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Hematopoietic stem cells (HSCs) are tightly controlled to maintain a balance between myeloid and lymphoid cell differentiation. Gata2 is a pivotal hematopoietic transcription factor required for HSC generation and maintenance. We generated a zebrafish mutant for the mouse Gata2 orthologue, gata2b. We found that in adult zebrafish, gata2b is required for both neutrophilic-and monocytic lineage differentiation. Single cell transcriptome analysis revealed that the myeloid defect present in Gata2b deficient zebrafish arise in the most immature hematopoietic stem and progenitor cell (HSPC) compartment and that this population is instead committed towards the lymphoid and erythroid lineage. Taken together, we find that Gata2b is vital for the fate choice between the myeloid and lymphoid lineages.

Background: Paroxysmal nocturnal haemoglobinuria (PNH) is a life-threatening clinical syndrome caused by acquired mutations in PIGA in the haemopoietic compartment. The clinicopathological features of PNH can overlap with aplastic anaemia (AA) and myelodysplastic syndrome (MDS) and these entities may represent different manifestations of a common underlying pathological process. To date, the landscape of acquired mutations in de novo PNH (i.e. not preceded by a formal diagnosis of AA/MDS) remains incompletely characterised. Aims: To characterise the spectrum of acquired mutations in the cellular and cell-free compartments in a cohort of patients with de novo PNH. Methods: DNA was extracted from peripheral blood leukocytes (n = 19) or bone marrow (n = 2) from 21 patients with a clinicopathological diagnosis of PNH (defined as a detectable PNH clone by flow cytometry, active haemolysis and not meeting formal diagnostic criteria for MDS or AA). Patients with a pre-existing diagnosis of AA or MDS were excluded. Cell-free DNA (cfDNA) was also collected from 19 patients. DNA sequencing libraries were prepared from cellular DNA using both the Peter MacCallum Cancer Centre PanHaem hybridisation and Myeloid amplicon panels (sensitivity 5%) which includes sequence variant detection for recurrently mutated genes associated with haematological malignancy and whole genome copy number assessment. A custom panel using Anchored Multiplex PCR target enrichment chemistry and unique molecular barcodes was used for cfDNA sequencing (sensitivity <1%). Results: At time of sampling 18 of 21 patients were two or more years post diagnosis (median time since diagnosis of 8 years) with a median age of 41 years (range 23 -82). 16 patients were being treated with complement inhibitor therapy. PIGA mutations were detected in 19 patients, with multiple mutations detected in half the cohort (11 patients, range 2 -9 PIGA mutations per patient). 21 mutations were detected in the cellular DNA, while cfDNA sequencing revealed 30 additional PIGA mutations at low allelic burden (median allele frequency 2.19%, range 0.57% -4.86%) (20 frameshift, 15 missense, 10 splice site, 6 nonsense). Two patients did not have a detectable PIGA mutation but instead harboured focal copy number losses at the PIGA locus, which also involved ZRSR2 in the minimal deleted region. Remarkably, 7 of 21 patients had truncating ASXL1 mutations detectable in both the cellular and cfDNA (9 frameshift, 3 nonsense); in 4 patients multiple ASXL1 mutations were detected. Clonal pathogenic mutations were also detected in U2AF1 (2 patients) and BCOR (1 patient). Patients with ASXL1 and/or U2AF1 mutations had significantly lower neutrophil counts (mean 1.7 vs 1.08, p = 0.021) compared to those without. Haemoglobin and platelet counts were not statistically different between the two groups. Summary/Conclusion: Multiple subclonal PIGA mutations are detectable in patients with PNH. In addition acquired pathogenic variants in ASXL1, U2AF1 and ZRSR2 were detected in a third of the cohort. L...

Introduction: Severe congenital neutropenia (SCN) is a genetically heterogeneous disease characterized by recurrent infections and a predisposition for malignant transformation. A wide variety of autosomal dominant or sporadic mutations in ELANE encoding neutrophil elastase (NE) are the most frequent cause of SCN, whereas recessive mutations in HAX1 are responsible for the autosomal recessive form of SCN known as Kostmann syndrome. How ELANE and HAX1 mutations cause SCN is still unclear. A prevailing hypothesis is that cellular stresses either caused by protein misfolding or malfunction in the case of ELANE-SCN, or by mitochondrial dysfunction in the case of HAX1-SCN, are drivers of the neutropenia. We focused on the role of the promyelocytic leukemia protein (PML) because PML is implicated in controlling cellular stress responses caused by reactive oxygen species (ROS) and protein misfolding and may exert both oncogenic and tumor-suppressive functions. Aims: (1) To elucidate which cellular stress mechanisms are involved in different genetic subtypes of SCN. (2) To assess the role of PML in SCN with a predicted ELANE misfolding mutation. Methods: We generated induced pluripotent stem cells (iPSCs) from healthy control and SCN patients with non-overlapping mutations: ELANE-I60F, ELANE-R103L and HAX1-W44X. CD34+CD45+ Hematopoietic Stem and Progenitor cells (HSPCs) were derived from iPSCs using the STEMdiff™ Hematopoietic Kit (STEMCELL Technologies). PML-/- iPSCs were created by introducing a stop codon in exon 3, shared by all PML isoforms, using CRISPR/Cas9 mediated genome editing. Results: HSPCs derived from the SCN-iPSCs showed increased ROS levels as measured with CellROX Deep Red. Consequently, nuclear translocation of the antioxidant regulatory factor NRF2 was significantly elevated in both ELANE- and HAX1-mutant SCN HSPCs relative to controls. Mutation prediction analysis (Venselaar, BMC Bioinformatics 2010) showed that ELANE-I60F likely causes NE protein misfolding, whereas the ELANE-R103L mutation predictably causes NE malfunction by disrupting interactions with other proteins. The mutation in HAX1 was predicted to result in nonsense-mediated mRNA decay. Transcriptome analysis using Gene Set Enrichment Analysis (GSEA) confirmed upregulation of the nonsense mediated decay pathway in HAX1 mutant HSPCs and in line with previous studies (Klein et al, 2008), FACS analysis using TMRM and Mitotracker Red showed that loss of HAX1 protein reduced mitochondrial membrane integrity. Surprisingly, and in apparent conflict with the mutation prediction analysis, GSEA on ELANE-I60F HSPCs did not show increased expression of the classical unfolded protein response (UPR) pathway. Because PML has been implicated as an alternative player involved in degrading misfolded proteins (Guo, Mol Cell 2014), we investigated a possible link between ELANE-I60F and PML. Immunofluorescent staining showed increased numbers of PML nuclear bodies (PML-NBs) in ELANE-I60F derived HPSCs, but not in ELANE-R103L or HAX1-W44X HSPCs. Furthermore, GSEA showed upregulation of transcripts associated with PML chromatin binding in ELANE-I60F, but not in ELANE-R103L or HAX1-W44X cells. Deletion of PML by CRISPR-Cas9 revealed that PML enhanced MYC and mTORC1-induced transcription and cell cycle signatures in HSPCs from ELANE-I60F, suggestive of an oncogenic role of PML by inducing proliferation and metabolism in ELANE-I60F. In contrast, PML inhibited these pathways in HSPCs derived from healthy control iPSCs, indicative of its tumor-suppressive function in normal HSPCs. Finally, and perhaps most intriguingly, transcriptome analysis revealed that ELANE-I60F HSPCs expressed 5-fold higher levels of (mutant) ELANE transcripts than control HSPCs, which were reduced to basal levels after deletion of PML. Conclusion: HAX1 and ELANE mutations cause oxidative stress in SCN-HSPCs by distinct mechanisms. We provide evidence for a dual role of PML in the pathogenesis of SCN caused by an ELANE mutation (I60F) associated with NE misfolding: (1) NE misfolding and increased oxidative stress cause elevated formation of PML-NBs, leading to increased expression of proliferation, cell cycle and metabolism associated transcripts, (2) PML strongly enhances the levels of ELANE transcripts, thus driving the expression of the disease causative ELANE mutant through a feed-forward mechanism. Disclosures No relevant conflicts of interest to declare.

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leukemias, whereas CIMPcases mainly belong to the TAL-LMO rearranged subtype of human T-ALL. Given that each molecular genetic subtype is linked to a specific T-cell differentiation arrest, we wondered whether CIMP status would merely be a reflection of T-cell maturation and/or cell of origin. However, and most notably, integration of these data with DNA methylation signatures obtained from 11 different subsets along the T-cell differentiation trajectory, revealed that DNA methylation profiles from both CIMP + and CIMP -T-ALLs are strongly perturbed as compared to all their normal T-cell counterparts. Indeed, human T-ALLs uniformly display a DNA hypermethylation phenotype as compared to their normal counterpart and putative cell of origin. Given this, we wondered if targeting aberrant DNA methylation could serve as a uniform therapeutic strategy for both CIMP + and CIMPhuman T-ALLs. To evaluate this, we used a xenograft model of luciferase-positive LOUCY cells and showed that Decitabine treatment caused a marked reduction in bioluminescence with a concomitant loss of leukemic blasts in peripheral blood and a significant increase in leukemia free survival. Next, we used patient derived xenograft models from 6 different T-ALL/T-LBL patients (different genetic subtypes, CIMP + and CIMPand including one relapse) to show that Decitabine treatment resulted in a significant increase in leukemia free survival for all samples analyzed (log-rank test, p < 0,05). Finally we used human T-ALL cell lines to investigate the molecular effects of 48 h Decitabine treatment. As expected Decitabine treatment was associated with a decreased in total DNA 5mC levels and induced up-regulation and down-regulation of 1623 and 452 genes, respectively (padj < 0,005, absolute Fc > 1,25). Enrichment analysis revealed that Decitabine most prominently caused downregulation of MYC and E2F gene signatures. Summary/Conclusion: All together, our data suggest that human T-ALL samples are uniformly characterized by aberrant DNA methylation signatures and that Decitabine might serve as a promising and uniform novel therapeutic strategy for all genetic subtypes of human T-ALL and T-LBL.