Kinase-Inactivating Braf Mutation Drives Aneuploidy In Splenic Myeloid Cells by Deregulation of Craf

Abstract 3138

BRAF belongs to RAF family serine/threonine kinases regulating the downstream MEK/ERK pathway, and is known as an oncogene mutated in ∼7% of human cancers, including some hematopoietic neoplasms (e.g. childhood ALL, therapy-related AML, and Langerhans cell histiocytosis). Although most oncogenic BRAF mutants aberrantly activate the MEK/ERK pathway, ^D594BRAF mutant, which represents ∼1% of oncogenic BRAF mutants, lacks in vitro kinase activity and in vivo ERK-activating potential when ectopically expressed, implying that this mutant might contribute to tumorigenesis through MEK/ERK-independent mechanisms. Using a compound knock-in mouse model, we recently reported that kinase-inactive ^D594ABraf cooperates with oncogenic Kras to promote tumorigenesis through another Raf family kinase, Craf (Cell. 2010; 140(2):209-221). To further understand how the Braf^D594A mutation contributes to tumorigenesis, we analyzed Braf^D594A/+ mice retaining wild-type ras alleles.

Braf^D594A/+ mice developed splenomegaly at 100% penetrance within three months after birth, with increased CD11b+ splenic myeloid (monocytic) cells (mutants: 4.50 ×10⁷/spleen, littermate controls: 0.96 ×10⁷/spleen, p=0.003, student's t-test). In vivo BrdU incorporation assay revealed that CD11b+ cells in the mutant spleen were actively cycling compared to littermate wild-type controls (mutants: 35.0 +/&− 6.2%, controls: 14.5 +/&− 4.0%, p=0.027, student's t-test). The aberrant myeloid expansion was restricted to spleen, and there was no significant difference in bone marrow cellularity and differential cell count between the mutants and wild-type controls.

Interestingly, DNA ploidy analysis of the mutant splenic CD11b+ cells frequently exhibited aneuploid populations. Clear aneuploid (hyperdiploid to near-triploid) peaks were detected in 4 out of 10 mutant spleens, while a minor aneuploid peak detected in only one out of 24 control spleens (p=0.007, chi-square test). Karyotyping of CD11b+ macrophages/monocytic cells developed from Braf^D594A/+ splenocytes in culture also revealed that more than 70% of mitotic cells were aneuploid (hypodiploid 55.6%, hyperdiploid 12.6%, near-tetraploid 5.2%, in total 135 metaphases from 3 independent cultures on day 10). These data indicate that ^D594ABRaf promotes myeloid expansion with compromising chromosome stability, specifically in the splenic microenvironment.

Craf kinase activity in Braf^D594A/+ splenocytes was about 2.5 times higher than littermate wild-type controls, suggesting that ^D594ABraf could transactivate Craf even in the absence of oncogenic Ras. Craf was also found to be essential for ^D594ABraf-induced aneuploidy because pharmacological inhibition of Craf by sorafenib or reduced Craf protein expression by genetic modification (Craf^D486A allele, Mol Cell. 2008; 31(6):862-72) rescued the aneuploidization of Braf^D594A/+ splenic myeloid cells. Unexpectedly, increased ERK phosphorylation was also found in Braf^D594A/+ splenocytes, suggesting that ^D594ABraf could activate the MEK/ERK pathway through Craf transactivation when endogenously expressed. However, MEK inhibition by UO126 rather facilitated tetraploidization of Braf^D594A/+ splenocytes in culture without improving aneuploidy, and constitutive MEK/ERK activation introduced by Braf^V600E mutation (Cancer Res. 2005; 65(24):11493-500) did not promote aneuploidization of splenocytes.

Collectively, we conclude that Craf transactivation by kinase-inactive Braf promotes aneuploidization of splenic myeloid cells in a MEK/ERK-independent manner. These results shed light on the potential involvement of Craf in the pathogenesis of aneuploid myeloid neoplasms.