Page 193 - 2019_07 resto del Mondo-web

Molecular characterization of GFI1BQ287* mutation
RCOR1, HDAC1/2, PHF21A (BHC80), and HMG20B (BRAF35).35 The other proteins found, GSE1, RCOR3, and ZMYM2/3, are also known LSD1 interactors,36 of which ZMYM2/3 have not been reported in complex with GFI1B before. ZMYM3 was identified as the only significantly different interacting protein between GFI1B and GFI1BQ287*. Whether this is relevant for disease pathogene- sis remains to be seen. Most interactors (LSD1, RCOR1/3, HDAC1/2, GSE1, ZMYM2/3) are detected in differentiat- ing megakaryocytes as well (Online Supplementary Table S4), suggesting that they may be relevant for megakaryo- cyte development.
Comparison of the proteome of different iPSC-derived megakaryocytic cell cultures showed a uniform expres- sion pattern in GFI1BQ287* cells compared to wildtype cells. The latter might be more variable due to differences in megakaryocytic differentiation stages between cultures. GFI1BQ287* megakaryocytes exhibit a more uniform, micromegakaryocyte-like appearance, suggesting that they are arrested before polyploidization. This is in con- trast with megakaryocytes observed in GFI1BQ287* individ- uals5 and conditional GFI1B knockout mice,37 in which a block after polyploidization but before cytoplasmic mat- uration is observed. Possibly, environmental cues that are absent in in vitro cultures may stimulate differentiation of GFI1BQ287* megakaryocytes in vivo.
In GFI1BQ287* iPSC-derived megakaryocytic cells we observed a downregulation of downstream interferon-γ signaling targets, such as STAT1, MX1, IFI16, IFI30, IFI35, IFIT3, OAS2 and OAS3. Interferon-γ and its downstream effector STAT1 stimulate megakaryocyte differentiation and platelet production. STAT1 promotes polyploidiza- tion and loss of STAT1 in JAK2V617F mice resulted in reduced platelet numbers through interference with megakaryocyte development.38,39 The failure to activate interferon target genes in GFI1BQ287* megakaryocytes may inhibit differentiation and explain the micromegakaryo- cyte-like appearance of iPSC-derived megakaryocytes.
GFI1BQ287* iPSC-derived megakaryocytic cells further revealed significantly increased levels of the MCM com- plex and MEIS1. This might be relevant as the MCM complex is involved in DNA replication/cell cycle pro- gression and, in accordance with several studies,40,41 we observed that MCM proteins are downregulated upon megakaryocyte differentiation (Online Supplementary Figure S6D).40,41 In addition, we showed earlier that forced MEIS1 expression in CD34+ cells results in larger and more colonies in colony-forming unit-megakaryocyte assays.30 Thus, failure of downmodulation of DNA repli- cation-associated genes by GFI1B as a consequence of sequestration of LSD1 and associated proteins by GFI1BQ287* might contribute to increased proliferation and lack of polyploidization.
The platelets of GFI1BQ287* patients showed a major reduction in a-granule proteins, ranging from a mild <2- fold decrease (e.g. for SPARC, fibrinogen a and β chains) to a severe >10-fold depletion (e.g. for VEGF and P- selectin). To our surprise, NBEAL2 showed significantly increased expression in both platelets and iPSC-derived cells harboring the GFI1BQ287* mutation. Inactivating muta- tions in NBEAL2 cause recessive gray platelet syndrome, a bleeding disorder characterized by a severe paucity in a- granules.31-33 Although its exact function is still unclear,
NBEAL2 is thought to be required for the biogenesis and/or retention of a-granules in megakaryocytes.42-44 Other proteins with putative roles in a-granule formation, such as VPS33B45 and VIPAS39,46 and proposed NBEAL2 interactors DOCK7, SEC16A and VAC1447 were mostly unaffected in both GFI1BQ287* platelets and iPSC-derived cells, except SEC16A and VAC14, which showed a mild (<2-fold) decrease and increase in GFI1BQ287* platelets, respectively. Possibly, these proteins are under differential control of GFI1B. In contrast to the GFI1BQ287* platelets there was no change in a-granule proteins observed in GFI1BQ287* iPSC-derived megakaryocytic cells. This could suggest that the observed a-granule defect in primary GFI1BQ287* platelets may not be caused by defective protein expression, but possibly originates from defective trans- port from proteins to a-granules and/or defective traffick- ing of a-granules to proplatelets.
In addition to the reduction in a-granule proteins, the proteome of GFI1BQ287* platelets showed other abnormali- ties including increased expression of ribosomal, proteaso- mal and mitochondrial proteins. These findings imply that proplatelet-forming megakaryocytes of GFI1BQ287* patients may differ from normal mature megakaryocytes at the metabolic level. Some of the upregulated platelet proteins, in particular the ribosome subunits, showed significant downregulation during in vitro megakaryocyte differentia- tion, in line with the presumed maturation defect in GFI1BQ287* megakaryocytes. Indeed, early megakaryocytes exhibit high protein synthesis rates to support their increasing cellular mass, while this ceases in the final stages of maturation.48 In addition, proteasomal and mito- chondrial activities are closely related to the regulation of cell fate decisions.49,50 Highly proliferating cells, including cancer cells and (hematopoietic) stem cells, show distinct mitochondrial activities, and proliferating hematopoietic stem cells have increased mitochondrial mass compared to quiescent hematopoietic stem cells.51,52 Thus, the increase in mitochondrial proteins might support the hyperproliferation of GFI1BQ287* megakaryocytes.
In conclusion, GFI1B regulates protein expression in megakaryocytes through recruitment of the LSD1- RCOR-HDAC co-repressor complex. During megakaryo- poiesis many proteins are regulated by GFI1B, which associate with expected but also new processes. GFI1BQ287* may inhibit GFI1B by specifically sequestering the LSD1-RCOR-HDAC complex, making it less avail- able for GFI1B. The normal and affected megakaryocyte and platelet proteomes reported here may serve as a ref- erence for better understanding of other platelet disorders and the molecular pathways that drive megakaryopoiesis and platelet development.
Acknowledgments
The authors would like to thank Clemens Mellink and Anne- Marie van der Kevie-Kersemaekers from the Academic Medical Center Amsterdam, Department of Clinical Genetics, Amsterdam, the Netherlands for performing the karyotyping of BEL-5-Cl2 and Konnie M. Hebeda from the Department of Pathology, Radboudumc, Nijmegen, the Netherlands for her expert opinion on the megakaryocyte electron microscopy results. This work was supported by the Landsteiner Foundation for Blood Transfusion Research (project 1531), Sanquin PPOC 15- 25p-2089 and the Radboudumc.
haematologica | 2019; 104(7)
1471