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Peptidic degron for IMiD-induced degradation ofheterologous
proteinsVidyasagar Koduria, Samuel K. McBrayera, Ella Liberzona,
Adam C. Wanga, Kimberly J. Briggsa,1, Hyejin Choa,2,and William G.
Kaelin Jr.a,b,3
aDepartment of Medical Oncology, Dana–Farber Cancer Institute
and Brigham and Womens Hospital, Harvard Medical School, Boston, MA
02215;and bHoward Hughes Medical Institute, Chevy Chase, MD
02185
Contributed by William G. Kaelin Jr., December 9, 2018 (sent for
review November 2, 2018; reviewed by Deepak Nijhawan and Charles L.
Sawyers)
Current systems for modulating the abundance of proteins
ofinterest in living cells are powerful tools for studying
proteinfunction but differ in terms of their complexity and ease of
use.Moreover, no one system is ideal for all applications, and the
bestsystem for a given protein of interest must often be
determinedempirically. The thalidomide-like molecules (collectively
called theIMiDs) bind to the ubiquitously expressed cereblon
ubiquitin ligasecomplex and alter its substrate specificity such
that it targets theIKZF1 and IKZF3 lymphocyte transcription factors
for destruction.Here, we mapped the minimal IMiD-responsive IKZF3
degron andshow that this peptidic degron can be used to target
heterologousproteins for destruction with IMiDs in a time- and
dose-dependentmanner in cultured cells grown ex vivo or in
vivo.
tunable proteins | protein stability | ubiquitylation |
thalidomide |proteasome
Systems for regulating the abundance of a given protein
ofinterest are useful for studying that protein’s function and
formodeling the consequences of pharmacologically inhibiting
thatprotein. The systems available today can be divided into
thosethat alter protein abundance through changes in mRNA
abundanceand those that act primarily at the level of protein
stability (1–3).Some of these systems require three components,
such as a suitablyengineered promoter, a drug-responsive
transcriptional effector, anda drug; others are two-component
systems, such as systems incor-porating polypeptides that confer
drug-induced stability or drug-induced instability when fused to
heterologous proteins. Each ofthese approaches has inherent
advantages and disadvantages. Theideal choice for any one protein
will often depend upon a number offactors, including the normal
turnover of that protein and its mRNA,tolerance of that protein to
in-frame fusion events, and function ofthe protein. Moreover, these
approaches differ with respect to thetechnical expertise and time,
as well as the ease and cost of obtainingthe reagents needed to
implement them. Finally, the degree ofcontrol achieved with these
approaches for any given protein cancurrently only be determined
empirically. In light of these consid-erations, we reasoned it
would be useful to have another system forcontrolling protein
abundance, especially one that was technicallysimple and used
readily available and inexpensive components.Thalidomide-like
drugs, such as lenalidomide and pomalidomide
[collectively referred to as immunomodulatory drugs (IMiDs)],
binddirectly to the cereblon ubiquitin ligase complex and alter its
sub-strate specificity such that it targets the lymphoid-restricted
tran-scription factors IKZF1 and IKZF3 for destruction (4–8). Loss
ofthese two transcription factors is both necessary and sufficient
toaccount for the antimyeloma activity of these compounds (4,
5).Many ubiquitin ligases can recognize and polyubiquitylate
theirsubstrates even if those substrates are fused to heterologous
re-porters (5, 9). Consistent with this observation, cereblon
targetsfusion proteins consisting of firefly luciferase fused to
either IKZF1or IKZF3 for destruction in an IMiD-dependent manner
(4, 5).Ebert and coworkers (4) showed that the IKZF3 degron
recognizedby cereblon in the presence of an IMiD is located in
IKZF3 zincfinger 2 and contained within residues 136–180 (Fig. 1A).
We did
fine mapping of this degron in hopes of identifying a short,
peptidic,degron that could be used to target heterologous proteins
for de-struction in an IMiD-dependent manner.
ResultsMapping and Functional Characterization of theMinimal
IMiD-ResponsiveIKZF3 Degron.Modeled on our earlier work, we made a
mammalianexpression plasmid that encodes two bioluminescent
proteins from asingle mRNA: (i) a protein of interest fused to the
C terminus offirefly luciferase (FLuc) and (ii) renilla luciferase
(RLuc), with bothprotein ORFs preceded by internal ribosome entry
site (IRES) el-ements. (Fig. 1B). Using this vector system, we
expressed variousfragments of IKZF3 fused to firefly luciferase in
transiently trans-fected 293T cells treated with pomalidomide or
vehicle. We thenmeasured the ratio of firefly to renilla luciferase
activity as a sur-rogate for destabilization of the fusion proteins
by pomalidomide.As expected, firefly luciferase fusion proteins
containing full-
length IKZF3 (1–509) or the IKZF3 degron identified by Ebertand
coworkers [IKZF3 (136–180)] were down-regulated bypomalidomide
(Fig. 1C). Analysis of N-terminal and C-terminaldeletions of IKZF3
(136–180) showed that this degron could benarrowed further to IKZF3
(146–168) (Fig. 1C). The C-terminalboundary of the degron is
clearly at residue 168 because elimi-nating this residue
inactivated the IKZF3 degron [Fig. 1C, seeIKZF3 (144–168) and IKF3
(144–167)]. The N-terminal boundary is
Significance
Systems for degrading proteins at will are useful for a variety
ofbiological experiments. Although a number of such systems
havebeen described, they vary widely in terms of complexity, ease
ofobtaining the necessary reagents, and costs. Moreover, no
onesystem seems to work for all proteins, and the ideal system
oftenmust be determined empirically. Thalidomide-like drugs
(IMiDs)reprogram the ubiquitiously expressed cereblon ubiquitin
ligasecomplex to degrade the lymphocyte transcription factors IKZF1
andIKZF3. Here, we show that an IKZF3-derived 25mer constitutes
amodular degron that can be used to target heterologous proteinsfor
destruction by IMiDs, which are widely available and cross
theblood–brain barrier, in cell culture and in mouse
experiments.
Author contributions: V.K., K.J.B., H.C., and W.G.K. designed
research; V.K., S.K.M., E.L.,A.C.W., K.J.B., and H.C. performed
research; V.K., S.K.M., and E.L. contributed new re-agents/analytic
tools; V.K., S.K.M., and W.G.K. analyzed data; and V.K. and W.G.K.
wrotethe paper.
Reviewers: D.N., University of Texas Southwestern; and C.L.S.,
Memorial Sloan–KetteringCancer Center.
The authors declare no conflict of interest.
Published under the PNAS license.1Present address: Discovery
Biology, Tango Therapeutics, Cambridge, MA 02141.2Present address:
Department of Biology, Peloton Therapeutics, Dallas, TX 75235.3To
whom correspondence should be addressed. Email:
[email protected].
This article contains supporting information online at
www.pnas.org/lookup/suppl/doi:10.1073/pnas.1818109116/-/DCSupplemental.
Published online January 25, 2019.
www.pnas.org/cgi/doi/10.1073/pnas.1818109116 PNAS | February 12,
2019 | vol. 116 | no. 7 | 2539–2544
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approximate because some of the N-terminal IKZF3 residuesin
IKZF3 (146–168) might be serving as spacers between the N-terminal
firefly luciferase and the degron itself rather than
directlycontacting cereblon. Unfortunately, the IKZF3 fragments we
stud-ied, including both IKZF3 (136–180) and IKZF3 (146–168),
weregrossly impaired as degrons relative to IKZF3 (1–509) when
fusedto the N terminus of firefly luciferase (SI Appendix, Fig.
S1), pre-venting us from further refining the N-terminal degron
boundaryfree of neighboring sequences (see also below). In
pomalidomidedose titration experiments, IKZF3 (144–168) slightly
outperformedIKZF3 (146–168), which corresponds exactly to IKZF3
Zinc Finger2, with respect to pomalidomide-induced degradation of
their cor-responding firefly luciferase fusion proteins (SI
Appendix, Fig. S2 Aand B) and so this sequence
(RPFQCNQCGASFTQKGNLLR-HIKLH) was used in the studies below.As
expected, based on the behavior of full-length IKFZ3, the
function of the IKZF3 (144–168) degron was crippled by
intro-ducing a Q147H mutation (5) and was blocked in cells in
whichcullin-dependent ligases were inactivated with the
NEDD8-activating enzyme inhibitor MLN4924 or in which
cereblonitself was deleted using CRISPR/Cas9 (Fig. 1 D and G).
TheIKZF3 (144–168) degron was at least as capable as
full-lengthIKZF3 at targeting firefly luciferase for destruction in
cis in dose-titration experiments with three progressively more
potent IMiDs(Fig. 1 E, F,H, and I). In time course experiments,
degradation was
apparent within the first 6 h and maintained for 24 h in cells
con-tinuously exposed to an IMiD (Fig. 1 J–M). Finally, we
confirmedthat varying the length of the linker between FLuc and
IKZF1between 3 and 27 amino acid residues did not affect
degradation bypomalidomide (SI Appendix, Fig. S2 C and D).
IMiD-Dependent Degradation of Degron Fusion Proteins Occurs in
Boththe Nucleus and Cytoplasm. The IKZF3 (144–168) degron
alsotargeted green fluorescent protein (GFP) for
pomalidomide-induced degradation when fused to the GFP C terminus
andcould do so in both the cytosol and in the nucleus in the
context ofGFP-IKZF3 (144–168) fusion proteins that were engineered
tocontain strong nuclear localization or nuclear export signals
(Fig. 2A and B) as determined by fluorescence activated cell
sorting (Fig.2C) and immunoblot analysis (Fig. 2D). The IKZF3
(144–168)polypeptide did not function as a degron when fused to the
Nterminus of GFP (SI Appendix, Fig. S3). Finally, this degron
tar-geted exogenous HIF2α for pomalidomide-induced degradationwhen
fused to the C terminus of HIF2α, leading to down-regulation of
HIF2α-responsive mRNAs in cells in which endog-enous HIF2α was
inactivated using CRISPR/Cas9 (Fig. 3).
IMiD-Dependent Degradation of Oncogenic Fusion Protein
InhibitsTransformation in Soft Agar Assay. Next, we sought to test
whetherour IMiD-dependent degradation strategy could be used to
modulate
DMSOPomMLN4924MLN4942 + Pom
0.0
0.5
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DMSO10 nM100 nM
Thal Len Pom0.0
0.5
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1.5IKZF3
1 μM10 μM
Thal Len Pom0.0
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IRES
FLuc POI
IRES
RLuc
FLuc
DMSOPomMLN
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FLuc
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DMSO 6h 12h 18h 24h0.0
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WT Degron
FLuc
Vinculin
FLuc
Vinculin
A B
C
D
G
E
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F
I
DMSO 6h 12h 18h 24h DMSO 6h 12h 18h 24h
L M
0.0 0.5 1.0
144-166144-167148-168147-168146-168145-168144-168136-180
1-509
0.0 0.5 1.0
136-160136-164136-168136-172136-176136-180
1-509
0.0 0.5 1.0
156-180152-180148-180144-180140-180136-180
1-509
FLuc/RLuc (Pom/DMSO)
DM
SO
Thal Len Pom
FLuc
Vinculin
Pom
Pom
Pom
Pom
FLuc/RLuc (Pom/DMSO) FLuc/RLuc (Pom/DMSO)
FLuc
/RLu
c (P
om/D
MS
O)
DegronCRBN
WT WTQ147H+/+ +/+ -/-
CRBN
WT Q147H WT Degron:
IKZF3
Fig. 1. Mapping of IMiD-responsive IKZF3 degron. (A) IKZF3
schematic. Shaded boxes, zinc finger domains; dotted box, region
containing the IMiD-responsivedegron. (B) Schematic for bicistronic
reporter vector. POI, protein of interest. (C) Firefly luciferase
(FLuc) values, normalized to Renilla luciferase values (RLuc),
of293T cells transfected to express the indicated fragments of
IKZF3 fused to the C terminus of FLuc using the vector in B and
treated with 1 μM pomalidomide for24 h relative to cells treated
with DMSO. (D) FLuc/Rluc values of 293T cells (CRBN+/+ or CRBN−/−)
transfected to express IKZF3 144–168 (WT Degron) or IKZF3
144–168;Q147H (Q147H Degron) fused to FLuc and treated with 1 μM
pomalidomide, 1 μM MLN4924, or both for 24 h relative to cells
treated with DMSO. (E and F)FLuc/RLuc values of 293T cells
transfected to express full-length IKZF3 (E) or IKZF3 144–168 (WT
Degron) (F) fused to FLuc and treated with the indicated
con-centrations of thalidomide, lenalidomide, or pomalidomide for
24 h relative to cells treated with DMSO. (G–I) Immunoblots of
cells treated as in D–F, respectively.(J and L) FLuc/Rluc values of
293T cells transfected to express full-length IKZF3 (J) or IKZF3
144–168 (WT Degron) (L) fused to FLuc and treated with 1
μMpomalidomide for the indicated duration relative to cells treated
with DMSO. (K and M) Immunoblots of cells treated as in J and L,
respectively.
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oncoprotein stability and function. We lentivirally infected
im-mortalized melanocytes, PmeL* cells (10), to express
themicrophthalmia-associated transcription factor (MITF), which
is
a known melanoma oncoprotein capable of inducing
anchorage-independent growth (11), fused to the WT degron, to
theQ147H degron, or unfused. Pomalidomide suppressed the
GFP GFPWT Degron
GFP-NLSWT Degron
GFP-NESWT Degron
GFP
H33352
A
C
GFP
GFP
GFP
GFP NLS
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GGS
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GFP
-WT
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-NES
-WT
Degr
on
GFP
-NLS
-WT
Degr
on
EV
Pom
Fig. 2. The core IMiD-responsive IKZF3 degron functions in
different cellular compartments. (A) GFP fusion protein schematics.
Degron, IKZF3 144–168; GGS,glycine-glycine-serine linker; NES,
nuclear export signal; NLS, nuclear localization signal. (B)
Fluorescence images of 293T cells stably infected to express
theindicated GFP fusion proteins. H33352, nuclear dye. (Images
captured with 60× objective lens.) (C and D) GFP fluorescence
intensity, as determined by FACS(C), and immunoblot analysis (D) of
293T cells stably infected as in B and treated with 1 μM
pomalidomide or DMSO for 24 h.
A B
C D
Fig. 3. The core-IMiD responsive IKZF3 degron functions when
fused to a heterologous transcription factor. (A) Immunoblot
analysis of 786-O renal car-cinoma cells that underwent
CRISPR/Cas9-mediated gene editing with a HIF2α sgRNA or control
sgRNA, where indicated, before being stably infected with
anlentivirus encoding sgRNA-resistant HIF2α-IKZF3(144–168)
(HIF2α-WT Degron) or HIF2α-IKZF3(144–168;Q147H) (HIF2α-Q147H
Degron) and then treated witheither DMSO or 1 μM pomalidomide for
24 h. (B–D) Abundance of the indicated mRNAs measured by qPCR in
786-O cells treated as in A after normalization toACTBmRNA and then
to the values of the 786-O cells treated with DMSO and the control
sgRNA. For all panels, data presented are means ± SD; **P <
0.01; ns,not significant. Two-tailed P values were determined by
unpaired t test.
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anchorage-independent growth of PmeL* cells expressing the
MITF-WT Degron fusion relative to cells expressing unfused MITF or
theMITF-Q147H Degron fusion (SI Appendix, Fig. S4). These
resultsindicate that our inducible strategy for heterologous
protein degra-dation might be useful for studying oncoprotein
function and de-pendence in preclinical cancer models.
IKZF3 (144–168) Degron Functions in Multiple Orthotopic
MouseModels. To ask if the IKZF3 (144–168) degron could functionin
vivo, we stably infected 786-O renal carcinoma cells, MDA-MB-231
breast carcinoma cells, and HOG oligodendrogliomacells to produce
Firefly Luciferase-IKZF3 (144–168) or FireflyLuciferase-IKZF3
(144–168; Q147H). Pomalidomide down-regulated the former and not
the latter in all three cellular con-texts and did not have any
effect on cellular viability at concen-trations up to 10 μM (Fig. 4
A–C and SI Appendix, Figs. S5 andS6A). Next these cells were grown
orthotopically in the kidneys,mammary fat pads, and brains,
respectively, of immunocompro-mised mice. Once tumors were
established, as determined by se-rial bioluminescence imaging
(BLI), mice were randomized toreceive 30 mg/kg pomalidomide or
vehicle by oral gavage twicedaily for a total of four doses. As
expected, pomalidomide causeda greater than 50% reduction in BLI
signals in tumors containingthe Firefly Luciferase-IKZF3 (144–168)
chimera, but not in tu-mors containing the Q147H chimera (Fig. 4
D–I). In drug washoutand rechallenge experiments, mice did not
exhibit tolerance topomalidomide, with each rechallenge producing
an ∼50% re-duction in Firefly Luciferase-IKZF3 (144–168) BLI signal
(Fig. 4 Jand K). The tumors formed by the two different chimeras
werecomparable in size at necropsy (SI Appendix, Fig. S6 B–D).
DiscussionThere are a number of methods to regulate the
transcription orstability of a protein of interest. Directly
regulating protein sta-bility, however, creates an opportunity to
more rapidly alter theabundance and, hence, function, of a protein
of interest com-pared with methods that act at the transcriptional
level. More-over, it will perhaps more faithfully mimic the effects
of smallmolecule protein antagonists, especially those that act
wholly orin part by destabilizing their targets. The approach
designed herecomplements several ingenious approaches that have
been de-scribed over the past decade for chemically stabilizing
orchemically destabilizing proteins of interest.One system for
chemically stabilizing a protein of interest in-
volves fusing it to a variant of human FKBP12 (FKBP12*) that
istargeted for degradation unless it is bound to an artificial
ligandcalled Shield-1 (12). FKBP12* also has a point mutation
(F36V)such that it binds to Shield-1 with 1,000-fold selectivity
comparedwith wild-type FKBP12. The FKBP cassette is
considerablylarger than the one described here (107 versus 25 amino
acidresidues) and so it might be more prone to alter protein
function.A modified version of this system allows the stabilization
andrelease of an unfused protein of interest (traceless shield),
but atthe expense of expressing two foreign proteins: an FRB
(FKBP-Rapamycin-Binding) domain-UbN fusion and a FKBP12*-UbCprotein
of interest fusion (13). In this embodiment, Shield-1stabilizes the
protein of interest, which can then be released byrapamycin-induced
reconstitution of the ubiquitin protease. Fi-nally, this technique
has been further modified by Nabet et al(14), who showed that a
heterobifunctional chemical ligandcomprised of AP1867 and an IMiD
could be used to trigger thedegradation of proteins of interest
fused to FKBP12*.A second method for chemically stabilizing
proteins involves
fusing the protein of interest to an unstable variant of
Escherichiacoli dihydrofolate reductase (ecDHFR) that is stabilized
in thepresence of trimethoprim (TMP) (15, 16). The biodistribution
ofTMP has been better studied than that of Shield-1 and is knownto
cross the blood–brain barrier. However, ecDHFR might prove
to be immunogenic. Moreover, both the FKBP12*/Shield-1
andecDHFR/TMP systems require that Shield-1 and TMP, re-spectively,
be continuously present until the moment when acuteprotein
destabilization is desired. This could prove cumbersomeand costly,
especially in animal models.To circumvent this problem, Wandless
and coworkers (17)
fused FKBP12 (F36V) to an additional 19 amino acids thatcreate a
cryptic degron that is displayed only after Shield-1 isadded and
showed that this chimera could be used to targetheterologous
proteins for destruction with Shield-1. In a com-plementary
approach, called “SMASh,” Lin and coworkers (18)fused a modular
degron to proteins of interest with interveningsequences encoding
the hepatitis C NS3 protease and an NS3protease cleavage site such
that the degron is constitutively re-leased unless cells are
treated with the protease inhibitorAsunaprevir. In this latter
system, unlike the former, the stableversion of the protein is
minimally altered, having only the short“stub” generated by
protease cleavage. However, Asunaprevircan only act on newly
synthesized proteins because mature formsof the protein will
already have excised the artificial degron. Inaddition, Asunaprevir
does not cross the blood–brain barrier.Another method for targeting
heterologous proteins for destruc-
tion exploits the naturally occurring plant hormones called
auxins,which bind to the ubiquitin ligase SCFTIR1 and trigger the
degrada-tion of members of the AUX/IAA family of transcriptional
repressors(19, 20). Natsume and coworkers showed that the fusion of
a 68-amino acid sequence derived from the IAA17 transcriptional
re-pressor to a target heterologous protein rendered that protein
sus-ceptible to rapid degradation in the presence of an auxin (19).
Anadvantage of this system is that plant auxins do not have
knowntargets in mammalian cells and are therefore unlikely to cause
tox-icity. However, use of the auxin system in mammalian cells
requiresthe enforced expression of the plant-derived TIR1 protein
in additionto tagging the protein of interest with a 68-amino acid
residue tag.The IKZF3 degron system described here has a number of
po-
tential advantages compared with previously described systems.
First,it requires only a simple genetically encoded modification of
a pro-tein of interest that can easily be introduced with a
synthetic oligo-nucleotide and that leads to a modest peptidic
addition analogous toappending an epitope tag. In fact, one can
envision the IKZF3 25merserving as both a degron and an epitope tag
if suitable antibodies canbe raised. Moreover, the IKZF3 degron
system utilizes IMiDs, whichare well-studied, bioavailable, and
easily obtainable. In particular,pomalidomide is bioavailable and
active in the CNS. Finally, theIKZF3 degron system is understood in
sufficient detail to allow forthe incorporation of additional
specificity controls, including, asshown here, loss of function
degron point mutants and pharmaco-logical and genetic inhibitors of
the cereblon ubiquitin ligase.So far it appears the IKZF3 25mer
degron functions best when
fused to a target protein’s C terminus rather than its N
terminus.As larger IKZF3 fragments do function as degrons when
fused tothe N terminus of firefly luciferase, we presume this
relates tosteric factors and protein folding. Determining whether
this ap-parent bias is true will require testing additional fusion
partnersin the future. For reasons we do not yet fully understand,
theIKZF3 25mer degron does not target all proteins for
destruction.For example, we have been unable to target the VHL
andFOXP3 proteins for destruction with the IKZF3 25mer
degron,whether fused at the N terminus or C terminus (data not
shown).The empirically determined percentage of endogenous
proteinsamenable to this approach after CRISPR/Cas9-mediated
in-troduction of an IKZF1 (or 3)-derived degron is ∼30–40%
(SIAppendix, Fig. S7). We hypothesize that the cereblon
ubiquitinligase complex cannot bind to the IKZF degron in some
fusionsdue to steric effects or, once bound, is not able to
productivelyreach a surface lysine residue that is capable of being
poly-ubiquitylated. Both of these issues might, theoretically,
beaddressed by exploring different spacers between the degron
and
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(Pos
ttrea
tmen
t : P
retre
atm
ent) *
MDA-MB-231
0.0
0.5
1.0
1.5
2.0
2.5
Biol
umin
esce
nce
(Pos
ttrea
tmen
t : P
retre
atm
ent) *
786-O
E
H
J K
0 3 6 9 12 15 18107
108
109
1010
Days
Biol
umin
esce
nce
(pho
tons
/sec
)
MDA-MB-231
WTQ147H
0 3 6 9 12 15 18108
109
1010
Days
Biol
umin
esce
nce
(pho
tons
/sec
)
786-O
WT
Q14
7H WT
Q14
7H
Pre Post786-O
WT
Q14
7H WT
Q14
7H
Pre PostMDA-MB-231
C
F
I
Pre Post
Pre Post
WT
Q147H
HOG
- + - +
FLuc
Pom
Vinculin
786-O
- + - +
FLuc
Pom
Vinculin
MDA-MB-231
- + - +
FLuc
Pom
Vinculin
0
2
4
6
8
HOG
**WTQ147H
Biol
umin
esce
nce
(Pos
ttrea
tmen
t : P
retre
atm
ent)
HOG
N=12 N=16 N=8
Degron WT Q147H Degron WT Q147H Degron WT Q147H
Fig. 4. The core IKZF3 IMiD-responsive degron functions in vivo.
(A–C) Immunoblot analysis of 786-O renal carcinoma cells (A),
MDA-MB-231 breast carci-noma cells (B), and HOG oligodendroglioma
cells (C) stably infected to express FLuc-IKZF3 144–168 (WT) or
FLuc-IKZF3 144–168;Q147H (Q147H) and treatedwith 1 μM pomalidomide
or DMSO for 24 h. (D–F) Representative BLI of orthotopic tumors
formed by cells as in A–C, respectively, before (pre) and after
(post)systemic administration of 30 mg/kg pomalidomide by oral
gavage twice a day for 48 h (4 doses). (G–I) Quantification of BLI
signals from mice treated as in D–F, respectively. (J and K).
Quantification of BLI signals from representative mice as in G and
H, respectively, over time. Yellow bars indicate treatment
withpomalidomide as above. Note logarithmic scale. For all panels,
data presented are means ± SD; **P < 0.05; **P < 0.01.
Two-tailed P values were determinedby unpaired t test.
Koduri et al. PNAS | February 12, 2019 | vol. 116 | no. 7 |
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the heterologous protein, changing the placement of the
degronrelative to the heterologous protein, extending the length of
thedegron, and/or incorporating lysine residues into the spacer
(seealso SI Appendix, Fig. S7). Similarly, we have not been able
totarget the VHL protein for degradation with a SMASh tag, de-spite
drug-induced degron retention, suggesting that differentsystems may
or may not work for any given protein. A strength orweakness of the
IKZF3 degron system, depending upon theapplication, is that mouse
cereblon does not engage IKZF3 whenbound to an IMiD. However, a
mouse with a humanized cere-blon has been developed (21).Two recent
studies showed that indisulam redirects an ubiq-
uitin ligase containing the substrate adaptor DCAF15 to
degradeRBM39, much as the IMiDs redirect the cereblon ligase to
targetIKZF1 and IKZF3 for destruction (22, 23). Moreover, it is
nowclear that some IMiDs can also direct the destruction of
caseinkinase 1α, ERF3, and SALL4 (24–26). Mapping the
relevantdegrons in RBM39, casein kinase 1α, and ERF3A, and
comparingtheir behavior to the IKZF1/3 degron, might yield
additionalportable degrons for creating tunable proteins. The
IKZF3
degron system might also be improved by random mutagenesis of
theIKZF3 degron followed by positive selection for enhanced binding
tocereblon in the presence of an IMiD, as well as by next
generationIMiDs that are more potent than existing compounds.
Materials and MethodsA detailed description of the cell lines,
plasmids, and antibodies used in thispaper can be found in the SI
Appendix. Detailed protocols for lentiviral in-fection,
immunoblotting, luciferase assays, FACS analysis, fluorescence
mi-croscopy, real-time qPCR, soft agar assays, and orthotopic
xenograftexperiments can also be found in SI Appendix. All
experimental proceduresrelated to orthotopic xenografts were
approved by the Institutional AnimalCare and Use Committee of
Dana–Farber Cancer Institute.
ACKNOWLEDGMENTS. We thank Benjamin Ebert, Eric Fischer, Egon
Ogris,and Gromoslaw Smolen for critical reading of the manuscript;
members ofthe W.G.K. laboratory for useful discussions and comments
on the manu-script; Matthew Oser for help with fluorescence
microscopy; Eric Fischer forsharing unpublished data; and Gang Lu
and Rizwan Haq for reagents. Thiswork was supported by NIH Grants
(to W.G.K.), T32 NIH Training GrantCA009172 and American Society of
Hematology Research Training Award(to V.K.). W.G.K. is a Howard
Hughes Medical Institute Investigator.
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