The programmes of work in the laboratory are currently aimed at understanding the molecular mechanisms and physiological significance of intracellular signalling networks which involve a family of enzymes called phosphoinositide 3OH-kinases (PI3Ks).
PI3Ks are now accepted to be critical regulators of numerous important and complex cell responses, including cell growth, division, survival and movement.
PI3Ks catalyse the formation of one or more critical phospholipid messenger molecules, which signal information by binding to specific domains in target proteins. Currently the best understood pathway involves the activation of Class I PI3Ks by cell surface receptors.
In recent years, the laboratory has increasingly focused on the role of PI3Ks in the signalling mechanisms which allow receptors on neutrophils (white blood cells) to control various aspects of neutrophil function.
Neutrophils are key players in the front line of our immune system, responsible primarily for the recognition and destruction of bacterial and fungal pathogens. However, they are also involved in the amplification cascades that underlie various inflammatory pathologies, e.g. Acute Respiratory Distress Syndrome (ARDS) and rheumatoid arthritis.
PTEN (phosphatase and tensin homolog deleted on chromosome ten) is a tumor suppressor, the function of which is impaired in many diverse cancers. It has phosphoinositide lipid phosphatase activity by which it suppresses activation of the oncogenic PI3K signaling network but in聽vitro also displays activity against protein substrates and is able to auto-dephosphorylate its Thr366 residue. Here we generate germline knock-in mice expressing PTEN-Y138L, a mutant enzyme which selectively lacks protein phosphatase activity and retains lipid phosphatase activity. Homozygous Pten mice die in utero before E10.5. Primary MEFs and thymocytes with only a single Pten allele display normal low levels of AKT phosphorylation indicating effective regulation of PI3K signaling by endogenous PTEN-Y138L in聽vivo. Heterozygous Pten mice have reduced overall survival compared to wild-type littermates and develop tumors in multiple organs. Our data imply that in addition to its lipid phosphatase activity, the protein phosphatase activity of PTEN is also required for normal embryonic development and tumor suppression.
Although the regulation of branching morphogenesis by spatially distributed cues is well established, the underlying intracellular signaling mechanisms are not well understood. The development of the lacrimal gland is driven by fibroblast growth factor (FGF) signaling, which activates phospholipase C gamma (PLC纬). Here, we showed that mutating the PLC纬1 binding site on Fgfr2 leads to ectopic branching and hyperplasia in the lacrimal gland, which was phenocopied by either deleting PLC纬1 or disabling any of its SH2 domains. PLC纬1 inactivation did not change the level of Fgfr2 or affect mitogen-activated protein kinase (MAPK) signaling but instead led to sustained AKT phosphorylation due to increased phosphatidylinositol 3,4,5-trisphosphate (PIP3) production. Consistent with this, the PLC纬1 mutant phenotype can be reproduced by the elevation of phosphatidylinositol 3-kinase (PI3K) signaling in Pten knockout and attenuated by blocking AKT signaling. Our findings demonstrate that FGF-activated PLC纬 modulates PI3K signaling by shifting phosphoinositide metabolism, revealing the crucial role of PLC纬 in branching morphogenesis and organ size control.
Metastatic uveal melanoma is an aggressive disease with limited effective therapeutic options. To comprehensively map monogenic and digenic dependencies, we performed CRISPR-Cas9 screening in ten extensively profiled human uveal melanoma cell line models. Analysis involved genome-wide single-gene and combinatorial paired-gene CRISPR libraries. Among our 76 uveal melanoma-specific essential genes and 105 synthetic lethal gene pairs, we identified and validated the CDP-diacylglycerol synthase 2 gene (CDS2) as a genetic dependency in the context of low CDP-diacylglycerol synthase 1 gene (CDS1) expression. We further demonstrate that CDS1/CDS2 forms a synthetic lethal interaction in vivo and reveal that CDS2 knockout results in the disruption of phosphoinositide synthesis and increased cellular apoptosis and that re-expression of CDS1 rescues this cell fitness defect. We extend our analysis using pan-cancer data, confirming increased CDS2 essentiality in diverse tumor types with low CDS1 expression. Thus, the CDS1/CDS2 axis is a therapeutic target across a range of cancers.