Establishment and Characterization of Bortezomib-Resistant Myeloma Cell Lines: A Possible Role of Mutated Proteasome β5 Subunit in Preventing the Accumulation of Unfolded Protein, Which Is Otherwise Followed by Catastrophic Endoplasmic Reticulum (ER) Stress.

We established two stable BTZ-resistant MM cell lines, KMS-11/BTZ and OPM-2/BTZ, whose IC50 values were respectively 24.7- and 16.6-fold higher than their parental cell lines, under continuous exposure to BTZ. Using these resistant cells, we investigated on their proteasome activity, the alteration of proteasome β5 subunit (PSMB5) gene, misfolded protein accumulation, endoplasmic reticulum (ER) stress, and apoptosis signals including BH3 only proteins in comparison with their parental cells at clinically achievable concentration of BTZ treatment.

No activation of caspase -3,-8, and -9 and BH3 only protein, Noxa, which were initially up-regulated in BTZ-treated cells, were noted in BTZ-resistant cells even in the presence of BTZ. These results indicate avoidance of fatal intracellular stress may block transcriptional activation of Noxa in resistant cells at an early phase after BTZ exposure. In gel shift assay detecting NF-kB-DNA complexes, BTZ-resistant cells maintained constitutive NF-kB activation, whereas their parental cells lost its activity in the presence of BTZ. In addition, cellular proteasome activities including chymotrypsin-like and caspase-like activity were markedly inhibited by BTZ treatment in parental cells, and moderately also in BTZ-resistant cells, when detected by fluorogenic substrates specific for each proteasome activity. While time-dependent accumulation of ubiquitinated proteins was prevented only in BTZ-resistant cells, but not in their parental cells after BTZ exposure. Resistance was partly explained by the presence of a unique point mutation, G322A, in the gene encoding PSMB5 in both BTZ-resistant cell lines, which substituted Thr for Ala at the codon 49 in amino acid level. This constitution has been reported to gives rise to the conformational change of BTZ-binding pocket in β5 subunit, which results in partial disruption of the contact between BTZ and chymotrypsin-like active site. Furthermore, BTZ-resistant and parental MM cells had nearly equal expression of cytoplasmic and ER chaperons, however, only BTZ-resistant cells could prevent misfolded protein accumulation and therefore avoid fatal ER stress represented as activation of CHOP and of caspase-4, -12 after BTZ treatment.

Two kinds of stable BTZ-resistant MM cell lines were established, which acquired the unique point mutation (G322A) in BTZ-binding pocket of PSMB5, prevented the accumulation of misfolded proteins probably via reduced affinity of 26S proteasome to BTZ and avoided the development of catastrophic ER stress unlike their parental cells. These cell lines will provide better understanding of the underlying mechanisms of the BTZ-resistance, and will lead to the development of novel treatment strategies for overcoming BTZ-resistance in the patients with MM.

Iida:JANSSEN PHARMACEUTICAL: Honoraria; KYOWA KIRIN: Research Funding. Nakashima:KYOWA KIRIN: Employment. Miyazaki:KYOWA KIRIN: Employment. Shiotsu:KYOWA KIRIN: Employment.