bluuebunny/crossref_metadata_2025_split huggingface dataset:
MUNC13–4 Mutations in Patients with Hemophagocytic Lymphohistiocytosis Are Scattered over the Functional Domains of the Protein.
Authors: Santoro Alessandra , Cannella Sonia , Bossi Giovanna , Gallo Federico , Trizzino Antonino , Pende Daniela , Dieli Francesco , Bruno Giuseppa , Stinchcombe Jane C. , Putti Caterina , De Fusco Carmela , Danesino Cesare , Moretta Lorenzo , Notarangelo Luigi D. , Griffiths Gillian , Aricò Maurizio 
Published: October 2019

Abstract
Mutations of PRF1 and MUNC13–4 genes, involved with cellular cytotoxicity, are associated with the familial form of Hemophagocytic Lymphohistiocytosis (HLH) type 2 (FHL2) and 3 (FHL3). In all patients with HLH, in which PRF1 mutations had been excluded, we screened MUNC13–4 mutations.
The 32 exons and their proximal flanking regions of MUNC13–4 gene were sequenced by “cycle sequencing” approach (BigDye Terminator, Applied Biosystems).
Of 60 patients, 21 had MUNC13–4 mutations. 5 were reported mutations: 753+1G>T, 817C>T (R273X); 1389+1G>A; 1822del 12 bp (del V608-A611); 2346–9delGGAG (R782FsX12). Of 16 novel mutations 2 (2212C>T and 2650C>T) introduce a stop codon (at Q738 and Q884); 4 cause frameshift: 441delA (P147fsX14), 532delC (Q178fsX70), 3082delC (L1028fsX), and 3226insG (H1076fsX51). Of 8 missense mutations [175G>A (A59T), 419T>C (I140T), 610A>G (M204B), 1241G>T (R414L), 1847A>G (E616G), 2039G>C (R680P), 2570T>G (F857C), 2782C>T (R928C)], 6 fall within the protein functional domains; each of the 2 falling outside was associated with 2 additional pathogenic mutations.
The remaining novel 1992+5 G>A and 2448insC −12 fall outside the coding region but are close enough to induce alternative splicing. We identified polymorphisms 279C>T and 3198A>G. The 2599A>G (K867E) transition was found in several unrelated families, including one homozygous parent. To assess its frequency we studied 50 consecutive newborns, of which 64% were heterozygous.
To understand the effects of the mutations we analysed Munc13–4 protein expression from CTL and/or NK cells by western blot using an antibody raised against amino acids 1–262. Trace amounts of protein could be detected only in 5 patients. Analysis of granule polarisation in 2 patients with trace amounts of Munc13–4 protein showed many more docked granules visible than in controls, consistent with a block in granule secretion in these patients.
Median age at diagnosis of FHL3 was 6.5 months, but 8/21 (38%) patients were diagnosed when older than 5 years, with one young adult of 18 years. CNS disease was present in 10 patients; NK was markedly reduced or absent in all patients tested. MUNC13–4 mutations were found in 35% of our HLH patients non type-2. Mutations were almost entirely different from those reported so far and scattered over different exons, but in far most cases they fall within the protein functional domain. Since these patients may develop the disease during adolescence or even later on, not only pediatric but also as adults hematologists should include FHL-2 and 3 in the differential diagnosis of children and young adults with fever, cytopenia, splenomegaly and hypercytokinemia.
Interaction and Regulation of HSC with Osteopontin Is Mediated through α4β1 and α9β1Integrins, Which Are Differentially Expressed in the HSC Pool.
Authors: Haylock David N. , Whitty Genevieve A. , Williams Brenda , Storan Melonie J. , Nilsson Susie K. 
Published: October 2019

Abstract
Osteoblasts are a key cellular component of the hemopoietic stem cell (HSC) niche and directly regulate the HSC pool. Molecules synthesised by osteoblasts both promote or inhibit HSC proliferation. Osteopontin (Opn) is an osteoblast produced, RGD containing protein with roles in cell adhesion and migration. Until recently, the role of Opn in hemopoiesis was seen as restricted to the regulation of bone turnover. However, from analysis of hemopoiesis in the Opn null mouse, we have demonstrated that Opn plays a critical role in regulating the HSC pool. Furthermore Opn is critical in trans-marrow migration and lodgement of HSC within the BM after transplantation. When added to in vitro HSC cultures, exogenous thrombin-cleaved Opn also inhibits cell proliferation and potently suppresses HSC differentiation. We have now demonstrated that this interaction occurs in an RGD-independent manner via the cryptic SVVYGLR epitope revealed on the N-terminal fragment of Opn following thrombin cleavage. This epitope has previously been shown to bind to α4β1 and α9β1. HSC are known to express α4β1, but we have now shown that within the HSC pool this occurs in a differential manner, mimicking that of CD38, with more committed CD34+CD38+ cord blood progenitors having the highest levels of expression. In addition, we have shown the previously unrecognised characteristic of human marrow and cord blood HSC, the expression of α9β1, which also occurs in a differential manner, but mimicking CD34. Expression of α9β1 is highest on cord blood CD34+CD38− cells, a population highly enriched for HSC. Using the synthetic SVVYGLR peptide in culture, we re-capitulated the thrombin-cleaved Opn induced suppression of HSC differentiation in a dose dependent manner. Antibody blocking experiments demonstrated that binding to this peptide was occurring through both α4β1 and α9β1. In contrast, suppression of HSC proliferation and differentiation did not occur through the upstream alternate α4β1 binding site. Furthermore, we have now demonstrated endogenous binding of Opn to α4β1 and α9β1 to cord blood HSC in vivo. Together, these data provide strong evidence that Opn is an important component of the HSC niche which acts as a physiological negative regulator. Furthermore, our studies identify the previously unrecognised characteristic of HSC, the expression of α9β1, which together with α4β1 provides two receptors on HSC with differing expression signatures and potentially a mechanism for fine tunning the physiological effects of Opn.
Synergistic Activity of the Aurora Kinase Inhibitor MK-0457 (VX-680) with Idarubicin, Ara-C, and Inhibitors of BCR-ABL.
Authors: Hoover Russell R. , Harding Matthew W. 
Published: October 2019

Abstract
MK-0457 (VX-680) is a reversible small molecule kinase inhibitor that targets Aurora A, B, and C with Ki values of 0.7, 18, and 4.6 nM, respectively. MK-0457 also inhibits Flt3 (Ki = 30 nM), and both the wild type and the T315I mutant of BCR-ABL (Ki = 30 and 40 nM, respectively). Clinical trials are ongoing in patients with solid tumors and hematological malignancies. Recent data show that MK-0457 is active in patients against subtypes of AML, BCR-ABL T315I mutant CML, and Philadelphia positive (Ph+) ALL. To support multi-agent clinical trials, the activity of MK-0457 in combination with idarubicin, Ara-C, and BCR-ABL inhibitors was investigated. The viability of a panel of AML, ALL, and CML cell lines was assessed following single agent and either simultaneous or sequential combinations of agents. Combination effects were evaluated using the Bliss Independence Model. MK-0457 as a single agent markedly inhibited leukemia cell viability (at 72 hrs) with an IC50 range of 20–300 nM for MV4-11, Molt-4, Molm-13, K562, LAMA-84, MEG-01, and KU812F cells. Additionally, MK-0457 inhibited the viability of BaF3 cells transformed by wild type, T315I, or Y253F mutants of BCR-ABL with similar IC50s (approximately 300 nM). The sequential combination of MK-0457 followed by either idarubicin or Ara-C showed greater synergy than simultaneous combinations in a cell line dependent manner. MK-0457 displayed strong synergy in simultaneous combination with Gleevec (imatinib mesylate) in a panel of human CML-derived cell lines and BaF3 cells expressing wild type BCR-ABL. MK-0457 enhanced the Gleevec-mediated cell death of K562 leukemia cells as evidenced by increased caspase activity, PARP cleavage, and induction of the sub-G1 population. At concentrations where synergy was observed by cell viability analysis, the MK-0457/Gleevec combination resulted predominantly in aneuploidy and G2/M arrest, consistent with inhibition of Aurora kinases by MK-0457. These results support the clinical evaluation of MK-0457 combined with idarubicin and Ara-C in AML and with BCR-ABL inhibitors in CML and Ph+ ALL.
NPM-ALK Fusion Tyrosine Kinase of Anaplastic Large Cell Lymphoma Exerts Its Transforming Potential by Increasing Translation of JUNB through mTOR and S6K1.
Authors: Staber Philipp B. , Vesely Paul , Ott Rene , Haq Naznin , Linkesch Werner , Schauer Silvia , Hrzenjak Andelko , Kenner Lukas , Dirks Wilhelm , Kadin Marshall E. , Sternberg David W. , Hoefler Gerald 
Published: October 2019

Abstract
The nucleophosmin (NPM) - anaplastic lymphoma kinase (ALK) fusion protein, which is the product of the balanced chromosomal rearrangement t(2;5)(p23;q35), occurs in about 50% of nodal anaplastic large cell lymphoma (ALCL). Expression of this fusion kinase results in neoplastic transformation by modulating multiple intracellular signaling molecules, such as the PI3-kinase and the ERK1/2 kinases.
Here we show high activation of JunB in various human ALCL cell lines. Moreover, using human embryonic kidney (HEK293) and murine hematopoietic Ba/F3 cells ectopically expressing NPM-ALK, we demonstrate that NPM-ALK is the trigger for JunB activation.
Knock down of JUNB in NPM-ALK expressing cells using RNA interference results in upregulation of p16INK4a and downregulation of Cyclin D1, causing G1/S cell cycle arrest. Thus, JunB plays a critical oncogenic role in NPM-ALK transformed cells.
Using the PI3-kinase inhibitor LY294002 and the MEK-inhibitor UO126, we demonstrate that NPM-ALK-induced JunB activity as well as cellular proliferation are dependent on both PI3-kinase and MEK-ERK signaling. Moreover, we illustrate that NPM-ALK and subsequently PI3-kinase activate AKT and mTOR/S6K1 which are implicated in the translational control of specific mRNA molecules containing a polypyrimidine motif (see pathway diagram). Since JUNB mRNA harbors such a motif we confirmed that JUNB mRNA is translated in large polysomes using ribosomal gradient preparations. Selective block of mTOR with the immunosuppressant rapamycin decreases proliferation and reduces protein but not mRNA levels of JunB in human and murine NPM-ALK positive cell lines. A similar effect is seen when inhibiting the upstream activator of mTOR, PI3-kinase. In contrast, a significant decrease of JUNB mRNA levels is shown in cells treated by the MEK-inhibitor UO126.
An analogous effect of NPM-ALK is observed in ALCL patient samples. Hyperphosphorylation of S6K1 at Thr389 indicating activated mTOR/S6K1 is seen in 9/10 NPM-ALK positive ALCL cases in contrast to only 1/15 of NPM-ALK negative ALCL samples (P < 0,001). Hence, these findings suggest that the signaling cascade NPM-ALK→PI3K→AKT→mTOR/S6K1 is crucial to induce JUNB translation in human NPM-ALK positive ALCL.
In conclusion, we reveal that JunB acts as an oncogene in NPM-ALK positive neoplastic cells and therefore represents a valid therapeutic target. Our data indicate a novel mechanism for regulation of AP-1 activity in general and suggest a new therapeutic approach to specifically modulate translation of an oncogene.
Figure Figure
Biochemical and Functional Analysis of Germline KRAS Mutations That Cause Disorders of the Noonan Syndrome Spectrum.
Authors: Schubbert Suzanne , Sovik Oddmund , Bollag Gideon , Nguyen Hoa , Rowe Sara L. , Kratz Christian , Zenker Martin M. , Niemeyer Charlotte Marie , Molven Anders , Shannon Kevin 
Published: October 2019

Abstract
Noonan syndrome is characterized by short stature, facial dysmorphism, and cardiac defects. We and our colleagues discovered novel de novo germline KRAS mutations that introduce V14I, T58I, or D153V amino acid substitutions in individuals with NS and a P34R alteration in an individual with cardio-facio-cutaneous (CFC) syndrome, which has overlapping phenotypic features with NS. Recombinant V14I and T58I K-Ras proteins display defective intrinsic GTP hydrolysis and impaired responsiveness to the GTPase activating proteins (GAPs) p120 GAP and neurofibromin. We also found that V14I and T58I K-Ras render primary hematopoietic progenitors hypersensitive to growth factors and deregulate signal transduction in a cell lineage specific manner (Nature Genetics38, 331, 2006). We recently began interrogating the P34R and D153V K-Ras mutant proteins and a novel CFC-associated F156L K-Ras mutant protein. Both P34R and D153V K-Ras display normal levels of intrinsic GTP hydrolysis. In contrast, F156L K-Ras displays defective intrinsic GTP hydrolysis that resembles oncogenic G12D K-Ras. P34R K-Ras is completely resistant to both GAPs, which is intriguing as these data suggest that the ability of GAPs to down-regulate Ras-GTP levels is dispensible for development. In contrast, D153V K-Ras is responsive to both GAPs. We expressed mutant Ras proteins in COS-7 monkey kidney cells to investigate activation of Ras and downstream effectors. Cells expressing P34R, D153V, F156L, and G12D K-Ras demonstrate elevated levels of Ras-GTP and phospho-MEK in basal and serum-starved conditions. We conclude that germline KRAS mutations that cause human disease encode proteins with distinct mechanisms of biochemical activation. Further investigation of this allele series will provide insights about the relative importance of the intrinsic Ras GTPase, GAPs, and other biochemical mechanisms in controlling the growth of different cell lineages.
Regulation of NOTCH2 Signaling in B-CLL: Involvement of PKC-δ.
Authors: Hubmann Rainer , Duechler Markus , Hilgarth Martin , Schnabl Susanne , Demirtas Dita , Schwarzmeier Josef D. , Jaeger Ulrich , Shehata Medhat 
Published: October 2019

Abstract
We have previously shown that NOTCH2 signaling is involved in the overexpression of CD23 in B-cell chronic lymphocytic leukemia (B-CLL) cells (Hubmann et al., BLOOD 2002 May 15;99(10):3742–7). NOTCH2 plays a determining role in the development/homeostasis of CD5+ B1 B-cells and of the related marginal zone (MZ) B2 B-cells, suggesting a potential role for NOTCH2 in B-CLL leukemogenesis. Using electrophoretic mobility shift assays (EMSA) we demonstrate that freshly isolated B-CLL patient samples (n=30) express an activated form of nuclear NOTCH2 (N2IC) irrespective of their prognostic marker profile (ie. IgVH mutational status, CD38 surface expression, cytogenetics).
Although the majority of cultured B-CLL samples lose their N2IC activity within one day, DNA-bound N2IC complexes could be maintained by low concentrations of the PKC-stimulating phorbol esther PMA (1ng/ml). This was accompanied by the upregulation of CD23. The effect of PMA on N2IC activation and CD23 expression was abrogated by the PKC-δ inhibitor Rottlerin. Since wild type NOTCH2 signaling is regulated through binding to its ligand followed by γ-secretase mediated cleavage and release of the intracellular domain (N2IC), we induced NOTCH2 signaling by PMA in the presence or absence of the γ-secretase inhibitors (GSI) DAPT and compound E. Results demonstrated that the PMA-induced NOTCH2 activity is resistant to GSI treatment in 24 out of 30 B-CLL cases (80%). This suggests that the leukemic cells from the majority of B-CLL patients express an activated form of N2IC which is independent from γ-secretase cleavage and, thus, do not reqire NOTCH2 ligands for signaling.
In conclusion, the results suggest that PKC-δ is involved in the activation/nuclear translocation of N2IC in B-CLL cells. The data may also suggest that deregulated NOTCH2 signaling is an early step in the development of B-CLL and might be critically involved in the aberrant response of the malignant clone to cell fate modulating stimuli acting through PKC.
Lymphoid Microenvironment Inhibits Apoptosis in B-CLL Cells: Involvement of PI3-K/AKT Pathway and PTEN.
Authors: Shehata Medhat , Schnabl Susanne , Demirtas Dita , Schwarzmeier Josef D. , Hilgarth Martin , Duechler Markus , Gaiger Alexander , Hubmann Rainer , Jaeger Ulrich 
Published: October 2019

Abstract
The activation of anti-apoptotic mechanisms in the leukemic B cells in chronic lymphocytic leukemia (B-CLL) through the interaction with their microenvironment may lead to prolonged survival and the accumulation of the malignant clone. The aim of this study is to elucidate the influence of the lymphoid microenvironment in the activation of the potent anti-apoptotic PI3-K/Akt pathway and to investigate the pattern of expression of the tumor suppressor PTEN in B-CLL. Stromal fibroblasts of bone marrow (BMF), spleen (SF) and lymph gland (LGF) were used as an in vitro model for lymphoid microenvironment. Pharmacological inhibitors and siRNAs against PI3-K and Akt were applied to explore the anti-apoptotic effect of this pathway in B-CLL. The results showed that co-cultivation of B-CLL cells with human BMF, LGF, and SF significantly inhibited apoptosis and prolonged survival of the leukemic cells in comparison to suspension cultures. To explore the involvement of PI3-K/Akt pathway in the anti-apoptotic effect of stromal fibroblasts, co-cultures were performed in presence of PI3-K inhibitors (wortmannin or LY294002) or siRNAs against PI3-K and Akt1. These inhibitors significantly reduced the supportive effect of stromal fibroblasts and induced apoptosis in B-CLL cells. The leukemic cells were more sensitive to PI3-K inhibition than T cells, monocytes and stromal fibroblasts. Induction of apoptosis was associated with a significant decrease in the intracellular levels of PIP3, PI3-K, PDK1, Akt1, NF-kappa B, IKK, and dephosphorylation (activation) of PTEN. Since PTEN activity, as a negative regulator for PI3-K signalling, is controlled by its phosphorylation at the tail domain, we studied the pattern of PTEN protein expression in B-CLL. Western blotting demonstrated that the total PTEN in PBMC of B-CLL patients (n=40) is comparable to healthy individuals (n=8). However, using phosphospecific anti-PTEN antibody demonstrated that samples of B-CLL patients highly express phosphorylated (inactive) forms of PTEN in comparison to healthy persons. In conclusion, the results demonstrate that PI3-K/Akt pathway is involved in inhibition of apoptosis of B-CLL cells and suggest that interaction of the leukemic cells with lymphoid microenvironment may lead to the activation of this pathway. The data also suggest that targeting this pathway represents a feasible therapeutic approach in B-CLL.
Oncogenic NRAS, KRAS, and HRAS Induce Distinct Hematological Diseases in Mice.
Authors: Parikh Chaitali , Subrahmanyam Ramesh , Ren Ruibao 
Published: October 2019

Abstract
RAS family proteins (NRAS, HRAS and KRAS 4B/4A) are small GTPases that play a central role in transducing signals that regulate cell proliferation, survival and differentiation. The RAS proteins interact with a common set of activators and effectors and, therefore, share many biochemical and biological functions. However, the RAS proteins associate with different microdomains of the plasma membrane as well as other internal cell membranes and are capable of generating distinct signal outputs. Mutations that result in constitutive activation of RAS proteins are associated with approximately 30% of all human cancers, including 20–30% acute myeloid leukemia (AML) and 50–70% chronic myelomonocytic leukemia (CMML). Different RAS oncogenes are preferentially associated with different types of human cancer. In myeloid malignancies, NRAS mutations occur (in approximately 70% of cases), more frequently than KRAS mutations, while HRAS mutations are rare. The mechanism underlying the different frequencies of RAS isoforms mutated in myeloid leukemia is not known. One possibility is that oncogenic RAS proteins have different leukemogenic potentials. To test this possibility, we compared the ability of the three oncogenic RAS proteins to induce leukemias using the same animal model. We have shown previously that oncogenic NRAS rapidly and efficiently induces CMML- or AML-like disease in an improved mouse bone marrow transduction and transplantation model. We found here that in the same model system, oncogenic KRAS invariably induces a CMML-like disease that is similar to what has been shown in a mouse conditional knock in model for oncogenic KRAS. Surprisingly, all mice receiving oncogenic HRAS infected bone marrow cells develop an AML-like disease. The HRAS mice have the shortest disease latency, followed by NRAS and then KRAS. HRAS induced disease also appears to be more aggressive than N or KRAS and is usually accompanied by massive pulmonary infiltration and hemorrhages. These studies demonstrate that all three RAS oncogenes have the potential to induce myeloid leukemias, yet have distinct leukemogenic potentials. The underlying mechanism of the discrepancy between the frequency of HRAS mutation in human myeloid leukemia and its leukemogenic potential in mice is not clear, but the models established here provide a system for further studying the molecular mechanisms in the pathogenesis of myeloid malignancies and for testing targeted therapies.
High-Throughput Genetic Profiling of Acute Myeloid Leukemias: Novel Mutations Detected in Signal Transduction Pathway Genes by a Strategy Utilizing Endonuclease-Based Heteroduplex Scanning and DNA Sequencing.
Authors: Lilleberg Stan L. , Hempel Jill , Durocher Jeff , Foster Damien , Hartman Christopher , Stoddard Jason , Walters Brooke , Nickerson Michael L. , Koul Manika 
Published: October 2019

Abstract
The Raf/MEK/ERK, Wnt/beta-catenin, JAK/STAT and PI3K/Akt signal transduction pathways have key roles in regulation of cell cycle progression and apoptosis, and are current focal points of therapeutic development and intervention strategies for hematopoietic neoplasias. Although mutations in several pathway-associated genes known to contribute to the malignant phenotype have been described, the mutation status of genes encoding components of these pathways as a whole remains to be determined. This has yet to be achieved in even a single type of cancer. In this study, we applied a novel mutation scanning strategy (HTMS) that utilizes SURVEYOR Endouclease heteroduplex cleavage analysis of patient derived PCR products, along with selective fluorescent DNA sequencing in a combinatorial fashion to achieve high-throughput, accuracy, and sensitivity. Comprehensive screening of the protein coding portions of 33 signal transduction pathway genes was performed on PCR products from 192 AML samples and 48 controls. Target genes included receptor kinases (FLT3, KIT, CSF1R, FGFR3, NOTCH1), cytoplasmic kinases (ABL1, SRC, PIK3CA, JAK2), GTPases (KRAS, NRAS, HRAS), transcription factors (MYC, GATA1, CEBPA) and tumor suppressors (PTEN, P53). The Raf/MEK/ERK (genes: BRAF, ARAF, CRAF, PTPN11, MEK1/2, ERK1/2) and Wnt/beta-catenin (genes: APC, CTNNB1, CDH1, AXIN1, AXIN2, GSK3B, PP2A) pathways were emphasized. Using this high-throughput mutation scanning (HTMS) methodology, over 10 million base pairs from 192 cancer genomes and 48 control genomes was analyzed for somatic alterations and inherited polymorphisms in these genes. In the AML sample set, we identified 393 somatic mutations (2.0 mutations/AML), of which 54 (13.7%) have not been previously reported. These novel variants included point mutations in key functional domains of FLT3, KIT, NRAS, BRAF, ARAF, PTPN11, ABL1, FGFR3, MYC, NOTCH1, APC, CTNNB1, and GSK3B. At least one mutation was found in 100% of the AML samples. The relevance of these somatic mutations to the AML pathogenesis awaits detailed functional studies. The approach demonstrated in this study represents an effective in-depth mining strategy for high-throughput mutation analysis, compared to the standard re-sequencing approach for profiling of complex genetic diseases like cancer. The HTMS approach alleviates the current bottleneck of rigorous, manual sequence analysis that is required to identify somatic mutations. By integrating mutational profiling with comparative genomic hybridization, high-density SNP screening, and RNA profiling, many genetic changes relevant to cancer diagnosis, treatment and patient management, will surely be discovered, along with new targets for therapeutic intervention.