EphrinB2 Inhibition and Pembrolizumab in Metastatic Urothelial Carcinoma.

PURPOSE: Patients with metastatic urothelial carcinoma have poor prognosis after failure of standard first-line chemotherapy. Immune check point programmed death 1-programmed death ligand 1 antibodies have low response rates and thus there exists a major unmet need. MATERIALS AND METHODS: In this phase II trial, patients with metastatic urothelial carcinoma that recurred or progressed after platinum-based chemotherapy received soluble EphB4-human serum albumin (sEphB4-HSA) in combination with pembrolizumab. The primary end points were tolerability and overall survival (OS). The secondary end points were progression-free survival (PFS), objective response rate (ORR), duration of response, and toxicity. The expression of sEphB4-HSA target EphrinB2 was correlated with outcomes. RESULTS: Seventy patients were enrolled. The median follow up was 22.9 months (range, 1.3-54.7). The regimen had acceptable toxicity. In the intent-to-treat analysis (N = 70), the median OS was 14.6 months (95% CI, 9.2 to 21.5). Twenty-six (37%) patients had an objective response (95% CI, 26 to 48). The median PFS was 4.1 (95% CI, 1.5 to 5.7) months. Forty-six (66%) patients expressed EphrinB2, and among them, the median OS was 21.5 months (95% CI, 12.4 to not reached), the ORR was 52% (95% CI, 37 to 67), including a complete response rate of 24% (11 of 46; 95% CI, 12 to 36). The median PFS was 5.7 (95% CI, 2.7 to 27.9) months. Response was maintained at 6, 12, and 24 months in 88%, 74%, and 69% of the patients, respectively. CONCLUSION: The combination of sEphB4-HSA and pembrolizumab appears synergistic with improved OS and ORR compared with historical data for programmed death 1/programmed death ligand 1 monotherapy.

PD-1 Blockade on Tumor Microenvironment-Resident ILC2s Promotes TNF-α Production and Restricts Progression of Metastatic Melanoma.

While pulmonary ILC2s represent one of the major tissue-resident innate lymphoid cell populations at steady state and are key drivers of cytokine secretion in their occupational niche, their role in pulmonary cancer progression remains unclear. As the programmed cell death protein-1 (PD-1) plays a major role in cancer immunotherapy and immunoregulatory properties, here we investigate the specific effect of PD-1 inhibition on ILC2s during pulmonary B16 melanoma cancer metastasis. We demonstrate that PD-1 inhibition on ILC2s suppresses B16 tumor growth. Further, PD-1 inhibition upregulates pulmonary ILC2-derived TNF-α production, a cytotoxic cytokine that directly induces cell death in B16 cells, independent of adaptive immunity. Together, these results highlight the importance of ILC2s and their anti-tumor role in pulmonary B16 cancer progression during PD-1 inhibitory immunotherapy.

Quantitative Tyrosine Phosphoproteome Profiling of AXL Receptor Tyrosine Kinase Signaling Network.

Overexpression and amplification of AXL receptor tyrosine kinase (RTK) has been found in several hematologic and solid malignancies. Activation of AXL can enhance tumor-promoting processes such as cancer cell proliferation, migration, invasion and survival. Despite the important role of AXL in cancer development, a deep and quantitative mapping of its temporal dynamic signaling transduction has not yet been reported. Here, we used a TMT labeling-based quantitative proteomics approach to characterize the temporal dynamics of the phosphotyrosine proteome induced by AXL activation. We identified >1100 phosphotyrosine sites and observed a widespread upregulation of tyrosine phosphorylation induced by GAS6 stimulation. We also detected several tyrosine sites whose phosphorylation levels were reduced upon AXL activation. Gene set enrichment-based pathway analysis indicated the activation of several cancer-promoting and cell migration/invasion-related signaling pathways, including RAS, EGFR, focal adhesion, VEGFR and cytoskeletal rearrangement pathways. We also observed a rapid induction of phosphorylation of protein tyrosine phosphatases, including PTPN11 and PTPRA, upon GAS6 stimulation. The novel molecules downstream of AXL identified in this study along with the detailed global quantitative map elucidating the temporal dynamics of AXL activation should not only help understand the oncogenic role of AXL, but also aid in developing therapeutic options to effectively target AXL.

Tyrosine Phosphoproteomics of Patient-Derived Xenografts Reveals Ephrin Type-B Receptor 4 Tyrosine Kinase as a Therapeutic Target in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma is a recalcitrant tumor with minimal response to conventional chemotherapeutic approaches. Oncogenic signaling by activated tyrosine kinases has been implicated in cancers resulting in activation of diverse effector signaling pathways. Thus, the discovery of aberrantly activated tyrosine kinases is of great interest in developing novel therapeutic strategies in the treatment and management of pancreatic cancer. Patient-derived tumor xenografts (PDXs) in mice serve as potentially valuable preclinical models as they maintain the histological and molecular heterogeneity of the original human tumor. Here, we employed high-resolution mass spectrometry combined with immunoaffinity purification using anti-phosphotyrosine antibodies to profile tyrosine phosphoproteome across 13 pancreatic ductal adenocarcinoma PDX models. This analysis resulted in the identification of 1199 tyrosine-phosphorylated sites mapping to 704 proteins. The mass spectrometric analysis revealed widespread and heterogeneous activation of both receptor and non-receptor tyrosine kinases. Preclinical studies confirmed ephrin type-B receptor 4 (EphB4) as a potential therapeutic target based on the efficacy of human serum albumin-conjugated soluble EphB4 in mice bearing orthotopic xenografts. Immunohistochemistry-based validation using tissue microarrays from 346 patients with PDAC showed significant expression of EphB4 in >70% of patients. In summary, we present a comprehensive landscape of tyrosine phosphoproteome with EphB4 as a promising therapeutic target in pancreatic ductal adenocarcinoma.

A Novel DNA Methylation Signature as an Independent Prognostic Factor in Muscle-Invasive Bladder Cancer.

BACKGROUND: Muscle-invasive bladder cancer (MIBC) accounts for approximately 20% of all urothelial bladder carcinomas (UBC) at time of diagnosis, and up to 30% of patients with non-muscle invasive UBC will progress to MIBC over time. An increasing body of evidence has revealed a strong correlation between aberrant DNA methylation and tumorigenesis in MIBC. RESULTS: Using The Cancer Genome Atlas (TCGA) molecular data for 413 patients, we described a DNA methylation-based signature as a prognostic factor for overall survival (OS) in MIBC patients. By using a least absolute shrinkage and selection operator (LASSO) model, differentially methylated regions were first identified using multiple criteria followed by survival and LASSO analyses to identify DNA methylation probes related to OS and build a classifier to stratify patients with MIBC. The prognostic value of the classifier, referred to as risk score (RS), was validated in a held-out testing set from the TCGA MIBC cohort. Finally, receiver operating characteristic (ROC) analysis was used to compare the prognostic accuracy of the models built with RS alone, RS plus clinicopathologic features, and clinicopathologic features alone. We found that our seven-probe classifier-based RS stratifies patients into high- and low-risk groups for overall survival (OS) in the testing set (n = 137) (AUC at 3 years, 0.65; AUC at 5 years, 0.65). In addition, RS significantly improved the prognostic model when it was combined with clinical information including age, smoking status, Tumor (T) stage, and Lymph node metastasis (N) stage. CONCLUSIONS: The DNA methylation-based RS can be a useful tool to predict the accuracy of preoperative and/or post-cystectomy models of OS in MIBC patients.

Tetraspanin18 regulates angiogenesis through VEGFR2 and Notch pathways.

The VEGF pathway is critically required for vasculogenesis, the formation of the primary vascular network. It is also required for angiogenesis resulting in sprouting and pruning of vessels to generate mature arborizing structures. The Notch pathway is essential for arterial-venous specification and the maturation of nascent vessels. We have determined that Tspan18, a member of the Tetraspanin family, is expressed in developing vessels but not in mature vasculature in zebrafish and mouse wound healing. Moreover, reduction at Tspan18 level resulted in aberrant vascular patterning, impaired vessel stability and defective arterial-venous specification. Tspan18 deficiency reduced VEGF, VEGFR2, Notch3 and EphrinB2, and increased EphB4, VEGFR3, Semaphorin3, Neuropilin and PlexinD1 expression. Furthermore, vascular defects of Tspan18 deficiency could be rescued by ectopic expression of VEGFR2 and Notch, but not by knockdown of Semaphorin or Plexin. Functional studies showed that knockdown of Tspan18 led to reduced endothelial cell migration, invasion and tube formation. Tspan18 has dynamic expression, regulates vascular development and maturation in the embryo with re-expression in adult life in wound healing.

Delta-like ligand 4-mediated Notch signaling controls proliferation of second heart field progenitor cells by regulating Fgf8 expression.

The role played by the Notch pathway in cardiac progenitor cell biology remains to be elucidated. Delta-like ligand 4 (Dll4), the arterial-specific Notch ligand, is expressed by second heart field (SHF) progenitors at time-points that are crucial in SHF biology. Dll4-mediated Notch signaling is required for maintaining an adequate pool of SHF progenitors, such that Dll4 knockout results in a reduction in proliferation and an increase in apoptosis. A reduced SHF progenitor pool leads to an underdeveloped right ventricle (RV) and outflow tract (OFT). In its most severe form, there is severe RV hypoplasia and poorly developed OFT resulting in early embryonic lethality. In its milder form, the OFT is foreshortened and misaligned, resulting in a double outlet right ventricle. Dll4-mediated Notch signaling maintains Fgf8 expression by transcriptional regulation at the promoter level. Combined heterozygous knockout of Dll4 and Fgf8 demonstrates genetic synergy in OFT alignment. Exogenous supplemental Fgf8 rescues proliferation in Dll4 mutants in ex-vivo culture. Our results establish a novel role for Dll4-mediated Notch signaling in SHF biology. More broadly, our model provides a platform for understanding oligogenic inheritance that results in clinically relevant OFT malformations.

Programmed Death 1 and Programmed Death Ligand 1 Inhibitors in Advanced and Recurrent Urothelial Carcinoma: Meta-analysis of Single-Agent Studies.

We performed a systematic review and meta-analysis on the response rates of patients with treatment-refractory urothelial carcinoma treated with programmed cell death 1 (PD-1) and programmed death ligand 1 (PD-L1) inhibitors. We reviewed the literature for prospective studies evaluating PD-1/PD-L1 inhibitors in refractory urothelial carcinoma patients, which formed the basis for US Food and Drug Administration approval of 5 different antagonistic antibodies targeting PD-1 or PD-L1 (atezolizumab, durvalumab, avelumab, nivolumab, and pembrolizumab). We considered studies examining PD-1/PD-L1-treated patients, which we identified using the following key terms in the Pubmed, Scopus, Web of Science, ClinicalTrial.gov, and Cochrane Library databases. Eligible studies had ≥ 20 patients each and reported response rates, duration of response, and overall survival (OS). We performed fixed and random-effects meta-analyses to model the point estimates for objective response rate and complete response. The median progression-free survival (PFS) and OS for studies reporting these statistics were evaluated. We found 10 eligible studies that met our inclusion criteria, providing extractable numerators and denominators for response rates, PFS, and OS for 1934 patients with metastatic urothelial carcinoma. The objective response rate was 18% (95% confidence interval, 15-22) for second-line or later therapies. The random-effects estimate for complete response was 4% (95% confidence interval, 3-5), including all disease locations and all PD-1 and PD-L1 inhibitors. Median OS and PFS were < 13 months and 3 months, respectively, across all studies, irrespective of PD-L1 expression. We found that the estimated response rates of agents included in this meta-analysis seem to be more favorable than other salvage therapies.

EPHB4 inhibition activates ER stress to promote immunogenic cell death of prostate cancer cells.

The EPHB4 receptor is implicated in the development of several epithelial tumors and is a promising therapeutic target, including in prostate tumors in which EPHB4 is overexpressed and promotes tumorigenicity. Here, we show that high expression of EPHB4 correlated with poor survival in prostate cancer patients and EPHB4 inhibition induced cell death in both hormone sensitive and castration-resistant prostate cancer cells. EPHB4 inhibition reduced expression of the glucose transporter, GLUT3, impaired glucose uptake, and reduced cellular ATP levels. This was associated with the activation of endoplasmic reticulum stress and tumor cell death with features of immunogenic cell death (ICD), including phosphorylation of eIF2α, increased cell surface calreticulin levels, and release of HMGB1 and ATP. The changes in tumor cell metabolism after EPHB4 inhibition were associated with MYC downregulation, likely mediated by the SRC/p38 MAPK/4EBP1 signaling cascade, known to impair cap-dependent translation. Together, our study indicates a role for EPHB4 inhibition in the induction of immunogenic cell death with implication for prostate cancer therapy.

GRP78 regulates CD44v membrane homeostasis and cell spreading in tamoxifen-resistant breast cancer.

GRP78 conducts protein folding and quality control in the ER and shows elevated expression and cell surface translocation in advanced tumors. However, the underlying mechanisms enabling GRP78 to exert novel signaling functions at cell surface are just emerging. CD44 is a transmembrane protein and an important regulator of cancer metastasis, and isoform switch of CD44 through incorporating additional variable exons to the extracellular juxtamembrane region is frequently observed during cancer progression. Using super-resolution dual-color single-particle tracking, we report that GRP78 interacts with CD44v in plasma membrane nanodomains of breast cancer cells. We further show that targeting cell surface GRP78 by the antibodies can effectively reduce cell surface expression of CD44v and cell spreading of tamoxifen-resistant breast cancer cells. Our results uncover new functions of GRP78 as an interacting partner of CD44v and as a regulator of CD44v membrane homeostasis and cell spreading. This study also provides new insights into anti-CD44 therapy in tamoxifen-resistant breast cancer.

Histological Validation of 11Carbon-Acetate Positron Emission Tomography/Computerized Tomography in Detecting Lymph Node Metastases in Prostate Cancer.

PURPOSE: Conventional imaging cannot definitively detect nodal metastases of prostate cancer. We histologically validated C-acetate positron emission tomography/computerized tomography to identify nodal metastases, examining prostate cancer factors that influence detection rates. MATERIALS AND METHODS: Patients with C-acetate avid positron emission tomography/computerized tomography imaged pelvic/retroperitoneal lymph nodes underwent high extended robotic lymphadenectomy. A standardized mapping template comprising 8 predetermined anatomical regions was dissected during lymphadenectomy, allowing for matched, region based analysis and comparison of imaging and histological data. RESULTS: In 25 patients a total of 2,149 lymph nodes were excised (mean 86 per patient, range 27 to 136) and 528 (22%) harbored metastases (mean 21 positive nodes per patient, range 0 to 109). A total of 174 anatomical regions had matching imaging histological data. C-acetate positron emission tomography/computerized tomography accurately identified 48 node-positive regions and accurately ruled out 88 regions as metastasis-free. C-acetate sensitivity, specificity, and positive and negative predictive values were 67%, 84%, 74% and 79%, respectively. An increasing, histologically measured metastatic lesion size in long axis diameter of 5 or less, 6 to 10, 11 to 15, 16 to 20 and 21 mm or greater correlated with improved C-acetate detection rates of 45%, 62%, 81%, 89% and 100%, respectively. Each standard uptake value unit increase correlated with a 1.9 mm increase in nodal long axis diameter (p <0.001) and a 1.2 mm increase in short axis diameter (p <0.001). Positive C-acetate positron emission tomography/computerized tomography findings correlated with histological lymph node size (long axis diameter 12 mm and short axis diameter 6 mm), metastatic lesion size (long axis diameter 11 mm and short axis diameter 6 mm) and extranodal extension (positive 88% vs false-negative 58%, p = 0.005). CONCLUSIONS: C-acetate positron emission tomography/computerized tomography can identify prostate cancer metastatic nodal disease. However, it underestimates the true cephalad extent of nodal involvement, performing better in the pelvis than in the retroperitoneum. Standard uptake value, histological nodal size, intranodal metastasis size and extranodal extension correlate with cancer bearing nodes.

Vascularization to preovulatory follicle and corpus luteum-a valuable predictor of fertility in dairy cows.

The aim of the present study was to predict pregnancy rate based on vascularization to follicle and corpus luteum (CL). 26 Holstein Friesian cows were synchronized using Ovsynch protocol. On day 10 of the protocol, vascularization and morphological characteristics [sectional area (SA), volume (V) and mean diameter] of follicle was assessed and animals underwent artificial insemination (AI). Morphological evaluation and vascularization to CL was assessed on day 12 and 21 following AI and blood samples were obtained for estimation of plasma progesterone (P4). Pregnancy diagnosis was performed on day 60 of AI and was classified as normal, complicated and non-pregnant. The overall conception rate was 76.92% (20/26); normal pregnancy was 53.85% (14/26). Complications observed in pregnancy were intrauterine growth retardation, late embryonic death and infection. Cows with a highly vascularized follicle (>550 pixel2) underwent a normal pregnancy, whereas those that had moderately (250-550 pixel2) and poorly (<250 pixel2) vascularized follicle experienced complicated pregnancy or remained non-pregnant, respectively. On day 12, there was no significant variation (P > 0.05) between mean diameter, SA, V, luteal blood flow (LBF) or plasma P4 concentration among CL of cows that remained pregnant (PCL), non-pregnant (NPCL) or that had a complicated pregnancy (CPCL). LBF alone was not beneficial in differentiating among the three groups (P > 0.05), but assessment of LBF along with turbulence to blood flow in day 21 CL proved highly valuable due to an increased turbulence in CPCL (66.67%) compared to PCL (16.67%). Assessment of turbulence and LBF on day 12 and 21 can also be used to predict luteolysis with accuracy.

GRP78 haploinsufficiency suppresses acinar-to-ductal metaplasia, signaling, and mutant Kras-driven pancreatic tumorigenesis in mice.

Pancreatic ductal adenocarcinoma (PDAC) remains a highly lethal disease in critical need of new therapeutic strategies. Here, we report that the stress-inducible 78-kDa glucose-regulated protein (GRP78/HSPA5), a key regulator of endoplasmic reticulum homeostasis and PI3K/AKT signaling, is overexpressed in the acini and PDAC of Pdx1-Cre;KrasG12D/+;p53f/+ (PKC) mice as early as 2 mo, suggesting that GRP78 could exert a protective effect on acinar cells under stress, as during PDAC development. The PKC pancreata bearing wild-type Grp78 showed detectable PDAC by 3 mo and rapid subsequent tumor growth. In contrast, the PKC pancreata bearing a Grp78f/+ allele (PKC78f/+ mice) expressing about 50% of GRP78 maintained normal sizes during the early months, with reduced proliferation and suppression of AKT, S6, ERK, and STAT3 activation. Acinar-to-ductal metaplasia (ADM) has been identified as a key tumor initiation mechanism of PDAC. Compared with PKC, the PKC78f/+ pancreata showed substantial reduction of ADM as well as pancreatic intraepithelial neoplasia-1 (PanIN-1), PanIN-2, and PanIN-3 and delayed onset of PDAC. ADM in response to transforming growth factor α was also suppressed in ex vivo cultures of acinar cell clusters isolated from mouse pancreas bearing targeted heterozygous knockout of Grp78 (c78f/+ ) and subjected to 3D culture in collagen. We further discovered that GRP78 haploinsufficiency in both the PKC78f/+ and c78f/+ pancreata leads to reduction of epidermal growth factor receptor, which is critical for ADM initiation. Collectively, our studies establish a role for GRP78 in ADM and PDAC development.

Post-mastectomy cancer recurrence with and without a continuous paravertebral block in the immediate postoperative period: a prospective multi-year follow-up pilot study of a randomized, triple-masked, placebo-controlled investigation.

PURPOSE: Retrospective studies have associated perioperative regional anesthesia/analgesia during mastectomy for breast cancer with a decreased incidence of cancer recurrence. However, to date, no prospective data from a randomized controlled trial have been reported. In a previous study we found that extending a single-injection paravertebral block with a multiple-day perineural local anesthetic infusion improves analgesia. This follow-up study investigates the rates of cancer recurrence for the single-injection and multiple-day infusion treatments. METHODS: Patients undergoing unilateral (n = 24) or bilateral mastectomy (n = 36) were included in the study. All patients had been diagnosed with breast cancer or tumor in situ, except for six patients who were receiving prophylactic bilateral mastectomy and were excluded from analyses. Patients received unilateral or bilateral single-injection thoracic paravertebral block(s) corresponding to their surgical site(s) with ropivacaine and perineural catheter(s). Subsequently, patients were randomized to receive either ropivacaine 0.4% (n = 30) or normal saline (n = 30) via their catheter(s) until catheter removal on postoperative day 3. Cancer recurrence from the date of surgery until at least 2 years post surgery was investigated via chart review. RESULTS: Five of the 54 (9.2%) patients experienced a cancer recurrence following mastectomy-3 of 26 (11.5%) of the patients with perineural ropivacaine and 2 of 28 (7.1%) of the patients with perineural saline. CONCLUSIONS: This pilot study found no evidence that extending a single-injection paravertebral block with a multi-day perineural local anesthetic infusion decreases the risk of post-mastectomy cancer recurrence. However, due to the small sample size of this investigation, further research is needed to draw definitive conclusions.

EPHB4 is a therapeutic target in AML and promotes leukemia cell survival via AKT.

EPHB4, an ephrin type B receptor, is implicated in the growth of several epithelial tumors and is a promising target in cancer therapy; however, little is known about its role in hematologic malignancies. In this article, we show that EPHB4 is highly expressed in ∼30% of acute myeloid leukemia (AML) samples. In an unbiased RNA interference screen of primary leukemia samples, we found that EPHB4 drives survival in a subset of AML cases. Knockdown of EPHB4 inhibits phosphatidylinositol 3-kinase/AKT signaling, and this is accompanied by a reduction in cell viability, which can be rescued by a constitutively active form of AKT. Finally, targeting EPHB4 with a highly specific monoclonal antibody (MAb131) is effective against AML in vitro and in vivo. EPHB4 is therefore a potential target in AML with high EPHB4 expression.

Enhancing radiosensitization in EphB4 receptor-expressing Head and Neck Squamous Cell Carcinomas.

Members of the Eph family of receptor tyrosine kinases have been implicated in a wide array of human cancers. The EphB4 receptor is ubiquitously expressed in head and neck squamous cell carcinoma (HNSCC) and has been shown to impart tumorigenic and invasive characteristics to these cancers. In this study, we investigated whether EphB4 receptor targeting can enhance the radiosensitization of HNSCC. Our data show that EphB4 is expressed at high to moderate levels in HNSCC cell lines and patient-derived xenograft (PDX) tumors. We observed decreased survival fractions in HNSCC cells following EphB4 knockdown in clonogenic assays. An enhanced G2 cell cycle arrest with activation of DNA damage response pathway and increased apoptosis was evident in HNSCC cells following combined EphB4 downregulation and radiation compared to EphB4 knockdown and radiation alone. Data using HNSCC PDX models showed significant reduction in tumor volume and enhanced delay in tumor regrowth following sEphB4-HSA administration with radiation compared to single agent treatment. sEphB4-HSA is a protein known to block the interaction between the EphB4 receptor and its ephrin-B2 ligand. Overall, our findings emphasize the therapeutic relevance of EphB4 targeting as a radiosensitizer that can be exploited for the treatment of human head and neck carcinomas.

Global phosphotyrosine survey in triple-negative breast cancer reveals activation of multiple tyrosine kinase signaling pathways.

Breast cancer is the most prevalent cancer in women worldwide. About 15-20% of all breast cancers are triple negative breast cancer (TNBC) and are often highly aggressive when compared to other subtypes of breast cancers. To better characterize the biology that underlies the TNBC phenotype, we profiled the phosphotyrosine proteome of a panel of twenty-six TNBC cell lines using quantitative high resolution Fourier transform mass spectrometry. A heterogeneous pattern of tyrosine kinase activation was observed based on 1,789 tyrosine-phosphorylated peptides identified from 969 proteins. One of the tyrosine kinases, AXL, was found to be activated in a majority of aggressive TNBC cell lines and was accompanied by a higher level of AXL expression. High levels of AXL expression are correlated with a significant decrease in patient survival. Treatment of cells bearing activated AXL with a humanized AXL antibody inhibited cell proliferation and migration in vitro, and tumor growth in mice. Overall, our global phosphoproteomic analysis provided new insights into the heterogeneity in the activation status of tyrosine kinase pathways in TNBCs. Our approach presents an effective means of identifying important novel biomarkers and targets for therapy such as AXL in TNBC.

PET Imaging of Dll4 Expression in Glioblastoma and Colorectal Cancer Xenografts Using (64)Cu-Labeled Monoclonal Antibody 61B.

Delta-like ligand 4 (Dll4) expressed in tumor cells plays a key role to promote tumor growth of numerous cancer types. Based on a novel antihuman Dll4 monoclonal antibody (61B), we developed a (64)Cu-labeled probe for positron emission tomography (PET) imaging of tumor Dll4 expression. In this study, 61B was conjugated with the (64)Cu-chelator DOTA through lysine on the antibody. Human IgG (hIgG)-DOTA, which did not bind to Dll4, was also prepared as a control. The Dll4 binding activity of the probes was evaluated through the bead-based binding assay with Dll4-alkaline phosphatase. The resulting PET probes were evaluated in U87MG glioblastoma and HT29 colorectal cancer xenografts in athymic nude mice. Our results demonstrated that the 61B-DOTA retained (77.2 ± 3.7) % Dll4 binding activity of the unmodified 61B, which is significantly higher than that of hIgG-DOTA (0.06 ± 0.03) %. Confocal microscopy analysis confirmed that 61B-Cy5.5, but not IgG-Cy5.5, predominantly located within the U87MG and HT29 cells cytoplasm. U87MG cells showed higher 61B-Cy5.5 binding as compared to HT29 cells. In U87MG xenografts, 61B-DOTA-(64)Cu demonstrated remarkable tumor accumulation (10.5 ± 1.7 and 10.2 ± 1.2%ID/g at 24 and 48 h postinjection, respectively). In HT29 xenografts, tumor accumulation of 61B-DOTA-(64)Cu was significantly lower than that of U87MG (7.3 ± 1.3 and 6.6 ± 1.3%ID/g at 24 and 48 h postinjection, respectively). The tumor accumulation of 61B-DOTA-(64)Cu was significantly higher than that of hIgG-DOTA-(64)Cu in both xenografts models. Immunofluorescence staining of the tumor tissues further confirmed that tumor accumulation of 61B-Cy5.5 was correlated well with in vivo PET imaging data using 61B-DOTA-(64)Cu. In conclusion, 61B-DOTA-(64)Cu PET probe was successfully synthesized and demonstrated prominent tumor uptake by targeting Dll4. 61B-DOTA-(64)Cu has great potential to be used for noninvasive Dll4 imaging, which could be valuable for tumor detection, Dll4 expression level evaluation, and Dll4-based treatment monitoring.

Novel EPHB4 Receptor Tyrosine Kinase Mutations and Kinomic Pathway Analysis in Lung Cancer.

Lung cancer outcomes remain poor despite the identification of several potential therapeutic targets. The EPHB4 receptor tyrosine kinase (RTK) has recently emerged as an oncogenic factor in many cancers, including lung cancer. Mutations of EPHB4 in lung cancers have previously been identified, though their significance remains unknown. Here, we report the identification of novel EPHB4 mutations that lead to putative structural alterations as well as increased cellular proliferation and motility. We also conducted a bioinformatic analysis of these mutations to demonstrate that they are mutually exclusive from other common RTK variants in lung cancer, that they correspond to analogous sites of other RTKs' variations in cancers, and that they are predicted to be oncogenic based on biochemical, evolutionary, and domain-function constraints. Finally, we show that EPHB4 mutations can induce broad changes in the kinome signature of lung cancer cells. Taken together, these data illuminate the role of EPHB4 in lung cancer and further identify EPHB4 as a potentially important therapeutic target.