The Vitamin C Enantiomers Possess a Comparable Potency in the Induction of Oxidative Stress in Cancer Cells but Differ in Their Toxicity.

The use of vitamin C (VC) in high doses demonstrates a potent tumor suppressive effect by mediating a glucose-dependent oxidative stress in Kirsten rat sarcoma (KRAS) mutant cancer cells. VC with arsenic trioxide (ATO) is a promising drug combination that might lead to the development of effective cancer therapeutics. Considering that a tumor suppressive effect of VC requires its high-dose administration, it is of interest to examine the toxicity of two enantiomers of VC (enantiomer d-optical isomer D-VC and natural l-optical isomer L-VC) in vitro and in vivo. We show that the combinations of L-VC with ATO and D-VC with ATO induced a similar cytotoxic oxidative stress in KrasG12D-expressing mutant cancer cells as indicated by a substantial increase in reactive oxidative species (ROS) production and depolarization of mitochondria. To examine the L-VC and D-VC toxicity effects, we administered high doses of D-VC and L-VC to CD1 mice and carried out an evaluation of their toxic effects. The daily injections of L-VC at a dose of 9.2 g/kg for 18 days were lethal to mice, while 80% of mice remained alive following the similar high-dose administration of D-VC. Following the drug injection courses and histopathological studies, we determined that a natural form of VC (L-VC) is more harmful and toxic to mice when compared to the effects caused by the similar doses of D-VC. Thus, our study indicates that the two enantiomers of VC have a similar potency in the induction of oxidative stress in cancer cells, but D-VC has a distinctive lower toxicity in mice compared to L-VC. While the mechanism of a distinctive toxicity between D-VC and L-VC is yet to be defined, our finding marks D-VC as a more preferable option compared to its natural enantiomer L-VC in clinical settings.

Universal whole-genome Oxford nanopore sequencing of SARS-CoV-2 using tiled amplicons.

We developed a comprehensive multiplexed set of primers adapted for the Oxford Nanopore Rapid Barcoding library kit that allows universal SARS-CoV-2 genome sequencing. This primer set is designed to set up any variants of the primers pool for whole-genome sequencing of SARS-CoV-2 using single- or double-tiled amplicons from 1.2 to 4.8 kb with the Oxford Nanopore. This multiplexed set of primers is also applicable for tasks like targeted SARS-CoV-2 genome sequencing. We proposed here an optimized protocol to synthesize cDNA using Maxima H Minus Reverse Transcriptase with a set of SARS-CoV-2 specific primers, which has high yields of cDNA template for RNA and is capable of long-length cDNA synthesis from a wide range of RNA amounts and quality. The proposed protocol allows whole-genome sequencing of the SARS-CoV-2 virus with tiled amplicons up to 4.8 kb on low-titer virus samples and even where RNA degradation has occurred. This protocol reduces the time and cost from RNA to genome sequence compared to the Midnight multiplex PCR method for SARS-CoV-2 genome sequencing using the Oxford Nanopore.

Whole-Genome Sequencing Among Kazakhstani Children with Early-Onset Epilepsy Revealed New Gene Variants and Phenotypic Variability.

In Kazakhstan, there is insufficient data on genetic epilepsy, which has its own clinical and management implications. Thus, this study aimed to use whole genome sequencing to identify and evaluate genetic variants and genetic structure of early onset epilepsy in the Kazakhstani pediatric population. In this study, for the first time in Kazakhstan, whole genome sequencing was carried out among epilepsy diagnosed children. The study involved 20 pediatric patients with early onset epilepsy and no established cause of the disease during the July-December, 2021. The average age at enrolment was 34.5 months, with a mean age at seizure onset of 6 months. Six patients (30%) were male, and 7 were familial cases. We identified pathogenic and likely pathogenic variants in 14 (70%) cases, among them, 6 novel disease gene variants (KCNQ2, CASK, WWOX, MT-CO3, GRIN2D, and SLC12A5). Other genes associated with the disease were SCN1A (x2), SLC2A1, ARX, CACNA1B, PCDH19, KCNT1, and CHRNA2. Identification of the genetic causes in 70% of cases confirms the general structure of the etiology of early onset epilepsy and the necessity of using NGS in diagnostics. Moreover, the study describes new genotype-phenotypic correlations in genetic epilepsy. Despite certain limitations of the study, it can be concluded that the genetic etiology of pediatric epilepsy in Kazakhstan is very broad and requires further research.

Enhancing an Oxidative "Trojan Horse" Action of Vitamin C with Arsenic Trioxide for Effective Suppression of KRAS-Mutant Cancers: A Promising Path at the Bedside.

The turn-on mutations of the KRAS gene, coding a small GTPase coupling growth factor signaling, are contributing to nearly 25% of all human cancers, leading to highly malignant tumors with poor outcomes. Targeting of oncogenic KRAS remains a most challenging task in oncology. Recently, the specific G12C mutant KRAS inhibitors have been developed but with a limited clinical outcome because they acquire drug resistance. Alternatively, exploiting a metabolic breach of KRAS-mutant cancer cells related to a glucose-dependent sensitivity to oxidative stress is becoming a promising indirect cancer targeting approach. Here, we discuss the use of a vitamin C (VC) acting in high dose as an oxidative "Trojan horse" agent for KRAS-mutant cancer cells that can be potentiated with another oxidizing drug arsenic trioxide (ATO) to obtain a potent and selective cytotoxic impact. Moreover, we outline the advantages of VC's non-natural enantiomer, D-VC, because of its distinctive pharmacokinetics and lower toxicity. Thus, the D-VC and ATO combination shows a promising path to treat KRAS-mutant cancers in clinical settings.

Akt: a key transducer in cancer.

Growth factor signaling plays a pivotal role in diverse biological functions, such as cell growth, apoptosis, senescence, and migration and its deregulation has been linked to various human diseases. Akt kinase is a central player transmitting extracellular clues to various cellular compartments, in turn executing these biological processes. Since the discovery of Akt three decades ago, the tremendous progress towards identifying its upstream regulators and downstream effectors and its roles in cancer has been made, offering novel paradigms and therapeutic strategies for targeting human diseases and cancers with deregulated Akt activation. Unraveling the molecular mechanisms for Akt signaling networks paves the way for developing selective inhibitors targeting Akt and its signaling regulation for the management of human diseases including cancer.

A high scale SARS-CoV-2 profiling by its whole-genome sequencing using Oxford Nanopore Technology in Kazakhstan.

Severe acute respiratory syndrome (SARS-CoV-2) is responsible for the worldwide pandemic, COVID-19. The original viral whole-genome was sequenced by a high-throughput sequencing approach from the samples obtained from Wuhan, China. Real-time gene sequencing is the main parameter to manage viral outbreaks because it expands our understanding of virus proliferation, spread, and evolution. Whole-genome sequencing is critical for SARS-CoV-2 variant surveillance, the development of new vaccines and boosters, and the representation of epidemiological situations in the country. A significant increase in the number of COVID-19 cases confirmed in August 2021 in Kazakhstan facilitated a need to establish an effective and proficient system for further study of SARS-CoV-2 genetic variants and the development of future Kazakhstan's genomic surveillance program. The SARS-CoV-2 whole-genome was sequenced according to SARS-CoV-2 ARTIC protocol (EXP-MRT001) by Oxford Nanopore Technologies at the National Laboratory Astana, Kazakhstan to track viral variants circulating in the country. The 500 samples kindly provided by the Republican Diagnostic Center (UMC-NU) and private laboratory KDL "Olymp" were collected from individuals in Nur-Sultan city diagnosed with COVID-19 from August 2021 to May 2022 using real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR). All samples had a cycle threshold (Ct) value below 20 with an average Ct value of 17.03. The overall average value of sequencing depth coverage for samples is 244X. 341 whole-genome sequences that passed quality control were deposited in the Global initiative on sharing all influenza data (GISAID). The BA.1.1 (n = 189), BA.1 (n = 15), BA.2 (n = 3), BA.1.15 (n = 1), BA.1.17.2 (n = 1) omicron lineages, AY.122 (n = 119), B.1.617.2 (n = 8), AY.111 (n = 2), AY.126 (n = 1), AY.4 (n = 1) delta lineages, one sample B.1.1.7 (n = 1) belongs to alpha lineage, and one sample B.1.637 (n = 1) belongs to small sublineage were detected in this study. This is the first study of SARS-CoV-2 whole-genome sequencing by the ONT approach in Kazakhstan, which can be expanded for the investigation of other emerging viral or bacterial infections on the country level.

The Oxidative Drug Combination for Suppressing KRAS G12D Inducible Tumour Growth.

Background: Kirsten rat sarcoma (KRAS) protein is an essential contributor to the development of pancreatic ductal adenocarcinoma (PDAC). KRAS G12D and G12V mutant tumours are significant challenges in cancer therapy due to high resistance to the treatment. Objective: To determine how effective is the ATO/D-VC combination in suppression of PDAC the mouse transgenic model. This study investigated the antitumour effect of a novel combination of arsenic trioxide (ATO) and D-ascorbic acid isomer (D-VC). Such a combination can be used to treat KRAS mutant cancer by inducing catastrophic oxidative stress. Methods: In this study, we examined the effectiveness of ATO and D-VC on xenograft models-AK192 cells transplanted into mice. Previously, it has been shown that a high concentration of Vitamin C (VC) selectively can kill the cells expressing KRAS. Results: The results of this study demonstrated that the combination of VC with a low dose of the oxidizing drug ATO led to the enhancement of the therapeutic effect. These findings suggest that the combined treatment using ATO and D-VC is a promising approach to overcome the limitation of drug selectivity and efficacy.

Wheat Germination Is Dependent on Plant Target of Rapamycin Signaling.

Germination is a process of seed sprouting that facilitates embryo growth. The breakdown of reserved starch in the endosperm into simple sugars is essential for seed germination and subsequent seedling growth. At the early stage of germination, gibberellic acid (GA) activates transcription factor GAMYB to promote de novo synthesis of isoforms of α-amylase in the aleurone layer and scutellar epithelium of the embryo. Here, we demonstrate that wheat germination is regulated by plant target of rapamycin (TOR) signaling. TOR is a central component of the essential-nutrient-dependent pathway controlling cell growth in all eukaryotes. It is known that rapamycin, a highly specific allosteric inhibitor of TOR, is effective in yeast and animal cells but ineffective in most of higher plants likely owing to structural differences in ubiquitous rapamycin receptor FKBP12. The action of rapamycin on wheat growth has not been studied. Our data show that rapamycin inhibits germination of wheat seeds and of their isolated embryos in a dose-dependent manner. The involvement of Triticum aestivum TOR (TaTOR) in wheat germination was consistent with the suppression of wheat embryo growth by specific inhibitors of the TOR kinase: pp242 or torin1. Rapamycin or torin1 interfered with GA function in germination because of a potent inhibitory effect on α-amylase and GAMYB gene expression. The TOR inhibitors selectively targeted the GA-dependent gene expression, whereas expression of the abscisic acid-dependent ABI5 gene was not affected by either rapamycin or torin1. To determine whether the TaTOR kinase activation takes place during wheat germination, we examined phosphorylation of a ribosomal protein, T. aestivum S6 kinase 1 (TaS6K1; a substrate of TOR). The phosphorylation of serine 467 (S467) in a hydrophobic motif on TaS6K1 was induced in a process of germination triggered by GA. Moreover, the germination-induced phosphorylation of TaS6K1 on S467 was dependent on TaTOR and was inhibited by rapamycin or torin1. Besides, a gibberellin biosynthesis inhibitor (paclobutrazol; PBZ) blocked not only α-amylase gene expression but also TaS6K1 phosphorylation in wheat embryos. Thus, a hormonal action of GA turns on the synthesis of α-amylase in wheat germination via activation of the TaTOR-S6K1 signaling pathway.

Author Correction: A hypoxia-responsive TRAF6-ATM-H2AX signalling axis promotes HIF1α activation, tumorigenesis and metastasis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A chirality-dependent action of vitamin C in suppressing Kirsten rat sarcoma mutant tumor growth by the oxidative combination: Rationale for cancer therapeutics.

Kirsten rat sarcoma (KRAS) mutant cancers, which constitute the vast majority of pancreatic tumors, are characterized by their resistance to established therapies and high mortality rates. Here, we developed a novel and extremely effective combinational therapeutic approach to target KRAS mutant tumors through the generation of a cytotoxic oxidative stress. At high concentrations, vitamin C (VC) is known to provoke oxidative stress and selectively kill KRAS mutant cancer cells, although its effects are limited when it is given as monotherapy. We found that the combination of VC and the oxidizing drug arsenic trioxide (ATO) is an effective therapeutic treatment modality. Remarkably, its efficiency is dependent on chirality of VC as its enantiomer d-optical isomer of VC (d-VC) is significantly more potent than the natural l-optical isomer of VC. Thus, our results demonstrate that the oxidizing combination of ATO and d-VC is a promising approach for the treatment of KRAS mutant human cancers.

Uncovering the Role of RNA-Binding Protein hnRNP K in B-Cell Lymphomas.

BACKGROUND: Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is an RNA-binding protein that is aberrantly expressed in cancers. We and others have previously shown that reduced hnRNP K expression downmodulates tumor-suppressive programs. However, overexpression of hnRNP K is the more commonly observed clinical phenomenon, yet its functional consequences and clinical significance remain unknown. METHODS: Clinical implications of hnRNP K overexpression were examined through immunohistochemistry on samples from patients with diffuse large B-cell lymphoma who did not harbor MYC alterations (n = 75). A novel transgenic mouse model that overexpresses hnRNP K specifically in B cells was generated to directly examine the role of hnRNP K overexpression in mice (three transgenic lines). Molecular consequences of hnRNP K overexpression were determined through proteomics, formaldehyde-RNA-immunoprecipitation sequencing, and biochemical assays. Therapeutic response to BET-bromodomain inhibition in the context of hnRNP K overexpression was evaluated in vitro and in vivo (n = 3 per group). All statistical tests were two-sided. RESULTS: hnRNP K is overexpressed in diffuse large B-cell lymphoma patients without MYC genomic alterations. This overexpression is associated with dismal overall survival and progression-free survival (P < .001). Overexpression of hnRNP K in transgenic mice resulted in the development of lymphomas and reduced survival (P < .001 for all transgenic lines; Line 171[n = 30]: hazard ratio [HR] = 64.23, 95% confidence interval [CI] = 26.1 to 158.0; Line 173 [n = 31]: HR = 25.27, 95% CI = 10.3 to 62.1; Line 177 [n = 25]: HR = 119.5, 95% CI = 42.7 to 334.2, compared with wild-type mice). Clinical samples, mouse models, global screening assays, and biochemical studies revealed that hnRNP K's oncogenic potential stems from its ability to posttranscriptionally and translationally regulate MYC. Consequently, Hnrnpk overexpression renders cells sensitive to BET-bromodomain-inhibition in both in vitro and transplantation models, which represents a strategy for mitigating hnRNP K-mediated c-Myc activation in patients. CONCLUSION: Our findings indicate that hnRNP K is a bona fide oncogene when overexpressed and represents a novel mechanism for c-Myc activation in the absence of MYC lesions.

Formation of mammalian preribosomes proceeds from intermediate to composed state during ribosome maturation.

In eukaryotes, ribosome assembly is a rate-limiting step in ribosomal biogenesis that takes place in a distinctive subnuclear organelle, the nucleolus. How ribosomes get assembled at the nucleolar site by forming initial preribosomal complexes remains poorly characterized. In this study, using several human and murine cell lines, we developed a method for isolation of native mammalian preribosomal complexes by lysing cell nuclei through mild sonication. A sucrose gradient fractionation of the nuclear lysate resolved several ribonucleoprotein (RNP) complexes containing rRNAs and ribosomal proteins. Characterization of the RNP complexes with MS-based protein identification and Northern blotting-based rRNA detection approaches identified two types of preribosomes we named here as intermediate preribosomes (IPRibs) and composed preribosome (CPRib). IPRib complexes comprised large preribosomes (105S to 125S in size) containing the rRNA modification factors and premature rRNAs. We further observed that a distinctive CPRib complex consists of an 85S preribosome assembled with mature rRNAs and a ribosomal biogenesis factor, Ly1 antibody-reactive (LYAR), that does not associate with premature rRNAs and rRNA modification factors. rRNA-labeling experiments uncovered that IPRib assembly precedes CPRib complex formation. We also found that formation of the preribosomal complexes is nutrient-dependent because the abundances of IPRib and CPRib decreased substantially when cells were either deprived of amino acids or exposed to an mTOR kinase inhibitor. These findings indicate that preribosomes form via dynamic and nutrient-dependent processing events and progress from an intermediate to a composed state during ribosome maturation.

Two distinct mTORC2-dependent pathways converge on Rac1 to drive breast cancer metastasis.

BACKGROUND: The importance of the mTOR complex 2 (mTORC2) signaling complex in tumor progression is becoming increasingly recognized. HER2-amplified breast cancers use Rictor/mTORC2 signaling to drive tumor formation, tumor cell survival and resistance to human epidermal growth factor receptor 2 (HER2)-targeted therapy. Cell motility, a key step in the metastatic process, can be activated by mTORC2 in luminal and triple negative breast cancer cell lines, but its role in promoting metastases from HER2-amplified breast cancers is not yet clear. METHODS: Because Rictor is an obligate cofactor of mTORC2, we genetically engineered Rictor ablation or overexpression in mouse and human HER2-amplified breast cancer models for modulation of mTORC2 activity. Signaling through mTORC2-dependent pathways was also manipulated using pharmacological inhibitors of mTOR, Akt, and Rac. Signaling was assessed by western analysis and biochemical pull-down assays specific for Rac-GTP and for active Rac guanine nucleotide exchange factors (GEFs). Metastases were assessed from spontaneous tumors and from intravenously delivered tumor cells. Motility and invasion of cells was assessed using Matrigel-coated transwell assays. RESULTS: We found that Rictor ablation potently impaired, while Rictor overexpression increased, metastasis in spontaneous and intravenously seeded models of HER2-overexpressing breast cancers. Additionally, migration and invasion of HER2-amplified human breast cancer cells was diminished in the absence of Rictor, or upon pharmacological mTOR kinase inhibition. Active Rac1 was required for Rictor-dependent invasion and motility, which rescued invasion/motility in Rictor depleted cells. Rictor/mTORC2-dependent dampening of the endogenous Rac1 inhibitor RhoGDI2, a factor that correlated directly with increased overall survival in HER2-amplified breast cancer patients, promoted Rac1 activity and tumor cell invasion/migration. The mTORC2 substrate Akt did not affect RhoGDI2 dampening, but partially increased Rac1 activity through the Rac-GEF Tiam1, thus partially rescuing cell invasion/motility. The mTORC2 effector protein kinase C (PKC)α did rescue Rictor-mediated RhoGDI2 downregulation, partially rescuing Rac-guanosine triphosphate (GTP) and migration/motility. CONCLUSION: These findings suggest that mTORC2 uses two coordinated pathways to activate cell invasion/motility, both of which converge on Rac1. Akt signaling activates Rac1 through the Rac-GEF Tiam1, while PKC signaling dampens expression of the endogenous Rac1 inhibitor, RhoGDI2.

High-throughput profiling of signaling networks identifies mechanism-based combination therapy to eliminate microenvironmental resistance in acute myeloid leukemia.

The bone marrow microenvironment is known to provide a survival advantage to residual acute myeloid leukemia cells, possibly contributing to disease recurrence. The mechanisms by which stroma in the microenvironment regulates leukemia survival remain largely unknown. Using reverse-phase protein array technology, we profiled 53 key protein molecules in 11 signaling pathways in 20 primary acute myeloid leukemia samples and two cell lines, aiming to understand stroma-mediated signaling modulation in response to the targeted agents temsirolimus (MTOR), ABT737 (BCL2/BCL-XL), and Nutlin-3a (MDM2), and to identify the effective combination therapy targeting acute myeloid leukemia in the context of the leukemia microenvironment. Stroma reprogrammed signaling networks and modified the sensitivity of acute myeloid leukemia samples to all three targeted inhibitors. Stroma activated AKT at Ser473 in the majority of samples treated with single-agent ABT737 or Nutlin-3a. This survival mechanism was partially abrogated by concomitant treatment with temsirolimus plus ABT737 or Nutlin-3a. Mapping the signaling networks revealed that combinations of two inhibitors increased the number of affected proteins in the targeted pathways and in multiple parallel signaling, translating into facilitated cell death. These results demonstrated that a mechanism-based selection of combined inhibitors can be used to guide clinical drug selection and tailor treatment regimens to eliminate microenvironment-mediated resistance in acute myeloid leukemia.

A hypoxia-responsive TRAF6-ATM-H2AX signalling axis promotes HIF1α activation, tumorigenesis and metastasis.

The understanding of how hypoxia stabilizes and activates HIF1α in the nucleus with related oncogenic signals could revolutionize targeted therapy for cancers. Here, we find that histone H2AX displays oncogenic activity by serving as a crucial regulator of HIF1α signalling. H2AX interacts with HIF1α to prevent its degradation and nuclear export in order to allow successful VHL-independent HIF1α transcriptional activation. We show that mono-ubiquitylation and phosphorylation of H2AX, which are strictly mediated by hypoxia-induced E3 ligase activity of TRAF6 and ATM, critically regulate HIF1α-driven tumorigenesis. Importantly, TRAF6 and γH2AX are overexpressed in human breast cancer, correlate with activation of HIF1α signalling, and predict metastatic outcome. Thus, TRAF6 and H2AX overexpression and γH2AX-mediated HIF1α enrichment in the nucleus of cancer cells lead to overactivation of HIF1α-driven tumorigenesis, glycolysis and metastasis. Our findings suggest that TRAF6-mediated mono-ubiquitylation and subsequent phosphorylation of H2AX may serve as potential means for cancer diagnosis and therapy.

Rictor/mTORC2 Drives Progression and Therapeutic Resistance of HER2-Amplified Breast Cancers.

HER2 overexpression drives Akt signaling and cell survival and HER2-enriched breast tumors have a poor outcome when Akt is upregulated. Akt is activated by phosphorylation at T308 via PI3K and S473 via mTORC2. The importance of PI3K-activated Akt signaling is well documented in HER2-amplified breast cancer models, but the significance of mTORC2-activated Akt signaling in this setting remains uncertain. We report here that the mTORC2 obligate cofactor Rictor is enriched in HER2-amplified samples, correlating with increased phosphorylation at S473 on Akt. In invasive breast cancer specimens, Rictor expression was upregulated significantly compared with nonmalignant tissues. In a HER2/Neu mouse model of breast cancer, genetic ablation of Rictor decreased cell survival and phosphorylation at S473 on Akt, delaying tumor latency, penetrance, and burden. In HER2-amplified cells, exposure to an mTORC1/2 dual kinase inhibitor decreased Akt-dependent cell survival, including in cells resistant to lapatinib, where cytotoxicity could be restored. We replicated these findings by silencing Rictor in breast cancer cell lines, but not silencing the mTORC1 cofactor Raptor (RPTOR). Taken together, our findings establish that Rictor/mTORC2 signaling drives Akt-dependent tumor progression in HER2-amplified breast cancers, rationalizing clinical investigation of dual mTORC1/2 kinase inhibitors and developing mTORC2-specific inhibitors for use in this setting. Cancer Res; 76(16); 4752-64. ©2016 AACR.

ATF4 induction through an atypical integrated stress response to ONC201 triggers p53-independent apoptosis in hematological malignancies.

The clinical challenge posed by p53 abnormalities in hematological malignancies requires therapeutic strategies other than standard genotoxic chemotherapies. ONC201 is a first-in-class small molecule that activates p53-independent apoptosis, has a benign safety profile, and is in early clinical trials. We found that ONC201 caused p53-independent apoptosis and cell cycle arrest in cell lines and in mantle cell lymphoma (MCL) and acute myeloid leukemia (AML) samples from patients; these included samples from patients with genetic abnormalities associated with poor prognosis or cells that had developed resistance to the nongenotoxic agents ibrutinib and bortezomib. Moreover, ONC201 caused apoptosis in stem and progenitor AML cells and abrogated the engraftment of leukemic stem cells in mice while sparing normal bone marrow cells. ONC201 caused changes in gene expression similar to those caused by the unfolded protein response (UPR) and integrated stress responses (ISRs), which increase the translation of the transcription factor ATF4 through an increase in the phosphorylation of the translation initiation factor eIF2α. However, unlike the UPR and ISR, the increase in ATF4 abundance in ONC201-treated hematopoietic cells promoted apoptosis and did not depend on increased phosphorylation of eIF2α. ONC201 also inhibited mammalian target of rapamycin complex 1 (mTORC1) signaling, likely through ATF4-mediated induction of the mTORC1 inhibitor DDIT4. Overexpression of BCL-2 protected against ONC201-induced apoptosis, and the combination of ONC201 and the BCL-2 antagonist ABT-199 synergistically increased apoptosis. Thus, our results suggest that by inducing an atypical ISR and p53-independent apoptosis, ONC201 has clinical potential in hematological malignancies.

Skp2-Mediated RagA Ubiquitination Elicits a Negative Feedback to Prevent Amino-Acid-Dependent mTORC1 Hyperactivation by Recruiting GATOR1.

The regulation of RagA(GTP) is important for amino-acid-induced mTORC1 activation. Although GATOR1 complex has been identified as a negative regulator for mTORC1 by hydrolyzing RagA(GTP), how GATOR1 is recruited to RagA to attenuate mTORC1 signaling remains unclear. Moreover, how mTORC1 signaling is terminated upon amino acid stimulation is also unknown. We show that the recruitment of GATOR1 to RagA is induced by amino acids in an mTORC1-dependent manner. Skp2 E3 ligase drives K63-linked ubiquitination of RagA, which facilitates GATOR1 recruitment and RagA(GTP) hydrolysis, thereby providing a negative feedback loop to attenuate mTORC1 lysosomal recruitment and prevent mTORC1 hyperactivation. We further demonstrate that Skp2 promotes autophagy but inhibits cell size and cilia growth through RagA ubiquitination and mTORC1 inhibition. We thereby propose a negative feedback whereby Skp2-mediated RagA ubiquitination recruits GATOR1 to restrict mTORC1 signaling upon sustained amino acid stimulation, which serves a critical mechanism to maintain proper cellular functions.

The nuclear import of ribosomal proteins is regulated by mTOR.

Mechanistic target of rapamycin (mTOR) is a central component of the essential signaling pathway that regulates cell growth and proliferation by controlling anabolic processes in cells. mTOR exists in two distinct mTOR complexes known as mTORC1 and mTORC2 that reside mostly in cytoplasm. In our study, the biochemical characterization of mTOR led to discovery of its novel localization on nuclear envelope where it associates with a critical regulator of nuclear import Ran Binding Protein 2 (RanBP2). We show that association of mTOR with RanBP2 is dependent on the mTOR kinase activity that regulates the nuclear import of ribosomal proteins. The mTOR kinase inhibitors within thirty minutes caused a substantial decrease of ribosomal proteins in the nuclear but not cytoplasmic fraction. Detection of a nuclear accumulation of the GFP-tagged ribosomal protein rpL7a also indicated its dependence on the mTOR kinase activity. The nuclear abundance of ribosomal proteins was not affected by inhibition of mTOR Complex 1 (mTORC1) by rapamycin or deficiency of mTORC2, suggesting a distinctive role of the nuclear envelope mTOR complex in the nuclear import. Thus, we identified that mTOR in association with RanBP2 mediates the active nuclear import of ribosomal proteins.