Tuning apicobasal polarity and junctional recycling in the hemogenic endothelium orchestrates the morphodynamic complexity of emerging pre-hematopoietic stem cells.

Hematopoietic stem cells emerge in the embryo from an aortic-derived tissue called the hemogenic endothelium (HE). The HE appears to give birth to cells of different nature and fate but the molecular principles underlying this complexity are largely unknown. Here we show, in the zebrafish embryo, that two cell types emerge from the aortic floor with radically different morphodynamics. With the support of live imaging, we bring evidence suggesting that the mechanics underlying the two emergence types rely, or not, on apicobasal polarity establishment. While the first type is characterized by reinforcement of apicobasal polarity and maintenance of the apical/luminal membrane until release, the second type emerges via a dynamic process reminiscent of trans-endothelial migration. Interfering with Runx1 function suggests that the balance between the two emergence types depends on tuning apicobasal polarity at the level of the HE. In support of this and unexpectedly, we show that Pard3ba - one of the four Pard3 proteins expressed in the zebrafish - is sensitive to interference with Runx1 activity, in aortic endothelial cells. This supports the idea of a signaling cross talk controlling cell polarity and its associated features, between aortic and hemogenic cells. In addition, using new transgenic fish lines that express Junctional Adhesion Molecules and functional interference, we bring evidence for the essential role of ArhGEF11/PDZ-RhoGEF in controlling the HE-endothelial cell dynamic interface, including cell-cell intercalation, which is ultimately required for emergence completion. Overall, we highlight critical cellular and dynamic events of the endothelial-to-hematopoietic transition that support emergence complexity, with a potential impact on cell fate.

In mammals and other animals with backbones, the cells that will make up blood and immune cells are generated during a very narrow timeframe in embryonic development. These cells, called hematopoietic stem cells and progenitors (or HSPCs for short), emerge from tissue known as hemogenic endothelium that makes up the floor of early blood vessels. For HPSCs to eventually specialise into different types of blood and immune cells, they require diverse migratory and homing properties that, ultimately, will determine the specific type of functions they exert. An important question for scientists studying the development of different blood and immune cell types is when this commitment to functional diversity is established. It could, for example, arise due to cells in the hemogenic endothelium having different origins. Alternatively, the signals that generate hemogenic endothelium cells could be responsible. It is also possible that both explanations are true, and that having different mechanisms involved ensures diversity in populations of HSPCs. To investigate differences between the HSPCs emerging from the hemogenic endothelium, Torcq et al. studied zebrafish embryos that had been modified so that one of the proteins involved in sensing cell polarity ­ where the top and bottom of the cell are located ­ was fluorescent. Live imaging of the embryos showed that two types of cells, with striking differences in morphology, emerge from the hemogenic tissue. In addition, one cell type displays the same polarity as the other vessel cells, whereas the other does not. Torcq et al. also present evidence suggesting that the signals responsible for controlling this cell polarity are provided by surrounding blood vessel cells, supporting the idea of an interplay between the different cell types. The finding that two different cell types emerge from the hemogenic endothelium, reveals a potential new source of diversity in HSPCs. Ultimately, this is expected to contribute to their functional complexity, resulting in both long-term stem cells that retain their full regenerative potential into adulthood and more specialized blood and immune cells.

A multiomic characterization of the leukemia cell line REH using short- and long-read sequencing.

The B-cell acute lymphoblastic leukemia (ALL) cell line REH, with the t(12;21) ETV6::RUNX1 translocation, is known to have a complex karyotype defined by a series of large-scale chromosomal rearrangements. Taken from a 15-yr-old at relapse, the cell line offers a practical model for the study of pediatric B-ALL. In recent years, short- and long-read DNA and RNA sequencing have emerged as a complement to karyotyping techniques in the resolution of structural variants in an oncological context. Here, we explore the integration of long-read PacBio and Oxford Nanopore whole-genome sequencing, IsoSeq RNA sequencing, and short-read Illumina sequencing to create a detailed genomic and transcriptomic characterization of the REH cell line. Whole-genome sequencing clarified the molecular traits of disrupted ALL-associated genes including CDKN2A, PAX5, BTG1, VPREB1, and TBL1XR1, as well as the glucocorticoid receptor NR3C1 Meanwhile, transcriptome sequencing identified seven fusion genes within the genomic breakpoints. Together, our extensive whole-genome investigation makes high-quality open-source data available to the leukemia genomics community.

RUNX1-induced upregulation of PTGS2 enhances cell growth, migration and invasion in colorectal cancer cells.

Colorectal cancer (CRC) arises via the progressive accumulation of dysregulation in key genes including oncogenes and tumor-suppressor genes. Prostaglandin-endoperoxide synthase 2 (PTGS2, also called COX2) acts as an oncogenic driver in CRC. Here, we explored the upstream transcription factors (TFs) responsible for elevating PTGS2 expression in CRC cells. The results showed that PTGS2 silencing repressed cell growth, migration and invasion in HCT116 and SW480 CRC cells. The two fragments (499-981 bp) and (1053-1434 bp) were confirmed as the core TF binding profiles of the PTGS2 promoter. PTGS2 expression positively correlated with RUNX1 level in colon adenocarcinoma (COAD) samples using the TCGA-COAD dataset. Furthermore, RUNX1 acted as a positive regulator of PTGS2 expression by promoting transcriptional activation of the PTGS2 promoter via the 1086-1096 bp binding motif. In conclusion, our study demonstrates that PTGS2 upregulation induced by the TF RUNX1 promotes CRC cell growth, migration and invasion, providing an increased rationale for the use of PTGS2 inhibitors in CRC prevention and treatment.

The role of RUNX1/NF-κB in regulating PVAT inflammation in aortic dissection.

The pathogenesis of aortic dissection (AD), an aortic disease associated with high mortality, involves significant vascular inflammatory infiltration. However, the precise relationship between perivascular adipose tissue (PVAT) and aortic dissection remains incompletely understood. The objective of this study is to investigate the role of PVAT inflammation in the pathogenesis of aortic dissection and identify novel therapeutic targets for this disease. The mouse model of aortic dissection was established in this study through intraperitoneal injection of Ang II and administration of BAPN in drinking water. Additionally, control groups were established at different time points including the 2-week group, 3-week group, and 4-week group. qPCR and immunohistochemistry techniques were employed to detect the expression of inflammatory markers and RUNX1 in PVAT surrounding the thoracic aorta in mice. Additionally, an aortic dissection model was established using RUNX1 knockout mice, and the aforementioned indicators were assessed. The 3T3-L1 cells were induced to differentiate into mature adipocytes in vitro, followed by lentivirus transfection for the knockdown or overexpression of RUNX1. The study aimed to investigate the potential cell-to-cell interactions by co-culturing 3T3-L1 cells with A7r5 or RAW264.7 cells. Subsequently, human aortic PVAT samples were obtained through clinical surgery and the aforementioned indicators were detected. In comparison to the control group, the aortic dissection model group exhibited decreased expression of MMP-2 and NF-κB in PVAT, while TNF-α and RUNX1 expression increased. Suppression of RUNX1 expression resulted in increased MMP-2 and NF-κB expression in PVAT, along with decreased TNF-α expression. Overexpression of RUNX1 upregulated the expression levels of NF-Κb, MMP-2, and TNF-α in adipocytes, whereas knockdown of RUNX1 exerted an opposite effect. Macrophages co-cultured with adipocytes overexpressing RUNX1 exhibited enhanced CD86 expression, while vascular smooth muscle cells co-cultured with these adipocytes showed reduced α-SMA expression. In human samples, there was an increase in both RUNX1 and MMP-2 expression levels, accompanied by a decrease in TNF-α and NF-Κb expression. The presence of aortic dissection is accompanied by evident inflammatory alterations in the PVAT, and this phenomenon appears to be associated with the involvement of RUNX1. It is plausible that the regulation of PVAT's inflammatory changes by RUNX1/NF-κB signaling pathway plays a role in the pathogenesis of aortic dissection.

RUNX transcription factors are essential in maintaining epididymal epithelial differentiation.

Apart from the androgen receptor, transcription factors (TFs) that are required for the development and formation of the different segments of the epididymis have remained unknown. We identified TF families expressed in the developing epididymides, of which many showed segment specificity. From these TFs, down-regulation of runt related transcription factors (RUNXs) 1 and 2 expression coincides with epithelial regression in Dicer1 cKO mice. Concomitant deletion of both Runx1 and Runx2 in a mouse epididymal epithelial cell line affected cell morphology, adhesion and mobility in vitro. Furthermore, lack of functional RUNXs severely disturbed the formation of 3D epididymal organoid-like structures. Transcriptomic analysis of the epididymal cell organoid-like structures indicated that RUNX1 and RUNX2 are involved in the regulation of MAPK signaling, NOTCH pathway activity, and EMT-related gene expression. This suggests that RUNXs are master regulators of several essential signaling pathways, and necessary for the maintenance of proper differentiation of the epididymal epithelium.

A senescence restriction point acting on chromatin integrates oncogenic signals.

We identify a senescence restriction point (SeRP) as a critical event for cells to commit to senescence. The SeRP integrates the intensity and duration of oncogenic stress, keeps a memory of previous stresses, and combines oncogenic signals acting on different pathways by modulating chromatin accessibility. Chromatin regions opened upon commitment to senescence are enriched in nucleolar-associated domains, which are gene-poor regions enriched in repeated sequences. Once committed to senescence, cells no longer depend on the initial stress signal and exhibit a characteristic transcriptome regulated by a transcription factor network that includes ETV4, RUNX1, OCT1, and MAFB. Consistent with a tumor suppressor role for this network, the levels of ETV4 and RUNX1 are very high in benign lesions of the pancreas but decrease dramatically in pancreatic ductal adenocarcinomas. The discovery of senescence commitment and its chromatin-linked regulation suggests potential strategies for reinstating tumor suppression in human cancers.

Tyrosine phosphorylation of CARM1 promotes its enzymatic activity and alters its target specificity.

An important epigenetic component of tyrosine kinase signaling is the phosphorylation of histones, and epigenetic readers, writers, and erasers. Phosphorylation of protein arginine methyltransferases (PRMTs), have been shown to enhance and impair their enzymatic activity. In this study, we show that the hyperactivation of Janus kinase 2 (JAK2) by the V617F mutation phosphorylates tyrosine residues (Y149 and Y334) in coactivator-associated arginine methyltransferase 1 (CARM1), an important target in hematologic malignancies, increasing its methyltransferase activity and altering its target specificity. While non-phosphorylatable CARM1 methylates some established substrates (e.g. BAF155 and PABP1), only phospho-CARM1 methylates the RUNX1 transcription factor, on R223 and R319. Furthermore, cells expressing non-phosphorylatable CARM1 have impaired cell-cycle progression and increased apoptosis, compared to cells expressing phosphorylatable, wild-type CARM1, with reduced expression of genes associated with G2/M cell cycle progression and anti-apoptosis. The presence of the JAK2-V617F mutant kinase renders acute myeloid leukemia (AML) cells less sensitive to CARM1 inhibition, and we show that the dual targeting of JAK2 and CARM1 is more effective than monotherapy in AML cells expressing phospho-CARM1. Thus, the phosphorylation of CARM1 by hyperactivated JAK2 regulates its methyltransferase activity, helps select its substrates, and is required for the maximal proliferation of malignant myeloid cells.

PU.1 and BCL11B sequentially cooperate with RUNX1 to anchor mSWI/SNF to poise the T cell effector landscape.

Adaptive immunity relies on specialized effector functions elicited by lymphocytes, yet how antigen recognition activates appropriate effector responses through nonspecific signaling intermediates is unclear. Here we examined the role of chromatin priming in specifying the functional outputs of effector T cells and found that most of the cis-regulatory landscape active in effector T cells was poised early in development before the expression of the T cell antigen receptor. We identified two principal mechanisms underpinning this poised landscape: the recruitment of the nucleosome remodeler mammalian SWItch/Sucrose Non-Fermentable (mSWI/SNF) by the transcription factors RUNX1 and PU.1 to establish chromatin accessibility at T effector loci; and a 'relay' whereby the transcription factor BCL11B succeeded PU.1 to maintain occupancy of the chromatin remodeling complex mSWI/SNF together with RUNX1, after PU.1 silencing during lineage commitment. These mechanisms define modes by which T cells acquire the potential to elicit specialized effector functions early in their ontogeny and underscore the importance of integrating extrinsic cues to the developmentally specified intrinsic program.

A novel t(X;21)(p11.4;q22.12) translocation adds to the role of BCOR and RUNX1 in myelodysplastic syndromes and acute myeloid leukemias.

In myeloid neoplasms, both fusion genes and gene mutations are well-established events identifying clinicopathological entities. In this study, we present a thus far undescribed t(X;21)(p11.4;q22.12) in five cases with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). The translocation was isolated or accompanied by additional changes. It did not generate any fusion gene or gene deregulation by aberrant juxtaposition with regulatory sequences. Molecular analysis by targeted next-generation sequencing showed that the translocation was accompanied by at least one somatic mutation in TET2, EZH2, RUNX1, ASXL1, SRSF2, ZRSR2, DNMT3A, and NRAS genes. Co-occurrence of deletion of RUNX1 in 21q22 and of BCOR in Xp11 was associated with t(X;21). BCOR haploinsufficiency corresponded to a significant hypo-expression in t(X;21) cases, compared to normal controls and to normal karyotype AML. By contrast, RUNX1 expression was not altered, suggesting a compensatory effect by the remaining allele. Whole transcriptome analysis showed that overexpression of HOXA9 differentiated t(X;21) from both controls and t(8;21)-positive AML. In conclusion, we characterized a new recurrent reciprocal t(X;21)(p11.4;q22.12) chromosome translocation in MDS and AML, generating simultaneous BCOR and RUNX1 deletions rather than a fusion gene at the genomic level.

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Runt-related transcription factor (RUNX1) is a transcription factor closely involved in hematopoiesis. RUNX1 gene mutation plays an essential pathogenic role in the initiation and development of hematological tumors, especially in acute myeloid leukemia. Recent studies have shown that RUNX1 is also involved in the regulation of bone development and the pathological progression of bone-related diseases. RUNX1 promotes the differentiation of mesenchymal stem cells into chondrocytes and osteoblasts and modulates the maturation and extracellular matrix formation of chondrocytes. The expression of RUNX1 in mesenchymal stem cells, chondrocytes, and osteoblasts is of great significance for maintaining normal bone development and the mass and quality of bones. RUNX1 also inhibits the differentiation and bone resorptive activities of osteoclasts, which may be influenced by sexual dimorphism. In addition, RUNX1 deficiency contributes to the pathogenesis of osteoarthritis, delayed fracture healing, and osteoporosis, which was revealed by the RUNX1 conditional knockout modeling in mice. However, the roles of RUNX1 in regulating the hypertrophic differentiation of chondrocytes, the sexual dimorphism of activities of osteoclasts, as well as bone loss in diabetes mellitus, senescence, infection, chronic inflammation, etc, are still not fully understood. This review provides a systematic summary of the research progress concerning RUNX1 in the field of bone biology, offering new ideas for using RUNX1 as a potential target for bone related diseases, especially osteoarthritis, delayed fracture healing, and osteoporosis.

Flow cytometric minimal residual disease measurement accounting for cytogenetics in children with non-high-risk acute lymphoblastic leukemia treated according to the ALL-MB 2008 protocol.

BACKGROUND: Quantitative measurement of minimal residual disease (MRD) is the "gold standard" for estimating the response to therapy in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Nevertheless, the speed of the MRD response differs for different cytogenetic subgroups. Here we present results of MRD measurement in children with BCP-ALL, in terms of genetic subgroups with relation to clinically defined risk groups. METHODS: A total of 485 children with non-high-risk BCP-ALL with available cytogenetic data and MRD studied at the end-of-induction (EOI) by multicolor flow cytometry (MFC) were included. All patients were treated with standard-risk (SR) of intermediate-risk (ImR) regimens of "ALL-MB 2008" reduced-intensity protocol. RESULTS AND DISCUSSION: Among all study group patients, 203 were found to have low-risk cytogenetics (ETV6::RUNX1 or high hyperdiploidy), while remaining 282 children were classified in intermediate cytogenetic risk group. For the patients with favorable and intermediate risk cytogenetics, the most significant thresholds for MFC-MRD values were different: 0.03% and 0.04% respectively. Nevertheless, the most meaningful thresholds were different for clinically defined SR and ImR groups. For the SR group, irrespective to presence/absence of favorable genetic lesions, MFC-MRD threshold of 0.1% was the most clinically valuable, although for ImR group the most informative thresholds were different in patients from low-(0.03%) and intermediate (0.01%) cytogenetic risk groups. CONCLUSION: Our data show that combining clinical risk factors with MFC-MRD measurement is the most useful tool for risk group stratification of children with BCP-ALL in the reduced-intensity protocols. However, this algorithm can be supplemented with cytogenetic data for part of the ImR group.

AML with RUNX1::RUNX1T1 Cooperating two Mutations Relapsed Quickly after Achieving CR.

BACKGROUND: Acute myeloid leukemia (AML) with t(8;21)(q22;q22.1); RUNX1::RUNX1T1 has a relatively favorable prognosis with a high complete remission rate and long disease-free survival. METHODS AND RESULTS: Here we describe a patient who had AML with t(8;21)(q22;q22.1); RUNX1::RUNX1T1. Cooperating mutations including KRAS and ASXL1, and with other abnormal karyotype del(17) and with a myelomonocytic differentiation. CONCLUSIONS: The patient relapsed despite achieving a morphologic complete remission (CR).

Gene Expression Studies in Down Syndrome: What Do They Tell Us about Disease Phenotypes?

Down syndrome is a well-studied aneuploidy condition in humans, which is associated with various disease phenotypes including cardiovascular, neurological, haematological and immunological disease processes. This review paper aims to discuss the research conducted on gene expression studies during fetal development. A descriptive review was conducted, encompassing all papers published on the PubMed database between September 1960 and September 2022. We found that in amniotic fluid, certain genes such as COL6A1 and DSCR1 were found to be affected, resulting in phenotypical craniofacial changes. Additionally, other genes such as GSTT1, CLIC6, ITGB2, C21orf67, C21orf86 and RUNX1 were also identified to be affected in the amniotic fluid. In the placenta, dysregulation of genes like MEST, SNF1LK and LOX was observed, which in turn affected nervous system development. In the brain, dysregulation of genes DYRK1A, DNMT3L, DNMT3B, TBX1, olig2 and AQP4 has been shown to contribute to intellectual disability. In the cardiac tissues, dysregulated expression of genes GART, ETS2 and ERG was found to cause abnormalities. Furthermore, dysregulation of XIST, RUNX1, SON, ERG and STAT1 was observed, contributing to myeloproliferative disorders. Understanding the differential expression of genes provides insights into the genetic consequences of DS. A better understanding of these processes could potentially pave the way for the development of genetic and pharmacological therapies.

[Use of the ETV6/RUNX1 probe to verify the performance of the fluorescence in situ hybridization probe before clinical detection].

Objective: To explore the standardized performance of a FISH probe before clinical detection. Methods: The probe sensitivity and specificity of ETV6/RUNX1 were analyzed via interphase and metaphase FISH in 20 discarded healthy bone marrow samples. The threshold system of the probe was established using an inverse beta distribution, and an interpretation standard was established. Finally, a parallel-controlled polymerase chain reaction detection study was conducted on 286 bone marrow samples from patients at our hospital. The clinical sensitivity, specificity, and diagnostic coincidence rate of ETV6/RUNX1 FISH detection were analyzed, and the diagnostic consistency of the two methods was analyzed by the kappa test. Results: The probe sensitivity and specificity of the ETV6/RUNX1 probe were 98.47% and 100%, respectively. When 50, 100, and 200 cells were counted, the typical positive signal pattern cutoffs were 5.81%, 2.95%, and 1.49%, respectively, and the atypical positive signal pattern cutoffs were 13.98%, 9.75%, and 6.26%, respectively. The clinical sensitivity of FISH was 96.1%, clinical specificity was 99.6%, diagnostic coincidence rate was 99.00%, diagnostic consistency test kappa value was 0.964, and P value was <0.001. Conclusion: For FISH probes without a national medical device registration certificate, standardized performance verification and methodology performance verification can be performed using laboratory developed test verification standards to ensure a reliable and accurate reference basis for clinical diagnosis and treatment.

Usefulness of KIT mutant transcript levels for monitoring measurable residual disease in t (8;21) acute myeloid leukemia.

In addition to RUNX1::RUNX1T1 transcript levels, measurable residual disease monitoring using KIT mutant (KITmut ) DNA level is reportedly predictive of relapse in t (8; 21) acute myeloid leukemia (AML). However, the usefulness of KITmut transcript levels remains unknown. A total of 202 bone marrow samples collected at diagnosis and during treatment from 52 t (8; 21) AML patients with KITmut (D816V/H/Y or N822K) were tested for KITmut transcript levels using digital polymerase chain reaction. The individual optimal cutoff values of KITmut were identified by performing receiver operating characteristics curve analysis for relapse at each of the following time points: at diagnosis, after achieving complete remission (CR), and after Course 1 and 2 consolidations. The cutoff values were used to divide the patients into the KITmut -high (KIT_H) group and the KITmut -low (KIT_L) group. The KIT_H patients showed significantly lower relapse-free survival (RFS) and overall survival (OS) rates than the KIT_L patients after Course 1 consolidation (p = 0.0040 and 0.021, respectively) and Course 2 consolidation (p = 0.018 and 0.011, respectively) but not at diagnosis and CR. The <3-log reduction in the RUNX1::RUNX1T1 transcript levels after Course 2 consolidation was an independent adverse prognostic factor for RFS and OS. After Course 2 consolidation, the KIT_H patients with >3-log reduction in the RUNX1::RUNX1T1 transcript levels (11/45; 24.4%) had similar RFS as that of patients with <3-log reduction in the RUNX1::RUNX1T1 transcript levels. The combination of KITmut and RUNX1::RUNX1T1 transcript levels after Course 2 consolidation may improve risk stratification in t (8; 21) AML patient with KIT mutation.

FTO-mediated m6A mRNA demethylation aggravates renal fibrosis by targeting RUNX1 and further enhancing PI3K/AKT pathway.

Chronic kidney disease (CKD) is a global health burden, with ineffective therapies leading to increasing morbidity and mortality. Renal interstitial fibrosis is a common pathway in advanced CKD, resulting in kidney function and structure deterioration. In this study, we investigate the role of FTO-mediated N6-methyladenosine (m6A) and its downstream targets in the pathogenesis of renal fibrosis. M6A modification, a prevalent mRNA internal modification, has been implicated in various organ fibrosis processes. We use a mouse model of unilateral ureteral obstruction (UUO) as an in vivo model and treated tubular epithelial cells (TECs) with transforming growth factor (TGF)-ß1 as in vitro models. Our findings revealed increased FTO expression in UUO mouse model and TGF-ß1-treated TECs. By modulating FTO expression through FTO heterozygous mutation mice (FTO+/- ) in vivo and small interfering RNA (siRNA) in vitro, we observed attenuation of UUO and TGF-ß1-induced epithelial-mesenchymal transition (EMT), as evidenced by decreased fibronectin and N-cadherin accumulation and increased E-cadherin levels. Silencing FTO significantly improved UUO and TGF-ß1-induced inflammation, apoptosis, and inhibition of autophagy. Further transcriptomic assays identified RUNX1 as a downstream candidate target of FTO. Inhibiting FTO was shown to counteract UUO/TGF-ß1-induced RUNX1 elevation in vivo and in vitro. We demonstrated that FTO signaling contributes to the elevation of RUNX1 by demethylating RUNX1 mRNA and improving its stability. Finally, we revealed that the PI3K/AKT pathway may be activated downstream of the FTO/RUNX1 axis in the pathogenesis of renal fibrosis. In conclusion, identifying small-molecule compounds that target this axis could offer promising therapeutic strategies for treating renal fibrosis.

Functional characterization of cooperating MGA mutations in RUNX1::RUNX1T1 acute myeloid leukemia.

MGA (Max-gene associated) is a dual-specificity transcription factor that negatively regulates MYC-target genes to inhibit proliferation and promote differentiation. Loss-of-function mutations in MGA have been commonly identified in several hematological neoplasms, including acute myeloid leukemia (AML) with RUNX1::RUNX1T1, however, very little is known about the impact of these MGA alterations on normal hematopoiesis or disease progression. We show that representative MGA mutations identified in patient samples abolish protein-protein interactions and transcriptional activity. Using a series of human and mouse model systems, including a newly developed conditional knock-out mouse strain, we demonstrate that loss of MGA results in upregulation of MYC and E2F targets, cell cycle genes, mTOR signaling, and oxidative phosphorylation in normal hematopoietic cells, leading to enhanced proliferation. The loss of MGA induces an open chromatin state at promoters of genes involved in cell cycle and proliferation. RUNX1::RUNX1T1 expression in Mga-deficient murine hematopoietic cells leads to a more aggressive AML with a significantly shortened latency. These data show that MGA regulates multiple pro-proliferative pathways in hematopoietic cells and cooperates with the RUNX1::RUNX1T1 fusion oncoprotein to enhance leukemogenesis.

RUNX1 C-terminal mutations impair blood cell differentiation by perturbing specific enhancer-promoter networks.

ABSTRACT: The transcription factor RUNX1 is a master regulator of hematopoiesis and is frequently mutated in myeloid malignancies. Mutations in its runt homology domain (RHD) frequently disrupt DNA binding and result in loss of RUNX1 function. However, it is not clearly understood how other RUNX1 mutations contribute to disease development. Here, we characterized RUNX1 mutations outside of the RHD. Our analysis of the patient data sets revealed that mutations within the C-terminus frequently occur in hematopoietic disorders. Remarkably, most of these mutations were nonsense or frameshift mutations and were predicted to be exempt from nonsense-mediated messenger RNA decay. Therefore, this class of mutation is projected to produce DNA-binding proteins that contribute to the pathogenesis in a distinct manner. To model this, we introduced the RUNX1R320∗ mutation into the endogenous gene locus and demonstrated the production of RUNX1R320∗ protein. Expression of RUNX1R320∗ resulted in the disruption of RUNX1 regulated processes such as megakaryocytic differentiation, through a transcriptional signature different from RUNX1 depletion. To understand the underlying mechanisms, we used Global RNA Interactions with DNA by deep sequencing (GRID-seq) to examine enhancer-promoter connections. We identified widespread alterations in the enhancer-promoter networks within RUNX1 mutant cells. Additionally, we uncovered enrichment of RUNX1R320∗ and FOXK2 binding at the MYC super enhancer locus, significantly upregulating MYC transcription and signaling pathways. Together, our study demonstrated that most RUNX1 mutations outside the DNA-binding domain are not subject to nonsense-mediated decay, producing protein products that act in concert with additional cofactors to dysregulate hematopoiesis through mechanisms distinct from those induced by RUNX1 depletion.

Natural history of clonal haematopoiesis seen in real-world haematology settings.

Recursive partitioning of healthy consortia led to the development of the Clonal Hematopoiesis Risk Score (CHRS) for clonal haematopoiesis (CH); however, in the practical setting, most cases of CH are diagnosed after patients present with cytopenias or related symptoms. To address this real-world population, we characterize the clinical trajectories of 94 patients with CH and distinguish CH harbouring canonical DNMT3A/TET2/ASXL1 mutations alone ('sole DTA') versus all other groups ('non-sole DTA'). TET2, rather than DNMT3A, was the most prevalent mutation in the real-world setting. Sole DTA patients did not progress to myeloid neoplasm (MN) in the absence of acquisition of other mutations. Contrastingly, 14 (20.1%) of 67 non-sole DTA patients progressed to MN. CHRS assessment showed a higher frequency of high-risk CH in non-sole DTA (vs. sole DTA) patients and in progressors (vs. non-progressors). RUNX1 mutation conferred the strongest risk for progression to MN (odds ratio [OR] 10.27, 95% CI 2.00-52.69, p = 0.0053). The mean variant allele frequency across all genes was higher in progressors than in non-progressors (36.9% ± 4.62% vs. 24.1% ± 1.67%, p = 0.0064). This analysis in the post-CHRS era underscores the natural history of CH, providing insight into patterns of progression to MN.

Differential prognostic values of the three AKT isoforms in acute myeloid leukemia.

The PI3K-AKT-mTOR pathway lies at the confluence of signaling pathways in which various components are subjected to activating genetic alterations in acute myeloid leukemia (AML), thus contributing to oncogenesis. Three AKT isoforms exist in humans. However, whether one isoform predominates in AML remains unknown. This study reveals that AKT3 behaves very distinctly than AKT1 or AKT2 in both normal myeloid differentiation and AML. During normal differentiation, AKT3 is preferentially expressed in hematopoietic stem cells whilst AKT1 becomes preferentially expressed as cells differentiate into granulocytes or monocytes. AKT2 expression remains unchanged. In AML, AKT3 expression varies widely among patient samples and is counterintuitively high in mature/monocytic leukemia. Furthermore, a low level of AKT3 expression is strongly correlated to genetic alterations associated with a better outcome (NPM1 mutations and RUNX1-RUNX1T1 translocation), while a high level is correlated to alterations associated to a bad outcome (RUNX1 mutations; and SRSF2, U2AF1, SF3B1, ASXL1 and BCOR mutations occurring frequently in MDS and MPN). Consistently, a high AKT3 expression level appears as a very strong predictor of poor survival. Curiously, although modestly varying among AML samples, a high AKT1 expression shows in contrast as a strong predictor of a better patient outcome. These data suggest that AKT3 and AKT1 expressions have strong, yet opposite, prognostic values.