Loss of Ubr1 Cooperatively Synergizes with HOX11 in B Cell Lymphomagenesis.

The HOX11 homeobox gene was originally identified at the recurrent t(10;14)(q24;q11) translocation breakpoint, a chromosomal abnormality observed in 5–7% of T cell acute lymphoblastic leukemias (T-ALLs). Transgenic mice ectopically expressing HOX11 in the B cell compartment die in their second year of life due to the onset of mature B cell lymphomas. However, the long latency prior to the development of leukemia has led to the hypothesis that additional mutations are necessary prior to the onset of full-blown malignancy. To identify collaborating genetic loci responsible for HOX11-induced B cell lymphomagenesis, proviral insertional mutagenesis, using the mature B cell-specific retrovirus, the murine AIDS (mAIDS) virus, was used. In eight of ten animals, there was an acceleration of development of B cell lymphomas, manifested by the development of a mediastinal mass comprising predominantly of mature IgM⁺ IgD⁺ B cells. Histological analysis revealed expansion of splenic germinal centres and hyperplasia of adjacent lymph nodes, consistent with diffuse large B cell lymphoma. Using the provirus as a molecular tag, we identified Ubr1 as a frequent site of proviral insertion. Three mice exhibited an insertion into the 10^th exon of the Ubr1 gene, with two animals exhibiting an identical insertion at nucleotide 1295 and another animal exhibiting an insertion at nucleotide 1251. Insertion into this genomic region was confirmed by Southern blotting demonstrating the presence of a rearranged Ubr1 allele, and by the ability to generate a PCR amplicon across the viral-genome junction. Western immunoblot analysis revealed down-regulated expression of the Ubr1 gene product subsequent to viral integration. Ubr1 is a member of the E3 ubiquitin ligase family and participates in the ubiquitin-dependent proteolytic pathway. Among its numerous targets, Ubr1 controls the timely degradation of cohesin subunits during mitosis. Consequently, Ubr1^−/−S. cerevisiae are prone to chromosome loss due to chromosome malsegregation during anaphase. We sought to investigate possible similar effects in primary B lymphocyte cultures derived from HOX11^Tg/Ubr1^+/+ and HOX11^Tg/Ubr1^−/− mice, and to determine whether HOX11 overexpression in such a Ubr1-null background possesses any synergizing effects on the ploidy of these cells. Direct counting of chromosome numbers from chromosome spreads prepared from HOX11^Tg/Ubr1^−/− primary B lymphocytes cultured in vitro for 4–5 days revealed increased incidences of aneuploidy and chromosome loss relative to HOX11^wt/Ubr1^−/− controls (2n=39.51 vs. 2n=39.98). Similarly, micronucleus assays indicated increased presence of micronuclei in HOX11^Tg/Ubr1^−/− primary B lymphocyte cultures (5.2% vs 0.8%). Additionally, HOX11^Tg B lymphocytes exhibit increased cyclin B1 expression and an ability to bypass G2-M arrest induced by the tyrosine kinase inhibitor, genistein. Therefore, the effect of HOX11 in exacerbating chromosome loss in these cultures may be associated with its ability to allow cells to bypass the G2-M cell cycle checkpoint, permitting the accrual of additional chromosome losses and cytogenic abnormalities en route to malignancy.