Akt-independent Mechanism Research Articles

Activation of Membrane Estrogen Receptors Attenuates NOP-Mediated Tactile Antihypersensitivity in a Rodent Model of Neuropathic Pain.

Women manifest a higher prevalence of several chronic pain disorders compared to men. We demonstrated earlier that estrogen rapidly attenuates nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP)-mediated thermal antinociception through the activation of membrane estrogen receptors (mERs). However, the effect of mER activation on NOP-mediated attenuation of tactile hypersensitivity in a neuropathic model of pain and the underlying mechanisms remain unknown. Following spared nerve injury (SNI), male and ovariectomized (OVX) female rats were intrathecally (i.t.) injected with a selective mER agonist and nociceptin/orphanin FQ (N/OFQ), the endogenous ligand for NOP, and their effects on paw withdrawal thresholds (PWTs) were tested. In addition, spinal cord tissue was used to measure changes in phosphorylated extracellular signal regulated kinase (ERK), protein kinase A (PKA), protein kinase C (PKC), and protein kinase B (Akt) levels. SNI significantly reduced PWTs in males and OVX females, indicating tactile hypersensitivity. N/OFQ restored PWTs, indicating an antihypersensitive effect. Selective mER activation attenuated the effect of N/OFQ in an antagonist-reversible manner. SNI led to a robust increase in the phosphorylation of ERK, PKA, PKC, and Akt. However, mER activation did not further affect it. Thus, we conclude that activation of mERs rapidly abolishes NOP-mediated tactile antihypersensitivity following SNI via an ERK-, PKA-, PKC-, and Akt-independent mechanism.

AKT-independent activation of p38 MAP kinase promotes vascular calcification

Vascular calcification is prevalent in patients with atherosclerosis, and oxidative stress promotes pathogenesis of atherosclerosis. We have previously reported that activation of AKT by oxidative stress induces vascular calcification. Using sodium dichloroacetate (DCA), a previously reported small molecule inhibitor of AKT, the present studies uncovered an AKT-independent mechanism in regulating vascular calcification.We found that DCA dose-dependently induced calcification of vascular smooth muscle cells (VSMC) in vitro and aortic rings ex vivo. Furthermore, DCA markedly enhanced vascular calcification in atherosclerotic ApoE knockout mice in vivo. DCA-induced VSMC calcification was associated with increased Runx2, but not via activation of AKT, a key upstream signal that upregulates Runx2 during VSMC calcification. In contrast, DCA inhibited AKT activation and induced activation of p38 MAPK in calcified atherosclerotic lesions in vivo and calcified VSMC in vitro. Using a pharmacological inhibitor and shRNA for p38 MAPK, we demonstrated that inhibition of p38 MAPK blocked DCA-induced Runx2 upregulation and VSMC calcification. Furthermore, Runx2 deletion attenuated DCA-induced VSMC calcification. Immunoprecipitation analysis revealed association of p38 MAPK with Runx2, which was enhanced by DCA treatment. Knockdown p38 MAPK inhibited DCA-induced Runx2 transactivity, supporting the function of p38 MAPK in regulating Runx2 transactivity.Our studies have uncovered a new function of DCA in regulating vascular calcification, via AKT-independent activation of p38 MAPK. Furthermore, we have identified novel interaction between p38 MAPK and Runx2 enhances Runx2 transactivity, thus promoting VSMC calcification. These results revealed a novel signaling mechanism underlying DCA-induced vascular calcification, and offer opportunities to identify new therapeutic targets.

Tenascin C to regulate the function of sHER3, an endogenous inhibitor of neuregulin signaling.

e23002 Background: HER3 expression is associated with poor prognosis in melanoma patients but the mechanistic basis has not been determined. An endogenous secreted isoform of HER3, p85-sHER3, has been shown by us to inhibit neuregulin-1 mediated, Akt-dependent breast and ovarian cancer cell growth in vitro. Here we demonstrate that p85-sHER3 also regulates neuregulin-dependent melanoma cell migration via an Akt- independent, tenascin C-dependent mechanism. Methods: Immunoblot analysis and RT-PCR were used to study HER3 and p85-sHER expression. Cell proliferation and chamber assays were conducted to determine the effect of p85-sHER3 on melanoma cell growth and migration. Mass spectrometry (MS) was performed to identify sHER3-interacting proteins. CRSPR in vitro editing methods were used to knock out tenascin C (TNC) expression. Results: HER3 and p85-sHER3 were expressed in 5 of 6 human melanoma-derived cell lines tested, with exception of SK-MEL-103 which did not express either gene product. Exogenous addition of 10 nM p85-sHER3 inhibited growth in 3 cells lines, although Akt phosphorylation was not reduced. Protein extracts from M14-MEL cell conditioned media were characterized by MS, and identified p85-sHER3 and TNC. Their association was confirmed by co-immunoprecipitation. sHER3 was further shown to inhibit neuregulin stimulated cell migration in 2 cell lines which exhibited high levels of TNC. CRSPR knockout of TNC in these cell lines (but not in control lines) eliminated sHER3 inhibition of neuregulin stimulated melanoma cell migration. Conclusions: Our results suggest that p85-sHER3, predominantly secreted by HER3-expressing melanoma cells, inhibits melanoma cell proliferation and migration via an Akt-independent mechanism, which is likely achieved through interaction(s) with TNC. TNC promotes an intermediate adhesive state favoring cell migration and this state appears to be mediated by Rho-associated kinase signaling. The Rho inhibitor, CCG-203971 has been shown to inhibit melanoma cell migration. Together, these results suggest the existence of a TNC and HER3-dependent signaling pathway in melanoma that regulates cell migration, and therefore may be correlated with poor patient survival.

Simultaneous Inhibition of PI3Kδ and PI3Kα Induces ABC-DLBCL Regression Via Attenuation of BCR-Dependent and Independent Activation of NF-Kb and AKT

Introduction: ABC-DLBCL is a subtype of DLBCL with less favorable clinical outcomes to the standard of care (SoC) therapies. Constitutive activation of NF-κB by various genetic alterations in ABC-DLBCL has been identified as one of the key mechanisms driving chemotherapy resistance. Inhibition of B cell receptor (BCR) signaling with BTK (Bruton’s tyrosine kinase) inhibitor ibrutinib demonstrated encouraging clinical responses in ABC-DLBCL. However, patients with CD79wt/MyD88mut, or CARD11mut did not respond to ibrutinib, indicating the need for new therapies targeting ibrutinib refractory ABC-DLBCL. Recent approval of PI3Kδ selective inhibitor idelalisib for the treatment of indolent NHL (iNHL) as monotherapy highlighted selective inhibition of PI3Kδ as an effective therapeutic strategy. However, idelalisib did not show clinical activity in DLBCL in a Phase I expansion cohort study. Here we report immunohistochemical (IHC) analysis of clinical tumor samples from follicular lymphoma (FL) and DLBCL patients and a series of in vitro and in vivo mechanistic and functional studies to explore the importance of PI3K isoforms in regulating key signaling pathways in ABC-DLBCL.Methods: Expression of PI3K isoforms and PTEN was assessed by IHC and western blot from a panel of 45 FL and 45 DLBCL primary tumors. Effects of PI3K inhibitors (PI3Kδ-selective inhibitor idelalisib, PI3Kα-selective inhibitor BLY-719, PI3Kβ-selective inhibitor TGX-221, predominant PI3Kα/δ inhibitor copanlisib and BTK inhibitor ibrutinib) on nuclear NF-κB activation were determined using stable cell lines expressing NF-κB-luciferase reporter (obtained by lentiviral infection), IHC staining of p65 NF-κB, expression of CCL4, IL-6, and IL-10 by RT-PCR and protein production by ELISA assays. In vitro and in vivo mechanisms of action were addressed by assessing the activities of the key survival signaling pathways. In vitro and in vivo anti-tumor activities were investigated using cell lines and patient derived xenograft ABC-DLBCL models representing the key molecular features of BCR-dependent and independent ABC-DLBCL.Results: Although PI3Kδ was predominantly expressed in both FL and DLBCL, high PI3Kα expression was more prevalent in DLBCL (60% vs 18%), a patient population resistant to PI3Kδ-selective inhibition in the clinic. Simultaneous inhibition of PI3Kα and PI3Kδ with BYL-719 plus idelalisib or copanlisib alone dramatically enhanced anti-tumor profile in ABC-DLBCL models compared to selective inhibition of PI3Kδ, PI3Kα or BTK. The anti-tumor activity was associated with suppression of p-AKT and a novel mechanism of blocking NF-κB activation driven by CD79mut, CARD11mut, A20mut or MyD88mutin vitro and in vivo. Suppression of NF-kB activation by PI3K inhibition is independent from AKT, but involves a novel mechanism of modulating c-IAP expression. Inhibition of PI3Kα/δ resulted in complete tumor regression in an ibrutinib-resistant MyD88mut-LY0257 patient-derived (PDx) ABC-DLBCL model. Furthermore, rebound activation of BTK and AKT was identified as a mechanism limiting CD79mut ABC-DLBCL to show robust response to PI3K and BTK inhibitors, respectively, as single agents in vivo. Combination of ibrutinib with PI3Kα/δ inhibitor copanlisib dosed intermittently iv was well tolerated and produced complete tumor regression in CD79Bmut TMD-8 cell line and Ly2298 PDx ABC-DLBCL models.Conclusions: High expression of PI3Kα in addition to PI3Kδ in ABC-DLBCL is associated with intrinsic resistance to PI3Kδ selective inhibition. Simultaneous inhibition of PI3Kα/δ by copanlisib modulates not only the PI3K/AKT pathway but also BCR-dependent and independent activation of nuclear NF-κB via a novel AKT-independent mechanism, indicating a promising utility for the treatment of clinically relevant ibrutinib-resistant ABC-DLBCL patients with CD79wt/MyD88mut, A20mut, or CARD11mut tumor genotypes. Combination of PI3Kα/δ and BTK inhibitors demonstrated promising potential for ibrutinib-responsive ABC-DLBCL to achieve complete tumor regression by blocking rebound activation of BTK and AKT. Thus, our findings presented here provide additional insights on intrinsic and acquired resistance mechanisms to selective PI3Kδ and BTK inhibitors and provide rationale for clinical development of PI3K inhibitors with specific isoform profile in combination for the treatment of ABC-DLBCL. DisclosuresPaul:Bayer AG: Employment. Sojoun:Bayer AG: Employment. Wengner:Bayer AG: Employment. Zitzmann-Kolbe:Bayer AG: Employment. Sturz:Bayer AG: Employment. Haike:Bayer AG: Employment. Martin:Bayer AG: Employment. Mumberg:Bayer AG: Employment. Ziegelbauer:Bayer AG: Employment. Liu:Bayer AG: Employment.

Both mTORC1 and mTORC2 are involved in the regulation of cell adhesion.

mTOR is a central controller for cell growth/proliferation and survival. Recent studies have shown that mTOR also regulates cell adhesion, yet the underlying mechanism is not known. Here we found that inhibition of mTOR by rapamycin reduced the basal or type I insulin-like growth factor (IGF-1)-stimulated adhesion of cancer cells. Further research revealed that both mTORC1 and mTORC2 were involved in the regulation of cell adhesion, as silencing expression of raptor or rictor inhibited cell adhesion. Also, PP242, an mTORC1/2 kinase inhibitor, inhibited cell adhesion more potently than rapamycin (mTORC1 inhibitor). Of interest, ectopic expression of constitutively active and rapamycin-resistant mutant of p70 kinase 1 (S6K1) or downregulation of eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) conferred resistance to rapamycin inhibition of cell adhesion, whereas expression of constitutively hypophosphorylated 4E-BP1 (4EBP1-5A) or downregulation of S6K1 suppressed cell adhesion. In contrast, neither genetic manipulation of Akt activity nor pharmacological inhibition of Akt affected cell adhesion. The results suggest that both mTORC1 and mTORC2 are involved in the regulation of cell adhesion; and mTORC1 regulates cell adhesion through S6K1 and 4E-BP1 pathways, but mTORC2 regulates cell adhesion via Akt-independent mechanism.

Perifosine treatment in chronic lymphocytic leukemia: results of a phase II clinical trial and in vitro studies

Because of the importance of the phosphoinositide 3-kinase (PI3K)/AKT pathway in chronic lymphocytic leukemia (CLL), we evaluated in vitro cytotoxicity induced by perifosine, an AKT inhibitor, in CLL lymphocytes and found that the mean 50% effective dose (ED50) was 313 nM. We then performed a phase II trial of perifosine in patients with relapsed/refractory CLL to assess response, outcomes, toxicity and ex vivo correlative measures. After 3 months of treatment, six of eight patients showed stable disease, one achieved a partial response and one had progressive disease. Median event-free survival and overall survival in all patients treated were 3.9 and 9.7 months. Adverse events included hematologic, infectious/fever, pain, gastrointestinal and constitutional toxicities. Unexpectedly, AKT phosphorylation in CLL lymphocytes from treated patients was not correlated with response. Additionally, perifosine did not inhibit AKT phosphorylation in cultured CLL lymphocytes. Perifosine is cytotoxic to CLL cells in vitro, and largely induces stabilized disease in vivo, with an AKT-independent mechanism.

MUC1 induces drug resistance in pancreatic cancer cells via upregulation of multidrug resistance genes

MUC1 (CD227), a membrane tethered mucin glycoprotein, is overexpressed in >60% of human pancreatic cancers (PCs), and is associated with poor prognosis, enhanced metastasis and chemoresistance. The objective of this study was to delineate the mechanism by which MUC1 induces drug resistance in human (BxPC3 and Capan-1) and mouse (KCKO, KCM) PC cells. We report that PC cells that express high levels of MUC1 exhibit increased resistance to chemotherapeutic drugs (gemcitabine and etoposide) in comparison with cells that express low levels of MUC1. This chemo resistance was attributed to the enhanced expression of multidrug resistance (MDR) genes including ABCC1, ABCC3, ABCC5 and ABCB1. In particular, levels of MRP1 protein encoded by the ABCC1 gene were significantly higher in the MUC1-high PC cells. In BxPC3 and Capan-1 cells MUC1 upregulates MRP1 via an Akt-dependent pathway, whereas in KCM cells MUC1-mediated MRP1 upregulation is via an Akt-independent mechanism. In KCM, BxPC3 and Capan-1 cells, the cytoplasmic tail motif of MUC1 associates directly with the promoter region of the Abcc1/ABCC1 gene, indicating a possible role of MUC1 acting as a transcriptional regulator of this gene. This is the first report to show that MUC1 can directly regulate the expression of MDR genes in PC cells, and thus confer drug resistance.

Abstract 1732: PI3K inhibition potentiates ABT-737 induced apoptosis in colorectal cancer cells independent of MCL1 downregulation or AKT/mTOR inhibition.

Abstract Background Colorectal cancer (CRC) is the third most common cancer and is a leading cause of morbidity worldwide. The five year survival rate of patients with advanced or metastatic CRC is less than 5%, identifying the need for new and improved therapies in the treatment of advanced and metastatic CRC. Phosphoinositide 3-kinase (PI3K) signaling has been implicated in several hallmarks of cancer, such as sustained proliferation, resisting cell death and invasion, and is frequently deregulated in CRC via a mutation of effectors such as PIK3CA, PIK3R1 and PTEN, making it an attractive therapeutic drug target. Despite this, inhibition of PI3K signaling does not cause cell death in CRC cells. Furthermore, CRC cells are relatively resistant to the BH3 mimetic ABT-737, which directly targets the BCL2 family of proteins inducing apoptosis via the mitochondrial pathway. Therefore, we investigated whether combining PI3K inhibitors and ABT-737 in CRC cells might synergise to induce apoptosis. Results PI3K inhibition enhanced ABT-737 induced apoptosis by 2.3-4.5 fold and reduced expression levels of Mcl-1, an established resistance biomarker for ABT-737. PI3K inhibition enhanced ABT-737-induced apoptosis a further 1.4-2.4 fold in CRC cells with siRNA depleted Mcl-1 suggesting contributory mechanisms additional to Mcl-1 down regulation. PI3K inhibition increased levels of Bcl-xL bound BimEL independently of Mcl-1 depletion. Further investigations are being carried out to determine whether this is the cause of increase ABT-737 sensitivity. ABT-737-induced apoptosis was unaffected by inhibition of PI3K downstream effectors AKT and mTOR. A siRNA library screen has been conducted to identify proteins downstream of PI3K which affect ABT-737 sensitivity and the results will be reported. Conclusions These data reveal a PI3K inhibition enhanced, ABT-737 driven apoptosis that occurs via Mcl-1, AKT and mTOR independent mechanism(s) consistent with increased association of BimEL and Bcl-xL. Further work is ongoing to elucidate the molecular mechanisms that underpin enhanced response to this BH-3 mimetic in CRC. Citation Format: Danielle S. Potter, Paul J. Kelly, Olive A. Denneny, Caroline Dive, Christopher J. Morrow. PI3K inhibition potentiates ABT-737 induced apoptosis in colorectal cancer cells independent of MCL1 downregulation or AKT/mTOR inhibition. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1732. doi:10.1158/1538-7445.AM2013-1732

Interleukin 1β Regulation of FoxO1 Protein Content and Localization: EVIDENCE FOR A NOVEL CERAMIDE-DEPENDENT MECHANISM

FoxO1 transcription factor controls the glucose and lipid metabolism, as well as cell proliferation and stress response. Akt, activated by insulin and other growth factors, phosphorylates FoxO1 causing its nuclear export and activity suppression. In this manuscript, we show that IL-1β, a pro-inflammatory cytokine, has the opposite effects on FoxO1. IL-1β stimulation of primary rat hepatocytes and HEK293 cells overexpressing the IL-1β receptor (293-IL-1RI) results in increased nuclear and cytosolic FoxO1 protein but not mRNA levels. IL-1β stimulation also elevates the levels of a mutant FoxO1 that is resistant to Akt phosphorylation. This suggests that an Akt-independent mechanism is involved. Co-stimulation with insulin does not affect the IL-1β induction of FoxO1. The IL-1β effects on FoxO1 are counteracted, however, by the silencing or inhibition of neutral sphingomyelinase 2 (nSMase-2) using shRNAi, scyphostatin, or GW4869, as well as by the pharmacological inhibition of JNK and ERK. Reversely, the overexpression of nSMase-2 through adenovirus-mediated gene transfer potentiates, in a JNK- and ERK-dependent manner, the IL-1β effects. We also show that transcription of insulin-like growth factor-binding protein-1 mRNA, which requires active FoxO1, is stimulated by IL-1β and is suppressed by the inhibition of nSMase-2 and JNK. In conclusion, we propose that IL-1β regulates FoxO1 activity through a novel nSMase-2-dependent pathway.

Evidence of mTOR Activation by an AKT-Independent Mechanism Provides Support for the Combined Treatment of PTEN-Deficient Prostate Tumors with mTOR and AKT Inhibitors

Activation of the phosphoinositide 3-kinase pathway is commonly observed in human prostate cancer. Loss of function of phosphatase and tensin homolog (PTEN) is associated with the activation of AKT and mammalian target of rapamycin (mTOR) in many cancer cell lines as well as in other model systems. However, activation of mTOR is also dependent of kinases other than AKT. Here, we show that activation of mTOR is not dependent on AKT in a prostate-specific PTEN-deficient mouse model of prostate cancer. Pathway bifurcation of AKT and mTOR was noted in both mouse and human prostate tumors. We demonstrated for the first time that cotargeting mTOR and AKT with ridaforolimus/MK-8669 and M1K-2206, respectively, delivers additive antitumor effects in vivo when compared to single agents. Our preclinical data suggest that the combination of AKT and mTOR inhibitors might be more effective in treating prostate cancer patients than current treatment regimens or either treatment alone.

The Effects of IL‐6 Dosage and Duration on the Regulation of Protein Turnover in C2C12 Myotubes

One of hallmarks of cachexia is skeletal muscle wasting caused by an imbalance between the rates of protein synthesis and degradation. Circulating IL‐6 is chronically elevated in many types of cachexia, and has emerged as a key regulator of muscle mass. The purpose of this study was to investigate the influence of IL‐6 dosage and duration of exposure on signaling pathways regulating protein turnover in cultured C2C12 myotubes. Differentiated C2C12 myotubes were treated with two doses (20ng/ml and 100ng/ml) of IL‐6 for 4h, 24h and 48h. Protein samples were analyzed by Western blots. The low dose (20ng/ml) IL‐6 treatment transiently (4h) increased phosphorylation of ERK1/2 (T202/Y204), mTOR (S2448) and p70S6K (T389), through an Akt independent mechanism. Inhibition of Stat3 signaling by pyrrolidine dithioicarbamate (PDTC) abolished these anabolic effects of IL‐6. Chronic treatment (24h) of IL‐6 at this dosage caused reduced activation of p70S6K phosphorylation, and inhibition of mTOR phosphorylation. However, 100ng/ml IL‐6 showed an inhibitory effect on p70S6K phosphorylation at 4h, 24h and 48h, and 4EBP‐1 phosphorylation (T37/46) at 48h. Finally, Atrogin‐1 protein expression was elevated by 100ng/ml IL‐6 at all three time points. These results demonstrate IL‐6 dosage and duration of exposure can differentially effect the regulation of protein turnover in C2C12 muscle cells.

Genetic depletion of the phosphatase PHLPP1 upregulates receptor tyrosine kinase signaling

The PH‐domain leucine rich repeat protein phosphatase PHLPP1 is a Ser/Thr phosphatase that negatively regulates Akt signaling. This pathway controls many cellular processes; loss of PHLPP leads to hyperactivation of Akt and an imbalance in cell homeostasis, triggering many diseases. Using cells derived from a phlpp1−/− mouse, we show that PHLPP1 suppresses the amplitude of the ERK pathway through an Akt‐independent mechanism. phlpp1−/− cells have increased receptor tyrosine kinase levels leading to augmented activation of downstream signaling. In these cells, the effects on ERK are due to an increased rate of receptor biosynthesis, not defective receptor degradation. Neither pharmacological inhibition of the PI3K‐Akt pathway, nor the MEK‐Erk pathway affects receptor levels, indicating that the PHLPP1 target is not Akt nor ERK. Furthermore, the catalytic and leucine rich repeat regions are critical to regulate receptor levels in normal cells. To find the mechanism controlling receptor levels, we performed pharmacological and PCR arrays to dissect the components responsible for this phenotype in phlpp1−/− cells. Our data indicate that general transcriptional machinery rather than only one transcriptional factor are involved. These data unveil an additional function of PHLPP1 in suppressing receptor tyrosine kinase signaling in cells by a transcriptional mechanism.This work was funded by NIH GM067946.

Mechanistic Analysis of a DNA Damage-Induced, PTEN-Dependent Size Checkpoint in Human Cells

Following DNA damage, human cells undergo arrests in the G(1) and G(2) phases of the cell cycle and a simultaneous arrest in cell size. We previously demonstrated that the cell size arrest can be uncoupled from the cell cycle arrest by mutational inactivation of the PTEN tumor suppressor gene. Here we show that the cell size checkpoint is inducible by DNA-damaging chemotherapeutic agents as well as by ionizing radiation and is effectively regulated by PTEN but not by its oncogenic counterpart, PIK3CA. Mutational analysis of PTEN and pharmacological inhibition of Akt revealed that modulation of Akt phosphorylation is unnecessary for cell size checkpoint control. To discover putative PTEN regulators and/or effectors involved in size checkpoint control, we employed a novel endogenous epitope tagging (EET) approach, which revealed that endogenous PTEN interacts at the membrane with an actin-remodeling complex that includes actin, gelsolin, and EPLIN. Pharmacological inhibition of actin remodeling in PTEN(+/+) cells recapitulated the lack of size checkpoint control seen in PTEN(-/-) cells. Taken together, these results provide further support for the existence of a DNA damage-inducible size checkpoint that is regulated by a major tumor suppressor, and they provide a novel Akt-independent mechanism by which PTEN controls cell size.

Targeting mTOR Signaling Is An Effective Treatment Strategy for IKAROS and JAKKinase Mutated Acute Lymphoblastic Leukemia

AbstractAbstract 3251Recently, an extremely poor risk subset of Philadelphia chromosome (9;22) negative ALL patients was identified that nonetheless have a gene expression profile similar to BCR-ABL positive ALL. The majority of cases in this ALL subset were found to have genetic alterations in IKZF1 (IKAROS). In addition, a significant proportion of these cases had CRLF2 rearrangements, and over half of the CRLF2 rearranged cases also had activating mutations in a JAK kinase. Identifying a biologically-targeted therapy for these patients is important as they are poor responders to conventional chemotherapeutics. Unfortunately, IKZF1 and CRLF2 are not currently targetable, and less than one half of these patients have JAK mutations.mTOR signaling has been shown to be activated in patients with BCR-ABL+ leukemia, in part through an Akt-independent mechanism. Accordingly, mTOR inhibition is effective in BCR-ABL+ ALL, while Akt inhibition is not. As patients with IKZF1 mutations have a gene expression profile similar to BCR-ABL+ ALL, we hypothesized that mTOR may be activated and that targeting mTOR signaling would be effective in this poor risk ALL subtype, whereas targeting Akt would be ineffective.To test this hypothesis, we established xenografts from 21 patients with this poor risk ALL subtype using NOG mice. 17 of 21 samples successfully engrafted with sufficient disease burden to generate second and third generation xenografts. We randomized second-generation xenografts from 4 patient samples to treatment with sirolimus (mTOR inhibitor), perifosine (Akt inhibitor), or vehicle control. Disease was evaluated at weekly intervals by FACS of peripheral blood for human CD19+/CD45+ ALL cells. To assess if there was a differential effect based on JAK mutations and/or CRLF2 rearrangements, we treated mice bearing patient ALL samples from each of the following sub-types: Jak+/CRLF2+, Jak+/CRLF2-, Jak-/CRLF2+, and Jak-/CRLF2-.Mice treated with sirolimus had a profound response with markedly decreased disease in blood and in spleen at sacrifice in all 4 samples (Table 1). Perifosine had no anti-leukemic effect. Both drugs were well tolerated. We confirmed the drugs were hitting target pathways in treated animals by immunoblot for pS6 and pAKT.SampleAvg (range) pb blasts control mice at sacrificeAvg (range) pb blasts sirolimus treated at sacrificeAvg (range) pb blasts perifosine treatedsirolimus vs control; perifosine vs control (t-test)Jak-/CRLF2-5,596 (4,203 – 7,922)120 (61 – 248)4,498 (3,522 – 5,474)p = 0.01; p = 0.11Jak-/CRLF2+30,009 (21,173 – 41,166)12,764 (7,171 – 14,945)24,587 (12,980 – 44,498)p = 0.01; p = 0.32Jak+/CRLF2-50,6730 (24,415 – 90,144)15,523 (13,814 – 17,915)31,512 (22,621 – 43,669)p = 0.02; p = 0.10Jak+/CRLF2+6,799 (2,165 – 13,095)1,676 (398 – 3,431)5,042 (1,632 – 9,442)p = 0.05; p = 0.31In conclusion, targeting mTOR with sirolimus maybe an effective treatment for IKZF1 and JAK mutated ALL. Disclosures:No relevant conflicts of interest to declare.

Resveratrol Inhibits mTOR Signaling by Promoting the Interaction between mTOR and DEPTOR

Resveratrol (RSV) is a naturally occurring polyphenol that has been found to exert antioxidant, anti-inflammatory, and neuroprotective properties. However, how RSV exerts its beneficial health effects remains largely unknown. Here, we show that RSV inhibits insulin- and leucine-stimulated mTOR signaling in C2C12 fibroblasts via a Sirt1-independent mechanism. Treating C2C12 cells with RSV dramatically inhibited insulin-stimulated Akt, S6 kinase, and 4E-BP1 phosphorylation but had little effect on tyrosine phosphorylation of the insulin receptor and activation of the p44/42 MAPK signaling pathway. RSV treatment also partially blocked mTOR and S6 kinase phosphorylation in TSC1/2-deficient mouse embryonic fibroblasts, suggesting the presence of an inhibitory site downstream of TSC1/2. Knocking out PDK1 or suppressing AMP-activated protein kinase had little effect on leucine-stimulated mTOR signaling. On the other hand, RSV significantly increased the association between mTOR and its inhibitor, DEPTOR. Furthermore, the inhibitory effect of RSV on leucine-stimulated mTOR signaling was greatly reduced in cells in which the expression levels of DEPTOR were suppressed by RNAi. Taken together, our studies reveal that RSV inhibits leucine-stimulated mTORC1 activation by promoting mTOR/DEPTOR interaction and thus uncover a novel mechanism by which RSV negatively regulates mTOR activity.

Lopinavir inhibits meningioma cell proliferation by Akt independent mechanism

Recent studies suggest that HIV-1 protease inhibitors may have anti-neoplastic effects on some malignancies. The anti-neoplastic effects of lopinavir have not been established or studied in brain tumors. Primary cultures of three fetal leptomeninges and 18 meningiomas were treated with lopinavir alone or with PDGF-BB. DNA synthesis was assessed by CyQUANT. Lopinavir effects on basal and PDGF-stimulated phosphorylation of the Akt-mTOR, MEK1/2-MAPK and STAT3 pathways, phosphorylation of Rb, Caspase 3 activation and reductions in survivin were assessed by Western blots. Lopinavir produced a significant reduction in PDGF-BB stimulation of DNA synthesis in a leptomeningeal culture (P = 0.0013) and 1 of 6 WHO grade I and 1 of 4 grade II meningiomas at 24 h and in 3 of 6 WHO grade I, 4 of 4 grade II and 1 of 1 grade III cell cultures (P = 0.0001) at 72 h. Lopinavir reduced PDGF-BB stimulation of phosphorylation/activation of MAPK in the 22 week fetal leptomeningeal cell cultures and in cells from 1 grade I meningioma at 24 h, but in none of 4 grade I and 5 grade II meningiomas at 6 h. Lopinavir had no notable effect on basal or PDGF-stimulated p-mTOR, p-MEK1/2, or p-STAT3, activation of Caspase 3 or survivin levels. Lopinavir treatment for 24 h had no effect on basal Rb phosphorylation but reduced Rb phosphorylation in all four meningioma cultures. These studies suggest that lopinavir may inhibit meningioma growth, and does so in part by cell cycle arrest. Additional evaluation of lopinavir as a potential adjunct chemotherapy is warranted.

The Catalytic Subunit of Protein Phosphatase 1 Gamma Regulates Thrombin-Induced Murine Platelet αIIbβ3 Function

BackgroundHemostasis and thrombosis are regulated by agonist-induced activation of platelet integrin αIIbβ3. Integrin activation, in turn is mediated by cellular signaling via protein kinases and protein phosphatases. Although the catalytic subunit of protein phosphatase 1 (PP1c) interacts with αIIbβ3, the role of PP1c in platelet reactivity is unclear.Methodology/Principal FindingsUsing γ isoform of PP1c deficient mice (PP1cγ−/−), we show that the platelets have moderately decreased soluble fibrinogen binding and aggregation to low concentrations of thrombin or protease-activated receptor 4 (PAR4)-activating peptide but not to adenosine diphosphate (ADP), collagen or collagen-related peptide (CRP). Thrombin-stimulated PP1cγ−/− platelets showed decreased αIIbβ3 activation despite comparable levels of αIIbβ3, PAR3, PAR4 expression and normal granule secretion. Functions regulated by outside-in integrin αIIbβ3 signaling like adhesion to immobilized fibrinogen and clot retraction were not altered in PP1cγ−/− platelets. Thrombus formation induced by a light/dye injury in the cremaster muscle venules was significantly delayed in PP1cγ−/− mice. Phosphorylation of glycogen synthase kinase (GSK3)β-serine 9 that promotes platelet function, was reduced in thrombin-stimulated PP1cγ−/− platelets by an AKT independent mechanism. Inhibition of GSK3β partially abolished the difference in fibrinogen binding between thrombin-stimulated wild type and PP1cγ−/− platelets.Conclusions/SignificanceThese studies illustrate a role for PP1cγ in maintaining GSK3β-serine9 phosphorylation downstream of thrombin signaling and promoting thrombus formation via fibrinogen binding and platelet aggregation.

IκB Kinase Overcomes PI3K to Control FoxO3a Subcellular Localization in Acute Myeloid Leukemia.

Abstract 478The prognosis of Acute Myeloid Leukaemia (AML) remains globally poor. Aberrant activation of signalling pathways is frequently found in AML and leads to uncontrolled cell growth and survival. Effective targeting of these pathways with new therapeutics may result in suppression of leukemic cell proliferation and survival. In AML, constitutive PI3K activity, mainly due to the expression of the class IA PI3K p110δ isoform, is detected in 50% of patients at diagnosis (PI3K+ AML samples) (Sujobert, Bardet et al., Blood 2005; Tamburini, Elie et al., Blood 2007). The PI3K/Akt axis represents therefore an attractive therapeutic target in this disease. Accordingly, inhibition of PI3K activity with IC87114, a specific p110δ inhibitor decreases AML blast cells proliferation (Sujobert, Bardet et al., Blood 2005). However, IC87114 failed to induce significant apoptosis (Park, Chapuis et al., Leukemia 2008). The PI3K/Akt network controls different targets implicated in the regulation of cell survival. Among them, the FoxO transcription factors which include FoxO1, FoxO3a, FoxO4 and FoxO6 up regulate the expression of target genes involved in apoptosis, such Fas-L and Bim. Akt, by phosphorylating FoxO proteins at three conserved sites (T32, S253 and S315 on FoxO3a), negatively regulates their transcriptional activity by inducing their nuclear export. Given that IC87114 did not induce apoptosis, we hypothesized that FoxO proteins may escape to PI3K/Akt control in AML cells. For that purpose, we investigated their mechanisms of regulation in both primary AML cells and a MV4-11 human leukemic cell line engineered to stably overexpress a FoxO3awt-GPF fusion protein introduced by lentiviral infection. We focused especially on FoxO3a as we found, using real-time RT-PCR and WB analysis, that it was the only FoxO protein constantly expressed in primary AML cells. First, we examined the relationship between FoxO3a subcellular localization using immunofluorescence and PI3K activity in primary AML cells. As expected in PI3K+ AML cells (n=6), FoxO3a was phosphorylated on T32 and S253 and constantly localized in the cytoplasm. However, although Akt S473 and FoxO3a T32 and S253 phosphorylations were fully inhibited by IC87114, FoxO3a did not translocate into the nucleus of blast cells. Accordingly, Fas-L and Bim expression tested by qRT-PCR were not induced by IC87114. Identical results were obtained from the same analysis of the MV4-11FoxO3awt-GFP cell line. Furthermore, in PI3K- AML cells (n=5), neither Akt S473 nor FoxO3a T32 and S253 phosphorylations were detected but FoxO3a was still confined in the cytoplasm of these cells. Altogether, these results clearly demonstrate that an Akt-independent mechanism contributes to the nuclear exclusion of FoxO3a in AML cells. In addition to Akt, the IκB Kinase (IKK) may regulate FoxO3a subcellular localization by phosphorylation on S644 in AML, as it has previously been shown in breast cancer (Hu, Lee et al., Cell 2004). As the oncogenic IκB Kinase is known to be activated in almost all AML samples (Guzman, Neering et al., Blood 2001), we investigated whether IKK could maintain FoxO3a in the cytoplasm of AML cells. Interestingly, we found that inhibition of IKK with a specific IKKγ/NEMO-antagonistic peptide (Agou, Courtois et al., J Biol Chem 2004) induced a strong nuclear translocation of FoxO3a and an efficient apoptosis in both primary AML blast cells (n=5) and MV4-11FoxO3awt-GFP cell line. To confirm this result, MV4-11 cells were infected with a lentivirus expressing a FoxO3aS644A-GFP mutant protein in which the IKK phosphorylation is abrogated. We found that the FoxO3aS644A-GFP mutant protein primarily localized within the nucleus of MV4-11 cells and induced a decrease of cell proliferation and a moderate pro-apoptotic effect. We thus conclude that IKK, by phosphorylating FoxO3a on S644, leads to the constant nuclear export of FoxO3a in AML cells. Consequently, the therapeutical potential value of IKK targeted inhibition in AML could be due not only to the inhibition of NF-κB activity but also to the FoxO3a nuclear import. Moreover, the PI3K-independent FoxO3a regulation probably contributes to the low potential of specific PI3K inhibitors to induce apoptosis in AML cells. Disclosures:No relevant conflicts of interest to declare.

Effects of PI3K catalytic subunit and Akt isoform deficiency on mTOR and p70S6K activation in myoblasts

The PI3K/Akt/mTOR signaling pathway is critical for cellular growth and survival in skeletal muscle, and is activated in response to growth factors such as insulin-like growth factor-I (IGF-I). We found that in C2C12 myoblasts, deficiency of PI3K p110 catalytic subunits or Akt isoforms had distinct effects on phosphorylation of mTOR and p70S6K. siRNA-mediated knockdown of PI3K p110α, p110β, and simultaneous knockdown of p110α and p110β resulted in increased basal and IGF-I-stimulated phosphorylation of mTOR S2448 and p70S6K T389; however, phosphorylation of S6 was reduced in p110β-deficient cells, possibly due to reductions in total S6 protein. We found that IGF-I-stimulated Akt1 activity was enhanced in Akt2- or Akt3-deficient cells, and that knockdown of individual Akt isoforms increased mTOR/p70S6K activation in an isoform-specific fashion. Conversely, levels of IGF-I-stimulated p70S6K phosphorylation in cells simultaneously deficient in both Akt1 and Akt3 were increased beyond those seen with loss of any single Akt isoform, suggesting an alternate, Akt-independent mechanism that activates mTOR/p70S6K. Our results collectively suggest that mTOR/p70S6K is activated in a PI3K/Akt-dependent manner, but that in the absence of p110α or Akt, alternate pathway(s) may mediate activation of mTOR/p70S6K in C2C12 myoblasts.

Akt inhibitor Akt-IV blocks virus replication through an Akt-independent mechanism.

Many viruses activate the phosphatidylinositol 3'-kinase (PI3k)/Akt intracellular signaling pathway to promote viral replication. We have analyzed whether a rapidly replicating rhabdovirus, vesicular stomatitis virus (VSV), requires the PI3k/Akt signaling pathway for its replication. Through the use of chemical inhibitors of PI3k and Akt, we show that VSV replication and cytopathic effects do not require activation of these kinases. Inhibitors that block the activating phosphorylations of Akt at threonine 308 (Thr308) and serine 473 (Ser473) did not inhibit VSV protein expression or the induction of the cytopathic effects of VSV. One compound, Akt inhibitor Akt-IV, inhibited the replication of VSV, respiratory syncytial virus, and vaccinia virus but increased the phosphorylation of Akt at positions Thr308 and Ser473 and did not inhibit Akt kinase activity in vitro. Together, our data suggest that the PI3k/Akt pathway is of limited relevance to the replication of VSV but that Akt inhibitor Akt-IV is a novel broad-spectrum antiviral compound with a mechanism differing from that of its previously reported effect on the PI3k/Akt pathway. Identification of other targets for this compound may define a new approach for blocking virus replication.