No abstract

SummaryWnt proteins are secreted post-translationally modified proteins that signal locally to regulate development and proliferation. The production of bioactive Wnts requires a number of dedicated factors in the secreting cell whose coordinated functions are not fully understood. A screen for small molecules identified inhibitors of vacuolar acidification as potent inhibitors of Wnt secretion. Inhibition of the V-ATPase or disruption of vacuolar pH gradients by diverse drugs potently inhibited Wnt/-catenin signaling both in cultured human cells and in vivo, and impaired Wnt-regulated convergent extension movements in Xenopus embryos. WNT secretion requires its binding to the carrier protein wntless (WLS); we find that WLS is ER-resident in human cells and WNT3A binding to WLS requires PORCN-dependent lipid modification of WNT3A at serine 209. Inhibition of vacuolar acidification results in accumulation of the WNT3A-WLS complex both in cells and at the plasma membrane. Modeling predictions suggest that WLS has a lipid-binding -barrel that is similar to the lipocalin-family fold. We propose that WLS binds Wnts in part through a lipid-binding domain, and that vacuolar acidification is required to release palmitoylated WNT3A from WLS in secretory vesicles, possibly to facilitate transfer of WNT3A to a soluble carrier protein.

No abstract

Transfer RNA (tRNA)‐derived small RNAs (tsRNAs) have recently emerged as important regulators of protein translation and shown to have diverse biological functions. However, the underlying cellular and molecular mechanisms of tsRNA function in the context of dynamic cell‐state transitions remain unclear. Expression analysis of tsRNAs in distinct heterologous cell and tissue models of stem vs. differentiated states revealed a differentiation‐dependent enrichment of 5′‐tsRNAs. We report the identification of a set of 5′‐tsRNAs that is upregulated in differentiating mouse embryonic stem cells (mESCs). Notably, interactome studies with differentially enriched 5′‐tsRNAs revealed a switch in their association with “effector” RNPs and “target” mRNAs in different cell states. We demonstrate that specific 5′‐tsRNAs can preferentially interact with the RNA‐binding protein, Igf2bp1, in the RA‐induced differentiated state. This association influences the transcript stability and thereby translation of the pluripotency‐promoting factor, c‐Myc, thus providing a mechanistic basis for how 5′‐tsRNAs can modulate stem cell states in mESCs. Together our study highlights the role of 5′‐tsRNAs in defining distinct cell states.

Casein kinase 1 delta and epsilon (CK1δ/ɛ) are key regulators of diverse cellular growth and survival processes including Wnt signaling, DNA repair and circadian rhythms. Recent studies suggest that they have an important role in oncogenesis. RNA interference screens identified CK1ɛ as a pro-survival factor in cancer cells in vitro and the CK1δ/ɛ-specific inhibitor IC261 is remarkably effective at selective, synthetic lethal killing of cancer cells. The recent development of the nanomolar CK1δ/ɛ-selective inhibitor, PF670462 (PF670) and the CK1ɛ-selective inhibitor PF4800567 (PF480) offers an opportunity to further test the role of CK1δ/ɛ in cancer. Unexpectedly, and unlike IC261, PF670 and PF480 were unable to induce cancer cell death. PF670 is a potent inhibitor of CK1δ/ɛ in cells; nanomolar concentrations are sufficient to inhibit CK1δ/ɛ activity as measured by repression of intramolecular autophosphorylation, phosphorylation of disheveled2 proteins and Wnt/β-catenin signaling. Likewise, small interfering RNA knockdown of CK1δ and CK1ɛ reduced Wnt/β-catenin signaling without affecting cell viability, further suggesting that CK1δ/ɛ inhibition may not be relevant to the IC261-induced cell death. Thus, while PF670 is a potent inhibitor of Wnt signaling, it only modestly inhibits cell proliferation. In contrast, while sub-micromolar concentrations of IC261 neither inhibited CK1δ/ɛ kinase activity nor blocked Wnt/β-catenin signaling in cancer cells, it caused a rapid induction of prometaphase arrest and subsequent apoptosis in multiple cancer cell lines. In a stepwise transformation model, IC261-induced killing required both overactive Ras and inactive p53. IC261 binds to tubulin with an affinity similar to colchicine and is a potent inhibitor of microtubule polymerization. This activity accounts for many of the diverse biological effects of IC261 and, most importantly, for its selective cancer cell killing.

SUMMARY Obesity develops due to altered energy homeostasis favoring fat storage. Here we describe a novel transcription co-regulator for adiposity and energy metabolism, TRIP-Br2 (also called SERTAD2). TRIP-Br2 null mice are resistant to obesity and obesity-related insulin resistance. Adipocytes of the knockout (KO) mice exhibited greater stimulated lipolysis secondary to enhanced expression of hormone sensitive lipase (HSL) and β3-adrenergic (Adrb3) receptors. The KOs also exhibit higher energy expenditure due to increased adipocyte thermogenesis and oxidative metabolism by up-regulating key enzymes in respective processes. Our data show for the first time that a cell cycle transcriptional co-regulator, TRIP-Br2, modulates fat storage through simultaneous regulation of lipolysis, thermogenesis and oxidative metabolism. These data together with the observation that TRIP-BR2 expression is selectively elevated in visceral fat in obese humans suggests that this transcriptional co-regulator is a novel therapeutic target for counteracting the development of obesity, insulin resistance and hyperlipidemia.

No abstract

The use of BCR-ABL1 tyrosine kinase inhibitors (TKI) has led to excellent clinical responses in patients with chronic phase chronic myeloid leukemia (CML). However these inhibitors have been less effective as single agents in the terminal blast phase (BP). We show that pyrvinium, a FDA-approved anthelminthic drug, selectively targets BP-CML CD34+ progenitor cells. Pyrvinium is effective in inducing apoptosis, inhibiting colony formation and self-renewal capacity of CD34+ cells from TKI-resistant BP-CML patients, while cord blood CD34+ are largely unaffected. The effects of pyrvinium are further enhanced upon combination with dasatinib, a second generation BCR-ABL1 TKI. In a CML xenograft model pyrvinium significantly inhibits tumor growth as a single agent, with complete inhibition in combination with dasatinib. While pyrvinium has been shown to inhibit the Wnt/β-catenin signalling pathway via activation of casein kinase 1α, we find its activity in CML is not dependent on this pathway. Instead, we show that pyrvinium localizes to mitochondria and induces apoptosis by inhibiting mitochondrial respiration. Our study suggests that pyrvinium is a useful addition to the treatment armamentarium for BP-CML and that targeting mitochondrial respiration may be a potential therapeutic strategy in aggressive leukemia.