P53 tumor suppressor is required for efficient execution of the death program following treatment with a cytotoxic limonoid obtained from Melia azedarach

Joray, Mariana Belén; Villafañez, Florencia; González, María Laura; Crespo, María Inés; Laiolo, Jerónimo; Palacios, Sara M.; Bocco, José Luis; Soria, Gastón; Carpinella, María Cecilia

doi:10.1016/j.fct.2017.04.039

Cited by 14 publications

(20 citation statements)

References 44 publications

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“…After 10 days of culture, the media was removed, and crystal violet staining solution was added for colony visualization. The surviving fraction was determined as described in (20).…”

Section: Clonogenic Assaysmentioning

confidence: 99%

Polo-like Kinase 1 Inhibition as a Therapeutic Approach to Selectively Target BRCA1-Deficient Cancer Cells by Synthetic Lethality Induction

Carbajosa

Pansa

Paviolo

et al. 2019

Clinical Cancer Research

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Purpose: BRCA1 and BRCA2 deficiencies are widespread drivers of human cancers that await the development of targeted therapies. We aimed to identify novel synthetic lethal relationships with therapeutic potential using BRCA-deficient isogenic backgrounds.Experimental Design:We developed a phenotypic screening technology to simultaneously search for synthetic lethal (SL) interactions in BRCA1-and BRCA2-deficient contexts. For validation, we developed chimeric spheroids and a dualtumor xenograft model that allowed the confirmation of SL induction with the concomitant evaluation of undesired cytotoxicity on BRCA-proficient cells. To extend our results using clinical data, we performed retrospective analysis on The Cancer Genome Atlas (TCGA) breast cancer database.Results: The screening of a kinase inhibitors library revealed that Polo-like kinase 1 (PLK1) inhibition triggers strong SL induction in BRCA1-deficient cells. Mechanistically, we found no connection between the SL induced by PLK1 inhibition and PARP inhibitors. Instead, we uncovered that BRCA1 downregulation and PLK1 inhibition lead to aberrant mitotic phenotypes with altered centrosomal duplication and cytokinesis, which severely reduced the clonogenic potential of these cells. The penetrance of PLK1/BRCA1 SL interaction was validated using several isogenic and nonisogenic cellular models, chimeric spheroids, and mice xenografts. Moreover, bioinformatic analysis revealed high-PLK1 expression in BRCA1-deficient tumors, a phenotype that was consistently recapitulated by inducing BRCA1 deficiency in multiple cell lines as well as in BRCA1-mutant cells.Conclusions: We uncovered an unforeseen addiction of BRCA1-deficient cancer cells to PLK1 expression, which provides a new means to exploit the therapeutic potential of PLK1 inhibitors in clinical trials, by generating stratification schemes that consider this molecular trait in patient cohorts.

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“…After 10 days of culture, the media was removed, and crystal violet staining solution was added for colony visualization. The surviving fraction was determined as described in (20).…”

Section: Clonogenic Assaysmentioning

confidence: 99%

Polo-like Kinase 1 Inhibition as a Therapeutic Approach to Selectively Target BRCA1-Deficient Cancer Cells by Synthetic Lethality Induction

Carbajosa

Pansa

Paviolo

et al. 2019

Clinical Cancer Research

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“…The treatment resulted in a concomitant slight but not significantly different ( p > 0.05) decrease in both the G1 and the S phase in K562 cells and a significant decrease in the G1 phase ( p < 0.001) in Lucena 1 cells. This effect was also observed with DOX (Figure ), which is known to induce G 2 /M arrest . Hydroxyurea (HU) was introduced as a second reference compound, since it is well-established that it induces S-phase cell cycle arrest …”

Section: Resultsmentioning

confidence: 72%

“…As observed in Table and Figure S1 (Supporting Information), compound 1 showed a cytotoxic effect against K562 and CCRF-CEM leukemia cells and their respective MDR/P-gp counterparts, Lucena 1 and CEM/ADR5000 cell lines, in the micromolar range, as is evident from the IC 50 values obtained, ranging from 0.40 to 5.1 μM. The criterion of activity was proven according to the threshold of IC 50 value established by the U.S. National Cancer Institute for considering a pure compound as cytotoxic, which corresponds to 10 μM . Compounds 2 – 4 were in general less potently cytotoxic against the cell lines used than compound 1 (Table ).…”

Section: Resultsmentioning

confidence: 99%

Cytotoxic Activity of Germacrane-Type Sesquiterpene Lactones from Dimerostemma aspilioides

et al. 2020

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The need for effective candidates as cytotoxic drugs that at the same time challenge cancer multidrug resistance encouraged a search for these in plants of central Argentina. Bioassay-guided fractionation of the cytotoxic extract from Dimerostemma aspilioides led to the isolation of the germacranolide tomenphantin A (1), along with three new analogues (2–4). These efficiently inhibited the proliferation of the leukemia cell lines K562 and CCRF-CEM and their resistant variants, Lucena 1 and CEM/ADR5000, respectively, with IC50 values ranging from 0.40 to 7.7 μM. The structures and relative configurations of compounds 1–4 were elucidated by analysis of the spectroscopic data, in particular NMR spectroscopy. The most active among these was compound 1 (IC50 = 0.40–5.1 μM), and, therefore, this was selected as a model for a mechanistic study, which revealed that its antiproliferative effect was mediated by cell cycle arrest in the G2/M phase followed by apoptosis. The activity of compound 1 was selective, given the absence of cytotoxicity toward peripheral blood mononuclear cells. The results show the potential of these compounds, and in particular of compound 1, as leads for the development of drug candidates to fight sensitive and resistant leukemia cells.

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“…For cycle analysis, cells were prepared as described previously [53]. SYTOX Red (Thermo Fisher Scientific) was used for dead cell staining according as previously described [54]. Stained samples were subjected to fluorescence-activated cell sorting (FACS) (Attune NxT, Thermo Fisher Scientific) and data were analyzed using FlowJo software (FlowJo LLC).…”

Section: Methodsmentioning

confidence: 99%

AKT inhibition impairs PCNA ubiquitylation and triggers synthetic lethality in homologous recombination-deficient cells submitted to replication stress

et al. 2019

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Translesion DNA synthesis (TLS) and homologous recombination (HR) cooperate during S-phase to safeguard replication forks integrity. Thus, the inhibition of TLS becomes a promising point of therapeutic intervention in HR-deficient cancers, where TLS impairment might trigger synthetic lethality (SL). The main limitation to test this hypothesis is the current lack of selective pharmacological inhibitors of TLS. Herein, we developed a miniaturized screening assay to identify inhibitors of PCNA ubiquitylation, a key post-translational modification required for efficient TLS activation. After screening a library of 627 kinase inhibitors, we found that targeting the pro-survival kinase AKT leads to strong impairment of PCNA ubiquitylation. Mechanistically, we found that AKT-mediated modulation of Proliferating Cell Nuclear Antigen (PCNA) ubiquitylation after UV requires the upstream activity of DNA PKcs, without affecting PCNA ubiquitylation levels in unperturbed cells. Moreover, we confirmed that persistent AKT inhibition blocks the recruitment of TLS polymerases to sites of DNA damage and impairs DNA replication forks processivity after UV irradiation, leading to increased DNA replication stress and cell death. Remarkably, when we compared the differential survival of HR-proficient vs HR-deficient cells, we found that the combination of UV irradiation and AKT inhibition leads to robust SL induction in HR-deficient cells. We link this phenotype to AKT ability to inhibit PCNA ubiquitylation, since the targeted knockdown of PCNA E3-ligase (RAD18) and a non-ubiquitylable (PCNA K164R) knock-in model recapitulate the observed SL induction. Collectively, this work identifies AKT as a novel regulator of PCNA ubiquitylation and provides the proof-of-concept of inhibiting TLS as a therapeutic approach to selectively kill HR-deficient cells submitted to replication stress.

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P53 tumor suppressor is required for efficient execution of the death program following treatment with a cytotoxic limonoid obtained from Melia azedarach

Cited by 14 publications

References 44 publications

Polo-like Kinase 1 Inhibition as a Therapeutic Approach to Selectively Target BRCA1-Deficient Cancer Cells by Synthetic Lethality Induction

Polo-like Kinase 1 Inhibition as a Therapeutic Approach to Selectively Target BRCA1-Deficient Cancer Cells by Synthetic Lethality Induction

Cytotoxic Activity of Germacrane-Type Sesquiterpene Lactones from Dimerostemma aspilioides

AKT inhibition impairs PCNA ubiquitylation and triggers synthetic lethality in homologous recombination-deficient cells submitted to replication stress

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