XRCC4 suppresses medulloblastomas with recurrent translocations in p53-deficient mice

Inactivation of the XRCC4 nonhomologous end-joining factor in the mouse germ line leads to embryonic lethality, in association with apoptosis of newly generated, postmitotic neurons. We now show that conditional inactivation of the XRCC4 in nestin-expressing neuronal progenitor cells, although leading to no obvious phenotype in a WT background, leads to early onset of neuronally differentiated medulloblastomas (MBs) in a p53-deficient background. A substantial proportion of the XRCC4/p53-deficient MBs have high-level N-myc gene amplification, often intrachromosomally in the context of complex translocations or other alterations of chromosome 12, on which N-myc resides, or extrachromosomally within double minutes. In addition, most XRCC4/p53-deficient MBs harbor clonal translocations of chromosome 13, which frequently involve chromosome 6 as a partner. One copy of the patched gene (Ptc), which lies on chromosome 13, was deleted in all tested XRCC4/p53-deficient MBs in the context of translocations or interstitial deletions. In addition, Cyclin D2, a chromosome 6 gene, was amplified in a subset of tumors. Notably, amplification of Myc-family or Cyclin D2 genes and deletion of Ptc also have been observed in human MBs. We therefore conclude that, in neuronal cells of mice, the nonhomologous end-joining pathway plays a critical role in suppressing genomic instability that, in a p53-deficient background, routinely contributes to genesis of MBs with recurrent chromosomal alterations.     

Synergistic induction of apoptosis by combination of BTK and dual mTORC1/2 inhibitors in diffuse large B cell lymphoma

Diffuse large B cell lymphoma is generally treated by chemotherapy and there is an unmet medical need for novel targeted therapies or combination therapies. Using in vitro screening, we have identified the combination of ibrutinib, an inhibitor of the tyrosine kinase BTK, and AZD2014, an mTOR catalytic inhibitor, as being highly synergistic in killing ABC-subtype DLBCL cell lines. Simultaneous inhibition of BTK and mTOR causes apoptosis both in vitro and in vivo and results in tumor regression in a xenograft model. We identify two parallel mechanisms that underlie apoptosis in this setting: cooperative inhibition of cap-dependent translation, and the inhibition of an NF-κB/IL10/STAT3 autocrine loop. Combined disruption of these pathways is required for apoptosis. These data represent a rational basis for the dual inhibition of BTK and mTOR as a potential treatment for ABC-subtype DLBCL.     

ATM-deficient thymic lymphoma is associated with aberrant tcrd rearrangement and gene amplification

Ataxia telangiectasia mutated (ATM) deficiency predisposes humans and mice to T lineage lymphomas with recurrent chromosome 14 translocations involving the T cell receptor α/δ (Tcra/d) locus. Such translocations have been thought to result from aberrant repair of DNA double-strand breaks (DSBs) during Tcra locus V(D)J recombination, and to require the Tcra enhancer (Eα) for Tcra rearrangement or expression of the translocated oncogene. We now show that, in addition to the known chromosome 14 translocation, ATM-deficient mouse thymic lymphomas routinely contain a centromeric fragment of chromosome 14 that spans up to the 5′ boundary of the Tcra/d locus, at which position a 500-kb or larger region centromeric to Tcra/d is routinely amplified. In addition, they routinely contain a large deletion of the telomeric end of one copy of chromosome 12. In contrast to prior expectations, the recurrent translocations and amplifications involve V(D)J recombination–initiated breaks in the Tcrd locus, as opposed to the Tcra locus, and arise independently of the Eα. Overall, our studies reveal previously unexpected mechanisms that contribute to the oncogenic transformation of ATM-deficient T lineage cells.Immature T cell lymphomas represent a significant portion of human lymphoid malignancies (Ferrando and Look, 2000; Armstrong and Look, 2005). Approximately 50% of these tumors harbor recurrent chromosomal translocations (Aifantis et al., 2008), which likely arise through errors in the repair of DNA double-strand breaks (DSBs). In this context, normal mouse and human thymocyte differentiation requires V(D)J recombination, a process that involves the introduction of DSBs at T cell receptor (Tcr) loci by the recombination-activating gene (RAG) 1 and 2 endonuclease (Bassing et al., 2002). If such programmed Tcr DSBs are repaired incorrectly, they can generate chromosomal translocations that involve Tcr loci. In fact, 70% of recurrent translocations in human T cell acute lymphoblastic leukemias (T-ALL) involve TCR loci (Aifantis et al., 2008). Furthermore, RAG-initiated DSBs in and around T cell oncogene loci may also contribute to clonal translocations in T cell lymphoma (Aifantis et al., 2008). In addition, developing T cells are also subject to general DSBs that occur during S phase in association with periods of rapid cellular division. These DSBs apparently can occur in numerous chromosomal locations, and potentially participate in Tcr locus-related translocations (Bassing et al., 2003).During T cell development in the thymus, TCR variable region gene exons are assembled by V(D)J recombination that is initiated by RAG-generated DSBs between V, D, and J segments and their flanking recombination signal sequences (RSSs; Rooney et al., 2004a). To complete V(D)J recombination, RAG-initiated coding sequence DSBs and RSS DSBs are joined to each other by the classical nonhomologous end-joining pathway (C-NHEJ; Rooney et al., 2004a; Lieber, 2008). The V(D)J recombination process is also influenced by general DSB response factors such as the ataxia telangiectasia mutated (ATM) protein (Liyanage et al., 2000; Bredemeyer et al., 2006; Callén et al., 2007; Huang et al., 2007). In humans and mice, the two distinct lineages of T lymphocytes are distinguished by the surface expression of either αβ or γδ TCRs (Bhandoola et al., 2007). TCRβ, γ, and δ variable region exons are assembled in CD4⁻/CD8⁻ (double-negative) thymocytes (von Boehmer, 2004). Productive VδDδJδ and VγJγ rearrangements generate TCR δ and γ chains that form cell surface γδ TCR and prescribe γδ T cell expansion and development (Krangel et al., 1998). Productive VβDβJβ rearrangements generate TCRβ chains, which, in conjunction with Notch1 signaling, promote differentiation to the CD4⁺/CD8⁺ (double positive; DP) stage, in which Tcra rearrangements occur (Garbe and von Boehmer, 2007). Productive VαJα rearrangements generate TCRα chains that associate with TCRβ and promote further differentiation to CD4⁺ or CD8⁺ single-positive (SP) mature T cells.In both humans and mice, the TCRA and TCRD genes are encoded within the same locus (TCRA/D; Krangel et al., 2004). The mouse Tcra/d locus lies on chromosome 14, with the Cα exons, which lie at the 3′ (telomeric) end of Tcra/d, being preceded upstream by ∼60 Jα segments (Krangel et al., 2004; Fig. S1). The Cδ exons lie upstream of the Jα segments and are flanked downstream by the Vδ5 segment and upstream by a Vδ4 segment, two Dδ segments, and two Jδ segments. Most Vα and Vδ segments lie upstream of Vδ4 with Vα segments lying at the distal (5′ or centromeric) end of the cluster, Vα/Vδ segments in the center, and Vδ segments at the downstream (telomeric) end (Krangel et al., 2004). Because of the unique Tcra/d organization, joining of Vα and Jα segments deletes the Tcrd portion of the locus (including Cδ), permanently committing to the TCRα/β lineage. Vα to Jα rearrangement requires the Tcra enhancer (Eα) at the 3′ end of Tcra/d locus (downstream of Cα) to promote V(D)J recombinational accessibility of Jα segments lying up to 100-kb away (Sleckman et al., 1997). Eα also activates TCRα expression (Sleckman et al., 1997). The human TCRA/D locus is similarly organized and lies on human chromosome 14 (Fig. S1).DNA DSBs, including V(D)J recombination DSBs, activate the ATM kinase, which then phosphorylates substrates involved in DNA repair and cell cycle control (Kastan et al., 2001). Although not required for V(D)J recombination, ATM helps promote normal DSB repair during this process (Bredemeyer et al., 2006; Callén et al., 2007; Huang et al., 2007). Human ATM mutations cause AT, which is characterized by immunodeficiency, genomic instability, and lymphoid malignancies (Lavin, 2008). Most T cell malignancies in AT patients are T-ALLs, which have not been well characterized cytogenetically. However, older AT patients who harbor clonal expansions of peripheral T cells with inv(14)(q11;q32), t(14;14)(q11;32.1), or rarely, t(X;14)(q28;q11) translocations, sometimes develop T cell prolymphocytic leukemia (T-PLL; Pekarsky et al., 2001). The inv(14)(q11;q32), t(14;14)(q11;32.1), or t(X;14)(q28;q11) translocations appear to involve TCRA/D at 14q11 in humans and the T cell leukemia/lymphoma 1 (TCL1) (14q32.1) and MTCP1 (Xq28) oncogene loci, respectively (Russo et al., 1989; Fig. S1). ATM-deficient mice recapitulate aspects of human AT (Barlow et al., 1996; Elson et al., 1996; Xu et al., 1996; Herzog et al., 1998; Borghesani et al., 2000). In this regard, ATM-deficient mice have impaired thymocyte development with reduced CD3 expression on DP thymocytes and decreased SP thymocyte numbers (Borghesani et al., 2000; Huang et al., 2007), which is consistent with defective Tcra V(D)J recombination. Moreover, ATM-deficient mice succumb to DP thymic lymphomas with RAG-dependent translocations, which often join the Tcra/d on chromosome 14 to the telomeric end of chromosome 12, where mouse Tcl1 is located. Thus, it has been speculated that the inv(14) or t(14;14) in human T-PLL and the t(12;14) in mouse ATM-deficient thymic lymphoma both may activate TCL1 or another dominant oncogene in this chromosomal region (Liyanage et al., 2000; Callén et al., 2009; Fig. S1).We now characterize recurrent translocations in mouse ATM-deficient thymic lymphomas in depth and also test for roles of Eα in tumor development. Surprisingly, we find that the recurrent chromosome 14 translocations observed in ATM-deficient thymic lymphomas are associated with V(D)J recombination errors at Tcrd, as opposed to Tcra locus. In addition, we show that Eα is completely dispensable for the oncogenic processes leading to these tumors. Finally, we show that most of these tumors amplify a set of genes lying just upstream of the Tcra/d locus. Based on our findings, we propose a model for the generation of the recurrent genomic abnormalities in ATM-deficient thymic lymphomas (Fig. S1).