Letters to the Editor 162 3 Laboratoire de Mathe´matiques Nicolas Oresme CNRS UMR 4 Roulland S, Lebailly P, Roussel G, Briand M, Cappellen D, Pottier D 6139, Caen, France and et al. BCL-2/JH translocation in peripheral blood lymphocytes of 4Centre d’Immunologie de Marseille-Luminy (CIML), CNRS- unexposed individuals: lack of seasonal variations in frequency and INSERM-Universite´ de la Me´diterrane´e, Marseille, France molecular features. Int J Cancer 2003; 104: 695–698. E-mail: [email protected] 5 Liang KY, Zeger SL. Longitudinal data-analysis using generalized linear-models. Biometrika 1986; 73: 13–22. 6 McDonnell TJ, Deane N, Platt FM, Nunez G, Jaeger U, McKearn JP References et al. bcl-2-immunoglobulin transgenic mice demonstrate extended B cell survival and follicular lymphoproliferation. Cell 1989; 57: 1 Kuppers R. Mechanisms of B-cell lymphoma pathogenesis. Nature 79–88. Reviews Cancer 2005; 5: 251–262. 7 Giannelli F, Moscarella S, Giannini C, Caini P, Monti M, Gragnani L 2 Janz S, Potter M, Rabkin CS. Lymphoma- and leukemia-associated et al. Effect of antiviral treatment in patients with chronic chromosomal translocations in healthy individuals. Chromo- HCV infection and t(14;18) translocation. Blood 2003; 102: somes Cancer 2003; 36: 211–223. 1196–1201. 3 Ladetto M, Drandi D, Compagno M, Astolfi M, Volpato F, Voena C 8 Jager U, Bocskor S, Le T, Mitterbauer G, Bolz I, Chott A et al. et al. PCR-detectable nonneoplastic Bcl-2/IgH rearrangements are com- Follicular lymphomas’ BCL-2/IgH junctions contain templated mon in normal subjects and cancer patients at diagnosis but rare in nucleotide insertions: novel insights into the mechanism of subjects treated with chemotherapy. JClinOncol2003; 21: 1398–1403. t(14;18) translocation. Blood 2000; 95: 3520–3529.

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu/).

Assessment of F-MuLV-induced tumorigenesis reveals new candidate tumor genes including Pecam1, St7, and Prim2

Leukemia (2006) 20, 162–165. doi:10.1038/sj.leu.2404034; in F-MuLV-induced nonerythroid tumors arising in C57BL/6 published online 24 November 2005 mice. Nontransgenic (six mice) and gp91phox-SV40-transgenic (80 mice) C57BL/6 mice were injected as neonates with The ability to achieve advances in the treatment of hemato- F-MuLV and aged for tumor development. Among the resulting poietic malignancies depends on better insight into the lesions were 75 hematopoietic malignancies and 12 preleuke- molecular mechanisms underlying their deregulated growth. mias. The neoplasms included histiocytic sarcomas, myeloid Critical insight into this is afforded by identifying genetic lesions leukemias and lymphoid leukemia/lymphomas (Table 1). From within these neoplasms. In mice, nononcogene-containing tumor DNA, proviral insertion sites were determined by inverse retroviruses can induce a variety of hematopoietic neoplasms, PCR followed by molecular cloning and DNA sequence the genesis of which is dependent on proviral integration analysis. A total of 762 proviral integration sites (PIS) were resulting in dysregulated expression of key growth regulatory characterized. An examination of the compiled PIS allowed us genes. The characterization of proviral insertion sites constitutes to determine 52 common insertion sites (CIS) present in three or a powerful approach to the identification of genes involved in more tumors, when considering our data and that present in the neoplastic process. The pathogenesis of retrovirally-induced hematopoietic tumors in the mouse involves a complex interaction between the virus and host, the outcome of which is determined by virus type and Table 1 Histopathologic diagnoses for lesions analyzed mouse strain. The Friend murine leukemia virus is the helper virus component of the Friend complex and proviral insertional Number of cases Diagnosis activation or inactivation of specific growth regulatory genes such as Trp53 and Fli1 leads to a clonal erythroleukemias. 35 Histiocytic sarcoma Certain mouse strains, such as C57BL6, are resistant to F-MuLV- 20 Myelomonocytic leukemia 12 Lymphoid leukemia or lymphoma induced erythroleukemia, and develop myeloid leukemias and 12 Preleukemia lymphomas upon infection, with a latency nearly twice as long 2 Myelodysplasia as for the F-MuLV-induced erythroleukemias. The sites of 1 Erythroleukemia proviral insertion in F-MuLV-induced myeloid leukemias and 7 No tumor lymphomas have been largely unreported but these sites are of Total number of diagnoses is greater than the number of lesions (75 considerable interest since they are likely to yield insight into specimens) since some lesions had more than one tumor type. Tumors the corresponding human diseases. In our previous work, we were generated as described1. Tumors were diagnosed by histo- demonstrated that F-MuLV accelerated myelomonocytic neo- pathologic examination of H+E-stained spleen, liver, bone marrow and plasms in transgenic mice harboring an SV40 early region lymph nodes. Preleukemias were diagnosed as a moderate (two- to transgene under the control of a myelomonocytic promoter three-times normal weight) increase in spleen weight, with expansion 1 of either red or white pulp areas of the spleen, but no marked infiltration region (gp91phox): F-MuLV shortened the latency of tumor- into liver or evident circulating blasts on Giemsa-stained smear of igenesis from 167 to 103 days, and this was associated with peripheral blood. Also taken into consideration for the diagnosis of the presence of proviral insertions at Fli1, and a novel site, preleukemia was the finding of proviral insertion into common sites or Fim4. Here, we further analyze the sites of proviral insertion into cancer-related genes.

Leukemia Table 2 (a) Novel common retroviral insertion sites and (b) Known common retroviral insertion sites

CIS Protein product Function Chr. # hits CIS Provirus location, our data Provirus location Tumor type name others’ data a b 1234567

(a) Novel common retroviral insertion sites 1 Prim2 DNA , p58 subunit Cell Div 1 3 Intron 7, std 1 1 2 Lcp1 Lymphocyte cytosolic protein 1/actin binding Cytoskel 14 2 2 Intron 1, otd, 30 of ATG & intron 2 Intron 2 1 1 3 Scd2 Steroyl-coenzyme A desaturase 2 Metabolism 19 2 2 40 & 12.3 kb up, otd 12 & 0.2 kb ups, otd 2 4 Gdi3 Rab GDP Dissociation inhibitor RAS family 13 2 1 Intron 1, std, 30 of ATG & 19 kb up 12.4 kb up, otd 2 5 Pecam1 Platelet/endothelial cell adhesion molecule Receptor 11 2 1 Intron 15, otd Intron 15 1 2 6 Scap2 src family associated phosphoprotein 2 Signaling 6 2 1 Intron 9, otd Intron 9 2 7 Nkx2-3 Homeobox TF/M 19 3 5; 5; 4 kb up 2 8 ST7/St7 Suppressor of tumorigenicity Uncharacter 6 3 Intron 1, otd 1 3 9 GENSCAN00000117499 Hypothetical protein Uncharacter 6 4 Fim4 7 kb up, std 2 2 1 10 GENSCAN00000137744 Hypothetical protein Uncharacter 8 5 Fim5 Intron 6; intron 3; 3 & 4 kb up 1 2 1 11 5830405N20Rik Hypothetical protein NP_899087 Uncharacter 10 3 Fim10 34 & 16 kb up, std 1 1 12 BC031781 Hypothetical protein MGC30618 Uncharacter 1 1 2 0.5 kb up, otd 1.3 & 0.7 kb up, otd 1

(b) Known common retroviral insertion sites 1 Sept9 Cell division control Cell Div 11 1 6 Sint1 Intron 4, otd 1 2 Capg Capping protein/actin modification Cytoskel 6 1 3 Capg 1 3 Lcp1 Lymphocyte cytosolic protein 1/actin binding Cytoskel 14 2 2 Intron 1, otd, 30 of ATG & intron 2 Intron 2 1 1 4 Brd2 Bromodomain containing nuclear Kinase 17 1 2 Dkmi26 1 5 Camk2d Calcium/calmodulin-depend Kinase 3 1 4 Evi55 1 1 II delta

6 Map3k8 Mitogen activated protein kinase kinase Kinase 18 2 12 Tpl2 2 Editor the to Letters kinase 8 7 Hmgcr 3-hydroxy-3-methylglutaryl coenzyme Metabolism 13 1 2 Evi19 1 1 A reductase 50 8 Scd2 Steroyl-coenzyme A desaturase 2 Metabolism 19 2 2 40 & 12.3 kb up, otd 12 & 0.2 kb ups, otd 2 9 Ugcg UDP-glucose ceramide glucosyltransferase Metabolism 4 2 4 18 kb ds 52; 99; 114.8; 117 kb ds 2 10 Ptpn1 Protein tyrosin phosphatase, nonreceptor PO4-tase 2 2 5 Dkmi3 1 1 type 1 11 Gdi3 Rab GDP Dissociation inhibitor RAS family 13 2 1 Intron 1, std, 30 of ATG & 19 kb up 12.4 kb up, otd 2 12 Nf1 RasGAP RAS family 11 1 14 Evi2 1 13 Rasgrp1 Ras guanyl releasing protein 1 RAS family 2 1 24 Evi18 1 14 Csf1r Colony stimulating factor 1 receptor Receptor 18 1 9 Fim2 1 15 Kit Stem cell factor receptor Receptor 5 1 9 Dkmi9 1 16 Mpl Thrombopoietin receptor Receptor 4 2 2 6 & 8 kb up, otd 0.25 &10 kb up 1 1 1 17 Notch2 Notch-related Receptor 3 3 6 Evi54 11 18 Fnbp1 Formin binding protein 1 Signaling 2 1 2 Evi43 1 19 Sh3kbp1 SH3-domain kinase binding protein 1 Signaling X 2 3 10 kb up; intron 2, std 30 & 30 kb up; intron 1 1 20 Ptbp1 Polypyrimidine tract binding protein 1 Splicing 10 2 4 Evi103 2 21 Ahi1/Myb Transcription factor TF/M 10 2 46 Myb 1 1 22 Bach2 BTB and CNC homology 2/repressor TF/M 4 2 6 Evi59 2 23 Bcl11a B-cell CLL/lymphoma 11A TF/M 11 1 7 Evi9 1 1 24 Erg Ets related TF/M 16 2 2 Intron 2, otd Intron 2 2 25 Evi1 Transcription factor/repressor TF/M 3 17 20 Evi1 2 6172 26 Fli1 Ets related TF/M 9 1 7 Ets1 1 1 27 Fos Transcription factor TF/M 12 2 4 Si4 11 28 Gfi1 Zinc finger TF/M 5 3 64 Evi5 1 1 1 29 Hoxa7 Homeobox TF/M 6 1 13 Evi7 2 kb up Leukemia 163 Letters to the Editor 164 other studies (Mouse Retroviral Tagged Cancer Gene Database;2 http://rtcgd.ncifcrf.gov/index.html; Table 2a and b). Of these CIS same 1

¼ 40 have previously been characterized as CIS representing

1 known, suspected or homologs of human cancer genes (Table 2b), while 12 CIS represent novel sites of insertion Tumor type (Table 2a). These new insertion site genes represent excellent cancer gene candidates (Prim2, Gdi3, Scap2, Nkx2–3, and St7). Four CISs encode novel uncharacterized genes (No.’s 9–12, Table 2a). All three Prim2 proviral insertions were in the opposite transcriptional orientation within intron 7, thus raising the possibility that the net effect could be loss of gene function, R reaction were cloned into pAMP1- truncation of an overexpressed N-terminal portion, or over-

y (http://info.med.yale.edu/wmkeck/). The expression of a C-terminal fragment via transcriptional initiation

ddbj.nig.ac.jp/E-mail/homology.html). All PIS at a cryptic promoter downstream of the proviral insertion. others’ data

astic syndrome; 7: erythroleukemia. Prim2, located in human at 6p12–p11.1, encodes a 58 kDa protein, which, together with the p49 subunit, forms the heterodimeric DNA primase . DNA primase plays a downstream of the polyadenylation signal; std

¼ key role in both the initiation of DNA replication and the synthesis of for lagging strand synthesis, specifically by synthesizing short RNA molecules that prime DNA synthesis. While p49 possesses the phosphodiester bond formation activity by itself, p58 plays a critical regulatory role via protein-protein interactions, mediating complex formation with DNA a, as well as binding to the protein, ORC2. Primase is involved in checkpoint 3

Provirus location, our data Provirus location pathway coupling DNA replication to repair. It is possible that within the context of such regulatory functions p58, or a fragment thereof that is generated by the provirally activated

name , may have a dominant stimulatory effect on cell growth. This will be the focus of future studies. Two tumors were identified with proviral insertions into intron a b15 of Pecam1 1234567 , encoding platelet endothelial cell adhesion upstream of the transcription start site; ds

¼ molecule-1 (or PECAM1), a member of the cell adhesion molecule (CAM) subgroup of the immunoglobulin (Ig) super- family. PECAM1 is expressed on the surface of circulating hematopoietic cells. It is also a major constituent of the endothelial cell intercellular junction. PECAM1 is implicated in several functions including transendothelial migration of leukocytes, angiogenesis, integrin activation and cell survival. In some cases of AML and ALL, leukemic cells have low levels of PECAM1 expression which may contribute to the decreased adhesiveness of leukemia cells.4 Consistent with this, endothe- lial cells from Pecam1À/À mice proliferate more rapidly than similarly transformed cells from normal mice and are less adherent and migrate more slowly.5 Pecam1 is expressed in hematopoietic progenitor cells capable of repopulating irra- diated recipients, but its role in these cells is not known. It may be that loss of Pecam1 expression confers a growth advantage on cells but not enough to induce leukemia. The human homolog is located on chromosome 17q23. The Ray1/St7 locus harbors two different starts of transcription and several alternatively spliced exons, thus possessing the possibility of generating multiple protein isoforms including

opposite transcriptional direction; (TF/M) transcription factor/modulator. RAY1 and ST7, which bear little homology to known proteins ¼ and whose function is not known. RAY1 and ST7 are generated from two different transcription start sites and we identified three proviral insertions in the interval between the first exon of RAY1 and that of ST7. ST7 is a candidate tumor suppressor gene with proposed involvement in primary ovarian carcinomas, based on 6

Continued allelic loss at 7q31.1 in such carcinomas. However, this conclusion is disputed by two independent studies (e.g., 7). Our data indicate that overexpression rather than loss of RAY1/ST7

CIS gene Protein product Function Chr. # hits CIS expression contribute to malignancy. Studies to show this is 30 Klf331 Klf432 Lmo233 Nfkb134 Runx135 Sfpi136 Tcf1237 Trp5338 Zfpn1a139 Kruppel-like Dlst40 Kruppel-like Cystein-rich Ifi47 and Lim-domain Rel/Dorsal relatedKey to Runt abbreviations: related # hits: a, PU.1; among Ets our related proviral Helix-loop-helix insertions; b, among Transformation Zinc related results finger protein of (Ikaros) 53 others; up Dihydrolipoamide S-succinyltransferase TF/M Interferon gamma inducible protein/GTP-binding Unchar Unchar TF/M 2 11 TF/M TF/M 1 1 TF/M 12 2 2 Lmo2 Evi107 11 1 TF/M TF/M 65 kb 2 up 2 5 TF/M 3 TF/M 6 4 Trp53 2 1 2 5 4 16 Evi64 Evi157 2 3 Intron Evi162 11 1 9 8, otd 2 53 kb 8 up, 1 1 std 2 Evi13 Spi1 7 4 Dkmi17 10 69 kb & ds, 0.946 std kb up Intron 10 & 91 0.2 kb; kb 286 up kb; 291 kb up 1 1 1 Intron 1 2 1 1 1 1 1 1 1 transcriptional direction; otd Tumor types: 1: preleukemia;High-molecular-weight 2: DNA myeloid was leukemia; extracted 3:cloning from vector monocytic frozen (Gibco leukemia; spleen BRL). 4: tumors Plasmidsresultant histiocytic and were sequences sequenced sarcoma; inverse were using PCR 5: edited M13 was lymphocyticwere forward and performed primers leukemia/lymphoma; compared then by as 6: with compared automated described and myelodyspl against sequencing (1). the submitted at PCR public the to products Keck sequence the Biotechnology from databases Mouse Resource the (http://www.ensembl.org/Mus_musculus/ Laborator Retroviral and secondary Tagged http://www. PC Cancer Gene Database (http://wwwgenome2.ncifcrf.gov/RTCGD). Table 2 actually the case will be the focus of future work.

Leukemia Letters to the Editor 165 The goal of this study was to gain further insight into the Acknowledgements mechanisms involved in F-MuLV-induced neoplastic transfor- mation of nonerythroid hematopoietic lineages. We have This work was supported by NIH Grant no. R01-CA081216 and utilized the unique combination of F-MuLV and C57BL/6 in American Cancer Society Institutional Research Grant IRG-58- order to select for nonerythroid tumors. In addition, most of the 012-45. We thank Joseph Chang, PhD, for his review of our tumors were generated in mice with myelomonocytic-specific statistical approach and comments on the manuscript. We thank expression of SV40 early region, which produces SV40T William Copeland, PhD for helpful discussion regarding Prim2. antigen, a multifunctional protein that can bind to and inactivate p53 and Rb. Thus, we are obviating the need for inactivation of B Yatsula, C Galvao, M McCrann and AS Perkins Department of Pathology, Yale School of Medicine, PO Box either Rb or p53. One of the expectations in this study was to 208023, New Haven, CT 06520-8023, USA identify a small set of genes that cooperate with SV40 early E-mail: [email protected] region.1 However, the results showed a large number of different insertions, with only three genes being found more than three times: Evi1 (17 times), Fim4 (four times) and Fim5 (five times). This suggests that a large number of different genes References can cooperate with SV40 early region in the generation of myelomonocytic neoplasms, but it also precludes one from 1 Kone J, Arroyo J, Savinelli T, Lin S, Boyd K, Wu Y et al. F-MuLV discerning a set of necessary or sufficient cooperating oncogenes Acceleration of myelomonocytic tumorigenesis in SV40T antigen transgenic mice is accompanied by retroviral insertion at Fli1 and a or pathways in this model system. It is currently postulated that novel locus, Fim-4. Leukemia 2002; 16: 1827–1834. integration of ecotropic proviruses tends to occur at or near the 2 Akagi K, Suzuki T, Stephens R, Jenkins N, Copeland N. RTCGD: 50 ends of transcriptionally active genes. Recent studies of retroviral tagged cancer gene database. Nucleic Acids Res 2004; proviral insertions occurring in retroviral marking studies in 32: D523. hematopoietic stem cells showed that proviral insertions occur 3 Michael WM, Ott R, Fanning E, Newport J. Activation of the DNA in genes with known roles in growth regulation;8 even in the replication checkpoint through RNA synthesis by primase. Science 2000; 289: 2133–2137. absence of leukemia and using a replication defective virus. 4 De Waele M, Renmans W, Jochmans K, Schots R, Lacor P, There is likely a selection process in which dominant clones Trullemans F et al. Different expression of adhesion molecules on harboring growth-promoting proviral insertions emerge out of CD34+ cells in AML and B-lineage ALL and their normal bone the population of infected cells. In this study,8 the retrovirus was marrow counterparts. Eur J Haematol 1999; 63: 192–201. nonreplicating, thus precluding acquisition of additional leuke- 5 Rothermel T, Engelhardt B, Sheibani N. Polyoma virus middle-T- mogenic insertions that would complete the transformation transformed PECAM-1 deficient mouse brain endothelial cells process. That we identified 12 new CIS genes in this study proliferate rapidly in culture and form hemangiomas in mice. J Cell Physiol 2005; 202: 230–239. probably reflects our use of a distinctive transgenic strain 6 Zenklusen JC, Weitzel JN, Ball HG, Conti CJ. Allelic loss at 7q31.1 harboring SV40 early region under the control of the myelomo- in human primary ovarian carcinomas suggests the existence of a nocyte-specific gp91phox promoter, which likely caused tumor suppressor gene. Oncogene 1995; 11: 359–363. expansion of specific cells within the host hematopoietic 7 Thomas NA, Choong DYH, Jokubaitis VJ, Neville PJ, Campbell IG. system. Thus, the particular spectrum of insertions we obtained Mutation of the ST7 tumor suppressor gene on 7q31.1 is rare likely reflects the complement of genes that is transcriptionally in breast, ovarian and colorectal cancers. Nat Genet 2001; 29: active in the partially transformed myelomonocytic progenitors 379–380. 8 Kustikova O, Fehse B, Modlich U, Yang M, Du¨llmann J, within the transgenic, preleukemic mouse. If true, this may yield Kamino K et al. Clonal dominance of hematopoietic stem insight into the pattern of in the myelomono- cells triggered by retroviral gene marking. Science 2005; 308: cytic progenitor compartment. 1171–1174.

Expression of Tel-Aml1 transcript variants in pediatric acute lymphoblastic leukemia

Leukemia (2006) 20, 165–167. doi:10.1038/sj.leu.2404028; responsive to Gleevec therapy comparing with Bcr-Ablp190 published online 24 November 2005 positive cells.2 There are data concerning differences in prognosis of patients with various transcripts of Pml-Rara, expressed in acute promyelocytic leukemia.3 Biological or clinical signifi- Translocation t(12;21)(p13;q22), associated with favorable prog- cance of various Tel-Aml1 transcripts is still unknown. Therefore, nosis, is one of the most frequent structural chromosomal we evaluated expression of Tel-Aml1 transcript variants and their aberrations found in pediatric B-lineage acute lymphoblastic relationship to laboratory features of childhood ALL. leukemia (ALL). This aberration leads to the fusion of exon 5 of A total of 172 patients with B-lineage ALL (age 0.8–18.0 Tel gene with exons 2 or 3 of Aml1 gene and results in long and years) were included into this study. Diagnosis of ALL was short transcripts of Tel-Aml1. The short variant is differed from long verified according to FAB-classification. Immunological diag- form by 39 base pairs, that reflects the loss of the exon 2 of Aml1 nosis was defined using scoring system devised by the European 1 gene during rearrangement or in the process of alternative splicing. Group for the Immunological Classification of Leukemia (EGIL). It is known that various splice-variants/transcripts of fusion Cytogenetic study was conducted using standard G-banding on genes can be associated with different clinical features. For slides with cultured leukemic cells. Expression of Tel-Aml1 was p210 example, leukemic clone expressing Bcr-Abl is more evaluated using nested RT-PCR.4

Leukemia