Affinitätschromatographie mit orientiert und nicht-orientiert immobilisiertem Calmodulin am Beispiel der Aufreinigung der Ubiquityl-Calmodulin-Synthetase Faktor 2

Im Rahmen dieser Dissertation gelang es eine zielgerichtete Immobilisierung des Calmodulins durch Verwendung des Glutathion-S-Transferase-Calmodulin-Fusionspro-teins (GST-Calmodulin) zu erreichen. Das neue GST-Calmodulin-Gel wurde zur Auf-reinigung von Ubiquityl-Calmodulin-Synthetase Faktor 2 (uCaM Syn-F2) eingesetzt. Da-durch konnte die Ausbeute um Faktor zwei gegenüber der bisher verwendeten Methode mit ungerichtet immobilisiertem Divinylsulfon-Calmodulin gesteigert werden. Von den Ausgangsüberlegungen her hätte dieser Faktor aber etwa sechs betragen müssen, da den sechs verschiedenen Lysinen des Calmodulins im Falle der Divinylsulfon-Kopplung nur ein N-Terminus im Falle der Immobilisierung des GST-Calmodulins gegenüber stand. Die Diskrepanz zwischen dem erwarteten Ergebnis und dem tatsächlich beobachteten Befund wurde so gedeutet, dass im Falle der ungerichteten Immobilisierung möglicherweise nur zwei Lysine des Calmodulins an der Kopplung teilnehmen. Von diesen sich ergebenden Orientierungen würde nur eine die Interaktion mit calmodulinbindenden Proteinen ermöglichen. Diese Vorstellung wiederum erklärt das ähnliche Verhalten im Vergleich zur streng orientierten Immobilisierung des Fusionsproteins am N-Terminus. Auf der Basis bioinformatorischer Untersuchungen wurden verschiedene Modellvorstellungen, wie z.B. Force-Field und Zugänglichkeit, überprüft um so die Bevorzugung bestimmter Lysine zu erklären. Als Anwendungsbeispiel diente hierbei die Ribonuclease, von der bekannt war, dass sie nur über zwei Lysine an die Sepharose gebunden wird. Jedoch konnte keine Modellvorstellung die Bevorzugung einzelner Lysine erklären. Als abschließender Ansatz könnte die unterschiedliche Aktivierungsenergie der einzelnen Lysinseitengruppen eine Erklärung für die eingeschränkte Zahl reaktiver Lysine liefern. Die Aufreinigung der uCaM Syn-F2 an dem neuen Gel ergab, dass uCaM Syn-F2 durch proteolytische Einflüsse während des Lagerns in seinen biochemischen Eigenschaften (z.B. Molekulargewicht und Affinität zum Anionenaustauschergel MonoQ) verändert wurde. Die Proteolyse wurde durch die Anwesenheit von Calcium und der damit bedingten Aktivierung von Calpain verstärkt. Erst durch den Einsatz von Proteaseinhibitoren und kurzen Lagerungszeiten konnten die proteolytischen Einflüsse minimiert werden. Auf Grund hoher Verlusten bei der Anionenaustauschchromatographie wurde versucht, uCaM Syn-F2 durch die, in der Literatur beschriebenen, Blot-Overlay-Verfahren nachzuweisen. Diese Methoden ließen sich im Falle der uCaM Syn-F2 nicht reproduzieren, erst durch eine, in dieser Arbeit neue entwickelten, Overlay-Methode ließ sich eine 31 kDa große Bande als mögliche calmodulinbindende Komponente der uCaM Syn-F2 identifizieren

The Role of Clonal Evolution on Progression, Blood Parameters, and Response to Therapy in Multiple Myeloma

IntroductionA variety of biomarkers are considered for diagnosis (e.g., β2-microgobulin, albumin, or LDH) and prognosis [e.g., cytogenetic aberrations detected by fluorescence in situ hybridization (FISH)] of multiple myeloma (MM). More recently, clonal evolution has been established as key. Little is known on the clinical implications of clonal evolution.MethodsWe performed in-depth analyses of 25 patients with newly diagnosed MM with respect to detailed clinical information analyzing blood samples collected at several time points during follow-up (median follow-up: 3.26 years since first diagnosis). We split our cohort into two subgroups: with and without new FISH clones developing in the course of disease.ResultsEach subgroup showed a characteristic chromosomal profile. Forty-three percent of patients had evidence of appearing new clones. The patients with new clones showed an increased number of translocations affecting chromosomes 14 (78% vs. 33%; p = 0.0805) and 11, and alterations in chromosome 4 (amplifications and translocations). New clones, on the contrary, were characterized by alterations affecting chromosome 17. Subsequent to the development of the new clone, 6 out of 9 patients experienced disease progression compared to 3 out of 12 for patients without new clones. Duration of the therapy applied for the longest time was significantly shorter within the group of patients developing new clones (median: 273 vs. 406.5 days; p = 0.0465).DiscussionWe demonstrated that the development of new clones, carrying large-scale alterations, was associated with inferior disease course and shorter response to therapy, possibly affecting progression-free survival and overall survival as well. Further studies evaluating larger cohorts are necessary for the validation of our results

Activity of tafasitamab in combination with rituximab in subtypes of aggressive lymphoma

BackgroundDespite recent advances in the treatment of aggressive lymphomas, a significant fraction of patients still succumbs to their disease. Thus, novel therapies are urgently needed. As the anti-CD20 antibody rituximab and the CD19-targeting antibody tafasitamab share distinct modes of actions, we investigated if dual-targeting of aggressive lymphoma B-cells by combining rituximab and tafasitamab might increase cytotoxic effects.MethodsAntibody single and combination efficacy was determined investigating different modes of action including direct cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) in in vitro and in vivo models of aggressive B-cell lymphoma comprising diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL).ResultsThree different sensitivity profiles to antibody monotherapy or combination treatment were observed in in vitro models: while 1/11 cell lines was primarily sensitive to tafasitamab and 2/11 to rituximab, the combination resulted in enhanced cell death in 8/11 cell lines in at least one mode of action. Treatment with either antibody or the combination resulted in decreased expression of the oncogenic transcription factor MYC and inhibition of AKT signaling, which mirrored the cell line-specific sensitivities to direct cytotoxicity. At last, the combination resulted in a synergistic survival benefit in a PBMC-humanized Ramos NOD/SCID mouse model.ConclusionThis study demonstrates that the combination of tafasitamab and rituximab improves efficacy compared to single-agent treatments in models of aggressive B-cell lymphoma in vitro and in vivo

Efficacy of COVID-19 Booster Vaccines in Patients with Hematologic Malignancies: Experiences in a Real-World Scenario.

BACKGROUND Two-dose COVID-19 vaccination often results in poor humoral response rates in patients with hematologic malignancies (HMs); yet responses to COVID-19 booster vaccines and the risk of COVID-19 infection post-booster are mostly uncertain. METHODS We included 200 outpatients with HMs and predominantly lymphoid neoplasms (96%, 191/200) in our academic center and reported on the humoral responses, which were assessed by measurement of anti-spike IgG antibodies in peripheral blood as early as 14 days after mRNA-based prime-boost vaccination, as well as factors hampering booster efficacy. Previous basic (double) immunization was applied according to the local recommendations with mRNA- and/or vector-based vaccines. We also report on post-booster COVID-19 breakthrough infections that emerged in the Omicron era and the prophylaxis strategies that were applied to poor and non-responders to booster vaccines. RESULTS A total of 55% (110/200) of the patients achieved seroconversion (i.e., anti-spike protein IgG antibody titer > 100 AU/mL assessed in median 48 days after prime-boost vaccination) after prime-boost vaccination. Multivariable analyses revealed age, lymphocytopenia, ongoing treatment and prior anti-CD20 B-cell depletion to be independent predictors for booster failure. With each month between anti-CD20-mediated B-cell depletion and booster vaccination, the probability of seroconversion increased by approximately 4% (p < 0.001) and serum-antibody titer (S-AbT) levels increased by 90 AU/mL (p = 0.011). Notably, obinutuzumab treatment was associated with an 85% lower probability for seroconversion after prime-boost vaccination compared to rituximab (p = 0.002). Of poor or non-responders to prime-boost vaccination, 41% (47/114) underwent a second booster and 73% (83/114) underwent passive immunization. COVID-19 breakthrough infections were observed in 15% (29/200) of patients after prime-boost vaccination with predominantly mild courses (93%). Next to seroconversion, passive immunization was associated with a significantly lower risk of COVID-19 breakthrough infections after booster, even in vaccine non-responders (all p < 0.05). In a small proportion of analyzed patients with myeloid neoplasms (9/200), the seroconversion rate was higher compared to those with lymphoid ones (78% vs. 54%, accordingly), while the incidence rate of COVID-19 breakthrough infections was similar (22% vs. 14%, respectively). Following the low frequency of myeloid neoplasms in this study, the results may not be automatically applied to a larger cohort. CONCLUSIONS Patients with HMs are at a high risk of COVID-19 booster vaccine failure; yet COVID-19 breakthrough infections after prime-boost vaccination are predominantly mild. Booster failure can likely be overcome by passive immunization, thereby providing immune protection against COVID-19 and attenuating the severity of COVID-19 courses. Further sophistication of clinical algorithms for preventing post-vaccination COVID-19 breakthrough infections is urgently needed

Clinical Post-SARS-CoV-2 Infection Scenarios in Vaccinated and Non-Vaccinated Cancer Patients in Three German Cancer Centers: A Retrospective Analysis.

COVID-19 vaccines have become an integral element in the protection of cancer patients against SARS-CoV-2. To date, there are no direct comparisons of the course of COVID-19 infection in cancer patients between the pre- and post-vaccine era. We analyzed SARS-CoV-2 infections and their impact on cancer in COVID-19 vaccinated and non-vaccinated patients from three German cancer centers. Overall, 133 patients with SARS-CoV-2 were enrolled in pre- and post-vaccine eras: 84 non-vaccinated and 49 vaccinated, respectively. A mild course of COVID-19 was documented more frequently in vaccinated patients (49% vs. 29%), while the frequency of severe and critical courses occurred in approximately one-half of the non-vaccinated patients (22% vs. 42%, p = 0.023). Particularly, patients with hematologic neoplasms benefited from vaccination in this context (p = 0.031). Admissions to intermediate- and intensive-care units and the necessity of non-invasive and invasive respiratory support were reduced by 71% and 50% among vaccinated patients, respectively. The median length of admission was 11 days for non-vaccinated and 5 days for vaccinated patients (p = 0.002). COVID-19 mortality was reduced by 83% in vaccinated patients (p = 0.046). Finally, the median time from SARS-CoV-2 infection to restarting cancer therapy was 12 and 26 days among vaccinated and non-vaccinated groups, respectively (p = 0.002). Although this study does not have enough power to perform multivariate analyses to account for confounders, it provides data on COVID-19 in non-vaccinated and vaccinated cancer patients and illustrates the potential benefits of COVID-19 vaccines for these patients

Structural Variants as a Basis for Targeted Therapies in Hematological Malignancies

Structural variants (SV) are changes in the genomic landscape that can alter gene expression levels and thus lead to disease development. The most common and best studied SVs in hematological malignancies are chromosomal translocations. Here, parts of two genes that are normally on different chromosomes come into close proximity due to a failure in DNA repair. As a consequence, fusion proteins which show a different function and/or cellular localization compared to the two original proteins are expressed, sometimes even at different levels. The identification of chromosomal translocations is often used to identify the specific disease a patient is suffering from. In addition, SVs such as deletions, duplications, inversions and single nucleotide polymorphisms (SNPs) can occur in hematopoietic cells and lead to their malignant transformations. Changes in the 3D genome structure have also recently been shown to impact disease development. In this review, we describe a variety of SVs occurring in different subtypes of hematological malignancies. Currently, most therapeutic approaches target fusion proteins which are the cellular product of chromosomal translocations. However, amplifications and SNPs also play a role in disease progression and can be targeted. We present some examples for different types of structural variants and how they are currently treated

Combination Treatment Targeting mTOR and MAPK Pathways Has Synergistic Activity in Multiple Myeloma

Multiple myeloma (MM) is an incurable, malignant B cell disorder characterized by frequent relapses and a poor prognosis. Thus, new therapeutic approaches are warranted. The phosphatidylinositol-3-kinase (PI3K) pathway plays a key role in many critical cellular processes, including cell proliferation and survival. Activated PI3K/AKT (protein kinases B)/mTOR (mammalian target of rapamycin) signaling has been identified in MM primary patient samples and cell lines. In this study, the efficacy of PI3K and mTOR inhibitors in various MM cell lines representing three different prognostic subtypes was tested. Whereas MM cell lines were rather resistant to PI3K inhibition, treatment with the mTOR inhibitor temsirolimus decreases the phosphorylation of key molecules in the PI3K pathway in MM cell lines, leading to G0/G1 cell cycle arrest and thus reduced proliferation. Strikingly, the efficacy of temsirolimus was amplified by combining the treatment with the Mitogen-activated protein kinase kinase (MEK) inhibitor trametinib. Our findings provide a scientific rationale for the simultaneous inhibition of mTOR and MEK as a novel strategy for the treatment of MM

High Metabolic Dependence on Oxidative Phosphorylation Drives Sensitivity to Metformin Treatment in MLL/AF9 Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a group of hematological cancers with metabolic heterogeneity. Oxidative phosphorylation (OXPHOS) has been reported to play an important role in the function of leukemic stem cells and chemotherapy-resistant cells and are associated with inferior prognosis in AML patients. However, the relationship between metabolic phenotype and genetic mutations are yet to be explored. In the present study, we demonstrate that AML cell lines have high metabolic heterogeneity, and AML cells with MLL/AF9 have upregulated mitochondrial activity and mainly depend on OXPHOS for energy production. Furthermore, we show that metformin repressed the proliferation of MLL/AF9 AML cells by inhibiting mitochondrial respiration. Together, this study demonstrates that AML cells with an MLL/AF9 genotype have a high dependency on OXPHOS and could be therapeutically targeted by metformin