Genome sequencing has uncovered a new mutational phenomenon in cancer and congenital disorders called chromothripsis. Chromothripsis is characterized by extensive genomic rearrangements and an ...oscillating pattern of DNA copy number levels, all curiously restricted to one or a few chromosomes. The mechanism for chromothripsis is unknown, but we previously proposed that it could occur through the physical isolation of chromosomes in aberrant nuclear structures called micronuclei. Here, using a combination of live cell imaging and single-cell genome sequencing, we demonstrate that micronucleus formation can indeed generate a spectrum of genomic rearrangements, some of which recapitulate all known features of chromothripsis. These events are restricted to the mis-segregated chromosome and occur within one cell division. We demonstrate that the mechanism for chromothripsis can involve the fragmentation and subsequent reassembly of a single chromatid from a micronucleus. Collectively, these experiments establish a new mutational process of which chromothripsis is one extreme outcome.
The Hippo pathway maintains tissue homeostasis by negatively regulating the oncogenic transcriptional co-activators YAP and TAZ. Though functional inactivation of the Hippo pathway is common in ...tumors, mutations in core pathway components are rare. Thus, understanding how tumor cells inactivate Hippo signaling remains a key unresolved question. Here, we identify the kinase STK25 as an activator of Hippo signaling. We demonstrate that loss of STK25 promotes YAP/TAZ activation and enhanced cellular proliferation, even under normally growth-suppressive conditions both in vitro and in vivo. Notably, STK25 activates LATS by promoting LATS activation loop phosphorylation independent of a preceding phosphorylation event at the hydrophobic motif, which represents a form of Hippo activation distinct from other kinase activators of LATS. STK25 is significantly focally deleted across a wide spectrum of human cancers, suggesting STK25 loss may represent a common mechanism by which tumor cells functionally impair the Hippo tumor suppressor pathway.
Artifacts introduced in whole-genome amplification (WGA) make it difficult to derive accurate genomic information from single-cell genomes and require different analytical strategies from bulk genome ...analysis. Here, we describe statistical methods to quantitatively assess the amplification bias resulting from whole-genome amplification of single-cell genomic DNA. Analysis of single-cell DNA libraries generated by different technologies revealed universal features of the genome coverage bias predominantly generated at the amplicon level (1-10 kb). The magnitude of coverage bias can be accurately calibrated from low-pass sequencing (∼0.1 × ) to predict the depth-of-coverage yield of single-cell DNA libraries sequenced at arbitrary depths. We further provide a benchmark comparison of single-cell libraries generated by multi-strand displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC). Finally, we develop statistical models to calibrate allelic bias in single-cell whole-genome amplification and demonstrate a census-based strategy for efficient and accurate variant detection from low-input biopsy samples.
Human NDR1 and 2 (NDR1/2) are serine-threonine protein kinases in a subgroup of the AGC kinase family 1. The mechanisms of physiological NDR1/2 activation and their function remain largely unknown. ...Here we report that Fas and TNF-α receptor stimulation activates human NDR1/2 by promoting phosphorylation at the hydrophobic motif (Thr444/442). Moreover, NDR1/2 are essential for Fas receptor-induced apoptosis as shown by the fact that NDR knockdown significantly reduced cell death whereas overexpression of the NDR1 kinase further potentiated apoptosis. Activation of NDR1/2 by death receptor stimulation is mediated by the tumor suppressor RASSF1A. Furthermore, RASSF1A-induced apoptosis largely depends on the presence of NDR1/2. Fas receptor stimulation promoted direct phosphorylation and activation of NDR1/2 by the mammalian STE20-like kinase 1 (MST1), a downstream effector of RASSF1A. Concurrently, the NDR1/2 coactivator MOB1 induced MST1-NDR-MOB1 complex formation, which is crucial for MST1-induced NDR1/2 phosphorylation upon induction of apoptosis. Our findings identify NDR1/2 as novel proapoptotic kinases and key members of the RASSF1A/MST1 signaling cascade.
Human MST/hSAV/LATS/hMOB tumor suppressor cascades are regulators of cell death and proliferation; however, little is known about other functions of MST/hMOB signaling. Mob1p, one of two MOB proteins ...in yeast, appears to play a role in spindle pole body duplication (the equivalent of mammalian centrosome duplication). We therefore investigated the role of human MOB proteins in centrosome duplication. We also addressed the regulation of human centrosome duplication by mammalian serine/threonine Ste20-like (MST) kinases, considering that MOB proteins can function together with Ste20-like kinases in eukaryotes.
By studying the six human MOB proteins and five MST kinases, we found that MST1/hMOB1 signaling controls centrosome duplication. Overexpression of hMOB1 caused centrosome overduplication, whereas RNAi depletion of hMOB1 or MST1 impaired centriole duplication. Significantly, we delineated an hMOB1/MST1/NDR1 signaling pathway regulating centrosome duplication. More specifically, analysis of shRNA-resistant hMOB1 and NDR1 mutants revealed that a functional NDR/hMOB1 complex is critical for MST1 to phosphorylate NDR on the hydrophobic motif that in turn is required for human centrosome duplication. Furthermore, shRNA-resistant MST1 variants revealed that MST1 kinase activity is crucial for centrosome duplication whereas MST1 binding to the hSAV and RASSF1A tumor suppressor proteins is dispensable. Finally, by studying the PLK4/HsSAS-6/CP110 centriole assembly machinery, we also observed that normal daughter centriole formation depends on intact MST1/hMOB1/NDR signaling, although HsSAS-6 centriolar localization is not affected.
Our observations propose a novel pathway in control of human centriole duplication after recruitment of HsSAS-6 to centrioles.
The mammalian genome encodes four members of the NDR/LATS kinase family: NDR1 (STK38), NDR2 (STK38L), LATS1 and LATS2, which are highly conserved from yeast to man. Members of the NDR/LATS kinase ...family have been implicated in a variety of biological processes ranging from cell division and morphology to apoptosis and tumor suppression. In mammals, LATS1/2 function as central parts of the HIPPO tumor suppressor pathway by restricting the activity of the YAP/TAZ proto-oncogenes. Recent evidence suggested that NDR1/2 are also part of an extended HIPPO tumor suppressor pathway. Apart from functions in apoptosis signaling and tumor suppression, NDR1/2 have been implicated in controlling centrosome duplication and mitotic chromosome alignment downstream of the HIPPO kinase homologs MST1 and MST2. Significantly, we also reported recently that NDR1/2 are controlling G
1
/S transition downstream of a third MST family member MST3. Intriguingly, this newly described MST3-NDR1/2 axis promotes G
1
progression by stabilizing c-myc and preventing p21 accumulation, indicating a potential pro-tumorigenic role for NDR kinases. Here, we discuss these novel cell cycle functions of NDR kinases in a broader context and elaborate on possible explanations for the opposing functions of NDR kinases in normal and tumor biology.
The NDR (nuclear Dbf2-related) family of kinases is highly conserved from yeast to human, and has been classified as a subgroup of the AGC group of protein kinases based on the sequence of the ...catalytic domain. Like all other members of the AGC class of protein kinases, NDR kinases require the phosphorylation of conserved Ser/Thr residues for activation. Importantly, NDR family members have two unique stretches of primary sequence: an N-terminal regulatory (NTR) domain and an insert of several residues between subdomains VII and VIII of the kinase domain. The kinase domain insert functions as an auto-inhibitory sequence (AIS), while binding of the co-activator MOB (Mps-one binder) proteins to the NTR domain releases NDR kinases from inhibition of autophosphorylation. However, despite such advances in our understanding of the molecular activation mechanism(s) and physiological functions of NDR kinases in yeast and invertebrates, most biological NDR substrates still remain to be identified. Nevertheless, by showing that the centrosomal subpopulation of human NDR1/2 is required for proper centrosome duplication, the first biological role of human NDR1/2 kinases has been defined recently. How far NDR-driven centrosome overduplication could actually contribute to cellular transformation will also be discussed.
The mechanism of cell transformation by Fms-like tyrosine kinase 3 (FLT3) in acute myeloid leukemia (AML) is incompletely understood. The most prevalent activated mutant FLT3 ITD exhibits an altered ...signaling quality, including strong activation of the STAT5 transcription factor. FLT3 ITD has also been found partially retained as a high-mannose precursor in an intracellular compartment. To analyze the role of intracellular retention of FLT3 for transformation, we have generated FLT3 versions that are anchored in the perinuclear endoplasmic reticulum (ER) by appending an ER retention sequence containing a RRR (R3) motif. ER retention of R3, but not of corresponding A3 FLT3 versions, is shown by biochemical, fluorescence-activated cell sorting, and immunocytochemical analyses. ER anchoring reduced global autophosphorylation and diminished constitutive activation of ERK1/2 and AKT of the constitutively active FLT3 versions. ER anchoring was, however, associated with elevated signaling to STAT3. Transforming activity of the FLT3 D835Y mutant was suppressed by ER anchoring. In contrast, ER-anchored FLT3 ITD retained STAT5-activating capacity and was transforming in vitro and in vivo. The findings highlight another aspect of the different signaling quality of FLT3 ITD: It can transform cells from an intracellular location.
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