Bortezomib-induced Neurotoxicity Research Articles

Bortezomib-induced neurotoxicity in human neurons is the consequence of nicotinamide adenine dinucleotide depletion.

The proteosome inhibitor bortezomib has revolutionized the treatment of multiple hematologic malignancies, but in many cases, its efficacy is limited by a dose-dependent peripheral neuropathy. We show that human induced pluripotent stem cell (hiPSC)-derived motor neurons and sensory neurons provide a model system for the study of bortezomib-induced peripheral neuropathy, with promising implications for furthering the mechanistic understanding of and developing treatments for preventing axonal damage. Human neurons in tissue culture displayed distal-to-proximal neurite degeneration when exposed to bortezomib. This process coincided with disruptions in mitochondrial function and energy homeostasis, similar to those described in rodent models of bortezomib-induced neuropathy. Moreover, although the degenerative process was unaffected by inhibition of caspases, it was completely blocked by exogenous nicotinamide adenine dinucleotide (NAD+), a mediator of the SARM1-dependent axon degeneration pathway. We demonstrate that bortezomib-induced neurotoxicity in relevant human neurons proceeds through mitochondrial dysfunction and NAD+ depletion-mediated axon degeneration, raising the possibility that targeting these changes might provide effective therapeutics for the prevention of bortezomib-induced neuropathy and that modeling chemotherapy-induced neuropathy in human neurons has utility.

Inhibition of Neurotoxicity/Anticancer Activity of Bortezomib by Caffeic Acid and Chlorogenic Acid.

Bortezomib, used for the treatment of multiple myeloma, has been reported to induce potent neurotoxicity. The present study investigated whether eight popular polyphenols inhibit bortezomib-induced neurotoxicity without affecting its anticancer activity. Viable cell number was determined with the MTT method. Tumor-specificity was determined by the relative cytotoxicity in human oral squamous cell carcinoma vs. normal oral cells. Neurotoxicity was determined by the relative cytotoxicity in differentiated rat neuronal PC12 cells vs. normal cells. Apoptotic cells were quantified by cell cycle analysis. Bortezomib induced cell shrinkage, disruption of neurites, and accumulation of PC-12 cells in subG1. Only chlorogenic acid and caffeic acid protected PC-12 cells from bortezomib-induced neurotoxicity. Ferulic acid that has one of the two hydroxyl groups replaced by a methoxy group showed a significantly reduced neuroprotective effect. Caffeic acid and the chlorogenic acid also neutralized the anticancer potential of bortezomib. Caffeic acid and the chlorogenic acid may reduce the biological activity of bortezomib by forming a conjugate.

Reversal of Bortezomib-Induced Neurotoxicity by Suvecaltamide, a Selective T-Type Ca-Channel Modulator, in Preclinical Models.

Simple SummaryChemotherapy-induced peripheral neurotoxicity (CIPN) is a side-effect of anti-cancer medications, which can lead to pain, disruptions to movement, and eventually results in the need to interrupt or stop chemotherapy. This study sought to test whether the drug suvecaltamide could help to reduce the impact of the chemotherapy agent bortezomib (BTZ) on symptoms of CIPN using animal models and human cells. Suvecaltamide did reverse negative changes in nerve conduction velocity and intraepidermal nerve fiber density indicative of CIPN in rats, and did not interfere with the anti-cancer effect of BTZ. These results indicate that suvecaltamide could potentially be useful for patients experiencing CIPN, although further mechanistic and molecular studies in vitro and in vivo are required before clinical trials.This study evaluated suvecaltamide, a selective T-type calcium channel modulator, on chemotherapy-induced peripheral neurotoxicity (CIPN) and anti-cancer activity associated with bortezomib (BTZ). Rats received BTZ (0.2 mg/kg thrice weekly) for 4 weeks, then BTZ alone (n = 8) or BTZ+suvecaltamide (3, 10, or 30 mg/kg once daily; each n = 12) for 4 weeks. Nerve conduction velocity (NCV), mechanical threshold, β-tubulin polymerization, and intraepidermal nerve fiber (IENF) density were assessed. Proteasome inhibition was evaluated in peripheral blood mononuclear cells. Cytotoxicity was assessed in human multiple myeloma cell lines (MCLs) exposed to BTZ alone (IC50 concentration), BTZ+suvecaltamide (10, 30, 100, 300, or 1000 nM), suvecaltamide alone, or vehicle. Tumor volume was estimated in athymic nude mice bearing MCL xenografts receiving vehicle, BTZ alone (1 mg/kg twice weekly), or BTZ+suvecaltamide (30 mg/kg once daily) for 28 days, or no treatment (each n = 8). After 4 weeks, suvecaltamide 10 or 30 mg/kg reversed BTZ-induced reduction in NCV, and suvecaltamide 30 mg/kg reversed BTZ-induced reduction in IENF density. Proteasome inhibition and cytotoxicity were similar between BTZ alone and BTZ+suvecaltamide. BTZ alone and BTZ+suvecaltamide reduced tumor volume versus the control (day 18), and BTZ+suvecaltamide reduced tumor volume versus BTZ alone (day 28). Suvecaltamide reversed CIPN without affecting BTZ anti-cancer activity in preclinical models.

Proteomic analysis of human iPSC-derived sensory neurons implicates cell stress and microtubule dynamics dysfunction in bortezomib-induced peripheral neurotoxicity

The neurotoxic effects of the chemotherapeutic agent bortezomib on dorsal root ganglia sensory neurons are well documented, yet the mechanistic underpinnings that govern these cellular processes remain incompletely understood. In this study, system-wide proteomic changes were identified in human induced pluripotent stem cell-derived sensory neurons (iSNs) exposed to a clinically relevant dose of bortezomib. Label-free mass spectrometry facilitated the identification of approximately 2800 iSN proteins that exhibited differential levels in the setting of bortezomib. A significant proportion of these proteins affect the cellular processes of microtubule dynamics, cytoskeletal and cytoplasmic organization, and molecular transport, and pathway analysis revealed an enrichment of proteins in signaling pathways attributable to the unfolded protein response and the integrated stress response. Alterations in microtubule-associated proteins suggest a multifaceted relationship exists between bortezomib-induced proteotoxicity and microtubule cytoskeletal architecture, and MAP2 was prioritized as a topmost influential candidate. We observed a significant reduction in the overall levels of MAP2c in somata without discernable changes in neurites. As MAP2 is known to affect cellular processes including axonogenesis, neurite extension and branching, and neurite morphology, its altered levels are suggestive of a prominent role in bortezomib-induced neurotoxicity.

Protection of Bortezomib-induced Neurotoxicity by Antioxidants.

Although chemotherapy agents, such as oxaliplatin, cisplatin, paclitaxel and bortezomib frequently cause severe peripheral neuropathy, very few studies have reported the effective strategy to prevent this side effect. In this study, we first investigated whether these drugs show higher neuropathy compared to a set of 15 other anticancer drugs, and then whether antioxidants, such as sodium ascorbate, N-acetyl-L-cysteine, and vitamin B12 have any protective effect against them. Rat PC12 cells were induced to differentiate into neuronal cells by repeated overlay of serum-free medium supplemented with nerve growth factor. The cytotoxic levels of anticancer drugs against four human oral squamous cell carcinoma cell lines, three normal oral cells, and undifferentiated and differentiated PC12 cells were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Cells were sorted for apoptotic cells (distributed into subG1 phase) and cells at different stages of cell cycle (G1, S and G2/M). All 19 anticancer drugs showed higher cytotoxicity against PC12 compared to oral normal cells. Among them, bortezomib showed the highest cytotoxicity against both undifferentiated and differentiated PC12 cell and, committed them to undergo apoptosis. Sodium ascorbate and N-acetyl-L-cysteine, but not vitamin B12, completely reversed the cytotoxicity of bortezomib. Bortezomib-induced neuropathy might be ameliorated by antioxidants.

Molecular Mechanisms of Bortezomib Action: Novel Evidence for the miRNA-mRNA Interaction Involvement.

Bortezomib is an anti-tumor agent, which inhibits 26S proteasome degrading ubiquitinated proteins. While apoptotic transcription-associated activation in response to bortezomib has been suggested, mechanisms related to its influence on post-transcriptional gene silencing mediated regulation by non-coding RNAs remain not fully elucidated. In the present study, we examined changes in global gene and miRNA expression and analyzed the identified miRNA–mRNA interactions after bortezomib exposure in human neuroblastoma cells to define pathways affected by this agent in this type of cells. Cell viability assays were performed to assess cytotoxicity of bortezomib. Global gene and miRNA expression profiles of neuroblastoma cells after 24-h incubation with bortezomib were determined using genome-wide RNA and miRNA microarray technology. Obtained results were then confirmed by qRT-PCR and Western blot. Further bioinformatical analysis was performed to identify affected biological processes and pathways. In total, 719 genes and 28 miRNAs were downregulated, and 319 genes and 61 miRNAs were upregulated in neuroblastoma cells treated with bortezomib. Possible interactions between dysregulated miRNA/mRNA, which could be linked to bortezomib-induced neurotoxicity, affect neurogenesis, cellular calcium transport, and neuron death. Bortezomib might exert toxic effects on neuroblastoma cells and regulate miRNA–mRNA interactions influencing vital cellular functions. Further studies on the role of specific miRNA–mRNA interactions are needed to elucidate mechanisms of bortezomib action.

MOLECULAR FEATURES OF BORTEZOMIB-INDUCED NEUROPATHY IN PATIENTS WITH MULTIPLE MYELOMA

Background. The regimens of therapy with bortezomib have significantly improved the survival among patients with multiple myeloma (MM). However, the development of peripheral polyneuropathy (PP) resulting from treatment using proteasome inhibitors is still an undesirable event. Risk factors for PP in MM patients include old age, previous neuropathy and use of neurotoxic drugs. Recent studies have established the presence of a genetic component in the mechanism of developing bortezomib-induced neurotoxicity. However, there are conflicting opinions on the role of genetic characteristics in predicting the risk of treatment-induced neuropathy development.Aim. To identify the risk group of bortezomib-induced PP based on the analysis of gene polymorphism of the immune response in patients with newly-diagnosed MM.Materials and methods. A study of the association of 20 polymorphic loci of 14 immune response genes in 46 MM patients was conducted using a candidate gene identification approach. All the patietns were receiving VCD therapy with bortezomib.Results. The distribution of single nucleotide polymorphisms was compared in groups of patients with the presence and absence of PP. It is found that homozygous carriers of the wild type allele of the genes TLR6 (Ser249Pro) (p = 0.006), IL1β (G-1473C) (p = 0.04), IL4 (C-589T) (p = 0.04), as well as haplotype carriers with the mutant allele of the gene IL10 (G-1082A) (p = 0.04) and with the wild type allele gene IL2 (T-330G) (p = 0.01) were significantly more frequent among PP patients.Сonclusion. Our results have confirmed the contribution of the genetic component to the risk of developing bortezomibinduced neuropathy. These findings can be used for individualization of therapeutic approaches to the treatment of MM patients.

Kinesin-5 Blocker Monastrol Protects Against Bortezomib-Induced Peripheral Neurotoxicity.

Neurotoxicity is a relevant side effect of bortezomib treatment. Previous reports have shown that the development of peripheral neuropathy caused by anti-neoplastic agents may be a result of reduced axonal transport. Based on evidence from prior studies that the kinesin-5 inhibitor monastrol enhances axonal transport and improves neuronal regeneration, we focused on the neuroprotective role of monastrol during the chemotherapeutic treatment with bortezomib. Prolonged treatment of C57BL/6 mice with bortezomib induced a length-dependent small-fiber neuropathy with axonal atrophy and loss of sensory nerve fibers. The administration of monastrol substantially alleviated morphological features of axonal injury and functional measures of sensory neuropathy. Cytotoxicity studies in leukemia and multiple myeloma cell lines showed no interference of monastrol with the cytostatic effects of bortezomib. Our data indicate that the novel approach of targeting microtubule turnover by monastrol provides protection against bortezomib-induced neurotoxicity. The favorable cytotoxic profile of monastrol makes it an interesting candidate as neuroprotective agent in combined chemotherapy regimens that warrants further consideration.

Neurotoxicity to DRG neurons varies between rodent strains treated with cisplatin and bortezomib

Chemotherapy-induced peripheral neuropathy (CIPN) is a major dose limiting side effect that can lead to long-term morbidity. Approximately one-third of patients receiving chemotherapy with taxanes, vinca alkaloids, platinum compounds or proteasome inhibitors develop this toxic side effect. It is not possible to predict who will get CIPN, however, genetic susceptibility may play a role. We explored this hypothesis using an established in vitro dorsal root ganglia neurite outgrowth (DRG-NOG) assay to assess possible genetic influences for cisplatin- and bortezomib-induced neurotoxicity. Almost all previous in vitro studies have used rats or mice. We compared DRG-NOG between four genetically defined, inbred mouse strains (C57BL/6J, DBA/2J, BALB/cJ, and C3H/HeJ) and one rat strain (Sprague Dawley). Our studies found differences in cisplatin and bortezomib-induced neurotoxicity between mouse and rat strains and between the different mouse strains. C57BL/6J and Balb/cJ DRG-NOG was more sensitive to cisplatin than DBA/2J and C3H/HeJ DRG-NOG, and all mouse strains were more sensitive to cisplatin than rat. Bortezomib induced a biphasic dose response in DBA/2J and C3H/H3J mice. C57BL/6J DRG-NOG was most sensitive and Balb/cJ DRG-NOG was least sensitive to bortezomib. Our animal data supports the hypothesis that genetic background may play a role in CIPN and care must be taken when rodent models are used to better understand the contribution of genetics in patient susceptibility to CIPN.

Stem Cell Mobilization Chemotherapy with Gemcitabine Is Effective and Safe in Myeloma Patients with Bortezomib Induced Neurotoxicity

Background: Vinorelbine chemotherapy with G-CSF stimulation is a widely applied non-myelosuppressive mobilization regimen in Switzerland for myeloma patients, but its neurotoxic potential limits its use in patients with bortezomib induced polyneuropathy. Methods: In this single-center study, we alternatively evaluated safety and effectiveness of gemcitabine chemotherapy with G-CSF for mobilization of autologous stem cells. Results: Between 03/2012 and 02/2013, all bortezomib pretreated myeloma patients planned to undergo first-line high-dose melphalan chemotherapy received a single dose of 1250mg/m2 gemcitabine, with G-CSF started on day four. The 24 patients in this study have received a median of four cycles of bortezomib-dexamethason based induction. Bortezomib-caused polyneuropathy was identified in 21 patients (88%) by clinical evaluation and a standardized questionnaire. Administration of gemcitabine mobilization did not induce novel or aggravate preexisting neuropathy. Stem cell mobilization was successful in all 24 patients, with a single day of apheresis being sufficient in 19 patients (78%). The median yield was 9.51 x 106 CD34+ cells/kg. Stem collection could be accomplished at day 8 in 67%. Conclusion: Our data suggest that single-dose gemcitabine together with G-CSF is an effective mobilization regimen in myeloma patients and a safe alternative non-myelosuppressive mobilization chemotherapy for myeloma patients with bortezomib induced polyneuropathy. DisclosuresNo relevant conflicts of interest to declare.

An Overview of Bortezomib-Induced Neurotoxicity.

The boronic acid dipeptide bortezomib, able to induce tumor cell death by degradation of key proteins, is the first proteasome inhibitor drug to enter clinical practice. It is employed as first-line treatment in relapsed or resistant multiple myeloma (MM) patients. However, bortezomib often induces a dose-limiting toxicity in the form of painful sensory neuropathy, which can mainly be reduced by subcutaneous administration or dose modification. In this review we focus on the current understanding of the pathophysiological mechanisms of bortezomib-induced neuropathy to allow further studies in animal models and humans, including analysis of clinical and pharmacogenetic aspects, to optimize the treatment regimens.

Reactive nucleolar and Cajal body responses to proteasome inhibition in sensory ganglion neurons

The dysfunction of the ubiquitin proteasome system has been related to a broad array of neurodegenerative disorders in which the accumulation of misfolded protein aggregates causes proteotoxicity. The ability of proteasome inhibitors to induce cell cycle arrest and apoptosis has emerged as a powerful strategy for cancer therapy. Bortezomib is a proteasome inhibitor used as an antineoplastic drug, although its neurotoxicity frequently causes a severe sensory peripheral neuropathy. In this study we used a rat model of bortezomib treatment to study the nucleolar and Cajal body responses to the proteasome inhibition in sensory ganglion neurons that are major targets of bortezomib-induced neurotoxicity. Treatment with bortezomib induced dose-dependent dissociation of protein synthesis machinery (chromatolysis) and nuclear retention of poly(A) RNA granules resulting in neuronal dysfunction. However, as a compensatory response to the proteotoxic stress, both nucleoli and Cajal bodies exhibited reactive changes. These include an increase in the number and size of nucleoli, strong nucleolar incorporation of the RNA precursor 5′-fluorouridine, and increased expression of both 45S rRNA and genes encoding nucleolar proteins UBF, fibrillarin and B23. Taken together, these findings appear to reflect the activation of the nucleolar transcription in response to proteotoxic stress Furthermore, the number of Cajal bodies, a parameter related to transcriptional activity, increases upon proteasome inhibition. We propose that nucleoli and Cajal bodies are important targets in the signaling pathways that are activated by the proteotoxic stress response to proteasome inhibition. The coordinating activity of these two organelles in the production of snRNA, snoRNA and rRNA may contribute to neuronal survival after proteasome inhibition. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease.

Tanespimycin Prevents Bortezomib Toxicity and Preserves Neuronal Morphology in Primary Rat Dorsal Root Ganglion Cultures.

Abstract 2847Poster Board II-823 INTRODUCTION:Tanespimycin, an inhibitor of Hsp90, is in phase 3 clinical trials in combination with bortezomib in patients with relapsed/refractory multiple myeloma (MM). The combination of tanespimycin and bortezomib produces synergistic antitumor activity and enhanced proteasome inhibition in primary MM cells (Mitsiades, Blood, 2006). In a phase 1/2 study in 72 patients with relapsed/refractory myeloma, tanespimycin + bortezomib produced durable responses in patients including bortezomib-refractory patients. Bortezomib-induced peripheral neuropathy (PN) is the key dose-limiting toxicity in patients with MM. In rat models of bortezomib-induced PN, tanespimycin is neuroprotective and can reverse bortezomib-induced PN. No Grade 3/4 PN was reported in the phase 1/2 study. OBJECTIVE:To explore the mechanism of tanespimycin-mediated neuroprotection from bortezomib-induced neurotoxicity in primary rat dorsal root ganglion (DRG) cells. METHODS:Differentiated DRG cultures prepared from rat fetuses were treated for up to 24 hours with tanespimycin alone, bortezomib alone, or the combination of tanespimycin + bortezomib. Total cell ATP, caspase 3/7 induction, calpain activity, and chymotrypsin-like activity of the proteasome were analyzed as measures of cell viability, apoptosis, neuron-specific protease activity, and proteasome function, respectively. Neuronal morphology was evaluated by light microscopy. RESULTS:At concentrations ≥100 nM, both bortezomib and tanespimycin induced a rapid increase in apoptosis (up to a fivefold increase in caspase activity) associated with reduced cell viability (based on ATP levels) to 20% of control values. In addition, bortezomib at ≥100 nM visibly shortened neurite extensions from DRG cell bodies. When DRG cells were co-exposed to both tanespimycin (500 nM) and bortezomib (320 nM–2.5 μM), the inhibitory effects of bortezomib on cell viability and neurite extension were ameliorated. At concentrations as low as 100 nM, tanespimycin completely and reproducibly abrogated the induction of caspase activity at all bortezomib concentrations tested (ie, 0.15–100 μM). When administered alone, bortezomib and tanespimycin had opposite effects on proteasome and calpain activity; bortezomib inhibited all proteasome and calpain activity at concentrations >1 μM, while tanespimycin (100 nM–10 μM) induced calpain and proteasome activity by up to fivefold and threefold, respectively. When DRG cells were exposed to both bortezomib (<1 μM) and tanespimycin, tanespimycin (10 nM–1 μM) induction of the proteasome ameliorated bortezomib proteasome inhibition. Similarly, but to a lesser extent, tanespimycin also overcame the bortezomib-induced inhibition of calpain activity. CONCLUSION:In primary rat DRG cells, tanespimycin prevented bortezomib-induced apoptosis and loss of cell viability in cultures and restored neuronal morphology. These neuroprotective effects of tanespimycin are consistent with the effects observed in rat models of bortezomib-induced PN and with the lack of severe PN observed in patients in the phase 1/2 study of tanespimycin + bortezomib in MM. The mechanism for the protective effect of tanespimycin on bortezomib-induced neuronal toxicity in MM is currently being explored. Disclosures:Flint:Bristol-Myers Squibb: Employment, Equity Ownership. Moulin:Bristol-Myers Squibb: Employment. Oberdoerster:Bristol-Myers Squibb: Employment. Berman:Bristol-Myers Squibb: Employment.