Differential regulation of mTOR signaling determines sensitivity to AKT inhibition in diffuse large B cell lymphoma.

Agents that target components of the PI3K/AKT/mTOR pathway are under investigation for the treatment of diffuse large B cell lymphoma (DLBCL). Given the highly heterogeneous nature of DLBCL, it is not clear whether all subtypes of DLBCL will be susceptible to PI3K pathway inhibition, or which kinase within this pathway is the most favorable target. Pharmacological profiling of a panel of DLBCL cell lines revealed a subset of DLBCL that was resistant to AKT inhibition. Strikingly, sensitivity to AKT inhibitors correlated with the ability of these inhibitors to block phosphorylation of S6K1 and ribosomal protein S6. Cell lines resistant to AKT inhibition activated S6K1 independent of AKT either through upregulation of PIM2 or through activation by B cell receptor (BCR) signaling components. Finally, combined inhibition of AKT and BTK, PIM2, or S6K1 proved to be an effective strategy to overcome resistance to AKT inhibition in DLBCL.

Developmental propagation of V(D)J recombination-associated DNA breaks and translocations in mature B cells via dicentric chromosomes.

Mature IgM(+) B-cell lymphomas that arise in certain ataxia telangiectasia-mutated (ATM)-deficient compound mutant mice harbor translocations that fuse V(D)J recombination-initiated IgH double-strand breaks (DSBs) on chromosome 12 to sequences downstream of c-myc on chromosome 15, generating dicentric chromosomes and c-myc amplification via a breakage-fusion-bridge mechanism. As V(D)J recombination DSBs occur in developing progenitor B cells in the bone marrow, we sought to elucidate a mechanism by which such DSBs contribute to oncogenic translocations/amplifications in mature B cells. For this purpose, we applied high-throughput genome-wide translocation sequencing to study the fate of introduced c-myc DSBs in splenic IgM(+) B cells stimulated for activation-induced cytidine deaminase (AID)-dependent IgH class switch recombination (CSR). We found frequent translocations of c-myc DSBs to AID-initiated DSBs in IgH switch regions in wild-type and ATM-deficient B cells. However, c-myc also translocated frequently to newly generated DSBs within a 35-Mb region downstream of IgH in ATM-deficient, but not wild-type, CSR-activated B cells. Moreover, we found such DSBs and translocations in activated B cells that did not express AID or undergo CSR. Our findings indicate that ATM deficiency leads to formation of chromosome 12 dicentrics via recombination-activating gene-initiated IgH DSBs in progenitor B cells and that these dicentrics can be propagated developmentally into mature B cells where they generate new DSBs downstream of IgH via breakage-fusion-bridge cycles. We propose that dicentrics formed by joining V(D)J recombination-associated IgH DSBs to DSBs downstream of c-myc in ATM-deficient B lineage cells similarly contribute to c-myc amplification and mature B-cell lymphomas.

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.

Mechanisms that can promote peripheral B-cell lymphoma in ATM-deficient mice.

The Ataxia Telangiectasia-mutated (ATM) kinase senses DNA double-strand breaks (DSB) and facilitates their repair. In humans, ATM deficiency predisposes to B- and T-cell lymphomas, but in mice it leads only to thymic lymphomas. We tested the hypothesis that increased DSB frequency at a cellular oncogene could promote B-cell lymphoma by generating ATM-deficient mice with a V(D)J recombination target (DJß cassette) within c-myc intron 1 ("DA" mice). We also generated ATM-deficient mice carrying an Eµ-Bcl-2 transgene (AB mice) to test whether enhanced cellular survival could promote B-cell lymphomas. About 30% of DA or AB mice and nearly 100% of mice harboring the combined genotypes (DAB mice) developed mature B-cell lymphomas. In all genotypes, B-cell tumors harbored oncogenic c-myc amplification generated by breakage-fusion-bridge (BFB) from dicentric chromosomes formed through fusion of IgH V(D)J recombination-associated DSBs on chromosome 12 to sequences downstream of c-myc on chromosome 15. AB tumors demonstrate that B lineage cells harboring spontaneous DSBs leading to IgH/c-myc dicentrics are blocked from progressing to B-cell lymphomas by cellular apoptotic responses. DA and DAB tumor translocations were strictly linked to the cassette, but occurred downstream, frequently in a 6-kb region adjacent to c-myc that harbors multiple cryptic V(D)J recombination targets, suggesting that bona fide V(D)J target sequences may activate linked cryptic targets. Our findings indicate that ATM deficiency allows IgH V(D)J recombination DSBs in developing B cells to generate dicentric translocations that, via BFB cycles, lead to c-myc-activating oncogenic translocations and amplifications in mature B cells.

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 alpha/delta (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 (Ealpha) 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 Ealpha. Overall, our studies reveal previously unexpected mechanisms that contribute to the oncogenic transformation of ATM-deficient T lineage cells.

MAIDS resistance-associated gene expression patterns in secondary lymphoid organs.

Murine acquired immunodeficiency syndrome (MAIDS) is caused by exposure to murine leukemia virus and serves as a model to study human AIDS. In MAIDS-susceptible C57BL/6 mice, virus exposure leads to progressive immune deficiency, while resistant strains such as BALB/c recover from infection and develop protective immunity. The goal of this study was to identify early gene expression patterns that may be important in establishing this strain-specific differential response. Total RNA was isolated from spleens and pooled lymph nodes of both mouse strains at 3 and 7 days post virus infection. The complementary DNA generated from this RNA was hybridized to mouse oligonucleotide DNA microarrays using a strategy that controlled for inherent variability and highlighted only virus-induced changes. Fluorescent intensities were normalized and analyzed for statistically significant differential expression between strains across both time points and lymphoid organs. The majority of the resistance-associated genes was identified at day 3 post-infection and demonstrated the highest fold differences between strains, while more susceptibility-associated sequences were seen at 7 days post-infection. Among the most highly differentially expressed sequences seen at the earlier time point were genes related to protein metabolism, especially serine proteases. Differential patterns of chemokine-related genes were observed at the later time point. The overall pattern of expression suggests strain-specific differences in proteases and chemokines within secondary lymphoid organs shortly after infection influence the likelihood of disease progression.

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.

H2AX prevents DNA breaks from progressing to chromosome breaks and translocations.

Histone H2AX promotes DNA double-strand break (DSB) repair and immunoglobulin heavy chain (IgH) class switch recombination (CSR) in B-lymphocytes. CSR requires activation-induced cytidine deaminase (AID) and involves joining of DSB intermediates by end joining. We find that AID-dependent IgH locus chromosome breaks occur at high frequency in primary H2AX-deficient B cells activated for CSR and that a substantial proportion of these breaks participate in chromosomal translocations. Moreover, activated B cells deficient for ATM, 53BP1, or MDC1, which interact with H2AX during the DSB response, show similarly increased IgH locus breaks and translocations. Thus, our findings implicate a general role for these factors in promoting end joining and thereby preventing DSBs from progressing into chromosomal breaks and translocations. As cellular p53 status does not markedly influence the frequency of such events, our results also have implications for how p53 and the DSB response machinery cooperate to suppress generation of lymphomas with oncogenic translocations.