Therapeutic Targeting of PTEN in Duchenne Muscular Dystrophy

Main Text

Duchenne muscular dystrophy (DMD), caused by a mutation in the dystrophin gene, is one of the most common forms of muscular dystrophy during childhood.¹ The skeletal muscles of DMD patients undergo cycles of muscle degeneration and regeneration, inflammation, and fibrosis, leading to progressive muscle deterioration and eventually death due to cardiorespiratory failure.¹ Although gene therapy to repair or replace the defective dystrophin gene represents a promising approach, the large size of the dystrophin gene becomes a big obstacle because it exceeds the packaging limit of viral vectors. Therefore, palliative strategies are needed to treat the already damaged muscle using pharmacological agents. Chronic deficiency of dystrophin leads to dysregulated signaling in skeletal muscle, which contributes considerably to the disease progression in DMD patients. In this issue of Molecular Therapy, Yue et al.² provide initial evidence that phosphatase and tensin homolog (PTEN), a dual-specificity lipid and protein phosphatase, is dysregulated in dystrophic muscles and that targeted inhibition of PTEN using genetic or pharmacological approaches reduces skeletal muscle injury and inflammation and improves membrane repair in a preclinical mouse model of DMD.

PTEN is a potent tumor suppressor that regulates a variety of biological processes, such as cell survival, migration, proliferation, metabolism, and maintenance of genome stability.³ PTEN dephosphorylates phosphatidylinositol- 3,4,5-trisphosphate (PIP3) to phosphatidylinositol-4,5-bisphosphate (PIP2), thereby negatively regulating the activation of phosphoinositide 3-kinase (PI3K) and downstream signaling pathways.³ While activation of PI3K/Akt pathway downstream of PTEN improves survival and proliferation of many cell types, the same pathway inhibits catabolic signaling and promotes skeletal muscle growth through the activation of an mTOR complex.⁴ Studies employing genetic mouse models have shown that forced activation of this pathway causes skeletal muscle hypertrophy and ameliorates dystrophinopathy.⁵ A recent study has also suggested that the targeted deletion of PTEN during embryonic stage enhances the proliferation and differentiation of muscle progenitor cells, leading to muscle hypertrophy during post-natal growth.⁶ Interestingly, the loss of PTEN also reduces the self-renewing capacity of muscle stem cells, leading to progressive loss in their pool and diminished skeletal muscle regeneration capacity at older ages, suggesting that long-term inhibition of PTEN can have negative consequences on muscle regeneration.⁶ While the mutations of PTEN in stem cells, including embryonal rhabdomyosarcoma, have been associated with tumorigenesis, ablation of PTEN in muscle progenitor cells or myofibers does not lead to tumor formation, even in older mice.⁶

In the recent past, a few studies have investigated the regulation and potential role of the PI3K/Akt signaling pathway in dystrophic muscles. Phosphatidylinositol transfer protein-α (PITPNA) plays important roles in lipid-mediated signaling as well as in membrane traffic. Levels of PITPNA are reduced in “escaper” Golden retriever muscular dystrophy dogs, which have considerably milder symptoms, despite being dystrophin deficient. PITPNA appears to repress PTEN levels, which leads to increased phosphorylation of Akt kinase and improvement in muscle pathology and function. This was confirmed by the findings that knockdown of PITPNA improves muscle structure, survival, and swim function in dystrophin-deficient zebrafish. Similarly, small interfering RNA (siRNA)-mediated knockdown of PITPNA in cultured human DMD myoblasts represses the levels of PTEN and enhances the levels of phosphorylated Akt, which results in the formation of larger myotubes.⁷

MicroRNAs (miRs) are 21–24 nucleotide small RNAs that play a major role in post-transcriptional gene regulation in mammals. Several miRs, including miR-486, is reduced in the skeletal muscles of dystrophin-deficient mdx mice and in muscle biopsies from DMD patients. Importantly, muscle-specific overexpression of miR-486 ameliorates dystrophic phenotype and improves muscle strength in a mouse model of DMD.⁸ Of interest are the findings that miR-486 directly targets dedicator of cytokinesis 3 (DOCK3), a negative regulator of the PI3K/Akt pathway. Overexpression of miR-486 reduces the levels of PTEN and increases the levels of phosphorylated Akt, resulting in improvement of muscle pathology in dystrophin-deficient mdx mice.⁸ Another study reported that the forced activation of Staufen1, an RNA-binding protein, in the skeletal muscle of mice increases the expression of PTEN, thereby inhibiting the activity of PI3K/Akt signaling and ensuing muscle pathology.⁹ Altogether, these reports suggest that repression of PTEN levels mitigates the dystrophic phenotype, whereas its elevated levels can lead to muscle degeneration and myopathy.

An abundance of PTEN has been found to be increased in the skeletal muscle of DMD patients and the dystrophic muscle of animal models.^2,7 Yue et al.² have now directly studied the effects of muscle-specific ablation of PTEN on the severity of disease progression in the D2.B10 DMD^mdx (D2-mdx) model of DMD. Their study showed that targeted deletion of PTEN in D2-mdx remarkably improves skeletal muscle mass and strength and ameliorates various features of dystrophic muscle, such as myofiber necrosis, inflammatory response, and fibrosis.² An interesting finding of the new study was that the ablation of PTEN improved membrane integrity potentially through upregulation of the components of the sarcolemma repair machinery (Figure 1). MG53 protein is a key component of sarcolemmal membrane repair that functions through active trafficking of intracellular vesicles to damaged sites of the sarcolemma. The localization of MG53 to the sarcolemmal membrane as well as increased abundance of caveolae on the muscle membrane were found to be considerably enhanced in the skeletal muscle of Pten-deficient D2-mdx mice, further suggesting that inhibition of PTEN stimulates sarcolemma repair mechanisms.² Therefore, the beneficial effects of inhibition of PTEN in DMD could be attributed to increased activation of growth stimulatory PI3K/Akt signaling and improvement in sarcolemma repair mechanisms (Figure 1).

Figure 1.

Schematic Representation of Mode of Action of PTEN Inhibitors in Duchenne Muscular Dystrophy (DMD)

While genetic studies help in identification of novel molecular targets, eventually a drug is needed that can be used in clinical settings. Several pharmacological compounds have now been developed that target specific components of the PI3K/Akt pathway. Yue et al.² reported that VO-OHpic trihydrate (VO-OHpic), a pharmacological inhibitor of PTEN, is effective in ameliorating dystrophinopathy and improving muscle function in D2-mdx mice.² However, treating DMD patients with pharmacological inhibitors of PTEN can be quite challenging because of the potential high risk of off-target side effects in other tissues and organs due to broad targeting spectrums. Moreover, PTEN is an important tumor suppressor; therefore, its sustained inhibition can also lead to tumorigenesis in some non-muscle cells and tissues.³ Recent advancements in drug delivery methods have suggested that synthetic nanoparticles can be used for temporal and spatial control of drug release in vivo. In this regard, Huang et al.¹⁰ have reported that nanoparticles consisting of poly(lactide- co -glycolide)- b -poly(ethylene glycol) and a muscle-homing peptide, M12, can be useful in delivering VO-OHpic to dystrophic muscle in vivo. Altogether, these new genetic and pharmacological studies and development of nanoparticles for targeted delivery of small molecules in skeletal muscle provide initial evidence that the inhibition of PTEN can be a potential approach for treatment of DMD patients in the future.

In summary, PTEN has now emerged as an important disease modifier that shows therapeutic promise for DMD patients. While initial results are encouraging, long-term studies in larger animal models are needed to ascertain that inhibition of PTEN does not lead to any major side effects in muscle or non-muscle tissues in DMD patients. There are also many outstanding questions that need to be addressed. For example, the mechanisms by which PTEN improves myofiber repair and function in dystrophic muscle remain to be investigated. Given that PTEN inhibition increases Akt activation, the possibility of upregulated muscle growth should also be examined as a potential mechanism for the improvement of dystrophic phenotype in models of DMD. Moreover, transcriptional and post-transcriptional mechanisms that regulate the levels of PTEN and its interacting proteins in dystrophic muscles remain to be investigated. Finally, more studies are warranted to identify downstream targets of PTEN-mediated signaling that promote muscle repair and can be targeted with pharmacological compounds without any deleterious consequences.