ATP-competitive mTOR Inhibitor Research Articles

Abstract 69: In vitro and in vivo tumor cell radiosensitization by the competitive mTOR inhibitor INK128.

Abstract Radiation-induced translational control of gene expression has been shown to be a component of the cellular radioresponse. Furthermore, recent data suggest strategies targeting eIF4E may be effective for tumor cell radiosensitization. The mechanistic target of rapamycin (mTOR) is a critical kinase in the regulation of eIF4E and mRNA translation. The newly developed ATP-competitive mTOR inhibitor, INK128, inhibits both mTORC1 and mTORC2 activity, and is in currently undergoing evaluation in Phase 1 clinical trials. In the study described here we evaluated the effects of INK128 treatment on tumor cell radiosensitivity in vitro using a clonogenic survival assay and in vivo according to tumor growth delay. For tumor cell lines initiated from different histologic origins in vitro exposure to INK128 resulted in the complete inhibition of both mTORC1 and 2. Treatment with the same concentration of INK128 immediately after irradiation increased tumor cell radiosensitivity with DEFs greater than 1.3. To determine whether the INK128 induced radiosensitization could be extended to an in vivo model we used subcutaneous xenografts initiated from U251 or PSN1 tumor cells. INK128 treatment (oral gavage) inhibited mTOR activity in tumors established from both cell lines as defined by loss of 4E-BP1 phosphorylation. Furthermore, m7-GTP batch chromatography showed that INK128 treatment decreased cap-binding activity in xenografts. To evaluate the effects of INK128 on in vivo tumor radiosensitivity a tumor growth delay assay was performed using mice bearing U251 tumor xenografts. When tumors reached approximately 210mm3 mice were randomized into 4 groups: vehicle, radiation, INK128, and INK128 plus radiation. INK128 was delivered once a day (3 mg/kg, oral gavage) for four days with the tumor locally irradiated (2 Gy) 2h after each of the four drug treatments. Absolute growth delays were calculated as the time in days for tumors in treated mice to grow from 210 to 1000mm3 minus the time in days for tumors to reach the same size in vehicle treated mice. The absolute growth delays for INK128 alone and radiation alone groups were 1.7 and 12.7 days respectively. The growth delay in mice treated with the combination of INK128 and radiation was 22.2 days, which is greater than the sum of the growth delays caused by INK128 and radiation alone. The DEF, obtained by dividing the normalized tumor growth delay in mice treated with the radiation/PP242 combination (20.5) by the absolute growth delay in mice treated with radiation only (12.7), was 1.6. These results suggest that mTOR inhibition by the clinically relevant ATP-competitive mTOR inhibitor, INK128, enhances tumor cell radiosensitivity both in vitro and in vivo. Citation Format: Thomas J. Hayman, Tamalee Kramp, Kevin Camphausen, Philip J. Tofilon. In vitro and in vivo tumor cell radiosensitization by the competitive mTOR inhibitor INK128. [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 69. doi:10.1158/1538-7445.AM2013-69

Incomplete inhibition of phosphorylation of 4E-BP1 as a mechanism of primary resistance to ATP-competitive mTOR inhibitors

The mammalian target of rapamycin (mTOR) regulates cell growth by integrating nutrient and growth factor signaling and is strongly implicated in cancer. But mTOR is not an oncogene, and which tumors will be resistant or sensitive to new ATP-competitive mTOR inhibitors now in clinical trials remains unknown. We screened a panel of over 600 human cancer cell lines to identify markers of resistance and sensitivity to the mTOR inhibitor PP242. RAS and PIK3CA mutations were the most significant genetic markers for resistance and sensitivity to PP242, respectively; colon origin was the most significant marker for resistance based on tissue type. Among colon cancer cell lines, those with KRAS mutations were most resistant to PP242, while those without KRAS mutations most sensitive. Surprisingly, cell lines with co-mutation of PIK3CA and KRAS had intermediate sensitivity. Immunoblot analysis of the signaling targets downstream of mTOR revealed that the degree of cellular growth inhibition induced by PP242 was correlated with inhibition of phosphorylation of the translational repressor 4E-BP1, but not ribosomal protein S6. In a tumor growth inhibition trial of PP242 in patient-derived colon cancer xenografts, resistance to PP242 induced inhibition of 4E-BP1 phosphorylation and xenograft growth was again observed in KRAS mutant tumors without PIK3CA co-mutation, compared to KRAS WT controls. We show that, in the absence of PIK3CA co-mutation, KRAS mutations are associated with resistance to PP242 and that this is specifically linked to changes in the level of phosphorylation of 4E-BP1.

Anti-Fibrotic Effect of Combined mTORc1 and mTORc2 Inhibition in Bronchiolitos Obliterans

Purpose The mammalian target of rapamycin (mTOR) pathway plays an important role in regulating protein translation. The aim of this study was to evaluate the contribution of mTORc1 and mTORc2 signaling in fibrotic functions of mesenchymal cell and the potential use of mTOR inhibitors as anti-fibrotic agents in bronchiolitis obliterans syndrome (BOS). Methods and Materials Lung-resident mesenchymal stromal cells (LR-MSCs) were isolated from bronchoalveolar lavage of patients±BOS. PI3K/PTEN/Akt/mTOR pathway was studied by western analysis of whole-cell lysates for total PTEN, phospho-Akt, and phospho-p70S6K1. Effect of mTOR inhibitors on collagen I and α-smooth muscle actin (α-SMA) expression was assessed. Results A significantly higher expression of p70S6Kinase (Thr389) and 4E-BP1 (Thr37/46) was noted in BOS LR-MSCs compared to non-BOS LR-MSCs. BOS LR-MSCs also exhibited increased expression of phosphorylated AKT (S473). A decrease in collagen I protein expression was seen in the presence of rapamycin, while α-SMA expression was not changed. Rapamycin inhibited p70S6Kinase but failed to decrease 4E-BP1 expression. An increased in pAKT(S473) in response to rapamycin was noted. A high baseline expression of pAKT(S473) in BOS LR-MSCs and its further upregulation by mTORc1 inhibition, led us to study AZD8055, an ATP-competitive mTOR inhibitor which inhibits downstream signaling from both mTORc1 and mTORc2. Treatment with AZD8055 led to a 96% collagen suppression compared to 62% decrease by rapamycin (p Conclusions mTOR signaling pathway is activated in BOS LR-MSCs. While treatment with rapamycin modestly inhibits fibrotic phenotype of LR-MSCs, its also results in augmented mTORc2 signaling. AZD8055, an ATP-competitive mTOR inhibitor, inhibits collagen, α-SMA, and mTOR substrate phosphorylation more significantly than rapamycin, suggesting AZD8055 as a promising therapeutic in the context of lung transplantation.

MTOR Inhibitors Alone and in Combination with JAK2 Inhibitors Effectively Inhibit Cells of Myeloproliferative Neoplasms

BackgroundDysregulated signaling of the JAK/STAT pathway is a common feature of chronic myeloproliferative neoplasms (MPN), usually associated with JAK2V617F mutation. Recent clinical trials with JAK2 inhibitors showed significant improvements in splenomegaly and constitutional symptoms in patients with myelofibrosis but meaningful molecular responses were not documented. Accordingly, there remains a need for exploring new treatment strategies of MPN. A potential additional target for treatment is represented by the PI3K/AKT/mammalian target of rapamycin (mTOR) pathway that has been found constitutively activated in MPN cells; proof-of-evidence of efficacy of the mTOR inhibitor RAD001 has been obtained recently in a Phase I/II trial in patients with myelofibrosis. The aim of the study was to characterize the effects in vitro of mTOR inhibitors, used alone and in combination with JAK2 inhibitors, against MPN cells.FindingsMouse and human JAK2V617F mutated cell lines and primary hematopoietic progenitors from MPN patients were challenged with an allosteric (RAD001) and an ATP-competitive (PP242) mTOR inhibitor and two JAK2 inhibitors (AZD1480 and ruxolitinib). mTOR inhibitors effectively reduced proliferation and colony formation of cell lines through a slowed cell division mediated by changes in cell cycle transition to the S-phase. mTOR inhibitors also impaired the proliferation and prevented colony formation from MPN hematopoietic progenitors at doses significantly lower than healthy controls. JAK2 inhibitors produced similar antiproliferative effects in MPN cell lines and primary cells but were more potent inducers of apoptosis, as also supported by differential effects on cyclinD1, PIM1 and BcLxL expression levels. Co-treatment of mTOR inhibitor with JAK2 inhibitor resulted in synergistic activity against the proliferation of JAK2V617F mutated cell lines and significantly reduced erythropoietin-independent colony growth in patients with polycythemia vera.Conclusions/SignificanceThese findings support mTOR inhibitors as novel potential drugs for the treatment of MPN and advocate for clinical trials exploiting the combination of mTOR and JAK2 inhibitor.

MTOR inhibitor efficacy is determined by the eIF4E/4E-BP ratio

The mammalian/mechanistic target of rapamycin (mTOR) is a multifunctional serine/threonine kinase that is hyperactivated in cancer [1]. mTOR forms two distinct complexes, mTORC1 and 2. mTORC1 stimulates translation and perturbs energy metabolism to drive cell proliferation and growth, whereas mTORC2 regulates cytoskeletal organization and cell survival by stimulating AGC kinases (e.g. AKT). Therefore, suppressing mTOR's activity is widely considered as a very attractive anti-cancer therapy. Accordingly, mTOR inhibitors are increasingly used as anti-neoplastic agents in the clinic. Rapamycin is a naturally occurring allosteric inhibitor of mTORC1 and its analogs (rapalogs) are used as anti-cancer agents for the treatment of refractory renal cell carcinomas, mantle cell lymphomas, and pancreatic neuroendocrine tumors. However, success of rapalogs as anti-cancer monotherapies is limited [2]. This has been attributed to the activation of AKT signaling resulting from the loss of a negative-feedback mechanism, as well as rapamycin-resistant mTORC1 outputs, such as the phosphorylation of the eukaryotic initiation factor 4E-binding proteins (4E-BPs). To overcome these deficiencies a new generation of ATP-competitive mTOR inhibitors [also referred to as dual mTORC1/2 inhibitors or active-site mTOR inhibitors (asTORi)] was developed [2]. asTORi suppress AKT signaling by inhibiting mTORC2, and abrogate rapamycin-resistant mTORC1 outputs including the phosphorylation of 4E-BPs. Accordingly, asTORi exhibit stronger anti-proliferative and anti-tumorigenic effects as compared to rapalogs, and are currently in multiple clinical trials aiming to target aberrant mTOR signaling in cancer [2]. However, a major obstacle to applying asTORi to the clinic is a lack of predictive biomarkers that would facilitate the stratification of the patients that are most likely to respond to these drugs. Discovery of predictive biomarkers indicative of the efficacy of mTOR inhibitors is hampered by the complexity of the mTOR pathway, given that mTOR controls a variety of cellular processes via a multitude of substrates. 4E-BPs are a family of small translational repressors, which sequester the 5' mRNA cap-binding protein eukaryotic translation initiation factor (eIF)-4E (eIF4E), thereby impeding the assembly of the eIF4F complex. The eIF4F complex, which consists of eIF4E, scaffolding protein eIF4G and the DEAD-box helicase eIF4A, recruits the mRNA to the ribosome to initiate translation. mTORC1 phosphorylates and inactivates 4E-BPs, thereby facilitating the assembly of the eIF4F complex and translation initiation (See Figure ​Figure1).1). eIF4E acts as a general translation initiation factor, but a subset of mRNAs referred to as “eIF4E-sensitive” are particularly sensitive to changes in eIF4E activity. These mRNAs encode tumor-promoting factors, such as cyclin D3, ornithine decarboxylase and myc. The inability of rapalogs to completely suppress 4E-BP phosphorylation and translation of “eIF4E-sensitive” mRNAs is thought to be the reason for their relatively limited anti-tumorigenic efficacy in the clinic [2, 3]. Figure 1 Sensitivity of tumor to asTORi as a function of eIF4E/4E-BP ratio Overexpression of eIF4E and variations in 4E-BP levels and phosphorylation are frequently observed in tumors, thereby suggesting that eIF4E/4E-BP stoichiometry may significantly differ among patients, or even within a single tumor [4]. We demonstrated that the eIF4E/4E-BP ratio determines the ability of asTORi to suppress neoplastic growth [5]. Resistance of malignant cells with high eIF4E/4E-BP ratio to asTORi can be explained by their deficiency to inhibit eIF4F complex assembly and translation of “eIF4E-sensitive” mRNAs (Figure ​(Figure1).1). We also showed that cancer cells acquire resistance to asTORi by increasing eIF4E availability via downregulation of 4E-BP1 and 2. These data corroborate earlier findings showing that the amplification of the eIF4E gene underlies resistance to the dual PI3K/mTOR inhibitor BEZ235 [6], and that eIF4E translation activity can predict sensitivity to rapalogs [3]. Therefore, eIF4E/4E-BP ratio, rather than individual levels or phosphorylation status of these proteins, is more likely to serve as a prognostic biomarker to select the patients for clinical trials using asTORi and to tailor personalized mTOR targeted therapies. Recently, several mechanisms potentiating resistance to mTOR inhibitors emerged, including the activation of alternative signaling pathways such as the MAPK pathway [7]. Therefore, combined targeting of the mTOR and MAPK pathways has been suggested as a promising approach to overcome resistance to mTOR inhibitors in the clinic. In addition to this strategy, our findings suggest that combining therapeutic approaches that suppress eIF4E expression or activity (e.g. ISIS-EIF4ERx, 4EGI-1, or 4E1RCat [8]) with mTOR targeted therapies should be beneficial in patients bearing tumors with elevated eIF4E/4E-BP ratio.

Discovery and Biological Profiling of Potent and Selective mTOR Inhibitor GDC-0349.

Aberrant activation of the PI3K-Akt-mTOR signaling pathway has been observed in human tumors and tumor cell lines, indicating that these protein kinases may be attractive therapeutic targets for treating cancer. Optimization of advanced lead 1 culminated in the discovery of clinical development candidate 8h, GDC-0349, a potent and selective ATP-competitive inhibitor of mTOR. GDC-0349 demonstrates pathway modulation and dose-dependent efficacy in mouse xenograft cancer models.

RAD001 Enhances the Potency of BEZ235 to Inhibit mTOR Signaling and Tumor Growth

The mammalian target of rapamycin (mTOR) is regulated by oncogenic growth factor signals and plays a pivotal role in controlling cellular metabolism, growth and survival. Everolimus (RAD001) is an allosteric mTOR inhibitor that has shown marked efficacy in certain cancers but is unable to completely inhibit mTOR activity. ATP-competitive mTOR inhibitors such as NVP-BEZ235 can block rapamycin-insensitive mTOR readouts and have entered clinical development as anti-cancer agents. Here, we show the degree to which RAD001 and BEZ235 can be synergistically combined to inhibit mTOR pathway activation, cell proliferation and tumor growth, both in vitro and in vivo. RAD001 and BEZ235 synergized in cancer lines representing different lineages and genetic backgrounds. Strong synergy is seen in neuronal, renal, breast, lung, and haematopoietic cancer cells harboring abnormalities in PTEN, VHL, LKB1, Her2, or KRAS. Critically, in the presence of RAD001, the mTOR-4EBP1 pathway and tumorigenesis can be fully inhibited using lower doses of BEZ235. This is relevant since RAD001 is relatively well tolerated in patients while the toxicity profiles of ATP-competitive mTOR inhibitors are currently unknown.

Inhibitory Effect of mTOR Activator MHY1485 on Autophagy: Suppression of Lysosomal Fusion

Autophagy is a major degradative process responsible for the disposal of cytoplasmic proteins and dysfunctional organelles via the lysosomal pathway. During the autophagic process, cells form double-membraned vesicles called autophagosomes that sequester disposable materials in the cytoplasm and finally fuse with lysosomes. In the present study, we investigated the inhibition of autophagy by a synthesized compound, MHY1485, in a culture system by using Ac2F rat hepatocytes. Autophagic flux was measured to evaluate the autophagic activity. Autophagosomes were visualized in Ac2F cells transfected with AdGFP-LC3 by live-cell confocal microscopy. In addition, activity of mTOR, a major regulatory protein of autophagy, was assessed by western blot and docking simulation using AutoDock 4.2. In the result, treatment with MHY1485 suppressed the basal autophagic flux, and this inhibitory effect was clearly confirmed in cells under starvation, a strong physiological inducer of autophagy. The levels of p62 and beclin-1 did not show significant change after treatment with MHY1485. Decreased co-localization of autophagosomes and lysosomes in confocal microscopic images revealed the inhibitory effect of MHY1485 on lysosomal fusion during starvation-induced autophagy. These effects of MHY1485 led to the accumulation of LC3II and enlargement of the autophagosomes in a dose- and time- dependent manner. Furthermore, MHY1485 induced mTOR activation and correspondingly showed a higher docking score than PP242, a well-known ATP-competitive mTOR inhibitor, in docking simulation. In conclusion, MHY1485 has an inhibitory effect on the autophagic process by inhibition of fusion between autophagosomes and lysosomes leading to the accumulation of LC3II protein and enlarged autophagosomes. MHY1485 also induces mTOR activity, providing a possibility for another regulatory mechanism of autophagy by the MHY compound. The significance of this study is the finding of a novel inhibitor of autophagy with an mTOR activating effect.

The ALKF1174L Mutation Potentiates the Oncogenic Activity of MYCN in Neuroblastoma

The ALK(F1174L) mutation is associated with intrinsic and acquired resistance to crizotinib and cosegregates with MYCN in neuroblastoma. In this study, we generated a mouse model overexpressing ALK(F1174L) in the neural crest. Compared to ALK(F1174L) and MYCN alone, co-expression of these two oncogenes led to the development of neuroblastomas with earlier onset, higher penetrance, and enhanced lethality. ALK(F1174L)/MYCN tumors exhibited increased MYCN dosage due to ALK(F1174L)-induced activation of the PI3K/AKT/mTOR and MAPK pathways, coupled with suppression of MYCN pro-apoptotic effects. Combined treatment with the ATP-competitive mTOR inhibitor Torin2 overcame the resistance of ALK(F1174L)/MYCN tumors to crizotinib. Our findings demonstrate a pathogenic role for ALK(F1174L) in neuroblastomas overexpressing MYCN and suggest a strategy for improving targeted therapy for ALK-positive neuroblastoma.

Discovery and optimization of potent and selective imidazopyridine and imidazopyridazine mTOR inhibitors

mTOR is a critical regulator of cellular signaling downstream of multiple growth factors. The mTOR/PI3K/AKT pathway is frequently mutated in human cancers and is thus an important oncology target. Herein we report the evolution of our program to discover ATP-competitive mTOR inhibitors that demonstrate improved pharmacokinetic properties and selectivity compared to our previous leads. Through targeted SAR and structure-guided design, new imidazopyridine and imidazopyridazine scaffolds were identified that demonstrated superior inhibition of mTOR in cellular assays, selectivity over the closely related PIKK family and improved in vivo clearance over our previously reported benzimidazole series.

Abstract B58: The ATP-competitive mammalian target of rapamycin (mTOR) inhibitors suppress proliferation and induce apoptosis in the acquired rapamycin resistant breast cancer cells

Abstract The mammalian target of rapamycin (mTOR) is the major regulator of cell growth, metabolism and survival. The anti-cancer effect of allosteric mTOR-inhibitors, rapamycin and its analogs (rapalogs), has been tested extensively in different types of cancer cells. However, there is only limited clinical efficacy observed by treatment of these traditional mTOR inhibitors, partly due to the occurring of acquired rapamycin resistance (ARR). Several new mTOR inhibitors have been developed which specifically target to the catalytic sites of mTOR by competing with ATP binding, and are suggested to be more effective in inhibiting mTOR signaling and cell growth. However, their effect and mechanisms in ARR cells are not clear yet. In the present study, we used an in vitro model of acquired rapamycin-resistant (ARR) breast cancer cells to evaluate the efficacy of two major classes of ATP-competitive mTOR inhibitors, PI3K/mTOR dual and mTORC1/mTORC2 inhibitors. We found that ATP-competitive mTOR inhibitors acutely suppress the proliferation and prominently inhibit PI3K/mTOR signaling of ARR breast cancer cells. In contrast to the cytostatic effect of rapamycin, the ATP-competitive mTOR inhibitors induced apoptosis in ARR breast cancer cells. Unexpectedly, one of PI3K/mTOR dual inhibitors we tested, PI-103, only transiently inhibits its proposed signaling targets and the anti-proliferation effects are possibly mediated through the off-target mechanism. Our results support the rationale to use ATP-competitive mTOR inhibitors to treat rapamycin-resistant breast cancer cells and highlight the putative off-target effect of the new generation of mTOR inhibitor on cancer cells.

Abstract PR4: Incomplete inhibition of phosphorylation of 4EBP1 is a mechanism for KRAS driven resistance to the ATP-competitive mTOR inhibitor PP242 in colorectal cancer

Abstract The mammalian target of rapamycin (mTOR) regulates cell growth by integrating nutrient and growth factor signaling and has been strongly implicated in cancer. But mTOR is not an oncogene, and which tumors will be resistant and sensitive to new ATP-competitive mTOR inhibitors now in clinical trials remains unknown. We screened a panel of 357 human cancer cell lines to identify common markers of resistance and sensitivity to the mTOR inhibitor PP242. Analysis by tissue of origin and genetic mutation revealed colorectal origin as the most statistically significant marker for resistance to therapy, while KRAS and PI3K mutations were the most significant genetic markers for resistance and sensitivity respectively. Notably, KRAS mutations are overrepresented in colorectal cancer (CRC) cell lines. Within CRC cell lines, those with KRAS mutations were most resistant to PP242 while those with other mutations were more sensitive; cell lines with co-mutation of PI3K and KRAS had intermediate sensitivity. Immunoblot analysis of the signaling networks downstream of mTOR revealed that the degree of cellular growth inhibition induced by PP242 was directly correlated with inhibition of phosphorylation of the translational repressor eIF4E binding protein 1 (4EBP1), but not ribosomal protein S6. We were able to recapitulate these observations using a pair of KRAS mutant isogenic cell lines differing only by a PI3K mutation. PP242 treatment of the PI3K mutant cell line increased the observed inhibition of 4EBP1 phosphorylation and improved the drug response. In an efficacy trial of PP242 in primary CRC xenograft tumors, resistance to inhibition of 4EBP1 phosphorylation was again observed in KRAS mutant tumors and these tumors responded poorly compared to KRAS WT controls. We show that, in the absence of PI3K co-mutation, KRAS mutations are associated with resistance to PP242 and that this is specifically linked to changes in the 4EBP1 phosphorylation state in both CRC cell lines and primary patient xenografts. This proffered talk is also presented as Poster A35.

Abstract 3761: Efficacy of the ATP-competitive mTOR inhibitor AZD8055 in PTEN-wild type cancer cells

Abstract Although the PI3K/Akt/mTOR pathway is frequently hyperactivated in human cancers due to the loss of function of the PTEN tumor suppressor, a proportion of cancer cells still carries wild type PTEN and thus the PI3K/Akt/mTOR pathway in these cells may remain unaltered. While it has been widely accepted that these cancer cells are less susceptible to the allosteric mTOR inhibitor rapamycin, it is unclear whether this is also the case for an ATP-competitive mTOR inhibitor. To address this question we evaluated the effects of the mTOR kinase inhibitor AZD8055 in a panel of wild type PTEN expressing cancer cells (6 cell lines of 4 tumor types). The phosphorylated Akt (S473) level was undetectable in most of these cells, suggesting the regular mTOR complex 1 (mTORC1) pathway and low mTOR complex 2 (mTORC2) activity. Cell proliferation was determined by WST-1 (MTT) assay and clonogenic survival was tested by colony formation assay. AZD8055 suppressed cell proliferation at the nanomolar level in a dose dependent manner, with IC50 values in a range of 6∼35 nM for different cell types. In contrast, rapamycin exhibited limited anti-proliferative effect, with less than 50% of inhibition observed even with higher doses in most cell lines. Similarly, clonogenic capacity was impaired by AZD8055 to a greater extent than was the case for rapamycin. To detect the mTOR pathway alterations by drug treatment, two cell lines with different expression levels of mTORC1 downstream pathways were selected: murine mammary tumor 4T1 cells (low phosphorylated S6 and high phosphorylated 4E-BP1) and human breast cancer MDA-MB-231 cells (normal phosphorylated S6 and moderate phosphorylated 4E-BP1). While the phospho-S6K (T389) and phospho-S6 (S235/236) pathway was potently inhibited by both agents, AZD8055 displayed more striking inhibition of the hierarchical 4E-BP1 phosphorylation (T37/46, T70 and S65) than rapamycin in both cells. In addition, both rapamycin and AZD8055 slightly increased Akt phosphorylation (T308 or S473), suggesting that the activation of the feedback loop controlled by S6K was functional. However, the PI3K inhibitor wortmannin did not sensitize cells to either agent, implying such feedback-dependent activation is dispensable for attenuating the drug response. Taken together, these findings indicate that AZD8055 significantly inhibits growth and survival of cancer cells that have wild type PTEN and normal PI3K/Akt/mTOR signaling, an observation mainly associated with the comprehensive activation of 4E-BP1. These data provide in vitro evidence in support of broadening the application of ATP-competitive mTOR inhibitors in cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3761. doi:1538-7445.AM2012-3761

Abstract 2935: The ATP-competitive mTOR inhibitor Torin2 enhances sensitivity of the ALK F1174L mutation to crizotinib in neuroblastoma

Abstract Mutations in the anaplastic lymphoma kinase (ALK) receptor represent an important therapeutic target in neuroblastoma. The most frequently occurring somatic mutation, ALK F1174L, is sensitive to the ALK inhibitor crizotinib only at higher doses in vitro, and not at all in in vivo. Moreover, ALK F1174L mediates acquired resistance to crizotinib in ALK-rearranged cancers, posing a therapeutic challenge in these diseases. To identify critical components of ALK F1174L-associated signaling pathways that contribute to neuroblastoma cell survival and whose simultaneous inhibition with ALK F1174L could increase sensitivity to crizotinib, we exposed neuroblastoma cell lines expressing ALK F1174L (Kelly and SHSY-5Y) to crizotinib (at treatment doses and exposures times titrated to abolish ALK phosphorylation, but in the absence of any detectable commitment to apoptosis), and compared their “ALK-inactive” gene expression signatures with vehicle-treated control cells in which the ALK F1174L signature remained “active”. We noted differential expression of genes involved in the PI3K/AKT/mTOR pathway in cells in which ALK F1174L was inhibited with crizotinib, with downregulation of AKT1, 2 and 3 and elevated expression of mTOR. Immunoblotting confirmed that downregulation of pALK upon exposure to crizotinib was accompanied by downregulation of pAKT473, with unchanged or elevated pRPS6. To test the possibility that the cytotoxicity of crizotinib could be enhanced by simultaneous inhibition of this ALK-driven pathway, we tested crizotinib in combination with the ATP-competitive mTOR inhibitor, Torin2, both in cell lines and in xenograft models of neuroblastoma expressing ALK F1174L. Treatment with crizotinib had no standalone activity as demonstrated by lack of effects on tumor volume or survival. Treatment with Torin2 significantly suppressed tumor growth, but this did not translate into a prolongation of survival. Combined treatment with Torin2 and crizotinib resulted in significant attenuation of tumor growth (p<0.001), and prolongation of survival in comparison to control animals, as well as single agent treatment with either Torin2 or crizotinib. RPS6 remained phosphorylated in neuroblastoma xenografts of mice treated exclusively with crizotinib but was downregulated in cells treated with Torin 2, and in those treated with the combination. Together, these results suggest that single agent treatment with crizotinib at standard doses is less efficacious due to persistent activation of mTOR signaling and that mTOR pathway inhibition should augment the activity of crizotinib in the treatment of patients with ALK F1174L-expressing neuroblastomas and may even delay the onset of resistance in ALK-rearranged cancers in which this pathway is activated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2935. doi:1538-7445.AM2012-2935

Antitumor activities of ATP-competitive inhibitors of mTOR in colon cancer cells

BackgroundThe mammalian target of rapamycin (mTOR) is frequently activated in colon cancers due to mutations in the phosphatidylinositol 3-kinase (PI3K) pathway. Targeting mTOR with allosteric inhibitors of mTOR such as rapamycin reduces colon cancer progression in several experimental models. Recently, a new class of mTOR inhibitors that act as ATP-competitive inhibitors of mTOR, has been developed. The effectiveness of these drugs in colon cancer cells has however not been fully characterized.MethodsLS174T, SW480 and DLD-1 colon cancer cell lines were treated with PP242 an ATP-competitive inhibitor of mTOR, NVP-BEZ235, a dual PI3K/mTOR inhibitor or rapamycin. Tumor cell growth, proliferation and survival were assessed by MTS assay, 5-bromo-2'-deoxyuridine (BrDU) incorporation or by quantification of DNA fragmentation respectively. In vivo, the anticancer activity of mTOR inhibitors was evaluated on nude mice bearing colon cancer xenografts.ResultsPP242 and NVP-BEZ235 reduced the growth, proliferation and survival of LS174T and DLD-1 colon cancer cells more efficiently than rapamycin. Similarly, PP242 and NVP-BEZ235 also decreased significantly the proliferation and survival of SW480 cells which were resistant to the effects of rapamycin. In vivo, PP242 and NVP-BEZ235 reduced the growth of xenografts generated from LS174T and SW480 cells. Finally, we also observed that the efficacy of ATP-competitive inhibitors of mTOR was enhanced by U0126, a MEK inhibitor.ConclusionsTaken together, these results show that ATP-competitive inhibitors of mTOR are effective in blocking colon cancer cell growth in vitro and in vivo and thus represent a therapeutic option in colon cancer either alone or in combination with MEK inhibitors.

Kinome-wide Selectivity Profiling of ATP-competitive Mammalian Target of Rapamycin (mTOR) Inhibitors and Characterization of Their Binding Kinetics

An intensive recent effort to develop ATP-competitive mTOR inhibitors has resulted in several potent and selective molecules such as Torin1, PP242, KU63794, and WYE354. These inhibitors are being widely used as pharmacological probes of mTOR-dependent biology. To determine the potency and specificity of these agents, we have undertaken a systematic kinome-wide effort to profile their selectivity and potency using chemical proteomics and assays for enzymatic activity, protein binding, and disruption of cellular signaling. Enzymatic and cellular assays revealed that all four compounds are potent inhibitors of mTORC1 and mTORC2, with Torin1 exhibiting ∼20-fold greater potency for inhibition of Thr-389 phosphorylation on S6 kinases (EC(50) = 2 nM) relative to other inhibitors. In vitro biochemical profiling at 10 μM revealed binding of PP242 to numerous kinases, although WYE354 and KU63794 bound only to p38 kinases and PI3K isoforms and Torin1 to ataxia telangiectasia mutated, ATM and Rad3-related protein, and DNA-PK. Analysis of these protein targets in cellular assays did not reveal any off-target activities for Torin1, WYE354, and KU63794 at concentrations below 1 μM but did show that PP242 efficiently inhibited the RET receptor (EC(50), 42 nM) and JAK1/2/3 kinases (EC(50), 780 nM). In addition, Torin1 displayed unusually slow kinetics for inhibition of the mTORC1/2 complex, a property likely to contribute to the pharmacology of this inhibitor. Our results demonstrated that, with the exception of PP242, available ATP-competitive compounds are highly selective mTOR inhibitors when applied to cells at concentrations below 1 μM and that the compounds may represent a starting point for medicinal chemistry efforts aimed at developing inhibitors of other PI3K kinase-related kinases.

Effect of PIK3CA and KRAS mutations on sensitivity to ATP-competitive mTOR inhibitors in a primary xenograft model of colorectal carcinoma.

483 Background: The mammalian target of rapamycin (mTOR) regulates cell growth by integrating nutrient and growth factor signaling and has been strongly implicated in cancer. Mutations in KRAS and the PI3K pathway are upstream of mTOR and are common in colorectal carcinoma (CRC). Currently approved mTOR inhibitors are derivatives of the natural product rapamycin and have shown little clinical efficacy in CRC. A new and potentially more efficacious class of ATP-competitive mTOR inhibitors has now entered clinical trials. Methods: Seeking a more representative preclinical model of CRC, we generated primary xenografts in nude mice of surgically resected specimens of human hepatic colorectal cancer metastases. We then treated xenograft tumors with the selective ATP-competitive mTOR inhibitor PP242 and monitored response by inhibition of tumor growth, changes in histopatholgy, and alterations in signaling pathways. Cell line experiments were performed to support observations made in the patient derived xenografts. Results: We demonstrate that in contrast to rapamycin, the mTOR inhibitor PP242 is highly effective at inhibiting tumor growth in both the primary xenograft model and in colorectal cancer cell lines. The inhibition of tumor growth in the xenografts and cell lines depended upon the strong inhibition of phosphorylation of mTOR substrate eIF4E binding protein 1 (4EBP1) but was not correlated with inhibition of phosphorylation of S6 kinase (S6K). Cells with mutant KRAS were relatively resistant to PP242 induced growth inhibition and this correlated with reduced inhibition of 4EBP1 phosphorylation. However, these effects were partially rescued in cells in which a co-mutation in PIK3CA resulted in AKT activation. Conclusions: Our studies reveal the first mTOR inhibitor resistant cell line, the genetic basis for its resistance and most importantly, these findings were revealed through the use of a primary xenograft mouse model which recapitulates morphologically the features of the tumors isolated from patients. We believe ATP-competitive inhibitors may be of limited clinical utility in mutant KRAS tumors, except for those that have concomitant activation of AKT.

Combination of ATP-competitive mammalian target of rapamycin inhibitors with standard chemotherapy for colorectal cancer

ATP-competitive mammalian target of rapamycin (mTOR) inhibitors are in early phase clinical trials. These novel targeted agents, including PP242, are mechanistically distinct from the allosteric, partial mTOR inhibitor, rapamycin. The goal of this study was to evaluate how PP242 best combines with standard chemotherapies for colorectal cancer (CRC), and which subsets of patients are most likely to benefit. The combination index for PP242 plus 5-fluorouracil, oxaliplatin, or irinotecan was determined in CRC cell lines with different mutational backgrounds. In KRAS mutant CRC cell lines, sensitivity to PP242 increases with co-mutation of PIK3CA. Mutation of p53 predicts resistance to chemotherapy, but not PP242. Efficacy of PP242 was comparable to that of standard chemotherapies over the dose range tested. Sensitivity or resistance to PP242 dictates relative synergy or antagonism, respectively, when PP242 is combined with 5-fluorouracil. The same trend exists for PP242 + oxaliplatin, but with a narrower dynamic range. Conversely potency of PP242 and the combination index for PP242 + irinotecan were unrelated, but synergy exists across all dose levels in PP242 and irinotecan sensitive, p53 wild-type cell lines. Overall, our in vitro analysis predicts that mutational status can be used to rank sensitivity to PP242 and standard chemotherapies. Single agent potency can in turn be used to predict the combination index in a drug-specific manner. Our data suggest a clinical trial to determine whether ATP-competitive mTOR inhibitors provide benefit in combination with standard chemotherapies for patients with PIK3CA mutant metastatic CRC, stratified by the presence or absence of KRAS co-mutation.

The mammalian target of rapamycin regulates cholesterol biosynthetic gene expression and exhibits a rapamycin-resistant transcriptional profile

The mammalian target of rapamycin (mTOR) is a central regulator of cell growth and proliferation in response to growth factor and nutrient signaling. Consequently, this kinase is implicated in metabolic diseases including cancer and diabetes, so there is great interest in understanding the complete spectrum of mTOR-regulated networks. mTOR exists in two functionally distinct complexes, mTORC1 and mTORC2, and whereas the natural product rapamycin inhibits only a subset of mTORC1 functions, recently developed ATP-competitive mTOR inhibitors have revealed new roles for both complexes. A number of studies have highlighted mTORC1 as a regulator of lipid homeostasis. We show that the ATP-competitive inhibitor PP242, but not rapamycin, significantly down-regulates cholesterol biosynthesis genes in a 4E-BP1-dependent manner in NIH 3T3 cells, whereas S6 kinase 1 is the dominant regulator in hepatocellular carcinoma cells. To identify other rapamycin-resistant transcriptional outputs of mTOR, we compared the expression profiles of NIH 3T3 cells treated with rapamycin versus PP242. PP242 caused 1,666 genes to be differentially expressed whereas rapamycin affected only 88 genes. Our analysis provides a genomewide view of the transcriptional outputs of mTOR signaling that are insensitive to rapamycin.

MTOR Inhibition, the Second Generation: ATP-Competitive mTOR Inhibitor Initiates Unexpected Receptor Tyrosine Kinase–Driven Feedback Loop

mTOR inhibition with the ATP-competitive kinase inhibitor AZD8055 induces receptor tyrosine kinase-dependent feedback activation of AKT.