Akt Hyperactivation Research Articles

PKMYT1 inhibits lung adenocarcinoma progression by abrogating AKT1 activity.

AKT hyperactivation drives malignant phenotypes in lung cancer via promoting tumor cell proliferation and survival. However, the relationship between dysregulation of cell cycle progression and AKT1 kinase activity is still not clear. Following the expression level of PKMYT1 in lung cancer, we performed cell proliferation, migration, invasion, and xenograft assays to determine the function of PKMYT1. We used RNA-seq to explore the anti-tumor mechanism of PKMYT1 and examined the effect of PKMYT1 on AKT1 activity. In this study, we report that PKMYT1 is downregulated in lung adenocarcinoma (LUAD) tissues and its low expression predicts a poor prognosis in LUAD patients. PKMYT1 exerts potent tumor-suppressive functions in LUAD cells by inhibiting AKT1 activation and thereby repressing cell cycle progression, which depends on its tyrosine and threonine protein kinase activity. Interestingly, PKMYT1 could directly bind AKT1 to abrogate AKT1 activation. Moreover, silencing AKT1 and inhibitors targeting the AKT pathway effectively reverse the promoting effects of PKMYT1 knockdown on proliferation, migration and invasion of LUAD cells. This work reveals the anti-tumor effect of PKMYT1 in LUAD and provides evidence to clarify the dual roles of PKMYT1 in tumor progression. Moreover, our findings broaden the current understandings on AKT1 activation and identify PKMYT1 as a potential negative regulator of AKT1 kinase activity, providing further insights into targeting the AKT pathway in LUAD.

AKT Regulation of ORAI1-Mediated Calcium Influx in Breast Cancer Cells.

Simple SummaryA remodeling in calcium homeostasis and the protein kinase AKT signaling pathway often promotes tumorigenic traits in cancer cells. Changes in calcium signaling can be mediated through altered expression or activity of calcium channels and pumps, which constitute a class of targetable therapeutic targets. Currently, the interplay between the two signaling pathways in breast cancer cells is unclear. A better understanding of the association between calcium and AKT signaling, and the molecular players involved may identify novel therapeutic strategies for breast cancers with abnormal AKT signaling. Using fluorescence calcium imaging and gene silencing/knockout techniques, we showed that increased AKT activation results in increased calcium entry, and that this is mediated through ORAI1 calcium channels. Future studies exploring therapeutic strategies to target PTEN-deficient or hyperactivated AKT cancers should consider this novel correlation between AKT activation and ORAI1-mediated calcium influx.Although breast cancer cells often exhibit both abnormal AKT signaling and calcium signaling, the association between these two pathways is unclear. Using a combination of pharmacological tools, siRNA and CRISPR/Cas9 gene silencing techniques, we investigated the association between PTEN, AKT phosphorylation and calcium signaling in a basal breast cancer cell line. We found that siRNA-mediated PTEN silencing promotes AKT phosphorylation and calcium influx in MDA-MB-231 cells. This increase in AKT phosphorylation and calcium influx was phenocopied by the pharmacological AKT activator, SC79. The increased calcium influx associated with SC79 is inhibited by silencing AKT2, but not AKT1. This increase in calcium influx is suppressed when the store-operated calcium channel, ORAI1 is silenced. The results from this study open a novel avenue for therapeutic targeting of cancer cells with increased AKT activation. Given the association between ORAI1 and breast cancer, ORAI1 is a possible therapeutic target in cancers with abnormal AKT signaling.

Richter Syndrome: From Molecular Pathogenesis to Druggable Targets.

Simple SummaryChronic lymphocytic leukemia represents the most frequent leukemia in adults and evolves from an indolent phase to an aggressive lymphoma in 5–10% of the cases. Such clinico-pathologic transformation is known as Richter syndrome and represents a major unmet medical need because of treatment refractoriness, lack of targeted therapeutic strategies and unsatisfactory survival rates. Molecular investigations by innovative approaches have clarified in detail the pathogenesis of Richter syndrome and have led to the identification of the genetic and biologic changes associated with its evolution from indolent to aggressive disease. Knowledge of the molecular profile of Richter syndrome has revealed several molecular targets that may be exploited for devising novel therapeutic strategies, both with small molecules acting as pathway inhibitors and with monoclonal antibodies, that also include drug immunoconjugates. Clinical trials with these novel medicines are ongoing and may pave the way to a precision medicine approach to Richter syndrome.Richter syndrome (RS) represents the occurrence of an aggressive lymphoma, most commonly diffuse large B-cell lymphoma (DLBCL), in patients with chronic lymphocytic leukemia (CLL). Most cases of RS originate from the direct transformation of CLL, whereas 20% are de novo DLBCL arising as secondary malignancies. Multiple molecular mechanisms contribute to RS pathogenesis. B-cell receptor (BCR) overreactivity to multiple autoantigens is due to frequent stereotyped BCR configuration. Genetic lesions of TP53, CDKN2A, NOTCH1 and c-MYC deregulate DNA damage response, tumor suppression, apoptosis, cell cycle and proliferation. Hyperactivation of Akt and NOTCH1 signaling also plays a role. Altered expression of PD-1/PD-L1 and of other immune checkpoints leads to RS resistance to cytotoxicity exerted by T-cells. The molecular features of RS provide vulnerabilities for therapy. Targeting BCR signaling with noncovalent BTK inhibitors shows encouraging results, as does the combination of BCL2 inhibitors with chemoimmunotherapy. The association of immune checkpoint inhibitors with BCL2 inhibitors and anti-CD20 monoclonal antibodies is explored in early phase clinical trials with promising results. The development of patient-derived xenograft mice models reveals new molecular targets for RS, exemplified by ROR1. Although RS still represents an unmet medical need, understanding its biology is opening new avenues for precision medicine therapy.

Identification and development of a subtype-selective allosteric AKT inhibitor suitable for clinical development

The serine/threonine protein kinase AKT plays a pivotal role within the PI3K pathway in regulating cellular proliferation and apoptotic cellular functions, and AKT hyper-activation via gene amplification and/or mutation has been implicated in multiple human malignancies. There are 3 AKT isoenzymes (AKT1-3) which mediate critical, non-redundant functions. We present the discovery and development of ALM301, a novel, allosteric, sub-type selective inhibitor of AKT1/2. ALM301 binds in an allosteric pocket created by the combined movement of the PH domain and the catalytic domain, resulting in a DFG out conformation. ALM301 was shown to be highly selective against a panel of over 450 kinases and potently inhibited cellular proliferation. These effects were particularly pronounced in MCF-7 cells containing a PI3KCA mutation. Subsequent cellular downstream pathway analysis in this sensitive cell line revealed potent inhibition of pAKT signalling up to 48 h post dosing. ALM301 treatment was well tolerated in an MCF-7 xenograft model and led to a dose-dependent reduction in tumour growth. Enhanced efficacy was observed in combination with tamoxifen. In summary, ALM301 is a highly specific AKT 1/2 inhibitor with an excellent pharmacological profile suitable for further clinical development.

KP372-1-Induced AKT Hyperactivation Blocks DNA Repair to Synergize With PARP Inhibitor Rucaparib via Inhibiting FOXO3a/GADD45α Pathway.

Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) have exhibited great promise in the treatment of tumors with homologous recombination (HR) deficiency, however, PARPi resistance, which ultimately recovers DNA repair and cell progress, has become an enormous clinical challenge. Recently, KP372-1 was identified as a novel potential anticancer agent that targeted the redox enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO1), to induce extensive reactive oxygen species (ROS) generation that amplified DNA damage, leading to cancer cell death. To overcome PARPi resistance and expand its therapeutic utility, we investigated whether a combination therapy of a sublethal dose of KP372-1 with a nontoxic dose of PARPi rucaparib would synergize and enhance lethality in NQO1 over-expressing cancers. We reported that the combination treatment of KP372-1 and rucaparib induced a transient and dramatic AKT hyperactivation that inhibited DNA repair by regulating FOXO3a/GADD45α pathway, which enhanced PARPi lethality and overcame PARPi resistance. We further found that PARP inhibition blocked KP372-1-induced PARP1 hyperactivation to reverse NAD+/ATP loss that promoted Ca2+-dependent autophagy and apoptosis. Moreover, pretreatment of cells with BAPTA-AM, a cytosolic Ca2+ chelator, dramatically rescued KP372-1- or combination treatment-induced lethality and significantly suppressed PAR formation and γH2AX activation. Finally, we demonstrated that this combination therapy enhanced accumulation of both agents in mouse tumor tissues and synergistically suppressed tumor growth in orthotopic pancreatic and non-small-cell lung cancer xenograft models. Together, our study provides novel preclinical evidence for new combination therapy in NQO1+ solid tumors that may broaden the clinical utility of PARPi.

Phosphate Metabolic Inhibition Contributes to Irradiation-Induced Myelosuppression through Dampening Hematopoietic Stem Cell Survival.

Myelosuppression is a common and intractable side effect of cancer therapies including radiotherapy and chemotherapy, while the underlying mechanism remains incompletely understood. Here, using a mouse model of radiotherapy-induced myelosuppression, we show that inorganic phosphate (Pi) metabolism is acutely inhibited in hematopoietic stem cells (HSCs) during irradiation-induced myelosuppression, and closely correlated with the severity and prognosis of myelosuppression. Mechanistically, the acute Pi metabolic inhibition in HSCs results from extrinsic Pi loss in the bone marrow niche and the intrinsic transcriptional suppression of soluble carrier family 20 member 1 (SLC20A1)-mediated Pi uptake by p53. Meanwhile, Pi metabolic inhibition blunts irradiation-induced Akt hyperactivation in HSCs, thereby weakening its ability to counteract p53-mediated Pi metabolic inhibition and the apoptosis of HSCs and consequently contributing to myelosuppression progression. Conversely, the modulation of the Pi metabolism in HSCs via a high Pi diet or renal Klotho deficiency protects against irradiation-induced myelosuppression. These findings reveal that Pi metabolism and HSC survival are causally linked by the Akt/p53–SLC20A1 axis during myelosuppression and provide valuable insights into the pathogenesis and management of myelosuppression.

Targeting Neuropilin‐2 in Lymphatic Malformation

Lymphatic malformations (LM) are sporadic, non‐familial congenital lesions consisting of dilated, abnormal lymphatic vessels that can cause severe lymphedema, disfigurement, organ dysfunction, and in some cases morbidity. These vascular anomalies grow slowly but progress over time and recur after surgery; no effective drugs or preventions currently exist for LM. Genome sequencing identified that approximately 80% of LM patients have somatic activating mutations in the PIK3CA gene. Lymphatic endothelial cells (LEC) expressing mutant PIK3CAH1047Rinduce both macrocystic and microcystic LM in mouse models. Our goal is to understand the cellular and molecular processes that regulate LM growth in order to develop novel therapeutic strategies for patients with LM. Neuropilin‐2 (NRP2) is an essential co‐receptor for VEGFC/VEGFR3 signaling through the PI3K/AKT pathway which is crucial for LEC viability and motility, yet NRP2 also mediates inhibitory signals from its Semaphorin‐3F (SEMA3F) ligand to attenuate activation of AKT and induce cytostasis. Our results using immunohistochemistry to detect NRP2 protein in paraffin‐embedded human samples indicate that NRP2 is highly upregulated in LM lesions compared to normal lymphatic vessels. LM‐LEC primary cell lines, derived separately from several patients, show increased NRP2 levels and hyper‐activated AKT compared to primary LEC derived from healthy human dermis. Additionally, LEC isolated from the skin of transgenic mice (prox1‐creERT2;Nrp2 f/f) specifically lacking Nrp2 in lymphatic vessels following tamoxifen induction (LEC‐Nrp2‐KO) show suppressed proliferation and migration following VEGF‐C stimulation compared to normal LEC isolated from wildtype mice. Importantly, SEMA3F exogenous protein, in a dose‐dependent manner, inhibits the proliferation of normal LEC and LM‐LEC and attenuates the phosphorylation of AKT. Taken together, our study suggests that NRP2 may be an important target for therapy in LM patients. Future studies using SEMA3F protein or specific antibodies targeting the VEGF‐binding domain of NRP2 will be tested in preclinical trials.

Inhibition of BTK and PI3Kδ impairs the development of human JMML stem and progenitor cells

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasia that lacks effective targeted chemotherapies. Clinically, JMML manifests as monocytic leukocytosis, splenomegaly with consequential thrombocytopenia. Most commonly, patients have gain-of-function (GOF) oncogenic mutations in PTPN11 (SHP2), leading to Erk and Akt hyperactivation. Mechanism(s) involved in co-regulation of Erk and Akt in the context of GOF SHP2 are poorly understood. Here, we show that Bruton's tyrosine kinase (BTK) is hyperphosphorylated in GOF Shp2-bearing cells and utilizes B cell adaptor for PI3K to cooperate with p110δ, the catalytic subunit of PI3K. Dual inhibition of BTK and p110δ reduces the activation of both Erk and Akt. Invivo, individual targeting of BTK or p110δ in a mouse model of human JMML equally reduces monocytosis and splenomegaly; however, the combined treatment results in a more robust inhibition and uniquely rescues anemia and thrombocytopenia. RNA-seq analysis of drug-treated mice showed a profound reduction in the expression of genes associated with leukemic cell migration and inflammation, leading to correction in the infiltration of leukemic cells in the lung, liver, and spleen. Remarkably, in a patient derived xenograft model of JMML, leukemia-initiating stem and progenitor cells were potently inhibited in response to the dual drug treatment.

Meroterpenoid-Rich Ethanoic Extract of Sargassum macrocarpum Ameliorates Dextran Sulfate Sodium-Induced Colitis in Mice.

Colitis is a colon mucosal disorder characterized by intestinal damage and inflammation. This current study aimed to evaluate the effect of meroterpenoid-rich ethanoic extract of a brown algae, Sargassum macrocarpum (MES) on dextran sulfate sodium (DSS)-induced colitis in mice and explore the possible mechanisms. Mice were given 4% DSS in drinking water for 7 days to induce colitis, followed by 3 days of regular water. MES (12 mg/kg body weight) or celecoxib (10 mg/kg body weight) was administrated orally to mice on a daily basis during these 10 days. Both MES and celecoxib supplementations significantly attenuated DSS-induced weight loss, shortening of colon length, elevated myeloperoxidase activity as well as histomorphological changes of colon. MES and celecoxib reduced the inflammation level of colon tissue, as indicated by its suppression on a panel of pro-inflammatory cytokines, including interleukin (IL)-1β, IL-17, tumor necrosis factor α, and interferon γ, and a group of inflammatory proteins, including intracellular adhesion molecule 1, vascular adhesion molecule 1, matrix metalloproteinase (MMP)-2, MMP-9, MMP-13, and inducible nitric oxidase. In addition, their administration down-regulated pro-inflammatory cytokines in serum. Moreover, the supplementation of MES suppressed the DSS-induced hyperactivation of Akt, JNK, and NF-κB signaling pathways. Taken together, our results demonstrate that MES ameliorates DSS-induced colitis in mice, suggesting that MES may have therapeutic implications for the treatment of colitis.

CRISPR/Cas9-mediated Bag-1 knockout increased mesenchymal characteristics of MCF-7 cells via Akt hyperactivation-mediated actin cytoskeleton remodeling.

Bag-1 protein is a crucial target in cancer to increase the survival and proliferation of cells. The Bag-1 expression is significantly upregulated in primary and metastatic cancer patients compared to normal breast tissue. Overexpression of Bag-1 decreases the efficiency of conventional chemotherapeutic drugs, whereas Bag-1 silencing enhances the apoptotic efficiency of therapeutics, mostly in hormone-positive breast cancer subtypes. In this study, we generated stable Bag-1 knockout (KO) MCF-7 breast cancer cells to monitor stress-mediated cellular alterations in comparison to wild type (wt) and Bag-1 overexpressing (Bag-1 OE) MCF-7 cells. Validation and characterization studies of Bag-1 KO cells showed different cellular morphology with hyperactive Akt signaling, which caused stress-mediated actin reorganization, focal adhesion decrease and led to mesenchymal characteristics in MCF-7 cells. A potent Akt inhibitor, MK-2206, suppressed mesenchymal transition in Bag-1 KO cells. Similar results were obtained following the recovery of Bag-1 isoforms (Bag-1S, M, or L) in Bag-1 KO cells. The findings of this study emphasized that Bag-1 is a mediator of actin-mediated cytoskeleton organization through regulating Akt activation.

AD-linked R47H-TREM2 mutation induces disease-enhancing microglial states via AKT hyperactivation.

The hemizygous R47H variant of triggering receptor expressed on myeloid cells 2 (TREM2), a microglia-specific gene in the brain, increases risk for late-onset Alzheimer’s disease (AD). Using transcriptomic analysis of single nuclei from brain tissues of patients with AD carrying the R47H mutation or the common variant (CV)–TREM2, we found that R47H-associated microglial subpopulations had enhanced inflammatory signatures reminiscent of previously identified disease-associated microglia (DAM) and hyperactivation of AKT, one of the signaling pathways downstream of TREM2. We established a tauopathy mouse model with heterozygous knock-in of the human TREM2 with the R47H mutation or CV and found that R47H induced and exacerbated TAU-mediated spatial memory deficits in female mice. Single-cell transcriptomic analysis of microglia from these mice also revealed transcriptomic changes induced by R47H that had substantial overlaps with R47H microglia in human AD brains, including robust increases in proinflammatory cytokines, activation of AKT signaling, and elevation of a subset of DAM signatures. Pharmacological AKT inhibition with MK-2206 largely reversed the enhanced inflammatory signatures in primary R47H microglia treated with TAU fibrils. In R47H heterozygous tauopathy mice, MK-2206 treatment abolished a tauopathy-dependent microglial subcluster and rescued tauopathy-induced synapse loss. By uncovering disease-enhancing mechanisms of the R47H mutation conserved in human and mouse, our study supports inhibitors of AKT signaling as a microglial modulating strategy to treat AD.

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

AbstractHuman 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.

Comprehensive in Vitro Characterization of CXCR4WHIM variants to Decipher Genotype-Phenotype Correlations in WHIM Syndrome

▪Background: WHIM (Wart, Hypogammaglobulinemia, Infections, Myelokathexis) syndrome is a rare, autosomal-dominant primary immunodeficiency with neutropenia and lymphopenia. The clinical presentation may include recurrent infections, and increased susceptibility to human papillomavirus. In >80% of cases, WHIM syndrome is caused by heterozygous gain-of-function (GOF) mutations in C-X-C chemokine receptor 4 (CXCR4), with >12 variants reported in WHIM syndrome to date (nonsense [NS], frameshift [FS], and missense[MS]) spanning 27 C-terminal amino acids . These mutations cause hyperactivation of downstream signaling and retention of WBC in the bone marrow (McDermott D, et al. Immunol Rev. 2019;287(1):91-102; Beaussant S, et al. Orphanet J Rare Dis. 2012;7(71):1-14). To date, a comprehensive study characterizing the functional abnormalities caused by pathogenic CXCR4 mutations and correlating these measures with clinical presentation in patients has not been conducted. Here, we aimed to establish genotype-phenotype correlations for all known pathogenic variants using in vitro functional assays. These assays characterize CXCR4 receptor trafficking and downstream signaling, which will enable the long-term goal of assessing pathogenicity of novel CXCR4 variants of uncertain significance (VUS). We further aimed to assess the in vitro response of each variant to mavorixafor, an investigational CXCR4 antagonist.Methods: We used the CXCR4-negative K562 cell line as a model system to express all 14 known CXCR4 variants identified in patients diagnosed with WHIM syndrome (previous reports, ClinVar, and genetic screening initiatives [Invitae PATH4WARD]). The effects of the mutations on CXCR4 receptor trafficking (internalization and degradation), downstream signaling (Ca 2+ mobilization, cAMP inhibition, ERK and AKT activation), and chemotaxis were studied in parallel in a series of assays in cells stimulated with the natural ligand CXCL12. All in vitro functional parameters characterized were investigated for potential correlations with the clinical phenotypes reported for each variant, including disease manifestations and biomarkers.Results: The most conserved feature of the 14 CXCR4 variants was an impaired receptor internalization in response to CXCL12, evidenced by higher percentage of CXCR4 receptors remaining on the cell surface compared to untreated control, with truncated variants showing maximum impairment and the MS variant E343K being least affected. The decreased CXCR4 internalization correlated with both decreased CXCR4 degradation and increased cAMP inhibition. When stimulated with CXCL12, most variants demonstrated a higher amplitude and duration of ERK and AKT activation. Chemotactic responses to CXCL12 were diverse, depending on the variant sequence and subtype. While Ca 2+ mobilization was not enhanced compared to wild-type (WT) CXCR4-expressing cells in this assay, mavorixafor demonstrated inhibition of Ca 2+ mobilization in all CXCR4 mutant cells with a trend toward greater effect in the NS variants. In addition, both ERK and AKT activation decreased with increasing concentration of mavorixafor. Correlation analyses of the functional parameters in cells expressing mutated or WT CXCR4 and clinical manifestations or WBC counts in patients with these mutations revealed that the CXCR4 internalization defect strongly correlated with severity of peripheral blood cytopenias (ie, decreases in absolute neutrophil counts, and in CD3+ and CD4+ T-cell levels), which was paralleled by an increased susceptibility to recurrent infections. In addition, AKT hyperactivation correlated with lower IgA and decreased CD4+ T-cell levels (Figure).Conclusions: In the current study, we performed a detailed functional analysis of the entire spectrum of CXCR4 WHIM mutations known to date. In vitro CXCR4 receptor internalization correlates with WBC cytopenias and an increased susceptibility to recurrent infections in patients with CXCR4 GOF mutations. These data suggest that CXCR4 internalization and AKT activation may be used as key assays for the assessment of CXCR4 variant pathogenicity in vitro and potentially as WHIM-related disease biomarkers. Additionally, all tested CXCR4 variant cell lines were sensitive to mavorixafor at clinically relevant concentrations, rescuing defective GOF signaling toward that of WT CXCR4-expressing cells. [Display omitted] DisclosuresZmajkovicova: X4 Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Maier-Munsa: X4 Pharmaceuticals: Current Employment. Maierhofer: X4 Pharmaceuticals: Current Employment. Taveras: X4 Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Badarau: X4 Pharmaceuticals: Current equity holder in publicly-traded company, Ended employment in the past 24 months.

Discovery and Characterization of the Potent and Highly Selective 1,7-Naphthyridine-Based Inhibitors BAY-091 and BAY-297 of the Kinase PIP4K2A.

PIP4K2A is an insufficiently studied type II lipid kinase that catalyzes the conversion of phosphatidylinositol-5-phosphate (PI5P) into phosphatidylinositol 4,5-bisphosphate (PI4,5P2). The involvement of PIP4K2A/B in cancer has been suggested, particularly in the context of p53 mutant/null tumors. PIP4K2A/B depletion has been shown to induce tumor growth inhibition, possibly due to hyperactivation of AKT and reactive oxygen species-mediated apoptosis. Herein, we report the identification of the novel potent and highly selective inhibitors BAY-091 and BAY-297 of the kinase PIP4K2A by high-throughput screening and subsequent structure-based optimization. Cellular target engagement of BAY-091 and BAY-297 was demonstrated using cellular thermal shift assay technology. However, inhibition of PIP4K2A with BAY-091 or BAY-297 did not translate into the hypothesized mode of action and antiproliferative activity in p53-deficient tumor cells. Therefore, BAY-091 and BAY-297 serve as valuable chemical probes to study PIP4K2A signaling and its involvement in pathophysiological conditions such as cancer.

Proteotoxic Stress as an Exploitable Vulnerability in Cells with Hyperactive AKT.

Hyperactivity of serine-threonine kinase AKT is one of the most common molecular abnormalities in cancer, where it contributes to poor outcomes by facilitating the growth and survival of malignant cells. Despite its well-documented anti-apoptotic effects, hyperactivity of AKT is also known to be stressful to a cell. In an attempt to better elucidate this phenomenon, we observed the signs of proteotoxic stress in cells that harbor hyperactive AKT or have lost its principal negative regulator, PTEN. The activity of HSF1 was predictably elevated under these circumstances. However, such cells proved more sensitive to various regimens of heat shock, including the conditions that were well-tolerated by syngeneic cells without AKT hyperactivity. The sensitizing effect of hyperactive AKT was also seen in HSF1-deficient cells, suggesting that the phenomenon does not require the regulation of HSF1 by this kinase. Notably, the elevated activity of AKT was accompanied by increased levels of XBP1, a key component of cell defense against proteotoxic stress. Interestingly, the cells harboring hyperactive AKT were also more dependent on XBP1 for their growth. Our observations suggest that proteotoxic stress conferred by hyperactive AKT represents a targetable vulnerability, which can be exploited by either elevating the stress above the level tolerated by such cells or by eliminating the factors that enable such tolerance.

Retinoid Metabolism in the Degeneration of Pten-Deficient Mouse Retinal Pigment Epithelium.

In vertebrate eyes, the retinal pigment epithelium (RPE) provides structural and functional homeostasis to the retina. The RPE takes up retinol (ROL) to be dehydrogenated and isomerized to 11-cis-retinaldehyde (11-cis-RAL), which is a functional photopigment in mammalian photoreceptors. As excessive ROL is toxic, the RPE must also establish mechanisms to protect against ROL toxicity. Here, we found that the levels of retinol dehydrogenases (RDHs) are commonly decreased in phosphatase tensin homolog (Pten)-deficient mouse RPE, which degenerates due to elevated ROL and that can be rescued by feeding a ROL-free diet. We also identified that RDH gene expression is regulated by forkhead box O (FOXO) transcription factors, which are inactivated by hyperactive Akt in the Pten-deficient mouse RPE. Together, our findings suggest that a homeostatic pathway comprising PTEN, FOXO, and RDH can protect the RPE from ROL toxicity.

Ipatasertib plus abiraterone and prednisolone in metastatic castration-resistant prostate cancer (IPATential150): a multicentre, randomised, double-blind, phase 3 trial

The PI3K/AKT and androgen-receptor pathways are dysregulated in metastatic castration-resistant prostate cancers (mCRPCs); tumours with functional PTEN-loss status have hyperactivated AKT signalling. Dual pathway inhibition with AKT inhibitor ipatasertib plus abiraterone might have greater benefit than abiraterone alone. We aimed to compare ipatasertib plus abiraterone with placebo plus abiraterone in patients with previously untreated mCRPC with or without tumour PTEN loss. We did a randomised, double-blind, phase 3 trial at 200 sites across 26 countries or regions. Patients aged 18 years or older with previously untreated asymptomatic or mildly symptomatic mCRPC who had progressive disease and Eastern Collaborative Oncology Group performance status of 0 or 1 were randomly assigned (1:1; permuted block method) to receive ipatasertib (400 mg once daily orally) plus abiraterone (1000 mg once daily orally) and prednisolone (5 mg twice a day orally) or placebo plus abiraterone and prednisolone (with the same dosing schedule). Patients received study treatment until disease progression, intolerable toxicity, withdrawal from the study, or study completion. Stratification factors were previous taxane-based therapy for hormone-sensitive prostate cancer, type of progression, presence of visceral metastasis, and tumour PTEN-loss status by immunohistochemistry. Patients, investigators, and the study sponsor were masked to the treatment allocation. The coprimary endpoints were investigator-assessed radiographical progression-free survival in the PTEN-loss-by-immunohistochemistry population and in the intention-to-treat population. This study is ongoing and is registered with ClinicalTrials.gov, NCT03072238. Between June 30, 2017, and Jan 17, 2019, 1611 patients were screened for eligibility and 1101 (68%) were enrolled; 554 (50%) were assigned to the placebo-abiraterone group and 547 (50%) to the ipatasertib-abiraterone group. At data cutoff (March 16, 2020), median follow-up duration was 19 months (range 0-33). In the 521 (47%) patients who had tumours with PTEN loss by immunohistochemistry (261 in the placebo-abiraterone group and 260 in the ipatasertib-abiraterone group), median radiographical progression-free survival was 16·5 months (95% CI 13·9-17·0) in the placebo-abiraterone group and 18·5 months (16·3-22·1) in the ipatasertib-abiraterone group (hazard ratio [HR] 0·77 [95% CI 0·61-0·98]; p=0·034; significant at α=0·04). In the intention-to-treat population, median progression-free survival was 16·6 months (95% CI 15·6-19·1) in the placebo-abiraterone group and 19·2 months (16·5-22·3) in the ipatasertib-abiraterone group (HR 0·84 [95% CI 0·71-0·99]; p=0·043; not significant at α=0·01). Grade 3 or higher adverse events occurred in 213 (39%) of 546 patients in the placebo-abiraterone group and in 386 (70%) of 551 patients in the ipatasertib-abiraterone group; adverse events leading to discontinuation of placebo or ipatasertib occurred in 28 (5%) in the placebo-abiraterone group and 116 (21%) in the ipatasertib-abiraterone group. Deaths due to adverse events deemed related to treatment occurred in two patients (<1%; acute myocardial infarction [n=1] and lower respiratory tract infection [n=1]) in the placebo-abiraterone group and in two patients (<1%; hyperglycaemia [n=1] and chemical pneumonitis [n=1]) in the ipastasertb-abiraterone group. Ipatasertib plus abiraterone significantly improved radiographical progression-free survival compared with placebo plus abiraterone among patients with mCRPC with PTEN-loss tumours, but there was no significant difference between the groups in the intention-to-treat population. Adverse events were consistent with the known safety profiles of each agent. These data suggest that combined AKT and androgen-receptor signalling pathway inhibition with ipatasertib and abiraterone is a potential treatment for men with PTEN-loss mCRPC, a population with a poor prognosis. F Hoffmann-La Roche and Genentech.

PRMT5-mediated arginine methylation activates AKT kinase to govern tumorigenesis

AKT is involved in a number of key cellular processes including cell proliferation, apoptosis and metabolism. Hyperactivation of AKT is associated with many pathological conditions, particularly cancers. Emerging evidence indicates that arginine methylation is involved in modulating AKT signaling pathway. However, whether and how arginine methylation directly regulates AKT kinase activity remain unknown. Here we report that protein arginine methyltransferase 5 (PRMT5), but not other PRMTs, promotes AKT activation by catalyzing symmetric dimethylation of AKT1 at arginine 391 (R391). Mechanistically, AKT1-R391 methylation cooperates with phosphatidylinositol 3,4,5 trisphosphate (PIP3) to relieve the pleckstrin homology (PH)-in conformation, leading to AKT1 membrane translocation and subsequent activation by phosphoinositide-dependent kinase-1 (PDK1) and the mechanistic target of rapamycin complex 2 (mTORC2). As a result, deficiency in AKT1-R391 methylation significantly suppresses AKT1 kinase activity and tumorigenesis. Lastly, we show that PRMT5 inhibitor synergizes with AKT inhibitor or chemotherapeutic drugs to enhance cell death. Altogether, our study suggests that R391 methylation is an important step for AKT activation and its oncogenic function.

Cleavage of Syndecan-1 Promotes the Proliferation of the Basal-Like Breast Cancer Cell Line BT-549 Via Akt SUMOylation.

Basal-like breast cancer is characterized by an aggressive clinical outcome and presence of metastasis, for which effective therapies are unavailable. We have previously shown that chondroitin 4-O-sulfotransferase-1 (C4ST-1) controls the invasive properties of the basal-like breast cancer cell line BT-549 by inducing matrix metalloproteinase (MMP) expression through the N-cadherin/β-catenin pathway. Here we report that C4ST-1 controls the proliferation of BT-549 cells via the MMP-dependent cleavage of syndecan-1. Syndecan-1 is a membrane-bound proteoglycan associated with an aggressive phenotype and poor prognosis in breast cancer. In addition, the cleavage of syndecan-1 at a specific juxtamembrane cleavage site is implicated in the pathophysiological response in breast cancer. Knockout of C4ST-1 remarkably suppressed both the cleavage of syndecan-1 and proliferation of BT-549 cells. Kinases (AKT1, ERK1/2, PI3K, and STAT3) comprising cancer proliferative pathways are phosphorylated in C4ST-1 knockout cells at a level similar to that in parental BT-549 cells, whereas levels of phosphorylated S6 kinase and SUMOylated AKT (hyperactivated AKT observed in breast cancer) decreased in C4ST-1 knockout cells. An MMP inhibitor, GM6001, suppressed the small ubiquitin-like modifier (SUMO) modification of AKT, suggesting that cleavage of syndecan-1 by MMPs is involved in the SUMO modification of AKT. Forced expression of the cytoplasmic domain of syndecan-1, which is generated by MMP-dependent cleavage, increased the SUMO modification of AKT and global protein SUMOylation. Furthermore, syndecan-1 C-terminal domain-expressing BT-549 cells were more proliferative and sensitive to a potent SUMOylation inhibitor, tannic acid, compared with BT-549 cells transfected with an empty expression vector. These findings assign new functions to the C-terminal fragment of syndecan-1 generated by MMP-dependent proteolysis, thereby broadening our understanding of their physiological importance and implying that the therapeutic inhibition of syndecan-1 cleavage could affect the progression of basal-like breast cancer.

Tnfaip8 Regulates Intestinal Stemness and Terminal Differentiation of Secretory Progenitors

The intestine is a dynamic environment that relies on constant self-renewal of the intestinal epithelium to maintain homeostasis. Maintenance of stem cell populations as well as coordinated cell fate determination is necessary for coordinating intestinal function and regeneration. Tumor necrosis factor-alpha-induced protein 8 (TIPE0) is a regulator of phosphoinositide 3-kinase (PI3K)-mediated signaling through the sequestration of membranous PIP2. The PI3K/protein kinase B (Akt)/ꞵ-catenin pathway regulates proliferation in the intestine in conjunction with Wnt signalling. We recently found that loss of Tipe0 leads to hyperactivation of microbiome-mediated Akt/ꞵ-catenin signaling in the intestine and a single-cell RNA sequencing survey of Tipe0-/- small intestine found that Tipe0-/- intestinal cells were shifted towards an undifferentiated state, with the notable exception of goblet cells, which were expanded.Here, we performed more in depth analysis of our existing sc-RNASeq data set to better characterize the changes in intestinal differentiation seen with loss of TIPE0, using pseudo-time based approaches. We hypothesized that various transcription factors/epigenetic regulators would be differentially expressed in the Tipe0-/- intestine, underlying the expansion of goblet cells we had previously seen. In these studies, we confirmed that loss of Tipe0 causes a shift towards an undifferentiated state with an increase in Lgr5+ stem cells and transitional enterocytes and a consequent decrease in terminally differentiated enterocytes with the exception of goblet cells which were also enriched. Additionally, in this process we identified three possible novel regulators of terminal cell fate decisions in the intestinal secretory lineage: Nupr1, Kdm4a, and Gatad1. We propose that these novel regulators drive epigenetic changes involved in goblet cell (Nupr1) or tuft cell (Kdm4a and Gatad1) fate commitment and that TIPE0 may play a role in orchestrating terminal differentiation, likely through its regulation of homeostatic Akt/ꞵ-catenin signaling.