Carfilzomib can induce tumor cell death through selective inhibition of the chymotrypsin-like activity of the proteasome

Abstract

Carfilzomib is a proteasome inhibitor in clinical development that primarily targets the chymotrypsin-like (CT-L) subunits in both the constitutive proteasome (c20S) and the immunoproteasome (i20S). To investigate the impact of inhibiting the CT-L activity with carfilzomib, we set out to quantitate the levels of CT-L subunits ␤5 from the c20S and LMP7 from the i20S in normal and malignant hematopoietic cells. We found that the i20S is a major form of the proteasome expressed in cells of hematopoietic origin, including multiple myeloma (MM) CD138 ؉ tumor cells. Although specific inhibition of either LMP7 or ␤5 alone was insufficient to produce an antitumor response, inhibition of all proteasome subunits was cytotoxic to both hematologic tumor cells and peripheral blood mononuclear cells. However, selective inhibition of both ␤5 and LMP7 was sufficient to induce an antitumor effect in MM, non-Hodgkin lymphoma, and leukemia cells while minimizing the toxicity toward nontransformed cells. In MM tumor cells, CT-L inhibition alone was sufficient to induce proapoptotic sequelae, including proteasome substrate accumulation, Noxa and caspase 3/7 induction, and phospho-eIF2␣ suppression. These data support a hypothesis that hematologic tumor cells are uniquely sensitive to CT-L inhibition and provide a mechanistic understanding of the clinical safety profile and antitumor activity of proteasome inhibitors. (Blood.

Clinical activity of carfilzomib correlates with inhibition of multiple proteasome subunits: application of a novel pharmacodynamic assay

“…Previously, the pharmacodynamic profile of carfilzomib was assessed in vitro using the ProCISE assay (Parlati et al , 2009). In tumour cell lines, carfilzomib has been shown to have equivalent and potent activity against both β5 and LMP7 and to be at least 10‐fold less potent against the other subunits of the c20S and i20S (Parlati et al , 2009).…”

“…We previously published the methodology of the ProCISE assay for evaluation of samples from primary cells and tumour cell lines (Parlati et al , 2009). The clinical application of the ProCISE assay involves, in brief, the following steps: (i) incubation of PABP with whole blood, PBMC or bone marrow lysates; (ii) denaturation with 5∙6 M guanidine hydrochloride; (iii) addition of streptavidin‐coated beads; (iv) extensive bead washing; (v) addition of proteasome subunit‐specific primary antibody followed by a secondary HRP‐conjugated antibody; and (vi) luminescence‐based detection.…”

“…MM cells express both proteasome types, and the role of individual subunits in the anti‐tumour effect of clinically relevant proteasome inhibitors remains an area of active research (Kuhn et al , 2009; Parlati et al , 2009; Wehenkel et al , 2012). Meanwhile, the role of the non–CT‐L active sites and their inhibition by proteasome inhibitors remain poorly understood, mainly due to a lack of suitable substrates for measuring enzymatic activity.…”

“…Other assessment tools are needed to fully characterize the role of c20S and i20S active sites in the anti‐cancer activity of proteasome inhibitors. Considering these limitations, we developed the proteasome constitutive/immunoproteasome subunit enzyme‐linked immunosorbent (ProCISE) assay to measure the amounts and occupancy of proteasome active sites in samples with heterogeneous proteasome composition (Parlati et al , 2009). We previously reported the use of the ProCISE assay in quantifying c20S and i20S subunit levels in primary cells and cell lines derived from haematopoietic and solid tumours (ST) (Parlati et al , 2009).…”

“…Considering these limitations, we developed the proteasome constitutive/immunoproteasome subunit enzyme‐linked immunosorbent (ProCISE) assay to measure the amounts and occupancy of proteasome active sites in samples with heterogeneous proteasome composition (Parlati et al , 2009). We previously reported the use of the ProCISE assay in quantifying c20S and i20S subunit levels in primary cells and cell lines derived from haematopoietic and solid tumours (ST) (Parlati et al , 2009). Herein, we present the first clinical application of ProCISE, demonstrating its utility in determining the level of occupancy of all six proteasome active sites in samples derived from MM and ST patients treated with carfilzomib from five phase I/II and phase II clinical trials.…”