Use Of Bortezomib In Combination Research Articles

The efficacy of bortezomib in human multiple myeloma cells is enhanced by combination with omega-3 fatty acids DHA and EPA: Timing is essential

Although bortezomib as one of the first line medicines that has greatly improved the overall survival of patients with multiple myeloma (MM), undesired drug resistance is frequently observed. Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) have been shown to be able to enhance the efficacy of chemotherapeutic drugs in many cancer types. The aim of the present study was to further evaluate the anticancer activity of DHA and EPA in relation to bortezomib chemosensitivity in human MM cells. The potential involvement of NF-κB signaling pathway was studied. MM cells were treated with DHA/EPA with or without bortezomib. Cell viability was estimated by WST-1 assay. Apoptotic cells were determined through flow cytometry using annexin V and propidium iodide (PI) staining. Protein expression and phosphorylation was investigated by western blotting. Cell type dependent anticancer potential of DHA and EPA was observed in the cell viability assay. DHA and EPA induced apoptosis in L363, OPM2, MM.1S and U266cell lines through both mitochondrial and death receptor pathways. Treating MM cells with DHA and EPA significantly downregulated IκBα and upregulated phosphorylation of p65, indicating that they triggered NF-κB activation in MM cells. Treating cells with DHA or EPA prior to bortezomib enhanced the induced cell death. However, concomitant use of bortezomib in combination with either of DHA or EPA decreased the cell death induced by bortezomib, indicating that timing of coincubation is important for the effects on chemosensitivity. The present study provides novel evidence for the anticancer effects of DHA and EPA, and highlights their rational utilization in combination with bortezomib to achieve improved therapeutic outcome for MM.

Durable complete remission with combination chemotherapy and bortezomib in HIV-associated plasmablastic lymphoma

Plasmablastic lymphoma (PBL) is an aggressive form of non-Hodgkin’s lymphoma (NHL) classically seen in patients infected with the human immunodeficiency virus, but can also be seen in other immunocompromised states...

Bortezomib as a new therapeutic approach for blastic plasmacytoid dendritic cell neoplasm.

Blastic plasmacytoid dendritic cell neoplasm is an aggressive hematologic malignancy with a poor prognosis. No consensus regarding optimal treatment modalities is currently available. Targeting the nuclear factor-kappa B pathway is considered a promising approach since blastic plasmacytoid dendritic cell neoplasm has been reported to exhibit constitutive activation of this pathway. Moreover, nuclear factor-kappa B inhibition in blastic plasmacytoid dendritic cell neoplasm cell lines, achieved using either an experimental specific inhibitor JSH23 or the clinical drug bortezomib, interferes in vitro with leukemic cell proliferation and survival. Here we extended these data by showing that primary blastic plasmacytoid dendritic cell neoplasm cells from seven patients were sensitive to bortezomib-induced cell death. We confirmed that bortezomib efficiently inhibits the phosphorylation of the RelA nuclear factor-kappa B subunit in blastic plasmacytoid dendritic cell neoplasm cell lines and primary cells from patients in vitro and in vivo in a mouse model. We then demonstrated that bortezomib can be associated with other drugs used in different chemotherapy regimens to improve its impact on leukemic cell death. Indeed, when primary blastic plasmacytoid dendritic cell neoplasm cells from a patient were grafted into mice, bortezomib treatment significantly increased the animals’ survival, and was associated with a significant decrease of circulating leukemic cells and RelA nuclear factor-kappa B subunit expression. Overall, our results provide a rationale for the use of bortezomib in combination with other chemotherapy for the treatment of patients with blastic plasmacytoid dendritic cell neoplasm. Based on our data, a prospective clinical trial combining proteasome inhibitor with classical drugs could be envisaged.

FLT3-ITD Confers Resistance to Bortezomib By Protecting the mTOR/4EBP1 Pathway through Activation of STAT5 and Induction of Pim-1 Expression

FLT3-ITD and FLT3-TKD are the most frequent tyrosine kinase mutations in AML, with the former strongly associated with a poor prognosis. We have recently revealed that FLT3-ITD confers resistance to the PI3K/AKT pathway inhibitors by protecting the mTOR/4EBP1/Mcl-1 pathway through STAT5 activation in AML. The proteasomal inhibitor bortezomib (BZM) has recently been reported as a promising agent for treatment of AML. We examine here the molecular mechanisms involved in induction of apoptosis by BZM in hematopoietic cells, including AML cells, and evaluate the effects of FLT3-ITD and TKD on these mechanisms and on the sensitivity of cells to BZM-induced apoptosis.We first comparatively examined the effect of BZM on survival of hematopoietic 32D cells and human leukemic UT7 cells driven by FLT3-ITD (32D/ITD and UT7/ITD) or FLT3-D835Y (32D/TKD and UT7/TKD). BZM induced activation of Bax, decline in mitochondrial membrane potential, and activation of caspase-9, thus leading to apoptosis, more conspicuously in cells driven by FLT3-ITD than FLT3-TKD. These results indicate that FLT3-ITD, as compared with FLT3-TKD, conferred resistance to apoptosis induced by BZM through the intrinsic pathway in these cells.To elucidate the molecular mechanisms involved in BZM-induced apoptosis in these cells, we next examined the effect of BZM on expression levels of FLT3 in these cells as well as in human leukemic MV4-11 cells expressing FLT3-ITD. Intriguingly, treatment of these cells with BZM conspicuously reduced expression levels of FLT3. It was further reveled that BZM further facilitated the decline in FLT3-ITD expression in MV4-11 cells treated with actinomycin D to shut down the transcriptional activity. Furthermore, BZM significantly retarded the recovery of FLT3-ITD expression in MV4-11 cells washed out from the translation inhibitor cycloheximide. These results suggest that BZM may downregulate the FLT3-ITD expression mainly at the translational level. However, we did not observe any significant difference in extent of the BZM-induced decline in expression levels between FLT3-ITD and FLT3-TKD.We next examined the effect of BZM on the mTOR/4EBP1 pathway, which we have shown to play important roles in regulation of apoptosis downstream of FLT3-ITD. It was found that BZM downregulated this pathway more significantly in 32D/TKD cells than in 32D/ITD cells. Because we have also previously found that STAT5 activated robustly by FLT3-ITD plays an important role in modulation of the mTOR/4EBP1 pathway, we examined the effects of BZM on 32D/TKD cells forced to express the constitutively activated STAT5 mutant, STAT5A1*6. As expected, STAT5A1*6 conferred resistance to BZM-induced downregulation of the mTOR/4EBP1 pathway as well as apoptosis in 32D/TKD cells. Consistent with this, the STAT5 inhibitor pimozide, clinically in use for neuropsychiatric disorders, abrogated the resistance of 32D/ITD, UT7/ITD, and MV4-11 cells to BZM-induced inhibition of the mTOR/4EBP1 pathway and apoptosis.We finally examined the possible involvement of the STAT5 target gene product Pim-1 in acquisition of resistance to BZM by cells expressing FLT3-ITD. We fist confirmed that Pim-1 was expressed at a higher level in 32D/ITD cells than in 32D/TKD cells and that STAT5A1*6 increased the expression level of Pim-1 in 32D/TKD cells. We then examined the effects of a specific Pim kinase inhibitor, AZD-1208, and found that it synergistically downregulated the mTOR/4EBP1 pathway and induced apoptosis with BZM in 32D/ITD cells as well as 32D/TKD cells expressing STAT5A1*6. We also examined the effects of a BET bromodomain inhibitor, JQ1, which has recently been shown to inhibit the STAT5 activity and to reduce specifically the expression level of Pim-1 as well as c-Myc in MV4-11 cells. As expected, pretreatment of 32D/ITD or MV4-11 cells with JQ1 conspicuously sensitized these cells to BZM-induced apoptosis.These results suggest that BZM downregulates FLT3 expression and the mTOR/4EBP1 pathway to activate the intrinsic apoptotic pathway and that robust STAT5 activation by FLT3-ITD confers resistance to BZM on AML cells through protection of the mTOR/4EBP1 pathway at least partly by inducing Pim-1 expression. The present study may contribute to development of novel therapeutic strategies against FLT3-ITD-positive AML by combined use of BZM and the STAT5/Pim-1 pathway inhibitors. DisclosuresNo relevant conflicts of interest to declare.

Profiling human protein degradome delineates cellular responses to proteasomal inhibition and reveals a feedback mechanism in regulating proteasome homeostasis.

Global change in protein turnover (protein degradome) constitutes a central part of cellular responses to intrinsic or extrinsic stimuli. However, profiling protein degradome remains technically challenging. Recently, inhibition of the proteasome, e.g., by using bortezomib (BTZ), has emerged as a major chemotherapeutic strategy for treating multiple myeloma and other human malignancies, but systematic understanding of the mechanisms for BTZ drug action and tumor drug resistance is yet to be achieved. Here we developed and applied a dual-fluorescence-based Protein Turnover Assay (ProTA) to quantitatively profile global changes in human protein degradome upon BTZ-induced proteasomal inhibition. ProTA and subsequent network analyses delineate potential molecular basis for BTZ action and tumor drug resistance in BTZ chemotherapy. Finally, combined use of BTZ with drugs targeting the ProTA-identified key genes or pathways in BTZ action reduced BTZ resistance in multiple myeloma cells. Remarkably, BTZ stabilizes proteasome subunit PSMC1 and proteasome assembly factor PSMD10, suggesting a previously under-appreciated mechanism for regulating proteasome homeostasis. Therefore, ProTA is a novel tool for profiling human protein degradome to elucidate potential mechanisms of drug action and resistance, which might facilitate therapeutic development targeting proteostasis to treat human disorders.

The Order of Exposure to Cytotoxic Chemotherapy and the Proteasome Inhibitor Bortezomib Dictates the Extent of Reversal of Chemotherapy Resistance in T-ALL

AbstractAbstract 1355 Introduction:While most children with T-cell acute lymphoblastic leukemia (T-ALL) are cured, the outcome for children who respond poorly to therapy or relapse is almost universally fatal. Our laboratory is interested in understanding the mechanisms contributing to therapy resistance and has developed a mutliparameter phosphoflow cytometry assay to study signal transduction events at a single cell level.Nuclear Factor kappa B (NF-κB) is a family of transcription factors that plays an important role in cancer development by preventing apoptosis and facilitating tumor cell growth. Consistent with this, constitutive NF-κB activity has been reported in T-ALL, and has been suggested as a predictor of relapse. We hypothesized that the proteasome inhibitor Bortezomib, which blocks NF-κB activation, induces apoptosis in T-ALL. We further hypothesized that combining Bortezomib with conventional chemotherapeutic agents sensitizes T-ALL cells to apoptosis and overcomes chemotherapy resistance by altering the balance of downstream pro- and anti- apoptotic mediators. Methods:A panel of T-ALL cell lines was cultured in the presence of vehicle alone, conventional cytotoxic drugs (Dexmethasone, Etoposide, Cyclophosphamide, Cytarabine), Bortezomib, or combinations of these. Cytotoxic effect was measured by trypan blue staining and flow cytometric detection of cleaved caspase-3. Multiparameter phosphoflow cytometry was used to measure expression levels of downstream apoptosis mediators. An advantage of multiparameter phosphoflow cytometry is the ability to gate on caspase 3-negative (i.e., chemotherapy resistant) cells, allowing us to interrogate potential mechanisms of resistance by comparing expression profiles of cell survival proteins before and after treatment. Results:T-ALL cells were exposed to cytotoxic chemotherapy or vehicle for 24 to 48 hours and subjected to phosphoflow cytometry. Analysis of caspase-3 negative cells indicated that chemotherapy resistant cells rewired their signaling networks by upregulating their MAPK, NF-κB, and AKT/PI3K/S6 signaling networks in response to genotoxic stress. This correlated with upregulation of the pro-survival protein survivin, suggesting a potential mechanism for chemotherapy resistance. To test whether Bortezomib could reverse this resistance, cells were exposed to subtoxic concentrations of Bortezomib alone or in combination with cytotoxic drugs. Combination therapy produced only a modest synergistic effect. We then wondered whether the sequence of administration of cytotoxic drug and Bortezomib could potentiate this effect. T-ALL cell lines were therefore 1) pretreated with Bortezomib for 24 hours followed by cytotoxic drug for an additional 24 hours, 2) pretreated with cytotoxic drug for 24 hours followed by addition of Bortezomib for an additional 24 hours, or 3) treated with both agents simultaneously. Surprisingly, the most pronounced cytotoxic synergy was observed when T-ALL cells were pretreated with cytotoxic drug followed by Bortezomib. While simultaneous or pretreatment with Bortezomib had no effect on upregulation of surivin in response to cytoxic chemotherapy, addition of Bortezomib post genotoxic stress resulted in markedly decreased levels of survivin. Conclusions:We found that Bortezomib lowered the apoptotic threshold to conventional cytotoxic drugs and can reverse drug resistance in T-ALL cell lines. Interestingly, the order of exposure to cytotoxic chemotherapy and Bortezomib determines the extent of reversal of chemotherapy resistance. Our data is in agreement with studies in multiple myeloma and provides evidence supporting the use of Bortezomib in combination with conventional cytotoxic agents in the treatment of relapsed or refractory disease. Disclosures:No relevant conflicts of interest to declare.

Bortezomib In Combination with Rituximab, Cyclophosphamide, and Prednisone with or without Doxorubicin Followed by Rituximab Maintenance In Patients with Relapsed or Refractory Follicular Lymphoma: Results of a Phase 2 Study.

AbstractAbstract 2798 Background:Non-Hodgkin's lymphoma is the most common hematologic malignancy, with an estimated >65,000 new cases in the US in 2010. The indolent follicular lymphoma (FL) and marginal zone lymphoma (MZL) subtypes account for approximately 22% and 10% of cases, respectively. Rituximab, cyclophosphamide, vincristine, and prednisone, with doxorubicin (R-CHOP) or without (R-CVP) are the most common regimens used for frontline and relapsed/refractory FL. Bortezomib (VELCADE®) has shown activity alone and with rituximab in relapsed/refractory FL/MZL, and also in combination with chemo-immunotherapy regimens such as R-CHOP. However, overlapping peripheral neuropathy (PN) with vincristine may limit bortezomib dosing. Thus, the aim of this phase 2, two-arm, non-randomized, multicenter study was to evaluate the safety and efficacy of administering bortezomib in place of vincristine in the R-CHOP and R-CVP regimens. Methods:Patients aged ≥18 years with relapsed or refractory FL (including transformed FL) or MZL following ≥1 prior chemotherapy, ≥1 measurable tumor mass, ECOG performance status ≤2, no grade ≥2 residual toxicity from previous therapy/surgery, no grade ≥2 PN, and adequate hematologic, renal, and hepatic function were eligible. Based on investigator preference, patients received up to six 21-day cycles of bortezomib 1.6 mg/m2 IV on days 1 and 8, rituximab 375 mg/m2 IV on day 1, and prednisone 100 mg PO on days 1–5, plus either cyclophosphamide 1000 mg/m2 IV (VR-CP arm), or cyclophosphamide 750 mg/m2 IV and doxorubicin 50 mg/m2 IV (VR-CAP arm) on day 1. All patients then received rituximab 375 mg/m2 IV every 3 months for ≤2 years as maintenance. The primary endpoint was complete response (CR) rate, determined after all patients completed the 12-week follow-up visit. Secondary endpoints included overall response rate (ORR; CR + partial response [PR]), duration of response (DOR), progression-free survival (PFS), and safety/tolerability. Response was assessed by investigators using modified International Workshop Response Criteria. Toxicities were graded using National Cancer Institute Common Terminology Criteria for Adverse Events version 3.0. Results:A total of 55 patients have been enrolled, 48 to VR-CP and 7 to VR-CAP; reasons for selecting VR-CP included extent of prior anthracycline therapy (56%), study center preference (25%), response to prior R-CVP (13%), and age (10%). Of the 48 VR-CP patients, 50% were male, 88% were white, median age was 63.5 years (range 40–85), 47 had FL (52/33/15% grade 1/2/3; 19/43/38% low/intermediate/high risk by FL International Prognostic Index [FLIPI]), and 1 had MZL. Median number of prior lines of therapy was 2 (range 1–10), which included R-CHOP in 27 (56%), rituximab in 19 (40%), and R-CVP in 7 (15%) patients. Of 47 evaluable patients, 14 (30%) achieved CR and 23 (49%; including 1 MZL) PR (ORR 79%). Median DOR and PFS have not been reached. Patients received a median of 6 (range 2–6) cycles of VR-CP; 40 (83%) received all 6 cycles, and 28 (58%) have continued into the rituximab maintenance phase. All patients reported ≥1 adverse event (AE), including 65% with grade ≥3 AEs, and 25% with serious AEs. Most common AEs included diarrhea (52%), nausea (46%), fatigue (44%), neutropenia (31%), constipation, and vomiting (each 23%); grade ≥3 AEs included neutropenia (27%), lymphopenia (10%), and thrombocytopenia (6%). The incidence of PN was 21% (2% grade ≥3). Four (8%) patients discontinued due to AEs. Of 7 VR-CAP patients (2 male, 7 white, median age 57 years [range 55–73]), 4 had FL (3 grade 1, 1 unknown; all FLIPI high-risk), 2 MZL, and 1 chronic lymphocytic leukemia. Median number of prior lines of therapy was 1 (range 1–8). Of 6 response-evaluable patients, 0/4 achieved CR/PR (3 FL, 1 MZL; ORR 67%). Five patients received all 6 cycles; 4 have continued into rituximab maintenance. AEs included fatigue (n=5), alopecia, neutropenia (each n=4), anemia, constipation, nausea, rigors, and thrombocytopenia (each n=3). Neutropenia (n=4) was the only grade ≥3 AE reported in >1 patient. PN was seen in 2 patients (no grade 3). Two patients had serious AEs. There have been no on-study deaths in either arm. Conclusion:These results indicate that VR-CP is active, with a 30% CR rate, in patients with relapsed or refractory FL/MZL, including those who had received prior chemo-immunotherapy. Prolonged follow-up is required to evaluate DOR and PFS; updated results will be presented. Disclosures:Craig:Genentech: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Off Label Use: Use of bortezomib in combination in follicular lymphoma and marginal zone lymphoma. Cabanillas:Celgene: Consultancy, Research Funding; Bayer: Research Funding; Pfizer: Research Funding. Parasuraman:Millennium Pharmaceuticals, Inc.: Employment, Equity Ownership. Neuwirth:Millennium Pharmaceuticals, Inc.: Employment. O'Connor:Millennium Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

The combination of bortezomib and resveratrol may prevent muscle wasting in diabetes

Hypercatabolic syndrome is a biochemical state characterized by a imbalance between catabolism and anabolism in favor of catabolism. Diabetes is an example of hypercatabolic syndrome with presence of decreased insulin level or impaired insulin signaling besides increased inflammatory cytokines. One of the significant outcomes of this state is accelerated protein degradation and muscle wasting. Increased ubiquitin-proteasomal system activity is the major responsible for the muscle wasting. Increase in expression and activities of proteasomal proteins in diabetes had been determined. NF-κB transcription factor mediated inflammation and oxidative stress accompanies proteasomal activity increase. Oxidative stress continuously produces substrate for proteasomes by causing protein oxidation. An intervention that inhibits proteasomal activity, suppressing inflammation and oxidative stress may form a solution in order to prevent muscle wasting. Therefore, I am considering that the combined use of bortezomib, a proteasome inhibitor and an anti-inflammatory with resveratrol, an antioxidant and anti-inflammatory, could prevent diabetes induced muscle wasting. This combination may be a novel therapeutic approach for muscle wasting.

Combination Melphalan and Bortezomib Conditioning with Autologous Hematopoietic Stem Cell Support in Patients with Advanced Multiple Myeloma. A Phase I/II Study.

Abstract 1214Poster Board I-236Bortezomib is an active agent in the treatment of multiple myeloma (MM) and shows additive or synergistic effects when combined with various chemotherapeutics in pre-clinical and clinical models. We are conducting a phase I/II clinical study of bortezomib with dose-intense melphalan with autologous peripheral blood stem cell transplantation (PBSCT) in patients (pts) with disease progression or less than partial response after a prior PBSCT (tandem transplant). Primary exclusion criteria are active infection at time of PBSCT, cardiac amyloid deposition, and creatinine clearance of <20 ml/min. Peripheral neuropathy of less than grade 4 is not an exclusion. Bortezomib is given on days -4 and -1 with melphalan 200 mg/m2 (actual weight) given on day -2 before PBSCT. All pts are given oral ice during the melphalan infusion to prevent oral mucositis. For the phase I study, bortezomib was given at rising doses of 1.0, 1.3, and 1.6 mg/m2. Three pts were to be enrolled at each dose level, with an additional 3 pts enrolled in case of a serious toxicity event at any level. An additional 20 pts are being enrolled in the phase II portion of the study.Twelve pts (median age, 59 yrs) were treated in the phase I study with 6 pts treated at the 1.0 mg/m2 level after 1 pt experienced an SAE of prolonged diarrhea, and 3 pts treated at each of the subsequent levels. Eleven pts had 1 prior and 1 pt tandem prior cycles of dose-intense melphalan with PBSCT. All pts experienced the expected pancytopenia requiring red cell and/or platelet support. Ten pts had febrile neutropenia with bacteremia identified in 3 pts. One pt had mild tumor lysis. Mucositis was minimal and comparable to PBSCT with melphalan alone. All pts engrafted at a median time to ANC>500/uL of 11 days (range, 9-19) and plt>20,000/uL of 14 days (range, 11-27). No other SAEs occurred in the phase I study beyond the usual events of high-dose therapy. No neurological SAEs, including severe peripheral neuropathies, were observed. A bortezomib dose of 1.6 mg/m2 was chosen for the phase II study.Eleven pts (median age, 57 yrs) are now treated in the phase II study. Three pts had disease progression and 8 pts had less than PR after a prior PBSCT. All pts experienced pancytopenia and 10 engrafted with median time to ANC>500/uL of 10 days (range, 8-13) and plt>20,000 11 days (range, 9-65). Seven pts had febrile neutropenia with 3 pts with positive blood cultures and 1 pt with RSV bronchitis. Three SAEs are reported in the phase II portion: 1 pt expired of complications of Candida krusei infection and 1 of MRSA sepsis (before ANC recovery). A third pt developed tumor lysis requiring dialysis. One pt had a dysphoric reaction to anti-emetics and did not receive the 2nd bortezomib dose. No neurological SAEs attributable to this regimen were observed in this population.Pts underwent restaging studies at monthly intervals after transplantation with marrow examination at 3 and 12 months (mos). Response classification is in accordance to standard definitions. Three pts underwent subsequent allogeneic PBSC and 2 pts died of transplant-related complications and are not evaluable for response. Two pts succumbed to progressive disease. The remaining 16 pts are in ongoing follow-up. Eight pts including 6 of 11 pts with stable disease or minimal response after prior dose-intense melphalan achieved a CR. Six pts remain in continuous CR at 11+ to 23+ mos (median, 15+ mos) after PBSCT with 2 pts having disease progression at 12 and 26 mo after PBSCT.These data indicate that bortezomib can be added to dose-intense melphalan in this schedule with acceptable toxicities. Strikingly, 8 of 20 pts achieved CR after minimal response to dose-intense melphalan alone or disease progression after a prior PBSCT, and 2 pts showed tumor lysis, indicating a synergistic effect of adding bortezomib to dose-intense melphalan. This regimen will now be studied in a larger cross-over study of tandem PBSCT to assess relative response to the two regimens. Disclosures:Off Label Use: Bortezomib is indicated in the treatment of multiple myeloma. This study involved the use of bortezomib in combination with dose-intense melphalan with stem cell transplantation.. Siegel:Millenium: Consultancy, Research Funding, Speakers Bureau. Goy:Millenium: Speakers Bureau.

Oncogenic transformation confers a selective susceptibility to the combined suppression of the proteasome and autophagy

The proteasome and the autophagy systems are two evolutionarily conserved mechanisms for degrading intracellular materials. They are functionally coupled and suppression of the proteasome promotes autophagy. Although suppression of the proteasome leads to cell death, suppression of autophagy can be either prodeath or prosurvival. To understand the underlining mechanism of this dichotomy and its potential clinical implications, we treated various transformed and nontransformed human cells with proteasome inhibitors. We found that whether autophagy served a prosurvival role in this scenario was contingent on the cellular oncogenic status. Thus, autophagy suppression enhanced apoptosis induced by proteasome inhibitors in transformed cells, but not in nontransformed cells. Oncogenic transformation enhanced the ability of cells to initiate autophagy in response to stress, reflecting a stronger dependence of transformed cells on autophagy for survival. Indeed, a combined use of bortezomib, the only Food and Drug Administration-approved proteasome inhibitor for clinical use, and chloroquine, which inhibits autophagy by disturbing lysosomal functions, suppressed tumor growth more significantly than either agent alone in a xenograft model. These findings indicate that suppression of both intracellular degradation systems could constitute a novel strategy for enhanced cancer control in a tumor-specific way.

Cytotoxic Effect of Proteasome Inhibitor Bortezomib in Combination with Purine Nucleoside Analogues on Chronic Lymphocytic Leukemia Cells in Vitro.

Background: Bortezomib (Velcade, PS-341), a selective inhibitor of the 26S proteasome which was recently approved for the treatment of patients with refractory multiple myeloma, showed also anti-leukemic activity in several studies. Bortezomid alone was found to induce apoptosis of chronic lymphocytic leukemia (CLL) cells in vitro. So far, there was no data on effects of its combination with other drugs routinely used in this disease.Aim: Cytotoxic activity of bortezomib in combination with purine nucleoside analogues, fludarabine (FA) and cladribine (2-CdA) was tested in CLL cells in vitro.Methods: The study was performed on CLL lymphocytes derived from 26 patients. Cell viability was assessed by propidium iodide staining. Apoptosis was evaluated by both Annexin-V and caspase activation flow cytometry assays. Additionally, expression of the apoptosis-regulating proteins Bax, Bak, Bid, Bcl-w, Bcl-2, XIAP and Mcl-1 was evaluated in CLL cells.Results: Bortezomib alone induced significant, dose-dependent cytotoxicity, inducing apoptosis of CLL cells. The viability of CLL cells treated with bortezomib decreased from 73.4%±14.9 (at 1.25nM) to 39.1%±15.4% (at 10nM). The mean apoptotic index estimated after 24h of treatment was in a range from 29.9%±18.0% at the dose of 1.25nM to 66.1%±21.8% at 10nM of bortezomib. Combination of this agent with FA or 2-CdA resulted in an increased cytotoxicity, comparing single drugs. It was especially evident when 5nM of bortezomib was combined with suboptimal doses of FA or 2-CdA. Cytotoxic effect of 5nM bortezomib +FA was significantly higher than 5nM bortezomib (p=0.0004) or FA (p<0.0001) alone. Similarly, cytotoxicity of 5nM bortezomib +2-CdA combination was significantly higher than 5nM bortezomib or 2-CdA used as a single agents (p=0.001 and p<0.0001, respectively). The combination index (CI) was 0.82 for bortezomib+FA and 0.87 for bortezomib+2-CdA, indicating the additive effect of these combinations. Corresponding to enhanced apoptosis, the expression of some apoptosis-regulating proteins was altered. Namely, XIAP protein was moderately downregulated by 2-CdA and FA, but not changed by bortezomib alone. However, combination of bortezomib with 2-CdA or FA significantly decreased XIAP expression (0.42-fold and 0.53-fold decrease, respectively; p=0.0007 and p=0.003, respectively). Bortezomib alone did not influence Bid levels, but its combination with 2-CdA induced a significant, 1.58-fold increase of Bid expression, comparing to the untreated control (p=0.028). Combination with FA also upregulated Bid protein (mean increase 1.39-fold vs. control), however, this difference was not statistically significant (p=0.582). The level of Mcl-1 protein was moderately elevated in the cells treated with bortezomib alone (1.41-fold increase; p=0.501). 2-CdA or FA only slightly decreased Mcl-1expression, but both purine nucleoside analogues prevented the Mcl-1 upregulation when combined with bortezomib.Conclusion: This study provides the strong rationale for the use of bortezomib in combination with purine nucleoside analogues for the treatment of CLL. This combination downregulates the expression of apoptosis inhibitors XIAP and Mcl-1 and upregulates proapoptotic Bid protein in CLL cells.

Sensitivity, Schedule-Dependence and Molecular Effects of the Proteasome Inhibitor Bortezomib in Non-Hodgkin's Lymphoma Cells.

Bortezomib, the first of a new class of agents called proteasome inhibitors, has shown promising activity in several tumor types, especially in hematological malignancies. To further characterize its activity in Non Hodgkin Lymphoma (NHL), we determined the efficacy of bortezomib, in vitro in 19 cell lines representing five subtypes of lymphoma and in ex vivo culture of patients samples. Using an MTS cell cytotoxicity assay, bortezomib was found to induce apoptosis at lower doses (IC50 ranging from 13.2 to 19.4 nM) in Mantle Cell Lymphoma (MCL) and Diffuse Large B-Cell Lymphoma (DLBCL) cells than in Burkitt lymphoma, Primary Effusion Lymphoma (PEL) and Anaplastic Large Cell Lymphoma (ALCL). In ex vivo culture of patients samples treated with 5 or 10 nM of bortezomib, the percentage of cells undergoing apoptosis as determined by annexin V-FITC staining was greater in MCL (+28.2% ± 12 with 10nM; +12.5% ± 13.8 with 5 nM) than in DLCL samples (+13.4% ± 6.2 with 10nM; +4.9% ± 1.5 with 5nM) when compared to spontaneous apoptosis. Bortezomib also showed a much lower IC50 than conventional cytotoxic agents (doxorubicin, vincristine and 4-hydroperoxycyclophosphamide) in 4 MCL cell lines. The potential synergy between cytotoxic agents and bortezomib was studied using annexin FITC staining as well as PARP cleavage analysis. Co-treatment of MCL cell lines with bortezomib and any of these chemotherapy agents showed a synergistic effect. However, the synergy was greater if the cells were sequentially treated with doxorubicin or vincristine followed by bortezomib. On the opposite the synergistic effect was greatly reduced or abolished when the cells were pretreated with bortezomib before exposure to doxorubicin or vincristine. We then used gene expression profiling to characterize the molecular consequences of bortezomib treatment in both MCL and DLBCL lymphoma cell lines. Using a 2 fold cut-off and a p value of <0.05, a list of 120 genes was generated. The transcriptional profile of bortezomib treated cells identified genes involved in apoptosis and cell cycle in the bortezomib sensitive cell lines we tested; these changes were not observed in a bortezomib resistant PEL cell line. The transcription of several heat-shock proteins (HSP70, 90 and 105) as well as genes involved in the ubiquitin proteasome pathway was significantly increased as previously described. These expression changes were validated on all 37 genes tested using QRT-PCR. We then tested the genes expression changes in the sequential combinations on MCL cell line exposed to subtoxic doses of doxorubicin with pre, concomittant or post treatment with subtoxic dose of bortezomib as well. Results showed that the level of expression of genes involved in key cellular functions (CDK2AP1, TYMS, CHEK1, CD81, RASSF2, CCNB2, AURKB, CNAP1, PIK3CD, TRIB3, CDK2, SQSTM1) was found modified only when MCL cells were sequentially treated with doxorubicin followed by bortezomib. Altogether these results might explain why in a sequential drug administration, pre-treatment of MCL cell lines with bortezomib did not sensitize the cells to doxorubicin or vincristine. These results also provide insight into the mechanism of bortezomib activity in NHL cells as well as a rationale for the use of bortezomib in combination with conventional therapy in future clinical trials.