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Cancer is driven by somatic mutations that provide a fitness advantage. While targeted therapies often focus on the mutated gene or its direct downstream effectors, imbalances brought on by cell-state alterations may also confer unique vulnerabilities. In myeloproliferative neoplasms (MPN), somatic mutations in the calreticulin (CALR) gene are disease-initiating through aberrant binding of mutant CALR to the thrombopoietin receptor MPL and ligand-independent activation of JAK-STAT signaling. Despite these mechanistic insights into the pathogenesis of CALR-mutant MPN, there are currently no mutant CALR-selective therapies available. Here, we identified differential upregulation of unfolded proteins, the proteasome and the ER stress response in CALR-mutant hematopoietic stem cells (HSCs) and megakaryocyte progenitors. We further found that combined pharmacological inhibition of the proteasome and IRE1-XBP1 axis of the ER stress response preferentially targets Calr-mutated HSCs and megakaryocytic-lineage cells over wild-type cells in vivo, resulting in an amelioration of the MPN phenotype. In serial transplantation assays following combined proteasome/IRE1 inhibition for six weeks, we did not find preferential depletion of Calr-mutant long-term HSCs. Together, these findings leverage altered proteostasis in Calr-mutant MPN to identify combinatorial dependencies that may be targeted for therapeutic benefit and suggest that eradicating disease-propagating Calr-mutant LT-HSCs may require more sustained treatment.

Calreticulin (CALR) mutations are frequent, disease-initiating events in myeloproliferative neoplasms (MPNs). Although the biological mechanism by which CALR mutations cause MPNs has been elucidated, there currently are no clonally selective therapies for CALR-mutant MPNs. To identify unique genetic dependencies in CALR-mutant MPNs, we performed a whole-genome clustered regularly interspaced short palindromic repeats (CRISPR) knockout depletion screen in mutant CALR-transformed hematopoietic cells. We found that genes in the N-glycosylation pathway (among others) were differentially depleted in mutant CALR-transformed cells as compared with control cells. Using a focused pharmacological in vitro screen targeting unique vulnerabilities uncovered in the CRISPR screen, we found that chemical inhibition of N-glycosylation impaired the growth of mutant CALR-transformed cells, through a reduction in MPL cell surface expression. We treated Calr-mutant knockin mice with the N-glycosylation inhibitor 2-deoxy-glucose (2-DG) and found a preferential sensitivity of Calr-mutant cells to 2-DG as compared with wild-type cells and normalization of key MPNs disease features. To validate our findings in primary human cells, we performed megakaryocyte colony-forming unit (CFU-MK) assays. We found that N-glycosylation inhibition significantly reduced CFU-MK formation in patient-derived CALR-mutant bone marrow as compared with bone marrow derived from healthy donors. In aggregate, our findings advance the development of clonally selective treatments for CALR-mutant MPNs.

The majority of JAK2V617F-negative myeloproliferative neoplasms (MPNs) have disease-initiating frameshift mutations in calreticulin (CALR), resulting in a common carboxyl-terminal mutant fragment (CALRMUT), representing an attractive source of neoantigens for cancer vaccines. However, studies have shown that CALRMUT-specific T cells are rare in patients with CALRMUTMPN for unknown reasons. We examined class I major histocompatibility complex (MHC-I) allele frequencies in patients with CALRMUTMPN from two independent cohorts. We observed that MHC-I alleles that present CALRMUTneoepitopes with high affinity are underrepresented in patients with CALRMUTMPN. We speculated that this was due to an increased chance of immune-mediated tumor rejection by individuals expressing one of these MHC-I alleles such that the disease never clinically manifested. As a consequence of this MHC-I allele restriction, we reasoned that patients with CALRMUTMPN would not efficiently respond to a CALRMUTfragment cancer vaccine but would when immunized with a modified CALRMUTheteroclitic peptide vaccine approach. We found that heteroclitic CALRMUTpeptides specifically designed for the MHC-I alleles of patients with CALRMUTMPN efficiently elicited a CALRMUTcross-reactive CD8+T cell response in human peripheral blood samples but not to the matched weakly immunogenic CALRMUTnative peptides. We corroborated this effect in vivo in mice and observed that C57BL/6J mice can mount a CD8+T cell response to the CALRMUTfragment upon immunization with a CALRMUTheteroclitic, but not native, peptide. Together, our data emphasize the therapeutic potential of heteroclitic peptide–based cancer vaccines in patients with CALRMUTMPN.

There is prevailing evidence to suggest a decisive role for platelet-derived growth factors (PDGF) and their receptors in primary myelofibrosis. While PDGF receptor β (PDGFRβ) expression is increased in bone marrow stromal cells of patients correlating with the grade of myelofibrosis, knowledge on the precise role of PDGFRβ signaling in myelofibrosis is sparse. Using the Gata-1low mouse model for myelofibrosis, we applied RNA sequencing, protein expression analyses, multispectral imaging and, as a novel approach in bone marrow tissue, an in situ proximity ligation assay to provide a detailed characterization of PDGFRβ signaling and regulation during development of myelofibrosis. We observed an increase in PDGFRβ and PDGF-B protein expression in overt fibrotic bone marrow, along with an increase in PDGFRβ–PDGF-B interaction, analyzed by proximity ligation assay. However, PDGFRβ tyrosine phosphorylation levels were not increased. We therefore focused on regulation of PDGFRβ by protein tyrosine phosphatases as endogenous PDGFRβ antagonists. Gene expression analyses showed distinct expression dynamics among PDGFRβ-targeting phosphatases. In particular, we observed enhanced T-cell protein tyrosine phosphatase protein expression and PDGFRβ–T-cell protein tyrosine phosphatase interaction in early and overt fibrotic bone marrow of Gata-1low mice. In vitro, T-cell protein tyrosine phosphatase (Ptpn2) knockdown increased PDGFRβ phosphorylation at Y751 and Y1021, leading to enhanced downstream signaling in fibroblasts. Furthermore, Ptpn2 knockdown cells showed increased growth rates when exposed to low-serum growth medium. Taken together, PDGF signaling is differentially regulated during myelofibrosis. Protein tyrosine phosphatases, which have so far not been examined during disease progression, are novel and hitherto unrecognized components in myelofibrosis.

Myelofibrosis is a progressive bone marrow malignancy associated with monocytosis, and is believed to promote the pathological remodelling of the extracellular matrix. Here we show that the mechanical properties of myelofibrosis, namely the liquid-to-solid properties (viscoelasticity) of the bone marrow, contribute to aberrant differentiation of monocytes. Human monocytes cultured in stiff, elastic hydrogels show proinflammatory polarization and differentiation towards dendritic cells, as opposed to those cultured in a viscoelastic matrix. This mechanically induced cell differentiation is blocked by inhibiting a myeloid-specific isoform of phosphoinositide 3-kinase, PI3K-γ. We further show that murine bone marrow with myelofibrosis has a significantly increased stiffness and unveil a positive correlation between myelofibrosis grading and viscoelasticity. Treatment with a PI3K-γ inhibitor in vivo reduced frequencies of monocyte and dendritic cell populations in murine bone marrow with myelofibrosis. Moreover, transcriptional changes driven by viscoelasticity are consistent with transcriptional profiles of myeloid cells in other human fibrotic diseases. These results demonstrate that a fibrotic bone marrow niche can physically promote a proinflammatory microenvironment.

Inherited platelet disorders are a heterogeneous group of rare diseases, caused by inherited defects in platelet production and/or function. Their genetic diagnosis would benefit clinical care, prognosis and preventative treatments. Until recently, this diagnosis has usually been performed via Sanger sequencing of a limited number of candidate genes. High-throughput sequencing is revolutionizing the genetic diagnosis of diseases, including bleeding disorders. We have designed a novel high-throughput sequencing platform to investigate the unknown molecular pathology in a cohort of 82 patients with inherited platelet disorders. Thirty-four (41.5%) patients presented with a phenotype strongly indicative of a particular type of platelet disorder. The other patients had clinical bleeding indicative of platelet dysfunction, but with no identifiable features. The high-throughput sequencing test enabled a molecular diagnosis in 70% of these patients. This sensitivity increased to 90% among patients suspected of having a defined platelet disorder. We found 57 different candidate variants in 28 genes, of which 70% had not previously been described. Following consensus guidelines, we qualified 68.4% and 26.3% of the candidate variants as being pathogenic and likely pathogenic, respectively. In addition to establishing definitive diagnoses of well-known inherited platelet disorders, high-throughput sequencing also identified rarer disorders such as sitosterolemia, filamin and actinin deficiencies, and G protein-coupled receptor defects. This included disease-causing variants in DIAPH1 (n=2) and RASGRP2 (n=3). Our study reinforces the feasibility of introducing high-throughput sequencing technology into the mainstream laboratory for the genetic diagnostic practice in inherited platelet disorders., This study was supported by research grants from the Gerencia Regional de Salud (GRS 1370/A/16), ISCIII & Feder (PI14/01956), CIBERER CB15/00055, Fundación Séneca (19873/GERM/15) and Sociedad Española de Trombosis y Hemostasia (SETH). SPW holds a British Heart Foundation chair.

Myelofibrosis (MF) is a progressive, myeloid malignancy characterized by deposition of collagen and reticulin fibers in the bone marrow (BM). Previous studies have shown that monocytosis is associated with poor prognosis in MF, highlighting a potential pathogenic role for monocytes in MF. Although many studies have addressed the role of cell-intrinsic and soluble extracellular factors in MF development, it is currently unknown if mechanical properties of fibrotic BM contribute to aberrant differentiation of myeloid cells and of monocytes in particular. We first defined the stiffness and viscoelastic properties of healthy and fibrotic BM. Stiffness is defined as the resistance of a matrix to deformation, while viscoelasticity is the rate of dissipation of an applied stress over time. Independent of stiffness, an applied stress relaxes rapidly in a more viscous, liquid-like matrix, whereas in a more elastic, solid-like material, stress relaxes slowly. We next generated a cohort of fibrotic and non-fibrotic mice by transplanting retrovirally transduced JAK2V617F or empty vector (EV) control hematopoietic stem and progenitor cells (HSPCs) into lethally irradiated recipients. Femurs from these mice were harvested seven months post-transplant, as well as from age- and sex-matched healthy primary mice. Nanoindentation was performed to measure BM stiffness and viscoelasticity. Fibrotic BM showed higher stiffness, as well as trending higher elastic, solid-like properties, compared to BM of control mice. We then aimed to study the effect of matrix stiffness and viscoelasticity on monocytes. Human BM-derived monocytes were encapsulated in stiff, viscous or stiff, elastic hydrogels and cultured in the presence of GM-CSF, IL-4, and PGE2 for 3 days, followed by nanoString and flow cytometry analyses. Cells in elastic gels upregulated gene sets associated with co-stimulatory molecules and cytokine receptor signaling, MHC class II antigen presentation, and regulation of extracellular matrix (ECM), compared to cells in viscous gels of the same stiffness. The fraction of dendritic cells (DCs) was significantly upregulated, as indicated by double-positive CD11c+CD1c+ (40.9% viscous vs 69.5% elastic of CD11b+HLA-DR+ cells) and CD80+ cells (20.9% viscous vs 62.7% elastic of CD11b+HLA-DR+ cells), and surface expression of HLA-DR (gMFI 2587 viscous vs 6334 elastic). Consistent with these findings, the fraction of pro-fibrotic SLAMF7+ cells (4.2% viscous vs 17.3% elastic) were also significantly higher in elastic gels. Together, these data suggest that stiff, elastic ECM drives pro-inflammatory polarization and differentiation of monocytes into antigen-presenting cells. Next, we examined the role of the cytoskeleton on human monocyte differentiation. Cortical F-actin was significantly upregulated in cells in stiff, elastic gels compared to viscous gels. Cells were exposed to a highly selective small molecular inhibitor of the γ-isoform of PI3K. Treatment with the PI3Ky inhibitor significantly reduced F-actin staining of cells in elastic gels, upregulated immature monocyte markers, reduced surface expression of HLA-DR, and downregulated the cytokines IL6, IL8, CCL4, which have previously been associated with disease progression in myelofibrosis. In line with the above human ex vivo data, BM isolated from fibrotic mice (described above) showed skewing towards Ly6G-Ly6C+ monocytes (a population enriched for inflammatory monocytes) within the CD11b myeloid compartment compared to control transplanted mice or to non-fibrotic mice that were transplanted with endogenously expressing Jak2V617F cells. Additionally, the percentage of conventional DCs (cDCs) was increased in fibrotic Jak2V617F mice compared to control mice. Importantly, 16 day in vivo treatment with the PI3Ky inhibitor significantly reduced the fraction of Ly6G-Ly6C+ monocytes within the CD11b compartment as well as the fraction of cDCs, compared to vehicle-treated Jak2V617F mice. In summary, fibrotic BM is stiffer and more elastic than normal BM. Our studies show that a stiff, elastic BM environment drives monocytes towards a more pro-inflammatory state which can in part be suppressed by PI3K-γ inhibition. Our results have relevance for human MF by demonstrating that a fibrotic BM niche is not just a consequence of chronic inflammation but is also inflammation-promoting. KHV and AEM contributed equally to this work. Disclosures Pozdnyakova: Scopio Labs: Consultancy. Mullally: Janssen, PharmaEssentia, Constellation and Relay Therapeutics: Consultancy. Wucherpfennig: Novartis: Research Funding; Nextechinvest: Membership on an entity's Board of Directors or advisory committees; Immunitas Therapeutics: Current holder of individual stocks in a privately-held company; TScan Therapeutics: Membership on an entity's Board of Directors or advisory committees; TCR2 Therapeutics: Membership on an entity's Board of Directors or advisory committees; SQZ Biotech: Membership on an entity's Board of Directors or advisory committees. Mooney: Novartis: Patents & Royalties: Licensed IP, Research Funding; Sirenex: Patents & Royalties: Licensed IP; Samyang Corp: Membership on an entity's Board of Directors or advisory committees; IVIVA: Membership on an entity's Board of Directors or advisory committees; Attivare: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Revela: Membership on an entity's Board of Directors or advisory committees; Amend Surgical: Patents & Royalties: Licensed IP; Lyell: Current equity holder in publicly-traded company, Patents & Royalties.

Somatic mutations in the ER chaperone calreticulin (CALR) are frequent and disease-initiating in myeloproliferative neoplasms (MPN). Although the mechanism of mutant CALR-induced MPN is known to involve pathogenic binding between mutant CALR and MPL, this insight has not yet been exploited therapeutically. Consequently, a major deficiency is the lack of clonally selective therapeutic agents with curative potential. Hence, we set out to discover and validate unique genetic dependencies for mutant CALR-driven oncogenesis. We first performed a whole-genome CRISPR knockout screen in CALR Δ52 MPL-expressing hematopoietic cells to identify genes that were differentially required for the growth of cytokine-independent, transformed CALR Δ52 cells as compared to control cells. Using gene-set enrichment analyses, we identified the N-glycan biosynthesis, unfolded protein response, and the protein secretion pathways to be amongst the most significantly differentially depleted pathways (FDR q values Strikingly, seven of the top ten hits in both screens were linked to protein N-glycosylation. Four of those genes encode proteins involved in the enzymatic activity of dolichol-phosphate mannose synthase (DPM1, DPM2, DPM3, and MPDU1). This enzyme synthesizes dolichol D-mannosyl phosphate, an essential substrate for protein N-glycosylation. Importantly, these findings from an unbiased whole-genome screen align with prior mechanistic studies demonstrating that both the N-glycosylation sites on MPL and the lectin-binding sites on CALR Δ52 are required for mutant CALR-driven oncogenesis. We next performed single gene CRISPR Cas9 validation studies and found that DPM2 is required for CALR Δ52-mediated transformation, as demonstrated by increased cell death, reduced p-STAT5 and decreased MPL cell-surface levels, when Dpm2 is knocked out. Importantly, cells cultured in cytokine-rich medium were unaffected by DPM2 loss. Upon cytokine withdrawal, a sub-clone of non-edited Dpm2WT CALR Δ52 cells grew out, further demonstrating requirement for DPM2 for the survival of CALR Δ52 cells. Additionally, we observed a >50% reduction in ex vivo myeloid colony formation of murine CalrΔ52 Dpm2 ko bone marrow (BM) compared with CRISPR-Cas9 non-targeting controls, with non-significant effects on CalrWT BM cells. To enable clinical translation, we performed a pharmacological screen targeting pathways significantly depleted in our CRISPR screens. Screening 70 drugs, we found that the N-glycosylation pathway was the only pathway in which all tested compounds preferentially killed CALR Δ52 transformed cells. We then treated primary Calr Δ52/+ mice with a clinical grade N-glycosylation (N-Gi) inhibitor and found platelet counts (Sysmex) to be significantly reduced (vehicle 3x10 6/mL, N-Gi 1x10 6/mL after 18 days, p We next performed competitive BM transplantation assays using CD45.2 UBC-GFP MxCre CalrΔ52 knockin and CD45.1 mice. We found that mice treated with N-Gi had significantly reduced platelet counts (vehicle 1440x10 6/mL, N-Gi 845x10 6/mL, p=0.005) as well as significantly reduced platelet chimerism (vehicle 55%, N-Gi 27%, p Finally, we interrogated RNA-sequencing data from primary human MPN platelets. We found N-glycosylation-related pathways to be significantly upregulated in CALR-mutated platelets (n = 13) compared to healthy control platelets (n = 21), highlighting the relevance of our findings to human MPN. In summary, using unbiased genetic and focused pharmacological screens, we identified the N-glycan biosynthesis pathway as essential for mutant CALR-driven oncogenesis. Using a pre-clinical MPN model, we found that in vivo inhibition of N-glycosylation normalizes key features of MPN and preferentially targets CalrΔ52 over WT cells. These findings have therapeutic implications through inhibiting N-glycosylation alone or in combination with other agents to advance the development of clonally selective therapeutic approaches in CALR-mutant MPN. AEM and JSJ contributed equally. Figure 1 Figure 1. Disclosures Mullally: Janssen, PharmaEssentia, Constellation and Relay Therapeutics: Consultancy.

Overexpression of the BRE (brain and reproductive organ-expressed) gene defines a distinct pediatric and adult acute myeloid leukemia (AML) subgroup. Here we identify a promoter enriched for active chromatin marks in BRE intron 4 causing strong biallelic expression of a previously unknown C-terminal BRE transcript. This transcript starts with BRE intron 4 sequences spliced to exon 5 and downstream sequences, and if translated might code for an N terminally truncated BRE protein. Remarkably, the new BRE transcript was highly expressed in over 50% of 11q23/KMT2A (lysine methyl transferase 2A)-rearranged and t(8;16)/KAT6A-CREBBP cases, while it was virtually absent from other AML subsets and normal tissues. In gene reporter assays, the leukemia-specific fusion protein KMT2A-MLLT3 transactivated the intragenic BRE promoter. Further epigenome analyses revealed 97 additional intragenic promoter marks frequently bound by KMT2A in AML with C-terminal BRE expression. The corresponding genes may be part of a context-dependent KMT2A-MLLT3-driven oncogenic program, because they were higher expressed in this AML subtype compared with other groups. C-terminal BRE might be an important contributor to this program because in a case with relapsed AML, we observed an ins(11;2) fusing CHORDC1 to BRE at the region where intragenic transcription starts in KMT2A-rearranged and KAT6A-CREBBP AML.Leukemia advance online publication, 24 October 2017; doi:10.1038/leu.2017.280.

Activated JAK-STAT signaling is central to the pathogenesis of BCR-ABL-negative myeloproliferative neoplasms (MPNs) and occurs as a result of MPN phenotypic driver mutations in JAK2, CALR, or MPL. The spectrum of concomitant somatic mutations in other genes has now largely been defined in MPNs. With the integration of targeted next-generation sequencing (NGS) panels into clinical practice, the clinical significance of concomitant mutations in MPNs has become clearer. In this review, we describe the consequences of concomitant mutations in the most frequently mutated classes of genes in MPNs: (1) DNA methylation pathways, (2) chromatin modification, (3) RNA splicing, (4) signaling pathways, (5) transcription factors, and (6) DNA damage response/stress signaling. The increased use of molecular genetics for early risk stratification of patients brings the possibility of earlier intervention to prevent disease progression in MPNs. However, additional studies are required to decipher underlying molecular mechanisms and effectively target them.

To identify novel therapeutic targets in acute myeloid leukemia (AML), we examined kinase expression patterns in primary AML samples. We found that the serine/threonine kinase IKBKE, a noncanonical IkB kinase, is expressed at higher levels in myeloid leukemia cells compared with normal hematopoietic cells. Inhibiting IKBKE, or its close homolog TANK-binding kinase 1 (TBK1), by either short hairpin RNA knockdown or pharmacological compounds, induces apoptosis and reduces the viability of AML cells. Using gene expression profiling and gene set enrichment analysis, we found that IKBKE/TBK1-sensitive AML cells typically possess an MYC oncogenic signature. Consistent with this finding, the MYC oncoprotein was significantly downregulated upon IKBKE/TBK1 inhibition. Using proteomic analysis, we found that the oncogenic gene regulator YB-1 was activated by IKBKE/TBK1 through phosphorylation, and that YB-1 binds to the MYC promoter to enhance MYC gene transcription. Momelotinib (CYT387), a pharmacological inhibitor of IKBKE/TBK1, inhibits MYC expression, reduces viability and clonogenicity of primary AML cells, and demonstrates efficacy in a murine model of AML. Together, these data identify IKBKE/TBK1 as a promising therapeutic target in AML.

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.

Author(s): Marneth, Anna E; Botezatu, Lacramioara; Hones, Judith M; Israel, Jimmy CL; Schutte, Judith; Vassen, Lothar; Lams, Robert F; Bergevoet, Saskia M; Groothuis, Laura; Mandoli, Amit; Martens, Joost HA; Huls, Gerwin; Jansen, Joop H; Duhrsen, Ulrich; Berg, Tobias; Moroy, Tarik; Wichmann, Christian; Lo, Mia-Chia; Zhang, Dong-Er; van der Reijden, Bert A; Khandanpour, Cyrus

In 11q23 leukemias, the N-terminal part of the mixed lineage leukemia (MLL) gene is fused to >60 different partner genes. In order to define a core set of MLL rearranged targets, we investigated the genome-wide binding of the MLL-AF9 and MLL-AF4 fusion proteins and associated epigenetic signatures in acute myeloid leukemia (AML) cell lines THP-1 and MV4-11. We uncovered both common as well as specific MLL-AF9 and MLL-AF4 target genes, which were all marked by H3K79me2, H3K27ac and H3K4me3. Apart from promoter binding, we also identified MLL-AF9 and MLL-AF4 binding at specific subsets of non-overlapping active distal regulatory elements. Despite this differential enhancer binding, MLL-AF9 and MLL-AF4 still direct a common gene program, which represents part of the RUNX1 gene program and constitutes of CD34+ and monocyte-specific genes. Comparing these data sets identified several zinc finger transcription factors (TFs) as potential MLL-AF9 co-regulators. Together, these results suggest that MLL fusions collaborate with specific subsets of TFs to deregulate the RUNX1 gene program in 11q23 AMLs.

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 gray platelet syndrome is a hereditary, usually autosomal recessive bleeding disorder caused by a deficiency of alpha granules in platelets. We detected a nonsense mutation in the gene encoding the transcription factor GFI1B (growth factor independent 1B) that causes autosomal dominant gray platelet syndrome. Both gray platelets and megakaryocytes had abnormal marker expression. In addition, the megakaryocytes had dysplastic features, and they were abnormally distributed in the bone marrow. The GFI1B mutant protein inhibited nonmutant GFI1B transcriptional activity in a dominant-negative manner. Our studies show that GFI1B, in addition to being causally related to the gray platelet syndrome, is key to megakaryocyte and platelet development. A large family is described with gray platelet syndrome due to an autosomal dominant inheritance pattern related to a dominant-negative mutation in GFI1B. The mutation leads to a loss in gene repression during megakaryocyte development. Platelets are formed through fragmentation of megakaryocytes that reside in the bone marrow.(1),(2) Platelet alpha granules, which are by far the most abundant platelet organelles, store proteins that stimulate platelet adhesiveness, hemostasis, and wound healing.(3),(4) The gray platelet syndrome is an inherited bleeding disorder characterized by defective production of alpha granules.(5),(6) Patients with this syndrome have reduced numbers of larger-than-normal platelets, and on light microscopy these platelets have a typical gray appearance caused by the lack of alpha granules. For a final diagnosis, the lack of alpha granules must be confirmed by means of electron microscopy.(7) Clinically, ...

To the editor: Mutations in the transcription factor Growth Factor Independent 1B ( GFI1B ) are causal to an autosomal dominant inherited bleeding disorder characterized by macrothrombocytopenia and reduced platelet α-granule numbers (platelet-type bleeding disorder-17, Online Mendelian

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.