Nancy Mackay

Runx2 : A novel oncogenic effector revealed by in vivo complementation and retroviral tagging

The Runx2 (Cbfa1, Pebp2αA, Aml3) gene was previously identified as a frequent target for transcriptional activation by proviral insertion in T-cell lymphomas of CD2-MYC transgenic mice. We have recently shown that over-expression of the full-length, most highly expressed Runx2 isoform in the thymus perturbs T-cell development, leads to development of spontaneous lymphomas at low frequency and is strongly synergistic with Myc. To gain further insight into the relationship of Runx2 to other lymphomagenic pathways, we tested the effect of combining the CD2-Runx2 transgene either with a Pim1 transgene (Eμ-Pim1) or with the p53 null genotype, as each of these displays independent synergy with Myc. In both cases we observed synergistic tumour development. However, Runx2 appeared to have a dominant effect on the tumour phenotype in each case, with most tumours conforming to the CD3+, CD8+, CD4+/− phenotype seen in CD2-Runx2 mice. Neonatal infection of CD2-Runx2 mice with Moloney murine leukaemia virus (Moloney MLV) also led to a dramatic acceleration of tumour onset. Analysis of known Moloney MLV target genes in these lymphomas showed a high frequency of rearrangement at c-Myc or N-Myc (82%), and a significant number at Pim1 or Pim2 (23%), and at Pal1/Gfi1 (18%). These results indicate that Runx2 makes a distinct contribution to T-cell lymphoma development which does not coincide with any of the oncogene complementation groups previously identified by retroviral tagging.

The Runx genes as dominant oncogenes

We have shown previously that Runx2 is a frequent target (approximately equal to 30%) for proviral insertion in murine leukemia virus (MLV) induced T cell tumors in CD2-MYC transgenic mice. Further investigation of a large panel of these tumors revealed that a small number also contain insertions at either Runx3 or Runx1. None of the tumors contained insertions at more than one family member, but in each case proviral insertion was associated with a high level of expression from the upstream (P1) promoter of the respective target gene. Moreover, we confirmed that transcriptional activation of Runx1 does not affect the integrity of the coding sequence, as previously observed for Runx2. These observations suggest that the three Runx genes act as functionally redundant oncogenes in T-cell lymphoma development. To explore the oncogenic potential of Runx2 further we created transgenic mice that over-express this gene in the T cell compartment. These CD2-Runx2 animals show a preneoplastic enlargement of the CD8 immature single positive (ISP) thymocyte pool and develop lymphomas at a low incidence. Although the CD8 ISP population is greatly increased, unlike their wild type counterparts these cells are largely non-cycling. Co-expression of c-MYC in this lineage accentuates the CD8 ISP skew and induces rapid tumor development, confirming the potent synergy that exists between these two oncogenes. Experiments designed to understand the nature of the observed synergy are ongoing and are based on the hypothesis that Runx2 may exert a survival effect in c-MYC expressing tumors in vivo while c-MYC may rescue cells from the antiproliferative effects of Runx2. The oncogenic potential of Runx1 is also being assessed using primary murine embryonic fibroblasts (MEFs). These studies have revealed that while Runx1 exerts a growth suppressive effect in wild type cells a growth promoting effect is seen in the absence of p53, suggesting that the Runx genes may harbor latent oncogene-like properties.

The common retroviral insertion locus Dsi1 maps 30 kilobases upstream of the P1 promoter of the murine Runx3/Cbfa3/Aml2 gene.

ABSTRACT The Dsi1 locus was identified as a common integration site for Moloney murine leukemia virus (MLV) in rat thymic lymphomas, but previous efforts to identify a gene affected by these insertions were unsuccessful. We considered the Runx3 gene a potential candidate on the basis of genetic mapping which showed that Dsi1 and Runx3 are closely linked on mouse chromosome 4 and the precedent of the related Runx2 gene, which emerged recently as a Myc-collaborating gene activated by retroviral insertion in thymic lymphomas of CD2-MYC mice. We now report the physical mapping of the Dsi1 locus to a site 30 kb upstream of the distal (P1) promoter of the murine Runx3 gene. Comparison with the syntenic region of human chromosome 1 shows that the next gene is over 250 kb 5′ to Runx3, suggesting that Runx3 may be the primary target of retroviral insertions at Dsi1. Screening of CD2-MYC lymphomas for rearrangements at Dsi1 revealed a tumor cell line harboring an MLV provirus at this locus, in the orientation opposite that of Runx3. Proviral insertion was associated with very high levels of expression of Runx3, with a preponderance of transcripts arising at the P1 promoter. These results confirm that Runx3 is a target of retroviral insertions at Dsi1 and indicate that Runx3 can act as an alternative to Runx2 as a Myc-collaborating gene in thymic lymphoma.

Vaccination with Inactivated Virus but Not Viral DNA Reduces Virus Load following Challenge with a Heterologous and Virulent Isolate of Feline Immunodeficiency Virus

ABSTRACT It has been shown that cats can be protected against infection with the prototypic Petaluma strain of feline immunodeficiency virus (FIVPET) using vaccines based on either inactivated virus particles or replication-defective proviral DNA. However, the utility of such vaccines in the field is uncertain, given the absence of consistent protection against antigenically distinct strains and the concern that the Petaluma strain may be an unrepresentative, attenuated isolate. Since reduction of viral pathogenicity and dissemination may be useful outcomes of vaccination, even in the absence of complete protection, we tested whether either of these vaccine strategies ameliorates the early course of infection following challenge with heterologous and more virulent isolates. We now report that an inactivated virus vaccine, which generates high levels of virus neutralizing antibodies, confers reduced virus loads following challenge with two heterologous isolates, FIVAM6 and FIVGL8. This vaccine also prevented the marked early decline in CD4/CD8 ratio seen in FIVGL8-infected cats. In contrast, DNA vaccines based on either FIVPET or FIVGL8, which induce cell-mediated responses but no detectable antiviral antibodies, protected a fraction of cats against infection with FIVPET but had no measurable effect on virus load when the infecting virus was FIVGL8. These results indicate that the more virulent FIVGL8 is intrinsically more resistant to vaccinal immunity than the FIVPET strain and that a broad spectrum of responses which includes virus neutralizing antibodies is a desirable goal for lentivirus vaccine development.

Insertional mutagenesis reveals progression genes and checkpoints in MYC/Runx2 lymphomas

In this study, we have exploited the power of insertional mutagenesis to elucidate tumor progression pathways in mice carrying two oncogenes (MYC/Runx2) that collaborate to drive early lymphoma development. Neonatal infection of these mice with Moloney murine leukemia virus resulted in accelerated tumor onset with associated increases in clonal complexity and lymphoid dissemination. Large-scale analysis of retroviral integration sites in these tumors revealed a profound bias towards a narrow range of target genes, including Jdp2 (Jundm2), D cyclin, and Pim family genes. Remarkably, direct PCR analysis of integration hotspots revealed that every progressing tumor consisted of multiple clones harboring hits at these loci, giving access to large numbers of independent insertion events and uncovering the contrasting mutagenic mechanisms operating at each target gene. Direct PCR analysis showed that high-frequency targeting occurs only in the tumor environment in vivo and is specific for the progression gene set. These results indicate that early lymphomas in MYC/Runx2 mice remain dependent on exogenous growth signals, and that progression can be achieved by constitutive activation of pathways converging on a cell cycle checkpoint that acts as the major rate-limiting step for lymphoma outgrowth.

Runx1 promotes B-cell survival and lymphoma development.

Runx1 is essential for the homeostatic control of normal hematopoiesis and is required for lymphoid development. Translocations or point mutations that result in RUNX1 loss or disrupted function predispose to leukemia but data derived from model systems suggests that Runx genes can also be pro-oncogenic. Here we investigate the effects of enforced Runx1 expression in lymphoid lineages both in vivo and in vitro and show that transgene expression enhanced cell survival in the thymus and bone marrow but strongly inhibited the expansion of hematopoietic and B cell progenitors in vitro. Despite this, modestly enhanced levels of Runx1 accelerated Myc-induced lymphomagenesis in both the B cell and T cell lineages. Together these data provide formal proof that wild type Runx1 can promote oncogenesis in lymphoid tissues and that, in addition to loss of function, gain of function may have an aetiological role in leukemia.

Protection against feline immunodeficiency virus using replication defective proviral DNA vaccines with feline interleukin-12 and -18.

A molecular clone of the Glasgow-8 isolate of FIV (FIVGL8) was rendered replication defective by an in-frame deletion in either reverse transcriptase (deltaRT) or integrase (deltaIN) genes for use as DNA vaccines. To test the ability of these multi-gene vaccines to protect against two feline immunodeficiency virus (FIV) isolates of differing virulence, cats were immunized using either DNA vaccine alone or co-administered with interleukin-12 (IL-12) and/or interleukin-18 (IL-18) cytokine DNA. Animals were challenged sequentially with FIV-Petaluma (FIVPET) an FIV isolate of relatively low virulence and subsequently with the more virulent FIVGL8. A proportion of vaccinates (5/18 deltaIN and 2/12 deltaRT) were protected against primary challenge with FIV(PET). Five of the vaccinated-protected cats were re-challenged with FIV(PET); four (all deltaIN) remained free of viraemia whilst all naive controls became viraemic. Following subsequent challenge with the more virulent FIVGL8 these four vaccinated-protected animals all became viraemic but showed lower proviral loads than naive cats. This study suggests that while our current DNA vaccines may not produce sterilizing immunity against more virulent isolates of FIV, they may nevertheless significantly reduce the impact of infection.

Immunogenicity of a peptide from a major neutralising determinant of the feline immunodeficiency virus surface glycoprotein

The third variable region (V3) of the feline immunodeficiency virus (FIV) surface glycoprotein is predicted to have similar physical properties to that of HIV and has been shown to contain immunodominant and neutralizing epitopes. Immunological characteristics of this region were investigated further using a peptide corresponding to the middle of the putative FIV V3 loop. The peptide was recognized in ELISA by sera from the majority of naturally FIV-infected cats, and absorbed a significant fraction of the virus neutralizing activity from a pool of sera of cats naturally infected with FIV, confirming the immunogenic nature of this region. A sheep immunized with an octameric form of the peptide (multiple antigenic peptide; MAP) in Freunds complete adjuvant generated neutralizing antibody to a higher titre than infected cats. However, immunization of cats with the same MAP in an acceptable adjuvant formulation (Quil A) induced antibody and cytotoxic T-cell responses to the immunizing peptides but only minimal neutralizing activity. These responses did not significantly alter the kinetics of infection or the proviral load after challenge with a homologous strain of FIV, compared with naive controls. While the potential efficacy of peptide vaccines to lentiviruses remains to be determined, this study shows that the immune response evoked may be highly dependent on the delivery and adjuvant regime used.

Insertional mutagenesis and deep profiling reveals gene hierarchies and a Myc/p53-dependent bottleneck in lymphomagenesis.

Retroviral insertional mutagenesis (RIM) is a powerful tool for cancer genomics that was combined in this study with deep sequencing (RIM/DS) to facilitate a comprehensive analysis of lymphoma progression. Transgenic mice expressing two potent collaborating oncogenes in the germ line (CD2-MYC, -Runx2) develop rapid onset tumours that can be accelerated and rendered polyclonal by neonatal Moloney murine leukaemia virus (MoMLV) infection. RIM/DS analysis of 28 polyclonal lymphomas identified 771 common insertion sites (CISs) defining a ‘progression network’ that encompassed a remarkably large fraction of known MoMLV target genes, with further strong indications of oncogenic selection above the background of MoMLV integration preference. Progression driven by RIM was characterised as a Darwinian process of clonal competition engaging proliferation control networks downstream of cytokine and T-cell receptor signalling. Enhancer mode activation accounted for the most efficiently selected CIS target genes, including Ccr7 as the most prominent of a set of chemokine receptors driving paracrine growth stimulation and lymphoma dissemination. Another large target gene subset including candidate tumour suppressors was disrupted by intragenic insertions. A second RIM/DS screen comparing lymphomas of wild-type and parental transgenics showed that CD2-MYC tumours are virtually dependent on activation of Runx family genes in strong preference to other potent Myc collaborating genes (Gfi1, Notch1). Ikzf1 was identified as a novel collaborating gene for Runx2 and illustrated the interface between integration preference and oncogenic selection. Lymphoma target genes for MoMLV can be classified into (a) a small set of master regulators that confer self-renewal; overcoming p53 and other failsafe pathways and (b) a large group of progression genes that control autonomous proliferation in transformed cells. These findings provide insights into retroviral biology, human cancer genetics and the safety of vector-mediated gene therapy.

Addiction to Runx1 is partially attenuated by loss of p53 in the Eµ-Myc lymphoma model

The Runx genes function as dominant oncogenes that collaborate potently with Myc or loss of p53 to induce lymphoma when over-expressed. Here we examined the requirement for basal Runx1 activity for tumor maintenance in the Eμ-Myc model of Burkitts lymphoma. While normal Runx1fl/fl lymphoid cells permit mono-allelic deletion, primary Eμ-Myc lymphomas showed selection for retention of both alleles and attempts to enforce deletion in vivo led to compensatory expansion of p53null blasts retaining Runx1. Surprisingly, Runx1 could be excised completely from established Eμ-Myc lymphoma cell lines in vitro without obvious effects on cell phenotype. Established lines lacked functional p53, and were sensitive to death induced by introduction of a temperature-sensitive p53 (Val135) allele. Transcriptome analysis of Runx1-deleted cells revealed a gene signature associated with lymphoid proliferation, survival and differentiation, and included strong de-repression of recombination-activating (Rag) genes, an observation that was mirrored in a panel of human acute leukemias where RUNX1 and RAG1,2 mRNA expression were negatively correlated. Notably, despite their continued growth and tumorigenic potential, Runx1null lymphoma cells displayed impaired proliferation and markedly increased sensitivity to DNA damage and dexamethasone-induced apoptosis, validating Runx1 function as a potential therapeutic target in Myc-driven lymphomas regardless of their p53 status.