KMS-12-BM

Creation of a Resistant KMS-12-BM Subline

Proteasome inhibitors bortezomib (Btz), carfilzomib (Cfz), and ixazomib (Ixz) are the backbones of the treatment of multiple myeloma (MM). To develop a Btz-resistant cell line using clinically relevant pulse treatment, KMS-12-BM cells were treated twice weekly with 1 h pulses of Btz. As resistance developed, the dose stepwise was increased from 900 nM to 7.2 µM Btz (Fig. 1a) over 6 months, until a sub-line was approximately tenfold more resistant to a 1 h-pulse (Fig. 1b) and continuous Btz treatment. It is named KMS-12-BM-BPR, where BPR stands for "bortezomib-pulse resistant". For brevity, it also refers to BPR. This sub-line did not require culturing in the presence of Btz to maintain resistance and grew at the same rate as the parental line.

There were no specific mutations in the resistant cells (Fig. 1c) and no significant changes in the activities of any of the proteasome's three catalytic subunits (Fig. 1d). 1 h pulse treatment with Btz caused similar inhibition of β5 (i.e., the combined activity of β5c and β5i) and β1 (i.e., combined activity of β1c and β1i) activities in the parental cells and the resistant subclone (Fig. 1e). Thus, resistance in the BPR sub-line is not due to decreased affinity of Btz to the active sites.

Fig. 1 Development and characterization of Btz-resistant KMS-12-BM-BPR cells. (Downey-Kopyscinski SL, et al., 2022)

Characterization of Resistance to APO010 in KMS-12-BM Cell Lines

APO010 is a hexameric Fas-ligand that mimics cytotoxic T-lymphocyte signaling through the Fas-receptor to induce apoptosis. APO010-resistant variants of human multiple myeloma cell lines (KMS-12-BM) were developed. Stable APO010-resistant cancer cell lines were established by exposing the cells to gradually increasing concentrations of APO010 over 6-12 months (Fig. 2). Half-maximal inhibitory concentrations (IC₅₀) for APO010 in each parental cell line were chosen as the starting concentrations of APO010 exposure (Table 1). Cell lines were maintained at each APO010 concentration until there was no further effect of APO010, and then drug concentration was doubled at this point. The developed resistant cell lines were designated as KMS-12-BM_APO.

Development of resistance to APO010 had no significant effect on the proliferation rate of resistant cell lines in comparison to their controls. The KMS-12-BM_APO (33.7 h) cell line had a DT almost the same as the DT of its parental control (34.2 hours). To evaluate the role of Fas in acquiring resistance to APO010, Western blotting of Fas protein expression was performed. A complete downregulation of Fas expression was observed in the KMS-12-BM_APO cell lines (Fig. 3). Reduction in the membrane levels of Fas was observed between the APO010-resistant cell lines and their parental controls by ICC staining. Almost no membrane staining of Fas was seen in the KMS-12-BM_APO cell lines compared with their parental controls (Fig. 4).

Fig. 2 MTT assay revealing reduced APO010 sensitivity in KMS-12-BM_APO cell lines compared with their parental controls. (Jandu H, et al., 2020)

Table 1. Initial and final concentrations of APO010 exposure during the development of resistance to APO010. (Jandu H, et al., 2020)

Fig. 3 Fas protein expression in APO010-resistant cell lines. (Jandu H, et al., 2020)

Fig. 4 Immunocytochemistry staining of Fas expression in APO010-resistant cell lines. (Jandu H, et al., 2020)