Jeffrey D. Neighbors

Background: Glioblastoma (GBM) is the most common malignant primary brain tumor and is associated with a poor prognosis. Repurposed posaconazole (PCZ) has demonstrated efficacy in vitro by targeting key metabolic pathways. The aim of this study was to determine the neuro-pharmacological profile of PCZ in patients with GBM through a Phase 0 first-in-human trial for this indication (NCT04825275). Methods: We conducted an open-label, non-randomized, parallel-arm trial in participants with primary or recurrent GBM. Patients in the treatment arm received oral PCZ for 7–10 days presurgically to achieve steady-state concentrations. Microdialysis catheters were implanted to measure interstitial drug levels. Postoperative tissue and fluid samples were analyzed to evaluate PCZ pharmacokinetics (mass spectrometry) and pharmacodynamics, including vascular density, apoptosis, and metabolite concentrations (lactate and pyruvate) compared to controls. Results: The trial was closed early due to slow accrual, enrolling two participants in the PCZ arm and three in the control arm. No drug-related safety issues were observed. Although PCZ was undetectable in microdialysate fluid, it accumulated in tumor tissue, with concentrations showing an association with CD31-measured endothelial content. PCZ-treated tumors exhibited trends toward lower BCL2 expression indicative of increased apoptosis, and significantly lower pyruvate levels in the tumor periphery compared to controls. Plasma lactate levels in treated patients normalized to control levels over 24 hours. Conclusions: Despite limited accrual, this trial provides the first clinical evidence that oral PCZ accumulates in human GBM tissue and shows preliminary signals of metabolic modulation. These findings provide biological rationale warranting further clinical investigation, potentially utilizing direct tissue sampling rather than microdialysis.

Oncogenic viruses, which are causative for some cancers, are typically acquired in youth and suppressed until older age. These malignancies account for over 10% of the cancer burden and often reprogram cellular metabolic pathways to promote their own survival and proliferation, often targeting lipogenic pathways to increase bioavailability of cellular products. The isoprenoid biosynthesis pathway (IBP) is an important lipogenic pathway that has been explored extensively for its dysregulation contributing to carcinogenesis, with notable discussion of statin therapy as a means of perturbing neoplasia. To our knowledge, we are the first group to provide a comprehensive discussion of the seven known oncogenic viruses and their reliance upon the IBP to promote tumorigenesis. As knowledge on this topic is limited, we aim to draw attention to a neglected area of the oncogenic research space, while also highlighting the use of inhibitors of the IBP as potential avenues for novel treatment options.

Background: Merkel cell carcinoma (MCC) is a rare malignancy of the skin caused by Merkel cell polyomavirus (80% of cases) or by ultraviolet (UV) sun exposure (20% of cases). The isoprenoid biosynthesis pathway (IBP) is an essential metabolic pathway shown to be upregulated in tumorigenesis, causing aberrant activation of Ras and Rho GTPases and resulting in unregulated cellular proliferation and survival. Methods: Through the use of pharmacological inhibitors of the IBP, we assessed the role of the IBP and its downstream targets in viral-positive and viral-negative MCC cell lines. To identify the most critical IBP intermediates, cellular metabolic activity, cell death and cell cycle distribution were measured after IBP perturbation. Results: Across all cell lines, treatment with IBP inhibitors, especially fluvastatin, decreased metabolic activity; however, perturbation of different intermediates resulted in variable responses between viral-positive and viral-negative cell lines. Conclusions: Our findings demonstrate varying dependence on the IBP between viral-positive and viral-negative MCC and highlight the importance of cellular dynamics when determining a treatment regimen for patients with MCC.

Schweinfurthin analogues – SAR, target engagement, MoA.

Merkel cell carcinoma (MCC) is a rare but aggressive form of skin cancer predominantly caused by the human Merkel cell polyomavirus (MCPyV). Treatment for MCC includes excision and radiotherapy of local disease, and chemotherapy or immunotherapy for metastatic disease. The schweinfurthin family of natural compounds previously displayed potent and selective growth inhibitory activity against the NCI-60 panel of human-derived cancer cell lines. Here, we investigated the impact of schweinfurthin on human MCC cell lines. Treatment with the schweinfurthin analog, 5′-methylschweinfurth G (MeSG also known as TTI-3114), impaired metabolic activity through induction of an apoptotic pathway. MeSG also selectively inhibited PI3K/AKT and MAPK/ERK pathways in the MCPyV-positive MCC cell line, MS-1. Interestingly, expression of the MCPyV small T (sT) oncogene selectively sensitizes mouse embryonic fibroblasts to MeSG. These results suggest that the schweinfurthin family of compounds display promising potential as a novel therapeutic option for virus-induced MCCs.

BACKGROUND: Glioblastoma (GBM) is the most common malignant primary brain tumor with a universally poor prognosis. GBMs express elevated levels of hexokinase 2 (HK2), catalyzing the critical step in glycolysis and influencing several oncogenic pathways. Previous preclinical work has suggested a role for repurposed posaconazole (PCZ) in downregulating HK2 activity, reducing lactate and pyruvate production, interfering with tumor cell metabolism, and increasing mouse survival. OBJECTIVE: To establish brain tumor penetrance, neuropharmacokinetic profile, and mechanistic effect on tumor cell metabolism of PCZ in adults with GBM. METHODS: This is an open label, nonrandomized, parallel arm trial involving patients with GBM. Cohorts will receive PCZ (intervention, n = 5) or will not receive PCZ (control, n = 5), followed by tumor resection and microdialysis catheter placement. Dialysate, plasma, and tumor samples will be analyzed for lactate and pyruvate concentrations. Tumor samples will also be assessed for PCZ concentration, HK2 expression, angiogenesis, and apoptosis. PCZ's neuropharmacokinetics will be determined based on the concentration vs time profile and area under the curve 0 to 24 hours of PCZ concentration in the brain interstitium. EXPECTED OUTCOMES: (1) Increased PCZ concentration in contrast-enhancing brain regions compared with nonenhancing regions; (2) inverse correlation between lactate/pyruvate and PCZ concentrations in dialysate samples from treated patients, over time; and (3) decreased HK2 activity in PCZ-treated tumor samples. DISCUSSION: A successful trial will support the decision to proceed to advanced phase trials. Any tumor penetration by PCZ, with concomitant effect on glycolysis, warrants further in-depth analysis, as therapeutic options for these deadly tumors are currently limited.

Introduction: Multiple myeloma (MM) is a B plasma cell malignancy currently incurable, and novel therapeutics are needed. Evidences regarding the effect of natural compound schweinfurthins suggest that hematological cancers showed growth inhibitory effects to this family of compounds at single nanomolar concentrations. In this study, we evaluated the cytotoxicity of the schweinfurthin synthetic analog 5′-methylschweinfurthin G (MeSG) in MM cell lines, to better understand the validity of this compound as a therapeutic candidate for further studies in MM. Methods: MeSG toxicity against MM cell lines RPMI-8226, MM.1S, and H-929 was evaluated. Trypan blue exclusion and MTT assays measured cell viability and mitochondrial activity, respectively. Flow cytometry was performed to detect apoptotic mitochondria. Flow cytometry and Western blotting techniques were used to investigate apoptosis and to examine the cell cycle. Western blotting was used to determine AKT activation upon MeSG treatment. Results: We provide evidence that in all MM cells analyzed, MeSG exerts diverse cytotoxic effects. MeSG treatment of MM.1S and H-929, but not in RPMI-8226, causes a loss of mitochondria membrane potential. MeSG causes an arrest in G₂/M, especially in RPMI-8226, supported by decreased levels of cyclin-B1 and early increased levels of p21. Finally, there is a diverse response to the MeSG treatment for AKT phosphorylation. MM.1S and H-929 showed a marked decrease in AKT phosphorylation at earlier time points compared to the RPMI-8226 line. Conclusions: MeSG cytotoxicity has been confirmed in all of 3 cell lines studied. Results suggest an early event of increased reactive oxygen species, and/or involvement of cholesterol homeostasis via decreased AKT activation, both of which are currently under investigation.

Dopamine D₂‐like receptor antagonists have been suggested as being potential anticancer therapeutics with specific utility for central nervous system cancers due to their ability to cross the blood‐brain barrier. Despite a plethora of data reporting anticancer effects for D₂R antagonists in cell or animal studies, the ligand concentrations or doses required to achieve such effects greatly exceed the levels known to cause high degrees of occupancy of the D₂ receptor. To resolve this conundrum, we interrogated a panel of glioblastoma multiforme (GBM) cell lines using D₂ antagonists of varying chemotype. We studied the cytotoxic effects of these compounds, and also ascertained the expression of D₂ receptors (D₂R) on these cells. Although several chemotypes of D₂R antagonists, including phenothiazines and phenylbutylpiperidines, were effective against GBM cell line cultures, the highly selective antagonist remoxipride had no anticancer activity at biologically relevant concentrations. Moreover the D₂R antagonist‐induced cytotoxicity in monolayer cultures was independent of whether the cells expressed D₂R. Instead, cytotoxicity was associated with a rapid, high‐magnitude calcium flux into the cytoplasm and mitochondria, which then induced depolarization and apoptosis. Blocking this flux protected the GBM cell lines U87MG, U251MG, and A172. Together, these data suggest that the cytotoxicity of these D₂R antagonists involves calcium signaling mechanisms, not D₂R antagonism. Repurposing of existing drugs should focus on the former, not latter, mechanism.

Background: Glioblastoma multiforme (GBM) is a common and lethal cancer of the central nervous system. This cancer is difficult to treat because most anticancer therapeutics do not readily penetrate into the brain due to the tight control at the cerebrovascular barrier. Numerous studies have suggested that dopamine D2 receptor (D2R) antagonists, such as first generation antipsychotics, may have anticancer efficacy in vivo and in vitro. The role of the D2R itself in the anticancer effects is unclear, but there is evidence suggesting that D2R activation promotes stem-like and spheroid forming behaviors in GBM. Objectives: We aimed to observe the role of the dopamine D2R and its modulators (at selective concentrations) in spheroid formation and stemness of GBM cell line, U87MG, to clarify the validity of the D2R as a therapeutic target for cancer therapy. Methods: Spheroid formation assays and Western blotting of the glioblastoma cell line, U87MG, were used to observe responses to treatment with the D2R agonists sumanirole, ropinirole, and 4-propyl-9-hydroxynaphthoxazine (PHNO); and the D2R antagonists thioridazine, pimozide, haloperidol, and remoxipride. Extreme limiting dilution analysis was done to determine the impact of sumanirole and remoxipride treatment on sphere-forming cell frequency. Proliferation was also measured by crystal violet staining. Stable lentiviral transduction of DRD2 or shDRD2 was used to validate the role of the D2R in assay behaviors. Results: D2R antagonists thioridazine, pimozide, haloperidol, and remoxipride decrease spheroid formation behaviors at a selective 100 nmol/L concentration, while D2R agonists PHNO, sumanirole, and ropinirole increase the formation of spheroids. Similarly, 100 nmol/L remoxipride decreased sphere-forming cell frequency. These results were recapitulated with genetic overexpression and knockdown of the D2R, and combination experiments indicate that the D2R is required for the effects of the pharmacological modulators. Furthermore, spheroid proliferation and invasive capacity increased under treatment with 100 nmol/L sumanirole and decreased under treatment with 100 nmol/L thioridazine. Expression levels of the stemness markers Nestin and Sox2, as well as those of differentiation marker glial fibrillary acidic protein, were not altered by 100 nmol/L thioridazine or sumanirole for 72 h or continuous treatment with these compounds for 7 days during a spheroid formation assay. Conclusions: Signaling activity of the dopamine D2R may be involved in the spheroid formation phenotype in the context of the U87MG cell line. However, this modulation may not be due to alterations in stemness marker expression, but due to other factors that may contribute to spheroid formation, such as cell-cell adhesion or EGFR signaling.

The dopamine D₂ receptor (D₂R) family is upregulated in many cancers and tied to stemness. Reduced cancer risk has been correlated with disorders such as schizophrenia and Parkinson’s disease, in which dopaminergic drugs are used. D₂R antagonists are reported to have anticancer efficacy in cell culture and animal models where they have reduced tumor growth, induced autophagy, affected lipid metabolism, and caused apoptosis, among other effects. This has led to several hypotheses, the most prevalent being that D₂R ligands may be a novel approach to cancer chemotherapy. This hypothesis is appealing because of the large number of approved and experimental drugs of this class that could be repurposed. We review the current state of the literature and the evidence for and against this hypothesis. When the existing literature is evaluated from a pharmacological context, one of the striking findings is that the concentrations needed for cytotoxic effects of D₂R antagonists are orders of magnitude higher than their affinity for this receptor. Although additional definitive studies will provide further clarity, our hypothesis is that targeting D₂-like dopamine receptors may only yield useful ligands for cancer chemotherapy in rare cases.