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Abstract Background Lymphangiogenesis, the formation of lymphatic vessels, is tightly linked to the development of the venous vasculature, both at the cellular and molecular levels. Here, we identify a novel role for Sorbs1, the founding member of the SoHo family of cytoskeleton adaptor proteins, in vascular and lymphatic development in the zebrafish. Results We show that Sorbs1 is required for secondary sprouting and emergence of several vascular structures specifically derived from the axial vein. Most notably, formation of the precursor parachordal lymphatic structures is affected in sorbs1 mutant embryos, severely impacting the establishment of the trunk lymphatic vessel network. Interestingly, we show that Sorbs1 interacts with the BMP pathway and could function outside of Vegfc signaling. Mechanistically, Sorbs1 controls FAK/Src signaling and subsequently impacts on the cytoskeleton processes regulated by Rac1 and RhoA GTPases. Inactivation of Sorbs1 altered cell-extracellular matrix (ECM) contacts rearrangement and cytoskeleton dynamics, leading to specific defects in endothelial cell migratory and adhesive properties. Conclusions Overall, using in vitro and in vivo assays, we identify Sorbs1 as an important regulator of venous and lymphatic angiogenesis independently of the Vegfc signaling axis. These results provide a better understanding of the complexity found within context-specific vascular and lymphatic development.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive type of leukemia caused by accumulation of multiple genetic alterations in T-cell progenitors. However, for many genes it remains unknown how their mutations contribute to disease development. Therefore, we performed two single-cell CRISPR screens in primary pro-T cells ex vivo to study the transcriptional impact of loss-of-function alterations in T-ALL and correlate this with effects on cell fitness. The various perturbations were clustered based on their effects on E2F/MYC or STAT/NOTCH signatures, which play a defining role in driving T-cell proliferation. Many of the perturbations resulted in positive effects on the STAT and NOTCH signatures and were predicted to behave as haploinsufficient tumor suppressors in T-ALL. Additionally, Spi1 was identified as an essential gene for pro-T cell survival, associated with deregulation of the MYC signature and epigenetic consequences. In contrast, Bcl11b was identified a strong tumor suppressor gene in immature T lymphocytes, associated with deregulation of NF-kB and JAK/STAT signaling. We found a correlation between BCL11B expression level and JAK/STAT pathway mutations in T-ALL patients and demonstrated oncogenic cooperation between Bcl11b inactivation and JAK3 hyperactivation in pro-T cells. Altogether, these single-cell CRISPR screens in pro-T cells provide fundamental insights in the mechanisms of transcriptional deregulation caused by genetic alterations in T-ALL.

Abstract The CRISPR genome editing technology has revolutionized the way gene function is studied. Genome editing can be achieved in single genes or for thousands of genes simultaneously in sensitive genetic screens. While conventional genetic screens are limited to bulk measurements of cell behavior, recent developments in single-cell technologies make it possible to combine CRISPR screening with single-cell profiling. In this way, cell behavior and gene expression can be monitored simultaneously, with the additional possibility of including data on chromatin accessibility and protein levels. Moreover, the availability of various Cas proteins leading to inactivation, activation, or other effects on gene function further broadens the scope of such screens. The integration of single-cell multi-omics approaches with CRISPR screening open the path to high-content information on the impact of genetic perturbations at single-cell resolution. Current limitations in cell throughput and data density need to be taken into consideration, but new technologies are rapidly evolving and are likely to easily overcome these limitations. In this review, we discuss the use of bulk CRISPR screening in hematology research, as well as the emergence of single-cell CRISPR screening and its added value to the field.

Plasmodium parasites cause malaria, a global health disease that is responsible for more than 200 million clinical cases and 600 000 deaths each year. Most deaths are caused by various complications, including malaria-associated acute respiratory distress syndrome (MA-ARDS). Despite the very rapid and efficient killing of parasites with antimalarial drugs, 15% of patients with complicated malaria succumb. This stresses the importance of investigating resolution mechanisms that are involved in the recovery from these complications once the parasite is killed. To study the resolution of MA-ARDS, P. berghei NK65-infected C57BL/6 mice were treated with antimalarial drugs after onset of symptoms, resulting in 80% survival. Micro-computed tomography revealed alterations of the lungs upon infection, with an increase in total and non-aerated lung volume due to edema. Whole body plethysmography confirmed a drastically altered lung ventilation, which was restored during resolution. Single-cell RNA sequencing indicated an increased inflammatory state in the lungs upon infection, which was accompanied by a drastic decrease in endothelial cells, consistent with CD8+ T cell-mediated killing. During resolution, anti-inflammatory pathways were upregulated and proliferation of endothelial cells was observed. MultiNicheNet interactome analysis identified important changes in the ligand-receptor interactions during disease resolution that warrant further exploration in order to develop new therapeutic strategies. In conclusion, our study provides insights in pro-resolving pathways that limit inflammation and promote endothelial cell proliferation in experimental MA-ARDS. This information may be useful for the design of adjunctive treatments to enhance resolution after Plasmodium parasite killing by antimalarial drugs.

SPI1 fusion genes in T-cell acute lymphoblastic leukemia (T-ALL) are commonly found with co-occurring NRAS mutations. Here, the authors show that the combination of these oncogenes is necessary to drive T-ALL in a murine model and that the oncogenic activity of the SPI1 fusion is dependent on β-catenin.

Summary: Although chemotherapy induces complete remission in the majority of acute myeloid leukemia (AML) patients, many face a relapse. This relapse is caused by survival of chemotherapy-resistant leukemia (stem) cells (measurable residual disease; MRD). Here, we demonstrate that the anthracycline doxorubicin epigenetically reprograms leukemia cells by inducing histone 3 lysine 27 (H3K27) and H3K4 tri-methylation. Within a doxorubicin-sensitive leukemia cell population, we identified a subpopulation of reversible anthracycline-tolerant cells (ATCs) with leukemic stem cell (LSC) features lacking doxorubicin-induced H3K27me3 or H3K4me3 upregulation. These ATCs have a distinct transcriptional landscape than the leukemia bulk and could be eradicated by KDM6 inhibition. In primary AML, reprogramming the transcriptional state by targeting KDM6 reduced MRD load and survival of LSCs residing within MRD, and enhanced chemotherapy response in vivo. Our results reveal plasticity of anthracycline resistance in AML cells and highlight the potential of transcriptional reprogramming by epigenetic-based therapeutics to target chemotherapy-resistant AML cells.

Peripheral T cell lymphoma (PTCL) not otherwise specified (NOS) is a subgroup of PTCL, which has no distinctive features and is poorly characterized at the genetic level. Here, the authors identify two fusion transcripts that activate T cell receptor complex signalling and confer therapeutic vulnerability in PTCL-NOS.

Acute lymphoblastic leukemia (ALL) is characterized by the presence of chromosomal changes, including numerical changes, translocations, and deletions, which are often associated with additional single-nucleotide mutations. In this study, we used single cell–targeted DNA sequencing to evaluate the clonal heterogeneity of B-ALL at diagnosis and during chemotherapy treatment. We designed a custom DNA amplicon library targeting mutational hotspot regions (in 110 genes) present in ALL, and we measured the presence of mutations and small insertions/deletions (indels) in bone marrow or blood samples from 12 B-ALL patients, with a median of 7973 cells per sample. Nine of the 12 cases showed at least 1 subclonal mutation, of which cases with PAX5 alterations or high hyperdiploidy (with intermediate to good prognosis) showed a high number of subclones (1 to 7) at diagnosis, defined by a variety of mutations in the JAK/STAT, RAS, or FLT3 signaling pathways. Cases with RAS pathway mutations had multiple mutations in FLT3, NRAS, KRAS, or BRAF in various clones. For those cases where we detected multiple mutational clones at diagnosis, we also studied blood samples during the first weeks of chemotherapy treatment. The leukemia clones disappeared during treatment with various kinetics, and few cells with mutations were easily detectable, even at low frequency (2 subclones at diagnosis and that even very rare mutant cells can be detected at diagnosis or during treatment by single cell–targeted DNA sequencing.

TAL1 is ectopically expressed in about 30% of T-cell acute lymphoblastic leukemia (T-ALL) due to chromosomal rearrangements leading to the STIL-TAL1 fusion genes or due to non-coding mutations leading to a de novo enhancer driving TAL1 expression. Analysis of sequence data from T-ALL cases demonstrates a significant association between TAL1 expression and activating mutations of the PI3K-AKT pathway. We investigated the oncogenic function of TAL1 and the possible cooperation with PI3K-AKT pathway activation using isogenic pro-T-cell cultures ex vivo and in vivo leukemia models. We found that TAL1 on its own suppressed T-cell growth, in part by affecting apoptosis genes, while the combination with AKT pathway activation reduced apoptosis and was strongly driving cell proliferation ex vivo and leukemia development in vivo. As a consequence, we found that TAL1+AKTE17K transformed cells are more sensitive to PI3K-AKT pathway inhibition compared to AKTE17K transformed cells, related to the negative effect of TAL1 in the absence of activated PI3K-AKT signaling. We also found that both TAL1 and PI3K-AKT signaling increased the DNA-repair signature in T cells resulting in synergy between PARP and PI3K-AKT pathway inhibition. In conclusion, we have developed a novel mouse model for TAL1+AKTE17K driven T-ALL development and have identified a vulnerability of these leukemia cells to PI3K-AKT and PARP inhibitors.

Abstract Background The CNOT3 protein is a subunit of the CCR4-NOT complex, which is involved in mRNA degradation. We recently identified CNOT3 loss-of-function mutations in patients with T-cell acute lymphoblastic leukemia (T-ALL). Methods Here, we use different Drosophila melanogaster eye cancer models to study the potential tumor suppressor function of Not3, the CNOT3 orthologue, and other members of the CCR4-NOT complex. Results Our data show that knockdown of Not3, the structural components Not1/Not2, and the deadenylases twin/Pop2 all result in increased tumor formation. In addition, overexpression of Not3 could reduce tumor formation. Not3 downregulation has a mild but broad effect on gene expression and leads to increased levels of genes involved in DNA replication and ribosome biogenesis. CycB upregulation also contributes to the Not3 tumor phenotype. Similar findings were obtained in human T-ALL cell lines, pointing out the conserved function of Not3. Conclusions Together, our data establish a critical role for Not3 and the entire CCR4-NOT complex as tumor suppressor.

Mammalian genomes encode a plethora of long non-coding RNA (lncRNA). These transcripts are thought to regulate gene expression, influencing biological processes from development to pathology. Results from the few lncRNA that have been studied in the context of the immune system have highlighted potentially critical functions as network regulators. Here we explored the nature of the lncRNA transcriptome in regulatory T cells (Tregs), a subset of CD4+ T cells required to establish and maintain immunological self-tolerance. The identified Treg lncRNA transcriptome showed distinct differences from that of non-regulatory CD4+ T cells, with evidence of direct shaping of the lncRNA transcriptome by Foxp3, the master transcription factor driving the distinct mRNA profile of Tregs. Treg lncRNA changes were disproportionally reversed in the absence of Foxp3, with an enrichment for colocalisation with Foxp3 DNA binding sites, indicating a direct coordination of transcription by Foxp3 independent of the mRNA coordination function. We further identified a novel lncRNA Flatr, as a member of the core Treg lncRNA transcriptome. Flatr expression anticipates Foxp3 expression during in vitro Treg conversion, and Flatr-deficient mice show a mild delay in in vitro and peripheral Treg induction. These results implicate Flatr as part of the upstream cascade leading to Treg conversion, and may provide clues as to the nature of this process.

Despite therapeutic improvements, a sizable number of patients with T-cell acute lymphoblastic leukemia still have a poor outcome. To unravel the genomic background associated with refractoriness, we evaluated the transcriptome of 19 cases of refractory/early relapsed T-cell acute lymphoblastic leukemia (discovery cohort) by performing RNA-sequencing on diagnostic material. The incidence and prognostic impact of the most frequently mutated pathways were validated by Sanger sequencing on genomic DNA from diagnostic samples of an independent cohort of 49 cases (validation cohort), including refractory, relapsed and responsive cases. Combined gene expression and fusion transcript analyses in the discovery cohort revealed the presence of known oncogenes and identified novel rearrangements inducing overexpression, as well as inactivation of tumor suppressor genes. Mutation analysis identified JAK/STAT and RAS/PTEN as the most commonly disrupted pathways in patients with chemorefractory disease or early relapse, frequently in association with NOTCH1/FBXW7 mutations. The analysis on the validation cohort documented a significantly higher risk of relapse, inferior overall survival, disease-free survival and event-free survival in patients with JAK/STAT or RAS/PTEN alterations. Conversely, a significantly better survival was observed in patients harboring only NOTCH1/FBXW7 mutations: this favorable prognostic effect was abrogated by the presence of concomitant mutations. Preliminary in vitro assays on primary cells demonstrated sensitivity to specific inhibitors. These data document the negative prognostic impact of JAK/STAT and RAS/PTEN mutations in T-cell acute lymphoblastic leukemia and suggest the potential clinical application of JAK and PI3K/mTOR inhibitors in patients harboring mutations in these pathways.

Recurrent deletions of the long arm of chromosome 5 were detected in 23/200 cases of T-cell acute lymphoblastic leukemia. Genomic studies identified two types of deletions: interstitial and terminal. Interstitial 5q deletions, found in five cases, were present in both adults and children with a female predominance (chi-square, P=0.012). Interestingly, these cases resembled immature/early T-cell precursor acute lymphoblastic leukemia showing significant down-regulation of five out of the ten top differentially expressed genes in this leukemia group, including TCF7 which maps within the 5q31 common deleted region. Mutations of genes known to be associated with immature/early T-cell precursor acute lymphoblastic leukemia, i.e. WT1, ETV6, JAK1, JAK3, and RUNX1, were present, while CDKN2A/B deletions/mutations were never detected. All patients had relapsed/resistant disease and blasts showed an early differentiation arrest with expression of myeloid markers. Terminal 5q deletions, found in 18 of patients, were more prevalent in adults (chi-square, P=0.010) and defined a subgroup of HOXA-positive T-cell acute lymphoblastic leukemia characterized by 130 up- and 197 down-regulated genes. Down-regulated genes included TRIM41, ZFP62, MAPK9, MGAT1, and CNOT6, all mapping within the 1.4 Mb common deleted region at 5q35.3. Of interest, besides CNOT6 down-regulation, these cases also showed low BTG1 expression and a high incidence of CNOT3 mutations, suggesting that the CCR4-NOT complex plays a crucial role in the pathogenesis of HOXA-positive T-cell acute lymphoblastic leukemia with terminal 5q deletions. In conclusion, interstitial and terminal 5q deletions are recurrent genomic losses identifying distinct subtypes of T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia is caused by the accumulation of multiple oncogenic lesions, including chromosomal rearrangements and mutations. To determine the frequency and co-occurrence of mutations in T-cell acute lymphoblastic leukemia, we performed targeted re-sequencing of 115 genes across 155 diagnostic samples (44 adult and 111 childhood cases). NOTCH1 and CDKN2A/B were mutated/deleted in more than half of the cases, while an additional 37 genes were mutated/deleted in 4% to 20% of cases. We found that IL7R-JAK pathway genes were mutated in 27.7% of cases, with JAK3 mutations being the most frequent event in this group. Copy number variations were also detected, including deletions of CREBBP or CTCF and duplication of MYB. FLT3 mutations were rare, but a novel extracellular mutation in FLT3 was detected and confirmed to be transforming. Furthermore, we identified complex patterns of pairwise associations, including a significant association between mutations in IL7R-JAK genes and epigenetic regulators (WT1, PRC2, PHF6). Our analyses showed that IL7R-JAK genetic lesions did not confer adverse prognosis in T-cell acute lymphoblastic leukemia cases enrolled in the UK ALL2003 trial. Overall, these results identify interconnections between the T-cell acute lymphoblastic leukemia genome and disease biology, and suggest a potential clinical application for JAK inhibitors in a significant proportion of patients with T-cell acute lymphoblastic leukemia.

Subgraph matching algorithms are used to find and enumerate specific interconnection structures in networks. By enumerating these specific structures/subgraphs, the fundamental properties of the network can be derived. More specifically in biological networks, subgraph matching algorithms are used to discover network motifs, specific patterns occurring more often than expected by chance. Finding these network motifs yields information on the underlying biological relations modelled by the network. In this work, we present the Index-based Subgraph Matching Algorithm with General Symmetries (ISMAGS), an improved version of the Index-based Subgraph Matching Algorithm (ISMA). ISMA quickly finds all instances of a predefined motif in a network by intelligently exploring the search space and taking into account easily identifiable symmetric structures. However, more complex symmetries (possibly involving switching multiple nodes) are not taken into account, resulting in superfluous output. ISMAGS overcomes this problem by using a customised symmetry analysis phase to detect all symmetric structures in the network motif subgraphs. These structures are then converted to symmetry-breaking constraints used to prune the search space and speed up calculations. The performance of the algorithm was tested on several types of networks (biological, social and computer networks) for various subgraphs with a varying degree of symmetry. For subgraphs with complex (multi-node) symmetric structures, high speed-up factors are obtained as the search space is pruned by the symmetry-breaking constraints. For subgraphs with no or simple symmetric structures, ISMAGS still reduces computation times by optimising set operations. Moreover, the calculated list of subgraph instances is minimal as it contains no instances that differ by only a subgraph symmetry. An implementation of the algorithm is freely available at https://github.com/mhoubraken/ISMAGS.

Subgraph matching algorithms are designed to find all instances of predefined subgraphs in a large graph or network and play an important role in the discovery and analysis of so-called network motifs, subgraph patterns which occur more often than expected by chance. We present the index-based subgraph matching algorithm (ISMA), a novel tree-based algorithm. ISMA realizes a speedup compared to existing algorithms by carefully selecting the order in which the nodes of a query subgraph are investigated. In order to achieve this, we developed a number of data structures and maximally exploited symmetry characteristics of the subgraph. We compared ISMA to a naive recursive tree-based algorithm and to a number of well-known subgraph matching algorithms. Our algorithm outperforms the other algorithms, especially on large networks and with large query subgraphs. An implementation of ISMA in Java is freely available at http://sourceforge.net/projects/isma/.