERIVED CELL LINES
Authors: Mann Breanna , Zhang Xiaopei , Bell Noah , Adefolaju Adebimpe , Dasari Rajaneekar , Hingtgen Shawn , Satterlee Andrew
Published: November 2022

The lack of experimentally tractable models that recapitulate brain structure and function represents a major impediment in the development of novel treatment options for brain cancer. In vitro assays, though fast and high throughput, produce artificial results which do not fully encompass the clinically relevant outcome. Low passage patient derived cell lines offer an advantage over immortalized cell lines in terms of relation to the clinical presentation. Low passage patient derived cell lines, however, show slower growth and decreased tumorgenicity. We have previously shown that the use of organotypic brain slice culture (OBSCs) can recapitulate growth patterns and migration which is seen in vivo. We have further expanded the OBSCs to study their use as a model for patient derived cell lines. Some cell lines such as MS21, a human glioblastoma, or IFF109-DMG, a human pediatric diffuse midline glioma, are simply supported over a four-day time period, whereas other lines such as IFF105-DIPG, a pediatric human diffuse intrinsic pontine glioma, can grow over 3-fold in a 4-day time period. Additionally, we can assess treatment response to a variety of clinically relevant chemotherapeutics. The decreased variability with the OBSCs allows for the assessment of minute differences in treatment response. Overall, these results suggest the OBSC with patient derived cell lines have the potential to be effective models to accelerate preclinical evaluation of therapeutics and guide drug development towards more effective treatment strategies.
CNSC-37. ATYPICAL GERMLINE MUTATIONS AND PRIMARY CNS TUMORS: A SINGLE-CENTER CASE SERIES AND LITERATURE REVIEW
Authors: Shah Trusha , Ruiz Amber , Ross Che , Dubard-Gault Marianne , Graber Jerome
Published: November 2022

A growing spectrum of germline mutations and their association with primary CNS tumors has been described over the last decade. These germline mutations are implicated in regulation of DNA integrity and repair, telomere maintenance, and cell cycle regulation resulting in aberrant cellular proliferation and immune surveillance evasion. To date, there are no well-defined guidelines for primary CNS tumor surveillance for these patients. Moreover, the exact role of traditional chemotherapy, immunotherapy, and radiation is unclear. We present a single-institution case series of adult patients with primary CNS tumors with atypical germline mutations and literature review. The most common germline mutations encountered involved heterozygous mutations of one of the mismatch repair (MMR) genes (MSH2, PMS2, MLH1). All patients developed high-grade gliomas defined as astrocytoma or glioblastoma WHO grade 4 based on the WHO 2021 classification. Germline mutations involving RAD51C, POT1, IDH1/2, MutyH, NTHL1, PALB2, and CHEK2 were associated with gliomas of variable grades and molecular subtypes. Non-glial tumors, including atypical rhabdoid tumors, medulloblastomas, hemangioblastomas, and meningiomas were found in association with germline SMARCB1, ATM, BRCA1, and CHEK2 mutations respectively. Review of the existing literature and knowledge obtained from our case series suggests several important concepts: (1) there is an increased risk of both glioma and non-glial tumor types in several atypical germline mutations, (2) multiple risk genes or variants of unknown significance likely contribute to primary brain tumor formation, (3) germline MMR mutations associated with high-grade glioma may be MMR gene-specific with potential increased risk of resistance and/or disease progression in the presence of alkylating agents such as temozolomide, (4) several germline mutations may increase risk of secondary tumor development after radiation, and (5) the role of screening for atypical germline mutations and subsequent disease surveillance remain unanswered.
STEM-01. GLIOMA STEM CELLS ACTIVATE PLATELETS BY PLASMA-INDEPENDENT THROMBIN PRODUCTION TO PROMOTE GBM TUMORIGENESIS
Authors: Sloan Anthony , Lee-Poturalski Christine , Hoffman Harry , Harris Peggy , Elder Theresa , Cioffi Gino , Barnholtz-Sloan Jill , Rich Jeremy N , Jankowsky Eckhard , Gupta Anirban Sen , Sloan Andrew
Published: November 2022

The interaction between platelets and cancer cells has been underexplored in solid tumor models that do not metastasize, for example glioblastoma (GBM) for which metastasis is rare. Histologically, it is known that glioma stem cells (GSCs) are found in perivascular and pseudsopalisading regions of GBM, which are also areas of platelet localization. High platelet counts have been associated with poor clinical outcome in many cancers. While platelets are known to promote progression of other tumors, mechanisms by which platelets influence GBM oncogenesis are unknown. Here, we aimed to understand how the bidirectional interaction between platelets and GSCs drives GBM oncogenesis. Male and female NSG mice were transplanted with patient derived GSC lines and treated with antiplatelet and anti-thrombin inhibitors. Immunofluorescence, qPCR, and Western blots were used to determine expression of coagulation mechanism in GBM tissue and subsequent GSC lines. We demonstrate that GSCs co-opt and activate platelets to promote GBM tumor progression. GSCs endogenously produce all coagulation factors of the intrinsic and extrinsic cascade generating thrombin and activating platelets in the absence of plasma. Conversely, inhibition of platelet activation and of thrombin production by GSCs abrogates platelet-mediated GSC self-renewal and growth (p< 0.0005). Similarly, inhibiting intratumoral thrombin production and function decreases tumor formation in vivo (p< 0.005). These studies challenge and revise the longstanding view that the complete coagulation cascades are found only in the liver and plasma demonstrating that cancer stem cells readily execute a highly liver-specific gene expression program that is mechanistically linked to GBM oncogenesis. We show that cancer cells can co-opt non-cancer cells to promote tumor growth, by uncovering the preferential relationship between platelets and GSCs that drive GBM malignancies and identifies a therapeutically targetable novel interaction.
STEM-06. NUCLEAR STRUCTURE MAINTAINS GLIOBLASTOMA STEM CELLS
Authors: Duan Tingting , Bhargava Shruti , Rich Jeremy
Published: November 2022

Glioblastoma stem cells (GSCs) contribute to therapeutic resistance, promote tumor angiogenesis, and evade anti-tumor immune responses. We and others have shown that GSCs display differential regulation of their epigenetic landscapes and metabolic profiles and contribute to the striking cellular heterogeneity and complex tumor microenvironments. Here, we find that GSCs undergo dramatic changes in chromatin organization and nuclear morphology upon differentiation. Leveraging unbiased RNA sequencing and protein profiling screens comparing GSCs and differentiated glioma cells, we identified nuclear membrane molecular regulation that defines nuclear size and lipid regulation. Genetic or pharmacologic targeting of nuclear envelope function reduced cancer stem cell proliferation, self-renewal, and in vivo tumor growth. Collectively, we define a novel role of the nuclear envelope in maintaining chromatin state and cancer stem cell self-renewing, informing the development of novel potential therapeutic paradigms for glioblastoma.
STEM-07. DEFINING THE ROLE OF HUMAN ENDOGENOUS RETROVIRUS-K (HML-2) IN THE GLIOBLASTOMA STEM-CELL NICHE
Authors: Shah Ashish , Rivas Sarah , Doucet-Ohare Tara , Govindarajan Vaidya , DeMarino Catherine , Ampie Leo , Banasavadi-Siddegowda Yeshavanath , Maric Dragan , Garcia-Montojo Marta , Suter Rob , Lee Myoung-Hwa , Walbridge Stuart , Zaghloul Kareem , Steiner Joseph , Johnson Kory , Gilbert Mark , Heiss John , Nath Avindra
Published: November 2022

Human Endogenous Retrovirus (HERV) are ancestral viral relics that comprise nearly 8% of the human genome. Although silenced in normal tissues, the most recently integrated provirus HERV-K (HML-2) can be pathologically reactivated in certain cancers. Here, we report pathological expression of HML-2 transcripts in human malignant gliomas in cerebrospinal fluid (HERV-K DNA/RPP30=35.2±8.8 vs 23.1±6.7, n=18, p=0.02) and tumors (HERV-K RNA/HPRTmean=1.15±0.2 vs. 0.5±0.2, p=0.01, n=20) compared to epilepsy controls. Aberrant HML-2 expression corresponded to a unique stem-cell niche using multivoxel automated segmentation. Using a tailored single-cell RNA sequencing pipeline to detect retrotransposons, we identified glioblastoma cellular populations with elevated HML-2 transcripts in neural progenitor-like cells that can drive cellular plasticity (ANOVA, multiple-testing correction, p< 0.001). Using CRISPR technology, we demonstrate that HML-2 is critical to maintenance of glioblastoma stemness and tumorigenesis in both glioblastoma neurospheres and intracranial orthotopic murine models (OS: 26 days vs. 18.6, p=0.0008, n=20). Downregulation of HERV-K using CRISPRi reduces the glioblastoma neurosphere formation (2-way ANOVA, p< 0.0001). and HERV-K env, Polymerase, OCT4 and Nestin transcripts (2-way ANOVA p< 0.001). Using Chromatin Immunoprecipitation, we determined that this interaction in gliomas is likely mediated by the nuclear transcription factor OCT4 which binds to an HML-2 specific Long-Terminal Repeat (LTR5Hs). Moreover, using Transmission Electron Microscopy, we discovered that some glioma stem-cells form immature retroviral virions in glioblastoma. Inhibiting HML-2 expression with nucleoside reverse transcriptase inhibitors reduces extracellular reverse transcriptase (One-way ANOVA, p< 0.05), tumor viability (IC50=75.8-123.1 uM), and pluripotency (One-way ANOVA, p< 0.01). Our results suggest that HML-2 is overexpressed in the cancer stem-cell niche of glioblastoma. Since persistence of glioblastoma stem-cells is considered responsible for treatment resistance and recurrence, HML-2 may serve as a unique therapeutic target in glioblastoma.
STEM-10. TEMPORAL MULTI-MODAL SINGLE-CELL ANALYSES REVEAL DYNAMIC INTERACTIONS BETWEEN GLIOBLASTOMA AND CAR-T CELLS AND IMMUNOLOGIC MODULATORS OF CANCER STEM CELL STATE
Authors: Zhang Daniel , Wang Xin , Sun Yusha , O'Rourke Donald , Ming Guo-Li , Song Hongjun
Published: November 2022

CAR-T cell therapy is a promising new immunotherapy for a number of difficult-to-treat cancers, however, it has yet to yield broad success in glioblastoma (GBM). In particular, tumor heterogeneity presents a major therapeutic challenge, and a detailed understanding of the complex interplay between different neoplastic, non-neoplastic, and CAR-T cells is critical for developing better treatments. Using a patient-derived GBM organoid model of CAR-T cell therapy, we performed single-cell multi-omics to examine the longitudinal dynamics of the adaptive tumor response, changes in cell states, and evolution of cell-to-cell interaction networks. We find that all tumor cell types - neoplastic and non-neoplastic - respond to CAR-T cell activity, and they generate to an initially anti-tumor, but subsequently pro-tumor and immune-inhibitory microenvironment, which is accompanied by eventual CAR-T cell dysfunction and exhaustion. Unexpectedly, CAR-T cell activity also leads to attenuation of glioma stem-like states in both antigen-positive and antigen-negative neoplastic cells and reduces their proliferation via diffusible factors, of which IFNɣ is required but not sufficient. These findings are supported by analyses in patient samples from CAR-T cell therapy clinical trials, and they are consistent across both de novo and recurrent tumors with different somatic mutational landscapes. Our study unravels how the complex heterogeneity of GBM interacts with CAR-T cell therapy, and we identify previously unappreciated possibilities to affect antigen-negative neoplastic cells in ways that may be further augmented for enhanced therapeutic efficacy.
STEM-11. POLYAMINE METABOLISM AND INFLAMMATORY STATE CONTROL A VIRAL MIMICRY-DRIVEN QUIESCENT PHENOTYPE IN GLIOMA STEM CELLS (GSCS)
Authors: Chesnelong Charles , Charton Connor , Dirks Peter
Published: November 2022

The rate limiting enzyme of the polyamine pathway, SAT1, is significantly upregulated in non-cycling GSCs. However, higher SAT1 expression predicts poorer survival in GBM suggesting a role in quiescence and recurrence post-treatment. Treating GSCs with N1N11-Diethylnorspermine (DENSpm) recapitulates SAT1 upregulation and triggers a chemoprotective, long-term reversible growth arrest, while not affecting expression of stem, differentiation, nor senescence-associated markers. Remarkably, DENSpm strongly induces double-stranded RNA in GSCs. Interestingly, S-Adenosyl methionine (SAM) is an essential aminopropyl donor for polyamine biosynthesis. We found a significant correlation between DNA methylation, SAM, and polyamines across GSCs suggesting that SAM depletion may cause epigenetic and post-transcriptional alterations leading to re-activation of endogenous retroviral elements (ERVs) and viral mimicry-driven quiescence. However, while we confirmed DENSpm-induced SAM depletion, we did not detect DNA, histone, nor RNA demethylation. Noteworthy, ERVs are the most accessible transposable elements in GSCs indicative of a poised state. Furthermore, the correlation between expression of ERVs and interferon stimulated genes reveals a spectrum of viral mimicry amongst GSCs. Using a larger cohort of GSCs and hfNSCs, we identified three DENSpm response subgroups. A resistant subgroup comprised of cells able to proliferate through treatment, a reversible one whereby cells become quiescent and readily re-enter cell cycle post-treatment, and an irreversible consisting of cells that enter a deep quiescent state which they cannot escape. Importantly, we found that DENSpm response correlates with the cell's basal inflammatory state and the activity of viral defense pathways. These results indicate that a viral mimicry-driven quiescence may be specific to slower-growing neurodevelopmental GSCs, while more proliferative, inflammatory-poised GSCs are impervious to these additional cues. We propose that polyamine metabolism, glioma stem cell hierarchy and tumour inflammatory context play a central role in regulating quiescence and have important clinical implications in the initial response to treatment, aggressiveness, and ultimately recurrence of GBM.
STEM-14. NEAR-INSTANT DETECTION OF GLIOMA STEM CELLS IN LIVE HUMAN GBM-TISSUE
Authors: Persson Oscar , Neofytou Christina , Keane Lily , Jensdottir Margret , Joseph Bertrand , Hermanson Ola , Ilkhanizadeh Shirin
Published: November 2022

Emerging research suggests that failure to target glioma stem cells (GSCs) rather than the inability to remove tumors through surgery, radiation, or chemotherapy, explains the poor survival of GBM patients. In this study, a luminescent conjugated oligothiophene (LCO), named GlioStem (p-HTMI), is used for non-invasive and non-amplified real-time detection of GSCs in live human GBM-tissue. More than 90 patient samples were stained, quantified, and analyzed by fluorescent microscopy for the presence of Gliostem-positive (GS+) cells. Approximately 30 of the samples were FACS-sorted for GS+ and GS- cells where quantification by FACS could verify microscopy results. In addition, GS+ cells were shown to express significantly higher levels of stem cell markers (CD271, CD133, PDGFRa, CD44) in FACS-experiments. Bulk RNA sequencing of 7 GBM patient samples with paired GS+ and GS- sorted cells revealed that GS+ patient samples clustered together, whereas the GS- populations did not cluster together neither with each other nor with the GS+ populations. These data suggest a distinct heterogeneity in the GS- samples and a certain level of homogeneity regarding the GS+ populations, independent of intra-patient or patient-to-patient heterogeneity. Moreover, the GS+ samples were found to express significantly higher levels of stem cell markers including SOX10, OLIG1/2, and ASCL1, compared to the GS- samples. More specifically, the GS+ samples exhibited significantly higher expression of 35 genes associated with stemness compared to the GS- samples, with most markers being associated with pre-oligodendrocyte precursor cells (pre-OPCs) and pro-neural subtypes. Our results suggest that GlioStem is a versatile tool for near-instant and selective detection of GSCs in live tumor tissue. Detecting and eliminating these cells during tumor resection may therefore be an important aim in efficiently preventing tumor regrowth and would mean a crucial step towards increased patient survival.
STEM-19. LIVE DETECTION OF NEURAL STEM AND GLIOBLASTOMA CELLS BY A LUMINESCENT CONJUGATED OLIGOTHIOPHENE DERIVATIVE
Authors: Ilkhanizadeh Shirin , Gracias Aileen , Åslund Andreas K O , Bäck Marcus , Simon Rozalyn , Neofytou Christina , Rraklli Vilma , Migliori Bianca , Kavanagh Edel , Nelander Sven , Westermark Bengt , Uhrbom Lene , Forsberg-Nilsson Karin , Teixeira Ana I , Konradsson Peter , Uhlén Per , Holmberg Johan , Joseph Bertrand , Nilsson K Peter R , Hermanson Ola
Published: November 2022

Glioblastoma (GBM) is an aggressive nervous system tumor with a mean survival time of 12-14 months. Cells with neural stem cell-like properties can be derived from GBM tumors. These cells seem to escape conventional irradiation treatment, chemotherapy, and surgery, and may play a crucial role for relapse. It is therefore urgent to develop novel approaches for reliable detection of neural stem cell-like cells in GBM. Here we report a luminescent conjugated oligothiophene (LCO), named GlioStem (p-HTMI), for non-invasive and non-amplified real-time detection of live human patient-derived GBM cells and embryonic neural stem/progenitor cells (NSPCs). Within a maximum of 10 minutes after administration of the molecule in vitro, in the existing media, fluorescence emission was observed without any modulation of the cells or additional vehicle, resulting in efficient detection of cytoplasmic luminescent signal in NSPCs or GBM cells from rodents and humans, detectable at Alexa488/GFP wavelength. GlioStem is functionalized with a methylated imidazole moiety resembling the side chain of histidine/histamine, and non-methylated analogues were not functional. In vitro, GlioStem was shown to identify fetal cortical NSPCs from rat (FGF2-expanded), embryonic stem cell-derived NSPCs from mouse (FGF2/EGF-expanded), and FGF2-exposed C6 glioma cell cultures from rat, but not any other cell types investigated. Cell sorting experiments of patient-derived, FGF2/EGF-expanded GBM cells demonstrated that GlioStem in addition to NSPC-markers like Nestin and Sox2 labeled the same population (overlap > 90%) of cells as CD271, a proposed marker for stem cell-like cells and rapidly migrating cells in glioblastoma. Our results suggest that the LCO GlioStem is a versatile tool for immediate and selective detection of subpopulations of neural stem and glioma cells.
STEM-22. EFFECT OF A SRC INHIBITORY PEPTIDE BASED ON CONNEXIN43 ON NEURAL STEM CELLS WITH GLIOMA-DRIVER MUTATIONS
Authors: Álvarez-Vázquez Andrea , San-Segundo Laura , Cerveró-García Pilar , Flores-Hernández Raquel , Ollauri-Ibáñez Claudia , Segura-Collar Berta , Sánchez-Gómez Pilar , Pollard Steven M , Tabernero Arantxa
Published: November 2022

Glioblastomas are one of the most malignant tumors. Glioma Stem Cells (GSCs) are a subpopulation of cells resistant to standard treatments, responsible for tumor recurrence. Strong evidence supports that Neural Stem Cells (NSCs) from the subventricular zone (SVZ) are the origin of GSCs. This transition frequently concurs with epidermal growth factor receptor (EGFR) overexpression or mutations, such as EGFRvIII. Our group designed a cell penetrating peptide based on connexin43 (TAT-Cx43266-283) that inhibits the oncoprotein c-Src and therefore targets GSCs, increasing survival in glioma-bearing mice. Because c-Src and EGFR signaling are closely related, we explored the effect of TAT-Cx43266-283 in the transition of NSCs to GSCs. We compared the effect of TAT-Cx43266-283, control peptides, temozolomide and erlotinib, a common EGFR inhibitor, in SVZ NSCs (Control-NSCs) and murine NSC and patient-derived GSCs with key glioma-driver mutations in Nf1, PTEN and EGFR. EGFR signaling pathway was analyzed by Western blot. Furthermore, we are analyzing the effect of TAT-Cx43266-283 in a mouse model where tumors originate from SVZ NSCs with glioma-driver mutations. Our results showed a strong effect of TAT-Cx43266-283 in cell viability of NSCs and patient-derived GSCs with EGFR amplification or active EGFRvIII, without significant effects in Control-NSCs. Importantly, we found a lower effect of temozolomide, erlotinib, and other control peptides in these cells. Western blot analyses suggest that TAT-Cx43266-283 decreases EGFR, EGFRvIII and c-Src activity. Preliminary in vivo results suggest that TAT-Cx43266-283 decreases tumor size and enhances survival in mice bearing gliomas initiated by altered NSCs. So far, our results show that TAT-Cx43266-283 impairs EGFR signaling pathway with the subsequent reduction in the proliferation and survival of NSCs or GSCs with EGFR alterations. These results stress the relevance of TAT-Cx43266-283 as a future therapy against glioblastoma, alone or in combination with other treatments.
STEM-26. ASTROCYTIC SOX2+ CELLS RELAY IN NON-CANONICAL GLI SIGNALING TO FACILITATE MEDULLOBLASTOMA RELAPSE
Authors: Swiderska-Syn Marzena , Mir-Pedrol Julia , Oles Alexander , Babcock Benjamin , Martin Vanesa , Krieg Carsten , Gershon Timothy , McCrea Heather , Rodriguez-Blanco Jezabel
Published: November 2022

Medulloblastoma is the most common malignant pediatric brain tumor. Despite the fair efficacy of current treatment approaches, 30 % of patients with medulloblastoma relapse. Cells expressing the stemness biomarker SRY (sex determining region Y)-box 2 (SOX2) are known to play key roles in sustaining medulloblastoma growth, providing chemo-resistance, and driving tumor relapse. It is therefore critical to elucidate the underlying mechanisms that propagate these cells and ensure therapies to target them. Single cell sequencing analyses revealed the existence of a subset of astrocyte-like SOX2+ cells expressing biomarkers indicative of SHH signaling activation. Intriguingly, such SOX2+ cells were not affected by the upstream SHH inhibitor vismodegib. Using SOX2+ enriched cultures, we observed that astrocyte-like SOX2 cells not only express SHH effectors, but require GLI signaling to proliferate, and that GLI is activated in a non-canonical and MYC dependent manner. Importantly, in vivo inhibition of SHH signaling downstream of SMO depleted the vismodegib resistant SOX2+ cell pool, while reduced the ability of residual medulloblastoma tissues to engraft in vivo. Our data show that in medulloblastoma, a subset of SOX2+ tumor cells rely on non-canonical GLI signaling to propagate, and emphasizes the importance of using therapies that deplete SOX2+ cells to prevent tumor recurrence.
CCRG-03. STUDY OF PIWI-INTERACTING RNAS IN GLIOBLASTOMA PATHOLOGY: A NEW LEVEL OF REGULATION OF GLIOBLASTOMA STEM CELLS?
Authors: Fadrus Pavel , Vybihal Vaclav , Siegl Frantisek , Smrcka Martin , Slaby Ondrej , Sana Jiri
Published: November 2022

BACKGROUND

One of the putative mechanisms of early recurrence of GBM is the existence of glioblastoma stem cells (GSCs) that can resist therapy and subsequently establish new GBM foci. PIWI-interacting RNAs (piRNAs), responsible for maintaining genome stability, may then play a key role in their biology, particularly in germ and stem cells. A number of piRNA molecules are also promising biomarkers useful for prognosis and prediction of disease progression.Material and

METHODS

Glioblastoma stem cells were cultured in DMEM/F12 medium supplemented with bFGF and EGF growth factors. DMEM medium containing 10% fetal bovine serum (FBS) was used to culture differentiated cells. RNA was isolated from the selected paired primocultures and subsequently checked for quality by capillary electrophoresis. NEBNext Small RNA Library Prep Set was used for the preparation of cDNA libraries. Sequencing analysis was performed using the Next500/550 High Output v2 Kit for 75 cycles and the NextSeq500 sequencer.

RESULTS

Twenty paired primary cultures were prepared to verify the expression of CD133 and SOX2 in GSCs, the ability to establish tumors in immunodeficient mice, and the ability to differentiate. At this time, we finished sequencing of piRNA molecules and acquired data are bioinformatically analysed. Subsequently, we will perform analysis of selected piRNAs to be diagnostic and prognostic biomarkers on the GBM samples and non-tumor brain controls. All results will be presented at the conference.

CONCLUSION

Based on recent studies, it is evident that piRNA expression can have a significant impact on GBM biology. Their expression within GSCs may then provide a tool for identifying and targeting these cells and could be usable as a diagnostic and prognostic biomarker in GBM patients.The study was supported by the programme project of the Ministry of Health of the Czech Republic with reg. no. NV19-03-00501, NV19-03-00559, Conceptual development of research organization FNBr, 65269705
CCRG-04. TARGETING THE CELL-CYCLE EFFECTS OF TUMOR TREATING FIELDS (TTFIELDS) TO INCREASE ITS EFFICACY
Authors: Slangen Paul , Elbakary Basma , van Mil Lotte , de Gooijer Mark , Krenning Lenno , van den Broek Bram , Mertz Marjolijn , Van Tellingen Olaf , Borst Gerben
Published: November 2022

Tumor Treating Fields (TTFields) are a novel, non-invasive FDA-approved treatment modality for glioblastoma (GBM) that utilises alternating electric fields. Our research aims to elucidate the effect of TTFields on the cell cycle to advance the understanding of TTFields and find novel targets for increasing its efficacy. We studied the cell cycle progression using live-cell imaging (inovitro Live™) of PIP-FUCCI-transduced GBM cells, EdU incorporation, RPA foci and fiber assay (in U251-MG, SNB-19, A172). These tools allow us to track individual cells evaluate the time spent in each cell cycle phase, and study the ultimate cell fate in control and treatment conditions concerning the cell cycle and replication fork dynamics. Statistical testing was performed by 2-way ANOVA, Tukey-corrected for multiple comparisons, and Kruskal-Wallis test. TTFields causes an immediate delay in S and G2 cell cycle phase, with a subsequent G1 arrest. Wee1 inhibition by AZD1775 forces cells through S-phase and abrogates the subsequent TTFields-induced G2-checkpoint, preventing DNA repair. Combined treatment with TTFields and AZD1775 led to decreased incorporation of EdU and an increase in S-phase-specific phospho-RPA foci. TTFields reduced significantly the DNA fiber length after 12, 24, 48 and 72 hrs of exposure (10.2, 8.3, 7.1 and 5.6 vs 13 um), with also an increase in replication fork asymmetry at these time points (0.56, 0.45, 0.49, and 0.47 vs 0.9 ratio) respectively. CombiningTTFields with either Wee1, ATR, or Chk1 inhibition synergistically decreased clonogenic survival. TTFields induced cell cycle effects with the induction of replication stress, which is aggravated by inhibition of Wee1. Additionally, Wee1, ATR, and Chk1 inhibition dramatically boost the efficacy of TTFields in vitro. This combination strategy has promising clinical potential and will be tested in vivo in different GBM xenografts.

Cell signaling and signaling pathways
CSIG-04. BMP4 INDUCTION OF A SENESCENCE-LIKE PHENOTYPE IN HUMAN GLIOBLASTOMA CELLS IS PARTIALLY DEPENDENT ON P21
Authors: Niklasson Mia , Dalmo Erika , Westermark Bengt
Published: November 2022

Bone morphogenetic protein 4 (BMP4) was initially suggested as a potential differentiation-inducing factor to be used in the therapy of glioblastoma. We and others have however demonstrated that the response is reversible, variable among patient samples, and heterogeneous within the same cell line. To deepen our knowledge on how BMP4 affects different types of glioblastoma cells, we treated phenotypically different clones from the same patient tumor—a multitherapy-sensitive/proneural-like (SENS/PN) clone and a multitherapy-resistant/mesenchymal-like (RES/MES) clone—with BMP4. In the SENS/PN clone, BMP4 turned on a mesenchymal-related gene program, whereas this response was less prominent in the RES/MES clone. Untreated SENS/PN cells were smaller than RES/MES cells, but both clones responded to BMP4 by cell size enlargement. Increase in cell size has been suggested to precede senescence; young cells are smaller than old cells that eventually enter replicative senescence. BMP4 induced a senescence-like phenotype in a subpopulation of cells, demonstrated by induction of senescence-associated (SA)-β-gal, p21 up-regulation, lamin B1 down-regulation, as well as increased lysosomal mass and granularity. This was more pronounced in the RES/MES clone than in the SENS/PN clone, and it was dependent on canonical SMAD signaling. Senolytic treatment ablated the SA-β-gal positive cells and reduced the p21 level at the population level. Targeted deletion of p21 abolished BMP4-induced SA-β-gal and increase in cell growth, while lamin B1 down-regulation remained, demonstrating that p21 signaling is crucial for one part of the senescence induction by BMP4. We are currently further investigating a connection between cell size, mesenchymality and senescence. We hypothesize that large mesenchymal-like cells are closer to senescence than smaller proneural cells within the cell culture. A combination of senescence-induction and senolytic treatment may open treatment opportunities to target therapy-resistant glioblastoma cells.
CSIG-07. GAIN-OF-FUNCTION MUTANT P53 REGULATES LONG-NONCODING RNAS IN GLIOBLASTOMA
Authors: Zhang Ying , Yuan Feng , Grello Cassandra , Reon Brian , Gibert Myron , Dube Collin , Dutta Anindya , Holland Eric , Abounader Roger
Published: November 2022

P53 is frequently mutated in most human cancers, including glioblastoma (GBM). Many p53 mutants acquire gain-of-function oncogenic effects through only partially understood mechanisms. To investigate the role of gain-of-function mutant p53 (MUT-p53) in GBM, we performed ChIP-seq of wildtype p53 (WT-p53) and MUT-p53 GBM cell lines. Among 2834 unique peaks reads in MUT-p53 cells, we found 242 long non-coding RNAs (lncRNAs) with up to 145 fold enrichment relative to WT-p53. LncRNAs regulate many molecular and cellular functions, including gene expression, cell proliferation, death, cancer stem cell renewal and differentiation. We selected lncRNAs SOX21-AS1 and LINC00643 with highly enriched binding by MUT-p53 and investigated their expressions and functions in the p53 pathway. We performed ChIP confirmation of MUT-p53 binding to the promoters of these lncRNAs. We found that these lncRNAs are deregulated in GBM and correlated with GBM patient survival in the TCGA database. To investigate the functions of these LncRNAs, we knocked down their expressions by siRNA, and found significant cell death induced by si-SOX21-AS1, but not by si-LINC00643. Overexpression of LINC00643 in GBM cells led to inhibition of GBM cell proliferation, migration, invasion and in vivo xenograft growth. LINC00643 mediated the effects of MUT-p53. Co-expression of human LINC00643 and its mouse homologous in a RCAS transgenic mouse model of GBM reduced tumor growth and improved animal survival. To elucidate the mechanisms of action of the lncRNA, we performed Chromatin Isolation by RNA purification high-throughput sequencing (CHIRP-seq) to identify its binding targets. We found that LINC00643 binds to HIF1a 5’ promoter/enhancer region. Overexpression of LINC00643 in GBM cells at hypoxia growth condition reduced HIF1a mRNA and protein expression. Our study shows for the first time that gain-of-function mutant p53 regulates a subset of lncRNAs and that the lncRNAs mediate the oncogenic effects of the MUT-p53 in GBM.
CSIG-08. MASS CYTOMETRY IDENTIFIES LOCATION-SPECIFIC TUMOR POPULATION ASSOCIATED WITH CLINICAL PROGRESSION IN GLIOBLASTOMA
Authors: Alvarado Alvaro , Bastola Soniya , Muthukrishnan Sree Deepthi , Alban Tyler , Tessema Kaleab , Nakano Ichiro , Lathia Justin , Goldman Steven , Kornblum Harley
Published: November 2022

Recent advances in single cell expression technology have allowed us to appreciate the complexity in tumoral heterogeneity of solid tumors such as glioblastoma (GBM), which remains uniformly fatal despite aggressive therapies. Currently, there is no knowledge about cell identities and functional characteristics of the different tumoral populations. To this end, we created a 28-marker mass cytometry panel composed of signaling markers, including phospho-proteins, and lineage markers that have been associated with GBM or are considered putative cancer stem cell markers. We evaluated lines established from core and edge areas from the same patient and validated to be functionally different with RNA expression and in vivo experiments. We found clusters shared by cells from both regions as well as region-specific ones. In order to better characterize these, we used marker enrichment modeling (MEM) and generated machine-readable labels. These were compared to MEM labels generated after using the same signaling markers to cluster primary samples from newly diagnosed or recurrent patients. Similarly, MEM labels were obtained from patients in a clinical trial treated with capecitabine that had positive or negative responses. Clusters from edge cells were found in higher proportion in recurrent tumors and were correlated with negative responses to chemotherapy. Initial analyses have also been done correlating immune clusters with tumor clusters in the same samples. Edge clusters are characterized by a CD44+ SOX2+ Nestin+ population. Treatment of tumor cells with temozolomide and irradiation results in an increase of this population in vitro. We are currently conducting single cells analyses to characterize this population as well as in vivo studies to validate the tumor-immune interactions. Our studies exploit single cell cytometry to underscore pathways that are preferentially use by different populations and affect immune cells with the goal of establishing therapeutic efforts to extend patient survival.
CSIG-12. BXQ-350 INDUCES MITOPHAGY IN GBM CELLS LEADING TO CELL DEATH
Authors: Wilkins Nikhil , Furnish Robin , Stephens Tim
Published: November 2022

BACKGROUND

Mitophagy is an autophagic phenomenon where defective mitochondria are selectively degraded. When mitochondria need recycling, the cell upregulates the production of the lipid C-18 ceramide on the mitochondria and lipoprotein LC3B-II on the lysosome, which transitions to an autophagolysosome. This creates a signal causing the degradation of mitochondria. Here we show that BXQ-350, a novel protein-lipid small molecule consisting of the ceramide creating protein Saposin C and the lipid Dioleoyl Phosphatidylserine (DOPS), induces glioblastoma cells into performing mitophagy. Increased production of mitophagy associated proteins, C-18, and LC3B-II leads to the degradation of healthy mitochondria followed by cell death.

METHODS

The GBM cell lines, Gli36 and U87 cells, were used to determine if increased mitophagy was occurring in response to BXQ-350 treatment. Protein abundance was measured using SILAC proteomics. Ceramide levels were analyzed using LC/MS-MS. Western Blot analysis was used to measure differences in LC3B-II levels. Mitophagy was measured using Mtphagy Dye from Dojindo and the BioTek Cytation 5 microscope.

RESULTS

Proteomics showed cells treated with BXQ-350 had an increase in proteins associated with overexpression of C-18 ceramide or autophagolysosome maturation. Western blots analysis showed a marked increase in LC3B-II in BXQ-350 treated cells. Furthermore, cells treated with BXQ-350 had increased levels of C-18 ceramide. The Mtphagy dye showed that as BXQ-350 concentrations increased, the percentage of cells positive for Mitophagy increased. To determine if mitophagy occurs in association with BXQ-350 cytotoxicity, medias with varying BXQ-350 cytotoxicity were used. The Mtphagy dye showed cytotoxic BXQ-350 medias increased levels of mitophagy, while noncytotoxic medias display no increase in mitophagy.

CONCLUSION

BXQ-350 causes the increase of mitophagy signaling proteins, C-18 ceramide and LC3B-II production, resulting in the formation of autophagolysosomes which track towards the mitochondria initiating mitophagy. This leads to aberrant mitophagy where cells degrade healthy mitochondria leading to mitophagic cell death.
CSIG-16. EXPLOITING A NEGATIVE FEEDBACK LOOP LINKING S6 KINASES AND AXL TO REDUCE PYRIMIDINE BIOSYNTHESIS IN PTEN-DEFICIENT GLIOBLASTOMA
Authors: Behrmann Catherine , Sarma Pranjal , Ennis Kelli , Wetzel Collin , Clark Nicholas , Vallabhapurapu Subrahmanya , Reigle James , Meller Jarek , Qi Xiaoyang , Medvedovic Mario , Sengupta Soma , Dasgupta Biplab , Plas David
Published: November 2022

PTEN inactivation triggers oncodependent signal transduction through the PI3K/Akt signal transduction pathway in glioblastoma (GBM). Targeting the PI3K/Akt pathway to counteract PTEN loss in GBM has been impeded by negative feedback signal transduction networks mediated by the downstream protein kinases S6K1 and S6K2. Here, we show that the receptor tyrosine kinase AXL is a major target of that S6K feedback signaling and that combined inactivation of just S6K1 and AXL is an effective therapeutic strategy for treatment of PTEN-deficient GBM. Chemical-genetic interaction studies in gliomasphere and GBM cell lines revealed critical and independent roles for S6K1 and S6K2 in mediating GBM growth in PTEN-deficient cells. Interestingly, S6K2 exerted a dual role in signal transduction: it sustained GBM growth while also exerting negative feedback control on the upstream receptor tyrosine kinase AXL. Genetic inactivation of S6K2 sensitized PTEN-deficient GBMs to AXL inhibition, indicating that derepressed AXL is required for compensatory signaling upon targeting of the PI3K/Akt pathway. Combining the AXL inhibitor BMS-777607 with S6K1 inhibitor LY-2584702 also prevented compensatory AXL signaling and triggered cytotoxic responses selectively in the PTEN-deficient condition. Importantly, combination inhibition of AXL and S6K1 reduced pyrimidine biosynthesis, a known vulnerability of PTEN-deficient GBMs. The oral inhibitors of S6K1 and AXL were found to be brain-penetrant and effective in reducing GBM growth in several mouse models. These results establish that targeting of AXL can circumvent S6 Kinase dependent feedback to reduce pyrimidine biosynthesis and trigger cytotoxic responses in PTEN-deficient GBMs.
CSIG-20. PROTEOGENOMICS PROFILING REVEALS ENRICHED PROTEIN TRANSLATION REGULATORS AS NEW THERAPEUTIC TARGETS IN DIPG
Authors: Vadivel Arun Anguraj , Pajovic Sanja , Phillips Lauren , Simpson Craig , Zhang Cunjie , Bu Ying , Nitz Mark , Moran Michael , Hawkins Cynthia
Published: November 2022

Diffuse intrinsic pontine glioma (DIPG) is a devastating brain tumor arising in the brainstems of children. Current therapies are ineffective resulting in a median survival rate of less than one year and it is the leading cause of brain tumor-related death in children. A novel mutation in histone H3 protein (H3K27M) was recently identified as a genetic initiation event and affects global K27 trimethylation on histone H3 proteins and DNA methylation. The epigenetic changes caused by H3K27M mutation suggest the existence of an H3K27M-specific transcriptome and proteome. We investigated DIPG tissues at the multi-omics level including total proteome, phosphoproteome, methylproteome and metabolome by mass spectrometry. A total of 30 patient tumours were profiled to identify differentially expressed proteins, differentially phosphorylated proteins, and differentially methylated proteins in tumour tissues compared to normal brains. We identified 2995 proteins that are differentially regulated, suggesting changes in key oncogenic pathways including negative regulation of apoptosis, translation, and metabolic pathways such as methionine salvage and TCA cycle in DIPG. The deregulation of these metabolic pathways due to the differentially expressed proteins in DIPG cells was confirmed by metabolomics studies. Protein phosphorylation and protein methylation profiling of DIPG implicated that translation-related proteins were the most highly modified (post-translationally) proteins in DIPG tissues. Furthermore, protein translation measured by CyTOF showed higher translation rates in DIPG and immortalized astrocytes carrying H3K27M than their WT counterparts. We investigated the functional consequence of knockdown of the highest methylated translation regulator EEF1A1 and its methyltransferase METTL13. ShRNA knockdown of both, EEF1A and METTL13 in DIPG cells significantly reduced the cell growth. Multi-omics analysis of DIPG highlighted regulation of protein translation as a potential therapeutic target.
CSIG-21. EXPLORING THE PHENOTYPIC EFFECTS OF TOLL SIGNALING PATHWAYS IN TUMOR PROGRESSION IN EGFR-DRIVEN GLIOBLASTOMA MODELS
Authors: Saborio Julia G , Gulbronson Riley , Young Elizabeth E , Hsu Irene , Read Renee D
Published: November 2022

The treatment options for Glioblastoma (GBM) are currently limited to resection and chemotherapy approaches, but resistance to chemotherapy is common in patients. There is an urgent need for further understanding of the underlying biology of tumorigenesis in order to develop new treatment options. Previous genomic work on GBMs has shown that the epidermal growth factor receptor (EGFR) is frequently amplified and constitutively active in GBM tumors. To discover new factors that contribute to progression of EGFR-driven GBMs, we used our Drosophila GBM model in which GBM-like glial neoplasia can be induced by glial-specific overexpression of constitutively active forms of EGFR and dp110, a subunit of Phosphoinositide 3-kinase (PI3K), using the UAS-GAL4 gene expression system. Using our Drosophila GBM model system, we performed a modifier screen in which we searched for genes and pathways that worsened or ‘enhanced’ glial neoplasia when specifically overexpressed or activated in neoplastic glia. From our screen, we identified the Toll pathway, which in Drosophila activates an NF-κB signaling pathway. Furthermore, the Toll pathway normally functions in innate immune and injury responses as well as in development and cell proliferation. Using our Drosophila GBM model, we are studying how the Toll signaling pathway and its effectors, including NF-κB, contribute to enhanced growth of neoplastic glial cells. Furthermore, using well-defined mouse genetic models and human patient-derived GBM models, we are examining whether Toll pathway regulatory and signaling mechanisms that we have uncovered in Drosophila function in EGFR-mutant GBM tumor stem cells to promote GBM progression and chemotherapy resistance.
CSIG-26. PROTEIN KINASE B (PKB/AKT) PROTECTS IDH-MUTATED GLIOMA FROM FERROPTOSIS VIA NRF2
Authors: Liu Yang , Lang Fengchao , Yang Chunzhang
Published: November 2022

BACKGROUND

Mutations of the isocitrate dehydrogenase (IDH) gene are highly prevalent in WHO II/III glioma. IDH-mutated glioma frequently exhibits strong molecular signatures of PI3K/AKT/mTOR signaling, whereas the precise roles of this oncogenic pathway in IDH-mutated glioma remain elusive.

METHODS

In this study, we performed unbiased RNA sequencing and gene set enrichment analysis (GSEA) to investigate the relevant molecular pathways governed by the Phosphoinositide 3-kinase (PI3K)/AKT in IDH-mutated cancer cells. The role of nuclear factor erythroid 2-related factor 2 (Nrf2) and glutathione de novo synthesis were further analyzed through biochemistry assays. Moreover, the role of AKT in glioma disease outcomes was evaluated in a preclinical animal model bearing IDH-mutated orthoptic intracranial xenograft.

RESULTS

RNA sequencing and GSEA analysis indicated that the AKT pathway significantly correlated with Nrf2-guided gene expression and ferroptosis-related pathways. Mechanistically, AKT activation compromised the phosphorylation of Nrf2 at its SDS domain, which is a critical post-translational modification that determines Nrf2 degradation through an E3 ligase β-transducin repeat-containing protein (β-TrCP). Thus, the activation of the AKT pathway limited the proteolysis of Nrf2 and a remarkably enhanced Nrf2 transcriptional activity, which enables cytoprotective pathways such as antioxidant, tumor microenvironment remodeling, and ferroptosis protection. Moreover, a combination of AKT inhibitor ipatasertib with genotoxic agent temozolomide resulted in potent synthetic lethality in IDH-mutated cancer cells, highlighted with enhanced ferroptotic cell death. Further, the AKT inhibitor and TMZ combination regimen improved disease outcomes in a preclinical animal model with IDH-mutated orthotopic intracranial xenograft, with significantly prolonged overall survival.

CONCLUSION

Overall, our findings revealed an uncharted oncogenic mechanism in IDH-mutated glioma, highlighted with AKT/Nrf2-guided cytoprotective pathway. Our study also provides critical preclinical evidence for a synthetic lethality approach for IDH mutated cancers.