Magnetic Resonance Imaging of Iron Metabolism with T2* Mapping Predicts an Enhanced Clinical Response to Pharmacologic Ascorbate in Patients with GBM.

PURPOSE: Pharmacologic ascorbate (P-AscH-) is hypothesized to be an iron (Fe)-dependent tumor-specific adjuvant to chemoradiation in treating glioblastoma (GBM). This study determined the efficacy of combining P-AscH- with radiation and temozolomide in a phase II clinical trial while simultaneously investigating a mechanism-based, noninvasive biomarker in T2* mapping to predict GBM response to P-AscH- in humans. PATIENTS AND METHODS: The single-arm phase II clinical trial (NCT02344355) enrolled 55 subjects, with analysis performed 12 months following the completion of treatment. Overall survival (OS) and progression-free survival (PFS) were estimated with the Kaplan-Meier method and compared across patient subgroups with log-rank tests. Forty-nine of 55 subjects were evaluated using T2*-based MRI to assess its utility as an Fe-dependent biomarker. RESULTS: Median OS was estimated to be 19.6 months [90% confidence interval (CI), 15.7-26.5 months], a statistically significant increase compared with historic control patients (14.6 months). Subjects with initial T2* relaxation < 50 ms were associated with a significant increase in PFS compared with T2*-high subjects (11.2 months vs. 5.7 months, P < 0.05) and a trend toward increased OS (26.5 months vs. 17.5 months). These results were validated in preclinical in vitro and in vivo model systems. CONCLUSIONS: P-AscH- combined with temozolomide and radiotherapy has the potential to significantly enhance GBM survival. T2*-based MRI assessment of tumor iron content is a prognostic biomarker for GBM clinical outcomes. See related commentary by Nabavizadeh and Bagley, p. 255.

Differential H2O2 Metabolism among Glioblastoma Subtypes Confers Variable Responses to Pharmacological Ascorbate Therapy Combined with Chemoradiation.

Glioblastoma (GBM), a highly lethal and aggressive central nervous system malignancy, presents a critical need for targeted therapeutic approaches to improve patient outcomes in conjunction with standard-of-care (SOC) treatment. Molecular subtyping based on genetic profiles and metabolic characteristics has advanced our understanding of GBM to better predict its evolution, mechanisms, and treatment regimens. Pharmacological ascorbate (P-AscH-) has emerged as a promising supplementary cancer therapy, leveraging its pro-oxidant properties to selectively kill malignant cells when combined with SOC. Given the clinical challenges posed by the heterogeneity and resistance of various GBM subtypes to conventional SOC, our study assessed the response of classical, mesenchymal, and proneural GBM to P-AscH-. P-AscH- (20 pmol/cell) combined with SOC (5 µM temozolomide and 4 Gy of radiation) enhanced clonogenic cell killing in classical and mesenchymal GBM subtypes, with limited effects in the proneural subtype. Similarly, following exposure to P-AscH- (20 pmol/cell), single-strand DNA damage significantly increased in classical and mesenchymal but not proneural GBM. Moreover, proneural GBM exhibited increased hydrogen peroxide removal rates, along with increased catalase and glutathione peroxidase activities compared to mesenchymal and classical GBM, demonstrating an altered H2O2 metabolism that potentially drives differential P-AscH- toxicity. Taken together, these data suggest that P-AscH- may hold promise as an approach to improve SOC responsiveness in mesenchymal GBMs that are known for their resistance to SOC.

Evaluating the iron chelator function of sirtinol in non-small cell lung cancer.

A distinctive feature of cancer is the upregulation of sirtuin proteins. Sirtuins are class III NAD+-dependent deacetylases involved in cellular processes such as proliferation and protection against oxidative stress. SIRTs 1 and 2 are also overexpressed in several types of cancers including non-small cell lung cancer (NSCLC). Sirtinol, a sirtuin (SIRT) 1 and 2 specific inhibitor, is a recent anti-cancer agent that is cytotoxic against several types of cancers including NSCLC. Thus, sirtuins 1 and 2 represent valuable targets for cancer therapy. Recent studies show that sirtinol functions as a tridentate iron chelator by binding Fe3+ with 3:1 stoichiometry. However, the biological consequences of this function remain unexplored. Consistent with preliminary literature, we show that sirtinol can deplete intracellular labile iron pools in both A549 and H1299 non-small cell lung cancer cells acutely. Interestingly, a temporal adaptive response occurs in A549 cells as sirtinol enhances transferrin receptor stability and represses ferritin heavy chain translation through impaired aconitase activity and apparent IRP1 activation. This effect was not observed in H1299 cells. Holo-transferrin supplementation significantly enhanced colony formation in A549 cells while increasing sirtinol toxicity. This effect was not observed in H1299 cells. The results highlight the fundamental genetic differences that may exist between H1299 and A549 cells and offer a novel mechanism of how sirtinol kills NSCLC cells.

Pharmacological ascorbate as a novel therapeutic strategy to enhance cancer immunotherapy.

Pharmacological ascorbate (i.e., intravenous infusions of vitamin C reaching ~ 20 mM in plasma) is under active investigation as an adjuvant to standard of care anti-cancer treatments due to its dual redox roles as an antioxidant in normal tissues and as a prooxidant in malignant tissues. Immune checkpoint inhibitors (ICIs) are highly promising therapies for many cancer patients but face several challenges including low response rates, primary or acquired resistance, and toxicity. Ascorbate modulates both innate and adaptive immune functions and plays a key role in maintaining the balance between pro and anti-inflammatory states. Furthermore, the success of pharmacological ascorbate as a radiosensitizer and a chemosensitizer in pre-clinical studies and early phase clinical trials suggests that it may also enhance the efficacy and expand the benefits of ICIs.

Detection of Ferritin Expression in Soft Tissue Sarcomas With MRI: Potential Implications for Iron Metabolic Therapy.

Background: Cancer cells often have altered iron metabolism relative to non-malignant cells with increased transferrin receptor and ferritin expression. Targeting iron regulatory proteins as part of a cancer therapy regimen is currently being investigated in various malignancies. Anti-cancer therapies that exploit the differences in iron metabolism between malignant and non-malignant cells (e.g. pharmacological ascorbate and iron chelation therapy) have shown promise in various cancers, including glioblastoma, lung, and pancreas cancers. Non-invasive techniques that probe tissue iron metabolism may provide valuable information for the personalization of iron-based cancer therapies. T2* mapping is a clinically available MRI technique that assesses tissue iron content in the heart and liver. We aimed to investigate the capacity of T2* mapping to detect iron stores in soft tissue sarcomas (STS). Methods: In this study, we evaluated T2* relaxation times ex vivo in five STS samples from subjects enrolled on a phase Ib/IIa clinical trial combining pharmacological ascorbate with neoadjuvant radiation therapy. Iron protein expression levels (ferritin, transferrin receptor, iron response protein 2) were evaluated by Western blot analysis. Bioinformatic data relating clinical outcomes in STS patients and iron protein expression levels were evaluated using the KMplotter database. Results: There was a high level of inter-subject variability in the expression of iron protein and T2* relaxation times. We identified that T2* relaxation time is capable of accurately detecting ferritin-heavy chain expression (r = -0.96) in these samples. Bioinformatic data acquired from the KMplot database revealed that transferrin receptor and iron-responsive protein 2 may be negative prognostic markers while ferritin expression may be a positive prognostic marker in the management of STS. Conclusion: These data suggest that targeting iron regulatory proteins may provide a therapeutic approach to enhance STS management. Additionally, T2* mapping has the potential to be used a clinically accessible, non-invasive marker of STS iron regulatory protein expression and influence cancer therapy decisions that warrants further investigation. Level of Evidence: IV.

Oxidation of ferumoxytol by ionizing radiation releases iron. An electron paramagnetic resonance study.

Ferumoxytol (FMX) is an iron oxide nanoparticle that is FDA approved for the treatment of iron deficiency anemia. FMX contains an Fe3O4 core. Currently, the redox chemistry of Fe3O4 nanoparticles remains relatively unexplored. FMX has recently gained interest as an anti-cancer agent. Ionizing radiation (IR) is a treatment modality employed to treat several types of cancer. Utilizing electron paramagnetic resonance (EPR) spectroscopy, we found that the products produced from the radiolysis of water can oxidize the Fe3O4 core of FMX. Because of the limited diffusion of the HO2• and HO• produced, these highly oxidizing species have little direct effect on FMX oxidation. We have determined that H2O2 is the primary oxidant of FMX. In the presence of labile Fe2+, we found that reducing species generated from the radiolysis of H2O are able to reduce the Fe3+ sites of the Fe3O4 core. Importantly, we also have shown that IR stimulates the release of ferric iron from FMX. Because of its release of iron, FMX may serve as an adjuvant to enhance radiotherapy.

Utilization of Pharmacological Ascorbate to Enhance Hydrogen Peroxide-Mediated Radiosensitivity in Cancer Therapy.

Interest in the use of pharmacological ascorbate as a treatment for cancer has increased considerably since it was introduced by Cameron and Pauling in the 1970s. Recently, pharmacological ascorbate has been used in preclinical and early-phase clinical trials as a selective radiation sensitizer in cancer. The results of these studies are promising. This review summarizes data on pharmacological ascorbate (1) as a safe and efficacious adjuvant to cancer therapy; (2) as a selective radiosensitizer of cancer via a mechanism involving hydrogen peroxide; and (3) as a radioprotector in normal tissues. Additionally, we present new data demonstrating the ability of pharmacological ascorbate to enhance radiation-induced DNA damage in glioblastoma cells, facilitating cancer cell death. We propose that pharmacological ascorbate may be a general radiosensitizer in cancer therapy and simultaneously a radioprotector of normal tissue.

An integrated physico-chemical approach for explaining the differential impact of FLASH versus conventional dose rate irradiation on cancer and normal tissue responses.

For decades the field of radiation oncology has sought to improve the therapeutic ratio through innovations in physics, chemistry, and biology. To date, technological advancements in image guided beam delivery techniques have provided clinicians with their best options for improving this critical tool in cancer care. Medical physics has focused on the preferential targeting of tumors while minimizing the collateral dose to the surrounding normal tissues, yielding only incremental progress. However, recent developments involving ultra-high dose rate irradiation termed FLASH radiotherapy (FLASH-RT), that were initiated nearly 50 years ago, have stimulated a renaissance in the field of radiotherapy, long awaiting a breakthrough modality able to enhance therapeutic responses and limit normal tissue injury. Compared to conventional dose rates used clinically (0.1-0.2 Gy/s), FLASH can implement dose rates of electrons or X-rays in excess of 100 Gy/s. The implications of this ultra-fast delivery of dose are significant and need to be re-evaluated to appreciate the fundamental aspects underlying this seemingly unique radiobiology. The capability of FLASH to significantly spare normal tissue complications in multiple animal models, when compared to conventional rates of dose-delivery, while maintaining persistent growth inhibition of select tumor models has generated considerable excitement, as well as skepticism. Based on fundamental principles of radiation physics, radio-chemistry, and tumor vs. normal cell redox metabolism, this article presents a series of testable, biologically relevant hypotheses, which may help rationalize the differential effects of FLASH irradiation observed between normal tissue and tumors.