Tumor Suppressors BTG1 and BTG2 Fulfill Both Unique and Overlapping Functions During Normal B Lymphocyte Development

Abstract 1303

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common form of cancer in children, characterized by genetic aberrations affecting master regulators of lymphoid differentiation, such as RUNX1, IKZF1, TCF3, and PAX5, as well as tumor suppressor genes that control the cell cycle, including RB1 and CDKN2A. Another gene frequently altered in BCP-ALL is BTG1, which displays highly clustered mono-allelic deletions in childhood BCP-ALL (9%) and adult ALL (6%). The frequency of BTG1 deletions is two- to three-fold higher in ETV6-RUNX1- and BCR-ABL1-positive leukemias. BTG1, and its close homologue BTG2 regulate gene expression, for instance by associating with protein arginine methyltransferase 1 (PRMT1), affecting the activity of a variety of transcription factors, including several nuclear hormone receptors and HoxB9. In addition, BTG1 and BTG2 have been implicated in regulating mRNA stability by interacting with the Ccr4-Not complex. Recent studies have also identified missense point mutations in BTG1 and BTG2 in about 20% of non-Hodgkin lymphomas, arguing that altered function of these genes contributes to B cell malignancies.

To investigate a role of BTG1 and BTG2 in B cell development, we studied the phenotype of Btg1 and Btg2 single knockout (KO) and Btg1;Btg2 double KO mice. Animals deficient for either BTG1 or BTG2 displayed a mild B cell phenotype with a moderate reduction of ∼20% in the total amount of B220⁺ progenitor B cells in bone marrow, while splenic B cells were present at normal frequencies. More detailed analyses revealed that Btg1^−/− and Btg2^−/− mice both showed a partial block at the pre-pro-B cell stage (Hardy fraction A). Methylcellulose colony assays in the presence of interleukin-7 (IL-7) demonstrated 30% fewer colonies using bone marrow from Btg2^−/− mice, whereas 70% fewer colonies were obtained using bone marrow derived from Btg1^−/− mice. To assess whether BTG1 and BTG2 fulfill redundant functions during B cell development, we analyzed the phenotype of Btg1^−/−;Btg2^−/− mice. Hence we observed that the combined loss of BTG1 and BTG2 led to a much stronger block in B cell differentiation, with the majority of progenitor B cells arrested at the pre-pro-B cell stage. In the spleens of these double knockout mice we observed a roughly 50% reduction in B220+ IgM+ B cells, suggesting that these genes act to modify the activity of B lineage transcription factors rather than to fully block their activities. This is consistent with a role for these genes as modifiers of transcriptional activity. Current studies are aimed at defining the molecular targets regulated by BTG1 and BTG2 during early B cell development using RNA sequencing and protein interaction experiments.

In conclusion, our data demonstrate that BTG1 and BTG2 act as important regulators of normal B cell differentiation, and that this function might be critical for their role as tumor suppressors in (early) B cell malignancies.