Growth Factor Independence 1b Research Articles

GFI1B and LSD1 repress myeloid traits during megakaryocyte differentiation

The transcription factor Growth Factor Independence 1B (GFI1B) recruits Lysine Specific Demethylase 1 A (LSD1/KDM1A) to stimulate gene programs relevant for megakaryocyte and platelet biology. Inherited pathogenic GFI1B variants result in thrombocytopenia and bleeding propensities with varying intensity. Whether these affect similar gene programs is unknow. Here we studied transcriptomic effects of four patient-derived GFI1B variants (GFI1BT174N,H181Y,R184P,Q287*) in MEG01 megakaryoblasts. Compared to normal GFI1B, each variant affected different gene programs with GFI1BQ287* uniquely failing to repress myeloid traits. In line with this, single cell RNA-sequencing of induced pluripotent stem cell (iPSC)-derived megakaryocytes revealed a 4.5-fold decrease in the megakaryocyte/myeloid cell ratio in GFI1BQ287* versus normal conditions. Inhibiting the GFI1B-LSD1 interaction with small molecule GSK-LSD1 resulted in activation of myeloid genes in normal iPSC-derived megakaryocytes similar to what was observed for GFI1BQ287* iPSC-derived megakaryocytes. Thus, GFI1B and LSD1 facilitate gene programs relevant for megakaryopoiesis while simultaneously repressing programs that induce myeloid differentiation.

Hemoglobin Response to Zinc Supplementation in Patients with Zinc Deficiency and Chronic Anemia

Background: Zinc is a necessary micronutrient used as a catalyst, structural element, and regulatory ion in many metabolic processes including erythropoiesis. Chronic anemia has an estimated prevalence of 5.6% within the US according to NHANES. Iron deficiency is considered the most common nutritional deficiency leading to anemia, but other nutritional deficiencies have been implicated in anemia including deficiencies of Vitamin A, B12, B6, C, D, and E, folate, Riboflavin, Copper, and Zinc. While zinc fortification has been studied in developing countries and has been associated with a decrease in the overall prevalence of anemia within the pediatric and maternal populations, there have been no studies in adults with chronic anemic in the developed world. The mechanism of Zinc in erythropoiesis is likely multifactorial with zinc acting as a catalyst in alpha-aminolevulinic acid dehydratase enzyme in iron metabolism and heme synthesis. Zinc's incorporation in the structure of the growth factor independent 1B (Gfi-1B) Zinc finger protein and GATA-1 Zinc finger transcription factor, which functions as a regulator in erythroid cell growth by modulating gene expression specific to erythroid series including the SMAD pathways and TGF-β signaling. Zinc's interaction in signaling cascades such as the growth hormone and insulin-like growth factor-1 pathways also result in increased erythropoietin transcription and therefore increased erythropoiesis. In addition, zinc is regarded as an important erythroid differentiation factor. This descriptive, retrospective study describes zinc deficient patients with chronic anemia, who underwent zinc supplementation, and their changes in hemoglobin (HGB) over a 12-month period in an academic outpatient hematology center in California, United States of America. Objectives: Primary endpoints: determine the rate of anemia resolution over 12 months following supplementation of zinc in patients with zinc deficiency and chronic anemia. Secondary endpoints: determine if zinc supplementation in zinc deficient patients with chronic anemia will improve HGB by at least 1.0 g.dL by 1, 3, 6, and 12 month time points. Methods: Ninety Three patients with chronic anemia (HGB <12 for women, HGB <13 for men for >3 months) over the age of 18 were identified to have zinc deficiency (plasma zinc concentration <65 ug/dL) in an academic outpatient hematology center in California. Patients did not start new treatment for their anemia, including initiating new treatments for malignancy 3 months preceding, and within the data collection period. Patients were supplemented with Zinc Sulfate 50 mg daily and their HGB was followed over 12 months. A retrospective analysis was completed for prior medical history, lab data, and HGB was tabulated at the time markers of 1, 3, 6, and 12 months following initiation of zinc supplementation. ANOVA, Wilcoxon rank-sum, and unpaired Students and t tests were performed to determine significant differences where p <0.05 is considered statistically significant. Results Prior to supplementation, average HGB among patients was 9.83 g/dL, WBC was 6.8 g/dL, MCV was 89 fl, PLTS were 211 g/dL. Following 1 month of supplementation average HGB was 10.46 g/dL (p = 0.015, n = 82), at 3 months average HGB was 10.75 g/dL (p = 0.0002, n = 83), at 6 months average HGB was 11.34 g/dL (p= 0.000000004, n = 74), and at 12 months average HGB was 11.48 g/dL (p = 0.00000002, n= 57). Primary endpoint of anemia resolution was found in 39 of 93 patients (42%). Secondary endpoint of HGB improvement by at least 1.0 g/dL was reached in 31% of patients at 1 month, 36% of patients at 3 months, 58% of patients at 6 months and 68% of patients at 12 months. Conclusions: Zinc supplementation significantly improved HGB levels in patients with zinc deficiency and chronic anemia, with resolution of anemia in 42% of patients. There was statistically significant improvement from baseline HGB at the1, 3, 6, and 12 month time markers. Further study is warranted to determine the specific mechanism of zinc deficiency in anemia, as well as clinical studies determining zinc supplementation doses, monitoring, and toxicity.

Hemoglobin Response to Zinc Supplementation in Chronic Kidney Disease in Patients with Zinc Deficiency and Anemia

Background: Chronic anemia has an estimated prevalence of 5.6% within the US, where 25.3% of patients with chronic kidney disease (CKD) 3-5 have anemia of chronic kidney disease according to NHANES. Anemia in CKD has long been attributed to a decrease in EPO production due to their kidney disease. While iron deficiency is treated in anemia of chronic kidney disease, other micronutrients are less commonly tested or supplemented. Zinc may be an additional micronutrient of interest in anemia of chronic kidney disease as zinc has numerous proposed mechanisms in erythropoeisis including acting as a catalyst in the iron metabolism and heme synthesis through the activity of alpha-aminolevulinic acid dehydratase enzyme. Zinc is found in the structure of the growth factor independent 1B (Gfi-1B) Zinc finger protein and GATA-1 Zinz finger transcription factor, which functions as a regulator in erythroid cell growth by modulating gene expression specific to erythroid series including the SMAD pathways and TGF-β signaling. The interaction with zinc in signaling cascades such as the growth hormone and insulin-like growth factor-1 pathways also result in increased erythropoietin transcription and therefore increased erythropoiesis. In addition, zinc is regarded as an important erythroid differentiation factor which progress maturation of erythrocytes. Zinc has also shown to stimulate EPO production in nephrectomized rats and Zinc supplementation increased reticulocyte production in human subjects. This descriptive, retrospective study describes zinc deficient patients with CKD 3-5 and chronic anemia; who underwent oral zinc supplementation, and their changes in hemoglobin (HGB) over a 12 month period in an academic outpatient hematology clinic in California, United States of America. Objectives: Primary endpoints: determine the rate of anemia resolution over 12 months following supplementation of zinc in patients with zinc deficiency, CKD, and anemia of chronic kidney disease. Secondary endpoints: determine if zinc supplementation in zinc deficient patients with chronic anemia and CKD 3-5 will improve HGB by at least 1.0 g.dL by 1, 3, 6, and 12 month time points. Methods: Hematology patients from Loma Linda University Cancer Center over the age of 18 who had CKD 3 or above (GFR <59 ml/min) with chronic anemia (HGB <12 for women, HGB <13 for men for >3 months) were identified to have zinc deficiency (plasma zinc concentration <65 ug/dL). Patients did not start new treatment for their anemia, including initiating new treatments for malignancy 3 months preceding, and within the data collection period. Patients were supplemented with Zinc Sulfate 50 mg daily and their HGB was followed over 12 months. A retrospective analysis was completed for prior medical history, lab data, and hemoglobin was tabulated at the time markers of 1, 3, 6, and 12 months following initiation of zinc supplementation. ANOVA, Wilcoxon rank-sum, and unpaired Students and t tests were performed to determine significant differences. p <0.05 is considered statistically significant. Results Thirty-Four patients were identified to have CKD 3-5 with chronic anemia and zinc deficiency. They were found to have no statistically significant baseline characteristics, hematologic values at time of diagnosis, or micronutrient and hormonal etiologies for anemia. HGB prior to supplementation averaged 9.96 g/dL, WBC averaged 5.8, and PLTS averaged 149. Following supplementation HGB at 1 month average was 10.47 g/dL (p=0.19 n=30), at 3 months average of 10.49 (p=0.12 n=31), at 6 months average of 11.03 (p=0.011 n=27), at 1 year 10.93 (p=0.03, n=17). Primary endpoint of HGB resolution was found in 12 of the 34 patients (35%). Secondary endpoint of HGB improvement by at least 1.0 g/dL was reached in 27% of patients at 1 month, 32% of patients at 3 months, 59% of patients at 6 months and 64% of patients at 12 months. Conclusions: Zinc supplementation improved patients' anemia with resolution of anemia in 35% of patients with incremental improvement in HGB over a 1 year time frame. There was statistically significant improvement from baseline HGB at the 6 and 12 month time markers. Further study is warranted to determine the specific mechanism of zinc deficiency in anemia with chronic kidney disease, as well as clinical studies determining zinc supplementation doses, monitoring, and toxicity.

Diagnostic value of multigene sequencing for inherited thrombocytopenia.

Multigene testing for inherited thrombocytopenia can detect an unexpected diagnosis as demonstrated in the case report from Ming-Ping et al., where the mode of inheritance and clinical phenotype did not point towards dominant Schlafen family member 14 (SLFN14) moderate thrombocytopenia. They describe two siblings with very severe thrombocytopenia due a pathogenic SLFN14 variant and gonosomal mosaicism in their healthy mother. In their paper Ming-Ping et al. describe two siblings with severe thrombocytopenia (platelets counts <15 × 109/l) and bleeding symptoms, born from healthy non-consanguineous parents.1 The first child died at the age of 5 months due to intracranial haemorrhage. This child also had larger platelets, while this was not found for his affected sister. She has received regular platelet transfusions since birth. Neonatal and immune thrombocytopenia were ruled out. Their diagnosis was made after using whole-exome sequencing with the detection of a heterozygous pathogenic SLFN14 variant that is responsible for dominant thrombocytopenia. Interestingly, gonosomal mosaicism for this variant was detected in the mother who has normal platelet counts. This case report nicely demonstrates the power of multigene sequencing for the diagnosis of inherited thrombocytopenia, which is an extremely heterogenous disorder with already 40 diagnostic-grade genes as potential underlying cause (Figure 1).2, 3 Using only textbook knowledge, the diagnosis of SLFN14-related thrombocytopenia could probably never have been made for these patients. Based on the clinical phenotypes present in the family members, a recessive form of inherited (macro)thrombocytopenia was expected as parents have a normal platelet count. In addition, SLFN14-related thrombocytopenia is an ultrarare disorder and clinical and laboratory data have only been described for four unrelated pedigrees.4 All patients with pathogenic SLFN14 variants have platelet counts >65 × 109/l (moderate thrombocytopenia) and highly variable degrees of bleeding symptoms and International Society of Thrombosis and Haemostasis (ISTH) bleeding scores ranging between 2 and 20. The children in this case report have a much more severe phenotype and still they carry the same genetic variant as previously described.4 In the past, several diagnostic algorithms have been proposed to assist clinicians in the differential diagnosis of inherited thrombocytopenia. The classification of inherited thrombocytopenia was typically driven by the presence or absence of other clinical features besides a platelet defect (syndromic versus non-syndromic), the mode of inheritance (recessive, dominant and X-linked) and the platelet size (macro, micro and normal size platelets) (Figure 1).5 Applying such an algorithm was very useful, as the numbers of genes implicated in inherited thrombocytopenia was less than half of the genes we know today. However, this algorithm would have missed the diagnosis in the family. Since the discovery of many novel genes for inherited thrombocytopenia using next-generation sequencing approaches over the last decade, phenotype–genotype associations for this disorder have become very complex.6 In addition, the mode of inheritance has expanded for well-known genes (e.g., dominant mild Bernard–Soulier syndrome versus the classical recessive form and recessive Fli-1 proto-oncogene, ETS transcription factor [FLI1] and growth factor-independent 1B transcriptional repressor [GFI1B] variants versus the known dominant forms of this type of thrombocytopenia). To date, multigene panel testing is typically used to diagnose inherited thrombocytopenia by investigating all the known genes involved in this pathology in a single analysis.7 Providing detailed clinical and laboratory information to genetic laboratories remains very important for the interpretation of this test as it assists in the variant classification to decide if a variant is pathogenic or not. The major drawbacks of performing a multigene panel test for inherited thrombocytopenia are the detection of many variants of unknown significance than cannot be used for clinical management and unsolicited findings. If for example pathogenic variants are detected in either RUNX family transcription factor 1 (RUNX1), ETS variant transcription factor 6 (ETV6) or ankyrin repeat domain-containing 26 (ANKRD26) in a patient with thrombocytopenia without a family history of leukaemia, such variants can be considered as an unsolicited finding if the patient was not aware about these genetic risk factors for leukaemia before initiating the multigene test. On the other hand, a multigene test allows the detection of ‘the unexpected diagnosis’. This case report provides such an example as a SLFN14 defect was not really expected based on the known disease phenotypes associated with pathogenic variants in this gene.

P1649: GFI1B AND LSD1 REPRESS A MYELOID DIFFERENTIATION PROGRAM DURING INDUCED PLURIPOTENT STEM CELL DERIVED MEGAKARYOCYTE DIFFERENTIATION

Background: Megakaryocytic development is driven by the transcription factor Growth Factor Independence 1B (GFI1B) and its interaction with the chromatin remodeler Lysine Specific Demethylase 1 (LSD1/KDM1A). GFI1B aberrations are linked to inherited bleeding disorders, resulting in macro-thrombocytopenia, gray platelets and increased expression of CD34. One such mutation, which was previously show to be the disease-causing mutation in a family with gray-platelet syndrome, introduces a premature stop codon (Q287*) in the fifth zinc finger. Aims: Here, we study the effect of the Q287* mutation on gene expression regulation, as well as GFI1B interaction with LSD1, during induced pluripotent stem cells (IPSC) derived megakaryocytic development. Methods: We differentiated IPSCs that contain wildtype GFI1B or GFI1BQ287* into megakaryocytes. During differentiation, megakaryocytes derived from GFI1B wildtype IPSCs were also treated with an LSD1 inhibitor that disrupts GFI1B-LSD1 interaction for 24 hours. The transcriptomic landscape of differentiated IPSCs was explored using single-cell RNA sequencing and downstream computational analysis. We applied a novel filtering strategy, removing cells based on doublet annotation, mitochondrial gene count and exclusive differential expression of ribosomal or mitochondrial genes. Results: After filtering and data integration we retained 14,848 cells. Within this population we were able to annotate six different cell types using single-cell GSEA and publicly available reference datasets. Megakaryocytes and megakaryocyte progenitors made up roughly 75% of all cells differentiated from IPSCs containing wildtype GFI1B. They are characterized by approximately 220 differentially expressed genes (p-value < 0.05, log-fold change > 0.69), including common megakaryocyte markers such as CD41 and CD42b. Strikingly, we also observed a population of myeloid and myeloid-progenitor cells after differentiation towards megakaryocytes. This population specifically expressed common myeloid markers, such as VIM, CD81, and SPI1 as well as approximately 200 other genes (p-value < 0.05, log-fold change > 0.69) and made up around 10% of the entire GFI1B wildtype cell population. This population was expanded two-fold in cells differentiated from GFI1B-wildtype IPSCs treated with the LSD1 inhibitor and three-fold in GFI1BQ287* IPSCs. Notably, in the GFI1BQ287* cell population megakaryocytes were reduced by 2-fold. Pseudotime analysis showed a differentiation trajectory starting at megakaryocytes and going towards myeloid cells. Megakaryocyte and platelet associated genes, such as CD41, CD42b and PF4, are gradually downregulated along this trajectory while myeloid associated genes exhibited a mirrored expression pattern. Thus, LSD1i treated and GFI1BQ287* IPSC-derived cells showed an increase in myeloid marker gene expression after differentiation and downregulation of coagulation-associated genes, hinting at a decreased potential to differentiate into megakaryocytes. Summary/Conclusion: Our data show that GFI1B and LSD1 function to repress a myeloid cell fate during IPSC-induced megakaryocytic development. Disruption of the interaction between GFI1B and LSD1 and a dominant-negative GFI1B mutation results in myeloid differentiation at the expense of megakaryocyte development.

Characterization of a genomic region 8 kb downstream of GFI1B associated with myeloproliferative neoplasms

A genomic locus 8 kb downstream of the transcription factor GFI1B (Growth Factor Independence 1B) predisposes to clonal hematopoiesis and myeloproliferative neoplasms. One of the most significantly associated polymorphisms in this region is rs621940-G. GFI1B auto-represses GFI1B, and altered GFI1B expression contributes to myeloid neoplasms. We studied whether rs621940-G affects GFI1B expression and growth of immature cells. GFI1B ChIP-seq showed clear binding to the rs621940 locus. Preferential binding of various hematopoietic transcription factors to either the rs621940-C or -G allele was observed, but GFI1B showed no preference. In gene reporter assays the rs621940 region inhibited GFI1B promoter activity with the G-allele having less suppressive effects compared to the C-allele. However, CRISPR-Cas9 mediated deletion of the locus in K562 cells did not alter GFI1B expression nor auto-repression. In healthy peripheral blood mononuclear cells GFI1B expression did not differ consistently between the rs621940 alleles. Long range and targeted deep sequencing did not detect consistent effects of rs621940-G on allelic GFI1B expression either. Finally, we observed that myeloid colony formation was not significantly affected by either rs621940 allele in 193 healthy donors. Together, these findings show no evidence that rs621940 or its locus affect GFI1B expression, auto-repression or growth of immature myeloid cells.

Specific proteome changes in platelets from individuals with GATA1-, GFI1B-, and RUNX1-linked bleeding disorders

AbstractMutations in the transcription factors GATA binding factor 1 (GATA1), growth factor independence 1B (GFI1B), and Runt-related transcription factor 1 (RUNX1) cause familial platelet and bleeding disorders. Mutant platelets exhibit common abnormalities including an α-granule reduction resulting in a grayish appearance in blood smears. This suggests that similar pathways are deregulated by different transcription factor mutations. To identify common factors, full platelet proteomes from 11 individuals with mutant GATA1R216Q, GFI1BQ287*, RUNX1Q154Rfs, or RUNX1TD2-6 and 28 healthy controls were examined by label-free quantitative mass spectrometry. In total, 2875 platelet proteins were reliably quantified. Clustering analysis of more than 300 differentially expressed proteins revealed profound differences between cases and controls. Among cases, 44 of 143 significantly downregulated proteins were assigned to platelet function, hemostasis, and granule biology, in line with platelet dysfunction and bleedings. Remarkably, none of these proteins were significantly diminished in all affected cases. Similarly, no proteins were commonly overrepresented in all affected cases compared with controls. These data indicate that the studied transcription factor mutations alter platelet proteomes in distinct largely nonoverlapping manners. This work provides the quantitative landscape of proteins that affect platelet function when deregulated by mutated transcription factors in inherited bleeding disorders.

Platelet CD34 Expression and a Congenital Collar Bone Malformation Associated with a Partial CBFB Deletion in a Case with a Bleeding Disorder

Mutations in the transcription factor Growth Factor Independence 1B (GFI1B) and Runt-related transcription factor 1 (RUNX1) are causal to familial bleeding disorders. Mutant RUNX1 and GFI1B affect megakarocyte development, resulting in decreased numbers of platelets with significantly reduced α-granules and platelets that express the hematopoietic stem and progenitor cell marker CD34. A case that presented in our clinic with a bleeding disorder (Tosetto bleeding score of 11) was diagnosed with a δ-granule secretion defect. Platelet light transmission aggregometry revealed an aggregation defect upon stimulation with epinephrine and low concentrations of collagen and ADP. ATP release from δ-granules was normal after stimulation with a high concentration of collagen, however, no ATP was released upon stimulation with epinephrine and low levels were measured after stimulation with the thromboxane analogue U46619. Whole mount electron microscopy showed increased numbers of δ-granules in platelets derived from the index case compared to controls (average 5.84 δ-granules vs 3.66, per platelet respectively). To identify a genetic cause for the disease, a bleeding disorder gene panel was screened following whole exome sequencing. No disease-causing variants were called. Open exome analysis detected a ~100Kb heterozygous chromosome 16q22.1 deletion that was confirmed by SNP array. The observed deletion covers nine genes, including the last exon of CBFB. To date, none of the deleted genes have been implicated in inherited bleeding or platelet disorders. However, conditional knockout of Cbfb in mice results in a plethora of hematopoietic abnormalities including disturbed megakaryopoiesis and thrombocytopenia. CBFB is part of a heterodimeric transcription factor complex with either RUNX1, -2 or -3. Because of the CBFB-RUNX1 interaction and the fact that bleeding disorders caused by RUNX1 mutations result in platelet CD34 expression, we tested the platelets of the case reported here. Using flow cytometry, we clearly detected platelet CD34 expression compared to healthy controls, albeit to a lower level compared to RUNX1 and GFI1B affected cases. Mutations in RUNX2 may cause cleidocranial dysplasia and entire CBFB deletions have been reported in rare cases with congenital bone abnormalities. The medical record of the case presented here indicated that she had undergone surgery at the age of 13 for a congenital malformed collar bone. These data suggest a relation between the 16q/CBFB deletion and the observed abnormalities. To address this, we determined whether the abnormalities segregated with the 16q deletion. The mother and son of the index case had no history of clinical bleedings, no signs of bone malformations and did not have the 16q deletion. The father who was deceased was reported by the family not to have a bleeding propensity nor bone abnormalities. Platelets from the son did not express CD34. Remarkably, platelets from the son showed an aggregation defect upon stimulation with epinephrine and ADP, similar to the index patient. No ATP was released from δ-granules following stimulation with epinephrine. Thus, the 16q deletion associates with platelet CD34 expression and a congenital bone abnormality but is not responsible for the δ -granule secretion defect as the latter was observed in the son who did not have the 16q deletion, nor bleedings. Defective downmodulation of CD34 during platelet formation in the index case reported here suggests abnormal megakaryocyte development that might contribute in a multifactorial manner to bleedings. Recently, we reported CD34 expression on platelets from patients with rare GFI1B variants that may predispose to a bleeding propensity, but do not cause bleedings on their own. Thus, it will be important to determine whether the 16q deletion (and C-terminal truncation of CBFB) reported here affects megakaryocyte development and if so, contributes to the bleeding disorder in the index case. In contrast to Cbfb null mice, Cbfb heterozygous mice do not exhibit discernable hematopoietic phenotypes. Thus, if the C-terminal CBFB truncation contributes to the disease characteristics, it is likely that this occurs through a dominant-negative mechanism rather than haploinsufficiency. The findings reported here warrant further studies into the role of 16q (and CBFB) deletions in megakaryocyte development. Disclosures No relevant conflicts of interest to declare.

Inhibition of the MiR-185-PAK6-Mediated Survival and Metabolic Pathways Selectively Targets Drug-Resistant CML Stem/Progenitor Cells

Overcoming drug resistance and targeting leukemic stem cells (LSCs) remain major challenges for curative treatment of human leukemia, including chronic myeloid leukemia (CML). Indeed, most patients with CML require life-long therapy with ABL1 tyrosine kinase inhibitors (TKIs), due to the persistence of residual LSCs that maintain the potential for relapse. Increasing evidence also indicates that LSCs are susceptible to cellular metabolic changes and seem to have a greater dependence on mitochondrial oxidative phosphorylation (OXPHOS) for survival. Previously, through global transcriptome profiling, we identified a key microRNA (miRNA), miR-185 as a predictive biomarker and it is also required for CML LSC survival. Its expression was significantly reduced in CD34+treatment-naïve CML cells and predictive of therapy response. Conversely, restored expression of miR-185 by lentiviral transduction in CD34+TKI-nonresponder cells significantly impaired survival of these cells, sensitizing them to TKIs in vitroand in pre-clinical xenotransplantation models, indicating that miR-185 acts as a tumor suppressor and is critical in regulating TKI response/resistance of CML stem/progenitor cells. PAK6, a serine/threonine-protein kinase, was identified as a target gene of miR-185; it is upregulated in CD34+TKI-nonresponder cells vs. TKI-responders, correlating with reduced miR-185 expression. To further investigate the molecular and biological roles of the miR-185-PAK6 axisin the regulation of survival of drug-resistant cells, including LSCs, we performed RNA-seq and gene set enrichment analysis (GSEA) in the same CD34+patient cells where miR-185 and PAK6 were identified as being differentially expressed. Interestingly, this analysis has now identified a significant gene set enrichment of OXPHOS, reactive oxygen species (ROS), and adipogenesis pathways in CD34+CML cells compared to healthy CD34+cells (Normalized Enrichment Scores (NES): 2.44, 1.65 and 1.8). Moreover, these changes were significantly higher in TKI-nonresponder cells than in TKI-responders (NES: 1.73, 0.49 and 0.34). We have thus hypothesized that the miR-185-PAK6 axis may contribute to the perturbation of specific metabolic pathways in TKI-nonresponder LSC/progenitor cells and confer therapy-resistance to these cells. Indeed, a pre-clinically validated pan-PAK inhibitor (PF-3758309) alone, or in combination with a TKI, greatly reduced mitochondrial activity in TKI-nonresponder cells, in MitoTracker analysis, an effect that was not seen in the same cells treated with a TKI. Notably, ROS production was also significantly reduced in these cells treated with PF-3758309 and further reduction was observed with a combination of PF-3758309 and TKIs. Notably, PF-3758309 significantly reduced the growth of IM-resistant cell lines (IC50 25-70 nM) and CD34+TKI-nonresponder cells, as assessed by viability and colony-forming cell assays, and increased their apoptosis; these effects were greatly enhanced by TKIs (2-fold, P&lt;0.05). These results were further confirmed in TKI-resistant cells using a lentiviral CRISPR/Cas9 knockout system that specifically targets PAK6. In addition, specific molecular changes associated with PF-3758309 treatment were also investigated using the PharmacoDB database and PharmacoGx R-package. Several candidates were identified, including growth factor independent 1B transcriptional repressor (GFI1B), a myeloid-enriched transcription factor. Its expression was reduced by PF-3758309 treatment and significantly increased in CML compared to healthy controls (&gt;2-fold). Interestingly, expression of GFI1B is further upregulated in CD34+TKI-nonresponders compared to responders. Taken together, these findings indicate that dual targeting of miR-185-PAK6-mediated survival and metabolic pathways, along with BCR-ABL, selectively eradicates therapy-resistant LSC/progenitors, providing a valuable therapeutic strategy for improved treatment and care. Disclosures No relevant conflicts of interest to declare.

Growth Factor Independence 1B-Mediated Transcriptional Repression and Lineage Allocation Require Lysine-Specific Demethylase 1-Dependent Recruitment of the BHC Complex.

Growth factor independence 1B (GFI1B) coordinates assembly of transcriptional repressor complexes comprised of corepressors and histone-modifying enzymes to control gene expression programs governing lineage allocation in hematopoiesis. Enforced expression of GFI1B in K562 erythroleukemia cells favors erythroid over megakaryocytic differentiation, providing a platform to define molecular determinants of binary fate decisions triggered by GFI1B. We deployed proteome-wide proximity labeling to identify factors whose inclusion in GFI1B complexes depends upon GFI1B's obligate effector, lysine-specific demethylase 1 (LSD1). We show that GFI1B preferentially recruits core and putative elements of the BRAF-histone deacetylase (HDAC) (BHC) chromatin-remodeling complex (LSD1, RCOR1, HMG20A, HMG20B, HDAC1, HDAC2, PHF21A, GSE1, ZMYM2, and ZNF217) in an LSD1-dependent manner to control acquisition of erythroid traits by K562 cells. Among these elements, depletion of both HMG20A and HMG20B or of GSE1 blocks GFI1B-mediated erythroid differentiation, phenocopying impaired differentiation brought on by LSD1 depletion or disruption of GFI1B-LSD1 binding. These findings demonstrate the central role of the GFI1B-LSD1 interaction as a determinant of BHC complex recruitment to enable cell fate decisions driven by GFI1B.

S847 DISTINCT AND COMMON DEREGULATED PATHWAYS IN RUNX1, GATA1 AND GFI1B ASSOCIATED BLEEDING DISORDERS

Background:Inherited mutations in the transcription factors (TF) Runt‐related transcription factor‐1 (RUNX1), Growth factor independence 1B (GFI1B) and GATA Binding Protein1 (GATA1) cause megakaryocyte and platelet dysfunction resulting in bleeding disorders. Several platelet abnormalities overlap (e.g. platelet α‐granule paucity) suggesting commonly affected pathways.Aims:The functional effects of many pathogenic mutations remain largely unknown. To further the understanding of the pathobiology of TF mutated bleeding disorders, we identified the affected pathways and proteins important during megakaryopoiesis.Methods:We identified variants in RUNX1 (TD2‐6, Q154fs, G165S), GFI1B (Q287∗, T174N) and GATA1 (R216Q) by next generation sequencing of DNA from cases with thrombocytopenia/pathy and bleedings. According to AMCG guidelines all but the RUNX1 G165S and GFI1B T174N mutations were classified as pathogenic. To identify (commonly) affected biological pathways downstream of mutated TFs, we analyzed platelets from these cases and five controls with label free quantification (LFQ) mass spectrometry.Results:Proteomes from controls were highly similar and clearly distinct from all cases with bleedings. Besides, the non‐pathogenic variants in RUNX1 (G165S) and GFI1B (T174N) clustered together but separate from controls and cases with pathogenic mutations. Pathogenic RUNX1, GFI1B and GATA1 proteomes were also clearly distinct from each other (P &lt; 0.001, between 220 and 520 proteins significantly differentially expressed). For cases with pathogenic TF mutations, gene ontology (GO) pathway analysis detected significantly downregulated platelet a‐granule, ‐ degranulation and ‐ activation pathways. Platelet α‐granule proteins such as platelet factor 4V1, SERPINE2 and F5, but also the structural MYL9 protein were all significantly downregulated. In addition, all pathogenic TF patient samples had negative enrichment for coagulation proteins, compared to control samples. Remarkably, in all TF affected platelets we observed a negative enrichment for JAK‐STAT signaling (AKT1, JAK2, JAK3), normally required for proper megakaryocyte differentiation. To address this finding in more detail, we determined the proteomes of normal megakaryocytes at different stages of differentiation derived from primary immature CD34 positive cells. TF mutated platelet proteomes clearly resembled immature rather than mature megakaryocytes, in line with defective megakaryocyte maturation. For example, in affected platelets an increase in oxidative phosphorylation proteins (ATP6V1A, NDUFS3, NDUFA2) was observed, that are downregulated upon normal megakaryocyte maturation. Many other affected pathways, including the ubiquitin‐proteasome pathway were deregulated in affected platelets.Summary/Conclusion:Pathogenic TF mutations have dominant distinctive effects on platelet proteome composition. Platelets from cases without a proven TF mutation have similar affected proteomes distinct from controls and cases with pathogenic TF mutations. Thus, the studied TF mutations have a dramatic effect on the platelet proteome, in line with the plethora of abnormalities in megakaryocytes and platelets. Identifying which pathways and proteins act during megakaryopoiesis and which are affected by TF mutations allows identification of biological pathways previously unknown to be fundamental to platelet development.

Molecular mechanisms of bleeding disorderassociated GFI1BQ287* mutation and its affected pathways in megakaryocytes and platelets.

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.

T-448, a specific inhibitor of LSD1 enzyme activity, improves learning function without causing thrombocytopenia in mice.

Dysregulation of histone H3 lysine 4 (H3K4) methylation has been implicated in the pathogenesis of several neurodevelopmental disorders. Targeting lysine-specific demethylase 1 (LSD1), an H3K4 demethylase, is therefore a promising approach to treat these disorders. However, LSD1 forms complexes with cofactors including growth factor independent 1B (GFI1B), a critical regulator of hematopoietic differentiation. Known tranylcypromine-based irreversible LSD1 inhibitors bind to coenzyme flavin adenine dinucleotide (FAD) and disrupt the LSD1-GFI1B complex, which is associated with hematotoxicity such as thrombocytopenia, representing a major hurdle in the development of LSD1 inhibitors as therapeutic agents. To discover LSD1 inhibitors with potent epigenetic modulation and lower risk of hematotoxicity, we screened small molecules that enhance H3K4 methylation by the inhibition of LSD1 enzyme activity in primary cultured rat neurons but have little impact on LSD1-GFI1B complex in human TF-1a erythroblasts. Here we report the discovery of a specific inhibitor of LSD1 enzyme activity, T-448 (3-((1S,2R)-2-(cyclobutylamino)cyclopropyl)-N-(5-methyl-1,3,4-thiadiazol-2-yl)benzamide fumarate). T-448 has minimal impact on the LSD1-GFI1B complex and a superior hematological safety profile in mice via the generation of a compact formyl-FAD adduct. T-448 increased brain H3K4 methylation and partially restored learning function in mice with NMDA receptor hypofunction. T-448-type LSD1 inhibitors with improved safety profiles may provide unique therapeutic approaches for central nervous system disorders associated with epigenetic dysregulation.

3120 - Dominant-Negative GFI1B Mutations are Causal in Rare Inherited Platelet Disorders and Cause Defects in Stress Thrombopoiesis

Inherited platelet disorders represent a heterogeneous group of rare diseases often characterized by lifelong bleeding diatheses. Specific therapies are lacking because the affected molecular processes are poorly understood. Previously, heterozygous nonsense mutations in Growth Factor Independence 1b (GFI1B) in families with autosomal-dominant bleeding disorders were identified. This disease is characterized by a plethora of megakaryocyte (MK) and platelet abnormalities, including macro thrombocytopenia, dysmorphic megakaryocytes, paucity of platelet /-granules, CD34 expression on MKs and platelets, increased MK numbers and their dislocation in the marrow. GFI1B is a transcription factor that contains an evolutionary conserved N-terminal SNAG domain and six C-terminal zinc fingers of which zinc finger three to five are crucial for DNA binding. Through the SNAG domain GFI1B recruits chromatin-modifying enzymes to promoters of target genes to regulate gene expression. Many mutations in the GFI1B gene found in inherited platelet disorders are located in zinc finger five, resulting in a DNA binding defective protein. To study the consequences of these mutations in vivo, we used Crispr/Cas technology and established three independent mouse lines that carry one Gfi1b allele in which the 5th and 6th zinc fingers are truncated or eliminated. All three lines of GFI1B DN mice had a mild but significant reduction of platelet counts resembling the situation found in patients. Moreover, GFI1B DN mice showed an expansion MKs and Mk precursors as well as hematopoietic stem cells. To test whether the DN mutations affect platelet recovery, we injected GFI1B DN mice and controls with an anti GP-IB antibody, which leads to an almost complete depletion of platelets. Clearly, GFI1B DN mice showed a delayed recovery of platelet numbers compared to controls. In addition, in many cases the platelets of DN mice were larger and appeared less granular after recovery than their counterparts in wt mice. Our data provide evidence for a causal relationship between inherited mutations in GFI1B in patients with bleeding disorders and reveal a new role for GFI1B in stress thrombopoiesis.

Gfi1b: a key player in the genesis and maintenance of acute myeloid leukemia and myelodysplastic syndrome.

Differentiation of hematopoietic stem cells is regulated by a concert of different transcription factors. Disturbed transcription factor function can be the basis of (pre)malignancies such as myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Growth factor independence 1b (Gfi1b) is a repressing transcription factor regulating quiescence of hematopoietic stem cells and differentiation of erythrocytes and platelets. Here, we show that low expression of Gfi1b in blast cells is associated with an inferior prognosis of MDS and AML patients. Using different models of human MDS or AML, we demonstrate that AML development was accelerated with heterozygous loss of Gfi1b, and latency was further decreased when Gfi1b was conditionally deleted. Loss of Gfi1b significantly increased the number of leukemic stem cells with upregulation of genes involved in leukemia development. On a molecular level, we found that loss of Gfi1b led to epigenetic changes, increased levels of reactive oxygen species, as well as alteration in the p38/Akt/FoXO pathways. These results demonstrate that Gfi1b functions as an oncosuppressor in MDS and AML development.

Thrombocytopenia and CD34 expression is decoupled from α‐granule deficiency with mutation of the first growth factor‐independent 1B zinc finger

Background Mutation of the growth factor-independent 1B (GFI1B) fifth DNA-binding zinc-finger domain causes macrothrombocytopenia and α-granule deficiency leading to clinical bleeding. The phenotypes associated with GFI1B variants disrupting non-DNA-binding zinc-fingers remain uncharacterized. Objectives To determine the functional and phenotypic consequences of GFI1B variants disrupting non-DNA-binding zinc-finger domains. Methods The GFI1B C168F variant and a novel GFI1B c.2520 + 1_2520 + 8delGTGGGCAC splice variant were identified in four unrelated families. Phenotypic features, DNA-binding properties and transcriptional effects were determined and compared with those in individuals with a GFI1B H294 fs mutation of the fifth DNA-binding zinc-finger. Patient-specific induced pluripotent stem cell (iPSC)-derived megakaryocytes were generated to facilitate disease modeling. Results The DNA-binding GFI1B variant C168F, which is predicted to disrupt the first non-DNA-binding zinc-finger domain, is associated with macrothrombocytopenia without α-granule deficiency or bleeding symptoms. A GFI1B splice variant, c.2520 + 1_2520 + 8delGTGGGCAC, which generates a short GFI1B isoform that lacks non-DNA-binding zinc-fingers 1 and 2, is associated with increased platelet CD34 expression only, without quantitative or morphologic platelet abnormalities. GFI1B represses the CD34 promoter, and this repression is attenuated by different GFI1B zinc-finger mutations, suggesting that deregulation of CD34 expression occurs at a direct transcriptional level. Patient-specific iPSC-derived megakaryocytes phenocopy these observations. Conclusions Disruption of GFI1B non-DNA-binding zinc-finger 1 is associated with mild to moderate thrombocytopenia without α-granule deficiency or bleeding symptomatology, indicating that the site of GFI1B mutation has important phenotypic implications. Platelet CD34 expression appears to be a common feature of perturbed GFI1B function, and may have diagnostic utility.

Gene expression profiling of idiopathic interstitial pneumonias (IIPs): identification of potential diagnostic markers and therapeutic targets

BackgroundChronic fibrosing idiopathic interstitial pneumonia (IIP) is characterized by alveolar epithelial damage, activation of fibroblast proliferation, and loss of normal pulmonary architecture and function. This study aims to investigate the genetic backgrounds of IIP through gene expression profiling and pathway analysis, and to identify potential biomarkers that can aid in diagnosis and serve as novel therapeutic targets.MethodsRNA extracted from lung specimens of 12 patients with chronic fibrosing IIP was profiled using Illumina Human WG-6 v3 BeadChips, and Ingenuity Pathway Analysis was performed to identify altered functional and canonical signaling pathways. For validating the results from gene expression analysis, immunohistochemical staining of 10 patients with chronic fibrosing IIP was performed.ResultsNinety-eight genes were upregulated in IIP patients relative to control subjects. Some of the upregulated genes, namely desmoglein 3 (DSG3), protocadherin gamma-A9 (PCDHGA9) and discoidin domain-containing receptor 1 (DDR1) are implicated in cell-cell interaction and/or adhesion; some, namely collagen type VII, alpha 1 (COL7A1), contactin-associated protein-like 3B (CNTNAP3B) and mucin-1 (MUC1) are encoding the extracellular matrix molecule or the molecules involved in cell-matrix interactions; and the others, namely CDC25C and growth factor independent protein 1B (GFI1B) are known to affect cell proliferation by affecting the progression of cell cycle or regulating transcription. According to pathway analysis, alternated pathways in IIP were related to cell death and survival and cellular growth and proliferation, which are more similar to cancer than to inflammatory response and immunological diseases. Using immunohistochemistry, we further validate that DSG3, the most highly upregulated gene, shows higher expression in chronic fibrosing IIP lung as compared to control lung.ConclusionWe identified several genes upregulated in chronic fibrosing IIP patients as compared to control, and found genes and pathways implicated in cancer, rather than in inflammatory or immunological disease to play important roles in the pathogenesis of IIPs. Moreover, DSG3 is a novel potential biomarker for chronic fibrosing IIP with its significantly high expression in IIP lung.

A Novel LSD1 Inhibitor T-3775440 Disrupts GFI1B-Containing Complex Leading to Transdifferentiation and Impaired Growth of AML Cells.

Dysregulation of lysine (K)-specific demethylase 1A (LSD1), also known as KDM1A, has been implicated in the development of various cancers, including leukemia. Here, we describe the antileukemic activity and mechanism of action of T-3775440, a novel irreversible LSD1 inhibitor. Cell growth analysis of leukemia cell lines revealed that acute erythroid leukemia (AEL) and acute megakaryoblastic leukemia cells (AMKL) were highly sensitive to this compound. T-3775440 treatment enforced transdifferentiation of erythroid/megakaryocytic lineages into granulomonocytic-like lineage cells. Mechanistically, T-3775440 disrupted the interaction between LSD1 and growth factor-independent 1B (GFI1B), a transcription factor critical for the differentiation processes of erythroid and megakaryocytic lineage cells. Knockdown of LSD1 and GFI1B recapitulated T-3775440-induced transdifferentiation and cell growth suppression, highlighting the significance of LSD1-GFI1B axis inhibition with regard to the anti-AML effects of T-3775440. Moreover, T-3775440 exhibited significant antitumor efficacy in AEL and AMKL xenograft models. Our findings provide a rationale for evaluating LSD1 inhibitors as potential treatments and indicate a novel mechanism of action against AML, particularly AEL and AMKL. Mol Cancer Ther; 16(2); 273-84. ©2016 AACR.

Megakaryocyte Expansion and Platelet CD34 Expression Observed in GFI1BQ287*-Related Bleeding and Platelet Disorder Is Caused By Quenching of the Lysine Specific Demethylase LSD1/KDM1A

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. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

An Autosomal-Recessive GFI1B Mutation Defines the Splice Isoform p37 As Essential for Biogenesis of Functional Human Platelets, but Dispensable for Erythropoiesis

Growth factor independent 1 (GFI1) and Growth Factor Independent 1B (GFI1B) are zinc finger transcriptional repressors that share about 90% homology on amino acid sequence and are expressed during hematopoiesis. While GFI1 is most important for granulocyte-monocyte lineage commitment, GFI1B is an essential master regulator of erythroid and megakaryocytic lineages. Mice lacking Gfi1b are embryonic lethal due to anemia and thrombocytopenia. In humans, alternative splicing leads to a shorter p32 isoform that lacks the first 2 of 6 zinc fingers. GFI1B germline mutations have been reported to cause autosomal-dominant macrothrombocytopenia with a grey-platelet syndrome phenotype, implying that the mutant protein acts in a dominant-negative manner.We report on a Chechen family from eastern Georgia whose affected family members all present with severe, life-threatening bleeding diathesis. The female index patient had recurrent hematomata and multiple petechiae since childhood. Both of her children (age 9 and 7) present with very low platelet counts (below 45/nL) and a similar cutaneous bleeding pattern like her mother. The brother also had thrombocytopenia and died at age 33 in reponse to a spontaneous cerebral hemorrhage. In contrast, the index patient's husband, her parents and the children of the deceased brother were clinically unaffected.Blood smears of affected patients showed macrothrombocytopenia with reduced May-Grünwald-Giema staining and decreased staining for alpha-granule markers von Willebrand factor (vWF) and P-selectin (CD62P). Platelet function testing revealed reduced responses to ADP, collagen, TRAP-6 and arachidonic acid. White and red blood cell parameters were overall normal in the index patient and the two affected children. We analyzed DNA from the index patient by targeted next generation sequencing for 59 genes relevant for platelet formation or function. We found a novel homozygous single nucleotide insertion in GFI1B (NM_004188.5; c.551insG), which was confirmed by Sanger sequencing and is expected to cause a premature stop-codon. The homozygous mutation co-segregated with the phenotype. The unaffected mother, the husband and two unaffected nephews were heterozygous, suggesting a local founder variant and an unexpected autosomal-recessive trait. Bone marrow analysis showed unaffected myeloid and erythroid cells, but dysplastic micromegakaryocytes with increased CD34 staining. Peripheral blood platelets were also positive for CD34. We performed quantitative real-time PCR of platelet RNA and found residual homozygous c.551_G insertion in the p37 transcript and an unexpected expression of the p32 variant. The p37 transcript was markedly reduced in context with an increased p32/p37 ratio compared to controls. Our findings indicate that the mutated transcript was not completely degraded by nonsense-mediated decay, but mostly subjected to alternative splicing skipping the mutated exon 9.Our findings imply that the first two zinc fingers of GFI1B are dispensable for human erythropoiesis, but essential for normal megakaryopoiesis and the production of functional platelets. While previous mutations affect both isoforms, the insertion variant presented here, results in a premature stop-codon and affects only the p37 isoform due to alternative splicing. This splice variant defines an important node at the megakaryocytic-erythroid progenitor stage and we conclude that the transcriptional regulation of erythropoiesis is uncoupled from that of megakaryopoiesis through alternative splicing of GFI1B. DisclosuresNo relevant conflicts of interest to declare.