Collateral deletion of the mitochondrial AAA+ ATPase ATAD1 sensitizes cancer cells to proteasome dysfunction

  1. Jacob M Winter
  2. Heidi L Fresenius
  3. Corey N Cunningham
  4. Peng Wei
  5. Heather R Keys
  6. Jordan Berg
  7. Alex Bott
  8. Tarun Yadav
  9. Jeremy Ryan
  10. Deepika Sirohi
  11. Sheryl R Tripp
  12. Paige Barta
  13. Neeraj Agarwal
  14. Anthony Letai
  15. David M Sabatini
  16. Matthew L Wohlever
  17. Jared Rutter  Is a corresponding author
  1. Department of Biochemistry, University of Utah, United States
  2. Department of Chemistry & Biochemistry, University of Toledo, United States
  3. Whitehead Institute for Biomedical Research, United States
  4. Dana-Farber Cancer Institute, Harvard Medical School, United States
  5. University of Utah and ARUP Laboratories, United States
  6. Huntsman Cancer Institute, University of Utah, United States
  7. Department of Biology, Massachusetts Institute of Technology, United States
  8. Howard Hughes Medical Institute, United States
17 figures, 5 tables and 4 additional files

Figures

Figure 1 with 4 supplements
ATAD1 is co-deleted with PTEN in cancer and its loss confers synthetic lethal vulnerabilities.

(A) Schematic of PTEN and ATAD1 loci. (B) Oncoprint plots from three TCGA studies of cancer. ATAD1 and PTEN alteration frequencies are shown, with blue bars indicating deep deletions. (C) Frequency of ATAD1 deep deletions across various cancer types; data from cBioPortal. (D) CRISPR screen design for wild-type (WT) and ATAD1∆ Jurkat cells. (E) Jurkat CRISPR screen results; each point represents one gene. CRISPR score (CS) values were calculated by taking the average log2 fold-change in relative abundance of all sgRNAs targeting a given gene over 14 population doublings. WT CS values are shown on the y-axis. The CS values per gene for each of the two ATAD1∆ clones were averaged and are plotted on the x-axis. The top 10 genes that were differentially essential between WT and ATAD1∆ are labeled in blue, with MARCH5 labeled in red. (F) CRISPR screen design for HGC27 cells (Chr10q23 deletion, ATAD1-null) comparing gene essentiality in ATAD1 complemented cells or empty vector (EV) (ATAD1-null) control. (G) HGC27 CRISPR screen results; CS values are as described for (E). The x-axis depicts CS for the ATAD1-null condition of EV-transduced cells, and the y-axis depicts CS for the ATAD1-complemented (+ATAD1) condition. Labels are as described for (E).

Figure 1—figure supplement 1
ATAD1 is co-deleted with PTEN as a passenger.

(A) Summary of IHC study on PTEN-null prostate adenocarcinoma (PrAd). (B) Representative histology of tumor samples from patients with PTEN-null tumors. (C) Representative histology of PTEN-positive tumors. (D) Somatic mutations in the PTEN (D) or ATAD1 (E) loci, from TCGA Pan-Cancer Atlas studies (32 studies; n=10,528 samples); note logarithmic scale on y-axis.

Figure 1—figure supplement 2
Characterization of co-deleted genes on Chr10q23.

(A) Schematic of human chr10, with the 10q23 region highlighted with a red box, and CNV of 698 tumors from patients with metastatic castrate-resistant prostate cancer. Blue horizontal bars indicate deletion of the corresponding region of the chromosome, with darker blue indicating deeper deletion (i.e. lower copy number). Red indicates amplification. (B) Plot of deep deletion frequency vs. chromosomal location. Each point corresponds to the genomic coordinates of the start codon for the corresponding gene, as annotated in cBioPortal, and the frequency of deep deletions in the cohort shown in (A). The x-axis is to scale, but only approximately to scale in relation to (A).

Figure 1—figure supplement 3
Supporting data for Jurkat CRISPR screens.

(A) Western blot demonstrating PTEN and ATAD1 status across cell lines. (B) Western blot verification of ATAD1 deficiency of ATAD1∆ cell lines. (C) Proliferation of wild-type (WT) and ATAD1∆ cell lines over 4 days; mean ± SD for n=3 independent experiments. (D) Differential CRISPR scores for the two ATAD1∆ clonal cell lines relative to WT; Pearson coefficient = 0.51, p=2.16 × 10–16.

Figure 1—figure supplement 4
Estimated number of deaths worldwide by cancer type.

Both sexes and all ages are included. Data from GLOBOCAN2020; http://gco.iarc/fr/.

Figure 2 with 9 supplements
ATAD1/MARCH5 synthetic lethality is partially mediated by BIM, which is a novel ATAD1 substrate.

(A) Western blot of Jurkat cell lines, with quantification of BIMEL levels normalized to alpha-tubulin; one sample t and Wilcoxon test. (B) Western blot of whole cell lysates from wild-type (WT) or ATAD1∆ Jurkat cells stably expressing sgNT or sgBIM with Cas9-T2A-GFP. Lysates were mock treated or treated with lambda phosphatase (λ PPase) and analyzed by PhosTag/SDS-PAGE. (C) Western blots of Jurkat cell lines stably expressing Cas9-T2A-GFP with sgNT or sgBIM, harvested 4 days after transduction with additional indicated sgRNAs. (D) Viability of Jurkat cells after deletion of MARCH5, using different genetic backgrounds. Viability at 4 days post-transduction was normalized to that of cells transduced with sgRNA targeting AAVS1. Data analyzed by two-way ANOVA with Tukey’s multiple comparisons. (E) Viability of Jurkat cells stably expressing GFP or Myc-tagged MCL1 after deletion of MARCH5 and normalized as in (D). (F) Viability of Jurkat cells transduced with tetracycline-inducible GFP or GFP-BIMEL fusion; t=48 hr, normalized to viability of cells without doxycycline. (G) Western blot of cell lines as described in (D), treated with doxycycline (Dox) for 24 hr. (H) Schematic of in vitro extraction assay; ‘Ni2+ lipos’ indicates the use of nickel chelating headgroups of lipids in the liposomes; the star symbolizes a GST tag on the soluble chaperones, calmodulin (CaM) and SGTA, which are included to catch extracted TA substrates. (I) Extraction assay using His-ATAD1 and 3xFLAG-BIML (lanes 1–8, 13–20) or the negative control yeast TA protein, 3xFLAG-Fis1p (lanes 9–12); E193Q indicates the use of a catalytically inactive mutant of ATAD1; in samples shown in lanes 5–8, Ni2+ chelating lipids were omitted; in samples shown in lanes 17–20, ATP was omitted; ‘I’=Input, ‘FT’=flow-through, ‘W’=final wash, ‘E’=elution. Eluted fractions represent TA proteins extracted by ATAD1 and bound by GST-tagged chaperones; compare elution ‘E’ to input ‘I’. (J) Extraction assay as described in (H) but comparing different BH3-only proteins, BIM, BIK, and PUMA. (K) Quantification of assays as shown in (I), n=6 independent experiments.

Figure 2—figure supplement 1
BIM phosphorylation in Jurkat cells.

(A) Western blot of lysates separated by SDS-PAGE or Phos-Tag SDS-PAGE. Image representative of two independent experiments. (B) Western blot of anti-BIM immunoprecipitates, probing with phospho-specific antibodies to BIM. Representative of two independent experiments.

Figure 2—figure supplement 2
Synthetic lethality of ATAD1/MARCH5 partially depends on BIM and can be suppressed by MCL1.

(A) Western blot of whole cell lysates from Jurkat cells transduced with sgAAVS1, sgMARCH5, or sgPCNA at 4 days post-transduction. Wild-type (WT) and ATAD1∆ cells here stably express Cas9-T2A-GFP+sgNT, and either GFP or Myc-MCL1. (B) Viability of Jurkat cells stably expressing Cas9-T2A-GFP+sgNT/sgBIM, transduced with mCherry+sgMARCH5/sgPCNA and normalized to the same cells transduced instead with mCherry+sgAAVS1 at day 7, because PCNA deletion is not immediately toxic to cells. N=5 biological replicates. (C) Viability of Jurkat cells as described in (A), measured at day = 7, because PCNA deletion is not immediately toxic to cells. N=5 biological replicates.

Figure 2—figure supplement 3
Functional and physical interaction of ATAD1 and BIM.

(A) Fold-change in confluence measured by Incucyte software, of Jurkat cell lines transduced with tetracycline-inducible GFP-BIMEL, treated with indicated concentrations of doxycycline; mean ± SEM from n=3 biological replicates, representative of three independent experiments. (B) Western blot of H4 cells transduced with indicated constructs. Multiple ATAD1 bands correspond to cleavage of C-terminal epitope tags, which is sometimes seen in cell lines re-expressing ATAD1-FLAG/HA. (C) BH3 profiling data on H4 glioma cells (Del10q23) transduced with the indicated constructs (rows). A133 indicates A1331852. FMO: Fluorescence Minus One FACS control; mean of n=3 biological replicates is shown; **** or ** indicates p<0.001 or p<0.01 by unpaired two-sided t-test. (D) Western blot of co-immunoprecipitation from H4 cells transduced with empty vector (EV) or ATAD1-FLAG/HA and transfected with GFP-BIMEL in the presence of zVAD-FMK. Representative of two independent experiments. (E) Western blot of co-immunoprecipitation from membrane fractions of H4 cells, precipitating endogenous BIM (anti-BIM or IgG as control) and immunoblotting for FLAG-tagged ATAD1. Representative of three independent experiments.

Figure 2—figure supplement 4
Validation of proteoliposome extraction assay.

(A) Soluble His-Msp1 and full-length Msp1 (B) extract BIML from proteoliposomes. (C) The extraction assay recapitulates physiological substrate selectivity of Msp1. Fis1 is extracted when a known Msp1 recognition motif consisting of a hydrophobic patch of residues from Pex15 is inserted N-terminal to the TMD (‘Fis1 - patch’). Sec22 and Sec61b are positive controls demonstrating that Msp1 can recognize ER-native TA proteins.

Figure 2—figure supplement 5
Comparing intrinsically disordered regions of BIML, BIK, and PUMA.

Y-axis is a measure of disorder, with amino acid position on the x-axis; FASTA sequences were obtained from UniProt and analyzed with IUPred2A and ANCHOR2.

Figure 2—figure supplement 6
ATAD1 promotes non-mitochondrial localization of GFP-tagged BIMEL∆BH3.

(A) Confocal microscopy of live cells transduced with empty vector (EV) or ATAD1-FLAG, plus TetON(GFP-BIMEL∆BH3) and treated with 100 ng/mL doxycycline for 24 hr. Bortezomib treatment was used at a concentration of 100 nM for 2 hr. Mitochondria were visualized with MitoTracker Red. Images are representative of at least three independent experiments, and the microscopist was blinded. Arrows indicate GFP+ MTRed puncta. Scale bar = 20 µm. (B) Quantification of GFP+ MTRed puncta in BTZ-treated cells, as shown in (A); n=132 (EV) or 127 cells (ATAD1) compiled from three independent experiments; unpaired, two-sided t-test, **** indicates p<0.001. (C) Additional examples of GFP-positive puncta induced by bortezomib treatment in SW1088 cells expressing ATAD1 and GFP-BIMEL∆BH3. Images were taken by a blinded investigator and are representative of three independent experiments.

Figure 2—figure supplement 7
GFP-BIM puncta do not colocalize with mitochondria labeled with mito-mCherry.

(A) Confocal microscopy of live SW1088 cells transduced with empty vector (EV)/ATAD1, mito-mCherry, and TetON(GFP-BIMEL∆BH3). BIM expression was induced with 100 ng/mL doxycycline for 24 hr, and bortezomib was used for 2 hr prior to imaging at 100 nM. Mitochondria were labeled by expressing mCherry with an N-terminal fusion of the mitochondrial targeting sequence of COX8. N≥30 cells per condition, imaged by a blinded microscopist. (B) Quantification of colocalization between GFP (BIM) and mCherry (mitochondria) in the presence and absence of ATAD1, with and without bortezomib treatment. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test. Colocalization analysis was conducted using Coloc2 in FIJI, and the region of interest was defined as a single cell, excluding the nucleus.

Figure 2—figure supplement 8
GFP-BIM puncta do not colocalize with peroxisomes.

Confocal microscopy of SW1088 cells transduced with empty vector (EV) or ATAD1, monomeric RFP-SKL (peroxisome marker), and TetON(GFP-BIMEL∆BH3). BIM expression was induced with 100 ng/mL doxycycline for 24 hr, and bortezomib was used for 2 hr prior to imaging at 100 nM. Mitochondria were labeled with Mitotracker Deep Red. N≥30 cells per condition, imaged by a blinded microscopist.

Figure 2—figure supplement 9
GFP-BIM puncta do not colocalize with lysotracker blue.

Confocal microscopy of SW1088 cells transduced with empty vector (EV) or ATAD1, and TetON(GFP-BIMEL∆BH3). BIM expression was induced with 100 ng/mL doxycycline for 24 hr, and bortezomib was used for 2 hr prior to imaging at 100 nM. Mitochondria were labeled with Mitotracker Deep Red. Lysosomes were labeled with LysoTracker Blue. N≥30 cells per condition, imaged by a blinded microscopist.

Figure 3 with 4 supplements
ATAD1 protects cells from apoptosis triggered by proteasome inhibition.

(A) Viability of HGC27 cells treated with bortezomib (BTZ) for 16 hr. (B) Viability of SW1088 cells treated with BTZ for 24 hr. (C) Viability of SW1088 cells treated with carfilzomib, a different proteasome inhibitor, for 24 hr. (D) Viability of PC3 cells treated with BTZ for 16 hr. (E) Western blots of HGC27 cells screen treated with 1 µM BTZ for 16 hr. (F) Western blots of SW1088 cells transduced with empty vector (EV) or ATAD1-FLAG and treated with 100 nM BTZ for 16 hr. (G) Western blots of PC3 cells transduced with non-targeting sgRNA or ATAD1 sgRNA and treated with 1 µM BTZ for 16 hr. (H) Viability as measured by normalized crystal violet staining (Abs 590 nm) in PC3 cells transduced with sgNT vs. sgATAD1, treated with BTZ for 16 hr in the presence or absence of 40 µM zVAD-FMK. Data analyzed by two-way ANOVA with Tukey’s multiple comparisons.

Figure 3—figure supplement 1
ATAD1 promotes BIMEL phosphorylation in response to proteasome inhibition.

(A) Western blot of SW1088 cells (Del10q23; basally ATAD1-null) transduced with empty vector (EV) or ATAD1-FLAG and treated with 100 nM bortezomib overnight. Whole cell lysates were analyzed by SDS-PAGE and Phos-Tag SDS-PAGE; representative of two independent experiments. (B) Expression levels of ATAD1 across cell lines in DepMap (log2(TPM+1)). (C) Western blot of PC3 cells transduced with LentiCRISPRv2 sgNT or sgATAD1. (D) Western blot of PC3 cells transduced with non-targeting gRNA (sgNT) or sgATAD1 and treated with 1 µM bortezomib overnight. Whole cell lysates were analyzed by SDS-PAGE and Phos-Tag SDS-PAGE; representative of four independent experiments. (E) Quantification of BIM phosphorylation in PC3 cells treated as in (B), n=4 independent experiments. Data were compared by one-way ANOVA with Tukey’s multiple comparisons test. (F) Enlarged blot from (B) above used to clearly demonstrate phosphorylation status of BIM: ‘o’=unphosphorylated; ‘i’=monophosphorylated; ‘ii’=di-phosphorylated; ‘iii’=poly-phosphorylated.

Figure 3—figure supplement 2
ATAD1 status and proteasome inhibition in cancer cell lines.

(A) Viability of RPMI7951 cells treated with carfilzomib or bortezomib (B) for 16 hr, n=3 biological replicates, two independent experiments. (C) Viability of SW1088 cells treated with marizomib for 24 hr, n=3 biological replicates, two independent experiments. (D) Viability of SW1088 cells treated with bortezomib (3.9 nM) for the indicated durations, n=3 biological replicates. (E) Viability of RPMI7951 cells treated with bortezomib (3.1 nM) for the indicated durations, n=3 biological replicates. (F) Viability of PC3 cells transduced with empty vector (EV), ATAD1WT, or ATAD1E193Q and treated with bortezomib for 24 hr. n=2 biological replicates, 3 independent experiments. Data were analyzed by two-way ANOVA with Tukey’s multiple comparisons test.

Figure 3—figure supplement 3
ATAD1 protects cells from proteasome inhibition by blocking apoptosis, specifically.

(A) Quantification of PARP cleavage from experiments; PC3 cells treated with bortezomib (BTZ) for 16 hr. (B) Western blot of whole cell lysates from RPMI7951 cells transduced with empty vector (EV) or ATAD1-FLAG and treated with BTZ (100 nM) for 8 hr; representative of three independent experiments. (C) Quantification of PARP cleavage from experiments represented by (A). (D) Viability of RPMI7951 cells transduced with EV/ATAD1, treated with DMSO (0.1%) or ZVAD-FMK (20 µM), and varying doses of BTZ for 16 hr. n=3 biological replicates, 2 independent experiments. (E) Representative image of crystal violet staining of PC3 cells transduced with sgNT/sgATAD1, treated with DMSO (0.1%) or ZVAD-FMK (40 µM), and indicated doses of BTZ for 16 hr. n=3 independent experiments. Quantification shown in main figure. (F) Representative image of crystal violet staining of RPMI7951 cells transduced with EV/ATAD1, treated with DMSO (0.1%) or ZVAD-FMK (20 µM), and indicated doses of BTZ for 16 hr. n=4 independent experiments. (G) Quantification of eluted crystal violet from RPMI7951 cells treated as described in (E). Values were normalized to that of DMSO-treated cells from the same plate. Data analyzed by two-way ANOVA with Tukey’s multiple comparisons test. (H) Schematic depicting that proteasome inhibition can decrease cell fitness via caspase-independent and caspase-dependent (apoptosis) pathways. ATAD1, like ZVAD-FMK, only affects the caspase-dependent pathway, an unexpected insight into how ATAD1 protects cells from protein stress. Data were analyzed by two-way ANOVA with Tukey’s multiple comparisons test.

Figure 3—figure supplement 4
Effect of BIM knockout in PC3 cells treated with bortezomib (BTZ).

(A) Viability of PC3 cells transduced with lentiCRISPRv2-GFP+sgNT/sgATAD1 with or without lentiCRISPRv2-Puro sgBIM, treated with BTZ for 16 hr. n=3 biological replicates, 2 independent experiments. (B) Western blot of whole cell lysates from PC3 cells as described in (A) treated with BTZ (1 µM, 16 hr) and probed for BIM. (C) Western blot of whole cell lysates from PC3 cells. Cells were treated with or without 1 µM BTZ for 16 hr. n=2 independent experiments. BIK and PUMA were undetectable by western blot. (D) Quantification of blots shown in (C) normalized to beta-actin.

Figure 4 with 2 supplements
ATAD1 loss sensitizes PC3 xenografts to proteasome inhibition and predicts improved survival in patients with metastatic prostate cancer.

(A) Tumor volume over time for mice with flank xenografts of PC3 cells treated with saline (vehicle) or 1 mg/kg bortezomib (BTZ). (B) Tumor volume over time for mice with flank xenografts of ATAD1-knockout PC3 cells treated with saline (vehicle) or 1 mg/kg BTZ. (C) Western blots of whole cell lysates from tumor samples taken from animals as in (A,B) sacrificed 24 hr after receiving saline/BTZ. (D) Kaplan-Meier curve of overall survival from patients with metastatic, castrate-resistant prostate cancer (mCRPC), stratified based on tumor genotype at the ATAD1 and PTEN loci, with accompanying table below. (E) Survival (months) after initiating chemotherapy or hormone therapy (F) in patients with mCRPC, based on tumor genotype. (G) Graphical summary.

Figure 4—figure supplement 1
Quantification of western blots from PC3 xenograft lysates.

(A) ATAD1 normalized to alpha-tubulin; PC3 xenografts. (B) NRF1/TCF11 normalized to alpha-tubulin; PC3 xenografts. (C) NOXA normalized to nonspecific band with molecular weight of ≈15 kD; PC3 xenografts. (D) BIMEL normalized to alpha-tubulin; PC3 xenografts. (E) MCL1 normalized to alpha-tubulin; PC3 xenografts. (F) BAK normalized to alpha-tubulin; PC3 xenografts. Data were analyzed by two-way ANOVA with Tukey’s multiple comparisons test.

Figure 4—figure supplement 2
ATAD1 re-expression confers tumorigenicity to SW1088 cells.

(A) Tumor volume as a function of time for SW1088 flank xenografts; n=17 mice injected with SW1088 cells transduced with empty vector (EV); n=21 mice injected with SW1088 cells transduced with ATAD1-FLAG. (B) Tumor-free survival over time for the two groups of mice. (C) Crystal violet staining of SW1088 cells transduced with EV/ATAD1 and cultured for 3 days; representative of two independent experiments.

Author response image 1
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MARCH5 vs ATAD1 expression across all DepMap cell lines.
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LEFT: PARP inhibitor sensitivity (Olaparib, AUC) in DepMap cell lines based on BRCA1/2 status.

“In group” consists of all DepMap cell lines harboring BRCA1 and/or BRCA2 mutation. The “out group” is all remaining cell lines. The parameter shown is sensitivity to Olaparib, as measured by area under the curve (AUC) from the CTD2 dataset. Lower values (left direction) on the x-axis indicate lower AUC and increased sensitivity. Based on extensive literature, we would expect BRCA mutant cell lines to be more sensitive than the “out group.” That is not the case (Q = 0.867; Author response table 3).

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Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Cell line (human)HEK293T cellsATCC#CRL-11268, RRID:CVCL_1926
Cell line (human)Jurkat E6.1ATCCTIB-152
Cell line (human)H4ATCCHTB-148
Cell line (human)RPMI-7951ATCCHTB-66
Cell line (human)SW1088ATCCHTB-12
Cell line (human)PC3ATCCCRL-1435
Cell line (human)HGC27HGC2794042256
AntibodyAnti-Flag
Mouse mAB
Sigma-Aldrich#F7425, RRID:AB_4396871:5000
AntibodyAnti-V5
Mouse mAB
Abcam#ab9116, RRID:AB_3070241:5000
AntibodyAnti-GFP
Rabbit mAB
Cell Signaling#2956S1:5000
AntibodyAnti-ATAD1
Mouse mAB
NeuroMab75–1571:1000
AntibodyAnti-PTEN
Rabbit mAB
CST#91881:1000
AntibodyAnti-beta actin
Rabbit mAB
CST#49701:20,000
AntibodyAnti-alpha tubulin
Mouse mAB
CST#38731:20,000
AntibodyAnti-MCL1
Rabbit mAB
CST942961:1000
AntibodyAnti-BCLXL
Rabbit mAB
CST27641:1000
AntibodyAnti-pBIM(Ser69)
Rabbit mAB
CST45851:1000
AntibodyAnti-pBIM(Ser77)
Rabbit mAB
CST124331:1000
AntibodyAnti-pBIM(Thr112)
Rabbit mAB
Thermo FisherPA5-646551:1000
AntibodyAnti-NRF1/TCF11
Rabbit mAB
CST80521:1000
AntibodyAnti-BID
Rabbit mAB
CST20021:1000
AntibodyAnti-MAVS
Rabbit mAB
CST249301:1000
AntibodyAnti-MFF
Rabbit mAB
AbcamAB1290751:1000
AntibodyAnti-FIS1
Rabbit mAB
AbcamAB1568561:1000
AntibodyAnti-BIM
Rabbit mAB
CST#29331:1000
AntibodyAnti-BAK
Rabbit mAB
CST#121051:1000
AntibodyAnti-ubiquitin
Rabbit mAB
CST#431241:1000
AntibodyAnti-ubiquitin
Mouse mAB
Abcam#ab72541:1000
AntibodyAnti-GAPDH
Mouse mAB
CST#97166S1:5000
AntibodyAnti-PARP
Rabbit mAB
CST#95321:1000
AntibodyAnti-Caspase 3
Rabbit mAB
CST#14220S1:1000
AntibodyGoat Anti-Mouse IgG (H&L) Antibody Dylight 800 ConjugatedRockland#610-145-002-0.51:10,000
AntibodyDonkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 680Invitrogen#A100431:10,000
AntibodyGoat Anti-Mouse IgG (H+L) Antibody, Alexa Fluor 680 ConjugatedInvitrogen#A21057, RRID:AB_1414361:10,000
AntibodyDonkey Anti-Rabbit IgG (H&L) Antibody Dylight 800 ConjugatedRockland#611-145-002-0.5, AB_111835421:10,000
AntibodyGoat anti-Mouse IgG (H+L), Superclonal Recombinant Secondary Antibody, HRPThermo Fisher#A28177, RRID:AB_25361631:10,000
AntibodyGoat anti-Rabbit IgG (H+L), HRPProteinTechRRID:AB_27225641:10,000
Recombinant DNA reagentpsPAX2Addgene
Recombinant DNA reagentpMD2.GAddgene
Recombinant DNA reagentpSpCas9(BB)–2A-GFP (PX458)Addgene
Recombinant DNA reagentLentiCRISPRv2GFP-sgNTAddgene
Recombinant DNA reagentPx458_sgATAD1_1This studysgRNA targeting ATAD1 in Px458 vector; see Materials and methods
Recombinant DNA reagentPx458_sgATAD1-2This studysgRNA targeting ATAD1 in Px458; see Materials and methods
Recombinant DNA reagentLRCherry2.1-sgMARCH5_10This studysgRNA targeting MARCH5 in LRCherry2.1; see Materials and methods
Recombinant DNA reagentLRCherry2.1-sgPCNAAddgene
Recombinant DNA reagentLRCherry2.1-sgAAVS1Addgene
Recombinant DNA reagentpLenti-BlastAddgene
Recombinant DNA reagentpQCXIPClontech
Recombinant DNA reagentpQCXIP-ATAD1-FLAG/HAChen et al., 2014
Recombinant DNA reagentpQCXIP-ATAD1^E193Q-FLAG/HAChen et al., 2014
Recombinant DNA reagentpQCXIP-mito-mCherryThis studyCox8-MTS upstream of mCherry, in PQCXIP
Recombinant DNA reagentpQCXIP-mRFP-SKLThis studySKL amino acids fused to C-terminus of mRFP in pQCXIP
Recombinant DNA reagentpLenti-Blast-ATAD1-FLAGThis studyATAD1 CDS with C-terminal FLAG tag cloned into pLenti-Blast
Recombinant DNA reagentpLVX-TetOne-PuroTakara
Recombinant DNA reagentpLVX-TetOne-Puro-GFPThis studyGFP CDS cloned into pLVX-TetOne-Puro
Recombinant DNA reagentpLVX-TetOne-Puro-GFP-BIMELThis studyGFP-BIMEL fusion cloned into pLVX-TetOne-Puro
Recombinant DNA reagentpLVX-TetOne-Puro-GFP-BIMEL∆BH3This studyGFP-BIMEL fusion with 3 amino acids in BH3 domain mutated, cloned into pLVX-TetOne-Puro
Recombinant DNA reagentpLenti-GFP-PuroAddgene
Recombinant DNA reagentpLenti-Myc-MCL1This studyMCL1 with N-terminal Myc tag swapped with GFP in pLenti-GFP-Puro
Recombinant DNA reagentBrunello CRISPR knockout sgRNA libraryAddgene
Recombinant DNA reagentpET21: Msp1-His (S. cerevisiae)Wohlever et al., 2017
Recombinant DNA reagentpET28: His-TEV-∆1–32-Msp1 (S. cerevisiae)Wohlever et al., 2017
Recombinant DNA reagentpET28: His-TEV-∆1-39-ATAD1 (R. norvegicus)This studyWohlever Lab; see Materials and methods
Recombinant DNA reagentpET28: His-Flag-Sumo-Sec22 TMD-OpsinWang et al., 2010
Recombinant DNA reagentpET28: His-Flag-Sumo-BimL-OpsinThis studyWohlever Lab; see Materials and methods
Recombinant DNA reagentpET28: His-Flag-Sumo-Fis1 TMD-OpsinThis studyWohlever Lab; see Materials and methods
Recombinant DNA reagentpET28: His-Flag-Sumo-Bik-OpsinThis studyWohlever Lab; see Materials and methods
Recombinant DNA reagentpET28: His-Flag-Sumo-Puma-OpsinThis studyWohlever Lab; see Materials and methods
Recombinant DNA reagentpGEX6p1: GST-SGTAMateja et al., 2015
Recombinant DNA reagentpGEX6p1: GST-CalmodulinShao and Hegde, 2011
Commercial assay or kitPierce BCAThermo23225
Commercial assay or kitCellTiterGlo Luminescent Viability AssayPromegaG7572
Chemical compound, drugDDMGoldBioDDM25
Chemical compound, drugLipofectamine 3000Thermo FisherL3000008
Chemical compound, drugLipofectamine RNAiMAXInvitrogen13778150
Chemical compound, drugMitoTracker Red CMXRosInvitrogenM7512
Chemical compound, drugLysoTracker Blue DND-22InvitrogenL7525
Chemical compound, drugMitoTracker Deep Red FMInvitrogenM22426
Chemical compound, drugCrystal VioletSigmaC0775
Chemical compound, drugRIPA BufferCell Signaling9806
Chemical compound, drugSE Cell Line 4D-Nucleofector X Kit LLonzaV4XC-1012
Chemical compound, drugAdenosine TriphosphateAcros OrganicsAC10280-0100
Chemical compound, drugBovine liver phosphatidyl inositolAvanti840042C-10mg
Chemical compound, drugSynthetic DOPSAvanti840035C-10mg
Chemical compound, drugSynthetic DOGS-Ni-NTAAvanti790404C-5mg
Chemical compound, drugChicken egg phosphatidyl ethanolamineAvanti840021C-25mg
Chemical compound, drugChicken egg phosphatidyl cholineAvanti840051C-200mg
Chemical compound, drugSynthetic TOCLAvanti710335C-25mg
Chemical compound, drugBortezomibEMD Millipore5043140001
Chemical compound, drugCarfilzomibSelleck ChemS2853
Chemical compound, drugMarizomibSelleck ChemS7504
Chemical compound, drugzVAD-FMKSigma-AldrichV116
Software, algorithmmetapMichael Dewey, 2020
Software, algorithmRR Core Team
Software, algorithmGgplot2Wickham, 2009
OtherSuperSep PhosTag precast gels, 12.5% acWako/Fujifilm195-17991
Author response table 1
GeneDeep DeletionShallow DeletionAmplificationGainNo CNAs
PTEN81553233
ATAD13453249
Author response table 2
Jurkat ScreenHGC27 Screen
Tissue of originBlood; T-ALLGastric cancer
Growth substrateSuspensionAdherent
MediaRPMI1640EMEM
sgRNA librarySabatini/Lander (10 sgRNA/gene)Brunello (4 sgRNA/gene)
LocationWhitehead InstituteUniversity of Utah
Year20182021
CNA (Del)CDKN2A/B, MSH2, MSH6
CNA (Amp)TERTMYC, AKT1
MutationsBAX,PIK3CA, APC, JAK1
Author response table 3
Olaparib sensitivity (PRISM AUC)
In GroupOut GroupEffect sizeP-
Value
Q-
Value
Number of cell lines
BRCA1 and/or BRCA2 mutantAll other cell lines-0.0007690.2030.826338
Olaparib sensitivity (CTD2 AUC)
In GroupOut GroupEffect sizeP-
Value
Q-
Value
Number of cell lines
BRCA1 and/or BRCA2 mutantAll other cell lines0.005370.9050.867772
Author response table 4
DepMap: Expression of Gene 1 vs.Dependency on Gene 2.
PaperGene 1 (Deletedgene)Gene 2 (Syntheticlethal partner)PearsonSpearmanP-Value
Dey et al. Nature 2017ME2ME3-0.01107.46E-01
Zhao et al., Nature 2017PTENCHD10.0490.0391.36E-01
Fan et al., eLife
2017FXR2FXR10.022-0.0115.07E-01
Kryukov et al.,
Science 2016; Mavrakis et al.,
Science 2016MTAPPRMT50.1700.1621.52E-7
Muller et al., Nature
2012ENO2ENO10.2790.2962.27E-18
BRCA1PARP10.1510.1593.14E-06
BRCA2PARP10.0960.0903.12E-03
Winter et al.ATAD1MARCH50.0860.1168.14E-03

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  1. Jacob M Winter
  2. Heidi L Fresenius
  3. Corey N Cunningham
  4. Peng Wei
  5. Heather R Keys
  6. Jordan Berg
  7. Alex Bott
  8. Tarun Yadav
  9. Jeremy Ryan
  10. Deepika Sirohi
  11. Sheryl R Tripp
  12. Paige Barta
  13. Neeraj Agarwal
  14. Anthony Letai
  15. David M Sabatini
  16. Matthew L Wohlever
  17. Jared Rutter
(2022)
Collateral deletion of the mitochondrial AAA+ ATPase ATAD1 sensitizes cancer cells to proteasome dysfunction
eLife 11:e82860.
https://doi.org/10.7554/eLife.82860