bluuebunny/crossref_metadata_2025 huggingface dataset:
Transgenic Mouse Modeling of Therapeutic Responses in Myeloma
Authors: Van Ness Brian G. , Ramos Christine , Gosse Mary
Published: April 2020

While myeloma is still considered a fatal disease, new therapeutic approaches, including combination of drugs that target different intracellular pathways, have shown marked improvements in response and survival. Yet there are wide variations in responses seen in the patient population. One major source is the variation in chromosomal abnormalities and the genes deregulated among the plasma cell (PC) tumors. Another may be in inherited variations (SNPs) that can have a major influence on drug distribution, transport and metabolism. Our laboratory has developed a transgenic mouse model targeting overexpression of c-myc and bcl-xL in late B cells, that results in early expansion of non-malignant PCs, followed by development of clonal plasma cell malignancies in 100% of the mice. We have reported on cell surface, chromosomal abnormalities, and gene expression profiles of the mouse plasma cell tumors, that demonstrate a very similar profile and heterogeneity to human myelomas. The mouse PC tumors can be adoptively transferred to syngenic recipient mice, as well as grown in culture. The advantage of this model is that the impact of tumor heterogeneity can be assessed in a common germline genetic background in the mice; and an intact immune system is maintained, unlike many common xenograft models. We find that disease progression is variable among tumors and have extended this observed heterogeneity of progression to analysis of drug sensitivity; for example, identifying tumors that are highly sensitive to dexamethasone, and others extremely resistant, and others that show protection in the presence of IL-6. Although bcl-xL expression is important in the initiation of the transgenic malignancy, it was not clear if Bcl-xL was required to maintain the transformed phenotype. To test this, we blocked Bcl-xL with the inhibitor BH3I-2′(Calbiochem). We find that inhibition of Bcl-xL is effective in killing the PC tumors in vitro, and dose is directly related to levels of bcl-xL expression. We also examined various dose combinations of dexamethasone and BH3I-2′ and found an antagonistic relationship of the two drugs. We are in the process of expanding the tumor mouse population to target other therapeutic approaches in the transgenic mouse that models tumor heterogeneity in a common genetic background.
Opposing Effects of Dexamethasone on Lenalidomide Activity in Multiple Myeloma: Additive/Synergistic Effects on Anti-Proliferative Activity on Myeloma Cells and Antagonistic Effects on Immune Function
Authors: Schafer Peter H , Gandhi Anita K , Zhang Ling-Hua , Kang Jian , Capone Lori , Parton Stacey , Wu Lei , Bartlett Blake
Published: April 2020

Lenalidomide (Revlimid®) is approved for the treatment of previously treated multiple myeloma (MM) in combination with dexamethasone. Single agent lenalidomide has direct antiproliferative activity against MM cells and direct immunomodulatory activity via costimulation of T cell and natural killer (NK) cell responses. The ECOG Phase III trial in newly diagnosed MM (E4A03) reported that low dose (40 mg/d on days 1, 8, 15 & 22 of a 28-day cycle) dexamethasone in combination with lenalidomide showed an overall survival advantage when compared with the higher, standard-dose (40 mg/d on days 1-,4, 9–12 & 17–20 of a 28-day cycle) of dexamethasone that is used in combination with lenalidomide. We have examined the pharmacological activity of lenalidomide in combination with dexamethasone with respect to direct MM antiproliferative and T/NK cell stimulation in vitro. Antiproliferative activity against various MM cell lines was associated with increased expression of tumor suppressor genes, including Egr1, Egr3, p21, and p27. The specific tumor suppressor genes induced varied among the MM cell lines, even among those that were sensitive to lenalidomide. Lenalidomide and dexamethasone induced cell cycle arrest in G0/G1 phase, activation of caspases 3/7, and apoptosis in the sensitive cell lines. The combination of lenalidomide and dexamethasone was partially additive in most MM cell lines, while the most dramatic combination effect observed was induction of p21 gene expression and apoptosis in LP-1 MM cells. In contrast to its synergistic effect on MM cell proliferation, dexamethasone was strongly antagonistic to the T and NK cell costimulatory effects of lenalidomide. Specifically, in primary human T cells dexamethasone inhibited lenalidomide-enhanced IL-2 production. Furthermore, in primary human NK cells dexamethasone inhibited lenalidomide-enhanced IFN-γ production. These findings suggest that the lenalidomide plus low-dose dexamethasone regimen may be able to facilitate the MM cell killing via induction of tumor suppressor gene expression, cell cycle arrest, and apoptosis, while allowing lenalidomide to enhance T and NK cell function to a greater magnitude. The enhanced survival with the low-dose dexamethasone schedule in MM patients suggests exploring further dosing schedules of dexamethasone in combination with lenalidomide in this population, with measurement of biomarkers of T and NK cell function.
Targeting PKC: A Novel Role for Beta-catenin in ER Stress and Apoptotic Signaling
Authors: Raab Marc S , Breitkreutz Iris , Tonon Giovanni , Zhang Jing , Fruehauf Johannes , Lin Boris K , Chauhan Dharminder , Hideshima Teru , Munshi Nikhil , Anderson Kenneth C. , Podar Klaus
Published: April 2020

Targeting protein kinase C (PKC) isoforms by the small molecule inhibitor enzastaurin has shown promising pre-clinical activity in a wide range of tumor cells. In this study, we further delineated its mechanism of action in multiple myeloma (MM) cells and found a novel role of b-catenin in regulating growth and survival of tumor cells. Inhibition of PKC leads to rapid accumulation of b-catenin by preventing the phosphorylation required for its proteasomal degradation. Specifically, b-catenin was dephosphorylated at Ser33,37,41 and accumulated in a dose- and time-dependent manner in all cell lines tested (including primary MM cells and 10 MM cell lines, 3 colon cancer, HeLa, as well as HEK 293 cells). Microarray analysis and siRNA-mediated gene silencing in MM cells revealed that accumulated b-catenin activates early ER stress signaling via eIF2a, CHOP and p21, leading to immediate inhibition of proliferation. Conversely, knock-down of components of the ER stress response pathway by siRNA (i.e., CHOP) abrogated the inhibitory effect of enzastaurin on MM cell proliferation. Importantly, accumulated b-catenin also contributes to enzastaurin-induced cell death, since inhibition of b-catenin by siRNA partially rescued HeLa, HEK 293, and MM cells from cell death induced by enzastaurin. Analysis of downstream target molecules revealed a b-catenin -dependent up-regulation of c-Jun, but not of c-Myc or Cyclin D1. c-Jun has been reported to stabilize p73, a pro-apoptotic p53-family member; b-catenin induction by enzastaurin led to p73 (but not p53) activation, which was also abrogated by b-catenin -specific siRNA. In turn, specific knockdown of p73 by siRNA rescued cells from enzastaurin-induced apoptosis. Finally, ectopic overexpression of b-catenin in HeLa and MM cells induced c-Jun expression and p73 activation, followed by apoptotic cell death. In summary, our data reveal a novel role of b-catenin in ER stress-mediated growth inhibition and a new pro-apoptotic mechanism triggered by b-catenin upon inhibition of PKC isoforms, and further demonstrate that p73 represents a novel therapeutic target in MM. Based on these and previous data, enzastaurin is currently under clinical investigation in a variety of hematologic malignancies including MM.
Phase I Study of Elotuzumab (HuLuc63) in Phase I Study of Elotuzumab (HuLuc63) in Relapsed/Refractory Multiple Myeloma
Authors: Zonder Jeffrey A. , Singhal Seema , Bensinger William , Mohrbacher Ann , Hussein Mohamad A. , Munshi Nikhil C. , Caras Ingrid , Singhal Anil , van Rhee Frits
Published: April 2020

Elotuzumab is a humanized monoclonal antibody directed against CS1, a cell surface glycoprotein, which is highly and uniformly expressed in multiple myeloma. Elotuzumab has demonstrated significant anti-tumor activity in pre-clinical mouse models of multiple myeloma (MM). The primary objective of this Phase I study is to evaluate the maximum tolerated dose (MTD) of elotuzumab in patients with relapsed/refractory MM. The study also evaluates the overall safety, pharmacokinetics (PK), biologic activity and clinical response to elotuzumab. Elotuzumab is administered every 2 weeks × 4 doses in six escalating dose cohorts ranging from 0.5 mg/kg to 20 mg/kg. An additional 4 doses may be administered if the patient demonstrates at least stable disease during the initial 4 doses of therapy.

To date, a total of 23 patients, 52% male, with a median age of 64 years and a median of seven prior treatments, have been treated in the six cohorts. In the 2.5 mg/kg cohort, one out of six patients experienced a DLT of Grade 4 increase in serum creatinine which was concomitant to an unrelated serious adverse event (SAE) of sepsis. Four additional elotuzumab-related SAEs in two patients (Grade 2 chills and fever in one subject in the 2.5 mg/kg cohort, Grade 2 chest pressure and bradycardia in another subject in the 10 mg/kg cohort, all requiring overnight hospitalization) have been observed. However, the MTD for elotuzumab has not been reached up to the 10 mg/kg dose. Several patients (10/16) experienced Grade 1 and 2 AEs occurring during or immediately after the first dose including chills, flushing, pyrexia, rigors, dyspnea and fatigue. An increase in serum levels of a number of cytokines such as interferon-gamma and TNF-alpha, and chemokines including IP10 and MCP1 was observed for all patients following the first dose of elotuzumab, peaking between 2–4h after the end of infusion and returning to near baseline levels by 24h. These infusion-related symptoms were not observed during the subsequent dosing of elotuzumab, possibly due to premedication with antihistamines and acetaminophen. Preliminary PK analysis suggests a short serum half-life of 2–4 days at low doses of elotuzumab (0.5–2.5 mg/kg), with an increase to 10–11 days at higher doses (5–10 mg/kg). Cmax levels of 53–114 mcg/mL and 291–384 mcg/mL were observed in the 5 mg/kg and 10 mg/kg cohorts, respectively. Following the completion of 4 doses of elotuzumab, 6 patients demonstrated stable disease, four of these at 5 mg/kg or higher dose levels, according to the EBMT response criteria.

In summary, elotuzumab appears to have a manageable safety profile in patients with relapsed/refractory MM. MTD for elotuzumab has not been reached up to the 10 mg/kg dose. Higher doses of elotuzumab lead to a significant increase in Cmax and half-life. Evaluation of the 20 mg/kg dose cohort is ongoing.
Mef2C Is a Lineage-Restricted Target Gene of Scl/Tal1 and Regulates Megakaryopoiesis and B-Cell Homeostasis
Authors: Rhodes Katrin E , Gekas Christos , Gereige Laurraine , Helgadottir Hildur , Ferrari Roberto , Kurdistani Siavash , Montecino-Rodriguez Encarnacion , Basel-Duby Rhonda , Olson Eric , Orkin Stuart H. , Pellegrini Matteo , Mikkola Hanna
Published: April 2020

The bHLH transcription factor stem cell leukemia/T-cell acute leukemia gene (Scl/Tal1) is a master regulator for hematopoiesis, essential for hematopoietic specification and proper differentiation of the erythroid and megakaryocyte lineages. However, the critical downstream targets of Scl remain undefined. To identify Scl target genes in hematopoietic cells, we performed gene expression analysis on HOX11-immortalized Sclfl/fl fetal liver cell lines. Analysis of the top 50 downregulated genes revealed several genes related to hematopoiesis including erythroid and megakaryocyte development, vasculogenesis, as well as genes/unknown ESTs that have not been previously linked to blood development. One of the top downregulated genes was transcription factor myocyte enhancer factor 2C (Mef2C). Mef2C−/− embryos die at E9.5, the same time as Scl−/− embryos, and exhibit severe defects in cardiac and muscle development. Analysis of Mef2C−/− embryos showed that, Mef2C, in contrast to Scl, is not required for specification into primitive or definitive hematopoietic lineages. To bypass the embryonic lethality, we utilized a conditionally targeted Mef2Cfl/fl strain and crossed it with a hematopoietic cell-specific VavCre strain that deactivates Mef2C shortly after the emergence of HSCs. Interestingly, adult VavCre+Mef2Cfl/fl mice exhibited severe platelet defects highly reminiscent to those observed in Scl deficient mice. The platelet counts were reduced, while platelet size was increased and the platelet shape and granularity was altered. Furthermore, megakaryopoiesis was severely impaired in vitro. ChIP-on-chip analysis revealed that Mef2C is directly regulated by Scl in megakaryocytic cells, but not in erythroid cells. In addition, an Scl independent requirement for Mef2C in B-lymphoid homeostasis was observed in Mef2C-deficient mice, characterized as severe age-dependent reductions of specific B-cell progenitor populations reminiscent of premature aging. In summary, this work identifies Mef2C as an integral member of hematopoietic transcription factors with distinct upstream regulatory mechanisms and functional requirements in megakaryocyte and B-lymphoid lineages.
Differentiation-Dependent Interactions Between Runx-1 and Fli-1 during Megakaryocyte Development
Authors: Huang Hui , Yu Ming , Moran Tyler B , Tu Nathan , Akie Thomas E , Cantor Alan B
Published: April 2020

The transcription factor Runx-1 is required for the ontogeny of all definitive hematopoiesis, and plays a specific role in megakaryopoiesis during later stages of development. Germline mutations in Runx-1 cause Familial Platelet Disorder with Propensity to Develop AML (FPD/AML), and acquired mutations occur in a subset of patients with myelodysplastic syndrome (MDS) and acute myelogenous leukemia. Although many of the reported Runx-1 mutations affect DNA binding and/or its interaction with the cofactor CBF-beta, other mutations occur outside of these binding regions and have unknown mechanistic effects. In this study, we purified Runx-1 containing multiprotein complexes from murine megakaryocytic cells in order to identify potential novel Runx-1 associated factors whose interaction may be altered by Runx-1 mutations. Here we report the identification of the key megakaryocyte ets transcription factor Fli- 1 as a direct Runx-1 binding partner. This interaction involves the negative regulatory DNA binding and activation domains of Runx-1 (amino acids 179–370), and a region around the Ets DNA binding region of Fli-1 (amino acids 281–361). The interaction is lost in the MDS- associated Y254X Runx-1 mutation. We also show that Runx-1 and Fli-1 co-occupy the c-mpl promoter in primary megakaryocytes and act synergistically in transcriptional reporter assays. Interestingly, the interaction between Runx-1 and Fli- 1 occurs in murine L8057 megakaryoblastic cells only after they have been induced to differentiate, even though both proteins are expressed abundantly in uninduced cells. The interaction correlates with assembly of a large multiprotein complex that also includes the key megakaryocyte transcription factor GATA-1 and its cofactor Friend of GATA-1 (FOG- 1) based on gel filtration chromatography experiments. Furthermore, we show that Fli-1 from this large complex lacks phosphorylation of a specific residue that is phosphorylated on non-complexed Fli-1. Mutation of this site to aspartic acid, which mimics constitutive phosphorylation, disrupts the interaction between Fli-1 and Runx-1 and abrogates their transcriptional synergy. We propose that dephosphorylation of Fli-1 is a key event in the transcriptional activation of megakaryocyte terminal maturation by facilitating the assembly of a RUNX-1/FLI-1/GATA-1/FOG-1 enhancesome complex. These findings have implications for the differentiation of other cell types where interactions between Runx and ets family proteins occur.
Elevated Procoagulant Microparticles Expressing Endothelial and Platelet Markers in Essential Thrombocythemia
Authors: Trappenburg Marijke , van Schilfgaarde Muriel , Marchetti Marina , Spronk Henri , ten Cate Hugo , Leyte Anja , Terpstra Wim , Falanga Anna
Published: April 2020

Background: Most cell types, including blood - and vascular cells, produce microparticles (MPs) upon activation. Since cellular MPs are known to be elevated in thromboembolic diseases, we hypothesized a role for MPs in the pathogenesis of thrombosis in Essential Thrombocythemia (ET).

Design and methods: In plasma samples from 21 ET patients and 10 healthy subjects, the levels and the cellular origin of MPs were determined by flowcytometric analysis, while the MP-associated procoagulant activity was measured by the thrombin generation assay.

Results: ET patients had significantly higher numbers of circulating AnnexinV-positive MPs than controls (median 4500 vs 2500×106 events/L; p=0.039), including significantly higher number of MPs positive for the platelet marker CD61 (median 4000 vs 2400×106/L; p=0.043) the endothelial marker CD62E (median 875 vs 14×106/L; p=0.009), and for Tissue Factor (median 1.8 vs 0.9×106/L; p=0.036). CD62E was co-expressed with the platelet marker CD41 on MPs, suggesting a bilineal origin of such MPs, that were observed only in patients with risk factors for thrombosis. ET patients had higher plasma mature von Willebrand factor (vWF) levels (median 50 vs 35 nM, p=0.045) but similar propeptide levels (median 7 vs 5 nM, p=0,07) compared to controls, indicating chronic endothelial activation. In thrombin generation analyses, MP rich plasma from ET patients had a shorter lag time (9.7 min, 95%CI: 8.7–10.7 versus 15.9 min, 95%CI: 10.9–20.9, p=0.001) and higher peak height (215 nM, 95%CI: 189–241 versus 142 nM, 95%CI: 87–189, p=0.038) than from controls. Peak height correlated significantly with the total number of MPs (R=0.634, p<0.001).

Conclusions: ET patients showed higher number of circulating MPs with platelet and endothelial markers, suggesting ongoing platelet and endothelial activation. This is confirmed by an increased mature vWF level and an abnormal mature vWF/propeptide ratio, and a hypercoagulable state reflected in thrombin generation. These findings suggest a role for MPs in thrombosis in ET.
Allogeneic Hematopoietic Cell Transplants in Patients with Myelofibrosis Age 60 and Older
Authors: Samuelson Scott James , Sandmaier Brenda M. , Heslop Helen E. , Popat U. , Boyer Michael , Hickman Kimberly , Prchal Josef T. , Deeg H. Joachim
Published: April 2020

Allogeneic Hematopoietic Cell Transplants in Patients with Myelofibrosis ≥ 60 years of age. Primary myelofibrosis (PMF) and myelofibrosis arising from polycythemia vera (PV) and essential thrombocythemia (ET) are challenging clinical entities and many patients have a very poor prognosis. For the most part, treatment is palliative and often of limited efficacy. Allogeneic hematopoietic cell transplants (HCT) can effectively control the disease, and reduced intensity conditioning regimens have reduced regimen related complications. However, many studies have excluded patients older than age 65 (or even 60) years. This is problematic as the median age at diagnosis is approximately 67. We retrospectively evaluated a cohort of 26 patients age ≥ 60 years with PMF or post PV or ET myelofibrosis who underwent allogeneic HCT between 1999 and 2007. The median age was 63 (range 60–75) years; 9 of these patients were older than 65 (range 66–75) years. Donors were HLA-matched siblings (n=14) or unrelated (n=12). Conditioning regimens included Fludarabine (Flu)/2 Gy TBI (n=16), Flu/Campath(Cam)/TBI (n=2), Flu/Melphalan/ATG (n=2), Cam/Flu/CD45Mab/4.5 Gy TBI (n=2), Flu/Busulfan (Bu) (n=2), Flu/Bu/ATG (n=1), Flu/Mel/Cam (n=1). The median follow up for the cohort was 200 (range 17 – 1322) days. Eleven of 26 patients were alive at the date of last followup, and 9 were relapse free. The median follow up for living patients was 251 (range 100 – 1322) days. Of the 15 patients who had died, 5 died of disease progression and the remaining 10 of non-relapse causes: infection (n=4), GVHD (n=2), respiratory failure (n=2), multi-organ failure (n=1), dissecting aneurysm (n=1). At last follow up, 4 of 9 patients age >65 were alive a median of 374 (range 132 – 896) days without disease relapse. Five of the nine patients died, 4 from non-relapse causes and one with disease progression. In summary, reduced intensity conditioning and allogeneic transplantation in patients with myelofibrosis more than 60 years of age resulted in relapse-free survival of 40–45%. While the mortality rate was high, the success rate was remarkable in view of the patient age and the frequent presence of co-morbid conditions. We suggest, therefore, that ongoing and future clinical trials of allogeneic HCT for myelofibrosis should allow enrollment of older patients.
Gene Expression Profiling Reveals a Crucial Role for C/EBPbeta in Proliferation Pathways of ALK+ ALCL Cell Lines
Authors: Bonzheim Irina , Irmler Martin , Anastasov Natasa , Klier Margit , Beckers Johannes , Pittaluga Stefania , Fend Falko , Raffeld Mark , Quintanilla-Martinez Leticia
Published: April 2020

Introduction: ALK+ anaplastic large cell lymphomas (ALCL) overexpress C/EBPβ, as a consequence of NPM-ALK kinase activity. C/EBPβ is a leucine zipper transcription factor, which plays a major role in cellular differentiation, inflammation, proliferation and metabolism control. To determine the role of C/EBPβ in ALK+ ALCL transformation, and to identify its downstream targets, a highly specific C/EBPβ-shRNA was used to knockdown C/EBPβ. The consequences of C/EBPβ gene-silencing were analyzed by gene expression profiling.

Materials and Methods: Four ALK+ ALCL cell lines, SUDHL-1, Kijk, Karpas 299 and SUP-M2 were transfected with lentivirus containing the C/EBPβ shRNA or the vector without shRNA in triplicates. Western Blot analysis and qRT-PCR were performed to quantify the knockdown effect. At day three after infection, RNA was extracted and used for Gene Chip expression analysis (Affymetrix). Using Anova software for statistical analysis, we identified genes, which were regulated in all four cell lines. The effect of C/EBPβ knockdown on proliferation, cell cycle, and viability was analyzed by MTT assay and FACS analysis.

Results: In all four ALK+ ALCL, efficient C/EBPβ knockdown resulted in profound growth retardation (up to 84%) compared to control cells after 6 days of infection, and a clear shift from the S phase to the G1 phase in the cell cycle was observed. To identify genes regulated by C/EBPβ in all four cell lines, we performed statistical analysis applying a false discovery rate of 20%, and accepted only genes with a >1,1 and <0,9 fold ratio. We identfied 435 genes regulated after C/EBPβ knockdown (117 upregulated, 318 downregulated). Classification of the differentially expressed genes into biological categories revealed overrepresentation of genes involved in the regulation of kinase activity, cell cycle and proliferation, lymphocyte differentiation, and metabolic processes. In particular, kinases involved in the regulation of JNK activity, which have been shown previously to be involved in proliferation of ALCL, were highly affected by C/EBPβ knockdown. Genomatix Bibliosphere Pathway Analysis revealed C/EBPβ to be connected to pathways involving cell cycle (RUNX3, CCNG1, CDKN2A), apoptosis (FAS, PTPRC, BCL2A1, BIRC3) and MAPK cascades (TRIB1 and several MAP3Ks). Several of the genes identified contain known C/EBPβ binding sites.

Conclusions: C/EBPβ silencing induces growth arrest in ALK+ALCL, which correlates with differential expression of genes involved in cell cycle, apoptosis and differentiation. This study reveals C/EBPβ as a master transcription regulator of NPM-ALK induced cellular proliferation, and therefore, an ideal candidate for targeted therapeutic intervention.
Prognostic Impact of Microvessel Density in Follicular Lymphoma Patients Treated with Immunochemotherapy
Authors: Taskinen Minna , Bono Petri , Leppä Sirpa
Published: April 2020

Tumor infiltrating mast cells have at least two potential roles in remodeling tumor microenvironment and tumor growth: suppression of immune responses and potentiation of angiogenesis. We have recently shown that accumulation of mast cells in follicular lymphoma (FL) contributes to unfavorable survival after immunochemotherapy. Interestingly, high mast cell content eliminates the positive prognostic value of tumor-associated macrophages (TAMs). Here we investigated whether tumor vascularity, as determined by CD31 immunostaining, is associated with mast cell content and outcome of primary FL patients treated with combination of rituximab (R) and CHOP chemotherapy. We found that increased microvessel density (MVD) correlates positively with the number of tumor infiltrating mast cells (r=0.266, p=0.011). In a cohort of 95 patients, the patients with high MVD (>the lowest tertile) had a worse 4-year progression free survival (PFS) (44% vs 82%, p=0.001). In multivariate analysis with FLIPI, MVD retained its negative predictive value. Additional prognostic impact was especially provided in patients with low FLIPI scores. When MVD related outcome was estimated separately for patients with high and low mast cell contents, the adverse effect of high MVD on the PFS was seen in both subgroups. Taken together, the data demonstrate that MVD is associated with mast cell content and adverse outcome in R-CHOP treated FL patients. In contrast to TAMs, the predictive value of MVD is independent of tumor infiltrating mast cells.
NPM-ALK Modulates the p53 Tumour Suppressor Pathway in a JNK and PI 3-Kinase Dependent Manner: MDM-2 Is a Potential Therapeutic Target for the Treatment of ALK-Expressing Malignancies
Authors: Cui Yuxin , McDuff Fiona , Kerby Alan , Ye Hongtau , Turner Suzanne Dawn
Published: April 2020

Anaplastic large cell lymphoma (ALCL) is in the majority of cases a paediatric disease of a T- or null-cell phenotype and is characterised by the presence of the t(2;5)(p23;q35) or variant translocations involving the ALK gene on chromosome 2. This chromosomal translocation generates the Nucleophosmin-Anaplastic Lymphoma Kinase (NPM-ALK) fusion protein, a hyperactive kinase with transforming properties. The p53 tumour suppressor gene is rarely mutated in ALK-expressing ALCL, perhaps one reason why this disease has a good prognosis. However, the mechanism controlling p53 activity in ALCL has not been fully elucidated. We show in patient-derived ALCL cell lines and NPM-ALK transformed BaF3 cells that NPM-ALK induces post-translational modification of the p53 antagonist MDM2, leading to inactivation of the p53 tumour suppressor pathway. Furthermore, we demonstrate that the PI 3-Kinase-Akt pathway downstream of NPM-ALK is responsible for this activity. It therefore follows that inactivation of MDM2 with the specific inhibitor nutlin-3 results in a decrease in proliferation and subsequently apoptosis of NPM-ALK-expressing ALCL cells, a response that is enhanced when cells are exposed to nutlin-3 in conjunction with the PI 3-Kinase inhibitor LY294003. We also demonstrate that NPM-ALK activates JNK by phosphorylation in turn leading to JNK-mediated sequestration and degradation of p53. This activity can be attenuated following administration of a specific JNK inhibitor. We conclude that NPM-ALK regulates the activity of the p53 tumour suppressor pathway via sequestration by JNK and MDM2 leading to its degradation. MDM2 antagonists in combination with JNK/PI 3-Kinase inhibitors may therefore be potential targets for the treatment of ALK-expressing malignancies such as ALCL.
ALL-Associated JAK1 Mutants Activate the JAK/STAT Pathway Via IL-9Rα Homodimers
Authors: Hornakova Tekla , Staerk Judith , Royer Yohan , Flex Elisabetta , Tartaglia Marco , Constantinescu Stefan N. , Renauld Jean-Christophe , Knoops Laurent
Published: April 2020

Point mutations in the JAK2 tyrosine kinase play a major role in the development of myeloproliferative syndromes by activating preformed homodimers of cytokine receptors such as EpoR, TpoR and GM-CSFR. More recently, a series of activating mutations in JAK1 have also been shown to be associated with B and T cell acute lymphoblastic leukemias, but little is known about the mechanisms involved in the constitutive activation of these mutants and about their mode of interaction with cytokine receptors. Here, we studied the ability of several ALL-associated JAK1 mutants (V658F, A634D, R724H, R879C) to activate the JAK/STAT pathway upon ectopic expression in HEK293 cells, alone or together with the other components of the IL-9 receptor complex (IL-9Rα, γc and JAK3). Expression of the JAK1 mutants alone failed to trigger STAT activation, but coexpression of the IL-9Rα chain promoted JAK1 mutant phosphorylation and STATs activation, even without γc and JAK3 which are required for IL-9 responsiveness. Mutation of the FERM domain of JAK1, which is critical for cytokine receptor association, or of the single tyrosine of IL-9Rα involved in STAT recruitment abolished this activity, indicating that JAK1 mutants need to associate with IL-9R and phosphorylate this tyrosine residue to activate STAT factors. Several lines of evidence indicated that IL-9Rα homodimerization was involved in this process. IL-9Rα variants with deletion or mutation of the JAK-interacting BOX1 region not only failed to promote JAK1 activation, but acted as dominant negative forms reverting the effect of wild-type IL-9Rα. Coimmunoprecipitation experiments also showed the formation of IL-9Rα homodimers. Interestingly, this process was partially inhibited by the expression of γc, suggesting that overlapping residues are involved in IL-9Rα homodimerization and IL-9Rα/γc heterodimerization. Coexpression of wild-type JAK3 partially reverted the inhibitory effect of γc, indicating that JAK3 can cooperate with JAK1 mutants within the IL-9 receptor complex, even in the absence of the cytokine. Similar results were also observed with IL-2Rβ, which binds JAK1, but not for JAK2-associated homodimeric receptors such as TpoR or EpoR. Taken together, our results show that IL-9Rα and IL-2Rβ homodimers can efficiently mediate constitutive activation of ALL-associated JAK1 mutants, even in cells lacking the expression of γc, which is required for the activation of wild-type JAK1 to their respective physiological ligands, IL-9 and IL-2.
Role of CalDAG-GEFI in Calcium-Dependent Thromboxane A2 Generation
Authors: Stefanini Lucia , Roden R. Claire , Bergmeier Wolfgang
Published: April 2020

The second messenger Ca2+ is essential for various platelet functions, including integrin activation, thromboxane A2 (TxA2) generation and granule release. In previous studies, we have identified Ca2+- and diacylglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI) as a key molecule regulating Ca2+-dependent activation of Rap1 and integrins in platelets. Here we demonstrate that CalDAG-GEFI/Rap1 signaling is required for Ca2+-dependent ERK 1/2 activation and TxA2 generation in platelets. Impaired TxA2 formation was observed in collagen- and convulxin-activated CalDAG-GEFI deficient platelets, both at high and low doses of agonists. In contrast, no defect in TxA2 generation was observed in mutant platelets stimulated with high doses of PAR4-activating peptide, suggesting that CalDAG-GEFI may be particularly important for TxA2 generation in platelets activated through the major collagen receptor, GPVI. Our data further demonstrate a strong correlation between reduced TxA2 generation and impaired activation of ERK1/2 and Rap1 in mutant platelets. Pretreatment of CalDAG-GEFI−'/− platelets with 2-MesAMP, an inhibitor of P2Y12/Gi signaling, blocked the residual TxA2 production. These data suggest that CalDAG-GEFI synergizes with the Gi signaling pathway to control TxA2 generation. Finally, addition of thromboxane A2 analog, U46619, restored dense granule release in CalDAG-GEFI-deficient platelets stimulated with low doses of convulxin, suggesting that impaired TxA2 generation contributes to the defect in granule release previously described for CalDAG-GEFI−/− platelets. In conclusion, our studies strongly support that CalDAG-GEFI is the elusive Ca2+ sensor that links increases in intracellular Ca2+ to the signaling pathways regulating integrin activation and TxA2 formation in stimulated platelets.
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

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

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

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

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

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 (<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 >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

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.