Targeting the proteasome system with bortezomib (BTZ) results in anti-tumour activity and potentiates the effects of chemotherapy/biological agents in multiple myeloma and B-cell lymphoma. Carfilzomib (CFZ) is a more selective proteasome inhibitor that is structurally distinct from BTZ. In an attempt to characterize its biological activity, we evaluated CFZ in several lymphoma pre-clinical models. Rituximab-sensitive cell lines (RSCL), rituximab-resistant cell lines (RRCL), and primary tumour cells derived from B-cell lymphoma patients were exposed to CFZ or BTZ. Cell viability and changes in cell cycle were determined. Western blots were performed to detect PARP-cleavage and/or changes in Bcl-2 (BCL2) family members. CFZ was 10 times more active than BTZ and exhibited dose- and time-dependent cytotoxicity. CFZ exposure induced apoptosis by upregulation of Bak (BAK1) and subsequent PARP cleavage in RSCL and RRCL; it was also partially caspase-dependent. CFZ induced G2/M phase cell cycle arrest in RSCL. CFZ demonstrated the ability to overcome resistance to chemotherapy in RRCL and potentiated the anti-tumour activity of chemotherapy agents. Our data suggest that CFZ is able to overcome resistance to chemotherapeutic agents, upregulate pro-apoptotic proteins to promote apoptosis, and induce G2/M cell cycle arrest in lymphoma cells. Our pre-clinical data supports future clinical evaluation of CFZ in B-cell lymphoma.
Key Points• Pevonedistat (MLN4924), a NEDD8-activating enzyme inhibitor, is active in MCL preclinical models and potentiates rituximab activity.• Our findings support further investigation of pevonedistat with or without rituximab in the treatment of MCL.Mantle cell lymphoma (MCL) is characterized by an aggressive clinical course and inevitable development of refractory disease, stressing the need to develop alternative therapeutic strategies. To this end, we evaluated pevonedistat (MLN4924), a novel potent and selective NEDD8-activating enzyme inhibitor in a panel of MCL cell lines, primary MCL tumor cells, and 2 distinct murine models of human MCL. Pevonedistat exposure resulted in a dose-, time-, and caspase-dependent cell death in the majority of the MCL cell lines and primary tumor cells tested. Of interest, in the MCL cell lines with lower half-maximal inhibitory concentration (0.1-0.5 mM), pevonedistat induced G1-phase cell cycle arrest, downregulation of Bcl-xL levels, decreased nuclear factor (NF)-kB activity, and apoptosis. In addition, pevonedistat exhibited additive/synergistic effects when combined with cytarabine, bendamustine, or rituximab. In vivo, as a single agent, pevonedistat prolonged the survival of 2 MCL-bearing mouse models when compared with controls. Pevonedistat in combination with rituximab led to improved survival compared with rituximab or pevonedistat monotherapy. Our data suggest that pevonedistat has significant activity in MCL preclinical models, possibly related to effects on NF-kB activity, Bcl-xL downregulation, and G1 cell cycle arrest. Our findings support further investigation of pevonedistat with or without rituximab in the treatment of MCL. (Blood. 2016;127(9):1128-1137
To further develop therapeutic strategies targeting the proteasome system, we studied the antitumor activity and mechanisms of action of MLN2238, a reversible proteasome inhibitor, in preclinical lymphoma models. Experiments were conducted in rituximab-chemotherapy-sensitive cell lines, rituximab-chemotherapy-resistant cell lines (RRCL), and primary B-cell lymphoma cells. Cells were exposed to MLN2238 or caspase-dependent inhibitors, and differences in cell viability, alterations in apoptotic protein levels, effects on cell cycle, and the possibility of synergy when combined with chemotherapeutic agents were evaluated. MLN2238 showed more potent dose-dependent and time-dependent cytotoxicity and inhibition of cell proliferation in lymphoma cells than bortezomib. Our data suggest that MLN2238 can induce caspase-independent cell death in RRCL. MLN2238 (and to a much lesser degree bortezomib) reduced RRCL S phase and induced cell cycle arrest in the G2/M phase. Exposure of rituximab-chemotherapy-sensitive cell lines and RRCL to MLN2238 potentiated the cytotoxic effects of gemcitabine, doxorubicin, and paclitaxel and overcame resistance to chemotherapy in RRCL. MLN2238 is a potent proteasome inhibitor active in rituximab-chemotherapy-sensitive and rituximab-chemotherapy-resistant cell models and potentiates the antitumor activity of chemotherapy agents and has the potential of becoming an effective therapeutic agent in the treatment of therapy-resistant B-cell lymphoma.
2761 Clinical outcome of patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) remains poor with currently available therapies. Recently, the ubiquitin-proteasome system (UPS) appears to play an important role in the development of resistance in MCL and some subtypes of DLBCL. Targeting UPS represents a rational approach in an attempt to eradicate drug-resistant lymphoma clones. MLN4924 is a novel, potent and selective inhibitor of the NEDD8-activating enzyme (NAE) that is necessary for the modification of cullin-RING ubiquitin ligases. We evaluated the anti-tumor activity of MLN4924 against a panel of rituximab-sensitive (RSCL) or rituximab/chemotherapy–resistant (RCRCL) DLBCL and Burkitt lymphoma cell lines, cytarabine-sensitive or -resistant (AraCR) MCL cell lines, and primary tumor cells freshly isolated from lymphoma patients (n=13). Lymphoma cells were exposed to escalating doses of MLN4924 alone or in combination with selected chemotherapy agents for up to 72 hrs. Changes in the cell viability or ATP content were determined by alamar Blue reduction or CellTiterGlo assays, respectively. Induction of apoptosis and changes in the levels of NFkB and UPS regulatory proteins were analyzed by Western blotting. Cell cycle alterations were determined by propidium iodide staining and NFkB activity was quantified by flow cytometry using the Imagestream technology. MLN4924 demonstrated time- and dose-dependent anti-lymphoma activity in all cell lines tested. The IC50 in RSCLs were Raji=400nM, RL=1uM and U2932=>3uM. All RCRCLs were less responsive to MLN4924 as a single agent with IC50 concentrations 4–10× those of their respective sensitive parental cell lines. The MCL cell lines Mino, MinoAraCR, Z-138, HBL-2 and HBL-2AraCR were most sensitive to MLN4924 anti-tumor effects (IC50=250nM) with no significant difference between cytarabine-sensitive and -resistant cell lines; while the MCL cell lines Rec-1, Rec-1AraCR, Jeko-1 and Jeko-1AraCR were less sensitive (IC50=500–1000nM). A variable degree of anti-tumor activity was also observed in primary lymphoma cells. In addition to single-agent activity, MLN4924 plus selected anti-lymphoma chemotherapy agents (bortezomib, bendamustine and cytarabine) demonstrated synergy in cytarabine-sensitive and (to a lesser degree) cytarabine-resistant MCL cell lines. Combinations with additional chemotherapeutic agents (doxorubicin and vincristine) resulted in additive effects. Exposure of MCL cells to MLN4924 resulted in G1 cell cycle arrest. In vitro exposure of the more sensitive MCL cell lines Mino and MinoAraCR to MLN4924 resulted in an increase in p-IkBα and down-regulation of both total and nuclear NFkB. The less sensitive cell lines Rec-1 and Rec-1AraCR demonstrated little to no change in NFkB activation following exposure to MLN4924. Additional studies are ongoing to further define the molecular mechanisms of the anti-tumor activity observed following NAE inhibition by MLN4924 in these pre-clinical models and to further evaluate the activity of MLN4924 in in vivo SCID mouse models of B-cell lymphoma. Our data suggests that MLN4924, a novel NAE inhibitor, is active against B-cell lymphomas, particularly MCL, and is a promising agent warranting further investigation in relapsed/refractory aggressive B-cell lymphomas. Disclosures: No relevant conflicts of interest to declare.
Multi-agent chemotherapy regimens have significantly improved long-term survival of pediatric Burkitt Lymphoma (BL) to over 80%. However, patients with refractory or relapsed disease have a dismal prognosis, stressing the need to identify mechanisms of therapeutic resistance and develop novel therapeutic approaches. Recent data demonstrated tonic B-cell receptor (BCR) activation of PI3K in BL (Srinivisan et al, Cell 2009); and implicated PI3K, in coordination with MYC, in Burkitt lymphomagenesis (Sander et al, Cancer Cell 2012). In order to investigate mechanisms of resistance, our laboratory generated several resistant BL cell lines. We exposed Raji cells to escalating doses of rituximab +/- human serum and generated several BL rituximab-resistant (Raji 7R and Raji 8RH) (RRCL) or therapy (rituximab-chemotherapy) resistant (Raji 2R and Raji 4RH) (TRCL) cell lines. We then screened for aberrant activation of signal transduction pathways using western blot, phospho-flow cytometry and phosphoproteomics to define pathways associated with the development of resistance. While total Akt expression was similar between all cell lines tested, Raji 2R and Raji 4RH cells had increased phosphorylation of Akt at Ser473 and Thr308, indicating Akt activation, when compared to Raji, Raji 7R and Raji 8RH by western blot and phospho-flow cytometry. Phosphoproteomic analysis comparing Raji and Raji 4RH identified an increase of at least 2 fold in phosphorylation of 315 proteins in Raji 4RH cells, including multiple direct targets of AKT (e.g. GSK3B, WEE1, FOXO1 and PRAS40). Altered phosphorylation of proteins downstream of Akt (e.g. BAD, 4EBP1, GSK3B and ERK) was also detected by western blot in Raji 2R and Raji 4RH compared to Raji, Raji 7R and Raji 8RH. These findings suggest that activation of PI3K/Akt may play a role in the development of chemoresistance noted in our cell line model. KEGG analysis of phosphoproteome and gene expression comparison of Raji to Raji 4RH cells identified the BCR pathway as one of the top pathways altered. Additionally, the microRNA (miR) 17-92 has been noted to increase Akt activation through its inhibitory effects on PTEN, a key regulator of Akt activation. A recent copy number analysis indicated that a recurrent gain in 13q, containing the MIR17HG locus, was also associated with higher mir17 RNA levels and early relapse in pediatric BL patients (Schiffman et al, Br J Haematol 2011). An analysis of miR expression in Raji 2R and Raji 4RH compared to Raji noted a 1.93 and 3.77 fold increase in miR17 respectively. These findings suggest possible mechanisms for increased PI3K/Akt activation in our chemoresistant cell lines. In vitro exposure to MK-2206, a pan-Akt inhibitor, resulted in dose- and time-dependent decreases in viability of TRCLs (IC50 at 72 hours: Raji 2R = 2.6µM; Raji 4RH = 3.2µM) and to a slightly lesser degree Raji and RRCL (Raji= 4.4µM; Raji 7R = 4.0µM; Raji 8RH = 5.2µM) (TRCL vs. Raji/RRCL, p<0.05), by alamarBlu assay. Cell cycle analysis following exposure to MK2206 identified G1 cell cycle arrest in Raji/RRCL, but G2/M cell cycle arrest was observed in TRCL, suggesting differing mechanisms of anti-proliferative effect between sensitive and resistant cells. The combination of MK2206 and either doxorubicin or dexamethasone resulted in synergistic decreases in cell viability at a variety of dose combinations in both sensitive and resistant cells, as well as in BL Ramos and Daudi cells, using the Chou-Talalay method to measure synergy. In Raji and Ramos, the combination of MK2206 and doxorubicin also resulted in a higher degree of PARP cleavage compared to either agent alone, though no PARP cleavage was noted in Raji 2R and Raji 4RH cells likely related to impaired apoptotic potential previously noted in these cell lines. An siRNA knockdown of Akt in Raji cells resulted in a decrease in viable cells compared to controls following exposure to doxorubicin. Similar effects on cell proliferation, cell cycle and synergy were seen with upstream inhibition of PI3K by the PI3Kδ inhibitor idelalisib. Our data suggests that constitutive activation of the PI3K/Akt pathway, through activation of the BCR pathway or over-expression of miR17, is associated with the development of resistance in BL cell lines, and inhibition of PI3K/Akt can sensitize BL cells to the effects of some chemotherapeutic agents. Targeting the PI3K/Akt signaling pathway may be a clinically relevant approach in pediatric BL. Disclosures Cairo: Celgene: Research Funding.
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