Joanna Lisztwan

Comprehensive mapping of p53 pathway alterations reveals an apparent role for both SNP309 and MDM2 amplification in sarcomagenesis

Purpose: Reactivation of p53 tumor suppressor activity in diseases such as soft-tissue sarcoma is considered an attractive means of targeted therapy. By systematically assessing alterations affecting the p53 pathway, we aimed to (a) classify sarcoma subtypes, (b) define a potential role in malignancy, and (c) identify potential patient biomarkers in this heterogeneous disease. Experimental Design: We have mapped mutational events in a panel of 192 benign or malignant bone and soft-tissue sarcomas. Analyses included TP53 and CDKN2A mutational and SNP status, MDM2 and MDM4 amplification and MDM2 SNP309 status. Results: We found an inverse relationship between MDM2 amplification and TP53 mutations, with a predominantly wild-type CDKN2A background. A high rate of point mutations in TP53 was observed uniquely in leiomyosarcoma, osteosarcoma, and MFH. Both MDM2 and MDM4 were also amplified in a subtype-specific manner, which was frequently seen as a coamplification event. We have also analyzed the risk allele frequencies for MDM2 SNP309, and show that the G allele was strongly associated with both liposarcomas and MDM2 amplification. Conclusions: Our data emphasize the critical role of p53 inactivation in sarcomagenesis, whereby different pathway alterations may be related to the heterogeneity of the disease. Moreover, we observed a strong association of malignancy with TP53 mutation, or MDM2 amplification and the presence of a G allele in SNP309, especially in lipoma versus liposarcoma. We propose, therefore, that MDM2 markers along with TP53 sequencing should be considered as patient biomarkers in clinical trials of sarcomas using MDM2 antagonists. Clin Cancer Res; 17(3); 416–26. ©2010 AACR.

Crystal Structures of Human MdmX (HdmX) in Complex with p53 Peptide Analogues Reveal Surprising Conformational Changes

p53 tumor suppressor activity is negatively regulated through binding to the oncogenic proteins Hdm2 and HdmX. The p53 residues Leu26, Trp23, and Phe19 are crucial to mediate these interactions. Inhibiting p53 binding to both Hdm2 and HdmX should be a promising clinical approach to reactivate p53 in the cancer setting, but previous studies have suggested that the discovery of dual Hdm2/HdmX inhibitors will be difficult. We have determined the crystal structures at 1.3 Å of the N-terminal domain of HdmX bound to two p53 peptidomimetics without and with a 6-chlorine substituent on the indole (which binds in the same subpocket as Trp23 of p53). The latter compound is the most potent peptide-based antagonist of the p53-Hdm2 interaction yet to be described. The x-ray structures revealed surprising conformational changes of the binding cleft of HdmX, including an “open conformation” of Tyr99 and unexpected “cross-talk” between the Trp and Leu pockets. Notably, the 6-chloro p53 peptidomimetic bound with high affinity to both HdmX and Hdm2 (Kd values of 36 and 7 nm, respectively). Our results suggest that the development of potent dual inhibitors for HdmX and Hdm2 should be feasible. They also reveal possible conformational states of HdmX, which should lead to a better prediction of its interactions with potential biological partners.

The central valine concept provides an entry in a new class of non peptide inhibitors of the p53-MDM2 interaction.

Disrupting the interaction between the p53 tumor suppressor and its regulator MDM2 is a promising therapeutic strategy in anticancer drug research. In our search for non peptide inhibitors of this protein-protein interaction, we have devised a ligand design concept exploiting the central position of Val 93 in the p53 binding pocket of MDM2. The design of molecules based on this concept has allowed us to rapidly identify compounds having a 3-imidazolyl indole core structure as the first representatives of a new class of potent inhibitors of the p53-MDM2 interaction.

The aromatase inhibitor letrozole and inhibitors of insulin-like growth factor I receptor synergistically induce apoptosis in in vitro models of estrogen-dependent breast cancer

IntroductionEndocrine-dependent, estrogen receptor positive breast cancer cells proliferate in response to estrogens, synthesized by the cytochrome p450 aromatase enzyme. Letrozole is a potent nonsteroidal aromatase inhibitor that is registered for the treatment of postmenopausal women with advanced metastatic breast cancers and in the neoadjuvant, early, and extended adjuvant indications. Because crosstalk exists between estrogen receptor and insulin-like growth factor I receptor (IGF-IR), the effect of combining a selective IGF-IR inhibitor (NVP-AEW541) with letrozole was assessed in two independent in vitro models of estrogen-dependent breast cancer.MethodsMCF7 and T47D cells stably expressing aromatase (MCF7/Aro and T47D/Aro) were used as in vitro models of aromatase-driven breast cancer. The role of the IGF-IR pathway in breast cancer cells stimulated only by 17β-estradiol or androstenedione was assessed by proliferation assays. The combination of letrozole and NVP-AEW541 was assessed for synergy in inhibiting cell proliferation using Chou-Talalay derived equations. Finally, combination or single agent effects on proliferation and apoptosis were assessed using proliferation assays, flow cytometry, and immunoblotting.ResultsBoth MCF7 and T47D cells, as well as MCF7/Aro and T47D/Aro, exhibited sensitivity to inhibition of 17β-estradiol dependent proliferation by NVP-AEW541. Letrozole combined with NVP-AEW541 synergistically inhibited androstenedione-dependent proliferation in aromatase-expressing cells with combination index values of 0.6 or less. Synergistic combination effects correlated with higher levels of apoptosis as compared with cells treated with the single agent alone. Treatment with either agent also appeared to inhibit IGF-IR signalling via phosphoinositide 3-kinase. Notably, IGF-IR inhibition had limited effect on estrogen-dependent proliferation in the cell lines, but was clearly required for survival, suggesting that the combination of letrozole and IGF-IR inhibition sensitizes cells to apoptosis.ConclusionInhibition of the IGF-IR pathway and aromatase was synergistic in two independent estrogen-dependent in vitro models of breast cancer. Moreover, synergism of NVP-AEW541 and letrozole correlated with induction of apoptosis, but not cell cycle arrest, in the cell lines tested. Combination of IGF-IR inhibitors and letrozole may hold promise for the treatment of patients with estrogen-dependent breast cancers.

Knowledge-based virtual screening: application to the MDM4/p53 protein-protein interaction.

Chemogenomics knowledge-based drug discovery approaches aim to extract the knowledge gained from one target and to apply it for the discovery of ligands and hopefully drugs of a new target which is related to the parent target by homology or conserved molecular recognition. Herein, we demonstrate the potential of knowledge-based virtual screening by applying it to the MDM4-p53 protein-protein interaction where the MDM2-p53 protein-protein interaction constitutes the parent reference system; both systems are potentially relevant to cancer therapy. We show that a combination of virtual screening methods, including homology based similarity searching, QSAR (Quantitative Structure-Activity Relationship) methods, HTD (High Throughput Docking), and UNITY pharmacophore searching provide a successful approach to the discovery of inhibitors. The virtual screening hit list is of the magnitude of 50,000 compounds picked from the corporate compound library of approximately 1.2 million compounds. Emphasis is placed on the facts that such campaigns are only feasible because of the now existing HTCP (High throughput Cherry-Picking) automation systems in combination with robust MTS (Medium Throughput Screening) fluorescence-based assays. Given that the MDM2-p53 system constitutes the reference system, it is not surprising that significantly more and stronger hits are found for this interaction compared to the MDM4-p53 system. Novel, selective and dual hits are discovered for both systems. A hit rate analysis will be provided compared to the full HTS (High-throughput Screening).

Tetra-substituted imidazoles as a new class of inhibitors of the p53–MDM2 interaction

Capitalizing on crystal structure information obtained from a previous effort in the search for non peptide inhibitors of the p53-MDM2 interaction, we have discovered another new class of compounds able to disrupt this protein-protein interaction, an important target in oncology drug research. The new inhibitors, based on a tetra-substituted imidazole scaffold, have been optimized to low nanomolar potency in a biochemical assay following a structure-guided approach. An appropriate strategy has allowed us to translate the high biochemical potency in significant anti-proliferative activity on a p53-dependent MDM2 amplified cell line.

Abstract 3970: Targeting the adenosine immunosuppressive pathway for cancer immunotherapy with small molecule agents

Standard Immune Checkpoint Therapies, such as anti-CTLA-4 or anti-PD-1/PD-L1 mAb, demonstrate long progression-free-survival for responding patients but with a low response rate. This can be due to additional immunosuppressive mechanisms developed by resistant tumors. The adenosine pathway is one of the main drivers of tumor progression by acting on two key features of the TME (Tumor Micro-Environment); immunity and angiogenesis. Expression of CD73 on tumor cells and on immunosuppressive cell subsets leads to the generation of adenosine from AMP. Adenosine inhibits the biological functions of T lymphocytes infiltrating the tumor by binding to the A2AR (receptor). Evotec and Exscientia are collaborating to accelerate small molecule drug discovery in the field of Immuno-Oncology by integrating a unique biophysical screening approach to the adenosine receptors and CD73 to drive automated medicinal chemistry design with a translational-focused screening cascade, to discover novel agents that target the adenosinergic pathway in immuno-oncology. We have firstly sought to generate novel A2AR antagonists and secondly to create A2AR/CD73 bi-specific inhibitory molecules with a dual pharmacological profile, from fragment screening and de novo design approaches. In vivo combination study demonstrate the advantages of targeting CD73 and A2AR. We have obtained active molecules illustrated by in vitro data on primary human T lymphocytes. These results, have paved the way to an optimized process to identify adenosinergic pathway inhibitors with properties optimized for immuno-oncology. Citation Format: Pierre Fons, Michael Esquerre, Stephanie Versluys, Gigliola Mambrini, Michael Paillasse, Andy Bell, Adrian Schreyer, Richard Cox, Richard Bickerton, Joanna Lisztwan, Mark Whittaker, Francoise Bono, Craig Johnstone, Andrew Hopkins. Targeting the adenosine immunosuppressive pathway for cancer immunotherapy with small molecule agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3970. doi:10.1158/1538-7445.AM2017-3970

Abstract 1797: Discovery of NVP-CGM097, a highly potent and optimized small molecule inhibitor of Mdm2 under evaluation in a Phase I clinical trial

Activation of p53 by blocking the p53-Mdm2 interaction using non-peptidic small-molecule inhibitors is being pursued as a promising cancer therapeutic strategy. In the present study, we show the identification of NVP-CGM097, a novel, highly optimized, and selective inhibitor of the p53-Mdm2 interaction. NVP-CGM097 binds to human Mdm2 protein with a Ki value of 1.3 nM, activates p53 in human cells and induces robust p53-dependent cell cycle arrest and apoptosis in human p53 wild-type tumor cells. Its activity and selectivity has been tested and confirmed across a large panel of cancer cell lines from the Cancer Cell Line Encyclopedia. Importantly, NVP-CGM097 displays desirable pharmacokinetic and pharmacodynamic profiles in animals together with excellent oral bioavailability, which triggers rapid and sustained activation of p53-dependent pharmacodynamic biomarkers resulting in tumor regression in multiple xenografted models of p53 wild-type human cancer. The validation and understanding of its mechanism of action, the overall favorable drug-like properties and the characterization of its on-target toxicological profile in preclinical species strongly supported the initiation of Phase I clinical trials with NVP-CGM097 in pre-selected patients with p53 wild-type tumors. Citation Format: Sebastien Jeay, Joerg Berghausen, Nicole Buschmann, Patrick Chene, Robert Cozens, Dirk Erdmann, Stephane Ferretti, Pascal Furet, Tobias Gabriel, Francois Gessier, Diana Graus-Porta, Francesco Hofmann, Philipp Holzer, Moriko Ito, Edgar Jacoby, Michael Jensen, Joerg Kallen, Marc Lang, Joanna Lisztwan, Masato Murakami, Carole Pissot-Soldermann, Stephan Ruetz, Caroline Rynn, Dario Sterker, Stefan Stutz, Therese Valat, Marion Wiesmann, Keiichi Masuya. Discovery of NVP-CGM097, a highly potent and optimized small molecule inhibitor of Mdm2 under evaluation in a Phase I clinical trial. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1797. doi:10.1158/1538-7445.AM2014-1797

Abstract 2634: EVT801: Standalone cancer immunotherapy in VEGFR3+tumors and combination with immune checkpoint therapies in VEGFR3-tumors

In collaboration with Sanofi, we previously showed that inhibition of the VEGFR3 pathway by SAR131675 leads to reduction of tumor growth by acting on the tumor microenvironment. We have identified a new drug candidate, EVT801, and evaluated its activity on tumor models expressing VEGFR3. For proof-of-concept, we transfected the mouse BNL hepatoma cell line with VEGFR3. EVT801 displayed a strong therapeutic activity by acting on both VEGFR3+ tumor cells and on the Tumor Microenvironment (TME). By deciphering the mechanism of action of the compounds we showed that EVT801 strongly decreased tumor-associated immunosuppression by decreasing MDSCs (Myeloid Derived Suppressor cells) and CD4+ regulatory T cells and by increasing macrophages with a M1 phenotype inside the tumor. As a consequence the T-cell:MDSC ratios were increased in the TME and also in peripheral blood. In addition to its immunomodulatory properties, EVT801 decreased angiogenesis without increasing hypoxia. We have evaluated EVT801 therapeutic activity in syngeneic tumor mouse models which are not expressing VEGFR3 such as 4T1 mammary carcinoma and CT26 colon carcinoma models. As expected, we observed an intermediate therapeutic activity of the compound on both tumor models. Positive modulation of the TME was equivalent to what we observed with the VEGFR3+ tumor model. With this unique mechanism of action of EVT801 on the TME, we evaluated its ability to increase therapeutic activity of standard Immune Checkpoint Therapies (ICT) such as anti-CTLA-4 and anti-PD-1 mAbs. Strong additive therapeutic activities were observed with EVT801 in combination with these ICT as illustrated by the development of long-term tumor-specific memory CD8+ T cell responses. Toxicological data show that EVT801 has a favorable pharmacological profile consistent with its entry into pre-clinical development. Taken together, these results indicate that EVT801 represents an innovative drug for cancer Immunotherapy which provide a favorable microenvironment to promote tumor regression. In addition, EVT801 may improve the frequency of response to ICT. Citation Format: Michael Esquerre, Pierre Fons, Gaelle Badet, Pauline Barron, Jeremy Kagan, Antoine Alam, Jerome Meneyrol, Isabelle Blanc, Roselyne Broussy, Florence Gaujarengues, Joanna Lisztwan, Michael Paillasse, Mark Whittaker, Francoise Bono. EVT801: Standalone cancer immunotherapy in VEGFR3+ tumors and combination with immune checkpoint therapies in VEGFR3- tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2634. doi:10.1158/1538-7445.AM2017-2634