Abstract 750: JNK-IN-8: a novel covalent inhibitor targeting JNK signaling in triple-negative breast cancer
Authors: Xie Xuemei , Kaoud Tamer S. , Edupunganti Ramakrishna , Zhang Tinghu , Kogawa Takahiro , Chauhan Gaurav B. , Gray Nathanael S. , Bartholomeusz Chandra , Dalby Kevin N. , Ueno Naoto T. 
Published: May 2015

Abstract
Background: Patients with triple-negative breast cancer (TNBC) have a very poor prognosis due to metastasis and limited targeted therapies. Basal-like TNBC has hyperactivated JNK (c-Jun N-terminal kinase). JNK plays a vital role in malignant transformation in different cancers and self-renewal and maintenance of glioma cancer stem cells (CSCs). However, it is not known whether JNK signaling is a clinically relevant target in TNBC. We tested the hypotheses that JNK signaling plays a fundamental role in TNBC tumorigenesis and metastasis by promoting CSCs and that suppressing JNK signaling using JNK-IN-8 inhibits tumorigenicity and metastasis of TNBC cells.
Methods: We examined the effects of JNK knockdown or overexpression on cellular functions of TNBC cells and CSC subpopulations in vitro. We also evaluated the anti-TNBC efficacy of JNK-IN-8, an ATP-directed covalent small molecule inhibitor of JNK that specifically binds the Cys116 on JNK.
Results: In patients with TNBC (n=79), low c-Jun mRNA level was associated with better disease-free survival (DFS) (P=0.025), and there was a tendency (P=0.167) toward better DFS with low JNK1 mRNA level. No similar trend was observed for JNK2. These results suggest that JNK1 and c-Jun may have clinical significance in TNBC.
Knockdown of JNK1 or JNK2 led to reductions in mammosphere formation, migration, and invasion in HCC70 and SUM149 cells. In contrast, overexpression of constitutively active MKK7-JNK1 or -JNK2 enhanced these cellular activities and increased the ALDH1+ subpopulation. These data indicate a potential role of JNK signaling in regulating TNBC tumorigenesis and metastasis, possibly by promoting CSCs.
JNK-IN-8 suppressed proliferation, anchorage-independent growth, migration, and invasion in HCC70 and SUM149 cells. In addition, JNK-IN-8 also reduced mammosphere formation and the CD44+/CD24- and ALDH1+ subpopulations, suggesting that JNK-IN-8 can inhibit JNK signaling-mediated CSC self-renewal and maintenance. Furthermore, overexpression of mutant JNK1- or JNK2-C116S, in which Cys116 was replaced with Ser116, partially abolished the inhibitory effects of JNK-IN-8 on cellular functions, suggesting that JNK-IN-8 executes antitumor activity by specifically targeting JNK. Lastly, JNK inhibition by JNK-IN-8 led to down-regulation of Notch1, suggesting involvement of Notch1 in JNK signaling-mediated CSC regulation.
Conclusion: JNK signaling regulates TNBC tumorigenesis and metastasis by promoting CSCs partly via Notch signaling. Our data demonstrate that JNK is an important therapeutic target in TNBC, providing a rationale for further preclinical and clinical development of a JNK-targeted therapy for TNBC. JNK-IN-8 is an effective inhibitor targeting JNK signaling in TNBC. Our studies justify searching for JNK isoform selective inhibitors since there is evidence that JNK1 and JNK2 can have opposing roles in certain settings.
Citation Format: Xuemei Xie, Tamer S. Kaoud, Ramakrishna Edupunganti, Tinghu Zhang, Takahiro Kogawa, Gaurav B. Chauhan, Nathanael S. Gray, Chandra Bartholomeusz, Kevin N. Dalby, Naoto T. Ueno. JNK-IN-8: a novel covalent inhibitor targeting JNK signaling in triple-negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 750. doi:10.1158/1538-7445.AM2014-750