*Note:You currently have Javscript Disabled.
You are also using an older version of Internet Explorer
. This page is optimized for Google Chrome and Mozilla Firefox
or Internet Explorer plus Javascript
. While we recommend that you enable javascript or use a different browser, you can view all relevant information on your current browser.

Sean W. Fanning, Ph.D.

Postdoctoral Research

The University of Chicago Ben May Department for Cancer Research
(May 2012 – Present)

I am very fortunate to currently work for Dr. Geoffrey Greene at the Ben May Department for Cancer Research at the University of Chicago. I work on the estrogen receptor (ER), an important protein for breast cancer. Specifically, I investigate how mutations to the estrogen receptor lead to metastatic breast tumors that are resistant to anti-estrogen therapies.

The estrogen receptor alpha (ERα) is critical for the etiology and treatment of breast cancer. In fact, up to 70% of breast cancers express ERα and are sensitive to anti-estrogen therapies. Tamoxifen and raloxifene represent members of a family of anti-estrogen drugs, termed selective estrogen receptor modulators (SERMs), which are approved to treat or reduce the risk of ER-dependent breast cancers. Classical SERMs act by competitively binding to the ERα ligand-binding domain (LBD). Unfortunately, SERM resistance is acquired in about half of ER-positive patients after five years of treatment1. We recently identified three somatic mutations to ESR1 (537S, 538G and 463P) in metastatic breast tumors from patients who previously received SERM/SERD and/or AI treatment for 1.5 to 10 years (average of 5 years) FOUND HERE. Because these mutations are present in a significant population (>20%), generate an ERα which is active independent of hormone, and were not observed in primary tumors, we hypothesize that this represents a possible mechanism for acquired SERM-resistance. Our work will provide a comprehensive understanding of how these mutations impact the ERα function from intimate molecular interactions to the downstream effects on ERα signaling within the tumor environment and metastasis. Importantly, our research will both lay the foundation for the rapid generation of improved drugs and provide guidance to clinicians treating patients possessing these mutations in metastatic, SERM resistant, breast tumors.

The ER is a ligand-activated transcription factor, belongs to the nuclear hormone receptor family (NHR), and exists in two isoforms, ERα and ERβ. ERα predominates in ER+ breast cancers and is a dominant regulator of cell growth, survival and metastasis via genomic and non-genomic actions. Once estrogen binds to the receptor, a cascade of cellular events ensue which ultimately lead to increased tumor growth and metastasis. Because of its key role in breast cancer tumorgenesis, inhibition of ER via selective estrogen receptor modulators (SERMs) remains one of the most successful targeted therapeutic strategies. Due to their wide therapeutic index patients may receive SERM treatments for as long as ten years. However, the majority of breast cancers become refractory to SERM treatment after prolonged exposure. Too often, once a patient has acquired SERM-resistant disease they are moved onto cytotoxic therapies, which are less efficacious and convey a dramatic reduction in quality of life, before ultimately succumbing to the disease. SERM resistance represents a significant clinical challenge which must be overcome to improve the prognosis and quality of life for a significant population of breast cancer patients.

Despite its pervasiveness, the mechanisms by which SERM resistance is acquired are not well understood. Numerous mechanisms have been proposed to explain how progressive ER-positive breast cancers acquire SERM resistance. These include avoidance of SERM-induced tumor apoptosis through the up regulation of MAP kinase, AKT or HER-2/neu signaling. However, these mechanisms fail to account for all acquired anti-estrogen resistance and do not represent viable routes for therapeutic intervention using existing treatment strategies. One possible mechanism of SERM resistance is by somatic mutation to residues near the ERα ligand binding site. In fact, somatic mutations are a common mechanism for drug resistance in other cancers. For prostate cancer patients with prolonged exposure to anti-androgen therapies (e.g. enzalutamide), the androgen receptor (AR), another NHR, becomes insensitive to anti-androgen treatments through mutations in the AR-LBD. In light of these findings, comprehensive studies have been undertaken to explore whether somatic mutations in ERα are present in SERM resistant metastatic breast tumors2. We recently identified three ESR1 (the gene for ERα) mutations (S463P, D538G, and Y537S) in 9/36 breast tumors from patients who previously received SERM and/or aromatase inhibitor (AI) treatment for an average of 5 years in collaboration with Dr. Sarat Chandarlapaty at Memorial Sloan-Kettering Cancer Center (MSKCC). Compellingly, these mutations were not identified in a study of over 800 primary breast tumors (e.g. no previous exposure to SERM/AI) investigated by TCGA. In contrast, other common mutations such as TP53 and PIK3CA were observed at comparable levels between the two datasets. Further, we showed that these mutations produce a constitutively active ERα. Importantly, additional studies have subsequently confirmed our findings by identifying Y537S and D538G activating somatic mutations in AI/SERM-resistant metastatic breast tumors.

Doctoral Research

Northern Illinois University (August 2008 – May 2012)

My graduate research was conducted at Northern Illinois University between 2008 and 2012 advised by Dr. James R. Horn in the Department of Chemistry and Biochemistry. During this time, I used protein engineering, biophysics and structural biology to advance our understanding of the camelid heavy chain-only single domain (VHH) antibody. Antibodies are invaluable affinity reagents in many diagnostic and therapeutic applications. Camelid heavy chain-only single domain (VHH) antibodies are of particular interest because despite lacking a variable light chain, they possess affinities and specificities rivaling full length IgG antibodies. In addition, their relative simplicity and ideal biophysical properties make the VHH antibody an excellent scaffold for protein engineering.

Study 1: The anti-Methotrexate VHH antibody recognizes MTX with a cryptic binding mechanism:
Antibodies are well known for their ability to recognize target molecules with high affinity and specificity. Although many antibodies recognize protein antigens, those that bind haptens (e.g. small molecule drugs) hold some of the greatest potential. While the biophysical and structural details of hapten binding have been well characterized for many anti-hapten antibodies, detailed studies of anti-hapten single-domain VHH antibodies were lacking. In this work I used isothermal titration calorimetry and x-ray crystallography to investigate the binding affinity and specificity for a VHH antibody which recognizes the anti-metabolic drug Methotrexate (MTX). When I obtained the first x-ray crystal structure I was very surprised to discover that the anti-MTX VHH recognized the drug with a completely novel binding mechanism (top right). The MTX drug is shown as green sticks with the blue and red sticks indicating nitrogen and oxygen atoms respectively. The red, blue, orange and yellow highlight the regions of the protein called complementarity determining regions which recognize the drug. While other VHHs which target small molecules bind them on the surface of the protein, the anti-MTX VHH undergoes a dramatic rearrangement to form a binding pocket for the drug in the interior of the protein. The second and third images shown to the right display the protein before and after MTX binding. The work described here was published in Protein Science in July, 2011 and was the featured cover art (let me know if you want a signed copy). If you are interested in reading the full manuscript it can be FOUND HERE

Study 2: A combinatorial approach towards the introduction of a metal-switch to control protein-protein interactions:
The second focus of my graduate research was the development of novel synthetic combinatorial library techniques to engineer multi-specificity into an anti-RNase A VHH antibody. To make that last sentence a little easier to understand: a synthetic combinatorial library (in protein science) is a library of variants of one protein. You can use these techniques to create billions of variants of one protein. As such, rather than rationally designing mutants of one protein which can be time consuming, you take a "shotgun" approach where you make tens of billions of variants of the protein then select for those which demonstrate your desired properties. Here our goal was to create libraries which could be used to tune the protein-binding affinity of an antibody using a metal but with minimal impact to its native affinity. We used an existing VHH antibody which recognized the protein RNase A as our model. The scheme below demonstrates how a metal could be used to tune the binding affinity:

A combinatorial phage display library was generated which sampled histidine and the wild-type residue across the anti-RNase A VHH antigen binding interface (CDRs 1 and 3). Histidine was chosen because bi-histidines represent a minimalist protein-metal binding motif. This histidine-scanning library, when combined with a novel co-selection strategy, produced a VHH variant termed "Metal VHH", which incorporated three histidines on the periphery of the antigen-binding interface. Biophysical investigations of Metal VHH revealed that it was specific for Ni2+ versus other transition metal ions. Furthermore, the Metal VHH's RNase A affinity could be tuned based on the amount of metal ion present while retaining near-native binding affinity towards RNase A. Further structural and biophysical investigations demonstrated that metalloregulation of VHH/RNase A binding was facilitated by a dual-function histidine and a flexible CDR1. The images on the right show the Metal VHH binding to Ni2+. If you are interested in reading more you can check out the initial publication from this project. The follow up manuscript describing the structural details of this metalloregulation should be submitted soon!