TOP2A a New Predictive Marker of Response to Carfilzomib in Multiple Myeloma

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 (CZ), a second-generation proteasome inhibitor (PI) currently used in clinical settings, could be identified.

Materials and Methods: Nine human myeloma cell lines (HMCLs) were treated with carfilzomib and a cell viability profile was assessed categorizing the HMCLs as sensitive or resistant to carfilzomib. Subsequently, the gene expression profiles (GEP) of untreated resistant versus sensitive HMCLs were compared. GEP-identified differences were validated on a panel of 17 HMCLs with q-RT-PCR and siRNA knockdown assays. Topoisomerase 2-alpha (TOP2A) immunohistochemistry was performed on a panel of fully annotated trephine biopsy specimens acquired prior to treatment with PIs.

Results: 206 genes were differentially expressed between the sensitive and resistant HMCLs. Gene ontology analysis identified two pathways that were significantly different: 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 overexpressed in carfilzomib-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 has also been noted to be overexpressed in the 'proliferation cluster' of published MM GEP profiles and associated with poor outcome. Following suppression of TOP2A by siRNA, carfilzomib-resistant HMCLs were re-sensitized to carfilzomib, moreover, the combination of carfilzomib with topoisomerase inhibitors known to target directly TOP2A, demonstrated synergistic cytotoxic effects against HMCLs. Trephine-derived TOP2A protein expression levels were shown to be higher in patients treated with PIs including bortezomib, carfilzomib, ixazomib, marizomib, failing to achieve a response when compared to responding patients. Finally, logistic regression analysis confirmed that TOP2A protein expression was a highly significant predictor of response to PIs (AUC 0.764, p=0.048).

Conclusion: TOP2A status may be used as a predictive factor for patient response to PIs including carfilzomib.