Bryan T. Hennessy

Exploiting the PI3K/AKT Pathway for Cancer Drug Discovery

Evolving studies with several different targeted therapeutic agents are demonstrating that patients with genomic alterations of the target, including amplification, translocation and mutation, are more likely to respond to the therapy. Recent studies indicate that numerous components of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway are targeted by amplification, mutation and translocation more frequently than any other pathway in cancer patients, with resultant activation of the pathway. This warrants exploiting the PI3K/AKT pathway for cancer drug discovery.

The biology of ovarian cancer: new opportunities for translation

Over the past two decades, the 5-year survival for ovarian cancer patients has substantially improved owing to more effective surgery and treatment with empirically optimized combinations of cytotoxic drugs, but the overall cure rate remains approximately 30%. Many investigators think that further empirical trials using combinations of conventional agents are likely to produce only modest incremental improvements in outcome. Given the heterogeneity of this disease, increases in long-term survival might be achieved by translating recent insights at the molecular and cellular levels to personalize individual strategies for treatment and to optimize early detection.

An Integrative Genomic and Proteomic Analysis of PIK3CA, PTEN, and AKT Mutations in Breast Cancer

Phosphatidylinositol 3-kinase (PI3K)/AKT pathway aberrations are common in cancer. By applying mass spectroscopy-based sequencing and reverse-phase protein arrays to 547 human breast cancers and 41 cell lines, we determined the subtype specificity and signaling effects of PIK3CA, AKT, and PTEN mutations and the effects of PIK3CA mutations on responsiveness to PI3K inhibition in vitro and on outcome after adjuvant tamoxifen. PIK3CA mutations were more common in hormone receptor-positive (34.5%) and HER2-positive (22.7%) than in basal-like tumors (8.3%). AKT1 (1.4%) and PTEN (2.3%) mutations were restricted to hormone receptor-positive cancers. Unlike AKT1 mutations that were absent from cell lines, PIK3CA (39%) and PTEN (20%) mutations were more common in cell lines than tumors, suggesting a selection for these but not AKT1 mutations during adaptation to culture. PIK3CA mutations did not have a significant effect on outcome after adjuvant tamoxifen therapy in 157 hormone receptor-positive breast cancer patients. PIK3CA mutations, in comparison with PTEN loss and AKT1 mutations, were associated with significantly less and inconsistent activation of AKT and of downstream PI3K/AKT signaling in tumors and cell lines. PTEN loss and PIK3CA mutation were frequently concordant, suggesting different contributions to pathophysiology. PTEN loss rendered cells significantly more sensitive to growth inhibition by the PI3K inhibitor LY294002 than did PIK3CA mutations. Thus, PI3K pathway aberrations likely play a distinct role in the pathogenesis of different breast cancer subtypes. The specific aberration present may have implications for the selection of PI3K-targeted therapies in hormone receptor-positive breast cancer.

Measurement of Residual Breast Cancer Burden to Predict Survival After Neoadjuvant Chemotherapy

PURPOSE To measure residual disease after neoadjuvant chemotherapy in order to improve the prognostic information that can be obtained from evaluating pathologic response. PATIENTS AND METHODS Pathologic slides and reports were reviewed from 382 patients in two different treatment cohorts: sequential paclitaxel (T) then fluorouracil, doxorubicin, and cyclophosphamide (FAC) in 241 patients; and a single regimen of FAC in 141 patients. Residual cancer burden (RCB) was calculated as a continuous index combining pathologic measurements of primary tumor (size and cellularity) and nodal metastases (number and size) for prediction of distant relapse-free survival (DRFS) in multivariate Cox regression analyses. RESULTS RCB was independently prognostic in a multivariate model that included age, pretreatment clinical stage, hormone receptor status, hormone therapy, and pathologic response (pathologic complete response [pCR] v residual disease [RD]; hazard ratio = 2.50; 95% CI 1.70 to 3.69; P < .001). Minimal RD (RCB-I) in 17% of patients carried the same prognosis as pCR (RCB-0). Extensive RD (RCB-III) in 13% of patients was associated with poor prognosis, regardless of hormone receptor status, adjuvant hormone therapy, or pathologic American Joint Committee on Cancer stage of residual disease. The generalizability of RCB for prognosis of distant relapse was confirmed in the FAC-treated validation cohort. CONCLUSION RCB determined from routine pathologic materials represented the distribution of RD, was a significant predictor of DRFS, and can be used to define categories of near-complete response and chemotherapy resistance.

The energy sensing LKB1-AMPK pathway regulates p27kip1 phosphorylation mediating the decision to enter autophagy or apoptosis

Nutrients and bioenergetics are prerequisites for proliferation and survival of mammalian cells. We present evidence that the cyclin-dependent kinase inhibitor p27Kip1, is phosphorylated at Thr 198 downstream of the Peutz-Jeghers syndrome protein–AMP-activated protein kinase (LKB1–AMPK) energy-sensing pathway, thereby increasing p27 stability and directly linking sensing of nutrient concentration and bioenergetics to cell-cycle progression. Ectopic expression of wild-type and phosphomimetic Thr 198 to Asp 198 (T198D), but not unstable Thr 198 to Ala 198 (p27T198A) is sufficient to induce autophagy. Under stress conditions that activate the LKB1–AMPK pathway with subsequent induction of autophagy, p27 knockdown results in apoptosis. Thus LKB1–AMPK pathway-dependent phosphorylation of p27 at Thr 198 stabilizes p27 and permits cells to survive growth factor withdrawal and metabolic stress through autophagy. This may contribute to tumour-cell survival under conditions of growth factor deprivation, disrupted nutrient and energy metabolism, or during stress of chemotherapy.

Reverse phase protein array: validation of a novel proteomic technology and utility for analysis of primary leukemia specimens and hematopoietic stem cells

Proteomics has the potential to provide answers in cancer pathogenesis and to direct targeted therapy through the comprehensive analysis of protein expression levels and activation status. The realization of this potential requires the development of new, rapid, high-throughput technologies for performing protein arrays on patient samples, as well as novel analytic techniques to interpret them. Herein, we describe the validation and robustness of using reverse phase protein arrays (RPPA) for the analysis of primary acute myelogenous leukemia samples as well as leukemic and normal stem cells. In this report, we show that array printing, detection, amplification, and staining precision are very high, reproducible, and that they correlate with traditional Western blotting. Using replicates of the same sample on the same and/or separate arrays, or using separate protein samples prepared from the same starting sample, the intra- and interarray reproducibility was extremely high. No statistically significant difference in protein signal intensities could be detected within the array setups. The activation status (phosphorylation) was maintained in experiments testing delayed processing and preparation from multiple freeze-thawed samples. Differences in protein expression could reliably be detected in as few as three cell protein equivalents. RPPA prepared from rare populations of normal and leukemic stem cells were successfully done and showed differences from bulk populations of cells. Examples show how RPPAs are ideally suited for the large-scale analysis of target identification, validation, and drug discovery. In summary, RPPA is a highly reliable, reproducible, high-throughput system that allows for the rapid large-scale proteomic analysis of protein expression and phosphorylation state in primary acute myelogenous leukemia cells, cell lines, and in human stem cells. [Mol Cancer Ther 2006;5(10):2512–21]

AKT-independent signaling downstream of oncogenic PIK3CA mutations in human cancer

Dysregulation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway occurs frequently in human cancer. PTEN tumor suppressor or PIK3CA oncogene mutations both direct PI3K-dependent tumorigenesis largely through activation of the AKT/PKB kinase. However, here we show through phosphoprotein profiling and functional genomic studies that many PIK3CA mutant cancer cell lines and human breast tumors exhibit only minimal AKT activation and a diminished reliance on AKT for anchorage-independent growth. Instead, these cells retain robust PDK1 activation and membrane localization and exhibit dependency on the PDK1 substrate SGK3. SGK3 undergoes PI3K- and PDK1-dependent activation in PIK3CA mutant cancer cells. Thus, PI3K may promote cancer through both AKT-dependent and AKT-independent mechanisms. Knowledge of differential PI3K/PDK1 signaling could inform rational therapeutics in cancers harboring PIK3CA mutations.

Hyperactivation of phosphatidylinositol-3 kinase promotes escape from hormone dependence in estrogen receptor–positive human breast cancer

Many breast cancers exhibit a degree of dependence on estrogen for tumor growth. Although several therapies have been developed to treat individuals with estrogen-dependent breast cancers, some tumors show de novo or acquired resistance, rendering them particularly elusive to current therapeutic strategies. Understanding the mechanisms by which these cancers develop resistance would enable the development of new and effective therapeutics. In order to determine mechanisms of escape from hormone dependence in estrogen receptor-positive (ER-positive) breast cancer, we established 4 human breast cancer cell lines after long-term estrogen deprivation (LTED). LTED cells showed variable changes in ER levels and sensitivity to 17beta-estradiol. Proteomic profiling of LTED cells revealed increased phosphorylation of the mammalian target of rapamycin (mTOR) substrates p70S6 kinase and p85S6 kinase as well as the PI3K substrate AKT. Inhibition of PI3K and mTOR induced LTED cell apoptosis and prevented the emergence of hormone-independent cells. Using reverse-phase protein microarrays, we identified a breast tumor protein signature of PI3K pathway activation that predicted poor outcome after adjuvant endocrine therapy in patients. Our data suggest that upon adaptation to hormone deprivation, breast cancer cells rely heavily on PI3K signaling. Our findings also imply that acquired resistance to endocrine therapy in breast cancer may be abrogated by combination therapies targeting both ER and PI3K pathways.

Incidence and Outcome of BRCA Mutations in Unselected Patients with Triple Receptor-Negative Breast Cancer

Purpose: To investigate the incidence of germline and somatic BRCA1/2 mutations in unselected patients with triple-negative breast cancer (TNBC) and determine the prognostic significance of carrying a mutation. Methods: DNA was obtained from 77 TNBC and normal tissues. BRCA1/2 exons/flanking regions were sequenced from tumor and patients classified as mutant or wild type (WT). Sequencing was repeated from normal tissue to identify germline and somatic mutations. Patient characteristics were compared with chi-square. Survival was estimated by Kaplan–Meier method and compared with log-rank. Cox proportional hazards models were fit to determine the independent association of mutation status with outcome. Results: Median age was 51 years (27–83 years). Fifteen patients (19.5%) had BRCA mutations: 12 (15.6%) in BRCA1 (one somatic), and 3 (3.9%) in BRCA2. Patients with BRCA mutations tended to be younger than WT, (P = 0.005). Grade, histology, and stage were not associated with mutation status. At a median follow-up of 43 months (7–214 months), there were 33 (42.9%) recurrences and 35 (45.5%) deaths. Five-year recurrence-free survival estimates were 51.7% for WT versus 86.2% for patients with mutations, (P = 0.031); and 5-year overall survival estimates were 52.8% for WT versus 73.3% for patients with mutations (P = 0.225). After adjustment, patients with BRCA mutations had a significantly better RFS (HR: 0.19, 95% CI: 0.045–0.79, P = 0.016) compared with WT. Conclusions: In this unselected cohort of TNBC, we found a 19.5% incidence of BRCA mutations. Genetic testing should be discussed with patients with TNBC. Patients with TNBC with BRCA mutations had a significantly lower risk of relapse. Clin Cancer Res; 17(5); 1082–9. ©2011 AACR.

Next-generation mTOR inhibitors in clinical oncology: how pathway complexity informs therapeutic strategy

Mammalian target of rapamycin (mTOR) is a PI3K-related kinase that regulates cell growth, proliferation, and survival via mTOR complex 1 (mTORC1) and mTORC2. The mTOR pathway is often aberrantly activated in cancers. While hypoxia, nutrient deprivation, and DNA damage restrain mTORC1 activity, multiple genetic events constitutively activate mTOR in cancers. Here we provide a brief overview of the signaling pathways up- and downstream of mTORC1 and -2, and discuss the insights into therapeutic anticancer targets - both those that have been tried in the clinic with limited success and those currently under clinical development - that knowledge of these pathways gives us.