Reversible Tumorigenesis by MYC in Hematopoietic Lineages

Molecular Cell, Vol. 4, 199–207, August, 1999, Copyright 1999 by Cell Press

Reversible Tumorigenesis by MYC in Hematopoietic Lineages Dean W. Felsher*†‡ and J. Michael Bishop† * Division of Hematology-Oncology Department of Medicine † G. W. Hooper Foundation and Department of Microbiology and Immunology University of California San Francisco, California 94143-0552

Summary The targeted repair of mutant protooncogenes or the inactivation of their gene products may be a specific and effective therapy for human neoplasia. To examine this possibility, we have used the tetracycline regulatory system to generate transgenic mice that conditionally express the MYC protooncogene in hematopoietic cells. Sustained expression of the MYC transgene culminated in the formation of malignant T cell lymphomas and acute myleoid leukemias. The subsequent inactivation of the transgene caused regression of established tumors. Tumor regression was associated with rapid proliferative arrest, differentiation and apoptosis of tumor cells, and resumption of normal host hematopoiesis. We conclude that even though tumorigenesis is a multistep process, remediation of a single genetic lesion may be sufficient to reverse malignancy.

Introduction Cancer is commonly associated with gain-of-function mutations affecting protooncogenes (Bishop, 1991). Therapies that repair these mutant genes or inactivate their protein products may be useful in the treatment of cancer. Since cancer is caused by multiple genetic abnormalities, targeting the inactivation of one oncogene may not be effective in inducing tumor regression. Moreover, since the genomes of tumors are typically unstable, they may easily accommodate therapies that target a single mutant gene (Lengauer et al., 1998). There is presently no method to systematically identify which oncogenes may be most useful to pharmacologically target for the therapy of neoplasia. The development of the tetracycline regulatory system (tet system) provides the ability to generate transgenic mice that can conditionally express particular oncogenes in specific tissues (Gossen and Bujard, 1992; Furth et al., 1994; Efrat et al., 1995; Gossen et al., 1995; Ewald et al., 1996; Kistner et al., 1996; Tremblay et al., 1998). We reasoned that if the inactivation of one oncogene were sufficient to induce tumor regression, then tumors induced by a conditionally expressed transgenic oncogene should regress if that transgene were inactivated. MYC is frequently overexpressed in human neoplasia (Marcu et al., 1992; Dang, 1999). Transgenic models ‡ To whom correspondence should be addressed (e-mail: felsher@

itsa.ucsf.edu).

have confirmed that MYC overexpression causes tumorigenesis (Adams et al., 1985; Leder et al., 1986; Harris et al., 1988; Morgenbesser and DePinho, 1994). Thus, MYC may be a good target for therapy of many types of human malignancies. MYC encodes a transcription factor that is likely to contribute to tumorigenesis through its sustained effects on cellular proliferation and differentiation (Marcu et al., 1992; Bouchard et al., 1998; Facchini and Penn, 1998). Therefore, it seemed plausible that the loss of MYC overexpression may cause tumors to regress. In this study, we used the tet system to generate mice that conditionally expressed a MYC transgene in their hematopoietic cells. Expression of the transgene led to malignant lymphoid and myeloid tumors. Inactivation of the transgene prevented development of tumors and also caused sustained regression of established tumors. Regression apparently resulted from the differentiation of tumor cells to a nonproliferative stage. We conclude that despite the multistep nature of tumorigenesis, remediation of a single genetic lesion can have therapeutic value. Results Conditional Tumorigenesis of a MYC Transgene in Hematopoietic Cells We used a version of the tet system in which the tetracycline-transactivating protein (tTA) mediates the transcription of a transgene placed under the control of the tetracycline-responsive promoter (tet-o). The presence of tetracycline or doxycycline inactivates transcription mediated by tTA. Thus, to regulate MYC transcription in hematopoietic cells, we generated two sorts of transgenic mice (Figure 1A). The first contained the human MYC cDNA under the control of the tetracycline-responsive minimal promoter (tet-o-MYC). The second expressed the tTA under the control the immunoglobulin heavy chain enhancer and the SRa promoter (EmSR-tTA). We identified multiple independent transgenic founder lines for each construct. When lines from each construct were crossed, they yielded progeny that conditionally expressed the MYC transgene. Four out of the five teto-MYC lines when crossed with one of the EmSR-tTA lines resulted in tumorigenesis. Five out of the eight EmSR-tTA lines when crossed with one of the tet-oMYC lines resulted in tumorigenesis. Unless otherwise specified, the results described below were for transgenic mice that are from a cross between EmSR-tTA line 83 and tet-o-MYC line 36, which produced hematopoietic tumors at a frequency of 100%. Splenic lymphocytes from mice transgenic for both EmSR-tTA and tet-o-MYC were found to express MYC in the absence but not the presence of doxycycline (Figure 1B). Mice transgenic for either EmSR-tTA or teto-MYC, or mice transgenic for both constructs but treated continuously with doxycycline, developed no tumors, had a normal life expectancy, exhibited normal histology of hematopoietic tissues, and contained normal numbers of immature and differentiated lymphoid

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Figure 1. A Conditional Transgenic Model for MYC-Induced Tumorigenesis (A) Tetracycline regulatory system constructs. (B) Mice transgenic for both EmSR-tTA and tet-o-MYC conditionally express MYC protein in splenic lymphocytes. Western analysis was performed on spleen lymphocytes from transgenic mice in the absence (2) or the presence (1) of doxycycline in drinking water. Similar results were seen in four independent experiments. (C) Survival curves for transgenic mice. Open squares, mice transgenic for EmSR-tTA and tet-o-MYC; closed squares, mice transgenic for EmSR-tTA and tet-o-MYC with doxycycline treatment or either EmSR-tTA or tet-o-MYC alone. There were at least 20 animals per group. (D) Deceased animals had an enlarged thymus, spleen, liver, and mesenteric lymph nodes. Histology demonstrated invasion of (E) spleen (403), (F) blood (1003), (G) kidney (403), and (H) liver (203).

and myeloid cells (Figure 1C and data not shown). In the absence of doxycycline treatment, mice transgenic for both constructs died within 5 months of age (Figure 1C) of invasive tumors (Figures 1D–1H). We confirmed that tumor cells exhibited abundant levels of transgenic MYC protein expression (see below, Figure 3D). The tumors were manifested by gross enlargement of the thymus, liver, spleen, and the gastrointestine (Figure 1D). Tumors histologically resembled a high grade, diffuse large cell lymphoma (Figure 1E), which effaced the architecture of thymus (data not shown), spleen (Figure 1E), and bone marrow (data not shown). Tumor cells also invaded the peripheral blood (Figure 1F), kidney (Figure 1G, see arrows), and liver (Figure 1H, see arrow). Gastrointestinal invasion of mesenteric lymph nodes was accompanied by invasion into the lamina propria of the small and large intestine (see below, Figure 5D, and data not shown). Of 16 tumors analyzed, 14 were found to be immature CD41 CD81 T cell lymphomas. These tumors were also surface positive for other T cell markers, including T cell receptor a/b, Thy1, CD3, and CD5. Tumors were negative for the B cell markers IgM and B220 and for the myeloid marker GR1 (data not shown). Of 16 tumors analyzed, 2 were found to be acute myeloid leukemias

bearing the surface markers Gr-11 and Mac-1, but negative for T cell and B cell markers (data not shown). To assess their clonality, tumors were analyzed for T cell receptor rearrangements in their b loci and the surface expression of specific Vb family members (Figure 2). All tumors analyzed exhibited no more than two clonal rearrangements at a given locus, suggesting that they were derived from a single cell (Figure 2A, see asterisks). At the Jb1 locus, tumor 967 exhibited one rearranged and one deleted allele, and tumors 1232 and 1137 exhibited two rearrangements (Figure 2A, C1b blot). At the Jb2 locus, tumor 967 has rearranged one allele, tumor 1232 was germline for both alleles, and tumor 1137 had rearranged both alleles (Figure 2A, J2b blot). In addition, nine tumors were analyzed by FACS and found to be surface positive for only 1 of a panel of 15 antibodies specific for distinct Vb families (Figure 2B and data not shown). These results indicate that the tumors were clonal. Tumor Regression with the Inactivation of the MYC Transgene To determine whether inactivation of the MYC transgene was sufficient to cause regression of tumors, transgenic mice moribund with tumor burden were treated with doxycycline (Figure 3A). Of the 20 mice treated, 2 mice

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Figure 2. Clonality of Tumors (A) Tumors exhibited clonal T cell receptor b chain rearrangements. Southern blot of HindIII-digested genomic DNA hybridized with probes to Cb1 or Jb2, as shown in the figure. Arrows at left indicate the position of germline fragments. Asterisks indicate rearranged fragments. (B) Tumor cells were surface positive for a single T cell receptor, Vb. Tumors were analyzed by FACS analysis for the surface expression of T cell receptor Vb2, 3, 4, 5.1 and 5.2, 6, 7, 8.1 and 8.2, 8.3, 9, 10b, 11, 12, 13, 14, and 17a (Pharmingen, TCR screening panel). Tumors were surface positive only for the indicated Vb.

died within 2 days after commencing treatment (perhaps because treatment was initiated in mice with too advanced disease). Within 3 days of treatment, the other 18 mice became more physically active and exhibited reduction in abdominal girth, loss of lymph node enlargement, and absence of a palpable spleen. After 6 weeks, 2 of these mice relapsed with tumor and died, but the other 16 mice exhibited a sustained remission observed for up to 30 weeks after the initiation of treatment (Figure 3A). Thus, inactivation of the MYC transgene was sufficient to induce tumor regression in 90% of treated mice, with sustained remission in 80% of mice for as long as 30 weeks. Tumor regression was observed in transgenic mice derived from two different EmSR-tTA lines and four different tet-o-MYC lines. Tumor regression could be demonstrated by Gallium scan (Figure 3B). Gallium-67 is a radioisotope avid for tumor cells that is useful in the evaluation of the therapeutic response in human lymphoma patients (Vose et al., 1996). As predicted, mice incorporated gallium at sites of tumor burden. Two weeks after the initiation of

doxycycline treatment, tumors had regressed, and the Gallium scan appeared identical to a control mouse. We also demonstrated tumor regression by magnetic resonance imaging (data not shown). Similarly, we found that inactivation of the MYC transgene induced regression of tumors that had been transplanted into syngeneic mice (Figure 3C). Six different transgenic tumors were prepared as single cell suspensions, then directly transplanted. Animals bearing transplanted tumors recapitulated the pattern of tumor invasion of primary transgenic tumors, with marked enlargement of thymus, spleen, and gastrointestine (compare Figure 1D with Figure 4A). Transplant recipients exhibited profound reduction in abdominal girth, palpable spleen, and lymphadenopathy within 3 days of treatment with doxycycline. Within 2 weeks of treatment, tumors regressed with loss of the thymic tumor, reduction in liver and spleen size, and the absence of a gastrointestinal mass (Figure 4A versus 4B, see arrows with labels). Histologic regression of tumor cells was observed in liver (Figure 4C versus 4D, see arrows), kidney

Figure 3. Inactivation of the MYC Transgene Resulted in Tumor Regression (A) Survival of transgenic mice with tumors that were then treated (closed squares, n 5 20) or not treated (open squares, n 5 5) with doxycycline. (B) Gallium imaging of control mouse (Control), a mouse moribund from tumor prior to doxycycline treatment (Pre-Rx), or 2 weeks after doxycycline treatment (Doxycycline). Identical results were seen in eight other mice. (C) Survival of transplant recipients of transgenic tumors treated with doxycycline when moribund with tumor burden. Open squares portray results with tumors transplanted directly into syngeneic hosts (tumors 966, 967, 1232, 1137, 1979, and 2263) or after in vitro passage for 2 months (tumors 1137 and 1232). Open circles portray results of tumor 966 after in vitro passage for 2 months. In all cases, transplanted tumors were inoculated into at least 12 syngeneic mice intraperitoneally, 107 cells per mouse. (D) MYC expression in relapsed tumors. Western analysis in the absence (2) or presence (1) of doxycycline treatment of tumor 966 and two doxycycline-resistant variants (R1 and R2).

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did not relapse unless they were first passaged in vitro prior to inoculation into syngeneic hosts (tumor 966 and 967, Figure 3C and data not shown). Some transplanted tumors did not relapse even after being passaged in vitro for 2 months (tumor 1232 and 1137, Figure 3C). Next, we investigated the mechanisms of tumor relapse. These tumors had an identical morphology and FACS phenotype to the parent tumors when analyzed (data not shown). Relapsed tumors remained transplantable and were invasive (data not shown). Some relapsed tumors exhibited loss of expression of transgenic MYC in the presence of doxycycline (see lane R1, Figure 3D). Presumably, these tumors had undergone secondary genomic events that phenocopied MYC. Other relapsed tumors expressed the MYC transgene despite the presence of doxycycline (see lane R2, Figure 3D). Apparently, the latter group of relapsed tumors had escaped the conditional regulation imposed by the tet system.

Figure 4. Inactivation of the MYC Transgene Resulted in Sustained Tumor Regression in Transplant Recipients Tumor 966 was transplanted into syngeneic mice, and when moribund, the mice were treated with doxycycline for 2 weeks. (A) Prior to doxycycline treatment, mice exhibited enlargement of thymus, spleen, liver, and a gastrointestinal mass (see labels with arrows). (B) Doxycycline treatment resulted in the apparent absence of tumor in thymus, liver, spleen, and gastrointestine (see labeled arrows). Doxycycline treatment resulted in the histologic regression of tumors in liver ([C] versus [D], 203) and kidney ([E] versus [F], 203). Identical results were seen in five other mice. The same results were seen in syngeneic mice transplanted with tumor 967, 1137, 1232, 1979, or 2263.

(Figure 4E versus 4F, see arrow), gastrointestine, bone marrow, and blood (data not shown). Treated transplant recipients exhibited sustained tumor regression for as long as 60 weeks (Figure 3C). Tumor Relapse despite Inactivation of the MYC Transgene Although 90% of transgenic mice with tumors initially responded to doxycycline treatment, 10% of the mice relapsed after 6 weeks of continuous doxycycline treatment (Figure 3A). This suggested to us that some tumors could escape the requirement for MYC activation. We wondered if passage of tumors in syngeneic hosts could increase the frequency of relapse. Transplanted tumors

Mechanism of Tumor Elimination To examine the kinetics and mechanism of tumor elimination, mice that were moribund with tumor were treated with doxycycline, then sacrificed at different times for analysis. As noted above, tumors invaded the spleen, bone marrow, and lamina propia of the intestine (Figures 5A and 5D, and data not shown). After 3 days of doxycycline treatment, tumor cells uniformly became smaller with a higher nuclear-to-cytoplasmic ratio, consistent with their differentiation from blast cells to more mature lymphocytes, which was also notable in the lamina propia (Figure 5F versus 5G). The primary follicles reappeared in the spleen (Figure 5A versus 5B), and loss of tumor volume was associated with the formation of vacuoles in the lamina propia (see arrow, Figure 5E). After 6 days of doxycycline treatment, there was restoration of normal splenic architecture with the presence of primary follicles with germinal centers (see arrows, Figure 5C). Correspondingly, after 3 or 6 days of doxycycline treatment, we found by FACS analysis that tumor regression was accompanied by the appearance in the spleen of normal T cells, B cells, and myeloid cells (data not shown). Thus, MYC inactivation appears to lead to tumor cell differentiation associated with the restoration of normal host hematopoiesis. The appearance of differentiated lymphocytes in the spleen and bone marrow also could be explained by the restoration of normal host hematopoiesis in areas previously occupied by tumor cells. However, this is an unlikely account for the observation that tumors in the lamina propia differentiated in mature lymphocytes (Figure 5F versus 5G). The lamina propia does not normally contain hematopoetic cells, so there is little reason to suppose the abundant population of mature lymphocytes corresponded to normal host lymphocytes that were induced to migrate and proliferate there. The rapidity and magnitude of reduction in tumor burden suggested to us that not only were the tumor cells differentiating, but many of the tumor cells were being eliminated. To assess whether any of the tumor cells were undergoing apoptosis, we used the Tunel assay to analyze tumors before or after doxycycline treatment. Prior to treatment, tumors exhibited no evidence for Tunel-positive cells (Figure 5H). After 3 or 6 days of

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Figure 5. Kinetics of Tumor Regression Tumor 966 was transplanted into syngeneic mice, and when moribund, the mice were treated with doxycycline. Animals were sacrificed before treatment, or either 3 or 6 days after instituting treatment with doxycycline. (A–C) Histology of the spleen after 0, 3, or 6 days of doxycycline treatment. Histology of the gastrointestine 0 or 3 days after doxycycline treatment ([D] versus [E], 203; [H] versus [I], 1003). (F and G) Tunel assay of a gastointestinal tumor, either 0 or 3 days after doxycycline treatment. (J and K) DAPI staining of a gastrointestinal tumor 0 and 3 days after doxycycline treatment. Three animals were analyzed per time point. Identical results were seen with tumor 967.

treatment, many areas of the tumors exhibited frequent Tunel-positive cells (Figure 5H versus 5I, and data not shown). Therefore, apoptosis may contribute to the loss of some of the tumor cells. We also used short-term in vitro cultures of tumor cells to further address the cellular consequences of the inactivation of the MYC transgene. Treatment of lymphomas or myeloid leukemias with doxycycline resulted in a loss of MYC expression (Figure 3C and data not shown). In the absence of doxycycline, tumor cells exponentially increased in number (Figure 6A, open squares). In contrast, when treated with doxycycline, tumor cells no longer expanded in number (Figure 6A, closed squares). FACS analysis for DNA content demonstrated that tumor lines were highly proliferative with the majority of cells synthesizing DNA, as illustrated by the incorporation of BRDU (Figure 6B). Moreover, tumor cells exhibited a substantial proportion of cells accumulating in G2/M and a polyploid subpopulation (Figure

6B, left panels). Tumor cells treated in vitro with doxycycline for 2 days underwent proliferative arrest in G1 with a diploid DNA content and the absence of BRDU incorporation (Figure 6B, right panels). There was no longer a polyploid subpopulation. Many cells were subdiploid, consistent with apoptosis. These results are consistent with our observations in vivo that MYC inactivation leads to the proliferative arrest of tumor cells. Loss of MYC overexpression in tumor cells in vitro was also associated with morphologic and phenotypic changes consistent with differentiation (Figure 6C). Lymphomas exhibited differentiation from lymphoblasts to mature lymphocytes (T cell, Figure 6C). Acute myeloid leukemia tumor lines similarly matured from myeloblasts to differentiated myeloid cells with the characteristic donutshaped nucleus seen in murine neutrophils (AML, Figure 6C). Removing doxycycline from differentiated tumors restored MYC expression, but tumor cells remained quiescent and morphologically unchanged (data not shown).

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Figure 6. Inactivation of MYC Resulted in Differentiation (A) Relative cell number in the absence (open squares) or presence (closed squares) of doxycycline (tumor 966). Cell lines derived from tumor were grown in vitro in the presence or absence of doxycylcine and counted in triplicate every 2 days in the presence of trypan blue to determine the total number of viable cells. Data are expressed as the ratio between the mean number of viable cells and the total number of cells prior to instituting treatment with doxycycline. Identical results were seen with tumors 967, 1137, and 1232. (B) Cell cycle arrest with doxycycline treatment (tumor 966). FACS and PI staining are shown in the bottom two panels. BRDU incorporation and PI staining are shown in the top two panels. Identical results were seen with tumors 967, 1137, and 1232. (C) Differentiation of tumors after doxycycline treatment. Giemsa staining of cytospun preparations of tumor cells treated in vitro with doxycycline for 2 days is shown. T cell lymphoma (T cell) was tumor 967. Acute myeloid leukemia (AML) was tumor 1137. Identical results were seen with T cell lymphomas 966 and 1232, and acute myeloid leukemia 785.

Discussion Reversible Malignancies Induced by the MYC Protooncogene We have found that highly invasive and lethal hematopoietic tumors induced by a conditionally transcribed

transgene of MYC will regress if the transgene is inactivated. The reversibility of the neoplastic phenotype of these tumors raises two issues that bear on the significance of our findings. First, the tumors might not be authentic malignancies but instead result from the polyclonal proliferation of

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otherwise normal lymphoid cells and, thus, may be more readily reversible. We do not believe this is the case. The tumor cells are highly invasive, transplantable, immortal, immature, aneuploid, and clonal. This combination of properties bespeaks a malignant phenotype. Second, the tumors might be attributable solely to the overexpression of MYC, in contrast to most other malignancies, which arise from a combination of genetic anomalies (Bishop, 1991; Hunter, 1991). Two observations argue against this possibility: the latent period of several months that precedes appearance of the tumors and the clonality of the tumors. Both observations suggest that once tumorigenesis has been initiated by a transgene, further progression occurs in the form of additional genetic mutations that engender the eventual malignancy. In the face of this inference, it is provocative that the two types of hematopoietic malignancies could be reversed by mitigating the effect of the initiating lesion; it appears that in this instance, at least, the malignant cells have not evolved away from dependence upon that lesion. Variations in the Hematopoietic Tumors Elicited by a MYC Transgene In previous work, a MYC transgene driven by the EmVH control element produced lymphomas of pre–B cells (Adams et al., 1985; Harris et al., 1988). In the present instance, a MYC transgene driven by the EmSRa control element resulted in T cell lymphomas and occasional acute myeloid leukemias. There are at least two possible explanations for this difference. First, chromosomal position of the transgenes and strain differences among mice alter the tissue specificity of Em-VH (Yukawa et al., 1989). Second, the SRa promoter is more likely to produce high levels of gene expression in T cells (Bodrug et al., 1994). In any event, elevated expression of MYC has been reported in T cell lymphomas and acute myeloid leukemias of humans (Marcu et al., 1992), so the findings in the present study are potentially relevant to human disease. Tumors Can Escape Dependence upon MYC We identified occasional tumors that evolved to become independent of the MYC transgene. The propensity to evolve to independence appeared to be an intrinsic property of certain tumors, and propagation of these tumors in vitro appeared to increase that propensity. Subsequently, inactivation of the MYC transgene no longer elicited tumor regression. Other tumors retained their dependence upon MYC despite cultivation in vitro. We presume that some tumors became independent of MYC by acquiring genetic lesions that substituted for the requirement of MYC. The ability of tumors to gain independence from MYC may be attributable to the action of MYC itself. MYC has the ability to destabilize the cellular genome (Cerni et al., 1986; Mai et al., 1996; Chernova et al., 1998; Felsher and Bishop, 1999), which could facilitate the accumulation of tumorigenic mutations. There is a precedent for this proposal in our finding that the transient overexpression of MYC in Rat1A cells concomitantly destabilizes the genome and substantially increases their predisposition to tumorigenesis (Felsher and Bishop,

1999). In this case, the resulting tumors are no longer dependent upon continued overexpression of MYC. The Mechanism by which MYC Induces Tumorigenesis The action of MYC has been implicated in a variety of cellular functions, including progression through the cell cycle (Marcu et al., 1992), differentiation (Marcu et al., 1992; Broussard-Diehl et al., 1996; Facchini and Penn, 1998), and apoptosis (Evan et al., 1995). Distortion of any of these cellular functions is potentially tumorigenic. Hematopoietic tumors elicited by MYC have been generally attributed to impedance of differentiation, which creates an expanding population of immature cells that are at risk of accumulating additional tumorigenic mutations (Harris et al., 1988; Marcu et al., 1992). The T cell lymphomas and acute myeloid leukemias elicited by the conditional transgenic MYC used in our studies conform to this idea. The malignant tumor cells were immature. Loss of the overexpression of MYC was associated with the differentiation of tumor cells and a loss of their malignant potential. Arrest of proliferation is a natural accompaniment of differentiation. However, the apoptosis observed here is more difficult to explain. Perhaps the great surfeit of newly differentiated hematopoietic cells outstripped the supportive capacity of the cellular microenvironment. Alternatively, differentiated T cells would not have undergone appropriate selection in the thymus and thus may have been marked for destruction by apoptosis (Broussard-Diehl et al., 1996). Possibly, MYC countered the proapoptotic effects of other unknown genetic lesions in the tumor cells. As for the specific case of the acute myeloid leukemias, differentiated neutrophils are normally short-lived. Remediation of One Genetic Event Can Be Sufficient to Reverse Tumorigenesis We conclude that at least some types of neoplasia can be reversed by remediating only one of the multiple genetic anomalies that were responsible for tumorigenesis. There have been previous reports that anticipated this view: pharmacologic inhibitors of single oncoproteins can inhibit the growth of some tumor cells (Kohl et al., 1995; Deininger et al., 1997; Barrington et al., 1998); antisense oligonucleotides directed against MYC and other oncogenes have caused tumor regression (Webb et al., 1997; Kronenwett and Haas, 1998; Smith and Wickstrom, 1998), and the tumorigenicity of certain human cell lines could be reduced by complementing the defect in a single tumor suppressor gene (Baker et al., 1990; Bookstein et al., 1990). On the other hand, neither pharmacological agents nor antisense oligonucleotides are necessarily specific for their supposed targets and may influence expression of endogenous protooncogenes (Kronenwett and Haas, 1998; Lebowitz and Prendergast, 1998). Constitutive overexpression of a tumor suppressor gene is not necessarily equivalent to restoring endogenous tumor suppressor function. In contrast, the strategy we employed specifically regulates the expression of a MYC transgene and would not be expected to interfere with the function of the endogenous MYC’s expression or function.

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There are also examples where the inactivation of an oncoprotein or oncogene failed to reverse the malignant phenotype (Ewald et al., 1996; Plattner et al., 1996). Thus, it seems likely that the response of tumors to remediation of a single genetic lesion will vary with the nature of the lesions and their contribution to tumorigenesis, the cellular lineage of the tumors, and the particular combination of genetic lesions responsible for tumorigenesis. It remains to be determined whether the targeted inactivation of a single oncogenic event is sufficient to induce tumor regression in human neoplasia. Experimental Procedures Transgenic Mice Transgenic mice were generated using conventional techniques as described (Hogan et al., 1986). Founders were derived in FVB/N. Human MYC cDNA exons 2 and 3 were cloned into the EcoR1 site of the polylinker of pUHD10-3 (provided by H. Bujard), which contains the tetracycline response element generating tet-o-MYC. tTA was cloned into the EcoRV site of EmSRa (provided by J. Adams). Tumorigenicity Assays To suppress MYC transgene expression, mice were administered doxycycline in their drinking water, changed once per week, at a concentration of 100 mg/ml. For transplantation experiments, tumors were prepared as single cell suspensions by crushing thymic tumors between frosted slides. RBCs were removed by hypotonic lysis. Cells were resuspended in PBS, then 107 cells were inoculated intraperitoneally into syngeneic mice. In some cases, tumor cells were first adapted to in vitro growth, then harvested when in log phase, and 107 cells were injected intraperitoneally into syngeneic mice. Histology Tissues were fixed in 10% buffered formalin, and 5 mm paraffin sections were stained with hematoxylin and eosin. Western Blots Western analysis was performed using conventional techniques. MYC protein expression was detected using the 9E10 antibody that is specific for human MYC protein (Evan et al., 1985). Southern Blots Southern analysis was performed using conventional techniques. T cell receptor b chain rearrangements were detected by probing Southern blots of HindIII-digested genomic DNA with T cell receptor probes for Cb1 (from pUC-C1A) and Jb2 (from pUC-J2b) kindly provided by Astar Winoto (University of California, Berkeley). FACS Analysis Antibodies for FACS were obtained from Pharmingen and used as described by the supplier. Anti-BRDU was obtained from Boehringer Mannheim. BRDU incorporation and PI staining were measured as described by the manufacturer. Tumor Cell Lines Tumors readily adapted to in vitro growth in RPMI1640 supplemented with 10% FCS, beta-mercaptoethanol, glutamine, and penstrep. Proliferation of tumor-derived cell lines was measured by counting cells that excluded trypan blue. Giemsa staining was performed on cytospun preparations using traditional techniques. Gallium Scans Gallium Scans were performed using conventional techniques by Dr. David Price in the Department of Nuclear Medicine, University of California, San Francisco. Tunel Assay Apoptosis was measured using the In Situ Death Detection Kit from Boehringer Mannheim as described by the supplier. Cells were counterstained with DAPI (0.2 mg/ml).

Acknowledgments We thank Bruce Conklin, Anthony DeFranco, Nigel Killeen, Arthur Weiss, Krishna Komanduri, and the members of the Bishop laboratory for their helpful suggestions; Doug Hanahan, Scott Kogan, Andreas Trump, and William Weiss for a critical reading of the manuscript; David Price for performing gallium imaging; Paul Dazin for assistance performing FACS analysis; Ken Aldape, Brian Herndier, and Scott Kogan for assistance in examining the histology of tumor specimens; Jerry Adams for providing EmSRa; Hermann Bujard for providing phCMV-1; and Astar Winoto for providing pUC-C1A and pUC-J2B. D. W. F. is presently supported by the National Cancer Institute, grant number K08 CA75967-01, and was supported by a Howard Hughes Medical Institute Postdoctoral Fellowship, a Lymphoma Research Foundation Fellowship, and a Pfizer Postdoctoral Fellowship. Work was also supported by funds from the National Institutes of Health, grant number CA 44338, and the G. W. Hooper Research Foundation. Received April 23, 1999; revised June 14, 1999. References Adams, J.M., Harris, A.W., Pinkert, C.A., Corcoran, L.M., Alexander, W.S., Cory, S., Palmiter, R.D., and Brinster, R.L. (1985). The c-myc oncogene driven by immunoglobulin enhancers induces lymphoid malignancy in transgenic mice. Nature 318, 533–538. Baker, S.J., Markowitz, S., Fearon, E.R., Willson, J.K., and Vogelstein, B. (1990). Suppression of human colorectal carcinoma cell growth by wild-type p53. Science 249, 912–915. Barrington, R.E., Subler, M.A., Rands, E., Omer, C.A., Miller, P.J., Hundley, J.E., Koester, S.K., Troyer, D.A., Bearss, D.J., Conner, M.W., et al. (1998). A farnesyltransferase inhibitor induces tumor regression in transgenic mice harboring multiple oncogenic mutations by mediating alterations in both cell cycle control and apoptosis. Mol. Cell. Biol. 18, 85–92. Bishop, J.M. (1991). Molecular themes in oncogenesis. Cell 64, 235–248. Bodrug, S.E., Warner, B.J., Bath, M.L., Lindeman, G.J., Harris, A.W., and Adams, J.M. (1994). Cyclin D1 transgene impedes lymphocyte maturation and collaborates in lymphomagenesis with the myc gene. EMBO J. 13, 2124–2130. Bookstein, R., Shew, J.Y., Chen, P.L., Scully, P., and Lee, W.H. (1990). Suppression of tumorigenicity of human prostate carcinoma cells by replacing a mutated RB gene. Science 247, 712–715. Bouchard, C., Staller, P., and Eilers, M. (1998). Control of cell proliferation by Myc. Trends Cell Biol. 8, 202–206. Broussard-Diehl, C., Bauer, S.R., and Scheuermann, R.H. (1996). A role for c-myc in the regulation of thymocyte differentiation and possibly positive selection. J. Immunol. 156, 3141–3150. Cerni, C., Mougneau, E., Zerlin, M., Julius, M., Marcu, K.B., and Cuzin, F. (1986). c-myc and functionally related oncogenes induce both high rates of sister chromatid exchange and abnormal karyotypes in rat fibroblasts. Curr. Top. Microbiol. Immunol. 132, 193–201. Chernova, O.B., Chernov, M.V., Ishizaka, Y., Agarwal, M.L., and Stark, G.R. (1998). MYC abrogates p53-mediated cell cycle arrest in N-(phosphonacetyl)-L-aspartate-treated cells, permitting CAD gene amplification. Mol. Cell. Biol. 18, 536–545. Dang, C.V. (1999). c-myc target genes involved in cell growth, apoptosis, and metabolism. Mol. Cell. Biol. 19, 1–11. Deininger, M.W., Goldman, J.M., Lydon, N., and Melo, J.V. (1997). The tyrosine kinase inhibitor CGP57148B selectively inhibits the growth of BCR-ABL-positive cells. Blood 90, 3691–3698. Efrat, S., Fusco-DeMane, D., Lemberg, H., al Emran, O., and Wang, X. (1995). Conditional transformation of a pancreatic beta-cell line derived from transgenic mice expressing a tetracycline-regulated oncogene. Proc. Natl. Acad. Sci. USA 92, 3576–3580. Evan, G.I., Lewis, G.K., Ramsay, G., and Bishop, J.M. (1985). Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol. Cell. Biol. 5, 3610–3616.

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Evan, G.I., Brown, L., Whyte, M., and Harrington, E. (1995). Apoptosis and the cell cycle. Curr. Opin. Cell Biol. 7, 825–834. Ewald, D., Li, M., Efrat, S., Auer, G., Wall, R.J., Furth, P.A., and Hennighausen, L. (1996). Time-sensitive reversal of hyperplasia in transgenic mice expressing SV40 T antigen. Science 273, 1384– 1386. Facchini, L.M., and Penn, L.Z. (1998). The molecular role of Myc in growth and transformation: recent discoveries lead to new insights. FASEB J. 12, 633–651. Felsher, D.W., and Bishop, J.M. (1999). Transient excess of MYC activity can elicit genomic instability and tumorigenesis. Proc. Natl. Acad. Sci. USA 96, 3940–3944. Furth, P.A., St. Onge, L., Bo¨ger, H., Gruss, P., Gossen, M., Kistner, A., Bujard, H., and Hennighausen, L. (1994). Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter. Proc. Natl. Acad. Sci. USA 91, 9302–9306. Gossen, M., and Bujard, H. (1992). Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. USA 89, 5547–5551. Gossen, M., Freundlieb, S., Bender, G., Mu¨ller, G., Hillen, W., and Bujard, H. (1995). Transcriptional activation by tetracyclines in mammalian cells. Science 268, 1766–1769. Harris, A.W., Pinkert, C.A., Crawford, M., Langdon, W.Y., Brinster, R.L., and Adams, J.M. (1988). The E mu-myc transgenic mouse. A model for high-incidence spontaneous lymphoma and leukemia of early B cells. J. Exp. Med. 167, 353–371. Hogan, B., Costantini, F., and Lacy, E. (1986). Manipulating the mouse embryo: a laboratory manual (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory). Hunter, T. (1991). Cooperation between oncogenes. Cell 64, 249–270. Kistner, A., Gossen, M., Zimmermann, F., Jerecic, J., Ullmer, C., Lu¨bbert, H., and Bujard, H. (1996). Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice. Proc. Natl. Acad. Sci. USA 93, 10933–10938. Kohl, N.E., Omer, C.A., Conner, M.W., Anthony, N.J., Davide, J.P., deSolms, S.J., Giuliani, E.A., Gomez, R.P., Graham, S.L., Hamilton, K., et al. (1995). Inhibition of farnesyltransferase induces regression of mammary and salivary carcinomas in ras transgenic mice. Nat. Med. 1, 792–797. Kronenwett, R., and Haas, R. (1998). Antisense strategies for the treatment of hematological malignancies and solid tumors. Ann. Hematol. 77, 1–12. Lebowitz, P.F., and Prendergast, G.C. (1998). Non-Ras targets of farnesyltransferase inhibitors: focus on Rho. Oncogene 17, 1439– 1445. Leder, A., Pattengale, P.K., Kuo, A., Stewart, T.A., and Leder, P. (1986). Consequences of widespread deregulation of the c-myc gene in transgenic mice: multiple neoplasms and normal development. Cell 45, 485–495. Lengauer, C., Kinzler, K.W., and Vogelstein, B. (1998). Genetic instabilities in human cancers. Nature 396, 643–649. Mai, S., Hanley-Hyde, J., and Fluri, M. (1996). c-Myc over-expression associated DHFR gene amplification in hamster, rat, mouse and human cell lines. Oncogene 12, 277–288. Marcu, K.B., Bossone, S.A., and Patel, A.J. (1992). myc function and regulation. Annu. Rev. Biochem. 61, 809–860. Morgenbesser, S.D., and DePinho, R.A. (1994). Use of transgenic mice to study myc family gene function in normal mammalian development and in cancer. Sem. Cancer Biol. 5, 21–36. Plattner, R., Anderson, M.J., Sato, K.Y., Fasching, C.L., Der, C.J., and Stanbridge, E.J. (1996). Loss of oncogenic ras expression does not correlate with loss of tumorigenicity in human cells. Proc. Natl. Acad. Sci. USA 93, 6665–6670. Smith, J.B., and Wickstrom, E. (1998). Antisense c-myc and immunostimulatory oligonucleotide inhibition of tumorigenesis in a murine B-cell lymphoma transplant model. J. Natl. Cancer Inst. 90, 1146– 1154. Tremblay, P., Meiner, Z., Galou, M., Heinrich, C., Petromilli, C., Lisse,

T., Cayetano, J., Torchia, M., Mobley, W., Bujard, H., et al. (1998). Doxycycline control of prion protein transgene expression modulates prion disease in mice. Proc. Natl. Acad. Sci. USA 95, 12580– 12585. Vose, J.M., Bierman, P.J., Anderson, J.R., Harrison, K.A., Dalrymple, G.V., Byar, K., Kessinger, A., and Armitage, J.O. (1996). Single-photon emission computed tomography gallium imaging versus computed tomography: predictive value in patients undergoing highdose chemotherapy and autologous stem-cell transplantation for non-Hodgkin’s lymphoma. J. Clin. Oncol. 14, 2473–2479. Webb, A., Cunningham, D., Cotter, F., Clarke, P.A., di Stefano, F., Ross, P., Corbo, M., and Dziewanowska, Z. (1997). BCL-2 antisense therapy in patients with non-Hodgkin lymphoma. Lancet 349, 1137– 1141. Yukawa, K., Kikutani, H., Inomoto, T., Uehira, M., Bin, S.H., Akagi, K., Yamamura, K., and Kishimoto, T. (1989). Strain dependency of B and T lymphoma development in immunoglobulin heavy chain enhancer (E mu)-myc transgenic mice. J. Exp. Med. 170, 711–726.