The potent and selective RIPK2 inhibitor BI 706039 improves intestinal inflammation in the TRUC mouse model of inflammatory bowel disease.

Mouse and human data implicate the NOD1 and NOD2 sensors of the intestinal microbiome and the associated signal transduction via the receptor interacting protein kinase 2 (RIPK2) as a potential key signaling node for the development of inflammatory bowel disease (IBD) and an attractive target for pharmacological intervention. The TRUC mouse model of IBD was strongly indicated for evaluating RIPK2 antagonism for its effect on intestinal inflammation based on previous knockout studies with NOD1, NOD2, and RIPK2. We identified and profiled the BI 706039 molecule as a potent and specific functional inhibitor of both human and mouse RIPK2 and with favorable pharmacokinetic properties. We dosed BI 706039 in the spontaneous TRUC mouse model from age 28 to 56 days. Oral, daily administration of BI 706039 caused dose-responsive and significant improvement in colonic histopathological inflammation, colon weight, and terminal levels of protein-normalized fecal lipocalin (all P values <0.001). These observations correlated with dose responsively increasing systemic levels of the BI 706039 compound, splenic molecular target engagement of RIPK2, and modulation of inflammatory genes in the colon. This demonstrates that a relatively low oral dose of a potent and selective RIPK2 inhibitor can modulate signaling in the intestinal immune system and significantly improve disease associated intestinal inflammation.NEW & NOTEWORTHY The RIPK2 kinase at the apex of microbiome immunosensing is an attractive target for pharmacological intervention. A low oral dose of a RIPK2 inhibitor leads to significantly improved intestinal inflammation in the murine TRUC model of colitis. A selective and potent inhibitor of the RIPK2 kinase may represent a new class of therapeutics that target microbiome-driven signaling for the treatment of IBD.

Correlation of High-Risk Soft Tissue Sarcoma Biomarker Expression Patterns with Outcome following Neoadjuvant Chemoradiation.

BACKGROUND: Sarcoma mortality remains high despite adjuvant chemotherapy. Biomarker predictors of treatment response and outcome could improve treatment selection. METHODS: Tissue microarrays (TMAs) were created using pre- and posttreatment tumor from two prospective trials (MGH pilot and RTOG 9514) of neoadjuvant/adjuvant MAID chemotherapy and preoperative radiation. Biomarkers were measured using automated computerized imaging (AQUA or ACIS). Expression was correlated with disease-free survival (DFS), distant disease-free survival (DDFS), and overall survival (OS). RESULTS: Specimens from 60 patients included 23 pretreatment (PRE), 40 posttreatment (POST), and 12 matched pairs (MPs). In the MP set, CAIX, GLUT1, and PARP1 expression significantly decreased following neoadjuvant therapy, but p53 nuclear/cytoplasmic (N/C) ratio increased. In the PRE set, no biomarker expression was associated with DFS, DDFS, or OS. In the POST set, increased p53 N/C ratio was associated with a significantly decreased DFS and DDFS (HR 4.13, p=0.017; HR 4.16, p=0.016), while increased ERCC1 and XPF expression were associated with an improved DFS and DDFS. No POST biomarkers were associated with OS. CONCLUSIONS: PRE biomarker expression did not predict survival outcomes. Expression pattern changes after neoadjuvant chemoradiation supports the concepts of tumor reoxygenation, altered HIF-1α signaling, and a p53 nuclear accumulation DNA damage response. CLINICAL TRIAL REGISTRATION: NRG Oncology RTOG 9514 is registered with ClinicalTrials.gov. The ClinicalTrials.gov Identifier is NCT00002791.

GLI1, CTNNB1 and NOTCH1 protein expression in a thymic epithelial malignancy tissue microarray.

BACKGROUND/AIM: Thymic epithelial tumors (TET) are rare. Wingless and INT (WNT), NOTCH and sonic hedgehog pathway interactions between thymocytes and thymic stroma are important to thymus and T-cell development. We analyzed a thymoma tissue microarray (TMA) for glioma associated oncogene homolog 1 (GLI1), NOTCH1 and catenin (cadherin-associated protein, beta 1) (CTNNB1) expression as surrogate markers of sonic hedgehog, NOTCH and WNT pathway activity. MATERIALS AND METHODS: GLI1, NOTCH1 and CTNNB1 expression were assayed in a tissue microarray of 68 TET and eight benign thymus by fluorescent immunohistochemistry (AQUA) as surrogates for activity of the sonic hedgehog, NOTCH and WNT pathways respectively. RESULTS: No difference in tumor GLI1 (mean 201 vs. 211, p=0.31), CTNNB1 (mean 222 vs. 306, p=0.66) or NOTCH1 expression (mean 317 vs. 325, p=0.82) was noted between thymic tumor and benign thymus. CONCLUSION: No evidence for preferential expression of GLI1, NOTCH1 or CTNNB1 was noted. High-throughput immunofluorescence using AQUA technology can help overcome limitations of small sample size and tissue heterogeneity when analyzing protein expression in thymic tumors.

Interaction of Hsp90 with the nascent form of the mutant epidermal growth factor receptor EGFRvIII.

EGFRvIII is a mutant epidermal growth factor that promotes aggressive growth of glioblastomas. We made a plasmid that directed the expression of an EGFRvIII with three copies of the Flag epitope at its amino terminus. Flag-tagged EGFRvIII was expressed at the same levels as unmodified EGFRvIII, and showed the same subcellular localization. However, the Flag epitope could only be detected on EGFRvIII present in the endoplasmic reticulum; the epitope was covalently modified during trafficking of the receptor through the Golgi so that it was no longer recognized by anti-Flag antibody. This property was exploited to selectively purify nascent EGFRvIII from glioblastoma cells. Nascent EGFRvIII was found to copurify with a set of other proteins, identified by mass spectrometry as the two endoplasmic reticulum chaperones Grp94 and BiP, and the two cytosolic chaperones Hsc70 and Hsp90. The Hsp90-associated chaperone Cdc37 also co-purified with EGFRvIII, suggesting that Hsp90 binds EGFRvIII as a complex with this protein. Geldanamycin and radicicol, two chemically unrelated inhibitors of Hsp90, decreased the expression of EGFRvIII in glioblastoma cells. These studies show that nascent EGFRvIII in the endoplasmic reticulum associates with Hsp90 and Cdc37, and that the Hsp90 association is necessary to maintain expression of EGFRvIII.