bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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.
1 UBE2G1 Governs the Destruction of Cereblon Neomorphic Substrates
2
3 Gang Lu1,*, Stephanie Weng1,¶, Mary Matyskiela1,¶, Xinde Zheng1, Wei Fang1, Scott Wood1,
4 Christine Surka1, Reina Mizukoshi1, Chin-Chun Lu1, Derek Mendy1, In Sock Jang1, Kai Wang1,
5 Mathieu Marella1, Suzana Couto1, Brian Cathers1, James Carmichael1, Philip Chamberlain1,
6 Mark Rolfe1
7
8 Affiliations:
9 1Celgene Corporation, 10300 Campus Point Drive, Suite 100, San Diego, CA 92121, USA
10
11
12 * For correspondence: E-mail: [email protected]
13 ¶ These authors contributed equally to this work.
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19 bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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.
20 Abstract
21 The immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide as well as
22 the novel cereblon modulating agents (CMs) including CC-122, CC-220 and cereblon-based
23 proteolysis-targeting chimaeras (PROTACs) repurpose the Cul4-RBX1-DDB1-CRBN
24 (CRL4CRBN) E3 ubiquitin ligase complex to induce the degradation of specific neomorphic
25 substrates via polyubiquitination in conjunction with an E1 ubiquitin-activating enzyme and E2
26 ubiquitin-conjugating enzymes, which have until now remained elusive. Here we show that the
27 ubiquitin-conjugating enzymes UBE2G1 and UBE2D3 cooperatively promote the
28 polyubiquitination of CRL4CRBN neomorphic substrates in a cereblon- and CM-dependent
29 manner via a sequential ubiquitination mechanism: UBE2D3 transforms the neomorphic
30 substrates into mono-ubiquitinated forms, upon which UBE2G1 catalyzes K48-linked
31 polyubiquitin chain extension. Blockade of UBE2G1 diminishes the ubiquitination and
32 degradation of neomorphic substrates, and consequent antitumor activities elicited by all tested
33 CMs. For example, UBE2G1 inactivation significantly attenuated the degradation of myeloma
34 survival factors IKZF1 and IKZF3 induced by lenalidomide and pomalidomide, hence conferring
35 drug resistance. UBE2G1-deficient myeloma cells, however, remained sensitive to a more potent
36 IKZF1/3 degrader CC-220. Collectively, these findings suggest that loss of UBE2G1 activity
37 might be a resistance mechanism to drugs that hijack the CRL4CRBN to eliminate disease-driving
38 proteins, and that this resistance mechanism can be overcome by next-generation CMs that
39 destroy the same targeted protein more effectively.
40
41 bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
42 Introduction
43 The ubiquitin-proteasome system (UPS) is a highly regulated component of the protein
44 homeostasis network that dictates multiple cellular processes in eukaryotes (Hershko and
45 Ciechanover, 1998). Through the orchestrated actions of ubiquitin-activating enzymes (E1),
46 ubiquitin-conjugating enzymes (E2) and ubiquitin-ligating enzymes (E3), the ε-amine of a lysine
47 residue in a target protein is covalently conjugated with K48- or K11-linked poly-ubiquitin
48 chains, thereby marking the target protein for proteasomal degradation (Jin et al., 2008;
49 Komander and Rape, 2012; Pickart, 2001). Recently, repurposing the Cullin-Ring E3 ligase
50 complexes CRL4CRBN (Cul4-RBX1-DDB1-CRBN) and CRL2VHL (Cul2-RBX1-EloB/C-VHL)
51 with small-molecule degraders to remove disease-driving proteins otherwise considered
52 ‘undruggable’ has emerged as a novel therapeutic modality that has the potential to transform
53 drug discovery and development (Bondeson and Crews, 2017; Huang and Dixit, 2016; Lebraud
54 and Heightman, 2017).
55 There are two types of small-molecule degraders that have been exploited used to date. The first
56 is represented by thalidomide (THAL), lenalidomide (LEN) and pomalidome (POM), as well as
57 other cereblon modulating agents CC-122, CC-220, and CC-885. This class of molecule docks
58 into a tri-tryptophan pocket in the thalidomide-binding domain of cereblon, the substrate receptor
59 of CRL4CRBN, to create a hotspot for protein-protein interactions thereby enhancing the binding
60 of unique neomorphic substrate to cereblon, resulting in substrate ubiquitination and degradation
61 (Fischer et al., 2014) (Chamberlain et al., 2014) (Matyskiela et al., 2018) (Petzold et al., 2016).
62 THAL, LEN and POM promote the degradation of two hematopoietic transcription factors
63 IKZF1 and IKZF3 to achieve anti-myeloma activity (Kronke et al., 2014) (Lu et al., 2014a)
64 (Gandhi et al., 2014), whereas only LEN targets CK1α for effective degradation (Kronke et al., bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
65 2015), which is presumably linked to its efficacy in myelodysplastic syndrome with chromosome
66 5q deletion. CC-220 is a significantly more potent IKZF1 and IKZF3 degrader than IMiD drugs
67 (Matyskiela et al., 2018) (Nakayama et al., 2017) (Schafer et al., 2018), and it is currently in
68 clinical trials for relapsed/refractory multiple myeloma and systemic lupus erythematosus. By
69 contrast, CC-885 is the only aforementioned cereblon modulating agent that allows cereblon to
70 recognize translation termination factor GSPT1 for ubiquitination and degradation (Matyskiela et
71 al., 2016). The second type of small-molecule degraders is generally referred to as a proteolysis-
72 targeting chimera (PROTAC) (Sakamoto et al., 2001), which is composed of two linked protein
73 binding ligands, with one engaging a target protein and the other interacting with an E3 ubiquitin
74 ligase such as CRL4CRBN or CRL2VHL to trigger proximity-induced substrate ubiquitination and
75 degradation (Deshaies, 2015) (Neklesa et al., 2017). Many PROTACs have been described
76 recently, but the clinical value of this approach has not yet been established.
77 CRL4CRBN belongs to the multi-subunit cullin-RING E3 ubiquitin ligase family containing over
78 200 members (Petroski and Deshaies, 2005). The mammalian cullin scaffold proteins (including
79 Cul1, Cul2, Cul3, Cul4A, Cul4B, Cul5, and Cul7) bring their substrates into close proximity with
80 E2 ubiquitin-conjugating enzymes, thereby enabling effective substrate ubiquitination (Petroski
81 and Deshaies, 2005). SCF(Skp1-Cul1-F-box)Cdc4, the founding member of the cullin-RING E3
82 ligase family, was first discovered in the budding yeast Saccharomyces cerevisiae, in which
83 SCFCdc4 works in conjunction with a single E2 ubiquitin-conjugating enzyme Cdc34 to promote
84 the polyubiquitination of a variety of SCF substrates (Feldman et al., 1997) (Skowyra et al.,
85 1997) (Blondel et al., 2000) (Jang et al., 2001; Perkins et al., 2001). Human Cdc34/UBE2R1 can
86 substitute for yeast Cdc34 in Saccharomyces cerevisiae underscoring their functional
87 conservation (Plon et al., 1993). However, in contrast to its dominant role in catalyzing the bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
88 ubiquitination of SCF substrates in yeast, Cdc34 coordinates ubiquitination with
89 UBE2D3/UbcH5c via a sequential ubiquitination mechanism to improve reaction rate and
90 efficiency in human cells. In brief, Cdc34 acts as an ubiquitin chain elongation enzyme that
91 assembles the K48-linked ubiquitin chains on mono-ubiquitins pre-conjugated to SCF substrates
92 by UBE2D3 (Pan et al., 2004). Such sequential ubiquitination by two E2 enzymes was first
93 reported for the anaphase-promoting complex ubiquitin ligase (Rodrigo-Brenni and Morgan,
94 2007). Several ubiquitin conjugation E2 enzymes have been reported to regulate CRL4
95 substrates as well. For instance, in response to UV irradiation, the CRL4cdt2 ligase complex
96 mediates the proteolysis of Cdt1 with the help of E2 enzymes UBE2G1 and its paralog UBE2G2,
97 while working together with a different E2 enzyme UbcH8/UBEL6 to trigger the degradation of
98 p21 and Set8 in human cells (Shibata et al., 2011). Despite the proven cellular efficacy and
99 clinical success of many cereblon modulating agents, it remain unknown whether unique
100 ubiquitin E2 enzymes control the ubiquitination of each specific cereblon neomorphic substrate,
101 and whether loss of E2 enzymes contributes to resistance to these agents.
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108 bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
109 Results
110 UBE2G1 is the dominant ubiquitin E2 enzyme that governs the destruction of cereblon
111 neomorphic substrates induced by cereblon modulating agents
112 The clinical course of multiple myeloma typically follows a recurring pattern of remission and
113 relapse with resistance to IMiD drugs based combination regimens (Harousseau and Attal, 2017).
114 Such relapse is not frequently associated with cereblon downregulation and/or mutation (Kortum
115 et al., 2016; Qian et al., 2018) (Zhu et al., 2011)Hence, we reasoned that resistance to IMiD
116 drugs in myeloma could be ascribed to reduced degradation of IKZF1 and IKZF3 as a result of
117 inactivation of other essential components of the CRL4CRBN ligase complex, for instance the E2
118 ubiquitin conjugation enzyme. To look for such proteins, we devised a high-throughput CRISPR-
119 Cas9 screen approach to monitor the effect of individual knockout of a gene of interest on POM-
120 induced degradation of IKZF1 protein tagged with enhanced ProLabel (ePL), a small β-
121 galactosidase N-terminal fragment (Figure 1A), and created a single guide RNA (sgRNA) library
122 containing three sgRNAs for each of the 41 annotated E2 enzymes in the human genome, as well
123 as three non-targeting sgRNAs in arrayed format (Supplemental Table 1). The ePL tag
124 complements with the large β-galactosidase C-terminal fragment to form an active enzyme that
125 hydrolyzes substrate to emit a chemiluminescent signal, allowing the measurement of ePL-
126 IKZF1 fusion protein level in a high-throughput fashion.
127 To determine the robustness of this screening approach, we transduced U937 cells stably
128 expressing Cas9 and ePL-IKZF1 (U937_Cas9_ePL-IKZF1) with lentiviral vector expressing a
129 non-targeting or CRBN-specific sgRNA. Four days post transduction, cells were seeded into 384-
130 well plates pre-dispensed with either DMSO vehicle control or POM at varying concentrations.
131 Sixteen hours after incubation, IKZF1 degradation was assessed using the ePL luminescent bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
132 assay. As expected, cereblon knockout completely abrogated the degradation of ePL-tagged
133 IKZF1 fusion protein (Figure S1A). Using this approach we then evaluated the effect of
134 individual knockout of each E2 enzyme on ePL-IKZF1 degradation induced by POM. Out of 41
135 E2 enzymes, UBE2G1 and to a lesser extent UBE2M, UBE2D3, and UBE2D2/UbcH5b, when
136 depleted, imposed statistically significant inhibition on the ePL-IKZF1 degradation (Figures 1B,
137 S2D and S2I).
138 UBE2M, also called UBC12, is a NEDD8-conjuating enzyme, which regulates, via neddylation,
139 the activity of all Cullin Ring E3 ligases including CRL4CRBN (Petroski and Deshaies, 2005)
140 (Gong and Yeh, 1999) (Pan et al., 2004). Indeed, co-treatment with MLN4924, an inhibitor of
141 NEDD8-activating enzyme (Soucy et al., 2009), prevented the degradation of ePL-IKZF1
142 induced by POM (Figure S1C). The effect of UBE2M knockout, however, was much less
143 pronounced (Figure S2I). Given the well-established role of UBE2M in cell proliferation and
144 survival, we reasoned that this difference could be explained by that U937 cells only with partial
145 UBE2M loss survived four days after CRISPR gene editing. Consistent with this notion, cellular
146 fitness was markedly reduced by 48-hour treatment of MLN2924 at concentrations that elicited
147 near-complete blockage of POM-induced IKZF1 degradation in the U937_Cas9_ePL- IKZF1
148 cell line used in the screen (Figures S1C and S1D).
149 CRISPR knockout of UBE2G1 also attenuated the destabilization of endogenous IKZF1 by POM
150 in U937 cells, and this defect could be rescued by wild-type UBE2G1, but not a UBE2G1
151 enzymatically-dead mutant (C90S, Figure 1C). Knockout of UBE2D3, on the other hand,
152 showed very little effect on the degradation of endogenous IKZF1 (Figure 2C). Thus, we
153 reasoned that there are additional E2 enzyme(s) modulating the degradation of IKZF1
154 cooperatively with UBE2G1. To identify such E2(s), we evaluated the effect of double knockout bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
155 of UBE2G1 and one of the 41 E2 enzymes on IKZF1 degradation using a dual gRNA-directed
156 gene knockout approach (Figures 2A and S3). Notably, double knockout of UBE2G1 and
157 UBE2D3 produced more inhibition of POM-induced degradation of ePL-tagged and endogenous
158 IKZF1 than either single knockout alone (Figures 2B and 2C). Combinatorial ablation of
159 UBE2G1 with UBE2E1 or UBE2M also demonstrated subtle but noticeable further inhibition on
160 IKZF1 degradation (Figures S3E and S3I). Although UBE2D2 knockout slightly attenuated the
161 POM-induced ePL-IKZF1 degradation (Figure S2D), double knockout of UBE2D2 and
162 UBE2G1 did not significantly augment the inhibition of ePL-IKZF1 degradation imposed by
163 UBE2G1 single knockout (Figure S3D).
164 To assess the substrate selectivity of UBE2G1, we determined the effect of UBE2G1 knockout
165 on the degradation of IKZF1, its paralog IKZF3 and other well-characterized cereblon
166 neomorphic substrates, triggered by their respective cereblon modulating agents including
167 cereblon-based PROTACs. Ablation of UBE2G1 significantly diminished the degradation of
168 IKFZ1, IKZF3 and ZFP91 by LEN, POM, and CC-220, as well as CK1α degradation by LEN in
169 OPM2, DF15 and MM1S myeloma cells (Figures 3A, S4A, S4B, 7B, S8A and S8B). UBE2G1
170 loss also reduced the degradation of GSPT1 induced by CC-885 in myeloma cell lines OPM2,
171 DF15 and MM1S (Figures 3B, S4C and S3D), AML cell lines OCI-AML2, U937, MOLM-13
172 and MV4-11 (Figures S4E-H), as well as 293T human embryonic kidney cells (Figure S4I). The
173 GSPT1 degradation defect conferred by UBE2G1 depletion could also be rescued by UBE2G1
174 wild-type but not C90S mutant in 293T cells (Figure S4I). UBE2G1 loss also blocked the
175 degradation of Brd4 induced by the cereblon-based BET PROTAC dBET1 (Winter et al., 2015),
176 but not the VHL-based BET PROTAC MZ1 (Zengerle et al., 2015) in 293T cells. UBE2G1
177 depletion prolonged the protein half-lives of IKZF1 and IKZF3 in OPM2 cells treated with bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
178 POM. Lastly, UBE2G1 loss did not affect stability of SCF substrates p27 and c-Myc, suggesting
179 that UBE2G1 might be specific to CRL4 (Figure 3C).
180 UBE2G1 mediates the ubiquitination of cereblon neomorphic substrates
181 UBE2G1 and its paralog UBE2G2 share similar domain structures with CDC34. A common
182 structural feature of these three E2 enzymes is an acidic loop (Figure S5A, highlighted with red)
183 in the vicinity of their respective active site cysteines (Figure S5A, highlighted with blue), which
184 facilitates the direct binding with ubiquitin and enables K48-linked ubiquitin chain assembly in
185 the absence of associated E3 ligases (Choi et al., 2015). In association with gp78, an ER
186 membrane bound RING finger E3 ubiquitin ligase, UBE2G2 directly tags misfolded proteins
187 with K48-linked ubiquitin chains preassembled on the catalytic cysteine of UBE2G2, resulting in
188 ER associated protein degradation (ERAD) (Li et al., 2007). Although it has been shown that
189 UBE2G1 and UBE2G2 redundantly mediate the destabilization of the CRL4cdt2 substrate Cdt1 in
190 response to UV irradiation, the direct transfer of ubiquitin to Cdt1 by UBE2G1 or UBE2G2 has
191 not been demonstrated (Shibata et al., 2011).
192 Next, we employed a reconstituted in vitro ubiquitination assay to address the role of UBE2G1
193 and UBE2D3 in ubiquitination of IKZF1 and GSPT1 induced by POM and CC-885,
194 respectively. We monitored the production of ubiquitin conjugates of IKZF1 and GSPT1
195 catalyzed by UBE2D3 alone, UBE2G1 alone, or in combination, in the presence of Ube1 (E1),
196 Cul4-Rbx1, cereblon-DDB1, ubiquitin and ATP with or without POM or CC-885. UBE2D3
197 alone produced ubiquitin conjugates of IKZF1 or GSPT1 mainly with a single or di-ubiquitin
198 moiety in a POM-or CC-885- dependent manner (Figures 4A and 4B). By contrast, UBE2G1
199 alone failed to display any ubiquitin conjugating activity for both IKZF1 and GSPT1 (Figures 4A
200 and 4B, lanes 3 and 4). However, when combined with UBE2D3, UBE2G1 significantly bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
201 promoted the extent of ubiquitination of both substrates (Figures 4A and 4B, lanes 2 and 6).
202 Moreover, the ubiquitin conjugates of IKZF1 or GSPT1 formed with both UBE2G1 and
203 UBE2D3, but not UBE2D3 alone, were exclusively K48-linked, because the wild-type ubiquitin
204 used in the reconstituted ubiquitination reaction could be functionally replaced by ubiquitin
205 mutant K48-only (with 6 remaining lysine residues mutated to arginine), but not by K48R
206 (remaining lysine residues were intact), and the ubiquitination pattern of IKZF1 or GSPT1
207 catalyzed by UBE2D3 exhibited no obvious difference between wild-type ubiquitin and K48-
208 only or K48R mutant (Figures 4A and 4B, lane 2; Figures 4C and 4D, lanes 2 and 8).
209 To further explore the mechanism underlying the cooperativity between UBE2G1 and UBE2D3,
210 we separated the ubiquitination reaction of GSPT1 into two steps. First, following GSPT1
211 ubiquitination by UBE2D3 alone, we isolated GSPT1 ubiquitin conjugates from the rest of
212 reaction components using a size-exclusion column (Figure 4E, lanes 1 and 2). We then
213 incubated the purified GSPT1 ubiquitin conjugates with UBE2G1, Ube1 (E1), cereblon-DDB1,
214 ubiquitin and ATP with or without CC-885 and Cul4-Rbx1. We found that UBE2G1 was capable
215 of catalyzing the further ubiquitination of GSPT1 only with prior-conjugated ubiquitin (Figure
216 4E, lanes 1-4, note the conversion of the mono-ubiquitinated GSPT1 into di- or tri-ubiquitinated
217 forms), and this action required the presence of CC-885 and Cul4A-Rbx1 (Figures 4E).
218 To rule out the possibility that the UBE2G1 function observed above is simply an artifact of
219 bacterial recombinant protein, we reconstituted the ubiquitination reaction using FLAG-tagged
220 UBE2G1 and FLAG-tagged UBE2D3 proteins purified from 293T UBE2G1-/- cells, in which
221 ectopic overexpression of FLAG-tagged UBE2G1 and UBE2D3, but not their respective
222 enzymatically-dead mutant, could partially rescue the defect in CC-885-induced GSPT1
223 degradation elicited by UBE2G1 loss (Figure S5B). In agreement with our previous findings, bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
224 GSPT1 ubiquitination catalyzed in vitro by FLAG-UBE2G1 and FLAG-UBE2D3, alone or in
225 combination, was similar to what was observed with bacterial recombinant UBE2G1 and
226 UBE2D3 (Figure S5C). In addition, we examined the in vivo function of UBE2G1 and UBE2D3
227 in the regulation of POM-induced ubiquitination of IKZF1 ectopically expressed in 293T cells.
228 Ablation of both UBE2G1 and UBE2D3 significantly reduced, but did not completely block, the
229 mono- and polyubiquitination of IKZF1 induced by POM, indicating the existence of additional
230 E2 enzymes with redundant function (Figure 5A and S6A). Reintroduction of UBE2G1 alone via
231 transient transfection in 293T UBE2G1-/- ; UBE2D3-/- cells dramatically enhanced the extent of
232 POM-induced IKZF1 polyubiquitination (Figure 5B, lanes 5-8 vs 1-4). Transient overexpression
233 of UBE2D3 alone promoted IKZF1 monoubiquitination as well as polyubiquitination but to a
234 lesser extent (Figure 5B, lanes 9-12 vs 1-4). Overexpression of both achieved an additive or
235 possibly synergistic, effect on IKZF1 ubiquitination (Figure 5B, lanes 13-16 vs 5-12, note the
236 conversion of monoubiquinated IKZF1 into its polyubiquitinated forms). In keeping with the in
237 vitro finding (Figure S5C), IKZF1 was mainly mono-ubiquitinated in 293T UBE2G1-/- cells
238 following POM treatment, and UBE2G1 wild-type but not C90S mutant could restore IKZF1
239 polyubiquitination (Figure 5C and S6C).
240 UBE2G1 loss confers resistance to cereblon modulating agents
241 Since UBE2G1 depletion significantly attenuated the degradation of all cereblon neomorphic
242 substrates, we reasoned that UBE2G1 protein downregulation, gene deletion or mutation might
243 lead to reduced CRL4CRBN activity, thereby leading to resistance to cereblon modulating agents.
244 To test this hypothesis, we surveyed the protein expression level of UBE2G1 in myeloma cell
245 lines with variable sensitivity to LEN and POM (Figure 6A). LEN sensitive cell lines MM1S,
246 OPM2, DF15 and NCI-H929 displayed higher expression level of UBE2G1 than LEN medium- bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
247 sensitive cell line ANABL-6 and LEN resistant cell lines EJM, L363, SKMM2, CAG and ARH-
248 77, and more strikingly UBE2G1 expression in SKMM2 was undetectable (Figure 6B). As
249 expected, reintroduction of UBE2G1 wild-type but not C90S mutant significantly augmented the
250 antiproliferative effect of both LEN and POM, which was linked to the enhanced degradation of
251 IKZF1 and IKZF3 (Figures 6C and 6D). UBE2G1 loss also conferred resistance to CC-885 in
252 OCI-AML2, U937, MOLM-13, and MV4-11 AML cells (Figures S7A-D), as well as 293T cells,
253 and this defect could be rescued by UBE2G1 wild-type but not C90S mutant (Figure S7E).
254 Moreover, UBE2G1 deletion conferred resistance to BET PROTAC dBET1 but not MZ1 in
255 293T cells. Lastly, UBE2G1 knockout in DF15, MM1S and OPM2 myeloma cells conferred
256 significant resistance to LEN and POM. Importantly, these cells were only partially resistant to
257 CC-220, in keeping with the increased efficiency in triggering IKZF1 and IKZF3 degradation
258 (Figures 7A, 7B and S8A-D). These results indicate that UBE2G1 deficiency may be a key
259 differentiator in the clinical success of cereblon modulating agents.
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267 bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
268 Discussion
269 The sequential recruitment of two functionally distinct E2s by a single E3 is a general
270 mechanism for substrate ubiquitination conserved from yeast to human (Rodrigo-Brenni and
271 Morgan, 2007) (Wu et al., 2010) (Kleiger and Deshaies, 2016). In this work, we provide
272 evidence that CRL4CRBN deploys the same mechanism to mark its neomorphic substrates with
273 K48-linked poly-ubiquitin chains for proteasomal degradation. Mechanistically, UBE2D3
274 transfers the first ubiquitin onto the lysine residue(s) of the cereblon neomophic substrate,
275 thereby enabling UBE2G1 to assemble the K48-linked ubiquitin chains onto the initial anchor
276 ubiquitin (Figure 4F). This orchestrated action between UBE2D3 and UBE2G1 closely
277 resembles the cooperativity of UBE2D3 and Cdc34 in promoting IκBα polyubiquitination
278 mediated by SCFβTRCP2 (Wu et al., 2010), except that unlike Cdc34, UBE2G1 does not possess
279 the ability to transfer ubiquitin onto substrates without prior ubiquitin conjugation. UBE2G1
280 was known to produce K48-linked poly-ubiquitin chains in the absence of an E3 ubiquitin ligase
281 (Choi et al., 2015), but UBE2G1 cannot promote GSPT1 ubiquitination in the absence of CC-
282 885 or Cul4A-Rbx1 (Figure 4E), indicating that close proximity of UBE2G1 to cereblon
283 neomorphic substrates bridged by CRL4CRBN is required to increase the processivity of UBE2G1
284 at physiological concentrations. This provides an explanation to how cereblon modulating agents
285 can induce effective substrate degradation via K48-linked polyubiquitination, as well as speaks
286 to the potential role of UBE2G1 in mediating the ubiquitination and degradation of other CRL4
287 cognate and/or neomophic substrates. This is supported by the impaired degradation of p21 and
288 RBM39 induced by UV irradiation and E7070 treatment, respectively, in 293T UBE2G1-/- cells
289 (Figures S9A and S9B). bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
290 Although UBE2D3 and UBE2G1 cooperatively regulate the ubiquitination of cereblon
291 neomorphic substrates, ablation of UBE2D3 exhibited very little impact on substrate degradation
292 as compared to loss of UBE2G1, suggesting that additional E2s might fulfill the role of
293 UBE2D3, e.g., other UBE2D family proteins with a high degree of sequence homology
294 including UBE2D1/UbcH5a, UBE2D3, and UBE2D4 (Figure S10A). This idea is supported by
295 the following observations. First, just like UBE2D3, UBE2D1 and UBE2D2 acted synergistically
296 with UBE2G1 in catalyzing the ubiquitination of GSPT1 in the presence of CC-885, whereas
297 cooperativity among UBE2D1, UBE2D2 and UBE2D3 cannot be detected (Figure S10B).
298 Second, UBE2D1 and to a lesser extent UBE2D2 were far more efficient than UBE2D3 in
299 catalyzing CC-885-dependent GSPT1 polyubiquitination with or without the help of UBE2G1
300 (Figure S10B, lanes 2, 4 and 6). Third, UBE2D2 knockout impaired the POM-induced
301 degradation of ePL-tagged IKZF1 (Figure S2D). Although the synergistic interaction between
302 UBE2D2 and UBE2G1 was not evident in the dual-gRNA CRISPR screen (Figure S3D), their
303 cooperativity might still exist but fall below the detection limit of this assay. Regardless, it is
304 more likely that UBE2D2 could generate extensive substrate polyubiquitination in the absence of
305 UBE2G1 in vivo, leading to substrate degradation. Taken together, we speculate that the CM-
306 induced proteolysis of cereblon neomorphic substrates is both redundantly and cooperatively
307 regulated by UBE2D family proteins and UBE2G1, with UBE2G1 playing a key role in
308 enhancing the rate and extent of K48-linked substrate ubiquitination, resulting in rapid and
309 efficient substrate degradation.
310 Although loss of UBE2G1 conferred resistance to LEN and POM in human myeloma cell lines,
311 it remains to be seen whether UBE2G1 deficiency occurs in human myeloma patients with
312 inherent or acquired resistance to IMiD drug treatment, especially those with normal cereblon bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
313 expression. The myeloma cell line SKMM2, which lost both copies of UBE2G1 gene (based on
314 CCLE gene copy number characterization), and has undetectable UBE2G1 protein expression
315 (Figure 6B), was derived from a human myeloma patient who never received any prior treatment
316 with IMiD drugs (Eton et al., 1989), warranting the further clinical evaluation of UBE2G1
317 activity in myeloma patients. Given that UBE2G1 inactivation conferred resistance to all CMs
318 tested and also to cereblon-based PROTACs, patient stratification approaches based on UBE2G1
319 status might be applicable to the development of IMiD drugs and other novel cereblon
320 modulating agents for a variety of human diseases. Lastly, CC-220, a novel CM that targets
321 IKZF1 and IKZF3 for degradation much more effectively than does LEN or POM, retained
322 strong antitumor activity at clinically achievable concentrations (Schafer et al., 2018) in
323 UBE2G1-deficient myeloma cells, suggesting that human patients with resistance to CM drugs
324 owing to diminished UBE2G1 function may be responsive to next-generation CMs that possess
325 higher efficiency and/or potency for degrading the same target protein.
326
327 Acknowledges
328 The authors thank members of the early drug discovery team at Celgene for helpful discussion
329 and technical support.
330 Competing interests
331 The authors declare competing financial interests: G.L., S.W., M.M., X.Z., W.F., S.W., C.S., C-
332 C. L., D.M., I.S.J., K.W., M.M., S.C., B.C., J.M., P.M., M.R. are employees of Celgene.
333 bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
334 Material and Methods
335 Purification of In Vitro Ubiquitination Assay Components
336 Cereblon-DDB1 purification: ZZ-domain-6xHis-thrombin-tagged human cereblon (amino
337 acids 40 – 442) and full length human DDB1 were co-expressed in SF9 insect cells in ESF921
338 medium (Expression Systems), in the presence of 50 uM zinc acetate. Cells were resuspended in
339 buffer containing 50 mM Tris-HCl pH 7.5, 500 mM NaCl, 10 mM imidazole, 10% glycerol, 2
340 mM TCEP, 1X Protease Inhibitor Cocktail (San Diego Bioscience), and 40,000 U Benzonase
341 (Novagen), and sonicated for 30 s. Lysate was clarified by high speed centrifugation at 30,000
342 rpm for 30 minutes, and clarified lysate was incubated with Ni-NTA affinity resin (Qiagen) for 1
343 hour. Complex was eluted with buffer containing 500 mM imidazole, and the ZZ-domain-6xHis
344 tag removed by thrombin cleavage (Enzyme Research) overnight, combined with dialysis in 10
345 mM imidazole buffer. Cleaved eluate was incubated with Ni-NTA affinity resin (Qiagen), and
346 the flow-through diluted to 200 mM NaCl for further purification over an ANX HiTrap ion
347 exchange column (GE Healthcare). The ANX column was washed with 10 column volumes 50
348 mM Tris-HCl pH 7.5, 200 mM NaCl, 3 mM TCEP, followed by 10 column volumes of 50 mM
349 Bis-Tris pH 6.0, 200 mM NaCl, 3 mM TCEP, and the cereblon-DDB1 peak eluted at 210 mM
350 NaCl. This peak was collected and further purified by size exclusion chromatography using a
351 Sephacryl S-400 16/60 column (GE Healthcare) in buffer containing 10 mM HEPES pH 7.0, 240
352 mM NaCl, and 3 mM TECP. The cereblon-DDB1 complex was concentrated to 30 mg/mL.
353 Cul4-Rbx1 purification: Human full length Cul4A and Rbx1 were co-expressed in SF9 insect
354 cells. Cells were resuspended in buffer containing 50 mM Tris-HCl pH 7.5, 500 mM NaCl, 10
355 mM imidazole, 10% glycerol, 2 mM TCEP, 1X Protease Inhibitor Cocktail (San Diego
356 Bioscience), and 40,000 U Benzonase (Novagen), and sonicated for 30 s. Lysate was clarified by bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
357 high speed centrifugation at 30,000 rpm for 30 minutes, and clarified lysate was incubated with
358 Ni-NTA affinity resin (Qiagen) for 1 hour. Complex was eluted with buffer containing 500 mM
359 imidazole, and concentrated for size exclusion chromatography. Complex was further purified
360 over an S200 Superdex 200 16/600 column (GE Healthcare) in buffer containing 200 mM NaCl,
361 50 mM Tris pH 7.5, 3 mM TCEP, and 10% glycerol.
362 Substrate purification: MBP-IKZF1 (amino acids 140-168) or MBP-GSPT1 (amino acids 437-
363 633) was expressed in E.coli BL21 (DE3) Star cells (Life Technologies) using 2XYT media
364 (Teknova). Cells were induced at OD600 0.6 for 18 hours at 16 ˚C, with 150 uM zinc acetate
365 added upon induction for IKZF1 expression. Cells were pelleted and resuspended in buffer
366 containing 200 mM NaCl, 50 mM Tris pH 7.5, 3 mM TCEP, 10% glycerol, 150 µM zinc acetate,
367 0.01 mg/mL lysozyme (Sigma), 40,000 U benzonase (Novagen), and 1X protease inhibitor
368 cocktail (San Diego Bioscience). Resuspended cells were frozen, thawed for purification, and
369 sonicated for 30 s before high speed centrifugation at 30,000 rpm for 30 minutes. Clarified lysate
370 was incubated with maltose affinity resin (NEB) at 4 ˚C for 1 hour before beads were washed.
371 Protein was eluted with buffer containing 200 mM NaCl, 50 mM Tris pH 7.5, 3 mM TCEP, 10%
372 glycerol, 150 µM zinc acetate, and 10 mM maltose. Eluate was concentrated and further purified
373 by size exclusion chromatography over a Superdex 200 16/600 column (GE Healthcare) in
374 buffer containing 200 mM NaCl, 50 mM Tris pH 7.5, 3 mM TCEP, 10% glycerol, and 150 µM
375 zinc acetate.
376 UBE2G1 purification from E. coli: Human full length UBE2G1 with an N-terminal 6XHis-
377 thrombin tag was expressed in E.coli BL21 (DE3) Star cells (Life Technologies) using 2XYT
378 media (Teknova). Cells were induced at OD600 0.6 for 18 hours at 16 ˚C. Cells were pelleted and
379 resuspended in buffer containing 50 mM Tris pH7.5, 250 mM NaCl, 3mM TCEP, 1X Protease bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
380 Inhibitor Cocktail (San Diego Bioscience), 20 mM imidazole, and 40,000 U Benzonase
381 (Novagen), and sonicated for 3 times for 30 s. Lysate was clarified by high speed centrifugation
382 at 30,000 rpm for 30 minutes, and clarified lysate was incubated with Ni-NTA affinity resin
383 (Qiagen) for 1 hour at 4C. The protein was eluted with buffer containing 500 mM imidazole. The
384 6XHis tag was then removed by thrombin cleavage (Enzyme Research) overnight, combined
385 with dialysis into 50 mM Tris pH7.5, 250 mM NaCl, 3mM TCEP, 1X Protease Inhibitor Cocktail
386 (San Diego Bioscience), 20 mM imidazole. The cleaved protein was then loaded onto a 5ml
387 HiTrap Ni column (GE Healthcare), and cleaved protein collected in the flow-through. The
388 flow-through was then concentrated and further purified by size exclusion chromatography over
389 a Superdex 75 16/600 column (GE Healthcare) in buffer containing 20mM Tris pH7.5, 150mM
390 NaCl, 1mM DTT, and concentrated to 25 μM.
391 E2 purification from 293T cells: FLAG tagged UBE2D3 wild-type and C85S mutant, and
392 FLAG-tagged UBE2G1 wild-type and C905S mutant were purified from 293T UBE2G1-/- cell
393 lines stably expressing the respective protein. A pellet of ~5000 million cells expressing a
394 FLAG-tagged E2 was re-suspended in buffer containing 50 mM Tris-HCl pH 7.5, 250 mM
395 NaCl, 1 mM TCEP, 1X Protease Inhibitor Cocktail (San Diego Bioscience), and Phosphatase
396 inhibitor cocktail (Sigma (Roche), REF 04 906 837 001), and sonicated for 15s. Lysate was
397 clarified by high speed centrifugation at 30,000 rpm for 30 minutes, and clarified lysate was
398 incubated with anti-FLAG M2 Affinity Gel (A2220 Sigma) for 2 hour. The E2 was eluted with
399 buffer containing 50 mM Tris pH7.5, 250 mM NaCl, 0.15 mg/ml FLAG peptide (3X FLAG
400 Peptide F4799, Sigma Aldrich). The protein was then dialyzed into 4 L of buffer containing 50
401 mM Tris pH 7.5 and 250 mM NaCl for one hour twice. The protein was then concentrated and
402 further purified by size exclusion chromatography over a Superdex 75 16/600 column (GE bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
403 Healthcare) in buffer containing 20mM Tris pH 7.5, 150 mM NaCl. All 4 proteins were then
404 concentrated to 25 μM final concentration.
405
406 In Vitro Ubiquitination Assays
407 Purified E1, E2, ubiquitin, Cul4A-Rbx1, and cereblon-DDB1 proteins were used to reconstitute
408 the ubiquitination of MBP-fused GSPT1 or IKZF1 substrates in vitro. Purified recombinant
409 human Ube1 E1 (E-305), UbcH5a/UBE2D1 (E2-616-100), UbcH5b/UBE2D2 (E2-622-100),
410 UbcH5c/UBE2D3 (E2-627-100), wild-type ubiquitin (U-100H), K48R ubiquitin (UM-K48R-
411 01M), and K48-only ubiquitin (UM-K480-01M) were purchased from R&D systems. For the
412 ubiquitination of IKZF1 or GSPT1 shown in Figures 4A, 4B S5C and S10B, reaction
413 components were mixed to final concentrations of 80 mM ATP, 1.5 µM Ube1, 275 µM Ub, 2
414 µM Cul4-Rbx1, 2 µM cereblon-DDB1, 5 uM IKZF1 (a.a. 140-168) or 5 µM MBP-GSPT1 (a.a.
415 437-633) as indicated, and then 5 µM UBE2D1, 5 µM UBE2D2, 5 µM UBE2D3 (purified from
416 either from E.coli or human cells), or 7.5 µM UBE2G1 (purified from either from E.coli or
417 human cells), was added alone or in combination, as indicated. Reactions were incubated in the
418 presence of either DMSO or 80 µM compound (pomalidomide or CC-885) in ubiquitination
419 assay buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 10 mM MgCl2). To start the reactions, E1,
420 E2, ATP and ubiquitin were pre-incubated for 30 minutes, and separately MBP-substrate,
421 CRBN-DDB1, Cul4-Rbx1, and compound were pre-incubated for 5 minutes at room
422 temperature, before ubiquitination reactions were started by mixing the two pre-incubations.
423 Reactions were incubated at 30ºC for 2 hours before separation by SDS-PAGE followed by
424 immunoblot analysis using anti-MBP antibody (MBP-probe R29.6, Santa Cruz). For the
425 ubiquitin mutant reactions shown in Figures 4C and 4D, 275 µM K48-only or K48R ubiquitin bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
426 was substituted for wild-type ubiquitin as indicated, and the E. coli-purified UBE2G1 was used.
427 Reactions were incubated at 30ºC for 2 hours before separation by SDS-PAGE followed by
428 immunoblot analysis using anti-MBP antibody (MBP-probe R3.2, Santa Cruz).
429 For the ubiquitination of a pre-ubiquitinated substrate shown in figure 4E, MBP-GSPT1 (a.a.
430 437-633) was incubated with 80 mM ATP, 3 µM Ube1, 600 µM Ub, 4 µM Cul4-Rbx1, 4 µM
431 cereblon-DDB1, 5 µM UBE2D3 (purified from E. coli), and 80 uM CC-885 for 4 hours before
432 separation of the reaction over a 10/300 S200 GL (GE 17-5175-01) size exclusion
433 chromatography column to separate the substrate from the rest of the ubiquitination reaction
434 components. 1.25 µM purified MBP-GSPT1 was then used as the substate in ubiquitnation
435 reactions including 80 mM ATP, 1.5 µM Ube1, 600 µM Ub, 2 µM Cul4-Rbx1, 2 µM cereblon-
436 DDB1, 80 µM CC-885, and 7 µM UBE2G1 (purified from E. coli). Reactions were incubated at
437 30ºC for 2 hours before separation by SDS-PAGE followed by immunoblot analysis using anti-
438 MBP antibody (MBP-probe R29.6, Santa Cruz).
439
440 Cell Culture and Materials
441 Human embryonic kidney cell line 293T (Clontech) was maintained in Dulbecco’s Modified
442 Eagle’s medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS;
443 Invitrogen), 1x sodium pyruvate (Invitrogen), 1x non-essential amino acids (Invitrogen), 100
444 U/mL penicillin (Invitrogen), and 100 µg/mL streptomycin (Invitrogen). Acute myeloid
445 leukemia cell lines U937, MOLM-13, and MV4-11 and myeloma cell line MM1S were
446 purchased from American Tissue Culture Collection (ATCC). Acute myeloid leukemia cell line
447 OCI-AML2 cell line and myeloma cell lines OPM2 was purchased from Deutsche Sammlung bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
448 von Mikroorganismen und Zellkulturen GmbH (DSMZ). Myeloma cell line DF15 was obtained
449 from Dr John Shaughnessy (University of Arkansas, Little Rock, AR, USA). U937, MOLM-13,
450 OPM2, MM1S and DF15 cell lines were maintained in Roswell Park Memorial Institute (RPMI)
451 1640 tissue culture medium (Invitrogen) supplemented with 10% FBS, 1x sodium pyruvate, 1x
452 non-essential amino acids, 100 U/mL penicillin, and 100 µg/mL streptomycin. MV4-11 cell line
453 was maintained in Iscove’s Modified Dulbecco’s medium (IMDM; (Invitrogen) supplemented
454 with 10% FBS, 1x sodium pyruvate, 1x non-essential amino acids, 100 U/mL penicillin, and 100
455 µg/mL streptomycin. OCI-AML2 cell line was maintained in minimal essential medium (MEM;
456 Invitrogen) supplemented with 10% FBS, 1x sodium pyruvate, 1x non-essential amino acid, 100
457 U/mL penicillin, and 100 µg/mL streptomycin. All cell lines were cultured at 37ºC with 5% CO2
458 in the relevant media mentioned above.
459
460 Plasmids
461 UBE2G1 and UBE2D3 complimentary deoxyribonucleic acid (cDNA) clones were purchased
462 from Dharmacon. The coding regions of UBE2G1 and UBE2D3 were polymerase chain reaction
463 (PCR)-amplified and shuttled into pDONR223 via BP (attB and attP) recombination to generate
464 pDONR223-UBE2G1, pDONR223-FLAG-UBE2G1, and pDONR223-FLAG-UBE2D3. Site-
465 directed mutagenesis using overlapping PCR was then carried out to generate pDONR223-
466 UBE2G1-CR (CRISPR resistant), pDONR223-UBE2G1-C90S-CR, pDONR223-FLAG-
467 UBE2G1-CR, and pDONR223-FLAG-UBE2G1-C90S-CR, and pDONR223-FLAG-UBE2D3-
468 C85S. Next, gateway donor vectors pDONR223-UBE2G1-CR and pDONR223-UBE2G1-C90S-
469 CR were shuttled into plenti-Ubcp-gateway-IRES-Pur or plenti-PGK-gateway-IRES-Pur via LR
470 (attL and attR) recombination to generate plenti-Ubcp-UBE2G1-CR-IRES-Pur, plenti-Ubcp- bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
471 UBE2G1-C90S-CR-IRES-Pur, plenti-PGK-UBE2G1-CR-IRES-Pur, and plenti-PGK-UBE2G1-
472 C90S-CR-IRES-Pur. Gateway donor vectors pDONR223-FLAG-UBE2G1-CR, pDONR223-
473 FLAG-UBE2G1-C90S-CR, pDONR223-FLAG-UBE2D3, and pDONR223-FLAG-UBE2D3-
474 C85S were shuttled into plenti-EF1α-gateway-IRES-Pur via LR recombination to generate
475 plenti-EF1α-FLAG-UBE2G1-CR-IRES-Pur, plenti- EF1α-FLAG-UBE2G1-C90S-CR-IRES-
476 Pur, plenti-EF1α-FLAG-UBE2D3-IRES-Pur, and plenti- EF1α-FLAG-UBE2D3-C85S-IRES-
477 Pur. Constructs pDONR221-U6-sgRNA-EF1a-Cas9-P2A-GFP, plenti-EF1α-Cas9-IRES-Bla,
478 pDONR223-IKZF1, pcDNA3-IKZF1-V5, pcDNA3- CRBN, and pcDNA3-8 x His-Ub were
479 described previously. The Cas9-P2A-GFP coding region of pDONR221-U6-sgRNA-EF1a-Cas9-
480 P2A-GFP was subcloned into pcDNA3.1 (Invitrogen) to generate pcDNA3.1-Cas9-P2A-GFP.
481 The IKZF1 coding region of pDONR223-IKZF1 was shuttle into plenti-EF1α -ePL-gateway-
482 IRES-Bla via LR recombination to generate plenti-EF1α -ePL-IKZF1-IRES-Bla.
483 Complementary oligonucleotides containing three non-targeting sgRNAs or three gene-specific
484 sgRNAs targeting CRBN or each of the 41 annotated E2 enzymes were annealed and cloned into
485 pRSG16-U6-sgEV-UbiC-TagRFP-2A-Puro or pRSG16-U6-sgEV-UbiC-Hyg, both of which
486 were modified from pRSG16-U6-sg-HTS6C-UbiC-TagRFP-2A-Puro (Cellecta). All sgRNA
487 sequences used in this report (see supplemental table 1) were selected from the human genome-
488 wide CRISPR sgRNA library (Cellecta).
489
490 Lentiviral Production and Transduction
491 Lentiviral plasmid was cotransfected with the 2nd Generation packaging system (ABM) into
492 293T cells (Clontech) using Lipofectamine® 2000. After 16 hours of incubation, media was
493 changed to fresh DMEM media supplemented with 20% FBS. At 48 hours post transfection, bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
494 viral supernatant was collected and cleared via centrifugation at 2000 rpm for 5 minutes, and
495 then filtered through a 0.45 micron cellulose acetate or nylon filter unit. Acute myeloid
496 leukemia and myeloma cell lines were spin-inoculated with lentivirus at 2500 rpm for
497 120 minutes. After twelve hours, viral supernatant was removed and complete culture media
498 was added to the cells. Forty-eight hours later, cells were incubated with 1~2 μg/mL puromycin
499 (Thermofisher), 10~20 μg/mL blasticidin (Thermofisher), or 250~500 μg/mL hygromycin B
500 (Thermofisher) for an additional 2~7 days to select cells stably integrated with lentiviral vectors.
501
502 CRISPR Gene Editing
503 AML and MM cell lines were transduced with plenti-EF1a-Cas9-IRES-Bla, followed by limiting
504 dilution and blasticidin selection in 96-well plates (Corining) to generate single clones stably
505 expressing Cas9. The expression of Cas9 in stable clones were validated by immunoblot
506 analysis. Next, Cas9-expressing cells were transduced with pRSG16-U6-sgNT-1-UbiC-TagRFP-
507 2A-Puro, pRSG16-U6-sgNT-3-UbiC-TagRFP-2A-Puro, pRSG16-U6-sgUBE2G1-1-UbiC-
508 TagRFP-2A-Puro, pRSG16-U6-sgUBE2G1-5-UbiC-TagRFP-2A-Puro, pRSG16-U6-
509 sgUBE2D3-4-UbiC-TagRFP-2A-Puro, or pRSG16-U6-sgCRBN-8-UbiC-TagRFP-2A-Puro. One
510 days after transduction, cells were selection with puromycin for 2 days. Then, gene editing
511 efficiency was verified by immunoblot analysis with antibodies recognizing the targeted
512 proteins. For gene editing of both UBE2G1 and UBE2D3, U937-Cas9 cells were first transduced
513 with pRSG16-U6-sgUBE2G1-5-UbiC-Hyg. After 24 hours, cells were selected with hygromycin
514 B selection for additional 5 days, and then transduced with pRSG16-U6-sgUBE2D3-4-UbiC-
515 TagRFP-2A-Puro, followed by puromycin selection for 2 days and immunoblot analysis. bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
516 293T cells were transiently transfected with pcDNA3.1-Cas9-P2A-GFP, pRSG16-U6-
517 sgUBE2G1-5-UbiC-TagRFP-2A-Puro with or without pRSG16-U6-sgUBE2D3-4-UbiC-
518 TagRFP-2A-Puro. Three days after transfection, cells were subjected to limiting dilution into 96-
519 well plates. After two weeks, stable clones were cherry-picked, expanded and subjected to
520 immunoblot analysis. 293T UBE2G1-/- clone 13 was validated to be UBE2G1 deficient, and
521 UBE2G1-/-;UBE2D3-/- clone 4 was proven deficient for both UBE2G1 and UBE2D3.
522 For the single-guide RNA directed E2 CRISPR screen, U937 Cas9 cells were first transduced
523 with plenti-EF1a-ePL-IKZF1-IRES-Bla to generate U937_Cas9_ePL-IKZF1 cells, which were
524 then transduced with the focused lentiviral CRISPR library containing 3 non-targeting control
525 sgRNAs and 3 gene-specific sgRNAs targeting each of the 41 E2 enzymes.
526 For the dual-guide RNA directed E2 CRISPR screen, U937_Cas9_ePL-IKZF1 were transduced
527 with pRSG16-U6-sgUBE2G1-5-UbiC-Hyg, followed by hygromycin B selection for additional 5
528 days. Then, cells were transduced with the focused lentiviral CRISPR library targeting all
529 annotated E2s as described above.
530
531 IKZF1-ePL Degradation Assay
532 Four days after transduction with lentiviral vectors expressing non-targeting, CRBN-specific or
533 UBE2-specific sgRNA, U937_Cas9_ePL-IKZF1 cells were dispensed into a 384-well plate
534 (Corning) pre-spotted with pomalidomide at varying concentrations. Twenty-five microliters of
535 RPMI-1640 growth media containing 5000 cells was dispensed into each well. After incubation
™ 536 at 37°C with 5% CO2 for 16 hrs, 25 µL of the InCELL Hunter Detection Reagent Working
537 Solution (DiscoverX) was added to each well and incubated at RT for 30 minutes protected from bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
538 light. After 30 minutes, luminescence was read on an EnVision Multimode Plate Reader (Perkin
539 Elmer). For each cell line, The IKZF1 degradation induced by pomalidomide at each indicated
540 concentration was normalized with the DMSO control. Then, a four parameter logistic model
541 (sigmoidal dose-response model) was used to plot the IKZF1 destruction curves. All percentage
542 of control IKZF1 destruction curves were processed and graphed using GraphPad Prism,Version
543 7.
544
545 Cell Proliferation Assay
546 AML cell lines (5000 cells per well), MM cell lines (5000 cells per well) or 293T cells (2000
547 cells per well) in 50 μL complete culture media were seeded into black 384-well plates
548 containing with DMSO or test compounds. After 3 or 5 days, cell proliferation was assessed
549 using CTG according to manufacturer’s instructions. Relative cell proliferation was normalized
550 against the DMSO control. The growth inhibitory curve of each test compound was processed
551 and graphed using GraphPad Prism,Version 7.
552
553 Immunoblot Analysis
554 Following treatment with test compounds at 37°C for the indicated time, cells were washed in
555 ice-cold 1X PBS twice before harvest in Buffer A [50 mM Tris.Cl (pH 7.6), 150 mM NaCl, 1%
556 Triton X-100, 1 mM EDTA, 1 mM EGTA, 1 mM β-glycerophosphate, 2.5 mM sodium
557 pyrophosphate, 1 mM Na3VO4, 1 µg/mL leupeptin, one tablet of Complete ULTRA protease
558 inhibitor cocktail (Roche), and one tablet of PhosSTOP phosphatase inhibitor cocktail (Roche)].
559 Whole cell extracts were collected after centrifugation at top speed for 10 minutes, resolved by bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
560 SDS-PAGE gel electrophoresis, transferred onto a nitrocellulose membrane using the Turboblot
561 system (Bio-Rad), and probed with the indicated primary antibodies. Bound antibodies were
562 detected with IRDye-680 or -800 conjugated secondary antibodies using a LI-COR scanner.
563
564 Protein Half Life Analysis
565 293T parental and UBE2G1-/- cells were pretreated with DMSO or pomalidomide (1 μM) for 30
566 minutes, followed by the addition of 100 μg/ml cycloheximide (EMD) into the culture medium.
567 At various time points as indicated in Fig. 3D, cells were collected and subjected to immunoblot
568 analysis.
569
570 In Vivo Ubiquitination Assay
571 The ubiquitination assays were carried out as described previously (Lu et al., 2014b). In brief,
572 293T parental, UBE2G1-/-, and UBE2G1-/-;UBE2D3-/- cells seeded in 6 well plates were
573 transiently transfected with pcDNA3-IKZF1-V5, pcDNA3- CRBN, pcDNA3-8 x His-Ub, or
574 pcDNA3 empty vector. In Figures 5B and S6B-D, 293T UBE2G1-/- and UBE2G1-/-;UBE2D3-/-
575 cells were also transfected with plenti-EF1a-UBE2G1-IRES-Pur, plenti-EF1a-UBE2D3-IRES-
576 Pur or both. Forty-eight hours post transfection, cells were treated with 10 μM MG132 with or
577 without an increased concentrations of pomalidomide as indicated. Eight hours later, cells were
578 washed twice with ice cold PBS and resuspended in 1 mL PBS. Twenty uL of the cell suspension
579 was boiled in LDS loading buffer, and the remaining cells were collected via centrifugation and
580 lysed in Buffer C (6M guanidine-HCL, 0.1M Na2HPO4/NaH2PO4, 20 mM imidazole, pH 8.0).
581 Next, whole cell extracts were sonicated for 12 pulses, and mixed with 20 μL of HisPur™ Ni-
582 NTA Magnetic Beads (Thermofisher) at 37°C for 4 hours. Ni-NTA beads were then washed bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
583 three times with Buffer C, three times with Buffer D (1 Volume of Buffer C: 3 volumes of
584 Buffer E), and three times with Buffer E (25 mM Tris.CL, 20 mM imidazole, pH 6.8). Bound
585 proteins were eluted by boiling in 2x LDS loading buffer and subjected to immunoblot analysis.
586
587 Antibodies
588 Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-
589 human GSPT1 polyclonal antibody (pAb; Abcam), rabbit anti-human IKZF1 mAb (Cell
590 Signaling), rabbit anti-human IKZF3 mAb (Cell Signaling), rabbit anti-human CK1α pAb
591 (Abcam), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences), mouse anti-human UBE2G1
592 mAb (Santa Cruz), rabbit anti-human UBE2D3 pAb (Sigma), rabbit anti-human Cul4A pAb
593 (Cell Signaling), rabbit anti-human DDB1 pAb (Cell Signaling), rabbit anti-human Rbx1 pAb
594 (Cell Signaling), rabbit anti-human Cdt1 pAb (Cell Signaling), rabbit anti-human Cdt2 pAb (Cell
595 Signaling), rabbit anti-human Set8 pAb (Cell Signaling), rabbit anti-human RBM39 pAb
596 (Sigma), rabbit anti-human p21 pAb (Cell Signaling), rabbit anti-human p27 pAb (Cell
597 Signaling), rabbit anti-human c-Myc pAb (Cell Signaling), mouse anti-penta-HIS mAb (Qiagen),
598 and mouse anti-human Actin and Tubulin antibodies (Sigma) were used as primary antibodies.
599 Goat anti-mouse 800 antibody (LI•COR Biosciences), goat anti-rabbit 680 antibody (LI•COR
600 Biosciences), goat anti-mouse 800 antibody (LI•COR Biosciences) and goat anti-rabbit 680
601 antibody (LI-COR Biosciences) were used as secondary antibodies.
602
603
604
605 bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
606 Figure Legend
607 Figure 1. Identification of UBE2G1 as the most critical ubiquitin E2 enzyme that mediates the
608 pomalidomide-induced degradation of IKZF1
609 (A) Schematic showing the design of the CRISPR screen to identify E2 enzyme(s)
610 regulating the CM-induced degradation of ePL-tagged IKZF1 in 384-well array format.
611 (B) Chemiluminescent measurement of ePL-IKZF1 protein expression level in U937-
612 Cas9_ePL-IKZF1 parental cells or cells expressing non-targeting or UBE2G1-specific
613 sgRNAs. Cells were treated with POM at the indicated concentrations for 16 hours. Data
614 are presented as mean ± SD (n = 4).
615 (C) Immunoblot analysis of U937-Cas9 parental or UBE2G1-/- cells with or without
616 stable expression of UBE2G1 wild-type or C90S mutant. Cells were treated with POM at
617 the indicated concentrations for 16 hours.
618
619 Figure 2. UBE2G1 and UBE2D3 redundantly regulate the pomalidomide-induced degradation of
620 IKZF1
621 (A) Schematic showing the design of dual-gRNA directed CRISPR screen of E2s
622 regulating the CM-induced degradation of ePL-tagged IKZF1 in 384-well array format.
623 (B) Chemiluminescent measurement of ePL-IKZF1 protein expression level in U937-
624 Cas9_ePL-IKZF1 parental cells or cells expressing UBE2G1-specfic sgRNA alone or in
625 combination with non-targeting or UBE2D3-specific sgRNA. Cells were treated with bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
626 POM at the indicated concentrations for 16 hours. Data are presented as mean ± SD (n =
627 4).
628 (C) Immunoblot analysis of U937-Cas9 parental cells or cells expressing non-targeting
629 sgRNA, UBE2G1-specific sgRNA, UBE2D3-specfic sgRNA, or both UBE2G1 and
630 UBE2D3 sgRNAs. Cells were treated with POM at the indicated concentrations for 16
631 hours. SE, short exposure; LE, long exposure.
632
633 Figure 3. Loss of UBE2G1 blocked the degradation of cereblon neomorphic substrates induced
634 by cereblon-modulating agents
635 (A and B) Immunoblot analysis of OPM2-Cas9 cells expressing non-targeting, UBE2G1-
636 specific or CRBN-specific sgRNA. Cells were treated with LEN, POM or CC-220 for 16
637 hrs (A) or CC-885 for 4 hours (B) at the indicated concentrations.
638 (C) Immunoblot analysis of 293T parental or UBE2G1-/- cells treated with Brd4
639 PROTACs dBET1 or MZ1 at the indicated concentrations for 16 hours.
640 (D) Immunoblot analysis of OPM2 parental or UBE2G1-/- cells treated with 100 µg/ml
641 cycloheximide with or without 1 µM POM pretreatment for half an hour. Cells were
642 harvested at the indicated time points.
643
644 Figure 4. UBE2G1 and UBE2D3 sequentially catalyze the in vitro ubiquitination of IKZF1 and
645 GSPT1 in the presence of pomalidomide and CC-885, respectively bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
646 (A- D) In vitro ubiquitination of IKZF1 (A and C) and GSPT1 (B and D) MBP fusion
647 proteins by recombinant CRL4CRBN complex. Recombinant protein products as
648 indicated were incubated with or without 80 µM POM (A and C) or 80 µM CC-885 (B
649 and D) in the ubiquitination assay buffer containing 80 mM ATP at 30 °C for 2 hours,
650 and then analyzed by immunoblotting.
651 (E) Sequential in vitro ubiquitination of GSPT1 by recombinant CRL4CRBN complex.
652 MBP-GSPT1 recombination protein was incubated with Ube1, UBE2D3, Cul4-Rbx1,
653 DDB1-cereblon, Ubiquitin, ATP and CC-885 in the ubiquitination assay at 30 °C for
654 4 hours. After purification over size-exclusion chromatography, pre-ubiquitinated MBP-
655 GSPT1 protein was then incubated with Ube1, DDB1-cereblon, Ubiquitin, ATP and
656 UBE2G1 with or without CC-885 or Cul4A-Rbx1 in the ubiquitination assay at 30 °C for
657 2 hours, followed by immunoblot analysis.
658 (F) Schematic showing the sequential ubiquitination of CRBN neomorphic substrates by
659 UBE2D3 and UBE2G1.
660
661 Figure 5. UBE2G1 and UBE2D3 cooperatively promote the in vivo ubiquitination of IKZF1
662 (A and B) 293T parental and UBE2G1-/-;UBE2D3-/- (clone 4) cells were transiently
663 transfected with plasmids expressing cereblon, V5-tagged IKZF1 and 8xHis-Ub with or
664 without UBE2G1, UBE2D3 or both. (C) 293T parental and UBE2G1-/- (clone 13) cells
665 were transiently transfect with plasmids expressing cereblon, IKZF1-V5, 8xHis-Ub with
666 or without UBE2G1 wild-type or C90S mutant. In (A), (B) and (C), 48 hours after
667 transfection, cells were treated with MG-132 (10 µM) and POM at the indicated bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
668 concentrations for additional 8 hours. Ubiquitinated protein products enriched with
669 magnetic nickel sepharose were subjected to immunoblot analysis. Immunoblot analysis
670 of whole cell extracts showing equal input proteins is shown in Supplemental Figures 6A,
671 6B and 6C.
672
673 Figure 6. UBE2G1 loss confers resistance to lenalidomide and pomalidomide in myeloma cell
674 lines
675 (A) Effect of lenalidomide (top panel) and pomalidomide (bottom panel) on proliferation
676 of myeloma cell lines. Cell proliferation was determined by CTG. Data are presented as
677 mean ± SD (n=3).
678 (B) Immunoblot analysis of myeloma cell lines.
679 (C and D) Proliferation (C) and immunoblot analysis (D) of SKMM2 cells transduced
680 with lentiviral vectors encoding GFP, UBE2G1 and UBE2G1-C90S. Cells were treated
681 with DMSO vehicle control, LEN or POM at the indicated concentrations for 5 days (C)
682 or 16 hours (D). In (C), cell proliferation was determined by CTG, and data are presented
683 as mean ± SD (n=3).
684
685 Figure 7. UBE2G1-deficient OPM2 myeloma cells are resistant to lenalidomide and
686 pomalidomide but remain sensitive to CC-220 at clinical relevant concentrations
687 (A and B) Cell proliferation (A) and immunoblot analysis (B) of OPM2-Cas9 cells
688 transduced with lentiviral vectors expressing non-targeting, UBE2G1-specific or CRBN- bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
689 specific sgRNAs. Cells were treated DMSO vehicle control, LEN, POM or CC-220 at the
690 indicated concentrations for 5 days (A) or 16 hours (B). In (A), cell proliferation was
691 determined by CTG, and data are presented as mean ± SD (n=3).
692
693 Figure S1. Pomalidomide-induced destruction of IKZF1 requires CRL4CRBN
694 (A and B) Chemiluminescent measurement (A) or immunoblot analysis (B) of ePL-
695 IKZF1 protein expression level in U937_Cas9_ePL-IKZF1 parental cells or cells
696 expressing non-targeting or CRBN-specific sgRNA. Cells were treated with DMSO or an
697 increasing concentrations of POM for 16 hours. In (A), data are presented as mean ± SD
698 (n = 4). In (B), note that the degradation efficiency of ePL-tagged and endogenous IKZF1
699 is comparable.
700 (C) Chemiluminescent measurement of ePL-IKZF1 protein expression level in
701 U937_Cas9_ePL-IKZF1 cells treated with DMSO or an increasing concentrations of
702 POM in the presence or absence or MLN4924 at the indicated concentrations for 16
703 hours. Data are presented as mean ± SD (n=4).
704 (D) Cell proliferation of U937_Cas9_ePL-IKZF1 cells treated with DMSO or MLN4924
705 at the indicated concentrations for 48 hours. Cell proliferation was determined by CTG.
706 Data are presented as mean ± SD (n=3).
707
708 Figure S2. The effect of individual knockout of each of the 41 annotated E2 enzymes on
709 pomalidomide-induced destruction of IKZF1 bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
710 (A-U) Chemiluminescent measurement of ePL-IKZF1 protein expression level in
711 U937_Cas9_ePL-IKZF1 parental cells or cells expressing non-targeting or E2-specific
712 sgRNA. Cells were treated with DMSO or an increasing concentrations of POM for 16
713 hours. Data are presented as mean ± SD (n = 4).
714
715 Figure S3. The effect of double knockout of UBE2G1 and one of the 41 E2 enzymes on
716 pomalidomide-induced destruction of IKZF1
717 (A-U) Chemiluminescent measurement of ePL-IKZF1 protein expression level in
718 U937_Cas9_ePL-IKZF1 parental cells or cells expressing both UBE2G1-specific and
719 non-targeting or E2-specific sgRNA. Cells were treated with DMSO or an increasing
720 concentrations of POM for 16 hours. Data are presented as mean ± SD (n = 4).
721
722 Figure S4. Elimination of UBE2G1 blocks the degradation of cereblon neomorphic substrates
723 recruited by lenalidomide and CC-885
724 (A-F) Immunoblot analysis of DF15-Cas9 cells (A and C), MM1S-Cas9 cells (B and D),
725 OCI-AML2-Cas9 cells (E), U937-Cas9 cells (F), MOLM-13-Cas9 cells (G) and MV4-
726 11-Cas9 cells (H) transduced with lentiviral vectors expressing non-targeting, UBE2G1-
727 specific or CRBN-specific sgRNAs. Cells were treated with lenalidomide for 16 hours (A
728 and B), or CC-885 for 4 hours (C-H) at the indicated concentrations.
729 (I) Immunoblot analysis of 293T parental or UBE2G1-/- cells stably expressing UBE2G1
730 wild-type or C90S mutant. Cells were treated with CC-885 at the indicated
731 concentrations for 4 hours. bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
732
733 Figure S5. UBE2G1 catalyzes the ubiquitin chain assembly on GSPT1 pre-conjugated with
734 ubiquitin
735 (A) Sequence alignment of human UBE2G1, human UBE2G2 and human CDC34 using
736 Clustal W 2.1. The acidic loops indispensable for the assembly of K48-linked ubiquitin
737 chains are highlighted with red, and the catalytic cysteines are highlighted with blue.
738 (B) Immunoblot analysis of 293T parental or UBE2G1-/- cells transduced with lentiviral
739 vectors expressing FLAG-tagged UBE2G1 wild-type or C90S mutant, or FLAG-tagged
740 UBE2D3 wild-type or C85S mutant. Cells were treated with CC-885 at the indicated
741 concentrations for 4 hours. Note that overexpression of wild-type FLAG-UBE2G1 or
742 FLAG-UBE2D3 partially rescued the GSPT1 degradation defect caused by UBE2G1
743 deficiency, while overexpression of catalytically-dead mutant FLAG-UBE2G1-C90S or
744 FLAG-UBE2D3-C85S further blocked the degradation of GSPT1.
745 (C) In vitro ubiquitination of GSPT1 by CRL4CRBN with or without CC-885 and indicated
746 E2 variants. Consistent with results observed with bacterial recombinant UBE2G1 and
747 UBE2D3 proteins, FLAG-UBE2G1 and FLAG-UBE2D3 proteins purified from human
748 cells acted in concert to promote the ubiquitination of GSPT1.
749
750 Figure S6. Input protein levels for the in vivo ubiquitinaiton studies corresponding to Figure 5
751 (A) Total input for Figures 5A. Immunoblot analysis of 293T parental and UBE2G1-/-
752 ;UBE2D3-/- (Clone 4) cells transfected to produce 8xHis-Ubiquitin, cereblon and IKZF1-
753 V5. (B) Total input for Figure 5B. Immunoblot analysis of 293T parental and UBE2G1-/- bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
754 ;UBE2D3-/- (Clone 4) cells transfected to produce 8xHis-Ubiquitin, CRBN, IKZF1-V5
755 with or without UBE2G1 and/or UBE2D3. (C) Total input for Figure 5C. Immunoblot
756 analysis of 293T parental and UBE2G1-/- (Clone 13) cells transfected to produce 8xHis-
757 Ubiquitin, CRBN, IKZF1-V5 with or without UBE2G1 wildtype or C90S mutant. In (A),
758 (B) or (C), 48 hours after transfection, cells were treated with 10 µM MG132 and POM at
759 the indicated concentrations for additional 8 hours.
760
761 Figure S7. The growth-inhibitory effect of CC-885 and Brd4 PROTACs in AML cell lines and
762 293T cells
763 (A-D) Cell proliferation of AML cell lines OCI-AML2 (A), U937 (B), MOLM-13 (C)
764 and MV4-11 (D) treated with DMSO or CC-885 at the indicated concentrations for 72
765 hours. Cell proliferation was determined by CTG. Data are presented as mean ± SD
766 (n=4).
767 (E and F) Cell proliferation of 293T parental and UBE2G1-/- (clone 13) cells with or
768 without ectopic overexpression of UBE2G1 wild-type or C90S mutant. Cells were treated
769 with CC-885 (E), dBET1 (F) or MZ-1 (F). Cell proliferation was determined by CTG.
770 Data are presented as mean ± SD (n=4).
771
772 Figure S8. UBE2G1 knockout diminished the responses to lenalidomide, pomalidomide and CC-
773 220 in myeloma cell lines DF15 and MM1S bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
774 (A-D) Cell proliferation (A and C) and immunoblot analysis (B and D) of MM1S-Cas9
775 (A and B) and DF15-Cas9 (C and D) cells infected with lentiviral vectors expressing non-
776 targeting, UBE2G1-specific or CRBN-specific sgRNAs. Cells were treated DMSO
777 vehicle control, LEN, POM or CC-220 at the indicated concentrations for 5 days (A and
778 C) or 16 hours (B and D). In (A and C), cell proliferation was determined by CTG, and
779 data are presented as mean ± SD (n=3).
780
781 Figure S9. Depletion of UBE2G1 attenuated the degradation of p21 and RMB39 induced by UV
782 irradiation and sulfonamide treatment, respectively.
783 (A and B) Immunoblot analysis of 293T parental and UBE2G1-/- (clone 13) cells treated
784 with UV irradiation (A) or E7070 (B). In (A), cells were UV irradiated at 50 J/m2 using a
785 Stratalinker, and collected at the indicated time points thereafter. In (B), cells were
786 treated with DMSO or an increasing concentration of E7070 for 16 hours.
787
788 Figure S10. UBE2D family proteins redundantly promote the ubiquitination of GSPT1
789 (A) Sequence alignment of human UBE2D family proteins using Clustal W 2.1. Note that
790 the amino acid sequence identity among all 4 family proteins is close to 90%.
791 (B) In vitro ubiquitination of GSPT1 MBP fusion protein by recombinant CRL4CRBN
792 complex in the presence of UBE2G1, UBE2D1, UBE2D2, or UBE2D3, alone or in
793 combination. Recombinant protein products as indicated were incubated with or without bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
794 80 µM CC-885 in the ubiquitination assay buffer at 30 °C for 2 hours, and then analyzed
795 by immunoblotting. SE, short exposure; LE, long exposure
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812 bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
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899 Schafer, P.H., Ye, Y., Wu, L., Kosek, J., Ringheim, G., Yang, Z., Liu, L., Thomas, M., Palmisano, M., and
900 Chopra, R. (2018). Cereblon modulator iberdomide induces degradation of the transcription factors
901 Ikaros and Aiolos: immunomodulation in healthy volunteers and relevance to systemic lupus
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903 Shibata, E., Abbas, T., Huang, X., Wohlschlegel, J.A., and Dutta, A. (2011). Selective ubiquitylation of p21
904 and Cdt1 by UBCH8 and UBE2G ubiquitin-conjugating enzymes via the CRL4Cdt2 ubiquitin ligase
905 complex. Mol Cell Biol 31, 3136-3145. bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
906 Skowyra, D., Craig, K.L., Tyers, M., Elledge, S.J., and Harper, J.W. (1997). F-box proteins are receptors
907 that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex. Cell 91, 209-219.
908 Soucy, T.A., Smith, P.G., Milhollen, M.A., Berger, A.J., Gavin, J.M., Adhikari, S., Brownell, J.E., Burke, K.E.,
909 Cardin, D.P., Critchley, S., et al. (2009). An inhibitor of NEDD8-activating enzyme as a new approach to
910 treat cancer. Nature 458, 732-736.
911 Winter, G.E., Buckley, D.L., Paulk, J., Roberts, J.M., Souza, A., Dhe-Paganon, S., and Bradner, J.E. (2015).
912 DRUG DEVELOPMENT. Phthalimide conjugation as a strategy for in vivo target protein degradation.
913 Science 348, 1376-1381.
914 Wu, K., Kovacev, J., and Pan, Z.Q. (2010). Priming and extending: a UbcH5/Cdc34 E2 handoff mechanism
915 for polyubiquitination on a SCF substrate. Mol Cell 37, 784-796.
916 Zengerle, M., Chan, K.H., and Ciulli, A. (2015). Selective Small Molecule Induced Degradation of the BET
917 Bromodomain Protein BRD4. ACS Chem Biol 10, 1770-1777.
918 Zhu, Y.X., Braggio, E., Shi, C.X., Bruins, L.A., Schmidt, J.E., Van Wier, S., Chang, X.B., Bjorklund, C.C.,
919 Fonseca, R., Bergsagel, P.L., et al. (2011). Cereblon expression is required for the antimyeloma activity of
920 lenalidomide and pomalidomide. Blood 118, 4771-4779.
921
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926 bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
927 Supplemental Table 1. Guide RNA sequences targeting 41 annotated ubiquitin-conjugating
928 enzymes.
sgRNA Target Sequence sgUBE2A_1 TGGACATACTTCAGAACCGT sgUBE2A_2 ATGGAAGACACATCATAGGT sgUBE2A_3 TCCGTCCGAGAACAACATAA sgUBE2B_1 CTGGATTTCCAGGTTACAAG sgUBE2B_2 GGCTCATGCGGGATTTCAAG sgUBE2B_5 CAAATAAACCACCAACTGTT sgUBE2C_1 GCAGGAGCTGATGACCCTCA sgUBE2C_3 GCTGGTGACCTGCTTTGAGT sgUBE2C_6 AGCGTCGCCGCCGCCCGTAA sgUBE2D1_1 TCGATATTCTGAGGTCACAA sgUBE2D1_2 CCACCTTGATATGCGCTATC sgUBE2D1_3 CAGCCTGATAGCGCATATCA sgUBE2D2_1 ATCACACAACAGAGAACAGA sgUBE2D2_2 CGAGCAATCTCAGGCACTAA sgUBE2D2_4 CCATTATTGTAGCTTGCCAA sgUBE2D3_4 GGTGTAGGAACTTAGTGATT sgUBE2D3_5 TACTTACTATCATCCCCAAC sgUBE2D3_7 CAGAATGACAGCCCATATCA sgUBE2D4_1 ACTACCTACCGGGCCCATGA sgUBE2D4_2 AGGAATTAACCGACTTGCAG sgUBE2D4_3 CCAGCGTTGACTGTGTCAAA sgUBE2E1_1 GAATACACCACCCTCATACA sgUBE2E1_3 ATATCCCTTCAAGCCTCCAA sgUBE2E1_7 GCAACTCACCTGCAATTAGG sgUBE2E3_1 CCTTCATATACAGAACCCGG sgUBE2E3_2 TCTTACCTGCAATTAGGAGG sgUBE2E3_3 TTCAAGCTTCTTACCTTTGG sgUBE2G1_1 ATAGGGAGCCAGCGTTCCTC sgUBE2G1_5 TAACCATACTTATCTTCCCC sgUBE2G1_6 ACTGCTACTGCGAAGACAGC sgUBE2G2_3 GTTGGGATGAAACATCTCAC sgUBE2G2_4 AGAATTAACACTGAATCCTC sgUBE2G2_7 GCTACGAGAGCAGCGCGGAG sgUBE2H_2 TAGCATCGAGAGTAAACATG sgUBE2H_5 CAAATTCATTAAGTCCTCCC sgUBE2H_8 CAGTCCGGGCAAGAGGCGGA sgUBE2I_5 ACAGGCACACTGTCCCCGAA sgUBE2I_7 TCACCCGAATGTGTACCCTT sgUBE2I_8 ACAGATCCTATTAGGAATAC bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
sgUBE2L3_2 CCAGCAACCAAAACCGACCA sgUBE2L3_3 CCCAGCCTGAGCACCCGCTT sgUBE2L3_7 CCGAAGCGGGTGCTCAGGCT sgUBE2L6_6 CATTGGCATCATCGCTGGAC sgUBE2L6_7 CGTTCTCGTCCACGTTGGGG sgUBE2L6_8 CCTTGCACCAAGACTTGCCA sgUBE2M_1 GTTGCCCTCGAGGTCAATGT sgUBE2M_2 TCACCAAGAAGAGATACTGC sgUBE2M_3 ACGTCTGCCTCAACATCCTC sgUBE2N_2 ACTGTGCGGATCTGCAGTGC sgUBE2N_3 CGGTTACCTTGATGATCCTG sgUBE2N_4 CGGGTTCTGACAAGATGGCC sgUBE2V1_4 ACGCAAGATGGCAGCCACCA sgUBE2V1_6 TACAGCCTTAAAATAGAATG sgUBE2V1_8 CTGTAACACTGTCCTTCGGG sgUBE2V2_4 TAGGCGACGGTACAGTTAGC sgUBE2V2_7 TAGGTCCACATTCTACTTTC sgUBE2V2_8 GGCGACGGTACAGTTAGCTG sgUBE2S_1 GGGCATCCGACACGTACTGC sgUBE2S_2 AACTCACCAGCAGTACGTGT sgUBE2S_7 AAACTCACCAGGGCCCTCGA sgCDC34_1 GGGCACTAGCGGCCGAGCCA sgCDC34_3 GCGCTCACCTCGTAGATGTT sgCDC34_4 CACCTTCTCGCCCGCAAACG sgUBE2K_1 GCAATGACAATAATACCGTG sgUBE2K_4 AGCTGCAATGACTCTCCGCA sgUBE2K_7 AATCAAGCGGGAGTTCAAGG sgUBE2R2_2 GGTGAATAGGGGTAGTCAAT sgUBE2R2_4 GCAGTGAGATTACACTTAAT sgUBE2R2_8 GCAGATGACCAGCTCGCAGA sgUBE2J1_2 TAACCTCTAAAGGCTGCGCA sgUBE2J1_3 GGCTAATGGTCGATTTGAAG sgUBE2J1_4 GGTTTCATGGGATACTCTGG sgUBE2J2_2 CCGACGTCTCTATACTGCCC sgUBE2J2_3 CCACCCGGACACGTGGAACC sgUBE2J2_6 AGAATCCTTACCTTCATAAG sgUBE2W_6 GCACCAGGTACCTTATATGA sgUBE2W_7 TTCTAGGTGGATTGTAGACA sgUBE2W_8 TTGAACACTCTTCTCATTTA sgUBE2E2_1 CTATCCGTTTAAACCCCCTA sgUBE2E2_7 GACAGTCCAAGCACTAGTGG sgUBE2E2_8 GGAACTTGCAGAAATCACAT sgUEVLD_3 ATTCTTACCATAGGTGTCCA sgUEVLD_7 AAAATTACTGTGGTTGGAGG bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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.
sgUEVLD_8 AGGGACTAAAGGAGCCACGA sgUBE2Z_1 GGAGAGTCCGACTGAGGAGG sgUBE2Z_2 GTTGTCATCAGTTTGACCCG sgUBE2Z_5 CAACGGGCAATAACACAGTG sgAKTIP_3 TAGAAGACCCCTATGCAATT sgAKTIP_4 CTTAATGCAGAGCGATAAGA sgAKTIP_5 GGTGAAGAGAAGACATTAAC sgUBE2O_2 CGTGCGCGTCCAGTGGTACC sgUBE2O_4 GACTGTGCCGTCAAGCTCAT sgUBE2O_7 GGTGCGCCTCATCCACGGCG sgUBE2Q1_2 GGAGGTTATAGAGTTTACAC sgUBE2Q1_4 GGAGTCTGATGACCCTAACT sgUBE2Q1_7 GAAAGAGGAAGAGCCAGCTG sgUBE2T_1 GGAGTGAGAAATCGGATCTG sgUBE2T_7 TCCTCAAGAATGCCAGACAG sgUBE2T_8 ACAGTTGCGATGTTGAGGGA sgUBE2F_3 TTTCAGCTAACAGTAACCCC sgUBE2F_4 TGAAAACAATGAAGCTTGTT sgUBE2F_5 TTCCCCTGTCTCTGTGATGT sgBIRC6_1 GAGTGGCTGGTGCTGCGGGA sgBIRC6_2 GTAGTGTATGCCTCGTTTGT sgBIRC6_8 GAGGCTGCCAAAGTTTGCAG sgUBE2U_3 TCCATCATATCTTCACTTAC sgUBE2U_6 AAGGTCTACAGAATTCAGTT sgUBE2U_8 CTACAGAATTCAGTTTGGCA sgUBE2Q2_4 ACTTACCCTCCTGAGAGAAC sgUBE2Q2_6 AGATACTAAGAACAACAATT sgUBE2Q2_8 GGTAACACCACTCGAACAAA sgTSG101_3 TCCAGTAGCCATAGGCATAT sgTSG101_5 ACAATCCCTGTGCCTTATAG sgTSG101_6 TCACCATATGAATCCAAAAC sgUBE2Q2L_6 TGCTGGGTCAAGGCCAGGAG sgUBE2Q2L_7 GTGCTGGGTCAAGGCCAGGA sgUBE2Q2L_8 CGGGTGCGCAGGGGGCGCGC sgUBE2QL1_4 CAGACATCAGCCGTCGGACA sgUBE2QL1_6 GCTTCACGTTCCAGTCGAAC sgUBE2QL1_8 GAACGTGAAGCTGCACCAGG sgUBE2NL_1 ATCAAAGGATTCCCCCTTTG sgUBE2NL_2 TGATGGCCGAGCTGCCCCAC sgUBE2NL_6 GTTTCCTTGATGATCCTGTG 929 bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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 1 A B U 9 3 7 _ C a s 9 _ e P L - I K Z F 1
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0 EF1a ePL IKZF1 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 P O M ( M ) EF1a NLS-Cas9-NLS U6 sgRNA
C sgNT-3 sgUBE2G1-5 UBE2G1 C90S UBE2G1 C90S DMSO + + + + + + POM ( 0.1 µM) + + + + + + POM ( 1 µM) + + + + + + POM ( 10 µM) + + + + + + (16 hrs)
IKZF1
UBE2G1
CRBN
Actin
U937-Cas9 bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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 A B U 9 3 7 _ C a s 9 _ e P L - I K Z F 1
1 5 0
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EF1a ePL IKZF1 P O M ( M ) EF1a NLS-Cas9-NLS U6 sgUBE2G1 U6 sgRNA sgUBE2G1-5 + C sgNT-3 sgUBE2G1-5 sgUBE2D3-4 sgUBE2D3-4 DMSO + + + + + POM ( 0.1 µM) + + + + + + + + + + POM ( 1 µM) (16hrs) POM ( 10 µM) + + + + +
IKZF1 (SE)
IKZF1 (LE)
UBE2G1
UBE2D3
Actin U937-Cas9 bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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
B sgNT-1 sgUBE2G1-5 sgCRBN-8 A sgNT-1 sgCRBN-4 sgCRBN-8 sgUBE2G1-1 sgUBE2G1-5 885 885 885 885 885 885 ------885 885 885 - - - 220(5nM) 220(5nM) 220(5nM) 220(5nM) 220(5nM) -
- (4 hrs) - - - (16 Hrs) 3 nM CC 30 nM CC 30 nM CC 30 nM CC 10 nM CC 10 nM CC 10 nM CC 3 nM CC DMSO DMSO DMSO 3 nM CC LEN LEN (200nM) CC LEN LEN (200nM) LEN LEN (200nM) CC POM (100nM) CC CC LEN LEN (200nM) POM (100nM) CC POM (100nM) DMSO LEN LEN (200nM) DMSO POM (100nM) POM (100nM) DMSO DMSO DMSO GSPT1 IKZF1 IKZF3 CRBN
CRBN UBE2G1
UBE2G1 Actin Actin OPM2-Cas9 OPM2-Cas9
C D DMSO 1 µM POM DMSO 1 µM POM pre 0.5hr pre 0.5 hr pre 0.5hr pre 0.5 hr CHX 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 (16 hrs) 1 µM 1 µM dBET1 DMSO 1 nM dBET1 10 µM dBET1 1 µM 1 µM dBET1 1 nM dBET1 10 nM dBET1 100 nM dBET1 100 nM dBET1 10 nM dBET1 DMSO 10 µM dBET1 1 nM MZ1 1 nM MZ1 1 µM 1 µM MZ1 1 µM MZ1 10 nM MZ1 10 nM MZ1 DMSO 10 µM MZ1 DMSO 10 µM MZ1 100 nM MZ1 100 nM MZ1 IKZF1 Brd4 IKZF3 CRBN UBE2G1
VHL c-Myc
UBE2G1 p27
Actin CRBN Actin 293T 293T 293T 293T Parental UBE2G1-/- Parental UBE2G1-/- OPM2_Cas9_sgNT-1 OPM2_Cas9_sgUBE2G1-5 bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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
A + + + + + + B C MBP-IKZF1 MBP-GSPT1 + + + + + + MBP-IKZF1 + + + + + + + + + + + + + + + + + + Ubiquitin Ubiquitin + + + + + + K48 only Ub + + + + + + + + + + + + Cul4A-Rbx1 Cul4A-Rbx1 + + + + + + K48R Ub + + + + + + + + + + + + DDB-cereblon DDB-cereblon + + + + + + Cul4A-Rbx1 + + + + + + + + + + + + + + + + + + Ube1 Ube1 + + + + + + DDB-cereblon + + + + + + + + + + + + + + + + UBE2G1 UBE2G1 + + + + Ube1 + + + + + + + + + + + + + + + + UBE2D3 UBE2D3 + + + + UBE2G1 + + + + + + + + + + + DMSO DMSO + + + UBE2D3 + + + + + + + + + + + POM CC-885 + + + DMSO + + + + + + POM + + + + + +
IKZF1 GSPT1
IKZF1 DDB1 DDB1
Cul4 Cul4 DDB1 CRBN CRBN Cul4 UBE2G1 UBE2G1 CRBN UBE2D3 UBE2D3 UBE2G1 Rbx1 Rbx1
UBE2D3
Rbx1 bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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
D E F MBP-GSPT1 + + + + + + + + + + + + Ub K48 only Ub + + + + + + K48R Ub + + + + + + Cul4A-Rbx1 + + + + + + + + + + + + DDB1 DDB1-cereblon + + + + + + + + + + + + Rbx1 Ube1 + + + + + + + + + + + + UBE2D3 UBE2G1 + + + + + + + + DDB1-cereblon + Ub + UBE2D3 + + + + + + + + + + + + Ube1+ UBE2G1
Ub Cul4 DMSO + + + + + + - + + Cul4A- Rbx1 CC-885 + + + + + + + + DMSO GSPT1 GSPT1 + + CC-885 Cereblon Modulating Agents
GSPT1
GSPT1 Ub DDB1 Ub DDB1 Ub Cul4 Ub CRBN UbUb
CRBN Cul4 DDB1 UBE2G1 Rbx1 Rbx1 UEB2G1 UBE2D3 UBE2G1 Cul4 Rbx1 UBE2D3 bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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 5
B C A UBE2D3 Empty + UBE2G1 UBE2D3 Vector UBE2G1 UBE2G1 C90S 1 µM 1 µM POM DMSO 0.01 µM POM 0.01 µM POM DMSO DMSO 1 µM POM 0.1 µM POM 0.01 µM POM 0.1 µM POM DMSO 1 µM 1 µM POM 0.01 µM POM 0.1 µM POM 1 µM 1 µM POM 1 µM 1 µM POM 0.1 µM POM 0.1 µM POM 0.1 µM POM 1 µM 1 µM POM 0.01 µM POM 0.01 µM POM DMSO DMSO DMSO DMSO 1 µM POM DMSO 1 µM POM 1 µM 1 µM POM
poly-Ub- poly-Ub-IKZF1 IKZF1 poly-Ub-IKZF1 mono-Ub-IKZF1 mono-Ub-IKZF1 mono-Ub-IKZF1
CRBN CRBN CRBN NTA Denaturing Denaturing Pull Down NTA NTA Denaturing Denaturing Pull Down NTA - - Ni Ni NTA Denaturing Denaturing Pull Down NTA - HIS-Ub HIS-Ub Ni
HIS-Ub 293T 293T 293T UBE2G1-/- Parental UBE2G1-/- ; UBE2D3-/-
293T UBE2G1-/- ; UBE2D3-/- bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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 6 A C 1 2 5 M M 1 S
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o 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0
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P O M ( M ) M M POM M M POM M POM
B M POM M M POM M POM M M LEN M M LEN M LEN M M LEN M M LEN M LEN M M POM M M POM M POM M M LEN M M LEN M LEN (16 hrs) 0.01 0.1 1 0.01 0.1 1 0.01 0.1 1 DMSO 0.1 1 10 DMSO 0.1 1 10 DMSO 0.1 1 10 IKZF1
IKZF1 IKZF3
IKZF3 UBE2G1 CRBN CRBN UBE2G1 Actin UBE2D3 Tubulin SKMM-2 bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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 7 O P M 2 - C a s 9 O P M 2 - C a s 9 1 2 0 A O P M 2 - C a s 9 1 2 0
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B sgNT-1 sgUBE2G1-5 sgCRBN-8 220 220 220 220 220 - - 220 - - - - 220 220 220 - - -
(16hrs) 1 µM 1 µM LEN 1 µM LEN DMSO DMSO 0.1 µM POM 0.1 µM POM 1 µM 1 µM POM 1 µM POM 0.1 µM LEN 0.1 µM LEN 1 nM CC 1 nM CC 1 µM 1 µM LEN 0.01 µM POM 0.01 µM POM 10 nM CC 10 nM CC 100 nM CC 100 nM CC DMSO 0.1 µM POM 10 µM LEN 10 µM LEN 1 µM 1 µM POM 0.1 µM LEN 1 nM CC 0.01 µM POM 10 nM CC 100 nM CC 10 µM LEN IKZF1
IKZF3
ZFP91
CK1α
UBE2G1
CRBN
Actin
OPM2-Cas9 bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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 S1
A B Parental sgNT-3 sgCRBN-8 U 9 3 7 _ C a s 9 _ e P L - I K Z F 1 + + + 1 5 0 DMSO
POM ( 0.01 µM) + + + o
t + +
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P O M ( M ) Actin U937_Cas9_ePL-IKZF1
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P O M ( M ) bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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 S2
C s g U B E 2 C & s g U B E 2 D 1 A s g U B E 2 A & s g U B E 2 B
N o n - T a r g e t i n g & s g U B E 2 N L B o
t 1 5 0
o
o
t 1 5 0
t 1 5 0
d C o n t r o l
C o n t r o l l
C o n t r o l d
e
d
l
l
o
e z
e s g U B E 2 C _ 1
i
r
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o s g N T _ 1
l z
s g U B E 2 A _ 1 t
i
r
i
r
l
t
l
a t n 1 0 0
a s g U B E 2 C _ 3 a
s g N T _ 2 n n 1 0 0 s g U B E 2 A _ 2 o
1 0 0 m
o
o
c
r
m
m
c
s g N T _ 3 r s g U B E 2 C _ 6
c
r
o
s g U B E 2 A _ 3
o
O
o
n
O
O
s g U B E 2 N L _ 1 n S
n s g U B E 2 D 1 _ 1
S
S s g U B E 2 B _ 1
U M
U 5 0
U s g U B E 2 N L _ 2 L M 5 0
M 5 0 s g U B E 2 D 1 _ 2
D L
L s g U B E 2 B _ 2
D
R D
s g U B E 2 N L _ 6
R
R
s g U B E 2 D 1 _ 3
s g U B E 2 B _ 5 %
% % 0 0 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 P O M ( M ) P O M ( M ) P O M ( M )
s g U B E 2 D 2 & s g U B E 2 D 3 s g U B E 2 E 3 & s g U B E 2 G 1
D E s g U B E 2 D 4 & s g U B E 2 E 1 F
o
o t
o 1 5 0 t
1 5 0 t
1 5 0 C o n t r o l
C o n t r o l d l
d C o n t r o l
d
e
l
l
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o
e
z
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z s g U B E 2 E 3 _ 1 i
s g U B E 2 D 2 _ 1 o
z
i
t r
s g U B E 2 D 4 _ 1 l
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r
l
t
l
t
n
a
a n a s g U B E 2 E 3 _ 2 1 0 0 s g U B E 2 D 2 _ 2 n 1 0 0
1 0 0 s g U B E 2 D 4 _ 2 o
o
m
o
m
c
m
r
c
r
c r
s g U B E 2 D 2 _ 4 s g U B E 2 E 3 _ 3
o
o s g U B E 2 D 4 _ 3
O
o
O
n
O
n
n
S
S s g U B E 2 D 3 _ 4 s g U B E 2 G 1 _ 1
S s g U B E 2 E 1 _ 1
U
U M
U 5 0 M 5 0
M 5 0
L
L D s g U B E 2 D 3 _ 5 L s g U B E 2 G 1 _ 5
D s g U B E 2 E 1 _ 3
D
R
R
R
s g U B E 2 D 3 _ 7 s g U B E 2 E 1 _ 7 s g U B E 2 G 1 _ 6
%
% %
0 0 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0
P O M ( M ) P O M ( M ) P O M ( M )
G H I s g U B E 2 L 6 & s g U B E 2 M
s g U B E 2 G 2 & s g U B E 2 H s g U B E 2 I & s g U B E 2 L 3
o t
1 5 0
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o t 1 5 0 d t C o n t r o l
1 5 0
l C o n t r o l e
d C o n t r o l
d
o
z
l
e
l i
r s g U B E 2 L 6 _ 6
e
l
t
o
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s g U B E 2 G 2 _ 3 z i
r s g U B E 2 I _ 5
a
i
r
l
n
t l
t 1 0 0 s g U B E 2 L 6 _ 7
a
o
n
a m
1 0 0 s g U B E 2 G 2 _ 4 n 1 0 0 s g U B E 2 I _ 7
r
c
o
o
m
m s g U B E 2 L 6 _ 8
o
c
r
c
r
s g U B E 2 G 2 _ 7 s g U B E 2 I _ 8 O
o
n
o
O
S O
n s g U B E 2 M _ 1
n
U S s g U B E 2 H _ 2 S s g U B E 2 L 3 _ 2
M 5 0
U
L U
M 5 0 s g U B E 2 M _ 2
M 5 0
D
L L
s g U B E 2 H _ 5 R
D s g U B E 2 L 3 _ 3
D
R
R
s g U B E 2 M _ 3
s g U B E 2 H _ 8 s g U B E 2 L 3 _ 7 %
% % 0 0 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 P O M ( M ) P O M ( M ) P O M ( M )
L s g C D C 3 4 & s g U B E 2 K J K s g U B E 2 V 2 & s g U B E 2 S
s g U B E 2 N & s g U B E 2 V 1
o
o t
o 1 5 0 t
1 5 0
t
C o n t r o l
1 5 0 C o n t r o l d
d
d
l e
C o n t r o l l
l
e
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z s g C D C 3 4 _ 1
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o
z
r o
z s g U B E 2 V 2 _ 4
l
i
t
i
r
r l
s g U B E 2 N _ 2 l
t
t
a
n a
a 1 0 0 s g C D C 3 4 _ 3 n
n 1 0 0 s g U B E 2 V 2 _ 7
o m
1 0 0 s g U B E 2 N _ 3 o
o
m
c
m
r
c
r r
c s g C D C 3 4 _ 4
o
s g U B E 2 V 2 _ 8
o o
s g U B E 2 N _ 4 O
n
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n n
S s g U B E 2 K _ 1
S s g U B E 2 S _ 1 S s g U B E 2 V 1 _ 4 U
M 5 0
U
U L M 5 0
M 5 0 s g U B E 2 K _ 4
D L
L s g U B E 2 S _ 2 D
s g U B E 2 V 1 _ 6 R
D
R
R
s g U B E 2 K _ 7
s g U B E 2 V 1 _ 8 s g U B E 2 S _ 7 %
% % 0 0 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 P O M ( M ) P O M ( M ) P O M ( M ) bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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 S2
N O M s g U B E 2 J 2 & s g U B E 2 W s g U B E 2 E 2 & s g U E V L D s g U B E 2 R 2 & s g U B E 2 J 1
1 5 0 1 5 0 1 5 0
C o n t r o l C o n t r o l o
C o n t r o l o
t
t
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d s g U B E 2 J 2 _ 2 d
l s g U B E 2 E 2 _ 1 l
d s g U B E 2 R 2 _ 2
l
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t r s g U B E 2 J 2 _ 3 t
l 1 0 0 i
l 1 0 0 s g U B E 2 E 2 _ 7 t
l 1 0 0 s g U B E 2 R 2 _ 4
n
n
a
a
n
a
o
o
o m
s g U B E 2 J 2 _ 6 m
c s g U B E 2 E 2 _ 8 c
m s g U B E 2 R 2 _ 8
c
r
r
r
o
o
O
O
o O
n s g U B E 2 W _ 6
n s g U E V L D _ 3 n
s g U B E 2 J 1 _ 2 S
S
S
5 0 5 0
U
M U
5 0 M U M s g U B E 2 W _ 7
L s g U E V L D _ 7
s g U B E 2 J 1 _ 3 L
L
D
D
D
R
R
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s g U B E 2 J 1 _ 4 s g U B E 2 W _ 8 s g U E V L D _ 8
%
% %
0 0 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0
P O M ( M ) P O M ( M ) P O M ( M ) Q R P s g U B E 2 O & s g U B E 2 Q 1 s g U B E 2 Z & s g A K T I P s g U B E 2 T & s g U B E 2 F
1 5 0 1 5 0 C o n t r o l 1 5 0
C o n t r o l o o
t C o n t r o l
o
t
t
d s g U B E 2 O _ 2
l d
l s g U B E 2 Z _ 1
d s g U B E 2 T _ 1
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l
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o
o
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z
r
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r
i
t
i
z r
t s g U B E 2 O _ 4
l 1 0 0 l 1 0 0 s g U B E 2 Z _ 2 i
t s g U B E 2 T _ 7 n
l 1 0 0
a
n
a
n
a
o
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s g U B E 2 O _ 7 o
c m
c s g U B E 2 Z _ 5 s g U B E 2 T _ 8
r
m
r
c
r
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O
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n s g U B E 2 Q 1 _ 2
n s g A K T I P _ 3 s g U B E 2 F _ 3
S
S
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5 0 U
5 0 M U
M 5 0 U
s g U B E 2 Q 1 _ 4 M s g U B E 2 F _ 4 L
L s g A K T I P _ 4
D
D
L
D
R
R
R
s g A K T I P _ 5 s g U B E 2 Q 1 _ 7 s g U B E 2 F _ 5
%
% % 0 0 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 P O M ( M ) P O M ( M ) P O M ( M ) T U S s g U B E 2 Q 2 & s g T S G 1 0 1 s g U B E 2 Q 2 L & s g U B E 2 Q L 1 s g B I R C 6 & s g U B E 2 U
1 5 0 1 5 0 1 5 0 C o n t r o l C o n t r o l
C o n t r o l o
t
o
o
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s g U B E 2 Q 2 _ 4
d
l s g U B E 2 Q 2 L _ 6
s g B I R C 6 _ 1 d
l
d
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t l
1 0 0 n
l
a
n
a
n o a s g U B E 2 Q 2 _ 8
o s g U B E 2 Q 2 L _ 8
o m
s g B I R C 6 _ 8 c
m
c
r
m
c
r
r
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s g T S G 1 0 1 _ 3 o O
o s g U B E 2 Q L 1 _ 4 O
s g U B E 2 U _ 3 n
S
n
S
n
S
5 0 M
U 5 0 M
5 0 s g T S G 1 0 1 _ 5 U s g U B E 2 Q L 1 _ 6 M
U s g U B E 2 U _ 6
L
D
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L
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R
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R s g T S G 1 0 1 _ 6 s g U B E 2 Q L 1 _ 8
s g U B E 2 U _ 8
%
% %
0 0 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0
P O M ( M ) P O M ( M ) P O M ( M ) bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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 S3
B s g U B E 2 C & s g U B E 2 D 1 A N o n - T a r g e t i n g & s g U B E 2 N L C s g U B E 2 A & s g U B E 2 B
1 5 0 o
o 1 5 0 t
t 1 5 0
s g U B E 2 G 1 _ 5 o s g U B E 2 G 1 _ 5 t
s g U B E 2 G 1 _ 5 d
d
l
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l
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o s g U B E 2 G 1 _ 5 + s g U B E 2 C _ 1
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r i
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r
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l
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n s g U B E 2 G 1 _ 5 + s g U B E 2 C _ 3
t 1 0 0
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n s g U B E 2 G 1 _ 5 + s g U B E 2 A _ 2
m
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c
r
s g U B E 2 G 1 _ 5 + s g N T _ 3 m
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s g U B E 2 G 1 _ 5 + s g U B E 2 A _ 3
o
O
n O
o s g U B E 2 G 1 _ 5 + s g U B E 2 D 1 _ 1
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s g U B E 2 G 1 _ 5 + s g U B E 2 N L _ 1 S
n
S
S s g U B E 2 G 1 _ 5 + s g U B E 2 B _ 1 U
M 5 0 U s g U B E 2 G 1 _ 5 + s g U B E 2 D 1 _ 2
M 5 0 L s g U B E 2 G 1 _ 5 + s g U B E 2 N L _ 2 U
M 5 0
D
L
D L
s g U B E 2 G 1 _ 5 + s g U B E 2 B _ 2 R
D
R s g U B E 2 G 1 _ 5 + s g U B E 2 N L _ 6 s g U B E 2 G 1 _ 5 + s g U B E 2 D 1 _ 3
R
s g U B E 2 G 1 _ 5 + s g U B E 2 B _ 5 %
% % 0 0 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 P O M ( M ) P O M ( M ) P O M ( M )
s g U B E 2 E 3 & s g U B E 2 G 1 s g U B E 2 D 4 & s g U B E 2 E 1 F
D s g U B E 2 D 2 & s g U B E 2 D 3 E o
t 1 5 0 1 5 0
o s g U B E 2 G 1 _ 5 o
1 5 0 d t
t s g U B E 2 G 1 _ 5
l
s g U B E 2 G 1 _ 5 e
o
z d
d s g U B E 2 G 1 _ 5 + s g U B E 2 E 3 _ 1
l
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l s g U B E 2 G 1 _ 5 + s g U B E 2 D 4 _ 1
e
l
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s g U B E 2 G 1 _ 5 + s g U B E 2 D 2 _ 1 o
o
z
z
a
r
n
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i s g U B E 2 G 1 _ 5 + s g U B E 2 E 3 _ 2 t
l 1 0 0 l t s g U B E 2 G 1 _ 5 + s g U B E 2 D 4 _ 2
1 0 0 o
n
m a
a s g U B E 2 G 1 _ 5 + s g U B E 2 D 2 _ 2
n 1 0 0
c r
o s g U B E 2 G 1 _ 5 + s g U B E 2 E 3 _ 3
o
m o
m s g U B E 2 G 1 _ 5 + s g U B E 2 D 4 _ 3
c
r
c r
s g U B E 2 G 1 _ 5 + s g U B E 2 D 2 _ 4 O
n
o s g U B E 2 G 1 _ 5 + s g U B E 2 G 1 _ 1
o O
s g U B E 2 G 1 _ 5 + s g U B E 2 E 1 _ 1 S
n
O
n S
s g U B E 2 G 1 _ 5 + s g U B E 2 D 3 _ 4 U
M 5 0
S s g U B E 2 G 1 _ 5 + s g U B E 2 G 1 _ 5 L U 5 0
M s g U B E 2 G 1 _ 5 + s g U B E 2 E 1 _ 3
U D
M 5 0
L R
s g U B E 2 G 1 _ 5 + s g U B E 2 D 3 _ 5 D L
s g U B E 2 G 1 _ 5 + s g U B E 2 G 1 _ 6
D R
s g U B E 2 G 1 _ 5 + s g U B E 2 E 1 _ 7
R %
s g U B E 2 G 1 _ 5 + s g U B E 2 D 3 _ 7 %
% 0 0 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 P O M ( M ) P O M ( M ) P O M ( M )
s g U B E 2 L 6 & s g U B E 2 M s g U B E 2 I & s g U B E 2 L 3 I s g U B E 2 G 2 & s g U B E 2 H H G o
t 1 5 0
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l
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d s g U B E 2 G 1 _ 5
d
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l s g U B E 2 G 1 _ 5 + s g U B E 2 L 6 _ 6
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s g U B E 2 G 1 _ 5 + s g U B E 2 I _ 5 t
o
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s g U B E 2 G 1 _ 5 + s g U B E 2 G 2 _ 3 z
i
a
r
r
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n
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t s g U B E 2 G 1 _ 5 + s g U B E 2 L 6 _ 7
t 1 0 0
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a s g U B E 2 G 1 _ 5 + s g U B E 2 I _ 7
n n
1 0 0 s g U B E 2 G 1 _ 5 + s g U B E 2 G 2 _ 4 1 0 0 m
c
r
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s g U B E 2 G 1 _ 5 + s g U B E 2 L 6 _ 8
m
m
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s g U B E 2 G 1 _ 5 + s g U B E 2 G 2 _ 7
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n
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s g U B E 2 G 1 _ 5 + s g U B E 2 M _ 1
O
S
O n n s g U B E 2 G 1 _ 5 + s g U B E 2 L 3 _ 2
s g U B E 2 G 1 _ 5 + s g U B E 2 H _ 2
U
S S
M 5 0 s g U B E 2 G 1 _ 5 + s g U B E 2 M _ 2
U
L U
M 5 0 M 5 0
s g U B E 2 G 1 _ 5 + s g U B E 2 H _ 5 s g U B E 2 G 1 _ 5 + s g U B E 2 L 3 _ 3 D
L
L
R
D D
s g U B E 2 G 1 _ 5 + s g U B E 2 M _ 3
R
R
s g U B E 2 G 1 _ 5 + s g U B E 2 H _ 8 s g U B E 2 G 1 _ 5 + s g U B E 2 L 3 _ 7
%
% % 0 0 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 P O M ( M ) P O M ( M ) P O M ( M )
s g U B E 2 N & s g U B E 2 V 1 s g C D C 3 4 & s g U B E 2 K
J K s g U B E 2 V 2 & s g U B E 2 S L o
o 1 5 0 1 5 0
t
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1 5 0 s g U B E 2 G 1 _ 5
s g U B E 2 G 1 _ 5 o
d
t d
s g U B E 2 G 1 _ 5
l
l
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o s g U B E 2 G 1 _ 5 + s g C D C 3 4 _ 1
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s g U B E 2 G 1 _ 5 + s g U B E 2 N _ 2 l
z
r
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i s g U B E 2 G 1 _ 5 + s g U B E 2 V 2 _ 4
l
t
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l
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n s g U B E 2 G 1 _ 5 + s g C D C 3 4 _ 3
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t l n s g U B E 2 G 1 _ 5 + s g U B E 2 N _ 3 1 0 0
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a n
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m
m
c
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c s g U B E 2 G 1 _ 5 + s g C D C 3 4 _ 4
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n s g U B E 2 G 1 _ 5 + s g U B E 2 K _ 1
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M 5 0
M 5 0 s g U B E 2 G 1 _ 5 + s g U B E 2 K _ 4
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M 5 0 L
s g U B E 2 G 1 _ 5 + s g U B E 2 V 1 _ 6 s g U B E 2 G 1 _ 5 + s g U B E 2 S _ 2 D
D
L
R
D
R
s g U B E 2 G 1 _ 5 + s g U B E 2 K _ 7 R
s g U B E 2 G 1 _ 5 + s g U B E 2 V 1 _ 8 s g U B E 2 G 1 _ 5 + s g U B E 2 S _ 7
%
% % 0 0 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 P O M ( M ) P O M ( M ) P O M ( M ) bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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 S3
s g U B E 2 J 2 & s g U B E 2 W s g U B E 2 E 2 & s g U E V L D M s g U B E 2 R 2 & s g U B E 2 J 1 N O 1 5 0 1 5 0 1 5 0 s g U B E 2 G 1 _ 5 s g U B E 2 G 1 _ 5
s g U B E 2 G 1 _ 5 o
o
t
t
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s g U B E 2 G 1 _ 5 + s g U B E 2 E 2 _ 1
t s g U B E 2 G 1 _ 5 + s g U B E 2 J 2 _ 2
d
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l
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r i z 1 0 0
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m
c
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s g U B E 2 G 1 _ 5 + s g U B E 2 J 1 _ 2 n
s g U B E 2 G 1 _ 5 + s g U B E 2 W _ 6 S
n
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5 0 M 5 0 U
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U s g U B E 2 G 1 _ 5 + s g U B E 2 J 1 _ 3 s g U B E 2 G 1 _ 5 + s g U E V L D _ 7 L
s g U B E 2 G 1 _ 5 + s g U B E 2 W _ 7 D
L
D
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D
R
R
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s g U B E 2 G 1 _ 5 + s g U B E 2 J 1 _ 4 s g U B E 2 G 1 _ 5 + s g U E V L D _ 8
s g U B E 2 G 1 _ 5 + s g U B E 2 W _ 8
%
% % 0 0 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0
P O M ( M ) P O M ( M ) P O M ( M )
s g U B E 2 O & s g U B E 2 Q 1 s g U B E 2 T & s g U B E 2 F P s g U B E 2 Z & s g A K T I P Q R 1 5 0 1 5 0 1 5 0 s g U B E 2 G 1 _ 5
s g U B E 2 G 1 _ 5 o
s g U B E 2 G 1 _ 5 t
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t s g U B E 2 G 1 _ 5 + s g U B E 2 T _ 1
d
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s g U B E 2 G 1 _ 5 + s g U B E 2 O _ 2 l
t
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s g U B E 2 G 1 _ 5 + s g U B E 2 Z _ 1 l
z
r
d
l
e o i s g U B E 2 G 1 _ 5 + s g U B E 2 T _ 7
t 1 0 0
l
e
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o s g U B E 2 G 1 _ 5 + s g U B E 2 O _ 4 i
t 1 0 0
n
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s g U B E 2 G 1 _ 5 + s g U B E 2 Z _ 2 a l
i 1 0 0
t
n
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a s g U B E 2 G 1 _ 5 + s g U B E 2 T _ 8
n
m
c a
o s g U B E 2 G 1 _ 5 + s g U B E 2 O _ 7
r
o s g U B E 2 G 1 _ 5 + s g U B E 2 Z _ 5
m
c
m
c
o
r O
s g U B E 2 G 1 _ 5 + s g U B E 2 F _ 3
r
o n
O s g U B E 2 G 1 _ 5 + s g U B E 2 Q 1 _ 2
S
o
O s g U B E 2 G 1 _ 5 + s g A K T I P _ 3
n
S
n
S 5 0
M U
5 0 s g U B E 2 G 1 _ 5 + s g U B E 2 F _ 4 M
5 0 U
s g U B E 2 G 1 _ 5 + s g U B E 2 Q 1 _ 4 L
M D
U s g U B E 2 G 1 _ 5 + s g A K T I P _ 4
L
D
L
D R
s g U B E 2 G 1 _ 5 + s g U B E 2 F _ 5
R R
s g U B E 2 G 1 _ 5 + s g U B E 2 Q 1 _ 7
s g U B E 2 G 1 _ 5 + s g A K T I P _ 5
%
% % 0 0 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 P O M ( M ) P O M ( M ) P O M ( M ) S T V s g U B E 2 Q 2 L & s g U B E 2 Q L 1 s g B I R C 6 & s g U B E 2 U s g U B E 2 Q 2 & s g T S G 1 0 1 1 5 0 1 5 0 1 5 0 s g U B E 2 G 1 _ 5
s g U B E 2 G 1 _ 5 s g U B E 2 G 1 _ 5
o
t
o o
s g U B E 2 G 1 _ 5 + s g U B E 2 Q 2 L _ 6
t
t
d
s g U B E 2 G 1 _ 5 + s g B I R C 6 _ 1 s g U B E 2 G 1 _ 5 + s g U B E 2 Q 2 _ 4 l
d
l
e
d
o
l
e
r
z o
e 1 0 0 s g U B E 2 G 1 _ 5 + s g U B E 2 Q 2 L _ 7
o
t
i r
z s g U B E 2 G 1 _ 5 + s g U B E 2 Q 2 _ 6
l r z s g U B E 2 G 1 _ 5 + s g B I R C 6 _ 2
i 1 0 0
t n
i 1 0 0
t
l
a
l
n
o
n a
a s g U B E 2 G 1 _ 5 + s g U B E 2 Q 2 L _ 8
o c s g U B E 2 G 1 _ 5 + s g U B E 2 Q 2 _ 8 m
o s g U B E 2 G 1 _ 5 + s g B I R C 6 _ 8
r
m
c
m
c
r
r o
O s g U B E 2 G 1 _ 5 + s g U B E 2 Q L 1 _ 4
o O
o s g U B E 2 G 1 _ 5 + s g T S G 1 0 1 _ 3
n O
s g U B E 2 G 1 _ 5 + s g U B E 2 U _ 3 S
n
S
n S
5 0
M 5 0 U
5 0 M s g U B E 2 G 1 _ 5 + s g U B E 2 Q L 1 _ 6 U
M s g U B E 2 G 1 _ 5 + s g T S G 1 0 1 _ 5
U D
s g U B E 2 G 1 _ 5 + s g U B E 2 U _ 6 L
L
D
L
D
R
R R
s g U B E 2 G 1 _ 5 + s g T S G 1 0 1 _ 6 s g U B E 2 G 1 _ 5 + s g U B E 2 Q L 1 _ 8
s g U B E 2 G 1 _ 5 + s g U B E 2 U _ 8
%
% % 0 0 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 P O M ( M ) P O M ( M ) P O M ( M ) bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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 S4
A sgNT-1 sgCRBN-4 sgCRBN-8 sgUBE2G1-1 sgUBE2G1-5 C sgNT-1 sgUBE2G1-1 sgCRBN-8 885 885 885 885 - 885 - 885 - - - - 885 885 885 - - - 220(5nM) 220(5nM) 220(5nM) 220(5nM) 220(5nM) - - - - - (4 hrs) (16 Hrs) LEN LEN (200nM) LEN LEN (200nM) CC LEN LEN (200nM) CC DMSO CC POM (100nM) DMSO CC DMSO LEN LEN (200nM) CC DMSO POM (100nM) POM (100nM) LEN (200nM) DMSO DMSO 1 nM CC POM (100nM) POM (100nM) 0.5 nM CC 1nM CC 0.1 nM CC DMSO 1 nM CC 0.1nM CC DMSO 0.5nM CC 0.1 nM CC 0.5 nM CC IKZF1 GSPT1 IKZF3 UBE2G1 CRBN CRBN UBE2G1 Actin Actin MM1S-Cas9 MM1S Cas9
B D
sgNT-1 sgCRBN-4 sgCRBN-8 sgUBE2G1-1 sgUBE2G1-5 sgNT-1 sgUBE2G1-1 sgCRBN-8 885 885 885 885 885 885 ------885 885 885 - - - 220(5nM) 220(5nM) 220(5nM) 220(5nM) 220(5nM) - - - - - (16 Hrs) LEN LEN (200nM) LEN LEN (200nM) CC DMSO LEN LEN (200nM) DMSO CC CC POM (100nM) LEN LEN (200nM) CC CC DMSO POM (100nM) POM (100nM) LEN LEN (200nM) POM (100nM) POM (100nM) DMSO DMSO (4 hrs) 1 nM CC 1 nM CC 1 nM CC 0.1 nM CC 0.5 nM CC 0.1 nM CC 0.5 nM CC 0.1 nM CC 0.5 nM CC DMSO DMSO IKZF1 DMSO IKZF3 GSPT1 CRBN UBE2G1 UBE2G1 CRBN Actin Actin DF15-Cas9 DF15-Cas9 bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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 S4
E Parental sgNT-1 sgUBE2G1-5 sgCRBN-8 F Parental sgNT-1 sgUBE2G1-5 sgCRBN-8 885 885 885 885 885 885 885 885 885 885 885 885 885 885 885 885 ------885 - - 885 - - 885 885 885 885 885 885 ------
(4 hrs) (4 hrs) 3 nM CC 30 nM CC 3 nM CC DMSO DMSO DMSO DMSO 30 nM CC 3 nM CC 3 nM CC 30 nM CC 3 nM CC 3 nM CC 30 nM CC DMSO 10 nM CC DMSO 30 nM CC 10 nM CC 30 nM CC 10 nM CC 3 nM CC 30 nM CC 10 nM CC 30 nM CC 3 nM CC DMSO DMSO 10 nM CC 10 nM CC 10 nM CC 10 nM CC GSPT1 GSPT1
UBE2G1 UBE2G1
CRBN CRBN I UBE2G1 C90S Actin Actin 885 885 885 885 885 885 885 885 ------885 885 885 885 - - - OCI-AML-2-Cas9 U937-Cas9 -
(4 hrs)
H 3 nM CC 30 nM CC 3 nM CC 30 nM CC DMSO 10 nM CC DMSO DMSO 10 nM CC 30 nM CC 30 nM CC 3 nM CC 3 nM CC 10 nM CC 10 nM CC G Parental sgNT-1 sgUBE2G1-5 sgCRBN-8 Parental sgNT-1 sgUBE2G1-5 sgCRBN-8 DMSO 885
885 885 885 GSPT1 885 - 885 885 885 885 885 - - - 885 885 ------885 885 885 885 885 885 885 885 885 885 885 885 ------CRBN
(4 hrs) (4 hrs) 0.3 nM CC 3 nM CC 30 nM CC 3 nM CC 30 nM CC 3 nM CC 3 nM CC DMSO 10 nM CC DMSO DMSO 10 nM CC DMSO 30 nM CC 30 nM CC 3 nM CC DMSO 1 nM CC 1 nM CC 0.3 nM CC 0.3 nM CC 0.3 nM CC 3 nM CC 3 nM CC 3 nM CC DMSO 1 nM CC 1 nM CC 10 nM CC 10 nM CC DMSO DMSO UBE2G1 GSPT1 GSPT1
UBE2G1 UBE2G1 UBE2D3
CRBN CRBN Actin Actin Actin 293T 293T Parental UBE2G1-/- MOLM-13-Cas9 MV4-11-Cas9 bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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 S5 A UBE2G1 ---MTELQSALLLRRQLAELNKNPVEGFSAGLIDDNDLYRWEVLIIGPPDTLYEGGVFKA UBE2G2 ----MAGTALKRLMAEYKQLTLNPPEGIVAGPMNEENFFEWEALIMGPEDTCFEFGVFPA C Cdc34 MARPLVPSSQKALLLELKGLQEEPVEGFRVTLVDEGDLYNWEVAIFGPPNTYYEGGYFKA Cul4A-Rbx1 + + + + + + + + : * : * :* **: . ::: :::.**. *:** :* :* * * * DDB1-cereblon + + + + + + + + UBE2G1 HLTFPKDYPLRPPKMKFITEIWHPNVDKNGDVCISILHEPGEDKYGYEKPEERWLPIHTV MBP-GSPT1 + Ube1 + Ub + + + + + + + + UBE2G2 ILSFPLDYPLSPPKMRFTCEMFHPNIYPDGRVCISILHAPGDDPMGYESSAERWSPVQSV FLAG-UBE2D3 + + + + + + Cdc34 RLKFPIDYPYSPPAFRFLTKMWHPNIYETGDVCISILHPPVDDPQSGELPSERWNPTQNV FLAG-UBE2G1 + + *.** *** ** ::* :::***: * ******* * :* . * . *** * :.* FLAG-UBE2G1-C90S + + DMSO + + + + UBE2G1 ETIMISVISMLADPNGDSPANVDAAKEWREDRNGEFKRKVARCVRKSQETAFE------CC-885 + + + + UBE2G2 EKILLSVVSMLAEPNDESGANVDASKMWRDDRE-QFYKIAKQIVQKSLGL------Cdc34 RTILLSVISLLNEPNTFSPANVDASVMYRKWKESKGKDREYTDIIRKQVLGTKVDAERDG ..*::**:*:* :** * *****: :*. :: : : :. GSPT1 UBE2G1 ------UBE2G2 ------Cdc34 VKVPTTLAEYCVKTKAPAPDEGSDLFYDDYYEDGEVEEEADSCFGDDEDDSGTEES DDB1 FLAG- FLAG- B FLAG- UBE2G1 FLAG- UBE2D3 CRBN UBE2G1 C90S UBE2D3 C85S 885 885 885 885 885 885 ------
885 885 Cul4 885 885 885 885 885 885 885 885 885 885 ------
UBE2G1 (4 hrs) FLAG 30 nM CC 30 nM CC DMSO DMSO 10 nM CC 30 nM CC DMSO DMSO 30 nM CC 100 nM CC 10 nM CC 10 nM CC 30 nM CC DMSO 100 nM CC 100 nM CC 100 nM CC 10 nM CC 100 nM CC 10 nM CC 30 nM CC DMSO 100 nM CC 10 nM CC UBE2D3 GSPT1 RBX1 CRBN FLAG-UBE2G1 endogenous-UBE2G1 UBE2G1 FLAG-UBE2D3 UBE2D3 endogenous-UBE2D3
FLAG-UBE2G1 FLAG FLAG-UBE2D3
Actin
293T 293T Parental UBE2G1-/- Figure S6 bioRxiv preprint A WCL
Parental DMSO doi: 293T certified bypeerreview)istheauthor/funder.Allrightsreserved.Noreuseallowedwithoutpermission.
0.01 µM POM https://doi.org/10.1101/389098 0.1 µM POM
UBE2G1 1 µM POM
DMSO - / 293T 0.01 µM POM - ; ; UBE2D3 0.1 µM POM this versionpostedAugust9,2018. 1 µM POM - / CRBN IKZF1 UBE2D3 UBE2G1 Actin - The copyrightholderforthispreprint(whichwasnot
WCL B
DMSO Vector Empty 0.01 µM POM 0.1 µM POM 1 µM POM DMSO UBE2G1 UBE2G1 0.01 µM POM 0.1 µM POM - / 293T 293T 1 µM POM - ; UBE2D3 DMSO 0.01 µM POM UBE2D3
- 0.1 µM POM / - 1 µM POM DMSO UBE2G1 0.01 µM POM UBE2D3 + 0.1 µM POM 1 µM POM CRBN UBE2D3 UBE2G1 IKZF1 Actin C
WCL DMSO 1 µM POM UBE2G1 UBE2G1
293T 293T DMSO
- 1 µM POM / -
DMSO C90S 1 µM POM UBE2G1 IKZF1 Actin CRBN bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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 S7
A O C I - A M L 2 - C a s 9 B
U 9 3 7 - C a s 9
o o
t 1 5 0
t
1 5 0
d
d
l
l
e
e
o
o
z
z
r
r
i
i
t
t
l l
n 1 0 0
a n a 1 0 0 P a r e n t a l
P a r e n t a l o
o
m
c
m
c
r
r s g N T - 1
s g N T - 1
o
O
o O
n s g U B E 2 G 1 - 5
S n
s g U B E 2 G 1 - 5 5 0 S
5 0
M
U U
M s g C R B N - 8
s g C R B N - 8 L
D
L
D
R
R 0 0 % % 0 . 0 0 0 1 0 . 0 0 1 0 . 0 1 0 . 1 1 0 . 0 0 0 1 0 . 0 0 1 0 . 0 1 0 . 1 1 C C - 8 8 5 ( M ) C C - 8 8 5 ( M ) C D M V 4 - 1 1 - C a s 9
M O L M - 1 3 - C a s 9 o t
1 5 0
o
t
d
l
1 5 0 e
o
d
z
l
r
i
e
t
l
o
z
r
n a
i 1 0 0 t
l P a r e n t a l
o
n
a m
1 0 0 P a r e n t a l c
r
o s g N T - 1
m
o
c
O r
s g N T - 1
n
o S s g U B E 2 G 1 - 5
O 5 0
n s g U B E 2 G 1 - 5
U
M
S 5 0 s g C R B N - 8
L
D U
M s g C R B N - 8
R
L
D
R 0
% 0
% 0 . 0 0 0 1 0 . 0 0 1 0 . 0 1 0 . 1 1 0 . 0 0 0 1 0 . 0 0 1 0 . 0 1 0 . 1 1 C C - 8 8 5 ( M ) C C - 8 8 5 ( M )
F 1 5 0 2 9 3 T P a r e n t a l + d B E T 1 o
E t o
2 9 3 T P a r e n t a l 1 5 0
t 2 9 3 T U B E 2 G 1 - / - + d B E T 1
d
l e
d 2 9 3 T U B E 2 G 1 - / -
o
l
z r
e 2 9 3 T P a r e n t a l + M Z 1
i
t
o
l z
r 1 0 0 i
2 9 3 T U B E 2 G 1 - / - U B C - U B E 2 G 1 n
a
t l
1 0 0 o 2 9 3 T U B E 2 G 1 - / - + M Z 1
n
a m
2 9 3 T U B E 2 G 1 - / - U B C - C 9 0 S c
r
o
m
o
c
r
O
n
o S
O 5 0
n
U M
S 5 0
L
D
U
M
R
L
D
R
%
0 0 % 0 . 0 0 0 1 0 . 0 0 1 0 . 0 1 0 . 1 1 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0
C C - 8 8 5 ( M ) C o m p o u n d ( M ) bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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 S8 A M M 1 S - C a s 9 M M 1 S - C a s 9 M M 1 S - C a s 9 1 2 0 1 2 0 1 2 0 s g N T - 1 s g N T - 1
s g N T - 1 o t
o 1 0 0
o
t 1 0 0 t
1 0 0 s g U B E 2 G 1 - 1
l
s g U B E 2 G 1 - 1 d
l
d l
d s g U B E 2 G 1 - 1
o
e
o
r
e
o e
z 8 0
t
r i
r s g U B E 2 G 1 - 5
z 8 0
z
t
l i
t 8 0 s g U B E 2 G 1 - 5
i n
s g U B E 2 G 1 - 5 l
l
a
n
n
o
a a
o s g C R B N - 4 o
c 6 0 m
s g C R B N - 4 c
m 6 0
r m c s g C R B N - 4
6 0
r
r
o
O o
O s g C R B N - 8
o
n
O S
s g C R B N - 8 4 0
n S
n s g C R B N - 8
4 0
S
M
4 0 U
M
U
U
L
M
D
L D
L 2 0
R D
2 0
R R
2 0
% %
% 0 0 0 0 . 0 0 0 1 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 0 . 0 0 0 1 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 0 . 0 0 0 1 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 C C - 2 2 0 ( M ) L E N ( M ) P O M ( M )
B sgNT-1 sgUBE2G1-5 sgCRBN-8 220 220 220 - 220 - 220 - 220 - - - 220 220 220 - - -
(16hrs) 0.1 µM POM 0.01 µM POM 0.01 µM POM 0.1 µM POM 0.1 µM LEN 1 µM LEN 1 µM LEN 1 µM 1 µM POM 100 nM CC 100 nM CC 0.1 µM LEN 1 nM CC 10 nM CC DMSO 10 µM LEN 10 µM LEN 1 µM 1 µM POM DMSO 1 µM POM 0.1 µM POM 0.1 µM LEN 10 nM CC 1 nM CC 100 nM CC 10 nM CC DMSO 10 µM LEN 0.01 µM POM 1 µM 1 µM LEN 1 nM CC IKZF1 IKZF3 ZFP91 CK1 CRBN
UBE2G1
Actin
MM1S-Cas9 bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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 S8
C D F 1 5 - C a s 9 D F 1 5 - C a s 9 D F 1 5 - C a s 9 1 2 0 1 2 0
1 2 0 o
s g N T - 1 o s g N T - 1
t
t
o
t s g N T - 1 1 0 0
1 0 0
d
l d s g U B E 2 G 1 - 1 l
d 1 0 0
l e
o s g U B E 2 G 1 - 1
e
o
r z
e s g U B E 2 G 1 - 1
o
r
z
i
t
r
z
i l
8 0 t i
l 8 0
t s g U B E 2 G 1 - 5
n l
a s g U B E 2 G 1 - 5
8 0 n a
n s g U B E 2 G 1 - 5
o
a
o
m o
c s g C R B N - 4 m
6 0
r
c m
c 6 0 s g C R B N - 4
r r
s g C R B N - 4
6 0 o
O
o
o
n O
O s g C R B N - 8
S
n
n 4 0 s g C R B N - 8
S
s g C R B N - 8 S
U 4 0
4 0 M
U
U
L
M
M
D
L
L R
2 0 D
D
R
2 0 R 2 0
% %
0 % 0 0 0 . 0 0 0 1 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 0 . 0 0 0 1 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 0 . 0 0 0 1 0 . 0 0 1 0 . 0 1 0 . 1 1 1 0 P O M ( M ) L E N ( M ) C C - 2 2 0 ( M )
D sgNT-1 sgUBE2G1-5 sgCRBN-8 220 220 220 - 220 - 220 220 - - - - 220 220 220 - - -
(16hrs) 0.1 µM POM 0.01 µM POM 0.01 µM POM 0.1 µM POM 0.1 µM LEN 1 µM LEN 1 µM LEN 1 µM 1 µM POM 100 nM CC 100 nM CC 1 nM CC 0.1 µM LEN 10 nM CC DMSO 10 µM LEN 10 µM LEN 1 µM 1 µM POM DMSO 1 µM POM 0.1 µM POM 0.1 µM LEN 10 nM CC 1 nM CC 100 nM CC 10 nM CC DMSO 10 µM LEN 0.01 µM POM 1 µM 1 µM LEN 1 nM CC IKZF1 IKZF3
ZFP91 CK1
CRBN UBE2G1
Actin
DF15-Cas9 bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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 S9
A B
(16 Hrs) UV (50 J/m2 ) 0 15 30 60 90 0 15 30 60 90 (mins) RBM39 Cdt1
UBE2G1 Set8
Actin p21
293T 293T Cdt2 Parental UBE2G1-/-
UBE2G1
Actin
293T 293T Parental UBE2G1-/- bioRxiv preprint doi: https://doi.org/10.1101/389098; this version posted August 9, 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 S10 A >UBE2D1 MALKRIQKELSDLQRDPPAHCSAGPVGDDLFHWQATIMGPPDSAYQGGVFFLTVHFPTDY >UBE2D2 MALKRIHKELNDLARDPPAQCSAGPVGDDMFHWQATIMGPNDSPYQGGVFFLTIHFPTDY >UBE2D3 MALKRINKELSDLARDPPAQCSAGPVGDDMFHWQATIMGPNDSPYQGGVFFLTIHFPTDY >UBE2D4 MALKRIQKELTDLQRDPPAQCSAGPVGDDLFHWQATIMGPNDSPYQGGVFFLTIHFPTDY ******:***.** *****:*********:********** **.*********:******
>UBE2D1 PFKPPKIAFTTKIYHPNINSNGSICLDILRSQWSPALTVSKVLLSICSLLCDPNPDDPLV >UBE2D2 PFKPPKVAFTTRIYHPNINSNGSICLDILRSQWSPALTISKVLLSICSLLCDPNPDDPLV >UBE2D3 PFKPPKVAFTTRIYHPNINSNGSICLDILRSQWSPALTISKVLLSICSLLCDPNPDDPLV >UBE2D4 PFKPPKVAFTTKIYHPNINSNGSICLDILRSQWSPALTVSKVLLSICSLLCDPNPDDPLV ******:****:**************************:*********************
>UBE2D1 PDIAQIYKSDKEKYNRHAREWTQKYAM >UBE2D2 PEIARIYKTDREKYNRIAREWTQKYAM >UBE2D3 PEIARIYKTDRDKYNRVSREWTQKYAI >UBE2D4 PEIAHTYKADREKYNRLAREWTQKYAM *:**: **:*::**** :********:
B MBP-GSPT1 + + + + + + + + + + + + + + + + + + + + Ub + + + + + + + + + + + + + + + + + + + + Cul4A-Rbx1 + + + + + + + + + + + + + + + + + + + + DDB1-cereblon + + + + + + + + + + + + + + + + + + + + Ube1 + + + + + + + + + + + + + + + + + + + + UBE2D1 + + + + + + + + UBE2D2 + + + + + + + + UBE2D3 + + + + + + + + UBE2G1 + + + + + + + + DMSO + + + + + + + + + + CC-885 + + + + + + + + + +
GSPT1 (SE)
GSPT1
GSPT1 (LE)