Greg Coffey

Cutting Edge: The Transmembrane E3 Ligase GRAIL Ubiquitinates the Costimulatory Molecule CD40 Ligand during the Induction of T Cell Anergy

Activation of naive T lymphocytes is regulated through a series of discrete checkpoints that maintain unresponsiveness to self. During this multistep process, costimulatory interactions act as inducible signals that allow APCs to selectively mobilize T cells against foreign Ags. In this study, we provide evidence that the anergy-associated E3 ubiquitin ligase GRAIL (gene related to anergy in lymphocytes) regulates expression of the costimulatory molecule CD40L on CD4 T cells. Using its luminal protease-associated domain, GRAIL binds to the luminal/extracellular portion of CD40L and facilitates transfer of ubiquitin molecules from the intracellular GRAIL RING (really interesting new gene) finger to the small cytosolic portion of CD40L. Down-regulation of CD40L occurred following ectopic expression of GRAIL in naive T cells from CD40−/− mice, and expression of GRAIL in bone marrow chimeric mice was associated with diminished lymphoid follicle formation. These data provide a model for intrinsic T cell regulation of costimulatory molecules and a molecular framework for the initiation of clonal T cell anergy.

Engagement of CD81 induces ezrin tyrosine phosphorylation and its cellular redistribution with filamentous actin

CD81 is a tetraspanin family member involved in diverse cellular interactions in the immune and nervous systems and in cell fusion events. However, the mechanism of action of CD81 and of other tetraspanins has not been defined. We reasoned that identifying signaling molecules downstream of CD81 would provide mechanistic clues. We engaged CD81 on the surface of B-lymphocytes and identified the induced tyrosine-phosphorylated proteins by mass spectrometry. This analysis showed that the most prominent tyrosine phosphorylated protein was ezrin, an actin-binding protein and a member of the ezrin-radixin-moesin family. We also found that CD81 engagement induces spleen tyrosine kinase (Syk) and that Syk was involved in tyrosine phosphorylation of ezrin. After engagement of CD81, it colocalized with ezrin and F-actin, and this association was disrupted when Syk activation was blocked. Taken together, these studies suggest a model in which CD81 interfaces between the plasma membrane and the cytoskeleton by activating Syk, mobilizing ezrin, and recruiting F-actin to facilitate cytoskeletal reorganization and cell signaling. This mechanism might explain the pleiotropic effects induced in response to stimulation of cells by anti-CD81 antibodies or by the hepatitis C virus, which uses this molecule as its key receptor.

Pre-BCR signaling in precursor B-cell acute lymphoblastic leukemia regulates PI3K/AKT, FOXO1 and MYC, and can be targeted by SYK inhibition

Precursor-B-cell receptor (pre-BCR) signaling and spleen tyrosine kinase (SYK) recently were introduced as therapeutic targets for patients with B-cell acute lymphoblastic leukemia (B-ALL), but the importance of this pathway in B-ALL subsets and mechanism of downstream signaling have not fully been elucidated. Here, we provide new detailed insight into the mechanism of pre-BCR signaling in B-ALL. We compared the effects of pharmacological and genetic disruption of pre-BCR signaling in vitro and in mouse models for B-ALL, demonstrating exquisite dependency of pre-BCR+ B-ALL, but not other B-ALL subsets, on this signaling pathway. We demonstrate that SYK, PI3K/AKT, FOXO1 and MYC are important downstream mediators of pre-BCR signaling in B-ALL. Furthermore, we define a characteristic immune phenotype and gene expression signature of pre-BCR+ ALL to distinguish them from other B-ALL subsets. These data provide comprehensive new insight into pre-BCR signaling in B-ALL and corroborate pre-BCR signaling and SYK as promising new therapeutic targets in pre-BCR+ B-ALL.

Dual SYK/JAK inhibition overcomes ibrutinib resistance in chronic lymphocytic leukemia: Cerdulatinib, but not ibrutinib, induces apoptosis of tumor cells protected by the microenvironment

Ibrutinib (BTK inhibitor) has generated remarkable responses in CLL. However, the drug, to a large extent, does not cause cell death directly and does not eradicate CLL malignant clones. Inability to eradicate CLL has fostered resistance generation. Once patients become resistant, they do poorly with a median survival of 3-4 months. Novel therapeutic strategies are needed to prevent resistance, improve treatment outcome and ultimately cure the disease. Herein, we explore dual targeting of the BCR and JAK-STAT pathways with a novel single agent, cerdulatinib, which selectively inhibits both SYK (a BCR component) and JAK kinases. We demonstrated that cerdulatinib delivered potent tumor inhibition in 60 primary CLL patient samples, especially in those with poor prognostic indicators. Importantly, cerdulatinib, but not ibrutinib, is able to overcome the support of microenvironment and induces CLL cell death at clinically achievable concentrations. Notably, cerdulatinib blocked proliferation of ibrutinib-resistant primary CLL cells and of BTKC481S-transfected/ibrutinib-resistant lymphoma cells. These anti-tumor effects are well correlated with the inhibition of BCR and JAK-STAT signaling and downstream inhibition of the functions of AKT, ERK and NF?B. Collectively, our results show that simultaneous targeting of BCR and JAK-STAT pathways is a more effective strategy relative to single BTK inhibition.

Abstract CT144: Preclinical and clinical studies and modeling and simulation to identify phase II dose for cerdulatinib: a dual SYK/JAK inhibitor for the treatment of B-cell malignancies

Background. Subsets of B cell lymphomas appear to rely on B-cell receptor (BCR) and/or cytokine JAK/STAT signaling for survival. SYK is upstream of BTK, PI3Kδ, and PLCγ2 on the BCR signaling pathway, and thus a potential therapeutic target. Additional survival support appears to be mediated by cytokine-induced JAK/STAT pathways. Cerdulatinib is a dual SYK/JAK inhibitor being evaluated in patients with relapsed/refractory B cell malignancies in a dose escalation study. Patient PK and PD parameters indicated nonlinearity at doses above 30 mg QD. Exposure at the 45 mg dose was 3-fold higher than at 30 mg QD. As the dose was further increased up to 100 mg QD, the Cssmax and AUC values plateaued. PD results also appeared to plateau achieving approximately 50-90% target inhibition in peripheral blood assays at steady-state Cmin to Cmax, respectively. The target therapeutic exposure for oncology, based on pre-clinical models, is a Cssmin >1.5uM which would lead to >90% SYK/JAK inhibition at the trough concentration Objective. Pre-clinical and clinical PK/PD relationships and correlations between SYK/JAK inhibition and tumor response will be presented. A physiological-based PK (PBPK) model was developed to elucidate the relationship between dose, dosing schedule, and exposure. The goal of developing a PBPK model was to provide a strategy to increase exposure in patients to therapeutic target levels. Methods. This is a 3+3 dose escalation study with 28-day cycles and doses studied from 15 mg to 100 mg QD, and up to 45 mg BID. PK, PD, and safety were monitored. SYK and JAK inhibition was determined by multiple whole blood assays measuring signaling via the B cell antigen receptor, IL2, IL4, IL6, and GM-CSF. Serum markers of tumor burden - CCL3, CCL4, and other markers of inflammation, were also measured. Physiochemical parameters and in vitro ADME parameters were used to construct a PBPK model. Results. Exposure correlated well with PD and tumor responses in the dose escalation study. Low pH dependent solubility was identified as the rate-limiting factor in increasing plasma concentrations. The PBPK model predicted BID dosing would result in significantly higher plasma levels. The Cssmin concentration of QD doses from 40 to 100 mg QD was ∼0.70 uM while for the 45 mg BID dose for the first 3 patients the Cssmin was ∼1.5 uM. Complete target inhibition at this Cssmin was achieved with good tolerability. Conclusions. The preliminary PBPK model identified the pH dependent low solubility of cerdulabinib as the rate-limiting factor of absorption with increasing doses. Subsequent evaluation of 45 mg BID doses in patients provided higher Cmin, Cmax, and AUC values for all patients treated at this dose level and PD markers indicated complete inhibition of both pathways. We are now enrolling additional patients at this dose, in preparation for selecting our final phase II dose. Citation Format: Janet M. Leeds, Greg Coffey, Anjali Pandey, Pam B. Conley, Karen Rowland Yeo, Alice B. Ke, John T. Curnutte, Glenn Michelson. Preclinical and clinical studies and modeling and simulation to identify phase II dose for cerdulatinib: a dual SYK/JAK inhibitor for the treatment of B-cell malignancies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT144.