Activation of the c-myb locus is insufficient for the rapid induction of disseminated avian B-cell lymphoma.

We have previously reported that infection of 9- to 13-day-old chicken embryos with RAV-1 results in rapid development of a novel B-cell lymphoma in which proviral insertion has activated expression of the c-myb gene (E. Pizer and E. H. Humphries, J. Virol. 63:1630-1640, 1989). The biological properties of these B-cell lymphomas are distinct from those associated with the B-cell lymphomas that develop following avian leukosis virus proviral insertion within the c-myc locus. In an extension of this study, more than 200 chickens, infected as 10- to 11-day-old embryos, were examined for development of lymphomas that possess disrupted c-myb loci. Fourteen percent developed disseminated B-cell lymphoma. In the majority of these tumors, the RAV-1 provirus had inserted between the first and second exons that code for p75c-myb. However, insertions between the second and third exons and between the third and fourth exons were also detected. In situ analysis of myb protein expression in tumor tissue revealed morphological features suggesting that the tumor originates in the bursa. Within the bursa, the lymphoma appeared to spread from follicle to follicle without compromising the structural integrity of the organ. Tumor masses in liver demonstrated heterogeneous levels of myb protein suggestive of biologically distinct subpopulations. In contrast to the morbidity data, immunohistological analysis of bursae from 4- to 6-week-old chickens at risk of developing lymphomas bearing altered c-myb loci revealed lesions expressing elevated levels of myb in 16 of 19 birds. The activated myb lymphoma displayed very poor capacity to proliferate outside its original host. Only 1 of 33 in vivo transfers of tumor to recipient hosts established a transplantable tumor. None of the primary tumor tissue nor the transplantable tumor exhibited the capacity for in vitro proliferation. Similar experimental manipulation has yielded in vitro lines established from avian B-cell lymphomas expressing elevated levels of c-myc or v-rel. The dependence on embryonic infection for development of activated-myb lymphoma suggests a requirement for a specific target cell in which c-myb is activated by proviral insertion. It is likely, moreover, that continued tumor development requires elevated expression of myb proteins within a specific cell population in a restricted stage of differentiation.     

Rorgamma (Rorc) is a common integration site in type B leukemogenic virus-induced T-cell lymphomas

The retrovirus type B leukemogenic virus (TBLV) causes T-cell lymphomas in mice. We have identified the Rorgamma locus as an integration site in 19% of TBLV-induced tumors. Overexpression of one or more Rorgamma isoforms in >77% of the tumors tested may complement apoptotic effects of c-myc overexpression.     

The 3' IgH locus control region is sufficient to deregulate a c-myc transgene and promote mature B cell malignancies with a predominant Burkitt-like phenotype

Burkitt lymphoma (BL) features translocations linking c-myc to an Ig locus. Breakpoints in the H chain locus (IgH) stand either close to J(H) or within switch regions and always link c-myc to the 3' IgH locus control region (3' LCR). To test the hypothesis that the 3' LCR alone was sufficient to deregulate c-myc, we generated mice carrying a 3' LCR-driven c-myc transgene and specifically up-regulating c-myc in B cells. Splenic B cells from mice proliferated exaggeratedly in response to various signals had an elevated apoptosis rate but normal B220/IgM/IgD expression. Although all Ig levels were lowered in vivo, class switching and Ig secretion proved normal in vitro. Beginning at the age of 12 wk, transgenic mice developed clonal lymphoblastic lymphomas or diffuse anaplastic plasmacytomas with an overall incidence of 80% by 40 wk. Lymphoblastic lymphomas were B220(+)IgM(+)IgD(+) with the BL "starry sky" appearance. Gene expression profiles revealed broad alterations in the proliferation program and the Ras-p21 pathway. Our study demonstrates that 3' IgH enhancers alone can deregulate c-myc and initiate the development of BL-like lymphomas. The rapid and constant occurrence of lymphoma in this model makes it valuable for the understanding and the potential therapeutic manipulation of c-myc oncogenicity in vivo.     

Interleukin-6- and leukemia inhibitory factor-induced terminal differentiation of myeloid leukemia cells is blocked at an intermediate stage by constitutive c-myc.

Interleukin-6 (IL-6) and leukemia inhibitory factor (LIF), two multifunctional cytokines, recently have been identified as physiological inducers of hematopoietic cell differentiation which also induce terminal differentiation and growth arrest of the myeloblastic leukemic M1 cell line. In this work, it is shown that c-myc exhibited a unique pattern of expression upon induction of M1 terminal differentiation by LIF or IL-6, with an early transient increase followed by a decrease to control levels by 12 h and no detectable c-myc mRNA by 1 day; in contrast, c-myb expression was rapidly suppressed, with no detectable c-myb mRNA by 12 h. Vectors containing the c-myc gene under control of the beta-actin gene promoter were transfected into M1 cells to obtain M1myc cell lines which constitutively synthesized c-myc. Deregulated and continued expression of c-myc blocked terminal differentiation induced by IL-6 or LIF at an intermediate stage in the progression from immature blasts to mature macrophages, precisely at the point in time when c-myc is normally suppressed, leading to intermediate-stage myeloid cells which continued to proliferate in the absence of c-myb expression.     

Analysis of wild-type and mutant SL3-3 murine leukemia virus insertions in the c-myc promoter during lymphomagenesis reveals target site hot spots, virus-dependent patterns, and frequent error-prone gap repair

The murine leukemia retrovirus SL3-3 induces lymphomas in the T-cell compartment of the hematopoetic system when it is injected into newborn mice of susceptible strains. Previously, our laboratory reported on a deletion mutant of SL3-3 that induces T-cell tumors faster than the wild-type virus (S. Ethelberg, A. B. Sorensen, J. Schmidt, A. Luz, and F. S. Pedersen, J. Virol. 71:9796-9799, 1997). PCR analyses of proviral integrations in the promoter region of the c-myc proto-oncogene in lymphomas induced by wild-type SL3-3 [SL3-3(wt)] and the enhancer deletion mutant displayed a difference in targeting frequency into this locus. We here report on patterns of proviral insertions into the c-myc promoter region from SL3-3(wt), the faster variant, as well as other enhancer variants from a total of approximately 250 tumors. The analysis reveals (i) several integration site hot spots in the c-myc promoter region, (ii) differences in integration patterns between SL3-3(wt) and enhancer deletion mutant viruses, (iii) a correlation between tumor latency and the number of proviral insertions into the c-myc promoter, and (iv) a [5'-(A/C/G)TA(C/G/T)-3'] integration site consensus sequence. Unexpectedly, about 12% of the sequenced insertions were associated with point mutations in the direct repeat flanking the provirus. Based on these results, we propose a model for error-prone gap repair of host-provirus junctions.     

Radiation leukemia virus common integration at the Kis2 locus: simultaneous overexpression of a novel noncoding RNA and of the proximal Phf6 gene

Retroviral tagging has been used extensively and successfully to identify genes implicated in cancer pathways. In order to find oncogenes implicated in T-cell leukemia, we used the highly leukemogenic radiation leukemia retrovirus VL3 (RadLV/VL3). We applied the inverted PCR technique to isolate and analyze sequences flanking proviral integrations in RadLV/VL3-induced T lymphomas. We found retroviral integrations in c-myc and Pim1 as already reported but we also identified for the first time Notch1 as a RadLV common integration site. More interestingly, we found a new RadLV common integration site that is situated on mouse chromosome X (XA4 region, bp 45091000). This site has also been reported as an SL3-3 and Moloney murine leukemia virus integration site, which strengthens its implication in murine leukemia virus-induced T lymphomas. This locus, named Kis2 (Kaplan Integration Site 2), was found rearranged in 11% of the tumors analyzed. In this article, we report not only the alteration of the Kis2 gene located nearby in response to RadLV integration but also the induction of the expression of Phf6, situated about 250 kbp from the integration site. The Kis2 gene encodes five different alternatively spliced noncoding RNAs and the Phf6 gene codes for a 365-amino-acid protein which contains two plant homology domain fingers, recently implicated in the B?rjeson-Forssman-Lehmann syndrome in humans. With the recent release of the mouse genome sequence, high-throughput retroviral tagging emerges as a powerful tool in the quest for oncogenes. It also allows the analysis of large DNA regions surrounding the integration locus.     

Deregulated c-myb disrupts interleukin-6- or leukemia inhibitory factor-induced myeloid differentiation prior to c-myc: role in leukemogenesis.

The c-myb proto-oncogene is abundantly expressed in tissues of hematopoietic origin, and changes in endogenous c-myb genes have been implicated in both human and murine hematopoietic tumors. c-myb encodes a DNA-binding protein capable of trans-activating the c-myc promoter. Suppression of both of these proto-oncogenes was shown to occur upon induction of terminal differentiation but not upon induction of growth inhibition in myeloid leukemia cells. Myeloblastic leukemia M1 cells that can be induced for terminal differentiation with the physiological hematopoietic inducers interleukin-6 and leukemia inhibitory factor were genetically manipulated to constitutively express a c-myb transgene. By using immediate-early to late genetic and morphological markers, it was shown that continuous expression of c-myb disrupts the genetic program of myeloid differentiation at a very early stage, which precedes the block previously shown to be exerted by deregulated c-myc, thereby indicating that the c-myb block is not mediated via deregulation of c-myc. Enforced c-myb expression also prevents the loss in leukemogenicity of M1 cells normally induced by interleukin-6 or leukemia inhibitory factor. Any changes which have taken place, including induction of myeloid differentiation primary response genes, eventually are reversed. Also, it was shown that suppression of c-myb, essential for terminal differentiation, is not intrinsic to growth inhibition. Taken together, these findings show that c-myb plays a key regulatory role in myeloid differentiation and substantiate the notion that deregulated expression of c-myb can play an important role in leukemogenicity.     

ALY is a common coactivator of RUNX1 and c-Myb on the type B leukemogenic virus enhancer

Type B leukemogenic virus (TBLV), a mouse mammary tumor virus (MMTV) variant, often induces T-cell leukemias and lymphomas by c-myc activation following viral DNA integration. Transfection assays using a c-myc reporter plasmid indicated that the TBLV long terminal repeat (LTR) enhancer is necessary for T-cell-specific increases in basal reporter activity. The sequence requirements for this effect were studied using mutations of the 62-bp enhancer region in an MMTV LTR reporter vector. Deletion of a nuclear factor A-binding site dramatically reduced reporter activity in Jurkat T cells. However, a 41-bp enhancer missing the RUNX1 site still retained minimal enhancer function. DNA affinity purification using a TBLV enhancer oligomer containing the RUNX1 binding site followed by mass spectrometry resulted in the identification of ALY. Subsequent experiments focused on the reconstitution of enhancer activity in epithelial cells. ALY overexpression synergized with RUNX1B on TBLV enhancer activity, and synergism required the RUNX1B-binding site. A predicted c-Myb binding site in the enhancer was confirmed after c-myb overexpression elevated TBLV LTR reporter activity, and overexpression of c-Myb and RUNX1B together showed additive effects on reporter gene levels. ALY also synergized with c-Myb, and coimmunoprecipitation experiments demonstrated an interaction between ALY and c-Myb. These experiments suggest a central role for ALY in T-cell enhancer function and oncogene activation.     

Cellular oncogene expression in human tumors transplantable to athymic mice

Expression of 3 cellular oncogenes among 7 ones under investigation is identified in the majority of 20 strains of human tumors, passaged in nude mice without significant specificity as far as the type of the tumor is concerned. The levels of the expression of these 3 oncogenes (c-myc, c-fos, c-ras) were higher than the ones in primary human tumors except for the human melanoma Mel-2 strain, where the expression of c-myb oncogene was identified. All the rest oncogenes (c-mos, B-lym, c-sis, c-myb) showed no expression in human tumors of the examined strains.     

Genetic mapping of a cellular DNA region involved in induction of thymic lymphomas (Mlvi-1) to mouse chromosome 15.

Mlvi-1 defines a genetic locus representing a common domain for proviral DNA integration in Moloney murine leukemia virus-induced rat thymic lymphomas. Cellular sequences homologous to Mlvi-1 are present in mouse DNA, and we have used hamster-mouse somatic cell hybrids to chromosomally map Mlvi-1 in the mouse genome. Results demonstrated that Mlvi-1 maps to mouse chromosome 15 and that it is distinct from the Mlvi-2 integration region and from the cellular oncogenes c-myc and c-sis, which also map to this chromosome. Therefore, Mlvi-1 may contain novel sequences involved in the establishment and maintenance of virus-induced murine tumors, many of which contain abnormalities of chromosome 15.