In vivo Mechanismen des axonalen Kalziumeinstroms im Tiermodell der Multiplen Sklerose

Die Multiple Sklerose (MS) ist eine entzündliche Erkrankung des zentralen Nervensystems, die in Deutschland eine Prävalenz von knapp 300 pro 100.000 Einwohner aufweist und am häufigsten Frauen im Alter von 20-40 Jahren betrifft. Lange Zeit wurde die Demyelinisierung als wichtigster Faktor der Pathophysiologie der MS erachtet. Allerdings haben Forschungsarbeiten in den vergangenen Jahren immer deutlicher gezeigt, dass die irreversible Schädigung von Axonen von entscheidender Bedeutung für das Ausmaß der dauerhaften Behinderung von MS-Patienten ist. Trotz intensiver Bemühungen sind die Prozesse, die zum Verlust von Axonen führen, bislang nur unzureichend verstanden. Besonders in der chronisch progredienten Phase der Erkrankung, die sich bei der Mehrheit der Patienten im Krankheitsverlauf entwickelt, stehen die neurodegenerativen Aspekte im Vordergrund. Während in den vergangenen Jahren mehrere, in erster Linie immunmodulatorisch wirksame Medikamente zur Behandlung der schubförmigen MS zugelassen wurden, sind die Behandlungsmöglichkeiten der chronisch progredienten MS immer noch stark limitiert. Es ist daher von hoher Relevanz, die der axonalen Schädigung zugrundeliegenden Abläufe möglichst detailliert zu verstehen, um daraus in der Zukunft erfolgversprechende neuroprotektive Therapieansätze ableiten zu können. Die Experimentelle Autoimmune Enzephalomyelitis (EAE) ist das am weitesten verbreitete Tiermodell der MS in Ratten und Mäusen und hat trotz aller Limitationen wertvolle Erkenntnisse zur Pathogenese der MS geliefert. Aus diesem Modell wurde eine Vielzahl an Hypothesen zur Pathophysiologie der axonalen Degeneration im Kontext autoimmunentzündlicher ZNS-Erkrankungen abgeleitet. In unserem Labor konnte vor einigen Jahren der Prozess der axonalen Schädigung im Rahmen der EAE mittels in-vivo Mikroskopie in Echtzeit im intakten Gewebe untersucht werden. Dieser Prozess der sogenannten fokalen axonalen Degeneration (FAD) lässt sich auch durch exogene reaktive Sauerstoff- (ROS) und Stickstoffspezies (RNS) induzieren und ähnliche Veränderungen finden sich ebenfalls in Hirnautopsien von MS-Patienten. In Vorarbeiten zu der hier vorliegenden Arbeit konnte mittels in-vivo 2-Photonen-Kalziummikroskopie weiterhin gezeigt werden, dass Axone in akuten EAE-Läsionen oftmals eine erhöhte intrazelluläre Kalzium (Ca2+)-Konzentration aufweisen und dass diese Störung der Ca2+-Homöostase ein wichtiger Indikator dafür ist, ob ein Axon im weiteren zeitlichen Verlauf einen irreversiblen Degenerationsprozess durchläuft oder ob es der entzündlichen Schädigung widerstehen kann. Zur in-vivo 2-Photonen-Ca2+-Mikroskopie werden bei uns im Labor transgene Mäuse verwendet, die unter Kontrolle des neuronenspezifischen Promoters Thy-1 einen FRET-basierten, ratiometrischen Ca2+-Sensor exprimieren. Nachdem aber bislang kein Auslöser dieser pathophysiologisch relevanten Erhöhung der intraaxonalen Ca2+-Konzentration identifiziert werden konnte, befasste ich mich in der hier vorliegenden Arbeit mit der Frage, ob es im Rahmen der akuten Entzündungsreaktion durch die Produktion von ROS/RNS zu einer oxidativen Schädigung der axonalen Zellmembran kommt und ob diese zu einer nachweisbaren Störung der Membranintegrität führt. Hierzu entwickelte ich einen neuartigen Versuchsaufbau, bei dem ich verschiedene Fluoreszenzfarbstoffe intrathekal bei gesunden und akut an EAE erkrankten Mäusen injizierte. Dadurch gelang es mir zu zeigen, dass es im Rahmen der akuten EAE zu einer axonalen Aufnahme von Fluoreszenzfarbstoffen als Hinweis auf Membranläsionen kommt und dass diese mit dem Ausmaß der morphologischen Schädigung zusammenhängt, aber nicht zwangsläufig eine Demyelinisierung voraussetzt. Durch die Kombination von in-vivo 2-Photonen-Ca2+-Mikroskopie und intrathekaler Farbstoffinjektion konnte ich nachweisen, dass Axone mit einer erhöhten Ca2+-Konzentration signifikant häufiger farbstoffpositiv sind als Axone mit einer physiologisch niedrigen Ca2+-Konzentration. Es liegt also der Schluss nahe, dass die Membranläsionen eine mögliche Ursache für die Störung der axonalen Ca2+-Homöostase sein könnten

Calcium Influx through Plasma-Membrane Nanoruptures Drives Axon Degeneration in a Model of Multiple Sclerosis

Axon loss determines persistent disability in multiple sclerosis patients. Here, we use in vivo calcium imaging in a multiple sclerosis model to show that cytoplasmic calcium levels determine the choice between axon loss and survival. We rule out the endoplasmic reticulum, glutamate excitotoxicity, and the reversal of the sodium-calcium exchanger as sources of intra-axonal calcium accumulation and instead identify nanoscale ruptures of the axonal plasma membrane as the critical path of calcium entry

Toward a more patient‐centered drug development process in clinical trials for patients with myelodysplastic syndromes/neoplasms (MDS): Practical considerations from the International Consortium for MDS (icMDS)

Notable treatment advances have been made in recent years for patients with myelodysplastic syndromes/neoplasms (MDS), and several new drugs are under development. For example, the emerging availability of oral MDS therapies holds the promise of improving patients' health‐related quality of life (HRQoL). Within this rapidly evolving landscape, the inclusion of HRQoL and other patient‐reported outcomes (PROs) is critical to inform the benefit/risk assessment of new therapies or to assess whether patients live longer and better, for what will likely remain a largely incurable disease. We provide practical considerations to support investigators in generating high‐quality PRO data in future MDS trials. We first describe several challenges that are to be thoughtfully considered when designing an MDS‐focused clinical trial with a PRO endpoint. We then discuss aspects related to the design of the study, including PRO assessment strategies. We also discuss statistical approaches illustrating the potential value of time‐to‐event analyses and their implications within the estimand framework. Finally, based on a literature review of MDS randomized controlled trials with a PRO endpoint, we note the PRO items that deserve special attention when reporting future MDS trial results. We hope these practical considerations will facilitate the generation of rigorous PRO data that can robustly inform MDS patient care and support treatment decision‐making for this patient population

Modulation of RNA splicing enhances response to BCL2 inhibition in leukemia.

Therapy resistance is a major challenge in the treatment of cancer. Here, we performed CRISPR-Cas9 screens across a broad range of therapies used in acute myeloid leukemia to identify genomic determinants of drug response. Our screens uncover a selective dependency on RNA splicing factors whose loss preferentially enhances response to the BCL2 inhibitor venetoclax. Loss of the splicing factor RBM10 augments response to venetoclax in leukemia yet is completely dispensable for normal hematopoiesis. Combined RBM10 and BCL2 inhibition leads to mis-splicing and inactivation of the inhibitor of apoptosis XIAP and downregulation of BCL2A1, an anti-apoptotic protein implicated in venetoclax resistance. Inhibition of splicing kinase families CLKs (CDC-like kinases) and DYRKs (dual-specificity tyrosine-regulated kinases) leads to aberrant splicing of key splicing and apoptotic factors that synergize with venetoclax, and overcomes resistance to BCL2 inhibition. Our findings underscore the importance of splicing in modulating response to therapies and provide a strategy to improve venetoclax-based treatments

Risk Prediction for Clonal Cytopenia: Multicenter Real-World Evidence.

Clonal cytopenia of undetermined significance (CCUS) represents a distinct disease entity characterized by myeloid-related somatic mutations with a variant allele fraction of ≥2% in individuals with unexplained cytopenia(s) but without a myeloid neoplasm (MN). Notably, CCUS carries a risk of progressing to MN, particularly in cases featuring high-risk mutations. Understanding CCUS requires dedicated studies to elucidate its risk factors and natural history. Our analysis of 357 CCUS patients investigated the interplay between clonality, cytopenia, and prognosis. Multivariate analysis identified 3 key adverse prognostic factors: the presence of splicing mutation(s) (score = 2 points), platelet count <100×109/L (score = 2.5), and ≥2 mutations (score = 3). Variable scores were based on the coefficients from the Cox proportional hazards model. This led to the development of the Clonal Cytopenia Risk Score (CCRS), which stratified patients into low- (score <2.5 points), intermediate- (score 2.5-<5), and high-risk (score ≥5) groups. The CCRS effectively predicted 2-year cumulative incidence of MN for low- (6.4%), intermediate- (14.1%), and high- (37.2%) risk groups, respectively, by Gray's test (P <.0001). We further validated the CCRS by applying it to an independent CCUS cohort of 104 patients, demonstrating a c-index of 0.64 (P =.005) in stratifying the cumulative incidence of MN. Our study underscores the importance of integrating clinical and molecular data to assess the risk of CCUS progression, making the CCRS a valuable tool that is practical and easily calculable. These findings are clinically relevant, shaping the management strategies for CCUS and informing future clinical trial designs

Special considerations in the management of adult patients with acute leukaemias and myeloid neoplasms in the COVID-19 era: recommendations from a panel of international experts

This article is made available for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.The ongoing COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 is a global public health crisis. Multiple observations indicate poorer post-infection outcomes for patients with cancer than for the general population. Herein, we highlight the challenges in caring for patients with acute leukaemias and myeloid neoplasms amid the COVID-19 pandemic. We summarise key changes related to service allocation, clinical and supportive care, clinical trial participation, and ethical considerations regarding the use of lifesaving measures for these patients. We recognise that these recommendations might be more applicable to high-income countries and might not be generalisable because of regional differences in health-care infrastructure, individual circumstances, and a complex and highly fluid health-care environment. Despite these limitations, we aim to provide a general framework for the care of patients with acute leukaemias and myeloid neoplasms during the COVID-19 pandemic on the basis of recommendations from international experts

Hyperleukocytosis and Leukostasis in Acute Myeloid Leukemia: Can a Better Understanding of the Underlying Molecular Pathophysiology Lead to Novel Treatments?

Up to 18% of patients with acute myeloid leukemia (AML) present with a white blood cell (WBC) count of greater than 100,000/µL, a condition that is frequently referred to as hyperleukocytosis. Hyperleukocytosis has been associated with an adverse prognosis and a higher incidence of life-threatening complications such as leukostasis, disseminated intravascular coagulation (DIC), and tumor lysis syndrome (TLS). The molecular processes underlying hyperleukocytosis have not been fully elucidated yet. However, the interactions between leukemic blasts and endothelial cells leading to leukostasis and DIC as well as the processes in the bone marrow microenvironment leading to the massive entry of leukemic blasts into the peripheral blood are becoming increasingly understood. Leukemic blasts interact with endothelial cells via cell adhesion molecules such as various members of the selectin family which are upregulated via inflammatory cytokines released by leukemic blasts. Besides their role in the development of leukostasis, cell adhesion molecules have also been implicated in leukemic stem cell survival and chemotherapy resistance and can be therapeutically targeted with specific inhibitors such as plerixafor or GMI-1271 (uproleselan). However, in the absence of approved targeted therapies supportive treatment with the uric acid lowering agents allopurinol and rasburicase as well as aggressive intravenous fluid hydration for the treatment and prophylaxis of TLS, transfusion of blood products for the management of DIC, and cytoreduction with intensive chemotherapy, leukapheresis, or hydroxyurea remain the mainstay of therapy for AML patients with hyperleukocytosis

Risk-Adapted, Individualized Treatment Strategies of Myelodysplastic Syndromes (MDS) and Chronic Myelomonocytic Leukemia (CMML)

Myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) are two distinct blood cancers with a variable clinical symptom burden and risk of progression to acute myeloid leukemia. Management decisions should be guided by individual patient and disease characteristics and based on validated risk stratification tools. While supportive care with red blood cell transfusions, erythropoiesis-stimulating agents, and iron chelation remains the mainstay of therapy for lower-risk (LR)-MDS patients, luspatercept has recently been approved for transfusion-dependent anemic LR-MDS patients ending a decade without any new drug approvals for MDS. For higher-risk patients, allogeneic hematopoietic cell transplant (allo-HCT) remains the only curative therapy for both MDS and CMML but most patients are not eligible for allo-HCT. For those patients, the hypomethylating agents (HMA) azacitidine and decitabine remain standard of care with azacitidine being the only agent that has shown an overall survival benefit in randomized trials. Although early results from novel molecularly driven agents such as IDH1/2 inhibitors, venetoclax, magrolimab, and APR-246 for MDS as well as tagraxofusp, tipifarnib, and lenzilumab for CMML appear encouraging, confirmatory randomized trials must be completed to fully assess their safety and efficacy prior to routine clinical use. Herein, we review the current management of MDS and CMML and conclude with a critical appraisal of novel therapies and general trends in this field