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The natural product (+)-discodermolide (DDM) is a microtubule stabilizing agent and potent inducer of senescence. We refined the structure of DDM and evaluated the activity of novel congeners in triple negative breast and ovarian cancers, malignancies that typically succumb to taxane resistance. Previous structure-activity analyses identified the lactone and diene as moieties conferring anticancer activity, thus identifying priorities for the structural refinement studies described herein. Congeners possessing the monodiene with a simplified lactone had superior anticancer efficacy relative to taxol, particularly in resistant models. Specifically, one of these congeners, B2, demonstrated 1) improved pharmacologic properties, specifically increased maximum response achievable and area under the curve, and decreased EC(50); 2) a uniform dose-response profile across genetically heterogeneous cancer cell lines relative to taxol or DDM; 3) reduced propensity for senescence induction relative to DDM; 4) superior long-term activity in cancer cells versus taxol or DDM; and 5) attenuation of metastatic characteristics in treated cancer cells. To contrast the binding of B2 versus DDM in tubulin, X-ray crystallography studies revealed a shift in the position of the lactone ring associated with removal of the C2-methyl and C3-hydroxyl. Thus, B2 may be more adaptable to changes in the taxane site relative to DDM that could account for its favorable properties. In conclusion, we have identified a DDM congener with broad range anticancer efficacy that also has decreased risk of inducing chemotherapy-mediated senescence. SIGNIFICANCE STATEMENT: Here, we describe the anticancer activity of novel congeners of the tubulin-polymerizing molecule (+)-discodermolide. A lead molecule is identified that exhibits an improved dose-response profile in taxane-sensitive and taxane-resistant cancer cell models, diminished risk of chemotherapy-mediated senescence, and suppression of tumor cell invasion endpoints. X-ray crystallography studies identify subtle changes in the pose of binding to β-tubulin that could account for the improved anticancer activity. These findings support continued preclinical development of discodermolide, particularly in the chemorefractory setting.

Objective Treatment options are limited for patients with uterine serous carcinoma (USC). Knowledge of USC's somatic mutation landscape is rapidly increasing, but its role in hereditary cancers remains unclear. We aim to evaluate the frequency and characteristics of germline mutations in genes commonly implicated in carcinogenesis, including those within homologous recombination (HR) and mismatch repair (MMR) pathways in patients with pure USC. Methods By using targeted capture exome sequencing, 43 genes were analyzed in a cohort of 7 consecutive patients with paired tumor and non-tumor USC samples in our institutional tumor repository. Mutations predicted to have damaging effects on protein function are validated by Sanger Sequencing. Results We found 21 germline mutations in 11 genes in our USC cohort. Five patients harbored 7 germline mutations (33.3%) within genes involved in the HR pathway, RAD51D being the most common. Four patients had 9 (42.8%) germline mutations in hereditary colon cancer genes, most commonly MLH. All patients (42.7%) who are platinum-sensitive had HR germline mutations (RAD50, NBN, ATM). Patients with HER2 overexpression (2/7, 28.6%) had germline HR mutations and were platinum-sensitive. Three patients in our cohort reported a personal history of breast cancer, one with HR germline mutation, and 2 in patients with germline mutations in HCC genes. In addition, 5 out of 7 patients had germline mutations in genes associated with growth factor signaling pathway. Conclusions A significant proportion of our cohort harbor germline mutations in DNA repair genes. This may be associated with the high rate of breast cancer in our patients and their family, and suggests a targeted cohort for genetic counseling. If validated in a larger cohort, our findings may allow clinicians to expand therapeutic options to include targeted therapies and inclusion of USC patient in preventative and genetic counseling.

Background: Activated MAPK and PI3K/AKT/mTOR pathway signaling are associated with poor prognosis in triple negative Breast cancer (TNBC). Therefore, suppression of both arms of the MAPK/PI3K/AKT/mTOR network is a promising strategy for targeting TNBC. Here we explore the anti-tumor effects of combinations of MEK inhibitor with PI3K, AKT, or mTOR inhibitors with a focus on cell fate and biomarker development in two major subtypes of TNBC, basal-like and mesenchymal. Methods: Combinations of a MEK inhibitor with a PI3K inhibitor, AKT inhibitor, dual mTORC 1/2 inhibitor, or rapalog were evaluated in TNBC cell lines and an orthotopically implanted patient-derived xenograft (PDX) model of TNBC. We utilized reverse-phase protein array (RPPA) and quantitative real-time PCR to interrogate the signaling architecture and transcriptional activity of the treated cell lines. Results: All combinations synergistically suppress the growth of basal-like and mesenchymal TNBC models. RPPA confirms that all combinations suppress common network targets including pERK Th202/T204, pPRAS40 T246, pS6rp S235/236, and p4E-BP1 S65 in all cell lines. Notably, however, comparable repression of MAPK/PI3K/AKT/mTOR signaling produces distinct fates in basal-like and mesenchymal subtypes. Basal-like cell lines preferentially undergo delayed cell death with surviving cells displaying profound growth arrest. In contrast, mesenchymal cell lines respond with uniform quiescence exhibiting little or no cell death. Transcriptional analysis corroborates these phenotypic effects demonstrating differential modulation of genes regulating apoptosis and proliferation in basal-like versus mesenchymal cell lines. Drug treated mesenchymal cells exhibit transcriptional ‘reprogramming’ of epithelial-mesenchymal status as evidenced by reduced mesenchymal gene expression inferring potential effects on invasion and metastasis. In a PDX model of basal-like TNBC that is paclitaxel resistant, early and sustained suppression of tumor growth was achieved by treating once daily with the MEK inhibitor, GDC-0973, in combination with either the PI3K inhibitor, GDC-0941, or the rapalog, Temsirolimus. Concurrent dosing on this intense schedule was associated with some toxicity that could likely be diminished with dose modifications. Conclusions: These data highlight the therapeutic potential of combined MEK and PI3K/AKT/mTOR inhibition in chemo-resistant TNBC. Importantly, they demonstrate innate differences in the response of basal-like and mesenchymal subtypes of TNBC to these combinations, supporting the concept that molecular subtype will be an important predictor of response both in this setting and for other targeted therapies. The combination of GDC-0973 and GDC-0941 performed particularly well in a taxane resistant PDX model and deserves further evaluation in clinical trials for the treatment of TNBC. Citation Format: Sarah J. Schweber, Alicia Rodriguez-Gabin, Jinghang Zhang, Valerie Calvert, Huiping Liu, Emanuel M. Petricoin, III, Eleni Andreopoulou, Susan Band Horwitz, Hayley M. McDaid. Proteomic and transcriptional profiling reveal differential responses to combined MEK and PI3K-mTOR network inhibition in basal-like and mesenchymal subtypes of triple negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A35.