mTOR Kinase Inhibitor AZD8055 Research Articles

Abstract 5357: mTOR kinase inhibitor AZD8055 reverses acquired rapamycin resistance in prostate cancer cells

Abstract The mTOR pathway is frequently over-activated in human cancers. However, classic allosteric mTOR inhibitors rapamycin only exerts limited clinical benefits. It has been shown that prostate cancer are able to acquire resistance after rapamycin treatment for a period of time. Such secondary resistance remains as major concerns in rapamycin-based anti-cancer therapies that may inevitably lead to therapeutic failure. In the current study, we evaluated treatment effects of ATP-competitive dual mTOR kinase inhibitor AZD8055 on cancer cells that have acquired resistance to rapamycin, and underpinning molecular mechanisms. To establish cells with acquired rapamycin resistance, PTEN-mt prostate cancer PC-3 cells were gradually treated with increasing concentrations of rapamycin (up to 10 μM) until the cells were completely unresponsive to rapamycin (PC-3RR). Cell proliferation was determined by MTT assay and clonogenic survival was tested by colony formation assay. m7G pull-down assay was employed to detect the cap-dependent translation. mTOR downstream pathways, including p-S6K (T389)/p-S6 (S235/236), p-4EBP1 (T37/S46) and p-Akt (S473) were detected by Western Blotting. PC-3RR cells essentially failed to respond to rapamycin, with IC50 values greater than 1 μM. However, AZD8055 suppressed PC-3RR proliferation in a dose dependent manner, with IC50 < 30 nM. Similarly, clonogenic capacity was impaired by AZD8055 to a greater extent than was the case for rapamycin. At the molecular level, AZD8055, but not rapamycin, strongly prevents the dissociation of 4E-BP1 from eIF4E. Further, while the p-S6K and p-S6 pathway was potently inhibited by both agents, AZD8055 displayed more striking inhibition on p-4E-BP1 and p-Akt than rapamycin. Interestingly, similar to rapamycin, AZD8055 slightly increased p-Akt (T308), suggesting the existence of the PI3K-mediated feedback loop. These findings indicate that AZD8055 significantly inhibits malignant functions in cancer cells that are highly resistant to rapamycin. Such effects are likely to be associated with the deactivation of rapamycin-insensitive mTOR downstream pathways. These data provide preliminary evidence for future evaluation of ATP-competitive mTOR inhibitors in cancer models that are resistant to rapamycin analogs. Citation Format: Yao Dai, Dietmar Siemann. mTOR kinase inhibitor AZD8055 reverses acquired rapamycin resistance in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5357.

Abstract 660: Combination of mTOR kinase inhibitors with Src inhibitors in PTEN-mt prostate cancer cells

Abstract Although Src inhibitors are able to suppress malignant functions, their clinical outcomes are modest for the treatment of advanced prostate cancer, many of which harbor mutated PTEN. mTOR pathway is commonly hyperactivated in prostate cancer due to frequent PTEN mutation, and it is known that two mTOR complexes are both critical in prostate cancer progression. However, the functional role of mTOR kinase in Src inhibitor-mediated response remains unknown. In the current study, we combined mTOR kinase inhibitor AZD8055 and Src inhibitor dasatinib to evaluate functional behaviors in PTEN-deleted prostate cancer cells. Gene silencing was achieved by siRNA or shRNA transfection. Cell proliferation and clonogenic survival were performed by MTT and colony formation assay, respectively. “Wound-healing” scratch assay was conducted to monitor cell migration, and Matrigel-based transwell assay was applied to assess cell invasion. Protein expression was detected by Western blotting. We found that in PTEN-mt cells, C4-2B showed greater expression of p-Akt, p-S6, p-4EBP1 and p-eIF4E, all of which are mTOR complexes downstream components. Such molecular profile is consistent with functional observation that C4-2B cells are more resistant to dasatinib. Interestingly, single knockdown of either 4EBP1, S6RP or AKT showed little effects of inhibiting cell survival, while double knockdown of both 4EBP1 and AKT, cell survival was significantly inhibited. Consistently, two agents together showed significantly greater inhibition on proliferation and clonogenic survival than either single agent. Such combination effects were either additive or synergistic depending on the cell types, as reflected by combination index calculation. In addition, cell migration and invasion were inhibited in a greater extent by AZD8055 plus dasatinib, compared to either treatment alone. At the molecular level, co-targeting mTOR and Src kinases leads to strong inhibition of mTOR and Src pathways, such as FAK, Akt, S6RP and 4E-BP1. These data indicate that adding AZD8055 to dasatinib may achieve better treatment outcomes in PTEN-mt prostate cancer models. Such combination modality hold potential for future development as a novel strategy for PTEN-mutated prostate cancer patients who are less responsive to Src inhibitors. Citation Format: Yao Dai, Dietmar Siemann. Combination of mTOR kinase inhibitors with Src inhibitors in PTEN-mt prostate cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 660.

Identification of Genotype-Specific Therapeutic Vulnerabilities By Comparative Dynamic BH3 Profiling Analysis of Human and Murine CLL

Identification of Genotype-Specific Therapeutic Vulnerabilities By Comparative Dynamic BH3 Profiling Analysis of Human and Murine CLL

A pharmacokinetic-pharmacodynamic model predicting tumour growth inhibition after intermittent administration with the mTOR kinase inhibitor AZD8055.

AZD8055 is a potent orally available mTOR kinase inhibitor with in vitro and in vivo antitumour activity against a range of tumour types. Preclinical studies showed that AZD8055 induced a dose-dependent pharmacodynamic effect in xenograft models in vivo, but a lack of understanding of the relative contributions of the maximum inhibition of the biomarkers and the duration of inhibition to the antitumour effect, limited the rational design of experiments to optimize the dose and schedules of treatment. In this study, a mathematical modelling approach was developed to relate pharmacodynamics and antitumour activity using preclinical data generated in mice bearing U87-MG xenografts. Refinement and validation of the model was carried out in a panel of additional human tumour xenograft models with different growth rates and different sensitivity to AZD8055 (from partial growth inhibition to regression). Finally, the model was applied to accurately predict the efficacy of high, intermittent dosing schedules of AZD8055. Overall, this new model linking pharmacokinetics, pharmacodynamic biomarkers and efficacy across several tumour xenografts with different sensitivity to AZD8055 was able to identify the optimal dose and route of administration to maximize the antitumour efficacy in preclinical models and its potential for translation into man.

MTOR Is Dispensable in Hematopoietic Stem Cells and GMP Progenitors, but Plays an Essential Role in the CMP Population, for Autophagy Regulation

Macroautophagy, or autophagy, is an intracellular self-degrading and recycling process that maintains cellular homeostasis. In blood system, autophagy is essential for life-long support and maintenance of hematopoietic stem cells (HSCs). Defect in autophagy process may lead to HSC aging, abnormal development in both myeloid and lymphoid lineages, and failure of blood reconstitution after transplantation. Current biochemical model of autophagy views the mTOR kinase activity, a therapeutic target for a variety of hematologic diseases including leukemia, as a critical inhibitory mechanism of autophagy initiation through phosphorylation of ULK1 and Atg13. Previously we showed that mTOR gene deletion in the blood system lead to hematopoietic failure, with a transient expansion of the HSC pool and differential blockage of various progenitor populations without detectable effects on survival. Since cellular responses to mTOR deletion vary in different blood cell populations, we hypothesize that autophagy dependence on mTOR may also differ among different HSC and progenitor populations. To examine the basal state and mTOR regulated autophagy flux in hematopoiesis, we used a reporter mouse carrying an autophagy reporter GFP-LC3 that allows a readout of autophagy flux in live or fixed cells. Crossing it to an Mx-Cre;mTORflox/flox or an Mx-Cre;mTORflox/knockin genetic background produced the mTOR null or the mTOR kinase-activity deficient D2338A mutant knockin genotype upon poly I:C induced Cre recombination in all blood lineages. Subpopulations of mTOR wild-type (WT), mTOR knockout (KO) and mTOR kinase-dead knockin (KI) cells from freshly harvested mouse bone marrow, including HSC (Lin-Sca-1+c-Kit+CD135-), CMP (Lin-Sca-1-c-Kit+CD34+CD16/32lo), and GMP (Lin-Sca-1-c-Kit+CD34+CD16/32hi, were isolated by flow cytometry. The sorted cells were treated with autophagy inhibitor Bafilomycin A1 (BA), mTOR kinase inhibitor AZD8055 (AZD), or a nonspecific AMPK inhibitor Compound C, and the autophagy flux activities of the various populations of HSC and progenitors were quantified by immunofluorescent microscopy of the GFP-LC3 puncta or FACS analysis. In wild-type mice, HSC and CMP populations had significantly higher basal autophagy flux than the GMP cells, with ~55% LC3 puncta positive cells in HSC and CMP versus ~15% in GMP. Surprisingly, the HSC and GMP populations of cells did not respond to either mTOR KO or KI in their autophagy activities while the CMP cells were highly responsive to the mTOR KO or KI showing a significant increase of autophagy flux in the KO and KI cells, compared with wild-type CMP cells. In the GMP enriched Lin-c-Kit+ population, p-Akt308 was significantly upregulated upon mTOR loss, possibly due to a compensatory mechanism, which may in turn suppress autophagy through Beclin 1. Upon treatment by the mTOR inhibitor AZD, autophagy flux was induced in wild-type HSC, CMP and GMP populations but not in the corresponding mTOR KO or KI populations, whereas Compound C, a non-specific kinase inhibitor, induced LC3 puncta accumulation in HSC, CMP and GMP populations of all three mTOR WT, KO, and KI genotypes. Our data shows that in HSPCs, basal autophagy flux varies in different populations, with higher autophagy activity found in the most primitive HSCs and lower autophagy activity in the GMP population. The dependence on mTOR in autophagy regulation also differs amongst different HSC and progenitor populations, with HSC and GMP populations showing a surprising independence whereas CMP population a dependence on mTOR in autophagy suppression. The autophagy flux of the mTOR KO genotype are similar to that of the mTOR KI genotype in the stem/progenitor populations, suggesting that the mTOR kinase activity, not a kinase-independent mTOR function, regulates autophagy. Lastly, the mTOR WT, KO and KI CMP cells respond to a non-specific kinase inhibitor Compound C in autophagy induction, suggesting that mTOR-independent kinases are involved in autophagy suppression. Thus, despite that the mTOR KO and KI HSPCs show similar deficiencies in the canonical mTOR signaling including dampened p-S6 and p-4EBP1, the molecular mechanisms underlying autophagy regulation is diverse amongst distinct blood stem/progenitor cell types. DisclosuresNo relevant conflicts of interest to declare.

Loss of ARID1A Activates ANXA1, which Serves as a Predictive Biomarker for Trastuzumab Resistance.

Despite the substantial progress in the development of targeted anticancer drugs, treatment failure due to primary or acquired resistance is still a major hurdle in the effective treatment of most advanced human cancers. Understanding these resistance mechanisms will be instrumental to improve personalized cancer treatment. Genome-wide loss-of-function genetic screens were performed to identify genes implicated in resistance to HER2/PI3K/mTOR targeting agents in HER2+ breast cancer cell lines. Expression and adjuvant trastuzumab response data from the HER2+ breast cancer trials FinHer and Responsify were used to validate our findings in patient series. We find that reduced ARID1A expression confers resistance to several drugs that inhibit the HER2/PI3K/mTOR signaling cascade at different levels. We demonstrate that ARID1A loss activates annexin A1 (ANXA1) expression, which is required for drug resistance through its activation of AKT. We find that the AKT inhibitor MK2206 restores sensitivity of ARID1A knockdown breast cancer cells to both the mTOR kinase inhibitor AZD8055 and trastuzumab. Consistent with these in vitro data, we find in two independent HER2+ breast cancer patient series that high ANXA1 expression is associated with resistance to adjuvant trastuzumab-based therapy. Our findings provide a rationale for why tumors accumulate ARID1A mutations and identify high ANXA1 expression as a predictive biomarker for trastuzumab-based treatment. Our findings also suggest strategies to treat breast cancers with elevated ANXA1 expression. Clin Cancer Res; 22(21); 5238-48. ©2016 AACR.

Abstract B59: Dual mTOR kinase inhibitor reverses rapamycin resistance in prostate cancer cells

Abstract The mTOR pathway is frequently over-activated in human cancers. However, classic allosteric mTOR inhibitors rapamycin and its analogs only exert limited clinical benefits in patients. It has been shown that many tumors either fail to respond to rapamycin initially or are able to acquire resistance after the initial treatment of rapamycin. Such primary and secondary resistances remain as major concerns in rapamycin-based anti-cancer therapies that may inevitably lead to therapeutic failure. We have previously shown that the ATP-competitive dual mTOR kinase inhibitor AZD8055 can significantly inhibit malignant behaviors in parental cells however its functional efficacies in the rapamycin-resistance setting remains unclear. Cancer cells with wildtype PTEN were selected as models of primary rapamycin resistance since the mTOR pathway is usually less abnormal in these cells. To establish cells with acquired rapamycin resistance, PTEN-null prostate cancer PC-3 cells were gradually treated with increasing concentrations of rapamycin (up to 10 μM) until the cells were completely unresponsive to rapamycin (PC-3RR). Cell proliferation was determined by MTT assay and clonogenic survival was tested by colony formation assay. m7G pull-down assay was employed to detect the cap-dependent translation. mTOR downstream pathways were detected by Western blotting. PTEN wildtype and PC-3RR cells essentially failed to respond to rapamycin; IC50 values were greater than 100 nM in PTEN wildtype cells and 1 μM in PC-3RR cells, respectively. However, for both cell types AZD8055 suppressed proliferation at the nanomolar level in a dose dependent manner, with IC50 values less than 30 nM. Similarly, cellular clonogenic capacity was impaired by AZD8055 to a greater extent than was the case for rapamycin. At the molecular level, AZD8055, but not rapamycin, strongly prevents the dissociation of 4E-BP1 from eIF4E, therefore blocking cap-dependent translation. Further, while the p-S6K (T389) and p-S6 (S235/236) pathway was potently inhibited by both agents, AZD8055 displayed more striking inhibition on p-4E-BP1 (T37/46) and p-Akt (S473) than did rapamycin in all cells tested. Interestingly, similar to rapamycin, AZD8055 slightly increased p-Akt (T308), suggesting the existence of the PI3K-mediated feedback loop. However, the PI3K inhibitor wortmannin failed to sensitize cell response to AZD8055, implying such feedback-dependent activation has little impact on the drug efficacy. These findings indicate that dual mTOR kinase inhibitor AZD8055 significantly inhibits malignant functions in cancer cells that are highly resistant to rapamycin. Such effects are likely to be associated with deactivation of rapamycin-insensitive mTOR downstream components such as Akt and 4E-BP1, but not the upstream feedback pathway. These data provide in vitro evidence in support of future evaluation of ATP-competitive mTOR inhibitors in cancer models that are resistant to rapamycin analogs. Citation Format: Yao Dai, Li Zhao, Dietmar W. Siemann. Dual mTOR kinase inhibitor reverses rapamycin resistance in prostate cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B59.

Abstract 3578: Dual mTOR kinase inhibitor overcomes rapamycin resistance in vitro

Abstract The mTOR pathway is frequently over-activated in human cancers. However, classic allosteric mTOR inhibitors rapamycin and its analogs only exert limited clinical benefits in patients. It has been shown that many tumors either fail to respond to rapamycin initially or are able to acquire resistance after the initial treatment of rapamycin. Such primary and secondary resistances remain as major concerns in rapamycin-based anti-cancer therapies that may inevitably lead to therapeutic failure. We have previously shown that the ATP-competitive dual mTOR kinase inhibitor AZD8055 can significantly inhibit malignant behaviors however its functional efficacies in the rapamycin-resistance setting remains unclear. Cancer cells with wildtype PTEN were selected as models of primary rapamycin resistance since the mTOR pathway is usually less abnormal in these cells. To establish cells with acquired rapamycin resistance, PTEN-null prostate cancer PC-3 cells were gradually treated with increasing concentrations of rapamycin (up to 10 μM) until the cells were completely unresponsive to rapamycin (PC-3RR). Cell proliferation was determined by MTT assay and clonogenic survival was tested by colony formation assay. m7G pull-down assay was employed to detect the cap-dependent translation. mTOR downstream pathways, including p-S6K (T389)/p-S6 (S235/236), p-4EBP1 (T37/S46) and p-Akt (S473) were detected by Western Blotting. PTEN wildtype and PC-3RR cells essentially failed to respond to rapamycin; IC50 values were greater than 100 nM in PTEN wildtype cells and 1 μM in PC-3RR cells. However, for both cell types AZD8055 suppressed proliferation at the nanomolar level in a dose dependent manner, with IC50 values less than 30 nM. Similarly, cellular clonogenic capacity was impaired by AZD8055 to a greater extent than was the case for rapamycin. At the molecular level, AZD8055, but not rapamycin, strongly prevents the dissociation of 4E-BP1 from eIF4E, therefore blocking cap-dependent translation. Further, while the p-S6K and p-S6 pathway was potently inhibited by both agents, AZD8055 displayed more striking inhibition on p-4E-BP1 and p-Akt than did rapamycin in all cells tested. Interestingly, similar to rapamycin, AZD8055 slightly increased p-Akt (T308), suggesting the existence of the PI3K-mediated feedback loop. However, the PI3K inhibitor wortmannin did not further sensitize cells to AZD8055, implying such feedback-dependent activation is less responsible for weakening the drug efficacy. These findings indicate that AZD8055 significantly inhibits malignant functions in cancer cells that are highly resistant to rapamycin. Such effects are likely to be associated with the deactivation of rapamycin-insensitive mTOR downstream components such as Akt and 4E-BP1, but not the upstream feedback pathway. These data provide in vitro evidence in support of future evaluation of ATP-competitive mTOR inhibitors in cancer models that are resistant to rapamycin analogs. Citation Format: Yao Dai, Li Zhao, Dietmar Siemann. Dual mTOR kinase inhibitor overcomes rapamycin resistance in vitro. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3578. doi:10.1158/1538-7445.AM2015-3578

PI3K-independent mTOR activation promotes lapatinib resistance and IAP expression that can be effectively reversed by mTOR and Hsp90 inhibition

Although HER2 targeted therapies have substantially improved outcomes in HER2 overexpressing (HER2+) breast cancer, resistance to these therapies remains a clinical challenge. To better understand the mechanisms of resistance to lapatinib, a HER2 and EGFR dual kinase inhibitor, we treated HER2+ breast cancer cells with lapatinib for an extended period to generate a lapatinib-resistant (LapR) cell line model and examined cancer-promoting signaling activation in LapR cells. We found that LapR cells possess enhanced mTOR activation, which was independent of PI3K and other known mTOR activators. Lapatinib resistance could be reversed by mTOR kinase inhibition. Intriguingly, LapR cells had constitutive cytosolic cytochrome C, indicating that LapR cells suppress lapatinib-induced apoptosis downstream of cytochrome C release from mitochondria into the cytosol rather than by preventing its release into the cytosol. Consistent with this notion, LapR cells possessed increased levels of 2 of the inhibitors of apoptosis (IAPs), survivin and c-IAP-2, which are reported to block caspase activation downstream of cytosolic cytochrome C release. Further, treatment with the mTOR kinase inhibitor AZD8055 or the Hsp90 inhibitor 17-AAG reversed expression of IAPs and overcame lapatinib resistance in LapR cells. Together, these data suggest that suppression of apoptosis downstream of cytosolic cytochrome C release, possibly through increased expression of IAPs or other caspase-suppressing proteins, may promote lapatinib resistance. Further, PI3K is thought to be the main driver of lapatinib resistance, but our findings indicate that PI3K inhibitors may be ineffective in some lapatinib-resistant HER2+ breast cancers with PI3K-independent activation of mTOR kinase, which may instead benefit from mTOR or Hsp90 inhibitors.

Vertical Targeting of AKT and mTOR as Well as Dual Targeting of AKT and MEK Signaling Is Synergistic in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the sixth most common cancer, and the third most common cause of cancer related death worldwide. The multi-kinase inhibitor Sorafenib represents the only systemic treatment option until today, and results from clinical trials with allosteric mTOR inhibitors were sobering. Since the PI3K/AKT/mTOR and RAF/MEK/ERK signaling pathways are frequently upregulated in HCC, we have analyzed the effects of AKT inhibitor MK-2206, MEK inhibitor AZD6244 (ARRY 142886) and mTOR kinase inhibitor AZD8055, given as single drugs or in combination, on proliferation and apoptosis of three HCC cell lines in vitro. We show that all three inhibitor combinations synergistically inhibit proliferation of the three HCC cell lines, with the strongest synergistic effect observed after vertical inhibition of AKT and mTORC1/2. We demonstrate that AKT kinase activity is restored 24h after blockade of mTORC1/2 by increased phosphorylation of T308, providing a rationale for combined targeting of AKT and mTOR inhibition in HCC. Our data suggest that a combination of inhibitors targeting those respective pathways may be a viable approach for future application in patients with hepatocellular carcinoma.

Delineating the mTOR kinase pathway using a dual TORC1/2 inhibitor, AZD8055, in multiple myeloma.

Despite promising preclinical results with mTOR kinase inhibitors in multiple myeloma, resistance to these drugs may arise via feedback activation loops. This concern is especially true for insulin-like growth factor 1 receptor (IGF1R), because IGF1R signaling is downregulated by multiple AKT and mTOR feedback mechanisms. We have tested this hypothesis in multiple myeloma using the novel selective mTOR kinase inhibitor AZD8055. We evaluated p-mTOR S(2481) as the readout for mTORC2/Akt activity in multiple myeloma cells in the context of mTOR inhibition via AZD8055 or rapamycin. We next validated AZD8055 inhibition of mTORC1 and mTORC2 functions in multiple myeloma cells alone or in culture with bone marrow stroma cells and growth factors. Unlike rapamycin, AZD8055 resulted in apoptosis of multiple myeloma cells. AZD8055 treatment, however, induced upregulation of IGF1R phosphorylation in p-Akt S(473)-expressing multiple myeloma cell lines. Furthermore, exposure of AZD8055-treated cells to IGF1 induced p-Akt S(473) and rescued multiple myeloma cells from apoptosis despite mTOR kinase inhibition and TORC2/Akt blockage. The addition of blocking IGF1R antibody resulted in reversing this effect and increased AZD8055-induced apoptosis. Our study suggests that combination treatment with AZD8055 and IGF1R-blocking agents is a promising strategy in multiple myeloma with potential IGF1R/Akt signaling-mediated survival.

Dual Inhibition of PI3K-AKT-mTOR- and RAF-MEK-ERK-signaling is synergistic in cholangiocarcinoma and reverses acquired resistance to MEK-inhibitors.

Until today, there is no systemic treatment available for advanced cholangiocarcinoma (CCA). Recent studies have shown a frequent upregulation of the PI3K-AKT-mTOR and RAF-MEK-ERK pathways in this type of cancer. However, considering their high extend of redundancy and cross-talk, targeting only one pathway is likely to result in therapy failure and emergence of resistances. To provide a rationale for treatment of CCA with inhibitors of these respective pathways, we analyzed the effects of AKT inhibitor MK-2206, MEK inhibitor AZD6244 (ARRY-142886) and mTOR kinase inhibitor AZD8055 on three CCA cell lines in vitro, concerning proliferation, cell signaling and apoptosis. Furthermore, AZD6244 resistant cell lines have been generated to investigate, how their response may be affected by prolonged treatment with only a single inhibitor. Our data demonstrates that co-targeting of both, the PI3K/AKT/mTOR and RAF-MEK-ERK pathway, as well as vertical targeting of AKT and mTOR results in strong synergistic effects on proliferation and cell survival with combination indices below 0.3. Mechanistically, the combinatorial treatment with MK-2206 in addition to AZD8055 is necessary because AKT kinase activity was quickly restored after mTOR kinase inhibition. Interestingly, acquired MEK inhibitor resistance to AZD6244 was reversed by combined treatment with AZD6244 and either MK-2206 or AZD8055. Our data suggest that a combination of inhibitors targeting those respective pathways may be a viable approach for future application in patients with cholangiocarcinoma. AKT, mTOR and MEK are promising targets for a combinatorial treatment of cholangiocarcinoma cells even after acquisition of MEK inhibitor resistance.

Switch in signaling control of mTORC1 activity after oncoprotein expression in thyroid cancer cell lines.

Thyroid growth is regulated by TSH and requires mammalian target of rapamycin (mTOR). Thyroid cancers frequently exhibit mutations in MAPK and/or phosphoinositol-3-kinase-related kinase effectors. The objective of the study was to explore the contribution of RET/PTC, RAS, and BRAF to mTOR regulation and response to mTOR inhibitors. PCCL3 cells conditionally expressing RET/PTC3, HRAS(G12V), or BRAF(V600E) and human thyroid cancer cells harboring mutations of these genes were used to test pathways controlling mTOR and its requirement for growth. TSH/cAMP-induced growth of PCCL3 cells requires mTOR, which is stimulated via protein kinase A in a MAPK kinase (MEK)- and AKT-independent manner. Expression of RET/PTC3, HRAS(G12V), or BRAF(V600E) in PCCL3 cells induces mTOR but does not entirely abrogate the cAMP-mediated control of its activity. Acute oncoprotein-induced mTOR activity is regulated by MEK and AKT, albeit to differing degrees. By contrast, mTOR was not activated by TSH/cAMP in human thyroid cancer cells. Tumor genotype did not predict the effects of rapamycin or the mTOR kinase inhibitor AZD8055 on growth, with the exception of a PTEN-null cell line. Selective blockade of MEK did not influence mTOR activity of BRAF or RAS mutant cells. Combined MEK and mTOR kinase inhibition was synergistic on growth of BRAF- and RAS-mutant thyroid cancer cells in vitro and in vivo. Thyroid cancer cells lose TSH/cAMP dependency of mTOR signaling and cell growth. mTOR activity is not decreased by the MEK or AKT inhibitors in the RAS or BRAF human thyroid cancer cell lines. This may account for the augmented effects of combining the mTOR inhibitors with selective antagonists of these oncogenic drivers.

Impact of dual mTORC1/2 mTOR kinase inhibitor AZD8055 on acquired endocrine resistance in breast cancer in vitro

IntroductionUpregulation of PI3K/Akt/mTOR signalling in endocrine-resistant breast cancer (BC) has identified mTOR as an attractive target alongside anti-hormones to control resistance. RAD001 (everolimus/Afinitor®), an allosteric mTOR inhibitor, is proving valuable in this setting; however, some patients are inherently refractory or relapse during treatment requiring alternative strategies. Here we evaluate the potential for novel dual mTORC1/2 mTOR kinase inhibitors, exemplified by AZD8055, by comparison with RAD001 in ER + endocrine resistant BC cells.MethodsIn vitro models of tamoxifen (TamR) or oestrogen deprivation resistance (MCF7-X) were treated with RAD001 or AZD8055 alone or combined with anti-hormone fulvestrant. Endpoints included growth, cell proliferation (Ki67), viability and migration, with PI3K/AKT/mTOR signalling impact monitored by Western blotting. Potential ER cross-talk was investigated by immunocytochemistry and RT-PCR.ResultsRAD001 was a poor growth inhibitor of MCF7-derived TamR and MCF7-X cells (IC50 ≥1 μM), rapidly inhibiting mTORC1 but not mTORC2/AKT signalling. In contrast AZD8055, which rapidly inhibited both mTORC1 and mTORC2/AKT activity, was a highly effective (P <0.001) growth inhibitor of TamR (IC50 18 nM) and MCF7-X (IC50 24 nM), and of a further T47D-derived tamoxifen resistant model T47D-tamR (IC50 19 nM). AZD8055 significantly (P <0.05) inhibited resistant cell proliferation, increased cell death and reduced migration. Furthermore, dual treatment of TamR or MCF7-X cells with AZD8055 plus fulvestrant provided superior control of resistant growth versus either agent alone (P <0.05). Co-treating with AZD8055 alongside tamoxifen (P <0.01) or oestrogen deprivation (P <0.05) also effectively inhibited endocrine responsive MCF-7 cells. Although AZD8055 inhibited oestrogen receptor (ER) ser167 phosphorylation in TamR and MCF7-X, it had no effect on ER ser118 activity or expression of several ER-regulated genes, suggesting the mTOR kinase inhibitor impact was largely ER-independent. The capacity of AZD8055 for ER-independent activity was further evidenced by growth inhibition (IC5018 and 20 nM) of two acquired fulvestrant resistant models lacking ER.ConclusionsThis is the first report demonstrating dual mTORC1/2 mTOR kinase inhibitors have potential to control acquired endocrine resistant BC, even under conditions where everolimus fails. Such inhibitors may prove of particular benefit when used alongside anti-hormonal treatment as second-line therapy in endocrine resistant disease, and also potentially alongside anti-hormones during the earlier endocrine responsive phase to hinder development of resistance.

Examining Changes in [18 F]FDG and [18 F]FLT Uptake in U87-MG Glioma Xenografts as Early Response Biomarkers to Treatment with the Dual mTOR1/2 Inhibitor AZD8055

The mTOR kinase inhibitor AZD8055 inhibits both mTORC1 and mTORC2 leading to disruption of glucose metabolism and proliferation pathways. This study assessed the impact of single and multiple doses of AZD8055 on the uptake of the glucose metabolism marker 2-deoxy-2-[(18) F]fluoro-D-glucose ([(18) F]FDG) and the proliferation marker 3'-deoxy-3'-[(18) F]fluorothymidine ([(18) F]FLT) in U87-MG glioma xenografts. Mice bearing U87-MG tumours received either vehicle or AZD8055 (20 mg/kg) once daily p.o. Mice were imaged with either [(18) F]FDG or [(18) F]FLT PET to assess treatment response. Comparisons were made between in vivo imaging and ex vivo histopathology data. Tumour uptake of [(18) F]FDG was reduced by 33 % 1 h after a single dose of AZD8055 and by 49 % following 4 days of dosing. These changes coincided with suppression of the mTOR pathway biomarkers pS6 and pAKT. In contrast, the effect of AZD8055 on [(18) F]FLT uptake was inconsistent. The very rapid change in [(18) F]FDG uptake following acute AZD8055 treatment suggests that this could be used as an early mechanistic biomarker of metabolic changes resulting from mTOR inhibition. The utility of [(18) F]FLT for measuring the anti-proliferative effect of AZD8055 remains unclear.

Abstract 909: Hsp90 inhibitor AUY922 abrogates mTOR inhibitor AZD8055-induced up-regulation of HER2/HER3 and potentiates antiproliferative activity in human breast cancer cells.

Abstract Mammalian target of rapamycin (mTOR) has been validated as a promising target for cancer therapy. Several mTOR kinase inhibitors displaying potent antitumor activity due to their capability to inhibit both mTORC1 and mTORC2 have entered clinical trials. It has been recently reported that long term inhibition of mTOR led to activation of upstream receptor tyrosine kinases and Akt, which may attenuate the efficacy of mTOR kinase inhibitors. As a number of receptor tyrosine kinases and AKT are client proteins of Hsp90, we hypothesized that combination of inhibitors targeting mTOR and Hsp90 respectively would potentiate the anticancer activity. By calculating the Combination Index, mTOR kinase inhibitor AZD8055 and Hsp90 inhibitor AUY922 displayed synergistic effect against a panel of human breast cancer cells irrespective their ER, HER2 or PI3K status. The combination of sub-optimal concentration of these two drugs exerted significant antiproliferative activity, which is associated with enhanced cell cycle arrest but not induction of apoptosis. Down-regulation of either raptor or rictor by specific shRNA reinforced the antitumor activity of AUY922, indicating restraint of both mTORC1 and mTORC2 contribute to the synergistic activity of AZD8055 with AUY922. In T47D cells,long time exposure to AZD8055 led to restoration of phosphorylated AKT at T308 and GSK3β at S9, indicating reactivation of AKT, which was abrogated by AUY922 co-treatment. Moreover, phosphorylation of AKT at S473 was more significantly inhibited when cells were co-treated with AZD8055 and AUY922 than single agent treatment. We further revealed that AUY922 prevented feedback activation of PI3K upon AZD8055 treatment. As PI3K is not a client protein of Hsp90, block of PI3K activation by AUY922 might be due to down-regulation of upstream tyrosine kinase receptors. Accordingly, though the mRNA levels of EGFR, HER2, HER3 and HER4 increased after either single agent treatment or co-treatment, AUY922 not only inhibited the expression of tested tyrosine kinases, but more importantly, abrogated activation of HER2/HER3 induced by AZD8055 . In summary, the combination of catalytic mTOR inhibitor and Hsp90 inhibitor demonstrated synergistic activity against breast cancer cells, which is due to inhibition of tyrosine kinases expression and abrogation of feedback activation of HER2/HER3. As both AZD8055 and AUY922 are in clinical trials for breast cancer therapy, this study established a mechanistic rationale for a combination approach using AZD8255 and AUY922 in the treatment of breast cancer. Citation Format: Simeng Chen, Chenliang Guo, Jian Ding, Linghua Meng. Hsp90 inhibitor AUY922 abrogates mTOR inhibitor AZD8055-induced up-regulation of HER2/HER3 and potentiates antiproliferative activity in human breast cancer cells. [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 909. doi:10.1158/1538-7445.AM2013-909

Adaptation to mTOR kinase inhibitors by amplification of eIF4E to maintain cap-dependent translation

The mechanistic target of rapamycin (mTOR) protein kinase coordinates responses to nutrients and growth factors and is an anti-cancer drug target. To anticipate how cells will respond and adapt to chronic mTOR complex (mTORC)1 and mTORC2 inhibition, we have generated SW620 colon cancer cells with acquired resistance to the ATP-competitive mTOR kinase inhibitor AZD8055 (SW620:8055R). AZD8055 inhibited mTORC1 and mTORC2 signalling and caused a switch from cap-dependent to internal ribosome entry site (IRES)-dependent translation in parental SW620 cells. In contrast, SW620:8055R cells exhibited a loss of S6K signalling, an increase in expression of the eukaryotic translation initiation factor eIF4E and increased cap-dependent mRNA translation. As a result, the expression of CCND1 and MCL1, proteins encoded by eIF4E-sensitive and cap-dependent transcripts, was refractory to AZD8055 in SW620:8055R cells. RNAi-mediated knockdown of eIF4E reversed acquired resistance to AZD8055 in SW620:8055R cells; furthermore, increased expression of eIF4E was sufficient to reduce sensitivity to AZD8055 in a heterologous cell system. Finally, although the combination of MEK1/2 inhibitors with mTOR inhibitors is an attractive rational drug combination, SW620:8055R cells were actually cross-resistant to the MEK1/2 inhibitor selumetinib (AZD6244). These results exemplify the convergence of ERK1/2 and mTOR signalling at eIF4E, and the key role of eIF4E downstream of mTOR in maintaining cell proliferation. They also have important implications for therapeutic strategies based around mTOR and the MEK1/2-ERK1/2 pathway.

Rational Combination Treatment of a Novel Selective mTOR Kinase Inhibitor AZD8055 with IGF-1R Inhibitors in Multiple Myeloma

AbstractAbstract 4023 Background:mTOR kinase-targeted therapy is in the early phase of clinical evaluation in multiple myeloma (MM). Despite promising preclinical results with mTOR inhibitors, resistance to this class of drugs in MM patients may occur due to feedback Akt activation by mTORC1. This led to the development of mTORC1/2 inhibition strategies in the treatment for MM, predicated upon the rationale that mTORC2 inhibitors prevent inhibition of mTORC1 blockade-induced feedback AKT activation by mTORC1 inhibitors. Indeed, our previous studies using a novel dual mTORC1 and mTORC2 selective inhibitor AZD8055 show MM cell growth inhibition via apoptosis, associated with inhibition of mTORC1 and mTORC2 signaling, including rapamycin-resistant 4E-BP1 (downstream of mTORC1) and Akt as well as NDRG1 (an effector of mTORC2). Importantly, AZD8055 also inhibited PI3K/Akt signaling and related MM cell growth induced by cytokines (i.e., IL-6, IGF-1) or co-culture with bone marrow stromal cells (BMSCs). Recent studies, however, reveal that constitutively activated Akt signaling negatively regulates IGF-1 receptor (IGF-1R) at the transcriptional level, independent of mTOR activity. Moreover, AKT-induced IGF-1R down-regulation reduces sensitivity of IRS1 to IGF-1 stimulation. We have also shown that IGF-1R inhibitor triggers significant MM cell toxicity. Methods and Results:In this study, we therefore hypothesized that mTORC2 blockade may upregulate IGF-1R expression and/or activity via Akt modulation in MM cells, and that IGF-1R blockade may enhance the cytotoxic effects of mTOR kinase inhibition in MM cells. We first examined the tyrosine phosphorylation sites (Y1135/1136) in the activation loop of the IGF-1R kinase domain in three MM cell lines (MM.1S, OPM1 and RPMI8226) treated with AZD8055 or rapamycin. AZD8055 induced more pronounced upregulation of p-IGF-1R in MM.1S and OPM1 MM cells than rapamycin at earlier time periods. IGF-1 clearly upregulated Akt phosphorylation in MM cells; however, it had no effect on mTOR phosphorylation (Ser2481). Moreover, AZD8055-treated cells exposed to IGF-1 sustained p-Akt (Ser473) expression, while p-mTOR (Ser2481) remained fully inhibited. These results suggest that IGF-1/IGF-1R signaling may bypass mTORC2/Akt when mediating p-Akt (Ser473) upregulation. Moreover, reactivation of IGF-1 signaling in MM cells in the context of mTOR kinase inhibitors suggests that MM may survive in an IGF-1 R–dependent fashion. We therefore next treated MM.1S, OPM1 and RPMI8226 cells with AZD8055, in the presence or absence of IGF-1. MM.1S and OPM1 MM cells (with higher Akt baseline activity) partially escaped AZD8055 cytotoxicity, while RPMI8226 MM cells (with lower Akt activity) did not. Moreover, the addition of blocking IGF-1R antibody or of IGF-1R inhibitor enhanced AZD8055 cytotoxicity in MM.1S and OPM1 cells. Conclusions:Our study therefore shows interaction of mTOR/Akt and IGF-1R/Akt pathways in MM tumors with IGF-1-enabled Akt activation. Importantly, they suggest that combination treatment with AZD8055 and IGF-1R inhibitor is a promising strategy to mTOR kinase inhibition in MM with potential IGF-1R/Akt signaling mediated survival. Disclosures:Hideshima:Acetylon Pharmaceuticals, Inc.: Consultancy. Guichard:AstraZeneca: Employment, Shareholder Other. Anderson:Onyx: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Raje:Onyx: Consultancy; Celgene: Consultancy; Millenium: Consultancy; Acetylon: Research Funding; Amgen: Research Funding; Eli-Lilly: Research Funding.

Impact of Combined mTOR and MEK Inhibition in Uveal Melanoma Is Driven by Tumor Genotype

Uveal melanomas possess activation of the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT/mammalian Target of Rapamycin (mTOR) pathways. MAPK activation occurs via somatic mutations in the heterotrimeric G protein subunits GNAQ and GNA11 for over 70% of tumors and less frequently via V600E BRAF mutations. In this report, we describe the impact of dual pathway inhibition upon uveal melanoma cell lines with the MEK inhibitor selumetinib (AZD6244/ARRY-142886) and the ATP-competitive mTOR kinase inhibitor AZD8055. While synergistic reductions in cell viability were observed with AZD8055/selumetinib in both BRAF and GNAQ mutant cell lines, apoptosis was preferentially induced in BRAF mutant cells only. In vitro apoptosis assay results were predictive of in vivo drug efficacy as tumor regressions were observed only in a BRAF mutant xenograft model, but not GNAQ mutant model. We went on to discover that GNAQ promotes relative resistance to AZD8055/selumetinib-induced apoptosis in GNAQ mutant cells. For BRAF mutant cells, both AKT and 4E-BP1 phosphorylation were modulated by the combination; however, decreasing AKT phosphorylation alone was not sufficient and decreasing 4E-BP1 phosphorylation was not required for apoptosis. Instead, cooperative mTOR complex 2 (mTORC2) and MEK inhibition resulting in downregulation of the pro-survival protein MCL-1 was found to be critical for combination-induced apoptosis. These results suggest that the clinical efficacy of combined MEK and mTOR kinase inhibition will be determined by tumor genotype, and that BRAF mutant malignancies will be particularly susceptible to this strategy.

Abstract 4835: Genetic determinants of mTOR inhibitor response in breast and endometrial cancer

Abstract Background: Breast and endometrial cancers frequently harbour genetic aberrations that result in increased activation of the PI3K-AKT-mTOR signalling pathway including amplification and overexpression of receptor tyrosine kinases, activating PIK3CA mutations, and inactivating PTEN mutations or loss of PTEN function. Given the pivotal role of the PI3K-AKT-mTOR pathway in cancer cell growth and survival, inhibitors of several of its components are currently being tested in clinical trials. The aim of this study was to define the genetic determinants of mTOR inhibitor response in breast and endometrial cancer cells. Methods: A panel of 31 breast and 26 endometrial cancer cell lines was treated with serial dilutions of ‘rapalogs’ (i.e. allosteric inhibitors of mTOR complex 1 (mTORC1)) and mTOR kinase inhibitors targeting mTORC1 and mTORC2. Cell viability was determined using CellTiter Blue. PTEN protein levels and mutations in PTEN transcripts were assessed in all cell lines using western blotting and Sanger sequencing, respectively. PIK3CA mutations were assessed using Sanger sequencing in the breast cancer cells, and hot-spot mutations in PIK3CA and 18 additional oncogenes using the Sequenom OncoCarta Panel v1.0 in the endometrial cancer cells. Results: Response of breast cancer cells to both the rapalog everolimus and the mTOR kinase inhibitor PP242 was determined by the presence of activating PIK3CA mutations but not PTEN loss of function. In addition, PP242 treatment induced a more efficient response in HER2-amplified breast cancer cell lines, irrespective of the PIK3CA mutation status. By contrast, endometrial cancer cell lines not only harbouring mutations in PIK3CA but also in PTEN were sensitive to treatment with the rapalog temsirolimus. The mTOR kinase inhibitor AZD8055 induced a strong reduction in cell viability in all endometrial cancer cell lines tested. Conclusions: Our results suggest that response to mTOR inhibitors in breast and endometrial cancer cells may be determined by the presence of alterations affecting PIK3CA and PTEN. Our data further indicate, however, that the epistatic interactions between different components of the PI3K-AKT-mTOR pathway are distinct in cancers of the breast and endometrium. 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 4835. doi:1538-7445.AM2012-4835