Phosphorylated protein chip combined with artificial intelligence tools for precise drug screening

Cellular function, proliferation, differentiation and death are regulated through a number of extracellular stimuli which mediate the transcription of specific genes, either directly or indirectly via specific intracellular pathways (Alberts et al, 1994). In leukaemia the mediators at work are primarily haematopoietic growth factors and cytokines (Table I). These small polypeptides or glycoproteins play pivotal roles in normal and neoplastic cell proliferation by binding to their cell surface receptors and thus activating signal transduction pathways leading to gene transcription (Carpenter et al, 1999; Shuai, 1999). Three general families of cell-surface receptors have been characterized by their modes of action: ion channel-linked receptors, G-protein-linked receptors and enzyme-linked receptors. The first two families are involved in processes such as neurotransmitter signalling and are of little known significance in cytokine action. The enzyme-linked receptors, however, acting either directly as enzymes or in association with enzymes, activate intracellular events and play important roles in cytokine function. Five known classes of enzyme-linked receptors have been described: (1) receptor guanylyl cyclases, (2) receptor tyrosine phosphatases, (3) receptor serine/threonine kinases, (4) receptor tyrosine kinases, and (5) tyrosine kinase-associated receptors (Alberts et al, 1994). Most growth factors and cytokines bind to receptor tyrosine kinases and tyrosine kinase-associated receptors on the cell surface. Some tyrosine kinase-associated receptors have no inher- ent kinase activity but can regulate the haematopoietic system by activating a variety of non-receptor protein tyrosine kinases; these have been named the cytokine receptor superfamily (Bazan, 1990; Ihle et al, 1998). As aberrant signalling through these receptors and their downstream pathways plays a significant role in leukaemogenesis, their modulation will probably result in therapeutic benefits (Frank, 1999). Studies directed at understanding interferon (IFN) function have provided crucial information on the signalling pathways activated by IFNs and several other cytokines (Darnell et al, 1994; Ihle et al, 1998; Frank, 1999; Shuai, 1999). These pathways involve the activation of a family of non-receptor tyrosine kinases called Janus kinases (Jaks), and the recruitment by Jaks of another family of proteins called signal transducer and activator of transcription (Stat) that induce gene transcription (Decker, 1999; Shuai, 1999). As Jak-Stat pathways are utilized by a variety of growth factors, cytokines and hormones (Darnell et al, 1994; Taniguchi, 1995; Shuai, 1999), better understanding of their role in normal haematopoiesis and in leukaemogenesis will probably lead to more selective and less toxic therapies. This approach has already been proven to be effective in initial studies of the drug STI571 in chronic myelogenous leukaemia (CML) (Druker et al, 1999; Talpaz et al, 2000). The cytokine receptor superfamily is divided into four subfamilies on the basis of the characteristic structural motifs found in their extracellular binding domains (Fig 1A) (Bazan, 1990; Alberts et al, 1994; Taniguchi, 1995; Ihle et al, 1998). These extracellular domains are characterized by the presence of conserved motifs, suggesting their common evolution to utilize common signalling pathways. Many cytokine receptors consist of two or more subunits (named α, β and γ) with one subunit common to several cytokine receptors (Taniguchi, 1995). For example, in the gp130 subfamily, the receptors for interleukin 6 (IL-6), IL-11, oncostatin M (OSM), leukaemia inhibitory factor (LIF) and ciliary neurotropic factor (CNTF) all signal through a common βc-chain called gp130 (Fig 1B). The sharing of receptor subunits allows the cytokine receptors to initiate biological cytokine activities that are both specific and redundant (Taniguchi, 1995; Carpenter et al, 1999). The cytokine receptor superfamily. (A) Cytokine receptors subfamilies. Receptor components shared by different subtypes are shown in the same colour. For example, members of the type 1 receptor group are characterized by the presence of multiple fibronectin type III repeats in the extracellular domain. Type 1 receptors include the gp130 family, the IL-2 receptor, the gp140 family and the GH receptor. Type 2 receptors include interferon and IL-10 receptors. Type 3 receptors include members of tumour necrosis factor (TNF) and Fas receptors. Type 4 includes the IL-1 receptor. (B) The gp130 receptor family are compared with receptor tyrosine kinases (RTK). Note that some members of the RTK family, independently of Jaks, can mediate Stat activation. The receptors for IL-12, granulocyte colony-stimulating factor (G-CSF) and leptin have a similar extracellular binding domain and are therefore classed in the same family, although no clear association of these receptors with gp130 has been clearly demonstrated. All receptors of the gp130 family, with the exception of IL-12 receptor, activate Stat3. Dimerization of the cytoplasmic component of the cytokine receptor is the necessary next step in the initiation of cellular signalling (Carpenter et al, 1999). The dimerized units then associate with intracellular non-receptor tyrosine kinases such as members of the Src or the Jak families of kinases to further propagate the signal (Fig 1A). Different Src family members are associated with different receptors and phosphorylate distinct but overlapping sets of proteins (Stahl et al, 1993; Alberts et al, 1994). For example, Lck, Lyn and Fyn have been shown to be activated by IL-2 and to associate with the same region of the IL-2 receptor (Kobayashi et al, 1993). Src kinases can also associate with receptors having intrinsic kinase activity, thus accounting for the overlap seen between the pathways activated by the Src family of tyrosine kinase-associated receptors and by the receptor tyrosine kinases. The Jak family of kinases (Jak1, Jak2, Jak3 and Tyk2) includes relatively large proteins that are able to bind cytokine receptor β components. (Duhe & Farrer, 1998; Ihle et al, 1998; Yeh & Pellegrini, 1999; Ward et al, 2000). The highly conserved structure of the Jak family members consists of seven Jak homology (JH) domains (Taniguchi, 1995; Yeh & Pellegrini, 1999). The JH1 domain at the carboxy terminus of the protein includes a domain with tyrosine kinase activity (Fig 2A). In general, Jak proteins are divided into an amino-terminal region (N), a catalytically inactive kinase-like (KL) domain and the tyrosine kinase (TK) domain (Yeh & Pellegrini, 1999). The cytokines, growth factors and hormones that activate Jaks are listed in Table II. Jaks are able to associate non-covalently with the cytokine receptors through their cytoplasmic domain as well as with each other (Livnah et al, 1999; Remy et al, 1999). This association results in transphosphorylation and activation of the kinase activity of Jaks, and phosphorylation of other downstream targets (Fig 2B). The Jak-Stat pathway. (A) Domains of Jak protein. JH1 is the catalytic domain with tyrosine kinase (TK) activity. KL is a catalytically inactive kinase-like domain with unknown function. SOCS are suppressors of cytokine signalling which inhibit Jaks. RBR is the receptor binding region. 'SH2' is a SH2-like domain with unidentified function. (B) Mechanism of Stat activation by cytokine receptors and their negative regulation by SOCS. Ligand binding and receptor dimerization result in Jak activation, Stat dimerization, and translocation into the nucleus and transcription of genes including those coding for SOCS. The latter have a negative regulatory effect on the pathway. The crucial role of Jaks in cytokine signalling has been established in mouse knockout models. Newborn mice with homozygously disrupted Jak1 are small, die early, and are profoundly deficient in thymocyte production and B-lymphocyte differentiation (Rodig et al, 1998). Jak2-deficient mice embryos do not respond to various haematopoietic cytokines, respond poorly to colony-stimulating factor 1 (CSF-1) and stem cell factor (SCF), and die of severe anaemia as a result of ineffective erythropoiesis (Neubauer et al, 1998; Parganas et al, 1998). Jak3-deficient mice have profoundly defective immune systems characterized by a hypoplastic thymus, non-functional peripheral T cells and a lack of natural killer cells among other defects (Thomis et al, 1995; Eynon et al, 1996). Several disorders are associated with Jak-related abnormalities. These include severe combined immunodeficiency (SCID) due to Jak3 mutations (Notarangelo, 1996), leukaemia due to chromosomal translocations resulting in TEL/JAK2 constructs causing constitutive Stat5 activation, and IL-3-independent cellular proliferation (Lacronique et al, 1997; Peeters et al, 1997). Furthermore, infection with human T-lymphotropic virus 1 (HTLV-1) and Abelson murine leukaemia viruses results in enhanced tyrosine kinase activity of Jaks, which may account for their leukaemogenic potential (Danial et al, 1995; Migone et al, 1995). Jak proteins interact with a number of intracellular signalling proteins, most of which undergo tyrosine phosphorylation upon cytokine stimulation and are therefore likely to be substrates and downstream effectors of Jaks (Table III). Although the biological effects of most of these interactions are still unknown, it is hypothesized that Jaks regulate many cytokine signalling events. The Stat proteins were first identified in the late 1980s in studies aimed at understanding the molecular mechanisms of IFN-mediated effects. These studies identified a number of genes whose transcription was induced by IFN-α and IFN-γ. Specific DNA elements were found to bind proteins such as interferon-α-stimulated gene factor-3 (ISGF-3) (Fu et al, 1992; Khan et al, 1993). These factors, located in the cytoplasm, are rapidly translocated to the nucleus and bind to their cognate DNA sites upon IFN stimulation (Shuai et al, 1992). ISGF-3 consists of three proteins: p48, p91/p84 and p113. The latter two are related proteins also known as Stat1α/β and Stat 2 respectively (Schindler et al, 1992). Five additional Stat proteins (Stat3, Stat4, Stat5A, Stat5B and Stat6) have been identified and their activation by various cytokines documented (Table II) (Hou et al, 1994; Yamamoto et al, 1994; Zhong et al, 1994; Mui et al, 1995). Stats are transcription factors whose activities are modified and regulated by tyrosine phosphorylation (Shuai et al, 1993a; Darnell, 1997). This process has been shown to be necessary for Stat homo- or heterodimerization, translocation to the nucleus, DNA binding and gene activation (Fig 2B) (Shuai et al, 1994; Zhong et al, 1994). All Stat proteins share conserved domains important for their function (Shuai, 1999). The SH2 domain is necessary for Stat tyrosine phosphorylation (Shuai et al, 1993b). The binding of a cytokine to the α subunit of the cytokine receptor rapidly induces tyrosine phosphorylation of the cytoplasmic domains of the receptor β subunit by activated Jak kinases, thus providing a docking site for Stat proteins (as well as other SH2-containing proteins such as the growth factor receptor binding protein 2, or Grb2). The SH2 domain of a Stat protein is thought to bind the tyrosine-phosphorylated residue of the cytokine β receptor with specific cytokine receptors activating specific Stats (Uddin et al, 1995; Yan et al, 1996). The SH2 domains are also necessary for Stat dimer formation (Heim et al, 1995; Carpenter et al, 1999; Decker, 1999; Shuai, 1999) and the N-terminal region of the Stat molecule for DNA binding (Vinkemeier et al, 1996; Xu et al, 1996). The differential DNA binding affinities of different Stat proteins are important in achieving specificity in Stat-mediated gene activation. To induce gene transcription, Stat proteins interact with other transcription factors such as the p300/CREB binding protein (CBP) family of co-activators, which requires histone acetyltransferase activity (Bhattacharya et al, 1996; Horvai et al, 1997; Decker & Kovarik, 1999; Heim, 1999). The transcriptional activity of Stats can also bemodulated by phosphorylation of their serine and threonine residues, although the mechanistic implications of such regulation are not entirely clear (Eilers et al, 1995; Decker, 1999). While serine phosphorylation is not sufficient to induce Stat activation, it can enhance it. Kinases involved in this process include Jun NH-2 kinase (JNK), mitogen-activated protein kinase (MAPK), protein kinase C and protein kinase A (Frank, 1999). The function of various Stats has been elucidated through the creation of mutant mice with disrupted Stat genes (Levy, 1999; Ward et al, 2000). Mice deficient in Stat1 are extremely sensitive to viral infections, consistent with Stat1's presumed importance in IFN function. (Durbin et al, 1996; Meraz et al, 1996) Mice deficient in Stat3, however, demonstrate early embryonic lethality (Takeda et al, 1997). Stat4-deficient mice have no developmental abnormalities but are completely resistant to IL-12 stimulation and fail to induce Th1 polarization of T-helper lymphocytes (Kaplan et al, 1996a; Thierfelder et al, 1996). Stat5A/B knockout mice develop normally except for defects in adult mammary gland development and lactogenesis (Liu et al, 1997; Udy et al, 1997). Stat6-deficient mice are not overtly abnormal but do exhibit defects in IL-4 function (e.g. a complete lack of IL-4-induced Th2 polarization of T-helper cells) (Shimoda et al, 1996). Stat activation and signalling are involved in diverse and sometimes opposite cellular events affecting growth, differentiation and apoptosis (Schindler, 1998; Frank, 1999; Mui, 1999). Stat activation, for example, may result in both growth arrest and cellular proliferation. Stat1 mediates the growth-inhibitory effects of IFN-γ by inducing the cyclin-dependent kinase inhibitor p21waf1, whereas Stat5 induces the proliferative effects of IL-3 and granulocyte–macrophage colony-stimulating factor (GM-CSF) (Chin et al, 1996; Mui et al, 1996). Similarly, in myeloid cells, phosphorylation of Stat3 can lead to IL-6 and IL-10-induced growth arrest (via induction of the cyclin-dependent kinase inhibitor p19INK4d) on the one hand and to GM-CSF- and IL-3-induced proliferation on the other (Mui et al, 1996; Chaturvedi et al, 1998; O'Farrell et al, 1998). Whether these diverse effects are tissue-specific or mediated by other factors is yet to be determined. Stats also mediate transcriptional events associated with cellular differentiation (Mui, 1999). Stat3 and Stat5 have been implicated in myeloid differentiation and Stat4 and Stat6 in Th cell differentiation. Stat3 is involved in IL-6-induced differentiation of M1 myeloid leukaemia cells, but suppresses differentiation in other cell lines (Yamanaka et al, 1996). Stat5 has been implicated in cell differentiation in a variety of systems, including thrombopoietin-dependent maturation of megakaryocytes and erythropoietin-dependent maturation of erythroid cell lines (Mui, 1999). When activated by IL-4, Stat6 helps regulate the expression of cell-surface proteins such as major histocompatibility complex (MHC) class II antigens, immunoglobulin class switching and Th2 differentiation (Rothman et al, 1991; Kaplan et al, 1996b). Therefore, Stat activation may play a key role in overcoming differentiation arrest. Stats are also important in regulating programmed cell death. In fact, constitutive expression of caspases necessary for induction of apoptosis in fibroblasts may depend on Stat1 (Kumar et al, 1997). For example, reconstitution of Stat1 in Stat1-null U3A cells, which do not respond to tumour necrosis factor α (TNF-α), restores basal caspase expression and sensitivity to TNF-induced apoptosis (Kumar et al, 1997). Conversely, Stat3 and Stat5 contribute to the antiapoptotic effects of IL-6 and IL-2 respectively (Fukada et al, 1996; Zamorano & Keegan, 1998). In response to IFN-γ, Stat1 mediates the upregulated expression of Fas expression and FasL, thereby activating the caspase cascade (Xu et al, 1998). Activation of the Jak-Stat pathway is both rapid and transient. Following stimulation by IFNs, Stat1 phosphorylation peaks within 15–30 min and returns to basal levels after 1–2 h. Three possible mechanisms of Stat inactivation following stimulation have been postulated: proteasome-mediated degradation, tyrosine dephosphorylation, and inhibition by various inhibitory proteins (Shuai, 1999). The first mechanism involves the ubiquitin-proteasome pathway, which is responsible for the degradation of many proteins involved in fundamental cellular processes (Kim & Maniatis, 1996). The second mechanism, tyrosine dephosphorylation, involves cytoplasmic phosphatases such as SH-2-domain-containing protein tyrosine phosphatase-1 (SHP)-1 that are believed to inhibit cytokine-activated Jak-Stat pathways through their dephosphorylation and inactivation of Jak (Hilton, 1999; Starr & Hilton, 1999). SHP-1-deficient mice demonstrate multiple haematopoietic abnormalities, including hyperproliferation and abnormal activation of granulocytes and macrophages in the lungs and in the skin, resulting in the characteristic moth-eaten appearance of their coats (Jiao et al, 1997; Takahashi et al, 1998). They also produce fewer B-cell progenitors and natural killer cells and exhibit premature thymic involution leading to immune deficiency (Jiao et al, 1997). This suggests that SHP-1 is a negative regulator of both myeloidand lymphoid lineages (Van Zant & Shultz, 1989; Yoshimura et al, 1990; de la Chapelle et al, 1993; Klingmuller et al, 1995; Furukawa et al, 1997). Ofparticular interest is a report on families with polycythaemia in whom the C-terminal region of the erythropoietin (EPO) receptor (which includes the docking site for SHP-1) is deleted (de la Chapelle et al, 1993; Furukawa et al, 1997). The third mechanism of Stat inactivation involves various inhibitory proteins including the suppressors of cytokine signalling (SOCS) and the protein inhibitors of activated Stats (PIAS) (Alexander et al, 1999; Shuai, 1999; Starr & Hilton, 1999). The SOCS proteins (eight in number) are alternatively known as JAB (Jak-binding protein), SSI (Stat-induced Stat inhibitor) or CIS (cytokine-inducible SH2-containing protein) (Yoshimura et al, 1995; Endo et al, 1997; Naka et al, 1997; Starr et al, 1997). Recent studies have established the SOCS proteins as negative regulators of cytokines (Yoshimura et al, 1995; Endo et al, 1997; Naka et al, 1997; Adams et al, 1998; Song & Shuai, 1998; Nicholson et al, 1999). Several lines of evidence also suggest that Stats are important in the induction of SOCS expression, although it is likely that Stat proteins are capable of inducing the expression of several SOCS and that each SOCS protein is capable of being induced by several Stats (Hilton, 1999). The PIAS family includes five proteins with significant sequence homology and several highly conserved domains (Chung et al, 1997; Liu et al, 1998). They are specific inhibitors of the Stat signalling pathway, with PIAS1 and PIAS3 being associated with Stat1 and Stat3 respectively (Shuai, 1999). The IFN-induced phosphorylation of Stat1 on Tyr701 is necessary for the PIAS–Stat1 interaction. Thus, the interaction of PIAS-Stat is highly specific and dependent on cytokine stimulation (Shuai, 1999). As cellular signalling pathways are intimately involved in the physiological regulation of cell survival, proliferation and differentiation, their abnormal function can lead to neoplastic changes. Studies in the Drosophila fly, which expresses a Jak and a Stat homologue, have shown that gain-of-function mutations affecting Jak cause leukaemia-like haematopoietic defects (Harrison et al, 1995). Over recent years, several investigators have reported that inappropriate activation of Stats may have a significant role in the pathogenesis of human malignancies and leukaemia in particular (Table IV) (Frank, 1999). This inappropriate activation is initiated by the abnormal hyperfunction or mutation of physiological Stat activators (such as Jaks) or by mutation of kinases not usually involved in Stat phosphorylation (such as abl), thus causing increased cellular proliferation (Frank, 1999). Various activated cellular tyrosine kinases phosphorylate Stats, either directly or via Jak activation, and induce cellular transformation. Activation of Jak1 by the oncogenic tyrosine kinase v-abl induces malignant transformation of haematopoietic cells, whereas constitutive Stat activation by mutated members of the Src kinase family induces cellular transformation (Danial et al, 1998; Frank, 1999). For example, v-src, v-fps and v-sis activate Stat3 and induce fibroblast transformation (Yu et al, 1995; Chaturvedi et al, 1998). Similarly, Lck, another Src tyrosine kinase, is overexpressed in a mouse T-cell lymphoma and is associated with Stat3, Stat5, Jak1 and Jak2 activation (Yu et al, 1997). Stat activation plays a key role in the pathogenesis of human leukaemia. Stat5 and Stat1 for example are constitutively activated in acute lymphoblastic leukaemia (ALL) and Stat1, Stat3, and Stat5 in acute myelogenous leukaemia (AML) (Gouilleux-Gruart et al, 1996; Weber-Nordt et al, 1996; Xia et al, 1998). The chromosomal translocation t(9:12)(p24:p13) found in acute lymphoblastic leukaemia (ALL) results in the fusion of the helix-loop-helix domain of the transcription factor of the family of transcription with the catalytic domain of Jak2, thus activating Jak2 and inducing gene transcription et al, 1998; et al, 1999). of the fusion protein cells growth factor and of mice with a this protein induces a et al, 2000). Activation of these pathways is also crucial in the pathogenesis of chronic myelogenous leukaemia of gene constructs into haematopoietic cell lines induces growth proliferation & transformation of haematopoietic cells to tyrosine phosphorylation of Stat1 and Stat5 in cell lines and cells with & 1996; et al, 1996; et al, 1997). As these cells do not demonstrate Jak activation, it is likely that the enhanced kinase activity of is directly responsible for Stat phosphorylation and cellular transformation (Fig In fact, recent on the activity of the specific kinase inhibitor STI571 and to induce and in have provided evidence for the role of Stat signalling in the pathogenesis of this and suggest that inhibition of Stat activity may be (Druker et al, of on signal transduction pathways implicated in mechanisms include activation of Stat1 and Stat5, activation of pathway through and activation of via proteins such as growth factor and virus and the and protein may also the complex function through a et chromosomal translocations leading to the fusion of kinases and transcription factors include and (Frank, 1999). In the of activation of the receptor tyrosine kinase is mediated by the domain of the transcription factor leading to constitutive Stat activation and growth of haematopoietic cells et al, 1996; et al, 1997; et al, 1998). Although cellular proliferation associated with Jak-Stat pathway is seen in acute and no constitutive tyrosine phosphorylation of Stats has been seen to in chronic leukaemia et al, 1997). serine phosphorylation of Stat1 and Stat3, which is known to their function, has been seen in cells but not in normal peripheral lymphocytes et al, 1997). This serine which can enhance the transcriptional response mediated by tyrosine-phosphorylated Stats and lead to of B-cell function, may the of (Frank, 1999). viruses may also play a role in Stat activation and In the example, T cells with IL-2 and exhibit enhanced Stat3 and Stat5 activity et al, 1995). In with adult T-cell Stat1, Stat3 Stat5, as well as Jak kinases, are constitutively activated et al, 1995; Xu et al, 1995; et al, 1997). Although the mechanisms of Jak activation in cells are still unknown, it has been hypothesized that a viral protein with the IL-2 receptor to activate the pathway. the effect of on T cells is in T cells et al, 1999). Receptor tyrosine kinases are enzymes with an extracellular a and a highly conserved intracellular domain that mediates phosphorylation of tyrosine on substrates in a similar to the Jak-Stat pathway 1996; & 1998). These receptors are not activated by binding but also by such as interactions via cell in response to cellular and by stimulation of receptors et al, 1997). have been into four classes on the basis of their structural class includes the receptors for growth factor class those for growth class those for growth factor colony-stimulating factor or receptor and tyrosine receptor and class those for fibroblast growth factor (Fig et al, 1991; 1990; 1996). and significant homology in their tyrosine kinase domains but less homology in their extracellular domains 1996). All cells levels of and most et al, 1995; et al, 1995; 1996). Several studies have the role of class III receptors and their in the regulation of survival, proliferation and maturation of early haematopoietic usually in with other cytokines 1990; et al, 1991; et al, 1995; et al, 1995; et al, 1995). Several have the role of these growth factors in the pathogenesis of leukaemia and et al, 1992; et al, 1993; et al, 1995; et al, 1996; 1996; et al, 1997; et al, 1999; et al, 1999). is at levels by and lymphoid or cells, but at levels by cells 1996). levels have been shown to with in leukaemia cell growth in et al, 1999; et al, 1999). mutation of has been reported in with disorders et al, and activation of by has been shown to the proliferation of some human leukaemia cell lines et al, 1993). In expression has been reported in all subtypes of and in myeloid but not in et al, 1996; et al, 1998; et al, 1999). A large number of intracellular signalling proteins bind the on the activated including the protein and non-receptor (Alberts et al, 1994; & 1998; et al, 2000). The activation of these proteins then serine/threonine phosphorylation resulting in activation of transcription factors and modulation of cellular processes by gene transcription et al, 2000). Many major cellular signalling pathways involve tyrosine phosphorylation of Src and Jak kinases 1998). several other major such as the signalling involve activation of serine/threonine kinases et al, 2000). The proteins to the large superfamily of which also several other subfamilies including the and proteins involved in cell surface receptors to the and the family involved in the of intracellular (Alberts et al, 1994; & 1999). The three human genes and four that to the surface of the and function as et al, 1998). proteins are to a number of signalling pathways including those mediated by non-receptor tyrosine kinase-associated receptors and

Leptin controls body weight by activating the long form of the leptin receptor (LEPRb). Janus kinase 2 (JAK2) is associated with LEPRb and autophosphorylates in response to leptin. JAK2 also phosphorylates LEPRb, STAT3, and multiple other downstream molecules. Surprisingly, here we show that JAK2 is not required for leptin stimulation of STAT3 phosphorylation. Leptin time- and dose-dependently stimulated tyrosine phosphorylation of STAT3 in both human and mouse JAK2-null cells. Leptin also increased the viability of JAK2-null cells. Overexpression of c-Src or Fyn, two Src family members, promoted STAT3 phosphorylation, whereas inhibition of the endogenous Src family members by either pharmacological inhibitors or dominant negative Src(K298M) decreased the ability of leptin to stimulate the phosphorylation of STAT3 and ERK1/2. Leptin also stimulated tyrosine phosphorylation of kinase-inactive JAK2(K882E) in JAK2-null cells. Overexpression of JAK2(K882E) enhanced the ability of leptin to stimulate STAT3 phosphorylation in JAK2-null cells. Tyr1138 in LEPRb was required for leptin-stimulated phosphorylation of STAT3 but not JAK2(K882E). These data suggest that leptin stimulates non-JAK2 tyrosine kinase(s), including the Src family members, which phosphorylate JAK2, STAT3, and other molecules downstream of LEPRb. JAK2 mediates leptin signaling by both phosphorylating its substrates and forming a signaling complex as a scaffolding/adaptor protein. The non-JAK2 kinase(s) and JAK2 may act coordinately and synergistically to mediate leptin response.

Bcr-Abl Directly Activates Stat5 Independent of Jak2

Previous studies have shown that the kinase activation loop (KAL) of the oncogenic fusion protein NPM-ALK regulates its overall tyrosine phosphorylation status and tumorigenicity. Using tandem affinity purification-mass spectrometry, we assessed how the KAL of NPM-ALK regulates the phosphorylation status of its individual tyrosines. Using the lysates of GP293 cells transfected with NPM-ALK, our highly reproducible results showed evidence of phosphorylation in all 3 tyrosines in KAL and 8 tyrosines outside KAL. We created 7 KAL mutants, each of which carried a Tyr-to-Phe mutation of >or=1 of the 3 tyrosines in KAL. A complete loss of the 8 phosphotyrosines outside KAL was found in 3 KAL mutants, and their oncogenicity (assessed by cell viability, colony formation, and the ability to phosphorylate effector proteins) was abrogated. A partial loss of the 8 phosphotyrosines was found in 4 KAL mutants, but their oncogenicity did not show simple correlation with the number of residual phosphotyrosines. Tyr-to-Phe mutations of each of the 8 phosphotyrosines outside KAL did not result in a significant decrease in the oncogenicity. In conclusion, we have provided details of how the KAL in NPM-ALK regulates its tyrosine phosphorylation pattern. Our results challenge some of the current concepts regarding the relationship between the tyrosine phosphorylation and oncogenicity of NPM-ALK.

Exposure to high glucose and insulin inhibits surfactant synthesis in vitro. We have demonstrated that fetal lung insulin receptor tyrosine kinase (TK) activity is downregulated after culture in high glucose plus insulin, with a resultant decrease in glucose uptake. To see whether relative substrate depletion following substrate excess would further diminish surfactant synthesis, 20-day fetal rat lung explants were initially cultured for 44 hours in media containing 100 mM glucose with or without 0.1 U/mL insulin, followed by a 4-hour pulse in 10 mM glucose +/- insulin or 100 mM glucose +/- insulin, after which the rate of choline incorporation into phosphatidylcholine (PC) or disaturated PC was measured. Choline incorporation was significantly lower after a 4-hour pulse in low glucose (+/- insulin) than under continuing high glucose (+/- insulin) conditions (P<.01). To determine the time required for reversal of TK downregulation in lung explants, insulin receptor TK activity was assayed after 44 hours in high glucose + insulin, followed by an additional 4, 8, 12, or 24 hours in either 10 mM glucose or continued 100 mM glucose + insulin. After 44 hours in 50 mM or 100 mM glucose + insulin, TK activity was significantly decreased (68 +/- 9 % and 57 +/- 9% of control; P<.01). When explants cultured in 100 mM glucose + insulin for 44 hours were subsequently placed in 10 mM glucose for an additional 4 or 8 hours, TK activity remained significantly downregulated (70.2 +/- 7.0% and 84.9 +/- 5.5% of control, respectively, P<.05) compared to explants cultured in low glucose throughout. However, after 12 or 24 hours in low glucose, TK activity was no longer significantly different from control values (106.5 +/- 2.1% and 106.0 +/- 19.6%, respectively). Culture of type II cells for 44 hours in 25 mM glucose + insulin, followed by an additional 4 or 24 hours in either low glucose (5.5 mM) + insulin or high glucose (25 mM) + insulin yielded similar results: TK activity was decreased 20-30% by culture in 25 mM glucose + insulin conditions (P<.05) and this downregulation continued for 4 (but not 24) hours after switching to lower glucose conditions (P<.05). Continued receptor downregulation during a period of relative substrate deprivation may adversely affect surfactant synthesis, as in some infants of diabetic mothers who experience hypoglycemia (following intrauterine hyperglycemia) in the immediate postnatal period.

The binding of epidermal growth factor (EGF) to epidermal growth factor receptor (EGF receptor) induces dimerization of the receptor and activation of its protein tyrosine kinase. Each of these three steps was followed as a function of the concentrations of EGF and of EGF receptor. Binding of EGF was followed by sedimentation of the complex between [3H]EGF and EGF receptor, dimerization was measured by quantitative cross-linking with glutaraldehyde, and the activation of the protein tyrosine kinase was monitored under the same conditions by following the initial velocity of the phosphorylation of peptides containing tyrosine. The binding of epidermal growth factor to its receptor was measured as a function of the concentration of epidermal growth factor, and the relationship was sigmoid with an average value of 1.7 for the Hill coefficient. Both dimerization and the activation of the tyrosine kinase displayed saturation as a function of the concentration of EGF. The ranges of the concentrations of EGF where dimerization and activation of the tyrosine kinase activity were half-maximal were 15-30 and 50-200 nM, respectively, but the value for the concentration of EGF at the half-maximum for the activation of the tyrosine kinase was a complex function of the concentration of EGF receptor. The observed behavior of the binding of EGF, the dimerization of EGF receptor, and the activation of the tyrosine kinase were used as criteria against which to test mechanisms for the process of activation. Equations were derived for various reversible and irreversible mechanisms and used to calculate the theoretical behaviors of the three properties. In direct comparisons of the experimental and the theoretical data, several of the previously proposed reversible and irreversible mechanisms for the activation of EGF receptor were found to be inadequate, but a reasonable mechanism was formulated that was compatible with the experimental data. In this mechanism, dimeric EGF receptor must be occupied by two molecules of EGF for enzymatic activity to be expressed.

Pharmacological and biochemical approaches were used to elucidate the involvement of growth factor signaling pathways mediating estrogen neuroprotection in primary cortical neurons after glutamate excitotoxicity. We addressed the activation of mitogen-activated protein kinase (MAPK) signaling pathways, which are activated by growth factors such as nerve growth factor (NGF). Inhibition of MAPK signaling with the MAPK kinase inhibitor PD98059 blocks both NGF and estrogen neuroprotection in these neurons. These results correlate with a rapid and sustained increase in MAPK activity within 30 min of estrogen exposure. The involvement of signaling molecules upstream from MAPK was also examined to determine whether activation of MAPK by estrogen is mediated by tyrosine kinase activity. Estrogen produces a rapid, transient activation of src-family tyrosine kinases and tyrosine phosphorylation of p21(ras)-guanine nucleotide activating protein. Effects of estrogen on neuroprotection, as well as rapid activation of tyrosine kinase and MAPK activity, are blocked by the anti-estrogen ICI 182,780. This provides evidence that activation of the MAPK pathway by estrogen participates in mediating neuroprotection via an estrogen receptor. These results describe a novel mechanism by which cytoplasmic actions of the estrogen receptor may activate the MAPK pathway, thus broadening the understanding of effects of estrogen in neurons.

RNAi Functional Screening of the Tyrosine Kinome Identifies Therapeutic Targets in Acute Myeloid Leukemia Patients.

BCR-ABL is a constitutively activated tyrosine kinase that causes chronic myeloid leukemia (CML). Since tyrosine kinase activity is essential to the transforming function of BCR-ABL, an inhibitor of the kinase could be an effective treatment for CML. We conducted a phase 1, dose-escalating trial of STI571 (formerly known as CGP 57148B), a specific inhibitor of the BCR-ABL tyrosine kinase. STI571 was administered orally to 83 patients with CML in the chronic phase in whom treatment with interferon alfa had failed. Patients were successively assigned to 1 of 14 doses ranging from 25 to 1000 mg per day. Adverse effects of STI571 were minimal; the most common were nausea, myalgias, edema, and diarrhea. A maximal tolerated dose was not identified. Complete hematologic responses were observed in 53 of 54 patients treated with daily doses of 300 mg or more and typically occurred in the first four weeks of therapy. Of the 54 patients treated with doses of 300 mg or more, cytogenetic responses occurred in 29, including 17 (31 percent of the 54 patients who received this dose) with major responses (0 to 35 percent of cells in metaphase positive for the Philadelphia chromosome); 7 of these patients had complete cytogenetic remissions. STI571 is well tolerated and has significant antileukemic activity in patients with CML in whom treatment with interferon alfa had failed. Our results provide evidence of the essential role of BCR-ABL tyrosine kinase activity in CML and demonstrate the potential for the development of anticancer drugs based on the specific molecular abnormality present in a human cancer.

Liposarcoma is a rare fat cell adult tumor with high risk of recurrence and metastasis, largely underserved by research community and till now limited progress has been made in treatment of this aggressive disease. We used a strategy to identify effective and potential therapeutic kinase inhibitors, irrespective of the activated kinase pathway, using small molecule kinase inhibitor panel. We screened liposarcoma cell lines of different histotypes to a panel of small molecule kinase inhibitors and analyzed using cell proliferation assay. In vitro cell proliferation assay, colony formation, cell cycle analysis, apoptosis assay and western blotting analysis were performed to investigate the effect and mechanism of inhibitor treatment on liposarcoma. Liposarcoma xenograft model system was also employed to investigate anti-tumor in vivo effects of inhibitor treatment. We observed that cell lines demonstrated diverse in vitro drug sensitivity patterns to various kinase inhibitors. Most of the cell lines showed very high sensitivity towards inhibitors targeting proteasome, protein kinase C, Hsp90, PI3K, mTOR and CDKs. Among the receptor tyrosine kinase inhibitors; Ponatinib, Dasatinib, and Sunitinib are the top most sensitive drugs affecting liposarcoma cells lines’ viability irrespective of subtypes and all these are already approved by the U.S. Food and Drug Administration to treat various cancers. We studied multi-targeted tyrosine kinase inhibitor ponatinib as an effective potential drug molecule in liposarcoma. We demonstrated that ponatinib treatment in liposarcoma cell lines reduced the levels of phosphorylated KIT compared to total KIT protein levels in dose dependent manner. Further, western blotting experiments revealed effect of ponatinib treatment on KIT downstream signalling by inhibiting the phosphorylation of AKT, ERK1/2, STAT3, mTOR, P70S6K and RB without affecting their total protein levels. Significant reduction in cell number and colonies with ponatinib treatment implicates anti-neoplastic effects in liposarcoma. Ponatinib treatment causes cell cycle arrest at G0/G1 phase by regulating CDK4 and cyclinD1 proteins levels. It also induces apoptosis of treated cells by downregulating AKT and ERK signaling pathway leading to dephosphorylation of BAD. Similar growth inhibiting effect of ponatinib was demonstrated in in vivo liposarcoma xenograft model. In vitro drug sensitivity profiling of liposarcoma highlighted Ponatinib, an oral FDA approved drug, as potential therapeutic drug candidate for treatment and management of this deadly tumor. Citation Format: Deepika Kanojia, Manoj Garg, Jakki Martinez, Anand M.T., Samuel B Luty, Ngan B Doan, Jonathan W Said, Jeffrey W Tyner, H Phillip Koeffler. Multitargeted tyrosine kinase inhibitor identified as potential therapeutic intervention for liposarcoma using high-throughput profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4188. doi:10.1158/1538-7445.AM2017-4188

The prognosis of melanoma strongly correlates with the disease stage. Patients with local operable disease have a favorable prognosis, whereas <10% of patients with late-stage metastatic melanoma survive for more than 5 yr (Tsao et al., 2004). Current therapies for metastatic melanoma remain inefficient for the majority of patients and have failed to significantly improve survival in controlled randomized trials. New strategies for treating metastatic melanoma are more than welcome, also because rates of new diagnoses have been rising in Western countries. Mutational activation of ERBB receptor tyrosine kinases (EGFR, ERBB2, ERBB3, and ERBB4) has received much attention after the finding that activating mutations in EGFR sensitize lung cancer patients to tyrosine kinase inhibitors, such as gefitinib and erlotinib. Since then, numerous studies have screened ERBB genes for mutations, in particular in lung cancer, and concentrating mainly on the kinase domains of EGFR and ERBB2. Recently, a few studies have discovered mutations also in ERBB4 in various human cancers (Figure 1) albeit with relatively low frequencies ranging from ∼1 to 5%. Somatic mutations of ERBB4 in human cancers. ERBB4 is a transmembrane receptor tyrosine kinase with an N-terminal extracellular domain and a C-terminal intracellular domain. The extracellular domain divided to subdomains I, II, III, and IV is mainly involved in ligand binding and receptor dimerization, whereas the tyrosine kinase domain and the C-terminal tail regulate the intracellular signal transduction. P-loop, αC helix, C-loop and A-loop indicate functional structures within the kinase domain. Mutations found in metastatic melanoma (Prickett et al., 2009) are indicated with red, mutations found in non-small cell lung cancer (NSCLC) (Davies et al., 2005. Cancer Res. 65, 7591–7595; Soung et al., 2006. Int. J. Cancer 118, 1426−1429; Ding et al., 2008. Nature 455, 1069–1075) with gray, mutations found in colorectal cancer (Parsons et al., 2005. Nature 436, 792; Soung et al., 2006; Greenman et al., 2007. Nature 446, 153–158) with blue, mutations found in breast cancer (Soung et al., 2006) with black, and mutations found in gastric cancer (Soung et al., 2006) with green arrows. Asterisks indicate the mutations found in melanoma that were functionally analyzed (Prickett et al., 2009). In the latest issue of Nature Genetics, Prickett et al. make a significant contribution to the molecular genetics of melanoma, reporting somatic ERBB4 mutations in a relatively high percentage, 19%, of patients with metastatic melanoma (Prickett et al., 2009). The authors first analyzed the coding exons that comprise the kinase domains of 86 human protein tyrosine kinases (PTK) in 29 melanomas, finding 30 kinase domain mutations in 19 PTKs. The investigators then analyzed all coding exons of these 19 PTKs for mutations in a series of 79 samples of metastatic melanoma. In the total screen, ERBB4 was the most frequently mutated gene, with a total of 24 missense mutations in 15 different patients (FLT1 and PT2B shared the second position with eight mutations in eight patients found in each). The sites of ERBB4 mutations showed no apparent mutational hot spots, in contrast to other well-characterized oncogenes, such as KRAS or BRAF. The identified mutations were dispersed throughout the ERBB4 gene affecting all functional domains of ERBB4 including areas with putative roles in dimerization (extracellular domain) and activity (kinase domain). However, all the seven ERBB4 mutations selected for functional analyses (marked with an asterisk in Figure 1) demonstrated enhanced basal tyrosine phosphorylation and kinase activity as compared with the wild-type ERBB4 when transiently expressed in HEK 293T cells. Furthermore, melanoma cell lines naturally harboring ERBB4 mutations exhibited increased ERBB4 tyrosine phosphorylation as compared with lines expressing wild-type ERBB4. The authors further investigated the transformation ability of the activating mutations, and found that the mutations induced focus formation as well as anchorage-independent growth when ectopically expressed in either NIH 3T3 mouse fibroblasts or the human melanoma cell line SK-Mel-2. For potential therapeutic applicability, an important question is whether the mutated ERBB4 is driving the growth of melanoma, and if so, can one suppress the growth by pharmacological inhibition of ERBB4? To address the first part of the question, the authors studied the effect of stable knock-down of ERBB4 by shRNA in human melanoma cell lines endogenously expressing wild-type or mutant ERBB4. Whereas the cell lines expressing wild-type ERBB4 did not react to ERBB4 knock-down, the proliferation rate of the cell lines expressing mutant ERBB4 was dramatically reduced, indicating that mutant ERBB4 was functioning as a driver of proliferation. To address, whether pharmacological inhibition of ERBB4 signaling repressed the growth of melanoma cells, the authors used an FDA-approved ERBB kinase inhibitor lapatinib. Lapatinib indeed did repress the growth of cell lines endogenously expressing mutant ERBB4 significantly more than control lines endogenously expressing wild-type ERBB4. The mutant cell lines were estimated to be 10–250 fold more sensitive to lapatinib when compared to the wild-type cell lines, based on IC50 calculations from in vitro growth assays. Curiously, as EGFR and ERBB2 are much more sensitive to lapatinib than ERBB4, concentrations used to inhibit mutant ERBB4-driven growth of melanoma cells should also inhibit EGFR and ERBB2. The authors recognize this and discuss that observed ERBB4 inhibition might not result from direct inhibition of ERBB4 kinase activity, but from indirect inhibiton via suppression of EGFR and ERBB2 kinase activities. Yet the authors report no apparent inhibition of ERBB2 phosphorylation in melanoma cells upon lapatinib administration, while EGFR expression is not detected, supporting a hypothesis that the mutations may sensitize the intrinsic ERBB4 kinase to lapatinib. Clearly, more studies are needed to unravel the underlying mechanisms of lapatinib response, as the authors themselves point out. Experiments with other ERBB4 inhibiting compounds, such as pan-ERBB tyrosine kinase inhibitors with spectrums favoring ERBB4 or anti-ERBB4 monoclonal antibodies, are expected to provide important mechanistic information for developing future clinical applications. Activation of the signaling pathway involving AKT, a protein kinase generally implicated in the regulation of cancer cell survival, was proposed to be the mechanism by which mutant ERBB4 drives the growth of melanoma cells. Loss of AKT activation and subsequent apoptosis was seen upon lapatinib treatment of melanoma cells expressing mutant ERBB4. Confirming the role of ERBB4, AKT activation was also reduced upon shRNA-mediated ERBB4 knock-down in melanoma cells expressing mutant ERBB4. Neither lapatinib nor shRNA significantly suppressed ERK phosphorylation. While the frequency of currently known mutations affecting the PI3-K/AKT pathway in melanoma is relatively low (e.g., PTEN mutations have been described in 5–20% of patients) the RAS-RAF pathway is commonly hyperactivated (BRAF mutated in 50–70% and NRAS in 15–30% of patients) (Gray-Schopfer et al., 2007). Thus, the activating ERBB4 mutations may specifically add to the repertoire of mechanisms by which the melanoma cells can activate the PI3-K/AKT pathway, which in turn may drive tumorigenesis in co-operation with the hyperactive RAS-RAF pathway. Interestingly, among the ERBB4 mutations discovered by Prickett et al. three are intronic mutations putatively affecting splicing of the ERBB4 transcript. Two of these mutations may affect the exons 15 and 26 which both are involved in the production of alternatively spliced ERBB4 isoforms (Junttila et al., 2003). This is intriguing, since the ERBB4 splice variants have different functions and differ in their ability to promote growth. The authors used an ERBB4 isoform JM-a CYT-1 as the plasmid backbone to which the ERBB4 mutations were cloned. Whether this represents an ERBB4 isoform also expressed in melanoma tissue in vivo was, however, not reported. Possible consequences of these mutations for actual ERBB4 splicing also remain to be examined. Prickett et al. present the first report of activating transforming mutations of ERBB4 in melanoma but also in cancer in general. This highlights the importance of the study, in addition to the melanoma field, for our understanding about the biological significance of ERBB4 in human cancer. Functional effects of somatic ERBB4 mutations on ERBB4 signaling were recently also analyzed by Tvorogov et al. (2009). This work on ERBB4 mutations harboring the kinase domain and discovered in human neoplasias other than melanoma revealed two kinase-dead mutations (G802dup and D861Y; Figure 1). Instead of inducing a loss-of-function phenotype, the kinase-dead mutations were as effective as wild-type ERBB4 in heterodimerizing with ERBB2 and promoting mitogenic signaling via AKT and ERK pathways. However, the mutants had lost their ability to induce differentiation via a STAT5-dependent pathway (requiring intrinsic ERBB4 kinase activity) shifting the balance of signaling output to favor growth (Tvorogov et al., 2009). Taken together with these earlier findings, the report by Prickett et al. indicate that ERBB4 could be listed as a human cancer gene (Futreal et al., 2004). The impressive effort by Pricket et al. provides compelling evidence about mutated ERBB4 as a promising novel drug target for metastatic melanoma. Given the relatively high incidence of discovered ERBB4 mutations in metastatic melanoma (19%) and the currently inefficient arsenal of tools to fight this disease, further preclinical and clinical studies addressing the effects of ERBB4 targeting compounds in melanoma are definitely warranted.

The failure of conventional chemotherapeutic regimes to produce any meaningful impact on survival in patients with pancreatic cancer highlights a desperate need for novel treatment strategies. The purpose of this study was to translate the current understanding of activated tyrosine kinase signaling pathways in pancreas cancer into improved patient therapies by targeting two tyrosine kinases, the epidermal growth factor receptor (EGFR) and the non-receptor Src kinase, which act in complementary pathways. The biochemical effects of individual and combined drug treatment with dasatinib (Sprycel, BMS-354825), a potent Src kinase inhibitor, erlotinib (Tarceva, OSI-774), a tyrosine kinase inhibitor to EGFR and the nucleoside analog gemcitabine were examined in pancreas cancer cell lines in vitro and in vivo. IC50 for dasatinib (DTB), erlotinib and gemcitabine was determined by the MTT proliferation assay against 9 human pancreas cancer cell lines, and sensitive and resistant cell lines were identified. In vitro regulation of cell motility, proliferation, survival, migration, invasion and in vivo tumor growth inhibition was determined. Expression of Ki67, cleaved caspase 3, phosphorylated Src and AKT in tumor xenografts was determined following exposure to combination of drugs. The combined inhibition of Src with DTB and EGFR with erlotinib results in cooperative attenuation of cell migration, invasion, proliferation and increases in apoptosis of pancreatic cancer cells. Combined inhibition of Src and EGFR also results in cooperative inhibition of multiple signaling pathways including FAK, AKT, Stat3, ERK, JNK, MAPK and decreases cyclin D1 at concentrations that are ineffective as individual agents. The addition of gemcitabine enhances the effects of combined inhibition of Src and EGFR compared to that seen with individual agents alone or in double combination. Mice with tumor xenografts of BxPC3 (sensitive cell line) and Panc1 (resistant cell line) cells treated with DTB, erlotinib or gemcitabine either individually or as two drug or three drug combinations showed that the most effective in vivo anti-tumor effects were seen with the triple combination of DTB, erlotinib and gemcitabine even in xenografts of resistant pancreatic cells. Pharmacological inhibition of EGFR and Src, in addition to gemcitabine treatment, results in cooperative inhibition of pancreatic tumor cell growth and appears to overcome resistance of single and double agent therapy. These results show that activation of Src and EGFR signaling pathways is evident in pancreas cancer and may be a driver of poor prognosis and chemotherapy resistance and provide a rationale for combination therapy with erlotinib, DTB and gemcitabine in clinical trials of pancreas cancer. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A258.

Epidermal growth factor receptor (EGFR) signaling inhibition by monoclonal antibodies and EGFR-specific tyrosine kinase inhibitors has shown clinical efficacy in cancer by restoring susceptibility of tumor cells to therapeutic apoptosis induction. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer agent with tumor-selective apoptotic activity. Here we present a novel approach that combines EGFR-signaling inhibition with target cell-restricted apoptosis induction using a TRAIL fusion protein with engineered specificity for EGFR. This fusion protein, scFv425:sTRAIL, comprises the EGFR-blocking antibody fragment scFv425 genetically fused to soluble TRAIL (sTRAIL). Treatment with scFv425:sTRAIL resulted in the specific accretion to the cell surface of EGFR-positive cells only. EGFR-specific binding rapidly induced a dephosphorylation of EGFR and down-stream mitogenic signaling, which was accompanied by cFLIP(L) down-regulation and Bad dephosphorylation. EGFR-specific binding converted soluble scFv425:sTRAIL into a membrane-bound form of TRAIL that cross-linked agonistic TRAIL receptors in a paracrine manner, resulting in potent apoptosis induction in a series of EGFR-positive tumor cell lines. Co-treatment of EGFR-positive tumor cells with the EGFR-tyrosine kinase inhibitor Iressa resulted in a potent synergistic pro-apoptotic effect, caused by the specific down-regulation of c-FLIP. Furthermore, in mixed culture experiments binding (L)of scFv425:sTRAIL to EGFR-positive target cells conveyed a potent apoptotic effect toward EGFR-negative bystander tumor cells. The favorable characteristics of scFv425:sTRAIL, alone and in combination with Iressa, as well as its potent anti-tumor bystander activity indicate its potential value for treatment of EGFR-expressing cancers.

Depending on their structure, some polyphenols (e.g. flavonoids) abundantly found in plant-derived beverages such as green tea can efficiently inhibit tyrosine kinase and serine/threonine kinase activities. Extra-virgin olive oil (EVOO - the juice of the olive obtained solely by pressing and consumed without any further refining process) is unique among other vegetable oils because of the high level of naturally occurring phenolic compounds. We explored the ability of EVOO polyphenols to modulate HER2 tyrosine kinase receptor-induced in vitro transformed phenotype in human breast epithelial cells. Using MCF10A normal breast epithelial cells retrovirally engineered to overexpress the wild-type sequence of human HER2, we further determined the relationship between chemical structures of EVOO-derived phenolics and their inhibitory activities on the tyrosine kinase activity of the HER2 oncoprotein. When the activation (phosphorylation) status of HER2 was semi-quantitatively measured the secoiridoids blocked HER2 signaling by rapidly reducing the activation status of the 1248 tyrosine residue (Y1248), the main autophosphorylation site of HER2. EVOO-derived single phenols tyrosol and hydroxytyrosol and the phenolic acid elenolic acid failed to significantly decrease HER2 tyrosine kinase activity. The anti-HER2 tyrosine kinase activity IC50 values were up to 5-times lower in the presence of EVOO-derived lignans and secoiridoids than in the presence of EVOO-derived single phenols and phenolic acids. EVOO polyphenols induced strong tumoricidal effects by selectively triggering high levels of apoptotic cell death in HER2-positive MCF10A/HER2 cells but not in MCF10A/pBABE matched control cells. EVOO lignans and secoiridoids prevented HER2-induced in vitro transformed phenotype as they inhibited colony formation of MCF10A/HER2 cells in soft-agar. Our current findings not only molecularly support recent epidemiological evidence revealing that EVOO-related anti-breast cancer effects primarily affect the occurrence of breast tumors over-expressing the type I receptor tyrosine kinase HER2 but further suggest that the stereochemistry of EVOO-derived lignans and secoiridoids might provide an excellent and safe platform for the design of new HER2 targeted anti-breast cancer drugs.

Tyrosine phosphorylation has recently been associated with capacitation and suggested as a regulator of sperm movement, especially characterizing hyperactivation. The objective of this study was to verify if tyrosine phosphorylation of human sperm proteins was essentially required for the maintenance of motility as well as the development of hyperactivation. Washed sperm were incubated for 6 h in Ham's F10 + 0.35% HSA at 37 degrees C in 5% CO(2), with and without the tyrosine kinase inhibitors genistein, tyrphostin, erbstatin, or herbimycin A and the wide-spectrum kinase inhibitor staurosporin. The concentrations of the inhibitors used in the experiments did not induce sperm toxicity, as measured by membrane integrity and mitochondrial function assays. Samples incubated without the inhibitors (control), increased their tyrosine kinase activity (ELISA), the number and intensity of tyrosine-phosphorylated (PY) protein bands (Western blot), the incidence of PY-immunoreactive sperm (immunofluorescence), and some of the sperm motion characteristics (CASA), such as velocity (VEL), amplitude of lateral head displacement (ALH), and hyperactivation. Among the selective protein tyrosine kinase inhibitors, genistein was the most active and consistent, inhibiting sperm tyrosine kinase activity, PY proteins, incidence of PY sperm, and sperm motility and motion parameters, such as VEL, ALH, and hyperactivation. The rest of the kinase inhibitors decreased motion characteristics to a varied extent and had different effects on phosphorylation parameters. In general, they decreased PY phosphorylation of 2 proteins (83 and 54 kDa) present in whole sperm extracts, and two sets of proteins of low (39-49 kDa) and medium (55-87 kDa) molecular weight present in the Triton X-100-solubilized sperm protein fraction. This inhibition was evident regardless of the total tyrosine kinase activity of the samples or the incidence of PY-immunoreactive sperm. The described findings further support the association between motility and protein tyrosine phosphorylation in human sperm and point to certain proteins as the main linkers.