(5-(2-4-bis((3s)-3-methylmorpholin-4-yl)pyrido(2-3-d)pyrimidin-7-yl)-2-methoxyphenyl)methanol and Colorectal-Neoplasms

The mechanistic target of rapamycin complex 2 (mTORC2) is a potentially novel and promising anticancer target due to its critical roles in proliferation, apoptosis, and metabolic reprogramming of cancer cells. However, the activity and function of mTORC2 in distinct cells within malignant tissue in vivo is insufficiently explored. Surprisingly, in primary human and mouse colorectal cancer (CRC) samples, mTORC2 signaling could not be detected in tumor cells. In contrast, only macrophages in tumor-adjacent areas showed mTORC2 activity, which was downregulated in stromal macrophages residing within human and mouse tumor tissues. Functionally, inhibition of mTORC2 by specific deletion of Rictor in macrophages stimulated tumorigenesis in a colitis-associated CRC mouse model. This phenotype was driven by a proinflammatory reprogramming of mTORC2-deficient macrophages that promoted colitis via the cytokine SPP1/osteopontin to stimulate tumor growth. In human CRC patients, high SPP1 levels and low mTORC2 activity in tumor-associated macrophages correlated with a worsened clinical prognosis. Treatment of mice with a second-generation mTOR inhibitor that inhibits mTORC2 and mTORC1 exacerbated experimental colorectal tumorigenesis in vivo. In conclusion, mTORC2 activity is confined to macrophages in CRC and limits tumorigenesis. These results suggest activation but not inhibition of mTORC2 as a therapeutic strategy for colitis-associated CRC.

Topics: Animals; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Cells, Cultured; Colitis, Ulcerative; Colon; Colorectal Neoplasms; Dextran Sulfate; Disease Models, Animal; Female; Humans; Intestinal Mucosa; Kaplan-Meier Estimate; Macrophages; Male; Mechanistic Target of Rapamycin Complex 2; Mice; Mice, Transgenic; Morpholines; Osteopontin; Primary Cell Culture; Prognosis; Survival Rate

The use of targeted agents to impel dual inhibition of anti-apoptotic mechanisms and mTOR-mediated pro-survival signaling in colorectal carcinoma (CRC) cell lines with KRAS or BRAF mutation has been shown to induce apoptosis, a timely result given CRC entities harboring such mutations are in need of new therapies. Since CRC comprises heterogeneous tumors with predominant hypoxic components, we investigated effects of an inhibitor of anti-apoptotic Bcl-2 family proteins (ABT-737) in combination with an mTOR inhibitor (AZD8055)-collectively referred to as combo-Rx, in hypoxic CRC cell lines.. Cell viability measures, expression of proteins implicated in apoptosis and MAPK/PI3K-AKT/mTOR pathway signaling, and profiling of composite kinase activities were undertaken in a panel of 14 cell lines.. In hypoxic conditions, combo-Rx suppressed viability of 13 of the cell lines, albeit ABT-737 did not significantly potentiate the inhibitory effect of single-agent AZD8055 in six of the models. Hypoxic KRAS/PIK3CA-mutant HCT-116 and HCT-15 cell lines (both with low endogenous expression of the anti-apoptotic Mcl-1 protein and showing augmented inhibition of viability following the addition of ABT-737 to AZD8055) responded to combo-Rx by induction of apoptosis but with the simultaneous strong Mcl-1 up-regulation and activation of MAPK/PI3K-conducted signaling. In contrast, in hypoxic KRAS-mutant LoVo (devoid of PIK3CA mutation), BRAF/PIK3CA-mutant RKO, and wild-type Colo320DM cell lines (all with high endogenous Mcl-1 expression and being resistant to the additional effect of ABT-737 to AZD8055), combo-Rx did not elicit apoptotic or pro-survival responses.. The concurrent inhibition of anti-apoptotic proteins and mTOR-mediated signaling in hypoxic KRAS/PIK3CA-mutant CRC cell lines resulted in pro-survival responses in parallel with the intended anti-proliferative effects, a finding that should be of note if considering combinatory targeting of multiple pathways in this CRC entity.

Topics: Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Cell Line, Tumor; Cell Survival; Class I Phosphatidylinositol 3-Kinases; Colorectal Neoplasms; Enzyme Inhibitors; Humans; Mitogen-Activated Protein Kinases; Morpholines; Mutation; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins p21(ras); Signal Transduction; Sulfonamides; TOR Serine-Threonine Kinases; Tumor Hypoxia; Up-Regulation

We here tested the anti-colorectal cancer (CRC) activity by a first-in-class small molecule TRAIL inducer ONC201. The potential effect of mTOR on ONC201's actions was also examined. ONC201 induced moderate cytotoxicity against CRC cell lines (HT-29, HCT-116 and DLD-1) and primary human CRC cells. Significantly, AZD-8055, a mTOR kinase inhibitor, sensitized ONC201-induced cytotoxicity in CRC cells. Meanwhile, ONC201-induced TRAIL/death receptor-5 (DR-5) expression, caspase-8 activation and CRC cell apoptosis were also potentiated with AZD-8055 co-treatment. Reversely, TRAIL sequestering antibody RIK-2 or the caspase-8 specific inhibitor z-IETD-fmk attenuated AZD-8055 plus ONC201-induced CRC cell death. Further, mTOR kinase-dead mutation (Asp-2338-Ala) or shRNA knockdown significantly sensitized ONC201's activity in CRC cells, leading to profound cell death and apoptosis. On the other hand, expression of a constitutively-active S6K1 (T389E) attenuated ONC201-induced CRC cell apoptosis. For the mechanism study, we showed that ONC201 blocked Akt, but only slightly inhibited mTOR in CRC cells. Co-treatment with AZD-8055 also concurrently blocked mTOR activation. These results suggest that mTOR could be a primary resistance factor of ONC201 in CRC cells.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Colorectal Neoplasms; Enzyme Activation; Gene Knockdown Techniques; Gene Silencing; Heterocyclic Compounds, 4 or More Rings; Humans; Imidazoles; Morpholines; Mutation; Proto-Oncogene Proteins c-akt; Pyridines; Pyrimidines; TOR Serine-Threonine Kinases

Colorectal cancers harboring KRAS or BRAF mutations are refractory to current targeted therapies. Using data from a high-throughput drug screen, we have developed a novel therapeutic strategy that targets the apoptotic machinery using the BCL-2 family inhibitor ABT-263 (navitoclax) in combination with a TORC1/2 inhibitor, AZD8055. This combination leads to efficient apoptosis specifically in KRAS- and BRAF-mutant but not wild-type (WT) colorectal cancer cells. This specific susceptibility results from TORC1/2 inhibition leading to suppression of MCL-1 expression in mutant, but not WT, colorectal cancers, leading to abrogation of BIM/MCL-1 complexes. This combination strategy leads to tumor regressions in both KRAS-mutant colorectal cancer xenograft and genetically engineered mouse models of colorectal cancer, but not in the corresponding KRAS-WT colorectal cancer models. These data suggest that the combination of BCL-2/BCL-XL inhibitors with TORC1/2 inhibitors constitutes a promising targeted therapy strategy to treat these recalcitrant cancers.

Topics: Aniline Compounds; Animals; Antineoplastic Agents; bcl-X Protein; Cell Line, Tumor; Colorectal Neoplasms; Humans; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Mice; Mice, Mutant Strains; Mice, Nude; Morpholines; Multiprotein Complexes; Mutation; Myeloid Cell Leukemia Sequence 1 Protein; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins p21(ras); ras Proteins; Sulfonamides; TOR Serine-Threonine Kinases

The mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase/AKT signaling pathways interact at multiple nodes in cancer, including at mTOR complexes, suggesting an increased likelihood of redundancy and innate resistance to any therapeutic effects of single pathway inhibition. In this study, we investigated the therapeutic effects of combining the MAPK extracellular signal-regulated kinase (MEK)1/2 inhibitor selumetinib (AZD6244) with the dual mTORC1 and mTORC2 inhibitor (AZD8055). Concurrent dosing in nude mouse xenograft models of human lung adenocarcinoma (non-small cell lung cancers) and colorectal carcinoma was well tolerated and produced increased antitumor efficacy relative to the respective monotherapies. Pharmacodynamic analysis documented reciprocal pathway inhibition associated with increased apoptosis and Bim expression in tumor tissue from the combination group, where key genes such as DUSP6 that are under MEK functional control were also modulated. Our work offers a strong rationale to combine selumetinib and AZD8055 in clinical trials as an attractive therapeutic strategy.

Topics: Animals; Apoptosis; Benzimidazoles; Carcinoma, Non-Small-Cell Lung; Colorectal Neoplasms; Female; Gene Expression Profiling; Humans; Lung Neoplasms; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase Kinases; Morpholines; Mutation; Neoplasms, Experimental; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); ras Proteins; Signal Transduction; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays