Modeling Proteasome Inhibition in Lymphoma

Abstract Abstract 4946 The proteasome inhibitor Bortezomib (Bz) has been widely used to treat multiple myeloma, relapsed mantle cell lymphoma and is undergoing clinical evaluation for other B cell malignancies including non-Hodgkin lymphoma. Despite its initial success, patients treated with Bz eventually relapse due to the development of drug resistance. Therefore, understanding the basis of drug resistance is a critical component for improved therapy. The acquisition of Bz resistance in lymphomas, particularly those with constitutive expression of the B cell-specific DNA mutator, activation-induced cytidine deaminase (AID), has not been previously characterized. We have utilized the AID-expressing human non-Hodgkin Burkitt's lymphoma as a model system for this study. Burkitt's lymphoma lines (Ramos and BL-2) are suitable because they are highly sensitive to Bz induced apoptosis with an IC50 of approximately 11 nM after 48 hrs of treatment. In order to generate Bz resistant Burkitt's lymphoma lines, Ramos cells were treated weekly with increasing concentrations of Bz for 3 months. Compared to the parental line, this newly formed line displayed an approximately 2.5–3-fold increase in IC50 to Bz as well as to three other proteasome inhibitors (next-generation proteasome inhibitor, MLN 2238, epoxomicin and carfilzomib), while maintaining sensitivity to different chemotherapeutic agents (PD 0332991 cyclin 4/6 dependent kinase inhibitor and melphalan). In this model system, resistance to Bz conferred a general cross-resistance to other proteasome inhibitors, a phenotype that has been stably maintained for 5 months. We next asked whether AID plays a role in the acquisition of Bz resistance in Ramos cells by promoting hypermutation and genomic instability. In support of this hypothesis, the G322A and C326T mutations in the gene encoding the proteasome subunit and target of Bz, psmb5, occur within AID hotspots raising the possibility that AID could directly mutate psmb5. Furthermore, like many proteins, AID is degraded by the proteasome arguing that proteasome inhibition further stabilizes AID protein resulting in aberrant hypermutation. Consistent with this, we detected by flow cytometry (intracellular staining) a 2-fold increase in AID protein following a 24-hour, 20 nM Bz treatment of Ramos cells. AID normally mutates immunoglobulin (Ig) genes and these mutations are necessary for the production of protective antibodies, while aberrant AID activity leads to mutations in non-Ig genes. Surprisingly, despite the increase in AID protein, we observe reduced mutation frequency within the functional IgH gene following 3-month Bz treatment compared to untreated controls. Despite this reduction, array comparative genomic hybridization (a-CGH) studies indicate copy number abnormalities in Bz resistant cells and details of the chromosomal abnormalities and target genes deregulated will be presented. These data demonstrate that Burkitt's lymphoma cells are sensitive to Bz and drug resistance can be readily achieved in vitro. Furthermore, Bz treatment stabilizes AID protein and promotes increased genomic instability. Disclosures: Stessman: Millennium: The Takeda Oncology Company: Research Funding. Mansoor:Millennium: The Takeda Oncology Company: Research Funding. Van Ness:Millennium: The Takeda Oncology Company: Research Funding.

Download Full-text

Rapid induction of p62 and GABARAPL1 upon proteasome inhibition promotes survival before autophagy activation

The Journal of Cell Biology ◽

10.1083/jcb.201708168 ◽

2018 ◽

Vol 217 (5) ◽

pp. 1757-1776 ◽

Cited By ~ 29

Author(s):

Zhe Sha ◽

Helena M. Schnell ◽

Kerstin Ruoff ◽

Alfred Goldberg

Keyword(s):

Proteasome Inhibitor ◽

Neuroblastoma Cells ◽

Proteasome Inhibitors ◽

Proteasome Inhibition ◽

Nuclear Factor ◽

Ubiquitinated Proteins ◽

Rapid Induction ◽

Independent Mechanism ◽

Transcription Factor Nuclear Factor ◽

Inhibitor Treatment

Proteasome inhibitors are used as research tools and to treat multiple myeloma, and proteasome activity is diminished in several neurodegenerative diseases. We therefore studied how cells compensate for proteasome inhibition. In 4 h, proteasome inhibitor treatment caused dramatic and selective induction of GABARAPL1 (but not other autophagy genes) and p62, which binds ubiquitinated proteins and GABARAPL1 on autophagosomes. Knockdown of p62 or GABARAPL1 reduced cell survival upon proteasome inhibition. p62 induction requires the transcription factor nuclear factor (erythroid-derived 2)-like 1 (Nrf1), which simultaneously induces proteasome genes. After 20-h exposure to proteasome inhibitors, cells activated autophagy and expression of most autophagy genes by an Nrf1-independent mechanism. Although p62 facilitates the association of ubiquitinated proteins with autophagosomes, its knockdown in neuroblastoma cells blocked the buildup of ubiquitin conjugates in perinuclear aggresomes and of sumoylated proteins in nuclear inclusions but did not reduce the degradation of ubiquitinated proteins. Thus, upon proteasome inhibition, cells rapidly induce p62 expression, which enhances survival primarily by sequestering ubiquitinated proteins in inclusions.

Download Full-text

Mechanistic Insights into the Enhancement of Adeno-Associated Virus Transduction by Proteasome Inhibitors

Journal of Virology ◽

10.1128/jvi.01826-13 ◽

2013 ◽

Vol 87 (23) ◽

pp. 13035-13041 ◽

Cited By ~ 25

Author(s):

Angela M. Mitchell ◽

R. Jude Samulski

Keyword(s):

Cysteine Protease ◽

Proteasome Inhibitor ◽

Proteasome Inhibitors ◽

Proteasome Inhibition ◽

Proteasome Activity ◽

Protease Inhibition ◽

Adeno Associated Virus

Proteasome inhibitors (e.g., bortezomib, MG132) are known to enhance adeno-associated virus (AAV) transduction; however, whether this results from pleotropic proteasome inhibition or off-target serine and/or cysteine protease inhibition remains unresolved. Here, we examined recombinant AAV (rAAV) effects of a new proteasome inhibitor, carfilzomib, which specifically inhibits chymotrypsin-like proteasome activity and no other proteases. We determined that proteasome inhibitors act on rAAV through proteasome inhibition and not serine or cysteine protease inhibition, likely through positive changes late in transduction.

Download Full-text

Neurodegeneration Induced by Bortezomib Exposure in Vitro Occurs Via Proteasome Independent Mechanisms.

Blood ◽

10.1182/blood.v114.22.2859.2859 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2859-2859

Author(s):

Shirin Arastu-Kapur ◽

Andrew J. Ball ◽

Janet L. Anderl ◽

Mark K Bennett ◽

Christopher J Kirk

Keyword(s):

Peripheral Nerve ◽

Proteasome Inhibitor ◽

Serine Proteases ◽

Proteasome Inhibitors ◽

Proteasome Inhibition ◽

Nerve Degeneration ◽

Differentiated Cells ◽

Favorable Safety ◽

Neurite Degeneration

Abstract Abstract 2859 Poster Board II-835 BACKGROUND: The dipeptide boronate proteasome inhibitor bortezomib (BTZ; Velcade®) is approved for the treatment of multiple myeloma and non-Hodgkin's lymphoma. Bortezomib-induced peripheral neuropathy (BIPN, Blood (2008)112:1593-1599) is seen in ∼30% of BTZ-treated patients and can result in dose reductions and discontinuations that may result in suboptimal levels of proteasome inhibition. Carfilzomib (CFZ), a tetrapeptide epoxyketone, is a selective and irreversible proteasome inhibitor that is structurally and mechanistically distinct from bortezomib. Single agent treatment with CFZ has demonstrated strong activity in relapsed and refractory myeloma and a favorable safety profile in Phase 2 trials (ASH2008:864 & 865). Importantly treatment-emergent PN was seen at low levels and did not result in dose modifications or discontinuations. The disparate safety data for these proteasome inhibitors suggest that non-proteasomal mechanisms may underlie BIPN. Using activity-based probes in peripheral blood mononuclear cell (PBMC) lysates, we previously demonstrated inhibition of non-proteasomal proteases by BTZ and other proteasome inhibitors with a boronate pharmacophore (EHA2009:0939). However, the involvement of the proteasome in the peripheral nerve degeneration and BIPN in BTZ-treated myeloma patients remains to be established. AIMS: To establish an in vitro model of peripheral nerve degeneration and to determine the effects of proteasome inhibition by BTZ and CFZ on neurite outgrowth and cell survival. METHODS: SH-SY5Y neuroblastoma cells were differentiated by long term culture in retinoic acid and brain derived nerve growth factor to induce neurite outgrowth. The effects of proteasome inhibitors were measured by high content image analysis of fluorescent images for cell survival (Hoechst nuclear counterstain) and neurite degeneration (FITC-mouse anti-beta-III-tubulin). Phase contrast images were also collected to observe morphological effects and gross cell death. Cell viability and proteasome inhibition was measured in undifferentiated and differentiated cells. The MEROPS (peptidase) database was mined for candidate serine proteases with a P1 selectivity of Leu/Phe/Tyr to identify candidate off-targets CFZ and BTZ and candidate proteases were validated by standard biochemical and cell biology techniques. RESULTS: In differentiated SH-SY5Y cells, the average neurite length decreased by 33% following 24 hr exposure to 10nM BTZ but was unaffected by the same concentration of CFZ. Proteasome inhibition as determined by a fluorescent substrate for the chymotrypsin-like activity was equivalent (∼70%) after a 24 hr exposure for both compounds in differentiated cells, suggesting that neurodegeneration involves non-proteasomal pathways. With 72 hrs continuous exposure, BTZ was 10-fold more potent than CFZ at inducing neurodegeneration. Furthermore, in both undifferentiated and differentiated SH-SY5Y cells, BTZ was 5-fold more cytotoxic than CFZ. Database mining for serine proteases with a selectivity for Leu/Phe/Tyr at P1 was used to identify other potential BTZ targets that might underlie neurotoxicity. One candidate is HtrA2 (also called Omi), an inducible mitochondrial serine protease whose activity protects neurons from stress induced apoptosis (Hum Mol Genet (2005) 14(5):2099-2111). HtrA2 levels increased 2-fold in SH-SY5Y cells treated with either BTZ or CFZ for 6 hrs at 40 nM. Using a gel based assay and purified enzyme preparations, BTZ inhibited HtrA2 activity with an IC50 ∼ 4 nM, equivalent to its activity against the proteasome. In contrast, Carfilzomib did not inhibit HtrA2 at the highest concentration tested (10 mM). CONCLUSIONS: These data demonstrate that BTZ induces neuronal cell death and neurite degeneration in vitro by proteasome-independent mechanisms. We propose that combined inhibition of the proteasome and HtrA2 by BTZ may underlie peripheral nerve toxicities in vitro and may be involved in BIPN in myeloma patients. In this model, CFZ, which mediates equivalent proteasome inhibition to BTZ in neurons, does not induce neurodegeneration due to inactivity against HtrA2. Future profiling of non-proteasomal targets of BTZ, including HtrA2 activity, in patient samples is merited. These results suggest that the favorable safety profile of CFZ in myeloma patients may be a result of its high selectivity for proteasomal proteases. Disclosures: Arastu-Kapur: Proteolix, Inc: Employment. Ball:Millipore Corp: Employment. Anderl:Millipore Corp: Employment. Bennett:Proteolix: Employment. Kirk:Proteolix, Inc: Employment.

Download Full-text

Efficacy of a Brief, Cyclophosphamide-Intensive Regimen for Older Patients with Newly Diagnosed Burkitt's or Atypical Burkitt's Lymphoma/Leukemia.

Blood ◽

10.1182/blood.v114.22.2685.2685 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2685-2685

Author(s):

Yvette L. Kasamon ◽

Robert A. Brodsky ◽

Michael J. Borowitz ◽

Pamela A. Crilley ◽

Richard F. Ambinder ◽

...

Keyword(s):

Overall Survival ◽

Median Time ◽

Older Patients ◽

Research Funding ◽

Tumor Lysis Syndrome ◽

B Cell Lymphoma ◽

Burkitt’S Lymphoma ◽

Burkitt's Lymphoma ◽

High Dose ◽

Newly Diagnosed

Abstract Abstract 2685 Poster Board II-661 Background: Although standard therapies cure most adolescents and young adults with Burkitt's lymphoma/leukemia (BL), older patients (pts) have an inferior prognosis with an estimated 1-year survival of 50%. The inferior outcome is attributable to both insufficient efficacy and excess toxicity. Cyclophosphamide (Cy) has long been recognized to be arguably the most active agent in BL. Prior work at our institution showed that high-dose Cy, equivalent to transplantation doses, could be given without stem cell rescue with minimal toxicity even in older pts. Patients and Methods: A phase II trial for pts age ≥ 30, based on intensive Cy and incorporating rituximab but no anthracycline, was developed with a primary endpoint of 1-year overall survival. Entry requirements included newly diagnosed BL or atypical BL; any performance status (PS); HIV negative; and no significant cardiac dysfunction. Renal failure, even if necessitating dialysis, was permitted if it was acute. Treatment consisted of 3 cycles, with successive cycles beginning on day 15 or when ANC was ≥ 500/μL. Cycles 1 and 2 consisted of Cy 1500 mg/m2 IV day 1; vincristine 1.4 mg/m2 (2 mg cap) day 1; prednisone 100 mg days 1-5; rituximab 375 mg/m2 IV days 1 and 8; methotrexate 3 g/m2 IV day 8 with leucovorin rescue; cytarabine 100 mg intrathecally days 1, 4, and 11; and filgrastim. Cycle 3 consisted of rituximab 375 mg/m2 IV day 1; high-dose Cy (50 mg/kg IV days 2, 3, 4, and 5) with uroprotection; filgrastim; and rituximab 375 mg/m2 IV weekly for 4 weeks once ANC was ≥ 1000/μL. Eligibility for cycle 3 included ECOG PS < 4; no disease progression or uncontrolled meningeal disease; not on dialysis; and transaminases ' 5X upper limit of normal. Results: A prespecified interim analysis of the first 12 of a planned 20 evaluable pts is presented. Diagnosis was BL in 8 and atypical BL/unclassifiable high-grade lymphoma with features intermediate between BL and diffuse large B-cell lymphoma in 4. Median age was 56 (range 34 – 75), 8/12 (67%) had Ann Arbor stage III/IV disease, and all were high-risk by Magrath's criteria. PS ranged from 0 to 4. Two pts received hemodialysis on presentation. For all pts, actuarial event-free survival and overall survival (Figure) are 66% and 75%, respectively, at both 1 year and 2 years after treatment initiation. Three pts died during cycle 1: tumor lysis syndrome on day 1, neutropenic sepsis on day 8, multiorgan failure on day 46 after respiratory arrest on day 20. All of the other 9 pts completed protocol therapy: 8 (89%) achieved anatomic CR/CRu as well as a complete metabolic response by PET, and are event-free at a median of 29 months (range < 1 – 44 months) after therapy completion. The remaining pt had residual marrow disease followed by progression and is in remission 1 year after myeloablative allogeneic BMT. Adverse events in these 9 pts included 7 neutropenic fevers; 1 non-neutropenic bacteremia; and 1 self-limited episode of pericarditis with rapid atrial fibrillation. Grade 3 peripheral neuropathy was limited to 2 pts. The planned dose intensity was achievable: median time to cycle 2 was 15 days (14 – 21), and median time from start of cycle 1 to start of cycle 3 was 31 days (28 – 35). Median time to neutrophil recovery after the last dose of Cy was 16 days (10 – 21); median time to platelets ≥ 20,000/μL, without transfusion in the preceding week, was 22 days (0 – 30). Early stopping criteria for response or all-cause mortality have not been met. Conclusion: A very short regimen based on intensive Cy without anthracycline produces a high rate of durable CR's in older, poorer-risk pts with BL or atypical BL. Disclosures: Kasamon: Genentech: Research Funding. Swinnen:Genentech: Consultancy, Research Funding; Enzon: Consultancy; Abbot: Consultancy, Research Funding.

Download Full-text

Phase 1 Clinical Trial of the Novel Structure Proteasome Inhibitor NPI-0052.

Blood ◽

10.1182/blood.v114.22.2693.2693 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2693-2693 ◽

Cited By ~ 2

Author(s):

Andrew Spencer ◽

Michael Millward ◽

Paul Mainwaring ◽

Simon Harrison ◽

Laurence Catley ◽

...

Keyword(s):

Solid Tumor ◽

Whole Blood ◽

Proteasome Inhibitor ◽

Mononuclear Cells ◽

Proteasome Inhibitors ◽

Proteasome Inhibition ◽

Pharmacokinetic Data ◽

Employment Equity ◽

Cognitive Changes ◽

Equity Ownership

Abstract Abstract 2693 Poster Board II-669 Background: NPI-0052 is a proteasome inhibitor with a novel bicyclic structure (other proteasome inhibitors in clinical use are peptide based). Preclinical studies indicate rapid, broad and prolonged inhibition of all 3 catalytic sites of the proteasome, and subsequently unique proteasome inhibition, signal transduction, toxicology and efficacy profiles. Taken together these suggest the potential for improvements in therapeutic ratio and activity in hematologic and solid tumor malignancies. Materials and Methods: Patients with solid tumor, lymphoma, leukemia or myeloma diagnoses without standard treatment options have been treated with IV NPI-0052 on one of two arms (weekly or twice weekly) in this 3+3 design dose escalation study. This is followed by 10 patient Recommended Phase 2 dose Cohorts of patients with lymphomas, CLL and myeloma respectively. Proteasome inhibition (pharmacodynamics) and pharmacokinetics are also assayed in whole blood, and proteasome inhibition in peripheral blood mononuclear cells (PBMC). Results: 44 patients have been treated with NPI-0052 at doses ranging from 0.075 mg/m2 to 0.9 mg/m2. Common adverse events include fatigue, parosmia/dysgeusia, transient peri-infusion site pain, lymphopenia, headaches, dizziness / unsteady gait, closed-eye visuals, cognitive changes. Incidence and grade of these events correlate with dose, being quite tolerable at the MTD of 0.7 mg/m2 on the weekly dosing arm. An MTD has not yet been determined for the twice weekly dosing arm. Pharmacokinetic data has demonstrated a rapid elimination half-life (<20 minutes) and relatively large volume of distribution. Assessment of proteasome inhibition has demonstrated increasing inhibition of chymotrypsin-like activity of up to 88% Day 1 and 100% Day 15. Inhibition of caspase-like and trypsin-like activity of up to 52% and 71% respectively has also been seen. Inhibition remains between doses in whole blood (principally RBC), but recovers between doses in PBMC. Clinical benefit, including stable disease, regression or response, was reported in patients with mantle cell lymphoma, myeloma, Hodgkin's lymphoma, cutaneous marginal zone lymphoma, follicular lymphoma, sarcoma, prostate carcinoma and melanoma. Conclusions: NPI-0052 produces dose-dependent pharmacologic effects through the predicted efficacious range, while producing a toxicity profile that is dissimilar to what is reported with other proteasome inhibitors (notably deficient in peripheral neuropathy, neutropenia and thrombocytopenia) in spite of producing equal or greater proteasome inhibition. These data indicate a broad range of potential uses, and led to additional studies in hematologic malignancies and solid tumors alone and in combination. Disclosures: Longenecker: Nereus Pharmaceuticals: Employment. Palladino:Nereus Pharmaceuticals: Employment, Equity Ownership. Lloyd:Nereus Pharmaceuticals: Employment, Equity Ownership. Neuteboom:Nereus Pharmaceuticals: Employment, Equity Ownership. Spear:Nereus Pharmaceuticals: Employment, Equity Ownership.

Download Full-text

The Proteasome Inhibitor Bortezomib Stimulates Osteoblastic Differentiation of Human Osteoblast Precursor Cells Via Upregulation of Vitamin D Receptor Signaling

Blood ◽

10.1182/blood.v116.21.1899.1899 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1899-1899

Author(s):

Martin Kaiser ◽

Maren Mieth ◽

Orhan Sezer ◽

Ulrike Heider

Keyword(s):

Multiple Myeloma ◽

Vitamin D ◽

Proteasome Inhibitor ◽

Research Funding ◽

Fold Increase ◽

Osteoblastic Differentiation ◽

Luciferase Reporter ◽

Human Osteoblast ◽

Myeloma Cells ◽

Osteoblast Precursors

Abstract Abstract 1899 Introduction In multiple myeloma (MM), interactions of the malignant plasma cell clone with the bone marrow microenvironment lead to an enhanced osteoclast recruitment and impaired osteoblast activity. The proteasome inhibitor bortezomib has been shown to suppress osteoclast activity, and there is recent evidence that bortezomib enhances osteoblast differentiation. The aim of this study was to investigate the effects of bortezomib on human osteoblast precursors, focusing on vitamin D (VD) dependent osteoblastic differentiation. Since vitamin D receptor (VDR) is degraded by the proteasome, we hypothesized that bortezomib could influence its signaling and hence vitamin D induced osteoblastic differentiation. This might be of clinical importance, since an increased rate of vitamin D deficiency has recently been reported in patients with MM. Methods Primary human mesenchymal stem cells (hMSC) and primary human osteoblasts (hOB) were isolated from bone marrow aspirates or from bone fragments of healthy donors undergoing orthopedic surgery, respectively. Ascorbic acid and β-glycerolphosphate were used for osteoblastic stimulation (OS), either in combination with or without vitamin D. In order to analyze the effects of proteasome inhibition on osteoblastic differentiation and activity, hMSC and hOB were incubated with bortezomib at subapoptotic doses (1 - 5 nM). In addition, coculture experiments of hMSC, hOB and myeloma cells were performed. Expression of osteocalcin and osteopontin (OPN) were quantified by real-time PCR as markers of osteoblastic lineage differentiation. Expression of VDR was analyzed by western blot in subcellular fractions and VDR signaling was investigated using luciferase reporter assays. Results In coculture experiments, myeloma cells inhibited the vitamin D dependent differentiation and activity of osteoblast precursors, e.g. coculture of hMSC with the myeloma cell line LP-1 for 4 days decreased their osteocalcin expression by 58%. Treatment with bortezomib led to an increased osteoblastic differentiation of hMSC and hOB by OS, represented by an enhanced expression of osteoblast markers osteocalcin and OPN. Importantly, this effect could be further increased, when vitamin D was added. In hMSC stimulated with OS only, addition of 5 nM bortezomib led to an 18.3 fold increase in OPN mRNA expression. In comparison, hMSC stimulated with OS + vitamin D showed a 27.5 fold increase in OPN mRNA with the addition of bortezomib. Osteocalcin expression was increased 1.9 fold by bortezomib in the presence of OS and vitamin D, but not with OS alone. Similar results were obtained with osteoblasts: Incubation with bortezomib slightly increased osteocalcin and OPN mRNA expression in cells stimulated with OS only (1.3 fold and 2.4 fold, respectively). In comparison, in cells stimulated with OS and vitamin D, bortezomib elevated osteocalcin and OPN expression 2.9 fold and 5.5 fold, respectively. Bortezomib led to an increase in nuclear VDR levels in hMSC in western blot analyses. Primary hMSC transfected with a VDR luciferase reporter construct showed a 3.7 fold increase in VDR signaling with bortezomib. Conclusion Our data show that bortezomib stimulates osteoblastic differentiation of hMSCs and hOBs and acts, at least in part, through VDR signaling. Substitution of vitamin D in multiple myeloma patients treated with bortezomib may be beneficial for bone turnover and needs clinical evaluation. Disclosures: Kaiser: Johnson & Johnson: Research Funding. Mieth:Johnson & Johnson: Research Funding. Sezer:Johnson & Johnson: Research Funding. Heider:Johnson & Johnson: Research Funding.

Download Full-text

Investigational Agent MLN2238/MLN9708, a Specific, Orally Available, Small Molecule Proteasome Inhibitor, Shows Promising In Vitro Activity Against Multiple Myeloma Cell Lines

Blood ◽

10.1182/blood.v116.21.3014.3014 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3014-3014

Author(s):

Giada Bianchi ◽

Vijay G. Ramakrishnan ◽

Teresa Kimlinger ◽

Jessica Haug ◽

S. Vincent Rajkumar ◽

...

Keyword(s):

Multiple Myeloma ◽

Er Stress ◽

Cell Lines ◽

Small Molecule ◽

Proteasome Inhibitor ◽

Research Funding ◽

Proteasome Inhibitors ◽

Biologically Active ◽

Investigational Agent

Abstract Abstract 3014 Background: Proteasome inhibitors have proven particularly effective in treatment of multiple myeloma, the second most frequent hematologic malignancy in the western world. Bortezomib, the first in class proteasome inhibitor in clinical use, was first approved in 2003 via fast FDA track, given the remarkable activity shown during phase II clinical trials. Nevertheless, more than 50% of multiple myeloma patients did not respond to single agent bortezomib when administered as second line agent. Moreover, bortezomib is only available for intravenous administration, representing a cumbersome therapy for patients, and its use is limited by significant toxicities (especially peripheral neuropathy). MLN9708 (Millennium Pharmaceuticals, Inc.), an investigational orally available, small molecule, is a potent, specific and reversible inhibitor of the 20S proteasome. It is currently under clinical investigation for the treatment of hematologic and non-hematologic malignancies. Upon exposure to aqueous solutions or plasma, MLN9708 rapidly hydrolyzes to MLN2238, the biologically active form, and MLN2238 was used for all of the preclinical studies reported here. In vitro biochemistry studies have shown that MLN2238 has a faster dissociation rate from the proteasome compared to bortezomib, and in vivo studies of MLN2238 have shown antitumor activity in a broader range of tumor xenografts when compared to bortezomib. Given these encouraging preclinical results, we set to investigate the anti-myeloma activity of MLN2238 in vitro. Results: MLN2238 proved to have anti-proliferative and pro-apoptotic activity against a broad range of MM cell lines with EC50 at 24 hours ranging between 10 and 50 nM, even in relatively resistant MM cell lines (OPM2, DOX6, RPMI, etc.). In MM.1S cells, induction of apoptosis was time and dose dependent and related to activation of both caspase 8 and 9. When compared to MM.1S treated for 24 hours with EC50 dose of bortezomib, treatment with EC50 dose of MLN2238 resulted in the same extent of caspases cleavage occurring at an earlier time point (8-12 hours), possibly suggesting more rapid onset and/or irreversibility of apoptosis in cells treated with MLN2238. Treatment with MLN2238 was associated with early, but persistent induction of endoplasmic reticulum (ER) stress with BiP being induced 2–4 hours after treatment with EC50 dose and gradually increasing over time. While bortezomib has been associated with early induction and late decrease in proteins involved in ER stress, MLN2238 appears to induce a persistent rise in these factors, suggesting either more sustained proteasome blockade with stabilization of proteasome substrates or de-novo induction of unfolded protein response (UPR) genes. MLN2238 also proved effective in reducing phosphorylation of ERK1-2 with no overall alteration in the total ERK level, thus accounting for the observed reduction in proliferation upon treatment. Preliminary data indicate potential for additive and synergistic combination with widely used drugs, including doxorubicin and dexamethasone. Conclusion: While further clinical data are needed to establish the effectiveness of MLN2238 in the treatment of multiple myeloma, these preliminary nonclinical data, together with the favorable biochemical and pharmacokinetic properties, including oral bioavailability, make the investigational agent MLN9708 an appealing candidate for treatment of multiple myeloma. Further in vitro data could help establish whether a difference in the apoptotic mechanisms exist between MLN2238 and other proteasome inhibitors, primarily bortezomib, and could also help inform combination treatment approaches aimed at increasing effectiveness, overcoming bortezomib resistance and decreasing toxicity. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Research Funding; Novartis: Research Funding; Genzyme: Consultancy, Research Funding; Cephalon: Research Funding.

Download Full-text

Selective Inhibition of the proteasome's β2 Catalytic Subunit Alone Does Not Induce Cytotoxicity, but Resensitizes Bortezomib-Refractory Myeloma Cells for Bortezomib Treatment

Blood ◽

10.1182/blood.v118.21.2915.2915 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2915-2915

Author(s):

Marianne Kraus ◽

Bobby Florea ◽

Jürgen Bader ◽

Nan Li ◽

Paul Geurink ◽

...

Keyword(s):

Er Stress ◽

Cell Lines ◽

Active Site ◽

Proteasome Inhibitor ◽

Proteasome Inhibitors ◽

Proteasome Inhibition ◽

Selective Inhibition ◽

Catalytic Site ◽

Myeloma Cells ◽

Bortezomib Treatment

Abstract Abstract 2915 Bortezomib is a reversible first-generation proteasome inhibitor that inhibits the β5 and to a lesser extent the β1 catalytic site of the proteasome. However, bortezomib does not inhibit the β2 catalytic proteasomal site at clinically relevant concentrations, and bortezomib-resistance is accompanied by upregulation of the β2 subunit, suggesting that increased β2 activity may compensate for the loss of β1/ β5 activity during bortezomib-treatment. The second generation proteasome inhibitor carfilzomib, due to the chemistry of its epoxyketone warhead, has a higher substrate specificity and functions as an irreversible proteasome inhibitor, but is still a β1/ β5 inhibitor that does not affect the β2 active site. We investigated the effect of β2-specific proteasome inhibition on myeloma and acute myeloid leukemia (AML) cells and tested the hypothesis that β2-selective proteasome inhibition may overcome bortezomib-resistance. To this end we have developed a set of epoxyketone- and vinylsulfone-based, cell permeable proteasome inhibitors of which we selected the compounds PR523A and PR671A for further testing in cell-based assays. PR671A is a peptide-vinylsulfone that selectively inhibits the proteasome's β2/ β2i subunit in an irreversible fashion in human cell lines and primary cells at low micromolar concentrations without inhibition of other protease species. PR523A is a β5-selective peptide-epoxyketone with otherwise similar properties. Treatment of myeloma and AML cell lines (AMO-1, U-266, HL-60, THP-1) with PR523A induced ER-stress mediated apoptosis, very similar to bortezomib. The combination of bortezomib with PR523A led to additive, but not synergistic induction of apoptosis, as expected. Selective β2 inhibition by PR671A resulted in the induction of ER stress and the accumulation of poly-ubiquitinated protein, however, this was not effectively translated into apoptotic cell death. This indicates that selective inhibition of the β2 proteasome subunit alone has only a poor cytotoxic effect on myeloma and AML cell lines, suggesting that the function of β2 is largely redundant and can be compensated when the remaining proteasome catalytic subunits (β1 and β5) remain active. However, when the β2 inhibitor PR671A was combined with agents that target the proteasome's β5 active site (PR523A) or the β5 and the β1 site (bortezomib), the combination of either inhibitor with the β2 inhibitor PR671A was highly synergistic for both activation of ER stress and the induction of apoptotic death. Importantly, the bortezomib-resistance in bortezomib-adapted myeloma and AML cell lines could be overcome by combining PR671A with either bortezomib or PR523A, while β2 inhibition by PR671A alone had no effect on the viability of bortezomib-adapted cells. We conclude that PR671A is a β2 selective proteasome inhibitor. Selective Inhibition of the proteasome's β2 subunit has little effect on viability or ER stress both in normal and bortezomib-resistant myeloma and leukemia cells, suggesting that the function of the β2 catalytic site is largely redundant. However, when β1/ β5 proteasome activity is inhibited by drugs like bortezomib or carfilzomib, proper function of the β2 proteasome active site is crucial for cell survival, also in bortezomib-resistant myeloma cells. The use of specific β2 inhibitors like PR671A in combination with β1/ β5 inhibitors like bortezomib or carfilzomib is therefore a promising strategy to overcome resistance against β1/ β5-selective proteasome inhibitors. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

The Novel Proteasome Inhibitors Carfilzomib and Oprozomib Induce Milder Degenerative Effects Compared To Bortezomib When Administered Via Oral Feeding In An In Vivo Drosophila Experimental Model: A Biological Platform To Evaluate Safety/Efficacy Of Proteasome Inhibitors

Blood ◽

10.1182/blood.v122.21.1930.1930 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1930-1930

Author(s):

Evangelos Terpos ◽

Eleni N. Tsakiri ◽

Efstathios Kastritis ◽

Tina Bagratuni ◽

Vassilis G. Gorgoulis ◽

...

Keyword(s):

Oral Administration ◽

Proteasome Inhibitor ◽

Germ Line ◽

Conflicts Of Interest ◽

Proteasome Inhibitors ◽

Proteasome Inhibition ◽

Somatic Tissue ◽

Oral Feeding ◽

Somatic Tissues

Abstract The proteasome is involved in the degradation of both normal, short-lived ubiquitinated proteins and mutated or damaged proteins. Carfilzomib is a tetrapeptide epoxyketone–based proteasome inhibitor and oprozomib is an orally bioavailable tripeptide epoxyketone-based proteasome inhibitor. The primary target for both agents is the chymotrypsin-like β5 subunit of the constitutive proteasome and immunoproteasome. Oprozomib is 5-fold less potent than carfilzomib, but displays similar cytotoxic potential with longer exposure times due to its time-dependent proteasome inhibition. In contrast, bortezomib is a slowly reversible proteasome inhibitor with potency of proteasome inhibition similar to carfilzomib. We propose the fruit fly Drosophila melanogaster as an in vivo platform for screening and characterizing proteasome inhibitors at the whole organism level. Drosophilais well-suited to this line of investigation, due to its powerful genetics, its similarities in key metabolic and aging pathways with humans, the fact that it expresses proteasomes that structurally resemble those from mammals, and also because it comprises a soma-germ line demarcation composed of both post-mitotic and mitotic cells. Moreover, flies live for few months and thus, drug screening on large cohorts can be completed in a reasonable time. We validate our model by investigating the effects of orally administered carfilzomib and oprozomib vs. bortezomib. In isolated Drosophila proteasome in vitroassays, carfilzomib showed a pattern of inhibitory activity similar to bortezomib, whereas oprozomib was less effective. After continuous oral administration of the inhibitors (∼50 μM of carfilzomib and ∼300-400 μM of oprozomib) to young flies (by adding the inhibitor in the flies’ culture medium) a proteasome inhibitory effect in somatic tissues roughly similar to 1 μM bortezomib was induced. Similar findings were noted when we analyzed distinct somatic tissue parts (i.e., head, thorax and abdomen), indicating that orally administered proteasome inhibitors are equally distributed to different body parts. As in the case of bortezomib, the effects of the inhibitors were less pronounced in the reproductive tissues. At the molecular level, carfilzomib (as compared to bortezomib) induced a milder disruption of fly somatic tissue proteostasis, lower rates of somatic tissue oxidative stress and less intense activation of genomic antioxidant response elements that correlated with reduced intensities of proteasome genes and protein subunit upregulation. Proteasome subunit induction was found to depend on the activity of the transcription factor Nrf2, a master regulator of cellular anti-oxidant responses. Furthermore, carfilzomib promoted the induction of lysosomal enzymes (e.g. cathepsins) and autophagy-related genes but less intensively compared to bortezomib. At concentrations that induced rates of proteasome inhibition that were similar to bortezomib, there were no significant toxic effects of either carfilzomib or oprozomib to oogenesis or to embryogenesis. Compared to bortezomib, both inhibitors exerted a significantly milder impact on the neuromusculatory system (locomotor performance) of the flies. Finally, we found that sustained oral administration of either carfilzomib or oprozomib exerted significantly milder effects (as compared to bortezomib) on flies’ mortality rate, healthspan and overall longevity. Our in vivo data support that carfilzomib is significantly less toxic compared to bortezomib, including neuromusculatory toxicity. Oprozomib was also less toxic but it is worth noting that it showed reduced activity against fly proteasomes. In support, our preliminary analyses indicated that in comparison to bortezomib and carfilzomib, oprozomib was less potent when tested in human osteosarcoma cancer cell lines. The validity of our in vivo pharmacological model is exemplified by the observed similarities with the reported clinical adverse effects, while the ratio of the different doses used to achieve similar rates of proteasome inhibition in Drosophila somatic tissues (i.e. ∼1 μM bortezomib, ∼50 μM carfilzomib) is reminiscent of the doses used in the clinic (i.e. ∼1.3 mg/m2 bortezomib and ∼25-56 mg/m2 carfilzomib). We conclude that fruit flies represent a valid biological platform for evaluating the efficacy and toxicity of proteasome inhibitors. Disclosures: No relevant conflicts of interest to declare.

Download Full-text