Small Molecule Proteasome Inhibitor Research Articles

Clickable Probes for Pathogen Proteasomes: Synthesis and Applications.

The 20S proteasome is a multimeric protease complex that is essential for proteostasis in the cell. Small molecule proteasome inhibitors are approved drugs for various cancers and are advancing clinically as antiparasitics. Although tools and technologies to study the 20S proteasome have advanced, only one probe is commercially available to image proteasome activity. This probe consists of a fluorescently labeled, peptidyl vinyl sulfone that binds to one or more of the catalytic proteasome subunits. Here, we synthesized two, active site-directed epoxyketone probes, LJL-1 and LJL-2, that were based on the peptidyl backbones of the anticancer drugs, carfilzomib and bortezomib, respectively. Each probe was conjugated, via click chemistry, to a bifunctional group comprising 5-carboxytetramethylrhodamine (TAMRA) and biotin to, respectively, visualize and enrich the 20S proteasome from protein extracts of two eukaryotic pathogens, Leishmania donovani and Trichomonas vaginalis. Depending on species, each probe generated a different subunit-binding profile by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and the biotin tag enabled the enrichment of the bound subunits which were then formally identified by proteomics. Species differences in the order of electrophoretic migration by the β subunits were also noted. Finally, both probes reacted specifically with the 20S subunits in contrast to the commercial vinyl sulfone probe that cross reacted with cysteine proteases. LJL-1 and LJL-2 should find general utility in the identification and characterization of pathogen proteasomes, and serve as reagents to evaluate the specificity and mechanism of binding of new antiparasitic proteasome inhibitors.

CTNI-58. UPDATED RESULTS AND MOLECULAR SUBGROUP ANALYSES FROM THE RANDOMIZED PHASE 3 MIRAGE TRIAL ON MARIZOMIB IN PATIENTS WITH NEWLY DIAGNOSED GLIOBLASTOMA

Abstract BACKGROUND The standard of care for patients with newly diagnosed glioblastoma includes maximum safe resection followed by radiotherapy (RT) with concomitant and maintenance temozolomide (TMZ) chemotherapy (TMZ/RT→TMZ). Among potential new therapeutic strategies, targeting the proteasome has been considered promising because of its implication in numerous biological processes in tumors cells. Marizomib is a novel small molecule proteasome inhibitor that crosses the blood brain barrier and showed signs of activity in recurrent glioblastoma. METHODS EORTC 1709/CCTG CE.8 was a randomized, controlled, multicenter, open label phase 3 superiority trial. Major inclusion criteria were histologically confirmed newly diagnosed glioblastoma, age of 18 years or older and a Karnofsky performance status (KPS) > 70. Patients with tumors known to harbor an isocitrate dehydrogenase mutation were not eligible. RESULTS The trial was opened at 82 institutions in Europe, Canada and the US and 749 patients (of 750 planned) were randomized in a 1:1 ratio. Following a pre-planned interim analysis and a recommendation of the independent data monitoring committee, enrolment was discontinued. No significant difference in the median OS (mOS) was observed (standard arm: 17 months, marizomib arm: 16.5 months; p = 0.64; HR = 1.04). Median PFS was 6.0 vs. 6.3 months (p = 0.67; HR = 0.97). Patients with tumors harboring an unmethylated O6-methylguanine DNA methyltransferase (MGMT) promoter had a mOS of 14.5 months in the standard therapy group and 15.1 months in the marizomib arm (p = 0.27, HR = 1.13). Among patients with an MGMT promoter-methylated tumor, median OS was 29.4 months in the marizomib arm and 25.5 months in the standard arm (p = 0.41, HR = 0.86). More CTCAE grade 3/4 treatment-emergent adverse events were noticed in the marizomib arm than in the standard arm. CONCLUSIONS The addition of marizomib to standard temozolomide-based radiochemotherapy was associated with more toxicity but did not confer a survival benefit in glioblastoma patients, independent of the MGMT promoter methylation status.

Smurf2 inhibition enhances chemotherapy and radiation sensitivity in non-small-cell lung cancer

Lung cancer has been the most common cancer worldwide for several decades. The outcomes of patients with locally advanced lung cancer remain dismal, and only a minority of patients survive more than 5 years. However, tumor therapeutic resistance mechanisms are poorly studied. Identification of therapeutic resistance pathways in lung cancer in order to increase the sensitivity of lung tumor cells to therapeutic agents is a crucial but challenging need. To identify novel genes that modulate the response to platinum-based therapy, we performed a genome-wide high-throughput ribonucleic acid interference (RNAi) screen via transfection of human lung cancer (PC9) cells with a viral short hairpin RNA (shRNA) library. We further validated a potential target via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clonogenic survival assays on PC9 and A549 lung tumor cells transfected with small interfering RNAs (siRNAs) to successfully downregulate protein expression and then treated with increasing doses of cisplatin or X-ray radiation. We determined protein expression by immunohistochemistry (IHC) after chemoradiotherapy and analyzed gene expression-based survival outcomes in two cohorts of human non-small-cell lung cancer (NSCLC) patients. The screen identified several targets involved in epithelial-to-mesenchymal transition (EMT), including Smurf1, Smurf2, YAP1, and CEBPZ, and glycolytic pathway proteins, including PFKFB3. Furthermore, we found that the small molecule proteasome inhibitor bortezomib significantly downregulated Smurf2 in lung cancer cells. The addition of bortezomib in combination with cisplatin and radiation therapy in PC9 and A549 cells led to an increase in deoxyribonucleic acid (DNA) double-strand breaks with increased numbers of γ-H2AX-positive cells and upregulation of apoptosis. Finally, we found that Smurf2 protein expression was upregulated in situ after treatment with cisplatin and radiation therapy in a relevant cohort of patients with stage III NSCLC. Additionally, Smurf2 gene expression was the strongest predictor of survival in patients with squamous NSCLC after chemotherapy or chemoradiotherapy. We successfully identified and validated Smurf2 as both a common modulator of resistance and an actionable target in lung cancer. These results suggest the urgent need to investigate clinical Smurf2 inhibition via bortezomib in combination with cisplatin and radiation for patients with locally advanced NSCLC.

Daratumumab as Salvage Therapy in Pediatric Thoracic Organ Transplantation: An Addition to the Antibody Mediated Rejection Toolbox?

Purpose Antibody mediated rejection (AMR) is difficult to treat and often results in death or graft loss. Therapies targeted at antibodies or B cells are inadequate for decreasing donor specific antibodies (DSA), particularly to MHC class II. Bortezomib, a small molecule proteasome inhibitor targets plasma cells, but has many side effects and unsatisfactory results. Alternatively, we used Daratumumab (DAR), an anti-CD38 monoclonal antibody, in two pediatric transplant (Tx) recipients. Methods Chart review. Results An 18-year-old female with cystic fibrosis had a lung-liver Tx at 12-years-old. Twenty months post-Tx she developed severe AMR and Acute cellular rejection (ACR) treated with ATG, rituximab, TPE, photopheresis, and steroids. She ultimately required redo lung Tx. She did well for three years, but then developed AMR. She again received TPE, ATG, photopheresis, and rituximab. She was not considered a candidate for third lung Tx so all attempts were made to salvage her graft. She had no B cells, but continued to make DSA, so a plasma cell targeted therapy was desired. She received three doses of DAR 16 mg/kg on days 0, 7 and 21 with no infusion reactions. She developed neutropenia 2 months later which resolved with 3 weeks of GCSF. Both total PRA and DSA significantly decreased after DAR and have not returned 7 months later. PRA class I dropped from peak of 96% to 0, class II from peak 34% to 3%. She is maintained on subcutaneous IgG replacement for secondary hypogammaglobulinemia. Her graft function is poor, but has stabilized since DAR. She had one episode of presumed line associated sepsis and MSSA pneumonia without sequalae. A 3-year-old girl two years post heart Tx developed refractory AMR (by C4d and elevated DSA) despite ATG, TPE, IVIG, eculizumab, and ofatumumab. She then received two doses of DAR, 16 mg/kg each on days 0 and 14. She tolerated the infusions without any initial side effects. Not enough time has elapsed to follow up antibody levels or graft function. Conclusion Treatment for AMR remains challenging, especially when due to class II HLA antibodies. We safely administered DAR to two pediatric patients without infusion reactions or significant infectious complications to date. One patient had a decline in antibodies, while the other is too soon to determine. While more studies are needed, DAR may be a promising addition to the AMR toolbox.

Preclinical evaluation of TQB3602, a small molecule proteasome inhibitor clinical candidate.

e20546 Background: Proteasome inhibitors, selectively inhibit the activity of proteasome in ubiquitin proteasome system, together with immunomodulatory drugs formed the basis of many treatment regimens for multiple myeloma in first-line therapy as well as for the treatment of relapsed disease. Bortezomib (Velcade), the first approved therapeutic proteasome inhibitor with a unique boronic acid as warhead, limited by its requirement for parenteral administration (intravenous or subcutaneous). The 2nd generation boronic acid – orally bioavailable Ixazomib (Ninlaro) was approved in 2015 by FDA greatly improved patient’s compliance. However, its adverse effect remained an issue in clinical application. The purpose of this study is to investigate the in vitro and in vivo antitumor activity of TQB3602, a novel orally bioavailable small molecule proteasome inhibitor, in preclinical models of multiple myeloma. Methods: The enzyme inhibition and anti-proliferative activity of TQB3602 was evaluated in 20S proteasome and MM.1S cell line. Oral administration of TQB3602 at 3 doses was used to evaluate the in vivo anti-tumor activity in MM.1S multiple myeloma xenograft (CDX) models. Results: TQB3602 displayed potent kinase inhibiting activity for 20S proteasome enzyme with IC50 15.3 nM. TQB3602 inhibited cell proliferation in MM.1S multiple myeloma cell line with IC50 10.1 nM. In the MM.1S multiple myeloma CB-17 CDX model, TQB3602 showed antitumor efficacy with TGI > 100% @7 mpk, BIW (tumor can’t be measured from day 17). Conclusions: We have identified a novel potent proteasome inhibitor TQB3602. Preclinical studies showed antitumor efficacy of TQB3602 in MM.1S multiple myeloma model. TQB3602 represents a promising clinical candidate for treating multiple myeloma and the First-in-human trial is expected in the middle of 2020.

Novel proteasome inhibitor delanzomib sensitizes cervical cancer cells to doxorubicin-induced apoptosis via stabilizing tumor suppressor proteins in the p53 pathway.

Cervical cancer, the third most commonly occurring cancer, is the second leading cause of cancer related mortality among women. Aberrant ubiquitination and proteasome activity, both human papillomavirus and tumor derived, have been shown to contribute to tumor angiogenesis, proliferation, and invasion in many cancers, including cervical cancer. Thus, small molecule proteasome inhibitors are a potential and strategic treatment option for cervical cancer. In this study, novel proteasome inhibitor delanzomib (CEP-18770) exhibited potent pro-apoptotic and cytotoxic effects on a panel of cervical cancer cell lines by blocking proteasomal activity. Delanzomib also significantly sensitized cervical cancer cells to treatment of doxorubicin (Dox), a traditional chemotherapeutic agent. Furthermore, proteasome inhibition revealed stabilization of p53 and p53 transcriptional targets and induction of p38/JNK phosphorylation. Additionally, delanzomib worked synergistically with Dox to further upregulate p53 and its downstream targets and enhanced Dox-induced p38 phosphorylation. Our study strongly supports the 26S proteasome as a potential therapeutic target in cervical cancer and proteasome inhibition by delanzomib may be a potential treatment strategy for cervical cancer patients.

Abstract 5060: Activity of the EZH2 inhibitor tazemetostat as a monotherapy and in combination with multiple myeloma therapies in preclinical models

Abstract The EZH2 inhibitor tazemetostat (EPZ-6438) is currently being evaluated in phase 2 clinical trials for the treatment of non-Hodgkin’s Lymphoma (NHL). EZH2 inhibitors have shown anti-proliferative effects in multiple preclinical models of NHL and objective clinical responses have been reported in patients with B-cell lymphomas in phase 1 and phase 2 studies of tazemetostat. Mounting evidence suggests that EZH2 is an important regulator of B cell differentiation, both in normal B-cells and in B-cell lymphoma, and may be an important mediator of cell fate in B-cell malignancies in the clinic. Consistent with its essential role in regulating B cell differentiation, recent studies have also shown a dependence on EZH2 activity in multiple myeloma (MM), a disease arising from terminally-differentiated B-cell lymphocyte plasmablasts. Frequent genetic alterations of epigenetic modulators are observed in MM, pointing towards an important role in the initiation and maintenance of this disease. Dysregulation of the H3K27 methyltransferase EZH2, its corresponding histone demethylase UTX and the H3K36 methyltransferase WHSC1 in MM suggest that disruption of the balance of histone methylation may be fundamental to MM pathogenesis in a subset of cases. Indeed, inhibition of EZH2 alone has shown potent anti-proliferative effects both in in vitro and in vivo preclinical models of MM. Here, we describe the effects of small molecule EZH2 inhibitors as monotherapy and in combination with standard of care agents in preclinical models of MM. Tazemetostat selectively inhibits intracellular H3K27 methylation in MM cell lines and elicits a robust anti-proliferative effect in 14-day assays. Following demonstration of single agent activity, we then investigated potential for combinatorial activity of tazemetostat with first and second line therapies for multiple myeloma as well as other non-approved but emerging therapies. Synergistic anti-proliferative activity was observed when tazemetostat was combined with glucocorticoid receptor agonists (dexamethasone, prednisolone), small molecule immune system modulators (lenalidomide, pomalidomide) and proteasome inhibitors (bortezomib, ixazomib) when cells were primed with tazemetostat for seven days prior to the addition of the standard of care drugs. Combination activity was also observed with an alternate treatment schedule where cells were co-treated with tazemetostat along with the combination partner for seven days. Studies with selected therapeutic modalities were expanded into in vivo xenograft models to further evaluate monotherapy and combination activity of EZH2 inhibitors in MM. Citation Format: Allison E. Drew, Vinny Motwani, John E. Campbell, Cuyue Tang, Jesse J. Smith, Richard Chesworth, Robert A. Copeland, Alejandra Raimondi, Scott Ribich. Activity of the EZH2 inhibitor tazemetostat as a monotherapy and in combination with multiple myeloma therapies in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5060. doi:10.1158/1538-7445.AM2017-5060

Targeting the interaction of HDAC2 and MYC in Group 3 medulloblastoma

Patients with high-risk subgroup medulloblastoma (MB) show poor overall survival rates despite aggressive treatment while survivors suffer from important treatment-associated morbidity. Particularly poor outcome is observed in patients with tumors of the molecular subgroup Group 3 with an amplification and high levels of the transcription factor MYC. We have previously shown that Group 3 MB cell lines with MYC amplification are highly susceptible to pan- and class I-histone deacetylase (HDAC) inhibitor treatment. We here explore the molecular interaction of the well known oncogenic driver MYC and class I HDAC2. Six established MB cell lines were used for experiments in cell culture, including MYC amplified cell lines. The functional interaction between MYC and HDAC2 was investigated by enzymatic inhibition of class I HDACs using class I-selective HDAC inhibitors (HDACi) and by siRNA-mediated knockdown of HDAC2. Observed protein degradation was rescued with proteasome inhibition using small molecule proteasome inhibitor MG132. The direct interaction of HDAC2 and MYC was analyzed in co-immunoprecipitation and mass spectrometry studies. The pharmacological inhibition of class I HDACs including HDAC2 as well as artificial HDAC2 protein depletion lead to reduced MYC protein levels, most likely due to increased proteasomal degradation. MYC and HDAC2 are localized in a protein complex together with other potential drug targets. Our data suggest that class I HDAC inhibition leads to destabilization of MYC protein depriving the cells from one of their main oncogenic drivers. The MYC/HDAC2 multi-protein complex represents an attractive target for novel combinatorial treatment approaches. Funding: MYCNet (BMBF Germany), Verein fur krebskranke Kinder Odenwald e.V.

Proteasomal Inhibition by Ixazomib Induces CHK1 and MYC-Dependent Cell Death in T-cell and Hodgkin Lymphoma.

Proteasome-regulated NF-κB has been shown to be important for cell survival in T-cell lymphoma and Hodgkin lymphoma models. Several new small-molecule proteasome inhibitors are under various stages of active preclinical and clinical development. We completed a comprehensive preclinical examination of the efficacy and associated biologic effects of a second-generation proteasome inhibitor, ixazomib, in T-cell lymphoma and Hodgkin lymphoma cells and in vivo SCID mouse models. We demonstrated that ixazomib induced potent cell death in all cell lines at clinically achievable concentrations. In addition, it significantly inhibited tumor growth and improved survival in T-cell lymphoma and Hodgkin lymphoma human lymphoma xenograft models. Through global transcriptome analyses, proteasomal inhibition showed conserved overlap in downregulation of cell cycle, chromatin modification, and DNA repair processes in ixazomib-sensitive lymphoma cells. The predicted activity for tumor suppressors and oncogenes, the impact on "hallmarks of cancer," and the analysis of key significant genes from global transcriptome analysis for ixazomib strongly favored tumor inhibition via downregulation of MYC and CHK1, its target genes. Furthermore, in ixazomib-treated lymphoma cells, we identified that CHK1 was involved in the regulation of MYC expression through chromatin modification involving histone H3 acetylation via chromatin immunoprecipitation. Finally, using pharmacologic and RNA silencing of CHK1 or the associated MYC-related mechanism, we demonstrated synergistic cell death in combination with antiproteasome therapy. Altogether, ixazomib significantly downregulates MYC and induces potent cell death in T-cell lymphoma and Hodgkin lymphoma, and we identified that combinatorial therapy with anti-CHK1 treatment represents a rational and novel therapeutic approach. Cancer Res; 76(11); 3319-31. ©2016 AACR.

Ixazomib, an Oral Proteasome Inhibitor, Induces Rapid Mobilization of Hematopoietic Stem Cells in Mice

Introduction: Granulocyte colony-stimulating factor (G-CSF) is the most commonly used drug for stem cell mobilization. Unfortunately, 5-30% of patients fail to collect sufficient hematopoietic stem/progenitor cells (HSPCs) to proceed to transplant. New strategies are needed to increase HSPCs collection in these patients. We previously reported that bortezomib directly and rapidly mobilizes HSPCs in mice by modulating the VLA-4/VCAM-1 axis (Ghobadi A et al. Blood 2014, 124: 2742-2753). Ixazomib (MLN9708) is a next-generation small-molecule proteasome inhibitor that has several potential advantages over bortezomib including oral route of administration. Here we study the effect of ixazomib on stem cell mobilization in mice.Methods: DBA mice were treated with: (1) ixazomib via oral gavage (o.g., 8 mg/kg), (2) a single intravenous (iv) dose of bortezomib (0.8 mg/kg), (3) AMD3100 (5 mg/kg) SC, and (4) control diluent for ixazomib (2-hydroxypropyl-β-cyclodextrin [HPBCD]). Blood was harvested at baseline and 12, 15, and 24 hours (h) after ixazomib and bortezomib administration and at 3 hours and 12 hours after AMD3100 administration. Harvested peripheral blood (PB) was plated on MethoCult media (Stem Cell Technologies) for colony forming unit-cells (CFU-C) enumeration.Results: Compared to vehicle (ixazomib diluent, 2-hydroxypropyl-β-cyclodextrin [HPBCD]), ixazomib mobilized significantly more peripheral blood colony forming unit-cells (CFU-C) at 12 - 15 h after administration (mean peak CFU-C: 105/ml vs. 870/ml respectively, i.e., a 0.6 vs. 5-fold increase in CFU-C , P < 0.02; Figure 1A). The majority of CFU-Cs mobilized by ixazomib were CFU-GM (CFU-granulocyte and monocyte, Figure 1B). The magnitude and kinetics of HSPC mobilization in the single ixazomib oral gavage group was identical to a single dose of IV bortezomib (Figure 1C, mean peak of 870/ml vs. 975/ml respectively, P > 0.66). There was no statistically significant difference in peak HSPC mobilization between the ixazomib (12 h after ixazomib administration) and AMD3100 groups (3 h after AMD3100 administration) (mean peak of 870/ml vs. 1240/ml respectively, P = 0.36) suggesting that ixazomib is a potent HSPC mobilizing agent (Figure 1D) with intermediate mobilization kinetics compared to AMD3100 (2-3 hours) and G-CSF (5-6 days) and with identical kinetics to IV bortezomib (12-15 hours).Conclusion: Ixazomib is a potent and modestly rapid HSPC mobilizer agent in mice. Kinetics and magnitude of HSPC mobilization by ixazomib is identical to bortezomib. Trials to assess proteasome inhibitors as mobilizing agents are currently underway at Washington University. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Targeted Delivery of Proteasome Inhibitors to Somatostatin-Receptor-Expressing Cancer Cells by Octreotide Conjugation.

Clinical application of proteasome inhibitors (PIs) is so far limited to peripheral blood cancers due to the pronounced cytotoxicity towards all cell types. Targeted delivery of PIs could permit the treatment of other cancers along with decreasing side effects. Herein we describe the first small-molecule proteasome inhibitor conjugate for targeted delivery, created by fusing PIs to a synthetic ligand of somatostatin receptors, which are highly expressed in a variety of tumors. X-ray crystallographic studies and in vitro IC50 measurements demonstrated that addition of the cyclopeptide octreotide as a targeting vehicle does not affect the PI's binding mode. The cytotoxicity of the conjugate against somatostatin-receptor-expressing cells was up to 11-fold higher than that of a non-targeting surrogate. We have therefore established PIs as a new payload for drug conjugates and have shown that targeted delivery thereof could be a promising approach for the broader application of this FDA-approved class of compounds.

Development of Small Molecular Proteasome Inhibitors Using a Caenorhabditis elegans Screen.

We have developed a screening protocol to identify compounds with characteristics of small molecule proteasome inhibitors using the real-time analysis of the Caenorhabditis elegans germ line. This screen is able to identify compounds that induce germ line phenotypes characteristic of a reduction in proteasome function such as changes in polarity, aberrant nuclear morphology, and stimulation of apoptosis. This basic protocol is amenable to a high throughput (96-well) format and has been used successfully to identify multiple compounds for further analysis based on structural elements from the naturally occurring compounds lactacystin and the β-lactone homologs omuralide and salinosporamide A. The further development of this assay system should allow for the generation of novel small molecule proteasome inhibitors in a genetically tractable whole animal amenable to biochemical analysis.

Results from a phase 2 trial of bortezomib (B) and sorafenib (S) in unresectable or metastatic renal cell cancer.

495 Background: B is a small molecule proteasome inhibitor that affects multiple signaling pathways. S is an orally active multikinase inhibitor with known activity in renal cell cancer. In vitro data has shown that S and B interact synergistically in a number of neoplastic cell lines to cause apoptosis. A phase 1 trial of S and B demonstrated no unexpected toxicities. We now report the results of this phase 2 efficacy trial. Methods: Eligibility included cytologically confirmed clear cell cancer with no prior chemotherapy, PS 0-1, Cr &lt; 1.5 mg/dl, normal LFTs. Regimen: S 200 mg PO BID and B 1mg/m2 IV days 1,4,8, &amp; 11 every 21 days. Patients were treated until disease progression or unacceptable toxicity. The primary objective of the study was to achieve a PFS of 70 weeks. Results: Seventeen patients were enrolled between April of 2011 and January of 2013. Median age was 62y (range 44-75). Four of 17 patients had known brain metastasis on entry to the trial. Median number of cycles = 4 (range 1-45+). Response: CR/PR/SD/PD =0/1/12/4 (RR: 6%, 95% CI = 0%, 29%) Median progression free survival was 13.7 weeks; median overall survival was 110 weeks Toxicity: See table. Only 1 pts. treatment was stopped due to an AE of pancreatitis. There were no toxic deaths. The study was halted for futility. Conclusions: 1.The combination of S and B was well tolerated. 2. The RR of 6% and PFS of 13.7 weeks is not superior to S as a single agent. 3. The combination of SB is not recommended for further development. Clinical trial information: NCT 01100242.

Abstract P2-09-17: Allosteric inhibitors of the proteasome: A new concept for breast cancer treatment

Abstract Proteasome is an essential protease of the proteasome-ubiquitin pathway, responsible for maintaining intracellular proteostasis. It is a multifunctional, multisubunit enzyme built from interchangeable modules and intricately regulated on the molecular and cellular levels. Proteasome inhibitors display proapoptotic properties, preferentially aimed at cancerous cells. Consequently, targeted therapy with bortezomib, the competitive inhibitor of the proteasome, has been very successful with refractory/relapsed multiple myeloma and with lymphomas. Unfortunately, breast cancers performed disappointingly in clinical trials with bortezomib. We propose that a new generation of small-molecule proteasome inhibitors distinct from bortezomib may prove successful with breast cancers. In contrast to bortezomib which directly blocks proteasome active sites, the new inhibitors are allosteric ligands binding to noncatalytic sites and disrupting the enzyme's function by targeting long-distance transfer of structural signals within the enzyme's molecule. The advantages of allosteric ligands, now rapidly gaining popularity in targeted drug design, include versatility of molecular actions, defying drug resistance mechanisms, and usually high specificity with low toxicity. For example, one of the small-molecule allosteric ligands we created compromises interactions between the catalytic core proteasome and its regulatory sub-assembly 19S (“cap”, regulatory particle) responsible for recognizing and processing the majority of intracellular protein substrates tagged for degradation by polyubiquitin. We found that treatment with nanomolar concentration of the compound induced cell death in Mcf7 human breast cancer cultured cells. Interestingly, this cell line is known for its resistance to bortezomib treatment. Even more, treatment with the allosteric ligand together with bortezomib produced a synergistic effect and sensitized the Mcf7 cells to the actions of competitive inhibitor. Remarkable, the cytotoxic effects of allosteric ligand or its combination with bortezomib were not observed with the noncancerous human breast cell line, Mcf10A. Summarizing, allosteric regulation of the proteasome by small-molecule ligands, alone or in combination with established drugs competitively inhibiting proteasome, seem to be a promising strategy to utilize proteasome as a drug target in breast cancers. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-17.

A novel tamoxifen derivative, ridaifen-F, is a nonpeptidic small-molecule proteasome inhibitor

In a survey of nonpeptide noncovalent inhibitors of the human 20S proteasome, we found that a novel tamoxifen derivative, RID-F (compound 6), inhibits all three protease activities of the proteasome at submicromolar levels. Structure–activity relationship studies revealed that a RID-F analog (RID-F-S*4, compound 25) is the smallest derivative compound capable of inhibiting proteasome activity, with a potency similar to that of RID-F. Kinetic analyses of the inhibition mode and competition experiments involving biotin-belactosin A (a proteasome inhibitor) binding indicated that the RID-F derivatives interact with the protease subunits in a different manner. Culturing of human cells with these compounds resulted in accumulation of ubiquitinated proteins and induction of apoptosis. Thus, the RID-F derivatives may be useful lead chemicals for the generation of a new class of proteasome inhibitors.

Pseudomonas syringae pv. syringae Uses Proteasome Inhibitor Syringolin A to Colonize from Wound Infection Sites

Infection of plants by bacterial leaf pathogens at wound sites is common in nature. Plants defend wound sites to prevent pathogen invasion, but several pathogens can overcome spatial restriction and enter leaf tissues. The molecular mechanisms used by pathogens to suppress containment at wound infection sites are poorly understood. Here, we studied Pseudomonas syringae strains causing brown spot on bean and blossom blight on pear. These strains exist as epiphytes that can cause disease upon wounding caused by hail, sand storms and frost. We demonstrate that these strains overcome spatial restriction at wound sites by producing syringolin A (SylA), a small molecule proteasome inhibitor. Consequently, SylA-producing strains are able to escape from primary infection sites and colonize adjacent tissues along the vasculature. We found that SylA diffuses from the primary infection site and suppresses acquired resistance in adjacent tissues by blocking signaling by the stress hormone salicylic acid (SA). Thus, SylA diffusion creates a zone of SA-insensitive tissue that is prepared for subsequent colonization. In addition, SylA promotes bacterial motility and suppresses immune responses at the primary infection site. These local immune responses do not affect bacterial growth and were weak compared to effector-triggered immunity. Thus, SylA facilitates colonization from wounding sites by increasing bacterial motility and suppressing SA signaling in adjacent tissues.

Overcoming bortezomib resistance in human B cells by anti-CD20/rituximab-mediated complement-dependent cytotoxicity and epoxyketone-based irreversible proteasome inhibitors

BackgroundIn clinical and experimental settings, antibody-based anti-CD20/rituximab and small molecule proteasome inhibitor (PI) bortezomib (BTZ) treatment proved effective modalities for B cell depletion in lymphoproliferative disorders as well as autoimmune diseases. However, the chronic nature of these diseases requires either prolonged or re-treatment, often with acquired resistance as a consequence.MethodsHere we studied the molecular basis of acquired resistance to BTZ in JY human B lymphoblastic cells following prolonged exposure to this drug and examined possibilities to overcome resistance by next generation PIs and anti-CD20/rituximab-mediated complement-dependent cytotoxicity (CDC).ResultsCharacterization of BTZ-resistant JY/BTZ cells compared to parental JY/WT cells revealed the following features: (a) 10–12 fold resistance to BTZ associated with the acquisition of a mutation in the PSMB5 gene (encoding the constitutive β5 proteasome subunit) introducing an amino acid substitution (Met45Ile) in the BTZ-binding pocket, (b) a significant 2–4 fold increase in the mRNA and protein levels of the constitutive β5 proteasome subunit along with unaltered immunoproteasome expression, (c) full sensitivity to the irreversible epoxyketone-based PIs carfilzomib and (to a lesser extent) the immunoproteasome inhibitor ONX 0914. Finally, in association with impaired ubiquitination and attenuated breakdown of CD20, JY/BTZ cells harbored a net 3-fold increase in CD20 cell surface expression, which was functionally implicated in conferring a significantly increased anti-CD20/rituximab-mediated CDC.ConclusionsThese results demonstrate that acquired resistance to BTZ in B cells can be overcome by next generation PIs and by anti-CD20/rituximab-induced CDC, thereby paving the way for salvage therapy in BTZ-resistant disease.

Bendamustine, Bortezomib, and Rituximab in Patients with Previously Untreated Low Grade Lymphoma: A Phase II Trial of the Sarah Cannon Research Institute

AbstractAbstract 1624 Background:Rituximab and bendamustine combination regimens have demonstrated high activity in non-Hodgkin's lymphoma (NHL). A phase III trial compared bendamustine plus R-CHOP as first-line treatment for follicular, indolent, and mantle cell lymphoma (Rummel ASH 2009). Bendamustine plus rituximab had an overall response rate of 94% and a complete remission rate of 40% compared to 94% and 31%, respectively, for R-CHOP. Phase III trials evaluating bendamustine plus rituximab as first-line therapy for patients (pts) with indolent and mantle cell lymphoma have shown equivalent or superior results when compared to R-CHOP or R-CVP as first-line treatment for indolent lymphoma. Bortezomib is a small molecule proteasome inhibitor that has shown substantial activity in multiple myeloma and lymphoma. Pre-clinical studies demonstrate significant synergy and/or ability to overcome resistance to a variety of current and investigational treatments for lymphoma. This trial evaluated the activity of bendamustine, bortezomib, and rituximab (BBR) in patients with previously untreated low-grade lymphoma. Methods:Eligible patients had histologically-confirmed follicular center cell (FCC) lymphoma (grade 1 or 2), marginal zone lymphoma (MZL), small lymphocytic lymphoma (SLL), or lymphoplasmacytic lymphoma (LPL); lymph node biopsy containing CD20+ B-cells; Ann-Arbor Stage 2, 3, or 4 disease; no previous systemic treatment for lymphoma; bi-dimensional measurable disease with at least 1 lesion measuring > 2.0 cm in a single dimension; ECOG PS 0–2. Treatment for all patients was given in 28-day cycles for a maximum of 6 cycles. Patients received rituximab 375 mg/m2 IV on days 1, 8, and 15 of cycle 1 (cycles 2–6, rituximab only on day 1); bendamustine 90 mg/m2 IV on days 1 and 2; and bortezomib 1.6 mg/m2IV on days 1, 8, and 15. Response evaluations were performed after cycle 3 and cycle 6. Patients were permitted to begin maintenance treatment with rituximab 6 months after completion of study treatment and after 6-month follow-up assessments have been conducted. Results:Between 1/2010 and 11/2011, 55 patients were enrolled with a median age of 62 years (range 30–89). Fifty one percent were male, 85% stage III or IV. Diagnoses were: FCC, 38pts (69%); MZL, 8pts (15%); SLL, 5pts (9%) and LPL, 4pts (7%). The median follow-up is 13 months (range: 6–26). At the time of this analysis, 78% of pts had completed 6 cycles of BRR, and 56% had continued to maintenance rituximab. Two pts remain in the first 6 cycles of BRR. Five pts (9%) discontinued treatment due to toxicity (1pt G2 neuropathy, 1 pt G2 atrial fibrillation, 1 pt G3 pancreatitis, 1 pt G3 diarrhea, 1pt G2 rash). The remaining 5 pts discontinued prematurely (1pt death due to stroke, 1pt wound complication, 1 pt lost to follow-up, 2 pts off due to request). Objective response was achieved in 89% of pts; 26pts (47%) complete response, 23pts (42%) partial response, 2pts (4%) stable disease, and 4pts (7%) unevaluable at the time of this analysis. Related grade 3/4 hematologic toxicities were: neutropenia (25%), febrile neutropenia (2%), thrombocytopenia (5%), and anemia (4%). We observed no grade 4 treatment related non -hematologic adverse events, the most common grade 3 were neuropathy (9%), diarrhea (7%), fatigue (7%), constipation (5%), and rash (5%). Time-to-event data are immature and will be the subject of a later analysis. At the time of data cut-off, 3 pts (5.5%) had progressed or relapsed and 3pts (5.5%) had died. Conclusion:BBR was well tolerated and produced high CR and OR rates. Toxicities including neuropathy were modest. Further study of this regimen in patients with previously untreated lymphoma is warranted. Disclosures:Off Label Use: Off-label bendamustine and bortezomib in first-line treatment for lymphoma. Boccia:Cephalon: Research Funding.

Abstract 5669: Mechanisms of activity and drug resistance with proteasome inhibitors in multiple myeloma: Comparison of bortezomib and the investigational drug MLN9708

Abstract Background: Therapy with proteasome inhibitors (PIs) is an effective strategy against multiple myeloma (MM), but a significant proportion of patients is primarily resistant or eventually becomes refractory. Paradoxical activation of phospho-Akt, induction of HSP27 and excessive proteasome activity in relation to polyubiquitinated protein burden are all mechanisms for bortezomib resistance in MM cells in vitro. Preclinical data showed investigational drug MLN9708, a specific, reversible, orally bioavailable, small molecule PI, to be active in MM cell lines, patient cells and mouse models and is currently under clinical investigation. We compared the signaling pathways induced by MLN2238, the biologically active form of MLN9708, and bortezomib to outline differences that could be employed to design rational combinations to overcome resistance. Results: We performed time course experiments in MM.1S cells with EC50 doses of MLN2238 and bortezomib and analyzed signaling pathways via western blotting. Compared to bortezomib, MLN2238 induced earlier cleavage of caspase 8, 9, 3 and PARP and a significant downregulation of XIAP starting at 12 hours. Early and persistent phosphorylation of Bad, known to protect from mitochondrial apoptosis, was observed in bortezomib-, but not MLN2238-treated cells, in which phospho-Bad was reduced at 24 hours. Similarly, MLN2238 decreased phosphorylated Akt and mTOR at 24 hours, while bortezomib-treated cells only showed subtle changes. Interestingly, both drugs caused downregulation of the mTOR downstream targets phospho-p70 S6 kinase and phospho-4E-BP1, albeit of different intensity. Induction of heat shock proteins was comparable with both PI, opening the possibilities for combination therapy of MLN2238 with HSP90 inhibitors. Importantly, the inhibitory effect on phosphorylation of Akt, mTOR and Bad persisted when MM.1S cells were treated with MLN2238 in the presence of bone marrow stromal cells (BMSC). Conclusion: We observed differences in several crucial signaling pathways upon treatment of MM.1S cells with bortezomib or MLN2238. In particular, treatment with the latter inhibited phosphorylation of Bad, Akt and mTOR, suggesting a broader pro-apoptotic and anti-proliferative profile, compared to bortezomib. MLN2238 retained the capability of targeting these substrates also in the presence of BMSC. Confirmatory data on patient-derived cells are under current investigation in our lab. We believe that these data help understanding the differences in PI pharmacodynamics and bortezomib escape. This will allow us to design new drug combinations in the future that overcome clinical resistance to PIs and improve MM patient survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5669. doi:1538-7445.AM2012-5669

Proteasomal inhibition after injury prevents fibrosis by modulating TGF-β1 signalling

BackgroundThe development of organ fibrosis after injury requires activation of transforming growth factor β1 which regulates the transcription of profibrotic genes. The systemic administration of a proteasomal inhibitor has been...