36 research outputs found

    Regulation of RasGRP via a Phorbol Ester-Responsive C1 Domain

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    As part of a cDNA library screen for clones that induce transformation of NIH 3T3 fibroblasts, we have isolated a cDNA encoding the murine homolog of the guanine nucleotide exchange factor RasGRP. A point mutation predicted to prevent interaction with Ras abolished the ability of murine RasGRP (mRasGRP) to transform fibroblasts and to activate mitogen-activated protein kinases (MAP kinases). MAP kinase activation via mRasGRP was enhanced by coexpression of H-, K-, and N-Ras and was partially suppressed by coexpression of dominant negative forms of H- and K-Ras. The C terminus of mRasGRP contains a pair of EF hands and a C1 domain which is very similar to the phorbol ester- and diacylglycerol-binding C1 domains of protein kinase Cs. The EF hands could be deleted without affecting the ability of mRasGRP to transform NIH 3T3 cells. In contrast, deletion of the C1 domain or an adjacent cluster of basic amino acids eliminated the transforming activity of mRasGRP. Transformation and MAP kinase activation via mRasGRP were restored if the deleted C1 domain was replaced either by a membrane-localizing prenylation signal or by a diacylglycerol- and phorbol ester-binding C1 domain of protein kinase C. The transforming activity of mRasGRP could be regulated by phorbol ester when serum concentrations were low, and this effect of phorbol ester was dependent on the C1 domain of mRasGRP. The C1 domain could also confer phorbol myristate acetate-regulated transforming activity on a prenylation-defective mutant of K-Ras. The C1 domain mediated the translocation of mRasGRP to cell membranes in response to either phorbol ester or serum stimulation. These results suggest that the primary mechanism of activation of mRasGRP in fibroblasts is through its recruitment to diacylglycerol-enriched membranes. mRasGRP is expressed in lymphoid tissues and the brain, as well as in some lymphoid cell lines. In these cells, RasGRP has the potential to serve as a direct link between receptors which stimulate diacylglycerol-generating phospholipase Cs and the activation of Ras

    Irs2 Silencing Increases Apoptosis And Potentiates The Effects Of Ruxolitinib In Jak2v617f-positive Myeloproliferative Neoplasms.

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    The recurrent V617F mutation in JAK2 (JAK2V617F) has emerged as the primary contributor to the pathogenesis of myeloproliferative neoplasms (MPN). However, the lack of complete response in most patients treated with the JAK1/2 inhibitor, ruxolitinib, indicates the need for identifying pathways that cooperate with JAK2. Activated JAK2 was found to be associated with the insulin receptor substrate 2 (IRS2) in non-hematological cells. We identified JAK2/IRS2 binding in JAK2V617F HEL cells, but not in the JAK2WT U937 cell line. In HEL cells, IRS2 silencing decreased STAT5 phosphorylation, reduced cell viability and increased apoptosis; these effects were enhanced when IRS2 silencing was combined with ruxolitinib. In U937 cells, IRS2 silencing neither reduced cell viability nor induced apoptosis. IRS1/2 pharmacological inhibition in primary MPN samples reduced cell viability in JAK2V617F-positive but not JAK2WT specimens; combination with ruxolitinib had additive effects. IRS2 expression was significantly higher in CD34+ cells from essential thrombocythemia patients compared to healthy donors, and in JAK2V617F MPN patients when compared to JAK2WT. Our data indicate that IRS2 is a binding partner of JAK2V617F in MPN. IRS2 contributes to increased cell viability and reduced apoptosis in JAK2-mutated cells. Combined pharmacological inhibition of IRS2 and JAK2 may have a potential clinical application in MPN.

    Bayesian multi-source regression and monocyte-associated gene expression predict BCL-2 inhibitor resistance in acute myeloid leukemia

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    The FDA recently approved eight targeted therapies for acute myeloid leukemia (AML), including the BCL-2 inhibitor venetoclax. Maximizing efficacy of these treatments requires refining patient selection. To this end, we analyzed two recent AML studies profiling the gene expression and ex vivo drug response of primary patient samples. We find that ex vivo samples often exhibit a general sensitivity to (any) drug exposure, independent of drug target. We observe that this "general response across drugs" (GRD) is associated with FLT3-ITD mutations, clinical response to standard induction chemotherapy, and overall survival. Further, incorporating GRD into expression-based regression models trained on one of the studies improved their performance in predicting ex vivo response in the second study, thus signifying its relevance to precision oncology efforts. We find that venetoclax response is independent of GRD but instead show that it is linked to expression of monocyte-associated genes by developing and applying a multi-source Bayesian regression approach. The method shares information across studies to robustly identify biomarkers of drug response and is broadly applicable in integrative analyses

    The PI3K/Akt1 pathway enhances steady-state levels of FANCL

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    Fanconi anemia hematopoietic stem cells display poor self-renewal capacity when subjected to a variety of cellular stress. This phenotype raises the question of whether the Fanconi anemia proteins are stabilized or recruited as part of a stress response and protect against stem cell loss. Here we provide evidence that FANCL, the E3 ubiquitin ligase of the Fanconi anemia pathway, is constitutively targeted for degradation by the proteasome. We confirm biochemically that FANCL is polyubiquitinated with Lys-48-linked chains. Evaluation of a series of N-terminal-deletion mutants showed that FANCL's E2-like fold may direct ubiquitination. In addition, our studies showed that FANCL is stabilized in a complex with axin1 when glycogen synthase kinase-3ÎČ is overexpressed. This result leads us to investigate the potential regulation of FANCL by upstream signaling pathways known to regulate glycogen synthase kinase-3ÎČ. We report that constitutively active, myristoylated-Akt increases FANCL protein level by reducing polyubiquitination of FANCL. Two-dimensional PAGE analysis shows that acidic forms of FANCL, some of which are phospho-FANCL, are not subject to polyubiquitination. These results indicate that a signal transduction pathway involved in self-renewal and survival of hematopoietic stem cells also functions to stabilize FANCL and suggests that FANCL participates directly in support of stem cell function

    31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

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    Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

    Omecamtiv mecarbil in chronic heart failure with reduced ejection fraction, GALACTIC‐HF: baseline characteristics and comparison with contemporary clinical trials

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    Aims: The safety and efficacy of the novel selective cardiac myosin activator, omecamtiv mecarbil, in patients with heart failure with reduced ejection fraction (HFrEF) is tested in the Global Approach to Lowering Adverse Cardiac outcomes Through Improving Contractility in Heart Failure (GALACTIC‐HF) trial. Here we describe the baseline characteristics of participants in GALACTIC‐HF and how these compare with other contemporary trials. Methods and Results: Adults with established HFrEF, New York Heart Association functional class (NYHA) ≄ II, EF ≀35%, elevated natriuretic peptides and either current hospitalization for HF or history of hospitalization/ emergency department visit for HF within a year were randomized to either placebo or omecamtiv mecarbil (pharmacokinetic‐guided dosing: 25, 37.5 or 50 mg bid). 8256 patients [male (79%), non‐white (22%), mean age 65 years] were enrolled with a mean EF 27%, ischemic etiology in 54%, NYHA II 53% and III/IV 47%, and median NT‐proBNP 1971 pg/mL. HF therapies at baseline were among the most effectively employed in contemporary HF trials. GALACTIC‐HF randomized patients representative of recent HF registries and trials with substantial numbers of patients also having characteristics understudied in previous trials including more from North America (n = 1386), enrolled as inpatients (n = 2084), systolic blood pressure &lt; 100 mmHg (n = 1127), estimated glomerular filtration rate &lt; 30 mL/min/1.73 m2 (n = 528), and treated with sacubitril‐valsartan at baseline (n = 1594). Conclusions: GALACTIC‐HF enrolled a well‐treated, high‐risk population from both inpatient and outpatient settings, which will provide a definitive evaluation of the efficacy and safety of this novel therapy, as well as informing its potential future implementation

    Regulation of RasGRP via a Phorbol Ester-Responsive C1 Domain

    Get PDF
    As part of a cDNA library screen for clones that induce transformation of NIH 3T3 fibroblasts, we have isolated a cDNA encoding the murine homolog of the guanine nucleotide exchange factor RasGRP. A point mutation predicted to prevent interaction with Ras abolished the ability of murine RasGRP (mRasGRP) to transform fibroblasts and to activate mitogen-activated protein kinases (MAP kinases). MAP kinase activation via mRasGRP was enhanced by coexpression of H-, K-, and N-Ras and was partially suppressed by coexpression of dominant negative forms of H- and K-Ras. The C terminus of mRasGRP contains a pair of EF hands and a C1 domain which is very similar to the phorbol ester- and diacylglycerol-binding C1 domains of protein kinase Cs. The EF hands could be deleted without affecting the ability of mRasGRP to transform NIH 3T3 cells. In contrast, deletion of the C1 domain or an adjacent cluster of basic amino acids eliminated the transforming activity of mRasGRP. Transformation and MAP kinase activation via mRasGRP were restored if the deleted C1 domain was replaced either by a membrane-localizing prenylation signal or by a diacylglycerol- and phorbol ester-binding C1 domain of protein kinase C. The transforming activity of mRasGRP could be regulated by phorbol ester when serum concentrations were low, and this effect of phorbol ester was dependent on the C1 domain of mRasGRP. The C1 domain could also confer phorbol myristate acetate-regulated transforming activity on a prenylation-defective mutant of K-Ras. The C1 domain mediated the translocation of mRasGRP to cell membranes in response to either phorbol ester or serum stimulation. These results suggest that the primary mechanism of activation of mRasGRP in fibroblasts is through its recruitment to diacylglycerol-enriched membranes. mRasGRP is expressed in lymphoid tissues and the brain, as well as in some lymphoid cell lines. In these cells, RasGRP has the potential to serve as a direct link between receptors which stimulate diacylglycerol-generating phospholipase Cs and the activation of Ras

    Bayesian multi-source regression and monocyte-associated gene expression predict BCL-2 inhibitor resistance in acute myeloid leukemia

    Get PDF
    The FDA recently approved eight targeted therapies for acute myeloid leukemia (AML), including the BCL-2 inhibitor venetoclax. Maximizing efficacy of these treatments requires refining patient selection. To this end, we analyzed two recent AML studies profiling the gene expression and ex vivo drug response of primary patient samples. We find that ex vivo samples often exhibit a general sensitivity to (any) drug exposure, independent of drug target. We observe that this "general response across drugs" (GRD) is associated with FLT3-ITD mutations, clinical response to standard induction chemotherapy, and overall survival. Further, incorporating GRD into expression-based regression models trained on one of the studies improved their performance in predicting ex vivo response in the second study, thus signifying its relevance to precision oncology efforts. We find that venetoclax response is independent of GRD but instead show that it is linked to expression of monocyte-associated genes by developing and applying a multi-source Bayesian regression approach. The method shares information across studies to robustly identify biomarkers of drug response and is broadly applicable in integrative analyses.Peer reviewe

    Targeting mitochondria in cancer therapy could provide a basis for the selective anti-cancer activity.

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    To determine the target of the recently identified lead compound NSC130362 that is responsible for its selective anti-cancer efficacy and safety in normal cells, structure-activity relationship (SAR) studies were conducted. First, NSC13062 was validated as a starting compound for the described SAR studies in a variety of cell-based viability assays. Then, a small library of 1,4-naphthoquinines (1,4-NQs) and quinoline-5,8-diones was tested in cell viability assays using pancreatic cancer MIA PaCa-2 cells and normal human hepatocytes. The obtained data allowed us to select a set of both non-toxic compounds that preferentially induced apoptosis in cancer cells and toxic compounds that induced apoptosis in both cancer and normal cells. Anti-cancer activity of the selected non-toxic compounds was confirmed in viability assays using breast cancer HCC1187 cells. Consequently, the two sets of compounds were tested in multiple cell-based and in vitro activity assays to identify key factors responsible for the observed activity. Inhibition of the mitochondrial electron transfer chain (ETC) is a key distinguishing activity between the non-toxic and toxic compounds. Finally, we developed a mathematical model that was able to distinguish these two sets of compounds. The development of this model supports our conclusion that appropriate quantitative SAR (QSAR) models have the potential to be employed to develop anti-cancer compounds with improved potency while maintaining non-toxicity to normal cells
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