Autoimmunity Against Surfactant Protein B Is Associated with Pneumonitis During Checkpoint Blockade.

RATIONALE: Immune checkpoint inhibitor-related pneumonitis is a serious autoimmune event affecting up to 20% of patients with non-small cell lung cancer, yet the factors underpinning its development in some patients and not others are poorly understood. OBJECTIVES: To investigate the role of autoantibodies and autoreactive T cells against surfactant-related proteins in the development of pneumonitis. METHODS: The study cohort consisted of non-small cell lung cancer patients who gave blood samples before and during immune checkpoint inhibitor treatment. Serum was used for proteomics analyses and to detect autoantibodies present during pneumonitis. T cell stimulation assays and single-cell RNA sequencing were performed to investigate the specificity and functionality of peripheral autoreactive T cells. The findings were confirmed in a validation cohort comprising patients with non-small cell lung cancer and patients with melanoma. MEASUREMENTS AND MAIN RESULTS: Across both cohorts, patients who developed pneumonitis had higher pre-treatment levels of immunoglobulin G autoantibodies targeting surfactant protein-B. At the onset of pneumonitis, these patients also exhibited higher frequencies of CD4+ interferon-gamma-positive surfactant protein B-specific T cells, and expanding T cell clonotypes recognizing this protein, accompanied by a pro-inflammatory serum proteomic profile. CONCLUSIONS: Our data suggest that the co-occurrence of surfactant protein-B-specific immunoglobulin G autoantibodies and CD4+ T cells is associated with the development of pneumonitis during ICI therapy. Pre-treatment levels of these antibodies may represent a potential biomarker for elevated risk of developing pneumonitis and on-treatment levels may provide a diagnostic aid. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

A first-in-class Wiskott-Aldrich syndrome protein activator with anti-tumor activity in hematologic cancers.

Hematological cancers are among the most common cancers in adults and children. Despite significant improvements in therapies, many patients still succumb to the disease. Therefore, novel therapies are needed. The Wiskott-Aldrich syndrome protein (WASp) family regulates actin assembly in conjunction with the Arp2/3 complex, a ubiquitous nucleation factor. WASp is expressed exclusively in hematopoietic cells and exists in two allosteric conformations: autoinhibited or activated. Here, we describe the development of EG-011, a first-in-class small molecule activator of the WASp auto-inhibited form. EG-011 possesses in vitro and in vivo anti-tumor activity as a single agent in lymphoma, leukemia, and multiple myeloma, including models of secondary resistance to PI3K, BTK, and proteasome inhibitors. The in vitro activity was confirmed in a lymphoma xenograft. Actin polymerization and WASp binding was demonstrated using multiple techniques. Transcriptome analysis highlighted homology with drugs-inducing actin polymerization.

Prevention and management of adverse events during treatment with bispecific antibodies and CAR T cells in multiple myeloma: a consensus report of the European Myeloma Network.

T-cell redirecting bispecific antibodies (BsAbs) and chimeric antigen receptor T cells (CAR T cells) have revolutionised multiple myeloma therapy, but adverse events such as cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome (ICANS), cytopenias, hypogammaglobulinaemia, and infections are common. This Policy Review presents a consensus from the European Myeloma Network on the prevention and management of these adverse events. Recommended measures include premedication, frequent assessing for symptoms and severity of cytokine release syndrome, step-up dosing for several BsAbs and some CAR T-cell therapies; corticosteroids; and tocilizumab in the case of cytokine release syndrome. Other anti-IL-6 drugs, high-dose corticosteroids, and anakinra might be considered in refractory cases. ICANS often arises concomitantly with cytokine release syndrome. Glucocorticosteroids in increasing doses are recommended if needed, as well as anakinra if the response is inadequate, and anticonvulsants if convulsions occur. Preventive measures against infections include antiviral and antibacterial drugs and administration of immunoglobulins. Treatment of infections and other complications is also addressed.

High-dose carfilzomib achieves superior anti-tumor activity over low-dose and recaptures response in relapsed/refractory multiple myeloma resistant to lowdose carfilzomib by co-inhibiting the ß2 and ß1 subunits of the proteasome complex.

Optimal carfilzomib dosing is a matter of debate. We analyzed the inhibition profiles of proteolytic proteasome subunits ß5, ß2 and ß1 after low-dose (20/27 mg/m2) versus high-dose (≥36 mg/m2) carfilzomib in 103 pairs of peripheral blood mononuclear cells from patients with relapsed/refractory (RR) multiple myeloma (MM). ß5 activity was inhibited (median inhibition >50%) in vivo by 20 mg/m2, whereas ß2 and ß1 were co-inhibited only by 36 and 56 mg/m2, respectively. Coinhibition of ß2 (P=0.0001) and ß1 activity (P=0.0005) differed significantly between high-dose and low-dose carfilzomib. Subsequently, high-dose carfilzomib showed significantly more effective proteasome inhibition than low-dose carfilzomib in vivo (P=0.0003). We investigated the clinical data of 114 patients treated with carfilzomib combinations. High-dose carfilzomib demonstrated a higher overall response rate (P=0.03) and longer progression-free survival (PFS) (P=0.007) than low-dose carfilzomib. Therefore, we escalated the carfilzomib dose to ≥36 mg/m2 in 16 patients who progressed during low-dose carfilzomib-containing therapies. High-dose carfilzomib recaptured response (≥ partial remission) in nine (56%) patients with a median PFS of 4.4 months. Altogether, we provide the first in vivo evidence in RRMM patients that the molecular activity of high-dose carfilzomib differs from that of low-dose carfilzomib by coinhibition of ß2 and ß1 proteasome subunits and, consequently, high-dose carfilzomib achieves a superior anti-MM effect than low-dose carfilzomib and recaptures the response in RRMM resistant to low-dose carfilzomib. The optimal carfilzomib dose should be ≥36 mg/m2 to reach a sufficient anti-tumor activity, while the balance between efficacy and tolerability should be considered in each patient.

Alcohol-abuse drug disulfiram targets cancer via p97 segregase adaptor NPL4.

Cancer incidence is rising and this global challenge is further exacerbated by tumour resistance to available medicines. A promising approach to meet the need for improved cancer treatment is drug repurposing. Here we highlight the potential for repurposing disulfiram (also known by the trade name Antabuse), an old alcohol-aversion drug that has been shown to be effective against diverse cancer types in preclinical studies. Our nationwide epidemiological study reveals that patients who continuously used disulfiram have a lower risk of death from cancer compared to those who stopped using the drug at their diagnosis. Moreover, we identify the ditiocarb-copper complex as the metabolite of disulfiram that is responsible for its anti-cancer effects, and provide methods to detect preferential accumulation of the complex in tumours and candidate biomarkers to analyse its effect on cells and tissues. Finally, our functional and biophysical analyses reveal the molecular target of disulfiram's tumour-suppressing effects as NPL4, an adaptor of p97 (also known as VCP) segregase, which is essential for the turnover of proteins involved in multiple regulatory and stress-response pathways in cells.

Outcome of older myeloma patients in relationship to comorbidities and availability of second-generation novel agents in a real-life setting.

Major improvements in outcome of older patients with multiple myeloma (MM) have been achieved with the introduction of novel agents. Their impact in real-life treatment of older patients is unclear. In this single center retrospective study, we analyzed the outcome of patients >65 years treated with first-generation (FGNA) and second-generation novel agents (SGNA) within two time periods 2012-2014 and 2015-2017. Patients were analyzed based on age, Charlson Comorbidity Index (CCI), International Staging System stage, year of diagnosis and withdrawal of agents due to toxicities. Overall 96 patients were included for analysis. Median age was 73 years (range 65-90), 55 (57%) patients were 65-75 years and 41 (43%) were >75 years old. 84 patients received a first-line therapy, whereas 45 patients had ≥2 lines of systemic therapy. 20 patients were consolidated with autologous stem cell transplantation. 12 patients had no systemic therapy at all. In 17 of 21 cases a FGNA and in 4 of 21 a SGNA was withdrawn due to toxicity. Median overall survival (OS) for all patients with systemic therapy was 4.75 years (95% CI, 3.05-NA). Borderline significant improvement of OS was observed in patients diagnosed 2015-2017 compared to 2012-2014 with HR 0.57 (95% CI, 0.31-1.02) p = 0.06. OS significantly differed for comorbid patients with low and intermediate risk CCI, HR 1.94 (95% CI, 1.07-3.54), p = 0.03 in the overall population. OS in patients treated with SGNA was not significantly different in patients with intermediate versus low risk CCI (HR 1.48 (95% CI 0.43-5.14, p = 0.54)). In conclusion, we found a trend toward improved survival for older MM patients after the introduction of novel agents during the observed time period. In patients treated with SGNA a smaller effect that comorbidity negatively affects survival was observed.

Treatment of relapsed and refractory multiple myeloma: recommendations from the International Myeloma Working Group.

This Policy Review presents the International Myeloma Working Group's clinical practice recommendations for the treatment of relapsed and refractory multiple myeloma. Based on the results of phase 2 and phase 3 trials, these recommendations are proposed for the treatment of patients with relapsed and refractory disease who have received one previous line of therapy, and for patients with relapsed and refractory multiple myeloma who have received two or more previous lines of therapy. These recommendations integrate the issue of drug access in both low-income and middle-income countries and in high-income countries to help guide real-world practice and thus improve patient outcomes.

Impaired proteolysis by SPPL2a causes CD74 fragment accumulation that can be recognized by anti-CD74 autoantibodies in human ankylosing spondylitis.

Ankylosing spondylitis (AS) is associated with autoantibody production to class II MHC-associated invariant chain peptide, CD74/CLIP. In this study, we considered that anti-CD74/CLIP autoantibodies present in sera from AS might recognize CD74 degradation products that accumulate upon deficiency of the enzyme signal peptide peptidase-like 2A (SPPL2a). We analyzed monocytes from healthy controls (n = 42), psoriatic arthritis (n = 25), rheumatoid arthritis (n = 16), and AS patients (n = 15) for SPPL2a enzyme activity and complemented the experiments using SPPL2a-sufficient and -deficient THP-1 cells. We found defects in SPPL2a function and CD74 processing in a subset of AS patients, which culminated in CD74 and HLA class II display at the cell surface. These findings were verified in SPPL2a-deficient THP-1 cells, which showed expedited expression of MHC class II, total CD74 and CD74 N-terminal degradation products at the plasma membrane upon receipt of an inflammatory trigger. Furthermore, we observed that IgG anti-CD74/CLIP autoantibodies recognize CD74 N-terminal degradation products that accumulate upon SPPL2a defect. In conclusion, reduced activity of SPPL2a protease in monocytes from AS predisposes to endosomal accumulation of CD74 and CD74 N-terminal fragments, which, upon IFN-γ-exposure, is deposited at the plasma membrane and can be recognized by anti-CD74/CLIP autoantibodies.

HIV protease inhibitors for the treatment of multiple myeloma.

Outcomes in multiple myeloma (MM) patients have improved in recent years owing to the introduction of new drugs. Among them, proteasome inhibitors and immunomodulatory imide drugs have become central in the management of newly diagnosed and relapsed MM. However, resistance to these classes of agents develops in most patients and ultimately leads to death from relapsed/refractory disease. A need exists for new classes of antimyeloma drugs, especially ones that are active in the multirefractory setting. The conventional drug development process, which involves extensive preclinical and clinical testing prior to assessment of clinical activity, has fallen short in delivering adequately safe and active novel drug candidates. HIV protease inhibitors such as nelfinavir are safe, US Food and Drug Administration-approved agents that have been shown to have potent antimyeloma activity in both preclinical models and patients with refractory disease. The repurposing of HIV protease inhibitors for treatment of MM is promising in light of their antimyeloma activity in conjunction with their global availability, established safety, and relatively low cost. This review will summarize the preclinical and clinical data available on HIV protease inhibitors for the treatment of refractory MM.

European Myeloma Network recommendations on tools for the diagnosis and monitoring of multiple myeloma: what to use and when.

The diagnosis of multiple myeloma can be challenging, even for experienced physicians, and requires close collaboration between numerous disciplines (orthopedics, radiology, nuclear medicine, radiation therapy, hematology and oncology) before the final diagnosis of myeloma is made. The definition of multiple myeloma is based on the presence of clinical, biochemical, histopathological, and radiological markers of disease. Specific tests are needed both at presentation and during follow-up in order to reach the correct diagnosis and characterize the disease precisely. These tests can also serve prognostic purposes and are useful for follow-up of myeloma patients. Molecular analyses remain pivotal for defining high-risk myeloma and are used in updated patient stratifications, while minimal residual disease assessment via flow cytometry, molecular techniques and radiological approaches provides additional prognostic information on patients' long-term outcome. This pivotal information will guide our future treatment decisions in forthcoming clinical trials. The European Myeloma Network group updated their guidelines on different diagnostic recommendations, which should be of value to enable appropriate use of the recommendations both at diagnosis and during follow-up.

Improving the efficacy of proteasome inhibitors in the treatment of renal cell carcinoma by combination with the human immunodeficiency virus (HIV)-protease inhibitors lopinavir or nelfinavir.

OBJECTIVES: To assess the potential of second-generation proteasome inhibition by carfilzomib and its combination with the human immunodeficiency virus (HIV) protease inhibitors (HIV-PIs) lopinavir and nelfinavir in vitro for improved treatment of clear cell renal cell cancer (ccRCC). MATERIALS AND METHODS: Cytotoxicity, reactive oxygen species (ROS) production, and unfolded protein response (UPR) activation of proteasome inhibitors, HIV-PIs, and their combination were assessed in three cell lines and primary cells derived from three ccRCC tumours by MTS assay, flow cytometry, quantitative reverse transcriptase-polymerase chain reaction and western blot, respectively. Proteasome activity was determined by activity based probes. Flow cytometry was used to assess apoptosis by annexin V/propidium iodide assay and ATP-binding cassette sub-family B member 1 (ABCB1) activity by MitoTracker™ Green FM efflux assay (Thermo Fisher Scientific, MA, USA). RESULTS: Lopinavir and nelfinavir significantly increased the cytotoxic effect of carfilzomib in all cell lines and primary cells. ABCB1 efflux pump inhibition, induction of ROS production, and UPR pre-activation by lopinavir were identified as underlying mechanisms of this strong synergistic effect. Combined treatment led to unresolved protein stress, increased activation of pro-apoptotic UPR pathway, and a significant increase in apoptosis. CONCLUSION: The combination of the proteasome inhibitor carfilzomib and the HIV-PIs lopinavir and nelfinavir has a strong synergistic cytotoxic activity against ccRCCin vitro at therapeutically relevant drug concentrations. This effect is most likely explained by synergistic UPR triggering and ABCB1-modulation caused by HIV-PIs. Our findings suggest that combined treatment of second-generation proteasome inhibitors and HIV-PIs should be investigated in patients with metastatic RCC within a clinical trial.

Bendamustine, lenalidomide and dexamethasone (BRd) has high activity as 2nd -line therapy for relapsed and refractory multiple myeloma - a phase II trial.

The combination of lenalidomide (Revlimid® , R) and dexamethasone (d) is a standard regimen for patients with relapsed/refractory multiple myeloma (rrMM). With this regimen, only a small fraction of patients will achieve high quality responses [≥ very good partial response (VGPR)]. The combination of bendamustine (B), lenalidomide and dexamethasone (BRd) has shown high efficacy in patients with advanced rrMM. However, dose-limiting haematotoxicity restricted its use in extensively pre-treated patient populations. This prospective, multicentre Phase II study evaluated the efficacy and safety of BRd in rrMM patients with one prior line of therapy. Fifty patients were enrolled (median age 68·5 years [range 46-83]) and were treated with B 75 mg/m2  days 1, 2; R 25 mg days 1-21 and d (40/20 mg) days 1, 8, 15 and 22, for 6 28-day induction cycles, followed by 12 cycles with Rd alone. Pegfilgrastim was administered according to protocol-defined criteria. The study aimed to demonstrate a complete response (CR)/VGPR rate of >40% after induction therapy. Of 45 evaluable patients, 23 (51%) achieved a CR/VGPR. Grade 4 neutropenia or thrombocytopenia occurred in 17 (34%) and 8 (16%) of patients, respectively. BRd is a safe and efficacious regimen as a second line treatment for rrMM, leading to high quality responses in a considerable proportion of patients.

Multiple myeloma: practice patterns across Europe.

Real-world data describing management of patients with multiple myeloma are limited. A European (Belgium, France, Germany, Italy, Spain, Switzerland, UK) observational chart review was conducted to address this. Physicians completed questionnaires for every patient seen during a 2-4-week observation period, regardless of treatment status. A total of 435 physicians completed 7635 cross-sectional chart reviews. Overall, 47% of patients were undergoing anti-tumour drug treatment, 42% had previously received ≥1 line of treatment and 12% had never received anti-tumour drug treatment. Of the patients treated by oncologists, onco-haematologists or internists, 95% received, or were expected to receive, at least one line of anti-tumour drug treatment, 61% received ≥2 lines of therapy and 38% received ≥3 lines. Except in the UK, the most commonly used induction therapies contained bortezomib (48%); lenalidomide was the most commonly used first-line maintenance therapy (45%) and second- and third-line agent overall (60% and 52% of patients at those lines, respectively). Bortezomib retreatment was used in 47% of patients who received it first line. Treatment patterns became more diverse with subsequent treatment lines. This study provides insight into real-world treatment patterns in Europe. While treatment practices are broadly similar across countries, some notable differences in the agents used exist.

Multiple myeloma: patient outcomes in real-world practice.

With increasing number of therapies available for the treatment of multiple myeloma, it is timely to examine the course of patients' journeys. We investigated patient characteristics, treatment durations and outcomes, and symptom burden across the treatment pathway in Belgium, France, Germany, Italy, Spain, Switzerland and the UK. In total, 435 physicians retrospectively reviewed 4997 patient charts. Profiles of patients diagnosed with multiple myeloma during the last 12 months were similar across countries; bone pain was the most common presentation. Median duration of first-line therapy was 6 months, followed by a median treatment-free interval of 10 months; both these decreased with increasing lines of therapy, as did time to progression. Depth of response, as assessed by the treating physician, also decreased with each additional line of therapy: 74% of patients achieved at least a very good partial response at first line, compared with only 11% at fifth line. Deeper responses were associated with longer time to progression, although these were physician-judged. Toxicities and co-morbidities increased with later treatment lines, and were more likely to have led to discontinuation of treatment. These real-world data provide an insight into patient outcomes and treatment decisions being made in clinical practice.

Treatment with the HIV protease inhibitor nelfinavir triggers the unfolded protein response and may overcome proteasome inhibitor resistance of multiple myeloma in combination with bortezomib: a phase I trial (SAKK 65/08).

Downregulation of the unfolded protein response mediates proteasome inhibitor resistance in multiple myeloma. The Human Immunodeficieny Virus protease inhibitor nelfinavir activates the unfolded protein response in vitro. We determined dose-limiting toxicity and recommended dose for phase II of nelfinavir in combination with the proteasome inhibitor bortezomib. Twelve patients with advanced hematologic malignancies were treated with nelfinavir (2500-5000 mg/day p.o., days 1-14, 3+3 dose escalation) and bortezomib (1.3 mg/m(2), days 1, 4, 8, 11; 21-day cycles). A run in phase with nelfinavir monotherapy allowed pharmakokinetic/pharmakodynamic assessment of nelfinavir in the presence or absence of concomittant bortezomib. End points included dose-limiting toxicity, activation of the unfolded protein response, proteasome activity, toxicity and response to trial treatment. Nelfinavir 2×2500 mg was the recommended phase II dose identified. Nelfinavir alone significantly up-regulated expression of proteins related to the unfolded protein response in peripheral blood mononuclear cells and inhibited proteasome activity. Of 10 evaluable patients in the dose escalation cohort, 3 achieved a partial response, 4 stable disease for 2 cycles or more, while 3 had progressive disease as best response. In an exploratory extension cohort with 6 relapsed, bortezomib-refractory, lenalidomide-resistant myeloma patients treated at the recommended phase II dose, 3 reached a partial response, 2 a minor response, and one progressive disease. The combination of nelfinavir with bortezomib is safe and shows promising activity in advanced, bortezomib-refractory multiple myeloma. Induction of the unfolded protein response by nelfinavir may overcome the biological features of proteasome inhibitor resistance. (clinicaltrials.gov identifier: 01164709).

Improved survival of older patients with multiple myeloma in the era of novel agents.

Multiple myeloma (MM) is the most common hematologic malignancy in Europe. Although remaining an incurable disease, substantial progress has been made within the last two decades. However, until recently, improvement in overall survival (OS) was only documented in younger, transplant-eligible patients. In this analysis, we retrospectively investigated the outcome of older patients with newly diagnosed MM in an unselected patient population with a special focus on the use of novel agents in a routine care community-based, non-university setting. A total of 107 patients older than 65 years of age or patients aged 60-65 years with relevant comorbidities precluding the use of autologous stem cell transplantation diagnosed with MM between 2000 and 2011 at the two largest non-university hospitals of Eastern Switzerland were analyzed. Patients were grouped into two six-year periods by date of initial diagnosis, 2000-2005 and 2006-2011. The median follow-up was 6.9 (range of 2.1 to 9.4) years. The median OS for the entire cohort was 3.0 years (95% confidence interval, 2.4-4.4). The median OS was significantly longer for patients in the 2006-2011 group (4.3 years) compared with the 2000-2005 group (2.6 years, p = 0.04). The 5-year estimated OS improved from 26% to 38%; 1-year survival was similar in both groups (86% in the 2000-2005 group and 84% in the 2006-2011 group respectively). The use of novel agents showed a statistically significant correlation with OS, whereas the impact of age was only of borderline significance. In conclusion, we demonstrate improved OS outcomes in an unselected population of older patients with MM during the last decade. This improvement is associated with an increased use of novel agents for the treatment of transplant-ineligible MM patients in daily clinical practice. Copyright © 2015 John Wiley & Sons, Ltd.

Development of new Malt1 inhibitors and probes.

Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (Malt1) is a promising therapeutic target for the treatment of activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL). Several research groups have reported on the development of Malt1 inhibitors and activity-based probes for in vitro and in situ monitoring and modulating Malt1 activity. In this paper, we report on two activity-based Malt1 probes (6 and 7) and a focused library of 19 new Malt1 inhibitors. Our peptide-based probe 6 labels Malt1 in an activity-based manner. In contrast, probe 7, derived from the known covalent inhibitor MI-2, labels both wild type and catalytically inactive Cys to Ala mutant Malt1, suggesting that MI-2 inhibits Malt1 by reacting with a nucleophilic residue other than the active site cysteine. Furthermore, two of our inhibitors (9, apparent IC50 3.0µM, and 13, apparent IC50 2.1µM) show good inhibitory activity against Malt1 and outperform MI-2 (apparent IC50 7.8µM) in our competitive activity-based protein profiling assay.

A Set of Activity-Based Probes to Visualize Human (Immuno)proteasome Activities.

Proteasomes are therapeutic targets for various cancers and autoimmune diseases. Constitutively expressed proteasomes have three active sites, ß1c, ß2c, and ß5c. Lymphoid tissues also express the immunoproteasome subunits ß1i, ß2i, and ß5i. Rapid and simultaneous measurement of the activity of these catalytic subunits would assist in the discovery of new inhibitors, improve analysis of proteasome inhibitors in clinical trials, and simplify analysis of subunit expression. In this work, we present a cocktail of activity-based probes that enables simultaneous gel-based detection of all six catalytic human proteasome subunits. We used this cocktail to develop specific inhibitors for ß1c, ß2c, ß5c, and ß2i, to compare the active-site specificity of clinical proteasome inhibitors, and to demonstrate that many hematologic malignancies predominantly express immunoproteasomes. Furthermore, we show that selective and complete inhibition of ß5i and ß1i is cytotoxic to primary cells from acute lymphocytic leukemia (ALL) patients.

The novel ß2-selective proteasome inhibitor LU-102 synergizes with bortezomib and carfilzomib to overcome proteasome inhibitor resistance of myeloma cells.

Proteasome inhibitor resistance is a challenge for myeloma therapy. Bortezomib targets the ß5 and ß1 activity, but not the ß2 activity of the proteasome. Bortezomib-resistant myeloma cells down-regulate the activation status of the unfolded protein response, and up-regulate ß2 proteasome activity. To improve proteasome inhibition in bortezomib-resistant myeloma and to achieve more efficient UPR activation, we have developed LU-102, a selective inhibitor of the ß2 proteasome activity. LU-102 inhibited the ß2 activity in intact myeloma cells at low micromolar concentrations without relevant co-inhibition of ß1 and ß5 proteasome subunits. In proteasome inhibitor-resistant myeloma cells, significantly more potent proteasome inhibition was achieved by bortezomib or carfilzomib in combination with LU-102, compared to bortezomib/carfilzomib alone, resulting in highly synergistic cytotoxic activity of the drug combination via endoplasmatic reticulum stress-induced apoptosis. Combining bortezomib/carfilzomib with LU-102 significantly prolonged proteasome inhibition and increased activation of the unfolded protein response and IRE1-a activity. IRE1-α has recently been shown to control myeloma cell differentiation and bortezomib sensitivity (Leung-Hagesteijn, Cancer Cell 24:3, 289-304). Thus, ß2-selective proteasome inhibition by LU-102 in combination with bortezomib or carfilzomib results in synergistic proteasome inhibition, activation of the unfolded protein response, and cytotoxicity, and overcomes bortezomib/carfilzomib resistance in myeloma cells in vitro.