Christian Tovar

Phosphorylation of p53 on Key Serines Is Dispensable for Transcriptional Activation and Apoptosis

The p53 tumor suppressor is a key mediator of the cellular response to stress. Phosphorylation induced by multiple stress-activated kinases has been proposed to be essential for p53 stabilization, interaction with transcriptional co-activators, and activation of p53 target genes. However, genetic studies suggest that stress-activated phosphorylation may not be essential for p53 activation. We therefore investigated the role of p53 phosphorylation on six key serine residues (Ser6, Ser15, Ser20, Ser37, Ser46, and Ser392) for p53 activation using nutlin-3, a recently developed small molecule MDM2 antagonist. We show here that nutlin does not induce the phosphorylation of p53. Comparison of the activity of unphosphorylated and phosphorylated p53 induced by the genotoxic drugs doxorubicin and etoposide in HCT116 and RKO cells revealed no difference in their sequence-specific DNA binding and ability to transactivate p53 target genes and to induce p53-dependent apoptosis. We conclude that p53 phosphorylation on six major serine sites is not required for activation of p53 target genes or biological responses in vivo.

Discovery of RG7388, a Potent and Selective p53–MDM2 Inhibitor in Clinical Development

Restoration of p53 activity by inhibition of the p53-MDM2 interaction has been considered an attractive approach for cancer treatment. However, the hydrophobic protein-protein interaction surface represents a significant challenge for the development of small-molecule inhibitors with desirable pharmacological profiles. RG7112 was the first small-molecule p53-MDM2 inhibitor in clinical development. Here, we report the discovery and characterization of a second generation clinical MDM2 inhibitor, RG7388, with superior potency and selectivity.

Activation of the p53 pathway by small-molecule-induced MDM2 and MDMX dimerization

Activation of p53 tumor suppressor by antagonizing its negative regulator murine double minute (MDM)2 has been considered an attractive strategy for cancer therapy and several classes of p53-MDM2 binding inhibitors have been developed. However, these compounds do not inhibit the p53-MDMX interaction, and their effectiveness can be compromised in tumors overexpressing MDMX. Here, we identify small molecules that potently block p53 binding with both MDM2 and MDMX by inhibitor-driven homo- and/or heterodimerization of MDM2 and MDMX proteins. Structural studies revealed that the inhibitors bind into and occlude the p53 pockets of MDM2 and MDMX by inducing the formation of dimeric protein complexes kept together by a dimeric small-molecule core. This mode of action effectively stabilized p53 and activated p53 signaling in cancer cells, leading to cell cycle arrest and apoptosis. Dual MDM2/MDMX antagonists restored p53 apoptotic activity in the presence of high levels of MDMX and may offer a more effective therapeutic modality for MDMX-overexpressing cancers.

Discovery of RG7112: A Small-Molecule MDM2 Inhibitor in Clinical Development.

The p53 tumor suppressor is a potent transcription factor that plays a key role in the regulation of cellular responses to stress. It is controlled by its negative regulator MDM2, which binds directly to p53 and inhibits its transcriptional activity. MDM2 also targets p53 for degradation by the proteasome. Many tumors produce high levels of MDM2, thereby impairing p53 function. Restoration of p53 activity by inhibiting the p53-MDM2 interaction may represent a novel approach to cancer treatment. RG7112 (2g) is the first clinical small-molecule MDM2 inhibitor designed to occupy the p53-binding pocket of MDM2. In cancer cells expressing wild-type p53, RG7112 stabilizes p53 and activates the p53 pathway, leading to cell cycle arrest, apoptosis, and inhibition or regression of human tumor xenografts.

In vitro and in vivo activity of R547: a potent and selective cyclin-dependent kinase inhibitor currently in phase I clinical trials.

The cyclin-dependent protein kinases are key regulators of cell cycle progression. Aberrant expression or altered activity of distinct cyclin-dependent kinase (CDK) complexes results in escape of cells from cell cycle control, leading to unrestricted cell proliferation. CDK inhibitors have the potential to induce cell cycle arrest and apoptosis in cancer cells, and identifying small-molecule CDK inhibitors has been a major focus in cancer research. Several CDK inhibitors are entering the clinic, the most recent being selective CDK2 and CDK4 inhibitors. We have identified a diaminopyrimidine compound, R547, which is a potent and selective ATP-competitive CDK inhibitor. In cell-free assays, R547 effectively inhibited CDK1/cyclin B, CDK2/cyclin E, and CDK4/cyclin D1 (Ki = 1–3 nmol/L) and was inactive (Ki > 5,000 nmol/L) against a panel of >120 unrelated kinases. In vitro, R547 effectively inhibited the proliferation of tumor cell lines independent of multidrug resistant status, histologic type, retinoblastoma protein, or p53 status, with IC50s ≤ 0.60 μmol/L. The growth-inhibitory activity is characterized by a cell cycle block at G1 and G2 phases and induction of apoptosis. R547 reduced phosphorylation of the cellular retinoblastoma protein at specific CDK phosphorylation sites at the same concentrations that induced cell cycle arrest, suggesting a potential pharmacodynamic marker for clinical use. In vivo, R547 showed antitumor activity in all of the models tested to date, including six human tumor xenografts and an orthotopic syngeneic rat model. R547 was efficacious with daily oral dosing as well as with once weekly i.v. dosing in established human tumor models and at the targeted efficacious exposures inhibited phosphorylation of the retinoblastoma protein in the tumors. The selective kinase inhibition profile and the preclinical antitumor activity of R547 suggest that it may be promising for development for use in the treatment of solid tumors. R547 is currently being evaluated in phase I clinical trials. [Mol Cancer Ther 2006;5(11):2644–58]

Elevated MDM2 boosts the apoptotic activity of p53-MDM2 binding inhibitors by facilitating MDMX degradation

The p53 tumor suppressor is a powerful growth suppressive and pro-apoptotic molecule frequently inactivated in human cancer. Many tumors overproduce its negative regulator MDM2, a specific p53 ubiquitin ligase and transcriptional inhibitor, to disable p53 function. Therefore, p53 activation by inhibiting MDM2 has been proposed as a novel strategy for cancer therapy in tumors expressing wild-type p53. Recently developed small-molecule p53-MDM2 binding inhibitors, the nutlins, selectively activate p53 function and induce cell cycle arrest and apoptosis in cancer cells. By stabilizing p53, nutlins also elevate the cellular level of its transcriptional target MDM2. Here, we present evidence that nutlin-induced MDM2 retains its ubiquitin ligase activity and contributes to the anti-tumor activity of p53-MDM2 binding inhibitors by facilitating the degradation of another p53 inhibitor, MDMX. MDM2 and MDMX levels were analyzed in a panel of 12 randomly selected solid tumor cell lines. In the presence of nutlin-3, MDM2 increased in all and MDMX decreased in most of the cell lines. MDMX was resistant to nutlin-induced degradation in 2/12 cell lines. In these cells, MDMX appears to be a major suppressor of the apoptotic response to p53 activation although this effect was only partially p53-dependent. Doxorubicin facilitated MDMX degradation through DNA damage response pathways and restored their sensitivity to nutlin, suggesting that combination therapy may be an effective way to overcome nutlin resistance in cancers with MDMX aberrations.

Discovery of Potent and Orally Active p53-MDM2 Inhibitors RO5353 and RO2468 for Potential Clinical Development

The development of small-molecule MDM2 inhibitors to restore dysfunctional p53 activities represents a novel approach for cancer treatment. In a previous communication, the efforts leading to the identification of a non-imidazoline MDM2 inhibitor, RG7388, was disclosed and revealed the desirable in vitro and in vivo pharmacological properties that this class of pyrrolidine-based inhibitors possesses. Given this richness and the critical need for a wide variety of chemical structures to ensure success in the clinic, research was expanded to evaluate additional derivatives. Here we report two new potent, selective, and orally active p53-MDM2 antagonists, RO5353 and RO2468, as follow-ups with promising potential for clinical development.

Preclinical Optimization of MDM2 Antagonist Scheduling for Cancer Treatment by Using a Model-Based Approach

Purpose: Antitumor clinical activity has been demonstrated for the MDM2 antagonist RG7112, but patient tolerability for the necessary daily dosing was poor. Here, utilizing RG7388, a second-generation nutlin with superior selectivity and potency, we determine the feasibility of intermittent dosing to guide the selection of initial phase I scheduling regimens. Experimental Design: A pharmacokinetic–pharmacodynamic (PKPD) model was developed on the basis of preclinical data to determine alternative dosing schedule requirements for optimal RG7388-induced antitumor activity. This PKPD model was used to investigate the pharmacokinetics of RG7388 linked to the time-course of the antitumor effect in an osteosarcoma xenograft model in mice. These data were used to prospectively predict intermittent and continuous dosing regimens, resulting in tumor stasis in the same model system. Results: RG7388-induced apoptosis was delayed relative to drug exposure with continuous treatment not required. In initial efficacy testing, daily dosing at 30 mg/kg and twice a week dosing at 50 mg/kg of RG7388 were statistically equivalent in our tumor model. In addition, weekly dosing of 50 mg/kg was equivalent to 10 mg/kg given daily. The implementation of modeling and simulation on these data suggested several possible intermittent clinical dosing schedules. Further preclinical analyses confirmed these schedules as viable options. Conclusion: Besides chronic administration, antitumor activity can be achieved with intermittent schedules of RG7388, as predicted through modeling and simulation. These alternative regimens may potentially ameliorate tolerability issues seen with chronic administration of RG7112, while providing clinical benefit. Thus, both weekly (qw) and daily for five days (5 d on/23 off, qd) schedules were selected for RG7388 clinical testing. Clin Cancer Res; 20(14); 3742–52. ©2014 AACR.

Small-molecule inducer of cancer cell polyploidy promotes apoptosis or senescence: Implications for therapy

Polyploidy results from deregulated cell division and has been considered an undesirable event leading to increased mutation rate and cancer development. However, polyploidy may also render cancer cells more vulnerable to chemotherapy. Here, we identify a small-molecule inducer of polyploidy, R1530, which interferes with tubulin polymerization and mitotic checkpoint function in cancer cells, leading to abortive mitosis, endoreduplication and polyploidy. In the presence of R1530, polyploid cancer cells underwent apoptosis or became senescent which translated into potent in vitro and in vivo efficacy. Normal proliferating cells were resistant to R1530-induced polyploidy thus supporting the rationale for cancer therapy by induced polyploidy. Mitotic checkpoint kinase BubR1 was found downregulated during R1530-induced exit from mitosis, a likely consequence of PLK4 inhibition. BubR1 knockdown in the presence of nocodazole induced an R1530-like phenotype, suggesting that BubR1 plays a key role in polyploidy induction by R1530 and could be exploited as a target for designing more specific polyploidy inducers.

Discovery of a Highly Potent, Orally Active Mitosis/Angiogenesis Inhibitor R1530 for the Treatment of Solid Tumors

A new series of 7,8-disubstituted pyrazolobenzodiazepines based on the lead compound 1 have been synthesized and evaluated for their effects on mitosis and angiogenesis. Described herein is the design, synthesis, SAR, and antitumor activity of these compounds leading to the identification of R1530, which was selected for clinical evaluation.