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. 2021 Dec 2;11:23280. doi: 10.1038/s41598-021-02695-0

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Validation of NEDD8 as a synergistic target in AML. (A) mRNA levels of NEDD8, RBX1, and PSMD11 were assessed in HL-60 cells transfected with corresponding shRNA and control shRNA. (B) Protein levels were measured in HL-60 with Western blotting of whole-cell lysates. (C) Proliferation rates of HL-60 cells transfected with shRNAs targeting either NEDD8 or RBX1 and Luciferase as control. Transfected cells were exposed to 500 nM azacytidine (AZA) or DMSO for 24 h. Proliferation rates were calculated by cell counting after 72 h and normalized to the proliferation rate of DMSO treated cells. (D–I) Proliferation rate of HL-60 and Kasumi-1 exposed to NEDD8 inhibitor pevonedistat (PEV) in combination with either azacytidine (AZA) or cytarabine (AraC). Proliferation rate was measured by cell counting and calculated as explained above. (J–K) Methylcellulose colony-forming assays were performed in HL-60 cells after 72 h daily treatment in vitro with either azacytidine (AZA) or cytarabine (AraC) in combination with pevonedistat (PEV). Controls were treated with DMSO.