ATP-competitive mTOR Inhibitor Research Articles

Abstract PR04: mTOR mutations in cancer

Abstract Genes in the PI3K-Akt-mTOR signaling axis are frequently mutated in cancer but relatively few mutations have been observed in the MTOR gene itself. By mining publicly available cancer sequencing data, we recently reported that MTOR is commonly mutated in cancer, with more than 35 previously uncharacterized pathway activating mutations. Most were recurrent, localized within distinct clusters in the C-terminal half of the protein, and induced activation of mTOR Complex 1 (mTORC1), mTORC2, or both. The mutations did not prevent mTOR inhibition by rapamycin or ATP-competitive inhibitors, although several conferred resistance to nutrient withdrawal. Importantly, cancer cell lines with activating mTOR mutations are especially sensitive to rapamycin both in vitro and in vivo, indicating pathway dependency. In a companion report, we also demonstrated that the tumor of a bladder cancer patient with exquisite sensitivity to a rapamycin analog (rapalog) harbored two activating mTOR mutations, likely driving mTOR pathway dependency. However, after 14 months of rapalog response, the patient displayed progressive disease, indicating that the tumor had acquired resistance to the targeted therapy. Here, we demonstrate two mechanisms by which cancers may display resistance to rapamycin through mTOR mutation. In the first example, we acquired a tumor biopsy from an anaplastic thyroid carcinoma patient before they were enrolled on a rapalog clinical trial. The patient displayed a durable 18 month response to the rapalog before progressing, at which time we acquired a second biopsy. Whole exome sequencing of the biopsies revealed the presence of a loss-of-function mutation in the mTORC1 negative regulator Tuberin (TSC2), likely driving pathway addiction and thus sensitivity to the rapalog. Sequencing of the post-resistance biopsy revealed that the tumor had acquired a mutation in the rapamycin-binding domain of mTOR. Cells made to exogenously express this mutation displayed potent resistance to rapamycin-mediated inhibition of cell proliferation and phosphorylation of the mTORC1 substrate S6K1. Importantly, the cells remained sensitive to an ATP-competitive mTOR inhibitor because the two classes of mTOR inhibitors rely upon completely different mechanisms of action. Thus, we show that as patients acquire resistance to rapalogs, they may still be effectively treated with ATP-competitive mTOR inhibitors. The rapalog-resistant mutation described above is an example of an acquired mutation, although due to the genetic instability of cancer cells, a tumor may harbor pre-existing rapamycin-resistant mutations. To determine the prevalence of such mutations, we again turned to publicly available cancer sequencing data, identifying approximately 30 mutations in the rapamycin-binding domain of mTOR. The majority these mutations did not alter mTOR pathway activation and remained rapamycin sensitive. Importantly, one breast cancer sample harbored a rapamycin-resistant mTOR mutation, although it remained sensitive to an ATP-competitive mTOR inhibitor. This rare event is highly relevant given that rapalogs are currently approved for the treatment of breast cancer. Had this patient been treated with a rapalog, they would have only experienced the side-effects and none of the benefits of treatment. Collectively, we demonstrate that mTOR mutations are common in cancer, many of which confer pathway activation and addiction. Additionally, both acquired and pre-existing mTOR mutations can confer resistance towards rapamycin treatment, although this resistance can be overcome with ATP-competitive inhibitors. This work has important ramifications for the treatment of cancer patients with mTOR inhibitors, by identifying both those patients likely to respond or display resistance to such treatments. This abstract is also being presented as Poster A44. Citation Format: Brian Grabiner, Nikhil Wagle, Eliezer Van Allen, Levi Garraway, Jochen Lorch, David Sabatini. mTOR mutations in cancer. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr PR04.

Abstract B46: Activity of the novel mTOR inhibitor Torin-2 in B-precursor acute lymphoblastic leukemia and its therapeutic potential to prevent AKT reactivation

Abstract mTOR is a highly conserved and widely expressed serine/threonine kinase, that is a member of the phosphatidylinositol-3 kinase–like kinase (PIKK) family. mTOR plays a pivotal role in the PI3K/Akt/mTOR signaling pathway, which senses growth factor and serves as a central regulator of fundamental cellular processes such cell growth/apoptosis, autophagy, translation, and metabolism. Hyperactivation of this pathway through loss of negative regulators, such as PTEN, or mutational activation of receptor tyrosine kinases of phosphoinositide 3-kinase (PI3K) is a frequent occurrence in leukemia patients, where it negatively influences response to therapeutic treatments. In B-precursor acute lymphoblastic leukemia (B-pre ALL) many research efforts are currently devoted to the development of targeted therapies to limit side effects of chemotherapy and to increase treatment efficacy for poor prognosis patients, i.e. poor outcome following relapse. Thus, targeting the PI3K/AKT /mTOR pathway is an attractive therapeutic strategy. Numerous inhibitors targeting these kinases are currently undergoing clinical evaluation in hematological malignancies including ALL, mostly in combination with conventional chemotherapy. Prolonged suppression of mTORC1 also results in disruption of a negative feedback loop and often results in re/hyper-phosphorylation of Akt through activation of IRS1 and PI3K, which may counteract rapalogs activity. A new class of ATP-competitive mTOR inhibitors, such as Torin-2, has revealed that these inhibitors potently targets mTORC1 and mTORC2. Together, these features have generated hope that this new generation of inhibitors will exhibit broader clinical efficacy when compared to the rapalogs. We therefore hypothesized that dual inhibition of mTORC1 and mTORC2 by Torin-2 would provide a superior outcome in ALL as compared to inhibition of mTORC1 alone with RAD001 in B-pre-ALL. We tested the capability of Torin-2 to prevent AKT reactivation after mTORC1 and mTORC2 inhibition. Furthermore we explored if dual targeting of mTORC1 and AKT may achieve results similar to those obtained with Torin-2 alone. Drugs cytotoxic activity were analyzed in a panel of B-pre-ALL cells by MTT Assay and Western Blotting at different time points. Cell cycle, apoptosis and autophagy were analyzed by flow cytometry, Western Blotting and fluorescent staining. In all the B-pre-ALL cell lines Torin-2 showed a powerful cytotoxic activity, inhibiting the growth of each cell line in a dose-dependent manner, with an IC50 in the nanomolar range as assessed by MTT assays. The major proteins along the PI3K/AKT/mTOR signaling pathway where heavily dephosphorylated after 2 hrs of drug exposure. This inhibition lasted up to at least 48 hrs at variance to RAD001, that already after 24 hrs was unable to prevent AKT reactivation. However the association of RAD001 with MK-2206, an allosteric AKT inhibitor, prevented AKT reactivation and reached a significant cytotoxicity. These data suggest an interesting cytotoxicity of Torin-2 in B-pre-ALL acting on both mTORC1 and mTORC2 as assessed by their specific substrate inhibition. Feedback activation of PI3K/AKT was suppressed by Torin-2 alone, whereas RAD001 required the addition of MK-2206 to achieve the same efficacy. These two pharmacological strategies targeting PI3K/Akt/mTOR at different points of the signaling pathway cascade might represent a new therapeutic option in B-pre-ALL patients also preventing Akt reactivation. Citation Format: Carolina Simioni, Alice Cani, Alberto M. Martelli, Giorgio Zauli, Silvano Capitani, Luca M. Neri. Activity of the novel mTOR inhibitor Torin-2 in B-precursor acute lymphoblastic leukemia and its therapeutic potential to prevent AKT reactivation. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B46.

Abstract B53: Effects of metformin and ATP-competitive inhibitor of mTOR on targeted-metabolomic profile in HPAF-II pancreatic cancer cell lines

Abstract Introduction: Pancreatic cancer (PC) is a devastating disease attributable to late diagnosis and is resistant to conventional treatment. Thus, there is an urgent need to develop new strategies for intervention of this lethal disease. Pancreatic ductal adenocarcinoma (PDAC) is the most common and most aggressive type of PC. Recent reports indicate the 50% of PDAC patients are diabetic at the time of diagnosis. To that end, tumor-related diabetes is now considered Type 3cDM. The mammalian Target of Rapamycin mTOR (also known as mechanistic TOR) is a conserved serine/threonine kinase and master regulator of metabolism, cell growth and proliferation. mTOR is dysregulated in a variety of tumors including pancreatic cancer. Furthermore, the anti-diabetic drug, metformin, which indirectly inhibits mTOR by activation of AMPK, has emerged as a potential therapeutic target in the treatment of PC, particularly when associated with type 3c Diabetes Mellitus. Mounting evidence links glycolytic metabolic disturbances to the adverse pancreatic cancer prognosis. The objective of this study is to determine the targeted-metabolomic profile in human PDAC cell line (HPAF-II) and the possible synergism between pan mTOR inhibition by ATP competitors (which inhibits both mTORC1 and mTORC2) and metformin administration as potential targets for therapeutic intervention in pancreatic cancer. Methods: Well differentiated pancreatic cancer cell line HPAF-II (CRL# 1997) were cultured in DMEM media supplemented with 10% fetal bovine serum in the presence of either Torin 2 (ATP-competitive mTOR inhibitor), metformin, both, or vehicle control for 1 hour and 24 hours in five replicate samples per group. We utilized targeted LC/MS/MS to characterize the alterations in the glycolytic and tricarboxylic acid (TCA) cycle metabolomics, and employed Western Blot analysis for cell signaling activation by phosphorylation. Comparisons between groups were analyzed using one-way Analysis of Variance (ANOVA) for detection of the overall statistical significance followed by secondary post-hoc analysis to determine differences between groups. The significance level was assessed at p < 0.05. Results: After 1 hour incubation with Torin 2, AMP concentration was reduced compared to the control and other treatment groups (p < 0.03). After 24 hours incubation, Torin-2 significantly decreased the glycolysis intermediates (fructose 1, 6 bisphosphate (FBP), and 2 phosphoglycerate/3 phosphoglycerate (2PG/3PG), TCA intermediate metabolites (acetyl CoA, citrate/isocitrate, and malate), as well as the electron acceptors (NAD+, and FAD). Metformin treatment alone reduced FBP and citrate/ citrate/isocitrate ratio. However, when HPAF-II cells were incubated with both Torin-2 and metformin, there was significant reduction in nicotinamide adenine dinucleotide (NAD+) and flavin adenine dinucleotide (FAD), suggesting decreased levels of energy equivalents available to the electron transport chain. To confirm that Torin 2 blocked both mTORC1 and mTORC2, whole cell lysates from all treatment and control groups where subjected to Western Blot analysis. Torin 2 treatment decreased the autophosphoyration of mTOR (S2481), and phosphorylation of its downstream target phospho S6 (pS6) confirming the inhibition of mTORC1, and decreased Akt phosphorylation at S473 (phospho-Akt-S473) indicating the inhibition of mTORC2, while rapamycin had no effect on Akt phosphorylation. Conclusion: The targeted metabolomics data indicate that mTORC1 and mTORC2 inhibition by Torin 2 reduced glycolytic intermediates and TCA metabolites in HPAF- II and may synergize with metformin to decrease the electron donors NAD+ and FAD by the TCA cycle which may lead to reduced energy production. The work on this abstract utilized Metabolomics Core Services supported by grant U24 DK097153 of NIH to the University of Michigan. Citation Format: Ghada A. Soliman, Asserewou Etekpo. Effects of metformin and ATP-competitive inhibitor of mTOR on targeted-metabolomic profile in HPAF-II pancreatic cancer cell lines. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B53.

Abstract P6-11-09: A preclinical study to demonstrate the utility of fulvestrant and MLN0128 combination against HR+ and HER2+ breast cancer

Abstract Background: Up to 10% of total breast cancers are positive for both hormone receptor [HR: estrogen receptor (ER) and/or progesterone receptor (PR)] and HER2. These patients belong mainly to the luminal B subtype, which exhibits resistance to endocrine therapy. Currently, systemic treatment for HR+/HER2+ breast cancer patients involves a combination of chemotherapy and HER2-directed therapy. While these therapies improve outcomes, they are associated with pernicious side effects. Results and Discussion: As demonstrated by studies from a number of laboratories, in HR+/HER2+ cancer, ER is constitutively activated. In other words, the estrogen ligand is not needed for ER activation. The constitutively activated ER, through its non-genomic pathways, can stimulate both HER2 (a feed forward loop) and associated kinases. In patients with HR+/HER2+ tumors, genes in the PI3K and ER pathways have been altered. Activation of PI3K/Akt/mTOR by HER2 overexpression predicts tumor progression in breast cancer [Zhou et al., Clin Cancer Res. 10: 6779 (2004)]. Therefore, PI3k/AKT/mTOR is considered as an attractive therapeutic target for HR+/HER2+ breast cancer. Allosteric inhibitors of mTOR, such as RAD001 (Everlimus), only target mTORC1 but not mTORC2, relieving the negative feedback loop in this pathway, and leading to the activation of AKT. Our results suggest that to effectively treat HR+/HER2+ cancers, both mTORC1 and mTORC2 signaling must be suppressed. In these contexts; MLN0128 (i.e., INK128 or "MLN") is a new ATP-competitive inhibitor of mTOR. It targets both mTORC1 and mTORC2; and it does not interact with FKBP12 (an immunoregulatory protein). Consequently, MLN produces weaker immune-suppressing effects than everolimus. Results from our cell culture experiments reveal that MLN is twenty times more potent than everolimus against ER+/HER2+ cells. Furthermore, while MLN alone has been demonstrated to inhibit the proliferation of ER+/HER2+ cells, we have observed more benefits when it is used as part of a combination. Fulvestrant (ICI) by itself only suppresses the proliferation of ER+/HER2+ cells partially. However, when MLN and ICI are used together, our studies revealed a synergistic effect. In addition, HR+/HER2+ PDX models have been generated to identify novel molecular networks and to examine the in vivo action of new targeted therapies against HR+/HER2+ cancer. We not only have verified the synergistic effect of MLN and ICI combination in vivo, we also identified a set of important genes playing roles in the growth of HR+/HER2+ tumors through RNA-Seq analysis. Conclusion. Due to their synergistic and targeted action, this MLN and ICI combination could provide better clinical outcome and less side-effects to HR+/HER2+ breast cancer patients, compared to the currently available options in the clinics. Citation Format: Shang Victoria Wu, Hannah Lu, Masaya Kai, Noriko Kanaya, Thenhang Luu, Courtney Vito, Laura Kruper, Joanne Mortimer, Shiuan Chen. A preclinical study to demonstrate the utility of fulvestrant and MLN0128 combination against HR+ and HER2+ breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-11-09.

The preclinical evaluation of the dual mTORC1/2 inhibitor INK-128 as a potential anti-colorectal cancer agent

The colorectal cancer is the leading contributor of cancer-related mortality. Mammalian target of rapamycin (mTOR), existing in 2 complexes (mTORC1/2), is frequently dysregulated and constitutively activated in colorectal cancers. It represents an important drug target. Here we found that INK-128, the novel ATP-competitive kinase inhibitor of mTOR, blocked both mTORC1 and mTORC2 activation in colorectal cancer cells (both primary and transformed cells). The immunoprecipitation results showed that the assembly of mTORC1 (mTOR-Raptor association) and mTORC2 (mTOR-Rictor-Sin1 association) was also disrupted by INK-128. INK-128 inhibited colorectal cancer cell growth and survival, and induced both apoptotic and non-apoptotic cancer cell death. Further, INK-128 showed no effect on Erk/MAPK activation, while MEK/Erk inhibition by MEK-162 enhanced INK-128-induced cytotoxicity in colorectal cancer cells. Meanwhile, INK-128 downregulated Fascin1 (FSCN1)/E-Cadherin expressions and inhibited HT-29 cell in vitro migration. In vivo, daily INK-128 oral administration inhibited HT-29 xenograft growth in mice, which was further enhanced by MEK-162 administration. Finally, we found that INK-128 sensitized 5-fluorouracil-(5-FU)-mediated anti-HT-29 activity in vivo and in vitro. Thus, our preclinical studies strongly suggest that INK-128 might be investigated for colorectal cancer treatment in clinical trials.

Metabolic Re-Programming in Notch-Activated T-ALL By mTOR Inhibitor AZD2014 Combined with L-Asparaginase

Adult T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis associated with resistance and rapid relapses, necessitating novel therapeutic strategies. Malignant T cells require high concentrations of nutrients to sustain their increased rates of proliferation, and frequent activation of mTOR signaling, a central regulator of cell metabolism, has been shown in a high-risk subgroup of T-ALL with constitutive Notch activation (Chan, Blood, 2007;110,278).In this study, we analyzed the metabolic response mechanisms of Notch-activated aggressive T-ALL to the ATP-competitive mTOR inhibitor AZD2014 and to the combination of AZD2014 with a conventional chemotherapeutic agent, L-asparaginase (ASNase). To investigate the molecular modifications of key cellular metabolic processes induced by these agents, we undertook a comprehensive and quantitative analysis of charged metabolites by capillary electrophoresis mass spectrometry (CE-MS, Agilent Technologies) for metabolic profiling.We first assessed the anti-proliferative effects of the combination of AZD2014 and ASNase in CUTLL1, a T-ALL cell line with a Notch gain-of-function mutation. The medianinhibitory concentrations of AZD-2014 and ASNase were 250 nM and 0.005 U/mL, respectively (MTT assay, 48 hours). The combination of AZD2014 (100 nM) and ASNase (0.001 U/mL) caused apoptosis induction with increase in the % of sub-G1 fraction (Control: 5.5±1.8, AZD-2014: 9.5±2.1, ASNase: 10.9 ±1.5, AZD-2014/ASNase: 17.9 ±4.9; p<0.05). Inhibition of mTOR kinase activity by AZD2014 resulted in downregulation of mTORC1 substrates phospho-(p-)S6K (Ser240-244) and p-4EBP1 (Thr37/46) and mTORC2 target p-Akt (Ser473) and induced autophagy, as shown by the conversion of the autophagosomal marker LC3-I to LC-II. AZD2014 further impaired the expression of oncogenic transcription factor c-Myc, an intermediary between Notch and mTOR signaling. In turn, ASNase downregulated c-Myc expression without inhibiting mTOR signaling.We next investigated the metabolic changes in CUTLL1 cells after AZD2014 (100 nM) and/or ASNase (0.001 U/mL) treatment (24 hours). CE-MS analysis demonstrated differences in 66 polar metabolites (p<0.05). As expected, ASNase downregulated L-asparagine monohydrate levels. Although AZD2014 did not change the level of L-asparagine monohydrate by itself, it significantly enhanced its depletion by ASNase. The AZD2014 and ASNase combination also impaired aspartate synthesis. In the TCA cycle, the AZD2014 / ASNase combination significantly downregulated gluconeogenesis by decreasing malate and fructose-6-phosphate and inhibited fatty acid synthesis with citrate depletion. In nucleotide metabolism, the combination treatment suppressed guanine and hypoxanthine levels, which led to decreases in uric acid. Finally, glutamate metabolism was markedly downregulated by depleting intracellular glutamine. All these changes were less prominent in CUTLL1 cells treated with single-agent AZD2014 or ASNase.These results indicate that AZD2014 effectively facilitates the ASNase-induced blockade of glycolysis/gluconeogenesis and fatty acid synthesis and represses asparate and glutamate metabolism in Notch-activated T-ALL cells. The multiple molecular alterations resulting from mTOR blockade by AZD2014, such as repression of translation initiation through potent inhibition of S6K and 4EBP1, induction of autophagy, and inhibition of c-Myc, may further impair the cellular metabolism altered by ASNase in aggressive T-ALL. In conclusion, metabolic re-programming by the combination of AZD2014 and L-asparaginase in Notch-activated T-ALL provides avenues for novel rationally designed combination regimens in ALL. DisclosuresNo relevant conflicts of interest to declare.

S136 Mtor Signalling Is An Essential Pathway For Tgf- 1 Induced sma And Collagen Gene Expression

<h3></h3> Emerging evidence suggests a role for PI3K/mTOR signalling in the pathology of organ fibrosis. The aim of this study was to delineate PI3K/mTOR signalling in response to TGF-β₁stimulation of primary human lung fibroblasts (HLFs), and to investigate the role of this pathway in TGF-β₁ mediated myofibroblast differentiation and collagen synthesis. A time-course of SMAD 2/3 and Akt (Ser473) phosphorylation, the major downstream effector of the PI3K/mTOR pathway, was constructed to assess TGF-β₁ induced signalling kinetics in HLFs. TGF-β₁(1 ng/ml) induced rapid phosphorylation of SMAD2/3, peaking at 1 h, followed by Akt phosphorylation which peaked 12 h after initial stimulation. Maximal expression of <i>ACTA2</i> and <i>COL1A1</i> was observed 36 h after TGF-β₁ stimulation, correlating with the delayed time-course of Akt phosphorylation. To investigate the role of the PI3K/mTOR pathway in TGF-β₁ induced myofibroblast differentiation and collagen gene expression, HLFs were treated with pharmacological titrations of potent pathway inhibitors. Maximal Akt signalling and expression of <i>ACTA2 </i>and <i>COL1A1</i> were significantly inhibited by a dual PI3K/mTOR inhibitor, while SMAD phosphorylation was unaffected. Treatment with an ATP competitive mTOR inhibitor also resulted in significantly reduced Akt phosphorylation and expression of <i>ACTA2</i> and <i>COL1A1</i>, in response to TGF-β₁. In contrast, treatment of HLFs with either an allosteric or ATP competitive Akt inhibitor showed no inhibitory effect on TGF-β₁induced gene expression. These data suggest PI3 kinase/mTOR signalling is an important component in TGF-β₁ induced αSMA and collagen gene expression and that an mTOR dependent, Akt independent pathway mediates this functional response in primary HLFs.

Abstract SY42-01: Translational control and tumor stem cell radioresponse

Abstract SY42-01: Translational control and tumor stem cell radioresponse

A critical role for mTORC1 in erythropoiesis and anemia.

Red blood cells (RBC) must coordinate their rate of growth and proliferation with the availability of nutrients, such as iron, but the signaling mechanisms that link the nutritional state to RBC growth are incompletely understood. We performed a screen for cell types that have high levels of signaling through mTORC1, a protein kinase that couples nutrient availability to cell growth. This screen revealed that reticulocytes show high levels of phosphorylated ribosomal protein S6, a downstream target of mTORC1. We found that mTORC1 activity in RBCs is regulated by dietary iron and that genetic activation or inhibition of mTORC1 results in macrocytic or microcytic anemia, respectively. Finally, ATP competitive mTOR inhibitors reduced RBC proliferation and were lethal after treatment with phenylhydrazine, an inducer of hemolysis. These results identify the mTORC1 pathway as a critical regulator of RBC growth and proliferation and establish that perturbations in this pathway result in anemia.

Adenosine Triphosphate-Competitive mTOR Inhibitors: A New Class of Immunosuppressive Agents That Inhibit Allograft Rejection

The mechanistic/mammalian target of rapamycin (mTOR) is inhibited clinically to suppress T cell function and prevent allograft rejection. mTOR is the kinase subunit of two mTOR-containing complexes, mTOR complex (mTORC) 1 and 2. Although mTORC1 is inhibited by the macrolide immunosuppressant rapamycin (RAPA), its efficacy may be limited by its inability to block mTORC1 completely and its limited effect on mTORC2. Adenosine triphosphate (ATP)-competitive mTOR inhibitors are an emerging class of mTOR inhibitors that compete with ATP at the mTOR active site and inhibit any mTOR-containing complex. Since this class of compounds has not been investigated for their immunosuppressive potential, our goal was to determine the influence of a prototypic ATP-competitive mTOR inhibitor on allograft survival. AZD8055 proved to be a potent suppressor of T cell proliferation. Moreover, a short, 10-day course of the agent successfully prolonged murine MHC-mismatched, vascularized heart transplant survival. This therapeutic effect was associated with increased graft-infiltrating regulatory T cells and reduced CD4(+) and CD8(+) T cell interferon-γ production. These studies establish for the first time, that ATP-competitive mTOR inhibition can prolong organ allograft survival and warrant further investigation of this next generation mTOR inhibitors.

Molecular rationale for the use of PI3K/AKT/mTOR pathway inhibitors in combination with crizotinib in ALK-mutated neuroblastoma

Mutations in the ALK tyrosine kinase receptor gene represent important therapeutic targets in neuroblastoma, yet their clinical translation has been challenging. The ALK(F1174L) mutation is sensitive to the ALK inhibitor crizotinib only at high doses and mediates acquired resistance to crizotinib in ALK-translocated cancers. We have shown that the combination of crizotinib and an inhibitor of downstream signaling induces a favorable response in transgenic mice bearing ALK(F1174L)/MYCN-positive neuroblastoma. Here, we investigated the molecular basis of this effect and assessed whether a similar strategy would be effective in ALK-mutated tumors lacking MYCN overexpression. We show that in ALK-mutated, MYCN-amplified neuroblastoma cells, crizotinib alone does not affect mTORC1 activity as indicated by persistent RPS6 phosphorylation. Combined treatment with crizotinib and an ATP-competitive mTOR inhibitor abrogated RPS6 phosphorylation, leading to reduced tumor growth and prolonged survival in ALK(F1174L)/MYCN-positive models compared to single agent treatment. By contrast, this combination, while inducing mTORC1 downregulation, caused reciprocal upregulation of PI3K activity in ALK-mutated cells expressing wild-type MYCN. Here, an inhibitor with potency against both mTOR and PI3K was more effective in promoting cytotoxicity when combined with crizotinib. Our findings should enable a more precise selection of molecularly targeted agents for patients with ALK-mutated tumors.

PGE2-induced colon cancer growth is mediated by mTORC1

The inflammatory prostaglandin E2 (PGE2) cytokine plays a key role in the development of colon cancer. Several studies have shown that PGE2 directly induces the growth of colon cancer cells and furthermore promotes tumor angiogenesis by increasing the production of the vascular endothelial growth factor (VEGF). The signaling intermediaries implicated in these processes have however not been fully characterized. In this report, we show that the mechanistic target of rapamycin complex 1 (mTORC1) plays an important role in PGE2-induced colon cancer cell responses. Indeed, stimulation of LS174T cells with PGE2 increased mTORC1 activity as observed by the augmentation of S6 ribosomal protein phosphorylation, a downstream effector of mTORC1. The PGE2 EP4 receptor was responsible for transducing the signal to mTORC1. Moreover, PGE2 increased colon cancer cell proliferation as well as the growth of colon cancer cell colonies grown in matrigel and blocking mTORC1 by rapamycin or ATP-competitive inhibitors of mTOR abrogated these effects. Similarly, the inhibition of mTORC1 by downregulation of its component raptor using RNA interference blocked PGE2-induced LS174T cell growth. Finally, stimulation of LS174T cells with PGE2 increased VEGF production which was also prevented by mTORC1 inhibition. Taken together, these results show that mTORC1 is an important signaling intermediary in PGE2 mediated colon cancer cell growth and VEGF production. They further support a role for mTORC1 in inflammation induced tumor growth.

Picking the point of inhibition: a comparative review of PI3K/AKT/mTOR pathway inhibitors.

The frequent activation of the PI3K/AKT/mTOR pathway in cancer, and its crucial role in cell growth and survival, has made it a much desired target for pharmacologic intervention. Following the regulatory approval of the rapamycin analogs everolimus and temsirolimus, recent years have seen an explosion in the number of phosphoinositide 3-kinase (PI3K) pathway inhibitors under clinical investigation. These include: ATP-competitive, dual inhibitors of class I PI3K and mTORC1/2; "pan-PI3K" inhibitors, which inhibit all four isoforms of class I PI3K (α, β, δ, γ); isoform-specific inhibitors of the various PI3K isoforms; allosteric and catalytic inhibitors of AKT; and ATP-competitive inhibitors of mTOR only (and thus mTORC1 and mTORC2). With so many agents in development, clinicians are currently faced with a wide array of clinical trials investigating a multitude of inhibitors with different mechanisms of action, being used both as single agents and in combination with other therapies. Here, we provide a review of the literature, with the aim of differentiating the genomic contexts in which these various types of inhibitors may potentially have superior activity.

Species Difference in Glucuronidation Formation Kinetics with a Selective mTOR Inhibitor

The mammalian target of rapamycin (mTOR) is a protein kinase that shows key involvement in age-related disease and promises to be a target for treatment of cancer. In the present study, the elimination of potent ATP-competitive mTOR inhibitor 3-(6-amino-2-methylpyrimidin-4-yl)-N-(1H-pyrazol-3-yl)imidazo[1,2-b]pyridazin-2-amine (compound 1) is studied in bile duct-cannulated rats, and the metabolism of compound 1 in liver microsomes is compared across species. Compound 1 was shown to undergo extensive N-glucuronidation in bile duct-catheterized rats. N-glucuronides were detected on positions N1 (M2) and N2 (M1) of the pyrazole moiety as well as on the primary amine (M3). All three N-glucuronide metabolites were detected in liver microsomes of the rat, dog, and human, while primary amine glucuronidation was not detected in cynomolgus monkey. In addition, N1- and N2-glucuronidation showed strong species selectivity in vitro, with rat, dog, and human favoring N2-glucuronidation and monkey favoring N1-glucuronide formation. Formation of M1 in monkey liver microsomes also followed sigmoidal kinetics, singling out monkey as unique among the species with regard to compound 1 N-glucuronidation. In this respect, monkeys might not always be the best animal model for N-glucuronidation of uridine diphosphate glucuronosyltransferase (UGT) 1A9 or UGT1A1 substrates in humans. The impact of N-glucuronidation of compound 1 could be more pronounced in higher species such as monkey and human, leading to high clearance in these species. While compound 1 shows promise as a candidate for investigating the impact of pan-mTOR inhibition in vivo, opportunities may exist through medicinal chemistry efforts to reduce metabolic liability with the goal of improving systemic exposure.

The ATP-Competitive mTOR Inhibitor INK128 Enhances In Vitro and In Vivo Radiosensitivity of Pancreatic Carcinoma Cells

Radiotherapy remains a primary treatment modality for pancreatic carcinoma, a tumor characterized by aberrant mTOR activity. Given the regulatory role of mTOR in gene translation, in this study, we defined the effects of the clinically relevant, ATP-competitive mTOR inhibitor, INK128 on the radiosensitivity of pancreatic carcinoma cell lines. Clonogenic survival was used to determine the effects of INK128 on in vitro radiosensitivity of three pancreatic carcinoma cell lines and a normal fibroblast cell line with mTOR activity defined using immunoblots. DNA double-strand breaks were evaluated according to γH2AX foci. The influence of INK128 on radiation-induced gene translation was determined by microarray analysis of polysome-bound mRNA. Leg tumor xenografts grown from pancreatic carcinoma cells were evaluated for mTOR activity, eIF4F cap complex formation, and tumor growth delay. INK128, while inhibiting mTOR activity in each of the cell lines, enhanced the in vitro radiosensitivity of the pancreatic carcinoma cells but had no effect on normal fibroblasts. The dispersal of radiation-induced γH2AX foci was inhibited in pancreatic carcinoma cells by INK128 as were radiation-induced changes in gene translation. Treatment of mice with INK128 resulted in an inhibition of mTOR activity as well as cap complex formation in tumor xenografts. Whereas INK128 alone had no effect of tumor growth rate, it enhanced the tumor growth delay induced by single and fractionated doses of radiation. These results indicate that mTOR inhibition induced by INK128 enhances the radiosensitivity of pancreatic carcinoma cells and suggest that this effect involves the inhibition of DNA repair.

Pan-Mammalian Target of Rapamycin (mTOR) Inhibitor AZD8055 Primes Rhabdomyosarcoma Cells for ABT-737-induced Apoptosis by Down-regulating Mcl-1 Protein

The PI3K/mammalian Target of Rapamycin (mTOR) pathway is often aberrantly activated in rhabdomyosarcoma (RMS) and represents a promising therapeutic target. Recent evaluation of AZD8055, an ATP-competitive mTOR inhibitor, by the Preclinical Pediatric Testing Program showed in vivo antitumor activity against childhood solid tumors, including RMS. Therefore, in the present study, we searched for AZD8055-based combination therapies. Here, we identify a new synergistic lethality of AZD8055 together with ABT-737, a BH3 mimetic that antagonizes Bcl-2, Bcl-xL, and Bcl-w but not Mcl-1. AZD8055 and ABT-737 cooperate to induce apoptosis in alveolar and embryonal RMS cells in a highly synergistic fashion (combination index < 0.2). Synergistic induction of apoptosis by AZD8055 and ABT-737 is confirmed on the molecular level, as AZD8055 and ABT-737 cooperate to trigger loss of mitochondrial membrane potential, activation of caspases, and caspase-dependent apoptosis that is blocked by the pan-caspase inhibitor Z-VAD-fmk. Similar to AZD8055, the PI3K/mTOR inhibitor NVP-BEZ235, the PI3K inhibitor NVP-BKM120 and Akt inhibitor synergize with ABT-737 to trigger apoptosis, whereas no cooperativity is found for the mTOR complex 1 inhibitor RAD001. Interestingly, molecular studies reveal a correlation between the ability of different PI3K/mTOR inhibitors to potentiate ABT-737-induced apoptosis and to suppress Mcl-1 protein levels. Importantly, knockdown of Mcl-1 increases ABT-737-induced apoptosis similar to AZD8055/ABT-737 cotreatment. This indicates that AZD8055-mediated suppression of Mcl-1 protein plays an important role in the synergistic drug interaction. By identifying a novel synergistic interaction of AZD8055 and ABT-737, our findings have important implications for the development of molecular targeted therapies for RMS.

Efficacy and Mechanisms Of The mTOR Inhibitor AZD2014 Combined With L-Asparaginase Or JAK2 Inhibitor TG101348 In ALL

Despite impressive outcomes achieved with combination chemotherapy regimens in acute lymphoblastic leukemia (ALL), resistance and relapses occur frequently necessitating a quest for novel therapeutic targets. PI3K/AKT/mTOR signalling is a central regulator of cell metabolism, growth, proliferation and survival and has been shown to be constitutively activated in the aggressive T-ALL with Notch gain-of-function mutations (Palomero, Leukemia, 2006;20:1279). In a high-risk subgroup of B-ALL harbouring mutations in the CRLF2 gene, the frequent activation of mTOR and JAK/STAT pathways was recently reported (Roberts, Cancer Cell, 2012;22:153).In this study, we tested the pre-clinical efficacy and mechanisms of action of the novel ATP-competitive mTOR inhibitor AZD2014 which effectively inhibits both TORC1 and TORC2 complexes. Pharmacological interactions with chemotherapeutic agent L-asparaginase (L-Asp) or JAK2 inhibitor TG101348 were further evaluated in high-risk immature T-ALL (PF-382, CCRF-CEM, RPMI-8402 cells, Notch1-mutated CUTLL1) and CRLF2-mutated B-ALL cells (MUTZ-5). Inhibition of mTOR kinase activity by AZD2014 used at sub-micromolar concentrations resulted in downregulation of mTORC1 substrates phospho-(p-)S6K (Ser240-244), p-4EBP1 (Thr37/46), mTORC2 target p-Akt (Ser473), and of the oncogenic transcription factor c-Myc. This translated into reduction of cell proliferation with G0/G1 cell cycle arrest in all tested ALL cells, with IC50s ranging from 70 nM to 1,000 nM. In Notch-activated CUTLL1 T-ALL cells, L-Asp enhanced the cell growth inhibition by AZD2014 and further decreased p-S6K levels. In CRLF2-rearranged MUTZ-5 B-ALL cells, the AZD2014/TG-101348 combination enhanced the anti-proliferative effects of AZD2014 with further decrease of p-4EBP1 and down-regulation of Pim-1, a downstream target of Akt and JAK/STAT signaling, more prominently than in other tested pre-B-ALL cells, Nalm6 and REH. AZD2014 induced autophagy as shown by the conversion of the autophagosomal marker LC3-I to LC-II in CUTLL1 and MUTZ-5 cells.To investigate the molecular modifications of the key cellular metabolic processes in response to mTOR blockade, we employed the proteomic technology of isobaric tags for relative and absolute quantitation (iTRAQ) with two-dimensional-liquid chromatography-tandem mass spectrometry. Using iTRAQ platform, more than 1,770 unique proteins were identified in each of the four cell lines tested (T-ALL, PF-382 and CUTLL1; B-ALL, Nalm6 and MUTZ-5). AZD2014 frequently and significantly downregulated anti-apoptotic chaperone heat shock proteins HSP70 and HSP90 (p < 0.05) along with upregulation of the voltage-dependent anion channels (VDAC) (p < 0.01) and histone H1 (p < 0.05), both functioning as dynamic initiators of mitochondria-mediated apoptosis. In T-ALL cells, L-Asp alone or in combination with AZD2014 further upregulated VDAC, histone H1, and apoptosis related mitochondrial chaperone HSP10 (p < 0.05), and repressed translation initiation factors and proteasome-related proteins (p < 0.05). Interestingly, in Notch-mutant CUTLL1 cells L-Asp downregulated DNA methyltransferase Dnmt1 (p < 0.05), and the combination with AZD2014 further decreased histone-binding protein RBBP4 (p < 0.05) which interacts with histone deacetylase, suggesting previously unrecognized epigenetic anti-tumor modifications by AZD2014/L-Asp treatment. In B-ALL cells, the AZD2014/TG101348 combination induced significant repression of ribosomal and spliceosomal pathways (p < 0.001) along with marked repression of HSP70 and ubiquitin related protein E3 ubiquitin-protein ligase (p < 0.05), and increased expression of the apoptosis regulators VDAC (p < 0.01), histone H1 (p < 0.05) and chaperone HSP60 (p < 0.05).In summary, these results suggest that mTOR blockade by AZD2014 inhibits ALL cell growth through multiple mechanisms, which include repression of translation initiation through potent inhibition of S6K and 4EBP1, induction of autophagy, modulation of HSP chaperone proteins and epigenetic modifications. These cellular alterations are further impaired by the concomitant inhibition of asparagine metabolism with L-Asp in T-ALL or through blockade of JAK2 signaling in B-ALL, providing avenues for novel rationally designed combinatorial regimens in ALL. Disclosures:No relevant conflicts of interest to declare.

Targeting mTOR to Overcome Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance in Non-Small Cell Lung Cancer Cells

AimsEpidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown dramatic clinical benefits in advanced non-small cell lung cancer (NSCLC); however, resistance remains a serious problem in clinical practice. The present study analyzed mTOR-associated signaling-pathway differences between the EGFR TKI-sensitive and -resistant NSCLC cell lines and investigated the feasibility of targeting mTOR with specific mTOR inhibitor in EGFR TKI resistant NSCLC cells.MethodsWe selected four different types of EGFR TKI-sensitive and -resistant NSCLC cells: PC9, PC9GR, H1650 and H1975 cells as models to detect mTOR-associated signaling-pathway differences by western blot and Immunoprecipitation and evaluated the antiproliferative effect and cell cycle arrest of ku-0063794 by MTT method and flow cytometry.ResultsIn the present study, we observed that mTORC2-associated Akt ser473-FOXO1 signaling pathway in a basal state was highly activated in resistant cells. In vitro mTORC1 and mTORC2 kinase activities assays showed that EGFR TKI-resistant NSCLC cell lines had higher mTORC2 kinase activity, whereas sensitive cells had higher mTORC1 kinase activity in the basal state. The ATP-competitive mTOR inhibitor ku-0063794 showed dramatic antiproliferative effects and G1-cell cycle arrest in both sensitive and resistant cells. Ku-0063794 at the IC50 concentration effectively inhibited both mTOR and p70S6K phosphorylation levels; the latter is an mTORC1 substrate and did not upregulate Akt ser473 phosphorylation which would be induced by rapamycin and resulted in partial inhibition of FOXO1 phosphorylation. We also observed that EGFR TKI-sensitive and -resistant clinical NSCLC tumor specimens had higher total and phosphorylated p70S6K expression levels.ConclusionOur results indicate mTORC2-associated signaling-pathway was hyperactivated in EGFR TKI-resistant cells and targeting mTOR with specific mTOR inhibitors is likely a good strategy for patients with EGFR mutant NSCLC who develop EGFR TKI resistance; the potential specific roles of mTORC2 in EGFR TKI-resistant NSCLC cells were still unknown and should be further investigated.

Rapamycin-resistant mTORC1 restrains dendritic cell B7-H1 expression that requires IL-1β to enhance regulatory T cell induction (P1349)

Abstract Introduction: The mammalian Target of Rapamycin (mTOR) is a central regulator of dendritic cell (DC) function that performs the catalytic activity of mTOR complex (mTORC)1 and 2. mTORC2 functions independently from mTORC1 and is resistant to inhibition by rapamycin (RAPA); however, mTORC1 has both RAPA-sensitive and -resistant outputs. Our goal was to ascertain the role of RAPA-resistant mTOR in DC. Methods: WT C57BL/6 or B7-H1-/- bone marrow-derived DC were generated with the addition of RAPA or ATP-competitive mTOR inhibitor, which blocks all mTOR signaling. DC lacking rictor, an mTORC2-specific subunit, were generated from conditional rictor KO mice. DC induction of regulatory T cells (Treg) was determined in MLR, using BALB/c CD4+CD25- T cell responders. Results and Conclusion: RAPA and mTORC2 deletion reduced DC B7-H1 expression, but ATP-competitive mTOR inhibitors enhanced B7-H1 expression. Augmented B7-H1 expression was blocked by STAT3 inhibition and correlated with reduced expression of the STAT3 negative regulator, SOCS3. DC exposed to ATP-competitive mTOR inhibitors increased Treg induction, which was dependent on DC B7-H1. IL-1β neutralization additionally reduced Treg induction by B7-H1-/- ATP-competitive mTOR inhibitor-exposed DC, suggesting that IL-1β and B7-H1 act additively to promote Treg induction by these DC. These findings establish a RAPA-resistant mTORC1 pathway that acts through SOCS3 and STAT3 to regulate DC B7-H1 expression and Treg induction.

Characterization of Torin2, an ATP-Competitive Inhibitor of mTOR, ATM, and ATR

mTOR is a highly conserved serine/threonine protein kinase that serves as a central regulator of cell growth, survival, and autophagy. Deregulation of the PI3K/Akt/mTOR signaling pathway occurs commonly in cancer and numerous inhibitors targeting the ATP-binding site of these kinases are currently undergoing clinical evaluation. Here, we report the characterization of Torin2, a second-generation ATP-competitive inhibitor that is potent and selective for mTOR with a superior pharmacokinetic profile to previous inhibitors. Torin2 inhibited mTORC1-dependent T389 phosphorylation on S6K (RPS6KB1) with an EC(50) of 250 pmol/L with approximately 800-fold selectivity for cellular mTOR versus phosphoinositide 3-kinase (PI3K). Torin2 also exhibited potent biochemical and cellular activity against phosphatidylinositol-3 kinase-like kinase (PIKK) family kinases including ATM (EC(50), 28 nmol/L), ATR (EC(50), 35 nmol/L), and DNA-PK (EC(50), 118 nmol/L; PRKDC), the inhibition of which sensitized cells to Irradiation. Similar to the earlier generation compound Torin1 and in contrast to other reported mTOR inhibitors, Torin2 inhibited mTOR kinase and mTORC1 signaling activities in a sustained manner suggestive of a slow dissociation from the kinase. Cancer cell treatment with Torin2 for 24 hours resulted in a prolonged block in negative feedback and consequent T308 phosphorylation on Akt. These effects were associated with strong growth inhibition in vitro. Single-agent treatment with Torin2 in vivo did not yield significant efficacy against KRAS-driven lung tumors, but the combination of Torin2 with mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor AZD6244 yielded a significant growth inhibition. Taken together, our findings establish Torin2 as a strong candidate for clinical evaluation in a broad number of oncologic settings where mTOR signaling has a pathogenic role.