Low-Level Detection of Poly(amidoamine) PAMAM Dendrimers Using Immunoimaging Scanning Probe Microscopy

Immunoimaging scanning probe microscopy was utilized for the low-level detection and quantification of biotinylated G4 poly(amidoamine) PAMAM dendrimers. Results were compared to those of high-performance liquid chromatography (HPLC) and found to provide a vastly improved analytical method for the low-level detection of dendrimers, improving the limit of detection by a factor of 1000 (LOD = 2.5 × 10−13 moles). The biorecognition method is reproducible and shows high specificity and good accuracy. In addition, the capture assay platform shows a promising approach to patterning dendrimers for nanotechnology applications

Selective USP7 inhibition elicits cancer cell killing through a p53-dependent mechanism

Ubiquitin specific peptidase 7 (USP7) is a deubiquitinating enzyme (DUB) that removes ubiquitin tags from specific protein substrates in order to alter their degradation rate and sub-cellular localization. USP7 has been proposed as a therapeutic target in several cancers because it has many reported substrates with a role in cancer progression, including FOXO4, MDM2, N-Myc, and PTEN. The multisubstrate nature of USP7, combined with the modest potency and selectivity of early generation USP7 inhibitors, has presented a challenge in defining predictors of response to USP7 and potential patient populations that would benefit most from USP7-targeted drugs. Here, we describe the structureguided development of XL177A, which irreversibly inhibits USP7 with sub-nM potency and selectivity across the human proteome. Evaluation of the cellular effects of XL177A reveals that selective USP7 inhibition suppresses cancer cell growth predominantly through a p53-dependent mechanism: XL177A specifically upregulates p53 transcriptional targets transcriptome-wide, hotspot mutations in TP53 but not any other genes predict response to XL177A across a panel of similar to 500 cancer cell lines, and TP53 knockout rescues XL177A-mediated growth suppression of TP53 wild-type (WT) cells. Together, these findings suggest TP53 mutational status as a biomarker for response to USP7 inhibition. We find that Ewing sarcoma and malignant rhabdoid tumor (MRT), two pediatric cancers that are sensitive to other p53-dependent cytotoxic drugs, also display increased sensitivity to XL177A

Germinal Center Kinase Regulates The Proliferation and Survival Of Diffuse Large B-Cell Lymphoma

Abstract Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL). The pathogenesis of DLBCL represents a multi-step process that involves the accumulation of multiple genetic and molecular lesions. Marked advances in the understanding of DLBCL pathobiology have been made by the application of gene expression arrays, comparative genomic hybridization arrays and “next” generation sequencing. This led to the identification of previously unrecognized DLBCL subtypes (germinal center-like (GCB) and activated B cell-like (ABC)) as well as type specific-deregulation of particular signaling pathways. These approaches focused on genetic aberrations and mRNA expression profiles, whereas the crucial events transforming normal cells are executed by proteins. Kinases play an important role in neoplastic transformation. Herein, we have undertaken the task of profiling kinase activity in DLBCL to further delineate potential mechanisms of DLBCL pathogenesis and develop novel therapeutic agents. A comprehensive analysis of global kinase activity/protein expression was performed using KiNativ technology. Kinomic analysis of 8 DLBCL cell lines, as compared to non-cancerous primary B-cells, led to the discovery of 13 members of the MAPK cascade which were activated and/or overexpressed in DLBCL. Only three of the detected MAPK members were inactive or had reduced expression compared to their non-cancerous counterparts. To determine whether these findings could be extended to de novo primary human DLBCL tumors, we performed immunohistochemistry (IHC) of the proximally activated kinase, MAP4K2 or “Germinal Center Kinase” (GCK) and the phosphorylated forms of its downstream targets: MAP3K1, MAP2K4, MAP2K7, and C-jun N-terminal Kinase 1 (JNK1). Analyzed kinases were expressed and activated in more than 80% of primary DLBCL tumors, confirming the KiNativ cell line data. The kinase array data was further corroborated with classical immunoprecipitation-based JNK and p38 assays. Hierarchical clustering analysis of 36 DLBCL specimens stained for GCB and ABC markers demonstrated that GCK expression/activation is not DLBCL subtype specific. Notably, in a cohort of 151 primary DLBCL cases, we found that patients whose tumors did not express GCK had an estimated progression free survival (PFS) of 85% at 10 years of follow up, whereas those tumors expressing GCK were associated with significantly reduced PFS of 53% (p=0.04). While there was a similar trend in overall survival, it did not reach statistical significance, which may be due to the relatively small number of DLBCL cases not expressing GCK and the potential rescue of these patients with second line treatments. RNA interference studies in DLBCL cell lines confirmed the importance of GCK for the survival of these tumors, resulting in reduced viability and G0/G1 arrest. We next developed a small molecule inhibitor, HG6-64-1. KiNativ, Ambit and Invitrogen profiling of HG6-64-1 targets revealed that it potently inhibited GCK. In vitro treatment with the novel GCK inhibitor, HG6-64-1, led to cell cycle arrest and the induction of apoptosis in DLBCL cell lines and primary DLBCL tumors. G452, a DLBCL cell line minimally expressing GCK, was not affected by HG6-64-1. In vivo treatment with HG6-64-1, via intratumoral and intraperitoneal injections, significantly decreased the tumor growth rate resulting in a significantly extended lifespan of DLBCL xenograft mouse models. Overall our results identified a previously unrecognized activation of the GCK pathway which contributes to the proliferation and survival of DLBCLs and can be used as a therapeutic target using novel GCK inhibitors. Disclosures: Patricelli: ActivX Biosciences: Employment. Nomanbhoy:ActivX Biosciences: Employment

Macrocyclic Hedgehog Pathway Inhibitors: Optimization of Cellular Activity and Mode of Action Studies

Macrocyclic Hedgehog (Hh) pathway inhibitors have been discovered with improved potency and maximal inhibition relative to the previously reported macrocycle robotnikinin. Analogues were prepared using a modular and efficient build-couple-pair (BCP) approach, with a ring-closing metathesis step to form the macrocyclic ring. Varying the position of the macrocycle nitrogen and oxygen atoms provided inhibitors with improved activity in cellular assays; the most potent analogue was <b>29</b> (BRD-6851), with an IC₅₀ of 0.4 μM against C3H10T1/2 cells undergoing Hh-induced activation, as measured by <i>Gli1</i> transcription and alkaline phosphatase induction. Studies with Patched knockout (<i>Ptch</i>^–/–) cells and competition studies with the Smoothened (Smo) agonists SAG and purmorphamine demonstrate that in contrast to robotnikinin, select analogues are Smo antagonists

Pathophysiological significance and therapeutic targeting of germinal center kinase in diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma, yet 40% to 50% of patients will eventually succumb to their disease, demonstrating a pressing need for novel therapeutic options. Gene expression profiling has identified messenger RNAs that lead to transformation, but critical events transforming cells are normally executed by kinases. Therefore, we hypothesized that previously unrecognized kinases may contribute to DLBCL pathogenesis. We performed the first comprehensive analysis of global kinase activity in DLBCL, to identify novel therapeutic targets, and discovered that germinal center kinase (GCK) was extensively activated. GCK RNA interference and small molecule inhibition induced cell-cycle arrest and apoptosis in DLBCL cell lines and primary tumors in vitro and decreased the tumor growth rate in vivo, resulting in a significantly extended lifespan of mice bearing DLBCL xenografts. GCK expression was also linked to adverse clinical outcome in a cohort of 151 primary DLBCL patients. These studies demonstrate, for the first time, that GCK is a molecular therapeutic target in DLBCL tumors and that inhibiting GCK may significantly extend DLBCL patient survival. Because the majority of DLBCL tumors (∼80%) exhibit activation of GCK, this therapy may be applicable to most patients