HDAC inhibitors as pharmacological treatment for Duchenne muscular dystrophy: a discovery journey from bench to patients.

Earlier evidence that targeting the balance between histone acetyltransferases (HATs) and deacetylases (HDACs), through exposure to HDAC inhibitors (HDACis), could enhance skeletal myogenesis, prompted interest in using HDACis to promote muscle regeneration. Further identification of constitutive HDAC activation in dystrophin-deficient muscles, caused by dysregulated nitric oxide (NO) signaling, provided the rationale for HDACi-based therapeutic interventions for Duchenne muscular dystrophy (DMD). In this review, we describe the molecular, preclinical, and clinical evidence supporting the efficacy of HDACis in countering disease progression by targeting pathogenic networks of gene expression in multiple muscle-resident cell types of patients with DMD. Given that givinostat is paving the way for HDACi-based interventions in DMD, next-generation HDACis with optimized therapeutic profiles and efficacy could be also explored for synergistic combinations with other therapeutic strategies.

CRIPTO-based micro-heterogeneity of mouse muscle satellite cells enables adaptive response to regenerative microenvironment.

Skeletal muscle repair relies on heterogeneous populations of satellite cells (SCs). The mechanisms that regulate SC homeostasis and state transition during activation are currently unknown. Here, we investigated the emerging role of non-genetic micro-heterogeneity, i.e., intrinsic cell-to-cell variability of a population, in this process. We demonstrate that micro-heterogeneity of the membrane protein CRIPTO in mouse-activated SCs (ASCs) identifies metastable cell states that allow a rapid response of the population to environmental changes. Mechanistically, CRIPTO micro-heterogeneity is generated and maintained through a process of intracellular trafficking coupled with active shedding of CRIPTO from the plasma membrane. Irreversible perturbation of CRIPTO micro-heterogeneity affects the balance of proliferation, self-renewal, and myogenic commitment in ASCs, resulting in increased self-renewal in vivo. Our findings demonstrate that CRIPTO micro-heterogeneity regulates the adaptative response of ASCs to microenvironmental changes, providing insights into the role of intrinsic heterogeneity in preserving stem cell population diversity during tissue repair.

EV-mediated promotion of myogenic differentiation is dependent on dose, collection medium, and isolation method.

Extracellular vesicles (EVs) have been implicated in the regulation of myogenic differentiation. C2C12 murine myoblast differentiation was reduced following treatment with GW4869 or heparin (to inhibit exosome biogenesis and EV uptake, respectively). Conversely, treatment with C2C12 myotube-conditioned medium enhanced myogenic differentiation. Ultrafiltration-size exclusion liquid chromatography (UF-SEC) was used to isolate EVs and non-EV extracellular protein in parallel from C2C12 myoblast- and myotube-conditioned medium. UF-SEC-purified EVs promoted myogenic differentiation at low doses (≤2 × 108 particles/mL) and were inhibitory at the highest dose tested (2 × 1011 particles/mL). Conversely, extracellular protein fractions had no effect on myogenic differentiation. While the transfer of muscle-enriched miRNAs (myomiRs) has been proposed to mediate the pro-myogenic effects of EVs, we observed that they are scarce in EVs (e.g., 1 copy of miR-133a-3p per 195 EVs). Furthermore, we observed pro-myogenic effects with undifferentiated myoblast-derived EVs, in which myomiR concentrations are even lower, suggestive of a myomiR-independent mechanism underlying the observed pro-myogenic effects. During these investigations we identified technical factors with profound confounding effects on myogenic differentiation. Specifically, co-purification of insulin (a component of Opti-MEM) in non-EV LC fractions and polymer precipitated EV preparations. These findings provide further evidence that polymer-based precipitation techniques should be avoided in EV research.

Muscle denervation promotes functional interactions between glial and mesenchymal cells through NGFR and NGF.

We performed scRNA-seq/snATAC-seq of skeletal muscles post sciatic nerve transection to delineate cell type-specific patterns of gene expression/chromatin accessibility at different time points post-denervation. Unlike myotrauma, denervation selectively activates glial cells and Thy1/CD90-expressing mesenchymal cells. Glial cells expressed Ngf receptor (Ngfr) and were located near neuromuscular junctions (NMJs), close to Thy1/CD90-expressing cells, which provided the main cellular source of NGF post-denervation. Functional communication between these cells was mediated by NGF/NGFR, as either recombinant NGF or co-culture with Thy1/CD90-expressing cells could increase glial cell number ex vivo. Pseudo-time analysis in glial cells revealed an initial bifurcation into processes related to either cellular de-differentiation/commitment to specialized cell types (e.g., Schwann cells), or failure to promote nerve regeneration, leading to extracellular matrix remodeling toward fibrosis. Thus, interactions between denervation-activated Thy1/CD90-expressing and glial cells represent an early abortive process toward NMJs repair, ensued by the conversion of denervated muscles into an environment hostile for NMJ repair.

LncRNA EPR regulates intestinal mucus production and protects against inflammation and tumorigenesis.

The long non-coding RNA EPR is expressed in epithelial tissues, binds to chromatin and controls distinct biological activities in mouse mammary gland cells. Because of its high expression in the intestine, in this study we have generated a colon-specific conditional targeted deletion (EPR cKO) to evaluate EPR in vivo functions in mice. EPR cKO mice display epithelium hyperproliferation, impaired mucus production and secretion, as well as inflammatory infiltration in the proximal portion of the large intestine. RNA sequencing analysis reveals a rearrangement of the colon crypt transcriptome with strong reduction of goblet cell-specific factors including those involved in the synthesis, assembly, transport and control of mucus proteins. Further, colon mucosa integrity and permeability are impaired in EPR cKO mice, and this results in higher susceptibility to dextran sodium sulfate (DSS)-induced colitis and tumor formation. Human EPR is down-regulated in human cancer cell lines as well as in human cancers, and overexpression of EPR in a colon cancer cell line results in enhanced expression of pro-apoptotic genes. Mechanistically, we show that EPR directly interacts with select genes involved in mucus metabolism whose expression is reduced in EPR cKO mice and that EPR deletion causes tridimensional chromatin organization changes.

Conserved transcription factors promote cell fate stability and restrict reprogramming potential in differentiated cells.

Defining the mechanisms safeguarding cell fate identity in differentiated cells is crucial to improve 1) - our understanding of how differentiation is maintained in healthy tissues or altered in a disease state, and 2) - our ability to use cell fate reprogramming for regenerative purposes. Here, using a genome-wide transcription factor screen followed by validation steps in a variety of reprogramming assays (cardiac, neural and iPSC in fibroblasts and endothelial cells), we identified a set of four transcription factors (ATF7IP, JUNB, SP7, and ZNF207 [AJSZ]) that robustly opposes cell fate reprogramming in both lineage and cell type independent manners. Mechanistically, our integrated multi-omics approach (ChIP, ATAC and RNA-seq) revealed that AJSZ oppose cell fate reprogramming by 1) - maintaining chromatin enriched for reprogramming TF motifs in a closed state and 2) - downregulating genes required for reprogramming. Finally, KD of AJSZ in combination with MGT overexpression, significantly reduced scar size and improved heart function by 50%, as compared to MGT alone post-myocardial infarction. Collectively, our study suggests that inhibition of barrier to reprogramming mechanisms represents a promising therapeutic avenue to improve adult organ function post-injury.

Smarcd3 is an epigenetic modulator of the metabolic landscape in pancreatic ductal adenocarcinoma.

Pancreatic cancer is characterized by extensive resistance to conventional therapies, making clinical management a challenge. Here we map the epigenetic dependencies of cancer stem cells, cells that preferentially evade therapy and drive progression, and identify SWI/SNF complex member SMARCD3 as a regulator of pancreatic cancer cells. Although SWI/SNF subunits often act as tumor suppressors, we show that SMARCD3 is amplified in cancer, enriched in pancreatic cancer stem cells and upregulated in the human disease. Diverse genetic mouse models of pancreatic cancer and stage-specific Smarcd3 deletion reveal that Smarcd3 loss preferentially impacts established tumors, improving survival especially in context of chemotherapy. Mechanistically, SMARCD3 acts with FOXA1 to control lipid and fatty acid metabolism, programs associated with therapy resistance and poor prognosis in cancer. These data identify SMARCD3 as an epigenetic modulator responsible for establishing the metabolic landscape in aggressive pancreatic cancer cells and a potential target for new therapies.

A post-middle-age crisis for CD47 and THBS1 that turns into a vicious cycle.

In this issue of Cell Stem Cell, Porpiglia et al.1 report on alterations in CD47 and THBS1 expression and function in aged muscle stem cells that disrupt their regeneration capacity. Targeting THBS1-CD47 cross-signaling is sufficient to reverse sarcopenia and restore muscle mass and function in aged mice.

SerpinE1 drives a cell-autonomous pathogenic signaling in Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare, fatal disease caused by Lamin A mutation, leading to altered nuclear architecture, loss of peripheral heterochromatin and deregulated gene expression. HGPS patients eventually die by coronary artery disease and cardiovascular alterations. Yet, how deregulated transcriptional networks at the cellular level impact on the systemic disease phenotype is currently unclear. A genome-wide analysis of gene expression in cultures of primary HGPS fibroblasts identified SerpinE1, also known as Plasminogen Activator Inhibitor (PAI-1), as central gene that propels a cell-autonomous pathogenic signaling from the altered nuclear lamina. Indeed, siRNA-mediated downregulation and pharmacological inhibition of SerpinE1 by TM5441 could revert key pathological features of HGPS in patient-derived fibroblasts, including re-activation of cell cycle progression, reduced DNA damage signaling, decreased expression of pro-fibrotic genes and recovery of mitochondrial defects. These effects were accompanied by the correction of nuclear abnormalities. These data point to SerpinE1 as a novel potential effector and target for therapeutic interventions in HGPS pathogenesis.

LncRNA EPR-induced METTL7A1 modulates target gene translation.

EPR is a long non-coding RNA (lncRNA) that controls cell proliferation in mammary gland cells by regulating gene transcription. Here, we report on Mettl7a1 as a direct target of EPR. We show that EPR induces Mettl7a1 transcription by rewiring three-dimensional chromatin interactions at the Mettl7a1 locus. Our data indicate that METTL7A1 contributes to EPR-dependent inhibition of TGF-ß signaling. METTL7A1 is absent in tumorigenic murine mammary gland cells and its human ortholog (METTL7A) is downregulated in breast cancers. Importantly, re-expression of METTL7A1 in 4T1 tumorigenic cells attenuates their transformation potential, with the putative methyltransferase activity of METTL7A1 being dispensable for its biological functions. We found that METTL7A1 localizes in the cytoplasm whereby it interacts with factors implicated in the early steps of mRNA translation, associates with ribosomes, and affects the levels of target proteins without altering mRNA abundance. Overall, our data indicates that METTL7A1-a transcriptional target of EPR-modulates translation of select transcripts.

Determinants of epigenetic resistance to HDAC inhibitors in dystrophic fibro-adipogenic progenitors.

Pharmacological treatment of Duchenne muscular dystrophy (DMD) with histone deacetylase inhibitors (HDACi) is currently being tested in clinical trials; however, pre-clinical studies indicated that the beneficial effects of HDACi are restricted to early stages of disease. We show that FAPs from late-stage mdx mice exhibit aberrant HDAC activity and genome-wide alterations of histone acetylation that are not fully reversed by HDACi. In particular, combinatorial H3K27 and/or H3K9/14 hypo-acetylation at promoters of genes required for cell cycle activation and progression, as well as glycolysis, are associated with their downregulation in late-stage mdx FAPs. These alterations could not be reversed by HDACi, due to a general resistance to HDACi-induced H3K9/14 hyperacetylation. Conversely, H3K9/14 hyper-acetylation at promoters of Senescence Associated Secretory Phenotype (SASP) genes is associated with their upregulation in late-stage mdx FAPs; however, HDACi could reduce promoter acetylation and blunt SASP gene activation. These data reveal that during DMD progression FAPs develop disease-associated features reminiscent of cellular senescence, through epigenetically distinct and pharmacologically dissociable events. They also indicate that HDACi might retain anti-fibrotic effects at late stages of DMD.

Human skeletal muscle CD90+ fibro-adipogenic progenitors are associated with muscle degeneration in type 2 diabetic patients.

Type 2 diabetes mellitus (T2DM) is associated with impaired skeletal muscle function and degeneration of the skeletal muscles. However, the mechanisms underlying the degeneration are not well described in human skeletal muscle. Here we show that skeletal muscle of T2DM patients exhibit degenerative remodeling of the extracellular matrix that is associated with a selective increase of a subpopulation of fibro-adipogenic progenitors (FAPs) marked by expression of THY1 (CD90)-the FAPCD90+. We identify platelet-derived growth factor (PDGF) as a key FAP regulator, as it promotes proliferation and collagen production at the expense of adipogenesis. FAPsCD90+ display a PDGF-mimetic phenotype, with high proliferative activity, clonogenicity, and production of extracellular matrix. FAPCD90+ proliferation was reduced by in vitro treatment with metformin. Furthermore, metformin treatment reduced FAP content in T2DM patients. These data identify a PDGF-driven conversion of a subpopulation of FAPs as a key event in the fibrosis development in T2DM muscle.

MyoD induces ARTD1 and nucleoplasmic poly-ADP-ribosylation during fibroblast to myoblast transdifferentiation.

While protein ADP-ribosylation was reported to regulate differentiation and dedifferentiation, it has so far not been studied during transdifferentiation. Here, we found that MyoD-induced transdifferentiation of fibroblasts to myoblasts promotes the expression of the ADP-ribosyltransferase ARTD1. Comprehensive analysis of the genome architecture by Hi-C and RNA-seq analysis during transdifferentiation indicated that ARTD1 locally contributed to A/B compartmentalization and coregulated a subset of MyoD target genes that were however not sufficient to alter transdifferentiation. Surprisingly, the expression of ARTD1 was accompanied by the continuous synthesis of nuclear ADP ribosylation that was neither dependent on the cell cycle nor induced by DNA damage. Conversely to the H2O2-induced ADP-ribosylation, the MyoD-dependent ADP-ribosylation was not associated to chromatin but rather localized to the nucleoplasm. Together, these data describe a MyoD-induced nucleoplasmic ADP-ribosylation that is observed particularly during transdifferentiation and thus potentially expands the plethora of cellular processes associated with ADP-ribosylation.

Activation of skeletal muscle-resident glial cells upon nerve injury.

Here, we report on the identification of Itga7-expressing muscle-resident glial cells activated by loss of neuromuscular junction (NMJ) integrity. Gene expression analysis at the bulk and single-cell level revealed that these cells are distinct from Itga7-expressing muscle satellite cells. We show that a selective activation and expansion of Itga7+ glial cells occur in response to muscle nerve lesion. Upon activation, muscle glial-derived progenies expressed neurotrophic genes, including nerve growth factor receptor, which enables their isolation by FACS. We show that activated muscle glial cells also expressed genes potentially implicated in extracellular matrix remodeling at NMJs. We found that tenascin C, which was highly expressed by muscle glial cells, activated upon nerve injury and preferentially localized to NMJ. Interestingly, we observed that the activation of muscle glial cells by acute nerve injury was reversible upon NMJ repair. By contrast, in a mouse model of ALS, in which NMJ degeneration is progressive, muscle glial cells steadily increased over the course of the disease. However, they exhibited an impaired neurotrophic activity, suggesting that pathogenic activation of glial cells may be implicated in ALS progression.

HDAC inhibitors tune miRNAs in extracellular vesicles of dystrophic muscle-resident mesenchymal cells.

We show that extracellular vesicles (EVs) released by mesenchymal cells (i.e., fibro-adipogenic progenitors-FAPs) mediate microRNA (miR) transfer to muscle stem cells (MuSCs) and that exposure of dystrophic FAPs to HDAC inhibitors (HDACis) increases the intra-EV levels of a subset of miRs, which cooperatively target biological processes of therapeutic interest, including regeneration, fibrosis, and inflammation. Increased levels of miR-206 in EVs released by FAPs of muscles from Duchenne muscular dystrophy (DMD) patients or mdx mice exposed to HDACi are associated with enhanced regeneration and decreased fibrosis. Consistently, EVs from HDACi-treated dystrophic FAPs can stimulate MuSC activation and expansion ex vivo, and promote regeneration, while inhibiting fibrosis and inflammation of dystrophic muscles, upon intramuscular transplantation in mdx mice, in vivo. AntagomiR-mediated blockade of individual miRs reveals a specific requirement of miR-206 for EV-induced expansion of MuSCs and regeneration of dystrophic muscles, and indicates that cooperative activity of HDACi-induced miRs accounts for the net biological effect of these EVs. These data point to pharmacological modulation of EV content as novel strategy for therapeutic interventions in muscular dystrophies.

Revealing the Therapeutic Potential of Botulinum Neurotoxin Type A in Counteracting Paralysis and Neuropathic Pain in Spinally Injured Mice.

Botulinum neurotoxin type A (BoNT/A) is a major therapeutic agent that has been proven to be a successful treatment for different neurological disorders, with emerging novel therapeutic indications each year. BoNT/A exerts its action by blocking SNARE complex formation and vesicle release through the specific cleavage of SNAP-25 protein; the toxin is able to block the release of pro-inflammatory molecules for months after its administration. Here we demonstrate the extraordinary capacity of BoNT/A to neutralize the complete paralysis and pain insensitivity induced in a murine model of severe spinal cord injury (SCI). We show that the toxin, spinally administered within one hour from spinal trauma, exerts a long-lasting proteolytic action, up to 60 days after its administration, and induces a complete recovery of muscle and motor function. BoNT/A modulates SCI-induced neuroglia hyperreactivity, facilitating axonal restoration, and preventing secondary cells death and damage. Moreover, we demonstrate that BoNT/A affects SCI-induced neuropathic pain after moderate spinal contusion, confirming its anti-nociceptive action in this kind of pain, as well. Our results provide the intriguing and real possibility to identify in BoNT/A a therapeutic tool in counteracting SCI-induced detrimental effects. Because of the well-documented BoNT/A pharmacology, safety, and toxicity, these findings strongly encourage clinical translation.

Acute conversion of patient-derived Duchenne muscular dystrophy iPSC into myotubes reveals constitutive and inducible over-activation of TGFß-dependent pro-fibrotic signaling.

BACKGROUND: In Duchenne muscular dystrophy (DMD), DYSTROPHIN deficiency exposes myofibers to repeated cycles of contraction/degeneration, ultimately leading to muscle loss and replacement by fibrotic tissue. DMD pathology is typically exacerbated by excessive secretion of TGFß and consequent accumulation of pro-fibrotic components of the extra-cellular matrix (ECM), which in turn impairs compensatory regeneration and complicates the efficacy of therapeutic strategies. It is currently unclear whether DMD skeletal muscle fibers directly contribute to excessive activation of TGFß. Development of skeletal myofibers from DMD patient-derived induced pluripotent stem cells (iPSC), as an "in dish" model of disease, can be exploited to determine the myofiber contribution to pathogenic TGFß signaling in DMD and might provide a screening platform for the identification of anti-fibrotic interventions in DMD. METHODS: We describe a rapid and efficient method for the generation of contractile human skeletal muscle cells from DMD patient-derived hiPSC, based on the inducible expression of MyoD and BAF60C (encoded by SMARCD3 gene), using an enhanced version of piggyBac (epB) transposone vectors. DMD iPSC-derived myotubes were tested as an "in dish" disease model and exposed to environmental and mechanical cues that recapitulate salient pathological features of DMD. RESULTS: We show that DMD iPSC-derived myotubes exhibit a constitutive activation of TGFß-SMAD2/3 signaling. High-content screening (HCS)-based quantification of nuclear phosphorylated SMAD2/3 signal revealed that DMD iPSC-derived myotubes also exhibit increased activation of the TGFß-SMAD2/3 signaling following exposure to either recombinant TGFß or electrical pacing-induced contraction. CONCLUSIONS: Acute conversion of DMD patient-derived iPSC into skeletal muscles, by the ectopic expression of MyoD and BAF60C, provides a rapid and reliable protocol for an "in dish" DMD model that recapitulates key pathogenic features of disease pathology, such as the constitutive activation of the TGFß/SMAD signaling as well as the deregulated response to pathogenic stimuli, e.g., ECM-derived signals or mechanical cues. Thus, this model is suitable for the identification of new therapeutic targets in DMD patient-specific muscles.

Lack of PKCθ Promotes Regenerative Ability of Muscle Stem Cells in Chronic Muscle Injury.

Duchenne muscular dystrophy (DMD) is a genetic disease characterized by muscle wasting and chronic inflammation, leading to impaired satellite cells (SCs) function and exhaustion of their regenerative capacity. We previously showed that lack of PKCθ in mdx mice, a mouse model of DMD, reduces muscle wasting and inflammation, and improves muscle regeneration and performance at early stages of the disease. In this study, we show that muscle regeneration is boosted, and fibrosis reduced in mdxθ-/- mice, even at advanced stages of the disease. This phenotype was associated with a higher number of Pax7 positive cells in mdxθ-/- muscle compared with mdx muscle, during the progression of the disease. Moreover, the expression level of Pax7 and Notch1, the pivotal regulators of SCs self-renewal, were upregulated in SCs isolated from mdxθ-/- muscle compared with mdx derived SCs. Likewise, the expression of the Notch ligands Delta1 and Jagged1 was higher in mdxθ-/- muscle compared with mdx. The expression level of Delta1 and Jagged1 was also higher in PKCθ-/- muscle compared with WT muscle following acute injury. In addition, lack of PKCθ prolonged the survival and sustained the differentiation of transplanted myogenic progenitors. Overall, our results suggest that lack of PKCθ promotes muscle repair in dystrophic mice, supporting stem cells survival and maintenance through increased Delta-Notch signaling.