Adaptor Protein CrkL Research Articles

Cell shape-based chemical screening reveals an epigenetic network mediated by focal adhesions.

Adapter proteins CRK and CRKL participate in a variety of signaling pathways, including cell adhesion, and fate regulation of mammalian cells. However, the molecular functions of CRK/CRKL in epigenetic regulation remain largely unknown. Here, we developed a pipeline to evaluate cell morphology using high-content image analysis combined with chemical screening of kinase and epigenetic modulators. We found that CRK/CRKL modulates gene regulatory networks associated with cell morphology through epigenetic alteration in mouse embryonic fibroblasts. Integrated epigenome and transcriptome analyses revealed that CRK/CRKL is involved in super-enhancer activity and upregulation of Cdt1, Rin1, and Spp1 expression for the regulation of cell morphology. Screening of a library of 80 epigenetic inhibitors showed that histone H3 modifiers, euchromatic histone methyltransferase 2 and mitogen- and stress-activated kinase 1, may be important for CRK/CRKL-mediated morphological changes. Taken together, our results indicate that CRK/CRKL plays a critical role in gene regulatory networks through epigenetic modification. DATABASES: Chromatin immunoprecipitation sequencing and RNA sequencing data were deposited in the DNA Data Bank of Japan under DRA011080 and DRA011081 accession numbers, respectively.

MiR-124-3p Suppresses the Invasiveness and Metastasis of Hepatocarcinoma Cells via Targeting CRKL.

Abnormal expressions of microRNAs are involved in growth and progression of human cancers including hepatocellular carcinoma (HCC). An adaptor protein CRKL plays a pivotal role in HCC growth, whereas miR-124-3p downregulation is associated with clinical stage and the poor survival of patients. However, the relationship between miR-124-3p and CRKL and the molecular mechanisms through which they regulate HCC metastasis remains unclear. In the current work, we explored miR-124-3p and its correlation with CRKL expression in HCC patient tissues. We found that miR-124-3p deficiency is inversely co-related with CRKL overexpression in tumorous tissues of HCC patients, which was also consistent in HCCLM3 and Huh7 HCC cell lines. Target validation data shows that miR-124-3p directly targets CRKL. The overexpression of miR-124-3p reverses the CRKL expression at both mRNA and protein levels and inhibits the cell development, migration, and invasion. Mechanistic investigations showed that CRKL downregulation suppresses the ERK pathway and EMT process, and concomitant decrease in invasion and metastasis of HCC cells. The expressions of key molecules in the ERK pathway such as RAF, MEK, ERK1/2, and pERK1/2 and key promoters of EMT such as N-cadherin and vimentin were downregulated, whereas E-cadherin, a key suppression indicator of EMT, was upregulated. MiR-124-3p-mediated CRKL suppression led to BAX/BCL-2 increase and C-JUN downregulation, which inhibited the cell proliferation and promoted the apoptosis in HCC cells. Collectively, our data illustrates that miR-124-3p acts as an important tumor-suppressive miRNA to suppress HCC carcinogenesis through targeting CRKL. The miR-124-3p-CRKL axial regulated pathway may offer valuable indications for cancer research, diagnosis, and treatment.

Requirement for Crk and CrkL during postnatal lens development

The Crk and CrkL adaptor proteins have SH2 and SH3 domains and play essential overlapping, as well as distinct, roles in many biological processes, ranging from cell structure and motility to proliferation. Conditional ablation of both Crk and CrkL in neuronal progenitor cells, using a Nestin-Cre transgene, resulted in severe defects in postnatal eye development, including progressive eye closure, lens rupture, and retinal malformation. These phenotypes were not observed in the presence of a single wild-type allele of either Crk or CrkL. We found that the lens in knockout mice started to rupture and disintegrate between postnatal days 7 and 12, although the structure of the retina was relatively well maintained. As the lens deteriorated further, the outer nuclear layer in the posterior of the retina enlarged and developed ruffles. Cre recombination occurred in the lens and retina of the knockout mice. Furthermore, the posterior lens capsule of the knockout mouse was thinner at postnatal days 0.5 and 3, suggesting that the defective lens capsule caused rupturing of the lens near the posterior pole. These results indicate that Crk and CrkL play essential overlapping roles in postnatal lens development.

CrkL is required for donor T cell migration to GvHD target organs.

The success of cancer therapies based on allogeneic hematopoietic stem cell transplant relies on the ability to separate graft-versus-host disease (GvHD) from graft-versus-tumor (GVT) responses. Controlling donor T cell migration into peripheral tissues is a viable option to limit unwanted tissue damage, but a lack of specific targets limits progress on this front. Here, we show that the adaptor protein CrkL, but not the closely related family members CrkI or CrkII, is a crucial regulator of T cell migration. In vitro, CrkL-deficient T cells fail to polymerize actin in response to the integrin ligand ICAM-1, resulting in defective migration. Using a mouse model of GvHD/GVT, we found that while CrkL-deficient T cells can efficiently eliminate hematopoietic tumors they are unable to migrate into inflamed organs, such as the liver and small intestine, and thus do not cause GvHD. These results suggest a specific role for CrkL in trafficking to peripheral organs but not the lymphatic system. In line with this, we found that although CrkL-deficient T cells could clear hematopoietic tumors, they failed to clear the same tumor growing subcutaneously, highlighting the role of CrkL in controlling T cell migration into peripheral tissues. Our results define a unique role for CrkL in controlling T cell migration, and suggest that CrkL function could be therapeutically targeted to enhance the efficacy of immunotherapies involving allogeneic donor cells.

Overexpression of CrkL as a novel biomarker for poor prognosis in gastric cancer

The signaling adapter protein CrkL plays vital roles in multiple cancers. However, the expression pattern of CrkL protein and its clinical significance have not been well characterized in human gastric cancer (GC) so far. To investigate the association of tissue-based CrkL protein expression level with the clinicopathological characteristics and prognosis of GC patients. The expression level of CrkL protein in 380 GC patients was analyzed by immunohistochemistry. The associations of CrkL protein expression level with clinicopathologicalal characteristics and clinical outcome were evaluated. Compared with the matched adjacent non-tumor tissues, CrkL protein expression level was significantly up-regulated in tumor tissues. In addition, there was a positive correlation between CrkL and Ki67 expression levels in GC patients. An elevated CrkL level statistically correlated with aggressive clinicopathologicalal characteristics, such as larger tumor size, deeper local invasion, more lymph node metastasis, advanced TNM stage, and poorer prognosis. Notably, multivariate analysis identified tissue-based CrkL level as an independent predictor for the unfavorable prognosis of GC. These results indicate that CrkL protein may serve as a novel prognostic biomarker in GC.

PF‑114, a novel selective inhibitor of BCR‑ABL tyrosine kinase, is a potent inducer of apoptosis in chronic myelogenous leukemia cells.

A t(9;22) chromosomal translocation which forms the chimeric tyrosine kinase breakpoint cluster region (BCR)‑Abelson murine leukemia viral oncogene homolog1(ABL) is a key mechanism underlying the pathogenesis of chronic myelogenous leukemia(CML). Pharmacological inhibition of BCR‑ABL with imatinib (Gleevec) has been reported as an effective targeted therapy; however, mutations (including the kinase domain of ABL) suppress the efficacy of inhibitors. PF‑114, a derivative of the third generation BCR‑ABL inhibitor ponatinib, demonstrated a high inhibitory activity against wild-type and mutant BCR‑ABL forms, such as the clinically important T315I. Furthermore, PF‑114 exhibited preferential kinase selectivity, safety, notable pharmacokinetic properties and therapeutic efficacy in a murine model. Investigation into the mechanisms of CML cell death revealed an exceptional potency of PF‑114 (at low nanomolar concentrations) for the CML‑derived K562 cell line, whereas leukemia cell lines that lack the chimeric tyrosine kinase were markedly more refractory. The molecular ordering of events mechanistically associated with K562 cell death included the dephosphorylation of CrkL adaptor protein followed by inhibition of ERK1/2 and Akt, G1 arrest, a decrease of phosphorylated Bcl‑2‑associated death promoter, Bcl‑2‑like protein11, BH3 interacting‑domain death agonist, Bcl‑extra large and Bcl‑2 family apoptosis regulator, and reduced mitochondrial transmembrane potential. Increased AnnexinV reactivity, activation of caspases and poly(ADP‑ribose)polymerase cleavage were proposed to lead to internucleosomal DNA fragmentation. Thus, PF‑114 may be a potent inducer of apoptosis in CML cells. Nevertheless, activation of STAT3 phosphorylation in response to PF‑114 may permit cell rescue; thus, a combination of BCR‑ABL and STAT3 inhibitors should be considered for improved therapeutic outcome. Collectively, the targeted killing of BCR‑ABL‑positive cells, along with other beneficial properties, such as invivo characteristics, suggests PF‑114 as a potential candidate for analysis in clinical trials with CML patients.

Abstract B183: LFA-1 signals via Crk adapter proteins to induce actin-dependent T-cell migration and mechanosensing

Abstract Allogeneic hematopoietic stem cell transplants (HSCT) are used to treat many malignancies, but the prevalence of graft-versus-host disease (GvHD) limits their overall success. Manipulating T-cell trafficking has emerged as an effective countermeasure, yet downstream integrin signaling pathways have yet to be targeted. We previously found that T-cells lacking the adapter proteins Crk and CrkL exhibit a robust antitumor response while causing little GvHD. We now find that T-cells from mice lacking Crk adapter proteins exhibit defects in LFA-1/ICAM-1 induced actin polymerization, leading edge formation, 2D migration, and integrin-mediated mechanosensing. Under shear flow, Crk/CrkL deficient T-cells fail to migrate upstream on ICAM-1 but migrate normally on VCAM-1, suggesting these integrin ligands relay different outside-in signals. Analysis of LFA-1 signaling reveals that Crk protein expression is required for phosphorylation of c-Cbl and its subsequent interaction with the PI3K subunit p85. Through this mechanism, Crk proteins promote PI3K activity and cytoskeletal remodeling downstream of LFA-1 engagement. Interestingly, this signaling pathway was largely specific to the LFA-1/ICAM-1 interaction, as WT-cells plated on VCAM-1 (which binds and signals through VLA-4) failed to induce high levels of phospho-Cbl, showed diminished PI3K activity, and did not migrate with a defined actin-rich leading edge. Finally, we show that knocking out CrkL alone is sufficient to alleviate GvHD, pointing toward unique roles of the Crk isoforms in T-cell biology. Together, these studies identify key signaling differences downstream of β2 vs β1 integrins that drive T-cell migratory behavior, and identify CrkL as an important factor during GvHD. Importantly, these data provide insight into integrin signaling that could be used to manipulate T-cell trafficking. Citation Format: Nathan H. Roy, Joanna L. MacKay, Alexander Buffone Jr., Krista Newell, Tanner F. Robertson, Sangya Agarwal, Mobin Karimi, Daniel A Hammer, Janis K. Burkhardt. LFA-1 signals via Crk adapter proteins to induce actin-dependent T-cell migration and mechanosensing [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B183.

GNB1 K89M Drives TKI Resistance in ETV6-ABL1-Positive Leukemia

Leukemias harboring ETV6-ABL1 fusion represent a rare prognostically unfavorable subgroup of hematological malignancies. To a great extent they share biological and clinical characteristics with more common BCR-ABL1+ leukemias. Both fusion genes encode constitutively activated aberrant tyrosine kinases which can be targeted by specific tyrosine kinase inhibitors (TKI). The efficiency of TKI to inhibit ETV6-ABL1 kinase activity was proven in vitro and substantiated their use in clinical practice. However, there is still a lack of knowledge on potential induction and mechanisms of TKI resistance in ETV6-ABL1+ malignancies.To address this issue we established a multi-TKI resistant ETV6-ABL1+ lymphoblastic cell line via a long term cultivation with TKI imatinib mesylate (IM) and investigated the mechanisms of the acquired TKI resistance. We excluded the most common mechanisms known from BCR-ABL1+ leukemias (kinase domain mutations, genomic amplification and enhanced kinase expression/activation) as causes of resistance in our model. Importantly, using shRNA mediated silencing we showed that unlike their sensitive counterparts, the resistant cells did not respond to the ETV6-ABL1 knockdown by growth suppression or cell death and, therefore, became independent on pro-survival signaling from the chimeric kinase.To further elucidate molecular mechanisms underlying the TKI resistance, we compared biological profiles of resistant and parental sensitive cells. Molecular changes potentially associated with the TKI resistance were analyzed via genomic (single nucleotide polymorphism array, whole exome sequencing), gene expression and proteomic profiling. We integrated data from all platforms and identified K89M mutation in GNB1 gene as the top candidate causative lesion. A mutation affecting the same GNB1 codon, K89E, was recently identified in a blastic plasmacytoid dendritic cell neoplasm by Yoda et al. (Nature Medicine, 2015) and was shown to result in the upregulation of PI3K/Akt/mTOR and MAPK pathways signaling. Interestingly, Yoda et al. also identified functionally similar mutations of GNB1 and GNB2 in several TKI-resistant primary malignancies including a single case with BCR-ABL1+ leukemia.In accordance with these findings we showed that unlike the parental sensitive cells, the resistant ETV6/ABL1+ cells were able to restore PI3K/Akt/mTOR and p44/MAPK signaling after IM treatment.To further confirm the key role of GNB1 K89M mutation in our TKI resistance model we transduced the parental sensitive ETV6/ABL1+ cell line with wild type (WT) GNB1, GNB1-K89M, GNB1-K89E or with empty vector, and studied the impact on TKI sensitivity and kinase signaling. While the cells transduced with WT-GNB1 or with empty vector remained sensitive to TKI, the cells transduced with mutated GNB1 (both K89E and K89M)became resistant to IM, nilotinib, dasatinib and ponatinib. Similarly, while the repeated application of IM resulted in cell growth arrest and cell death in the cells with WT-GNB1 and empty vector, the cells transduced with mutated GNB1 showed only subtle changes in proliferation and viability. Administration of IM resulted in the inhibition of ETV6-ABL1-mediated signaling in all cell lines as demonstrated by diminished phosphorylation of the ETV6-ABL1 direct substrate - CRKL adaptor protein. However, while the IM induced a prolonged inhibition of p44/MAPK and PI3K/Akt/mTOR pathway signaling in the cells with WT-GNB1 and with empty vector, the cells transduced by mutated GNB1 (both K89E and K89M) were able to restore activation of both pathways within 24 hours after IM treatment (similarly to the original multi-TKI resistant cell line).In summary, we showed that the long-term IM treatment of ETV6-ABL1+ leukemia resulted in a gain of TKI-resistance which was driven by the acquired GNB1 mutation activating pro-proliferative and pro-survival signaling, independent on the ETV6-ABL1 kinase activity. Importantly, leukemic cells with mutated GNB1 were resistant not only to IM, but also to the next generation TKI.The frequency of GNB1/GNB2 mutations in primary ETV6-ABL1+ and BCR-ABL1+ leukemias and its clinical relevance is to be determined in the study currently initiated within an international collaboration.Supported by grants GACR P302/12/G10 and GAUK 554214. DisclosuresNo relevant conflicts of interest to declare.

A pre-metazoan origin of the CRK gene family and co-opted signaling network.

CRK and CRKL adapter proteins play essential roles in development and cancer through their SRC homology 2 and 3 (SH2 and SH3) domains. To gain insight into the origin of their shared functions, we have investigated their evolutionary history. We propose a term, crk/crkl ancestral (crka), for orthologs in invertebrates before the divergence of CRK and CRKL in the vertebrate ancestor. We have isolated two orthologs expressed in the choanoflagellate Monosiga brevicollis, a unicellular relative to the metazoans. Consistent with its highly-conserved three-dimensional structure, the SH2 domain of M. brevicollis crka1 can bind to the mammalian CRK/CRKL SH2 binding consensus phospho-YxxP, and to the SRC substrate/focal adhesion protein BCAR1 (p130CAS) in the presence of activated SRC. These results demonstrate an ancient origin of the CRK/CRKL SH2-target recognition specificity. Although BCAR1 orthologs exist only in metazoans as identified by an N-terminal SH3 domain, YxxP motifs, and a C-terminal FAT-like domain, some pre-metazoan transmembrane proteins include several YxxP repeats in their cytosolic region, suggesting that they are remotely related to the BCAR1 substrate domain. Since the tyrosine kinase SRC also has a pre-metazoan origin, co-option of BCAR1-related sequences may have rewired the crka-dependent network to mediate adhesion signals in the metazoan ancestor.

Antibody-based detection of protein phosphorylation status to track the efficacy of novel therapies using nanogram protein quantities from stem cells and cell lines.

This protocol describes a highly reproducible antibody-based method that provides protein level and phosphorylation status information from nanogram quantities of protein cell lysate. Nanocapillary isoelectric focusing (cIEF) combines with UV-activated linking chemistry to detect changes in phosphorylation status. As an example application, we describe how to detect changes in response to tyrosine kinase inhibitors (TKIs) in the phosphorylation status of the adaptor protein CrkL, a major substrate of the oncogenic tyrosine kinase BCR-ABL in chronic myeloid leukemia (CML), using highly enriched CML stem cells and mature cell populations in vitro. This protocol provides a 2.5 pg/nl limit of protein detection (<0.2% of a stem cell sample containing <10(4) cells). Additional assays are described for phosphorylated tyrosine 207 (pTyr207)-CrkL and the protein tyrosine phosphatase PTPRC/CD45; these assays were developed using this protocol and applied to CML patient samples. This method is of high throughput, and it can act as a screen for in vitro cancer stem cell response to drugs and novel agents.

CrkL regulates SDF-1-induced breast cancer biology through balancing Erk1/2 and PI3K/Akt pathways.

The adapter protein CrkL is required for regulating the malignant potential of human cancers. However, the regulatory mechanisms of CrkL on the stromal cell-derived factor 1 (SDF-1)/CXCR4 signaling pathways in breast cancer are not well characterized. Here, CXCR4 and CrkL proteins were tested in breast cancer cell lines and 60 primary breast cancer tissues. In vitro, the roles of CrkL in SDF-1-induced MDA-MB-231 cell cycle, invasion and migration were investigated. In the present study, CXCR4 and CrkL were highly expressed in MCF-7, MDA-MB-231, MDA-MB-231HM MDA-MB-468 and tumor tissues (80 and 60%, respectively) and closely correlated with lymph node metastasis. In vitro studies revealed that SDF-1 induced the activation of CrkL, Erk1/2, Akt and matrix metallopeptidase 9 (MMP9) in MDA-MB-231 cells. The si-CrkL treatment significantly down-regulated the phosphorylated Erk1/2 (p-Erk1/2) and MMP9, but up-regulated p-Akt, compared with control. Importantly, wound-healing and transwell invasion assays showed that si-CrkL significantly impaired the wound closure and inhibited the SDF-1-induced invasion; similarly, flow cytometry showed that si-CrkL affected cell cycle. In conclusion, these results suggest that CrkL plays a regulatory role in the SDF-1-induced Erk1/2 and PI3K/Akt pathways and further managed the invasion and migration of breast cancer cells. Thus, CrkL may be recommended as an interesting therapeutic target for breast cancer.

Adaptor protein Crk-like (CrkL) controls natural killer cell function via regulating the accumulation of activating receptors and phosphorylated Vav-1 molecules at immunological synapses (INM2P.433)

Abstract Very few studies have addressed how CrkL, a ubiquitous adaptor protein, regulates the immune response, especially in NK cells. Previous studies indicate that CrkL is required for NKG2D-mediated cellular cytotoxicity (NC) and CD16 signaling triggered antibody mediated cellular cytotoxicity (ADCC). However, how CrkL protein controls NK cell cytotoxicity remains unclear. Here we used lentiviral inducible shRNA to silence CrkL in human NK cell line and primary NK cells. Inducible knockdown of CrkL significantly reduced NK-target conjugation, degranulation, and cytotoxicity in both NC- and ADCC-mediated immune responses. Further investigation on the function and signaling transduction at the NK cytotoxic immunological synapse (IS) reveals that polarization of lytic granules, and accumulation of both F-actin and phosphorylated Vav-1 at the IS were greatly reduced in CrkL-silenced NK cells. NK cell activation involves in actin-dependent activating receptor clustering. CrkL silencing resulted in reduced accumulation of ligands for CD16 and NKG2D on the lipid bilayer. Similar results were obtained in NK cells from DiGeorge Syndrome patients (CrkL gene deletion). We thus propose a new model for adaptor protein CrkL function in NK cell activation: CrkL controls NK cell cytotoxicity via promoting F-actin accumulation and lytic granule polarization by modulating the accumulation of activating receptors and phosphorylated Vav-1 molecules at the IS.

Monocyte‐derived dendritic cells from chronic myeloid leukaemia have abnormal maturation and cytoskeletal function that is associated with defective localisation and signalling by normal ABL1 protein

Mature dendritic cells (DCs) may be derived from the BCR/ABL1 expressing monocytes in chronic myeloid leukaemia. These cells have potential therapeutic applications, but are recognised to have defective function. In normal DCs, activation and maturation depend on ABL1 dependent signals. We therefore tested the hypothesis that in the DCs of chronic myeloid leukaemia, the presence of the BCR/ABL1 molecule disrupts normal ABL1 signal pathways, and contributes to the observed functional defects of the cells. We employed in vitro culture of clinical samples, combining microscopic and biochemical techniques with a phosphoproteomic approach to compare and characterise DCs from normal individuals and chronic myeloid leukaemia patients. We identified an altered intracellular localisation for ABL1 within DCs derived from the monocytes of chronic myeloid leukaemia. The protein was found in the perinuclear region co-distributed with the adapter-protein CRKL and the BCR/ABL1 protein. This altered distribution was associated with defective generation of ABL1-dependent maturation signals, and a dislocation of ABL1 from the F-actin cytoskeleton. We suggest that abnormal ABL1-dependent signals contribute to the recognised functional defects affecting chronic myeloid leukaemia DCs.

Src-inducible association of CrkL with procaspase-8 promotes cell migration

Procaspase-8, the zymogen form of the apoptosis-initiator caspase-8, undergoes phosphorylation following integrin-mediated cell attachment to an extracellular matrix substrate. Concordant with cell attachment to fibronectin, a population of procaspase-8 becomes associated with a peripheral insoluble compartment that includes focal complexes and lamellar microfilaments. Phosphorylation of procaspase-8 both impairs its maturation to the proapoptotic form and can promote cell migration. Here we show that the cytoskeletal adaptor protein CrkL promotes caspase-8 recruitment to the peripheral spreading edge of cells, and that the catalytic domain of caspase-8 directly interacts with the SH2 domain of CrkL. We show that the interaction is abolished by shRNA-mediated silencing of Src, in Src-deficient MEFs, and by pharmacologic inhibitors of the kinase. The results provide insight into how tyrosine kinases may act to coordinate the suppression caspase-8 mediated apoptosis, while promoting cell invasion.

Circulating CLL Cells Expressing CD49d Display a Phospho-Proteomic Profile Consistent with a Constitutive Receptor Engagement by Blood-Borne Ligands

AbstractAbstract 930Chronic lymphocytic leukemia (CLL) cell growth and survival occur in defined microenvironment niches controlled by several receptor/ligand interactions including those mediated by the α4β1 integrin. This integrin, in particular, is known to interact with both the extracellular matrix component fibronectin, via the non-RGD CS-1 fragments, and the bone marrow/stromal components, via the vascular-cell adhesion molecule-1 (VCAM-1). The α4 integrin chain (a.k.a. CD49d) has been previously shown to be associated with poor prognosis in CLL by marking a subset of CLL patients characterized by a more aggressive clinical course both in term of disease progression and overall survival. Despite the great deal of studies investigating CLL cell microenvironmental interactions in tissue sites, little is known regarding the constitutive engagement of adhesion receptors in circulating CLL cells, and the role in this context of plasma/plasma components. To address this issue, the proteomic profiles of circulating leukemic cells from 80 CLL cases expressing or not CD49d were explored using a reverse phase protein microarray (RPMA) approach quantitatively analysing 77 proteins (61 phosphoproteins) known to be involved in translational control, cell growth, apoptosis, B cell receptor and cytoskeletal signaling. Comparison of the signaling activation portrait between 40 CD49d pos and 40 CD49d neg CLL cases highlighted the over-expression in CD49d pos CLL of proteins involved in the regulation of integrin-mediated cytoskeletal dynamics, such as phospho-p21-activated kinase (PAK1 Ser199-204/PAK2 Ser192-197; p=0.0005), phospho-LIM kinase (LIMK1 Thr508/LIMK2 Thr505; p=0.00001) and the adaptor protein CrkII Tyr221 (p=0.039). Moreover, CD49d pos CLL cells were characterized by a high correlation between proteins involved in integrin-mediated signal trasduction, including the focal-adhesion kinase (FAK Tyr576-Ser577), the tyrosine kinase Src Tyr527 and the adaptor protein CrkL Tyr207. Since PAK and LIMK represent key players in the modulation of the structure and the activity of actin cytoskeleton, we focused on these proteins for validation experiments. The constitutive pPAK and pLIMK overexpression in the CD49d pos CLL group was confirmed by western blot analysis comparing purified CLL cells from 3 CD49d pos versus 3 CD49d neg cases (mean fold increase >100 for both proteins). The results obtained suggest that integrin signalling is constitutively active in CD49d-expressing circulating CLL cells, pointing to a constitutive receptor engagement by CD49d ligands allegedly present in plasma. To test whether plasma constituents could modulate integrin-signaling proteins, CLL cells from 5 cases, expressing or not CD49d, were challenged with autologous plasma (1:3 dilution) for 30 seconds, 1 and 3 minutes. The presence of plasma induced a strong increase of PAK and LIMK phosphorylation intensities in CD49d pos CLL cells, starting at 30 seconds upon stimulation (mean fold increase >10 as compared to control for both proteins), and increasing after 1 and 3 minutes (mean fold increase >40 and >50 for both the proteins, respectively). Conversely, plasma stimulation did not induce pPAK and pLIMK expression modulation in CD49d neg CLL cells. Of note, pretreatment of CD49d-expressing CLL cells with the anti-CD49d HP1/2 blocking antibody, resulted in lower up-regulation of pPAK and pLIMK with an overall 60% inhibition for both the proteins (p=0.01), confirming the involvement of CD49d triggering in the observed activated signaling. Given the above results, we investigated plasma from 24 CLL patients, expressing CD49d at different levels, for the presence of the CD49d ligand fibronectin using an ELISA detection kit. All samples tested showed more than 1 mg/ml fibronectin concentration, without differences between CD49d-expressing and CD49d neg CLL cases. Altogether these results sustain the hypothesis of an active role of plasma/plasma components in the activation of CD49d-mediated integrin pathway, thus favoring the delivery of chemoresistance/pro-survival signals even in the context of circulating CLL cells. Our results may be of interest in the perspective of novel therapies (e.g. Bruton tyrosine kinase inhibitors) known to provoke a massive egress of neoplastic cells from tissue sites into the blood stream. Disclosures:No relevant conflicts of interest to declare.

SHP-1 protein tyrosine phosphatase associates with the adaptor protein CrkL

SHP-1, encoded by the PTPN6 gene, is a protein tyrosine phosphatase with two src-homology-2 (SH2) domains that is implicated as providing suppression of hematopoietic malignancies. A number of reports have shown protein-protein interactions between SHP-1 SH2 domains and tyrosine-phosphorylated proteins. However, despite its having three proline-rich, potential SH3-binding motifs, no reports of protein-protein interactions through src-homology-3 (SH3)-binding domains with SHP-1 have been described. Herein we show that the SH3 domain-containing CT10 regulator of kinase-like (CrkL) adaptor protein associates with SHP-1. We also provide results that suggest this association is due to CrkL binding to PxxP domains located at amino acid residues 158-161 within the SHP-1 C-terminal SH2 domain, and amino acid residues 363-366 within its phosphatase domain. This study is the first to identify and define an interaction between SHP-1 and an SH3 domain-containing protein. Our findings provide an alternative way that SHP-1 can be linked to potential substrates.

Variations in Platelet Proteins Associated With ST-Elevation Myocardial Infarction

Our aim in this study was to provide novel information on the molecular mechanisms playing a major role in the unwanted platelet activation associated with ST-elevation myocardial infarction (STEMI). We compared the platelet proteome of 11 STEMI patients to a matched control group of 15 stable chronic ischemic cardiopathy patients. In addition, we did a prospective study to follow the STEMI patients over time. Proteins were separated by high-resolution 2D gel electrophoresis, identified by mass spectrometry, and validated by Western blotting. Platelets from STEMI patients on admission displayed 56 protein spot differences (corresponding to 42 unique genes) compared with the control group. The number of differences decreased with time during the patients' follow-up. Interestingly, the adapter protein CrkL and the active form of Src (phosphorylated in Tyr418) were found to be upregulated in platelets from STEMI patients. Major signaling pathways related to the proteins identified include integrin, integrin-linked kinase, and glycoprotein VI (GPVI) signaling. Interestingly, a study on an independent cohort of patients showed a higher degree of activation of GPVI signaling in STEMI patients. CrkL, the active form of Src, and GPVI signaling are upregulated in platelets from STEMI patients.

Syk Regulates Multiple Signaling Pathways Leading to CX3CL1 Chemotaxis in Macrophages

Several studies have clearly established the importance of the interaction between macrophages and CX3CL1 in the progression of disease. A previous study demonstrated that Syk was required for CX3CL1-mediated actin polymerization and chemotaxis. Here, we delineated the signaling cascade of Syk-mediated cell migration in response to CX3CL1. Inhibition of Syk in bone marrow-derived macrophages or reduction of Syk expression using siRNA in RAW/LR5 cells indicated that Syk was required for the activation of PI3K, Cdc42, and Rac1. Also, reduction in WASP or WAVE2 levels, common downstream effectors of Cdc42 or Rac1, resulted in impaired cell migration to CX3CL1. Syk indirectly regulated WASP tyrosine phosphorylation through Cdc42 activation. Altogether, our data identify that Syk mediated chemotaxis toward CX3CL1 by regulating both Rac1/WAVE2 and Cdc42/WASP pathways, whereas Src family kinases were required for proper WASP tyrosine phosphorylation.

Potentiation of the Antileukemic Effects of Imatinib through the Modulation of BCRP/ABCG2 Activity.

AbstractAbstract 3400Resistance to tyrosine kinase inhibitors (TKI) constitutes a growing problem in chronic myeloid leukemia (CML) therapy. Variations of intracellular concentration of TKIs, dependable on the activity of drug transporters, are among major mechanisms responsible for the development of resistance. Intracellular imatinib concentration depends on the balance of active transport of the drug into the cell, mediated mainly by OCT-1 transporter, and drug efflux out of the cell, mediated by transmembrane pumps belonging to ABC transporters family. While there are reports that imatinib is a substrate for ABCG2 efflux pump, other studies show that it rather acts as an inhibitor of this drug transporter. According to the latest reports both views may be true – in low concentration imatinib seems to be a substrate of ABCG2 pump while in high concentrations it can inhibit activity of the pump. Drugs able to modulate membrane-bound proteins conformation, including drug transporters, may influence intracellular concentration of imatinib. Statins, inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase, widely used in the treatment of hypercholesterolemia, can modulate activity of ABC transporters. Therefore, we evaluated the potential influence of statins on the antileukemic activity of imatinib in cell lines transformed with BCR/ABL oncoprotein and primary human CML CD34+ cells. Lovastatin, a member of statin family, not only exerted antileukemic activity alone, but also significantly enhanced cytotoxicity of imatinib in BCR/ABL-positive murine 32Dcl3 cells and in human K562 cells as compared to 32Dcl3 wild-type counterparts and HL-60 cells respectively, which was measured in cytotoxic assays (i.e. trypan blue, XTT) and clonogenic tests. Similar effects were observed for other statins (incl. atorvastatin, simvastatin and mevastatin). Potentiation of antileukemic activity of imatinib by lovastatin was also observed for primary CML CD34+ cells from patients in different stages of the disease. In contrast, this antileukemic effect was not observed in peripheral blood leukocytes from healthy blood donors. Moreover, in comparison to monotherapy the co-treatment induced cell cycle arrest and increased percentage of apoptotic leukemic cells measured using flow cytometric analysis after propidium iodide staining. Lovastatin also increased imatinib-induced reduction of the phosphorylated form of the adaptor protein CrkL, which serves as a marker for efficient BCR/ABL kinase inhibition in both stable cell lines and primary CML CD34+ cells. Additionally, co-treatment with statin and imatinib has modified cell cycle/apoptosis controlling proteins profile. Cytometric analysis of intracellular concentration of ABCG2 substrate (fluorescent dye BODIPY-prazosin), revealed that lovastatin significantly decreased ABCG2 mediated efflux capacity in BCR/ABL-positive cells. The effect was completely reversed by the addition of cholesterol to the sample previously incubated with lovastatin. These results suggest that statin-mediated depletion of cholesterol may influence conformational status of the efflux pumps and hence can modulate their activity. Such phenomenon was observed both for stable cell lines and primary CD34+ CML cells including cells from patients clinically resistant to imatinib with no detectable ABL kinase domain mutations. To confirm the results of indirect experiments with BODIPY-prazosin we examined whether statins modulate intracellular level of imatinib itself, employing radiolabeled 14C-imatinib. Pretreatment of K562 cells with lovastatin resulted in significant increase in intracellular labeled drug concentration (at least 2-fold). This effect was due to diminished efflux of the drug, since we did not observe changes in initial influx of imatinib between statin-treated and control cells. This suggests that statins may exert significant effect on activity of ABCG2, but not Oct1 transporter.Our results indicate that statins can positively influence the therapeutic potency of imatinib and modification of drug efflux may be a major mechanism responsible for such effect. We conclude that addition of statins to the treatment regimen may become an effective treatment modality capable of potentiating the antileukemic effectiveness of tyrosine kinase inhibitors. Disclosures:No relevant conflicts of interest to declare.

CrkL is a novel target of Sprouty2 in fibroblast growth factor signaling

Sprouty, an inhibitor of receptor tyrosine kinase signaling, plays an important role in the regulation of a wide variety of biological processes. Although it is established that the Sprouty inhibitory activity is induced by tyrosine phosphorylation in response to stimuli, its action mechanisms have not been fully elucidated. Here, we report identification of a novel target of Sprouty. We find that Sprouty1 and Sprouty2 bind to the adaptor protein CrkL in a stimulus-dependent manner. Biochemical analyses show that the binding requires tyrosine phosphorylation of Sprouty and that both the SH2 domain and the N-terminal SH3 domain of CrkL are necessary for the binding. In fibroblast growth factor-stimulated NIH3T3 cells, CrkL binding to Sprouty2 occurs concomitantly with tyrosine phosphorylation of Sprouty2, which occurs slowly but is sustained. Importantly, our results show that tyrosine-phosphorylated Sprouty2 suppresses Rap1 activation. These results taken together indicate that Sprouty2 acts as an inhibitor of CrkL-Rap1 signaling.