Mechanisms of cancer cell death by mutant p53-reactivating compound APR-246
Author: Ceder, Sophia
Date: 2021-03-05
Location: BioClinicum, J3:11 Birger & Margareta Blombäck, Karolinska University Hospital, Solna, Sweden
Time: 09.00
Department: Inst för onkologi-patologi / Dept of Oncology-Pathology
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Thesis (11.39Mb)
Abstract
Tumor suppressor TP53 is the most frequently mutated gene in cancer. A majority of TP53 mutations result in a mutant p53 that disrupts its DNA binding capabilities but may also acquire novel gain-of-function activities that contribute to tumor growth. The investigational drug APR-246 (Eprenetapopt) is the most clinically advanced compound to target mutant p53 and is being tested in a phase III clinical trial in mutant TP53 myelodysplastic syndrome (MDS). APR-246 is converted to its active product methylene quinuclidinone (MQ). MQ binds to cysteines in p53 promoting a folded structure and DNA binding, leading to cancer cell death. MQ also targets thiols or selenols in e.g. glutathione (GSH) or various enzymes. Depletion of glutathione and inhibition of antioxidant enzymes increase oxidative stress contributing to APR-246-induced cancer cell death.
In Project I, combination treatment of APR-246 and multidrug resistance protein 1 (MRP1) inhibitor resulted in synergistic growth suppression in vitro in tumor cell lines, in vivo in esophageal cancer xenografts, and ex vivo in esophageal and colorectal cancer patient-derived organoids (PDO). We show that inhibition of MRP1 results in increased intracellular 14C- content after 14C-APR-246 treatment. This was attributed to retention of GSH-conjugated MQ (GS-MQ). We demonstrate that GS-MQ binding is reversible and that retention of GS-MQ creates an intracellular MQ pool that may target numerous thiols contributing to APR-246- induced growth suppression. In Project II we studied the spectrum of MQ-targeted cysteines in p53. This was enabled by first establishing a method utilizing the reducing agent NaBH4 to lock the MQ cysteine adducts into a stable form, overcoming reversibility. Cys182, Cys229 and Cys277 in the p53 core domain showed most prominent MQ modification. Additional modification at Cys124 and Cys141 was found in mutant p53. The electrophilic properties of MQ enables targeting of multiple cellular thiols. In Project III we identified novel MQ targets using CEllular Thermal Shift Assay (CETSA). Asparaginase synthetase (ASNS) was stabilized upon MQ treatment and thus is a potential MQ target. In acute lymphoblastic leukemia (ALL), ASNS is associated with resistance to standard treatment asparaginase. Asparaginase depletes extracellular asparagine which renders asparagine-auxotrophic ALL cells sensitive and therefore ASNS expression allows ALL cell survival. We found that combination treatment of APR-246 and asparaginase leads to synergistic growth suppression in ALL cells and may offer a novel treatment strategy for ALL. Lastly, in Project IV we assessed the functional activity of novel germline TP53 mutations identified in a Swedish cohort of families with Li-Fraumeni syndrome (LFS) or hereditary breast cancer (HrBC). Assessing the pathological outcome of TP53 mutations is important for understanding the cancer risk of these families.
The first three projects are aimed at improving our understanding of mutant p53-reactivating compound APR-246. They suggest approaches for increasing treatment efficacy and novel combination strategies. The thesis has also addressed the role of mutant p53 in response to APR-246 and pathological properties in families with LFS or HrBC. All in all, these studies provide novel preclinical understanding of the role of mutant p53 in cancer and response to treatment, both highly relevant in the combat against cancer.
In Project I, combination treatment of APR-246 and multidrug resistance protein 1 (MRP1) inhibitor resulted in synergistic growth suppression in vitro in tumor cell lines, in vivo in esophageal cancer xenografts, and ex vivo in esophageal and colorectal cancer patient-derived organoids (PDO). We show that inhibition of MRP1 results in increased intracellular 14C- content after 14C-APR-246 treatment. This was attributed to retention of GSH-conjugated MQ (GS-MQ). We demonstrate that GS-MQ binding is reversible and that retention of GS-MQ creates an intracellular MQ pool that may target numerous thiols contributing to APR-246- induced growth suppression. In Project II we studied the spectrum of MQ-targeted cysteines in p53. This was enabled by first establishing a method utilizing the reducing agent NaBH4 to lock the MQ cysteine adducts into a stable form, overcoming reversibility. Cys182, Cys229 and Cys277 in the p53 core domain showed most prominent MQ modification. Additional modification at Cys124 and Cys141 was found in mutant p53. The electrophilic properties of MQ enables targeting of multiple cellular thiols. In Project III we identified novel MQ targets using CEllular Thermal Shift Assay (CETSA). Asparaginase synthetase (ASNS) was stabilized upon MQ treatment and thus is a potential MQ target. In acute lymphoblastic leukemia (ALL), ASNS is associated with resistance to standard treatment asparaginase. Asparaginase depletes extracellular asparagine which renders asparagine-auxotrophic ALL cells sensitive and therefore ASNS expression allows ALL cell survival. We found that combination treatment of APR-246 and asparaginase leads to synergistic growth suppression in ALL cells and may offer a novel treatment strategy for ALL. Lastly, in Project IV we assessed the functional activity of novel germline TP53 mutations identified in a Swedish cohort of families with Li-Fraumeni syndrome (LFS) or hereditary breast cancer (HrBC). Assessing the pathological outcome of TP53 mutations is important for understanding the cancer risk of these families.
The first three projects are aimed at improving our understanding of mutant p53-reactivating compound APR-246. They suggest approaches for increasing treatment efficacy and novel combination strategies. The thesis has also addressed the role of mutant p53 in response to APR-246 and pathological properties in families with LFS or HrBC. All in all, these studies provide novel preclinical understanding of the role of mutant p53 in cancer and response to treatment, both highly relevant in the combat against cancer.
List of papers:
I. Ceder, S., Eriksson, S. E., Cheteh, E. H., Dawar, S., Benitez, M. C., Bykov, V. J. N., Fujihara, K. M., Grandin, M., Li, X., Ramm, S., Behrenbruch, C., Simpson, K. J., Hollande, F., Abrahmsen, L., Clemons, N. J. and Wiman, K. G. A thiol-bound drug reservoir enhances APR-246-induced mutant p53 tumor cell death. EMBO Mol Med. 2020: e10852.
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II. Ceder, S., Bykov, V. J. N., Hagberg, L., Mermelekas, G., Jafari, R., Abrahmsen, L. and Wiman, K. G. Spectrum of p53 cysteines targeted by APR-246 active product MQ. [Manuscript]
III. Ceder, S., Eriksson, S. E., Yu, L. Y., Cheteh, E.H., Zhang, S. M., Fujihara, K. M., Bianchi, J., Bykov, V. J. N., Abrahmsen, L., Clemons, N. J., Nordlund, P., Rudd, S. and Wiman, K. G. Mutant p53-reactivating compound APR-246 synergizes with asparaginase in inducing growth suppression in acute lymphoblastic leukemia cells. [Manuscript]
IV. Kharaziha, P., Ceder, S., Axell, O., Krall, M., Fotouhi. O., Böhm, S., Lain, S., Borg, A., Larsson, C., Wiman, K. G., Tham, E. and Bajalica-Lagercrantz, S. Functional characterization of novel germline TP53 variants in Swedish families. Clin Genet. 2019, 96(3): 216-225.
Fulltext (DOI)
Pubmed
View record in Web of Science®
I. Ceder, S., Eriksson, S. E., Cheteh, E. H., Dawar, S., Benitez, M. C., Bykov, V. J. N., Fujihara, K. M., Grandin, M., Li, X., Ramm, S., Behrenbruch, C., Simpson, K. J., Hollande, F., Abrahmsen, L., Clemons, N. J. and Wiman, K. G. A thiol-bound drug reservoir enhances APR-246-induced mutant p53 tumor cell death. EMBO Mol Med. 2020: e10852.
Fulltext (DOI)
Pubmed
View record in Web of Science®
II. Ceder, S., Bykov, V. J. N., Hagberg, L., Mermelekas, G., Jafari, R., Abrahmsen, L. and Wiman, K. G. Spectrum of p53 cysteines targeted by APR-246 active product MQ. [Manuscript]
III. Ceder, S., Eriksson, S. E., Yu, L. Y., Cheteh, E.H., Zhang, S. M., Fujihara, K. M., Bianchi, J., Bykov, V. J. N., Abrahmsen, L., Clemons, N. J., Nordlund, P., Rudd, S. and Wiman, K. G. Mutant p53-reactivating compound APR-246 synergizes with asparaginase in inducing growth suppression in acute lymphoblastic leukemia cells. [Manuscript]
IV. Kharaziha, P., Ceder, S., Axell, O., Krall, M., Fotouhi. O., Böhm, S., Lain, S., Borg, A., Larsson, C., Wiman, K. G., Tham, E. and Bajalica-Lagercrantz, S. Functional characterization of novel germline TP53 variants in Swedish families. Clin Genet. 2019, 96(3): 216-225.
Fulltext (DOI)
Pubmed
View record in Web of Science®
Institution: Karolinska Institutet
Supervisor: Wiman, Klas
Co-supervisor: Bykov, Vladimir; Eriksson, Sofi
Issue date: 2021-02-11
Rights:
Publication year: 2021
ISBN: 978-91-8016-124-4
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