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The function of the pleckstrin homology domain in BCR–ABL-mediated leukemogenesis

Leukemia volume 24, pages 226–229 (2010)Cite this article

We introduced three BCR–ABL variants (p190BCR–ABL, p200BCR–ABL and p210BCR–ABL) into 32D cells and found that all three BCR–ABL proteins transformed the cells to growth factor independence (data not shown). Immunoblot analysis of bulk cultures expressing comparable levels of p190BCR–ABL, p200BCR–ABL or p210BCR–ABL (Figure 1b) showed similar global levels of cellular phosphotyrosine, as detected using an anti-phosphotyrosine antibody (Figure 1c). Similar findings were observed in a lymphoid setting using Ba/F3 cells (data not shown). Differences in kinase activity have been previously reported for the p190BCR–ABL and p210BCR–ABL variants.2, 3 We therefore compared the tyrosine kinase activity of anti-ABL immunoprecipitates from 32D cells expressing p190BCR–ABL, p200BCR–ABL or p210BCR–ABL using GST-Crk (120–225) as a substrate. Results from multiple experiments (N=16) revealed on average 1.8-fold and 2.3-fold increase in kinase activity of p190BCR–ABL and p200BCR–ABL compared with p210BCR–ABL (P=0.07 and P<0.05, respectively; Supplementary Figure 1). These rather subtle differences are in contrast with an earlier study that showed a substantial increase in autophosphorylation and substrate phosphorylation in recombinant p190BCR–ABL protein compared with p210BCR–ABL.3 However, our data are comparable to those reported by Li et al.,2 who observed a 1.5-fold increase in p190BCR–ABL kinase activity compared with p210BCR–ABL in a similar experimental system. These slight differences in kinase activity between the three BCR–ABL variants are consistent with the lack of changes in global phosphotyrosine levels, which are apparent on immunoblots of total cellular extracts. In light of earlier reports that have suggested differences in STAT6 phosphorylation in Ba/F3 cells expressing p190BCR–ABL or p210BCR–ABL,5, 6 we performed immunoblot experiments to determine whether this difference was upheld in p200BCR–ABL. We found STAT6 phosphorylation to be much higher in BaF/3 cells expressing p190BCR–ABL or p200BCR–ABL in comparison with those expressing p210BCR–ABL (Figure 1d), indicating that deletion of the PH domain is sufficient to lead to STAT6 activation in a lymphoid cell setting.

An initial comparison of the three BCR–ABL variants in murine bone marrow colony formation assays indicated that the p190BCR–ABL and p200BCR–ABL variants are only slightly more transforming than p210BCR–ABL in myeloid colony assays (Supplementary Figure 2a) and substantially more transforming in B-cell colony assays (Supplementary Figure 2b). To explore the role of the PH domain of BCR–ABL in vivo, we first compared p190BCR–ABL, p200BCR–ABL and p210BCR−ABL in a well-established mouse model system in which transplantation of bone marrow from 5-FU-treated mice infected with BCR–ABL retrovirus into lethally irradiated recipients leads to a myeloproliferative disease resembling CML (Figure 1e). All mice transplanted with BCR–ABL-infected marrow (N=6 per group) developed a myeloproliferative syndrome, as evidenced by fluorescence-activated cell sorting analysis of blood, bone marrow, spleen and liver tissues (Figure 1g). Mice transplanted with p190BCR–ABL- or p200BCR–ABL-infected marrow showed a median disease latency (the interval between transplantation and the first abnormal white blood cell count) of 12 days, whereas the median latency was 14 days for mice transplanted with p210BCR–ABL-infected marrow (P<0.05). Median survival was also slightly shorter in recipients of p190BCR–ABL- and p200BCR–ABL-infected marrow (14 and 14.5 days, respectively) compared with recipients of p210BCR–ABL-infected marrow (16 days, P<0.05). Together, these results suggest that BCR–ABL lacking the PH domain shows similar oncogenicity to that of p190BCR–ABL, inducing a slightly more aggressive CML-like myeloproliferative disease in mice than the p210BCR–ABL variant.

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Acknowledgements

This study was supported in part by the Leukemia and Lymphoma Society (BJD, MWD), the TJ Martell Foundation (BJD) and NHLBI grant HL082978-01 (MWD). JWT is supported by the William Lawrence Foundation and the Oregon Clinical and Translational Research Institute. MWD is a Scholar in Clinical Research of the Leukemia and Lymphoma Society. BJD is an investigator of the Howard Hughes Medical Institute.

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  1. Division of Hematology and Medical Oncology, Oregon Health and Science University Knight Cancer Institute, Portland, OR, USA

    S Demehri, T O'Hare, C A Eide, C A Smith, J W Tyner, B J Druker & M W N Deininger

  2. Howard Hughes Medical Institute, Portland, OR, USA

    T O'Hare, C A Eide & B J Druker

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  1. S Demehri
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Demehri, S., O'Hare, T., Eide, C. et al. The function of the pleckstrin homology domain in BCR–ABL-mediated leukemogenesis. Leukemia 24, 226–229 (2010). https://doi.org/10.1038/leu.2009.196

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