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.
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.
Abstract
Peripheral T-cell lymphoma (PTCL) is a heterogeneous group of non-Hodgkin lymphomas with poor prognosis. Up to 30% of PTCL lack distinctive features and are classified as PTCL, not otherwise ...specified (PTCL-NOS). To further improve our understanding of the genetic landscape and biology of PTCL-NOS, we perform RNA-sequencing of 18 cases and validate results in an independent cohort of 37 PTCL cases. We identify
FYN-TRAF3IP2
,
KHDRBS1-LCK
and
SIN3A-FOXO1
as new in-frame fusion transcripts, with
FYN-TRAF3IP2
as a recurrent fusion detected in 8 of 55 cases. Using ex vivo and in vivo experiments, we demonstrate that
FYN-TRAF3IP2
and
KHDRBS1-LCK
activate signaling pathways downstream of the T cell receptor (TCR) complex and confer therapeutic vulnerability to clinically available drugs.
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 noncoding 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 find that TAL1 on its own is suppressing 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 find 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 find that both TAL1 and PI3K-AKT signaling increase the DNA-repair signature in T cells resulting in synergy between PARP and PI3KAKT pathway inhibition. In conclusion, we have developed a novel mouse model for TAL1+AKTE17K driven T-ALL development and identify a vulnerability of these leukemia cells to PI3K-AKT and PARP inhibitors.
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.
Abstract
Spi-1 Proto-Oncogene (SPI1)
fusion genes are recurrently found in T-cell acute lymphoblastic leukemia (T-ALL) cases but are insufficient to drive leukemogenesis. Here we show that
SPI1
...fusions in combination with activating
NRAS
mutations drive an immature T-ALL in vivo using a conditional bone marrow transplant mouse model. Addition of the oncogenic fusion to the
NRAS
mutation also results in a higher leukemic stem cell frequency. Mechanistically, genetic deletion of the β-catenin binding domain within
Transcription factor 7
(
TCF7)-SPI1
or use of a TCF/β-catenin interaction antagonist abolishes the oncogenic activity of the fusion. Targeting the
TCF7-SPI1
fusion in vivo with a doxycycline-inducible knockdown results in increased differentiation. Moreover, both pharmacological and genetic inhibition lead to down-regulation of
SPI1
targets. Together, our results reveal an example where
TCF7-SPI1
leukemia is vulnerable to pharmacological targeting of the TCF/β-catenin interaction.
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.
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.
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.
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.
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
, as a member of the core Treg lncRNA transcriptome.
expression anticipates Foxp3 expression during
Treg conversion, and
-deficient mice show a mild delay in
and peripheral Treg induction. These results implicate
as part of the upstream cascade leading to Treg conversion, and may provide clues as to the nature of this process.
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.
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