CaMKII suppresses proteotoxicity by phosphorylating BAG3 in response to proteasomal dysfunction.

Protein quality control serves as the primary defense mechanism for cells against proteotoxicity induced by proteasome dysfunction. While cells can limit the build-up of ubiquitinated misfolded proteins during proteasome inhibition, the precise mechanism is unclear. Here, we find that protein kinase Ca2+/Calmodulin (CaM)-dependent protein kinase II (CaMKII) maintains proteostasis during proteasome inhibition. We show that proteasome inhibition activates CaMKII, which phosphorylates B-cell lymphoma 2 (Bcl-2)-associated athanogene 3 (BAG3) at residues S173, S377, and S386. Phosphorylated BAG3 activates the heme-regulated inhibitor (HRI)- eukaryotic initiation factor-2α (eIF2α) signaling pathway, suppressing protein synthesis and the production of aggregated ubiquitinated misfolded proteins, ultimately mitigating the proteotoxic crisis. Inhibition of CaMKII exacerbates the accumulation of aggregated misfolded proteins and paraptosis induced by proteasome inhibitors. Based on these findings, we validate that combined targeting of proteasome and CaMKII accelerates tumor cell death and enhances the efficacy of proteasome inhibitors in tumor treatment. Our data unveil a new proteasomal inhibition-induced misfolded protein quality control mechanism and propose a novel therapeutic intervention for proteasome inhibitor-mediated tumor treatment.

The Proteasome and Cul3-Dependent Protein Ubiquitination Is Required for Gli Protein-Mediated Activation of Gene Expression in the Hedgehog Pathway.

Many cellular processes are regulated by proteasome-mediated protein degradation, including regulation of signaling pathways and gene expression. Among the pathways regulated by the ubiquitin-proteasome system is the Hedgehog pathway and its downstream effectors, the Gli transcription factors. Here we provide evidence that proteasomal activity is necessary for maintaining the activation of the Hedgehog pathway, and this crucial event takes place at the level of Gli proteins. We undertook extensive work to demonstrate the specificity of the observed phenomenon by ruling out the involvement of primary cilium, impaired nuclear import, failed dissociation from Sufu, microtubule stabilization, and stabilization of Gli repressor forms. Moreover, we showed that proteasomal-inhibition-mediated Hedgehog pathway downregulation is not restricted to the NIH-3T3 cell line. We demonstrated, using CRISPR/Ca9 mutagenesis, that neither Gli1, Gli2, nor Gli3 are solely responsible for the Hedgehog pathway downregulation upon proteasome inhibitor treatment, and that Cul3 KO renders the same phenotype. Finally, we report two novel E3 ubiquitin ligases, Btbd9 and Kctd3, known Cul3 interactors, as positive Hedgehog pathway regulators. Our data pave the way for a better understanding of the regulation of gene expression and the Hedgehog signaling pathway.

Marizomib (Salinosporamide A) Promotes Apoptosis in A375 and G361 Melanoma Cancer Cells.

Malignant melanoma-a tumor originating from melanocytes-is characterized by dynamic growth and frequent metastases in the early stage of development. Current therapy methods are still insufficient, and there is a need to search for new ways of treating this malady. The induction of apoptosis-physiological cell death-by proteasome inhibitors is recognized as an effective method of non-invasive elimination of cancer cells. In our research, we wanted to check the potential of marizomib (MZB, salinosporamide A, NPI-0052)-an irreversible proteasome inhibitor derived from the marine actinomycete Salinispora tropica-to induce apoptosis in A375 and G361 malignant melanoma cells. We determined the cytotoxic activity of marizomib by performing an MTT test. Ethidium bromide and acridine orange staining demonstrated the disruption of membrane integrity in the examined cell lines. We confirmed the proapoptotic activity of marizomib by flow cytometry with the use of an FITC-Annexin V assay. A Western blot analysis presented an increase in the expression of proteins related to endoplasmic reticulum (ER) stress as well as markers of the apoptosis. The gathered findings suggest that marizomib induced the ER stress in the examined melanoma cancer cells and directed them towards the apoptosis pathway.

Proteasome inhibitors induce apoptosis by superoxide anion generation via NADPH oxidase 5 in human neuroblastoma SH-SY5Y cells.

The ubiquitin-proteasome system (UPS) is a major proteolytic system that plays an important role in the regulation of various cell processes, such as cell cycle, stress response, and transcriptional regulation, especially in neurons, and dysfunction of UPS is considered to be a cause of neuronal cell death in neurodegenerative diseases. However, the mechanism of neuronal cell death caused by UPS dysfunction has not yet been fully elucidated. In this study, we investigated the mechanism of neuronal cell death induced by proteasome inhibitors using human neuroblastoma SH-SY5Y cells. Z-Leu-D-Leu-Leu-al (MG132), a proteasome inhibitor, induced apoptosis in SH-SY5Y cells in a concentration- and time-dependent manner. Antioxidants N-acetylcysteine and EUK-8 attenuated MG132-induced apoptosis. Apocynin and diphenyleneiodonium, inhibitors of NADPH oxidase (NOX), an enzyme that produces superoxide anions, also attenuated MG132-induced apoptosis. It was also found that MG132 treatment increased the expression of NOX5, a NOX family member, and that siRNA-mediated silencing of NOX5 and BAPTA-AM, which inhibits NOX5 by chelating calcium, suppressed MG132-induced apoptosis and production of reactive oxygen species in SH-SY5Y cells. These results suggest that MG132 induces apoptosis in SH-SY5Y cells through the production of superoxide anion by NOX5.

TRAIL-induced apoptosis and proteasomal activity - Mechanisms, signalling and interplay.

Programmed cell death, in particular apoptosis, is essential during development and tissue homeostasis, and also is the primary strategy to induce cancer cell death by cytotoxic therapies. Precision therapeutics targeting TRAIL death receptors are being evaluated as novel anti-cancer agents, while in parallel highly specific proteasome inhibitors have gained approval as drugs. TRAIL-dependent signalling and proteasomal control of cellular proteostasis are intricate processes, and their interplay can be exploited to enhance therapeutic killing of cancer cells in combination therapies. This review provides detailed insights into the complex signalling of TRAIL-induced pathways and the activities of the proteasome. It explores their core mechanisms of action, pharmaceutical druggability, and describes how their interplay can be strategically leveraged to enhance cell death responses in cancer cells. Offering this comprehensive and timely overview will allow to navigate the complexity of the processes governing cell death mechanisms in TRAIL- and proteasome inhibitor-based treatment conditions.

Iron and copper: critical executioners of ferroptosis, cuproptosis and other forms of cell death.

Regulated cell death (RCD) is a regulable cell death that involves well-organized signaling cascades and molecular mechanisms. RCD is implicated in fundamental processes such as organ production and tissue remodeling, removing superfluous structures or cells, and regulating cell numbers. Previous studies have not been able to reveal the complete mechanisms, and novel methods of RCD are constantly being proposed. Two metal ions, iron (Fe) and copper (Cu) are essential factors leading to RCDs that not only induce ferroptosis and cuproptosis, respectively but also lead to cell impairment and eventually diverse cell death. This review summarizes the direct and indirect mechanisms by which Fe and Cu impede cell growth and the various forms of RCD mediated by these two metals. Moreover, we aimed to delineate the interrelationships between these RCDs with the distinct pathways of ferroptosis and cuproptosis, shedding light on the complex and intricate mechanisms that govern cellular survival and death. Finally, the prospects outlined in this review suggest a novel approach for investigating cell death, which may involve integrating current therapeutic strategies and offer a promising solution to overcome drug resistance in certain diseases. Video Abstract.

Inhibition of SQSTM1 S403 phosphorylation facilitates the aggresome formation of ubiquitinated proteins during proteasome dysfunction.

BACKGROUND: Ubiquitin-proteasome-system-mediated clearance of misfolded proteins is essential for cells to maintain proteostasis and reduce the proteotoxicity caused by these aberrant proteins. When proteasome activity is inadequate, ubiquitinated proteins are sorted into perinuclear aggresomes, which is a significant defense mechanism employed by cells to combat insufficient proteasome activity, hence mitigating the proteotoxic crisis. It has been demonstrated that phosphorylation of SQSTM1 is crucial in regulating misfolded protein aggregation and autophagic degradation. Although SQSTM1 S403 phosphorylation is essential for the autophagic degradation of ubiquitinated proteins, its significance in proteasome inhibition-induced aggresome formation is yet unknown. Herein, we investigated the influence of SQSTM1 S403 phosphorylation on the aggresome production of ubiquitinated proteins during proteasome suppression. METHODS: We examined the phosphorylation levels of SQSTM1 S403 or T269/S272 in cells after treated with proteasome inhibitors or/and autophagy inhibitors, by western blot and immunofluorescence. We detected the accumulation and aggresome formation of ubiquitinated misfolded proteins in cells treated with proteasome inhibition by western blot and immunofluorescence. Furthermore, we used SQSTM1 phosphorylation-associated kinase inhibitors and mutant constructs to confirm the regulation of different SQSTM1 phosphorylation in aggresome formation. We examined the cell viability using CCK-8 assay. RESULTS: Herein, we ascertained that phosphorylation of SQSTM1 S403 did not enhance the autophagic degradation of ubiquitinated proteins during proteasome inhibition. Proteasome inhibition suppresses the phosphorylation of SQSTM1 S403, which facilitated the aggresome production of polyubiquitinated proteins. Interestingly, we found proteasome inhibition-induced SQSTM1 T269/S272 phosphorylation inhibits the S403 phosphorylation. Suppressing S403 phosphorylation rescues the defective aggresome formation and protects cells from cell death caused by unphosphorylated SQSTM1 (T269/S272). CONCLUSIONS: This study shows that inhibition of SQSTM1 S403 phosphorylation facilitates the aggresome formation of ubiquitinated proteins during proteasome dysfunction. SQSTM1 T269/S272 phosphorylation inhibits the S403 phosphorylation, boosting the aggresome formation of ubiquitinated protein and shielding cells from proteotoxic crisis.

The prognostic value of 19S ATPase proteasome subunits in acute myeloid leukemia and other forms of cancer.

Introduction: The ubiquitin-proteasome system (UPS) is an intracellular organelle responsible for targeted protein degradation, which represents a standard therapeutic target for many different human malignancies. Bortezomib, a reversible inhibitor of chymotrypsin-like proteasome activity, was first approved by the FDA in 2003 to treat multiple myeloma and is now used to treat a number of different cancers, including relapsed mantle cell lymphoma, diffuse large B-cell lymphoma, colorectal cancer, and thyroid carcinoma. Despite the success, bortezomib and other proteasome inhibitors are subject to severe side effects, and ultimately, drug resistance. We recently reported an oncogenic role for non-ATPase members of the 19S proteasome in chronic myeloid leukemia (CML), acute myeloid leukemia (AML), and several different solid tumors. In the present study, we hypothesized that ATPase members of the 19S proteasome would also serve as biomarkers and putative therapeutic targets in AML and multiple other cancers. Methods: We used data from The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC) available at UALCAN and/or GEPIA2 to assess the expression and prognostic value of proteasome 26S subunit, ATPases 1-6 (PSMC1-6) of the 19S proteasome in cancer. UALCAN was also used to associate PSMC1-6 mRNA expression with distinct clinicopathological features. Finally, cBioPortal was employed to assess genomic alterations of PSMC genes across different cancer types. Results: The mRNA and protein expression of PSMC1-6 of the 19S proteasome were elevated in several cancers compared with normal controls, which often correlated with worse overall survival. In contrast, AML patients demonstrated reduced expression of these proteasome subunits compared with normal mononuclear cells. However, AML patients with high expression of PSMC2-5 had worse outcomes. Discussion: Altogether, our data suggest that components of the 19S proteasome could serve as prognostic biomarkers and novel therapeutic targets in AML and several other human malignancies.

Methylome Response to Proteasome Inhibition by Pseudomonas syringae Virulence Factor Syringolin A.

DNA methylation is an important epigenetic mark required for proper gene expression and silencing of transposable elements. DNA methylation patterns can be modified by environmental factors such as pathogen infection, in which modification of DNA methylation can be associated with plant resistance. To counter the plant defense pathways, pathogens produce effector molecules, several of which act as proteasome inhibitors. Here, we investigated the effect of proteasome inhibition by the bacterial virulence factor syringolin A (SylA) on genome-wide DNA methylation. We show that SylA treatment results in an increase of DNA methylation at centromeric and pericentromeric regions of Arabidopsis chromosomes. We identify several CHH differentially methylated regions (DMRs) that are enriched in the proximity of transcriptional start sites. SylA treatment does not result in significant changes in small RNA composition. However, significant changes in genome transcriptional activity can be observed, including a strong upregulation of resistance genes that are located on chromosomal arms. We hypothesize that DNA methylation changes could be linked to the upregulation of some atypical members of the de novo DNA methylation pathway, namely AGO3, AGO9, and DRM1. Our data suggests that modification of genome-wide DNA methylation resulting from an inhibition of the proteasome by bacterial effectors could be part of an epi-genomic arms race against pathogens. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Chloroquine and Hydroxychloroquine, as Proteasome Inhibitors, Upregulate the Expression and Activity of Organic Anion Transporter 3.

Organic anion transporter 3 (OAT3), at the basolateral membrane of kidney proximal tubule cells, facilitates the elimination of numerous widely used drugs. Earlier investigation from our laboratory revealed that ubiquitin conjugation to OAT3 leads to OAT3 internalization from the cell surface, followed by degradation in the proteasome. In the current study, we examined the roles of chloroquine (CQ) and hydroxychloroquine (HCQ), two well-known anti-malarial drugs, in their action as proteasome inhibitors and their effects on OAT3 ubiquitination, expression, and function. We showed that in cells treated with CQ and HCQ, the ubiquitinated OAT3 was considerably enhanced, which correlated well with a decrease in 20S proteasome activity. Furthermore, in CQ- and HCQ-treated cells, OAT3 expression and OAT3-mediated transport of estrone sulfate, a prototypical substrate, were significantly increased. Such increases in OAT3 expression and transport activity were accompanied by an increase in the maximum transport velocity and a decrease in the degradation rate of the transporter. In conclusion, this study unveiled a novel role of CQ and HCQ in enhancing OAT3 expression and transport activity by preventing the degradation of ubiquitinated OAT3 in proteasomes.

Proteasome inhibition reduces plasma cell and antibody secretion, but not angiotensin II-induced hypertension.

Introduction: Depletion of mature B cells affords protection against experimental hypertension. However, whether B cell-mediated hypertension is dependent on differentiation into antibody-secreting cells (ASCs) remains unclear. Using the proteasome inhibitor, bortezomib, the present study tested the effect of ASC reduction on angiotensin II-induced hypertension. Methods: Male C57BL6/J mice were infused with angiotensin II (0.7 mg/kg/day; s.c.) for 28 days via osmotic minipump to induce hypertension. Normotensive control mice received saline infusion. Bortezomib (750 µg/kg) or vehicle (0.1% DMSO) was administered (i.v.) 3 days prior to minipump implantation, and twice weekly thereafter. Systolic blood pressure was measured weekly using tail-cuff plethysmography. Spleen and bone marrow B1 (CD19+B220-), B2 (B220+CD19+) and ASCs (CD138hiSca-1+Blimp-1+) were enumerated by flow cytometry. Serum immunoglobulins were quantified using a bead-based immunoassay. Results: Bortezomib treatment reduced splenic ASCs by ∼68% and ∼64% compared to vehicle treatment in normotensive (2.00 ± 0.30 vs. 0.64 ± 0.15 × 105 cells; n = 10-11) and hypertensive mice (0.52 ± 0.11 vs. 0.14 ± 0.02 × 105 cells; n = 9-11), respectively. Bone marrow ASCs were also reduced by bortezomib in both normotensive (4.75 ± 1.53 vs. 1.71 ± 0.41 × 103 cells; n = 9-11) and hypertensive mice (4.12 ± 0.82 vs. 0.89 ± 0.18 × 103 cells; n = 9-11). Consistent with ASC reductions, bortezomib reduced serum IgM and IgG2a in all mice. Despite these reductions in ASCs and antibody levels, bortezomib did not affect angiotensin II-induced hypertension over 28 days (vehicle: 182 ± 4 mmHg vs. bortezomib: 177 ± 7 mmHg; n = 9-11). Conclusion: Reductions in ASCs and circulating IgG2a and IgM did not ameliorate experimental hypertension, suggesting other immunoglobulin isotypes or B cell effector functions may promote angiotensin II-induced hypertension.

Cardiovascular Toxicity of Proteasome Inhibitors: Underlying Mechanisms and Management Strategies: JACC: CardioOncology State-of-the-Art Review.

Proteasome inhibitors (PIs) are the backbone of combination treatments for patients with multiple myeloma and AL amyloidosis, while also indicated in Waldenström's macroglobulinemia and other malignancies. PIs act on proteasome peptidases, causing proteome instability due to accumulating aggregated, unfolded, and/or damaged polypeptides; sustained proteome instability then induces cell cycle arrest and/or apoptosis. Carfilzomib, an intravenous irreversible PI, exhibits a more severe cardiovascular toxicity profile as compared with the orally administered ixazomib or intravenous reversible PI such as bortezomib. Cardiovascular toxicity includes heart failure, hypertension, arrhythmias, and acute coronary syndromes. Because PIs are critical components of the treatment of hematological malignancies and amyloidosis, managing their cardiovascular toxicity involves identifying patients at risk, diagnosing toxicity early at the preclinical level, and offering cardioprotection if needed. Future research is required to elucidate underlying mechanisms, improve risk stratification, define the optimal management strategy, and develop new PIs with safe cardiovascular profiles.

PEGylated Liposomes Loaded with Carbamate Inhibitor ANP0903 Trigger Apoptosis by Enhancing ER Stress in HepG2 Cancer Cells.

Liver cancer is one of the most common causes of cancer death worldwide. In recent years, substantial progress has been made in the development of systemic therapies, but there is still the need for new drugs and technologies that can increase the survival and quality of life of patients. The present investigation reports the development of a liposomal formulation of a carbamate molecule, reported as ANP0903, previously tested as an inhibitor of HIV-1 protease and now evaluated for its ability to induce cytotoxicity in hepatocellular carcinoma cell lines. PEGylated liposomes were prepared and characterized. Small, oligolamellar vesicles were produced, as demonstrated by light scattering results and TEM images. The physical stability of the vesicles in biological fluids was demonstrated in vitro, alongside the stability during storage. An enhanced cellular uptake was verified in HepG2 cells treated with liposomal ANP0903, resulting in a greater cytotoxicity. Several biological assays were performed to elucidate the molecular mechanisms explaining the proapoptotic effect of ANP0903. Our results allow us to hypothesize that the cytotoxic action in tumor cells is probably due to the inhibition of the proteasome, resulting in an increase in the amount of ubiquitinated proteins within the cells, which in turn triggers activation of autophagy and apoptosis processes, resulting in cell death. The proposed liposomal formulation represents a promising approach to deliver a novel antitumor agent to cancer cells and enhance its activity.

Case report: Preclinical efficacy of NEDD8 and proteasome inhibitors in patient-derived models of signet ring high-grade mucinous colorectal cancer from a Lynch syndrome patient.

High-grade mucinous colorectal cancer (HGM CRC) is particularly aggressive, prone to metastasis and treatment resistance, frequently accompanied by "signet ring" cancer cells. A sizeable fraction of HGM CRCs (20-40%) arises in the context of the Lynch Syndrome, an autosomal hereditary syndrome that predisposes to microsatellite instable (MSI) CRC. Development of patient-derived preclinical models for this challenging subtype of colorectal cancer represents an unmet need in oncology. We describe here successful propagation of preclinical models from a case of early-onset, MSI-positive metastatic colorectal cancer in a male Lynch syndrome patient, refractory to standard care (FOLFOX6, FOLFIRI-Panitumumab) and, surprisingly, also to immunotherapy. Surgical material from a debulking operation was implanted in NOD/SCID mice, successfully yielding one patient-derived xenograft (PDX). PDX explants were subsequently used to generate 2D and 3D cell cultures. Histologically, all models resembled the tumor of origin, displaying a high-grade mucinous phenotype with signet ring cells. For preclinical exploration of alternative treatments, in light of recent findings, we considered inhibition of the proteasome by bortezomib and of the related NEDD8 pathway by pevonedistat. Indeed, sensitivity to bortezomib was observed in mucinous adenocarcinoma of the lung, and we previously found that HGM CRC is preferentially sensitive to pevonedistat in models with low or absent expression of cadherin 17 (CDH17), a differentiation marker. We therefore performed IHC on the tumor and models, and observed no CDH17 expression, suggesting sensitivity to pevonedistat. Both bortezomib and pevonedistat showed strong activity on 2D cells at 72 hours and on 3D organoids at 7 days, thus providing valid options for in vivo testing. Accordingly, three PDX cohorts were treated for four weeks, respectively with vehicle, bortezomib and pevonedistat. Both drugs significantly reduced tumor growth, as compared to the vehicle group. Interestingly, while bortezomib was more effective in vitro, pevonedistat was more effective in vivo. Drug efficacy was further substantiated by a reduction of cellularity and of Ki67-positive cells in the treated tumors. These results highlight proteasome and NEDD8 inhibition as potentially effective therapeutic approaches against Lynch syndrome-associated HGM CRC, also when the disease is refractory to all available treatment options.

Thrombospondin-1 induction and VEGF reduction by proteasome inhibition.

The present study focuses on investigating the expression of thrombospondin-1 (TSP-1), a natural inhibitor of neovascularization. Immunofluorescent staining was used to detect the expression of TSP-1 in rabbit corneal tissue with vascularization induced by limbectomy. TSP-1 was detected in healthy and Cultured Autologous Oral Mucosal Epithelial Cell Sheet (CAOMECS) grafted rabbit corneas. TSP-1 was not detected in diseased corneas. Rabbit and human primary oral mucosal and corneal epithelial cells were cultured and treated with proteasome inhibitor (PI) in vitro. Changes in the expression of TSP-1, HIF-1 alpha and 2 alpha, VEGF-A, and VEGF receptor were analyzed by Western blotting. Neovascularization developed in rabbits' corneas as early as 1 month after limbectomy and was stable for at least 3 months. HIF-1 alpha and VEGF-A expression was reduced in CAOMECS grafted corneas, as compared to sham corneas. While TSP-1 expression was decreased in injured corneas, it was expressed in CAOMECS grafted corneas, but still less expressed compared to healthy corneas. PI treatment, of human oral mucosal and corneal epithelial cells increased TSP-1 expression and reduced VEGF-A expression. The results showed that TSP-1 expression was lost in injured corneal surface and that CAOMECS grafting restored TSP-1 expression to certain extent. Proteasome inhibition treatment increased TSP-1 and decreased VEGF-A expression in human oral mucosal and corneal epithelial cells. The result suggests that corneal neovascularization could be managed with the inhibition of the proteasome after CAOMECS grafting and increase corneal transparency.

Dynamic Metabolic and Transcriptional Responses of Proteasome-Inhibited Neurons.

Proteasome inhibition is associated with parkinsonian pathology in vivo and degeneration of dopaminergic neurons in vitro. We explored here the metabolome (386 metabolites) and transcriptome (3257 transcripts) regulations of human LUHMES neurons, following exposure to MG-132 [100 nM]. This proteasome inhibitor killed cells within 24 h but did not reduce viability for 12 h. Overall, 206 metabolites were changed in live neurons. The early (3 h) metabolome changes suggested a compromised energy metabolism. For instance, AMP, NADH and lactate were up-regulated, while glycolytic and citric acid cycle intermediates were down-regulated. At later time points, glutathione-related metabolites were up-regulated, most likely by an early oxidative stress response and activation of NRF2/ATF4 target genes. The transcriptome pattern confirmed proteostatic stress (fast up-regulation of proteasome subunits) and also suggested the progressive activation of additional stress response pathways. The early ones (e.g., HIF-1, NF-kB, HSF-1) can be considered a cytoprotective cellular counter-regulation, which maintained cell viability. For instance, a very strong up-regulation of AIFM2 (=FSP1) may have prevented fast ferroptotic death. For most of the initial period, a definite life-death decision was not taken, as neurons could be rescued for at least 10 h after the start of proteasome inhibition. Late responses involved p53 activation and catabolic processes such as a loss of pyrimidine synthesis intermediates. We interpret this as a phase of co-occurrence of protective and maladaptive cellular changes. Altogether, this combined metabolomics-transcriptomics analysis informs on responses triggered in neurons by proteasome dysfunction that may be targeted by novel therapeutic intervention in Parkinson's disease.

Dinuclear and mononuclear metal(II) polypyridyl complexes against drug-sensitive and drug-resistant Plasmodium falciparum and their mode of action.

BACKGROUND: Malaria remains one of the most virulent and deadliest parasitic disease in the world, particularly in Africa and Southeast Asia. Widespread occurrence of artemisinin-resistant Plasmodium falciparum strains from the Greater Mekong Subregion is alarming. This hinders the national economies, as well as being a major drawback in the effective control and elimination of malaria worldwide. Clearly, an effective anti-malarial drug is urgently needed. METHODS: The dinuclear and mononuclear copper(II) and zinc(II) complexes were synthesized in ethanolic solution and characterized by various physical measurements (FTIR, CHN elemental analysis, solubility, ESI-MS, UV-Visible, conductivity and magnetic moment, and NMR). X-ray crystal structure of the dicopper(II) complex was determined. The in vitro haemolytic activities of these metal complexes were evaluated spectroscopically on B+ blood while the anti-malarial potency was performed in vitro on blood stage drug-sensitive Plasmodium falciparum 3D7 (Pf3D7) and artemisinin-resistant Plasmodium falciparum IPC5202 (Pf5202) with fluorescence dye. Mode of action of metal complexes were conducted to determine the formation of reactive oxygen species using PNDA and DCFH-DA dyes, JC-1 depolarization of mitochondrial membrane potential, malarial 20S proteasome inhibition with parasite lysate, and morphological studies using Giemsa and Hoechst stains. RESULTS: Copper(II) complexes showed anti-malarial potency against both Pf3D7 and Pf5202 in sub-micromolar to micromolar range. The zinc(II) complexes were effective against Pf3D7 with excellent therapeutic index but encountered total resistance against Pf5202. Among the four, the dinuclear copper(II) complex was the most potent against both strains. The zinc(II) complexes caused no haemolysis of RBC while copper(II) complexes induced increased haemolysis with increasing concentration. Further mechanistic studies of both copper(II) complexes on both Pf3D7 and Pf5202 strains showed induction of ROS, 20S malarial proteasome inhibition, loss of mitochondrial membrane potential and morphological features indicative of apoptosis. CONCLUSION: The dinuclear [Cu(phen)-4,4'-bipy-Cu(phen)](NO3)4 is highly potent and can overcome the total drug-resistance of Pf5202 towards chloroquine and artemisinin. The other three copper(II) and zinc(II) complexes were only effective towards the drug-sensitive Pf3D7, with the latter causing no haemolysis of RBC. Their mode of action involves multiple targets.

19S Proteasome Subunits as Oncogenes and Prognostic Biomarkers in FLT3-Mutated Acute Myeloid Leukemia (AML).

26S proteasome non-ATPase subunits 1 (PSMD1) and 3 (PSMD3) were recently identified as prognostic biomarkers and potential therapeutic targets in chronic myeloid leukemia (CML) and multiple solid tumors. In the present study, we analyzed the expression of 19S proteasome subunits in acute myeloid leukemia (AML) patients with mutations in the FMS-like tyrosine kinase 3 (FLT3) gene and assessed their impact on overall survival (OS). High levels of PSMD3 but not PSMD1 expression correlated with a worse OS in FLT3-mutated AML. Consistent with an oncogenic role for PSMD3 in AML, shRNA-mediated PSMD3 knockdown impaired colony formation of FLT3+ AML cell lines, which correlated with increased OS in xenograft models. While PSMD3 regulated nuclear factor-kappa B (NF-κB) transcriptional activity in CML, we did not observe similar effects in FLT3+ AML cells. Rather, proteomics analyses suggested a role for PSMD3 in neutrophil degranulation and energy metabolism. Finally, we identified additional PSMD subunits that are upregulated in AML patients with mutated versus wild-type FLT3, which correlated with worse outcomes. These findings suggest that different components of the 19S regulatory complex of the 26S proteasome can have indications for OS and may serve as prognostic biomarkers in AML and other types of cancers.

Selinexor inhibits growth of patient derived chordomas in vivo as a single agent and in combination with abemaciclib through diverse mechanisms.

Chordoma is a rare cancer that grows in the base of the skull and along the mobile spine from remnants of embryonic notochord tissue. The cornerstone of current treatments is surgical excision with adjuvant radiation therapy, although complete surgical removal is not always possible. Chordomas have high rates of metastasis and recurrence, with no approved targeted agents. Selinexor and eltanexor are selective inhibitors of nuclear export (SINE) that prevent the karyopherin protein exportin-1 (XPO1) from shuttling its cargo proteins through nuclear pore complexes out of the nucleus and into the cytoplasm. As cancer cells overexpress XPO1, and many of its cargos include tumor suppressor proteins and complexes bound to oncogene mRNAs, XPO1 inhibition can suppress oncogene translation and restore tumor suppressor protein activity in different cancer types. SINE compounds have exhibited anti-cancer activity in a wide range of hematological and solid tumor malignancies. Here we demonstrate the preclinical effectiveness of SINE compounds used as single agents or in combination with either the proteasome inhibitor, bortezomib, or the CDK4/6 inhibitor, abemaciclib, against various patient- derived xenograft (PDX) mouse models of chordoma, which included clival and sacral chordomas from adult or pediatric patients with either primary or metastatic disease, with either differentiated or poorly differentiated subtypes. SINE treatment significantly impaired tumor growth in all five tested chordoma models, with the selinexor and abemaciclib combination showing the strongest activity (tumor growth inhibition of 78-92%). Immunohistochemistry analysis of excised tumors revealed that selinexor treatment resulted in marked induction of apoptosis and reduced cell proliferation, as well as nuclear accumulation of SMAD4, and reduction of Brachyury and YAP1. RNA sequencing showed selinexor treatment resulted in differences in activated and repressed signaling pathways between the PDX models, including changes in WNT signaling, E2F pathways and glucocorticoid receptor signaling. This is consistent with SINE-compound mediated XPO1 inhibition exhibiting anti-cancer activity through a broad range of different mechanisms in different molecular chordoma subsets. Our findings validate the need for further investigation into selinexor as a targeted therapeutic for chordoma, especially in combination with abemaciclib.

Targeting autophagy increases the efficacy of proteasome inhibitor treatment in multiple myeloma by induction of apoptosis and activation of JNK.

BACKGROUND: The therapeutic armamentarium in multiple myeloma has been significantly broadened by proteasome inhibitors, highly efficient means in controlling of multiple myeloma. Despite the developments of therapeutic regimen in treatment of multiple myeloma, still the complete remission requires a novel therapeutic strategy with significant difference in outcomes. Proteasome inhibitors induce autophagy and ER stress, both pivotal pathways for protein homeostasis. Recent studies showed that the IRE1α-XBP1 axis of the unfolded protein response (UPR) is up-regulated in multiple myeloma patients. In addition, XBP1 is crucial for the maintenance of viability of acute lymphoblastic leukemia (ALL). RESULTS: We analyzed the efficacy of targeting IRE1α-XBP1 axis and autophagy in combination with proteasome inhibitor, ixazomib in treatment of multiple myeloma. In this present study, we first show that targeting the IRE1α-XBP1 axis with small molecule inhibitors (STF-083010, A106) together with the ixazomib induces cell cycle arrest with an additive cytotoxic effect in multiple myeloma. Further, we examined the efficacy of autophagy inhibitors (bafilomycin A, BAF and chloroquine, CQ) together with ixazomib in multiple myeloma and observed that this combination treatment synergistically reduced cell viability in multiple myeloma cell lines (viable cells Ixa: 51.8 ± 3.3, Ixa + BAF: 18.3 ± 7.2, Ixa + CQ: 38.4 ± 3.7) and patient-derived multiple myeloma cells (Ixa: 59.6 ± 4.4, Ixa + CQ: 7.0 ± 2.1). We observed, however, that this combined strategy leads to activation of stress-induced c-Jun N-terminal kinase (JNK). Cytotoxicity mediated by combined proteasome and autophagy inhibition was reversed by addition of the specific JNK inhibitor JNK-In-8 (viable cells: Ixa + BAF: 11.6 ± 7.0, Ixa + BAF + JNK-In-8: 30.9 ± 6.1). CONCLUSION: In this study we showed that combined inhibition of autophagy and the proteasome synergistically induces cell death in multiple myeloma. Hence, we consider the implication of pharmaceutical inhibition of autophagy together with proteasome inhibition and UPR-directed therapy as promising novel in vitro treatment strategy against multiple myeloma.