Directly targeting G-quadruplexes contributes to the anti-multiple myeloma efficacy of Epimedokoreanin B

Abstract

Multiple myeloma (MM) is a hematological malignancy for which novel therapeutic strategies are urgently needed. Epimedokoreanin B (EKB), an isoprenylated flavonoid compound derived from the medicinal plant Epimedium koreanum, has demonstrated promising antitumor activity. However, its effects on MM have not been previously investigated. This study explores the anti-MM activity and the molecular interaction mechanisms between EKB and G-quadruplexes (G4) through a combination of biological activity assessments and computer-aided methodologies. EKB exhibits potent cytotoxicity against the MM cell lines U266 and RPMI-8226, with IC50 values of 5.28 μM and 6.81 μM, respectively. It induces apoptosis in a concentration-dependent manner and specifically stabilizes the G4 structures of oncogenes such as c-Myc, c-KIT, Bcl-2, and k-RAS, as confirmed by BG4 immunofluorescence staining and fluorescence resonance energy transfer (FRET) assays. Additionally, EKB significantly suppresses the mRNA and protein expression levels of these genes in myeloma cells. Computational studies, including molecular docking, molecular dynamics (MD) simulations, and MM/GBSA calculations, confirm the strong binding affinity and stabilizing effects of EKB on G4s, revealing a mechanism involving π-π stacking and hydrogen bonding. This discovery underscores the unique ability of EKB to increase the stability of G4 structures, which are critical for regulating gene expression and inhibiting cancer cell proliferation. This research highlights the therapeutic potential of EKB in targeting these specific molecular structures, thereby offering a more effective approach to managing MM.

1. Introduction

Multiple myeloma (MM) is a hematologic malignancy characterized by the abnormal proliferation of plasma cells within the bone marrow and excessive production of monoclonal immunoglobulin or light chains (M protein). It is the second most common hematologic malignancy [1]. MM predominantly affects elderly individuals, with a higher incidence in males than in females. As the population ages, the incidence of MM is expected to increase annually [2]. Despite significant advancements in treatment modalities, including proteasome inhibitors, immunomodulatory drugs, and monoclonal antibodies, MM remains incurable, with drug resistance posing a major challenge. Furthermore, relapsed/refractory multiple myeloma (RRMM) has emerged as a critical focus, necessitating the development of novel therapeutic agents and strategies [3,4].

Aberrant activation of the oncogene c-Myc plays a critical role in various types of cancer, including serous ovarian cancer, breast cancer, lung cancer, leukemia, and multiple myeloma [5–7]. Furthermore, the nuclease hypersensitive element III1 (NHE III1) region upstream of the P1 promoter of the c-Myc gene, which contains a purine-rich DNA sequence, can form a specialized secondary structure known as a G-quadruplex (G4) [8]. This structure is well known for its role as a transcriptional repressor [8]. Previous studies have demonstrated that the stabilization of these structures through interactions with small molecules could lead to the downregulation of oncogene expression [5]. Therefore, the G4 structure of the cancer-related gene c-Myc has long been considered a potential drug target [9]. Subsequently, numerous G4 structures have been identified in the promoters of various oncogenes, including c-KIT, Bcl-2, and k-RAS (Figure 1). These discoveries have since positioned G4s as highly attractive targets for the development of antitumor therapeutics [10–13].

G4 structures are widely distributed in the promoter regions of various oncogenes and play crucial regulatory roles in tumor initiation and progression [14]. As such, they have emerged as highly promising targets for anticancer therapy. With increasing research, it has become evident that G4 structures are formed by the folding of single-stranded DNA into highly ordered conformations, exhibiting spatial specificity akin to the binding pockets found in globular proteins. This structural feature provides a precise platform for small-molecule binding, enabling targeted stabilization of G4 structures to achieve transcriptional repression of oncogenes [15]. Accordingly, small-molecule targeting of G4s has become a key strategy in anticancer drug discovery [16]. This approach not only addresses the