Cellular hierarchy insights reveal leukemic stem-like cells and early death risk in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) represents a paradigm for targeted differentiation therapy, with a minority of patients experiencing treatment failure and even early death. We here report a comprehensive single-cell analysis of 16 APL patients, uncovering cellular compositions and their impact on all-trans retinoic acid (ATRA) response in vivo and early death. We unveil a cellular differentiation hierarchy within APL blasts, rooted in leukemic stem-like cells. The oncogenic PML/RARα fusion protein exerts branch-specific regulation in the APL trajectory, including stem-like cells. APL cohort analysis establishes an association of leukemic stemness with elevated white blood cell counts and FLT3-ITD mutations. Furthermore, we construct an APL-specific stemness score, which proves effective in assessing early death risk. Finally, we show that ATRA induces differentiation of primitive blasts and patients with early death exhibit distinct stemness-associated transcriptional programs. Our work provides a thorough survey of APL cellular hierarchies, offering insights into cellular dynamics during targeted therapy.

Transcription factor 12-mediated self-feedback regulatory mechanism is required in DUX4 fusion leukaemia.

BACKGROUND: IGH::DUX4 is frequently observed in 4% B-cell acute lymphoblastic leukaemia patients. Regarding the IGH::DUX4-driven transactivation and alternative splicing, which are the main reasons behind this acute leukaemia outbreak, it remains unclear how transcriptional cofactors contribute to this oncogenic process. Further investigation is required to elucidate their specific role in leukaemogenesis. METHODS: In order to investigate the cofactors of IGH::DUX4, integrated mining of Chromatin immunoprecipitation (ChIP)-sequencing and RNA-sequencing of leukaemia cells and patient samples were conducted. Furthermore, to elucidate the synergistic interaction between transcription factor 12 (TCF12) and IGH::DUX4, knockdown and knockout experiment, mammalian two-hybridisation assay, co-immunoprecipitation and in situ proximity ligation assays were carried out. Additionally, to further investigate the direct interaction between TCF12 and IGH::DUX4, AI-based structural simulations were utilised. Finally, to validate the synergistic role of TCF12 in promoting IGH::DUX4 leukaemia, cell proliferation, apoptosis and drug sensitivity experiments were performed. RESULTS: In this study, we observed that the IGH::DUX4 target gene TCF12 might be an important cofactor/helper for this oncogenic driver. The co-expression of IGH::DUX4 and TCF12 resulted in enhanced DUX4-driven transactivation. Supportively, knockdown and knockout of TCF12 significantly reduced expression of IGH::DUX4-driven target genes in leukaemia REH (a precursor B-cell leukaemia cell line) and NALM-6 cells (a precursor B-cell leukaemia cell line). Consistently, in TCF12 knockout cells, the expression of structure-based TCF12 mutant, but not wild-type TCF12, failed to restore the TCF12-IGH::DUX4 crosstalk and the synergistic transactivation. More importantly, the breakdown in TCF12-IGH::DUX4 cooperation impaired IGH::DUX4-driven leukaemia cell survival, caused sensitivity to the chemotherapy. CONCLUSIONS: Altogether, these results helped to define a previously unrecognised TCF12-mediated positive self-feedback regulatory mechanism in IGH::DUX4 leukaemia, which holds the potential to function as a pivotal drug target for the management of this particular form of leukaemia. HIGHLIGHTS: Transcription factor 12 (TCF12) is a new novel cofactor in IGH::DUX4 transcriptional complexes/machinery. TCF12 mediates a positive self-feedback regulatory mechanism in IGH::DUX4-driven oncogenic transaction. IGH::DUX4-TCF12 structure/cooperation might represent a potent target/direction in future drug design against B-cell acute lymphoblastic leukaemia.

Transcription factor abnormalities in B-ALL leukemogenesis and treatment.

The biological regulation of transcription factors (TFs) and repressor proteins is an important mechanism for maintaining cell homeostasis. In B cell acute lymphoblastic leukemia (B-ALL) TF abnormalities occur at high frequency and are often recognized as the major driving factor in carcinogenesis. We provide an in-depth review of molecular mechanisms of six major TF rearrangements in B-ALL, including DUX4-rearranged (DUX4-R), MEF2D-R, ZNF384-R, ETV6-RUNX1 and TCF3-PBX1 fusions, and KMT2A-R. In addition, the therapeutic options and prognoses for patients who harbor these TF abnormalities are discussed. This review aims to provide an up-to-date panoramic view of how TF-based oncogenic fusions might drive carcinogenesis and impact on potential therapeutic exploration of B-ALL treatments.

Phase separation is required for PML nuclear body biogenesis and function.

PML nuclear body (NB) malfunction often leads to acute leukemia outbreaks and other severe diseases. PML NB rescue is the molecular basis of arsenic success in acute promyelocytic leukemia (APL) treatment. However, it is unclear how PML NBs are assembled. Here, we observed the presence of liquid-liquid phase separation (LLPS) in NB formation by fluorescence recovery after photobleaching (FRAP) experiment. Compared with the wild-type (WT) NBs, PML A216V derived from arsenic-resistant leukemia patients markedly crippled LLPS, but not altered the overall structure and PML RBCC oligomerization. In parallel, we also reported several Leu to Pro mutations that were critical to PML coiled-coil domain. FRAP characterization and comparison between L268P and A216V revealed markedly different LLPS activities in these mutant NBs. Transmission electron microscopy (TEM) inspections of LLPS-crippled and uncrippled NBs showed aggregation- and ring-like PML packing in A216V and WT/L268P NBs, respectively. More importantly, the correct LLPS-driven NB formation was the prerequisite for partner recruitment, post-translational modifications (PTMs), and PML-driven cellular regulations, such as ROS stress control, mitochondria production, and PML-p53-mediated senescence and apoptosis. Altogether, our results helped to define a critical LLPS step in PML NB biogenesis.

A comprehensive benchmarking of differential splicing tools for RNA-seq analysis at the event level.

RNA alternative splicing, a post-transcriptional stage in eukaryotes, is crucial in cellular homeostasis and disease processes. Due to the rapid development of the next-generation sequencing (NGS) technology and the flood of NGS data, the detection of differential splicing from RNA-seq data has become mainstream. A range of bioinformatic tools has been developed. However, until now, an independent and comprehensive comparison of available algorithms/tools at the event level is still lacking. Here, 21 different tools are subjected to systematic evaluation, based on simulated RNA-seq data where exact differential splicing events are introduced. We observe immense discrepancies among these tools. SUPPA, DARTS, rMATS and LeafCutter outperforme other event-based tools. We also examine the abilities of the tools to identify novel splicing events, which shows that most event-based tools are unsuitable for discovering novel splice sites. To improve the overall performance, we present two methodological approaches i.e. low-expression transcript filtering and tool-pair combination. Finally, a new protocol of selecting tools to perform differential splicing analysis for different analytical tasks (e.g. precision and recall rate) is proposed. Under this protocol, we analyze the distinct splicing landscape in the DUX4/IGH subgroup of B-cell acute lymphoblastic leukemia and uncover the differential splicing of TCF12. All codes needed to reproduce the results are available at https://github.com/mhjiang97/Benchmarking_DS.

A typical bedside-to-bench investigation of leukemogenic driver MEF2D fusion reveals new targeted therapy in B-cell acute lymphoblastic leukemia.

B-cell acute lymphoblastic leukemia (B-ALL) is a malignant tumor originating from B-lineage lymphoid precursor cells. The incidence of B-ALL is about 80% in childhood acute leukemia and 20% in adults. In recent years, with standardized treatment guided by risk stratification, the long-term disease-free survival rate of children is about 80%, while that of adults is less than 40%. However, the specific pathogenesis of the newly identified B-ALL and the targeted therapy strategies have not been vigorously investigated. In this review, we highlight the recent breakthroughs in mechanistic studies and novel therapeutic options in DUX4- and MEF2D-subtype B-ALLs.

Functional, structural, and molecular characterizations of the leukemogenic driver MEF2D-HNRNPUL1 fusion.

Recurrent MEF2D fusions with poor prognosis have been identified in B-cell precursor ALL (BCP-ALL). The molecular mechanisms underlying the pathogenic function of MEF2D fusions are poorly understood. Here, we show that MEF2D-HNRNPUL1 (MH) knock-in mice developed a progressive disease from impaired B-cell development at the pre-pro-B stage to pre-leukemia over 10 to 12 months. When cooperating with NRASG12D, MH drove an outbreak of BCP-ALL, with a more aggressive phenotype than the NRASG12D-induced leukemia. RNA-sequencing identified key networks involved in disease mechanisms. In chromatin immunoprecipitation-sequencing experiments, MH acquired increased chromatin-binding ability, mostly through MEF2D-responsive element (MRE) motifs in target genes, compared with wild-type MEF2D. Using X-ray crystallography, the MEF2D-MRE complex was characterized in atomic resolution, whereas disrupting the MH-DNA interaction alleviated the aberrant target gene expression and the B-cell differentiation arrest. The C-terminal moiety (HNRNPUL1 part) of MH was proven to contribute to the fusion protein's trans-regulatory activity, cofactor recruitment, and homodimerization. Furthermore, targeting MH-driven transactivation of the HDAC family by using the histone deacetylase inhibitor panobinostat in combination with chemotherapy improved the overall survival of MH/NRASG12D BCP-ALL mice. Altogether, these results not only highlight MH as an important driver in leukemogenesis but also provoke targeted intervention against BCP-ALL with MEF2D fusions.

DNA crosslinking and recombination-activating genes 1/2 (RAG1/2) are required for oncogenic splicing in acute lymphoblastic leukemia.

BACKGROUND: Abnormal alternative splicing is frequently associated with carcinogenesis. In B-cell acute lymphoblastic leukemia (B-ALL), double homeobox 4 fused with immunoglobulin heavy chain (DUX4/IGH) can lead to the aberrant production of E-26 transformation-specific family related gene abnormal transcript (ERGalt ) and other splicing variants. However, the molecular mechanism underpinning this process remains elusive. Here, we aimed to know how DUX4/IGH triggers abnormal splicing in leukemia. METHODS: The differential intron retention analysis was conducted to identify novel DUX4/IGH-driven splicing in B-ALL patients. X-ray crystallography, small angle X-ray scattering (SAXS), and analytical ultracentrifugation were used to investigate how DUX4/IGH recognize double DUX4 responsive element (DRE)-DRE sites. The ERGalt biogenesis and B-cell differentiation assays were performed to characterize the DUX4/IGH crosslinking activity. To check whether recombination-activating gene 1/2 (RAG1/2) was required for DUX4/IGH-driven splicing, the proximity ligation assay, co-immunoprecipitation, mammalian two hybrid characterizations, in vitro RAG1/2 cleavage, and shRNA knock-down assays were performed. RESULTS: We reported previously unrecognized intron retention events in C-type lectin domain family 12, member A abnormal transcript (CLEC12Aalt ) and chromosome 6 open reading frame 89 abnormal transcript (C6orf89alt ), where also harbored repetitive DRE-DRE sites. Supportively, X-ray crystallography and SAXS characterization revealed that DUX4 homeobox domain (HD)1-HD2 might dimerize into a dumbbell-shape trans configuration to crosslink two adjacent DRE sites. Impaired DUX4/IGH-mediated crosslinking abolishes ERGalt , CLEC12Aalt , and C6orf89alt biogenesis, resulting in marked alleviation of its inhibitory effect on B-cell differentiation. Furthermore, we also observed a rare RAG1/2-mediated recombination signal sequence-like DNA edition in DUX4/IGH target genes. Supportively, shRNA knock-down of RAG1/2 in leukemic Reh cells consistently impaired the biogenesis of ERGalt , CLEC12Aalt , and C6orf89alt . CONCLUSIONS: All these results suggest that DUX4/IGH-driven DNA crosslinking is required for RAG1/2 recruitment onto the double tandem DRE-DRE sites, catalyzing V(D)J-like recombination and oncogenic splicing in acute lymphoblastic leukemia.

Crystal structure of the GTP-binding protein-like domain of AGAP1.

AGAP1 is often considered to regulate membrane trafficking, protein transport and actin cytoskeleton dynamics. Recent studies have shown that aberrant expression of AGAP1 is associated with many diseases, including neurodevelopmental disorders and acute lymphoblastic leukemia. It has been proposed that the GTP-binding protein-like domain (GLD) is involved in the binding of cofactors and thus regulates the catalytic activity of AGAP1. To obtain a better understanding of the pathogenic mechanism underpinning AGAP1-related diseases, it is essential to obtain structural information. Here, the GLD (residues 70-235) of AGAP1 was overexpressed in Escherichia coli BL21 (DE3) cells. Affinity and gel-filtration chromatography were used to obtain AGAP1GLD with high purity for crystallization. Using the hanging-drop vapor-diffusion method with the protein at a final concentration of 20 mg ml-1, AGAP1GLD protein crystals of suitable size were obtained. The crystals were found to diffract to 3.0 Šresolution and belonged to space group I4, with unit-cell parameters a = 100.39, b = 100.39, c = 48.08 Å. The structure of AGAP1GLD exhibits the highly conserved functional G1-G5 loops and is generally similar to other characterized ADP-ribosylation factor (Arf) GTPase-activating proteins (GAPs), implying an analogous function to Arf GAPs. Additionally, this study indicates that AGAP1 could be classified as a type of NTPase, the activity of which might be regulated by protein partners or by its other domains. Taken together, these results provide insight into the regulatory mechanisms of AGAP1 in cell signaling.

Purification, crystallization, and X-ray diffraction analysis of myocyte enhancer factor 2D and DNA complex.

MEF2D-fusions have recently been identified as one of the major oncogenic drivers in precursor B-cell acute lymphoblastic leukemia (B-ALL). More importantly, they are often associated with patients with poor prognosis in B-ALL. To have a better understanding of the pathogenic mechanism underpinning MEF2D-fusions-driven leukemogenesis, it's essential to uncover the related structure information. In this study, we expressed and purified the MEF2D N-terminal DNA binding domain. The recombinant protein was engineered by cloning the encoding gene into the expression vector pET-32 m. A series of chromatographic steps involving affinity, ion-exchange and gel-filtration chromatography were used to achieve a final purity of >95%. For the crystallization of the MEF2D-DNA complex, a double-stranded DNA encoding 5'-AACTATTTATAAGA-3' and 5'-TTCTTATAAATAGT-3' was used (Wu et al., 2010) [1]. The MEF2D-DNA crystal with the size of about 20 µm × 20 µm × 20 µm was obtained at a final concentration of 12 mg/ml at the reservoir condition containing 30% PEG1500. The X-ray examination showed that the MEF2D-DNA crystal diffracted to 4.5 Å resolution, and belonged to space group P1, with unit-cell parameters of a = 77.2 Å, b = 77.2 Å, c = 231.4 Å.

PML Nuclear Body Biogenesis, Carcinogenesis, and Targeted Therapy.

Targeted therapy has become increasingly important in cancer therapy. For example, targeting the promyelocytic leukemia PML protein in leukemia has proved to be an effective treatment. PML is the core component of super-assembled structures called PML nuclear bodies (NBs). Although this nuclear megaDalton complex was first observed in the 1960s, the mechanism of its assembly remains poorly understood. We review recent breakthroughs in the PML field ranging from a revised assembly mechanism to PML-driven genome organization and carcinogenesis. In addition, we highlight that oncogenic oligomerization might also represent a promising target in the treatment of leukemias and solid tumors.

PML nuclear body biogenesis and oligomerization-driven leukemogenesis.

PML nuclear bodies (NBs), which are increasingly recognized as the central hub of many cellular signaling events, are superassembled spherical complexes with diameters of 0.1-2 µm. Recent studies reveal that RING tetramerization and B1-box polymerization are key factors to the overall PML NBs assembly. The productive RBCC oligomerization allows subsequent PML biogenesis steps, including the PML auto-sumoylation and partners recruitment via SUMO-SIM interactions. In promyelocytic leukemia, the oncoprotein PML/RARα (P/R) inhibits PML NBs assembly and leads to a full-fledged leukemogenesis. In this review, we review the recent progress in PML and acute promyelocytic leukemia fields, highlighting the protein oligomerization as an important direction of future targeted therapy.

How BamA recruits OMP substrates via poly-POTRAs domain.

Almost all the outer membrane proteins (OMPs) fold into an invariant ß-barrel fold via the polypeptide-transport-associated (POTRA) motif and ß-barrel assembly machinery (BAM). However, whether and how poly-POTRAs interact with OMPs remain largely unknown. Here, we have characterized the structures of Haemophilus influenzae poly-POTRAs via X-ray crystallography, small angle X-ray scattering, and molecular dynamics simulation. Unexpectedly, crystal packing reveals a putative OMP travel pathway spiraled by the conserved α2-ß2 edges in poly-POTRAs. Supportively, the structure-based mutations targeting the OMP binding sites significantly disrupt OMP biogenesis, resulting in severe cell growth defects. Another notable feature in H. influenzae POTRA structures is flexibility. As characterized by ELISA assays, poly-POTRAs could recruit OMP substrates in a step-wise manner. More importantly, the restriction of POTRA-POTRA linkage and flexibility significantly impairs the BamA function and causes cell growth defect. Altogether, these results suggest that the ß-strand augmentations and intrinsic flexibility are important factors for BamA-OMP recruitment.-Ma, X., Wang, Q., Li, Y., Tan, P., Wu, H., Wang, P., Dong, X., Hong, L., Meng, G. How BamA recruits OMP substrates via poly-POTRAs domain.

B1 oligomerization regulates PML nuclear body biogenesis and leukemogenesis.

ProMyelocyticLeukemia (PML) protein can polymerize into a mega-Dalton nuclear assembly of 0.1-2 µm in diameter. The mechanism of PML nuclear body biogenesis remains elusive. Here, PMLRBCC is successfully purified. The gel filtration and ultracentrifugation analysis suggest a previously unrecognized sequential oligomerization mechanism via PML monomer, dimer, tetramer and N-mer. Consistently, PML B1-box structure (2.0 Å) and SAXS characterization reveal an unexpected networking by W157-, F158- and SD1-interfaces. Structure-based perturbations in these B1 interfaces not only impair oligomerization in vitro but also abolish PML sumoylation and nuclear body biogenesis in HeLaPml-/- cell. More importantly, as demonstrated by in vivo study using transgenic mice, PML-RARα (PR) F158E precludes leukemogenesis. In addition, single cell RNA sequencing analysis shows that B1 oligomerization is an important regulator in PML-RARα-driven transactivation. Altogether, these results not only define a previously unrecognized B1-box oligomerization in PML, but also highlight oligomerization as an important factor in carcinogenesis.

Structural basis of DUX4/IGH-driven transactivation.

Oncogenic fusions are major drivers in leukemogenesis and may serve as potent targets for treatment. DUX4/IGHs have been shown to trigger the abnormal expression of ERGalt through binding to DUX4-Responsive-Element (DRE), which leads to B-cell differentiation arrest and a full-fledged B-ALL. Here, we determined the crystal structures of Apo- and DNADRE-bound DUX4HD2 and revealed a clamp-like transactivation mechanism via the double homeobox domain. Biophysical characterization showed that mutations in the interacting interfaces significantly impaired the DNA binding affinity of DUX4 homeobox. These mutations, when introduced into DUX4/IGH, abrogated its transactivation activity in Reh cells. More importantly, the structure-based mutants significantly impaired the inhibitory effects of DUX4/IGH upon B-cell differentiation in mouse progenitor cells. All these results help to define a key DUX4/IGH-DRE recognition/step in B-ALL.

RING tetramerization is required for nuclear body biogenesis and PML sumoylation.

ProMyelocyticLeukemia nuclear bodies (PML NBs) are stress-regulated domains directly implicated in acute promyelocytic leukemia eradication. Most TRIM family members bind ubiquitin E2s and many acquire ligase activity upon RING dimerization. In contrast, PML binds UBC9, the SUMO E2 enzyme. Here, using X-ray crystallography and SAXS characterization, we demonstrate that PML RING tetramerizes through highly conserved PML-specific sequences, which are required for NB assembly and PML sumoylation. Conserved residues implicated in RING dimerization of other TRIMs also contribute to PML tetramer stability. Wild-type PML rescues the ability of some RING mutants to form NBs as well as their sumoylation. Impaired RING tetramerization abolishes PML/RARA-driven leukemogenesis in vivo and arsenic-induced differentiation ex vivo. Our studies thus identify RING tetramerization as a key step in the NB macro-molecular scaffolding. They suggest that higher order RING interactions allow efficient UBC9 recruitment and thus change the biochemical nature of TRIM-facilitated post-translational modifications.

HSP70-Hrd1 axis precludes the oncorepressor potential of N-terminal misfolded Blimp-1s in lymphoma cells.

B lymphocyte-induced maturation protein-1 (Blimp-1) ensures B-cell differentiation into the plasma cell stage, and its instability constitutes a crucial oncogenic element in certain aggressive cases of activated B cell-like diffuse large B-cell lymphoma (ABC-DLBCL). However, the underlying degradation mechanisms and their possible therapeutic relevance remain unexplored. Here, we show that N-terminal misfolding mutations in ABC-DLBCL render Blimp-1 protein susceptible to proteasome-mediated degradation but spare its transcription-regulating activity. Mechanistically, whereas wild-type Blimp-1 metabolism is triggered in the nucleus through PML-mediated sumoylation, the degradation of lymphoma-associated mutants is accelerated by subversion of this pathway to Hrd1-mediated cytoplasmic sequestration and ubiquitination. Screening experiments identifies the heat shock protein 70 (HSP70) that selects Blimp-1 mutants for Hrd1 association, and HSP70 inhibition restores their nuclear accumulation and oncorepressor activities without disrupting normal B-cell maturation. Therefore, HSP70-Hrd1 axis represents a potential therapeutic target for restoring the oncorepressor activity of unstable lymphoma-associated Blimp-1 mutants.The transcriptional repressor Blimp-1 has an important role in B-cell differentiation. Here the authors show that lymphoma-associated Blimp-1 mutants are selectively recognized by HSP70-Hrd1, which leads to their accelerated degradation and propose HSP70 inhibition as a therapeutic approach for certain lymphomas.

MARK4 regulates NLRP3 positioning and inflammasome activation through a microtubule-dependent mechanism.

Excessive activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome is involved in many chronic inflammatory diseases, including cardiovascular and Alzheimer's disease. Here we show that microtubule-affinity regulating kinase 4 (MARK4) binds to NLRP3 and drives it to the microtubule-organizing centre, enabling the formation of one large inflammasome speck complex within a single cell. MARK4 knockdown or knockout, or disruption of MARK4-NLRP3 interaction, impairs NLRP3 spatial arrangement and limits inflammasome activation. Our results demonstrate how an evolutionarily conserved protein involved in the regulation of microtubule dynamics orchestrates NLRP3 inflammasome activation by controlling its transport to optimal activation sites, and identify a targetable function for MARK4 in the control of innate immunity.

Exome sequencing identifies somatic mutations of DDX3X in natural killer/T-cell lymphoma.

Natural killer/T-cell lymphoma (NKTCL) is a malignant proliferation of CD56(+) and cytoCD3(+) lymphocytes with aggressive clinical course, which is prevalent in Asian and South American populations. The molecular pathogenesis of NKTCL has largely remained elusive. We identified somatic gene mutations in 25 people with NKTCL by whole-exome sequencing and confirmed them in an extended validation group of 80 people by targeted sequencing. Recurrent mutations were most frequently located in the RNA helicase gene DDX3X (21/105 subjects, 20.0%), tumor suppressors (TP53 and MGA), JAK-STAT-pathway molecules (STAT3 and STAT5B) and epigenetic modifiers (MLL2, ARID1A, EP300 and ASXL3). As compared to wild-type protein, DDX3X mutants exhibited decreased RNA-unwinding activity, loss of suppressive effects on cell-cycle progression in NK cells and transcriptional activation of NF-κB and MAPK pathways. Clinically, patients with DDX3X mutations presented a poor prognosis. Our work thus contributes to the understanding of the disease mechanism of NKTCL.