High FDX1 expression enhances the sensitivity of multiple myeloma cells to elesclomol Free

Background:Copper is an essential micronutrient required for regular physiological functions in the body. Dysregulated copper metabolism plays a crucial role in the progression of various types of cancer. However, excessive copper accumulation leads to the aggregation of lipoylated proteins and inhibits the biosynthesis of iron-sulfur cluster proteins, ultimately triggering cuproptosis, a copper-dependent form of programmed cell death. Cuproptosis has demonstrated promising therapeutic potential in multiple cancer types. Therefore, investigating copper metabolism in multiple myeloma (MM) may contribute to the development of novel anti-MM therapeutic strategies.

Materials and Methods:A colorimetric assay was employed to quantify copper ion levels in the peripheral serum of healthy individuals (n = 25) and MM patients (n = 40), as well as intracellular copper levels in CD138⁺ and CD138⁻ cells isolated from the bone marrow of MM patients (n = 4). The subcellular localization of ATPase copper transporter 1 (ATP7A) in these CD138⁺ and CD138⁻ cells was analyzed by immunofluorescence staining. Cell proliferation in MM cell lines was assessed using the CCK-8 assay, EdU staining, and soft agar colony formation assay. Cell viability in MM cell lines and primary CD138⁺ and CD138⁻ cells from MM patients was evaluated by Calcein-AM/PI double staining. The expression levels of copper metabolism–related and cuproptosis–related genes were examined using qPCR and Western blotting.

Results:Our findings showed that serum copper levels were significantly elevated in MM patients compared to those in healthy donors. However, intracellular copper levels in CD138⁺ cells were markedly lower than those in CD138⁻ cells derived from the same MM patients. We also showed that ATP7A, a transmembrane protein involved in copper efflux and homeostasis, was overexpressed in MM cells compared to normal cells. Moreover, ATP7A was localized in the cytoplasmic membrane of primary CD138⁺ cells, whereas it was predominantly found in the Golgi apparatus of CD138⁻ cells, which may account for the reduced intracellular copper levels observed in primary CD138⁺ cells. Knockdown of ATP7A significantly increased intracellular copper levels, inhibited cell proliferation, and induced cuproptosis in MM cells. In addition, elesclomol, a copper ionophore, effectively inhibited cell proliferation, reduced cell viability, and triggered cuproptosis in MM cells. Notably, primary CD138⁺ cells exhibited greater sensitivity to elesclomol compared to CD138⁻ cells. While tetrathiomolybdate (ATM), a copper chelator, promoted MM cell proliferation and effectively neutralized elesclomol-induced cell death and cuproptosis in MM cells. These findings indicated that MM cells preferentially maintain low intracellular copper levels and are highly sensitive to elesclomol.

FDX1, a mitochondrial iron-sulfur cluster-containing protein, acts as a key mediator of cuproptosis through the regulation of protein lipoylation and the reduction of Cu²⁺ to Cu¹⁺. We found that FDX1 was dramatically overexpressed in primary CD138⁺ cells compared to CD138^- cells derived from the same MM patients. Furthermore, overexpression of FDX1 sensitized MM cells to the elesclomol-Cu²⁺ complex, while knockdown of FDX1 attenuated the inhibitory effect of this complex on MM cells. Therefore, high FDX1 expression is a key determinant in sensitizing MM cells to elesclomol, leading to reduced intracellular copper levels in these cells.

Conclusions: Our findingsindicated that MM cells maintain low intracellular copper levels through expressing ATP7A in the cytoplasmic membrane. High FDX1 expression led to increased sensitivity of MM cells to elesclomol. Overall, this study reveals the copper metabolism characteristics of MM cells and provides a novel therapeutic strategy for the treatment of MM.

Disclosures: No relevant conflicts of interest to declare.