Kinome directed target discovery and validation in unique ovarian clear cell carcinoma models

Advanced stage ovarian clear cell carcinoma (OCCC) is unresponsive to conventional platinum-based chemotherapy. Efforts to improve OCCC chemotherapy responses have focused on combining platinum with other chemotherapeutic agents or targeted therapies, but have unfortunately not led to higher survival rates. SWI/SNF chromatin remodeling complexes are important regulators of chromatin structure and gene transcription. Multiple SWI/SNF subunits are genetically altered in cancer. The SWI/SNF DNA targeting subunit ARID1A is frequently mutated with the highest mutation frequency found in OCCC. Kinases are important for the regulation of proliferation and apoptosis signaling. A number of kinases are mutated in OCCC, including PIK3CA and KRAS, and are known to promote tumor growth. The aim of this thesis was to identify new therapeutic targets for OCCC patients. To this end, we searched for specific kinase vulnerabilities in OCCC with deleterious mutations in ARID1A using kinome-centered shRNA based synthetic lethality screening in ARID1A wild-type and mutant OCCC cell lines. The identified synthetic lethal hit was validated in vitro and in vivo and we found causal evidence for the observed synthetic lethal interaction with ARID1A loss. In addition, we searched for new kinase alterations in a large group of OCCC tumors and cell lines and subsequently tested the druggability of downstream affected pathways in vitro and in patient-derived xenograft (PDX) models of OCCC. Based on the heterogeneous pattern of mutations across PI3K/AKT/mTOR and MAPK proliferation pathways in OCCC, we searched for effective combinations of PI3K/AKT/mTOR and MAPK kinase inhibitors in low-dose concentrations to simultaneously target key kinases in OCCC. Finally, we described the establishment of OCCC PDX models and compared genomic and histopathology characteristics between paired patient and PDX OCCC tumors to determine the level of similarity. In conclusion, new therapeutic targets in OCCC have been identified and a low-dose treatment strategy was preclinically tested in unique OCCC models in this thesis. These results may advance the treatment of OCCC

Enhanced Recovery after Intensive Care (ERIC): study protocol for a German stepped wedge cluster randomised controlled trial to evaluate the effectiveness of a critical care telehealth program on process quality and functional outcome

Introduction: Survival after critical illness has noticeably improved over the last decades due to advances in critical care medicine. Besides, there is an increasing number of elderly patients with chronic diseases being treated in the intensive care unit (ICU). More than half of the survivors of critical illness suffer from medium-term or long-term cognitive, psychological and/or physical impairments after ICU discharge, which is recognised as post-intensive care syndrome (PICS). There are evidence-based and consensus-based quality indicators (QIs) in intensive care medicine, which have a positive influence on patients' long-term outcomes if adhered to. Methods and analysis: The protocol of a multicentre, pragmatic, stepped wedge cluster randomised controlled, quality improvement trial is presented. During 3 predefined steps, 12 academic hospitals in Berlin and Brandenburg, Germany, are randomly selected to move in a one-way crossover from the control to the intervention condition. After a multifactorial training programme on QIs and clinical outcomes for site personnel, ICUs will receive an adapted, interprofessional protocol for a complex telehealth intervention comprising of daily telemedical rounds at ICU. The targeted sample size is 1431 patients. The primary objective of this trial is to evaluate the effectiveness of the intervention on the adherence to eight QIs daily measured during the patient's ICU stay, compared with standard of care. Furthermore, the impact on long-term recovery such as PICS-related, patient-centred outcomes including health-related quality of life, mental health, clinical assessments of cognition and physical function, all-cause mortality and cost-effectiveness 3 and 6 months after ICU discharge will be evaluated. Ethics and dissemination: This protocol was approved by the ethics committee of the Charité-Universitätsmedizin, Berlin, Germany (EA1/006/18). The results will be published in a peer-reviewed scientific journal and presented at international conferences. Study findings will also be disseminated via the website (www.eric-projekt.net). Trial registration number: ClinicalTrials.gov Registry (NCT03671447)

Low-dose triple drug combination targeting the PI3K/AKT/mTOR pathway and the MAPK pathway is an effective approach in ovarian clear cell carcinoma

Advanced stage ovarian clear cell carcinoma (OCCC) is poorly responsive to platinum-based chemotherapy and has an unfavorable prognosis. Previous studies revealed heterogeneous mutations in PI3K/AKT/mTOR and MAPK pathway nodules converging in mTORC1/2 activation. Here, we aimed to identify an effective low-dose combination of PI3K/AKT/mTOR pathway and MAPK pathway inhibitors simultaneously targeting key kinases in OCCC to preclude single-inhibitor initiated pathway rewiring and limit toxicity. Small molecule inhibitors of mTORC1/2, PI3K and MEK1/2 were combined at monotherapy IC20 doses in a panel of genetically diverse OCCC cell lines (n = 7) to determine an optimal low-dose combination. The IC20 dose triple combination reduced kinase activity in PI3K/AKT/mTOR and MAPK pathways, prevented single-inhibitor induced feedback mechanisms and inhibited short and long-term proliferation in all seven cell lines. Finally, this low-dose triple drug combination treatment significantly reduced tumor growth in two genetically characterized OCCC patient-derived xenograft (PDX) models without resulting in weight loss in these mice. The effectiveness and tolerability of this combined therapy in PDX models warrants clinical exploration of this treatment strategy for OCCC and might be applicable to other cancer types with a similar genetic background

Role of the cytosolic loop C2 and the C-terminus of YidC in ribosome binding and insertion activity

Members of the YidC/Oxa1/Alb3 protein family mediate membrane protein insertion, and this process is initiated by the assembly of YidC·ribosome nascent chain complexes at the inner leaflet of the lipid bilayer. The positively charged C terminus of Escherichia coli YidC plays a significant role in ribosome binding but is not the sole determinant because deletion does not completely abrogate ribosome binding. The positively charged cytosolic loops C1 and C2 of YidC may provide additional docking sites. We performed systematic sequential deletions within these cytosolic domains and studied their effect on the YidC insertase activity and interaction with translation-stalled (programmed) ribosome. Deletions within loop C1 strongly affected the activity of YidC in vivo but did not influence ribosome binding or substrate insertion, whereas loop C2 appeared to be involved in ribosome binding. Combining the latter deletion with the removal of the C terminus of YidC abolished YidC-mediated insertion. We propose that these two regions play an crucial role in the formation and stabilization of an active YidC·ribosome nascent chain complex, allowing for co-translational membrane insertion, whereas loop C1 may be involved in the downstream chaperone activity of YidC or in other protein-protein interactions