Kaijun Di

Low-doses of cisplatin injure hippocampal synapses: A mechanism for ‘chemo’ brain?

Chemotherapy-related cognitive deficits are a major neurological problem, but the underlying mechanisms are unclear. The death of neural stem/precursor cell (NSC) by cisplatin has been reported as a potential cause, but this requires high doses of chemotherapeutic agents. Cisplatin is frequently used in modern oncology, and it achieves high concentrations in the patients brain. Here we report that exposure to low concentrations of cisplatin (0.1μM) causes the loss of dendritic spines and synapses within 30min. Longer exposures injured dendritic branches and reduced dendritic complexity. At this low concentration, cisplatin did not affect NSC viability nor provoke apoptosis. However, higher cisplatin levels (1μM) led to the rapid loss of synapses and dendritic disintegration, and neuronal-but not NSC-apoptosis. In-vivo treatment with cisplatin at clinically relevant doses also caused a reduction of dendritic branches and decreased spine density in CA1 and CA3 hippocampal neurons. An acute increase in cell death was measured in the CA1 and CA3 neurons, as well as in the NSC population located in the subgranular zone of the dentate gyrus in the cisplatin treated animals. The density of dendritic spines is related to the degree of neuronal connectivity and function, and pathological changes in spine number or structure have significant consequences for brain function. Therefore, this synapse and dendritic damage might contribute to the cognitive impairment observed after cisplatin treatment.

TRIM11 is overexpressed in high-grade gliomas and promotes proliferation, invasion, migration and glial tumor growth

TRIM11 (tripartite motif-containing protein 11), an E3 ubiquitin ligase, is known to be involved in the development of the central nervous system. However, very little is known regarding the role of TRIM11 in cancer biology. Here, we examined the expression profile of TRIM11, along with two stem cell markers CD133 and nestin, in multiple glioma patient specimens, glioma primary cultures derived from tumors taken at surgery and normal neural stem/progenitor cells (NSCs). The oncogenic function of TRIM11 in glioma biology was investigated by knockdown and/or overexpression in vitro and in vivo experiments. Our results showed that TRIM11 expression levels were upregulated in malignant glioma specimens and in high-grade glioma-derived primary cultures, whereas remaining low in glioblastoma multiforme (GBM) stable cell lines, low-grade glioma-derived primary cultures and NSCs. The expression pattern of TRIM11 strongly correlated with that of CD133 and nestin and differentiation status of malignant glioma cells. Knock down of TRIM11 inhibited proliferation, migration and invasion of GBM cells, significantly decreased epidermal growth factor receptor (EGFR) levels and mitogen-activated protein kinase activity, and downregulated HB-EGF (heparin-binding EGF-like growth factor) mRNA levels. Meanwhile, TRIM11 overexpression promoted a stem-like phenotype in vitro (tumorsphere formation) and enhanced glial tumor growth in immunocompromised mice. These findings suggest that TRIM11 might be an indicator of glioma malignancy and has an oncogenic function mediated through the EGFR signaling pathway. TRIM11 overexpression potentially leads to a more aggressive glioma phenotype, along with increased malignant tumor growth and poor survival. Taken together, clarification of the biological function of TRIM11 and pathways it affects may provide novel therapeutic strategies for treating malignant glioma patients.

Cisplatin-induced mitochondrial dysfunction is associated with impaired cognitive function in rats

Purpose Chemotherapy‐related cognitive impairment (CRCI) is commonly reported following the administration of chemotherapeutic agents and comprises a wide variety of neurological problems. No effective treatments for CRCI are currently available. Here we examined the mechanisms involving cisplatin‐induced hippocampal damage following cisplatin administration in a rat model and in cultured rat hippocampal neurons and neural stem/progenitor cells (NSCs). We also assessed the protective effects of the antioxidant, N‐acetylcysteine in mitigating these damages. Experimental design Adult male rats received 6 mg/kg cisplatin in the acute studies. In chronic studies, rats received 5 mg/kg cisplatin or saline injections once per week for 4 weeks. N‐acetylcysteine (250 mg/kg/day) or saline was administered for five consecutive days during cisplatin treatment. Cognitive testing was performed 5 weeks after treatment cessation. Cisplatin‐treated cultured hippocampal neurons and NSCs were examined for changes in mitochondrial function, oxidative stress production, caspase‐9 activation, and neuronal dendritic spine density. Results Acute cisplatin treatment reduced dendritic branching and spine density, and induced mitochondrial degradation. Rats receiving the chronic cisplatin regimen showed impaired performance in contextual fear conditioning, context object discrimination, and novel object recognition tasks compared to controls. Cisplatin induced mitochondrial DNA damage, impaired respiratory activity, increased oxidative stress, and activated caspase‐9 in cultured hippocampal neurons and NSCs. N‐acetylcysteine treatment prevented free radical production, ameliorated apoptotic cellular death and dendritic spine loss, and partially reversed the cisplatin‐induced cognitive impairments. Conclusions Our results suggest that mitochondrial dysfunction and increased oxidative stress are involved in cisplatin‐induced cognitive impairments. Therapeutic agents, such as N‐acetylcysteine, may be effective in mitigating the deleterious effects of cisplatin. HighlightsCisplatin induces cognitive impairments.Cisplatin reduces hippocampal dendritic branching and spine density.Cisplatin causes neural mitochondrial dysfunction and apoptosis.NAC reduces cisplatin‐induced cognitive impairments.NAC ameliorates cisplatin‐mediated mitochondrial damage by reducing oxidative stress.

Marizomib activity as a single agent in malignant gliomas: ability to cross the blood-brain barrier

BACKGROUND The proteasome plays a vital role in the physiology of glioblastoma (GBM), and proteasome inhibition can be used as a strategy for treating GBM. Marizomib is a second-generation, irreversible proteasome inhibitor with a more lipophilic structure that suggests the potential for penetrating the blood-brain barrier. While bortezomib and carfilzomib, the 2 proteasome inhibitors approved for treatment of multiple myeloma, have little activity against malignant gliomas in vivo, marizomib could be a novel therapeutic strategy for primary brain tumors. METHODS The in-vitro antitumor activity of marizomib was studied in glioma cell lines U-251 and D-54. The ability of marizomib to cross the blood-brain barrier and regulate proteasome activities was evaluated in cynomolgus monkeys and rats. The antitumor effect of marizomib in vivo was tested in an orthotopic xenograft model of human GBM. RESULTS Marizomib inhibited the proteasome activity, proliferation, and invasion of glioma cells. Meanwhile, free radical production and apoptosis induced by marizomib could be blocked by antioxidant N-acetyl cysteine. In animal studies, marizomib distributed into the brain at 30% of blood levels in rats and significantly inhibited (>30%) baseline chymotrypsin-like proteasome activity in brain tissue of monkeys. Encouragingly, the immunocompromised mice, intracranially implanted with glioma xenografts, survived significantly longer than the control animals (P < .05) when treated with marizomib. CONCLUSIONS These preclinical studies demonstrated that marizomib can cross the blood-brain barrier and inhibit proteasome activity in rodent and nonhuman primate brain and elicit a significant antitumor effect in a rodent intracranial model of malignant glioma.

Profiling Hsp90 differential expression and the molecular effects of the Hsp90 inhibitor IPI-504 in high-grade glioma models

Retaspimycin hydrochloride (IPI-504), an Hsp90 (heat shock protein 90) inhibitor, has shown activity in multiple preclinical cancer models, such as lung, breast and ovarian cancers. However, its biological effects in gliomas and normal brain derived cellular populations remain unknown. In this study, we profiled the expression pattern of Hsp90α/β mRNA in stable glioma cell lines, multiple glioma-derived primary cultures and human neural stem/progenitor cells. The effects of IPI-504 on cell proliferation, apoptosis, motility and expression of Hsp90 client proteins were evaluated in glioma cell lines. In vivo activity of IPI-504 was investigated in subcutaneous glioma xenografts. Our results showed Hsp90α and Hsp90β expression levels to be patient-specific, higher in high-grade glioma-derived primary cells than in low-grade glioma-derived primary cells, and strongly correlated with CD133 expression and differentiation status of cells. Hsp90 inhibition by IPI-504 induced apoptosis, blocked migration and invasion, and significantly decreased epidermal growth factor receptor levels, mitogen-activated protein kinase and/or Akt activities, and secretion of vascular endothelial growth factor in glioma cell lines. In vivo study showed that IPI-504 could mildly attenuate tumor growth in immunocompromised mice. These findings suggest that targeting Hsp90 by IPI-504 has the potential to become an active therapeutic strategy in gliomas in a selective group of patients, but further research into combination therapies is still needed.

3D Mathematical modeling of glioblastoma suggests that transdifferentiated vascular endothelial cells promote resistance to current standard-of-care therapy.

Glioblastoma (GBM), the most aggressive brain tumor in human patients, is decidedly heterogeneous and highly vascularized. Glioma stem/initiating cells (GSC) are found to play a crucial role by increasing cancer aggressiveness and promoting resistance to therapy. Recently, cross-talk between GSC and vascular endothelial cells has been shown to significantly promote GSC self-renewal and tumor progression. Furthermore, GSC also transdifferentiate into bona fide vascular endothelial cells (GEC), which inherit mutations present in GSC and are resistant to traditional antiangiogenic therapies. Here we use three-dimensional mathematical modeling to investigate GBM progression and response to therapy. The model predicted that GSCs drive invasive fingering and that GEC spontaneously form a network within the hypoxic core, consistent with published experimental findings. Standard-of-care treatments using DNA-targeted therapy (radiation/chemo) together with antiangiogenic therapies reduced GBM tumor size but increased invasiveness. Anti-GEC treatments blocked the GEC support of GSCs and reduced tumor size but led to increased invasiveness. Anti-GSC therapies that promote differentiation or disturb the stem cell niche effectively reduced tumor invasiveness and size, but were ultimately limited in reducing tumor size because GECs maintain GSCs. Our study suggests that a combinatorial regimen targeting the vasculature, GSCs, and GECs, using drugs already approved by the FDA, can reduce both tumor size and invasiveness and could lead to tumor eradication. Cancer Res; 77(15); 4171-84. ©2017 AACR.

Mitochondrial Lon is over-expressed in high-grade gliomas, and mediates hypoxic adaptation: potential role of Lon as a therapeutic target in glioma

Mitochondrial dysfunction is a hallmark of cancer biology. Tumor mitochondrial metabolism is characterized by an abnormal ability to function in scarce oxygen conditions through glycolysis (the Warburg effect), and accumulation of mitochondrial DNA defects are present in both hereditary neoplasia and sporadic cancers. Mitochondrial Lon is a major regulator of mitochondrial metabolism and the mitochondrial response to free radical damage, and plays an essential role in the maintenance and repair of mitochondrial DNA. Despite these critical cellular functions of Lon, very little has been reported regarding its role in glioma. Lon expression in gliomas and its relevance with patient survival was examined using published databases and human tissue sections. The effect of Lon in glioma biology was investigated through siRNA targeting Lon. We also tested the in vitro antitumor activity of Lon inhibitor, CC4, in the glioma cell lines D-54 and U-251. High Lon expression was associated with high glioma tumor grade and poor patient survival. While Lon expression was elevated in response to a variety of stimuli, Lon knockdown in glioma cell lines decreased cell viability under normal conditions, and dramatically impaired glioma cell survival under hypoxic conditions. Furthermore, the Lon inhibitor, CC4, efficiently prohibited glioma cell proliferation and synergistically enhanced the therapeutic efficacy of the chemotherapeutic agents, temozolomide (TMZ) and cisplatin. We demonstrate that Lon plays a key role in glioma cell hypoxic survival and mitochondrial respiration, and propose Lon as a promising therapeutic target in the treatment of malignant gliomas.

Abstract 1809: Marizomib (NPI-0052) activity as a single agent in malignant glioma

Background: Glioblastoma multiforme (GBM) is a highly aggressive brain tumor, which displays innate resistance to multiple treatment modalities. Previous studies have shown that the proteasome plays a vital role in the physiology of GBM, and that proteasome inhibition can be used as a strategy for treating malignant gliomas. Marizomib (NPI-0052) is a second generation irreversible proteasome inhibitor, which has a more lipophilic structure and has a broader and more prolonged inhibition profile for 20S proteasome activities compared to bortezomib and carfilzomib, two proteasome inhibitors approved by FDA for treatment of multiple myeloma. While bortezomib and carfilzomib have only modest activity as a treatment of malignant gliomas, marizomib might potentially be a novel therapeutic strategy for primary brain tumors. Unfortunately, to date, the number of studies that have analyzed the effect of marizomib on glioma is limited. Methods: In these studies, we investigated the in vitro activities of marizomib in primary cell cultures derived from a multitude of human brain tumors (high-grade and low-grade gliomas and meningiomas), normal neural stem/progenitor cells (NSC) and as well as in the established human malignant glioma lines U251-MG and D54-MG. The effect of marizomib on cell proliferation, proteasome activity, motility, apoptosis and Reactive Oxygen Species (ROS) were evaluated in glioma cell lines. The inhibition of marizomib by the ROS quenching agent, N-acetyl cysteine (NAC) was also tested. Results: The sensitivities varied in function of the pathology of the tumor, with the malignant glioma stem-like cells being the most severely affected, in contrast with the low-grade glioma, meningioma and NSC-derived cultures. Marizomib inhibited the proliferation of U251-MG and D54-MG cell lines with a half maximal effective concentration (EC50) of 52nM and 20nM respectively, along with a significant decrease in cell migration and invasion. Treatment with marizomib at a concentration of 60nM for 4 hours inhibited proteasome chymotrypsin-like (CT-L, β5) activity by 85% in U251-MG and D54-MG cells. Marizomib treatment of human glioma cells was associated with increased free radical production and apoptosis, along with activation of caspase-3 and cleavage of PARP. Those effects of marizomib can be suppressed by exposure to the ROS quenching agent N-acetyl cysteine (NAC). Conclusion: These preclinical studies demonstrate a significant anti-tumor effect of marizomib in malignant glioma cells. Marizomib has relatively little effect on neural stem/progenitor cells suggesting minimal neurotoxicity, while severely affecting both malignant glioma stem cells and glioma cell lines. But importantly, unlike bortezomib and carfilzomib, marizomib can cross the blood brain barrier. Additional research into the use of marizomib as a potential treatment for malignant gliomas as a single agent or in combination with SOC therapies for glioma is warranted. Citation Format: Kaijun Di, Xing Gong, Dana M. Curticiu, Michael A. Palladino, Daniela A. Bota. Marizomib (NPI-0052) activity as a single agent in malignant glioma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1809. doi:10.1158/1538-7445.AM2014-1809

Abstract 3069: Investigation of pharmacodynamic and predictive biomarkers to define response to proteasome inhibitor marizomib in glioma

Proteasome inhibitors (PIs) have been employed with clinical success in multiple myeloma, but have been much less effective in solid tumors, despite the central role of the proteasome in controlling cellular metabolism. Marizomib (MRZ) is a novel second generation proteasome inhibitor which binds irreversibly to and inhibits the enzymatic activity of all three subunits of the proteasome. The unique ability of MRZ among PIs to cross the blood-brain barrier, combined with its pan-proteasome activity, suggest that MRZ may have distinct therapeutic advantages over the approved PIs in the treatment of glioma. Preclinical studies with MRZ have demonstrated anti-tumor activity in intracranial glioma studies, and MRZ is currently being evaluated in a Phase I clinical trial in WHO Grade IV recurrent glioma in combination with bevacizumab (NCT02330562). The aim of this study was to identify pharmacodynamic and predictive biomarkers of response to marizomib in glioma patients. Analysis of the pharmacodynamic profile of MRZ in packed whole blood from MRZ-treated glioma patients demonstrated >70% inhibition of the chymotrypsin-like (CT-L) activity as early as day 1 of cycle1 at 1 hr post-infusion, with 100% inhibition post-infusion in all patients by the end of cycle 1. Pre-infusion data demonstrate a prolonged effect, with >60% inhibition of CT-L persistent between day 15 of each cycle and day 1 of the next cycle. Trypsin-like (T-L) and caspase-like (C-L) activities increased after the first 1-2 MRZ doses, presumably due to compensatory hyperactivation of these subunits triggered by CT-L inhibition, which was subsequently overcome by repeated MRZ infusion, resulting in 40-60% inhibition of T-L and 10-30% inhibition of C-L evident through cycle 5. Analysis of proteasome enzymatic activity in archival glioma tumor tissue revealed that levels of all three proteasome activities are variable between high grade glioma samples, suggesting the potential for differential sensitivity to proteasome inhibition in glioma patients. Further, there is a linear correlation between CT-L activity (the rate limiting enzyme for proteasomal proteolysis) and C-L activity in these samples, suggesting that a PI such as MRZ with pan-proteasome specificity could potentially exhibit more activity in glioma compared to CT-L specific PIs. The data are currently being expanded to evaluate both proteasome enzymatic activity and subunit mRNA levels, to establish whether these endpoints might serve as a proteasome based biomarker. In conclusion, this study demonstrates that packed whole blood may be suitable as a pharmacodynamic biomarker for proteasome inhibition. This biomarker strategy may be crucial to stratify MRZ responsive patients in glioma. Citation Format: Daniela Bota, Annick Desjardins, Warren Mason, Kaijun Di, Ann P. MacLaren, Nancy Levin, Mohit Trikha. Investigation of pharmacodynamic and predictive biomarkers to define response to proteasome inhibitor marizomib in glioma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3069.

Abstract 4782: Cisplatin induces mitochondrial damage and hippocampal neurotoxicity: a potential mechanism for chemotherapy-related cognitive impairment

Advances in cancer treatment, chemotherapy in particular, have substantially increased the number of long-term cancer survivors. However, these drugs often have neurotoxic effects that impair cognitive function, thereby diminishing the quality of life of millions of cancer survivors. Chemotherapy-related cognitive impairment (CRCI, chemo-brain) is commonly reported following the administration of chemotherapeutic agents and comprises a wide variety of neurological problems. Cisplatin is used to treat breast cancer and advanced ovarian cancer among other malignancies. Notably, more than 30% of advanced ovarian cancer patients develop CRCI during and after cisplatin-based chemotherapy. A plausible explanation for CRCI is that cisplatin might impair the structure and functions of neurons in brain regions involved in learning and memory, such as the hippocampus. We have recently identified mitochondrial dysfunction and increased oxidative stress as a mechanism through which cisplatin causes hippocampal cell death, and severe dendritic damage in surviving neurons. The aims of this study were to examine the effect of the antioxidant N-acetylcysteine (NAC) in mitigating cisplatin-induced hippocampal damage and assesse the effect of cisplatin on cognitive performance in a rat model. At a high dose, cisplatin (1μM) induced ∼35% increase in caspase-9 activation in primary rat hippocampal neurons, whereas at a substantially lower dose, cisplatin (0.1μM) induced non-reversible damage to dendritic spines and branches. Both doses produced severe mitochondrial respiratory deficits and significant ROS production. Delayed treatment with NAC partially mitigated neuronal apoptosis and ameliorated cisplatin induced dendritic spine loss. When administered to adult Sprague Dawley rats, cisplatin (3 mg/kg) administered for two consecutive days caused ∼40% reduction in the number of dendritic spines in CA1 and CA3 hippocampal neurons. Lastly, cognitive testing of rats treated with a chronic cisplatin regimen, revealed significant deficits in hippocampus-dependent tasks. Rats were given weekly cisplatin (5mg/kg, i.p.) or saline injections for 4 weeks and then trained in Context-Object Discrimination, 6 weeks later (n = 7,8). Cisplatin-treated rats were impaired in discriminating between the out-of-context and in-context object. Mitochondrial dysfunction provokes free radical production, with resulting loss of dendritic spines. When administered to rats, cisplatin causes hippocampal neuronal and mitochondrial damage, as well as cognitive deficits, supporting that role of mitochondrial toxicity in the mechanisms of cisplatin-induced CRCI. Importantly the data demonstrates that these processes can be potentially mitigated with administration of the clinically available antioxidant, NAC. The effect of NAC in ameliorating cisplatin induced CRCI in rats is being evaluated. Citation Format: Naomi Lomeli, Jennifer Czerniawski, Kaijun Di, John Guzowski, Daniela Bota. Cisplatin induces mitochondrial damage and hippocampal neurotoxicity: a potential mechanism for chemotherapy-related cognitive impairment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4782.