TOP2A Knockdown Resensitizes Carfilzomib-Resistant Hmcls to Carfilzomib

Introduction/background: Multiple myeloma (MM) remains incurable despite the introduction of novel therapeutic agents. Microarray-based technologies were adopted in our study to determine if a genetic signature associated with resistance to carfilzomib, a second-generation proteasome inhibitor already in use in clinical settings, could be identified.

Materials and Methods: 18 genetically heterogeneous human myeloma cell lines (HMCLs) were treated with carfilzomib and a cell viability profile was assessed categorizing the HMCLs as sensitive, intermediate or resistant to carfilzomib. Following categorization gene expression profiling was performed and validated with q-RT-PCR and knockdown assays.

Results: 29 genes were differentially regulated between the sensitive and resistant cell lines. Top genes based on intensity values and biological significance were: LOC731314, TSPAN13, APH1B, TSPYL5, COX7B2, PCSK1N, LRRC38, TCIRG1, TOP2A, ADM2, ITM2A, TSPAN13, STOM, UBE2C, SNHG8. Gene ontology (GO) enrichment analysis identified two pathways that were significantly different between the resistant and sensitive HMCLs; pathogenic escherichia coli infection (p=0.002) and lysosome (p=0.006). Eight GO terms were enriched: 4 related to biological processes and 4 related to cellular components.

TOP2A, an enzyme that controls and alters the topologic states of DNA during transcription and is involved in cell cycle and proliferation, was identified to be overexpressed in resistant HMCLs. It functions as the target for several anticancer agents and a variety of mutations in this gene have been associated with the development of drug resistance. TOP2A may be used as a predictive factor for patient selection for specific protocols or as independent prognostic marker in solid tumors. TOP2A was also overexpressed in the 'proliferation cluster' associated with greater proliferation rate and poor outcome in newly diagnosed MM patients. Suppression of TOP2A by siRNA in carfilzomib-resistant HMCLs significantly resensitised the cell lines to carfilzomib.

Conclusion: Our results suggest that TOP2A is overexpressed in carfilzomib-resistant HMCLs indicating a possible role as a predictive marker of response to carfilzomib in MM.