SummaryChronic myeloid leukaemia (CML) arises following transformation of a haemopoietic stem cell (HSC) by protein-tyrosine kinase BCR-ABL1. Direct inhibition of BCR-ABL1 kinase has revolutionized disease management, but fails to eradicate leukaemic stem cells (LSC), which maintain CML. LSC are independent of BCR-ABL1 for survival, providing a rationale to identify and target kinase-independent pathways. Here we show using proteomics, transcriptomics and network analyses, that in human LSC aberrantly expressed proteins, in both imatinib-responder and non-responder patients are modulated in concert with p53 and c-Myc regulation. Perturbation of both p53 and c-Myc, not BCR-ABL1 itself, leads to synergistic kill, differentiation and near elimination of transplantable human LSC in mice, whilst sparing normal HSC. This unbiased systems approach targeting connected nodes exemplifies a novel precision medicine strategy providing evidence that LSC can be eradicated.

SUMMARY Many signals must be integrated to maintain self-renewal and pluripotency in embryonic stem cells (ESCs) and to enable induced pluripotent stem cell (iPSC) reprogramming. However, the exact molecular regulatory mechanisms remain elusive. To unravel the essential internal and external signals required for sustaining the ESC state, we conducted a short hairpin (sh) RNA screen of 104 ESC-associated phosphoregulators. Depletion of one such molecule, aurora kinase A (Aurka), resulted in compromised self-renewal and consequent differentiation. By integrating global gene expression and computational analyses, we discovered that loss of Aurka leads to up-regulated p53 activity that triggers ESC differentiation. Specifically, Aurka regulates pluripotency through phosphorylation-mediated inhibition of p53-directed ectodermal and mesodermal gene expression. Phosphorylation of p53 not only impairs p53-induced ESC differentiation but also p53-mediated suppression of iPSC reprogramming. Our studies demonstrate an essential role for Aurka-p53 signaling in the regulation of self-renewal, differentiation, and somatic cell reprogramming.

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Hematopoiesis and commitment to a restricted lineage are guided by a timely expressed set of cytokine receptors and their downstream transcription factors. A member of the mRNA export complex, THOC5 (suppressors of the transcriptional defects of hpr1 delta by overexpression complex 5) is a substrate for several tyrosine kinases such as macrophage colony-stimulating factor (M-CSF) receptor and various leukemogenic tyrosine kinases, such as Bcr-Abl, or NPM-ALK. THOC5 tyrosine phosphorylation is elevated in stem cells from patients with chronic myeloid leukemia, suggesting that THOC5 may be involved in leukemia development. THOC5 is also an essential element in the maintenance of hematopoiesis in adult mice. In this report, we show that THOC5 is located in the nuclear speckles, and that it is translocated from the nucleus to cytoplasm during M-CSF-induced bone marrow-derived macrophage differentiation. Furthermore, we have identified THOC5 target genes by trancriptome analysis, using tamoxifen-inducible THOC5 knockout macrophages. Although only 99 genes were downregulated in THOC5-depleted macrophages, half of the genes are involved in differentiation and/or migration. These include well-known regulators of myeloid differentiation inhibitor of DNA binding (Id)1, Id3, Smad family member 6 (Smad6) and Homeobox (Hox)A1. In addition, a subset of M-CSF-inducible genes, such as Ets family mRNAs are THOC5 target mRNAs. Upon depletion of THOC5, unspliced v-ets erythroblastosis virus E26 oncogene homolog (Ets1) mRNA was accumulated in the nucleus. Furthermore, THOC5 was recruited to chromatin where Ets1 was transcribed and bound to unspliced and spliced Ets1 transcripts, indicating that THOC5 has a role in processing/export of M-CSF-inducible genes. In conclusion, regulation of immediate-early gene response by THOC5, a member of mRNA export complex contributes to the M-CSF-induced macrophage differentiation.

Human papillomaviruses (HPV) are unique in that they generate mRNAs that apparently can express multiple proteins from tandemly arranged open reading frames. The mechanisms by which this is achieved are uncertain and are at odds with the basic predictions of the scanning model for translation initiation. We investigated the unorthodox mechanism by which the E6 and E7 oncoproteins from human papillomavirus type 16 (HPV-16) can be translated from a single, bicistronic mRNA. The short E6 5 untranslated region (UTR) was shown to promote translation as efficiently as a UTR from Xenopus ␤-globin. Insertion of a secondary structural element into the UTR inhibited both E6 and E7 expression, suggesting that E7 expression depends on ribosomal scanning from the 5 end of the mRNA. E7 translation was found to be cap dependent, but E6 was more dependent on capping and eIF4F activity than E7. Insertion of secondary structural elements at various points in the region upstream of E7 profoundly inhibited translation, indicating that scanning was probably continuous. Insertion of the E6 region between Renilla and firefly luciferase genes revealed little or no internal ribosomal entry site activity. However when E6 was located at the 5 end of the mRNA, it permitted over 100-fold-higher levels of downstream cistron translation than did the Renilla open reading frame. Internal AUGs in the E6 region with strong or intermediate Kozak sequence contexts were unable to inhibit E7 translation, but initiation at the E7 AUG was efficient and accurate. These data support a model in which E7 translation is facilitated by an extreme degree of leaky scanning, requiring the negotiation of 13 upstream AUGs. Ribosomal initiation complexes which fail to initiate at the E6 start codon can scan through to the E7 AUG without initiating translation, but competence to initiate is achieved once the E7 AUG is reached. These findings suggest that the E6 region of HPV-16 comprises features that sponsor both translation of the E6 protein and enhancement of translation at a downstream site.

Embryonic stem (ES) cells can differentiate in vitro toproduce the endothelial and hematopoietic precursor, the hemangioblasts, which are derived from the mesoderm germ layer. Differentiation of Bry GFP/؉ ES cell to hemangioblasts can be followed by the expression of the Bry GFP/؉ and Flk1 genes. Proteomic and transcriptomic changes during this differentiation process were analyzed to identify mechanisms for phenotypic change during early differentiation. Three populations of differentiating Bry GFP ES cells were obtained by flow cytometric sorting, GFP ؊ Flk1؊ (epiblast), GFP ؉ Flk1؊ (mesoderm), and GFP ؉ Flk1؉ (hemangioblast). Microarray analyses and relative quantification twodimensional LCLC-MS/MS on nuclear extracts were performed. We identified and quantified 2389 proteins, 1057 of which were associated to their microarray probe set. These included a variety of low abundance transcription factors, e.g. UTF1, Sox2, Oct4, and E2F4, demonstrating a high level of proteomic penetrance. When paired comparisons of changes in the mRNA and protein expression levels were performed low levels of correlation were found. A strong correlation between isobaric tag-derived relative quantification and Western blot analysis was found for a number of nuclear proteins. Pathway and ontology analysis identified proteins known to be involved in the regulation of stem cell differentiation, and proteins with no described function in early ES cell development were also shown to change markedly at the proteome level only. ES cell development is regulated at the mRNA and protein level. Molecular & Cellular Proteomics 7:459 -472, 2008.In the mouse embryo the process of gastrulation generates the mesodermal layer. During embryo development the mesoderm is generated from the epiblast or embryonic ectoderm.The cells involved in mesodermal commitment show a marked increase in Brachyury (Bry) 1 expression (1). The mesoderm migrates and develops into tissues including the endothelium as well as blood (2, 3). A common progenitor for these cell types, the hemangioblast, is first detected at the midstreak stage of gastrulation (4). These processes of early cell development within the embryo are not easily assessed because of limitations on the material available for study. This can be overcome by using embryonic stem (ES) cell lines derived from the inner cell mass of the blastocyst-stage embryo (5). ES cells can be allowed to differentiate in vitro to form three-dimensional spheroid cultures known as embryoid bodies (EBs). These structures contain derivatives of all three germ cell layers: mesoderm, endoderm, and ectoderm (6, 7). Mesoderm-derived populations contained within the EBs include hematopoietic and endothelial progeny. This model has been likened to the temporal pattern of development of these populations in embryogenesis (8 -10), and EB cultures have been shown to contain cells with hemangioblast potential (11,12).Differentiating ES cells in vitro with the use of the EB model system have been valuable in identifying a number of gen...

Transcription of immediate early genes (IEGs) in response to extrinsic and intrinsic signals is tightly regulated at multiple stages. It is known that untranslated regions of the RNA can play a role in these processes. Here we show that THOC5, a member of the TREX (transcription/export) complex, plays a role in expression of only a subset of constitutively active genes, however transcriptome analysis reveals that more than 90% of IEG were not induced by serum in THOC5 depleted cells. Furthermore, THOC5 depletion does not influence the expression of the most rapidly induced IEGs, e.g. Fos and Jun. One group of THOC5 target genes, including Id1, Id3 and Wnt11 transcripts, were not released from chromatin in THOC5 depleted cells. Genes in another group, including Myc and Smad7 transcripts, were released with shortening of 3′UTR by alternative cleavage, and were spliced but export was impaired in THOC5 depleted cells. By interactome analysis using THOC5 as bait, we show that upon stimulation with serum THOC5 forms a complex with polyadenylation-specific factor 100 (CPSF100). THOC5 is required for recruitment of CPSF100 to 3′UTR of THOC5 target genes. These data suggest the presence of a novel mechanism for the control of IEG response by THOC5 via 3′end-processing.

The glucocorticoid receptor (GR) is a member of the nuclear receptor superfamily, which controls programs regulating cell proliferation, differentiation, and apoptosis. We have identified an unexpected role for GR in mitosis. We discovered that specifically modified GR species accumulate at the mitotic spindle during mitosis in a distribution that overlaps with Aurora kinases. We found that Aurora A was required to mediate mitosis-driven GR phosphorylation, but not recruitment of GR to the spindle. GR was necessary for mitotic progression, with increased time to complete mitosis, frequency of mitotic aberrations, and death in mitosis observed following GR knockdown. Complementation studies revealed an essential role for the GR ligand-binding domain, but no clear requirement for ligand binding in regulating chromosome segregation. The GR N-terminal domain, and specifically phosphosites S203 and S211, were not required. Reduced GR expression results in a cell cycle phenotype, with isolated cells from mouse and human subjects showing changes in chromosome content over prolonged passage. Furthermore, GR haploinsufficient mice have an increased incidence of tumor formation, and, strikingly, these tumors are further depleted for GR, implying additional GR loss as a consequence of cell transformation. We identified reduced GR expression in a panel of human liver, lung, prostate, colon, and breast cancers. We therefore reveal an unexpected role for the GR in promoting accurate chromosome segregation during mitosis, which is causally linked to tumorigenesis, making GR an authentic tumor suppressor gene.glucocorticoid receptor | mitosis | aneuploidy | DNA damage | cancer G lucocorticoids (Gcs) act through the glucocorticoid receptor (GR), a member of the nuclear hormone receptor superfamily, and a ligand-activated transcription factor (1-4). The GR is ubiquitously expressed and regulates energy metabolism, immunity, and cell fate decisions. The quiescent GR resides in the cytoplasm in a complex with heat shock proteins and immunophilins, attached to the microtubule architecture of the cell in a heat shock protein 90-dependent manner (5). Ligand binding drives GR transformation, involving N-terminal phosphorylation on S203 and S211 and rapid translocation to the nucleus requiring attachment to dynein by heat shock protein 90, immunophilins, and dynamitin (6). Once in the nucleus, GR binds directly to DNA to regulate transcription, or tethers to other DNA-bound transcription factors, such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and activator protein 1 (AP1), to regulate their function (7-10).In mitosis, GR is phosphorylated on both S203 and S211, but in a ligand-independent manner (11), and more comprehensive phosphoproteomic analyses identify the presence of multiple N-terminal GR phosphoforms in purified mitotic spindle fractions (12). Although altered GR function in mitosis has been shown (11, 13, 14), the kinases responsible and cellular consequences have not been defined. The role of G...