B cell receptor signaling and associated pathways in the pathogenesis of chronic lymphocytic leukemia

Abstract

B cell antigen receptor (BCR) signaling is a key driver of growth and survival in both normal and malignant B cells. Several lines of evidence support an important pathogenic role of the BCR in chronic lymphocytic leukemia (CLL). The significant improvement of CLL patients' survival with the use of various BCR pathway targeting inhibitors, supports a crucial involvement of BCR signaling in the pathogenesis of CLL. Although the treatment landscape of CLL has significantly evolved in recent years, no agent has clearly demonstrated efficacy in patients with treatment-refractory CLL in the long run. To identify new drug targets and mechanisms of drug action in neoplastic B cells, a detailed understanding of the molecular mechanisms of leukemic transformation as well as CLL cell survival is required. In the last decades, studies of genetically modified CLL mouse models in line with CLL patient studies provided a variety of exciting data about BCR and BCR-associated kinases in their role in CLL pathogenesis as well as disease progression. BCR surface expression was identified as a particularly important factor regulating CLL cell survival. Also, BCR-associated kinases were shown to provide a crosstalk of the CLL cells with their tumor microenvironment, which highlights the significance of the cells' milieu in the assessment of disease progression and treatment. In this review, we summarize the major findings of recent CLL mouse as well as patient studies in regard to the BCR signalosome and discuss its relevance in the clinics.

Keywords: B cell receptor (BCR) signaling; BTK; CD79a/b (Igα/Igβ); IGHV; PI3K/AKT; SYK; chronic lymphocytic leukemia (CLL).

Figure 1

Igβ signaling tail deficiency in Eµ-TCL1 mice leads to CLL cell reduction. (A) Flow cytometry assessment was performed on B cells from the peripheral blood (PBL) of diseased mb1-CreER^T2;Igβ^Δc/fl;Eµ-TCL1 mice, 1 week (left), 3 weeks (middle), plus 8 weeks (right) following the initiation of Tamoxifen (Tam) treatment. The dot plots of the anti-CD19 versus GFP staining are shown. The CD19⁺GFP⁺ gated region indicates Igβ tail deficient B cells, while the CD19⁺GFP^- gated region marks the Igβ tail sufficient B cell population. The B220 vs CD5 staining of the CD19⁺GFP⁺ gated cells is depicted below. The B220^-CD5⁺ population represents diseased CLL cells, while the B220⁺CD5⁺ gated region exhibits healthy cells. The average relative frequency of the cells within the gate is indicated by the numbers in the dot plots. The data is presentable for three independent mouse analyses. (B) Eight weeks after administering Tam treatment to mb1CreER^T2;Igβ^Δc/fl;Eµ-TCL1 mice, the percentage of B cells in the CD19⁺GFP⁺ and CD19⁺GFP⁻ B cell populations was quantified. The graphs display the respective average percentage of B cells ± SEM, while p-values were determined by a Student’s t-test (two-tailed; ** p < 0.01). The cell count for each group consists of data from three mice. (C) After two weeks of Tam treatment, the fluorescence intensity of IgM BCR expression in CD19⁺GFP⁻ (red) or CD19⁺GFP⁺ (blue) B cells of mb1-CreER^T2;Igβ^Δc/fl;Eµ-TCL1 CLL mice was determined. The results presented in the histogram are representative of three self-contained experiments. (D) Flow cytometry was used to analyze the expression of GFP in mb1-CreER^T2;Igβ^Δc/fl;Eµ-TCL1 CLL cells that were either treated with 4-OHT in vitro (+4-OHT blue) or kept untreated (-4-OHT red) for 5 days (5d). The fluorescence intensity of CD19⁺ B cells was determined and indicated in histograms. The data is presentable of three self-sufficient experiments. (E) The survival of B cells in mb1-CreER^T2;Igβ^Δc/fl;Eµ-TCL1 CLL cells was statistically analyzed on day 5, 7, 9, and 12 following in vitro 4-OHT treatment (+4-OHT; light grey). The control remained without treatment (-4-OHT; dark grey). The graphs display the mean ± SEM and p-values were determined by the Student’s t-test (two-tailed; **** p < 0.0001; ** p < 0.01). The results of three independent analyses are presented.

Figure 2

Igβ-tail deficiency in a Eµ-TCL1 mouse model results in CLL development. (A) Flow cytometry analysis was conducted on B cells isolated from the spleen of 14-month-old Igβ^Δc/Δc;Eμ-TCL1 mice and WT control mice. The dot plot depicts the B220 vs CD5 staining of CD19⁺CD93⁻ gated mature B cells, and the IgM vs IgD staining of CD5⁺B220^low CLL cells of Igβ^Δc/Δc;Eμ-TCL1 mice or on normal CD5⁻ B220⁺ B cells of WT mice. The data presented is representative of three self-contained mouse analyses. (B) Flow cytometry was used to analyze isolated splenic B cells from Igβ^Δc/Δc;Eμ-TCL1 mice and WT mice. The fluorescent intensity of Igβ expression is represented in a histogram. (C) The absolute number of B cells in the peritoneal cavity of 14-month-old Igβ^Δc/Δc;Eμ-TCL1 mice and the control mice of the same age were quantified. The graphs represent the average count ± SEM. A two-tailed Student’s t-test was conducted to obtain the p-values. The cell count for each group comprises two mice. (D) The images show the spleen of a mb1- Igβ^Δc/Δc;Eμ-TCL1 mouse and a WT mouse.

Figure 3

Anti-Igβ treatment does not affect the progression of CLL cells lacking Igβ-tail. CLL cell survival in Eµ-TCL1 and Igβ^Δc/Δc;Eμ-TCL1 mice was analyzed using flow cytometry one day before and two weeks after administering an anti-Igβ antibody. The dot plot illustrates the staining of anti-B220 vs anti-CD5 mature B cells after gating on the CD19⁺CD93⁻ cell population. CLL cells can be identified by expression of the characteristic markers CD19⁺CD93⁻CD5⁺B220^low.

Figure 4

BCR signaling pathway and BCR-associated CXCR4 signaling in CLL. Activated Src family kinases (SFKs), such as LYN, stimulate the phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) located in the cytoplasmic Igα/Igβ signaling subunit of the BCR. This results in the recruitment and activation of SH2 domain-containing effectors, like SYK and ZAP-70, which phosphorylate BTK and CD19. SHP-1 inhibits the phosphorylation of the Igα ITAMs and SYK. Phosphorylated CD19 recruits the PI3K to the cell membrane, where it phosphorylates PIP₂ to generate PIP₃. Thereby, PI3K creates an essential docking platform for PH domain-containing signaling factors, such as PDK1, BTK and AKT. Binding to PIP₃ results in membrane recruitment and activation of PDK1, BTK and AKT, which mediate the initiation of several BCR downstream signaling cascades, such as RAS/RAF/MEK/ERK signaling, NFAT, NF-κB and mTORC1 signaling. The phosphatase PTEN represses PI3K signaling by PIP₃ dephosphorylation, generating PIP₂. NFAT is activated by increased cytoplasmic Ca²⁺ concentrations, which are induced by PLCγ. NF-κB is retained in an inactive state by the inhibitor IκB. Phosphorylation of IKK leads to IκBs phosphorylation and degradation, finally resulting in NF-κB activation. TSC1/2 inhibits RHEB GTPase activity, which is required to induce mTORC1 activation. AKT- or ERK-mediated inhibition of TSC2, results in mTORC1 activation. In addition, binding of CXCL12 to CXCR4 induces CLL cell migration, survival and chemotaxis via the activation of the downstream signaling pathways MAPK/ERK, PI3K/AKT, PLCγ/Ca²⁺ and NF-κB. This figure was created with BioRender.com. BCR, B cell receptor; LYN, LCK/YES novel kinase; SYK, spleen tyrosine kinase; ZAP-70, CD3ζ-chain-associated protein of 70 kDa; SHP-1, Src homology region 2 domain-containing phosphatase-1; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatase and tensin homolog; PIP₂, phosphatidylinositol-4,5-bisphosphate; PIP₃, phosphatidylinositol-3,4,5-triphosphate; PDK1, 3-phosphoinositide-dependent protein kinase 1; PKB/AKT, protein kinase B; BTK, Bruton’s tyrosine kinase; PLCγ, phospholipase Cγ; PKC, protein kinase C; CXCL12, chemokine C-X-C motif ligand 12; CXCR4, C-X-C motif chemokine receptor; RAS, RAF, Rat sarcoma protein family; MEK; ERK, extracellular-signal-regulated kinase; IKK, IκB kinase complex; IκB, inhibitor of nuclear factor κB; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; NFAT, nuclear factor of activated T-cells; TSC1/2, tuberous sclerosis complex 1/2; RHEB, Ras homolog enriched in brain; mTOR complex mTORC1, mechanistic target of rapamycin; Ca²⁺, Calcium-Ion; GTP, Guanosine-5′-triphosphate; GDP, Guanosindiphosphat; P, phosphorylation.