Rational Targeting of Notch in Breast Cancer

Rational Targeting of Notch in Breast Cancer Paola Rizzo, Clodia Osipo, Kimberly Foreman, Jieun Yun, Todd Golde, Barbara Osborne, Lucio Miele Abstract The importance of Notch signaling in solid tumors and particularly breast cancer is being increasingly appreciated. This has led to the development of Notch inhibitors, such as g-secretase inhibitors (GSIs), as candidate therapeutic agents. To determine whether and how these agents will be useful in the clinic it will be necessary to identify which patients are most likely to benefit from them, which agents should be combined with them and which agents have the best safety and efficacy profiles. Notch signaling intersects with multiple other pathways which include other therapeutic targets, and understanding these cross-talk interactions will be critical to the design of rational combination regimens. We have determined that estrogen and Her2/Neu regulate Notch signaling in breast cancer. Our data indicate that combinations of anti-estrogens and Notch inhibitors may be effective in ERa-positive breast cancers and possibly safer than GSI alone. Our data also indicate that combinations of trastuzumab or RTKis with Notch inhibitors may be effective in Her2/Neu positive cancers. Moreover, our data confirm the importance of Notch signaling in putative breast cancer stem cells. This brief presentation will summarize the current state of the field with emphasis on breast cancer, and describe future challenges and opportunities for Notch-targeted therapies. Background Among solid tumors, breast cancer may be the one in which the clinical relevance of Notch signaling is most firmly established (Fig. 1). Notch-1 is commonly expressed in breast cancer and can transform immortalized MCF-10A cells in vitro (1). Expression of Notch-1 and Jagged-1 correlates with poor prognosis and is an independent predictor of outcome in breast cancer (2, 3). This is in agreement with preclinical data that clearly show that at least 2 Notch homologs (Notch-1 and Notch-4) have oncogenic activity in vitro and in mice. Notch-2, on the other hand, may antagonize the effects of Notch-1 and -4 in breast and seems to have positive prognostic significance in clinical specimens and (4). Additionally, normal mammary stem cells and putative “breast cancer stem cells” isolated from DCIS require Notch signaling for mammosphere formation (5, 6). This has attracted considerable interest in targeting Notch signaling in breast cancer for therapeutic purposes. Author Affiliations: Loyola University Chicago, Maywood, Illinois, The Mayo Clinic, Jacksonville, Florida, The University of Massachusetts at Amherst, Amherst, Massachusetts ©2008 American Association for Cancer Research AACR Education Book • http://educationbook.aacrjournals.org 491 Emerging Targets: Wnt, Notch, Sonic Hedgehog Fig. 1. Diagram of putative therapeutic targets in the Notch pathway. Asterisks indicate targets for which there is molecular and/or genetic evidence but no specific agents have been developed yet. Question marks indicate potential targets, yet unverified. Theoretically, inhibition of Notch signaling could be achieved by targeting ligand ubiqutination (UQ), ligand-receptor interaction, NEC/ NIC dissociation, ADAM-mediated cleavage, receptor ubiquitination/endocytosis, g-secretase cleavage, assembly of the co-activator complex with Notch and CBF-1 and heterodimerization of DNA-bound Notch transcriptional complexes (NTC). For some of these steps agents have been produced that are in pre-clinical or clinical development. 492 AACR Annual Meeting 2008 • April 12–16, 2008 Rational Targeting of Notch in Breast Cancer Clinical and Translational Leads Based on preclinical and clinical evidence, the current consensus is that inhibitors of Notch signaling are attractive candidate agents for the treatment of some breast cancers. Inhibition of Notch signaling can be achieved theoretically by targeting ligand-receptor interactions (7), ligand ubiquitination/trans-endocytosis (8) ligand-induced conformational changes in Notch receptors (9), receptor cleavage by ADAM proteases (10) and g-secretase (11, 12), Notch monoubiquitination (13) or protein-protein interactions involved in Notch-dependent nuclear events (14, 15), including assembly of co-activators with the Notch transcriptional complex (NTC) and formation of higher order DNA-bound complexes. As of this writing, GSIs are in early clinical trials in various institutions (12), and monoclonal antibodies (mAb) that lock Notch receptors in an inactive conformation are in preclinical development (16). Early clinical experience with GSIs indicates that the main adverse event in patients is dose-limiting secretory diarrhea caused by goblet cell metaplasia of the small intestine, which was first observed in preclinical models (17). Our group is attempting to systematically dissect the cross-talk between Notch signaling and other pathways known to be relevant to breast cancer. The goal is to identify subsets of patients who may benefit the most from treatment with Notch inhibitors, and to generate proof of concept for rationally designed combination regimens including Notch inhibitors to be used in breast cancer patients. Our data indicate that: 1) Notch signaling is prominently regulated by estrogen and Her2/Neu; 2) estrogen receptor a (ERa)-negative and Her2/Neu negative cancers have higher Notch activity and may respond to Notch inhibitors; 3) Combinations of SERMS plus GSIs are particularly effective in ERa positive tumors; 4) Combinations of trastuzumab plus GSIs or receptor tyrosine kinase (RTK) inhibitors plus GSIs are likely to be effective in Her2/Neu overexpressing tumors; and 5) Notch activation is associated with some cases of tamoxifen resistance. An unexpected but potentially useful observation was that the intestinal toxicity of GSIs appears to be estrogen-dependent in mice. Co-treatment with tamoxifen greatly ameliorated the intestinal side effects of orally administered GSIs, suggesting that this combination may be not only more effective but also safer than single agent GSI treatment. Based on these data, a clinical trial of neoadjuvant antiestrogen plus GSI combination is currently underway at our institution. Challenges and Future Directions The development of cancer treatments targeting Notch signaling is in its infancy. What we know so far is that several solid tumors and breast cancer in particular may potentially benefit from Notch inhibition. However, we do not yet know how to select the patients who are most likely to benefit from Notch-targeted therapeutics, what agents or classes of agents are most likely to synergize with Notch inhibitors in vivo, which Notch inhibitors will have the best activity and safety profiles, or how to manage the mechanism-based toxicities of Notchtargeted therapeutics. To identify patients who are likely to benefit from Notch inhibitors, simply determining expression of receptors and ligands will not suffice. Because of extensive cross-talk between Notch and other pathways, as well as differential effects of different Notch receptors and ligands, there is no simple correlation between expression and Notch activity. It will be necessary to develop and validate accurate molecular tests that assess the level of AACR Education Book • http://educationbook.aacrjournals.org 493 Emerging Targets: Wnt, Notch, Sonic Hedgehog pathway activity in vivo, possibly based on expression levels of multiple genes in the pathway. To design rational combination regimens, the cross-talk between Notch and other potential therapeutic targets will have to be elucidated in preclinical models and tested in pilot trials. Each class of candidate Notch inhibitors has pros and cons. As of this writing, GSIs are the leading class of Notch inhibitors in clinical development. These are relatively simple drugs, they are orally bioavailable and they are relatively safe and well tolerated in short-term treatment regimens. Regimens that minimize intestinal toxicity can be utilized (by administering the drugs for a few days followed by a few days off). Intestinal toxicity may be prevented by co-administration of intestine-protecting drugs such as tamoxifen or glucocorticoids. On the other hand, targeting g-secretase is bound to have off-target effects on Notch-unrelated substrates, and chronic oral administration may lead to unacceptable toxicity. Agents that do not inhibit the enzyme competitively but rather target the interaction of specific substrates with it are being actively sought (18). These “g-secretase modifiers” (GSMs) may turn out to be more specific, and possibly less toxic, than GSIs. It is not clear that oral administration is optimal for Notch inhibitors, given that the intestine is the main toxicity target. Parenteral pharmaceutical formulations of GSIs (or GSMs), perhaps in the context of nanocarriers containing other agents, may turn out to be preferable. Biologics such as mAbs have the advantage of high specificity, and could be targeted to a single Notch receptor or ligand. However, this would not necessarily prevent mechanism-based toxicities, and the long biological half-life of mAbs may make it more difficult to fine-tune exposure time. This problem could be managed by using smaller mAb derivatives such as scFVs or F(ab)s. Finally, it is conceivable that targeting other components of the Notch pathway necessary for Notch activity, such as co-activators (19) or targeting non-canonical Notch signaling (20), may be preferable in some cases to targeting receptors, ligands or g-secretase. References 1. Stylianou S, Clarke RB, Brennan K. Aberrant activation of notch signaling in human breast cancer. Cancer Res. 2006;66:1517–25. 2. Reedijk M, Odorcic S, Chang L, et al. High-level coexpression of JAG1 and NOTCH1 is observed in human breast cancer and is associated with poor overall survival. Cancer Res. 2005;65:8530–7. 3. Dickson BC, Mulligan AM, Zhang H, et al. High-level JAG1 mRNA and protein predict poor outcome in breast cancer. 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Nam Y, Sliz P, Pear WS, Aster JC, Blacklow SC. Cooperative assembly of higher-order Notch complexes functions as a switch to induce transcription. Proc Natl Acad Sci U S A. 2007;104:2103–8. 16. Li K, Li Y, Wu W, et al. Modulation of notch signaling by antibodies specific for the extracellular negative regulatory region of Notch3. J Biol Chem. 2008. In press. 17. Wong GT, Manfra D, Poulet FM, et al. Chronic treatment with the gamma-secretase inhibitor LY411,575 inhibits beta-amyloid peptide production and alters lymphopoiesis and intestinal cell differentiation. J Biol Chem. 2004;279:12876–82. 18. Bakshi P, Liao YF, Gao J, Ni J, Stein R, Yeh LA, Wolfe MS. A high-throughput screen to identify inhibitors of amyloid beta-protein precursor processing. J Biomol Screen. 2005;10:1–12. 19. Allenspach EJ, Maillard I, Aster JC, Pear WS. Notch signaling in cancer. Cancer Biol Ther. 2002;1:466–76. 20. Osipo C, Golde TE, Osborne BA, Miele L. Off the beaten pathway: the complex cross talk between Notch and NF-kB. Lab Invest. 2008;88:11–7. AACR Education Book • http://educationbook.aacrjournals.org 495