Efficacious targeting of TERT oncogene rearrangement with BET bromodomain inhibitor and proteasome inhibitor combination therapy

Background: TERT oncogene rearrangement with transcriptional super-enhancers leads to TERT over-expression in one third of high-risk neuroblastoma patients, and this subtype of neuroblastoma patients shows very poor prognosis. Transcriptional super-enhancers of MYC oncogene are controlled by the BET bromodomain protein BRD4. However, factors important for super-enhancer activity in TERT-rearranged neuroblastoma are unknown. Aims: To examine the role of BRD4 in regulating TERT over-expression and cell proliferation in TERT-rearranged neuroblastoma cells, and to identify the best combination therapy with BRD4 inhibitors and other anticancer agents against TERT-rearranged neuroblastoma. Methods and Results: Knocking down BRD4 with BRD4 siRNAs decreased TERT mRNA and protein expression as well as telomerase activity in TERT-rearranged neuroblastoma cell lines. Alamar blue assays showed that transfection with TERT siRNAs or BRD4 siRNAs considerably reduced cell proliferation. After screening of the Food and Drug Administration-approved oncology drug library, we identified the proteasome inhibitor, carfilzomib, as the compound exerting the best synergistic anticancer effects, in combination with the BRD4 inhibitor OTX-015, against TERT-rearranged neuroblastoma cells. Immunoblot and telomerase activity assays showed that OTX-015 and carfilzomib synergistically decreased TERT protein expression and telomerase activity. Beta-galactosidase and Annexin-V staining of TERT-rearranged neuroblastoma cells revealed that OTX-015 and carfilzomib did not induce senescence but synergistically caused apoptosis, and that transfection with a TERT-over-expression construct rescued the tumour cells from the combination treatment. Importantly, in mice xenografted with TERT-rearranged neuroblastoma cells, combination therapy considerably suppressed tumour progression. Immunoblot analysis of mouse tumour tissues revealed that OTX-015 and carfilzomib dramatically reduced TERT protein expression. Immunohistochemistry analysis showed that OTX-015 and carfilzomib co-operatively reduced the proportion of neuroblastoma cells positively stained with PCNA, indicating that the combination treatment synergistically repressed tumour proliferation. Conclusions: The BET bromodomain protein BRD4 is essential for TERT oncogene expression and cell proliferation in TERT-rearranged neuroblastoma cells. Combination therapy with OTX-015 and carfilzomib considerably reduces TERT expression, induces TERT-rearranged neuroblastoma cell death and tumour growth inhibition, and is a very promising novel strategy for the therapy of TERT-rearranged neuroblastoma. Citation Format: Jingwei Chen, Christopher Nelson, Peiyan Liu, Andrew E. Tee, Bernard Atmadibrata, Karen MacKenzie, Jamie Fletcher, Toby Trahair, Patsie Polly, Roger Reddel, Hilda Pickett, Tao Liu. Efficacious targeting of TERT oncogene rearrangement with BET bromodomain inhibitor and proteasome inhibitor combination therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5209.