Multiple myeloma (MM), which is the second most common malignant hematologic disease, is a challenging disease to treat. The COQ2 gene is believed to have important implications within myeloma cells. Authors of a study published in the British Journal of Haematology aimed to validate whether COQ2 is an essential gene in the MM survival process using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screening, and whether eliminating the gene will lead to defects in signaling or increased lipid peroxidation (LPO).
The study evaluated the HEK293T cells and myeloma cells (eg., MM1 cells, H929 cells), which were cultured in DMEM medium that contained 10% FBS and RPMI-1640 medium containing 10% FBS, respectively. The primary MM cells were obtained from BM aspirates of patients with MM, and the cells were further purified by CD138-positive selection that utilized anti-CD138 magnetic-activated cell separation. In addition, Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK) was used to identify essential genes because of its strong performance when controlling the false discovery rate and high sensitivity, as well as its ability to simultaneously perform both positive and negative selection screens.
For LPO detection, live cells were harvested, resuspended, and prepped, and using the FSC/SSC gating, debris was removed by gating on the main cell population. In addition, isotype control-stained cells were used to differentiate between background staining and specific staining to reflect LPO. An apoptosis analysis was conducted on MM1, H929, primary cells were isolated from the bone marrow of patients with MM (eg, CD138+, CD138-), and peripheral blood mononuclear cells.
In order to identify genes and signaling pathways that have significant roles in the proliferation and growth of MM cells, both MM1s and H929 cells were infected with CRISPR-Cas9, and the genomic DNA of 1-time cells were collected. Each cell line had more than 200 genes that positively regulated cell growth identified and included 3 major pathways that were proven to be involved in the development of MM: the cell cycle, MAPK, and nuclear factor-κB, and the enrichment of the genes related to the pathways was observed.
According to the findings, COQ2 was expressed in the MM cell lines that were included in the analysis, and mRNA and protein levels of COQ2 were relatively high in MM1s and H929 cells. Cell functional experiments had also demonstrated that eliminating CoQ2 significantly reduced the cell viability of MM cells while simultaneously promoting G1/S phase arrest. The function of COQ2 was also validated in an in vivo, subcutaneous tumor model, and the elimination of COQ2 inhibited the subcutaneous tumor-forming ability of MM1s cells, with immunohistochemical results also showing the proportion of Ki67-positive cells in those with low COQ2 expression decreased significantly.
Key Takeaways
- COQ2 as an Essential Gene in Multiple Myeloma (MM) Survival: The study investigates the role of the COQ2 gene in MM cell proliferation and survival through CRISPR-Cas9 screening. Results indicate that COQ2 is highly expressed in MM cells and its elimination significantly reduces cell viability, suggesting its importance in MM survival.
- Mechanistic Insights into COQ2 Function: The study identifies the molecular mechanisms underlying COQ2's role in MM proliferation, and demonstrates that COQ2's involvement in mitochondrial biogenesis and its regulation of the MEK/ERK signaling pathway, highlighting the pathways through which COQ2 promotes MM cell growth.
- Therapeutic Implications: Understanding the significance of COQ2 in MM proliferation provides valuable insights for developing targeted therapies. COQ2's influence on MM cell sensitivity suggests its potential as a therapeutic target for designing future treatment strategies against MM.
The investigators also determined the specific molecular mechanism of COQ2 when promoting the proliferation and growth of MM cells. The authors found that the protein interaction network of COQ2 through proteomics and a bioinformatics analysis revealed a substantial enrichment of related proteins in the mitochondrial biogenesis. Further, the MAPK signaling pathway was also enriched, and according to the authors, this suggests that CIQ2 may help regulate the MEK/ERK signaling pathway in MM cells. COQ2 in MM1s and H929 had also reduced phosphorylation levels of both MEK1/2 and ERK1/2, whereas their total protein levels did not change which indicates COQ2 is able to activate MEK/ERK phosphorylation signaling pathway in MM cells.
The findings also verified that COQ2 was able to promote the growth of MM cells by helping regulate MYC. According to the findings, removing COQ2 in MM1s and H929 cells had reduced the phosphorylation levels of MYC at transcriptional activity and significantly decreased its protein level, and alternatively, overexpression of COQ2 increased the phosphorylation levels of MYC and its proteins levels. This means that COQ2 can stabilize MYC protein and activates its transcriptional activity through the activation of MEK/ERK. Further, the results also found that the COQ2 expression was upregulated in relapse patients compared with patients who did not have relapse, and it negatively correlated to SLC39A14 expression.
The study authors emphasize that screening in MM cells was essential in determining the significance of the COQ2 gene in MM proliferation. Because COQ2 affects the sensitivity of MM cells, this can provide with valuable information and insights when developing more treatment strategies for MM, according to the authors.
Reference
Li M, Zhang C-l, Zhou D-s, et al. Identification of COQ2 as a regulator of proliferation and lipid peroxidation through genome-scale CRISPR-Cas9 screening in myeloma cells. Br J Haematol. 2024; 00: 1–18. doi:10.1111/bjh.19375
