Pediatric Glioblastoma Cell Research Articles

IMMU-21. B7-H3 NANOBODY CAR-T CELL THERAPY IN PEDIATRIC GLIOBLASTOMA

Abstract BACKGROUND Immunotherapy is a developing but challenging field of research for the treatment of central nervous system malignancies. Chimeric antigen receptor (CAR)-T cell therapy has been effective in treating hematologic malignancies, but that success has not been clinically translated to solid tumors in children. B7-H3 has been identified as a promising immunotherapy target as it is highly expressed in pediatric glioblastoma and correlates with tumor progression and poor prognosis. We aim to evaluate a nanobody-based CAR-T cell therapy that binds B7-H3 with high affinity and hypothesize that anti-B7-H3 nanobody CAR-T cells can effectively kill B7-H3-expressing tumor cells. METHODS B7-H3 nanobodies were isolated from camel phage libraries and cloned into lentiviral CAR-T cell constructs. Human T cells were transduced by lentivirus to express CAR on the surface. Nanobody CAR-T cell functionality was evaluated by an impedance-based cytotoxicity assay and cytokine release was measured by ELISA. CAR-T avidity was quantified by the Lumicks z-Movi assay. In vivo studies are evaluating the efficacy of B7-H3 CAR-T cells in an orthotopic glioblastoma model in immunodeficient mice. Mice were treated with B7-H3 CAR-T cells by intracerebroventricular or intravenous delivery. RESULTS We confirmed high expression of B7-H3 on the surface of three pediatric glioblastoma cell lines by flow cytometry and Quantibrite staining. We confirmed effective killing of B7-H3-expressing tumor cells and the release of high levels of cytokines. B7-H3 nanobody CAR-T cells were observed to have high avidity compared to control CAR-T cells. In vivo studies are currently ongoing and treatment response will be evaluated by luminescence imaging and endpoint histologic analysis. CONCLUSION We have shown that B7-H3 nanobody CAR-T cells demonstrate significant preclinical efficacy. It is critical to study CAR-T cell therapy in immunocompetent in vivo models for further development and clinical application.

IMMU-20. EVALUATION OF B7-H3 NANOBODY CAR-T CELLS IN PEDIATRIC GLIOBLASTOMA

Abstract Chimeric antigen receptor (CAR)-T cell therapies are an undoubted success in childhood acute lymphoblastic leukemia; however, this success has not translated clinically to any childhood solid tumor. B7-H3 has been found to be highly expressed in pediatric brain tumors and correlates with tumor progression and poor prognosis. We utilized a novel nanobody based CAR-T cell which can bind tumor antigens with high affinity. We hypothesize that anti-B7-H3 nanobody CAR-T cells mediate more potent antitumor effects both in vitro and in vivo compared to existing antibody-based B7-H3 CAR-T cells. High expression of B7-H3 was confirmed on the surface of two pediatric glioblastoma cell lines by flow cytometry and Quantibrite staining. B7-H3 nanobodies isolated from camel phage libraries were cloned into lentiviral CAR-T cell constructs. We manufactured CAR-T cells by lentiviral transduction of human T cells. Glioblastoma tumor cells were co-cultured with B7-H3 CAR-T cells vs. control T cells. We confirmed B7-H3 specific cytokine release by CAR-T cells via ELISA and effective tumor cell killing via two different cytotoxicity assays (impedance or luciferase based). Nanobody based B7-H3 CAR-T cells exhibited statistically significant higher cytokine release and cytotoxicity compared to antibody based B7-H3 CAR-T cells. In vivo studies are currently evaluating the efficacy of intracerebroventricular delivery of B7-H3 CAR-T cells against two orthotopic glioblastoma models utilizing multiple human donor T cells in immunocompromised mice. We are simultaneously developing a syngeneic orthotopic glioblastoma model to evaluate the nanobody based B7-H3 CAR-T cell cross-reactive in mice utilizing an immunocompetent model. Careful investigation of CAR-T cell therapy in appropriate immunocompetent in vivo models is needed to develop effective immunotherapy for clinical application.

Abstract 3568: Developing pediatric glioblastoma microtumors with a three-dimensional bioprinter

Abstract Background:Glioblastoma multiforme is one of the most devastating forms of brain cancer seen in children. Children diagnosed with high-grade gliomas have poor prognosis, and there remains a need for improvement in the standard of care. In order to improve the standard of care for this condition, it is necessary to develop in vitro models which better recapitulate the nature of the tumors. Three-dimensional bioprinting has proven to be an effective technique in creating microtumors using adult glioblastoma cell lines but remains unexplored in pediatric glioblastoma. As such, we sought to develop a protocol which would allow for the creation of three-dimensionally bioprinted microtumors using patient-derived pediatric glioblastoma cells. Methods:A Bio X three-dimensional bioprinter (Cellink) was utilized to create the microtumor constructs. The composition of the bioink was modified through varying concentrations of sodium alginate and gelatin, or gelatin-methacrylate (Gel-MA). The microtumor size and volume was optimized through the variation of extrusion time, nozzle pressure, and both nozzle and print bed temperature. The crosslinking technique was also optimized through the utilization of 2% calcium chloride for varying exposure times, and also a 405-nm light (for Gel-MA bioink) at varied light intensity, length of time of application to, and distance from, the printed structure. Bioprinted pediatric XD456 brain tumor initiating cell constructs were treated with both chemotherapeutics and radiation to determine the model’s capacity to respond to treatment. PamStation 12 (PamGene Inc.) Kinomic data and RNA-seq (Illumina) data were generated for the constructs. Imaging was performed using Cytation 5 imager (Agilent). Results:Fluorescent microscopy with Calcein AM and Sytox™ Orange (ThermoFisher), along with viability assessments via CellTiter-Glo®, demonstrated expected cytotoxic effects after exposure to 10 μM Cisplatin. Additionally, PamStation 12 Kinomic and RNA-seq data indicated a difference in kinomic signaling between the bioprinted constructs as compared to the XD456 cells cultured as neurospheres. Conclusions:The utilization of a three-dimensional bioprinter allows for the creation of microtumors using patient-derived pediatric glioblastoma cells. Future studies will investigate the microtumors’ capacity as a drug screening model. The model will also be developed to further recapitulate the native tumor microenvironment of pediatric glioblastoma. Citation Format: Andee M. Beierle, Taylor L. Schanel, Hasan Alrefai, Joshua C. Anderson, Patricia H. Hicks, Lauren C. Nassour, Christopher D. Willey. Developing pediatric glioblastoma microtumors with a three-dimensional bioprinter. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3568.

RTID-04. CLINICAL POTENTIAL OF TARGETING TRANSFORMING GROWTH FACTOR BETA 2 WITH OT-101 FOR POST-RADIATION CONSOLIDATION IN DIFFUSE INTRINSIC PONTINE GLIOMA

Abstract Diffuse intrinsic pontine glioma (DIPG) in children has a dismal prognosis with a median overall survival (OS) of 10 months and a 2-year overall survival rate of < 10% after standard radiation therapy. Chemotherapy does offer clinically meaningful benefits. Therefore, there is an urgent need for therapeutic innovations for treatment of pediatric DIPG. High-grade glioma cells, including pediatric glioblastoma and DIPG cells have been shown to produce transforming growth factor beta 2 (TGFB2) which has been implicated both as promoter of glioma cells and as a key contributor to the T-cell hyporesponsiveness of the tumor microenvironment (TME) towards glioma cells. OT-101 is a first-in-class RNA therapeutic designed to abrogate the immunosuppressive and tumor promoting actions of TGF-B2. At low micromolar concentrations, OT-101 reduces the TGFB2 secretion by human glioma cells, blocks their proliferation as well as migration, and restores the anti-glioma cytolytic function of patient-derived T-cells. In a Phase 2 clinical study, intracerebrally applied OT-101 administered via convection-enhanced delivery showed promising single agent activity in Recurrent/Refractory (R/R) high-grade glioma (HGG) (Uckun et al., Cancers. 2019 28;11(12):1892). The intrathecal administration of antineoplastic drugs directly in the CSF allows to bypass the selective filter of BBB, achieving significant concentrations of the antineoplastic agents in CSF, while reducing the likelihood of systemic toxicity. Informed by favorable safety pharmacology studies of intrathecally delivered OT-101 in rabbits and primates and encouraged by its single agent activity in adult patients with HGG, we decided to embark upon a multi-center, two-part, randomized Phase 1-2 study of OT-101 in pediatric patients with DIPG. Multiple doses of OT-101 will be administered after completion of radiation therapy as intrathecal bolus injections. The study is designed to determine: 1) the maximum tolerated dose (MTD) or recommended Phase 2 dose (RP2D) of OT-101, and 2) its efficacy in children with DIPG.

Pediatric glioblastoma cells are sensitive to drugs that inhibit eIF2α dephosphorylation and its phosphomimetic S51D variant.

We found that pediatric glioblastoma (PED-GBM) cell lines from diffuse intrinsic pontine glioma (DIPG) carrying the H3K27M mutation or from diffuse hemispheric glioma expressing the H3G34R mutation are sensitive to the combination of vorinostat (a histone deacetylase inhibitor) and PARP-1 inhibitors. The combined treatment increased the phosphorylation of eIF2α (P-eIF2α) relative to each drug alone and enhanced the decrease in cell survival. To explore the role played by increased P-eIF2α in modulating PED-GBM survival and response to treatments, we employed brain-penetrating inhibitors of P-eIF2α dephosphorylation: salubrinal and raphin-1. These drugs increased P-eIF2α, DNA damage, and cell death, similarly affecting the sensitivity of DIPG cells and derived neurospheres to PARP-1 inhibitors. Interestingly, these drugs also decreased the level of eIF2Bϵ (the catalytic subunit of eIF2B) and increased its phosphorylation, thereby enhancing the effect of increased P-eIF2α. Transient transfection with the S51D phosphomimetic eIF2α variant recapitulated the effect of salubrinal and raphin-1 on PED-GBM survival and sensitivity to PARP-1 inhibitors. Importantly, either salubrinal or raphin-1 dramatically increased the sensitivity of DIPG cells to radiation, the main treatment modality of PED-GBM. Finally, PED-GBM was more sensitive than normal human astrocytes to salubrinal, raphin-1, and the treatment combinations described herein. Our results indicate that combinations of histone deacetylase inhibitors and PARP-1 inhibitors should be evaluated for their toxicity and efficacy in PED-GBM patients and point to drugs that increase P-eIF2α or modulate its downstream effectors as a novel means of treating PED-GBM.

Different Effects of Valproic Acid on SLC12A2, SLC12A5 and SLC5A8 Gene Expression in Pediatric Glioblastoma Cells as an Approach to Personalised Therapy.

Valproic acid (VPA) is a histone deacetylase inhibitor with sex-specific immunomodulatory and anticancer effects. This study aimed to investigate the effect of 0.5 and 0.75 mM VPA on NKCC1 (SLC12A2), KCC2 (SLC12A5) and SLC5A8 (SLC5A8) co-transporter gene expressions in pediatric PBT24 (boy’s) and SF8628 (girl’s) glioblastoma cells. The SLC12A2, SLC12A5 and SLC5A8 RNA expressions were determined by the RT-PCR method. The SLC12A2 and SLC5A8 expressions did not differ between the PBT24 and SF8628 controls. The SLC12A5 expression in the PBT24 control was significantly higher than in the SF8628 control. VPA treatment significantly increased the expression of SLC12A2 in PBT24 but did not affect SF8628 cells. VPA increased the SLC12A5 expression in PBT24 and SF8628 cells. The SLC12A5 expression of the PBT24-treated cells was significantly higher than in corresponding SF8628 groups. Both VPA doses increased the SLC5A8 expression in PBT24 and SF8628 cells, but the expression was significantly higher in the PBT24-treated, compared to the respective SF8628 groups. The SLC5A8 expression in PBT24-treated cells was 10-fold higher than in SF8628 cells. The distinct effects of VPA on the expression of SLC12A2, SLC12A5 and SLC5A8 in PBT24 and SF8628 glioblastoma cells suggest differences in tumor cell biology that may be gender-related.

Structural Basis of RACK7 PHD Domain to Read a Pediatric Glioblastoma‐Associated Histone Mutation H3.3G34R

Main observation and conclusionHistone point mutations, including missense mutations on histone H3 at positions 27 (K27M), 34 (G34R/V, G34W, G34L) and 36 (K36M), were identified as potential cancer driver mutations. H3.3G34R/V mutations account for pediatric glioblastomas (GBM). RACK7 (also known as ZMYND8, PRKCBP1) was recently reported to specifically bind H3.3G34R through its PHD (plant homedomain) domain (PHDRACK7) in vitro and in H3.3G34R pediatric glioblastoma cells, playing key roles in H3.3G34R‐mediated gene transcription. Herein, we provided both biochemical and NMR structural evidences that PHDRACK7 recognized histone H3.3G34R mutant via a mechanism distinct from all other reported PHD domains. Except the reported residue D104, two new sites D108 and L121 of PHDRACK7 were found necessary for the interactions between PHDRACK7 and histone H3.3G34R peptide. Our results provided a potential molecular basis for pediatric GBM driven by the H3.3G34R mutation.

HGG-04. ZINC ENHANCES TEMOZOLOMIDE CYTOTOXICITY IN PEDIATRIC GLIOBLASTOMA MULTIFORME MODEL SYSTEM

BACKGROUNDTemozolomide (TMZ) is an alkylating agent that has become the mainstay treatment of the most malignant brain cancer, glioblastoma multiforme (GBM). Unfortunately only a limited number of patients respond to it positively. We have shown that zinc metal reestablishes chemosensitivity in adult GBM in vitro and also in vivo but this effect has not been tested with pediatric GBM.METHODSUsing Human pediatric glioblastoma cell lines- KNS42 (mutant p53/ MGMT [+]) and SF188 (mutant p53/ MGMT [-]), we investigated whether addition of zinc to TMZ enhances its cytotoxicity against GBM.RESULTS In vitro cell viability analysis showed that the cytotoxic activity of TMZ was substantially increased with addition of zinc and this response was accompanied by an elevation of p21, PUMA, BAX and a decrease in growth fraction as manifested by low ki67. Beta gal analysis showed that most of the remaining cells after the combination therapy are in senescence state. In order to eliminate the senescent population created as a result of the combined treatment of TMZ and Zinc, we decided to use a senolytic agent Navitoclax (ABT-263) that was demonstrated to be effective in reducing senescent cells by specific inhibition of Bcl-2, Bcl-XL and Bcl-w. Following the addition of Navitoclax to the combined treatment, SF188 cells, but not KNS42, show a significance reduction in viability compare to the combination treatment.CONCLUSIONSOur results suggest that zinc may serve as a potentiator of TMZ therapy in pediatric GBM patients and using a second hit with senolytic drug in some cases may be even more beneficial.

A comparative study of brain tumor cells from different age and anatomical locations using 3D biomimetic hydrogels

Brain tumors exhibit vast genotypic and phenotypic diversity depending on patient age and anatomical location. Hydrogels hold great promise as 3D in vitro models for studying brain tumor biology and drug screening, yet previous studies were limited to adult glioblastoma cells, and most studies used immortalized cell lines. Here we report a hydrogel platform that supports the proliferation and invasion of patient-derived brain tumor cell cultures (PDCs) isolated from different patient age groups and anatomical locations. Hydrogel stiffness was tuned by varying poly(ethylene-glycol) concentration. Cell adhesive peptide (CGRDS), hyaluronic acid, and MMP-cleavable crosslinkers were incorporated to facilitate cell adhesion and cell-mediated degradation. Three PDC lines were compared including adult glioblastoma cells (aGBM), pediatric glioblastoma cells (pGBM), and diffuse pontine intrinsic glioma (DIPG). A commonly used immortalized adult glioblastoma cell line U87 was included as a control. PDCs displayed stiffness-dependent behavior, with 40 Pa hydrogel promoting faster tumor proliferation and invasion. Adult GBM cells exhibited faster proliferation than pediatric GBM, and DIPG showed slowest proliferation. These results suggest both patient age and tumor location affects brain tumor behaviors. Adult GBM PDCs also exhibited very different cell proliferation and morphology from U87. The hydrogel reported here can provide a useful tool for future studies to better understand how age and anatomical locations impacts brain tumor progression using 3D in vitro models.

Abstract 4176: Involvement of the lysosome in the cytotoxicity of Gallium Maltolate in human pediatric brain tumor cell lines

Abstract Introduction: Gallium maltolate (GaM) is a novel small molecule metallocompound that acts as an iron mimetic Trojan Horse to disrupt iron-dependent proteins essential in tumor biology. We recently reported that GaM retards the growth of orthotopically implanted adult human glioblastoma (GBM) xenografts in a rodent brain tumor model and is cytotoxic to glioblastoma stem cells (Mol Cancer Ther 17:1240, 2018). GaM's mechanisms of action include inhibition of both mitochondrial function and iron-dependent ribonucleotide reductase. In the present study, we further explored whether GaM has antitumor activity in pediatric brain tumor cells. Experimental procedures: The cytotoxicity of GaM in pediatric GBM and Atypical Teratoid Rhabdoid Tumor (ATRT) cell lines was examined by MTT and clonogenic assays. Apoptotic cell death was assessed by flow cytometry with annexin V (AV) and propidium iodide (PI) staining. Cellular bioenergetics was measured using a Seahorse XF96 Extracellular Flux Analyzer. GaM interaction with lysosomes was analyzed by immunofluorescence and electron microscopy (EM). Results: In a panel of pediatric brain tumor cell lines, CHLA-266 ATRT and GBM cells displayed the greatest sensitivity to GaM. Complete inhibition of colony growth was achieved with 6 - 7 µM GaM over a 96-hour incubation. With a shorter incubation of 48-h, 25 µM GaM induced apoptosis in > 60% of CHLA-266 ATRT cells. In contrast, a longer incubation of 96-h with 25 µM GaM was required to induce apoptosis in > 50% of CHLA-200 GBM, 69% of SF188 GBM and 60% of SJ-GBM2 cell lines. After a 24-h of incubation with GaM, prior to detectable cell death, CHLA-266 and GBM cells displayed a marked reduction in mitochondrial reserve capacity, glycolysis, and glycolytic capacity. Immunofluorescence staining for the lysosomal marker LAMP1 showed that GaM produced an increase in lysosome size and number after a 24-h or 48-h incubation relative to control cells. EM studies demonstrated a gallium signal within the lysosomal compartment as early as 6-h of incubation. EM findings consistent with GaM-induced apoptosis were noted after a 24-h incubation, thus confirming the results of the AV/PI assay. After 24-h however, gallium was no longer detectable in the lysosomes, suggesting that lysosomal disruption led to the release of gallium into the cytoplasm. Conclusions: Our results identify GaM as a novel compound with significant activity against a panel of cell lines representative of pediatric brain tumors. Most of the cell lines used in our study were derived from tumors taken from heavily pre-treated patients and were resistant to conventional chemotherapeutic drugs. Their sensitivity of GaM suggests that GaM does not share cross-resistance with other antineoplastic drugs. Our study also provides new insight into the lysosomes as a target for gallium. The prognosis of adult and pediatric brain tumors is dismal and there is an urgent need to develop newer drugs for this malignancy. Further investigation of GaM as a therapeutic agent for brain tumors is warranted. Citation Format: Salvatore Molino, Christopher R. Chitambar. Involvement of the lysosome in the cytotoxicity of Gallium Maltolate in human pediatric brain tumor cell lines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4176.

Abstract B09: Epigenetic changes mediated by H3.3 G34R mutation in a CRISPR-edited pediatric glioblastoma cell line

Abstract Background: Pediatric glioblastomas (pGBM) are aggressive and lethal brain tumors with few effective treatment options. Since the discovery of recurrent histone H3 mutations in pGBMs, multiple groups have sought to uncover the mechanism by which these mutations drive tumorigenesis. One striking aspect of histone H3 mutations is their tumor location and age-specificity: the K27M mutation occurs in midline brain structures in infants and young children, whereas the G34R/V mutations occur in the cerebral hemisphere of adolescents. Efforts to elucidate the mechanism underlying K27M tumorigenesis have centered on K27M-mediated inhibition of the PRC2 complex and the resulting drastic loss of the repressive H3K27me3 mark in these tumors. Studies have shown that G34 mutations affect methylation of the adjacent K36 residue, resulting in a local decrease of the active H3K36me3 mark on the mutant histone. It remains uncertain whether the G34R/V mutations promote global changes in the epigenome that drive tumorigenesis. Methods: To investigate the epigenetic consequences of the G34R mutation, we generated an isogenic in vitro model using CRISPR/Cas9 to correct the G34R mutation in a pGBM-derived cell line. We then comprehensively characterized and compared the epigenome and transcriptome of CRISPR-unedited clones (carrying the G34R mutation) and CRISPR-edited (wild-type) clones. Using ChIP-seq, we profiled H3K36me3 and H3K27me3 marks to study G34R-mediated regulation of these antagonistic marks. Results: ChIP-seq profiling revealed that CRISPR editing of the G34R mutation had minimal effects on total levels or genomic distribution and profile of H3K36me3 and H3K27me3 marks. In contrast, we previously showed that CRISPR editing of the K27M mutation in pGBM cell lines rescued total H3K27me3 levels and genomic distribution to that of histone wild-type pGBMs. In agreement with their similar epigenetic profiles, there were very few transcriptomic differences between G34R and wildtype cells. Conclusions: Our results suggest that the epigenome in G34R-mutant pGBM cells may be less dynamic or amenable to epigenetic perturbation from removal of the initial driver oncomutation than K27M-mutant tumors. These findings underscore that different strategies may be required to target and treat G34R and K27M-mutant pGBM tumors. Citation Format: Shriya Deshmukh, Carol C.L. Chen, Amira Ouanouki, Nada Jabado. Epigenetic changes mediated by H3.3 G34R mutation in a CRISPR-edited pediatric glioblastoma cell line [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B09.

Profiling cytotoxic microRNAs in pediatric and adult glioblastoma cells by high-content screening, identification, and validation of miR-1300

MicroRNAs play an important role in the regulation of mRNA translation and have therapeutic potential in cancer and other diseases. To profile the landscape of microRNAs with significant cytotoxicity in the context of glioblastoma (GBM), we performed a high-throughput screen in adult and pediatric GBM cells using a synthetic oligonucleotide library representing all known human microRNAs. Bioinformatics analysis was used to refine this list and the top seven microRNAs were validated in a larger panel of GBM cells using state-of-the-art in vitro assays. The cytotoxic effect of our most relevant candidate was assessed in a preclinical model. Our screen identified ~100 significantly cytotoxic microRNAs with 70% concordance between cell lines. MicroRNA-1300 (miR-1300) was the most potent and robust candidate. We observed a striking binucleated phenotype in miR-1300 transfected cells due to cytokinesis failure followed by apoptosis. This was also observed in two stem-like patient-derived cultures. We identified the physiological role of miR-1300 as a regulator of endomitosis in megakaryocyte differentiation where blockade of cytokinesis is an essential step. In GBM cells, where miR-1300 is normally not expressed, the oncogene Epithelial Cell Transforming 2 (ECT2) was validated as a direct key target. ECT2 siRNA phenocopied the effects of miR-1300, and ECT2 overexpression led to rescue of miR-1300 induced binucleation. We showed that ectopic expression of miR-1300 led to decreased tumor growth in an orthotopic GBM model. Our screen provides a resource for the neuro-oncology community and identified miR-1300 as a novel regulator of endomitosis with translatable potential for therapeutic application.

Pediatric glioblastoma target-specific efficient delivery of gemcitabine across the blood-brain barrier via carbon nitride dots.

Pediatric glioblastomas are known to be one of the most dangerous and life-threatening cancers among many others regardless of the low number of cases reported. The major obstacles in the treatment of these tumors can be identified as the lack of prognosis data and the therapeutic requirement to be able to cross the blood-brain barrier (BBB). Due to this lack of data and techniques, pediatric patients could face drastic side effects over a long-time span even after survival. Therefore, in this study, the capability of non-toxic carbon nitride dots (CNDs) to selectively target pediatric glioblastoma cells was studied in vitro. Furthermore, the nanocarrier capability and efficiency of CNDs were also investigated through conjugation of a chemotherapeutic agent and transferrin (Tf) protein. Gemcitabine (GM) was introduced into the system as a chemotherapeutic agent, which has never been successfully used for the treatment of any central nervous system (CNS) cancer. More than 95% of selective damage of SJGBM2 glioma cells was observed at 1 μM of CN-GM conjugate with almost 100% viability of non-cancerous HEK293 cells, although this ability was diminished at lower concentrations. However, further conjugation of Tf to obtain CN-GM-Tf allowed the achievement of selective targeting and prominent anti-cancer activity at a 100-fold lower concentration of 10 nM. Furthermore, both conjugates were capable of effectively damaging several other brain tumor cells, which were not well responsive towards the single treatment of GM. The capability of BBB penetration of the conjugates was observed using a zebrafish model, which confirms the CNDs' competence as an excellent nanocarrier to the CNS.

Abstract A109: Repurposing low dose of quinoline methanol derivatives as a novel treatment for pediatric glioblastoma

Abstract Both primary brain tumors and secondary brain metastases that start in another part of the body and spreads to the brain, such as lung, breast, melanoma, kidney, nasopharynx, and colon cancers have limited or no response to chemotherapy. Through repurposing drug screen using pediatric glioblastoma cells established from newly diagnosed and recurrent patients, we identified that FDA-approved compound mefloquine have significant cancer killing properties. A growing body of evidence suggests that this extensively used anti-malaria drug may cause adverse neurological effects after crossing the blood-brain barrier and accumulates in the brain at relatively high concentration. To the amelioration of this problem, an analog campaign was conducted in selecting compounds lacking penetration but maintaining the anti-malarial properties using mefloquine as the leading compound. We therefore thought to take advantage of this unique characteristic and use low dose of these compounds and target the infiltrative disease reservoir while avoiding the neurological side effects. While this program focuses on high anti-malaria potency, low neurotoxicity (against rat neurons), and low brain absorption, we are interested in high anti-malaria potency, low general toxicity, and high brain/tumor concentration. Among these compounds, we selected TQM due to its promising results in efficacy, potency, and selectivity in killing brain tumor cells at a much lower range compared to mefloquine based on our quantitative structure-activity relationship model. Studies suggested that this group of drugs induce cell apoptosis in host and parasites through increased production of reactive oxygen species (ROS). Even though for a long time ROS is believed to promote cell proliferation; however, brain cancer cells, due to their high basal metabolic rate, are more susceptible to ROS. Inducing an increased intracellular ROS levels in brain cancer cells may trigger cell death and destroy malignant cells, while sparing normal cells. Our preliminary results confirmed that mefloquine and its derivatives induce apoptosis through autophagy and ROS signaling in brain cancer cells at a much lower concentration, and these compounds reduces tumor growth and extends animal survival in mouse models. Our results pave the way for translation of those compounds to the clinic. Citation Format: Jian Teng, Litia Carvalho, Elie Tabet, Bakhos Tannous. Repurposing low dose of quinoline methanol derivatives as a novel treatment for pediatric glioblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A109. doi:10.1158/1535-7163.TARG-19-A109

Abstract 5215: Targeting of HOX-PBX binding in glioblastoma multiforme as a novel therapeutic treatment

Abstract The HOX genes encode a family of transcription factors that play an essential role in embryonic patterning. They are also aberrantly expressed in numerous cancers, including glioblastoma (GBM). Three Amino Acid Loop Extension Homeobox (TALE) proteins act as important co-factors for HOX proteins, modulating their binding affinities to genomic targets. TALE proteins include the Pre-B-cell leukemia homeobox (PBX) proteins 1-4, which bind anterior HOX proteins, facilitating their nuclear entry and limiting their degradation. HTL00-1 is a 2nd generation hexapeptide drug that inhibits HOX-PBX dimer formation, and has been shown to induce rapid apoptosis in cancer cells, but not normal cells, through the rapid upregulation of genes including cFos, DUSP1, and EGR1. We have found that both HOX and TALE genes are markedly dysregulated in primary GBM tumors as well as in murine (GL261), adult (LN18, U87-MG, U251-MG) and pediatric (KNS42, SF188) GBM cell lines, all of which are sensitive to HTL-001. These genes were even more highly expressed in experimentally induced GBM cancer stem cells (CSCs) compared with parental lines, with a corresponding increase in sensitivity to HTL-001. We also investigated the in vivo activity of HTL-001 in Black 6 mice carrying GL-261 subcutaneous and orthotropic tumors with twice weekly intraperitoneal delivery. HTL-001 was shown to cross the blood brain barrier using Alexa660 labelled peptide, and significant anti-tumor activity was observed in both subcutaneous and orthotropic models with increased survival (p<0.02 and p<0.0078, respectively). Resected tumors from HTL-001 treated mice showed marked evidence of apoptosis, tumor vasculature disruption and focal necrosis compared to untreated tumors. Taken together, our findings indicate that HOX-PBX inhibition is a potential therapeutic target for adult and pediatric GBM patients. Citation Format: Richard Morgan, Monika Primon, Steven Shnyder, Susan Short, Balveen Kaur, Bangxing Hong, Izhar Bagwan, William Rogers, Hardev S. Pandha. Targeting of HOX-PBX binding in glioblastoma multiforme as a novel therapeutic treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5215.

Abstract 3499: Dianhydrogalactitol (VAL-083) in combination with AZD1775 increases survival in diffuse intrinsic pontine glioma (DIPG), in vivo

Abstract OBJECTION: VAL-083 is a structurally unique bi-functional DNA targeting agent that readily crosses the blood-brain barrier and accumulates in brain tumor tissue. VAL-083 induces interstrand crosslinks leading to DNA double-strand breaks and S/G2 cell cycle arrest. By inhibiting the G2 checkpoint regulator kinase Wee1, AZD1775 allows a cancer cell with DNA damage to progress past the G2 checkpoint and into premature mitosis leading to cancer cell death. Herein we assess the activity of VAL-083 as single agent as well as in combination with Wee1 Kinase inhibitor AZD1775 in patient derived model systems of DIPG. METHODS: DIPG derived cell lines SF8628 and NEM157 (H3.3K27) as well as SF10693 (H3.1K27M) and pediatric glioblastoma cell lines SF188 (H3.3K27 wildtype) were treated with increasing concentrations of single agent VAL-083 as well as in combination with AZD1775. To determine potential synergistic activity, we applied the Chou-Talalay method, which allows the quantitative determination of drug interactions by calculating a combination index (CI). In vivo activity of VAL-083 (3 mg/kg) as single agent as well as in combination with AZD1775 (60 mg/kg) was assessed in an orthotopic engraftment model of pediatric DIPG (SF8628). RESULTS: The IC50 of VAL-083 as single agent ranged from 1µM to 10µM. VAL-083 exhibited synergistic activity in combination with AZD1775 in cell lines SF8628 and SF188 and additive effect in NEM157 with CI values ranging from 0.04 to 0.95, with CI < 1 indicating synergy. In vivo, treatment with VAL-083 as single agent and in combination with AZD1775 conferred significant survival benefit to mice with engrafted DIPG tumors compared to control as well as single agent treatment with AZD1775. The median survival for mice treated with VAL-083 alone was 54.5 days (p=0.0004, VAL-083 vs. control) and for the VAL-083/AZD1775 combination it was 62 days (p<0.0001, VAL-083/AZD1775 vs. control), compared to 44 days for control and 47 days for mice treated with AZD1775 alone (p=0.0839, AZD1775 vs. control). CONCLUSION: Our present study highlights that the combination of VAL-083 and AZD1775 might be a promising new therapeutic strategy for children with DIPG. Ongoing studies continue to assess the in vivo activity in other DIPG models as well explore the underlying mechanism of action of the combination strategy. Citation Format: Anne Steino, Xiaodong Yang, Cassie Kline, Jeffrey Bacha, Dennis M. Brown, Sabine Mueller. Dianhydrogalactitol (VAL-083) in combination with AZD1775 increases survival in diffuse intrinsic pontine glioma (DIPG), in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3499.

Using PIK3CB and connexin-43 inhibition to sensitize pediatric glioblastoma cells to temozolomide.

e13572 Background: Glioblastoma (GBM) is one of the most common high grade gliomas in pediatric patients, accounting for 2.8% of central nervous system tumors. Standard of care for glioblastoma includes surgical resection, radiation, and temozolomide chemotherapy. However, many pediatric patients are not suitable candidates for surgery, and their tumors inevitably develop temozolomide resistance. Despite aggressive treatment, temozolomide (TMZ) resistance leads to tumor recurrence in 90% of patients. Thus, outcome for children with pediatric GBM remains poor with a 2-year survival rate ranging from 10-30%, highlighting the need for new therapeutic approaches. Connexin-43 (Cx43) and PIK3CB are proteins involved in conferring temozolomide resistance in glioblastoma. Combined inhibition of these proteins could improve temozolomide sensitivity and help develop a selective therapy that significantly improves quality of life and prognosis in pediatric glioblastoma patients. Methods: Primary GBM cells were plated in a 96-well plate and treated with vehicle (DMSO), TMZ (50 μmol/L), TGX-221 (20 μmol/L), αCT1 (30 μmol/L), or a combination of the latter three drugs. Cell viability was measured using the MTS assay. Primary pediatric GBM cell lines SJ-GBM2 and CHLA-200 were tested. These drug combinations were also tested in normal human astrocytes to make sure there were no off-target effects. Results: We found that targeting PIK3CB and Cx43 in primary pediatric GBM increases their sensitivity to TMZ. The triple combination of TMZ, TGX-221, and αCT1 significantly decreased cell viability than each drug alone while sparing normal human astrocytes. Conclusions: Our findings suggest that inhibiting PIK3CB and Cx43 enhances therapeutic response to TMZ and limits off-target effects in pediatric GBM. These findings closely mirror our results using the triple combination in adult glioblastoma as well, thus laying the groundwork for a novel combination therapeutic that can be used across age groups.

DTCM-glutarimide Delays Growth and Radiosensitizes Glioblastoma.

Glioblastoma (GBM) is the most aggressive brain tumor. Even with the advent of temozolomide, patient survival remains poor, with expected median survival around 1 year from diagnosis. Consequently, the relentless search for new therapeutic strategies able to increase patient outcome persists. 3-[(dodecylthiocarbonyl) methyl] glutarimide (DTCM-g) is a new anti-inflammatory compound that already showed antitumor effects. Clonogenic survival, proliferation, apoptosis, cell cycle progression and invasion capacity of pediatric and adult GBM cell lines (U87MG, U251MG, SF188 and KNS-42) were evaluated under treatment with DTCM-g. The combined treatment with radiation was also evaluated in vitro and in vivo through xerographic models. DTCM-g is able to impair proliferation, reduce clonogenic capacity and induce cell cycle arrest in GBM cell lines. No alteration in apoptosis rates was found after treatment. DTCM-g also reduces the invasion capacity of all GBM cell lines without alterations in MMP2 and uPa expression. Moreover, the drug radiosensitized GBM in vitro and in vivo. Although additional studies are still necessary to support our findings, our results suggest that DTCM-g may be a promising drug on the adjuvant treatment of GBM exhibiting antitumor effects, especially through radiosensitization.

PDTM-33. ATRX LOSS CONFERS ENHANCED SENSITIVITY TO COMBINED PARP INHIBITION AND RADIOTHERAPY IN PAEDIATRIC GLIOBLASTOMA MODELS

Paediatric high grade glioma (pHGG) are defined by recurrent mutations in H3 histones, as well as frequent alterations in the SWI/SNF chromatin remodelling gene ATRX (α-thalassemia mental retardation X-linked), although the precise role of ATRX in tumorigenesis remains unclear. We sought to explore this using genomic analysis of patient samples, CRISPR-Cas9 engineered isogenic ATRX knockout (KO) cell lines and primary-patient-derived cultures. In combined retrospective and prospective cohorts of pHGG samples, we found ATRX mutations in 95/510 (18.6%) cases (27% hemispheric glioblastoma, 13% diffuse midline glioma), with the majority of truncating mutations found in the ADD domain, and missense mutations almost exclusively in the helicase domain. ATRX mutations commonly co-segregate with H3.3G34 and TP53 mutations, and define a subgroup of patients with a longer overall survival, though with a greater number of somatic mutations and copy number alterations than wild-type cases. CRISPR/Cas9-mediated ATRX KO targeting the ADD domain in TP53 mutant paediatric glioblastoma cells lead to loss of imprinting at the H19 locus in concert with upregulation of a pro-invasive transcriptional programme, though a slower rate of orthotopic tumour growth in vivo. ATRX deficient cells showed an abrogated DNA damage response, with prolonged accumulation of gH2AX foci after irradiation, and an increased dependency on PARP1 through persistent parylation and stalled replication forks. Screening ATRX-deficient isogenics and patient-derived cells against a library of >400 chemotherapeutics and small molecules identified a specific dependency for ATRX loss and sensitivity to distinct PARP inhibitor chemotypes, including catalytic inhibitors (olaparib, rucaparib), and PARP trappers (talazoparib). ATRX deficiency further conferred an enhanced radiosensitization of olaparib in vitro and a prolonged survival of mice treated with combined PARP inhibition and radiotherapy in vivo. These data suggest a synthetic lethality for PARP inhibitors in ATRX-deficient pHGG, and may represent a novel therapeutic strategy for these highly aggressive tumours.

Irradiation of pediatric glioblastoma cells promotes radioresistance and enhances glioma malignancy via genome-wide transcriptome changes.

Pediatric glioblastoma (GBM) is a relatively rare brain tumor in children that has a dismal prognosis. Surgery followed by radiotherapy is the main treatment protocol used for older patients. The benefit of adjuvant chemotherapy is still limited due to a poor understanding of the underlying molecular and genetic changes that occur with irradiation of the tumor. In this study, we performed total RNA sequencing on an established stable radioresistant pediatric GBM cell line to identify mRNA expression changes following radiation. The expression of many genes was altered in the radioresistant pediatric GBM model. These genes have never before been reported to be associated with the development of radioresistant GBM. In addition to exhibiting an accelerated growth rate, radioresistant GBM cells also have overexpression of the DNA synthesis-rate-limiting enzyme ribonucleotide reductase, and pro-cathepsin B. These newly identified genes should be concertedly studied to better understand their role in pediatric GBM recurrence and progression after radiation. It was observed that the changes in multiple biological pathways protected GBM cells against radiation and transformed them to a more malignant form. These changes emphasize the importance of developing a treatment regimen that consists of a multiple-agent cocktail that acts on multiple implicated pathways to effectively target irradiated pediatric GBM. An alternative to radiation or a novel therapy that targets differentially expressed genes, such as metalloproteases, growth factors, and oncogenes and aim to minimize oncogenic changes following radiation is necessary to improve recurrent GBM survival.