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Background The therapeutic use of [131I]meta-iodobenzylguanidine ([131I]MIBG) is often accompanied by hematological toxicity, primarily consisting of severe and persistent thrombocytopenia. We hypothesize that this is caused by selective uptake of MIBG via the serotonin transporter (SERT) located on platelets and megakaryocytes. In this study, we have investigated whether in vitro cultured human megakaryocytes are capable of selective plasma membrane transport of MIBG and whether pharmacological intervention with selective serotonin reuptake inhibitors (SSRIs) may prevent this radiotoxic MIBG uptake. Methods Peripheral blood CD34+ cells were differentiated to human megakaryocytic cells using a standardized culture protocol. Prior to [3H]serotonin and [125I]MIBG uptake experiments, the differentiation status of megakaryocyte cultures was assessed by flow cytometry. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to assess SERT and NET (norepinephrine transporter) mRNA expression. On day 10 of differentiation, [3H]serotonin and [125I]MIBG uptake assays were conducted. Part of the samples were co-incubated with the SSRI citalopram to assess SERT-specific uptake. HEK293 cells transfected with SERT, NET, and empty vector served as controls. Results In vitro cultured human megakaryocytes are capable of selective plasma membrane transport of MIBG. After 10 days of differentiation, megakaryocytic cell culture batches from three different hematopoietic stem and progenitor cell donors showed on average 9.2 ± 2.4 nmol of MIBG uptake per milligram protein per hour after incubation with 10–7 M MIBG (range: 6.6 ± 1.0 to 11.2 ± 1.0 nmol/mg/h). Co-incubation with the SSRI citalopram led to a significant reduction (30.1%—41.5%) in MIBG uptake, implying SERT-specific uptake of MIBG. A strong correlation between the number of mature megakaryocytes and SERT-specific MIBG uptake was observed. Conclusion Our study demonstrates that human megakaryocytes cultured in vitro are capable of MIBG uptake. Moreover, the SSRI citalopram selectively inhibits MIBG uptake via the serotonin transporter. The concomitant administration of citalopram to neuroblastoma patients during [131I]MIBG therapy might be a promising strategy to prevent the onset of thrombocytopenia.

Dominant-negative mutations in the transcription factor Growth Factor Independence-1B (GFI1B), such as GFI1BQ287*, cause a bleeding disorder characterized by a plethora of megakaryocyte and platelet abnormalities. The deregulated molecular mechanisms and pathways are unknown. Here we show that both normal and Q287* mutant GFI1B interacted most strongly with the lysine specific demethylase-1 – REST corepressor - histone deacetylase (LSD1-RCOR-HDAC) complex in megakaryoblasts. Sequestration of this complex by GFI1BQ287* and chemical separation of GFI1B from LSD1 induced abnormalities in normal megakaryocytes comparable to those seen in patients. Megakaryocytes derived from GFI1BQ287*-induced pluripotent stem cells also phenocopied abnormalities seen in patients. Proteome studies on normal and mutant-induced pluripotent stem cell-derived megakaryocytes identified a multitude of deregulated pathways downstream of GFI1BQ287* including cell division and interferon signaling. Proteome studies on platelets from GFI1BQ287* patients showed reduced expression of proteins implicated in platelet function, and elevated expression of proteins normally downregulated during megakaryocyte differentiation. Thus, GFI1B and LSD1 regulate a broad developmental program during megakaryopoiesis, and GFI1BQ287* deregulates this program through LSD1-RCOR-HDAC sequestering.

Peripheral blood mononuclear cells were isolated from an individual harboring a heterozygous c.859C -> T p.Q287* mutation in GFI1B, causing an autosomal dominant bleeding disorder, platelet type, 17 (BDPLT17). PBMCs were differentiated to erythroblasts and reprogrammed by lentiviral delivery of a self-silencing hOKSM polycistronic vector. Pluripotency of iPSC line was confirmed by expression of associated markers and by in vitro spontaneous differentiation towards the 3 germ layers. Normal karyotype confirmed the genomic integrity of iPSCs and the presence of disease causingmutationwas shown by Sanger sequencing. The generated iPSCs can be used to study BDPLT17 pathophysiology and basic functions of GFI1B. (c) 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license

The heterozygous Q287* mutation in Growth Factor Independence 1B (GFI1B) causes an autosomal-dominant bleeding disorder characterized by gray platelets as a result of reduced α-granule content.Affected individuals also exhibited macro-thrombocytopenia, increased megakaryocyte numbers and platelet CD34 expression. GFI1B functions as transcriptional repressor by recruiting the histone modifying enzyme LSD1/KDM1A. The C-terminally truncated GFI1B-Q287* mutant has lost its repressive function and inhibits the function of wild type GFI1B in a dominant-negative manner. To study how mutant GFI1B affects megakaryopoiesis, we expressed it in megakaryoblastic MEG01 cells by retroviral transduction. Compared to empty vector transduced cells, expression of GFI1B-Q287* caused a significant growth advantage, which is in line with the strong increase in megakaryocytes in the bone marrow of affected individuals. In contrast to GFI1B-Q287*, expression of GFI1B-WT significantly impaired MEG01 expansion (Figure 1). GFI1B recruits LSD1 through its proline and lysine amino acids at positions 2 and 8, respectively. Individual alanine mutations at these positions disturb the LSD1 interaction. We tested whether the LSD1 interaction was required for the growth phenotypes induced by GFI1B-WT and GFI1B-Q287* by separately introducing alanine mutations (P2A and K8A). This showed that both mutations nullified the growth advantage and disadvantage of GFI1B-Q287* and GFI1B-WT, respectively (Figure 1). Thus, GFI1B-WT may limit megakaryoblast expansion through LSD1 recruitment. The mutant protein may inhibit wild type GFI1B by quenching LSD1. Indeed, preliminary results show that co-expression of LSD1 and GFI1B-Q287* resulted in loss of the proliferative advantage as seen for GFI1B-Q287* alone. Figure 1: P2A and K8A mutations in GFI1B nullified the growth advantage of GFI1B-Q287* and the and growth disadvantage GFI1B-WT. GFP% of MEG01 cells was followed for 26 days. Data is normalized to day 5. To address this finding in more detail, we treated normal human CD34+ bone marrow cells with 4 µM LSD1 inhibitor GSK2879552 and induced megakaryocyte differentiation. This showed an 1.4-fold increased CD34+/CD41+ megakaryoblast expansion after 2 days of treatment. In addition, we observed that CD34 expression was retained and elevated. Thus, LSD1 inhibition of primary CD34+ cells during in vitro megakaryocyte differentiation phenocopies disease characteristics observed in individuals with the GFI1B-Q287* mutation. To further study megakaryopoiesis, we generated induced pluripotent stem (IPS) cells from two individuals with GFI1B-Q287* plus a healthy control. Hematopoietic differentiation of IPS colonies was induced using Stemline II, VEGF and BMP4 for the first 6 days and from day 6 onward in CellQuin, VEGF, BMP4, SCF, IL-1β, IL-3, IL-6 and TPO. Non-adherent hematopoietic cells where harvested and differentiated towards megakaryocytes using CellQuin, IL-1β and TPO. Upon megakaryocyte differentiation a 10-fold increase in expansion of megakaryocytes, sustained CD34 expression and increased hypolobulation was observed compared to GFI1B-WT differentiated IPS cells. Thus, GFI1B-Q287* IPS originated megakaryocytes phenocopy disease characteristics observed in individuals with the GFI1B-Q287* mutation. Our data indicate that the LSD1-GFI1B interaction is key to controlling megakaryoblast expansion and that it is important for proper megakaryocyte maturation as assessed by CD34 expression. These data also suggest that mutant GFI1B-Q287* inhibits the function of wild type GFI1B by quenching the demethylase LSD1. Induced LSD1 expression in the megakaryocyte lineage may be of therapeutic relevance for GFI1B related bleeding and platelet disorders. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.