IL-27 maintains cytotoxic Ly6C+ γδ T cells that arise from immature precursors.

In mice, γδ-T lymphocytes that express the co-stimulatory molecule, CD27, are committed to the IFNγ-producing lineage during thymic development. In the periphery, these cells play a critical role in host defense and anti-tumor immunity. Unlike αß-T cells that rely on MHC-presented peptides to drive their terminal differentiation, it is unclear whether MHC-unrestricted γδ-T cells undergo further functional maturation after exiting the thymus. Here, we provide evidence of phenotypic and functional diversity within peripheral IFNγ-producing γδ T cells. We found that CD27+ Ly6C- cells convert into CD27+Ly6C+ cells, and these CD27+Ly6C+ cells control cancer progression in mice, while the CD27+Ly6C- cells cannot. The gene signatures of these two subsets were highly analogous to human immature and mature γδ-T cells, indicative of conservation across species. We show that IL-27 supports the cytotoxic phenotype and function of mouse CD27+Ly6C+ cells and human Vδ2+ cells, while IL-27 is dispensable for mouse CD27+Ly6C- cell and human Vδ1+ cell functions. These data reveal increased complexity within IFNγ-producing γδ-T cells, comprising immature and terminally differentiated subsets, that offer new insights into unconventional T-cell biology.

Gut γδ T cells as guardians, disruptors, and instigators of cancer.

Colorectal cancer is the third most common cancer worldwide with nearly 2 million cases per year. Immune cells and inflammation are a critical component of colorectal cancer progression, and they are used as reliable prognostic indicators of patient outcome. With the growing appreciation for immunology in colorectal cancer, interest is growing on the role γδ T cells have to play, as they represent one of the most prominent immune cell populations in gut tissue. This group of cells consists of both resident populations-γδ intraepithelial lymphocytes (γδ IELs)-and transient populations that each has unique functions. The homeostatic role of these γδ T cell subsets is to maintain barrier integrity and prevent microorganisms from breaching the mucosal layer, which is accomplished through crosstalk with enterocytes and other immune cells. Recent years have seen a surge in discoveries regarding the regulation of γδ IELs in the intestine and the colon with particular new insights into the butyrophilin family. In this review, we discuss the development, specialities, and functions of γδ T cell subsets during cancer progression. We discuss how these cells may be used to predict patient outcome, as well as how to exploit their behavior for cancer immunotherapy.

Disrupting MLV integrase:BET protein interaction biases integration into quiescent chromatin and delays but does not eliminate tumor activation in a MYC/Runx2 mouse model.

Murine leukemia virus (MLV) integrase (IN) lacking the C-terminal tail peptide (TP) loses its interaction with the host bromodomain and extraterminal (BET) proteins and displays decreased integration at promoter/enhancers and transcriptional start sites/CpG islands. MLV lacking the IN TP via an altered open reading frame was used to infect tumorigenesis mouse model (MYC/Runx2) animals to observe integration patterns and phenotypic effects, but viral passage resulted in the restoration of the IN TP through small deletions. Mice subsequently infected with an MLV IN lacking the TP coding sequence (TP-) showed an improved median survival by 15 days compared to wild type (WT) MLV infection. Recombination with polytropic endogenous retrovirus (ERV), Pmv20, was identified in seven mice displaying both fast and slow tumorigenesis, highlighting the strong selection within the mouse to maintain the full-length IN protein. Mapping the genomic locations of MLV in tumors from an infected mouse with no observed recombination with ERVs, TP-16, showed fewer integrations at TSS and CpG islands, compared to integrations observed in WT tumors. However, this mouse succumbed to the tumor in relatively rapid fashion (34 days). Analysis of the top copy number integrants in the TP-16 tumor revealed their proximity to known MLV common insertion site genes while maintaining the MLV IN TP- genotype. Furthermore, integration mapping in K562 cells revealed an insertion preference of MLV IN TP- within chromatin profile states associated with weakly transcribed heterochromatin with fewer integrations at histone marks associated with BET proteins (H3K4me1/2/3, and H3K27Ac). While MLV IN TP- showed a decreased overall rate of tumorigenesis compared to WT virus in the MYC/Runx2 model, MLV integration still occurred at regions associated with oncogenic driver genes independently from the influence of BET proteins, either stochastically or through trans-complementation by functional endogenous Gag-Pol protein.

Collaboration of MYC and RUNX2 in lymphoma simulates T-cell receptor signaling and attenuates p53 pathway activity.

MYC and RUNX oncogenes each trigger p53-mediated failsafe responses when overexpressed in vitro and collaborate with p53 deficiency in vivo. However, together they drive rapid onset lymphoma without mutational loss of p53. This phenomenon was investigated further by transcriptomic analysis of premalignant thymus from RUNX2/MYC transgenic mice. The distinctive contributions of MYC and RUNX to transcriptional control were illustrated by differential enrichment of canonical binding sites and gene ontology analyses. Pathway analysis revealed signatures of MYC, CD3, and CD28 regulation indicative of activation and proliferation, but also strong inhibition of cell death pathways. In silico analysis of discordantly expressed genes revealed Tnfsrf8/CD30, Cish, and Il13 among relevant targets for sustained proliferation and survival. Although TP53 mRNA and protein levels were upregulated, its downstream targets in growth suppression and apoptosis were largely unperturbed. Analysis of genes encoding p53 posttranslational modifiers showed significant upregulation of three genes, Smyd2, Set, and Prmt5. Overexpression of SMYD2 was validated in vivo but the functional analysis was constrained by in vitro loss of p53 in RUNX2/MYC lymphoma cell lines. However, an early role is suggested by the ability of SMYD2 to block senescence-like growth arrest induced by RUNX overexpression in primary fibroblasts.

RUNX-mediated growth arrest and senescence are attenuated by diverse mechanisms in cells expressing RUNX1 fusion oncoproteins.

RUNX gene over-expression inhibits growth of primary cells but transforms cells with tumor suppressor defects, consistent with reported associations with tumor progression. In contrast, chromosomal translocations involving RUNX1 are detectable in utero, suggesting an initiating role in leukemias. How do cells expressing RUNX1 fusion oncoproteins evade RUNX-mediated growth suppression? Previous studies showed that the TEL-RUNX1 fusion from t(12;21) B-ALLs is unable to induce senescence-like growth arrest (SLGA) in primary fibroblasts while potent activity is displayed by the RUNX1-ETO fusion found in t(8;21) AMLs. We now show that SLGA potential is suppressed in TEL-RUNX1 but reactivated by deletion of the TEL HLH domain or mutation of a key residue (K99R). Attenuation of SLGA activity is also a feature of RUNX1-ETO9a, a minor product of t(8;21) translocations with increased leukemogenicity. Finally, while RUNX1-ETO induces SLGA it also drives a potent senescence-associated secretory phenotype (SASP), and promotes the immortalization of rare cells that escape SLGA. Moreover, the RUNX1-ETO SASP is not strictly linked to growth arrest as it is largely suppressed by RUNX1 and partially activated by RUNX1-ETO9a. These findings underline the heterogeneous nature of premature senescence and the multiple mechanisms by which this failsafe process is subverted in cells expressing RUNX1 oncoproteins.

Barriers to Infection of Human Cells by Feline Leukemia Virus: Insights into Resistance to Zoonosis.

The human genome displays a rich fossil record of past gammaretrovirus infections, yet no current epidemic is evident, despite environmental exposure to viruses that infect human cells in vitro Feline leukemia viruses (FeLVs) rank high on this list, but neither domestic nor workplace exposure has been associated with detectable serological responses. Nonspecific inactivation of gammaretroviruses by serum factors appears insufficient to explain these observations. To investigate further, we explored the susceptibilities of primary and established human cell lines to FeLV-B, the most likely zoonotic variant. Fully permissive infection was common in cancer-derived cell lines but was also a feature of nontransformed keratinocytes and lung fibroblasts. Cells of hematopoietic origin were generally less permissive and formed discrete groups on the basis of high or low intracellular protein expression and virion release. Potent repression was observed in primary human blood mononuclear cells and a subset of leukemia cell lines. However, the early steps of reverse transcription and integration appear to be unimpaired in nonpermissive cells. FeLV-B was subject to G→A hypermutation with a predominant APOBEC3G signature in partially permissive cells but was not mutated in permissive cells or in nonpermissive cells that block secondary viral spread. Distinct cellular barriers that protect primary human blood cells are likely to be important in protection against zoonotic infection with FeLV.IMPORTANCE Domestic exposure to gammaretroviruses such as feline leukemia viruses (FeLVs) occurs worldwide, but the basis of human resistance to infection remains incompletely understood. The potential threat is evident from the human genome sequence, which reveals many past epidemics of gammaretrovirus infection, and from recent cross-species jumps of gammaretroviruses from rodents to primates and marsupials. This study examined resistance to infection at the cellular level with the most prevalent human cell-tropic FeLV variant, FeLV-B. We found that blood cells are uniquely resistant to infection with FeLV-B due to the activity of cellular enzymes that mutate the viral genome. A second block, which appears to suppress viral gene expression after the viral genome has integrated into the host cell genome, was identified. Since cells derived from other normal human cell types are fully supportive of FeLV replication, innate resistance of blood cells could be critical in protecting against cross-species infection.

The RUNX Genes as Conditional Oncogenes: Insights from Retroviral Targeting and Mouse Models.

The observation that the Runx genes act as targets for transcriptional activation by retroviral insertion identified a new family of dominant oncogenes. However, it is now clear that Runx genes are 'conditional' oncogenes whose over-expression is growth inhibitory unless accompanied by another event such as concomitant over-expression of MYC or loss of p53 function. Remarkably, while the oncogenic activities of either MYC or RUNX over-expression are suppressed while p53 is intact, the combination of both neutralises p53 tumour suppression in vivo by as yet unknown mechanisms. Moreover, there is emerging evidence that endogenous, basal RUNX activity is important to maintain the viability and proliferation of MYC-driven lymphoma cells. There is also growing evidence that the human RUNX genes play a similar conditional oncogenic role and are selected for over-expression in end-stage cancers of multiple types. Paradoxically, reduced RUNX activity can also predispose to cell immortalisation and transformation, particularly by mutant Ras. These apparently conflicting observations may be reconciled in a stage-specific model of RUNX involvement in cancer. A question that has yet to be fully addressed is the extent to which the three Runx genes are functionally redundant in cancer promotion and suppression.

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.

Specialized Tfh cell subsets driving type-1 and type-2 humoral responses in lymphoid tissue.

Effective antibody responses are essential to generate protective humoral immunity. Different inflammatory signals polarize T cells towards appropriate effector phenotypes during an infection or immunization. Th1 and Th2 cells have been associated with the polarization of humoral responses. However, T follicular helper cells (Tfh) have a unique ability to access the B cell follicle and support the germinal center (GC) responses by providing B cell help. We investigated the specialization of Tfh cells induced under type-1 and type-2 conditions. We first studied homogenous Tfh cell populations generated by adoptively transferred TCR-transgenic T cells in mice immunized with type-1 and type-2 adjuvants. Using a machine learning approach, we established a gene expression signature that discriminates Tfh cells polarized towards type-1 and type-2 response, defined as Tfh1 and Tfh2 cells. The distinct signatures of Tfh1 and Tfh2 cells were validated against datasets of Tfh cells induced following lymphocytic choriomeningitis virus (LCMV) or helminth infection. We generated single-cell and spatial transcriptomics datasets to dissect the heterogeneity of Tfh cells and their localization under the two immunizing conditions. Besides a distinct specialization of GC Tfh cells under the two immunizations and in different regions of the lymph nodes, we found a population of Gzmk+ Tfh cells specific for type-1 conditions. In human individuals, we could equally identify CMV-specific Tfh cells that expressed Gzmk. Our results show that Tfh cells acquire a specialized function under distinct types of immune responses and with particular properties within the B cell follicle and the GC.

PD-1 and TIM-3 differentially regulate subsets of mouse IL-17A-producing γδ T cells.

IL-17A-producing γδ T cells in mice consist primarily of Vγ6+ tissue-resident cells and Vγ4+ circulating cells. How these γδ T cell subsets are regulated during homeostasis and cancer remains poorly understood. Using single-cell RNA sequencing and flow cytommetry, we show that lung Vγ4+ and Vγ6+ cells from tumor-free and tumor-bearing mice express contrasting cell surface molecules as well as distinct co-inhibitory molecules, which function to suppress their expansion. Vγ6+ cells express constitutively high levels of PD-1, whereas Vγ4+ cells upregulate TIM-3 in response to tumor-derived IL-1ß and IL-23. Inhibition of either PD-1 or TIM-3 in mammary tumor-bearing mice increased Vγ6+ and Vγ4+ cell numbers, respectively. We found that genetic deletion of γδ T cells elicits responsiveness to anti-PD-1 and anti-TIM-3 immunotherapy in a mammary tumor model that is refractory to T cell checkpoint inhibitors, indicating that IL-17A-producing γδ T cells instigate resistance to immunotherapy. Together, these data demonstrate how lung IL-17A-producing γδ T cell subsets are differentially controlled by PD-1 and TIM-3 in steady-state and cancer.

ß-Catenin Drives Butyrophilin-like Molecule Loss and γδ T-cell Exclusion in Colon Cancer.

Intraepithelial lymphocytes (IEL) expressing γδ T-cell receptors (γδTCR) play key roles in elimination of colon cancer. However, the precise mechanisms by which progressing cancer cells evade immunosurveillance by these innate T cells are unknown. Here, we investigated how loss of the Apc tumor suppressor in gut tissue could enable nascent cancer cells to escape immunosurveillance by cytotoxic γδIELs. In contrast with healthy intestinal or colonic tissue, we found that γδIELs were largely absent from the microenvironment of both mouse and human tumors, and that butyrophilin-like (BTNL) molecules, which can critically regulate γδIEL through direct γδTCR interactions, were also downregulated in tumors. We then demonstrated that ß-catenin activation through loss of Apc rapidly suppressed expression of the mRNA encoding the HNF4A and HNF4G transcription factors, preventing their binding to promoter regions of Btnl genes. Reexpression of BTNL1 and BTNL6 in cancer cells increased γδIEL survival and activation in coculture assays but failed to augment their cancer-killing ability in vitro or their recruitment to orthotopic tumors. However, inhibition of ß-catenin signaling via genetic deletion of Bcl9/Bcl9L in either Apc-deficient or mutant ß-catenin mouse models restored Hnf4a, Hnf4g, and Btnl gene expression and γδ T-cell infiltration into tumors. These observations highlight an immune-evasion mechanism specific to WNT-driven colon cancer cells that disrupts γδIEL immunosurveillance and furthers cancer progression.

NKG2D signaling regulates IL-17A-producing γδT cells in mice to promote cancer progression.

γδT cells are unconventional T cells particularly abundant in mucosal tissues that play an important role in tissue surveillance, homeostasis, and cancer. γδT cells recognize stressed cells or cancer cells through the NKG2D receptor to kill these cells and maintain normality. Contrary to the well-established anti-tumor function of these NKG2D-expressing γδT cells, we show here that, in mice, NKG2D regulates a population of pro-tumor γδT cells capable of producing IL-17A. Germline deletion of Klrk1, the gene encoding NKG2D, reduced the frequency of γδT cells in the tumor microenvironment and delayed tumor progression. We further show that blocking NKG2D reduced the capability of γδT cells to produce IL-17A in the pre-metastatic lung and that co-culture of lung T cells with NKG2D ligand-expressing tumor cells specifically increased the frequency of γδT cells. Together, these data support the hypothesis that, in a tumor microenvironment where NKG2D ligands are constitutively expressed, γδT cells accumulate in an NKG2D-dependent manner and drive tumor progression by secreting pro-inflammatory cytokines, such as IL-17A.

The MSP-RON axis stimulates cancer cell growth in models of triple negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with poor prognosis and high rates of relapse. The lack of actionable targets for TNBC has contributed to the high mortality rates of this disease, and new candidate molecules for potential manipulation are urgently required. Here, we show that macrophage-stimulating protein (MSP) and its tyrosine kinase receptor, Recepteur d'origine nantais (RON), are potent drivers of cancer cell growth and tumor progression in a mouse model of TNBC driven by the loss of Trp53 and Brca1. After comparison of two genetically engineered mouse models of TNBC, we found that mammary tumors from K14-Cre;Brca1F/F ;Trp53F/F (KB1P) mice exhibit high endogenous levels of MSP and RON expression. We show that MSP stimulates serine/threonine kinase 1 and extracellular regulated MAPK activation as well as cancer cell growth in cell lines derived from the two mouse models, while genetic and pharmacological inhibition of RON prevents these effects. Similarly, KB1P tumor progression in mice was robustly attenuated by treatment with a RON inhibitor with accompanied reduction in the proliferation marker, Ki-67. Analysis of human gene expression data confirmed that the genes encoding MSP and RON are robustly expressed in human TNBC as well as other subsets of breast cancer. Our findings uncover a mouse model where MSP expression and RON expression are naturally increased, and they provide evidence that this receptor and its ligand are viable candidate molecules for targeted treatment of breast cancer.

Conservation and expression of a novel alternatively spliced Evi1 exon.

The Evi1 transcriptional repressor protein is expressed in a developmentally regulated manner, is essential for normal development, participates in regulating cell proliferation and differentiation of cells of haemopoietic and neuronal origin and contributes to the progression of leukaemia. In this report we describe a new murine Evi1 gene transcript (Delta105) that contains two alternatively spliced regions encoding a 9 amino acid insertion (Rp+9) within the repressor domain (Rp) and a 105 amino acid C-terminal truncation. Abundant levels of the 105 amino acid truncated protein are observed in murine leukaemia cells. The combined primary sequence alterations do not affect the DNA binding, transcriptional repressor or CtBP2 protein binding properties of Evi1 but they do reduce its transforming and cell proliferation stimulating activities. Reduced transforming activity is most likely due to the C-terminal truncation as the activity of Evi1 containing either Rp or Rp+9 is indistinguishable. Both isoforms exist in all murine tissues and cell lines examined. However, only the Rp+9 alternative splice variant is also found in humans and other vertebrates. Murine and human forms of Evi1 with Rp or Rp+9 exist. The additional 9 amino acids are encoded by a conserved 27 nucleotide exon, the overall structural organisation of the gene being preserved in the two species. The function of the Rp+9 and Delta105 splice variants is unknown although the conservation of Rp+9 throughout evolution in vertebrate species suggests it is essential to the broad spectrum of biological activities attributed to this developmentally essential protein.

Proviral insertion indicates a dominant oncogenic role for Runx1/AML-1 in T-cell lymphoma.

The RUNX1/AML1 gene is a frequent target for chromosomal translocations in human leukemia. The biological properties of the resulting fusion products and the finding that haploinsufficiency increases the risk of developing leukemia (W-J. Song et al., Nat. Genet., 23: 166-175, 1999; M. Osata et al., Blood, 93: 1817-1824, 1999) have led to the widely held view that RUNX1 loss-of-function is a key event. However, we now report that the gene is a target for insertional mutagenesis in T-cell lymphomas of mice carrying a MYC oncogene, where promoter insertion results in overexpression without affecting the integrity of the coding sequence. Moreover, Runx1 haploinsufficiency does not accelerate lymphoma development in MYC/Runx2 transgenic or murine leukemia virus-infected mice. These findings reveal that the Runx1 gene can also act as a dominant oncogene and suggest that the involvement of the Runx gene family in human leukemia may be more widespread and complex than previously realized.

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 Burkitt's 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.

Gamma-Retrovirus Integration Marks Cell Type-Specific Cancer Genes: A Novel Profiling Tool in Cancer Genomics.

Retroviruses have been foundational in cancer research since early studies identified proto-oncogenes as targets for insertional mutagenesis. Integration of murine gamma-retroviruses into the host genome favours promoters and enhancers and entails interaction of viral integrase with host BET/bromodomain factors. We report that this integration pattern is conserved in feline leukaemia virus (FeLV), a gamma-retrovirus that infects many human cell types. Analysis of FeLV insertion sites in the MCF-7 mammary carcinoma cell line revealed strong bias towards active chromatin marks with no evidence of significant post-integration growth selection. The most prominent FeLV integration targets had little overlap with the most abundantly expressed transcripts, but were strongly enriched for annotated cancer genes. A meta-analysis based on several gamma-retrovirus integration profiling (GRIP) studies in human cells (CD34+, K562, HepG2) revealed a similar cancer gene bias but also remarkable cell-type specificity, with prominent exceptions including a universal integration hotspot at the long non-coding RNA MALAT1. Comparison of GRIP targets with databases of super-enhancers from the same cell lines showed that these have only limited overlap and that GRIP provides unique insights into the upstream drivers of cell growth. These observations elucidate the oncogenic potency of the gamma-retroviruses and support the wider application of GRIP to identify the genes and growth regulatory circuits that drive distinct cancer types.

RUNX1 transformation of primary embryonic fibroblasts is revealed in the absence of p53.

The mammalian Runx gene family (Runx1-3) are transcription factors that play essential, lineage-specific roles in development. A growing body of evidence implicates these genes as mutational targets in cancer where, in different contexts, individual family members have been reported to act as tumour suppressors, dominant oncogenes or mediators of metastasis. We are exploring these paradoxical observations by ectopic expression of RUNX genes in primary murine embryonic fibroblasts where, in common with a number of other dominant oncogenes, RUNX1 induces senescence-like growth arrest in the presence of an intact p19(ARF)-p53 pathway. We now report that, in MEFs lacking functional p53, RUNX1 has apparently pro-oncogenic effects on cell growth that include cytoskeletal reorganization, reduced contact inhibition at confluence and accelerated tumour expansion in vivo. On the other hand, RUNX1 conferred no obvious growth advantage at low cell density and actually delayed entry of primary MEFs into S phase. We also found that ectopic RUNX1 interferes with the morphological and growth responses of p53-null MEFs to TGFbeta indicating that these effects are mediated by overlapping pathways. These observations help to elucidate the context-dependent consequences of loss and gain of Runx activity.

Frequent infection of human cancer xenografts with murine endogenous retroviruses in vivo.

Infection of human cancer xenografts in mice with murine leukemia viruses (MLVs) is a long-standing observation, but the likelihood of infection in vivo and its biological consequences are poorly understood. We therefore conducted a prospective study in commonly used xenograft recipient strains. From BALB/c nude mice engrafted with MCF7 human mammary carcinoma cells, we isolated a virus that was virtually identical to Bxv1, a locus encoding replication-competent xenotropic MLV (XMLV). XMLV was detected in 9/17 (53%) independently isolated explants. XMLV was not found in primary leukemias or in THP1 leukemia cells grown in Bxv1-negative NSG (NOD/SCID/γCnull) mice, although MCF7 explants harbored replication-defective MLV proviruses. To assess the significance of infection for xenograft behavior in vivo, we examined changes in growth and global transcription in MCF7 and the highly susceptible Raji Burkitt lymphoma cell line chronically infected with XMLV. Raji cells showed a stronger transcriptional response that included up-regulation of chemokines and effectors of innate antiviral immunity. In conclusion, the risk of de novo XMLV infection of xenografts is high in Bxv1 positive mice, while infection can have positive or negative effects on xenograft growth potential with significant consequences for interpretation of many xenograft studies.

Conservation and expression of an alternative 3' exon of Runx2 encoding a novel proline-rich C-terminal domain.

The Runx2 (Cbfa1, Aml3, PEBP2alphaA) gene plays an essential role in bone development and is one of a three-member family of closely related genes that encode the alpha-chain DNA binding components of the heterodimeric core binding factor complex. While all three mammalian Runx genes share a complex dual promoter structure (P1, P2) and display alternative splicing, a distinctive feature of Runx2 is the potential to encode larger isoforms in which the C-terminal domain encoded by the standard 3' terminal exon (exon 6) is replaced by an extended 200-201 amino acid C-terminal sequence including an extensive proline-rich domain and a C-terminal amphipathic helix. We report that the novel exon that gives rise to these variants (exon 6.1) is located over 100 kb downstream of exon 6 in the mouse, rat and human genomes. Exon 6.1 spans a CpG-rich island, and human/rodent conservation is evident through the coding sequence and the 3' untranslated region (UTR). Reverse transcriptase polymerase chain reaction (RT-PCR) and blot hybridisation analyses reveal that exon 6.1 is utilised at low levels in all mouse tissues and cell lines that express Runx2, regardless of which promoter is active, giving Runx2 the potential to encode more than 12 distinct isoforms. RT-PCR analysis of human RUNX2 exon 6.1 expression shows that utilisation of this exon is also conserved. In vitro transcription/translation of cDNAs encoding several exon 6.1 isoforms reveals that the novel Runx proteins are able to bind specifically to canonical Runx DNA target sequences. Antibodies raised to the unique C-terminal domain were shown to be reactive by immunoprecipitation and immunoblot assay, and were used in confocal immunofluorescence microscopy to reveal low level cytoplasmic staining in osteosarcoma and lymphoma cells that express high levels of Runx2 mRNA. However, reactive protein could not be detected in immunoblots of extracts from either cell type, suggesting that these proteins are unstable in lymphoid and osteosarcoma cells. In conclusion, the conservation and widespread utilisation of Runx2 exon 6.1 suggest that its encoded isoforms play an as yet undetermined role in mammalian development.