Selective Proteasome Inhibitor Research Articles

Abstract B238: A phase 1 study of belinostat (PXD101) in combination with bortezomib in patients with advanced solid tumors or lymphoma

Abstract Background: Belinostat (B) is a low molecular weight, synthetic hydroxamic acid derivative that inhibits class I and II histone deacetylases. Bortezomib (Bz) is a potent, selective inhibitor of the 26S proteasome. In preclinical studies, synergistic antiproliferative and pro-apoptotic effects were observed with the combination, providing the rationale for this study. Methods: This phase 1 study is designed to determine the maximum tolerated dose (MTD) and to evaluate the safety and pharmacokinetic (PK) behavior of the combination of B and Bz. Each 21-day treatment cycle consists of B (600–1000 mg/m2) IV daily over 30 minutes on days 1–5 and Bz (0.7–1.5 mg/m2) IVP over 3–5 seconds on days 1, 4, 8, and 11 (days 2, 5, 8 and 11, cycle 1 only). Results: To date, 26 patients have been enrolled (median age 59 [range 27–72]; median PS 1 [range 0–1]). Twenty-two patients were evaluable for toxicity and received a total of 58 treatment cycles (median 2 [range 1–6]). Four patients, 2 at dose level (DL) 3 (B: 600 mg/m2)/Bz: 1.3 mg/m2) and 2 at DL 5 (B: 1000 mg/m2)/Bz: 1.5 mg/m2) experienced a dose limiting toxicity (DLT). At DL 3, the DLTs were grade (gr) 3 dehydration and gr 4 thrombocytopenia. The exact relationship of the gr 3 dehydration and study combination is unclear as the patient had uncontrolled constipation and decreased oral intake as a result of a change in pain medications on cycle 1 day 1. This cohort was expanded to 9 pts to further determine tolerability of the study combination. No other DLTs were identified and dose escalation continued. At DL 5, DLTs included gr 4 thrombocytopenia and gr 4 fatigue. Most adverse events (AEs) have been mild to moderate. Gr 1–2 AEs (number of cycles) include anorexia (8), acute infusion reaction (5), fatigue (14), nausea (19), neutropenia (1), pain (12), phlebitis (6), thrombocyctopenia (11), and vomiting (12). Gr 3 AEs (occurring after course 1) include anorexia, dehydration, fatigue, nausea, vomiting, hypoalbuminemia, and elevation of alkaline phosphatase. Analysis of B PK demonstrates no statistical differences in the parameters between days 1 (B only) and 2 (B + Bz) for AUC, Cmax, clearance, or t1/2. Likewise, increasing Bz doses have no effect on B PK, whereas doses of B from 600 to 1000 mg/m2 result in dose-proportional increases in drug exposure. Four patients have maintained stable disease for 4–6 cycles of therapy. Conclusions: Belinostat and bortezomib is a reasonable combination with a tolerable toxicity profile and no evidence of pharmacological interactions. Accrual is ongoing at the MTD, DL 4 (B: 1000 mg/m2)/Bz: 1.3 mg/m2). Additional biological assessments are planned in an expanded cohort of patients. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B238.

Selective Inhibitor of Proteasome's Caspase-like Sites Sensitizes Cells to Specific Inhibition of Chymotrypsin-like Sites

Proteasomes degrade most proteins in mammalian cells and are established targets of anticancer drugs. All eukaryotic proteasomes have three types of active sites: chymotrypsin-like, trypsin-like, and caspase-like. Chymotrypsin-like sites are the most important in protein degradation and are the primary target of most proteasome inhibitors. The biological roles of trypsin-like and caspase-like sites and their potential as cotargets of antineoplastic agents are not well defined. Here we describe the development of site-specific inhibitors and active-site probes of chymotrypsin-like and caspase-like sites. Using these compounds, we show that cytotoxicity of proteasome inhibitors does not correlate with inhibition of chymotrypsin-like sites and that coinhibition of either trypsin-like and/or caspase-like sites is needed to achieve maximal cytotoxicity. Thus, caspase-like and trypsin-like sites must be considered as cotargets of anticancer drugs.

Selective inhibition of CDK4/CDK6 sensitizes bone marrow myeloma cells for killing by proteasome inhibitors carfilzomib and PR-047 through cell cycle-dependent expression of pro-apoptotic Noxa and Bim.

Abstract 2854Poster Board II-830Targeting the cell cycle in combination with cytotoxic killing is a rational approach to cancer therapy. Progression in multiple myeloma (MM) stems from both loss of apoptotic control in the bone marrow (BM) microenvironment and dysregulation of the cyclindependent kinases (CDK)4 and CDK6, which precedes uncontrolled proliferation of myeloma cells in vivo in particular during relapse. This reinforces the critical importance of targeting CDK4/CDK6 in MM. Through selective and reversible inhibition of CDK4/CDK6 with PD 0332991, the only known CDK4/6-specific inhibitor, we have recently developed a novel strategy to sensitize primary myeloma cells for cytotoxic killing by diverse cytotoxic drugs. These include carfilzomib (PR-171), an irreversible selective inhibitor of the chymotrypsin-like activity of the proteasome, and PR-047, an orally bioavailable analog of carfilzomib. We showed that induction of prolonged early G1 arrest following inhibition of CDK4/CDK6 markedly enhances cytotoxic killing of primary BM myeloma cells by either carfilzomib or PR-047 despite protection by BM stromal cells. The enhancement of cytotoxic killing is further augmented during synchronous S phase entry upon removal of PD 0332991 subsequent to induction of prolonged G1 arrest in myeloma cell lines. In both cases, the enhancement in carfilzomib (or PR-047) mediated killing is not associated with cell cycle regulation of the proteasome activity. It is caspase-dependent, requiring only a brief (one hour) exposure to the proteasome inhibitor at concentrations as low as 30 nM. This killing is mediated by synergistic and rapid induction of mitochondrial membrane depolarization and activation of downstream caspase-9. Further, it is apparently initiated by cell cycle-dependent expression of the pro-apoptotic BH3-only proteins, which neutralize the anti-apoptotic Bcl-2 family proteins upstream of mitochondrial depolarization. Bim is upregulated during early G1 arrest to neutralize the anti-apoptotic MCL-1 and Bcl-2. By contrast, Noxa is silenced in G1 but dramatically upregulated in S phase, in particular when combined with carfilzomib. Importantly, targeting CDK4/CDK6 with PD 0332991 in combination with either carfilzomib or PR-047 leads to complete eradication of myeloma cells ex vivo, in contrast to the combination of PD 0332991 with other proteasome inhibitors. Selective inhibition of CDK4/CDK6 in combination with carfilzomib (or PR-047), therefore, not only halts tumor cell proliferation but also potently induces synergistic killing that is likely to profoundly inhibit cell cycle reentry and self-renewal in MM. PD 0332991 is a small molecule with bio-availability and proven tumor suppressing activity in both human myeloma xenograft and immunocompetent mouse myeloma models. It is well tolerated in humans as indicated by the ongoing Phase I/II clinical trials in myeloma and previous phase I trials in mantle cell lymphoma and solid tumors. Evidence from Phase 2 trials of carfilzomib indicates that it is also well tolerated, in fact, the peripheral neuropathy that is commonly observed with the proteasome inhibitor bortezomib appears to be less severe and possibly less frequent. Mechanism-based targeting of CDK4/6 in combination with selective proteasome inhibitors, like carfilzomib and PR-047, thus represents a new and promising therapeutic strategy for multiple myeloma and potentially other hematopoietic malignancies. Disclosures:Off Label Use: PD 0332991 is going to be used as a CDK4/6-specific inhibitor. Parlati:Proteolix, Inc.: Employment, Equity Ownership. Aujay:Proteolix, Inc.: Employment, Equity Ownership. Niesvizky:Millenium: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Seattle Genetics, Inc: Research Funding; Proteolix: Research Funding, data monitoring committee.

Dose Intensive Administration of PR-047, a Novel Orally Bioavailable Inhibitor of the 20S Proteasome, Is Well Tolerated in Experimental Animals.

Abstract 4910Proteasome inhibition is a validated therapeutic strategy for the treatment of multiple myeloma. Carfilzomib (CFZ) is a selective and irreversible proteasome inhibitor that is intravenously administered and an has shown an encouraging safety and efficacy profile in myeloma patients from multiple Phase 2 studies (ASH2008:864&865). CFZ induces prolonged proteasome inhibition in vivo and can be safely administered to patients on dose intensive schedules (e.g. QDx5); however, such schedules lack patient convenience. PR-047 is a novel inhibitor in the same chemical class as carfilzomib that is orally bioavailable Similar to carfilzomib, PR-047 potently targets the chymotrypsin-like activity of the proteasome and induces anti-tumor responses in vitro and in vivo across a range of tumor cell types including myeloma cells. We describe here the proteasome selectivity of PR-047 in vitro and the pharmacodynamics and safety of repeat dose oral administration in experimental animals. We found PR-047, like CFZ, to be highly selective for proteasomal proteases as demonstrated by a lack of inhibitory activity against a panel of non-proteasomal proteases, including serine proteases targeted by dipeptide boronates such as bortezomib, (J. Med. Chem. 2008 51:1068). In rats, well tolerated oral doses resulted in rapid and potent (>80%) inhibition of proteasome activity when the compound was administered either as a solution or in a capsule formulation suitable for clinical use. Similar to carfilzomib, PR-047 was well tolerated when administered on a dose intense schedule. Oral dosing of PR-047 to mice and rats daily for 5 consecutive days resulted in proteasome inhibition following the 5th dose that was equivalent to or greater than inhibition seen after the 1st dose in whole blood and tissues. This suggests that PR-047 promotes prolonged proteasome inhibition in vivo. These doses were well tolerated in GLP-compliant toxicity studies in rats and dogs where PR-047 was administered for 2, 14-day cycles of QDx5 followed by nine days rest. The MTD in rats was ∼6 fold greater than the dose required for >80% proteasome inhibition, suggesting a significant therapeutic window. At the MTD, PR-047 did not affect renal or liver function and was not associated with anemia or leukopenia. In addition, PR-047 administration did not result in behavioral or histologic signs of neurotoxicity. Comparable doses were well tolerated in dogs on the same dose schedule. Similar to CFZ, PR-047 is a selective and irreversible proteasome inhibitors that has potent anti-tumor activity and is well tolerated on dose intensive schedules that result in prolonged proteasome inhibition. These studies support a safe starting dose for Phase 1 clinical trials with PR-047, a novel agent for the treatment of malignant diseases. DisclosuresMuchamuel:Proteolix Inc.: Employment. Kapur:Proteolix, Inc.: Employment. Kirk:Proteolix, Inc: Employment. Jiang:Proteolix, Inc.: Employment. Lee:Proteolix, Inc.: Employment. Bennett:Proteolix: Employment. Lewis:Proteolix, Inc.: Employment. Yang:Proteolix, Inc.: Employment. Jumaa:Proteolix, Inc.: Employment. Ring:Proteolix Inc.: Employment. Phiasivongsa:Proteolix Inc.: Employment. Zhou:Proteolix Inc.: Employment. Wang:Proteolix, Inc.: Employment.

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

Abstract 2859Poster 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.

Bortezomib-Resistant Cell Lines Have Increased Proteasome Levels but Remain Sensitive to Carfilzomib.

Abstract 2852Poster Board II-828 Introduction:Carfilzomib (CFZ) is a selective and irreversible proteasome inhibitor that induces anti-tumor activity through inhibition of the proteasome. In vitro, CFZ induces cell death in CD138+ myeloma cells from patients refractory to the dipeptide boronate proteasome inhibitor bortezomib (BTZ) and, in Phase 2 trials, CFZ has demonstrated objective and durable responses in BTZ relapsed and refractory patients. To understand the mechanism underlying the ability of carfilzomib to overcome BTZ resistance, we compared the activity of the two inhibitors in vitro using tumor cells conditioned to be resistant to BTZ. Methods:HT-29, human colorectal adenocarcinoma cells, were cultured in the continuous presence of BTZ by stepwise increases in concentration (20nM to 200nM) over 7 months. Clonal isolates were derived by limiting dilution analysis and cultured in BTZ at 100nM and 200nM for an additional 4 months. Cytotoxic responses were measured as 50% inhibitory concentrations (IC50) following 72 hr compound exposure. Proteasome activity was measured using fluorogenic substrates for chymotrypsin-like, caspase-like and trypsin-like activities and by an active site ELISA (ProCISE) that quantitates the catalytic subunits of the constitutive proteasome (beta5, beta1, beta2) and immunoproteasome (LMP7, LMP2, MECL1). To assess recovery of proteasome activity, cells were exposed to a brief (1hr) “pulse” of either BTZ or CFZ, at concentrations resulting in 80% inhibition, and the chymotrypsin-like proteasome activity was measured at 4 and 24hrs after compound washout. We sequenced the cDNA encoding chymotrypsin-like proteasome subunits, beta5 and LMP7, along with a structural subunit, beta6, to identify potential mutations in the clonal BTZ resistant cell lines. Results:Compared to parental cells, clones grown in 100nM (BR100) or 200nM (BR200) BTZ were 15 – 25-fold less sensitive to BTZ-induced cytotoxicity and remained resistant after long-term culture without BTZ exposure. The cytotoxic response to CFZ was largely unchanged in these cells with <5-fold differences in IC50 values. Basal proteasome catalytic activity was increased by 7-11 fold in both BR100 and BR200 cells. This corresponded to an increase (5-8 fold) in expression of all catalytic subunits except for LMP2. No difference in the initial inhibition of proteasome activity by either compound was seen in resistant cells. However, 4 hr post BTZ exposure, resistant cells showed a more rapid recovery of proteasome activity relative to parental cells. Compared to BTZ, CFZ induced a prolonged proteasome inhibition that was not altered in resistant cells and may therefore relate to the preserved cytotoxic activity of CFZ in these cells. Interestingly, after 24 hrs of recovery from a 1hr exposure to either BTZ or CFZ, resistant cells did not recover their proteasome activity to levels seen parental cells, suggesting alterations in proteasome assembly. Sequence analysis of clonal BR100 and BR200 cell lines identified a mutation in the propeptide of beta5 (Arg24Cys). This mutation, which is in a region required for proper proteasome assembly, has previously been identified at a high frequency in myeloma patients (Wang L., et al Clin Cancer Res 2008;14:3503)). Mutations in beta5 near the interface with the alpha ring (Cys63Phe) and in the propeptide portion of LMP7 (Phe50Ile) were also found in BR200 cells but were absent in BR100 cells. We did not detect mutations in beta6 or a mutation in the active site of beta5 described by other groups. Conclusions:BTZ resistance in vitro is associated with increased proteasome subunit levels and a rapid recovery of proteasome activity at early (4hr) timepoints following BTZ exposure. Our data, which includes the first report of a mutation in LMP7, support a hypothesis that resistance is mediated by a more rapid and efficient proteasome assembly process. Since one of the mutations is prevalent at a high frequency in myeloma patients, this process may be involved in the etiology of disease and/or clinical resistance to BTZ treatment. CFZ overcomes BTZ resistance in these cells, due in part to prolonged inhibition of the proteasome activity, providing a possible mechanism for the activity of CFZ in myeloma patients that are refractory to BTZ therapy. Disclosures:Suzuki:Proteolix, Inc: Employment, Equity Ownership. Demo:Proteolix: Employment, Equity Ownership. Arastu-Kapur:Proteolix, Inc: Employment, Equity Ownership. Kirk:Proteolix, Inc: Employment, Equity Ownership. Bennett:Proteolix: Employment.

Carfilzomib (CFZ), a Novel Proteasome Inhibitor for Relapsed or Refractory Multiple Myeloma, Is Associated with Minimal Peripheral Neuropathic Effects.

Carfilzomib (CFZ), a Novel Proteasome Inhibitor for Relapsed or Refractory Multiple Myeloma, Is Associated with Minimal Peripheral Neuropathic Effects.

The Proteasome Is an Integral Part of Solar Ultraviolet A Radiation-induced Gene Expression

Solar ultraviolet (UV) A radiation is a well known trigger of signaling responses in human skin fibroblasts. One important consequence of this stress response is the increased expression of matrix metalloproteinase-1 (MMP-1), which causes extracellular protein degradation and thereby contributes to photoaging of human skin. In the present study we identify the proteasome as an integral part of the UVA-induced, intracellular signaling cascade in human dermal fibroblasts. UVA-induced singlet oxygen formation was accompanied by protein oxidation, the cross-linking of oxidized proteins, and an inhibition of the proteasomal system. This proteasomal inhibition subsequently led to an accumulation of c-Jun and phosphorylated c-Jun and activation of activator protein-1, i.e. transcription factors known to control MMP-1 expression. Increased transcription factor activation was also observed if the proteasome was inhibited by cross-linked proteins or lactacystin, indicating a general mechanism. Most importantly, inhibition of the proteasome was of functional relevance for UVA-induced MMP-1 expression, because overexpression of the proteasome or the protein repair enzyme methionine sulfoxide reductase prevented the UVA-induced induction of MMP-1. These studies show that an environmentally relevant stimulus can trigger a signaling pathway, which links intracellular and extracellular protein degradation. They also identify the proteasome as an integral part of the UVA stress response.

1207 Results of study PX-171–007 a phase 1b/2 study of carfilzomib, a selective proteasome inhibitor, in patients with selected advanced metastatic solid tumors

1207 Results of study PX-171–007 a phase 1b/2 study of carfilzomib, a selective proteasome inhibitor, in patients with selected advanced metastatic solid tumors

Proteasome inhibitor bortezomib promotes a rupture-prone plaque phenotype in ApoE-deficient mice

The ubiquitin-proteasome system is involved in the development and progression of atherosclerosis. The aim of this study was to investigate whether plaque composition is affected by proteasome function. In vitro, the potent and selective proteasome inhibitor bortezomib induced apoptosis in both cultured smooth muscle cells (SMCs) and activated macrophages. This effect was associated with increased expression of C/EBP homologous protein and cleavage of caspase-12, indicative of endoplasmic reticulum stress. The sensitivity to the proapoptotic effects of proteasome inhibition correlated with the protein synthesis rate. Proteasome inhibition in explanted atherosclerotic plaques of ApoE-deficient mice resulted in a significant decrease in SMCs and macrophages, indicating that both cell types in the atherosclerotic plaque were susceptible to the proapoptotic effects of proteasome inhibition. In vivo proteasome inhibition in ApoE-deficient mice did not affect plaque size or composition of early atherosclerotic plaques, but resulted in a significant decrease in collagen content as well as a significant enlargement of the necrotic core in advanced atherosclerotic plaques. In conclusion, our results indicate that an impaired proteasome function promotes features of a more rupture-prone plaque phenotype.

Phase II results of Study PX-171–007: A phase Ib/II study of carfilzomib (CFZ), a selective proteasome inhibitor, in patients with selected advanced metastatic solid tumors

3515 Background: CFZ is a novel proteasome inhibitor of the peptide epoxyketone class that exhibits a high level of selectivity for proteasome active sites. This phase 1/2 study assessed the maximum tolerated dose (MTD), safety, efficacy, pharmacokinetics (PK), and pharmacodynamics (PD) of CFZ in patients (pts) with advanced metastatic solid tumors. Methods: Pts failing ≥ 2 prior treatments were enrolled in the phase 1 3+3 dose escalation study. Pts received CFZ IV Day (D) 1, 2, 8, 9, 15 and 16 every 28 d for up to 12 cycles (C) Cycle 1 D1, D2 dosing in all cohorts was at 20 mg/m2. Subsequent doses remained at 20 mg/m2 or were escalated to 27 or 36 mg/m2 in a stepped up regimen on D8. At 20/36 mg/m2, 1 pt had a DLT (Grade 3 fatigue) and established the phase 2 dose. Phase 2 is designed as a Simon 2 stage of 70 pts split into 5 subgroups (small cell lung [SCLC], non-small cell lung [NSCLC], ovarian, renal, and other cancer). Tumor response was measured every 2 C. ORR, defined as CR+PR+SD, to 16 wks of CFZ. Stage 2 will open if a 1 PR or better at 16 wks occurs in a selected subgroup. Results: 14 pts in phase 1 and 51 pts in phase 2 (23M/28F, mean age 61 yrs) received a total of 154.5 cycles of CFZ. Median cycles administered was 1.7 (range 1 to 12). To date, there were 6 SCLC, 10 NSCLC, 11 ovarian, 6 renal, and 18 other cancer patients enrolled to the Simon stage 1. Efficacy of SD or better is detailed in the table. The most common AEs included fatigue headache, diarrhea, nausea and constipation. Notable was the absence of grade &gt; 1 peripheral neuropathy and severe hematologic toxicities. Final results of the PK and PD will be reported. Conclusions: CFZ is active as a single agent in relapsed solid tumors demonstrating PR in both renal and SCLC; and SD &gt;16 wks in mesothelioma, ovarian, renal and NSCLC. The 20/36 mg/m2 QDx2 dose schedule was well tolerated and lacks severe myelosuppression, hepatotoxicity and neuropathy which make CFZ an attractive agent to combine with traditional or novel targeted agents. [Table: see text] [Table: see text]

The Role of ATF4 Stabilization and Autophagy in Resistance of Breast Cancer Cells Treated with Bortezomib

The ubiquitin-proteasome system plays a key regulatory role in cellular homeostasis. The inhibition of the 26S proteasome by Bortezomib leads to the accumulation of misfolded proteins, resulting in endoplasmic reticulum stress followed by a coordinated cellular response called unfolded protein response (UPR). Endoplasmic reticulum stress is also a potent inducer of macroautophagy. Bortezomib is a selective and potent inhibitor of the 26S proteasome and is approved for the treatment of multiple myeloma. Clinical trials with Bortezomib have shown promising results for some types of cancers, but not for some others, including those of the breast. In this study, we show that Bortezomib induces the UPR and autophagy in MCF7 breast cancer cells. Surprisingly, Bortezomib did not induce phosphorylation of PERK, a key initial step of the UPR. We show that induction of autophagy by Bortezomib is dependent on the proteasomal stabilisation of ATF4 and up-regulation of LC3B by ATF4. We show that ATF4 and LC3B play a critical role in activating autophagy and protecting cells from Bortezomib-induced cell death. Our experiments also reveal that HDAC6 knockdown results in decreased LC3B protein and reduced autophagy. Our work shows that the induction of autophagy through ATF4 may be an important resistance mechanism to Bortezomib treatment in breast cancer, and targeting autophagy may represent a novel approach to sensitize breast cancers to Bortezomib.

Design and Synthesis of an Orally Bioavailable and Selective Peptide Epoxyketone Proteasome Inhibitor (PR-047)

Proteasome inhibition has been validated as a therapeutic modality in the treatment of multiple myeloma and non-Hodgkin's lymphoma. Carfilzomib, an epoxyketone currently undergoing clinical trials in malignant diseases, is a highly selective inhibitor of the chymotrypsin-like (CT-L) activity of the proteasome. A chemistry effort was initiated to discover orally bioavailable analogues of carfilzomib, which would have potential for improved dosing flexibility and patient convenience over intravenously administered agents. The lead compound, 2-Me-5-thiazole-Ser(OMe)-Ser(OMe)-Phe-ketoepoxide (58) (PR-047), selectively inhibited CT-L activity of both the constitutive proteasome (beta5) and immunoproteasome (LMP7) and demonstrated an absolute bioavailability of up to 39% in rodents and dogs. It was well tolerated with repeated oral administration at doses resulting in >80% proteasome inhibition in most tissues and elicited an antitumor response equivalent to intravenously administered carfilzomib in multiple human tumor xenograft and mouse syngeneic models. The favorable pharmacologic profile supports its further development for the treatment of malignant diseases.

Ongoing DNA synthesis in the rat cerebral cortex is regulated by a proteolytic pathway independent of the proteasome and calpains

By using mini-units of tissue and protease inhibitors in short term incubation (0-180 min), we studied the role of proteolysis for ongoing DNA replication in the developing rat cerebral cortex. The protease inhibitors TLCK, TPCK, PMSF, MG-132 and PSI markedly inhibited DNA synthesis. The inhibitory effects were concentration-dependent and of early onset (within 60 min). The most selective proteasome inhibitors lactacystin and clasto-lactacystin-beta-lactone as well as the calpain inhibitor I and II had no or minimal effects on DNA synthesis. Only high concentrations of calpain inhibitor I (>or= 250 microM) and calpain inhibitor II (>or= 500 microM) gave a DNA synthesis inhibition. These results suggest that (1) ongoing DNA replication is regulated by proteolysis and (2) the proteolytic pathways involved are neither the proteasome nor the calpains.

Nigral Degeneration with Inclusion Body Formation and Behavioral Changes in Rats after Proteasomal Inhibition

Objective: We were interested in studying nigral degeneration with inclusion body formation and behavioral changes in rats after proteasomal inhibition. Methods: Observation of progressive behavioral and pathological changes in rats following a unilateral nigral injection of lactacystin, a selective proteasome inhibitor. Results: After administration at a concentration of 10 μg (2 μl) of lactacystin, when tyrosine hydroxylase (TH) immunostaining decreased gradually in the substantia nigra pars compacta (SNc) and corpus striatum, α-synuclein-immunopositive inclusion appeared extensively in the surviving neurons. We also observed the degeneration of diverse cellular organelles by transmission electron microscopy. The effect of cellular organelle degeneration on behavior, a clinical index, was striking and was statistically significant. Over the 3 weeks following the administration of lactacystin, a highly significant decrease in TH immunostaining was observed and α-synuclein-immunopositive inclusions gradually appeared. Interestingly, there was a strong correlation in behavioral changes and the increase in α-synuclein-immunopositive inclusions whereas the decrease in TH immunostaining did not seem to induce any behavioral changes. Conclusions: Our results reveal that unilateral nigral proteasome inhibition induces degeneration in the SNc and corpus striatum as well as behavioral changes demonstrating strong time dependence. Behavioral changes were driven by the formation of α-synuclein inclusions, but not by decreased TH neurons.

Role of oxidative stress and intracellular glutathione in the sensitivity to apoptosis induced by proteasome inhibitor in thyroid cancer cells

BackgroundThe proteasome inhibitor bortezomib has shown impressive clinical activity alone and in combination with conventional and other novel agents for the treatment of multiple myeloma (MM) and some solid cancers. Although bortezomib is known to be a selective proteasome inhibitor, the downstream mechanisms of cytotoxicity and drug resistance are poorly understood.MethodsProteasome activity, intracellular glutathione (GSH) and ROS levels, as well as activities of GSH synthesis enzymes were measured using spectrophotometric methods. Cell death was analyzed using flow cytometry and caspase activity assay. The expression level of GSH synthesis enzymes were measured using real-time RT-PCR.ResultsAt concentrations that effectively inhibited proteasome activity, bortezomib induced apoptosis in FRO cells, but not in ARO cells. Bortezomib elevated the amount of glutathione (GSH) and the treatment with bortezomib increased the level of mRNA for GCL, a rate-limiting enzyme in glutathione synthesis. Furthermore, depletion of GSH increases apoptosis induced by bortezomib, in contrast, repletion of GSH decreases bortezomib-mediated cell death.ConclusionGSH protects cells from proteasome inhibition-induced oxidative stress and glutathione-dependent redox system might play an important role in the sensitivity to proteasome inhibition-induced apoptosis.

Bortezomib-induced perivascular dermatitis in a patient with multiple myeloma

Bortezomib is a potent, selective proteasome inhibitor. It is usually well tolerated, and bortezomib-induced skin lesions are less well known. A 59-year-old man with multiple myeloma underwent autologous hematopoetic stem cell transplantation (AHSCT). Administration of bortezomib was started after AHSCT as his disease progressed despite thalidomide maintenance. He developed a rash afer the third cycle of bortezomib. A biopsy showed superficial and deep perivascular dermatitis. The rash resolved within 4 days; complete response of myeloma was obtained after 8 cycles of bortezomib. The benign clinical course did not require interruption of the treatment, which in turn resulted in complete remission of myeloma.

Induction of Sustained Early G1 Arrest by Selective Inhibition of CDK4 and CDK6 Primes Myeloma Cells for Synergistic Killing by Proteasome Inhibitors Carfilzomib and PR-047

Targeting the cell cycle in combination with cytotoxic killing is a rational approach to cancer therapy. Dysregulation of the cyclin-dependent kinases CDK4 and CDK6 precedes uncontrolled proliferation of myeloma cells in vivo, in particular during relapse and drug resistance. This finding reinforces the critical importance of targeting CDK4/6 in myeloma, but success with broad-spectrum CDK inhibitors has been modest. Using the only known selective inhibitor of CDK4/6, PD 0332991, we have developed a novel approach to prime chemoresistant myeloma cells for synergistic killing by diverse cytotoxic agents. We show that selective inhibition of CDK4/6 by PD 0332991 leads to sustained cell cycle arrest in early G1 in the absence of apoptosis. However, it markedly augments cytotoxic killing by PR-171 (carfilzomib), a selective inhibitor of the chymotrypsin-like activity of the proteasome, or PR-047, an orally bioavailable analog of carfilzomib. Synergistic killing of myeloma cells arrested in early G1 by carfilzomib (or PR-047) is caspase-dependent, and requires only a brief (one hour) exposure to the proteasome inhibitor at concentrations as low as 60 nM. This effect is mediated by synergistic and rapid induction of mitochondrial membrane depolarization and activation of downstream caspase-9 within 6 hours of removal of carfilzomib or PR-047. As PD 0332991 acts as an ATP-competitive inhibitor of the CDK4/6 kinase domain, inhibition of CDK4/6 and the cell cycle by PD 0332991 is reversible. Importantly, targeting CDK4/6 with PD 0332991 in combination with either carfilzomib or PR-047 leads to complete eradication of myeloma cells ex vivo, in contrast to the combination of PD 0332991 with other proteasome inhibitors. Selective inhibition of CDK4/6 in combination with carfilzomib (or PR-047), therefore, not only halts cell proliferation but also potently induces synergistic killing that is likely to eliminate cell cycle reentry and generation of resistant cells. PD 0332991 is a small molecule with bio-availability and proven tumor suppressing activity in both human myeloma xenograft and immunocompetent mouse myeloma models. It is well tolerated in humans as shown by the ongoing Phase I/II clinical trials in myeloma and previous phase I trials in mantle cell lymphoma and solid tumors. Evidence from Phase I trials of carfilzomib indicates that it is also well tolerated, in fact, the peripheral neuropathy that is commonly observed with proteasome inhibitor bortezomib appears to be less severe and possibly less frequent. Mechanism-based targeting of CDK4/6 in combination with selective proteasome inhibitors, like carfizomib and PR-047, thus represents a new and promising therapeutic strategy for multiple myeloma and potentially other hematopoietic malignancies.

The Selective Proteasome Inhibitor Carfilzomib Is Well Tolerated in Experimental Animals with Dose Intensive Administration

Carfilzomib (CFZ) is a selective inhibitor of the chymotrypsin-like (CT-L) activity of the proteasome. Phase I trials have demonstrated that doses of carfilzomib from 11 – 20 mg/m2 were well tolerated, inhibited blood CT-L activity by ≥80%, and resulted in objective anti-tumor responses in patients with multiple myeloma (MM) and non-Hodgkin's lymphoma (NHL) (Blood 110:409 & 411). In order to further characterize the safety profile of CFZ, studies in mice, rats and monkeys were conducted to assess the toxicologic consequences of acute and chronic administration. In both rats and monkeys, a single dose of CFZ (equivalent to 24 mg/m2) resulted in > 80% inhibition of CT-L activity in blood and tissues with recovery occurring in most tissues with a t1/2 of 24 hr. At this dose, monkeys developed a pre-renal azotemia within 24 hr, which was characterized by increases in serum creatinine, blood urea nitrogen (BUN) levels and urine specific gravity. These changes resolved in 72 hr, were not associated with histologic changes in the kidney, and were not exacerbated by consecutive daily administrations, suggesting that CFZ may not have a direct nephrotoxic effect. A single dose in either rats or monkeys induced a transient drop in platelets within 48 hr that resolved within 7 days and neither the level of nor recovery from this thrombocytopenia were affected by consecutive daily administration. This phenomenon has also been noted with bortezomib (BTZ), and is observed clinically with both compounds, suggesting that thrombocytopenia is a hallmark of inhibition of proteasome CT-L activity. However, in contrast to BTZ, which has been reported to induce both lymphopenia and neutropenia in animals, CFZ treatment in both rats and monkeys resulted in acute increases in circulating neutrophils and monocytes which rose within 24 hr of a single dose and returned to baseline in 3 – 4 days. We were unable to detect a significant induction of cytokines or chemokines capable of mediating leukocyte fluxes. However, since the acute phase proteins fibrinogen and C-reactive protein were elevated during the same time period of leukocyte flux, cytokines may play a role in this response. In chronic studies, CFZ was administered on Days 1, 2, 8, 9, 15, and 16 of a 28-day cycle to rats and monkeys for 6 and 9 cycles, respectively. This dosing schedule results in anti-tumor activity in mouse models of MM, NHL and solid tumors and is currently being utilized in Phase 2 studies in MM and solid tumors. In these chronic studies, transient pre-renal azotemia and increases in circulating neutrophils were manifest in monkeys at 24 mg/m2 following the first dose of both the first and last cycles of treatment. However, no histologic signs of nephropathy were seen in the kidneys of rats receiving 6 cycles of treatment. In addition, chronic administration of CFZ at doses equivalent to those currently administered in the clinic did not impact on the neurobehavioral function of rats and monkeys. There was no severe degeneration in the dorsal root ganglion or in the sciatic, sural, and peroneal nerves of CFZ treated rats. The data with CFZ are in distinct contrast to the axonal swelling, demyelination and severe neural degeneration that is induced by BTZ in rodents and monkeys. In the clinic, peripheral neuropathy associated with CFZ treatment appears to be less severe and possibly less frequent than what is seen with BTZ. Overall, studies in rodents and monkeys have been largely predictive of the clinical experience to date with CFZ. Together with anti-tumor activity in human tumor xenograft models, the toxicologic data support that CFZ administered at therapeutic doses is well tolerated and prolonged treatment of patients with this agent will be feasible.

NF-κB signaling, liver disease and hepatoprotective agents

The NF-kappaB signaling pathway has particular relevance to several liver diseases including hepatitis (liver infection by Helicobacter, viral hepatitis induced by HBV and HCV), liver fibrosis and cirrhosis and hepatocellular carcinoma. Furthermore, the NF-kappaB signaling pathway is a potential target for development of hepatoprotective agents. Several types of drugs including: selective estrogen receptor modulators (SERMs), antioxidants, proteasome inhibitors, IKK inhibitors and nucleic acid-based decoys have been shown to interfere with NF-kappaB activity at different levels and may be useful for the treatment of liver diseases. However, NF-kappaB also plays an important hepatoprotective function that needs to be taken into consideration during development of new therapeutic regimens.