HDAC inhibitors for muscular dystrophies: progress and prospects

Duchenne muscular dystrophy (DMD) is the most common and severe form of muscular dystrophy (MD) that affects 1 in 3500–6000 live male births. This lethal X-linked genetic disease is caused by mutations in dystrophin gene that leads to a complete absence of the protein, thereby compromising the structural and functional integrity of the dystrophin-associated protein complex (DAPC).[1] The DAPC links the actin cytoskeleton to the extracellular matrix and has an essential role in stabilizing the sarcolemma during repeated cycles of contraction.[2] As such, dystrophin-deficient muscles are vulnerable to mechanical damage and develop progressive muscular weakness, as a consequence of muscle degeneration, leading to loss of myofibers, which are ultimately replaced by fibrotic scars and fat deposition.[3] There is still no available cure for DMD, but only palliative treatments that aim to counter muscle loss, thereby extending patient mobility and delaying the onset of respiratory and cardiac problems.[4]

The progression of DMD is highly influenced by the ability of dystrophin-deficient muscles to counter muscle loss by a regenerative response, which typically defines a clinical latency observed during the early stages of the disease. The gradual exhaustion of such compensatory response coincides with changes in the muscle tissue composition, leading to the progressive formation of fibrotic and adipose tissue in place of contractile fibers. This ‘restriction point’ in the natural history of DMD is due to alterations in the interactions between the cell types that contribute to promote regeneration, eventually culminating with a switch of muscle repair toward fibrotic and fat deposition.[5] In particular, studies in the last decade have revealed the importance of changes in the identity and functional properties of the cellular components of the muscle stem (satellite) cell (MuSC) niche. For instance, the interactions between inflammatory cells (e.g. macrophages), interstitial ‘support’ cells (e.g. fibro-adipogenic progenitors – FAPs) and MuSCs appear a key determinant to drive the skeletal muscle toward a regeneration or degeneration process.[6,7] This notion has inspired a number of current pharmacological strategies aimed at targeting these cells and the functional networks they establish,[8,9] as an alternative to gene replacement and cell-based strategies.

Among the pharmacological interventions that aim to slow down the disease progression by targeting key events downstream of the genetic defect, the histone deacetylase inhibitors (HDACi) have recently been translated into a clinical trial, based on the encouraging preclincial data generated in the mouse model of DMD – the mdx mice.[10,11]

In principle, the rationale for the use of HDACi in the treatment of DMD was provided by the finding that dystrophin-deficient muscles display an aberrant, constitutive activation of HDAC2, as a consequence of reduced nitric oxide (NO)-mediated NO-dependent S-nitrosylation in myofibers.[12] Moreover, recent studies have revealed complex epigenetic networks targeted by HDACi in MuSCs and FAPs.[13,14] In particular, studies from Saccone et al. have identified an HDAC-regulated network that controls the functional phenotype of FAPs from dystrophic muscles. Treatment with HDACi promotes the expression of two core components of the myogenic transcriptional machinery, MyoD and BAF60c [15] and upregulates three myogenic microRNA involved into muscle differentiation (myomiRs; miR-1.2, miR-133 and miR-206) in FAPs. MyomiRs in turn target two alternative BAF60 variants – BAF60 A and B – that when incorporated into the Switch/Sucrose NonFermentable (SWI/SNF) chromatin-remodeling complex would otherwise promote the activation of the fibro-adipogenic program.[14] Thus, HDACi-mediated selection of BAF60 C-based SWI/SNF complex appears to mediate a therapeutic switch of FAP phenotype from pro-fibroadipogenic to pro-myogenic one. Interestingly, FAPs from dystrophic muscles at advanced stage of disease are resistant to the beneficial effects of HDACi, accounting for the stage-specific effect of HDACi previously observed by Mozzetta et al.[9] Genome-wide studies of chromatin accessibility showed that at early stages of the disease, HDACi promote an extensive chromatin remodeling, which was not observed at late stage of the disease.[14] This finding indicates that pharmacological interventions that target FAPs could be used to promote compensatory regeneration, while preventing fibro-adipogenic degeneration of DMD muscles. It also suggests that FAPs of dystrophic muscles at late stages of DMD progression might acquire a chromatin conformation that confers resistance to treatments that are otherwise effective at early stages of disease. Understanding the molecular basis of such resistance could reveal novel targets for interventions aimed at restoring the ability of FAPs to support muscle regeneration rather than fibrosis and fat infiltration in a larger population of patients.

Conclusion

As preclinical evidence supported the rationale for the use of HDACi in the treatment of DMD, the HDACi Givinostat has been launched as the first epigenetic drug tested in a Phase I/II clinical trial on DMD boys between 8 and 10 years of age that is currently under investigation. Given the potential of HDACi to promote regeneration at the expense of fibro-adipogenic degeneration, one predicted effect of Givinostat on DMD muscles is to extend the compensatory regeneration and delay the disease progression in patients at early stages of disease progression, Future research should evaluate the efficacy of Givinostat in a larger population of DMD patients and identify molecular criteria and noninvasive biomarkers that define patient responsiveness and eventually inspire further interventions that sensitize to HDACi unresponsive patients.

Expert opinion

Three fundamental aspects for the future development of HDACi-based therapies concern a) the identification of biomarkers that help selecting patients for clinical studies and monitoring their responsiveness to the treatments, b) the discovery of complementary approaches that synergize with HDACi to provide an optimal combined therapeutic intervention and c) the potential extension of HDACi-based treatments to other forms of muscular dystrophies.

While the analysis of muscle biopsies is currently regarded as the most reliable readout of the histological effects of HDACi, this procedure is invasive, is limited to one specific muscle and cannot be repeated multiple times during the course of clinical trials. As such, it is currently missing an effective readout of the efficacy of treatment with HDACi or similar pharmacological approaches. Identification of circulating biomarkers and/or setting noninvasive or semi-invasive procedures for the evaluation of the effects of pharmacological interventions is clearly a fundamental step to further move forward pro-clinical and clinical research on DMD.

Moreover, beyond the beneficial effects on muscle histology, the clinical efficacy of HDACi treatment needs to be correlated with functional tests. Studies with HDACi in mdx mice demonstrated that the morphological recovery is accompanied by increased muscle strength and exercise performance.[10,11] Thus, matching functional tests, such as 6 Minute Walk Test (6MWT) and North Star Ambulatory Assessment, with the histological evaluation of treated muscles, will be mandatory to reveal the efficacy of HDACi and other pharmacological interventions in DMD patients.

Regarding the identification of complementary approaches that synergize with HDACi, the most urgent questions relate to the functional interactions between HDACi and steroids – the standard treatment currently used in the treatment of DMD. In this case, it is interesting to note that steroids are typically acting through a nuclear receptor – the glucocorticoid receptor (GR) – whose activity is negatively regulated by an HDAC-containing repressive complex. Moreover, the possibility to combine HDACi-based treatment with emerging strategies toward re-introducing functional dystrophin in DMD muscles (i.e. exon skipping or future CRISPR-based interventions) is supported by a strong rationale consisting of the necessity to protect the newly formed myofibers, induced by HDACi-promoted regeneration, from contraction-mediated regeneration, via dystrophin re-expression.

Finally, future research should determine the potential efficacy of HDACi on other forms of muscular dystrophies that share pathogenic events with DMD, e.g. the shift from compensatory regeneration to fibrosis and fat deposition.

Declaration of interest

Funding has been received from AFM Telethon and Duchenne Parent Project (DPP-NL). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.