ALBERT

Description: Due to early implementation of public health measures, Greece had low number of SARS-CoV-2 infections and COVID-19 severe incidents in hospitalized patients. The National and Kapodistrian University of Athens (ΝΚUA), especially its health-care/medical personnel, has been actively involved in the first line of state responses to COVID-19. To estimate the prevalence of antibodies (Igs) against SARS-CoV-2 among NKUA members, we designed a five consecutive monthly serosurvey among randomly selected NKUA consenting volunteers. Here, we present the results from the first 2500 plasma samples collected during June–July 2020. Twenty-five donors were tested positive for anti-SARS-CoV-2 Igs; thus, the overall seroprevalence was 1.00%. The weighted overall seroprevalence was 0.93% (95% CI: 0.27, 2.09) and varied between males [1.05% (95% CI: 0.18, 2.92)] and females [0.84% (95% CI: 0.13, 2.49)], age-groups and different categories (higher in participants from the School of Health Sciences and in scientific affiliates/faculty members/laboratory assistants), but no statistical differences were detected. Although focused on the specific population of NKUA members, our study shows that the prevalence of anti-SARS-CoV-2 Igs for the period June–July 2020 remained low and provides knowledge of public health importance for the NKUA members. Given that approximately one in three infections was asymptomatic, continuous monitoring of the progression of the pandemic by assessing Ig seroprevalence is needed.

Description: Chemical study of the CH2Cl2−MeOH (1:1) extract from the sponge Haliclona sp. collected in Mayotte highlighted three new long-chain highly oxygenated polyacetylenes, osirisynes G-I (1–3) together with the known osirisynes A (4), B (5), and E (6). Their structures were elucidated by 1D and 2D NMR spectra and HRESIMS and MS/MS data. All compounds were evaluated on catalase and sirtuin 1 activation and on CDK7, proteasome, Fyn kinase, tyrosinase, and elastase inhibition. Five compounds (1; 3–6) inhibited proteasome kinase and two compounds (5–6) inhibited CDK7 and Fyn kinase. Osirisyne B (5) was the most active compound with IC50 on FYNB kinase, CDK7 kinase, and proteasome inhibition of 18.44 µM, 9.13 µM, and 0.26 µM, respectively.

Description: Organisms require efficient surveillance of proteome functionality to prevent disruption of proteostasis. Central to the proteostasis ensuring network is the proteasome, which degrades both normal short-lived ubiquitinated proteins and damaged or mutated proteins. Over-activation of the proteasome seems to represent a hallmark of advanced tumors and thus, its selective inhibition provides a strategy for the development of novel anti-tumor therapies. This approach is applied in multiple myeloma (MM) that represents the second most common hematological malignancy. Specifically, proteasome inhibitors have demonstrated clinical efficacy in the treatment of MM and mantle cell lymphoma and are evaluated for the treatment of other malignancies. Nevertheless, the impact of proteasome dysfunction in normal human tissues (which relates to side effects in the clinic) remains poorly understood. By using the fruit fly Drosophila melanogaster as an in vivo experimental platform to study proteasome physiology we found that proteasome functionality is sex-, tissue- and age-dependent. Oral administration of proteasome inhibitors (e.g. Bortezomib or Carfilzomib) in young flies suppressed proteasome activities in the somatic tissues; reduced motor function (recapitulating peripheral neuropathy of Bortezomib treatment in the clinic) and caused premature aging. It also increased oxidative stress and activated an Nrf2-dependent feedback regulatory circuit that upregulated proteasome genes in order to restore normal proteasome functionality. Moreover, in line with observations in the clinic, Carfilzomib was found to cause milder (as compared to Bortezomib) neuromusculatory toxicity and reduction of flies' lifespan. To address the question whether these findings can be translated in humans we started characterizing proteasome physiology in both healthy donors, as well as in MM patients treated with therapeutic proteasome inhibitors. For our studies we used isolated red blood cells (RBCs; represent an anucleate relatively "long-lived" proteome) and peripheral blood mononucleated cells (PBMCs; represent cell lineages with active genomic responses). Our analyses in healthy donors of different ages revealed significant variability of basal proteasome peptidase activities in both cell types. PBMCs expressed (as compared to RBCs) higher basal proteasome activities and RBCs from females had higher chymotrypsin-like activity as compared to RBCs from males of similar age. Furthermore, as in the flies' somatic tissues, proteasome activities were found (independently of sex and cell type) to decline during aging. Studies in RBCs and PBMCs isolated from MM patients treated with Bortezomib revealed donor-, cell type- and drug-specific readouts. In most (but not all) cases proteasome activities were suppressed in both cell types at 24-hrs post-drug administration. RBCs were particularly sensitive to the inhibitor and their proteasome activities remained low during the entire course of treatment. On the contrary, PBMCs were characterized by phases of rebound proteasome activities during the periods of no drug administration; these phases correlated with upregulation of proteasome genes expression, indicating that the feedback regulatory circuit which functions to restore proteasome activities in flies is also operational in humans. Additional gene expression analyses in PBMCs showed that proteasome inhibition also triggers the induction of genes involved in chaperon, autophagy, unfolded protein- and antioxidant-responses pathways; while, as in the fly model, the intensity of genes induction seems to decline during aging. Interestingly, in those patients who (despite treatment) showed no reduction of proteasome activities we found marginal gene expression alterations, suggesting that the observed gene induction largely depends on proteasome loss of function. Importantly, at the clinical level we observed a positive correlation between the degree of proteasome inhibition (in PBMCs or RBCS) and the depth of disease responses. The similarities between the Drosophila pharmacological model and the MM patients indicate that the molecular responses to proteasome malfunction are largely conserved in higher metazoans. We foresee that our ongoing studies will support a more personalized clinical therapeutic approach in hematological malignancies. Disclosures Terpos: Amgen: Honoraria, Other: Travel expenses, Research Funding; Takeda: Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses; Novartis: Honoraria; Celgene: Honoraria, Other: Travel expenses. Dimopoulos:Celgene: Honoraria; Onyx: Honoraria; Novartis: Honoraria; Genesis: Honoraria; Janssen-Cilag: Honoraria; Janssen: Honoraria; Amgen: Honoraria.

Description: Carfilzomib (Cfz), an irreversible proteasome inhibitor licensed for relapsed/refractory myeloma, is associated with cardiotoxicity in humans. We sought to establish the optimal protocol of Cfz-induced cardiac dysfunction, to investigate the underlying molecular-signaling and, based on the findings, to evaluate the cardioprotective potency of metformin (Met). Mice were randomized into protocols 1 and 2 (control and Cfz for 1 and 2 consecutive days, respectively); protocols 3 and 4 (control and alternate doses of Cfz for 6 and 14 days, respectively); protocols 5A and 5B (control and Cfz, intermittent doses on days 0, 1 [5A] and 0, 1, 7, and 8 [5B] for 13 days); protocols 6A and 6B (pharmacological intervention; control, Cfz, Cfz+Met and Met for 2 and 6 days, respectively); and protocol 7 (bortezomib). Cfz was administered at 8 mg/kg (IP) and Met at 140 mg/kg (per os). Cfz resulted in significant reduction of proteasomal activity in heart and peripheral blood mononuclear cells in all protocols except protocols 5A and 5B. Echocardiography demonstrated that Cfz led to a significant fractional shortening (FS) depression in protocols 2 and 3, a borderline dysfunction in protocols 1 and 4, and had no detrimental effect on protocols 5A and 5B. Molecular analysis revealed that Cfz inhibited AMPKα/mTORC1 pathways derived from increased PP2A activity in protocol 2, whereas it additionally inhibited phosphatidylinositol 3-kinase/Akt/endothelial nitric oxide synthase pathway in protocol 3. Coadministration of Met prevented Cfz-induced FS reduction and restored AMPKα phosphorylation and autophagic signaling. Conclusively, Cfz decreased left ventricular function through increased PP2A activity and inhibition of AMPKα and its downstream autophagic targets, whereas Met represents a novel promising intervention against Cfz-induced cardiotoxicity.

Description: Cellular proteostasis (homeostasis of the proteome) is ensured by the efficient maintenance of proteome quality and functionality. Central to the proteostasis process is the proteasome, which is involved in the degradation of both normal short-lived ubiquitinated proteins and mutated or damaged proteins. Recent findings indicate that the over-activation of the proteostasis ensuring mechanisms represents a hallmark of advanced tumors, and thus their inhibition provides a strategy for the development of novel anti-tumor therapies. In line with this concept, proteasome inhibitors (i.e. bortezomib and carfilzomib) have demonstrated clinical efficacy in the treatment of multiple myeloma (MM) and mantle cell lymphoma and are under evaluation for the treatment of other malignancies. By using the fruit fly Drosophila melanogaster as an in vivo platform for screening the effects of proteasome inhibitors at the whole organism level we recently reported that proteasome functionality in flies’ somatic tissues is sex- and tissue- dependent and that it declines with aging. Administration of bortezomib to young flies caused dose-dependent decrease of proteasome activities in the somatic tissues; induction of the proteasome genes expression, disruption of proteostasis, reduced motor function (a phenotype that recapitulates peripheral neuropathy of bortezomib treatment in the clinic) and a marked reduction of flies’ lifespan. Our in vivo data also showed that carfilzomib was less toxic compared to bortezomib, including neuromusculatory toxicity and effects on flies’ longevity. To address the question whether these findings can be translated to humans we started characterizing proteasome regulation and functionality in both healthy donors, as well as in MM patients treated with either bortezomib or carfilzomib. Initially, we screened isolated red blood cells (RBCs) and peripheral blood mononucleated cells (PBMCs) from male and female healthy donors of different ages; these two cell types represent either an anucleate relatively “long-lived” proteome (RBCs) or cell lineages with the capacity to mobilize genome responses after proteasome inhibition (PBMCs). Our analyses revealed significant variability of basal proteasome peptidase activities among different donors in both RBCs and PBMCs. PBMCs expressed (independently of sex) higher basal proteasome activities as compared to RBCs. Moreover, RBCs isolated from female donors had elevated (as compared to males’ RBCs) basal chymotrypsin-like activity; whereas, males’ PBMCs exhibited higher trypsin-like and caspase-like enzymatic activities as compared to PBMCs from females of similar age. In line with our observations in flies’ somatic tissues, we also found that the proteasome peptidase activities decrease significantly during aging (in a sex-independent manner) in both RBCs and PBMCs. Our studies in isolated RBCs and PBMCs from MM patients treated with either bortezomib or carfilzomib revealed drug-, donor- and cell type-specific readouts. Specifically, in most cases proteasome activities were suppressed in both RBCs and PBMCs after drug administration. Also, we noted that RBCs were particularly sensitive to both inhibitors and their proteasome activities remained low during the entire course of treatment. On the other hand, PBMCs were characterized by phases of relapsed proteasome activities during the periods of no drug administration. Finally, as in the case of the in vivo Drosophila experimental model, proteasome dysfunction in PBMCs triggered in most patients a significant upregulation of the proteasome 20S and 19S genes expression. Moreover, we noted an induction of genes involved in cellular antioxidant responses; this finding is in line with our observations in flies showing that administration of proteasome inhibitors results in increased cellular oxidative stress that mobilizes genomic antioxidant responses. Data on the clinical outcomes of the treated patients in correlation with the recorded molecular responses will be presented at the meeting. Overall, our findings indicate that the molecular-cellular responses to proteasome inhibitors observed at the in vivo Drosophila Experimental model are largely translatable to humans. Moreover, we anticipate that our employed methodologies will set the basis towards a more personalized clinical therapeutic approach for multiple myeloma patients. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Carfilzomib (Cfz) significantly prolongs progression-free survival in relapsed or refractory multiple myeloma patients, as highlighted in the ENDEAVOR trial. However, Cfz has high incidences of cardiotoxicity and heart failure, leading to treatment cessation. Thus, there is an imperative need for preventive therapies. The study aimed to i) establish an in vivo Cfz cardiotoxicity protocol, ii) investigate the molecular mechanism, identify molecular targets and iii) based on initial results, investigate the potential protective effect and mechanism of Metformin (Met). Methods: Male, C57BL/6 mice, were randomized in groups as following: Acute protocol (6 days): Control (n=7), Cfz (n=8); Sub-chronic protocol (14 days): Control (n=5), Cfz (n=8); Pharmacological intervention protocol (6 days): Control (n=8), Cfz (n=8), Cfz+Met (n=8), Met (n=4). Cfz (8 mg/kg, ip) was administered on alternate days and Met (140 mg/kg, po) daily. Glucose levels were monitored following Met administration. Mice underwent echocardiography on baseline and at the end of treatments. Blood and myocardial tissue samples were obtained for histology, proteasome activity, PP2A activity and signaling pathways focused on PI3K/Akt/eNOS axis, NO homeostasis and AMPKα-mTOR-mediated autophagy. Results: Following acute administration, echocardiography in Cfz group presented a significant reduction in fractional shortening (FS%) vs. Control group (39.87±0.47 vs. 43.03±0.50 respectively, p

Description: Carfilzomib (CFZ) is a proteasome inhibitor associated with cardiovascular (CV) adverse events (AEs) mainly hypertension (HTN) and cardiac dysfunction. Endothelial dysfunction is a major mediating mechanism in cardiovascular diseases and endothelial function may be adversely affected by proteasome inhibition. However, CFZ effects on endothelial and vascular function and associations with inhibition of proteasome activity have not been explored in humans. We prospectively evaluated CFZ effects on endothelial function and underlying mechanisms as well as baseline vascular function and its response to treatment as potential predictors of CFZ-associated CV toxicity. In this prospective study (NCT03543579), 48 relapsed/refractory myeloma patients (median 1 (1-3) lines of therapy, median age: 67.5, 67% men) received Kd [CFZ 20/56 mg/m2 and dexamethasone] in routine practice. A detailed medical history and evaluation of risk factors for cardiotoxicity [age≥65 years, obesity, smoking, HTN, hypercholesterolemia, diabetes mellitus, previous anthracyclin use, chest or mediastinum radiotherapy and current myocardial disease] were recorded. Before CFZ start and at prespecified timepoints cardiac echo, hemodynamic parameters and vascular function that reflect pivotal mechanisms involved in development of HTN and CV events were non-invasively assessed [aortic blood pressure and arterial wave reflections, aortic stiffness and endothelial function using flow-mediated dilatation of the brachial artery (FMD)] along with 24h ambulatory blood pressure monitoring. Proteasome activity (PrA) was measured in peripheral blood mononuclear cells (PBMC) before and 2 hours post CFZ infusion on days 1 (FMDbaseD1 and FMDpostCFZD1) and 2 (FMDbaseD2 and FMDpostCFZD2) of cycle 1 and at prespecified time points concurrently with vascular function assessment. CV AEs were recorded and rated according to CTCAE v4.03. The prevalence of risk factors for cardiotoxicity was high (median 3 factors, IQR 2-4). After first CFZ dose, FMD decreased acutely at 2 hours (FMDbaseD1: 5.2%, IQR: 3.2-7.4 vs FMDpostCFZD1: 3.6%, IQR: 1.5-4.7, p=0.008), which partially recovered before and after second CFZ infusion (FMDbaseD2: 4.1% and FMDpostCFZD2: 4%, as compared to FMDbaseD1) (Figure1A). However, FMD decrease was more pronounced among patients who subsequently had lower recovery rate of PBMC PrA 24 hours after first CFZ administration (FMDbaseD1: 5.1%, vs FMDpostCFZD1: 3%, p=0.002) while FMD was not decreased significantly in those who had higher recovery rates of PrA (FMDbaseD1: 5.3%, vs FMDpost CFZD1: 4.5%, p=0.197, Figure 1B), suggesting that the ability to recover PrA is implicated in CFZ induced endothelial dysfunction. At the time of analysis, enrolment has completed; 35 patients are still receiving therapy, 13 have discontinued Kd and median follow up is 4.73 months. CV AEs were recorded in 17 (35.4%) patients [HTN(Gr3): 20.8%, Gr3 Left Ventricular dysfunction : 8.3%, Gr3 acute coronary syndrome: 4.2%, Gr3 pulmonary embolism: 2.1%] and generally occurred early (median time to CV event 2.9 months). Kd was discontinued in 2 patients (4.2%) due to cardiotoxicity and in 2 patients (4.2%) CFZ dose was reduced due to cardiotoxicity; no CFZ dose reduction or discontinuation was needed for HTN. Among clinical and hemodynamic parameters assessed at baseline, patients with higher aortic SBP (i.e at higher quartile) had a higher risk of HTN even after adjustment for age, gender and baseline HTN (HR=8, 95%CI 2.4-26, p=0.001 and HR=4.9, 95%CI 1.5-15, p=0.007 respectively). Lower PrA recovery rate was associated with increased risk of developing Gr3 HTN (log-rank test p=0.01). Higher post-CFZ reduction of FMD was associated with HTN in patients not receiving statins but not in those receiving statins (a drug class strongly affecting FMD), indicating that statin treatment may exert protective effects against CFZ-related CV toxicity. In conclusion, CFZ causes acute endothelial dysfunction; however, higher recovery of proteasome activity is associated with ameliorated depression of endothelial function. Given that poor proteasome recovery rate and acute deterioration of endothelial function are both associated with development of HTN, these findings may improve our understanding of CFZ-related CV toxicity and its prevention. The study is ongoing and more data will be presented at the meeting. Disclosures Kastritis: Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Prothena: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Terpos:Amgen Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel grant, steering committee member, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: member of DMC, Research Funding; Genesis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel grant, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: member of steering committee, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel grant, Research Funding; BMS: Consultancy; Novartis: Consultancy. Dimopoulos:Bristol-Myers Squibb: Honoraria; Amgen: Honoraria; Takeda: Honoraria; Celgene: Honoraria; Janssen: Honoraria.

Description: Introduction: Carfilzomib is a second-generation irreversible proteasome inhibitor that has been shown to improve overall survival in patients with relapsed and/or refractory multiple myeloma (RRMM). Carfilzomib may exert cardiovascular adverse events (CVAEs), although related mechanisms, prognostic markers and precipitating factors have not been fully characterized. In the prospective randomized phase 3 CANDOR study, comparing daratumumab in combination with carfilzomib and dexamethasone (DaraKd) versus Kd alone, a lower rate of cardiac failure events was observed in the DaraKd arm. The aim of this study was to assess whether treatment with daratumumab may ameliorate carfilzomib-related cardiovascular toxicity. Patients and Methods: This is an ongoing, prospective, observational study on the effects of Kd with or without daratumumab on cardiac function of patients with RRMM. All patients attended an initial visit, which included recording of medical history and cardiotoxicity risk factors (age ≥65 years, obesity, smoking, hypertension, hypercholesterolemia, diabetes mellitus, previous anthracycline use, previous chest or mediastinum radiotherapy and current myocardial disease). At baseline, cardiac ultrasound was performed and images were acquired for standard echocardiographic analysis and for speckle tracking offline analysis. As in ENDEAVOR study, carfilzomib was administered at 20 mg/m2 on days 1 (C1D1) and 2 (C1D2) of cycle 1 and at 56 mg/m2 thereafter, with dexamethasone 40 mg on days 1, 8 & 15 of 28-day cycles (Kd regimen). In the DaraKd regimen, daratumumab was administered at a weekly dose of 16 mg/kg, iv, for cycles 1-2, every 2 weeks for cycles 3-6 and every 4 weeks thereafter, while Kd was given at the dose described previously. A follow-up cardiac ultrasound study, as described above, was performed on the last day of cycle 6 (C6D16) or earlier if carfilzomib interruption was indicated. Patients were followed for a median of 10 months for carfilzomib-related CVAEs [hypertension (HTN), heart failure (HF) and acute coronary syndrome (ACS)]. Results: In this preliminary report, we evaluated 25 patients with relapsed or refractory MM who received either DaraKd or Kd in the everyday clinical practice; 11 patients received DaraKd and 14 received Kd. Patients' mean (±SD) age was 67.8±7.6 years and 60% were men. The two treatment groups did not significantly differ in baseline characteristics including age, gender and prevalence of hypertension, hyperlipidemia, smoking, diabetes mellitus and cardiovascular disease (p〉0.1 for all). In the DaraKd group, we did not observe any significant change of markers of left and right ventricular systolic function; however, these markers deteriorated in the Kd group. Specifically, in the Kd group, among left ventricular (LV) systolic function markers, average LV ejection fraction (LVEF) decreased from 59.6±4.8 to 56.6±5.4% (p=0.026) and LV global longitudinal strain (GLS) from -22.5±2.9 to -19.4±3.1 (p=0.007). Similarly, among right ventricular (RV) function markers, tricuspid annular plane systolic excursion (TAPSE) decreased from 23.8±3.6 to 20.4±2.8 mm (p=0.020) and RV free wall longitudinal strain from -31.9±3.4 to -28.2±4.3 (p=0.012). A significant group interaction (p

Description: Introduction: Carfilzomib (Cfz) is an approved irreversible proteasome inhibitor for the treatment of patients with relapsed/refractory multiple myeloma (R/R MM). Despite remarkable efficacy in R/R MM, Cfz clinical use is hampered by the incidence of cardiotoxicity. Age is recognized as an independent factor for the manifestation of cardiac failure and cardiovascular events. We have previously established a translational in vivo model of Cfz-induced cardiotoxicity, in which metformin (Met) had a potent prophylactic therapy, as it restored AMP-activated kinase α (AMPKα)-dependent autophagy in the myocardium of young mice, which had been inhibited by carfilzomib treatment (Efentakis P et al. Blood. 2019;133(7):710-723). Taking into consideration that MM is primarily a disease of the elderly, we sought to investigate whether our previous findings in young mice could be recapitulated in an aging in vivo model. Methods: Ten young C57Bl/6 mice (12-14 weeks of age) and thirty aged C57Bl/6 mice (15-17 months of age) were randomly assigned as follows: (i) Control group [Normal Saline (N/S) 0.9%, n=6]; (ii) Cfz group (8 mg/kg, n=6); (iii) Met group (140mg/kg, n=6); (iv) Cfz+Met group (8 mg/kg and 140 mg/kg respectively, n=6). N/S and Cfz were administered intraperitoneally on alternate days, while Met was administered per os daily for 7 days. At baseline and at the end of the experiments, mice were anesthetized with isoflurane (2% in 100% O2) and underwent echocardiography in order to investigate cardiac contractility markers (fractional shortening, FS%) and carotid plasticity markers (pulsatility index, PI% and resistance index, RI%). Subsequently mice were sacrificed for blood and myocardial tissue collection. Peripheral blood mononuclear cell (PBMCs), isolated from the whole blood, as well as myocardial tissue underwent proteasome activity assessment. Snap-frozen myocardial tissue underwent molecular immunoblotting analysis for the investigation of the molecular signaling. Results: Aged mice did not show any decreased proteasomal activity neither in the PBMCs or in the myocardium versus young C57Bl/6 mice. Cfz decreased proteasomal activity both in the PBMCs and the myocardium independently of Met administration. Aged mice presented a significant reduction of the FS% compared to the young mice at baseline, which represents an already established cardiac dysfunction in the elderly mice (mean FS%±SD: 37.40±1.6 vs. 45.62±0.8, respectively, p