As was the case for the plasma cell samples, exposure to IPSI-001 in 2 patient CLL isolates resulted in activation of apoptosis (Figure 4E,F). IPSI-001 overcomes chemotherapy resistance and synergizes with dexamethasone Novel agents that can overcome chemoresistance to other drugs may have greater potential for clinical antitumor activity, and we therefore studied IPSI-001 in several drug-resistance models. was able to overcome conventional and novel drug resistance, including resistance to bortezomib. These findings provide a rationale for the translation of IPSIs to the clinic, where they may provide antimyeloma activity with greater specificity and less toxicity than current inhibitors. Introduction The ubiquitin-proteasome pathway is used by eukaryotes as the major mechanism for regulated intracellular proteolysis. As such, it plays an important role in cellular homeostasis, and is critical to several processes, including angiogenesis, cell-cycle progression, DNA repair, programmed cell death, and the stress response.1,2 This pathway has become a validated target for cancer therapy with the approval of the proteasome inhibitor bortezomib as a single agent for mantle cell lymphoma3C5 and multiple myeloma (MM).6C8 Modulation of proteasome function is also a rational approach to achieve chemosensitization, and bortezomib with pegylated liposomal doxorubicin has recently been shown to be superior to bortezomib alone against MM.9 These successes have led to studies of the role of bortezomib in the up-front setting in myeloma, and to the introduction in early phase studies of a second generation of irreversible proteasome inhibitors, such as carfilzomib10 and NPI-0052.11,12 In its assembled form, the proteasome consists of a core 20S particle with 4 stacked rings of 7 subunits each. Most cells express the constitutive proteasome variant, in which the 20S MDL 28170 core is capped on both ends by a 19S activator complex, which aids in stabilization, ATP-dependent removal of ubiquitin, and unfolding/shuttling of proteins into the core. The inner 2 rings contain subunits numbered 1 through 7, of which 1, 2, and 5 participate in proteolysis.11C13 MDL 28170 These encode the 3 major catalytic activities of the proteasome: chymotrypsin-like (ChT-L), trypsin-like (T-L), and postCglutamyl peptide hydrolyzing, or caspase-like (C-L) activities. One variant of the proteasome that has been described is the immunoproteasome, which differs from the constitutive proteasome in part through the replacement of 1 1, 2, and 5, with FKBP4 distinct subunits known as 1i, 2i, and 5i, to form the 20Si core.14,15 This variant, which also contains different regulatory subunits known as 11S or PA28, 16 has been found to be highly expressed in cells of hematopoietic origin in several species.17C19 Studies of 20Si function have revealed that it generates peptide fragments with more hydrophobic and basic amino acids at the C-terminus,20 which are better suited for presentation to major histocompatability class I molecules, providing the rationale for its name. However, the 20Si also participates in many constitutive proteolytic processes,17,19,21C24 and conversely the 20S proteasome may in some cases generate immunogenic epitopes, 25 suggesting that each proteasome variant may provide both housekeeping and specialized functions. The role that the 20Si plays outside of the immune system is just beginning to be elucidated, with expression levels being correlated to several disease states, including Huntington disease,26 Alzheimer disease,27 and macular degeneration.28 Diseases associated with an aberrant immune response, such as inflammatory bowel disease29,30 and rheumatoid arthritis,31 have also been identified as having increased 20Si expression. This may be due in part to the ability of inflammatory cytokines, such as tumor necrosis factor and interferon-, to induce expression and preferential incorporation of 1i, 2i, and 5i into the proteasome.16 Preferential expression of 20Si has also been observed in MM,32 suggesting that specific immunoproteasome inhibition could be a promising therapeutic strategy. While bortezomib, carfilzomib, and NPI-0052, as well as other proteasome inhibitors, have been validated in MDL 28170 myeloma and other model systems, all of these appear to target both the constitutive and immunoproteasomes indiscriminately. This lack of specificity may in part explain some of the side effects of these agents, such as peripheral neuropathy and gastrointestinal effects, which may be due to targeting of the constitutive proteasome in these tissues. In contrast, by selectively inhibiting the 20Si, it may be possible to maintain antimyeloma and antilymphoma efficacy while reducing these toxicities, thereby increasing the therapeutic index. In this work, we describe the identification.