Bimiralisib. Phosphatidylinositol 3-kinase (PI3K)/mechanistic target of rapamycin (mTOR) inhibitor, Treatment of solid tumors, Treatment of lymphoma

Mammalian target of rapamycin (mTOR) is a cytoplasmic kinase that is a downstream component of the phosphatidylinositol 3-kinase (PI3K)/Akt kinase cascade. Together with its associated proteins, it can affect mRNA transcription, protein translation, protein degradation and changes in the actin cytoskeleton. Since mTOR has the ability to influence cell proliferation and apoptosis, it plays an important role in carcinogenesis. The existence of specific inhibitors of mTOR in the form of the antibiotic rapamycin, and its analogs, make the mTOR signaling pathway an attractive target for anti-cancer therapy. Rapamycin (sirolimus, Wyeth Pharmaceuticals, Philadelphia, PA) is a macrocyclic lactone that forms a complex with the intracellular immunophilin FKBP12 and inhibits mTOR. Rapamycin derivatives that are being evaluated in clinical trials include CCI-779 (temsirolimus), RAD001 (everolimus) and AP23573. Rapamycin inhibits T-cell proliferation and is thus used as an immunosuppressive agent. Temsirolimus (Wyeth Pharmaceuticals, Philadelphia, PA) has been evaluated in renal cell carcinoma (RCC), endometrial carcinoma, glioblastoma, breast cancer and mantle cell lymphoma. Everolimus (Novartis, East Hanover, NJ) is a macrolide derivative of rapamycin that has been evaluated in patients with imatinibrefractory gastrointestinal stromal tumors. AP23573 (ARIAD Pharmaceuticals, Cambridge, MA) is an intravenous (iv) mTOR inhibitor that has demonstrated antitumor activity against sarcomas. These examples illustrate the fact that mTOR inhibition is a viable therapeutic option in the treatment of solid tumors and further development of this new class of antineoplastic drugs should be pursued. Keywords: Solid Tumors, Mammalian Target of Rapamycin (mTOR), carcinogenesis, apoptosis, cell proliferation, antibiotic rapamycin, anti-cancer therapy, immunosuppressive agent

The mammalian target of rapamycin (mTOR) is a multifunctional protein involved in the regulation of cell growth, proliferation, and differentiation. The goal of this study was to determine the role of mTOR in type I collagen regulation. The pharmacological inhibitor of phosphatidylinositol (PI) 3-kinase, LY294002, significantly inhibited collagen type I protein and mRNA levels. The effects of LY294002 were more pronounced on the collagen alpha1(I) chain, which was inhibited at the transcriptional and mRNA stability levels versus collagen alpha2(I) chain, which was inhibited through a decrease in mRNA stability. In contrast, addition of the PI 3-kinase inhibitor, wortmannin, did not alter type I collagen steady-state mRNA levels. This observation and further experiments using an inactive LY294002 analogue suggested that collagen mRNA levels are inhibited independent of PI 3-kinase. Additional experiments have established that mTOR positively regulates collagen type I synthesis in human fibroblasts. These conclusions are based on results demonstrating that inhibition of mTOR activity using a specific inhibitor, rapamycin, reduced collagen mRNA levels. Furthermore, decreasing mTOR expression by about 50% by using small interfering RNA resulted in a significant decrease of collagen mRNA (75% COL1A1 decrease and 28% COL1A2 decrease) and protein levels. Thus, mTOR plays an essential role in regulating basal expression of collagen type I gene in dermal fibroblasts. Together, our data suggest that the classical PI 3-kinase pathway, which places mTOR downstream of PI 3-kinase, is not involved in mTOR-dependent regulation of type I collagen synthesis in dermal fibroblasts. Because collagen overproduction is a main feature of fibrosis, identification of mTOR as a critical mediator of its regulation may provide a suitable target for drug or gene therapy.

Delayed-onset psoriasiform eruption secondary to a phosphoinositide 3-kinase inhibitor: A case report and literature review

The phosphoinositide 3-kinase (PI3K) family of signalling enzymes play a key role in the transduction of signals from activated cell surface receptors controlling cell growth and proliferation, survival, metabolism, and migration. The intracellular signalling pathway from activated receptors to PI3K and its downstream targets v-akt murine thymoma viral oncogene homolog (Akt) and mechanistic target of rapamycin (mTOR) is very frequently deregulated by genetic and epigenetic mechanisms in human cancer, including leukaemia and lymphoma. In the past decade, an arsenal of small molecule inhibitors of key enzymes in this pathway has been developed and evaluated in pre-clinical studies and clinical trials in cancer patients. These include pharmacological inhibitors of Akt, mTOR, and PI3K, some of which are approved for the treatment of leukaemia and lymphoma. The PI3K family comprises eight different catalytic isoforms in humans, which have been subdivided into three classes. Class I PI3K isoforms have been extensively studied in the context of human cancer, and the isoforms p110α and p110δ are validated drug targets. The recent approval of a p110δ-specific PI3K inhibitor (idelalisib/Zydelig®) for the treatment of selected B cell malignancies represents the first success in developing these molecules into anti-cancer drugs. In addition to PI3K inhibitors, mTOR inhibitors are intensively studied in leukaemia and lymphoma, and temsirolimus (Torisel®) is approved for the treatment of a type of lymphoma. Based on these promising results it is hoped that additional novel PI3K pathway inhibitors will in the near future be further developed into new drugs for leukaemia and lymphoma.

Cell Signaling/Signal Transduction

Signaling through the mammalian target of rapamycin (mTOR) is hyperactivated in many human tumors, including hamartomas associated with tuberous sclerosis complex (TSC). Several small molecules such as LY294002 inhibit mTOR kinase activity, but they also inhibit phosphatidylinositol 3-kinase (PI3K) at similar concentrations. Compound 401 is a synthetic inhibitor of DNA-dependent protein kinase (DNA-PK) that also targets mTOR but not PI3K in vitro (Griffin, R. J., Fontana, G., Golding, B. T., Guiard, S., Hardcastle, I. R., Leahy, J. J., Martin, N., Richardson, C., Rigoreau, L., Stockley, M., and Smith, G. C. (2005) J. Med. Chem. 48, 569-585). We used 401 to test the cellular effect of mTOR inhibition without the complicating side effects on PI3K. Treatment of cells with 401 blocked the phosphorylation of sites modified by mTOR-Raptor and mTOR-Rictor complexes (ribosomal protein S6 kinase 1 Thr(389) and Akt Ser(473), respectively). By contrast, there was no direct inhibition of Akt Thr(308) phosphorylation, which is dependent on PI3K. Similar effects were also observed in cells that lack DNA-PK. The proliferation of TSC1-/- fibroblasts was inhibited in the presence of 401, but TSC1+/+ cells were resistant. In contrast to rapamycin, long-term treatment of TSC1-/- cells with 401 did not up-regulate phospho-Akt Ser(473). Because increased Akt activity promotes survival, this may explain why the level of apoptosis was increased in the presence of 401 but not rapamycin. These results suggest that mTOR kinase inhibitors might be more effective than rapamycins in controlling the growth of TSC hamartomas and other tumors that depend on elevated mTOR activity.

Background The discovery and development of inhibitors for the PI3K/AKT/mTOR signaling pathway is an attractive area of research due to its association with several oncogenic malignancies. This signaling pathway controls cellular growth as well as survival via regulation of widely divergent physiological processes, i.e. cell cycle progression, differentiation, transcription, translation and apoptosis. Constitutive activation of the PI3 kinase alpha and/or the downstream protein mTOR has been implicated in the progression of a large variety of solid tumors. Literature reports suggest the importance of developing combined and specific inhibitor of both PI3K and mTOR kinases. Described herein is the discovery of a novel small molecule, SPR965, a potent, and orally bioavailable inhibitor for class 1 PI3 Kinase and mTOR kinases with the potential for the clinical treatment of various solid tumors, specifically prostate, ovarian and colon cancers. The complete preclinical profile of SPR965 is presented in detail. Methods Drug candidates were evaluated in an in vitro enzyme assays to determine their inhibitory activity PI3 and mTOR C1/ C2 kinases. Promising candidates were then evaluated in proliferation assays using various human cancer cell lines (PC3 - prostate, SKOV3 - ovarian, HCT-116 - colon and A2780 - ovarian). The most promising leads were then evaluated against a panel of 456 kinases to determine the level of selectivity for our primary targets - PI3K and mTOR C1/C2. Compounds meeting the desired threshold of potency and selectivity were evaluated for their in vivo pharmacokinetic profile (iv/ po) in rodents followed by studies in mouse xenograft models (SKOV3 and HCT-116). Results SPR965 is a potent inhibitor of PI3K alpha and mTOR with an IC50 of 24 and 25 nM respectively. SPR965 is also a highly selective inhibitor of PI3 and mTOR C1/C2 kinases when evaluated in a screen against 456 kinases. Further studies demonstrated that SPR965 is a potent inhibitor of proliferation in a multiple cell lines and in several xenograft models. The proliferation inhibition activity (EC50) in A2780 is 17 nM, PC3 is 30 nM, SKOV3 is 74 nM, and HCT-116 is 163 nM. SPR965 is one of the most efficacious PI3/mTOR kinase inhibitor yet reported, with ED50 = 0.5 mg/Kg as determined in a SKOV3 xenograft mouse model and ED50 = 0.6 mg/ Kg in HCT-116 xenograft mouse model. This dose level is markedly lower than those reported for other reported inhibitors of this pathway. Pharmacokinetic studies in Sprague Dawley rats and in NUDE mice indicated that the oral bioavailability of SPR965was ∼100% and 75% respectively. Conclusion SPR965 is one of the most efficacious inhibitor of the PI3 and mTOR kinases when compared to the reported ED50s of other reported compounds. It also is one of the most selective and highly bioavailable inhibitor of the PI3 and mTOR kinases. Further studies are underway to support first-in-human trials with SPR965 where we hope to demonstrate its unique therapeutic benefits. Citation Format: Reena Arora, Bakul K. Dutta, Ravinder Goel, Frank P. Hollinger, Bilash Kulia, Dinesh Mahajan, Amal R. Mahapatra, Milind Sagar, Somdutta Sen, Amit Sharma, Sundeep Dugar. SPR965: an oral PI3K/ mTOR C1/C2 inhibitor for the treatment of solid tumors. [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 4515. doi:10.1158/1538-7445.AM2014-4515

Revision of immunosuppression in a solid organ transplant recipient leads to complete remission of metastatic undifferentiated carcinoma

Mammalian Target of Rapamycin (mTOR) Regulates Cellular Proliferation and Tumor Growth in Urothelial Carcinoma

Autophagy is an alternative cell death pathway that is induced by mammalian target of rapamycin (mTOR) inhibitors and up-regulated when apoptosis is defective. We investigated radiation-induced autophagy in the presence or absence of Bax/Bak with or without an mTOR inhibitor, Rad001. Two isogenic cell lines, wild type (WT) and Bak/Bak(-/-) mouse embryonic fibroblasts and tumor cell lines were used for this study. Irradiated Bak/Bak(-/-) cells had a decrease of Akt/mTOR signaling and a significant increase of pro-autophagic proteins ATG5-ATG12 COMPLEX and Beclin-1. These molecular events resulted in an up-regulation of autophagy. Bax/Bak(-/-) cells were defective in undergoing apoptosis but were more radiosensitive than the WT cells in autophagy. Both autophagy and sensitization of Bak/Bax(-/-) cells were further enhanced in the presence of Rad001. In contrast, inhibitors of autophagy rendered the Bak/Bax(-/-) cells radioresistant, whereas overexpression of ATG5 and Beclin-1 made the WT cells radiosensitive. When this novel concept of radiosensitization was tested in cancer models, small interfering RNAs against Bak/Bax also led to increased autophagy and sensitization of human breast and lung cancer cells to gamma radiation, which was further enhanced by Rad001. This is the first report to demonstrate that inhibition of pro-apoptotic proteins and induction of autophagy sensitizes cancer cells to therapy. Therapeutically targeting this novel pathway may yield significant benefits for cancer patients.

Insulin receptors are highly enriched at neuronal synapses, but whose function remains unclear. Here we present evidence that brief incubations of rat hippocampal slices with insulin resulted in an increased protein expression of dendritic scaffolding protein postsynaptic density-95 (PSD-95) in area CA1. This insulin-induced increase in the PSD-95 protein expression was inhibited by the tyrosine kinase inhibitor, AG1024, phosphatidylinositol 3-kinase (PI3K) inhibitors, LY294002 and wortmannin, translational inhibitors, anisomycin and rapamycin, but not by LY303511 (an inactive analogue of LY294002), and transcriptional inhibitor, actinomycin D, suggesting that insulin regulates the translation of PSD-95 by activating the receptor tyrosine kinase-PI3K-mammalian target of rapamycin (mTOR) signaling pathway. A similar insulin-induced increase in the PSD-95 protein expression was detected after stimulation of the synaptic fractions isolated from the hippocampal neurons. Furthermore, insulin treatment did not affect the PSD-95 mRNA levels. In agreement, insulin rapidly induced the phosphorylation of 3-phosphoinositide-dependent protein kinase-1 (PDK1), protein kinase B (Akt), and mTOR, effects that were prevented by the AG1024 and LY294002. We also show that insulin stimulated the phosphorylation of 4E-binding protein 1 (4E-BP1) and p70S6 kinase (p70S6K) in a mTOR-dependent manner. Finally, we demonstrate the constitutive expression of PSD-95 mRNA in the synaptic fractions isolated from hippocampal neurons. Taken together, these findings suggest that activation of the PI3K-Akt-mTOR signaling pathway is essential for the insulin-induced up-regulation of local PSD-95 protein synthesis in neuronal dendrites and indicate a new molecular mechanism that may contribute to the modulation of synaptic function by insulin in hippocampal area CA1.

Cellular mechanisms that regulate the replication of hepatitis C virus (HCV) RNA are poorly understood. p21-activated kinase 1 (PAK1) is a serine/threonine kinase that has been suggested to participate in antiviral signaling. We studied its role in the cellular control of HCV replication. Transfection of PAK1-specific small interfering RNA enhanced viral RNA and protein abundance in established replicon cell lines as well as cells infected with chimeric genotype 1a/2a HCV, despite reducing cellular proliferation, suggesting specific regulation of HCV replication. PAK1 knockdown did not reduce interferon regulatory factor 3-dependent gene expression, indicating that this regulation is independent of the retinoic acid-inducible gene I/interferon regulatory factor 3 pathway. On the other hand, LY294002 and rapamycin abolished PAK1 phosphorylation and enhanced HCV abundance, suggesting that the mammalian target of rapamycin (mTOR) is involved in PAK1 regulation of HCV. Small interfering RNA knockdown of the mTOR substrate p70 S6 kinase abrogated PAK1 phosphorylation and enhanced HCV RNA abundance, whereas overexpression of a constitutively active alternate substrate, eukaryotic translation initiation factor 4E-binding protein 1, increased cap-independent viral translation and viral RNA abundance without influencing PAK1 phosphorylation. Similar data indicated that mTOR is regulated by both phosphatidylinositol 3-kinase/Akt and ERK. Taken together, the data indicate that p70 S6 kinase activates PAK1 and contributes to phosphatidylinositol 3-kinase- and ERK-mediated regulation of HCV RNA replication.

The phosphoinositide 3-kinase (PI3K) family of signalling enzymes play a key role in the transduction of signals from activated cell surface receptors controlling cell growth and proliferation, survival, metabolism, and migration. The intracellular signalling pathway from activated receptors to PI3K and its downstream targets v-akt murine thymoma viral oncogene homolog (Akt) and mechanistic target of rapamycin (mTOR) is very frequently deregulated by genetic and epigenetic mechanisms in human cancer, including leukaemia and lymphoma. In the past decade, an arsenal of small molecule inhibitors of key enzymes in this pathway has been developed and evaluated in pre-clinical studies and clinical trials in cancer patients. These include pharmacological inhibitors of Akt, mTOR, and PI3K, some of which are approved for the treatment of leukaemia and lymphoma. The PI3K family comprises eight different catalytic isoforms in humans, which have been subdivided into three classes. Class I PI3K isoforms have been extensively studied in the context of human cancer, and the isoforms p110α and p110δ are validated drug targets. The recent approval of a p110δ-specific PI3K inhibitor (idelalisib/Zydelig®) for the treatment of selected B cell malignancies represents the first success in developing these molecules into anti-cancer drugs. In addition to PI3K inhibitors, mTOR inhibitors are intensively studied in leukaemia and lymphoma, and temsirolimus (Torisel®) is approved for the treatment of a type of lymphoma. Based on these promising results it is hoped that additional novel PI3K pathway inhibitors will in the near future be further developed into new drugs for leukaemia and lymphoma.

Chaperonin containing TCP-1 (CCT) is a large multisubunit complex that mediates protein folding in eukaryotic cells. CCT participates in the folding of newly synthesized polypeptides, including actin, tubulin, and several cell cycle regulators; therefore, CCT plays an important role in cytoskeletal organization and cell division. Here we identify the chaperonin CCT as a novel physiological substrate for p90 ribosomal S6 kinase (RSK) and p70 ribosomal S6 kinase (S6K). RSK phosphorylates the beta subunit of CCT in response to tumor promoters or growth factors that activate the Ras-mitogen-activated protein kinase (MAPK) pathway. CCTbeta Ser-260 was identified as the RSK site by mass spectrometry and confirmed by site-directed mutagenesis. RSK-dependent Ser-260 phosphorylation was sensitive to the MEK inhibitor UO126 and the RSK inhibitor BID-1870. Insulin weakly activates RSK but strongly activates the phosphoinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) pathway and utilizes S6K to regulate CCTbeta phosphorylation. Thus, the Ras-MAPK and PI3K-mTOR pathways converge on CCTbeta Ser-260 phosphorylation in response to multiple agonists in various mammalian cells. We also show that RNA interference-mediated knockdown of endogenous CCTbeta causes impaired cell proliferation that can be rescued with ectopically expressed murine CCTbeta wild-type or phosphomimetic mutant S260D, but not the phosphorylation-deficient mutant S260A. Although the molecular mechanism of CCTbeta regulation remains unclear, our findings demonstrate a link between oncogene and growth factor signaling and chaperonin CCT-mediated cellular activities.

Activation of Mammalian Target of Rapamycin Signaling Promotes Cell Cycle Progression and Protects Cells from Apoptosis in Mantle Cell Lymphoma