CV Pier Lorenzo Puri (inglés)

NAME: Pier Lorenzo Puri, M.D.

CURRENT POSITION: Professor

PROGRAM: Development, Aging and Regeneration

CENTER:Sanford Children’ Health Center

DATE OF HIRE OR LAST PROMOTION:Date of hire Sept 2004; Last promotionJune 2015

EDUCATION:

University of Rome La Sapienza      Undergrad      1986-1991      Medicine & Surgery

University of Rome La Sapienza      M.D.                1991                Medicine & Surgery

University of Rome La Sapienza      Postgrad &      1992-1997      Internal Medicine

Clinical Internship

University of Rome La Sapienza      Residency      1997                Internal Medicine

Univ. of California, San Diego           Postdoctoral   1997-2000      Department of Biology

PROFESSIONAL EXPERIENCE:

2001-2004      Staff Scientist, Peptide Biology Laboratory, Salk Institute, La Jolla, CA

2002-2007      Assistant Telethon Scientist, Dulbecco Telethon Institute (DTI), Roma, Italy

2004-2009      Assistant Professor, Burnham Institute for Medical Research, La Jolla, CA

2007-2012      Associate Telethon Scientist, Dulbecco Telethon Institute (DTI), Roma, Italy

2008-2014      Adjunct Professor of Pediatrics, UCSD, La Jolla, CA

2008-present  Associate Scientist Sanford Children’ Health Center

2010-present Associate Professor Sanford-Burnham Medical Research Institute

2012                Senior Telethon Scientist, Dulbecco Telethon Institute (DTI), Roma, Italy (position declined)

2012-present  Lab Director, Fondazione Santa Lucia, Roma, Italy

2015                Professor Sanford-Burnham Medical Research Institute

HONORS AND AWARDS:

1992-1997      Recipient of a CEE Fellowship for a Postgraduate School of Internal Medicine

1998-2000      Recipient of a Human Frontier Fellowship

2000-2003      MDA Development Grant

2002-2004      American Heart Association Beginning Grant-in-Aid

OVERVIEW OF RESEARCH ACTIVITIES

 Recent research:

 General introduction

My lab investigatesthe molecular and epigenetic basisof skeletal muscle development and regeneration in health and disease, with the final goal of translating this knowledge into pre-clinical studies and eventually clinical trials for neuromuscular diseases.The projects listed below have been funded by national and international agencies (as acknowledged for each project)and successfully renewed during the last 5 years. Collectively they have advanced our understanding on how skeletal muscles regenerate following physical injury or in disease conditions.

One research line has led to the identification of the functional interactions between twokey cellular components (i.e. muscle stem satellite cells – MuSCs; and fibroadipogenic progenitors – FAPs) of the regeneration machinery activated in damaged muscles. We have elucidated themolecular and epigenetic networks that regulate two alternative functionalinteractions betweenthese cell types resulting incompensatory regeneration or pathogenic degeneration, whichtypically underlie early and late stages of muscular disease progression, respectively. Importantly, these studies have inspiredthe rationale for therapeutic interventions in muscular dystrophies (MD), and in particular Duchenne Muscular Dystrophy (DMD), that culminated with the pre-clinical development of a pharmacological treatment with epigenetic drugs (HDAC inhibitors – HDACi) that has untimately been translated into a clinical trial with DMD boys.

In a parallel effort, we have studied the epigenetic determinants of hESC commitment to skeletal myogenesis, by investigated the well known hESC resistance to direct conversion into skeletal muscle upon ectopic expression of MyoD, which otherwise reprograms somatic cells into the skeletal muscle lineage. Thisbottleneck has prevented the generation of “in dish” models of human neuromuscular diseases by hiPSC, at variance with the majority of other cell types. We have discovered that hESC resistance to myogenic conversion is caused by the lack of expression of one structural component of the SWI/SNF chromatin remodelling complex – BAF60C – which is specifically induced in embryoid bodies We showed that ectopic expression of BAF60C enables MyoD-mediated activation of skeletal myogenesis in hESCs and hiPSCs.This discovery eventually inspired a protocol for direct generation of 3D contractile myospheres from hESCs and hiPSCs, providing the first evidence of“in dish” disease models of neuromuscular disorders, such as DMD.

Project 1-Muscle regeneration:interplay between adult stem cells for compensatory or pathogenic regeneration, and identification of targets for pharmacological treatment of neuromuscular disorders by epigenetic drugs(funded by NIHgrants 5 R01 AR052779-09and P30AR061303-03; MDA; Sanford Association Member Award).

1a – p38-BAF60C/SWI/SNF and p38-PRC2 signaling in satellite cells

We have revealed the mechanism by which muscle environmental cues are converted into epigenetic changes that regulate gene expression in healthy and diseased muscles, via extracellular signal-activated kinase targeting of chromatin-modifying enzymes. We have shown that in regeneration-activated satellite cells,p38 alpha kinase simoultaneously directs both SWI/SNF-mediated chromatin remodeling at muscle loci, via phosphorylation of BAF60C (Simone et al. 2004 Nat Genetics; Serra et al. 2007 Mol Cell; Forcales et al. 2012 EMBO J.) and Polycomb Repressory Complex (PCR2)-mediated repression of stemness and cell cycle genes, via phosphorylation of the meyhyltransferase Ezh2 (Palacios et al. 2010 Cell Stem Cell). Further investigation revealed that the distinct SWI/SNF complexes, distinguished by the alternative incorporation of the ATPases Brg1 and Brm, coordinate repression of cell cycle genes and activation of muscle genes in myogenic progenitors at sequential stages of skeletal myogenesis (Albini et al. J Cell Biol. in revision).

1b – HDAC-miR-BAF60 network in FAPs and stage dependent effect of HDACi in DMD

Our previousdiscovery that HDACicounter disease progression in the mouse model of DMD (mdx mice) (Minetti et al. Nature Medicine 2006) and that HDAC2 activity is regulated by dystrophin-activated nNOS signaling (Colussi et al. PNAS 2008), haverevealed a previously unrecognized link between constitutive activation of HDAC2 and alterations of the epigenetic landscape in dystrophic muscles. Our recent studies showed that HDACi promote compensatory regeneration and prevent fibro-adipogenic degeneration in mdx mice at early stages of diseases, by targeting a population of muscle interstitial cells – FAPs (Mozzetta et al. EMBO Mol Med. 2013). We have identified a novel network by which HDAC-regulated expression of myomiRs controls the alternative incorporation of BAF60 variants into SWI/SNF complexes that direct the ability of FAPs to adopt a pro-myogenic or fibro-adipogenic phenotype (reviewed in Puri and Mercola genes & Dev. 2013). HDACi promote expression of myomiRs and formation of BAF60C-based SWI/SNF complex that directs the pro-myogenic phenotype in FAPs of muscle from young, but not old, mdx mice, and this explains why the beneficial effect of HDACi is restricted to early stages of disease (Saccone et al. Genes & Dev. 2014). Importantly, this work provided the foundation for pre-clinical studies (Consalvi et al. Mol Med 2013) that led to the ongoing clinical trial with the HDACi Givinostat in DMD boys, and provided the criteria for the “early stage” patient enrollment to the trial. Of note, we are currently analyzing the patient’s muscle biopsies (obtained before and after the treatment) to investigate whether changes in HDAC-miR-BAF60 network detected in FAPs can provide molecular biomarkers of disease progression andcan accurately monitor treatment efficacy.

 Project 2 – Generation of hESC- and hiPSC-derived 3D contractile myospheres, (funded by NIH grant 2 R01 AR056712-06)

2a – Discovery of BAF60C as key regulator of skeletal and cardiac myogenesis in hESCs

We have discovered that the SWI/SNF component BAF60C is the limiting factor for activation of skeletal and cardiac myogenesis in hESCs, by directing tissue-specifc transcription factor (TF)-binding and chromatin remodelling, and thereby promoting the selective transcription at skeletal and cardiac loci. The ability to activate skeletal myogenesis or cardiogenesis is determined by the expression of skeletal muscle-specific TFs (such as MyoD) (Albini et al. Cell Report 2013) or cardiac-restricted TFs (such as GATA4, Nkx2.5)in combination with cardiogenic signals (Cai et al. Genes & Dev 2013).

2b–Setting a new protocol for hESC- and hiPSC-derived 3D contractile myospheres

Based on these studies, we have recently established a protocol of hESC-derived 3D contractile myospheres (Albini et al. 2013; see also Albini et al. 2014 JoVE) that offers the unprecedented opportunity to dissect and analyze the epigenetic dynamics that underlie the formation of skeletal muscles and to identify changes in the epigenome induced by contractile activity in healthy vs dystrophin-deficient myofibers. When applied to human induced pluripotent stem cells (hiPSCs) derived from patients affected by muscular dystrophies, this technology has the potential to unravel the relationship between the consequences of the genetic defects (i.e. absence of dystrophin in DMD patients) and pathological alterations of the epigenetic landscape that contribute to DMD pathogenesis on a patient-specific basis.

 Other Relevant Projects

Project 3 –TBP vs TBP2-activated transcription of muscle gene expression in muscle regeneration.

This project originally stemmed from our interest in determining the relationship between SWI/SNF-mediated nucleosomal depletion at the transcription start site (TSS) of muscle genes and the basal transcriptional machinery. After a breackthrough publication from Tijan labreporting on the switching of the core transcription machinery, with TBP2 replacing the canonical TBP/TFIID complex to activate muscle gene transcription in myogenic progenitors induced to differentiate (Deato et al. Genes & Dev 2007), we analyzed muscle development and regeneration in TBP2 null mice. The intact developmental myogenesis and regeneration potential of these mice prompted a re-analysis of the role of TBP vs TBP2 in skeletal myogenesis, eventually leading to the discovery that TBP2 is not expressed in muscle cells and does not play any role in muscle gene transcription, which is instead entirely dependant on canonical TBP/TFIID complex. This manuscript, because of the controversy with previous Tijan  publication, is currently under analysis of Genes & Dev editorial staff.

Project 4 – DNA damage signaling to coordinate gene expression and repair through ABL-mediated phosphorylation of transcriptional activators and co-activators.

Following our long-standing interest in the relationship between DNA damage, repair, transcription and maintenance of genome stability in terminally differentiated tissues (such as skeletal muscle), we have continued to investigate the ABL-mediated signaling to MyoD that transiently prevents muscle gene expression in myoblasts exposed to genotoxic stress (Puri et al. Nature Genetics 2002; Innocenzi et al. 2011). Our recent studies have revealed that ABL-mediated phosphorylation of MyoD represses its transcriptional activity, while promoting a previously unrecognized DNA repair function of MyoD (Simonatto et al. Cell Death and Diff. 2013). This finding suggests that ABL signaling to MyoD coordinates the timing of DNA repair and muscle gene expression in muscle progenitors exposed to DNA damage, presumably to protect the genome integrity, by avoiding formation of differentiated progenies with unrepaired DNA lesions.

In a parallel study, we have identified the histone acetyltransferase (HAT) p300 as another direct target of DNA damage-activated ABL tyrosine kinase. ABL-mediated p300 tyrosine phosphorylation promotes HAT activity,and ChIP-seq data show that in fibroblasts exposed to genotoxins, ABL-phosphorylated p300 is redistributed to the regulatory regions of apoptotic genes. This study is revealing a nuclear signaling that determines how the promiscuous transcriptional co-activator p300 activates a specific DNA damage response – i.e. apoptosis.

 Future research:

Our future research will continue to investigate the biological and epigenetic basis of skeletal myogenesis and muscle regeneration, by taking the challenge of extending our studies from mouse models of diseases to human patients. Most of the experiments will be set toward this attempt, in order to move forward the field of skeletal myogenesis to an unprecedented dimension that is mandatory tofill the current gap of knowledge between the existing animal experimental models of DMD and the human disease. In parallel, we will continue to use mouse models of disease and molecular/epigenetic analysis of mouse-derived primary cells, with the ultimate goal of generating the initial knowledge that will be applied to human cells. This transition will improve our understanding of the molecular pathogenesisofhuman dystrophies, and will help to identify novel treatments, their translation into clinical trials and the identification of relieable biomarkers of disease progression and treatment efficacy.This operation is made possible by the availability of patient muscle biopsies, by our ability to isolate different muscle-derived cellular populations and analyze their biologiy and epigenome, as well as byusing hiPSC patient-derived contractile 3D myospheres as “in dish” disease model of neuromuscular diseases.

According to this purpose, we have started to request and receive funding based on proposals dealing with human-related studies (NIH grant 2 R01 AR056712-06) and will continue on this direction with applications to NIH, HHMI, DOD and CIRM agencies.

Below, I provide some details on how we intend to further extend our current projects toward human patients.

 Project 1 -Muscle regeneration: interplay between adult stem cells for compensatory or pathogenic regeneration, and identification of targets for pharmacological treatment of neuromuscular disorders by epigenetic drugs.

We are currently using genome-wide gene expression and chromatin analysis coupled toNextGenerationSequencing (NGS) and integrated with bio-informatic analysis, to identifiy and elucidate key regulatory networks that control gene expressionin muscle-derived cell populations from mouse models of human muscular diseases, with the ultimate goal of transferring this knowledge to human patients.This approach is based on FACS-mediated isolation of cell types that contribute to muscle regeneration, such as MuSCs, FAPs and macrophages, in mouse models of muscular dystrophies, and is finalized toidentify cell type-specific transcriptional profiles (by RNA-seqanalysis) and epigenetic/chromatin transitions (by ChIP-seq and MNase- or FAIRE-seq analysis) during disease progression and treatement. Through an integrated bio-informatic analysis, this approach has already revealed functional networks between these cell types, thatare implicated in disease progression and response to HDACi. For instance, we found that FAP- and macrophage-derived inflammatory cytokines elicit the STAT3-signalling in MuSCs. This finding prompted our recent collaboration with Dr. Sacco(Tierney et al. Nature Medicine 2014) and will be further exploited to elucidate the relationship betweenSTAT3 and Notch pathways and the downstream chromatin events mediated by PRC2 and Brg1-or Brm-based SWI/SNF that has been recently described by Crabtree lab (Ho et al. Nat Cell Biol. 2011; Kadam and Emerson mol Cell 2003) and is predicted to regulate gene expression in quiescent and activated MuSCs (Conboy et al. Nature 2005; Juan et al. Genes & Dev 2011).While this approach has the potential to unveil functional interactions between the epigenomes of distinct cell types, we are aware that it will require some essential improvement in protocols and analysis. Among them, we will continue to focus on technologies that allow gene expression and chromatin analysis of limited number of cells. In this regard, we have started a gene expression analysis of muscle-derived FAPs at single cell level that should enable future improvement in the analysis of patient-derived muscle biopsiesand could reveal novel insight into DMD pathogenesis. This project has been supported with funds awarded by CIRM fellowship and P30 NIH pilot grant to Dr Malecova – post-doc in my lab. Likewise, we will continue to improve our potential of bio-informatic analysis, with the recruitment of anoher bio-informatician with a specific expertise in system biology that will integrate the expertise in NGS data analysis of Dr Gatto (current post-doc in my lab).

Project 2 – Generation of hESC- and hiPSC-derived 3D contractile myospheres

In this project we plan to apply our studies to human subjects by taking advantage from patient hiPSC-derived 3D myospheres. To this purpose we have established a consortium for the study of rare (orphan) muscular disease through a “strategic” SanfordBurnham bi-costal synergy with Dr Wood and Florida hospitals. This operation is based on the generation in Dr Wood lab of hiPSCs from patient derived fibroblasts received by the hospitals. Moreover, I plan to further improve this “in dish”disease models by adding two currently missing key components of skeletal muscles – the extracellular matrix (ECM) and neural innervation. To this purpose we have started collaboration with Dr Engler (UCSD) to devise biomaterials that can recapitulate functional and physical features of ECM and will be used to encapsulate myospheres. I have also recently recruited a new post-doc that will be dedicated to setting the optimal conditions for co-culture of hiPSC-derived motoneurons and myospheres in order to form neuro-muscular junctions(NMJ) that will allowmyospheres contraction from neural stimulation.This setting will be particularly suitable to understand the effect of contraction on dystrophin-deficient myospheres, at different levels. In addition to the structural and metabolic responses, I am particularly interested in evaluating the effect of contraction on the epigenome and nuclear architecture of myospheres from generated from DMD hiPSCs. To this purpose, we have an ongoing collaboration with Bing Ren lab (USCD), by which we are exploiting Hi-C technology to map long-distance interactions between regulatory elements of the genome.

Other Relevant Projects

Will will continue our studies on DNA damage-activated ABL signaling to MyoD and p300. The next step for this project will be to obtain significant funding to understand how ABL-mediated phosphorylation changes MyoD and p300 interactions with regulatory proteins.This approach will be based on mass-spectometry studies, in collaboration with the SBMRI proteomic facility, and should provide biochemical and mechanistic insights into the phosphorylation-mediated functional changes of these proteins.  As a long term goal of this project, we would like to understand whetheran altered DNA damage-activated repair/transcription and the ensuing genetic instability can explain the relationship between cellular senescence and the age-related impairement of muscle regeneration potential.