Next-generation Proteasome Inhibitor Research Articles

Carfilzomib shows therapeutic potential for reduction of liver fibrosis by targeting hepatic stellate cell activation

Because hepatic stellate cells (HSCs) play a major role in fibrosis, we focused on HSCs as a potential target for the treatment of liver fibrosis. In this study, we attempted to identify drug candidates to inactivate HSCs and found that several proteasome inhibitors (PIs) reduced HSC viability. Our data showed that a second-generation PI, carfilzomib (CZM), suppressed the expression of fibrotic markers in primary murine HSCs at low concentrations of 5 or 10 nM. Since CZM was not toxic to HSCs up to a concentration of 12.5 nM, we examined its antifibrotic effects further. CZM achieved a clear reduction in liver fibrosis in the carbon tetrachloride (CCl4)-induced mouse model of liver fibrosis without worsening of liver injury. Mechanistically, RNA sequence analysis of primary HSCs revealed that CZM inhibits mitosis in HSCs. In the CCl4-injured liver, amphiregulin, which is known to activate mitogenic signaling pathways and fibrogenic activity and is upregulated in murine and human metabolic dysfunction-associated steatohepatitis (MASH), was downregulated by CZM administration, leading to inhibition of mitosis in HSCs. Thus, CZM and next-generation PIs in development could be potential therapeutic agents for the treatment of liver fibrosis via inactivation of HSCs without liver injury.

Carfilzomib Use Among Patients with Relapsed/Refractory Multiple Myeloma in the Asia Pacific Region: Characteristics and Outcomes By Regimen from a Prospective, Real-World Study

Background: Carfilzomib (CFZ) is a next-generation proteasome inhibitor currently approved in combination with lenalidomide and dexamethasone (KRd) or with dexamethasone alone (Kd) for treatment of multiple myeloma (MM) patients with ≥1 prior line of therapy in the Asia-Pacific (APAC) region. Here, we describe utilization of KRd and Kd in routine clinical practice in APAC, including treatment patterns, patient profile, reasons for discontinuation, clinical outcomes, and frequency of adverse events (AEs). Methods: This prospective, single-arm cohort study recruited adults with relapsed/refractory MM who received ≥1 dose of CFZ in routine clinical practice as either a combination or monotherapy regimen and had received ≥1 prior line of MM treatment before CFZ initiation. Between July 2019 and June 2021, approximately 300 patients were planned to be enrolled in this study. Medical history, patient characteristics, and clinical data are collected at baseline and throughout the 2-year observation period or until death, withdrawal of consent, or loss of follow-up. Follow-up data are collected through chart reviews every 3 months. AEs leading to treatment discontinuation are collected; common AEs associated with drugs/disease were excluded from collection by the protocol (all serious AEs were collected). The study is ongoing, and last patient record for the analysis will be collected no later than Q1 2023. Results: As of data cutoff April 1, 2022, 311 patients were enrolled, of whom 38 are excluded from the present analysis (11 died before waiver of informed consent approval and 27 patients had missing data for the first medical chart review or first CFZ administration). The 273 patients included in this ad hoc interim analysis were enrolled from 5 countries/regions: Australia (n=46), Hong Kong (n=17), Korea (n=172), Singapore (n=13), and Taiwan (n=25). To date, the regimens prescribed were KRd (n=160, 59%), Kd (n=103, 38%), and other CFZ-based regimens (n=10, 4%; monotherapy [n=1]; not classified [1]; or with dexamethasone and cyclophosphamide [5], pomalidomide [2], or daratumumab [1]). Table 1 displays detailed patient and disease characteristics, and Table 2 displays CFZ utilization and response. Overall, 33% of patients reported a history of hypertension, 11% of cardiac disorders, 7% of diabetes mellitus, and 3% of pulmonary embolism, prior to CFZ initiation. On average, patients prescribed KRd were slightly younger than those prescribed Kd. The majority of KRd patients (68%) had received 1 prior line of treatment, whereas one-third of Kd patients (36%) had received ≥4 prior lines of treatment. Similar proportions of KRd (33%) and Kd (34%) populations were bortezomib refractory, and 73% of Kd patients were lenalidomide refractory. The planned CFZ dose at first cycle was in accordance with the local product information and was used in 89% of KRd and 69% of Kd patients (27 and 56 mg/m2 twice weekly, respectively). At data cutoff, 43.1% and 30.1% of KRd and Kd patients, respectively, remained on CFZ treatment. The most common reason for CFZ discontinuation was disease progression/refractory disease. With KRd and Kd, respectively, the best overall response rate was 79.0% (n=94/119) and 54.7% (n=29/53) and median overall survival was 60.9 (95% confidence interval [CI], 55.9-not estimable [NE]) and 54.5 (95% CI, 33.3-NE) months. CFZ-treatment-related AEs of interest occurred in 10 (3.7%) total patients (cardiac arrhythmia, n=1 Kd; cardiac failure, 1 KRd; dyspnea, 2 KRd, 2 Kd, 1 other CFZ; hematopoietic thrombocytopenia, 1 KRd and 1 Kd; ischemic heart disease, 1 KRd; no incidence of treatment-related acute renal failure, hypertension, and peripheral neuropathy). Overall, 10 (3.7%) patients discontinued CFZ owing to an AE. Conclusions: This study investigates the real-world use of CFZ for relapsed/refractory MM in a predominantly Asian patient population across the APAC region. Interim results confirm that the standard dosing schedules for KRd and Kd are well tolerated in real-world practice and suggest a very low rate of discontinuation due to CFZ-related AEs, even in heavily pretreated MM. Further prospective data collection of patients in routine practice is ongoing to assess patterns of longer-term patient management, patterns after CFZ treatment failure, CFZ toxicity according to number of previous lines of therapy, response, survival outcome, and AEs, which will inform optimal use of CFZ. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

MLN2238 exerts its anti-tumor effects via regulating ROS/JNK/mitochondrial signaling pathways in intrahepatic cholangiocarcinoma.

Background: Intrahepatic Cholangiocarcinoma (iCCA) is a highly malignant tumor with limited treatment options that contributes largely to cancer-related deaths worldwide. Compared with traditional transcriptomic analysis, single-cell RNA sequencing (scRNA-seq) is emerging as a more advanced and popular tool for the in-depth exploration of cellular diversity and molecular complexity. As a next-generation proteasome inhibitor, MLN2238 presents better pharmacodynamics, pharmacokinetics, and therapeutic responses in various cancers. However, its effects and mechanisms of action in iCCA remain unknown. Methods: iCCA tumor heterogeneity was determined based on 4,239 qualified scRNA-seq data from 10 iCCA samples. The potential biological roles of proteasome-related genes in iCCA were investigated using a pseudo-trajectory reconstruction. The effect of MLN2238 on iCCA cell proliferation was estimated using the CCK-8, EdU, and clone formation assays. Flow cytometry was used to examine the effect of added MLN2238 on cell cycle and apoptosis levels. Autophagic flux was detected using AdPlus-mCherry-GFP-LC3B cells. ROS levels and mitochondrial membrane potential were determined using DCFH-DA probing and JC-1 staining. JNK activation and mitochondrial apoptosis were observed using western blotting and immunofluorescence microscopy, respectively. Finally, we used a tumor-bearing mouse model to validate its efficacy in vivo for iCCA treatment. Results: Proteasome-related genes were dysregulated in iCCA progression and expressed at higher levels in tumor tissues. MLN2238 suppressed cell proliferation, blocked the cell cycle in the G2/M phase, promoted apoptosis, and induced cytoprotective autophagy in iCCA cells. Furthermore, MLN2238 increased ROS levels and activated the JNK signaling pathway. Inhibition of ROS and JNK activation by NAC and SP600125 significantly reversed MLN2238-induced apoptosis. MLN2238 also suppressed the growth of iCCA tumors in vivo. Conclusion: Proteasome-related genes play pivotal roles in iCCA development. MLN2238, as a proteasome inhibitor, induces apoptosis in iCCA cells through ROS/JNK/mitochondrial signaling pathways, and hence, making MLN2238 a potential therapeutic choice for iCCA.

Resensitising proteasome inhibitor-resistant myeloma with sphingosine kinase 2 inhibition.

The introduction of the proteasome inhibitor bortezomib into treatment regimens for myeloma has led to substantial improvement in patient survival. However, whilst bortezomib elicits initial responses in many myeloma patients, this haematological malignancy remains incurable due to the development of acquired bortezomib resistance. With other patients presenting with disease that is intrinsically bortezomib resistant, it is clear that new therapeutic approaches are desperately required to target bortezomib-resistant myeloma. We have previously shown that targeting sphingolipid metabolism with the sphingosine kinase 2 (SK2) inhibitor K145 in combination with bortezomib induces synergistic death of bortezomib-naïve myeloma. In the current study, we have demonstrated that targeting sphingolipid metabolism with K145 synergises with bortezomib and effectively resensitises bortezomib-resistant myeloma to this proteasome inhibitor. Notably, these effects were dependent on enhanced activation of the unfolded protein response, and were observed in numerous separate myeloma models that appear to have different mechanisms of bortezomib resistance, including a new bortezomib-resistant myeloma model we describe which possesses a clinically relevant proteasome mutation. Furthermore, K145 also displayed synergy with the next-generation proteasome inhibitor carfilzomib in bortezomib-resistant and carfilzomib-resistant myeloma cells. Together, these findings indicate that targeting sphingolipid metabolism via SK2 inhibition may be effective in combination with a broad spectrum of proteasome inhibitors in the proteasome inhibitor resistant setting, and is an approach worth clinical exploration.

Measuring Cardiopulmonary Complications of Carfilzomib Treatment and Associated Risk Factors Using the SEER-Medicare Database

Background. Next-generation proteasome inhibitor carfilzomib (CFZ) increases frequency and depth of responses, and improves survival in heavily pretreated patients with multiple myeloma (MM). In the clinical trials that led to the approval of CFZ, its use was associated with cardiac and pulmonary adverse events (CAEs and PAEs). However, the actual frequency of these events in the overall population is unknown because strict eligibility criteria in the clinical trials result in enrolling healthier patients. Moreover, the risk factors for developing these adverse events (AEs) remain to be established. Our objective was to estimate the incidence of CAEs and PAEs through administrative claims in the SEER-Medicare linked database. In addition, we aimed to identify the risk factors associated with developing these AEs.Methods. We extracted all plasma cell myeloma cases (ICD-O-3 histology codes 9731, 9732, and 9734) in the SEER-Medicare linked database from 2000-2013 and corresponding claims through 2014. We then used the Healthcare Common Procedure Coding System (HCPCS) codes for CFZ (C9295 and J9047) to identify patients who have undergone treatment with CFZ at any time point during their disease course. Patients receiving additional anti-MM therapy along with CFZ, except for corticosteroids, were excluded. Breaks in CFZ of more than 60 days were considered separate regimens and only data from first CFZ regimen was included in the analysis. Subsequently, we used the International Classification of Disease, ninth revision (ICD-9) to identify all the codes for CAEs and PAEs associated with CFZ usage that have previously been reported in the peer-reviewed literature. AEs were collected from first CFZ dose through 28 days following last dose or the start of additional anti-MM agents. AEs that were present within 90 days prior to CFZ were considered comorbidities and not included as toxicity. The frequency of CAEs and PAEs were reported using descriptive statistics. Multivariate Cox regression was performed to determine the variables that were independently associated with development of CAEs of interest (hypertension [HTN], ischemic heart disease, or congestive heart failure [CHF]), PAEs of interest (deep venous thrombosis [DVT], pulmonary embolism [PE], or pulmonary HTN), and respiratory infections (cough, upper respiratory infection, or pneumonia). All statistical analyses were performed using SAS Enterprise Guide 5.1. A p value of < 0.05 was considered statistically significant.Results. Six hundred and thirty-five patients were included in the final analysis. The median age was 72 (range 36-94). Fifty-five percent of the patients were male, 79% Caucasian, 17% African-American, and 4% belonged to other races. The median duration of CFZ treatment was 58 days (range 1-716), and the median duration from MM diagnosis to CFZ treatment was 54 months (range 3-170).In terms of CAEs, 22% of the patients developed new HTN; 15% and 13% developed peripheral edema and systolic/diastolic heart failure, respectively. New chest pain and ischemic heart disease affected 10% and 6% of the patients, respectively. The incidence of new cardiac dysrhythmias was 7%. In terms of PAEs, 25% of patients developed dyspnea and 15% developed a cough. Pneumonia and respiratory tract infections were each registered in 14% of patients. Venous thromboembolic events were documented in 6% of patients and 1% of the patients were afflicted with pulmonary HTN. The frequency of toxicities is reported in Table 1.With regard to the risk factors associated with developing toxicities, only COPD was found to be independently associated with developing CAEs of interest. Patients with pre-existing COPD had a 37% increase in hazard for developing HTN, ischemic heart disease, or CHF (aHR 1.37, 95% CI 1.01-1.87). The multivariate analysis is summarized in Table 2.Conclusions. With the expanding role of CFZ in the treatment of newly diagnosed and relapsed/refractory patients with MM, it is essential to identify the incidence of adverse events associated with its use in real-life practice settings. It is also critical to recognize any risk factors that predispose patients to developing these AEs to mitigate toxicity.To the best of our knowledge, this is the first and the largest SEER-Medicare linked study estimating the incidence of cardiopulmonary AEs and associated risk factors in patients with MM who received CFZ. [Display omitted] DisclosuresVij:Takeda, Onyx: Research Funding; Celgene, Onyx, Takeda, Novartis, BMS, Sanofi, Janssen, Merck: Consultancy. Wildes:Carevive Systems Inc: Consultancy; Janssen: Consultancy.

Renal failure among multiple myeloma patients utilizing carfilzomib and associated factors in the "real world".

Carfilzomib, a next-generation proteasome inhibitor, improves outcomes in patients with multiple myeloma (MM); however, a proportion of those treated develop renal failure due to adverse event, comorbidity, or myeloma progression. The rate of renal failure and associated risk factors remains unknown in real-world populations. Adults with relapsed/refractory MM who received carfilzomib between the years 2013 and 2016 were identified in the Surveillance, Epidemiology, and End Results (SEER)-Medicare linked databases. Renal failure was defined using the corresponding International Classification of Diseases, Ninth Revision (ICD-9) and Tenth Revision (ICD-10) diagnostic codes and procedure codes for dialysis. Patients with a pre-existing diagnosis of renal failure were excluded to distinguish an adverse event from comorbidity. Multivariate cox regression analysis was performed to identify the variables independently associated with the development of renal failure among MM patients utilizing carfilzomib. A total of 1950 patients were included in the analysis. Renal failure developed in 22% of patients during the study period. The median time to development of renal failure from first carfilzomib administration was 1.6 months (range < 0.1-23.3). Increasing age (adjusted hazard ratio [aHR] 1.01 per year, p = 0.018), pre-existing heart failure (aHR 1.50, p = 0.005), and pre-existing chronic kidney disease (aHR 2.00, p < 0.001) were associated with a higher risk of developing renal failure. Renal failure occurred in up to 22% of patients on carfilzomib therapy. The exact cause and mechanism of renal failure cannot be determined from our study and may be multifactorial. Future studies are needed to further understand the cause of renal failure among patients on carfilzomib and devise strategies to mitigate the risk.

Novel strategies to target the humoral alloimmune response.

Antibody-mediated rejection (ABMR) represents a major cause of late allograft loss in solid organ transplantation worldwide. This process is driven by donor-specific antibodies (DSA), which develop either de-novo or, in sensitized patients, are preformed at the time of transplantation. Effective targeting of ABMR has been hampered by a lack of robust randomized controlled trials (RCT), required for the regulatory approval of new therapeutics. In this review, we discuss the evidence behind the present "standard" of care and recent progress in the development of novel strategies targeting different aspects of the alloimmune humoral response, including naïve and memory B-cell activation, the germinal centre reaction, plasma cell survival and antibody effector functions. In particular, we focus on co-stimulation blockade and its combination with next-generation proteasome inhibitors, new depleting monoclonal antibodies (anti-CD19, anti-BCMA, anti-CD38, anti-CD138), interleukin-6 blockade, complement inhibition and DSA degradation. These treatment modalities, when used in the appropriate clinical context and combination, have the potential to finally improve long-term allograft survival.

Amino acid depletion triggered by ʟ-asparaginase sensitizes MM cells to carfilzomib by inducing mitochondria ROS-mediated cell death

AbstractMetabolic reprogramming is emerging as a cancer vulnerability that could be therapeutically exploitable using different approaches, including amino acid depletion for those tumors that rely on exogenous amino acids for their maintenance. ʟ-Asparaginase (ASNase) has contributed to a significant improvement in acute lymphoblastic leukemia outcomes; however, toxicity and resistance limit its clinical use in other tumors. Here, we report that, in multiple myeloma (MM) cells, the DNA methylation status is significantly associated with reduced expression of ASNase-related gene signatures, thus suggesting ASNase sensitivity for this tumor. Therefore, we tested the effects of ASNase purified from Erwinia chrysanthemi (Erw-ASNase), combined with the next-generation proteasome inhibitor (PI) carfilzomib. We observed an impressive synergistic effect on MM cells, whereas normal peripheral blood mononuclear cells were not affected. Importantly, this effect was associated with increased reactive oxygen species (ROS) generation, compounded mitochondrial damage, and Nrf2 upregulation, regardless of the c-Myc oncogenic-specific program. Furthermore, the cotreatment resulted in genomic instability and DNA repair mechanism impairment via increased mitochondrial oxidative stress, which further enhanced its antitumor activity. Interestingly, carfilzomib-resistant cells were found to be highly dependent on amino acid starvation, as reflected by their higher sensitivity to Erw-ASNase treatment compared with isogenic cells. Overall, by affecting several cellular programs, Erw-ASNase makes MM cells more vulnerable to carfilzomib, providing proof of concept for clinical use of this combination as a novel strategy to enhance PI sensitivity in MM patients.

The proteostasis guardian HSF1 directs the transcription of its paralog and interactor HSF2 during proteasome dysfunction.

Protein homeostasis is essential for life in eukaryotes. Organisms respond to proteotoxic stress by activating heat shock transcription factors (HSFs), which play important roles in cytoprotection, longevity and development. Of six human HSFs, HSF1 acts as a proteostasis guardian regulating stress-induced transcriptional responses, whereas HSF2 has a critical role in development, in particular of brain and reproductive organs. Unlike HSF1, that is a stable protein constitutively expressed, HSF2 is a labile protein and its expression varies in different tissues; however, the mechanisms regulating HSF2 expression remain poorly understood. Herein we demonstrate that the proteasome inhibitor anticancer drug bortezomib (Velcade), at clinically relevant concentrations, triggers de novo HSF2 mRNA transcription in different types of cancers via HSF1 activation. Similar results were obtained with next-generation proteasome inhibitors ixazomib and carfilzomib, indicating that induction of HSF2 expression is a general response to proteasome dysfunction. HSF2-promoter analysis, electrophoretic mobility shift assays, and chromatin immunoprecipitation studies unexpectedly revealed that HSF1 is recruited to a heat shock element located at 1.397bp upstream from the transcription start site in the HSF2-promoter. More importantly, we found that HSF1 is critical for HSF2 gene transcription during proteasome dysfunction, representing an interesting example of transcription factor involved in controlling the expression of members of the same family. Moreover, bortezomib-induced HSF2 was found to localize in the nucleus, interact with HSF1, and participate in bortezomib-mediated control of cancer cell migration. The results shed light on HSF2-expression regulation, revealing a novel level of HSF1/HSF2 interplay that may lead to advances in pharmacological modulation of these fundamental transcription factors.

Carfilzomib, lenalidomide, and dexamethasone (KRd) versus bortezomib, lenalidomide, and dexamethasone (VRd) for initial therapy of newly diagnosed multiple myeloma (NDMM): Results of ENDURANCE (E1A11) phase III trial.

LBA3 Background: Bortezomib (btz) combined with lenalidomide (len) and dexamethasone (dex) (VRd) is a standard initial therapy for NDMM. Carfilzomib (cfz), a next-generation proteasome inhibitor, in combination with len-dex (KRd) has shown higher efficacy in phase II trials. This randomized phase III trial was designed to examine if KRd improves progression free survival (PFS) compared to VRd in NDMM (current results), and whether indefinite maintenance with len improves OS compared with two-year maintenance (to be analyzed once data matures). Methods: Patients (Pts) with NDMM, were randomized to receive VRd or KRd in a 1:1 fashion for 36 weeks followed by a second randomization (1:1) to indefinite versus two years of len maintenance. Pts without del17p, t (14;16), t(14;20), plasma cell leukemia or high-risk GEP70 profile, were enrolled. VRd arm included btz 1.3 mg/m2 on days(d) 1, 4, 8, and 11 (d 1, 8 for cycles 9-12), len 25 mg d 1-14, and dex 40 mg d 1, 2, 4, 5, 8, 9, 11, 12 of a 3-week (wk) cycle for 12 cycles, while pts in the KRd arm received cfz 36 mg/m2 d 1, 2, 8, 9, 15, 16 with len 25 mg daily on d 1-21 and dex 40 mg wkly, in 4 wk cycles for 9 cycles. Maintenance included len 15mg d 1-21 every 4 wks. The study was designed to detect a hazard ratio (HR)=0.75 with 80% power at 1-sided 2.5% alpha and 399 PFS events (progression or death regardless of intervening therapy). Results: The study accrued 1087 pts (VRd=542, KRd=545). The median age was 65y. Treatment, efficacy, and toxicity data are in the table. At the second of 3 planned interim analyses, with PFS HR=1.04 (95% CI, 0.8 to 1.3, p=0.74), futility was met. Median PFS was VRd=34.4m and KRd=34.6m; no differences were seen based on age (&lt;65 or ≥65), presence or absence of t(4;14) or ISS stage. The three-year OS (95% CI) was similar: VRd 84% (80 to 88) and KRd 86% (82 to 89). Conclusions: In this randomized phase 3 trial, KRd did not improve PFS compared with VRd in NDMM. A significantly higher rate of cardio-pulmonary and renal toxicity was observed with KRd, while neuropathy rates were higher with VRd. VRd remains the standard triplet induction regimen in standard and intermediate risk NDMM, and a suitable backbone for 4 drug combinations. Clinical trial information: NCT01863550 . [Table: see text]

Relapse after Allogeneic Hematopoietic Cell Transplantation for Multiple Myeloma: Survival Outcomes and Factors Influencing Them.

Many patients with multiple myeloma (MM) eventually relapse even after allogeneic hematopoietic cell transplantation (alloHCT) for curative intent.Over the past decade, outcomes for patients with MM have improved significantly with the availability of new therapies, including next-generation proteasome inhibitors, immunomodulatory agents, and, more recently, monoclonal antibodies. Although several published studies have evaluated the outcomes of alloHCT for MM, the data on survival outcomes in patients with MM experiencing disease relapse following alloHCT are limited. In addition, the predictors for postrelapse survival in these patients are not known. In this study, we examined the outcomes of a single-center cohort of 60 patients with MM who experienced relapse or progression after alloHCT. In addition, we evaluated the use of salvage regimens for treatment of relapsed MM and analyzed the predictors for improved postrelapse survival. After a median follow-up of 2.2 years from the time of relapse, the median duration of postrelapse survival was 1.8 years (95% confidence interval [CI], 1.2 to 5.0 years). Patients received a median of 3 lines of therapy (range, 0 to 10) for treatment of MM beyond the post-alloHCT relapse/progression. Multivariate analysis identified cytogenetic risk (standard risk versus high risk; hazard ratio [HR], .34; P=.01), time to relapse after alloHCT (>12 months versus ≤12 months: HR, .41; P=.04), and occurrence of acute graft-versus-host disease (GVHD) before relapse (GVHD versus no GVHD: HR, 2.89; P=.01) significantly affected postrelapse survival. These data illustrate that long-term myeloma control and survival is attainable in those relapsing/progressing after alloHCT and suggest that the synergism between novel therapies and the allogeneic immune platform is the key to improved survival in this high-risk patient population.

Proteasome Inhibitors for the Treatment of Multiple Myeloma.

Use of proteasome inhibitors (PIs) has been the therapeutic backbone of myeloma treatment over the past decade. Many PIs are being developed and evaluated in the preclinical and clinical setting. The first-in-class PI, bortezomib, was approved by the US food and drug administration in 2003. Carfilzomib is a next-generation PI, which selectively and irreversibly inhibits proteasome enzymatic activities in a dose-dependent manner. Ixazomib was the first oral PI to be developed and has a robust efficacy and favorable safety profile in patients with multiple myeloma. These PIs, together with other agents, including alkylators, immunomodulatory drugs, and monoclonal antibodies, have been incorporated into several regimens. This review summarizes the biological effects and the results of clinical trials investigating PI-based combination regimens and novel investigational inhibitors and discusses the future perspective in the treatment of multiple myeloma.

The Zinc-Finger AN1-Type Domain 2a Gene Acts as a Regulator of Cell Survival in Human Melanoma: Role of E3-Ligase cIAP2.

The zinc-finger AN1-type domain-2a gene, also known as AIRAP (arsenite-inducible RNA-associated protein), was initially described as an arsenite-inducible gene in Caenorhabditis elegans and mammalian cells. Differently from the AIRAP worm homologue, aip-1, a gene known to play an important role in preserving animal lifespan and buffering arsenic-induced proteotoxicity, mammals have a second, constitutively expressed, AIRAP-like gene (AIRAPL), recently implicated in myeloid transformation. We have identified human AIRAP as a canonical heat-shock gene, whose expression, differently from AIRAPL, is strictly dependent on the proteotoxic-stress regulator heat-shock factor 1 (HSF1). AIRAP function is still not well defined and there is no information on AIRAP in cancer. Herein we show that bortezomib and next-generation proteasome inhibitors ixazomib and carfilzomib markedly induce AIRAP expression in human melanoma at concentrations comparable to plasma-levels in treated patients. AIRAP-downregulation leads to bortezomib sensitization, whereas AIRAP-overexpression protects melanoma cells from the drug, identifying AIRAP as a novel HSF1-regulated marker of chemotherapy resistance. More importantly, this study unexpectedly revealed that, also in the absence of drugs, AIRAP-silencing hinders melanoma clonogenic potential and spheroid growth, promoting caspase activation and apoptotic cell death, an effect independent of AIRAPL and linked to downregulation of the antiapoptotic protein cIAP2. Interestingly, AIRAP was found to interact with cIAP2, regulating its stability in melanoma. Taken together, the results identify AIRAP as a novel HSF1-dependent regulator of prosurvival networks in melanoma cells, opening new therapeutic perspectives in chemoresistant melanoma treatment. IMPLICATIONS: The findings identify ZFAND2A/AIRAP as a novel stress-regulated survival factor implicated in the stabilization of the antiapoptotic protein cIAP2 and as a new potential therapeutic target in melanoma.

Monoclonal Antibody Therapies in Multiple Myeloma: A Challenge to Develop Novel Targets.

The treatment options in multiple myeloma (MM) has changed dramatically over the past decade with the development of novel agents such as proteasome inhibitors (PIs); bortezomib and immunomodulatory drugs (IMiDs); thalidomide, and lenalidomide which revealed high efficacy and improvement of overall survival (OS) in MM patients. However, despite these progresses, most patients relapse and become eventually refractory to these therapies. Thus, the development of novel, targeted immunotherapies has been pursued aggressively. Recently, next-generation PIs; carfilzomib and ixazomib, IMiD; pomalidomide, histone deacetylase inhibitor (HDADi); panobinostat and monoclonal antibodies (MoAbs); and elotuzumab and daratumumab have emerged, and especially, combination of mAbs plus novel agents has led to dramatic improvements in the outcome of MM patients. The field of immune therapies has been accelerating in the treatment of hematological malignancies and has also taken center stage in MM. This review focuses on an overview of current status of novel MoAb therapy including bispecific T-cell engager (BiTE) antibody (BsAb), antibody-drug conjugate (ADC), and chimeric antigen receptor (CAR) T cells, in relapsed or refractory MM (RRMM). Lastly, investigational novel MoAb-based therapy to overcome immunotherapy resistance in MM is shown.

Overexpression of immunoproteasome low-molecular-mass polypeptide 7 and inhibiting role of next-generation proteasome inhibitor ONX 0912 on cell growth in glioma.

The aim of this study was to determine the expression level of immunoproteasome and its clinical significance in glioma preliminarily. Furthermore, we studied the function and molecular mechanism of proteasome inhibitor ONX 0912 on glioma cell. The expression of immunoproteasome in glioma and tumor-adjacent brain tissues was detected by western blot. Immunohistochemical technique was used to detect the expression of low-molecular-mass polypeptide 7 in 55 cases of glioma tissues and 6 cases of tumor-adjacent brain tissues. Chi-square test was used to analyze the relationship between the expression level of low-molecular-mass polypeptide 7 and clinical characteristics. Kaplan-Meier method and Cox regression analysis were applied to analyze the correlation between low-molecular-mass polypeptide 7 expression and prognosis of patients. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium) (MTS) proliferation assay was introduced to detect the impact of ONX 0912 on proliferation of glioma cells. Western blot was used to detect the apoptosis- and autophagy-related protein in glioma cell treated with ONX 0912. Our results showed that only low-molecular-mass polypeptide 7 expression was notably upregulated in gliomas in comparison with tumor-adjacent brain tissues and further increased in malignant gliomas compared with benign gliomas (P < 0.01). In the multivariate Cox proportional regression analyses, it was evident that low-molecular-mass polypeptide 7 was an independent unfavorable prognostic factor (P < 0.05). The results of MTS assay showed that ONX 0912 could inhibit the proliferation of glioma cell. Besides, we found that ONX 0912 could prompt apoptosis and autophagosome accumulation, which may be responsible for inhibiting glioma cell proliferation. In conclusion, our results indicated that low-molecular-mass polypeptide 7 might be a candidate prognostic biomarker, and proteasome inhibitor ONX 0912 might act as a potential treatment agent for glioma.

New proteasome inhibitors in the management of multiple myeloma

The landscape of multiple myeloma treatment transformed at the last 15 years by the introduction of novel agents (bortezomib, lenalidomide) and wide application of autologous hematopoietic stem cell transplantation, which have prolonged the survival of multiple myeloma patients. Despite the fact that multiple myeloma remains an incurable disease due to the new options, the median overall survival of patients with multiple myeloma in Russia in 2006–2016 was about 55–68 months. Drug resistance and clonal evolution remain a problem. The novel proteasome inhibitors (carfilzomib, ixazomib) differ in chemical structure and pharmacological characteristics. Thereby the next-generation proteasome inhibitor (IPs)-based regimens (KRd (carfilzomib, lenalidomide, dexamethasone), IRd (ixazomib, lenalidomide, dexamethasone), and Kd (carfilzomib, dexamethasone)) are emerging as new standards for the treatment of patients with relapsed and/or refractory multiple myeloma. In a randomized trial phase 3 ENDEAVOR, carfilzomib demonstrated improved survival in direct comparison to bortezomib. The dose-dependent activity of carfilzomib demonstrated in the study of A.R.R.O.W. Аctivity of ixazomib is comparable to that of bortezomib, the oral method of administration and the absence of neurological toxicity, allow for long-term control of the disease. The new PIs are an important advance in relapsed and/or refractory multiple myeloma treatment, increasing survival, response rate and quality of life, even in subgroups of patients with poor prognosis. This review summarizes the main pharmacological properties, mechanisms of action and clinical outcomes of major clinical studies with these agents. A separate issue discusses the problem of overcoming new proteasome inhibitors of drug resistance to bortezomib.

The Deubiquitinase Inhibitor b-AP15 and Its Effect on Phenotype and Function of Monocyte-Derived Dendritic Cells

The ubiquitin-proteasome system is elementary for cellular protein degradation and gained rising attention as a new target for cancer therapy due to promising clinical trials with bortezomib, the first-in class proteasome inhibitor meanwhile approved for multiple myeloma and mantle cell lymphoma. Both bortezomib and next-generation proteasome inhibitors mediate their effects by targeting the 20S core particle of the 26S proteasome. The novel small molecule inhibitor b-AP15 affects upstream elements of the ubiquitin-proteasome cascade by suppressing the deubiquitinase activity of both proteasomal regulatory 19S subunits and showed promising anticancer activity in preclinical models. Nonetheless, effects of inhibitors on the ubiquitin-proteasome system are not exclusively restricted to malignant cells: alteration of natural killer cell-mediated immune responses had already been described for drugs targeting either 19S or 20S proteasomal subunits. Moreover, it has been shown that bortezomib impairs dendritic cell (DC) phenotype and function at different levels. In the present study, we comparatively analyzed effects of bortezomib and b-AP15 on monocyte-derived DCs. In line with previous results, bortezomib exposure impaired maturation, antigen uptake, migration, cytokine secretion and immunostimulation, whereas treatment with b-AP15 had no compromising effects on these DC features. Our findings warrant the further investigation of b-AP15 as an alternative to clinically approved proteasome inhibitors in the therapy of malignancies, especially in the context of combinatorial treatment with DC-based immunotherapies.

Development of Novel Epoxyketone-Based Proteasome Inhibitors as a Strategy To Overcome Cancer Resistance to Carfilzomib and Bortezomib.

Over the past 15 years, proteasome inhibitors (PIs), namely bortezomib, carfilzomib (Cfz) and ixazomib, have significantly improved the overall survival and quality-of-life for multiple myeloma (MM) patients. However, a significant portion of MM patients do not respond to PI therapies. Drug resistance is present either de novo or acquired after prolonged therapy through mechanisms that remain poorly defined. The lack of a clear understanding of clinical PI resistance has hampered the development of next-generation PI drugs to treat MM patients who no longer respond to currently available therapies. Here, we designed and synthesized novel epoxyketone-based PIs by structural modifications at the P1' site. We show that a Cfz analog, 9, harboring a hydroxyl substituent at its P1' position was highly cytotoxic against cancer cell lines displaying de novo or acquired resistance to Cfz. These results suggest that peptide epoxyketones incorporating P1'-targeting moieties may have the potential to bypass resistance mechanisms associated with Cfz and to provide additional clinical options for patients resistant to Cfz.

MP51-09 COBICISTAT, A POTENT CYP3A4 INHIBITOR, ACTS SYNERGISTICALLY WITH OPROZOMIB TO CAUSE ENDOPLASMIC RETICULUM STRESS IN BLADDER CANCER CELLS

INTRODUCTION AND OBJECTIVES:Inducing endoplasmic reticulum (ER) stress is a novel approach to cancer treatment. The next-generation proteasome inhibitor oprozomib acts against cancer by increasing ...

Carfilzomib for Treatment of Refractory Chronic Gvhd: A Chronic GVHD Consortium Pilot Trial

Advances are needed in the therapy of treatment-refractory chronic graft-vs.-host disease (GVHD). Prior experimental and clinical data suggest proteasome inhibition may have immunomodulatory activity relevant to GVHD. Carfilzomib is a specific, irreversible, next-generation proteasome inhibitor with extensive experience in the therapy of multiple myeloma. To explore the safety and activity of carfilzomib in advanced chronic GVHD, we conducted a pilot multi-center phase II trial through the Chronic GVHD Consortium. Carfilzomib was administered at 20 mg/m2 on day 1, and then 36mg/m2 on days 8 and 15 of a 28-day treatment cycle (cycle 1), and then 36 mg/m2 on days 1, 8, and 15 of a 28-day treatment cycle (cycles 2-6). The primary endpoint was 6 month treatment failure, a composite endpoint including death, relapse, and requirement for an additional line of systemic immune suppressive (IS) therapy. Considering a historical 6 month treatment failure rate of 44%, this 20 subject pilot trial allowed estimation of the 6 month treatment failure rate with a standard error of 10 percentage points. Secondary objectives included safety, physician- and NIH-based response rates, failure-free survival, and patient-reported outcomes. Key eligibility criteria included age ≥ 18, NIH Consensus defined chronic GVHD, and failure of at least one prior line of systemic IS therapy. A total of 20 subjects were enrolled at 4 institutions. Median age was 53 years. Median time from transplant to enrollment was 2.8 years (IQR 1.7-5), and from chronic GVHD onset to enrollment 1.5 years (IQR 0.5-3.7). Chronic GVHD was NIH moderate (30%) or severe (70%), predominantly classic (90% vs. overlap 10%), and involved multiple diverse organ sites. Prior lines of systemic therapy for chronic GVHD were ≤ 2 (n=6, 30%) or ≥ 3 (n=14, 70%). Doses were held primarily for infection (50% of total held doses); only 4 patients (20%) completed all planned 6 months of carfilzomib. SAEs occurred in 40% of subjects, and 7 deaths occurred among study participants occurring between 0.3-9 months after last carfilzomib dose (none attributed to carfilzomib). The 6 month treatment failure rate was not significantly improved vs. the historical benchmark (40% vs. 44%, p=0.36), figure 1. NIH response rates at 3, 6, and 12 months are presented in figure 2. Overall survival at 6 and 12 months was 79% and 58%. Failure-free survival at 12 months was 32%. These pilot phase II data suggest that carfilzomib therapy in this very advanced chronic GVHD population did not improve expected 6 month treatment failure rates achieved under conventional practices and is not recommended for further study for this indication.