ALBERT

Description: 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

Description: The adaptor protein SLP-65 plays an essential role during B cell differentiation. A crucial consequence of SLP-65 deficiency in mice is a high incidence of pre-B-cell leukemia, suggesting a tumor suppressor role for SLP-65 in pre-B-cells. While the link between SLP-65 deficiency and leukemia development is established in mice, experiments mainly using microarrays for gene expression profiling suggested normal expression of SLP-65 in human precursor B-cell ALL. This analysis however does not discriminate between normal and aberrant SLP-65 transcripts with the latter being unable to generate functional protein. To examine the correlation between SLP-65 deficiency and childhood precursor B-cell ALL, we determined SLP-65 expression in 119 precursor B-cell ALL samples by both RNA and protein methods. The expression of SLP-65 was compared to clinical and laboratory findings, cytogenetics as well as to the outcome data within this uniformly treated cohort of patients. Loss of slp-65 protein was significantly associated with the occurrence of the TEL/AML1 rearrangement (p=0.026) but not with any other clinical or cytogenetic feature. We found a profound disconnection between slp-65 mRNA and protein expression in 38 out of the 119 leukemic samples pointing to a posttranscriptional regulation of slp-65 (Table). To confirm that SLP-65 transcript expression does not automatically correlate with its protein expression, we analyzed a panel of human cell lines derived from precursor B-cell ALL patients. The cell lines HPB-NULL and BV-173 showed a deficiency in SLP-65 protein expression, although SLP-65 transcripts can easily be detected in both lines. Together, the data suggest that SLP-65 expression might be regulated at the posttranscriptional level and that the presence of SLP-65 transcripts does not necessarily lead to SLP-65 protein and function. In one particular patient, we found a truncated slp-65 transcript and the predicted slp-65 protein lacks its SH2 domain. We tested whether this SLP-65 protein lacking the SH2 domain is functional in pre-B cells. To this end, we transfected murine SLP-65 −/− pre-B cells with retroviral constructs for either wild-type (wt SLP-65) or truncated SLP-65 (SLP-65delSH2) and analysed pre-BCR downregulation, Ca2+ release and pre-B cell differentiation. The results showed that, in contrast to wt SLP-65, SLP-65delSH2 failed to induce any effects in the performed experiments. Together with previous findings showing that SLP-65-deficient mice develop pre-B cell leukemia, the data suggest that SLP-65 acts as a tumor suppressor that limits pre-B cell proliferation by inducing differentiation. Disconnection between slp-65 transcripts and protein expression total slp-65 protein+ (51 patients) slp-65 protein weak (19 patients) slp-65 protein- (49 patients) PCR+ 108 51(9 TEL/AML+, 42 TEL/AML-) 19 (9 TEL/AML+, 10 TEL/AML-) 38 (15 TEL/AML+, 23 TEL/AML-) PCR- 11 0 0 11 (T-ALL)