Rahman, Masum

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 BACKGROUND The microenvironment of glioblastoma is complex and defined in part by protumorigenic metabolic changes. Within this microenvironment, blood-brain barrier disruption is often associated with the most aggressive portions of the tumor. To date, the metabolic tumor microenvironments of enhancing, blood-brain barrier (BBB) disrupted portions of gliomas, as compared to BBB-intact tumor, remain poorly understood. Specifically, it is unclear if the associated blood-brain barrier disruption merely reflects or functionally supports tumor aggressiveness. METHODS Intra-operative high-molecular weight microdialysis was utilized under an investigational device exemption (NCT NCT04047264) to sample the extracellular metabolome of radiographically diverse regions during fifteen neurosurgical resections. The global extracellular metabolome of recovered microdialysate from contrast-enhancing and non-enhancing tumor, as well as brain adjacent to tumor, was evaluated via ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) on the Metabolon platform and assessed using enrichment and correlation analyses. RESULTS Among 162 named metabolites identified via ultra-performance liquid chromatography tandem mass spectrometry, a novel glioma-associated metabolite, guanidinoacetate (GAA), was 126.32x higher in enhancing tumor than in adjacent brain and 13.63x higher in non-enhancing tumor than brain. Forty-eight additional metabolites were 2.05-10.18x more abundant in enhancing tumor than brain, including carnitine family members and all nine large neutral amino acids. Except for GAA and 2-hydroxyglutarate in IDH-mutant gliomas, differences between non-enhancing tumor and brain microdialysate were comparatively modest and less consistent, suggesting heterogeneity in the glioma metabolic microenvironment of blood-brain barrier (BBB) intact areas as compared to BBB-disrupted portions of gliomas. Interestingly, the enhancing but not the non-enhancing glioma metabolome was significantly enriched for plasma-associated metabolites, largely comprising amino acids and carnitines. CONCLUSION Our patient-derived data suggest that the BBB-disrupted metabolome of high-grade gliomas is characterized by metabolite diffusion from circulation into the tumor microenvironment. Further studies are ongoing to determine how this alteration in the glioma extracellular metabolome impacts glioma behavior and how it could be therapeutically targeted via BBB-modulating drugs.

Abstract Background Bevacizumab is commonly used to manage cerebral edema associated with brain tumors. However, its long half-life poses challenges for patients requiring urgent surgery due to wound complications. We present a case of utilizing therapeutic plasma exchange (TPE) to remove bevacizumab in a patient with recurrent glioblastoma requiring urgent surgery. Methods A 58-year-old male with recurrent glioblastoma, IDH-wildtype, presented with clinical and radiographic concern for ventriculitis requiring urgent wound washout only 4 days after his last bevacizumab infusion. TPE was performed for 3 sessions after surgery using a centrifugation-based cell separator. Replacement fluids included normal serum albumin, normal saline, and fresh frozen plasma. Bevacizumab levels were quantified using an enzyme-linked immunoabsorbent assay before and after each TPE session. Results TPE effectively removed bevacizumab, enabling safe surgery without new complications. Plasma bevacizumab levels decreased from 1087.63 to 145.35 ng/mL (13.4% of original) by the end of the last TPE session. This decline is consistent with nearly 3 half-lives, which compares favorably to the expected timeline of natural decline given the 21-day half-life. Conclusions We report a complex clinical scenario of a patient requiring urgent wound washout 4 days after last bevacizumab infusion for CNS infection. Surgery was successfully performed without new complications with use of TPE to remove bevacizumab immediately following surgery. This case highlights the feasibility of this approach, which may be utilized effectively in patients requiring surgery after having recently received bevacizumab.

High-grade gliomas are the most common and aggressive adult primary brain tumors with a median survival of only 12–15 months. Current standard therapy consists of maximal safe surgical resection followed by DNA-damaging agents, such as irradiation and chemotherapy that can delay but not prevent inevitable recurrence. Some have interpreted glioma recurrence as evidence of glioma stem cells which persist in a relatively quiescent state after irradiation and chemotherapy, before the ultimate cell cycle re-entry and glioma recurrence. Conversely, latent cancer cells with a therapy-induced senescent phenotype have been shown to escape senescence, giving rise to more aggressive stem-like tumor cells than those present in the original tumor. Therefore, approaches are needed to either eliminate or keep these glioma initiating cells in a senescent state for a longer time to prolong survival. In our current study, we demonstrate that the radiation-induced cell cycle inhibitor P21 can provide a powerful route to induce cell death in short-term explants of PDXs derived from three molecularly diverse human gliomas. Additionally, cells not killed by P21 overexpression were maintained in a stable senescent state for longer than control cells. Collectively, these data suggest that P21 activation may provide an attractive therapeutic target to improve therapeutic outcomes.

Abstract Gliomas are genomically heterogeneous tumors that may harness a convergent and therapeutically targetable set of metabolic pathways. At present, the metabolic landscape of in situ human gliomas remains incompletely characterized, hampering translational progress. To that end, we are leveraging the previously untapped potential of high molecular weight microdialysis to determine the global extracellular metabolic profiles of live human gliomas. Under an investigational device exemption, HMW microdialysis (< 100 kDa) was performed at 2.0 μL/min in an initial discovery cohort of five patients in glioma and adjacent brain during neurosurgical resection; a subsequent cohort of five patients was independently analyzed to critically evaluate results from the discovery group. Untargeted metabolomics via ultra-performance liquid chromatography-tandem mass spectrometry revealed over 300 named metabolites and five drugs from only 20 μL of microdialysate, representing a short and feasible 10 minutes of intraoperative collection time. Enrichment analysis of each patient’s tumor vs. brain ranked extracellular metabolome highlighted marked metabolic convergence within the most aggressive regions of molecular diverse tumors (FDR = 0). Pathway analysis revealed significant enrichment for large neutral amino acid pathways, including valine, leucine, and isoleucine biosynthesis (p=1.6E-9) and degradation (p=0.001) and glycine, serine, and threonine metabolism (p=4.7E-5). Notably, this amino acid signature was not as abundantly present in nonenhancing tumor when compared to enhancing tumor (Average tumor/brain: 1.9x vs. 4.3x, respectively), suggesting upregulation of neutral amino acid transporters in enhancing tumor or delivery from plasma into the CNS via a disrupted BBB. Interestingly, guanidinoacetate (GAA) was our most highly conserved and upregulated metabolite (128.9x in tumor vs. brain). Given its co-production with ornithine, the precursor to protumorigenic polyamines, we posit that GAA may serve as a biomarker of increased ODC activity in live human gliomas. In conclusion, intraoperative HMW microdialysis feasibly offers improved opportunities to perform glioma metabolic biomarker and therapeutic discovery.

Abstract The relative inaccessibility of a patient’s glioma throughout their disease course precludes individualized insights into tumor biology and responses thereof to therapeutic challenge. We propose that serial cerebrospinal fluid (CSF) samples can be used to perform glioma espionage in the form of biomarker discovery for analytes that are sensitive to tumor burden (for monitoring) and candidate therapies (for biological impact). To that end, we are routinely leveraging neurosurgical access to the CNS to collect CSF samples intraoperatively and from chronically implanted Ommaya reservoirs for longitudinal CSF access (NCT04692337, NCT04692324). Global proteomic analysis of CSF was performed on the Somalogic platform – an aptamer-based technology for highly sensitive and specific analysis of over 7,000 proteins. Using each patient’s ranked protein list (glioma vs. 3 control patients), enrichment analysis was performed in a discovery cohort of five patients to identify conserved proteins of glioma. To reveal candidate glioma monitoring, further enrichment analysis was performed to identify proteins that decreased with resection and increased with recurrence, using two sets of paired patient samples. Forty-four proteins met these enrichment criteria; this candidate high-grade glioma proteomic signature was highly significantly enriched in an independent cohort of twelve glioma patients (FDR=0 for all patients). The HGG signature decreased in abundance following resection in serial samples from two other patients, confirming decreased tumor burden. Top proteins included LANC2, DPYL3, Sorcin, Integrin A1B1, and lactate dehydrogenase (FC of glioma vs. control: 145.3x, 113.9x, 78.0x, 71.6x, and 62.7x, respectively). Interestingly, despite having explicitly chosen samples with little-to-no hemoglobin in the discovery cohort, this HGG signature was highly enriched for plasma-like proteins (FDR=0), suggesting a potential biological impact of blood-brain barrier disruption on the glioma extracellular proteome. In conclusion, our data demonstrate that the glioma extracellular proteome can feasibly be accessed to perform biomarker discovery (“espionage”) via serially-acquired CSF samples.

Abstract Gliomas may leverage alternate metabolic pathways in response to metabolism-targeted therapeutic intervention, all of which remain unexplored in the live human glioma, in situ. Defining emergent mechanisms of metabolic resistance in response to therapeutic challenge can help guide rational combinatorial therapies. To date, the metabolic response of gliomas in response to therapeutic intervention has remained poorly understood due to the relative inaccessibility of the live human tumor, in situ. Microdialysis is an underutilized tool that could be leveraged to overcome this longstanding challenge. Data from our ongoing intraoperative microdialysis trial have revealed an upregulation of polyamine metabolism and a novel glioma-associated metabolite, guanidinoacetate (GAA) -- a metabolite co-produced with ornithine, which is required for polyamine synthesis. In a Phase 0 trial, we will evaluate in situ glioma responses to polyamine depletion (difluoromethylornithine, DFMO) with or without blockade of polyamine uptake (AMXT 1501) to identify candidate extracellular biomarkers of target engagement and cytotoxicity in fifteen post-operative patients who have undergone a standard-of-care planned subtotal resection for high-grade glioma. Intraoperatively, high-molecular-weight catheters will be implanted into the residual tumor and brain adjacent to the resection cavity for post-operative longitudinal monitoring of extracellular metabolites via microdialysis. Polyamines and guanidinoacetate, a candidate biomarker of glioma-upregulated polyamine synthesis, will be monitored throughout therapeutic intervention from post-operative day (POD) 1 to POD5 via longitudinal microdialysis to determine live in situ glioma pharmacodynamic responses to polyamine depletion. Catheters will be removed on post-operative day five prior to discharge. We hypothesize that GAA will reflect local tumor production of polyamine metabolism. Additionally, in situ microdialysis in Phase 0 trials will allow for pharmacodynamic and pharmacokinetic, in addition to metabolic, monitoring, an opportunity which is rarely afforded in most clinical trials due to lack of access to the CNS.

Abstract INTRODUCTION Rapid, detailed feedback is needed to understand the individualized biological impacts of novel glioma therapies. We are performing glioma biomarker discovery by serial cerebrospinal fluid (CSF) sampling from Ommaya reservoirs to determine how the CSF proteome can reveal early longitudinal intelligence regarding glioma status, biology, and therapeutic response. METHODS Global proteomic analysis of CSF was performed on the Somalogic platform − an aptamer-based technology for highly sensitive and specific analysis of over 7,000 proteins. Discovery analysis comprised of the top-500 ranked proteins in CSF from seven patients with high-grade gliomas (HGG) versus non-glioma controls. The top-500 HGG proteins were then preliminarily filtered to include only proteins that met two additional criteria of decrease with resection and increase with recurrence in individual paired patient samples. RESULTS Proteomic enrichment analysis revealed a conserved HGG CSF proteomic signature defined by 79 proteins, including ones known to be over-expressed in solid tumor malignancies, such as retinoblastoma binding protein 4, heat shock protein 90, and sorcin. The HGG proteomic signature was consistently enriched in an independent validation cohort consisting of 13 gliomas diverse in primary versus recurrent status, subtype, and grade, when compared to control CSF samples. Encouragingly, proteins in the HGG signature decreased in the two patients for whom CSF was collected prior to and after resection (both at POD16 and POD18) with decreased tumor burden. CONCLUSION Our preliminary data demonstrate the ability to gain detailed, individualized insights regarding glioma biology, tumor burden, and evolution through global CSF proteomics acquired from longitudinal neurosurgical access to unique gliomas.

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.

In this study, we demonstrate that Raman microscopy combined with computational analysis is a useful approach to discriminating accurately between brain tumor bio-specimens and to identifying structural changes in glioblastoma (GBM) bio-signatures after nordihydroguaiaretic acid (NDGA) administration. NDGA phenolic lignan was selected as a potential therapeutic agent because of its reported beneficial effects in alleviating and inhibiting the formation of multi-organ malignant tumors. The current analysis of NDGA’s impact on GBM human cells demonstrates a reduction in the quantity of altered protein content and of reactive oxygen species (ROS)-damaged phenylalanine; results that correlate with the ROS scavenger and anti-oxidant properties of NDGA. A novel outcome presented here is the use of phenylalanine as a biomarker for differentiating between samples and assessing drug efficacy. Treatment with a low NDGA dose shows a decline in abnormal lipid-protein metabolism, which is inferred by the formation of lipid droplets and a decrease in altered protein content. A very high dose results in cell structural and membrane damage that favors transformed protein overexpression. The information gained through this work is of substantial value for understanding NDGA’s beneficial as well as detrimental bio-effects as a potential therapeutic drug for brain cancer.