Targeting Mitochondrial Structure Sensitizes Acute Myeloid Leukemia to Venetoclax Treatment

Published OnlineFirst May 2, 2019; DOI: 10.1158/2159-8290.CD-19-0117

RESEARCH ARTICLE

Xufeng Chen 1 ,2 , Christina Glytsou 1 ,2 , Hua Zhou 3 , Sonali Narang 2 , Denis E. Reyna 4 ,5 ,6 , Andrea Lopez 4 ,5 ,6 , Theodore Sakellaropoulos 1 ,2 , Yixiao Gong 1 ,2 ,7 , Andreas Kloetgen 1 ,2 , Yoon Sing Yap 1 ,2 , Eric Wang 1 ,2 , Evripidis Gavathiotis 4 ,5 ,6 , Aristotelis Tsirigos 1 ,2 ,3 , Raoul Tibes 2 , and Iannis Aifantis 1 ,2

ABSTRACT The BCL2 family plays important roles in acute myeloid leukemia (AML). Veneto- clax, a selective BCL2 inhibitor, has received FDA approval for the treatment of AML. However, drug resistance ensues after prolonged treatment, highlighting the need for a greater understanding of the underlying mechanisms. Using a genome-wide CRISPR/Cas9 screen in human AML, we identifi ed genes whose inactivation sensitizes AML blasts to venetoclax. Genes involved in mitochondrial organization and function were signifi cantly depleted throughout our screen, including the mitochondrial chaperonin CLPB. We demonstrated that CLPB is upregulated in human AML, it is further induced upon acquisition of venetoclax resistance, and its ablation sensitizes AML to venetoclax. Mechanistically, CLPB maintains the mitochondrial cristae structure via its interaction with the cristae-shaping protein OPA1, whereas its loss promotes apoptosis by inducing cristae remodeling and mitochondrial stress responses. Overall, our data suggest that targeting mitochondrial architecture may provide a promising approach to circumvent venetoclax resistance.

SIGNIFICANCE: A genome-wide CRISPR/Cas9 screen reveals genes involved in mitochondrial biological processes participate in the acquisition of venetoclax resistance. Loss of the mitochondrial protein CLPB leads to structural and functional defects of mitochondria, hence sensitizing AML cells to apoptosis. Targeting CLPB synergizes with venetoclax and the venetoclax/azacitidine combination in AML in a p53-independent manner.

See related commentary by Savona and Rathmell, p. 831

1Department of Pathology, NYU Langone Health and NYU School of Medi- X. Chen and C. Glytsou contributed equally to this work. 2 cine, New York, New York. Laura and Isaac Perlmutter Cancer Center, Corresponding Authors: Iannis Aifantis, Department of Pathology, NYU NYU Langone Health and NYU School of Medicine, New York, New York. School of Medicine, 522 First Avenue, SRB 1304, New York, NY 10016. 3 Applied Bioinformatics Laboratories, NYU School of Medicine, New York, Phone: 212-263-9898; Fax: 212-263-8211; E-mail: Ioannis.Aifantis@ 4 New York. Department of Biochemistry, Albert Einstein College of Medi- nyumc.org ; and Raoul Tibes, Perlmutter Cancer Center, NYU Langone 5 cine, Bronx, New York. Department of Medicine, Albert Einstein College of Health, 522 First Avenue, SRB 1311, New York, NY 10016; E-mail: Raoul. 6 Medicine, Bronx, New York. Albert Einstein Cancer Center, Albert Einstein [email protected] College of Medicine, Bronx, New York. 7 Marie-Josée and Henry R. Kravis Center for Molecular Oncology, Memorial Sloan Kettering Cancer Center, Cancer Discov 2019;9:890–909 New York, New York. doi: 10.1158/2159-8290.CD-19-0117 Note: Supplementary data for this article are available at Cancer Discovery ©2019 American Association for Cancer Research. Online (http://cancerdiscovery.aacrjournals.org/).

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INTRODUCTION (4). Following a death signal, the proapoptotic factors are released from BCL2 and induce the oligomerization of BAX Acute myeloid leukemia (AML) is a hematopoietic neo- and BAK on the outer mitochondrial membrane (OMM; ref. plasm characterized by the proliferation and accumulation 5). This is a crucial step for the release of cytochrome c from of aberrant immature myeloid progenitor cells. AML is asso- mitochondria to the cytosol where, together with dATP, ciated with poor clinical outcome and high mortality, with APAF1, and caspase 9, it forms the apoptosome, a complex an overall five-year survival rate of 15% to 30%. For patients that subsequently activates the executioner caspases and thus with AML, standard therapies often fail to achieve complete apoptosis (6, 7). remission, disease relapse is often fatal, and salvage and In cancer cells, enhanced BCL2 expression supports cell bone marrow transplantation are applicable to only a few survival, as it leads to the suppression of mitochondrial- select patients with AML, highlighting the need for novel mediated apoptosis. Inhibiting BCL2 via a selective BH3 targeted treatments. A number of such emerging treatments mimetic such as venetoclax has proven to be an efficient have targeted essential “hallmarks” of cancer, including the strategy to promote caspase-dependent cell death in AML regulation of cancer cell survival by members of the BCL2 (8). Venetoclax is an orally bioavailable drug that is approved protein family. Among these members, B-cell lymphoma 2 for chronic lymphocytic leukemia and other hematologic (BCL2) is found upregulated in AML cells (1) and, specifically malignancies. Venetoclax recently received FDA approval for in leukemic stem cells (LSC; ref. 2). BCL2 overexpression is a the treatment of newly diagnosed elderly patients with AML poor-risk factor in AML and is associated with poor response in combination with hypomethylating agents (azacitidine to standard cytotoxic therapy (1, 3). or decitabine; ref. 9), as proposed by our group (10, 11), or Mechanistically, BCL2 is the founding member of the with low-dose cytarabine. However, approximately 30% of BCL2 protein family, and prevents programmed cell death patients do not respond up front, and many patients with by binding to proapoptotic members of the same family AML still develop resistance while on treatment (12). This

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RESEARCH ARTICLE Chen et al. highlights the need for a greater mechanistic understanding top sgRNAs for each gene and monitoring their ability to of venetoclax resistance, both in combination and as mono- confer resistance to or synergize with venetoclax in AML cells therapy. To that end, multiple combinational therapies for performing a competition-based assay (Supplementary Fig. venetoclax have been proposed, including CDK (CDK9) and S1C). Our results confirmed that all the sgRNAs targeting the MCL1 inhibition, as we and others have suggested (13–15), positively selected genes demonstrated significant resistance with several of these combinations having entered clinical to venetoclax, whereas the sgRNAs targeting the negatively trials (16). selected genes strongly sensitized MOLM-13 cells to the To shed light on the mechanisms of resistance to vene- drug treatment (Fig. 1E; Supplementary Fig. S1D). Likewise, toclax, and to propose novel venetoclax treatment com- a significant increase of IC50 in KASUMI-1 and MOLM-13 binations, we performed a genome-wide CRISPR/Cas9 cells upon deletion of BAX and PMAIP1 validated the gain loss-of-function screen in human AML cells in the pres- of resistance of venetoclax in BAX- and PMAIP1-deficient ence or absence of venetoclax and identified CLPB as one AML cells. Notably, TP53 sgRNAs increased the venetoclax of the top candidates whose ablation sensitizes AML cells IC50 in p53 wild-type MOLM-13 but not in TP53-mutant to the drug treatment. We show that the mitochondrial KASUMI-1, confirming the specificity of the guides used in protein CLPB is upregulated in patients with AML and this study (Supplementary Fig. S1E and S1F). Together, our protects AML cells against caspase-dependent apoptosis genome-wide CRISPR/Cas9 loss-of-function screen success- and mitochondrial dysfunction. In particular, we demon- fully revealed potential modes of resistance to venetoclax as strate that CLPB is essential for sustaining the correct well as synthetic lethal partners in AML. mitochondrial cristae morphology by its direct interaction Next, we aimed to identify key pathways and biological with OPA1, the master regulator of mitochondrial dynam- processes that are enriched in our screen. We performed Gene ics. Moreover, CLPB deficiency leads to mitochondrial dys- Ontology (GO) analysis focusing first on positively selected function, which causes ATF4-mediated mitochondrial stress genes that were at least 8-fold [log fold change (LFC) > 3] responses and alterations at the cell transcriptome and enriched. The majority of these genes significantly clustered metabolome. Therefore, depleting CLPB sensitizes AML into key biological processes, categories that regulate the cells to venetoclax-induced programmed cell death in vitro intrinsic apoptotic signaling pathway, including cytochrome and in vivo. Our work suggests that targeting mitochondrial c release and mitochondrial outer membrane permeabiliza- structure could be a promising strategy to overcome veneto- tion (Supplementary Fig. S1G). STRING protein–protein clax resistance in patients with AML. interaction network analysis was then performed using the positively selected genes at both days 8 and 16 of venetoclax RESULTS treatment (Supplementary Fig. S1H). One of the major clusters from this network was the p53-mediated apoptotic sign- CRISPR Screen Identifies Synthetic Lethal aling pathway in mitochondria (Supplementary Fig. S1I). We Vulnerabilities for Venetoclax in AML were particularly interested in the negatively selected genes, To systematically identify key factors that regulate veneto- as they are potential therapeutic targets for circumventing clax resistance in human AML, we performed a genome-wide venetoclax resistance. GO analysis of the “sensitizer” genes CRISPR/Cas9 loss-of-function screen (17). We transduced revealed strong enrichment for mitochondrial processes, MOLM-13 AML cells with the Brunello single-guide RNA such as mitochondrial transport, organization, and oxida- (sgRNA) library (18), cultured them in the presence of vene- tive phosphorylation (Supplementary Fig. S1J). Moreover, toclax or DMSO for 16 days, and sequenced the distribution when we combined the two time points of our screen, of sgRNAs at days 8 and 16 after drug treatment (Fig. 1A; we successfully identified 353 common negatively selected Supplementary Fig. S1A). Our strategy ensured that genes genes (Fig. 1F). Among these, 55 were genes that encode that confer resistance to or synergize with venetoclax would proteins functioning in mitochondria, highlighting the rel- be positively or negatively selected, respectively, following 8 evance of mitochondrial processes in the sensitization of and 16 days of drug treatment compared with DMSO treat- AML cells to venetoclax. Consistently, mapping this 353- ment (Fig. 1B–D; Supplementary Fig. S1B and Supplemen- gene set on the STRING database generated a highly con- tary Table S1). BAX and PMAIP1 (NOXA) were among the nected network consisting mainly of genes that regulate positively selected genes (genes whose loss confers resistance), mitochondrial functions, as well as chromatin remodelers, a finding consistent with the mechanism of action of the ubiquitin ligases, signal transducers, and components of drug (14, 19, 20). Interestingly, the tumor suppressor p53 mRNA processing (Fig. 1G). Therefore, our genome-wide (TP53) was also positively selected in the screen, in agreement CRISPR/Cas9 loss-of-function screen identified mitochon- with recent studies (15) as well as the accompanying CRISPR drial processes as key potential synthetic lethal vulnerabili- screen performed by Nechiporuk and colleagues in this issue. ties for venetoclax in AML. MDM2, encoding an E3 ubiquitin ligase that targets p53 for degradation (15, 21) and MCL1, a primary mode of resistance Venetoclax Treatment Leads to to venetoclax (13, 22), were among the negatively selected Aberrant Mitochondrial Structure genes (genes whose loss synergizes with venetoclax) at both and Depolarization in AML days 8 and 16 of drug treatment (Fig. 1B–D; Supplementary Given that venetoclax action converges on mitochondria Fig. S1B). We further validated a number of positively and which actively participate in programmed cell death, our negatively selected genes (GID8, BAX, TP53, PMAIP1, SLC25A1, screen results intrigued us to further investigate how BCL2 RTN4IP1, and CLPB) throughout the screen by selecting the inhibition affects mitochondrial structure and function

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Targeting Mitochondria Sensitizes AML to Venetoclax RESEARCH ARTICLE

A B Negatively selected Positively selected DMSO 5 (synergy) (resistance)

MOLM-13-Cas9-dBSD TMEM14E 4 BAX Puromycin DMSO CCNC selection

3 CLC CLPB

Drug 2 DCAF15 PMAIP1 Venetoclax Harvest samples at Deep sequencing RTN4IP1 ZNF670 − lg ( P -value) MDM2 Genome-wide sgRNA library days 8 and 16 1 TP53 • Genes targeted: 19,114 after treatment MCL1 • gRNA no.: 76,441 • Control gRNA no.: 1,000 0 −4 −3 −2 −10 1234 Delta CRISPR score

CD Delta CRISPR score Delta CRISPR score Negative control Negative control

−10 −50 510 Positively selected −10 −50 510 Positively selected

BAX (resistance) BAX (resistance) PMAIP1 PMAIP1 TP53 TP53 Negatively selected MCL1 Negatively selected MCL1 Known regulators of Known regulators of Known venetoclax resistance venetoclax resistance venetoclax (synergy) MDM2 (synergy) MDM2

RTN4IP1 RTN4IP1

CLPB CLPB Day 8 after drug treatment Day Day 16 after drug treatment Day SLC25A1 SLC25A1 selected genes selected genes Other negatively Other negatively

E G Positively selected genes Negatively selected genes 4 Post drug * treatment Day 0 3 Day 2 * *** *** Day 4 2 * * Day 6 *** *** ** ** * *** *** 1 Normalized enrichment 0

sgBAXsg #1BAX #2 sgGID8sg #1GID8 #2 sgTP53 #1 sgCLPBsg #1CLPB #2 sgPMAIP1sgPMAIP1 #1 #2 sgSLC25A1sgSLC25A1 #1sgRTN4IP1 #2sgRTN4IP1 #1 #2

F Day 16 Day 8 1,121 genes 678 genes (LFC < −3) (LFC < −1)

768 353 325 Mitochondrial functions mRNA processing Protein polyubiquitination

Intracellular signal transduction Chromatin remodeling Others

Figure 1. Genome-wide CRISPR screen identifies genes controlling mitochondrial physiology as synthetic lethal with venetoclax treatment in AML. A, Schematic outline of the viability-based, genome-wide CRISPR/Cas9 loss-of-function screen. B, Volcano plot showing both positively and negatively selected genes in the CRISPR screen at day 8 after drug treatment. A number of positively and negatively selected genes are shown in red and green, respectively. Known regulators of venetoclax resistance are shown in orange (positively selected) and blue (negatively selected), respectively. C and D, Frequency histograms of the delta CRISPR score of the negative control guides (top), and selected genes at days 8 (C) and 16 (D) after drug treatment. E, Validation of selected genes in the CRISPR screen using a competition-based survival assay in MOLM-13 cells. The normalized enrichment scores were calculated as shown in Supplementary Fig. S1C. Data, mean ± SEM (n = 4 for each sgRNA). F, Venn diagram of the negatively selected genes (“sensitizers”) in the CRISPR screen at day 8 (log fold change < −1) and day 16 (log fold change < −3) after drug treatment. G, STRING protein–protein interaction network of the 353 common negatively selected genes as defined inF . The minimum required interaction score was set to 0.5, and the disconnected dots were removed. k-means clustering was applied with the number of clusters set to 6. Data with statistical significance are as indicated: *,P < 0.05; **, P < 0.01; ***, P < 0.001.

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RESEARCH ARTICLE Chen et al. in AML. Mitochondria change their shape early during Venetoclax Resistance Is Coupled to OPA1 the process of apoptosis; the individual inner membrane Overexpression and Tighter Mitochondrial Cristae lamellae, named cristae, fuse, and the opposing faces of To further investigate the mechanisms of venetoclax resist- the cristae membrane (cristae junctions) open, allowing the ance, we generated four human AML clones highly resistant redistribution of cytochrome c from the cristae lumen to the to the drug, by growing the parental MOLM-13 and MV4-11 intermembrane space (23, 24). To determine the mitochon- cells in increasing doses of the drug for over 8 weeks. IC50 drial structure alterations in AML, we performed electron analysis verified more than 100-fold increase of venetoclax microscopy–based morphometric analysis of AML cells concentration required for the 50% cell death in the veneto- (THP-1 and MOLM-13) treated with venetoclax. Indeed, clax-resistant (VR) cell lines in respect to the original clones mitochondria exhibit abnormal ultrastructure after veneto- (Par; Fig. 2G). Driven by the role of BCL2 inhibition in mito- clax treatment, characterized by a lower number of cristae chondrial architecture, we aimed to closely examine the orga- and increase of the cristae lumen width (Fig. 2A–C; Supple- nelle’s structure in the venetoclax-resistant AML cells. We mentary Fig. S2A–S2C). The normal mitochondrial struc- discovered that venetoclax-resistant AML cells exhibit tighter ture is crucial for the maintenance of the mitochondrial cristae (approximately 14–15 nm in diameter) and a higher membrane potential, which is the driving force behind ATP number of cristae per mitochondrion than the parental sensi- production and mitochondrial homeostasis. Early during tive clones (Fig. 2H and I; Supplementary Fig. S2G–S2I). This apoptosis, the mitochondrial membrane potential collapses, phenotype could be explained by the significant induction leading to the complete cytochrome c release from mito- of OPA1 protein expression in the AML venetoclax-resistant chondria to the cytosol (25). We therefore predicted that cell lines (Fig. 2J; Supplementary Fig. S2J). Notably, this is venetoclax, as an inducer of apoptosis, causes loss of mito- not simply the result of a general increase of mitochondrial chondrial membrane potential in leukemic cells. Indeed, biogenesis, as the mitochondrial protein TOM20 does not staining of AML cells with the cell-permeant, cationic fluo- display similar expression changes (Supplementary Fig. S2K). rescent dye tetramethylrhodamine methyl-ester (TMRM) These data suggested that mitochondria change their shape revealed depolarization of mitochondria after treatment in venetoclax-resistant AML cells, most likely to antagonize with the BCL2 inhibitor (Fig. 2D; Supplementary Fig. S2D). the cell-intrinsic apoptotic signaling cascade initiated by To further investigate how venetoclax treatment may be BCL2 inhibition. causing widening of the cristae, we turned to the mitochon- Because MCL1 upregulation has been previously impli- drial protein Optic Atrophy 1 (OPA1), which functions as a cated in the development of venetoclax resistance (12), we molecular staple at cristae junctions to prevent cytochrome aimed to examine the protein levels of the major BCL2 fam- c mobilization and release (26). In physiologic conditions, ily members (MCL1, BCL2, and BCL-XL) in the generated membrane-associated (long) and soluble, cleaved (short) venetoclax-resistant cell lines. We observed a slight increase forms of OPA1 coexist to form oligomers and complexes of MCL1 and BCL2 protein levels in MOLM-13-VR cells. that contribute to cristae maintenance. Upon an intrinsic Also, we found no increase of either BCL-XL protein or any death stimulus, OPA1 proteolytic cleavage is enhanced, of the examined BH3 antiapoptotic proteins in the MV4- allowing the “opening” of the cristae junctions and the 11-VR cell lines. These results suggest that BCL2 and MCL1 cytochrome c redistribution (25). We observed a decrease upregulation may serve as one of the potential mechanisms in the long (L-OPA1) forms and accumulation of short of cellular resistance to venetoclax; however, at least in (S-OPA1) forms of OPA1 in response to venetoclax treat- AML, there appear to exist alternative modes of resistance ment, suggesting its proteolysis following BCL2 inhibition acquisition (Supplementary Fig. S2L). In agreement with (Fig. 2E and F). Venetoclax treatment also led to caspase that notion, and based on the generated resistant AML lines, activation, as indicated by caspase-3 cleavage (Supplemen- OPA1 upregulation and mitochondrial structural adapta- tary Fig. S2E) and subsequently cell death, as demonstrated tions appear to represent a more universal mechanism of using Annexin V staining (Supplementary Fig. S2F). venetoclax resistance.

Figure 2. Mitochondrial response upon venetoclax treatment and after acquisition of drug resistance. A, Representative electron micrographs of THP-1 treated with 4 μmol/L venetoclax or DMSO for 16 hours. Scale bars, 5 μm (top) and 0.5 μm (bottom). B, Quantification of the maximal cristae width in 60 randomly selected mitochondria from 15 cells in experiments as in A (n = 200 cristae per condition). Data represent mean ± SEM of 3 independent experiments. C, Quantification of the percentage of mitochondria with abnormal ultrastructure in experiments as inA (n = 100 mitochondria per condition). Data represent mean ± SEM of 3 independent experiments. D, Quantification of the membrane potential loss after staining with TMRM in THP-1 cells treated with 4 μmol/L venetoclax or DMSO for 16 hours. Data represent mean ± SD (n = 3). E and F, THP-1 were treated with 4 μmol/L venetoclax for 16 hours, and equal amounts (30 μg) of cell lysates were separated by SDS-PAGE and immunoblotted using the indicated antibodies (E). L-OPA1, long forms of OPA1; S-OPA1, short forms of OPA1. The bar plot (F) shows the quantitative densitometric analysis of the ratio of long OPA1 forms to the total OPA1. Data, mean ± SEM of 3 independent experiments. G, IC50 curves of venetoclax in parental or venetoclax-resistant (VR) AML cell lines. Data represent mean ± SD (n = 3 for each cell line). H, Representative electron micrographs of mitochondria from parental (Par) or venetoclax-resistant MOLM-13. Scale bar, 0.5 μm. I, Quantification of the maximal cristae width in 60 randomly selected mitochondria from 15 cells in experiment as in H (n = 282 cristae per condition). Data, mean ± SEM. J, Western blot analysis in cell lysates from parental (Par) or venetoclax-resistant AML cell lines. K, GO analysis of differentially expressed genes involved in mitochondrial processes in venetoclax-resistant cells (MOLM-13 VR1) compared with the parental cell line (log fold change > 1 or < −1, FDR < 0.05). L, Scatter plot presenting the delta CRISPR score (Fig. 1B) plotted against the log fold change from MOLM-13 VR RNA-seq (Supplementary Fig. S3A). Colors correspond to the common −log (P value) that was generated using the geometric mean of the CRISPR screen P value and the RNA-seq FDR. Selected genes are highlighted. Data with statistical significance are as indicated. *, P < 0.05; ***, P < 0.001.

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Targeting Mitochondria Sensitizes AML to Venetoclax RESEARCH ARTICLE

A DMSO Venetoclax BC 30 *** 50 *** 40 20 30 (%) (nm) 20 10 10 Mitochondria with Maximal cristae width 0 abnormal ultrastructure 0 DMSO DMSO Venetoclax Venetoclax

D EF G IC50 of venetoclax 25 0.6 ) 150 n − *** MOLM-13 MOLM-13 VR DMSO Ve 20 MV4-11 MV4-11 VR L-OPA1 0.4 , DAPI 100 15 S-OPA1 −

10 Ratio

SDHA rms/total OPA1 0.2 * 50 5 Cell viability

% TMRM-low cells TMRM-low % Actin Long fo 0 0.0 V (% Annexin 0 DMSO DMSO −1 012345 Venetoclax Venetoclax log (conc.) (nmol/L)

H IJ MOLM-13 Par MOLM-13 VR 24 *** MOLM-13 MV4-11

18 r r Pa VR1 VR2 Pa VR1 VR2 12

(nm) OPA1 6 Actin

Maximal cristae width 0 MOLM-13 Par MOLM-13 VR1 MOLM-13 VR2

KLGenes encoding mitochondrial proteins Fold change (LFC > 1 or LFC < −1, FDR < 0.05) CRISPR screen vs. RNA-seq in VR cells GO biological process complete 02040 60 80 Response to type I interferon 5.0 Cellular response to type I interferon Type I interferon signaling pathway HAX1 Regulation of mitochondrial depolarization MCL1 Mitochondrial depolarization 2.5 MSTO1 Defense response to virus ATPAF1 Regulation of membrane depolarization CLPB −log P Cellular amino acid metabolic process 3 SLC25A1 Regulation of mitochondrial membrane potential 0 2 Response to virus TP53 PMAIP1 BAX 1 Coenzyme metabolic process BCL2L11 Negative regulation of ATP biosynthetic process BAD Negative regulation of purine nucleotide biosynthetic process SLC27A2 Negative regulation of nucleotide biosynthetic process −2.5 Membrane depolarization −log P LFC of FPKM in RNA-seq Neurotransmitter catabolic process 3.5 Alpha-amino acid metabolic process 3.0 Negative regulation of ATP metabolic process 2.5 −5.0 Negative regulation of purine nucleotide metabolic process 2.0 Negative regulation of nucleotide metabolic process 1.5 −4 −2 024 Delta CRISPR score

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RESEARCH ARTICLE Chen et al.

To further explore the molecular mechanisms underpin- we also focused on the upregulated genes in MOLM-13-VR ning venetoclax resistance, RNA sequencing (RNA-seq) was cells compared with MOLM-13-Par cells, whose ablation sen- performed. Indeed, the transcriptional landscape of the AML sitizes cells to the drug (negatively selected genes). We identi- cells changed significantly upon acquisition of resistance fied genes encoding the mitochondrial proteins CLPB and to venetoclax (Supplementary Fig. S3A–S3B). Interestingly, SLC25A1, as well as antiapoptotic proteins such as MCL1 and among the differentially expressed genes, there were many HAX1. We also found ATPAF1 and MSTO1, whose products involved in various mitochondrial processes. We focused on are involved in the maintenance of mitochondrial structure such genes (“Genes encoding mitochondrial proteins,” using and functions (Fig. 2L). the MitoMiner data set; Supplementary Fig. S3A–S3B) and Driven by these findings, we hypothesized that target- performed GO analysis to dissect the functional changes ing proteins regulating mitochondrial cristae maintenance in mitochondria upon acquisition of venetoclax resistance. and function might open up potential synthetic lethal vul- Strikingly, pathways that regulate mitochondrial membrane nerabilities for venetoclax treatment. To further refine the organization, potential, and depolarization were strongly candidate list from the screen, we excluded 97 core cellular enriched in both MOLM-13-VR and MV4-11-VR, though fitness genes (17), genes that are generally required for all strong variances were observed at the global transcriptomic cell types and therefore may not serve as ideal future thera- level in these cell lines (Fig. 2K; Supplementary Fig. S3C– peutic targets. From the remaining 256 genes, we focused S3D), which is consistent with our findings that mitochon- on genes that displayed higher mRNA expression levels in drial structure adaptation is universally required for gain AML patient samples compared with healthy primary CD34+ of venetoclax resistance in AML cells. Besides, metabolic hematopoietic stem and progenitor cells (HSPC) according processes of key cellular components, such as amino acids, to The Cancer Genome Atlas (TCGA). After applying these coenzyme, ATP, and nucleotides, were also enriched within criteria, we identified 18 candidates (Fig. 3A). One of the top- the top 20 enriched pathways, suggesting the metabolic adap- scoring candidates was CLPB, which encodes a mitochondrial tation is likewise important for venetoclax resistance. Finally, AAA+ ATPase chaperonin. Our expression studies revealed we observed enhanced interferon responses in MOLM-13-VR that CLPB gene expression is significantly higher in AML cells, which might be a result of mitochondrial DNA release patient samples when compared with normal CD34+ HSPCs to the cytosol as a consequence of incomplete apoptosis and (Fig. 3B). Moreover, CLPB median expression in AML was the partial mitochondrial dysfunction during the persistent vene- second highest among all other cancer subtypes in the TCGA toclax treatment (Fig. 2K; Supplementary Fig. S3D; ref. 27). data set and the third highest among all cancer cell lines in Overall, these data suggested that AML blasts require tran- the Cancer Cell Line Encyclopedia database (Fig. 3C; Supple- scriptional and post-translational mitochondrial adaptation mentary Fig. S4A). to acquire resistance to venetoclax. As a further suggestion of a role in this process, we observed higher levels of CLPB protein in venetoclax-resistant Targeting the Mitochondrial Protein CLPB human AML cell lines when compared with the parental Overcomes Venetoclax Resistance in AML sensitive clones (Fig. 3D and E). Based on our RNA-seq data We next attempted to determine whether there is a correla- (Supplementary Fig. S4C), CLPB transcripts are only slightly tion between genes differentially expressed in the resistant cell upregulated in venetoclax-resistant cells, indicating that this lines and gene hits in our CRISPR screen. We initially focused overexpression is post-translational. Importantly, the protein on the downregulated genes in MOLM-13-VR cells compared levels of the mitochondrial transcription factor A (TFAM) with MOLM-13-Par cells, whose ablation confers resistance were unchanged, indicating that the total mitochondrial mass to venetoclax (positively selected genes). We identified the was not altered. Furthermore, when we treated AML cells with validated genes TP53, BAX, and PMAIP1, as well as the proap venetoclax, we observed a significant increase in the CLPB optotic genes BAD and BCL2L11 (also known as BIM), and protein, indicating that cells that survive venetoclax treat- SLC27A2, a gene involved in fatty-acid metabolism. Moreover, ment have higher CLPB levels (Supplementary Fig. S4D–S4E).

Figure 3. Targeting the mitochondrial protein CLPB synergizes with venetoclax in AML. A, Venn diagram of the 353 common negatively selected genes (“sensitizers”) throughout the screen, the 2,221 core essential genes defined by Hart and colleagues (17) as well as 2,220 genes that are upregulated in patients with AML compared with healthy CD34+ HSPCs. Among these, 18 genes (listed in the table) were found nonessential and upregulated in patients with AML. B, Violin plot of CLPB mRNA expression level (FPKM) from RNA-seq in TCGA patients with AML (200 patients) and normal human + CD34 HSPCs (6 healthy donors). C, CLPB mRNA expression levels across diverse cancers from TCGA (log2 FPKM). Sorted by median expression level. D–E, Western blotting (D) and quantitative densitometric analysis (E) of CLPB protein levels of whole cell lysates from parental (Par) and venetoclax- resistant AML cell lines. F–G, IC50 curves of venetoclax in parental AML cells (F) and venetoclax-resistant AML cells (G) transduced with CLPB sgRNAs or negative control (sgRosa). Transduced AML cells were selected with puromycin and then treated with venetoclax for 48 hours. Viable cells were measured by CellTiter-Glo. Data, mean ± SD (n = 3 for each group). H, Bioluminescent images of mice transplanted with MOLM-13 cells transduced with sgRosa or sgCLPB #1. Mice were administrated with vehicle or venetoclax (Ven) from days 6 to 20 after transplantation as described in Supplementary Fig. S5E. The same mice are depicted at each time point (n = 4 mice per group). I, Quantification of bioluminescence emitted from the whole body of each mouse described in H at the indicated time points. J, Flow cytometry analysis of GFP+ sgRNA-expressing leukemia cells in the peripheral blood of MOLM-13 leukemia recipient mice described in H at the indicated time points. K, Kaplan–Meier survival curves of the MOLM-13 leukemia recipient mice described in H. The P values were determined using the log-rank Mantel–Cox test. L, Western blotting in whole cell lysates from sorted GFP+ sgRosa-expressing leukemia cells in the bone marrow of MOLM-13 leukemia recipient mice treated with vehicle or venetoclax (Ven), as described in H. Animals were sacrificed when they showed signs of late-stage leukemia. Data with statistical significance are as indicated. *,P < 0.05; **, P < 0.01; ***, P < 0.001; N.S., not significant.

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Targeting Mitochondria Sensitizes AML to Venetoclax RESEARCH ARTICLE

AC Upregulated in Common negatively Common hits AML patients vs. selected genes NCKIPSD 12 Not sequenced SPATA20 No mutation CD34+ HSPCs in the screen Missense CCDC88B 11 Nonsense 2,220 genes 353 genes CLPB Frameshift 10 USP3

SEMA4D 9 2,056 18 238 SLC30A1 OSCAR 8 3 TRIM8 143 94 CLSTN3 7 MTMR10 CLPB Expression–RNA-seq V2 (log) 6 1,981 LILRB3 ARHGEF2 5 ACSM1 PancreasThyroid (TCGA)Cholangiocarcinoma (TCGA)ProstateThymoma (TCGA)Cervical (TCGA) (TCGA)Mesothelioma (TCGA)SarcomaUterine (TCGA) (TCGA)Lung CS (TCGA) adenoccRCC (TCGA)pRCC (TCGA) Breast(TCGA) Testicular(TCGA)Uterine germColorectal (TCGA) cell (TCGA)Lung (TCGA) squHead (TCGA) &Bladder neck DLBC(TCGA) (TCGA) (TCGA)MelanomaGlioma (TCGA)Ovarian (TCGA)GBM (TCGA) (TCGA)ACC (TCGA)Liver (TCGA)Uveal melanomaPCPG AML(TCGA) (TCGA) (TCGA)chRCC (TCGA) DCAF15 Core essential genes BTBD7 RNF213 2,221 genes MED25 (numTKO hits > 1)

CLPB band intensity MOLM-13 MV4-11 B DE(relative to Actin) FPKM of CLPB mRNA VR2 * 5101520 Par VR1 VR2 Par VR1 VR2 VR1 * CLPB CD34+ HSPCs Par VR2 *** *** TFAM Patients with AML VR1 *** Par Actin MOLM-13 MV4-11 0 2468

F G MOLM-13 MV4-11 MOLM-13-VR1 MV4-11-VR1 100 120 120 120 80 100 100 100 80 80 80 60 60 60 60 40 40 40 40

% Viable cells 20 % Viable cells 20 % Viable cells 20 % Viable cells 20 0 0 0 0 −10 12 −10 12 −10 1432 5 −10 1432 5 log (conc.) (nmol/L) log (conc.) (nmol/L) log (conc.) (nmol/L) log (conc.) (nmol/L) sgRosa sgCLPB #1 sgCLPB #2 Par_sgRosa VR_sgRosa VR_sgCLPB #1 VR_sgCLPB #2

HIJ sgRosa sgRosa sgCLPB #1 1010 *** 10 0.068 ** *** Vehicle Ven Ven 8 0.15 109 * ** 6 5.00 0.056 108 cells in PB Day 6 + 4 N.S. * 1.70 7 4 10 N.S. * 0.13 × 10 2 0.32 % GFP (photons/sec/mouse) 3.30 6 Bioluminescence intensity 10 0 Day 11 6 11 14 18 11 14 18 0.005 Days after injection Days after injection sgRosa_VehiclesgRosa_Ven sgCLPB #1_Ven 2.00

1.30 5 KL Day 14 × 10 100 Vehicle Ven 0.67 0.005 80 #1 #2 #1 #2 1.00 60 CLPB 0.67 × 106 40 sgRosa_Vehicle Day 18 OPA1 0.34 Percent survival 20 sgRosa_Ven sgCLPB #1_Ven ** 0.005 0 GAPDH 0510 15 18 20 22 24 26 28 30 32 Days after injection

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RESEARCH ARTICLE Chen et al.

In addition, we quantified the CLPB protein levels in 4 dif- Finally, we sought to examine the synergistic effects of ferent AML cell lines (MOLM-13, MV4-11, OCI-AML3, and CLPB deletion with venetoclax in vivo by using preclinical THP-1) and found that cell lines with high CLPB protein animal models. Specifically, NOD/SCID gamma (NSG) mice levels were more resistant to venetoclax than those with low were transplanted with MOLM-13-Par cells or MOLM-13-VR CLPB levels (Supplementary Fig. S4F–S4H); however, the cells transduced with sgRosa or sgCLPB. After transplanta- small statistical sample of cell lines analyzed precludes us tion, we treated the mice with venetoclax or vehicle from from suggesting a definite predictive role of CLPB protein days 6 to 20. Bioluminescence imaging and examination of levels in venetoclax sensitivity. peripheral blood circulating leukemic cells (GFP+) indicated To further evaluate the importance of CLPB function that CLPB deletion synergizes efficiently with venetoclax in across different human AML cells, we depleted CLPB in both venetoclax-sensitive and venetoclax-resistant xenografts multiple AML cell lines using CLPB-targeting sgRNAs (Sup- (Fig. 3H–K; Supplementary Fig. S5E–S5J). In the course of plementary Fig. S6A), and we subsequently measured the these in vivo experiments, we determined that mice bearing venetoclax IC50. CLPB depletion significantly reduced the wild-type tumors and being treated with venetoclax exhibit IC50 of venetoclax in all the cell lines tested (Fig. 3F; Sup- significantly elevated CLPB and OPA1 protein levels at late plementary Fig. S5A). Additionally, CLPB-deficient AML cells stages of tumor progression compared with the vehicle- were negatively selected upon venetoclax addition at a much treated mice (Fig. 3L), suggesting once more that mitochon- faster pace than the DMSO control groups in a competition- drial structure adaptation also occurs in vivo as a mechanism based viability assay (Supplementary Fig. S5B), confirming of resistance to the drug. that CLPB ablation can sensitize AML cells to venetoclax treatment. Considering that CLPB is stabilized in venetoclax- CLPB Ablation Impairs Mitochondrial Structure in resistant AML cells (Fig. 3D and E), we sought to validate AML Cells, Rendering Them More Susceptible to the sensitization effect of CLPB depletion in our venetoclax- Apoptosis resistant cells. Similar to the results observed in the parental Next, we aimed to investigate how CLPB loss promotes clones (Fig. 3F), ablation of CLPB significantly resensitized venetoclax-induced programmed cell death in AML cells. VR cells to the drug treatment (Fig. 3G; Supplementary Fig. First, to confirm the subcellular localization of CLPB, we S5C–S5D). The synthetic lethal relationship between CLPB performed confocal imaging, which indicated that endog- ablation and venetoclax is specific to venetoclax mechanisms enous CLPB colocalizes with the mitochondrial marker of action, because no synergistic effect was detected in AML TOM20 in THP-1 and HeLa cells (Supplementary Fig. S7A). cells treated with cytarabine, idarubicin, or JQ-1 treatment Notably, CLPB expression in patients with AML correlates (Supplementary Fig. S4I–S4J). Moreover, in contrast to vene- with the transcription of genes whose products partici- toclax, treatment of MOLM-13 cells with idarubicin did not pate in the control of mitochondrial organization, such lead to increase of CLPB protein levels in the surviving cells as OPA3 and ATAD3A (Supplementary Fig. S7B and S7C), (Supplementary Fig. S4K). Finally, to avoid potential side suggesting a similar function of CLPB in leukemic cells. To effects of CRISPR-mediated gene deletion, silencing of CLPB test the hypothesis that CLPB participates in the organiza- using four distinct shRNAs with different efficacies further tion of mitochondrial ultrastructure, we performed electron validated the dose-dependent competitive disadvantage of microscopy in CLPB-knockout AML cells. Mitochondria CLPB loss in MOLM-13 cells treated with venetoclax (Sup- lacking CLPB exhibited an aberrant structure, characterized plementary Fig. S6B–S6D). Altogether, our findings revealed by wider cristae lumen (maximal cristae width—from a mean a functional role of mitochondrial CLPB in the response of of 18 nm in the controls to 27 nm in the knockout). As AML cells to venetoclax and suggested that targeting CLPB expected, the mitochondrial structure abnormalities were can overcome venetoclax resistance in AML. exacerbated when the cells were treated overnight with

Figure 4. CLPB loss induces mitochondrial ultrastructure defects sensitizing AML cells to mitochondria-mediated cell death. A, Representative electron micrographs of THP-1 transduced with sgRNAs targeting CLPB or control (sgRosa) and treated with 4 μmol/L venetoclax or DMSO for 16 hours. Scale bars, 2 μm (top) and 500 nm (bottom). B, Quantification of the percentage of mitochondria with abnormal ultrastructure in experiments as inA (n = 100 mitochondria per condition; top). Quantification of the maximal cristae width in 60 randomly selected mitochondria from 15 cells in experiments as in A (n = 200 cristae per condition; bottom). C, Equal amounts (30 μg) of protein from THP-1 infected with sgRNAs targeting CLPB or control (sgRosa) were separated by SDS-PAGE and immunoblotted using the indicated antibodies. D, Quantitative densitometric analysis of the ratio of OPA1 long (L-OPA1) versus short (S-OPA1) forms in experiments as in C. Data represent mean ± SEM of 8 independent experiments. E, Representative electron micrographs and morphometric analysis of BAX/BAK double-knockout MOLM-13 cells transduced with sgRNAs targeting CLPB or control (sgRosa). Scale bars, 500 nm. Morphometric analysis was performed in 60 randomly selected mitochondria (n = 200 cristae per condition) in two independent experiments. F, Quantification of the membrane potential loss after staining with TMRM in THP-1 infected with sgRNAs targetingCLPB or control (sgRosa) and treated with 4 μmol/L venetoclax or DMSO for 16 hours. Data, mean ± SD (n = 3 for each group). G, BH3 profiling: mitochondrial depolarization measured by JC-1 in permeabilized THP-1 transduced with sgRosa or sgCLPB upon stimulation with BIM and BID peptides. Depolarization (%) was calculated based on the area under the curve for each condition and normalized to CCCP-positive control and 1% DMSO as negative control as previously described (48). H, Isolated mitochondria from THP-1 transduced as indicated were treated with recombinant cBID for 30 minutes and centrifuged at 12,000 × g for 10 minutes. Pellet and supernatant (SN) of each sample were separated with SDS-PAGE and immunoblotted for cytochrome c (cyt c). % Cyt c release is calculated as the percentage of the supernatant to the total (pellet and supernatant) cytochrome c band intensity. I, AML cells were transduced with sgRNAs targeting CLPB or control (Rosa), treated with venetoclax or DMSO for 16 hours, and cell death was determined by flow cytometry using Annexin V. Data represent mean ± SD (n = 3). J, Liver mitochondrial lysates were immunoprecipitated with anti-CLPB coupled to magnetic protein G beads. Copre- cipitated proteins were separated by SDS-PAGE and immunoblotted against CLPB and OPA1. Input was diluted 1:10. Data with statistical significance are as indicated. *, P < 0.05; **, P < 0.01; ***, P < 0.001; N.S., not significant.

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Targeting Mitochondria Sensitizes AML to Venetoclax RESEARCH ARTICLE

A DMSO Venetoclax B 100 *** *** sgRosa sgCLPB sgRosa sgCLPB 80 60 ***

(%) 40 20 Mitochondria with

abnormal ultrastructure 0 50 ** 40 * 30 ***

(nm) 20 10 Max cristae width 0 sgRosa sgRosa_Ven sgCLPB sgCLPB_Ven

C DEsgRosa sgCLPB sgRosa sgRosa sgCLPB #1 sgCLPB #2 sgCLPB

s 1.5 L-OPA1 rm S-OPA1

1.0 TFAM

0.5 BAX/BAK DKO ** sgCLPB *** Actin sgRosa 0.0 0102030 Ratio L-OPA1/S-OPA1 fo Ratio L-OPA1/S-OPA1 Max cristae width (nm) F sgRosa GH sgCLPB #1 110 sgRosa sgCLPB #1 sgCLPB #2 100 sgRosa sgCLPB 50 *** 90 80 Untr cBID Untr cBID 40 ** 70 60 30 50 *** Pellet SN Pellet SN Pellet SN Pellet SN 20 40 30 % depolarization 10 20 10 TMRM low cells (%) TMRM low 0 0 18% 27% 6% 64% DMSO Ven % Cyt c release

5 mol/L 1BID mol/L BID 5 µmol/L 1BIM µmol/L BIM 10 µmol/L BIDµ µ 10 µmol/L BIM 0.5 µmol/L BIM 0.5 µmol/L BID

20 µmol/L PUMA 2A 25 µmol/L alamethicin

I MOLM-13 MV4-11 THP-1 J 100 100 100 *** 80 80 80 ** Input IP CLPB control Neg. *** sgRosa *** ** 60 ** 60 60 *** *** sgCLPB #1 CLPB

cells, % of GFP) cells, sgCLPB #2

+ 40 40 40 Cell death N.S. 20 20 ** 20 N.S. 0 0 0 OPA1 (Annexin V (Annexin

DMSO DMSO DMSO

n 10 µmol/L Ven 5 mol/L Ven 17 nmol/LVen 35 nmol/L Ven 17 nmol/L µ Ve

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RESEARCH ARTICLE Chen et al. venetoclax, and they were more significant in mitochondria CLPB Interacts with the Antiapoptotic lacking CLPB compared with wild-type (Fig. 4A and B; Mitochondrial Proteins HAX1 and OPA1 Supplementary Fig. S7D). The structural perturbations of To investigate how CLPB participates in the preservation CLPB-ablated mitochondria were accompanied by a promi- of the proper mitochondrial ultrastructure and the regula- nent accumulation of short OPA1 forms, indicating exces- tion of apoptosis, we sought to identify the interacting part- sive OPA1 processing, as demonstrated by Western blotting ners of this chaperonin in human AML cells. We performed (Fig. 4C and D). These phenomena were also observed in immunoprecipitation (IP) of the endogenous human CLPB cells deficient for BAX and BAK, which do not respond followed by mass spectrometry (MS) in whole THP-1 cell to cell death stimuli, suggesting that the mitochondrial lysates (Supplementary Fig. S7I). Our analysis uncovered morphologic defects are not a simple result of undergo- 64 mitochondrial proteins found exclusively in the CLPB ing apoptosis (Fig. 4E). In addition, TMRM staining after pulldown and not in the IgG sample (negative control), venetoclax treatment indicated an elevated percentage of indicating their potential association with CLPB. Among the cells with depolarized mitochondria in AML cells lacking top enriched CLPB interactors, we identified two proteins CLPB, suggesting that CLPB-knockout cells are more prone involved in the regulation of mitochondrial morphology to venetoclax-induced mitochondrial depolarization (Fig. and apoptosis, HAX1 and OPA1 (26, 28), ranked No. 1 and 4F; Supplementary Fig. S7E). To expand on these results, 3, respectively (Supplementary Table S2). Western blotting we performed a “BH3 profiling” in which permeabilized of the CLPB immunoprecipitation in THP-1 lysates and sgCLPB- and sgRosa-expressing AML cells were treated with mouse liver mitochondria confirmed the physical interaction increasing doses of BH3 peptides, and mitochondrial mem- of CLPB with HAX1 and OPA1 (Fig. 4J; Supplementary Fig. brane potential was monitored over time using the JC-1 S7J–S7K). Altogether, these data suggest that CLPB regulates dye. As expected, CLPB-deficient AML cells undergo faster mitochondrial cristae morphology and apoptosis in AML depolarization and are more primed for cell death than cells, via its specific interactions with OPA1 and HAX1. wild-type counterparts upon treatment with the BH3-only “activators” BIM and BID (Fig. 4G; Supplementary Fig. S7F–S7G). Given the wider cristae and the higher sensitivity CLPB Loss Suppresses AML Cell Growth to mitochondrial membrane potential loss upon apoptosis In Vitro and In Vivo stimulation, we speculated that CLPB-deficient mitochon- Given the mitochondrial structure defects, we hypothe- dria would readily release cytochrome c following a death sized that CLPB ablation directly affects mitochondrial func- stimulus. To address this hypothesis, we isolated functional tions. Indeed, using the live-cell metabolic assay platform mitochondria from wild-type and CLPB-knockout THP-1 Seahorse XF, we were able to show impaired basal and maxi- cells, stimulated them with the recombinant proapoptotic mal mitochondrial respiration, as well as defective glycolysis caspase-8 cleaved BID (cBID), and centrifuged them to in CLPB-deleted AML, suggesting a more quiescent metabolic retrieve both the pellet (mitochondria) and supernatant state compared with the highly energetic wild-type AML cells (released factors) fractions. Western blot analysis indicated (Fig. 5A). We then asked whether such bioenergetic defects that CLPB-deficient mitochondria are more sensitive to have an impact on cell proliferation. EdU incorporation cytochrome c release upon cBID stimulation compared with assays suggested a suppression of cell replication in AML the wild-type organelles (Fig. 4H). All of the above suggest cells deficient of CLPB (Fig. 5B). We verified these cell growth an increased responsiveness of CLPB-ablated AML cells to defects measuring cell doublings across time (Fig. 5C). More programmed cell death. Indeed, immunoblotting against importantly, suppression of cell growth was not seen or was pro- and cleaved caspase-3 verified a prevalent caspase-3 less pronounced in a number of non-AML cancer cell lines, activation upon BCL2 inhibition in AML cells lacking including T-ALL, B-ALL, CML, and melanoma (Supplemen- CLPB relative to the wild-type (Supplementary Fig. S7H). tary Fig. S6E), highlighting once more the specificity of CLPB Moreover, Annexin V staining in puromycin-selected AML dependency in AML cells (Supplementary Fig. S4B). cells transduced with sgRNAs targeting CLPB confirmed Finally, we examined the in vivo significance of CLPB hypersensitivity of these cells to venetoclax compared with in AML progression using a preclinical mouse model. We the negative controls (Fig. 4I). Taken together, these data labeled the Cas9-competent MOLM-13 cells with luciferase suggest that CLPB is an important protein for the mainte- (Luci) and transduced them with sgRNA targeting CLPB or nance of physiologic mitochondrial cristae, as well as the negative control (sgRosa). sgRNA-infected MOLM-13 cells control of cytochrome c release and ultimately the execution (GFP+) were then sorted by FACS and injected intravenously of cell death. into NSG mice (Fig. 5D). We observed a significant delay

Figure 5. CLPB ablation leads to cell growth suppression in AML in vitro and in vivo. A, Oxygen consumption rate (OCR), respiration (bar plot), and extracellular acidification rate (ECAR) of wild-type andCLPB -knockout MOLM-13 determined by Seahorse Extracellular Flux Analysis. Data, mean ± SEM (n = 5). B, Representative flow cytometry plots showing EdU cell-cycle analysis of wild-type andCLPB -knockout MV4-11 (left). Bar charts, mean percentage of cell populations ± SD (n = 3) in MV4-11 or THP-1 AML cells (right). C, Cell growth analysis of AML cells transduced with sgRNAs targeting CLPB or control (sgRosa; mean ± SD, n = 3). D, Schematic outline of the mouse model of CLPB dependency in AML. E, Bioluminescent images of mice transplanted with MOLM-13 cells transduced with sgRosa (n = 3) or sgCLPB #1 (n = 6). Representative images of two mice per sgRNA construct are shown. The same mice are depicted at each time point. F, Quantification of bioluminescence emitted from the whole body of each mouse transduced with sgRosa or sgCLPB #1 construct at the indicated time points. G, Flow cytometry analysis of GFP+ sgRNA-expressing leukemia cells in the peripheral blood of MOLM-13 leukemia recipient mice at indicated time points. H, Kaplan–Meier survival curves of recipient mice transduced with sgRosa and sgCLPB #1 are plotted. The P values were determined using the log-rank Mantel–Cox test. Data with statistical significance are as indicated. *,P < 0.05; **, P < 0.01; ***, P < 0.001; N.S., not significant.

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Targeting Mitochondria Sensitizes AML to Venetoclax RESEARCH ARTICLE

A Oligomycin FCCP Rot/AA Oligomycin FCCP Rot/AA 1,000 1,000 40 * 800 800 ** 30 *** 600 600 *** 20 400 400 sgRosa 10 OCR (pmol/min) 200

200 ECAR (mpH/min) sgCLPB #1 Respiration (pmol/min) 0 0 sgCLPB #2 0 020 40 60 80 100 Basal Max 020406080 100 Time (minutes) Time (minutes) sgRosa sgCLPB #1 sgCLPB #2 sgRosa sgCLPB #1 sgCLPB #2

B sgRosa sgCLPB 75 MV4-11 75 THP-1 5 5 *** 10 36.8% 10 24.8% ** 104 104 50 50

3 3 10 11.2% 10 8.7% ** *** % Cells 25 25 EdU 46.5% 53.9% 102 2.8% 102 9% 0 0 0 0 1 1 0 50K 100K 150K 250K200K0 50K 100K 150K 250K200K -G S 1 -G S 1 0 -M 0 -M G G 2 G G 2 Sub G Sub G DAPI sgRosa sgCLPB #1 sgCLPB #2

C E sgRosa sgCLPB #1 MOLM-13 MV4-11 THP-1 6 4 6 ) ) ) 6 6 6 Day 10 3 4 *** 4 . (10 . (10 . (10 ** 2 *** 2 2 Cell no Cell no Cell no 1 Day 14 0 0 0 012 345 012345 012 3456 Days Days Days sgRosa sgCLPB #1 sgCLPB #2 Day 18 D

Measuring survival Day 20 Day 0 Day 10 Day 14 Day 18 Day 20

0.5M GFP+ MOLM-13 5 cells per NSG mice Bioluminescence imaging Bleeding × 10 0.5 2.0 3.5 5.0

F G H sgRosa ** 1011 30 sgRosa *** sgCLPB #1 100 1010 * sgCLPB #1 20 80 109 ** * 10 60 N.S. cells in PB

8 + 10 N.S. ** 40 sgRosa 0 sgCLPB #1 ** 107 20 Percent survival Percent %GFP (photons/sec/mouse) 106 −10 0

Bioluminescence intensity 10 14 18 20 10 14 18 20 0510 15 20 25 30 Days after injection Days after injection Days after injection

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RESEARCH ARTICLE Chen et al. in leukemia progression in mice receiving CLPB-deficient cells, we measured mitochondrial reactive oxygen species MOLM-13 cells, as determined by bioluminescent imaging (ROS) upon treatment with the complex III inhibitor antimy- (Fig. 5E and F). FACS analysis of peripheral blood from cin A using MitoSOX Red. CLPB deficiency in MOLM-13 cells recipient mice detected a significantly lower percentage of led to a significant elevation of the mean MitoSOX fluo- circulating leukemia cells harboring CLPB sgRNAs compared rescent intensity after antimycin A addition relative to the with the negative control (Fig. 5G), which correlated with the steady-state levels, indicating greater ROS accumulation significant prolonged survival seen in mice harboring CLPB- inside CLPB-ablated mitochondria upon challenge (Supple- deficient MOLM-13 cells (Fig. 5H). These data suggested that mentary Fig. S8B), thus demonstrating that AML cells are CLPB is required for AML maintenance, which is consistent under mitochondrial stress upon CLPB depletion. Given that with the physiologic function of CLPB in regulating key proapoptotic BH3-only proteins can be induced by ATF4 mitochondrial biological processes, including cristae struc- (31), we wondered if these pro-death sensitizers were upregu- ture, membrane potential, and oxidative phosphorylation. lated in response to CLPB depletion. Indeed, PMAIP1, BBC3, and HRK gene expression was significantly upregulated in Loss of CLPB Induces Mitochondrial Stress CLPB-deficient AML cells (Fig. 6F; Supplementary Fig. S8G). Response In line with this finding, BH3 profiling revealed increased To further focus on the underlying mechanisms of CLPB sensitivity of CLPB-ablated cells to the peptide “sensitizers” function, we performed RNA-seq in AML cells upon CLPB PUMA and BMFγ, as well as to the MCL1 inhibitor (MS1; deletion. We used two CLPB-targeting sgRNAs and pro- Supplementary Fig. S8H). Such findings were also consistent filed cells at days 6 and 8 after CLPB depletion (Fig. 6A). with our previous results showing that CLPB-ablated AML Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway cells are more primed to apoptosis (Fig. 4G and H). analysis of the differentially expressed genes in both CLPB- Considering that CLPB-deficient AML cells are under mito- deficient MOLM-13 cells (sgCLPB #1 and #2) showed strong chondrial stress and harbor dysfunction in the biosynthesis of enrichment on metabolic pathways, such as biosynthesis of several metabolites (Fig. 6B), a more detailed metabolic profil- amino acids and one carbon metabolism (Fig. 6B), all consisting was performed using mass spectroscopy. Among the 150 ent with the functions of CLPB in regulating mitochondrial metabolites tested, the intensity of 16 substances was signifi- processes (Fig. 5). Ingenuity pathway analysis (IPA) was per- cantly changed in CLPB-deficient MOLM-13 cells (Fig. 6G; Sup- formed to reveal the upstream transcriptional regulators that plementary Table S3). Pathway analysis of the metabolomics are responsible for the transcriptional landscape changes data revealed significant enrichment in amino acid–related upon CLPB ablation. Interestingly, ATF4, a key transcription pathways (glycine, serine, and threonine metabolism, arginine factor induced by the integrative stress response (29), ranked and proline metabolism, aminoacyl-tRNA biosynthesis; Fig. first in MOLM-13 cells transduced with any of the CLPB- 6H). Indeed, the glycine, serine, and threonine biosynthesis targeting sgRNAs (Fig. 6C). Indeed, qPCR analysis verified is one of the most altered metabolic pathways in cells under the upregulation of ATF4 and its downstream effector CHOP mitochondrial stress (30). Notably, the majority of the altered in AML cells lacking CLPB (Fig. 6D; Supplementary Fig. metabolites upon CLPB depletion were strongly correlated with S8D–S8F). Considering our previous results and given that the differentially expressed genes shown in Fig. 6A, highlighting ATF4 has been identified as a key regulator of the mitochon- the consistency of our RNA-seq and metabolomics data (Sup- drial stress response in mammals (30), we speculated that plementary Fig. S8C). These findings suggest that CLPB defi- CLPB-deficient AML cells are under mitochondrial stress. To ciency amplifies proapoptotic signals through the induction of this end, we performed gene-set enrichment analysis (GSEA) the ATF4-mediated mitochondrial stress response. using a combined mitochondrial stress expression gene signature extracted from the RNA-seq studies described by CLPB Targeting Overcomes p53-Mediated Quirós and colleagues (30). Such analysis revealed that genes Venetoclax Resistance and Sensitizes AML associated with mitochondrial stress response were indeed Cells to Combined Venetoclax and Azacitidine strongly enriched in CLPB-deficient MOLM-13 cells at both Treatment time points (Fig. 6E; Supplementary Fig. S8A). To function- Stabilization of the p53 protein synergizes with veneto- ally confirm the mitochondrial stress in CLPB-deficient AML clax in AML (15). Based on data included here and in the

Figure 6. CLPB deficiency amplifies proapoptotic signals by inducing mitochondrial stress response. A, Heat map showing the differentially expressed genes in MOLM-13 transduced with sgRNAs targeting CLPB or control (sgRosa) at days 6 and 8 after transduction (log fold change > 1 or < −1, false discovery rate < 0.05 in all samples). Common genes are shown in the heat map. B, KEGG pathway enrichment analysis of the differentially expressed genes in MOLM-13 transduced with sgRNAs targeting CLPB or control (sgRosa) at day 6 after transduction. C, IPA of the differentially expressed genes (log fold change > 1 or < −1, false discovery rate < 0.05 in all samples) in MOLM-13 transduced with two independent sgRNAs targeting CLPB (left, sgCLPB #1; right, sgCLPB #2) or control (sgRosa) at day 6 after transduction revealing activation of the ATF4 upstream pathway. D, qPCR analysis of ATF4 transcripts in MOLM-13 transduced with sgRNAs targeting CLPB or control (sgRosa; mean ± SEM, n = 3). E, Enrichment score plots from GSEA using the mitochondrial stress expression signature defined by Quirós and colleagues (30) and the RNA-seq data of MOLM-13 transduced with sgRNAs targeting CLPB or control (sgRosa) at day 6 after transduction. FDR, false discovery rate; NES, normalized enrichment score. F, qPCR analysis of the relative expression levels of PMAIP1 (NOXA) mRNA, PMAIP1 (NOXA) mRNA, and HRK mRNA in MOLM-13 transduced with CLPB-targeting sgRNAs or control (sgRosa) at day 6 after transduction (mean ± SD, n = 3). G, Heat map showing the significantly differentially detected metabolites in MOLM- 13 transduced with two independent sgRNAs targeting CLPB or control (sgRosa) at day 6 or 8 after transduction (P < 0.05). Average intensity of each metabolite was shown. H, Metabolome pathway enrichment analysis of top altered metabolites in G. Data with statistical significance are as indicated. *, P < 0.05; **, P < 0.01.

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Targeting Mitochondria Sensitizes AML to Venetoclax RESEARCH ARTICLE

A B KEGG pathway analysis Fold change sgRosa sgCLPB #1 sgCLPB #2 LFC > 0.5 or LFC < −0.5, FDR < 0.05 02.5 5.0 7.5 Day 6DDay 8 ay 6DDay 8 ay 6 Day 8 Biosynthesis of amino acids GLI1 ULBP1 Glycine, serine, and threonine metabolism BEND6 ALDH1L2 SLC6A9 Phagosome −log P PTPN13 FYN PPM1E Carbon metabolism RP11–293M10.6 5 GYPC CHAC1 mTOR signaling pathway INHBE 4 ALPK2 GDAP1L1 Malaria FUT1 3 S100P PLEKHA4 One carbon pool by folate SPOCK1 2 C5AR1 IGFBP2 Staphylococcus aureus infection NXPH4 GABRG1 PAPPA2 Legionellosis FAM84B FZD4 DFNA5 Acute myeloid leukemia HRK SPTB NDUFA4L2 SLC6A12 CADM1 LPO NLRP12 Activation Z-score Activation Z-score HERC5 ASNS C (sgCLPB #2) ORM1 (sgCLPB #1) KDM7A RPS6KA2 0 123 012 3 STC2 ASS1P7 KCNG1 ATF4 ATF4 TTC39B BATF3 ZBED3 IRF7 IRF5 CTH −log P ASS1 PHGDH SPI1 MRTFB ATP6V1C2 TMEM45A IRF5 SLPI MYCN 10 ABCA1 ROS1 IRF1 MRTFA SLC35F1 5 TRGC1 ECM1 IRF3 SMAD7 RP11–162D9.3 ADAMTS5 CLEC5A STAT1 FAM46C AE000661.37 POU2AF1 −log P ATF4 mRNA expression CTD–2231E14.8 D TLR4 BRCA1 16 (relative to sgRosa) 0 246 MYC 12 −2 −10 12 MSC 8 sgRosa NFE2L2 4 sgCLPB #1 * sgCLPB #2 *

E F sgCLPB #1 vs. sgRosa sgCLPB #2 vs. sgRosa 0.9 0.9 0.8 NES = 2.81 0.8 NES = 2.80 PMAIP1 BBC3 HRK 0.7 p-val = 0.0 0.7 p-val = 0.0 0.6 0.6 12 8 4 0.5 FDR = 0.0 0.5 FDR = 0.0 ** 0.4 0.4 * * ** 0.3 0.3 0.2 0.2 6 3 0.1 0.1 8 Enrichment score (ES) 0.0 Enrichment score (ES) 0.0 2 to sgRosa) to sgRosa) * 4 * to sgRosa) 4 4 ‘na_pos’ (positively correlated) 5.0 ‘na_pos’ (positively correlated) 3 2 1 2 2.5 mRNA expression mRNA expression 1 Zero cross at 5,909 Zero cross at 5,514 mRNA expression (relati ve (relati ve 0 0.0 (relati ve −1 0 0 0 −2 ‘na_neg’ (negatively correlated) −2.5 ‘na_neg’ (negatively correlated) 002,500 5,000 7,500 10,000 2,500 5,000 7,500 10,000 Rank in ordered dataset Rank in Ordered Dataset Enrichment profile Hits Ranking metric scores Enrichment profile Hits Ranking metric scores Ranked list metric (PreRanked) Ranked list metric (PreRanked) Ranked sgRosa sgRosa sgRosa sgCLPBsgCLPB #1 #2 sgCLPBsgCLPB #1 #2 sgCLPBsg CLPB#1 #2

G H sgCLPB Arginine and proline metabolism sgRosa #1 #2 Day 6 Day 8 Day 6 Day 8 Day 6 Day 8

Glycine, serine, and threonine metabolism Hydroxyproline

C5–carnitines 56 Propionylcarnitine Serine Aminoacyl-tRNA biosynthesis Methionine Leucine N–acetyllysine Cysteine and methionine metabolism UMP − log (P) Phosphocreatine 34 Pyrimidine metabolism Sarcosine Creatine UDP–GlcNAc Butyrylcarnitine Amino sugar and nucleotide sugar metabolism Glucose_6–phosphate

Hypoxanthine 12 Carbamoyl_aspartate 0.05 0.1 0.15 −2 −1 012 0 Pathway impact

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RESEARCH ARTICLE Chen et al. related article by Nechiporuk and colleagues in this issue, CRISPR/Cas9 screen identified both possible mechanisms p53 deletion can confer resistance to the drug in AML of resistance and synthetic lethal combinations with vene- cells (Fig. 1B–E; Supplementary Fig. S1B and S1D–S1F). toclax in AML, including the targeting of a number of Therefore, the key question of obvious clinical significance proteins regulating mitochondrial structure. These proteins is whether CLPB deletion can sensitize p53-deficient AML can control the architecture of both the OMM and the cells to venetoclax treatment. To this end, we deleted p53 in mitochondrial cristae. Interestingly, both these structures MOLM-13 cells using two independent sgRNAs targeting are essential for BCL2 family function (5), cytochrome c TP53 (Supplementary Fig. S9A). Our studies showed that release, and cell death (34). For the complete release of the CLPB-deficient AML cells were more sensitive to venetoclax cristae-endowed cytochrome c stores, the inner mitochon- treatment, even in a TP53-knockout (p53-KO) background drial membrane needs to be remodeled and the cristae wid- (Fig. 7A). More importantly, CLPB depletion was able to ened by the proteolytic cleavage of OPA1 and the disruption fully resensitize p53-deficient MOLM-13 cells to venetoclax of its complexes, which normally keep two opposing cristae treatment by inducing the ATF4-mediated upregulation of membranes tight (26, 35). Both OMM rupture and cristae proapoptotic sensitizers (Fig. 7B; Supplementary Fig. S9D– remodeling is what we observed by electron microscopy S9E). Likewise, CLPB ablation led to a synergistic effect with upon venetoclax treatment in AML cells. The changes in venetoclax also in an AML cell line with naturally occurring mitochondrial architecture could, at least partially, explain TP53 mutation (KASUMI-1; Supplementary Fig. S9B–S9C). the reported impairment in oxidative phosphorylation fol- Collectively, CLPB-mediated resensitization of venetoclax- lowing BCL2 inhibition in the LSC derived from patients resistant AML cells is p53-independent. with AML (2). On the opposite side, venetoclax-resistant Because resistance to venetoclax monotherapy rapidly AML cells display mitochondria with narrow cristae lumen ensues in AML (14, 32), multiple combinational therapies and increased protein levels of the cristae “staple” OPA1, for venetoclax have been proposed and tested, some of which possibly rendering them more resistant to the cytochrome c are in clinical trials (https://www.cancer.gov/about-cancer/ release upon stimulation. treatment/clinical-trials/intervention/Venetoclax). Among One of the top-ranked genes revealed by our screen to act these, the combination of venetoclax with hypomethylating synergistically with venetoclax is the mitochondrial chaper- agents (HMA), such as azacitidine, is currently in clinics due onin CLPB. Although a plethora of studies in bacteria and to its favorable responses in the clinical trials (33). To inves- yeast have extensively characterized the homolog of CLPB as tigate if CLPB inhibition can enhance the efficacy of vene- a protein chaperone that disaggregates misfolded proteins toclax and azacitidine combined treatment, we tested the (36–38), the exact function of the mammalian CLPB has possibility that CLPB depletion can further sensitize AML not been investigated thoroughly. Of note, the amino acid cells to the combined treatment. Indeed, CLPB-deficient sequence of the human CLPB is only about 20% identical AML cells were negatively selected upon combined treat- to the ortholog in Escherichia coli (38). CLPB belongs to the ment at a much faster pace than the DMSO control groups large AAA+ ATPase superfamily, whose members act both (Fig. 7C), confirming that CLPB ablation can sensitize AML as general protein chaperones and as targeted proteases cells to combined venetoclax/azacitidine treatment. Alto- that degrade specific substrates. Other mitochondrial AAA+ gether, our findings highlight that loss of the mitochondrial ATPases, such as YME1L, AFG3L2, ATAD3, and m-AAA protein CLPB leads to structural and functional defects protease, participate in the organelle’s protein quality con- of mitochondria, hence sensitizing AML cells to apopto- trol, mitochondrial protein synthesis, and maintenance of sis. Therefore, targeting CLPB synergizes with venetoclax mitochondrial architecture (39–43). Wortmann and col- and venetoclax/azacitidine combination in AML in a p53- leagues, who studied the mutations in CLPB in patients independent manner (Fig. 7D). with an autosomal recessive metabolic syndrome, hypothesized a possible role for CLPB in apoptosis, due to its in situ–predicted interaction with HAX1 (44). Our study pro- DISCUSSION vides the biochemical evidence of the association of CLPB The recent FDA approval for venetoclax introduces an with HAX1 and uncovers its function in the cell protection exciting and novel strategy to target AML. In the initial from intrinsic mitochondria-mediated apoptosis. Further- single-agent venetoclax studies in AML, response rates more, our work reveals that CLPB physically interacts with were modest and rather short-lived (12). Our genome-wide OPA1—possibly as a means to protect it from its proteolytic

Figure 7. CLPB targeting overcomes p53-mediated venetoclax resistance and sensitizes AML cells to combined venetoclax and azacitidine treatment. A, Synergistic effect of CLPB depletion and BCL2 inhibition in TP53-knockout (KO) MOLM-13 cell lines (two clones, B10 and B11) generated as shown in Supplementary Fig. S9. Plotted are GFP+ percentages measured during 4 days in culture and normalized to day 0 of drug treatment. Negative control (sgRosa) and two independent sgRNAs targeting CLPB are shown in the graphs. Data represent mean ± SD (n = 4). B, IC50 curves of venetoclax in p53 wild-type (WT) and p53-deficient (KO; two clones, B10 and B11) MOLM-13 cells transduced with sgCLPB #1 or sgRosa. Transduced AML cells were selected with puromycin and then treated with venetoclax for 48 hours. Viable cells were measured by CellTiter-Glo. Data, mean ± SD (n = 6 for each group). C, Validation of the synergistic effect of CLPB depletion and venetoclax + azacitidine combined treatment using a competition-based survival assay in MOLM-13 (left) and MV4-11 (right) cells. Plotted are GFP+ percentages measured during 6 days (for MOLM-13) or 4 days (for MV4-11) in culture and normalized to day 0 of drug treatment. Negative control (sgRosa) and two independent sgRNAs targeting CLPB are shown in the graphs. Data, mean ± SD (n = 6 for MOLM-13 and n = 3 for MV4-11). D, Schematic outline of this study. Targeting the mitochondrial CLPB sensitizes AML cells to venetoclax treatment by (i) promoting apoptotic cristae remodeling and (ii) inducing mitochondrial stress response which will amplify the programmed cell death pathway. Data with statistical significance are as indicated. ***,P < 0.001.

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Targeting Mitochondria Sensitizes AML to Venetoclax RESEARCH ARTICLE

A p53-KO-MOLM-13 (B10) p53-KO-MOLM-13 (B11) p53-WT-MOLM-13 120 120 120 100 100 100 80 80 80 cells cells cells + + 60 60 + 60 40 40 40 % GFP % GFP % GFP 20 *** 20 *** 20 *** 0 0 0 0 12345 0 12345 012345 Days after drug treatment Days after drug treatment Days after drug treatment sgRosa_DMSO sgCLPB #1_DMSO sgCLPB #2_DMSO sgRosa_Ven sgCLPB #1_Ven sgCLPB #2_Ven

B IC50 of venetoclax C MOLM-13 MV4-11 100 120 120

80 100 100 80 80

60 cells cells + 60 + 60 40 40 40 % Viable cells Viable % % GFP % GFP 20 20 *** 20 *** 0 0 0 −1 0123 02468 012345 log (conc.) (nmol/L) Days after drug treatment Days after drug treatment p53-WT sgRosa sgRosa_DMSO sgRosa_Ven+Aza p53-KO (B10) sgRosa sgCLPB #1_DMSO sgCLPB #1_Ven+Aza p53-KO (B11) sgRosa sgCLPB #2_DMSO sgCLPB #2_Ven+Aza p53-KO (B10) sgCLPB #1 p53-KO (B11) sgCLPB #1

D Drug resistance Drug sensitization CLPB high CLPB low Cristae remodeling Mitochondrial stress Venetoclax Venetoclax Nucleus

BCL2 BCL2 Proapoptotic sensitizers Proapoptotic proteins Proapoptotic proteins ATF4

BAX BAX BAX BAX Tight cristae Wide cristae

Cytochrome c Cytochrome c

Activation of Activation of executioner caspases executioner caspases

Resisting cell death Cell death

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RESEARCH ARTICLE Chen et al. cleavage—and thus demonstrates a role for CLPB in the cell lines transduced with retroviral Cas9-2A-blast (Addgene, plas- maintenance of the physiologic mitochondrial structure. mid no. 73310) were selected with blasticidin (InvivoGen) 48 hours Loss of CLPB results in abnormal mitochondria with wider after transduction. All transfections were performed in HEK293T cristae than the normal; hence, such organelles are more cells using polyethylenimine reagent at 4:2:3 ratios of sgRNA prone to release their cytochrome c following a death sig- or shRNA construct (Supplementary Table S4): pVSVG: pPAX2 in OPTI-MEM solution. Viral supernatant was collected 36 and nal, such as venetoclax treatment. Our work proposes a 48 hours after transfection. Spin infections were performed at room specific CLPB dependency of AML cells. Its depletion leads temperature at 1,500 × g for 30 minutes with polybrene reagent to cell-cycle suppression, possibly due to the defects in both (1:2,000; Fisher Scientific). oxidative phosphorylation and glycolysis. As a consequence For the generation of the venetoclax-resistant cell lines, MOLM- of the mitochondrial structural and functional impairment, 13 and MV4-11 cells were cultured in media containing increasing cells lacking CLPB are more sensitive to oxidative stress. As doses of venetoclax (from 5 to 1,000 nmol/L) for more than 8 weeks a response, the CLPB-deficient AML cells alter their tran- to achieve complete resistance to the drug. scriptional program and reprogram their metabolism in an Cell line information is as follows: MOLM-13, DSMZ, ACC554; ATF4-dependent manner, thus leading to cell-cycle arrest THP-1, ATCC, TIB-202; MV4-11, ATCC, CRL-9591; CuTLL-1, Adolfo and enhancement of apoptotic pathways. Overall, we show Ferrando Lab; Nalm-6, ATCC, CRL-3273; K562, ATCC, CCL-243; HEK293T, ATCC, CRL-1573; SK-MEL-239, Eva Hernando Lab; and that CLPB is most likely required for AML progression and, HeLa, ATCC, CCL-2. importantly, can synergize with venetoclax to induce rapid All the cell lines were determined negative for Mycoplasma, as cell death both in vivo and in vitro. indicated by the latest Mycoplasma test (December 2018) using the Recently, Pollyea and colleagues demonstrated that the LookOut Mycoplasma PCR Detection Kit (Sigma). Cells were used FDA-approved combined treatment of venetoclax with for experiments within 15 to 20 passages from thawing. azacitidine disrupts the tricarboxylic acid cycle in LSCs of patients with AML and that relapsed patients with AML CRISPR Screen remodel their metabolism (45, 46). These studies support Cas9-expressing MOLM-13 cells were transduced with the the notion that targeting mitochondrial functions concomi- Brunello sgRNA library (18) virus at a low multiplicity of infection tantly with venetoclax and azacitidine could have a clinical (∼0.3). On day 2 after transduction, GFP+ percentage was assessed to relevance for patients with AML. In line with this idea, we determine infection efficiency and sgRNA coverage ∼( 1,000×). Then, + show that depleting CLPB, which affects the cellular meta- puromycin (1 μg/mL) was added for 5 days to select infected (GFP ) bolic status of the AML cells, sensitizes them to the com- cells. After selection, viable infected cells were isolated by Histopaque 1077 (Sigma-Aldrich) and grown without antibiotics for 2 days. After bined treatment in a p53-independent way. Interestingly, a recovery, 100 × 106 infected cells were cultured with 10 nmol/L vene- bacterial CLPB inhibitor has been developed and proposed toclax (Selleckchem) or DMSO. Another 100 × 106 cells were used to be utilized as an antimicrobial agent (47). Because this for genomic DNA (gDNA) extraction and served as an initial refer- inhibitor targets the conserved domain of the bacteria and ence (day 0 of drug treatment). The concentration of venetoclax was the human orthologs (44) and displays moderate toxicity increased during the screen (from 10 nmol/L to 2 μmol/L) to avoid in human cell lines (47), it could synergize with venetoclax spontaneous gain of drug resistance. For each passage, 100 × 106 treatment in AML cells. In general, targeting mitochondrial cells were placed back into culture until 16 days after drug treatment. proteins that would promote apoptotic cristae remodeling gDNA of cells containing ∼1,000× coverage was harvested on days 8 should be investigated as a potential way to overcome vene- and 16 after drug treatment using a Qiagen DNA kit according to the toclax resistance in AML, in addition to ongoing trials manufacturer’s protocol. Further details on library construction and data analysis can be found in the Supplementary Data. targeting other BCL2 family members, such as MCL1 (R. Tibes, personal communication), either directly with novel Drug Treatment and IC Measurements MCL1 inhibitors, or indirectly via cyclin-dependent kinase 50 inhibition affecting MCL1 transcript expression, as we and Cells were plated in 96-well plates and exposed to venetoclax (Selleckchem) at concentrations ranging from 0.4 to 300 nmol/L others have proposed (11, 16). In conclusion, targeting CLPB (for MOLM-13 and MV4-11) or 150 nmol/L to 10 μmol/L (for and possibly its interactome in combination with venetoclax venetoclax-resistant cell lines and THP-1) with a minimum of three treatment directly promotes the apoptotic cristae remod- technical replicates per concentration per cell line. Cell viability eling and indirectly induces mitochondrial stress responses, was measured with the CellTiter-Glo reagent (Promega) according which will both amplify the programmed cell death pathway to the manufacturer’s instructions. Absolute viability values were and suppress the growth of AML cells. Our data open a converted to percentage viability versus DMSO control treatment, potential avenue for novel venetoclax drug combinations in and then nonlinear fit of log(inhibitor) versus response (three AML. parameters) was performed in GraphPad Prism v7.0 to obtain the IC50 values. For venetoclax and azacitidine combined treatment, two drugs were mixed at the fixed ratio (ven:aza= 1:4) before dilut- METHODS ing to certain concentrations. Cell Lines and Cell Culture Transmission Electron Microscopy All human leukemia cells (MOLM-13, THP-1, MV4-11, CuTLL-1, Cultured cells were fixed in 0.1 mol/L sodium cacodylate buffer Nalm-6, and K562) were cultured in RPMI medium supplemented (pH 7.4) containing 2.5% glutaraldehyde and 2% paraformaldehyde with 10% FBS and 1% penicillin and streptomycin. The adherent cell for 2 hours and post-fixed with 1% osmium tetroxide and 1% potas- lines HeLa and HEK293T were grown in DMEM with 10% FBS and sium ferrocyanide for 1 hour at 4°C. Then, block was stained in 1% penicillin and streptomycin. SK-MEL-239 cells were cultured in 0.25% aqueous uranyl acetate, processed in a standard manner, and RPMI, 10% FBS, and 1% penicillin and streptomycin. Cas9-expressing embedded in EMbed 812 (Electron Microscopy Sciences). Ultrathin

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Targeting Mitochondria Sensitizes AML to Venetoclax RESEARCH ARTICLE sections (60 nm) were cut, mounted on copper grids, and stained Development of methodology: X. Chen, C. Glytsou, D.E. Reyna, with uranyl acetate and lead citrate. Stained grids were examined Y. Gong, Y.S. Yap, E. Wang, I. Aifantis under Philips CM-12 electron microscope and photographed with a Acquisition of data (provided animals, acquired and managed Gatan (4k × 2.7k) digital camera. For morphometric analysis, maxi- patients, provided facilities, etc.): X. Chen, C. Glytsou, A. Lopez, mal cristae width was measured using ImageJ (NIH) as shown in Sup- E. Gavathiotis, R. Tibes plementary Fig. S2A in at least 50 randomly selected mitochondria Analysis and interpretation of data (e.g., statistical analysis, bio- from a minimum of 15 cells/experiment. statistics, computational analysis): X. Chen, C. Glytsou, H. Zhou, S. Narang, D.E. Reyna, A. Lopez, T. Sakellaropoulos, Y. Gong, Mitochondrial Respiration A. Kloetgen, E. Gavathiotis, A. Tsirigos, R. Tibes Oxygen consumption rate (pmol/min) was determined using the Writing, review, and/or revision of the manuscript: X. Chen, XFe24 Extracellular Flux Analyzer and the XF Cell Mito Stress Test C. Glytsou, H. Zhou, D.E. Reyna, A. Lopez, Y. Gong, E. Gavathiotis, kit (Agilent). Details can be found in the Supplementary Data. R. Tibes, I. Aifantis Administrative, technical, or material support (i.e., reporting Mitochondrial Membrane Potential or organizing data, constructing databases): X. Chen, C. Glytsou, H. Zhou, Y. Gong Mitochondrial membrane potential was monitored cytofluorimet- Study supervision: X. Chen, C. Glytsou, I. Aifantis rically using TMRM. Cells were loaded with 10 nmol/L TMRM in the presence of 2 μmol/L cyclosporin H in Hank’s Balanced Salt Solution Acknowledgments supplemented with 10 mmol/L HEPES, 30 minutes at 37°C. When We would like to thank all members of the Aifantis lab for useful indicated, the experiments were performed in cells pretreated with discussions and comments on the manuscript; A. Heguy and the NYU venetoclax for 16 hours at the indicated concentrations. Genome Technology Center (supported in part by NIH/NCI grant P30CA016087-30) for expertise with sequencing experiments; M. Cam- Metabolomics mer and the NYU Langone Microscopy Laboratory (grant NCRR S10 Cells (7 × 105) were flash-frozen as pellets and processed using the RR023704-01A1) for assistance with confocal microscopy; A. Liang, C. LC/MS-MS with the hybrid metabolomics method. Details can be Petzold, and K. Dancel-Manning and the NYU Langone Health DART found in the Supplementary Data. Microscopy Lab for their assistance with TEM work; NYU Langone Health’s Metabolomics Laboratory for their help in acquiring and ana- Animal Experiments lyzing the data presented; and NYU High Throughput Biology Labo- For in vivo experiments, Cas9 and luciferase-expressing parental and ratory, which is partially supported by the Laura and Isaac Perlmutter venetoclax-resistant MOLM-13 cells were transduced with sgRosa or Cancer Center Support Grant (NIH/NCI P30CA16087) and NYSTEM sgCLPB #1 constructs. At day 2 after transduction, sgRNA-positive Contract C026719. The proteomics studies were supported in part by cells (GFP+) were sorted by FACS. A half million sgRNA-expressing the NYU School of Medicine and the Laura and Isaac Perlmutter Can- leukemia cells were intravenously injected into recipient NSG mice. cer Center Support Grant P30CA016087 from the NCI. The Orbitrap For experiments that required drug administration, recipient mice Fusion Lumos mass spectrometer used in this study was purchased were treated with vehicle or venetoclax (100 mg/kg; Selleckchem) with a shared instrumentation grant (1S10OD010582-01A1) from daily by oral gavage from days 6 to 20. Venetoclax was prepared in the NIH. Also, we thank C. Quirin and L. Scorrano laboratory for the 10% ethanol, 30% polyethyleneglycol-400 (Sigma), and 60% phosal recombinant cBID. Finally, we thank L. Pernas and M.E. Soriano for 50 propylene glycol (Lipoid). Whole-body bioluminescent imaging critical reading of the manuscript. I. Aifantis was supported by the was performed at indicated time points by intraperitoneal injection NIH and the NCI (R01CA173636, R01CA216421, R01CA133379, and of luciferin (Goldbio) at a 50 mg/kg concentration, and imaging was R01CA169784), the Leukemia and Lymphoma Society (TRP#6340-11 performed after 5 minutes using an IVIS imager. Bioluminescent and LLS#6373-13), the Chemotherapy Foundation, the Taub Founda- signals (radiance) were quantified using Living Image software with tion, and The Alex’s Lemonade Stand Foundation for Childhood Can- standard regions of interest rectangles. Peripheral blood of recipient cer. The work was also supported by the New York State Department NSG mice was collected at indicated time points after transplanta- of Health (#CO030132, C32587GG, and C32563GG). R. Tibes was tion, and sgRNA-positive cells (GFP+) were analyzed by flow cytom- supported by the Leukemia and Lymphoma Society (CDA#2312-15 etry. All animal experiments were performed in accordance with and TRP#6080-12) and NCI grant R01-CA17897. protocols approved by the New York University Institutional Animal Care and Use Committee (ID: IA16-00008_TR1). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby Data and Software Availability marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Gene Expression Omnibus: All newly generated RNA-seq data were deposited under accession number GSE125403. Received January 27, 2019; revised April 15, 2019; accepted April 30, 2019; published first May 2, 2019. Disclosure of Potential Conflicts of Interest E. Gavathiotis has ownership interest (including stock, patents, etc.) in Stelexis Therapeutics and Selphagy Therapeutics, and is a consultant/advisory board member for Stelexis Therapeutics, Selphagy REFERENCES Therapeutics, and Life Biosciences. R. Tibes is a consultant/advisory 1. Campos L, Rouault JP, Sabido O, Oriol P, Roubi N, Vasselon C, board member for AbbVie. No potential conflicts of interest were et al. High expression of bcl-2 protein in acute myeloid leuke- disclosed by the other authors. mia cells is associated with poor response to chemotherapy. 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Targeting Mitochondria Sensitizes AML to Venetoclax RESEARCH ARTICLE

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Targeting Mitochondrial Structure Sensitizes Acute Myeloid Leukemia to Venetoclax Treatment

Xufeng Chen, Christina Glytsou, Hua Zhou, et al.

Cancer Discov 2019;9:890-909. Published OnlineFirst May 2, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/2159-8290.CD-19-0117

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