Type I PRMT inhibitor MS023 promotes SMN2 exon 7 inclusion and synergizes with nusinersen to rescue the phenotype of SMA mice

  1. Anna J Kordala
  2. Nina Ahlskog
  3. Muhammad Hanifi
  4. Amarjit Bhomra
  5. Jessica Stoodley
  6. Wooi Fang Lim
  7. Suzan M Hammond
  8. Matthew JA Wood
  9. Carlo Rinaldi

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Abstract

Spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality. The advent of approved treatments for this devastating condition has significantly changed SMA patients’ life expectancy and quality of life. Nevertheless, these are not without limitations, and research efforts are underway to develop new approaches to be used alone and in combination, to ensure improved and long-lasting benefits for SMA patients. Protein arginine methyltransferases (PRMT) are emerging as druggable epigenetic targets, with several small molecule PRMT inhibitors already in clinical trial stage. From a screen of highly potent and selective next generation epigenetic small molecules, we have identified MS023, a potent and selective type I PRMT inhibitor, able to promote SMN2 exon 7 inclusion and increase SMN protein levels in preclinical SMA model, by inhibiting the binding of splicing factor hnRNPA1 to SMN2 pre-mRNA. Treatment of SMA mice with MS023 results in amelioration of the disease phenotype, with strong synergistic amplification of the positive effect when delivered in combination with the SMN2 -targeting antisense oligonucleotide nusinersen. Moreover, transcriptomic analysis revealed that MS023 treatment has very minimal off-target effects and that the added benefit of the combination therapy is mainly attributable to targeting neuroinflammation. Our study warrants further clinical investigation of PRMT inhibition both as a stand-alone and add-on therapy for SMA patients.

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    Referee #2

    Evidence, reproducibility and clarity

    This study by Kordala et al reports the identification of new therapeutics to further improve the clinical outcomes of spinal muscular atrophy (SMA) patients. SMA is childhood neurological disease that is caused by insufficient levels of the survival motor neuron (SMN) protein. There are currently three approved therapies for SMA, yet the disease is not cured and many patients remain severely disabled. The authors conducted a screen of known epigenetic regulators to identify molecules that increase SMN protein. They identified MS023, which is a selectively PRMT inhibitor, that promotes SMN exon 7 inclusion and thus full length SMN protein. Importantly, MS023 improves the SMA phenotype alone or in combination with the SMN2 antisense oligonucleotide suggesting it can potentially be used by itself or in combinatorial approaches. While this is a generally well written paper with relatively straight forward experimental design there remains some concerns that should be addressed.

    Major Concerns:

    1. The mechanism of action needs further clarification. Does MS023 work similarly to Risdaplam? Also, if Nusinersen is already interfering with hnRNPA1 how does MS023 augment the splicing.
    2. MS023 alone did not increase improve exon 7 inclusion in the spinal cord of treated SMA mice yet the protein levels were increased (Fig. 3D,F). Are there alternative mechanisms through which MS023 is acting?
    3. Further explanation of why MS023 did not improve exon 7 inclusion in the spinal cord but enhanced the effect of the ASO (Fig. 4B) is needed.
    4. Nusinersen has been shown to almost completely rescue the SMA phenotype in mice. Was the dose used here chosen to be suboptimal?

    Minor Concern:

    Many neurological diseases are now moving to a multimodal approach. The manuscript could be improved with further discussion of why MS023 would be an attractive option compared to other synergistic strategies being employed for SMA, including the most obvious of combining some of the already approved therapies.

    Significance

    This is a generally well done study that works through a screening methodology to identify a molecule that increase the levels of SMN. Mechanistic studies suggest that the compound works through inhibiting the recruitment of hnRNPA1 to the SMN2 gene, thus promoting inclusion of exon 7 and the production of full-length SMN protein.

    The study does not provide definitive data that methyl transferase activity of PRMT promotes exon 7 exclusion or that the inhibitor changes the methylation state of any of the proteins involved. However, knockdown experiments does not exclude this possibility.

    This study would be of general interest to wider audience if more detail was included regarding the current SMA landscape and how MS023 fits in with what is currently available. The transcriptome data was potentially very interesting since it provided clues on how MS023 is exerting its synergistic effect(neuroinflammation angle is relatively unique), but that data was only briefly discussed.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    SMA is a severe, monogenetic, progressive neuromuscular disease that mostly affects young children. SMA is cause by homozygous loss of function of SMN1. The main modifier of disease severity - that ranges widely, from neonatal to adult onset - is the presence of a varying number of SMN2 copies in the human genome. Recently, several gene-targeting therapies for SMA have been approved. This has changed the outcome of SMA drastically for many patients, but, surprisingly, the effect of these treatments varies strongly between patients. This leads to significant uncertainty for patients, families and clinicians and poses a challenge to reliable prognosis.

    One of the drugs that has been approved for treating SMA is the antisense oligonucleotide nusinersen (Spinraza). Nusinersen improves SMA outcomes by enhancing the splicing of SMN2 -transcribed pre-mRNA, leading to an increase in the inclusion of exon 7 leading to an increase in the availability of full-length, functional SMN protein. In the current manuscript, Kordala and colleagues investigate the effect of a library of 54 compounds (focused on modulating epigenetic regulators) on SMN2 splicing and SMN protein in an SMA type II patient fibroblast cell line. They found the Type I PMRT inhibitor, MS023 to dose-dependently increase full length SMN2 splicing SMN protein levels, by decreasing hnRNPA1 binding to SMN2 pre-mRNA. Next, they show that MS023 monotreatment of the severe 'Taiwanese' (+/- SMA type I) mouse model of SMA leads to improved survival and weight gain. Moreover, they show that combinatorial treatment of the same mouse model with MS023 and nusinersen, significantly further improves survival compared to both nusinersen and MS023 monotreatment. Finally, transcriptome analysis suggests that the majority of misregulated transcripts in SMA is rescued by both nusinersen an combinatorial treatment but, importantly, the rescue observed in the combinatorial group seems more complete.

    Suggestions

    The authors state in the abstract that "transcriptomic analysis revealed that MS023 treatment has very minimal off-target effects". However, their transcriptomic analyses do not contain a condition that investigates the effects of MS023 on the transcriptome in WT and SMA animals on its own. I belief this would have been an essential addition to support the conclusion on off-target effects of MS023, especially considering the benefits that the authors list in the discussion when compared to e.g. VPA. I agree with their comments about the unspecificity of such drugs; however, I don't belief their current transcriptomics analysis on MS023 fully support this conclusion either. It may not be feasible to include such an experiment in a revised version of the manuscript, but in this case the authors should reflect on the wording of their conclusions.

    I agree with the authors that the effect of MS023 on SMN-FL RNA appears to be dose-dependent but I don't think the data fully supports that conclusion for SMN protein levels (compare e.g. 250 nM and 2.5 µM quantification). In fact, there are many inconsistencies between SMN RNA and SMN protein levels: in figure 3 (MS023 monotreatment), the authors observe in spinal cord no change in SMN RNA but a significant increase in SMN protein. In contrast, in the same figure, in muscle, both SMN RNA and protein increase significantly. This is a bit confusing and to me mostly means that the regulation of SMN RNA and protein expression in complex and likely depends on many more factors than PMRT activity and hnRNPA1 arginine methylation status. Indeed, the authors pick hnRNPA1 as a promising target from a list of proteins that contains 72 in total. Are there no other promising candidates in this list that would be able to explain the unclear and inconsistent correlation between SMN RNA splicing and SMN protein levels?

    The in vitro work was based on the use of one primary fibroblast cell line. It would be relatively straightforward to characterized the effect of MS023 on e.g. type 1 and type 3 patient-derived lines, thus providing a clearer overview of the use of this type of drug in SMA patients of different types. Both through the Corriell repository (as used in the current paper) and surely also through biobanks at Oxford it should be relatively straightforward to obtain such cell lines and for the authors to extend their analyses to include patients of different types (and with varying SMN2 copy number).

    The mechanism that the authors suggest in e.g. Fig. 2D about the interaction of hnRNPA1 with the SMN2-ISSN1 in relation to PMRT inhibition is very similar to how nusinersen prevents SMN2-ISSN1 binding of hnRNPA1 (as the authors mention in the discussion). How do the authors suggest this would work? Do they have suggestions for further experiments to investigate this interaction (e.g. using hnRNPA1 and nusinersen molecules with point mutations?)

    Minor comments

    Do I understand correctly that none of the screened molecules in figure 1 lead to significantly unregulated SMN protein levels (including MS023)? What causes the difference between figure 1 (no signficicant upregulation of SMN protein) vs figure 2 (a dose dependent increase of SMN protein)? Do the authors have an explanation for this difference? In relation to this point, I am somewhat surprised at the variability in protein quantifications in especially figures 1 and 2. In these figures, biological replicates are obtained from one cell line. Although I understand that there is not necessarily much benefit to including all western blots used for quantification in for example the supplementary files with the paper, it would be useful to see some examples for e.g. the western blots for the quantifications in fig. 1C. Similarly, I appreciate the complexity of the IPs and arginine-methylation specific blotting in fig 2E, but the current tightly cropped blots are not super convincing and the uncropped blots are not included in the supplementary data. Also how was this quantified; fig 2F lacks some indication of standard deviation or other indicator of reproducibility between measurements.

    There are some what appear to be reference formatting errors (e.g. lines 17 and 20 on page 15 of the manuscript PDF amongst others).

    The PDF version of Supplementary table 2 in its current format is not really usable or readable; an Excel version would be preferable.

    Significance

    The paper addresses an interesting question: it aims to improve the efficacy of existing drugs for SMA by identifying novel molecules that may improve the working mechanism of, in this case, nusinersen. Others have tried this before by using VPA, but the current molecule appears to be more specific. However, it would have been interesting to get more details on the effect of this novel compound: a wider range of cell lines, further mouse experiments (a control group in figs. 3 and 5) and analysis (e.g. pathological analysis of the neuromuscular system). It would in fact have been interesting to combine some of the analyses in the current work also with the other available SMN2 splicing modifier risdiplam: as risdiplam also modulates SMN2 splicing, MS023 might also have been suitable to improve risdiplam efficacy. Especially in the cell line the authors have used this would have been a relatively straightforward addition. I believe the paper may provide an interesting start, but without further analysis remains at that stage.

    The audience to likely be most interest are mostly colleagues from the SMA field, as the mechanisms in the current manuscript focus very much on ISS-N1-regulated SMN2 splicing which is highly specific for SMA.

  4. Version published to 10.1101/2022.10.18.512489v3 on bioRxiv
  5. Version published to 10.1101/2022.10.18.512489v2 on bioRxiv
  6. Version published to 10.1101/2022.10.18.512489v1 on bioRxiv