mTOR Inhibitor MLN0128 Research Articles

Cytochrome b561 regulates iron metabolism by activating the Akt/mTOR pathway to promote Breast Cancer Cells proliferation

Breast cancer (BC) is the leading cause of cancer-related mortality in women, necessitating the development of novel therapeutic targets. While cytochrome b561 (CYB561) expression is associated with poor prognosis in BC, the precise role of CYB561 in BC and its potential mechanisms remain unclear. In the present study, we found that CYB561 plays an essential role in BC growth. CYB561 expression was up-regulated in surgically resected cancerous tissues and in six BC cell lines. Lentivirus-mediated CYB561 knockdown in BC cells significantly reduced their proliferation, migration, and invasiveness. CYB561 participates in the regulation of iron metabolism in BC. CYB561 knockdown reduced total iron content, increased ferrous iron content, and down-regulated the expression of proteins associated with iron metabolism (transferrin receptor 1, divalent metal transporter 1, and ferritin heavy chain 1). Conversely, up-regulation of CYB561 through co-incubation with exogenous iron (ferric ammonium citrate) produced contrary outcomes. Additionally, CYB561 activated the protein kinase B/mammalian target of rapamycin (Akt/mTOR) signaling pathway in BC cells. Down-regulation of CYB561 expression inhibited the Akt/mTOR signaling pathway activity. The application of an mTOR agonist (MHY1485) rescued this negative effect, as well as the inhibitory effect of CYB561 knockdown on cell proliferation. Importantly, the dual mTOR inhibitor MLN0128 (50 nM, 48 h) down-regulated CYB561 expression and the iron metabolism-related proteins transferrin receptor, divalent metal transporter 1, and ferritin heavy chain 1, whereas the mTOR agonist MHY1485 rescued the down-regulation of CYB561 knockdown on iron metabolism-related proteins. We conclude that CYB561 promotes the proliferation of BC cells by regulating iron metabolism through the activation of the Akt/mTOR signaling pathway.

Discovery of novel hybrids of mTOR inhibitor and NO donor as potential anti-tumor therapeutics

Nitric oxide (NO) may be beneficial to overcoming drug resistance resulting from mutation of mTOR kinases and bypass mechanisms. In this study, a novel structural series of hybrids of mTOR inhibitor and NO donor were designed and synthesized via structure-based drug design (SBDD). Throughout the 20 target compounds, half of the compounds (13a, 13b, 19a-19d, 19f-19j) demonstrated attractive mTOR inhibitory activity with IC50 at single-digit nanomolar level. In particular, 19f exerted superior anti-proliferative activity against HepG2, MCF-7, HL-60 cells (HepG2, IC50 = 0.24 μM; MCF-7, IC50 = 0.88 μM; HL-60, IC50 = 0.02 μM) to that of the clinical investigated mTOR inhibitor MLN0128, and show mild cytotoxicity against normal cells with IC50 over 10 μM. 19a, with the most potent mTOR inhibitory activity in this series (IC50 = 3.31 nM), also displayed attractive cellular potency. In addition, 19f treatment in HL-60 reduces the levels of Phos-Akt and Phos-S6 in a dose-dependent manner, and releases NO in cells. In summary, 19f deserves further development as a novel mTOR-based multi-target anti-cancer agent.

Alpelisib combination treatment as novel targeted therapy against hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the sixth most common primary cancer with an unsatisfactory long-term survival. Gain of function mutations of PIK3CA occur in a subset of human HCC. Alpelisib, a selective PIK3CA inhibitor, has been approved by the FDA to treat PIK3CA mutant breast cancers. In this manuscript, we evaluated the therapeutic efficacy of alpelisib, either alone or in combination, for the treatment of HCC. We tested alpelisib in mouse HCC induced by hydrodynamic injection of c-Met/PIK3CA(H1047R) (c-Met/H1047R), c-Met/PIK3CA(E545K) (c-Met/E545K), and c-Met/sgPten gene combinations. Alpelisib slowed down the growth of c-Met/H1047R and c-Met/E545K HCC but was ineffective in c-Met/sgPten HCC. Mechanistically, alpelisib inhibited p-ERK and p-AKT in c-Met/H1047R and c-Met/E545K HCC progression but did not affect the mTOR pathway or genes involved in cell proliferation. In human HCC cell lines transfected with PIK3CA(H1047R), alpelisib synergized with the mTOR inhibitor MLN0128 or the CDK4/6 inhibitor palbociclib to suppress HCC cell growth. In c-Met/H1047R mice, alpelisib/MLN0128 or alpelisib/palbociclib combination therapy caused tumor regression. Our study demonstrates that alpelisib is effective for treating PIK3CA-mutated HCC by inhibiting MAPK and AKT cascades. Furthermore, combining alpelisib with mTOR or CDK4/6 inhibitors has a synergistic efficacy against PIK3CA-mutated HCC, providing novel opportunities for precision medicine against HCC.

Discovery of novel pyrazolopyrimidine derivatives as potent mTOR/HDAC bi-functional inhibitors via pharmacophore-merging strategy

The mTOR and HDAC dual suppression is meaningful for counteracting drug resistance resulted from kinase mutation and bypass mechanisms. Herein, we communicate our recent discovery of a novel structural series of mTOR/HDAC bi-functional inhibitors featuring the pyrazolopyrimidine core via pharmacophore-merging strategy. More than half of them exerted potent dual-target inhibitory activities. In particular, compound 50 exhibited IC50 values of 0.49 and 0.91 nM against mTOR and HDAC1, respectively, along with remarkably enhanced anti-proliferative activity (IC50 = 1.74 μM) against MV4-11 cell line than mTOR inhibitor MLN-0128 (IC50 = 5.84 μM) and HDAC inhibitor SAHA (IC50 = 8.44 μM). Its intracellular intervention of both mTOR signaling and HDAC was validated by the Western blot analysis. Moreover, as the first disclosed mTOR/HDAC dual inhibitor with selectivity for some specific HDAC subtypes, it has the potential to alleviate the adverse effects resulted from pan-HDAC inhibition. Attributed to its favorable in vitro performance, compound 50 is valuable for further functional investigation as a polypharmacological anti-cancer agent.

Phase 1 Trial of MLN0128 (Sapanisertib) and CB-839 HCl (Telaglenastat) in Patients With Advanced NSCLC (NCI 10327): Rationale and Study Design

IntroductionThere are currently no approved targeted therapies for lung squamous-cell carcinoma (LSCC) and KRAS-mutant lung adenocarcinoma (LUAD). About 30% of LSCC and 25% of KRAS-mutant LUAD exhibit hyperactive NRF2 pathway activation through mutations in NFE2L2 (the gene encoding NRF2) or its negative regulator, KEAP1. Preclinical data demonstrate that these tumors are uniquely sensitive to dual inhibition of glycolysis and glutaminolysis via mammalian target of rapamycin (mTOR) and glutaminase inhibitors. This phase 1 study was designed to assess safety and preliminary activity of the mTOR inhibitor MLN0128 (sapanisertib) in combination with the glutaminase inhibitor CB-839 HCl. MethodsPhase 1 dose finding will use the queue-based variation of the 3 + 3 dose escalation scheme with the primary endpoint of identifying the recommended expansion dose. To confirm the acceptable tolerability of the recommended expansion dose, patients will subsequently enroll onto 1 of 4 expansion cohorts (n = 14 per cohort): (1) LSCC harboring NFE2L2 or (2) KEAP1 mutations, or (3) LUAD harboring KRAS/(KEAP1 or NFE2L2) coalterations, or (4) LSCC wild type for NFE2L2 and KEAP1. The primary endpoint of the dose expansion is to determine the preliminary efficacy of MLN0128/CB-839 combination therapy. ConclusionThis phase 1 study will determine the recommended expansion dose and preliminary efficacy of MLN0128 and CB-839 in advanced non–small-cell lung cancer with a focus on subsets of LSCC and KRAS-mutant LUAD harboring NFE2L2 or KEAP1 mutations.

Utilizing <sup>18</sup>F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET) imaging. Co-mutations in the KRAS proto-oncogene and the LKB1 tumor suppressor gene are frequent events in lung cancer that drive hypermetabolic, glycolytic tumor growth. A critical pathway regulating the growth and metabolism of these tumors is the mechanistic target of the rapamycin (mTOR) pathway, which can be effectively targeted using selective catalytic mTOR kinase inhibitors. The mTOR inhibitor MLN0128 suppresses glycolysis in mice bearing tumors with Kras and Lkb1 co-mutations, referred to as KL mice. The therapy response in KL mice is first measured by 18F-FDG PET and computed tomography (CT) imaging before and after the delivery of MLN0128. By utilizing 18F-FDG PET/CT, researchers are able to measure dynamic changes in the glucose metabolism in genetically engineered mouse models (GEMMs) of lung cancer following a therapeutic intervention with targeted therapies. This is followed by ex vivo autoradiography and a quantitative immunohistochemical (qIHC) analysis using morphometric software. The use of qIHC enables the detection and quantification of distinct changes in the biomarker profiles following treatment as well as the characterization of distinct tumor pathologies. The coupling of PET imaging to quantitative histology is an effective strategy to identify metabolic and therapeutic responses in vivo in mouse models of disease.

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer.

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET) imaging. Co-mutations in the KRAS proto-oncogene and the LKB1 tumor suppressor gene are frequent events in lung cancer that drive hypermetabolic, glycolytic tumor growth. A critical pathway regulating the growth and metabolism of these tumors is the mechanistic target of the rapamycin (mTOR) pathway, which can be effectively targeted using selective catalytic mTOR kinase inhibitors. The mTOR inhibitor MLN0128 suppresses glycolysis in mice bearing tumors with Kras and Lkb1 co-mutations, referred to as KL mice. The therapy response in KL mice is first measured by 18F-FDG PET and computed tomography (CT) imaging before and after the delivery of MLN0128. By utilizing 18F-FDG PET/CT, researchers are able to measure dynamic changes in the glucose metabolism in genetically engineered mouse models (GEMMs) of lung cancer following a therapeutic intervention with targeted therapies. This is followed by ex vivo autoradiography and a quantitative immunohistochemical (qIHC) analysis using morphometric software. The use of qIHC enables the detection and quantification of distinct changes in the biomarker profiles following treatment as well as the characterization of distinct tumor pathologies. The coupling of PET imaging to quantitative histology is an effective strategy to identify metabolic and therapeutic responses in vivo in mouse models of disease.

Use of dual mTOR inhibitor MLN0128 against everolimus-resistant breast cancer.

HR+/HER2- aromatase inhibitor-resistant metastatic breast cancer can be treated with everolimus and a second AI until the cancer recurs. Targeting these everolimus-resistant patients with the latest standard of care, CDK4/6 inhibitors, has not been clearly addressed. Understanding the signaling transduction pathways, which everolimus resistance activates, will elucidate the mechanisms and offer treatment strategies of everolimus resistance. To mimic the clinical setting, letrozole-resistant cells were used to generate an everolimus-resistant model (RAD-R). Reverse phase protein array (RPPA) was performed to reveal changes in the signaling transduction pathways, and expression levels of key proteins were analyzed. Inhibitors targeting the major signaling pathways, a CDK4/6 inhibitor palbociclib and a mTORC1/2 inhibitor (MLN0128), were evaluated to establish resistance mechanisms of RAD-R. RPPA results from RAD-R indicated changes to significant regulatory pathways and upregulation of p-AKT expression level associating with everolimus resistance. MLN0128, that inhibits the AKT phosphorylation, effectively suppressed the proliferation of RAD-R cells while treatment with palbociclib had no effect. Among the many signaling transduction pathways, which are altered post everolimus resistance, targeting dual mTORC1/2 is a possible option for patients who have recurrent disease from previous everolimus treatment.

The CDK4/6 inhibitor Palbociclib and the mTOR inhibitor MLN0128 are effective for the treatment of experimental intrahepatic cholangiocarcinoma

The CDK4/6 inhibitor Palbociclib and the mTOR inhibitor MLN0128 are effective for the treatment of experimental intrahepatic cholangiocarcinoma

Central role of mTORC1 downstream of YAP/TAZ in hepatoblastoma development

Hepatoblastoma (HB) is the most common type of liver malignancy in children. Recent studies suggest that activation of Yes-associated protein (YAP) is a major molecular event in HB development, as activated YAP synergizes with mutant β-catenin to promote HB formation in mice (YAP/β-catenin). However, how YAP regulates HB development remains poorly defined. Similarly, de-regulation of mammalian target of rapamycin complex 1 (mTORC1) signaling has been implicated in multiple tumor types, but its functional role in HB development is scarcely understood. In the present study, we found that mTORC1 is activated in human HB cells and YAP/β-catenin-induced mouse HB tumor tissues. mTOR inhibitor MLN0128 significantly inhibits human HB cell growth in vitro. Furthermore, ablation of Raptor, the unique subunit of mTORC1, strongly delayed YAP/β-catenin-induced HB development in mice. At the molecular level, we found that expression of the amino acid transporter SLC38A1 is induced in mouse HB tissues, and amino acid deprivation leads to mTORC1 suppression in HB cell lines. Silencing of YAP and/or its paralog, transcriptional co-activator with PDZ binding motif (TAZ), decreased SLC38A1 expression as well as mTORC1 activation in HB cells. Furthermore, a frequent and concomitant upregulation of mTORC1 and SLC38A1 was detected in a collection of human HB specimens. Altogether, our study demonstrates the key role of mTORC1 in HB development. YAP and TAZ promote HB development via inducing SLC38A1 expression, whose upregulation leads to mTORC1 activation. Targeting mTOR pathway or amino acid transporters may represent novel therapeutic strategies for the treatment of human HB.

Pan-mTOR inhibitor MLN0128 is effective against intrahepatic cholangiocarcinoma in mice

Intrahepatic cholangiocarcinoma (ICC) is a lethal malignancy without effective treatment options. MLN0128, a second generation pan-mTOR inhibitor, shows efficacy for multiple tumor types. We evaluated the therapeutic potential of MLN0128 vs. gemcitabine/oxaliplatin in a novel ICC mouse model. We established a novel ICC mouse model via hydrodynamic transfection of activated forms of AKT (myr-AKT) and Yap (YapS127A) protooncogenes (that will be referred to as AKT/YapS127A). Genetic approaches were applied to study the requirement of mTORC1 and mTORC2 in mediating AKT/YapS127A driven tumorigenesis. Gemcitabine/oxaliplatin and MLN0128 were administered in AKT/YapS127A tumor-bearing mice to study their anti-tumor efficacy in vivo. Multiple human ICC cell lines were used for in vitro experiments. Hematoxylin and eosin staining, immunohistochemistry and immunoblotting were applied for the characterization and mechanistic study. Co-expression of myr-AKT and YapS127A promoted ICC development in mice. Both mTORC1 and mTORC2 complexes were required for AKT/YapS127A ICC development. Gemcitabine/oxaliplatin had limited efficacy in treating late stage AKT/YapS127A ICC. In contrast, partial tumor regression was achieved when MLN0128 was applied in the late stage of AKT/YapS127A cholangiocarcinogenesis. Furthermore, when MLN0128 was administered in the early stage of AKT/YapS127A carcinogenesis, it led to disease stabilization. Mechanistically, MLN0128 efficiently inhibited AKT/mTOR signaling both in vivo and in vitro, inducing strong ICC cell apoptosis and only marginally affecting proliferation. This study suggests that mTOR kinase inhibitors may be beneficial for the treatment of ICC, even in tumors that are resistant to standard of care chemotherapeutics, such as gemcitabine/oxaliplatin-based regimens, especially in the subset of tumors exhibiting activated AKT/mTOR cascade. Lay summary: We established a novel mouse model of intrahepatic cholangiocarcinoma (ICC). Using this new preclinical model, we evaluated the therapeutic potential of mTOR inhibitor MLN0128 vs. gemcitabine/oxaliplatin (the standard chemotherapy for ICC treatment). Our study shows the anti-neoplastic potential of MLN0128, suggesting that it may be superior to gemcitabine/oxaliplatin-based chemotherapy for the treatment of ICC, especially in the tumors exhibiting activated AKT/mTOR cascade.

Preclinical drug screen reveals topotecan, actinomycin D, and volasertib as potential new therapeutic candidates for ETMR brain tumor patients.

Embryonal tumor with multilayered rosettes (ETMR) is a rare and aggressive embryonal brain tumor that solely occurs in infants and young children and has only recently been recognized as a separate brain tumor entity in the World Health Organization classification for CNS tumors. Patients have a very dismal prognosis with a median survival of 12 months upon diagnosis despite aggressive treatment. The aim of this study was to develop novel treatment regimens in a preclinical drug screen in order to inform potentially more active clinical trial protocols. We have carried out an in vitro and in vivo drug screen using the ETMR cell line BT183 and its xenograft model. Furthermore, we have generated the first patient-derived xenograft (PDX) model for ETMR and evaluated our top drug candidates in an in vitro drug screen using this model. BT183 cells are very sensitive to the topoisomerase inhibitors topotecan and doxorubicin, to the epigenetic agents decitabine and panobinostat, to actinomycin D, and to targeted drugs such as the polo-like kinase 1 (PLK1) inhibitor volasertib, the aurora kinase A inhibitor alisertib, and the mammalian target of rapamycin (mTOR) inhibitor MLN0128. In xenograft mice, monotherapy with topotecan, volasertib, and actinomycin D led to a temporary response in tumor growth and a significant increase in survival. Finally, using multi-agent treatment regimens of topotecan or doxorubicin combined with methotrexate and vincristine, the response in tumor growth and survival was further increased compared with mice receiving single treatments. We have identified several promising candidates for combination therapies in future clinical trials for ETMR patients.

A Phase II Study of the Dual mTOR Inhibitor MLN0128 in Patients with Metastatic Castration-Resistant Prostate Cancer (mCRPC).

e16529Background: MLN0128 is a first-in-class, dual mTOR inhibitor that has the potential to overcome resistance seen with standard rapalogs through inhibition of both TORC1 and TORC2. This phase I...

Alterations in cellular metabolome after pharmacological inhibition of Notch in glioblastoma cells.

Notch signaling can promote tumorigenesis in the nervous system and plays important roles in stem-like cancer cells. However, little is known about how Notch inhibition might alter tumor metabolism, particularly in lesions arising in the brain. The gamma-secretase inhibitor MRK003 was used to treat glioblastoma neurospheres, and they were subdivided into sensitive and insensitive groups in terms of canonical Notch target response. Global metabolomes were then examined using proton magnetic resonance spectroscopy, and changes in intracellular concentration of various metabolites identified which correlate with Notch inhibition. Reductions in glutamate were verified by oxidation-based colorimetric assays. Interestingly, the alkylating chemotherapeutic agent temozolomide, the mTOR-inhibitor MLN0128, and the WNT inhibitor LGK974 did not reduce glutamate levels, suggesting that changes to this metabolite might reflect specific downstream effects of Notch blockade in gliomas rather than general sequelae of tumor growth inhibition. Global and targeted expression analyses revealed that multiple genes important in glutamate homeostasis, including glutaminase, are dysregulated after Notch inhibition. Treatment with an allosteric inhibitor of glutaminase, compound 968, could slow glioblastoma growth, and Notch inhibition may act at least in part by regulating glutaminase and glutamate.

Abstract A49: Inhibition of mTOR induces chemoresistance in B-cell acute lymphoblastic leukemia

Abstract B-ALL is the most prominent cancer in pediatric patients. Most patients are responsive to the current chemotherapy, but B-ALLs with activated oncogenic kinases including BCR-ABL, PDGFR and JAK have a high risk of relapse. Currently, patients with the BCR-ABL translocation are successfully treated when the BCL-ABL inhibitor dasatinib is added to their chemotherapy regimen. In accord, we found that dasatinib sensitizes B-ALL cell lines to drugs used during induction and consolidation phase of chemotherapy. However, dasatinib protects BCR-ABL leukemia cell lines from killing by maintenance phase drugs methotrexate and 6-mercaptopurine. This protection only occurs in cells where dasatinib reduces downstream mTOR activity. To directly test if reduced mTOR activity can account for the protection, we assessed the effects of the allosteric mTOR inhibitor rapamycin and active-site mTOR inhibitor MLN0128. Indeed, both mTOR inhibitors consistently protect B-ALL cells from killing by methotrexate and 6-mercaptopurine. Inhibitors of AKT and PI3K also result in varying degrees of chemoprotection, which correlates strongly with the degree to which they downregulate mTOR activity. The chemoprotective effect of mTOR inhibition correlates with cell cycle arrest but other mechanisms may also be involved. These data suggest that mTOR inhibition is detrimental in some chemotherapeutic combinations, whether by inhibitors directly targeting mTOR or indirectly targeting upstream effectors. The identification of such drug combination effects is an important consideration for the current use of dasatinib and future use of PI3K/AKT/mTOR inhibitors. Citation Format: Thanh-Trang Vo, Duc Nguyen, Brandon Lui, Mengrou Lu, Sharmila Mallya, David Fruman. Inhibition of mTOR induces chemoresistance in B-cell acute lymphoblastic leukemia. [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 A49.

Generation of a patient-derived chordoma xenograft and characterization of the phosphoproteome in a recurrent chordoma

The management of patients with locally recurrent or metastatic chordoma is a challenge. Preclinical disease models would greatly accelerate the development of novel therapeutic options for chordoma. The authors sought to establish and characterize a primary xenograft model for chordoma that faithfully recapitulates the molecular features of human chordoma. Chordoma tissue from a recurrent clival tumor was obtained at the time of surgery and implanted subcutaneously into NOD-SCID interleukin-2 receptor gamma (IL-2Rγ) null (NSG) mouse hosts. Successful xenografts were established and passaged in the NSG mice. The recurrent chordoma and the derived human chordoma xenograft were compared by histology, immunohistochemistry, and phospho-specific immunohistochemistry. Based on these results, mice harboring subcutaneous chordoma xenografts were treated with the mTOR inhibitor MLN0128, and tumors were subjected to phosphoproteome profiling using Luminex technology and immunohistochemistry. SF8894 is a novel chordoma xenograft established from a recurrent clival chordoma that faithfully recapitulates the histopathological, immunohistological, and phosphoproteomic features of the human tumor. The PI3K/Akt/mTOR pathway was activated, as evidenced by diffuse immunopositivity for phospho-epitopes, in the recurrent chordoma and in the established xenograft. Treatment of mice harboring chordoma xenografts with MLN0128 resulted in decreased activity of the PI3K/Akt/mTOR signaling pathway as indicated by decreased phospho-mTOR levels (p = 0.019, n = 3 tumors per group). The authors report the establishment of SF8894, a recurrent clival chordoma xenograft that mimics many of the features of the original tumor and that should be a useful preclinical model for recurrent chordoma.

Abstract 2448: mTOR inhibitor MLN0128 Has antitumor efficacy in cell lines with intrinsic and acquired rapamycin resistance in vitro and in vivo.

Abstract Introduction: The mammalian target of rapamycin (mTOR) pathway plays a central role in cancer biology. mTOR forms two multiprotein complexes, mTOR complex 1 and 2 (mTORC1 and mTORC2). Rapamycin and analogues are allosteric mTOR inhibitors that predominantly inhibit mTORC1. Rapamycin analogues have been FDA approved for several cancer types and are in clinical trials for treatment of many others. MLN0128 is a mTOR kinase inhibitor, that inhibit s both mTORC1 and mTORC2. We hypothesized that MLN0128 may have antitumor efficacy in cancer cell lines with intrinsic or acquired rapamycin-resistance. Methods: Twenty immortalized human cancer cell lines representing various cancer types and a range of rapamycin sensitivity were treated with Rapamycin, MLN0128 or vehicle. Sulforhodamine B assay was used to determine rapamycin and MLN0128 half maximal inhibitory concentrations (IC50), which were later confirmed with colony formation assay (CFA). Acquired resistance models were created in BT-474 and MCF-7 cell lines through culturing in progressively increasing doses of rapamycin until sustained growth at supratherapeutic (10uM) doses was achieved. Western blotting was used to characterize PI3K/mTOR signaling pathway expression. Flow cytometry and cell sorting (FACS) was used to detect and quantify apoptosis of Annexin-V-FLUOS stained cancer cell lines. Xenograft models of nu/nu mice were treated with MLN0128 and rapamycin or everolimus for 28 days. Results: Cell lines that were intrinsically rapamycin sensitive as well as those that were intrinsically rapamycin resistant were sensitive to MLN0128. MLN0128 induced cell cycle arrest; Induction of apoptosis or autophagy with MLN0128 was not detected with western blotting and annexin labeling in most cell lines tested. MLN0128 demonstrated greater inhibition of mTORC1 signaling, especially with a decrease in p4E-BP1 across all cell lines. In addition, MLN0128 demonstrated inhibition of mTORC2, with decline in pAKT473 and pAKT450. However a feedback-dependent phosphorylation of AKT S308 was observed. In cell lines with acquired rapamycin resistance, treatment with MLN0128 produced a significantly higher growth inhibition compared to rapamycin. Thus far, we have demonstrated that MLN0128 treatment resulted in increased tumor growth inhibition compared to rapamycin in xenograft models of BT474 cells with acquired rapamycin resistance. Conclusion: Cancer cell lines with intrinsic as well as acquired resistance to rapamycin are responsive to mTOR kinase inhibitor MLN0128. Further investigations are needed to study the efficacy of MLN0128 in clinical trials and to evaluate its potential as a treatment option in cases of resistance to rapalogs. Citation Format: Burhan Hassan, Argun Akcakanat, Takafumi Sangai, Farrell Adkins, Kurt Evans, Gordon B. Mills, Funda Meric-Bernstam. mTOR inhibitor MLN0128 Has antitumor efficacy in cell lines with intrinsic and acquired rapamycin resistance in vitro and in vivo. [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 2448. doi:10.1158/1538-7445.AM2013-2448

MTOR Inhibitor MLN0128 has Antitumor Efficacy in Cell Lines With Intrinsic and Acquired Rapamycin-Resistance.

The mammalian target of rapamycin (mTOR) pathway plays a central role in cancer biology. mTOR forms two multiprotein complexes, mTOR complex 1 and 2 (mTORC1 and mTORC2). Rapamycin and analogues are allosteric mTOR inhibitors that predominantly inhibit mTORC1. Rapamycin analogues have been FDA approved for several cancer types and are in clinical trials for treatment of many others. MLN0128 is a mTOR kinase inhibitor, that inhibit s both mTORC1 and mTORC2. We hypothesized that MLN0128 may have antitumor efficacy in cancer cell lines with intrinsic or acquired rapamycin-resistance.