Src Kinase Family Members Research Articles

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.

Axl Receptor Tyrosine Kinase Signaling Pathway and the p53 Tumor Suppressor Protein Exist In A Novel Regulatory Loop In B-Cell Chronic Lymphocytic Leukemia Cells

Abstract 799 Background:Axl is a constitutively active receptor tyrosine kinase (RTK) in CLL B-cells. We have shown that Axl is physically associated with multiple non-receptor cellular kinases and regulates their activities in CLL B-cells including Lyn kinase (a member of Src-family kinases reported to be overexpressed in CLL), PI3K, Syk and phospholipase γ2 (PLCγ2). These aspects likely relate to drug resistance and survival. Importantly, inhibition of Axl by pharmacological inhibitor (R428) induces massive cell death suggesting its critical role in CLL B-cell survival. We hypothesized that in addition to its role in regulating cellular kinases, Axl might modulate the tumor suppressor p53 to make CLL B-cells more resistant to conventional therapeutic agents. This is based on our unpublished findings that CLL B-cells from patients who have undergone treatment without any reduction in tumor burden expressed increased levels of Axl vs. their pre-therapy levels. In addition, we have detected higher levels of Axl in 17p-deleted CLL patients. Thus to understand the mechanism of Axl RTK-mediated regulation of CLL B-cell growth and survival, we interrogated the functional association between Axl and the HDM2 (human homolog of mouse MDM2)/p53 axis. Methods:We cloned the entire (∼2 kb) 3’-untranslated region (UTR) of the Axl transcript into pMIR-Report vector downstream of the fire-fly luciferase gene. Human embryonic kidney cells (HEK293) were transfected with a full length Axl gene containing the first 500 nucleotides of its 3’-UTR or the pMIR-Report plasmid with or without miR-34a. Cell lysates were examined for the expression of Axl by Western blot or performed luciferase assay. Purified CLL B-cells were also treated with various kinase inhibitors including, Wortmannin (pan-PI3K-inhibitor), PP1 (Src-inhibitor) and SKI-606 (targets both Src and Axl) to detect signaling pathway components downstream of Axl involved in regulating HDM2 function. Results:CLL B-cells from majority of CLL patients (n=15) showed constitutive levels of Axl phosphorylation which we found was associated with the phosphorylation level of HDM2 at Ser-166. HDM2 has an important function in controlling p53 stability in response to genotoxic stress. Recent studies have shown that HDM2 can be phosphorylated on Ser-166 by AKT, MAPKs, and other kinases and this phosphorylation regulates HDM2 nuclearcytoplasmic shuttling under certain conditions. However, we found that HDM2 phosphorylation in CLL is independent of PI3K/AKT, p70S6K, and Erk1/2 activation. Interestingly, we found that inhibition of Axl and/or Src kinases (using SKI-606 and PP1) inhibited HDM2 phosphorylation suggesting that Axl and Src kinases are critically involved in regulating HDM2 affinity towards p53 in CLL B-cells. We also identified a complementary binding sequence of miR-34a microRNA on the first 100 nucleotides of Axl 3’-UTR, suggesting a possible post-translational regulation of Axl. Interestingly, miR-34 family transcription was reported to be regulated by p53, and miR-34a was found differentially expressed in CLL versus normal CD5+ B-cells and is linked to response to therapy in CLL. Indeed, exogenous expression of the Axl gene containing the first 500 nucleotides of the Axl 3’-UTR in HEK293 cells exhibited a dose-dependent down regulation of the Axl protein upon co-transfection with miR-34a using Western blot analysis. Furthermore, activation of p53 in CLL B-cells upon treatment with the DNA-damage agent doxorubicin showed a 4–5 fold decrease of Axl protein by flow cytometry. Activation of p53 by doxorubicin was verified by detecting increased expression of p53 and its acetylation at lysine-382 by Western blot. In addition, luciferase assay data using the Axl-3’-UTR reporter plasmid DNA and miR-34a revealed a dose-dependent inhibition of the luciferase activity. Further studies are underway to fully dissect these novel regulatory signaling pathways. Conclusions: Together, these findings suggest that the two functionally distinct cell growth regulatory pathways can modulate CLL B-cell survival in response to various stimuli. This study has established a link between Axl RTK cell-survival signaling pathway and the tumor suppressor p53-mediated apoptosis-signaling pathway in CLL. These first of a kind observations also underscore that the relative balance of p53 status and Axl levels in tumor cells could relate to CLL therapeutic outcome. Disclosures:Kay:Biothera: Research Funding; Clegene: Research Funding; Cephalon: Research Funding; Genentech: Research Funding; Glaxo Smith Kline: Research Funding; Hospira: Research Funding; Novartis: Research Funding; Supergen: Research Funding; Calistoga: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Emergent Biosolutions (Formerly Trubion): Membership on an entity's Board of Directors or advisory committees.

Receptor-like Tyrosine Phosphatases CD45 and CD148 Have Distinct Functions in Chemoattractant-Mediated Neutrophil Migration and Response to S. aureus

Neutrophils, critical innate immune effectors, use bacterial-derived chemoattractant-induced G protein-coupled receptor (GPCR) signaling for their pursuit of bacteria. Tyrosine phosphorylation pathways and receptor-like tyrosine phosphatases (RPTPs) are rarely considered in chemoattractant-mediated GPCR signaling. Here, we report that two RPTPs, CD45 and CD148, previously shown to share redundant roles in positively regulating Src family kinases (SFKs) in immunoreceptor signaling pathways in B cells and macrophages, are critical in the neutrophil response to S. aureus infection and, surprisingly, in chemoattractant-mediated chemotaxis. Remarkably, deficiency in either of these RPTPs influenced neutrophil GPCR responses in unique ways. Our results reveal that CD45 positively while CD148 positively and negatively regulate GPCR function and proximal signals including Ca(2+), phosphatidylinositol 3'OH kinase (PI3K), and phospho-extracellular regulated kinase (pERK) activity. Moreover, our results suggest that CD45 and CD148 preferentially target different SFK members (Hck and Fgr versus Lyn, respectively) to positively and negatively regulate GPCR pathways.

Inhibition of cell growth and up-regulation of MAD2 in human oesophageal squamous cell carcinoma after treatment with the Src/Abl inhibitor dasatinib

Aberrant expression and/or activity of the non-receptor protein tyrosine kinase SFK (Src family kinase) members are commonly observed in progressive stages of human tumours. The aim of the present study was to investigate whether Src is a potential drug target for treating oesophageal squamous cell carcinoma. Compared with the human immortalized oesophageal epithelial cell line SHEE, oesophageal squamous cell carcinoma cells have increased tyrosine phosphorylation activities. We have explored the therapeutic potential of dasatinib, a small-molecule inhibitor that targets multiple cytosolic and membrane-bound tyrosine kinases, for the treatment of oesophageal squamous cell carcinoma. We examined that the effects of dasatinib on proliferation, invasion, apoptosis, spindle checkpoint, cell-cycle arrest and kinase activity in vitro using three human oesophageal carcinoma cell lines KYSE30, KYSE180 and EC109. In nude mouse models, dasatinib treatment effectively inhibited the expression of activated Src, resulting in the inhibition of tumour growth. Multiple drug effect isobologram analysis was used to study interactions with the chemotherapeutic drug docetaxel. As expected, the three oesophageal carcinoma cell lines were highly sensitive to dasatinib, but SHEE cells were not sensitive to this drug. Concentration-dependent anti-proliferative effects of dasatinib were observed in the three oesophageal carcinoma cell lines. Dasatinib significantly inhibited oesophageal carcinoma cell invasion and up-regulation of MAD2 (mitotic arrest-deficient 2), as well as inducing cell apoptosis and cell-cycle arrest. Additive and synergistic interactions were observed for the combination of dasatinib and docetaxel. Therefore it was concluded that dasatinib blocks the G₁/S transition and inhibits cell growth. These results provided a clear biological rationale to test dasatinib as a single agent or in combination with chemotherapy in oesophageal squamous cell carcinoma. Moreover, we have shown in vitro and in vivo that dasatinib might have therapeutic benefit for patients with oesophageal squamous cell carcinoma who are not eligible for surgery.

Ouabain Stimulates a Na+/K+-ATPase-Mediated SFK-Activated Signalling Pathway That Regulates Tight Junction Function in the Mouse Blastocyst

The Na+/K+-ATPase plays a pivotal role during preimplantation development; it establishes a trans-epithelial ionic gradient that facilitates the formation of the fluid-filled blastocyst cavity, crucial for implantation and successful pregnancy. The Na+/K+-ATPase is also implicated in regulating tight junctions and cardiotonic steroid (CTS)-induced signal transduction via SRC. We investigated the expression of SRC family kinase (SFK) members, Src and Yes, during preimplantation development and determined whether SFK activity is required for blastocyst formation. Embryos were collected following super-ovulation of CD1 or MF1 female mice. RT-PCR was used to detect SFK mRNAs encoding Src and Yes throughout preimplantation development. SRC and YES protein were localized throughout preimplantation development. Treatment of mouse morulae with the SFK inhibitors PP2 and SU6656 for 18 hours resulted in a reversible blockade of progression to the blastocyst stage. Blastocysts treated with 10−3 M ouabain for 2 or 10 minutes and immediately immunostained for phosphorylation at SRC tyr418 displayed reduced phosphorylation while in contrast blastocysts treated with 10−4 M displayed increased tyr418 fluorescence. SFK inhibition increased and SFK activation reduced trophectoderm tight junction permeability in blastocysts. The results demonstrate that SFKs are expressed during preimplantation development and that SFK activity is required for blastocyst formation and is an important mediator of trophectoderm tight junction permeability.

Differential transformation capacity of Src family kinases during the initiation of prostate cancer

Src family kinases (SFKs) are pleiotropic activators that are responsible for integrating signal transduction for multiple receptors that regulate cellular proliferation, invasion, and metastasis in a variety of human cancers. Independent groups have identified increased expression of individual SFK members during prostate cancer progression, raising the question of whether SFKs display functional equivalence. Here, we show that Src kinase, followed by Fyn kinase and then Lyn kinase, exhibit ranked tumorigenic potential during both paracrine-induced and cell-autonomous-initiated prostate cancer. This quantitative variation in transformation potential appears to be regulated in part by posttranslational palmitoylation. Our data indicate that development of inhibitors against specific SFK members could provide unique targeted therapeutic strategies.

Functional roles of Src and Fgr in ovarian carcinoma.

Src is an attractive target because it is overexpressed in a number of malignancies, including ovarian cancer. However, the effect of Src silencing on other Src family kinases (SFKs) is not known. We hypothesized that other SFK members could compensate for the lack of Src activity. Cell viability after either Src or Fgr silencing was examined in ovarian cancer cell lines by MTT assay. Expression of SFKs after Src silencing in ovarian cancer cells was examined by real-time reverse transcriptase (RT)-PCR. Therapeutic effect of in vivo Src and/or Fgr silencing was examined using siRNA incorporated into chitosan nanoparticles (siRNA/CH-NP). Microvessel density, cell proliferation, and apoptosis markers were determined by immunohistochemical staining in ovarian tumor tissues. Src silencing enhanced cytotoxicity of docetaxel in both SKOV3ip1 and HeyA8 cells. In addition, Src silencing using siRNA/CH-NP in combination with docetaxel resulted in significant inhibition of tumor growth compared with control siRNA/CH-NP (81.8% reduction in SKOV3ip1, P = 0.017; 84.3% reduction in HeyA8, P < 0.005). These effects were mediated by decreased tumor cell proliferation and angiogenesis, and increased tumor cell apoptosis. Next, we assessed the effects of Src silencing on other SFK members in ovarian cancer cell lines. Src silencing resulted in significantly increased Fgr levels. Dual Src and Fgr silencing in vitro resulted in increased apoptosis that was mediated by increased caspase and AKT activity. In addition, dual silencing of Src and Fgr in vivo using siRNA/CH-NP resulted in the greatest reduction in tumor growth compared with silencing of either Src or Fgr alone in the HeyA8 model (68.8%, P < 0.05). This study demonstrates that, in addition to Src, Fgr plays a biologically significant role in ovarian cancer growth and might represent an important target.

Tyrosine Phosphorylation Profiling in FGF-2 Stimulated Human Embryonic Stem Cells

The role of fibroblast growth factor-2 (FGF-2) in maintaining undifferentiated human embryonic stem cells (hESC) was investigated using a targeted phosphoproteomics approach to specifically profile tyrosine phosphorylation events following FGF-2 stimulation. A cumulative total number of 735 unique tyrosine phosphorylation sites on 430 proteins were identified, by far the largest inventory to date for hESC. Early signaling events in FGF-2 stimulated hESC were quantitatively monitored using stable isotope dimethyl labeling, resulting in temporal tyrosine phosphorylation profiles of 316 unique phosphotyrosine peptides originating from 188 proteins. Apart from the rapid activation of all four FGF receptors, trans-activation of several other receptor tyrosine kinases (RTKs) was observed as well as induced tyrosine phosphorylation of downstream proteins such as PI3-K, MAPK and several Src family members. Both PI3-K and MAPK have been linked to hESC maintenance through FGF-2 mediated signaling. The observed activation of the Src kinase family members by FGF-2 and loss of pluripotent marker expression post Src kinase inhibition may point to the regulation of cytoskeletal and actin depending processes to maintain undifferentiated hESC.

Role of Abl and Src family kinases in actin-cytoskeletal rearrangements induced by the Helicobacter pylori CagA protein

The clinical outcome of infections with Helicobacter pylori is determined by a complex interplay of host–pathogen interactions, and persistent infection with this pathogen is the major cause of developing chronic gastritis, peptic ulcers and gastric cancer. Highly virulent strains encode a so-called type IV secretion system which translocates the CagA effector protein into gastric epithelial target cells. Injected CagA becomes tyrosine-phosphorylated on EPIYA sequence motifs by Src and Abl family kinase members. CagA then binds to and activates/inactivates various signalling proteins in a phosphorylation-dependent and phosphorylation-independent manner. In this way injected CagA can act as a master key that evolved during evolution the ability to highjack multiple downstream signalling cascades. Here we review our knowledge on the tyrosine phosphorylation motifs in CagA, the recent advances in the interaction of CagA with Src and Abl tyrosine kinases and their role in signalling events leading to changes of the phosphorylation status of actin-binding proteins cortactin, ezrin and vinculin followed by actin-cytoskeletal rearrangements, cell scattering and elongation. Detailed investigation of these pathways will help to yield novel insights and to elucidate the mechanisms of H. pylori-induced pathogenesis.

Fyn is induced by Ras/PI3K/Akt signaling and is required for enhanced invasion/migration

Src family kinases (SFKs) are frequently over-expressed and/or activated in human cancers, and play key roles in cancer cell invasion, metastasis, proliferation, survival, and angiogenesis. Allosteric activation of SFKs occurs through well-defined post-translational mechanisms, however the SFK member Fyn is over-expressed in multiple human cancers (prostate, melanoma, pancreatic, glioma, chronic myelogenous leukemia) and the mechanism of increased Fyn expression is unclear. Since activation of Ras oncogenes is a common oncogenic event leading to the activation of multiple effector pathways, we explored if Ras could induce Fyn expression. Retroviral transduction of the human keratinocyte cell line HaCaT with oncogenic H-Ras dramatically up-regulated Fyn mRNA (>100-fold, P < 0.001), protein, and kinase activity without affecting Src levels or activity. Activation of Akt, but not MAPK or EGFR, was necessary and sufficient for induction of Fyn by H-Ras. Expression of active Fyn was sufficient to increase HaCaT cell migration and invasion, and the enhanced migration and invasion induced by H-Ras could be significantly blocked (70% reduction, P < 0.01) by knockdown of Fyn with a specific siRNA or inhibition of SFKs with PP2. In addition, expression of Fyn in MDA-MB-231 breast cancer cells was dependent on PI3K activity and was involved in their invasive phenotype. Thus, the Ras/PI3K/Akt pathway can account for Fyn over-expression in cancers, and Fyn is a critical mediator of the Ras-stimulated invasive cell phenotype. These results support the development of therapeutic strategies targeting Akt/Fyn pathway to block migration and invasion of tumor cells.

Tyrosine phosphorylation controls nuclear export of Fyn, allowing Nrf2 activation of cytoprotective gene expression

Fyn, an Src kinase family member, acts as a negative regulator of NF-E2-related factor 2 (Nrf2). Under stressful conditions, Nrf2 translocates into the nucleus and binds to the antioxidant response element (ARE), activating defensive gene expression. Once Nrf2 completes activation, Fyn phosphorylates tyrosine 568 of Nrf2, resulting in the nuclear export and degradation of Nrf2. The present studies demonstrate that within 0.5 h of antioxidant treatment in human hepatoblastoma (HepG2) cells, Fyn exports out of the nucleus, allowing Nrf2 unimpeded movement to the ARE. Mutation of tyrosine 213 of Fyn stymied nuclear export, suggesting that tyrosine phosphorylation controls nuclear export. Mass spectrometry confirmed tyrosine 213 as the site of phosphorylation. ChIP and real-time PCR assays revealed that FynY213A mutant caused decreased binding of Nrf2 to the promoter of defensive gene NAD(P)H:quinone oxidoreductase 1 (NQO1) and decreased NQO1 expression by 5-fold (P<0.0001) compared to wild-type Fyn. In addition, a putative nuclear export signal (NES) was identified, and mutation of it also inhibited nuclear export of Fyn. Furthermore, FynY213A caused an increased susceptibility to cell death following treatment with etoposide in mouse hepatoma (Hepa-1) cells. The preinduction regulation of Nrf2 is controlled by the nuclear export of Fyn, allowing for activation of defensive gene expression.

Virtual and Physical Analysis of the Proximal T Cell Signalosome Reveals PP2A Inhibition Is Important for Co-Stimulatory Function of Immunomodulatory (IMiD) Drugs

AbstractAbstract 281Lenalidomide generates high rates of transfusion independence and complete cytogenetic response in del(5q) MDS patients by selective clonal suppression. This family of immunomodulatory drugs (IMiDs) including lenalidomide (CC5013, Revlimid®) and pomalidomide (CC4047, Actimid®) are thalidomide analogues that also induce T cell receptor (TCR) co-stimulation, as well as augment NK and NKT cells by an unknown mechanism of action. Signaling by the T cell receptor (TCR) involves activation of a multi-subunit complex known as the Proximal Signalosome that orchestrates the ordered sequential events leading to phosphorylation of Src-family kinase members Lck and Fyn, phosphorylation of ZAP-70/Syk protein tyrosine kinases, recruitment of SLP-76, Grb-2, and Vav1. IMiDs operate through the B7-CD28 pathway, which provides co-stimulation and enhancement to the Signalosome pathway. To determine the molecular target, we evaluated the activation status of proteins involved in the TCR/CD28 Signalosome after lenalidomide treatment using a murine CD28-/- thymoma cell line transfected with full-length, wild type CD28 or a mutant (tailless) CD28 molecule lacking the intracellular signaling domain (Dennehy et al, 2007, J. of Immunology). We found that lenalidomide augments phosphorylation components downstream of CD28 in WT, but not in tailless mutant cells, suggesting that the intracytoplasmic domain of CD28 is necessary for drug response. We have previously shown that lenalidomide inhibits the activity of two haplodeficient phosphatases on chromosome 5q, Protein Phosphatase 2A (PP2A) and Cdc25c, located within the commonly deleted region (CDR). PP2Ac is known to bind to CD28 and is hypothesized to inhibit T cell co-stimulation. Therefore, it is plausible that lenalidomide and other IMiDs inhibit PP2A in T cells to augment proximal T cell signals. We examined this using molecular modeling and virtual screening. The application GLIDE (Schrödinger, L.L.C.) was used to estimate the free energy of binding for thalidomide, lenalidomide, and pomalidomide to the PP2A-ca heterodimer. The PP2A crystal structure (PDB 3K7V) was analyzed by SiteMap (Schrödinger, L.L.C.) to identify potential small-molecule binding sites. SiteMap identified the catalytic site which was then used for our binding simulations. The IMiD compounds and a known PP2A inhibitor, Fostriecin, were prepared for docking using LigPrep (Schrödinger, L.L.C.) which creates 3D geometry for the structures, provides alternative tautomers, ionization states (for pH values between 5.0 and 9.0), alternative ring conformations, and diastereomers. Free energies of binding and poses for the molecules suggest that both the R and S enantiomers of all three immunomodulatory drugs bind to the same position within the catalytic pocket of the c subunit of the PP2A heterodimer and thereby inhibit phosphatase activity. The S enantiomers of both lenalidomide and pomalidomide have better binding poses and energies (-5.80 kcal/mol and -6.06 kcal/mol respectively) than S thalidomide which has one hydrogen bond, instead of two, and a worse binding free energy than its enantiomer (-5.34 kcal/mol vs. -6.38 kcal/mol) (Figure 1). This increase in binding energy, when coupled with the change in binding pose suggests that S thalidomide would be an inferior inhibitor. The R enantiomers of all three drugs bind with nearly identical posing and binding free energy. Therefore, we hypothesized that, as a racemate, thalidomide may have reduced inhibitory potency compared to the other IMiDs. This was tested in vitro using hemaglutinin (HA)-tagged PP2A immunoprecipitated from stably transfected ad293 cells in a Malachite Green phosphatase assay. All of the drugs showed inhibition of phosphatase activity compared to control but interestingly, PP2A treated with 10μ M of lenalidomide and pomalidomide showed 40.4%±1.93 and 42.6%±0.29 decreased activity respectively compared with thalidomide (24.6%±7.5), which validated the modeling predictions. Together, these results suggest that IMiDs inhibit the enzymatic activity of PP2A, which augments TCR/CD28 Signalosome activity. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Breast cancer patients’ clinical outcome measures are associated with Src kinase family member expression

Background:This study determined mRNA expression levels for Src kinase family (SFK) members in breast tissue specimens and assessed protein expression levels of prominent SFK members in invasive breast cancer to establish associations with clinical outcome. Ki67 was investigated to determine association between SFK members and proliferation.Methods:The mRNA expression levels were assessed for eight SFK members by quantitative real-time PCR. Immunohistochemistry was performed for c-Src, Lyn, Lck and Ki67.Results:mRNA expression was quantified in all tissue samples. SRC and LYN were the most highly expressed in malignant tissue. LCK was more highly expressed in oestrogen receptor (ER)-negative, compared with ER-positive tumours. High cytoplasmic Src kinase protein expression was significantly associated with decreased disease-specific survival. Lyn was not associated with survival at any cellular location. High membrane Lck expression was significantly associated with improved survival. Ki67 expression correlated with tumour grade and nuclear c-Src, but was not associated with survival.Conclusions:All eight SFK members were expressed in different breast tissues. Src kinase was highest expressed in breast cancer and had a negative impact on disease-specific survival. Membrane expression of Lck was associated with improved clinical outcome. High expression of Src kinase correlated with high proliferation.

Well-Known Signaling Proteins Exert New Functions in the Nucleus and Mitochondria

One distinguishing feature of eukaryotic cells is their compartmentalization into organelles, which all have a unique structural and functional identity. Some proteins are exclusively localized in a single organelle, whereas others are found in more than one. A few proteins, whose function was thought to be completely understood, were only recently found to be present in the mitochondria. Although these proteins come from diverse functional classes, their common new denominator is the regulation of respiratory chain activity. Therefore, this review focuses on new functions of the Signal Transducer and Activator of Transcription 3, originally described as a transcription factor, the most prominent Src kinase family members, Src, Fyn, and Yes, which were so far known as plasma membrane-associated molecular effectors of a variety of extracellular stimuli, the tyrosine phosphatase Shp-2 previously characterized as a modulator of cytosolic signal transduction involved in cell growth, development, inflammation, and chemotaxis, and Telomerase Reverse Transcriptase, the key enzyme preventing telomere erosion in the nucleus. Their unexpected localization in other organelles and regulation of mitochondrial and/or nuclear functions by them adds a new layer of regulatory complexity. This extends the flexibility to cope with changing environmental demands using a limited number of genes and proteins.

Role of the Kaposi's Sarcoma-Associated Herpesvirus K15 SH3 Binding Site in Inflammatory Signaling and B-Cell Activation

The Kaposi's sarcoma-associated herpesvirus (KSHV) contains several open reading frames (ORFs) that encode proteins capable of initiating and modulating cellular signaling pathways. Among them is ORF K15, encoding a 12-transmembrane-spanning protein with a cytoplasmic C-terminal domain. Through conserved binding motifs, such as Src homology 2 (SH2) and SH3 binding sites, K15 interacts with cellular proteins, activates the NF-kappaB, MEK/Erk, and Jun N-terminal protein kinase (JNK) pathways, and induces the expression of several inflammatory and angiogenic genes. In this study, we investigated the role of an SH3 domain binding site centered on a PPLP motif in K15. We screened libraries of cellular SH3 domains to identify signaling molecules interacting with the KSHV PPLP motif. We found its affinities for two Src kinase family members, Lyn and Hck, to exceed those of other viral proteins. While the SH2 binding motif YEEV is essential for the inflammatory response induced by KSHV K15, recruitment of Lyn and Hck to the K15 PPLP motif seems to be dispensable for this inflammatory response. However, the PPLP motif is essential for the decrease in B-cell receptor-mediated signaling induced by K15, as measured by calcium mobilization assays.

Structural elements and allosteric mechanisms governing regulation and catalysis of CSK-family kinases and their inhibition of Src-family kinases

C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK) are endogenous inhibitors constraining the activity of the oncogenic Src-family kinases (SFKs) in cells. Both kinases suppress SFKs by selectively phosphorylating their consensus C-terminal regulatory tyrosine. In addition to phosphorylation, CHK can suppress SFKs by a unique non-catalytic inhibitory mechanism that involves tight binding of CHK to SFKs to form stable complexes. In this review, we discuss how allosteric regulators, phosphorylation, and inter-domain interactions interplay to govern the activity of CSK and CHK and their ability to inhibit SFKs. In particular, based upon the published results of structural and biochemical analysis of CSK and CHK, we attempt to chart the allosteric networks in CSK and CHK that govern their catalysis and ability to inhibit SFKs. We also discuss how the published three-dimensional structure of CSK complexed with an SFK member sheds light on the structural basis of substrate recognition by protein kinases.

Abstract 3687: Expression of a Src-family kinase in CML cells induces resistance to imatinib in a kinase-dependent manner

Abstract Chronic Myelogenous Leukemia (CML) is characterized by the presence of Philadelphia Chromosome, which results in the expression of the 210 kDa Bcr-Abl tyrosine kinase. Bcr-Abl constitutively activates signaling pathways important for the proliferation, survival and cytokine independence of myeloid progenitors, including the Src family kinases (SFKs). Imatinib, an inhibitor of Bcr-Abl tyrosine kinase activity, is the frontline therapy for CML. Although imatinib is remarkably effective in the chronic phase of the disease, patients with accelerated or blast crisis CML often develop resistance. Several recent studies performed on clinical specimens from imatinib-resistant patients with wild-type Bcr-Abl found that the Src family kinase members Hck and Lyn are overexpressed or highly active, suggesting that Src kinases may play a role in imatinib resistance. While the role of Lyn has been addressed in some detail, the contribution of Hck to this type of imatinib resistance is less well understood. To test whether Hck overexpression in CML cells induces resistance to imatinib in a kinase-dependent manner, we employed a Hck mutant (Hck-T338A) that is uniquely sensitive to the pyrazolo-pyrimidine inhibitor, NaPP1. In vitro, Hck-T338A was 48 times more sensitive to NaPP1 than the wild-type kinase. Importantly, this “gatekeeper” mutation was functionally silent and did not confer a loss or gain of function on Hck in a control fibroblast transformation assay. Expression of wild-type Hck or the T338A mutant in K562 CML cells resulted in resistance to imatinib-induced apoptosis and inhibition of soft-agar colony formation. Treatment with NaPP1 restored sensitivity to imatinib in a concentration-dependent manner only in cells expressing the Hck-T338A mutant. In contrast, cells expressing wild-type Hck were not affected by NaPP1, demonstrating the selectivity of NaPP1 for the Hck-T338A mutant. This result shows that Hck-induced imatinib resistance requires Hck kinase activity. In addition, NaPP1 also reduced Hck-mediated phosphorylation of Bcr-Abl at sites that may affect imatinib binding exclusively in cells expressing Hck-T338A. Together, these data establish a direct cause and effect relationship between Hck overexpression and imatinib resistance in CML cells. Furthermore, our results show that imatinib resistance requires Hck kinase function and may occur via direct Hck-induced trans-phosphorylation of Bcr-Abl. Selective drug targeting of Hck may be of therapeutic benefit in imatinib-resistant CML patients with increased Hck expression or activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3687.

A chemical and phosphoproteomic characterization of dasatinib action in lung cancer

We describe a strategy to comprehend signaling pathways active in lung cancer cells and targeted by dasatinib employing chemical proteomics to identify direct interacting proteins combined with immunoaffinity purification of tyrosine phosphorylated peptides corresponding to activated tyrosine kinases. We identified nearly 40 different kinase targets of dasatinib. These include SFK members (LYN, SRC, FYN, LCK, YES), non-receptor tyrosine kinases (FRK, BRK, ACK), and receptor tyrosine kinases (Ephrin receptors, DDR1, EGFR). Using quantitative phosphoproteomics we identified peptides corresponding to autophosphorylation sites of these tyrosine kinases that are inhibited in a concentration-dependent manner by dasatinib. Using drug resistant gatekeeper mutants, we show that SFK kinases, particularly SRC and FYN, as well as EGFR are relevant targets for dasatinib action. The combined mass spectrometry based approach described here provides a system-level view of dasatinib action in cancer cells and suggests both functional targets and rationale combinatorial therapeutic strategies.

Dasatinib promotes ATRA-induced differentiation of AML cells

Dasatinib is a US Food and Drug Administration-approved compound that was developed as an inhibitor of ABL and Src family kinases (SFKs).1 Dasatinib is substantially more potent than imatinib against wild-type BCR–ABL. Moreover, with the exception of the T315I mutant, dasatinib retains activity against most imatinib-resistant BCR–ABL mutants, helping to explain the effectiveness of dasatinib in all stages of imatinib-resistant CML. The efficacy of dasatinib in CML is furthered by the ability of this compound to inhibit the SFK member LYN, which is persistently hyperactivated in imatinib-resistant disease.2

Src Kinase Family Members Expression in Human Breast Tissue and Their Association to Clinical Outcome of Breast Cancer Patients.

Abstract Background: There is a paucity in the literature about expression levels of Src kinase family members (SKFMs) in human breast tissue. The aim of this study was to assess m-RNA SKFM expression levels in human breast specimens and to assess protein expression of the most significant SKFMs in invasive breast cancer to establish their association to clinical outcome.Material and Methods: m-RNA expression of eight SKFMs (Src, Lck, Lyn, Fgr, Fyn, Hck, Blk, Yes) was assessed by quantitative real time PCR. IHC was performed using antibodies to c-Src, Y419Src, Lck and Lyn. Expression was assessed using the weighted histoscore method.Results: Cohort one consisted of 52 invasive breast cancers (M), 36 non-malignant (NM) and 10 normal (N) breast tissue samples. Median age of the patients was 60 years. Median size of breast cancer was 25mm (IQR 20-40mm). ER status was not significantly different between tissue types (p=0.50). Every SKFM expression was quantified in all tissue samples. Fyn was the most expressed SKFM in normal tissue and Lyn in the NM breast tissue. Blk was the least expressed SKFM in all breast tissues. In malignant breast tissue Src and Lyn were most expressed. Higher expression levels of Lck were observed in invasive breast cancers compared to NM and N (p=&amp;lt;0.001). SKFMs Lck and Lyn were higher expressed in ER negative compared to ER positive tumours. c-Src (p=0.01) and Fyn (p=0.03) were expressed at higher levels in lobular compared to ductal carcinomas. There were no correlations observed between c-Src and SKFMs in N breast tissue. However in NM and M tissue, all seven SKFMs correlated with c-Src expression; strongest correlation observed was with Lyn (p&amp;lt;0.001, c.c 0.805) and least with Yes (p=0.003, c.c.=0.489). No association was noticed between SKFM expression, tumour grade, size and LN positivity.Cohort two consisted of 320 patients with median follow-up of 6.3 years. Median age was 58 years (IQR 24-90). Median tumour size was 20mm (IQR 15-30mm). In both cohorts majority of the cancer specimens were pathologically graded as G2 and G3. 49% of the patients were axillary lymph node positive. High cytoplasmic Src and membrane Y419Src kinase expression levels were significantly associated with decreased disease specific survival (p=0.028, p=0.023). Lyn was not associated with survival at any cellular location. High membrane Lck expression was significantly associated with improved survival (p=0.039).Patient Cohort 2 Patient NumbersP valueAge&amp;lt;50 yrs810.351 &amp;gt;50 yrs239 Tumour typeDuctal2980.714 Lobular18 Others6 Grade1220.014 2152 3146 Size&amp;lt;20mm126&amp;lt;0.001 20-50mm173 &amp;gt;50mm21 ER statusPositive212&amp;lt;0.001 Negative108 PR statusPositive1520.006 Negative168 HER2 statusPositive550.010 Negative265 c-Src cytoplasmPositive1410.023 Negative179 Y419Src membranePositive1540.028 Negative166 Lck membranePositive140.039 Negative266 Discussion: All eight SKFMs are expressed in different breast tissues. In invasive breast cancer Src kinase is highest expressed and seems to have a negative impact on disease specific survival. Whereas, high membrane expression of Lck provides better clinical outcome in those breast cancer patients. Further investigations are required to determine underlying mechanisms for this observation. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6130.