Oncogene (2006) 25, 2575–2587 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc ORIGINAL ARTICLE Dysregulated expression of mitotic regulators is associated with B-cell lymphomagenesis in HOX11-transgenic mice EChen1,2, MS Lim3, S Rosic-Kablar2, J Liu2,4, P Jolicoeur5, ID Dube´ 1,2,6 and MR Hough1,2,6 1Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada; 2Department of Molecular and Cellular Biology, Sunnybrook and Women’s College Health Sciences Centre, Toronto, Ontario, Canada; 3Department of Pathology, University of Utah, Salt Lake City, UT, USA; 4Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada; 5Clinical Research Institute of Montre´al, McGill University, Montre´al, Quebec, Canada and 6Department of Laboratory Medicine & Pathobiology, University of Toronto, Toronto, Ontario, Canada Dysregulated expression of the homeobox gene, HOX11 Keywords: HOX11; homeobox; aneuploidy; insertional is a frequent etiologic event in T-cell acute lymphoblastic mutagenesis leukemias. HOX11-transgenic mice (IgHl-HOX11Tg)- expressing HOX11 in the B-cell compartment develop B-cell lymphomas with extended latency. The latency Introduction suggests that additional genetic events are required prior to the onset of malignant lymphoma. We report the Dysregulated expression of the orphan homeobox gene, identification of 17 HOX11 collaborating genes, revealed HOX11 is a frequent etiologic event in pediatric T-cell through their propensity to be targeted in a proviral acute lymphoblastic leukemias (T-ALLs). Aberrant acti- insertional mutagenesis screen. Seven integrations dis- vation of HOX11 can be achieved by a t(10;14)(q24;q11) rupted genes in mitotic spindle checkpoint control, chromosome rearrangement, juxtaposing the HOX11 suggesting that cells with elevated HOX11 expression gene downstream of the T-cell receptor (TCR) a/d are especially sensitive to dysregulation of chromo- regulatory elements (Dube´ et al., 1986, 1991). While the some segregation during mitosis. IgHl-HOX11Tg primary t(10;14) translocation is seen in only 4–7% of reported B-lymphocyte cultures exposed to the aneugenic agents, T-ALL cases, a recent study revealed elevated HOX11 colchicine and colcemid, exhibited increased incidences of expression levels in leukemic blasts in B50% (37/76) of chromosome missegregation as assessed by cytokinesis- pediatric T-ALL cases (Kees et al., 2003). Moreover, block micronucleus assays. Additionally, IgHl-HOX11Tg an additional 30% of pediatric T-ALLs exhibited cultures were shown to exhibit aberrant bypass of spindle inappropriate activation of the related HOX11 gene, checkpoint arrest, as assessed by the increased presence of HOX11L2 (Mauvieux et al., 2002). Clinically, patients cycling cells determined by assessment of DNA content with HOX11-positive lymphoblasts have a better prog- and by BrdU immunolabelling. Western immunoblotting nosis when treated with modern combination chemother- revealed elevated expression of the mitotic effector mole- apy than patients with T-ALLs involving activation of cules, cyclin A, cyclin B1 and cdc20 in IgHl-HOX11Tg HOX11L2 (Ferrando et al., 2002, 2004). The incidence of cultures. Moreover, spontaneously arising lymphoid HOX11-positive lymphomas correlates with increasing neoplasms in IgHl-HOX11Tg mice frequently exhibit age, indicating a longer latency for disease manifestation aberrant expression of mitotic regulators, concomitant and suggesting the requirement of additional genetic with increased development of micronuclei, abnormal events prior to malignancy (Asnafi et al., 2004). mitotic checkpoint control and increased incidences of Early attempts at generating clinically relevant mouse abnormal karyotypes when expanded in culture. Collec- models of HOX11-related disease by using the Lck tively, these findings indicate that abnormal regulation of proximal promoter to drive expression of HOX11 spindle checkpoint control as a result of HOX11 over- during early stages of thymocyte development were expression leads to a heightened predisposition for unsuccessful and resulted in embryonic lethality. The development of aneuploidy, contributing to oncogenesis. first in vivo model of HOX11-associated lymphoma- Oncogene (2006) 25, 2575–2587. doi:10.1038/sj.onc.1209285; genesis was generated by ectopically expressing HOX11 published online 9 January 2006 in the B-cell compartment by placing the transgene under the control of the immunoglobulin heavy chain (IgH) promoter and enhancer sequences. Within 2 years, Correspondence: Dr M Hough, Department of Molecular and Cellular HOX11-transgenic mice (IgHm-HOX11Tg) developed Biology, Sunnybrook and Women’s College Health Sciences Centre, mature marginal zone B-cell lymphomas, originating 2075 Bayview Avenue, Room S232, Toronto, Ontario, Canada M4N in the spleen with frequent dissemination of lympho- 3M5. E-mail: [email protected] matous tissue to distant sites, including the thymus, Received 29 June 2005; revised 22 September 2005; accepted 26 October lymph nodes, lungs and kidneys (Hough et al., 1998). 2005; published online 9 January 2006 These mice represented a relevant model of B-lymphoma Dysregulated expression of mitotic regulators E Chen et al 2576 disease progression in which to develop immuno- zone B-cell malignancies with extended latency (Hough therapies (Rosic-Kablar et al., 2000). Moreover, the et al., 1998), indicating the necessity of additional extended latency prior to the development of lymphoma genetic lesions to cooperate with HOX11 in the onco- indicated that additional genetic events were necessary genic process. Attempts to accelerate B-cell lymphoma prior to the onset of malignancy, and therefore, these in these mice by infection of neonates with the mice represented a powerful system in which to identify Moloney murine leukemia virus (MMLV) were unsuc- genetic pathways underlying the pathogenesis of cessful, as all mice injected with the virus developed HOX11-mediated lymphomagenesis. T-cell lymphomas at B3 months postinjection, with Retroviral insertional mutagenesis in mice is a no effects on the B-cell compartment (Thy1 þ popu- powerful approach for the identification of genes that lation in spleen: 68.9% in wild type versus 67.9% in participate in tumorigenesis. It is particularly useful IgHm-HOX11Tg mice, n ¼ 6) (Table S1 in Supplementary for the identification of genes that are downstream, Information). This lack of effect was likely due to the cooperating events in multistep malignant transforma- preference of the MMLV to target early thymocyte tion processes (Berns, 1991; Jonkers and Berns, 1996). progenitors. Inoculation of transgenic mice with slow, nononcogene- An alternative novel approach of utilizing the Du5H bearing retroviruses results in disease acceleration by mAIDS virus as an insertional mutagen was developed random proviral insertions within the host genome and given the virus preference for infecting mature B-cells subsequent disruption of adjacent genomic regions. (Huang et al., 1989, 1991). However, mutagenesis Retroviral-derived DNA sequences that integrate studies in the CD1 IgHm-HOX11Tg mice using the adjacent to cellular oncogenes or within coding regions Du5H mAIDS virus were hampered by the decreased of tumor suppressors, can result in altered host gene susceptibility of CD1 mice to infection with the Du5H expression contributing, ultimately, to clonal expansion mAIDS virus (Huang et al., 1992). To overcome this of the infected cell. While the proviral tagging approach difficulty, the CD1 IgHm-HOX11Tg mice were back- has resulted in the discovery of many genes involved in crossed with C57BL6/J mice for >20 generation to lymphoid and myeloid leukemias (Li et al., 1999; Hwang give rise to heterozygous IgHm-HOX11Tg mice on a et al., 2002; Kim et al., 2003; Erkeland et al., 2004; mAIDS-susceptible, inbred C57BL6/J genetic back- Iwasaki et al., 2004; Shin et al., 2004), less work has been ground. To confirm that this line of C57BL6/J conducted on mature B-cell lymphomas. IgHm-HOX11Tg mice (hereafter referred to as the In this report, we employ a novel approach to identify IgHm-HOX11Tg mice) also developed B-cell malignan- genes that collaborate with HOX11 in tumorigenesis in cies, a cohort of 25 IgHm-HOX11Tg mice together with a mature B-lymphocyte target population by using the a control cohort of 25 age- and sex-matched C57BL6/J Du5H murine AIDS (mAIDS) virus (Aziz et al., 1989; control littermates were followed for 24 months. At Chattopadhyay et al., 1989) to accelerate lymphoma the end of this period, all but one of the control mice development in IgHm-HOX11Tg animals. Most cells were healthy, whereas 60% (15/25) of the IgHm- infected by the Du5H-defective virus are mature HOX11Tg mice had developed mature B-cell malig- IgM þ IgD þ B-cells where the virus has been shown to nancies. The median age of disease onset for the 15 be capable of functioning as an insertional mutagen affected mice was 603 days (Figure 1a, blue curve). (Huang et al., 1991, 1995). In one study, inoculation of The latency period was longer and the pene- susceptible C57BL6/J mice with helper-free stocks of trance lower than that seen with IgHm-HOX11Tg mice Du5H mAIDS virus identified two common integra- on a CD1 genetic background (>80% developed tion sites, Dis-1 and Dis-2, in 20 and 13% of infected marginal zone lymphomas by B420 days), indicating mice, respectively (Huang et al., 1995). This approach, strain-specific variability in disease penetrance