David P. LeBrun

E2A-PBX1 Interacts Directly with the KIX Domain of CBP/p300 in the Induction of Proliferation in Primary Hematopoietic Cells

The E2A gene encodes DNA-binding transcription factors, called E12 and E47, involved in cell specification and maturation. E2A is also involved in a chromosomal translocation that leads to the expression of an oncogenic transcription factor called E2A-PBX1 in cases of acute leukemia. In the work described here, we elucidate the interaction between E2A-PBX1 and transcriptional co-activators. We confirm that the E2A portion can interact with CBP and PCAF and map required elements on E2A and CBP. On CBP, the interaction involves the KIX domain, a well characterized domain that mediates interactions with several other oncogenic transcription factors. On E2A, the interaction with CBP requires conserved α-helical domains that reside within activation domains 1 and 2 (AD1 and AD2, respectively). Using purified, recombinant proteins, we show that the E2A-CBP interaction is direct. Notwithstanding the previously demonstrated ability of AD1 and AD2 to function independently, some of our findings suggest functional cooperativity between these two domains. Finally, we show that the CBP/p300-interactive helical domains of E2A are important in the induction of proliferation in cultured primary bone marrow cells retrovirally transduced with E2A-PBX1. Our findings suggest that some aspects of E2A-PBX1 oncogenesis involve a direct interaction with the KIX domain of CBP/p300.

Restricted Expression of E2A Protein in Primary Human Tissues Correlates with Proliferation and Differentiation

E2A is a basic helix-loop-helix (bHLH) transcription factor required for B cell lymphopoiesis and implicated in myogenesis and the regulation of insulin expression. As E2A is expressed widely in tissues, tissue-specific downstream effects are thought to result primarily from dimerization with other bHLH proteins. To investigate the degree to which regulation of E2A protein abundance may serve to regulate E2A function, expression of E2A was evaluated using immunohistochemistry on histological sections of primary human tissues. Somewhat surprisingly, nuclear staining for E2A was restricted in all tissues examined, often to a small subpopulation of cells. In some tissues, such as adult liver, expression was absent or limited to rare infiltrating lymphocytes. E2A-expressing cells were most abundant in lymphoid tissues. In tonsil, lymph node, and spleen, expression appeared most abundant and prevalent among rapidly proliferating centroblasts of the germinal center dark zone. Scattered E2A-expressing thymocytes were more numerous in the thymic cortex than medulla. In developing skeletal muscle, E2A was detectable in striated myotubes but not in more primitive mononucleated progenitors or mature muscle. Differential E2A expression was also noted in proliferating periventricular neuroepithelial cells in the developing brain. These results suggest that regulation of E2A abundance complements protein-protein interactions in modulating E2A function.

Critical Role for a Single Leucine Residue in Leukemia Induction by E2A-PBX1

ABSTRACT In roughly 5% of cases of acute lymphoblastic leukemia, a chromosomal translocation leads to expression of the oncogenic protein E2A-PBX1. The N-terminal portion of E2A-PBX1, encoded by the E2A gene, is identical in sequence to the corresponding portion of the E proteins E12/E47 and includes transcriptional activation domains. The C terminus consists of most of the HOX interacting transcription factor PBX1, including its DNA-binding homeodomain. Structure-function correlative experiments have suggested that oncogenesis by E2A-PBX1 requires an activation domain, called AD1, at the extreme N terminus. We recently demonstrated that a potentially helical portion of AD1 interacts directly with the transcriptional coactivator protein cyclic AMP response element-binding protein (CBP) and that this interaction is essential in the immortalization of primary bone marrow cells in tissue culture. Here we show that a conserved LXXLL motif within AD1 is required in the interaction between E2A-PBX1 and the KIX domain of CBP. We show by circular dichroism spectroscopy that the LXXLL-containing portion of AD1 undergoes a helical transition upon interacting with the KIX domain and that amino acid substitutions that prevent helix formation prevent both the KIX interaction and cell immortalization by E2A-PBX1. Perhaps most strikingly, substitution of a single, conserved leucine residue (L20) within the LXXLL motif impairs leukemia induction in mice after transplantation with E2A-PBX1-expressing bone marrow. The KIX domain of CBP mediates well-characterized interactions with several transcription factors of relevance to leukemia induction. Circumstantial evidence suggests that the side chain of L20 might interact with a deep hydrophobic pocket in the KIX domain. Therefore, our results serve to identify a potential new drug target.

Structural basis of CBP/p300 recruitment in leukemia induction by E2A-PBX1

E-proteins are critical transcription factors in B-cell lymphopoiesis. E2A, 1 of 3 E-protein-encoding genes, is implicated in the induction of acute lymphoblastic leukemia through its involvement in the chromosomal translocation 1;19 and consequent expression of the E2A-PBX1 oncoprotein. An interaction involving a region within the N-terminal transcriptional activation domain of E2A-PBX1, termed the PCET motif, which has previously been implicated in E-protein silencing, and the KIX domain of the transcriptional coactivator CBP/p300, critical for leukemogenesis. However, the structural details of this interaction remain unknown. Here we report the structure of a 1:1 complex between PCET motif peptide and the KIX domain. Residues throughout the helical PCET motif that contact the KIX domain are important for both binding KIX and bone marrow immortalization by E2A-PBX1. These results provide molecular insights into E-protein-driven differentiation of B-cells and the mechanism of E-protein silencing, and reveal the PCET/KIX interaction as a therapeutic target for E2A-PBX1-induced leukemia.

Transplacental benzene exposure increases tumor incidence in mouse offspring: possible role of fetal benzene metabolism

Childhood cancer is the leading cause of disease-related death in children aged 1-14 years in Canada and the USA and it has been hypothesized that transplacental exposure to environmental carcinogens such as benzene may contribute to the etiology of these cancers. Our objectives were to determine if transplacental benzene exposure increased tumor incidence in mouse offspring and assess fetal benzene metabolism capability. Pregnant CD-1 and C57Bl/6N mice were given intraperitoneal injections of corn oil, 200 mg/kg, or 400 mg/kg benzene on gestational days 8, 10, 12 and 14. A significant increase in tumor incidence was observed in CD-1, but not C57BL/6N, 1-year-old offspring exposed transplacentally to 200 mg/kg benzene. Hepatic and hematopoietic tumors were predominantly observed in male and female CD-1 offspring, respectively. Female CD-1 offspring exposed transplacentally to 200 mg/kg benzene had significantly suppressed bone marrow CD11b(+) cells 1 year after birth, correlating with reduced colony-forming unit granulocyte/macrophage numbers in 2-day-old pups. CD-1 and C57Bl/6N maternal blood benzene levels and fetal liver benzene, t, t-muconic acid, hydroquinone and catechol levels were analyzed by gas chromatography/mass spectrometry. Significant strain-, gender- and dose-related differences were observed. Male CD-1 fetuses had high hydroquinone levels, whereas females had high catechol levels after maternal exposure to 200 mg/kg benzene. This is the first demonstration that transplacental benzene exposure can induce hepatic and hematopoietic tumors in mice, which may be dependent on fetal benzene metabolism capability.

Interleukin-6 is required for pristane-induced plasma cell hyperplasia in mice.

Intraperitoneal injection of pristane induces production of interleukin‐6 (IL‐6) and either plasmacytosis or plasmacytoma in mice, depending upon the genetic background. Pristane does not induce plasmacytoma in IL‐6 knockout (IL‐6−/−) mice, suggesting that IL‐6 is required for this process. In the present study we determined whether IL‐6 is also required for pristane‐induced hyperplasia of normal plasma cells. Pristane was injected intraperitoneally into IL‐6−/− and IL‐6 wild‐type (IL‐6+/+) mice. Overall there were more deaths in IL‐6+/+ mice (85%) than in IL‐6−/− mice (40%), P=0.024. Hyperplastic lymph node and spleen weight did not differ (P=0.82 and P=0.15, respectively) in IL‐6−/− versus IL‐6+/+ mice. Lymphocytosis with similar patterns of expression of B‐cell (B220) and T‐cell (Thy‐1) antigens was noted in both IL‐6−/− and IL‐6+/+ mice. However, morphological studies, dual fluorescent staining for Syn‐1 and B220 antigens (syn‐1+ B220+ cells), and intracytoplasmic Ig staining revealed plasma cell hyperplasia in lymph node and spleen from IL‐6+/+, but not IL‐6−/−, mice. These plasma cells from IL‐6+/+ mice were polyclonal and unable to induce tumour formation in severe combined immunodeficient mice. These data demonstrate that IL‐6 is required for pristane‐induced hyperplasia of polyclonal plasma cells in mice.

FLI-1 distinguishes Ewing sarcoma from small cell osteosarcoma and mesenchymal chondrosarcoma.

Small cell osteosarcoma and mesenchymal chondrosarcoma are 2 primary bone tumors with a small round blue cell component, which can mimic the appearance of Ewing sarcoma. Distinguishing these tumors from each other on biopsy material is important clinically, as optimal therapy differs according to the tumor type. However, separating these entities on morphology alone can be challenging. FLI-1 has been described to be a useful marker for Ewing sarcoma, particularly when hematolymphoid markers are negative. In small cell osteosarcoma and mesenchymal chondrosarcoma, the FLI-1 staining pattern has not been adequately characterized. Using a monoclonal FLI-1 antibody, nuclear immunoreactivity in tumor cells was evaluated in 10 small cell osteosarcomas, 10 mesenchymal chondrosarcomas, and 8 Ewing sarcomas, together with a number of other small, round, blue cell tumors. None of the small cell osteosarcomas or mesenchymal chondrosarcomas exhibited FLI-1 staining in the tumor cells, in contrast to the positive nuclear FLI-1 staining in the stromal endothelial cells. In comparison, 6 of the 8 Ewing sarcomas showed moderate-to-strong nuclear FLI-1 staining of the tumor cells in addition to strong staining of the stromal endothelial cell nuclei. With the exception of lymphoblastic lymphomas, FLI-1 positivity was not seen in the other small round blue cell tumors examined. These findings show that, in contrast to Ewing sarcoma, small cell osteosarcoma and mesenchymal chondrosarcoma lack FLI-1 immunoreactivity. FLI-1 is therefore useful in the differential diagnosis of small round blue cell tumors of the bone.

Inactivation of the CDKN2A Tumor-Suppressor Gene by Deletion or Methylation Is Common at Diagnosis in Follicular Lymphoma and Associated with Poor Clinical Outcome

Purpose: Follicular lymphoma, the most common indolent lymphoma, is clinically heterogeneous. CDKN2A encodes the tumor suppressors p16INK4a and p14ARF and frequently suffers deleterious alterations in cancer. We investigated the hypothesis that deletion or hypermethylation of CDKN2A might identify follicular lymphoma cases with distinct clinical or pathologic features potentially amenable to tailored clinical management. Experimental Design: Deletion of CDKN2A was detected in pretreatment biopsy specimens using a single nucleotide polymorphism–based approach or endpoint PCR, and methylation of CpG elements in CDKN2A was quantified by methylation-specific PCR. Correlations between CDKN2A status and pathologic or clinical characteristics, including overall survival (OS), were investigated in 106 cases using standard statistical methods. Results: Deletion of CDKN2A was detected in 9 of 111 samples (8%) and methylation was detectable in 22 of 113 (19%). CDKN2A was either deleted or methylated in 29 of 106 cases (27%) and this status was associated with inferior OS especially among patients treated with rituximab (P = 0.004). CDKN2A deletion or methylation was associated with more advanced age (P = 0.012) and normal hemoglobin (P = 0.05) but not with sex, FLIPI score, ECOG stage, LDH, performance status, number of involved nodal sites, B symptoms, histologic grade, the presence of a component of diffuse large B-cell lymphoma, proliferation index, or other pathologic factors. Conclusions: Our results show that deletion or methylation of CDKN2A is relatively common in pretreatment follicular lymphoma biopsy specimens and defines a group of cases associated with reduced survival in the rituximab era presumably on the basis of more aggressive disease biology. Clin Cancer Res; 20(6); 1676–86. ©2014 AACR.

MicroRNA Signature Obtained From the Comparison of Aggressive With Indolent Non-Hodgkin Lymphomas: Potential Prognostic Value in Mantle-Cell Lymphoma

PURPOSE Mantle-cell lymphoma (MCL) has a variable natural history but is incurable with current therapies. MicroRNAs (miRs) are useful in prognostic assessment of cancer. We determined an miR signature defining aggressiveness in B-cell non-Hodgkin lymphomas (NHL) and assessed whether this signature aids in MCL prognosis. METHODS We assessed miR expression in a training set of 43 NHL cases. The miR signature was validated in 44 additional cases and examined on a training set of 119 MCL cases from four institutions in Canada. miRs significantly associated with overall survival were examined in an independent cohort of 114 MCL cases to determine association with patient outcome. miR expression was combined with current clinical prognostic factors to develop an enhanced prognostic model in patients with MCL. RESULTS Fourteen miRs were differentially expressed between aggressive and indolent NHL; 11 of 14 were validated in an independent set of NHL (excluding MCL). miR-127-3p and miR-615-3p were significantly associated with overall survival in the MCL training set. Their expression was validated in an independent MCL patient set. In comparison with Ki-67, expression of these miRs was more significantly associated with overall survival among patients with MCL. miR-127-3p was combined with Ki-67 to create a new prognostic model for MCL. A similar model was created with miR-615-3p and Mantle Cell Lymphoma International Prognostic Index scores. CONCLUSION Eleven miRs are differentially expressed between aggressive and indolent NHL. Two novel miRs were associated with overall survival in MCL and were combined with clinical prognostic models to generate novel prognostic data for patients with MCL.

Role for Homodimerization in Growth Deregulation by E2a Fusion Proteins

ABSTRACT The oncogenic transcription factor E2a-Pbx1 is expressed in some cases of acute lymphoblastic leukemia as a result of chromosomal translocation 1;19. The early observation that E2a-Pbx1 incorporates transcriptional activation domains from E2a and a DNA-binding homeodomain from Pbx1 inspired a model in which E2a-Pbx1 promotes leukemogenic transformation of lymphoid progenitor cells through transcriptional induction of target genes defined by the Pbx1 portion of the molecule. However, the subsequent demonstration that the only known DNA-binding module on the molecule, the Pbx1 homeodomain, is dispensable for the induction of lymphoblastic lymphoma in transgenic mice called into question the contribution made by the Pbx1 portion. In this study, we have used a domain swap approach coupled with a fibroblast-based focus formation assay to evaluate further the requirement for PBX1-encoded peptide elements in growth deregulation by E2a-Pbx1. No impairment of focus formation was observed when the entire Pbx1 portion was replaced with DNA-binding/dimerization domains derived from yeast transcription factor GAL4 or GCN4. Furthermore, replacement of Pbx1 with tandem FKBP domains that mediate homodimerization in the presence of a synthetic ligand led to striking growth deregulation exclusively in the presence of the dimerizing agent. N-terminal elements encoded by E2A, including the AD1 transcriptional activation domain, were required for dimerization-induced focus formation. We conclude that transcriptional target genes defined by heterologous C-terminal DNA-binding modules are not required in growth deregulation by E2a fusion proteins. We speculate that interactions between N-terminal E2a elements and undefined proteins that could function as components of a transcriptional coactivator complex may be more important.