The AKT kinase signaling network is rewired by PTEN to control proximal BCR signaling in germinal center B cells.

B cell antigen receptor (BCR) and CD40 signaling are rewired in germinal center (GC) B cells (GCBCs) to optimize selection for high-affinity B cells. In GCBC, BCR signals are constrained, but the mechanisms are not well understood. Here we describe a GC-specific, AKT-kinase-driven negative feedback loop that attenuates BCR signaling. Mass spectrometry revealed that AKT target activity was altered in GCBCs compared with naive B cells. Retargeting was linked to differential AKT T308 and S473 phosphorylation, in turn controlled by GC-specific upregulation of phosphoinositide-dependent protein kinase PDK1 and the phosphatase PTEN. In GCBCs, AKT preferentially targeted CSK, SHP-1 and HPK1, which are negative regulators of BCR signaling. We found that phosphorylation enhances enzymatic activity of these proteins, creating a negative feedback loop that dampens upstream BCR signaling. AKT inhibition relieved this negative feedback and enhanced activation of BCR-proximal kinase LYN, as well as downstream BCR signaling molecules in GCBCs.

Restoration of Visual Function by Enhancing Conduction in Regenerated Axons.

Although a number of repair strategies have been shown to promote axon outgrowth following neuronal injury in the mammalian CNS, it remains unclear whether regenerated axons establish functional synapses and support behavior. Here, in both juvenile and adult mice, we show that either PTEN and SOCS3 co-deletion, or co-overexpression of osteopontin (OPN)/insulin-like growth factor 1 (IGF1)/ciliary neurotrophic factor (CNTF), induces regrowth of retinal axons and formation of functional synapses in the superior colliculus (SC) but not significant recovery of visual function. Further analyses suggest that regenerated axons fail to conduct action potentials from the eye to the SC due to lack of myelination. Consistent with this idea, administration of voltage-gated potassium channel blockers restores conduction and results in increased visual acuity. Thus, enhancing both regeneration and conduction effectively improves function after retinal axon injury.

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.

The functions and regulation of the PTEN tumour suppressor: new modes and prospects.

PTEN is a potent tumour suppressor, and its loss of function is frequently observed in both heritable and sporadic cancers. PTEN has phosphatase-dependent and phosphatase-independent (scaffold) activities in the cell and governs a variety of biological processes, including maintenance of genomic stability, cell survival, migration, proliferation and metabolism. Even a subtle decrease in PTEN levels and activity results in cancer susceptibility and favours tumour progression. Regulation of PTEN has therefore emerged as a subject of intense research in tumour biology. Recent discoveries, including the existence of distinct PTEN isoforms and the ability of PTEN to form dimers, have brought to light new modes of PTEN function and regulation. These milestone findings have in turn opened new therapeutic avenues for cancer prevention and treatment through restoration of PTEN tumour suppressor activity.

Circling back to PTEN: Fumarate inhibits canonical tumor suppressor.

Ge et al. (2022) describes an inhibitory, post-translational modification of PTEN at C211 by fumarate, which offers new insight into the integration of PI3K signaling and metabolism via a potential feedforward regulatory mechanism involving a PI3K-glucose-fumarate-PTEN axis.

Mutant PTEN in Cancer: Worse Than Nothing.

Tumor suppressors block the development of cancer and are often lost during tumor development. Papa et al. show that partial loss of normal PTEN tumor suppressor function can be compounded by additional disruption caused by the expression of inactive mutant PTEN protein. This has significant implications for patients with PTEN gene mutations.

Genetic and clonal dissection of murine small cell lung carcinoma progression by genome sequencing.

Small cell lung carcinoma (SCLC) is a highly lethal, smoking-associated cancer with few known targetable genetic alterations. Using genome sequencing, we characterized the somatic evolution of a genetically engineered mouse model (GEMM) of SCLC initiated by loss of Trp53 and Rb1. We identified alterations in DNA copy number and complex genomic rearrangements and demonstrated a low somatic point mutation frequency in the absence of tobacco mutagens. Alterations targeting the tumor suppressor Pten occurred in the majority of murine SCLC studied, and engineered Pten deletion accelerated murine SCLC and abrogated loss of Chr19 in Trp53; Rb1; Pten compound mutant tumors. Finally, we found evidence for polyclonal and sequential metastatic spread of murine SCLC by comparative sequencing of families of related primary tumors and metastases. We propose a temporal model of SCLC tumorigenesis with implications for human SCLC therapeutics and the nature of cancer-genome evolution in GEMMs.

Cancer-associated PTEN mutants act in a dominant-negative manner to suppress PTEN protein function.

PTEN dysfunction plays a crucial role in the pathogenesis of hereditary and sporadic cancers. Here, we show that PTEN homodimerizes and, in this active conformation, exerts lipid phosphatase activity on PtdIns(3,4,5)P3. We demonstrate that catalytically inactive cancer-associated PTEN mutants heterodimerize with wild-type PTEN and constrain its phosphatase activity in a dominant-negative manner. To study the consequences of homo- and heterodimerization of wild-type and mutant PTEN in vivo, we generated Pten knockin mice harboring two cancer-associated PTEN mutations (PtenC124S and PtenG129E). Heterozygous Pten(C124S/+) and Pten(G129E/+) cells and tissues exhibit increased sensitivity to PI3-K/Akt activation compared to wild-type and Pten(+/-) counterparts, whereas this difference is no longer apparent between Pten(C124S/-) and Pten(-/-) cells. Notably, Pten KI mice are more tumor prone and display features reminiscent of complete Pten loss. Our findings reveal that PTEN loss and PTEN mutations are not synonymous and define a working model for the function and regulation of PTEN.

Accelerated plasma-cell differentiation in Bach2-deficient mouse B cells is caused by altered IRF4 functions.

Transcription factors BACH2 and IRF4 are both essential for antibody class-switch recombination (CSR) in activated B lymphocytes, while they oppositely regulate the differentiation of plasma cells (PCs). Here, we investigated how BACH2 and IRF4 interact during CSR and plasma-cell differentiation. We found that BACH2 organizes heterochromatin formation of target gene loci in mouse splenic B cells, including targets of IRF4 activation such as Aicda, an inducer of CSR, and Prdm1, a master plasma-cell regulator. Release of these gene loci from heterochromatin in response to B-cell receptor stimulation was coupled to AKT-mTOR pathway activation. In Bach2-deficient B cells, PC genes' activation depended on IRF4 protein accumulation, without an increase in Irf4 mRNA. Mechanistically, a PU.1-IRF4 heterodimer in activated B cells promoted BACH2 function by inducing gene expression of Bach2 and Pten, a negative regulator of AKT signaling. Elevated AKT activity in Bach2-deficient B cells resulted in IRF4 protein accumulation. Thus, BACH2 and IRF4 mutually modulate the activity of each other, and BACH2 inhibits PC differentiation by both the repression of PC genes and the restriction of IRF4 protein accumulation.

The microRNA miR-148a functions as a critical regulator of B cell tolerance and autoimmunity.

Autoreactive B cells have critical roles in a large diversity of autoimmune diseases, but the molecular pathways that control these cells remain poorly understood. We performed an in vivo functional screen of a lymphocyte-expressed microRNA library and identified miR-148a as a potent regulator of B cell tolerance. Elevated miR-148a expression impaired B cell tolerance by promoting the survival of immature B cells after engagement of the B cell antigen receptor by suppressing the expression of the autoimmune suppressor Gadd45α, the tumor suppressor PTEN and the pro-apoptotic protein Bim. Furthermore, increased expression of miR-148a, which occurs frequently in patients with lupus and lupus-prone mice, facilitated the development of lethal autoimmune disease in a mouse model of lupus. Our studies demonstrate a function for miR-148a as a regulator of B cell tolerance and autoimmunity.

Control of Glucocorticoid Receptor Levels by PTEN Establishes a Failsafe Mechanism for Tumor Suppression.

The PTEN tumor suppressor controls cell death and survival by regulating functions of various molecular targets. While the role of PTEN lipid-phosphatase activity on PtdIns(3,4,5)P3 and inhibition of PI3K pathway is well characterized, the biological relevance of PTEN protein-phosphatase activity remains undefined. Here, using knockin (KI) mice harboring cancer-associated and functionally relevant missense mutations, we show that although loss of PTEN lipid-phosphatase function cooperates with oncogenic PI3K to promote rapid mammary tumorigenesis, the additional loss of PTEN protein-phosphatase activity triggered an extensive cell death response evident in early and advanced mammary tumors. Omics and drug-targeting studies revealed that PI3Ks act to reduce glucocorticoid receptor (GR) levels, which are rescued by loss of PTEN protein-phosphatase activity to restrain cell survival. Thus, we find that the dual regulation of GR by PI3K and PTEN functions as a rheostat that can be exploited for the treatment of PTEN loss-driven cancers.

PTEN deficiency exposes a requirement for an ARF GTPase module for integrin-dependent invasion in ovarian cancer.

Dysregulation of the PI3K/AKT pathway is a common occurrence in high-grade serous ovarian carcinoma (HGSOC), with the loss of the tumour suppressor PTEN in HGSOC being associated with poor prognosis. The cellular mechanisms of how PTEN loss contributes to HGSOC are largely unknown. We here utilise time-lapse imaging of HGSOC spheroids coupled to a machine learning approach to classify the phenotype of PTEN loss. PTEN deficiency induces PI(3,4,5)P3 -rich and -dependent membrane protrusions into the extracellular matrix (ECM), resulting in a collective invasion phenotype. We identify the small GTPase ARF6 as a crucial vulnerability of HGSOC cells upon PTEN loss. Through a functional proteomic CRISPR screen of ARF6 interactors, we identify the ARF GTPase-activating protein (GAP) AGAP1 and the ECM receptor ß1-integrin (ITGB1) as key ARF6 interactors in HGSOC regulating PTEN loss-associated invasion. ARF6 functions to promote invasion by controlling the recycling of internalised, active ß1-integrin to maintain invasive activity into the ECM. The expression of the CYTH2-ARF6-AGAP1 complex in HGSOC patients is inversely associated with outcome, allowing the identification of patient groups with improved versus poor outcome. ARF6 may represent a therapeutic vulnerability in PTEN-depleted HGSOC.

Control of PI(3) kinase in Treg cells maintains homeostasis and lineage stability.

Foxp3(+) regulatory T cells (Treg cells) are required for immunological homeostasis. One notable distinction between conventional T cells (Tconv cells) and Treg cells is differences in the activity of phosphatidylinositol-3-OH kinase (PI(3)K); only Tconv cells downregulate PTEN, the main negative regulator of PI(3)K, upon activation. Here we found that control of PI(3)K in Treg cells was essential for lineage homeostasis and stability. Mice lacking Pten in Treg cells developed an autoimmune-lymphoproliferative disease characterized by excessive T helper type 1 (TH1) responses and B cell activation. Diminished control of PI(3)K activity in Treg cells led to reduced expression of the interleukin-2 (IL-2) receptor α subunit CD25, accumulation of Foxp3(+)CD25(-) cells and, ultimately, loss of expression of the transcription factor Foxp3 in these cells. Collectively, our data demonstrate that control of PI(3)K signaling by PTEN in Treg cells is critical for maintaining their homeostasis, function and stability.

Treg cells require the phosphatase PTEN to restrain TH1 and TFH cell responses.

The interplay between effector T cells and regulatory T cells (Treg cells) is crucial for adaptive immunity, but how Treg cells control diverse effector responses is elusive. We found that the phosphatase PTEN links Treg cell stability to repression of type 1 helper T cell (TH1 cell) and follicular helper T cell (TFH cell) responses. Depletion of PTEN in Treg cells resulted in excessive TFH cell and germinal center responses and spontaneous inflammatory disease. These defects were considerably blocked by deletion of interferon-γ, indicating coordinated control of TH1 and TFH responses. Mechanistically, PTEN maintained Treg cell stability and metabolic balance between glycolysis and mitochondrial fitness. Moreover, PTEN deficiency upregulates activity of the metabolic checkpoint kinase complex mTORC2 and the serine-threonine kinase Akt, and loss of this activity restores functioning of PTEN-deficient Treg cells. Our studies establish a PTEN-mTORC2 axis that maintains Treg cell stability and coordinates Treg cell-mediated control of effector responses.

Systemic elevation of PTEN induces a tumor-suppressive metabolic state.

Decremental loss of PTEN results in cancer susceptibility and tumor progression. PTEN elevation might therefore be an attractive option for cancer prevention and therapy. We have generated several transgenic mouse lines with PTEN expression elevated to varying levels by taking advantage of bacterial artificial chromosome (BAC)-mediated transgenesis. The "Super-PTEN" mutants are viable and show reduced body size due to decreased cell number, with no effect on cell size. Unexpectedly, PTEN elevation at the organism level results in healthy metabolism characterized by increased energy expenditure and reduced body fat accumulation. Cells derived from these mice show reduced glucose and glutamine uptake and increased mitochondrial oxidative phosphorylation and are resistant to oncogenic transformation. Mechanistically we find that PTEN elevation orchestrates this metabolic switch by regulating PI3K-dependent and -independent pathways and negatively impacting two of the most pronounced metabolic features of tumor cells: glutaminolysis and the Warburg effect.

PTEN Suppresses Glycolysis by Dephosphorylating and Inhibiting Autophosphorylated PGK1.

The PTEN tumor suppressor is frequently mutated or deleted in cancer and regulates glucose metabolism through the PI3K-AKT pathway. However, whether PTEN directly regulates glycolysis in tumor cells is unclear. We demonstrate here that PTEN directly interacts with phosphoglycerate kinase 1 (PGK1). PGK1 functions not only as a glycolytic enzyme but also as a protein kinase intermolecularly autophosphorylating itself at Y324 for activation. The protein phosphatase activity of PTEN dephosphorylates and inhibits autophosphorylated PGK1, thereby inhibiting glycolysis, ATP production, and brain tumor cell proliferation. In addition, knockin expression of a PGK1 Y324F mutant inhibits brain tumor formation. Analyses of human glioblastoma specimens reveals that PGK1 Y324 phosphorylation levels inversely correlate with PTEN expression status and are positively associated with poor prognosis in glioblastoma patients. This work highlights the instrumental role of PGK1 autophosphorylation in its activation and PTEN protein phosphatase activity in governing glycolysis and tumorigenesis.

Distinct early role of PTEN regulation during HCMV infection of monocytes.

Human cytomegalovirus (HCMV) infection of monocytes is essential for viral dissemination and persistence. We previously identified that HCMV entry/internalization and subsequent productive infection of this clinically relevant cell type is distinct when compared to other infected cells. We showed that internalization and productive infection required activation of epidermal growth factor receptor (EGFR) and integrin/c-Src, via binding of viral glycoprotein B to EGFR, and the pentamer complex to ß1/ß3 integrins. To understand how virus attachment drives entry, we compared infection of monocytes with viruses containing the pentamer vs. those without the pentamer and then used a phosphoproteomic screen to identify potential phosphorylated proteins that influence HCMV entry and trafficking. The screen revealed that the most prominent pentamer-biased phosphorylated protein was the lipid- and protein-phosphatase phosphatase and tensin homolog (PTEN). PTEN knockdown with siRNA or PTEN inhibition with a PTEN inhibitor decreased pentamer-mediated HCMV entry, without affecting trimer-mediated entry. Inhibition of PTEN activity affected lipid metabolism and interfered with the onset of the endocytic processes required for HCMV entry. PTEN inactivation was sufficient to rescue pentamer-null HCMV from lysosomal degradation. We next examined dephosphorylation of a PTEN substrate Rab7, a regulator of endosomal maturation. Inhibition of PTEN activity prevented dephosphorylation of Rab7. Phosphorylated Rab7, in turn, blocked early endosome to late endosome maturation and promoted nuclear localization of the virus and productive infection.

Pten inhibition dedifferentiates long-distance axon-regenerating intrinsically photosensitive retinal ganglion cells and upregulates mitochondria-associated Dynlt1a and Lars2.

Central nervous system projection neurons fail to spontaneously regenerate injured axons. Targeting developmentally regulated genes in order to reactivate embryonic intrinsic axon growth capacity or targeting pro-growth tumor suppressor genes such as Pten promotes long-distance axon regeneration in only a small subset of injured retinal ganglion cells (RGCs), despite many RGCs regenerating short-distance axons. A recent study identified αRGCs as the primary type that regenerates short-distance axons in response to Pten inhibition, but the rare types which regenerate long-distance axons, and cellular features that enable such response, remained unknown. Here, we used a new method for capturing specifically the rare long-distance axon-regenerating RGCs, and also compared their transcriptomes with embryonic RGCs, in order to answer these questions. We found the existence of adult non-α intrinsically photosensitive M1 RGC subtypes that retained features of embryonic cell state, and showed that these subtypes partially dedifferentiated towards an embryonic state and regenerated long-distance axons in response to Pten inhibition. We also identified Pten inhibition-upregulated mitochondria-associated genes, Dynlt1a and Lars2, which promote axon regeneration on their own, and thus present novel therapeutic targets.

Coding-independent regulation of the tumor suppressor PTEN by competing endogenous mRNAs.

Here, we demonstrate that protein-coding RNA transcripts can crosstalk by competing for common microRNAs, with microRNA response elements as the foundation of this interaction. We have termed such RNA transcripts as competing endogenous RNAs (ceRNAs). We tested this hypothesis in the context of PTEN, a key tumor suppressor whose abundance determines critical outcomes in tumorigenesis. By a combined computational and experimental approach, we identified and validated endogenous protein-coding transcripts that regulate PTEN, antagonize PI3K/AKT signaling, and possess growth- and tumor-suppressive properties. Notably, we also show that these genes display concordant expression patterns with PTEN and copy number loss in cancers. Our study presents a road map for the prediction and validation of ceRNA activity and networks and thus imparts a trans-regulatory function to protein-coding mRNAs.

Nuclear PTEN regulates the APC-CDH1 tumor-suppressive complex in a phosphatase-independent manner.

PTEN is a frequently mutated tumor suppressor gene that opposes the PI3K/AKT pathway through dephosphorylation of phosphoinositide-3,4,5-triphosphate. Recently, nuclear compartmentalization of PTEN was found as a key component of its tumor-suppressive activity; however its nuclear function remains poorly defined. Here we show that nuclear PTEN interacts with APC/C, promotes APC/C association with CDH1, and thereby enhances the tumor-suppressive activity of the APC-CDH1 complex. We find that nuclear exclusion but not phosphatase inactivation of PTEN impairs APC-CDH1. This nuclear function of PTEN provides a straightforward mechanistic explanation for the fail-safe cellular senescence response elicited by acute PTEN loss and the tumor-suppressive activity of catalytically inactive PTEN. Importantly, we demonstrate that PTEN mutant and PTEN null states are not synonymous as they are differentially sensitive to pharmacological inhibition of APC-CDH1 targets such as PLK1 and Aurora kinases. This finding identifies a strategy for cancer patient stratification and, thus, optimization of targeted therapies. PAPERCLIP: