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The procoagulant thrombin promotes the adhesion of polymorphonuclear leukocytes to endothelial cells by a mechanism involving expression of intercellular adhesion molecule 1 (ICAM-1) via an NF-kappaB-dependent pathway. We now provide evidence that protein kinase C-delta (PKC-delta) and the p38 mitogen-activated protein (MAP) kinase pathway play a critical role in the mechanism of thrombin-induced ICAM-1 gene expression in endothelial cells. We observed the phosphorylation of PKC-delta and p38 MAP kinase within 1 min after thrombin challenge of human umbilical vein endothelial cells. Pretreatment of these cells with the PKC-delta inhibitor rottlerin prevented the thrombin-induced phosphorylation of p38 MAP kinase, suggesting that p38 MAP kinase signals downstream of PKC-delta. Inhibition of PKC-delta or p38 MAP kinase by pharmacological and genetic approaches markedly decreased the thrombin-induced NF-kappaB activity and resultant ICAM-1 expression. The effects of PKC-delta inhibition were secondary to inhibition of IKKbeta activation and of subsequent NF-kappaB binding to the ICAM-1 promoter. The effects of p38 MAP kinase inhibition occurred downstream of IkappaBalpha degradation without affecting the DNA binding function of nuclear NF-kappaB. Thus, PKC-delta signals thrombin-induced ICAM-1 gene transcription by a dual mechanism involving activation of IKKbeta, which mediates NF-kappaB binding to the ICAM-1 promoter, and p38 MAP kinase, which enhances transactivation potential of the bound NF-kappaB p65 (RelA).

The mechanisms by which interferon-alpha (IFN-alpha) mediates its anti-leukemic effects in chronic myelogenous leukemia (CML) cells are not known. We determined whether p38 MAPK is activated by IFN-alpha in BCR-ABL-expressing cells and whether its function is required for the generation of growth inhibitory responses. IFN-alpha treatment induced phosphorylation/activation of p38 in the IFN-alpha-sensitive KT-1 cell line, but not in IFN-alpha-resistant K562 cells. Consistent with this, IFN-alpha treatment of KT-1 (but not K562) cells induced activation of the small GTPase Rac1, which functions as an upstream regulator of p38. In addition, IFN-alpha-dependent phosphorylation/activation of p38 was induced by treatment of primary granulocytes isolated from the peripheral blood of patients with CML. To define the functional role of the Rac1/p38 MAPK pathway in IFN-alpha signaling, the effects of pharmacological inhibition of p38 on the induction of IFN-alpha responses were determined. Treatment of KT-1 cells with the p38-specific inhibitors SB203580 and SB202190 reversed the growth inhibitory effects of IFN-alpha. On the other hand, the MEK kinase inhibitor PD098059 had no effects, further demonstrating the specificity of these findings. To directly determine the significance of IFN-alpha-dependent activation of p38 in the induction of the anti-leukemic effects of IFN-alpha, we evaluated the effects of p38 inhibition on leukemic colony formation in bone marrow samples of patients with CML. IFN-alpha inhibited leukemic granulocyte/macrophage colony formation in a dose-dependent manner, whereas concomitant treatment with p38 inhibitors reversed such an inhibition. Thus, the Rac1/p38 MAPK pathway is activated by IFN-alpha in BCR-ABL-expressing cells and appears to play a key role in the generation of the growth inhibitory effects of IFN-alpha in CML cells.

Interferon α (IFNα) has significant clinical activity in the treatment of patients with chronic myelogenous leukaemia (CML), but the mechanisms of its selective efficacy in the treatment of the disease are unknown. The CrkL adaptor protein interacts directly with the BCR–ABL fusion protein that causes the malignant transformation and is constitutively phosphorylated in BCR–ABL-expressing cells. In the present study, we provide evidence that CrkL was engaged in IFNα-signalling in the CML-derived KT-1 cell line, which expresses BCR–ABL and is sensitive to the growth inhibitory effects of IFNα. CrkL is constitutively associated with BCR–ABL in these cells and treatment with IFNα had no effect on the BCR–ABL/CrkL interaction. After IFNα stimulation, CrkL associated with Stat5, which also underwent phosphorylation in an IFNα-dependent manner. The interaction of CrkL with Stat5 was facilitated by the function of both the SH2 and the N-terminus SH3 domains of CrkL. The resulting CrkL–Stat5 complex translocated to the nucleus and could be detected in gel shift assays using elements derived from either the β-casein promoter or the promoter of the PML gene, an IFNα-inducible gene that mediates growth inhibitory responses. In addition to its interaction with Stat5, CrkL interacts with C3G in KT-1 cells and such an interaction regulates the downstream activation of the small GTPase Rap1, which also mediates inhibition of cell proliferation. Thus, despite its engagement by BCR–ABL in CML-derived cells, CrkL mediates activation of downstream signalling pathways in response to the activated type I IFN receptor and such signals may contribute to the generation of the anti-proliferative effects of IFNα in CML.

Interferons (IFNs) regulate the expression of a number of cellular genes by activating the JAK-STAT pathway. We have recently discovered that CCAAAT/enhancer-binding protein-beta (C/EBP-beta) induces gene transcription through a novel IFN response element called the gamma-IFN-activated transcriptional element (Roy, S. K., Wachira, S. J., Weihua, X., Hu, J., and Kalvakolanu, D. V. (2000) J. Biol. Chem. 275, 12626-12632. Here, we describe a new IFN-gamma-stimulated pathway that operates C/EBP-beta-regulated gene expression independent of JAK1. We show that ERKs are activated by IFN-gamma to stimulate C/EBP-beta-dependent expression. Sustained ERK activation directly correlated with C/EBP-beta-dependent gene expression in response to IFN-gamma. Mutant MKK1, its inhibitors, and mutant ERK suppressed IFN-gamma-stimulated gene induction through the gamma-IFN-activated transcriptional element. Ras and Raf activation was not required for this process. Furthermore, Raf-1 phosphorylation negatively correlated with its activity. Interestingly, C/EBP-beta-induced gene expression required STAT1, but not JAK1. A C/EBP-beta mutant lacking the ERK phosphorylation site failed to promote IFN-stimulated gene expression. Thus, our data link C/EBP-beta to IFN-gamma signaling through ERKs.

Interleukin-5 (IL-5) drives the terminal differentiation of myeloid progenitors to the eosinophil lineage; blocks eosinophil apoptosis; and primes eosinophils for enhanced functional activities in allergic, parasitic, and other eosinophil-associated diseases. Here we describe a novel signaling pathway activated by the IL-5 receptor in eosinophils involving the CrkL adapter protein. We determined whether IL-5 induces activation of CrkL and STAT5 in eosinophils using both the human eosinophil-differentiated AML14.3D10 cell line and purified peripheral blood eosinophils from normal donors. Stimulation of AML14.3D10 cells or blood eosinophils with IL-5 induced rapid tyrosine phosphorylation of the CrkL adapter and STAT5 and the association of CrkL and STAT5 in vivo as evidenced by the detection of STAT5 in anti-CrkL immunoprecipitates. The resulting CrkL.STAT5 complexes translocated to the nucleus and bound STAT5 consensus DNA-binding sites present in the promoters of IL-5-regulated genes, as shown in gel mobility and antibody supershift assays. IL-5 also induced marked activity of an 8X-GAS (interferon gamma-activated site)-luciferase reporter construct in transient transfections of AML14.3D10 eosinophils, demonstrating that these complexes play a functional role in IL-5 signaling. CrkL was also found to interact, via its N-terminal SH3 domain, with C3G, a guanine exchange factor for the small G-protein Rap1, which was also rapidly activated in an IL-5-dependent manner in these cells, establishing that CrkL mediates downstream activation of at least two signaling cascades in IL-5-stimulated eosinophils. Thus, the CrkL adapter plays an important role in IL-5 signaling in the eosinophil, acting as a nuclear adapter for STAT5 and as an upstream regulator of the C3G-Rap1 signaling pathway.

The p38 mitogen-activated protein (MAP) kinase is activated during engagement of the type I interferon (IFN) receptor and mediates signals essential for IFNalpha-dependent transcriptional activation via interferon-stimulated response elements without affecting formation of the ISGF3 complex. In the present study, we provide evidence that the small GTPase Rac1 is activated in a type I IFN-dependent manner and that its function is required for downstream engagement of the p38 MAP kinase pathway. We also demonstrate that p38 is required for IFNalpha-dependent gene transcription via GAS elements and regulates activation of the promoter of the PML gene that mediates growth inhibitory responses. In studies to determine whether the regulatory effects of p38 are mediated by serine phosphorylation of Stat1 or Stat3, we found that the p38 kinase inhibitors SB203580 or SB202190 or overexpression of a dominant negative p38 mutant do not inhibit phosphorylation of Stat1 or Stat3 on Ser-727 in several IFNalpha-sensitive cell lines. Altogether these data demonstrate that the Rac1/p38 MAP kinase signaling cascade plays a critical role in type I IFN signaling, functioning in cooperation with the Stat-pathway, to regulate transcriptional regulation of IFNalpha-sensitive genes and generation of growth inhibitory responses.

Erythropoietin (Epo), stem cell factor (SCF), and insulin-like growth factor-1 (IGF-1) are key regulators of erythroid cell proliferation and differentiation. To understand the mechanisms of generation of signals by each of these growth factors, we determined the activation of the PI3-kinase/Akt pathway during proliferation and differentiation of primary human erythroid progenitors. Our results demonstrate that PKB/Akt is activated by Epo and SCF, but not by IGF-1 in human primary erythroid progenitors. In addition, Epo treatment of erythroid progenitors induces phosphorylation of a member of the Forkhead family (FH) of transcription factors FKHRL1, downstream of activation of the Akt kinase. Such Epo-dependent activation of FKHRL1 apparently regulates the generation of Epo-dependent antiapoptotic signals as evidenced by the induction of apoptosis of erythroid progenitors during treatment of cells with the PI3-kinase (PI3K) inhibitor LY294002. Thus, the PI3K/Akt/FKHRL1 pathway is essential for inhibition of apoptosis in response to Epo and SCF, while the IGF-1 receptor utilizes a different pathway.

IFN-γ transduces signals by activating the IFN-γ receptor-associated Jak-1 and Jak-2 kinases and by inducing tyrosine phosphorylation and activation of the Stat-1 transcriptional activator. We report that IFN-γ activates a distinct signaling cascade involving the c-cbl protooncogene product, CrkL adapter, and small G protein Rap1. During treatment of NB-4 human cells with IFN-γ, c-cbl protooncogene product is rapidly phosphorylated on tyrosine and provides a docking site for the src homology 2 domain of CrkL, which also undergoes IFN-γ-dependent tyrosine phosphorylation. CrkL then regulates activation of the guanine exchange factor C3G, with which it interacts constitutively via its N terminus src homology 3 domain. This results in the IFN-γ-dependent activation of Rap1, a protein known to exhibit tumor suppressor activity and mediate growth inhibitory responses. In a similar manner, Rap1 is also activated in response to treatment of cells with type I IFNs (IFN-α, IFN-β), which also engage CrkL in their signaling pathways. On the other hand, IFN-γ does not induce formation of nuclear CrkL-Stat5 DNA-binding complexes, which are induced by IFN-α and IFN-β, indicating that pathways downstream of CrkL are differentially regulated by different IFN subtypes. Taken altogether, our data demonstrate that, in addition to activating the Stat pathway, IFN-γ activates a distinct signaling cascade that may play an important role in the generation of its growth inhibitory effects on target cells.

We report the case of a patient with rare CD5+ hairy cell leukemia, which was unusual, as there was also a discrepancy between the bone marrow and peripheral blood immunophenotypes. We propose that some degree of maturation within the malignant clone and the predominance of more mature CD5– hairy cells in the peripheral blood can explain this observation. The patient was treated with a conventional course of cladribine (2′-chlorodeoxyadenosine) and achieved complete remission. We conclude that the unusual CD5+ immunophenotype of this patient did not affect prognosis or change the response to standard therapy.

Interferons are potent regulators of normal and malignant hematopoietic cell proliferation in vitro and in vivo, but the signaling mechanisms by which they exhibit their growth inhibitory effects are unknown. We have recently shown that CrkL is engaged in Type I IFN signaling, as shown by its rapid tyrosine phosphorylation during engagement of the Type I IFN receptor. In the present study, we provide evidence that the related CrkII protein is also rapidly phosphorylated on tyrosine during treatment of U-266 and Daudi cells with IFNalpha or IFNbeta. We also show that both members of the Crk-family, CrkL and CrkII, are phosphorylated in an interferon-dependent manner in primary hematopoietic progenitors. Furthermore, inhibition of CrkL or CrkII protein expression by antisense oligonucleotides, reverses the inhibitory effects of IFNalpha or IFNgamma on the proliferation of normal bone marrow progenitor cells (colony forming units-granulocytic/monocytic [CFU-GM] and burst-forming units-erythroid [BFU-E]). Thus, both CrkL and CrkII are engaged in a signaling pathway (s) that mediates interferon-regulated inhibition of hematopoietic cell proliferation.

Several signaling cascades are activated during engagement of the erythropoietin receptor to mediate the biological effects of erythropoietin. The members of the insulin receptor substrate (IRS) family of proteins play a central role in signaling for various growth factor receptors and cytokines by acting as docking proteins for the SH2 domains of signaling elements, linking cytokine receptors to diverse downstream pathways. In the present study we provide evidence that the recently cloned IRS-related proteins, Gab1 and Gab2, of the Gab family of proteins, are rapidly phosphorylated on tyrosine during erythropoietin treatment of erythropoietin-responsive cells and provide docking sites for the engagement of the SHP2 phosphatase and the p85 subunit of the phosphatidylinositol 3′-kinase. Furthermore, our data show that Gab1 is the primary IRS-related protein activated by erythropoietin in primary erythroid progenitor cells. In studies to identify the erythropoietin receptor domains required for activation of Gab proteins, we found that tyrosines 425 and 367 in the cytoplasmic domain of the erythropoietin receptor are required for the phosphorylation of Gab2. Taken together, our data demonstrate that Gab proteins are engaged in erythropoietin signaling to mediate downstream activation of the SHP2 and phosphatidylinositol 3′-kinase pathways and possibly participate in the generation of the erythropoietin-induced mitogenic responses.

We determined whether selective activation of components of the Jak-Stat pathway by different type I interferons (IFN) occurs in human myocardial fibroblasts that exhibit much higher sensitivity to the antiviral effects of IFN-beta than of IFN-alpha. Similar levels of activation of the Tyk2 kinase and the Stat3 transcription factor were induced in response to either IFN-beta or IFN-alpha treatment. However, activation of the Jak1 tyrosine kinase was detectable only in IFN-beta-treated but not IFN-alpha-treated cells. Consistent with this, tyrosine phosphorylation of Stat1 and Stat2 and formation of the IFN-stimulated gene factor 3 (ISGF3) complex occurred to a much higher degree in response to IFN-beta stimulation. These findings demonstrate that differential activation of distinct components of the Jak-Stat pathway by different type I IFN can occur. Furthermore, they strongly suggest that such selective activation accounts for the occurrence of differences in the antiviral properties of distinct type I IFN in certain cell types.

The precise role of the different subunits (alpha/IFNAR1 and betaL/IFNAR2) of the type I interferon receptor (IFN-R) in the activation of signal transducer and activator of transcription (Stat) 1, Stat2, and Stat3 has not yet been established. In this report we demonstrate that there are functionally redundant phosphotyrosine-dependent and -independent binding sites for Stat2 in the alpha and beta subunits of the type I IFN-R. Expression of a type I IFN-R containing only the constitutive Stat2 site or the proximal tyrosines of betaL, but not the docking site on the alpha chain (Tyr466 and Tyr481), supported low levels of Stat2 activation. However, the presence of only one intact Stat2 site did not lead to induction of interferon-stimulated gene factor 3 (ISGF3) or an antiviral state. Normal levels of Stat2 tyrosine phosphorylation, induction of ISGF3, and an antiviral effect always required the proximal tyrosines of betaL and at least one of the other Stat2 sites (Tyralpha466, 481 or betaL404-462). These data suggest that a threshold of Stat2 tyrosine phosphorylation is required for complete activation of ISGF3. Interestingly, a receptor in which all tyrosines were mutated to phenylalanine shows normal Stat3 phosphorylation and low levels of activation of Stat1.

Type I interferon (IFN) signaling leads to transcription and translation of key IFN-stimulated genes (ISGs), whose protein products exhibit anti-tumorigenic, anti-viral, and immunomodulatory functions [1–3]. These responses are triggered by the interaction of type I IFNs (IFNα, IFNβ, IFNω) with a unique cell surface receptor composed by two subunits: IFNα receptor 1 (IFNAR1) and IFNAR2 [1–3]. After the Type I IFN receptor is targeted by different members of this cytokine family, there is induction of the kinase activities of two known kinases; tyrosine kinase 2 (TYK2) and janus kinase 1 (JAK1), which activate several downstream signaling pathways, including STAT (signal transducer and activator of transcription), MAPKKK (mitogen-activated protein kinase kinase kinase), PI3K (phosphatidylinositol 3-kinase)-AKT, mTORC1 (mammalian target of rapamycin complex 1), and mTORC2 signaling cascades [1–3]. There has been rapid accumulation of information in the IFN-signaling field for over two decades. A detailed map of signaling events and elements that ultimately control IFN-responses has emerged [1–3]. Yet, unique mechanisms accounting for activation and coordination of distinct IFN signaling pathways still need to be defined. Similarly, the elements that account for optimized cellular responses and adjust the balance between negative and positive cellular control in the IFN-response, remain to be established. In a recently published report, we identified Unc-51-like kinase 1 (ULK1) as a novel positive regulator of type I IFN signaling, which is essential for both type I IFN-induced anti-viral and anti-proliferative responses [4]. Additionally, we presented evidence showing that engagement of type I IFN receptor activates a novel pathway, possibly involving both PI3K-AKT-dependent and independent meditators, culminating in activation of ULK1 [4]. Importantly, ULK1 kinase activity seems to cooperate, either directly or through intermediate kinases, with the MAPKKK-MKK3/6 signaling cascade for optimal IFN-induced activation of p38 MAPK [4]. Thus, ULK1 activation appears to mediate transcription of ISGs, in part, via regulation of the p38 MAPK pathway [4]. In a recent report, ULK1 was shown to localize to the nucleus and promote cell death by increasing poly (ADP-ribose) polymerase 1 (PARP1) activity in response to reactive oxygen species-induced cellular damage [5]. These new findings, taken together with our recently published report [4], raise the intriguing possibility that ULK1 translocates into the nucleus in response to type I IFNs, and could directly or indirectly control transcription of ISGs, but this remains to be explored in future studies. Type I IFNs can be produced by all nucleated cell types in response to pathogen infection, physiological signals, and other inducers and stimuli [6, 7]. However, the induction of an IFN response involves expression and activation of both positive and negative regulators of Type I IFN production and signaling [6, 7]. Many of these negative regulators are ISGs and IFN-regulated proteins, which control the time and magnitude of an IFN response, preventing inflammation and tissue damage [6, 7]. Interestingly, this seems to be the case also for ULK1 [8]. The stimulator of IFN genes (STING) is activated by cyclic dinucleotides, leading to activation of TBK1 (TRAF family member associated NF-κB activator-binding kinase 1) and phosphorylation of IRF3 [6, 7]. Active IRF3 translocates into the nucleus and induces expression of IFN genes, initiating an IFN response [6, 7]. Our data suggests that ULK1 is activated downstream of the type I IFN receptor, leading to transcription of key ISGs and positively mediating IFN responses [4]. However, based on other recent work, at a later stage ULK1 is activated by cyclic dinucleotides and acts as a negative regulator of STING, resulting in inhibition of IRF3, and subsequent blockage of type I IFN production, thus shutting down the IFN response [8]. Thus, taking together the evidence presented by Kono et al. [8] and our findings [4], one can propose a model in which ULK1 may initially promote IFN responses against pathogens, but it later inhibits STING activity and IFN production, thus limiting/optimizing the IFN response (Figure ​(Figure1).1). Further studies are warranted to fully understand this dual role of ULK1 in the IFN system. This has important implications in the IFN-signaling field and may provoke other questions in the future. For example, it would be interesting to identify putative phosphorylation sites of ULK1 that may correlate with negative versus positive regulatory effects on Type I IFN responses. A complete understanding of the mechanisms of action of ULK1 during IFN-signaling may also prove fundamental for the development of better therapies designed to either promote or inhibit type I IFN-dependent responses. Figure 1 Regulatory roles of ULK1 in Type I IFN responses

Background: DAB 389 IL-2 is a novel fusion toxin that retargets the cytotoxic A-chain of diphtheria toxin to interleukin-2 (IL-2) receptor–expressing tumors. Objective: The purpose of this phase I trial was to study the toxicity, maximum tolerated dose, and clinical efficacy of DAB 389 IL-2 in IL-2 receptor expressing lymphoproliferative malignancies, including cutaneous T-cell lymphoma. Methods: DAB 389 IL-2 was administered intravenously daily for 5 days every 3 weeks. Dose escalation occurred between patient groups. Patients were monitored for laboratory and clinical toxicity, kinetics, immune response, and clinical efficacy. Results: Thirty-five patients with cutaneous T-cell lymphoma (including 30 patients with mycosis fungoides) were treated. Previously, conventional therapy had not worked for 34 of the patients. Thirteen patients (37%) achieved an objective response, including a complete response in five patients (14%). Complete response was achieved in patients with extensive erythroderma and tumor stage mycosis fungoides. Adverse events consisted of reversible fever/chills, hypotension, nausea/vomiting, and elevation of hepatic transaminase. Doses of less than 31 μg/kg per day were well tolerated. Clinical responses were observed at all dose levels. Conclusion: DAB 389 IL-2 is well tolerated at doses of less than 31 μg/kg per day, and it induced clinical responses in previously treated mycosis fungoides, providing evidence for the antitumor activity of this molecule. (J Am Acad Dermatol 1998;39:63-73.)

Familial hemophagocytic lymphohistiocytosis (FHLH) is an autosomal recessive disease with features similar to those of the murine motheaten phenotype resulting from mutations of protein tyrosine phosphatase SHP-1. This has raised the possibility that defects in SHP-1 or SHP-1-regulated signaling molecules may be present in FHLH. In this study, we examined SHP-1 protein and transcript in the peripheral blood mononuclear cells (PBMC) of an FHLH family. Our results show that the FHLH patient and the parents express comparable levels of a single SHP-1 protein and that the SHP-1 cDNA clone from the patient contains no mutation in the coding region. Interestingly, a reduced association of SHP-1 with the Jak family kinase Tyk2 was detected in the patient and the defect appears to have been inherited from one of the parents. This reduced SHP-1/Tyk2 association is likely due to a defect in Tyk2 or in cellular factors regulating Tyk2, because we found no abnormalities in SHP-1 or in SHP-1 association with the other Jak kinases. These data demonstrate that the SHP-1 gene is intact in FHLH and that the defect in some cases with this disease may involve signaling molecules regulated by SHP-1.

The signaling specificity for cytokines that have common receptor subunits is achieved by the presence of additional cytokine-specific receptor components. In the type I interferon (IFN) family, all 14 subtypes of IFNalpha, IFNbeta, and IFNomega bind to the same alpha and betaL subunits of the type I IFN-R, yet differences in signaling and biological effects exist among them. Our data demonstrate that IFNalpha2 and IFNbeta utilize different regions of the betaL subunit for signaling. Thus, in contrast to other cytokine systems, signal diversity in the type I IFN system can be accomplished within the same receptor complex by utilizing different regions of the same receptor subunits.

Interferon-alpha (IFNα) mediates its biological effects through activation of the JAK-STAT signaling pathway and it has been shown to be one of most effective therapeutic agents for a number of hematological malignancies, including cutaneous T-cell lymphoma (CTCL). Nevertheless, its efficacy is limited by the development of clinical resistance but the reasons for resistance in CTCL are unknown. Here, we report the development of an IFNα-resistant CTCL cell line (HUT78R), characterized by its ability to proliferate in high concentration of recombinant IFNα, which can be used as a model system to study IFN resistance. The levels of IFN receptor expression and binding affinity were found to be comparable between the parental sensitive (HUT78S) and resistant (HUT78R) cells. However, IFNα stimulation failed to induce interferon-stimulated gene factor 3 (ISGF3) complex formation in HUT78R cells. In addition, the expression of the IFN-inducible 2-5 OAS gene was significantly reduced in HUT78R cells, suggesting the presence of a defect in the Jak-STAT signaling pathway. Our results showed that the IFNα-activated form of a latent transcriptional factor STAT1 was not found in HUT78R cells, whereas activated STAT2 and STAT3 were clearly detectable. By Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) analyses, we found that HUT78R cells do not express any STAT1 protein or mRNA, suggesting the possibility of a null mutation in the STAT1 gene. Resistance to the growth inhibitory effect of IFNα in CTCL cells may result from lack of STAT1 expression.

Interferon-alpha (IFNα) mediates its biological effects through activation of the JAK-STAT signaling pathway and it has been shown to be one of most effective therapeutic agents for a number of hematological malignancies, including cutaneous T-cell lymphoma (CTCL). Nevertheless, its efficacy is limited by the development of clinical resistance but the reasons for resistance in CTCL are unknown. Here, we report the development of an IFNα-resistant CTCL cell line (HUT78R), characterized by its ability to proliferate in high concentration of recombinant IFNα, which can be used as a model system to study IFN resistance. The levels of IFN receptor expression and binding affinity were found to be comparable between the parental sensitive (HUT78S) and resistant (HUT78R) cells. However, IFNα stimulation failed to induce interferon-stimulated gene factor 3 (ISGF3) complex formation in HUT78R cells. In addition, the expression of the IFN-inducible 2-5 OAS gene was significantly reduced in HUT78R cells, suggesting the presence of a defect in the Jak-STAT signaling pathway. Our results showed that the IFNα-activated form of a latent transcriptional factor STAT1 was not found in HUT78R cells, whereas activated STAT2 and STAT3 were clearly detectable. By Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) analyses, we found that HUT78R cells do not express any STAT1 protein or mRNA, suggesting the possibility of a null mutation in the STAT1 gene. Resistance to the growth inhibitory effect of IFNα in CTCL cells may result from lack of STAT1 expression.

During IFN alpha stimulation, p59(fyn) associates with the Type I IFNR-associated Tyk-2 kinase in several human hematopoietic cell lines in vivo. This interaction is direct, and is mediated by the SH2 domain in p59(fyn), as shown by binding studies using glutathione-S-transferase fusion proteins and far western blots. Furthermore, in response to IFN alpha-treatment of cells, the SH2 domain of Fyn interacts with the Tyk-2-associated c-cbl proto-oncogene product. In a similar manner, during IFN gamma stimulation, p59(fyn) associates via its SH2 domain with the activated form of the IFN gamma-dependent Jak-2 kinase. These data suggest that p59(fyn) is a common element in IFN alpha and IFN gamma signaling, and is selectively engaged by the Type I or II IFN receptors via specific interactions with distinct Jak kinases.

The vav proto-oncogene product participates in the signaling pathways activated by various cell-surface receptors, including the type I IFN receptor. During engagement of the type I IFN receptor, p95 vav is phosphorylated on tyrosine residues, but the kinase regulating its phosphorylation has not been identified to date. Our studies demonstrate that p95 vav forms a stable complex with the IFN-receptor-associated Tyk-2 kinase in vivo, and strongly suggest that this kinase regulates its phosphorylation on tyrosine. Thus, p95 vav is engaged in IFN-signaling by a direct interaction with the functional type I IFN receptor complex to transduce downstream signals. © 1997 Federation of European Biochemical Societies.

Upregulation of Pim kinases is observed in several types of leukemias and lymphomas. Pim-1, -2, and -3 promote cell proliferation and survival downstream of cytokine and growth factor signaling pathways. AZD1208 is a potent, highly selective, and orally available Pim kinase inhibitor that effectively inhibits all three isoforms at

There is emerging evidence that the IFN-inducible family of Slfn genes and proteins play important roles in cell cycle progression and control of cellular proliferation, but the precise functional roles of different Slfn members in the regulation of tumorigenesis remain unclear. In the present study, we undertook a systematic analysis on the expression and functional relevance of different mouse Slfn genes in malignant melanoma and renal cell carcinoma cells. Our studies demonstrate that several mouse Slfn genes are up-regulated in response to IFN treatment of mouse melanoma and renal cell carcinoma cells, including Slfn1, Slfn2, Slfn4, Slfn5, and Slfn8. Our data show that Slfn2 and Slfn3 play essential roles in the control of mouse malignant melanoma cell proliferation and/or anchorage-independent growth, suggesting key and non-overlapping roles for these genes in the control of malignant melanoma tumorigenesis. In renal cell carcinoma cells, in addition to Slfn2 and Slfn3, Slfn5 also exhibits important antineoplastic effects. Altogether, our findings indicate important functions for distinct mouse Slfn genes in the control of tumorigenesis and provide evidence for differential involvement of distinct members of this gene family in controlling tumorigenesis. They also raise the potential of future therapeutic approaches involving modulation of expression of members of this family of genes in malignant melanoma and renal cell carcinoma.

Arsenic compounds exert important biological effects and arsenic trioxide has been approved by the Food and Drug Administration (FDA) for the treatment of patients with acute promyelocytic leukemia (APL). Much of arsenic’s actions in cells reflect its ability to bind thiol groups in cellular proteins or to affect the production of reactive oxygen species (ROS), leading to the engagement and regulation of several cellular signaling pathways. Arsenic has been also shown to degrade abnormal fusion proteins found in myeloid leukemias. It has also been shown to effect NFκB, MAPK, mTOR and Hedgehog pathways which can modulate the viability of cancer cells. Many clinical trials have been performed to examine the clinical efficacy of arsenic trioxide alone or in combination with other agents in the treatment of various hematological malignancies. The continuous advances in basic and translational research and the better understanding of the mechanisms of action of arsenic should lead to more effective combinations with other agents that could result in better clinical outcomes.

Chronic myeloid leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL) are characterized by the presence of the BCR-ABL oncoprotein, which leads to activation of a plethora of pro-mitogenic and pro-survival pathways, including the mTOR signaling cascade. We provide evidence that in BCR-ABL expressing cells, treatment with tyrosine kinase inhibitors (TKIs) results in upregulation of mRNA levels and protein expression of sestrin3 (SESN3), a unique cellular inhibitor of mTOR complex 1 (mTORC1). Such upregulation appears to be mediated by regulatory effects on mTOR, as catalytic inhibition of the mTOR kinase also induces SESN3. Catalytic mTOR inhibition also results in upregulation of SESN3 expression in cells harboring the TKI-insensitive T315I-BCR-ABL mutant, which is resistant to imatinib mesylate. Overexpression of SESN3 results in inhibitory effects on different Ph+ leukemic cell lines including KT-1-derived leukemic precursors, indicating that SESN3 mediates anti-leukemic responses in Ph+ cells. Altogether, our findings suggest the existence of a novel mechanism for the generation of antileukemic responses in CML cells, involving upregulation of SESN3 expression.

SUMMARY Despite advances in molecular research related to acute myeloid leukemia (AML) and a better understanding of the mechanisms of leukemogenesis and pathophysiology of the disease, the pharmacological agents used in the treatment of AML have remained essentially unchanged for the last three decades. Advances in the clinical management of AML patients have been achieved by defining better molecular prognostic markers, but there remains a need for new targeted drugs that disrupt non-overlapping pathways in leukemia cells. The mTOR cellular cascade is critical for cell metabolism, growth, proliferation and survival. Extensive preclinical work suggests that targeting mTOR may provide a powerful approach to block AML precursor cells, while other findings suggest enhanced antileukemic effects by combining mTOR inhibitors with traditional chemotherapy. Such combinations may increase antileukemic responses further, offering unique ways to overcome leukemic cell resistance and to eliminate primitive leukemic precursors.

The kinases Mnk1 and Mnk2 are activated downstream of the p38 MAPK and MEK/ERK signaling pathways. Extensive work over the years has shown that these kinases control phosphorylation of the eukaryotic initiation factor 4E and regulate engagement of other effector elements, including hnRNPA1 and PSF. Mnk kinases are ubiquitously expressed and play critical roles in signaling for various cytokine receptors, and there is emerging evidence that they have important functions as mediators of proinflammatory cytokine production. In this review, the mechanisms of activation of MNK pathways by cytokine receptors are addressed and their roles in diverse cytokine-dependent biological processes are reviewed. The clinical-translational implications of such work and the relevance of future development of specific MNK inhibitors for the treatment of malignancies and autoimmune disorders are discussed.

Acute promyelocytic leukemia (APL) is a biologically distinct form of acute myeloid leukemia (AML), characterized by a translocation of the retinoic acid receptor α (RAR α ) gene on chromosome 17 [1]. Over 95% of cases result from t(15;17) (q22;q21), which allows fusion of the promyelocytic leukemia (PML) gene on chromosome 15 to RAR α , although a small minority of cases arise from variant translocations including t(11;17) and t(5;17) [1]. RAR α is believed to be important in the proliferation and diff erentiation of promyelocytes into neutrophils, and thus APL is characterized by the halting of granulocytes at the promyelocytic stage of diff erentiation in the bone marrow and peripheral blood. Also unique to APL compared to other forms of myeloid leukemia is the dramatic improvement in response with the use of diff erentiation agents. Th e combination of all- trans retinoic acid (ATRA) and arsenic trioxide (ATO), with or without chemotherapy, has achieved complete remission rates of 95% with 2-year overall survival 90% [2,3]. Th e prognosis of APL with additional cytogenetic abnormalities has also been studied. De Botton et al . [4] reported on the prognostic signifi cance of secondary chromosomal changes in 292 patients treated with ATRA and chemotherapy, fi nding no signifi cant diff erence in complete remission, event-free survival, relapse or overall survival at 2 years between patients with t(15;17) alone and those with other cytogenetic abnormalities. Twenty-six percent of patients included in this study were found to have additional chromosomal abnormalities, the most common of which was trisomy 8 in 45% of patients, followed by deletions or translocations involving chromosomes 9, 7, 21 and 17 [4]. In 2010, Cervera et al . [5] published data on 495 patients with APL treated with ATRA and chemotherapy, fi nding a similar overall rate of additional chromosomal abnormalities (28%), again with trisomy 8 as the most frequently noted. Similar complete remission rates were again observed, although patients with additional cytogenetic abnormalities had higher rates of coagulopathy, lower platelet counts and higher relapse-risk scores compared to those with t(15;17) alone [5]. Although additional cytogenetic abnormalities were signifi cantly associated with lower relapse-free survival, they were not identifi ed as independent risk factors for relapse [5].

The Schlafen (SLFN) family of proteins includes several mouse and human members. There is emerging evidence that members of this family of proteins are involved in important functions, such as the control of cell proliferation, induction of immune responses, and the regulation of viral replication. These proteins span across all species with great diversity, with 10 murine and 5 human isoforms. Recent work has established that mouse and human SLFN proteins are regulated by interferons (IFNs). Several Slfn genes were shown to be induced as classical interferon-stimulated genes, and emerging evidence suggests that these proteins play important roles in the growth inhibitory and antineoplastic effects of IFNs. In the current review, the known properties of mouse and human SLFNs are reviewed, and the implications of their emerging functions are discussed.

As signal transducers and transcription factors, signal transducer and activator of transcriptions (STATs) exert a critical role in cell growth, differentiation, and cell death. Their dysregulation that may lead to constitutive activation, no activation, or functional change, is associated with many different cancers. In this review, the contribution of STATs to cancer growth and progression is presented, and then the context in which interferon regulation of STAT activation provides a mechanism for therapeutic intervention for specific cancers is discussed.

Because of the central role of the mammalian target of rapamycin (mTOR) pathway in the control and distribution of signals essential for mRNA translation of mitogenic genes and generation of oncogenic proteins, effective targeting of mTOR remains a major goal in medical oncology.This article summarizes preclinical and clinical studies relating to the next generation of mTOR inhibitors. While rapalogs have shown activity in the treatment of breast, renal and neuroendocrine tumors, these agents do not block mTORC2, one of the two major protein complexes in which mTOR participates. In addition, there is emerging evidence that these agents only partially block mTORC1, underscoring the need for more effective mTOR inhibitors. In recent years, catalytic mTOR inhibitors have been developed, which block both mTORC1 and mTORC2. Such inhibitors show generally better activity in preclinical models than rapalogs and some of them have been or are in clinical trials in humans.It is anticipated that with the continuous expansion of work in this research field, the therapeutic potential of targeting the mTOR pathway for the treatment of several malignancies will reach a maximum point in the next few years and may ultimately change the way we treat several malignant tumors.

Mnk kinases regulate the phosphorylation and activation of the eukaryotic initiation factor 4E (eIF4E), a protein that plays key roles in the initiation of messenger RNA translation and whose activity is critical for various cellular functions. eIF4E is deregulated in acute myeloid leukemia (AML), and its aberrant activity contributes to leukemogenesis. We determined whether cercosporamide, an antifungal agent that was recently shown to act as a unique Mnk inhibitor, exhibits antileukemic properties. Treatment of AML cells with cercosporamide resulted in a dose-dependent suppression of eIF4E phosphorylation. Such suppression of Mnk kinase activity and eIF4E phosphorylation by cercosporamide resulted in dose-dependent suppressive effects on primitive leukemic progenitors (CFU-L) from AML patients and enhanced the antileukemic properties of cytarabine (Ara-C) or mammalian target of rapamycin (mTOR) complex 1 inhibition. Similarly, the combination of cercosporamide with cytarabine resulted in enhanced antileukemic responses in a xenograft mouse model in vivo. Altogether, this work demonstrates that the unique Mnk inhibitor cercosporamide suppresses phosphorylation of eIF4E and exhibits antileukemic effects, in support of future clinical-translational efforts involving combinations of Mnk inhibitors with cytarabine and/or mTOR inhibitors for the treatment of AML.

Therapeutic success and prognosis of patients with acute myeloid leukemia (AML) largely depend on a variety of patient- and disease-specific characteristics. For over 20 years the recurrent chromos...

All Type I interferons (IFNα, IFNβ, IFNω) bind to the Type I IFN receptor (IFNR) and elicit a common set of signaling events, including activation of the Jak/Stat and IRS pathways. However, IFNβ selectively induces the association of the α subunit of the Type I IFNR with p100, a tyrosyl phosphoprotein, to transduce IFNβ-specific signals. Using antibodies raised against the different components of the Type I IFNR, we identified p100 as the long form of the β subunit (βL subunit) of the Type I IFNR. This was also confirmed in experiments with mouse L-929 cells transfected with truncated forms of βL. Thus, IFNβ stimulation of human cells or mouse L-929 transfectants expressing the human α and βL subunits, selectively induces the formation of a signaling complex containing the α and βL subunits of the receptor. The IFNβ-regulated interaction of the α and βL chains is rapid and transient and follows a similar time course with the tyrosine phosphorylation of these receptor components. These data demonstrate that the signaling specificity for different Type I IFNs is established early in the signaling cascade, at the receptor level, and results from distinct interactions between components of the Type I IFNR.

Binding of interferon alpha (IFN alpha) to its receptor induces activation of the Tyk-2 and Jak-1 tyrosine kinases and tyrosine phosphorylation of multiple downstream signaling elements, including the Stat components of the interferon-stimulated gene factor 3 (ISGF-3). IFN alpha also induces tyrosine phosphorylation of IRS-1, the principle substrate of the insulin receptor. In this study we demonstrate that various Type I IFNs rapidly stimulate tyrosine phosphorylation of IRS-2. This is significant since IRS-2 is the major IRS protein found in hematopoietic cells. The IFN alpha-induced phosphorylated form of IRS-2 associates with the p85 regulatory subunit of the phosphatidylinositol 3'-kinase, suggesting that this kinase participates in an IFN alpha-signaling cascade downstream of IRS-2. We also provide evidence for an interaction of IRS-2 with Tyk-2, suggesting that Tyk-2 is the kinase that phosphorylates this protein during IFN alpha stimulation. A conserved region in the pleckstrin homology domain of IRS-2 may be required for the interaction of IRS-2 with Tyk-2, as shown by the selective binding of glutathione S-transferase (GST) fusion proteins containing the IRS-2-IH1PH or IRS-1-IH1PH domains to Tyk-2 but not other Janus kinases in vitro.

The tyrosine kinase Tyk-2 is physically associated with the Type I interferon (IFN) receptor complex and is rapidly activated during IFN alpha stimulation. We report that Tyk-2 forms stable complexes with the SH2-containing hematopoietic cell phosphatase (HCP) in several hematopoietic cell lines in vivo, and that the IFN alpha-induced tyrosine-phosphorylated form of Tyk-2 is a substrate for the phosphatase activity of HCP in in vitro assays. Furthermore, treatment of cells with the phosphatase inhibitor sodium orthovanadate induces tyrosine phosphorylation of Tyk-2 and an associated 115-kDa protein. Altogether, these data suggest that HCP regulates tyrosine phosphorylation of the Tyk-2 kinase, and thus its function may be important in the transmission of signals generated at the Type I IFN receptor level.

Introduction: Genomic analysis of circulating tumor cell-free DNA (cfDNA) represents a non-invasive method of assessing genomic alterations using peripheral blood. We compared the concordance between cfDNA and tissue biopsies across genomic alterations in 65 genes in this pilot retrospective study. Methods: 54 consecutive patients with cfDNA NGS testing (Guardant360) were identified. Of these, 28 had tissue DNA NGS testing (Foundation One). 65 genes were common to both assays. Concordance was defined as the absence or presence of the identical genomic sequencing alteration(s) in a single gene. The analysis included non-synonymous DNA mutations, rearrangements, and copy number variants regardless of clone percentage. Results: There were 14 lung cancers and 14 other advanced solid tumors. Including all alterations and variants of unknown significance (VUS), the average number of alterations per patient for tissue and cfDNA was 4.82 (SD 3.02) and 2.96 (SD 3.01), respectively. Concordance between the two assays was 91.9%. Among only genes with reported genomic alterations in either assay (n = 170), concordance was 12.4%. Concordance was similar when stratifying based on timeframe between biopsies (median time interval between assays; 89 days). 21.5% of alterations found in tissue were detected in cfDNA. 34.9% of alterations found in cfDNA were detected in tissue. Across the 5 most common genes, sensitivity and specificity were 40.0% and 94.2%, respectively (Table 1). Discussion: Concordance between tumor tissue and cfDNA NGS was 91.9%. The cfDNA assay had high specificity, but low sensitivity. In addition, more mutations were detected in tissue. Reasons for these findings may include different sequencing techniques, spatial and temporal factors, tumor heterogeneity, and inclusion of subclones. [AD and YC contributed equally.] Table 1: Diagnostic Accuracy across 5 Most Common Genes Tissue MutationsSensitivity (%)Specificity (%)PPV (%)NPV (%)Diagnostic Accuracy (%)cfDNA mutations(+)(-)TP53(+)82(-)8850.080.080.050.057.1EGFR(+)12(-)22333.392.033.392.085.7KRAS(+)41(-)22166.795.580.091.389.3APC(+)01(-)4230.095.80.085.282.1CDKN2A(+)10(-)52216.7100.0100.081.582.1Total positive146Total negative2197Total (positive + negative)3510340.094.270.082.280.4 PPV: positive predictive value NPV: negative predictive value Citation Format: Young Kwang Chae, Andrew A. Davis, Benedito A. Carneiro, Sunandana Chandra, Nisha Mohindra, Aparna Kalyan, Jason Kaplan, Maria Matsangou, Leonidas C. Platanias, Massimo Cristofanilli, Francis J. Giles. Concordance of genomic alterations by next generation sequencing (NGS) in tumor tissue vs. cell-free DNA. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5233.

Our recent work suggested that inhibition of mTORC1 activates Mnk in a PI3K-dependent manner, thereby providing a survival mechanism for medulloblastoma cells. The PI3K-Akt axis represents an important survival pathway that also confers therapy resistance to medulloblastoma stem cells, resulting in tumor recurrence. Here, we investigated a role for p110 isoforms of PI3K in medulloblastoma stem-like cancer cell biology and studied the potential of Mnk inhibition for sensitizing medulloblastoma stem-like cancer cells and orthotopic xenograft tumors to PI3K inhibition. We used medulloblastoma cell lines Daoy and D556 grown as conventional 2-D cultures or under stem-cell conditions as 3-D neurospheres to elucidate the roles of PI3K-Akt signaling in medulloblastoma proliferation, colony formation and stem cell function. We employed extreme limiting dilution analysis (ELDA) to ask whether concomitant Mnk inhibition enhances antineoplastic effects of PI3K inhibition on cancer stem cell growth. Additionally, in a preliminary intracereballar xenograft mouse study, we investigated the effects of pharmacologic PI3K and Mnk inhibition. We found that Akt activity greatly increased when 2-D cultures were converted into 3-D neurospheres. This Akt activation coincided with increased expression of CD133 and nestin, suggesting an important role for PI3K-Akt signaling in medulloblastoma stem cells. The p110a specific inhibitor BYL-719 blocked this Akt activation in neurospheres indicating this Akt activation is mediated by p110a. Consistently, of all class I PI3K catalytic isoforms (p110a, p110b, p110d and p110g) only knockdown of p110a disrupted stem cell frequencies in ELDA. We previously reported that mTORC1 inhibition engages Mnk signaling in a negative feedback manner to promote survival. Here we show that Mnk inhibition by CGP57380 sensitized medulloblastoma cells for pharmacologic inhibition and siRNA-mediated knockdown of p110a both in 2-D cancer cells and 3-D stem-like cancer cell cultures. After intracerebellar injection of medulloblastoma cells in nude mice, we found that combined targeting of PI3K-p110a and Mnk results in inhibition of tumor growth and increased survival. In summary, pharmacologic inhibition of PI3K-p110a by BYL-719 showed potent activity against medulloblastoma cells and stem-like cancer cells. Knockdown of p110a disrupted cancer stem cell frequency in ELDA and this effect was greatly enhanced by pharmacologic inhibition of Mnk. Finally, in an orthotopic mouse model we found that concomitant inhibition of p110a and Mnk prolonged survival and reduced tumor size. The striking effects of concomitant Mnk inhibition on stem-like cancer cells, neurospheres and tumors is particularly interesting as it suggests enhanced vulnerability of the therapy-resistant, tumor-initiating cancer stem cell population to p110a inhibition in medulloblastoma. Citation Format: Jonathan B. Bell, Frank Eckerdt, Quanhong Ma, Jessica R. Clymer, Stewart Goldman, Rintaro Hashizume, Leonidas C. Platanias. Mnk targeting enhances vulnerability of medulloblastoma stem-like cancer cells to PI3K-p110alpha inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-009.

MB-67. DUAL TARGETING OF PI3K AND mTOR SIGNALING IN MEDULLOBLASTOMA Jessica Clymer1,2, Frank Eckerdt2, Jonathan Bell2, Rishi Lulla1,2, Stewart Goldman1,2, and Leonidas Platanias2,3; Division of Hematology/ Oncology/Stem Cell Transplantation, Department of Pediatrics, Ann & Robert H. Lurie Children’s Hospital, Chicago, IL, USA; Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA; Division of Hematology/Oncology, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA BACKGROUND: Aberrant activation of the PI3K pathway has been shown to play a role in medulloblastoma cell proliferation, while mTOR activation contributes to therapy resistance. Here we sought to evaluate the effects of combined targeting of the PI3K and mTOR pathways in medulloblastoma. METHODS: Medulloblastoma cell lines Daoy and D556 were grown in 2-D cultures to investigate the effects of pharmacologic inhibition of PI3K and mTORon kinase signaling, medulloblastomacell proliferation, colony formation, and apoptosis. After knockdown of p110 isoforms, cells were also grown under stem cell conditions as 3-D neurospheres and subjected to extreme limiting dilution analysis (ELDA) for assessment of stem-like cancer cell growth. RESULTS: Of all class I PI3K isoforms (p110a, p110b, p110d, and p110g), only p110a was essential for medulloblastoma cell proliferation, colony formation and neurosphere growth. Accordingly, pharmacologic targeting of the p110a isoform of PI3K with BYL-719 synergizedwith the mTOR inhibitor OSI-027, as it resulted in inhibition of effector pathways, blocked cell proliferation and colony formation, and increased malignant cell death by apoptosis. Finally,BYL-719and OSI-027 greatly impairedgrowthof stem-likecancer cells as 3-D neurospheres. CONCLUSIONS: The p110a isoform of PI3K exerts crucial roles in medulloblastoma. Inhibition of p110a and mTOR exerts potent antineoplastic effects on medulloblastoma cells and also blocks neurosphere growth. Dual inhibition of p110a and mTOR might be promising for targeting both malignant medulloblastoma cells and the therapy-resistant cancer stem cell population. Neuro-Oncology 18:iii97–iii122, 2016. doi:10.1093/neuonc/now076.63 #The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

3042Background: Mutations in DNA repair genes are common in cancer, and may induce a state of hypermutability. Importantly, mutational burden is linked to enhanced response to immunotherapies, as p...

11528Background: Cell-free DNA (cfDNA) is a non-invasive method of assessing mutations in the blood. This study compared the concordance between cfDNA and tissue biopsies across DNA genomic alterations in patients with advanced breast cancer (ABC). Methods: Forty-five patients with ABC who had both NGS testing with cfDNA (Guardant) and tissue DNA (Foundation One) for clinical management were included. 45-67 genes were common to both assays depending on when the assays were performed. Concordance was defined as the presence or absence of the identical genomic sequencing alteration(s) in a single gene. Results: Median time between assays was 3.3 months [range, 0-67 months]. Including all alterations and variants of unknown significance, the average number per patient for tissue and cfDNA was 4.50 (SD 2.96) and 1.98 (SD 2.13), respectively. Concordance between the two assays was 91.2%. Among only genes with reported genomic alterations in either assay (N=232), concordance was 10.3%. 15.0% of alterations foun...

Insulin activates the ras signaling pathway and promotes hematopoietic cell proliferation. One possible mediator in such signaling is the vav proto-oncogene product (p95vav), which is specifically expressed in cells of hematopoietic origin and contains domains typical of guanine nucleotide exchange factors as well as Src homology 2 and Src homology 3 domains. We studied the tyrosine phosphorylation of p95vav in hematopoietic cells expressing insulin receptors. Immunoblotting experiments with an antiphosphotyrosine monoclonal antibody disclosed that insulin induces rapid and transient tyrosine phosphorylation of p95vav in the human U-266 myeloma cell line. These findings were confirmed by immunoprecipitation experiments performed with 32P-labeled cells and phosphoamino acid analysis of the bands corresponding to p95vav. Similarly, insulin-dependent tyrosine phosphorylation of p95vav was observed in the human IM-9 and mouse J558L hematopoietic cell lines. Furthermore, insulin treatment of cells led to the association of the Src homology 2 domain of p95vav with the activated beta-subunit of the insulin receptor in vitro. Altogether, these data suggest that p95vav is a substrate for the insulin receptor tyrosine kinase and may be involved in an insulin signaling pathway linking receptor-generated signals to Ras or other GTP-binding proteins in cells of hematopoietic origin.

Interferons (IFNs) have important antiviral and antineoplastic properties, but the precise mechanisms required for generation of these responses remain to be defined. We provide evidence that during engagement of the Type I IFN receptor (IFNR), there is up-regulation of expression of Sprouty (Spry) proteins 1, 2, and 4. Our studies demonstrate that IFN-inducible up-regulation of Spry proteins is Mnk kinase-dependent and results in suppressive effects on the IFN-activated p38 MAP kinase (MAPK), the function of which is required for transcription of interferon-stimulated genes (ISGs). Our data establish that ISG15 mRNA expression and IFN-dependent antiviral responses are enhanced in Spry1,2,4 triple knock-out mouse embryonic fibroblasts, consistent with negative feedback regulatory roles for Spry proteins in IFN-mediated signaling. In other studies, we found that siRNA-mediated knockdown of Spry1, Spry2, or Spry4 promotes IFN-inducible antileukemic effects in vitro and results in enhanced suppressive effects on malignant hematopoietic progenitors from patients with polycythemia vera. Altogether, our findings demonstrate that Spry proteins are potent regulators of Type I IFN signaling and negatively control induction of Type I IFN-mediated biological responses.

We have recently shown that arsenic trioxide (As 2O 3) is a potent inducer of autophagic degradation of the BCR-ABL1 oncoprotein, which is the cause of chronic myeloid leukemia (CML) and Ph+ acute lymphoid leukemia (Ph+ ALL). Our recently published work has shown that pharmacological inhibition of autophagy or molecularly targeting of elements of the autophagic machinery partially reverses the suppressive effects of As 2O 3 on primitive leukemic precursors from CML patients. Altogether, our studies have provided direct evidence that arsenic-induced, autophagy-mediated, degradation of BCR-ABL1 is an important mechanism for the generation of the effects of As 2O 3 on BCR-ABL1 transformed leukemic progenitors. These studies raise the potential of future clinical-translational efforts employing combinations of arsenic trioxide with autophagy-modulating agents to promote elimination of early leukemic progenitors and, possibly, leukemia-initiating stem cells.

The target of rapamycin (TOR) pathway is highly conserved among eukaryotes and has evolved to couple nutrient sensing to cellular growth. TOR is found in two distinct signaling complexes in cells, TOR complex 1 (TORC1) and TOR complex 2 (TORC2). These complexes are differentially regulated and act as effectors for the generation of signals that drive diverse cellular processes such as growth, proliferation, protein synthesis, rearrangement of the cytoskeleton, autophagy, metabolism and survival. Mammalian TOR (mTOR) is very important for development in embryos, while in adult organisms it is linked to aging and lifespan effects. In humans, the mTOR pathway is implicated in the tumorigenesis of multiple cancer types and its deregulation is associated with familial cancer syndromes. Because of its high biological relevance, different therapeutic strategies have been developed to target this signaling cascade, resulting in the emergence of unique pharmacological inhibitors that are either already approved for use in clinical oncology or currently under preclinical or clinical development. Multimodal treatment strategies that simultaneously target multiple nodes of the pathway and/or negative feedback regulatory loops may ultimately provide the best therapeutic advantage in targeting this pathway for the treatment of malignancies.

We provide evidence that arsenic trioxide (As2O3) targets the BCR-ABL oncoprotein via a novel mechanism involving p62/SQSTM1-mediated localization of the oncoprotein to the autolysosomes and subsequent degradation mediated by the protease cathepsin B. Our studies demonstrate that inhibitors of autophagy or cathepsin B activity and/or molecular targeting of p62/SQSTM1, Atg7, or cathepsin B result in partial reversal of the suppressive effects of AS2O3 on BCR-ABL expressing leukemic progenitors, including primitive leukemic precursors from chronic myelogenous leukemia (CML) patients. Altogether, these findings indicate that autophagic degradation of BCR-ABL is critical for the induction of the antileukemic effects of As2O3 and raise the potential for future therapeutic approaches to target BCR-ABL expressing cells by modulating elements of the autophagic machinery to promote BCR-ABL degradation.

Background A number of constitutively activated signaling pathways play critical roles in the survival and growth of primary effusion lymphoma cells (PELs) including NFkB and PI3/AKT kinase cascades. NFkBis constitutively activated in a number of malignancies, including multiple myeloma, Burkitt’s lymphoma and diffuse large cell B-cell lymphoma. However, its role in primary effusion lymphoma has not been fully explored. Methodology/Principal Findings We used pharmacological inhibition and gene silencing to define the role of NFkB in growth and survival of PEL cells. Inhibition of NFkB activity by Bay11-7085 resulted in decreased expression of p65 in the nuclear compartment as detected by EMSA assays. In addition, Bay11-7085 treatment caused de-phosphorylation of AKT and its downstream targets suggesting a cross-talk between NFkB and the PI3-kinase/AKT pathway. Importantly, treatment of PEL cells with Bay11-7085 led to inhibition of cell viability and induced apoptosis in a dose dependent manner. Similar apoptotic effects were found when p65 was knocked down using specific small interference RNA. Finally, co-treatment of PEL cells with suboptimal doses of Bay11-7085 and LY294002 led to synergistic apoptotic responses in PEL cells. Conclusion/Significance These data support a strong biological-link between NFkB and the PI3-kinase/AKT pathway in the modulation of anti-apoptotic effects in PEL cells. Synergistic targeting of these pathways using NFKB- and PI3-kinase/AKT- inhibitors may have a therapeutic potential for the treatment of PEL and possibly other malignancies with constitutive activation of these pathways.

The precise mechanisms by which the activation of interferon (IFN) receptors (IFNRs) ultimately controls mRNA translation of specific target genes to induce IFN-dependent biological responses remain ill defined. We provide evidence that IFN-α induces phosphorylation of programmed cell death 4 (PDCD4) protein on Ser67. This IFN-α-dependent phosphorylation is mediated by either the p70 S6 kinase (S6K) or the p90 ribosomal protein S6K (RSK) in a cell-type-specific manner. IFN-dependent phosphorylation of PDCD4 results in downregulation of PDCD4 protein levels as the phosphorylated form of PDCD4 interacts with the ubiquitin ligase β-TRCP (β-transducin repeat-containing protein) and undergoes degradation. This process facilitates IFN-induced eukaryotic translation initiation factor 4A (eIF4A) activity and binding to translation initiation factor eIF4G to promote mRNA translation. Our data establish that PDCD4 degradation ultimately facilitates expression of several ISG protein products that play important roles in the generation of IFN responses, including IFN-stimulated gene 15 (ISG15), p21(WAF1/CIP1), and Schlafen 5 (SLFN5). Moreover, engagement of the RSK/PDCD4 pathway by the type I IFNR is required for the suppressive effects of IFN-α on normal CD34(+) hematopoietic precursors and for antileukemic effects in vitro. Altogether, these findings provide evidence for a unique function of PDCD4 in the type I IFN system and indicate a key regulatory role for this protein in mRNA translation of ISGs and control of IFN responses.

IFNs transduce signals by binding to cell surface receptors and activating cellular pathways and regulatory networks that control transcription of IFN-stimulated genes (ISGs) and mRNA translation, leading to generation of protein products that mediate biological responses. Previous studies have shown that type I IFN receptor-engaged pathways downstream of AKT and mammalian target of rapamycin complex (mTORC) 1 play important roles in mRNA translation of ISGs and the generation of IFN responses, but the roles of mTORC2 complexes in IFN signaling are unknown. We provide evidence that mTORC2 complexes control IFN-induced phosphorylation of AKT on serine 473 and their function is ultimately required for IFN-dependent gene transcription via interferon-stimulated response elements. We also demonstrate that such complexes exhibit regulatory effects on other IFN-dependent mammalian target of rapamycin-mediated signaling events, likely via engagement of the AKT/mTORC1 axis, including IFN-induced phosphorylation of S6 kinase and its effector rpS6, as well as phosphorylation of the translational repressor 4E-binding protein 1. We also show that induction of ISG protein expression and the generation of antiviral responses are defective in Rictor and mLST8-KO cells. Together, our data provide evidence for unique functions of mTORC2 complexes in the induction of type I IFN responses and suggest a critical role for mTORC2-mediated signals in IFN signaling.