The Expression of the Adaptor Protein SLP-65 Is Freqently Lost in Childhood B-Lineage Acute Lymphoblastic Leukemia (ALL).

Objective: The adaptor protein SLP-65 (also known as BLNK or BASH) plays an essential role in B cell differentiation. A crucial consequence of SLP-65 deficiency in mice is the high incidence of pre-B cell leukemia, suggesting a tumor suppressor role for SLP-65 in pre-B cells. We recently demonstrated deficiency slp-65 in 16 out of 34 pre-B ALL samples from children (Jumaa et al., Nature, 2003). These data were recently questioned by another study in which slp-65 levels were found to be as high as in normal B cells (Imai, et al., Leukemia, 2004). To resolve this issue we investigated another series of 148 primary ALL-samples by quantitative RT-PCR, Western-blotting and sequencing of aberrant slp-65 transcripts. We provide evidence that a newly identified slp-65 aberration, with inclusion of a newly identified exon 11a, leads to a functionless slp-65 protein.

Materials: Samples were collected at diagnosis and contained at least 80 % marrow blasts. Screening for the presence of the fusion genes BCR/ABL, MLL/AF4, or TEL/AML1 was done by standard RT-PCR. For immunophenotyping and classification into pro-B, pre-B, c-, B- or T-cell ALL we used the standard criteria. Slp-65 mRNA was measured by taqman technology, protein expression by Western blotting with the B-211 antibody. To assess the functional consequences of a truncated form of slp-65, we measured Ca-responsiveness of the pre-B cell receptor. Pre-B cell differentiation properties of the wildtype and the truncated isoform of slp-65 were functionally tested as described previously (Flemming et al., Nat. Immunol, 2003).

Results: The slp-65 mRNA expression ranged widely among the samples but was not correlated to any of the clinical or genetically parameters tested, such as sex, Age, WBC, or event-free survival. With respect to the immunophenotype, slp-65 mRNA was extremely low in T-ALL and but highly expressed in pro-B-ALL (p<0.001). We found the inclusion of additional sequences between these exons named exon 3a (n=9), 3b (n= 16). By primers that specifically amplify exon 3a+b, we found 2 patients with exceptionally high expression of this aberrant slp-65 transcript. One drawback of quantitative RT-PCR analysis, however, is that aberrant transcripts and alternatively spliced forms are detected as normal transcripts, although they are unable to generate a functional protein. To check for full-length SLP-65 protein expression we applied Western-botting. We found truncated spl-65 protein variants in 23 samples and a total loss of the SLP 65 protein in 41 cases. We identified a novel transcript of slp-65 in which an additional exon (exon 11a) was inserted into the mRNA. Although low amounts of this 11a-transcript were also seen in normal B-cells, this aberrantly spliced slp-65 mRNA was dominantly expressed in 18 patients (2 pro-B-ALL, 12 c-ALL, 4 pre-B-ALL). Inclusion of exon 11a leads to a truncated slp-65 protein that lacks the SH-2 domain. The SH2 domain lacking SLP-65 form completely failed to induce calcium response and pre-B cell differentiation in vitro. Our data confirm the correlation between SLP-65 deficiency and leukemia and suggest a tumor suppressor role for SLP-65 in human pre-B cells.