Abstract LB-258: The NAE inhibitor pevonedistat (MLN4924) interacts with the HDAC inhibitor belinostat via disruption of the intra-S checkpoint and both HR and NHEJ DNA repair in AML cells
Authors: Zhou Liang , Chen Shuang , Zhang Yu , Leng Yun , Li Lihong , Lin Hui , Kmieciak Maciej , Rizzo Kathryn A. , Dumur Catherine I. , Ferreira-Gonzalez Andrea , Dai Yun , Grant Steven 
Published: August 2015

Protein degradation via the ubiquitin proteasome system (UPS) is regulated by cullin-RING E3-ligases (CRLs). The first-in-class investigational Nedd8-activating enzyme (NAE) inhibitor MLN4924 (pevonedistat) blocks CRL activity by inhibiting their neddylation, thereby disrupting the turnover of a subset of proteins with broad functions associated with cancer cell growth and survival pathways, including NF-κB, DNA replication, and DNA damage checkpoints, among others. Such findings provide a rationale for combining MLN4924 with HDAC inhibitors (HDACIs e.g., the recently FDA-approved belinostat) that interrupt DNA replication, checkpoints and repair. Notably, both single-agent MLN4924 and HDACIs (e.g., pracinostat) have induced CRs in AML patients. Consequently, interactions between MLN4924 and belinostat were investigated in AML. MLN4924/belinostat co-administration synergistically induced apoptosis in AML cells with various genetic backgrounds (e.g., wt- or mutant- p53 or FLT3-ITD), while p53 knock-down or enforced FLT3-ITD expression increased sensitivity to this regimen. MLN4924 blocked belinostat-induced NF-κB activation and belinostat co-administration up-regulated Bim, while Bim shRNA knockdown abrogated lethality. Following MLN4924/belinostat co-exposure, an RT2 Array revealed a distinct expression profile of DNA damage response (DDR) network genes. Significantly, belinostat diminished MLN4924-induced ATR/Chk1/Wee1 activation and Cdt1 up-regulation, abrogating the intra-S checkpoint. Notably, shRNA knockdown of Chk1 or Wee1 significantly sensitized cells to MLN4924 in the absence of belinostat. Moreover, belinostat also blocked MLN4924-mediated up-regulation of multiple proteins involved in HR (e.g., CtIP, BRCA1 S1524 and S1423 phosphorylation, FANCD2-L), while co-administration increased H3K56 and H4K16 acetylation, indicating defective NHEJ repair. As a consequence, co-treatment induced robust DSBs, chromosome pulverization, and apoptosis. This regimen sharply induced apoptosis in primary MDS/AML blasts (n = 47) bearing various poor-prognostic mutations, defined by NGS using the Cancer Hotspot Panel v2, as well as CD34+/CD38-/CD123+ populations enriched for leukemia-initiating cells (n = 25), but was minimally toxic to normal CD34+ cells. Finally, MLN4924/belinostat co-treatment profoundly diminished tumor burden and prolonged survival in both s.c. and i.v. AML xenograft models, with minimal toxicity, via the PD events observed in vitro. Collectively, these findings indicate that MLN4924 and HDACIs interact reciprocally to disable the DDR and promote DNA damage and cell death in AML cells, including those with unfavorable genetic features and primitive progenitors, while sparing their normal counterparts. This strategy warrants further consideration in AML.

Citation Format: Liang Zhou, Shuang Chen, Yu Zhang, Yun Leng, Lihong Li, Hui Lin, Maciej Kmieciak, Kathryn A. Rizzo, Catherine I. Dumur, Andrea Ferreira-Gonzalez, Yun Dai, Steven Grant. The NAE inhibitor pevonedistat (MLN4924) interacts with the HDAC inhibitor belinostat via disruption of the intra-S checkpoint and both HR and NHEJ DNA repair in AML cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-258. doi:10.1158/1538-7445.AM2015-LB-258