bioRxiv preprint doi: https://doi.org/10.1101/476283; this version posted November 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Title: The CIC-DUX4 fusion oncoprotein drives metastasis and tumor growth via
distinct downstream regulatory programs and therapeutic targets in sarcoma
Authors: Ross A. Okimoto1,2, Wei Wu1, Shigeki Nanjo1, Victor Olivas1, Yone K.
Lin1, Rieko Oyama3, Tadashi Kondo3, and Trever G. Bivona1,2,4
Affiliations:
1.Department of Medicine, University of California, San Francisco, San Francisco,
California, USA
2. Helen Diller Family Comprehensive Cancer Center, University of California, San
Francisco, San Francisco, California, USA
3. Division of Rare Cancer Research, National Cancer Center Research Institute,
Tokyo, Japan
4. Cellular and Molecular Pharmacology, University of California, San Francisco,
San Francisco, California, USA
Correspondence to: Ross A. Okimoto (email: [email protected]) or Trever
G. Bivona (email: [email protected])
Potential conflicts of interest: The authors declare no conflicts of interest.
1 bioRxiv preprint doi: https://doi.org/10.1101/476283; this version posted November 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Abstract. Transcription factor fusion genes create oncoproteins that drive
oncogenesis, and represent challenging therapeutic targets. Understanding the
molecular targets by which such fusion oncoproteins promote malignancy offers
an approach to develop rational treatment strategies to improve clinical outcomes.
CIC-DUX4 is a transcription factor fusion that defines certain undifferentiated
round cell sarcomas with high metastatic propensity and poor clinical outcomes.
The molecular targets regulated by the CIC-DUX4 oncoprotein that promote this
aggressive malignancy remain largely unknown. We show that increased
expression of ETV4 and CCNE1 occurs via neo-morphic, direct effects of CIC-
DUX4 and drives tumor metastasis and survival, respectively. We demonstrate a
molecular dependence on the CCNE-CDK2 cell cycle complex that renders CIC-
DUX4 tumors sensitive to inhibition of the CCNE-CDK2 complex, highlighting a
therapeutic strategy for CIC-DUX4 tumors. Our findings highlight a paradigm of
functional diversification of transcriptional repertoires controlled by a genetically-
aberrant transcriptional regulator, with therapeutic implications.
2 bioRxiv preprint doi: https://doi.org/10.1101/476283; this version posted November 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Introduction. The biological regulation of transcription factors and repressor
proteins is an essential mechanism to maintain cellular homeostasis, and is often
dysregulated in human cancer 1. Indeed, chromosomal rearrangements involving
transcriptional regulatory genes that lead to transcriptional dysregulation are
present in many cancers, including ~30% of all soft-tissue sarcomas 2. The
majority of these oncogenic fusions involve transcription factors or regulators that
are not readily “druggable” in a direct pharmacologic manner, and thus have
proven difficult to therapeutically target in the clinic. A prime example is the CIC-
DUX4 fusion oncoprotein, which fuses Capicua (CIC) to the double homeobox 4
gene, DUX4. CIC-DUX4-positive soft-tissue tumors are an aggressive subset of
undifferentiated round cell sarcomas that arise in children and young adults.
Despite histological similarities to Ewing sarcoma, CIC-DUX4-positive sarcomas
are clinically-distinct and typically characterized by the rapid development of lethal
metastatic disease and chemo-resistance 3. The unique cytogenetic and clinical
features that distinguish CIC-DUX4-positive sarcomas from other small round cell
tumors provides an opportunity for specific precision medicine-based therapies to
improve clinical outcomes.
CIC is a transcriptional repressor protein 4. The CIC-DUX4 fusion structurally
retains >90% of native CIC, yet it functions as a transcriptional activator instead of
a transcriptional repressor 5,6. This property suggests that the C-terminal DUX4
binding partner may confer neo-morphic transcriptional regulatory properties to
CIC, while retaining wild-type CIC DNA binding specificity. ETV4 is one of the
most well-characterized transcriptional targets of CIC-DUX4 5,7,8. Over 90% of
3 bioRxiv preprint doi: https://doi.org/10.1101/476283; this version posted November 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
CIC-DUX4 tumors show ETV4 upregulation by immunohistochemistry (IHC),
which distinguishes them from other small round blue cell sarcomas 8. We and
others have demonstrated a pro-metastatic function for ETV4 overexpression in
tumors with inactivation of wild-type (WT) CIC 9,10. The functional role of ETV4 in
CIC-DUX-positive tumors is unknown.
Beyond ETV4, the identity and function of other CIC target genes are less well-
defined. Intriguingly, recent studies showed increased expression of cell-cycle
regulatory genes in CIC-rearranged sarcomas, although the functional relevance
of this observation for oncogenesis and cancer growth in this context is unclear 6,7.
Here, we develop a range of in vitro and in vivo cancer model systems to define
the mechanism by which CIC-DUX4 co-opts transcriptional pathways that native
CIC controls to promote hallmark features of malignancy, including tumor cell
survival, growth, and metastasis.
4 bioRxiv preprint doi: https://doi.org/10.1101/476283; this version posted November 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Results and Discussion.
We recently reported that inactivation of native CIC de-represses an ETV4-MMP24
mediated pro-metastatic circuit 9. Since ETV4 is a direct transcriptional target of
CIC-DUX4 (Fig. S1A) 6,7, we hypothesized that the high metastatic rate observed
in patients harboring CIC-DUX4 fusions was dependent on ETV4 expression. To
explore this hypothesis, we developed an orthotopic soft-tissue metastasis model
that utilizes luciferase-based imaging to track tumor dissemination in vivo (Fig. 1A).
This system produces rapid pulmonary metastases that accurately recapitulates
metastatic tumor dissemination in human patients 3. Using this in vivo system, we
engineered NIH-3T3 mouse fibroblasts that offer the advantage of a genetically-
controlled system (as with the study of other oncoproteins), to express the CIC-
DUX4 fusion oncoprotein11–13. We observed rapid primary tumor formation at the
site of implantation in 100% of the injected mice (Fig. 1B). Genetic silencing of
ETV4 decreased expression of its established target MMP24 9 (Fig. 1C), and
significantly impaired metastatic efficiency in vivo and invasive capacity of cells in
vitro, compared to mice bearing a control silencing vector (Fig. 1B, 1D, and 1E).
While ETV4 suppression decreased distant pulmonary metastases, it did not have
a profound impact on tumor growth when CIC-DUX4 expressing cells were
implanted either orthotopically or subcutaneously into the flank of
immunocompromised mice (Fig. 1F-G). These findings suggest that the primary
function of CIC-DUX4-mediated ETV4 upregulation is to promote invasion and
metastasis, but not tumor cell proliferation or tumor growth per se.
5 bioRxiv preprint doi: https://doi.org/10.1101/476283; this version posted November 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
We next investigated transcriptional targets and programs that could regulate other
key aspects of tumor biology beyond metastasis, including tumor cell proliferation
and growth. Prior studies suggested that the CIC-DUX4 fusion can regulate cell-
cycle gene expression 6,7. We established that ectopic expression of CIC-DUX4
in NIH-3T3 cells increased tumor growth in vitro and in vivo through enhanced cell-
cycle progression (Fig. S1B-S1E). The CIC-DUX4 fusion increased the number of
cells progressing through S-phase, as reflected by an increased G2/M fraction
compared to control (Fig. S1F-G). This CIC-DUX4 mediated cell-cycle progression
was reversed upon 5-fluorouracil (5-FU) treatment (Fig. S1F-G). Since 5-FU
induces a G1/S arrest, we hypothesized that the CIC-DUX4 fusion regulates late
G1 and S phase-promoting genes. In order to identify these CIC-DUX4 regulated
cell-cycle genes, we leveraged a publicly available microarray–based dataset
(GSE60740) to perform a comparative transcriptional analysis between CIC-DUX4
replete (IB120 EV cell line) and two independent CIC-DUX4 knockdown (KD)
patient-derived cell lines (IB120 shCIC-DUX4a and IB120 shCIC-DUX4b) 14. Upon
CIC-DUX4 KD, we observed 409 and 205 down-regulated genes (logFC <-2, FDR
<0.05) in IB120 shCICDUX4a and IB120 shCIC-DUX4b, respectively (Fig. 2A).
There were 165 shared genes between the two shCIC-DUX4 datasets. Functional
clustering of the 165 putative CIC-DUX4 target genes revealed enrichment for
genes that regulate DNA replication and cell-cycle machinery (Fig. 2B). By
comparing the differential expression of these 165 genes between 14 CIC-DUX4
and 6 EWS-NFATC2 patient-derived tumors (GSE60740), we observed a
significant increase in expression of multiple cell-cycle regulatory genes in CIC-
6 bioRxiv preprint doi: https://doi.org/10.1101/476283; this version posted November 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
DUX4 tumors (Fig. 2C). Our findings extend recent studies6,7, and indicate that
targeting the cell-cycle in CIC-DUX4 tumors is a potential therapeutic approach.
In order to identify direct transcriptional targets of the CIC-DUX4 fusion, we first
surveyed the promoters (within -2kb and +150bp of the transcriptional start site) of
all 165 putative response genes for the highly conserved CIC binding motif
(T(G/C)AATG(A/G)A)5. Using this systematic approach, we identified 37 genes,
including the known CIC target genes ETV1/4/5 and multiple regulators of the cell-
cycle (Table S1). Since ectopic expression of CIC-DUX4 promoted S-phase
progression (Fig. S1F-G), we focused on genes that directly regulated the G1/S
transition. Of these genes, CCNE1 expression was consistently upregulated in
CIC-DUX4 tumors (Fig. 2C). We therefore investigated whether CIC-DUX4
transcriptionally controls CCNE1 expression. To explore this hypothesis, we first
localized two tandem CIC-binding motifs within 1kb of the CCNE1 transcriptional
start site (Fig. 3A). We next performed chromatin immunoprecipitation PCR (ChIP-
PCR) analysis, which revealed CCNE1 promoter occupancy by the CIC-DUX4
fusion (Fig. 3B-C). Additionally, a luciferase-based reporter assay demonstrated
enhanced promoter activity by the ectopic expression of CIC-DUX4 compared to
CIC wild-type and empty-vector (EV) control (Fig. 3D). These data show that
CCNE1 is a direct transcriptional target that is upregulated by CIC-DUX4.
To explore the functional role of CCNE1 in CIC-DUX4-expressing tumors, we
genetically-silenced CCNE1 in CIC-DUX4 expressing NIH-3T3 cells (a genetically-
controlled system) and observed decreased tumor growth in vitro and in vivo
7 bioRxiv preprint doi: https://doi.org/10.1101/476283; this version posted November 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
compared to control (Fig. 3E-G and S2A-C). While there is no validated
pharmacologic strategy to directly block CCNE1, inhibition of the CCNE1 binding
partner CDK2 has therapeutic efficacy in other cancer types 15–17. We
hypothesized that inhibiting CDK2 in CIC-DUX4 expressing tumors with the
established small molecule drug dinaciclib15 could limit tumor growth. To explore
this hypothesis, we implanted CIC-DUX4 expressing NIH-3T3 cells
subcutaneously into immune-deficient mice and treated with low-dose dinaciclib
(20mg/kg/day) and observed decreased tumor growth compared to vehicle control
(Fig. 4A-C) 15. These findings suggest that pharmacologic inhibition of the CCNE-
CDK2 complex is a potential therapeutic strategy in CIC-DUX4 expressing tumors.
There are few patient-derived models of CIC-DUX4-positive tumors.
Nevertheless, in order to increase the clinical relevance of our findings we obtained
rare, established patient-derived CIC-DUX4 expressing cells (NCC-CDS1-X1 and
NCC-CDS-X3) 18 to test the functional impact of ETV4 KD and CDK2 inhibition.
Consistent with the findings arising from our isogenic NIH-3T3 system, we found
that genetic silencing of ETV4 decreased invasiveness, but did not impact tumor
growth (Fig. S3A-B). Additionally, we found that CIC-DUX4 expressing cells were
exquisitely sensitive to nanomolar (nM) concentrations of two established,
independent CDK2 inhibitors, dinaciclib and SNS-032 15,19 (Fig. 4D, S3C-E). The
effects on tumor viability were largely mediated through apoptotic cell death as
measured by caspase activity and PARP cleavage, again indicating that CIC-
DUX4 tumors are dependent on the CCNE-CDK2 complex for survival (Fig. 4E-
G).
8 bioRxiv preprint doi: https://doi.org/10.1101/476283; this version posted November 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
We next tested whether CDK2 inhibition could specifically suppress the growth of
patient-derived CIC-DUX4-expressing cells in vivo. To address this hypothesis,
we generated subcutaneous tumor xenografts of the patient-derived CIC-DUX4
expressing cells in immune-deficient mice and found that dinaciclib limited tumor
growth compared to vehicle control treatment (Fig. 4H-J). The decreased tumor
growth observed in dinaciclib-treated mice was accompanied by increased PARP
cleavage in tumor explants, consistent with the apoptotic effect observed in vitro
(Fig. 4K). The impact on tumor growth following CDK2 inhibition was relatively
specific for CIC-DUX4 tumors, as we did not observe similar therapeutic responses
to dinaciclib in other sarcoma subtypes that harbor distinct transcriptional factor
fusion oncoproteins (Rhabdomyosarcoma or Ewing Sarcoma), in vitro or in vivo
(Fig. S4A-B). Consistent with these findings, we observed low levels of CCNE1 in
PAX3-FOXO1-positive RH30 rhabdomyosarcoma cells compared to CIC-DUX4
expressing NCC-CDS-X1 cells (Fig. S4C). These findings further suggest that the
CCNE-CDK2 complex is a specific molecular dependence and therapeutic target
in CIC-DUX4-expressing tumors.
To further demonstrate the therapeutic specificity of targeting CDK2, we used the
clinical CDK4/6 inhibitor palbociclib that has no substantial activity against CDK2,
and found no impact on tumor growth or apoptosis in NCC-CDS-X1 cells (Fig.
S5A). Moreover, while genetic silencing of CDK2 with siRNAs did not impact cell
invasion, it did decrease cell number and viability in CIC-DUX4-expressing NCC-
CDS-X1 cells compared to control (Fig. S5B-D). While we did not observe an
effect on viability with CCNE1 knockdown (KD) alone, combined CCNE1 and
9 bioRxiv preprint doi: https://doi.org/10.1101/476283; this version posted November 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
CCNE2 KD reduced viability to similar levels as siCDK2 cells (Fig. S5C-D). These
findings are consistent with prior observations that both CCNE1 and CCNE2
converge on, and activate CDK2, which our pharmacologic and genetics studies
described above indicate is required for CIC-DUX4-expressing tumor survival20.
Our data suggest that in patient-derived NCC-CDS-X1 cells CCNE2 can
compensate for CCNE1 loss. Consistent with this finding, we observed significant
(~100-fold) upregulation of CCNE2 mRNA upon genetic silencing of CCNE1 (Fig.
S5F) in NCC-CDS-X1 cells. In contrast to these findings, we did not observe a
compensatory increase in CCNE2 expression with CCNE1 KD in NIH-3T3 (mouse)
cells expressing CIC-DUX4 (Fig. S5G). The lack of CCNE2 upregulation in CIC-
DUX4-expressing NIH-3T3 cells is a plausible explanation for the growth
suppressive effect of CCNE1 KD in our initial NIH-3T3 system (Fig. 3E), a
phenotype that was not shared with patient-derived NCC-CDS cells (Fig. S5B).
Collectively, our data suggest that human CIC-DUX4-expressing cells have a
specific dependence on the CCNE-CDK2 cell-cycle regulatory complex. Our
mechanistic dissection of CIC-DUX4 tumors reveals a unique molecular and
therapeutic dependence on the CCNE-CDK2 cell cycle complex. Our data show
that this dependence can be exploited with clinical CDK2 inhibitors that limit tumor
growth through apoptotic induction in a tumor type with few effective therapies.
The CIC-DUX4 fusion oncoprotein is a relatively understudied molecular entity that
characterizes a rare, albeit lethal subset of undifferentiated round cell sarcomas.
We undertook a mechanistic dissection of the molecular function of the CIC-DUX4
10 bioRxiv preprint doi: https://doi.org/10.1101/476283; this version posted November 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
fusion oncoprotein and report unique dependencies on specific transcriptional
targets of the fusion oncoprotein that promote either tumor growth or metastasis.
We reveal that ETV4 is a conserved target gene that enhances the metastatic
capacity of CIC-DUX4 tumors, without significantly impacting tumor growth. These
findings extend previous data that demonstrate a pro-metastatic role for ETV4 in
certain human cancer s9,21. Targeting an ETV4-mediated transcriptional program
downstream of CIC-DUX4 can potentially limit tumor dissemination.
Further analysis of CIC-DUX4-bearing tumors coupled with our functional studies
also identified a dependence on specific cell-cycle machinery to enhance tumor
growth, but not invasive capacity. We reveal that the CCNE-CDK2 complex is a
molecular target of the CIC-DUX4 oncoprotein that controls tumor growth and
survival. While others have observed transcriptional upregulation of cell-cycle
genes in CIC-DUX4 tumors6,7, our data establish the CCNE-CDK2 complex as a
direct molecular target that can be pharmacologically exploited with clinically-
developed CDK2 inhibitors. The therapeutic impact may extend beyond CIC-
DUX4 fusion oncoproteins, to include other CIC-fused oncoproteins. Recent
findings reveal a shared transcriptional program downstream of all known CIC
fusions, including CIC-FOXO4 and CIC-NUTM122. These findings suggest that
many CIC-fused tumors retain their CIC binding specificity while converting native
CIC into a transcriptional activator instead of a repressor in a neo-morphic
functional manner. It would be compelling to explore if the CCNE-CDK2 complex
or other components of the cell-cycle machinery drive tumor growth in these other
CIC-fused tumor types, an area for future investigation.
11 bioRxiv preprint doi: https://doi.org/10.1101/476283; this version posted November 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Our mechanistic dissection of the downstream molecular targets of CIC-DUX4
provides therapeutically-relevant insight for targeting ETV4-mediated metastasis
and CCNE-CDK2-regulated tumor growth. Our data enforce a broader conceptual
paradigm in which certain transcription factor fusion oncoproteins, such as CIC-
DUX4, utilize neo-morphic and distinct downstream regulatory programs to control
divergent cancer hallmarks, such as proliferative capacity and metastatic
competency. The data offer a potential mechanistic explanation for the pleiotropic
functions of this important class of oncoproteins (i.e. transcription factor fusion
oncoproteins such as CIC-DUX4).
Our findings highlight the clinical importance of molecular sub-classification of
morphologically-similar tumor types, such as small round cell sarcomas.
Identifying the different fusion oncoproteins present in clinical samples paves the
way for oncoprotein fusion-specific therapeutic targeting to improve patient
outcomes. Our study highlights the utility of elucidating the mechanistic features
of tumors that are driven by transcription factor fusion oncoproteins to identify
precision medicine-based, molecular therapeutic strategies.
12 bioRxiv preprint doi: https://doi.org/10.1101/476283; this version posted November 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Methods.
Orthotopic and subcutaneous soft tissue xenografts in immunodeficient
mice. Six to eight-week old female SCID mice were purchased from Taconic
(Germantown, NY). Specific pathogen-free conditions and facilities were
approved by the American Association for Accreditation of Laboratory Animal
Care. Surgical procedures were reviewed and approved by the UCSF Institutional
Animal Care and Use Committee (IACUC), protocol #AN107889-03A.
To prepare cell suspensions for quadriceps injection, adherent tumor cells were
briefly trypsinized, quenched with 10% FBS RPMI media and resuspended in PBS.
Cells were pelleted again and mixed with Matrigel matrix (BD Bioscience 356237)
on ice for a final concentration of 1.0x105 cells/μl. The Matrigel-cell suspension
was transferred into a 1ml syringe and remained on ice until the time of
implantation.
For orthotopic injection, mice were placed in the right lateral decubitus position and
anesthetized with 2.5% inhaled isoflurane. A 0.5 cm surgical incision was made
along the posterior medial line of the left hindlimb, fascia and adipose tissue layers
were dissected and retracted to expose the quadriceps femoris muscle. A 30-
guage hypodermic needle was used to advance through the muscular capsule.
For all cell lines, care was taken to inject 10μl (1.0x106 cells) of cell suspension
directly into the left quadriceps femoris. The needle was rapidly withdrawn and
mice were observed for bleeding. Visorb 4/0 polyglycolic acid sutures were used
for primary wound closure of the fascia and skin layer. Mice were observed post-
13 bioRxiv preprint doi: https://doi.org/10.1101/476283; this version posted November 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
procedure for 1-2 hours and body weights and wound healing were monitoring
weekly. For PDX subcutaneous xenotransplantation, 3.0x106 NCC_CDS1_X3
cells were resuspended in 50% PBS/50% Matrigel matrix and injected into the
flanks of immunodeficient mice.
In-vivo bioluminescence imaging. Mice were imaged at the UCSF Preclinical
Therapeutics Core starting on post-injection day 7 with a Xenogen IVIS 100
bioluminescent imaging system. Prior to imaging, mice were anesthetized with
isoflurane and intraperitoneal injection (IP) of 200μl of D-Luciferin at a dose of
150mg/kg body weight was administered. Weekly monitoring of bioluminescence
of the engrafted hindlimb tumors was performed until week 5. Radiance was
calculated automatically using Living Image Software following demarcation of the
thoracic cavity (ROI) in the supine position. The radiance unit of
photons/sec/cm2/sr is the number of photons per second that leave a square
centimeter of tissue and radiate into a solid angle of one steradian (sr).
Ex-vivo bioluminescence imaging. Mice were injected IP with 200 μl
(150mg/kg) of D-Luciferin and subsequently sacrificed at 5 weeks, en-bloc
resection of the heart and lungs was performed. The heart was removed and the
lungs were independently imaged. Imaging was performed in a 12 well tissue
culture plate with Xenogen IVIS 100 bioluminescent imaging.
Cell lines and culture reagents. Cell lines were cultured as recommended by
the American Type Culture Collection (ATCC). NIH-3T3, 293T, A673, SAOS2 cells
14 bioRxiv preprint doi: https://doi.org/10.1101/476283; this version posted November 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
were obtained ATCC. NCC_CDS1_X1 and NCC_CDS_X3 were obtained from
Tadashi Kondo at the National Cancer Center, Tokyo, Japan. The presence of the
CIC-DUX4 fusion was confirmed through RNAseq analysis using the “grep”
command as previously described (Panagopoulos et al, Plos One 2014). All cell
lines were maintained at 37 °C in a humidified atmosphere at 5% CO2 and grown
RPMI 1640 media supplemented with 10% FBS, 100 IU/ml penicillin and 100ug/ml
streptomycin.
Dinaciclib, palbociclib, SNS-032 was purchased from SelleckChem.
Gene knockdown and over-expression assays. All shRNAs were obtained from
Sigma Aldrich. Sequences for individual shRNAs are as follows:
shETV4a: catalog # TRCN0000055132.
shETV4b: catalog # TRCN0000295522.
shCCNE1a: catalog # TRCN0000222722
shCCNE1b: catalog # TRCN0000077776
shCCNE1b: catalog # TRCN0000077777
ON-TARGET plus ETV4, Scramble, CDK2, CDK1, CCNE1, CCE2 siRNA was
obtained from GE Dharmacon and transfection performed with Dharmafect
transfection reagent. The HA-tagged CIC-DUX4 plasmid was obtained from
Takuro Nakarmua, Tokyo, Japan. Sequence verification was performed using
sander sequencing. The lentiviral GFP-Luciferase vector was a kind gift from
Michael Jensen (Seattle Children’s Research Institute, Seattle). Fugene 6
15 bioRxiv preprint doi: https://doi.org/10.1101/476283; this version posted November 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
transfection reagent was used for all virus production and infection was carried out
with polybrene.
Chromatin immunoprecipitation and PCR. CIC null cells (H1975 M1) were
transfected with either GFP control, wild-type CIC, or CIC-DUX4 for 48 hours.
SimpleCHIP Enzymatic Chromatin IP Kit (Cell Signaling Technology) was used
with IgG (Cell signaling Technology) and CIC (Acris) antibodies per the
manufactures protocol.
ETV4 PCR primers were previously described (Okimoto et al., Nature Genetics
2016). The ETV4 primer sequences were as follows:
ETV4_Foward 5’-CGCATCAGACCCAAGACCGTGG-3’
ETV4_Reverse 5’-CCGGAGAGTCGTCCGGCCTGG-3’
CCNE1 PCR primers were designed to flank a tandem TGAATGAA/TGAATGAA
sequence from positions -635 to -628 and -627 to -620 in the CCNE1 promoter.
The primer sequences were as follows:
CCNE1_1F CGTCTCGGCCTCCCACAATGCTGGG and
CCNE1_1R CGCGCCTGTGCCTTGGCCTAGAACC.
Luciferase promoter assay. 293T cells were obtained from ATCC. Cells were
grown in Dulbecco’s modified Eagle Medium (DMEM), supplemented with 10%
FBS, 100 IU/ml penicillin and 100ug/ml streptomycin in a 5% CO2 atmosphere.
Cells were split into a 96 well plate to achieve 50% confluence the day of
16 bioRxiv preprint doi: https://doi.org/10.1101/476283; this version posted November 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
transfection. LightSwitch luciferase assay system (SwitchGear Genomics
S720355) was used per the manufactures protocol. Briefly, a mixture containing
FuGENE 6 transfection reagent, 50ng Luciferase GoClone CCNE1 promoter
(#S720355) plasmid DNA, 50ng of either control (empty) vector or CIC-DUX4 or
wild-type CIC was added to each well. All transfections were performed in
quintuplicate.
Western blot and qRT-PCR. All immunoblots represent at least two independent
experiments. Adherent cells were washed and lysed with RIPA buffer
supplemented with proteinase and phosphatase inhibitors. Proteins were
separated by SDS-PAGE, transferred to Nitrocellulose membranes, and blotted
with antibodies recognizing: GFP (Cell Signaling), E-Cadherin (Cell Signaling),
HSP90 (Cell Signaling), ETV4 (Lifespan), CCNE1 (Cell Signaling), PARP (Cell
Signaling), Phosphor-RB (Cell Signaling), Actin (Sigma), HA-tag (Cell Signaling).
Xenograft tumors. Subcutaneous xenografts were explanted on day 4 of
treatment. Tumor explants were immediately immersed in liquid nitrogen and
stored at -80 degrees. Tumors were disrupted with a mortar and pestle, followed
by sonication in RIPA buffer supplemented with proteinase and phosphatase
inhibitors. Proteins were separated as above. Antibodies to PARP and phosphor-
RB were both from Cell Signaling.
Isolation and purification of RNA was performed using RNeasy Mini Kit (Qiagen).
500 ng of total RNA was used in a reverse transcriptase reaction with the
17 bioRxiv preprint doi: https://doi.org/10.1101/476283; this version posted November 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
SuperScript III first-strand synthesis system (Invitrogen). Quantitative PCR
included four replicates per cDNA sample. Human (CDK1, CDK2, CCNE1,
CCNE2, GAPDH, and TBP) and mouse (CCNE1, CCNE2, and GAPDH) were
amplified with Taqman gene expression assays (Applied Biosystems). Expression
data was acquired using an ABI Prism 7900HT Sequence Detection System
(Applied Biosystems). Expression of each target was calculated using the 2-ΔΔCt
method and expressed as a relative mRNA expression.
Transwell migration and invasion assays. RPMI with 10% FBS was added to
the bottom well of a trans-well chamber. 2.5x104 cells resuspended in serum free
media was then added to the top 8 μM pore matrigel coated (invasion) or non-
coated (migration) trans-well insert (BD Biosciences). After 20 hours, non-invading
cells on the apical side of inserts were scraped off and the trans-well membrane
was fixed in methanol for 15 minutes and stained with Crystal Violet for 30 minutes.
The basolateral surface of the membrane was visualized with a Zeiss Axioplan II
immunofluorescent microscope at 10X. Each trans-well insert was imaged in five
distinct regions at 10X and performed in triplicate. % invasion was calculated by
dividing the mean # of cells invading through Matrigel membrane / mean # of cells
migrating through control insert.
Immunostaining (IHC): subcutaneous xenografts. Formalin fixed, paraffin
embedded (FFPE) patient derived tumor specimens were obtained under the
auspices of institutional review board (IRB)-approved clinical protocols.
Specimens were de-paraffinized and stained with an antibody against Cleaved
18 bioRxiv preprint doi: https://doi.org/10.1101/476283; this version posted November 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Caspase 3 (Cell Signaling) and phosphorylated RB (Cell Signaling).
Establishment of a CIC responsive gene set and identification of CIC-DUX4
target genes. A publically curated Affymetrix mRNA dataset (GSE60740) of 14
CIC-DUX4, 7 EWSR1-NFATc2 tumors, and a CIC-DUX4 expressing cell line
(IB120) expressing either shRNA’s targeting CIC-DUX4 or control was used to
generate a list of CIC-DUX4 responsive genes. Specifically, we first independently
compared IB120 cells expressing either EV control to each individual shRNA
targeting the CIC-DUX4 fusion (shCIC-DUX4a and shCICDUX4b). Using logFC <-
2 and FDR<0.05 to identify the most down-regulated genes, we then generated a
shared gene list (N = 165) that we referred to as “CIC-DUX4 responsive genes”.
We then used the CIC-DUX4 responsive gene set to perform functional clustering
with Database for Annotation, Visualization, and Integrated Discovery (DAVID).
Using the CIC-DUX4 responsive gene set, we generated a gene expression heat
map comparing: 1) IB120 cells expressing control vector and; 2) the two
independent shRNAs targeting CIC-DUX4; 3) the 14 CIC-DUX4 patient derived
tumors and; 4) the 7 EWSR1-NFATc2 tumors. Hierarchical clustering was
performed using the differentially expressed CIC-DUX4 responsive gene set. We
performed a similar hierarchical comparison of PAX3-FOXO1 positive cell lines
(RH30) to CIC-DUX4 positive NCC-CDS-X1 cells as documented above.
To identify putative CIC-DUX4 target genes, we surveyed all 165 CIC responsive
genes for the CIC-binding motif (TGAATGAA) within -2000bp and +150bp of the
19 bioRxiv preprint doi: https://doi.org/10.1101/476283; this version posted November 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
transcriptional start site. 37 of the 165 genes contained the CIC-binding motif
(supplementary table). Promoter sequences were download from eukaryotic
promoter database (http://epd.vital-it.ch/).
Cell cycle analysis. To determine the effect of CIC-DUX4 expression on cell
cycle, NIH-3T3 cell lines were cultured to ~70% confluence and transfected with
CIC-DUX4 or a GFP control vector for 48 hours. Cells were trypsinized and fixed
in ice cold ethanol for 10 minutes and subsequently stained with propidium iodide
(PI) solution (Sigma Aldrich) at room temperature for 15 minutes. Cells were
analyzed on a BD LSRII flow cytometer.
Statistical analysis. Experimental data are presented as mean +/- SEM. P-
values derived for all in-vitro experiments were calculated using two-tailed
Student’s t test.
Author contributions. R.A.O. designed and performed the experiments,
analyzed the data, and wrote the manuscript. R.A.O., V.O., S.N., R. O. and T.K.
performed mouse studies. W.W. performed bioinformatics. T.K. performed
experiments and provided cell lines. T.G.B. directed the project, analyzed
experiments, and wrote the manuscript.
Acknowledgements. R.A.O. was supported by a grant from the A.P. Giannini
Foundation and 1K08CA222625-01. T.G.B acknowledges support from NIH / NCI
U01CA217882, NIH / NCI U54CA224081, NIH / NCI R01CA204302, NIH / NCI
20 bioRxiv preprint doi: https://doi.org/10.1101/476283; this version posted November 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
R01CA211052, NIH / NCI R01CA169338, and the Pew-Stewart Foundations. The
authors thank Dr. Takuro Nakamura for providing the HA-tagged CIC-DUX4
plasmid.
21 bioRxiv preprint doi: https://doi.org/10.1101/476283; this version posted November 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
References.
1. Bhagwat, A. S. & Vakoc, C. R. Targeting Transcription Factors in Cancer.
Trends in cancer 1, 53–65 (2015).
2. Mertens, F., Antonescu, C. R. & Mitelman, F. Gene fusions in soft tissue
tumors: Recurrent and overlapping pathogenetic themes. Genes.
Chromosomes Cancer 55, 291–310 (2016).
3. Antonescu, C. R. et al. Sarcomas With CIC-rearrangements Are a Distinct
Pathologic Entity With Aggressive Outcome. Am. J. Surg. Pathol. 41, 941–
949 (2017).
4. Jimenez, G., Shvartsman, S. Y. & Paroush, Z. The Capicua repressor--a
general sensor of RTK signaling in development and disease. J. Cell Sci.
125, 1383–1391 (2012).
5. Kawamura-Saito, M. et al. Fusion between CIC and DUX4 up-regulates
PEA3 family genes in Ewing-like sarcomas with t(4;19)(q35;q13)
translocation. Hum. Mol. Genet. 15, 2125–37 (2006).
6. Specht, K. et al. Distinct transcriptional signature and immunoprofile of
CIC-DUX4 fusion-positive round cell tumors compared to EWSR1-
rearranged Ewing sarcomas: further evidence toward distinct pathologic
entities. Genes. Chromosomes Cancer 53, 622–33 (2014).
7. Yoshimoto, T. et al. CIC-DUX4 Induces Small Round Cell Sarcomas
Distinct from Ewing Sarcoma. Cancer Res. 77, 2927–2937 (2017).
8. Le Guellec, S. et al. ETV4 is a useful marker for the diagnosis of CIC-
rearranged undifferentiated round-cell sarcomas: a study of 127 cases
including mimicking lesions. Mod. Pathol. 29, 1523–1531 (2016).
22 bioRxiv preprint doi: https://doi.org/10.1101/476283; this version posted November 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
9. Okimoto, R. A. et al. Inactivation of Capicua drives cancer metastasis. Nat.
Genet. 49, 87–96 (2017).
10. Kim, E. et al. Capicua suppresses hepatocellular carcinoma progression by
controlling the ETV4-MMP1 axis. Hepatology 67, 2287–2301 (2018).
11. May, W. A. et al. The Ewing’s sarcoma EWS/FLI-1 fusion gene encodes a
more potent transcriptional activator and is a more powerful transforming
gene than FLI-1. Mol. Cell. Biol. 13, 7393–8 (1993).
12. Lam, P. Y., Sublett, J. E., Hollenbach, A. D. & Roussel, M. F. The
oncogenic potential of the Pax3-FKHR fusion protein requires the Pax3
homeodomain recognition helix but not the Pax3 paired-box DNA binding
domain. Mol. Cell. Biol. 19, 594–601 (1999).
13. Barco, R. et al. The synovial sarcoma SYT-SSX2 oncogene remodels the
cytoskeleton through activation of the ephrin pathway. Mol. Biol. Cell 18,
4003–12 (2007).
14. Charville, G. W. et al. EWSR1 fusion proteins mediate PAX7 expression in
Ewing sarcoma. Mod. Pathol. an Off. J. United States Can. Acad. Pathol.
Inc 30, 1312–1320 (2017).
15. Parry, D. et al. Dinaciclib (SCH 727965), a Novel and Potent Cyclin-
Dependent Kinase Inhibitor. Mol. Cancer Ther. 9, 2344–2353 (2010).
16. Desai, B. M. et al. The anti-melanoma activity of dinaciclib, a cyclin-
dependent kinase inhibitor, is dependent on p53 signaling. PLoS One 8,
e59588 (2013).
17. Fu, W. et al. The Cyclin-Dependent Kinase Inhibitor SCH 727965
(Dinacliclib) Induces the Apoptosis of Osteosarcoma Cells. Mol. Cancer
23 bioRxiv preprint doi: https://doi.org/10.1101/476283; this version posted November 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Ther. 10, 1018–1027 (2011).
18. Oyama, R. et al. Generation of novel patient-derived CIC- DUX4 sarcoma
xenografts and cell lines. Sci. Rep. 7, 4712 (2017).
19. Misra, R. N. et al. N -(Cycloalkylamino)acyl-2-aminothiazole Inhibitors of
Cyclin-Dependent Kinase 2. N -[5-[[[5-(1,1-Dimethylethyl)-2-
oxazolyl]methyl]thio]-2-thiazolyl]-4- piperidinecarboxamide (BMS-387032),
a Highly Efficacious and Selective Antitumor Agent. J. Med. Chem. 47,
1719–1728 (2004).
20. Caldon, C. E. & Musgrove, E. A. Distinct and redundant functions of cyclin
E1 and cyclin E2 in development and cancer. Cell Div. 5, 2 (2010).
21. Aytes, A. et al. ETV4 promotes metastasis in response to activation of PI3-
kinase and Ras signaling in a mouse model of advanced prostate cancer.
Proc. Natl. Acad. Sci. U. S. A. 110, E3506-15 (2013).
22. Watson, S. et al. Transcriptomic definition of molecular subgroups of small
round cell sarcomas. J. Pathol. 245, 29–40 (2018).
24 bioRxiv preprint doi: https://doi.org/10.1101/476283; this version posted November 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Figures and Legends.
Figure 1. ETV4 promotes metastasis in CIC-DUX4 sarcoma. A B C Orthotopic Soft Tissue Sarcoma Model! NIH-3T3 + CIC-DUX4! shCtrl! shETV4! ! !
Lung Metastases! Lung (Metastases) !
R! L! muscle Sponataneous ! ! Metastasis! femoris (Primary)
Right! Left! Implantation site ! (left quadriceps femoris)! Quadriceps Post-implantation day 20!
D E F G p=0.002 NIH-3T3 8 Orthotopic soft-tissue Subcutaneous xenograft 20 p=0.04 15 EV CIC-DUX4 + shCtrl 600 EV )
3 CIC-DUX4 6 CIC-DUX4 CIC-DUX4 + shETV4a 15 CIC-DUX4 +shETV4a 10 400 CIC-DUX4 +shETV4b 4 10
ns ns 5 200 2 % Invasion 5 Relative photon flux Tumor volume (mm volume Tumor Number of lung metastases 0 0 0 0 0 5 10 15 20 20 23 26 29 32 EV shCtrl shCtrl Days post-implantation Days post-implantation shETV4ashETV4b shETV4a
Figure 1. ETV4 promotes metastasis in CIC-DUX4 sarcoma. A) Schematic of
the orthotopic soft-tissue metastasis model. B) Representative bioluminscent (BLI)
images from mice bearing CIC-DUX4 NIH-3T3 cells with shCtrl (n=9) or shETV4
(n=7). C) Immunoblot of ETV4 and MMP24 from NIH3T3 cells expressing EV,
shCtrl, shETV4a, or shETV4b. D) Number of lung metastases in mice bearing CIC-
DUX4 NIH-3T3 cells with shCtrl or shETV4. E) Transwell invasion assay
comparing CIC-DUX4 expressing NIH-3T3 cells with either EV, shCtrl, shETV4a,
or shETV4b. F) Relative photon flux from mice orthotopically implanted with CIC-
DUX4 NIH-3T3 cells expressing either shCtrl or shETV4. G) Subcutaneously
implanted NIH-3T3 cells with either EV, CIC-DUX4, or CIC-DUX4 with shETV4a
or shETVb.
25 bioRxiv preprint doi: https://doi.org/10.1101/476283; this version posted November 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Figure 2. CIC-DUX4 regulates cell-cycle genes
Color Key Color Key IB120 cells ! Color Key A (CIC-DUX4 fusion postive)! C −4 2 EV! EV! Row Z−Score vs ! vs ! −-44! 2+4! shCIC-DUX4a! shCIC-DUX4b! Z-score!
AURKB FOXM1 AURKB BIRC5 MND1FOXM1 TCF19BIRC5 GTSE1MND1 KIF2CTCF19 Row Z−Score KIFC1 GTSE1 E2F8 KIF2C CHAF1A CENPUKIFC1 CDC45E2F8 POLQCHAF1A RAD54LCENPU KIF15 CDC45 BLM POLQ DTL RAD54L KIF4A CDC45! 409! 205! FAM83DKIF15 HMMRBLM HJURPDTL AURKB CDCA5KIF4A CENPM FOXM1 FAM83D FEN1 BIRC5 HMMR logFC < - 2! HELLS Down! Down! HJURP MND1 DEPDC1B TCF19 KIF14CDCA5 CDC6 GTSE1 CENPM MCM10FEN1 165! KIF2C CENPE CDCA5! HELLS FDR < 0.05! KIFC1 CEP152 DEPDC1B E2F8 RHEBL1 Regulated! Regulated! CITKIF14 CHAF1A SAPCD2CDC6 CENPU TGFBR3MCM10 CDC45 LRRC1CENPE CDC7 CDC6! POLQ CEP152 RMI2 RAD54L RHEBL1 POLE2 genes! genes! KIF15 EZH2CIT BLM KIF18BSAPCD2 DTL MCM5TGFBR3 FIGNL1 KIF4A LRRC1 FANCI CDC7 FAM83D RFC5 RMI2 HMMR POLA1 POLE2 HJURP MCM2 CDC7! MCM7EZH2 CDCA5 RNASEH2AKIF18B CENPM SGOL2MCM5 FEN1 NID2FIGNL1 GLCCI1 HELLS FANCI ID2 DEPDC1B RFC5 CCDC3 KIF14 FLRT3POLA1 CDC6 ADAMTS9MCM2 MCM10 NRARPMCM7 ETV1 CENPE RNASEH2A TET1 SGOL2 CEP152 PKNOX2 NID2 RHEBL1 THSD7B GLCCI1 CIT SHC3 BPIID2 SAPCD2 EFR3BCCDC3 TGFBR3 CHTF18FLRT3 BMP4 LRRC1 ADAMTS9 PRICKLE1 CDC7 NRARP MFSD2A RMI2 FGFBP3ETV1 CIC-DUX4 activated genes! POLE2 LINC00473TET1 EZH2 TESCPKNOX2 ETV1! KIF18B CALB2THSD7B ANGPT2 MCM5 SHC3 COLEC11 BPI FIGNL1 LHX1 EFR3B FANCI LINC00911 CHTF18 RFC5 SYBU VGLL2BMP4 POLA1 MYH13PRICKLE1 MCM2 CYP2S1MFSD2A MCM7 SOSTDC1 FGFBP3 SLC6A15 RNASEH2A LINC00473 MYH8 SGOL2 PIK3AP1TESC NID2 TYRP1CALB2 GLCCI1 ACVRL1ANGPT2 ID2 BTBD11COLEC11 AGR2 CCDC3 LHX1 TSPAN11 LINC00911 FLRT3 STARD8 ADAMTS9 GALNT16SYBU VGLL2 NRARP MYBPC2 SULT1E1MYH13 ETV1 IL18R1CYP2S1 TET1 HCRTR2 SOSTDC1 PKNOX2 PCSK1 SLC6A15 THSD7B NCAPH 37 genes with CIC binding motif! MYH8 CKAP2L SHC3 EXO1PIK3AP1 BPI CDCA8TYRP1 EFR3B SKA3ACVRL1 CHTF18 CCNE2BTBD11 CDT1 BMP4 AGR2 T(G/C)AATG(A/G)A! HAUS8 PRICKLE1 NEIL3TSPAN11 MFSD2A DDIASSTARD8 FGFBP3 SPC25GALNT16 APOBEC3BMYBPC2 LINC00473 ! ZNF804ASULT1E1 TESC CENPK IL18R1 CALB2 RAD51AP1 HCRTR2 ANGPT2 MAD2L1 CENPWPCSK1 COLEC11 NCAPGNCAPH LHX1 KIAA0101CKAP2L LINC00911 ORC6EXO1 NUSAP1 SYBU CDCA8 CDCA8! ZWINT SKA3 VGLL2 MCM3 MYH13 CDCA7CCNE2 CYP2S1 UBE2CCDT1 RRM2 SOSTDC1 HAUS8 LMNB1NEIL3 SLC6A15 BUB1B CCNE2! DDIAS MYH8 KIF11 SPC25 PIK3AP1 CDK1 ATAD2APOBEC3B TYRP1 NUF2ZNF804A ACVRL1 KIF20ACENPK BTBD11 SPAG5RAD51AP1 GINS2 AGR2 MAD2L1 CDC20 TSPAN11 CENPW TRIP13 STARD8 DLGAP5NCAPG GALNT16 AURKAKIAA0101 B MYBPC2 FAM64AORC6 ELOVL6 SULT1E1 NUSAP1 CCNE1ZWINT IL18R1 LOC100506718 MCM3 HCRTR2 CDH4 CDCA7 PCSK1 PRKAR2B ID1 UBE2C CDCA7! NCAPH SLC2A3RRM2 CKAP2L SNX30LMNB1 EXO1 LPCAT1BUB1B PLSCR1 CDCA8 KIF11 SPP1 SKA3 CDK1 VCAN CCNE2 TGFB3ATAD2 CDK1! CDT1 FOSNUF2 HAUS8 MAFBKIF20A ENPP2 NEIL3 SPAG5 HEY1 GINS2 DDIAS LBH CDC20 DNA replication SPC25 ETV4 TRIP13 APOBEC3B MAN1A1 ETV5DLGAP5 CDC20! ZNF804A POLEAURKA CENPK CRHFAM64A RAD51AP1 IRX1ELOVL6 SHC4 MAD2L1 CCNE1 VGF CENPW SCARA5LOC100506718 NCAPG NPTX2CDH4 CCNE1! KIAA0101 DIO3PRKAR2B ORC6 ID1 NUSAP1 SLC2A3 ZWINT SNX30 MCM3 LPCAT1 Cell cycle PLSCR1 CDCA7 SPP1 UBE2C VCAN RRM2 TGFB3 LMNB1 FOS BUB1B MAFB KIF11 ENPP2 CDK1 HEY1 ATAD2 LBH NUF2 ETV4 MAN1A1 ETV4! KIF20A SPAG5 ETV5 GINS2 POLE CRH Base excision repair CDC20 IRX1 ETV5! TRIP13 SHC4 DLGAP5 VGF AURKA SCARA5 FAM64A GSM1486551 GSM1486555 GSM1486554 GSM1486557 GSM1486556 GSM1486559 GSM1486558 GSM1486560 GSM1486561 GSM1486548 GSM1486550 GSM1486549 GSM1486552 GSM1486553 GSM1486547 GSM1486533 GSM1486539 GSM1486535 GSM1486562 GSM1486563 GSM1486537 GSM1486544 GSM1486543 GSM1486540 GSM1486538 GSM1486546 GSM1486545 GSM1486536 GSM1486542 GSM1486541 GSM1486534 NPTX2 ELOVL6 DIO3 CCNE1 ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! LOC100506718 CDH4 PRKAR2B ID1 SLC2A3 TGF-beta pathway SNX30 LPCAT1 PLSCR1 SPP1 VCAN TGFB3 FOS MAFB ENPP2 HEY1 LBH ETV4 0 5 10 MAN1A1 ETV5 POLE CRH IRX1 SHC4
GSM1486551 GSM1486555 GSM1486554 GSM1486557 GSM1486556 GSM1486559 GSM1486558 GSM1486560 GSM1486561 GSM1486548 GSM1486550 GSM1486549 GSM1486552 GSM1486553 GSM1486547 GSM1486533 GSM1486539 GSM1486535 GSM1486562 GSM1486563 GSM1486537 GSM1486544 GSM1486543 GSM1486540 GSM1486538 GSM1486546 VGFGSM1486545 GSM1486536 GSM1486542 GSM1486541 GSM1486534 CIC-DUX4(33) CIC-DUX4(39) CIC-DUX4(35) CIC-DUX4(37) CIC-DUX4(44) CIC-DUX4(43) CIC-DUX4(40) CIC-DUX4(38) CIC-DUX4(46) CIC-DUX4(45) CIC-DUX4(36) CIC-DUX4(42) CIC-DUX4(41) CIC-DUX4(34) SCARA5 NPTX2 -log10 (p-value) DIO3 +Empty Vector b Vector +Empty a Vector +Empty +EWSR1-NFATc2b +EWSR1-NFATc2a IB120(CIC-DUX4) a IB120(CIC-DUX4) b EWSR1-NFATc2 (51) EWSR1-NFATc2 (48) EWSR1-NFATc2 (50) EWSR1-NFATc2 (49) EWSR1-NFATc2 (52) EWSR1-NFATc2 (53) EWSR1-NFATc2 (47) EWSR1-NFATc2 IB120shCIC-DUX4 2b IB120shCIC-DUX4 2a IB120shCIC-DUX4 1b IB120shCIC-DUX4 1a hMSC hMSC hMSC hMSC GSM1486551 GSM1486555 GSM1486554 GSM1486557 GSM1486556 GSM1486559 GSM1486558 GSM1486560 GSM1486561 GSM1486548 GSM1486550 GSM1486549 GSM1486552 GSM1486553 GSM1486547 GSM1486533 GSM1486539 GSM1486535 GSM1486562 GSM1486563 GSM1486537 GSM1486544 GSM1486543 GSM1486540 GSM1486538 GSM1486546 GSM1486545 GSM1486536 GSM1486542 GSM1486541 GSM1486534
Figure 2. CIC-DUX4 regulates cell-cycle genes. A) Schematic algorithm to
identify putative CIC-DUX4 target genes. B) Functional annotation of CIC-DUX4
activated genes in IB120 cells. C) Heatmap depicting the 165 CIC-DUX4 activated
genes identified in Fig. 2A across 14 CIC-DUX4 and 7 EWSR1-NFATc2 patient
derived tumors. Cell-cycle genes are magnified.
26 bioRxiv preprint doi: https://doi.org/10.1101/476283; this version posted November 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Figure 3. CCNE1 is a molecular target of CIC-DUX4
H1975 M1 (+ CIC-DUX4) 293T p = 0.0003 4 p=0.0001 A B C 60 α-IgG D α-CIC 3 ! ! promoter TGAATGAA! ! 40 +1! -CIC 2 input α IgG (-1kB)! CCNE1! + CIC-DUX4! CCNE1 CCNE1! 20 1 -635! -627! activity (Luciferase) activity
H1975 M1 ChIP (% Input)
(CIC null)! Relative 0 0 CCNE1 EV CIC WT CIC-DUX4
E F G CIC-DUX4 NIH3T3 p < 0.00001 EV 2.4 3000 CIC-DUX4 2.2 )
3 CIC-DUX4 +shCCNE1a 2500 2.0 CIC-DUX4 +shCCNE1b shCtrl 1.8 2000 1.6 1.4 1500 shCCNE1a 1.2 P<0.0001 1.0
1000 CIC-DUX4 0.8 shCCNE1b 0.6 500 0.4 Tumor Weight (grams) Weight Tumor Tumor volume (mm 0.2 0 0.0 20 23 26 29 32
Days post-implantation shCtrl
shCCNE1a shCCNE1b
Figure 3. CCNE1 is a molecular target of CIC-DUX4. A) Schematic of two CIC
biding motifs in the CCNE1 promoter. B-C) ChIP-PCR from H1975 M1 (CIC wild-
type null) cells reconstituted with CIC-DUX4 showing CIC-DUX4 occupancy on the
CCNE1 promoter. D) CCNE1 luciferase promoter assay in 293T cells comparing
EV, CIC wild-type, or CIC-DUX4. E) Subcutaneously implanted NIH-3T3 cells
expressing either EV, CIC-DUX4, or CIC-DUX4 with shCCNE1a or shCCNE1b. F)
Tumor explants from mice in 3E. G) Tumor weights from mice in 3E.
27 bioRxiv preprint doi: https://doi.org/10.1101/476283; this version posted November 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Figure 4. CCNE-CDK2 complex is a therapeutic target in CIC-DUX4 sarcoma CIC-DUX4 A NIH-3T3 + CIC-DUX4 B C p = 0.003 1.2 600 Vehicle 1.1
) 1.0
3 Dinaciclib 20mg/kg/d NIH-3T3CIC-DUX4 + CIC-DUX4! 0.9 0.8 400 0.7 Vehicle! Vehicle! 0.6 P < 0.0001 0.5 0.4 200 Dinaciclib! Dinaciclib! 0.3 0.2
Tumor Weight (grams) Weight Tumor 0.1
Tumor volume (mm volume Tumor NIH-3T3 + CICDUX4! 0.0 0 1 3 6 9 12 15 Vehicle Treatment (Days) Dinaciclib F NCC_CDS1_X1 G NCC_CDS1_X3 D E ** Dinaciclib Dinaciclib (50nM)! 50 50 ** ** DMSO 10nM 50nM 0h 1h 4h 8h 16h ! ** * p=0.01 * p=0.01 40 40 phos-RB!
X1 30 30 1 0.8 1.4 1.2 0.02 ** p=0.0001 NCC-CDS1 ** p=0.0001 1 0 0 PARP! 20 20
CIC-DUX4 cl-PARP!
X3 1 0.8 4.3 8.6 57 10 10 (Fold of DMSO control) DMSO (Fold of (Fold of DMSO control) DMSO (Fold of NCC-CDS1 1 0 0 Actin! * * Relative Caspase 3/7 activity 3/7 Caspase Relative Relative Caspase 3/7 activity 3/7 Caspase Relative 0 0 DMSO 10 100 1000 DMSO 10 100 1000 Dinaciclib (nM) Dinaciclib (nM) H NCC_CDS1_X3 I NCC_CDS1_X3 p = 0.0006 ! 6 Vehicle 2.0 K ! 5 Dinaciclib 20mg/kg/d Dinaciclib J Veh 1.5 4 phos-RB! 1 0.36 3 P = 0.0006 1.0 Vehicle! PARP! 2 cl-PARP!
Tumor volume Tumor 0.5 Dinaciclib! 1 1.6 (Relativeto day 0) 1 Tumor Weight (grams) Weight Tumor NCC_CDS1_X3! Actin! 0 0.0 0 3 6 9 12 NCC_CDS1_X3! Xenograft! Treatment (Days) Vehicle Dinaciclib
* P = 0.002 Figure 4. CCNE-CDK2 complex is a therapeutic target in CIC-DUX4 sarcoma.
A) Subcutaneously implanted NIH-3T3 cells expressing CIC-DUX4 and treated
with vehicle or dinaciclib. B) Tumor explants from mice in 4A. C) Tumor weights
from mice in 4A. D) Patient derived CIC-DUX4 expressing cells (NCC_CDS1_X1
and NCC_CDS_X3) treated with dinaciclib or DMSO. E) Immunoblot of
phosphorylated-Rb, PARP, and actin control in NCC_CDS1_X3 cells treated with
dinaciclib. F) Relative caspase 3/7 activity in NCC_CDS1_X1 and; G)
NCC_CDS_X3 cells treated with dinaciclib or DMSO. **p-value < 0.0001. H)
Subcutaneously implanted NCC_CDS1_X3 cells treated with vehicle control or
dinaciclib. I) Tumor weights from mice treated in 4H. J) Tumor explants from mice
in 4H. K) Immunoblot of phosphorylated-RB, total and cleaved PARP, and actin
control from a NCC_CDS1_X3 tumor explant treated with vehicle or dinaciclib.
28