Publications by authors named "Ernesto Guccione"

89 Publications

Integrative RNA-omics discovers GNAS alternative splicing as a phenotypic driver of splicing factor-mutant neoplasms.

Cancer Discov 2021 Oct 7. Epub 2021 Oct 7.

Oncological Sciences, Icahn School of Medicine at Mount Sinai

Mutations in splicing factors (SFs) are the predominant class of mutations in myelodysplastic syndrome (MDS), but convergent downstream disease drivers remain elusive. To identify common direct targets of mis-splicing by mutant U2AF1 and SRSF2, we performed RNA-Seq and eCLIP in human hematopoietic stem/progenitor cells (HSPCs) derived from isogenic induced pluripotent stem cell (iPSC) models. Integrative analyses of alternative splicing and differential binding converged on a long isoform of GNAS (GNAS-L), promoted by both mutant factors. MDS population genetics, functional and biochemical analyses support that GNAS-L is a driver of MDS and encodes a hyperactive long form of the stimulatory G protein alpha subunit, Gas-L, that activates ERK/MAPK signaling. SF-mutant MDS cells have activated ERK signaling and consequently are sensitive to MEK inhibitors. Our findings highlight an unexpected and unifying mechanism by which SRSF2 and U2AF1 mutations drive oncogenesis with potential therapeutic implications for MDS and other SF-mutant neoplasms.
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http://dx.doi.org/10.1158/2159-8290.CD-21-0508DOI Listing
October 2021

Differential effects of the second SARS-CoV-2 mRNA vaccine dose on T cell immunity in naive and COVID-19 recovered individuals.

Cell Rep 2021 08 4;36(8):109570. Epub 2021 Aug 4.

Programme in Emerging Infectious Diseases, Duke-NUS Medical School, Singapore 169547, Singapore.

The rapid development of mRNA-based vaccines against the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) led to the design of accelerated vaccination schedules that have been extremely effective in naive individuals. While a two-dose immunization regimen with the BNT162b2 vaccine has been demonstrated to provide a 95% efficacy in naive individuals, the effects of the second vaccine dose in individuals who have previously recovered from natural SARS-CoV-2 infection has not been investigated in detail. In this study, we characterize SARS-CoV-2 spike-specific humoral and cellular immunity in naive and previously infected individuals during and after two doses of BNT162b2 vaccination. Our results demonstrate that, while the second dose increases both the humoral and cellular immunity in naive individuals, COVID-19 recovered individuals reach their peak of immunity after the first dose. These results suggests that a second dose, according to the current standard regimen of vaccination, may be not necessary in individuals previously infected with SARS-CoV-2.
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http://dx.doi.org/10.1016/j.celrep.2021.109570DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8332924PMC
August 2021

PRMT1-dependent regulation of RNA metabolism and DNA damage response sustains pancreatic ductal adenocarcinoma.

Nat Commun 2021 07 30;12(1):4626. Epub 2021 Jul 30.

Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer that has remained clinically challenging to manage. Here we employ an RNAi-based in vivo functional genomics platform to determine epigenetic vulnerabilities across a panel of patient-derived PDAC models. Through this, we identify protein arginine methyltransferase 1 (PRMT1) as a critical dependency required for PDAC maintenance. Genetic and pharmacological studies validate the role of PRMT1 in maintaining PDAC growth. Mechanistically, using proteomic and transcriptomic analyses, we demonstrate that global inhibition of asymmetric arginine methylation impairs RNA metabolism, which includes RNA splicing, alternative polyadenylation, and transcription termination. This triggers a robust downregulation of multiple pathways involved in the DNA damage response, thereby promoting genomic instability and inhibiting tumor growth. Taken together, our data support PRMT1 as a compelling target in PDAC and informs a mechanism-based translational strategy for future therapeutic development.Statement of significancePDAC is a highly lethal cancer with limited therapeutic options. This study identified and characterized PRMT1-dependent regulation of RNA metabolism and coordination of key cellular processes required for PDAC tumor growth, defining a mechanism-based translational hypothesis for PRMT1 inhibitors.
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http://dx.doi.org/10.1038/s41467-021-24798-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8324870PMC
July 2021

HNRNPM controls circRNA biogenesis and splicing fidelity to sustain cancer cell fitness.

Elife 2021 06 2;10. Epub 2021 Jun 2.

Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.

High spliceosome activity is a dependency for cancer cells, making them more vulnerable to perturbation of the splicing machinery compared to normal cells. To identify splicing factors important for prostate cancer (PCa) fitness, we performed pooled shRNA screens in vitro and in vivo. Our screens identified heterogeneous nuclear ribonucleoprotein M (HNRNPM) as a regulator of PCa cell growth. RNA- and eCLIP-sequencing identified HNRNPM binding to transcripts of key homeostatic genes. HNRNPM binding to its targets prevents aberrant exon inclusion and backsplicing events. In both linear and circular mis-spliced transcripts, HNRNPM preferentially binds to GU-rich elements in long flanking proximal introns. Mimicry of HNRNPM-dependent linear-splicing events using splice-switching-antisense-oligonucleotides was sufficient to inhibit PCa cell growth. This suggests that PCa dependence on HNRNPM is likely a result of mis-splicing of key homeostatic coding and non-coding genes. Our results have further been confirmed in other solid tumors. Taken together, our data reveal a role for HNRNPM in supporting cancer cell fitness. Inhibition of HNRNPM activity is therefore a potential therapeutic strategy in suppressing growth of PCa and other solid tumors.
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http://dx.doi.org/10.7554/eLife.59654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8346284PMC
June 2021

Cancer synthetic vulnerabilities to protein arginine methyltransferase inhibitors.

Curr Opin Pharmacol 2021 08 27;59:33-42. Epub 2021 May 27.

Department of Oncological Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA; Mount Sinai Center for Therapeutics Discovery, Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

Protein arginine methylation is an abundant post-translational modification involved in the modulation of essential cellular processes ranging from transcription, post-transcriptional RNA metabolism, and propagation of signaling cascades to the regulation of the DNA damage response. Excitingly for the field, in the past few years there have been remarkable advances in the development of molecular tools and clinical compounds able to selectively and potently inhibit protein arginine methyltransferase (PRMT) functions. In this review, we first discuss how the somatic mutations that confer advantages to cancer cells are often associated with vulnerabilities that can be exploited by PRMTs' inhibition. In a second part, we discuss strategies to uncover synthetic lethal combinations between existing therapies and PRMT inhibitors.
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http://dx.doi.org/10.1016/j.coph.2021.04.004DOI Listing
August 2021

TOP1 inhibition therapy protects against SARS-CoV-2-induced lethal inflammation.

Cell 2021 05 30;184(10):2618-2632.e17. Epub 2021 Mar 30.

Department of Genetics and Genomics, Icahn School of Medicine at Mount Sinai, New York, NY, USA; Icahn Institute of Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

The ongoing pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently affecting millions of lives worldwide. Large retrospective studies indicate that an elevated level of inflammatory cytokines and pro-inflammatory factors are associated with both increased disease severity and mortality. Here, using multidimensional epigenetic, transcriptional, in vitro, and in vivo analyses, we report that topoisomerase 1 (TOP1) inhibition suppresses lethal inflammation induced by SARS-CoV-2. Therapeutic treatment with two doses of topotecan (TPT), an FDA-approved TOP1 inhibitor, suppresses infection-induced inflammation in hamsters. TPT treatment as late as 4 days post-infection reduces morbidity and rescues mortality in a transgenic mouse model. These results support the potential of TOP1 inhibition as an effective host-directed therapy against severe SARS-CoV-2 infection. TPT and its derivatives are inexpensive clinical-grade inhibitors available in most countries. Clinical trials are needed to evaluate the efficacy of repurposing TOP1 inhibitors for severe coronavirus disease 2019 (COVID-19) in humans.
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http://dx.doi.org/10.1016/j.cell.2021.03.051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8008343PMC
May 2021

The duality of PRDM proteins: epigenetic and structural perspectives.

FEBS J 2021 Mar 28. Epub 2021 Mar 28.

Department of Oncological Sciences and Pharmacological Sciences, Center for Therapeutics Discovery, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

PRDF1 and RIZ1 homology domain containing (PRDMs) are a subfamily of Krüppel-like zinc finger proteins controlling key processes in metazoan development and in cancer. PRDMs exhibit unique dualities: (a) PR domain/ZNF arrays-their structure combines a SET-like domain known as a PR domain, typically found in methyltransferases, with a variable array of C2H2 zinc fingers (ZNF) characteristic of DNA-binding transcription factors; (b) transcriptional activators/repressors-their physiological function is context- and cell-dependent; mechanistically, some PRDMs have a PKMT activity and directly catalyze histone lysine methylation, while others are rather pseudomethyltransferases and act by recruiting transcriptional cofactors; (c) oncogenes/tumor suppressors-their pathological function depends on the specific PRDM isoform expressed during tumorigenesis. This duality is well known as the 'Yin and Yang' of PRDMs and involves a complex regulation of alternative splicing or alternative promoter usage, to generate full-length or PR-deficient isoforms with opposing functions in cancer. In conclusion, once their dualities are fully appreciated, PRDMs represent a promising class of targets in oncology by virtue of their widespread upregulation across multiple tumor types and their somatic dispensability, conferring a broad therapeutic window and limited toxic side effects. The recent discovery of a first-in-class compound able to inhibit PRDM9 activity has paved the way for the identification of further small molecular inhibitors able to counteract PRDM oncogenic activity.
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http://dx.doi.org/10.1111/febs.15844DOI Listing
March 2021

Npac Is a Co-factor of Histone H3K36me3 and Regulates Transcriptional Elongation in Mouse Embryonic Stem Cells.

Genomics Proteomics Bioinformatics 2021 Mar 3. Epub 2021 Mar 3.

Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore; The State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Macau 999078, China. Electronic address:

Chromatin modification contributes to pluripotency maintenance in embryonic stem cells (ESCs). However, the related mechanisms remain obscure. Here, we show that Npac, a "reader" of histone H3 lysine 36 trimethylation (H3K36me3), is required to maintain mouse ESC (mESC) pluripotency since knockdown of Npac causes mESC differentiation. Depletion of Npac in mouse embryonic fibroblasts (MEFs) inhibits reprogramming efficiency. Furthermore, our Npac ChIP-seq results reveal that Npac co-localizes with histone H3K36me3 in gene bodies of actively transcribed genes in mESCs. Interestingly, we find that Npac interacts with p-TEFb, RNA Pol II Ser2, and Ser5. Depletion of Npac disrupts transcriptional elongation of pluripotency genes Nanog and Rif1. Taken together, we propose that Npac is essential for transcriptional elongation of pluripotency genes by recruiting of p-TEFb and interacting with RNA Pol II Ser2 and Ser5.
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http://dx.doi.org/10.1016/j.gpb.2020.08.004DOI Listing
March 2021

Mutations in Are a Novel Cause of Galloway-Mowat Syndrome.

J Am Soc Nephrol 2021 03 16;32(3):580-596. Epub 2021 Feb 16.

Institute of Human Genetics, Faculty of Medicine, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.

Background: Galloway-Mowat syndrome (GAMOS) is characterized by neurodevelopmental defects and a progressive nephropathy, which typically manifests as steroid-resistant nephrotic syndrome. The prognosis of GAMOS is poor, and the majority of children progress to renal failure. The discovery of monogenic causes of GAMOS has uncovered molecular pathways involved in the pathogenesis of disease.

Methods: Homozygosity mapping, whole-exome sequencing, and linkage analysis were used to identify mutations in four families with a GAMOS-like phenotype, and high-throughput PCR technology was applied to 91 individuals with GAMOS and 816 individuals with isolated nephrotic syndrome. and studies determined the functional significance of the mutations identified.

Results: Three biallelic variants of the transcriptional regulator were detected in six families with proteinuric kidney disease. Four families with a variant in the protein's zinc-finger (ZNF) domain have additional GAMOS-like features, including brain anomalies, cardiac defects, and skeletal defects. All variants destabilize the PRDM15 protein, and the ZNF variant additionally interferes with transcriptional activation. Morpholino oligonucleotide-mediated knockdown of Prdm15 in embryos disrupted pronephric development. Human wild-type RNA rescued the disruption, but the three variants did not. Finally, CRISPR-mediated knockout of in human podocytes led to dysregulation of several renal developmental genes.

Conclusions: Variants in can cause either isolated nephrotic syndrome or a GAMOS-type syndrome on an allelic basis. PRDM15 regulates multiple developmental kidney genes, and is likely to play an essential role in renal development in humans.
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http://dx.doi.org/10.1681/ASN.2020040490DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7920168PMC
March 2021

Splice-Switching Antisense Oligonucleotides as a Targeted Intrinsic Engineering Tool for Generating Armored Redirected T Cells.

Nucleic Acid Ther 2021 04 9;31(2):145-154. Epub 2021 Feb 9.

Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.

Modification of specificity of T cells for the use in adoptive transfer (CAR- or TCR-redirected T cells) has revolutionized the therapy of liquid tumors and some infectious diseases. However, several obstacles are still hampering the efficacy of such potent therapy, hence concurrent modification of the function is also required to obtain successful results. Here we show the use of splice-switching antisense oligonucleotides (SSOs) as a tool to transiently modify T cell function. We demonstrate the possibility to transfect SSOs and an exogenous TCR into primary human T cells in the same electroporation reaction, without affecting viability and function of the transfected T lymphocytes. Moreover, we show that SSOs targeting T cell-specific mRNAs induce the skipping of the targeted exons, and the reduction of the protein and consequent modification of T cell function. This technical work paves the way to the use of SSOs in immune cells, not only for the knockdown of the functional isoform of the targeted proteins, but also for the protein manipulation by elimination of specific domains encoded by targeted exons.
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http://dx.doi.org/10.1089/nat.2020.0905DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7997720PMC
April 2021

Topoisomerase 1 inhibition therapy protects against SARS-CoV-2-induced inflammation and death in animal models.

bioRxiv 2020 Dec 1. Epub 2020 Dec 1.

The ongoing pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is currently affecting millions of lives worldwide. Large retrospective studies indicate that an elevated level of inflammatory cytokines and pro-inflammatory factors are associated with both increased disease severity and mortality. Here, using multidimensional epigenetic, transcriptional, and analyses, we report that Topoisomerase 1 (Top1) inhibition suppresses lethal inflammation induced by SARS-CoV-2. Therapeutic treatment with two doses of Topotecan (TPT), a FDA-approved Top1 inhibitor, suppresses infection-induced inflammation in hamsters. TPT treatment as late as four days post-infection reduces morbidity and rescues mortality in a transgenic mouse model. These results support the potential of Top1 inhibition as an effective host-directed therapy against severe SARS-CoV-2 infection. TPT and its derivatives are inexpensive clinical-grade inhibitors available in most countries. Clinical trials are needed to evaluate the efficacy of repurposing Top1 inhibitors for COVID-19 in humans.
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http://dx.doi.org/10.1101/2020.12.01.404483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7724667PMC
December 2020

EHMT2 epigenetically suppresses Wnt signaling and is a potential target in embryonal rhabdomyosarcoma.

Elife 2020 11 30;9. Epub 2020 Nov 30.

Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.

Wnt signaling is downregulated in embryonal rhabdomyosarcoma (ERMS) and contributes to the block of differentiation. Epigenetic mechanisms leading to its suppression are unknown and could pave the way toward novel therapeutic modalities. We demonstrate that EHMT2 suppresses canonical Wnt signaling by activating expression of the Wnt antagonist . Inhibition of EHMT2 expression or activity in human ERMS cell lines reduced expression and elevated canonical Wnt signaling resulting in myogenic differentiation in vitro and in mouse xenograft models in vivo. Mechanistically, EHMT2 impacted Sp1 and p300 enrichment at the promoter. The reduced tumor growth upon EHMT2 deficiency was reversed by recombinant DKK1 or LGK974, which also inhibits Wnt signaling. Consistently, among 13 drugs targeting chromatin modifiers, EHMT2 inhibitors were highly effective in reducing ERMS cell viability. Our study demonstrates that ERMS cells are vulnerable to EHMT2 inhibitors and suggest that targeting the EHMT2-DKK1-β-catenin node holds promise for differentiation therapy.
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http://dx.doi.org/10.7554/eLife.57683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7728445PMC
November 2020

Trained Immunity-Promoting Nanobiologic Therapy Suppresses Tumor Growth and Potentiates Checkpoint Inhibition.

Cell 2020 10;183(3):786-801.e19

Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.

Trained immunity, a functional state of myeloid cells, has been proposed as a compelling immune-oncological target. Its efficient induction requires direct engagement of myeloid progenitors in the bone marrow. For this purpose, we developed a bone marrow-avid nanobiologic platform designed specifically to induce trained immunity. We established the potent anti-tumor capabilities of our lead candidate MTP-HDL in a B16F10 mouse melanoma model. These anti-tumor effects result from trained immunity-induced myelopoiesis caused by epigenetic rewiring of multipotent progenitors in the bone marrow, which overcomes the immunosuppressive tumor microenvironment. Furthermore, MTP-HDL nanotherapy potentiates checkpoint inhibition in this melanoma model refractory to anti-PD-1 and anti-CTLA-4 therapy. Finally, we determined MTP-HDL's favorable biodistribution and safety profile in non-human primates. In conclusion, we show that rationally designed nanobiologics can promote trained immunity and elicit a durable anti-tumor response either as a monotherapy or in combination with checkpoint inhibitor drugs.
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http://dx.doi.org/10.1016/j.cell.2020.09.059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8074872PMC
October 2020

Mitchell-Riley syndrome iPSCs exhibit reduced pancreatic endoderm differentiation due to a mutation in .

Development 2020 11 5;147(21). Epub 2020 Nov 5.

Institute of Molecular and Cellular Biology, Agency for Science Technology and Research (A*STAR), 61 Biopolis Drive, 138673, Singapore.

Mitchell-Riley syndrome (MRS) is caused by recessive mutations in the regulatory factor X6 gene () and is characterised by pancreatic hypoplasia and neonatal diabetes. To determine why individuals with MRS specifically lack pancreatic endocrine cells, we micro-CT imaged a 12-week-old foetus homozygous for the nonsense mutation c.1129C>T, which revealed loss of the pancreas body and tail. From this foetus, we derived iPSCs and show that differentiation of these cells proceeds normally until generation of pancreatic endoderm, which is significantly reduced. We additionally generated an reporter allele by gene targeting in wild-type H9 cells to precisely define RFX6 expression and in parallel performed hybridisation for in the dorsal pancreatic bud of a Carnegie stage 14 human embryo. Both and , we find that RFX6 specifically labels a subset of PDX1-expressing pancreatic endoderm. In summary, is essential for efficient differentiation of pancreatic endoderm, and its absence in individuals with MRS specifically impairs formation of endocrine cells of the pancreas head and tail.
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http://dx.doi.org/10.1242/dev.194878DOI Listing
November 2020

METTL6 is a tRNA mC methyltransferase that regulates pluripotency and tumor cell growth.

Sci Adv 2020 Aug 26;6(35):eaaz4551. Epub 2020 Aug 26.

Institute of Functional Epigenetics, Helmholtz Zentrum München, 85764 Neuherberg, Germany.

Recently, covalent modifications of RNA, such as methylation, have emerged as key regulators of all aspects of RNA biology and have been implicated in numerous diseases, for instance, cancer. Here, we undertook a combination of in vitro and in vivo screens to test 78 potential methyltransferases for their roles in hepatocellular carcinoma (HCC) cell proliferation. We identified methyltransferase-like protein 6 (METTL6) as a crucial regulator of tumor cell growth. We show that METTL6 is a bona fide transfer RNA (tRNA) methyltransferase, catalyzing the formation of 3-methylcytidine at C32 of specific serine tRNA isoacceptors. Deletion of in mouse stem cells results in changes in ribosome occupancy and RNA levels, as well as impaired pluripotency. In mice, knockout results in reduced energy expenditure. We reveal a previously unknown pathway in the maintenance of translation efficiency with a role in maintaining stem cell self-renewal, as well as impacting tumor cell growth profoundly.
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http://dx.doi.org/10.1126/sciadv.aaz4551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7449687PMC
August 2020

Discovery of First-in-Class Protein Arginine Methyltransferase 5 (PRMT5) Degraders.

J Med Chem 2020 09 21;63(17):9977-9989. Epub 2020 Aug 21.

Mount Sinai Center for Therapeutics Discovery, Departments of Pharmacological Sciences and Oncological Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029, United States.

The aberrant expression of protein arginine methyltransferase 5 (PRMT5) has been associated with multiple cancers. Using the proteolysis targeting chimera technology, we discovered a first-in-class PRMT5 degrader (MS4322). Here, we report the design, synthesis, and characterization of compound and two structurally similar controls (MS4370) and (MS4369), with impaired binding to the von Hippel-Lindau E3 ligase and PRMT5, respectively. Compound , but not and , effectively reduced the PRMT5 protein level in MCF-7 cells. Our mechanism studies indicate that compound degraded PRMT5 in an E3 ligase- and proteasome-dependent manner. Compound also effectively reduced the PRMT5 protein level and inhibited growth in multiple cancer cell lines. Moreover, compound was highly selective for PRMT5 in a global proteomic study and exhibited good plasma exposure in mice. Collectively, compound and its two controls and are valuable chemical tools for exploring the PRMT5 functions in health and disease.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7486226PMC
September 2020

Global translation during early development depends on the essential transcription factor PRDM10.

Nat Commun 2020 07 17;11(1):3603. Epub 2020 Jul 17.

Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.

Members of the PR/SET domain-containing (PRDM) family of zinc finger transcriptional regulators play diverse developmental roles. PRDM10 is a yet uncharacterized family member, and its function in vivo is unknown. Here, we report an essential requirement for PRDM10 in pre-implantation embryos and embryonic stem cells (mESCs), where loss of PRDM10 results in severe cell growth inhibition. Detailed genomic and biochemical analyses reveal that PRDM10 functions as a sequence-specific transcription factor. We identify Eif3b, which encodes a core component of the eukaryotic translation initiation factor 3 (eIF3) complex, as a key downstream target, and demonstrate that growth inhibition in PRDM10-deficient mESCs is in part mediated through EIF3B-dependent effects on global translation. Our work elucidates the molecular function of PRDM10 in maintaining global translation, establishes its essential role in early embryonic development and mESC homeostasis, and offers insights into the functional repertoire of PRDMs as well as the transcriptional mechanisms regulating translation.
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http://dx.doi.org/10.1038/s41467-020-17304-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7368010PMC
July 2020

PRDM15 is a key regulator of metabolism critical to sustain B-cell lymphomagenesis.

Nat Commun 2020 07 14;11(1):3520. Epub 2020 Jul 14.

Division of Cellular and Molecular Research, National Cancer Centre Singapore, Singapore, Singapore.

PRDM (PRDI-BF1 and RIZ homology domain containing) family members are sequence-specific transcriptional regulators involved in cell identity and fate determination, often dysregulated in cancer. The PRDM15 gene is of particular interest, given its low expression in adult tissues and its overexpression in B-cell lymphomas. Despite its well characterized role in stem cell biology and during early development, the role of PRDM15 in cancer remains obscure. Herein, we demonstrate that while PRDM15 is largely dispensable for mouse adult somatic cell homeostasis in vivo, it plays a critical role in B-cell lymphomagenesis. Mechanistically, PRDM15 regulates a transcriptional program that sustains the activity of the PI3K/AKT/mTOR pathway and glycolysis in B-cell lymphomas. Abrogation of PRDM15 induces a metabolic crisis and selective death of lymphoma cells. Collectively, our data demonstrate that PRDM15 fuels the metabolic requirement of B-cell lymphomas and validate it as an attractive and previously unrecognized target in oncology.
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http://dx.doi.org/10.1038/s41467-020-17064-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7360777PMC
July 2020

Posttranslational Regulation of the Exon Skipping Machinery Controls Aberrant Splicing in Leukemia.

Cancer Discov 2020 09 22;10(9):1388-1409. Epub 2020 May 22.

H3 Biomedicine, Inc., Cambridge, Massachusetts.

Splicing alterations are common in diseases such as cancer, where mutations in splicing factor genes are frequently responsible for aberrant splicing. Here we present an alternative mechanism for splicing regulation in T-cell acute lymphoblastic leukemia (T-ALL) that involves posttranslational stabilization of the splicing machinery via deubiquitination. We demonstrate there are extensive exon skipping changes in disease, affecting proteasomal subunits, cell-cycle regulators, and the RNA machinery. We present that the serine/arginine-rich splicing factors (SRSF), controlling exon skipping, are critical for leukemia cell survival. The ubiquitin-specific peptidase 7 (USP7) regulates SRSF6 protein levels via active deubiquitination, and USP7 inhibition alters the exon skipping pattern and blocks T-ALL growth. The splicing inhibitor H3B-8800 affects splicing of proteasomal transcripts and proteasome activity and acts synergistically with proteasome inhibitors in inhibiting T-ALL growth. Our study provides the proof-of-principle for regulation of splicing factors via deubiquitination and suggests new therapeutic modalities in T-ALL. SIGNIFICANCE: Our study provides a new proof-of-principle for posttranslational regulation of splicing factors independently of mutations in aggressive T-cell leukemia. It further suggests a new drug combination of splicing and proteasomal inhibitors, a concept that might apply to other diseases with or without mutations affecting the splicing machinery..
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http://dx.doi.org/10.1158/2159-8290.CD-19-1436DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7483384PMC
September 2020

PRDM15 loss of function links NOTCH and WNT/PCP signaling to patterning defects in holoprosencephaly.

Sci Adv 2020 01 10;6(2):eaax9852. Epub 2020 Jan 10.

Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (ASTAR), Singapore.

Holoprosencephaly (HPE) is a congenital forebrain defect often associated with embryonic lethality and lifelong disabilities. Currently, therapeutic and diagnostic options are limited by lack of knowledge of potential disease-causing mutations. We have identified a new mutation in the gene (C844Y) associated with a syndromic form of HPE in multiple families. We demonstrate that C844Y is a loss-of-function mutation impairing PRDM15 transcriptional activity. Genetic deletion of murine causes anterior/posterior (A/P) patterning defects and recapitulates the brain malformations observed in patients. Mechanistically, PRDM15 regulates the transcription of key effectors of the NOTCH and WNT/PCP pathways to preserve early midline structures in the developing embryo. Analysis of a large cohort of patients with HPE revealed potentially damaging mutations in several regulators of both pathways. Our findings uncover an unexpected link between NOTCH and WNT/PCP signaling and A/P patterning and set the stage for the identification of new HPE candidate genes.
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http://dx.doi.org/10.1126/sciadv.aax9852DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6954057PMC
January 2020

Epigenetic Silencing of CDR1as Drives IGF2BP3-Mediated Melanoma Invasion and Metastasis.

Cancer Cell 2020 01;37(1):55-70.e15

Department of Pathology, New York University Langone Medical Center, New York, NY, USA; Interdisciplinary Melanoma Cooperative Group, New York University Langone Medical Center, New York, NY, USA. Electronic address:

Metastasis is the primary cause of death of cancer patients. Dissecting mechanisms governing metastatic spread may uncover important tumor biology and/or yield promising therapeutic insights. Here, we investigated the role of circular RNAs (circRNA) in metastasis, using melanoma as a model aggressive tumor. We identified silencing of cerebellar degeneration-related 1 antisense (CDR1as), a regulator of miR-7, as a hallmark of melanoma progression. CDR1as depletion results from epigenetic silencing of LINC00632, its originating long non-coding RNA (lncRNA) and promotes invasion in vitro and metastasis in vivo through a miR-7-independent, IGF2BP3-mediated mechanism. Moreover, CDR1as levels reflect cellular states associated with distinct therapeutic responses. Our study reveals functional, prognostic, and predictive roles for CDR1as and expose circRNAs as key players in metastasis.
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http://dx.doi.org/10.1016/j.ccell.2019.12.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184928PMC
January 2020

Discovery of a chemical probe for PRDM9.

Nat Commun 2019 12 17;10(1):5759. Epub 2019 Dec 17.

Structural Genomics Consortium, University of Toronto, Toronto, ON, M5G 1L7, Canada.

PRDM9 is a PR domain containing protein which trimethylates histone 3 on lysine 4 and 36. Its normal expression is restricted to germ cells and attenuation of its activity results in altered meiotic gene transcription, impairment of double-stranded breaks and pairing between homologous chromosomes. There is growing evidence for a role of aberrant expression of PRDM9 in oncogenesis and genome instability. Here we report the discovery of MRK-740, a potent (IC: 80 ± 16 nM), selective and cell-active PRDM9 inhibitor (Chemical Probe). MRK-740 binds in the substrate-binding pocket, with unusually extensive interactions with the cofactor S-adenosylmethionine (SAM), conferring SAM-dependent substrate-competitive inhibition. In cells, MRK-740 specifically and directly inhibits H3K4 methylation at endogenous PRDM9 target loci, whereas the closely related inactive control compound, MRK-740-NC, does not. The discovery of MRK-740 as a chemical probe for the PRDM subfamily of methyltransferases highlights the potential for exploiting SAM in targeting SAM-dependent methyltransferases.
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http://dx.doi.org/10.1038/s41467-019-13652-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6917776PMC
December 2019

Discovery of a first-in-class EZH2 selective degrader.

Nat Chem Biol 2020 02 9;16(2):214-222. Epub 2019 Dec 9.

Mount Sinai Center for Therapeutics Discovery, Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

The enhancer of zeste homolog 2 (EZH2) is the main enzymatic subunit of the PRC2 complex, which catalyzes trimethylation of histone H3 lysine 27 (H3K27me3) to promote transcriptional silencing. EZH2 is overexpressed in multiple types of cancer including triple-negative breast cancer (TNBC), and high expression levels correlate with poor prognosis. Several EZH2 inhibitors, which inhibit the methyltransferase activity of EZH2, have shown promise in treating sarcoma and follicular lymphoma in clinics. However, EZH2 inhibitors are ineffective at blocking proliferation of TNBC cells, even though they effectively reduce the H3K27me3 mark. Using a hydrophobic tagging approach, we generated MS1943, a first-in-class EZH2 selective degrader that effectively reduces EZH2 levels in cells. Importantly, MS1943 has a profound cytotoxic effect in multiple TNBC cells, while sparing normal cells, and is efficacious in vivo, suggesting that pharmacologic degradation of EZH2 can be advantageous for treating the cancers that are dependent on EZH2.
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http://dx.doi.org/10.1038/s41589-019-0421-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6982609PMC
February 2020

Identification of Quinolinols as Activators of TEAD-Dependent Transcription.

ACS Chem Biol 2019 12 2;14(12):2909-2921. Epub 2019 Dec 2.

Department of Multi-Modal Molecular (M3) Biology , Institute of Molecular and Cell Biology , 61 Biopolis Drive , 138673 Singapore.

The transcriptional co-regulators YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif) are the vertebrate downstream effectors of the Hippo signaling pathway that controls various physiological and pathological processes. YAP and TAZ pair with the TEAD (TEA domain) family of transcription factors to initiate transcription. We previously identified a tractable pocket in TEADs, which has been physiologically shown to bind palmitate. Herein, a TEAD-palmitate interaction screen was developed to select small molecules occupying the palmitate-binding pocket (PBP) of TEADs. We show that quinolinols were TEAD-binding compounds that augment YAP/TAZ-TEAD activity, which was verified using TEAD reporter assay, RT-qPCR, and RNA-Seq analyses. Structure-activity relationship investigations uncovered the quinolinol substituents that are necessary for TEAD activation. We reveal a novel mechanism where quinolinols stabilize YAP/TAZ protein levels by occupying the PBP. The enhancement of YAP activity by quinolinols accelerates the wound closure in a mouse wound-healing model. Although small molecules that occupy the PBP have been shown to inhibit YAP/TAZ-TEAD activity, leveraging PBP to activate TEADs is a novel approach.
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http://dx.doi.org/10.1021/acschembio.9b00786DOI Listing
December 2019

The metabolic sensor PASK is a histone 3 kinase that also regulates H3K4 methylation by associating with H3K4 MLL2 methyltransferase complex.

Nucleic Acids Res 2019 11;47(19):10086-10103

Department of Anatomy, College of Medicine and Health Sciences, United Arab Emirates University, PO Box 17666, Al Ain, Abu Dhabi, UAE.

The metabolic sensor Per-Arnt-Sim (Pas) domain-containing serine/threonine kinase (PASK) is expressed predominantly in the cytoplasm of different cell types, although a small percentage is also expressed in the nucleus. Herein, we show that the nuclear PASK associates with the mammalian H3K4 MLL2 methyltransferase complex and enhances H3K4 di- and tri-methylation. We also show that PASK is a histone kinase that phosphorylates H3 at T3, T6, S10 and T11. Taken together, these results suggest that PASK regulates two different H3 tail modifications involving H3K4 methylation and H3 phosphorylation. Using muscle satellite cell differentiation and functional analysis after loss or gain of Pask expression using the CRISPR/Cas9 system, we provide evidence that some of the regulatory functions of PASK during development and differentiation may occur through the regulation of these histone modifications.
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http://dx.doi.org/10.1093/nar/gkz786DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821284PMC
November 2019

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

Cancer Cell 2019 08;36(2):194-209.e9

Ontario Cancer Institute/Princess Margaret Hospital, Toronto, ON M5G 2M9, Canada.

Cancer-associated mutations in genes encoding RNA splicing factors (SFs) commonly occur in leukemias, as well as in a variety of solid tumors, and confer dependence on wild-type splicing. These observations have led to clinical efforts to directly inhibit the spliceosome in patients with refractory leukemias. Here, we identify that inhibiting symmetric or asymmetric dimethylation of arginine, mediated by PRMT5 and type I protein arginine methyltransferases (PRMTs), respectively, reduces splicing fidelity and results in preferential killing of SF-mutant leukemias over wild-type counterparts. These data identify genetic subsets of cancer most likely to respond to PRMT inhibition, synergistic effects of combined PRMT5 and type I PRMT inhibition, and a mechanistic basis for the therapeutic efficacy of PRMT inhibition in cancer.
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http://dx.doi.org/10.1016/j.ccell.2019.07.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7194031PMC
August 2019

Author Correction: The regulation, functions and clinical relevance of arginine methylation.

Nat Rev Mol Cell Biol 2019 Sep;20(9):567

Segal Cancer Center, Lady Davis Institute for Medical Research, SMBD Jewish General Hospital and Gerald Bronfman Department of Oncology and Departments of Medicine, Biochemistry and Human Genetics, McGill University, Montréal, Québec, Canada.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41580-019-0166-7DOI Listing
September 2019

The role of GRHL2 and epigenetic remodeling in epithelial-mesenchymal plasticity in ovarian cancer cells.

Commun Biol 2019 24;2:272. Epub 2019 Jul 24.

1Cancer Science Institute of Singapore, National University of Singapore, Singapore, 117599 Singapore.

Cancer cells exhibit phenotypic plasticity during epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) involving intermediate states. To study genome-wide epigenetic remodeling associated with EMT plasticity, we integrate the analyses of DNA methylation, ChIP-sequencing of five histone marks (H3K4me1, H3K4me3, H3K27Ac, H3K27me3 and H3K9me3) and transcriptome profiling performed on ovarian cancer cells with different epithelial/mesenchymal states and on a knockdown model of EMT suppressor Grainyhead-like 2 (GRHL2). We have identified differentially methylated CpG sites associated with EMT, found at promoters of epithelial genes and GRHL2 binding sites. GRHL2 knockdown results in CpG methylation gain and nucleosomal remodeling (reduction in permissive marks H3K4me3 and H3K27ac; elevated repressive mark H3K27me3), resembling the changes observed across progressive EMT states. Epigenetic-modifying agents such as 5-azacitidine, GSK126 and mocetinostat further reveal cell state-dependent plasticity upon GRHL2 overexpression. Overall, we demonstrate that epithelial genes are subject to epigenetic control during intermediate phases of EMT/MET involving GRHL2.
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http://dx.doi.org/10.1038/s42003-019-0506-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6656769PMC
May 2020

The regulation, functions and clinical relevance of arginine methylation.

Nat Rev Mol Cell Biol 2019 10 26;20(10):642-657. Epub 2019 Jul 26.

Segal Cancer Center, Lady Davis Institute for Medical Research, SMBD Jewish General Hospital and Gerald Bronfman Department of Oncology and Departments of Medicine, Biochemistry and Human Genetics, McGill University, Montréal, Québec, Canada.

Methylation of arginine residues by protein arginine methyltransferases (PRMTs) is involved in the regulation of fundamental cellular processes, including transcription, RNA processing, signal transduction cascades, the DNA damage response and liquid-liquid phase separation. Recent studies have provided considerable advances in the development of experimental tools and the identification of clinically relevant PRMT inhibitors. In this review, we discuss the regulation of PRMTs, their various cellular roles and the clinical relevance of PRMT inhibitors for the therapy of neurodegenerative diseases and cancer.
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http://dx.doi.org/10.1038/s41580-019-0155-xDOI Listing
October 2019
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