A p210 BCR/ABL Mutant That Lacks Guanine Nucleotide Exchange Factor Activity Induces Erythroleukemia in a Murine Bone Marrow Transplantation Model,

Abstract 3756

The p210 BCR/ABL and p190 BCR/ABL fusion proteins are constitutively active tyrosine kinases that are associated with chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL) respectively. Whereas both of these fusion proteins contain equivalent contributions from ABL, they differ in the amount of BCR sequence at the NH2-terminus. We have identified a domain with guanine nucleotide exchange factor (GEF) activity which is contained within the BCR sequences of p210 BCR/ABL that distinguishes it from p190 BCR/ABL. This activity is subject to autoinhibition in the context of BCR, but is constitutively active in p210 BCR/ABL, and cannot be blocked using the tyrosine-kinase inhibitor, Imatinib mesylate. The activities of small GTPases are regulated by GEFs, and are essential for the regulation of hematopoietic stem cell differentiation and proliferation. In a human Ph-positive erythroleukemia cell line (K562), inhibiting p210 BCR/ABL-mediated Ras activation induces erythroid differentiation, while abnormal activation of Cdc42 reduces erythroid progenitors and erythroid colony-forming units in bone marrow. In order to directly investigate the contribution of the GEF activity to p210 BCR/ABL-mediated leukemogenesis, we have introduced a point mutation into p210 BCR/ABL (p210 BCR/ABL(S509A)) that eliminates the activity. The mutation has no discernable effect on tyrosine kinase activity, which is consistent with previous data that the two activities are functionally independent. We have compared p190 BCR/ABL, p210 BCR/ABL, and p210 BCR/ABL(S509A) in a murine bone marrow transplantation (BMT) model. Although all transplanted mice develop splenomegaly with leukocytosis, the p190 BCR/ABL and p210 BCR/ABL(S509A) transplanted mice exhibit a more rapid onset of disease than mice transplanted with p210 BCR/ABL (15–18 days vs 23–38 days). This difference is observed regardless of whether the donor mice are pre-treated with 5-Fluorouracil. The more rapid onset of disease induced by p190 BCR/ABL and p210 BCR/ABL(S509A) is associated with reticulocytosis, and increased numbers of nucleated red blood cells (nRBC), but no anemia. The nRBC/100 WBC counts are 0, 4.75±2.21, 277.5±190.6, and 119.5±66.0 for MIG, p210 BCR/ABL, p190 BCR/ABL and p210 BCR/ABL (S509A) respectively, thus suggesting leukemic expansion of the erythroid lineage. To confirm this difference in lineage expansion, clonogenicity assays were performed. Consistent with previous reports, p210 BCR/ABL supports the growth of CFU G/M, but not BFU-E, and colony growth is completely inhibited by Nilotinib. In contrast, p190 BCR/ABL and p210 BCR/ABL(S509A) support the growth of BFU-E (but not CFU G/M), and these colonies are completely insensitive to Nilotinib treatment. In summary, our observations suggest that a point mutation that eliminates the GEF activity of p210 BCR/ABL produces a phenocopy of p190 BCR/ABL, without affecting the tyrosine kinase activity. These results support a model in which the GEF activity that distinguishes p210 BCR/ABL from p190 BCR/ABL actively regulates disease progression by determining lineage-specific leukemic expansion.