Phosphoinositides in cell proliferation and metabolism

Journal Pre-proof Phosphoinositides in cell proliferation and metabolism Emilio Hirsch, Federico Gulluni, Miriam Martini

PII:

S2212-4926(20)30004-X

DOI:

https://doi.org/10.1016/j.jbior.2020.100693

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JBIOR 100693

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Advances in Biological Regulation

Received Date: 11 November 2019 Revised Date:

16 December 2019

Accepted Date: 16 January 2020

Please cite this article as: Hirsch E, Gulluni F, Martini M, Phosphoinositides in cell proliferation and metabolism, Advances in Biological Regulation, https://doi.org/10.1016/j.jbior.2020.100693. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Phosphoinositides in cell proliferation and metabolism Emilio Hirsch1, Federico Gulluni1 and Miriam Martini1* 1

Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Torino, Torino, Italy

Phosphoinositides (PI) are key players in many trafficking and signaling pathways. Recent advances regarding the synthesis, location and functions of these lipids have improved our understanding of how and when these lipids are generated and what their roles are in physiology and disease. In particular, PI play a central role in the regulation of cell proliferation and metabolism. Here, we will review recent advances in our understanding of PI function, regulation, and importance in different aspects of proliferation and energy metabolism.

Keywords PI3K; Phosphoinositides; mTOR,; cell metabolism; proliferation

Introduction Phosphoinositides (PIs) consist of a family of lipids that act as second messengers regulating many intracellular signalling and subcellular biological processes including cell growth, proliferation and energy metabolism control (Balla, 2013) (Gulluni et al., 2019). Based on the inositol ring structure (Fig. 1), PI can be phosphorylated at hydroxyl positions 3, 4, and 5, which gives rise to seven possible PIs comprising PI(3)P, PI(4)P, PI(5)P, PI(3,4)P2, PI(3,5)P2, PI(4,5)P2 and PI(3,4,5)P3 (Balla, 2013) (Ciraolo et al., 2014). In this review we will focus on 3phosphorilated PIs and their functions in cell division and energy metabolism. PIs can be interconverted by numerous cytosolic enzymes which selectively add or remove phosphates from the inositol ring acting as kinase or phosphatases, respectively. Among the kinases involved in 3-phosphorilated PIs, phosphoinositide 3-kinases (PI3Ks) are the most important players. PI3K can be divided in three classes bases on structure similarities and common substrate specificity (Martini et al., 2014). Class I PI3K mainly recognize PI(4,5)P2 as a substrate, thus resulting in PI(3,4,5)P3 production (Rathinaswamy and Burke, 2019). In some conditions, scaffold proteins, like IQGAP1 organize the different phosphatidylinositol kinases required for the sequential formation of PI(4,5)P2 and PI(3,4,5)P3 (Choi et al., 2018). Given that class I PI3K are mainly activated downstream receptor tyrosine kinase (RTK) and G-protein coupled receptors (GPRC), PI(3,4,5)P3 is particularly abundant at plasma membrane. Conversely, class II PI3K has evolved increased specificity towards PI and PI(4)P as substrate and are responsible of the production of PI(3)P on endosomal vesicles and PI(3,4)P2 at plasma membrane (Gulluni et al., 2019). Lastly, class III PI3K, also referred to VPS34, only recognizes PI as substrate and thus produces PI(3)P on early and late endosomal membrane (Funderburk et al., 2010). Besides PI3Ks, PIKfyve, a FYVE finger-containing phosphoinositide kinase, is also involved in the production of 3-phosphoinositides by catalysing the phosphorylation of PI(3)P on the fifth hydroxyl of the inositol ring and generating PI(3,5)P2 which mainly localizes on endosome carrier vesicles (ECV) and multivesicular bodies (MVB) (Dove et al., 2009). Interconversion among these four 3-phosphoinositide PI is made possible by the phosphatase activity of enzymes that selectively remove 4- or 5-phosphate

from the inositol ring. Particularly, 5-phosphates such as SHIP1/2, INPP5D/J/K and SKIP can convert PI(3,4,5)P3 to PI(3,4)P2 and INPP4A/B can produce PI(3)P by removing the 4-phosphate from PI(3,4)P2 (Balla, 2013). The activity of PI(3,4,5)P3 and PI(3,4)P2 phosphatases has been initially thought to be required to shut down PI3K signaling, however, emerging evidence have now established a direct role for PI(3,4)P2 and PI(4)P in many cellular processes including endo- and exocytosis, cell movement, mTOR pathway repression and cell growth (Gulluni et al., 2019).

3-phosphoinositides in cell division and proliferation Among the other functions, spatiotemporal control of 3-phosphoinosites synthesis has clear impact on cell division and proliferation. PI(3,4,5)P3 has been widely linked to the activation of the AKT-dependent signaling cascade that ultimately result in cell growth, survival and proliferation. Following RTK and GPCR activation and downstream signalling events, activated receptors are internalized and PI(3,4,5)P3 is hydrolysed to prevent receptor hyperactivation and hyperproliferation of cells as seen in many forms of cancer (Sheppard et al., 2012). Besides the canonical RTK/GPCR-AKT pathway, PI(3,4,5,)P3 is also involved in membrane-domain specification during cells division. During mitosis, the assembly of the metaphase spindle defines the axis of cell division (Kapoor, 2017). That axis mainly depends on interactions between astral microtubules with the cell cortex (Siller and Doe, 2009) and among other proteins, the dynein-dynactin motor complexes mediate this interaction. Particularly, during metaphase, dyneindynactin complex localizes to the cell cortex in a PI(3,4,5)P3 dependent manner thus controlling spindle orientation (Toyoshima et al., 2007). Accordingly, lack of class I PI3Ks results in the altered localization of dynactin to the cortex. This alteration can be rescued by the exogenous addition of PI(3,4,5)P3 but not PI(3,4)P2 or PI(4,5)P2, demonstrating a selective requirement of PI(3,4,5)P3 during the definition of cell division plane in mitosis (Toyoshima et al., 2007). To allow proper PI(3,4,5)P3 enrichment at the cell cortex, the class I PI3K lipid product is counter balanced by the expression of the PTEN phosphatase which converts PI(3,4,5)P2 to PI(4,5)P2 thus spatially restricting the localization of PI(3,4,5)P3 in mitosis. In line with that,

depletion of PTEN causes the extension of the PI(3,4,5)P3 domain over the whole cortex and is associated with dynactin mislocalization and spindle orientation defects (Toyoshima et al., 2007). Among the different stimuli that can induce class I PI3K activation, a member of the Rho GTPase family named Cdc42 has been linked to PtdIns(3,4,5)P3 production and the consequently localization of dynactin to the cell cortex (EtienneManneville and Hall, 2002), (Etienne-Manneville, 2006). The junctional adhesion molecule-A (JAM-A) acts upstream Cdc42 and activates Cdc42 itself that stimulates PI3K activation and PtdIns(3,4,5)P3 accumulation at the midcortex, regulating the formation of the cortical actin cytoskeleton and the cortical localization of dynein during mitosis (Tuncay et al., 2015). Accordingly, depletion of Cdc42 during mitosis, suppresses PI3K activation leading to a rapid decrease in cortical dyneindynactin complex accumulation (Mitsushima et al., 2009). In addition to the key role of class I PI3K in defining cell polarity during metaphase (Toyoshima et al., 2007), the PTEN phosphatase has also been involved in controlling the mitotic checkpoint (Gupta et al., 2009) and consequent mitotic progression (Choi et al., 2014). Particularly, phosphorylation of PTEN at Ser-380 causes PTEN association with. Expression of phospho-deficient mutant, but not wild-type PTEN, caused enhanced mitotic exit, suggesting that Ser-380 phosphorylation may play a role in stabilizing PTEN during mitosis (Choi et al., 2014). Differently from PI(3,4,5)P3 that is mainly enriched at plasma membrane, PI(3)P is a well-established membrane determinant of early endosomes, controlling endosomal trafficking and vesicle fusion during autophagy. However, similar to PI(3,4,5)P3, PI(3)P plays a crucial role during mitosis, particularly during the last step of cell division when the two daughter cells need to divide by cutting the intercellular bridge that still keep them together, in a process called abscission (Gulluni et al., 2017b). During late mitosis, PI(3)P positive endosomes travel along the intercellular bridge transporting proteins required for abscission including CHMP4B, one of the key component of the ESCRT machinery involved in the final cut during late cytokinesis (Sagona et al., 2010) (Gulluni et al., 2017b). This source of PI(3)P mainly derives from the catalytic activity of VPS34 (Sagona et al., 2010).

VPS34 non only controls early endosome trafficking but it is also involved in autophagy and cell growth through the mTOR pathway regulation. Interestingly, recent development of inhibitors targeting the catalytic pocket of VPS34 showed decreased cell proliferation and synergize with the mTOR inhibitor everolimus (Jhanwar-Uniyal et al., 2019; Pavel et al., 2018) (Bago et al., 2014) (Ronan et al., 2014). A synergistic effect of VPS34 inhibition in BRAFV600E autophagy-dependent brain tumor cells has been also recently reported (Zahedi et al., 2019). Along this line, loss of VPS34 and its lipid product PI(3)P results in reduced cell proliferation and lethality in mice (Zhou et al., 2011). Similarly to VPS34, the two class II PI3K alpha and beta contribute to PI(3)P levels on early endosomal membranes. Several studies suggested that loss of PI3KC2α and PI3K-C2β catalytic activity is linked to reduced cell proliferation and cell growth (Gulluni et al., 2019) (De Santis et al., 2019), although at least for PI3KC2α, a kinase unrelated function in the control of cell proliferation has been recently reported (Gulluni et al., 2017a). Interestingly, loss of PI3K-C2α in human patients resulted in growth retardation and reduced height, demonstrating a crucial function for this enzyme not only in the proliferation of cultured cells but also in humans (Tiosano et al., 2019). In addition to PI3P production, its hydrolysis is likely required for cell division and particularly cytokinesis completion. In Drosophila S2 cells, removal of the myotubularin mtm gene, the analogous of MTM1 in mammals, that converts PI(3)P into PI, leads to giant and binucleated cells (Ben El Kadhi et al., 2011). Recently, also in mammalian cells, both depletion or overexpression of either myotubularin-related protein 3 (MTMR3) or myotubularin-related protein 4 (MTMR4) results in abnormal midbody morphology and cytokinesis failure (StDenis et al., 2015). A direct function for PI(3,4)P2 in the control of cell division has been never reported so far. However, recent studies suggest that PI3K-C2β derived PI(3,4)P2 acts as a negative regulator of the mTORC1 pathway (Marat et al., 2017), possibly affecting cell growth and survival. In line with that, INPP4B phosphatase, that converts PI(3,4)P2 in PI(3)P has been linked to cancer cell proliferation. Particularly, INPP4B impedes the proliferation and invasiveness of cervical cancer

cells by inhibiting the activation of two downstream molecules of the PI3K pathway, AKT and SGK3 (Chen et al., 2018). High expression of INPP4B was also observed in NPM1-mutated AML. Knockdown of INPP4B repressed cell proliferation in OCI-AML3 cells, whereas recovered INPP4B rescued this inhibitory effect in vitro (Jin et al., 2018). There is debate concerning whether PI(3,4)P2 contributes to AKT and downstream signalling pathway

activation together with PI(3,4,5)P3. If

PI(3,4)P2 acts as a positive effector, INPP4B would be a negative regulator of PI3K signaling, and there is some evidence to support this. In line with that, in PTEN-null triple-negative breast cancer cell lines, it has been shown that silencing INPP4B decreased basal phospho-AKT and cellular proliferation, and in most cases sensitized cells to class I PI3K inhibitors (Reed and Shokat, 2017). Conversely, overexpression of INPP4B desensitized cells to PI3K inhibitors in a phosphatase activity-dependent manner (Reed and Shokat, 2017). Recent evidence also showed that INPP4B inhibits PI(3,4)P2-mediated AKT activation in thyroid, breast and prostate cancer. On the contrary, INPP4B expression is increased in acute myeloid leukaemia (AML), melanoma and colon cancer where it paradoxically promotes cell proliferation, transformation and/or drug resistance (Li Chew et al., 2015) (Gasser et al., 2014) (Guo et al., 2016) (Woolley et al., 2015) (Rodgers et al., 2017)]. Further studies are required to better understand the precise function of PI(3,4)P2 in cell growth and proliferation and the molecular mechanisms behind that.

Energy metabolism mTOR is a serine/threonine protein kinase in the PI3K-related kinase (PIKK) family that forms the catalytic subunit of two distinct protein complexes, known as mTOR Complex 1 (mTORC1) and 2 (mTORC2). The mTORC1 (mechanistic target of rapamycin complex 1) complex is charachterized by three core components: mTOR, Raptor (regulatory protein associated with mTOR), and mLST8 (mammalian lethal with Sec13 protein 8) (Saxton and Sabatini, 2017). Raptor facilitates substrate recruitment to mTORC1 through binding to the TOR signaling (TOS) motif found on several canonical

mTORC1 substrates (Nojima et al., 2003) and it is required for the correct subcellular localization of mTORC1. mLST8 associates with the catalytic domain of mTORC1 stabilizing the kinase activation loop (Yang and Chi, 2013), nevertheless several genetic studies suggest that mLST8 is dispensable for mTORC1 essential functions (Guertin et al., 2006). In addition to these three core components, mTORC1 also contains the two inhibitory subunits PRAS40 (proline-rich Akt substrate of 40 kDa) (Sancak et al., 2007; Vander Haar et al., 2007; Wang et al., 2007) and DEPTOR (DEP domain containing mTOR interacting protein) (Peterson et al., 2009). mTORC1 garners much attention as a signaling hub that coordinates input from growth-factor receptors and nutrients availability with metabolism and cell growth and proliferation. mTOR signaling is essential for proper metabolic regulation by coordinating anabolic and catabolic metabolism at the cellular and organismal level. The activation of mTOR signaling is associated with several physiological outcomes, indicating that the proper modulation of mTOR signaling in response to changing environmental conditions is crucial. mTORC1 positively regulates cell growth and proliferation by promoting many anabolic processes, including biosynthesis of proteins, lipids and organelles, and by limiting catabolic processes such as autophagy. mTORC1 activation is mediated by class I PI3K signaling during times of nutrient abundance. Active mTORC1 phosphorylates and thereby inactivates the serine/threonine kinase ULK1, resulting in suppression of starvation-induced autophagy (F et al., 2013). The localization of mTORC1 to the lysosome is critical for its activation. Rags and the vacuolar ATPase are also important regulators of TORC1 activation and lysosomal localization (Sancak et al., 2010). The loss of PtdIns(3,5)P2 disrupts the localization of TORC1 to the lysosome in both yeast and mammalian systems potentially via direct binding of Raptor to this lipid (Jin et al., 2014). The loss of either PtdIns(3)P or PtdIns(3,5)P2 has been associated with reduced TORC1 activity in both yeast and mammalian systems (Jin et al., 2014). The precise molecular events by which these lipids modulate the activation cascade of TORC1 are still under intense investigation.

In cancer, mTORC1 functions as a downstream effector for many frequently mutated oncogenic pathways including the PI3K/Akt pathway as well as the Ras/Raf/Mek/Erk (MAPK) pathway, resulting in mTORC1 hyperactivation in a high percentage of human cancers. The most important regulator of mTORC1 activity is the tuberous sclerosis complex (TSC), which is a heterodimer that comprises TSC1 (hamartin) and TSC2 (tuberin) subunits. TSC1/2 functions as a GTPase-activating protein (GAP) for the small Ras-related GTPase Rheb (Ras homolog enriched in brain) that directly stimulates mTORC1 activity (Long et al., 2005). The exact mechanism by which Rheb activates mTORC1 remains to be determined. TSC1/2 is GAP of Rheb and negatively regulates mTORC1 signaling by converting Rheb into GDP-bound status (Inoki et al., 2003). Consistent with a role of TSC1/2 in the negative regulation of mTORC1, inactivating mutations or loss of heterozygosity of TSC1/2 give rise to tuberous sclerosis, a disease associated with the presence of numerous benign tumors. Furthermore, the tumor suppressors TP53 and LKB1 are negative regulators of mTORC1 upstream of TSC1 and TSC2 tumor suppressors. Several components of the nutrient sensing input to mTORC1 have also been implicated in cancer progression, including all three subunits of the GATOR1 complex, which are mutated with low frequency in glioblastoma (Bar-Peled et al., 2013), as well as RagC, which was recently found to be mutated at high frequency (~18%) in follicular lymphoma (Okosun et al., 2016). Additionally, mutations in the gene encoding folliculin (FLCN) are the causative lesion in the Birt-Hogg-Dube hereditary cancer syndrome (Nickerson et al., 2002), which manifests similarly to TSC. Finally, mutations in MTOR itself are also found in a variety of cancer subtypes, consistent with a role for mTOR in cancer (Saxton and Sabatini, 2017). Various inhibitors against mTOR have been designed and tested for cancer treatment, with either specific selectivity against one or the other complex or together with class I PI3K targeting. Several clinical trials concluded that available drugs blocking this pathway are unsatisfactory in most of the malignancies assessed. More studies are needed to better define where and when mTOR targeting is beneficial in cancer therapy (Jhanwar-Uniyal et al., 2019).

Conclusions Since their discovery as major modulators of insulin action, PI have been under intensive investigation (Yang and Chi, 2013). Molecules involved in PI signaling pathways are considered attractive drug targets for human disease therapy. Thus, there is an absolute need to precisely define the spatial organization of PIs and their metabolism l. Further studies using PI probes (Balla and Varnai, 2009) will make major strides in unraveling the contribution of each PI-metabolizing enzyme to PI turnover in a specific cellular compartment. Only with such knowledge can PIs be successfully manipulated for therapy.

Acknowledgments This work was supported by Associazione Italiana per la Ricerca sul Cancro (AIRC21875). FG (19421) is supported by AIRC/FIRC fellowships. References Bago, R., Malik, N., Munson, M.J., Prescott, A.R., Davies, P., Sommer, E., Shpiro, N., Ward, R., Cross, D., Ganley, I.G., et al. (2014). Characterization of VPS34-IN1, a selective inhibitor of Vps34, reveals that the phosphatidylinositol 3-phosphate-binding SGK3 protein kinase is a downstream target of class III phosphoinositide 3-kinase. Biochem J 463, 413-427. Balla, T. (2013). Phosphoinositides: tiny lipids with giant impact on cell regulation. Physiol Rev 93, 1019-1137. Balla, T., and Varnai, P. (2009). Visualization of cellular phosphoinositide pools with GFP-fused protein-domains. Curr Protoc Cell Biol Chapter 24, Unit 24 24. Bar-Peled, L., Chantranupong, L., Cherniack, A.D., Chen, W.W., Ottina, K.A., Grabiner, B.C., Spear, E.D., Carter, S.L., Meyerson, M., and Sabatini, D.M. (2013). A Tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1. Science 340, 11001106. Ben El Kadhi, K., Roubinet, C., Solinet, S., Emery, G., and Carreno, S. (2011). The inositol 5-phosphatase dOCRL controls PI(4,5)P2 homeostasis and is necessary for cytokinesis. Current biology : CB 21, 1074-1079.

Chen, Y., Sun, Z., Qi, M., Wang, X., Zhang, W., Chen, C., Liu, J., and Zhao, W. (2018). INPP4B restrains cell proliferation and metastasis via regulation of the PI3K/AKT/SGK pathway. J Cell Mol Med 22, 2935-2943. Choi, B.H., Pagano, M., and Dai, W. (2014). Plk1 protein phosphorylates phosphatase and tensin homolog (PTEN) and regulates its mitotic activity during the cell cycle. The Journal of biological chemistry 289, 14066-14074. Choi, S., Houdek, X., and Anderson, R.A. (2018). Phosphoinositide 3-kinase pathways and autophagy require phosphatidylinositol phosphate kinases. Adv Biol Regul 68, 31-38. Ciraolo, E., Gulluni, F., and Hirsch, E. (2014). Methods to measure the enzymatic activity of PI3Ks. Methods in enzymology 543, 115-140. De Santis, M.C., Gulluni, F., Campa, C.C., Martini, M., and Hirsch, E. (2019). Targeting PI3K signaling in cancer: Challenges and advances. Biochim Biophys Acta Rev Cancer 1871, 361-366. Dove, S.K., Dong, K., Kobayashi, T., Williams, F.K., and Michell, R.H. (2009). Phosphatidylinositol 3,5-bisphosphate and Fab1p/PIKfyve underPPIn endo-lysosome function. Biochem J 419, 1-13. Etienne-Manneville, S. (2006). In vitro assay of primary astrocyte migration as a tool to study Rho GTPase function in cell polarization. Methods in enzymology 406, 565-578. Etienne-Manneville, S., and Hall, A. (2002). Rho GTPases in cell biology. Nature 420, 629-635. F, O.F., Rusten, T.E., and Stenmark, H. (2013). Phosphoinositide 3-kinases as accelerators and brakes of autophagy. FEBS J 280, 6322-6337. Funderburk, S.F., Wang, Q.J., and Yue, Z. (2010). The Beclin 1-VPS34 complex--at the crossroads of autophagy and beyond. Trends Cell Biol 20, 355-362. Gasser, J.A., Inuzuka, H., Lau, A.W., Wei, W., Beroukhim, R., and Toker, A. (2014). SGK3 mediates INPP4B-dependent PI3K signaling in breast cancer. Molecular cell 56, 595-607. Guertin, D.A., Stevens, D.M., Thoreen, C.C., Burds, A.A., Kalaany, N.Y., Moffat, J., Brown, M., Fitzgerald, K.J., and Sabatini, D.M. (2006). Ablation in mice of the mTORC components raptor, rictor, or mLST8 reveals that mTORC2 is required for signaling to Akt-FOXO and PKCalpha, but not S6K1. Developmental cell 11, 859-871. Gulluni, F., De Santis, M.C., Margaria, J.P., Martini, M., and Hirsch, E. (2019). Class II PI3K Functions in Cell Biology and Disease. Trends Cell Biol 29, 339-359. Gulluni, F., Martini, M., De Santis, M.C., Campa, C.C., Ghigo, A., Margaria, J.P., Ciraolo, E., Franco, I., Ala, U., Annaratone, L., et al. (2017a). Mitotic Spindle Assembly and Genomic Stability in Breast Cancer Require PI3KC2alpha Scaffolding Function. Cancer cell 32, 444-459 e447. Gulluni, F., Martini, M., and Hirsch, E. (2017b). Cytokinetic Abscission: Phosphoinositides and ESCRTs Direct the Final Cut. J Cell Biochem 118, 3561-3568. Guo, S.T., Chi, M.N., Yang, R.H., Guo, X.Y., Zan, L.K., Wang, C.Y., Xi, Y.F., Jin, L., Croft, A., Tseng, H.Y., et al. (2016). INPP4B is an oncogenic regulator in human colon cancer. Oncogene 35, 3049-3061.

Gupta, A., Yang, Q., Pandita, R.K., Hunt, C.R., Xiang, T., Misri, S., Zeng, S., Pagan, J., Jeffery, J., Puc, J., et al. (2009). Cell cycle checkpoint defects contribute to genomic instability in PTEN deficient cells independent of DNA DSB repair. Cell Cycle 8, 2198-2210. Inoki, K., Zhu, T., and Guan, K.L. (2003). TSC2 mediates cellular energy response to control cell growth and survival. Cell 115, 577-590. Jhanwar-Uniyal, M., Wainwright, J.V., Mohan, A.L., Tobias, M.E., Murali, R., Gandhi, C.D., and Schmidt, M.H. (2019). Diverse signaling mechanisms of mTOR complexes: mTORC1 and mTORC2 in forming a formidable relationship. Adv Biol Regul 72, 51-62. Jin, H., Yang, L., Wang, L., Yang, Z., Zhan, Q., Tao, Y., Zou, Q., Tang, Y., Xian, J., Zhang, S., et al. (2018). INPP4B promotes cell survival via SGK3 activation in NPM1-mutated leukemia. J Exp Clin Cancer Res 37, 8. Jin, N., Mao, K., Jin, Y., Tevzadze, G., Kauffman, E.J., Park, S., Bridges, D., Loewith, R., Saltiel, A.R., Klionsky, D.J., et al. (2014). Roles for PI(3,5)P2 in nutrient sensing through TORC1. Mol Biol Cell 25, 1171-1185. Kapoor, T.M. (2017). Metaphase Spindle Assembly. Biology (Basel) 6. Li Chew, C., Lunardi, A., Gulluni, F., Ruan, D.T., Chen, M., Salmena, L., Nishino, M., Papa, A., Ng, C., Fung, J., et al. (2015). In Vivo Role of INPP4B in Tumor and Metastasis Suppression through Regulation of PI3K-AKT Signaling at Endosomes. Cancer discovery 5, 740-751. Long, X., Lin, Y., Ortiz-Vega, S., Yonezawa, K., and Avruch, J. (2005). Rheb binds and regulates the mTOR kinase. Current biology : CB 15, 702-713. Marat, A.L., Wallroth, A., Lo, W.T., Muller, R., Norata, G.D., Falasca, M., Schultz, C., and Haucke, V. (2017). mTORC1 activity repression by late endosomal phosphatidylinositol 3,4-bisphosphate. Science 356, 968-972. Martini, M., De Santis, M.C., Braccini, L., Gulluni, F., and Hirsch, E. (2014). PI3K/AKT signaling pathway and cancer: an updated review. Annals of medicine 46, 372-383. Mitsushima, M., Toyoshima, F., and Nishida, E. (2009). Dual role of Cdc42 in spindle orientation control of adherent cells. Mol Cell Biol 29, 2816-2827. Nickerson, M.L., Warren, M.B., Toro, J.R., Matrosova, V., Glenn, G., Turner, M.L., Duray, P., Merino, M., Choyke, P., Pavlovich, C.P., et al. (2002). Mutations in a novel gene lead to kidney tumors, lung wall defects, and benign tumors of the hair follicle in patients with the Birt-Hogg-Dube syndrome. Cancer cell 2, 157-164. Nojima, H., Tokunaga, C., Eguchi, S., Oshiro, N., Hidayat, S., Yoshino, K., Hara, K., Tanaka, N., Avruch, J., and Yonezawa, K. (2003). The mammalian target of rapamycin (mTOR) partner, raptor, binds the mTOR substrates p70 S6 kinase and 4E-BP1 through their TOR signaling (TOS) motif. The Journal of biological chemistry 278, 15461-15464. Okosun, J., Wolfson, R.L., Wang, J., Araf, S., Wilkins, L., Castellano, B.M., Escudero-Ibarz, L., Al Seraihi, A.F., Richter, J., Bernhart, S.H., et al. (2016). Recurrent mTORC1-activating RRAGC mutations in follicular lymphoma. Nat Genet 48, 183-188. Pavel, M., Renna, M., Park, S.J., Menzies, F.M., Ricketts, T., Fullgrabe, J., Ashkenazi, A., Frake, R.A., Lombarte, A.C., Bento, C.F., et al. (2018).

Contact inhibition controls cell survival and proliferation via YAP/TAZautophagy axis. Nature communications 9, 2961. Peterson, T.R., Laplante, M., Thoreen, C.C., Sancak, Y., Kang, S.A., Kuehl, W.M., Gray, N.S., and Sabatini, D.M. (2009). DEPTOR is an mTOR inhibitor frequently overexpressed in multiple myeloma cells and required for their survival. Cell 137, 873-886. Rathinaswamy, M.K., and Burke, J.E. (2019). Class I phosphoinositide 3kinase (PI3K) regulatory subunits and their roles in signaling and disease. Adv Biol Regul, 100657. Reed, D.E., and Shokat, K.M. (2017). INPP4B and PTEN Loss Leads to PI3,4-P2 Accumulation and Inhibition of PI3K in TNBC. Mol Cancer Res 15, 765-775. Rodgers, S.J., Ferguson, D.T., Mitchell, C.A., and Ooms, L.M. (2017). Regulation of PI3K effector signalling in cancer by the phosphoinositide phosphatases. Biosci Rep 37. Ronan, B., Flamand, O., Vescovi, L., Dureuil, C., Durand, L., Fassy, F., Bachelot, M.F., Lamberton, A., Mathieu, M., Bertrand, T., et al. (2014). A highly potent and selective Vps34 inhibitor alters vesicle trafficking and autophagy. Nat Chem Biol 10, 1013-1019. Sagona, A.P., Nezis, I.P., Pedersen, N.M., Liestol, K., Poulton, J., Rusten, T.E., Skotheim, R.I., Raiborg, C., and Stenmark, H. (2010). PtdIns(3)P controls cytokinesis through KIF13A-mediated recruitment of FYVE-CENT to the midbody. Nat Cell Biol 12, 362-371. Sancak, Y., Bar-Peled, L., Zoncu, R., Markhard, A.L., Nada, S., and Sabatini, D.M. (2010). Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell 141, 290-303. Sancak, Y., Thoreen, C.C., Peterson, T.R., Lindquist, R.A., Kang, S.A., Spooner, E., Carr, S.A., and Sabatini, D.M. (2007). PRAS40 is an insulinregulated inhibitor of the mTORC1 protein kinase. Molecular cell 25, 903915. Saxton, R.A., and Sabatini, D.M. (2017). mTOR Signaling in Growth, Metabolism, and Disease. Cell 168, 960-976. Sheppard, K., Kinross, K.M., Solomon, B., Pearson, R.B., and Phillips, W.A. (2012). Targeting PI3 kinase/AKT/mTOR signaling in cancer. Crit Rev Oncog 17, 69-95. Siller, K.H., and Doe, C.Q. (2009). Spindle orientation during asymmetric cell division. Nat Cell Biol 11, 365-374. St-Denis, N., Gupta, G.D., Lin, Z.Y., Gonzalez-Badillo, B., Pelletier, L., and Gingras, A.C. (2015). Myotubularin-related proteins 3 and 4 interact with polo-like kinase 1 and centrosomal protein of 55 kDa to ensure proper abscission. Molecular & cellular proteomics : MCP 14, 946-960. Tiosano, D., Baris, H.N., Chen, A., Hitzert, M.M., Schueler, M., Gulluni, F., Wiesener, A., Bergua, A., Mory, A., Copeland, B., et al. (2019). Mutations in PIK3C2A cause syndromic short stature, skeletal abnormalities, and cataracts associated with ciliary dysfunction. PLoS Genet 15, e1008088.

Toyoshima, F., Matsumura, S., Morimoto, H., Mitsushima, M., and Nishida, E. (2007). PtdIns(3,4,5)P3 regulates spindle orientation in adherent cells. Developmental cell 13, 796-811. Tuncay, H., Brinkmann, B.F., and Ebnet, K. (2015). Mitotic spindle orientation: JAM-A can fix it. Cell Cycle 14, 3773-3774. Vander Haar, E., Lee, S.I., Bandhakavi, S., Griffin, T.J., and Kim, D.H. (2007). Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40. Nat Cell Biol 9, 316-323. Wang, P., Yang, X., Wu, P., Zhang, J., Sato, T., Yamagata, S., and Yamagata, T. (2007). GM3 signals regulating TNF-alpha expression are mediated by Rictor and Arhgdib in mouse melanoma B16 cells. Oncology 73, 430-438. Woolley, J.F., Dzneladze, I., and Salmena, L. (2015). Phosphoinositide signaling in cancer: INPP4B Akt(s) out. Trends Mol Med 21, 530-532. Yang, K., and Chi, H. (2013). Tuning mTOR activity for immune balance. J Clin Invest 123, 5001-5004. Zahedi, S., Fitzwalter, B.E., Morin, A., Grob, S., Desmarais, M., Nellan, A., Green, A.L., Vibhakar, R., Hankinson, T.C., Foreman, N.K., et al. (2019). Effect of early-stage autophagy inhibition in BRAF(V600E) autophagydependent brain tumor cells. Cell Death Dis 10, 679. Zhou, X., Takatoh, J., and Wang, F. (2011). The mammalian class 3 PI3K (PIK3C3) is required for early embryogenesis and cell proliferation. PLoS One 6, e16358.

E.H. is a co-founder of Kither Biotech, a company involved in the development of PI3K inhibitors. The other authors declare no conflict of interest.