Inhibition of Type I PRMTs Reforms Muscle Stem Cell Identity Enhancing their Therapeutic Capacity

Reviewer #1 (Public Review):

In this study, Dominici et. al. show that small molecule inhibition of Type I PRMTs in muscle stem cells (MSCs) can result in the expansion of this cell type in vitro, solving a major limitation in the field. Importantly, once the inhibitor is removed these stem cell differentiate "normally". This advance will likely facilitate CRISPR-based screening approaches and stem cell engraftment therapy. Furthermore, they show that when a mouse model of Duchenne muscular dystrophy is treated with these same inhibitors these mice rather rapidly gain grip strength, demonstrating the therapeutic value of these findings.

Strengths:

- Previous studies from the same group have shown that the conditional ablation of PRMT1 in MSCs results in the expansion of this cell type, but this expanded PRMT1-null MSC pool cannot terminate the myogenic differentiation program. This raises the question of whether PRMT1 small molecule inhibition of MSCs will also facilitate the expansion of these cells, and if the removal of the inhibitor after expansion will result in a large functional pool of MSCs, which could then be used for both in vitro and in vivo studies.

- Using a combination of muscle fiber culture, myoblast culture and single cell RNA-seq, this is indeed what they show.

- They also perform two types of in vivo experiments to validate their cell culture findings; 1) MSCs expanded under the treatment of MS023 were washed clean of the inhibitor and engrafted into the tibialis anterior muscle. These cells were marked with GFP to allow efficient tracking. Mice receiving the MS023-treated MSCs produced more than double the mature GFP+ muscle fibers than cells treated with DMSO. 2) A mouse model of Duchenne muscular dystrophy displayed grip strength improvement after just one treatment of MS023.

- MS023 is a Type I PRMT inhibitor and thus can also target CARM1. CARM1 has been implicated in MSC function by the Rudnicki group. Importantly, they exclude a role for CARM1 in the expansion of MSC cell number by treatment with a very specific CARM1 inhibitor, TP064. Thus, indicating that PRMT1 inhibition is likely the main driver of this expansion phenotype.

Weaknesses:

- Very few weaknesses.

- The in vivo efficacy of MS023 does not seem to be very great. The mice treated with MS023 display a very small reduction in ADMA levels and a small increase in SDMA levels (Fig S6A).

Reviewer #2 (Public Review):

In this manuscript, Dominici et al. aim to determine whether the reversible inhibition of the type I protein arginine methyltransferases (PRMT) would maintain the stemness of muscle stem cells in culture and enable subsequent regenerative capacities. They demonstrate that the type I PRMT inhibitor MS023 enhances self-renewal and in vitro expansion of muscle stemm cells isolated from mice. Using a very rigorous single cell RNA-sequencing approach, they further demonstrate that a distinct sub-populations of cells emerge under type I PRMT inhibition and that these cells entered the differentiation program more efficiently. Moreover, they revealed a shift in metabolism in these cells, which they confirmed in vitro. Finally, they demonstrate that MS023 enhances muscle stem cells engraftment in vivo and that the direct injection of MS023 increases muscle strength in a mice model of Duchenne muscular dystrophy.
This study will have a great impact in the field of stem cells and offer potential therapeutic avenues for diseases such as Duchenne muscular dystrophy.

Two weaknesses are noted which lie in overstatements of the findings. There are six type I PRMTs (PRMT1, 2, 3, 6, 8, and CARM1), all of which are inhibited by MS023. While the authors demonstrate that their observations are not due to the inhibition of CARM1, they do not demonstrate that it is due to the inhibition of PRMT1, as they suggest.

Furthermore, this study suggests that the switch and elevated cellular metabolism in muscle stem cells due to MS023 enhanced self-renewal and engraftment capabilities but does not demonstrate this fact directly as stated.

Reviewer #3 (Public Review):

Dominici et al studied the effects of the type I PRMT inhibitor MS023 on skeletal muscle stem cells (MuSCs) and on muscle strength in dystrophin-deficient mdx mice. The authors observed an enhanced proliferative capacity of cultured MuSCs with an increase of Pax7+/MyoD- cells. The observations are more or less in line with previous studies of the same group, describing reduced differentiation but enhanced proliferation of MuSCs after genetic inactivation of Prmt1. scRNA-seq identified different subpopulations of MuSCs, showing a shift to increased Pax7 expression and elevated oxidative phosphorylation and glycolysis after treatment with MS023. Treatment of MuSC with MS023 during expansion in vitro enhanced engraftment of MuSCs and treatment of dystrophic mdx mice increased muscle strength.

Overall, the manuscript provides new insights into the beneficial effects of the type I PRMT inhibitor MS023 for skeletal muscle regeneration. The description of the MS023-induced transcriptional and metabolic changes in MuSC is interesting and the effects on MuSC transplantation and muscle strength are stunning. However, the proposed underlying mechanism, which is claimed to rely on the expansion of MuSC and 'reprograming' of MuSCs towards a "unique and previously uncharacterized identity" is not sufficiently supported. The extent of the description of scRNA-seq data is inappropriate. Some conclusions from the scRNA-seq data appear to be overinterpreted or are rather trivial. It remains completely unclear whether the MS023-stimulated increase of metabolic pathway activity (OXPHOS, glycolysis) plays any role for preserving stem cell properties of MuSC during expansion and improves engraftment. Additional functional and mechanistic studies are required to explore the underlying molecular processes. Furthermore, it remains completely unclear whether the acclaimed increase in grip and tetanic strength of mdx mice after MS023 treatment relies on enhanced expansion of MuSC mediated by PRMT1 inhibition.

Author Response:

We’d like to thank the three reviewers for reviewing our work in depth and providing insightful comments and suggestions.

Reviewer 1

1. The in vivo efficacy of MS023 does not seem to be very great. The mice treated with MS023 display a very small reduction in ADMA levels and a small increase in SDMA levels (Fig S6A).

REPLY: We have quantified proteins with ADMA and SDMA by Western blotting tail clippings from mice treated with vehicle (n=6) and MS023 (n=6). These were normalized for equal loading to b-actin levels. The average ADMA relative expression was 0.92 for vehicle treated mice and 0.86 for MS023 treated mice (p < 0.044). The average SDMA relative expression was 0.89 for vehicle treated mice and 0.98 for MS023 treated mice (p < 0.000019). These whole-body measurements show MS023 promotes the decrease of proteins with ADMA and increasing proteins with SDMA, as observed before with inhibition of PRMT1 (Dhar et al, 2013).

Reviewer 2

1. Two weaknesses are noted which lie in overstatements of the findings. There are six type I PRMTs (PRMT1, 2, 3, 6, 8, and CARM1), all of which are inhibited by MS023. While the authors demonstrate that their observations are not due to the inhibition of CARM1, they do not demonstrate that it is due to the inhibition of PRMT1, as they suggest.

REPLY: MS023 has been shown to have in vitro activity for several type I enzymes (Eram et al, 2016) and the same goes for GSK3368712 (Fedoriw et al, 2019). MS023 IC50 in vitro 30nM PRMT1, 119 nM PRMT3, 83 nM CARM1, 4 nM PRMT6, and 5 nM PRMT8 (Eram et al., 2016). It was documented early that PRMT1 is the major cellular type I enzyme (Pawlak et al, 2000) and this is why PRMT1 and PRMT5, major type II, are embryonic lethal in mice (Guccione & Richard, 2019). In vivo data using MS023 is paralleled by using siPRMT1 (Gao et al, 2019; Plotnikov et al, 2020; Wu et al, 2022; Zhu et al, 2019). Thus in vivo, MS023 targets the main type I PRMT, PRMT1. Further, in support of our claim that MS023 targets PRMT1 in MuSCs is our previous observation that deleting PRMT1 stimulates MuSC proliferation. Since this effect was irreversible (Blanc et al, 2016) we pursued studies with the reversible MS023, the only compound to have significant activity towards PRMT1 in vivo. For these reasons, we are convinced that the effect of MS023 is mainly mediated by inhibiting PRMT1 in the MuSC.

To be thorough we should test all other type I PRMT inhibitors as they become available. CARM1 was shown to be a player in MuSC (Kawabe et al, 2012), but we excluded it using a CARM1 inhibitor TP-064 (Nakayama et al, 2018). PRMT6 mice that we generated are perfectly viable without overt phenotypes, suggesting PRMT6 is not involved (Neault et al, 2012), and PRMT8 is brain specific (Taneda et al, 2007).

2. Furthermore, this study suggests that the switch and elevated cellular metabolism in muscle stem cells due to MS023 enhanced self-renewal and engraftment capabilities but does not demonstrate this fact directly as stated.

REPLY: Agreed. The link between cellular metabolism and MS023 enhanced self-renewal and engraftment capabilities is correlative and we will edit the revised text to reflect this.

Reviewer 3

1. However, the proposed underlying mechanism, which is claimed to rely on the expansion of MuSC and 'reprograming' of MuSCs towards a "unique and previously uncharacterized identity" is not sufficiently supported. The extent of the description of scRNA-seq data is inappropriate. Some conclusions from the scRNA-seq data appear to be overinterpreted or are rather trivial.

REPLY: We presented the top marker genes for each subpopulation that was identified in our scRNAseq to aid the reader in establishing a broad view of whether a given subpopulation was quiescent-like, proliferating, or differentiating. M1-M5 clusters were all enriched for cell cycle markers (Mki67, Cdk1, etc), indicating a proliferative identity. The unique finding in our data is that treatment with MS023 resulted in a shift in identity as compared to the DMSO-treated proliferating MuSCs (M1, M2 and M4), creating transcriptionally distinct M3 and M5 clusters. M3 and M5 had elevated markers for metabolism (E.g. Eno1, Atp5k, etc) and early activation (E.g. Fos, Jun), while the untreated MuSCs in clusters M1, M2 and M4 did not. Furthermore, M3 and M5 had higher baseline levels of Pax7 expression when compared to untreated cells. Together, these findings describe a transitional subpopulation of MuSCs unique to MS023 treatment which not only harbour stem like/early activation markers Pax7, Fos and Jun, but also elevated proliferative markers related to cell cycle and energy metabolism. This particular combination of characteristics is unique to the MS023-treated MuSCs, thus identifying a novel subtype of MuSC identity. In accordance with our scRNAseq data, we validated experimentally that MS023-treated cells have higher energy metabolism and increased self-renewal potential, thereby confirming that the unique transcriptomic signature of these cells also lead to a different cell fate decision.

2. It remains completely unclear whether the MS023-stimulated increase of metabolic pathway activity (OXPHOS, glycolysis) plays any role for preserving stem cell properties of MuSC during expansion and improves engraftment. Additional functional and mechanistic studies are required to explore the underlying molecular processes.

REPLY: Agreed. The link between cellular metabolism and MS023 enhanced self-renewal and engraftment capabilities is correlative and we will edit the revised text to reflect this.

3. Furthermore, it remains completely unclear whether the acclaimed increase in grip and tetanic strength of mdx mice after MS023 treatment relies on enhanced expansion of MuSC mediated by PRMT1 inhibition.

REPLY: Agreed. We cannot exclude if the effect is mediated by an expansion of the MuSC pool or by an effect on other cell types, such as a direct impact on the myofibers. The goal of this figure was to provide a therapeutic perspective for the use of type I PRMT inhibitor for the treatment of DMD. Muscle wasting/weakness in DMD is a complex and multifactorial process (e.g., myofiber fragility, MuSC defects, chronic inflammation, fibrofatty accumulation). If MS023 can target multiple aspects of the physiopathology of the disease it would increase its therapeutic applicability. Further studies will be needed to determine the exact mechanism by which MS023 mediate its beneficial effect. The manuscript will be modified to reflect this.

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