Therapeutics, BioNTx, and Polaris. L.L. serves on advisory boards for Servier. S.C.W. and J.P.A. are inventors of a patent application submitted by The University of Texas MD Anderson Cancer Center related to a genetic mouse model of immune checkpoint blockade induced immune-related adverse events. S.C.W. is currently an employee of Spotlight Therapeutics. E.M.W has ownership interest in Pathogenesis, LLC. W.C.M. was supported by funding from the Niels Stensen Fellowship and the Netherlands Heart Institute. D.B.J serves on advisory boards for Array Biopharma, BMS, Merck, Novartis; research funding from BMS and Incyte. J.E.S serves on advisory boards for BMS. J.E.S, D.B.J and J.J.M are inventors of a patent application submitted by The Assistance Publique-Hopitaux de Paris related to abatacept for the treatment of immune-related adverse events associated with immune checkpoint inhibitors. Research.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a stress-induced cardiac channelopathy that has a high mortality in untreated patients. Our understanding has grown tremendously since CPVT was first described as a clinical syndrome in 1995. It is now established that the deadly arrhythmias are caused by unregulated 'pathological' calcium release from the sarcoplasmic reticulum (SR), the major calcium storage organelle in striated muscle. Important questions remain regarding the molecular mechanisms that are responsible Matthew Wleklinski is an MD/PhD student interested in studying how mutations in calcium handling proteins lead to cardiac arrhythmias and sudden death. His current work focuses on the protein calsequestrin and its role in catecholaminergic polymorphic ventricular tachycardia. Dr

Triple-negative breast cancer (TNBC) is a subclass of breast cancers (i.e. estrogen receptor negative, progesterone receptor negative, and HER2 negative) that have poor prognosis and very few identified molecular targets. Strikingly, a high percentage of TNBC’s overexpress the epidermal growth factor receptor (EGFR), yet EGFR inhibition has yielded little clinical benefit. Over the last decade, advances in EGFR biology have established that EGFR functions in two distinct signaling pathways: 1) classical membrane-bound signaling, and 2) nuclear signaling. Previous studies have demonstrated that nuclear EGFR (nEGFR) can enhance resistance to anti-EGFR therapies and is correlated with poor overall survival in breast cancer. Based on these findings we hypothesized that nEGFR may promote intrinsic resistance to cetuximab in TNBC. To examine this question, a battery of TNBC cell lines and human tumors were screened and found to express nEGFR. Knockdown of EGFR expression demonstrated that TNBC cell lines retained dependency on EGFR for proliferation, yet all cell lines were resistant to cetuximab. Further, Src Family Kinases (SFKs) influenced nEGFR translocation in TNBC cell lines and in vivo tumor models, where inhibition of SFK activity led to potent reductions in nEGFR expression. Inhibition of nEGFR translocation led to a subsequent accumulation of EGFR on the plasma membrane, which greatly enhanced sensitivity of TNBC cells to cetuximab. Collectively, these data suggest that targeting both the nEGFR signaling pathway, through the inhibition of its nuclear transport, and the classical EGFR signaling pathway with cetuximab may be a viable approach for the treatment of TNBC patients.

Nuclear localized HER family receptor tyrosine kinases (RTKs) have been observed in primary tumor specimens and cancer cell lines for nearly two decades. Inside the nucleus, HER family members (EGFR, HER2, and HER3) have been shown to function as co-transcriptional activators for various cancer-promoting genes. However, the regions of each receptor that confer transcriptional potential remain poorly defined. The current study aimed to map the putative transactivation domains (TADs) of the HER3 receptor. To accomplish this goal, various intracellular regions of HER3 were fused to the DNA binding domain of the yeast transcription factor Gal4 (Gal4DBD) and tested for their ability to transactivate Gal4 UAS-luciferase. Results from these analyses demonstrated that the C-terminal domain of HER3 (CTD, amino acids distal to the tyrosine kinase domain) contained potent transactivation potential. Next, nine HER3-CTD truncation mutants were constructed to map minimal regions of transactivation potential using the Gal4 UAS-luciferase based system. These analyses identified a bipartite region of 34 (B1) and 27 (B2) amino acids in length that conferred the majority of HER3’s transactivation potential. Next, we identified full-length nuclear HER3 association and regulation of a 122 bp region of the cyclin D1 promoter. To understand how the B1 and B2 regions influenced the transcriptional functions of nuclear HER3, we performed cyclin D1 promoter-luciferase assays in which HER3 deleted of the B1 and B2 regions was severely hindered in regulating this promoter. Further, the overexpression of HER3 enhanced cyclin D1 mRNA expression, while HER3 deleted of its identified TADs was hindered at doing so. Thus, the ability for HER3 to function as a transcriptional co-activator may be dependent on specific C-terminal TADs.

Artificial transcription factors (ATFs) are precision-tailored molecules designed to bind DNA and regulate transcription in a preprogrammed manner. Libraries of ATFs enable the high-throughput screening of gene networks that trigger cell fate decisions or phenotypic changes. We developed a genome-scale library of ATFs that display an engineered interaction domain (ID) to enable cooperative assembly and synergistic gene expression at targeted sites. We used this ATF library to screen for key regulators of the pluripotency network and discovered three combinations of ATFs capable of inducing pluripotency without exogenous expression of Oct4 (POU domain, class 5, TF 1). Cognate site identification, global transcriptional profiling, and identification of ATF binding sites reveal that the ATFs do not directly target Oct4; instead, they target distinct nodes that converge to stimulate the endogenous pluripotency network. This forward genetic approach enables cell type conversions without a priori knowledge of potential key regulators and reveals unanticipated gene network dynamics that drive cell fate choices.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmia syndrome caused by gene mutations that render RYR2 calcium release channels hyperactive, provoking spontaneous calcium release and delayed afterdepolarizations (DADs). What remains unknown is the cellular source of ventricular arrhythmia triggered by DADs -

Heart failure (HF) is a complex disease characterized by abnormal contraction, metabolic imbalance, and increased propensity for arrhythmias. Dysregulation of intracellular Na þ handling is a major (yet understudied) aspect of HF-induced remodeling of cardiac myocytes. Elevated late Na þ current (I NaL ) in HF prolongs the action potential (AP), thereby facilitating the development of arrhythmogenic early afterdepolarizations. Increased Na þ loading limits the ability of the Na þ /Ca 2þ exchanger to remove Ca 2þ , which along with the reduced sarcoplasmic reticulum (SR) Ca 2þ uptake and increased diastolic SR Ca 2þ leak leads to Ca 2þ overload, thus contributing to diastolic dysfunction and triggered arrhythmias (i.e., via delayed afterdepolarizations). Increased Ca 2þ signals enhance the activity of the Ca 2þ /calmodulin-dependent protein kinase II (CaMKII), which is upregulated and chronically active in HF and directly promotes I NaL , diastolic Na þ influx and SR Ca 2þ leak. To investigate quantitatively this vicious cycle of positive feedback in HF, we updated our computational model of the failing rabbit ventricular myocyte. We modified the main repolarizing and depolarizing currents to reproduce the HF-induced changes measured during AP-clamp experiments performed with physiologic Ca 2þ handling 5 CaMKII inhibition. We validated the cellular model using data describing the frequency-dependence of AP and Ca 2þ transient properties assessed in normal condition and when various branches of the feedback loop are blocked. This updated model serves as a framework to investigate the role of the CaMKII-Na þ -Ca 2þ -CaMKII feedback in promoting Ca 2þ and AP instabilities. Analysis of the relative roles of the interacting components that form the feedback loop within the integrated AP-Ca 2þ cycling-signaling model will allow the identification of the key relationships in the signaling network that could be targeted therapeutically to limit arrhythmias in HF.

The HER family of receptor tyrosine kinases consist of four receptors, epidermal growth factor receptor (EGFR/ErbB1/HER1), HER2 (ErbB2), HER3 (ErbB3), and HER4 (ErbB4). Collectively, this family of receptors plays a critical role in initiating proliferation and survival signals in several human cancers. It is well established that the HER family receptors rely on two distinct compartments of signaling: 1) Classical membrane bound signaling and 2) nuclear signaling. In the nucleus, HER family receptors can serve as co-transcription factors, mediated by their C-terminal domains, to promote transcription of several genes essential for cell proliferation and cell cycle regulation, including regulation of the gene cyclin D1. However, the domains within the C-terminus of EGFR, HER2 and HER3 that confer this transcriptional potential (transactivation domains) have yet to be defined. In the current study we aimed to minimally map the regions of the C-terminal domains of EGFR, HER2 and HER3 that function as transactivation domains. We first demonstrate that EGFR, HER2, and HER3 are nuclear localized in their full-length forms in various breast and lung cancer cell lines. Next, we fused various intracellular cytoplasmic domain regions of each receptor to the DNA binding domain of the yeast transcription factor Gal4, and measured the ability for each construct to transactivate Gal4 UAS-luciferase activity. This analysis demonstrated that the C-terminal region distal to the tyrosine kinase domain (CTD) of all HER family receptors have strong transactivation potential, with HER2 exhibiting the highest transactivation potential. Further deletion mapping analysis of each receptor's CTD identified two regions (bipartite) of approximately 25-40 amino acids in length that harbored the majority of the receptors transactivation potential. To understand how the identified bipartite C-terminal regions of each HER family receptor influenced their transcriptional functions we deleted these regions from each full-length receptor and performed cyclin D1 promoter-luciferase assays. While wild type EGFR, HER2, and HER3 overexpression was capable of transactivating cyclin D1-luciferase, bipartite deleted receptors were severely hindered in their ability to regulate the cyclin D1 promoter. Collectively, the findings presented herein suggest that the EGFR, HER2 and HER3 contain a bipartite C-terminal transactivation domain that may be responsible for their ability to function as co-transcription factors. Citation Format: Toni M. Brand, Mari Iida, Matthew J. Wleklinski, Neha Luthar, Megan M. Starr, Deric L. Wheeler. Mapping C-terminal transactivation domains of nuclear HER family receptor tyrosine kinases. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4276. doi:10.1158/1538-7445.AM2013-4276