Dynamic pre-BCR homodimers fine-tune autonomous survival signals in B cell precursor acute lymphoblastic leukemia

Abstract

The pre-B cell receptor (pre-BCR) is an immature form of the BCR critical for early B lymphocyte development. It is composed of the membrane-bound immunoglobulin (Ig) heavy chain, surrogate light chain components, and the signaling subunits Igα and Igβ. We developed monovalent Quantum Dot (QD)-labeled probes specific for Igβ to study the behavior of pre-BCRs engaged in autonomous, ligand-independent signaling in live B cells. Single-particle tracking revealed that QD-labeled pre-BCRs engaged in transient, but frequent, homotypic interactions. Receptor motion was correlated at short separation distances, consistent with the formation of dimers and higher-order oligomers. Repeated encounters between diffusing pre-BCRs appeared to reflect transient co-confinement in plasma membrane domains. In human B cell precursor acute lymphoblastic leukemia (BCP-ALL) cells, we showed that frequent, short-lived, homotypic pre-BCR interactions stimulated survival signals, including expression of BCL6, which encodes a transcriptional repressor. These survival signals were blocked by inhibitory monovalent antigen-binding antibody fragments (Fabs) specific for the surrogate light chain components of the pre-BCR or by inhibitors of the tyrosine kinases Lyn and Syk. For comparison, we evaluated pre-BCR aggregation mediated by dimeric galectin-1, which has binding sites for carbohydrate and for the λ5 component of the surrogate light chain. Galectin-1 binding resulted in the formation of large, highly immobile pre-BCR aggregates, which was partially relieved by the addition of lactose to prevent the crosslinking of galectin-BCR complexes to other glycosylated membrane components. Analysis of the pre-BCR and its signaling partners suggested that they could be potential targets for combination therapy in BCP-ALL.

Oncogene-independent BCR-like signaling adaptation confers drug resistance in Ph-like ALL

“…We thus hypothesized that BCR-like signaling in Ph-like ALL is activated by spontaneous association of cell surface proteins involved in normal BCR signaling. Previous studies in pre-BCR + B-ALL suggest that CD79A and CD79B can cross-link and activate signaling in the absence of external factors (60), potentially via short-lived, homotypic pre-BCR interactions that provide survival signals (61). Demonstration of the exact complex and its mechanism of interaction with downstream signaling was beyond the scope of this study.…”

Limitations of Qdot labelling compared to directly-conjugated probes for single particle tracking of B cell receptor mobility

“…The first reason relates to the potential for receptor crosslinking. We minimized this possibility by using limiting dilutions of Fabs and Qdots, but although it is possible to engineer monovalent Qdots with a single bound Fab 16 , 18 , 58 , monovalency is a significant benefit of directly-labelled Fab fragments. Qdots can also have cytotoxic effects that may influence the physiology of the cell and hence the mobility of the receptor 59 , 60 .…”

“…Importantly, methods for determining the diffusion coefficient while accounting for limited positional accuracy have recently been advanced 29 – 31 . A large number of models extending the Brownian paradigm have also been developed, allowing us to study transient changes in particle behaviour during individual tracks such as fast/slow apparent mobility 32 – 34 , directed motion 35 , 36 , transient binding 16 and/or anomalous diffusion 37 – 40 . Similarly, there has been gradual development in our ability to confidently detect zones of transient confinement on the basis of track behavior 41 – 43 .…”

VpreB surrogate light chain expression in B-lineage ALL: a report from the Children’s Oncology Group

“… 1 The pre-BCR is composed of 5 units (see visual abstract): a membrane-bound V-, D-, J-recombined immunoglobulin heavy chain, an invariably constant surrogate light chain (SLC), comprising VpreB (CD179a) and λ5 (CD179b), 1 and transmembrane immunoglobulin α (Igα) and Igβ accessory chains that coassemble to provide intracellular signaling through SRC and SYK family kinases. 2,3 Differentiation into mature B cells can only occur when immature B-precursors have undergone recombination of genes encoding κ or λ light chains, which dynamically replace the SLC in maturing B cells to create a functional BCR. 3 Without pre-BCR mediated “tonic” autonomous signaling, immature B cells undergo programmed cell death, but this critical selection step may be subverted by oncogenic transformation.…”