Examination of Clinically-Derived p210 BCR/ABL Rhogef Mutations in a Murine Bone Marrow Transplantation Model of CML

The Philadelphia chromosome is formed by a balanced, reciprocal translocation that pairs sequences from BCR on Chromosome 22 with sequences from ABL on Chromosome 9, resulting in the production of the constitutively active tyrosine kinase (TK) BCR-ABL. Depending on the location of the breakpoint within BCR, three different sizes of BCR-ABL may form, each associated with distinct clinical sequelae. Previous studies identified a functional domain within the BCR sequences preserved by the more-indolent forms of BCR-ABL (i.e., p210 and p230) that demonstrates a constitutive Rho guanine nucleotide exchange factor (RhoGEF) activity. Using the structurally derived S509A mutation, which does not affect TK activity but abrogates RhoGEF signaling, we, subsequently, showed that this feature regulates leukemia progression in mice. The RhoGEF domain was recently reported to contain two missense mutations (F547L and T654K) in a chronic myelogenous leukemia (CML) blast crisis (BC) patient, suggesting that it may play a role in human disease as well. To evaluate the clinical significance of this region, we, therefore, examined p210 BCR-ABL constructs containing these clinically derived RhoGEF mutations (CDRMs), both in isolation and in combination, using a murine bone marrow transplant (mBMT) model of CML. The mutations did not destabilize p210 BCR-ABL expression or TK activity but decreased RhoGEF signaling. Relative to mice transplanted with wild-type (WT) p210 BCR-ABL, those that received the CDRMs exhibited an earlier onset of disease, frequently developing previously unseen dermatologic involvement or myeloid sarcomas, but demonstrated significantly increased survival in an additive manner [Fig. 1]. While mice transplanted with WT p210 BCR-ABL exhibited neutrophilia that progresses to a less-differentiated phenotype at death, disease in the CDRM mice was characterized by eosinophilia and monocytosis with no maturation arrest. The most likely cause of death in mice transplanted with WT p210 BCR-ABL is widespread hematogenous involvement of the lungs resulting in acute respiratory distress. In contrast, mice receiving the CDRMs demonstrated pulmonary involvement which was limited to the bronchovascular bundles or subpleural space. This is consistent with a switch to lymphatogenous spread, likely secondary to skewed differentiation, and it implies an alternate cause(s) of death. To help determine the mechanism responsible for the observed differences in differentiation