Chemotherapeutics Used for High-Risk Neuroblastoma Therapy Improve the Efficacy of Anti-GD2 Antibody Dinutuximab Beta in Preclinical Spheroid Models.

Anti-disialoganglioside GD2 antibody ch14.18/CHO (dinutuximab beta, DB) improved the outcome of patients with high-risk neuroblastoma (HR-NB) in the maintenance phase. We investigated chemotherapeutic compounds used in newly diagnosed patients in combination with DB. Vincristine, etoposide, carboplatin, cisplatin, and cyclophosphamide, as well as DB, were used at concentrations achieved in pediatric clinical trials. The effects on stress ligand and checkpoint expression by neuroblastoma cells and on activation receptors of NK cells were determined by using flow cytometry. NK-cell activity was measured with a CD107a/IFN-γ assay. Long-term cytotoxicity was analyzed in three spheroid models derived from GD2-positive neuroblastoma cell lines (LAN-1, CHLA 20, and CHLA 136) expressing a fluorescent near-infrared protein. Chemotherapeutics combined with DB in the presence of immune cells improved cytotoxic efficacy up to 17-fold compared to in the controls, and the effect was GD2-specific. The activating stress and inhibitory checkpoint ligands on neuroblastoma cells were upregulated by the chemotherapeutics up to 9- and 5-fold, respectively, and activation receptors on NK cells were not affected. The CD107a/IFN-γ assay revealed no additional activation of NK cells by the chemotherapeutics. The synergistic effect of DB with chemotherapeutics seems primarily attributed to the combined toxicity of antibody-dependent cellular cytotoxicity and chemotherapy, which supports further clinical evaluation in frontline induction therapy.

BCL11B depletion induces the development of highly cytotoxic innate T cells out of IL-15 stimulated peripheral blood αß CD8+ T cells.

BCL11B, an essential transcription factor for thymopoiesis, regulates also vital processes in post-thymic lymphocytes. Increased expression of BCL11B was recently correlated with the maturation of NK cells, whereas reduced BCL11B levels were observed in native and induced T cell subsets displaying NK cell features. We show that BCL11B-depleted CD8+ T cells stimulated with IL-15 acquired remarkable innate characteristics. These induced innate CD8+ (iiT8) cells expressed multiple innate receptors like NKp30, CD161, and CD16 as well as factors regulating migration and tissue homing while maintaining their T cell phenotype. The iiT8 cells effectively killed leukemic cells spontaneously and neuroblastoma spheroids in the presence of a tumor-specific monoclonal antibody mediated by CD16 receptor activation. These iiT8 cells integrate the innate natural killer cell activity with adaptive T cell longevity, promising an interesting therapeutic potential. Our study demonstrates that innate T cells, albeit of limited clinical applicability given their low frequency, can be efficiently generated from peripheral blood and applied for adoptive transfer, CAR therapy, or combined with therapeutic antibodies.

Nuclear import of BCL11B is mediated by a classical nuclear localization signal and not the Krüppel-like zinc fingers.

The Krüppel-like transcription factor (KLF) BCL11B is characterized by a wide tissue distribution and crucial functions in key developmental and cellular processes, as well as in various pathologies including cancer and HIV infection. Although the basics of BCL11B activity and relevant interactions with other proteins have been uncovered, how this exclusively nuclear protein localizes to its compartment remained unclear. Here, we demonstrate that unlike other KLFs, BCL11B does not require the C-terminal DNA-binding domain to pass through the nuclear envelope but has an independent, previously unidentified, nuclear localization signal (NLS), which is located distantly from the zinc finger domains and fulfills the essential criteria of being an autonomous NLS. First, it can redirect a heterologous cytoplasmic protein to the nucleus. Second, its mutation causes aberrant localization of the protein of origin. Finally, we provide experimental and in silico evidences of the direct interaction with importin-α. The relative conservation of this motif allows formulating a consensus sequence (K/R)K-X13-14-KR+K++ ('+' indicates amino acids with similar chemical properties), which can be found in all BCL11B orthologs among vertebrates and in the closely related protein BCL11A.

Unveiling the N-Terminal Homodimerization of BCL11B by Hybrid Solvent Replica-Exchange Simulations.

Transcription factors play a crucial role in regulating biological processes such as cell growth, differentiation, organ development and cellular signaling. Within this group, proteins equipped with zinc finger motifs (ZFs) represent the largest family of sequence-specific DNA-binding transcription regulators. Numerous studies have proven the fundamental role of BCL11B for a variety of tissues and organs such as central nervous system, T cells, skin, teeth, and mammary glands. In a previous work we identified a novel atypical zinc finger domain (CCHC-ZF) which serves as a dimerization interface of BCL11B. This domain and formation of the dimer were shown to be critically important for efficient regulation of the BCL11B target genes and could therefore represent a promising target for novel drug therapies. Here, we report the structural basis for BCL11B-BCL11B interaction mediated by the N-terminal ZF domain. By combining structure prediction algorithms, enhanced sampling molecular dynamics and fluorescence resonance energy transfer (FRET) approaches, we identified amino acid residues indispensable for the formation of the single ZF domain and directly involved in forming the dimer interface. These findings not only provide deep insight into how BCL11B acquires its active structure but also represent an important step towards rational design or selection of potential inhibitors.