Orthotopic GBM Research Articles

Substrate stiffness regulates the recurrent glioblastoma cell morphology and aggressiveness

Recurrent glioblastoma is highly aggressive with currently no specific treatment regime. Therefore, to identify novel therapeutic targets for recurrent GBM, we used a cellular model developed in our lab from commercially available cell line U87MG and patient-derived cultures that allows the comparison between radiation naïve (Parent) and recurrent GBM cells generated after parent cells are exposed to lethal dose of radiation. Total RNA-seq of parent and recurrent population revealed significant upregulation of cell-ECM interactions pathway in the recurrent population. These results led us to hypothesize that the physical microenvironment contributes to the aggressiveness of recurrent GBM. To verify this, we cultured parent and recurrent GBM cells on collagen-coated polyacrylamide gels mimicking the stiffness of normal brain (Young's modulus E = 0.5kPa) or tumorigenic brain (E = 10kPa) and tissue culture plastic dishes (E ∼ 1 GPa). We found that compared to parent cells, recurrent cells showed higher proliferation, invasion, migration, and resistance to EGFR inhibitor. Using orthotopic GBM mouse model and resection model, we demonstrate that recurrent cells cultured on 0.5kPa had higher in vivo tumorigenicity and recurrent disease progression than parent cells, whereas these differences were insignificant when parent and recurrent cells were cultured on plastic substrates. Furthermore, recurrent cells on 0.5kPa showed high expression of ECM proteins like Collagen, MMP2 and MMP9. These proteins were also significantly upregulated in recurrent patient biopsies. Additionally, the brain of mice injected with recurrent cells grown on 0.5kPa showed higher Young's moduli suggesting the ability of these cells to make the surrounding ECM stiffer. Total RNA-seq of parent and recurrent cells grown on plastic and 0.5kpa identified PLEKHA7 significantly upregulated specifically in recurrent cells grown on 0.5 kPa substrate. PLEKHA7 was also found to be high in recurrent GBM patient biopsies. Accordingly, PLEKHA7 knockdown reduced invasion and survival of recurrent GBM cells. Together, these data provide an in vitro model system that captures the observed in vivo and clinical behavior of recurrent GBM by mimicking mechanical microenvironment and identifies PLEKHA7 as a novel potential target for recurrent GBM.

Refining the delivery and therapeutic efficacy of cetuximab using focused ultrasound in a mouse model of glioblastoma: An 89Zr-cetuximab immunoPET study

IntroductionGlioblastoma (GBM) is the most common and deadly form of primary brain tumor. Between 30 % and 60 % of GBM are characterized by overexpression of the Epidermal Growth Factor Receptor (EGFR). The anti-EGFR antibody Cetuximab (CTX) showed a favorable effect for EGFR+ colorectal cancer but failed to demonstrate efficacy for GBM. Insufficient CTX passage through the blood–brain barrier (BBB) and the blood-tumor barrier (BTB) is assumed to be the primary determinant of the limited efficacy of this immunotherapy. ObjectiveUsing positron emission tomography (PET) imaging, we have previously demonstrated that focused ultrasound (FUS) combined with microbubbles (µB) allowed significant and persistent delivery of CTX across the BBB in healthy mice. In the current study, we investigated by PET imaging the combination impact of CTX and FUS on orthotopic GBM preclinical model. MethodsAfter radiolabeling CTX with the long half-life isotope 89Zr, PET images have been acquired overtime in mice bearing U251 (EGFR+) with or without FUS treatment. Autoradiography combined with immunofluorescence staining was used to corroborate CTX delivery with EGFR expression. A survival study was conducted simultaneously to evaluate the therapeutic benefit of repeated CTX monotherapy associated or not with FUS. ResultsEx vivo analysis confirmed that FUS enhanced and homogenized the delivery of CTX into all the FUS exposure area, including the tumor and the contralateral hemisphere at the early-time-point. Interestingly, FUS did not improve the long-term accumulation and retention of CTX in the tumor compared with the control group (no FUS). No significant difference in the CTX treatment efficacy, determined by the survival between FUS and non-FUS groups, has been either observed. This result is consistent with the absence of change in the CTX distribution through the GBM tumor after FUS. The neuroinflammation induced by FUS is not significant enough to explain the failure of the CTX delivery improvement. ConclusionAll together, these data suggest that the role of FUS combined with µB on the CTX distribution, even after multiple therapeutic sessions and glial cell activation is insufficient to improve survival of GBM mice compared with CTX treatment alone in this model.

EXTH-26. MICROGLIA-SPECIFIC DISRUPTION OF SIALIC ACID-SIGLEC-9/E INTERACTIONS: A NOVEL IMMUNOTHERAPY AGAINST GLIOBLASTOMA?

Abstract Recently, ‘don’t eat me’-signals like CD47 have emerged as novel innate immune checkpoints, enabling cancer cells to evade clearance by phagocytes such as microglia (MG) or monocyte-derived cells (MdCs). Here, we aim at defining the role of inhibitory Siglec-9 in human and its mouse homologue Siglec-E in innate-centered immunotherapy against GBM. TCGA RNA-sequencing data revealed a significant correlation between high expression of immunoinhibitory SIGLEC9 and poor survival in GBM patients (log-rank p = 0.02). Using a CT-2A orthotopic GBM mouse model with MG-specific spatio-temporal deletion of Siglece (Sall1CreERT2 x Sigleceflox) , we observed high MG-proliferation upon Siglece knockout (Ki-67+ MG 14.8% in Crenegvs. 34.9% in Creposp < 0.0001) accompanied by an enhanced microglial GBM-cell uptake (5.6% in Crenegvs. 12.3% in Crepos, p < 0.001). By extending the Siglece knockout to the MdC compartment (Cx3cr1CreERT2x Sigleceflox) we observed a significantly prolonged survival in the Crepospopulation (21d in Crenegvs. 27d post-tumor injection in Crepos, p = 0.018), which could be further promoted by combining Siglece knockout with CD47 blockade (11% long-term remission in Crepos+ anti-CD47). Proteomics analysis revealed increased antigen processing and presentation capabilities of SigleceKOMdCs which was confirmed by ex-vivo OT-1 cross-presentation assays. This increased T cell priming upon MdC SigleceKOwas further boosted by addition of anti-PD1 antibody to the SigleceKO+ anti-CD47 combination. Resulting in 23% of animals experiencing long-term remission in the triple treatment arm, even after tumor re-challenge. Genetic targeting of sialic acids, the ligand for Siglec receptors, on CT-2A cells (GNE-KO), resulted in increased GBM-cell phagocytosis by MG and MdCs and less exhausted tumor-infiltrating CD8+ T cells (14.8% in WT vs. 5.9% in GNE-KO, p = 0.003). In a translational approach, we are currently testing anti-Siglec-9 treatment regimens in patient GBM explants, cultured for 5 days in perfusion bioreactors.

TMIC-76. HUMANIN RELEASE FROM TUMOR ASSOCIATED MYELOID CELLS PROMOTES GLIOMA CHEMORESISTANCE

Abstract Transcriptomic screens of brain tumor (glioblastoma; GBM) parenchymal cells indicated tumor supporting traits of the GBM microenvironment, but largely excluded mitochondrially enriched gene-sets. Here, we show that a mitochondrial transcript of GBM parenchymal cells contributes to therapy resistance. We inspected the non-coding transcriptome of human GBM associated myeloid cells (GAM) and observed an upregulation of the mitochondrial ribosomal subunit MT-RNR2, which contains an open reading frame for the signaling peptide humanin. Immunohistology disclosed that humanin was preponderant in GAM. In vitro assays with a range of human stem-like GBM cells (GSCs) revealed that nanomolar concentrations of humanin can drive tumor cell expansion and chemoresistance to temozolomide. A series of genetic, pharmacological and Western blotting experiments showed that extracellular humanin increased GSC viability via stimulation of the GP130/IL6ST receptor and MAPK (Erk) activation. Humanin responsiveness was subject to inter-individual heterogeneity and predominated in GSC with high expression levels of the IL6ST subunit. Mechanistically, humanin promoted the ATR-dependent DNA-repair machinery in GSCs via induction of the DNA-clamp component HUS1. A slice culture model containing human microglia and GSCs confirmed these observations. Humanin is absent from the mouse brain. Exogenous delivery of humanin into orthotopic GBM mouse models or over-expression specifically of a secreted humanin variant recapitulated the in vitro findings. Blockade of the trimeric interleukin receptor with the brain permeant, FDA-approved drug bazedoxifene blocked humanin-mediated chemoresistance in vitro and in vivo. Overall, we identified a clinically applicable compound together with a predictive marker to prevent chemoresistance in a human-specific GBM model.

A Chlorotoxin‐Directed Diselenide‐Bridged Tumor‐Homing Persistent Luminescence Nanoprobes Mediating Inhibition of Oxidative Phosphorylation for Long‐Term Near‐Infrared Imaging and Therapy of Glioblastoma

AbstractGlioblastoma (GBM) is the most prevailing malignant primary brain tumor, and the precise diagnosis of GBM has always been a challenge. Gboxin is a recently developed drug efficiently inhibiting the oxidative phosphorylation in GBM cells, and both the chlorotoxin (CLTX) and GBM cell membrane coating are capable of GBM targeting and tumor homing. Herein, the near‐infrared (NIR) persistent luminescence (PL) nanoparticle, CUDZG, with a dual function of imaging and therapy is developed based on ZnGa2O4:Cr3+,Sn4+. CUDZG exhibits superior rechargeable NIR PL for at least 48 h with excellent tissue penetration in vivo, which enables the longstanding autofluorescence‐free imaging of the orthotopic GBM. The tumor growth of both the orthotropic and subcutaneous GBM‐bearing mice are significantly suppressed by CUDZG. This is the first‐time report of 1) the integration of CLTX and cell membrane coating for drug delivery, 2) diselenide‐based trigger release for anti‐GBM therapy, and 3) the systemic delivery of Gboxin. This study also offers an example of the highly promising blood‐brain penetrable drug carriers for precise diagnosis and therapy of central nervous system diseases.

P06.08.B Radiation therapy enhances anti-tumor activity of a MET CAR T-based immunotherapy for glioblastoma

Abstract Background Glioblastoma is the most frequent primary brain tumor with dismal prognosis after standard treatment with surgery, and chemoradiation (the Stupp protocol). After a decade of failed clinical trials, tumor-treating fields have been first to show the added benefit of improved overall survival compared to the Stupp protocol (20.9 months vs 16.0 months). However, GBM remains a devastating disease, with almost inevitable recurrence, and limited options for second-line therapy. Radiation therapy (RT), is a standard therapy option for GBM, and it is used in most GBM cases affecting tumor through induction of DNA-damage. Recently, RT has been investigated as a mediator of T cell-based therapies in the context of immunosuppressive GBM microenvironment. The findings have shown promise in combination of T cell-based therapies, such as chimeric antigen receptor (CAR) T cell therapies, in improving the tumor infiltration, and penetration with immune cells. MET is a relevant oncogene in the context of GBM, being involved in stem-like properties, radiation response and resistance․ Hence, MET appeared to be a plausible target for combination with RT. In our research, we use MET-targeting CAR T cells (MET-CAR T cells) combined with radiation, and hypothesize synergistic interaction for GBM treatment. Material and Methods We used adherent (2D) and stem-like (3D) human GBM cell lines with different levels of MET expression. For MET-CAR T cell generation we did retrovirus-mediated transduction of activated human T cells and sorted the CAR-positive cells. We co-cultured MET-CAR T cells with GBM cells with or without RT, and assessed the killing and cytokine production in CAR T cells. Results Our results indicated that 5Gy radiation combined with MET-CAR T cells increases their potential in tumor cell killing. We observed increased CAR T cells effect at lower CAR T to target cells ratios when combined with radiation, even when radiation treatment did not lead to a significant decrease in viability. This phenomenon was similar across different types of cell lines (adherent, stem-like), different levels of MET expression, and different sensitivity to CAR T cells. We investigated the underlying mechanisms via intracellular cytokine measurement. We observed the most prominent response in TNF-α-expression. We also observed an increase in Granzyme B expression in co-culture with some of the GBM cell lines, especially in CD8+ subpopulation of CAR T cells. IFN-gamma expression increased in some adherent glioma cell lines but not in stem-like cell lines. Conclusion In conclusion, our data demonstrates the potency of MET-CAR T cells against GBM, and increased efficiency when combined with radiation. The suggested mechanism is the increased activation of T cells in TNF-α-dependent-manner. To validate these results we are testing our setup in an orthotopic mouse GBM model.

PL02.2.A Microglia-specific disruption of sialic acid-Siglec-9/E interactions. A novel immunotherapy against glioblastoma?

Abstract Background Recently, ‘don’t eat me’-signals like CD47 have emerged as novel innate immune checkpoints, enabling cancer cells to evade clearance by phagocytes such as monocyte-derived macrophages (MdM) or microglia (MG). Here, we aim at defining the role of inhibitory Siglec-9 in human and its mouse homologue Siglec-E in MG-centered immunotherapy against GBM. Material and Methods We employed a CT-2A orthotopic GBM mouse model with MG specific (Sall1CreERT2 x Sigleceflox) and whole innate-compartment (Cx3cr1CreERT2 x Sigleceflox) spatio-temporal deletion of Siglece. We applied multi-color flow cytometry, transcriptomics and proteomics analysis to decipher the immune response upon Siglece knockout. Results TCGA RNA-sequencing data revealed a significant correlation between high expression of immunoinhibitory SIGLEC9 and poor survival in GBM patients (log-rank p = 0.02). Siglec-E blockade increased murine MG mediated GBM cell in vitro phagocytosis (normalized phagocytosis of 1.00 in isotype vs. 1.76 in anti-Siglec-E antibody, p < 0.001). In the MG specific spatio-temporal deletion of Siglece (Sall1CreERT2 xSigleceflox), we observed high MG-proliferation upon Siglec-E knockout (Ki-67+ MG 14.8% in Cre- vs. 34.9% in Cre+, p < 0.0001) accompanied by an enhanced microglial GBM-cell uptake (5.6% in Cre- vs. 12.3% in Cre+, p < 0.001). By extending the Siglece knockout to the MdM compartment in our glioma mouse model (Cx3cr1CreERT2 x Sigleceflox) we observed a significantly prolonged survival in the Cx3cr1Cre+ population (21d in Cre- vs. 27d post-tumor injection in Cre+, p = 0.018), which could be further promoted by combining Siglece knockout with CD47 blockade (30d post-tumor injection in Cre+ + anti-CD47). Unbiased proteomics analysis revealed increased antigen processing and presentation capabilities of Siglece knockout MdMs which was confirmed by ex-vivo OT-1 cross-presentation assays. This bridging of innate and adaptive responses with increased T cell priming upon MdM Siglece knockout was further promoted by addition of anti-PD1 antibody to the combined Siglece knockout and anti-CD47 treatment arm. Animals harboring CT-2A tumors, exhibited a sustained survival benefit under the triple therapy, with 23% of animals experiencing long-term remission, even after tumor re-challenge into the contra-lateral hemisphere. By genetically targeting sialic acids, the ligand for Siglec receptors, on CT-2A cells (GNE-KO), we observed a strong innate and adaptive immune response with increased GBM-cell phagocytosis by MG and MdMs and less exhausted tumor-infiltrating CD8+ T-cells (14.8% in WT vs. 5.9% in GNE-KO, p = 0.003). Conclusion These data identify the sialic-acid-Siglec-E pathway as an anti-phagocytic signal in a pre-clinical GBM model, and demonstrate its therapeutic potential in GBM immunotherapy.

Abstract 2086: Intratumorally administrated encapsulated IL-12 (PCX12) promotes anti-tumor immune response in GL-261 murine orthotopic glioma model

Abstract Glioblastoma (GBM) is inherently immunosuppressed (cold). Short term localized controlled immune activation has been shown to increase cytotoxic T cell infiltrate, resulting in reduced tumor growth and prolonged survival (Barrett et al. 2018). We have developed an encapsulated-mouse IL-12 formulation (mPCX12), using a sustained IL-12 release platform (Egilmez et al. 2000) to facilitate short term localized immune activation in the tumor without systemic toxicity. mPCX12 elicited a sustained dose-related IL-12 release concomitant with downstream IFNγ in mouse splenocytes, demonstrating mPCX12’s biologic activity. Similar results were observed with hPCX12 in human PBMCs. In an orthotopic murine glioma model, C57B6/L mice were inoculated with 3 × 105 GL-261 glioma cells in the frontal lobe via intracranial injection with drug treatment on Day 5. Mice were randomly assigned (n=22/group) to receive a single intratumoral dose of mPCX12 at 0.5, 0.75 and 1 mg, or mrIL-12 1µg (equivalent to mPCX12 1mg), or empty shells or vehicle. In addition, lomustine (6 mg/kg, QD×5 i.p.) was also assessed. The results showed that mPCX12 treatment increased tumor IL-12 and downstream IFNγ levels in a dose dependent manner. At 1mg mPCX12, tumor IL-12 and IFNγ levels were 2069±631 and 646±8 pg/mg, respectively, on Day 3. Low levels of IL-12 and IFNγ were observed in the systemic circulation of mPCX12 and mrIL-12 groups. mPCX12 tumor IL-12 level persisted, while mrIL-12 returned to baseline by Day 7. Flowcytometric immunophenotyping of tumors showed increased cytotoxic T cells in mPCX12 treated gliomas. These increases in local cytokine levels translated into a dose-related prolongation of survival compared to the median survival in the groups treated with vehicle and empty shells (~19 days), lomustine (20 days), and mrIL-12 (37 days). mPCX12 elicited a dose-related increase in survival. For mPCX12 at 1mg, the median survival was >90 days. At Day 90, 41%, 45% and 55% of the animals that received doses of 0.5, 0.75 or 1mg mPCX12, respectively, were alive with median survival >90 days (last day of study). At the end of the study, a portion of the surviving mice subgroups were euthanized, and those animals treated with mPCX12 were tumor free. The remaining surviving mPCX12-treated animals from the 0.75 mg and 1 mg mPCX12 groups were reinoculated with 3 × 105 GL261 cells in the ipsilateral side of the brain and compared with age-matched control vehicle or empty shells. The median survival in control groups was 27 days, and all rechallenged mPCX12-treated groups survived at Day 90, demonstrating memory T cell activation. In summary, short-term controlled release of locally administered IL-12 via PCX12 resulted in turning cold tumors hot, resulting in enhanced overall survival in an orthotopic GBM model. Thus, short term controlled locally administered PCX12 warrants further investigation in GBM patients. Citation Format: Sribalaji Lakshmikanthan, Damaris Diaz, Prasad Kolli, Arunthi Thiagalingam, Paul M. Gonzales, Mario Sepulveda, Jessica Dalsing-Hernandez, Francois Lebel, John A. Barrett. Intratumorally administrated encapsulated IL-12 (PCX12) promotes anti-tumor immune response in GL-261 murine orthotopic glioma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2086.

Abstract 2663: Preclinical profiling of BAY 2666605: The first PDE3A-SLFN12 complex inducer for cancer therapy

Abstract BAY 2666605, co-developed by the Broad Institute and Bayer Pharmaceuticals, is a selective and potent molecular glue, part of a family of small molecules recently baptized as ‘velcrins’, that induces complex formation between phosphodiesterase 3A (PDE3A) and SLFN12. BAY 2666605 has recently entered a First-in-Human study (NCT04809805) in patients with advanced solid tumors and here we describe its pre-clinical pharmacology profile. DNMDP, the precursor to BAY 2666605, was discovered in a phenotypic screen of genomically annotated cancer cell lines and sensitivity to treatment correlated to high expression of PDE3A (1). Upon treatment, SLFN12 is recruited into a stable complex with PDE3A where its RNase activity is enhanced and required for response (2). BAY 2666605 is a potent complex inducer (EC50 = 7 nM) and cytotoxic in vitro with nanomolar potency (IC50 = 1nM, in the most sensitive cell lines). Cancer cells with high expression of PDE3A and co-expression of SLFN12 are killed by a mechanism independent of PDE3A enzymatic inhibition. PDE3A-SLFN12 binding is required for cytotoxicity. Biomarker-positive lines are enriched in the melanoma lineage and show dose-dependent sensitivity to BAY 2666605 both in vitro and in vivo. Notably, we have consistently observed tumor regression in biomarker-positive melanoma models, including in PDX models (10mg/kg po BID). Based on target expression data from TCGA and tumor arrays, various other tumor types also co-express PDE3A and SLFN12, such as sarcomas and ovarian cancer. To this end, we show that BAY 2666605 inhibits tumor growth of PDX models of sarcoma and ovarian cancer in vivo. BAY 2666605 has excellent brain penetration, making glioblastoma a promising indication. Biomarker-positive GBM models are sensitive to BAY 2666605 both in vitro and in vivo. In a subset of orthotopic GBM models BAY 2666605 treatment has significant impact on survival. In BAY 2666605 treated models we have observed MCL1 downregulation and this biomarker will be evaluated in clinical settings. Our pre-clinical data indicate that BAY 2666605 is a potent anti-tumor agent with first-in-class potential and broad indication space. 1. de Waal et al. Identification of cancer-cytotoxic modulators of PDE3A by predictive chemogenomics, Nat. Chem. Biol. 12, 102-108 (2016) 2. Garvie et al. Structure of PDE3A-SLFN12 complex reveals requirements for activation of SLFN12 RNase, Nat. Commun. 12, 4375 (2021) Citation Format: Silvia Goldoni, Martin Lange, Charlotte Kopitz, Stefan Kaulfuss, Sven Golfier, Adrian Tersteegen, Stefanie Bunse, Melanie Berthold, Thibaud Jordan, Philip Lienau, Franziska Siegel, Annette Walter, Henrik Seidel, Elisa Aquilanti, Andrew Baker, Xiaoyun Wu, Sooncheol Lee, Stefan Gradl, Emmanuelle di Tomaso, Matthew Meyerson, Knut Eis, Ashley Eheim, Heidi Greulich. Preclinical profiling of BAY 2666605: The first PDE3A-SLFN12 complex inducer for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2663.

Novel, small molecule inhibitors of PD-1/PD-L1 pathway.

2597 Background: Programmed Cell Death 1 (PD-1) protein plays a key role in inhibiting immune responses and enhancing self-tolerance via modulation of T-cell activity, inducing T-cell apoptosis and inhibiting apoptosis of regulatory T cells. PD-L1 also plays an important role in various malignancies where it can attenuate the host immune response to tumor cells thereby favouring tumor progression and metastasis. High expression of PD-L1 in glioblastoma tumor tissues is associated with poor survival of patients, and PD-L1 may act as a prognostic predictor and an effective therapeutic target for glioblastoma. A number of monoclonal antibodies targeting PD-1/PD-L1 have approved for various malignancies. Still, efficacy of these antibodies in glioblastoma and brain metastasis continues to be moderate potentially owing to lack of or poor brain penetrance of these agents. Therefore, there is still a need for potent, selective small molecule PD-1/PD-L1 inhibitors with enhanced brain penetration in the treatment of such cancers. Methods: Rational design approaches were used to design novel small molecule PD-1/PD-L1 pathway inhibitors; potency of these inhibitors was assessed in an in-vitro TR-FRET assay. Checkpoints signalling reporter assays as well cell based PD-L1 dimerization assays were used to assess the mechanistic and functional effects. In vivo efficacy was assessed in orthotopic GBM as well as in syngeneic and humanized subcutaneous tumor models in mice. Results: Our lead PD-L1 inhibitor JBI-2174 showed strong in vitro IC50 of ̃1 nM in TR-FRET assay that measures interaction between hPD-1 and hPD-L1 and a picomolar IC50 against monkey PD-L1. In selectivity assays for immunooncology targets, JBI-2174 was highly selective for PD-L1. JBI-2174 also inhibited PD-L1/PD-1 mediated signalling essential for T-cell modulation. JBI-2174 induced dimerization of PD-L1 as observed by size exclusion chromatography, which was confirmed by co-crystal structure and recapitulated in cell based dimerization assay. Elucidation of the co-crystal structure, clearly demonstrated that JBI-2174 clearly interacts with multiple amino acids on PD-L1 that are critical for PD-1 binding. JBI-2174 showed excellent oral bioavailability across pre-clinical species and sustained brain exposure. In the in vivo efficacy studies, JBI-2174 showed comparable efficacy to the anti-PD-L1 antibody or Atezolizumab in syngeneic (4T1, CT-26) and in partially humanized models (MC-38/hPD-L1). Further, oral administration of JBI-2174 resulted in statistically significant increase in survival (Day 27 in control vs day 38 in treated, p < 0.05) in a mouse glioma orthotopic model. Conclusions: The oral bioavailability and brain exposure of this molecule will make it attractive for cancers with unmet medical needs such as GBM and brain metastasis. IND enabling studies are being initiated for this compound.

Abstract P057: Targeting Treg cells with GITR activation alleviates resistance to immunotherapy in murine glioblastomas

Abstract Glioblastoma (GBM) shows high level of resistance to currently available treatments including the standard of care and immunotherapy, representing the most fatal cancer type. Our study revealed that immune suppression by regulatory T cells (Treg) secondary to therapy with immune checkpoint blocker (anti-PD1) confers this resistance. In the GBM tumor microenvironment, Treg cells with increased suppressive phenotype were found of which frequency and anergic phenotype increase after ICB therapy, potentially contributing to the resistance. Targeting Treg has a dual-barreled effect on enhancing anti-tumor immunity: GBM is highly infiltrated with Treg while CD8 T cells are excluded. In view of Treg's intrinsic reactivity to self-antigens, mobilizing converted Treg as effector T cells can be an effective strategy to a tumor type that expresses a low level of neoantigens including GBM. Our study revealed that GITR (glucocorticoid induced TNFR family related protein) is a desirable therapeutic target based on its increased expression on GBM Tregs as compared to peripheral Tregs. Engagement of GITR with agonistic antibody led to conversion of Treg to Th1-like effector T cells, which is accompanied with downregulation of Helios and IL-10 expression that are associated with Treg suppressive function. Through combining anti-GITR with anti-PD1 therapy, tumor recognition by converted Treg and CD8 T cells could be enhanced via IFNg induced promotion of MHC class I and II expression by GBM cells, which also resulted in T cell memory formation in the long-term survivors. To obtain clinically relevant information, we established a standard of care regimen consisting of surgery, radiation, and chemotherapy for orthotopic mouse GBM. We found that the anti-GITR +anti-PD1 therapy tailored to the GBM specific TME synergizes with the standard of care, suggesting a translational potential in patients. Citation Format: Zohreh Amoozgar, Jonas Kloepper, Jun Ren, Rong En Tay, Samuel W. Kazer, Evgeny Kiner, Shanmugarajan Krishnan, Jessica M. Posada, Mitrajit Ghosh, Emilie Mamessier, Christina Wong, Gino B. Ferraro, Ana Batista, Nancy Wang, Mark Badeaux, Sylvie Roberge, Lei Xu, Peigen Huang, Alex K. Shalek, Dai Fukumura, Hye-Jung Kim, Rakesh K. Jain. Targeting Treg cells with GITR activation alleviates resistance to immunotherapy in murine glioblastomas [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P057.

EXTH-18. IL-12 ARMORED CAR T CELL THERAPY FOR HETEROGENEOUS GLIOBLASTOMA

Abstract INTRODUCTION Glioblastoma (GBM) is a lethal primary malignant brain tumor with a median survival of < 20 months. Our next generation immunotherapy utilizes chimeric antigen receptor (CAR) T cells targeted to GBM-specific overexpression of epidermal growth factor receptor variant III (EGFRvIII), “armored” with IL-12, a stimulatory cytokine that enhances T cell persistence and function, to treat orthotopic heterogeneous GBM. METHODS C57Bl/6 mice were intracranially implanted with 5 x 105 of either CT2AvIII tumor cells or a 1:1 mixture of CT2AvIII and CT2A parental tumor cells. Seven days post-implant, mice were treated with 2 x 106 IL-12 CARvIII and monitored for survival. For endogenous T cell activity assessment, TCRα -/- mice were intracranially implanted with either CT2AvIII cells or a 1:1 mixture of CT2AvIII:CT2A parental cells and treated 7 days post implant with intracranial IL-12 CARvIII therapy. RESULTS IL-12 CARvIII therapy was curative in the treatment of CT2AvIII homogeneous tumors and confers a long-term survival in 50% of CT2AvIII:CT2A mice. Furthermore, IL-12 CARvIII therapy successfully eradicated the homogeneous CT2AvIII tumors in TCRα-/- mice but failed to produce any efficacy against the heterogeneous CT2AvIII:CT2A parental tumors, suggesting that endogenous T cells are required for IL-12 CARvIII therapeutic success against heterogeneous tumors. CONCLUSIONS Our findings suggest that IL-12 CARvIII may be an effective treatment against heterogeneous glioma. Furthermore, this data provides insight on treatment against the immunosuppressive tumor microenvironment and applications against other solid tumors. In anticipation of translating this therapy into a phase I clinical trial, we are also investigating adjuvant therapies such irradiation to improve antitumor efficacy of IL-12 CARvIII against heterogeneous glioma.

TMOD-16. HYPOXIA RESHAPES THE GLIOBLASTOMA MICROENVIRONMENT INVOKING NECROSIS AND TRIGGERING DISEASE PROGRESSION

Abstract Emerging necrosis within solid tumors corresponds with malignant progression. Current tumor model systems fail to adequately mimic the magnitude of post-necrotic restructuring within the microenvironment and remain overly reliant on post-mortem and descriptive analyses, obligating researchers to extrapolate causal relationships between necrosis and progression phenomena that emerge during tumor evolution. In glioblastoma (GBM; WHO grade 4), the most malignant primary brain tumor, vascular pathology and central necrosis precede rapid, radial expansion. Despite extensive genetic characterization in GBM, mechanisms enabling selective fitness within a hypoxic/anoxic setting remain poorly understood. Persistent nutrient deprivation culminates in necrosis, dramatically altering the tumor microenvironment (TME). We established mouse models that more aptly capture events found in human gliomas, exposing the dynamic temporal and spatial changes that facilitate expansive progression while including unique microenvironmental stressors typically absent from GBM animal models, specifically central necrosis. This model combines hypoxia-induced focal necrosis within GBM with real time intravital microscopy to capture TME restructuring and elucidate its impact on glioma progression. Our studies use genetically characterized patient-derived orthotopic GBM xenografts, alongside an immunocompetent RCAS/tv-a model, to determine how these processes impact disease progression and outcomes across multiple GBM molecular subtypes. Simultaneously, we employ in vitro models to scrutinize how hypoxia-related crosstalk between GBM, microglia and circulating monocytes alter tumor-associated macrophage (TAMs) recruitment and reprogramming. Our preliminary data suggests microenvironmental cues significantly alter microglia behavior and have demonstrated increased 3D invasion under hypoxic conditions as compared to normoxia. Monocyte invasion varies based on signals emanating from specific GBM subtypes, yet the signaling events that elicit differential responses remains unknown. However, exposure to any GBM conditioned media uniformly upregulates immunosuppressive TAM programming. Our ongoing investigations seek to uncover the mechanisms driving post-necrotic GBM evolution and reactive neuroinflammation

EXTH-45. MICROGLIA-SPECIFIC DISRUPTION OF SIALIC ACID-SIGLEC-9/E INTERACTIONS: A NOVEL IMMUNOTHERAPY AGAINST GLIOBLASTOMA?

Abstract Recently, ‘don’t eat me’-signals like CD47 have emerged as novel innate immune checkpoints, enabling cancer cells to evade clearance by phagocytes such as monocyte-derived macrophages (MDM) or microglia (MG). Here, we aim at defining the role of inhibitory Siglec-9 in human and its mouse homologue Siglec-E in MG-centered immunotherapy against GBM. TCGA RNA-sequencing data revealed a significant correlation between high expression of immunoinhibitory SIGLEC9 and poor survival in GBM patients (log-rank p = 0.02). Siglec-E blockade increased murine MG mediated GBM cell in vitro phagocytosis (normalized phagocytosis of 1.00 in isotype vs. 1.76 in anti-Siglec-E antibody, p < 0.001). By employing a CT-2A orthotopic GBM mouse model with MG-specific spatio-temporal deletion of Siglece (Sall1 CreERT2 x Siglece flox ), we observed high MG-proliferation upon Siglec-E knockdown (Ki-67+ MG 14.8% in Cre- vs. 34.9% in Cre+, p < 0.0001) accompanied by an enhanced microglial GBM-cell uptake (5.6% in Cre- vs. 12.3% in Cre+, p < 0.001). This beneficial response was counteracted by an accentuated influx of pro-tumorigenic MDM in the Cre+ group (CD163high CD86low MDM of total MDM 47.1% in Cre- vs. 65.3% in Cre+, p = 0.002), which prevented an efficient adaptive anti-tumor immune response and survival benefit. Currently, we are investigating the cross-talk between GBM-associated MG and MDM upon Siglec-E knockdown by scRNAseq of the tumor-infiltrating immune compartment, including TCR-clonotype tracking. By genetically targeting sialic acids, the ligand for Siglec receptors, on CT-2A cells (GNE-KO), we observed a strong innate and adaptive immune response with less exhausted tumor-infiltrating CD8+ T-cells (14.8% in WT vs. 5.9% in GNE-KO, p = 0.003), which resulted in a prolonged survival (30d in WT vs. 41d post-tumor-injection in GNE-KO, p = 0.03). These data identify the sialic-acid-Siglec-E pathway as an anti-phagocytic signal in a pre-clinical GBM model, and demonstrate its therapeutic potential in GBM immunotherapy.

IMMU-46. ARTIFICIAL INTELLIGENCE-DIRECTED, GENE THERAPY-BASED TRANSDIFFERENTIATION OF GLIOBLASTOMA TO FUNCTIONAL DENDRITIC CELLS AS NOVEL CANCER IMMUNOTHERAPY

Abstract BACKGROUND Despite recent advances in tumor immunotherapy in solid tumors, success in GBM remains elusive, likely due to its poor immunogenicity and CNS barriers limiting immune cell trafficking. Here we describe a novel approach of stimulating glioma-specific immunity by transdifferentiating GBM cells in situ to induced dendritic cells (iDCs). METHODS We applied NETZEN, an integrated deep-learning and gene network-based ranking computational platform and identified cell fate determinants (CFDs) to convert GBM cells to DCs. CFDs were delivered using a viral vector. Transdifferentiation was assessed by immunophenotyping and iDCs functionally validated by their ability to prime naive T cells. RESULTS A four CFDs subnetwork anchored by PU.1 was sufficient to transdifferentiate mouse GBM cells to CD45+MHCII+ cells with high co-stimulatory CD80 expression and to induce nearly 98% of GBM cells to express 100-fold higher levels of MHCI. Consistent with a new identity of antigen-presenting cells (APC), the induced immune cells are growth arrested, exhibit 3-fold higher phagocytic activity and upregulate the canonical antigen processing and presenting machineries by 10-40 folds, resulting in 40-fold greater efficiency at processing ovalbumin and presenting SIINFEKL on MHCI compared to native GBM cells. Importantly, SIINFEKL-loaded iAPCs are capable of activating naive OTII-CD4+ and OTI-CD8+ T cells, indicating that they are DC-like. In addition, iDCs efficiently present tumor cell-intrinsic antigens and elicit >20-fold higher activation and cytotoxicity in tumor-specific T cells compared to native GBM cells. Lastly, intratumoral GBM-DC transdifferentiation in a syngeneic orthotopic GBM model produces a robust memory T cell response in deep cervical draining lymph nodes compared to control animals. CONCLUSIONS Our results comfirm that GBM-derived iDCs acquire functions similar to native DCs, and thus, lay the foundation for a novel therapeutic approach in which poorly immunogenic tumors like GBM may be forced to generate their own immunity from within through cell fate transdifferentiation.

STEM-21. INHIBITING ADENINE SYNTHESIS ATTENUATES GLIOBLASTOMA CELL STEMNESS AND TEMOZOLOMIDE RESISTANCE

Abstract Glioblastoma (GBM) is a lethal brain cancer that exhibits high levels of drug resistance, a feature partially imparted by tumor cell stemness. Recent work has shown that homozygous MTAP deletion, a frequent genetic alteration in GBM, promotes the stemness of GBM cells. Here, exploiting the MTAP loss-conferred deficiency in adenine salvage, we demonstrated that subtle levels of adenine blockade via treatment with Alanosine, an inhibitor of de novo adenine synthesis, attenuate the stemness of MTAP-null GBM cells. Transcriptomic profiling performed on Alanosine-treated GBM cells revealed a reduction of mitochondrial DNA-encoded gene expression. Furthermore, Seahorse XF analysis showed that Alanosine treatment led to a reduction in mitochondrial respiration and eliminated GBM cells’ spare respiratory capacity, an important metabolic measure of cell fitness that is representative of their ability to respond to oxidative stress. Importantly, long term adenine shortage via treatment with low doses of Alanosine resulted in similarly compromised mitochondria functionality and attenuated GBM cells’ stemness. In further supporting this adenine blockade – compromised mitochondria – reduced stemness cascade, treatment with a mitochondrial respiration inhibitor attenuated the stemness of GBM cells, suggesting the importance of mitochondrial function in maintaining GBM stemness. Finally, in agreement with the diminished stemness and compromised mitochondrial function, we showed that Alanosine sensitized GBM cells to temozolomide (TMZ) in both in vitro cultures and in an orthotopic GBM model. Collectively, these results identify critical roles of adenine supply in maintaining stemness and mitochondrial function in GBM cells, suggest a targeted method of abrogating stemness and chemoresistance in GBMs, and support targeting adenine synthesis as a complementary approach for treating the half of GBMs harboring MTAP deletions.

A Microfluidics-Based Scalable Approach to Generate Extracellular Vesicles with Enhanced Therapeutic MicroRNA Loading for Intranasal Delivery to Mouse Glioblastomas.

Extracellular vesicles (EVs), including exosomes and microvesicles derived from different cell sources, are used as promising nanovesicles for delivering therapeutic microRNAs (miRNAs) and drugs in cancer therapy. However, their clinical translation is limited by the quantity, size heterogeneity, and drug or small RNA loading efficiency. Herein, we developed a scalable microfluidic platform that can load therapeutic miRNAs (antimiRNA-21 and miRNA-100) and drugs while controlling the size of microfluidically processed EVs (mpEVs) using a pressure-based disruption and reconstitution process. We prepared mpEVs of optimal size using microvesicles isolated from neural stem cells engineered to overexpress CXCR4 receptor and characterized them for charge and miRNA loading efficiency. Since the delivery of therapeutic miRNAs to brain cancer is limited by the blood-brain barrier (BBB), we adopted intranasal administration of miRNA-loaded CXCR4-engineered mpEVs in orthotopic GBM mouse models and observed a consistent pattern of mpEVs trafficking across the nasal epithelia, bypassing the BBB into the intracranial compartment. In addition, the CXCR4-engineered mpEVs manifested selective tropism toward GBMs by stromal-derived factor-1 chemotaxis to deliver their miRNA cargo. The delivered miRNAs sensitized GBM cells to temozolomide, resulting in prominent tumor regression, and improved the overall survival of mice. A simple and efficient approach of packaging miRNAs in mpEVs using microfluidics, combined with a noninvasive nose-to-brain delivery route presents far-reaching potential opportunities to improve GBM therapy in clinical practice.

Multiple Antitumor Molecular Mechanisms Are Activated by a Fully Synthetic and Stabilized Pharmaceutical Product Delivering the Active Compound Sulforaphane (SFX-01) in Preclinical Model of Human Glioblastoma

Frequent relapses and therapeutic resistance make the management of glioblastoma (GBM, grade IV glioma), extremely difficult. Therefore, it is necessary to develop new pharmacological compounds to be used as a single treatment or in combination with current therapies in order to improve their effectiveness and reduce cytotoxicity for non-tumor cells. SFX-01 is a fully synthetic and stabilized pharmaceutical product containing the α-cyclodextrin that delivers the active compound 1-isothiocyanato-4-methyl-sulfinylbutane (SFN) and maintains biological activities of SFN. In this study, we verified whether SFX-01 was active in GBM preclinical models. Our data demonstrate that SFX-01 reduced cell proliferation and increased cell death in GBM cell lines and patient-derived glioma initiating cells (GICs) with a stem cell phenotype. The antiproliferative effects of SFX-01 were associated with a reduction in the stemness of GICs and reversion of neural-to-mesenchymal trans-differentiation (PMT) closely related to epithelial-to-mesenchymal trans-differentiation (EMT) of epithelial tumors. Commonly, PMT reversion decreases the invasive capacity of tumor cells and increases the sensitivity to pharmacological and instrumental therapies. SFX-01 induced caspase-dependent apoptosis, through both mitochondrion-mediated intrinsic and death-receptor-associated extrinsic pathways. Here, we demonstrate the involvement of reactive oxygen species (ROS) through mediating the reduction in the activity of essential molecular pathways, such as PI3K/Akt/mTOR, ERK, and STAT-3. SFX-01 also reduced the in vivo tumor growth of subcutaneous xenografts and increased the disease-free survival (DFS) and overall survival (OS), when tested in orthotopic intracranial GBM models. These effects were associated with reduced expression of HIF1α which, in turn, down-regulates neo-angiogenesis. So, SFX-01 may have potent anti-glioma effects, regulating important aspects of the biology of this neoplasia, such as hypoxia, stemness, and EMT reversion, which are commonly activated in this neoplasia and are responsible for therapeutic resistance and glioma recurrence. SFX-01 deserves to be considered as an emerging anticancer agent for the treatment of GBM. The possible radio- and chemo sensitization potential of SFX-01 should also be evaluated in further preclinical and clinical studies.

Targeting UDP-α-d-glucose 6-dehydrogenase alters the CNS tumor immune microenvironment and inhibits glioblastoma growth

Glioblastoma (GBM, WHO grade IV glioma) is the most common and lethal malignant brain tumor in adults with a dismal prognosis. The extracellular matrix (ECM) supports GBM progression by promoting tumor cell proliferation, migration, and immune escape. Uridine diphosphate (UDP)-glucose 6-dehydrogenase (UGDH) is the rate-limiting enzyme that catalyzes the biosynthesis of glycosaminoglycans that are the principal component of the CNS ECM. We investigated how targeting UGDH in GBM influences the GBM immune microenvironment, including tumor-associated microglia/macrophages (TAMs) and T cells. TAMs are the main immune effector cells in GBM and can directly target tumor cells if properly activated. In co-cultures of GBM cells and human primary macrophages, UGDH knockdown in GBM cells promoted macrophage phagocytosis and M1-like polarization. In orthotropic human GBM xenografts and syngeneic mouse glioma models, targeting UGDH decreased ECM deposition, increased TAM phagocytosis marker expression, reduced M2-like TAMs and inhibited tumor growth. UGDH knockdown in GBM cells also promoted cytotoxic T cell infiltration and activation in orthotopic syngeneic mouse glioma models. The potent and in-human-use small molecule GAG synthesis inhibitor 4-methylumbelliferone (4-MU) was found to inhibit GBM cell proliferation and migration in vitro, mimic the macrophage and T-cell responses to UGDH knockdown in vitro and in vivo and inhibit growth of orthotopic murine GBM. Our study shows that UGDH supports GBM growth through multiple mechanisms and supports the development of ECM-based therapeutic strategies to simultaneously target tumor cells and their microenvironment.

P13.15 Pre-clinical trial of T601 oncolytic virus for high grade glima via intra-tumoral injection

Abstract BACKGROUND An effective therapeutic method still hasn’t been devised for lethal high grade glioma. Thus, a method with high anti-tumoral efficiency, tumoral targeting, and acceptable side effect needs to be designed. Oncolytic virotherapy which can specifically lyse tumor cells via mass replication and deleting nucleotide metabolism related gene, like TK, required in viral replication and overexpressed in tumor cells, provides hope for patients. However, the virus only contained TK deletion is unable to show sufficient specificity of anti-tumoral response in tumor cells. Here, the adapted strain of vaccinia virus with high tumoral specificity due to TK and RR deletion and FUC1 insertion, named T601, is chosen in this project. In clinical application, intra-tumoral injection showed improved anti-tumoral efficiency and acceptable side effect. However, intra-tumoral viral injection in orthotropic glioma model is rare. In this project, various biosafety and antitumoral efficiency parameter would be tested for confirming the biosafety and reliability of intra-tumoral T601 viral injection for future clinical trials. MATERIAL AND METHODS For measuring the IC50 of T601, 10 different amounts of virus was tested in vitro via calculating cell viability with CCK-8(cell counting kit-8). For measuring the further antitumoral response of FCU1, different concentration of the 5-FC was added into the medium with IC50 viral amount. To ensure the biosafety of T601, MTD (maximum tolerance dose) was measured. Based on the MTD result, for evaluating the anti-tumoral efficiency, 106 pfu,105 pfu,104 pfu of virus was intra-tumoral injected in orthotopic GBM bearing mice. Tumor size was measured once a week through in vivo bioimaging system. RESULTS 0.022 MOI, the IC50 of T601, showed high cytotoxicity of T601. Moreover, the significantly decreased cell viability under the combined treatment of 5-FC and 0.22MOI T601 showed intact anti-tumoral function. In MTD assay, except for 107 group, no significant weight loss was found. However, in 107 pfu group, mean body weight decreased around 10% and animal fatality happened on day 9. According to the MTD result, certain amount of virus was intra-tumorally injected. In all treatment group, the tumor size was significantly shrined. At the same time, the survival rate of mice under viral treatment was significantly extended. CONCLUSION In summary, T601 exhibited efficient anti-tumoral function and acceptable side effect. T601 treatment prolonged the survival period of GBM mice with acceptable neurotoxicity, demonstrating that T601 contains necessary criterial for intra-tumoral injection. Ultimately, this project provided basic reference information of dose for future clinical trial.