Although the role of Hedgehog (Hh) signalling in embryonic pattern formation is well established 1 , its functions in adult tissue renewal and maintenance remain unclear, and the relationship of these functions to cancer development has not been determined. Here we show that the lossof Smoothened (Smo), an essential component of the Hh pathway 2 , impairs haematopoietic stem cell renewal and decreases induction of chronic myelogenous leukaemia (CML) by the BCR-ABL1 oncoprotein 3 . Loss of Smo causes depletion of CML stem cells-the cells that propagate the leukaemia-whereas constitutively active Smo augments CML stem cell number and accelerates disease. As a possible mechanism for Smo action, we show that the cell fate determinant Numb, which depletes CML stem cells, is increased in the absence of Smo activity. Furthermore, pharmacological inhibition of Hh signalling impairs not only the propagation of CML driven by wildtype BCR-ABL1, but also the growth of imatinib-resistant mouse and human CML. These data indicate that Hh pathway activity is required for maintenance of normal and neoplastic stem cells of the haematopoietic system and raise the possibility that the drug resistance and disease recurrence associated with imatinib treatment of CML 4,5 might be avoided by targeting this essential stem cell maintenance pathway.

A key characteristic of stem cells and cancer cells is their ability to self-renew. To test if Wnt signaling can regulate the self-renewal of both stem cells and cancer cells in the hematopoietic system, we developed mice that lack beta-catenin in their hematopoietic cells. Here we show that beta-catenin-deficient mice can form HSCs, but that these cells are deficient in long-term growth and maintenance. Moreover, beta-catenin deletion causes a profound reduction in the ability of mice to develop BCR-ABL-induced chronic myelogenous leukemia (CML), while allowing progression of acute lymphocytic leukemia (ALL). These studies demonstrate that Wnt signaling is required for the self-renewal of normal and neoplastic stem cells in the hematopoietic system.

We report the de novo folding of three hyperstable RNA tetraloops to 1-3 Å rmsd from their experimentally determined structures using molecular dynamics simulations initialized in the unfolded state. RNA tetraloops with loop sequences UUCG, GCAA, or CUUG are hyperstable because of the formation of noncanonical loop-stabilizing interactions, and they are all faithfully reproduced to angstrom-level accuracy in replica exchange molecular dynamics simulations, including explicit solvent and ion molecules. This accuracy is accomplished using unique RNA parameters, in which biases that favor rigid, highly stacked conformations are corrected to accurately capture the inherent flexibility of ssRNA loops, accurate base stacking energetics, and purine syn-anti interconversions. In a departure from traditional quantum chemistrycentric approaches to force field optimization, our parameters are calibrated directly from thermodynamic and kinetic measurements of intra-and internucleotide structural transitions. The ability to recapitulate the signature noncanonical interactions of the three most abundant hyperstable stem loop motifs represents a significant milestone to the accurate prediction of RNA tertiary structure using unbiased all-atom molecular dynamics simulations.RNA folding | molecular simulations S tructured RNAs exhibit a distinct preference for loops of precisely 4 nt, which was originally noted by Woese et al. (1) using comparative sequence analysis of ribosomes. Approximately 70% of these tetraloops are comprised of just three specific loop sequences: UUCG, GCAA, or CUUG. The abundance of these sequences is thermodynamic in origin, because each motif forms a unique network of noncanonical interactions within their loops that stabilizes the folded state. The abundance of high-resolution structural and thermodynamic data available for these motifs coupled with their characteristic noncanonical signatures make them ideal for adjudicating the accuracy of RNA folding simulations.RNA folding is understood to be hierarchical in nature, with secondary and tertiary folds stabilized by distinct thermodynamic driving forces (2). Secondary structure (the formation of canonical helices stabilized by Watson-Crick base pairs) can be accurately predicted from the nucleotide sequence alone using simple nearest neighbor thermodynamic models (3). In contrast, tertiary structure formation is a subtle competition between intrinsic flexibility of single-stranded segments, rigidity imparted from base-stacking interactions, stabilization of noncanonical hydrogen bonding patterns, and site-specific ion binding. In principle, a molecular dynamics simulation using a properly calibrated force field should capture all of the physicochemical properties of ribonucleotides relevant to the RNA folding process. Up until now, however, even small, fast-folding tetraloops cannot be accurately and reversibly folded from the unfolded state (4-6). In contrast, numerous documented successes have been reported using de novo protein folding with all-ato...

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