Gene Summary

Gene:RAG2; recombination activating 2
Aliases: RAG-2
Summary:This gene encodes a protein that is involved in the initiation of V(D)J recombination during B and T cell development. This protein forms a complex with the product of the adjacent recombination activating gene 1, and this complex can form double-strand breaks by cleaving DNA at conserved recombination signal sequences. The recombination activating gene 1 component is thought to contain most of the catalytic activity, while the N-terminal of the recombination activating gene 2 component is thought to form a six-bladed propeller in the active core that serves as a binding scaffold for the tight association of the complex with DNA. A C-terminal plant homeodomain finger-like motif in this protein is necessary for interactions with chromatin components, specifically with histone H3 that is trimethylated at lysine 4. Mutations in this gene cause Omenn syndrome, a form of severe combined immunodeficiency associated with autoimmune-like symptoms. [provided by RefSeq, Jul 2008]
Databases:OMIM, HGNC, Ensembl, GeneCard, Gene
Protein:V(D)J recombination-activating protein 2
Source:NCBIAccessed: 31 August, 2019


What does this gene/protein do?
Show (21)

Cancer Overview

Research Indicators

Publications Per Year (1994-2019)
Graph generated 31 August 2019 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

  • Genes, Immunoglobulin
  • Neoplasm Proteins
  • Herpesvirus 4, Human
  • Molecular Sequence Data
  • Cell Proliferation
  • Chromosome 11
  • Cell Survival
  • Proteins
  • Leukemic Gene Expression Regulation
  • Gene Expression
  • Disease Models, Animal
  • Childhood Cancer
  • Cell Line
  • Biomarkers, Tumor
  • Base Sequence
  • Proto-Oncogene Proteins
  • B-Lymphocytes
  • Breast Cancer
  • Genes, RAG-1
  • Genetic Recombination
  • Cell Differentiation
  • Neoplastic Cell Transformation
  • DNA-Binding Proteins
  • Acute Lymphocytic Leukaemia
  • Leukaemia
  • Neoplasm Metastasis
  • Nuclear Proteins
  • Lung Cancer
  • Apoptosis
  • Adult T-Cell Leukemia-Lymphoma
  • Gene Expression Profiling
  • Knockout Mice
  • Polymerase Chain Reaction
  • Homeodomain Proteins
  • Mice, Transgenic
  • Mice, Inbred BALB C
  • Burkitt Lymphoma
  • Cancer Gene Expression Regulation
  • Immunohistochemistry
  • Flow Cytometry
Tag cloud generated 31 August, 2019 using data from PubMed, MeSH and CancerIndex

Specific Cancers (5)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: RAG2 (cancer-related)

Zhang S, Zhang H, Ghia EM, et al.
Inhibition of chemotherapy resistant breast cancer stem cells by a ROR1 specific antibody.
Proc Natl Acad Sci U S A. 2019; 116(4):1370-1377 [PubMed] Free Access to Full Article Related Publications
Breast cancers enduring treatment with chemotherapy may be enriched for cancer stem cells or tumor-initiating cells, which have an enhanced capacity for self-renewal, tumor initiation, and/or metastasis. Breast cancer cells that express the type I tyrosine kinaselike orphan receptor ROR1 also may have such features. Here we find that the expression of ROR1 increased in breast cancer cells following treatment with chemotherapy, which also enhanced expression of genes induced by the activation of Rho-GTPases, Hippo-YAP/TAZ, or B lymphoma Mo-MLV insertion region 1 homolog (BMI1). Expression of ROR1 also enhanced the capacity of breast cancer cells to invade Matrigel, form spheroids, engraft in Rag2

Endo S, Nishimura N, Kawano Y, et al.
MUC1/KL-6 expression confers an aggressive phenotype upon myeloma cells.
Biochem Biophys Res Commun. 2018; 507(1-4):246-252 [PubMed] Related Publications
The sialic glycoprotein, MUC1, is known to be involved in the pathogenesis of various types of cancers. KL-6 is one of the surface antigens of MUC1 and also a marker of interstitial pneumonitis. A fraction of patients with myeloma (3.9%) have elevated serum KL-6 levels without any evidence of interstitial pneumonitis and their myeloma cells have high MUC1 expression. We established a myeloma cell line designated EMM1 from a patient with multiple myeloma accompanied with elevated serum KL-6. EMM1 cells expressed high levels of MUC1 compared with other myeloma cell lines. Knockdown of MUC1 in EMM1 cells induced cell cycle arrest during S phase and apoptosis, suggesting that the MUC1 expression is involved in accelerated growth of EMM1 cells. RNA-seq analysis suggests that MUC1 expression activates k-ras and TNFα-induced NFκB pathways in EMM1 cells. We injected EMM1 cells subcutaneously into Rag2

Fullwood RA, Low GM, Chase EP, et al.
The Kaposi's sarcoma-associated herpesvirus viral interleukin 6 gene affects metastasis and expression of B cell markers in a murine xenograft model.
PLoS One. 2018; 13(9):e0204947 [PubMed] Free Access to Full Article Related Publications
Kaposi's sarcoma-associated herpesvirus (KSHV) is a cancer-causing virus in humans, primarily affecting AIDS patients. KSHV causes a range of cancers including Kaposi's sarcoma, pleural effusion lymphoma and multicentric Castleman's disease. Current methods available for treating these cancers are relatively ineffective, and new targets for therapy are needed. The KSHV viral homolog of interleukin-6 gene (vIL-6) may play a significant role in tumor development and may serve as a new anti-cancer target, but its role in tumor formation is only partially understood. Here, a novel animal model was used to study how vIL-6 affects tumor development. Highly immune-deficient Rag2-/-γc-/- mice were transplanted with an immortalized human B cell line (BJAB) harboring either wild-type (WT) KSHV or a mutant strain lacking vIL-6 ΔvIL-6). Solid tumors developed and total tumor mass and the number of tumors were characterized. The vIL-6 gene had no significant impact on tumor mass, but significantly more tumors were detected when vIL-6 was present. Significant differences in expression of B cell markers in cells from extracted tumors were detected based upon the presence of vIL-6. B cell markers in tumor cells were also compared to the same cell type in culture, prior to xenotransplantation; B cell markers were mostly downregulated during tumor formation and these changes did not differ based upon the presence of vIL-6. The only marker that significantly increased in expression during tumor development was CD30. Tumor blood vessels were quantified to determine if more angiogenesis occurred with vIL-6-expressing virus, but there was no significant difference. These data indicate that vIL-6 plays a role in KSHV tumor formation in B cells in vivo. Further investigation into how vIL-6 manipulates CD30 expression may shed insight into KSHV oncogenesis, and may identify how vIL-6 can be targeted.

Coleman CB, Lang J, Sweet LA, et al.
Epstein-Barr Virus Type 2 Infects T Cells and Induces B Cell Lymphomagenesis in Humanized Mice.
J Virol. 2018; 92(21) [PubMed] Free Access to Full Article Related Publications
Epstein-Barr virus (EBV) has been classified into two strains, EBV type 1 (EBV-1) and EBV type 2 (EBV-2) based on genetic variances and differences in transforming capacity. EBV-1 readily transforms B cells in culture while EBV-2 is poorly transforming. The differing abilities to immortalize B cells

Mansour MR, He S, Li Z, et al.
JDP2: An oncogenic bZIP transcription factor in T cell acute lymphoblastic leukemia.
J Exp Med. 2018; 215(7):1929-1945 [PubMed] Free Access to Full Article Related Publications
A substantial subset of patients with T cell acute lymphoblastic leukemia (T-ALL) develops resistance to steroids and succumbs to their disease.

Taurozzi AJ, Beekharry R, Wantoch M, et al.
Spontaneous development of Epstein-Barr Virus associated human lymphomas in a prostate cancer xenograft program.
PLoS One. 2017; 12(11):e0188228 [PubMed] Free Access to Full Article Related Publications
Prostate cancer research is hampered by the lack of in vivo preclinical models that accurately reflect patient tumour biology and the clinical heterogeneity of human prostate cancer. To overcome these limitations we propagated and characterised a new collection of patient-derived prostate cancer xenografts. Tumour fragments from 147 unsupervised, surgical prostate samples were implanted subcutaneously into immunodeficient Rag2-/-γC-/- mice within 24 hours of surgery. Histologic and molecular characterisation of xenografts was compared with patient characteristics, including androgen-deprivation therapy, and exome sequencing. Xenografts were established from 47 of 147 (32%) implanted primary prostate cancers. Only 14% passaged successfully resulting in 20 stable lines; derived from 20 independent patient samples. Surprisingly, only three of the 20 lines (15%) were confirmed as prostate cancer; one line comprised of mouse stroma, and 16 were verified as human donor-derived lymphoid neoplasms. PCR for Epstein-Barr Virus (EBV) nuclear antigen, together with exome sequencing revealed that the lymphomas were exclusively EBV-associated. Genomic analysis determined that 14 of the 16 EBV+ lines had unique monoclonal or oligoclonal immunoglobulin heavy chain gene rearrangements, confirming their B-cell origin. We conclude that the generation of xenografts from tumour fragments can commonly result in B-cell lymphoma from patients carrying latent EBV. We recommend routine screening, of primary outgrowths, for latent EBV to avoid this phenomenon.

Hennenberg EM, Eyking A, Reis H, Cario E
MDR1A deficiency restrains tumor growth in murine colitis-associated carcinogenesis.
PLoS One. 2017; 12(7):e0180834 [PubMed] Free Access to Full Article Related Publications
Patients with Ulcerative Colitis (UC) have an increased risk to develop colitis-associated colorectal cancer (CAC). Here, we found that protein expression of ABCB1 (ATP Binding Cassette Subfamily B Member 1) / MDR1 (multidrug resistance 1) was diminished in the intestinal mucosa of patients with active UC with or without CAC, but not in non-UC patients with sporadic colon cancer. We investigated the consequences of ABCB1/MDR1 loss-of-function in a common murine model for CAC (AOM/DSS). Mice deficient in MDR1A (MDR1A KO) showed enhanced intratumoral inflammation and cellular damage, which were associated with reduced colonic tumor size and decreased degree of dysplasia, when compared to wild-type (WT). Increased cell injury correlated with reduced capacity for growth of MDR1A KO tumor spheroids cultured ex-vivo. Gene expression analysis by microarray demonstrated that MDR1A deficiency shaped the inflammatory response towards an anti-tumorigenic microenvironment by downregulating genes known to be important mediators of cancer progression (PTGS2 (COX2), EREG, IL-11). MDR1A KO tumors showed increased gene expression of TNFSF10 (TRAIL), a known inducer of cancer cell death, and CCL12, a strong trigger of B cell chemotaxis. Abundant B220+ B lymphocyte infiltrates with interspersed CD138+ plasma cells were recruited to the MDR1A KO tumor microenvironment, concomitant with high levels of immunoglobulin light chain genes. In contrast, MDR1A deficiency in RAG2 KO mice that lack both B and T cells aggravated colonic tumor progression. MDR1A KO CD19+ B cells, but not WT CD19+ B cells, suppressed growth of colonic tumor-derived spheroids from AOM/DSS-WT mice in an ex-vivo co-culture system, implying that B-cell regulated immune responses contributed to delayed tumor development in MDR1A deficiency. In conclusion, we provide first evidence that loss of ABCB1/MDR1 function may represent an essential tumor-suppressive host defense mechanism in CAC.

Rodríguez-Hernández G, Hauer J, Martín-Lorenzo A, et al.
Infection Exposure Promotes
Cancer Res. 2017; 77(16):4365-4377 [PubMed] Related Publications

Carroll VA, Lafferty MK, Marchionni L, et al.
Expression of HIV-1 matrix protein p17 and association with B-cell lymphoma in HIV-1 transgenic mice.
Proc Natl Acad Sci U S A. 2016; 113(46):13168-13173 [PubMed] Free Access to Full Article Related Publications
HIV-1 infection is associated with increased risk for B-cell lymphomas. How HIV infection promotes the development of lymphoma is unclear, but it may involve chronic B-cell activation, inflammation, and/or impaired immunity, possibly leading to a loss of control of oncogenic viruses and reduced tumor immunosurveillance. We hypothesized that HIV structural proteins may contribute to lymphomagenesis directly, because they can persist long term in lymph nodes in the absence of viral replication. The HIV-1 transgenic mouse Tg26 carries a noninfectious HIV-1 provirus lacking part of the gag-pol region, thus constituting a model for studying the effects of viral products in pathogenesis. Approximately 15% of Tg26 mice spontaneously develop leukemia/lymphoma. We investigated which viral proteins are associated with the development of leukemia/lymphoma in the Tg26 mouse model, and performed microarray analysis on RNA from spleen and lymph nodes to identify potential mechanisms of lymphomagenesis. Of the viral proteins examined, only expression of HIV-1 matrix protein p17 was associated with leukemia/lymphoma development and was highly expressed in bone marrow before disease. The tumor cells resembled pro-B cells, and were CD19

Hunter ZR, Xu L, Yang G, et al.
Transcriptome sequencing reveals a profile that corresponds to genomic variants in Waldenström macroglobulinemia.
Blood. 2016; 128(6):827-38 [PubMed] Free Access to Full Article Related Publications
Whole-genome sequencing has identified highly prevalent somatic mutations including MYD88, CXCR4, and ARID1A in Waldenström macroglobulinemia (WM). The impact of these and other somatic mutations on transcriptional regulation in WM remains to be clarified. We performed next-generation transcriptional profiling in 57 WM patients and compared findings to healthy donor B cells. Compared with healthy donors, WM patient samples showed greatly enhanced expression of the VDJ recombination genes DNTT, RAG1, and RAG2, but not AICDA Genes related to CXCR4 signaling were also upregulated and included CXCR4, CXCL12, and VCAM1 regardless of CXCR4 mutation status, indicating a potential role for CXCR4 signaling in all WM patients. The WM transcriptional profile was equally dissimilar to healthy memory B cells and circulating B cells likely due increased differentiation rather than cellular origin. The profile for CXCR4 mutations corresponded to diminished B-cell differentiation and suppression of tumor suppressors upregulated by MYD88 mutations in a manner associated with the suppression of TLR4 signaling relative to those mutated for MYD88 alone. Promoter methylation studies of top findings failed to explain this suppressive effect but identified aberrant methylation patterns in MYD88 wild-type patients. CXCR4 and MYD88 transcription were negatively correlated, demonstrated allele-specific transcription bias, and, along with CXCL13, were associated with bone marrow disease involvement. Distinct gene expression profiles for patients with wild-type MYD88, mutated ARID1A, familial predisposition to WM, chr6q deletions, chr3q amplifications, and trisomy 4 are also described. The findings provide novel insights into the molecular pathogenesis and opportunities for targeted therapeutic strategies for WM.

Dong Y, Liu F, Wu C, et al.
Illegitimate RAG-mediated recombination events are involved in IKZF1 Δ3-6 deletion in BCR-ABL1 lymphoblastic leukaemia.
Clin Exp Immunol. 2016; 185(3):320-31 [PubMed] Free Access to Full Article Related Publications
Breakpoint cluster region-Abelson murine leukaemia viral oncogene homologue 1 (BCR-ABL1), encoded by the Philadelphia (Ph) chromosome, is the characteristic of chronic myeloid leukaemia (CML) and a subset of acute lymphoblastic leukaemia (ALL). We demonstrated that expression of the Ik6 transcript, which lacked exons 3-6, was observed exclusively in BCR-ABL1(+) B ALL and lymphoid blast crisis CML (BC-CML) patients harbouring the IKZF1 Δ3-6 deletion. To confirm the hypothesis that illegitimate recombination activating gene protein (RAG)-mediated recombination events are involved in IKZF1 Δ3-6 deletion in BCR-ABL1 lymphoblastic leukaemia, we first demonstrated that the expression rates of RAG1 and RAG2, collectively called RAG, were higher in ALL and BC-CML (lymphoid). Notably, analysis of relationships among RAG, BCR-ABL1 and Ikaros 6 (Ik6) showed that Ik6 can be generated only if RAG and BCR-ABL1 are co-existing. The sequencing data showed that the deleted segments of introns 2 and 6 contained cryptic recombination signal sequences (cRSSs) and frequently had non-template nucleotides inserted between breakpoints. Furthermore, we used chromatin immunoprecipitation (ChIP) technology and demonstrated that the sequences directly flanking IKZF1 Δ3-6 deletion breakpoints have significantly higher levels of histone H3 lysine 4 trimethylation (H3K4me3) modifications. Overall, RAG expression, good-quality cRSS and a specific chromatin modification, H3K4me3, satisfy the conditions of RAG's off-target effects on IKZF1. Our work provides evidence for RAG-mediated IKZF1 Δ3-6 deletion. Our results raise the prospect that RAG is a valuable biomarker in disease surveillance. Dissecting the contribution of RAG should not only provide valuable mechanistic insights, but will also lead to a new therapeutic direction.

Prasad A, Rabionet R, Espinet B, et al.
Identification of Gene Mutations and Fusion Genes in Patients with Sézary Syndrome.
J Invest Dermatol. 2016; 136(7):1490-1499 [PubMed] Related Publications
Sézary syndrome is a leukemic form of cutaneous T-cell lymphoma with an aggressive clinical course. The genetic etiology of the disease is poorly understood, with chromosomal abnormalities and mutations in some genes being involved in the disease. The goal of our study was to understand the genetic basis of the disease by looking for driver gene mutations and fusion genes in 15 erythrodermic patients with circulating Sézary cells, 14 of them fulfilling the diagnostic criteria of Sézary syndrome. We have discovered genes that could be involved in the pathogenesis of Sézary syndrome. Some of the genes that are affected by somatic point mutations include ITPR1, ITPR2, DSC1, RIPK2, IL6, and RAG2, with some of them mutated in more than one patient. We observed several somatic copy number variations shared between patients, including deletions and duplications of large segments of chromosome 17. Genes with potential function in the T-cell receptor signaling pathway and tumorigenesis were disrupted in Sézary syndrome patients, for example, CBLB, RASA2, BCL7C, RAMP3, TBRG4, and DAD1. Furthermore, we discovered several fusion events of interest involving RASA2, NFKB2, BCR, FASN, ZEB1, TYK2, and SGMS1. Our work has implications for the development of potential therapeutic approaches for this aggressive disease.

Hu J, Zhang Y, Zhao L, et al.
Chromosomal Loop Domains Direct the Recombination of Antigen Receptor Genes.
Cell. 2015; 163(4):947-59 [PubMed] Free Access to Full Article Related Publications
RAG initiates antibody V(D)J recombination in developing lymphocytes by generating "on-target" DNA breaks at matched pairs of bona fide recombination signal sequences (RSSs). We employ bait RAG-generated breaks in endogenous or ectopically inserted RSS pairs to identify huge numbers of RAG "off-target" breaks. Such breaks occur at the simple CAC motif that defines the RSS cleavage site and are largely confined within convergent CTCF-binding element (CBE)-flanked loop domains containing bait RSS pairs. Marked orientation dependence of RAG off-target activity within loops spanning up to 2 megabases implies involvement of linear tracking. In this regard, major RAG off-targets in chromosomal translocations occur as convergent RSS pairs at enhancers within a loop. Finally, deletion of a CBE-based IgH locus element disrupts V(D)J recombination domains and, correspondingly, alters RAG on- and off-target distributions within IgH. Our findings reveal how RAG activity is developmentally focused and implicate mechanisms by which chromatin domains harness biological processes within them.

Ochodnicka-Mackovicova K, Bahjat M, Bloedjes TA, et al.
NF-κB and AKT signaling prevent DNA damage in transformed pre-B cells by suppressing RAG1/2 expression and activity.
Blood. 2015; 126(11):1324-35 [PubMed] Free Access to Full Article Related Publications
In developing lymphocytes, expression and activity of the recombination activation gene protein 1 (RAG1) and RAG2 endonuclease complex is tightly regulated to ensure ordered recombination of the immunoglobulin genes and to avoid genomic instability. Aberrant RAG activity has been implicated in the generation of secondary genetic events in human B-cell acute lymphoblastic leukemias (B-ALLs), illustrating the oncogenic potential of the RAG complex. Several layers of regulation prevent collateral genomic DNA damage by restricting RAG activity to the G1 phase of the cell cycle. In this study, we show a novel pathway that suppresses RAG expression in cycling-transformed mouse pre-B cells and human pre-B B-ALL cells that involves the negative regulation of FOXO1 by nuclear factor κB (NF-κB). Inhibition of NF-κB in cycling pre-B cells resulted in upregulation of RAG expression and recombination activity, which provoked RAG-dependent DNA damage. In agreement, we observe a negative correlation between NF-κB activity and the expression of RAG1, RAG2, and TdT in B-ALL patients. Our data suggest that targeting NF-κB in B-ALL increases the risk of RAG-dependent genomic instability.

Pore N, Jalla S, Liu Z, et al.
In Vivo Loss of Function Screening Reveals Carbonic Anhydrase IX as a Key Modulator of Tumor Initiating Potential in Primary Pancreatic Tumors.
Neoplasia. 2015; 17(6):473-80 [PubMed] Free Access to Full Article Related Publications
Reprogramming of energy metabolism is one of the emerging hallmarks of cancer. Up-regulation of energy metabolism pathways fuels cell growth and division, a key characteristic of neoplastic disease, and can lead to dependency on specific metabolic pathways. Thus, targeting energy metabolism pathways might offer the opportunity for novel therapeutics. Here, we describe the application of a novel in vivo screening approach for the identification of genes involved in cancer metabolism using a patient-derived pancreatic xenograft model. Lentiviruses expressing short hairpin RNAs (shRNAs) targeting 12 different cell surface protein transporters were separately transduced into the primary pancreatic tumor cells. Transduced cells were pooled and implanted into mice. Tumors were harvested at different times, and the frequency of each shRNA was determined as a measure of which ones prevented tumor growth. Several targets including carbonic anhydrase IX (CAIX), monocarboxylate transporter 4, and anionic amino acid transporter light chain, xc- system (xCT) were identified in these studies and shown to be required for tumor initiation and growth. Interestingly, CAIX was overexpressed in the tumor initiating cell population. CAIX expression alone correlated with a highly tumorigenic subpopulation of cells. Furthermore, CAIX expression was essential for tumor initiation because shRNA knockdown eliminated the ability of cells to grow in vivo. To the best of our knowledge, this is the first parallel in vivo assessment of multiple novel oncology target genes using a patient-derived pancreatic tumor model.

Chun E, Lavoie S, Michaud M, et al.
CCL2 Promotes Colorectal Carcinogenesis by Enhancing Polymorphonuclear Myeloid-Derived Suppressor Cell Population and Function.
Cell Rep. 2015; 12(2):244-57 [PubMed] Free Access to Full Article Related Publications
Our study reveals a non-canonical role for CCL2 in modulating non-macrophage, myeloid-derived suppressor cells (MDSCs) and shaping a tumor-permissive microenvironment during colon cancer development. We found that intratumoral CCL2 levels increased in patients with colitis-associated colorectal cancer (CRC), adenocarcinomas, and adenomas. Deletion of CCL2 blocked progression from dysplasia to adenocarcinoma and reduced the number of colonic MDSCs in a spontaneous mouse model of colitis-associated CRC. In a transplantable mouse model of adenocarcinoma and an APC-driven adenoma model, CCL2 fostered MDSC accumulation in evolving colonic tumors and enhanced polymorphonuclear (PMN)-MDSC immunosuppressive features. Mechanistically, CCL2 regulated T cell suppression of PMN-MDSCs in a STAT3-mediated manner. Furthermore, CCL2 neutralization decreased tumor numbers and MDSC accumulation and function. Collectively, our experiments support that perturbing CCL2 and targeting MDSCs may afford therapeutic opportunities for colon cancer interception and prevention.

Sanmamed MF, Rodriguez I, Schalper KA, et al.
Nivolumab and Urelumab Enhance Antitumor Activity of Human T Lymphocytes Engrafted in Rag2-/-IL2Rγnull Immunodeficient Mice.
Cancer Res. 2015; 75(17):3466-78 [PubMed] Related Publications
A current pressing need in cancer immunology is the development of preclinical model systems that are immunocompetent for the study of human tumors. Here, we report the development of a humanized murine model that can be used to analyze the pharmacodynamics and antitumor properties of immunostimulatory monoclonal antibodies (mAb) in settings where the receptors targeted by the mAbs are expressed. Human lymphocytes transferred into immunodeficient mice underwent activation and redistribution to murine organs, where they exhibited cell-surface expression of hCD137 and hPD-1. Systemic lymphocyte infiltrations resulted in a lethal CD4(+) T cell-mediated disease (xenograft-versus-host disease), which was aggravated when murine subjects were administered clinical-grade anti-hCD137 (urelumab) and anti-hPD-1 (nivolumab). In mice engrafted with human colorectal HT-29 carcinoma cells and allogeneic human peripheral blood mononuclear cells (PBMC), or with a patient-derived gastric carcinoma and PBMCs from the same patient, we found that coadministration of urelumab and nivolumab was sufficient to significantly slow tumor growth. Correlated with this result were increased numbers of activated human T lymphocytes producing IFNγ and decreased numbers of human regulatory T lymphocytes in the tumor xenografts, possibly explaining the efficacy of the therapeutic regimen. Our results offer a proof of concept for the use of humanized mouse models for surrogate efficacy and histology investigations of immune checkpoint drugs and their combinations.

Agathangelidis A, Scarfò L, Barbaglio F, et al.
Establishment and Characterization of PCL12, a Novel CD5+ Chronic Lymphocytic Leukaemia Cell Line.
PLoS One. 2015; 10(6):e0130195 [PubMed] Free Access to Full Article Related Publications
Immortalized cell lines representative of chronic lymphocytic leukemia (CLL) can assist in understanding disease pathogenesis and testing new therapeutic agents. At present, very few representative cell lines are available. We here describe the characterization of a new cell line (PCL12) that grew spontaneously from the peripheral blood (PB) of a CLL patient with progressive disease and EBV infection. The CLL cell origin of PCL12 was confirmed after the alignment of its IGH sequence against the "original" clonotypic sequence. The IGH gene rearrangement was truly unmutated and no CLL-related cytogenetic or genetic lesions were detected. PCL12 cells express CD19, CD20, CD5, CD23, low levels of IgM and IgD and the poor-outcome-associated prognostic markers CD38, ZAP70 and TCL1. In accordance with its aggressive phenotype the cell line is inactive in terms of LYN and HS1 phosphorylation. BcR signalling pathway is constitutively active and anergic in terms of p-ERK and Calcium flux response to α-IgM stimulation. PCL12 cells strongly migrate in vitro in response to SDF-1 and form clusters. Finally, they grow rapidly and localize in all lymphoid organs when xenotrasplanted in Rag2-/-γc-/- mice. PCL12 represents a suitable preclinical model for testing pharmacological agents.

Swaminathan S, Klemm L, Park E, et al.
Mechanisms of clonal evolution in childhood acute lymphoblastic leukemia.
Nat Immunol. 2015; 16(7):766-774 [PubMed] Free Access to Full Article Related Publications
Childhood acute lymphoblastic leukemia (ALL) can often be traced to a pre-leukemic clone carrying a prenatal genetic lesion. Postnatally acquired mutations then drive clonal evolution toward overt leukemia. The enzymes RAG1-RAG2 and AID, which diversify immunoglobulin-encoding genes, are strictly segregated in developing cells during B lymphopoiesis and peripheral mature B cells, respectively. Here we identified small pre-BII cells as a natural subset with increased genetic vulnerability owing to concurrent activation of these enzymes. Consistent with epidemiological findings on childhood ALL etiology, susceptibility to genetic lesions during B lymphopoiesis at the transition from the large pre-BII cell stage to the small pre-BII cell stage was exacerbated by abnormal cytokine signaling and repetitive inflammatory stimuli. We demonstrated that AID and RAG1-RAG2 drove leukemic clonal evolution with repeated exposure to inflammatory stimuli, paralleling chronic infections in childhood.

Ba Z, Meng FL, Gostissa M, et al.
A Rapid Embryonic Stem Cell-Based Mouse Model for B-cell Lymphomas Driven by Epstein-Barr Virus Protein LMP1.
Cancer Immunol Res. 2015; 3(6):641-9 [PubMed] Free Access to Full Article Related Publications
The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) contributes to oncogenic human B-cell transformation. Mouse B cells conditionally expressing LMP1 are not predisposed to B-cell malignancies, as LMP1-expressing B cells are eliminated by T cells. However, mice with conditional B-cell LMP1 expression and genetic elimination of α/β and γ/δ T cells ("CLT" mice) die early in association with B-cell lymphoproliferation and lymphomagenesis. Generation of CLT mice involves in-breeding multiple independently segregating alleles. Thus, although introduction of additional activating or knockout mutations into the CLT model is desirable for further B-cell expansion and immunosurveillance studies, doing such experiments by germline breeding is time-consuming, expensive, and sometimes unfeasible. To generate a more tractable model, we generated clonal CLT embryonic stem (ES) cells from CLT embryos and injected them into RAG2-deficient blastocysts to generate chimeric mice, which, like germline CLT mice, harbor splenic CLT B cells and lack T cells. CLT chimeric mice generated by this RAG2-deficient blastocyst complementation ("RDBC") approach die rapidly in association with B-cell lymphoproliferation and lymphoma. Because CLT lymphomas routinely express the activation-induced cytidine deaminase (AID) antibody diversifier, we tested potential AID roles by eliminating the AID gene in CLT ES cells and testing them via RDBC. We found that CLT and AID-deficient CLT ES chimeras had indistinguishable phenotypes, showing that AID is not essential for LMP1-induced lymphomagenesis. Beyond expanding accessibility and utility of CLT mice as a cancer immunotherapy model, our studies provide a new approach for facilitating generation of genetically complex mouse cancer models.

Xu SQ, Buraschi S, Morcavallo A, et al.
A novel role for drebrin in regulating progranulin bioactivity in bladder cancer.
Oncotarget. 2015; 6(13):10825-39 [PubMed] Free Access to Full Article Related Publications
We recently established a critical role for the growth factor progranulin in bladder cancer insofar as progranulin promotes urothelial cancer cell motility and contributes, as an autocrine growth factor, to the transformed phenotype by modulating invasion and anchorage-independent growth. In addition, progranulin expression is upregulated in invasive bladder cancer tissues compared to normal controls. However, the molecular mechanisms of progranulin action in bladder cancer have not been fully elucidated. In this study, we searched for novel progranulin-interacting proteins using pull-down assays with recombinant progranulin and proteomics. We discovered that drebrin, an F-actin binding protein, bound progranulin in urothelial cancer cells. We characterized drebrin function in urothelial cancer cell lines and showed that drebrin is critical for progranulin-dependent activation of the Akt and MAPK pathways and modulates motility, invasion and anchorage-independent growth. In addition, drebrin regulates tumor formation in vivo and its expression is upregulated in bladder cancer tissues compared to normal tissue controls. Our data are translationally relevant as indicate that drebrin exerts an essential functional role in the regulation of progranulin action and may constitute a novel target for therapeutic intervention in bladder tumors. In addition, drebrin may serve as novel biomarker for bladder cancer.

Motoyama K, Onodera R, Tanaka N, et al.
Evaluation of antitumor effects of folate-conjugated methyl-β-cyclodextrin in melanoma.
Biol Pharm Bull. 2015; 38(3):374-9 [PubMed] Related Publications
Melanoma is a life-threatening disorder and its incidence is increasing gradually. Despite the numerous treatment approaches, conventional systemic chemotherapy has not reduced the mortality rate among melanoma patients, probably due to the induction of toxicity to normal tissues. Recently, we have developed folate-conjugated methyl-β-cyclodextrin (FA-M-β-CyD) and clarified its potential as a new antitumor agent involved in autophagic cell death. However, it remains uncertain whether FA-M-β-CyD exerts anticancer effects against melanomas. Therefore, in this study, we investigated the effects of FA-M-β-CyD on the folate receptor-α (FR-α)-expressing melanoma cell-selective cytotoxic effect. FA-M-β-CyD showed cytotoxic effects in Ihara cells, a human melanoma cell line expressing FR-α. In sharp contrast to methyl-β-cyclodextrin, FA-M-β-CyD entered Ihara cells [FR-α(+)] through FR-α-mediated endocytosis. Additionally, FA-M-β-CyD elicited the formation of autophagosomes in Ihara cells. Notably, FA-M-β-CyD suppressed melanoma growth in BALB/c nude recombinase-activating gene-2 (Rag-2)/Janus kinase 3 (Jak3) double deficient mice bearing Ihara cells. Therefore, these results suggest that FA-M-β-CyD could be utilized as a potent anticancer agent for melanoma chemotherapy by regulating autophagy.

Navarro JM, Touzart A, Pradel LC, et al.
Site- and allele-specific polycomb dysregulation in T-cell leukaemia.
Nat Commun. 2015; 6:6094 [PubMed] Free Access to Full Article Related Publications
T-cell acute lymphoblastic leukaemias (T-ALL) are aggressive malignant proliferations characterized by high relapse rates and great genetic heterogeneity. TAL1 is amongst the most frequently deregulated oncogenes. Yet, over half of the TAL1(+) cases lack TAL1 lesions, suggesting unrecognized (epi)genetic deregulation mechanisms. Here we show that TAL1 is normally silenced in the T-cell lineage, and that the polycomb H3K27me3-repressive mark is focally diminished in TAL1(+) T-ALLs. Sequencing reveals that >20% of monoallelic TAL1(+) patients without previously known alterations display microinsertions or RAG1/2-mediated episomal reintegration in a single site 5' to TAL1. Using 'allelic-ChIP' and CrispR assays, we demonstrate that such insertions induce a selective switch from H3K27me3 to H3K27ac at the inserted but not the germline allele. We also show that, despite a considerable mechanistic diversity, the mode of oncogenic TAL1 activation, rather than expression levels, impact on clinical outcome. Altogether, these studies establish site-specific epigenetic desilencing as a mechanism of oncogenic activation.

Hassan WA, Yoshida R, Kudoh S, et al.
Notch1 controls cell invasion and metastasis in small cell lung carcinoma cell lines.
Lung Cancer. 2014; 86(3):304-10 [PubMed] Related Publications
INTRODUCTION: Notch signaling plays a key role in a wide variety of human neoplasms, and it can be either oncogenic or anti-proliferative. Moreover, Notch function in regulating cancer is unpredictable, and its outcome is strictly context-dependent.
AIM: To study the role of Notch1 signaling in human small cell lung carcinoma (SCLC) and its effect on cell invasion and metastasis.
MATERIALS AND METHODS: We used small interfering RNA (siRNA) technology, to down-regulate the expression of Notch1 in H69AR and SBC3 SCLC cells. On the other hand, we up-regulated Notch1 in H69 and H1688 SCLC cells through transfection with venus Notch1 intracellular domain (v.NICD) plasmid. In addition, H69 cells with v.NICD were xenotransplanted into immune-compromised Rag2(-/-) Jak3(-/-) mice, for analysis of ex vivo tumor epithelial mesenchymal transition (EMT) phenotype and for detection of metastatic cancer cells in the lung tissues. Moreover, we examined the metastatic ability for H69AR and SBC3 cells transfected with siRNA against Notch1, compared to their subsequent controls, by use of tail vein xenograft mouse models.
RESULTS: Notch1 controls cell adhesion and EMT. Overexpression of Notch1 in SCLC switched off EMT, cell motility and cell metastatic potential.
CONCLUSION: Our results demonstrate that activation of Notch1 signaling pathway may represent a new strategy for treating human SCLC.

Tsukahara T, Iwase N, Kawakami K, et al.
The Tol2 transposon system mediates the genetic engineering of T-cells with CD19-specific chimeric antigen receptors for B-cell malignancies.
Gene Ther. 2015; 22(2):209-15 [PubMed] Free Access to Full Article Related Publications
Engineered T-cell therapy using a CD19-specific chimeric antigen receptor (CD19-CAR) is a promising strategy for the treatment of advanced B-cell malignancies. Gene transfer of CARs to T-cells has widely relied on retroviral vectors, but transposon-based gene transfer has recently emerged as a suitable nonviral method to mediate stable transgene expression. The advantages of transposon vectors compared with viral vectors include their simplicity and cost-effectiveness. We used the Tol2 transposon system to stably transfer CD19-CAR into human T-cells. Normal human peripheral blood lymphocytes were co-nucleofected with the Tol2 transposon donor plasmid carrying CD19-CAR and the transposase expression plasmid and were selectively propagated on NIH3T3 cells expressing human CD19. Expanded CD3(+) T-cells with stable and high-level transgene expression (~95%) produced interferon-γ upon stimulation with CD19 and specifically lysed Raji cells, a CD19(+) human B-cell lymphoma cell line. Adoptive transfer of these T-cells suppressed tumor progression in Raji tumor-bearing Rag2(-/-)γc(-/-) immunodeficient mice compared with control mice. These results demonstrate that the Tol2 transposon system could be used to express CD19-CAR in genetically engineered T-cells for the treatment of refractory B-cell malignancies.

Brodbeck T, Nehmann N, Bethge A, et al.
Perforin-dependent direct cytotoxicity in natural killer cells induces considerable knockdown of spontaneous lung metastases and computer modelling-proven tumor cell dormancy in a HT29 human colon cancer xenograft mouse model.
Mol Cancer. 2014; 13:244 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: For long, natural killer (NK) cells have been suspected to play a critical role in suppressing the development of spontaneous metastases in cancer patients. Despite a wide range of studies it remains unclear so far to what extent primary tumor growth together with formation of distant metastases and NK cell activity influence each other.
METHODS: To precisely investigate the role of NK cells with a perforin-deficiency in cancer growth and metastasis formation, human HT29 colon cancer cells were subcutaneously grafted into pore forming protein and recombination activating gene 2 double knock out (pfp/rag2) mice and in recombination activating gene 2 only knock out (rag2) mice both with black six background. Both mice lack B and T cell functions due to the absence of rag2.
RESULTS: Primary tumors developed in 16/16 in pfp/rag2 and 20/20 rag2 mice. At sacrifice primary tumor weight did not differ significantly. However, tumors grew faster in pfp/rag2 mice (50 days) than in pfp/rag2 mice (70 days). Circulating tumor cells (CTC) in murine blood were nearly three times higher in pfp/rag2 (68 cells/ml) than in rag2 mice (24 cells/ml). Lung metastases occurred frequently in pfp/rag2 mice (13/16) and infrequently in rag2 mice (5/20). The mean number of metastases was 789 in pfp/rag2 mice compared to 210 in rag2 mice. Lung metastases in pfp/rag2 mice consisted of 10-100 tumor cells while those in rag2 mice were generally disseminated tumor cells (DTCs).Computer modelling showed that perforin-dependent killing of NK cells decelerates the growth of the primary tumour and kills 80% of CTCs. Furthermore, perforin-mediated cytotoxicity hampers the proliferation of the malignant cells in host tissue forcing them to stay dormant for at least 30 days.
CONCLUSION: The results exactly quantified the effect of perforin-dependent direct cytotoxicity of NK cells on HT29 on primary tumor growth, number of CTCs in the blood and the number of metastases. The largest effects were seen in the number of mice developing spontaneous lung metastases and the mean number of lung metastases. Hence, perforin-mediated cytotoxicity used for direct killing by NK cells is more important than indirect killing by secretion of death-inducing ligands by NK cells.

Shimazaki N, Lieber MR
Histone methylation and V(D)J recombination.
Int J Hematol. 2014; 100(3):230-7 [PubMed] Related Publications
V(D)J recombination is the process by which the diversity of antigen receptor genes is generated and is also indispensable for lymphocyte development. This recombination event occurs in a cell lineage- and stage-specific manner, and is carefully controlled by chromatin structure and ordered histone modifications. The recombinationally active V(D)J loci are associated with hypermethylation at lysine4 of histone H3 and hyperacetylation of histones H3/H4. The recombination activating gene 1 (RAG1) and RAG2 complex initiates recombination by introducing double-strand DNA breaks at recombination signal sequences (RSS) adjacent to each coding sequence. To be recognized by the RAG complex, RSS sites must be within an open chromatin context. In addition, the RAG complex specifically recognizes hypermethylated H3K4 through its plant homeodomain (PHD) finger in the RAG2 C terminus, which stimulates RAG catalytic activity via that interaction. In this review, we describe how histone methylation controls V(D)J recombination and discuss its potential role in lymphoid malignancy by mistargeting the RAG complex.

Nagel S, Meyer C, Kaufmann M, et al.
Deregulated FOX genes in Hodgkin lymphoma.
Genes Chromosomes Cancer. 2014; 53(11):917-33 [PubMed] Related Publications
FOX genes encode transcription factors which regulate basic developmental processes during embryogenesis and in the adult. Several FOX genes show deregulated expression in particular malignancies, representing oncogenes or tumor suppressors. Here, we screened six Hodgkin lymphoma (HL) cell lines for FOX gene activity by comparative microarray profiling, revealing overexpression of FOXC1 and FOXD1, and reduced transcription of FOXN3, FOXO1, and FOXP1. In silico expression analyses of these FOX gene candidates in HL patient samples supported the cell line data. Chromosomal analyses demonstrated an amplification of the FOXC1 locus at 6p25 and a gain of the FOXR2 locus at Xp11, indicting genomic aberrations for their upregulation. Comparative expression profiling and ensuing stimulation experiments revealed implementation of the TGFβ- and WNT-signaling pathways in deregulation of FOXD1 and FOXN3. Functional analysis of FOXP1 implicated miR9 and miR34a as upstream regulators and PAX5, TCF3, and RAG2 as downstream targets. A similar exercise for FOXC1 revealed repression of MSX1 and activation of IPO7, both mediating inhibition of the B-cell specific homeobox gene ZHX2. Taken together, our data show that aberrantly expressed FOX genes and their downstream targets are involved in the pathogenesis of HL via deregulation of B-cell differentiation and may represent useful diagnostic markers and/or therapeutic targets.

Shultz LD, Goodwin N, Ishikawa F, et al.
Human cancer growth and therapy in immunodeficient mouse models.
Cold Spring Harb Protoc. 2014; 2014(7):694-708 [PubMed] Free Access to Full Article Related Publications
Since the discovery of the "nude" mouse more than 40 years ago, investigators have attempted to model human tumor growth in immunodeficient mice. Here, we summarize how the field has advanced over the ensuing years owing to improvements in the murine recipients of human tumors. These improvements include the discovery of the scid mutation and development of targeted mutations in the recombination-activating genes 1 and 2 (Rag1(null), Rag2(null)) that severely cripple the adaptive immune response of the murine host. More recently, mice deficient in adaptive immunity have been crossed with mice bearing targeted mutations designed to weaken the innate immune system, ultimately leading to the development of immunodeficient mice bearing a targeted mutation in the gene encoding the interleukin 2 (IL2) receptor common γ chain (IL2rg(null), also known in humans as cytokine receptor common subunit γ). The IL2rg(null) mutation has been used to develop several immunodeficient strains of mice, including the NOD-scid IL2rg(null) (NSG) strain. Using NSG mice as human xenograft recipients, it is now possible to grow almost all types of primary human tumors in vivo, including most solid tumors and hematological malignancies that maintain characteristics of the primary tumor in the patient. Programs to optimize patient-specific therapy using patient-derived xenograft tumor growth in NSG mice have been established at several institutions, including The Jackson Laboratory. Moreover, NSG mice can be engrafted with functional human immune systems, permitting for the first time the potential to study primary human tumors in vivo in the presence of a human immune system.

Daudigeos-Dubus E, LE Dret L, Rouffiac V, et al.
Establishment and characterization of new orthotopic and metastatic neuroblastoma models.
In Vivo. 2014 Jul-Aug; 28(4):425-34 [PubMed] Related Publications
BACKGROUND/AIM: Treatment of metastatic neuroblastoma remains a challenge in pediatric oncology. Relevant preclinical models may improve exploration of oncogenesis and new therapies. We developed new orthotopic and metastatic models derived from stage 4 neuroblastoma.
MATERIAL AND METHODS: Orthotopic and systemic models were established in BalbC Rag2(-/-)gammaC(-/-) mice following adrenal and intravenous injection of luciferase-transfected IMR-32 and IGR-N91 cells, respectively.
RESULTS: All four models exhibited 100% tumor take rate. Metastatic spread of orthotopic IMR-32-Luc cells was observed mainly to the lung, liver and bone; that of IGR-N91-Luc cells to liver, spleen and adrenals. Interestingly, systemic IMR-32-Luc cells metastasized rather to the lung, liver and bone, and IGR-N91-Luc to liver, lung, spleen and adrenals. Feasibility of non-invasive, real-time antitumor response evaluation was validated in the systemic models.
CONCLUSION: These neuroblastoma models with distinct patterns of metastatic spread represent relevant tools for exploring local and metastatic tumor cell tropism, mechanisms of spread and evaluating new cancer therapeutics.

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