Combined GLUT1 and OXPHOS inhibition eliminates acute myeloid leukemia cells by restraining their metabolic plasticity.

Acute myeloid leukemia (AML) is initiated and propagated by leukemia stem cells (LSCs), a self-renewing population of leukemia cells responsible for therapy resistance. Hence, there is an urgent need to identify new therapeutic opportunities targeting LSCs. Here, we performed an in vivo CRISPR knockout screen to identify potential therapeutic targets by interrogating cell surface dependencies of LSCs. The facilitated glucose transporter type 1 (GLUT1) emerged as a critical in vivo metabolic dependency for LSCs in a murine MLL::AF9-driven model of AML. GLUT1 disruption by genetic ablation or pharmacological inhibition led to suppression of leukemia progression and improved survival of mice that received transplantation with LSCs. Metabolic profiling revealed that Glut1 inhibition suppressed glycolysis, decreased levels of tricarboxylic acid cycle intermediates and increased the levels of amino acids. This metabolic reprogramming was accompanied by an increase in autophagic activity and apoptosis. Moreover, Glut1 disruption caused transcriptional, morphological, and immunophenotypic changes, consistent with differentiation of AML cells. Notably, dual inhibition of GLUT1 and oxidative phosphorylation (OXPHOS) exhibited synergistic antileukemic effects in the majority of tested primary AML patient samples through restraining of their metabolic plasticity. In particular, RUNX1-mutated primary leukemia cells displayed striking sensitivity to the combination treatment compared with normal CD34+ bone marrow and cord blood cells. Collectively, our study reveals a GLUT1 dependency of murine LSCs in the bone marrow microenvironment and demonstrates that dual inhibition of GLUT1 and OXPHOS is a promising therapeutic approach for AML.

IRSp53 is a novel interactor of SHIP2: A role of the actin binding protein Mena in their cellular localization in breast cancer cells.

A tight control of the machineries regulating membrane bending and actin dynamics is very important for the generation of membrane protrusions, which are crucial for cell migration and invasion. Protein/protein and protein/phosphoinositides complexes assemble and disassemble to coordinate these mechanisms, the scaffold properties of the involved proteins playing a prominent role in this organization. The PI 5-phosphatase SHIP2 is a critical enzyme modulating PI(3,4,5)P3, PI(4,5)P2 and PI(3,4)P2 content in the cell. The scaffold properties of SHIP2 contribute to the specific targeting or retention of the protein in particular subcellular domains. Here, we identified IRSp53 as a new binding interactor of SHIP2 proline-rich domain. Both proteins are costained in HEK293T cells protrusions, upon transfection. We showed that the SH3-binding polyproline motif recognized by IRSp53 in SHIP2 is different from the regions targeted by other PRR binding partners i.e., CIN85, ITSN or even Mena a common interactor of both SHIP2 and IRSp53. We presented evidence that IRSp53 phosphorylation on S366 did not influence its interaction with SHIP2 and that Mena is not necessary for the association of SHIP2 with IRSp53 in MDA-MB-231 cells. The absence of Mena in MDA-MB-231 cells decreased the intracellular content in F-actin and modified the subcellular localization of SHIP2 and IRSp53 by increasing their relative content at the plasma membrane. Together our data suggest that SHIP2, through interaction with the cell protrusion regulators IRSp53 and Mena, participate to the formation of multi-protein complexes. This ensures the appropriate modulations of PIs which is important for regulation of membrane dynamics.

Phosphoinositide 5-phosphatases SKIP and SHIP2 in ruffles, the endoplasmic reticulum and the nucleus: An update.

Phosphoinositides (PIs) are phosphorylated derivatives of phosphatidylinositol. They act as signaling molecules linked to essential cellular mechanisms in eukaryotic cells, such as cytoskeleton organization, mitosis, polarity, migration or invasion. PIs are phosphorylated and dephosphorylated by a large number of PI kinases and PI phosphatases acting at the 5-, 4- and 3- position of the inositol ring. PI 5-phosphatases i.e. OCRL, INPP5B, SHIP1/2, Synaptojanin 1/2, INPP5E, INPP5J, SKIP (INPP5K) are enzymes that dephosphorylate the 5-phosphate position of PIs. Several human genetic diseases such as the Lowe syndrome, some congenital muscular dystrophy and opsismodysplasia are due to mutations in PI phosphatases, resulting in loss-of-function. The PI phosphatases are also up or down regulated in several human cancers such as glioblastoma or breast cancer. Their cellular localization, that is dynamic and varies in response to stimuli, is an important issue to understand function. This is the case for two members of the PI 5-phosphatase SKIP and SHIP2. Both enzymes are in ruffles, plasma membranes, the endoplasmic reticulum, a situation that is unique for SKIP, and the nucleus. Following localization, PI 5-phosphatases act on specific cellular pools of PIs, which in turn interact with target proteins. Nuclear PIs have emerged as regulators of genome functions in different area of cell signaling. They often localize to nuclear speckles, as do several PI metabolizing kinases and phosphatases. We asked whether SKIP and SHIP2 could have an impact on nuclear PI(4,5)P2. In two glioblastoma cell models, lowering SKIP expression had an impact on nuclear PI(4,5)P2. In a model of SHIP2 deletion in MCF-7 cells, no change in nuclear PI(4,5)P2 was observed. Finally, we present evidence of an anti-tumoral role of SKIP in vivo, in xenografts using as model U87shSKIP cells.

Lipid phosphatases SKIP and SHIP2 regulate fibronectin-dependent cell migration in glioblastoma.

Cell migration is an important process that occurs during development and has also been linked to the motility of cancer cells. Cytoskeleton reorganization takes place in the migration process leading to lamellipodia formation. Understanding the molecular underpinnings of cell migration is particularly important in studies of glioblastoma, a highly invasive and aggressive cancer type. Two members of the phosphoinositide 5-phosphatase family, SKIP and SHIP2, have been associated with cell migration in glioblastoma; however, the precise role these enzymes play in the process-and whether they work in concert-remains unclear. Here, we compared phosphoinositide 5-phosphatases expression in glioblastoma primary cells and cell lines and showed that SHIP2 and SKIP expression greatly varies between different cell types, while OCRL, another phosphoinositide 5-phosphatase, is constitutively expressed. Upon adhesion of U-251 MG cells to fibronectin, SHIP2, SKIP, and PI(4,5)P2 colocalized in membrane ruffles. Upregulation of PI(4,5)P2 was observed in SKIP-depleted U-251 MG cells compared to control cells, but only when cells were adhered to fibronectin. Both PTEN-deficient (U-251) and PTEN-containing (LN229) glioblastoma cells showed a decrease in cell migration velocity in response to SKIP downregulation. Moreover, a SHIP2 catalytic inhibitor lowered cell migration velocity in the U-251 MG cell line. We conclude that integrin activation in U-251 cells leads to colocalization of both SKIP and SHIP2 in ruffles, where they act as potential drivers of cell migration. Depending on their expression levels in glioblastoma, phosphoinositide 5-phosphatases could cooperate and synergize in the regulation of cell migration and adhesion.

The impact of phosphoinositide 5-phosphatases on phosphoinositides in cell function and human disease.

Phosphoinositides (PIs) are recognized as major signaling molecules in many different functions of eukaryotic cells. PIs can be dephosphorylated by multiple phosphatase activities at the 5-, 4-, and 3- positions. Human PI 5-phosphatases belong to a family of 10 members. Except for inositol polyphosphate 5-phosphatase A, they all catalyze the dephosphorylation of PI(4,5)P2 and/or PI(3,4,5)P3 at the 5- position. PI 5-phosphatases thus directly control the levels of PI(3,4,5)P3 and participate in the fine-tuning regulatory mechanisms of PI(3,4)P2 and PI(4,5)P2 Second messenger functions have been demonstrated for PI(3,4)P2 in invadopodium maturation and lamellipodia formation. PI 5-phosphatases can use several substrates on isolated enzymes, and it has been challenging to establish their real substrate in vivo. PI(4,5)P2 has multiple functions in signaling, including interacting with scaffold proteins, ion channels, and cytoskeleton proteins. PI 5-phosphatase isoenzymes have been individually implicated in human diseases, such as the oculocerebrorenal syndrome of Lowe, through mechanisms that include lipid control. Oncogenic and tumor-suppressive functions of PI 5-phosphatases have also been reported in different cell contexts. The mechanisms responsible for genetic diseases and for oncogenic or tumor-suppressive functions are not fully understood. The regulation of PI 5-phosphatases is thus crucial in understanding cell functions.

The inositol phosphatase SHIP2 enables sustained ERK activation downstream of FGF receptors by recruiting Src kinases.

Sustained activation of extracellular signal-regulated kinase (ERK) drives pathologies caused by mutations in fibroblast growth factor receptors (FGFRs). We previously identified the inositol phosphatase SHIP2 (also known as INPPL1) as an FGFR-interacting protein and a target of the tyrosine kinase activities of FGFR1, FGFR3, and FGFR4. We report that loss of SHIP2 converted FGF-mediated sustained ERK activation into a transient signal and rescued cell phenotypes triggered by pathologic FGFR-ERK signaling. Mutant forms of SHIP2 lacking phosphoinositide phosphatase activity still associated with FGFRs and did not prevent FGF-induced sustained ERK activation, demonstrating that the adaptor rather than the catalytic activity of SHIP2 was required. SHIP2 recruited Src family kinases to the FGFRs, which promoted FGFR-mediated phosphorylation and assembly of protein complexes that relayed signaling to ERK. SHIP2 interacted with FGFRs, was phosphorylated by active FGFRs, and promoted FGFR-ERK signaling at the level of phosphorylation of the adaptor FRS2 and recruitment of the tyrosine phosphatase PTPN11. Thus, SHIP2 is an essential component of canonical FGF-FGFR signal transduction and a potential therapeutic target in FGFR-related disorders.

Inhibition of SHIP2 activity inhibits cell migration and could prevent metastasis in breast cancer cells.

Metastasis of breast cancer cells to distant organs is responsible for ∼50% of breast cancer-related deaths in women worldwide. SHIP2 (also known as INPPL1) is a phosphoinositide 5-phosphatase for phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2]. Here we show, through depletion of SHIP2 in triple negative MDA-MB-231 cells and the use of SHIP2 inhibitors, that cell migration appears to be positively controlled by SHIP2. The effect of SHIP2 on migration, as observed in MDA-MB-231 cells, appears to be mediated by PI(3,4)P2. Adhesion on fibronectin is always increased in SHIP2-depleted cells. Apoptosis measured in MDA-MB-231 cells is also increased in SHIP2-depleted cells as compared to control cells. In xenograft mice, SHIP2-depleted MDA-MB-231 cells form significantly smaller tumors than those formed by control cells and less metastasis is detected in lung sections. Our data reveal a general role for SHIP2 in the control of cell migration in breast cancer cells and a second messenger role for PI(3,4)P2 in the migration mechanism. In MDA-MB-231 cells, SHIP2 has a function in apoptosis in cells incubated in vitro and in mouse tumor-derived cells, which could account for its role on tumor growth determined in vivo.

Fibroblasts derived from patients with opsismodysplasia display SHIP2-specific cell migration and adhesion defects.

The SH2 domain containing inositol phosphatase 2 (SHIP2) dephosphorylates PI(3,4,5)P3 to generate PI(3,4)P2, a lipid involved in the control of cell migration and adhesion. The INPPL1 gene that encodes SHIP2 has been found to be mutated in several cases of opsismodysplasia (OPS), a rare autosomal recessive chondrodysplasia characterized by growth plate defects and delayed bone maturation. Reported mutations often result in premature stop codons or missense mutations in SHIP2 catalytic domain. SHIP2 biochemical properties are known from studies in cancer cells; its role in endochondral ossification is unknown. Here, we report two novel mutations in the INPPL1 gene and show that cell migration is very much decreased in fibroblasts derived from three OPS patients as compared with control individuals. In contrast, cell adhesion on fibronectin is increased in OPS fibroblasts. An inhibitory effect on migration was also observed when normal fibroblasts were incubated in the presence of a SHIP2 competitive inhibitor. We conclude that both migration and adhesion are very much disrupted in OPS-derived fibroblasts. It is suggested that signaling events linked to migration and particularly to adhesion, which are lost in OPS patients, would prevent normal endochondral ossification.

The SHIP2 interactor Myo1c is required for cell migration in 1321 N1 glioblastoma cells.

The phosphoinositide 5-phosphatases consist of several enzymes that have been shown to modulate cell migration and invasion. SHIP2, one family member, is known to interact with growth factor receptors and cytoskeletal proteins. In a human model of glioblastoma 1321 N1 cells, we recently identified Myo1c as a new interactor of SHIP2. This was shown in a complex of proteins also containing filamin A. We show here that SHIP2 localization at lamellipodia and ruffles is impaired in Myo1c depleted cells. In the absence of Myo1c, N1 cells tend to associate to form clusters. Cell migration is very much reduced in Myo1c depleted cells, concomitantly with a decrease in FAK Tyr397 phosphorylation, focal adhesion length and PI(4,5)P2 immunostaining. In N1 cells, Myo1c is thus important for lamellipodia formation to assemble a protein complex containing SHIP2 to facilitate cell migration.

New Functions of the Inositol Polyphosphate 5-Phosphatases in Cancer.

Inositol polyphosphate 5-phosphatases act on inositol phosphates and phosphoinositides as substrates. They are 10 different isoenzymes and several splice variants in the human genome that are involved in a series of human pathologies such as the Lowe syndrome, the Joubert and MORM syndromes, breast cancer, glioblastoma, gastric cancer and several other type of cancers. Inositol 5-phosphatases can be amplified in human cancer cells, whereas the 3- and 4- phosphatase tumor suppressor PTEN and INPP4B, repectively are often repressed or deleted. The inositol 5-phosphatases are critically involved in a complex network of higly regulated phosphoinositides, affecting the lipid content of PI(3, 4, 5)P3, PI(4, 5)P2 and PI(3, 4)P2. This has an impact on the normal behavior of many intracellular target proteins e.g. protein kinase B (PKB/Akt) or actin binding proteins and final biological responses. The production of PI(3, 4P)2 by dephosphorylation of the substrate PI(3, 4, 5)P3 is particularly important as it produces a new signal messenger in the control of cell migration, invasion and endocytosis. New inhibitors/activators of inositol 5- phosphatases have recently been identified for the possible control of their activity in several human pathologies such as inflamation and cancer.

SHIP2 controls plasma membrane PI(4,5)P2 thereby participating in the control of cell migration in 1321 N1 glioblastoma cells.

Phosphoinositides, particularly phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], are recognized by SHIP2 (also known as INPPL1) a member of the inositol polyphosphate 5-phosphatase family. SHIP2 dephosphorylates PI(3,4,5)P3 to form PI(3,4)P2; the latter interacts with specific target proteins (e.g. lamellipodin). Although the preferred SHIP2 substrate is PI(3,4,5)P3, PI(4,5)P2 can also be dephosphorylated by this enzyme to phosphatidylinositol 4-phosphate (PI4P). Through depletion of SHIP2 in the glioblastoma cell line 1321 N1, we show that SHIP2 inhibits cell migration. In different glioblastoma cell lines and primary cultures, SHIP2 staining at the plasma membrane partly overlaps with PI(4,5)P2 immunoreactivity. PI(4,5)P2 was upregulated in SHIP2-deficient N1 cells as compared to control cells; in contrast, PI4P was very much decreased in SHIP2-deficient cells. Therefore, SHIP2 controls both PI(3,4,5)P3 and PI(4,5)P2 levels in intact cells. In 1321 N1 cells, the PI(4,5)P2-binding protein myosin-1c was identified as a new interactor of SHIP2. Regulation of PI(4,5)P2 and PI4P content by SHIP2 controls 1321 N1 cell migration through the organization of focal adhesions. Thus, our results reveal a new role of SHIP2 in the control of PI(4,5)P2, PI4P and cell migration in PTEN-deficient glioblastoma 1321 N1 cells.

Inositol(1,4,5)P3 3-kinase isoenzymes: Catalytic properties and importance of targeting to F-actin to understand function.

Inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) 3-kinases (Itpks) catalyze the phosphorylation of inositol(1,4,5)trisphosphate into inositol(1,3,4,5)tetrakisphosphate (Ins(1,3,4,5)P4). Three isoenzymes Itpka/b and c have been identified in human, rat and mouse. They share a catalytic domain relatively well conserved at the C-terminal end and a quite isoenzyme specific regulatory domain at the N-terminal end of the protein. Activity determined in cell homogenates with Ins(1,4,5)P3 and ATP as substrate is generally very low compared to Ins(1,4,5)P3 5-phosphatase, except in a few tissues such as brain, testis, thymus or intestine. Activity is very much Ca(2+) sensitive and increased in the presence of Ca(2+)/calmodulin (CaM) as compared to EGTA alone. When challenged after receptor activation, activity could be further activated several fold, e.g. in rat brain cortical slices stimulated by carbachol or in human astrocytoma cells stimulated by purinergic agonists. Two of the three isoenzymes show an unexpected cytoskeletal localization for Itpka/b or at the leading edge for Itpkb. This is explained by the presence of an F-actin binding site at the N-terminal part of the two isoenzymes. This interaction confers to Itpka the properties of an F-actin bundling protein with two major consequences: i) it can reorganize the cytoskeletal network, particularly in dendritic spines, and ii) can provide an opportunity for Ins(1,3,4,5)P4 to act very locally as second messenger.

Inositol hexakisphosphate kinase 1 maintains hemostasis in mice by regulating platelet polyphosphate levels.

Polyphosphate (polyP), a polymer of orthophosphate moieties released from the dense granules of activated platelets, is a procoagulant agent. Inositol pyrophosphates, another group of phosphate-rich molecules, consist of mono- and diphosphates substituted on an inositol ring. Diphosphoinositol pentakisphosphate (IP7), the most abundant inositol pyrophosphate, is synthesized on phosphorylation of inositol hexakisphosphate (IP6) by IP6 kinases, of which there are 3 mammalian isoforms (IP6K1/2/3) and a single yeast isoform. Yeast lacking IP6 kinase are devoid of polyP, suggesting a role for IP6 kinase in maintaining polyP levels. We theorized that the molecular link between IP6 kinase and polyP is conserved in mammals and investigated whether polyP-dependent platelet function is altered in IP6K1 knockout (Ip6k1(-/-)) mice. We observe a significant reduction in platelet polyP levels in Ip6k1(-/-) mice, along with slower platelet aggregation and lengthened plasma clotting time. Incorporation of polyP into fibrin clots was reduced in Ip6k1(-/-) mice, thereby altering clot ultrastructure, which was rescued on the addition of exogenous polyP. In vivo assays revealed longer tail bleeding time and resistance to thromboembolism in Ip6k1(-/-) mice. Taken together, our data suggest a novel role for IP6K1 in regulation of mammalian hemostasis via its control of platelet polyP levels.