BRAFV600E mutations have recently been identified in nearly 100% of patients with the chronic lymphoproliferative disorder hairy cell leukemia (HCL), as well as a small percentage of patients with the plasma cell malignancy multiple myeloma. Despite extensive knowledge regarding the functional effects of BRAFV600E expression in epithelial tissues, very little is understood about the role of the BRAFV600E mutation in hematopoietic transformation. We therefore utilized a conditional BRafV600E murine model crossed with Mx1-cre, Vav-cre, Cd19-cre, and Cγ1-cre transgenic mice to delineate the effects of mutant BRaf expression in pre-natal and post-natal hematopoietic stem and progenitor cells (HSPCs), B-lineage cells, and germinal center B cells respectively. We also investigated the origin of the BRAFV600E mutation in HCL patient bone marrow samples using prospective isolation of sorted HSPC populations followed by quantitative sequencing for the BRAFV600E mutation.
Surprisingly, we identified the presence of the BRAFV600E mutation in long-term hematopoietic stem cells of HCL patients, and we also observed marked alterations in HSPC frequencies. Consistent with the human genetic data, expression of BRafV600E in HSPCs of mice resulted in a lethal transplantable hematopoietic disorder characterized by splenomegaly, anemia, thrombocytopenia, increased circulating soluble CD25, and increased clonogenic capacity of B-lineage cells- all classic features of human HCL. In contrast, restricting expression of BRafV600E to B-lineage cells did not result in disease even up to 1.5 years of age.
We next assessed the effects of the BRafV600E mutation on HSPC self-renewal and lineage specification. We plated whole BM cells from Mx1-cre BRafV600E mice in methylcellulose containing myeloid/erythroid cytokines or lymphopoietic cytokes (IL-7). BRafV600E cells demonstrated impaired colony formation in myeloid/erythroid conditions. However, BRafV600E HSPCs exhibited limitless replating capacity when plated in the presence of IL-7, indicating that the BRAF mutation induces aberrant B lineage cell self-renewal. A clear competitive advantage was also seen with competitive transplantation of BRafV600E BM cells, identifying an increase in HSPC self-renewal associated with the BRAF mutation.
Data from the murine models studied here and characterization of the BM compartment in HCL patients suggest that the cytopenias seen in HCL patients are due in part to HSPC-intrinsic effects of the BRAFV600E mutation on erythropoiesis, megakarypoiesis, and myelopoiesis. Moreover, these data suggest that the use of therapies targeting MAP-kinase signaling in HCL may lead to durable remissions not only through effects on mature leukemic cells, but also through targeted inhibition of signaling and survival in mutant HSPCs.
Citation Format: Eunhee Kim, Stephen S. Chung, Jae H. Park, Young Rock Chung, Piro Lito, Julie Feldstein, Wenhuo Hu, Wendy Beguilin, Sebastien Monette, Cihangir Duy, Raajit Rampal, Leon Telis, Minal Patel, Min Kyung Kim, Ari M. Melnick, Neal Rosen, Martin S. Tallman, Christopher Y. Park, Omar Abdel-Wahab. Context specific effects of the BRAFV600E mutation on hematopoiesis identifies novel models of BRAF mutant hematopoietic disorders. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3140. doi:10.1158/1538-7445.AM2014-3140