While mutations affecting protein-coding regions have been examined across many cancers, structural variants at the genome-wide level are still poorly defined. Through integrative deep whole-genome and -transcriptome analysis of 101 castration-resistant prostate cancer metastases (109X tumor/38X normal coverage), we identified structural variants altering critical regulators of tumorigenesis and progression not detectable by exome approaches. Notably, we observed amplification of an intergenic enhancer region 624 kb upstream of the androgen receptor (AR) in 81% of patients, correlating with increased AR expression. Tandem duplication hotspots also occur near MYC, in lncRNAs associated with post-translational MYC regulation. Classes of structural variations were linked to distinct DNA repair deficiencies, suggesting their etiology, including associations of CDK12 mutation with tandem duplications, TP53 inactivation with inverted rearrangements and chromothripsis, and BRCA2 inactivation with deletions. Together, these observations provide a comprehensive view of how structural variations affect critical regulators in metastatic prostate cancer.

Lymphomas arising from NK or gd-T cells are very aggressive diseases and little is known regarding their pathogenesis. Here we report frequent activating mutations of STAT3 and STAT5B in NK/T-cell lymphomas (n ¼ 51), gd-T-cell lymphomas (n ¼ 43) and their cell lines (n ¼ 9) through next generation and/or Sanger sequencing. STAT5B N642H is particularly frequent in all forms of gd-T-cell lymphomas. STAT3 and STAT5B mutations are associated with increased phosphorylated protein and a growth advantage to transduced cell lines or normal NK cells. Growth-promoting activity of the mutants can be partially inhibited by a JAK1/2 inhibitor. Molecular modelling and surface plasmon resonance measurements of the N642H mutant indicate a marked increase in binding affinity of the phosphotyrosine-Y699 with the mutant histidine. This is associated with the prolonged persistence of the mutant phosphoSTAT5B and marked increase of binding to target sites. Our findings suggest that JAK-STAT pathway inhibition may represent a therapeutic strategy.

Highlights d ARfl and ARv7 genomic binding is interdependent and colocalized d ARv7, unlike ARfl, preferentially represses transcription d Expression of ARv7-repressed genes negatively correlates with recurrence d Re-expression of ARv7-repressed genes may serve as a biomarker of ARv7 inhibition

In the recently identified cholesterol catabolic pathway of Mycobacterium tuberculosis, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (HsaD) is proposed to catalyze the hydrolysis of a carbon-carbon bond in 4,5-9,10-diseco-3-hydroxy-5,9,17-tri-oxoandrosta-1(10),2-diene-4-oic acid (DSHA), the cholesterol meta-cleavage product (MCP) and has been implicated in the intracellular survival of the pathogen. Herein, purified HsaD demonstrated 4 -33 times higher specificity for DSHA (k cat /K m ‫؍‬ 3.3 ؎ 0.3 ؋ 10 4 M ؊1 s ؊1 ) than for the biphenyl MCP 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) and the synthetic analogue 8-(2-chlorophenyl)-2-hydroxy-5-methyl-6-oxoocta-2,4-dienoic acid (HOPODA), respectively. The S114A variant of HsaD, in which the active site serine was substituted with alanine, was catalytically impaired and bound DSHA with a K d of 51 ؎ 2 M. The S114A⅐DSHA species absorbed maximally at 456 nm, 60 nm red-shifted versus the DSHA enolate. Crystal structures of the variant in complex with HOPDA, HOPODA, or DSHA to 1.8 -1.9 Å indicate that this shift is due to the enzyme-induced strain of the enolate. These data indicate that the catalytic serine catalyzes tautomerization. A second role for this residue is suggested by a solvent molecule whose position in all structures is consistent with its activation by the serine for the nucleophilic attack of the substrate. Finally, the ␣-helical lid covering the active site displayed a ligand-dependent conformational change involving differences in side chain carbon positions of up to 6.7 Å , supporting a two-conformation enzymatic mechanism. Overall, these results provide novel insights into the determinants of specificity in a mycobacterial cholesterol-degrading enzyme as well as into the mechanism of MCP hydrolases.Mycobacterium tuberculosis is the leading cause of bacterial mortality, causing an estimated 2 million deaths/year (1). The mechanisms underlying the remarkable ability of this pathogen to survive for long periods of time within the host are poorly understood (2). Although it was well known that saprophytic mycobacteria could metabolize cholesterol (3), it was only recently demonstrated that pathogenic strains can also utilize this nutrient as a growth substrate (4, 5). Interestingly, cholesterol has been found in high concentrations within caseating granulomas in both humans and mice (6, 7), and bacteria have been observed congregating around cholesterol foci (7). Highlighting the importance of cholesterol in bacterial pathogenesis, the deletion of genes involved in cholesterol metabolism reduces the virulence of M. tuberculosis (5,8). Therefore, further knowledge of cholesterol metabolism in M. tuberculosis is crucial to our understanding of bacterial virulence.M. tuberculosis catabolizes cholesterol using a metabolic pathway similar to that identified in Rhodococcus jostii RHA1 (4, 9). In this pathway, the aerobic degradation of the fourringed steroid nucleus occurs through the opening of ring B, aromatization of ring A, and hydroxyla...

The androgen receptor (AR) is the best studied drug target for the treatment of prostate cancer. While there are a number of drugs that target the AR, they all work through the same mechanism of action and are prone to the development of drug resistance. There is a large unmet need for novel AR inhibitors which work through alternative mechanism(s). Recent studies have identified a novel site on the AR called Binding Function 3 (BF3) that is involved into AR transcriptional activity. In order to identify inhibitors that target the BF3 site, we have conducted a large-scale in-silico screen followed by experimental evaluation. A number of compounds were identified that effectively inhibited the AR transcriptional activity with no obvious cytotoxicity. The mechanism of action of these compounds was validated by biochemical assays and x-ray crystallography. These findings lay a foundation for the development of alternative or supplementary therapies capable of combating prostate cancer even in its anti-androgen resistant forms.

Immune responses are initiated and primed by dendritic cells (DCs) that cross-present exogenous antigen. The CD74 (invariant chain) chaperone protein is thought to exclusively promote DC priming in the context of MHC class II. However, we demonstrate herein a CD74-dependent MHC class I cross-presentation pathway in DCs that plays a major role in the generation of MHC class I restricted, cytolytic T lymphocyte (CTL) responses against viral protein- and cell-associated antigens. CD74 associates with MHC class I molecules in the endoplasmic reticulum of DCs and mediates trafficking of MHC class I to endolysosomal compartments for loading with exogenous peptides. We conclude that CD74 plays a hitherto, undiscovered physiological function in endolysosomal DC cross-presentation for priming MHC class I-mediated CTL responses.

It has come to our attention that we inadvertently swapped the headings on the two columns of Table S4. From left to right, the headings should read ''No AR peak amplification'' and then ''AR peak amplification''. Only the headings were swapped. The manuscript reports the correct result, and the statistical tests we performed on the values (two-by-two contingency table tests) are unchanged. The error has been corrected online, and we apologize for any confusion it may have caused.

The androgen receptor (AR) is one of the most studied drug targets for the treatment of prostate cancer. However, all current anti-androgens directly interact with the AR at the androgen binding site, which is prone to resistant mutations, calling for new strategies of the AR inhibition. The current study represents the first attempt to use virtual screening to identify inhibitors of activation function-2 (AF2) of the human AR. By combining large-scale docking with experimental approaches, we were able to identify several small molecules that interact with the AF2 and effectively prevent the transcriptional activation of the AR. The crystallographic structure of one of these inhibitors in complex with the AR provides critical insight into the corresponding protein-ligand interactions and suitable for future hit optimization. Taken together, our results provide a promising ground for development of novel anti-androgens that can help to address the problem of drug resistance in prostate cancer.