Signaling and Function of Interleukin-2 in T Lymphocytes.

The discovery of interleukin-2 (IL-2) changed the molecular understanding of how the immune system is controlled. IL-2 is a pleiotropic cytokine, and dissecting the signaling pathways that allow IL-2 to control the differentiation and homeostasis of both pro- and anti-inflammatory T cells is fundamental to determining the molecular details of immune regulation. The IL-2 receptor couples to JAK tyrosine kinases and activates the STAT5 transcription factors. However, IL-2 does much more than control transcriptional programs; it is a key regulator of T cell metabolic programs. The development of global phosphoproteomic approaches has expanded the understanding of IL-2 signaling further, revealing the diversity of phosphoproteins that may be influenced by IL-2 in T cells. However, it is increasingly clear that within each T cell subset, IL-2 will signal within a framework of other signal transduction networks that together will shape the transcriptional and metabolic programs that determine T cell fate.

PI(4,5)P2 Clustering and Its Impact on Biological Functions.

Located at the inner leaflet of the plasma membrane (PM), phosphatidyl-inositol 4,5-bisphosphate [PI(4,5)P2] composes only 1-2 mol% of total PM lipids. With its synthesis and turnover both spatially and temporally regulated, PI(4,5)P2 recruits and interacts with hundreds of cellular proteins to support a broad spectrum of cellular functions. Several factors contribute to the versatile and dynamic distribution of PI(4,5)P2 in membranes. Physiological multivalent cations such as Ca2+ and Mg2+ can bridge between PI(4,5)P2 headgroups, forming nanoscopic PI(4,5)P2-cation clusters. The distinct lipid environment surrounding PI(4,5)P2 affects the degree of PI(4,5)P2 clustering. In addition, diverse cellular proteins interacting with PI(4,5)P2 can further regulate PI(4,5)P2 lateral distribution and accessibility. This review summarizes the current understanding of PI(4,5)P2 behavior in both cells and model membranes, with emphasis on both multivalent cation- and protein-induced PI(4,5)P2 clustering. Understanding the nature of spatially separated pools of PI(4,5)P2 is fundamental to cell biology.

The Oxysterol-Binding Protein Cycle: Burning Off PI(4)P to Transport Cholesterol.

To maintain an asymmetric distribution of ions across membranes, protein pumps displace ions against their concentration gradient by using chemical energy. Here, we describe a functionally analogous but topologically opposite process that applies to the lipid transfer protein (LTP) oxysterol-binding protein (OSBP). This multidomain protein exchanges cholesterol for the phosphoinositide phosphatidylinositol 4-phosphate [PI(4)P] between two apposed membranes. Because of the subsequent hydrolysis of PI(4)P, this counterexchange is irreversible and contributes to the establishment of a cholesterol gradient along organelles of the secretory pathway. The facts that some natural anti-cancer molecules block OSBP and that many viruses hijack the OSBP cycle for the formation of intracellular replication organelles highlight the importance and potency of OSBP-mediated lipid exchange. The architecture of some LTPs is similar to that of OSBP, suggesting that the principles of the OSBP cycle-burning PI(4)P for the vectorial transfer of another lipid-might be general.

ARMH3-mediated recruitment of PI4KB directs Golgi-to-endosome trafficking and activation of the antiviral effector STING.

The cGAS-STING pathway mediates cytoplasmic DNA-triggered innate immunity. STING activation is initiated by cyclic-GMP-AMP (cGAMP)-induced translocation from the endoplasmic reticulum and sulfated glycosaminoglycans-induced polymerization at the Golgi. Here, we examine the mechanisms underlying STING transport and activation beyond the Golgi. A genome-wide CRISPR-Cas9 screen identified Armadillo-like helical domain-containing protein 3 (ARMH3) as critical for STING activation. Upon cGAMP-triggered translocation, ARMH3 interacted with STING at the Golgi and recruited phosphatidylinositol 4-kinase beta (PI4KB) to synthesize PI4P, which directed STING Golgi-to-endosome trafficking via PI4P-binding proteins AP-1 and GGA2. Disrupting PI4P-dependent lipid transport through RNAi of other PI4P-binding proteins impaired STING activation. Consistently, disturbed lipid composition inhibited STING activation, whereas aberrantly elevated cellular PI4P led to cGAS-independent STING activation. Armh3fl/fllLyzCre/Cre mice were susceptible to DNA virus challenge in vivo. Thus, ARMH3 bridges STING and PIK4B to generate PI4P for STING transportation and activation, an interaction conserved in all eukaryotes.

cis-B7:CD28 interactions at invaginated synaptic membranes provide CD28 co-stimulation and promote CD8+ T cell function and anti-tumor immunity.

B7 ligands (CD80 and CD86), expressed by professional antigen-presenting cells (APCs), activate the main co-stimulatory receptor CD28 on T cells in trans. However, in peripheral tissues, APCs expressing B7 ligands are relatively scarce. This raises the questions of whether and how CD28 co-stimulation occurs in peripheral tissues. Here, we report that CD8+ T cells displayed B7 ligands that interacted with CD28 in cis at membrane invaginations of the immunological synapse as a result of membrane remodeling driven by phosphoinositide-3-kinase (PI3K) and sorting-nexin-9 (SNX9). cis-B7:CD28 interactions triggered CD28 signaling through protein kinase C theta (PKCθ) and promoted CD8+ T cell survival, migration, and cytokine production. In mouse tumor models, loss of T cell-intrinsic cis-B7:CD28 interactions decreased intratumoral T cells and accelerated tumor growth. Thus, B7 ligands on CD8+ T cells can evoke cell-autonomous CD28 co-stimulation in cis in peripheral tissues, suggesting cis-signaling as a general mechanism for boosting T cell functionality.

Dynamic Remodeling of Membrane Composition Drives Cell Cycle through Primary Cilia Excision.

The life cycle of a primary cilium begins in quiescence and ends prior to mitosis. In quiescent cells, the primary cilium insulates itself from contiguous dynamic membrane processes on the cell surface to function as a stable signaling apparatus. Here, we demonstrate that basal restriction of ciliary structure dynamics is established by the cilia-enriched phosphoinositide 5-phosphatase, Inpp5e. Growth induction displaces ciliary Inpp5e and accumulates phosphatidylinositol 4,5-bisphosphate in distal cilia. This change triggers otherwise-forbidden actin polymerization in primary cilia, which excises cilia tips in a process we call cilia decapitation. While cilia disassembly is traditionally thought to occur solely through resorption, we show that an acute loss of IFT-B through cilia decapitation precedes resorption. Finally, we propose that cilia decapitation induces mitogenic signaling and constitutes a molecular link between the cilia life cycle and cell-division cycle. This newly defined ciliary mechanism may find significance in cell proliferation control during normal development and cancer.

Haploinsufficiency of phosphodiesterase 10A activates PI3K/AKT signaling independent of PTEN to induce an aggressive glioma phenotype.

Glioblastoma is universally fatal and characterized by frequent chromosomal copy number alterations harboring oncogenes and tumor suppressors. In this study, we analyzed exome-wide human glioblastoma copy number data and found that cytoband 6q27 is an independent poor prognostic marker in multiple data sets. We then combined CRISPR-Cas9 data, human spatial transcriptomic data, and human and mouse RNA sequencing data to nominate PDE10A as a potential haploinsufficient tumor suppressor in the 6q27 region. Mouse glioblastoma modeling using the RCAS/tv-a system confirmed that Pde10a suppression induced an aggressive glioma phenotype in vivo and resistance to temozolomide and radiation therapy in vitro. Cell culture analysis showed that decreased Pde10a expression led to increased PI3K/AKT signaling in a Pten-independent manner, a response blocked by selective PI3K inhibitors. Single-nucleus RNA sequencing from our mouse gliomas in vivo, in combination with cell culture validation, further showed that Pde10a suppression was associated with a proneural-to-mesenchymal transition that exhibited increased cell adhesion and decreased cell migration. Our results indicate that glioblastoma patients harboring PDE10A loss have worse outcomes and potentially increased sensitivity to PI3K inhibition.

Engagement of the costimulatory molecule ICOS in tissues promotes establishment of CD8+ tissue-resident memory T cells.

Elevated gene expression of the costimulatory receptor Icos is a hallmark of CD8+ tissue-resident memory (Trm) T cells. Here, we examined the contribution of ICOS in Trm cell differentiation. Upon transfer into WT mice, Icos-/- CD8+ T cells exhibited defective Trm generation but produced recirculating memory populations normally. ICOS deficiency or ICOS-L blockade compromised establishment of CD8+ Trm cells but not their maintenance. ICOS ligation during CD8+ T cell priming did not determine Trm induction; rather, effector CD8+ T cells showed reduced Trm differentiation after seeding into Icosl-/- mice. IcosYF/YF CD8+ T cells were compromised in Trm generation, indicating a critical role for PI3K signaling. Modest transcriptional changes in the few Icos-/- Trm cells suggest that ICOS-PI3K signaling primarily enhances the efficiency of CD8+ T cell tissue residency. Thus, local ICOS signaling promotes production of Trm cells, providing insight into the contribution of costimulatory signals in the generation of tissue-resident populations.

A Small Molecule RAS-Mimetic Disrupts RAS Association with Effector Proteins to Block Signaling.

Oncogenic activation of RAS genes via point mutations occurs in 20%-30% of human cancers. The development of effective RAS inhibitors has been challenging, necessitating new approaches to inhibit this oncogenic protein. Functional studies have shown that the switch region of RAS interacts with a large number of effector proteins containing a common RAS-binding domain (RBD). Because RBD-mediated interactions are essential for RAS signaling, blocking RBD association with small molecules constitutes an attractive therapeutic approach. Here, we present evidence that rigosertib, a styryl-benzyl sulfone, acts as a RAS-mimetic and interacts with the RBDs of RAF kinases, resulting in their inability to bind to RAS, disruption of RAF activation, and inhibition of the RAS-RAF-MEK pathway. We also find that ribosertib binds to the RBDs of Ral-GDS and PI3Ks. These results suggest that targeting of RBDs across multiple signaling pathways by rigosertib may represent an effective strategy for inactivation of RAS signaling.

PLEKHS1 drives PI3Ks and remodels pathway homeostasis in PTEN-null prostate.

The PIP3/PI3K network is a central regulator of metabolism and is frequently activated in cancer, commonly by loss of the PIP3/PI(3,4)P2 phosphatase, PTEN. Despite huge research investment, the drivers of the PI3K network in normal tissues and how they adapt to overactivation are unclear. We find that in healthy mouse prostate PI3K activity is driven by RTK/IRS signaling and constrained by pathway feedback. In the absence of PTEN, the network is dramatically remodeled. A poorly understood YXXM- and PIP3/PI(3,4)P2-binding PH domain-containing adaptor, PLEKHS1, became the dominant activator and was required to sustain PIP3, AKT phosphorylation, and growth in PTEN-null prostate. This was because PLEKHS1 evaded pathway-feedback and experienced enhanced PI3K- and Src-family kinase-dependent phosphorylation of Y258XXM, eliciting PI3K activation. hPLEKHS1 mRNA and activating Y419 phosphorylation of hSrc correlated with PI3K pathway activity in human prostate cancers. We propose that in PTEN-null cells receptor-independent, Src-dependent tyrosine phosphorylation of PLEKHS1 creates positive feedback that escapes homeostasis, drives PIP3 signaling, and supports tumor progression.

Glycolytic ATP fuels phosphoinositide 3-kinase signaling to support effector T helper 17 cell responses.

Aerobic glycolysis-the Warburg effect-converts glucose to lactate via the enzyme lactate dehydrogenase A (LDHA) and is a metabolic feature of effector T cells. Cells generate ATP through various mechanisms and Warburg metabolism is comparatively an energy-inefficient glucose catabolism pathway. Here, we examined the effect of ATP generated via aerobic glycolysis in antigen-driven T cell responses. Cd4CreLdhafl/fl mice were resistant to Th17-cell-mediated experimental autoimmune encephalomyelitis and exhibited defective T cell activation, migration, proliferation, and differentiation. LDHA deficiency crippled cellular redox balance and inhibited ATP production, diminishing PI3K-dependent activation of Akt kinase and thereby phosphorylation-mediated inhibition of Foxo1, a transcriptional repressor of T cell activation programs. Th17-cell-specific expression of an Akt-insensitive Foxo1 recapitulated the defects seen in Cd4CreLdhafl/fl mice. Induction of LDHA required PI3K signaling and LDHA deficiency impaired PI3K-catalyzed PIP3 generation. Thus, Warburg metabolism augments glycolytic ATP production, fueling a PI3K-centered positive feedback regulatory circuit that drives effector T cell responses.

Mutations in the transcription factor FOXO1 mimic positive selection signals to promote germinal center B cell expansion and lymphomagenesis.

The transcription factor forkhead box O1 (FOXO1), which instructs the dark zone program to direct germinal center (GC) polarity, is typically inactivated by phosphatidylinositol 3-kinase (PI3K) signals. Here, we investigated how FOXO1 mutations targeting this regulatory axis in GC-derived B cell non-Hodgkin lymphomas (B-NHLs) contribute to lymphomagenesis. Examination of primary B-NHL tissues revealed that FOXO1 mutations and PI3K pathway activity were not directly correlated. Human B cell lines bearing FOXO1 mutations exhibited hyperactivation of PI3K and Stress-activated protein kinase (SAPK)/Jun amino-terminal kinase (JNK) signaling, and increased cell survival under stress conditions as a result of alterations in FOXO1 transcriptional affinities and activation of transcriptional programs characteristic of GC-positive selection. When modeled in mice, FOXO1 mutations conferred competitive advantage to B cells in response to key T-dependent immune signals, disrupting GC homeostasis. FOXO1 mutant transcriptional signatures were prevalent in human B-NHL and predicted poor clinical outcomes. Thus, rather than enforcing FOXO1 constitutive activity, FOXO1 mutations enable co-option of GC-positive selection programs during the pathogenesis of GC-derived lymphomas.

Fumarate inhibits PTEN to promote tumorigenesis and therapeutic resistance of type2 papillary renal cell carcinoma.

Fumarate is an oncometabolite. However, the mechanism underlying fumarate-exerted tumorigenesis remains unclear. Here, utilizing human type2 papillary renal cell carcinoma (PRCC2) as a model, we show that fumarate accumulates in cells deficient in fumarate hydratase (FH) and inhibits PTEN to activate PI3K/AKT signaling. Mechanistically, fumarate directly reacts with PTEN at cysteine 211 (C211) to form S-(2-succino)-cysteine. Succinated C211 occludes tethering of PTEN with the cellular membrane, thereby diminishing its inhibitory effect on the PI3K/AKT pathway. Functionally, re-expressing wild-type FH or PTEN C211S phenocopies an AKT inhibitor in suppressing tumor growth and sensitizing PRCC2 to sunitinib. Analysis of clinical specimens indicates that PTEN C211 succination levels are positively correlated with AKT activation in PRCC2. Collectively, these findings elucidate a non-metabolic, oncogenic role of fumarate in PRCC2 via direct post-translational modification of PTEN and further reveal potential stratification strategies for patients with FH loss by combinatorial AKTi and sunitinib therapy.

An Oncogenic Alteration Creates a Microenvironment that Promotes Tumor Progression by Conferring a Metabolic Advantage to Regulatory T Cells.

Only a small percentage of patients afflicted with gastric cancer (GC) respond to immune checkpoint blockade (ICB). To study the mechanisms underlying this resistance, we examined the immune landscape of GC. A subset of these tumors was characterized by high frequencies of regulatory T (Treg) cells and low numbers of effector T cells. Genomic analyses revealed that these tumors bore mutations in RHOA that are known to drive tumor progression. RHOA mutations in cancer cells activated the PI3K-AKT-mTOR signaling pathway, increasing production of free fatty acids that are more effectively consumed by Treg cells than effector T cells. RHOA mutant tumors were resistant to PD-1 blockade but responded to combination of PD-1 blockade with inhibitors of the PI3K pathway or therapies targeting Treg cells. We propose that the metabolic advantage conferred by RHOA mutations enables Treg cell accumulation within GC tumors, generating an immunosuppressive TME that underlies resistance to ICB.

Regulation of PTEN translation by PI3K signaling maintains pathway homeostasis.

The PI3K pathway regulates cell metabolism, proliferation, and migration, and its dysregulation is common in cancer. We now show that both physiologic and oncogenic activation of PI3K signaling increase the expression of its negative regulator PTEN. This limits the duration of the signal and output of the pathway. Physiologic and pharmacologic inhibition of the pathway reduces PTEN and contributes to the rebound in pathway activity in tumors treated with PI3K inhibitors and limits their efficacy. Regulation of PTEN is due to mTOR/4E-BP1-dependent control of its translation and is lost when 4E-BP1 is deleted. Translational regulation of PTEN is therefore a major homeostatic regulator of physiologic PI3K signaling and plays a role in reducing the pathway activation by oncogenic PIK3CA mutants and the antitumor activity of PI3K pathway inhibitors. However, pathway output is hyperactivated in tumor cells with coexistent PI3K mutation and loss of PTEN function.

The physiological relevance of autophagosome morphogenesis.

Autophagy sequesters cytoplasmic portions into autophagosomes. While selective cargo is engulfed by elongation of cup-shaped isolation membranes (IMs), the morphogenesis of non-selective IMs remains elusive. Based on recent observations, we propose a novel model for autophagosome morphogenesis wherein active regulation of the IM rim serves the physiological roles of autophagy.

Cancer-Derived Succinate Promotes Macrophage Polarization and Cancer Metastasis via Succinate Receptor.

Macrophages form a major cell population in the tumor microenvironment. They can be activated and polarized into tumor-associated macrophages (TAM) by the tumor-derived soluble molecules to promote tumor progression and metastasis. Here, we used comparative metabolomics coupled with biochemical and animal studies to show that cancer cells release succinate into their microenvironment and activate succinate receptor (SUCNR1) signaling to polarize macrophages into TAM. Furthermore, the results from in vitro and in vivo studies revealed that succinate promotes not only cancer cell migration and invasion but also cancer metastasis. These effects are mediated by SUCNR1-triggered PI3K-hypoxia-inducible factor 1α (HIF-1α) axis. Compared with healthy subjects and tumor-free lung tissues, serum succinate levels and lung cancer SUCNR1 expression were elevated in lung cancer patients, suggesting an important clinical relevance. Collectively, our findings indicate that the secreted tumor-derived succinate belongs to a novel class of cancer progression factors, controlling TAM polarization and promoting tumorigenic signaling.

Growth Factor Receptor Signaling Inhibition Prevents SARS-CoV-2 Replication.

SARS-CoV-2 infections are rapidly spreading around the globe. The rapid development of therapies is of major importance. However, our lack of understanding of the molecular processes and host cell signaling events underlying SARS-CoV-2 infection hinders therapy development. We use a SARS-CoV-2 infection system in permissible human cells to study signaling changes by phosphoproteomics. We identify viral protein phosphorylation and define phosphorylation-driven host cell signaling changes upon infection. Growth factor receptor (GFR) signaling and downstream pathways are activated. Drug-protein network analyses revealed GFR signaling as key pathways targetable by approved drugs. The inhibition of GFR downstream signaling by five compounds prevents SARS-CoV-2 replication in cells, assessed by cytopathic effect, viral dsRNA production, and viral RNA release into the supernatant. This study describes host cell signaling events upon SARS-CoV-2 infection and reveals GFR signaling as a central pathway essential for SARS-CoV-2 replication. It provides novel strategies for COVID-19 treatment.

Cerebral organoid and mouse models reveal a RAB39b-PI3K-mTOR pathway-dependent dysregulation of cortical development leading to macrocephaly/autism phenotypes.

Dysregulation of early neurodevelopment is implicated in macrocephaly/autism disorders. However, the mechanism underlying this dysregulation, particularly in human cells, remains poorly understood. Mutations in the small GTPase gene RAB39b are associated with X-linked macrocephaly, autism spectrum disorder (ASD), and intellectual disability. The in vivo roles of RAB39b in the brain remain unknown. We generated Rab39b knockout (KO) mice and found that they exhibited cortical neurogenesis impairment, macrocephaly, and hallmark ASD behaviors, which resembled patient phenotypes. We also produced mutant human cerebral organoids that were substantially enlarged due to the overproliferation and impaired differentiation of neural progenitor cells (NPCs), which resemble neurodevelopmental deficits in KO mice. Mechanistic studies reveal that RAB39b interacts with PI3K components and its deletion promotes PI3K-AKT-mTOR signaling in NPCs of mouse cortex and cerebral organoids. The mTOR activity is robustly enhanced in mutant outer radial glia cells (oRGs), a subtype of NPCs barely detectable in rodents but abundant in human brains. Inhibition of AKT signaling rescued enlarged organoid sizes and NPC overproliferation caused by RAB39b mutations. Therefore, RAB39b mutation promotes PI3K-AKT-mTOR activity and alters cortical neurogenesis, leading to macrocephaly and autistic-like behaviors. Our studies provide new insights into neurodevelopmental dysregulation and common pathways associated with ASD across species.

A sterol-PI(4)P exchanger modulates the Tel1/ATM axis of the DNA damage response.

Upon DNA damage, cells activate the DNA damage response (DDR) to coordinate proliferation and DNA repair. Dietary, metabolic, and environmental inputs are emerging as modulators of how DNA surveillance and repair take place. Lipids hold potential to convey these cues, although little is known about how. We observed that lipid droplet (LD) number specifically increased in response to DNA breaks. Using Saccharomyces cerevisiae and cultured human cells, we show that the selective storage of sterols into these LD concomitantly stabilizes phosphatidylinositol-4-phosphate (PI(4)P) at the Golgi, where it binds the DDR kinase ATM. In turn, this titration attenuates the initial nuclear ATM-driven response to DNA breaks, thus allowing processive repair. Furthermore, manipulating this loop impacts the kinetics of DNA damage signaling and repair in a predictable manner. Thus, our findings have major implications for tackling genetic instability pathologies through dietary and pharmacological interventions.