Modulation Of Akt Activity Research Articles

Increased in vitro production of bovine embryos resulting from oocyte maturation in the presence of triciribine, a specific inhibitor of AKT

The aim of this study was to evaluate the effect of different concentrations of triciribine, a selective Akt inhibitor, on various aspects of oocyte maturation and on the IVF of bovine embryos. Cumulus-oocyte complexes (COCs) were matured in vitro in medium supplemented with: 0 (control), 1, 5, 10, and 20 μM of triciribine. The nuclear maturation was assessed by staining with acetic orcein, while the cytoplasmic maturation was evaluated by mitochondrial (MitoTracker® Red CMXRos) and lipid droplets distribution (LipidTOX). COCs were fertilized in vitro and cultured for nine days. Cleavage rates, blastocyst production, and hatching rates were determined on days three, seven, and nine of in vitro culture, respectively. Oocytes from COCs treated with 1 μM of triciribine were stained at 3, 6, and 9 h of IVM to determine the inhibitor's involvement in germinal vesicle breakdown. Analysis of variance (ANOVA) of the data was performed and the means were compared using the SNK test at a 5 % significance level. Exposure of COCs to 1, 5, and 10 μM of triciribine did not alter the number of matured oocytes (P < 0.05), a concentration of 20 μM reduced the number of oocytes in MII with a consequent increase in oocytes in MI (P < 0.05). This concentration markedly reduced the number of oocytes with peripheral cortical granules and the rates of cleavage and blastocysts (P < 0.05). On the other hand, when COCs were matured in the presence of 1 μM, there was an increase in the blastocyst rate (P < 0.05), but without altering the timing of meiosis resumption (P < 0.05). It is concluded that the Akt pathway participates in the nuclear and cytoplasmic events of in vitro maturation of bovine oocytes, but through mechanisms that do not interfere with germinal vesicle breakdown. Modulation of Akt activity in bovine COCs during IVM with 1 μM of triciribine increases the in vitro production of bovine embryos.

Spatially restricted ecto-5'-nucleotidase expression promotes the growth of uterine leiomyomas by modulating Akt activity.

Found in as many as 80% of women, uterine leiomyomas are a frequent cause of abnormal uterine bleeding, pelvic pain, and infertility. Despite their significant clinical impact, the mechanisms responsible for driving leiomyoma growth remain poorly understood. After obtaining IRB permission, expression of ecto-5'-nucleotidase (NT5E, CD73) was assessed in matched specimens of myometrium and leiomyoma by real-time qPCR, Western blot, and immunohistochemistry (IHC). Adenosine concentrations were measured by enzyme-linked assay. Primary cultures were used to assess the impact of adenosine and/or adenosine receptor agonists on proliferation, apoptosis, and patterns of intracellular signaling invitro. When compared to matched specimens of healthy myometrium, uterine leiomyomas were characterized by reduced CD73 expression. Largely limited to thin-walled vascular structures and the pseudocapsule of leiomyomas despite diffuse myometrial distribution. Restricted intra-tumoral CD73 expression was accompanied by decreased levels of intra-tumoral adenosine. Invitro, incubation of primary leiomyoma cultures with adenosine or its hydrolysis-resistant analog 2-chloro-adenosine (2-CL-AD) inhibited proliferation, induced apoptosis, and reduced proportion of myocytes in S- and G2-M phases of the cell cycle. Decreased proliferation was accompanied by reduced expression of phospho-Akt, phospho-Cdk2-Tyr15, and phospho-Histone H3. Enforced expression of the A2B adenosine receptor (ADORA2B) and ADORA2B-selective agonists similarly suppressed proliferation and inhibited Akt phosphorylation. Collectively, these observations broadly implicate CD73 and reducedextracellular concentrations ofadenosine as key regulators of leiomyoma growth and potentiallyidentify novel strategies for clinically managing these common tumors.

The deubiquitinase OTUD1 noncanonically suppresses Akt activation through its N-terminal intrinsically disordered region

Akt is commonly activated and serves as a valuable target in human cancer. In this study, OTUD1 is identified as an Akt-associated protein and is downregulated upon Akt activation. Ectopic OTUD1 inhibits Akt phosphorylation; however, its deubiquitinase activity contributes only slightly to this effect. A short peptide (OUN-36) located in the OTUD1 N-terminal intrinsically disordered region strongly binds to the Akt PH domain. The residues in the PH domain, which are required for PtdIns(3,4,5)P3 recognition, are also essential for OUN-36 binding. OUN-36 preferentially inhibits Akt-hyperactive tumor cells' proliferation and interferes with Akt cell membrane localization, presumably by disrupting PH domain-PIP3 interaction. Importantly, OUN-36-based therapy efficiently abrogates Akt feedback reactivation in response to MK-2206 treatment and sensitizes cancer cells to chemotherapy and immunotherapy. We therefore show a mechanism by which OTUD1 modulates Akt activity and suggest a potential peptide-based cancer therapeutic strategy implemented by targeting the Akt PH domain.

DJ-1 alleviates anoxia and hypoglycemia injury in cardiac microvascular via AKT and GSH

Cardiac microvascular damage, which is often caused by anoxia and hypoglycemia, is associated with the development of cardiac injury. DJ-1 encodes a peptidase C56 protein family related protein, is has been linked to oxidative stress in various cells such as neurons, COPD epithelial cells, and macrophages. However, the effect of DJ-1 towards oxidative stress caused by anoxia and hypoglycemia of cardiac microvascular endothelial cells (CMEC) remains unclear. In this study, we investigated the role and underlying molecular mechanism of DJ-1 in CMEC with anoxia/hypoglycemic (A/H) injury. We found that the mRNA and the protein expression of DJ-1 in CMEC with A/H injury were significantly downregulated. DJ-1 overexpression by pcDNA.3.1-DJ-1 transfection elevated cell viability while it inhibited LDH leakage, cell apoptosis, caspase-3 activity, ROS level, and MDA contents, while knockdown of DJ-1 has the opposite results. In addition, tube formation was increased in DJ-1 overexpression, while it was decreased in DJ-1 knockdown CMEC with A/H injury. In addition, our results indicated that DJ-1 can regulate glutathione (GSH) levels by modulating AKT activity in CMEC with A/H injury. The downregulation of AKT and GSH may remove the protective role of DJ-1 against A/H injury in CMEC. Taken together, this study showed that DJ-1 upregulation protected CMEC against A/H injury via the AKT/GSH signaling pathway.

Autophagy inhibition with chloroquine reverts paclitaxel resistance and attenuates metastatic potential in human nonsmall lung adenocarcinoma A549 cells via ROS mediated modulation of β-catenin pathway.

Paclitaxel is one of the most commonly used drugs for the treatment of nonsmall cell lung cancer (NSCLC). However acquired resistance to paclitaxel, epithelial to mesenchymal transition and cancer stem cell formation are the major obstacles for successful chemotherapy with this drug. Some of the major reasons behind chemoresistance development include increased ability of the cancer cells to survive under stress conditions by autophagy, increased expression of drug efflux pumps, tubulin mutations etc. In this study we found that inhibition of autophagy with chloroquine prevented development of paclitaxel resistance in A549 cells with time and potentiated the effect of paclitaxel by increased accumulation of superoxide-producing damaged mitochondria, with elevated ROS generation, it also increased the apoptotic rate and sub G0/ G1 phase arrest with time in A549 cells treated with paclitaxel and attenuated the metastatic potential and cancer stem cell population of the paclitaxel-resistant cells by ROS mediated modulation of the Wnt/β-catenin signaling pathway, thereby increasing paclitaxel sensitivity. ROS here played a crucial role in modulating Akt activity when autophagy process was hindered by chloroquine, excessive ROS accumulation in the cell inhibited Akt activity. In addition, chloroquine pre-treatment followed by taxol (10nM) treatment did not show significant toxicity towards non-carcinomas WI38 cells (lung fibroblast cells). Thus autophagy inhibition by CQ pre-treatment can be used as a fruitful strategy to combat the phenomenon of paclitaxel resistance development as well as metastasis in lung cancer.

Prostate cancer: unmet clinical needs and RAD9 as a candidate biomarker for patient management.

Prostate cancer is a complex disease, with multiple subtypes and clinical presentations. Much progress has been made in recent years to understand the underlying genetic basis that drives prostate cancer. Such mechanistic information is useful for development of novel therapeutic targets, to identify biomarkers for early detection or to distinguish between aggressive and indolent disease, and to predict treatment outcome. Multiple tests have become available in recent years to address these clinical needs for prostate cancer. We describe several of these assays, summarizing test details, performance characteristics, and acknowledging their limitations. There is a pressing unmet need for novel biomarkers that can demonstrate improvement in these areas. We introduce one such candidate biomarker, RAD9, describe its functions in the DNA damage response, and detail why it can potentially fill this void. RAD9 has multiple roles in prostate carcinogenesis, making it potentially useful as a clinical tool for men with prostate cancer. RAD9 was originally identified as a radioresistance gene, and subsequent investigations revealed several key functions in the response of cells to DNA damage, including involvement in cell cycle checkpoint control, at least five DNA repair pathways, and apoptosis. Further studies indicated aberrant overexpression in approximately 45% of prostate tumors, with a strong correlation between RAD9 abundance and cancer stage. A causal relationship between RAD9 and prostate cancer was first demonstrated using a mouse model, where tumorigenicity of human prostate cancer cells after subcutaneous injection into nude mice was diminished when RNA interference was used to reduce the normally high levels of the protein. In addition to activity needed for the initial development of tumors, cell culture studies indicated roles for RAD9 in promoting prostate cancer progression by controlling cell migration and invasion through regulation of ITGB1 protein levels, and anoikis resistance by modulating AKT activation. Furthermore, RAD9 enhances the resistance of human prostate cancer cells to radiation in part by regulating ITGB1 protein abundance. RAD9 binds androgen receptor and inhibits androgen-induced androgen receptor's activity as a transcription factor. Moreover, RAD9 also acts as a gene-specific transcription factor, through binding p53 consensus sequences at target gene promoters, and this likely contributes to its oncogenic activity. Given these diverse and extensive activities, RAD9 plays important roles in the initiation and progression of prostate cancer and can potentially serve as a valuable biomarker useful in the management of patients with this disease.

SIRT1 promotes formation of breast cancer through modulating Akt activity.

The silent information regulation factor 1 (sirtuin Type 1, SIRT1), as a kind of NAD+ dependent class III histone deacetylation enzyme, has been found to be involved in tumor proliferation, invasion, and metastasis. The roles of SIRTl in breast cancer is multifaceted depending on its substrate from upstream or downstream signaling pathway. In this study, we sought to make clear the regulating effects of SIRT1 in breast cancer cells, and to explore the underlying mechanisms through which SIRT1 regulates breast cancer. First, our results showed that SIRT1 was significantly up-regulated in breast cancer tissues and cells, which correlated with histological grade, tumor size, as well as lymph node metastasis. Then we established SIRT1-overexpressed and SIRT1- knockdown breast cancer cell lines to investigate the functions of SIRT1 in regulating colony formation, cell proliferation, cell cycle, cell apoptosis and migration. We found that overexpression of SIRT1 significantly promoted breast cancer growth both in vitro and in vivo, whereas knockdown of SIRT1 inhibited these phenotypes. Furthermore, SIRT1 was found to interact with Akt directly, consequently promoting the activity of Akt in breast cancer cells in vitro and positively correlating with expression of Akt, P-Akt, in breast cancer tissues in vivo. Down regulation the activity of Akt partially weakened the proliferative effect mediated by SIRT1. Taken together, our results demonstrated SIRT1's tumor promotion function and potential mechanisms in breast cancer, thus providing valuable therapeutic targets for breast cancer.

The LINK-A lncRNA interacts with PtdIns(3,4,5)P3 tohyperactivate AKTand confer resistance to AKTinhibitors.

Phosphatidylinositol-3,4,5-trisphosphate (PIP3) mediates signaling pathways as a second messenger in response to extracellular signals. Although primordial functions of phospholipids and RNAs have been hypothesized in the “RNA world”, physiological RNA-phospholipid interactions and their involvement in essential cellular processes has remained a mystery. We explicate the contribution of lipid-binding long non-coding RNAs (lncRNAs) in cancer cells. Among them, Long Intergenic Noncoding RNA for Kinase Activation (LINK-A) directly interacts with AKT pleckstrin homology domain and PIP3 at the single nucleotide level, facilitating AKT-PIP3 interaction and consequent enzymatic activation. LINK-A-dependent AKT hyperactivation leads to tumorigenesis and resistance to AKT inhibitors. Genomic deletions of the LINK-A PIP3-binding motif dramatically sensitized breast cancer cells to AKT inhibitors. Furthermore, meta-analysis showed the correlation between LINK-A expression and incidence of a SNP (rs12095274: A>G), AKT phosphorylation status, and poor outcomes for breast and lung cancer patients. PIP3-binding lncRNA modulates AKT activation with broad clinical implications.

A Drosophila model of insulin resistance associated with the human TRIB3 Q/R polymorphism.

ABSTRACTMembers of the Tribbles family of proteins are conserved pseudokinases with diverse roles in cell growth and proliferation. Both Drosophila Tribbles (Trbl) and vertebrate Trib3 proteins bind to the kinase Akt (Akt1) to block its phosphorylation activation and reduce downstream insulin-stimulated anabolism. A single nucleotide polymorphism (SNP) variant in human TRIB3, which results in a glutamine (Q) to arginine (R) missense mutation in a conserved motif at position 84, confers stronger Akt binding, resulting in reduced Akt phosphorylation, and is associated with a predisposition to Type 2 diabetes, cardiovascular disease, diabetic nephropathy, chronic kidney disease and leukemogenesis. Here, we used a Drosophila model to understand the importance of the conserved R residue in several Trbl functions. In the fly fat body, misexpression of a site-directed Q mutation at position R141 resulted in weakened binding to Drosophila Akt (dAkt), leading to increased levels of phospho-dAkt, increased cell and tissue size, and increases in the levels of stored glycogen and triglycerides. Consistent with the functional conservation of this arginine in modulating Akt activity, mouse Trib3 R84 misexpressed in the fly fat body blocked dAkt phosphorylation with a strength similar to wild-type Trbl. Limited mutational analysis shows that the R141 site dictates the strength of Akt binding but does not affect other Trbl-dependent developmental processes, suggesting a specificity that could serve as a drug target for metabolic diseases.

Downregulation of Deptor By MiR-155 Promotes Cell Survival through Activation of PI3K/AKT and NFkB Signaling in ABC-Type Diffuse Large B-Cell Lymphomas

IntroductionMiR-155 expression in DLBCLs is induced by NFkB and directly targets key regulators of B-cell maturation, motility and BCR signaling. In this study, we searched for new targets of miR-155 potentially involved in deregulation of BCR-PI3K/AKT and NFkB signaling in DLBCLs. We identified two new miR-155 targets: c-CBL (SYK ubiquitin E3 ligase) and DEPTOR (mTOR phosphatase). DEPTOR suppresses mTOR activity and in different tumors plays a role of either a tumor suppressor or an oncogene. Since the role of DEPTOR in DLCBLs cells has not been determined, we assessed the consequences of its inhibition in this malignancy.MethodsPredicted miR-155 targets were validated with 3'UTR luciferase reporter assays. MiR-155 expression was modulated through transfection with miR-155 mimic or miR-155 inhibitor. The DEPTOR silencing in DLBCL cell was achieved with retroviral shRNA vector. DEPTOR mRNA expression and survival of DLBCL patients was determined using publicly available microarray data (Lenz et al, 2008, GEO accession GSE10846). Immunohistochemical assessment of DEPTOR expression was performed in 76 newly diagnosed DLBCL patients with available GCB/non-GCB designations based on Hans algorithm.ResultsUsing miRNA target finding algorithms, we identified miR-155-matching sequences in 3’UTRs of two genes involved in SYK/PI3K/AKT pathway regulation: c-CBL and DEPTOR. MiR-155 suppressed luciferase activities of vectors containing 3’UTR fragments from c-CBL and DEPTOR genes with wild-type, but not mutant miR-155 seed sequence. To establish a link between miR-155 and c-CBL or DEPTOR, DLBCL cell lines were transfected with a control non-targeting miR or miR-155 mimic. Introduction of miR-155 resulted in decreased expression of c-CBL and DEPTOR, accompanied by a marked increase in phospho-AKT level. Inhibition of endogenous miR-155 in U2932 cell line by anti-miR-155 exhibited opposite effects. Using publicly available gene expression data (Lenz et al, 2008, GEO accession GSE10846), we found that miR-155 exhibited a reciprocal expression pattern with DEPTOR and c-CBL (r =-0.15; p=.002 and r=-0.189; p<.0001), indicating that miR-155 likely modulates expression of c-CBL and DEPTOR in primary tumors.Since the role of DEPTOR in DLBCL has not been previously addressed, we investigated its expression in a series of 76 newly diagnosed DLBCL patients by immunohistochemistry. Complete loss of DEPTOR expression was noted solely in non-GCB tumors (9 of 37; 24%). Additional 12 patients exhibited low expression of this protein. In contrast, none of the 39 GCB tumors stained negative for DEPTOR (Chi-square, p=.005). DEPTOR mRNA greater than mean in primary DLBCL biopsies was associated with longer overall survival (OS; P=0.016).To elucidate the function of DEPTOR in DLBCL cells, we silenced the expression of this protein with shRNA. Attenuated protein level of DEPTOR markedly enhanced AKT activity and promoted proliferation of DHL4 cells. DHL4 cells with silenced DEPTOR were also less prone to starvation- or SYK inhibition- induced apoptosis. Since AKT has been reported to stimulate activity of NFκB, we hypothesized that miR-155-induced decrease in DEPTOR expression would affect NFκB signaling. Consistent with this, inhibition of endogenous miR-155 in U2932 cells led to marked downregulation of NFĸB-controlled genes (CD40, BFL-1, RelB, IĸBα, A20, MIR155HG) and sensitized U2932 cells to ibrutinib, indicating that miR-155 amplifies NFĸB signaling in these cells.ConclusionsTaken together, our data underscore the role of miR-155 in the regulation of AKT and NFkB prosurvival signaling in DLBCL. By targeting multiple negative regulators of PI3K/AKT pathway, miR-155 creates a feed - forward loop leading to increased NFkB activity. We also show that DEPTOR protein expression is decreased or lost selectively in a large fraction of ABC-DLBCLs. Since DEPTOR modulates AKT activity and its silencing promotes proliferation of DLBCL cells, these data suggest that DEPTOR functions as a tumor suppressor in ABC-DLBCLs. DisclosuresProchorec-Sobieszek:Roche: Other: travel, accommodation. Warzocha:BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Juszczynski:Selvita S.A.: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Abstract 2613: MEK is a therapeutic and chemopreventative target in squamous cell carcinoma

Abstract Purpose: While the prognosis of cutaneous squamous cell carcinoma (cuSCC) is excellent overall, advanced metastatic disease represents a substantial mortality burden for which no standard targeted therapy exists. Findings from genomic and proteomic studies and the observed induction of cuSCC by BRAF inhibitor driven MEK/ERK pathway engagement suggest that MEK/ERK activation is essential for cuSCC tumorigenesis and tumor proliferation. Most cuSCC arise from a clinically and histopathologically defined preneoplastic precursor, the actinic keratosis (AK). Molecular studies of early events in cuSCC pathogenesis strongly implicate MEK/ERK signaling at the proteomic and transcriptional level. This occurs at the earliest recognizable transitions from chronically UV-exposed skin to AK, with sharp elevation of ETS2/ELK1 transcriptional target expression. With these compelling rationales in mind, we tested whether MEK inhibitors (MEKi) are a clinically actionable treatment and chemoprevention approach for cuSCC. Given that several MEKi are approved, this is a readily translated strategy. Experimental Design: Preclinical testing was performed in 10 cuSCC cell lines and two mouse models of cuSCC using two distinct MEK inhibitors, trametinib and cobimetinib. We show that two MEKi, trametinib and cobimetinib are highly effective against cuSCC cell lines in culture, effectively engage MEK/ERK signaling in cells and in vivo, and potently induce cell cycle arrest and senescence. Both established and new tumors are powerfully inhibited in both xenograft and UV-driven autochthonous mouse models. This model, which utilizes immunocompetent SKH-1E mice exposed to chronic, low-dose, solar simulated UV light (12.5 kJ/m2 UVB weekly administered across three doses) more faithfully recapitulates human cuSCC molecularly than chemical carcinogenesis models. Lesions in this model exhibit heterogeneity in latency and responses to therapy, as do human tumors. Results: MEK inhibitor treatment of cuSCC lines strongly reduces proliferation and induces senescence markers in cells, including p21 and beta-galactosidase. This response was universal, but highly heterogeneous. Sensitivity to MEKi was determined, in part, by modulation of AKT activity, and combination MEKi and AKTi. In-vivo, an SRB1 xenograft model was exquisitely sensitive to oral trametinib treatment, and in our spontaneous UV-driven Hairless mouse model of cuSCC, treatment with the MEK inhibitors trametinib and cobimetinib strongly reduced tumor growth and almost completely abrogated tumor induction. We confirmed target engagement in-vivo showing that ERK signaling was significantly suppressed. Conclusions: Overall, we conclude MEK signaling is critical for cuSCC tumor induction and maintenance, and that MEK inhibition is an attractive approach for both advanced cuSCC treatment as well as chemoprevention. Citation Format: Charles H. Adelmann, Kimberly Truong, Roger Liang, Varun Bansal, Rachael Saporito, Woojin Lee, Lili Du, Courtney Nicholas, Marco Napoli, Elsa R. Flores, Kenneth Y. Tsai. MEK is a therapeutic and chemopreventative target in squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2613.

S6K1 regulates hematopoietic stem cell self-renewal and leukemia maintenance

Hyperactivation of the mTOR pathway impairs hematopoietic stem cell (HSC) functions and promotes leukemogenesis. mTORC1 and mTORC2 differentially control normal and leukemic stem cell functions. mTORC1 regulates p70 ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding (eIF4E-binding) protein 1 (4E-BP1), and mTORC2 modulates AKT activation. Given the extensive crosstalk that occurs between mTORC1 and mTORC2 signaling pathways, we assessed the role of the mTORC1 substrate S6K1 in the regulation of both normal HSC functions and in leukemogenesis driven by the mixed lineage leukemia (MLL) fusion oncogene MLL-AF9. We demonstrated that S6K1 deficiency impairs self-renewal of murine HSCs by reducing p21 expression. Loss of S6K1 also improved survival in mice transplanted with MLL-AF9-positive leukemic stem cells by modulating AKT and 4E-BP1 phosphorylation. Taken together, these results suggest that S6K1 acts through multiple targets of the mTOR pathway to promote self-renewal and leukemia progression. Given the recent interest in S6K1 as a potential therapeutic target in cancer, our results further support targeting this molecule as a potential strategy for treatment of myeloid malignancies.

TonEBP/NFAT5 inhibits adipocyte differentiation via modulation of mitotic clonal expansion during early phase of differentiation in 3T3‐L1 cells

Obesity resulting from excessive accumulation of white adipose tissue is closely related to the chronic diseases such as diabetes, hyperlipidemia, and hypertension. White adipose mass is determined by the number and size of adipocytes. Preadipocyte like 3T3‐L1 cells undergo terminal differentiation manifested by formation of lipid droplets as well as adipocyte specific protein. TonEBP/NFAT5 belongs to the Rel family of transcription factors and plays important roles in the development and maintenance of kidney, immunity, arthritis, and cancer metastasis. Although the functions of TonEBP/NFAT5 during osteogenesis, chondrogenesis and myogenesis are reported recently, its role in the adipogenesis is not well known. In this study, we analyzed the role of TonEBP/NFAT5 in adipogenesis using 3T3‐L1 cells. TonEBP/NFAT5 expression was dramatically reduced during adipocyte differentiation of 3T3‐L1 cells. RNAi‐mediated knock down of TonEBP/NFAT5 facilitated adipogenesis. Whereas sustained expression of TonEBP/NFAT5 using adenovirus suppressed the formation of lipid droplet and the expression of FABP4, marker for terminally differentiated adipocytes. Also, TonEBP/NFAT5 inhibited the expression of PPARγ, master regulator of terminal adipocytes. TonEBP/NFAT5 suppressed adipogenesis in the early phase of the differentiation via modulation of Akt activity and mitotic clonal expansion. These results suggest that TonEBP/NFAT5 may be an important regulatory factor in the differentiation of adipocytes.Support or Funding InformationThis research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education (NRF‐2013R1A1A2060736, NRF‐2014R1A6A1029617)

Successful Salvage Therapy By Idelalisib Followed By Haploidentical Allogenic Transplantation of an Alemtuzumab-Refractory T-Cell Prolymphocytic Leukemia

BackgroundT-cell prolymphocytic leukemia (T-PLL) is a rare disease of extremely poor outcome.The current standard of care involves intravenous Alemtuzumab with high response rates. Virtually all patients experience relapse and the 5-years overall survival is not higher than 5%. Allogenic bone marrow transplantation is the only curative option.Case presentationA 35 years-old male was diagnosed with T-PLL in early 2014 with skin infiltration, fatigue, hepatomegaly, splenomegaly (28 cm), and lymphadenopathies. Immunophenotypic features of circulating lymphocytes showed a T-cell lymphoproliferation (CD2+ CD3+ CD5+ CD7+ CD8+). TCL1 hyperexpression was assessed on bone marrow and lymph node biopsy samples. There was a rearrangement of chromosome 14 involving TCL1 on circulating cells.Pentostatin was initiated for 2 cycles without success. IV alemtuzumab (30mg/m2) was then introduced. After 8 weekly cycles, a CT scan showed a reduction of the splenomegaly (22 cm), a stable hepatomegaly, and no remaining lymphadenopathy. No abnormal circulating lymphocytes were detectable. After 14 cycles, the patient showed cervical painful lymph nodes. A needle aspiration revealed relapse with T-PLL cells. We then introduced idelalisib (150 mg, twice daily). Alemtuzumab was discontinued. One week after the introduction of idelalisib, physical examination revealed the disappearance of all peripheral lymph nodes. After 2 weeks, the spleen size was reduced to 16 cm and a body PET-CT was negative, proving metabolic remission. No circulating lymphocyte could be found. The patient was considered as being in complete remission (CR) and underwent haplo-identical transplantation. Idelalisib was discontinued after three weeks of exposure before transplantation and reintroduced from days 30 to 60. At day 100 after transplantation, the patient was in persistent CR: a peripheral immunophenotypic study was performed, no clonal T population was found, chimerism was 100% of donor origin. A bone marrow aspirate was quantitatively and qualitatively normal. Seven months after transplant, the patient remains in CR with a grade 1 skin chronic GVHD.Biological rationaleIdealisib is a delta isoform inhibitor of protein phosphatidylinositol 3 kinase (PI3K). TCL1 is an overexpressed oncoprotein described in the majority of T-PLL due to chromosomal 14 rearrangement. TCL1 modulates AKT activity, facilitating both AKT dimerization and phosphorylation upon PI3K stimulation. The PI3K produces PIP2 and PIP3, which are required to phosphorylate AKT. Even if TCL1 is not sufficient to transform T cells into T-PLL cells and requires secondary genetic events as suggested in murine models, AKT hyperactivation may participate in T-cell proliferation (through the GSK-3β and mTOR pathways), and survival (through the NFκB pathway). We hypothesized that the inhibition of the PI3K with idelalisib would inhibit growth and eventually cell survival of the T-PLL cell, depending on the secondary genetic events. In order to support this hypothesis, we investigated the biochemical and functional effects of idelalisib in vitro. Fresh primary T-PLL cells were obtained from the ascites of an another case of T-PLL (CD2+, CD3+, CD5+, CD7+, CD4/CD8+, complex caryotype involving chromosome 14). In vitro exposure of primary T-PLL cells to idelalisib led to a dramatic and dose-dependent decrease in AKT phosphorylation after 24 hours (figure 1) and ex vivo after 5 days (figure 2). However, no apoptotic response was observed, neither in vitro nor ex vivo, even though therapeutic levels of idelalisib were reached in the ascites.ConclusionWe report the first case of idealisib successfully used as a salvage therapy in T-PLL, leading to complete remission. It was used as a bridge to transplantation for a patient who is still in complete remission 7 months after transplantation.Our results provide a paradigm for the use of a PI3K inhibitor in T-PLL, which results in AKT dephosphorylation. The lack of functional response for the second patient suggests that the clinical response may depend on other oncogenic events, which could bypass AKT dependency. There is a need to identify a patient subgroup that may benefit from PI3K inhibition. In an attempt to identify molecular mechanisms underlying this resistance, comparative full exome sequencing is being performed for both patients and will be shown at the meeting. [Display omitted] DisclosuresOff Label Use: idelalisib, PI3-Kinase inhibitor. Tournilhac:Roche: Other: Travel support, Research Funding; Celgene: Other: Travel support; Mundiphrama: Honoraria, Other: Travel Support, Research Funding; GSK: Other: Travel Support, Research Funding; Janssen Cilag: Honoraria, Other: Travel support; Gilead: Other: Travel Funding.

MiR-155 Amplifies AKT and NFkB Signaling By Targeting Multiple Regulators of BCR Signal in DLBCL

IntroductionThe B-cell receptor (BCR)- activated PI3K pathway plays a critical pro-survival function in normal B-lymphocytes and certain B-cell malignancies. Multiple mechanisms are involved in triggering and modulation of the BCR signal amplitude. MiR-155 has emerged as a positive regulator of PI3K signaling in multiple malignancies, including DLBCL, through targeting negative modulator of this pathway, SHIP-1. In the present study we have searched for new targets of miR-155, which might play a role in the deregulation of AKT and NFkB signaling in DLBCL.MethodsMiR-155 target prediction was performed with PicTar, Miranda and TagetScan algorithms. Predicted miR-155 targets were validated with 3'UTR luciferase reporter assays in HEK293 cells. MiR-155 expression was modulated through transfection with miR-155 mimic or miR-155 inhibitor. The consequences of the miR-155 perturbations were assessed in DLBCL cell lines by proliferation assays (MTS) and immunoblotting with antibodies against predicted miR-155 targets and p-AKT. The DEPTOR silencing in DLBCL cells was achieved with retroviral shRNA vector and its consequences were assessed using proliferation assays and immunoblotting. DEPTOR mRNA expression and survival of DLBCL patients was determined using publicly available microarray data (Lenz et al, 2008, GEO accession GSE10846).ResultsUsing miRNA target finding algorithms, we identified miR-155-matching sequences in 3'UTRs of two genes involved in SYK/PI3K/AKT pathway regulation: c-CBL (SYK ubiquitin E3 ligase) and DEPTOR (an mTOR phosphatase). MiR-155 suppressed the luciferase activities of vectors containing 3'UTR fragments from SHIP-1, c-CBL and DEPTOR genes with wild-type, but not mutant miR-155 seed sequence. To establish a link between miR-155 and c-CBL or DEPTOR, DLBCL cell lines were transfected with a control non-targeting miR or miR-155 mimic. The control miR did not affect the protein level of SHIP-1, c-CBL or DEPTOR, whereas introduction of miR-155 resulted in a decrease in expression of these proteins. The repression of miR-155 target genes was accompanied by increased phosphorylation of AKT. As expected, neutralization of endogenous miR-155 in U2932 cell line by anti-miR-155 exhibited opposite effects. In addition to modulation of SYK/PI3K/AKT pathway, miR-155 has been reported to modulate NFĸB activity. To determine whether miR-155 affects NFĸB in DLBCL, we assessed transcript abundance of the genes regulated by NFĸB (CD40, BFL-1, RelB, IĸBα, A20, MIR155HG) in U2932 cell line with reduced miR-155 level. Repression of endogenous miR-155 in these cells led to marked downregulation of NFĸB-controlled genes, indicating that miR-155 amplifies NFĸB signaling in this DLBCL cell line. Consistent with this, anti-miR-155 sensitized U2932 cells to ibrutinib.Since the function of a newly identified miR-155 target, the mTOR phosphatase DEPTOR, in DLBCL has not been elucidated, we silenced the expression of this protein with shRNA. Attenuated protein level of DEPTOR enhanced activity of AKT and promoted proliferation of SU-DHL4 cell line. To determine whether expression level of DEPTOR correlates with patient survival, we analyzed data from gene expression studies. Higher DEPTOR mRNA level in primary DLBCL biopsies was associated with longer overall survival (log rank test, p=0.018). Collectively, these data suggest that DEPTOR plays a tumor suppressor function in DLBCL.ConclusionsOur data underscore the role of miR-155 in the regulation of AKT and NFkB prosurvival signaling in DLBCL. MiR-155 regulates AKT signaling not only by decreasing expression of SHIP-1, but also by modulating the abundance of c-CBL and DEPTOR. DEPTOR modulates AKT activity and its silencing promotes proliferation of DLBCL cells, suggesting that DEPTOR functions as a tumor suppressor in DLBCL. DisclosuresNo relevant conflicts of interest to declare.

Imbalanced PTEN and PI3K Signaling Impairs Class Switch Recombination

Class switch recombination (CSR) generates isotype-switched Abs with distinct effector functions. B cells express phosphatase and tensin homolog (PTEN) and multiple isoforms of class IA PI3K catalytic subunits, including p110α and p110δ, whose roles in CSR remain unknown or controversial. In this article, we demonstrate a direct effect of PTEN on CSR signaling by acute deletion of Pten specifically in mature B cells, thereby excluding the developmental impact of Pten deletion. We show that mature B cell-specific PTEN overexpression enhances CSR. More importantly, we establish a critical role for p110α in CSR. Furthermore, we identify a cooperative role for p110α and p110δ in suppressing CSR. Mechanistically, dysregulation of p110α or PTEN inversely affects activation-induced deaminase expression via modulating AKT activity. Thus, our study reveals that a signaling balance between PTEN and PI3K isoforms is essential to maintain normal CSR.

The interplay between GRP78 expression and Akt activation in human colon cancer cells under celecoxib treatment.

It has been reported previously that celecoxib shows antitumor effects in many types of cancers. Here, we detected its effects on DLD-1 and SW480 (two human colon cancer cell lines) and investigated the dynamic relationship between the 78-kDa glucose-regulatory protein (GRP78) and the phosphoinositide 3-kinase (PI3K)/Akt pathway. Gene expression was detected by real-time PCR and western blot analysis; the cytotoxicity was determined by the MTT assay and flow cytometry. First, the results showed that celecoxib induced cytotoxicity in a dose-dependent and time-dependent manner. Furthermore, we found the celecoxib-triggered unfolded protein response and the bidirectional regulation of Akt activation in both cell lines. Inhibiting the Akt activation by the PI3K inhibitor LY294002 markedly enhanced GRP78 expression. Besides, silencing the GRP78 expression regulated Akt activation in a time-dependent manner and increased the induction of the C/EBP homologous protein (CHOP) as well as considerably promoted celecoxib-induced apoptosis. In conclusion, these findings provide evidence that under the celecoxib treatment, GRP78 plays a protective role by modulating Akt activation and abrogating CHOP expression. However, Akt activation can provide a feedback loop to inhibit GRP78 expression. These studies can lead to novel therapeutic strategies for human colon cancer.

CDK5 Regulates Paclitaxel Sensitivity in Ovarian Cancer Cells by Modulating AKT Activation, p21Cip1- and p27Kip1-Mediated G1 Cell Cycle Arrest and Apoptosis.

Cyclin-dependent kinase 5 (CDK5) is a cytoplasmic serine/ threonine kinase. Knockdown of CDK5 enhances paclitaxel sensitivity in human ovarian cancer cells. This study explores the mechanisms by which CDK5 regulates paclitaxel sensitivity in human ovarian cancers. Multiple ovarian cancer cell lines and xenografts were treated with CDK5 small interfering RNA (siRNA) with or without paclitaxel to examine the effect on cancer cell viability, cell cycle arrest and tumor growth. CDK5 protein was measured by immunohistochemical staining of an ovarian cancer tissue microarray to correlate CDK5 expression with overall patient survival. Knockdown of CDK5 with siRNAs inhibits activation of AKT which significantly correlates with decreased cell growth and enhanced paclitaxel sensitivity in ovarian cancer cell lines. In addition, CDK5 knockdown alone and in combination with paclitaxel induced G1 cell cycle arrest and caspase 3 dependent apoptotic cell death associated with post-translational upregulation and nuclear translocation of TP53 and p27Kip1 as well as TP53-dependent transcriptional induction of p21Cip1 in wild type TP53 cancer cells. Treatment of HEYA8 and A2780 wild type TP53 xenografts in nu/nu mice with CDK5 siRNA and paclitaxel produced significantly greater growth inhibition than either treatment alone. Increased expression of CDK5 in human ovarian cancers correlates inversely with overall survival. CDK5 modulates paclitaxel sensitivity by regulating AKT activation, the cell cycle and caspase-dependent apoptosis. CDK5 inhibition can potentiate paclitaxel activity in human ovarian cancer cells.

Integrin α3β1 regulates kidney collecting duct development via TRAF6-dependent K63-linked polyubiquitination of Akt.

The collecting system of the kidney develops from the ureteric bud (UB), which undergoes branching morphogenesis, a process regulated by multiple factors, including integrin-extracellular matrix interactions. The laminin (LM)-binding integrin α3β1 is crucial for this developmental program; however, the LM types and LM/integrin α3β1-dependent signaling pathways are poorly defined. We show that α3 chain-containing LMs promote normal UB branching morphogenesis and that LM-332 is a better substrate than LM-511 for stimulating integrin α3β1-dependent collecting duct cell functions. We demonstrate that integrin α3β1-mediated cell adhesion to LM-332 modulates Akt activation in the developing collecting system and that Akt activation is PI3K independent but requires decreased PTEN activity and K63-linked polyubiquitination. We identified the ubiquitin-modifying enzyme TRAF6 as an interactor with the integrin β1 subunit and regulator of integrin α3β1-dependent Akt activation. Finally, we established that the developmental defects of TRAF6- and integrin α3-null mouse kidneys are similar. Thus K63-linked polyubiquitination plays a previously unrecognized role in integrin α3β1-dependent cell signaling required for UB development and may represent a novel mechanism whereby integrins regulate signaling pathways.

The Ca2+ sensor S100A1 modulates neuroinflammation, histopathology and Akt activity in the PSAPP Alzheimer's disease mouse model

The contribution of the Ca2+ sensor S100A1 to in vivo Alzheimer's disease (AD) pathobiology has not been elucidated although S100A1 regulates numerous cellular processes linked to AD. This study uses genetic ablation to ascertain the effects of S100A1 on neuroinflammation, beta-amyloid (Aβ) plaque deposition and Akt activity in the PSAPP AD mouse model. PSAPP/S100A1−/− mice exhibited decreases in astrocytosis (GFAP burden), microgliosis (Iba1 burden) and plaque load/number when compared to PSAPP/S100A1+/+ mice at six and twelve months of age. The presence of detectable S100A1 staining in human AD specimens is consistent with a detrimental gain of S100A1 function in AD. S100A1 ablation also reduced plaque associated and increased non-plaque associated PO4-Akt and PO4-GSK3β staining. S100A1·Akt complexes were undetectable in PC12 cells and AD brain tissue suggesting that S100A1 indirectly modulates Akt activity. In contrast, S100A1·RyR (ryanodine receptor) complexes were present in human/mouse AD brain and exhibited Ca2+-dependent formation in neuronal cells. This is the first direct demonstration of an S100A1· target protein complex in tissue/cells and identifies the RyR as a primary S100A1 target protein in the brain. Collectively, these data suggest that S100A1 inhibition may be a novel strategy for normalizing aberrant Ca2+ signaling in AD.