Discovery of Small Molecule WWP2 Ubiquitin Ligase Inhibitors

We have screened small molecule libraries specifically for inhibitors that target WWP2, an E3 ubiquitin ligase associated with tumour outgrowth and spread. Selected hits demonstrated dose‐dependent WWP2 inhibition, low micromolar IC50 values, and inhibition of PTEN substrate‐specific ubiquitination. Binding to WWP2 was confirmed by ligand‐based NMR spectroscopy. Furthermore, we used a combination of STD NMR, the recently developed DEEP‐STD NMR approach, and docking calculations, to propose for the first time an NMR‐validated 3D molecular model of a WWP2‐inhibitor complex. These first generation WWP2 inhibitors provide a molecular framework for informing organic synthetic approaches to improve activity and selectivity

Does the PI3K pathway promote or antagonize regulatory T cell development and function?

Regulatory T cells (Tregs) prevent autoimmunity and inflammation by suppressing the activation of other T cells and antigen presenting cells. The role of phosphoinositide 3-kinase (PI3K) signaling in Treg is controversial. Some studies suggest that inhibition of the PI3K pathway is essential for the development of Tregs whereas other studies have shown reduced Treg numbers and function when PI3K activity is suppressed. Here we attempt to reconcile the different studies that have explored PI3K and the downstream effectors Akt, Foxo, and mTOR in regulatory T cell development and function and discuss the implications for health and therapeutic intervention

Smad7 Binds Differently to Individual and Tandem WW3 and WW4 Domains of WWP2 Ubiquitin Ligase Isoforms

WWP2 is an E3 ubiquitin ligase that differentially regulates the contextual tumour suppressor/progressor TGFβ signalling pathway by alternate isoform expression. WWP2 isoforms select signal transducer Smad2/3 or inhibitor Smad7 substrates for degradation through different compositions of protein–protein interaction WW domains. The WW4 domain containing WWP2-C induces Smad7 turnover in vivo and positively regulates the metastatic epithelial–mesenchymal transition programme. This activity and the overexpression of these isoforms in human cancers make them candidates for therapeutic intervention. Here, we use NMR spectroscopy to solve the solution structure of the WWP2 WW4 domain and observe the binding characteristics of Smad7 substrate peptide. We also reveal that WW4 has an enhanced affinity for a Smad7 peptide phosphorylated at serine 206 adjacent to the PPxY motif. Using the same approach, we show that the WW3 domain also binds Smad7 and has significantly enhanced Smad7 binding affinity when expressed in tandem with the WW4 domain. Furthermore, and relevant to these biophysical findings, we present evidence for a novel WWP2 isoform (WWP2C-ΔHECT) comprising WW3–WW4 tandem domains and a truncated HECT domain that can inhibit TGFβ signalling pathway activity, providing a further layer of complexity and feedback to the WWP2 regulatory apparatus. Collectively, our data reveal a structural platform for Smad substrate selection by WWP2 isoform WW domains that may be significant in the context of WWP2 isoform switching linked to tumorigenesis

Inactivation of PI(3)K p110δ breaks regulatory T-cell-mediated immune tolerance to cancer.

Inhibitors against the p110δ isoform of phosphoinositide-3-OH kinase (PI(3)K) have shown remarkable therapeutic efficacy in some human leukaemias. As p110δ is primarily expressed in leukocytes, drugs against p110δ have not been considered for the treatment of solid tumours. Here we report that p110δ inactivation in mice protects against a broad range of cancers, including non-haematological solid tumours. We demonstrate that p110δ inactivation in regulatory T cells unleashes CD8(+) cytotoxic T cells and induces tumour regression. Thus, p110δ inhibitors can break tumour-induced immune tolerance and should be considered for wider use in oncology

Protein kinase B controls transcriptional programs that direct cytotoxic T cell fate but is dispensable for T cell metabolism

SummaryIn cytotoxic T cells (CTL), Akt, also known as protein kinase B, is activated by the T cell antigen receptor (TCR) and the cytokine interleukin 2 (IL-2). Akt can control cell metabolism in many cell types but whether this role is important for CTL function has not been determined. Here we have shown that Akt does not mediate IL-2- or TCR-induced cell metabolic responses; rather, this role is assumed by other Akt-related kinases. There is, however, a nonredundant role for sustained and strong activation of Akt in CTL to coordinate the TCR- and IL-2-induced transcriptional programs that control expression of key cytolytic effector molecules, adhesion molecules, and cytokine and chemokine receptors that distinguish effector versus memory and naive T cells. Akt is thus dispensable for metabolism, but the strength and duration of Akt activity dictates the CTL transcriptional program and determines CTL fate

Selective targeting of activating and inhibitory Smads by distinct WWP2 ubiquitin ligase isoforms differentially modulates TGFβ signalling and EMT

Ubiquitin-dependent mechanisms have emerged as essential regulatory elements controlling cellular levels of Smads and TGFß-dependent biological outputs such as epithelial–mesenchymal transition (EMT). In this study, we identify a HECT E3 ubiquitin ligase known as WWP2 (Full-length WWP2-FL), together with two WWP2 isoforms (N-terminal, WWP2-N; C-terminal WWP2-C), as novel Smad-binding partners. We show that WWP2-FL interacts exclusively with Smad2, Smad3 and Smad7 in the TGFß pathway. Interestingly, the WWP2-N isoform interacts with Smad2 and Smad3, whereas WWP2-C interacts only with Smad7. In addition, WWP2-FL and WWP2-C have a preference for Smad7 based on protein turnover and ubiquitination studies. Unexpectedly, we also find that WWP2-N, which lacks the HECT ubiquitin ligase domain, can also interact with WWP2-FL in a TGFß-regulated manner and activate endogenous WWP2 ubiquitin ligase activity causing degradation of unstimulated Smad2 and Smad3. Consistent with our protein interaction data, overexpression and knockdown approaches reveal that WWP2 isoforms differentially modulate TGFß-dependent transcription and EMT. Finally, we show that selective disruption of WWP2 interactions with inhibitory Smad7 can stabilise Smad7 protein levels and prevent TGFß-induced EMT. Collectively, our data suggest that WWP2-N can stimulate WWP2-FL leading to increased activity against unstimulated Smad2 and Smad3, and that Smad7 is a preferred substrate for WWP2-FL and WWP2-C following prolonged TGFß stimulation. Significantly, this is the first report of an interdependent biological role for distinct HECT E3 ubiquitin ligase isoforms, and highlights an entirely novel regulatory paradigm that selectively limits the level of inhibitory and activating Smads

How ubiquitination regulates the TGF-β signalling pathway: New insights and new players

Ubiquitination of protein species in regulating signal transduction pathways is universally accepted as of fundamental importance for normal development, and defects in this process have been implicated in the progression of many human diseases. One pathway that has received much attention in this context is transforming growth factor-beta (TGF-ß) signalling, particularly during the regulation of epithelial-mesenchymal transition (EMT) and tumour progression. While E3-ubiquitin ligases offer themselves as potential therapeutic targets, much remains to be unveiled regarding mechanisms that culminate in their regulation. With this in mind, the focus of this review highlights the regulation of the ubiquitination pathway and the significance of a recently described group of NEDD4 E3-ubiquitin ligase isoforms in the context of TGF-ß pathway regulation. Moreover, we now broaden these observations to incorporate a growing number of protein isoforms within the ubiquitin ligase superfamily as a whole, and discuss their relevance in defining a new ‘iso-ubiquitinome’

BCATc modulates crosstalk between the PI3K/Akt and the Ras/ERK pathway regulating proliferation in triple negative breast cancer

The cytosolic branched chain aminotransferase (BCATc) protein has been found to be highly expressed in breast cancer subtypes, including triple negative breast cancer (TNBC), compared with normal breast tissue. The catabolism of branched-chain amino acids (BCAAs) by BCATc leads to the production of glutamate and key metabolites which further drive the TCA cycle, important for cellular metabolism and growth. Upregulation of BCATc has been associated with increased cell proliferation, cell cycle progression and metastasis in several malignancies including breast, gliomas, ovarian and colorectal cancer but the underlying mechanisms are unclear. As nutrient levels of BCAAs, substrates of BCATc, regulate the P13K/Akt pathway we hypothesized that increased expression of BCATc would contribute to tumour cell growth through upregulation of the insulin/IGF-1 signalling pathway. This pathway is known to potentiate proliferation and metastasis of malignant cells through the activation of PI3K/Akt and the Ras/ERK signalling cascades. Here we show that knockdown of BCATc significantly reduced insulin and IGF-1-mediated proliferation, migration and invasion of TNBC cells. An analysis of this pathway showed that when overexpressed BCATc regulates proliferation through the PI3K/Akt axis, whilst simultaneously attenuating the Ras/Erk pathway indicating that BCATc acts as a conduit between these two pathways. This ultimately led to an increase in FOXO3a, a key regulator of cell proliferation and Nrf2, which mediates redox homeostasis. Together this data indicates that BCATc regulates TNBC cell proliferation, migration and invasion through the IGF-1/insulin PI3K/Akt pathway, culminating in the upregulation of FOXO3a and Nrf2, pointing to a novel therapeutic target for breast cancer treatment

PI3Kδ and primary immunodeficiencies.

Primary immunodeficiencies are inherited disorders of the immune system, often caused by the mutation of genes required for lymphocyte development and activation. Recently, several studies have identified gain-of-function mutations in the phosphoinositide 3-kinase (PI3K) genes PIK3CD (which encodes p110δ) and PIK3R1 (which encodes p85α) that cause a combined immunodeficiency syndrome, referred to as activated PI3Kδ syndrome (APDS; also known as p110δ-activating mutation causing senescent T cells, lymphadenopathy and immunodeficiency (PASLI)). Paradoxically, both loss-of-function and gain-of-function mutations that affect these genes lead to immunosuppression, albeit via different mechanisms. Here, we review the roles of PI3Kδ in adaptive immunity, describe the clinical manifestations and mechanisms of disease in APDS and highlight new insights into PI3Kδ gleaned from these patients, as well as implications of these findings for clinical therapy

Single CD28 stimulation induces stable and polyclonal expansion of human regulatory T cells

Contains fulltext : 170288.pdf (publisher's version ) (Open Access)CD4+FOXP3+ Treg are essential for immune tolerance. Phase-1 clinical trials of Treg-therapy to treat graft-versus-host-disease reported safety and potential therapeutic efficacy. Treg-based trials have started in organ-transplant patients. However, efficient ex vivo expansion of a stable Treg population remains a challenge and exploring novel ways for Treg expansion is a pre-requisite for successful immunotherapy. Based on the recent finding that CD28-signaling is crucial for survival and proliferation of mouse Treg, we studied single-CD28 stimulation of human Treg, without T cell receptor stimulation. Single-CD28 stimulation of human Treg in the presence of recombinant human IL-2(rhIL-2), as compared to CD3/CD28/rhIL-2 stimulation, led to higher expression levels of FOXP3. Although the single-CD28 expanded Treg population was equally suppressive to CD3/CD28 expanded Treg, pro-inflammatory cytokine (IL-17A/IFNgamma) production was strongly inhibited, indicating that single-CD28 stimulation promotes Treg stability. As single-CD28 stimulation led to limited expansion rates, we examined a CD28-superagonist antibody and demonstrate a significant increased Treg expansion that was more efficient than standard anti-CD3/CD28-bead stimulation. CD28-superagonist stimulation drove both naive and memory Treg proliferation. CD28-superagonist induction of stable Treg appeared both PI3K and mTOR dependent. Regarding efficient and stable expansion of Treg for adoptive Treg-based immunotherapy, application of CD28-superagonist stimulation is of interest