Dana-Farber/Boston Children's Cancer and Blood Disorders Center

Abstract Somatic mutations in cancer genomes are caused by multiple mutational processes, each of which generates a characteristic mutational signature 1 . Here, as part of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium 2 of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA), we characterized mutational signatures using 84,729,690 somatic mutations from 4,645 whole-genome and 19,184 exome sequences that encompass most types of cancer. We identified 49 single-base-substitution, 11 doublet-base-substitution, 4 clustered-base-substitution and 17 small insertion-and-deletion signatures. The substantial size of our dataset, compared with previous analyses 3–15 , enabled the discovery of new signatures, the separation of overlapping signatures and the decomposition of signatures into components that may represent associated—but distinct—DNA damage, repair and/or replication mechanisms. By estimating the contribution of each signature to the mutational catalogues of individual cancer genomes, we revealed associations of signatures to exogenous or endogenous exposures, as well as to defective DNA-maintenance processes. However, many signatures are of unknown cause. This analysis provides a systematic perspective on the repertoire of mutational processes that contribute to the development of human cancer.

Abstract Cancer is driven by genetic change, and the advent of massively parallel sequencing has enabled systematic documentation of this variation at the whole-genome scale 1–3 . Here we report the integrative analysis of 2,658 whole-cancer genomes and their matching normal tissues across 38 tumour types from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). We describe the generation of the PCAWG resource, facilitated by international data sharing using compute clouds. On average, cancer genomes contained 4–5 driver mutations when combining coding and non-coding genomic elements; however, in around 5% of cases no drivers were identified, suggesting that cancer driver discovery is not yet complete. Chromothripsis, in which many clustered structural variants arise in a single catastrophic event, is frequently an early event in tumour evolution; in acral melanoma, for example, these events precede most somatic point mutations and affect several cancer-associated genes simultaneously. Cancers with abnormal telomere maintenance often originate from tissues with low replicative activity and show several mechanisms of preventing telomere attrition to critical levels. Common and rare germline variants affect patterns of somatic mutation, including point mutations, structural variants and somatic retrotransposition. A collection of papers from the PCAWG Consortium describes non-coding mutations that drive cancer beyond those in the TERT promoter 4 ; identifies new signatures of mutational processes that cause base substitutions, small insertions and deletions and structural variation 5,6 ; analyses timings and patterns of tumour evolution 7 ; describes the diverse transcriptional consequences of somatic mutation on splicing, expression levels, fusion genes and promoter activity 8,9 ; and evaluates a range of more-specialized features of cancer genomes 8,10–18 .

BACKGROUND: Fusions involving one of three tropomyosin receptor kinases (TRK) occur in diverse cancers in children and adults. We evaluated the efficacy and safety of larotrectinib, a highly selective TRK inhibitor, in adults and children who had tumors with these fusions. METHODS: We enrolled patients with consecutively and prospectively identified TRK fusion-positive cancers, detected by molecular profiling as routinely performed at each site, into one of three protocols: a phase 1 study involving adults, a phase 1-2 study involving children, or a phase 2 study involving adolescents and adults. The primary end point for the combined analysis was the overall response rate according to independent review. Secondary end points included duration of response, progression-free survival, and safety. RESULTS: A total of 55 patients, ranging in age from 4 months to 76 years, were enrolled and treated. Patients had 17 unique TRK fusion-positive tumor types. The overall response rate was 75% (95% confidence interval [CI], 61 to 85) according to independent review and 80% (95% CI, 67 to 90) according to investigator assessment. At 1 year, 71% of the responses were ongoing and 55% of the patients remained progression-free. The median duration of response and progression-free survival had not been reached. At a median follow-up of 9.4 months, 86% of the patients with a response (38 of 44 patients) were continuing treatment or had undergone surgery that was intended to be curative. Adverse events were predominantly of grade 1, and no adverse event of grade 3 or 4 that was considered by the investigators to be related to larotrectinib occurred in more than 5% of patients. No patient discontinued larotrectinib owing to drug-related adverse events. CONCLUSIONS: Larotrectinib had marked and durable antitumor activity in patients with TRK fusion-positive cancer, regardless of the age of the patient or of the tumor type. (Funded by Loxo Oncology and others; ClinicalTrials.gov numbers, NCT02122913 , NCT02637687 , and NCT02576431 .).

The classification of myeloid neoplasms and acute leukemias was last updated in 2016 within a collaboration between the World Health Organization (WHO), the Society for Hematopathology, and the European Association for Haematopathology. This collaboration was primarily based on input from a clinical advisory committees (CACs) composed of pathologists, hematologists, oncologists, geneticists, and bioinformaticians from around the world. The recent advances in our understanding of the biology of hematologic malignancies, the experience with the use of the 2016 WHO classification in clinical practice, and the results of clinical trials have indicated the need for further revising and updating the classification. As a continuation of this CAC-based process, the authors, a group with expertise in the clinical, pathologic, and genetic aspects of these disorders, developed the International Consensus Classification (ICC) of myeloid neoplasms and acute leukemias. Using a multiparameter approach, the main objective of the consensus process was the definition of real disease entities, including the introduction of new entities and refined criteria for existing diagnostic categories, based on accumulated data. The ICC is aimed at facilitating diagnosis and prognostication of these neoplasms, improving treatment of affected patients, and allowing the design of innovative clinical trials.

As targets for the immunosuppressive drugs cyclosporin A and FK506, transcription factors of the NFAT (nuclear factor of activated T cells) family have been the focus of much attention. NFAT proteins, which are expressed in most immune-system cells, play a pivotal role in the transcription of cytokine genes and other genes critical for the immune response. The activity of NFAT proteins is tightly regulated by the calcium/calmodulin-dependent phosphatase calcineurin, a primary target for inhibition by cyclosporin A and FK506. Calcineurin controls the translocation of NFAT proteins from the cytoplasm to the nucleus of activated cells by interacting with an N-terminal regulatory domain conserved in the NFAT family. The DNA-binding domains of NFAT proteins resemble those of Rel-family proteins, and Rel and NFAT proteins show some overlap in their ability to bind to certain regulatory elements in cytokine genes. NFAT is also notable for its ability to bind cooperatively with transcription factors of the AP-1 (Fos/Jun) family to composite NFAT:AP-1 sites, found in the regulatory regions of many genes that are inducibly transcribed by immune-system cells. This review discusses recent data on the diversity of the NFAT family of transcription factors, the regulation of NFAT proteins within cells, and the cooperation of NFAT proteins with other transcription factors to regulate the expression of inducible genes.

Activation of the transcription factor nuclear factor kappa B (NF-kappaB) is controlled by sequential phosphorylation, ubiquitination, and degradation of its inhibitory subunit IkappaB. A large multiprotein complex, the IkappaB kinase (IKK) signalsome, was purified from HeLa cells and found to contain a cytokine-inducible IkappaB kinase activity that phosphorylates IkappaB-alpha and IkappaB-beta. Two components of the IKK signalsome, IKK-1 and IKK-2, were identified as closely related protein serine kinases containing leucine zipper and helix-loop-helix protein interaction motifs. Mutant versions of IKK-2 had pronounced effects on RelA nuclear translocation and NF-kappaB-dependent reporter activity, consistent with a critical role for the IKK kinases in the NF-kappaB signaling pathway.

The NFAT family of transcription factors encompasses five proteins evolutionarily related to the Rel/NF B family (Chytil and Verdine 1996; Graef et al. 2001b). The primordial family member is NFAT5, the only NFATrelated protein represented in the Drosophila genome. NFAT5 is identical to TonEBP (tonicity element binding protein), a transcription factor crucial for cellular responses to hypertonic stress (Lopez-Rodriguez et al. 1999; Miyakawa et al. 1999). We focus here on the remaining four NFAT proteins (NFAT1–NFAT4, also known as NFATc1–c4; see Table 1), referring to them collectively as NFAT. The distinguishing feature of NFAT is its regulation by Ca and the Ca/calmodulin-dependent serine phosphatase calcineurin. NFAT proteins are phosphorylated and reside in the cytoplasm in resting cells; upon stimulation, they are dephosphorylated by calcineurin, translocate to the nucleus, and become transcriptionally active, thus providing a direct link between intracellular Ca signaling and gene expression. NFAT activity is further modulated by additional inputs from diverse signaling pathways, which affect NFAT kinases and nuclear partner proteins. In the first part of this review, we describe the influence of these multiple inputs on the nuclear–cytoplasmic distribution and transcriptional function of NFAT. Recent structural data emphasize the remarkable versatility of NFAT binding to DNA. At composite NFAT:AP-1 elements found in the regulatory regions of many target genes, NFAT proteins bind cooperatively with an unrelated transcription factor, AP-1 (Fos–Jun; Chen et al. 1998). At DNA elements that resemble NF B sites, NFAT proteins bind DNA as dimers (Giffin et al. 2003; Jin et al. 2003). In the second section of this review, we describe these two modes of DNA binding by NFAT. NFAT also acts synergistically with transcription factors other than Fos and Jun, but the structural basis for synergy remains unknown. Drawing on published structures, we discuss the potential cooperation of NFAT with other classes of DNA-binding proteins. It is clear that NFAT activates transcription of a large number of genes during an effective immune response (Rao et al. 1997; Kiani et al. 2000; Serfling et al. 2000; Macian et al. 2001). In the third part of this review, we present information obtained from these studies, highlighting experimental and bioinformatics approaches to identifying NFAT target genes. We discuss the finding that NFAT and NFAT–Fos–Jun complexes activate distinct subsets of target genes in lymphocytes (Macian et al. 2002). We also describe a novel aspect of gene regulation by NFAT, in which this transcription factor participates in an early phase of chromatin remodeling that occurs at specific genetic loci in differentiating T cells (Avni et al. 2002). There is evidence that NFAT regulates cell differentiation programs in cell types other than immune cells (Crabtree and Olson 2002; Horsley and Pavlath 2002; Graef et al. 2003; Hill-Eubanks et al. 2003). In the last section of this review, we select three differentiation programs—fiber-type specification in differentiated skeletal muscle, cardiac valve development, and osteoclast differentiation—for detailed consideration. We evaluate the evidence for NFAT involvement, point out novel cellular and molecular mechanisms that might regulate this familiar transcription factor, and discuss how NFAT exerts its biological effects. Because the phenotypes of NFAT knockout mice have been reviewed elsewhere (Crabtree and Olson 2002; Horsley and Pavlath 2002), we refer to them only as necessary to illustrate specific points.

The circulatory and migratory properties of white blood cells have evolved to allow efficient surveillance of tissues for infectious pathogens and rapid accu­ mulation at sites of injury and infection. Lymphocytes continually patrol the body for foreign antigen by recirculating from blood, through tissue, into lymph, and back to blood. Lymphocytes acquire a predilection, based on the environment in which they first encounter foreign antigen, to home to or recirculate through that same environment (39, 40). Granulocytes and mono­ cytes cannot recirculate, but emigrate from the bloodstream in response to molecular changes on the surface of blood vessels that signal injury or infec­ tion. Lymphocytes can similarly accumulate in response to inflammatory stim­ uli. The nature of the inflammatory stimulus determines whether lymphocytes. monocytes, neutrophils, or eosinophils predominate, and thus exercises spec­ ificity in the molecular or codes that are displayed on endothe­ lium and control of particular leukocyte classes. Recent findings show that the traffic signals for lymphocyte recirculation and for neutrophil and monocyte localization in inflammation are strikingly similar at the molecular level. These signal or area code molecules are

Chemotactic factors are postulated to direct emigration of lymphocytes from the blood stream into sites of inflammation. Members of a family of chemotactic cytokines, termed chemokines, have been shown to attract lymphocytes but efficacy, i.e., the maximal percentage of attracted cells, has been low. We have identified a highly efficacious lymphocyte chemotactic activity in the supernatants of the murine bone marrow stroma cell line MS-5 which attracts 10-fold more lymphocytes in vitro than currently described lymphocyte chemoattractants. Purification of this chemotactic activity revealed identity to stromal cell-derived factor 1 (SDF-1). SDF-1 acts on lymphocytes and monocytes but not neutrophils in vitro and is both a highly efficacious and highly potent mononuclear cell attractant in vivo. In addition, SDF-1 induces intracellular actin polymerization in lymphocytes, a process that is thought to be a prerequisite for cell motility. Since SDF-1 is expressed constitutively in a broad range of tissues it may have a role in immune surveillance and in basal extravasation of lymphocytes and monocytes rather than in inflammation.

Since the pioneering work of Gowans and colleagues in the 1960s,1,2 much progress has been made in understanding the pivotal role of cell migration in immunity. We now have considerable knowledge of the way in which specialized leukocytes are channeled to distinct target tissues in immune responses and inflammation (Figure 1). This review will concentrate on the migration of T cells, which are at the heart of most adaptive immune responses.Since T cells respond to pathogens only on direct contact with pathogen-derived antigen, they must migrate to sites where antigen is found. The T-cell receptor recognizes a peptide . . .

Hematopoietic progenitor cells migrate in vitro and in vivo towards a gradient of the chemotactic factor stromal cell-derived factor-1 (SDF-1) produced by stromal cells. This is the first chemoattractant reported for human CD34+ progenitor cells. Concentrations of SDF-1 that elicit chemotaxis also induce a transient elevation of cytoplasmic calcium in CD34+ cells. SDF-1-induced chemotaxis is inhibited by pertussis toxin, suggesting that its signaling in CD34+ cells is mediated by seven transmembrane receptors coupled to Gi proteins. CD34+ cells migrating to SDF-1 include cells with a more primitive (CD34+/CD38- or CD34+/DR-) phenotype as well as CD34+ cells phenotypically committed to the erythroid, lymphoid and myeloid lineages, including functional BFU-E, CFU-GM, and CFU-MIX progenitors. Chemotaxis of CD34+ cells in response to SDF-1 is increased by IL-3 in vitro and is lower in CD34+ progenitors from peripheral blood than in CD34+ progenitors from bone marrow, suggesting that an altered response to SDF-1 may be associated with CD34 progenitor mobilization.

Invasive tumor dissemination in vitro and in vivo involves the proteolytic degradation of ECM barriers. This process, however, is only incompletely attenuated by protease inhibitor-based treatment, suggesting the existence of migratory compensation strategies. In three-dimensional collagen matrices, spindle-shaped proteolytically potent HT-1080 fibrosarcoma and MDA-MB-231 carcinoma cells exhibited a constitutive mesenchymal-type movement including the coclustering of beta 1 integrins and MT1-matrix metalloproteinase (MMP) at fiber bindings sites and the generation of tube-like proteolytic degradation tracks. Near-total inhibition of MMPs, serine proteases, cathepsins, and other proteases, however, induced a conversion toward spherical morphology at near undiminished migration rates. Sustained protease-independent migration resulted from a flexible amoeba-like shape change, i.e., propulsive squeezing through preexisting matrix gaps and formation of constriction rings in the absence of matrix degradation, concomitant loss of clustered beta 1 integrins and MT1-MMP from fiber binding sites, and a diffuse cortical distribution of the actin cytoskeleton. Acquisition of protease-independent amoeboid dissemination was confirmed for HT-1080 cells injected into the mouse dermis monitored by intravital multiphoton microscopy. In conclusion, the transition from proteolytic mesenchymal toward nonproteolytic amoeboid movement highlights a supramolecular plasticity mechanism in cell migration and further represents a putative escape mechanism in tumor cell dissemination after abrogation of pericellular proteolysis.

BACKGROUND: Despite an increase in the number of therapies available to treat patients with immune thrombocytopenia (ITP), there are minimal data from randomized trials to assist physicians with the management of patients. OBJECTIVE: These evidence-based guidelines of the American Society of Hematology (ASH) are intended to support patients, clinicians, and other health care professionals in their decisions about the management of ITP. METHODS: In 2015, ASH formed a multidisciplinary guideline panel that included 8 adult clinical experts, 5 pediatric clinical experts, 2 methodologists with expertise in ITP, and 2 patient representatives. The panel was balanced to minimize potential bias from conflicts of interest. The panel reviewed the ASH 2011 guideline recommendations and prioritized questions. The panel used the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach, including evidence-to-decision frameworks, to appraise evidence (up to May 2017) and formulate recommendations. RESULTS: The panel agreed on 21 recommendations covering management of ITP in adults and children with newly diagnosed, persistent, and chronic disease refractory to first-line therapy who have non-life-threatening bleeding. Management approaches included: observation, corticosteroids, IV immunoglobulin, anti-D immunoglobulin, rituximab, splenectomy, and thrombopoietin receptor agonists. CONCLUSIONS: There was a lack of evidence to support strong recommendations for various management approaches. In general, strategies that avoided medication side effects were favored. A large focus was placed on shared decision-making, especially with regard to second-line therapy. Future research should apply standard corticosteroid-dosing regimens, report patient-reported outcomes, and include cost-analysis evaluations.

squamous cell carcinoma antigen 1 or 2 α1 antitrypsin (α1 proteinase inhibitor) α2 antiplasmin amyloid-β α1 antichymotrypsin antithrombin III monocyte-neutrophil elastase inhibitor ovalbumin plasminogen activator inhibitor type 1 or 2 pigment epithelium-derived factor vascular endothelial growth factor reactive site loop tissue plasminogen activator urokinase plasminogen activator The serpins (serineproteinase inhibitors) are a superfamily of proteins (350–500 amino acids in size) that fold into a conserved structure and employ a unique suicide substrate-like inhibitory mechanism. The serpins were last reviewed in 1994 (1Potempa J. Korzus E. Travis J. J. Biol. Chem. 1994; 269: 15957-15960Abstract Full Text PDF PubMed Google Scholar). More recent studies show: 1) an expanded distribution within the kingdoms of metazoa and plantae, as well as certain viruses, 2) a surprising effect on the covalently bound target proteinase, and 3) novel biochemical and biological functions. Most serpins inhibit serine proteinases of the chymotrypsin family. However, cross-class inhibitors have been identified. The viral serpin CrmA and, to a lesser extent, PI9 (SERPINB9) inhibit the cysteine proteinase, caspase 1 (2Komiyama T. Ray C.A. Pickup D.J. Howard A.D. Thornberry N.A. Peterson E.P. Salvesen G. J. Biol. Chem. 1994; 269: 19331-19337Abstract Full Text PDF PubMed Google Scholar), and SCCA11 (SERPINB3) neutralizes the potent papain-like cysteine proteinases, cathepsins L, K, and S (3Schick C. Pemberton P.A. Shi G.-P. Kamachi Y. Cataltepe S. Bartuski A.J. Gornstein E.R. Bromme D. Chapman H.A. Silverman G.A. Biochemistry. 1998; 37: 5258-5266Crossref PubMed Scopus (259) Google Scholar). In addition, several members no longer function as proteinase inhibitors but perform other roles such as hormone transport (thyroid-binding globulin (SERPINA6), corticosteroid-binding globulin (SERPINA7)), and blood pressure regulation (angiotensinogen (SERPINA8)) (1Potempa J. Korzus E. Travis J. J. Biol. Chem. 1994; 269: 15957-15960Abstract Full Text PDF PubMed Google Scholar). Data base searching provides evidence for ∼500 serpins, with full-length coding sequences known or predicted for about one-half of those (4Irving J.A. Pike R.N. Lesk A.M. Whisstock J.C. Genome Res. 2000; 10: 1845-1864Crossref PubMed Scopus (523) Google Scholar). A phylogenetic analysis divides serpins into 16 clades (see Supplemental Data, Table A) and 10 highly diverged “orphans” (4Irving J.A. Pike R.N. Lesk A.M. Whisstock J.C. Genome Res. 2000; 10: 1845-1864Crossref PubMed Scopus (523) Google Scholar). These data facilitate the construction of a consistent expandable nomenclature (see Supplemental Data for Serpin Nomenclature Guidelines, Table B). The completed DNA sequences of several organisms have yielded insight into the complexity of the family. The Caenorhabditis elegans, Drosophila melanogaster, and Arabidopsis thalianagenomes encode for ∼20,000, 13,000, and 25,000 genes, respectively. However, these three species harbor ∼9, 32, and 13 serpin genes, respectively. The nonlinear relationship among the number of serpin genes, relative to the total gene number, suggests that at least a subset of serpins has evolved divergent functions despite a striking degree of sequence and structural conservation. Serpins adopt a metastable conformation that is required for their inhibitory activity (5Stein P.E. Carrell R.W. Nat. Struct. Biol. 1995; 2: 96-113Crossref PubMed Scopus (398) Google Scholar). This conformation consists of a conserved secondary structure comprised of β-sheets A, B, and C and at least 7 α-helices (most typically have 9, lettered A–I; Fig.1 A). The RSL, which contains the proteinase recognition site, is an exposed, flexible stretch of ∼17 residues tethered between β-sheets A and C. Serpins can undergo major structural rearrangements that involve alternative conformations for the RSL, β-sheet A, and the attached strand 1 of β-sheet C. Considering only intramolecular structural changes, serpins can convert to the more stable latent form (Fig. 1 B). The RSL inserts into the middle of β-sheet A to give a fully antiparallel β-sheet, and s1C is extracted from β-sheet C to provide an exposed “return” from the bottom of the serpin. Serpins in the latent conformation are noninhibitory but can be converted back to the active state by denaturation and refolding. The Tm for unfolding of latent PAI1 (SERPINE1) is 17 °C higher than that for the native state (reviewed in Ref. 6Gettins P.G.W. Patston P.A. Olson S.T. Serpins: Structure, Function and Biology, Molecular Biology Intelligence Unit. R. G. Landes Co., and Chapman & Hall, Austin, TX1996Google Scholar). The most stable state for inhibitory serpins is the RSL-cleaved form, in which the RSL has fully inserted into β-sheet A, as in the latent conformation, but without the need to extract s1C from β-sheet C (Fig. 1 C). Estimates of the Tm for unfolding of such conformations are >120 °C, compared with ∼60 °C for the native state (7Kaslik G. Kardos J. Szabo E. Szilagyi L. Zavodszky P. Westler W.M. Markley J.L. Graf L. Biochemistry. 1997; 36: 5455-5464Crossref PubMed Scopus (104) Google Scholar). The most informative serpin structures, from a mechanistic viewpoint, are those of a Michaelis complex between Serpin 1 and trypsin (Fig.1 D) and of a covalent complex between α1AT (SERPINA1) and trypsin (8Huntington J.A. Read R.J. Carrell R.W. Nature. 2000; 407: 923-926Crossref PubMed Scopus (970) Google Scholar) (Fig. 1 E). This latter structure represents the proteinase after it has been kinetically trapped in the acyl-enzyme intermediate that forms normally along the peptide bond cleavage pathway. Whereas the bound serpin is almost indistinguishable from that of the RSL-cleaved form (Fig. 1 C), the proteinase is grossly distorted (see below). Serpins inhibit serine proteinases by an irreversible suicide substrate mechanism when the interaction proceeds down the inhibitory arm of a branched pathway (Fig. 2) (6Gettins P.G.W. Patston P.A. Olson S.T. Serpins: Structure, Function and Biology, Molecular Biology Intelligence Unit. R. G. Landes Co., and Chapman & Hall, Austin, TX1996Google Scholar). In the inhibitory pathway, the proteinase initially forms a noncovalent Michaelis-like complex (Fig. 1 D) through interactions with residues flanking the scissile bond (P1–P1′). Attack of the active site serine on the scissile bond leads to a covalent ester linkage between Ser-195 of the proteinase and the backbone carbonyl of the P1 residue and cleavage of the peptide bond (6Gettins P.G.W. Patston P.A. Olson S.T. Serpins: Structure, Function and Biology, Molecular Biology Intelligence Unit. R. G. Landes Co., and Chapman & Hall, Austin, TX1996Google Scholar). It is likely that only at this stage, with removal of the restraint, does the RSL start to insert into β-sheet A and transport the covalently bound proteinase with it. Upon complete loop insertion the proteinase is translocated by over 70 Å, and its active site is distorted (Fig. 1 E). The alignment of the active site catalytic triad is altered by as much as 3 Å, and the P1 side chain is removed from the S1 pocket. Also, 40% of the body of the proteinase shows no traceable electron density. Proteinase distortion and hence inactivation results from compression of the proteinase against the base of the serpin as a consequence of the inserted RSL being just the right length. The energy needed to effect the distortion may come from the much greater stability of the cleaved loop-inserted conformation compared with the native-like conformation. The net result of this conformational rearrangement is kinetic trapping of the acyl intermediate due to slowing of the deacylation steps of the normal substrate reaction by 6–8 orders of magnitude (k5 in Fig. 2). Because of the small values for k5 (complex t12≅ hours to weeks), serpin-proteinase complexes in vivowould bind to their receptors and be cleared (complext12 ≅ minutes) long before significant complex decay could occur. The point in transit where the enzyme activity is reduced sufficiently to commit the intermediate to the kinetic trap is not known but in part contributes to the branched nature of the pathway and the ultimate fate of the complex. If, for example, RSL movement is impeded, the enzyme may successfully complete the deacylation step and escape before it is irreversibly trapped. This noninhibitory pathway yields an active proteinase and a cleaved, inactive serpin. The ratio of serpin products (complex versuscleaved) thus reflects a competition between the rate of ester hydrolysis (k3 in Fig. 2) and that of loop insertion (k4 in Fig. 2) to the point of proteinase distortion. This ratio is signified also by the stoichiometry of inhibition, which is defined as (k3 +k4)/k4, i.e.the number of moles of serpin needed to inhibit 1 mol of proteinase as a kinetically trapped complex. This mechanism accounts for the requirements for effective inhibition by serpins, which include a critical RSL length, appropriate residues within the loop that are compatible with rapid and favorable burial into β-sheet A, and the presence of Ser in the proteinase active site (6Gettins P.G.W. Patston P.A. Olson S.T. Serpins: Structure, Function and Biology, Molecular Biology Intelligence Unit. R. G. Landes Co., and Chapman & Hall, Austin, TX1996Google Scholar). Such a mechanism is adaptable to the inhibition of cysteine proteinases by serpins, with the difference being that the kinetically trapped intermediate is a thiol ester rather than an oxy ester. The detection of CrmA, a serpin that inhibits cysteine proteinases of the caspase family, in the loop-inserted cleaved conformation supports the feasibility of a common inhibitory mechanism (9Renatus M. Zhou Q. Stennicke H.R. Snipas S.J. Turk D. Bankston L.A. Liddington R.C. Salvesen G.S. Struct. Fold. Des. 2000; 8: 789-797Abstract Full Text Full Text PDF Scopus (55) Google Scholar), whereas the detection of an SDS-stable complex between SCCA1 and cathepsin S (a cysteine proteinase of the papain family) provides evidence for the formation of a stable, covalent thiol ester-type linkage (3Schick C. Pemberton P.A. Shi G.-P. Kamachi Y. Cataltepe S. Bartuski A.J. Gornstein E.R. Bromme D. Chapman H.A. Silverman G.A. Biochemistry. 1998; 37: 5258-5266Crossref PubMed Scopus (259) Google Scholar). The few convincing reports of reversible inhibition, such as of single-chain uPA by PCI (SERPINA5) (10Schwartz B.S. Espana F. J. Biol. Chem. 1999; 274: 15278-15283Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) or of chymotrypsin by α2AP (SERPINF2) (11Shieh B.H. Potempa J. Travis J. J. Biol. Chem. 1989; 264: 13420-13423Abstract Full Text PDF PubMed Google Scholar) may represent special cases in which unusual stabilization of the initial noncovalent Michaelis-like complex blocks progression to the substrate reaction. A negative consequence of the need for a metastable conformation in the active state is that natural mutations, either alone or in combination with environmental factors, can promote inappropriate loop insertion. When this occurs between the RSL of one molecule and the β-sheet of another, dimers and higher order oligomers can result. Either through depletion of active serpin or through pathological effects of the polymers themselves, such aggregate formation can lead to disease. The best characterized examples are the emphysema (serpin depletion) and cirrhosis (intracellular inclusions) associated with loop-sheet polymers of the Z or S variants of α1AT (12Elliott P.R. Lomas D.A. Carrell R.W. Abrahams J.P. Nat. Struct. Biol. 1996; 3: 676-681Crossref PubMed Scopus (249) Google Scholar) (see Supplemental Data, Fig. A) and the dementia associated with neuroserpin (SERPINI1) inclusion bodies (see below). Understanding the biologic function of serpins remains an ongoing challenge. For example, the biologic functions for many of the human serpins involved in the clotting and fibrinolytic cascades are well documented. However the role of human serpins in some other types of biologic processes awaits further validation (Fig.3). In 1993 amino acid similarities among chicken ovalbumin (ov), PAI2 (SERPINB2), and MNEI (SERPINB1) led to the identification of a subgroup of the serpin superfamily (13Remold-O'Donnell E. FEBS Lett. 1993; 315: 105-108Crossref PubMed Scopus (217) Google Scholar). The N and C termini of the ov-serpins are shorter than the prototypical serpin α1AT, and they also lack a classical secretory signal peptide. At present, there are 13 human ov-serpins (see Supplemental Data, Table B). They map to 6p25 and 18q21 and fall into two classes based on a single difference in gene structure (14Scott F.L. Eyre H.J. Lioumi M. Ragoussis J. Irving J.A. Sutherland G.A. Bird P.I. Genomics. 1999; 62: 490-499Crossref PubMed Scopus (37) Google Scholar). Like ovalbumin, many of the 18q21 serpin genes have an exon encoding a polypeptide loop between helices C and D (CD loop) that may contribute to accessory functions. Unlike ovalbumin itself, most ov-serpins reside intracellularly with a cytoplasmic or nucleocytoplasmic distribution. However, several ov-serpins (PAI2, megsin (SERPINB7), MNEI, maspin (SERPINB5), and the SCCAs (SERPINB3 and -4)) may function extracellularly as they are released from cells under certain conditions. Release may be facilitated by an embedded, noncleaved hydrophobic N-terminal signal sequence and appears to involve both conventional and non-endoplasmic reticulum-Golgi secretory pathways (15Belin D. Thromb. Haemostasis. 1993; 70: 144-147Crossref PubMed Scopus (40) Google Scholar). Regardless of how ov-serpins are released from cells, those with RSL cysteine or methionine residues are susceptible to oxidative inactivation and are likely to have a limited half-life in the extracellular milieu. With the possible exception of maspin, all human ov-serpins are functional, competitive inhibitors of serine or cysteine proteinases. Several members of the group inhibit more than one proteinase, and dual reactive sites (utilization of more than one P1 residue) have been described for PI6 (SERPINB6), PI8 (SERPINB8), PI9, SCCA1, SCCA2, and MNEI (for example see Ref. 16Riewald M. Schleef R.R. J. Biol. Chem. 1996; 271: 14526-14532Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). However, the CD loops of the ov-serpins have the potential to interact with other proteins. For example, the CD loop of PAI2 is required for its cell survival function (17Dickinson J.L. Bates E.J. Ferrante A. Antalis T.M. J. Biol. Chem. 1995; 270: 27894-27904Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar) and is a target for transglutamination (18Jensen P.H. Schuler E. Woodrow G. Richardson M. Goss N. Hojrup P. Petersen T.E. Rasmussen L.K. J. Biol. Chem. 1994; 269: 15394-15398Abstract Full Text PDF PubMed Google Scholar). Bomapin (SERPINB10; like the chicken ov-serpin, MENT, see below) carries a nuclear localization signal in its CD loop that presumably interacts with a nuclear importin (19Chuang T.L. Schleef R.R. J. Biol. Chem. 1999; 274: 11194-11198Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The physiological functions of ov-serpins are still emerging. PAI2 may play a role in the regulation of extracellular matrix remodeling through the inhibition of uPA, as high PAI2 and low uPA levels correlate with a positive prognosis in breast cancer (20Duggan C. Kennedy S. Kramer M.D. Barnes C. Elvin P. McDermott E. O'Higgins N. Duffy M.J. Br. J. Cancer. 1997; 76: 622-627Crossref PubMed Scopus (66) Google Scholar). Also, PAI2 may have a structural role inside some cells (perhaps keratinocytes) as suggested by its ability to spontaneously polymerize and undergo transglutamination (21Mikus P. Ny T. J. Biol. Chem. 1996; 271: 10048-10053Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Many ov-serpins reside in proteinase-secreting cells (22Bird P.I. Immunol. Cell Biol. 1999; 77: 47-57Crossref PubMed Scopus (58) Google Scholar). For example, PI9, a potent inhibitor of granzyme B, is also present in cytotoxic lymphocytes. Because PI9 can protect cells against granzyme B-mediated apoptosis, it probably protects cytotoxic lymphocytes from autodestruction due to misdirected granzyme B. A similar cytoprotective role can be envisaged for PI6, PI8, MNEI, PAI2, and the SCCAs. In addition, endogenous or exogenous ov-serpins may protect bystander cells and tissue from proteolytic damage. Studies in rats show that recombinant MNEI delivered to the airways prevents lung injury by neutrophil proteinases and point to its potential in treating inflammatory lung disease (23Rees D.D. Rogers R.A. Cooley J. Mandle R.J. Kenney D.M. Remold-O'Donnell E. Am. J. Respir. Cell Mol. Biol. 1999; 20: 69-78Crossref PubMed Scopus (43) Google Scholar). The ability of many ov-serpins to inhibit more than one proteinase and their presence in epithelial cells suggest that they play a role in barrier function or host defense against microbial or viral proteinases. For example, PI9 inhibits Bacillussubtilisin, and PI8 inhibits furin, a subtilisin-related enzyme (24Dahlen J.R. Foster D.C. Kisiel W. Biochem. Biophys. Res. Commun. 1997; 238: 329-333Crossref PubMed Scopus (19) Google Scholar,25Dahlen J.R. Jean F. Thomas G. Foster D.C. Kisiel W. J. Biol. Chem. 1998; 273: 1851Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Additional functions of ov-serpins include the regulation of: 1) cell growth or differentiation, as exemplified by the role of megsin in megakaryocyte differentiation (26Tsujimoto M. Tsuruoka N. Ishida N. Kurihara T. Iwasa F. Yamashiro K. Rogi T. Kodama S. Katsuragi N. Adachi M. Katayama T. Nakao M. Yamaichi K. Hashino J. Haruyama M. Miura K. Nakanishi T. Nakazato H. Teramura M. Mizoguchi H. Yamaguchi N. J. Biol. Chem. 1997; 272: 15373-15380Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar), 2) tumor cell invasiveness and motility, as shown by the inhibitory role of maspin in breast and prostate tumors (27Zou Z. Anisowicz A. Hendrix M.J. Thor A. Neveu M. Sheng S. Rafidi K. Seftor E. Sager R. Science. 1994; 263: 526-529Crossref PubMed Scopus (842) Google Scholar), and 3) angiogenesis (see below). Grigoryev et al. (28Grigoryev S.A. Bednar J. Woodcock C.L. J. Biol. Chem. 1999; 274: 5626-5636Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) isolated a novel serpin, MENT, from the nuclei of terminally differentiated chicken hematopoietic cells. MENT is an ov-serpin with a CD loop that contains a nuclear localization signal, a lamin-like chromatin binding domain, and an A-T hook DNA binding motif. The molecule has a relatively high pI (9 versus 5–6.5 for that of other serpins) with the majority of positive charges clustering near the CD loop. Thus, MENT appears to utilize the CD loop to bind tightly to nucleosomes with an apparent stoichiometry of 2:1. MENT is the major non-histone chromatin protein in differentiated nuclei and is concentrated in the heterochromatin. MENT induces higher order chromatin compaction when it is expressed ectopically in cells or added to isolated nuclei in vitro. Although MENT contains a viable RSL, target proteinases have yet to be identified. which inhibits uPA, and growth factor in is from and cells G.A. S. E.P. R. S. M. D.A. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). may play a role in the from In a neuroserpin in within the and of the protein reduced the by and the number of cells by M. M. E. D.A. 2000; PubMed Google Scholar). In a form of dementia and neuroserpin Molecular analysis in two and in the C. D. P. J. F. D. M. D.A. B. P.R. Carrell R.W. Lomas D.A. Nature. 1999; PubMed Google Scholar). These are similar to that in α1AT in which an of β-sheet A and the formation of loop-sheet In these polymers and in the normal function is and, to a lesser extent, other serpins are within the of from with one of the most common forms of dementia (reviewed in Ref. S. 1998; 20: PubMed Scopus Google Scholar). Although the of this is the extracellular of may be by binding to low receptors and with appears to facilitate formation by as a for the The peptide inserts into A and C of in which it a conformation. Upon RSL is released into the extracellular in which the peptide is more to is a noninhibitory serpin that isolated from pigment epithelial cells but is also in and (6Gettins P.G.W. Patston P.A. Olson S.T. Serpins: Structure, Function and Biology, Molecular Biology Intelligence Unit. R. G. Landes Co., and Chapman & Hall, Austin, TX1996Google Scholar). This factor the survival and differentiation of and et al. P. H. W. Science. 1999; PubMed Scopus Google Scholar) show that inhibits of the and endothelial cell vitro. In the cell as potent as other angiogenesis inhibitors such as and the effects of the angiogenesis growth growth and in the with the of Thus, and to blood growth in the by and angiogenesis and respectively. maspin, and RSL-cleaved have been shown to with angiogenesis in M. Shi N. Nat. 2000; PubMed Scopus Google S. E. G.A. D.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google S. J. Science. 1999; PubMed Scopus Google Scholar). However, it has yet to be of these are involved in the or regulation of blood A of function in serpin leads to the and of the E. C. D. M. J.A. Science. 1999; PubMed Scopus Google Scholar). of the pathway proteolytic cleavage of the In leads to an in both and the pathway. appears to in a negative loop by proteinases that Thus, the of and the to be secondary to proteolytic The function of these proteins remains Several studies show that serpins are of serine proteinase H. Rasmussen J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). However, with the exception of a proteinase in conventional serine proteinase are in of the sequence of trypsin as a to classical In studies by et al. K. Y. R.J. B. J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) show that of the with the of the to and on these a role for serpins in host Several of human serpins have been by in cells. of these have to an whereas show and structural as well as (see Supplemental Data, Table C). Serpins are within a number of within the of the and the (see Supplemental Data, Table of the serpins are required for growth in cell the and three highly conserved serpins, and types of proteinases The of the also three serpins, the contains serpin genes and lack serpin of encode serpins with P1 and the and all have a with a P1 residue the have a serpin with a at the P1 For more on serpins see Supplemental The serpins are a superfamily of genes that are the metazoa and Serpin members are by a conserved structure and a unique suicide substrate-like inhibitory mechanism. Serpins reside both intracellularly and extracellularly and are involved in a of biologic functions that the ability of these to irreversibly inhibit target proteinases. The of serpin function biological such as the Biochem. Full Text Full Text PDF PubMed Scopus Google Scholar) and the role that these play in and host

BACKGROUND AND METHODS: Most hemangiomas are small, harmless birthmarks that appear soon after birth, proliferate for 8 to 18 months, and then slowly regress over the next 5 to 8 years, leaving normal or slightly blemished skin. In rare cases, hemangiomas can endanger vital structures, with a mortality of up to 60 percent. About a third of these life-threatening hemangiomas respond to treatment with corticosteroids, but for the others there is no safe and effective treatment. We evaluated the effects of daily subcutaneous injections of interferon alfa-2a (up to 3 million units per square meter of body-surface area) in 20 neonates and infants with life-threatening or vision-threatening hemangiomas that failed to respond to corticosteroid therapy. RESULTS: In 18 of the 20 patients the hemangiomas regressed by 50 percent or more after an average of 7.8 months of treatment (range, 2 to 13). One infant died of refractory proliferation of a lesion and consumptive coagulopathy. The condition of three other patients who had large hemangiomas associated with consumptive coagulopathies that were unresponsive to conventional therapies stabilized after seven days of treatment with interferon alfa-2a alone. Transient side effects of treatment with interferon alfa-2a included fever, neutropenia (one patient), and skin necrosis (one patient). No long-term toxicity has been observed after a mean follow-up of 16 months. CONCLUSIONS: Interferon alfa-2a appears to induce the early regression of life-threatening corticosteroid-resistant hemangiomas of infancy.

Abstract A key mutational process in cancer is structural variation, in which rearrangements delete, amplify or reorder genomic segments that range in size from kilobases to whole chromosomes 1–7 . Here we develop methods to group, classify and describe somatic structural variants, using data from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA), which aggregated whole-genome sequencing data from 2,658 cancers across 38 tumour types 8 . Sixteen signatures of structural variation emerged. Deletions have a multimodal size distribution, assort unevenly across tumour types and patients, are enriched in late-replicating regions and correlate with inversions. Tandem duplications also have a multimodal size distribution, but are enriched in early-replicating regions—as are unbalanced translocations. Replication-based mechanisms of rearrangement generate varied chromosomal structures with low-level copy-number gains and frequent inverted rearrangements. One prominent structure consists of 2–7 templates copied from distinct regions of the genome strung together within one locus. Such cycles of templated insertions correlate with tandem duplications, and—in liver cancer—frequently activate the telomerase gene TERT . A wide variety of rearrangement processes are active in cancer, which generate complex configurations of the genome upon which selection can act.

Despite decades of research, brain tumours remain among the deadliest of all forms of cancer. The ability of these tumours to resist almost all conventional and novel treatments relates, in part, to the unique cell-intrinsic and microenvironmental properties of neural tissues. In an attempt to encourage progress in our understanding and ability to successfully treat patients with brain tumours, Cancer Research UK convened an international panel of clinicians and laboratory-based scientists to identify challenges that must be overcome if we are to cure all patients with a brain tumour. The seven key challenges summarized in this Position Paper are intended to serve as foci for future research and investment.

Tyrosine-based signals within the cytoplasmic domain of integral membrane proteins mediate clathrin-dependent protein sorting in the endocytic and secretory pathways. A yeast two-hybrid system was used to identify proteins that bind to tyrosine-based signals. The medium chains (mu 1 and mu 2) of two clathrin-associated protein complexes (AP-1 and AP-2, respectively) specifically interacted with tyrosine-based signals of several integral membrane proteins. The interaction was confirmed by in vitro binding assays. Thus, it is likely that the medium chains serve as signal-binding components of the clathrin-dependent sorting machinery.

The immune system is capable of recognizing tumors and eliminates many early malignant cells. However, tumors evolve to evade immune attack, and the tumor microenvironment is immunosuppressive. Immune responses are regulated by a number of immunological checkpoints that promote protective immunity and maintain tolerance. T cell coinhibitory pathways restrict the strength and duration of immune responses, thereby limiting immune-mediated tissue damage, controlling resolution of inflammation, and maintaining tolerance to prevent autoimmunity. Tumors exploit these coinhibitory pathways to evade immune eradication. Blockade of the PD-1 and CTLA-4 checkpoints is proving to be an effective and durable cancer immunotherapy in a subset of patients with a variety of tumor types, and additional combinations are further improving response rates. In this review we discuss the immunoregulatory functions of coinhibitory pathways and their translation to effective immunotherapies for cancer.

Genome sequencing is often pivotal in the diagnosis of rare diseases, but many of these conditions lack specific treatments. We describe how molecular diagnosis of a rare, fatal neurodegenerative condition led to the rational design, testing, and manufacture of milasen, a splice-modulating antisense oligonucleotide drug tailored to a particular patient. Proof-of-concept experiments in cell lines from the patient served as the basis for launching an "N-of-1" study of milasen within 1 year after first contact with the patient. There were no serious adverse events, and treatment was associated with objective reduction in seizures (determined by electroencephalography and parental reporting). This study offers a possible template for the rapid development of patient-customized treatments. (Funded by Mila's Miracle Foundation and others.).