Tyrosine Kinase Binding Domain Research Articles

B-363 Solid Lipid Nanoparticles Mediated CBLC siRNA Delivery as a Platform to Monitor and Probe Biological Functions

Abstract Background Nanoparticles have been used as a tool for delivering therapeutics since few decades. Researchers has been focusing on advances and modifications to improve the properties of these nanoparticles such as sustained drug delivery and bioavailability. This study focuses on modifying the conventional solid lipid nanoparticles (SLN) with Arginine-Glycine-Aspartic acid tripeptide (RGD). The peptidyl link in these SLN has an ability to bind to the integrin of the target cells, allowing solid adhesion to the cells for sustained drug delivery. The use of small interfering RNA (siRNA) allow better invasion through the cell membranes, silencing the intracellular targets. This study uses Casistas B-lymphoma lineage (CBLC) siRNA as the therapeutics for the treatment of non-small cell lung cancer (NSCLC). CBL family consisting of different proteins such as CBL, CBLB, and CBLC origins from a RING finger class. They are E3 ubiquitin ligases responsible for transferring ubiquitin from E2 to E3 substrate. They function as the negative regulator of Tyrosine Kinase activity in cell cycle signaling. CBLC is abundantly present in epithelial cells and so its expression is noteworthy in lung adenocarcinoma. CBL family also possess Tyrosine Kinase Binding domain (TKB) responsible to decrease the activated Epidermal Growth Factor Receptor (aEGFR). Although, CBLC was initially known to perform the same function as other CBL family members, reports suggest that it is a demethylated target. The expression of CBLC upregulates with the treatment of NSCLC by inhibiting agents for DNA methylation. This process increases the demethylation of the CBLC promoter upregulating CBLC. This causes CBLC to compete with CBL for binding to the aEGFR leading to polyubiquitination of aEGFR. Trafficking of aEGFR in the nucleus makes it more stable progressing lung cancer. CBLC silencing allows tumor cells to be sensitized to the therapy with tyrosine kinase inhibiting agents. Methods SLN were modified with RGDP followed by the loading of CBLC siRNA. To characterize the SLN within the diameter range of 20–100 nm, Scanning Electron Microscopy (SEM) was performed. The Energy-dispersive X-ray Spectroscopy was performed to confirm the presence of Phosphorous and Nitrogen in SLN coming from lipids and RNA. Dynamic Light Scattering (DLS) was done to verify the SEM results. To measure the efficacy of the SLN, the cells were treated with control SLN vs siRNA loaded SLN. The Sybr gold assay and invitro release study was done to confirm the release of siRNA from SLN. The human lung normal cell line NL-20 as control and lung cancer cell line A549 were cultured and treated with synthesized nanoparticles for knocking down CBLC gene. The assays such as MTT, western blot, flow cytometry and mass spectrometry-based proteomics were performed to confirm the knockdown of the CBLC gene. Results The SLN characterization confirmed the diameter in the expected range. The efficacy assays confirmed the encapsulation and release of CBLC siRNA. The results from cell studies showed reduction in tumor growth and viability confirming the knockdown of CBLC gene. Conclusion The data suggests significant effects for the use of synthesized nanoparticles as a therapeutic for the treatment of NSCLC.

Negative regulation of receptor tyrosine kinases by ubiquitination: Key roles of the Cbl family of E3 ubiquitin ligases.

Receptor tyrosine kinases (RTKs) serve as transmembrane receptors that participate in a broad spectrum of cellular processes including cellular growth, motility, differentiation, proliferation, and metabolism. Hence, elucidating the regulatory mechanisms of RTKs involved in an assortment of diseases such as cancers attracts increasing interest from researchers. Members of the Cbl family ubiquitin ligases (c-Cbl, Cbl-b and Cbl-c in mammals) have emerged as negative regulators of activated RTKs. Upon activation of RTKs by growth factors, Cbl binds to RTKs via its tyrosine kinase binding (TKB) domain and targets them for ubiquitination, thus facilitating their degradation and negative regulation of RTK signaling. RTKs such as epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGF), fibroblast growth factor receptor (FGFR) and hepatocyte growth factor receptor (HGFR) undergo ubiquitination upon interaction with Cbl family members. In this review, we summarize the current knowledge related to the negative regulation of RTKs by Cbl family proteins.

Integrated Phosphoproteomic and N-Glycoproteomic Landscape of Cutaneous T-Cell Lymphoma Reveals Distinctive Pathogenetic Insights and Therapeutic Vulnerabilities

Background: A critical goal of precision oncology is the identification of deregulated signaling pathways that serve as disease-specific biomarkers and vulnerabilities that may be exploited as therapeutic targets. Cutaneous T cell lymphomas (CTCLs) are heterogeneous neoplasms with variable clinical outcomes. Whereas the genomic landscapes of CTCLs are being characterized, a large scale analysis of their proteomic signatures and post-translational modifications (PTM) has not been performed. In this study, we sought to gain insights into the pathogenesis of CTCLs using high mass accuracy tandem mass spectrometry (HMA-MS/MS)-based phosphoproteomics and glycoproteomics.Methods: CTCL-derived cell lines representative of Sézary syndrome (SS, HUT78), mycosis fungoides (MF, HH), and ALK-negative anaplastic large cell lymphoma (ALCL) with PCM1-JAK2 or NPM1-TYK2 translocations (Mac1, Mac2a, and Myla) were analyzed in technical and biologic triplicates to perform unbiased proteome characterization. Phosphoproteins were enriched using metal oxide affinity chromatography and immunoprecipitation using 3 anti-phosphotyrosine antibodies and N-glycoproteins were enriched using solid phase extraction and hydrazide resin and an Orbitrap Fusion or LTQ OrbitrapXL in-line with a Paradigm MS2 HPLC. Precise site-mapping of the PTMs was assigned and quantitated using Maxquant. Hierarchical clustering was performed using Perseus. Whole exome sequencing (WES) was performed using an Illumina HiSeq 2500. Functional studies using tyrosine kinase inhibitors were performed to assess selective vulnerabilities.Results: Phosphoproteomic analysis identified 1899 tyrosine (Y) phosphoproteins and 6029 serine/threonine (S/T) phosphoproteins. Unsupervised hierarchical clustering of the Y-phosphoproteome data accurately grouped the CTCLs to those with and without underlying tyrosine kinase translocations. Unique driver kinase phosphosignaling signatures were identified in MYLA (NPM1-TYK2) and MAC1/2 (PCM1-JAK2) representing the ALCL subgroup, as well as close similarity between the signatures of MF and SS-derived cells (HH and HUT78). Notably, phosphoproteomic analysis of HUT78 (SS) yielded high spectral counts of Y-phosphorylated residues at IL2RG p.Y325, JAK3 p.Y980/Y981, and STAT5 p.Y699 indicative of constitutive activation of the JAK/STAT5 pathway corroborated by WES showing activating mutations in JAK3 p.A573V and JAK1 p.Y654F.Beyond driver kinases, oncogenic kinase-specific substrates with distinct site-specific phosphorylated residues were identified for each disease class. Phosphoproteins selectively expressed in MF/SS, (46 p-S/T and 25 p-Y proteins) included members of the killer-cell immunoglobulin-like receptor (KIR) family implicated in immune modulation through immune tyrosine-based inhibitor or TYRO protein tyrosine kinase binding domains for signal transduction. Additionally, proteins not previously associated with MF/SS including NKG2-D type integral membrane protein family members were discriminative of this subgroup. Phosphoprotein members of lysine demethylases (KDM family) and Aryl Hydrocarbon Receptor families were distinctively identified in MF/SS cells, while components of the T-cell receptor signaling pathways were common in all groups of CTCLs. By comparison, N-glycoproteomic analysis identified 811 unique N-glycoproteins. A total of 51 N-glycoproteins were found solely in HUT78 of which 46 were novel in their specificity and included 13 with a role in cell-adhesion while N-glycoproteins unique to HH revealed 12 novel candidate biomarkers (e.g. MERTK, CD97 and SLC44A2). Integrative phosphoproteomic and N-glycoproteomic analysis strikingly revealed convergent pathways implicating a prominent role for the KIR family proteins in SS. Orthogonal immunophenotyping by flow cytometry has shown high sensitivity (96%) detection of KIR3DL2 in primary human SS cases. Significantly, anti-KIR3DL2 inhibition using a humanized antibody revealed activity against SS cells. By comparison, TYK2-driven CTCL (MYLA) was sensitive to inhibitors of TYK2 and JAK.Conclusions: Integrated phosphoproteomic and N-glycoproteomic analysis identifies known and novel candidate biomarkers, unique PTM signatures, and therapeutic vulnerabilities implicating disease-specific pathogenetic mechanisms in CTCL. DisclosuresNo relevant conflicts of interest to declare.

SLAP2 Adaptor Binding Disrupts c-CBL Autoinhibition to Activate Ubiquitin Ligase Function

CBL is a RING type E3 ubiquitin ligase that functions as a negative regulator of tyrosine kinase signaling and loss of CBL E3 function is implicated in several forms of leukemia. The Src-like adaptor proteins (SLAP/SLAP2) bind to CBL and are required for CBL-dependent downregulation of antigen receptor, cytokine receptor, and receptor tyrosine kinase signaling. Despite the established role of SLAP/SLAP2 in regulating CBL activity, the nature of the interaction and the mechanisms involved are not known. To understand the molecular basis of the interaction between SLAP/SLAP2 and CBL, we solved the crystal structure of CBL tyrosine kinase binding domain (TKBD) in complex with SLAP2. The carboxy-terminal region of SLAP2 adopts an α-helical structure which binds in a cleft between the 4H, EF-hand, and SH2 domains of the TKBD. This SLAP2 binding site is remote from the canonical TKBD phospho-tyrosine peptide binding site but overlaps with a region important for stabilizing CBL in its autoinhibited conformation. In addition, binding of SLAP2 to CBL in vitro activates the ubiquitin ligase function of autoinhibited CBL. Disruption of the CBL/SLAP2 interface through mutagenesis demonstrated a role for this protein-protein interaction in regulation of CBL E3 ligase activity in cells. Our results reveal that SLAP2 binding to a regulatory cleft of the TKBD provides an alternative mechanism for activation of CBL ubiquitin ligase function.

Sleuthing biochemical evidence to elucidate unassigned electron density in a CBL-SLAP2 crystal complex.

The Src-like adaptor proteins (SLAP/SLAP2) bind to CBL E3 ubiquitin ligase to downregulate antigen, cytokine and tyrosine kinase receptor signalling. In contrast to the phosphotyrosine-dependent binding of CBL substrates through its tyrosine kinase-binding domain (TKBD), CBL TKBD associates with the C-terminal tail of SLAP2 in a phospho-independent manner. To understand the distinct nature of this interaction, a purification protocol for SLAP2 in complex with CBL TKBD was established and the complex was crystallized. However, determination of the complex crystal structure was hindered by the apparent degradation of SLAP2 during the crystallization process, such that only the CBL TKBD residues could initially be modelled. Close examination of the CBL TKBD structure revealed a unique dimer interface that included two short segments of electron density of unknown origin. To elucidate which residues of SLAP2 to model into this unassigned density, a co-expression system was generated to test SLAP2 deletion mutants and define the minimal SLAP2 binding region. SLAP2 degradation products were also analysed by mass spectrometry. The model-building and map-generation features of the Phenix software package were employed, leading to successful modelling of the C-terminal tail of SLAP2 into the unassigned electron-density segments.

CBL mutations drive PI3K/AKT signaling via increased interaction with LYN and PIK3R1

AbstractCasitas B-lineage lymphoma (CBL) encodes an E3 ubiquitin ligase and signaling adaptor that regulates receptor and nonreceptor tyrosine kinases. Recurrent CBL mutations occur in myeloid neoplasms, including 10% to 20% of chronic myelomonocytic leukemia (CMML) cases, and selectively disrupt the protein's E3 ubiquitin ligase activity. CBL mutations have been associated with poor prognosis, but the oncogenic mechanisms and therapeutic implications of CBL mutations remain incompletely understood. We combined functional assays and global mass spectrometry to define the phosphoproteome, CBL interactome, and mechanism of signaling activation in a panel of cell lines expressing an allelic series of CBL mutations. Our analyses revealed that increased LYN activation and interaction with mutant CBL are key drivers of enhanced CBL phosphorylation, phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) recruitment, and downstream phosphatidylinositol 3-kinase (PI3K)/AKT signaling in CBL-mutant cells. Signaling adaptor domains of CBL, including the tyrosine kinase–binding domain, proline-rich region, and C-terminal phosphotyrosine sites, were all required for the oncogenic function of CBL mutants. Genetic ablation or dasatinib-mediated inhibition of LYN reduced CBL phosphorylation, CBL-PIK3R1 interaction, and PI3K/AKT signaling. Furthermore, we demonstrated in vitro and in vivo antiproliferative efficacy of dasatinib in CBL-mutant cell lines and primary CMML. Overall, these mechanistic insights into the molecular function of CBL mutations provide rationale to explore the therapeutic potential of LYN inhibition in CBL-mutant myeloid malignancies.

Inhibition of Ehrlich ascites cancer, hypoxia-inducible factor-1 alpha, and the kinase insert domain-containing receptor/fms-like tyrosine kinase-binding domains of vascular endothelial growth factor by Thiazole Acetamide Derivatives.

Tumor cells that have the ability to express vascular endothelial growth factor (VEGF) are more competent to growth and metastasize by the adequate amount of blood and oxygen supply by the blood vessels to the growing mass of cells. Hypoxic tumors are known for its aggressiveness and resistance to the treatment. Targeting VEGF and hypoxia-inducible factor-1 alpha (HIF-1α) is an attractive strategy to interrupt the multiple pathways crucial for tumor growth. In the present study, two thiazole acetamide derivative's anticancer property, anti VEGF and HIF-1α inhibitory property were investigated. Two thiazole acetamide compounds were synthesized, TA1 and TA2 and its anticancer property was studied in Erlich's ascites cancer cells. To evaluate the anticancer property the assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, DNA diffusion assay for apoptosis, and lactate dehydrogenase leakage assay were carried out. The cell culture media was used to assess the secreted VEGF level. Molecular docking studies were performed to analyze the binding efficiency of the study compounds to the kinase insert domain-containing receptor (KDR) and fms-like tyrosine kinase (FLT)-binding domains of VEGF protein. HIF-1α inhibitory study was performed by flow cytometry analysis using HUVEC cell line. The study compounds inhibited HIF-1α and VEGF secretion, these data shown positive prop up for the anticancer property of the derivatives. The docking studies showed moderate binding of study compounds to KDR and FLT-binding domains of VEGF protein. These results conclude the anticancer and anti-angiogenic property of the synthesized thiazole-acetamide derivatives.

Oncogenic Mechanisms of CBL E3 Ubiquitin Ligase Mutations in Myeloid Malignancies

CBL encodes an E3 ubiquitin ligase and signaling adaptor that acts downstream of cytokine receptors. Recurrent CBL mutations are found in a variety of myeloid disorders, including 10-15% of chronic myelomonocytic leukemia (CMML) cases, and specifically disrupt the protein's RING domain, which is responsible for E3 ligase activity; adaptor domains of CBL, including the tyrosine kinase-binding domain (TKB), proline-rich region (PRR) and C-terminal phosphotyrosine (pY) residues, remain intact in the context of RING mutations. In prior studies, CBL RING mutations were associated with hyperactivation of signaling pathways that drive cell proliferation. However, the precise mechanism by which CBL mutants act remains incompletely understood. Here we combined functional assays and mass spectrometry (MS) to comprehensively define the phosphoproteome, CBL interactome and molecular mechanism of signaling hyperactivation in a panel of cell lines expressing an allelic series of CBL RING mutants. We identified the SRC family kinase LYN as a key driver of signaling by CBL RING mutants; furthermore, we demonstrated in vitro and in vivo efficacy of LYN inhibition by dasatinib in CBL-mutant cell lines and primary CMML patient samples. We generated cell lines expressing wild-type (WT) or RING-mutant CBL using IL3-dependent mouse 32D cells and GM-CSF-dependent human TF1 cells. Cells expressing CBL RING mutants Y371H, C384Y or R420Q had a proliferative advantage over CBL WT or CBL knockout cells. To determine the role of CBL's adaptor domains in the proliferative advantage conferred by CBL RING mutants, we generated double mutants comprising the C384Y RING mutation in cis with mutations in the TKB domain (G306E), PRR (Δ477-688) or pY residues (Y700/731/774F). The proliferative advantage of cells expressing CBL C384Y was significantly reduced with mutation of the TKB domain, PRR or pY residues, indicating that CBL's adaptor domains are critical for the proliferative advantage of cells expressing RING-mutant CBL. To assess the effects of CBL RING mutation on signaling, we used MS to measure global protein phosphorylation in 32D cells expressing CBL WT or CBL C384Y. Activation of LYN and the PI3 kinase (PI3K) pathway were most significantly increased in cells expressing CBL C384Y compared to CBL WT; western blot confirmed increased phosphorylation of LYN, the PI3K p85 subunit and AKT in cells expressing CBL Y371H, C384Y or R420Q. We next employed immunoprecipitation (IP) followed by MS to characterize the global CBL interactome in 32D cells expressing CBL WT or RING mutants Y371H, C384Y or R420Q. In line with the phosphoproteomic analysis, LYN showed significantly increased binding to CBL RING mutants; the PI3K p85 subunit also showed increased binding to CBL RING mutants. Thus, global proteomic analyses revealed that increased binding of LYN and p85 to CBL RING mutants was directly associated with hyperactivation of LYN and PI3K-AKT signaling pathways. Deletion of CBL's PRR reduced interactions with both LYN and p85, and the CBL-p85 interaction required CBL Y731. Genetic ablation or inhibition of LYN by dasatinib decreased binding of p85 to CBL, suggesting that increased CBL Y731 phosphorylation by LYN enabled the CBL-p85 interaction. Indeed, CBL Y731 phosphorylation and AKT activation were diminished by deletion of CBL's PRR, LYN knockout or LYN inhibition by dasatinib. Altogether, these data demonstrated that enhanced LYN activation in cells expressing RING-mutant CBL drives increased CBL phosphorylation, p85 recruitment and downstream AKT signaling. Given the central role of LYN in signaling by CBL RING mutants, we hypothesized that LYN inhibition by dasatinib would abrogate the hyperproliferation of cells expressing CBL RING mutants. Dasatinib blocked the proliferative advantage of 32D and TF1 cells expressing CBL RING mutants. In addition, dasatinib significantly reduced the number of methylcellulose colonies formed by bone marrow mononuclear cells from 2 patients with CBL-mutated CMML; dasatinib treatment of mice xenografted with the same CMML cells resulted in a substantial decrease in leukemia burden compared to vehicle-treated mice. In summary, we have defined a mechanism by which LYN promotes PI3K-AKT signaling through CBL RING mutants. Our data provide rationale for exploring the therapeutic potential of LYN and/or PI3K-AKT inhibition in patients with CBL-mutated myeloid malignancies. Disclosures Ebert: Celgene: Research Funding; Deerfield: Research Funding.

Molecular designing, virtual screening and docking study of novel curcumin analogue as mutation (S769L and K846R) selective inhibitor for EGFR.

The somatic mutations in ATP binding cleft of the tyrosine kinase binding domain of EGFR are known to occur in 15–40% of non-small cell lung cancer (NSCLC) patients. Although first and second generation anti-EGFR inhibitors are widely used to treat these patients, their therapeutic efficacy is modest and often results in adverse effects or drug resistance. Therefore, there is a need to develop novel as well as safe anti-EGFR drugs. The rapid emergence of computational drug designing provided a great opportunity to both discover and predict the efficacy of novel EGFR inhibitors from plant sources. In the present study, we designed several chemical analogues of edible curcumin (CUCM) compound and assessed their drug likeliness, ADME and toxicity properties using a diverse range of advanced computational methods. We also have examined the structural plasticity and binding characteristics of EGFR wild-type and mutant forms (S769L and K846R) against ligand molecules like Gefitinib, native CUCM, and different CUCM analogues. Through multidimensional experimental approaches, we conclude that CUCM-36 ((1E,4Z,6E)-1-(3,4-Diphenoxyphenyl)-5-hydroxy-7-(4-hydroxy-3-phenoxyphenyl)-1,4,6-heptatrien-3-one) is the best anti-EGFR compound with high drug-likeness, ADME properties, and low toxicity properties. CUCM-36 compound has demonstrated better affinity towards both wild-type (ΔG is −8.5 kcal/Mol) and mutant forms (V769L & K846R; ΔG for both is >−9.20 kcal/Mol) compared to natural CUCM and Gefitinib inhibitor. This study advises the future laboratory assays to develop CUCM-36 as a novel drug compound for treating EGFR positive non-small cell lung cancer patients.

Structural Determinants of the Gain-of-Function Phenotype of Human Leukemia-associated Mutant CBL Oncogene

Mutations of the tyrosine kinase-directed ubiquitin ligase CBL cause myeloid leukemias, but the molecular determinants of the dominant leukemogenic activity of mutant CBL oncogenes are unclear. Here, we first define a gain-of-function attribute of the most common leukemia-associated CBL mutant, Y371H, by demonstrating its ability to increase proliferation of hematopoietic stem/progenitor cells (HSPCs) derived from CBL-null and CBL/CBL-B-null mice. Next, we express second-site point/deletion mutants of CBL-Y371H in CBL/CBL-B-null HSPCs or the cytokine-dependent human leukemic cell line TF-1 to show that individual or combined Tyr → Phe mutations of established phosphotyrosine residues (Tyr-700, Tyr-731, and Tyr-774) had little impact on the activity of the CBL-Y371H mutant in HSPCs, and the triple Tyr → Phe mutant was only modestly impaired in TF-1 cells. In contrast, intact tyrosine kinase-binding (TKB) domain and proline-rich region (PRR) were critical in both cell models. PRR deletion reduced the stem cell factor (SCF)-induced hyper-phosphorylation of the CBL-Y371H mutant and the c-KIT receptor and eliminated the sustained p-ERK1/2 and p-AKT induction by SCF. GST fusion protein pulldowns followed by phospho-specific antibody array analysis identified distinct CBL TKB domains or PRR-binding proteins that are phosphorylated in CBL-Y371H-expressing TF-1 cells. Our results support a model of mutant CBL gain-of-function in which mutant CBL proteins effectively compete with the remaining wild type CBL-B and juxtapose TKB domain-associated PTKs with PRR-associated signaling proteins to hyper-activate signaling downstream of hematopoietic growth factor receptors. Elucidation of mutant CBL domains required for leukemogenesis should facilitate targeted therapy approaches for patients with mutant CBL-driven leukemias.

CblY371H Transgene Combined with Hematopoietic Deletion of the Endogenous c-Cbl Gene Results in GM-CSF Hypersensitivity and Leukocytosis

Juvenile Myelomonocytic Leukemia (JMML) is a mixed myeloproliferative/myelodysplastic disease that is rapidly fatal with infiltration of myeloid cells into multiple organs. About 15% of JMML patient samples contain a mutation in c-Cbl, and germline mutation results in the predisposition for developing JMML. The c-Cbl gene encodes a multifunctional adaptor protein that contains an N-terminal tyrosine-kinase binding (TKB) domain, a RING finger motif that contains E3 ligase activity, and a C-terminal ubiquitin-associated domain. The TKB domain is involved in adaptor functions of the protein, whereas the ubiquitin ligase domain results in mono-ubiquitination of receptors which promotes lysosomal mediated degradation of activated receptors. Interestingly, a hotspot for mutations at residue 371 exists in JMML patients, where 1/3 of the detected mutations are a tyrosine to histidine substitution, Y371H. This residue belongs in the linker region of the CBL protein, and it was previously observed that Tyr-371 plays key roles in activating the ubiquitin ligase activity of the protein. In vitro, CblY371H mutation does indeed destroy its ligase function, resulting in prolonged signaling through the Ras pathway only when the endogenous c-Cbl gene is silenced. How mutant Cbl gives rise to JMML, however, and how it acts in concert with other genes in the pathogenesis of JMML requires further study.To address these questions, we overexpressed the oncogenic CblY371H mutation using transgenic mice. As expected, overexpression of CblY371H by itself in wildtype mice had no apparent phenotype. Therefore, Cbl transgenic mice were bred to Cbl heterozygous knockout mice (Cbl+/-) followed by further breeding in an attempt to generate Cbl transgenic mice with the endogenous Cbl gene inactivated (CblY371H; Cbl-/-). Surprisingly, unlike Cbl null mice, which are viable, overexpression of mutant Cbl allele in Cbl null mice caused embryonic lethality between 11.5 dpc and 12.5 dpc. In order to circumvent the developmental effects of expressing the mutant Cbl protein, we used a conditional Cbl knockout mouse to tissue specifically delete the endogenous Cbl gene. We chose the MMTV-Cre strain, which expresses Cre recombinase in only 10% of hematopoietic stem cells (CD34-; Lin-; Sca-1+; c-Kit+). With subsequent breeding with the CblY371H transgenic mice, we were able to bypass the embryonic lethality and produce mice with the correct genotype (MMTV-Cre;CblY371H;Cblfl/fl). These mice look normal but develop significant leukocytosis and show GM-CSF hypersensitivity even though only 10% of hematopoietic stem cells are affected. These mice, however, appear unaffected by the leukocytosis, and show no obvious difference with its littermates up to one year of age. We conclude that mutant CblY371H by itself is not sufficient for the development of JMML in this model and requires additional cooperating events. Whether further aging of these mice will result in JMML remains to be seen.In conclusion, we have developed a mouse model overexpressing the CblY371H protein ubiquitously, which causes deleterious development when it is the only c-Cbl protein available. This confirms the important role of c-Cbl activity during development. In hematopoietic cells, the overexpression of CblY371H results in leukocytosis and GM-CSF hypersensitivity when the endogenous gene is inactivated. We are currently investigating the cooperating events that are required for the development of JMML in this mouse model. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 4441: Ube2e inhibits the ubiquitination and degradation of EGFR mediated by Cbl and Ube2d

Abstract Receptor tyrosine kinases (RTKs) have a demonstrated role as drivers of malignant transformation. Many RTKs (e.g., EGFR, MET, FLT3) are negatively regulated by ubiquitination and degradation mediated by Cbl proteins a family of RING finger (RF) ubiquitin ligases (E3s). There are three mammalian family members Cbl, Cbl-b and Cbl-c. All Cbl proteins have a highly conserved N-terminal tyrosine kinase binding domain and a C3HC4 (RF) domain. Loss of Cbl protein function has be associated with malignant transformation drive by increased RTK activity. E3s such as the Cbl proteins confer specificity to the ubiquitination process and direct the conjugation of ubiquitin to one or more lysines on the specific target proteins in collaboration with ubiquitin-conjugating enzymes (E2s). The E2s bind to the E3 and together they mediate ubiquitination of specific substrates. There are approximately 30 human E2s. All of the E2s have a highly conserved catalytic domain (UBC domain). In this study, we characterized the interaction of Cbl with E2s. Using an in vitro E3 assay, we found that only the Ube2d family (Ube2d1, Ube2d2, and Ube2d3) of E2s mediates autoubiquitination of the three Cbl proteins. Subsequently, using the yeast two-hybrid system, we found that Ube2e1, Ube2e2, Ube2e3, Ube2L3, Ube2u, Ube2w and Ube2z can interact with Cbl even though they do not support autoubiquitination of Cbl in vitro E3 assay. Among these E2s, three Ube2e family members and Ube2w bind to the RF domain of Cbl as demonstrated by the loss of interaction when the RF domain is disrupted. Further, we demonstrated that Ube2e family members are involved in regulation of EGFR activity mediated by Cbl in HeLa cells. Knockdown of Ube2e increases down-regulation and ubiquitination of EGFR in HeLa cells. We further showed that three Ube2e inhibit autoubiquitination of Cbl mediated by Ube2d2 in vitro. Together these data suggest that RTK degradation can be modulated by competition between E2s for the Cbl E3s. Citation Format: Ke Ma, Stanley Lipkowitz, Rachel Klevit, Mariya Sergeyevna Liyasova. Ube2e inhibits the ubiquitination and degradation of EGFR mediated by Cbl and Ube2d. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4441. doi:10.1158/1538-7445.AM2014-4441

Abstract 1287: Mutations in the CBL gene among breast cancer patients in an Asian clinic-based population

Abstract INTRODUCTION Although mutations in BRCA1 and BRCA2 are associated with familial hereditary breast and ovarian cancer, mutations are only found in 20 to 25% of these patients. This suggests that additional genes may be mutated. The Casitas B-lineage lymphoma (CBL) gene encodes a RING finger E3 ubiquitin ligase. Recent studies have shown that mutations in the CBL gene occurs in human acute myeloid leukemia and lung cancers. The relationship of the CBL gene with breast cancer, however, is not well characterized. The aim of this study was to identify mutations in the CBL gene in Asian patients with familial or early-onset breast cancer. METHODS All patients (n=75) were accrued at the risk assessment clinic at the National Cancer Centre Singapore, and presented with a family history of breast and/or ovarian cancer or early-onset breast cancer. Screening for mutations in the BRCA1 and BRCA2 genes was performed using Sanger sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA). Next generation sequencing was done using the Agilent SureSelect target-enrichment technology on the Illumina MiSeq platform, followed by validation using Sanger sequencing. RESULTS Two mis-sense variants in the CBL gene were identified. They are CBL c. 560C>T (p. A187V) and c. 1858C>T (p.L620F) occurring in 2/75 (3%) and 8/75 (11%) of the patients, respectively. Both mis-sense mutations were predicted to be damaging using Polyphen-2 and SIFT. The p.A187V alteration was predicted to interrupt the N-terminal tyrosine kinase binding domain (TKB) that is important for ligand-induced ubiquitination of receptor tyrosine kinases (RTKs). The CBL c. 1858C>T variant has previously been reported in the NCBI database (rs2227988). Three individuals, who carried this mutation, had early-onset breast cancer. In addition, the p.L620F alteration within the proline-rich region, may disrupt the interactions of binding partners to the c-CBL protein, which are required to recruit c-CBL to RTKs. All patients, except one, with germline CBL mutations were negative for BRCA1 and BRCA2 mutations. CONCLUSION This preliminary study has identified damaging CBL mutations in an Asian population with breast cancer and warrants further investigations in other breast cancer populations. Citation Format: Edward Wong, Yoon Sim Yap, Ying Ying Cheng, Delia Chua, Lewis Zuocheng Hong, William F. Burkholder, Gay Hui Ho, Min Han Tan, Peter Ang, Ann S.G. Lee. Mutations in the CBL gene among breast cancer patients in an Asian clinic-based population. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1287. doi:10.1158/1538-7445.AM2014-1287

Glucocorticoid regulates TrkB protein levels via c-Cbl dependent ubiquitination: a decrease in c-Cbl mRNA in the prefrontal cortex of suicide subjects.

Brain derived neurotrophic factor (BDNF) signaling through its receptor TrkB plays a crucial role in neurodevelopment and plasticity. Stress and glucocorticoids have been shown to alter TrkB signaling in neurons, and defects in TrkB expression have been reported in the prefrontal cortex of suicide subjects. Glucocorticoid treatment has been shown to induce deleterious effects on the neuronal maturation. However, the mechanisms involved in the regulation of TrkB by glucocorticoid during neurodevelopment are not clear. Here we show that acute corticosterone exposure induced posttranslational upregulation of TrkB in primary cortical neurons (days in vitro 4, DIV4), which was blocked by the proteasome inhibitors. Acute corticosterone-induced increase in TrkB protein levels was dependent on glucocorticoid receptor (GR). At the cellular level, ubiquitin E3 ligase c-Cbl mediates TrkB stabilization and corticosterone-induced TrkB levels. Moreover, the tyrosine kinase binding domain in c-Cbl plays a critical role in corticosterone-induced TrkB levels. Chronic treatment of neurons with corticosterone induced significant decreases in both TrkB and c-Cbl protein levels. Acute corticosterone treatment failed to induce any significant change in TrkB and c-Cbl protein levels in mature neurons (DIV 12), where as chronic corticosterone exposure reduced TrkB levels. Under an in vivo condition, chronic corticosterone exposure induced down-regulation of c-Cbl in mouse frontal cortex and hippocampus. Importantly, we demonstrate for the first time a significant decrease in c-Cbl mRNA levels in the prefrontal cortex of suicide subjects indicating the possible role of c-Cbl in the pathophysiology of suicidal behavior. Thus, ubiquitin-proteasome-mediated TrkB regulation may be an important mechanism for improving BDNF signaling and maintaining neuroplasticity in stress-related neuropsychiatric disorders.

Prognostic value of epidermal growth factor receptor mutations in resected lung adenocarcinomas

The purpose of this study was to evaluate the association between epidermal growth factor receptor (EGFR) mutations and prognosis in patients with completely resected lung adenocarcinoma. A total of 131 patients were included in this study. EGFR mutation status in exons 18-21 of the tyrosine kinase-binding domain was detected using nested PCR amplification of individual exon. The χ (2) test was used to analyze the associations between EGFR mutations and the different variables. The log-rank test and Cox regression model were used to evaluate the factors influencing disease-free survival (DFS) and overall survival (OS). EGFR mutations in 18-21 exons were detected in 58 of the 131 patients (44.3 %). Smoking status (P = 0.029), N stage (P = 0.021), and pathologic stage (P = 0.048) were significantly associated with EGFR mutations. The median DFS in mutant EGFR and wild-type EGFR groups was 36.6 and 25.7 months, respectively (P = 0.533). No significant correlation was observed between EGFR mutations and OS (P = 0.564). However, patients with exon 19 mutation tended to have longer DFS than those with exon 21 mutation (46.2 vs. 21.9 months, P = 0.056), and the 1-, 2-, and 3-year OS rates were significantly higher in patients with exon 19 mutation compared to patients with exon 21 mutation (100, 96.7, 93.3 vs. 91.3, 82.6, 60.9 %, respectively, P = 0.01). Our data demonstrated that EGFR mutations do not have significant prognostic value in primary resected lung adenocarcinomas, but patients with exon 19 mutation tended to have better prognostic value compared to patients with exon 21 mutation.

EphrinA2 Regulates Clathrin Mediated KSHV Endocytosis in Fibroblast Cells by Coordinating Integrin-Associated Signaling and c-Cbl Directed Polyubiquitination

Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with human dermal endothelial cell surface tyrosine kinase EphrinA2 (EphA2) and integrins (α3β1 and αVβ3) in the lipid raft (LR) region, and EphA2 regulates macropinocytic virus entry by coordinating integrin-c-Cbl associated signaling. In contrast, KSHV enters human foreskin fibroblast (HFF) cells by LR-independent clathrin mediated endocytosis. The present studies conducted to identify the key molecules regulating KSHV entry in HFF cells showed that KSHV induces association with integrins (αVβ5, αVβ3 and α3β1) and EphA2 in non-LR regions early during infection and activates EphA2, which in turn associates with phosphorylated c-Cbl, myosin IIA, FAK, Src, and PI3-K, as well as clathrin and its adaptor AP2 and effector Epsin-15 proteins. EphA2 knockdown significantly reduced these signal inductions, virus internalization and gene expression. c-Cbl knockdown ablated the c-Cbl mediated K63 type polyubiquitination of EphA2 and clathrin association with EphA2 and KSHV. Mutations in EphA2's tyrosine kinase domain (TKD) or sterile alpha motif (SAM) abolished its interaction with c-Cbl. Mutations in tyrosine kinase binding (TKB) or RING finger (RF) domains of c-Cbl resulted in very poor association of c-Cbl with EphA2 and decreased EphA2 polyubiquitination. These studies demonstrated the contributions of these domains in EphA2 and c-Cbl association, EphA2 polyubiquitination and virus-EphA2 internalization. Collectively, these results revealed for the first time that EphA2 influences the tyrosine phosphorylation of clathrin, the role of EphA2 in clathrin mediated endocytosis of a virus, and c-Cbl mediated EphA2 polyubiquitination directing KSHV entry in HFF cells via coordinated signal induction and progression of endocytic events, all of which suggest that targeting EphA2 and c-Cbl could block KSHV entry and infection.

Structural flexibility regulates phosphopeptide-binding activity of the tyrosine kinase binding domain of Cbl-c

Through their ubiquitin ligase activity, Cbl-family proteins suppress signalling mediated by protein-tyrosine kinases (PTKs), but can also function as adaptor proteins to positively regulate signalling. The tyrosine kinase binding (TKB) domain of this family is critical for binding with tyrosine-phosphorylated target proteins. Here, we analysed the crystal structure of the TKB domain of Cbl-c/Cbl-3 (Cbl-c TKB), which is a distinct member of the mammalian Cbl-family. In comparison with Cbl TKB, Cbl-c TKB showed restricted structural flexibility upon phosphopeptide binding. A mutation in Cbl-c TKB augmenting this flexibility enhanced its binding to target phosphoproteins. These results suggest that proteins, post-translational modifications or mutations that alter structural flexibility of the TKB domain of Cbl-family proteins could regulate their binding to target phosphoproteins and thereby, affect PTK-mediated signalling.

Peptide Truncation Leads to a Twist and an Unusual Increase in Affinity for Casitas B-Lineage Lymphoma Tyrosine Kinase Binding Domain

We describe truncation and SAR studies to identify a pentapeptide that binds Cbl tyrosine kinase binding domain with a higher affinity than the parental peptide. The pentapeptide has an alternative binding mode that allows occupancy of a previously uncharacterized groove. A peptide library was used to map the binding site and define the interface landscape. Our results suggest that the pentapeptide is an ideal starting point for the development of inhibitors against Cbl driven diseases.

Autoinhibition and phosphorylation-induced activation mechanisms of human cancer and autoimmune disease-related E3 protein Cbl-b

Cbl-b is a RING-type E3 ubiquitin ligase that functions as a negative regulator of T-cell activation and growth factor receptor and nonreceptor-type tyrosine kinase signaling. Cbl-b dysfunction is related to autoimmune diseases and cancers in humans. However, the molecular mechanism regulating its E3 activity is largely unknown. NMR and small-angle X-ray scattering analyses revealed that the unphosphorylated N-terminal region of Cbl-b forms a compact structure by an intramolecular interaction, which masks the interaction surface of the RING domain with an E2 ubiquitin-conjugating enzyme. Phosphorylation of Y363, located in the helix-linker region between the tyrosine kinase binding and the RING domains, disrupts the interdomain interaction to expose the E2 binding surface of the RING domain. Structural analysis revealed that the phosphorylated helix-RING region forms a compact structure in solution. Moreover, the phosphate group of pY363 is located in the vicinity of the interaction surface with UbcH5B to increase affinity by reducing their electrostatic repulsion. Thus, the phosphorylation of Y363 regulates the E3 activity of Cbl-b by two mechanisms: one is to remove the masking of the RING domain from the tyrosine kinase binding domain and the other is to form a surface to enhance binding affinity to E2.

An adjacent arginine, and the phosphorylated tyrosine in the c-Met receptor target sequence, dictates the orientation of c-Cbl binding

Previously, we have demonstrated that the tyrosine phosphorylated hepatocyte growth factor receptor (Met) binds to the c-Cbl phosphotyrosine-recognition, tyrosine kinase binding (TKB) domain in a reverse orientation compared to other c-Cbl binding partners. A Met peptide with the DpYR motif changed to RpYD (MetRD) retains a similar TKB binding affinity as the native Met peptide. However, the TKB: MetRD complex crystal structure reveals a complete reversal of the binding orientation. Collated data indicates that both binding and orientation is dictated by the phosphorylated tyrosine and an adjacent arginine forming intra-peptide hydrogen bonds and aligning unidirectionally with complementary charges in the phosphotyrosine binding pocket of c-Cbl. Structured summaryc-Cbl and MetRDbind: shown by x-ray crystallography (view interaction)c-Cbl and MetRDbind: shown by mass spectrometry studies of complexes (view interaction)c-Cblbind to Met: shown by surface plasmon resonance (view interactions 1,2)