bluuebunny/crossref_metadata_2025_split huggingface dataset:
Complex and Novel Associations of the Guanine Exchange Factors Cool- 1 and Cool-2 with the Scaffolding Proteins GIT1 and GIT2 in Human Platelets
Authors: Greenberg Daniel L. , Spencer Alex 
Published: April 2020

Abstract
A major signaling pathway that regulates platelet shape change and reorganization of the cytoskeleton involves the Rho family of GTPases; CDC42 (fillapodia), Rac1 (lamellapodia) and RhoA (focal adhesions). These GTPases are converted from their inactive or GDP-loaded state to the active or GTP-loaded state by a class of enzymes called Guanine Exchange Factors (GEFs). GEFs are a family of multi-domain proteins that contain a GDP-GTP exchange domain (DH-PH) as well as other protein interacting domains that are regulated by the activation of receptors present on the platelet surface. We used an affinity binding technique followed by mass spectroscopy to identify novel Rho family binding GEFs in platelet lysates. Recombinant GST-Rho fusion proteins bound to agarose beads were prepared in the GTP, GDP and nucleotide-free states and incubated with clarified human platelet lysates. Platelet lysate proteins associated with the different GST-Rho preparations were eluted and run on SDS-PAGE. Gel slices were then cut out, trypsin digested and analyzed by Mass Spectroscopy. Platelet GEFs were identified by the presence of a DH-PH domain. Using this technique we found Cool-1 and Cool-2, two closely related GEFs that regulate Rac1 and CDC42 activation in nucleated cells. Cool-1 has not been previously described in platelets. Homodimeric Cool-1 specifically activates CDC42, whereas Cool-2 is capable of activating Rac1 as a homodimer or CDC42 as a monomer. The substrate specificity of Cool-2 is regulated by complex interactions with p21-activated kinases (PAK) and Gb/g proteins. In nucleated cells, Cool-1 and Cool-2 are localized to focal adhesions by the GPCR-kinase-interacting proteins 1 and 2 (GIT1 and GIT2). GIT binding to the Cool proteins has been shown to inhibit their GEF activity. Here we show that Cool-1 is present only in the membrane fraction of resting platelet lysates while Cool-2 is present in both the membrane and cytoplasmic fractions. Upon activation with thrombin, about half the membrane bound Cool-1 and all the membrane bound Cool- 2 migrate to the cytoplasm. Precipitation experiments in thrombin activated platelets with anti-GIT1 and anti-GIT2 antibodies show GIT-1 avidly binds Cool-2 whereas GIT2 binds a small amount of Cool-1. These data suggest a complex mechanism of CDC42 and Rac1 activation in platelets by the Cool proteins. Based on our findings and the current understanding of the regulation of Cool proteins in nucleated cells, we hypothesize that early after thrombin activation, the CDC42 and Rac1 GEF activities of Cool-1 and Cool-2 result in fillapodia (CDC42) and lamellapodia (Rac1) formation. As integrant mediated focal adhesions are formed, the GEF activities of Cool-1 and Cool-2 are inhibited by the scaffolding proteins GIT2 and GIT1, respectively. In this manner, Rac1 and CDC42 mediated fillapodia and lamellapodia formation may be regulated by the extent of integrin engagement and platelet spreading.
Cross Talk Between Serine/Threonine Protein Phosphatase 2A and Tyrosine Kinase Src Regulates αIIbβ3 Function
Authors: Pradhan Subhashree , Patel Vimal , Vijayan K. Vinod 
Published: April 2020

Abstract
Protein phosphatase 2A (PP2A) is a ubiquitously expressed serine/threonine phosphatase that regulates a variety of cellular functions. In the context of the platelets, we have previously shown that a pool of the catalytic subunit of PP2A (PP2Ac) associates constitutively with the resting αIIbβ3 and negatively regulates αIIbβ3 signaling. However, the mechanism by which PP2Ac controls αIIbβ3 adhesive function is incompletely understood. In this study, we demonstrated that PP2Ac expressed as a GST fusion protein interacts with the tyrosine kinase Src. Activation of Src is essential to initiate αIIbβ3 outside-in signaling. Small interference RNA mediated knockdown of endogenous PP2Acα expression in 293 cells overexpressing αIIbβ3 (293-αIIbβ3) and murine megakaryocytes, resulted in the activation of Src, as evidenced by the dephosphorylation of Src Tyr-529 and phosphorylation of Src Tyr-418. In contrast to PP2Acα, knockdown of the catalytic subunit of protein phosphatase 1 (PP1cα) did not activate Src, indicating that the regulation of Src activity by PP2Ac is specific. Dephosphorylation of Src Tyr-529 was not observed in PP2Aca depleted 293 cells treated with sulfanamido-benzbromarone compound, a selective protein tyrosine phosphatase 1 (PTP-1) inhibitor. These results suggest that inhibition of PP2Ac may activate a tyrosine phosphatase, capable of dephosphorylating Src Tyr-529. Activation of Src in PP2Ac depleted 293-αIIbβ3 cells had functional consequences for integrin αIIbβ3. PP2Ac depleted 293-αIIbβ3 cells exhibited ~2 fold increased adhesion to immobilized fibrinogen. Inhibition of Src kinase with a pharmacological agent PP2 but not by PP3 an inactive analogue of PP2, abolished the increased adhesiveness of PP2Acα–depleted 293 cells to fibrinogen. Finally, the increased activation of extracellular signal-regulated kinase (ERK1/2) in PP2Acα-depleted cells that we previously demonstrated was also blocked by Src inhibitor PP2 but not by PP3. These results indicate that both Src and ERK1/2 are activated in response to PP2Ac inhibition, with activation of Src being upstream of ERK1/2. These studies illustrate that inhibition of PP2Ac promotes αIIbβ3 adhesiveness by activating Src, and imply that the control of αIIbβ3 adhesive function can be further fine-tuned by a cross talk between the serine/threonine phosphatase PP2A and the tyrosine kinase Src.
Liar, a Novel Lyn-Binding Nuclear/Cytoplasmic Shuttling Protein That Influences Erythropoietin-Induced Differentiation
Authors: Samuels Amy L , Klinken Svend Peter , Ingley Evan 
Published: April 2020

Abstract
Erythropoiesis is primarily controlled by erythropoietin (Epo), which stimulates proliferation, differentiation and survival of erythroid precursors. We have previously shown that the tyrosine kinase Lyn is critical for transducing differentiation signals emanating from the activated Epo receptor. A yeast two-hybrid screen for downstream effectors of Lyn identified a novel protein, Liar (Lyn interacting ankyrin repeat), which forms a multi-protein complex with Lyn and HS1 in erythroid cells. Interestingly, the ankyrin repeats of Liar define a novel SH3 binding region for Lyn and HS1. Liar also contains functional nuclear localisation and nuclear export sequences, and shuttles rapidly between the nucleus and cytoplasm. Ectopic expression of Liar inhibited the differentiation of normal erythroid progenitors, as well as immortalised erythroid cells. Significantly, Liar affected Epo-activated signaling molecules including Erk2, STAT5 and Lyn. These results show that Liar is a novel Lyn-interacting molecule that plays an important role in regulating intracellular signaling events associated with erythroid terminal differentiation.
M-CSF Preferentially Induces C-Fos Via ERK to Specify Monopoiesis Whereas G-CSF Directs Granulopoiesis Via SHP2
Authors: Jack Graham D. , Friedman Alan D. 
Published: April 2020

Abstract
Hematopoietic cytokines stimulate proliferation and survival, but their role in lineage commitment remains uncertain. To gain insight into the contribution of cytokine signaling to myeloid lineage specification, we compared G-CSF and M-CSF signaling in primary murine, lineage-negative marrow cells. G-CSF specifically activated STAT3 and induced increased SHP2 phosphorylation, whereas M-CSF preferentially activated PLC-γ and ERK and induced c-Fos in marrow progenitors. In contrast, phosphorylation of Jun kinase or c-Jun was similar in response to both cytokines, as was induction of PU.1. Inhibition of ERK by either of two chemical inhibitors prevented c-Fos induction and interfered with monocyte lineage specification, as assessed by enumerating monocytic versus granulocytic colony-forming units in methylcellulose culture with IL3/IL6/SCF. ERK inhibition also reduced C/EBPα21 phosphorylation, as seen previously in cell lines. M-CSF induced C/EBPαS21 modification more effectively than G-CSF, but the effect of this modification on C/EBPα activities and its role in myeloid lineage choice remains uncertain. Consistent with a role for ERK in monocyte specification, phorbol ester potently activated ERK and induced monocytic colonies in IL3/IL6/SCF or in GCSF/SCF methylcellulose culture. SHP2 inhibition reduced ERK activation by G-CSF but not by M-CSF and reduced granulopoiesis but not monopoiesis in CFU assays with IL3/IL6/SCF, indicating that an ERK-independent target of SHP2 helps specify granulopoiesis. Phorbol ester induction of ERK and monopoiesis may reflect preferential activation of PLC-γ by M-CSF compared to G-CSF, as we observed. In summary, M-CSF signals activate ERK more potently than G-CSF signals and thereby induce c-Fos to favor monopoiesis, whereas G-CSF signals activate the SHP2 tyrosine phosphatase, shifting the balance to granulopoiesis, via effects of SHP2 on additional regulators besides ERK. How cytokine signals cooperate with transcription factors to specify lineage determination is a fundamental issue in hematopoiesis, and elucidating these functional interactions will facilitate efforts to expand normal myeloid cells and to use cytokines or downstream effectors to induce malignant cells to differentiate.
Differential Effects of a Novel Non-Peptidic Thrombopoietin Mimetic on Proliferation and Differentiation of Human CD34+ Progenitor Cells
Authors: Punnonen Juha , Muench Marcus O. , Spencer Jeffrey R. 
Published: April 2020

Abstract
Thrombopoietin (TPO) and TPO mimetics have been shown to be beneficial in the treatment of thrombocytopenia. Low molecular weight, orally available compounds offer the advantage of convenience and lack of immunogenicity when compared to protein-based drugs. STS-T4 is a novel low-molecular weight (&lt;500 Da), non-peptidic TPO mimetic compound that is a 2nd generation agent in preclinical development with promising in vitro potency and aqueous solubility. We have studied the effects of STS-T4 on the growth and differentiation of primary human CD34+ progenitor cells. A dose-dependent proliferation of highly purified human CD34+ cells was observed with EC50 value of less than 1 μM. A bell-shaped dose-response curve was observed, which is consistent with previous reports on compounds stimulating homodimeric cytokine receptors. In addition, the phenotype of the cultured cells was analyzed by flow cytometry and monoclonal antibodies specific for markers of megakaryocyte differentiation. During a culture period of 10 days, 85% of the cells cultured in the presence of STS-T4 differentiated into CD41+ megakaryocytes in the absence of any other cytokines or mimetics. In addition, 30% of these CD41+ cells coexpressed CD42b as a marker of more mature cells. When compared to recombinant human TPO (rhTPO), a similar proportion of the cultured cells expressed CD41 and CD42b in response to STS-T4 and the levels of expression of these antigens on the surface of the megakaryocytes were similar, suggesting that the effects of STS-T4 and rhTPO on human megakaryocyte differentiation are comparable. However, the level of cell proliferation induced by STS-T4 in cultures of CD34+ cells was 50–60% of that induced by rhTPO, supporting the conclusion that the signals mediating proliferation and differentiation of CD34+ progenitor cells are differentially regulated. In addition to the activity profile, the physical properties of STS-T4 are desirable for further development. Aqueous solubility and gastrointestinal permeability are major contributors to oral absorption, and high solubility and permeability generally also reduce the risk of food effects. The aqueous solubility of STS-T4 was measured by a kinetic method with HPLC and visual detection and was determined to be &gt;1 mM. These data suggest that further evaluation of safety and efficacy of STS-T4 for the treatment of thrombocytopenia is warranted. In addition, the results imply that CD34+ progenitor cells can differentiate into megakaryocytes expressing high levels of CD41 and CD42b in the absence of a full rhTPO-like proliferative response, which may support further investigation of new therapies for the treatment of thrombocytopenia that provide the benefit of megakaryocyte maturation while avoiding excessive expansion of hematopoietic stem cells.
Ligand-Independent MPLW515L Activity Requires Cell Surface Localization
Authors: Marty Caroline , Chaligné Ronan , Lacout Catherine , Constantinescu Stefan N. , Vainchenker William , Villeval Jean-Luc 
Published: April 2020

Abstract
The activating mutation W515L in the thrombopoietin receptor Mpl has been identified in a minority of primitive myelofibrosis and essential thrombocythemia negative for the JAK2V617F mutation. Mpl and the erythropoietin receptor EpoR belong to a distinct subset of the cytokine receptor superfamily. Mpl and EpoR lack intrinsic kinase activity and rely on the activation of the cytoplasmic janus tyrosine kinases (JAKs) for signal transduction. Recently, it was shown that EpoR dimerizes in a ligand-independent fashion in the endoplasmic reticulum (ER), where it associates with JAK2. Normal receptor signaling occurs at the cell surface after ligand binding, believed to trigger changes in the dimeric receptor conformation allowing for JAK2 trans-phosphorylation and activation. Based on numerous functional and structural similarities between Mpl and EpoR, we examined the possible occurrence of abnormal intracellular signaling concerning the endogenous ligand-independent MplW515L that might be involved in myeloproliferative disorder pathogenesis. The murine factor-dependent FDC-P1 cell line was transduced with human HA-tagged MplW515L modified with the ER retention KDEL sequence, in order to prevent its cell surface expression, or with the human HA-tagged MplW515L, as a positive control. We isolated cells expressing either the normally-processed Mpl mutant or MplW515L locked within its natural ER/Golgi maturation pathway. MplW515L-KDEL was unable to support autonomous growth of the cytokine-dependent FDC-P1 cells. Co-immunoprecipitation analysis showed that MplW515L-KDEL associated with JAK2 in the ER without activating the kinase. Indeed, MplW515L-KDEL-expressing cells lacked both constitutive and TPO-dependent phosphorylation of STAT5, ERK1/2 and AKT signaling molecules. Moreover, FDC-P1 cells expressing the ER-retained MplW515L became tumorigenic in nude mice only after in vivo selection of cells leaking the receptor at their surface. Finally, covalent disulfide modification (S402C) of MplW515L-KDEL, resulting in homodimerization of the receptor led to autonomous FDC-P1 cell growth. MplW515L-S402C-KDEL exhibited enhanced cell surface localization, suggesting that homodimerization overrided the ER/Golgi retention induced by the KDEL motif. Our results indicated that forced dimerization might promote cell surface localization. We propose that MplW515L is bound to JAK2 intracellularly in the ER/Golgi in an inactive conformation. In order to induce ligand-independent phosphorylation and activation of JAK2 and consequently downstream signaling, the MplW515L mutant receptor strictly needs to reach the cell surface to probably acquire a dimeric conformation.
Combination of the hyperCVAD Regimen with Dasatinib Is Effective in Patients with Relapsed Philadelphia Chromosome (Ph) Positive Acute Lymphoblastic Leukemia (ALL) and Lymphoid Blast Phase Chronic Meyloid Leukemia (CML-LB)
Authors: Jabbour Elias , O’Brien Susan , Thomas Deborah A. , Faderl Stefan , Koller Charles Asa , Dara Samuel , Garris Rebecca , Cortes Jorge , Kantarjian Hagop M , Ravandi Farhad 
Published: April 2020

Abstract
The dual Src and Abl inhibitor dasatinib is ~325 times more potent in vitro against BCR-ABL kinase activity and has significant clinical activity in patients with imatinibresistant lymphoid blast phase CML (CML-LB) and Ph+ ALL. In this study, patients with relapsed Ph+ ALL or CML-LB received dasatinib 50 mg po bid (or 100 mg daily) for the first 14 days of each of 8 cycles of alternating hyperCVAD and high dose cytarabine and methotrexate. Patients in CR continued to receive dasatinib 50 mg po bid (or 100 mg daily) and vincristine and prednisone monthly for 2 years followed by dasatinib indefinitely. To date 14 patients with relapsed Ph+ ALL or CML-LB have received a median of 3 cycles (range 1–8); 1 patient in complete remission (CR) is receiving maintenance therapy. Median age was 43 years (range 21–69); 4 (29%) patients were older than 50 years. Median WBC at start of treatment was 6.8 × 109/L (range, 0.4 – 27.2 × 109/L). Median blast percentage was 45% (range 0–96%) Three patients had CNS involvement. Median number of previous therapies was 1 (range, 1–4) including hyperCVAD and imatinib, tyrosine kinase inhibitor monotherapy, investigational agents and transplantation. All patients were evaluable for response. 10 patients (71%) achieved CR and 4 patients (29%) achieved CR with incomplete platelet recovery (CRp). Twelve of 14 (86%) patients achieved a major cytogenetic response, complete in 11, 1 (7%) had insufficient metaphases, and 1 (7%) had no response. Overall, 9 (64%) patients had achieved a major molecular response, complete in 5. Median time to neutrophil and platelet recovery during the first course was 18 and 22 days and 18 and 26 days, respectively after subsequent courses. Four patients relapsed after median response duration of 19 weeks (range, 9–38); two of them had acquired ABL kinase domain mutations: One patient lost his baseline mutation, Y253H, and acquired T315I and E450G; the second lost his 3 baseline mutations, Y253H, F359V, and E459K, and acquired T315I.Two patients received an allogeneic transplantation and one patient received a donor lymphocyte infusion. Grade 3 and 4 toxicities included 7 episodes of bleeding (4 GI, 1 GU, and 2 subdural hematomas), 4 episodes of pleural effusions, 2 episodes of pericardial effusions, infections, diarrhea, hypophosphatemia, hypocalcemia, elevated transaminases, and hyperbilirubinemia. With a median follow up of 6 months (range, 1–13 months), 9 patients are alive; 7 in CR/CRp. Two patients died after disease relapse, 1 died post transplant and 2 died in CR/CRp from infections. In conclusion, the hyperCVAD regimen with dasatinib is feasible and effective in patients with relapsed Phpositive ALL and CML-LB.
Stapled Peptides: Leveraging the BH3 α-Helix to Create a New Class of Drugs to Treat Hematological Malignancies
Authors: Kapeller Rosana , Han Jiawen , Sun Kaiming , Gangurde Pranoti , Kawahata Nori , Iadanza Matt , Guerlevais Vincent , Horstick Jim , Noehre Justin , Annis Allen , Licklider Larry , Nash Huw M. , Kung Andrew , Sawyer Tomi K. 
Published: April 2020

Abstract
AILERON is developing a selective approach to restore programmed cell death in cancer cells by borrowing from nature the BH3 domain alpha-helical “keys” from pro-apoptotic members of the BCL-2 family that fit into the “locks” of other anti- and pro-apoptotic BCL-2 proteins. AILERON has applied its proprietary chemical strategy termed hydrocarbon stapling (J. Am. Chem. Soc., 2000 122:5891) to generate cell-permeable BH3 stapled peptides that modulate the intracellular protein-protein interactions of BCL-2 family members and selectively kill cancer cells both in vitro and in vivo (Science, 2004 305:1466). The lead compounds possess the innate characteristics of the endogenous peptides they mimic, including mechanism of action and target specificity. BID BH3 and BIM BH3 stapled peptides have recently been shown to directly activate the pro-death molecule BAX, a unique property that differentiates BH3 stapled peptides from all BCL-2 small molecule antagonists (Mol. Cell, 2006 24:199). BID BH3 and BIM BH3 stapled peptides were tested for their ability to induce programmed cell death in a panel of 12 lymphoid-derived tumor cell lines. T-ALL derived cell lines were the most sensitive to the compounds, followed by multiple myeloma lines. CML-derived cell lines were the least sensitive. Resting human peripheral blood lymphocytes (hPBLs) and normal human embryonic lung fibroblasts (WI-38) were found to be resistant to BH3 stapled peptides, indicating that the compound may target only cells that are “primed to die” and not normal “resting” cells. The anti-cancer activity of BH3 stapled peptides was further investigated in orthotopic xenograft models and shown to dramatically suppress tumor growth. In a mixed lineage leukemia model (SEMK2), a tumor over control (T/C) of 27% was observed after 13 days of treatment at 30 mg/Kg IV q.d. We also investigated whether BH3 stapled peptides elicit an antibody response in rodents. No antibody titer was detected, indicating that BH3 stapled peptides are non-antigenic in rodents. While peptides are oftentimes unstable in vivo, with half-lives typically in the range of a few minutes, BH3 stapled peptides were 100% stable in both mouse and human plasma ex vivo, and exhibited excellent PK profiles in rats with half-lives greater than three hours. BH3 stapled peptides were well tolerated in all animal models tested to date. In conclusion, we show that BH3 stapled peptides exhibit promising pharmacological properties and represent a novel class of drugs for the treatment of hematological malignancies.