Potent activity of carfilzomib, a novel, irreversible inhibitor of the ubiquitin-proteasome pathway, against preclinical models of multiple myeloma

Abstract

The proteasome has emerged as an important target for cancer therapy with the approval of bortezomib, a first-in-class, reversible proteasome inhibitor, for relapsed/refractory multiple myeloma (MM). However, many patients have disease that does not respond to bortezomib, whereas others develop resistance, suggesting the need for other inhibitors with enhanced activity. We therefore evaluated a novel, irreversible, epoxomicin-related proteasome inhibitor, carfilzomib. In models of MM, this agent potently bound and specifically inhibited the chymotrypsin-like proteasome and immunoproteasome activities, resulting in accumulation of ubiquitinated substrates. Carfilzomib induced a dose- and time-dependent inhibition of proliferation, ultimately leading to apoptosis. Programmed cell death was associated with activation of c-Jun-N-terminal kinase, mitochondrial membrane depolarization, release of cytochrome c, and activation of both intrinsic and extrinsic caspase pathways. This agent also inhibited proliferation and activated apoptosis in patient-derived MM cells and neoplastic cells from patients with other hematologic malignancies. Importantly, carfilzomib showed increased efficacy compared with bortezomib and was active against bortezomib-resistant MM cell lines and samples from patients with clinical bortezomib resistance. Carfilzomib also overcame resistance to other conventional agents and acted synergistically with dexamethasone to enhance cell death. Taken together, these data provide a rationale for the clinical evaluation of carfilzomib in MM.

Figure 1

Inhibition of the proteasome by carfilzomib. (A) Structures of epoxomicin (top) and carfilzomib (bottom) are shown. (B) Quantitative representation of the in vitro inhibition of the 20S proteasome catalytic activities in ANBL-6 cellular lysates (10 μg per reaction) in the absence or presence of carfilzomib with fluorogenic peptide substrates for the proteasomal ChT-L, PGPH, and T-L activity, as indicated, followed by the measurement of free AMC groups is shown. (C) In cellulo measurement using ELISA techniques of the 20S proteasome subunit targets of carfilzomib in cell extracts from ANBL-6 cells pulse treated for 1 hour with carfilzomib is shown. Error bars in panels B and C are SD. (D) Competitive binding experiment in ANBL-6 cells between carfilzomib (5-hour pretreatment) followed by VS-L₃-AHx₃-danysl (2 hours) determined the in cellulo specificity of carfilzomib to individual proteasome catalytic subunits. (E,F) Shown is Western blot analysis of the accumulation of ubiquitinated (Ub) substrates (E) and proapoptotic Bax (F) after 1-hour pulse exposure to carfilzomib (100 nM) in RPMI 8226 cells, followed by 24- and 48-hour recovery times. Actin was used as a loading control. Veh indicates vehicle.

Figure 2

Inhibition of proliferation and induction of apoptosis by carfilzomib. (A,C) IL-6–independent RPMI 8226 and IL-6–dependent ANBL-6 MM cells (2 × 10⁴) were treated continuously (A) or pulsed for 1 hour (C) with increasing concentrations of carfilzomib. Cellular viability was determined at 24 hours using the water-soluble tetrazolium salt WST-1. (B,D) IL-6–dependent (ANBL-6, KAS-6/1) and IL-6–independent (RPMI 8226, U266) myeloma cells were continuously exposed (B) or pulsed (D) with carfilzomib for 1 hour and allowed to recover for 24 hours. Programmed cell death was then evaluated using a DNA fragmentation ELISA. Results are expressed as a fold-increase of DNA fragmentation over DMSO control, and error bars are SD.

Figure 3

Molecular events associated with exposure to carfilzomib. (A) ANBL-6 cells were exposed to a pulse of 100 nM carfilzomib, bortezomib, or vehicle (Veh) control and allowed to recover for 8 or 24 hours. Cellular lysates (30 μg per reaction) were incubated with 40-μM fluorogenic substrates specific for caspase-3, caspase-8, and caspase-9. Results are expressed as fold relative fluorescence units over DMSO control. (B) ANBL-6 (2 × 10⁴ cells per reaction) were pretreated for 20 hours with caspase-3 (C-3)-, caspase-8 (C-8)-, and caspase-9 (C-9)-specific inhibitors, a negative control (Neg), a pan-caspase inhibitor (Pan), or no inhibitor (−), followed by a 1-hour pulse with 100 nM carfilzomib. Fresh media containing caspase inhibitors were then added, and cellular proliferation was determined after a 24-hour recovery period. Data were expressed as percent inhibition compared with vehicle (DMSO) controls. (C) Carfilzomib induces depolarization of mitochondria. The fluorescent shift of the JC-1 cationic dye from cytosol (green fluorescence) to mitochondria (red fluorescence) in live cells was analyzed by flow cytometry in ANBL-6 cells pulsed with 100 nM carfilzomib (Q1: red fluorescence; Q2: red and green fluorescence; Q3: green fluorescence). (D) ANBL-6 cells treated for 1 hour with 100 nM carfilzomib were subjected to centrifugal cellular fractionation into the cytosolic fraction and HMF that included mitochondria. Release of cytochrome c and Smac from the mitochondria was assessed by Western blot. Cox II, an intramitochondrial protein, was used as a control. WCEs were monitored as a control for protein expression. (E) Western blot analysis for the phosphorylation of JNK (ie, activated JNK) and cleavage of PARP in RPMI 8226 cells, which were pulsed with 100 nM carfilzomib and allowed to recover for the indicated time periods. HSC-70 was used as a loading control. To determine whether abrogation of JNK signaling through c-Jun affects carfilzomib's antiproliferative and proapoptotic action, ANBL-6 cells (2 × 10⁴ per well) were infected with DN-c-Jun adenovirus for 24 hours, followed by addition of 100 nM pulse with carfilzomib. (F) Cellular growth was assessed using the WST-1 reagent. (G) Apoptosis was measured by DNA fragment production. Error bars for panels A, B, F, and G are SD.

Figure 4

Activity of carfilzomib and bortezomib against myeloma models. (A) Several MM cell lines were treated with a 1-hour pulse of increasing concentrations of carfilzomib or bortezomib. After 24 hours, the number of live cells was determined with a WST-1 assay. (B) Activation of the stress response in RPMI 8226 cells pulse treated for 1 hour with proteasome inhibitors and allowed to recover for the indicated time points is shown. Protein expression levels of activated JNK were examined after treatment with carfilzomib or bortezomib. (C) ANBL-6 cells were exposed to a 1-hour pulse of 100 nM carfilzomib or bortezomib and allowed to recover for 8 hours. Cellular lysates (30 μg per reaction) were then incubated with 40-μM fluorogenic substrates specific for caspase-3, caspase-8, and caspase-9 activity. Results are expressed as fold relative fluorescence units over DMSO control and determined as described in Figure 1. Error bars for panels A and C are SD.

Figure 5

Activity of carfilzomib and bortezomib in patient samples. (A) Purified plasma cells were continuously treated with increasing doses of carfilzomib for 24 hours. Cells were then lysed, and the ChT-L activity was determined (10 μg per reaction). (B) CD138⁺ cells were treated with continuous exposure to the indicated concentrations of carfilzomib, followed by a WST-1 cell viability assay. (C) Purified plasma cells were pulse-treated with 100 nM carfilzomib or bortezomib, followed by recovery in drug-free media for 24 hours. WST-1 was used to assess proliferation. Several of the samples are from patients with chromosome 13 deletions (MM-13, MM-15, MM-17, MM-20, and MM-23). Results are expressed as the percentage (%) inhibition of proliferation of carfilzomib-treated cells relative to bortezomib-treated cells, which were set at 0, with a positive result indicating the amount of enhanced antiproliferative activity of carfilzomib over that of bortezomib. (D) Pulse carfilzomib and bortezomib exposure in an NHL patient sample and determination of antiproliferation activity by WST-1 assay is shown. (E) Flow cytometric analysis of carfilzomib-induced versus bortezomib-induced specific apoptosis in patient-derived CD19⁺ CLL B-cells is shown. Patient cells were pulsed (100 nM) for 1 hour with the indicated drug and allowed to recover for 24 hours. Apoptosis was assessed in cells stained with Annexin V/TO-PRO-3/anti-CD19. Specific apoptosis is shown in the CD19⁺ gated population relative to vehicle controls. (F) AML cells from a patient with progressive disease after multiple chemotherapeutic treatments were pulsed for 1 hour with 100 nM carfilzomib or bortezomib or continuously treated with 1 μM Dox. Apoptosis was measured by DNA fragmentation ELISA and expressed as fold induction over DMSO control in CD33⁺cells purified from peripheral blood mononuclear cells (PBMCs). Error bars in panels A,B,C,D, and F are SD.

Figure 6

Carfilzomib and chemotherapeutic resistance. (A) ANBL-6.BR cells, and their wt counterparts, were pulsed with carfilzomib for 1 hour, and proliferation was assessed using the WST-1 reagent after a 24-hour recovery period. The DOR to bortezomib was computed by comparing the IC₅₀ of bortezomib-sensitive and BR cells. (B) CD138⁺ cells from a MM patient with a chromosome 13 deletion who did not have a clinical response to bortezomib were treated with continuous exposure to the indicated concentrations of carfilzomib or bortezomib for 24 hours in triplicate, followed by measurement of proliferation with the WST-1 assay. (C) CD138⁺ cells from a MM patient who progressed while on bortezomib treatment were exposed to increasing concentrations of carfilzomib or bortezomib for 24 hours before assessment for cellular proliferation. (D) CD138⁺ plasma cells from a patient who progressed on bortezomib were exposed to continuous and pulse treatments with equivalent concentrations of carfilzomib and bortezomib, followed by a WST-1 cellular proliferation assay. (E) RPMI 8226 cells were treated continuously with 5 nM carfilzomib and 10 μM Dex for 48 hours to determine the effect of this combined therapy against cellular proliferation. Error bars are SD.