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Fulvestrant is an FDA-approved drug with a dual mechanism of action (MOA), acting as a full antagonist and degrader of the estrogen receptor protein. A significant limitation of fulvestrant is the dosing regimen required for efficacy. Due to its high lipophilicity and poor pharmacokinetic profile, fulvestrant needs to be administered through intramuscular injections which leads to injection site soreness. This route of administration also limits the dose and target occupancy in patients. We envisioned a best-in-class molecule that would function with the same dual MOA as fulvestrant, but with improved physicochemical properties and would be orally bioavailable. Herein we report our progress toward that goal, resulting in a new lead GNE-502 which addressed some of the liabilities of our previously reported lead molecule GNE-149.

The interconnected PI3K and MAPK signaling pathways are commonly perturbed in cancer. Dual inhibition of these pathways by the small-molecule PI3K inhibitor pictilisib (GDC-0941) and the MEK inhibitor cobimetinib (GDC-0973) suppresses cell proliferation and induces cell death better than either single agent in several preclinical models. Using mass spectrometry-based phosphoproteomics, we have identified the RING finger E3 ubiquitin ligase RNF157 as a target at the intersection of PI3K and MAPK signaling. We demonstrate that RNF157 phosphorylation downstream of the PI3K and MAPK pathways influences the ubiquitination and stability of RNF157 during the cell cycle in an anaphase-promoting complex/cyclosome–CDH1-dependent manner. Deletion of these phosphorylation-targeted residues on RNF157 disrupts binding to CDH1 and protects RNF157 from ubiquitination and degradation. Expression of the cyclin-dependent kinase 2 (CDK2), itself a downstream target of PI3K/MAPK signaling, leads to increased phosphorylation of RNF157 on the same residues modulated by PI3K and MAPK signaling. Inhibition of PI3K and MEK in combination or of CDK2 by their respective small-molecule inhibitors reduces RNF157 phosphorylation at these residues and attenuates RNF157 interaction with CDH1 and its subsequent degradation. Knockdown of endogenous RNF157 in melanoma cells leads to late S phase and G2/M arrest and induces apoptosis, the latter further potentiated by concurrent PI3K/MEK inhibition, consistent with a role for RNF157 in the cell cycle. We propose that RNF157 serves as a novel node integrating oncogenic signaling pathways with the cell cycle machinery and promoting optimal cell cycle progression in transformed cells.

Estrogen receptor-positive (ER+) breast cancers frequently remain dependent on ER signaling even after acquiring resistance to endocrine agents, prompting the development of optimized ER antagonists. Fulvestrant is unique among approved ER therapeutics due to its capacity for full ER antagonism, thought to be achieved through ER degradation. The clinical potential of fulvestrant is limited by poor physicochemical features, spurring attempts to generate ER degraders with improved drug-like properties. We show that optimization of ER degradation does not guarantee full ER antagonism in breast cancer cells; ER "degraders" exhibit a spectrum of transcriptional activities and anti-proliferative potential. Mechanistically, we find that fulvestrant-like antagonists suppress ER transcriptional activity not by ER elimination, but by markedly slowing the intra-nuclear mobility of ER. Increased ER turnover occurs as a consequence of ER immobilization. These findings provide proof-of-concept that small molecule perturbation of transcription factor mobility may enable therapeutic targeting of this challenging target class.

Although K-Ras, Cdc42, and PAK4 signaling are commonly deregulated in cancer, only a few studies have sought to comprehensively examine the spectrum of phosphorylation-mediated signaling downstream of each of these key signaling nodes. In this study, we completed a label-free quantitative analysis of oncogenic K-Ras, activated Cdc42, and PAK4-mediated phosphorylation signaling, and report relative quantitation of 2152 phosphorylated peptides on 1062 proteins. We define the overlap in phosphopeptides regulated by K-Ras, Cdc42, and PAK4, and find that perturbation of these signaling components affects phosphoproteins associated with microtubule depolymerization, cytoskeletal organization, and the cell cycle. These findings provide a resource for future studies to characterize novel targets of oncogenic K-Ras signaling and validate biomarkers of PAK4 inhibition.

Starting from HTS hit 1a, X-ray co-crystallization and molecular modeling were used to design potent and selective inhibitors of PI3-kinase. Bioavailablity in this series was improved through careful modulation of physicochemical properties. Compound 12 displayed in vivo knockdown of PI3K pharmacodynamic markers such as pAKT, pPRAS40, and pS6RP in a PC3 prostate cancer xenograft model.

The murine Brca2 gene encodes a nuclear protein implicated in DNA repair. Brca2 behaves as a tumor suppressor, but paradoxically, its truncation causes proliferative arrest and spontaneous chromosomal damage. Here, we report that inactivation of cell cycle checkpoints responsive to mitotic spindle disruption, by mutant forms of p53 or Bub1, relieves growth arrest and initiates neoplastic transformation in primary cells homozygous for truncated Brca2. Tumors from Brca2-deficient animals exhibit dysfunction of the spindle assembly checkpoint, accompanied by mutations in p53, Bub1, and Mad3L. The chromosomal aberrations precipitated by Brca2 truncation can be suppressed by mutant forms of Bub1 and p53. Thus, inactivating mutations in mitotic checkpoint genes likely cooperate with BRCA2 deficiency in the pathogenesis of inherited breast cancer, with important implications for treatment.

Abnormalities precipitated by a targeted truncation in the murine gene Brca2 define its involvement in DNA repair. In culture, cells harboring truncated Brca2 exhibit a proliferative impediment that worsens with successive passages. Arrest in the G1 and G2/M phases is accompanied by elevated p53 and p21 expression. Increased sensitivity to genotoxic agents, particularly ultraviolet light and methylmethanesulfonate, shows that Brca2 function is essential for the ability to survive DNA damage. But checkpoint activation and apoptotic mechanisms are largely unaffected, thereby implicating Brca2 in repair. This is substantiated by the spontaneous accumulation of chromosomal abnormalities, including breaks and aberrant chromatid exchanges. These findings define a function of Brca2 in DNA repair, whose loss precipitates replicative failure, mutagen sensitivity, and genetic instability reminiscent of Bloom syndrome and Fanconi anemia.

To facilitate the positional cloning of the breast-ovarian cancer gene BRCA1, we constructed a high-density genetic map of the 8.3-cM interval between D17S250 and GIP on chromosome 17q12-q21. Markers were mapped by linkage in tbe CEPH and in extended kindreds in our breast cancer series. The map comprises 33 ordered polymorphisms, including 12 genes and 21 anonymous markers, yielding an average of one polymorphism every 250 kb. Twenty-five of the markers are PCR-based systems. The order of polymorphic genes and markers is cen-D17S250-D17S518-HER2-THRA1-RARA-D17S80-KRT10-[D17S800-[D17S857]-GAS-D17S856-EDH17B-D17S855-D17S859-D17S858-[PPY-D17S78]-D17S183-EPB3-D17S579-D17S509-[D17S508-D17S190 = D17S810]-D17S791-[D17S181 = D17S806]-D17S797-HOX2B-GP3A-[D17S507 = GIP]-qter. BRCA1 lies in the middle of the interval, between THRA1 and D17S183. Markers from this map can be used to determine whether cancer is linked to BRCA1 in families, to evaluate whether tumors have lost heterozygoslty at loci in the region, and to identify probes for characterizing chromosomal rearrangements from patients and from tumors.

Aim: We evaluated the efficacy of the PI3K inhibitor GDC-0941 (GDC) in combination with the angiogenesis inhibitor pazopanib (PAZ) in DDLPS xenografts with proven PI3K/AKT pathway activation. Methods: NMRI nu/nu mice were either injected bilaterally with 5x106 SW872 cells or transplanted with human DDLPS (UZLX-STS3). Animals were randomized to 4 groups and treated for three weeks: vehicle, GDC (p.o., 75mg/kg/qd), PAZ (p.o., 40mg/kg/bid), GDC+PAZ (same dose/schedule as single agents). Efficacy was assessed by tumour volume, Western blotting of the signalling pathway and histological evaluation [mitosis, apoptosis, microvascular density (MVD)]. Statistics were performed using the Mann-Whitney U and Wilcoxon matched pairs tests. p Results: At the end of treatment, tumour shrinkage was observed in GDC and GDC+PAZ groups in SW872 bearing animals (38% and 52% decrease compared to baseline, p Conclusions: Combined inhibition of PI3K and angiogenesis results in enhanced antitumour efficacy in DDLPS mouse models which warrants further study in this highly aggressive disease. Disclosure: P. Schoffski: Patrick Schoffski did receive honoraria for advisory and consulting functions and educational activities from Glaxo Smith Kline; L. Friedman: Lori Friedman is an employee of Genentech, a member of the Roche group. All other authors have declared no conflicts of interest.

The above paper was published with errors in Figures 1B (page 47) and ​and11H (page 50). The complete and correct figures are reprinted below. We regret any inconvenience that this error has caused. Figure 1B Effect of GDC-0980 alone and in combination with ABT888 plus carboplatin on DNA damage (pγH2AXS139) in BRCA-competent MDA-MB468 and MDA-MB231 TNBC cell lines. Figure 1H Effect of GDC-0980 alone and in combination with ABT888 plus carboplatin on DNA damage (pγH2AXS139) in BRCA-competent HCC70 and BT20 TNBC cell lines.

Specific BRCA1 mutations, PCR primers and hybridization probes are used in nucleic acid-based methods for diagnostic of inheritable breast cancer susceptibility. Addtionally, binding agents, such as antibodies, specific for peptides encoded by the subject BRCA1 mutants are used to identify expression products of diagnostic mutations/rare alleles in patient derived fluid or tissue samples. Compositions with high binding affinity for transcription or translation products of the disclosed BRCA1 mutations and alleles are used in therapeutic intervention. Such products include anti-sense nucleic acids, peptides encoded by the subject nucleic acids, and binding agents such as antibodies, specific for such peptides.