Inhibition Of Proteasome Activity Research Articles

Bromoditerpenes from the Red Seaweed Sphaerococcus coronopifolius as Potential Cytotoxic Agents and Proteasome Inhibitors and Related Mechanisms of Action.

Seaweeds are a great source of compounds with cytotoxic properties with the potential to be used as anticancer agents. This study evaluated the cytotoxic and proteasome inhibitory activities of 12R-hydroxy-bromosphaerol, 12S-hydroxy-bromosphaerol, and bromosphaerol isolated from Sphaerococcus coronopifolius. The cytotoxicity was evaluated on malignant cell lines (A549, CACO-2, HCT-15, MCF-7, NCI-H226, PC-3, SH-SY5Y, and SK-MEL-28) using the MTT and LDH assays. The ability of compounds to stimulate the production of hydrogen peroxide (H2O2) and to induce mitochondrial dysfunction, the externalization of phosphatidylserine, Caspase-9 activity, and changes in nuclear morphology was also studied on MCF-7 cells. The ability to induce DNA damage was also studied on L929 fibroblasts. The proteasome inhibitory activity was estimated through molecular docking studies. The compounds exhibited IC50 values between 15.35 and 53.34 µM. 12R-hydroxy-bromosphaerol and 12S-hydroxy-bromosphaerol increased the H2O2 levels on MCF-7 cells, and bromosphaerol induced DNA damage on fibroblasts. All compounds promoted a depolarization of mitochondrial membrane potential, Caspase-9 activity, and nuclear condensation and fragmentation. The compounds have been shown to interact with the chymotrypsin-like catalytic site through molecular docking studies; however, only 12S-hydroxy-bromosphaerol evidenced interaction with ALA20 and SER169, key residues of the proteasome catalytic mechanism. Further studies should be outlined to deeply characterize and understand the potential of those bromoditerpenes for anticancer therapeutics.

ISG20L2 suppresses bortezomib antimyeloma activity by attenuating bortezomib binding to PSMB5.

The proteasome inhibitors (PIs) bortezomib and carfilzomib, which target proteasome 20S subunit beta 5 (PSMB5) in cells, are widely used in multiple myeloma (MM) treatment. In this study, we demonstrated the role of interferon-stimulated 20 kDa exonuclease-like 2 (ISG20L2) in MM PI resistance. Gain- and loss-of-function studies showed that ISG20L2 suppressed MM cell sensitivity to PIs in vitro and in vivo. Patients with ISG20L2lo MM had a better response to PIs and a longer overall survival than patients with ISG20L2hi MM. Biotinylated bortezomib pull-down assays showed that ISG20L2 competed with PSMB5 in binding to bortezomib. The surface plasmon resonance assay confirmed the direct binding of bortezomib to ISG20L2. In ISG20L2hi MM cells, ISG20L2 attenuated the binding of bortezomib to PSMB5, resulting in lower inhibition of proteasome activity and therefore less bortezomib-induced cell death. Overall, we identified a potentially novel mechanism by which ISG20L2 conferred bortezomib resistance on MM. The expression of ISG20L2 correlated with MM PI responses and patient treatment outcomes.

The protease DDI2 regulates NRF1 activation in response to cadmium toxicity

SummaryDNA-damage inducible 1 homolog 2 (DDI2) is a protease that activates the transcription factor NRF1. Cellular models have shown that this pathway contributes to cell-stress adaptation, for example, on proteasome inhibition. However, DDI2 physiological function is unknown. Ddi2 Knock-out (KO) mice were embryonic lethal. Therefore, we generated liver-specific Ddi2-KO animals and used comprehensive genetic analysis to identify the molecular pathways regulated by DDI2. Here, we demonstrate that DDI2 contributes to metallothionein (MT) expression in mouse and human hepatocytes at basal and upon cadmium (Cd) exposure. This transcriptional program is dependent on DDI2-mediated NRF1 proteolytic maturation. In contrast, NRF1 homolog NRF2 does not contribute to MT production. Mechanistically, we observed that Cd exposure inhibits proteasome activity, resulting in DDI2-mediated NRF1 proteolytic maturation. In line with these findings, DDI2 deficiency sensitizes cells to Cd toxicity. This study identifies a function for DDI2 that links proteasome homeostasis to heavy metal mediated toxicity.

Cystargolide-based amide and ester Pz analogues as proteasome inhibitors and anti-cancer agents.

A series of cystargolide-based β-lactone analogues containing nitrogen atoms at the Pz portion of the scaffold were prepared and evaluated as proteasome inhibitors, and for their cytotoxicity profile toward several cancer cell lines. Inclusion of one, two or even three nitrogen atoms at the Pz portion of the cystargolide scaffold is well tolerated, producing analogues with low nanomolar proteasome inhibition activity, in many cases superior to carfilzomib. Additionally, analogue 8g, containing an ester and pyrazine group at Pz, was shown to possess significant activity toward RPMI 8226 cells (IC50 = 21 nM) and to be less cytotoxic toward the normal tissue model MCF10A cells than carfilzomib.

A protein-protein interaction map reveals that the Coxiella burnetii effector CirB inhibits host proteasome activity.

Coxiella burnetii is the etiological agent of the zoonotic disease Q fever, which is featured by its ability to replicate in acid vacuoles resembling the lysosomal network. One key virulence determinant of C. burnetii is the Dot/Icm system that transfers more than 150 effector proteins into host cells. These effectors function to construct the lysosome-like compartment permissive for bacterial replication, but the functions of most of these effectors remain elusive. In this study, we used an affinity tag purification mass spectrometry (AP-MS) approach to generate a C. burnetii-human protein-protein interaction (PPI) map involving 53 C. burnetii effectors and 3480 host proteins. This PPI map revealed that the C. burnetii effector CBU0425 (designated CirB) interacts with most subunits of the 20S core proteasome. We found that ectopically expressed CirB inhibits hydrolytic activity of the proteasome. In addition, overexpression of CirB in C. burnetii caused dramatic inhibition of proteasome activity in host cells, while knocking down CirB expression alleviated such inhibitory effects. Moreover, we showed that a region of CirB that spans residues 91–120 binds to the proteasome subunit PSMB5 (beta 5). Finally, PSMB5 knockdown promotes C. burnetii virulence, highlighting the importance of proteasome activity modulation during the course of C. burnetii infection.

PSMB2 knockdown suppressed proteasome activity and cell proliferation, promoted apoptosis, and blocked NRF1 activation in gastric cancer cells.

Proteasome 20S Subunit Beta 2 (PSMB2) has been suggested to play several roles in cancer. However, the role of PSMB2 and its underlying mechanisms in gastric cancer have not been studied. In this study, qRT-PCR was employed to detect the expression of genes that encode for 26s proteasome subunit proteins. PSMB2 expression and its prognostic ability were assessed by collecting patient tissue samples and reviewing the TCGA and Kaplan-Meier Plotter databases. Immunofluorescence and western blotting experiments were performed to evaluate the expression of PSMB2 in human gastric cancer cells and normal gastric epithelial cells. Subsequently, PSMB2 was knocked down in HGC-27 and SNU-1 cells and overexpressed in N-87 and AGS cells. Proteasome activity assays, 5-Ethynyl-2'-deoxyuridine staining, and TUNEL assays were used to assess proteasome activity, cell proliferation, and apoptosis. Tumor xenograft assays were conducted to evaluate PSMB2 function in vivo. Our results showed that a total of 8 genes encoding for the 26s proteasome subunit protein were highly expressed in a variety of gastric cancer cells. Next, PSMB2 was selected as the focus of subsequent studies which showed that PSMB2 was highly expressed in samples of gastric cancer tissue. Furthermore, a review of the TCGA database revealed that a high level of PSMB2 expression was associated with a poor clinical prognosis. Our results indicated that PSMB2 overexpression promoted proteasome activity, cell proliferation, and suppressed the apoptosis of gastric cancer cells, while those effects were reversed by treatment with a proteasome inhibitor (MG132). In contrast, PSMB2 knockdown produced the opposite effects and also blocked NRF1 activation. Moreover, PSMB2 knockdown inhibited tumor growth in vivo, decreased PSMB2 expression and cell proliferation, and promoted apoptosis in tumor tissues. Our findings revealed the role played by PSMB2 in gastric cancer and suggest PSMB2 as a new target molecule for use in diagnosing and treating gastric cancer.

Melatonin inhibits angiotensin II–induced atrial fibrillation through preventing degradation of Ang II Type I Receptor–Associated Protein (ATRAP)

Angiotensin II (Ang II) induced Atrial fibrillation (AF) often accompanied with reduced ATRAP which is a negative modulator of Ang II type 1 receptor (AT1R). Melatonin can protect against AF, but the underlying molecular mechanism remains poorly understood. In this study, Ang II was used to induce AF, and AF inducibility and duration were documented telemetrically. Ang II-infused mice had a higher AF incidence, which was associated with atrial fibrosis, inflammation, and oxidative stress. Melatonin partially inhibited these effects, and enforced expression of siRNA-ATRAP in atria counteracted the beneficial role of melatonin. Specifically, melatonin inhibited expression of Ang II-induced proteasome and immunoproteasome subunits β2, β2i, β5, and β5i as well as their corresponding trypsin-like and chymotrypsin-like activities and blocked ATRAP degradation. In turn, this inhibited AT1R-mediated NF-κB signaling, transforming growth factor (TGF)-β1/Smad signaling in the atria, and thereby affected atrial remodeling and AF. Melatonin receptor inhibition by the chemical inhibitor luzindole partially inhibited the inhibitory effects of melatonin on proteasome activity and also Ang II-induced pathological changes in the atria. Overall, our study demonstrates that melatonin protects against Ang II-induced AF by inhibiting proteasome activity and stabilizing ATRAP expression, and these effects are partially dependent on melatonin receptor activation.

Anti-L1 antibody-bound HPV16 pseudovirus is degraded intracellularly via TRIM21/proteasomal pathway

BackgroundPersistent HPV16 infection is the leading risk factor for developing cervical cancer. Anti-L1 antibodies against HPV16 produced in HPV16 infections play diverse roles in the clearance of virus infection and prevention of persistence. It has been implicated that the cervicovaginal squamous epithelial cells actually express TRIM21 and that some HPV16 particles could escape leaky endosomal compartment into the cytosol and that Fc receptor TRIM21 directly neutralize infection by targeting antibody-opsonized viruses for proteasomal degradation. We explored whether anti-L1 antibody opsonized HPV16 pseudovirus (PsV) entered into the cytosol could be neutralized by TRIM21-mediated activation of a proteasomal pathway to reduce the chance of persistent HPV16 infection.MethodsHPV16 PsV were generated and extracted in HEK 293FT cells co-transfected with pcDNA3.1-eGFP and p16sheLL plasmids according to the standard protocol. The HPV16 PsV with capsid protein L1 was characterized by fluorescence microscopy and western blot, and the HPV16 PsV titer and anti-L1-bound PsV entry efficiency were detected by flow cytometry. The expressions of transcription factors (TF) and cytokines elicited by the TRIM21-activated proteasomal pathway were confirmed by dual-luciferase reporter assay and RT-qPCR. The changes in HPV16 PsV load with or without inhibitors in the infected HEK 293FT cells were determinated by qPCR.ResultsSimultaneous transfection with pcDNA3.1-eGFP and p16sheLL plasmids into the HEK 293FT cells resulted in the self-assembly of HPV16 PsV with capsid protein L1. Both HPV16 PsV and anti-L1-bound HPV16 PsV could infect HEK 293FT cells. Anti-L1-bound PsV up-regulated TRIM21 mediated-activation of proteasome and increased expressions of TF and cytokines in the infected cells where HPV16 PsV load reduced by ~ 1000-fold in the presence of anti-L1 antibody, but inhibition of proteasomal activity increased HPV16 PsV load.ConclusionOur preliminary results indicate that anti-L1 antibody entered with HPV16 PsV into the cells could mediate degradation of HPV16 PsV by TRIM21-activated proteasomal pathway intracellularly, giving anti-capsid protein L1 antibody a role in host defense of persistent HPV16 infection.

Long-term treatment with chloroquine increases lifespan in middle-aged male mice possibly via autophagy modulation, proteasome inhibition and glycogen metabolism.

Previous studies have shown that the polyamine spermidine increased the maximum life span in C. elegans and the median life span in mice. Since spermidine increases autophagy, we asked if treatment with chloroquine, an inhibitor of autophagy, would shorten the lifespan of mice. Recently, chloroquine has intensively been discussed as a treatment option for COVID-19 patients. To rule out unfavorable long-term effects on longevity, we examined the effect of chronic treatment with chloroquine given in the drinking water on the lifespan and organ pathology of male middle-aged NMRI mice. We report that, surprisingly, daily treatment with chloroquine extended the median life span by 11.4% and the maximum life span of the middle-aged male NMRI mice by 11.8%. Subsequent experiments show that the chloroquine-induced lifespan elevation is associated with dose-dependent increase in LC3B-II, a marker of autophagosomes, in the liver and heart that was confirmed by transmission electron microscopy. Quite intriguingly, chloroquine treatment was also associated with a decrease in glycogenolysis in the liver suggesting a compensatory mechanism to provide energy to the cell. Accumulation of autophagosomes was paralleled by an inhibition of proteasome-dependent proteolysis in the liver and the heart as well as with decreased serum levels of insulin growth factor binding protein-3 (IGFBP3), a protein associated with longevity. We propose that inhibition of proteasome activity in conjunction with an increased number of autophagosomes and decreased levels of IGFBP3 might play a central role in lifespan extension by chloroquine in male NMRI mice.

Forskolin Stimulates Estrogen Receptor (ER) α Transcriptional Activity and Protects ER from Degradation by Distinct Mechanisms.

Estradiol action is mediated by estrogen receptors (ERs), a and ß. Estradiol binding initiates ER-mediated transcription and ER degradation, the latter of which occurs via the ubiquitin-proteasome pathway. Inhibition of proteasome activity prevents estradiol-induced ERα degradation and transactivation. In ER-positive GH3 cells (a rat pituitary prolactinoma cell line), forskolin, acting via protein kinase A (PKA), stimulates ERα transcriptional activity without causing degradation, and proteasome inhibition does not block forskolin-stimulated transcription. Forskolin also protects liganded ERα from degradation. In the current study, we first examined ERα and ERβ transcriptional activity in ER-negative HT22 cells and found that forskolin stimulated ERα-, but not ERβ-dependent transcription, through the ligand-binding domain (LBD). We also identified four mutations (L396R, D431Y, Y542F, and K534E/M548V) on the ERα LBD that selectively obliterated the response to forskolin. In GH3 cells, transfected ERα mutants and ERβ were protected from degradation by forskolin. Ubiquitination of ERα and ERβ was increased by forskolin or estradiol. ERα ubiquitination was diminished by a mutated ubiquitin (K48R) that prevents elongation of polyubiquitin chains for targeting the proteasome. Increased ERα ubiquitination was not affected by the deletion of the A/B domain but significantly diminished in the F domain deletion mutant. Our results indicate distinct and novel mechanisms for forskolin stimulation of ERα transcriptional activity and protection from ligand-induced degradation. It also suggests a unique mechanism by which forskolin increases unliganded and liganded ERα and ERβ ubiquitination but uncouples them from proteasome-mediated degradation regardless of their transcriptional responses to forskolin.

Disclosing the antitumour potential of the marine bromoditerpene sphaerococcenol A on distinct cancer cellular models

Nature has revealed to be a key source of innovative anticancer drugs. This study evaluated the antitumour potential of the marine bromoditerpene sphaerococcenol A on different cancer cellular models. Dose-response analyses (0.1–100 µM; 24 h) were accomplished in eight different tumour cell lines (A549, CACO-2, HCT-15, MCF-7, NCI-H226, PC-3, SH-SY5Y, SK-MEL-28). Deeper studies were conducted on MFC-7 cells, namely, determination of hydrogen peroxide (H2O2) levels and evaluation of apoptosis biomarkers (phosphatidylserine membrane translocation, mitochondrial dysfunction, Caspase-9 activity, and DNA changes). The ability of the compound to induce genotoxicity was verified in L929 fibroblasts. Sphaerococcenol A capacity to impact colorectal-cancer stem cells (CSCs) tumourspheres (HT29, HCT116, SW620) was evaluated by determining tumourspheres viability, number, and area, as well as the proteasome inhibitory activity. Sphaerococcenol A hepatoxicity was studied in AML12 hepatocytes. The compound exhibited cytotoxicity in all malignant cell lines (IC50 ranging from 4.5 to 16.6 µM). MCF-7 cells viability loss was accompanied by H2O2 generation, mitochondrial dysfunction, Caspase-9 activation and DNA nuclear morphology changes. Furthermore, the compound displayed the lowest IC50 on HT29-derived tumourspheres (0.70 µM), followed by HCT116 (1.77 µM) and SW620 (2.74 µM), impacting the HT29 tumoursphere formation by reducing their number and area. Finally, the compound displayed low cytotoxicity on AML12 hepatocytes without genotoxicity. Overall, sphaerococcenol A exhibits broad cytotoxic effects on different tumour cells, increasing H2O2 production and apoptosis. It also affects colorectal CSCs-enriched tumoursphere development. These data highlight the relevance to include sphaerococcenol A in further pharmacological studies aiming cancer treatments.

PSMD14 Targeting Triggers Paraptosis in Breast Cancer Cells by Inducing Proteasome Inhibition and Ca2+ Imbalance.

PSMD14, a subunit of the 19S regulatory particles of the 26S proteasome, was recently identified as a potential prognostic marker and therapeutic target in diverse human cancers. Here, we show that the silencing and pharmacological blockade of PSMD14 in MDA-MB 435S breast cancer cells induce paraptosis, a non-apoptotic cell death mode characterized by extensive vacuolation derived from the endoplasmic reticulum (ER) and mitochondria. The PSMD14 inhibitor, capzimin (CZM), inhibits proteasome activity but differs from the 20S proteasome subunit-inhibiting bortezomib (Bz) in that it does not induce aggresome formation or Nrf1 upregulation, which underlie Bz resistance in cancer cells. In addition to proteasome inhibition, the release of Ca2+ from the ER into the cytosol critically contributes to CZM-induced paraptosis. Induction of paraptosis by targeting PSMD14 may provide an attractive therapeutic strategy against cancer cells resistant to proteasome inhibitors or pro-apoptotic drugs.

Metabolism guided optimization of peptidomimetics as non-covalent proteasome inhibitors for cancer treatment

A series of novel non-covalent peptidomimetic proteasome inhibitors possessing bulky group at the C-terminus and N-alkylation at the N-terminus were designed with the aim to increase metabolic stabilities in vivo. All the target compounds were screened for their inhibitory activities against human 20S proteasome, and most analogs exhibited notable potency compared with the positive control bortezomib with IC50 values lower than 10 nM, which also displayed potent cytotoxic activities against multiple myeloma (MM) cell lines and human acute myeloid leukemia (AML) cells. Furthermore, whole blood stability and in vivo proteasome inhibitory activity experiments of selected compounds were conducted for further evaluation, and the representative compound 43 (IC50 = 8.39 ± 2.32 nM, RPMI-8226: IC50 = 15.290 ± 2.281 nM, MM-1S: IC50 = 9.067 ± 3.103 nM, MV-4-11: IC50 = 2.464 ± 0.713 nM) revealed a half-life extension of greater than 9-fold (329.21 min VS 36.79 min) and potent proteasome inhibitory activity in vivo. The positive results confirmed the reliability of the metabolism guided optimization strategy, and the analogs discovered are potential leads for exploring new anti-MM drugs.

Angiotensin II Induces Cardiac Edema and Hypertrophic Remodeling through Lymphatic-Dependent Mechanisms.

Cardiac lymphatic vessel growth (lymphangiogenesis) and integrity play an essential role in maintaining tissue fluid balance. Inhibition of lymphatic lymphangiogenesis is involved in cardiac edema and cardiac remodeling after ischemic injury or pressure overload. However, whether lymphatic vessel integrity is disrupted during angiotensin II- (Ang II-) induced cardiac remodeling remains to be investigated. In this study, cardiac remodeling models were established by Ang II (1000 ng/kg/min) in VEGFR-3 knockdown (Lyve-1Cre VEGFR-3f/−) and wild-type (VEGFR-3f/f) littermates. Our results indicated that Ang II infusion not only induced cardiac lymphangiogenesis and upregulation of VEGF-C and VEGFR-3 expression in the time-dependent manner but also enhanced proteasome activity, MKP5 and VE-cadherin degradation, p38 MAPK activation, and lymphatic vessel hyperpermeability. Moreover, VEGFR-3 knockdown significantly inhibited cardiac lymphangiogenesis in mice, resulting in exacerbation of tissue edema, hypertrophy, fibrosis superoxide production, inflammation, and heart failure (HF). Conversely, administration of epoxomicin (a selective proteasome inhibitor) markedly mitigated Ang II-induced cardiac edema, remodeling, and dysfunction; upregulated MKP5 and VE-cadherin expression; inactivated p38 MAPK; and reduced lymphatic vessel hyperpermeability in WT mice, indicating that inhibition of proteasome activity is required to maintain lymphatic endothelial cell (LEC) integrity. Our results show that both cardiac lymphangiogenesis and lymphatic barrier hyperpermeability are implicated in Ang II-induced adaptive hypertrophic remodeling and dysfunction. Proteasome-mediated hyperpermeability of LEC junctions plays a predominant role in the development of cardiac remodeling. Selective stimulation of lymphangiogenesis or inhibition of proteasome activity may be a potential therapeutic option for treating hypertension-induced cardiac remodeling.

Activation of the ubiquitin-proteasome system contributes to oculopharyngeal muscular dystrophy through muscle atrophy.

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset disorder characterized by progressive weakness and degeneration of specific muscles. OPMD is due to extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). Aggregation of the mutant protein in muscle nuclei is a hallmark of the disease. Previous transcriptomic analyses revealed the consistent deregulation of the ubiquitin-proteasome system (UPS) in OPMD animal models and patients, suggesting a role of this deregulation in OPMD pathogenesis. Subsequent studies proposed that UPS contribution to OPMD involved PABPN1 aggregation. Here, we use a Drosophila model of OPMD to address the functional importance of UPS deregulation in OPMD. Through genome-wide and targeted genetic screens we identify a large number of UPS components that are involved in OPMD. Half dosage of UPS genes reduces OPMD muscle defects suggesting a pathological increase of UPS activity in the disease. Quantification of proteasome activity confirms stronger activity in OPMD muscles, associated with degradation of myofibrillar proteins. Importantly, improvement of muscle structure and function in the presence of UPS mutants does not correlate with the levels of PABPN1 aggregation, but is linked to decreased degradation of muscle proteins. Oral treatment with the proteasome inhibitor MG132 is beneficial to the OPMD Drosophila model, improving muscle function although PABPN1 aggregation is enhanced. This functional study reveals the importance of increased UPS activity that underlies muscle atrophy in OPMD. It also provides a proof-of-concept that inhibitors of proteasome activity might be an attractive pharmacological approach for OPMD.

Immune-mediated neuropathy related to bortezomib in a patient with multiple myeloma

Treatment options in multiple myeloma (MM) based on novel agents are often limited by dose-related neurotoxicity. Bortezomib, a highly active reversible proteasome inhibitor, frequently causes peripheral neuropathy (PN). Bortezomib-induced PN (BIPN) is characterized by a length-dependent, sensory, axonal polyneuropathy (PNP) with predominant small fiber-affection. Following dose reduction or drug discontinuation, BIPN resolves within 3-4 months in the majority of patients. The pathophysiological mechanisms of BIPN are unclear. Rare cases of a severe demyelinating or mixed BIPN with prominent motor involvement have been attributed to autoimmune or inflammatory reactions. A case report, including nerve pathology, is presented of a 59-year-old man with stage III IgG-κ MM who was treated with bortezomib on the occurrence of progressive disease. After the fourth cycle, he developed a painful distal symmetric sensory PNP followed by gait instability and muscle weakness increasing over 3 months despite early cessation of bortezomib. Neurological examination revealed a distal flaccid tetraparesis mainly of the lower limbs with sensory loss and severe ataxia, electrophysiological features of a mixed axonal-demyelinating PNP, and pathomorphological evidence of neuritis. Steroid treatment was initiated, and partial recovery of the neurological symptoms within 6 months was observed. While a neurotoxic effect may explain the initial distal sensory disturbances, the worsening of neurological dysfunction after bortezomib withdrawal and the clinical pattern with steroid-responsive muscle weakness predominantly of the legs are consistent with an immune-mediated mechanism. This is in line with the sural nerve biopsy findings. Toxic BIPN followed by an immune-mediated BIPN in the same patient has not been reported before.

A personalized molecular approach in multiple myeloma: the possible use of RAF/RAS/MEK/ERK and BCL-2 inhibitors.

Multiple myeloma (MM) is a blood cancer that derives from plasma cells (PCs), which will accumulate in the bone marrow (BM). Over time, several drugs have been developed to treat this disease that is still uncurable. The therapies used to treat the disease target immune activity, inhibit proteasome activity, and involve the use of monoclonal antibodies. However, MM is a highly heterogeneous disease, in fact, there are several mutations in signaling pathways that are particularly important for MM cell biology and that are possible therapeutic targets. Indeed, some studies suggest that MM is driven by mutations within the rat sarcoma virus (RAS) signaling cascade, which regulates cell survival and proliferation. The RAS/proto-oncogene, serine/threonine kinase (RAF)/mitogen-activated extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK signaling pathway is deregulated in several cancers, for which drugs have been developed to inhibit these pathways. In addition to the signaling pathways, the disease implements mechanisms to ensure the survival and consequently a high replicative capacity. This strategy consists in the deregulation of apoptosis. In particular, some cases of MM show overexpression of anti-apoptotic proteins belonging to the B cell lymphoma 2 (BCL-2) family that represent a possible druggable target. Venetoclax is an anti-BCL-2 molecule used in hematological malignancies that may be used in selected MM patients based on their molecular profile. We focused on the possible effects in MM of off-label drugs that are currently used for other cancers with the same molecular characteristics. Their use, combined with the current treatments, could be a good strategy against MM.

Abstract 10240: Key Pathomechanisms of Cardiomyopathy Due to TTN Truncation

Introduction: Heterozygous truncating variants in TTN (TTNtv) coding for titin cause dilated cardiomyopathy (DCM) but the underlying pathomechanisms are unclear and disease management remains uncertain. Here, we aim to elucidate the key pathomechanisms of TTNtv-DCM. Hypothesis: Stable expression of truncated titin proteins and titin haploinsufficiency characterize TTNtv-DCM and represent potential targets for therapy. Methods and Results: We studied left ventricular tissues from 14 nonfailing donor hearts and 113 endstage failing DCM patients and identified a TTNtv in 22 DCM patients (19.5%) by next generation sequencing. Using titin protein gel electrophoresis and western blot we demonstrate, for the first time, titin haploinsufficiency in TTNtv-DCM hearts. Strikingly, all 21 adult TTNtv-DCM hearts of our cohort showed stable expression of truncated titin proteins. Expression was variable, up to one-half of the total titin protein pool, and negatively correlated to patient age at heart transplantation. Truncated titin proteins were not detected in sarcomeres but were present in intracellular aggregates. Deregulated ubiquitin-dependent protein quality control was apparent. Next, we produced human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), comparing wildtype controls to cells with a patient-derived, prototypical A-band TTNtv or a CRISPR/Cas9-edited M-band TTNtv. TTNtv hiPSC-CMs showed reduced wildtype titin expression and contained truncated titin proteins, whose proportion increased upon inhibition of proteasomal activity. In engineered heart muscle generated from hiPSC-CMs, depressed contractility caused by TTNtv could be reversed by correction of the mutation using CRISPR/Cas9, thereby eliminating truncated titin proteins and raising wildtype titin content. Functional improvement also occurred when titin protein content was increased by proteasome inhibition. Conclusions: Our study reveals the major pathomechanisms of TTNtv-DCM, which include titin haploinsufficiency as a life-long condition and truncated titin protein enrichment with aggregate formation, along with aberrant protein quality control. Results can be exploited for new therapies of TTNtv cardiomyopathies.

Anti-Multiple Myeloma Potential of Secondary Metabolites from Hibiscus sabdariffa-Part 2.

Multiple Myeloma (MM) is an aggressive tumor causing millions of deaths every year and currently available therapies are often unsuccessful or correlated with severe side effects. In our previous work we demonstrated that the Hibiscus sabdariffa hydroalcoholic extract inhibits the growth of the MM cell line and we isolated two metabolites responsible for the activity: Hib-ester and Hib-carbaldehyde. Herein we report their interaction with proteasome, one of the main targets in the fight against MM. The molecular modelling study outlined a good interaction of both compounds with the target and these results prompted us to investigate their potential to inhibit proteasome. Metabolites were then isolated from the calyces and an extract with a high content of Hib-ester and Hib-carbaldehyde was prepared. An anticancer profile was drawn, evaluating apoptosis, autophagy and proteasome inhibition, with the anticancer properties being mainly attributed to the Hib-ester and Hib-carbaldehyde, while the proteasome inhibition of the extract could also be ascribed to the presence of anthocyanins, a class of secondary metabolites already known for their proteasome inhibitory activity.

High Immunoproteasome Activity and sXBP1 in Pediatric Precursor B-ALL Predicts Sensitivity towards Proteasome Inhibitors.

Proteasome inhibitors (PIs) are approved backbone treatments in multiple myeloma. More recently, inhibition of proteasome activity with the PI bortezomib has been clinically evaluated as a novel treatment strategy in pediatric acute lymphoblastic leukemia (ALL). However, we lack a marker that could identify ALL patients responding to PI-based therapy. By using a set of activity-based proteasome probes in conjunction with cytotoxicity assays, we show that B-cell precursor ALL (BCP-ALL), in contrast to T-ALL, demonstrates an increased activity of immunoproteasome over constitutive proteasome, which correlates with high ex vivo sensitivity to the PIs bortezomib and ixazomib. The novel selective PI LU015i-targeting immunoproteasome β5i induces cytotoxicity in BCP-ALL containing high β5i activity, confirming immunoproteasome activity as a novel therapeutic target in BCP-ALL. At the same time, cotreatment with β2-selective proteasome inhibitors can sensitize T-ALL to currently available PIs, as well as to β5i selective PI. In addition, levels of total and spliced forms of XBP1 differ between BCP-ALL and T-ALL, and only in BCP-ALL does high-spliced XBP1 correlate with sensitivity to bortezomib. Thus, in BCP-ALL, high immunoproteasome activity may serve as a predictive marker for PI-based treatment options, potentially combined with XBP1 analyses.