Rational Design of Novel Allosteric EYA2 Inhibitors as Potential Therapeutics for Multiple Brain Cancers.

The Eyes Absent (EYA) family of developmental proteins, often in partnership with the sine oculis (SIX) homeobox proteins, promote cancer metastasis and recurrence in numerous tumor types. In addition to being a transcriptional coactivator, EYA2 is a Tyr phosphatase that dephosphorylates H2AX which leads to repair instead of apoptosis upon DNA damage and ERß which inhibits the anti-tumor transcriptional activity of ERß. The SIX members of the EYA-SIX complex are difficult to target, therefore, we targeted the EYA2 to promote cell death and prevent cancer progression. We conducted structural optimization of a previously discovered allosteric inhibitor of EYA2, 9987, using the combination of in silico modeling, biochemical and cell-based assays. A new series of compounds was developed with significantly improved cellular activity and physiochemical properties desirable for brain targets. Specifically, compound 2 e showed >30-fold improvement in the medulloblastoma cell line D458, relative to 9987, while maintaining potent and selective inhibitory activity against EYA2 Tyr phosphatase activity and a good multiparameter optimization score for central nervous system drugs.

An Entrustable Professional Activities Model for Assessment of Undergraduate Competence in Anesthesia and Surgery: Performance of the Assessment Scheme and the Impact of Assessment Timing and Variation in Structure of Teaching Activities on Student Outcomes.

An entrustable professional activity (EPA) model was used to assess the anesthesia and surgery competence of year 4 students during elective neutering procedures over 3 academic years (cohort A, cohort B, and cohort C). Two competence thresholds were defined by an expert panel, the minimum acceptable standard (MAS) and the standard expected at the start of final-year rotations (SFR). The assessment scheme performed as expected, and the median level of supervision achieved by students either matched or exceeded the SFR for all EPAs except one, which matched the MAS. Semester of assessment was associated with student performance, with more students in semester 2 achieving the SFR. In the EPAs assessing pain management, documentation, and patient discharge, cohort A was associated with reduced student performance; this could be explained by changes in the delivery of teaching that enhanced performance in subsequent cohorts (academic years). For all EPAs combined and for EPAs 3, 5, 6, 8, and 9, student performance at the SFR was associated with academic year. For all EPAs combined and EPAs 3, 8, and 9, there was a reduction in the proportion of students achieving the SFR threshold in each successive year. At the MAS, the only association for all EPAs combined was with cohort C. This progressive reduction in performance may have been related to the negative effect of decreased time spent at the neutering clinic and loss of feedback opportunities outweighing the positive effects of increased staff:student ratio and improvements in the preparative phases.

Inhibition of CtBP-Regulated Proinflammatory Gene Transcription Attenuates Psoriatic Skin Inflammation.

Psoriasis is a chronic immune-mediated disease characterized by excessive proliferation of epidermal keratinocytes and increased immune cell infiltration to the skin. Although it is well-known that psoriasis pathogenesis is driven by aberrant production of proinflammatory cytokines, the mechanisms underlying the imbalance between proinflammatory and anti-inflammatory cytokine expression are incompletely understood. In this study, we report that the transcriptional coregulators CtBP1 and 2 can transactivate a common set of proinflammatory genes both in the skin of imiquimod-induced mouse psoriasis model and in human keratinocytes and macrophages stimulated by imiquimod. We find that mice overexpressing CtBP1 in epidermal keratinocytes display severe skin inflammation phenotypes with increased expression of T helper type 1 and T helper type 17 cytokines. We also find that the expression of CtBPs and CtBP-target genes is elevated both in human psoriatic lesions and in the mouse imiquimod psoriasis model. Moreover, we were able to show that topical treatment with a peptidic inhibitor of CtBP effectively suppresses the CtBP-regulated proinflammatory gene expression and thus attenuates psoriatic inflammation in the imiquimod mouse model. Together, our findings suggest to our knowledge previously unreported strategies for therapeutic modulation of the immune response in inflammatory skin diseases.

The time required to achieve endotracheal intubation in dogs: a comparison of veterinary students and qualified veterinary surgeons.

OBJECTIVE: To determine whether final year veterinary students take longer to perform endotracheal intubation than qualified veterinary surgeons. STUDY DESIGN: Observational cohort study. ANIMALS: A total of 38 healthy mesocephalic dogs undergoing general anaesthesia for a clinical purpose unrelated to this study. METHODS: Time to successful endotracheal intubation, measured from termination of intravenous induction drug administration to confirmation of endotracheal intubation, was recorded for two groups: final year veterinary students (group S) and qualified veterinary surgeons (group V). Animal age, breed and anaesthetic induction agent were also recorded. Following normality testing the groups were compared for each variable using the Student's t test or Mann-Whitney U test where appropriate. The level of significance was defined as p < 0.05. Timed data are presented as median and interquartile range. RESULTS: Time to successful intubation was 54.2 (31.3) seconds in group S and 11.7 (8.5) seconds in group V, the difference being significant (p < 0.001). There was also a significant difference between groups for animal age (p = 0.036) but not for breed (p = 0.573) or induction agent (p = 0.239). CONCLUSIONS: and clinical relevance Veterinary students take longer to achieve successful endotracheal intubation of anaesthetized healthy dogs compared with qualified veterinary surgeons. To mitigate any additional risk of dogs developing hypoxaemia, it is recommended that a 55 second time limit is set after which the supervisor intervenes and takes over the intubation procedure. Preoxygenation may be used as an additional mitigation strategy.

Identification of a Small-Molecule Inhibitor That Disrupts the SIX1/EYA2 Complex, EMT, and Metastasis.

Metastasis is the major cause of mortality for patients with cancer, and dysregulation of developmental signaling pathways can significantly contribute to the metastatic process. The Sine oculis homeobox homolog 1 (SIX1)/eyes absent (EYA) transcriptional complex plays a critical role in the development of multiple organs and is typically downregulated after development is complete. In breast cancer, aberrant expression of SIX1 has been demonstrated to stimulate metastasis through activation of TGFß signaling and subsequent induction of epithelial-mesenchymal transition (EMT). In addition, SIX1 can induce metastasis via non-cell autonomous means, including activation of GLI-signaling in neighboring tumor cells and activation of VEGFC-induced lymphangiogenesis. Thus, targeting SIX1 would be expected to inhibit metastasis while conferring limited side effects. However, transcription factors are notoriously difficult to target, and thus novel approaches to inhibit their action must be taken. Here we identified a novel small molecule compound, NCGC00378430 (abbreviated as 8430), that reduces the SIX1/EYA2 interaction. 8430 partially reversed transcriptional and metabolic profiles mediated by SIX1 overexpression and reversed SIX1-induced TGFß signaling and EMT. 8430 was well tolerated when delivered to mice and significantly suppressed breast cancer-associated metastasis in vivo without significantly altering primary tumor growth. Thus, we have demonstrated for the first time that pharmacologic inhibition of the SIX1/EYA2 complex and associated phenotypes is sufficient to suppress breast cancer metastasis. SIGNIFICANCE: These findings identify and characterize a novel inhibitor of the SIX1/EYA2 complex that reverses EMT phenotypes suppressing breast cancer metastasis.

C-terminal binding proteins 1 and 2 in traumatic brain injury-induced inflammation and their inhibition as an approach for anti-inflammatory treatment.

Traumatic brain injury (TBI) induces an acute inflammatory response in the central nervous system that involves both resident and peripheral immune cells. The ensuing chronic neuroinflammation causes cell death and tissue damage and may contribute to neurodegeneration. The molecular mechanisms involved in the maintenance of this chronic inflammation state remain underexplored. C-terminal binding protein (CtBP) 1 and 2 are transcriptional coregulators that repress diverse cellular processes. Unexpectedly, we find that the CtBPs can transactivate a common set of proinflammatory genes both in lipopolysaccharide-activated microglia, astrocytes and macrophages, and in a mouse model of the mild form of TBI. We also find that the expression of these genes is markedly enhanced by a single mild injury in both brain and peripheral blood leukocytes in a severity- and time-dependent manner. Moreover, we were able to demonstrate that specific inhibitors of the CtBPs effectively suppress the expression of the CtBP target genes and thus improve neurological outcome in mice receiving single and repeated mild TBIs. This discovery suggests new avenues for therapeutic modulation of the inflammatory response to brain injury.

Structural and Functional Analyses of an Allosteric EYA2 Phosphatase Inhibitor That Has On-Target Effects in Human Lung Cancer Cells.

EYA proteins (EYA1-4) are critical developmental transcriptional cofactors that contain an EYA domain (ED) harboring Tyr phosphatase activity. EYA proteins are largely downregulated after embryogenesis but are reexpressed in cancers, and their Tyr phosphatase activity plays an important role in the DNA damage response and tumor progression. We previously identified a class of small-molecule allosteric inhibitors that specifically inhibit the Tyr phosphatase activity of EYA2. Herein, we determined the crystal structure of the EYA2 ED in complex with NCGC00249987 (a representative compound in this class), revealing that it binds to an induced pocket distant from the active site. NCGC00249987 binding leads to a conformational change of the active site that is unfavorable for Mg2+ binding, thereby inhibiting EYA2's Tyr phosphatase activity. We demonstrate, using genetic mutations, that migration, invadopodia formation, and invasion of lung adenocarcinoma cells are dependent on EYA2 Tyr phosphatase activity, whereas growth and survival are not. Further, we demonstrate that NCGC00249987 specifically targets migration, invadopodia formation, and invasion of lung cancer cells, but that it does not inhibit cell growth or survival. The compound has no effect on lung cancer cells carrying an EYA2 F290Y mutant that abolishes compound binding, indicating that NCGC00249987 is on target in lung cancer cells. These data suggest that the NCGC00249987 allosteric inhibitor can be used as a chemical probe to study the function of the EYA2 Tyr phosphatase activity in cells and may have the potential to be developed into an antimetastatic agent for cancers reliant on EYA2's Tyr phosphatase activity.

Smad7 Promotes Healing of Radiotherapy-Induced Oral Mucositis without Compromising Oral Cancer Therapy in a Xenograft Mouse Model.

PURPOSE: We previously reported preventive and therapeutic effects of Smad7, a multifunctional protein, on radiotherapy (RT)-induced mucositis in mice without promoting human oral cancer cell survival or migration in vitro. The current study aims to determine whether a Smad7-based biologic can treat existing oral mucositis during radiotherapy for oral cancer and whether this treatment compromises RT-induced cancer cell killing in neighboring oral cancer.Experimental Design: We transplanted human oral cancer cells into the tongues of mice and applied craniofacial irradiation to simultaneously kill tumor cells and induce oral mucositis, thus modeling RT and mucositis in oral cancer patients. We topically applied a recombinant human Smad7 protein fused with the cell-penetrating Tat tag (Tat-Smad7) to the oral mucosa of tumor-bearing mice post RT when oral mucositis began to develop. RESULTS: Topically applied Tat-Smad7 penetrated cells in both the oral mucosa and oral cancer, attenuating TGFß and NF-κB signaling as well as inflammation at both sites. Tat-Smad7 treatment alleviated oral mucositis with reductions in DNA damage and apoptosis in keratinocytes, but increased keratinocyte proliferation compared with vehicle-treated mucositis lesions. In contrast, adjacent oral cancer exposed to Tat-Smad7 did not show alterations in proliferation or direct DNA damage, but showed increased oxidative stress damage and apoptosis compared with tumors treated with vehicle. CONCLUSIONS: Our results suggest that short-course Tat-Smad7 application to oral mucositis promotes its healing but does not compromise the cytotoxic effect of RT on oral cancer and has context-specific effects on oral mucosa versus oral cancer.

CPP-E1A fusion peptides inhibit CtBP-mediated transcriptional repression.

The carboxyl-terminal binding proteins (CtBP) are transcriptional corepressors that regulate the expression of multiple epithelial-specific and pro-apoptotic genes. Overexpression of CtBP occurs in many human cancers where they promote the epithelial-to-mesenchymal transition, stem cell-like features, and cell survival, while knockdown of CtBP in tumor cells results in p53-independent apoptosis. CtBPs are recruited to their target genes by binding to a conserved PXDLS peptide motif present in multiple DNA-binding transcription factors. Disrupting the interaction between CtBP and its transcription factor partners may be a means of altering CtBP-mediated transcriptional repression and a potential approach for cancer therapies. However, small molecules targeting protein-protein interactions have traditionally been difficult to identify. In this study, we took advantage of the fact that CtBP binds to a conserved peptide motif to explore the feasibility of using peptides containing the PXDLS motif fused to cell-penetrating peptides (CPP) to inhibit CtBP function. We demonstrate that these peptides disrupt the ability of CtBP to interact with its protein partner, E1A, in an AlphaScreen assay. Moreover, these peptides can enter both lung carcinoma and melanoma cells, disrupt the interaction between CtBP and a transcription factor partner, and inhibit CtBP-mediated transcriptional repression. Finally, the constitutive expression of one such peptide, Pep1-E1A-WT, in a melanoma cell line reverses CtBP-mediated oncogenic phenotypes including proliferation, migration, and sphere formation and limits tumor growth in vivo. Together, our results suggest that CPP-fused PXDLS-containing peptides can potentially be developed into a research tool or therapeutic agent targeting CtBP-mediated transcriptional events in various biological pathways.

The Role of CtBP1 in Oncogenic Processes and Its Potential as a Therapeutic Target.

Transcriptional corepressor proteins have emerged as an important facet of cancer etiology. These corepressor proteins are often altered by loss- or gain-of-function mutations, leading to transcriptional imbalance. Thus, research directed at expanding our current understanding of transcriptional corepressors could impact the future development of new cancer diagnostics, prognostics, and therapies. In this review, our current understanding of the CtBP corepressors, and their role in both development and disease, is discussed in detail. Importantly, the role of CtBP1 overexpression in adult tissues in promoting the progression of multiple cancer types through their ability to modulate the transcription of developmental genes ectopically is explored. CtBP1 overexpression is known to be protumorigenic and affects the regulation of gene networks associated with "cancer hallmarks" and malignant behavior, including increased cell survival, proliferation, migration, invasion, and the epithelial-mesenchymal transition. As a transcriptional regulator of broad developmental processes capable of promoting malignant growth in adult tissues, therapeutically targeting the CtBP1 corepressor has the potential to be an effective method for the treatment of diverse tumor types. Although efforts to develop CtBP1 inhibitors are still in the early stages, the current progress and the future perspectives of therapeutically targeting this transcriptional corepressor are also discussed. Mol Cancer Ther; 16(6); 981-90. ©2017 AACR.

The SIX1-EYA transcriptional complex as a therapeutic target in cancer.

INTRODUCTION: The SIX homeodomain proteins and the eyes absent (EYA) family of co-activators form a bipartite transcription factor complex that promotes the proliferation and survival of progenitor cells during organogenesis and is down-regulated in most adult tissues. Abnormal over-expression of SIX1 and EYA in adult tissue is associated with the initiation and progression of diverse tumor types. Importantly, SIX1 and EYA are often co-overexpressed in tumors, and the SIX1-EYA2 interaction has been shown to be critical for metastasis in a breast cancer model. The EYA proteins also contain protein tyrosine phosphatase activity, which plays an important role in breast cancer growth and metastasis as well as directing cells to the repair pathway upon DNA damage. AREAS COVERED: This review provides a summary of the SIX1/EYA complex as it relates to development and disease and the current efforts to therapeutically target this complex. EXPERT OPINION: Recently, there have been an increasing number of studies suggesting that targeting the SIX1/EYA transcriptional complex will potently inhibit tumor progression. Although current attempts to develop inhibitors targeting this complex are still in the early stages, continued efforts toward developing better compounds may ultimately result in effective anti-cancer therapies.

Small Molecule, NSC95397, Inhibits the CtBP1-Protein Partner Interaction and CtBP1-Mediated Transcriptional Repression.

Carboxyl-terminal binding protein (CtBP) is a transcriptional corepressor that suppresses multiple proapoptotic and epithelial genes. CtBP is overexpressed in many human cancers, and its overexpression increases stem cell-like features, epithelial-mesenchymal transition, and cancer cell survival. Knockdown of CtBP also increases apoptosis independent of p53 in cell culture. Therefore, targeting CtBP with small molecules that disrupt its interaction with transcription factor partners may be an effective cancer therapy. To elicit its corepressing effect, CtBP binds to a conserved peptide motif in each transcription factor partner. We developed an AlphaScreen high-throughput screening assay to monitor the interaction between CtBP and E1A (which mimics the interaction between CtBP and its transcriptional partners). We screened the LOPAC library of 1280 bioactive compounds and identified NSC95397, which inhibits the CtBP-E1A interaction (IC50 = 2.9 µM). The inhibitory activity of NSC95397 was confirmed using two secondary assays and a counterscreen. NSC95397 also behaved as a weak substrate of CtBP dehydrogenase activity and did not inhibit another dehydrogenase, lactase dehydrogenase. Finally, NSC95397 was able to disrupt CtBP-mediated transcriptional repression of a target gene. These studies present a new possibility for the development of a therapeutic agent targeting tumors through disrupting the CtBP transcriptional complex.

Allosteric inhibitors of the Eya2 phosphatase are selective and inhibit Eya2-mediated cell migration.

Eya proteins are essential co-activators of the Six family of transcription factors and contain a unique tyrosine phosphatase domain belonging to the haloacid dehalogenase family of phosphatases. The phosphatase activity of Eya is important for the transcription of a subset of Six1-target genes, and also directs cells to the repair rather than apoptosis pathway upon DNA damage. Furthermore, Eya phosphatase activity has been shown to mediate transformation, invasion, migration, and metastasis of breast cancer cells, making it a potential new drug target for breast cancer. We have previously identified a class of N-arylidenebenzohydrazide compounds that specifically inhibit the Eya2 phosphatase. Herein, we demonstrate that these compounds are reversible inhibitors that selectively inhibit the phosphatase activity of Eya2, but not Eya3. Our mutagenesis results suggest that this class of compounds does not bind to the active site and the binding does not require the coordination with Mg(2+). Moreover, these compounds likely bind within a site on the opposite face of the active site, and function as allosteric inhibitors. We also demonstrate that this class of compounds inhibits Eya2 phosphatase-mediated cell migration, setting the foundation for these molecules to be developed into chemical probes for understanding the specific function of the Eya2 phosphatase and to serve as a prototype for the development of Eya2 phosphatase specific anti-cancer drugs.

Complex formation among the RNA export proteins Nup98, Rae1/Gle2, and TAP.

Most nucleocytoplasmic traffic through the nuclear pore complex is mediated by soluble receptors of the importin/exportin or karyopherin family. mRNA export is unique in that no receptor of this family has been implicated in trafficking of the bulk of mRNAs. Instead, many diverse proteins have been linked to mRNA export, but an all-encompassing model remains elusive. Understanding how these proteins interact with each other is central to the development of such a model. Here, we have focused on the interactions between three proteins implicated in mRNA export, Nup98, Rae1/Gle2, and TAP. We have defined the binary complexes that form among these proteins. We find that Gle2 requires two sites within TAP for stable interaction. Strikingly, rather than a general affinity for all nucleoporin FG repeats, TAP has highest affinity for a specific region within the GLFG domain of Nup98, indicating that not all repeats are identical in function. We have established that the ternary complex can form through simultaneous binding of both Gle2 and TAP to adjacent sites on Nup98. In contrast, Nup98 competes with TAP for Gle2 binding; when bound to Nup98, Gle2 no longer interacts directly with TAP. From these interactions, we propose that Gle2 may act to deliver TAP to Nup98 and that this may represent the first in a series of interactions between an export complex and a nucleoporin.