The t(8;21)(q22;q22) translocation, occurring in 40% of patients with acute myeloid leukemia (AML) of the FAB-M2 subtype (AML with maturation), results in expression of the RUNX1-CBF2T1 [AML1-ETO (AE)] fusion oncogene. AML͞ETO may contribute to leukemogenesis by interacting with nuclear corepressor complexes that include histone deacetylases, which mediate the repression of target genes. However, expression of AE is not sufficient to transform primary hematopoietic cells or cause disease in animals, suggesting that additional mutations are required. Activating mutations in receptor tyrosine kinases (RTK) are present in at least 30% of patients with AML. To test the hypothesis that activating RTK mutations cooperate with AE to cause leukemia, we transplanted retrovirally transduced murine bone marrow coexpressing TEL-PDGFRB and AE into lethally irradiated syngeneic mice. These mice (19͞19, 100%) developed AML resembling M2-AML that was transplantable in secondary recipients. In contrast, control mice coexpressing with TEL-PDGFRB and a DNA-binding-mutant of AE developed a nontransplantable myeloproliferative disease identical to that induced by TEL-PDGFRB alone. We used this unique model of AML to test the efficacy of pharmacological inhibition of histone deacetylase activity by using trichostatin A and suberoylanilide hydroxamic acid alone or in combination with the tyrosine kinase inhibitor, imatinib mesylate. We found that although imatinib prolonged the survival of treated mice, histone deacetylase inhibitors provided no additional survival benefit. These data demonstrate that an activated RTK can cooperate with AE to cause AML in mice, and that this system can be used to evaluate novel therapeutic strategies.T he t(8;21)(q22;q22) translocation, which fuses the RUNX1 (AML1͞PEBP␣͞CBFA2) gene on chromosome 21 with the ETO (MTG8) gene on chromosome 8, is a common mutation associated with cases of acute myeloid leukemia (AML) of the FAB-M2 subtype (AML with maturation) (1). Expression of the resulting AML1-ETO (AE) fusion gene is detected in 40% of M2-AML patients and 12% of all newly diagnosed cases of AML (2, 3). The correlation between AE expression and the leukemic phenotype strongly suggests a causative role for AE in transformation. AE transcripts have been detected in nonneoplastic progenitors from AML patients in remission, suggesting that the translocation is an early event in the leukemogenic process (4). Furthermore, t(8;21) translocation and AE expression can be detected in neonatal Guthrie blood spots, implying an in utero origin of the translocation preceding development of AML in children by as much as 10 years (5,6).Several murine models have demonstrated that AE alone is not sufficient to induce leukemia. Mice expressing an inducible AE transgene in bone marrow cells remained disease-free for a normal life span of 24 mo (7). When expression of AE was targeted to the myeloid lineage by using the human MRP8 promoter, again the mice had no discernable phenotype (8). However, when additional random mu...

Acute promyelocytic leukemia (APML) most often is associated with the balanced reciprocal translocation t(15;17) (q22;q11.2) and the expression of both the PML-RAR␣ and RAR␣-PML fusion cDNAs that are formed by this translocation. In this report, we investigated the biological role of a bcr-3 isoform of RAR␣-PML for the development of APML in a transgenic mouse model. Expression of RAR␣-PML alone in the early myeloid cells of transgenic mice did not alter myeloid development or cause APML, but its expression significantly increased the penetrance of APML in mice expressing a bcr-1 isoform of PML-RAR␣ (15% of animals developed APML with PML-RAR␣ alone vs. 57% with both transgenes, P < 0.001). The latency of APML development was not altered substantially by the expression of RAR␣-PML, suggesting that it does not behave as a classical ''second hit'' for development of the disease. Leukemias that arose from doubly transgenic mice were less mature than those from PML-RAR␣ transgenic mice, but they both responded to all-trans retinoic acid in vitro. These findings suggest that PML-RAR␣ drives the development of APML and defines its basic phenotype, whereas RAR␣-PML potentiates this phenotype via mechanisms that are not yet understood.A cute promyelocytic leukemia (APML, or AML M3, based on the French-American-British classification system) comprises about 10% of all new cases of AML. This disease is characterized by the accumulation of promyelocytes in the marrow and the peripheral blood, and a predisposition for bleeding diatheses (reviewed in refs. 1 and 2). The most common genetic abnormality associated with APML is the balanced t(15;17) (q22;q11.2) reciprocal translocation that generates PML-retinoic acid receptor ␣ (RAR␣) and RAR␣-PML fusion cDNAs (1). Although it is common to detect expression of both PML-RAR␣ and RAR␣-PML mRNAs in primary APML cells, the direct link between PML-RAR␣ expression and the development of APML was not demonstrated formally until transgenic expression of PML-RAR␣ in early myeloid cells was shown to cause the development of APML in transgenic mouse models (3-5).The first-generation mouse models demonstrated that expression of PML-RAR␣ in early myeloid cells altered myeloid development in all mice and caused 15-20% of mice to develop a fatal APML-like disease after a latency of 6-18 months (3-5). These findings suggested that PML-RAR␣ directly alters myeloid development and, in doing so, predisposes early myeloid cells to acquire additional mutations (second or subsequent hits) that ultimately cause transformation. The nature of these additional mutations is not yet known, although many patients with APML express the reciprocal RAR␣-PML cDNA, and a significant fraction have loss-of-function mutations in the p53 gene as well (6). The role of the reciprocal fusion for the development of APML has not been addressed previously in transgenic systems, probably because this fusion cDNA is small, containing only a few putative functional domains.Expression analyses of patients with t(15;17) (q22...

Matrix metalloproteinases (MMP) have been implicated in virtually all aspects of tumor progression. However, the recent failure of clinical trials employing synthetic MMP inhibitors in cancer chemotherapy has led us to hypothesize that some MMPs may actually serve the host in its defense against tumor progression. Here we show that mice deficient in macrophage elastase (MMP-12) develop significantly more gross Lewis lung carcinoma pulmonary metastases than their wild-type counterparts both in spontaneous and experimental metastasis models. The numbers of micrometastases between the two groups are equivalent; thus, it seems that MMP-12 affects lung tumor growth, and not metastasis formation, per se. MMP-12 is solely macrophage derived in this model, being expressed by tumor-associated macrophages and not by tumor or stromal cells. The presence of MMP-12 is associated with decreased tumor-associated microvessel density in vivo and generates an angiostatic>angiogenic tumor microenvironment that retards lung tumor growth independent of the production of angiostatin. These data define a role for MMP-12 in suppressing the growth of lung metastases and suggest that inhibitors designed to specifically target tumor-promoting MMPs may yet prove effective as cancer therapeutics. (Cancer Res 2006; 66(12): 6149-55)

Acute promyelocytic leukemia (APML) is characterized by abnormal myeloid development, resulting an accumulation of leukemic promyelocytes that are often highly sensitive to retinoic acid. A balanced t(15; 17) (q22; q21) reciprocal chromosomal translocation is found in approximately 90% of APML patients; this translocation fuses the PML gene on chromosome 15 to the retinoic acid receptor α (RARα) gene on chromosome 17, creating two novel fusion genes, PML-RARα and RARα-PML. The PML-RARα fusion gene product, which is expressed in virtually all patients with t(15; 17), is thought to play a direct role in the pathogenesis of APML. To determine whether PML-RARα is sufficient to cause APML in an animal model, we used the promyelocyte-specific targeting sequences of the human cathepsin G (hCG) gene to direct the expression of a PML-RARα cDNA to the early myeloid cells of transgenic mice. Mice expressing the hCG–PML-RARα transgene were found to have altered myeloid development that was characterized by increased percentages of immature and mature myeloid cells in the peripheral blood, bone marrow, and spleen. In addition, approximately 30% of transgene-expressing mice eventually developed acute myeloid leukemia after a long latent period. The splenic promyelocytes of mice with both the nonleukemic and leukemic phenotypes responded to all-trans retinoic acid (ATRA) treatment, which caused apoptosis of myeloid precursors. Although low-level expression of the hCG–PML-RARα transgene is not sufficient to directly cause acute myeloid leukemia in mice, its expression alters myeloid development, resulting in an accumulation of myeloid precursors that may be susceptible to cooperative transforming events.

The human cathepsin G (CG) gene is expressed only in promyelocytes and encodes a neutral serine protease that is packaged in the azurophil (primary) granules of myeloid cells. To define the cis-acting DNA elements that are responsible for promyelocyte-speciflc "targeting," we nijected a 6-kb transgene containing the entire human CG gene, including coding sequences contained in a 2.7-kb region, -2.5 kb of 5' flanking sequence, and "0.8 kb of 3' flanking sequence. Seven of seven "transient transgenic" murine embryos revealed human CG expression in the fetal livers at embryonic day 15. Stable trangenic founder lines were created with the same 6-kb fragment; four of five founder lines expressed human CG in the bone marrow. The level of human CG expression was relatively low per gene copy when compared with the endogenous murine CG gene, and expression was integration-site dependent; however, the level of gene expression correlated roughly with gene copy number. The human CG transgene and the endogenous murine CG gene were coordinately expressed in the bone marrow and the spleen. Immunohistochemical analysis of trngenic bone marrow revealed that the human CG protein was expressed exclusively in myeloid cells. Expression of human CG protein was highest in myeloid precursors and declined in mature myelold cells.These data suggest that the human CG gene was appropriately targeted and developmentally regulated, demonstrating that the cis-acting DNA sequences required for the early myeloid cell-specific expression of human CG are present in this small genomic fragment.

The TEL-PDGFRB fusion oncogene is associated with chronic myelomonocytic leukemia (CMML) and results in the expression of a constitutively active tyrosine kinase. SU11657 is a multitargeted selective inhibitor of class III/V receptor tyrosine kinases, including the plateletderived growth factor (PDGF) and vascular endothelial growth factor (VEGF) receptors KIT and FLT3. SU11657 inhibited TEL/PDGF␤R kinase activity at nanomolar concentrations and inhibited TEL-PDGFRB-mediated factor-independent growth in myeloblastic 32D cells. Daily oral administration of SU11657 at 40 mg/kg suppressed myeloproliferation and significantly prolonged survival in TEL-PDGFRB mice treated prior to disease development, as well as in those with large tumor burdens. Our findings suggest that SU11657 or similar agents may have therapeutic potential in humans with hematologic malignancies expressing PDGFR fusion oncogenes. (Blood.

The human CGL-1/cytotoxic serine protease B gene (CSP-B; also known as granzyme B) is transcriptionally activated during cytotoxic T- lymphocyte maturation. Activation can be mimicked in the PEER T-cell leukemia cell line by treatment with 12-O-tetradecanoylphorbol-13- acetate (TPA) and N6–2′-O-dibutyryladenosine 3′,5′-cyclic monophosphate (bt2cAMP). In this report, we show that a consensus AP-1 element and a consensus cAMP response element (CRE) located 5′ to the CSP-B transcriptional start site are both required for transcriptional activation of the CPS-B promoter in TPA + bt2cAMP-stimulated PEER cells. A 94-bp fragment containing both elements activates a heterologous promoter in an orientation-independent fashion. Several single nucleotide substitutions in the AP-1 site abolish activity of the 94-bp fragment. Several point mutations in the consensus CRE substantially reduce promoter activity, but one CRE mutation increases activity fourfold. Replacement of the CRE with a second copy of the AP- 1 site results in a level of transcriptional activity comparable with that of the wild-type sequence, but replacement of the AP-1 site with a CRE abolishes activity. Neither the AP-1 site nor the CRE can be effectively replaced with an SP-1 site. Deletions between the AP-1 site and the CRE retain full activity only if helical spacing is preserved, suggesting that synergism between these two elements is either the result of cooperative binding of factors to the DNA or of cooperative binding of DNA-bound factors to another protein.

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