ZAP70 Integrates BCR-Signaling into Innate Signaling Pathways in CLL

The recent clinical success with small molecule inhibitors targeting the B cell receptor (BCR) pathway provides unambiguous evidence that signals engaging the BCR play a key role in the pathogenesis of CLL. The binding of CLL cells to a proposed auto-antigen has encouraged many researchers to identify these antigens. This has more recently been challenged by the identification of an internal epitope within the BCR leading to a cell autonomous, antigen-independent activation of the BCR. Overall, the response of CLL cells to a continuous activation of the BCR is anergy. Notably, with respect to different subgroup of patients, M-CLL/ZAP70 negative cases appear to be more "locked" in an anergic state than UM/ZAP70 positive cases. Since investigations in non-malignant B cells suggest that innate signaling-pathways integrate into the BCR-signaling cascade to overcome anergy, we investigated the responses to TLR9 ligation in 57 purified CLL patient samples with regard to the expression of ZAP70, which is considered to amplify signals funneled through the BCR:

Here we report that ZAP70 positive CLL shows an increased proliferative response (p=0.005) and enhanced survival (p=0.001) upon TLR9 stimulation when compared to ZAP70 negative CLL. Notably, compared to untreated controls, TLR9 ligation induced apoptosis in ZAP70 negative clones, as opposed to ZAP70 positive cells, in which TLR9 agonists inhibit cell death. TLR9-conditioned media from ZAP70 positive cells completely inhibited the pro-apoptotic effect of TLR9 on ZAP70 negative cells, indicating that soluble factors protect cells from TLR9 induced cell death.

Analyzing the expression and modulation of BH3 proteins in TLR9 activated CLL cells, we observed a loss of the pro-apoptotic protein BIM in ZAP70 positive cells, strongly correlating with prolonged survival. Notably, increased BIM expression upon TLR9 ligation was observed in ZAP70 negative cells. In non-malignant B cells BIM is degraded upon activation of the BCR, but not due to the ligation of TLR9. Therefore, we hypothesized that BCR-mediated signals are involved in the TLR9 response of ZAP70 positive CLL. This hypothesis is further supported by the finding that TLR9 mediated loss of BIM in ZAP70 positive CLL was inhibited by the highly specific Syk inhibitor P505-15. Accordingly, TLR9 mediated survival of CLL cells was decreased by P505-15.

Since the anti-apoptotic effect of TLR9 stimulation is mediated by soluble factors, we analyzed the secretom of TLR9-conditioned media from CLL samples by mass spectrometry. Surprisingly, we found secreted IgM in conditioned media from TLR9 stimulated, ZAP70 positive, but not negative CLL. TLR9 induced secretion of IgM could also be antagonized with P505-15, further supporting our finding that TLR9 ligation impinges on BCR signaling in ZAP70 positive CLL. In addition, TLR9 conditioned media depleted of soluble IgM, failed to protect cells from apoptosis, indicating that IgM is essential to rescue TLR9 stimulated cells from cell death.

In summary, our data strongly indicate that -in ZAP70 positive CLL- TLR9 ligation induces the secretion of IgM and down-regulation of BIM, both features of BCR activation. We propose the idea that the soluble IgM binds to the surface BCR through an internal epitope interaction, engaging a positive feed-back loop and further promoting the survival of ZAP70 positive CLL. Thus, ZAP70 may operate as an amplifier for TLR9 mediated activation of the BCR signaling-cascade.

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