Rapamycin-resistant Cells Research Articles

Assembly of mTORC3 Involves Binding of ETV7 to Two Separate Sequences in the mTOR Kinase Domain.

mTOR plays a crucial role in cell growth by controlling ribosome biogenesis, metabolism, autophagy, mRNA translation, and cytoskeleton organization. It is a serine/threonine kinase that is part of two distinct extensively described protein complexes, mTORC1 and mTORC2. We have identified a rapamycin-resistant mTOR complex, called mTORC3, which is different from the canonical mTORC1 and mTORC2 complexes in that it does not contain the Raptor, Rictor, or mLST8 mTORC1/2 components. mTORC3 phosphorylates mTORC1 and mTORC2 targets and contains the ETS transcription factor ETV7, which binds to mTOR and is essential for mTORC3 assembly in the cytoplasm. Tumor cells that assemble mTORC3 have a proliferative advantage and become resistant to rapamycin, indicating that inhibiting mTORC3 may have a therapeutic impact on cancer. Here, we investigate which domains or amino acid residues of ETV7 and mTOR are involved in their mutual binding. We found that the mTOR FRB and LBE sequences in the kinase domain interact with the pointed (PNT) and ETS domains of ETV7, respectively. We also found that forced expression of the mTOR FRB domain in the mTORC3-expressing, rapamycin-resistant cell line Karpas-299 out-competes mTOR for ETV7 binding and renders these cells rapamycin-sensitive in vivo. Our data provide useful information for the development of molecules that prevent the assembly of mTORC3, which may have therapeutic value in the treatment of mTORC3-positive cancer.

Abstract 1670: Mechanistic insight into mTOR signaling identifies RNA splicing machinery as a potential therapeutic target for cancer

Abstract Aberrant signal transduction regulating cell growth and proliferation is a hallmark of many cancers. Activation of mTOR signaling is common and is an attractive therapeutic target with mechanistically different mTOR inhibitors in clinical use. One consequence of exposure to the mTOR inhibitor rapamycin is an alteration of gene expression through a global increase in alternative exon inclusion (Cheng et al. 2022, doi.org/10.3390/ijms232012416). To examine the mechanisms underlying this effect, we utilized the TempO-Seq targeted sequencing platform to profile both the whole transcriptome and a selection of alternatively spliced RNA targets in the rapamycin sensitive and resistant breast cancer cell lines MCF-7 and MDA MB 231 following exposure to 13 mTOR inhibitors, some closely related and some from distinct chemical classes. Recent publications have demonstrated that high quality dose response data can be obtained using this sequencing platform (Harrill et al. 2021, doi: 10.1093/toxsci/kfab009; Ramaiahgari et al. 2019, doi: 10.1093/toxsci/kfz065) and TempO-Seq has been widely adopted in toxicology for deriving compound-specific expression signatures to identify mechanisms of action and long-term toxicity. Treated cells were also profiled using TempO-LINC, a novel high-throughput single-cell assay based on the same platform. We observed that certain mTOR inhibitors induced both common and cell-specific differential expression as well as widespread alternative exon inclusion, as expected. Intriguingly, transcripts with the most significant changes in splicing upon treatment were those encoding members of the SR family of splicing factors, often overexpressed or amplified in breast cancers (Park, et al. 2019, doi: 10.1016/j.celrep.2019.10.110). This is significant because SR protein expression is autoregulated by alternative inclusion of an exon containing a premature stop codon in a feedback loop leading to nonsense-mediated decay (NMD) and lowered protein levels. Indeed, NMD is known to be inhibited by rapamycin (Martinez-Nunez, et al. 2017, doi: 10.1093/nar/gkw1109), which would lead to the observed accumulation of these transcripts. Thus, monitoring a targeted selection of alternative splicing events and regulators revealed mTOR regulation of mRNA isoform abundance consistent with regulation of the NMD RNA turnover process and with overexpression of SR proteins in breast cancers. Importantly, targeted bulk and single-cell TempO-Seq assays enable efficient sequencing and simplified data analysis without the expense and bioinformatic burden of RNAseq, making them particularly well-suited for drug discovery. These results support the use of this approach for further characterization of cell-specific effects of mTOR inhibitors and their mechanisms of action. Citation Format: Gaelle Rondeau, Megan Opichka, Kevin White, Joanne M. Yeakley, Dennis J. Eastburn, Bruce Seligmann. Mechanistic insight into mTOR signaling identifies RNA splicing machinery as a potential therapeutic target for cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1670.

Abstract 3022: Dissecting rapamycin resistance in a Tsc2-null rat leiomyoma cell line developed in a murine xenograft

Abstract Despite the identification of mTOR hyperactivation as the main biochemical defect downstream of TSC1/TSC2 inactivation in Lymphangioleiomyomatosis (LAM) and Tuberous Sclerosis Complex (TSC), and the approval of rapamycin and rapalogs for the treatment of the related lesions, these drugs are cytostatic and continued treatment is required for clinical benefit. The latter raises the possibility of acquired drug resistance over long-term use. To explore the mechanisms leading to rapamycin resistance in LAM/TSC, we xenografted SCID mice with ELT3 cells (Tsc2-null derived from an Eker rat uterine leiomyoma), explanted a tumor that was not responsive to rapamycin, and generated cell line ELT3-245. The cell growth of ELT3-245 is not inhibited by rapamycin in vitro. SCID mice inoculated with ELT3-245 form palpable tumors significantly faster, compared to ELT3 cells, respond very poorly to rapamycin at the initial stages of treatment, but soon become non-responsive. In addition, these cells form macroscopically visible metastases to the lungs of ELT3-245 tumor-bearing mice. We performed global gene expression profiling, comparing ELT3-245 to ELT3 cells, and found that the rapamycin-resistant cells have decreased expression of genes associated with epithelial cells and increased expression of genes associated with mesenchymal-like characteristics. Relative gene expression of Myc, Egfr, Akt2, Jun, and Ocln were assessed by RT-qPCR in ELT3 vs ELT3-245 after 24h of treatment with 20 nM rapamycin or DMSO. Important discrepancies include a 2.8-fold increased expression of Egfr under rapamycin treatment in ELT3 cells but not in ELT3-245; however, ELT3-245 had a 15-fold higher expression of Egfr prior to treatment, compared to ELT3. In addition, ELT3-245 cells strikingly increase expression of Myc when challenged with rapamycin, compared to ELT3. Similarly, Akt2 is increased considerably under rapamycin treatment in ELT3-245 and is already higher prior to treatment compared to ELT3. Finally, Ocln expression was at least 100-fold reduced in ELT3-245, compared to ELT3, both under rapamycin treatment and no-treatment conditions. These results suggest that Myc, Egfr, Akt2, and Ocln are among the genes highly relevant to acquired rapamycin resistance in this Tsc2-null rat leiomyoma cell line and point toward the pathways that need to be interfered with in order to overcome it. Citation Format: Natalia Filippidou, Mathildi Valianou, Daniel L. Johnson, John J. Bissler, Aristotelis Astrinidis. Dissecting rapamycin resistance in a Tsc2-null rat leiomyoma cell line developed in a murine xenograft [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3022.

Rapalog resistance is associated with mesenchymal-type changes in Tsc2-null cells

Tuberous Sclerosis Complex (TSC) and Lymphangioleiomyomatosis (LAM) are caused by inactivating mutations in TSC1 or TSC2, leading to mTORC1 hyperactivation. The mTORC1 inhibitors rapamycin and analogs (rapalogs) are approved for treating of TSC and LAM. Due to their cytostatic and not cytocidal action, discontinuation of treatment leads to tumor regrowth and decline in pulmonary function. Therefore, life-long rapalog treatment is proposed for the control of TSC and LAM lesions, which increases the chances for the development of acquired drug resistance. Understanding the signaling perturbations leading to rapalog resistance is critical for the development of better therapeutic strategies. We developed the first Tsc2-null rapamycin-resistant cell line, ELT3-245, which is highly tumorigenic in mice, and refractory to rapamycin treatment. In vitro ELT3-245 cells exhibit enhanced anchorage-independent cell survival, resistance to anoikis, and loss of epithelial markers. A key alteration in ELT3-245 is increased β-catenin signaling. We propose that a subset of cells in TSC and LAM lesions have additional signaling aberrations, thus possess the potential to become resistant to rapalogs. Alternatively, when challenged with rapalogs TSC-null cells are reprogrammed to express mesenchymal-like markers. These signaling changes could be further exploited to induce clinically-relevant long-term remissions.

Abstract 4120: Acquisition of chemoresistance to mTORC1 inhibition due to activation of the GSK-3 β/4EBP1 pathway might predict poor prognosis of mRCC patients

Abstract Background: PI3K/Akt/mTORC1 signaling pathway is aberrantly activated in renal cell carcinoma (RCC). We previously demonstrated that glycogen synthase kinase-3β (GSK-3β) positively regulated RCC proliferation. Herein, we aimed to investigate how the mTORC1 downstream substrate 4EBP1 is directly regulated by GSK-3β leading to acquisition of clinical chemoresistance to mTORC1 inhibitor (mTORi). Methods: The expression and phosphorylation of molecules in the subcellular pathways were examined by immunoblotting in surgically resected RCC tissues and human RCC cell lines including ACHN. Rapamycin-resistant ACHN cells (ACHN/RR) were generated by chronic exposure to an mTORi, rapamycin. Cell viability was investigated by MTS assay. The direct role of GSK-3β in regulating 4EBP1 was evaluated by an in vitro kinase assay. Pharmacological inhibition of GSK-3β, PI3K/Akt/mTORC1 pathway, and mTORC1 was achieved by treatment with AR-A014418, LY294002, and rapamycin, respectively. The effects of drug combination were determined using CompuSyn software. GSK-3β expression was evaluated in pathological RCC tissues obtained by nephrectomy from patients with metastatic RCC (mRCC), nine of whom were treated with mTORi as systemic therapies. GSK-3β expression was immunohistochemically graded as high and low expression using a semi-quantitative method based on positive staining intensity. Survival intervals were evaluated using the Kaplan-Meier method and log-rank test. Significance level was set as 0.05 in statistical analysis using a freeware R. Results: Inhibition of PI3K/Akt/mTORC1 pathway and mTORi treatment sufficiently decreased pS6RP, but only moderately decreased p4EBP1. In contrast to the effect of rapamycin, AR- A014418 remarkably inhibited cell proliferation, and rapidly suppressed p4EBP1 in ACHN and ACHN/RR. In vitro kinase assays showed that recombinant GSK-3β phosphorylated recombinant 4EBP1 at Thr37, Thr46, Thr70 and Ser65, and these phosphorylations were blocked by GSK-3 inhibitor. A combination of AR- A014418 and rapamycin produced an additive effect at lower concentrations, and were antagonistic at higher concentrations. Immunoblotting demonstrated that 4EBP1 and p4EBP1 expression in RCC tissues was positively correlated with GSK-3β expression. Overall survival was significantly short in patients with high GSK-3β expression, compared with those having low expression (n = 25 and 14, respectively, p = 0.04). In addition, high GSK-3β expression in tumors tended to shorten progression-free intervals in mRCC patients treated with mTORi. Conclusions: High GSK-3ββ expression and direct GSK-3β/4EBP1 pathway can be a key mechanism for RCC acquiring clinical chemoresistance to mTORCi. Activation of the direct pathway might be predictive of poor prognosis of mRCC patients. Citation Format: Hiromi Ito, Sei Naito, Osamu Ichiyanagi, Hidenori Kanno, Yuta Kurota, Atushi Yamagishi, Vladimir Bilim, Yoshihiko Tomita, Tomoyuki Kato, Akira Nagaoka, Norihiko Tsuchiya. Acquisition of chemoresistance to mTORC1 inhibition due to activation of the GSK-3 β/4EBP1 pathway might predict poor prognosis of mRCC patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4120. doi:10.1158/1538-7445.AM2017-4120

Inhibition of MDM2 Re-Sensitizes Rapamycin Resistant Renal Cancer Cells via the Activation of p53

Background/Aims: Rapamycin is a potential anti-cancer agent, which modulates the activity of mTOR, a key regulator of cell growth and proliferation. However, several types of cancer cells are resistant to the anti-proliferative effects of rapamycin. In this study, we report a MDM2/p53-mediated rapamycin resistance in human renal cancer cells. Methods: Trypan blue exclusion tests were used to determine the cell viability. Changes in mRNA and protein expression were measured using real-time PCR and western blot, respectively. Xenograft models were established to evaluate the in vivo effects of rapamycin combined with a MDM2 inhibitor. Results: Rapamycin treatment suppresses the expression of MDM2 and exogenous overexpression of MDM2 in A498 cells contributes to rapamycin resistance. By establishing a rapamycin resistant cell line, we observed that MDM2 was significantly upregulated in rapamycin resistant cells than that in rapamycin sensitive cells. Importantly, the rapamycin resistant cells demonstrated attenuated accumulation of p53 in the nucleus in response to rapamycin treatment. Moreover, the inhibition of MDM2 by siMDM2 sensitizes A498 cells to rapamycin through the activation of p53. In both in vitro and in vivo models, the combination of rapamycin with the MDM2 inhibitor, MI-319, demonstrated a synergistic inhibitory effect on rapamycin resistant cells. Conclusion: Our study reports a novel mechanism for rapamycin resistance in human renal cancer and provides a new perspective for the development of anti-cancer drugs.

Abstract 1726: Acquired resistance to rapamycin and mTOR kinase inhibitors is mediated by non-overlapping mutations in distinct sites in the mTOR protein

Abstract The prevalence of mTOR activation in cancer has led to the development of two classes of inhibitors of the protein as therapeutics: the natural product rapamycin and its analogs as well as direct inhibitors of mTOR kinase. mTOR exists in at least two multi-protein complexes. The mTORC1 complex phosphorylates S6K and 4EBP and stimulates protein translation, metabolism as well as other processes. The mTORC2 complex phosphorylates and activates AKT. Rapamycin binds to the immunophillin FKBP12. Drug-bound FKBP12 complex binds to mTOR FRB domain and selectively inhibits the activity of mTORC1. However, rapalogs preferentially inhibit S6K phosphorylation compared to 4EBP phosphorylation. Rapalogs have undergone extensive clinical testing and have significant antitumor activity in renal cell and pancreatic neuroendocrine tumors and, in combination with aromatase inhibitors, in resistant, ER positive breast cancers. In contrast, mTOR kinase inhibitors suppress both mTORC1 and mTORC2 functions and potently inhibit S6K, 4EBP and AKT S473 phosphorylation, these drugs are in early clinical testing. In order to better understand the mechanism of action of these drugs and potential mechanisms of tumor escape from mTOR inhibition, we selected breast tumor cells for resistance to growth inhibition in cell culture by treatment with either rapamycin or an mTOR kinase inhibitor. In rapamycin resistant cells, phosphorylation of S6K and S6 were insensitive to the drug, but remained sensitive to mTOR kinase inhibitors. Conversely, in clones resistant to mTOR kinase inhibitors, mTORC1 and mTORC2 substrates were insensitive to the drugs, but S6K and S6 phosphorylation remained sensitive to rapamycin. Deep sequencing results explained these findings: rapamycin resistant clones harbored mutations in the FRB domain of mTOR, in the sites shown to interact with the FKBP12-rapamycin complex; mTOR kinase resistant clones harbored mutations in the mTOR catalytic domain. These mutations were not observed in the parental cells. It is likely that the mutations identified in each domain prevent binding of the drug. Consistent with these data, growth of rapamycin resistant cells retain sensitivity to mTOR kinase inhibitors and mTOR kinase resistant cells retain sensitivity to rapalogs, both in tissue culture and in vivo. The results suggest that tumor cells with acquired resistance to mTOR inhibitors retain a requirement for mTOR signaling for proliferation. Furthermore, tumors resistant to either class of drug may not be cross-resistant to the other and combined therapy with both might delay the onset of resistance. Further studies on the genetics of human tumors with acquired resistance to these agents will determine the clinical importance of these findings. Citation Format: Vanessa S. Rodrik-Outmezguine, Zhan Yao, Radha Mukherjee, Liqun Cai, Derek Barratt, Richard Ward, Teresa Klinowska, Elisa De Stanchina, Michael Berger, Jose Baselga, Neal Rosen. Acquired resistance to rapamycin and mTOR kinase inhibitors is mediated by non-overlapping mutations in distinct sites in the mTOR protein. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1726. doi:10.1158/1538-7445.AM2014-1726

Abstract PD1-8: Acquired resistance to rapamycin and mTOR kinase inhibitors is mediated by non-overlapping mutations in distinct sites in the mTOR protein

Abstract The mammalian target of rapamycin (mTOR) protein kinase regulates protein translation and proliferation by integrating the availability of energy, nutrients and the presence of growth factors. Dysregulation of mTOR is a common event in human tumors and results from mutational activation of receptor tyrosine kinases, PI3K signaling or inactivating mutations of the Tuberous Sclerosis or GATOR complexes as well as the LKB/AMP kinase pathway. The prevalence of mTOR activation in cancer has led to the development of two classes of inhibitors of the protein as therapeutics: the natural product rapamycin and its analogs as well as direct inhibitors of mTOR kinase. mTOR exists in at least two multi-protein complexes. The mTORC1 complex phosphorylates S6K and 4EBP and stimulates protein translation, metabolism as well as other processes. The mTORC2 complex phosphorylates and activates AKT and other AGC kinases. Rapamycin binds to the immunophilin FKBP12. Drug-bound FKBP12 complex binds to mTOR FRB domain and selectively inhibits the activity of mTORC1. However, rapalogs preferentially inhibit S6K phosphorylation compared to 4EBP phosphorylation. Rapalogs have undergone extensive clinical testing and have significant antitumor activity in renal cell and pancreatic neuroendocrine tumors and, in combination with aromatase inhibitors, in resistant, ER positive breast cancers. In contrast, mTOR kinase inhibitors suppress both mTORC1 and mTORC2 functions and potently inhibit S6K, 4EBP and AKT S473 phosphorylation. These drugs are in early clinical testing. In order to better understand the mechanism of action of these drugs and potential mechanisms of tumor escape from mTOR inhibition, we selected breast tumor cells for resistance to growth inhibition in cell culture by treatment with either rapamycin or an mTOR kinase inhibitor. In rapamycin resistant cells, phosphorylation of S6K and S6 were insensitive to the drug, but remained sensitive to mTOR kinase inhibitors. Conversely, in clones resistant to mTOR kinase inhibitors, mTORC1 and mTORC2 substrates were insensitive to the drugs, but S6K and S6 phosphorylation remained sensitive to rapamycin. Deep sequencing results explained these findings: rapamycin resistant clones harbored mutations in the FRB domain of mTOR, in the sites shown to interact with the FKBP12-rapamycin complex; mTOR kinase resistant clones harbored mutations in the mTOR catalytic domain. These mutations were not observed in the parental cells. It is likely that the mutations identified in each domain prevent binding of the drug. Consistent with these data, growth of rapamycin resistant cells retain sensitivity to mTOR kinase inhibitors and mTOR kinase resistant cells retain sensitivity to rapalogs, both in tissue culture and in vivo. The results suggest that tumor cells with acquired resistance to mTOR inhibitors retain a requirement for mTOR signaling for proliferation. Furthermore, tumors resistant to either class of drug may not be cross-resistant to the other and combined therapy with both might delay the onset of resistance. Further studies on the genetics of human tumors with acquired resistance to these agents will determine the clinical importance of these findings. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD1-8.

Rapamycin Resistant Murine Th9 Cells Have a Stable In Vivo Phenotype and Inhibit Graft-Versus-Host Reactivity

The cytokine micro-environment can direct murine CD4+ T cells towards various differentiation lineages such as Th1, Th2 and Tregs even in the presence of rapamycin, which results in T cells that mediate increased in vivo effects. Recently, a new lineage of T cells known as Th9 cells that secrete increased IL-9 have been described. However, it is not known whether Th9 differentiation occurs in the presence of rapamycin or whether adoptively transferred donor Th9 cells would augment or restrict alloreactivity after experimental bone marrow transplantation. We found that CD4+ T cells that were co-stimulated and polarized with TGF-β and IL-4 in the presence or absence of rapamycin each yielded effector cells of Th9 phenotype that secreted increased IL-9 and expressed a transcription factor profile characteristic of both Th9 and Th2 cells (high GATA-3/low T-bet). Augmentation of T cell replete allografts with manufactured rapamycin resistant Th9 cells markedly reduced both CD4+ and CD8+ T cell engraftment and strongly inhibited allo-specific T cell secretion of IFN-γ. The potency of Th9 cell inhibition of alloreactivity was similar to that of rapamycin resistant Th2 cells. Importantly, rapamycin resistant Th9 cells persisted and maintained their cytokine phenotype, thereby indicating limited differentiation plasticity of the Th9 subset. As such, Th9 differentiation proceeds in the presence of rapamycin to generate a cell therapy product that maintains high IL-9 expression in vivo while inhibiting IFN-γ driven alloreactivity.

Abstract 1496: Epithelial to mesenchymal transition and rapamycin sensitivity in cancer cell lines.

Abstract Activation of the mTOR signaling pathway is common in multiple cancers. Rapamycin has inhibitory effects on solid tumors through inhibition of the mTOR kinase and alteration of downstream signaling. Though rapamycin and its analogs have yielded promising results from in vitro studies in sensitive cell lines, they have generated only a modest effect in clinical application. Epithelial to mesenchymal transition (EMT) in cancer cells involves not only the loss of cell-cell adhesion and acquisition of migratory and invasive properties but also resistance to cell death, senescence, and chemotherapy. We hypothesized that the EMT status of cancer cells regulates their sensitivity to rapamycin. We classified a panel of immortalized cancer cell lines as resistant or sensitive to rapamycin based on their IC50 as determined by Sulforhodamine B (SRB) assay. Using reverse phase proteomic array, we found that EMT markers were differentially expressed in sensitive compared to resistant immortalized cell lines. Rapamycin sensitive (RS) cell lines had increased expression of E-cadherin and N-cadherin while rapamycin resistant (RR) cell lines had increased expression of Smad3 (p<0.02). To validate these findings, we performed western blotting for EMT markers in selected RR and RS cell lines. E-cadherin expression was higher in RS cell lines than RR cell lines; in turn, RR cell lines expressed increased levels of the mesenchymal marker Vimentin. To determine if inducing EMT decreases rapamycin sensitivity, we assessed E-cadherin and Vimentin expression by western blotting in the epithelial, immortalized breast cancer cell line, MCF7, which was transfected with a constitutively-active mutant of Snail, a known EMT driver. Constitutively-active Snail mutant MCF7 demonstrated loss of E-cadherin and gain of Vimentin expression compared to cells transfected with nonfunctional mutant Snail or Wild-type Snail. Furthermore, we found decreased growth inhibition with rapamycin by SRB assay in constitutively-active Snail mutant MCF7 compared to controls (p<0.05). To determine if modulating EMT increases rapamycin sensitivity, we induced MET using ZEB1 siRNA knockdown and miR200b/c transfection in two mesenchymal cancer cell lines_ACHN and MDA-MB-231. Although miR200b/c transfection induced E-cadherin expression by immunoblotting in both cell lines, there was no significant increase in growth inhibition by SRB assay. In contrast, ZEB1 siRNA knockdown partially reversed EMT in these cells, which exhibited increased sensitivity to rapamycin by SRB assay. Most but not all mechanisms of EMT modulation altered rapamycin sensitivity in immortalized cancer cell lines. Future studies to determine the mechanism of EMT modulation that results in increased sensitivity to rapamycin could have important clinical applications for combination therapies utilizing rapamycin and its analogs. Citation Format: Ashley M. Holder, Farrell Adkins, Argun Akcakanat, Huiqin Chen, Kim-Anh Do, Mien-Chie Hung, Funda Meric-Bernstam. Epithelial to mesenchymal transition and rapamycin sensitivity in cancer cell lines. [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 1496. doi:10.1158/1538-7445.AM2013-1496

Antimycin induces apoptosis in rapamycin resistant SW620 colorectal cancer cells

Mammalian target of rapamycin, (mTOR), a serine/threonine protein kinase is a central regulator of cell growth and proliferation and is observed to be hyperactive in many common cancers. Rapamycin, a natural macrolide immunosuppressant, forms a complex with FKBP12 and mTOR inhibiting mTOR activity. Classified as Duke's Type C level cancer, SW620s are colorectal, rapamycin resistant, adenocarcinoma cells with widespread metastasis. During a high through put screening of the National Cancer Institute's Mechanistic Diversity Set, we demonstrated that antimycin not only inhibits growth and proliferation but induces apoptosis in SW620 cells. Antimycin, a group of secondary metabolites produced by Streptomyces bacteria, disrupts the proton gradient across the inner mitochondrial membrane which results in the build‐up of the toxic, free radical superoxide. As a result, apoptosis was induced in several other colorectal cancer cell lines suggesting antimycin is a compound that could potentially be used for the treatment of colorectal cancer.

Abstract P4-08-02: Reactivation of oncogenic signaling through mTOR inhibitors-induced feedback adaptations.

Abstract mTORC1 inhibitors (or rapalogs) relieve feedback inhibition of receptor tyrosine kinases (RTKs) and cause mTORC2-dependent phosphorylation of AKT S473 and activation of AKT kinase and signaling. The relief of feedback inhibition of mTORC2/AKT signaling has been shown to abolish the antitumor activity of rapalogs and has often been proposed as a potential mechanism of resistance to these compounds. mTOR kinase inhibitors that inhibit both mTORC1 and mTORC2 complexes have now been developed to circumvent these problems. mTOR kinase inhibitors block mTORC1 and mTORC2 and thus, do not cause the mTORC2 activation of AKT observed with rapamycin. We now show, however, that these novel classes of drugs have a biphasic effect on AKT. Inhibition of mTORC2 leads to AKT S473 dephosphorylation and a rapid but transient inhibition of AKT T308 phosphorylation and AKT signaling. However, inhibition of mTOR kinase, as observed with rapalogs, also relieves feedback inhibition of RTKs leading to subsequent PI3K activation and rephosphorylation of AKT but only at T308, which is sufficient to reactivate AKT activity and signaling. Thus, catalytic inhibition of mTOR kinase leads to a new steady state characterized by profound inhibition of mTORC1 and accumulation of activated AKT phosphorylated on T308 but not S473. We now confirm that the reactivation of AKT signaling is a mechanism of acquired resistance to rapalogs. We observe an induction of phospho-RTKs in rapamycin-resistant cells leading to both AKT and ERK signaling pathway activation enhancing cell survival. Combined inhibition of mTOR and the induced RTKs can prevent AKT reactivation and fully abolishes AKT signaling. These results reveal the adaptive capabilities of oncogenic signaling networks, highlighting the possible need for combinatorial approaches to block feedback-regulated pathways. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-08-02.

Perifosine enhances mTORC1-targeted cancer therapy by activation of GSK3β in NSCLC cells

mTORC1 inhibitors, including rapamycin and its analogs, have been actively studied both pre-clinically and clinically. However, the single treatment of mTORC1 inhibitors has been modest in most cancer types. We have previously demonstrated that the activation of PI3K/Akt and MEK/ERK signaling pathways attenuates the anticancer efficacy of mTORC1 inhibitors. In this study, we report that mTORC1 inhibition also phosphorylates and inactivates GSK3β, which is a tumor suppressor in lung cancer. Moreover, we show that perifosine, as an Akt inhibitor, decreases rapamycin-induced phosphorylation of GSK3β and elevated p-GSK3β levels in rapamycin-resistant cell lines. Combination of perifosine with mTORC1 inhibitors showed enhanced anticancer efficacy both in cell cultures and in a xenograft mouse model. In addition, perifosine inhibits the growth of both rapamycin sensitive and resistant A549 cells. However, inhibition of GSK3β by a selective inhibitor- LiCl, or downregulation of GSK3β expression by siRNA, reverses the growth inhibitory effects of perifosine on rapamycin resistant cells, suggesting the important role of GSK3β activation in enhancing mTORC1 inhibitors efficacy by perifosine. Thus, our results provide a potential therapeutic strategy to enhance mTORC1-targeted cancer therapy by using perifosine or targeting GSK3β.

Abstract 5607: TLE4 is a key modulator of resistance to mTOR pathway specific inhibition in glioblastoma

Abstract Rapamycin, a selective inhibitor of TOR (target of rapamycin), has been used in clinical trials for the treatment of glioma (Cloughesy et al., 2008). However, even though some patients respond temporarily to rapamycin treatment, virtually all recur. The mechanisms underlying this resistance to therapy are unknown. Here, we utilized patient-derived glioblastoma (GBM) neurosphere cultures to examine these mechanisms. GBM cell cultures exposed to chronic rapamycin yield a population of rapamycin resistant cells with an increased malignant phenotype upon removal from treatment, enhanced sphere formation in vitro and enhanced tumorigenesis. We used an unbiased, proteomics-based approach to discover phosphopeptides that were upregulated during rapamycin treatment in vitro. Amongst the peptides that we uncovered was Tle4/Groucho, a global co-repressor and known modulator of several key signaling pathways, including the Wnt and Notch pathways. In an expression microarray, Tle4 was found to be significantly upregulated within chronic rapa treated GBM cells. We show that TLE4 is a key modulator of resistance to mammalian target of rapamycin (mTOR) pathway specific inhibition. While TLE4 knockdown has no clear effect on naive cells, this knockdown promotes sensitivity under chronic rapamycin treatment as evidenced by reduced neurosphere formation. TLE4 mediate rapamycin resistance through activation of the quiescent population. Our results indicate that chronic mTOR specific inhibition imparts TLE4 mediated resistance through both ERK activation and inhibition of mTOR. The identification of this role for TLE4 in rapamycin resistance provides a novel target for mTOR pathway-associated chemotherapeutic agents.

Inhibition of tumor cell growth, proliferation and migration by X-387, a novel active-site inhibitor of mTOR

The mammalian target of rapamycin (mTOR), is deregulated in about 50% of human malignancies and exists in two complexes: mTORC1 and mTORC2. Rapalogs partially inhibit mTORC1 through allosteric binding to mTORC1 and their efficacy is modest as a cancer therapy. A few mTOR kinase inhibitors that inhibit both mTORC1 and mTORC2 have been reported to possess potent anticancer activities. Herein, we designed and synthesized a series of pyrazolopyrimidine derivatives targeting mTOR kinase domain and X-387 was identified as a promising lead. X-387 selectively inhibited mTOR in an ATP-competitive manner while sparing a panel of kinases from the PIKK family. X-387 blocked mTORC1 and mTORC2-mediacted signaling pathway in cell lines with activated mTOR signaling and in rapamycin-resistant cells. Specifically, X-387 inhibited phosphorylation of AKT at T308, which is thought to be a target of PDK1 but not mTOR. Such activity was not due to inhibition of PI3K since X-387 did not inhibit translocation of AKT to the cell membrane. X-387 induced autophagy as observed for other mTOR inhibitors, while induced autophagy is pro-survival since concurrent inhibition of autophagy by 3-MA reinforced the antiproliferative activity of mTOR inhibitors. X-387 also inhibited cell motility, which is associated with decrease in activity of small GTPases such as RhoA, Rac1 and Cdc42. Taken together, X-387 is a promising compound lead targeting mTOR and with a wide spectrum anticancer activity among tumor cell lines. The data also underscores the complexity of the mTOR signaling pathways which are far from being understood.

Rapamycin Induces Bad Phosphorylation in Association with Its Resistance to Human Lung Cancer Cells

Inhibition of mTOR signaling by rapamycin has been shown to activate extracellular signal-regulated kinase 1 or 2 (ERK1/2) and Akt in various types of cancer cells, which contributes to rapamycin resistance. However, the downstream effect of rapamycin-activated ERKs and Akt on survival or death substrate(s) remains unclear. We discovered that treatment of human lung cancer cells with rapamycin results in enhanced phosphorylation of Bad at serine (S) 112 and S136 but not S155 in association with activation of ERK1/2 and Akt. A higher level of Bad phosphorylation was observed in rapamycin-resistant cells compared with parental rapamycin-sensitive cells. Thus, Bad phosphorylation may contribute to rapamycin resistance. Mechanistically, rapamycin promotes Bad accumulation in the cytosol, enhances Bad/14-3-3 interaction, and reduces Bad/Bcl-XL binding. Rapamycin-induced Bad phosphorylation promotes its ubiquitination and degradation, with a significant reduction of its half-life (i.e., from 53.3-37.5 hours). Inhibition of MEK/ERK by PD98059 or depletion of Akt by RNA interference blocks rapamycin-induced Bad phosphorylation at S112 or S136, respectively. Simultaneous blockage of S112 and S136 phosphorylation of Bad by PD98059 and silencing of Akt significantly enhances rapamycin-induced growth inhibition in vitro and synergistically increases the antitumor efficacy of rapamycin in lung cancer xenografts. Intriguingly, either suppression of Bad phosphorylation at S112 and S136 sites or expression of the nonphosphorylatable Bad mutant (S112A/S136A) can reverse rapamycin resistance. These findings uncover a novel mechanism of rapamycin resistance, which may promote the development of new strategies for overcoming rapamycin resistance by manipulating Bad phosphorylation at S112 and S136 in human lung cancer.

Abstract 2869: Activation of AMP-activated protein kinase(AMPK) enhances Radiosensitization via inhibition of mTOR pathway in Rapamycin-resistant non-small cell lung cancer cells

Abstract Purpose: Most drug resistance cancer cells have resisted to radiation therapy. In this study, we hypothesized that inhibition of mammalian target of rapamycin(mTOR) via activation of AMPK could potentially enhance radiosensitization in Rapamycin-resistant non- small cell lung cancer stem cells. Methods: Stem cell marker was detected with CD133 and ALDH antibodies. P-AKT, P-mTOR and P-S6R were detected by Western Blotting. Clonogenic assay was used to determine radiosensitivity. Apoptosis was measured using the Annexin V-fluorescent isothiocyante apoptosis detection kit I (Pharmingen) with flow cytometry in vitro, and cleaved capase 3 by Western botting. Autophagy was determined by punctuate localization of GFP-LC3 fusion protein, which observed under a confocal fluorescence microscope in vitro. ATP levels were measured using Bioluminescence Assay kit CLS II from Roche Scientific (Indianpolis, IN, USA) as per the manufacturer's protocol. Results: Level of CD133 and ALDH expression which are markers of stem cell, P-Akt, p-mTOR and P-S6R proteins were enhanced in Rapamycin-resistant non- small cell lung cancer stem cells (CSNSCLCSTC) when compared to parental non-small cell lung cancer (H460) cells. Rapamycin-resistant non- small cell lung cancer stem cells mediate less radiation sensitivity when compared with parental cells in clonogenic assay(DER=0.82, p=0.02). However Metformin, activator of AMPK, obviously enhanced sensitivity of Rapamycin-selected non- small cell lung cancer stem cells to radiation (DER=1.2, p=0.01). Therefore, this enhanced radiosensitization was associated with a downregulation of mTOR signaling pathway and an increase in characteristic punctuate localization of GFP-LC3 as a marker for autophagy. Conclusions: These findings may be utilized as a novel strategy to enhance radiation therapy in drug-selected non-small cell lung cancer stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2869. doi:10.1158/1538-7445.AM2011-2869

Inhibition of Akt (ser473) Phosphorylation and Rapamycin-Resistant Cell Growth by Knockdown of Mammalian Target of Rapamycin with Small Interfering RNA in Vascular Endothelial Growth Factor Receptor-1-Targeting Vector

Previously we developed dicetyl phosphate-tetraethylenepentamine-based polycation liposomes (TEPA-PCL) for use in small interfering RNA (siRNA) therapy. In the present study, mammalian target of rapamycin (mTOR) expression in cancer cells was silenced with mTOR-siRNA (simTOR) formulated in TEPA-PCL modified with Ala-Pro-Arg-Pro-Gly (APRPG), a peptide having affinity for vascular endothelial growth factor receptor-1 (VEGFR-1). We investigated the effects of inhibition of mTOR, focusing on the differences between cells treated with simTOR and those with rapamycin in terms of Akt (ser473) phosphorylation and antiproliferative effects. Rapamycin treatment is known to induce Akt (ser473) phosphorylation which attenuates the antiproliferative effects of rapamycin. As a result, knockdown of mTOR did not alter or only slightly reduced Akt (ser473) phosphorylation in phosphatase and tensin homolog deleted from chromosome 10 (PTEN)-null (LNCaP and MDA-MB-468 cells) and PTEN-positive (DU 145 and MDA-MB-231) cells, although rapamycin induced Akt (ser473) phosphorylation of these cells. Rapamycin suppressed the growth of PTEN-null cells, in which the rapamycin-sensitive mTOR complex 1 (mTORC1) is excessively activated. On the other hand, rapamycin did not suppress the growth of PTEN-positive cells possibly through a negative feedback mechanism via the rapamycin-insensitive mTOR complex 2 (mTORC2) signaling pathway. In contrast, simTOR significantly suppressed the growth of cancer cells regardless of the presence of PTEN, possibly through inhibition of both mTORC1 and mTORC2. These results indicate that mTOR knockdown using APRPG-TEPA-PCL/simTOR is likely to be an effective strategy for cancer siRNA therapy.

Resveratrol enhances the anti-tumor activity of the mTOR inhibitor rapamycin in multiple breast cancer cell lines mainly by suppressing rapamycin-induced AKT signaling

The anti-tumor activity of rapamycin is compromised by the feedback-loop-relevant hyperactive PI3K and ERK-MAPK pathway signaling. In breast cancer cells treated with rapamycin, we observed a moderate increase of AKT phosphorylation (P-AKT) in a rapamycin resistant cell line, MDA-MB-231, as well as a slight increase of P-AKT in a rapamycin sensitive cell line, MCF-7. We found that resveratrol, a natural phytoalexin, suppressed the phosphorylation and activation of the PI3K/AKT pathway in all the three breast cancer cell lines that we tested. It also had a weak inhibitory effect on the activation of the mTOR/p70S6K pathway in two cell lines expressing wildtype PTEN, MCF-7 and MDA-MB-231. The combined use of resveratrol and rapamycin resulted in modest additive inhibitory effects on the growth of breast cancer cells, mainly through suppressing rapamycin-induced AKT activation. We, therefore, reveal a novel combination whereby resveratrol potentiates the growth inhibitory effect of rapamycin, with the added benefit of preventing eventual resistance to rapamycin, likely by suppressing AKT signaling. We also present data herein that PTEN is an important contributor to resveratrol’s growth suppressive effects and its potentiation of rapamycin in this therapeutic scenario, as resveratrol’s suppression of rapamycin-mediated induction of P-AKT is both PTEN-dependent and -independent. Thus, the resveratrol–rapamycin combination may have therapeutic value in treating breast cancer and perhaps other processes where mTOR is activated.

Disruption of DEPTOR/mTORC1/mTORC2 Signaling Cascade Using a Novel Selective mTOR Kinase Inhibitor AZD8055 Results In Growth Arrest and Apoptosis In Multiple Myeloma Cells

AbstractAbstract 791Targeting PI3K/Akt/mTOR signaling is among one of the promising therapeutic strategies in multiple myeloma (MM), since it facilitates MM cell survival and development of drug resistance in the context of the bone marrow microenvironment. Specifically, regulation of PI3K activity, which mediates MM cell growth and drug resistance, by mTOR complex 1 (mTORC1) provides the rationale for use of rapamycin analogs for MM treatment. However, rapamycin alone fails to overcome bone marrow-induced proliferation of MM cells, at least in part, because of the mTORC1-dependent feedback loops which activate PI3K/Akt. More recently, extensive studies of the mTOR network have identified mTORC2 as a “rapamycin-insensitive” complex. Sharing mTOR kinase as a common catalytic subunit, mTORC1 and mTORC2 mediate two distinct pathways: mTORC1 controls cell growth by phosphorylating key regulators of protein synthesis S6 kinase 1 (P70S6K) and the eIF-4E-binding protein 1 (4E-BP1); mTORC2 modulates cell survival and drug resistance by phosphorylating target proteins including Akt and serum/glucocorticoid regulated kinase 1(SGK1)/N-myc downstream regulated 1 (NDRG1). Moreover, studies have also revealed overexpression of a novel mTOR-interacting protein DEP domain containing 6 (DEPTOR), which can modulate mTOR activity and promote PI3K/mTORC2 signaling in primary MM tumor cells and in MM cell lines while mTORC1 remains silenced. We therefore hypothesized that targeting mTOR may disrupt DEPTOR/mTOR interaction and silence mTORC1/mTORC2 signaling, thereby overcoming mTOR resistance in MM cells. To confirm this idea, we used AZD8055, an orally bioavailable selective ATP-competitive mTOR kinase inhibitor, in our MM preclinical models. AZD8055- treatment of MM.1S inhibited phosphorylation of both mTORC1 and mTORC2 substrates: P70S6K; 4E-BP1 including the rapamycin-resistant T37/46 – downstream targets of mTORC1; as well as Akt and NDRG1 – effectors of mTORC2 refractory to rapamycin. Interestingly, AZD8055-mediated mTORC1/mTORC2 downregulation was associated with DEPTOR upregulation, which is consistent with the finding that DEPTOR expression is negatively regulated by mTORC1 and mTORC2. Moreover, inhibition of mTORC1 alone by rapamycin resulted in reduction of DEPTOR, associated with Akt activation. Furthermore, we observed that DEPTOR expression was decreased in MM.1S cells cultured with IL-6, IGF-1 or bone marrow stromal cells (BMSCs), which stimulate PI3K/Akt/mTOR signaling, evidenced by enhanced P70S6K and Akt phosphorylation. Unlike rapamycin, AZD8055 reversed those effects and inhibited MM.1S proliferation, even in the presence of these cytokines or BMSCs. AZD8055-induced growth inhibition was associated with apoptosis, evidenced by caspase-9, -3 and PARP cleavage in a time-dependent fashion (80% apoptotic cells at 72 hour culture as detected by Annexin V/PI staining). Moreover, AZD8055 induced cytotoxicity even in rapamycin resistant MM cell lines and primary patient MM cells. Finally, AZD8055 demonstrated significant anti-MM activity in an in vivo human MM cell xenograft SCID mouse model. Taken together, our data show that disruption of DEPTOR/mTORC1/mTORC2 cascade in MM cells results in significant anti-tumor effects, providing the framework for future clinical trials of AZD8055 to improve patient outcome in MM. Disclosures:Guichard:AstraZeneca: Employment, Shareholder AstraZeneca. Anderson:Millenium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Onyx: Consultancy; Merck: Consultancy; BMS: Consultancy; Acetylon: Membership on an entity's Board of Directors or advisory committees, Ownership interest (inc stock options) in a Start up company. Raje:AstraZeneca: Research Funding; Acetylon: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.