ORP4L is a prerequisite for the induction of T-cell leukemogenesis associated with human T-cell leukemia virus 1.

Human T-cell leukemia virus 1 (HTLV-1) causes adult T-cell leukemia (ATL), but the mechanism underlying its initiation remains elusive. In this study, ORP4L was expressed in ATL cells but not in normal T-cells. ORP4L ablation completely blocked T-cell leukemogenesis induced by the HTLV-1 oncoprotein Tax in mice, whereas engineering ORP4L expression in T-cells resulted in T-cell leukemia in mice, suggesting the oncogenic properties and prerequisite of ORP4L promote the initiation of T-cell leukemogenesis. For molecular insight, we found that loss of miR-31 caused by HTLV-1 induced ORP4L expression in T-cells. ORP4L interacts with PI3Kδ to promote PI(3,4,5)P3 generation, contributing to AKT hyperactivation; NF-κB-dependent, p53 inactivation-induced pro-oncogene expression; and T-cell leukemogenesis. Consistently, ORP4L ablation eliminates human ATL cells in patient-derived xenograft ATL models. These results reveal a plausible mechanism of T-cell deterioration by HTLV-1 that can be therapeutically targeted.

OSBP-Related Protein 5L Maintains Intracellular IP3/Ca2+ Signaling and Proliferation in T Cells by Facilitating PIP2 Hydrolysis.

Phospholipase C (PLC) isoforms play central roles in signaling cascades by cleaving PIP2 into the second messengers IP3 and DAG. In this study, to our knowledge, we uncover that ORP5L interacts physically with PLCγ1 in T cells, extracts PIP2 from the plasma membrane via its ORD domain (OSBP-related domain), presents it to PLCγ1 (enabling IP3 generation), and eventually maintains intracellular Ca2+ homeostasis. Through this mechanism, ORP5L promotes T cell proliferation in a Ca2+-activated NFAT2-dependent manner. To our knowledge, our study uncovers a new key function of ORP5L as a critical cofactor for PLCγ1 catalysis and its crucial role in human T cell proliferation.

MicroRNA-31-5p enhances the Warburg effect via targeting FIH.

The enhanced expression of miR-31 has been observed in many human malignancies including lung cancer, and this microRNA regulates several aspects of oncogenesis. However, the role of miR-31-5p in energy metabolism remains elusive. Here, we confirm that H1299 and A549 cells, 2 lung cancer cell lines, relay on aerobic glycolysis as main source of ATP. Inhibition of miR-31-5p leads to decreased glycolysis and ATP production, while miR-31-5p overexpression increases them. Hypoxia inducible factor 1 (HIF-1) up-regulates the expression of glycolytic enzymes, and the HIF-1α inhibitor (FIH) inhibits HIF-1 activity. Because FIH is a direct target of miR-31-5p, inhibition of miR-31-5p results in enhanced FIH expression and suppression of HIF-1 signaling, while overexpression of miR-31-5p has the opposite effects. Via this mechanism, miR-31-5p up-regulates aerobic glycolytic genes and maintains energy homeostasis. To further validate the mechanism of miR-31-5p in glycolysis regulation, we show that overexpression or knockdown of FIH rescued the effects of miR-31-5p or miR-31-5p inhibitor on HIF activation and its target gene expression, respectively. Finally, by means of an A549 cell xenograft mouse model, we demonstrate that the miR-31-5p promotes cell proliferation via enhancing glycolysis. In summary, this study reveals that miR-31-5p promotes the Warburg effect via direct targeting of FIH.-Zhu, B., Cao, X., Zhang, W., Pan, G., Yi, Q., Zhong, W., Yan, D. MicroRNA-31-5p enhances the Warburg effect via targeting FIH.

ORP4L couples IP3 to ITPR1 in control of endoplasmic reticulum calcium release.

Oxysterol-binding protein-related protein (ORP) 4L acts as a scaffold protein assembling CD3-ε, G-αq/11, and PLC-ß3 into a complex at the plasma membrane that mediates inositol (1,4,5)-trisphosphate (IP3)-induced endoplasmic reticulum (ER) Ca2+ release and oxidative phosphorylation in T-cell acute lymphoblastic leukemia cells. Here, we offer new evidence that ORP4L interacts with the carboxyl terminus of the IP3 receptor type 1 (ITPR1) in Jurkat T cells. ORP4L enables IP3 binding to ITPR1; a truncated construct that lacks the ITPR1-binding region retains the ability to increase IP3 production but fails to mediate IP3 and ITPR1 binding. In association with this ability of ORP4L, it enhances Ca2+ release from the ER and subsequent cytosolic and mitochondrial parallel Ca2+ spike oscillations that stimulate mitochondrial energetics and thus maintains cell survival. These data support a novel model in which ORP4L is a cofactor of ITPR1, which increases ITPR1 sensitivity to IP3 and enables ER Ca2+ release.-Cao, X., Chen, J., Li, D., Xie, P., Xu, M., Lin, W., Li, S., Pan, G., Tang, Y., Xu, J., Olkkonen, V. M., Yan, D., Zhong, W. ORP4L couples IP3 to ITPR1 in control of endoplasmic reticulum calcium release.

OSBP-related protein 4L promotes phospholipase Cß3 translocation from the nucleus to the plasma membrane in Jurkat T-cells.

Phosphoinositide phospholipases C (PLCs) are a family of eukaryotic intracellular enzymes with important roles in signal transduction. In addition to their location at the plasma membrane, PLCs also exist within the cell nucleus where they are stored. We previously demonstrated that OSBP-related protein 4L (ORP4L) anchors cluster of differentiation 3ϵ (CD3ϵ) to the heterotrimeric G protein subunit (Gαq/11) to control PLCß3 relocation and activation. However, the underlying mechanism by which ORP4L facilitates PLCß3 translocation remains unknown. Here, using confocal immunofluorescence microscopy and coimmunoprecipitation assays, we report that ORP4L stimulates PLCß3 translocation from the nucleus to the plasma membrane in Jurkat T-cells in two steps. First, we found that ORP4L is required for the activation of Ras-related nuclear protein (RAN), a GTP-binding nuclear protein that binds to exportin 1 and eventually promotes the nuclear export of PLCß3. Second, we also observed that ORP4L interacts with vesicle-associated membrane protein-associated protein A (VAPA) through its two phenylalanines in an acidic tract (FFAT) motif. This complex enabled PLCß3 movement to the plasma membrane, indicating that PLCß3 translocation occurs in a VAPA-dependent manner. This study reveals detailed mechanistic insight into the role of ORP4L in PLCß3 redistribution from storage within the nucleus to the plasma membrane via RAN activation and interaction with VAPA in Jurkat T-cells.

ORP4L Facilitates Macrophage Survival via G-Protein-Coupled Signaling: ORP4L-/- Mice Display a Reduction of Atherosclerosis.

RATIONALE: Macrophage survival within the arterial wall is a central factor contributing to atherogenesis. Oxysterols, major components of oxidized low-density lipoprotein, exert cytotoxic effects on macrophages. OBJECTIVE: To determine whether oxysterol-binding protein-related protein 4 L (ORP4L), an oxysterol-binding protein, affects macrophage survival and the pathogenesis of atherosclerosis. METHODS AND RESULTS: By hiring cell biological approaches and ORP4L-/- mice, we show that ORP4L coexpresses with and forms a complex with Gαq/11 and phospholipase C (PLC)-ß3 in macrophages. ORP4L facilitates G-protein-coupled ligand-induced PLCß3 activation, IP3 production, and Ca2+ release from the endoplasmic reticulum. Through this mechanism, ORP4L sustains antiapoptotic Bcl-XL expression through Ca2+-mediated c-AMP responsive element binding protein transcriptional regulation and thus protects macrophages from apoptosis. Excessive stimulation with the oxysterol 25-hydroxycholesterol disassembles the ORP4L/Gαq/11/PLCß3 complexes, resulting in reduced PLCß3 activity, IP3 production, and Ca2+ release, as well as decreased Bcl-XL expression and increased apoptosis. Overexpression of ORP4L counteracts these oxysterol-induced defects. Mice lacking ORP4L exhibit increased apoptosis of macrophages in atherosclerotic lesions and a reduced lesion size. CONCLUSIONS: ORP4L is crucial for macrophage survival. It counteracts the cytotoxicity of oxysterols/oxidized low-density lipoprotein to protect macrophage from apoptosis, thus playing an important role in the development of atherosclerosis.

An acquired phosphatidylinositol 4-phosphate transport initiates T-cell deterioration and leukemogenesis.

Lipid remodeling is crucial for malignant cell transformation and tumorigenesis, but the precise molecular processes involved and direct evidences for these in vivo remain elusive. Here, we report that oxysterol-binding protein (OSBP)-related protein 4 L (ORP4L) is expressed in adult T-cell leukemia (ATL) cells but not normal T-cells. In ORP4L knock-in T-cells, ORP4L dimerizes with OSBP to control the shuttling of OSBP between the Golgi apparatus and the plasma membrane (PM) as an exchanger of phosphatidylinositol 4-phosphate [PI(4)P]/cholesterol. The PI(4)P arriving at the PM via this transport machinery replenishes phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and phosphatidylinositol (3,4,5) trisphosphate [PI(3,4,5)P3] biosynthesis, thus contributing to PI3K/AKT hyperactivation and T-cell deterioration in vitro and in vivo. Disruption of ORP4L and OSBP dimerization disables PI(4)P transport and T-cell leukemogenesis. In summary, we identify a non-vesicular lipid transport machinery between Golgi and PM maintaining the oncogenic signaling competence initiating T-cell deterioration and leukemogenesis.

Loss of miR-31-5p drives hematopoietic stem cell malignant transformation and restoration eliminates leukemia stem cells in mice.

Leukemia stem cells (LSCs) propagate leukemia and are responsible for the high frequency of relapse of treated patients. The ability to target LSCs remains elusive, indicating a need to understand the underlying mechanism of LSC formation. Here, we report that miR-31-5p is reduced or undetectable in human LSCs compared to hematopoietic stem progenitor cells (HSPCs). Inhibition of miR-31-5p in HSPCs promotes the expression of its target gene FIH, encoding FIH [factor inhibiting hypoxia-inducing factor 1α (HIF-1α)], to suppress HIF-1α signaling. Increased FIH resulted in a switch from glycolysis to oxidative phosphorylation (OXPHOS) as the predominant mode of energy metabolism and increased the abundance of the oncometabolite fumarate. Increased fumarate promoted the conversion of HSPCs to LSCs and initiated myeloid leukemia-like disease in NOD-Prkdcscid IL2rgtm1/Bcgen (B-NDG) mice. We further demonstrated that miR-31-5p inhibited long- and short-term hematopoietic stem cells with a high frequency of LSCs. In combination with the chemotherapeutic agent Ara-C (cytosine arabinoside), restoration of miR-31-5p using G7 poly (amidoamine) nanosized dendriplex encapsulating miR-31-5p eliminated LSCs and inhibited acute myeloid leukemia (AML) progression in patient-derived xenograft mouse models. These results demonstrated a mechanism of HSC malignant transformation through altered energy metabolism and provided a potential therapeutic strategy to treat patients with AML.

ORP4L Extracts and Presents PIP2 from Plasma Membrane for PLCß3 Catalysis: Targeting It Eradicates Leukemia Stem Cells.

Leukemia stem cells (LSCs) are a rare subpopulation of abnormal hematopoietic stem cells (HSCs) that propagates leukemia and are responsible for the high frequency of relapse in therapies. Detailed insights into LSCs' survival will facilitate the identification of targets for therapeutic approaches. Here, we develop an inhibitor, LYZ-81, which targets ORP4L with high affinity and specificity and selectively eradicates LCSs in vitro and in vivo. ORP4L is expressed in LSCs but not in normal HSCs and is essential for LSC bioenergetics and survival. It extracts PIP2 from the plasma membrane and presents it to PLCß3, enabling IP3 generation and subsequent Ca2+-dependent bioenergetics. LYZ-81 binds ORP4L competitively with PIP2 and blocks PIP2 hydrolysis, resulting in defective Ca2+ signaling. The results provide evidence that LSCs can be eradicated through the inhibition of ORP4L by LYZ-81, which may serve as a starting point of drug development for the elimination of LSCs to eventually cure leukemia.