Vladimir Bykov

Restoration of the tumor suppressor function to mutant p53 by a low-molecular-weight compound

The tumor suppressor p53 inhibits tumor growth primarily through its ability to induce apoptosis. Mutations in p53 occur in at least 50% of human tumors. We hypothesized that reactivation of mutant p53 in such tumors should trigger massive apoptosis and eliminate the tumor cells. To test this, we screened a library of low-molecular-weight compounds in order to identify compounds that can restore wild-type function to mutant p53. We found one compound capable of inducing apoptosis in human tumor cells through restoration of the transcriptional transactivation function to mutant p53. This molecule, named PRIMA-1, restored sequence-specific DNA binding and the active conformation to mutant p53 proteins in vitro and in living cells. PRIMA-1 rescued both DNA contact and structural p53 mutants. In vivo studies in mice revealed an antitumor effect with no apparent toxicity. This molecule may serve as a lead compound for the development of anticancer drugs targeting mutant p53.

PRIMA-1 Reactivates Mutant p53 by Covalent Binding to the Core Domain

Restoration of wild-type p53 expression triggers cell death and eliminates tumors in vivo. The identification of mutant p53-reactivating small molecules such as PRIMA-1 opens possibilities for the development of more efficient anticancer drugs. Although the biological effects of PRIMA-1 are well demonstrated, little is known about its molecular mechanism of action. We show here that PRIMA-1 is converted to compounds that form adducts with thiols in mutant p53. Covalent modification of mutant p53 per se is sufficient to induce apoptosis in tumor cells. These findings might facilitate the design of more potent and specific mutant p53-targeting anticancer drugs.

Targeting p53 in Vivo: A First-in-Human Study With p53-Targeting Compound APR-246 in Refractory Hematologic Malignancies and Prostate Cancer

PURPOSE APR-246 (PRIMA-1MET) is a novel drug that restores transcriptional activity of unfolded wild-type or mutant p53. The main aims of this first-in-human trial were to determine maximum-tolerated dose (MTD), safety, dose-limiting toxicities (DLTs), and pharmacokinetics (PK) of APR-246. PATIENTS AND METHODS APR-246 was administered as a 2-hour intravenous infusion once per day for 4 consecutive days in 22 patients with hematologic malignancies and prostate cancer. Acute myeloid leukemia (AML; n = 7) and prostate cancer (n = 7) were the most frequent diagnoses. Starting dose was 2 mg/kg with dose escalations up to 90 mg/kg. RESULTS MTD was defined as 60 mg/kg. The drug was well tolerated, and the most common adverse effects were fatigue, dizziness, headache, and confusion. DLTs were increased ALT/AST (n = 1), dizziness, confusion, and sensory disturbances (n = 2). PK showed little interindividual variation and were neither dose nor time dependent; terminal half-life was 4 to 5 hours. Tumor cells showed cell cycle arrest, increased apoptosis, and upregulation of p53 target genes in several patients. Global gene expression analysis revealed changes in genes regulating proliferation and cell death. One patient with AML who had a p53 core domain mutation showed a reduction of blast percentage from 46% to 26% in the bone marrow, and one patient with non-Hodgkins lymphoma with a p53 splice site mutation showed a minor response. CONCLUSION We conclude that APR-246 is safe at predicted therapeutic plasma levels, shows a favorable pharmacokinetic profile, and can induce p53-dependent biologic effects in tumor cells in vivo.

Reactivation of Mutant p53 and Induction of Apoptosis in Human Tumor Cells by Maleimide Analogs

Reactivation of mutant p53 is likely to provide important benefits for treatment of chemotherapy- and radiotherapy-resistant tumors. We demonstrate here that the maleimide-derived molecule MIRA-1 can reactivate DNA binding and preserve the active conformation of mutant p53 protein in vitro and restore transcriptional transactivation to mutant p53 in living cells. MIRA-1 induced mutant p53-dependent cell death in different human tumor cells carrying tetracycline-regulated mutant p53. The structural analog MIRA-3 showed antitumor activity in vivo against human mutant p53-carrying tumor xenografts in SCID mice. The MIRA scaffold is a novel lead for the development of anticancer drugs specifically targeting mutant p53.

Characterization of the p53-rescue drug CP-31398 in vitro and in living cells

The Pfizer compound CP-31398 has been reported to stabilize the core domain of the tumour suppressor p53 in vitro and be an effective anti-cancer drug by virtue of rescuing destabilized mutants of p53. We did not detect any interaction between the p53 core domain and CP-31398 in vitro by a wide range of quantitative biophysical techniques over a wide range of conditions. CP-31398 did not stabilize p53 in our experiments. However, we found that CP-31398 intercalated with DNA and also altered and destabilized the DNA-p53 core domain complex. We analysed by NMR TROSY the interaction of the domain with a DNA oligomer and identified the changes in the complex on the binding of CP-31398. CP-31398 also decreased sequence-specific DNA binding of wild-type p53 and His-273 mutant p53. CP-31398 had a non-specific toxic effect independent of mutant p53 expression in several cell lines carrying Tet-regulated mutant p53. CP-31398 caused a small increase in MDM-2 expression and a more pronounced p53-independent increase in Bax expression. CP-31398 did, however, induce the PAb1620 epitope (characteristic of native p53) in cells expressing His-175 mutant p53. This was prevented by cycloheximide, suggesting that any stabilizing action of CP-31398 would have to be on newly synthesized p53. One of the unstable mutants that was reported to have been rescued by CP-31398, R249S, does not bind DNA when folded at lower temperatures.

PRIMA-1 MET synergizes with cisplatin to induce tumor cell apoptosis

Mutant p53-carrying tumors are often more resistant to chemotherapeutical drugs. We demonstrate here that the mutant p53-reactivating compound PRIMA-1MET acts synergistically with several chemotherapeutic drugs to inhibit tumor cell growth. Combined treatment with cisplatin and PRIMA-1MET resulted in a synergistic induction of tumor cell apoptosis and inhibition of human tumor xenograft growth in vivo in SCID mice. The induction of mutant p53 levels by chemotherapeutic drugs is likely to increase the sensitivity of tumor cells to PRIMA-1MET. Thus, the combination of PRIMA-1MET with currently used chemotherapeutic drugs may represent a novel and more efficient therapeutic strategy for treatment of mutant p53-carrying tumors.

Small molecules that reactivate mutant p53.

Around half of all human tumours carry mutant p53. This allows escape from p53-induced cell cycle arrest and apoptosis. Many tumours express mutant p53 proteins at elevated levels. Restoration of wild-type p53 function should trigger massive apoptosis in tumour cells and thus eradicate tumours. Various types of small molecules have been identified that can restore native conformation and wild-type function to mutant p53. Such molecules may serve as leads for the development of novel efficient anticancer drugs.

Mutant p53 reactivation by small molecules makes its way to the clinic.

The TP53 tumor suppressor gene is mutated in many human tumors, including common types of cancer such as colon and ovarian cancer. This illustrates the key role of p53 as trigger of cell cycle arrest or cell death upon oncogenic stress. Most TP53 mutations are missense mutations that result in single amino acid substitutions in p53 and expression of high levels of dysfunctional p53 protein. Restoration of wild type p53 function in such tumor cells will induce robust cell death and allow efficient eradication of the tumor. Therapeutic targeting of mutant p53 in tumors is a rapidly developing field at the forefront of translational cancer research. Various approaches have led to the identification of small molecules that can rescue mutant p53. These include compounds that target specific p53 mutations, including PK083 and PK5174 (Y220C mutant p53) and NSC319726 (R175H mutant p53), as well as PRIMA‐1 and its analog APR‐246 that affect a wider range of mutant p53 proteins. APR‐246 has been tested in a Phase I/II clinical trial with promising results.

Mutant p53 reactivation by PRIMA-1MET induces multiple signaling pathways converging on apoptosis

The low molecular weight compound PRIMA-1MET reactivates mutant p53 and triggers mutant p53-dependent apoptosis in human tumor cells. We investigated the effect of PRIMA-1MET on global gene expression using microarray analysis of Saos-2 cells expressing His273 mutant p53 and parental p53 null Saos-2 cells. PRIMA-1MET affected transcription of a significantly larger number of genes in the mutant p53-expressing cells compared to the p53 null cells. Genes affected by PRIMA-1MET in a mutant p53-dependent manner include the cell-cycle regulators GADD45B and 14-3-3γ and the pro-apoptotic Noxa. Several of the affected genes are known p53 target genes and/or contain p53 DNA-binding motifs. We also found mutant p53-dependent disruption of the cytoskeleton, as well as transcriptional activation of the XBP1 gene and cleavage of its mRNA, a marker for endoplasmic reticulum stress. Our data show that PRIMA-1MET induces apoptosis through multiple transcription-dependent and -independent pathways. Such integral engagement of multiple pathways leading to apoptosis is consistent with restoration of wild-type properties to mutant p53 and is likely to reduce the risk of drug resistance development in clinical applications of PRIMA-1MET.

Mutant p53 targeting by the low molecular weight compound STIMA-1

Reactivation of mutant p53 in human tumor cells should induce cell death by apoptosis and thus eliminate the tumor. Several small molecules that reactivate mutant p53 have been identified. Here we show that STIMA‐1, a low molecular weight compound with some structural similarities to the previously identified molecule CP‐31398, can stimulate mutant p53 DNA binding in vitro and induce expression of p53 target proteins and trigger apoptosis in mutant p53‐expressing human tumor cells. Human diploid fibroblasts are significantly more resistant to STIMA‐1 than mutant or wild type p53‐carrying tumor cells. STIMA‐1 may provide new insights into possible mechanisms of mutant p53 reactivation and thus facilitate the development of novel anticancer drugs that target mutant p53‐carrying tumors.