Seth Ettenberg

Ubiquitin ligase activity and tyrosine phosphorylation underlie suppression of growth factor signaling by c-Cbl/Sli-1.

Receptor desensitization is accomplished by accelerated endocytosis and degradation of ligand-receptor complexes. An in vitro reconstituted system indicates that Cbl adaptor proteins directly control downregulation of the receptor for the epidermal growth factor (EGFR) by recruiting ubiquitin-activating and -conjugating enzymes. We infer a sequential process initiated by autophosphorylation of EGFR at a previously identified lysosome-targeting motif that subsequently recruits Cbl. This is followed by tyrosine phosphorylation of c-Cbl at a site flanking its RING finger, which enables receptor ubiquitination and degradation. Whereas all three members of the Cbl family can enhance ubiquitination, two oncogenic Cbl variants, whose RING fingers are defective and phosphorylation sites are missing, are unable to desensitize EGFR. Our study identifies Cbl proteins as components of the ubiquitin ligation machinery and implies that they similarly suppress many other signaling pathways.

Evaluation of Biologic End Points and Pharmacokinetics in Patients With Metastatic Breast Cancer After Treatment With Erlotinib, an Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor

PURPOSE To evaluate changes in epidermal growth factor receptor (EGFR) phosphorylation and its downstream signaling in tumor and surrogate tissue biopsies in patients with metastatic breast cancer treated with erlotinib, an EGFR tyrosine kinase inhibitor, and to assess relationships between biomarkers in tumor and normal tissues and between biomarkers and pharmacokinetics. PATIENTS AND METHODS Eighteen patients were treated orally with 150 mg/d of erlotinib. Ki67, EGFR, phosphorylated EGFR (pEGFR), phosphorylated mitogen-activated protein kinase (pMAPK), and phosphorylated AKT (pAKT) in 15 paired tumor, skin, and buccal mucosa biopsies (at baseline and after 1 month of therapy) were examined by immunohistochemistry and analyzed quantitatively. Pharmacokinetic sampling was also obtained. RESULTS The stratum corneum layer and Ki67 in keratinocytes of the epidermis in 15 paired skin biopsies significantly decreased after treatment (P = .0005 and P = .0003, respectively). No significant change in Ki67 was detected in 15 tumors, and no responses were observed. One was EGFR-positive and displayed heterogeneous expression of the receptor, and 14 were EGFR-negative. In the EGFR-positive tumor, pEGFR, pMAPK, and pAKT were reduced after treatment. Paradoxically, pEGFR was increased in EGFR-negative tumors post-treatment (P = .001). Although markers were reduced in surrogate and tumor tissues in the patient with EGFR-positive tumor, no apparent associations were observed in patients with EGFR-negative tumor. CONCLUSION Erlotinib has inhibitory biologic effects on normal surrogate tissues and on an EGFR-positive tumor. The lack of reduced tumor proliferation may be attributed to the heterogeneous expression of receptor in the EGFR-positive patient and absence of target in this cohort of heavily pretreated patients.

WW Domain HECT E3s Target Cbl RING Finger E3s for Proteasomal Degradation

Cbl proteins have RING finger-dependent ubiquitin ligase (E3) activity that is essential for down-regulation of tyrosine kinases. Here we establish that two WW domain HECT E3s, Nedd4 and Itch, bind Cbl proteins and target them for proteasomal degradation. This is dependent on the E3 activity of the HECT E3s but not on that of Cbl. Consistent with these observations, in cells expressing the epidermal growth factor receptor, Nedd4 reverses Cbl-b effects on receptor down-regulation, ubiquitylation, and proximal events in signaling. Cbl-b also targets active Src for degradation in cells, and Nedd4 similarly reverses Cbl-mediated Src degradation. These findings establish that RING finger E3s can be substrates, not only for autoubiquitylation but also for ubiquitylation by HECT E3s and suggest an additional level of regulation for Cbl substrates including protein-tyrosine kinases.

cbl-3: a new mammalian cbl family protein.

We have cloned a new human gene, cbl-3, which encodes a protein with marked homology to the cbl family of proteins. The predicted protein encoded by this gene retains the conserved phosphotyrosine binding domain (PTB) in the N-terminal and the zinc finger but is significantly shorter (MW 52.5 kDa) than the other mammalian cbl proteins. The protein lacks the extensive proline rich domain and leucine zipper seen in c-cbl and cbl-b and structurally most resembles the C. elegans and Drosophila cbl proteins. The gene is ubiquitously expressed with highest expression in the aerodigestive tract, prostate, adrenal gland, and salivary gland. The protein is phosphorylated and recruited to the EGFR upon EGF stimulation and inhibits EGF stimulated MAP kinase activation. In comparison to the other mammalian cbl proteins (e.g. cbl-b), cbl-3 interacts with a restricted range of proteins containing Src Homology 3 regions. An alternatively spliced form of the cbl-3 protein was also identified which deletes a critical region of the PTB domain and which does not interact with the EGFR nor inhibit EGF stimulated MAP kinase activation. These data demonstrate that cbl-3, a novel mammalian cbl protein, is a regulator of EGFR mediated signal transduction.

cbl -b inhibits epidermal growth factor receptor signaling

The role of cbl-b in signaling by the epidermal growth factor receptor (EGFR) was studied and compared with c-cbl. We demonstrate in vivo, that cbl-b, like c-cbl, is phosphorylated and recruited to the EGFR upon EGF stimulation and both cbl proteins can bind to the Grb2 adaptor protein. To investigate the functional role of cbl proteins in EGFR signaling, we transfected cbl-b or c-cbl into 32D cells overexpressing the EGFR (32D/EGFR). This cell line is absolutely dependent on exogenous IL-3 or EGF for sustained growth. 32D/EGFR cells overexpressing cbl-b showed markedly inhibited growth in EGF compared to c-cbl transfectants and vector controls. This growth inhibition by cbl-b was the result of a dramatic increase in the number of cells undergoing apoptosis. Consistent with this finding, cbl-b overexpression markedly decreased the amplitude and duration of AKT activation upon EGF stimulation compared to either vector controls or c-cbl overexpressing cells. In addition, the duration of EGF mediated MAP kinase and Jun kinase activation in cells overexpressing cbl-b is shortened. These data demonstrate that cbl-b inhibits EGF-induced cell growth and that cbl-b and c-cbl have distinct roles in EGF mediated signaling.

Inhibition of NF-κB Activity Enhances TRAIL Mediated Apoptosis in Breast Cancer Cell Lines

Most breast cancer cell lines are resistant to TNF-related apoptosis inducing ligand (TRAIL) induced apoptosis. In sensitive breast cancer cell lines TRAIL rapidly induces the cleavage and activation of caspases leading to the subsequent cleavage of downstream caspase substrates. In contrast, there is no caspase activation in the resistant cell lines. The transcription factor NF-κB can inhibit apoptosis induced by a variety of stimuli including activation of death receptors. We investigated whether NF-κB contributes to the resistance of breast cancer cells to TRAIL induced apoptosis. All of the resistant breast cancer cell lines expressed NF-κB and had detectable NF-κB activity in nuclear extracts prior to treatment with TRAIL. Upon TRAIL treatment, a significant increase in NF-κB activity was seen in most of the cell lines. To directly test if NF-κB activity contributes to the resistance of these cell lines to TRAIL, we transiently transfected the resistant cell lines with an inhibitor of NF-κB (IκBΔN) and measured TRAIL induced apoptosis in control and transfected cells. All of the resistant cell lines tested showed an increase in TRAIL induced apoptosis when transfected with the IκBΔN. These results demonstrate that TRAIL resistant breast cancer cells fail to rapidly activate the apoptotic machinery but they do activate NF-κB. Inhibition of NF-κB activity increases the sensitivity to TRAIL mediated apoptosis in resistant cells. These results suggest that agents which inhibit NF-κB should increase the clinical efficacy of TRAIL in breast cancer cells.

Cbl-b interacts with ubiquitinated proteins ; differential functions of the UBA domains of c-Cbl and Cbl-b

Cbl proteins are ubiquitin protein ligases, which ubiquitinate activated tyrosine kinases and target them for degradation. Both c-Cbl and Cbl-b have an ubiquitin associated (UBA) domain at their C-terminal end. We observed that high molecular weight ubiquitinated proteins constitutively coimmunoprecipitated with transfected and endogenous Cbl-b, but not c-Cbl. The binding site for these ubiquitinated proteins was mapped to the UBA domain of Cbl-b (UBAb). GST-fusion proteins containing the UBAb interacted with ubiquitinated proteins and polyubiquitin chains in vitro, whereas those containing the UBA domain of c-Cbl (UBAc) did not. The UBAb had a much greater affinity for polyubiquitin chains than for monoubiquitin. Analysis of the UBAb and UBAc demonstrate that the affinity for ubiquitin is determined by multiple amino-acid differences between the two domains. Overexpression of the UBAb, but not overexpression of the UBAc, inhibited a variety of ubiquitin-mediated processes such as degradation of ubiquitinated proteins (i.e. EGFR, Mdm-2, and Siah-1). This in vivo result is consistent with the differences in ubiquitin binding observed in vitro between the UBAb and UBAc. This difference in ubiquitin-binding may reflect distinct regulatory functions of c-Cbl and Cbl-b.

N -(4-hydroxyphenyl) retinamide (4HPR) enhances TRAIL-mediated apoptosis through enhancement of a mitochondrial-dependent amplification loop in ovarian cancer cell lines

AbstractThe majority of ovarian cancer cells are resistant to apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Subtoxic concentrations of the semisynthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) enhanced TRAIL-mediated apoptosis in ovarian cancer cell lines but not in immortalized nontumorigenic ovarian epithelial cells. The enhancement of TRAIL-mediated apoptosis by 4HPR was not due to changes in the levels of proteins known to modulate TRAIL sensitivity. The combination of 4HPR and TRAIL enhanced cleavage of multiple caspases in the death receptor pathway (including the two initiator caspases, caspase-8 and caspase-9). The 4HPR and TRAIL combination leads to mitochondrial permeability transition, significant increase in cytochrome c release, and increased caspase-9 activation. Caspase-9 may further activate caspase-8, generating an amplification loop. Stable overexpression of Bcl-xL abrogates the interaction between 4HPR and TRAIL at the mitochondrial level by blocking cytochrome c release. As a consequence, a decrease in activation of caspase-9, caspase-8, and TRAIL-mediated apoptosis occurs. These results indicate that the enhancement in TRAIL-mediated apoptosis induced by 4HPR is due to the increase in activation of multiple caspases involving an amplification loop via the mitochondrial-death pathway. These findings offer a promising and novel strategy for the treatment of ovarian cancer.

Abstract A77: Efficient targeting of HER-2-positive cancers by Antibody-Coupled T cell Receptor (ACTR) engineered autologous T cells

HER2 gene amplification occurs in 20-30% of aggressive breast and gastric cancer diagnoses, often signifying poor prognosis. The current standard of care for HER2 amplified cancer is the HER2 targeting antibody trastuzumab with chemotherapy. In the setting of HER2 amplified breast cancer, the combination of HER2 targeting antibodies trastuzumab and pertuzumab with the chemotherapeutic docetaxel is the frontline therapy. Despite the success of targeting the HER2 pathway, there are still many patients who are refractory or relapse following HER2 targeting regimens. Outside of the setting of HER2 overexpression or gene amplification, targeting HER2-positive cancers has thus far been ineffective. Engineered autologous T-cells, including chimeric antigen receptors (CARs) and high affinity T-cell receptors (TCRs), have gained attention recently due to their potent efficacy, with overall response rates reaching 80% and examples of long lasting remissions, particularly in advanced lymphoma and leukemia. However, initial clinical attempts to target HER2-amplified breast cancer with CAR-T cell therapy met with either acute toxicities or lack of efficacy. The Antibody-Coupled T-cell Receptor (ACTR) platform is a universal, engineered T cell therapy technology developed to mediate anti-tumor activity in combination with tumor-targeting antibodies. The ACTR chimeric construct is composed of the high-affinity CD16 (FCGR3A) V158 variant with the signaling domains from CD3z and 4-1BB. ACTR is designed to engage the Fc domain of human IgG1 antibodies when opsonized to the cognate target cells and deliver an activation signal to the engineered T cells. ACTR T cells combined with either HER2 targeting trastuzumab or pertuzumab exhibited potent cytotoxic activity, cytokine response and proliferation on a HER2 amplified tumor cell lines. ACTR activity was specific to antibody treated cells, and had little activity on HER2 low or negative tumor lines. Furthermore, the effectiveness of ACTR T cells in a non-amplified HER2 (HER2 low) setting was tested in the presence of a combination of trastuzumab and pertuzumab. This multi-antibody combination increased the cytotoxicity by ACTR T cells, whereas trastuzumab or pertuzumab as single antibody combinations with ACTR had little effect, suggesting a potential therapeutic approach for this disease setting with high unmet medical need. Importantly, the ACTR platform decouples the targeting moiety from the engineered T cell allowing for an antibody dose response which may spare cytotoxicity on normal cells. The specific response to HER2 positive tumor cells in the presence of trastuzumab, pertuzumab or the combination of HER2 directed antibodies demonstrates the potential therapeutic activity of ACTR T cells and supports consideration of clinical testing of ACTR T cells in HER2-positive cancers. Furthermore, combining multiple targeting antibodies to achieve greater potency and efficacy is unique to the ACTR technology and demonstrates the potential for such activity across many other tumor targets and tumor targeting antibodies. Citation Format: Casey B. Judge, Rachel DeBarge, Eugene Choi, Katie O9Callaghan, Lindsay Edwards, Birgit Schultes, Seth Ettenberg, Heather A. Huet. Efficient targeting of HER-2-positive cancers by Antibody-Coupled T cell Receptor (ACTR) engineered autologous T cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A77.

Abstract 3762: Superior T cell activity of a membrane-proximal binding antibody when targeting Glypican-3 with an antibody-coupled T-cell receptor (ACTR) armed T cell

Glypican-3 (GPC3) is a GPI-anchored member of the heparan sulfate proteoglycan family. GPC3 is an oncofetal antigen expressed transiently during fetal development with re-expression during malignant transformation. GPC3 is an ideal tumor target as expression has been found in numerous epithelial malignancies, with highest expression in hepatocellular carcinoma (HCC) and non-small cell lung carcinoma (NSCLC), and normal tissue expression is highly restricted (Baumhoer D., Am J Clin Pathol, 2008.). Adoptive T-cell therapy with single-chain variable fragment (scFv)-derived chimeric antigen receptors (CARs) has transformed cancer therapy, but the broad applicability of this approach has been limited in part by safety concerns due the constitutive expression of a biologically active targeting receptor. The Antibody-Coupled T-cell Receptor (ACTR) platform is a universal, engineered T-cell therapy designed to engage the Fc domain of therapeutic antibodies opsonized to tumor cells to mediate anti-tumor activity. ACTR activity is therefore both regulatable and flexible, providing enhanced therapeutic control and improved safety of the T cell therapy. Using both HCC and NSCLC tumor cell lines, we tested a panel of wild-type and afucosylated antibodies with similar binding affinities that bound to regions spanning the GPC3 protein across unique epitopes. We found that for GPC3 targeting antibodies, the greatest activity in a Jurkat-NFAT reporter assay was observed for the afucosylated antibody that bound GPC3 most proximal to the membrane. Further, the antibody that bound membrane proximal also had the most potent activity in primary ACTR T cell cytotoxicity and cytokine release assays. The physical distance between T cells and tumor targets has been previously determined to impact T cell activation for both peptide-MHC and CAR-T interactions. Similarly, our results demonstrate a potential relationship between spatial distance of tumor targets and T cells in determining the activity ACTR transduced T cells when targeting GPC3. Our data demonstrate that ACTR T cell activity is antibody-specific and dose-titratable, highlighting both efficacy and improved safety of the ACTR T cell platform when targeting GPC3+ solid tumor malignancies. Citation Format: Greg Motz, John Shin, Kathleen Whiteman, Birgit Schultes, Tapasya Pai, Lori Westendorf, Seth Ettenberg, Travis Biechele, Django Sussman, Heather Huet. Superior T cell activity of a membrane-proximal binding antibody when targeting Glypican-3 with an antibody-coupled T-cell receptor (ACTR) armed T cell [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3762. doi:10.1158/1538-7445.AM2017-3762