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 surrogate light chain λ5 component. 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 cross-linking 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.

Fig. 1. Direct evidence of pre-BCR dimerization on the surface of BCP-ALL cells

(A) Experimental model with estimation of the interaction distance based upon the pre-BCR crystal structure (PDB ID: 2H32), streptavidin (PDB ID: 1STP), and the estimated QD radii. (B) Time-lapse imaging (20 frames/s) of a single cell under resting conditions. (C) Viterbi plot of the most likely state (dimer, domain, and free) between two QD interactions as derived from hidden Markov model (HMM) with separation distance as the observed parameter. (D) Two-channel 3D trajectory of anti-Igβ Fab’-QD655 (magenta) and Fab’-QD585 (green) bound to two receptors that serially engaged repeatedly over 14.5 s, with several instances of correlated motion before separating toward the end of acquisition. Data are representative of 35 independent experiments with 697 cells. (E and F) Analysis of displacement (magenta, jump magnitude) and degree of uncorrelated motion (green) as a function of separation distance (between 150 to 200 nm), observed with both 697 cells (E) and Nalm6 cells (F). (G and H) Diffusion by state, as determined by HMM analysis. (I and J) Distribution of dimer lifetimes in 697 and Nalm6 cells, with estimated off-rates as predicted by HMM analysis. Interaction distances in the HMM were set to 100 and 300 nm, respectively. Data in (E) to (J) represent 26,447 trajectories in 697 cells and 2,800 trajectories in Nalm6 cells.

Fig. 2. Galectin-1 stabilizes receptor dimerization and promotes the formation of higher-order complexes

(A) Analysis of the binding of labeled galectin-1 (red) to the pre-BCR (green) in 697 cells. Arrows indicate cluster formation and the colocalization of galectin-1 with the pre-BCR. Images are representative of three experiments. (B and C) Blocking the carbohydrate-binding domain of galectin-1 with 10 mM lactose revealed differences in displacements for the uncorrelated jump distance and the jump magnitude of distinct anti-Igβ probes at different separation distances. (D) Analysis of the diffusion of the pre-BCR in 697 cells treated with galectin-1 (GAL-1) in the absence or presence of 10 mM lactose. Plots represent cumulative data from 1491 (galectin) and 1560 (galectin + lactose) trajectories tracked over five independent experiments.

Fig. 3. Comparison of the effects of VpreB Fabs and galectin-1 on pre-BCR diffusion and dimerization

(A and B) Ensemble MSD values (A) and diffusion coefficients (B) revealed changes in pre-BCRs after 10 min of treatment with anti-VpreB Fabs (1 μM, blue) or galectin-1 (10 μM, black). The two-tailed t test from MATLAB was used to confirm statistical significance (see table S2). (C and D) Changes in uncorrelated jump distance and jump magnitude after 10 min of treatment with either 1 μM anti-VpreB Fab (blue dotted lines) or 10 μM galectin-1 (black dotted lines). (E and F) 697 cells were left untreated or were treated for 24 hours with 5 μM anti-VpreB Fab (E) or with 10 μM galectin-1 in the presence or absence of 10 mM lactose (F) before being subjected to Western blotting analysis with antibodies against the indicated proteins. Each blot is representative of three or more independent experiments. Bar graphs report total densitometry data for the abundances of the indicated proteins normalized to the abundance of actin.

Fig. 4. Western blotting analysis of pre-BCR signaling

(A) Western blotting analysis of Nalm6 cell lysates (3 × 10⁷cells) for Syk phosphorylation at Tyr³⁴⁸ (pSyk Y348) after the indicated treatments. Blots were stripped and incubated with antibodies against total Syk and actin. Note that the pervanadate lane is shown after gray scale adjustment of the digital image, to adjust for the intensity of the signal for pSykY348 compared to that in resting cells and after treatment with inhibitors. (B and C) Western blotting analysis of 697 cells (1.0 × 10⁶) with antibodies specific for pSyk and pCD79a (Igα) after the cells were treated for 30 min with anti-VpreB Fab in the presence of pervanadate. (D) Western blotting analysis of 697 cells (1.0 × 10⁶) with antibodies against pSyk and pCD79a (Igα) after the cells were treatment with BAY61-3606 (Syk inhibitor) or dasatinib (Lyn and Abl inhibitor) in the presence of pervanadate. (E) Western blotting analysis of 697 cells (1.0 × 10⁶) with antibody specific for pSyk after the cells were treated for 30 min with 3AC (SHIP inhibitor) or tofacitinib (Jak inhibitor) in the presence of pervanadate. (F) Western blotting analysis of 697 cells (1.0 × 10⁶) with antibody specific for total BCL6 protein after the cells were treated for 24 hours with dasatinib, tofacitinib, or 3AC. All blots are representative of three or more independent experiments. Accompanying bar graphs report total densitometry data for all experiments for a given condition, with the abundance of the protein of interest normalized to that of actin or total Syk.

Fig. 5. Analysis of apoptosis and viability in BCP-ALL cell lines after treatment with pre-BCR pathway inhibitors

(A) Annexin V (FITC-conjugated) and 7-AAD labeling of 697 cells after 3 days of culture in the absence (autonomous) and presence of anti-VpreB Fabs or vincristine, as indicated. (B) 7-AAD and Annexin-V labeling of 697 cells treated for 3 days with a range of concentrations (0.1 to 100 μM) of tyrosine kinase inhibitors (BAY61-6306, dasatinib, tofacitinib) or inositol phosphatase (3AC). (C) 7-AAD and Annexin-V labeling of 697 cells treated for 3 days with or without a single dose (10 μM) of tyrosine kinase inhibitors (BAY61-6306, dasatinib, tofacitinib) in combination with increasing concentrations (0.1 to 100 ng/ml) of vincristine. (D) 7-AAD and Annexin-V labeling of 697 cells incubated for 3 days with or without 3AC (0.1 to 50 μM) in combination with increasing doses of vincristine (0.1 to 100 ng/ml). In all plots, the percentage of cells that were 7AAD⁺Annexin-V⁺ is plotted against increasing concentrations of vincristine or inhibitor and fit with a sigmoidal dose-response curve. All data were acquired by flow cytometric analysis of at least 10,000 events for each condition and are representative of xxx independent experiments.

Fig. 6. Expression profile and single particle tracking of pre-BCRs on different primary BCP-ALL cells

(A) Comparison of the cell surface abundances of VpreB and CD79b (Igβ) on 697 cells, Nalm6 cells, and blasts from patients #238 and #280, as a readout of pre-BCR on the membrane of BCP-ALL cells. Cells were incubated with saturating concentrations of labeled fluorophore-conjugated anti-VpreB or anti-CD79b antibodies and binding values were quantified by flow cytometry. Results were calibrated by saturation binding of each antibody to Simply Cellular anti-mouse IgG microspheres. Data are representative of at least three independent experiments. (B) Comparison of the mean fluorescent values after anti-VpreB and anti-CD79b labeling of the indicated cell types, plotted as a ratio of pre-BCR (2 VpreB = 1 pre-BCR) to CD79b. (C) Comparison of the diffusion coefficient (μm²/s) of the pre-BCR in 697 cells (n = 26,447 trajectories) and Nalm6 cells (n = 3,573 trajectories) to primary cells from patient 238 (304 trajectories) and patient 280 (353 trajectories); single particle tracking was performed on untreated cells in all cases and performed over multiple days. (D) Ensemble MSD plots derived from SPT of the pre-BCR on 697 cells with or without a 10-min treatment with the indicated tyrosine kinase inhibitors. (E) Ensemble MSD plots derived from SPT of the pre-BCR on the surface of primary cells from patient 280 with and without a 10-min treatment with dasatinib (1 μM) or anti-VpreB Fabs (1 μM). (F) Ensemble MSD plots derived from SPT of the pre-BCR on the surface of primary cells from patient 238 with and without a 10-min treatment with dasatinib (1 μM) or anti-VpreB Fabs (1 μM). Data in (D) to (F) are representative of xxx independent experiments.

Fig. 7. Flow cytometric and Western blot analyses of primary BCP-ALL cells after treatment with tyrosine kinase inhibitors or anti-VpreB Fab

(A and B) Left: Plots show Annexin V (FITC-conjugated) and 7-AAD labeling of primary cells from patient #280 (A) and patient #238 (B) after 24 hours of culture with or without anti-VPreB Fabs (5 μM) or vincristine (1 to 100 ng/ml) as indicated. Right: Scatter plots report flow-based measurements of primary cells from patient 280 (A) and patient 238 (B) that were double labeled for 7-AAD and Annexin-V after 24 hours of culture with or without a single dose of anti-VpreB Fab (5 μM), vincristine (10 ng/ml), or both, as indicated. All data in (A) and (B) were acquired by flow cytometric analysis of at least 10,000 events for each condition and are representative of xxx independent experiments. (C) Western blotting analysis of cell lysates (1.0 × 10⁶ primary cells each) for patients 280 or 238 to compare the relative abundance of BCL6 protein after the cells were treated for 24 hours with dasatinib (10 μM) or anti-VpreB Fab (5 μM). (D) Western blotting analysis of cell lysates (1.0 × 10⁶ cells) from patients 280 and 238 to compare the relative abundance of BCL6 protein after the cells were treated for 24 hours with BAY61-6306, dasatinib, tofacitinib, or 3AC. Each blot is representative of three or more independent experiments. Bar graphs show total densitometry data for all experiments for a given condition, with the abundance of the protein of interest normalized to that of actin.