Target evaluation of the non-coding csRNAs reveals a link of the two-component regulatory system CiaRH to competence control in Streptococcus pneumoniae R6.

The two-component regulatory system CiaRH of Streptococcus pneumoniae controls 25 genes, five of which specify homologous small non-coding csRNAs (cia-dependent small RNAs). The csRNAs were predicted to act regulatory as base-pairing sRNAs, but their targets have not been identified. By csRNA gene inactivations we established that the major phenotypes associated with a hyperactive CiaRH system, enhanced ß-lactam resistance and prevention of genetic competence, are dependent on the csRNAs. Computational target predictions and evaluations by translational fusions identified six genes to be under csRNA control: spr0081, spr0371, spr0159, spr0551, spr1097 and spr2043(comC). Measuring the effect of single csRNAs on three targets indicated that they acted additively. One of the targets, comC(spr2043), encoding the precursor of the competence stimulating pheromone CSP, constitutes a link of CiaRH to competence control. Partially disrupting predicted csRNA-comC complementarity led to strongly diminished repression by the csRNAs and to transformability in a strain with a hyperactive CiaRH. Thus, a hyperactive CiaRH system prevents competence development by csRNA-dependent post-transcriptional repression of CSP production. The csRNAs are also involved in competence regulation in the wild-type strain R6, but their activity is only apparent in the absence of the protease gene htrA, another CiaRH regulon member.

Frequency and clinical features of treatment-refractory myasthenia gravis.

BACKGROUND: To investigate the frequency and characterize the clinical features of treatment-refractory myasthenia gravis in an Austrian cohort. METHODS: Patient charts of 126 patients with generalized myasthenia gravis and onset between 2000 and 2016 were analyzed retrospectively. Patients were classified as treatment-refractory according to strict, predefined criteria. These mandated patients being at least moderately symptomatic (i.e., MGFA class III) or needing either maintenance immunoglobulins or plasma exchange therapy for at least 1 year in spite of two adequately dosed immunosuppressive drugs. Clinical features and outcome at last follow-up were compared to treatment-responsive patients. RESULTS: 14 out of 126 patients (11.1%) met these criteria of treatment-refractory myasthenia gravis. Treatment-refractory patients had more frequent clinical exacerbations and more often received rescue treatments or a further escalation of immunosuppressive therapies. They also remained more severely affected at last follow-up. An early onset of myasthenia gravis was associated with a higher risk for a refractory course. CONCLUSION: A small subgroup of patients with generalized myasthenia gravis do not respond sufficiently to standard therapies. Refractory disease has considerable implications for both patients and health care providers and highlights an unmet need for new treatment options.

Requirement for LIM kinases in acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive disease for which only few targeted therapies are available. Using high-throughput RNA interference (RNAi) screening in AML cell lines, we identified LIM kinase 1 (LIMK1) as a potential novel target for AML treatment. High LIMK1 expression was significantly correlated with shorter survival of AML patients and coincided with FLT3 mutations, KMT2A rearrangements, and elevated HOX gene expression. RNAi- and CRISPR-Cas9-mediated suppression as well as pharmacologic inhibition of LIMK1 and its close homolog LIMK2 reduced colony formation and decreased proliferation due to slowed cell-cycle progression of KMT2A-rearranged AML cell lines and patient-derived xenograft (PDX) samples. This was accompanied by morphologic changes indicative of myeloid differentiation. Transcriptome analysis showed upregulation of several tumor suppressor genes as well as downregulation of HOXA9 targets and mitosis-associated genes in response to LIMK1 suppression, providing a potential mechanistic basis for the anti-leukemic phenotype. Finally, we observed a reciprocal regulation between LIM kinases (LIMK) and CDK6, a kinase known to be involved in the differentiation block of KMT2A-rearranged AML, and addition of the CDK6 inhibitor palbociclib further enhanced the anti-proliferative effect of LIMK inhibition. Together, these data suggest that LIMK are promising targets for AML therapy.

Requirement for YAP1 signaling in myxoid liposarcoma.

Myxoid liposarcomas (MLS), malignant tumors of adipocyte origin, are driven by the FUS-DDIT3 fusion gene encoding an aberrant transcription factor. The mechanisms whereby FUS-DDIT3 mediates sarcomagenesis are incompletely understood, and strategies to selectively target MLS cells remain elusive. Here we show, using an unbiased functional genomic approach, that FUS-DDIT3-expressing mesenchymal stem cells and MLS cell lines are dependent on YAP1, a transcriptional co-activator and central effector of the Hippo pathway involved in tissue growth and tumorigenesis, and that increased YAP1 activity is a hallmark of human MLS Mechanistically, FUS-DDIT3 promotes YAP1 expression, nuclear localization, and transcriptional activity and physically associates with YAP1 in the nucleus of MLS cells. Pharmacologic inhibition of YAP1 activity impairs the growth of MLS cells in vitro and in vivo These findings identify overactive YAP1 signaling as unifying feature of MLS development that could represent a novel target for therapeutic intervention.