A cell-penetrating TRIM21-based targeted protein degrader enables Pan-PIM kinase degradation in cancer cells

The ubiquitin–proteasome system (UPS) is a critical regulator of cellular proteostasis and an important therapeutic pathway for eliminating oncogenic proteins. PIM kinases are aberrantly expressed in multiple cancers, where they promote tumor cell survival, proliferation, metastasis, stemness, and therapeutic resistance, making them attractive therapeutic targets. Here, we developed a novel autogenous, cell-penetrating targeted protein degrader (TPD) platform for pan-PIM degradation with broad anticancer potential. The construct comprises the cell-penetrating peptide Penetratin (PEN), the RING domain of TRIM21 as an E3 ubiquitin ligase, and a human single-chain variable fragment (HuscFv7) targeting PIM2. Unlike conventional PROTAC-based degraders, which recruit endogenous E3 ligases such as cereblon or VHL, this platform incorporates an intrinsic ubiquitination module, thereby minimizing dependence on the availability of endogenous E3 ligases and on canonical ternary complex formation. The resulting PEN–Ring–HuscFv7 fusion protein binds all three human PIM isoforms and exhibits pan-PIM specificity. It efficiently penetrates cells and co-localizes with PIM1, PIM2, and PIM3. In breast cancer models, including MCF-7 and MDA-MB-231 cells, PEN–Ring–HuscFv7 induced dose- and time-dependent apoptosis through both caspase 3-dependent and caspase-3-independent pathways. Moreover, the fusion protein enhanced the cytotoxic effects of cisplatin in breast cancer cells. Structural modeling, molecular dynamics simulations, and epitope mapping revealed interactions with key residues in the ATP-binding, activation, and substrate-binding regions of PIM kinases, supporting potential dual mechanisms: kinase inhibition and proteasomal degradation. Compared with existing TPD strategies, this platform offers several distinctive features, including E3-ligase independence, a linker-free design, dual functional activity, pan-isoform targeting, and intrinsic cell-penetrating capability. Collectively, the TRIM21-based TPD system represents a mechanistically distinct and versatile biologic platform for targeting PIM kinases and potentially other oncogenic drivers in cancer. Novel autogenous cell-penetrating targeted protein degrader (TPD) for oncology Targets cancer’s PIM kinases, enhances chemotherapy, and overcomes drug resistance E3-independent TRIM21 RING design reduces the risk of ligase variability in targets No ternary complex needed, direct PIM binding for ubiquitination, cancer apoptosis Dual action: catalytic inhibition and proteasomal degradation of PIM kinases