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.

A dominant-negative GFI1B mutation in the gray platelet syndrome.

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.

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.

Inducible MLL-AF9 Expression Drives an AML Program during Human Pluripotent Stem Cell-Derived Hematopoietic Differentiation.

A t(9;11)(p22;q23) translocation produces the MLL-AF9 fusion protein, which is found in up to 25% of de novo AML cases in children. Despite major advances, obtaining a comprehensive understanding of context-dependent MLL-AF9-mediated gene programs during early hematopoiesis is challenging. Here, we generated a human inducible pluripotent stem cell (hiPSC) model with a doxycycline dose-dependent MLL-AF9 expression. We exploited MLL-AF9 expression as an oncogenic hit to uncover epigenetic and transcriptomic effects on iPSC-derived hematopoietic development and the transformation into (pre-)leukemic states. In doing so, we observed a disruption in early myelomonocytic development. Accordingly, we identified gene profiles that were consistent with primary MLL-AF9 AML and uncovered high-confidence MLL-AF9-associated core genes that are faithfully represented in primary MLL-AF9 AML, including known and presently unknown factors. Using single-cell RNA-sequencing, we identified an increase of CD34 expressing early hematopoietic progenitor-like cell states as well as granulocyte-monocyte progenitor-like cells upon MLL-AF9 activation. Our system allows for careful chemically controlled and stepwise in vitro hiPSC-derived differentiation under serum-free and feeder-free conditions. For a disease that currently lacks effective precision medicine, our system provides a novel entry-point into exploring potential novel targets for personalized therapeutic strategies.

Expression of the BRCA1 complex member BRE predicts disease free survival in breast cancer.

Breast cancer is one of the leading causes of cancer mortality in women. Recent advances in gene expression profiling have indicated that breast cancer is a heterogeneous disease and the current prognostication using clinico-pathological features is not sufficient to fully predict therapy response and disease outcome. In this retrospective study, we show that expression levels of BRE, which encodes a member of the BRCA1 DNA damage repair complex, predicted disease-free survival (DFS) in non-familial breast cancer patients. The predictive value of BRE expression depended on whether patients received radiotherapy as a part of their primary treatment. In radiotherapy-treated patients, high BRE expression predicted a favorable DFS (hazard ratio (HR) = 0.47, 95 % confidence interval (CI) = 0.28-0.78, p = 0.004), while in non-treated patients, high BRE expression predicted an adverse prognosis (HR = 2.59, 95 % CI = 1.00-6.75, p = 0.05). Among radiotherapy-treated patients, the prognostic impact of BRE expression was confined to patients with smaller tumors (HR = 0.23, 95 % CI = 0.068-0.75, p = 0.015) and it remained an independent factor after correction for the other prognostic factors age, tumor size, lymph node involvement, and histological grade (HR = 0.50, CI = 0.27-0.90, p = 0.021). In addition, high BRE expression predicted a favorable relapse-free survival in a publicly available dataset of 2,324 breast cancer patients (HR = 0.59, CI = 0.51-0.68, p < 0.001). These data indicate that BRE is an interesting candidate for future functional studies aimed at developing targeted therapies.

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.

Genetic polymorphisms in GSTA1, GSTP1, GSTT1, and GSTM1 and gastric cancer risk in a Vietnamese population.

Glutathione S-transferases (GSTs) are a family of enzymes involved in the detoxification of noxious agents. Genes encoding for GSTA1, GSTP1, GSTT1, and GSTM1 proteins are polymorphic in humans, which can result in (partial) loss of enzyme activity. Previous epidemiologic studies have associated dysfunction of these GST genes with a higher risk of cancer, but this is still controversial. The aim of this study was to investigate the susceptibility to gastric cancer in relation to the above-mentioned GST polymorphisms. Patients visiting the Can Tho General Hospital in Vietnam between January 2004 and August 2004 for upper gastrointestinal endoscopy, who were diagnosed with gastric cancer, were compared with a control group of endoscoped dyspepsia patients with no history of malignancy. Genotypes of the GSTs mentioned above were assessed by multiplex PCR. Fifty-nine patients with gastric cancer (mean age: 63 years, 80% males), and 109 dyspeptic controls (mean age: 46 years, 69% males) were included in this study. The frequencies of the combined heterozygote and homozygote mutant GSTA1 and GSTP1 genotypes were 10% and 48% in patients with gastric cancer versus 28% and 40% in dyspeptic controls, respectively. GSTT1 and GSTM1 were deleted in 42% and 73% of patients with gastric cancer and in 35% and 69% of the controls, respectively. The GSTA1 homozygous wild-type genotype was significantly more often present in patients with gastric cancer compared with controls (odds ratio 4.3, 95% CI 1.2-17), which was even more apparent after adjustment for age, gender, current smoking, current alcohol consumption, and polymorphisms in GSTP1, GSTT1, or GSTM1 (odds ratio 5.0, 95% CI 1.2-25). The present work shows that the homozygous wild-type GSTA1 genotype is associated with gastric cancer in a Vietnamese population, whereas there was no relationship with polymorphisms in GSTP1, GSTT1, or GSTM1.

Hematopoietic stem cells exhibit a specific ABC transporter gene expression profile clearly distinct from other stem cells.

BACKGROUND: ATP-binding cassette (ABC) transporters protect cells against unrelated (toxic) substances by pumping them across cell membranes. Earlier we showed that many ABC transporters are highly expressed in hematopoietic stem cells (HSCs) compared to more committed progenitor cells. The ABC transporter expression signature may guarantee lifelong protection of HSCs but may also preserve stem cell integrity by extrusion of agents that trigger their differentiation. Here we have studied whether non-hematopoietic stem cells (non-HSCs) exhibit a similar ABC transporter expression signature as HSCs. RESULTS: ABC transporter expression profiles were determined in non-hematopoietic stem cells (non-HSCs) from embryonic, neonatal and adult origin as well as in various mature blood cell types. Over 11,000 individual ABC transporter expression values were generated by Taqman Low Density Arrays (TLDA) to obtain a sensitivity comparable with quantitative real-time polymerase chain reactions. We found that the vast majority of transporters are significantly higher expressed in HSCs compared to non-HSCs. Furthermore, regardless their origin, non-HSCs exhibited strikingly similar ABC transporter expression profiles that were distinct from those in HSCs. Yet, sets of transporters characteristic for different stem cell types could be identified, suggesting restricted functions in stem cell physiology. Remarkably, in HSCs we could not pinpoint any single transporter expressed at an evidently elevated level when compared to all the mature blood cell types studied. CONCLUSIONS: These findings challenge the concept that individual ABC transporters are implicated in maintaining stem cell integrity. Instead, a distinct ABC transporter expression signature may be essential for stem cell function. The high expression of specific transporters in non-HSCs and mature blood cells suggests a specialized, cell type dependent function and warrants further functional experiments to determine their exact roles in cellular (patho)physiology.

Toll-like receptor triggering in cord blood mesenchymal stem cells.

Recently, the antagonizing effect on the differentiation of mesenchymal stem cells (MSCs) by toll-like receptor (TLR) ligands, was described. Our study shows that on more primitive cord blood derived MSCs, the expression of TLRs and ligand-induced triggering differs from that of bone marrow derived MSCs. At the RNA level, cord blood MSCs (unrestricted somatic stem cells; USSCs) express low levels of TLR1,3,5,9 and high levels of TLR4 and TLR6. At the protein level expression of TLR5 and very low expression of TLR4 was observed. NF-kappaB translocation studies revealed that both TLR4 and TLR5 are functional, although signalling kinetics induced by the individual ligands differed. Stimulation of USSCs with either lipopolysaccharide (LPS) or flagellin resulted in a marked increase of interleukin (IL)-6 and/or IL-8 production although levels differed significantly between both stimuli. Interestingly, tumour necrosis factor (TNF)-alpha was undetectable after TLR stimulation, which appeared to be due to an inactivated TNF-alpha promoter in USSCs. Moreover, osteoblastic differentiation was enhanced after triggering USSCs with LPS and flagellin. In summary, TLR4 and 5 signalling in USSCs is slow and results in the up-regulation of a restricted number of pro-inflammatory cytokines and enhanced osteoblastic differentiation. Apparently, the outcome of TLR signalling depends on the cell type that expresses them.

Role of epoxide hydrolase, NAD(P)H:quinone oxidoreductase, cytochrome P450 2E1 or alcohol dehydrogenase genotypes in susceptibility to colorectal cancer.

Colorectal cancer (CRC) is one of the most common forms of cancer in Western countries. CRC has been associated with genetic and lifestyle factors. Individual susceptibility to CRC may be due partly to variations in detoxification capacity in the gastrointestinal tract. Genetic polymorphisms in detoxification enzymes may result in variations in detoxification activities, which subsequently might influence the levels of toxic/carcinogenic compounds, and this may influence the risk for CRC. Therefore, we determined whether polymorphisms in the genes coding for microsomal epoxide hydrolase (EPHX1), NAD(P)H:quinone oxidoreductase (NQO1), cytochrome P450 2E1 (CYP2E1) and alcohol dehydrogenase (ADH3) predispose to the development of CRC. DNA samples were obtained from 371 patients with sporadic CRC and 415 healthy controls. Patients and controls were all of Caucasian origin. All genetic polymorphisms were determined by polymerase chain reaction, eventually followed by restriction-fragment-length-polymorphism analyses, except for the EPHX1 codon 113 polymorphism, which was genotyped by an allele-specific discrimination assay. Calculation of crude Odds Ratios (ORs) revealed an increased risk for CRC associated with variant NQO1 (OR 1.5, 95% CI 1.1-2.0) and CYP2E1 intron 6 genotypes (OR 2.2, 95% CI 1.3-3.8). However, after adjustment for age and gender, logistic regression analyses only showed a statistically significant risk for CRC associated with variant NQO1 genotypes (OR 1.6, 95% CI 1.03-2.4). No associations were found between CRC and the other polymorphic genes as mentioned above. In conclusion, these data suggest that the presence of variant NQO1 genotypes, with expected reduced enzyme activities might enhance susceptibility to CRC.

GFI1(36N) as a therapeutic and prognostic marker for myelodysplastic syndrome.

Inherited gene variants play an important role in malignant diseases. The transcriptional repressor growth factor independence 1 (GFI1) regulates hematopoietic stem cell (HSC) self-renewal and differentiation. A single-nucleotide polymorphism of GFI1 (rs34631763) generates a protein with an asparagine (N) instead of a serine (S) at position 36 (GFI1(36N)) and has a prevalence of 3%-5% among Caucasians. Because GFI1 regulates myeloid development, we examined the role of GFI1(36N) on the course of MDS disease. To this end, we determined allele frequencies of GFI1(36N) in four independent MDS cohorts from the Netherlands and Belgium, Germany, the ICGC consortium, and the United States. The GFI1(36N) allele frequency in the 723 MDS patients genotyped ranged between 9% and 12%. GFI1(36N) was an independent adverse prognostic factor for overall survival, acute myeloid leukemia-free survival, and event-free survival in a univariate analysis. After adjustment for age, bone marrow blast percentage, IPSS score, mutational status, and cytogenetic findings, GFI1(36N) remained an independent adverse prognostic marker. GFI1(36S) homozygous patients exhibited a sustained response to treatment with hypomethylating agents, whereas GFI1(36N) patients had a poor sustained response to this therapy. Because allele status of GFI1(36N) is readily determined using basic molecular techniques, we propose inclusion of GFI1(36N) status in future prospective studies for MDS patients to better predict prognosis and guide therapeutic decisions.

MicroRNA hsa-miR-135b regulates mineralization in osteogenic differentiation of human unrestricted somatic stem cells.

Unrestricted somatic stem cells (USSCs) have been recently identified in human umbilical cord blood and have been shown to differentiate into lineages representing all 3 germ layers. To characterize microRNAs that may regulate osteogenic differentiation of USSCs, we carried out expression analysis for 157 microRNAs using quantitative RT-PCR before and after osteogenic induction (t = 0.5, 24, 72, 168, 216 h). Three microRNAs, hsa-miR-135b, hsa-miR-224, and hsa-miR-31, were consistently down-regulated during osteogenesis of USSC line 1. Hsa-miR-135b was shown to be the most profoundly down-regulated in osteogenesis of USSC line 1 and further confirmed to be down-regulated in the osteogenic differentiation of 2 additional USSC lines. Function of hsa-miR-135b in osteogenesis of USSCs was examined by retroviral overexpression, which resulted in an evident decreased mineralization, indicating that hsa-miR-135b down-regulation is functionally important for full osteogenic differentiation of USSCs. MicroRNAs have been shown to regulate negatively expression of their target gene(s). To identify putative targets of hsa-miR-135b, we performed cDNA microarray expression analysis. We selected in total 10 transcripts that were down-regulated (>or=2-fold) in response to hsa-miR-135b overexpression at day 7 and day 9 of osteogenic differentiation. The function of most of these targets in human osteogenesis is unknown and requires further investigation. Markedly, quantitative RT-PCR data showed decreased expression of osteogenic markers IBSP and Osterix, both known to be involved in bone mineralization, in osteogenesis of USSCs that overexpress hsa-miR-135b. This finding suggests that hsa-miR-135b may control osteoblastic differentiation of USSCs by regulating expression of bone-related genes.