Citations

Abstract Glioblastoma (GBM) presents as a highly aggressive and malignant brain tumor with limited treatment options. Despite the availability of existing therapies, the invasive characteristics of GBM frequently result in tumor recurrence and poor prognosis. We previously developed a B-cell-based therapy (BVax) that has shown promising results in its ability to induce therapeutic responses in preclinical models of GBM. Our research revealed potency of BVax can be attributed to its robust antigen-presenting capability, cognate antigen specificity with T cells, and ability to generate tumor-reactive antibodies. The present study aims to characterize the unique antigenic reactivity of BVax-derived antibodies by evaluating their immunological effects and therapeutic potential in both murine models and GBM patient-specific samples. Leveraging CD45.1 and CD45.2 congenic mouse models, we first revealed that BVax preferentially migrated to glioma-bearing brains upon intravenous injection, resulting in a CD38+CD20-CD19+ plasmablast phenotype. To further characterize the reactivity of BVax-derived immunoglobulins (Ig), we utilized proteomic analysis and identified preferential targets of Gelsolin, Fibronectin, Versican, Fibrinogen, and collagen type IV alpha, commonly associated with cell motility and the extracellular matrix. Microdialysis data confirmed the increased presence of many of these BVax-derived Igs-recognized antigens in tumor regions compared to normal brain regions in GBM patients. Utilizing purified BVax-derived antibodies from patient specimen, in vitro cell motility assays demonstrated a significant prevention of the invasion and migration of cell lines from patient derived xenograft (PDX). In this study, we concluded that BVax elicits antitumor activity through targeted migration to tumor-bearing regions, differentiation into plasmablasts, and robust secretion of specific Igs that retain the ability to inhibit functionalities associated with cell invasion and migration. Here, we present the first in-depth study that characterizes GBM patients’ antibody reactivity, specifically a subset of inflammatory B cells (BVax) with therapeutic effects that offer an indication for a new direction of immunotherapies.

Aberrant metabolism is a hallmark of malignancies including gliomas. Intracranial microdialysis enables the longitudinal collection of extracellular metabolites within CNS tissues including gliomas and can be leveraged to evaluate changes in the CNS microenvironment over a period of days. However, delayed metabolic impacts of CNS injury from catheter placement could represent an important covariate for interpreting the pharmacodynamic impacts of candidate therapies. Intracranial microdialysis was performed in patient-derived glioma xenografts of glioma before and 72 h after systemic treatment with either temozolomide (TMZ) or a vehicle. Microdialysate from GBM164, an IDH-mutant glioma patient-derived xenograft, revealed a distinct metabolic signature relative to the brain that recapitulated the metabolic features observed in human glioma microdialysate. Unexpectedly, catheter insertion into the brains of non-tumor-bearing animals triggered metabolic changes that were significantly enriched for the extracellular metabolome of glioma itself. TMZ administration attenuated this resemblance. The human glioma microdialysate was significantly enriched for both the PDX versus brain signature in mice and the induced metabolome of catheter placement within the murine control brain. These data illustrate the feasibility of microdialysis to identify and monitor the extracellular metabolome of diseased versus relatively normal brains while highlighting the similarity between the extracellular metabolome of human gliomas and that of CNS injury.

Abstract Glioblastoma (GBM) is a rapidly fatal malignancy typically treated with radiation and temozolomide (TMZ), an alkylating chemotherapeutic. These cytotoxic therapies cause oxidative stress and DNA damage, yielding a senescent-like state of replicative arrest in surviving tumor cells. Unfortunately, recurrence is inevitable and may be driven by surviving tumor cells eventually escaping senescence. A growing number of so-called “senolytic” drugs have been recently identified that are defined by their ability to selectively eliminate senescent cells. A growing inventory of senolytic drugs is under consideration for several diseases associated with aging, inflammation, DNA damage, as well as cancer. Ablation of senescent tumor cells after radiation and chemotherapy could help mitigate recurrence by decreasing the burden of residual tumor cells at risk of recurrence. This strategy has not been previously explored for GBM. We evaluated a panel of 10 previously described senolytic drugs to determine whether any could exhibit selective activity against human GBM persisting after exposure to radiation or TMZ. Three of the 10 drugs have known activity against BCL-XL and preferentially induced apoptosis in radiated or TMZ-treated glioma. This senolytic activity was observed in 12 of 12 human GBM cell lines. Efficacy could not be replicated with BCL-2 inhibition or senolytic agents acting against other putative senolytic targets. Knockdown of BCL-XL decreased survival of radiated GBM cells, whereas knockdown of BCL-2 or BCL-W yielded no senolytic effect. Implications: These findings imply that molecularly heterogeneous GBM lines share selective senescence-induced BCL-XL dependency increase the significance and translational relevance of the senolytic therapy for latent glioma.

Abstract BACKGROUND Progress for gliomas is slowed in part by the paucity of mechanistic feedback during treatment with experimental therapies. Access to extracellular tumor pharmacodynamic biomarkers could provide an avenue to accelerate progress. We have initiated a program of intra-operative microdialysis to accelerate biomarker discovery and to identify candidate outcome measures for translational therapies. METHODS Intraoperative microdialysis was performed with M-dialysis 100kDA catheters and 107 variable rate pumps under an IDE. Four IDH-mutant and two IDH-WT lesions were studied intraoperatively with 3 divergently placed catheters. Microperfusate (artifical CSF+ 3% dextran) was perfused at 2uL/min and collected in 20 min increments. Paired CSF was also obtained when accessible. A parallel cohort of nude mice bearing human IDH-mutant, IDH-WT, or sham intracranial xenografts (n=6-12) underwent intratumoral microdialysis. A pilot murine study of intracranial drug delivery was performed via concurrent microdialysis during convection-enhanced delivery (CED) of saline or the IDH-inhibitor AG120. RESULTS Microdialysate from IDH-mutant intracranial xenografts revealed >100 differentially abundant metabolites compared to sham or IDH-WT tumors, including D2-HG (21x) and MTA(18x), p< 10^-5. The most significantly abundant metabolite was DMA (4x, p< 10^-10). 15-1000uM D2HG was recovered from intra-operative human IDH-mutant tumors and 1-2uM from normal brain adjacent to IDH-WT gliomas and < 1uM in all IDH-WT samples. Forty metabolites differentiated enhancing tumor from adjacent brain in 3/3 paired human samples including upregulated Aminoacyl-tRNA biosynthesis and downregulated purine metabolism. Serial aliquots of microdialysate during saline CED yielded steady D2-HG levels whereas CED with AG120 yielded undetectable D2-HG within 6 hours. CONCLUSION The extracellular metabolic landscape of glioma is diverse, dynamic and reflects tumor biology and response to therapy. Collectively, these studies suggest that intra-tumoral drug testing should be feasible with realistic expectation of gaining metabolic feedback within a short timeframe. Leveraging this paradigm can provide opportunities to accelerate therapeutic translation for gliomas.

Abstract Glioblastoma (GBM) is a common deadly malignant brain cancer of the central nervous system, with a median survival of 12–15 months. Scientific advancements are lacking in developing effective therapies for both primary GBM, as well as secondary GBMs, that typically originate as malignant transformation of lower-grade isocitrate dehydrogenase (IDH) 1-mutant tumors. The unique metabolomic profile of IDH1-mutant tumors presents opportunities to develop biomarker signatures of therapeutic efficacy. Microdialysis is an extracellular fluid sampling collection technique utilizing a perfused semipermeable catheter to permit diffusion of molecules between brain interstitium and the perfusate. We hypothesized that microdialysis may identify a metabolomics-based biomarker response to therapy in IDH1-mutant tumors. To test this hypothesis, orthotopic xenografts were generated from patient-derived xenografts (PDX) harboring mutant IDH-1 (R132H). Perfusates were collected from intra-cranial tumors in athymic nude mice sampled at baseline and 72h post treatment with temozolomide (TMZ), an oral alkylating agent used to treat IDH1-mutant gliomas, compared with vehicle treatment. Perfusates were analyzed via untargeted metabolomic profiling using liquid chromatography-mass spectrometry. Tumor specific metabolites such as (D)-2 hydroxyglutarate, were detected in microdialysate from IDH-1 mutant tumor bearing mice compared to non-tumor bearing mice. We also found high levels of metabolites such as 5-methylthioadenosine, and dimethylarginine and wide range of amino acids in microdialysate from IDH-1 mutant tumor bearing mice. TMZ treatment induced changes to metabolites in creatine and histidine metabolism. Our results indicate that microdialysis is a feasible technology to identify metabolomics-based biomarkers in IDH1-mutant gliomas and their response to therapy. We suggest that in vivo intratumoral microdialysis over several days could yield metabolic pharmacodynamic biomarkers of value to therapeutic translation for IDH-mutant gliomas.

Abstract BACKGROUND NAD+ is required for cell metabolism and DNA repair. It is generated from nicotinic acid (NA) by NAPRT and from Nicotinamide (NAM) by NAMPT. D2HG in IDH-mutant tumors methylates and inactivates NAPRT, increasing dependence on NAMPT. Toxic side effects of NAMPT inhibition can be prevented by NA supplementation in healthy cells without NAPRT methylation. A1326133 is a recently described CNS-penetrant NAMPT inhibitor hypothesized to selectively eliminate IDH-mutant NAPRT-methylated gliomas, likely in combination with other therapies. Our group is looking for biomarkers of drug efficacy to augment individualized therapies. To that end, we sought to identify GBM cell lines with varying sensitivity to NAMPT inhibition. METHODS Human non-immortalized astrocytes and human GBM cell lines were utilized from the Mayo Clinic Glioma patient-derived xenograft resource, including IDH-R132Hmutant lines (GBM164, 196) and IDH-WT lines (GBM6, 12, 76). Cell viability was analyzed after 4days incubation with the NAMPT inhibitor, A1326133 +/- Temozolomide (TMZ) or NA. IC50 for A1326133 was estimated based on intracellular ATP using Cell-Titer-Glo. RESULTS Marked heterogeneity between lines was observed in response to A1326133 +/- NA or TMZ. Sensitive and resistant lines were identified among both IDH-mutant and IDH-WT cell lines. IC50s: GBM164, 12, 6, 196 and 76 were 5.6, 9.3, 39.2, 910, and 9455nM, respectively. NA partially rescued GBM164 by NA (IC50 increased to 20.8nM) but not GBM6 nor 12. IC50 for Human astrocytes was 221.7nM, but >10,000nM with NA. Addition of TMZ did not improve A1326133 efficacy. CONCLUSION Our data illustrate the potential utility of NAMPT inhibition to kill a subset of IDH-WT and IDH mutant lines, but conflict with previously reported TMZ synergy and correlation with mutant IDH. NA may increase safety but could decrease efficacy in certain lines. Ongoing studies seek metabolic biomarkers of therapeutic efficacy to guide individualized therapy with NAMPT inhibitors.

Abstract Glioblastoma multiforme (GBM) is the most aggressive CNS neoplasm with a mere 15 month progression-free survival following current standards-of-care. Furthermore, conventional therapies have been effective, in part, by inducing a senescent-like phenotype in at least a proportion of glioma, as well as within the tumor-adjacent, healthy brain cells. Through an N-glyco-capture large-scale screening method, we were able to identify 25 genes exclusively overexpressed on glioma cell surfaces. Of those 25 targets, CD38 was an attractive target due to pre-existing FDA approved therapeutics. Recent studies have shown, however, that senescent cells, such as those induced via chemo- and radiotherapies, secrete a pro-inflammatory array of cytokines, known collectively as senescence associated secretory phenotype (SASP), that are capable of increasing CD38 expression in monocytes. CD38 functions as an ectoenzyme to convert extracellular NAD+ to nicotinamide (required for glioma cell salvage pathway NAD+ synthesis) and ADPR/cADPR (a cellular proliferation signal). To examine the effects of radiation on human glioma tissue, we performed reverse cyclase assays (RCA) on paired primary and recurrent human glioma tissue samples and found an increase in CD38 activity in post-irradiated tissues (recurrent glioma) compared to pre-irradiated (primary glioma). Through proliferation assays, we also found an increase in glioma cell growth following treatment with cADPR compared to untreated. Our results demonstrate that CD38 activity is tumorgenic, and furthermore that conventional chemo- and radiotherapies increase this CD38 activity. This indicates that treating CD38 with previously FDA approved therapeutics may provide hope for increasing progression free survival in our GBM patient population, and moreover emphasize the importance of leveraging novel large scale screening methods for identifying additional opportunities for treatment.

Abstract BACKGROUND Radiotherapy, combined with surgical resection and chemotherapy, remains a first-line treatment for infiltrative gliomas. However, these tumors are not surgically curable, and often recur, even within the prior radiation field, and may demonstrate a more aggressive phenotype. Importantly, high grade gliomas display diverse molecular phenotypes, and whether this genetic variability leads to divergent behaviour in the radiated tumor microenvironment is unknown. Herein, we characterize the effects of the irradiated brain microenvinroment on nine additional unique GBM cell lines to better understand the nuances of how tumor molecular phenotypes influence cellular dynamics. METHODS Female athymic nude mice were randomly divided into cranial radiation (15 Gy) and non-radiated groups. Mice then underwent intracranial implantation with one of the selected patient-derived xenograft (PDX) GBM cell lines (GBM 6, 10, 12, 39, 46, 76, 123, 164, 196; total n=8-15, per group, per line). Kaplan-Meyer (K-M) and log-rank tests were performed to compare the survival between irradiated and non-irradiated groups. RESULT Of nine previously untested human GBM lines, we found that five demonstrated shorter survival in the pre-radiated brain (GBM 6, 46, 76, 164, 196). However, two lines yielded prolonged survival in the pre-radiated brain (GBM 10, 12); GBM 39, 123 whose rate of growth was not impacted by the radiated brain. CONCLUSION These results highlight the likely critical impact of the irradiated microenvironment on tumor behaviour, yet illustrate that different tumors may exhibit opposing responses. Although further evaluation will be needed to understand mechanisms of divergent behavior, our data suggest the increased rate of growth in the radiated microenvironment may not apply to the fastest-growing tumor lines, which could instead demonstrate a paradoxical response.

Abstract BACKGROUND Glioblastoma (GBM) is an infiltrative, uniformly fatal brain tumor, treated with surgery, radiation, and Temozolomide (TMZ). Chemoradiation induces a senescent-like phenotype, which contributes to disease recurrence. We recently found that, radiated GBM cells can be eliminated by inhibition of BCL-XL. However, it remains unknown whether p53 is involved in this process and whether the elimination of senescent cells by BCL-XL inhibition could be augmented by MDM2 inhibition, a negative p53 regulator. METHODS p53-mutant (GBM6/GBM123) and p53-WT (GBM39/GBM76) human GBM cells were treated with 5Gy or TMZ following 48 hours of MDM2 inhibitor (AMG232) or vehicle treatment and maintained for seven days to establish a senescent-like phenotype. We evaluated the IC50 for BCL-XL inhibitor (A1331852) in radiated vs. non-radiated cells with or without MDM2 inhibitor pre-treatment. RESULTS MDM2-inhibitor treatment prior to radiation increased the expression of p21 and lead to increased cell death when combined with BCL-XL inhibition in p53-WT GBM cells. IC50 of BCL-XL inhibitor after prior MDM2 pretreatment and radiation in GBM76 was 4.5□M compared to 33.5□M, 18.1□M, and 32.3□M in vehicle without radiation, vehicle with radiation, and MDM2 inhibitor-alone treatment groups, respectively(p=0.0036). The IC50 of BCL-XL inhibitor without MDM2-inhibition in non-irradiated and radiated, as well as pre-administration of MDM2 inhibitor in non-irradiated and radiated GBM39 cells was 5618nM, 16117nM, 3926nM and 276.5nM, respectively(p=0.0003). Conversely, MDM2 co-inhibition with BCL-XL did not lead to increased rates of cell death in p53-mutant cell(p>0.05). CONCLUSION We previously identified that BCL-XL inhibition promotes cell death in senescent GBM cells. We build upon that work, demonstrating the increased rates of cell death can be augmented by MDM2 inhibition, but only in p53-WT cells. These findings highlight a novel therapeutic target for treating latent GBM tumors prior to recurrence, with the additional of MDM2 inhibition greatly increasing the efficacy of BCL-XL targeting agents.

Abstract BACKGROUND Glioblastoma is a fatal infiltrative primary brain tumor, and standard care includes maximal safe surgical resection followed by radiation and Temozolomide (TMZ). Therapy-resistant residual cells persist in a latent state a long time before inevitable recurrence. Conventional radiation and Temozolomide (TMZ) treatment cause oxidative stress and DNA damage resulting senescent-like state of cell-cycle arrest. However, increasing evidence demonstrates escaping senescence leads to tumor recurrence. Thus, the ablation of senescent tumor cells after chemoradiation may be an avenue to limit tumor recurrence. METHODS 100uM TMZ for 7days or 10-20Gy radiation (cesium gamma radiator) was used for senescence induction in human glioblastoma in vitro and confirmed by SA-Beta gal staining and PCR. Replication arrest assessed by automated quantification of cellular confluence (Thermo Scientific Series 8000 WJ Incubator). We evaluated the IC50 for several senolytics targeting multiple SCAPs, including Dasatinib, Quercetin, AMG-232, Fisetin, Onalespib, Navitoclax, and A1331852, and in senescent vs. proliferating cells. RESULTS Among the senolytic tested, the Bcl-XL inhibitors A1331852 and Navitoclax both shown senolytic effect by selectively killing radiated, senescent tumor cells at lower concentrations as compared to 0Gy treated non-senescent cells. Across 12 GBM cell lines, IC50 for senescent cells was 6–500 times lower than non-senescent GBM(p< 0.005). Such differential sensitivity to Bcl-XL inhibition after radiation has also observed by BCL-XL knockdown in radiated glioma. CONCLUSION These findings suggest the potential to harness radiation-induced biology to ablate surviving quiescent cells and demonstrate Bcl-XL dependency as a potential vulnerability of surviving tumor cells after exposure to chemoradiation.