Proteasome Inhibitors against Glioblastoma-Overview of Molecular Mechanisms of Cytotoxicity, Progress in Clinical Trials, and Perspective for Use in Personalized Medicine.

Proteasome inhibitors are moieties targeting the proteolytic activity of a proteasome, with demonstrated efficacy in certain hematological malignancies and candidate drugs in other types of cancer, including glioblastoma (GBM). They disturb the levels of proteasome-regulated proteins and lead to the cell cycle inhibition and apoptosis of GBM cells. The accumulation of cell cycle inhibitors p21 and p27, and decreased levels of prosurvival molecules NFKB, survivin, and MGMT, underlie proteasome inhibitors' cytotoxicity when used alone or in combination with the anti-GBM cytostatic drug temozolomide (TMZ). The evidence gathered in preclinical studies substantiated the design of clinical trials that employed the two most promising proteasome inhibitors, bortezomib and marizomib. The drug safety profile, maximum tolerated dose, and interaction with other drugs were initially evaluated, mainly in recurrent GBM patients. A phase III study on newly diagnosed GBM patients who received marizomib as an adjuvant to the Stupp protocol was designed and completed in 2021, with the Stupp protocol receiving patients as a parallel control arm. The data from this phase III study indicate that marizomib does not improve the PFS and OS of GBM patients; however, further analysis of the genetic and epigenetic background of each patient tumor may shed some light on the sensitivity of individual patients to proteasome inhibition. The mutational and epigenetic makeup of GBM cells, like genetic alterations to TP53 and PTEN, or MGMT promoter methylation levels may actually determine the response to proteasome inhibition.

Proteasome inhibition protects blood-brain barrier P-glycoprotein and lowers Aß brain levels in an Alzheimer's disease model.

BACKGROUND: Loss of P-glycoprotein (P-gp) at the blood-brain barrier contributes to amyloid-ß (Aß) brain accumulation in Alzheimer's disease (AD). Using transgenic human amyloid precursor protein (hAPP)-overexpressing mice (Tg2576), we previously showed that Aß triggers P-gp loss by activating the ubiquitin-proteasome pathway, which leads to P-gp degradation. Furthermore, we showed that inhibiting the ubiquitin-activating enzyme (E1) prevents P-gp loss and lowers Aß accumulation in the brain of hAPP mice. Based on these data, we hypothesized that repurposing the FDA-approved proteasome inhibitor, bortezomib (Velcade®; BTZ), protects blood-brain barrier P-gp from degradation in hAPP mice in vivo. METHODS: We treated hAPP mice with the proteasome inhibitor BTZ or a combination of BTZ with the P-gp inhibitor cyclosporin A (CSA) for 2 weeks. Vehicle-treated wild-type (WT) mice were used as a reference for normal P-gp protein expression and transport activity. In addition, we used the opioid receptor agonist loperamide as a P-gp substrate in tail flick assays to indirectly assess P-gp transport activity at the blood-brain barrier in vivo. We also determined P-gp protein expression by Western blotting, measured P-gp transport activity levels in isolated brain capillaries with live cell confocal imaging and assessed Aß plasma and brain levels with ELISA. RESULTS: We found that 2-week BTZ treatment of hAPP mice restored P-gp protein expression and transport activity in brain capillaries to levels found in WT mice. We also observed that hAPP mice displayed significant loperamide-induced central antinociception compared to WT mice indicating impaired P-gp transport activity at the blood-brain barrier of hAPP mice in vivo. Furthermore, BTZ treatment prevented loperamide-induced antinociception suggesting BTZ protected P-gp loss in hAPP mice. Further, BTZ-treated hAPP mice had lower Aß40 and Aß42 brain levels compared to vehicle-treated hAPP mice. CONCLUSIONS: Our data indicate that BTZ protects P-gp from proteasomal degradation in hAPP mice, which helps to reduce Aß brain levels. Our data suggest that the proteasome system could be exploited for a novel therapeutic strategy in AD, particularly since increasing Aß transport across the blood-brain barrier may prove an effective treatment for patients.

Population Pharmacokinetics and Pharmacodynamics of Carfilzomib in Combination with Rituximab, Ifosfamide, Carboplatin, and Etoposide in Adult Patients with Relapsed/Refractory Diffuse Large B Cell Lymphoma.

BACKGROUND: In patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL), salvage chemotherapy regimens (e.g., rituximab, ifosfamide, carboplatin, and etoposide, R-ICE) yield poor outcomes. Carfilzomib, an irreversible proteasome inhibitor, can overcome acquired rituximab-chemotherapy resistance and, when combined with R-ICE, improves outcomes in patients with R/R DLBCL. OBJECTIVE: This analysis aimed to develop a population pharmacokinetic/pharmacodynamic (PK/PD) model for carfilzomib in R/R DLBCL patients. PATIENTS AND METHODS: In a single-center, open-label, prospective phase 1 study, patients received carfilzomib (10, 15, or 20 mg/m2) on days 1, 2, 8, and 9, and standard doses of R-ICE on days 3-6 every 21 days (maximum of three cycles). Carfilzomib plasma concentrations up to 24 h postinfusion were measured by liquid chromatography coupled with tandem mass spectrometry. Proteasome activity (PD biomarker) in peripheral blood mononuclear cells was assessed on days 1-2 with sparse sampling. PK/PD models were developed using NONMEM v7.4.1 interfaced with Finch Studio v1.1.0 and PsN v4.7.0. Model selection was guided by objective function value, goodness-of-fit, and visual predictive checks. Stepwise covariate modeling was used for covariate selection. RESULTS: Twenty-eight patients were enrolled in the PK/PD analysis, from whom 217 PK samples and 127 PD samples were included. Carfilzomib PK was best described by a two-compartment model with linear disposition (typical total clearance of 133 L/h). Proteasome activity was best characterized using a turnover model with irreversible inactivation. All parameters were estimated with good precision. No statistically significant covariates were identified. CONCLUSIONS: A validated population-based PK/PD model of carfilzomib was developed successfully. Further research is needed to identify sources of variability in response to treatment with carfilzomib in combination with R-ICE. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov identifier number NCT01959698.

Proteasome inhibition in combination with immunotherapies: State-of-the-Art in multiple myeloma.

Multiple myeloma (MM) is a malignant plasma cell disorder accounting for around 1.8% of all neoplastic diseases. Nowadays, clinicians have a broad arsenal of drugs at their disposal for the treatment of MM, such as proteasome inhibitors, immunomodulatory drugs, monoclonal antibodies, bispecific antibodies, CAR T-cell therapies and antibody-drug conjugates. In this paper we briefly highlight essential clinical elements relating to proteasome inhibitors, such as bortezomib, carfilzomib and ixazomib. Studies suggest that the early use of immunotherapy may improve outcomes significantly. Therefore, in our review we specifically focus on the combination therapy of proteasome inhibitors with novel immunotherapies and/or transplant. A high number of patients develop PI resistance. Thus, we also review new generation PIs, such as marizomib, oprozomib (ONX0912) and delanzomib (CEP-18770) and their combinations with immunotherapies.

Selective noncovalent proteasome inhibiting activity of trifluoromethyl-containing gem-quaternary aziridines.

The ubiquitin-proteasome pathway (UPP) represents the principal proteolytic apparatus in the cytosol and nucleus of all eukaryotic cells. Nowadays, proteasome inhibitors (PIs) are well-known as anticancer agents. However, although three of them have been approved by the US Food and Drug Administration (FDA) for treating multiple myeloma and mantel cell lymphoma, they present several side effects and develop resistance. For these reasons, the development of new PIs with better pharmacological characteristics is needed. Recently, noncovalent inhibitors have gained much attention since they are less toxic as compared with covalent ones, providing an alternative mechanism for solid tumors. Herein, we describe a new class of bis-homologated chloromethyl(trifluoromethyl)aziridines as selective noncovalent PIs. In silico and in vitro studies were conducted to elucidate the mechanism of action of such compounds. Human gastrointestinal absorption (HIA) and blood-brain barrier (BBB) penetration were also considered together with absorption, distribution, metabolism, and excretion (ADMET) predictions.

Mechanistic Pharmacokinetic/Pharmacodynamic Modeling in Support of a Patient-Convenient, Longer Dosing Interval for Carfilzomib, a Covalent Inhibitor of the Proteasome.

BACKGROUND: Carfilzomib is an irreversible second-generation proteasome inhibitor that has a short elimination half-life but much longer pharmacodynamic (PD) effect based on its irreversible mechanism of action, making it amenable to longer dosing intervals. A mechanistic pharmacokinetic/pharmacodynamic (PK/PD) model was built using a bottom-up approach, based on the mechanism of action of carfilzomib and the biology of the proteasome, to provide further evidence of the comparability of once-weekly and twice-weekly dosing. METHODS: The model was qualified using clinical data from the phase III ENDEAVOR study, where the safety and efficacy of bortezomib (a reversible proteasome inhibitor) and carfilzomib were compared. Simulations were performed to compare the average proteasome inhibition across five cycles of treatment for the 20/70 mg/m2 once-weekly (70 QW) and 20/56 mg/m2 twice-weekly (56 BIW) regimens. RESULTS: Results indicated that while 70 QW had a higher maximum concentration (Cmax) and lower steady-state area under the concentration-time curve (AUC) than 56 BIW, the average proteasome inhibition after five cycles of treatment between the regimens was comparable. Presumably, the higher Cmax of carfilzomib from 70 QW compensates for the lower overall AUC compared with 56 BIW, and hence 70 QW is expected to have comparable proteasome inhibition, and therefore comparable efficacy, to 56 BIW. The comparable model-predicted proteasome inhibition between 70 QW and 56 BIW also translated to comparable clinical response, in terms of overall response rate and progression-free survival. CONCLUSION: This work provides a framework for which mechanistic PK/PD modeling can be used to guide optimization of dosing intervals for therapeutics with significantly longer PD effects than PK, and help further justify patient-convenient, longer dosing intervals.

[Development of demetia therapeutics regurating synaptic plasticity].

Alzheimer's disease (AD) is the most common dementia in the world characterized by the neuropathological hallmarks consisting of an accumulation of extracellular amyloid-ß (Aß) plaques and intracellular neurofibrillary tangles (NFT). There is no fundamental therapeutic treatment. We have developed a novel AD therapeutic candidate SAK3 which improves neuronal plasticity in the brain. SAK3 enhanced the acetylcholine release via T-type calcium channels. T-type calcium channels is highly expressed in neuro-progenitor cells in the hippocampal dentate gyrus. SAK3 enhanced the proliferation and differentiation of the neuro-progenitor cells, thereby improving depressive behaviors. The Cav3.1 null mice impaired the proliferation and differentiation of the neuro-progenitor cells. In addition, SAK3 activated CaMKII involving neuronal plasticity, thereby improving spine regeneration and proteasome activities impaired in AD related AppNL-F/NL-F knock-in mice. The improvement of the decreased proteasome activity through enhancement CaMKII/Rpt6 signaling by SAK3 treatment contributed to the amelioration of synaptic abnormalities and cognitive decline. The increased proteasome activity also accounted for inhibition of Aß deposition. Taken together, the proteasome activation via enhancement of CaMKII/Rpt6 signaling is a new strategy for AD treatment, which rescues the AD pathology including cognitive impairments and Aß deposition. SAK3 may be a new hopeful drug candidate rescuing dementia patients.

Ligand-based virtual screening, molecular dynamics, and biological evaluation of repurposed drugs as inhibitors of Trypanosoma cruzi proteasome.

Chagas disease is a well-known Neglected Tropical Disease, mostly endemic in continental Latin America, but that has spread to North America and Europe. Unfortunately, current treatments against such disease are ineffective and produce known and undesirable side effects. To find novel effective drug candidates to treat Chagas disease, we uniquely explore the Trypanosoma cruzi proteasome as a recent biological target and, also, apply drug repurposing through different computational methodologies. For this, we initially applied protein homology modeling to build a robust model of proteasome ß4/ß5 subunits, since there is no crystallographic structure of this target. Then, we used it on a drug repurposing via a virtual screening campaign starting with more than 8,000 drugs and including the methodologies: ligand-based similarity, toxicity predictions, and molecular docking. Three drugs were selected concerning their favorable interactions at the protein binding site and subsequently submitted to molecular dynamics simulations, which allowed us to elucidate their behavior and compare such theoretical results with experimental ones, obtained in biological assays also described in this paper.Communicated by Ramaswamy H. Sarma.

A Comprehensive Study on the Anti-cancer Effects of Quercetin and Its Epigenetic Modifications in Arresting Progression of Colon Cancer Cell Proliferation.

Colon cancer etiology involves a wide spectrum of genetic and epigenetic alterations, finding it challenging to find effective therapeutic strategies. Quercetin exhibits potent anti-proliferative/apoptotic properties. In the present study, we aimed to elucidate the anti-cancer and anti-aging effect of quercetin in colon cancer cell lines. The anti-proliferative effect of quercetin was assessed in vitro by CCK-8 in normal and colon cancer cell lines. To check the anti-aging potential of quercetin, collagenase, elastase, and hyaluronidase inhibitory activity assays were performed. The epigenetic and DNA damage assays were performed using the human NAD-dependent deacetylase Sirtuin-6, proteasome 20S, Klotho, Cytochrome-C, and telomerase ELISA kits. Furthermore, the aging-associated miRNA expression profiling was performed on colon cancer cells. The treatment with quercetin inhibited cell proliferation of colon cancer cells in a dose-dependent manner. Quercetin arrested colon cancer cell growth by modulating expression of aging proteins including Sirtuin-6 and Klotho and also by inhibiting telomerase activity to restrict the telomere length which is evident from qPCR analysis. Quercetin also exhibited DNA damage protection by reducing proteasome 20S levels. The miRNA expression profiling results displayed differential expression of miRNA in colon cancer cell, and in addition, the highly upregulated miRNA was involved in the regulation of cell cycle, proliferation, and transcription. Our data suggest that quercetin treatment inhibited cell proliferation in colon cancer cells through regulating the anti-aging protein expression and provides better understanding for quercetin's potential use in colon cancer treatment.

Excessive proteostasis contributes to pathology in fragile X syndrome.

In fragile X syndrome (FX), the leading monogenic cause of autism, excessive neuronal protein synthesis is a core pathophysiology; however, an overall increase in protein expression is not observed. Here, we tested whether excessive protein synthesis drives a compensatory rise in protein degradation that is protective for FX mouse model (Fmr1-/y) neurons. Surprisingly, although we find a significant increase in protein degradation through ubiquitin proteasome system (UPS), this contributes to pathological changes. Normalizing proteasome activity with bortezomib corrects excessive hippocampal protein synthesis and hyperactivation of neurons in the inferior colliculus (IC) in response to auditory stimulation. Moreover, systemic administration of bortezomib significantly reduces the incidence and severity of audiogenic seizures (AGS) in the Fmr1-/y mouse, as does genetic reduction of proteasome, specifically in the IC. Together, these results identify excessive activation of the UPS pathway in Fmr1-/y neurons as a contributor to multiple phenotypes that can be targeted for therapeutic intervention.

Luteolin Attenuates Diabetic Myocardial Hypertrophy by Inhibiting Proteasome Activity.

INTRODUCTION: Luteolin is a flavonoid polyphenolic compound exerting broad pharmacological and medicinal properties. Diabetes-related obesity increases the total blood volume and cardiac output and may increase the myocardial hypertrophy progression. However, the mechanism of luteolin in diabetic myocardial hypertrophy remains uncertain. Therefore, this study aimed to evaluate whether luteolin improved diabetic cardiomyopathy (DCM) by inhibiting the proteasome activity. METHODS: Cardiomyopathy was induced in streptozotocin-treated diabetes mellitus (DM) and db/db mice. Luteolin (20 mg kg-1·day-1) was administrated via gavage for 12 weeks. In vitro, high glucose and high insulin (HGI, glucose at 25.5 mM and insulin at 0.1 µM) inducing primary neonatal rat cardiomyocytes (NRCMs) were treated with or without luteolin for 48 h. Echocardiography, reverse transcription quantitative polymerase chain reaction, histology, immunofluorescence, and Western blotting were conducted. Proteasome activities were also detected using a fluorescent peptide substrate. RESULTS: Luteolin administration significantly prevented the onset of cardiac hypertrophy, fibrosis, and dysfunction in type 1 DM (T1DM) and type 2 DM (T2DM). Compared with DCM mice, luteolin groups showed lower serum triglyceride and total cholesterol levels. Furthermore, luteolin attenuated HGI-induced myocardial hypertrophy and reduced atrial natriuretic factor mRNA level in NRCMs. Proteasome activities were inhibited by luteolin in vitro. Luteolin also reduces the proteasome subunit levels (PSMB) 1, PSMB2, and PSMB5 of the 20S proteasome, as well as proteasome-regulated particles (Rpt) 1 and Rpt4 levels of 19S proteasome. Furthermore, luteolin treatment increased protein kinase B (AKT) and GSK-3α/ß (inactivation of GSK-3) phosphorylation. The phosphorylation level of AMPK activity was also reversed after the treatment with luteolin in comparison with the HGI-treated group. CONCLUSION: This study indicates that luteolin protected against DCM in mice, including T1DM and T2DM, by upregulating phosphorylated protein AMPK and AKT/GSK-3 pathways while decreasing the proteasome activity. These findings suggest that luteolin may be a potential therapeutic agent for DCM.

Identification and validation of novel biomarkers for cold-dampness syndrome of rheumatoid arthritis based on integration of multiple bioinformatics methods / 中国中药杂志

This study aims to identify the novel biomarkers of cold-dampness syndrome(RA-Cold) of rheumatoid arthritis(RA) by gene set enrichment analysis(GSEA), weighted gene correlation network analysis(WGCNA), and clinical validation. Firstly, transcriptome sequencing was carried out for the whole blood samples from RA-Cold patients, RA patients with other traditional Chinese medicine(TCM) syndromes, and healthy volunteers. The differentially expressed gene(DEG) sets of RA-Cold were screened by comparison with the RA patients with other TCM syndromes and healthy volunteers. Then, GSEA and WGCNA were carried out to screen the key DEGs as candidate biomarkers for RA-Cold. Experimentally, the expression levels of the candidate biomarkers were determined by RT-qPCR for an independent clinical cohort(not less than 10 cases/group), and the clinical efficacy of the candidates was assessed using the receiver operating characteristic(ROC) curve. The results showed that 3 601 DEGs associated with RA-Cold were obtained, including 106 up-regulated genes and 3 495 down-regulated genes. The DEGs of RA-Cold were mainly enriched in the pathways associated with inflammation-immunity regulation, hormone regulation, substance and energy metabolism, cell function regulation, and synovial pannus formation. GSEA and WGCNA showed that recombinant proteasome 26S subunit, ATPase 2(PSMC2), which ranked in the top 50% in terms of coefficient of variation, representativeness of pathway, and biological modules, was a candidate biomarker of RA-Cold. Furthermore, the validation results based on the clinical independent sample set showed that the F1 value, specificity, accuracy, and precision of PSMC2 for RA-Cold were 70.3%, 61.9%, 64.5%, and 81.3%, respectively, and the area under the curve(AUC) value was 0.96. In summary, this study employed the "GSEA-WGCNA-validation" integrated strategy to identify novel biomarkers of RA-Cold, which helped to improve the TCM clinical diagnosis and treatment of core syndromes in RA and provided an experimental basis for TCM syndrome differentiation.

[Identification and validation of novel biomarkers for cold-dampness syndrome of rheumatoid arthritis based on integration of multiple bioinformatics methods].

This study aims to identify the novel biomarkers of cold-dampness syndrome(RA-Cold) of rheumatoid arthritis(RA) by gene set enrichment analysis(GSEA), weighted gene correlation network analysis(WGCNA), and clinical validation. Firstly, transcriptome sequencing was carried out for the whole blood samples from RA-Cold patients, RA patients with other traditional Chinese medicine(TCM) syndromes, and healthy volunteers. The differentially expressed gene(DEG) sets of RA-Cold were screened by comparison with the RA patients with other TCM syndromes and healthy volunteers. Then, GSEA and WGCNA were carried out to screen the key DEGs as candidate biomarkers for RA-Cold. Experimentally, the expression levels of the candidate biomarkers were determined by RT-qPCR for an independent clinical cohort(not less than 10 cases/group), and the clinical efficacy of the candidates was assessed using the receiver operating characteristic(ROC) curve. The results showed that 3 601 DEGs associated with RA-Cold were obtained, including 106 up-regulated genes and 3 495 down-regulated genes. The DEGs of RA-Cold were mainly enriched in the pathways associated with inflammation-immunity regulation, hormone regulation, substance and energy metabolism, cell function regulation, and synovial pannus formation. GSEA and WGCNA showed that recombinant proteasome 26S subunit, ATPase 2(PSMC2), which ranked in the top 50% in terms of coefficient of variation, representativeness of pathway, and biological modules, was a candidate biomarker of RA-Cold. Furthermore, the validation results based on the clinical independent sample set showed that the F1 value, specificity, accuracy, and precision of PSMC2 for RA-Cold were 70.3%, 61.9%, 64.5%, and 81.3%, respectively, and the area under the curve(AUC) value was 0.96. In summary, this study employed the "GSEA-WGCNA-validation" integrated strategy to identify novel biomarkers of RA-Cold, which helped to improve the TCM clinical diagnosis and treatment of core syndromes in RA and provided an experimental basis for TCM syndrome differentiation.

Pathologic complete response following low-dose radiation for advanced oral cavity cancer in a patient with human immunodeficiency virus.

BACKGROUND: Advanced squamous cell carcinoma (SCCa) of the oral cavity is often not amenable to curative-intent therapy due to tumor location, tumor size, or comorbidities. CASE PRESENTATION: A 51-year-old male patient with human immunodeficiency virus and on highly active antiretroviral therapy (HAART) presented with a cT4aN2c SCCa of the tongue. He received a preoperative single course of Quad-Shot radiation therapy to 14 Gy in 4 fractions followed by surgical resection. Patient had no residual carcinoma on surgical pathology and no evidence of disease on subsequent clinical and radiological exams. CONCLUSIONS: To our knowledge, this is the first case of pathologic complete response for a patient on HAART following a single cycle of the Quad-Shot regimen for advanced oral cavity SCCa. Protease inhibitors in HAART can induce spontaneous tumor regression via inhibition of proteasome function and activation of apoptosis, and thus act as a cancer therapeutic.

PSMG2-controlled proteasome-autophagy balance mediates the tolerance for MEK-targeted therapy in triple-negative breast cancer.

Although the MAPK pathway is aberrantly activated in triple-negative breast cancers (TNBCs), the clinical outcome of MEK-targeted therapy is still poor. Through a genome-wide CRISPR-Cas9 library screening, we find that inhibition of PSMG2 sensitizes TNBC cells BT549 and MB468 to the MEK inhibitor AZD6244. Mechanistically, PSMG2 knockdown impairs proteasome function, which in turn activates autophagy-mediated PDPK1 degradation. The PDPK1 degradation significantly enhances AZD6244-induced tumor cell growth inhibition by interrupting the negative feedback signals toward the AKT pathway. Consistently, co-targeting proteasomes and MEK with inhibitors synergistically suppresses tumor cell growth. The autophagy inhibitor chloroquine partially relieves the PDPK1 degradation and reverses the growth inhibition induced by combinatorial inhibition of MEK and proteasome. The combination regimen with the proteasome inhibitor MG132 plus AZD6244 synergistically inhibits tumor growth in a 4T1 xenograft mouse model. In summary, our study not only unravels the mechanism of MEK inhibitor resistance but also provides a combinatorial therapeutic strategy for TNBC in clinics.

Study on the Effects of Different Doses of Dahuang Zhechong Pills on the Ubiquitin Proteasome Pathway/Nuclear Factor-κB in Rats with Atherosclerosis and Its Mechanism.

In order to investigate the effects of different doses of Dahuang Zhechong pills on the ubiquitin proteasome pathway/nuclear factor-κB (UPP-NF-κB) in rats with atherosclerosis (AS), 58-week-old male Wistar rats were selected and randomly divided into the normal group, model group, control group, low-dose group, and high-dose group. The model group and the drug group are given intraperitoneal injections of vitamins, and the model group and the drug group are given a high-fat diet. Rats in the low-dose group and high-dose group are given low-dose and high-dose Dahuang Zhechong pill lavage solution, respectively. Besides, the control group is given simvastatin solution by gavage, and intervention is performed once a day for 12 weeks. Ubiquitin (Ub) protein expression, ubiquitin activase (UBE1), nuclear factor-κB, nuclear inhibitory factor-κB (IκB) gene expression, total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and serum tumor necrosis factor-α (TNF-α) are compared. The experimental result shows that Dahuang Zhechong pills can reduce inflammation and prevent and treat AS by blocking the activation of the UPP/NF-κB signaling pathway and can be used as a proteasome inhibitor in the clinical treatment of AS.

CDK4/6 inhibitors downregulate the ubiquitin-conjugating enzymes UBE2C/S/T involved in the ubiquitin-proteasome pathway in ER + breast cancer.

PURPOSE: Despite significant improvement in therapeutic development in the past decades, breast cancer remains a formidable cause of death for women worldwide. The hormone positive subtype (HR(+)) (also known as luminal type) is the most prevalant category of breast cancer, comprising ~70% of patients. The clinical success of the three CDK4/6 inhibitors palbociclib, ribociclib, and abemaciclib has revolutionized the treatment of choice for metastatic HR(+) breast cancer. Accumulating evidence demonstrate that the properties of CDK4/6 inhibitors extend beyond inhibition of the cell cycle, including modulation of immune function, sensitizing PI3K inhibitors, metabolism reprogramming, kinome rewiring, modulation of the proteosome, and many others. The ubiquitin-proteasome pathway (UPP) is a crucial cellular proteolytic system that maintains the homeostasis and turnover of proteins. METHODS: We performed transcriptional profiling of the HR(+) breast cancer cell lines MCF7 and T47D treated with Palbociclib. Differential expressed genes were analyzed for novel pathways enriched. The results were further validated with biochemical assays and with real world clinical database cohorts. RESULTS: We uncovered a novel mechanism that demonstrate the CDK4/6 inhibitors suppress the expression of three ubiquitin conjugating enzymes UBE2C, UBE2S, UBE2T. Further validation in the HR(+) cell lines show that Palbociclib and ribociclib decrease UBE2C at both the mRNA and protein level, but this phenomenon was not shared with abemaciclib. These three E2 enzymes modulate several E3 ubiquitin ligases, including the APC/C complex which plays a role in G1/S progression. We further demonstrate the UBE2C/UBE2T expression levels are associated with breast cancer survival, and HR(+) breast cancer cells demonstrate dependence on the UBE2C. CONCLUSIONS: Our study suggests a novel link between CDK4/6 inhibitor and UPP pathway, adding to the potential mechanisms of their clinical efficacy in cancer.

Exploiting the ubiquitin system in myeloid malignancies. From basic research to drug discovery in MDS and AML.

The ubiquitin-proteasome system is the crucial homeostatic mechanism responsible for the degradation and turnover of proteins. As such, alterations at this level are often associated with oncogenic processes, either through accumulation of undegraded pathway effectors or, conversely, excessive degradation of tumor-suppressing factors. Therefore, investigation of the ubiquitin- proteasome system has gained much attraction in recent years, especially in the context of hematological malignancies, giving rise to efficient therapeutics such as bortezomib for multiple myeloma. Current investigations are now focused on manipulating protein degradation via fine-tuning of the ubiquitination process through inhibition of deubiquitinating enzymes or development of PROTAC systems for stimulation of ubiquitination and protein degradation. On the other hand, the efficiency of Thalidomide derivates in myelodysplastic syndromes (MDS), such as Lenalidomide, acted as the starting point for the development of targeted leukemia-associated protein degradation molecules. These novel molecules display high efficiency in overcoming the limitations of current therapeutic regimens, such as refractory diseases. Therefore, in this manuscript we will address the therapeutic opportunities and strategies based on the ubiquitin-proteasome system, ranging from the modulation of deubiquitinating enzymes and, conversely, describing the potential of modern targeted protein degrading molecules and their progress into clinical implementation.

Targeting an alternate Wilms' tumor antigen 1 peptide bypasses immunoproteasome dependency.

Designing effective antileukemic immunotherapy will require understanding mechanisms underlying tumor control or resistance. Here, we report a mechanism of escape from immunologic targeting in an acute myeloid leukemia (AML) patient, who relapsed 1 year after immunotherapy with engineered T cells expressing a human leukocyte antigen A*02 (HLA-A2)-restricted T cell receptor (TCR) specific for a Wilms' tumor antigen 1 epitope, WT1126-134 (TTCR-C4). Resistance occurred despite persistence of functional therapeutic T cells and continuous expression of WT1 and HLA-A2 by the patient's AML cells. Analysis of the recurrent AML revealed expression of the standard proteasome, but limited expression of the immunoproteasome, specifically the beta subunit 1i (ß1i), which is required for presentation of WT1126-134. An analysis of a second patient treated with TTCR-C4 demonstrated specific loss of AML cells coexpressing ß1i and WT1. To determine whether the WT1 protein continued to be processed and presented in the absence of immunoproteasome processing, we identified and tested a TCR targeting an alternative, HLA-A2-restricted WT137-45 epitope that was generated by immunoproteasome-deficient cells, including WT1-expressing solid tumor lines. T cells expressing this TCR (TTCR37-45) killed the first patients' relapsed AML resistant to WT1126-134 targeting, as well as other primary AML, in vitro. TTCR37-45 controlled solid tumor lines lacking immunoproteasome subunits both in vitro and in an NSG mouse model. As proteasome composition can vary in AML, defining and preferentially targeting these proteasome-independent epitopes may maximize therapeutic efficacy and potentially circumvent AML immune evasion by proteasome-related immunoediting.

Nonselective proteasome inhibitors in multiple myeloma and future perspectives.

INTRODUCTION: Proteasome inhibition can lead to inhibition of tumor cell proliferation, and therefore it constitutes a potential therapeutic anticancer approach especially in the therapeutic algorithm of patients with multiple myeloma. AREAS COVERED: Three different proteasome inhibitors are currently approved, bortezomib, carfilzomib, and ixazomib, and they have been investigated in multiple myeloma and other hematological malignancies. However, these agents lack specificity, since they target both the constitutive proteasome and the immunoproteasome. Targeting the constitutive proteasome is the main reason for side toxicity due to the effect on normal tissues. In contrary, immunoproteasome inhibition may reduce the adverse events while maintaining the therapeutic efficacy. In this review, the authors present the role of the available proteasome inhibitors in myeloma therapeutics and future perspectives of both selective and nonselective proteasome inhibitors. EXPERT OPINION: The available nonselective proteasome inhibitors have changed the therapeutics of multiple myeloma the last 10 years and have significantly improved the clinical outcomes of the patients. Furthermore, selective proteasome inhibitors are now under preclinical investigation and there is hope that their optimization will come with an improved safety profile with at least comparable efficacy.