Mechanisms of cancer cell death by mutant p53-reactivating compound APR-246

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

Functional characterization of novel germline TP53

Pathogenic germline TP53 variants predispose to a wide range of early onset cancers, often recognized as the Li-Fraumeni syndrome (LFS). They are also identified in 1% of families with hereditary breast cancer (HrBC) that do not fulfill the criteria for LFS. In this study, we present a total of 24 different TP53 variants identified in 31 Swedish families with LFS or HrBC. Ten of these variants, nine exonic and one splice, have previously not been described as germline pathogenic variants. The nine exonic variants were functionally characterized and demonstrated partial transactivation activity compared to wild-type p53. Some show nuclear localization similar to wild-type p53 while others possess cytoplasmic or perinuclear localization. The four frameshift variants (W91Gfs*32, L111 Wfs*12, S227 Lfs*20 and S240Kfs*25) had negligible, while F134 L and T231del had low level of p53 activity. The L111 Wfs*12 and T231del variants are also deficient for induction of apoptosis. The missense variant R110C retain p53 effects and the nonsense E349* shows at least partial transcription factor activity but has reduced ability to trigger apoptosis. This is the first functional characterization of novel germline TP53 pathogenic or likely pathogenic variants in the Swedish cohort as an attempt to understand its association with LFS and HrBC, respectively

Functional characterization of novel germline TP53 variants in Swedish families

Pathogenic germline TP53 variants predispose to a wide range of early onset cancers, often recognized as the Li-Fraumeni syndrome (LFS). They are also identified in 1% of families with hereditary breast cancer (HrBC) that do not fulfill the criteria for LFS. In this study, we present a total of 24 different TP53 variants identified in 31 Swedish families with LFS or HrBC. Ten of these variants, nine exonic and one splice, have previously not been described as germline pathogenic variants. The nine exonic variants were functionally characterized and demonstrated partial transactivation activity compared to wild-type p53. Some show nuclear localization similar to wild-type p53 while others possess cytoplasmic or perinuclear localization. The four frameshift variants (W91Gfs*32, L111 Wfs*12, S227 Lfs*20 and S240Kfs*25) had negligible, while F134 L and T231del had low level of p53 activity. The L111 Wfs*12 and T231del variants are also deficient for induction of apoptosis. The missense variant R110C retain p53 effects and the nonsense E349* shows at least partial transcription factor activity but has reduced ability to trigger apoptosis. This is the first functional characterization of novel germline TP53 pathogenic or likely pathogenic variants in the Swedish cohort as an attempt to understand its association with LFS and HrBC, respectively

A thiol‐bound drug reservoir enhances APR‐246‐induced mutant p53 tumor cell death

Abstract The tumor suppressor gene TP53 is the most frequently mutated gene in cancer. The compound APR‐246 (PRIMA‐1Met/Eprenetapopt) is converted to methylene quinuclidinone (MQ) that targets mutant p53 protein and perturbs cellular antioxidant balance. APR‐246 is currently tested in a phase III clinical trial in myelodysplastic syndrome (MDS). By in vitro, ex vivo, and in vivo models, we show that combined treatment with APR‐246 and inhibitors of efflux pump MRP1/ABCC1 results in synergistic tumor cell death, which is more pronounced in TP53 mutant cells. This is associated with altered cellular thiol status and increased intracellular glutathione‐conjugated MQ (GS‐MQ). Due to the reversibility of MQ conjugation, GS‐MQ forms an intracellular drug reservoir that increases availability of MQ for targeting mutant p53. Our study shows that redox homeostasis is a critical determinant of the response to mutant p53‐targeted cancer therapy

Tumour exosome integrins determine organotropic metastasis.

Ever since Stephen Paget's 1889 hypothesis, metastatic organotropism has remained one of cancer's greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6β4 and α6β1 were associated with lung metastasis, while exosomal integrin αvβ5 was linked to liver metastasis. Targeting the integrins α6β4 and αvβ5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis

Tumour exosome integrins determine organotropic metastasis.

Ever since Stephen Paget's 1889 hypothesis, metastatic organotropism has remained one of cancer's greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6β4 and α6β1 were associated with lung metastasis, while exosomal integrin αvβ5 was linked to liver metastasis. Targeting the integrins α6β4 and αvβ5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis

Tumour exosome integrins determine organotropic metastasis

Ever since Stephen Paget’s 1889 hypothesis, metastatic organotropism has remained one of cancer’s greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α(6)β(4) and α(6)β(1) were associated with lung metastasis, while exosomal integrin α(v)β(5) was linked to liver metastasis. Targeting the integrins α(6)β(4) and α(v)β(5) decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis