Stapled Peptide-based radiotheranostics agent targeting MDM2 suppresses p53-mutant tumor growth

Objectives: MDM2 is a cellular phosphoprotein and has E3 ubiquitin ligase activity, which plays a critical role in the degradation of p53. MDM2 is overexpressed in many human malignancies, and it is a major mechanism utilized by cancer cells to escape p53 surveillance. A subset of cancers that overexpress the inhibitory proteins MDM2 also possess wild-type p53, and thus pharmacological disruption of the interactions between p53 and MDM2 offers the opportunity to restore p53-dependent cell-cycle arrest and apoptosis in this class of tumors. However, approximately 50% of human cancers’ p53 are mutated and therefore, inhibitors dedicated to p53-MDM2 interactions are invalid for these important classes of tumors. Other MDM2 targeted yet independent of p53 phenotype therapeutic modalities are critical but still absent. The goal of this work is to develop a stapled peptide-based radiotheranostic agents for PET-imaging guided radiotherapy of p53 mutant cancer.

Methods: Stapled peptide STP was synthesized on resin based on the solid-phase peptide synthesis strategy and the peptide crosslinker (staple) was formed by ruthenium-catalyzed RCM reaction. To the N terminal of STP, a chelator DOTA was conjugated for ⁶⁴Cu labeling. The binding affinity of DOTA-STP to MDM2 was assessed by SPR. A murine colon cell line, MC38 with mutant p53, was selected for study. Its MDM2 expression was measured by flow cytometry and immunofluorescence. The cellular uptake of [⁶⁴Cu]STP was assayed by coincubation with MC38 for 5, 20, 40, 60, and 80 mins. The [⁶⁴Cu]STP binding to the cell surface and internalizing into the cell plasma was separately counted. Then [⁶⁴Cu]STP PET imaging was performed in both C57BL/6J normal mice and MC38 tumor-bearing C57BL/6J mice. Ex vivo biodistribution was conducted in tumor mice at 1, 3, 20 hours after intravenous injection of the tracer (50 μCi/mouse). Therapeutic effects against MC38 tumor-bearing mice was evaluated by i.v. injection of [⁶⁴Cu]STP (2 mCi/mouse) for 2 times (on days 5 and 12, n = 6) or 4 times (on days 5, 12, 19, and 26, n = 6). For control groups, mice were injected with an equivalent volume of saline (n = 6) or STP peptide (10 mg/kg, n = 5, on days 5, 12, and 19). Mice’s bodyweights were recorded during the therapy course.

Results: DOTA-STP binds to MDM2 with a K_D of 1.2 nM. MC38 cell lines revealed a high expression of MDM2 protein. [⁶⁴Cu]STP was demonstrated excellent cellular uptake, showing a 25%AD/g protein at 60 min after co-incubation. The ratios of cell membrane-bounded [⁶⁴Cu]STP against intracellular [⁶⁴Cu]STP were 5:1, 1:1.2, 1:3.4, 1:6.3. and 1:8.5 at 5, 20, 40, 60, and 80 mins after co-incubation. In normal mice, PET/CT imaging showed that [⁶⁴Cu]STP majorly accumulated in the liver and kidney, suggesting these organs are the main place for metabolism. The uptake of [⁶⁴Cu]STP in MC38 allograft tumors was high and increased over time, with the liver showing the highest off-target accumulation at all time points. Ex vivo biodistribution studies further verified this trend, with tumor uptake of 6.7 ± 1.4 %ID/g at 1 h p.i. to 9.4 ± 1.9 %ID/g at 3 h and 6.0 ± 1.0 %ID/g at 20 h (n=3). In therapeutic studies, [⁶⁴Cu]STP was found to slow tumor growth relative to the control groups and had significantly smaller (p<0.05) tumor volumes at 16 days p.i. Moreover, Four times of injection is more effective than two times of injection to suppress tumor growth (p<0.05). Histological analysis of ex vivo tissues revealed significant damage to the treated tumors.

Conclusions: This is the first time to evaluate a stapled peptide-based radiotheranostic agent in animal models. Our results demonstrated that [⁶⁴Cu]STP is a promising radiotheranotic agent for PET-imaging guided radiotherapy of p53 mutant MDM2 overexpressed tumors. Acknowledgement: We sincerely thank the financial support from the JSPS KAKENHI grant no. 19K17156 and 18H06217. This research is also supported by QST President's Strategic Grant (Exploratory Research).

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