The Durden laboratory is interested in understanding the fundamental mechanisms by which signals are transmitted from cell surface receptors to the cytoplasm and nucleus. Our efforts are focused on the role of lipid and protein phosphorylation and dephosphorylation in the regulation of signal transduction and epigenetic effector mechanisms. My lab is particularly interested in converting target discovery and validation into drug discovery and drug development in particular as it relates to specific kinases and/or epigenetic effectors and translating this basic science information from "bench to bedside” in pediatric medicine.
The main areas of focus of the Durden lab are as follows:
1. “PI-3 kinase and PTEN signaling in mammalian systems”
The Durden lab reported that the tumor suppressor PTEN controls tumor-induced angiogenesis (PNAS: 98: 4622, 2001). Our results demonstrate the first direct evidence that PTEN controls the tumor-induced angiogenic response in this model system. More importantly, this work lead to the formulation of the “intercept node” hypothesis (Castellino and Durden, Nat.Clin. Practice Neurology 3:682,2007) for target discovery/identification (Fig. 1). We and others have shown that PTEN exerts a control over p53 transcription and many other downstream networks within the tumor and stromal cell through the phosphorylation of AKT and the control of MDM2 (J. Biol. Chem. 77: 777, 2002; Cancer Res.63: 3585, 2003). Recently we defined a novel role for AKT and MDM2 in the cytoplasmic regulation of the hypoxic stability of HIF1α (J Biol. Chem., 2014). This novel control circuit would coordinate proliferation and the induction of angiogenesis. We propose a model where PTEN and p53 exert a coordinate control over a large number of signaling events (Fig. 1). This novel control circuit would coordinate proliferation and the induction of angiogenesis. We propose a model where PTEN and p53 exert a coordinate control over a large number of signaling events (Fig. 1). Durden laboratory has shown that PTEN controls tumor cell migration, invasion and matrix degradation. Moreover PTEN contributes to chemoradioinsensitive state of tumor cells via its control of p53 survival and DNA repair signaling pathways. We have developed small molecule PTEN inhibitors in silico.
2. Drug Discovery and development; Targeted therapeutics using RGD-integrin targeted PI-3 kinase inhibitors and PTEN inhibitors; Discovery and Development of SF1126 “Bench to bedside.”
Durden laboratory in collaboration with SignalRx Pharmaceuticals has generated αv-targeted pan-PI-3 kinase inhibitors as potential antiangiogenic agents for cancer therapy. Based on these studies we have developed an RGD integrin targeted small molecule inhibitor, SF1126 for clinical application (Cancer Research 68: 206, 2008). It was one of the first pan PI-3 kinase inhibitors to enter human clinical trials in cancer. We have now executed and completed the Phase I trial of SF1126 (Eur. J. Cancer, 2012). In 2015, we executed the first Phase I trial of a PI-3 kinase inhibitor in pediatric oncology via the NANT consortium and a Phase II genome driven bucket trial in adult cancers with PIK3CA mutations. This was first time a child was treated with this class of a therapeutic agent.
Fig. 1. PTEN/PI-3K exerts coordinate “central nodal” control over a large number of mammalian signaling events. Hence it could be viewed as “master control switch” for proliferation, angiogenesis, migration, differentiation and metastasis. The concept of coordinated intracellular regulation of angiogenesis is proposed as a model. Hence the PI-3 kinase signaling axis is viewed as a central element of mammalian signaling. Both these pathways are mutated at high frequency in human neoplasia. These pathways are major targets for anticancer therapeutics. In collaboration with Semafore Pharmaceuticals we have successfully developed both PI-3 kinase and PTEN inhibitors to control the “Yin and Yang” of mammalian signaling for therapeutic gain. Castellino and Durden, Nat.Clin. Practice Neurology 3:682,2007.
We have now gone on to develop in silico a large number of dual targeted small molecules in a pipeline for the treatment of pediatric diseases. Currently, we have a large in silico drug discovery effort fueled by X-ray crystallography/molecular modeling and built around the PI-3K signaling pathway. We have hundreds of small molecules in our pipeline which inhibit these targets at nM potency with good safety profiles in animal models for toxicity (J Med. Chem 2012). Dual inhibitory chemotypes developed so far include: 1) PI3K/MEK 2) PI-3K/PARP 3) PI-3K/BRD4 (SF2523 (Fig 2) 4) PI-3K/HDAC 5) PI3K/CDK4/6 and others. We have solved the co-crystallized structure of our dual PI-3K/BRD4 inhibitor, SF2523, bound to the active site of BRD4 at 1.8 Angstroms (BD1) (Fig. 2).
Fig. 2. Dual inhibitor of PI-3K and BRD4, SF2523. We show the co-crystal of BRD4 protein (BD1) and SF2523 (magenta) or JQ1 (grey color) solved at 1.8A for the binding of SF2523 to the BRD4 binding domain 1 (BD1). Grey regions are hydrophobic, red regions are negatively charged and blue areas are positively charged domains of BRD4/BD1. This chemotype is one of many dual inhibitory small molecules developed in the Durden laboratory for different therapeutic areas (listed above).
Drug Discovery efforts focused on PI-3 kinase or phosphatases is a novel area of investigation in the Durden laboratory. In collaboration with the chemists at SignalRx Pharmaceuticals we have developed small molecule inhibitors of PI-3 kinase and the PTEN phosphatase. Using recombinant PTEN or mutants of PTEN we have screened for inhibitors of this important intercept node. Our idea is that a coordinated control of phosphatase/kinase pairs will yield a condition of “signal manipulation” or therapeutic gain. We have used a number of strategies for drug discovery and development built around our interest in signaling pathways which serve as major intercept components of pathophysiologic states. These drug targets include: 1) PTEN 2) PI-3 kinase 3) Syk and 4) Csk kinase and 5) epigenetic effectors e.g. BRD4.
3) Tumor microenvironment (TME) signaling pathway controlling tumor progression and metastasis via the provisional integrin signaling network. Durden laboratory recently identified a signal transduction pathway in macrophages which extends from the provisional integrin, α4β1 through a kinase/GTPase pair (Syk-Rac2) to control tumor metastasis in vivo (Fig. 3). This signaling network has been shown to control macrophage M1-M2 differentiation, tumor progression and metastasis in vivo (PLoS One, 2013). RNA sequencing and CHIPseq have defined an entire transcriptomic program for the control of macrophage M1 to M2 transition and metastasis.
Fig. 3. Rac2 controls metastasis in vivo. We have elucidated a M2 macrophage autonomous signaling pathway that regulates tumor metastasis. The B16F10 melanoma metastasis model is shown. Upper panel metastatic nodules (black) in wild type mice, lower panel 5 mouse lungs from Rac2-/- animal (no metastasis) detected).
4. Signaling pathways in cancer stem cells (CSCs) in medulloblastomagenesis. In 2009, we engaged in the study of cancer stem cells (CSCs) in a well-developed Smo transgenic mouse model (Castellino et al, PLoS One, 2010). We have now defined the fundamental molecular features of the CSC (“stemness signaling properties”) which encode sensitivity and resistance to targeted therapeutic agents including PI-3K inhibitors vs chemotherapeutic drugs. These observations will have therapeutic implications for the treatment of cancers which are stem cell driven.
5. Molecular immunology: Molecular basis for Fcγ receptor signaling in myeloid cells: Targets for drug discovery to control ITIM and ITAM motifs for therapeutic gain? The Durden laboratory has maintained its interest in molecular immunology since 1992 and continued to search for new immunomodulatory targets for immuno-therapeutic exploitation. Most recently in 2014, we reported evidence that the phosphatases SHP1 and PKC regulate the FcγR signaling axis (ECR, 2014). Evidence from several knockout mouse models has demonstrated conclusively that the Fcγ receptors are critical elements in the control of autoimmunity and inflammatory diseases. The FcγRs are involved in both positive and negative regulation of immune responses and inflammatory cascades. Our research primarily involves the study of how tyrosine phosphorylation and dephosphorylation of cell proteins and lipids drives myeloid signal relay pathways. Over the past 12 years, my laboratory has developed a model for how the Fc receptor for IgG transmits intracytoplasmic signals to the respiratory burst and other pathways. (see Fig. 4). Our lab is interested in studying role of protein tyrosine phosphorylation and dephosphorylation in FcγR/ITIM/ITAM signaling. Role of complex adapter proteins and protein and lipid phosphatases in FcγRI, ITIM and ITAM signaling.
Fig. 4. Model for ITAM and ITIM signaling in macrophages. As is now known the revolution in Oncology in 2014 came with the discovery that checkpoint blockade of ITIM receptors (CD28, PD1, PDL1) could activate the immune system for significant efficacy in the treatment of human cancer! Ligand (IgG) binds to the FcγRIα subunit resulting in a conformation change in the homodimeric ITAM or γ subunits. This change induces the activation of HCK kinase activity which results in the tyrosine phosphorylation of the ITAM motif of FcγRI. Phosphorylation of FcγRIγ subunit recruits the binding and activation of the HCK, LYN and SYK kinases. Other proteins are tyrosine phosphorylated including the CBL and SHC adapter protein. The tyrosine phosphorylation of SHC is noted to bind to GRB2 (not shown) and the SOS nucleotide exchange protein, thus activating small GTPases in the cell through the conversion of GDPras to GTPras. GTPras activates downstream cascades including PI-3 kinase which generates PIP3 and activates other pathways. The role of CBL phosphorylation is to recruit to the receptor complex the PI-3 kinase p85 subunit. Downstream targets for FcγRI stimulation are the small GTPases, RAS, RAP1A, and RAC2 which control the myeloid respiratory burst. The respiratory burst response involves the macromolecular assembly of p47phox, p67phox, p40phox, p91phox and p22phox along with RAP1A and RAC resulting in the generation of superoxide anions (O2 ) which is measured using the reduction of cytochrome c as an assay. Abbreviations: γ, γ subunit (ITAM) of high affinity Fc receptor for IgG or FcγRIγ subunit; SH2, src homology 2 domain; SH3, src homology 3 domain; SOS, "son of sevenless" protein
6. New agents for immuno-oncology; Checkpoint blockade or TLR agonists to activate the immune response by blocking ITIM signaling in immune effectors (Fig 4). The Durden laboratory has been involved in the study of ITAM and ITIM signaling for the last 20 years. Finally, and perhaps most importantly our laboratory effort has recently identified the Rac2 and PI-3 kinase signaling pathways as negative regulators of the innate and adaptive immune response as potential control points for augmenting innate and adaptive immunity as an anticancer strategy. Moreover, we have executed the use of a TLR3 agonist, polyICLC in a Phase II trial in pediatric high grade glioma (funded by FDA RO1, Durden PI). This will be combined with checkpoint PD1 blockade in future clinical trials using PI-3 kinase inhibitors.