Comparative Proteomic Profiling of Multiple Myeloma (MM) Cell Lines Identifies Mitochondrial Dysfunction As a Marker of Resistance to Proteasome Inhibitors (PIs) with Potential Therapeutic Implications for BH3-Mimetics
Authors: Kubicki Tadeusz , Bednarek Kinga , Kostrzewska-Poczekaj Magdalena , Luczak Magdalena , Visser Lydia , Lodewijk Monique , Żyłka Krzysztof , Gil Lidia , Jarmuż-Szymczak Małgorzata , Dytfeld Dominik 
Published: December 2024

Introduction
PIs form the backbone of most first-line regimens for MM, both in transplant-eligible and ineligible patients. Despite advances, MM remains incurable primarily due to the development of drug resistance. Many studies on this phenomenon in PI-resistant cells have been conducted in recent years; nevertheless, we still lack an explanation that would lead to clinically translatable findings. In this study, to achieve better insight into the process of acquiring resistance to bortezomib (BTZ) and carfilzomib (CFZ), we conducted comparative proteomic profiling of sensitive MM cell lines and their resistant counterparts at different stages of acquiring the resistant phenotype. To further enhance the generalizability of these findings, we performed the experiments on two different MM cell lines.
Methods
Resistant cell lines were generated by continuously culturing sensitive RPMI8226 and U266B1 MM cells with increasing concentrations of BTZ or CFZ. Resistance was defined by the ability to remain viable in continuous culture with increasing concentrations of the drugs. Comparative proteomic profiling of sensitive cells and cells resistant to increasing concentrations of both drugs was performed as described previously (Kubicki, Am J of Cancer Res 2022). The Western blot (WB) method was used to validate the proteomic results. Proteasome activity was measured by the Proteasome-Glo Assays (Promega, USA). Apoptosis was measured by BH3 profiling as in previous reports (Langedonk, Int J Mol Sci 2022).
Results
RPMI8226 cells included in this analysis were able to remain viable in culture with 3, 4, 6 and 13 nM of BTZ and 2, 4, 6 and 23 nM of CFZ. For U266B1 cell lines, the concentrations were 1, 2, 5 and 11 nM of BTZ and 2, 5, 6, 11 and 17 nM of CFZ. Both drugs were able to similarly suppress chymotrypsin-like proteasome activity in both sensitive and resistant RPMI8226 and U266B1 cells. Additionally, the baseline activity of all three proteasome catalytic domains was the same in all cell lines. Subsequently, we assessed the cells' global adaptations through comparative proteomic profiling. The top-ranked canonical pathway was oxidative phosphorylation inhibited in BTZ-resistant (B-H p &lt; 10-21; z-score = 5.7), CFZ-resistant RPMI8226 (B-H p &lt; 10-30; z-score = 5.9), BTZ-resistant (B-H p &lt; 10-2; z-score = 5.0), and CFZ-resistant U266B1 (B-H p &lt; 10-2; z-score = 5.3) cells. Accumulation of almost all proteins involved in this process was linearly downregulated with increasing drugs concentrations. In line with the well-described Warburg effect, cytoplasmic proteins responsible for gluconeogenesis and glycolysis were also consistently upregulated in the resistant U266B1 and RPMI8226 cells, whereas mitochondrial proteins involved in the tricarboxylic acid cycle were downregulated. These finding were further supported by the downregulation of mitochondrial proteins that may contribute to mitochondrial dysfunction in all resistant cell lines. Based on these results, we selected the mitochondrial transcription factor A (TFAM) protein as a potential marker of resistance and confirmed its lower accumulation in both resistant RPMI8226 and U266B1 cells using WB. We also identified significant differences in accumulation of proteins associated with apoptosis in BTZ-resistant RPMI8226 cells (B-H p &lt; 10-29; z-score = -2.9). After priming during BH3 profiling, mitochondrial outer membrane permeabilization was lower in BTZ-resistant cells compared to the sensitive ones. Synergy between bortezomib and different BH3-mimetics was confirmed (5.1 for 1 µM of venetoclax, 2.1 for 1 µM of navitoclax, 2.7 for 0.1 µM of BCL-XL inhibitor, 1.5 for 0.01 µM of MCL1 inhibitor); the combination treatment was capable of partially restoring sensitivity to BTZ. Of note, RPMI8226 cells do not harbor the translocation t(11;14). Intriguingly, we did not observe similar pattern for CFZ-resistant cells and for U266B1.
Conclusions
CFZ- and BTZ-resistant RPMI8226 and U266B1 MM cells exhibit a Warburg effect phenotype and signs of mitochondrial dysfunction. We identified TFAM as a potential biomarker of resistance to PIs. Combining BH3-mimetics with BTZ restored sensitivityin the resistant RPMI8226 cells. Further studies are needed to confirm the clinical significance of these results, explore the potential of targeting mitochondrial metabolism and identify patients who may benefit from BH3-mimetics.