ALBERT

Description: Levels of Mcl-1, an anti-apoptotic Bcl-2 family member that plays an important role in MM cell survival, are tightly regulated by the proteasome, and recent data has demonstrated that proteasome inhibition with bortezomib leads to Mcl-1 accumulation, thereby attenuating its activity. Previously, we reported that inhibition of IL-6 signaling with the chimeric antibody, CNTO328, potentiated the anti-myeloma activity of bortezomib in IL-6-dependent cell line models of MM, and that the enhanced activity was associated with inhibition of downstream IL-6 signaling pathways and repression of bortezomib-mediated induction of the anti-apoptotic heat shock response. Based on our promising results with this combination and the role that IL-6 plays in Mcl-1 regulation, we have extended our preclinical studies and investigated whether CNTO328 could inhibit bortezomib-mediated Mcl-1 induction and how the bone marrow microenvironment affects the activity of the combination. Pre-treatment of the IL-6-dependent MM cell lines KAS-6 and ANBL-6 with CNTO328, but not an isotype control antibody, blunted bortezomib-mediated induction of anti-apoptotic Mcl-1L, and enhanced the cytotoxicity and pro-apoptotic activity of bortezomib. In contrast, CNTO328 did not attenuate bortezomib-mediated Mcl-1L accumulation in the IL-6-independent MM cell line RPMI 8226, nor did it enhance the cytotoxicity of bortezomib in these cells. In the presence of patient-derived bone marrow stromal cells, CNTO328 and bortezomib resulted in a greater reduction of cell viability than with either agent alone in both ANBL-6 and KAS-6 cells. Furthermore, although CNTO328 alone did not lead to an increase in apoptosis of ANBL-6 cells in the presence of bone marrow stroma, it significantly potentiated the pro-apoptotic activity of bortezomib in a synergistic manner at clinically achievable concentrations. As opposed to our cell viability data using the co-culture system, CNTO328 was not able to increase levels of apoptosis in KAS-6 cells either as a single agent or in combination with bortezomib, suggesting that CNTO328 may be inducing cell cycle arrest of KAS-6 cells in the presence of bone marrow stroma but is not able to potentiate the apoptotic activity of bortezomib. Finally, given the important role of IL-6 in glucocorticoid resistance and the additive preclinical and clinical activity of bortezomib and dexamethasone in MM, we evaluated the activity of dexamethasone in combination with CNTO328 and bortezomib in ANBL-6 cells. Whereas the combination of bortezomib (2.5 nM) and CNTO328 reduced viability to 58%, the addition of dexamethasone (10 mM) reduced viability further to 26%. Taken together, the above data demonstrate that CNTO328 enhances the activity of bortezomib in part by attenuating bortezomib-mediated Mcl-1 accumulation, and provide the rationale for clinical evaluation of CNTO328 and bortezomib, with or without dexamethasone, in patients with MM.

Description: Inhibition of the proteasome, a multi-catalytic proteinase complex responsible for regulated intracellular proteolysis, with bortezomib (VELCADE®) is a rational strategy against relapsed/refractory multiple myeloma, and studies are ongoing to further define the efficacy of this agent in other settings. However, bortezomib is associated with several toxicities, such as peripheral neuropathy, which can limit the ability of patients to receive maximal doses, possibly compromising anti-tumor efficacy. Novel proteasome inhibitors with an improved toxicity profile and improved anti-tumor activity are therefore needed. Most cells contain the constitutive version of the proteasome with three catalytic subunits, designated X, Y, and Z. Exposure of these cells to cytokines, such as γ-interferon, causes replacement of these subunits with three others, referred to as low molecular weight proteins (LMP)−2, −7, and −10. This LMP-containing proteasome, known as the immunoproteasome because it plays a role in generating antigens as part of the immune response, is also the major proteasome isoform in hematopoietic-derived cells even in the absence of exogenous cytokines. Using purified preparations of the XYZ and LMP proteasomes, we identified a panel of peptidyl-aldehydes that preferentially inhibit the immunoproteasome with relative sparing of the constitutive isoform. Among these, the most immunoproteasome specific inhibitors (IPSI) included compounds 001 and 006. IPSI-001 demonstrated a Ki of 1.03 μM against the chymotryptic activity of the LMP proteasome but only a 105 μM Ki against the XYZ proteasome, while IPSI-006 demonstrated Kis of 8.4 μM and 460 μM, respectively. In contrast, other commonly used peptidyl aldehydes, such as MG-132 and the aldehyde version of bortezomib, showed no ability to discriminate between these two proteasomes. Using radiolabeled 3,4-dichloroisocoumarin as a probe, IPSIs were shown to bind only to LMP-2 of the immunoproteasome, but not to any subunits of the constitutive proteasome in vitro. Studies of cell lines expressing immunoproteasome subunits showed IPSI-001 inhibited the chymotryptic activity of the LMP proteasome, with relative sparing of this activity in XYZ proteasome-expressing cell lines. IPSI-001 was also able to induce apoptosis in LMP-proteasome-expressing cells, including IM-9 lymphoblastoid cells and ANBL-6 interleukin-(IL)-6-dependent and RPMI-8226 IL-6-independent multiple myeloma cells, while relatively sparing XYZ-proteasome-expressing cell lines. Bortezomib, in contrast, induced apoptosis indiscriminately in both LMP- and XYZ-proteasome expressing cells. Taken together, these studies suggest that immunoproteasome specific inhibitors represent a novel class of drugs with activity against hematologic malignancies that may have less toxicity by virtue of their ability to spare the proteasome in most other tissues in the body, including in neural tissues. These properties could lead to an improved therapeutic index and anti-tumor efficacy, providing a rationale for development of these agents into clinically relevant drug candidates.

Description: Given the critical role that IL-6 plays in MM cell proliferation, survival, and resistance to GCs, we evaluated the ability of CNTO328, a chimeric monoclonal IL-6 neutralizing antibody, to overcome GC resistance in cell line models of human MM. In the presence of IL-6, the MM cell lines ANBL-6 and KAS-6 were resistant to the cytotoxic activity of dexamethasone (Dex) as assessed by cell viability assays both in suspension culture and in the context of patient-derived stromal cells. Resistance to dexamethasone was readily reversed by CNTO328, but not an isotype control antibody, in suspension culture. For example, in the case of the ANBL-6 model, viability was reduced by 12% with CNTO328 alone, 8% with Dex, but 74% with the combination, consistent with a synergistic interaction Given the ability of other growth factors in the bone marrow microenvironment to confer GC resistance in preclinical models of MM, we evaluated the activity of the CNTO328 and Dex combination in ANBL-6 and KAS-6 cells using a physiologically-relevant MM cell/patient-derived bone marrow stromal cell co-culture system. Importantly, bone marrow stromal cells rendered ANBL-6 and KAS-6 cells resistant to Dex in cell viability assays, and CNTO328 was able to reestablish Dex sensitivity, thus confirming a central role of IL-6 in bone marrow stroma-mediated GC resistance. Furthermore, treatment of ANBL-6 and KAS-6 cells with Dex alone did not induce apoptosis in this co-culture system, whereas the combination of CNTO328 and Dex led to a synergistic induction of apoptosis. In KAS-6 cells, IL-6-mediated Dex resistance was not overcome using pharmacologic inhibitors to p38, PI-3 kinase, mTor or MEK, suggesting that other IL-6 signaling pathways are likely involved. In contrast, the mTor inhibitor rapamycin was capable of sensitizing ANBL-6 cells to Dex in the presence of IL-6, suggesting that this pathway may be relevant to IL-6-mediated GC resistance in these cells. Induction of the pro-apoptotic Bcl-2 family member, Bim, has been shown to play an important role in GC-mediated cell death in lymphocytes as well as preclinical lymphoma and acute lymphoblastic leukemia models. Interestingly, although treatment of ANBL-6 cells in the presence of IL-6 with either CNTO328 or dexamethasone did not lead to induction of Bim, the combination led to a 3.3-fold increase in its expression. Taken together, the above data demonstrate that inhibition of IL-6 signaling with CNTO328 can effectively overcome IL-6-mediated GC resistance even in the presence of bone marrow stroma, and provide a compelling rationale for translation of this combination into clinical trials for patients suffering from MM. Furthermore, we show that the ability of CNTO328 to overcome GC resistance may be mediated in part by its ability to reverse IL-6-mediated repression of GC-induced Bim expression. Studies evaluating the relevance of Bim modulation in IL-6-mediated GC resistance and the molecular pathways that mediate this effect are on-going.

Description: The proteasome inhibitor bortezomib represents a significant advance in the treatment of multiple myeloma, but its efficacy is limited by a number of resistance mechanisms. One of the most important is the heat shock protein (HSP) and stress response pathways which, through members such as HSP-70 and mitogen-activated protein kinase (MAPK) phosphatase (MKP)-1, oppose the pro-apoptotic activities of bortezomib. Because interleukin (IL)-6 signaling augments the heat shock response through signal transducer and activator of transcription (STAT)-1 and heat shock transcription factor (HSF)-1, we hypothesized that downregulation of IL-6 signaling would attenuate HSP induction by bortezomib, thereby enhancing its anti-myeloma activity. Treatment of the IL-6-dependent multiple myeloma cell lines KAS-6 and ANBL-6 with the combination of bortezomib and CNTO328, a chimeric monoclonal IL-6 neutralizing antibody, resulted in greater reduction of cell viability than with either drug alone in a time- and concentration-dependent manner. This was associated with an enhanced induction of apoptosis which, under some conditions, was greater than the sum of the two individual agents alone, suggesting a synergistic interaction. Similar findings were not seen when using isotype control antibodies, and in studies of the IL-6-independent RPMI 8226 myeloma cell line. Increased activity was seen when cells were pre-treated with CNTO328 followed by bortezomib, or when they were treated with both agents concurrently, compared to treatment with bortezomib followed by CNTO328. Treatment with CNTO328 potently inhibited IL-6-mediated downstream signaling pathways, as demonstrated by marked blockade of STAT-3 and p44/42 MAPK phosphorylation. Enhanced activity of the combination regimen correlated with attenuated induction by bortezomib of the heat shock and stress response proteins HSP-70 and MKP-1 by up to 45% and 90%, respectively. Notably, CNTO328 markedly reduced levels of transcriptionally active phospho-STAT-1 and hyperphosphorylated HSF-1. Other strategies to suppress the heat shock response, including the use of the pharmacologic inhibitor KNK437, also yielded evidence for a synergistic anti-myeloma effect in combination with bortezomib. The synergistic activity of KNK437 and bortezomib was reproduced in normal mouse embryo fibroblasts (MEFs), but blunted in HSF-1 knockout MEFs. Taken together, the above data demonstrate that inhibition of IL-6 signaling enhances the anti-myeloma activity of bortezomib. They also support the hypothesis that this occurs, at least in part, by attenuating proteasome inhibitor-mediated induction of the heat shock response through downregulation of transcriptionally active STAT-1 and HSF-1. These findings provide a strong rationale for future translation of the CNTO328/bortezomib combination into the clinic.