Development of an Ex Vivo Three Dimensional (3-D) Model of Acute Myeloid Leukaemia (AML)

Acute Myeloid Leukaemia (AML) is a cancer of hematopoietic stem cells that develops in the three-dimensional (3-D) niches provided by the bone marrow microenvironment in vivo. The study of AML has been hampered by the lack of appropriate ex vivo models, which can mimic this microenvironment. It was hypothesised that the fabrication of scaffolds for the biomimetic growth of leukemic cells ex vivo could facilitate the study of the disease in its native 3-D niche. The growth of different leukemic cell lines was first evaluated, namely K- 562, HL-60 and Kasumi-6 on highly porous scaffolds fabricated from biodegradable and non-biodegradable polymeric materials: poly (L-lactic-co-glycolic acid) (PLGA), polyurethane (PU), poly (methyl-methacrylate) (PMMA), poly (D, L-lactade) (PDLLA), poly (caprolactone) (PCL), and polystyrene (PS). These results were compared with two commercially available scaffolds from BD™ Biosciences. Overall, out of all the scaffolds, PLGA and PU displayed the best seeding efficiency and leukemic cellular growth, assessed by MTS assay, scanning electron microscopy and immunohistochemistry. In order to improve the ex vivo 3-D leukemic cell culture, PLGA and PU scaffolds were coated with bone marrow extracellular matrix (ECM) proteins, collagen (62.5 or 125 μg/ml) and fibronectin (25 or 50 μg/ml) and a combination of both proteins: collagen + fibronectin (62.5 + 25 μg/ml) respectively. Once the abnormal hematopoietic 3-D model was established, a new model to culture normal hematopoietic cord blood mononuclear cells was studied and compared. All 3 leukemic cell lines and cord blood cells grew better in PU scaffolds coated with collagen type I using the low concentration and sustained growth in the absence of exogenous cytokines. As a result, it was concluded that PU-collagen scaffold could provide a practical model with which to study the biology and treatment of primary AML in an ex vivo mimicry without the use of animal models

Development of an ex vivo three dimensional ( 3-D) model of acute myeloid leukemia ( AML)

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Development of an ex vivo three dimensional ( 3-D) model of acute myeloid leukemia ( AML)

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Pseudouridine-modified tRNA fragments repress aberrant protein synthesis and predict leukaemic progression in myelodysplastic syndrome

Transfer RNA-derived fragments (tRFs) are emerging small noncoding RNAs that, although commonly altered in cancer, have poorly defined roles in tumorigenesis1. Here we show that pseudouridylation (Ψ) of a stem cell-enriched tRF subtype2, mini tRFs containing a 5′ terminal oligoguanine (mTOG), selectively inhibits aberrant protein synthesis programmes, thereby promoting engraftment and differentiation of haematopoietic stem and progenitor cells (HSPCs) in patients with myelodysplastic syndrome (MDS). Building on evidence that mTOG-Ψ targets polyadenylate-binding protein cytoplasmic 1 (PABPC1), we employed isotope exchange proteomics to reveal critical interactions between mTOG and functional RNA-recognition motif (RRM) domains of PABPC1. Mechanistically, this hinders the recruitment of translational co-activator PABPC1-interacting protein 1 (PAIP1)3 and strongly represses the translation of transcripts sharing pyrimidine-enriched sequences (PES) at the 5′ untranslated region (UTR), including 5′ terminal oligopyrimidine tracts (TOP) that encode protein machinery components and are frequently altered in cancer4. Significantly, mTOG dysregulation leads to aberrantly increased translation of 5′ PES messenger RNA (mRNA) in malignant MDS-HSPCs and is clinically associated with leukaemic transformation and reduced patient survival. These findings define a critical role for tRFs and Ψ in difficult-to-treat subsets of MDS characterized by high risk of progression to acute myeloid leukaemia (AML)

Patient-specific MDS-RS iPSCs define the mis-spliced transcript repertoire and chromatin landscape of SF3B1-mutant HSPCs

: SF3B1K700E is the most frequent mutation in myelodysplastic syndrome (MDS), but the mechanisms by which it drives MDS pathogenesis remain unclear. We derived a panel of 18 genetically matched SF3B1K700E- and SF3B1WT-induced pluripotent stem cell (iPSC) lines from patients with MDS with ring sideroblasts (MDS-RS) harboring isolated SF3B1K700E mutations and performed RNA and ATAC sequencing in purified CD34+/CD45+ hematopoietic stem/progenitor cells (HSPCs) derived from them. We developed a novel computational framework integrating splicing with transcript usage and gene expression analyses and derived a SF3B1K700E splicing signature consisting of 59 splicing events linked to 34 genes, which associates with the SF3B1 mutational status of primary MDS patient cells. The chromatin landscape of SF3B1K700E HSPCs showed increased priming toward the megakaryocyte- erythroid lineage. Transcription factor motifs enriched in chromatin regions more accessible in SF3B1K700E cells included, unexpectedly, motifs of the TEA domain (TEAD) transcription factor family. TEAD expression and transcriptional activity were upregulated in SF3B1-mutant iPSC-HSPCs, in support of a Hippo pathway-independent role of TEAD as a potential novel transcriptional regulator of SF3B1K700E cells. This study provides a comprehensive characterization of the transcriptional and chromatin landscape of SF3B1K700E HSPCs and nominates novel mis-spliced genes and transcriptional programs with putative roles in MDS-RS disease biology

Supplementary Figure S14 from Integrated Genomic and Transcriptomic Analysis Improves Disease Classification and Risk Stratification of MDS with Ring Sideroblasts

The proportion of estimated erythroblast-like cells correlate positively with the percentage of RS in BM smears.</p

Supplementary Figure S17 from Integrated Genomic and Transcriptomic Analysis Improves Disease Classification and Risk Stratification of MDS with Ring Sideroblasts

Gene set enrichment analysis on differentially expressed genes in LIN- CD34+ cells. Three MDS with RSEMK were compared to 3 MDS without RSIMP and displayed an enrichment of genes expressed in early erythroblast/megakaryocyte progenitor genes and a depletion of genes expressed in myeloid progenitors.</p