Suzuki, Yuta

Abstract Background Imaging-based glioma monitoring is confounded by treatment-related changes. D-2-hydroxyglutarate (D-2-HG), produced by the isocitrate dehydrogenase (IDH) mutation, is detectable in cerebrospinal fluid (CSF), which can be sampled from cranial or lumbar compartments. We evaluated CSF D-2-HG as a serially accessible biomarker for IDH-mutant gliomas, including the optimal compartment for longitudinal sampling. Methods Lumbar and cranial CSF samples were collected from patients with IDH-mutant gliomas or IDH-wild-type central nervous system pathologies via surgical field collection, lumbar punctures, and CSF access devices. CSF D-2-HG was quantified via our CLIA-certified gas chromatography mass spectrometry assay. Results D-2-HG was significantly higher in cranial than lumbar CSF from IDH-mutant glioma patients. Consistent with low D-2-HG abundance in lumbar CSF, lumbar samples could not discriminate IDH-mutant gliomas from IDH-wild-type lesions. In contrast, cranial CSF D-2-HG was significantly higher in IDH-mutant gliomas than wild-type lesions, providing an adequate baseline for initial evaluations of monitoring capabilities. Across 75 samples from 7 consecutive patients with grade 4 IDH-mutant astrocytomas, serial cranial CSF D-2-HG decreased with cytoreduction, remained unchanged with stable disease, and increased with disease progression, but not pseudoprogression. Conclusions Serial cranial CSF D-2-HG shows promise as a monitoring biomarker for IDH-mutant gliomas.

Abstract Polyamines are small molecules that play a critical role in tumor progression and in the regulation of immune responses within the tumor microenvironment. Elevated levels of polyamines in cerebrospinal fluid and tumor tissue have been reported in multiple studies and are shown to correlate with glioma grade. These findings suggest that the polyamine metabolic pathway may represent a therapeutic target in gliomas. Difluoromethylornithine (DFMO) is an irreversible inhibitor of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, expected to exert antitumor effects through polyamine depletion. Although the impact of DFMO against glioblastoma as a monotherapy appears limited, opportunities for synergy with other therapies remain understudied. In this study, we evaluated the therapeutic efficacy of DFMO using both in vitro and in vivo experiments with a murine glioma model (CT-2A) and patient-derived xenograft (PDX) cell lines. Among the models tested, CT-2A cells exhibited the highest expression of ODC1 and demonstrated strong sensitivity to DFMO in vitro. We compared two CT-2A variants: a commercially available line responsive to immunotherapy, and a widely used subline resistant to PD-1 blockade. While DFMO did not improve survival in mice implanted with the immunotherapy-sensitive commercial CT-2A line, it extended median survival by 8 days in mice bearing tumors derived from the immunotherapy-resistant subline. Transcriptomic analysis revealed that the DFMO-responsive, immunotherapy-resistant tumors exhibited lower baseline expression of immune-related genes, including those involved in interferon-β, interferon-γ, and IL-12 signaling pathways, compared to the immunotherapy-sensitive tumors. The latter showed markedly higher expression of STAT1 and interferon-γ and were enriched for immune-related Gene Ontology terms. These findings suggest that polyamine-targeted therapy may be appropriate for gliomas with an immunosuppressive, “cold” phenotype. Ongoing RNA sequencing analyses aim to further elucidate the immunomodulatory mechanisms of DFMO, and other agents comprising an adaptive toolkit to optimize and sustain anti-tumoral immunity.

Abstract BACKGROUND Many cancers demonstrate upregulated amino acid transport. In glioblastoma (GBM), evidence for upregulated amino acid flux includes high signal on amino acid PET imaging, increased expression of LAT1 (L-type amino acid transporter 1), and elevated extracellular amino acid levels via microdialysis. LAT1-targeted therapies have shown antitumor effects in certain cancer models by restricting essential amino acid supply and inhibiting the mTOR pathway, but their utility for GBM remains unknown. In this study, we evaluated impacts of a competitive LAT1 inhibitor, nanvuranlat, and a non-competitive inhibitor, JPH034 against glioblastoma. METHODS Murine (CT-2A, GL261) and human (GBM6, GBM39) GBM cell lines were tested under varying concentrations of nanvuranlat or JPH034. The competitive impacts of added essential amino acids, serum, and albumin were evaluated in vitro. Survival impacts were evaluated in an intracranial CT-2A glioma model. RESULTS Supplementing essential amino acids in vitro significantly decreased tumor cell sensitivity to nanvuranlat but did not change the IC50 of JPH034. Conversely, supplementing serum or albumin significantly reduced tumor cell sensitivity to JPH034 but did not change the IC50 of nanvuranlat. These findings were reproducible in all human and murine cell lines tested. In the intracranial mouse model, JPH034 treatment prolonged median overall survival from 16 days in the control group to 18.5 days, representing a 2.5-day extension (p = 0.060). CONCLUSION The antitumor effects of nanvuranlat and JPH034 on glioma cells in vitro, are influenced by concentrations of essential amino acids and albumin. Specifically, amino acids decreased impact of the competitive inhibitor (nanvuranlat) while albumin decreased impact of the non-competitive inhibitor (JPH034). These results suggest that the composition of the tumor microenvironment, which is impacted by BBB-disruption within GBM may differentially attenuate the therapeutic efficacy of certain drugs including LAT1 inhibitors.

Abstract The RBBP4/p300 complex was recently shown to control genes critical for DNA damage repair and may play a role in glioblastoma (GBM) therapy responsiveness, especially to temozolomide (TMZ). However, the role of this complex in the modulation of the GBM tumor microenvironment (GBM-TME) remains elusive. Because pro-tumorigenic GBM-TME is primarily due to angiogenesis and immune tolerance, we evaluated whether the RBBP4/p300 complex may control these critical processes. To that end, we used shRNA to silence each RBBP4/p300 complex member in GBM43 cells and RNAseq to identify angiogenesis and immune genes regulated by this complex. Our data demonstrated that silencing RBBP4 or p300 suppressed the expression of HIF1α and several other hypoxia response genes, including VEGFA and DDIT4, suggesting that RBBP4/p300 complex may regulate angiogenesis. Consistent with a role in regulating angiogenesis, culture medium conditioned by GBM43 cells expressing the control non-targeting shRNA (shNT) significantly induced angiogenic tube formation by human umbilical vein endothelial cells (HUVEC). In contrast, this effect was blocked by the medium conditioned by the shRBBP4 or shp300 expressing cells. Both shRBBP4 and shp300 expressing cells failed to activate HIF1α accumulation and other signaling in response to hypoxia. Additionally, the control shNT-conditioned medium inhibited IFN-γ production while the medium conditioned by the shRBBP4 or shp300 expressing cells supported IFN-ɣ production from CD8+ T cells. Intriguingly, silencing RBBP4 or p300 also suppressed the expression of CCL2 and PD-L1 (CD274). CCL2 protein is a chemokine that promotes TME infiltration by myeloid-derived suppressor cells (MDSCs), while PD-L1 interacts with PD1 to suppress cytotoxic CD8+ T cell activation. Although this is an ongoing investigation, we conclude that RBBP4/p300 complex mediates the expression of hypoxia response genes, including HIF1α, and immunosuppressive genes, including PD-L1, and this activity may enhance angiogenic and immunosuppressed GBM-TME, making this complex a potential target for GBM therapy.

Abstract INTRODUCTION Glioblastoma (GBM) is the most common and aggressive form of adult brain tumor and is an incurable disease due to primary and secondary resistance to therapy. We recently examined the candidate genes modulating temozolomide (TMZ) resistance using genome-wide shRNA screening in TMZ-resistant U251 (U251TMZ) model. We identified a total of 134 TMZ-resistant candidate genes, including Karyopherin A1 (KPNA1) and Exportin 1 (XPO1), the genes critical for nucleocytoplasmic (NC)-transport. Interestingly, XPO1 inhibitor Selinexor is an FDA-approved agent for refractory multiple myeloma, and there is an ongoing interest in evaluating Selinexor for GBM therapy. Therefore, we hypothesized that Selinexor is a promising drug for overcoming TMZ resistance. METHODS and RESULTS We conducted a series of experiments in vitro using Selinexor with and without TMZ. Selinexor and TMZ alone didn’t significantly block the growth of U251TMZ cells. In contrast, Selinexor significantly re-sensitized resistant U251TMZ cells to TMZ. Interestingly, MGMT-methylated GBM39 cells were sensitive to Selinexor alone, TMZ alone, and to combined Selinexor/TMZ treatment. Contrarily, MGMT-unmethylated GBM43 cells were only sensitive to combined Selinexor/TMZ treatment. These findings suggested that similar to TMZ, Selinexor sensitivity may be influenced by MGMT expression status, with MGMT expressing cells showing resistance. In line with this notion, re-expression of exogeneous MGMT blocked sensitivity of MGMT-methylated U251 cells to Selinexor and TMZ, but such inhibition was not observed in U251 expressing vector alone. Interestingly, the concentration-dependent experiments demonstrated that Selinexor can induce MGMT expression in unmethylated T98G, GBM6, GBM14, and GBM43 cells. Selinexor-induced MGMT expression was accompanied with increased phosphorylation of CREB protein. CONCLUSION These results suggest that Selinexor sensitivity alone and with TMZ may be limited to MGMT-methylated GBM cells and may increase MGMT expression in unmethylated cells through activation of PKA-CREB pathway. Future studies will evaluate how MGMT may impact the sensitivity of GBM cells to Selinexor.