Autophagy controls everolimus (RAD001) activity in mantle cell lymphoma

Mantle cell lymphoma (MCL) is an aggressive neoplasm, which lacks effective therapy. The mechanistic target of rapamycin (MTOR) kinase inhibitor everolimus (RAD001) has shown activity in preclinical and clinical models of MCL, despite the fact that its mechanism of action has not been fully elucidated. We found that everolimus activity in MCL cells is closely linked to AKT phosphorylation status, and that the prevention of AKT rephosphorylation upon everolimus treatment by means of a selective AKT inhibitor, greatly enhances everolimus activity. Furthermore, our data show that an accumulation of autophagic vacuoles correlates with a lack of efficacy of dual AKT-MTOR targeting and that the complete therapeutic potential of this strategy can be restored by ATG gene selective knockdown or secondary inhibition of autolysosome formation by hydroxychloroquine. We thus demonstrated for the first time that the use of an autophagy inhibitor can overcome resistance to the combination of MTOR and AKT inhibitors in MCL cell lines and primary samples, demonstrating the prosurvival role of autophagy in AKT-MTOR compromised cells, and pointing out some potential opportunities using this triple combinational strategy in hematological malignancies.     

Combination bortezomib and rituximab treatment affects multiple survival and death pathways to promote apoptosis in mantle cell lymphoma

Mantle cell lymphoma (MCL) is a distinct histologic subtype of B cell non-Hodgkins lymphoma (NHL) associated with an aggressive clinical course. Inhibition of the ubiquitin-proteasome pathway modulates survival and proliferation signals in MCL and has shown clinical benefit in this disease. This has provided rationale for exploring combination regimens with B-cell selective immunotherapies such as rituximab. In this study, we examined the effects of combined treatment with bortezomib and rituximab on patient-derived MCL cell lines (Jeko, Mino, SP53) and tumor samples from patients with MCL where we validate reversible proteasome inhibition concurrent with cell cycle arrest and additive induction of apoptosis. When MCL cells were exposed to single agent bortezomib or combination bortezomib/rituximab, caspase dependent and independent apoptosis was observed. Single agent bortezomib or rituximab treatment of Mino and Jeko cell lines and patient samples resulted in decreased levels of nuclear NFκB complex(es) capable of binding p65 consensus oligonucleotides, and this decrease was enhanced by the combination. Constitutive activation of the Akt pathway was also diminished with bortezomib alone or in combination with rituximab. On the basis of in vitro data demonstrating additive apoptosis and enhanced NFκB and phosphorylated Akt depletion in MCL with combination bortezomib plus rituximab, a phase II trial of bortezomib-rituximab in patients with relapsed/refractory MCL is underway.Key words: mantle cell lymphoma, proteasome inhibition, CD20, survival and death pathways, apoptosis     

Inhibition of autophagy by autophagic inhibitors enhances apoptosis induced by bortezomib in non-small cell lung cancer cells

Bortezomib is a novel proteasome inhibitor that has promising antitumor activity against various cancer cells. We have assessed its antitumor activity in non-small cell lung cancer (NSCLC) A549 and H157 cells in vitro where it inhibited cell growth and induced apoptosis, which was associated with cytochrome c release and caspase-3 activation. Bortezomib upregulated autophagic-related proteins, the Atg12–Atg5 complex and LC3-II, which indicated autophagy had occurred. The combination of bortezomib with autophagic inhibitor 3-methyladenine or chloroquine significantly enhanced suppression of cell growth and apoptosis induced by bortezomib in A549 and H157 cells. Our study indicated that inhibition of both proteasome and autophagy has great potential for NSCLC treatment.     

Combination bortezomib and rituximab treatment affects multiple survival and death pathways to promote apoptosis in mantle cell lymphoma

Mantle cell lymphoma (MCL) is a distinct histologic subtype of B cell non-Hodgkins lymphoma (NHL) associated with an aggressive clinical course. Inhibition of the ubiquitin-proteasome pathway modulates survival and proliferation signals in MCL and has shown clinical benefit in this disease. This has provided rationale for exploring combination regimens with B-cell selective immunotherapies such as rituximab. In this study, we examined the effects of combined treatment with bortezomib and rituximab on patient-derived MCL cell lines (Jeko, Mino, SP53) and tumor samples from patients with MCL where we validate reversible proteasome inhibition concurrent with cell cycle arrest and additive induction of apoptosis. When MCL cells were exposed to single agent bortezomib or combination bortezomib/rituximab, caspase dependent and independent apoptosis was observed. Single agent bortezomib or rituximab treatment of Mino and Jeko cell lines and patient samples resulted in decreased levels of nuclear NF-kB complex(es) capable of binding p65 consensus oligonucleotides, and this decrease was enhanced by the combination. Constitutive activation of the AKT pathway was also diminished with bortezomib alone or in combination with rituximab. On the basis of in vitro data demonstrating additive apoptosis and enhanced NF-kB and phosphorylated AKT depletion in MCL with combination bortezomib plus rituximab, a phase II trial of bortezomib-rituximab in patients with relapsed/refractory MCL is underway.     

Bortezomib enhances the osteogenic differentiation capacity of human mesenchymal stromal cells derived from bone marrow and placental tissues

Bortezomib (BZB) is a chemotherapeutic agent approved for treating multiple myeloma (MM) patients. In addition, there are several reports showing that bortezomib can induce murine mesenchymal stem cells (MSCs) to undergo osteogenic differentiation and increase bone formation in vivo. MSCs are the multipotent stem cells that have capacity to differentiate into several mesodermal derivatives including osteoblasts. Nowadays, MSCs mostly bone marrow derived have been considered as a valuable source of cell for tissue replacement therapy. In this study, the effect of bortezomib on the osteogenic differentiation of human MSCs derived from both bone marrow (BM-MSCs) and postnatal sources such as placenta (PL-MSCs) were investigated. The degree of osteogenic differentiation of BM-MSCs and PL-MSCs after bortezomib treatment was assessed by alkaline phosphatase (ALP) activity, matrix mineralization by Alizarin Red S staining and the expression profiles of osteogenic differentiation marker genes, Osterix, RUNX2 and BSP. The results showed that 1 nM and 2 nM BZB can induce osteogenic differentiation of BM-MSCs and PL-MSCs as demonstrated by increased ALP activity, increased matrix mineralization and up-regulation of osteogenic differentiation marker genes, Osterix, RUNX2 and BSP as compared to controls. The enhancement of osteogenic differentiation of MSCs by bortezomib may lead to the potential therapeutic applications in human diseases especially patients with osteopenia.     

The influence of proteasome inhibitor bortezamib on ABC transporters’ expression and activity in tumor cells

The goal of this study was to investigate the role of the ABC transporters in the evolution of tumor cell populations treated with bortezomib. Bortezomib (PS-341, Velcade) is a proteasome inhibitor used for treatment of some malignancies. Several pairs of cell lines different in Pgp expression (P-glycoprotein transporter, ABCB1) have been used in the study. We showed that the influence of the Pgp hyperexpression on cell sensitivity to bortezomib was bidirectional and depended on the tissue type. Bortezomib changed the mRNA level of MDR1 (ABCB1 and MRP1 (ABCC1)) genes, suggesting that the proteasome inhibitor is able to decrease the activity of some regulators of genes/proteins implicated in MDR. Bortezomib treatment increased the levels of proteins (Pgp or MPR1) in 3 out of 4 cell populations studied. Pgp was shown to remain functionally active in the cells cultured in bortezamib-containing medium. The UIC2-shift assay has shown that bortezomib is able to activate Pgp. This means that bortezomib influences Pgp conformation, thus activating the protein (in K562/i-S9 cells). These experiments also demonstrate that bortezomib is Pgp substrate.     

Mucin 20 modulates proteasome capacity through c-Met signalling to increase carfilzomib sensitivity in mantle cell lymphoma

Mantle cell lymphoma (MCL) is a haematologic malignancy. The proteasome inhibitor (PI) bortezomib has been approved to treat MCL, but resistance has emerged through mechanisms that remain unclear. This study aimed to explore the mechanism of PI resistance in MCL and identify new targets for this patient subgroup. Carfilzomib-resistant (CR) MCL cell lines and primary samples were used for both in vitro and in vivo experiments to identify gene expression and explore their related signalling pathways. We first identified mucin 20 (MUC20) suppression in carfilzomib-resistant MCL models. MUC20 overexpression sensitized cells to carfilzomib in vitro and in vivo. MUC20 expression was inversely related to activation of c-Met and the downstream p44/42 MAPK pathway. c-Met activation with hepatocyte growth factor (HGF) induced PI resistance, while c-Met inhibition restored PI sensitivity. Carfilzomib resistance and depressed MUC20 expression were associated with enhanced proteasome activity and higher expression of proteassemblin (POMP), a chaperone for catalytically active proteasome assembly. c-Met and POMP were associated through binding and induction of MAPK-regulated ELK1 to the POMP promoter. Our data reveal that c-Met signalling activation enhanced proteasome capacity as a mechanism of PI resistance, and MUC20 expression may be a useful biomarker for PI therapy.     

Inhibition of Inducible Heat Shock Protein-70 (Hsp72) Enhances Bortezomib-Induced Cell Death in Human Bladder Cancer Cells

The proteasome inhibitor bortezomib (Velcade) is a promising new agent for bladder cancer therapy, but inducible cytoprotective mechanisms may limit its potential efficacy. We used whole genome mRNA expression profiling to study the effects of bortezomib on stress-induced gene expression in a panel of human bladder cancer cell lines. Bortezomib induced strong upregulation of the inducible HSP70 isoforms HSPA1A and HSPA1B isoforms of Hsp72 in 253J B-V and SW780 (HSPA1A^high) cells, but only induced the HSPA1B isoform in UM-UC10 and UM-UC13 (HSPA1A^low) cells. Bortezomib stimulated the binding of heat shock factor-1 (HSF1) to the HSPA1A promoter in 253JB-V but not in UM-UC13 cells. Methylation-specific PCR revealed that the HSPA1A promoter was methylated in the HSPA1A^low cell lines (UM-UC10 and UM-UC13), and exposure to the chromatin demethylating agent 5-aza-2′-deoxycytidine restored HSPA1A expression. Overexpression of Hsp72 promoted bortezomib resistance in the UM-UC10 and UM-UC13 cells, whereas transient knockdown of HSPA1B further sensitized these cells to bortezomib, and exposure to the chemical HSF1 inhibitor KNK-437 promoted bortezomib sensitivity in the 253J B-V cells. Finally, shRNA-mediated stable knockdown of Hsp72 in 253J B–V promoted sensitivity to bortezomib in vitro and in tumor xenografts in vivo. Together, our results provide proof-of-concept for using Hsp72 inhibitors to promote bortezomib sensitivity in bladder cancers and suggest that selective targeting of HSPA1B could produce synthetic lethality in tumors that display HSPA1A promoter methylation.     

Cobblestone-Area Forming Cells Derived from Patients with Mantle Cell Lymphoma Are Enriched for CD133+ Tumor-Initiating Cells

Mantle cell lymphoma (MCL) is associated with a significant risk of therapeutic failure and disease relapse, but the biological origin of relapse is poorly understood. Here, we prospectively identify subpopulations of primary MCL cells with different biologic and immunophenotypic features. Using a simple culture system, we demonstrate that a subset of primary MCL cells co-cultured with either primary human mesenchymal stromal cells (hMSC) or murine MS-5 cells form in cobblestone-areas consisting of cells with a primitive immunophenotype (CD19−CD133+) containing the chromosomal translocation t (11;14)(q13;q32) characteristic of MCL. Limiting dilution serial transplantation experiments utilizing immunodeficient mice revealed that primary MCL engraftment was only observed when either unsorted or CD19−CD133+ cells were utilized. No engraftment was seen using the CD19+CD133− subpopulation. Our results establish that primary CD19−CD133+ MCL cells are a functionally distinct subpopulation of primary MCL cells enriched for MCL-initiating activity in immunodeficient mice. This rare subpopulation of MCL-initiating cells may play an important role in the pathogenesis of MCL.     

The Anti-apoptotic Protein BCL2L1/Bcl-xL Is Neutralized by Pro-apoptotic PMAIP1/Noxa in Neuroblastoma, Thereby Determining Bortezomib Sensitivity Independent of Prosurvival MCL1 Expression

Neuroblastoma is the most frequent extracranial solid tumor in children. Here, we report that the proteasome inhibitor bortezomib (PS-341, Velcade) activated the pro-apoptotic BH3-only proteins PMAIP1/Noxa and BBC3/Puma and induced accumulation of anti-apoptotic MCL1 as well as repression of anti-apoptotic BCL2L1/Bcl-xL. Retroviral expression of Bcl-xL, but not of MCL1, prevented apoptosis by bortezomib. Gene knockdown of Noxa by shRNA technology significantly reduced apoptosis, whereas Puma knockdown did not affect cell death kinetics. Immunoprecipitation revealed that endogenous Noxa associated with both, Bcl-xL and MCL1, suggesting that in neuronal cells Noxa can neutralize Bcl-xL, explaining the pronounced protective effect of Bcl-xL. Tetracycline-regulated Noxa expression did not trigger cell death per se but sensitized to bortezomib treatment in a dose-dependent manner. This implies that the induction of Noxa is necessary but not sufficient for bortezomib-induced apoptosis. We conclude that MCL1 steady-state expression levels do not affect sensitivity to proteasome-inhibitor treatment in neuronal tumor cells, and that both the repression of Bcl-xL and the activation of Noxa are necessary for bortezomib-induced cell death.     

Proteasome inhibition with bortezomib depletes plasma cells and autoantibodies in experimental autoimmune myasthenia gravis

Bortezomib, an inhibitor of proteasomes, has been reported to reduce autoantibody titers and to improve clinical condition in mice suffering from lupus-like disease. Bortezomib depletes both short- and long-lived plasma cells; the latter normally survive the standard immunosuppressant treatments targeting T and B cells. These findings encouraged us to test whether bortezomib is effective for alleviating the symptoms in the experimental autoimmune myasthenia gravis (EAMG) model for myasthenia gravis, a disease that is characterized by autoantibodies against the acetylcholine receptor (AChR) of skeletal muscle. Lewis rats were immunized with saline (control, n = 36) or Torpedo AChR (EAMG, n = 54) in CFA in the first week of an experimental period of 8 wk. After immunization, rats received twice a week s.c. injections of bortezomib (0.2 mg/kg in saline) or saline injections. Bortezomib induced apoptosis in bone marrow cells and reduced the amount of plasma cells in the bone marrow by up to 81%. In the EAMG animals, bortezomib efficiently reduced the rise of anti-AChR autoantibody titers, prevented ultrastructural damage of the postsynaptic membrane, improved neuromuscular transmission, and decreased myasthenic symptoms. This study thus underscores the potential of the therapeutic use of proteasome inhibitors to target plasma cells in Ab-mediated autoimmune diseases.     

Telomerase Activity in Human Brain Tumors: Astrocytoma and Meningioma

Somatic cells do not have telomerase activity but immortalized cell lines and more than 85 % of the cancer cells show telomerase activation to prevent the telomere from progressive shortening. The activation of this enzyme has been found in a variety of human tumors and tumor-derived cell lines, but only few studies on telomerase activity in human brain tumors have been reported. Here, we evaluated telomerase activity in different grades of human astrocytoma and meningioma brain tumors. In this study, assay for telomerase activity performed on 50 eligible cases consisted of 26 meningioma, 24 astrocytoma according to the standard protocols. In the brain tissues, telomerase activity was positive in 39 (65 %) of 50 patients. One sample t test showed that the telomerase activity in meningioma and astrocytoma tumors was significantly positive entirely (P < 0.001). Also, grade I of meningioma and low grades of astrocytoma (grades I and II) significantly showed telomerase activity. According to our results, we suggest that activation of telomerase is an event that starts mostly at low grades of brain including meningioma and astrocytoma tumors.