Small Molecule Regulator of ENTPD5, and ER Enzyme in the PTEN/AKT Pathway

PI3K signaling plays a crucial role in effecting alterations in a broad range of cellular functions in response to diverse extracellular stimuli (insulin, growth factors, integrins and GPCRs etc.). A key downstream effector of PI3K is the serine-threonine kinase Akt, which in response to PI3K activation, phosphorylates and regulates the activity of a number of cellular targets, through which it modulates a variety of cellular functions, including glucose metabolism, protein synthesis, cell proliferation and survival. Tumor supressor gene PTEN encodes a lipid phosphatase that antagonizes PI3K function and consequently inhibits downstream signaling through Akt. Dysregulation of this pathway has been found in a variety of human cancer, mainly by loss of function of PTEN, or amplification and activating mutations of PI3K and/or Akt. We were interested in the anti-apoptotic mechanism of PI3K/Akt signaling. We observed a defect in apoptosome formation in PTEN-null (PTEN -/-) MEF cell lysate. This defect is due to rapid depletion of ATP by a strong ATP hydrolysis activity in PTEN-null MEF lysate, which is absent in PTEN heterozygous (PTEN +/-) lysate. Following this activity, we purified three enzymes, namely ENTPD5, CMPK1 and AK1, that together forms a coupled enzymatic cycle, hydrolyzing ATP to AMP. In the cell, ENTPD5 is an ER localized UDPase that hydrolyzes UDP, the by-product of glycosyl-transferase, into UMP. Only in its monophosphate form can Uridine nucleotide exits ER through an antiporter by exchanging a molecule of UDP-sugar from cytosol. Up-regulation of ENTPD5 in PTEN-null MEF cells accelerates glycosylation substrate replenishment, therefore promotes N-glycosylation and increases ER protein folding capacity to accommendate the increase of protein synthesis resulted from active PI3K/Akt signaling. Knockdown of ENTPD5 in PTEN-null cells suppresses global N-glycosylation, resulting in ER stress and degradation of several growth factor receptors. As a consequence, the growth of PTEN-null cells is inhibited both in vitro and in mouse xenograft tumor models. Given the essential role of ENTPD5 in PI3K/Akt pathway, we performed biochemical high-throughput screen for ENTPD5 inhibitors. The newly identified inhibitors recapitulate the phenotype of ENTPD5 knockdown in vitro. Interestingly, PTEN-null MEF cells are more susceptible to these inhibitors than PTEN heterozygous MEF cells, in terms of the intensity of induced ER stress and cell death. Inhibition of ENTPD5 produces synthetic lethality with PTEN loss or PI3K/Akt hyperactivation, therefore provides a potential therapy for the cancers harboring these lesions.