SYK inhibition thwarts the BAFF - B-cell receptor crosstalk and thereby antagonizes Mcl-1 in chronic lymphocytic leukemia

Abstract

Although small molecule inhibitors of B-cell receptor-associated kinases have revolutionized therapy in chronic lymphocytic leukemia (CLL), responses are incomplete. Pro-survival signaling emanating from the microenvironment may foster therapeutic resistance of the malignant B cells resident in the protective lymphoid niches. B-cell activating factor (BAFF) is critical to the survival of both healthy and neoplastic B cells. However, the pro-survival pathways triggered by BAFF have not been fully characterized. Here we show that BAFF elicited resistance to spontaneous and drug-induced apoptosis in stromal co-cultures, induced activation of both canonical and non-canonical NFκB signaling pathways, and triggered B-cell receptor signaling in CLL cells, independently of IGHV mutational status. SYK, a proximal kinase in the B-cell receptor signaling cascade, acted via STAT3 to bolster transcription of the anti-apoptotic protein Mcl-1, thereby contributing to apoptosis resistance in BAFF-stimulated cells. SYK inhibitor entospletinib downregulated Mcl-1, abrogating BAFF-mediated cell survival. BAFF-B-cell receptor crosstalk in neoplastic B cells was mediated by SYK interaction with TRAF2/TRAF3 complex. Thus, SYK inhibition is a promising therapeutic strategy uniquely poised to antagonize crosstalk between BAFF and B-cell receptor, thereby disrupting the pro-survival microenvironment signaling in chronic lymphocytic leukemia.

Figure 1.

B-cell activating factor (BAFF) promotes activation of pro-survival pathways in chronic lymphocytic leukemia (CLL) cells. (A) CLL cells (n=10) were cultured on BAFF-expressing or parental cells for 24 hours (h), followed by incubation with the indicated drugs, or vehicle control (ctrl), for an additional 24 h. As a reference, cells were treated off stroma. Apoptosis within CD19⁺ subset of cells was determined by Annexin V and 7-AAD staining. Data are presented as mean±Standard Error (SE). *P<0.05 compared to ‘off stroma’. (B and C) CLL cells from 3 individual samples were co-cultured with BAFF-expressing stroma for 24 h. RNA was isolated from the purified CLL B cells and microarray analysis was performed as described in the Methods section. Heatmap shows hierarchical clustering of expression profiles of the 122 differentially expressed NFκB target genes (yellow: upregulation; blue: downregulation). (D) CLL cells from 3 individual patients were co-cultured with BAFF-expressing stroma for 4–24 h. Whole-cell protein lysates were subjected to immunoblotting. (E) CLL cells (n=4) were co-cultured with control, BAFF- or CD40L-expressing stroma for 24 h. p52 and p65/RelA activity was determined in whole-cell protein lysates using the TransAM NFκB activity assay. The dotted line represents activity measured in freshly isolated cells (at 0 h), which has been set at 1 (*P<0.05, **P<0.01 compared against that). (F) CLL cells (n=4) were co-cultured under the indicated conditions for 24 h. Whole-cell protein lysates were subjected to immunoblotting.

Figure 2.

Expression of Mcl-1 and Bcl-xL in chronic lymphocytic leukemia (CLL) lymph nodes. Lymphatic tissue from patients with CLL (n=10) were subjected to immunocytochemistry for Mcl-1 (A) and Bcl-xL (B), as described in the Methods section (40×).

Figure 3.

B-cell activating factor (BAFF) activates BCR signaling in chronic lymphocytic leukemia (CLL) cells. (A–C) CLL cells were stimulated with 5 μg/μL sol-IgM or 25 ng/μL sol-BAFF. Cells were lysed at the indicated time points and subjected to immunoblotting. A representative result of 10 independent experiments is shown (includes 4 unmut-IGHV and 6 mut-IGHV). Densitometry (C) was performed on immunoblots from 10 individual CLL samples after 15 minutes (min) stimulation with IgM or BAFF. (D) CLL cells were incubated or not with idelalisib (5 μM) for 1 hour (h) and stimulated with sol-IgM or sol-BAFF for 30 min. Cell migration using 200 ng/mL CXCL12 was evaluated as described in the Methods section. P<0.05 versus control.

Figure 4.

Inhibitors of BCR-associated kinases abrogate B-cell activating factor (BAFF)-mediated canonical NFκB activation in chronic lymphocytic leukemia (CLL). (A) CLL cells (n=6) were cultured on BAFF-expressing stroma for 24 hours (h), followed by incubation with the indicated drugs, or vehicle control (ctrl), for an additional 24 h. Apoptosis within the CD19⁺ subset of cells was determined by Annexin V and 7-AAD staining. Data are presented as mean±Standard Error (SE). *P<0.05 compared to vehicle control. (B) CLL cells (n=4) were incubated with the indicated drugs or vehicle control for 1 h, followed by stimulation with 25 ng/mL sol-BAFF for 30 minutes (min). Cell migration using 200 ng/mL CXCL12 was evaluated as described in the Methods section. (C) CLL cells (n=4) were co-cultured with BAFF-expressing stroma for 24 h, and incubated with the indicated drugs for an additional 24 h. p52/RelA activity was determined in nuclear protein lysates using the TransAM NFκB activity assay (ActivMotif). **P<0.01 compared to untreated control.

Figure 5.

SYK inhibition down-regulates Mcl-1 via STAT3. (A) Chronic lymphocytic leukemia (CLL) cells were co-cultured with B-cell activating factor (BAFF)-expressing stroma for 24 hours (h), followed by incubation with the indicated drugs for 24 h. Cells were also treated off stroma for 24 h. Apoptosis within CD19⁺ subset of cells was determined by Annexin V and 7-AAD staining (n=6). Data are presented as mean±Standard Error (SE). *P<0.05, **P<0.01, compared to ‘off stroma’ control, or as shown. (B and C) CLL cells were co-cultured with BAFF-expressing stroma for 24 h, followed by incubation with the indicated drugs for 24 h in the presence of caspase inhibitor QVD-OPh (1 μM). Cells were lysed and subjected to immunoblotting. Densitometry chart (C) represents data from 6 individual CLL samples. Data are presented as mean±Standard Error (SE). *P<0.05 compared to control. (D) CLL cells (4 individual samples) were co-cultured with BAFF-expressing stroma for 24 h, followed by addition of 100 μg/mL cycloheximide and 1 μM entospletinib or vehicle control. Cells were lysed at the indicated time points and subjected to immunoblotting. (E and F) CLL cells (n=4) were co-cultured with BAFF-expressing stroma for 24 h, treated with SYK inhibitors (entospletinib, R406), JAK1/2 inhibitor (ruxolitinib), BTK inhibitor (ibrutinib) or PI-3Kδ inhibitor (idelalisib) for 24 h in the presence of caspase inhibitor QVD-OPh (1 μM), followed by collection of mRNA and protein.

Figure 6.

SYK interacts with TRAF2/TRAF3 in neoplastic B cells. Cells were stimulated with 25 ng/mL sol-B-cell activating factor (BAFF) for 30 minutes (min). Proteins lysates were subjected to immunoprecipitation experiments using indicated antibodies as described in the Methods section. A representative blot of 3 independent experiments is shown. CLL: chronic lymphocytic leukemia.

Figure 7.

B-cell activating factor (BAFF)-BCR crosstalk in chronic lymphocytic leukemia (CLL) cells. BAFF-R engagement stabilizes NIK within the NIK/TRAF2/TRAF3/cIAP1/2 complex, promoting the non-canonical NFκB pathway activity. SYK recruitment to TRAF2/TRAF3 assists BAFF-mediated activation of BCR signaling, which contributes to activation of the canonical NFκB. Concurrently, SYK induces STAT3 transcription factor, thereby up-regulating Mcl-1, a pro-survival Bcl-2 family member.