SLC7A11 is a superior determinant of APR-246 (Eprenetapopt) response than<i>TP53</i>mutation status

Fujihara, Kenji M.; Corrales-Benitez, Mariana; Cabalag, Carlos S.; Zhang, Bonnie Z.; Ko, Hyojin; Liu, David S.; Simpson, Kaylene J.; Haupt, Ygal; Haupt, Sue; Phillips, Wayne A.; Clemons, Nicholas J.

doi:10.1101/2020.11.29.398875

Cited by 3 publications

(1 citation statement)

References 38 publications

(46 reference statements)

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“…While negative results in the thermal shift assay do not explicitly exclude p53 target engagement by these compounds, they do raise questions around whether they are bona fide reactivators of p53. Recently, new data has also shown that expression of SLC7A11 rather than TP53 mutational status is a superior determinant of sensitivity to APR-246 treatment, and a comprehensive study of p53 reactivators (including SCH529074 and APR-246) failed to confirm their ability to stabilize p53 mutants, thus further confounding understanding of these compounds’ primary efficacy targets and mechanisms.…”

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confidence: 99%

Rapid Evaluation of Small Molecule Cellular Target Engagement with a Luminescent Thermal Shift Assay

Mortison

Cornella-Taracido

Venkatchalam³

et al. 2021

ACS Med. Chem. Lett.

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Determination of target engagement for candidate drug molecules in the native cellular environment is a significant challenge for drug discovery programs. The cellular thermal shift assay (CETSA) has emerged as a powerful tool for determining compound target engagement through measurement of changes to a protein's thermal stability upon ligand binding. Here, we present a HiBiT thermal shift assay (BiTSA) that deploys a quantitative peptide tag for determination of compound target engagement in the native cellular environment using a high throughput, plate-based luminescence readout. We demonstrate that BiTSA can rapidly assess cellular target engagement of small molecule ligands against their cognate targets and highlight two applications of BiTSA for differentiating small molecules targeting mutant KRAS and TP53.

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mentioning

confidence: 99%

Rapid Evaluation of Small Molecule Cellular Target Engagement with a Luminescent Thermal Shift Assay

Mortison

Cornella-Taracido

Venkatchalam³

et al. 2021

ACS Med. Chem. Lett.

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Mutant p53-reactivating compound APR-246 synergizes with asparaginase in inducing growth suppression in acute lymphoblastic leukemia cells

et al. 2021

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Asparaginase depletes extracellular asparagine in the blood and is an important treatment for acute lymphoblastic leukemia (ALL) due to asparagine auxotrophy of ALL blasts. Unfortunately, resistance occurs and has been linked to expression of the enzyme asparagine synthetase (ASNS), which generates asparagine from intracellular sources. Although TP53 is the most frequently mutated gene in cancer overall, TP53 mutations are rare in ALL. However, TP53 mutation is associated with poor therapy response and occurs at higher frequency in relapsed ALL. The mutant p53-reactivating compound APR-246 (Eprenetapopt/PRIMA-1Met) is currently being tested in phase II and III clinical trials in several hematological malignancies with mutant TP53. Here we present CEllular Thermal Shift Assay (CETSA) data indicating that ASNS is a direct or indirect target of APR-246 via the active product methylene quinuclidinone (MQ). Furthermore, combination treatment with asparaginase and APR-246 resulted in synergistic growth suppression in ALL cell lines. Our results thus suggest a potential novel treatment strategy for ALL.

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Genome-wide CRISPR screens reveal APR-246 (Eprenetapopt) triggers ferroptosis and inhibits iron-sulfur cluster biogenesis

Fujihara

Jackson

et al. 2020

Preprint

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The mechanisms by which cells respond and adapt to oxidative stress are largely unknown but are key to developing a rationale for cancer therapies that target antioxidant pathways. APR-246 is a mutant-p53 targeted therapeutic currently under clinical investigation in myeloid dysplastic syndrome (MDS) and acute myeloid leukemia1. Whilst the mechanism of action of APR-246 is thought to be reactivation of wild-type p53 activity through covalent modification of cysteine residues in the core domain of mutant-p53 protein2,3, here we report that the anti-neoplastic capacity of APR-246 lies predominantly in the conjugation of free cysteine. Genome-wide CRISPR perturbation screening, metabolite profiling and proteomics in response to APR-246 treatment in mutant-p53 cancer cells highlighted the role of GSH and mitochondrial metabolism in determining APR-246 efficacy. APR-246 sensitivity was increased through loss of key enzymes in mitochondrial one-carbon metabolism, SHMT2 and MTHFD1L, due to diminished glycine supply for de novo GSH synthesis. Critically, we show that APR-246 induces iron-dependent, apoptotic machinery-independent cell death, ferroptosis. Whole-cell proteomics analyses indicated an upregulation of proteins involved in iron-sulfur cluster biogenesis (eg. FDX1). GSH, acetyl-CoA and NADH levels were also depleted in APR-246 treated cells. Importantly, we found that APR-246 inhibits iron-sulfur cluster biogenesis in the mitochondria of cancer cells through cysteine conjugation. This work not only details novel determinants of APR-246 activity in cancer cells, but also provides a clinical roadmap for targeting antioxidant pathways in tumours - beyond targeting mutant-p53 tumours.

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SLC7A11 is a superior determinant of APR-246 (Eprenetapopt) response thanTP53mutation status

Cited by 3 publications

References 38 publications

Rapid Evaluation of Small Molecule Cellular Target Engagement with a Luminescent Thermal Shift Assay

Rapid Evaluation of Small Molecule Cellular Target Engagement with a Luminescent Thermal Shift Assay

Mutant p53-reactivating compound APR-246 synergizes with asparaginase in inducing growth suppression in acute lymphoblastic leukemia cells

Genome-wide CRISPR screens reveal APR-246 (Eprenetapopt) triggers ferroptosis and inhibits iron-sulfur cluster biogenesis

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