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CDK4 inhibition diminishes p53 activation by MDM2 antagonists.

ABSTRACT: The genes encoding MDM2 and CDK4 are frequently co-amplified in sarcomas, and inhibitors to both targets are approved or clinically tested for therapy. However, we show that inhibitors of MDM2 and CDK4 antagonize each other in their cytotoxicity towards sarcoma cells. CDK4 inhibition attenuates the induction of p53-responsive genes upon MDM2 inhibition. Moreover, the p53 response was also attenuated when co-depleting MDM2 and CDK4 with siRNA, compared to MDM2 single knockdown. The complexes of p53 and MDM2, as well as CDK4 and Cyclin D1, physically associated with each other, suggesting direct regulation of p53 by CDK4. Interestingly, CDK4 inhibition did not reduce p53 binding or histone acetylation at promoters, but rather attenuated the subsequent recruitment of RNA Polymerase II. Taken together, our results suggest that caution must be used when considering combined CDK4 and MDM2 inhibition for patient treatment. Moreover, they uncover a hitherto unknown role for CDK4 and Cyclin D1 in sustaining p53 activity.

SUBMITTER: Sriraman A

PROVIDER: S-EPMC6133967 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Project description:Peptide inhibition of the interactions of the tumor suppressor protein P53 with its negative regulators MDM2 and MDMX activates P53 in vitro and in vivo, representing a viable therapeutic strategy for cancer treatment. Using phage display techniques, we previously identified a potent peptide activator of P53, termed PMI (TSFAEYWNLLSP), with binding affinities for both MDM2 and MDMX in the low nanomolar concentration range. Here we report an ultrahigh affinity, dual-specificity peptide antagonist of MDM2 and MDMX obtained through systematic mutational analysis and additivity-based molecular design. Functional assays of over 100 peptide analogs of PMI using surface plasmon resonance and fluorescence polarization techniques yielded a dodecameric peptide termed PMI-M3 (LTFLEYWAQLMQ) that bound to MDM2 and MDMX with K d values in the low picomolar concentration range as verified by isothermal titration calorimetry. Co-crystal structures of MDM2 and of MDMX in complex with PMI-M3 were solved at 1.65 and 3.0 Å resolution, respectively. Similar to PMI, PMI-M3 occupied the P53-binding pocket of MDM2/MDMX, which was dominated energetically by intermolecular interactions involving Phe3, Tyr6, Trp7, and Leu10. Notable differences in binding between PMI-M3 and PMI were observed at other positions such as Leu4 and Met11 with MDM2, and Leu1 and Met11 with MDMX, collectively contributing to a significantly enhanced binding affinity of PMI-M3 for both proteins. By adding lysine residues to both ends of PMI and PMI-M3 to improve their cellular uptake, we obtained modified peptides termed PMI-2K (KTSFAEYWNLLSPK) and M3-2K (KLTFLEYWAQLMQK). Compared with PMI-2K, M3-2K exhibited significantly improved antitumor activities in vitro and in vivo in a P53-dependent manner. This super-strong peptide inhibitor of the P53-MDM2/MDMX interactions may become, in its own right, a powerful lead compound for anticancer drug development, and can aid molecular design of other classes of P53 activators as well for anticancer therapy.

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CDK4 inhibition diminishes p53 activation by MDM2 antagonists.

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