Neuronal Pentraxin2: a Novel Tumor-Specific Molecular Target That Mediates Clear Cell Renal Cell Carcinoma Malignancy Christina A

Neuronal Pentraxin2: A Novel Tumor-Specific Molecular Target That Mediates Clear Cell Renal Cell Carcinoma Malignancy Christina A. von Roemeling1, Derek Radisky1, Laura Marlow1, Simon J. Cooper1, Stefan K Grebe2, Panagiotis Z. Anastasiadis1, Han W. Tun1,3, John A. Copland1 1Department of Cancer Biology, Mayo Clinic Florida, Jacksonville, Florida; 2Laboratory Medicine and Pathology, Mayo Clinic Minnesota, Rochester, MN; 3Department of Hematology/Oncology, Mayo Clinic Florida, Jacksonville, Florida April 7, 2014: Abstract LB-111 y BACKGROUND RESULTS CONCLUSIONS

RCC: Figure 1: NPTX2 Expression Profile in ccRCC Figure 2: Role of NPTX2 in ccRCC Viability and Invasion th • NPTX2 is highly over-expressed in ccRCC at the transcriptional and protein level • Renal cell carcinoma (RCC) is the third most prevalent urological cancer, the 10 A498 KIJ265T Caki2 th A C % cell death • NPTX2 expression is specific for the clear cell subtype of RCC

A most common cause of cancer death in men and the 9 most common cause in A) Nextbio was used to assess publicly NT sh1316 available expression datasets for NPTX2 in NT A498 2.7 71.3 • NPTX2 consistently is the most differentially expressed gene transcript in ccRCC women. the context of normal vs. tumor, clear cell KIJ265T 2.7 25.5 Caki2 1.1 25.0 NPTX2 expression is strongly correlated with advanced and metastatic ccRCC • The clear cell variant of RCC (ccRCC) is the most common subtype accounting for vs. other subtypes, and localized vs. • advanced ccRCC. B) Meta-analysis of gene • Loss of NPTX2 expression leads to decreased tumor cell growth, loss of tumor ~80% of all renal cancers. expression in 13 RCC gene expression • Due to its asymptomatic nature, it is estimated that up to 30% of patients present datasets for NPTX2. C) IHC for NPTX2 B sh1316 cell viability, and decreased invasive capacity protein expression in patient normal and • We identify that GluR4 complexes with NPTX2 in ccRCC tumor cells with metastatic ccRCC at time of diagnosis. Furthermore, due to its highly matched ccRCC tissue across all stages of metastatic nature 20-30% of patients diagnosed with localized disease will relapse disease. Stage I, II, III, IV, met: normal D • GluR4 is over-expressed in ccRCC tissue, and demonstrates the highest level of n=44, 32, 35, 7, and 6 and tumor n= 41, 26, expression in metastatic tissues with metastatic ccRCC. 33, 10, and 17 respectively. Expression is • For individuals presenting with advanced disease, treatment options are limited with presented as mean H-score +/- standard • Loss of GluR4 expression similarly leads to decreased cell proliferation, loss of deviation. no current drug therapy leading to long term survival with the exception of 6-7% of tumor cell viability, and decreased invasive capacity of ccRCC cells E patients who respond to interleukin-2. F • ccRCC rarely responds to chemotherapy and radiation therapies, and drug B resistance develops rapidly with application of targeted therapies. • Genetic factors contributing to ccRCC development, progression, and metastasis are poorly defined. CLINICAL IMPACT NPTX2: • Neuronal pentraxin 2 (NPTX2) belongs to a class of secreted proteins characterized by their pentraxin domain, and are related to C-reactive protein (CRP). • We demonstrate an oncogenic role for NPTX2 for the first time in a cancer • NPTX2 is typically expressed in nervous system, testicular, pancreatic, skeletal system muscle, and hepatic tissues, with little to no expression observed in normal renal • As NPTX2 is a secreted protein that is consistently and specifically tissue. A, ccRCC vs normal; B, ccRCC vs chromophobe RCC; C, ccRCC vs granular RCC; D, ccRCC vs papillary RCC overexpressed in patient ccRCC samples, it presents as: C • NPTX2 is best characterized for its role in neurite outgrowth and synaptic plasticity A) Western blot of normal and ccRCC cells for NPTX2 expression. B) mRNA expression of lentiviral • A candidate diagnostic biomarker for patients with the clear cell subtype of of neuronal cells: mediates the clustering of the AMPA family of ionic glutamate NT control and NPTX2 targeted (sh1316) ccRCC cell lines. C) Propidium iodide stain for cell death RCC quantitation and D) western blot to examine apoptosis via PARP cleavage of NT and NPTX2 targeted receptors, forming ion permeable channels during excitatory synaptogenesis. ccRCC cells. E) Invasion assay evaluating invasive capacity in KIJ265T and A498 (high endogenous • A candidate prognostic marker for patients at risk of disease recurrence and/or NPTX2 expression) with loss of NPTX2, and overexpression of NPTX2 in Caki1 and RWV366T (low the development of metatstatic disease endogenous NPTX2 expression). Invasion is quantitated as number of invading cells per visual field. F) Immunofluorescence in RWV366T control and NPTX2 overexpression cells and A498 control and • An optimal therapeutic target whose inhibition may clinically benefit a broad NPTX2 knockdown cells for NPTX2 expression and VASP localization- a regulatory protein involved in actin-based motility. spectrum of patients STUDY DESIGN

Figure 4: Loss of GluR4 Recapitulates NPTX2 Knockdown Figure 3: GluR4 in ccRCC and Interaction with NPTX2 Translational Relevance: FUTURE DIRECTIONS B Phenotype Patients with metastatic ccRCC have poor prognoses, with an estimated 5 year overall A survival of less than 10%. This is due to lack of remedial therapies that produce durable A C • Development of a monoclonal antibody or small molecule inhibitor of NPTX2 to disease stabilization or tumor regression. Drug resistance is a hallmark be tested for therapeutic efficacy in vivo of ccRCC and is linked to cancer cell heterogeneity. Additionally, there is a lack of • Evaluate NPTX2 expression in patient blood samples and correlate to disease known molecular factors that can be targeted pharmacologically. The clear cell variant C staging and progression to evaluate efficacy as a diagnostic and prognostic of RCC also frequently manifests as metastatic disease, likely due to its disposition for B biomarker increased migratory capacity and the formation of undetectable micrometastases. A • Delineate the signaling mechanism of NPTX2 oncogenic activity focus on identifying both factors that contribute to ccRCC cell migration and those that • Evaluate the efficacy of AMPA antagonists in vivo are therapeutically targetable is paramount.

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