A SUMOylation-dependent transcriptional subprogram is required for Myc-driven tumorigenesis.
ABSTRACT Myc is an oncogenic transcription factor frequently dysregulated in human cancer. To identify pathways supporting the Myc oncogenic program, we used a genome-wide RNA interference screen to search for Myc-synthetic lethal genes and uncovered a role for the SUMO-activating enzyme (SAE1/2). Loss of SAE1/2 enzymatic activity drives synthetic lethality with Myc. Inactivation of SAE2 leads to mitotic catastrophe and cell death upon Myc hyperactivation. Mechanistically, SAE2 inhibition switches a transcriptional subprogram of Myc from activated to repressed. A subset of these SUMOylation-dependent Myc switchers (SMS genes) is required for mitotic spindle function and to support the Myc oncogenic program. SAE2 is required for growth of Myc-dependent tumors in mice, and gene expression analyses of Myc-high human breast cancers suggest that low SAE1 and SAE2 abundance in the tumors correlates with longer metastasis-free survival of the patients. Thus, inhibition of SUMOylation may merit investigation as a possible therapy for Myc-driven human cancers.
- SourceAvailable from: Laura Soucek[Show abstract] [Hide abstract]
ABSTRACT: Gliomas are the most common primary tumours affecting the adult central nervous system and respond poorly to standard therapy. Myc is causally implicated in most human tumours and the majority of glioblastomas have elevated Myc levels. Using the Myc dominant negative Omomyc, we previously showed that Myc inhibition is a promising strategy for cancer therapy. Here, we preclinically validate Myc inhibition as a therapeutic strategy in mouse and human glioma, using a mouse model of spontaneous multifocal invasive astrocytoma and its derived neuroprogenitors, human glioblastoma cell lines, and patient-derived tumours both in vitro and in orthotopic xenografts. Across all these experimental models we find that Myc inhibition reduces proliferation, increases apoptosis and remarkably, elicits the formation of multinucleated cells that then arrest or die by mitotic catastrophe, revealing a new role for Myc in the proficient division of glioma cells.Nature Communications 01/2014; 5:4632. · 10.74 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Colorectal cancer is the third most common cancer worldwide. Although the transcription factor c-MYC is misregulated in the majority of colorectal tumors, it is difficult to target directly. The deubiquitinase USP28 stabilizes oncogenic factors, including c-MYC; however, the contribution of USP28 in tumorigenesis, particularly in the intestine, is unknown. Here, using murine genetic models, we determined that USP28 antagonizes the ubiquitin-dependent degradation of c-MYC, a known USP28 substrate, as well as 2 additional oncogenic factors, c-JUN and NOTCH1, in the intestine. Mice lacking Usp28 had no apparent adverse phenotypes, but exhibited reduced intestinal proliferation and impaired differentiation of secretory lineage cells. In a murine model of colorectal cancer, Usp28 deletion resulted in fewer intestinal tumors, and importantly, in established tumors, Usp28 deletion reduced tumor size and dramatically increased lifespan. Moreover, we identified Usp28 as a c-MYC target gene highly expressed in murine and human intestinal cancers, which indicates that USP28 and c-MYC form a positive feedback loop that maintains high c-MYC protein levels in tumors. Usp28 deficiency promoted tumor cell differentiation accompanied by decreased proliferation, which suggests that USP28 acts similarly in intestinal homeostasis and colorectal cancer models. Hence, inhibition of the enzymatic activity of USP28 may be a potential target for cancer therapy.The Journal of clinical investigation. 06/2014;
- [Show abstract] [Hide abstract]
ABSTRACT: Tamoxifen has been a frontline treatment for estrogen receptor alpha (ERα)-positive breast tumors in premenopausal women. However, resistance to tamoxifen occurs in many patients. ER still plays a critical role in the growth of breast cancer cells with acquired tamoxifen resistance, suggesting that ERα remains a valid target for treatment of tamoxifen-resistant (Tam-R) breast cancer. In an effort to identify novel regulators of ERα signaling, through a small-scale siRNA screen against histone methyl modifiers, we found WHSC1, a histone H3K36 methyltransferase, as a positive regulator of ERα signaling in breast cancer cells. We demonstrated that WHSC1 is recruited to the ERα gene by the BET protein BRD3/4, and facilitates ERα gene expression. The small-molecule BET protein inhibitor JQ1 potently suppressed the classic ERα signaling pathway and the growth of Tam-R breast cancer cells in culture. Using a Tam-R breast cancer xenograft mouse model, we demonstrated in vivo anti-breast cancer activity by JQ1 and a strong long-lasting effect of combination therapy with JQ1 and the ER degrader fulvestrant. Taken together, we provide evidence that the epigenomic proteins BRD3/4 and WHSC1 are essential regulators of estrogen receptor signaling and are novel therapeutic targets for treatment of Tam-R breast cancer.Cell Research advance online publication 30 May 2014; doi:10.1038/cr.2014.71.Cell research. 05/2014;
, 348 (2012);
, et al.Jessica D. Kessler
for Myc-Driven Tumorigenesis
A SUMOylation-Dependent Transcriptional Subprogram Is Required
This copy is for your personal, non-commercial use only.
clicking here.colleagues, clients, or customers by
, you can order high-quality copies for your
If you wish to distribute this article to others
The following resources related to this article are available online at
here.following the guidelines
can be obtained by
Permission to republish or repurpose articles or portions of articles
Updated information and services,
): January 24, 2012 www.sciencemag.org (this infomation is current as of
version of this article at:
including high-resolution figures, can be found in the online
can be found at:
Supporting Online Material
related to this article
A list of selected additional articles on the Science Web sites
, 14 of which can be accessed free:
cites 39 articles
1 articles hosted by HighWire Press; see:
This article has been
This article appears in the following
registered trademark of AAAS.
is aScience2012 by the American Association for the Advancement of Science; all rights reserved. The title
CopyrightAmerican Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005.
(print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by theScience
on January 24, 2012
7. A. Pessino et al., J. Exp. Med. 188, 953 (1998).
8. E. Cagnano et al., J. Invest. Dermatol. 128, 972 (2008).
9. R. Gazit et al., Nat. Immunol. 7, 517 (2006).
10. C. Gur et al., Nat. Immunol. 11, 121 (2010).
11. G. G. Halfteck et al., J. Immunol. 182, 2221 (2009).
12. E. Narni-Mancinelli et al., Proc. Natl. Acad. Sci. U.S.A.
108, 18324 (2011).
13. C. Levy et al., Mol. Cell 40, 841 (2010).
14. L. Chiossone et al., Blood 113, 5488 (2009).
15. S. Dovat et al., J. Immunol. 175, 3508 (2005).
16. S. H. Lee, K. S. Kim, N. Fodil-Cornu, S. M. Vidal, C. A. Biron,
J. Exp. Med. 206, 2235 (2009).
17. S. N. Waggoner, M. Cornberg, L. K. Selin, R. M. Welsh,
Nature 10.1038/nature10624 (2011).
18. D. M. Andrews et al., J. Exp. Med. 207, 1333 (2010).
19. K. Soderquest et al., J. Immunol. 186, 3304 (2011).
20. C. S. Tripp, S. F. Wolf, E. R. Unanue, Proc. Natl. Acad. Sci.
U.S.A. 90, 3725 (1993).
21. S. Kim et al., Nature 436, 709 (2005).
22. N. C. Fernandez et al., Blood 105, 4416 (2005).
23. N. Anfossi et al., Immunity 25, 331 (2006).
24. P. Brodin, T. Lakshmikanth, S. Johansson, K. Kärre,
P. Höglund, Blood 113, 2434 (2009).
25. D. H.Raulet, R. E. Vance, Nat.Rev. Immunol. 6, 520(2006).
26. W. M. Yokoyama, S. Kim, Immunity 24, 249 (2006).
27. M. T. Orr, L. L. Lanier, Cell 142, 847 (2010).
28. D. E. Oppenheim et al., Nat. Immunol. 6, 928 (2005).
29. J. C. Sun, L. L. Lanier, J. Exp. Med. 205, 1819 (2008).
30. S. K. Tripathy et al., J. Exp. Med. 205, 1829 (2008).
31. B. Zafirova et al., Immunity 31, 270 (2009).
32. O. Mandelboim et al., Nature 409, 1055 (2001).
Acknowledgments: We thank J. Ewbank, M. Bléry, J.-C. Andrau
and P. Kruse for advice; D. Dilg for bioinformatics analysis;
M. Dalod for MCMV reagents; M. C. Carroll for H1N1; G. Lauvau
for Lm-OVA; M. Roger for mice handling; and the Centre
d'Immunologie de Marseille-Luminy (CIML) mouse house
and cytometry core facilities. This work was supported
by an European Research Council advanced grant (E.V.
and S.U.); Functional Genomics in Mutant Mouse Models as
Tools to Investigate the Complexity of Human Immunological
Disease (MUGEN), Network of Excellence and Mechanisms
to Attack Steering Effectors of Rheumatoid Syndromes with
Innovated Therapy Choices (MASTERSWITCH), Integrating
Project from European Union (B.M. and M.M.); University of
Manitoba Dean of Medicine Strategic Fund and Natural
Sciences and Engineering Research Council (S.K.); Agence
Nationale de la Recherche (E.V. and S.U.); Equipe labellisée
“La Ligue,” Ligue Nationale contre le Cancer (E.V. and S.U.);
Agence pour la Recherche sur le Cancer (E.M.N.); Axa
research fund (B.N.J.); and institutional grants from INSERM,
CNRS, and Aix-Marseille University to the CIML. E.V. is a
cofounder of and shareholder in Innate-Pharma. E.N.M.,
E.V., and S.U. designed, analyzed the experiments, and wrote
the paper; C.B. identified Noé mouse during ENU screen;
E.N.M. and A.F. performed and analyzed the experiments;
S.K. and S.M. provided lentiviral vectors and protocols to
transduce NK cells; A.D.G. generated Helios-expressing
vectors; B.N.J., F.V., M.G., I.G.G., J.E., and S.C.H. performed
the sequencing and the bioinformatics analysis; L.N.G.
generated the model for NKp46W32R; M.M. and B.M. initiated
and conducted the early phases of the ENU screen; and
B.B. and E.B. helped in the ENU screen. The data reported
in this paper are tabulated in the main paper and in the
Supporting Online Material. Microarray data have been
deposited at the National Center for Biotechnology
Information GEO repository under accession no. GSE13229
and have been reported elsewhere (13). Ensembl accession
number for Ikzf2 is ENSMUST00000027146. Material
Transfer Agreements are required for use of the following
reagents: Noé mice, NKp46-specific mAb, NKp46iCre/iCre
mice, Helios short hairpin RNA, and control lentiviral
vectors. The invention (U.S. Patent Application 61/499,485;
NKp46-mediated NK cell tuning; E.N.M., S.U., and E.V.) relates
to compounds that inhibit NKp46.
Supporting Online Material
Materials and Methods
Figs. S1 to S15
21 October 2011; accepted 12 December 2011
Transcriptional Subprogram Is Required
for Myc-Driven Tumorigenesis
Jessica D. Kessler,1,2Kristopher T. Kahle,3,4Tingting Sun,1Kristen L. Meerbrey,1,2
Michael R. Schlabach,3Earlene M. Schmitt,1,2Samuel O. Skinner,1,5Qikai Xu,3Mamie Z. Li,3
Zachary C. Hartman,6Mitchell Rao,2Peng Yu,2Rocio Dominguez-Vidana,1,2Anthony C. Liang,3
Nicole L. Solimini,3Ronald J. Bernardi,7Bing Yu,8Tiffany Hsu,1,2Ido Golding,1,5Ji Luo,8
C. Kent Osborne,9,10,11,12Chad J. Creighton,9,13Susan G. Hilsenbeck,9,10,13
Rachel Schiff,9,10,11,12Chad A. Shaw,2Stephen J. Elledge,3* Thomas F. Westbrook1,2,7,9*
Myc is an oncogenic transcription factor frequently dysregulated in human cancer. To identify
pathways supporting the Myc oncogenic program, we used a genome-wide RNA interference
screen to search for Myc–synthetic lethal genes and uncovered a role for the SUMO-activating
enzyme (SAE1/2). Loss of SAE1/2 enzymatic activity drives synthetic lethality with Myc. Inactivation
of SAE2 leads to mitotic catastrophe and cell death upon Myc hyperactivation. Mechanistically,
SAE2 inhibition switches a transcriptional subprogram of Myc from activated to repressed. A subset
of these SUMOylation-dependent Myc switchers (SMS genes) is required for mitotic spindle function
and to support the Myc oncogenic program. SAE2 is required for growth of Myc-dependent tumors
in mice, and gene expression analyses of Myc-high human breast cancers suggest that low SAE1
and SAE2 abundance in the tumors correlates with longer metastasis-free survival of the patients.
Thus, inhibition of SUMOylation may merit investigation as a possible therapy for Myc-driven
suppressor genes. Removing oncogene function
can often reverse the tumorigenic phenotype, a
phenomenon referred to as “oncogene addiction”
(1, 2), and cancer researchers have focused on
difficult to inhibit pharmacologically, highlighting
ancers are driven by genomic alterations
that result in the activation of proto-
oncogenes and the inactivation of tumor
mutations confer dependencies on cellular pro-
The genes and signaling pathways underlying
such oncogenic support processes are largely un-
explored, and because these genes are not them-
selves oncogenes or otherwise mutatedincancer,
they cannot be identified through direct analyses
of cancer genomes and epigenomes.
The NOA pathways supporting the classical
c-Myc oncogene (referred to herein as Myc) are
poorly understood. The Myc gene, which codes
for a basic helix-loop-helix zipper transcription
factor, is frequently dysregulated in cancer cells
tein stabilization (7). Amplification or overex-
(8–11) and are associated with poor prognosis
(12). Genetic experiments have shown that Myc
is required for tumor maintenance and progres-
Oncogenic activation of Myc promotes a del-
icate balance in cells, conferring both pro- and
these opposing properties could be influenced by
inhibiting Myc oncogenic support pathways. To
1Verna and Marrs McLean Department of Biochemistry and
Molecular Biology, Baylor College of Medicine, Houston, TX
77030, USA.2Department of Molecular and Human Genetics,
Hughes Medical Institute, Department of Genetics, Harvard
Medical School, Division of Genetics, Brigham and Women’s
Hospital, Boston, MA 02115, USA.4Department of Neurosur-
Cancer Center, Houston, TX 77030, USA.7Department of Pe-
diatrics, Baylor College of Medicine, Houston, TX 77030, USA.
8Medical Oncology Branch, National Cancer Institute, Center
Drive, Bethesda, MD 20892, USA.9Dan L. Duncan Cancer
Center, Baylor College of Medicine, Houston, TX 77030, USA.
10The Lester and Sue Smith Breast Center, Baylor College of
ment of Molecular and Cellular Biology, Baylor College of
Medicine, One Baylor Plaza, Houston, TX 77030, USA.13Divi-
sion of Biostatistics, Baylor College of Medicine, Houston, TX
*To whom correspondence should be addressed. E-mail:
firstname.lastname@example.org (T.F.W.); email@example.com.
20 JANUARY 2012VOL 335
on January 24, 2012
search for pathways required for cells to tolerate
genetic screen for Myc–synthetic lethal (MySL)
short hairpin RNAs (shRNAs) in human mam-
mary epithelial cells (HMECs) engineered with
gene (Myc-ER HMECs) (fig. S1A). Induction
resulted in increased expression of known Myc
proliferation rate (fig. S1C). HMECs are ER-
negative, and in the absence of Myc-ER, do not
we screened for shRNAs that alter cell fitness
only in the presence of aberrant Myc signaling
(screen design in Fig. 1A).
To identify MySL shRNAs, we transduced
shRNAs targeting 32,293 unique transcripts in
three independent replicates. Transduced Myc-ER
HMECs were propagated in the absence or pres-
in shRNA-barcode abundance was measured in
both cell states (Myc-off and Myc-on). We iden-
tified 403 MySL shRNAs exhibiting more than a
twofold decrease in abundance in the Myc-on
1B, fig. S3, and table S1). Content analysis [Gene
Ontology (GO)] indicated that these candidates
were highly enriched for ion channels and en-
gation (including SUMOylation) (P = 0.002).
Components of the mitotic spindle were also
Analysis of the MySL candidates using Human
Protein Reference Database (HPRD) revealed a
highly connected protein-protein interaction net-
work, with many components of this network
playing a role in the mitotic spindle (Fig. 1D;
protein labels shown in fig. S4), suggesting that
er mitotic progression (17).
Among MySL candidates were several genes
previously implicated in the survival of Myc hy-
peractivated cells (GSK3b, FBXW7, and PTK2;
fig.S5A)(18–20).In addition,shRNAs targeting
MDM2 exhibited Mycsyntheticlethality,where-
as p53-targeting shRNAs enhanced proliferation
Myc promotes p53 activation (2, 21–23), and
p53 defects in promoting Myc-induced tumori-
genesis (2, 21, 22).
We also identified many candidates with pre-
viously unknown roles in Myc biology. To pri-
oritize these, we rank ordered MySL genes using
a modified two-way analysis of variance that we
for a given gene. Using this method, we identi-
with a P ≤ 0.001 (table S2). The most signifi-
cant candidate from both of these analyses was
the SUMO-activating enzyme (SAE) subunit 2
(SAE2/UBA2) (P < 0.00001), a critical compo-
nent of the sole SUMO-activating enzyme nec-
essary for SUMO conjugation to proteins (24).
Multiple SAE2-shRNAs exhibited Myc synthet-
ic lethality in the primary screen (fig. S5C). The
primary screen also identified SAE1, the hetero-
dimeric partner of SAE2 (table S2). Thus, we
interaction between SUMOylation and Myc.
To explore the physiological importance of
the Myc-SAE2 synthetic lethal interaction, we
transduced Myc-ER HMECs with two indepen-
and measured the effect of Myc activation on
pleted SAE2 protein (fig. S6A) and profoundly
Fig. 1. Genome-wide screen for Myc–synthetic lethal
(MySL) candidates. (A) Identification of MySL genes.
Myc-ER HMECs were transduced with a genome-wide library of
retroviral shRNAs in triplicate. At population doubling 0 (PD0),
cells were cultured with or without Myc-ER induction for 12
population doublings (PD12). To identify MySL candidates, we compared
relative barcode abundance from both conditions to that of initial PD0
samples via barcode microarrays. (B) Identification of MySL shRNAs. The Myc-
selective effect of all shRNAs from the genome-wide library are graphed
[y axis represents median difference between Myc-ER-on and Myc-ER-off
groups (log 2)], with a ratio < –1.0 indicating a decrease of at least twofold.
shRNAs are shown on the x axis (rank ordered by MySL effect). (C) Multiple
cellular processes are required to tolerate the Myc oncogenic state. MySL
components and processes). (D) MySL proteins engage in a highly connected
interaction network that regulates the mitotic spindle. Protein-protein in-
teractions between the top 100 MySL proteins were analyzed via HPRD.
mitotic spindle function, red indicates a MySL protein with a known role in
spindle function, and gray indicates a protein that interacts with a MySL
VOL 335 20 JANUARY 2012
on January 24, 2012
increased doubling time upon Myc induction
panels). Similar results were observed when a
constitutive Myc transgene was expressed to-
gether with shSAE2, indicating that these obser-
vations are not an artifact of the Myc-ER fusion
shRNAs elicited a Myc–synthetic lethal pheno-
type (Fig. 2A), and restoration of SAE2 protein
abundance with a SAE2 wild-type cDNA sup-
2, C and D; described below), indicating that the
Myc-SAE2 synthetic phenotype is not due to an
RNA interference (RNAi) off-target effect. Fur-
thermore, multiple shRNAs targeting SAE1 and
the downstream SUMO E2-conjugating enzyme
UBE2I (UBC9) (fig. S6, B and C) were also
synthetically lethal with Myc hyperactivation
(Fig.2A,middle and right graphs), demonstrat-
ing that SUMOylation interference is syntheti-
cally lethal with hyperactivated Myc.
We next investigated whether SUMOylation
is required for cells to tolerate aberrant Myc ac-
tivation. Depletion of SAE2 decreased abundance
of SUMO1- or SUMO2/3-modified proteins (Fig.
2B), indicating global impairment of SUMOyla-
tion in these cells. To determine whether SAE2
enzymatic activity is required to support Myc, we
engineered Myc-ER HMECs with an inducible
SAE2-shRNA (pINDUCER11-shSAE2) (25) to-
gether with constitutive shRNA-resistant cDNAs
encoding wild-type (WT) SAE2, catalytically in-
active SAE2-C173S, or control enhanced green
fluorescent protein (eGFP). SAE2 WT and mu-
tant cDNAs restored SAE2 to endogenous SAE2
levels (Fig. 2C). Restoration of WT SAE2 sup-
pressed the MySL phenotype of SAE2 shRNA
(Fig. 2D). However, SAE2-C173S failed to sup-
press the synthetic lethality of SAE2-shRNA (Fig.
2D), indicating that SAE2 enzymatic activity is
required to prevent the Myc-SAE2 synthetic le-
thal interaction. Together, these data suggest that
SUMOylation is required for HMECs to tolerate
aberrant Myc signaling.
A key question is how SAE2 depletion in the
presence of Myc hyperactivation impairs prolif-
eration. This could be due to changes in the cell
cycle and/or cell death, so we examined the ef-
on these processes. In SAE2-depleted cells, Myc
induction increased the number of cells with a
content (Fig. 3A and fig. S8A). These cell cycle
defects were followed by a significant apoptotic
response (Fig. 3B and fig. S8, B and C). The
increase in G2/M and >2N DNA content is char-
acteristic of mitotic defects known to cause mi-
genes are enriched for genes involved in the mi-
totic spindle (Fig. 1D), suggesting that Myc hy-
peractive cells might experience mitotic stress.
To explore this possibility, we examined mitotic
spindles in Myc hyperactive cells in the presence
or absence of SAE2 depletion. As hypothesized,
nificantly more spindle defects (defects in >25%
alone (Fig. 3, C and D; P = 7 × 10−7). These de-
fects, which included abnormal spindle number
and lagging chromosomes, might explain the ex-
tensive aneuploidy and apoptosis observed. Col-
lectively, these data suggest that the Myc-SAE2
genetic interaction results in dysregulation of the
mitotic spindle, which may in turn contribute to
We next investigated how Myc hyperactiva-
tion and SAE2 depletion result in the mitotic
aberrations. Myc hyperactivation induces differ-
ent cellular consequences (e.g., proliferation,
apoptosis, senescence) depending on the genetic
and epigenetic context. If this is due to the ability
of Myc to regulate distinct transcriptional pro-
grams, loss of SAE2 may lead to mitotic dys-
Therefore, we used gene expression profiling to
define the transcriptional effects of Myc with or
without SAE2 inactivation. Myc activation alone
in HMECs led to significant changes in the level
Fig. 2. HMECs require the E1 SAE to tolerate on-
cogenic Myc. (A) Inactivation of SAE2, SAE1, or
UBE2I is synthetically lethal with Myc hyperac-
tivation. Myc-ER HMECs were infected with control,
SAE2, SAE1, or UBE2I-targeting shRNAs and cul-
tured in the absence or presence of Myc-ER induc-
tion. Cell number was quantified after 6 days. Data
versus Myc-off states and normalized to control shRNA (aver-
images demonstrating altered morphology and decreased cell number in Myc
and shSAE2 cells are shown on the right. (B) Total protein SUMOylation is
decreased uponSAE2knockdown.Myc-ERHMECs infected withcontrolorSAE2-
ing control) protein abundance. (C and D) SAE2 catalytic activity is required to
tolerate Myc hyperactivation. Myc-ER HMECs transduced with a doxycycline
SAE2 UTR-eGFP) were subsequently infected with a virus expressing GFP, SAE2
WT, or SAE2 C173S cDNAs. Western blots were performed to confirm depletion
of SAE2 (C). Cells were cultured with or without Dox and in the absence or
presence of Myc-ER induction, and cell number was quantified after 8 days.
They axisindicates the relativechangeingrowth ofshSAE2-expressing cells
eight replicates) (D). Error bars in (A) and (D) represent the SEM.
20 JANUARY 2012VOL 335
on January 24, 2012
of 605 mRNAs (P < 0.05; Fig. 3E, left panel).
Surprisingly, 22.5% (86/383) of Myc-induced
transcripts are not induced or become repressed
in response to Myc when SAE2 is depleted (Fig.
3E, right panel), suggesting that a portion of the
Myc transcriptional response is “switched” de-
pending on the status of SAE2 function. Because
induced to Myc-repressed in a SAE2-dependent
manner, we termed these genes SUMOylation-
dependent Myc switchers (or SMS genes).
MESH analysis revealed that SMS genes
were significantly enriched for regulators of the
mitotic spindle (P < 4.9 × 10−12) (fig. S9A), and
mining of published literature revealed that 17 of
86 SMS genes have been shown genetically to
participate in the assembly or integrity of mitotic
spindles (26–31). Each of these spindle-related
and fig. S9B, blue bars) but exhibits a strong
SAE2-dependent switch in their Myc response
(Fig. 3F and fig. S9B, red bars). These observa-
tions highlight regulation of spindle assembly as
a key vulnerability in cells harboring the Myc-
active, SAE2-inactive state, and suggest that
SMS genes may be linchpins in the Myc-SAE2
synthetic lethal relationship. To test this hypo-
thesis, we examined whether SMS genes known
to play a role in the mitotic spindle are synthetic-
ally lethal with Myc. We found that three of the
top four SMS genes (CASC5, BARD1, CDC20)
Fig. 3. Inactivation of SAE2 switches the Myc transcriptional pro-
gram and dysregulates mitotic fidelity and cell viability. (A) Ectopic
and aberrant chromosomal content. Myc-ER HMECs transduced with
inducibleshSAE2 were cultured in the absenceor presenceof Myc-ER
analyzed for DNA content by flow cytometry (quantification of cells
with >2N DNA, right panel). (B) Depletion of SAE2 induces apoptosis
in cooperation with Myc hyperactivation. pINDUCER-mir-SAE2-eGFP
Myc-ER HMECs were cultured in the absence or presence of Myc-ER-
induction and with or without shSAE2 induction (48 hours). The cells
Myc-SAE2 genetic interaction leads to defects in the mitotic spindle.
Myc-ER HMECs transduced with inducible shRNA-SAE2 were cultured
and abnormal mitotic events (D). Data are represented as the percentage of abnormal mitoses (at least 100 mitotic events were counted per condition; P values
are from Fisher’s exact test). Scale bar, 5 mM. (E) Loss of SAE2 alters the transcriptional response to Myc. HMECs expressing Myc-ER and dox-inducible SAE2-
by Myc-ER induction (P < 0.05, twofold) are shown. The effect of Myc-ER induction on mRNA levels in the absence or presence of shRNA-SAE2 induction are
shown (left and right panels,respectively).mRNAs that change their response to Mycin thepresence orabsence of shSAE2are termed“sumoylation-dependent
Myc switchers,” or SMS genes. (F) Loss of SAE2 alters Myc control of spindle-regulatory genes. The effect of Myc in the absence or presence of shSAE2 (blue and
red bars, respectively) is shown for the top 4 of 17 SMS genes with known roles in spindle integrity and function (see fig. S9B for the list of 17 SMS genes). (G)
(F) are the SE.
VOL 335 20 JANUARY 2012
on January 24, 2012
time (Fig. 3G and fig. S10, A to C). These results
suggest the SMS transcriptional subprogram may
be required to tolerate the Myc oncogenic state.
gests that Myc-driven cancers may be dependent
on SAE2 and SUMOylation to support their
tumorigenic phenotypes. To test this hypothesis,
cancer–derived cell lines on Myc function. The
shRNAviruses and tested for clonogenicity. The
clonogenicity of SUM159 and MDA-MB-231
breast cancer cells was significantly impaired by
cancer cells were unaffected (Fig. 4A). We there-
fore classified the SUM159 and MDA-MB-231
cells as Myc-independent.
To determine whether Myc-dependent breast
cancer cells are similarly dependent on SAE2
function, we transduced each breast cancer cell
line with an inducible SAE2-shRNA lentivirus.
ycycline (dox)–dependent manner in each cell
genicity of the Myc-dependent breast cancer
cells but had no effect on Myc-independent cells
(Fig. 4B). Similarly, depletion of SAE2 also re-
duced the growth rate of Myc-dependent breast
cancer cells, as determined by a multicolor com-
petition assay (fig. S11B). By contrast, SAE2-
shRNA had no effect or only modest effects on
the proliferation of several normal cell types (fig.
is required for the growth and fitness of Myc-
dependent breast cancer cells.
To determine if SAE2 is essential for the
tumorigenicity of Myc-dependent cancer cells
in vivo, we engineered Myc-dependent (SUM159
and MDA-MB-231) and Myc-independent (MCF7)
breast cancer cells with a dox-inducible SAE2-
shRNA, transplanted the cells into immunocom-
promised mice, and measured tumor volume over
time. To circumvent the effects of SAE2 de-
pletion on in vitro proliferation from confound-
ing the tumorigenicity analyses, we treated the
mice with or without dox only after tumor trans-
plantation. SAE2 depletion inhibited tumor growth
of Myc-dependent SUM159 and MDA-MB-231
tumors in vivo (Fig. 4C, left and middle panels),
but had no significant effect on Myc-independent
MCF7 tumors (right panel). SAE2 depletion also
increased survival time as compared to the ani-
mals that were not treated with dox (fig. S13).
Furthermore, the tumors emerging in dox-treated
mice contained fewer GFP/shSAE2-expressing
cells, consistent with a selection against tumor cells
depleted of SAE2 during tumor growth (fig. S14).
Together, these data suggest that SAE2 function
is required for tumorigenicity of Myc-dependent
The data derived from the above model
systems would predict that Myc-high human
breast cancers with low expression of the SUMO-
activating enzyme may exhibit a less aggressive
clinical behavior. To test this hypothesis, we
compiled breast cancer data sets (n = 1297 pa-
tients) for which there was gene expression data
(Affymetrix U133 platform only) and a common
Tumors were stratified on the basis of Myc
expression levels, with 432 and 429 tumors de-
fined as Myc-high and Myc-low, respectively.
We then determined if levels of SAE1 and SAE2
In patients with Myc-high tumors, those with
Fig. 4. The E1 SAE enzyme is required to support Myc-dependent human breast
analyzed for clonogenic growth. Macroscopic colonies were quantified and
normalized to control-shRNA–infected cells for each cell line. (B) Inactivation of
SAE2 inhibits clonogenicity in Myc-dependent breast cancer cells. Breast cancer–
derived cell lines infected with dox-inducible control- or SAE2-shRNA lentivirus
were analyzed for clonogenic growth in the absence or presence of dox. (C) In-
(SUM159 and MDA-MB-231; left and middle panels, respectively) or Myc-
independent (MCF7, right panel) breast cancer cells infected with dox-inducible
SAE2-targeting shRNA lentivirus were transplanted into nude mice. Recipient
time. (D) Low SAE gene expression correlates with patient metastasis-free survival
correlated with increased metastasis-free survival in patients with Myc-high tumors
(P = 0.01, log-rank test). Tumors with the highest and lowest tertile of Myc mRNA
expression were considered “Myc-high” and “Myc-low,” respectively. Patients with
and red lines, respectively. Error bars in (A) to (C) represent the SEM.
20 JANUARY 2012VOL 335
on January 24, 2012
lower-level expression of SAE1 and SAE2 had
significantly better metastasis-free survival than
those with higher SAE1 and SAE2 (Fig. 4D, left
level expression of SAE1 and SAE2 did not cor-
relate with outcome in patients with Myc-low
tumors (Fig. 4D, right panel). This suggests that
Myc hyperactivation leads to an increased de-
pendency on SAE1 and SAE2 in human breast
We have shown here that the E1 SAE 1 and 2
and SAE2 represent enzymatic examples of the
“non-oncogene addiction” concept, and their dis-
covery illustrates the power of unbiased genetic
screens for identifying potential new leads for
cancer therapeutics. Loss of SUMOylation leads
to substantial mitotic catastrophe and cell death
by switching a subprogram of Myc transcrip-
tional targets that support mitotic spindle func-
tion. Thus, inactivation of SAE2 mimics the
mitotic disruption caused by spindle poisons, but
in a genotype-specific way (i.e., selectively in
cells that harbor oncogenic Myc activation). No-
tably, mitotic interference is a mainstay of cancer
therapeutics, and agents such as taxanes that
disrupt proper spindle function are used to treat a
wide variety of cancers. However, a major lim-
to nontumor organ systems, thus limiting their
therapeutic window. Our observation that inhibi-
tion of SUMOylation can mimic spindle poisons
selectively in cells expressing hyperactivated Myc
raises the possibility that drugs targeting the
SUMO pathway may have the antitumor effects
of spindle poisons with fewer side effects.
Myc promotes a balance of pro- and anti-
tumorigenic properties, and mutations in Myc
can shift this balance in pro- and anti-oncogenic
Myc functions, demonstrating that distinct tran-
scriptional (or other biochemical) functions of
Myc may be segregated (15, 16). We propose
that the Myc transcriptional program can be
shifted to favor the anti-oncogenic state. Specif-
ically, our data suggest that the inactivation of
SAE2 drives synthetic lethality with the Myc
oncogene by altering a subprogram of Myc tran-
scriptional targets that supports proper mitosis
and thus cell viability, a subprogram we term
genes. This SMS program is highly enriched in
proteins that control spindle integrity, and the
Myc-SAE2 synthetic lethal interaction elicits fre-
quentaberrations in the mitotic spindle and even-
to Myc-induced oncogenesis at least in part by
cooperating with Myc to maintain expression of
observations highlight the idea that altering distinct
subprograms of Myc transcription (by SAE2 in-
activation or other mechanisms) may be exploited
more broadly, suggest that subverting transcrip-
tional programs may be a general strategy in
treating cancers driven by oncogenic transcrip-
tion factors that are notoriously difficult to target
References and Notes
1. I. B. Weinstein, Science 297, 63 (2002).
2. S. W. Lowe, E. Cepero, G. Evan, Nature 432, 307
3. S. Jones et al., Science 321, 1801 (2008).
4. J. Luo, N. L. Solimini, S. J. Elledge, Cell 136, 823
5. P. G. Richardson, C. Mitsiades, T. Hideshima,
K. C. Anderson, Annu. Rev. Med. 57, 33 (2006).
6. H. Farmer et al., Nature 434, 917 (2005).
7. L. Soucek, G. I. Evan, Curr. Opin. Genet. Dev. 20,
91 (2010) (Feb).
8. S. L. Deming, S. J. Nass, R. B. Dickson, B. J. Trock,
Br. J. Cancer 83, 1688 (2000).
9. M. J. van de Vijver et al., N. Engl. J. Med. 347, 1999
10. K. Chin et al., Cancer Cell 10, 529 (2006).
11. A. S. Adler et al., Nat. Genet. 38, 421 (2006).
12. Y. Chen, O. I. Olopade, Expert Rev. Anticancer Ther. 8,
13. L. Soucek et al., Nature 455, 679 (2008).
14. R. B. Boxer, J. W. Jang, L. Sintasath, L. A. Chodosh,
Cancer Cell 6, 577 (2004).
15. M. T. Hemann et al., Nature 436, 807 (2005).
16. D. W. Chang, G. F. Claassen, S. R. Hann, M. D. Cole,
Mol. Cell. Biol. 20, 4309 (2000).
17. A. Menssen et al., Cell Cycle 6, 339 (2007).
18. S. Rottmann, Y. Wang, M. Nasoff, Q. L. Deveraux,
K. C. Quon, Proc. Natl. Acad. Sci. U.S.A. 102, 15195
19. Y. Wang et al., Cancer Cell 5, 501 (2004).
20. E. A. Beierle et al., J. Biol. Chem. 282, 12503
21. F. Zindy et al., Genes Dev. 12, 2424 (1998).
22. O. Vafa et al., Mol. Cell 9, 1031 (2002).
23. H. Hermeking, D. Eick, Science 265, 2091 (1994).
24. R. T. Hay, Mol. Cell 18, 1 (2005).
25. K. L. Meerbrey et al., Proc. Natl. Acad. Sci. U.S.A. 108,
26. V. Joukov et al., Cell 127, 539 (2006).
27. L. Song, M. Rape, Mol. Cell 38, 369 (2010).
28. T. Kiyomitsu, C. Obuse, M. Yanagida, Dev. Cell 13, 663
29. M. Shuaib, K. Ouararhni, S. Dimitrov, A. Hamiche,
Proc. Natl. Acad. Sci. U.S.A. 107, 1349 (2010).
30. J. Higgins et al., BMC Cell Biol. 11, 85 (2010).
31. H. H. Silljé, S. Nagel, R. Körner, E. A. Nigg, Curr. Biol.
16, 731 (2006).
32. S. Loi et al., Proc. Natl. Acad. Sci. U.S.A. 107, 10208
33. Y. Wang et al., Lancet 365, 671 (2005).
34. C. Desmedt et al.; TRANSBIG Consortium, Clin. Cancer
Res. 13, 3207 (2007).
35. L. D. Miller et al., Proc. Natl. Acad. Sci. U.S.A. 102,
36. M. Schmidt et al., Cancer Res. 68, 5405 (2008).
37. Y. Zhang et al., Breast Cancer Res. Treat. 116, 303
38. A. J. Minn et al., Proc. Natl. Acad. Sci. U.S.A. 104,
39. A. J. Minn et al., Nature 436, 518 (2005).
Acknowledgments: We thank C. Bland for critical comments
on the manuscript and T. Mitchell, W. Choi, S. Songyang, and
the BCM C-BASS and CCSC Cores for reagents and technical
assistance. J.D.K and K.L.M. are supported by NIH training
grants T32HD05520/T32CA090221-09 and U.S. Department
of Defense predoctoral fellowship W81XWH-10-1-0354,
respectively. The Golding lab is supported by NIH grant
R01GM082837, Human Frontier Science Program grant
RGY70/2008, Welch Foundation grant Q-1759, and NSF
grant 082265 (PFC: Center for the Physics of Living Cells).
This work was supported by a Susan G. Komen for the Cure
grant (KG090355), CPRIT grant (RP120583), and NIH grant
(CA149196) to T.F.W., Specialized Program of Research
Excellence developmental grant (P50 CA058183) to T.F.W.,
SU2C–American Association for Cancer Research Breast
Cancer program grant to R.S. and C.K.O., and U.S. Army
Innovator Award (W81XWH0410197) to S.J.E. S.J.E. is an
Investigator with the Howard Hughes Medical Institute.
T.F.W. is a scholar of The V Foundation and The Mary Kay
Ash Foundation for Cancer Research. Gene Expression data are
deposited in GEO (accession no. GSE34055).
Supporting Online Material
Materials and Methods
Figs. S1 to S14
Tables S1 and S2
16 August 2011; accepted 22 November 2011
Published online 8 December 2011;
Locally Synchronized Synaptic Inputs
Naoya Takahashi,1Kazuo Kitamura,2,3Naoki Matsuo,3,4Mark Mayford,5Masanobu Kano,2
Norio Matsuki,1Yuji Ikegaya1,3*
Synaptic inputs on dendrites are nonlinearly converted to action potential outputs, yet the
spatiotemporal patterns of dendritic activation remain to be elucidated at single-synapse
resolution. In rodents, we optically imaged synaptic activities from hundreds of dendritic spines
in hippocampal and neocortical pyramidal neurons ex vivo and in vivo. Adjacent spines were
frequently synchronized in spontaneously active networks, thereby forming dendritic foci that
received locally convergent inputs from presynaptic cell assemblies. This precise subcellular
geometry manifested itself during N-methyl-D-aspartate receptor–dependent circuit remodeling.
Thus, clustered synaptic plasticity is innately programmed to compartmentalize correlated inputs
along dendrites and may reify nonlinear synaptic integration.
of downstream neurons. Dendrites are arborized
hibit local nonlinear membrane potential dynamics
twined and form cell assemblies that fire
(2–4)and to transformdifferentspatiotemporal se-
quences of incoming inputs into different output
patterns(5,6).Therefore,knowing whether synap-
at a given time (fig. S1) is critical for determining
the dendritic computational power (7, 8); howev-
er, these dynamics are still poorly understood.
VOL 33520 JANUARY 2012
on January 24, 2012