BCR-ABL Lacking the Pleckstrin Homology (PH) Domain of BCR Induces a More Aggressive Leukemia Than P210BCR-ABL in a Murine Model of CML.

Background: The two common types of BCR-ABL fusion protein, p190 and p210, are associated with Ph-positive ALL and CML, respectively. Compared to p190, p210 retains the Dbl-like, CDC24 homology and PH domains. Dbl-like and CDC24 homology domains are thought to serve as a guanidine exchange factor (GEF) for small G proteins, including Rac, CDC42 and RhoA. It has been suggested that the loss of GEF activity may be responsible for the more aggressive phenotype of p190-positive leukemia (McWhirter and Wang, Oncogene1997; 15(14):1625–34). PH domains bind phosphoinositides and have been implicated in targeting proteins to cellular membranes as well as modulating the activity of the adjacent Dbl-like GEFs. It is not known, however, if the PH domain of BCR plays a role in determining disease features. We therefore decided to study the biology of p200 BCR-ABL , a naturally occurring variant identified in several CML patients that lacks the PH domain but retains the Dbl-like and CDC24 homology domains. Materials and methods: p190, p210 and p200 BCR-ABL cDNAs were cloned into pSRa and MIGR1 for expression in cell lines and primary murine bone marrow (BM) cells, respectively. The activation of signaling pathways downstream of BCR-ABL was studied in Ba/F3 and 32D cells expressing the various constructs. For colony-forming assays, murine BM cells were infected with p210, p200 or p190 retroviral vectors and seeded in methylcellulose in the presence or absence of cytokines (SCF, IL3 & IL6) and colonies were counted on day 12. For in vivo studies, BM cells isolated from 5-fluorouracil treated 5-6 week-old female Balb/c mice were infected with the various retroviral constructs in the presence of cytokines (SCF, IL3 & IL6), and 4.0 x 10 5 cells were injected retro-orbitally into lethally irradiated recipient mice. Disease latency was determined by monitoring white blood counts at 48h intervals. Moribund animals were sacrificed and studied by FACS and histopathology. Results: As expected, p200 transformed Ba/F3 and 32D cells to IL-3 independence. Immunoblotting of whole cell lysates with a phosphotyrosine antibody revealed no significant differences in global protein phosphorylation between the three variants, but phosphorylation of BCR-ABL was consistently less sensitive to imatinib inhibition in cells expressing p190 compared to p200 and p210. Analysis of downstream signaling showed approximately 15 fold higher levels of phosphorylated STAT6 in Ba/F3 cells expressing p200 and p190 vs. p210, while STAT5 and AKT phophorylation were similar in all variants. In the absence of cytokines, BM cells from Balb/c mice transduced with p200 and p190 generated significantly more CFU-GM than cells transduced with p210 (p in vivo in a murine CML model. Mice transplanted with p210-transduced cells survived 16–20 days, significantly longer than mice transplanted with p200 or p190 expressing cells (13–15 days) (p = 0.01, log-rank test). Conclusion: p200 is closely related to p190 but very distinct from p210 with respect to downstream signaling and in vivo transforming potential. These data suggest a critical role for the PH domain of BCR in attenuating the phenotype of the leukemia. We speculate that deletion of the PH domain may disrupt the ability of the Dbl-like and CDC24 homology domains to function as a GEF towards small G proteins.