A genome-wide CRISPR/Cas9 screen in acute myeloid leukemia cells identifies regulators of TAK-243 sensitivity

TAK-243 is a first-in-class inhibitor of ubiquitin-like modifier activating enzyme 1 that catalyzes ubiquitin activation, the first step in the ubiquitylation cascade. Based on its preclinical efficacy and tolerability, TAK-243 has been advanced to phase I clinical trials in advanced malignancies. Nonetheless, the determinants of TAK-243 sensitivity remain largely unknown. Here, we conducted a genome-wide CRISPR/Cas9 knockout screen in acute myeloid leukemia (AML) cells in the presence of TAK-243 to identify genes essential for TAK-243 action. We identified BEN domain-containing protein 3 (BEND3), a transcriptional repressor and a regulator of chromatin organization, as the top gene whose knockout confers resistance to TAK-243 in vitro and in vivo. Knockout of BEND3 dampened TAK-243 effects on ubiquitylation, proteotoxic stress, and DNA damage response. BEND3 knockout upregulated the ATP-binding cassette efflux transporter breast cancer resistance protein (BCRP; ABCG2) and reduced the intracellular levelsof TAK-243. TAK-243 sensitivity correlated with BCRP expression in cancer cell lines of different origins. Moreover, chemical inhibition and genetic knockdown of BCRP sensitized intrinsically resistant high-BCRP cells to TAK-243. Thus, our data demonstrate that BEND3 regulates the expression of BCRP for which TAK-243 is a substrate. Moreover, BCRP expression could serve as a predictor of TAK-243 sensitivity.

Through this cascade, protein substrates are tagged with mono-or poly-ubiquitin to induce their proteasomal degradation or to modulate their functions (4,5). This process is executed through multi-step enzymatic reactions whereby ubiquitin is initially activated by the ubiquitin-activating enzyme (E1) in an ATP-dependent manner. This step is followed by the transfer of the activated ubiquitin from the catalytic cysteine site of E1 to the corresponding catalytic cysteine in one of the cognate ubiquitin-conjugating E2 enzymes (E2s). Ubiquitin is then transferred to protein substrates by E2s and this step is facilitated by ubiquitin ligases (E3s). While UBA1 is the major ubiquitin E1 in the cell, there are over 30 ubiquitin E2s and hundreds of ubiquitin E3 that mediate the ubiquitylation of substrates in a highly coordinated and specific manner (6).
We previously reported that acute myeloid leukemia (AML) cell lines and primary patient samples are more dependent on the activity of UBA1 compared to normal hematopoietic cells, and thus are more vulnerable to UBA1 inhibition (7). UBA1 was also reported by others to serve as a therapeutic target in cancer (8). Accordingly, we evaluated the selective UBA1 inhibitor, TAK-243, in preclinical models of AML and found that it displayed potent anti-leukemic activity in vitro and in vivo (9,10). Similar findings have also been reported with TAK-243 in solid tumors and other hematologic malignancies (2,(11)(12)(13). Nonetheless, the determinants of sensitivity to TAK-243 are still largely unknown.
To gain further insights into the mechanisms of sensitivity and resistance to TAK-243, we conducted a genome-wide CRISPR/Cas9 knockout screen in AML cells and identified the transcriptional repressor, BEN domain-containing protein 3 (BEND3) as the top gene whose knockout confers resistance to TAK-243.

A genome-wide CRISPR/Cas9 knockout screen identifies BEND3 as a regulator of TAK-243 sensitivity
TAK-243 is a first-in-class inhibitor of UBA1 that has been advanced to clinical trials (2,10). To identify genes that influence the cytotoxicity of TAK-243, we performed a genome-wide CRISPR/Cas9 knockout screen in AML cells. OCI-AML2-Cas9 cells were transduced with a library of 91,320 gRNAs in lentiviral vectors targeting 17,232 genes at a ratio of 6 gRNAs per gene (14). Three days after transduction, cells were treated with TAK-243 at concentrations of the drug corresponding to the IC90 and IC99. Thirty-two days after the addition of TAK-243, surviving cells were harvested and the gRNA bar codes identified by sequencing. We focused our analysis on genes whose knockout conferred resistance to TAK-243.
Of the 90k gRNAs in the CRISPR library, approximately 11.5k and 5.5k gRNAs were enriched at least 2-fold ( Figure 1A). Using the MAGeCK algorithm to rank enriched genes (15) and a false discovery rate (FDR) of < 0.2, 33 and 11 genes were identified as enriched in the populations of cells treated with TAK-243 at its IC90 and IC99 arms, respectively (Table S1 and S2). At both IC90 and IC99 concentrations, BEND3 ranked as the top hit (FDR = 0.001238; Figure 1B). Gene set enrichment analysis (GSEA) demonstrated that significantly enriched gRNAs corresponded to genes involved in diverse biological processes including chromatin organization, peptidyl lysine acetylation, histone methylation, TORC1 signaling and regulation of biosynthetic processes ( Figure 1C). All 6 gRNAs targeting BEND3 were enriched up to 1,222-and 9,136-fold after selection with TAK-243 at the IC90 and IC99 concentrations, respectively ( Figure 1D). Based on this analysis, we focused our investigation on the top hit BEND3.

BEND3 knockout confers resistance to TAK-243 in AML cells
To validate the screen results, we knocked out BEND3 using independent gRNAs. OCI-AML2-Cas9 cells were stably transduced with gRNAs targeting BEND3 or control sequences, and knockout of BEND3 was confirmed by immunoblotting (Figure 2A and C). BEND3 knockout cells were then treated with increasing concentrations of TAK-243 and growth and viability measured by the MTS assay. BEND3 knockout conferred resistance to TAK-243 with up to a 9fold increase in the IC50 of the drug (Figure 2B and D). BEND3 knockout also conferred resistance to TAK-243 as measured by Annexin V/propidium iodide (PI) staining, and proliferation assays using trypan blue dye exclusion ( Figure E and F). Finally, knockout of BEND3 reduced the ability of TAK-243 to target the colony-forming cells as measured by clonogenic assays ( Figure 2G). Of note, BEND3 knockout had little or no impact on cell proliferation rate in the absence of TAK-243 treatment ( Figure 2F).

BEND3 knockout confers resistance to TAK-243 in vivo
Next, we determined whether BEND3 regulates the sensitivity of AML cells to TAK-243 in vivo.
Control or BEND3 knockout OCI-AML2 cells were injected into severe combined immunodeficiency (SCID) mice. After the tumors became palpable, mice were treated with increasing doses of TAK-243 subcutaneously twice weekly (BIW). As previously described (10), TAK-243 produced dramatic reductions in tumor growth in wild-type OCI-AML2 cells ( Figure   3A-B and E). In contrast, BEND3 knockout rendered the tumors resistant to TAK243 and thus grew at a rate similar to control ( Figure 3C-D and E). Of note, BEND3 knockout cells exhibited a tumor growth rate in vivo comparable to that of control cells in vehicle-treated mice, which is consistent with proliferation data observed in vitro ( Figure 3A, C and E). All TAK-243 doses were tolerated as evidenced by non-significant changes in mice weights in TAK-243-versus vehicle-treated mice ( Figure 3F).

BEND3 knockout dampens TAK-243 effects on ubiquitylation, proteotoxic stress and DNA damage response in AML cells
TAK-243 inhibits UBA1 leading to reductions in poly-and mono-ubiquitylation, with the resultant induction of proteotoxic and DNA damage stress and subsequent cell death (2,10). To determine how BEND3 influences sensitivity to TAK-243, we treated control and BEND3 knockout OCI-AML2-Cas9 cells with TAK-243 and measured changes in the levels of UBA1, the abundance of ubiquitylated proteins and markers of proteotoxic and DNA double-strand break repair. BEND3 knockout did not change protein levels of UBA1 or other related E1 enzymes ( Figure 4A).
However, it attenuated TAK-243-induced reductions in both poly-ubiquitylation and H2A monoubiquitylation ( Figure 4A and B). In keeping with this finding, TAK-243-treated BEND3 knockout cells exhibited a little or no induction of markers of proteotoxic stress (ATF4, CHOP and p-JNK), DNA damage (γH2AX) and apoptosis (PARP cleavage) ( Figure 4A and B).

BEND3 knockout reduces the intracellular transport of TAK-243 into AML cells
TAK-243 is an adenosine monophosphate (AMP)-mimetic that binds to the nucleotide-binding site of the UBA1 enzyme in an ATP-competitive manner and then forms a covalent adduct with ubiquitin in a reaction requiring UBA1 activity. The resulting TAK-243-ubiquitin adduct inhibits UBA1 (2). We used the CETSA assay to evaluate the binding of TAK-243 to UBA1 in control versus BEND3 knockout OCI-AML2-Cas9 cells. Control and BEND3 knockout cells were treated with increasing concentrations of TAK-243 followed by measuring the thermal shift of UBA1 by immunoblotting. As assessed by this assay, BEND3 knockout reduced TAK-243 binding to UBA1 ( Figure 4C). However, it did not change the intracellular levels of ATP, indicating that resistance to TAK-243 could not be explained by increased levels of ATP that competes for UBA1 binding ( Figure 4D).
To assess the accumulation of TAK-243 into OCI-AML2-Cas9 cells, we measured intracellular TAK-243 concentrations following treatment with increasing concentrations of the drug for 1h.
As assessed by liquid chromatography-mass spectrometry (LC-MS), knockout of BEND3 reduced the intracellular concentrations of TAK-243 compared to control ( Figure 4E).

Upregulation of BCRP mediates TAK-243 resistance in vitro and in vivo
The emergence of multi-drug resistance (MDR) is a common problem with antineoplastic agents including cytotoxic drugs and molecularly targeted therapeutics (16). A major class of proteins mediating MDR are the ATP-binding cassette (ABC) transporters that act as efflux pumps to extrude drugs and xenobiotics out of the cells in an ATP-dependent manner (17). Since BEND3 knockout reduced the accumulation of TAK-243 into AML cells, we hypothesized that the upregulation of one or more of ABC transporters may be responsible for the resistance phenotype.
Of the 49 known human ABC transporters, 12 have been reported to be commonly implicated in MDR (17,18). To determine the most likely transporter for which TAK-243 might serve as a substrate, we correlated publicly available mRNA expression data of these 12 transporters and the IC50 of TAK-243 across 30 cancer cell lines for which TAK-243 sensitivity has been reported (Table S3) (2). Breast cancer resistance protein (BCRP) displayed the strongest correlation between expression and TAK-243 sensitivity with cells having the highest expression of BCRP being most resistant to the drug (r = 0.83; p < 0.0001). Multidrug resistance-associated protein 2 (MRP2) also displayed a weaker but statistically significant correlation (r = 0.51; p < 0.0038). All the other transporters in our analysis did not correlate with sensitivity to TAK-243 ( Figure 5A-B and S1).
These data suggest BCRP (encoded by ABCG2) and MRP2 may mediate TAK-243 efflux, and changes in BCRP and/or MRP2 expression may explain the resistance to TAK-243 after BEND3 knockout. To test this hypothesis, we measured mRNA expression of ABCG2, ABCC2 (encoding MRP2) as well as ABCB1 (encoding P-glycoprotein [P-gp]) in BEND3 knockout versus control OCI-AML2-Cas9 cells. As assessed by RT-qPCR, BEND3 knockout increased ABCG2 mRNA expression by 15-fold, while having no significant effect on ABCC2 nor ABCB1 expression ( Figure 5C). Thus, we decided to focus our investigation on BCRP. To test the functional importance of BCRP in explaining resistance to TAK-243 after BEND3 knockout, we treated BEND3 knockout and control OCI-AML2-Cas9 cells with increasing concentrations of TAK-243 alone and in combination with either the selective BCRP inhibitor Ko143 (19,20), or zosuquidar, a selective P-gp inhibitor (21). Inhibition of BCRP but not P-gp re-sensitized BEND3 knockout cells to TAK-243 ( Figure 5D-E).
To test the functional importance of BCRP in TAK-243 sensitivity in vivo, BEND3 knockout OCI-

TAK-243 is a substrate for BCRP in cell lines of different origin
To determine whether BCRP mediates resistance to TAK-243 in other cell lines, we treated A549 lung cancer cells, MCF7 breast cancer cells, MDAY-D2 lymphosarcoma cells (27) To confirm these findings using a genetic approach, we knocked down ABCG2 in A549 and RPMI 8226 cells using two distinct shRNAs and confirmed target knockdown by immunoblotting ( Figure 8E-F). Using the MTS assay, shRNA-mediated knockdown of ABCG2 sensitized A549 and RPMI 8226 cells to TAK-243 and reduced the IC50 of the drug by 7-and 9-fold, respectively ( Figure 8G-H).

Discussion
TAK-243 is a selective, mechanism-based UBA1 inhibitor with a broad preclinical efficacy in solid and hematologic malignancies and has entered phase 1 clinical trials (2,(10)(11)(12)(13). In this study, we evaluated the regulators of sensitivity to TAK-243 in AML with potential implications in other malignancies using a genome-wide CRISPR/Cas9 knockout screen. From this screen, we identified BEND3 as the top hit whose knockout conferred resistance to TAK-243.
BEND3 is a transcriptional repressor that interacts with chromatin-modifying complexes and induces repressive histone and DNA methylation changes resulting in transcriptional repression (28,29). While BEND3 knockout conferred resistance to TAK-243 in vitro and in vivo, it did not alter basal cell proliferation, consistent with publicly available data from pan-cancer RNAi and CRISPR/Cas9 dropout screens showing BEND3 is not an essential gene with no significant cell depletion upon knockdown or knockout (30).
Our study demonstrated that knockout of BEND3 attenuated TAK-243 effects on poly-and monoubiquitylation of protein substrates and alleviated ER stress. Previous studies have shown that the induction of ER stress by TAK-243 is functionally important for TAK-243-induced cell death (2, 10-12).
Through subsequent experiments, we demonstrated that knockout of BEND3 upregulates the MDR protein BCRP resulting in increased efflux of the drug, reduced binding to UBA1, and consequently reduced UBA1 inhibition. The upregulation of MDR proteins leads to excessive efflux of structurally and mechanistically diverse drugs and is an important mechanism of drug resistance (31). BCRP has been reported to mediate the resistance of many unrelated anticancer drugs including doxorubicin (23), etoposide (32), imatinib (33), methotrexate (34) and mitoxantrone (23, 35), among others (16, 17,31). In keeping with this, our results showed the TAK-243-resistant BEND3 knockout cells were cross-resistant to the known BCRP substrate, mitoxantrone. In AML, high expression of BCRP has been correlated to chemotherapy resistance, poor prognosis and unfavorable therapeutic outcomes (36)(37)(38)(39)(40).
To our knowledge, no prior studies have implicated drug efflux pumps as mechanisms of resistance to TAK-243 or the related adenosine sulfamates including pevonedistat and the SAE inhibitor ML-792 (41). Pevonedistat has been extensively studied in preclinical settings and in over 30 clinical trials; however, the upregulation of MDR proteins has not been reported as a mechanism of resistance to this drug. Instead, on-target missense mutations in UBA3 (the gene encoding the active NAE subunit) have been reported to mediate acquired resistance to pevonedistat in preclinical systems (42)(43)(44). Analogous on-target missense mutations in UBA1 have also been associated with TAK-243 resistance (10,45). Here, we report for the first time that TAK-243 serves as a substrate for BCRP whose upregulation upon BEND3 knockout confers resistance to the drug and potentially related adenosine sulfamates.
TAK-243 has been preclinically evaluated in several malignancies; however, the determinants of sensitivity remain largely unknown (2, 10-13). Hyer et al. investigated whether the sensitivity of TAK-243 was related to UBA1 expression levels or cell line proliferation rates as assessed by doubling time, but found no significant correlation (2). In our study, we demonstrated that TAK-243 sensitivity strongly correlated with BCRP expression levels in cancer cell lines of different origin. We also found that selectively targeting BCRP with chemical inhibitors or shRNAmediated knockdown sensitized cell lines intrinsically resistant to TAK-243 as a result of their high BCRP expression. Modulation of MDR proteins with inhibitors such as zosuquidar and tariquidar has been investigated in clinical trials as a strategy to sensitize certain malignancies to chemotherapy (46,47). In such settings, it should be noted that while BCRP inhibitors may Page 13 of 40 sensitize cancer cells to TAK-243, they may also lead to a narrower therapeutic window by exposing cells, normally protected from xenobiotics by high BCRP expression, to higher concentrations of the drug (48,49). Therefore, this strategy may be used with caution in cases where toxicity can be managed or minimized.
Expression of BCRP and other MDR proteins is regulated by multiple transcriptional and posttranscriptional mechanisms as well as alterations in cellular signaling. In this respect, the promoter methylation status of ABCG2 under basal conditions or in response to chemotherapy was reported to control BCRP expression levels in multiple myeloma cell lines and patient samples (50).
In addition, hormonal alterations have been reported to alter cell signaling and subsequently BCRP expression in breast cancer (54,55). In our study, we demonstrated that BEND3 is important for regulating BCRP expression. Given its role as a transcriptional repressor, we speculate BEND3 regulates BCRP expression by inducing histone and DNA methylation changes at the promoter region of ABCG2. As per our CRISPR/Cas9 screen data, the histone methyltransferase KMT5B (SUV420H1) ranked as a second hit after BEND3. A related enzyme, KMT5C (SUV4-20H2), has been reported to interact with BEND3 in co-immunoprecipitation assays (28). Loss of BEND3 has also been reported to increase histone H3K4 trimethylation and DNA methylation of the ribosomal DNA (rDNA) promoter, silencing rDNA expression (29). Therefore, it is possible that BEND3 may interact with KMT5B to alter the methylation of ABCG2 promoter, resulting in expression changes.
In summary, our study demonstrates TAK-243 is a substrate for the ABC efflux transporter BCRP.
Moreover, TAK-243 sensitivity correlates with BCRP expression levels in cancer cell lines of different origin, suggesting BCRP expression can serve as a predictive biomarker of TAK-243 response and a potential therapeutic target for synergistic combinations with TAK-243. Our study also reports, for the first time, BEND3 as a transcriptional regulator of BCRP expression and lack of BEND3 expression confers resistance to TAK-243 and potentially other BCRP substrates.
BEND3 was also knocked out using a single-plasmid system encoding additional gRNAs. To do so, OCI-AML2 cells were transduced with lentiCRISPR v2 vectors encoding Cas9 and 3 distinct For shRNA-mediated knockdown experiments, ABCG2-targeting shRNAs were obtained from Sigma-Aldrich (product# SHCLNG-NM_004827) and transduced into A549 and RPMI 8226 cells as described above. Sequences of BEND3-targeting gRNAs and ABCG2-targeting shRNAs are listed in Table S4.

Cytotoxicity assays
CellTiter 96 ® AQueous MTS Reagent Powder was purchased from Promega (catalog# G1111), and Annexin V-FITC apoptosis kit from Biovision (catalog# K101-400). The MTS and Annexin For the proliferation assays, DMSO-and TAK-243-treated OCI-AML2 cells were seeded at a density of 10 4 cells/mL and viable trypan blue-negative cells were counted every 2-3 days using a hemocytometer.

Cellular thermal shift assay (CETSA)
We conducted CETSA as previously described (59). In brief, cells were treated with increasing concentrations of TAK-243 for 1h. Cells were then washed with PBS and re-suspended in PBS containing protease inhibitor cocktail (Thermo Fisher Scientific). Cells were heated at 54°C for 3 min in a thermal cycler (SimpliAmp, Applied Biosystems). This temperature corresponds to the maximal thermal shift of UBA1 experimentally derived as previously described (10). Cell lysates were prepared by 4 freeze-thaw cycles in liquid nitrogen and a thermal cycler set at 25°C, respectively with vigorous vortexing in between. Lysates were then centrifuged at 20,000 g for 20 min and supernatants were collected and frozen at -70°C until immunoblotting.

Quantitative reverse transcription polymerase chain reaction (RT-qPCR)
Total RNA was isolated using the RNeasy Plus Mini Kit (QIAGEN), and reverse transcribed into cDNAs using SuperScript IV Reverse Transcriptase (ThermoreFisher, MA, USA). Equal cDNA amounts were then added to a PCR master mix (Power SYBR Green PCR Master mix; Applied Biosystems, CA, USA). RT-qPCR reactions were conducted using an ABI Prism 7900 sequence detection system (Applied Biosystems, CA, USA). The relative gene expression was calculated by the 2 -∆∆ Ct method using 18s rRNA as a control. Primer sequences used in the study are listed in Table S5.

Immunoblotting
To prepare whole cell lysates, cells were washed with PBS (pH=7.4) and lysed with radioimmunoprecipitation assay (RIPA) buffer followed by sonication and centrifugation at 13,000 rpm for 20 min at 4°C. Supernatants were collected and total protein quantified using the Bradford assay (Bio Rad, Hercules, CA). Samples were then denatured by boiling at 95°C for 5 min. For CETSA lysates, samples were not sonicated and were heated at 70°C for 10 min. Proteins were loaded in equal amounts and then fractionated by 10% gels (except otherwise specified) using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to polyvinylidene difluoride (PVDF) membranes and then probed using appropriate primary and secondary antibodies (Table S6).

Determination of intracellular ATP levels
Intracellular ATP levels were measured using a highly sensitive ATP Bioluminescence Assay Kit HS II (Sigma-Aldrich; catalog# 11-699-709-001) as per the manufacturer's guidelines. In brief, control and BEND3 knockout OCI-AML2 cells were washed with PBS and re-suspended in the manufacturer's dilution buffer, and then seeded in triplicate in white 96-well microtiter plates at a plating density of 25,000 cells and a volume of 25 μL per well. Cells were then lysed by adding an equal volume of cell lysis buffer and incubating for 5 min at RT. A 50 μL of the luciferase reagent was then dispensed by automated injection and luminescence was measured after a 1 s delay and integration for 1 s using Hidex Sense Microplate Reader (Hidex Inc.). Relative ATP intensities obtained from the assay to control OCI-AML2 cells.

Measurement of intracellular TAK-243 concentrations
To assess TAK-243 concentrations in the cells, BEND3 knockout and control OCI-AML2 cells were seeded in triplicate in a 12-well plate at a density of 10x10 6 /well and then treated with increasing concentrations of the drug. After 1h of incubation, cells were collected, centrifuged at 3,000 rpm for 5 min, and media removed by aspiration. The cells were then washed twice with drug-free PBS and kept on ice during processing. Cell pellets were then extracted with 50 µL of ice-cold acetonitrile containing internal standard. Cell extracts were centrifuged at 14,000 rpm for 10 min, followed by careful collection of 40 µL of the supernatant in HPLC vials and were stored at -20°C until liquid chromatography-mass spectrometry (LC-MS) analysis. To measure TAK-243 by LC-MS, we used an Acquity UPLC BEH C18 (2.1 X 50 mm, 1.7 µm) column using Acuity UPLC I-Class system. The mobile phase was 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). A gradient starting at 95% solvent A going to 5% in 4.5 min., holding for 0.5 min., going back to 95% in 0.5 min. and equilibrating the column for 1 min. was employed. A Waters Synapt G2S QTof mass spectrometer equipped with an electrospray ionization source was used for mass spectrometric analysis.

Animal studies
To assess effect of BEND3 knockout on TAK-243 response in vivo, control and BEND3 knockout OCI-AML2 cells (1x10 6 tryban-negative viable cells) were injected subcutaneously (sc) into the right and left flanks of male SCID mice (Ontario Cancer Institute, Toronto, Canada), respectively.
After the tumors became palpable, mice were randomly divided into 4 groups (n=5 per group) and treated with vehicle (10 % HPBCD in water) or TAK-243 at doses of 10, 15, and 20 mg/kg sc twice weekly (BIW) for 3 weeks. Mice were weighed and tumor volumes were measured by caliper measurements every 2-3 days using the following equation: [tumor volume (mm 3 ) = tumor length (mm) × width 2 (mm)× 0.5236] as previously described (60). At the end of the experiment, mice were euthanized, and tumors excised for weighing.
To assess the impact of Ko143 on TAK-243 response in vivo, BEND3 knockout OCI-AML2 cells were similarly injected as described above. After the tumors became palpable, mice were randomly divided into 5 groups (n=10 per group) and treated BIW with vehicle, TAK-243 at doses of 10 and 20 mg/kg sc, Ko143 (dissolved in 10% DMSO/10% cremophor in 0.9% NaCl) at a dose of 10 mg/kg intraperitoneally, or a combination of TAK-243 10 mg/kg + Ko143 10 mg/kg where mice were injected with Ko143 2h before TAK-243. The selected dose of Ko143 was the maximally tolerated dose that could be given in combination with TAK-243.

Data analysis
Gene ontology (GO) enrichment analysis was performed for the significantly enriched genes

Datasets
The CRISPR/Cas9 datasets have been deposited in Gene Expression Omnibus (GEO) database with accession number GSE164639 and can be accessed online at: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE164639

Statistics
GraphPad Prism software was used to perform all statistical analyses. To calculate the significance of differences between means, unpaired t-test (2 groups), one-way ANOVA and appropriate multiple comparisons test (˃ 2 groups), and two-way ANOVA (> independent variables) and appropriate multiple comparisons test were used. All experiments were performed in triplicate with at least 3 biological replicates unless otherwise specified.

Study approval
All animal studies were carried out according to the regulations of the Canadian Council on Animal Care and with the approval of the local ethics review board at University Health Network.     , and Ser 139 phosphorylated H2AX (γH2AX) were measured by immunoblotting. GAPDH and β-actin were used as loading controls. C) Control and BEND3 knockout OCI-AML2-Cas9 cells were treated with DMSO or increasing concentrations of TAK-243 at 15-120 nM for 1h followed by heating the intact cells at 54°C. After heating, whole cell lysates were prepared and levels of UBA1 and GAPDH were measured by immunoblotting. D) Control and BEND3 knockout OCI-AML2-Cas9 cells were washed, seeded in equal numbers, and lysed. Luminescence was then measured after adding an ATP-dependent luciferase reagent. Relative luminescence obtained from BEND3 knockout OCI-AML2-Cas9 cells was calculated by normalizing to control cells. Data points represent means ± SEM of 3-5 independent experiments. E) Control and BEND3 knockout OCI-AML2 cells were treated with increasing concentrations of TAK-243 (300-1200 nM) for 1h, washed and pellets were then extracted with acetonitrile. TAK-243 concentrations were then measured by LC-MS. Data points represent means ± SEM of triplicate data from a representative experiment (n=2). ** p ≤ 0.01; **** p ≤ 0.0001 using two-way ANOVA and Sidak's multiple comparisons test.