Stapled α-helical peptide drug development: a potent dual inhibitor of MDM2 and MDMX for p53-dependent cancer therapy.

Stapled α-helical peptides have emerged as a promising new modality for a wide range of therapeutic targets. Here, we report a potent and selective dual inhibitor of MDM2 and MDMX, ATSP-7041, which effectively activates the p53 pathway in tumors in vitro and in vivo. Specifically, ATSP-7041 binds both MDM2 and MDMX with nanomolar affinities, shows submicromolar cellular activities in cancer cell lines in the presence of serum, and demonstrates highly specific, on-target mechanism of action. A high resolution (1.7-Å) X-ray crystal structure reveals its molecular interactions with the target protein MDMX, including multiple contacts with key amino acids as well as a role for the hydrocarbon staple itself in target engagement. Most importantly, ATSP-7041 demonstrates robust p53-dependent tumor growth suppression in MDM2/MDMX-overexpressing xenograft cancer models, with a high correlation to on-target pharmacodynamic activity, and possesses favorable pharmacokinetic and tissue distribution properties. Overall, ATSP-7041 demonstrates in vitro and in vivo proof-of-concept that stapled peptides can be developed as therapeutically relevant inhibitors of protein-protein interaction and may offer a viable modality for cancer therapy.

MDM2 small-molecule antagonist RG7112 activates p53 signaling and regresses human tumors in preclinical cancer models.

MDM2 negatively regulates p53 stability and many human tumors overproduce MDM2 as a mechanism to restrict p53 function. Thus, inhibitors of p53-MDM2 binding that can reactivate p53 in cancer cells may offer an effective approach for cancer therapy. RG7112 is a potent and selective member of the nutlin family of MDM2 antagonists currently in phase I clinical studies. RG7112 binds MDM2 with high affinity (K(D) ~ 11 nmol/L), blocking its interactions with p53 in vitro. A crystal structure of the RG7112-MDM2 complex revealed that the small molecule binds in the p53 pocket of MDM2, mimicking the interactions of critical p53 amino acid residues. Treatment of cancer cells expressing wild-type p53 with RG7112 activated the p53 pathway, leading to cell-cycle arrest and apoptosis. RG7112 showed potent antitumor activity against a panel of solid tumor cell lines. However, its apoptotic activity varied widely with the best response observed in osteosarcoma cells with MDM2 gene amplification. Interestingly, inhibition of caspase activity did not change the kinetics of p53-induced cell death. Oral administration of RG7112 to human xenograft-bearing mice at nontoxic concentrations caused dose-dependent changes in proliferation/apoptosis biomarkers as well as tumor inhibition and regression. Notably, RG7112 was highly synergistic with androgen deprivation in LNCaP xenograft tumors. Our findings offer a preclinical proof-of-concept that RG7112 is effective in treatment of solid tumors expressing wild-type p53.

Regulation of p14ARF expression by miR-24: a potential mechanism compromising the p53 response during retinoblastoma development.

BACKGROUND: Most human cancers show inactivation of both pRB- and p53-pathways. While retinoblastomas are initiated by loss of the RB1 tumor suppressor gene, TP53 mutations have not been found. High expression of the p53-antagonist MDM2 in human retinoblastomas may compromise p53 tumor surveillance so that TP53 mutations are not selected for in retinoblastoma tumorigenesis. We previously showed that p14ARF protein, which activates p53 by inhibiting MDM2, is low in retinoblastomas despite high mRNA expression. METHODS: In human fetal retinas, adult retinas, and retinoblastoma cells, we determined endogenous p14ARF mRNA, ARF protein, and miR-24 expression, while integrity of p53 signalling in WERI-Rb1 cells was tested using an adenovirus vector expressing p14ARF. To study p14ARF biogenesis, retinoblastoma cells were treated with the proteasome inhibitor, MG132, and siRNA against miR-24. RESULTS: In human retinoblastoma cell lines, p14ARF mRNA was disproportionally high relative to the level of p14ARF protein expression, suggesting a perturbation of p14ARF regulation. When p14ARF was over-expressed by an adenovirus vector, expression of p53 and downstream targets increased and cell growth was inhibited indicating an intact p14ARF-p53 axis. To investigate the discrepancy between p14ARF mRNA and protein in retinoblastoma, we examined p14ARF biogenesis. The proteasome inhibitor, MG132, did not cause p14ARF accumulation, although p14ARF normally is degraded by proteasomes. miR-24, a microRNA that represses p14ARF expression, is expressed in retinoblastoma cell lines and correlates with lower protein expression when compared to other cell lines with high p14ARF mRNA. Transient over-expression of siRNA against miR-24 led to elevated p14ARF protein in retinoblastoma cells. CONCLUSIONS: In retinoblastoma cells where high levels of p14ARF mRNA are not accompanied by high p14ARF protein, we found a correlation between miR-24 expression and low p14ARF protein. p14ARF protein levels were restored without change in mRNA abundance upon miR-24 inhibition suggesting that miR-24 could functionally repress expression, effectively blocking p53 tumor surveillance. During retinal tumorigenesis, miR-24 may intrinsically compromise the p53 response to RB1 loss.

The TAg-RB murine retinoblastoma cell of origin has immunohistochemical features of differentiated Muller glia with progenitor properties.

PURPOSE: Human retinoblastoma arises from an undefined developing retinal cell after inactivation of RB1. This is emulated in a murine retinoblastoma model by inactivation of pRB by retinal-specific expression of simian virus 40 large T-antigen (TAg-RB). Some mutational events after RB1 loss in humans are recapitulated at the expression level in TAg-RB, supporting preclinical evidence that this model is useful for comparative studies between mouse and human. Here, the characteristics of the TAg-RB cell of origin are defined. METHODS: TAg-RB mice were killed at ages from embryonic day (E)18 to postnatal day (P)35. Tumors were analyzed by immunostaining, DNA copy number PCR, or real-time quantitative RT-PCR for TAg protein, retinal cell type markers, and retinoblastoma-relevant genes. RESULTS: TAg expression began at P8 in a row of inner nuclear layer cells that increased in number through P21 to P28, when clusters reminiscent of small tumors emerged from cells that escaped a wave of apoptosis. Early TAg-expressing cells coexpressed the developmental marker Chx10 and glial markers CRALBP, clusterin, and carbonic anhydrase II (Car2), but not TuJ1, an early neuronal marker. Emerging tumors retained expression of only Chx10 and carbonic anhydrase II. As with human retinoblastoma, TAg-RB tumors showed decreased Cdh11 DNA copy number and gain of Kif14 and Mycn. It was confirmed that TAg-RB tumors lose expression of tumor suppressor cadherin-11 and overexpress oncogenes Kif14, Dek, and E2f3. CONCLUSIONS: TAg-RB tumors displayed molecular similarity to human retinoblastoma and origin in a cell with features of differentiated Müller glia with progenitor properties.