Bruton's tyrosine kinase mediated signaling enhances leukemogenesis in a mouse model for chronic lymphocytic leukemia

Abstract

In chronic lymphocytic leukemia (CLL) signals from the B cell receptor (BCR) play a major role in disease development and progression. In this light, new therapies that specifically target signaling molecules downstream of the BCR continue to be developed. While first studies on the selective small molecule inhibitor of Bruton's tyrosine kinase (Btk), Ibrutinib (PCI-32765), demonstrated that Btk inhibition sensitizes CLL cells to apoptosis and alters their migratory behavior, these studies however did not address whether Btk-mediated signaling is involved in the process of CLL leukemogenesis. To investigate the requirement of Btk signaling for CLL development, we modulated Btk expression in the IgH.ETμ CLL mouse model, which is based on sporadic expression of the simian oncovirus SV40 T-antigen in mature B cells. To this end, we crossed IgH.ETμ mice on a Btk-deficient background or introduced a human Btk transgene (CD19-hBtk). Here we show that Btk deficiency fully abrogates CLL formation in IgH.ETμ mice, and that leukemias formed in Btk haplo-insufficient mice selectively expressed the wild-type Btk allele on their active X chromosome. Conversely, Btk overexpression accelerated CLL onset, increased mortality, and was associated with selection of non-stereotypical BCRs into CLL clones. Taken together, these data show that Btk expression represents an absolute prerequisite for CLL development and that Btk mediated signaling enhances leukemogenesis in mice. We therefore conclude that in CLL Btk expression levels set the threshold for malignant transformation.

Keywords: B cell receptor signaling; Chronic lymphocytic leukemia (CLL); bruton’s tyrosine kinase (Btk).

Figure 1

Altered activation of downstream BCR signaling pathways in IgH.ETμ tumors. Flow cytometric measurement of phosphorylation of Akt (A) and Erk (B) upon BCR stimulation in IgH.ETμ CLL cells versus wild-type (WT) B cells. MACS-purified CD19⁺ cells were stimulated for 5 minutes with 20 μg/mL goat anti-mouse Igκ and subsequently stained for phosphorylated Akt or Erk. C: Protein levels of c-Rel determined by western blotting in nuclear lysates of unstimulated (-) and αIgM stimulated (+) CD19⁺ MACS-sorted CLL cells and wild-type (WT) splenic B cells. D: Left: Btk expression levels in gated CD19⁺ wild-type (WT) splenic cells versus IgH.ETμ CLL cells were evaluated using flow cytometry. Background staining levels were determined in Btk^-/- cells. Right: flow cytometric determination of median Btk expression levels in wild-type (filled circles) and IgH.ETμ CLL cells (open circles). MFI: median fluorescence intensity. For all analyses at least 4 CLL samples and 3 wild-type splenic B cell fractions were included; representative results are shown.

Figure 2

Btk is essential for CLL development. A: Kaplan-Meier survival curves of IgH.ETμ mice (dotted line; n=40) and Btk-deficient IgH.ETμ littermates (continuous line; n=19). B: Kaplan-Meier survival curves of IgH.ETμ mice (dotted line; n=40) and Btk-haplo-insufficient IgH.ETμ littermates (continuous line; n=12). C: Comparison of Btk expression levels in IgH.ETμ;Btk^+/- CLL cells as in Figure 1D. D: Flow cytometric measurement of β-galactosidase expression from lacZ-inserted Btk-knockout alleles in IgH.ETμ;Btk^+/- CLL cells versus Btk^-/- B cells, as determined by release of fluorescein upon FDG substrate degradation. Wild-type (WT) B cells were used as negative control. In (C) and (D) representative results are shown of 4 CLL samples analyzed.

Figure 3

Btk overexpression enhances CLL formation. A: Incidence curve of CLL formation in IgH.ETμ mice (dotted line; n=40) versus CD19-hBtk transgenic IgH.ETμ mice (continuous line; n=20). CLL formation was defined by the first appearance of monoclonal IgMb⁺ B cells in peripheral blood of these mice (IgMb⁺:IgMa⁺ ratio > 80:20 [21]). B: Kaplan-Meier survival curve of IgH.ETμ;CD19-hBtk mice (continuous line; n=20) versus IgH.ETμ non-CD19-hBtk transgenic mice (dotted line; n=40).

Figure 4

High Btk levels promote atypical BCR clone selection in CLL. A: Proportions of Igλ⁺ (gray) and Igκ⁺ (black) CLLs in IgH.ETμ mice (including IgH.ETμ;Btk^+/- mice; n=25) versus IgH.ETμ;CD19-hBtk mice as determined by flow cytometry (n=16). B: Proportions of stereotypical (V_H11-2⁺/V_κ14-126⁺) BCR (gray) and non-stereotypical BCR (black) expressing tumors in IgH.ETμ mice (including IgH.ETμ;Btk^+/- mice; n=25) versus IgH.ETμ;CD19-hBtk mice (n=16). C: Graph summarizing IgH CDR3 length determined by IgH sequencing of tumors from IgH.ETμ mice (including IgH. ETμ;Btk^+/- mice; black bars, n=25) and IgH.ETμ;CD19-hBtk mice (white bars, n=16).