No abstract

As is the case for embryo-derived stem cells, application of reprogrammed human induced pluripotent stem cells is limited by our understanding of lineage specification. Here we demonstrate the ability to generate progenitors and mature cells of the haematopoietic fate directly from human dermal fibroblasts without establishing pluripotency. Ectopic expression of OCT4 (also called POU5F1)-activated haematopoietic transcription factors, together with specific cytokine treatment, allowed generation of cells expressing the pan-leukocyte marker CD45. These unique fibroblast-derived cells gave rise to granulocytic, monocytic, megakaryocytic and erythroid lineages, and demonstrated in vivo engraftment capacity. We note that adult haematopoietic programs are activated, consistent with bypassing the pluripotent state to generate blood fate: this is distinct from haematopoiesis involving pluripotent stem cells, where embryonic programs are activated. These findings demonstrate restoration of multipotency from human fibroblasts, and suggest an alternative approach to cellular reprogramming for autologous cell-replacement therapies that avoids complications associated with the use of human pluripotent stem cells.

Selective targeting of cancer stem cells (CSCs) offers promise for a new generation of therapeutics. However, assays for both human CSCs and normal stem cells that are amenable to robust biological screens are limited. Using a discovery platform that reveals differences between neoplastic and normal human pluripotent stem cells (hPSC), we identify small molecules from libraries of known compounds that induce differentiation to overcome neoplastic self-renewal. Surprisingly, thioridazine, an antipsychotic drug, selectively targets the neoplastic cells, and impairs human somatic CSCs capable of in vivo leukemic disease initiation while having no effect on normal blood SCs. The drug antagonizes dopamine receptors that are expressed on CSCs and on breast cancer cells as well. These results suggest that dopamine receptors may serve as a biomarker for diverse malignancies, demonstrate the utility of using neoplastic hPSCs for identifying CSC-targeting drugs, and provide support for the use of differentiation as a therapeutic strategy.

A long-standing paradox in the study of T cell antigen recognition is that of the high specificity–low affinity T cell receptor (TCR)–major histocompatibility complex peptide (MHCp) interaction. The existence of multivalent TCRs could resolve this paradox because they can simultaneously improve the avidity observed for monovalent interactions and allow for cooperative effects. We have studied the stoichiometry of the TCR by Blue Native–polyacrylamide gel electrophoresis and found that the TCR exists as a mixture of monovalent (αβγɛδɛζζ) and multivalent complexes with two or more ligand-binding TCRα/β subunits. The coexistence of monovalent and multivalent complexes was confirmed by electron microscopy after label fracture of intact T cells, thus ruling out any possible artifact caused by detergent solubilization. We found that although only the multivalent complexes become phosphorylated at low antigen doses, both multivalent and monovalent TCRs are phosphorylated at higher doses. Thus, the multivalent TCRs could be responsible for sensing low concentrations of antigen, whereas the monovalent TCRs could be responsible for dose-response effects at high concentrations, conditions in which the multivalent TCRs are saturated. Thus, besides resolving TCR stoichiometry, these data can explain how T cells respond to a wide range of MHCp concentrations while maintaining high sensitivity.

The CD3epsilon subunit of the T cell receptor (TCR) complex undergoes a conformational change upon ligand binding that is thought to be important for the activation of T cells. To study this process, we built a molecular dynamics model of the transmission of the conformational change within the ectodomains of CD3. The model showed that the CD3 dimers underwent a stiffening effect that was funneled to the base of the CD3epsilon subunit. Mutation of two relevant amino acid residues blocked transmission of the conformational change and the differentiation and activation of T cells. Furthermore, this inhibition occurred even in the presence of excess endogenous CD3epsilon subunits. These results emphasize the importance of the conformational change in CD3epsilon for the activation of T cells and suggest the existence of unforeseen cooperativity between TCR complexes.

Triggering of the T-cell receptor (TCR)can produce very different responses, depending on the nature of the major histocompatibility complex/antigen peptide (MHCp) ligand. The molecular mechanisms that permit such fine discrimination are still unknown. We show here that an epitope in the cytoplasmic tail of the TCR CD3⑀ subunit, recognized by antibody APA1/1, is only detected when the TCR is fully activated. Exposure of the APA1/1 epitope is shown to be fast and independent of tyrosine kinase activity and that it takes place even when T cells are stimulated at 0°C. These results suggest that APA1/1 detects a conformational change in the TCR. APA1/1 staining concentrates in a restricted area of the immunologic synapse. Most important, we show that full agonist, but not partial agonist, peptides induce exposure of the APA1/1 epitope, indicating a correlation between the induction of the conformational change in the TCR and full T-cell activation. Finally, the conformational change is shown to occur in T cells that are being stimulated by antigen in vivo. Therefore, these results demonstrate that the TCR undergoes a conformational change on MHCp binding in vitro and in vivo, and they establish a molecular correlate for productive TCR engagement. IntroductionWhen T cells recognize antigen-presenting cells (APCs) that bear an appropriate antigen, large-scale rearrangement of the T-cell cytoskeleton occurs, coupled to a redistribution of plasma membrane and cytoplasmic molecules. This reorganization results in the formation of what is known as an immunologic synapse (IS). 1,2 Antibody-staining experiments and time-lapse confocal microscopy have demonstrated that central and peripheral supramolecular activation clusters (cSMACs and pSMACs) form at the IS. 3 Although the cSMAC contains the T-cell receptor (TCR) and signaling molecules such as protein kinase C (PKC), the pSMAC is formed by a ring of integrins, such as leukocyte factor antigen 1 (LFA1) (CD11a/CD18), and accompanying cytoskeletal proteins, such as talin. 3 It has been proposed that the formation of the IS serves to sustain TCR signaling, which is necessary for full T-cell activation. 1,2 However, some studies indicate that TCR signaling takes place before the IS is formed, 4 whereas others indicate that the IS is a site for TCR internalization and downmodulation of the activation signals. 5 Much of the evidence supporting the need to form the IS for T-cell activation derives from the observation that antagonist and partial agonist peptides do not form bona fide ISs. 2,6,7 Furthermore, TCR engagement with a partial agonist peptide results in a pattern of intracellular signaling distinct from that induced with a fullagonist peptide. Thus, stimulation with a partial agonist induces only partial phosphorylation of the CD3 and CD3⑀ chains and the recruitment, but not the phosphorylation, of ZAP70 (zetaassociated protein of 70 kDa), 8,9 resulting in the activation of a subset of the downstream signaling pathways that are activated by an agonist peptide. 10 Anta...

Targeting of human cancer stem cells (CSCs) requires the identification of vulnerabilities unique to CSCs versus healthy resident stem cells (SCs). Unfortunately, dysregulated pathways that support transformed CSCs, such as Wnt/β-catenin signaling, are also critical regulators of healthy SCs. Using the ICG-001 and CWP family of small molecules, we reveal Sam68 as a previously unappreciated modulator of Wnt/β-catenin signaling within CSCs. Disruption of CBP-β-catenin interaction via ICG-001/CWP induces the formation of a Sam68-CBP complex in CSCs that alters Wnt signaling toward apoptosis and differentiation induction. Our study identifies Sam68 as a regulator of human CSC vulnerability.

Long non-coding RNAs (lncRNAs) are deregulated in several tumors, although their role in acute myeloid leukemia (AML) is mostly unknown.We have examined the expression of the lncRNA HOX antisense intergenic RNA myeloid 1 (HOTAIRM1) in 241 AML patients. We have correlated HOTAIRM1 expression with a miRNA expression profile. We have also analyzed the prognostic value of HOTAIRM1 expression in 215 intermediate-risk AML (IR-AML) patients.The lowest expression level was observed in acute promyelocytic leukemia (P < 0.001) and the highest in t(6;9) AML (P = 0.005). In 215 IR-AML patients, high HOTAIRM1 expression was independently associated with shorter overall survival (OR:2.04;P = 0.001), shorter leukemia-free survival (OR:2.56; P < 0.001) and a higher cumulative incidence of relapse (OR:1.67; P = 0.046). Moreover, HOTAIRM1 maintained its independent prognostic value within the favorable molecular subgroup (OR: 3.43; P = 0.009). Interestingly, HOTAIRM1 was overexpressed in NPM1-mutated AML (P < 0.001) and within this group retained its prognostic value (OR: 2.21; P = 0.01). Moreover, HOTAIRM1 expression was associated with a specific 33- microRNA signature that included miR-196b (P < 0.001). miR-196b is located in the HOX genomic region and has previously been reported to have an independent prognostic value in AML. miR-196b and HOTAIRM1 in combination as a prognostic factor can classify patients as high-, intermediate-, or low-risk (5-year OS: 24% vs 42% vs 70%; P = 0.004).Determination of HOTAIRM1 level at diagnosis provided relevant prognostic information in IR-AML and allowed refinement of risk stratification based on common molecular markers. The prognostic information provided by HOTAIRM1 was strengthened when combined with miR-196b expression. Furthermore, HOTAIRM1 correlated with a 33-miRNA signature.