Irreversible Proteasome Inhibitor Research Articles

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

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

Empagliflozin Alleviates Carfilzomib-Induced Cardiotoxicity in Mice by Modulating Oxidative Stress, Inflammatory Response, Endoplasmic Reticulum Stress, and Autophagy.

Carfilzomib is an irreversible proteasome inhibitor used for multiple myeloma patients. However, carfilzomib treatment is associated with cardiovascular complications. Empagliflozin, an Sodium Glucose Co-transporter 2 inhibitor (SGLT-2) inhibitor, is an oral antidiabetic drug with proven antioxidant and anti-inflammatory properties. The aim of the present study was to determine the cardioprotective effects of empagliflozin against carfilzomib-induced cardiotoxicity. C57BL/6 mice were randomly divided into four groups: control, empagliflozin, carfilzomib, and carfilzomib + empagliflozin. Empagliflozin prevented carfilzomib-induced cardiotoxicity by ameliorating histological alterations, CK-MB, and troponin-I. Moreover, it inhibited carfilzomib-induced oxidative damage and inflammation via its action on catalase activity, reduced glutathione levels and superoxide dismutase activity, and reduced nuclear factor-κB (p65) and cytokine levels. Mechanistically, empagliflozin abrogated endoplasmic reticulum stress induced by carfilzomib, as evidenced by the effect on the Glucose Regulated Protein-78 (GRP-78)/Activating Transcription Factor 6 (ATF6)/C/EBP homologous protein (CHOP) axis. Intriguingly, carfilzomib significantly induced autophagy, an effect that was further enhanced by empagliflozin, evidenced by increased LC3B and beclin-1 mRNA expression and reduced p62 expression. The effect of empagliflozin on apoptosis was confirmed by reduced expression of active caspase-3. Importantly, empagliflozin did not alter the cytotoxic effect of carfilzomib on human U266B1 multiple myeloma cells. our findings suggest that empagliflozin may provide a new therapeutic strategy to mitigate carfilzomib-induced cardiotoxicity in multiple myeloma patients.

Acute accumulation of PIM2 and NRF2 and recovery of β5 subunit activity mitigate multiple myeloma cell susceptibility to proteasome inhibitors.

Resistance to proteasome inhibitors (PIs) has emerged as an important clinical issue. We investigated the mechanisms underlying multiple myeloma (MM) cell resistance to PIs. To mimic their pharmacokinetic/pharmacodynamic (PK/PD) profiles, MM cells were treated with bortezomib and carfilzomib for 1h at concentrations up to 400 and 1,000nM, respectively. Susceptibility to these PIs markedly varied among MM cell lines. Pulsatile treatments with PIs suppressed translation, as demonstrated by incorporation of puromycin at 24h in PI-susceptible MM.1S cells, but not PI-resistant KMS-11 cells. Inhibition of β5 subunit activity decreased at 24h in KMS-11 cells, even with the irreversible PI carfilzomib, but not under suppression of protein synthesis with cycloheximide. Furthermore, the proteasome-degradable pro-survival factors PIM2 and NRF2 acutely accumulated in MM cells subjected to pulsatile PI treatments. Accumulated NRF2 was trans-localized into the nucleus to induce the expression of its target gene, HMOX1, in MM cells. PIM and Akt inhibition restored the anti-MM effects of PIs, even against PI-resistant KMS-11 cells. Collectively, these results suggest that increased synthesis of β5 proteasome subunit and acute accumulation of PIM2 and NRF2 reduce the anti-MM effects of PIs.

Development of First in Class DNA-Zip Code Drug Conjugate (ZDC) in Multiple Myeloma

Multiple myeloma is a heterogeneous malignant plasma cell disorder with complex molecular and genetic abnormalities. Significant advances have been made in the treatment of multiple myeloma; however, a large proportion of patients fail to attain a favorable response and relapse. Due to efficacy and toxicity, the development of drug conjugates for multiple myeloma has been limited. Our lab discovered that circulating tumor DNA (ctDNA) serves as a cell-specific mechanism for genetic transmission. Certain retrotransposon sequences within the ctDNA were identified as being crucial for the tropism of ctDNA to target tumor cells (Cinar et al bioRxiv.2022). We therefore query whether these transposons can be exploited for the targeted drug delivery. To do so, chemically synthetize and CY5-labeled multiple retrotransposons DNA sequences (a.k.a Zip Codes, ZC) to perform high through put screening assessing for cell targeting. Our findings showed that the AluSp and MER11c DNA sequences (MM-ZC1 and 2) with unique MM mutations were the most efficient and specific DNA sequences for targeting myeloma cells. Furthermore, Flow cytometry and CYTOF validated the specific MM cell targeting on full bone marrow samples from MM patients. Based on this analysis we used MMZC1 for further drug development. We design a drug conjugation approach for the MMZC and screen various drugs for their killing potential. Two potent MM ZC drug conjugates (ZDC) were identified by high throughput viability screening assay: ZC-DM4, a microtubule inhibitor, and ZC-Carfilzomib, an irreversible proteasome inhibitor. We move forward exploration with ZC-DM4 due to limited carfilzomib-linker drug availability. Experiments cancer cell lines in vitro demonstrated that MMZC-DM4 had a potent killing effect on MM cells (IC50: 3.25 nM) but not on other cancer cell lines. These results were the validated in vivo. Maximum tolerated dosing studies (MTD) demonstrated excellent tolerability in all doses (ranges from 1 to 20 mg of MMZC-DM4/Kg (equivalent to 0.007-0.149 mg of DM4/kg). To look for an efficacy signal of MMZC-DM4, MM xenograft mice were given MMZC-DM4 twice a week for three weeks. Due to limitations in production, we treated the animals biweekly with three distinct low doses of MMZC-DM4: 1.9, 3.75, and 7.5 mg/kg (equivalent to 0.014, 0.028, and 0.056 mg of DM4/kg) and compared their response to mice not treated with MMZC-DM4 and mice treated weekly with bortezomib. MMZC-DM4 at such small doses reduced the tumor by 30% without affecting body weight, WBC, HB, AST, or platelets. In contrast, Bortezomib was associated with 90% growth inhibition at the expense of significant weight loss that required treatment interruptions and thrombocytopenia. Currently, investigations are being conducted to examine the therapeutic efficacy of greater doses of MMZC-DM4. This study described the first-in-class MM targeted medicine delivery employing an MM zic code drug conjugate. Our findings suggest new method for targeted delivery therapeutic cargo material directly to cancer cells that could revolutionize cancer treatment.

Lactoferrin ameliorates carfilzomib-induced renal and pulmonary deficits: Insights to the inflammasome NLRP3/NF-κB and PI3K/Akt/GSK-3β/MAPK axes

AimsCarfilzomib, an irreversible proteasome inhibitor, has been increasingly used to treat multiple myeloma worldwide. However, case studies showed its treatment has been associated with cardiac, renal, and pulmonary deleterious effects. Lactoferrin is an iron-binding glycoprotein present in milk. It is a multifunctional protein with antimicrobial activity, antitumor, antioxidant, and anti-inflammatory effects. Thus, this study aimed to assess the protective effects of lactoferrin against carfilzomib-induced nephrotoxicity and pulmonary toxicity, in addition to identifying the possible underlying molecular mechanisms. Main methodsMice were treated with lactoferrin (300 mg/kg/day) concomitantly with carfilzomib (4 mg/kg, i.p.) twice weekly for three weeks. Kidney and lung indices, serum creatinine, blood urea nitrogen (BUN), uric acid, kidney injury molecule-1 (KIM-1), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and histological examination were assessed. In addition, biochemical analyses of the inflammasome NLRP3/NF-κB and PI3K/Akt/GSK-3β/MAPK axes were conducted. Key findingsTreatment with lactoferrin decreased serum levels of creatinine, BUN, uric acid, KIM-1, ALP, AST, and LDH and reversed carfilzomib-induced histological changes in both kidney and lung. The inflammatory markers NLRP3, p65 NF-kB, caspases1, interleukin-1β, and interleukin-18, as well as the MAPK signaling pathway, were significantly reduced in renal and pulmonary tissues of mice following lactoferrin administration. Moreover, lactoferrin significantly counteracted carfilzomib-induced reduced expression of pAkt and pGSK-3β in both renal and pulmonary tissues. SignificanceThe current study suggests lactoferrin might be a promising candidate for ameliorating carfilzomib-induced nephrotoxicity and pulmonary toxicity.

Cardiovascular Toxicity after Carfilzomib, Lenalidomide, and Dexamethasone Combination Chemotherapy in the Real-World 364 Asian Multiple Myeloma Patients on Behalf of the Korean Multiple Myeloma Working Party (KMM2201 study)

Background: Carfilzomib, a selective and irreversible epoxyketone proteasome inhibitor (PI), in combination with lenalidomide and dexamethasone (KRd) significantly improved the survival of relapsed and/or refractory multiple myeloma (RRMM) patients as well as newly diagnosed MM patients. The major dose-limiting toxicity of carfilzomib, cardiovascular adverse events (CVAE), have been reported in a variety of studies, which necessitates cautious risk management for patients who are treated with carfilzomib-based combination regimen. By far, the incidence and risk factors, as well as the outcome of CVAE in a large cohort of Asian patients after KRd regimen is lacking. Patient and method: Retrospective data of 364 RRMM patients who have received KRd regimen for RRMM in 21 centers participating in the Korean multiple myeloma working party (KMMWP) between February, 2018 and October, 2020, was analyzed. 41% of the patient was trial-ineligible, which was defined by patient-, disease-, and treatment-related features that were insufficient to meet the eligibility criteria for ASPIRE trial. CVAE was defined by dyspnea, hypertension (HTN), congestive heart failure (CHF), ischemic heart disease (IHD), arrhythmias, and venous thromboembolism (VTE). Time to onset of CVAE was defined by the first date of KRd therapy to the onset date of first CVAE. Results: The median age was 63 years (range, 28-85). CVAEs were observed in 16% of total analyzed patients after a median 9 months (range, 0-36 months) of KRd treatment, among which 3 patients (1%) showed fatal CVAEs. More specifically, dyspnea, HTN, CHF, IHD, arrhythmias, and VTE occurred in 11%, 3%, 3%, 1%, 2%, and 3% of the patients after a median of 9, 21, 16, 31, 8, and 16 months after KRd therapy. CVAE resulted in dose reduction and discontinuation of carfilzomib in 13 (4%) and 14 patients (4%), respectively. 5 patients (1%) discontinued KRd treatment due to any CVAE. By univariate analysis, among the factors affecting the cardiovascular events, such as age ≥75 years, ECOG performance status ≥ 3, creatinine clearance < 30 ml/min, Hb < 8g/dL, diabetes mellitus, HTN, underlying heart disease, cerebrovascular disease, prior bortezomib, thalidomide, doxorubicin, and cyclophosphamide therapy, and autologous stem cell transplantation, HTN was the only factor that significantly affected the occurrence of CVAEs after KRd therapy (odds ratio 1.877, 95% confidence interval, 1.025-3.440, P=0.041). CVAEs after KRd regimen did not affect progression-free or overall survival in the study cohort (Figure1A, and 1B). Conclusion: The incidence of CVAE after KRd therapy in this large cohort of real-world Asian RRMM patients was comparable with previous prospective and retrospective studies. Cardiovascular event after carfilzomib-based regimen did not affect progression-free or overall survival in RRMM patients. The recognition and management of underlying HTN has the utmost importance amongst the CV risk factors to mitigate CVAE in the Asian RRMM patients treated with KRd regimen. Acknowledgement This study would not have been possible without the collaboration of the Korean multiple myeloma working party. We especially thank the patients, families, caregivers who participated in this study. This work was supported by Amgen.

Mutations in the alternative complement pathway in multiple myeloma patients with carfilzomib-induced thrombotic microangiopathy

Thrombotic microangiopathy (TMA) has been reported to occur in multiple myeloma (MM) patients in association with treatment with carfilzomib, an irreversible proteasome inhibitor (PI). The hallmark of TMA is vascular endothelial damage leading to microangiopathic hemolytic anemia, platelet consumption, fibrin deposition and small-vessel thrombosis with resultant tissue ischemia. The molecular mechanisms underlying carfilzomib-associated TMA are not known. Germline mutations in the complement alternative pathway have been recently shown to portend increased risk for the development of atypical hemolytic uremic syndrome (aHUS) and TMA in the setting of allogeneic stem cell transplant in pediatric patients. We hypothesized that germline mutations in the complement alternative pathway may similarly predispose MM patients to carfilzomib-associated TMA. We identified 10 MM patients with a clinical diagnosis of TMA in the context of carfilzomib treatment and assessed for the presence of germline mutations in the complement alternative pathway. Ten, matched MM patients exposed to carfilzomib but without clinical TMA were used as negative controls. We identified a frequency of deletions in the complement Factor H genes 3 and 1 (delCFHR3-CFHR1) and genes 1 and 4 (delCFHR1-CFHR4) in MM patients with carfilzomib-associated TMA that was higher as compared to the general population and matched controls. Our data suggest that complement alternative pathway dysregulation may confer susceptibility to vascular endothelial injury in MM patients and predispose to development of carfilzomib-associated TMA. Larger, retrospective studies are needed to evaluate whether screening for complement mutations may be indicated to properly counsel patients about TMA risk with carfilzomib use.

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

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

Elucidating the cardiac outcomes of irreversible proteasome inhibitor Carfilzomib in in vivo models of HFpEF and HFrEF. Preventive role of Metformin

Elucidating the cardiac outcomes of irreversible proteasome inhibitor Carfilzomib in in vivo models of HFpEF and HFrEF. Preventive role of Metformin

204 Is the proteasome activity involved in filaggrin breakdown?

Epidermal hydration is maintained mainly by the retention of water in the stratum corneum through the natural moisturizing factor (NMF). NMF is a complex mixture of components, including pyrrolidone carboxylic acid (PCA) and urocanic acid (UCA), two amino acid derivatives. In the stratum corneum, breakdown of filaggrin (FLG) is the main source of PCA and UCA. The proteases involved in this degradation are still incompletely known. In the keratosis linearis with ichthyosis congenital and sclerosing keratoderma (KLICK) syndrome, a defect in the proteasome maturation protein POMP causes an altered epidermal distribution of this multi-protein complex, associated with enlarge and rounded shaped keratohyalin granules and a disturbed detection of FLG. We hypothesized that the proteasome is involved in FLG degradation. 3D reconstructed human epidermis, produced at the air-liquid interface in a low relative humidity condition (30-50%), were treated for 24 hours with various concentrations of the irreversible proteasome inhibitor clasto-lactacystin b-lactone, and analyzed using histochemistry, indirect immuno-fluorescence, transmission electron microscopy, western blotting and UHPLC linked to mass spectrometry. After clasto-lactacystin b-lactone treatment, as expected, a significant accumulation of ubiquitinated proteins was observed, without any major cytotoxic effects compared to controls. The morphological integrity of the epidermis was maintain, even if many small vesicles and spherical keratohyalin granules were observed in the cytoplasm of differentiated keratinocytes. The treatment induced a significant decrease of FLG immunodetection. However, the amount of PCA and UCA did not show any drastic differences between treated and untreated conditions. Therefore, proteasome activity seems not directly involved in FLG breakdown but may act during keratinocyte differentiation and epidermal homeostasis.

Liposome Formulation of Carfilzomib with Improved Activity As a Monotherapy and in Combination with Vinblastine

Introduction: Carfilzomib (CFZ) is an epoxyketone-based irreversible proteasome inhibitor that is one of the leading treatments for multiple myeloma (MM). Cardiotoxicity is the primary limitation for the clinical use of CFZ. We developed a novel liposome nanoparticle formulation of CFZ, a compound with low water solubility (<5 µg/mL) and low pKa (5.14) (LS-CFZ) that dramatically reduces the toxicity, increases the plasma half-life, and improves the antitumor efficacy compared to the commercial formulation. In addition, CFZ was combined in the same liposome, with vinblastine (VBL) an autophagy inhibitor. The combination exhibited synergistic anti-tumor cell activity in vitro and in vivo using a colon cancer (C26) mouse tumor model. Methods: CFZ was screened for cytotoxicity in combination with various cell lines including MM and C26 with a focus on autophagy inhibitors. VBL was co-loaded into the same liposomes with CFZ, and also loaded into separate particles. Formulations with various lipids, trapping agents and drug to lipid ratios were screened for drug loading efficiency. The lead formulation was selected based on mouse plasma half-life after IV injection in male CD1 mice. The plasma concentration of CFZ, VBL and a fluorescent lipid (liposome) label (DiIC18(5)) were measured by HPLC. The dose ratio of the CFZ and VBL was by determined by the MTD in tumor-free mice and the optimum dose and ratio was used for the efficacy study. The lead formulations were evaluated for anti-tumor efficacy in the C26 colon cancer using a single treatment (day 1) when the tumor volume was 200 mm3 for all liposome formulations and two treatments (day 1 and day 2) (optimum for CFZ) for the unencapsulated CFZ and VBL. Results: Cytotoxicty studies in C26 cells indicated the optimum ratio of CFX/VBL was between 5:1 and 20:1. All liposomes used had a diameter of 110-130 nm. Ammonium sucrose octasulfate yielded the longest retention of CFZ and VBL in the liposome after IV injection in mice. The best lipid composition was sphingomyelin/Cholesterol/PEG-2000-PE (55/45/2.8, mol/mol/mol) with a particle size of 110-130 nm in diameter. Cryo TEM images of the LS-CFZ show a dense amorphous mass of drug precipitate in the center of the liposomes. The drug to lipid ratio chosen for use in the efficacy study was 101 g/mol lipid for CFZ and 103 g/mol lipid for VBL with a loading efficiency >90%. Using the optimized trapping agent and lipid composition the %ID of CFZ is nearly identical to that of the liposome carrier up to 6 h; an average of 98.4±5.4% of the drug is retained up to 6 h. The %ID of CFZ decreases below that of the liposome between 6 and 24 h and most (>98%) of the CFZ has been released from the liposomes by the 24 h point. The AUC for LS-CFZ (2 mg/kg) is 1584 fold higher than CFZ administered as SBCD-CFZ (sulfobutyl cyclodextrin formulated, Kyprolis®) (5 mg/kg). CFZ administered as SBCD-CFZ has a t1/2 of 0.2 h compared to 3.0 h for LS-CFZ. VBL had slightly better stability than CFZ with a plasma half-life of 4.9 h. LS-CFZ had single dose MTD of 30 mg/kg for L-CFZ, while SBCD-CFZ given QDx2 had an MTD 6 mg/kg. The dramatic decrease in toxicity is likely due to lower Cmax of the bioavailable CFZ and reduced cardiotoxicity. The increased tolerability of LS-CFZ allowed for a CFZ/VBL ratio of 20:1 (18/0.9 mg/kg, CFZ/VBL) for the liposome formulation; the unencapsulated CFZ and VBL were administered at a ratio of 5:1 (5/1 mg/kg, CFZ/VBL). The anti-tumor efficacy of LS-CFZ/VBL combination was far superior to all other treatments. LS-CFZ was nearly identical to LS-VBL, and both single agents had more activity than any unencapsulated drug. As a monotherapy, LS-CFZ was far more efficacious than SBCD-CFZ, which is commonly used for treatment of multiple myeloma patients. Conclusion: Liposome formulation of CFZ increased the tolerability by 5-fold compared to the commercial CFZ formulation. The described liposome technology enables significant improvement in anti-tumor efficacy for CFZ as a monotherapy or in combination against a C26 mouse model. The improved activity in solid tumors warrants investigation of LS-CFZ for MM treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Mineralocorticoid Receptor Pathway Is a Key Mediator of Carfilzomib-induced Nephrotoxicity: Preventive Role of Eplerenone.

Carfilzomib is an irreversible proteasome inhibitor indicated for relapsed/refractory multiple myeloma. Carfilzomib toxicity includes renal adverse effects (RAEs) of obscure pathobiology. Therefore, we investigated the mechanisms of nephrotoxicity developed by Carfilzomib. In a first experimental series, we used our previously established in vivo mouse models of Carfilzomib cardiotoxicity, that incorporated 2 and 4 doses of Carfilzomib, to identify whether Carfilzomib affects renal pathways. Hematology and biochemical analyses were performed, while kidneys underwent histological and molecular analyses. In a second and third experimental series, the 4 doses protocol was repeated for 24 hours urine collection and proteomic/metabolomic analyses. To test an experimental intervention, primary murine collecting duct tubular epithelial cells were treated with Carfilzomib and/or Eplerenone and Metformin. Finally, Eplerenone was orally co-administered with Carfilzomib daily (165 mg/kg) in the 4 doses protocol. We additionally used material from 7 patients to validate our findings and patients underwent biochemical analysis and assessment of renal mineralocorticoid receptor (MR) axis activation. In vivo screening showed that Carfilzomib-induced renal histological deficits and increased serum creatinine, urea, NGAL levels, and proteinuria only in the 4 doses protocol. Carfilzomib decreased diuresis, altered renal metabolism, and activated MR axis. This was consistent with the cytotoxicity found in primary murine collecting duct tubular epithelial cells, whereas Carfilzomib + Eplerenone co-administration abrogated Carfilzomib-related nephrotoxic effects in vitro and in vivo. Renal SGK-1, a marker of MR activation, increased in patients with Carfilzomib-related RAEs. Conclusively, Carfilzomib-induced renal MR/SGK-1 activation orchestrates RAEs and water retention both in vivo and in the clinical setting. MR blockade emerges as a potential therapeutic approach against Carfilzomib-related nephrotoxicity.

Cardiac mechanics in response to proteasome inhibition: a prospective study.

Ubiquitin-Proteasome System (UPS) is of paramount importance regarding the function of the myocardial cell. Consistently, inhibition of this system has been found to affect myocardium in experimental models; yet, the clinical impact of UPS inhibition on cardiac function has not been comprehensively examined. Our aim was to gain insight into the effect of proteasome inhibition on myocardial mechanics in humans. We prospectively evaluated 48 patients with multiple myeloma and an indication to receive carfilzomib, an irreversible proteasome inhibitor. All patients were initially evaluated and underwent echocardiography with speckle tracking analysis. Carfilzomib was administered according to Kd treatment protocol. Follow-up echocardiography was performed at the 3rd and 6th month. Proteasome activity (PrA) was measured in peripheral blood mononuclear cells.At 3 months after treatment, we observed early left ventricular (LV) segmental dysfunction and deterioration of left atrial (LA) remodelling, which was sustained and more pronounced than that observed in a cardiotoxicity control group. At 6 months, LV and right ventricular functions were additionally attenuated (P < 0.05 for all). These changes were independent of blood pressure, endothelial function, inflammation, and cardiac injury levels. Changes in PrA were associated with changes in global longitudinal strain (GLS), segmental LV strain, and LA markers (P < 0.05 for all). Finally, baseline GLS < -18% or LA strain rate > 1.71 were associated with null hypertension events. Inhibition of the UPS induced global deterioration of cardiac function.

P-025: Investigating the effect of irreversible proteasome inhibitor carfilzomib in in vivo models of cardiometabolic syndrome and early stage HFrEFP: prophylactic role of metformin

P-025: Investigating the effect of irreversible proteasome inhibitor carfilzomib in in vivo models of cardiometabolic syndrome and early stage HFrEFP: prophylactic role of metformin

Prolonged proteasome inhibition antagonizes TGFβ1-dependent signalling by promoting the lysosomal-targeting of TGFβ receptors

Impairing autophagy disrupts transforming growth factor beta 1 (TGFβ1) signalling and epithelial-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC). Since autophagy and proteasome-mediated degradation are interdependent, we investigated how prolonged downregulation of proteasomal catalytic activity affected TGFβ1-dependent signalling and EMT. Proteasome-dependent degradation was inhibited in A549 and H1299 NSCLC cells using MG132 and lactacystin, which are reversible and irreversible proteasome inhibitors, respectively. We observed that inhibiting proteasomal activity for 24 h decreased TGFβ-dependent nuclear accumulation of Smad2/3. Time course studies were then carried out to characterize the time frame of this observation. Short-term (< 8 h) proteasome inhibition resulted in increased receptor regulated Smad (R-Smad) phosphorylation and steady-state TGFβ receptor type II (TGFβRII) levels. However, prolonged (8–24 h) proteasome inhibition decreased TGFβ1-dependent R-Smad phosphorylation and steady-state TGFβRI and TGFβRII levels. Furthermore, proteasome inhibition blunted TGFβ-dependent E- to N-Cadherin shift, stress fiber formation, and increased cellular apoptosis via the TAK-1-TRAF6-p38 MAPK pathway. Interestingly, proteasome inhibition also increased autophagic flux, steady-state microtubule-associated protein light chain 3B-II and active uncoordinated 51-like autophagy activating kinase 1 levels, and co-localization of lysosomes with autophagy cargo proteins and autophagy-related proteins. Finally, we observed that proteasome inhibition increased TGFβRII endocytosis and trafficking to lysosomes and we conclude that prolonged proteasome inhibition disrupts TGFβ signalling outcomes through altered TGFβ receptor trafficking.

Genetic and Functional Evidence of Complement Dysregulation in Multiple Myeloma Patients with Carfilzomib-Induced Thrombotic Microangiopathy Compared to Controls.

Background: Carfilzomib, an irreversible proteasome inhibitor approved for the treatment of relapsed/refractory Multiple Myeloma (MM) has been associated with Thrombotic Microangiopathy (TMA). Several pathogenetic mechanisms of carfilzomib-induced TMA have been proposed; however, recently, there has been a shift of focus on the potential contribution of complement dysregulation. Our aim was to explore whether patients with carfilzomib-induced TMA harbor germline variants of complement-related genes, which have been characterized as risk factors for TMA. Methods: We retrospectively recruited consecutive MM patients with carfilzomib-induced TMA and compared them to MM patients who received ≥4 cycles of carfilzomib and did not develop signs/symptoms of TMA, in a 1:2 ratio. Genomic DNA from peripheral blood was analyzed using next generation sequencing (NGS) with a complement-related gene panel; ADAMTS13 activity and soluble C5b-9 were measured using ELISA. Results: Complement-related variants were more common in patients with carfilzomib-induced TMA compared to non-TMA controls, regardless of patient and treatment characteristics; ADAMTS13 activity and C5b-9 were compatible with the phenotype of complement-related TMA. Conclusions: We confirmed the previous findings that implicated complement-related genes in the pathogenesis of carfilzomib-induced TMA. Most importantly, by incorporating a control group of non-TMA MM patients treated with carfilzomib-based regimens and functional complement assays, we enhanced the credibility of our findings.

Co-Operation of MCL-1 and BCL-X L Anti-Apoptotic Proteins in Stromal Protection of MM Cells from Carfilzomib Mediated Cytotoxicity

Introduction: Expression and function of BCL-2 family proteins in multiple myeloma (MM) is important for tumour cell survival and drug resistance, and may partly mediate the protective effect of bone marrow (BM) stroma. Proteasome inhibitors (PIs) are a major class of anti-myeloma drugs, however some patients are resistant and almost all eventually relapse. Carfilzomib is a second generation, irreversible proteasome inhibitor, which is effective in both newly diagnosed and relapsed/refractory disease, but drug resistance and relapse occur.Aim: We aimed to study the role of BCL-2 family proteins in Carfilzomib (CFZ) mediated cytotoxicity in the presence of stroma, focusing on priming of the pro-apoptotic protein, BIM.Methods: A panel of Human Myeloma Cell Lines (HMCL) and primary MM cells were exposed to Carfilzomib, and cell death assessed using Annexin V/PI staining and flow cytometry. Co-cultures employing the HS5 stromal cell line were used to model the interaction with stroma. MM cells were exposed to CFZ in a 1hr pulse, to recapitulate the pharmacokinetics of drug exposure in patients. The expression of BCL-2 family proteins was assessed by flow cytometry and Western Blot (WB). The compounds S63845, ABT-199 (Venetoclax) and A-1331852 were used to inhibit MCL-1, BCL-2 and BCL-X L pro-survival proteins respectively. Changes in BIM binding partners were examined by immunoprecipitation and WB.Results: CFZ induced dose and time-dependent cell death of HMCL and primary MM cells. This was primarily mediated by apoptosis as shown by AnV/PI staining (eg. MM1.s at 25nM CFZ: 34±6% apoptosis at 24h, 47±9% at 48h, p=0.016; 50nM CFZ: 60 ±3.8% at 24h vs 79±6% at 48h, p=0.004, mean ±SEM, n=3), and by reduced cytotoxicity in MM1.s BAX/BAK KO cells (100nM CFZ: 10±1% cell death vs 42±5% in WT at 6h, 33±14% vs 99±0.5% at 24h; p=0.0005, n=3). CFZ cytotoxicity was significantly reduced when MM1.s cells were co-cultured with HS-5 (50nM CFZ: 53±4% apoptosis vs 79±4% in suspension, at 48h, p=0.02, n=3) confirming the protective effect of stromal cells. Similar results were seen with H929, KMS27 and KMS12BM HMCL (Figure 1). Co-culture of MM1.s with HS-5 cells induced upregulation of MCL-1 and BCL-X L and downregulation of BCL-2. Combined inhibition of BCL-X L and MCL-1 at concentrations where single agents had minimal effect, resulted ina marked increase in apoptosis on stroma (Ctrl: 9±1%, S63845 100nM:12±1.4%, A-1331852 1nM: 17±3%, S63845+A-1331852: 60±9%; 48h, p=0.0001, n=3) (Figure 3). BIM immunoprecipitation showed that in the presence of the MCL-1 inhibitor, S63845, there was increased BCL-X L binding to BIM; conversely when BCL-X L was inhibited by A-1331852, an increase in MCL-1/BIM complexes was seen. Exposure of MM cells to CFZ in stromal co-cultures led to a further increase in the expression of BCL-X L and MCL-1. Because BCL-X L and MCL-1 were upregulated both by stromal interaction and by CFZ (Figure 2), we tested the cooperativity of these anti-apoptotic proteins on resistance to CFZ. CFZ-induced cytotoxicity was significantly enhanced by the addition of single BH3 mimetics in the presence of HS-5: cell death 48h, BCL-X L A-133182 5nM: 40±12%, CFZ 25nM: 57±10%, CFZ+A-1331852: 94±2%, p=0.0065, n=3. Cell death with MCL-1 inhibitor S63845 200nM: 12±3%, CFZ 25nM:40±13%, S63845+CFZ: 54±18%, p=0.02, n=3). However, combined BCL-X L and MCL-1 inhibition, using lower concentrations of BH3 mimetics, potently sensitized MM cells to CFZ-mediated cytotoxicity in the presence of stroma (% killing at CFZ 25nM + A-1331852 1nM: 48±8%; CFZ+S63845 100nM: 38±5%; CFZ+A-1331852+S63845: 92±2%, p<0.0001, n=3, Figure 3).Conclusion: BCL-X L and MCL-1 are upregulated in MM cells by stromal interaction, and also in response to CFZ. The upregulation of pro-survival proteins by stroma may mediate MM cell resistance to PIs, and blockade of both BCL-X L and MCL-1 is required in order to abrogate the protective effect of stroma and restore sensitivity to CFZ. Combination of BH-3 mimetic drugs at subtherapeutic doses with proteasome inhibitors may be a potential therapeutic strategy. [Display omitted] DisclosuresHuang: The Walter and Eliza Hall Institute of Medical Research: Patents & Royalties: Employee of the Walter and Eliza Hall Institute and eligilble for payments in relation to venetoclax. Khwaja: Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Yong: Janssen: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; Takeda: Honoraria; GSK: Honoraria; Amgen: Honoraria; BMS: Research Funding; Autolus: Research Funding.

Mutations in the Alternative Complement Pathway in Multiple Myeloma Patients with Carfilzomib-Induced Thrombotic Microangiopathy

IntroductionVascular endothelial injury related to treatment with proteasome inhibitors (PI) has been previously described. Carfilzomib is an irreversible PI and has been associated with cardiovascular toxicity, suggesting increased risk of endothelial injury. Thrombotic microangiopathy (TMA) has been described in multiple myeloma (MM) patients receiving PIs; more often with carfilzomib. In the pediatric transplant population, increased risk of TMA was related to heterozygous mutation in the alternative complement pathway. The homozygous deletions enable uncontrolled complement activation and was found three times more frequently in TMA population. We hypothesized that MM patients with complement mutation are at increased risk of PI-related TMA.Materials and MethodsWe identified ten cases of renal TMA in MM patients receiving carfilzomib from two medical institutions in Greece and in the United States. TMA was diagnosed based on either renal biopsy or acute kidney injury, new-onset anemia, thrombocytopenia and increased LDH in the absence of disease progression. We performed targeted sequencing of the twelve genes implicated in TMA: CFH, CFI, MCP, CFB, CFHR5, C3, THBD, DGKE, PLG, ADAMTS13, MMACHC and G6PD genes. Multiplex Ligation-Dependent Probe Amplification (MLPA) was performed for the analysis of the CFH-CFHR5 region.ResultsPatient characteristics and laboratory values at TMA diagnosis are presented in Table 1. The median age of patients was 68 years (range, 47-73), with slight male predominance (60%). Median laboratory values at diagnosis included hemoglobin 9.35 g/dL, platelet count 26,500 x 10 6/L, LDH 356,5 U/L and creatinine 2.25 mg/dL. Patients were treated with carfilzomib doses ranging 20-70 mg/m 2. Regimens included carfilzomib-dexamethasone (Kd, 7 patients), carfilzomib-lenalidomide-dexamethasone (KRd, 1 patient), carfilzomib-pomalidomide-dexamethasone (KPd, 1 patient), and carfilzomib-daratumumab-dexamethasone (DaraKd, 1 patient). All patients had previously received at least one line of therapy and seven patients had previously undergone autologous stem cell transplant (ASCT), 1-12 years prior to TMA diagnosis. The median time between carfilzomib initiation and TMA diagnosis was 4.5 months (range, 1-60 months). Diagnosis was confirmed with renal biopsy in four cases. Carfilzomib was discontinued in all patients and five patients were treated with plasma exchange (PLEX) while one patient received eculizumab. Seven patients demonstrated clinical improvement and resolution of TMA at 1 year after discontinuation of carfilzomib. Two patients progressed to end stage renal disease (ESRD) requiring intermittent hemodialysis, and one patient developed multiorgan failure. Results of genetic panel are shown in Table 2. Deletions of the CFHR3-CFHR5 region were present in seven cases (70%): two patients carrying a homozygous deletion of CFHR3-CFHR1, four patients with a heterozygous deletion of CFHR3-CFHR1, and one patient with heterozygous deletion of CFHR1-CFHR4. Direct gene sequencing revealed identifiable mutations in CD46 (MCP) and CFHR5 in two distinct patients. The functional correlation and clinical significance are yet to be investigated.ConclusionsIn our cohort of ten patients of carfilzomib-induced TMA, deletions of CFHR3-CFHR5 occurred frequently (70%). In the setting of carfilzomib use, heterozygous CFHR3-CFHR1 deletion may represent a risk factor for the development of TMA. Our data set the bases for larger studies assessing complement mutation as a predisposing factor for PI-induced TMA. [Display omitted] DisclosuresKastritis: Amgen: Consultancy, Honoraria, Research Funding; Genesis Pharma: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Consultancy, Honoraria, Research Funding. Dimopoulos: Janssen: Honoraria; BMS: Honoraria; Takeda: Honoraria; Beigene: Honoraria; Amgen: Honoraria. Richardson: AbbVie: Consultancy; Secura Bio: Consultancy; Sanofi: Consultancy; GlaxoSmithKline: Consultancy; Karyopharm: Consultancy, Research Funding; Regeneron: Consultancy; AstraZeneca: Consultancy; Oncopeptides: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Janssen: Consultancy; Celgene/BMS: Consultancy, Research Funding; Protocol Intelligence: Consultancy; Jazz Pharmaceuticals: Consultancy, Research Funding. Bianchi: Jacob D. Fuchsberg Law Firm: Consultancy; MJH: Honoraria; Karyopharm: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria.

Mineralocorticoid receptor signaling is implicated in carfilzomib-induced increase in blood pressure

Abstract Introduction Carfilzomib (Cfz), an irreversible proteasome inhibitor, is a first line antineoplastic agent indicated for relapsed/refractory multiple myeloma, with its clinical use being hampered by cardiovascular adverse effects. Hypertension, is the most common cardiovascular side effect of Cfz, remaining of unknown pathogenicity. Purpose Considering that management of Cfz-related hypertension remains an unmet clinical need and that renal function plays a pivotal role in blood pressure regulation we sought to investigate the renal contribution in Cfz-induced hypertension. Methods We have previously established a translational model of Cfz-induced cardiomyopathy, based on clinically applicable dose regimens and we have concluded that two and four dose protocols successfully resemble the clinical observations in vivo. Herein, sixty C57Bl/6 male mice (12–14 weeks old) were randomized to: 1. Two doses Protocol: i. Control (N/S 0.9%), ii. Cfz (8mg/kg) for two consecutive days; and 2. Four doses Protocol: i. Control (N/S 0.9%), ii. Cfz (8mg/kg) for seven days intraperitoneally. Systolic (SBP) and diastolic blood pressure (DBP) were measured by tail cuffs; the latter protocol was repeated and urine collection was performed via metabolic cages studies. Renal samples were collected for histological, proteomic, metabolomic and molecular signaling analyses. Finally, eplerenone, a mineralocorticoid receptor (MR) blocker, was orally co-administered with Cfz to the mice daily (165 mg/kg) in the four doses protocol. Results Cfz increased SBP only in the four doses protocol (78.50±2.05 vs 68.20±0.73 in the Control group, **P&amp;lt;0.01). Histological evaluation of the kidneys revealed a juxtaglomerular apparatus hyperplasia (JAH) in the same dose regimen. Proteomic analysis presented that metabolic and transport of small molecules pathways were differentially regulated in the Cfz treated murine kidneys. Metabolomic analysis revealed an increase in urea cycle metabolites (L-Alanine, L-Glutamine, glutamate, aspartate) and taurine content in the kidneys. Additionally, mice presented decreased diuresis without any differences in other metabolic parameters. In parallel an upregulation of β-ENaC expression and activation of MR/SGK-1 signaling in the kidneys was observed, indicating that Cfz activates MR signaling. Co-administration of eplerenone and Cfz, restored diuresis, decreased SBP and inhibited MR/SGK-1 signaling in the kidneys. Conclusions Activation of MR signaling by Cfz in the kidneys orchestrates renal water/salt retention and drives an increase in blood pressure in vivo. Histological and metabolomic analyses present that Cfz induces an acute kidney injury and a tonicity increase. Eplerenone reversed Cfz-induced blood pressure increase and restored diuresis by inhibiting MR/SGK-1 signaling. Therefore, MR blockade emerges as a potent therapeutic approach against Cfz-related cardiovascular adverse events. Funding Acknowledgement Type of funding sources: None.

Global deterioration of cardiovascular mechanics by carfilzomib treatment is associated with proteasome activity in patients with relapsed multiple myeloma

Abstract Background Carfilzomib (CFZ) is a second generation irreversible proteasome inhibitor that has been shown to improve overall survival in patients with relapsed or refractory multiple myeloma (MM) but is associated with cardiac toxicity in MM. Because ubiquitin-proteasome system (UPS) is involved in cardiovascular homeostasis exploring the effects of proteasome inhibition on cardiac mechanics, in a clinical setting, would provide novel insight of the clinical role of UPS in cardiovascular disease. Aim To gain a thorough understanding of proteasome inhibition on myocardial mechanics in a clinical setting. Methods We prospectively evaluated 48 patients with relapsed or refractory MM and an indication to receive CFZ treatment [median age: 67.5, interquartile range (64–73), 66.7% men]. All patients underwent cardiac ultrasonography at an initial visit, at the first day of the 3rd month (C3D1, n=25) and at the last day of cycle 6 (C6D16, n=48). Pulse wave velocity (PWV) was used to assess arterial stiffness and calculate arterial-ventricular coupling (VAC) by the ratio (PWV/global longitudinal strain (GLS)). Carfilzomib was administered at 27mg/m2 on days 1 (C1D1) and 2 (C1D2) of cycle 1 and at 56mg/m2 thereafter. Blood was drawn at baseline, C3D1 and C6D16 to measure proteasome activity (PrA). Patients were followed up for carfilzomib–related cardiovascular adverse events. Results Regarding systolic function, between baseline and C6D16, we noticed deterioration in left ventricular ejection fraction (LVEF) (57.7±5.4% vs 56.1±7.19%, p=0.022), GLS (−20.51±2.78 vs −18.88±3.45%, p=0.003), S wave of LV longitudinal strain rate and radial strain (p&amp;lt;0.05 for both). Markers of LV diastolic function also deteriorated at 6 months including early diastolic (Ea) mitral annular velocity (7.27±1.83cm/sec vs 6.7±1.64cm/sec, p=0.008) and E wave of LV longitudinal strain rate (1.1±0.37sec-1 vs 0.95±0.36sec-1, p=0.010). Left atrial volume (LAV) and index (LAVi) increased both at 3 and 6 months (p&amp;lt;0.05). Peak LA strain at reservoir phase (peak atrial longitudinal strain, PALS) was also decreased at 6th and 3rd month measurements (p&amp;lt;0.05), whereas LA strain rate at reservoir function (s wave) was decreased, compared to baseline, only at 6th month (p=0.001). RV longitudinal strain also decreased between 3rd and 6th month (p=0.039). Furthermore, PWV/GLS also deteriorated at 6 months (−0.56±0.13 vs −0.64±0.21, p=0.001). By linear mixed models analysis, we found that changes in proteasome activity were associated with changes in PWV/GLS (p=0.017), LAV (p=0.015) and LAVi (p=0.009), and PALS (p=0.014). Conclusion CFZ treatment was associated with global deterioration of markers of myocardial systolic and diastolic function and VAC. The observed association of changes in proteasome activity with VAC and left atrial function and structure supports a direct effect of proteasome inhibition on the cross-talk between LV and arterial function and LV diastolic function. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Amgen