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We have examined the possible role of neu differentiation factor (NDF) in mammary tumongenesis using transgenic mice. NDF is a ligand for ErbB4, a member of the ErbB family of tyrosine kinase receptors, of which two other members (EGFR and NEU) have been implicated in human breast cancer. Transgenic mice expressing a chimeric ligand having an NDFb-derived receptor binding domain within a TGFa backbone under an MMTV promote developed Harderian gland hyperplasias at high frequency. One mouse of 18 founders developed mammary carcinoma. Neither phenotype was transmissible to offspring. Other constructs, including NDFb2A, and mutant derived from that isoform, failed to express at significant levels in transgenic mice, precluding further analysis. In a separate Task, we attempted to identify genes that cooperate with c-neu in mammary tumorigenesis by proviral tagging using MMTV that is passed from female C3H mice to offspring via their milk. However, the strain of C3H that we used has experienced phenotypic drift since its original derivation, and was found to have a very low incidence of mammary carcinoma for reasons that are not entrely clear. We did find evidence suggesting the presence of modifier loci in the FVB and/or C3H strains that influence the latency of neu-induced mammary tumors.

The neurofibromatosis (NF1) gene shows significant homology to mammalian GAP and is an important regulator of the ras signal transduction pathway. To study the function of NF1 in normal development and to try and develop a mouse model of NF1 disease, we have used gene targeting in ES cells to generate mice carrying a null mutation at the mouse Nf1 locus. Although heterozygous mutant mice, aged up to 10 months, have not exhibited any obvious abnormalities, homozygous mutant embryos die in utero. Embryonic death is likely attributable to a severe malformation of the heart. Interestingly, mutant embryos also display hyperplasia of neural crest-derived sympathetic ganglia. These results identify new roles for NF1 in development and indicate that some of the abnormal growth phenomena observed in NF1 patients can be recapitulated in neurofibromin-deficient mice

Magnetic resonance imaging was used to compare male subjects seropositive for antibody to human immunodeficiency virus type 1 (HIV positive), with and without medical symptoms, with two groups of men who were seronegative (HIV negative). The control subjects included men at high risk for exposure to HIV-1 and those at low risk. None of the HIV-positive subjects met criteria for HIV-associated dementia or had detectable opportunistic brain disease. Quantitative image-analytic techniques were used to estimate volumes of ventricular and cortical cerebrospinal fluid, cerebral white matter, and cortical and subcortical gray matter structures. Relative to low-risk group control subjects and asymptomatic HIV-positive subjects, nondemented but medically symptomatic HIV-positive subjects showed significant increases in cerebrospinal fluid, reduced volume of cerebral white matter, and reduced cerebral gray matter volumes. Unexpectedly, however, some cerebrospinal fluid increases and gray matter volume decreases were present in the seronegative high-risk control subjects as well.

myc genes are thought to function in the processes of cellular proliferation and differentiation. To gain insight into the role of the N-myc gene during embryogenesis, we examined its expression in embryos during postimplantation development using RNA in situ hybridization. Tissue- and cell-specific patterns of expression unique to N-myc as compared with the related c-myc gene were observed. N-myc transcripts become progressively restricted to specific cell types, primarily to epithelial tissues including those of the developing nervous system and those in developing organs characterized by epithelio-mesenchymal interaction. In contrast, c-myc transcripts were confined to the mesenchymal compartments. These data suggest that c-myc and N-myc proteins may interact with different substrates in performing their function during embryogenesis and suggest further that there are linked regulatory mechanisms for normal expression in the embryo. We have mutated the N-myc locus via homologous recombination in embryonic stem (ES) cells and introduced the mutated allele into the mouse germ line. Live-born heterozygotes are under-represented but appear normal. Homozygous mutant embryos die prenatally at approximately 11.5 days of gestation. Histologic examination of homozygous mutant embryos indicates that several developing organs are affected. These include the central and peripheral nervous systems, mesonephros, lung, and gut. Thus, N-myc function is required during embryogenesis, and the pathology observed is consistent with the normal pattern of N-myc expression. Examination of c-myc expression in mutant embryos indicates the existence of coordinate regulation of myc genes during mouse embryogenesis.

Abstract 3652 Poster Board III-588 SRF is a MADS-box transcription factor first identified as an inducer of immediate-early gene expression, such as c-fos, Junb, Fosb and Egr1, in response to cytokines. It plays a critical role in cardiac and smooth muscle development and differentiation by regulating cell cycle, apoptosis, cell growth, and differentiation. SRF regulates expression of contractile and cytoskeletal genes. Its function in hematopoiesis has not yet been revealed. While no hematopoietic disease specific mutations of SRF have been identified, the region on chromosome 6 where the SRF gene is located, is a site of frequent chromosomal aberrations in MDS. MKL1, a target of the t(1;22) translocation in acute megakaryoblastic leukemia, is a cofactor for SRF mediated gene activation. In published work (Cheng EC et al., Blood 2009;113:2826), we showed that MKL1 expression increases with normal megakaryocytic (Mk) differentiation, that it promotes Mk differentiation by increasing the expression of Mk specific genes such as CD42b, and that mice lacking MKL1 have decreased platelet counts while showing increased numbers of low ploidy Mk in the BM. In addition, we showed that the effects of MKL1 on Mk differentiation require SRF. In order to test the role of SRF in Mk development, we crossed PF4-Cre mice (Tiedt R et al., Blood 2007;109:1503), which express Cre recombinase in cells committed to the Mk lineage, to SRFF/F mice (Miano JM et al., PNAS 2004;101:17132) in which functional SRF is no longer expressed after Cre-mediated excision. Our findings are quite surprising, as knockout of SRF in the Mk lineage leads to a far more profound phenotype than MKL1 KO. PF4-Cre x SRFF/F (PF4-Cre/SRF) mice are born with a normal mendelian frequency, but have significant macrothrombocytopenia. The platelet counts in WT and KO mice are 703 ± 33 × 103/μl vs 460 ± 23 × 103/μl, respectively. Despite the decreased platelet number, the BM has increased numbers and percentages of CD41+ Mk (WT: 0.41 ± 0.06% and KO: 1.92 ± 0.12%) with a predominance of Mk with hypolobated nuclei. Spleens of PF4-Cre/SRF mice show significantly increased numbers of Mk (mean of 4 and 15 Mk per high power field for WT and KO spleens, respectively). Ploidy as an assay of Mk maturation in the BM is significantly reduced in SRF −/− Mk. The percentage of CD41+ cells in SRF KO BM that is 2N is nearly 2-fold higher, and the percentage with greater than 8N ploidy is decreased by 70%. Hematopoietic stem and progenitor populations in PF4-Cre/SRF BM are unaffected, as expected due to stage and lineage specific knockout of SRF. In contrast, there is an increase in Mk progenitors in vivo in PF4-Cre/SRF mice, reflected both by FACS analysis and CFU-Mk in vitro analysis. The mechanism by which SRF disrupts Mk maturation was studied by assessment of Mk morphology, and gene expression analysis. Mk lacking SRF expression have abnormal stress fiber formation based on phalloidin staining, a phenotype parallel to that observed in mice lacking myosin heavy chain 9 (MYH9) expression, and MYH9 expression was decreased in SRF KO Mk. In summary, the data show that SRF is critical for normal Mk maturation including polyploidization and platelet production, and the mechanism by which loss of SRF disrupts megakaryocytopoiesis is, at least in part, by loss of normal expression of known targets of SRF, such as cytoskeletal genes including MYH9. Disclosures: No relevant conflicts of interest to declare.

The EVI1 gene at chromosome 3q26 is associated with acute myeloid leukemogenesis, due to both chromosomal rearrangement and to overexpression in the absence of rearrangement. Some rearrangements such as t(3;3) and inv(3) result in overexpression of EVI1 protein, while translocation t(3;21) yields an AML1-MDS1-EVI1 (AME) fusion protein. EVI1 possesses two zinc finger domains, an N-terminal domain with fingers 1–7, which binds to GACAAGATA, and a C-terminal domain (fingers 8–10) which binds GAAGATGAG. Inhibition of EVI1 function with a small molecule compound may provide a targeted therapy for EVI1-expressing leukemias. As a first step towards inhibiting the leukemogenic function of EVI1, we performed structure-function studies on both EVI1 and AME protein to determine what domains are critical for malignant transformation activity. Assays were Rat1 fibroblasts in a soft agar colony forming assay for EVI1; primary bone marrow cells in a serial replating assay for AME. Both assays revealed that mutation of arginine 205 in zinc finger 6 of EVI1, which completely abrogates sequencespecific DNA binding via the N-terminal zinc finger domain, resulted in complete loss of transforming activity; mutations in other domains, such as the C-terminal zinc finger domain, CtBP binding domain, and the domains of AML1 had less of an effect or no effect on transforming activity. In an effort to inhibit EVI1 leukemogenic function, we developed a polyamide, DH-IV-298, designed to block zinc fingers 1–7 binding to the GACAAGATA motif. DNAseI footprinting revealed a specific interaction between DH-IV-298 and the GACAAGATA motif; no significant interaction was observed elsewhere; a mismatch polyamide failed to footprint at equivalent concentrations; and DH-IV-298 failed to bind to a control DNA lacking the GACAAGATA motif. Electromobility shift assay showed that, at a 1:1 polyamide:DNA ratio, DH-IV-298 lowered EVI1:DNA affinity by over 98%, while mismatch was significantly less effective (74% reduction). To assess the effect of DH-IV-298 on EVI1 binding to DNA in vivo, we performed CAT reporter assays in a NIH-3T3-derived cell line with a chromosome-embedded tet-inducible EVI1-VP16 as well as a EVI1-responsive CAT reporter. Removal of tetracycline resulted in a four-fold increase in CAT activity that was completely blocked by DH-IV-298. The mismatch polyamide was significantly less effective than DH-IV-298. Further studies are being performed to assess the effect on endogenous gene expression, and on growth of leukemic cells that express EVI1. These studies provide evidence that a cell permeable small molecule compound may effectively block the activity of a leukemogenic transcription factor.

Retroviral insertion at the Evi1 locus causes high level of expression of the gene in myeloid neoplasms in mice and in nonmalignant expansions of myeloid cells in humans and monkeys. In these settings, it is suggested that EVI1 confers a survival advantage on myeloid cells. Here, we investigate the survival phenotype in DA-1 cells, a leukemic cell line with provirally activated Evi1. We report that short hairpin-mediated suppression of Evi1 in DA-1 cells induces apoptosis via the intrinsic/mitochondrial pathway: DNA fragmentation and histone release are both induced, as is reduction in mitochondrial membrane potential. In addition, procasapses 3 and 9, but not caspase 8 or Bid, are cleaved following Evi1 knockdown; phosphoAKT remains unchanged. Furthermore, mRNA expression profiling following Evi1 suppression show transcriptional changes in several apoptotic regulators, including a 3.5-fold decrease in Bcl2a1 (bfl/A1), a prosurvival member of the Bcl-2 family. To assess whether EVI1 regulates Bcl2a1 expression in a cell type other than DA-1 cells, we transduced primary murine Lin-/Sca-1+/c-Kit+ cells with EVI1 via retroviral vector, and then assessed the expression of Bcl2a1. This revealed that EVI1 induced a more than five-fold increase in Bcl2a1 mRNA expression, as measured by quantitative PCR. Furthermore, transduction of primary murine bone marrow cells with retroviruses bearing either Bcl2a1 or Evi1 resulted in a significant decrease in spontaneous apoptosis, as assessed by the activity of caspases 3 and 7. To further test if Bcl2a1 is necessary for leukemic transformation by Evi1, we assessed the ability of Evi1 to confer serial replating ability on primary bone marrow cells from Bcl2a1−/− mice. Bone marrow was harvested from Bcl2a1−/− and C57BL6 mice and transduced with retrovirus containing either no gene or Evi1. While in C57BL6 mice, Evi1 induced a significant increase in colonies, bone marrow from Bcl2a1−/− mice were resistant to transformation by Evi1. To show that this was due to the lack of Bcl2a1, the gene was added back via retrovirus. While Bcl2a1 by itself did not induce significant number of colonies over vector, when introduced into Bcl2a1−/− cells together with Evi1, there was a significant increase in colony formation. These data indicate that transformation of bone marrow cells by Evi1 depends on the presence of the Bcl2a1 gene. We further show EVI1 can transcriptionally upregulate a BCL2A1::luc reporter that harbors 1.37 kb of the human BCL2A1 upstream sequence. Our analysis of the Bcl2a1 promoter indicates that the effect of EVI1 is likely indirect. Based on our findings, we propose that EVI1 acts to block apoptosis in DA-1 cells by transcriptionally activating Bcl2a1.