Abstract 4109: Covalent JNK inhibitor, JNK-IN-8, suppresses tumor growth in triple-negative breast cancer in part by activating lysosome biogenesis and autophagy via TFEB and TFE3
Authors: Soleimani Milad , Kaoud Tamer , Somma Alex , Bustamante Jorge , Wylie Dennis C. , Holay Nisha , Looney Agnieszka , Giri Uma , Triplett Todd , Kowalski-Muegge Jeanne , Dalby Kevin N. , Eckhardt S. Gail , Van Den Berg Carla 
Published: June 2022

Abstract
The heterogeneity and aggressiveness of Triple-Negative Breast Cancer (TNBC) contribute to its early recurrence and metastasis. Despite substantial research to identify effective molecular targets for therapy, TNBC remains particularly elusive in terms of improving patient outcomes for advanced disease. Here, we report that a covalent JNK inhibitor, JNK-IN-8, suppresses TNBC growth in cell lines and patient-derived organoids in vitro and patient-derived xenograft tumors in vivo.
JNK-IN-8 reduced colony formation in TNBC cell lines and cell viability in both cell lines and patient-derived organoids in a concentration-dependent manner. Further, the compound significantly slowed tumor growth compared to vehicle in a patient-derived xenograft model in vivo. Cells treated with JNK-IN-8 exhibited large, cytoplasmic vacuoles with lysosomal markers. To examine the molecular mechanism of this phenotype, we looked at the master regulators of lysosome biogenesis and autophagy TFEB and TFE3. JNK-IN-8 inhibited TFEB phosphorylation and induced nuclear translocation of both TFEB and TFE3. This was accompanied by an upregulation of TFEB/TFE3 target genes associated with lysosome biogenesis and autophagy. Depletion of both TFEB and TFE3 diminished the JNK-IN-8-driven reduction of colony formation and the upregulation of lysosome biogenesis and autophagy markers. JNK-IN-8 also reduced the phosphorylation of other mTOR targets in a concentration-dependent manner. Ectopic expression of RagC (S75L) rescued mTOR signaling and restored TFEB phosphorylation and cytoplasmic localization. In contrast, knockout of JNK1 and/or JNK2 had no impact on TFEB/TFE3 activation or mTOR inhibition by JNK-IN-8, but did inhibit colony formation. Similarly, re-expression of either wildtype or drug-nonbinding JNK (C116S) in JNK knockout cells did not reverse JNK-IN-8-induced TFEB dephosphorylation.
In summary, JNK-IN-8 induced lysosome biogenesis and autophagy by activating TFEB and TFE3 via mTOR inhibition independently of JNK. Together, these findings demonstrate the in vitro and in vivo efficacy of JNK-IN-8 as a targeted therapy for TNBC and reveal its novel lysosome- and autophagy-mediated mechanism of action.
Citation Format: Milad Soleimani, Tamer Kaoud, Alex Somma, Jorge Bustamante, Dennis C. Wylie, Nisha Holay, Agnieszka Looney, Uma Giri, Todd Triplett, Jeanne Kowalski-Muegge, Kevin N. Dalby, S. Gail Eckhardt, Carla Van Den Berg. Covalent JNK inhibitor, JNK-IN-8, suppresses tumor growth in triple-negative breast cancer in part by activating lysosome biogenesis and autophagy via TFEB and TFE3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4109.