Abstract 4422: Targeting MLK4 in herceptin-resistant breast cancer

Abstract Mixed Lineage Kinases play a significant role in cancer biology, including breast cancer, by influencing key cell proliferation, survival, and metastasis signaling pathways. Targeting MLKs offers a promising therapeutic strategy, especially in cancers with dysregulated MLK activity. We have reported that one of the members of the mixed lineage kinase (MLK) family, MLK3 kinase activity, was necessary to mediate the cytotoxic effect of anti-HER2 therapies (i.e., Herceptin and Lapatinib). Activation of MLK3 using C6-ceramide nanoparticles caused massive cell death and apoptosis in Herceptin-resistant breast tumors. Recently, we observed that another MLK family member, MLK4, was overexpressed in human HER2+ breast cancer Herceptin-resistant breast tumors and Herceptin-resistant cancer cell lines, suggesting that MLK4 could play a functional role in developing HER2-therapy resistance. Interestingly, MLK4 gene knockdown in Herceptin-resistant breast cancer cell lines significantly caused cell death and apoptosis. Knockdown of MLK4 in Herceptin-resistant SkBr3 & BT474 cell lines downregulated p-HER2 (Y1221/1222, Y1248), p-AKT (S473), pCDC2 (Y15) and arrested cells in G2-M phase suggesting MLK4 regulates HER2 activation, and cell cycle progression. Overexpressing MLK4 in Herceptin-sensitive cells further activated the HER2-PI3K-Akt pathway, leading to Herceptin resistance. Surprisingly, MLK4 and HER2 endogenously interacted, and an in vitro kinase assay showed that MLK4 phosphorylates the intracellular domain of HER2 at Tyrosine 1221 and 1222 sites (Y1221/2), indicating that MLK4-induced phosphorylation could activate HER2 downstream signaling. Finally, MLK4 knockdown using siMLK4-loaded nanoparticles in an in vivo mouse PDX model demonstrated significant tumor regression and apoptosis. These results suggest that MLK4 upregulation is necessary for Herceptin resistance in HER2-positive breast tumors. Considering our in vitro and in vivo data, we plan to develop a specific inhibitor of MLK4, which could be used clinically to treat Herceptin-resistant HER2+ metastatic breast cancer. Citation Format: Subhasis Das, Jingjing Sun, Song Li, Basabi Rana, Ajay Rana. Targeting MLK4 in herceptin-resistant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4422.