Mechanistic validation of a clinical lead stapled peptide that reactivates p53 by dual HDM2 and HDMX targeting

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Hydrocarbon-stapled peptides that display key residues of the p53 transactivation domain have emerged as bona fide clinical candidates for reactivating the tumor suppression function of p53 in cancer by dual targeting of the negative regulators HDM2 and HDMX. A recent study questioned the mechanistic specificity of such stapled peptides based on interrogating their capacity to disrupt p53/HDM2 and p53/HDMX complexes in living cells using a new recombinase enhanced bimolecular luciferase complementation platform (ReBiL). Here, we directly evaluate the cellular uptake, intracellular targeting selectivity and p53-dependent cytotoxicity of the clinical prototype ATSP-7041. We find that under standard serum-containing tissue culture conditions, ATSP-7041 achieves intracellular access without membrane disruption, dose-dependently dissociates both p53/HDM2 and p53/HDMX complexes but not an unrelated protein complex in long-term ReBiL experiments, and is selectively cytotoxic to cancer cells bearing wild-type p53 by inducing a surge in p53 protein level. These studies underscore the importance of a thorough stepwise approach, including consideration of the time-dependence of cellular uptake and intracellular distribution, in evaluating and advancing stapled peptides for clinical translation.

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We thank E Smith for graphical assistance, Lisa Cameron for assistance with confocal microscopy, and Alexandra Zoraian and Katherine Franklin for technical support. We appreciate the generosity of Drs Y Li and G Wahl for providing the ReBiL cells for this study, hosting FW in their laboratory to conduct confirmatory experiments and for helpful feedback on the manuscript. This work was supported by a Hyundai Hope on Wheels Quantum Award, Alex’s Lemonade Stand Reach Grant, the Todd J Schwartz Memorial Fund, and Leukemia and Lymphoma SCOR and Scholar Awards to LDW. FW is supported by an Alexander von Humboldt Foundation Feodor Lynen Fellowship, AMM by NIH training grant T32GM007753, RM by Doctoral Foreign Studies Award DFS-134963 from the Canadian Institutes of Health Research, and AZ and KF by the William Lawrence and Blanche Hughes Foundation and a generous gift from Jim and Lisa LaTorre, respectively.

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Correspondence to L D Walensky.

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LDW is a scientific advisory board member and consultant for Aileron Therapeutics.

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Supplementary Information accompanies this paper on the Oncogene website

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