No abstract

AKT1/PKB is a serine/threonine protein kinase that regulates biological processes such as proliferation, apoptosis and growth in a variety of cell types. To assess the oncogenic capability of an activated form of AKT in vivo we have generated several transgenic mouse lines that overexpress in the mammary epithelium the murine Akt1 gene modified with a myristoylation signal, which renders active this protein by localizing it to the plasma membrane. We demonstrate that expression of myristoylated AKT in the mammary glands increases the susceptibility of these mice to the induction of mammary tumors of epithelial origin by the carcinogen 9,10-dimethyl-1,2 benzanthracene (DMBA). We have found that while carcinogen-treated wild-type mice show mostly mammary tumors of sarcomatous origin, AKT transgenic mice treated with DMBA developed mainly adenocarcinoma or adenosquamous tumors, all of them displaying activated AKT. We analyzed other possible molecular alterations cooperating with AKT and found that neither Ras nor beta-catenin/Wnt pathways seemed altered nor p53 mutated. We have found that 100% of mammary DMBA-induced tumors and benign lesions in myrAKT mice are estrogen receptor (ERalpha)-positive and are more frequent than in wild-type littermates. These data show that AKT activation cooperates with deregulation of the estrogen receptor in the DMBA-induced mammary tumorigenesis model and recapitulate two characteristics of some human breast tumors. Thus, our model might provide a preclinical relevant model system to study the role of AKT and ERalpha in breast tumorigenesis and the response of mammary gland tumors to chemotherapeutics.

Elevated expression of AKT has been noted in a significant percentage of primary human breast cancers, mainly as a consequence of the PTEN/PI3K pathway deregulation. To investigate the mechanistic basis of the AKT gain of function-dependent mechanisms of breast tumorigenesis, we explored the phenotype induced by activated AKT transgenes in a quantitative manner. We generated several transgenic mice lines expressing different levels of constitutively active AKT in the mammary gland. We thoroughly analyzed the preneoplastic and neoplastic mammary lesions of these mice and correlated the process of tumorigenesis to AKT levels. Finally, we analyzed the impact that a possible senescent checkpoint might have in the tumor promotion inhibition observed, crossing these lines to mammary specific p53(R172H) mutant expression, and to p27 knock-out mice. We analyzed the benign, premalignant and malignant lesions extensively by pathology and at molecular level analysing the expression of proteins involved in the PI3K/AKT pathway and in cellular senescence. Our findings revealed an increased preneoplastic phenotype depending upon AKT signaling which was not altered by p27 or p53 loss. However, p53 inactivation by R172H point mutation combined with myrAKT transgenic expression significantly increased the percentage and size of mammary carcinoma observed, but was not sufficient to promote full penetrance of the tumorigenic phenotype. Molecular analysis suggest that tumors from double myrAKT;p53(R172H) mice result from acceleration of initiated p53(R172H) tumors and not from bypass of AKT-induced oncogenic senescence. Our work suggests that tumors are not the consequence of the bypass of senescence in MIN. We also show that AKT-induced oncogenic senescence is dependent of pRb but not of p53. Finally, our work also suggests that the cooperation observed between mutant p53 and activated AKT is due to AKT-induced acceleration of mutant p53-induced tumors. Finally, our work shows that levels of activated AKT are not essential in the induction of benign or premalignant tumors, or in the cooperation of AKT with other tumorigenic signal such as mutant p53, once AKT pathway is activated, the relative level of activity seems not to determine the phenotype.

Apoptotic pathways, including the phosphatidylinositol-3-kinase (PI3K)/AKT survival pathway, are altered in most cancer cells in relation to their normal counterparts and these differences may present an excellent therapeutic window. To gain insight into the relevance of the PI3K pathway as a target for drug discovery we generated tumor cell lines from different tumor samples that we maintained at low passage. The characterization of these cell lines indicates that all of them have constitutively activated the PI3K pathway through different mechanisms. All cell lines were differentially sensitive to the PI3K inhibitor LY294002. Our data also support previous work indicating that PI3K inhibition might help classical chemotherapeutic treatments such as gemcitabine and strengthen suboptimal doses that might be effective for these purposes in decreasing the risk of side-effects. Finally, the analysis of the molecular markers that might be implicated in the synergism between LY294002 and gemcitabine suggests that PI3K inhibition might aid chemotherapeutic treatment, leading to changes in the balance between anti- and pro-apoptotic molecules of the Bcl-2 family, Bcl-XL and Bax. These results facilitate the exploration of potential synergism between chemotherapeutic treatment and the search for others that can account for similar molecular mechanisms of cooperation.

The PI3K/AKT pathway is commonly activated in human cancer. Multiple small-molecule inhibitors have been developed to target PI3K/mTOR or AKT kinases, but the efficacy of these drugs is compromised by the stimulation of compensatory signaling pathways. The redundancy of oncogenic signaling pathways provides back-up mechanisms that allow cancer cells to escape to targeted therapies. One example of such compensatory pathways is that driven by PIM kinases, which produce parallel oncogenic signals to AKT and mTOR and share several downstream molecular targets. In fact, in mouse lymphoma models chemoresistance to doxorubicin caused by AKT is readily reversed by rapamycin, but PIM mediated resistance is refractory to mTORC1 inhibition. Moreover, PIM2 kinase can confer resistance of primary hematopoietic cells to rapamycin treatment. Furthermore, PIM 1 mediates resistance to AKT and PI3K/mTOR inhibition and suggests co-targeting to improve the efficacy of PI3K/AKT/mTOR inhibitors in anticancer therapy. The PI3K/AKT/mTOR and PIM pathways are activated and seem to be relevant for tumor progression in many lymphomas, leukemias and in some solid tumors. We have previously reported that the combination of PI3K inhibitor GDC-0941 with a PIM selective inhibitor, ETP-45299, was strongly synergistic in antiproliferation experiments in MV-4-11 AML cells. Therefore, combination of such activities in a single molecule might have the potential to achieve better clinical efficacy and prevent/suppress resistance in comparison with classical PI3K/AKT/mTOR inhibitors. During the course of our investigations in this field we have identified a new chemical series of compounds with dual (PIM/PI3K) and triple activity (PIM/PI3K/mTOR inhibitors). (WO2012/156756). In this work, we show the biochemical, cellular and in vivo characterization of dual PIM/PI3K ETP-539/(IBL-202) and triple PIM/PI3K/mTOR ETP-339/(IBL-301) inhibitors. These optimized lead compounds are low nanomolar pan PIM/PI3K and pan PIM/PI3K/mTOR inhibitors respectively. These dual and triple inhibitors show excellent kinase selectivity profile against a panel of 456 kinases. Both compounds have been profiled for their antiproliferative behavior. We have identified leukemia, lymphoma, colon, and NSCLC lines which exhibit a strong sensitivity to dual and triple inhibition with GI80 between 5-10 times more potent than PIM or PI3K selective reference inhibitors. Mechanistically, cells respond to dual and triple inhibitors with a clear cell cycle arrest and marked apoptosis in AML and NSCLC cell lines, and strong down regulation of biomarkers. Our dual and triple inhibitors are optimized with respect to their in vitro ADME properties and have excellent oral bioavailability. These inhibitors have been tested in vivo in xenograft (MV4:11 AML) and transgenic (KRAsV12NSCLC) cancer mouse models. Both compounds have demonstrated down regulation of biomarkers associated with their targeted profile and significant antitumor efficacy in both models after oral administration. These inhibitors have been well tolerated, with no signs of toxicity even 20 times above the efficacious dose. These results provide the rationale for further preclinical development of ETP-539/(IBL-202 and ETP-339/(IBL-301) and the basis for a potential clinical use in AML and NSCLC tumors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A275. Citation Format: Carmen Blanco Aparicio, Oliver Renner, Elena Gomez-Casero, Antonio Cebriá, Nuria Ajenjo, Enara Aguirre, David Cebrián, Ma Carmen Rodriguez de Miguel, Belén Pequeño, MaIsabel Albarrán, Rosario Riesco, Ana Belén García, Rosa Alvarez, Michael O'Neill, Sonia Martinez, Joaquin Pastor. Co-targeting PIM and PI3K/mTOR pathways with a single molecule: Novel orally available combined PIM/PI3K and PIM/PI3K/mTOR kinase inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A275.