Repurposing of the ALK Inhibitor Crizotinib for Acute Leukemia and Multiple Myeloma Cells

Abstract

Crizotinib was a first generation of ALK tyrosine kinase inhibitor approved for the treatment of ALK-positive non-small-cell lung carcinoma (NSCLC) patients. COMPARE and cluster analyses of transcriptomic data of the NCI cell line panel indicated that genes with different cellular functions regulated the sensitivity or resistance of cancer cells to crizotinib. Transcription factor binding motif analyses in gene promoters divulged two transcription factors possibly regulating the expression of these genes, i.e., RXRA and GATA1, which are important for leukemia and erythroid development, respectively. COMPARE analyses also implied that cell lines of various cancer types displayed varying degrees of sensitivity to crizotinib. Unexpectedly, leukemia but not lung cancer cells were the most sensitive cells among the different types of NCI cancer cell lines. Re-examining this result in another panel of cell lines indeed revealed that crizotinib exhibited potent cytotoxicity towards acute myeloid leukemia and multiple myeloma cells. P-glycoprotein-overexpressing CEM/ADR5000 leukemia cells were cross-resistant to crizotinib. NCI-H929 multiple myeloma cells were the most sensitive cells. Hence, we evaluated the mode of action of crizotinib on these cells. Although crizotinib is a TKI, it showed highest correlation rates with DNA topoisomerase II inhibitors and tubulin inhibitors. The altered gene expression profiles after crizotinib treatment predicted several networks, where TOP2A and genes related to cell cycle were downregulated. Cell cycle analyses showed that cells incubated with crizotinib for 24 h accumulated in the G2M phase. Crizotinib also increased the number of p-H3(Ser10)-positive NCI-H929 cells illustrating crizotinib’s ability to prevent mitotic exit. However, cells accumulated in the sub-G0G1 fraction with longer incubation periods, indicating apoptosis induction. Additionally, crizotinib disassembled the tubulin network of U2OS cells expressing an α-tubulin-GFP fusion protein, preventing migration of cancer cells. This result was verified by in vitro tubulin polymerization assays. In silico molecular docking also revealed a strong binding affinity of crizotinib to the colchicine and Vinca alkaloid binding sites. Taken together, these results demonstrate that crizotinib destabilized microtubules. Additionally, the decatenation assay showed that crizotinib partwise inhibited the catalytic activity of DNA topoisomerase II. In conclusion, crizotinib exerted kinase-independent cytotoxic effects through the dual inhibition of tubulin polymerization and topoisomerase II and might be used to treat not only NSCLC but also multiple myeloma.

Molecular Modes of Action of an Aqueous Nerium oleander Extract in Cancer Cells In Vitro and In Vivo

“…RPMI8226 cells were purchased from the American Type Cell Culture Collection (ATCC CCL-155, Manassas, VA, USA). Their maintenance has been described [ 80 ].…”

“…The analyses were accomplished using a FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA). The readings were obtained by measuring the turbidity variation (light scattering) every 30 s for 3 h (360 measurements in total) at 350 nm [ 80 ].…”

Xylochemical Synthesis and Biological Evaluation of the Orchidaceous Natural Products Isoarundinin I, Bleochrin F, Blestanol K, and Pleionol

“…Human peripheral blood mononuclear cells (PBMCs) of a healthy blood donor were extracted with Histopaque (Sigma–Aldrich, Taufkirchen, Germany), as previously described . In brief, 3 mL of blood was cautiously overlaid on top of 3 mL of Histopaque.…”

“…Before the use in the assay, natural products 1 – 4 were further purified by preparative HPLC (see the Experimental Section). The sensitivity of CEM/ADR5000 and CCRF-CEM leukemia cells, as well as PBMCs to isoarundinin I ( 1 ), (±)-bleochrin F ((±)- 2 ), (±)-blestanol K ((±)- 3 ), and (±)-pleionol ((±)- 4 ) was assessed using the resazurin reduction viability assay as previously described . Cells (10 4 /well) were placed in a flat-bottom 96-well plate.…”

Drug repositioning in non-small cell lung cancer (NSCLC) using gene co-expression and drug–gene interaction networks analysis

“…In a similar study, Muthu Kumar and colleagues 29 introduced another drug repurposing method for NSCLC, and they hypothesized that Nebivolol is an excellent candidate for inhibiting MEK in NSCLC patients. In another study, Joelle C. Boulos and colleagues 30 repurposed ALK Inhibitor Crizotinib for NSCLC, Acute Leukemia, and Multiple Myeloma Cells. Compared to the mentioned methods, this study applies a novel computational model based on gene co-expression and TF–TG interaction networks.…”