Specific Tumor Suppressor Function for E2F2 in Myc-Induced T Cell Lymphomagenesis

Specific tumor suppressor function for E2F2 in Myc-induced T cell lymphomagenesis

Rene Opavsky*†, Shih-Yin Tsai*†, Martin Guimond*‡, Anjulie Arora*†, Jana Opavska*†, Brian Becknell*‡, Michael Kaufmann*†, Nathaniel A. Walton*†, Julie A. Stephens§, Soledad A. Fernandez§, Natarajan Muthusamy‡, Dean W. Felsher¶, Pierluigi Porcu‡, Michael A. Caligiuri*‡ʈ, and Gustavo Leone*†ʈ**

*Human Cancer Genetics Program, Department of Molecular Virology, Immunology, and Medical Genetics, College of Medicine and Public Health and †Department of Molecular Genetics, College of Biological Sciences, §Center for Biostatistics, ‡Division of Hematology and Oncology, Department of Internal Medicine, and ʈThe Comprehensive Cancer Center, Ohio State University, Columbus, OH 43210; and ¶Division of Oncology, Department of Medicine, Stanford University, CCSR 1105B, 269 Campus Drive, Stanford, CA 94305-5151

Edited by Tak Wah Mak, University of Toronto, Toronto, ON, Canada, and approved August 10, 2007 (received for review July 6, 2007) Deregulation of the Myc pathway and deregulation of the Rb has little consequence on the proliferative capacity of cells, but the pathway are two of the most common abnormalities in human combined disruption of E2f4 and E2f5 or E2f4 and E2f6 results in malignancies. Recent in vitro experiments suggest a complex cross- the inappropriate proliferation and expression of target genes in regulatory relationship between Myc and Rb that is mediated response to specific antiproliferative signals (16, 17). through the control of E2F. To evaluate the functional connection Recent observations in cell culture systems indicate extensive between Myc and E2Fs in vivo, we used a bitransgenic mouse cross-regulation between the action of Myc and E2Fs in coordi- model of Myc-induced T cell lymphomagenesis and analyzed tumor nating the control of cellular proliferation. Myc action can be progression in mice deficient for E2f1, E2f2,orE2f3. Whereas the funneled by a number of concerted mechanisms to control E2F targeted inactivation of E2f1 or E2f3 had no significant effect on activity (5, 18), including through the regulation of their expression tumor progression, loss of E2f2 accelerated lymphomagenesis. (7). Although Myc can certainly influence E2F activities, it is also Interestingly, loss of a single copy of E2f2 also accelerated tumor- true that Myc can be influenced by E2Fs. How this complex igenesis, albeit to a lesser extent, suggesting a haploinsufficient cross-regulatory relationship between Myc and E2F is effectively function for this locus. The combined ablation of E2f1 or E2f3, orchestrated in vivo remains poorly understood. In this study, we along with E2f2, did not further accelerate tumorigenesis. Myc- used a mouse model of Myc-induced T cell lymphomagenesis along overexpressing T cells were more resistant to apoptosis in the with mice deficient for each of the E2F activators to directly absence of E2f2, and the reintroduction of E2F2 into these tumor examine the connection between Myc action and these E2Fs in vivo. cells resulted in an increase of apoptosis and inhibition of tumorigenesis. These results identify the E2f2 locus as a tumor suppressor Results through its ability to modulate apoptosis. E2f2 Locus Harbors Tumor Suppressor Function. To examine the connection between Myc and E2F transcription factors in vivo,we transcription ͉ cancer used a conditional bitransgenic mouse model of MYC-induced T cell lymphomagenesis (19). In this system, expression of E␮SR-tTA YC is often amplified in human cancers, and mouse models mediates the transcription of the Teto-MYC transgene in B and T Mof cancer have demonstrated a causal role for MYC over- cells and results in the development of predominantly immature T expression in hematopoietic, mammary, and other cancer types cell lymphomas. These tumors invade the spleen, lymphatics, bone (1–3). Deregulation of the Rb/E2F gene networks also represents marrow, and blood and eventually lead to the death of mice by 4 common events in cancer (4). Like Myc, E2F can positively and months of age. negatively regulate the expression of hundreds of targets whose To explore the possibility that E2f1, E2f2, and E2f3 play a role in gene products are involved in a wide spectrum of biological MYC-induced lymphomagenesis, we first determined whether these processes, with a bias for genes that control cell cycle, apoptosis, and E2Fs are expressed in hematopoietic cell lineages. As assessed by differentiation (5–10). real-time RT-PCR assays, E2f1, E2f2, and E2f3 are expressed in all Based on amino acid sequence analysis and structure–function of the main hematopoietic organs, including bone marrow, spleen, studies in vitro, E2F family members can be artificially grouped into thymus, and lymph nodes (Fig. 1A). Most other organs tested activator (E2F1–3) and repressor (E2F4–8) subclasses (11). Be- expressed significantly lower levels of E2f1 and E2f3 and essentially cause of the intense interest in E2Fs as major regulators of the cell little or no E2f2. It is not clear whether the particular high cycle, individual E2F family members have also been extensively expression of E2f2 in hematopoietic organs is a reflection of the studied in vivo by gene-targeting approaches in mice. E2f1Ϫ/Ϫ mice high levels of E2f2 transcripts in these compartments or is just a are viable and suffer from impaired thymocyte apoptosis, defective reflection of the low basal levels of E2f2 in other tissues. We also Ϫ/Ϫ examined the expression of these three E2fs in T cell tumors that negative selection, and testicular atrophy. E2f2 mice are also ␮ viable and have a mild increase in hematopoietic and autoreactive developed in E SR-tTA;Teto-MYC mice. This analysis revealed a T cells. Much later in life, a portion of E2f1Ϫ/Ϫ and E2f2Ϫ/Ϫ mice

develop hematopoietic malignancies (12–15). These mutant phe- Author contributions: R.O. and G.L. designed research; R.O., S.-Y.T., M.G., A.A., J.O., M.K., notypes might reflect the particular bias for the expression of E2f1 N.A.W., and G.L. performed research; B.B., D.W.F., and P.P. contributed new reagents/ and E2f2 in hematopoietic tissues. Although disruption of the E2f3 analytic tools; R.O., J.A.S., S.A.F., N.M., M.A.C., and G.L. analyzed data; and R.O. and G.L. gene in a mixed genetic background yields viable mice, its disrup- wrote the paper. tion in pure strains results in embryonic lethality at around em- The authors declare no conﬂict of interest. bryonic day 12.5 (G.L., unpublished observation). Surprisingly, This article is a PNAS Direct Submission. embryos deficient for each of these E2Fs have no apparent defect Abbreviation: MS, median survival. in cellular proliferation, raising the possibility of functional redun- **To whom correspondence should be addressed. E-mail: [email protected]. dancy among members of the activator subclass of E2Fs. There also This article contains supporting information online at www.pnas.org/cgi/content/full/ appears to be functional redundancy among members of the 0706307104/DC1. repressor subclass, because disruption of E2f4, E2f5,orE2f6 in mice © 2007 by The National Academy of Sciences of the USA

15400–15405 ͉ PNAS ͉ September 25, 2007 ͉ vol. 104 ͉ no. 39 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0706307104 Downloaded by guest on September 30, 2021 significant reduction in E2f2 expression in most tumors tested but little effect on the expression of E2f1, E2f3a, and E2f3b (Fig. 1B and data not shown). The basis for the reduction in E2f2 expression remains to be elucidated, but we speculated that it might be related to its potential role in the MYC-induced tumorigenic process. To rigorously assess the physiological role of E2F activators in MYC-induced lymphomagenesis, we initially generated cohorts of E␮SR-tTA;Teto-MYC mice that lacked either one or both alleles of E2f1 or E2f2. These mice were monitored for tumor formation over a period of 1 year, as described (19). Time of death was noted and plotted on Kaplan–Meier survival graphs. As shown previously, E␮SR-tTA;Teto-MYC mice succumbed to tumors by 110 days of age with a median survival (MS) of 83 days [confidence interval (C.I.) 78–85 days; n ϭ 109]. The inactivation of E2f1 had no significant effect on the MS of these tumor mice (Fig. 1C). Strikingly, the inactivation of E2f2 significantly accelerated tumor onset and progression, resulting in their death at a median age of 67 days (C.I., 65–70 days; n ϭ 128; P Ͻ 0.001; Fig. 1C). Interestingly, loss of even one copy of E2f2 significantly accelerated disease progression when compared with the control wild-type cohort (E2f2ϩ/ϩ, P Ͻ 0.001), albeit to a lesser extent than when both alleles were deleted. Tumors derived from E2f2 heterozygous animals retained the wild-type allele, as assessed by Southern blot analysis [supporting information (SI) Fig. 5 and data not shown], suggesting that small changes in the levels of E2F2 protein could have a substantial impact in tumorigenesis. Characterization of tumors by cell surface marker expression confirmed that control and E2f2-deficient tumors were positive for CD3 and TCR␤ and negative for the B cell marker B220 or myeloid marker CD11b (data not shown), indicating tumors were of T cell origin. In each case, tumors consisted of either CD4/CD8 double-positive cells or CD4 single-positive cells (SI Fig. 6). Together, these results formally demonstrate a haploinsufficient tumor suppressor function for the E2f2 locus in T cell lymphomagenesis.

Germ-line inactivation of E2f3 in an FVB strain background GENETICS results in embryonic lethality at around embryonic day 12.5, precluding tumor studies with these mice (G.L., unpublished observations). To circumvent the problem of embryonic lethality, we analyzed tumor development in E␮SR-tTA;Teto-MYC mice containing the Teto-Cre transgene and a conditional allele of E2f3 (E2f3LoxP). In these mice, tTA driven from the E␮SR-tTA transgene results in the expression of both the Teto-MYC and Teto-Cre transgenes. Thus, MYC expression and Cre-mediated ablation of E2f3 can be achieved within the same subset of hematopoietic cells. Fig. 1. Loss of E2f2 accelerates Myc-induced T cell lymphomagenesis. (A) As shown in Fig. 1C, there was no significant difference in the MS ␮ LoxP/LoxP ␮ Real-time RT-PCR analysis of the expression of E2f1, E2f2, E2f3a, and E2f3b in time between the E SR-tTA;Teto-MYC;E2f3 and E SR- LoxP/LoxP normal mouse tissues (b.m., bone marrow; l.n., lymph node). The relative tTA;Teto-MYC;Teto-Cre;E2f3 groups of mice (P ϭ 0.375). values were determined by comparing the expression of the indicated mRNAs Southern blot analysis and PCR-based genotyping confirmed the to the expression of GAPDH. The data are shown as induction (n-fold) of gene complete ablation of E2f3 in tumors arising in E␮SR-tTA;Teto- expression in tissues relative to expression in the liver that was set to one. One MYC;Teto-Cre; E2f3LoxP/LoxP mice (Fig. 1F and data not shown), representative example of three independent experiments is shown. Error suggesting that loss of E2f3 function was not selected against during Ϯ bars represent mean SD. Values that exceeded the y-axis scale are shown on tumorigenesis. Moreover, Cre expression itself did not appear to the top of interrupted bars. (B) Real-time RT-PCR analysis of E2f1, E2f2, E2f3a, affect tumor outcome, because cohorts of mice containing the and E2f3b expression in normal mouse thymocytes (controls) and MYC- induced T cell lymphomas (tumors). (C) Kaplan–Meier survival curves (time Teto-Cre transgene had identical MS times as those lacking the from birth to death) of the E␮SR-tTA (tetracycline controlled transactivator); Teto-Cre transgene (data not shown). These results strongly suggest Teto-MYC (ϩ/ϩ) and E␮SR-tTA;Teto-MYC cohorts of mice containing het- that E2f3 does not significantly contribute to MYC-induced lym- erozygous (ϩ/Ϫ) or homozygous (Ϫ/Ϫ) mutations of indicated E2fs. Survival phomagenesis. Because E2f3 was conditionally deleted in hemato- curve for E␮SR-tTA;Teto-MYC is shown by solid black line. Curves for the poietic lineages, as opposed to the global deletion of E2f1 or E2f2, cohorts of mice containing deletion of E2f2 allele are shown by red lines, we cannot rule out the formal possibility that inactivation of E2f3 whereas curves for the cohorts of mice that did not involve deletion of E2f2 are shown in blue. Dashed lines and solid lines indicate heterozygous and homozygous deletion of E2fs, respectively. The number of mice used in each cohort is indicated by n. Student’s t test was used for statistical analyses as black line) and E␮SR-tTA;Teto-MYC;Teto-Cre;E2f1Ϫ/Ϫ;E2f2Ϫ/Ϫ;E2f3LoxP/LoxP ([ϩ/ described in Materials and Methods, and P values are shown. (D) Kaplan– ϩ;ϩ/ϩ;LoxP/LoxP]; red line). (F) Conditional deletion of the E2f3LoxP allele in T Meier survival curves of the E␮SR-tTA;Teto-MYC ([ϩ/ϩ;ϩ/ϩ]; black line), E␮SR- cell lymphomas derived from E␮SR-tTA;Teto-MYC;Teto-Cre;E2f3LoxP/LoxP or tTA;Teto-MYC;Teto-Cre;E2f1Ϫ/Ϫ;E2f3LoxP/LoxP ([Ϫ/Ϫ;LoxP/LoxP]; red line), E␮SR-tTA;Teto-MYC;Teto-Cre;E2f3ϩ/LoxP mice as analyzed by Southern blot- E␮SR-tTA;Teto-MYC;E2f1Ϫ/Ϫ;E2f2Ϫ/Ϫ ([Ϫ/Ϫ;Ϫ/Ϫ]; red line), or E␮SR-tTA;Teto- ting using an E2f3-speciﬁc probe. Nondeleted E2f3LoxP/LoxP genomic DNA MYC;Teto-Cre;E2f2Ϫ/Ϫ;E2f3LoxP/LoxP ([Ϫ/Ϫ;LoxP/LoxP]; red line) cohorts of mice. served as a control. The position of bands corresponding to E2f3 knockout (Ϫ), (E) Kaplan–Meier survival curves of the E␮SR-tTA;Teto-MYC ([ϩ/ϩ;ϩ/ϩ;ϩ/ϩ]; E2f3LoxP (LoxP), and E2f3 wild-type (ϩ/Ϫ) alleles is indicated.

Opavsky et al. PNAS ͉ September 25, 2007 ͉ vol. 104 ͉ no. 39 ͉ 15401 Downloaded by guest on September 30, 2021 in the germ line would have a different phenotypic consequence on T cell lymphomagenesis than observed here.

E2f1 and E2f3 Are Dispensable for MYC-Induced Lymphomagenesis. Functional compensation among E2F family members, as demonstrated in fibroblasts cultured in vitro (20), may explain the lack of an effect on tumor outcome imparted by the loss of either E2f1 or E2f3. We therefore analyzed disease progression in mice deleted for both E2f1 and E2f3. Surprisingly, E␮SR-tTA;Teto-MYC;E2f1Ϫ/Ϫ; E2f3LoxP/LoxP and E␮SR-tTA;Teto-MYC;Teto-Cre;E2f1Ϫ/Ϫ; E2f3LoxP/LoxP mice developed T cell lymphomas with similar kinetics as wild-type mice (Fig. 1D). Southern blot analysis of tumor DNA confirmed the complete deletion of E2f3 in tumors derived from mice expressing Cre (SI Fig. 7A and data not shown). From these in vivo studies, we conclude that E2f1 and E2f3 do not play a measurable role in MYC-induced lymphomagenesis.

E2f2’s Tumor Suppressor Function Is Independent of E2f1 and E2f3. The tumor studies described above demonstrate specificity among E2fs in the manifestation of tumor outcome; however, because of Fig. 2. Decreased apoptosis in mice deﬁcient for E2f2 during tumorigenesis. the incredible functional plasticity among E2F family members (A) Development of double-negative thymocytes in 21-day-old E2f2ϩ/ϩ (non- (17), it is difficult to ascertain the molecular basis for this specificity. transgenic; white bars), E2f2Ϫ/Ϫ (nontransgenic; black bars),E␮SR-tTA;Teto- Although no significant change in the expression of other E2F MYC;E2f2ϩ/ϩ (E␮SR-tTA;Teto-MYC; white bars), E␮SR-tTA;Teto-MYC;E2f2Ϫ/Ϫ (E␮SR-tTA;Teto-MYC; black bars) as assessed by FACS. Staining with anti-CD4 family members was observed in tumors deficient for E2f2 (data not Ϫ Ϫ shown), it is possible that E2F1 and/or E2F3 action might have been and -CD8 antibody was used to determine CD4 CD8 double-negative pop- E2f2 ulation within the live lymphoid gate. Double-negative populations were rerouted in -deficient cell to perform functions not normally subsequently analyzed for CD44 and CD25 surface expression. Data are pre- performed in cells containing all three activator E2Fs. These sented as an average percentage Ϯ SD for DN1 (CD44ϩ), DN2 (CD44ϩCD25ϩ), ‘‘acquired functions’’ could be responsible for diminishing or ac- DN3 (CD44ϪCD25ϩ), and DN4 (CD44ϪCD25Ϫ). (B) Graphic representation of centuating the manifestation of tumor outcome resulting from the tumor clonality in E␮SR-tTA;Teto-MYC mice of the following genotypes: loss of E2f2. To test this possibility, we created cohorts of E␮SR- E2f2ϩ/ϩ, E2f2ϩ/Ϫ, and E2f2Ϫ/Ϫ, as determined by analysis of different tissues tTA;Teto-MYC;E2f2Ϫ/Ϫ mice that were also deficient for either using a panel of monoclonal antibodies recognizing different TCR V␤ chains. ϩ ϩ E2f1, E2f3, or both. As shown in Fig. 1D, E␮SR-tTA;Teto- (C) BrdU incorporation and apoptosis assays of E␮SR-tTA;Teto-MYC;E2f2 / ␮ Ϫ/Ϫ MYC;E2f1Ϫ/Ϫ;E2f2Ϫ/Ϫ andE␮SR-tTA;Teto-MYC;Teto-Cre;E2f2Ϫ/Ϫ; (white bars) or E SR-tTA;Teto-MYC;E2f2 (black bars) mice at ﬁnal stages of E2f3LoxP/LoxP cohorts had a similar MS time as E␮SR-tTA;Teto- disease as determined by FACS using anti-BrdU or anti-Annexin V antibodies. Ϫ/Ϫ The number of mice used for each cohort is indicated by n. Student’s t test was MYC;E2f2 mice (66, 67, and 67 days, respectively). Surprisingly, used for statistical analyses, and P values are shown. the simultaneous inactivation of all three E2F activators in E␮SR- tTA;Teto-MYC;Teto-Cre;E2f1Ϫ/Ϫ;E2f2Ϫ/Ϫ;E2f3LoxP/LoxP mice resulted in a similar progression of disease as in E2f2-deficient mice within the hematopoietic compartment and is likely cell- (MS time of 65 and 67 days, respectively; Fig. 1E). Once again, autonomous. Southern blot analysis confirmed the complete deletion of E2f3LoxP/LoxP in the subset of tumors expressing Cre (SI Fig. 7 B and Tumor Analysis in E2f2-Deficient Mice. Loss of E2f2 has been shown C). Based on these data, we conclude that E2f2’s tumor suppressor to lead to autoimmune disease in older mice due to enhanced role in T cell lymphomagenesis is independent of E2f1 and E2f3. TCR-stimulated proliferation and the accumulation of autoreactive effector/memory T lymphocytes (14). We therefore explored Cell-Autonomous Tumor Suppressor Function of E2F2. The global whether E2f2’s tumor suppressor function described above might be inactivation of E2f2 precluded us from making any conclusions causally related to a T cell differentiation defect, which could be relating to where the critical action of E2F2 for suppressing divided into four substages depending on the expression of CD25 lymphomagenesis might reside. To examine whether loss of E2f2 and CD44 markers: DN1 (CD44ϩCD25Ϫ), DN2 (CD44ϩCD25ϩ), accelerated the tumorigenic process in a cell-autonomous manner, DN3 (CD44ϪCD25ϩ), and DN4 (CD44ϪCD25Ϫ) (21). To this end, we used an adoptive transfer strategy to introduce E2f2-deficient we analyzed the expression of CD44, CD25, CD4, and CD8 in fetal liver cells into wild-type irradiated animals. To this end, thymocytes derived from 21-day-old wild-type E2f2Ϫ/Ϫ, E␮SR- lethally irradiated recipient FVB mice were injected with fetal liver tTA;Teto-MYC, and E␮SR-tTA;Teto-MYC;E2f2Ϫ/Ϫ mice. Loss of cells isolated from E␮SR-tTA;Teto-MYC and E␮SR-tTA;Teto- E2f2 in either a nontumor or tumor setting had no appreciable MYC;E2f2Ϫ/Ϫ 15.5-day-old embryos. Injected mice were then mon- effect on the distribution of DN1–DN4 cells or the proportion of itored for tumor formation over a period of 1 year (25 mice per CD4 or CD8 single or CD4–CD8 double-positive cells (Fig. 2A and genetic group; see SI Fig. 8A). Approximately 60% of mice (14/25) data not shown). We did observe, however, that in contrast to the from the group that received E␮SR-tTA;Teto-MYC cells developed monoclonal nature of tumors derived from E␮SR-tTA;Teto-MYC T cell lymphomas, as confirmed by FACS-based immunopheno- mice (19), the vast majority of tumors in the E2f2ϩ/Ϫ and E2f2Ϫ/Ϫ typing and histological analysis (SI Fig. 8B and data not shown) and background were oligoclonal, with homozygously deleted mice succumbed to tumors with a MS time of 160 days. The other 40% having tumors composed of up to five different clones that ex- of mice remained healthy during the 300-day observation period. In pressed different TCR receptor isoforms (Fig. 2B). contrast, most mice (22/25) that received E␮SR-tTA;Teto- In view of the well established role of E2Fs in the control of MYC;E2f2Ϫ/Ϫ fetal liver cells developed T cell lymphomas and had cellular proliferation and apoptosis, we investigated whether a MS time of 110 days (SI Fig. 8A). Lymphomas that lacked E2f2 changes in these two processes may represent the basis for the were immunophenotypically indistinguishable from those that had tumor suppressor function of E2F2. To this end, we measured E2f2. Statistical comparison between these two groups showed a proliferation and apoptotic indices in control and E2f2-deficient significant difference in their MS (P ϭ 0.019). These studies littermate animals at early and late stages of tumor development. demonstrate that the tumor suppressor function of E2f2 resides Analysis of precancerous (21 days of age) or terminally sick mice

15402 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0706307104 Opavsky et al. Downloaded by guest on September 30, 2021 coding sequence (SI Text). We then analyzed the expression of p19ARF, whose induction had been shown to be associated with the overexpression of MYC (23). As might have been expected, we observed a dramatic induction of p19ARF expression in all tumors analyzed, but this induction was independent of the status of E2f2 (Fig. 3A). Consistent with the conclusion that the p53 pathway is not differentially impacted by the presence or absence of E2f2,the expression of one of its target genes, p21Cip1, did not change in response to the loss of E2f2 (Fig. 3A). Because the expression of p73, a proapoptotic member of the p53 family, has been implicated as a downstream target of E2F1 during TCR activation-induced cell death of peripheral T cells (24), we examined its expression in normal and tumor tissues from E2f2-deficient mice. We found that p73 transcripts were significantly decreased in tumor samples, but this decrease did not depend on the status of E2f2 (Fig. 3A). In contrast, the expression of the antiapoptotic version of p73, deltaNp73, was unchanged between normal and tumor samples (SI Fig. 9). These results suggest that E2F2’s tumor suppressor function is independent of the p53 apoptotic axis. The ability of Myc to impact the cell cycle at least partially depends on its ability to down-regulate p27Kip1 (25), a cyclin- dependent kinase inhibitor known to contribute to the regulation of the cell cycle and to be down-regulated in a number of tumor settings (26, 27). Consistent with a posttranscriptional mechanism of regulation, p27Kip1 protein levels but not its mRNA levels, were substantially decreased in tumor cells derived from E␮SR-tTA;Teto- MYC or E␮SR-tTA;Teto-MYC;E2f2Ϫ/Ϫ mice (Fig. 3 A and B). Although this down-regulation of p27Kip1 likely represents an important event in MYC-induced T cell lymphomagenesis, its regulation would appear not to be linked to E2F2’s tumor suppressor function. Fig. 3. Expression of cell cycle-regulated genes in E2f2-deleted cells. (A) Real-time RT-PCR analysis of MYC, ODC, p19ARF, p73, p21Cip1, and p27Kip1 Reintroduction of E2f2 Activity into E2F2-Deficient Cells Inhibits expression in normal thymocytes (E2f2ϩ/ϩ), E2f2-deﬁcient thymocytes Tumorigenesis. To further examine the role of E2f2 in lym- Ϫ/Ϫ ␮ ϩ/ϩ ␮ (E2f2 ), tumors derived from E SR-tTA;Teto-MYC;E2f2 or E SR- phomagenesis, we evaluated T cells isolated from late-stage tumors GENETICS Ϫ Ϫ tTA;Teto-MYC;E2f2 / mice, as indicated. Sequences of primers used for that were reconstituted with exogenous E2F2. Tumor cells derived tested genes are shown in SI Table 1.(B) Western blot analysis of p27Kip1 ␮ Ϫ/Ϫ ␮ from E SR-tTA;Teto-MYC;E2f2 mice readily adapted to in vitro expression in normal thymocytes (N) and E SR-tTA;Teto-MYC tumors nonde- growth and could be efficiently infected with retroviral expression leted or deleted for E2f2 as indicated. vectors (Fig. 4 A and B). Flow cytometric analysis in multiple experiments showed that 20–80% of tumor cells infected with a failed to reveal any substantial difference in the proliferation of control or E2F2-expressing MSCV-IRES-GFP vector were GFP- control and E2f2-deficient T cells (Fig. 2C and data not shown). In positive (Fig. 4B). Subcutaneous injection of these cells resulted in contrast, AnnexinV staining revealed that E2f2-deficient T cells the formation of tumors within 2 weeks. Essentially all of the tumor were more resistant to apoptosis than control cells at both early and cells that emerged from the injection of E2F2-transduced cells were late stages of tumor development, suggesting that a loss of E2f2 may GFP-negative (Ͼ99% GFP-negative; Fig. 4 B and C). In contrast, confer a selective advantage for the expansion of MYC-expressing tumors that emerged from the injection of control-transduced cells T cells. retained the same percentage of GFP-positive cells as observed before the injection. These experiments reveal a profound bias Molecular Characterization of T Cell Lymphomas in E2f2-Deficient against the formation of tumors originating from E2F2 overex- Mice. To identify relevant downstream activities that might be pressing T cells. responsible for the observed acceleration of tumorigenesis in Alternatively, control and E2F2-transduced cells were plated on E2f2-deficient mice, we analyzed the expression of cell cycle inhib- tissue culture plates, and their growth was monitored over the itors that have previously been implicated in cancer biogenesis. course of Ϸ8 days. In these assays, we could measure a moderate First, we performed real-time RT-PCR expression analysis of the but consistent reduction in the proliferation of cells infected with MYC-transgene itself and two known MYC-target genes, Ornithine E2F2-expressing vectors (Fig. 4D). Importantly, comparison of decarboxylase (Odc) and Nucleolin, in thymic tumor masses from GFP-positive and -negative cells by FACS analysis revealed a terminally sick E␮SR-tTA;Teto-MYC and E␮SR-tTA;Teto- profound decrease over time in the percentage of GFP-positive MYC;E2f2Ϫ/Ϫ mice. This analysis confirmed that the expression of cells transduced with E2F2-vectors (Fig. 4E). In contrast, GFP- the MYC transgene and its two target genes was similarly activated positive and -negative cells in control-treated samples proliferated in all tumors examined when compared with T cells from aged- equally well. BrdU incorporation assays indicated that the number matched normal control mice (Fig. 3A and data not shown). of GFP-positive cells entering S phase was not influenced by the Previous work using the E␮-MYC model of B cell lymphoma over-expression of E2F2 (Fig. 4F and data not shown). AnnexinV demonstrated that MYC-induced B cell lymphomagenesis requires assays, however, revealed that the ratio of GFP-positive/-negative the inactivation of apoptotic checkpoints, and that this is frequently apoptotic cells was markedly increased in populations transduced achieved through disabling the Arf-p53 pathway (22). We therefore with E2F2-expressing vectors but not with control vectors (Fig. 4G). assessed the status of p53 in tumors arising in E␮SR-tTA;Teto-MYC These results suggest a strong bias against the proliferation of cells and E␮SR-tTA;Teto-MYC;E2f2Ϫ/Ϫ mice. Direct sequencing of p53 expressing the E2F2 protein that is based on its ability to potently cDNA prepared from 10 tumors did not reveal any mutation in its induce apoptosis.

Opavsky et al. PNAS ͉ September 25, 2007 ͉ vol. 104 ͉ no. 39 ͉ 15403 Downloaded by guest on September 30, 2021 and Rb that is mediated through the control of E2F activities. Here, we used a bitransgenic mouse model of MYC-induced T cell lymphomagenesis and mice deficient for E2f1, E2f2,orE2f3 to evaluate the functional relationship between Myc and E2Fs in vivo. These experiments demonstrate a unique tumor suppressor role for E2F2 in T cell lymphomagenesis. Adoptive transfer experiments show that E2f2’s tumor suppressor function resides within the hematopoietic compartment and is therefore likely to be cell- autonomous. Loss of even one allele significantly accelerated tumorigenesis, indicating that tumor progression is sensitive to small changes in total E2F2 protein. This raises the possibility that polymorphisms in the genome that result in lower levels of E2F2 protein, directly or indirectly, may place individuals at a higher risk for cancer development.

E2f1 and E2f3 Are Not Required for Lymphomagenesis. Based on their shared abilities to control cell proliferation and apoptosis, a functional connection between the Myc and E2F pathways has been long speculated. Most recently, work in mouse embryo fibroblasts suggested that two important functions of Myc in the control of proliferation and apoptosis are mediated, at least in part, by E2Fs (28). This work showed that in fibroblasts, the execution of Myc’s proliferative arm requires E2f2 and E2f3, and the execution of its apoptotic arm requires E2f1. This bifurcation of Myc’s function at the level of E2F suggested that E2f1 could have tumor suppressor function and E2f2 and E2f3 could have oncogenic functions. These predictions were not born out by the in vivo studies of T cell lymphomagenesis presented here. In fact, mice lacking both E2f1 and E2f3 developed T cell lymphomas with similar kinetics as mice containing a full complement of E2Fs, suggesting that E2f1 and Fig. 4. Proapoptotic tumor suppressor function of E2f2. (A) Western blot E2f3 do not play a measurable role in MYC-induced lymphomagen- Ϫ Ϫ analysis of T cells derived from E␮SR-tTA;Teto-MYC;E2f2 / tumors infected esis. The observation that T cells devoid of E2f1, E2f2, and E2f3 with MSCV-IRES-EGFP empty vector (con) or MSCV-E2F2-IRES-EGFP (E2F2) proliferated and were fully transformed is quite surprising given our using anti-E2f2 antibody. Tubulin served as loading control. (B) FACS analysis of unselected cells infected with the indicated retroviruses before injection previous work showing that fibroblasts deficient for these E2Fs into nude mice (Upper). Representative examples of FACS analysis of individ- were unable to proliferate in vitro (20). We do not yet know whether ual tumors that developed in nude mice (Lower). The percentage of GFP- this difference between E2f1/2/3-deficient T cells and fibroblasts positive and -negative cells is indicated within the FACS diagrams. (C) Analysis reflects a tissue-specific requirement for E2Fs or a consequence of of tumors that developed in mice injected with E␮SR-tTA;Teto-MYC;E2f2Ϫ/Ϫ MYC overexpression in T cells. Clearly, important differences must tumor T cells that were infected either with control or E2F2 retroviruses. The exist between fibroblasts and T cells, and a generalized outcome data are presented as the average ratio of percent of GFP-positive cells in each stemming from the action of E2Fs across different cell contexts may individual tumor relative to the percent of GFP-positive cells before injection. be difficult to predict. n indicates a number of tumors analyzed for each group. (D) In vitro prolif- The observation that loss of E2f1 had little bearing on MYC- eration assay of unselected cells infected with the indicated retroviral con- structs. Cells were plated at a concentration of 0.2 ϫ 106 per ml (day 0) and induced T cell lymphomagenesis is also in direct contradiction to a counted every 24 h for 7 days. (E) Cells were treated as in D, and the percentage recent study by Baudino et al. (29), where the authors show that loss of GFP-positive cells was determined by FACS. The infection efﬁciency at day of E2f1 dramatically delayed MYC-induced B cell lymphomagen- 0 (28% for the MSCV-IRES-EGFP vector control and 39% for MSCV-E2F2-IRES- esis. These two different tumor outcomes resulting from the EGFP) was set to 100%. The values obtained for percentage of GFP-positive inactivation of E2f1 could reflect inherent differences in the biology cells at each time point were plotted relative to the percentage at day 0. (F and of B and T cells. Alternatively, differences in the two E2f1-null G) The percentage of BrdU- and Annexin V-positive cells infected with the alleles (12, 13) or in genetic backgrounds (30, 31) housing envi- indicated retroviruses was measured 2 and 4 days after infection. For D–G, ronments, methods of tumor analysis, and size of genetic cohorts representative examples from three independent experiments are shown. could account for the observed discrepancies.

Tumor Suppressor Role of E2F2 in Apoptosis. Two observations In parallel experiments, we could show that overexpression of indicate that the underlying basis for E2f2’s tumor suppressor E2F1 and E2F3a in E2f2-deficient cells could also induce apoptosis function is in the control of apoptosis rather than in cell prolifer- and preclude the growth and tumorigenicity of tumor cells (SI Fig. ation. First, we observed a decreased number of apoptotic tumor 10 A–F and data not shown). These results suggest that using cells in E2f2-null mice. Second, reexpression of E2F2 in E2f2- overexpression approaches, any of the three E2F activators can deficient tumor cells resulted in an increase of apoptosis and engage apoptotic pathways and thus eliminate tumor cells. We view abrogation of tumor cell expansion, demonstrating that the critical these results to indicate that gene ablation strategies can reveal components required to signal and execute apoptosis remain intact functional specificity with greater fidelity than by overexpression in these tumor cells. In contrast, these loss-of-function and over- strategies. expression studies failed to reveal any E2f2-dependent differences in the ability of MYC-expressing tumor cells to replicate their DNA. Discussion The role of E2F2 in apoptosis early on during tumorigenesis The E2f2 Locus Harbors Tumor Suppressor Function. Overexpression could be important in limiting the number of cells susceptible to of the MYC oncogene and inactivation of the RB tumor suppressor MYC-induced oncogenesis. This hypothesis would predict that loss pathway are hallmarks of human cancers. Recent in vitro experi- of E2f2 might increase the tumor-prone T cell population respon- ments suggest a complex cross-regulatory relationship between Myc sive to MYC overexpression and thus facilitate tumor development.

15404 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0706307104 Opavsky et al. Downloaded by guest on September 30, 2021 Consistent with this notion, the vast majority of E2f2ϩ/Ϫ and previously shown that in contrast to E2f1ϩ/ϪE2f2Ϫ/Ϫ or E2f2Ϫ/Ϫ mice developed tumors that were oligoclonal in nature, E2f1Ϫ/ϪE2f2ϩ/Ϫ cells, E2f1Ϫ/ϪE2f2Ϫ/Ϫ bone marrow cells are un- with homozygously deleted tumors expressing up to five different able to contribute to development of multiple hematopoietic lin- TCR receptor isoforms (Fig. 2B). This is in contrast to the typical eages (33), suggesting that a single allele of E2f1 or E2f2 contribute monoclonal nature of tumors found in E2f2ϩ/ϩ mice. These data significantly to this process. Third, in unpublished work from our suggest a role for E2f2 in both the early and late stages of tumor laboratory, we have observed that loss of E2f2 in tissues that progression. normally express much lower levels of it than in T cells also accelerates tumorigenesis (G.L., unpublished observations). It Specificity of Function Among E2Fs. The tumor analysis in E2f- would thus seem that the tumor suppressor role of E2f2 is not simply deficient mice clearly demonstrates a specific role for E2f2 in T cell due to its abundance in T cells but rather is likely a reflection of its lymphomagenesis. The molecular basis for this tumor suppressor specific function in T cell lymphomagenesis. function, however, is less clear. On the one hand, a decrease in E2F2 Whether E2F2 might also play a role in tumor maintenance protein levels but not in E2F1 and E2F3 through the targeted remains to be investigated. Further experiments will be necessary inactivation of E2f2 is sufficient to suppress apoptosis and permit to decipher the exact mechanism by which E2F2 exerts its tumor tumor progression. On the other hand, overexpression of E2F2 or suppressor action in vivo. In summary, these studies reveal speci- E2F1 and, to lesser degree, E2F3a, is able to induce apoptosis in ficity among E2F activators in MYC-induced lymphomagenesis, MYC-overexpressing tumor cells. Thus, loss-of-function and over- highlighting an apoptotic role for E2f2 in this process. expression studies appear to lead to contradictory results. Because Materials and Methods overexpression of E2Fs can potentially compete with and alter the Ϫ/Ϫ Ϫ/Ϫ binding of other E2Fs to target promoters and/or cofactors, we Generation and Maintenance of Mice. The E2f1 , E2f2 , and believe that gene ablation approaches are more adept at revealing Teto-Cre mice were generous gifts from Michael Greenberg (Chil- physiological differences between the function of E2F family dren’s Hospital, Boston, MA), Stuart Orkin (Harvard Medical School, Boston, MA), and Andreas Nagy (Samuel Lunenfeld members. Research Institute, Toronto, ON, Canada), respectively. The gen- What is the underlying reason for the unique tumor suppressor eration of the conditional E2f3 knockout mice (E2f3LoxP/LoxP) has function of E2f2 in MYC-induced lymphomagenesis that is observed been described (20). Genotyping of mice was performed by PCR in vivo? One possibility is that endogenous levels of E2F2 protein from genomic DNA isolated from mouse tails. All tumor studies regulate the expression of a specific set of apoptotic-related genes were performed with mice bred into FVB (fifth generation). Mice that can be similarly achieved only by other E2Fs when overex- for tumor studies generated using standard genetic procedures were pressed at supraphysiological levels. The alternative explanation is monitored for tumor formation over a period of 1 year, as de- that E2f2’s tumor suppressor role is not intrinsic to the function of scribed (19). its protein product but rather depends on the magnitude of expression imparted by its locus. In other words, the size of the Statistical Analysis. Kaplan–Meier curves were generated, and MS ‘‘total pool’’ of E2F activity may be the critical variable that times with 95% confidence intervals (32) were calculated. Propor- determines whether an apoptotic response can be surmounted in tional hazards assumptions were confirmed, and the log-rank test

face of an oncogenic insult. Although it is possible that the basis for was found to be appropriate to compare the survival curves in all GENETICS the unique role of E2f2 in MYC-induced lymphomagenesis may cases. Bonferroni adjustments for multiple comparisons were used. stem from quantitative differences in the expression of ‘‘activator’’ Although this is a conservative method of adjustment, the P values E2Fs in T cells, three main reasons argue against this latter found in these data were on the extreme ends, and a less- possibility. First, if ‘‘total’’ E2F activity was determining tumor conservative method would have lead to the same conclusions. In the case where the proportional hazard assumption was not met, suppression, it would be expected that loss of all three E2F Ϫ Ϫ Ϫ Ϫ activators would accelerate lymphomagenesis further than that such as for the E2f1 / E2f3 / cohort (Fig. 1D), the survival curves observed by the simple loss of E2f2. This prediction was not were compared between groups before and after the cross-over of realized; rather, a deficiency of E2f1/E2f2/E2f3 accelerated lym- the survival curves. phomagenesis to the same extent as by loss of E2f2 (Fig. 1 D and E). Second, the absence of ‘‘enhanced’’ tumorigenesis in T cells This work was funded by National Institutes of Health Grants R01 CA85619 and P01 CA097189 (both to G.L.) and by a translational award deficient for all three E2F activators is not simply because E2f1/3 by the Leukemia and Lymphoma Society of America (to G.L.). R.O. is are not expressed to sufficient levels to have a function in T cells. supported by a T32 CA106196 fellowship in Cancer Genetics, and G.L. In fact, E2F1 has been shown, as has E2F2, to contribute to normal is the recipient of the Pew Charitable Trusts Scholar Award and the hematopoiesis in a number of settings. For example, it has been Leukemia and Lymphoma Society Scholar Award.

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