Abstract 6954: Discovery and characterization of selective mixed-lineage kinase 3 PROTAC degraders for cancer therapy

• Published in: Cancer research (Chicago, Ill.) Vol. 85; no. 8_Supplement_1; p. 6954
• Main Authors: Mehlich, Dawid, Karpinska, Kamila, Sabbasani, Venkata R., Lomiak, Michal, Torres-Ayuso, Pedro, Wrobel, Katarzyna, Nguyen-Phuong Truong, Vi, Serwa, Remigiusz, Swenson, Rolf E., Brognard, John, Marusiak, Anna A.
• Format: Journal Article
• Language: English
• Published: 21.04.2025
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/1538-7445.AM2025-6954

Mixed-lineage kinase 3 (MLK3) is a member of the MLK family of serine/threonine kinases. MLK3 is well-known to regulate MAPK signaling by acting upstream of ERK, JNK, and p38 pathways. In addition to its canonical role in activating MAPK cascades, MLK3 regulates the activity of various proteins and signaling pathways, including NF-κB, Wnt/β-catenin, and PAK1. Previous studies demonstrated that MLK3 can promote the proliferation, invasive growth, and metastatic potential of human cancers. Triple-negative breast cancer (TNBC) is a subtype of breast cancer that is characterized by poor prognosis owing to its aggressive clinical course and limited targeted treatment options. MLK3 is often upregulated in TNBC and is involved in driving the tumorigenic potential of TNBC cells. Consequently, several small-molecule inhibitors targeting MLK3 were pursued as new investigational drugs for TNBC in preclinical studies. However, the utility of these compounds is limited by their low specificity and multiple off-target effects. Furthermore, recent studies suggested that the pro-oncogenic activity of MLK3 might result from its kinase-independent and scaffolding functions, which are not effectively targeted by conventional small-molecule inhibitors. To overcome the challenges associated with MLK3 inhibition, we developed and characterized first-in-class MLK3 Proteolysis Targeting Chimera (PROTAC) degraders. We synthesized a series of PROTAC compounds by connecting pan-MLK inhibitor CEP-1347 to molecules binding different E3 ligases (VHL, IAP, Cereblon). We found that CEP-1347-VHL-02, a PROTAC based on CEP-1347 and ligand for VHL E3 ligase, was the most potent MLK3 degrader among all candidate compounds. Next, we demonstrated that CEP-1347-VHL-02 effectively degraded MLK3 via the ubiquitin-proteasome system in breast cancer cell lines. By applying biochemical assays and proteomic approaches, we found that CEP-1347-VHL-02 selectively targeted MLK3 but no other MLK kinases. Furthermore, we showed that MLK3 degradation by CEP-1347-VHL-02 led to diminished oncogenic features of TNBC cells, as evidenced by reduced proliferation, migratory potential, cell cycle arrest, and increased apoptosis. These effects were not observed in other breast cancer subtypes upon MLK3 degradation, which indicates the subtype-specific oncogenic functions of this kinase. Collectively, our research provides new opportunities for the selective targeting of MLK3 in cancer and highlights MLK3 degradation as a potential novel treatment strategy in TNBC. In the ongoing studies, we further investigate the pharmacokinetic properties and therapeutic potential of MLK3-targeting PROTACs across various cancer cell lines with high expression of this kinase.