Molecular recognition of methylated amino acids and peptides by Pillar[6]MaxQ.

We report the molecular recognition properties of Pillar[n]MaxQ (P[n]MQ) toward a series of (methylated) amino acids, amino acid amides, and post-translationally modified peptides by a combination of 1H NMR, isothermal titration calorimetry, indicator displacement assays, and molecular dynamics simulations. We find that P6MQ is a potent receptor for N-methylated amino acid side chains. P6MQ recognized the H3K4Me3 peptide with Kd = 16 nM in phosphate buffered saline.

Polycomb Paralog Chromodomain Inhibitors Active against Both CBX6 and CBX8*.

Methyllysine reader proteins bind to methylated lysine residues and alter gene transcription by changing either the compaction state of chromatin or by the recruitment of other multiprotein complexes. The polycomb paralog family of methyllysine readers bind to trimethylated lysine on the tail of histone 3 (H3) via a highly conserved aromatic cage located in their chromodomains. Each of the polycomb paralogs are implicated in several disease states. CBX6 and CBX8 are members of the polycomb paralog family with two structurally similar chromodomains. By exploring the structure-activity relationships of a previously reported CBX6 inhibitor we have discovered more potent and cell permeable analogs. Our current report includes potent, dual-selective inhibitors of CBX6 and CBX8. We have shown that the -2 position in our scaffold is an important residue for selectivity amongst the polycomb paralogs. Preliminary cell-based studies show that the new inhibitors impact cell proliferation in a rhabdoid tumor cell line.

Impaired T cell proliferation by ex vivo BET-inhibition impedes adoptive immunotherapy in a murine melanoma model.

Activation of naïve CD8+ T cells stimulates proliferation and differentiation into cytotoxic T-lymphocytes (CTLs). Adoptive T Cell Therapy (ACT) involves multiple rounds of ex vivo activation to generate enough CTLs for reinfusion into patients, but this drives differentiation into terminal effector T cells. Less differentiated CTL populations, such as stem cell memory T cells, are more ideal candidates for ACT because of increased self-renewal and persistent properties. Ex vivo targeting of T cell differentiation with epigenetic modifiers is a potential strategy to improve cytotoxic T-lymphocyte (CTL) generation for ACT. We established a pipeline to assess the effects of epigenetic modifiers on CD8+ T cell proliferation, differentiation, and efficacy in a preclinical melanoma model. Single treatment with epigenetic modifiers inhibited T cell proliferation in vitro, producing CD44hiCD62Lhi effector-like T cells rather than a stem cell memory T cell phenotype. Most epigenetic modifying agents had no significant effect on ACT efficacy with the notable exception of the bromodomain and extraterminal (BET)-inhibitor JQ1 which was associated with a decrease in efficacy compared to unmodified T cells. These findings reveal the complexity of epigenetic targeting of T cell differentiation, highlighting the need to precisely define the epigenetic targeting strategies to improve CTL generation for ACT.

Pan-specific and partially selective dye-labeled peptidic inhibitors of the polycomb paralog proteins.

Epigenetic regulation of gene expression is in part controlled by post-translational modifications on histone proteins. Histone methylation is a key epigenetic mark that controls gene transcription and repression. There are five human polycomb paralog proteins (Cbx2/4/6/7/8) that use their chromodomains to recognize trimethylated lysine 27 on histone 3 (H3K27me3). Recognition of the methyllysine side chain is achieved through multiple cation-pi interactions within an 'aromatic cage' motif. Despite high structural similarity within the chromodomains of this protein family, they each have unique functional roles and are linked to different cancers. Selective inhibition of different CBX proteins is desirable for both fundamental studies and potential therapeutic applications. We report here on a series of peptidic inhibitors that target certain polycomb paralogs. We have identified peptidic scaffolds with sub-micromolar potency, and will report examples that are pan-specific and that are partially selective for individual members within the family. These results highlight important structure-activity relationships that allow for differential binding to be achieved through interactions outside of the methyllysine-binding aromatic cage motif.

Robotic extended pelvic lymphadenectomy for intermediate- and high-risk prostate cancer.

BACKGROUND: Accurate staging of prostate cancer is enhanced by a thorough evaluation of the pelvic lymph nodes. Limited data are available regarding robotic extended pelvic lymphadenectomy (PLA) in this setting. OBJECTIVE: Analyze our experience performing robotic extended PLA. DESIGN, SETTING, AND PARTICIPANTS: A total of 143 consecutive men with intermediate- or high-risk clinically localized adenocarcinoma of the prostate underwent robotic extended PLA and radical prostatectomy between September 2010 and November 2011 by a single surgeon. SURGICAL PROCEDURE: Lymph node packets were sent separately from bilateral common, external, and internal iliacs, obturators, node of Cloquet, and anterior prostatic fat. MEASUREMENTS: Descriptive statistics were used to summarize lymph node yields and positive nodes. Clinical variables were examined in logistic regression models to predict lymph node positivity. RESULTS AND LIMITATIONS: Median lymph node yield was 20 (range: 9-65, interquartile range: 15-25). Eighteen patients (13%) were found to have metastatic prostate cancer in the lymph nodes. The mean number of positive nodes found was 2.9 (range: 1-11). In 14 of 18 node-positive patients (78%), the extent of nodal invasion was outside the boundaries of a limited PLA. For four patients with positive nodes (22%), prostate biopsy predicted unilateral disease but PLA revealed contralateral positive lymph nodes. A total of 82% of patients experienced no complications, and most Clavien grade 1-2 complications consisted of anastomotic leakage, urinary retention, ileus, and lymphocele. Only 4% of patients experienced a grade 3 complication. Under multivariate regression analysis, prostate-specific antigen (PSA), clinical stage, and maximum biopsy core tumor volume were identified as significant predictors of finding positive pelvic lymph nodes (area under the curve: 91%). The main limitations include short follow-up and lack of randomization. CONCLUSIONS: Robotic extended bilateral PLA for prostate cancer up to the common iliac bifurcation increases nodal yield and positive nodal rate and can be performed safely. PSA, clinical stage, and maximum biopsy core volume are predictors for lymph node invasion. Long-term follow-up is needed to evaluate for therapeutic benefit.