Therapeutic Targeting of RNA Splicing Catalysis through Inhibition of Protein Arginine Methylation

Fong, Jia Yi; Pignata, Luca; Goy, Pierre-Alexis; Kawabata, Kimihito C.; Lee, Stanley Chun-Wei; Koh, Cheryl M.; Musiani, Daniele; Massignani, Enrico; Kotini, Andriana G.; Penson, Alex; Wun, Cheng Mun; Shen, Yudao; Schwarz, Megan; Low, Diana; Rialdi, Alexander; Ki, Michelle; Wollmann, Heike; Mzoughi, Slim; Thompson, Christine; Hart, Timothy K.; Barbash, Olena; Luciani, Genna M.; Szewczyk, Magdalena M.; Wouters, Bas J.; Delwel, Ruud; Papapetrou, Eirini P.; Baršytė-Lovejoy, Dalia; Arrowsmith, C.H.; Minden, Mark D.; Jin, Jian; Melnick, Ari; Bonaldi, Tiziana; Abdel‐Wahab, Omar; Guccione, Ernesto

doi:10.1016/j.ccell.2019.07.003

Cited by 202 publications

(254 citation statements)

References 90 publications

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“…To characterize PRMT enzymes' substrate motifs, a variety of targeted enzymatic and computational approaches have been used (Wooderchak et al 2008;Gathiaka et al 2016;Gayatri et al 2016). Finally, others have tested the proteomic and transcriptomic consequences of inhibition of various PRMTs Eram et al 2016;Fong et al 2019;Musiani et al 2019;Radzisheuskaya et al 2019).…”

Section: Introductionmentioning

confidence: 99%

“…For instance, PTMScan-previously developed to immunoprecipitate post-translationally modified peptides after lysate protease cleavage (Stokes et al 2012)-was used in HEK293 cells to identify thousands of PRMT-regulated Rme1 sites (Larsen et al 2016) and to identify PRMT1 substrates in Toxoplasma (Yakubu et al 2017). Others have used PTMScan with SILAC to identify sites of all three methylarginine states, mostly found to be enriched in RNA-binding proteins and regulating splicing (Fong et al 2019;Musiani et al 2019;Radzisheuskaya et al 2019). Middle-down proteomics coupled with electron-transfer dissociation (ETD) was used to specifically identify arginine methylation in arginine-and serine-rich domains in RNA-binding proteins (Kundinger et al 2020).…”

Section: Introductionmentioning

confidence: 99%

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Transcriptomic and proteomic regulation through abundant, dynamic, and independent arginine methylation by Type I and Type II PRMTs

Lehman

Chen

Burgos

et al. 2020

Preprint

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Arginine methylation is essential for both cellular viability and development and is also dysregulated in cancer. PRMTs catalyze the post translational monomethylation (Rme1/MMA, catalyzed by Type I-III), asymmetric (Rme2a/ADMA, Type I enzymes)-, or symmetric (Rme2s/SDMA, Type II enzymes) dimethylation of arginine. Despite many studies, a thorough integration of PRMT enzyme substrate determination and proteomic and transcriptomic consequences of inhibiting Type I and II PRMTs is lacking. To characterize cellular substrates for Type I (Rme2a) and Type II (Rme2s) PRMTs, human A549 lung adenocarcinoma cells were treated with either Type I (MS023) or Type II (GSK591) inhibitors. Using total proteome hydrolysis, we developed a new mass spectrometry approach to analyze total arginine and lysine content. We showed that Rme1 was a minor population (~0.1% of total arginine), Rme2a was highly abundant (~1.1%), and Rme2s was intermediate (~0.4%). While Rme2s was mostly eliminated by GSK591 treatment, total Rme1 and Rme2a were more resistant to perturbation. To quantitatively characterize substrate preferences of the major enzymes PRMT1, PRMT4(CARM1), and PRMT5, we used oriented peptide array libraries (OPAL) in methyltransferase assays. We demonstrated that while PRMT5 tolerates aspartic acid residues in the substrate, PRMT1 does not. Importantly, PRMT4 methylated previously uncharacterized hydrophobic motifs. To integrate our studies, we performed PTMScan on PRMT-inhibited A549 cells and enriched for methylated arginine containing tryptic peptides. For detection of highly charged peptides, a method to analyze the samples using electron transfer dissociation was developed. Proteomic analysis revealed distinct methylated species enriched in nuclear function, RNA-binding, intrinsically disordered domains, and liquid-liquid phase separation. Parallel studies with proteomics and RNA-Seq revealed distinct, but ontologically overlapping, consequences to PRMT inhibition. Overall, we demonstrate a wider PRMT substrate diversity and methylarginine functional consequence than previously shown.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Transcriptomic and proteomic regulation through abundant, dynamic, and independent arginine methylation by Type I and Type II PRMTs

Lehman

Chen

Burgos

et al. 2020

Preprint

View full text Add to dashboard Cite

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“…Recently, several complementary reports demonstrated that combining PRMT1 inhibitors with PRMT5 inhibitors resulted in synergistic anti-tumor activity (Fedoriw, et al 2019;Fong, et al 2019).…”

Section: Discussionmentioning

confidence: 99%

Targeted CRISPR screening identifies PRMT5 as synthetic lethality combinatorial target with gemcitabine in pancreatic cancer cells

Wei

Yang

Adair

et al. 2020

Preprint

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Pancreatic ductal adenocarcinoma (PDAC) remains one of the most challenging cancer to treat. Due to the asymptomatic nature of the disease and ineffective drug treatment modalities, the survival rate of PDAC patients remains one of the lowest. The recurrent genetic alterations in PDAC are yet to be targeted; therefore, identifying effective therapeutic combinations is desperately needed. Here, we performed an in vivo CRISPR screening in a clinically relevant patient-derived xenograft (PDX) model system to identify synergistic drug combinations for PDAC treatment. Our approach revealed protein arginine methyltransferase gene 5 (PRMT5) as a promising druggable candidate whose inhibition creates synergistic vulnerability of PDAC cells to gemcitabine. Genetic and pharmacological inhibition results indicate that of PRMT5 depletion results in synergistic cytotoxicity with Gem due to depleted replication protein A (RPA) levels and an impaired non-homology end joining (NHEJ) DNA repair.Thus, the novel combination creates conditional lethality through the accumulation of excessive DNA damage and cell death, both in vitro and in vivo. The findings demonstrate that unbiased genetic screenings combined with a clinically relevant model system is an effective approach in identifying synthetic lethal drug combinations for cancer treatment. STATEMENT of SIGNIFICANCEIdentify synergistic drug combinations for PDAC is a significant unmet need. Through CRISPR screening, we discovered and validated that PRMT5 depletion creates synergistic vulnerability of PDAC cells to gemcitabine. Mechanistically, the combination impairs DNA repair, synergistic accumulation of DNA damage and cell death in vitro and in vivo.

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“…Phosphorylation of splicing factors by kinases of the SRPK and CLK families control their enzymatic activity and subcellular localization 61 and AML cells are sensitive to pharmacological inhibition of these kinases 62 . Many RNA binding proteins are also methylated by the PRMT family of protein arginine methyltransferases and PRMT inhibition kills leukemic cells 63 . Furthermore, splicing alterations due to epigenetic or chromatin changes due to somatic mutations 44,64 or possibly as a consequence of aging 65 have also been recently reported.…”

Section: Discussionmentioning

confidence: 99%

RNA splicing alterations induce a cellular stress response associated with poor prognosis in AML

Anande

Deshpande

Mareschal

et al. 2020

Preprint

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Anande et al RNA splicing alterations in AML 3 Key Points • Widespread and recurrent alternative splicing differences exist between AML patients with good or poor prognosis • Missplicing of RNA splicing factors leads to altered splicing of their target transcripts • Aberrant splicing of protein translation genes triggers the induction of an integrated stress response and concomitant inflammatory response • Alternative RNA splicing information can be used to improve the accuracy of existing prognostic algorithms in AML Anande et al RNA splicing alterations in AML 4Abstract RNA splicing is a fundamental biological process that generates protein diversity from a finite set of genes. Recurrent somatic mutations of splicing factor genes are relatively uncommon in Acute Myeloid Leukemia (AML, < 20%). We examined whether RNA splicing differences exist in AML even in the absence of splicing factor mutations. Analyzing RNA-seq data from two independent cohorts of AML patients, we identified recurrent differential alternative splicing between patients with poor and good prognosis. These alternative splicing events occurred even in patients without any discernible splicing factor mutations. The alternative splicing events recurrently occurred in genes involved in specific molecular functions, primarily related to protein translation. Developing informatics tools to predict the functional impact of alternative splicing on the translated protein, we discovered that ~45% of the splicing events directly affected highly conserved protein domains.Several splicing factors were themselves misspliced in patients, and the splicing of their target transcripts were also altered. By studying differential gene expression in the same patients, we identified that alternative splicing of protein translation genes in ELN Adv patients resulted in the induction of an integrated stress response and upregulation of inflammation-related genes. Lastly, using machine learning techniques, we identified a set of four genes whose alternative splicing can refine the accuracy of existing risk prognosis schemes and validated it in a completely independent cohort.Our discoveries therefore identify aberrant alternative splicing as a molecular feature of adverse AML with clinical relevance. Anande et alRNA splicing alterations in AML 5

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Therapeutic Targeting of RNA Splicing Catalysis through Inhibition of Protein Arginine Methylation

Cited by 202 publications

References 90 publications

Transcriptomic and proteomic regulation through abundant, dynamic, and independent arginine methylation by Type I and Type II PRMTs

Transcriptomic and proteomic regulation through abundant, dynamic, and independent arginine methylation by Type I and Type II PRMTs

Targeted CRISPR screening identifies PRMT5 as synthetic lethality combinatorial target with gemcitabine in pancreatic cancer cells

RNA splicing alterations induce a cellular stress response associated with poor prognosis in AML

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