Chemical proteomics reveals interactors of the alarmone diadenosine triphosphate in the cancer cell line H1299.

Intracellular dinucleoside polyphosphates (Npn Ns) have been known for decades but the functional role remains enigmatic. Diadenosine triphosphate (Ap3 A) is one of the most prominent examples, and its intercellular concentration was shown to increase upon cellular stress. By employment of previously reported Ap3 A-based photoaffinity-labeling probes (PALPs) in chemical proteomics, we investigated the Ap3 A interactome in the human lung carcinoma cell line H1299. The cell line is deficient of the fragile histidine triade (Fhit) protein, a hydrolase of Ap3 A and tumor suppressor. Overall, the number of identified potential interaction partners was significantly lower than in the previously investigated HEK293T cell line. Gene ontology term analysis revealed that the identified proteins participate in similar pathways as for HEK293T, but the percentage of proteins involved in RNA-related processes is higher for H1299. The obtained results highlight similarities and differences of the Ap3 A interaction network in different cell lines and give further indications regarding the importance of the presence of Fhit.

Chemical proteomic profiling reveals protein interactors of the alarmones diadenosine triphosphate and tetraphosphate.

The nucleotides diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A) are formed in prokaryotic and eukaryotic cells. Since their concentrations increase significantly upon cellular stress, they are considered to be alarmones triggering stress adaptive processes. However, their cellular roles remain elusive. To elucidate the proteome-wide interactome of Ap3A and Ap4A and thereby gain insights into their cellular roles, we herein report the development of photoaffinity-labeling probes and their employment in chemical proteomics. We demonstrate that the identified ApnA interactors are involved in many fundamental cellular processes including carboxylic acid and nucleotide metabolism, gene expression, various regulatory processes and cellular response mechanisms and only around half of them are known nucleotide interactors. Our results highlight common functions of these ApnAs across the domains of life, but also identify those that are different for Ap3A or Ap4A. This study provides a rich source for further functional studies of these nucleotides and depicts useful tools for characterization of their regulatory mechanisms in cells.

Inducible knock-out of BCL6 in lymphoma cells results in tumor stasis.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphomas worldwide and is characterized by a high diversity of genetic and molecular alterations. Chromosomal translocations and mutations leading to deregulated expression of the transcriptional repressor BCL6 occur in a significant fraction of DLBCL patients. An oncogenic role of BCL6 in the initiation of DLBCL has been shown as the constitutive expression of BCL6 in mice recapitulates the pathogenesis of human DLBCL. However, the role of BCL6 in tumor maintenance remains poorly investigated due to the absence of suitable genetic models and limitations of pharmacological inhibitors. Here, we have utilized tetracycline-inducible CRISPR/Cas9 mutagenesis to study the consequences of BCL6 deletion in established DLBCL models in culture and in vivo. We show that BCL6 knock-out in SU-DHL-4 cells in vitro results in an anti-proliferative response 4-7 days after Cas9 induction that was characterized by cell cycle (G1) arrest. Conditional BCL6 deletion in established DLBCL tumors in vivo induced a significant tumor growth inhibition with initial tumor stasis followed by slow tumor growth kinetics. Our findings support a role of BCL6 in the maintenance of lymphoma growth and showcase the utility of inducible CRISPR/Cas9 systems for probing oncogene addiction.

Leptospira spp. in Small Mammals from Areas with Low and High Human Hantavirus Incidences in South-West Germany.

INTRODUCTION: Leptospirosis is caused by Leptospira spp. and is considered the most widespread zoonotic disease worldwide. It mimics nephropathia epidemica in humans, a disease mainly caused by Puumala hantavirus (PUUV). Small mammals are reservoirs for Leptospira spp. and PUUV. Seewis virus (SWSV) is a shrew-borne hantavirus with unknown pathogenicity. The objective of this study was to estimate the prevalence for Leptospira spp. and the frequency of Leptospira-hantavirus co-infections in small mammals collected at locations with high and low incidences in humans. MATERIALS AND METHODS: In 2012 and 2013, 736 small mammals belonging to seven species (Apodemus flavicollis, Microtus agrestis, Microtus arvalis, Myodes glareolus, Sorex araneus, S. coronatus, and S. minutus) were collected at four high incidence sites (H1-H4) and four low (L1-L4) incidence sites for PUUV infection in humans. Kidney-derived DNA samples were tested for Leptospira spp. by real-time PCR targeting the lipl 32 gene and further analyzed by duplex PCR targeting the flaB and the secY genes. For the detection of Seewis virus, lung-derived DNA was tested via RT-PCR targeting the nucleocapsid gene. RESULTS: Altogether, 42 of the 736 small mammals including 27 of 660 bank voles and 11 of 66 shrews, were positive for Leptospira spp., while Sorex spp. (14.7%) showed significantly higher prevalences compared to bank voles (4.1%). Detected Leptospira spp. were pathogenic species other than L. kirschneri. Significantly more Leptospira-positive bank voles were found at H sites than at L sites. Altogether 22.2% of positive bank voles were infected with PUUV. Double infection of PUUV and Leptospira spp. occurrence in bank voles is 1.86 times (OR = 1.86; 95% CI: 0.72-4.73) more likely than infections with each pathogen separately. DISCUSSION: Leptospira- positive bank voles are focally positively associated with PUUV infection in bank voles and with human hantavirus cases. It should be considered that shrews may serve as Leptospira spp. reservoirs.

Efficacy and mechanism of action of volasertib, a potent and selective inhibitor of Polo-like kinases, in preclinical models of acute myeloid leukemia.

Polo-like kinase 1 (Plk1), a member of the Polo-like kinase family of serine/threonine kinases, is a key regulator of multiple steps in mitosis. Here we report on the pharmacological profile of volasertib, a potent and selective Plk inhibitor, in multiple preclinical models of acute myeloid leukemia (AML) including established cell lines, bone marrow samples from AML patients in short-term culture, and subcutaneous as well as disseminated in vivo models in immune-deficient mice. Our results indicate that volasertib is highly efficacious as a single agent and in combination with established and emerging AML drugs, including the antimetabolite cytarabine, hypomethylating agents (decitabine, azacitidine), and quizartinib, a signal transduction inhibitor targeting FLT3. Collectively, these preclinical data support the use of volasertib as a new therapeutic approach for the treatment of AML patients, and provide a foundation for combination approaches that may further improve and prolong clinical responses.

MR-guided breast biopsy using an active marker: a phantom study.

PURPOSE: To evaluate the advantages of using an active marker (active micro coil) for MR-guided breast biopsy procedures. MATERIALS AND METHODS: An add-on breast biopsy guidance device used with a standard breast coil was equipped with an active marker. The marker's position was determined with a dedicated MRI sequence. In combination with custom software, the biopsy planning process was reduced basically to defining the target in the diagnostic MR images. Automatic control scans verified the settings of the biopsy guidance device. To measure the targeting accuracy, x-ray control of the needle placement was performed in phantoms containing 36 small titanium cylinders. The reliability of the procedure was evaluated in 24 core needle biopsies on phantoms. Workflow enhancements were analyzed. RESULTS: The root mean square deviation of the needle position from the target perpendicular to the needle axis was 1.25 mm, in three-dimensions it was 1.35 mm. All targets were sampled successfully. The duration of a phantom biopsy was nine minutes. CONCLUSION: The use of an active marker can offer advantages for MR-guided breast biopsies in terms of handling and procedure time as well as accuracy.