Lymphoid cell growth and transformation are suppressed by a key regulatory element of the gene encoding PU.1.

Tight regulation of transcription factors, such as PU.1, is crucial for generation of all hematopoietic lineages. We previously reported that mice with a deletion of an upstream regulatory element (URE) of the gene encoding PU.1 (Sfpi1) developed acute myeloid leukemia. Here we show that the URE has an essential role in orchestrating the dynamic PU.1 expression pattern required for lymphoid development and tumor suppression. URE deletion ablated B2 cells but stimulated growth of B1 cells in mice. The URE was a PU.1 enhancer in B cells but a repressor in T cell precursors. TCF transcription factors coordinated this repressor function and linked PU.1 to Wnt signaling. Failure of appropriate PU.1 repression in T cell progenitors with URE deletion disrupted differentiation and induced thymic transformation. Genome-wide DNA methylation assessment showed that epigenetic silencing of selective tumor suppressor genes completed PU.1-initiated transformation of lymphoid progenitors with URE deletion. These results elucidate how a single transcription factor, PU.1, through the cell context-specific activity of a key cis-regulatory element, affects the development of multiple cell lineages and can induce cancer.

Therapeutic antibody targeting of CD97 in experimental arthritis: the role of antigen expression, shedding, and internalization on the pharmacokinetics of anti-CD97 monoclonal antibody 1B2.

CD97 is a member of the EGF-TM7 family of adhesion class receptors, with a proposed role in inflammatory cell recruitment. Neutralization of murine CD97 with the anti-mCD97 mAb 1B2 was efficacious in prevention of murine collagen-induced arthritis, a model with features resembling rheumatoid arthritis. Here, the therapeutic potential of neutralizing CD97 in arthritis was studied with emphasis on the 1B2 pharmacokinetics. Mice with established arthritis were treated with anti-mCD97 or anti-TNF-alpha serum. Ab pharmacokinetics and biodistribution were studied in diseased and nondiseased mice using labeled 1B2. The impact of CD97 expression on Ab pharmacokinetics was studied using CD97 knockout mice. Treatment with 1B2 showed an efficacy comparable to anti-TNF-alpha treatment. Pharmacokinetic analysis of 1B2 in wild-type and CD97 knockout mice indicated a dose-dependent Ab clearance, due to specific interaction with CD97. Biodistribution studies showed accumulation of 1B2 in spleen and lung. In vitro studies using murine splenocytes revealed that CD97 when bound to Ab was internalized. Moreover, soluble CD97 was detected in the supernatant, suggesting Ag shedding. Finally, in arthritic mice, higher levels of soluble CD97 were found and 1B2 treatment led to specific targeting of inflamed paws, resulting in a higher clearance rate of 1B2 in arthritic mice than in wild-type mice. In conclusion, our data support a therapeutic value of CD97 neutralization in experimental arthritis. The pharmacokinetic profile of the 1B2 Ab illustrates the complexity of Ab elimination from an organism and stresses the importance of understanding Ag-Ab interactions when developing therapeutic mAbs.

Role of transcription factors C/EBPalpha and PU.1 in normal hematopoiesis and leukemia.

Differentiation of hematopoietic stem and progenitor cells is under strict control of a regulatory network orchestrated by lineage-specific transcription factors. A block in normal differentiation is a major contributing factor in the development of solid tumors and leukemias. Cells from patients with acute myeloid leukemia (AML) frequently harbor mutated or dysregulated transcription factor genes, suggesting their involvement in leukemogenesis. As a consequence, these alterations diminish the pool of available molecules of a small number of critical transcription factors, such as CCAAT enhancer binding proteins, PU.1, GATA-1, and AML-1. In this review, we focus on the mechanisms of how this functional pool of transcription factors is maintained during normal and malignant hematopoiesis, including direct protein-protein interactions, competition for DNA binding, and the control of transcription factor genes by proximal and distal regulatory elements. Results of recent studies of mice carrying hypomorphic PU.1 alleles have indicated that reduction in the expression of a single transcription factor is capable of predisposing mice to AML. The implications of these findings for the study of hematopoiesis in the future as well as novel approaches to more disease-specific therapies are discussed.

Retroviral transduction in fetal thymic organ culture.

T-cell development requires cytokines and intimate contact with stromal cells provided exclusively by the thymus. Consequently, an in vitro model of thymocyte differentiation, fetal thymic organ culture (FTOC), has been developed. FTOC recapitulates the normal development of T-cells derived from both mouse and human progenitor populations, providing a more rapid means to study T-cell development compared with alternative in vivo approaches. Furthermore, FTOC is easily amenable to genetic manipulation using retroviral gene transfer. In this chapter, we outline the basic FTOC technique and describe several applications, including retroviral transduction of mouse thymocyte subsets and human CD34+ stem/progenitor cells.

CD97 antibody depletes granulocytes in mice under conditions of acute inflammation via a Fc receptor-dependent mechanism.

Antibodies to the pan-leukocyte adhesion-GPCR CD97 efficiently block neutrophil recruitment in mice, thereby reducing antibacterial host defense, inflammatory disease, and hematopoietic stem cell mobilization. Here, we investigated the working mechanism of the CD97 antibody 1B2. Applying sterile models of inflammation, intravital microscopy, and mice deficient for the CD97L CD55, the complement component C3, or the FcR common γ-chain, we show that 1B2 acts in vivo independent of ligand-binding interference by depleting PMN granulocytes in bone marrow and blood. Granulocyte depletion with 1B2 involved FcR but not complement activation and was associated with increased serum levels of TNF and other proinflammatory cytokines. Notably, depletion of granulocytes by CD97 antibody required acute inflammation, suggesting a mechanism of conditional, antibody-mediated granulocytopenia.

TLX1/HOX11-mediated disruption of primary thymocyte differentiation prior to the CD4+CD8+ double-positive stage.

The TLX1/HOX11 homeobox gene is frequently activated in T-cell acute lymphoblastic leukaemia (T-ALL) by the t(10;14)(q24;q11) and t(7;10)(q35;q24) chromosomal translocations or by as yet unknown transcriptional mechanisms in the absence of 10q24 cytogenetic abnormalities. Almost all TLX1(+) T-ALLs exhibit a CD4(+)CD8(+) double-positive (DP) phenotype. To investigate the role of TLX1 as an initiating oncogene in T-ALL pathogenesis, we assessed the consequences of retroviral vector-directed TLX1 expression during the differentiation of murine and human thymocytes in fetal thymic organ cultures. Interestingly, enforced expression of TLX1 disrupted the differentiation of murine fetal liver precursors and human cord blood CD34(+) stem/progenitor cells prior to the DP thymocyte stage. Although differentiation arrest was associated with an increased percentage of apoptotic thymocytes, it could only be partially bypassed by coexpression of transgenic BCL2. Mutation of the invariant asparagine residue at position 51 of the homeodomain - which is required for efficient DNA binding - released the block, consistent with the notion that TLX1 inhibits thymocyte differentiation and promotes T-cell oncogenesis by functioning as a transcription factor. The relevance of these findings is discussed in the context of activating NOTCH1 mutations and the other genetic lesions implicated in the multistep transformation process of TLX1(+) T-ALL.

HOX and non-HOX homeobox genes in leukemic hematopoiesis.

Dysregulation of homeobox (HB)-containing genes is becoming increasingly recognized as the underlying basis of many hematologic malignancies. Expression of clustered HB (HOX) genes within the hematopoietic system, and enforced overexpression and knockout studies have provided support for the concept that these homeodomain-containing transcription factors play a significant role in the developmental biology of hematopoietic cells. Diverged HB (non-HOX) genes have recently been identified as either cofactors and/or accelerators of leukemic disease mediated by HOX genes or as bona fide oncogenes. In this review, we examine the evidence that supports a central role for HB genes in normal and malignant hematopoiesis, paying particular attention to the non-HOX class and the possible mechanisms through which they contribute to leukemic transformation.

Specific homeodomain-DNA interactions are required for HOX11-mediated transformation.

HOX11 encodes a homeodomain protein that is aberrantly expressed in T-cell acute lymphoblastic leukemia as a consequence of the t(10;14) and t(7;10) chromosomal translocations. We previously reported that HOX11 immortalizes murine hematopoietic progenitors and induces pre-T-cell tumors in mice after long latency. It has been demonstrated in a number of studies that HOX11, similar to other homeodomain proteins, binds DNA and transactivates transcription. These findings suggest that translocation-activated HOX11 functions as an oncogenic transcription factor. Here we report that HOX11 represses transcription through both TATA-containing and TATA-less promoters. Interestingly, transcriptional repression by HOX11 is independent of its DNA binding capability. Moreover, a systematic mutational analysis indicated that repressor activity was separable from immortalizing function, which requires certain residues within the HOX11 homeodomain that make base-specific or phosphate-backbone contacts with DNA. We further showed that the pathologic action of HOX11 involves DNA binding-dependent transcriptional pathways that are distinct from those controlling expression of a chromosomal target gene (Aldh-1). We conclude that dysregulated expression of a particular set of downstream target genes by DNA binding via the homeodomain is of central importance for leukemia initiation mediated by HOX11.

Enhancement of hematopoietic stem cell repopulating capacity and self-renewal in the absence of the transcription factor C/EBP alpha.

The transcription factor C/EBP alpha is required for granulopoiesis and frequently disrupted in human acute myeloid leukemia (AML). Here, we show disruption of C/EBP alpha blocks the transition from the common myeloid to the granulocyte/monocyte progenitor but is not required beyond this stage for terminal granulocyte maturation. C/EBP alpha-deficient hematopoietic stem cells (HSCs) have increased expression of Bmi-1 and enhanced competitive repopulating activity. Bone marrow in adult C/EBP alpha-deficient mice was filled with myeloblasts, similar to human AML, supporting the notion that disruption of C/EBP alpha cooperates with other events in the development of leukemia. Therefore, C/EBP alpha is not only essential for granulocyte development but, in addition, is a regulator of hematopoietic stem cell activity.