Page 96 - 2021_02-Haematologica-web

C. Lagresle-Peyrou et al.
with these data and suggest that the organization of the cytoskeleton is altered in these cells. However, the muta- tion described herein could not be classified as an actinopathy such as Wiskott-Aldrich syndrome,32 Dock8 deficiency33 or warts, hypogammaglobulinemia, infec- tions, and myelokathexis (WHIM) syndrome.34 Although these diseases are characterized by impaired leukocyte migration, autoimmunity, and/or malignancies, none is associated with BM hypoplasia or a severe SCID pheno- type. Therefore, our present observation is the first to have evidenced cytokinesis failure in SCID patients.
In cell lines, actin dynamics have been described as a key regulator of the mitochondrial network,35 suggesting that the mitochondrial defects observed in the G12R mutated HSPC might be a consequence of actin dysregu- lation. In patients bearing D57N or E62K Rac2 mutations, the fact that actin cycling was disrupted in neutrophils15,19 but that mitochondrial membrane depolarization was not evident in the HSPC challenges this argument and sug- gests that RAC2 could regulate mitochondrial integrity and function through an actin-independent mechanism. This assumption fits with previous reports demonstrating that: (i) RAC2 interacts with SAM50, a mitochondrial transporter involved in the regulation of mitochondrial membrane potential and ROS production:36,37 (ii) unregu- lated RAC2 activation disrupts ROS production,28,38,39 which is required for the regulation of mitochondrial dynamics.40 Moreover, it is now well established that mitochondrial activity is a key regulator of HSPC home- ostasis and functions,41,42 suggesting that the inappropriate RAC2 activation driven by G12 mutations directly affects mitochondria and ROS production.
The RAC2 signaling pathways targeted by the G12R mutation are not reported here; however, given that G12R and G12V mutations have the same impact on HSPC, we can reasonably assume that they have the same down- stream targets. This assumption is reinforced by the obser- vations in KRAS mutants; both G12R and G12V muta- tions impaired GTP hydrolysis,22 suggesting that the mutant proteins are catalytically inactive. Interestingly, the G12V GOF RAC2 mutation constitutively activates the phospholipases C (PLC) isoforms PLCβ2 and PLCγ.23,43 These two PLC interact with phosphatidylinositol 3 kinase (PI3K) and protein kinase C (PKC), both of which are involved in various signaling pathways (including the ones that regulate HSPC homeostasis).44,45
The comparison of RAC2 mutants also highlighted the complexity of an unbalanced RAC2 network, as the D57N and E62K variants had a limited impact on HSPC. This find- ing might be related to the position of the amino-acid sub- stitution, i.e., inside the GDP/GTP binding pocket (G12R) or in the G3 (D57N) or switch II (E62K) domains (Online Supplementary Figure S1A). In particular, the replacement of glutamate by a lysine at position 62 may impact the GTP bound RAC2 form stability and thus explain the absence of a GTP-bound RAC2 form and a lower level of RAC2 expression, relative to the G12R condition (Online Supplementary Figure S3D). These findings differ from the report of Hsu et al.,19 probably because we did not use the
same cells or the same techniques to measure RAC2 GTP activity. However, our conclusions fit with the patients’ clinical phenotype; we can reasonably assume that the highly active, constitutive GTP-bound form of RAC2 driv- en by the G12R mutation leads to BM failure and a SCID phenotype. In contrast, the transient overactivation of the RAC2-GTP form driven by the E62K RAC2 mutation is associated with lymphopenia19 but not with BM abnormal- ities.46 Taken as a whole, our findings emphasize that vari- ous RAC2 mutations differ in their effects on HSPC. These differences may account for the broad observed spectrum of clinical phenotypes, ranging from neutrophil defects to a severe form of SCID. This type of heterogeneity has already been reported for RAG1 mutations involved in common variable immunodeficiency or SCID phenotype.47
In summary, the p.G12R RAC2 mutation has a drastic impact on the homeostatic regulation of hematopoiesis, which explains the severity of the patients’ clinical and immunological phenotypes. To the best of our knowl- edge, the present study is the first to have described an AD form of a SCID. Physicians should consider RAC2 gene sequencing for patients with SCID and a clinical presentation of RD. This gene should also be included in newborn screening programs for SCID detection. Finally, future research should seek to characterize the RAC2 sig- naling pathway and novel downstream targets specifically involved in hematopoietic cell commitment.
Disclosures
No conflicts of interests to disclose.
Contributions
CLP and MC supervised and designed the project, CLP ana- lyzed and interpreted data and drafted the manuscript; AO and CT performed the biochemical studies and drafted part of the manuscript; HS, AG and CDS performed some experiments and analyzed data; PR and YC performed the protein homology modelling and drafted part of the manuscript; AF, DM, CP, JLC and MC provided patient care; CP, JLC, IA and MC helped to draft the manuscript.
Acknowledgments
We especially thank Prof. Stephane Blanche and the nursing staff of the Department of Pediatric Immunology, Hematology and Rheumatology at Necker-Enfants Malades Hospital for patient care. We also thank Gisèle Froment, Didier Nègre, and Caroline Costa for lentiviral vector production (UMS3444/US8, BioSciences Gerland, Lyon), Olivier Pellé for cell sorting (at the flow cytometry core facility, SFR Necker, Paris), Thibault Courtheoux for the 3D cell imager analysis (Nanolive SA), Myriam Chouteau for technical assistance, and Patrick Revy (from the Genome Dynamics in the Immune System group, Imagine Institute, Paris) for fruitful discussions.
Funding
This study was funded by the French National Institute of Health and Medical Research (INSERM). The work at the CRCT was funded by an “Equipe Labellisée par la Fondation pour la Recherche Médicale” grant.
410
haematologica | 2021; 106(2)