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Supplementary Figure 1: Epithelial-mesenchymal transition in HMLE Twist1-ER cells; Supplementary Figure 3: Archazolid A does not induce apoptosis in HMLE Twist1-ER cells;Supplementary Figure 2: Archazolid A inhibits V-ATPase activity of HMLE Twist1-ER cells; Supplementary Figure 4: Archazolid A does not influence self-renewal markers Sox2 and OCT-4;Supplementary Figure 5: Archazolid A does not influence EMT markers at mRNA level; Supplementary Figure 6: Vesicle Tracking

Sorafenib represents the current standard of care for patients with advanced-stage hepatocellular carcinoma (HCC). However, acquired drug resistance occurs frequently during therapy and is accompanied by rapid tumor regrowth after sorafenib therapy termination. To identify the mechanism of this therapy-limiting growth resumption, we established robust sorafenib resistance HCC cell models that exhibited mitochondrial dysfunction and chemotherapeutic crossresistance. We found a rapid relapse of tumor cell proliferation after sorafenib withdrawal, which was caused by renewal of mitochondrial structures alongside a metabolic switch toward high electron transport system (ETS) activity. The translation-inhibiting antibiotic tigecycline impaired the biogenesis of mitochondrial DNA-encoded ETS subunits and limited the electron acceptor turnover required for glutamine oxidation. Thereby, tigecycline prevented the tumor relapse in vitro and in murine xenografts in vivo. These results offer a promising second-line therapeutic approach for advanced-stage HCC patients with progressive disease undergoing sorafenib therapy or treatment interruption due to severe adverse events.

IntroductionPharmacotherapy is critical in geriatric fallers owing to the vulnerability of this population. Comprehensive medication management can be an important strategy to reduce the medication-related risk of falling in this patient group. Patient-specific approaches and patient-related barriers to this intervention have rarely been explored among geriatric fallers. This study will focus on establishing a comprehensive medication management process to provide better insights into patients’ individual perceptions regarding their fall-related medication as well as identifying organisational and medical-psychosocial effects and challenges of this intervention.Methods and analysisThe study design is a complementary mixed-methods pre-post study which follows the approach of an embedded experimental model. Thirty fallers aged at least 65 years who were on five or more self-managed long-term drugs will be recruited from a geriatric fracture centre. The intervention consists of a five-step (recording, reviewing, discussion, communication, documentation) comprehensive medication management, which focuses on reducing the medication-related risk of falling. The intervention is framed using guided semi-structured pre-post interventional interviews, including a follow-up period of 12 weeks. These interviews will assess patients’ perceptions of falls, medication-related risks and gauge the postdischarge acceptability and sustainability of the intervention. Outcomes of the intervention will be measured based on changes in the weighted and summated Medication Appropriateness Index score, number of fall-risk-increasing drugs and potentially inadequate medication according to the Fit fOR The Aged and PRISCUS lists. Qualitative and quantitative findings will be integrated to develop a comprehensive understanding of decision-making needs, the perspective of geriatric fallers and the effects of comprehensive medication management.Ethics and disseminationThe study protocol was approved by the local ethics committee of Salzburg County, Austria (ID: 1059/2021). Written informed consent will be obtained from all patients. Study findings will be disseminated through peer-reviewed journals and conferences.Trial registration numberDRKS00026739.

Identification of regulators of osteoblastogenesis that can be pharmacologically targeted is a major goal in combating osteoporosis, a common disease of the elderly population. Here, unbiased kinome RNAi screening in primary murine osteoblasts identified cyclin-dependent kinase 5 (Cdk5) as a suppressor of osteoblast differentiation in both murine and human preosteoblastic cells. Cdk5 knockdown by siRNA, genetic deletion using the Cre-loxP system, or inhibition with the small molecule roscovitine enhanced osteoblastogenesis in vitro. Roscovitine treatment significantly enhanced bone mass by increasing osteoblastogenesis and improved fracture healing in mice. Mechanistically, downregulation of Cdk5 expression increased Erk phosphorylation, resulting in enhanced osteoblast-specific gene expression. Notably, simultaneous Cdk5 and Erk depletion abrogated the osteoblastogenesis conferred by Cdk5 depletion alone, suggesting that Cdk5 regulates osteoblast differentiation through MAPK pathway modulation. We conclude that Cdk5 is a potential therapeutic target to treat osteoporosis and improve fracture healing., publishedVersion

The human mitochondrial ClpXP protease complex (HsClpXP) has recently attracted major attention as a target for novel anti-cancer therapies. Despite its important role in disease progression, the cellular role of HsClpXP is poorly characterized and only few small molecule inhibitors have been reported. Herein, we screened previously established S. aureus ClpXP inhibitors against the related human protease complex and identified potent small molecules against human ClpXP. The hit compounds showed anti-cancer activity in a panoply of leukemia, liver and breast cancer cell lines. We found that the bacterial ClpXP inhibitor 334 impairs the electron transport chain (ETC), enhances the production of mitochondrial reactive oxygen species (mtROS) and thereby promotes protein carbonylation, aberrant proteostasis and apoptosis. In addition, 334 induces cell death in re-isolated patient-derived xenograft (PDX) leukemia cells, potentiates the effect of DNA-damaging cytostatics and re-sensitizes resistant cancers to chemotherapy in non-apoptotic doses.

The X-ray structure of the catalytic domain of the EphA3 tyrosine kinase in complex with a previously reported type II inhibitor was used to design two novel quinoxaline derivatives, inspired by kinase inhibitors that have reached clinical development. These two new compounds were characterized by an array of cell-based assays and gene expression profiling experiments. A global chemical proteomics approach was used to generate the drug-protein interaction profile, which suggested suitable therapeutic indications. Both inhibitors, studied in the context of angiogenesis and in vivo in a relevant lymphoma model, showed high efficacy in the control of tumor size.

Therapeutic options for patients with advanced-stage hepatocellular carcinoma (HCC) are very limited. The only approved first-line treatment is the multi-tyrosine kinase inhibitor sorafenib, which shows low response rates and severe side effects. In particular, the compensatory activation of growth factor receptors leads to chemoresistance and limits the clinical impact of sorafenib. However, combination approaches to improve sorafenib have failed. Here we investigate the inhibition of cyclin-dependent kinase 5 (Cdk5) as a promising combination strategy to improve sorafenib response in HCC. Combination of sorafenib with Cdk5 inhibition (genetic knockdown by short hairpin RNA or CRISPR/Cas9 and pharmacologic inhibition) synergistically impaired HCC progression in vitro and in vivo by inhibiting both tumor cell proliferation and migration. Importantly, these effects were mediated by a mechanism for Cdk5: A liquid chromatography-tandem mass spectrometry-based proteomic approach revealed that Cdk5 inhibition interferes with intracellular trafficking, a process crucial for cellular homeostasis and growth factor receptor signaling. Cdk5 inhibition resulted in an accumulation of enlarged vesicles and respective cargos in the perinuclear region, considerably impairing the extent and quality of growth factor receptor signaling. Thereby, Cdk5 inhibition offers a comprehensive approach to globally disturb growth factor receptor signaling that is superior to specific inhibition of individual growth factor receptors. Conclusion: Cdk5 inhibition represents an effective approach to improve sorafenib response and to prevent sorafenib treatment escape in HCC. Notably, Cdk5 is an addressable target frequently overexpressed in HCC, and with Dinaciclib, a clinically tested Cdk5 inhibitor is readily available. Thus, our study provides evidence for clinically evaluating the combination of sorafenib and Dinaciclib to improve the therapeutic situation for patients with advanced-stage HCC.

Limited supply of nutrient normally causes cell growth arrest. Our recent study (Nat Cell Biol. (7):833-843) shows that fumarase (FH), a key enzyme responsible for the conversion between fumarate and malate in tricarboxylic acid cycle, is importantly involved in the cellular response to nutrient condition.

Human caseinolytic protease P (hClpP) is important for degradation of misfolded proteins in the mitochondrial unfolded protein response. We here introduce tailored hClpP inhibitors that utilize a steric discrimination in their core naphthofuran scaffold to selectively address the human enzyme. This novel inhibitor generation exhibited superior activity compared to previously introduced beta-lactones, optimized for bacterial ClpP. Further insights into the bioactivity and binding to cellular targets were obtained via chemical proteomics as well as proliferation- and migration studies in cancer cells.