mTOR Kinase Inhibitor Research Articles

Abstract IA17: Strategies to target the mTORC1/eIF4F axis in B-cell leukemia and lymphoma

Abstract mTORC1 signaling is elevated in most lymphoid malignancies and frequently is associated with poor prognosis. Targeting mTORC1 with rapalogs or mTOR kinase inhibitors (TOR-KIs) are two strategies that both have limitations. Rapalogs are weak inducers of apoptosis, mainly due to incomplete mTORC1 inhibition, resulting in reduced phosphorylation of some substrates such as S6K1, but not 4E-BP1. TOR-KIs inhibit mTORC1 more completely, but also inhibit mTORC2, which likely contributes to dose-limiting toxicities in TOR-KI clinical trials. We have evaluated alternative strategies to target mTORC1 and downstream survival pathways in B-cell leukemia and lymphoma. In one approach, we have used novel bi-steric inhibitors of mTORC1, generated using our RevBlocksTM modular synthesis platform. These inhibitors engage both the FRB- and ATP-binding sites of mTOR and exhibit potent and selective inhibition of mTORC1. We found that RM-001, a proprietary bi-steric compound ~13-fold-selective for mTORC1 over mTORC2 in vitro, enhances the efficacy of the BCR-ABL1 inhibitor dasatinib in Philadelphia chromosome-positive pre-B cell acute lymphoblastic leukemia (Ph+ B-ALL) cell lines, causing profound and sustained phosphorylation inhibition of 4E-BP1 and S6, cell growth inhibition, and cell cycle arrest. Once-weekly dosing with RM-001 (10 mg/kg, IP) in a Ph+ B-ALL mouse model was well tolerated and effectively reduced leukemic burden as a single agent. A corresponding inhibition of phosphorylation of 4E-BP1 and S6 was observed in bone marrow. A key downstream effector pathway of mTORC1 is formation of the eIF4F translation initiation complex, which consists of eIF4E, eIF4G, and eIF4A. To assess targeting eIF4F as an alternative approach, we evaluated the function of eIF4F in both tumors and normal cells of the B lymphocyte lineage. In both B-ALL and diffuse large B-cell lymphoma (DLBCL) cells, disruption of eIF4F with a constitutively active 4E-BP1 mutant phenocopied the effect of TOR-KIs and bi-steric inhibitors. Notably, reduced gene dosage of Eif4e (eIF4E+/-) impaired the initiation and maintenance of B-cell transformation without affecting normal B-cell development and function. Most existing chemical inhibitors of eIF4F target either the cap-binding protein eIF4E or the RNA helicase eIF4A. We tested a novel eIF4G-binding molecule, SBI-756, shown previously to overcome resistance to BRAF inhibitors in melanoma models. SBI-756 (250 – 500 nM) disrupted eIF4F formation and caused marked cytotoxic effects in B-ALL, DLBCL, and normal B lymphocytes, while sparing T cells and NK cells. SBI-756 also synergized with targeted agents including dasatinib in Ph+ B-ALL and venetoclax in DLBCL. The combination of SBI-756 with venetoclax was more effective than single agents in a DLBCL xenograft model. These findings establish proof of concept for molecules that selectively bind to the scaffolding protein eIF4G. Collectively, these data highlight distinct strategies to target the mTORC1/eIF4F axis in B-cell leukemia and lymphoma. Citation Format: Lee-or Herzog, Bianca J. Lee, Thanh-Trang Vo, Honyin Chiu, Sharmila Mallya, Amos Fung, Mallika Singh, James Aggen, Nidhi Tibrewal, Jacqueline A.M. Smith, David Wildes, Ze'ev Ronai, Davide Ruggero, David A. Fruman. Strategies to target the mTORC1/eIF4F axis in B-cell leukemia and lymphoma [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr IA17.

Characterization of SPK 98, a Torin2 analog, as ATR and mTOR dual kinase inhibitor

A series of Torin2, a second-generation ATP-competitive inhibitor, analogues were biologically characterized to identify their potential for ATR and mTOR kinase inhibition. Compound SPK 98 was observed to inhibit ATR/mTOR kinase selectively over ATM kinase in HCT 116 cell line. In addition to that, SPK 98 on 30 min incubation with human, mice and rat liver microsomes showed improved properties with an increased half-life (a maximum T ½ of 157 min) and internal clearance in mouse as compared to Torin2. Further, SPK 98 was also noticed to indulge in inducing premature chromatin condensation as a result of ATR/mTOR kinase inhibition at 50 nM. In a nutshell, our work presents the identification and characterization of SPK 98, a small molecule inhibitor, which exhibits improved specific inhibition for ATR at a lower concentration than Torin2.

Rictor promotes tumor progression of rapamycin-insensitive triple-negative breast cancer cells

Triple-negative breast cancer (TNBC), characterized by decreased expression of hormone receptors and human epidermal growth factor type 2 receptor, has poor prognosis and lacks effective therapeutics. Recently, the mTOR inhibitor rapamycin and its analogs have attracted growing interests and evaluated as therapeutic agents against TNBC, in which the PI3K/AKT/mTOR pathway is often activated. However, some TNBCs are less sensitive to these drugs. In this study, we found that the sensitivity of TNBC cells to rapamycin was highly dependent on the expression level of rapamycin-insensitive companion of mTOR (Rictor), a key component of the mTOR complex 2. Repression of the Rictor expression strongly suppressed the growth of rapamycin-insensitive tumor cells. Furthermore, we showed that the suppression of Rictor expression was also effective in rapamycin-insensitive cells that had acquired resistance to mTOR kinase inhibitors. These findings indicate that Rictor can be a predictive marker for the use of rapamycin analogs in TNBC and highlight the need to develop therapeutics targeting Rictor in the treatment of TNBC.

Abstract 657: Inhibition of mTORC1 by dihydroartemisinin

Abstract mTOR functions as two complexes, mTORC1 and mTORC2, and is a central controller for cell growth and metabolism. Both mTORC1 and mTORC2 are activated in numerous cancers, and have become the focus of anticancer drug development. Rapalogs and mTOR kinase inhibitors (TORKIs) have been developed. However, the anticancer efficacy of rapalogs is clinically weak or mild to most cancers, and the adverse effects of rapalogs and TORKIs are greatly concerned. So, it is imperative to discover and develop new generation of mTOR inhibitors. Dihydroartemisinin (DHA) is an anti-malarial agent showing good safety in both children and adults. Recently, we have shown that DHA, at pharmacological concentrations, inhibits proliferation and induces apoptosis in cells derived from various cancer types but not in normal or immortalized cell lines, implying that DHA is potentially a selective anticancer agent and may be repurposed for cancer therapy. Of interest, DHA potently suppresses mTORC1-mediated phosphorylation of S6K1 and 4E-BP1, but does not obviously affect mTORC2-mediated phosphorylation of Akt, which is often activated by classic mTOR inhibitors (rapalogs) via a feedback mechanism. The results suggest that DHA is a novel mTORC1 inhibitor. This study was set to determine how DHA inhibits mTORC1. Here we found that DHA did not bind to any mTORC1 components (mTOR, mLST8 or raptor) directly, as determined by in vivo [3H]-DHA radiolabeling. Besides, our molecular docking revealed that DHA did not have the ability to bind FKBP12 or mTOR kinase domain, in contrast to the positive controls rapamycin (binding FKBP12) and INK128 (binding mTOR kinase domain). These data suggest that DHA inhibits mTORC1 via indirect mechanism(s). Further research revealed that DHA altered neither the phosphorylation of IGF-1R/PI3K/PTEN and ERK, nor the expression of REDD1, but induced phosphorylation of AMPKα (T172). Interestingly, DHA-induced mTORC1 inhibition was profoundly attenuated by inhibition with Compound C (AMPK inhibitor) or ectopic expression of dominant negative AMPKα2 (AMPKα2-DN), indicating that activation of AMPK links to inhibition of mTORC1. Collectively, the results support that DHA inhibits mTORC1 by activating AMPK. (Supported by the Feist-Weiller Cancer Center, LSU Health Sciences Center, Shreveport, LA, USA.) Citation Format: Wang Liu, Jun Luo, Yoshinobu Odaka, Shile Huang. Inhibition of mTORC1 by dihydroartemisinin [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 657.

Abstract 1671: Deregulation of mTOR signaling contributes to chemoradiation resistance in lung squamous cell carcinoma

Abstract Non-small cell lung carcinoma (NSCLC) represents ~ 85% of lung cancer, and ~1/3 of these patients present with locally-advanced, unresectable/medically-inoperable disease. Patients who present with locally-advanced NSCLC are commonly treated with definitive chemoradiation. Even though an initial response to concurrent chemoradiation is commonly observed, disease relapse in the radiation fields occurs in 1/3 of cases, likely driven by therapy-resistant tumor cells. However, the underlying molecular mechanisms of chemoradiation resistance of NSCLC remains largely unknown. We herein report that upregulation of mTOR signaling plays an important role in the chemoradiation resistance of NSCLC. Patient-derived xenograft (PDX) models of lung squamous cell carcinoma (LUSC) were established in the flanks of NOD-SCID gamma (NSG) mice. Chemoradiation resistant tumor models were developed by treating the tumors with a regimen of 2-4 weeks of chemoradiation (20, 30 or 40 Gy at 2 Gy per day, concurrent with cisplatin). Recurrent tumor models were collected and subjected to functional proteomics using reverse phase protein analysis (RPPA). Several LUSC tumors recurred in the 20 Gy+cisplatin, and 40 Gy+cisplatin groups. RPPA analysis of the recurrent and parental (untreated) PDX tumors showed that there were enhanced mTOR signaling in the chemoradiation resistant 20 Gy+cisplatin and 40 Gy+cisplatin tumors compared to that in the control tumors. Of note, phospho-AKT-S473, phospho-AKT-S308, phospho-S6-S235/6 and phospho-GSK3β-S9/21 were the highest upregulated signaling and were validated by immunoblotting. Consistently, pharmacological inhibition of mTOR kinase by Torin2 significantly sensitized LUSC cell lines to ionizing radiation. Our results demonstrate that utilization of PDX models is a useful method to elucidate the molecular mechanisms of chemoradiation resistance of NSCLC and deregulation of mTOR signaling contributes to the chemoradiation resistance of NSCLC. Our findings suggest that clonal selection of subpopulations with high mTOR signaling in heterogeneous NSCLC tumors might contribute to the relapse of NSCLC after chemoradiation and that mTOR kinase inhibitors are promising agents for the upfront treatment of NSCLC patients in combination with chemoradiation. Citation Format: Changxian Shen, Duan-Liang Shyu, Min Xu, Linlin Yang, Wenrui Duan, Terence M. Williams. Deregulation of mTOR signaling contributes to chemoradiation resistance in lung squamous cell carcinoma [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 1671.

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.

Abstract 665: Discovery and preclinical characterization of PQR626: A potent, orally available, and brain-penetrant mTOR inhibitor for the treatment of tuberous sclerosis complex

Abstract Mechanistic target of rapamycin (mTOR), a key player in cell proliferation, growth and survival, is overactivated in tumors and neurological disorders.[1] Rapalogs have recently been explored to alleviate epileptic seizures in Tuberous Sclerosis Complex (TSC).[2] Herein, we combined pharmacophore features of PQR620[3], the first-in-class brain penetrant ATP-competitive mTOR kinase inhibitor showing efficacy in a TSC mouse model, and PQR617[4], a potent, highly selective mTOR inhibitor. An extensive chemical exploration of the morpholine ring led to the discovery of PQR626, a highly potent, selective, brain penetrant inhibitor of mTORC1/2 kinase. In male Sprague Dawley rats and female C57BL/6J mice, PQR626 displayed an excellent brain penetration compared to everolimus, which possessed a limited ability to cross the blood-brain-barrier (brain/plasma levels ~ 1.4:1 vs 1:92). An additional pharmacokinetic comparative study with everolimus and AZD2014 confirmed the superiority of PQR626 [brain/plasma levels: ~ 1:25 (AZD2014), ~ 1:61 (everolimus), ~ 1.8:1 (PQR626)]. PQR626 showed very good tolerability in mice (MTD 100-150 mg/kg). Efficacy studies, using mice with conditional inactivation of the Tsc1 gene primarily in glia (Tsc1GFAPCKO mice), were performed in a dose-range finding study. PQR626 (50 mg/kg, BID - twice a day) showed a significant effect on survival and significantly prevented/decreased mortality as compared to the vehicle. On the basis of its favorable pharmacological parameters, excellent brain penetration, safety profile and efficacy in Tsc1GFAPCKO mice, PQR626 qualifies as a novel mTOR inhibitor with potential application in the treatment of epilepsy and neurological disorders. [1] Wymann M. P. and Schneiter R. Nat. Rev. Mol. Cell Biol. 2008, 9, 162-176. [2] Krueger D. A. et. al. Neurology 2016, 87, 2408-2415. [3] Rageot D. et. al. J Med Chem. 2018, 61 (22), 10084-10105. [4] Borsari C. et. al. J Med Chem. 2019, 62 (18), 8609-8630. Citation Format: Chiara Borsari, Erhan Keles, Denise Rageot, Anna Melone, Thomas Bohnacker, Lucinda Kate Batchelor, Martina De Pascale, Paul Hebeisen, Petra Hillmann, Doriano Fabbro, Matthias Wymann. Discovery and preclinical characterization of PQR626: A potent, orally available, and brain-penetrant mTOR inhibitor for the treatment of tuberous sclerosis complex [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 665.

Treatment of ErbB2 breast cancer by mitochondrial targeting

BackgroundErbB2 breast cancer still remains an unmet need due to primary and/or acquired resistance to current treatment strategies. MEDICA compounds consist of synthetic long-chain α,ω-dicarboxylic acids previously reported to suppress breast cancer in PyMT transgenic mice.MethodsMEDICA efficacy and mode of action in the ErbB2 context was studied in ErbB2 transgenic mice and human breast cancer cells.ResultsMEDICA treatment is shown here to suppress ErbB2 breast tumors and lung metastasis in ErbB2/neu MMTV transgenic mice, to suppress ErbB2/neu xenografts in nod/scid mice, and to suppress survival of AU565 and BT474 human ErbB2 breast cancer cells. Suppression of ErbB2 breast tumors by MEDICA is due to lipid raft disruption with loss of ErbB family members, including EGFR, ErbB2, and ErbB3. In addition, MEDICA inhibits mTORC1 activity, independently of abrogating the ErbB receptors and their signaling cascades. The double hit of MEDICA in abrogating ErbB and mTORC1 is partly accounted for by targeting mitochondria complex I.ConclusionsMitochondrial targeting by MEDICA suppresses ErbB2 breast tumors and metastasis due to lipid raft disruption and inhibition of mTORC1 activity. Inhibition of mTORC1 activity by MEDICA avoids the resistance acquired by canonical mTORC1 inhibitors like rapalogs or mTOR kinase inhibitors.

Role of mTOR As an Essential Kinase in SCLC.

SCLC represents 15% of all lung cancer diagnoses in the United States and has a particularly poor prognosis. We hypothesized that kinases regulating SCLC survival pathways represent therapeutically targetable vulnerabilities whose inhibition may improve SCLC outcome. A short-hairpin RNA (shRNA) library targeting all human kinases was introduced in seven chemonaive patient-derived xenografts (PDX) and the cells were cultured invitro and invivo. On harvest, lost or depleted shRNAs were considered as regulating-cell survival pathways and deemed essential kinases. Unsupervised hierarchical cluster analysis of recovered shRNAs separated the PDXs into two clusters, suggesting kinase-based heterogeneity among the SCLC PDXs. A total of 23 kinases were identified as essential in two or more PDXs, with mechanistic Target of Rapamycin (mTOR) a candidate essential kinase in four. mTOR phosphorylation status correlated with PDX sensitivity to mTOR kinase inhibition, and mTOR inhibition sensitized the PDX to cisplatin and etoposide. In the PDX in which mTOR was defined as essential, mTOR inhibition caused a 43% decrease in tumor volume at 21 days (p < 0.01). Combining mTOR inhibition with cisplatin and etoposide decreased PDX tumor volume 96% compared with cisplatin and etoposide alone at 70 days (p < 0.002). Chemoresistance did not develop with the combination of mTOR inhibition and cisplatin and etoposide in mTOR-essential PDX over 105 days. The prevalence of phospho-mTOR-Ser-2448 in a tissue microarray of chemonaive SCLC was 27%, thus, identifying an important SCLC subtype that might benefit from the addition of mTOR inhibition to standard chemotherapy. These studies reveal that kinases can define SCLC subgroups, can identify therapeutic vulnerabilities, and can potentially be used to optimize therapeutic approaches. Significance We used functional genomics to identify kinases regulating SCLC survival. mTOR was identified as essential in a subset of PDXs. mTOR inhibition decreased PDX growth, sensitized PDX to cisplatin and etoposide, and prevented chemoresistance.

Advances in mammalian target of rapamycin kinase inhibitors: application to devices used in the treatment of coronary artery disease.

Mammalian target of rapamycin (mTOR) inhibitors have been applied to vascular coronary devices to avoid neointimal growth and have become the predominant pharmacological agents used to prevent restenosis. mTOR inhibitors can affect not only proliferating vascular smooth muscle cells but also endothelial cells and therefore can result in delayed healing of the vessel including endothelialization. Emerging evidence suggests accelerated atherosclerosis due to the downstream negative effects on endothelial barrier functional recovery. The development of neoatherosclerosis within the neointima of drug-eluting stents can result in late thrombotic events. This type of problematic healing response may open the way for specific mTOR kinase inhibitors, such as ATP-competitive mTOR inhibitors. These inhibitors demonstrate a better healing profile than traditional limus-based drug-eluting stent and their clinical efficacy remains unknown.

Immunotherapy in Bladder Cancer: Current Methods and Future Perspectives.

Bladder cancer is one of the most significant genitourinary cancer, causing high morbidity and mortality in a great number of patients. Over the years, various treatment methods for this type of cancer have been developed. The most common is the highly efficient method using Bacillus Calmette-Guerin, giving a successful effect in a high percentage of patients. However, due to the genetic instability of bladder cancer, together with individual needs of patients, the search for different therapy methods is ongoing. Immune checkpoints are cell surface molecules influencing the immune response and decreasing the strength of the immune response. Among those checkpoints, the PD-1 (programmed cell death protein-1)/PD-L1 (programmed cell death protein ligand 1) inhibitors aim at blocking those molecules, which results in T cell activation, and in bladder cancer the use of Atezolizumab, Avelumab, Durvalumab, Nivolumab, and Pembrolizumab has been described. The inhibition of another pivotal immune checkpoint, CTLA-4 (cytotoxic T cell antigen), may result in the mobilization of the immune system against bladder cancer and, among anti-CTLA-4 antibodies, the use of Ipilimumab and Tremelimumab has been discussed. Moreover, several different approaches to successful bladder cancer treatment exists, such as the use of ganciclovir and mTOR (mammalian target of rapamycin) kinase inhibitors, IL-12 (interleukin-12) and COX-2 (cyclooxygenase-2). The use of gene therapies and the disruption of different signaling pathways are currently being investigated. Research suggests that the combination of several methods increases treatment efficiency and the positive outcome in individual.

Targeting mTOR for fighting diseases: A revisited review of mTOR inhibitors

mTOR (mammalian target of rapamycin), which is a serine/threonine kinase, has been well-established as being closely correlated with the occurrence of various human diseases, such as tumors and neurodegenerative diseases. Inhibition of the mTOR signaling pathways may effectively block the abnormal signal transduction of various growth factors and thereby block the occurrence and development of diseases. Of note, first-generation mTOR inhibitors are mainly reported to be rapamycin and its derivatives and second-generation mTOR inhibitors that consist of several ATP-competitive kinase inhibitors. Interestingly, the third-generation mTOR inhibitor, RapaLink-1, mediates rapamycin and mTOR kinase inhibitors via the same molecule and shows much higher efficiency. In addition, there are many mTOR inhibitors that have potential therapeutic effects for tumors and other types of diseases. Thus, we focus on summarizing the basic structures of mTOR and its complexes, some key upstream and downstream signaling pathways, and the structures and characteristics of the three generations of mTOR inhibitors in disease. Together, these findings may provide a better understanding of mTOR-regulated mechanisms and their inhibitors for fighting human diseases in the near future.

Potential new therapy of Rapalink-1, a new generation mammalian target of rapamycin inhibitor, against sunitinib-resistant renal cell carcinoma.

Sunitinib, a multitargeted receptor tyrosine kinase inhibitor including vascular endothelial growth factor, has been widely used as a first‐line treatment against metastatic renal cell carcinoma (mRCC). However, mRCC often acquires resistance to sunitinib, rendering it difficult to treat with this agent. Recently, Rapalink‐1, a drug that links rapamycin and the mTOR kinase inhibitor MLN0128, has been developed with excellent therapeutic effects against breast cancer cells carrying mTOR resistance mutations. The aim of the present study was to evaluate the in vitro and in vivo therapeutic efficacy of Rapalink‐1 against renal cell carcinoma (RCC) compared to temsirolimus, which is commonly used as a small molecule inhibitor of mTOR and is a derivative of rapamycin. In comparison with temsirolimus, Rapalink‐1 showed significantly greater effects against proliferation, migration, invasion and cFolony formation in sunitinib‐naïve RCC cells. Inhibition was achieved through suppression of the phosphorylation of substrates in the mTOR signal pathway, such as p70S6K, eukaryotic translation initiation factor 4E‐binding protein 1 (4EBP1) and AKT. In addition, Rapalink‐1 had greater tumor suppressive effects than temsirolimus against the sunitinib‐resistant 786‐o cell line (SU‐R 786‐o), which we had previously established, as well as 3 additional SU‐R cell lines established here. RNA sequencing showed that Rapalink‐1 suppressed not only the mTOR signaling pathway but also a part of the MAPK signaling pathway, the ErbB signaling pathway and ABC transporters that were associated with resistance to several drugs. Our study suggests the possibility of a new treatment option for patients with RCC that is either sunitinib‐sensitive or sunitinib‐resistant.

Rapamycin restrains platelet procoagulant responses via FKBP-mediated protection of mitochondrial integrity

Background and PurposeRapamycin is a potent immunosuppressant and anti-proliferative agent used clinically to prevent organ transplant rejection and for coating coronary stents to counteract restenosis. Rapamycin complexes with the immunophilin FKBP12, which subsequently binds and inhibits mTORC1. Despite several reports demonstrating that rapamycin affects platelet-mediated responses, the underlying mechanism of how it alters platelet function is poorly characterised. This study aimed to elucidate the effect of rapamycin on platelet procoagulant responses. Experimental ApproachThe effect of rapamycin on platelet activation and signalling was investigated alongside the catalytic mTOR inhibitors KU0063794 and WYE-687, and the FKBP12-binding macrolide FK506. Key ResultsRapamycin affects platelet procoagulant responses by reducing externalisation of the procoagulant phospholipid phosphatidylserine, formation of balloon-like structures and local generation of thrombin. Catalytic mTOR kinase inhibitors did not alter platelet procoagulant processes, despite having a similar effect as rapamycin on Ca2+ signalling, demonstrating that the effect of rapamycin on procoagulant responses is independent of mTORC1 inhibition and not linked to a reduction in Ca2+ signalling. FK506, which also forms a complex with FKBP12 but does not target mTOR, reduced platelet procoagulant responses to a similar extent as rapamycin. Both rapamycin and FK506 prevented the loss of mitochondria integrity induced by platelet activation, one of the central regulatory events leading to PS externalisation. Conclusions and ImplicationsRapamycin suppresses platelet procoagulant responses by protecting mitochondrial integrity in a manner independent of mTORC1 inhibition. Rapamycin and other drugs targeting FKBP immunophilins could aid the development of novel complementary anti-platelet therapies.

Combination therapy with mTOR kinase inhibitor and dasatinib as a novel therapeutic strategy for vestibular schwannoma

Neurofibromatosis type 2 (NF2) is an inherited disorder characterized by bilateral vestibular schwannomas (VS) that arise from neoplastic Schwann cells (SCs). NF2-associated VSs are often accompanied by meningioma (MN), and the majority of NF2 patients show loss of the NF2 tumor suppressor. mTORC1 and mTORC2-specific serum/glucocorticoid-regulated kinase 1 (SGK1) are constitutively activated in MN with loss of NF2. In a recent high-throughput kinome screen in NF2-null human arachnoidal and meningioma cells, we showed activation of EPH RTKs, c-KIT, and SFK members independent of mTORC1/2 activation. Subsequently, we demonstrated in vitro and in vivo efficacy of combination therapy with the dual mTORC1/2 inhibitor AZD2014 and the multi-kinase inhibitor dasatinib. For these reasons, we investigated activated mTORC1/2 and EPH receptor-mediated signaling in sporadic and NF2-associated VS. Using primary human VS cells and a mouse allograft model of schwannoma, we evaluated the dual mTORC1/2 inhibitor AZD2014 and the tyrosine kinase inhibitor dasatinib as monotherapies and in combination. Escalating dose-response experiments on primary VS cells grown from 15 human tumors show that combination therapy with AZD2014 and dasatinib is more effective at reducing metabolic activity than either drug alone and exhibits a therapeutic effect at a physiologically reasonable concentration (~0.1 µM). In vivo, while AZD2014 and dasatinib each inhibit tumor growth alone, the effect of combination therapy exceeds that of either drug. Co-targeting the mTOR and EPH receptor pathways with these or similar compounds may constitute a novel therapeutic strategy for VS, a condition for which there is no FDA-approved pharmacotherapy.

Cooperative Blockade of PKCα and JAK2 Drives Apoptosis in Glioblastoma.

The mTOR signaling is dysregulated prominently in human cancers including glioblastoma, suggesting mTOR as a robust target for therapy. Inhibitors of mTOR have had limited success clinically, however, in part because their mechanism of action is cytostatic rather than cytotoxic. Here, we tested three distinct mTOR kinase inhibitors (TORKi) PP242, KU-0063794, and sapanisertib against glioblastoma cells. All agents similarly decreased proliferation of glioblastoma cells, whereas PP242 uniquely induced apoptosis. Apoptosis induced by PP242 resulted from off-target cooperative inhibition of JAK2 and protein kinase C alpha (PKCα). Induction of apoptosis was also decreased by additional on-target inhibition of mTOR, due to induction of autophagy. As EGFR inhibitors can block PKCα, EGFR inhibitors erlotinib and osimertinib were tested separately in combination with the JAK2 inhibitor AZD1480. Combination therapy induced apoptosis of glioblastoma tumors in both flank and in patient-derived orthotopic xenograft models, providing a preclinical rationale to test analogous combinations in patients. SIGNIFICANCE: These findings identify PKCα and JAK2 as targets that drive apoptosis in glioblastoma, potentially representing a clinically translatable approach for glioblastoma.

Transcriptome profiling of tolerogenic dendritic cells conditioned with dual mTOR kinase inhibitor, AZD8055

Dendritic cells (DCs) can initiate and regulate adaptive immunity depending on their maturation status. Many pharmacological and genetic means have been used in the generation of immature/tolerogenic DCs. However, the key factors controlling DCs tolerogenicity remain obscure. In this work, we demonstrated that AZD8055, an ATP-competitive inhibitor of mammalian target of rapamycin (mTOR), could also lead to a tolerogenic DC phenotype from several lines of evidence, such as suppression of T cell proliferation, promoting the generation of Tregs, and inducing allogeneic T cell apoptosis. Further studies using RNA-seq method identified 430, 1172 and 1436 differentially expressed genes (DEGs) between AZD-DCs vs. Control-DCs, LPS-DCs vs. Control-DCs, and AZD-DCs vs. LPS-DCs, respectively. The 5 most differentially expressed transcripts identified by RNA-seq expression profiles were validated by quantitative RT-PCR assays. NF-κB, p38MAPK, the ribosome and PPAR signaling pathways may be involved in the induction of tolerogenic DCs by AZD8055. Functional annotation showed some genes like MGL2, Cadherin-1, 4-1BB, RhoB and Pdpn, were quite different between AZD-DCs and Control-DCs/LPS-DCs, which might be related to the tolerogenic properties of AZD-DCs. Our work provided the potential underlying molecular mechanisms involved in the generation of tolerogenic DCs. Further functional characterization of individual target gene in DC tolerogenicity will help to develop novel therapeutic modalities in circumstances like transplant tolerance induction and autoimmunity.

A conjugated mTOR/MEK bifunctional inhibitor as potential polypharmacological anticancer agent: the prototype compound discovery.

mTOR/MEK bifunctional inhibitors have the potential to surmount the drug resistance aroused from cross talk between PI3K/Akt/mTOR (PAM) and Ras/MEK/ERK pathways. Herein, we report the discovery of a conjugated dual-targeted molecule, compound 13, as the prototype mTOR/MEK bifunctional inhibitor. It exhibited moderately high inhibitory activity against mTOR and MEK1 with IC50 values of 0.19 μM and 0.98 μM, respectively. In particular, it displayed attractive antiproliferative activity against both A549 (GI50 = 4.66 μM) and HCT116 (GI50 = 5.47 μM) cell lines. To our knowledge, it has been the first example of a conjugated mTOR/MEK bifunctional inhibitor. In addition, from this proof-of-principle study, it has become evident that the single-agent dual inhibition of mTOR and MEK can be fulfilled via covalently attaching mTOR kinase inhibitor to an allosteric MEK inhibitor.

Exploring Molecular Descriptors and Fingerprints to Predict mTOR Kinase Inhibitors using Machine Learning Techniques.

Mammalian Target of Rapamycin (mTOR) is a Ser/Thr protein kinase, and its role is integral to the autophagy pathway in cancer. Targeting mTOR for therapeutic interventions in cancer through autophagy pathway is challenging due to the dual roles of autophagy in tumor progression. The architecture of mTOR reveals two complexes - mTORC1 and mTORC2, each having multiple protein subunits. mTOR kinase inhibitors target the structurally and functionally similar catalytic subunits of both mTORC1 and mTORC2. In this paper, we have explored two different categories of molecular features - descriptors and fingerprints for developing predictive models using machine learning techniques. Random Forest variable importance measures and autoencoders are used to identify molecular descriptors and fingerprints, respectively. We have built various predictive models using identified features and their combination for predicting mTOR kinase inhibitors. Finally, the best model based on the Mathew correlation co-efficient value over the validation dataset is selected for screening kinase SARfari bioactivity dataset. In this study, we have identified twenty best performing descriptors for predicting mTOR kinase inhibitors. To the best of our knowledge, it is the first study on integrating traditional machine learning and deep learning-based approaches for feature extraction to predict mTOR kinase inhibitors.

The Dephosphorylation of p70S6 (Thr389) Kinase as a Marker of L-Glutamate-Induced Excitotoxicity Related to Diabetes Disturbances-an Unconventional In Vitro Model.

Excitotoxicity is a modern clinical condition included in the pathogenesis of Alzheimer's disease. It is connected with diabetic disturbance, and it is still being analyzed in the context of the participation of the PI3K/mTOR pathway. A very important protein belonging to this pathway is p70S6K, whose activation promotes the pathogenesis of type 2 diabetes by the induction of insulin resistance. The study model was based on a PC12 cell line, derived from the pheochromocytoma of a rat adrenal medulla, cultured in RPMI 1640. The three reagents were used in different concentrations to create the model of excitotoxicity related to diabetes disturbances: L-glutamate (2.5mM; 10mM), glucose (150mM; 200mM), and insulin (0.093mM; 0.371mM). The aim of our study was to examine and evaluate the levels of phosphorylation of proteins involved in signal transduction controlled by MAPK, PI3K/Akt, and mTOR signaling pathways in L-glutamate-induced excitotoxicity with comorbid hyperglycemia and hyperinsulinemia imitating diabetic disturbances in in vitro conditions on PC12 cells. The results we obtained demonstrated the increased phosphorylation of p70S6K in Thr389 residue in almost all combinations of reagents, except for those including the highest concentration of L-glutamate, in which dephosphorylation was confirmed. This confirms the inhibition of mTOR kinase and suggests that p70S6K (Thr389) plays a functional role in the regulation of the signaling pathway in excitotoxicity related to diabetic disturbances.