Introduction
The tumor suppressor gene
Only 40 kb upstream of
Results
ATAD1 and PTEN are co-deleted in many human cancers
Because the
Figure 1.
(A) Schematic of
Figure 1—figure supplement 1.
(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
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
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/.
ATAD1
We conducted genome-wide CRISPR knockout screens to identify genes that are selectively essential in
Many properties of cell lines can affect their particular genetic dependencies, including tissue of origin, driver, and passenger mutations, and even the media in which they grow (Hart et al., 2015; Rossiter et al., 2021). Therefore, we conducted an additional genetic screen in a different cellular context to gain a broader perspective on how
We made ATAD1-proficient and -deficient HGC27 lines by transducing with lentiviral ATAD1-FLAG or empty vector (EV), and subsequently conducted genome-wide CRISPR screens. In this case, genes that are selectively essential for EV cells (ATAD1-deficient) represent putative
We were particularly intrigued by the interaction of
An imbalance of BCL2 family proteins underlies the synthetic lethality of
It has recently become clear that the key function of MARCH5 is to suppress apoptosis (Djajawi et al., 2020; Haschka et al., 2020; Subramanian et al., 2016; Arai et al., 2020). Apoptosis is regulated by OMM-localized BCL2 family proteins and requires the permeabilization of the OMM by BAX/BAK (Kale et al., 2018). Pro-survival proteins such as MCL1 bind to and inhibit BAX/BAK to prevent inappropriate cell death (Greaves et al., 2019). A variety of stressors activate BH3-only proteins (e.g. BIM), which trigger apoptosis by binding and inhibiting pro-survival proteins like MCL1 and in some cases by directly activating BAX/BAK (Letai, 2017). BH3-only proteins serve as sentinels for cellular stress and, upon activation, initiate mitochondrial outer membrane permeabilization (Bhatt et al., 2020; Llambi et al., 2011).
MARCH5 acts as a ‘guardian’ of MCL1 through an incompletely understood mechanism that involves the degradation of the pro-apoptotic BH3-only proteins BIM and/or NOXA (Kale et al., 2018; Letai, 2017; Llambi et al., 2011; Czabotar et al., 2014; Lin et al., 2022). We hypothesized that ATAD1 antagonizes these OMM-localized pro-apoptotic factors in parallel to MARCH5, such that simultaneous loss of both ATAD1 and MARCH5 leads to a lethal accumulation of pro-apoptotic proteins on the OMM. Indeed, BIM structurally resembles known substrates of ATAD1/Msp1 in that it is TA, has an intrinsically disordered region N-terminal to the transmembrane domain, and has basic residues at the extreme C-terminus (Castanzo et al., 2020; Li et al., 2019). Consistent with this hypothesis, the abundance of BIMEL (the predominant isoform of BIM) was increased in
Figure 2.
(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
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
(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
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
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.
Since MARCH5 regulates the BIM/NOXA/MCL1 axis, we assessed abundance of these proteins in the context of single and double deletion of
We tested whether BIM was required for the synthetic lethal interaction of
If MCL1 antagonism underlies
Altogether, our CRISPR screens identified a synthetic lethal interaction between
We next asked if BIM was sufficient to trigger apoptosis preferentially in
ATAD1 directly and specifically extracts BIM from membranes
We hypothesized that BIM might be a direct substrate of the ATAD1 dislocase, which could explain how ATAD1 suppresses BIM-induced apoptosis. Consistent with BIM being an ATAD1 substrate, GFP-BIMEL co-immunoprecipitated with FLAG-tagged ATAD1 in H4 cells (Figure 2—figure supplement 3D). Inversely, ATAD1-FLAG co-immunoprecipitated with endogenous BIM (Figure 2—figure supplement 3E). Thus, reciprocal co-immunoprecipitation argues that ATAD1 and BIM physically interact in cells.
We further tested whether ATAD1 can directly extract BIM from a membrane using an in vitro system with purified components (Wohlever et al., 2017). We used BIML because it is more soluble than BIMEL but shares the key structural features that would likely mediate ATAD1 recognition, including the tail-anchor and juxtamembrane regions (Ley et al., 2005; Liu et al., 2019; Chi et al., 2020). We were unable to purify active, full-length ATAD1. Instead, we swapped the N-terminal transmembrane domain with a His6 tag, which anchored His6-ATAD1 to liposomes doped with phospholipids containing nickel-chelated headgroups (‘Ni Lipos’, Figure 2H). In this extraction assay, TA proteins that are extracted from liposomes by ATAD1 bind soluble GST-tagged chaperones (SGTA and calmodulin), which are purified on a glutathione column and detected by immunoblotting (Figure 2H; Wohlever et al., 2017). We validated the Ni-His anchoring strategy using full-length or truncated yeast Msp1 (the yeast homolog of ATAD1) and positive and negative control substrates (Figure 2—figure supplement 4A–C).
His6-ATAD1 directly and efficiently extracted 3xFLAG-BIML from liposomes in this assay (Figure 2I; compare lanes with elution ‘E’ to input ‘I’; lanes 13–16). As expected, this activity was ATP-dependent (Figure 2I, lanes 17–20) and was abolished when we used the catalytically inactive mutant, ATAD1E193Q (Figure 2I, lanes 1–4). Omission of Ni-chelating lipids from the liposomes (‘Mito Lipos’, which cannot anchor His6-ATAD1; Figure 2I, lanes 5–8) prevented ATAD1 from extracting BIM, demonstrating that ATAD1 requires membrane anchoring for its dislocase activity. Importantly, ATAD1 did not extract yeast Fis1, consistent with previous reports that Fis1 is not an Msp1 substrate (Li et al., 2019; Figure 2I, lanes 9–12).
Many BH3-only proteins share key structural features, including a tail-anchor, so we next asked whether ATAD1 could extract other members of this protein family (Wilfling et al., 2012). In addition to BIM, we tested BIK, PUMA, and NOXA, since these have been proposed to mediate apoptosis triggered by proteotoxic stress. NOXA did not incorporate into proteoliposomes, which precluded it from our assay, and is consistent with a report that it lacks a transmembrane domain (Andreu-Fernández et al., 2016). ATAD1 extracted BIM in an ATP-dependent manner, as expected, but failed to extract BIK or PUMA under the same conditions (Figure 2J and K). While it is not clear how ATAD1 distinguishes between these substrates, BIM differs from BIK and PUMA in that it has a positively charged C-terminus and an intrinsically disordered region N-terminal to the transmembrane domain, which are important features for substrate recognition by Msp1, the yeast homolog of ATAD1 (Figure 2—figure supplement 5; Castanzo et al., 2020; Li et al., 2019). Taken together, we demonstrate that ATAD1 and BIM physically and genetically interact in cells, and that ATAD1 can directly extract BIM – but not other, related, BH3-only proteins – from membranes in a reconstituted system.
These data raise the question of what happens to BIM after it has been extracted by ATAD1. We transduced SW1088 cells, which are a Del(10q23) cell line suitable for imaging, with either EV or ATAD1-FLAG. These cells were then transduced with TetON(GFP-BIMEL∆BH3), in which four point mutations in the BH3 domain of BIM neutralize its pro-apoptotic activity to permit live cell imaging. We assessed GFP-BIMEL∆BH3 localization using live cell confocal microscopy in the presence and absence of ATAD1, using MitoTracker Red to label mitochondria. ATAD1 altered the localization of BIM under basal conditions, generating GFP-positive puncta that did not colocalize with mitochondria (Figure 2—figure supplement 6A). Since BIM is regulated by proteasomal degradation, we additionally treated cells with bortezomib, a proteasome inhibitor. Treatment with bortezomib exacerbated this phenotype and resulted in larger, brighter GFP-positive puncta only in ATAD1 expressing cells (Figure 2—figure supplement 6B and C). We saw the same phenomenon when we genetically labeled mitochondria with mCherry, ruling out the possibility that these GFP-BIM puncta are merely depolarized mitochondria that cannot accumulate Mitotracker dye (Figure 2—figure supplement 7). GFP-positive puncta also did not colocalize with lysosomes or peroxisomes (Figure 2—figure supplements 8 and 9). In addition to localization, BIM is regulated by inhibitory phosphorylation, which we had previously observed to be affected by
Proteasome inhibition is preferentially toxic to ATAD1-deficient cells
We next sought to pharmacologically exploit
Re-expression of
Figure 3.
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
Figure 3—figure supplement 1.
ATAD1 promotes BIMEL phosphorylation in response to proteasome inhibition.
(A) Western blot of SW1088 cells (Del10q23; basally
Figure 3—figure supplement 2.
(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.
We hypothesized that apoptosis was responsible for the sensitivity of
Apoptotic cell death is only one of many mechanisms underlying proteasome inhibitor toxicity (Tsvetkov et al., 2019; Schneider and Bertolotti, 2015; Huang et al., 2020). We next asked whether ATAD1 affects proteasome inhibitor sensitivity via some apoptosis-independent pathway, in which case ATAD1 re-expression and caspase inhibition (which blocks apoptosis) would have an additive effect in mitigating proteasome inhibitor toxicity. Treatment of
Although caspase inhibition completely rescued the bortezomib phenotype of ATAD1-deficient cells, the same was not true for deletion of BIM in PC3 cells, implying that other factors in addition to BIM mediate this phenotype (Figure 3—figure supplement 4A and B). We examined several OMM-localized or TA proteins in PC3 cells with and without bortezomib, and reduced BIM phosphorylation was the only consistent change caused by
ATAD1 loss limits tumor progression, particularly under proteotoxic stress
We next tested whether deficiency of
Figure 4.
(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
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.
(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.
ATAD1 was not necessary for basal tumor growth in PC3 cells, so we assessed the effect of
Finally, we examined clinical outcomes in patients with tumors that were (i)
Discussion
In this work we describe how the collateral deletion of
The effect of ATAD1 on apoptosis likely extends beyond
One limitation of our study is that deletion of BIM does not completely rescue the effects of
Overall, these data show that cells lacking ATAD1 have an increased dependency on the ubiquitin proteasome system. We therefore propose a model in which mitochondrial TA proteins have (at least) two fates: extraction by ATAD1, and ubiquitination and proteasomal degradation (Figure 4G). This model is strongly supported by the finding that ATAD1 becomes important for cellular viability only in the context of a dysfunctional UPS, such as upon
That ATAD1 loss sensitizes to proteasome dysfunction could have therapeutic implications for hundreds of thousands of cancer patients with Del(10q23) tumors, especially considering that drugs targeting the proteasome are already approved for the treatment of cancer. Previous trials of proteasome inhibitors in unselected patients with prostate cancer have shown activity in a subset of patients, consistent with the possibility that this subset was enriched for patients with
Materials and methods
Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Jared Rutter ([email protected]).
Materials availability
All unique/stable reagents generated within this study are available from the lead contact upon request without restriction.
Data and code availability
The CRISPR screening datasets generated during this study are available in the supplemental materials. The human mCRPC survival data are available in supplemental materials. The uncropped source data files for every western blot image are included in the ‘source data’ files.
Experimental model and subject details
Jurkat E6.1 human T-ALL cells (ATCC TIB-152) were grown in RPMI1640 with 10% FBS and 100 U/mL Pen/Strep (Thermo Fisher). Cells were electroporated using Lonza SE Cell Line 4D-Nucleofector X Kit L according to the manufacturer’s specifications and protocol optimized for Jurkat E6.1 cells. Px458-derived plasmids encoding sgRNA targeting
PC3 cells were transduced with lentivirus encoding LentiCRISPRv2-GFP (LCv2G) with non-targeting sgRNA (sgNT, which contains a 15 nt sgRNA sequence, 5’-
H4 and PC3 cells were transduced with retrovirus (pQCXIP transfer plasmid) encoding ATAD1 with C-terminal FLAG and HA tags. Two days after transduction, cells were selected with 1 µg/mL puromycin for 4 days. RPMI7951, HGC27, and SW1088 cells were transduced with lentivirus (pLenti-Blast transfer plasmid) encoding ATAD1-FLAG, and selected with 8 µg/mL blasticidin for 6 days. Cells were grown in media containing the selective antibiotic upon thawing stocks, but no experiments were conducted using media that contained selective antibiotics.
Method details
Immunohistochemistry
ATAD1 (using NeuroMab #75-157 mouse monoclonal antibody)
The ATAD1 immunohistochemical staining was performed on 4 µm thick sections of formalin-fixed, paraffin-embedded tissues. Sections were air-dried and then melted in a 60°C oven for 30 min. Slides were loaded onto the Leica Bond III automated staining instrument (Leica Biosystems, Buffalo Grove, IL) and de-paraffinized with the Bond Dewax solution. The antigen retrieval performed was done with Bond Epitope Retrieval Buffer 2 (ER2, pH 8.0) for 20 min at 95°C. The ATAD1 primary antibody concentration of 1:400 was applied at an incubation time of 30 min at room temperature. Positive signal was visualized using the Bond Polymer Refine Detection kit-DAB, which is a goat anti-mouse/anti-rabbit secondary HRP/polymer detection system, utilizing DAB (3,3’ diaminobenzidine) as the chromogen. Tissue sections were counterstained with hematoxylin for 10 min. The slides were removed from the immunostainer and placed in a dH2O/DAWN mixture. The sections were gently washed in a mixture of deionized water and DAWN solution to remove any unbound reagent. The slides were gently rinsed in deionized water until all of wash mixture was removed. The slides were de-hydrated in graded ethanols, cleared in xylene and then coverslipped.
PTEN (using rabbit anti-human monoclonal antibody): clone 138G6, catalog #9559L, Cell Signaling, Danvers, MA
The PTEN immunohistochemical staining was performed on 4 µm thick sections of formalin-fixed, paraffin-embedded tissues. Sections were air-dried and then melted in a 60°C oven for 30 min. Slides were loaded onto the Ventana BenchMark Ultra automated staining instrument (Ventana Medical Systems, Tucson, AZ), de-paraffinized with the EZ Prep solution. The antigen retrieval performed was done with a citrate buffer (pH 6.0) in a pressure cooker (BioCare Medical, Concord, CA) for 4 min at 100°C then cooled in hot buffer for 30 min. The PTEN primary antibody concentration of 1:50 was applied at an incubation time of 2 hr at room temperature. The Ventana Amplification kit was applied to increase the antibody signal. Positive signal was visualized using the UltraView DAB detection kit, which is a goat anti-mouse/anti-rabbit secondary HRP/polymer detection system, utilizing DAB as the chromogen. Tissue sections were counterstained with hematoxylin for 16 min. The slides were removed from the immunostainer and placed in a dH2O/DAWN mixture. The sections were gently washed in a mixture of deionized water and DAWN solution to remove any unbound reagent and coverslip oil applied by the automated instrument. The slides were gently rinsed in deionized water until all of wash mixture was removed. The slides were de-hydrated in graded ethanols, cleared in xylene, and then coverslipped.
Cell culture
Jurkat cells were cultured in RPMI1640 with 10% FBS (Sigma) and 100 U/mL Pen/Strep. Cells were counted regularly and typically split at a concentration of approximately 1–1.5 × 106 cells/mL, but always before reaching a concentration of 3×106 cells/mL. Jurkat cells transduced with tet-inducible vectors were cultured in RPMI1640 with 10% ‘Tet System Approved FBS’ (Takara) instead of standard FBS. Adherent cell lines were maintained in subconfluent cultures in the following media: RPMI1640 (PC3), DMEM (H4 and SW1088), EMEM (RPMI7951 and HGC27), all with 10% FBS and 100 U/mL Pen/Strep. Cells were periodically tested for mycoplasma contamination using a MycoAlert kit and were negative.
Cloning
All cloning was conducted via traditional PCR/restriction enzyme ‘cut and paste’ methods and verified by Sanger sequencing.
ATAD1 constructs
Retroviral plasmids encoding ATAD1-FLAG/HA and ATAD1E193Q-FLAG/HA were published previously (Chen et al., 2014). Lentiviral vectors were made, using the pLenti-BLAST backbone, by PCR-amplifying the ATAD1 CDS from the above retroviral vectors, but truncating the construct by replacing the HA tag with a stop codon, and ligating between SalI and XbaI sites.
GFP-BIM constructs
The pLVXTet-One vector was purchased from Takara. The coding sequence for EGFP was PCR-amplified and ligated into the MCS using AgeI/BamHI sites. A fusion of EGFP-BIMEL was generated using SOEing PCR and ligated using AgeI/BamHI sites. EGFP-BIMEL was also ligated into pEGFP-C3 for transient transfection.
A gene fragment encoding BIMEL∆BH3 was synthesized by GeneWiz and subcloned into the pLVXTet-One vector described above to make an N-terminal GFP fusion.
CellTiterGlo viability assay
Viability was determined by CellTiterGlo (Promega) according to the manufacturer’s recommendation, with some modifications. Cells were plated at a density of 5×103 cells/well (adherent cell lines) or 2–4 × 104 cells/well (Jurkat) in 100 µL in 96-well plates with white walls and clear bottoms (Corning #3610). CellTiterGlo reagent was reconstituted, diluted 1:4 using sterile PBS, and stored at –20°C in 10 mL aliquots. The outer wells of the 96-well plates were filled with media but not with cells, due to concerns of edge effects. Luminescence was measured using a Biotek Synergy Neo2 microplate reader. Luminescence values were normalized on each plate to untreated cells on the same plate, and expressed as percent. Viability experiments were conducted with multiple biological replicates and repeated with independent experiments.
Incucyte
Jurkat cells stably transduced with TetON(GFP-BIMEL) were seeded at a density of 103 cells/well in clear-bottom, black 96-well plates (Corning) with different concentrations of doxycycline. Cells were imaged with an Incucyte SX5 system and monitored by phase contrast microscopy, with five images taken per well, every 4 hr, for approximately 4 days. Confluence was normalized to t=0 and is expressed as fold-change. Three replicate wells were used for each condition, and three independent experiments were conducted.
Spinfection
Jurkat cells were routinely spinfected in 12-well plates, with 2–5 × 106 cells per mL in 1 mL containing 10 µg/mL polybrene. Centrifugation was conducted at 30°C and 1100 ×
CRISPR-based genetic interaction experiments
Jurkat cells (
Viral particles were made using the LRCherry2.1 transfer plasmid, which encodes U6-sgRNA and mCherry. Cells were spinfected using 10 µg/mL polybrene and viral supernatant. The same number of cells was used for each transduction. One day after spinfection (day 1), cells were resuspended in fresh RPMI media and cultured until day 3, when they were split or seeded into 96-well plates for subsequent analysis by CellTiterGlo (Promega). Viability was calculated by dividing CellTiterGlo values (day 4 or day 7) for a given cell line transduced with sgMARCH5 (5’-
Crystal violet staining
Cells cultured in 12- or 6-well plates were washed twice with PBS then fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min at room temperature. Wells were washed with ddH2O three times, then stained with 0.1% (w/v) crystal violet solution in 20% methanol for 30 min at room temperature. Wells were again washed with ddH2O three times, inverted to dry, and plates were photographed against a white background using an iPhone X. For quantification, glacial acetic acid was added to each well to elute the dye, and plates were incubated at room temperature on a rotary shaker for 30 min. Absorbance was measured at 590 nm using a Biotek Synergy Neo2 microplate reader, and values were normalized to those from untreated cells of the same genotype on each plate.
SDS-PAGE and immunoblotting
Whole cell lysates were prepared by scraping cells directly into RIPA buffer (or adding RIPA buffer to Jurkat cell pellets) supplemented with protease and phosphatase inhibitors (Sigma-Aldrich P8340, Roche Molecular 04906845001), incubated on ice for 30 min with vortexing every 10 min, and then spun at 16,000 ×
Co-immunoprecipitation
H4 cells (expressing EV or ATAD1-FLAG/HA) were transfected with GFP-BIMEL in pEGFP-C3 (GFP-BIM), 10 µg plasmid for 10 cm plate, in the presence of 20 µM zVAD-fmk. Transient expression proceeded overnight (approximately 16 hr). Cells were washed with cold PBS and lysed with HN buffer supplemented with protease inhibitor cocktail and 1% CHAPS (HNC buffer). Magnetic anti-FLAG beads (Sigma-Aldrich) were equilibrated with HNC buffer and then mixed with lysate (after removing 10% volume as input). Bead-lysate mixtures were incubated on a rotator at 4°C for 2–4 hr. Beads were washed 3× with HNC buffer, then heated at 65°C in 30 µL 1× Laemmli buffer for 10 min.
Cell counting
Cells were counted using Bio-Rad TC20 cell counter. At least two samples were taken from a culture any time a count was to be made and the mean was recorded. For proliferation experiments with Jurkat cells (cell counts over time), a hemocytometer was used.
Jurkat CRISPR screen
Jurkat cells (WT parental,
Sequencing reads were aligned to the sgRNA library, given a pseudocount of 1, the counts from each sample were normalized for sequencing depth, and the relative abundance of each sgRNA was calculated as described previously (Wang et al., 2015; Kanarek et al., 2018). sgRNAs with fewer than 50 reads, and genes with fewer than 4 sgRNAs, in the initial reference dataset were omitted from downstream analyses. The log2 fold-change in abundance of each sgRNA between the final and initial reference populations was calculated and used to define a CS for each gene. The CS is the average log2 fold-change in abundance of all sgRNAs targeting a given gene. To achieve a direct comparison of gene essentiality in an
HGC27 CRISPR screen
The HGC27 CRISPR screen was conducted using the Brunello sgRNA library, which was obtained from Addgene and amplified according to the instructions provided by the depositing lab. HGC27 cells transduced with pLentiBLAST-EV or ATAD1-FLAG were selected and propagated as described above. Cells were spinfected with viral sgRNA library, and 1 day later were treated with puromycin (0.5 µg/mL) for 2 days. The following day (day 4), cell pellets were collected for the initial time point (CPD = 0). Mock-transduced cells were included as a control and demonstrated complete death in response to puromycin. Pellets (80e6 cells) were collected at CPDs of 0 and 14. Genomic DNA was harvested as described above. Sequencing libraries were prepared by University of Utah Genomics Core. Sequencing reads were aligned and quantified using BBtools Seal. Counts were normalized for library size and a pseudocount was added to each value. CS per gene were calculated as the mean log2-transformed fold-change in sgRNA abundance between the final time point (CPD 14) and the initial time point post-infection and antibiotic selection (CPD 0). Selectively essential genes were ranked by dCS:
BH3 profiling
BH3 profiling was conducted using a FACS-based method to directly monitor cytochrome C release/retention in cells, as described previously (Ryan and Letai, 2013).
Confocal microscopy
SW1088 cells transduced with EV or ATAD1-FLAG and TetON(GFP-BIMEL∆BH3) were seeded at a density of 3.5×104 cells per dish, in 35 mm Fluorodish plates (World Precision Instruments). Approximately 16 hr later, media was removed and replaced with media containing 100 ng/mL doxycycline. After 24 hr, cells were treated with 20 nM MitoTracker Red for 15 min and then imaged on a Zeiss LSM 880 confocal laser scanning microscope for 45 min in 5% CO2 at 37°C. Imaging on the Zeiss LSM 880 confocal laser scanning microscope was performed with a Plan-Apochromat 63×/1.40 Oil DIC f/ELYR objective. Alternatively, cells were treated with 100 nM bortezomib for 90 min prior to imaging, were stained with MitoTracker Red as described above, and imaged for 30 min. Doxycycline concentrations were maintained throughout the staining and imaging process. All images were Airyscan processed using the Zeiss Zen Desk software.
Microscopy was conducted by an investigator (C Cunningham) who was blinded to genotype (EV vs. ATAD1) and treatment (presence or absence of bortezomib). Experiments were repeated for three independent replicates (both with and without bortezomib) and two additional replicates (without bortezomib only). At least 30 images (representing approximately 40–60 cells) were taken per condition, per replicate. GFP-positive, MitoTracker Red-negative puncta were counted using FIJI with the multi-point tool and were graphed using GraphPad Prism 9 for MacOS.
Co-localization of GFP-BIMEL∆BH3 and mito-mCherry was analyzed by Coloc2 package (FIJI). Regions of interest were defined by selecting entire cells but excluding nuclei using the GFP channel. All images were identically processed by background subtraction and smoothening. Each data point represents the cytoplasmic region of interest for one cell.
Mouse xenografts
SW1088 cells (transduced with EV or ATAD1-FLAG) were grown under normal culture conditions, as described above. Cells (3×106) were mixed 1:1 with Matrigel (Corning) and injected into one flank per mouse. Mice were male NOD/SCID aged 13–15 weeks. Tumor volumes were monitored biweekly using a Biopticon TumorImager. Animal experiments were conducted in accordance with The University of Utah IACUC.
PC3 cells (transduced with LentiCRISPRv2-sgNT or sgATAD1) were grown as described above. Cells (1.8×106) were mixed 1:1 with Matrigel (Corning) and injected into one flank per mouse (12-week-old, male, NRG). Once tumors had established, mice were randomized into bortezomib or vehicle groups and treatment occurred by tail-vein injection twice weekly for 4 weeks. At the conclusion of the experiment, mice were sacrificed within 24 hr of receiving an IV injection. Tumors were harvested and snap-frozen on liquid nitrogen. Tumor fragments were homogenized in RIPA buffer using ceramic beads on a Omni Bead Ruptor 24 Bead Mill Homogenizer (2 cycles of 45 s at 6 m/s, 4°C). Homogenates were centrifuged (16,000 RCF, 10 min, 4°C) and supernatants were recovered, then used for downstream analysis by immunoblot. Data were analyzed by mixed effects model with Tukey’s multiple comparisons. Animal experiments were conducted in accordance with The University of Utah IACUC.
Patient data
Outcome data from patients with mCRPC were downloaded from TCGA via cBioPortal. Patients were stratified into three groups based on status of
Bacterial transformation
For cloning,
E. cloni cells
For cloning, E. cloni10G competent cells were transformed according to the manual provided by the manufacturer (Lucigen) and grown on LB agar plates at 37°C overnight.
BL21-DE3 pRIL cells
For protein expression,
Production of soluble constructs
Δ1-32 Msp1 and Δ1-39ATAD1
The gene encoding the soluble region of
Plasmids encoding soluble Msp1, ATAD1, or their mutants were purified as described previously (Wohlever et al., 2017). Plasmids were transformed into
The protein was further purified by size exclusion chromatography (SEC) (Superdex 200 Increase 10/300 GL, GE Healthcare) in 20 mM Tris pH 7.5, 200 mM KAc, 1 mM DTT. Peak fractions were pooled, concentrated to 5–15 mg/mL in a 30 kDa MWCO Amicon Ultra centrifugal filter (Pierce) and aliquots were flash-frozen in liquid nitrogen and stored at –80°C. Protein concentrations were determined by A280 using a calculated extinction coefficient (Expasy).
GST-SGTA and GST-calmodulin
GST-tagged SGTA was expressed and purified as described previously (Mateja et al., 2015). The original calmodulin plasmid was a kind gift of the Hegde lab (Shao and Hegde, 2011). Calmodulin was cloned into pGEX6p1 plasmid by standard methods. GST-SGTA and GST-calmodulin were expressed as described above for soluble Msp1 constructs. Cells were harvested by centrifugation and resuspended in SGTA Lysis Buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 0.01 mM EDTA, 1 mM DTT, 10% glycerol) supplemented with 0.05 mg/mL lysozyme (Sigma), 1 mM PMSF and 500 U of universal nuclease (Pierce), and lysed by sonication. The supernatant was isolated by centrifugation for 30 min at 4°C at 18,500 ×
Production of membrane proteins
BIM and Fis1
Expression plasmids for SumoTMD were transformed into
Ni-NTA resin was washed with 10 CV of SumoTMD Wash Buffer 1 (50 mM Tris pH 7.5, 500 mM NaCl, 10 mM MgCl2, 10 mM imidazole, 5 mM β-mercaptoethanol (BME), 10% glycerol, 0.1% DDM). Resin was then washed with 10 CV of SumoTMD Wash Buffer 2 (same as Wash Buffer 1 except with 300 mM NaCl and 25 mM imidazole) and 10 CV of SumoTMD Wash Buffer 3 (same as Wash Buffer 1 with 150 mM NaCl and 50 mM imidazole) and then eluted with 3 CV of SumoTMD Elution Buffer (same as Wash Buffer 3 except with 250 mM imidazole).
The protein was further purified by SEC (Superdex 200 Increase 10/300 GL, GE Healthcare) in 50 mM Tris pH 7.5, 150 mM NaCl, 10 mM MgCl2, 5 mM BME, 10% glycerol, 0.1% DDM. Peak fractions were pooled and concentrated in a 30 kDa MWCO spin concentrator (Pierce). Sample was then incubated with 3C Protease at a 1:100 ratio at 4°C overnight to remove the His tag. The following day, the sample was run over Ni-NTA resin equilibrated in Lysis Buffer to remove 3C protease, His tag, and uncleaved proteins. Flow through was collected, aliquoted, and flash-frozen in liquid nitrogen and stored at –80°C. Protein concentrations were determined by A280 using a calculated extinction coefficient (Expasy).
Msp1
Full-length
Reconstitution of Msp1 activity in proteoliposomes
Liposome preparation
Liposomes mimicking the lipid composition of the yeast OMM were prepared as described (Kale et al., 2014). Briefly, a 25 mg lipid film was prepared by mixing chloroform stocks of chicken egg phosphatidyl choline (Avanti 840051C), chicken egg phosphatidyl ethanolamine (Avanti 840021C), bovine liver phosphatidyl inositol (Avanti 840042C), synthetic DOPS (Avanti 840035C), and synthetic TOCL (Avanti 710335C) at a 48:28:10:10:4 molar ratio with 1 mg of DTT. Nickel liposomes were made as described above, except, 1,2-dioleoyl-
Chloroform was evaporated under a gentle steam of nitrogen and then left on a vacuum (<1 mTorr) overnight. Lipid film was resuspended in Liposome Buffer (50 mM HEPES KOH pH 7.5, 15% glycerol, 1 mM DTT) to a final concentration of 20 mg/mL and then subjected to five freeze-thaw cycles with liquid nitrogen. Liposomes were extruded 15 times through a 200 nm filter at 60°C, distributed into single-use aliquots, and flash-frozen in liquid nitrogen.
Proteoliposome preparation
For extraction assays with full-length Msp1, proteoliposomes were prepared by mixing 1 µM Msp1, 1 µM TA protein (SumoTMD), and 2 mg/mL of mitochondrial liposomes in Reconstitution Buffer (50 mM HEPES KOH pH 7.5, 200 mM potassium acetate, 7 mM magnesium acetate, 2 mM DTT, 10% sucrose, 0.01% sodium azide, and 0.1% deoxy big chaps). For extraction assays with soluble Msp1/ATAD1, proteoliposomes were prepared by mixing 1 µM TA protein (SumoTMD), and 2 mg/mL of Nickel liposomes in Reconstitution Buffer. Detergent was removed by adding 25 mg of biobeads and rotating the samples for 16 hr at 4°C. After removing biobeads, unincorporated TA protein was pre-cleared by incubating the reconstituted material with excess (5 µM) GST-SGTA and GST-calmodulin and passing over a glutathione spin column (Pierce #16103); the flow through was collected and used immediately for dislocation assays.
Extraction assay
Extraction assays contained 60 µL of pre-cleared proteoliposomes, 5 µM GST-SGTA, 5 µM calmodulin, and 2 mM ATP and the final volume was adjusted to 200 µL with Extraction Buffer (50 mM HEPES KOH pH 7.5, 200 mM potassium acetate, 7 mM magnesium acetate, 2 mM DTT, 0.1 µM calcium chloride). Samples were incubated at 30°C for 35 min and then loaded onto a glutathione spin column. Columns were washed 4× with Extraction Buffer and eluted with the same buffer supplemented with 20 mM glutathione pH 8.5. Samples were loaded onto stain-free gels, imaged, and then transferred to a PVDF membrane and blotted as indicated in the Key resources table. To account for variability in reconstitution efficiency and western blotting, a new reconstitution and dislocation assay with WT Msp1 was done in parallel with each mutant Msp1. Figures are representative of N>3 separate reconstitutions. Note that the ‘input’ lane is diluted 5× relative to the ‘elution’ lane.
Quantification and statistical analysis
To account for variability in reconstitution efficiency and western blotting, a new reconstitution and dislocation assay with WT Msp1 was done in parallel with each Msp1 mutant. Figures are representative of N>3 separate reconstitutions. Dislocation efficiency was quantified by comparing the amount TA protein in the ‘elution’ lane with the amount of substrate in the ‘input’ lane.
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Abstract
The tumor suppressor gene
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