There is strong evidence to support 'tonic signaling' as central feature in the developmental process leading from pre-B cells to mature, fully competent B cells. These signals are thought to be mediated through the pre-B Cell Receptor (pre-BCR) and essential for pre-B cell protection from apoptosis. This proposal is based upon the central hypothesis that B Cell Precursor Acute Lympoblastic Leukemia (BCP-ALL) blasts also require this pre-BCR survival mechanism, which may contribute to resistance to standard chemotherapeutic challenges and modulate proliferative rates. Moreover, tonic signaling from the pre-BCR is proposed to occur through 1) transient dimerization that occurs through self-association of the l5 component of the surrogate light chain and 2) galectin-mediated crosslinking of pre-BCR, forming more stable pre-BCR dimers and potentially higher- order oligomers. To test these concepts, this study will incorporate state-of-the-art imaging methods, including single particle tracking, FRAP and hyperspectral imaging. This pre-BCR dimerization initiates lyn-mediated phosphorylation of Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) on the cytoplasmic tails of pre- BCR signaling components (Ig? and Ig?). ITAM phosphorylation leads to recruitment of spleen tyrosine kinase (Syk) and activation of downstream signaling cascades involved in cell fate decisions. Readouts of these key events will be based on co-immunoprecipitation, anti-PY western blotting and proximity ligation. Another major goal of this proposal is to develop peptidomimetic inhibitors to block pre-BCR dimerization and tonic signaling. This phase of the project will use molecular dynamic approaches to design inhibitory peptides using a stringent scoring criteria, followed by experiments to optimize binding and to test for disruption of the pre-BCR self- association and/or galectin-binding interfaces using the advanced imaging technologies and cell signaling assays. Later, the generated peptides will be used to screen phage and yeast libraries to develop scFv that can bind to the pre-BCR subcomponents with high affinity. The overall goal is to develop monovalent, biologic agents (peptidomimetics, scFv-Fc) that bind with high-affinity to the pre-BCR and block prosurvival signaling. The scFv-Fc will be evaluated for the potential to recruit NK cells and macrophages for antibody-mediated cytotoxicity (ADCC) or phagocytosis (ADCP), a critical first step for development of anti-pre-BCR therapeutic antibodies.
This project focuses on the most common form of leukemia in children, which results in unrestricted proliferation of 'precursor' B leukemia cells. These cells have a unique receptor on their surface that is referred to as the pre- B Cell Receptor (pre-BCR) and the present study will validate the pre-BCR as a highly specific therapeutic target for treatment of pediatric precursor leukemia, as well as for young adults with the disease who traditionally have poor outcome. This project is particularly important because it can specifically target cancerous pre-B cells while protecting patients from depletion of their mature, antibody-producing B cells that occurs during treatment with other therapeutic choices.
| Erasmus, M Frank; Matlawska-Wasowska, Ksenia; Kinjyo, Ichiko et al. (2016) Dynamic pre-BCR homodimers fine-tune autonomous survival signals in B cell precursor acute lymphoblastic leukemia. Sci Signal 9:ra116 |
