Functional Genomic Screen in Mesothelioma Reveals that Loss of Function of BRCA1-Associated Protein 1 Induces Chemoresistance to Ribonucleotide Reductase Inhibition

Our plain-English summary of Functional Genomic Screen in Mesothelioma Reveals that Loss of Function of BRCA1-Associated Protein 1 Induces Chemoresistance to Ribonucleotide Reductase Inhibition, by A. Okonska, S. Bühler, V. Rao, et al (Molecular Cancer Therapeutics, 2020).

The study

• Authors: A. Okonska, S. Bühler, V. Rao, M. Ronner, M. Blijlevens, I.H. van der Meulen-Muileman, R.X. de Menezes, M. Wipplinger, K. Oehl, E.F. Smit, W. Weder, R.A. Stahel, L. Penengo, V.W. van Beusechem, E. Felley-Bosco
• Journal: Molecular Cancer Therapeutics
• Published: 2020;19(2):552-563
• Cited by: 24 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Loss of function of BRCA1-associated protein 1 (BAP1) is observed in about 50% of malignant pleural mesothelioma (MPM) cases. The aim of this study was to investigate whether this aspect could be exploited for targeted therapy. A genetically engineered model was established expressing either functional or nonfunctional BAP1, and whole-genome siRNA synthetic lethality screens were performed assessing differentially impaired survival between the two cell lines. The whole-genome siRNA screen unexpectedly revealed 11 hits (FDR < 0.05) that were more cytotoxic to BAP1-proficient cells. Two actionable targets, ribonucleotide reductase (RNR) catalytic subunit M1 (RRM1) and RNR regulatory subunit M2 (RRM2), were validated. In line with the screen results, primary mesothelioma (BAP1 +/-) overexpressing BAP1 C91A (catalytically dead mutant) was more resistant to RNR inhibition, while BAP1 knockdown in the BAP1-proficient cell lines rescued the cells from their vulnerability to RNR depletion. Gemcitabine and hydroxyurea were more cytotoxic in BAP1-proficient cell line-derived spheroids compared with BAP1 deficient. Upregulation of RRM2 upon gemcitabine and hydroxyurea treatment was more profound in BAP1 mut/del cell lines. Increased lethality mediated by RNR inhibition was observed in NCI-H2452 cells reconstituted with BAP1-WT but not with BAP1 C91A. Upregulation of RRM2 in NCI-H2452-BAP1 WT spheroids was modest compared with control or C91A mutant. Together, we found that BAP1 is involved in the regulation of RNR levels during replication stress. Our observations reveal a potential clinical application where BAP1 status could serve as predictive or stratification biomarker for RNR inhibition-based therapy in MPM.

©2019 American Association for Cancer Research.

In plain English

This laboratory study used malignant pleural mesothelioma cells grown in dishes and as three-dimensional clumps called spheroids. No patients were treated and no asbestos exposure was studied, but public gene data from 87 mesothelioma tumor samples were examined. The authors compared cells with and without working BAP1, using a screen that switched off genes one at a time across the genome. Unexpectedly, all 11 genes passing the screen's statistical threshold were more lethal to switch off in cells with working BAP1. Two, RRM1 and RRM2, make up an enzyme blocked by the drugs gemcitabine and hydroxyurea. The authors tested four cell lines with normal (wild-type) BAP1 and three with mutated or deleted BAP1. Silencing RRM1 or RRM2 reduced viability more in the BAP1-normal cells. In spheroids, gemcitabine was approximately 1000-fold more potent in the BAP1-normal group. Cells lacking working BAP1 raised RRM2 levels more after drug treatment. The authors concluded that BAP1 loss induces resistance to these drugs in mesothelioma cells. They said BAP1 status could serve as a predictive biomarker for gemcitabine, which current clinical trials will assess.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (cell culture) study: genome-wide siRNA screen in a genetically engineered mesothelioma cell line pair, followed by validation experiments in cell lines, a primary culture and spheroids, plus analysis of public tumor gene-expression data
• Who was studied: Human malignant pleural mesothelioma cell lines were studied. NCI-H2452 was engineered with or without working BAP1. Four lines were BAP1 wild-type (SPC111, ACC-Meso-1, NCI-H2052, Mero82) and three were BAP1 mutated or deleted (NCI-H226, NCI-H2452, ACC-Meso-4). The study also used the primary mesothelioma culture SDM81, which was compared with its parental tumor, and normal mesothelial LP9/TERT-1 cells. The authors also analysed public TCGA expression data from 87 MPM samples.
• Exposure: No asbestos or fiber exposure was studied. Cells were exposed to siRNA silencing of genes, including RRM1, RRM2 and BAP1. They were also exposed to the drugs gemcitabine and hydroxyurea, which inhibit ribonucleotide reductase.
• Main results:
  • In the screen, silencing 1,775 genes affected viability differently in BAP1-proficient versus BAP1-deficient cells, based on P less than 0.05 alone.
  • Under the stricter criteria (FDR less than 0.05), 11 genes differed between the two cell lines. Depleting each of the 11 was more cytotoxic to the BAP1-proficient cell line.
  • Across the panel, BAP1 wild-type mesothelioma cell lines lost more viability than BAP1 mutated or deleted lines when RRM1 and RRM2 were silenced.
  • In the SDM81 primary mesothelioma culture, overexpressing the catalytically dead BAP1 C91A mutant led to resistance to gemcitabine.
  • In BAP1 wild-type Mero-82 cells, silencing BAP1 reversed the cell death caused by silencing RRM2 together with gemcitabine.
  • In spheroids, gemcitabine was approximately 1000-fold more potent at reducing cell viability in the BAP1 wild-type group than in the BAP1 mutated or deleted group.
  • Gemcitabine and hydroxyurea had only a minor effect on normal mesothelial LP9/TERT-1 cells, reducing their viability only at the highest concentrations.
  • In spheroids, RRM2 rose significantly more after gemcitabine and hydroxyurea treatment in the BAP1 mutated or deleted group than in the BAP1 wild-type group.
  • NCI-H2452 cells given working (wild-type) BAP1 were more sensitive to gemcitabine than empty-vector cells. Cells given the C91A mutant were not.
  • Public TCGA mRNA data from 87 MPM samples showed an inverse relationship between BAP1 and RRM1 or RRM2, similar to that seen in the cell lines.
• The authors' conclusion: The authors concluded that BAP1 loss induces resistance to drugs that inhibit ribonucleotide reductase in mesothelioma cells. They said BAP1 status could serve as a predictive biomarker for gemcitabine treatment in MPM, which current clinical trials will assess.
• Limitations the authors state:
  • The authors did not measure cellular nucleotide (dNTP) pools. They assumed gemcitabine depleted nucleotides because adding nucleotides rescued its toxicity.
  • Adding wild-type BAP1 to NCI-H2452 cells increased drug sensitivity by at most 27%. The authors suggested this may be due to competition with the cells' own mutated BAP1.
  • Spheroids contain both dividing and non-dividing cells, and the non-dividing cells could be less sensitive to nucleotide shortage.
  • In the screen, silencing PARP1 and PARP2 had poor effects on viability compared with the drug olaparib.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Mesothelioma.

Medical terms in this study, explained in our Lexicon: Mesothelioma, BAP1, Pleural Mesothelioma, Targeted Therapy, Gemcitabine, Biomarker.

Related studies

• Importance of excision repair cross-complementation group 1 and ribonucleotide reductase M1 as prognostic biomarkers in malignant pleural mesothelioma treated with platinum-based induction chemotherapy followed by surgery — L. Frischknecht, M. Meerang, et al. (2015)
• A Novel BRCA1-Associated Protein-1 Isoform Affects Response of Mesothelioma Cells to Drugs Impairing BRCA1-Mediated DNA Repair — R. Parrotta, A. Okonska, et al. (2017)
• Functional differences between wild-type and mutant-type BRCA1-associated protein 1 tumor suppressor against malignant mesothelioma cells — S. Hakiri, H. Osada, et al. (2015)
• Loss of BRCA1-associated protein 1 (BAP1) expression is useful in diagnostic cytopathology of malignant mesothelioma in effusions — I. Cozzi, F.A. Oprescu, et al. (2018)
• Loss of BRCA1-associated protein 1 (BAP1) expression is rare in non-small cell lung cancer — D. Owen, B.S. Sheffield, et al. (2017)
• Prevalence of BRCA-1 associated protein 1 germline mutation in sporadic malignant pleural mesothelioma cases — A. Rusch, G. Ziltener, et al. (2015)

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