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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.

The effect of size and release kinetics of doxorubicin-nanoparticles on anti-tumor efficacy was evaluated in a panel of human cancer cell lines, including triple-negative breast cancer (TNBC) cells that frequently demonstrate resistance to doxorubicin. Different nano-formulations of sol-gel-based Doxorubicin containing nanoparticles were synthesized. Increased cell kill in chemorefractory triple-negative breast cancer cells was associated with the smallest size of nanoparticles and the slowest release of Dox. Modeling of dose-response parameters in Dox-sensitive versus Dox-resistant lines demonstrated increased EMax and area under the curve in Dox-resistant mesenchymal TNBC cells, implying potentially favorable activity in this molecular subtype of breast cancer. Mesenchymal TNBC cells demonstrated a high rate of fluorescent bead uptake suggestive of increased endocytosis, which may partially account for the enhanced efficacy of Dox-np in this subtype. Thus, manipulation of size and release kinetics of this nanoparticle platform is associated with enhanced dose-response metrics and tumor cell kill in therapeutically recalcitrant TNBC cell models. This platform is easily customizable and warrants further exploration.

Panobinostat (pano) is an FDA-approved histone deacetylase inhibitor. There is interest in evaluating alternate dosing schedules and novel combinations of pano for the treatment of upper aerodigestive and lung malignancies; thus we evaluated it in combination with Taxol, a chemotherapeutic with activity in both diseases. Dose-dependent synergy was observed in Non-Small Cell Lung Cancer (NSCLC) and Head and Neck Squamous Cell Carcinoma (HNSCC) cell lines and was due to senescence rather than potentiation of cell death. Senescence occurred following cisplatin- or Taxol-treatment in cell lines from both cancer types and was associated with decreased histone 3 (H3) acetylation and increased Bcl-xL expression: the latter a biomarker of senescence and target of anti-senescence therapeutics, or senolytics. Since H3 acetylation and Bcl-xL expression were altered in senescence, we subsequently evaluated pano as a senolytic in chemotherapy-treated cancer cells enriched for senescent cells. Pano caused cell death at significantly higher rates compared to repeat dosing with chemotherapy. This was associated with decreased expression of Bcl-xL and increased acetylated H3, reversing the expression patterns observed in senescence. These data support evaluating pano as a post-chemotherapy senolytic with the potential to kill persistent senescent cells that accumulate during standard chemotherapy in NSCLC and HNSCC.

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.

While the clinical benefit of MEK inhibitor (MEKi)-based therapy is well established in Raf mutant malignancies, its utility as a suppressor of hyperactive MAPK signaling in the absence of mutated Raf or Ras, is an area of ongoing research. MAPK activation is associated with loss of ERα expression and hormonal resistance in numerous malignancies. Herein, we demonstrate that MEKi induces a feedback response that results in ERα overexpression, phosphorylation and transcriptional activation of ER-regulated genes. Mechanistically, MEKi-mediated ERα overexpression is largely independent of erbB2 and AKT feedback activation, but is ERK-dependent. We subsequently exploit this phenomenon therapeutically by combining the ER-antagonist, fulvestrant with MEKi. This results in synergistic suppression of tumor growth, in vitro and potentiation of single agent activity in vivo in nude mice bearing xenografts. Thus, we demonstrate that exploiting adaptive feedback after MEKi can be used to sensitize ERα-positive tumors to hormonal therapy, and propose that this strategy may have broader clinical utility in ERα-positive ovarian carcinoma.

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.

Objectives: Our objective is to determine the effect of PI3K activity in mediating ligand-independent estrogen signaling and endocrine sensitivity in Uterine Papillary Serous Carcinoma (UPSC). Methods: Drug effect on cell proliferation was calculated via Calcusyn in patient-derived UPSC cell lines ARK 1 and 2. PI3K mutation was analyzed after laser capture microdissection of tumor DNA in 7 consecutive patients with UPSC. Protein expression, via Western Blot, was correlated prospectively with clinical parameters. Results: Table 1 shows cells line baseline characteristics and IC50 to drugs. Fulvestrant, an ER antagonist, rendered the cells significantly more resistant to taxol in ARK1 and 2(p=0.035, 0.021 respectively). The concomitant decrease in pERS167 and pAKTS473 with Fulvestrant treatment is independent of baseline ER expression and PI3K mutation. In ARK2, a cell line that is ER-, disrupting AKT signaling via the PI3K inhibitor GDC-0941, or with the lapatinib is synergistic with Fulvestrant with Combination Index (CI)50 of 0.441 and 0.229, respectively. Independent of HER2 amplification or PI3K mutation, lapatinib and GDC-0941 exhibited synergistic cytotoxicity in both UPSC cell lines (CI50 of 0.577, ARK 1 and 0.233, ARK2). Finally, PI3K mutation and pAKTS473 expression was analyzed in 7 tumor samples. While none harbored PI3K mutation, patients who are chemotherapy resistant had significantly lower expression of baseline pAKTS473 in their tumor samples than those who are chemosensitive, similar to our cell line data. Conclusions: PI3K pathway dysregulation, either via upstream erbB amplification, or downstream constitutive activation of AKT, may be important in mediating endocrine and taxol sensitivity in UPSC. pAKTS473 may be an important biomarker of drug sensitivity. The combination of PI3K inhibitor and lapatinib is synergistic and warrant further therapeutic investigation. Baseline characteristics and drug sensitivities in UPSC cell linesARK1ARK2PI3KCA mutationfExon 9NoERα (WB)YesNopERα167 (WB)YesNopHER (WB)NoYesIC50 nM (mean ± STD)GDC-094193.1±5.9271±116.5LapatinibNo effect135.6±29.2FulvestrantNo effectNo effectTaxol4.6±1.97.8±5.6Cisplatin1103±65.72632.6±1797.9 Citation Format: Kelly S. Levano, Alicia Rodriguez-Gabin, Gary L. Goldberg, Susan B. Horwitz, June Y. Hou. The PI3K inhibitor GDC-0941 is synergistic with lapatinib, and mediates endocrine sensitivity in uterine papillary serous carcinoma. [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 4687. doi:10.1158/1538-7445.AM2014-4687