Family Tyrosine Kinase Inhibitor Research Articles

Imatinib-Sensitive Tyrosine Kinases Regulate Mycobacterial Pathogenesis and Represent Therapeutic Targets against Tuberculosis

In Figure 2G of the above paper, the figure key was incorrectly labeled. In the figure key, the filled square symbol (■■■) was labeled as “ABL1−/−/ABL2−/− + Imatinib.” The filled square should be labeled as “ABL1−/−/ABL2−/−.” Please refer to the revised figure here. In addition, correct version of the article is now online. We regret any inconvenience this has caused. Imatinib-Sensitive Tyrosine Kinases Regulate Mycobacterial Pathogenesis and Represent Therapeutic Targets against TuberculosisNapier et al.Cell Host & MicrobeNovember 17, 2011In BriefThe lengthy course of treatment with currently used antimycobacterial drugs and the resulting emergence of drug-resistant strains have intensified the need for alternative therapies against Mycobacterium tuberculosis (Mtb), the etiologic agent of tuberculosis. We show that Mtb and Mycobacterium marinum use ABL and related tyrosine kinases for entry and intracellular survival in macrophages. In mice, the ABL family tyrosine kinase inhibitor, imatinib (Gleevec), when administered prophylactically or therapeutically, reduced both the number of granulomatous lesions and bacterial load in infected organs and was also effective against a rifampicin-resistant strain. Full-Text PDF Open Archive

LCB 03-0110, a Novel Pan-Discoidin Domain Receptor/c-Src Family Tyrosine Kinase Inhibitor, Suppresses Scar Formation by Inhibiting Fibroblast and Macrophage Activation

Wound healing generally induces an inflammatory response associated with tissue fibrosis in which activated macrophage and myofibroblast cells are primarily involved. Although this is known to be the underlying mechanism for scarring and various fibrotic pathologies, no effective intervention is currently available. We identified (3-(2-(3-(morpholinomethyl)phenyl)thieno[3,2-b]pyridin-7-ylamino)phenol (LCB 03-0110), a thienopyridine derivative, as a potent inhibitor of discoidin domain receptor family tyrosine kinases and discovered that this compound strongly inhibits several tyrosine kinases, including the c-Src family, spleen tyrosine kinase, Bruton's tyrosine kinase, and vascular endothelial growth factor receptor 2, which are important for immune cell signaling and inflammatory reactions. LCB 03-0110 suppressed the proliferation and migration of primary dermal fibroblasts induced by transforming growth factor β1 and type I collagen, and this result correlated with the inhibition ability of the compound against enhanced expression of α-smooth muscle actin and activation of Akt1 and focal adhesion kinase. In J774A.1 macrophage cells activated by lipopolysaccharide LCB 03-0110 inhibited cell migration and nitric oxide, inducible nitric-oxide synthase, cyclooxygenase 2, and tumor necrosis factor-α synthesis. LCB 03-0110 applied topically to full excisional wounds on rabbit ears suppressed the accumulation of myofibroblast and macrophage cells in the healing wound and reduced hypertrophic scar formation after wound closing, without delaying the wound closing process. Taken together, the pharmacological activities of LCB 03-0110 suggest that it could be an effective agent for suppressing fibroinflammation by simultaneously targeting activated fibroblasts and macrophages.

Clonal Dasatinib Large Granular Expansion Is Associated with Suboptimal and Optimal Leukemia Net Response Criteria in Chronic Myelogenous Leukemia

Abstract 1696 Introduction:Dasatinib a BCR/ABL and Src family tyrosine kinase inhibitor (TKI) used in the treatment of Chronic Myelogenous Leukemia (CML) has recently been associated with large granular (LG) expansion. Its clinical meaning and pathogenic mechanisms are still to be elucidated. Methodology: Based on the knowledge that both LG subsets (NK and T) have cytotoxic properties implicated in anti-tumoral and immune mediated activity, we conducted a prospective study from December 2009 to August 2011 with CML patients to evaluate the clinical significance of LG lymphocytosis (LGL) and T-LG clonality in dasatinib users and characterize if these conditions are specific for dasatinib when compared to imatinib. Imatinib patients (N=28) were vertically studied as a control group. Blood samples were analyzed by complete blood count, immunophenotyping with four-color flow cytometer and RT-PCR to detect T-receptor clonality. Dasatinib group (N=36) was prospectively followed for the same parameters and also evaluated for clinical response (according to the European LeukemiaNet 2009) and side effects. Results: The mean diagnosis time for the imatinib group was 6.2 years (0.3–17.4) and 5.9 years (1.2–18.5) for the dasatinib group. In both, the mean time since beginning current TKI therapy was over than 12 months (4.6 years on imatinib, 1.7 years on dasatinib). The incidence of LGL was higher in the dasatinib group compared to the imatinib group (86% × 53.5%, p=0.005) (table 1). T-LG clonality was statically associated with response by LeukemiaNet definition (p=0.0246) (table 2). It is worth to emphasize that one patient with oligoclonal LGL had major molecular response with just 50mg of dasatinib (dose reduction because of thrombocytopenia). LGL above 800/mm3 was statically associated with clonality (p=0,0224). Excluding hematologic toxicity, significant toxicity was observed in 30% (N=11) (colitis, pleural effusion, hepatic and pancreatic toxicity) of the dasatinib users (table 3). In these patients LGL was observed in 81.8% and clonality in 63.6%. Development of hemorrhagic colitis happened in the oligoclonal patient with the highest LG peak (NK=4224/mm3 and T-LG lymphocyte =1672/mm3).Table 1:Lymphocyte profile of patients treated with imatinib and dasatinibImatinib (n=28)Dasatinib (n=36)PAbsolute lymphocytosis (>3400/mm3)0%14 (39%)0.0001Media of Absolute lymphocytosis/mm31708 (800–2700)3055 (1100–9300)–T/ NK LGL (>400/ mm3)15 (53.5%)31 (86%)0.005Media of LGL (>400/ mm3)663 (440–1080)1427 (484–3280)**–Clonal lymphocyte population*6 (21%)17 (47%)0.04Monoclonality0%23.5%–Oligoclonality100%76.5%–*Oligo/monoclonal by RT-PCR.**Media of median LGL/patient.Table 2:Response and Clonality in dasatib patientsLeukemiaNet 2009 definitionResponse* (N=31)Failure (N=5)PLGL87%80%p=0.6746NK26.9%0%T-LG26.9%50%NK+T-LG46.2%50%Clonality54.8%0%p=0.0246Oligoclonal76.5%Monoclonal23.5%*Suboptimal/optimal responseTable 3:Clinical features of dasatinib patients and T-lymphocyte clonality statusClonal (n = 17)Policlonal (n = 19)PChronic phase14 (82%)11 (58%)0.21Hematological toxicity5 (29%)4 (21%)0.70Non-hematological toxicity7 (41%)4 (21%)0.28Median dose of dasatinib125mg/d (M)108mg/d—104mg/d (O)O=oligoclonal. M=monoclonal. Discussion:Previous articles suggest that T-LG lymphocytes expressed mainly two Src kinase family members, Fyn and Lck and that inhibition of this pathway (a feature of dasatinib) interfered with their development which goes against what we find in current practice. In someway dasatinib is capable of stimulating our innate defense and leads to a selection of a T-LG clone that is associated with a better response. Conclusion: We found an important association between absolute lymphocytosis, LGL and clonality in dasatinib group that was not evident with imatinib. We were able to demonstrate that LGL above 800/mm3 was associated with T-LG clonality and that clonality was statistically associated with response by LeukemiaNet criteria for CML. This suggests that clonal T-LG expansion has a role in improving dasatinib ITK function, a feature not observed with imatinib. Perspective: If dasatinib is able to stimulate our innate anti-tumoral defenses its use should be evaluated in other kind of tumors and the molecular way it occurs should be investigated. Disclosures:Off Label Use: Dasatinib dose reduction due to toxicity.

Imatinib-Sensitive Tyrosine Kinases Regulate Mycobacterial Pathogenesis and Represent Therapeutic Targets against Tuberculosis

The lengthy course of treatment with currently used antimycobacterial drugs and the resulting emergence of drug-resistant strains have intensified the need for alternative therapies against Mycobacterium tuberculosis (Mtb), the etiologic agent of tuberculosis. We show that Mtb and Mycobacterium marinum use ABL and related tyrosine kinases for entry and intracellular survival in macrophages. In mice, the ABL family tyrosine kinase inhibitor, imatinib (Gleevec), when administered prophylactically or therapeutically, reduced both the number of granulomatous lesions and bacterial load in infected organs and was also effective against a rifampicin-resistant strain. Further, when coadministered with current first-line drugs, rifampicin or rifabutin, imatinib acted synergistically. These data implicate host tyrosine kinases in entry and intracellular survival of mycobacteria and suggest that imatinib may have therapeutic efficacy against Mtb. Because imatinib targets host, it is less likely to engender resistance compared to conventional antibiotics and may decrease the development of resistance against coadministered drugs.

Alveolar Epithelial Cells Undergo Epithelial-to-Mesenchymal Transition in Response to Endoplasmic Reticulum Stress

Expression of mutant surfactant protein C (SFTPC) results in endoplasmic reticulum (ER) stress in type II alveolar epithelial cells (AECs). AECs have been implicated as a source of lung fibroblasts via epithelial-to-mesenchymal transition (EMT); therefore, we investigated whether ER stress contributes to EMT as a possible mechanism for fibrotic remodeling. ER stress was induced by tunicamyin administration or stable expression of mutant (L188Q) SFTPC in type II AEC lines. Both tunicamycin treatment and mutant SFTPC expression induced ER stress and the unfolded protein response. With tunicamycin or mutant SFTPC expression, phase contrast imaging revealed a change to a fibroblast-like appearance. During ER stress, expression of epithelial markers E-cadherin and Zonula occludens-1 decreased while expression of mesenchymal markers S100A4 and α-smooth muscle actin increased. Following induction of ER stress, we found activation of a number of pathways, including MAPK, Smad, β-catenin, and Src kinase. Using specific inhibitors, the combination of a Smad2/3 inhibitor (SB431542) and a Src kinase inhibitor (PP2) blocked EMT with maintenance of epithelial appearance and epithelial marker expression. Similar results were noted with siRNA targeting Smad2 and Src kinase. Together, these studies reveal that induction of ER stress leads to EMT in lung epithelial cells, suggesting possible cross-talk between Smad and Src kinase pathways. Dissecting pathways involved in ER stress-induced EMT may lead to new treatment strategies to limit fibrosis.

Suppression of autophagy sensitizes multidrug resistant cells towards Src tyrosine kinase specific inhibitor PP2

Upregulated Src activity has been implicated in a variety of cancers. Thus, Src family tyrosine kinase (SFK) inhibitors are often effective cancer treatments. Here, we employed 4-amino-5-(4-chlorophenyl)-7-( t-butyl)pyrazolo[3,4- d]pyrimidine (PP2), a selective SFK inhibitor, to determine the possible involvement of tyrosine phosphorylation in the modulation of autophagy, for overcoming multidrug resistance. We found that multidrug-resistant v-Ha- ras-transformed NIH 3T3 cells (Ras-NIH 3T3/Mdr) were more susceptible to PP2 treatment than were their parental cells (Ras-NIH 3T3). The antiproliferative activity of PP2 appeared to be due to cell-cycle arrest at G 1/S without induction of apoptosis. Interestingly, PP2 preferentially induced autophagy in Ras-NIH 3T3 cells but not in Ras-NIH 3T3/Mdr cells, which implies that a high level of autophagy may protect PP2-treated cells from undergoing cell death. PP2-induced autophagy in Ras-NIH 3T3 cells is accompanied by an inhibition of the mTOR signaling pathway. However, we found that in Ras-NIH 3T3/Mdr cells, PP2-induced mTOR inhibition was uncoupled from the induction of autophagy—likely due to the hyperactivation of AMPK by delayed Raf activation. We also found that PP2-induced dissociation of Beclin 1 from Bcl-2 leads to autophagy in Ras-NIH 3T3 cells. Taken together, these results suggest that functional autophagy in response to PP2 may lead to cell survival in Ras-NIH 3T3 cells, while defective autophagy may contribute to inhibition of growth in Ras-NIH 3T3/Mdr cells. Thus, modulators of autophagy may be used beneficially as adjunctive therapeutic agents during the treatment of cancers with SFK inhibitors.

Neutrophil-derived resistin release induced by Aggregatibacter actinomycetemcomitans

Resistin is an adipokine that induces insulin resistance in mice. In humans, resistin is not produced in adipocytes, but in various leukocytes instead, and it acts as a proinflammatory molecule. The present investigation demonstrated high levels of resistin in culture supernatants of neutrophils that are stimulated by a highly leukotoxic strain of Aggregatibacter actinomycetemcomitans. In contrast, the level of resistin was remarkably low when neutrophils were exposed to two other strains that produce minimal levels of leukotoxin and a further isogenic mutant strain incapable of producing leukotoxin. Pretreatment of neutrophils with a monoclonal antibody to CD18, β chain of lymphocyte function-associated molecule 1 (LFA-1), or an Src family tyrosine kinase inhibitor before incubation with the highly leukotoxic strain inhibited the release of resistin. These results show that A. actinomycetemcomitans-expressed leukotoxin induces extracellular release of human neutrophil-derived resistin by interacting with LFA-1 on the surface of neutrophils and, consequently, activating Src family tyrosine kinases.

Novel Agents and Future Directions for Refractory Breast Cancer

Tumor resistance remains a major clinical challenge. Numerous pathways are under investigation to determine how best to target therapies to specific mutations in tumor biology and circumvent resistance. Agents in development include inhibitors of the poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) pathway, such as iniparib, olaparib, and veliparib; the PI3K/Akt/mTOR pathway inhibitor everolimus; and the Src family tyrosine kinase inhibitor dasatinib. Research is ongoing to determine whether patients with specific biochemical attributes, such as the presence of a BRCA1 or BRCA2 mutation, will have a better response to targeted therapy and whether targeted agents act synergistically with chemotherapeutic agents.

High-Dimensional Analysis of Intracellular Signaling and Dasatinib Inhibition In High-Risk Pediatric Leukemia by 31-Parameter Mass Cytometry

AbstractAbstract 2761 Background:High-risk pediatric B precursor lymphoblastic leukemia (preB ALL) remains a serious cause of morbidity and mortality despite therapeutic advances with consequent improvements in survival over the past 30 years. Intracellular protein networks are the functional mediators of differentiation decisions. Perturbing these networks with chemical and biological agents to unveil their signaling potential has shed insight into key networks in other hematologic malignancies such as B cell lymphoma, AML, JMML, and myelofibrosis. The current study was designed to profile evoked signaling responses in NCI high-risk pediatric preB ALL using a next-generation ‘mass cytometry’ platform (CyTOF.., DVS Sciences). This new technology platform utilizes antibodies conjugated to chelated metal ion tags, and thereby circumvents the limitations of overlapping spectral emission associated with more conventional fluorochrome-conjugated antibodies. Thus, in this preliminary study we measured 13 surface markers and 18 functional intracellular proteins simultaneously at the single-cell level in primary patient samples, with the goal of correlating distinct signaling responses with surface marker expression and response to the Src family tyrosine kinase inhibitor, dasatinib. Methods:All procedures were approved by the Stanford University Human Subject Institutional Review Board. Signaling profiles were measured for cryopreserved diagnostic bone marrow samples from pediatric patients with NCI high-risk pediatric leukemia in the basal state and in response to a 15 minute treatment to 18 different extracellular modulators, with or without prior exposure to dasatinib for 30 minutes. An example is shown in Figure 1. Cell populations were gated based on surface marker expression. The activation states of 18 functional intracellular markers were measured within the leukemic blasts (Figure 1). Results:Within the bulk blast population, distinct signaling responses could be discerned correlating with surface marker heterogeneity. Blasts were manually gated into 5 distinct populations based on surface expression of CD45 and CD10. These populations demonstrated further heterogeneous expression of surface markers CD34, CD38 and CD19 as well as heterogeneous activation states of intracellular proteins such as pERK, NFkB, and pStat5. Robust activation of phospho-proteins proximal to the preB cell receptor was observed following treatment with preBCR cross-linking antibodies or with tyrosine phosphatase inhibitors. This robust signaling was abrogated by pre-treatment of leukemic blasts with dasatinib. Unexpectedly, concurrent treatment with IL-7 or preB cell receptor crosslinking antibodies and dasatinib sensitized cells to dasatinib inhibition, resulting in a synergistic suppression of phosphorylation below baseline values (Figure 2). Discussion:Together, these results suggest that intracellular signaling and response to therapeutic agents such as kinase inhibitors may differ based on the complex interaction of the leukemic cell and its microenvironment. This preliminary study shows the feasibility of using a novel mass cytometry platform to simultaneously monitor 18 intracellular signaling mediators on a per cell basis in primary samples from pediatric high risk ALL patients to a level of detail not previously possible. This approach allowed the identification of cells with a robust signaling phenotype, based on the activation of pERK, pStat5 and NFkB, members of pathways known to be involved proliferation and survival. These data identify potential targets for therapeutic agents such as the tyrosine kinase inhibitor dasatinib. inhibited preBCR signal transduction, and synergy with other biological agents. Further studies are ongoing to measure multidimensional signaling with CyTOF technology in additional high risk pediatric ALL samples with the goal of identifying and guiding the most beneficial therapeutic choices.EFS and KLD contributed equally to this work. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

A Refractory Stage IVb Thymoma Responded Completely to a Combination of Dasatinib and Prednisone but Poorly to Either One Alone

AbstractAbstract 4925 Introduction:Thymomas are rare tumors for which there is little randomized evidence to guide treatment and therefore, therapeutic options to cure advanced malignant thymomas are limited. We hereby report the first clinical outcome of combined dasatinib and prednisone therapy in a patient with heavily treated stage IVB malignant thymoma. Case Presentation:A 41 year old white male presented with fatigue, fever and granulocytopenia. He soon developed myasthenia gravis and aplastic anemia. Computer tomography showed a mediastinal mass, pleural thickening, splenomegaly and mesenteric nodes; biopsy of mediastinal mass revealed a malignant thymoma. Prednisone for 6 weeks along with antithymocyte globulin and cyclosporine were started for myasthenia gravis and aplastic anemia. Chemotherapy for unresectable thymoma was soon initiated with ADOC (Cisplatin, Doxorubicin, Vincristine, and Cyclophosphamide), but only small partial response was noted. After failing to show response to another three lines of therapy, we commenced therapy with dasatinib 75 mg twice daily. Thymoma showed partial response to dasatinib initially but soon became resistant and progressed. When prednisone 100 mg daily was added, a complete response was observed. Conclusion:A patient with stage IVB thymic carcinoma failed to respond well 3 lines of combination chemotherapy, and neither to prednisone or dasatinib alone. When dasatinib is combined with prednisone, it induced a complete response. Disclosures:Off Label Use: Dasatinib is an oral dual BCR/ABL and Src family tyrosine kinase inhibitor. It is a potent inhibitor of Imatinib resistant KIT activation loop mutants and induces apoptosis in mast cells and leukemic cells. At higher concentrations, Dasatinib also inhibits c-Kit, PDGFR and EphedrinA2. it has currently FDA approval for treatment of adults in all phases of chronic myeloid leukemias with resistance or intolerance to prior therapy, including Gleevec. The second indication for Dasatinib, is for the treatment of Ph+ALL. A number of Phase II trials have shown activity of this agent to various solid tumors including breast and lung cancer. Imatinib showed activity in thymomas, thus the rationale for using Dasatinib in the treatment of our patient.

Hypotonic swelling-induced activation of PKN1 mediates cell survival in cardiac myocytes

Hypotonic cell swelling in the myocardium is induced by pathological conditions, including ischemia-reperfusion, and affects the activities of ion transporters/channels and gene expression. However, the signaling mechanism activated by hypotonic stress (HS) is not fully understood in cardiac myocytes. A specialized protein kinase cascade, consisting of Pkc1 and MAPKs, is activated by HS in yeast. Here, we demonstrate that protein kinase N1 (PKN1), a serine/threonine protein kinase and a homolog of Pkc1, is activated by HS (67% osmolarity) within 5 min and reaches peak activity at 60 min in cardiac myocytes. Activation of PKN1 by HS was accompanied by Thr(774) phosphorylation and concomitant activation of PDK1, a potential upstream regulator of PKN1. HS also activated RhoA, thereby increasing interactions between PKN1 and RhoA. PP1 (10(-5) M), a selective Src family tyrosine kinase inhibitor, significantly suppressed HS-induced activation of RhoA and PKN1. Constitutively active PKN1 significantly increased the transcriptional activity of Elk1-GAL4, an effect that was inhibited by dominant negative MEK. Overexpression of PKN1 significantly increased ERK phosphorylation, whereas downregulation of PKN1 inhibited HS-induced ERK phosphorylation. Downregulation of PKN1 and inhibition of ERK by U-0126 both significantly inhibited the survival of cardiac myocytes in the presence of HS. These results suggest that a signaling cascade, consisting of Src, RhoA, PKN1, and ERK, is activated by HS, thereby promoting cardiac myocyte survival.

Src family tyrosine kinases activate thrombin-induced non-capacitative cation entry in human platelets

Platelet activation is critically regulated by an increase in intracellular calcium concentration ([Ca2+]i). Although Ca2+ release from intracellular Ca2+ stores and subsequent store-operated Ca2+ entry are often thought to be the major contributors to increases in [Ca2+]i evoked by most agonists, high concentrations of thrombin activate a Ca2+ entry pathway that is independent of Ca2+ store depletion (known as ‘non-capacitative cation entry’–NCCE). The channel that conducts NCCE has not previously been clearly identified, and the mechanisms that regulate its activation are also unknown. Here we have investigated NCCE using fura-2-loaded human platelets. To investigate NCCE independently of other Ca2+ signaling pathways, the intracellular Ca2+ stores were first rapidly depleted in the absence of extracellular Ca2+. Sr2+ was then added to monitor maximal store-operated cation influx. Thrombin was then added to stimulate NCCE. Flufenamic acid, which inhibits Ca2+ entry through most TRPC isoforms, but potentiates entry through TRPC6, was found to block store-operated cation entry. In contrast, thrombin-induced NCCE was increased, suggesting the possible involvement of TRPC6. Since TRPC6 is regulated by Src family tyrosine kinases in some cells, we investigated the possible role of this kinase family in NCCE. PP2, a Src family tyrosine kinase inhibitor, completely abolished thrombin-induced NCCE. Furthermore, NCCE was enhanced by phenylarsine oxide and could be directly induced by vanadyl hydroperoxide, both tyrosine phosphatase inhibitors. These data indicate that Src family tyrosine kinase activation is a required step in NCCE activation. In conclusion NCCE may be an important regulator of platelet activation when local thrombin concentrations are high.

Abstract B260: Antitumor activity of HM781-36B, an irreversible pan-HER inhibitor, alone or in combination with cytotoxic chemotherapeutic agents in gastric cancer cells

Abstract Background: HM781-36B is a novel quinazoline-based irreversible pan-HER inhibitor. Although the effects of many other pan-HER inhibitors (CI-1033, PF00299804, BMS-599626, BMS-690514 and JNJ-28871063) have been studied in various types of cancer, data regarding the use of pan-HER inhibitors to treat gastric cancers are limited. In this study, we evaluated the therapeutic potential of HM781-36B for the treatment of gastric cancers by testing its in vitro and in vivo efficacy. In addition, we further characterized its activity when administered in combination with chemotherapeutic agents. Methods: Experiments were conducted in gastric cancer cell lines. Enzyme activity assay was conducted to determine the IC50 values of HM781-36B for kinase inhibition and MTT assay was used for determining growth inhibitory effect. Cell cycle distribution was analyzed by flow cytometry and apoptotic effect was measured by annexin V assay. And N87 xenograft model was used to see tumor regression effect. Western blot analysis was used to verify mechanism of this compound. Results: We found that HM781-36B suppressed the proliferation of HER2-amplified gastric cancer cell lines (SNU216 and N87) potently when compared to the effects of other HER family tyrosine kinase inhibitors (gefitinib, lapatinib, BIBW-2992 and CI-1033). Furthermore, we found that HM781-36B inhibited the phosphorylation of HER family and their downstream signaling proteins, which resulted in apoptosis through the caspase-mediated mitochondrial pathway and G1 arrest. The apoptotic process requires the induction of the proapoptotic BH3-only BCL2 family member, BIM, and HM781-36B led to a dramatic increase in the BIM level in SNU216 and N87 cells. Consistent with these in vitro findings, HM781-36B showed exceptional anti-tumor activity in a xenograft model of N87 cells. Furthermore, HM781-36B exerted synergistic effects when administered in combination with 5-fluorouracil or cisplatin in both HER2 amplified cells and HER2 non-amplified gastric cancer cells. Conclusion: Taken together, these data suggest that HM781-36B may be useful as a targeted therapy for the treatment of HER2 amplified gastric cancer as well as a chemosensitizer of cytotoxic chemotherapeutic agents used to treat gastric cancer, even in HER2 non-amplified gastric cancer. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B260.

Angiotensin II receptor blocker attenuates PDGF-induced mesangial cell migration in a receptor-independent manner

Clinical studies have shown that angiotensin II (Ang II) type 1 (AT1) receptor blockers (ARBs) are able to provide renoprotection independent of their blood pressure lowering effects. ARBs also are reported to suppress oxidative stress, inflammation and certain other cellular responses in a receptor-independent manner. We investigated the effects of an ARB, olmesartan, on the cell migration induced by platelet-derived growth factor (PDGF), a major mitogen involved in the pathogenesis of glomerulonephritis in rat mesangial cells (RMCs). Cell migration was determined by a modified Boyden chamber assay. The intracellular signalling pathway was examined by western blotting. AT1 receptor expression was knocked down by small interfering RNAs. The intracellular reactive oxygen species (ROS) was measured by using a fluorescent probe. The O(2)(.-) scavenging activities were studied by the electron paramagnetic resonance-spin trapping method. PDGF-induced cell migration was inhibited by olmesartan in AT1 receptor knockdown RMCs. Olmesartan attenuated big mitogen-activated protein (MAP) kinase 1 (BMK1) and Src activation by PDGF in AT1 receptor knockdown RMCs. PDGF-induced BMK1 activation was suppressed by the Src family tyrosine kinase inhibitors, indicating that Src exists upstream of BMK1. The NADPH oxidase inhibitors inhibited not only PDGF-induced BMK1 and Src activation but also RMC migration. The elevation in ROS generation induced by PDGF was decreased by olmesartan. Olmesartan displayed neither directly ROS scavenging activity nor the inhibition of ROS-mediated intracellular signalling in RMCs. Olmesartan attenuates ROS generation by PDGF, leading to the subsequent inhibition of Src/ BMK1/migration in an AT1 receptor-independent manner in RMCs.

Involvement of protein kinase C and Src tyrosine kinase in acute tolerance to ethanol inhibition of spinal NMDA‐induced pressor responses in rats

The present study was carried out to examine the role of protein kinases in the development of acute tolerance to the effects of ethanol on spinal N-methyl-D-aspartate (NMDA) receptor-mediated pressor responses during prolonged ethanol exposure. Blood pressure responses induced by intrathecal injection of NMDA were recorded. The levels of several phosphorylated residues on NMDA receptor NR1 (GluN1) (NR1) and NMDA receptor NR2B (GluN2B) (NR2B) subunits were determined by immunohistochemistry and Western blot analysis. Ethanol inhibited spinal NMDA-induced pressor responses at 10 min, but the inhibition was significantly reduced at 40 min following continuous infusion. This effect was dose-dependently blocked by chelerythrine [a protein kinase C (PKC) inhibitor, 1-1000 pmol] or PP2 (a Src family tyrosine kinase inhibitor, 1-100 pmol) administered intrathecally 10 min following ethanol infusion. A significant increase in the immunoreactivity of phosphoserine 896 of NR1 subunits (pNR1-Ser896) and phosphotyrosine 1336 of NR2B subunits (pNR2B-Tyr1336) was found in neurons of intermediolateral cell column during the development of tolerance. Levels of pNR1-Ser896 and pNR2B-Tyr1336 were also significantly increased in lateral horn regions of the spinal cord slices incubated with ethanol for 40 min in vitro. The increases in pNR1-Ser896 and pNR2B-Tyr1336 levels were inhibited by post-treatment with chelerythrine and PP2, respectively, both in the in vivo and in vitro studies. The results suggest that activation of PKC and Src tyrosine kinase during prolonged ethanol exposure leading to increases in the levels of pNR1-Ser896 and pNR2B-Tyr1336 may contribute to acute tolerance to inhibition by ethanol of NMDA receptor function.

A phase I dose-escalation study of 5-day intermittent oral lapatinib therapy with biomarker analysis in patients with HER-2–overexpressing breast cancer

e14530 Background: Although current HER2 targeting agents have made measurable impact on the treatment of HER2overexpressing breast cancer, their effects have been modest. Recent evidence has redefined the target of interest as the HER2-HER3 signaling dimer and identified inherent signal buffering capacity that protects it against partial inhibitors of HER2 function. In preclinical studies, HER2 catalytic function and HER2-HER3 signaling can be suppressed, but this requires higher fully inactivating doses of HER- family tyrosine kinase inhibitors (TKIs) leading to apoptotic tumor cell death. We hypothesize that TKIs may be much more clinically effective if used at much higher and fully inactivating doses. Such high dosing may only be tolerable if given intermittently rather than continuously. Methods: To test this hypothesis clinically, we conducted a phase 1 dose-escalation study of a 5-day course of lapatinib (Lp) in women with advanced HER2-overexpressing breast cancer. Lp was administered on days 1–5 of a 14 day cycle. Starting Lp dose was 1750 mg/day and was escalated in cohorts of 3 until the maximum tolerated dose (MTD) was defined. The first cycle of Lp therapy was bracketed by tumor fine needle aspirations and serologic studies, performed at baseline and on day 5. Functional signaling pathway biomarker analysis was performed on tumor lysates using Reverse Phase Protein Microarrays to assay the inactivation of HER2-HER3 signaling. Plasma Lp concentration was assayed at all dose levels and cardiac function was rigorously monitored. Tumor core biopsies were performed on a subset of patients at the MTD to determine tissue Lp levels. Results: 17 patients have been treated to date. No grade 4 or serious adverse events (AEs) attributable to Lp have been noted. 1 patient experienced dose-limiting toxicity at dose level 6 (7000 mg/day) consisting of grade 3 diarrhea despite maximal anti-diarrheal support. There have been no cardiac AEs. The most common AEs include grade 2 diarrhea, nausea, vomiting, acne. Conclusions: The MTD and systemic exposure of Lp can be considerably increased throsugh intermittent dosing. [Table: see text]

Clinical recommendations for the use of lapatinib ditosylate plus capecitabine for patients with advanced or metastatic HER2‐positive breast cancer

AbstractPrimary and acquired resistance to trastuzumab pose a therapeutic challenge when treating patients with HER2 (erbB‐2)‐positive locally advanced or metastatic breast cancer (MBC). The recent introduction of lapatinib (Tykerb/Tyverb, GlaxoSmithKline, Brentford, UK) provides a new management option for such patients. A prospective, randomized phase III clinical trial has confirmed that lapatinib in combination with capecitabine extends time to progressive disease in HER2‐positive MBC, compared with capecitabine alone in patients with disease progression despite prior anthracycline, taxane and trastuzumab therapy. Preliminary data also indicate that lapatinib may exert a beneficial effect on brain metastases, a common sanctuary site for HER2‐positive breast cancer following trastuzumab treatment. The tolerability of lapatinib is commensurate with that of other erbB family tyrosine kinase inhibitors and no significant new adverse events have emerged following its introduction into clinical practice. In particular, no additive cardiotoxicity has been observed when lapatinib is prescribed after trastuzumab therapy. Based on the published literature and supplemented by clinical experience, this article provides practical management recommendations for the use of lapatinib plus capecitabine in patients with MBC. Issues addressed include patient selection, baseline evaluation and monitoring for clinical benefit. The minimization and management of adverse events is also discussed in detail, particularly the dermatological and gastrointestinal effects, which are the most clinically significant side‐effects of lapatinib therapy. Further recommendations cover the minimization of drug interactions, anticipated dosing alterations and the optimal employment of oral anticancer regimens.

Fibronectin promotes cell proliferation of human pre-B cell line via its interactions with VLA-4 and VLA-5

Fibronectin (FN) is thought to play an important role in various aspects of hematopoiesis through binding to very late antigen (VLA)-4 and VLA-5. Little is known, however, about the effects of FN on the proliferation of B cell progenitors. In this study, we investigated the effects of immobilized FN on the proliferation of the pre-B cell line, Nalm-6, which expresses both VLA-4 and VLA-5. Immobilized FN significantly promoted the proliferation of Nalm-6 cells through the synergistic effects of VLA-4 and VLA-5. Furthermore, FN induced the phosphorylation of mitogen-activated protein kinases (MAPKs) of Nalm-6 cells. The MAPK kinase 1 (MEK1) inhibitor, PD98059, and Src family tyrosine kinase inhibitor, herbimycin A, inhibited the FN-promoted proliferation of Nalm-6 cells. These results demonstrate that the interactions of FN and VLA-4/VLA-5 transmit the growth signals that are mediated through Src family tyrosine kinases and the MAPK cascade in Nalm-6 cells. The precise mechanism of synergistic effect of VLA-4 and VLA-5 on FN-promoted proliferation of Nalm-6 cells should be further investigated.

Increased Activity of the Human Sperm Tyrosine Kinase SRC by the cAMP-Dependent Pathway in the Presence of Calcium1

SRC-related tyrosine kinases are suggested to play a role in the increase of sperm protein phosphotyrosine content that occurs during capacitation. In our laboratory, we previously demonstrated that the SRC-related tyrosine kinase YES1 (also known as c-YES) is present in human spermatozoa. However, since it is negatively regulated by Ca(2+), whose intracellular concentration increases during capacitation, another kinase would most likely be involved in the capacitation-related increase in sperm protein tyrosine phosphorylation. The present study represents the first direct assessment of SRC tyrosine kinase activity in ejaculated mammalian sperm. By immunohistochemistry on human testis sections, it is clearly shown that SRC is expressed during spermatogenesis, mainly in round and elongating spermatids. Using an indirect immunofluorescence approach, SRC is detected in the acrosomal region of the head and in the sperm flagellum of ejaculated sperm. This tyrosine kinase is associated with the plasma membrane and with cytoskeletal elements, as suggested by its partial solubility in nonionic detergents. Despite its partial solubility, SRC kinase activity was assayed after immunoprecipitation using acid-denatured enolase as a substrate. It is clearly demonstrated that SRC activity is inhibited by SU6656 and PP1, selective SRC family tyrosine kinase inhibitors, and activated in a Ca(2+)-dependent manner. Furthermore, it is shown that SRC is activated in a cAMP/PRKA-dependent manner; SRC coimmunoprecipitates with the catalytic subunit of the cAMP-dependent protein kinase (PRKAC) and is phosphorylated by this latter kinase, resulting in an increase in enolase phosphorylation. All these results support the involvement of the tyrosine kinase SRC in the increase in sperm protein phosphotyrosine content observed during capacitation.

P21-activated Kinase-aberrant Activation and Translocation in Alzheimer Disease Pathogenesis

Defects in dendritic spines and synapses contribute to cognitive deficits in mental retardation syndromes and, potentially, Alzheimer disease. p21-activated kinases (PAKs) regulate actin filaments and morphogenesis of dendritic spines regulated by the Rho family GTPases Rac and Cdc42. We previously reported that active PAK was markedly reduced in Alzheimer disease cytosol, accompanied by downstream loss of the spine actin-regulatory protein Drebrin. beta-Amyloid (Abeta) oligomer was implicated in PAK defects. Here we demonstrate that PAK is aberrantly activated and translocated from cytosol to membrane in Alzheimer disease brain and in 22-month-old Tg2576 transgenic mice with Alzheimer disease. This active PAK coimmunoprecipitated with the small GTPase Rac and both translocated to granules. Abeta42 oligomer treatment of cultured hippocampal neurons induced similar effects, accompanied by reduction of dendrites that were protected by kinase-active but not kinase-dead PAK. Abeta42 oligomer treatment also significantly reduced N-methyl-d-aspartic acid receptor subunit NR2B phosphotyrosine labeling. The Src family tyrosine kinase inhibitor PP2 significantly blocked the PAK/Rac translocation but not the loss of p-NR2B in Abeta42 oligomer-treated neurons. Src family kinases are known to phosphorylate the Rac activator Tiam1, which has recently been shown to be Abeta-responsive. In addition, anti-oligomer curcumin comparatively suppressed PAK translocation in aged Tg2576 transgenic mice with Alzheimer amyloid pathology and in Abeta42 oligomer-treated cultured hippocampal neurons. Our results implicate aberrant PAK in Abeta oligomer-induced signaling and synaptic deficits in Alzheimer disease.