Establishing a chemical genetic link between Bruton tyrosine kinase activity in malignant B cells and cell functions involved in the micro-environmental dialogue.

To elucidate their mechanism of action, inhibitors of Bruton tyrosine kinase (BTK) and resistant BTK mutants were employed to dissect target-dependent cellular functions. BTK-C481S and -T474I, expressed in Ramos and NALM-6 cells, maintained BTK auto-phosphorylation under treatment with ibrutinib or dasatinib, respectively, which showed only modest cytotoxicity. Retained activity of BTK-T474 partially rescued cell migration from inhibition by dasatinib. Importantly, resistant BTK mutants reconstituted B cell receptor-triggered chemokine secretion in the presence of corresponding inhibitors, demonstrating that BTK activity is connected with cell-intrinsic functions of malignant B cells with importance for their dialogue with the micro-environment.

Functional impact and molecular binding modes of drugs that target the PI3K isoform p110δ.

Targeting the PI3K isoform p110δ against B cell malignancies is at the mainstay of PI3K inhibitor (PI3Ki) development. Therefore, we generated isogenic cell lines, which express wild type or mutant p110δ, for assessing the potency, isoform-selectivity and molecular interactions of various PI3Ki chemotypes. The affinity pocket mutation I777M maintains p110δ activity in the presence of idelalisib, as indicated by intracellular AKT phosphorylation, and rescues cell functions such as p110δ-dependent cell viability. Resistance owing to this substitution consistently affects the potency of p110δ-selective in contrast to most multi-targeted PI3Ki, thus distinguishing usually propeller-shaped and typically flat molecules. Accordingly, molecular dynamics simulations indicate that the I777M substitution disturbs conformational flexibility in the specificity or affinity pockets of p110δ that is necessary for binding idelalisib or ZSTK474, but not copanlisib. In summary, cell-based and molecular exploration provide comparative characterization of currently developed PI3Ki and structural insights for future PI3Ki design.

A teaching tool about the fickle p value and other statistical principles based on real-life data.

A poor understanding of statistical analysis has been proposed as a key reason for lack of replicability of many studies in experimental biomedicine. While several authors have demonstrated the fickleness of calculated p values based on simulations, we have experienced that such simulations are difficult to understand for many biomedical scientists and often do not lead to a sound understanding of the role of variability between random samples in statistical analysis. Therefore, we as trainees and trainers in a course of statistics for biomedical scientists have used real data from a large published study to develop a tool that allows scientists to directly experience the fickleness of p values. A tool based on a commonly used software package was developed that allows using random samples from real data. The tool is described and together with the underlying database is made available. The tool has been tested successfully in multiple other groups of biomedical scientists. It can also let trainees experience the impact of randomness, sample sizes and choice of specific statistical test on measured p values. We propose that live exercises based on real data will be more impactful in the training of biomedical scientists on statistical concepts.

Relevant Cytokines in the B Cell Lymphoma Micro-Environment.

Cytokines are soluble protein factors with importance in intercellular communication and, as such, play pivotal roles in the pathogenesis of B cell malignancies. Evidence from in vitro cultures permitted us to choose example cytokines that bind to different biochemical receptor types. Activated malignant B cells or stromal fibroblasts and macrophages prominently secrete the chemokines CCL3 or CXCL12 and CXCL13, respectively. Apart from helper T cells, various cell types of the B cell lymphoma microenvironment are capable of producing the cytokines IL-4, IL-6, IL-10 and TNFα. Owing to its impact on the development of myeloid cells, CSF-1 is among important soluble factors in the B cell lymphoma microenvironment. Inhibitors of B cell receptor-associated kinases often act via the blockade of cytokine production, but also prevent cytokine effects, e.g., chemotaxis. Increments in blood levels in chronic lymphocytic leukemia patients compared to healthy donors and normalization upon treatment with ibrutinib can be explained by producing cell types and modulation of cytokine production observed in vitro.

Copanlisib for treatment of B-cell malignancies: the development of a PI3K inhibitor with considerable differences to idelalisib.

On the occasion of its recent approval for relapsed follicular lymphoma, we review the design and development of the pan-class I PI3K inhibitor copanlisib as a drug for the treatment of B-cell malignancies in comparison with other kinase inhibitors targeting B-cell-receptor signaling, in particular with strictly isoform-δ-selective idelalisib. In agreement with previously defined PI3K-inhibitor chemotypes, the 2,3-dihydroimidazo[1,2-c]quinazoline scaffold of copanlisib adopts a flat conformation in the adenine-binding pocket of the catalytic p110 subunit and further extends into a deeper-affinity pocket in contrast to idelalisib, the quinazoline moiety of which is accommodated in a newly created selectivity pocket. Copanlisib shows higher potency than other clinically developed PI3K inhibitors against all four class I isoforms, with approximately tenfold preference for p110α and p110δ. Owing to its potency and isoform profile, copanlisib exhibits cell-type-specific cytotoxicity against primary chronic lymphocytic leukemia cells and diffuse large B-cell lymphoma (DLBCL) cell lines at nanomolar concentrations. Moreover, copanlisib differs from idelalisib in regard to intravenous versus oral administration and weekly versus twice-daily dosing. In regard to adverse effects, intermittent intravenous treatment with copanlisib leads to fewer gastrointestinal toxicities compared with continuous oral dosing of idelalisib. In relapsed follicular lymphoma, copanlisib appears more effective and especially better tolerated than other targeted therapies. Copanlisib extends existing treatment options for this subtype of indolent non-Hodgkin lymphoma and also shows promising response rates in DLBCL, especially of the activated B-cell type.

Sensitive Detection of the Natural Killer Cell-Mediated Cytotoxicity of Anti-CD20 Antibodies and Its Impairment by B-Cell Receptor Pathway Inhibitors.

The antibody-dependent cell-mediated cytotoxicity (ADCC) of the anti-CD20 monoclonal antibodies (mAbs) rituximab and obinutuzumab against the cell line Raji and isolated CLL cells and its potential impairment by kinase inhibitors (KI) was determined via lactate dehydrogenase release or calcein retention, respectively, using genetically modified NK92 cells expressing CD16-176V as effector cells. Compared to peripheral blood mononuclear cells, recombinant effector cell lines showed substantial alloreactivity-related cytotoxicity without addition of mAbs but afforded determination of ADCC with reduced interassay variability. The cytotoxicity owing to alloreactivity was less susceptible to interference by KI than the ADCC of anti-CD20 mAbs, which was markedly diminished by ibrutinib, but not by idelalisib. Compared to rituximab, the ADCC of obinutuzumab against primary CLL cells showed approximately 30% higher efficacy and less interference with KI. Irreversible BTK inhibitors at a clinically relevant concentration of 1 µM only weakly impaired the ADCC of anti-CD20 mAbs, with less influence in combinations with obinutuzumab than with rituximab and by acalabrutinib than by ibrutinib or tirabrutinib. In summary, NK cell line-based assays permitted the sensitive detection of ADCC of therapeutic anti-CD20 mAbs against CLL cells and of the interference of KI with this important killing mechanism.