Development of small molecules restoring the tumor suppression functions of p53 – Galina Selivanova Group

Our research focuses on the development of small molecules restoring the tumor suppression functions of p53, either by refolding mutant p53 to rescue its activity, or via preventing proteasomal degradation of p53 in tumors with non-mutated p53. Further, we use small molecules reactivating p53 as tools to study p53 biology.

Group picture of Galina Selivanova group, picture taken outside

Research

Manipulation of the p53 tumor suppressor pathway: from lab bench to clinic

p53 is the major tumor suppressor which eliminates damaged or oncogene-expressing cells by activating transcription of genes inducing apoptosis, cell cycle arrest or senescence. p53 inactivation via mutations or enhanced degradation by MDM2 is the most frequent alteration in human cancers, which underscores the key role of p53 in combating cancer. Reinstatement of p53 by genetic means have demonstrated remarkable tumor suppression in animal models, including inhibition of aggressive metastatic lesions. This inspires the idea of developing small molecules reactivating p53 to fight cancer (Figure 1).

p53 reactivation illustration
Figure 1

Depending on the type of p53 inactivation, there could be envisioned two major strategies of p53 reinstatement: restoring the function of mutant p53 and preventing p53 inactivation by MDM2 (Figure 2).

Illustration Figure 2. strategy of p53 restoration by small molecules depends on the type of p53 inactivation
Figure 2. strategy of p53 restoration by small molecules depends on the type of p53 inactivation. Photo: n/a

A number of MDM2 inhibitors reactivating wild type p53 have been discovered, including small molecule RITA discovered by us (Issaeva et al, Nature Medicine, 2004; Enge et al, Cancer Cell 2009; Grinkevich et al, Cancer Cell, 2009).  Several of these are currently being tested in clinical trials, including derivatives of nutlin and the stapled peptide ATSP-7041, which are activating wild type (wt) p53 are in Phase I/II trials (www.cilicaltrials.gov, see also our review Sanz G et al, J Mol Cell Biol, 2019).

Mutant p53 is expressed in cancers, where it adopts an unfolded conformation resulting in loss of DNA binding and in some cases gaining oncogenic function. Thus, our hypothesis is to stabilize p53 conformation by small molecule to restore the DNA binding tumor suppressor function of p53. Since around 50% of all human tumors carry mutations in p53, it could be widely applicable in clinic. We have identified a small molecule PRIMA-1MET by screening NCI chemical library using cell-based assay (Bykov et al, Nature Medicine, 2002). PRIMA-1MET(commercial name APR-246, please see www.aprea.com) restores the active conformation and DNA binding of mutant p53 in cells and in vitro, re-activates the function of mutant p53 in tumor cells of different origin and suppreses the growth of human xenograft tumors in mice.  

APR-246 has been tested in patients in Phase I clinical trial and in March 2020 APR-246 received Fast Track and Breakthrough Therapy designations from FDA based on outstanding results of Phase II trial in MDS patients. These prompted the start of Phase III randomized trial in MDS.

p53 targets are working on different levels: to prevent tumor development by inducing growth arrest and DNA repair as well as anioxidant response or to exterminate developed tumors by inducing senescence, apoptosis and immunologic cell death. Photo: Galina Selivanova och Alexandra Selivanova

In light of clinical developments of p53-targeting therapies, it is imperative to rationally design combinatorial treatments (since monotherapy cannot cure cancer) and find biomarkers to stratify patients. To achieve these, we need to get a deep understanding of the molecular mechanisms and pathways affected by p53 and p53-reactivating compounds.

If you would like to learn more about our research (or just to drop in to say hello!) you are welcome to visit us at Biomedicum, Quarter C8.

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Illustration
Figure 1. Pharmacological activation of p53 to kill cancer cells, reprogram CAFs and boost immune response against cancer. Photo: Galina Selinova

Targeting p53 to kill tumor cells, reprogram cancer-associated fibroblasts and boost anti-cancer immune response

Half of human tumors carry mutations in the p53 gene, resulting in the expression of inactive protein. Tumors that do not carry p53 mutations, develop an alternative mechanisms of p53 inactivation, converging on enhanced proteasomal degradation. Given the extraordinary high frequency of p53 inactivation in tumors and the high potency of p53 in elimination of tumors, it appears highly desirable to restore the tumor suppressor function of p53 as a strategy to combat cancer. A number of p53-targeting therapies are currently being tested in patients. 

We are addressing the fundamental questions that need to be solved for the efficient application of p53-targeting medicines and their combinations in clinic, i.e. thorough understanding of the mechanism of action of the identified compounds, including target specificity in vitro and in vivo and off-target effects.

While p53 reactivating molecules have been shown to kill cancer cells, the question remains open how p53 reinstatement will affect tumor microenvironment, cancer-associated fibroblasts in particular, as well as anti-cancer immune response. 

Our ambition is to provide basis for innovative p53-based treatment strategies targeting both cancer cells and tumor microenvironment to generate synergistic anti-tumor effects that result in long term survival of patients. Defining this concept, its mechanisms and implications for novel anti-cancer therapies form the main objectives of our studies.

In addition to applied aspects, we are actively working on important basic aspects of tumor biology, including genetic screens for p53 regulators and identification of novel factors which control p53 activity in tumor and in normal cells, which can serve as targets for therapeutic intervention in a future. In essence, to understand p53 is to understand how its interaction with proteins, and thus DNA, is controlled. In order to address these challenging questions we will employ a comprehensive and multidisciplinary approach. We will apply cutting-edge molecular and cell biology methodologies, multi-omics analyses and systems biology analysis, combined with ex vivo 3D cell & tissue culture and in vivo models. We combine hypothesis-driven strategy with unbiased multi-omics approach and apply the analysis of publicly available patient data sets (e.g., TCGA), as well as newly obtained data from patient material via my collaborations with clinicians in Sweden and abroad.

We perform highly parallel and comprehensive search for p53 modulators and identify their functional significance by inter-disciplinary integration of genome-wide expression profiling, ChIP-seq and proteomic approaches, followed by systems biology analysis. This will pave the way to the identification of key p53 target genes and factors contributing to alternative biological responses. These will be thoroughly validated in cells, mouse models and patient samples using functional genomics and protein-protein interaction assays. Selected factors will be used for chemical libraries screens to identify small molecules that target them.  We hope our studies will open the way for the development of novel therapeutic approaches.

In a recent study published in Cancer Discovery in 2021, we demonstrated that pharmacological activation of p53 induces the expression of ERVs and generation of double-stranded (ds) RNA which caused intracellular dsRNA stress leading to type 1 and type 3 IFN responses and induction of  antigen processing and presentation  (APP) genes.  Notably, we found that p53 activation promotes the recruitment of immune cells to tumors in vivo in mouse models and sensitizes refractory tumors to programmed cell death protein 1 (PD-1) blockade.  

Figure illustrating MDM2 inhibitors sensitize tumors to Immune checkpoint blockade.
MDM2 inhibitors sensitize tumors to Immune checkpoint blockade. Photo: Xiaolei Zhou

Importantly, the analysis of pre- and post-treatment tumor biopsy samples from melanoma patients treated with the experimental MDM2 inhibitor ALRN-6924,  revealed the induction of viral mimicry response genes, as well as immune function signatures suggesting infiltration of cytotoxic CD8+ T cells.

Our results suggest the potential role of  p53-reactivating compounds with checkpoint inhibitors. In conclusion, our data presented across cancer cell lines, tumor-bearing mouse models, and melanoma patients suggest that pharmacological p53 reactivation triggers the ERV-dsRNA-IFN pathway within tumor cells, thereby altering the tumor microenvironment evoking tumor immune surveillance.

Project groups within the Galina Selivanova group

Sylvain Peuget project group

p53 regulation by bacteria and its impact on tumorigenesis  

Increasing evidence from metagenomics study highlights the role of the microbiota composition in cancer initiation, progression and resistance to therapies. Cancers are clearly associated with bacterial dysbiosis, which could be a passenger but also a driver of the tumorigenesis process. However, little is known about how the bacteria species enriched during dysbiosis promote tumorigenesis.

We are investigating the molecular mechanisms of the host response to bacterial dysbiosis, with a focus on the tumor suppressor p53 pathway. p53 plays a central role in cell signaling and is therefore strongly regulated in response to commensal and pathogenic bacteria signaling during inflammation. Moreover, p53 encodes a transcriptional program which in turns regulates the innate and adaptive immune response. Therefore, it is not surprising that bacteria have evolved different mechanisms to target and manipulate the host p53 pathway in order to establish and keep their niche. Finally, p53 is the major barrier against cancer in humans and its role as a regulator of immune response is emerging as a key component for its tumor suppressive function. Hence, bacterial interference with the p53 pathway could have important consequences for tumorigenesis.

Our main questions are:  

  1. How the p53 pathway is involved in the physiological host response to bacteria, i.e. inflammation and innate immune response?  
  2. How some bacteria hijack the p53 pathway for their own benefits?  
  3. How bacteria-induced deregulation of the p53 pathway influence cancer initiation and progression?  

Our ultimate goal is to find new therapeutic targets in bacteria and/or the human host to help prevent of combat cancer.  

To investigate these fundamental questions at the molecular level both on the bacteria and host side, this project is a collaboration performed together with an expert in microbiology of pathogenic bacteria Ass. Prof. Marie-Stephanie Aschtgen in Prof Birgitta Henriques-Normark’s lab.

Ingemar Ernberg Group

Our primary research interest is infection and cancer in humans.  We particularly study Epstein-Barr Virus (EBV)-infection, which can also be used as a model to study early steps in in vitro transformation, viral genetic switches, non-genetic variation and cancer cell metabolism.

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Karolinska Institutet
Department of Microbiology, Tumor and Cell Biology
171 77 Stockholm

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Karolinska Institutet
Biomedicum, Quarter C8
Solnavägen 9
171 65 Solna

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Tomtebodavägen 16
1715 Solna

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Karolinska Institutet, Biomedicum, Solnavägen 9