Antibody specificity and diversity is generated in B cells during germinal center maturation through clonal expansion while they undergo class-switch recombination and somatic hypermutation. Here we demonstrate that the transcriptional repressor Bcl-6 mediates this phenotype by directly repressing ATR in centroblasts and lymphoma cells. ATR is critical in replication and DNA damage-sensing checkpoints. Bcl-6 allowed B cells to evade ATR-mediated checkpoints and attenuated the response of the B cells to exogenous DNA damage. Repression of ATR was necessary and sufficient for those Bcl-6 activities. CD40 signaling 'rescued' B cells from those effects by disrupting the Bcl-6 transcription-repression complex on the promoter of the gene encoding ATR. Our data demonstrate a transcriptional regulatory loop whereby Bcl-6 mediates the centroblast phenotype through transient silencing of ATR.

Our findings suggest that increased activity of the ubiquitin-proteasome pathway, marked decreases in MyHCs, and atrophic AKT-FOXO signaling play important roles in eliciting the myofiber atrophy and decreases in diaphragm force generation associated with prolonged human diaphragm disuse.

Chronic myelogeneous leukemia (CML) is a two-stage disease associated with expression of the BCR/ABL tyrosine kinase protein. However, whether BCR/ABL expression directly causes blast crisis, and if so by what mechanism, is unknown. We have found that BCR/ABL translocates from the cytoplasm to the nucleus after genotoxic stress. Furthermore, BCR/ABL increases DNA double-strand damage after etoposide treatment and leads to a defect in an intra-S phase checkpoint, causing a radioresistant DNA synthesis (RDS) phenotype. In the nucleus, BCR/ABL associates with the ataxia-telangiectasia and rad 3-related protein (ATR) and disrupts ATR-dependent signal transduction. Overexpression of ATR in a BCR/ABL-expressing cell line corrects the DNA damage phenotype. These results demonstrate a nuclear role for BCR/ABL in altering the cellular response to DNA damage.

Purpose: Inhibiting mammalian target of rapamycin (mTOR) signaling in acute myelogenous leukemia (AML) blasts and leukemic stem cells may enhance their sensitivity to cytotoxic agents. We sought to determine the safety and describe the toxicity of this approach by adding the mTOR inhibitor, sirolimus (rapamycin), to intensive AML induction chemotherapy. Experimental Design: We performed a phase I dose escalation study of sirolimus with the chemotherapy regimen MEC (mitoxantrone, etoposide, and cytarabine) in patients with relapsed, refractory, or untreated secondary AML. Results: Twenty-nine subjects received sirolimus and MEC across five dose levels. Dose-limiting toxicities were irreversible marrow aplasia and multiorgan failure. The maximum tolerated dose (MTD) of sirolimus was determined to be a 12 mg loading dose on day 1 followed by 4 mg/d on days 2 to 7, concurrent with MEC chemotherapy. Complete or partial remissions occurred in 6 (22%) of the 27 subjects who completed chemotherapy, including 3 (25%) of the 12 subjects treated at the MTD. At the MTD, measured rapamycin trough levels were within the therapeutic range for solid organ transplantation. However, direct measurement of the mTOR target p70 S6 kinase phosphorylation in marrow blasts from these subjects only showed definite target inhibition in one of five evaluable samples. Conclusions: Sirolimus and MEC is an active and feasible regimen. However, as administered in this study, the synergy between MEC and sirolimus was not confirmed. Future studies are planned with different schedules to clarify the clinical and biochemical effects of sirolimus in AML and to determine whether target inhibition predicts chemotherapy response. (Clin Cancer Res 2009;15(21):6732-9) Although intensive combination chemotherapy is effective in temporizing acute myelogenous leukemia (AML) in most treated patients, the majority of those who achieve complete remission (CR) subsequently relapse and ultimately die of their disease (1-3). More than 30 years of combination chemotherapy clinical trials have failed to substantially improve survival in AML, leading to efforts to develop targeted therapeutics for this disease. We and others have concentrated on identifying agents or signaling inhibitors that target AML cells and AML stem cells without additional toxicity to normal hematopoietic cells. Some of these agents seem to be cytotoxic by themselves (4-9), whereas others alter the response of AML cells to chemotherapy (10, 11). The most extensively studied drugs in this class are the FLT3 inhibitors, for which clinical trials are ongoing (12-16). Use of FLT3 inhibitors has been studied primarily in patients with FLT3 mutations, which are known to confer a (12,15,17,18).Phosphatidylinositol-3′-kinase (PI3K) is a potential novel therapeutic target in AML. The role of PI3K signaling in leukemia has been studied in both murine models and primary human cells. Yilmaz et al. studied a murine leukemia model that conditionally induces activated PI3K signaling in hematop...

Earlier reports have suggested that the BCR/ABL oncogene, associated with chronic myeloid leukemia, induces a mutator phenotype; however, it is unclear whether this leads to long-term changes in chromosomes and whether the phenotype is found in primary chronic myelogeneous leukemia (CML) cells. We have addressed both these issues. BCR/ABL-expressing cell lines show an increase in DNA breaks after treatment with etoposide as compared to control cells. However, although BCR/ABL-expressing cell lines have an equivalent cell survival, they have an increase in chromosomal translocations after DNA repair as compared to control cells. This demonstrates that BCR/ABL expression decreases the fidelity of DNA repair. To see whether this is true in primary CML samples, normal CD34 + progenitor cells and CML progenitor cells were treated with etoposide. CML progenitor cells have equivalent survival but have an increase in DNA double-strand breaks (DSBs). Spectral karyotyping demonstrates new chromosomal translocations in CML cells, but not normal progenitor cells, consistent with error-prone DNA repair. Taken together, these data demonstrate that BCR/ABL enhances the accumulation of DSBs and alters the apoptotic threshold in CML leading to error-prone DNA repair.

IntroductionTyrosine kinase fusion proteins are the products of a growing family of oncogenes associated with both solid tumors and hematologic malignancies. 1 The best known tyrosine kinase fusion protein is the BCR/ABL tyrosine kinase that results from a t(9;22) translocation in patients with chronic myeloid leukemia. 2 Previous work has demonstrated that BCR/ABL activates multiple signal transduction pathways, including the phosphatidylinositol-3 (PI3) kinase pathway and that transformation by BCR/ABL requires activation of PI3 kinase. [3][4][5][6] To determine if tyrosine kinase fusion proteins share common mechanisms of transformation or if each functions in unique ways, we have chosen to study the TEL/plateletderived growth factor receptor ␤ (PDGF␤R) fusion protein that results from the t(5;12) translocation in patients with chronic myelomonocytic leukemia. 7 TEL/PDGF␤R contains the amino-terminal 154 amino acids of TEL fused to the transmembrane and cytoplasmic domains of the PDGF␤R. TEL is a member of the ETS family of transcription factors and has been described as a common site of rearrangement in multiple forms of leukemia. [8][9][10] Structurally, wild-type TEL contains a 5Ј oligomerization domain, designated the PNT domain; this domain is retained in the fusion protein and is essential for the transforming activity of TEL/PDGF␤R as demonstrated by us and others. 11,12 Evidence suggests that the PNT domain may cause multimerization of the fusion protein, not simple dimerization. 13 TEL has been reported to induce G1 arrest in vitro 14 and to be required for yolk sac angiogenesis according to murine knockout experiments. 15 PDGF␤R is a well-characterized plasma membrane receptor with endogenous tyrosine kinase activity that is autophosphorylated in response to binding of dimeric PDGF ligand. 16 In the fusion protein there is retention of the transmembrane domain and the complete tyrosine kinase domain of PDGF␤R. An intact kinase activity is necessary for transforming activity. 17 The protein retains multiple tyrosine sites that act as binding sites for SH2-containing signaling molecules in the wild type PDGF␤R. Furthermore, immunolocalization of TEL/PDGF␤R has demonstrated that the protein is located primarily in the cytosol, retaining neither the nuclear localization of TEL or the plasma membrane localization of PDGF␤R. 12 Thus, initial models of transformation by TEL/ PDGF␤R have suggested that the protein is constitutively oligomerized through the TEL PNT domain, leading to constitutive activation of the kinase activity of the 3Ј PDGF␤R kinase domain and activation of critical signaling pathways. However, the signaling pathways that are necessary for transformation remain undefined.Several signaling pathways have been identified as being activated by TEL/PDGF␤R. In transformed cell lines, TEL/ PDGF␤R is known to associate with or cause phosphorylation of phospholipase C (PLC␥1), SHP2, and JNK. 11,18,19 In addition, we have recently shown that TEL/PDGF␤R activates STAT1 and STAT5 but have been unabl...

We have recently identified a novel candidate oncogene, MCT-1, in the HUT 78 T-cell line. When overexpressed in NIH3T3 fibroblasts, the MCT-1 gene shortens the G1 phase of the cell cycle and promotes anchorage-independent growth. Progression of cells through a late G1 phase restriction point is regulated by G1 cyclins whose phosphorylation of the retinoblastoma gene product facilitates entry into S phase. Deregulated expression of G1 cyclins and their cognate cdk partners is often found in human tumor cells. In order to address the potential relationship of MCT-1 to cell cycle regulatory molecules, we analyzed the ability of MCT-1 overexpression to modulate cdk4 and cdk6 kinase activity in NIH3T3 fibroblasts constitutively overexpressing MCT-1. We observed an increase in the kinase activity of both cdk4 and cdk6 in asynchronously growing transformed cells compared with the parent cells. This increased kinase activity was accompanied by an elevated level of cyclin D1 protein and increased G1 cyclin/cdk complex formation. We also observed a correlation between increased protein levels of MCT-1 with cyclin D1 expression in a panel of lymphoid cell lines derived from T-cell malignancies. These results demonstrate that constitutive expression of MCT-1 is associated with deregulation of protein kinase-mediated G1 phase checkpoints.

The BCL6 oncogenic transcriptional repressor is required for development of germinal center centroblasts, which undergo simultaneous genetic recombination and massive clonal expansion. Although BCL6 is required for survival of centroblasts, its expression in earlier B-cells is toxic. Understanding these opposing effects could provide critical insight into normal B-cell biology and lymphomagenesis. We examined the transcriptional and biological effects of BCL6 in various primary cells. BCL6 repression of ATR was previously shown to play a critical role in the centroblast phenotype. Likewise, we found that BCL6 could impose an ATR-dependent phenotype of attenuated DNA damage sensing and repair in primary fibroblasts and B-cells. BCL6 induced true genomic instability because DNA repair was delayed and was qualitatively impaired, which could be critical for BCL6-induced lymphomagenesis. Although BCL6 can directly repress TP53 in centroblasts, BCL6 induced TP53 expression in primary fibroblasts and B-cells, and these cells underwent p53-dependent growth arrest and senescence in the presence of physiological levels of BCL6. This differential ability to trigger a functional p53 response explains at least in part the different biological response to BCL6 expression in centroblasts versus other cells. The data suggest that targeted re-activation of TP53 could be of therapeutic value in centroblast-derived lymphomas.