MCF10A mammary epithelial cells form growth-arrested structures when cultured in three-dimensional basement membrane gels. Activation of the receptor tyrosine kinase ErbB2 induces formation of proliferative structures that share properties with noninvasive early stage lesions. We conducted a genetic screen to identify cDNAs that can cooperate with ErbB2 to induce migration in these cells, with the hypothesis that they would represent candidate ''second hits'' in the development of invasive breast carcinomas. We found that expression of transforming growth factor (TGF)␤1 and TGF␤3 in cells expressing activated ErbB2 induces migration in transwell chambers and invasive behavior in both basement membrane cultures and invasion chambers. The ability of ErbB2 to cooperate with TGF␤ correlated with sustained, elevated activation of extracellular signal-regulated kinase (Erk)-mitogen-activated protein kinase. Pharmacological reduction of Erk activity inhibited the cooperative effect of TGF␤ and ErbB2 on migration and expression of activated Erk kinase was sufficient to cooperate with TGF␤ to induce migration and invasion, suggesting that sustained Erk activation is critical for ErbB2͞TGF␤ cooperation. In addition, we show that costimulation of ErbB2 and TGF␤ induces autocrine secretion of factors that are sufficient to induce migration, but not invasion, by means of both epidermal growth factor receptor-dependent and -independent processes. These results support the role of TGF␤ as a pro-invasion factor in the progression of breast cancers with activated ErbB2 and suggest that activation of the Erk and epidermal growth factor receptor pathways are key in mediating these events. E rbB2 (HER2͞Neu) is overexpressed in 20% to 30% of invasive breast tumors and up to 85% of comedo type ductal carcinoma in situ (DCIS), an early stage in breast cancer (1, 2). The ErbB receptor family has four members: ErbB1 (EGFR͞ HER1), ErbB2, ErbB3, and ErbB4. When these receptors are stimulated with epidermal growth factor (EGF) family ligands, receptor activation occurs by means of both homo-and heterodimerization among the receptor family members (3). However, under conditions where ErbB2 is amplified or overexpressed, activation can occur by ligand-independent homodimerization (4, 5).Previously, we investigated the consequences of activation of ErbB2 homodimers in the context of a nontransformed human mammary epithelial cell line, MCF10A (6, 7). When grown in basement membrane cultures, MCF10A cells form growth arrested three-dimensional structures, termed acini (8). These structures are comprised of a single layer of polarized epithelial cells surrounding a hollow lumen and resemble the mammary acini that form terminal ductal lobular units in the adult breast (7,8).To study the contributions of ErbB receptors in these cells, we generated chimeras of ErbB1 (p75.B1) and ErbB2 (p75.B2), which are inducibly activated by means of homodimerization by using the dimeric FKBP ligand AP1510 (ARIAD Pharmaceuticals, Cambridge, MA; refs. 9 and 10). T...

The completion of the human genome project and the development of high-throughput approaches herald a dramatic acceleration in the pace of biological research. One of the most compelling next steps will be learning the functional roles of all proteins. Achievement of this goal depends in part on the rapid expression and isolation of proteins at large scale. We exploited recombinational cloning to facilitate the development of methods for the high-throughput purification of human proteins. cDNAs were introduced into a master vector from which they could be rapidly transferred into a variety of protein expression vectors for further analysis. A test set of 32 sequenceverified human cDNAs of various sizes and activities was moved into four different expression vectors encoding different affinity-purification tags. By means of an automatable 2-hr protein purification procedure, all 128 proteins were purified and subsequently characterized for yield, purity, and steps at which losses occurred. Under denaturing conditions when the His 6 tag was used, 84% of samples were purified. Under nondenaturing conditions, both the glutathione S-transferase and maltose-binding protein tags were successful in 81% of samples. The developed methods were applied to a larger set of 336 randomly selected cDNAs. Sixty percent of these proteins were successfully purified under denaturing conditions and 82% of these under nondenaturing conditions. A relational database, FLEXProt, was built to compare properties of proteins that were successfully purified and proteins that were not. We observed that some domains in the Pfam database were found almost exclusively in proteins that were successfully purified and thus may have predictive character.W ith the application of large-scale and high-throughput (HT) approaches to biological and medical questions, biology has embraced a new era of technology development and information collection. The great task lying ahead is to elucidate the functions of all proteins encoded in the genomes of sequenced model organisms. This process involves collection of information about the temporal, spatial, and physiological regulation of proteins, their interaction partners, biochemical activities, posttranslational modifications, and the mutual influence of all these parameters on the physiology of the organism. Over the past several decades, biologists and biochemists have amassed a large collection of powerful tools for the study of individual proteins. However, compared with the study of nucleic acids, the HT study of proteins is still in its infancy. The next great challenge in biology will be to adapt these tools and develop new ones that enable the simultaneous and parallel study of thousands of proteins.The elucidation of biochemical activity and protein-protein interactions are central aspects of understanding protein function. Protein microarrays provide one platform for biochemical experiments to be carried out at extraordinary pace (1-3). However, this exciting technology calls attention to the quest...

Activating KRAS mutations are major oncogenic drivers in multiple tumor types. Synthetic lethal screens have previously been used to identify targets critical for the survival of KRAS mutant cells, but their application to drug discovery has proven challenging, possibly due in part to a failure of monolayer cultures to model tumor biology. Here, we report the results of a high-throughput synthetic lethal screen for small molecules that selectively inhibit the growth of KRAS mutant cell lines in soft agar. Chemoproteomic profiling identifies the target of the most KRAS-selective chemical series as dihydroorotate dehydrogenase (DHODH). DHODH inhibition is shown to perturb multiple metabolic pathways. In vivo preclinical studies demonstrate strong antitumor activity upon DHODH inhibition in a pancreatic tumor xenograft model.

Functional proteomics approaches that comprehensively evaluate the biological activities of human cDNAs may provide novel insights into disease pathogenesis. To systematically investigate the functional activity of cDNAs that have been implicated in breast carcinogenesis, we generated a collection of cDNAs relevant to breast cancer, the Breast Cancer 1000 (BC1000), and conducted screens to identify proteins that induce phenotypic changes that resemble events that occur during tumor initiation and progression. Genes were selected for this set using bioinformatics and data mining tools that identify genes associated with breast cancer. Greater than 1000 cDNAs were assembled and sequence verified with high-throughput recombination-based cloning. To our knowledge, the BC1000 represents the first publicly available sequence-validated human disease gene collection. The functional activity of a subset of the BC1000 collection was evaluated in cellbased assays that monitor changes in cell proliferation, migration and morphogenesis in MCF10A mammary epithelial cells expressing a variant of ErbB2 that can be inducibly activated through dimerization. Using this approach, we identified many cDNAs, encoding diverse classes of cellular proteins, that displayed activity in one or more of the assays, thus providing insights into a large set of cellular proteins capable of inducing functional alterations associated with breast cancer development.

Large-scale functional genomics studies for malaria vaccine and drug development will depend on the generation of molecular tools to study protein expression. We examined the feasibility of a high-throughput cloning approach using the Gateway system to create a large set of expression clones encoding Plasmodium falciparum single-exon genes. Master clones and their ORFs were transferred en masse to multiple expression vectors. Target genes (n = 303) were selected using specific sets of criteria, including stage expression and secondary structure. Upon screening four colonies per capture reaction, we achieved 84% cloning efficiency. The genes were subcloned in parallel into three expression vectors: a DNA vaccine vector and two protein expression vectors. These transfers yielded a 100% success rate without any observed recombination based on single colony screening. The functional expression of 95 genes was evaluated in mice with DNA vaccine constructs to generate antibody against various stages of the parasite. From these, 19 induced antibody titers against the erythrocytic stages and three against sporozoite stages. We have overcome the potential limitation of producing large P. falciparum clone sets in multiple expression vectors. This approach represents a powerful technique for the production of molecular reagents for genome-wide functional analysis of the P. falciparum genome and will provide for a resource for the malaria resource community distributed through public repositories.

Primary treatment for estrogen receptor-positive (ER+) breast cancer is endocrine therapy. However, substantial evidence indicates a continued role for ER signaling in tumor progression. Selective estrogen receptor degraders (SERD), such as fulvestrant, induce effective ER signaling inhibition, although clinical studies with fulvestrant report insufficient blockade of ER signaling, possibly due to suboptimal pharmaceutical properties. Furthermore, activating mutations in the ER have emerged as a resistance mechanism to current endocrine therapies. New oral SERDs with improved drug properties are under clinical investigation, but the biological profile that could translate to improved therapeutic benefit remains unclear. Here, we describe the discovery of SAR439859, a novel, orally bioavailable SERD with potent antagonist and degradation activities against both wild-type and mutant Y537S ER. Driven by its fluoropropyl pyrrolidinyl side chain, SAR439859 has demonstrated broader and superior ER antagonist and degrader activities across a large panel of ER+ cells, compared with other SERDs characterized by a cinnamic acid side chain, including improved inhibition of ER signaling and tumor cell growth. Similarly, in vivo treatment with SAR439859 demonstrated significant tumor regression in ER+ breast cancer models, including MCF7-ESR1 wild-type and mutant-Y537S mouse tumors, and HCI013, a patient-derived tamoxifen-resistant xenograft tumor. These findings indicate that SAR439859 may provide therapeutic benefit to patients with ER+ breast cancer, including those who have resistance to endocrine therapy with both wild-type and mutant ER.

Estrogen receptor positive (ER+) breast cancer accounts for 70% of all breast cancers and is primarily treated with endocrine therapy. Approximately 40% of patients on endocrine therapy will become resistant via a number of mechanisms. There is evidence that in many cases ER continues to play a central role, including mutations in ER leading to a constitutively active receptor. Estrogen receptor degraders like fulvestrant are effective in shutting down ER signaling; however, poor pharmaceutical properties limit fulvestrant clinical activity and prevent it from achieving maximum receptor blockade. We describe the discovery of SAR439859, a novel, orally bioavailable SERD that is a potent antagonist and degrader of ER both in vitro and in vivo. SAR439859 has robust activity in multiple ER+ breast cancer cell lines including cells that are resistant to tamoxifen as well as cell lines harboring ER mutants. Across a large panel of ER+ cells, SAR439859 demonstrated broad and superior ER degradation activity than most SERDs undergoing clinical testing. This leads to a profound inhibition of ER signaling, better inhibition of cell growth and results in improved in vivo efficacy. SAR439859 demonstrated tumor regression in all ER+ BC models including MCF7-ESR1 mutant-Y537S model, as well as patient-derived xenograft model that is resistant to endocrine therapies. Furthermore, SAR439859 displays limited cross-resistance with other class of SERDs. Taken together, these results suggest that SAR439859 would be of therapeutic benefit in metastatic BC setting for patients harboring wild type or mutant ER. SAR439859 is being advanced toward the clinic. Citation Format: Shomali M, Cheng J, Koundinya M, Weinstein M, Malkova N, Sun F, Hebert A, Cindachao M, Hoffman D, McManus J, Levit M, Pollard J, Vincent S, Besret L, Adrian F, Winter C, El-Ahmad Y, Halley F, Hsu K, Lager J, Garcia-Echeverria C, Bouaboula M. Identification of SAR439859, an orally bioavailable selective estrogen receptor degrader (SERD) that has strong antitumor activity in wild-type and mutant ER+ breast cancer models [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-04-05.

Despite resistance to endocrine therapies, estrogen receptor-positive (ER+) breast cancers (BC) still rely on ER. Eliminating ER by inducing its degradation with selective ER downregulators (SERD) should induce complete ablation of ER pathways. The clinical SERD fulvestrant, although it has demonstrated clinical benefits, is hampered by its poor drug properties and undesirable pharmacokinetics, undermining its optimal clinical activity. Therefore there is an unmet need for an improved therapy targeting ER. Here we describe SAR439859, a novel, nonsteroidal, orally bioavailable SERD currently in clinical trials. SAR439859 has a potent ER antagonist and ER degrading activity that translates in a robust inhibition of ER signaling in multiple ER+ breast cancer cell lines, including tamoxifen-resistant lines as well as cell lines harboring ER mutations. SAR439859 displays a broad and superior ER degradation activity across a large panel of ER+ cells. Importantly, based on its mechanism of action SAR439859 shows limited cross-resistance with other clinical SERD molecules. SAR439859 induces strong in vivo antitumor activity against a variety of BC cell lines and patient-derived xenografts, including models that harbor ERα mutations. The transcriptional profile analysis highlighted a crosstalk of ER mutant signaling and other oncogenic pathways. Interestingly, CDK4/6 inhibition by palbociclib induces partial activation of ER pathways as potential mechanism of tumor escape, which is completely abolished by the combination of SAR439859 with palbociclib. Finally, we demonstrate that SAR439859 in combination with palbociclib can lead to higher in vivo efficacy. This study highlights novel mechanism of ER degradation by SAR439859 that leads to profound inhibition of ER signaling as well as modulation of other oncogenic pathways and provides rationale for the ongoing clinical investigation of SAR439859 in ER+ breast cancer patients, both as a single agent and in combination with approved agents, such as CDK4 inhibitor. Citation Format: Monsif Bouaboula, Maysoun Shomali, Jane Cheng, Natalia Malkova, Fangxian Sun, Malvika Koundinya, Zhuyan Guo, Stephane Poirier, Mikhail Levit, Dietmar Hoffman, Hui Cao, Laurent Bestret, Francisco Adrian, Christoph Winter, Youssef El-Ahmad, Sylvie Vincent, Frank Halley, Gary McCort, Laurent Schio, Vicky Richon, Hong Cheng, Karl Hsu, Chris Soria, Patrick Cohen, Joanne Lager, Carlos Garcia-Echeverria, Laurent Debussche. SAR439859, an orally bioavailable selective estrogen receptor degrader (SERD) that demonstrates robust antitumor efficacy and limited cross-resistance in ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 943.