Chromosomal rearrangements have been identified as the main drivers of pediatric Acute Megakaryoblastic Leukemia in absence of Down syndrome (non-DS-AMKL). The t(11;12) involving NUP98 and ...KDM5A/JARID1A (NJ) accounts for approximately 15% of pediatric non-DS-AMKL cases and correlates with poor prognosis. An important aspect of AMKL is the strong enrichment in pediatric patients with frequent occurrence in infants (disease within 2 years post birth). In this study, we aimed to explore the role of cellular ontogeny in NJ-driven non-DS-AMKL and highlight TRIP13 as a fetal-enriched NJ-specific vulnerability acting through P53 regulation. To investigate the mechanism of NJ transformation, we first transduced murine hematopoietic stem and progenitor cells (HSPCs) originating from fetal liver (mFL-HSPCs) and bone marrow of adult mice (mBM-HSPCs) with a lentiviral vector overexpressing the cDNA encoding human NJ. In line with other studies, NJacts as a potent oncogene in vitro, sustaining cell proliferation and arresting cell differentiation. Both mFL-HSPCs and mBM-HSPCs expressing NJ transformed and dominated the culture after 3-4 weeks with an immature immune-phenotype (Lin-, Sca1+ and c-Kit+). Notably, outgrowth of mFL-HSPCs was significantly accelerated compared to adult mBM-HSPCs, despite similar transduction rates. Furthermore, in vivo experiments revealed that NJ-expressing mFL-HSPCs displayed a more aggressive phenotype (latency of 36 days) compared to mBM-HSPCs cells expressing NJ (latency of 70 days; P Long-rank < 0.001). Thus, both our in vitro and in vivo experiments strongly indicate an impact of fetal cell background. Suspecting an impact of fetal gene programs on NJ-mediated transformation, we performed a CRISPR-Cas9 screening targeting fetal expression signatures with a library probing 880 genes found to be deregulated when comparing fetal versus adult murine and human primary HSPCs. By comparing our findings with two other fetal liver-derived leukemia models - representing DS AMKL and familial platelet disorder with predisposition to AML (FPD-AML) -, we identified TRIP13 as a high-confidence candidate gene with exclusive dependency in NJ-driven non-DS-AMKL. Comparing human AML cell lines and NJ-overexpressing human fetal liver cells (hFL-HSPCs) confirmed selective targeting of NJ-driven cells by TRIP13 loss. To explore the molecular mechanism of TRIP13 sensitivity, we performed RNAseq. Surprisingly, gene set expression analysis (GSEA) revealed one highly significantly enriched pathway, i.e. TP53 signaling. Rescue experiments of Trip13 knockout in NJ-driven non-DS-AMKL cells further supported this finding: the massive depletion upon loss of Trip13 was completely reverted by either re-expression of a human TRIP13 cDNA or by knockout of Trp53. Of note, neither activation of TP53 signaling nor cell depletion was seen in healthy hematopoietic cells from FL upon Trip13 perturbation. Finally, we aimed to leverage TRIP13 dependency therapeutically. To this end, we first explored Trip13-depletion in an in vivo setting. In a fluorescence-based competitive transplantation assay, Trip13-ablated leukemic blasts were significantly diminished in the bone marrow of recipient mice after 4 weeks, showing a mean depletion of 83%. Next, NJ-driven non-DS-AMKL mFL-HSPCs were treated with the TRIP13 inhibitor DCZ0415. Similarly to genetic TRIP13 ablation, DCZ0415-treated NJ mFL-HSPCs showed a significantly higher sensitivity to the drug compared to models of DS AMKL, FPD-AML, and healthy HSPCs with fold changes of 1.6 (p-value = 0.024), 2.3 (p-value = 0.001) and 2.0 (p-value = 0.008), respectively. To exploit our mechanistic knowledge on TRIP13 ablation-mediated P53 activation, we further combined DCZ0415 with the MDM2 inhibitor Idasanutlin. In line with our mechanistic data, a high synergy (Bliss synergy score = 9.7) of TRIP13 and MDM2 inhibition was observed. In conclusion, our study uncovers TRIP13 as a fetal-enriched vulnerability in NJ-driven non-DS-AMKL, mechanistically acting through P53, which we leveraged for a mechanism-driven treatment approach with dual TRIP13/MDM2 inhibition as a potential therapeutic strategy for the treatment of high-risk pediatric non-DS-AMKL.
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IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZRSKP
A hallmark of acute myeloid leukaemias (AMLs) are chromosomal rearrangements that give rise to novel leukaemia-specific fusion genes. Most of these fusion genes are both initiating and driving events ...in AML and therefore constitute ideal therapeutic targets but are challenging to target by conventional drug development. siRNAs are frequently used for the specific suppression of fusion gene expression but require special formulations for efficient in vivo delivery. Here we describe the use of siRNA-loaded lipid nanoparticles for the specific therapeutic targeting of the leukaemic fusion gene RUNX1/ETO. Transient knockdown of RUNX1/ETO reduces its binding to its target genes and alters the binding of RUNX1 and its co-factor CBFβ. Transcriptomic changes in vivo were associated with substantially increased median survival of a t(8;21)-AML mouse model. Importantly, transient knockdown in vivo causes long-lasting inhibition of leukaemic proliferation and clonogenicity, induction of myeloid differentiation and a markedly impaired re-engraftment potential in vivo. These data strongly suggest that temporary inhibition of RUNX1/ETO results in long-term restriction of leukaemic self-renewal. Our results provide proof for the feasibility of targeting RUNX1/ETO in a pre-clinical setting and support the further development of siRNA-LNPs for the treatment of fusion gene-driven malignancies.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
In the present study, the atomistic simulations were carried out to investigate the mechanical properties and deformation mechanisms of nanocrystalline magnesium with different grain sizes. Also, the ...effect of reinforcement particle position and structure on the mechanical behavior of nanocrystalline magnesium / (amorphous/crystalline) silica nanocomposite under uniaxial compression loading was studied by using the molecular dynamics method. Firstly, eight structure with the mean grain sizes from 5 to 24 nm of nanocrystalline magnesium were constructed using the Voronoi tessellation method, and used to study the deformation mechanism. The simulation results showed that the sample with an average grain size of 14 nm had the optimum mean flow stress. Furthermore, by increasing the average grain size to values close to coarse-grained structures (> 25 nm), an increasing in the dislocation density and elastic modulus was observed. Moreover, there are two distinct deformation mechanisms activated by decreasing grain size. The first one, the dislocation-dominated deformation mechanism, belonged to large average grain size (≥ 14 nm) samples, and the second one, the grain boundary sliding deformation mechanism, was observed in small grain-sized simulation samples. In the following, nanocrystalline Mg nanocomposite simulation samples with different amorphous silica nanoparticle positions were studied, and it was observed that the presence of nanoparticle in the triple junction among three grains had the main effect on the strength of nanocomposites compared to the samples with nanoparticle at the grain boundary or inside the grain. Accordingly, the maximum strength and fracture strain was observed in the simulation samples with nanoparticles in the triple junctions. The investigated structures showed that the partial dislocations and aggregation of FCC structure stacking faults as lamella structure defects were observed in all simulated nanocomposite samples. Also, the local atomic shear strain contour obtained by simulation of nanocrystalline magnesium reinforced by amorphous and crystalline silica nanoparticles showed different responses of amorphous and crystalline nanoparticles during compression loading. Furthermore, amorphous and crystalline silica with the same size have different effects on the mechanical properties of the magnesium: in which, amorphous silica not only increases the ultimate compressive strength but also increases the strain before fracture compared to crystalline silica. The results of this research can expand the use of amorphous ceramic nanoparticles as reinforcement instead of crystalline ceramic nanoparticles in the development of nanocomposite materials.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
•Genomic analysis of MF identifies alterations associated with high risk of progression and shorter overall survival.•Clonal evolution of MF shows acquisition of JUNB, gain of 10p15.1 (IL2RA/IL15RA), ...or del12p13.1 (CDKN1B) at progression.
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Mycosis fungoides (MF) is the most prevalent primary cutaneous T-cell lymphoma, with an indolent or aggressive course and poor survival. The pathogenesis of MF remains unclear, and prognostic factors in the early stages are not well established. Here, we characterized the most recurrent genomic alterations using whole-exome sequencing of 67 samples from 48 patients from Lille University Hospital (France), including 18 sequential samples drawn across stages of the malignancy. Genomic data were analyzed on the Broad Institute’s Terra bioinformatics platform. We found that gain7q, gain10p15.1 (IL2RA and IL15RA), del10p11.22 (ZEB1), or mutations in JUNB and TET2 are associated with high-risk disease stages. Furthermore, gain7q, gain10p15.1 (IL2RA and IL15RA), del10p11.22 (ZEB1), and del6q16.3 (TNFAIP3) are coupled with shorter survival. Del6q16.3 (TNFAIP3) was a risk factor for progression in patients at low risk. By analyzing the clonal heterogeneity and the clonal evolution of the cohort, we defined different phylogenetic pathways of the disease with acquisition of JUNB, gain10p15.1 (IL2RA and IL15RA), or del12p13.1 (CDKN1B) at progression. These results establish the genomics and clonality of MF and identify potential patients at risk of progression, independent of their clinical stage.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
In this study, a Mg-X wt.%SiO
2
(X = 1, 2) nanocomposite was developed using amorphous silica nanoparticles via the accumulative extrusion method. The reinforcement phase was added to the matrix ...between extrusion passes. The study evaluated the mechanical properties of the composite samples via compression and hardness tests, while the microstructure and texture were analyzed using optical microscopy, field emission scanning electron microscopy (FESEM) and X-ray diffractometry. To remove the deformation history and examine the effect of the reinforcement phase on mechanical properties, the samples were annealed in an argon atmosphere. In addition, monolithic magnesium samples were fabricated through the same process to serve as a basis for comparison. This study revealed that adding 1 wt% amorphous silica nanoparticles to the magnesium matrix improved the overall mechanical properties. However, the nanocomposites displayed varying properties in different directions. Along the extrusion direction, the yield strength and ductility increased up to 57% and 5%, respectively, while the ultimate compressive strength decreased by about 8%. Along the normal direction, the yield strength and ductility increased up to 37% and 45%, respectively, while the ultimate compressive strength decreased by about 9%. The Mg/2wt.%SiO
2
nanocomposite sample showed superior Brinell hardness. The number of extrusion passes had a significant impact on the distribution of nanoparticles within the matrix. The optical microscope micrographs revealed that the reinforcement phase was uniformly distributed throughout the matrix, and no agglomeration of nanoparticles was observed. The X-ray diffraction results demonstrated that the texture of nanocomposite samples weakened after adding nanoparticles, resulting in improved ductility.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Given the plasticity of hematopoietic stem and progenitor cells, multiple routes of differentiation must be blocked in the the pathogenesis of acute myeloid leukemia, the molecular basis of which is ...incompletely understood. We report that posttranscriptional repression of the transcription factor ARID3A by miR-125b is a key event in the pathogenesis of acute megakaryoblastic leukemia (AMKL). AMKL is frequently associated with trisomy 21 and GATA1 mutations (GATA1s), and children with Down syndrome are at a high risk of developing the disease. The results of our study showed that chromosome 21–encoded miR-125b synergizes with Gata1s to drive leukemogenesis in this context. Leveraging forward and reverse genetics, we uncovered Arid3a as the main miR-125b target behind this synergy. We demonstrated that, during normal hematopoiesis, this transcription factor promotes megakaryocytic differentiation in concert with GATA1 and mediates TGFβ-induced apoptosis and cell cycle arrest in complex with SMAD2/3. Although Gata1s mutations perturb erythroid differentiation and induce hyperproliferation of megakaryocytic progenitors, intact ARID3A expression assures their megakaryocytic differentiation and growth restriction. Upon knockdown, these tumor suppressive functions are revoked, causing a blockade of dual megakaryocytic/erythroid differentiation and subsequently of AMKL. Inversely, restoring ARID3A expression relieves the arrest of megakaryocytic differentiation in AMKL patient-derived xenografts. This work illustrates how mutations in lineage-determining transcription factors and perturbation of posttranscriptional gene regulation can interact to block multiple routes of hematopoietic differentiation and cause leukemia. In AMKL, surmounting this differentiation blockade through restoration of the tumor suppressor ARID3A represents a promising strategy for treating this lethal pediatric disease.
•miR-125b–mediated repression of the megakaryocytic transcription factor ARID3A synergizes with GATA1s to induce leukemia.•Restoring ARID3A expression relieves megakaryocytic differentiation arrest in megakaryoblastic leukemia.
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IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZRSKP
The noncoding genome presents a largely untapped source of biological insights, including tens of thousands of long noncoding RNA (lncRNA) loci. While some produce bona fide lncRNAs, others exert ...transcript-independent cis-regulatory effects, and the lack of predictive features renders their mechanistic dissection highly challenging. Here, we describe CTCF-enriched lncRNA loci (C-LNC) as a putative new subclass of functional genetic elements exemplified by MYNRL15 - myeloid leukemia noncoding regulatory locus on chromosome 15.
Initially identified by an expression-guided CRISPRi screen of hematopoietic stem and progenitor (HSPC) / acute myeloid leukemia (AML) lncRNA signatures (480 genes, 1545 sgRNAs), we found MYNRL15 dependency in myeloid leukemia cells of diverse genetic backgrounds. Interestingly, cis and trans perturbation approaches revealed both the MYNRL15 transcript and its flanking protein-coding genes to be dispensable. High density CRISPR tiling of a 15 kb area centered on MYNRL15 (1613 sgRNAs) instead uncovered two crucial, candidate cis-regulatory DNA elements in the locus, which drive the MYNRL15 perturbation phenotype. To determine the molecular basis of MYNRL15 dependence, we performed transcriptome, chromatin conformation, chromatin accessibility, and CTCF profiling. RNA-sequencing established MYNRL15's involvement in maintaining key cancer dependency pathways (e.g. cell cycle, ribosome, spliceosome). Further, MYNRL15 perturbation associated with the coordinated dysregulation of several chromosome 15 neighbourhoods, and formation of a long-range chromatin interaction between the locus and the base of a distal loop, as detected via next-generation Capture-C. The gained interaction was accompanied by diffuse gains in chromatin accessibility across the distal interaction sites (ATAC-seq) as well as reduced CTCF occupancy at the MYRNL15 locus (CTCF CUT&RUN), altogether indicating the 3D re-organization of chromosome 15 following MYNRL15 perturbation. Integrative analysis of the chromatin conformation and transcriptome data, combined with a small CRISPR-Cas9 knockout screen of protein-coding genes from the gained interaction region (29 genes, 149 sgRNAs), pinpointed two potent cancer dependency genes that are located in the region and downregulated following MYNRL15 perturbation: namely, WDR61 and IMP3. Individual knockout of both genes robustly depleted myeloid leukemia cells, recapitulating the MYNRL15 perturbation phenotype and positioning WDR61 and IMP3 as its regulatory targets. Importantly, in primary cells, MYNRL15 perturbation eradicated AML blasts while sparing 50-60% of CD34 + HSPCs in vitro, and reduced patient-derived AML xenografts up to 10-fold in vivo, indicating a potential therapeutic window.
Having implicated MYNRL15 in 3D genome organization and demonstrated its role in myeloid leukemia cells, we explored whether MYNRL15 may belong to a sub-category of biologically relevant lncRNA loci that have thus far been overlooked due to their lack of transcript-specific functions. Remarkably, elevated CTCF density (e.g. number of CTCF binding sites per kb of gene length) distinguishes MYNRL15 and 531 other lncRNA loci in K562 cells, of which 43-54% associate with genetic subgroups and/or survival in AML patient cohorts, and 18.4% are functionally required for leukemia maintenance as determined by CRISPR-Cas9 screening. The latter hit identification rate represents a substantial improvement over typical lncRNA essentiality screens (which range from 2-6%) - illustrating the effectiveness of CTCF density metrics in refining functional lncRNA candidate lists, and underlining the relevance such loci hold for AML and cancer pathophysiology in general. Curated C-LNC catalogs in other cell types will facilitate the search for noncoding oncogenic vulnerabilities in AML and other malignancies.
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Reinhardt: Celgene Corporation: Consultancy; Novartis: Consultancy; Bluebird Bio: Consultancy; Janssen: Consultancy; CLS Behring: Research Funding; Roche: Research Funding. Klusmann: Bluebird Bio: Consultancy; Novartis: Consultancy; Roche: Consultancy; Jazz Pharmaceuticals: Consultancy.
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IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZRSKP
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