Spatial organization and early signaling of the B-cell receptor in CLL.

Most chronic lymphocytic leukemia (CLL) clones express B-cell receptors (BcR) of both IgM/IgD isotypes; however, 5%-10% of CLL cases express isotype-switched immunoglobulin G (IgG). The early signaling and spatial patterning of the various BcRs at steady state and after activation are still fully unresolved. Herein, we show higher expression of the BcR signalosome elements and a more robust constitutive cell-intrinsic proximal BcR signaling in CLL with unmutated IGHV expressing IgM isotype (IgM U-CLL), compared with IGHV-mutated CLL (M-CLL) expressing either IgM or IgG isotypes. IgM in U-CLL is frequently located in the membrane plane in polarized patches, occasionally in caps, and sometimes inside the cells. Among M-CLL, IgM is scattered laterally in the membrane plane in a similar pattern as seen in normal B cells, whereas IgG is dispersed around the cell membrane in smaller clusters than in IgM U-CLL. Upon BcR engagement, both IgG and IgM expressing M-CLL showed attenuated signaling and only slight spatial reorganization dynamics of BcR microclusters and internalization, compared with the extensive reorganization and internalization of the BcR in IgM expressing U-CLL. The global gene signature of IgG M-CLL was closely related to that of IgM M-CLL rather than IgM U-CLL. Overall, we report fundamental differences in the basal composition, biochemical status, and spatial organization of the BcR in the three examined immunogenetic CLL subtypes that correlate with their clinical behavior. On the basis of our findings, IgG class-switched M-CLL likely represents the same disease as IgM M-CLL rather than a different biological and/or clinical entity.

Proteolysis Targeting Chimeras for BTK Efficiently Inhibit B-Cell Receptor Signaling and Can Overcome Ibrutinib Resistance in CLL Cells.

Proteolysis targeting chimeras (PROTACs) are small molecules that form ternary complexes between their target and E3 ligase, resulting in ubiquitination and proteasomal degradation of the target protein. Using our own designed Bruton's tyrosine kinase (BTK) PROTAC compounds, we show herein efficient BTK degradation in chronic lymphocytic leukemia (CLL) cells. The reversible non-covalent compound (NC-1) was the most potent and therefore we focused on this PROTAC to investigate its subsequent effects on the BCR pathway. NC-1 decreased baseline BTK phosphorylation as well as activation of BTK and other signaling molecules downstream of the BCR pathway, following IgM engagement. These effects were also obtained in samples from CLL patients with clinical resistance to ibrutinib and mutations at C481. NC-1 treatment further decreased baseline CD69 surface levels, completely abrogated its upregulation following IgM activation, decreased CLL cells migration toward SDF-1 and overcame stromal anti-apoptotic protection. In conclusion, our results indicate that targeting BTK using the PROTAC strategy could be a potential novel therapeutic approach for CLL.

Efficacy of the BNT162b2 mRNA COVID-19 vaccine in patients with chronic lymphocytic leukemia.

Patients with chronic lymphocytic leukemia (CLL) have an increased risk for severe COVID-19 disease and mortality. The goal of this study was to determine the efficacy of COVID-19 vaccine in patients with CLL. We evaluated humoral immune responses to the BNT162b2 messenger RNA (mRNA) COVID-19 vaccine in patients with CLL and compared responses with those obtained in age-matched healthy control subjects. Patients received 2 vaccine doses, 21 days apart, and antibody titers were measured by using the Elecsys Anti-SARS-CoV-2 S assay after administration of the second dose. In a total of 167 patients with CLL, the antibody response rate was 39.5%. A comparison between 52 patients with CLL and 52 sex- and aged-matched healthy control subjects revealed a significantly reduced response rate among patients (52% vs 100%, respectively; adjusted odds ratio, 0.010; 95% confidence interval, 0.001-0.162; P < .001). The response rate was highest in patients who obtained clinical remission after treatment (79.2%), followed by 55.2% in treatment-naive patients and 16.0% in patients under treatment at the time of vaccination. In patients treated with either Bruton's tyrosine kinase inhibitors or venetoclax ± anti-CD20 antibody, response rates were considerably low (16.0% and 13.6%). None of the patients exposed to anti-CD20 antibodies <12 months before vaccination responded. In a multivariate analysis, the independent predictors of response were younger age, female sex, lack of currently active treatment, immunoglobulin G levels ≥550 mg/dL, and immunoglobulin M levels ≥40 mg/dL. In conclusion, antibody-mediated response to the BNT162b2 mRNA COVID-19 vaccine in patients with CLL is markedly impaired and affected by disease activity and treatment. This trial was registered at www.clinicaltrials.gov as #NCT04746092.