Inactivation of Pre-B Cell Receptor-Mediated Tumor Suppression by Aberrant Splicing in Ph⁺ Acute Lymphoblastic Leukemia.

Abstract 579

The pre-B cell receptor instructs differentiation of normal pre-B cells and functions as a tumor suppressor in pre-B acute lymphoblastic leukemia (ALL). Its function is impaired in virtually all cases of pre-B ALL carrying the Philadelphia chromosome (Ph), which encodes the BCR-ABL1 kinase. However, genomic deletions in pre-B cell receptor pathway are rare and the mechanism of its inactivation in ALL is not known. Here we report that aberrant splicing of SLP65 (BLNK, BASH) and the pre-B cell receptor-related linker molecules LCP2, SYK and LAT represents a major mechanism of pre-B cell receptor-inactivation, which is consistent with deregulated expression of splice factors in Ph⁺ ALL. In contrast to normal pre-B cells, BCR-ABL1-transformed pre-B ALL expressed multiple splice variants of the pre-B cell receptor-related linker molecules SLP65, LCP2, SYK and LAT. A detailed sequence analysis of these splice variants revealed that aberrant splicing resulted in functional inactivation of important protein domains in SLP65, LCP2, SYK and LAT transcripts. Of note, the vast majority of SLP65 splice variants lacked a functional SH2 domain, which is required to anchor SLP65 to the Igαa signaling chain of the pre-B cell receptor. We then focused our analysis on the SH2 domain of SLP65 for the following reasons: (1) SLP65 functions as a powerful tumor suppressor downstream of the pre-B cell receptor. (2) Exon 16 which encodes the central part of the SH2 domain was affected in the vast majority of SLP65 splice variants. (3) The SH2 domain of SLP65 is required to link downstream effector molecules (BTK, SYK, PLCγ2) to the Igαa signaling chain of the pre-B cell receptor. We found that deletion of the SH2 domain represents a frequent feature in Ph⁺ ALL.

In reconstitution experiments with pre-B cells from SLP65^-/- mice, SLP65 forms lacking the SH2 domain failed to support pre-B cell receptor signaling. By coexpression with full-length SLP65, we demonstrated that SH2-deficient variants have a dominant-negative effect by reducing the Ca²⁺-release and the leukemia-suppressive function of SLP65. These effects are dose-dependent as demonstrated in a titration experiment, in which SLP65 and SH2-deficient SLP65 were expressed at different ratios. SH2-deficient SLP65 also compromised the leukemia-suppressive function of the pre-B cell receptor in vivo transplantation model: While reconstitution of full-length SLP65 in SLP65^-/- BCR-ABL1 ALL cells suppressed leukemia growth and bone marrow infiltration, loss of the SLP65 SH2 domain abolished the tumor suppressor function of the pre-B cell receptor.

To address potential mechanisms of aberrant pre-mRNA splicing, we studied expression levels of splicing factors in Ph⁺ ALL and searched for splice site mutations: Consistent with aberrant splicing of pre-B cell receptor-related linker molecules including SLP65, SYK, LAT and LCP2, we found evidence of splice factor deregulation in Ph⁺ ALL: Among the 94 pre-mRNA splice factors studied, 24 were differentially expressed between Ph⁺ ALL (n=15) and normal bone marrow B cell precursors (n=11). Of note, the SRPK1, hnRPC, hnRNP-E2 (PCBP2) and hnRNP-A1 pre-mRNA splice factors that were previously found as BCR-ABL1-regulated, were also identified in our analysis. Sequence analysis of the 3'prime splice site of exon 16 in three Ph⁺ ALL cell lines and six primary cases of Ph⁺ ALL revealed one clonal splice site mutation in one primary Ph⁺ ALL case. In one cell line and one primary Ph⁺ ALL case, each one additional mutation was found within 2 basepairs distance from the splice site. It is not clear, however, whether and to which extent these mutations affect splice site selection. Interestingly, we found that clones amplified from one primary Ph⁺ ALL case and one Ph⁺ ALL cell line harbor mutations encoding premature translation stops close to the end of exon 16. While these mutations do not lead to aberrant splicing of exon 16, they reinforce the notion that Ph⁺ ALL cells are selected for inactivation of the SLP65 SH2 domain.

We conclude that aberrant splicing frequently results in the expression of dominant-negative SH2-deficient SLP65, which is sufficient to compromise the tumor suppressor function of the pre-B cell receptor in Ph⁺ ALL. Whereas mutations and deletions of the SLP65 gene are rare, we conclude that aberrant splicing of the SLP65 SH2 domain represents a common mechanism in ALL cells to compromise the leukemia-suppressive function of the pre-B cell receptor.