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CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY

5-Azacitidine Induces NOXA to Prime AML Cells forVenetoclax-Mediated Apoptosis A C

Sha Jin1, Dan Cojocari1, Julie J. Purkal1, Relja Popovic2, Nari N. Talaty3, Yu Xiao1, Larry R. Solomon1,Erwin R. Boghaert1, Joel D. Leverson4, and Darren C. Phillips1

ABSTRACT◥

Purpose: Patients with acute myeloid leukemia (AML) frequent-ly do not respond to conventional therapies. Leukemic cell survivaland treatment resistance have been attributed to the overexpressionof B-cell lymphoma 2 (BCL-2) and aberrant DNA hypermethyla-tion. In a phase Ib study in elderly patients with AML, combiningthe BCL-2 selective inhibitor venetoclax with hypomethylatingagents 5-azacitidine (5-Aza) or decitabine resulted in 67% overallresponse rate; however, the underlying mechanism for this activityis unknown.

Experimental Design:We studied the consequences of combin-ing two therapeutic agents, venetoclax and 5-Aza, in AML preclin-ical models and primary patient samples. We measured expressionchanges in the integrated stress response (ISR) and the BCL-2 familyby Western blot and qPCR. Subsequently, we engineered PMAIP1(NOXA)- and BBC3 (PUMA)-deficient AML cell lines using

CRISPR-Cas9 methods to understand their respective roles indriving the venetoclax/5-Aza combinatorial activity.

Results: In this study, we demonstrate that venetoclax and 5-Azaact synergistically to kill AML cells in vitro and display combina-torial antitumor activity in vivo. We uncover a novel nonepigeneticmechanism for 5-Aza–induced apoptosis in AML cells throughtranscriptional induction of the proapoptotic BH3-only proteinNOXA. This induction occurred within hours of treatment and wasmediated by the ISR pathway. NOXA was detected in complex withantiapoptotic proteins, suggesting that 5-Aza may be “priming” theAML cells for venetoclax-induced apoptosis. PMAIP1 knockoutconfirmed its major role in driving venetoclax and 5-Aza synergy.

Conclusions: These data provide a novel nonepigenetic mech-anism of action for 5-Aza and its combinatorial activity withvenetoclax through the ISR-mediated induction of PMAIP1.

IntroductionAcute myeloid leukemia (AML) is a clonal hematologic malignancy

characterized by genomic heterogeneity and epigenetic changes,including aberrant DNA methylation (1). Hypomethylating agents(HMAs), such as the cytidine analogs 5-azacitidine (5-Aza) anddecitabine, demonstrate single-agent activity in 25% to 50% of patientswith myelodysplastic syndrome (MDS; ref. 2) and myeloproliferativeneoplasms (3), and are active in about 15% to 29% of AML (4, 5).Following cellular uptake, 5-Aza is metabolized and incorporated intoboth DNA and RNA to drive distinct cellular responses. Many of5-Aza's epigenetic effects can be attributed to its incorporation intoDNA to deplete DNA methyltransferase (DNMT) and drive DNAhypomethylation (6, 7). However, 80% to 90%of 5-Aza is incorporatedinto RNA (8–10), which may drive nonepigenetic effects (11, 12)including apoptosis (13, 14). This may account for some of 5-Aza'sclinical activity because DNA hypomethylation is not predictive ofclinical response to 5-Aza in myeloid malignancies (15).

Apoptosis is primarily regulated at the mitochondrial level by theB-cell lymphoma protein-2 (BCL-2) family of proteins. This col-lection of cell death regulators is divided into three groups that eachcontains at least one BCL-2 homology (BH) motif (BH1-4). Theproapoptotic “BH3-only” proteins BIM, BID, PUMA, NOXA, BAD,BIK, BMF, and HRK, and the “multidomain effector” proteins BAXand BAK are activated or induced by various cell death stimuli thatdrive mitochondrial outer membrane permeabilization and subse-quently apoptosis (16). The antiapoptotic members (BCL-2, BCL-XL, MCL-1, BCL-W, and BCL2-A1) possess BH3-binding groovesthat function to constrain the “BH3-only” and multidomain effec-tors. Aberrant expression and/or function of BCL-2 family proteinsare integral to tumorigenesis and therapeutic resistance by enablingmalignant cells to evade apoptosis (16, 17). Deletion of the genesencoding the BH3-only proteins BIM (BCL2L11), PUMA (BBC3),or NOXA (PMAIP1) can drive resistance to various apoptosisstimuli (18–22) and accelerates tumorigenesis in mouse models ofhematologic malignancies (23–25). Similarly, BCL-2 overexpressionis a clinical feature of human hematologic malignancies (26–28)that exacerbates the malignant state (29) and drives apoptosisresistance phenotypes (30), including that induced by 5-Aza (13).Consequently, targeting apoptosis signaling, and in particular BCL-2 itself, has emerged as a tractable therapeutic approach inoncology.

Venetoclax (ABT-199) is a highly selective, orally bioavailableBCL-2 inhibitor that induces apoptosis in BCL-2–dependent tumorcells (31), and is approved by the FDA in the United States for use inpatients with small lymphocytic lymphoma or chronic lymphocyticleukemia, whohave tried at least one therapy (32). Further, in a phase IImonotherapy study in patients with R/R AML, venetoclax has dem-onstrated an overall response rate (ORR) of 19% (33), providing thefoundation for combinational studies in AML. Subsequent phase Ibdata in treatment-na€�ve patients with AML indicate that combiningvenetoclax with 5-Aza or decitabine results in an ORR of 67% (34),

1Oncology Discovery, AbbVie Inc., North Chicago, Illinois. 2Genomics ResearchCenter, AbbVie Inc., North Chicago, Illinois. 3Drug Discovery Science andTechnologies, AbbVie Inc., North Chicago, Illinois. 4Oncology Development,AbbVie Inc., North Chicago, Illinois.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

S. Jin and D. Cojocari contributed equally to this article.

Corresponding Author: Darren C. Phillips, AbbVie Inc, 1 North Waukegan Road,North Chicago, IL 60064. Phone: 847-938-8508; Fax: 847-935-5165; E-mail:[email protected]

Clin Cancer Res 2020;26:3371–83

doi: 10.1158/1078-0432.CCR-19-1900

�2020 American Association for Cancer Research.

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comparedwith the historical ORRof 15% to 29%withHMA treatmentalone (5, 35–37). These encouraging data have led to the initiation ofrandomized phase III trials to evaluate venetoclax activity in combi-nation with 5-Aza in elderly treatment-na€�ve AML patients ineligiblefor standard induction therapy (M15-656, NCT02993523). However,the underlying mechanism for the combinational activity observedbetween venetoclax and 5-Aza is unknown.

Herein, we demonstrate that acute 5-Aza treatment drives com-binatorial activity with venetoclax independent of epigeneticeffects (6, 7). Specifically, we determine that, at pharmacologicallyrelevant concentrations (38), 5-Aza activates the integrated stressresponse (ISR) pathway to induce expression of the canonical targetDDIT3, as well as the BH3-only proteins NOXA (PMAIP1) andPUMA (BBC3) in human AML cell lines, priming them for apo-ptosis independent of DNA demethylation. We subsequently dem-onstrate that NOXA induction is not only required for the synergyobserved between 5-Aza and venetoclax, but PMAIP1 deletionsignificantly affects the kinetics and depth of response to eitheragent alone or in combination. Together, these data provide arationale for an ongoing randomized phase III clinical trial eval-uating venetoclax activity in combination with 5-Aza (M15-656,NCT02993523), and advocate for the assessment of PMAIP1 andDDIT3 gene induction in patients treated with 5-Aza and veneto-clax as potential biomarkers of response.

Materials and MethodsCell culture and reagents

AML cell lines OCI-AML5 (RRID:CVCL_1620), SET-2 (CVCL_2187), PL-21 (CVCL_2161), SKM-1 (CVCL_0098), MOLM-13 (CVCL_2119), SKNO-1 (CVCL_2196), SHI-1 (CVCL_2191), and NOMO-1(CVCL_1609) were purchased from DSMZ; and HEL (CVCL_2481),U-937 (CVCL_0007), MV4-11 (CVCL_0064), Kasumi-1 (CVCL_0589),KG-1 (CVCL_0374), and THP-1 (CVCL_0006) were purchasedfrom the ATCC and cultivated for 1 to 10 passages in RPMI-1640medium, 20 mmol/L HEPES (Gibco) supplemented with penicillin/streptomycin and 10% FBS (Invitrogen). Cells were grown at 37�Cin a humidified atmosphere with 5% CO2. All cell lines were testedfor authenticity by short tandem repeat profiling and mycoplasmaby the AbbVie Core Cell Line Facility. Primary AML cells fromperipheral blood were purchased from Discovery Life Sciences,collected with informed consent from patients, and cultured over-night in media described above, plus 30 U/mL of IL2, prior to 5-Azatreatment. AML-340 and AML-343 (stage M2) were newly diagnosed,

untreated; AML-69 had previous treatment: gemtuzumab ozogamicin,and decitabine, treatment at time of collection: venetoclax; AML-3667treatment at time of collection:mercaptopurine. Venetoclax and 5-Azawere obtained from AbbVie chemical library. ISRIB (trans-isomer)was purchased from Selleck Chemicals (S7400).

Cell viabilityAML cell lines were seeded at 10,000 cells per well in 96-well plates

and treated with 5-Aza and or venetoclax for 24 hours. For combi-natorial studies, cells were treated inmatrix of nine doses of venetoclax(10mmol/L, 1:3 dilutions) and threedoses of 5-Aza, 0.3, 1, and 3mmol/L.Cell viability was subsequently determined using CellTiter-Glo reagentas described by the manufacturer's instructions (Promega Inc.). Theeffective concentration to induce 50% cell death (EC50) was determinedby nonlinear regression algorithms using Prism 7.03 (GraphPadSoftware).

Western blottingAML cells were treated with 0.3, 1, 3, 10, or 30 mmol/L of 5-Aza

for 24 hours. To inhibit caspase activation, cells were pretreatedwith 40 mmol/L Z-VAD-fmk for 1 hour prior to treatment with5-Aza for a further 24 hours. Protein concentration was quantifiedusing Pierce bicinchoninic acid assay (ThermoFisher Scientific),and equal amounts of cellular protein samples were separated with4% to 12% SDS-PAGE (Invitrogen) and blotted to nitrocellulose orPVDF membranes (Invitrogen). The blots were incubated with thefollowing primary antibodies: anti-BIM (Cell Signaling Technology;catalog No. 2933, RRID:AB_1030947), anti–BCL-2 (Abcam; catalogNo. ab32124, RRID:AB_725644), anti–BCL-XL (Abcam; catalog No.ab32370, RRID:AB_725655), anti–MCL-1 (Cell Signaling Technol-ogy; catalog No. 94296, RRID:AB_2722740), anti-PUMA (Abcam;catalog No. ab9645, RRID:AB_296538), anti-NOXA (Abcam; cat-alog No. ab13654, RRID:AB_300536), anti-PARP (BD Biosciences;catalog No. 556494, RRID:AB_396433), anti–caspase-3 (Abcam;catalog No. ab13585, RRID:AB_300480), ani-DNMT1 (Cell Signal-ing Technology; catalog No. 5032, RRID:AB_10548197), anti-ATF4(Cell Signaling Technology; catalog No. 11815, RRID:AB_2616025),anti-CHOP (Cell Signaling Technology; catalog No. 2895, RRID:AB_2089254), anti-eIF2a (Cell Signaling Technology; catalog No.2103, RRID:AB_836874), anti–phospho-eIF2a (S51) (Cell SignalingTechnology; catalog No. 3398, RRID:AB_2096481), anti–b-actin(Sigma-Aldrich; catalog No. A2228, RRID:AB_476697), and anti-GAPDH (Abcam; catalog No. ab110305, RRID:AB_10861081). Theblots were imaged using the Odyssey CLx (Li-Cor) followingincubation in with the IRDye secondary antibodies (LI-COR Bio,AB_10795014; AB_10796098). Approximate protein molecularsize was calculated from protein size standards in Image Studio5.0 (LI-COR).

ImmunoprecipitationCells were pretreated with 40 mmol/L zVAD-fmk 1 hour prior to

treatment with 0.3, 1, 3, or 10 mmol/L of 5-Aza for a further 24 hours.Cellular proteins were extracted and centrifuged in 1% CHAPs buffer.Cell lysates (250 mg) were incubated with 3 mg of biotinylated anti–BCL-2 (US Biological, catalog No. B0807-06F) or BCL-XL (R&Dsystems, catalog No. DYC894-2) or MCL-1 (BD Bioscience, catalogNo. 624008 custom biotinylation) or IgG control (US Biological,B1750-06X) antibodies in the presence of protease inhibitors (com-plete tablets, Roche) overnight at 4�C. Streptavidin beads (Sigma) wereadded to precipitate complexes containing BCL-2, BCL-XL, or MCL-1prior to separation by SDS-PAGE.

Translational Relevance

The FDA recently approved the combination of venetoclax withhypomethylating agents in elderly patients with acute myeloidleukemia (AML); however, the molecular mechanism behind thecombinatorial activity is unknown. We show that 5-azacitidine(5-Aza) and venetoclax provided added antitumorigenic benefitrelative to either agent alone in preclinical models of AML. Wecharacterize the 5-Aza–mediated apoptotic priming of AML cellslinked to the induction of the proapoptotic protein NOXA and itsbinding to antiapoptotic BCL-2 family proteins. This studyuncovers a nonepigenetic mechanism for the combinatorial activ-ity between venetoclax and 5-Aza, and highlights a central role forNOXA in venetoclax-induced apoptosis.

Jin et al.

Clin Cancer Res; 26(13) July 1, 2020 CLINICAL CANCER RESEARCH3372

DNA methylation analysisAML cells were treatedwith 0.3 or 3mmol/L of 5-Aza for 24 hours or

7 days, replenished on days 0, 2, 4, and 6. For methylation analysis,500 ng ofDNAwas denatured by heating the sample at 100�C. Sampleswere treated with 5 units of Nuclease P1 (Sigma) in reaction buffer(5 mmol/L ZnCl2, 50 mmol/L NaCl) at 50�C for 1 hour. Samples werefurther treated with 0.002 units of phosphodiesterase I (Sigma) at 37�Cfor 2 hours followed by treatment with 0.5 U of alkaline phosphatase(Invitrogen) for 1 hour at 37�C. The samples were diluted threefold todilute out the salts and enzymes, and injected into a Acquity UPLCHSST3 2.1-mmx50-mmcolumn (1.8-mmparticle size;Waters catalogNo. 186003538). Samples were run on a ShimadzuNexera-X2UHPLCcoupled to an ABSciex 6500 Triple Quadrupole Mass Spectrometer.Quantification was performed using the Analyst 1.6.2 Workstationsoftware using the intelliQuan algorithm in a multiple reactionmonitoring mode. All runs included standard curves for 5-hmC,5-mC, and 5-dC. Linear regression was performed to obtain slopesand intercepts, whichwere used to calculate the actual % 5-mdC and%5-hmdC values. All standards and samples were matrix matched andtreated with same dilutions.

Cell line–derived xenograft models of AMLSKM1.FP1 is a cell line derived from a subcutaneous tumor

generated by the injection of 2 � 106 of SKM-1 cells (DSMZ).SKM1.FP1 and MV4-11 (ATCC) were cultured as described above.Female Fox Chase SCID Beige (RRID: IMSR_CRL:250; for SKM1.FP1;8–10 weeks, 18–20 g) and female Fox Chase SCID (RRID:IMSR_CRL:236; for MV4-11; 8–10 weeks, 18–20 g) mice were pur-chased from Charles River Laboratories. Body weights upon arrivalwere 18 to 20 g. Food and water were provided ad libitum. Animalswere on the light phase of a 12-hour light: 12-hour dark cycle. Allanimal studies were conducted in accordance with the guidelinesestablished by the AbbVie Institutional Animal Care and Use Com-mittee. In flank xenograft experiments, mice were inoculated with 2�106 (SKM1.FP1) or 5 � 106 (MV4-11) cells subcutaneously into theright flank. Mice were injected with a 0.1-mL inoculum of 1:1 cellmixture in culture media and Matrigel (BD Biosciences). Whentumors reached approximately 225 mm3, the mice were size-matched into treatment and control groups. Mice were treated QDx14PO 50 mg/kg with venetoclax, Q7Dx3 IV 8 mg/kg with 5-Aza, orcombination of both. Venetoclax was formulated in 60% phosal 50propylene glycol, 30% polyethylene glycol 400, and 10% ethanol, and5-Aza was formulated with 0.9% sodium chloride. Tumor volume wasmeasured twice per week with electronic calipers and calculatedaccording to the formula (L x W2)/2. All treatment groups consistedof eight mice per group.

Caspase-3/-7 activity time courseNote that 96-well clear-bottom black polystyrene microplates

(Corning) were coated with CellTAK (Corning) as recommended bythe manufacturer. For each well, 5� 104 cells were seeded in 50 mL ofRPMI media and then placed in the tissue culture incubator for 20minutes. The IncuCyte Caspase-3/-7 Red Apoptosis Assay Reagent(Sartorius) and the compounds were dispensed into the correspondingwells and dilutions using the D300 Digital Dispenser (Tecan). Theassay plate was then placed in the IncuCyte ZOOM (Sartorius) andprogrammed to take four images per well at a 1-hour interval for24 hours. Data were analyzed using the IncuCyte Zoom 2017 softwareand plotted as the number of red objects (active Caspase-3/7þ, aCasp-3/-7þ) per well divided by area (in mm2) occupied by the cells, per well.

RT-qPCRNote that 1.0� 107 cells, for AML cell lines, and 2.5� 105 cells per

well, for primary AML cells, were treated with DMSO or 0.3, 1, or3 mmol/L of 5-Aza, for 6 and 24 hours, at which point the cells werewashed with 10 mL of cold DPBS and spun down at 4�C, 300 rcf for5minutes. The supernatant was removed, and the cells were lysed with400 mL of RLT buffer containing b-mercaptoethanol. RNA wasisolated using Qiagen RNeasy Plus Mini Kit (74134) for cell lines,and Micro Kit (74034) for primary samples, as recommended bymanufacturer. cDNA was prepared from 3 mg of purified RNA usingiScript Reverse Transcription Supermix (Bio-Rad) protocol. qPCRwasperformed on 2 mL of cDNA reaction, TaqMan Fast Advanced(Life Technologies, No. 4444557) in 20-mL final volume. The followingPrimeTime qPCR Probes were manufactured by Integrated DNATechnologies (IDT): HEX dye probe RPLP0 (reference gene, Hs.PT.39a.22214824); 6-FAM probes: PMAIP1 (Hs.PT.58.21318159),BBC3 (Hs.PT.5839966045), DDIT3 (Hs.PT.58.39204289.g), CDKN1A(Hs.PT.47.2442322; Supplementary Table S1). The cDNA standards(fivefold dilutions) were prepared from AML5 and Kasumi-1 cellstreated with SN-38 for 24 hours. qPCR was performed in 96-well PCRplates (Bio-Rad) on the Bio-Rad CFX 96. Gene expression (DDCq) wasquantified on the Bio-Rad CFX Manager 3.1. Target 6-FAM probeswere normalized to HEX-GAPDH control gene and relative to vehiclecontrol treated 0 mmol/L 5-Aza.

Knockout cell linesGenomic editing for elimination of BBC3 (PUMA) and PMAIP1

(NOXA) was done using the ALT-R CRISPR-Cas9 system from IDT.All reagents were from IDT, and RNP complexes were formed asdescribed by IDT. Briefly, the CRISPR RNA (crRNA; SupplementaryTable S1)was annealed to tracRNA labeledwithATTO550fluorescentreagent. The annealed product was combined with Alt-R S.p. Cas9Nuclease V3 (IDT) and subsequently introduced into the OCI-AML5,Kasumi-1, and MV4-11 cells by electroporation. The electroporationwas donewith aNEONTransfection System (Thermo Fisher) contain-ing 2 � 106 cells using the following settings: OCI-AML5 (1 � 30 mspulse @ 1,500 V), Kasumi-1 (1� 20ms pulse @ 1,700 V), andMV4-11(1 � 20 ms pulse @ 1,700 V). Transfection efficiency was monitoredusing the ATTO 550 fluorescent reagent. At 24 hours followingtransfection, the top 5% ATTO 550þ cells were sorted by fluores-cence-activated flow cytometry (BD FACSAria Fusion) under sterileconditions as a single cell per well of a 96-well plate and were used toisolate clonal cell lines. The expression of either NOXA or PUMAprotein was monitored using Western blot analysis. Genomic DNAfrom clones that showed lack of expression of the intended target wasamplified with PCR primers flanking the site of the target CRISPRcrRNA. Primers for PCR and sequencing are shown in SupplementaryTable S2. The sequencing data were analyzed using Vector NTIExpress (Thermo Fisher) to confirm successful genomic editing.

Statistical analysisData are shown as mean and SEM of at least three independent

experiments and two technical replicates. Groups were statisticallycompared using the Student t test or one-way and two-way ANOVAfor multiple comparisons using Prism 7.03 (GraphPad Software).Asterisks on graphs denote a significant difference (�, P < 0.05), andns for not significant (ns, P > 0.05). Spearman correlation and linear fit� 95% confidence intervals was used for correlation of gene expressionand bliss sums. The Bliss independence model was used to evaluatecombinatorial activity, with positive integers indicating synergy (39).

Aza-Induced NOXA Prime AML for Venetoclax-Mediated Apoptosis

AACRJournals.org Clin Cancer Res; 26(13) July 1, 2020 3373

Figure 1.

Chronic or acute 5-Aza synergizeswith venetoclax inAML cell lines in vitro andprovides addedbenefit over either agent alone in xenograftmodels of AML.A,AMLcelllines MV4-11, OCI-AML5, SET-2, and SHI-1 were treatedwith 300 nmol/L of 5-Aza on days 0, 2, 4, and 6, followed bymeasurements of relative 5-methylcytosine (mC)in DNA on day 7 (n ¼ 3; � , P < 0.05; ns, not significant). B, The 5-Aza pretreated cells (7 days, 300 nmol/L) were washed and treated with the indicated doses ofvenetoclax for 24 hours, at which point cell viability was assayed (n ¼ 3). C, Cells with active Caspase-3/-7þ (aCasp-3/-7þ) were counted over time followingtreatment with venetoclax, 5-Aza (3 mmol/L), and venetoclax in combination with 5-Aza in OCI-AML5 and MV4-11 cell lines (n ¼ 3). D, AML cell lines SKM-1, PL-21,OCI-AML5, and MV4-11 were cotreated with the indicated doses of 5-Aza and venetoclax for 24 hours at which point cell viability was assayed (n ≥ 3). E, Synergisticactivity between 5-Aza and venetoclax was quantified using the Bliss independence model and the total synergy across the combination matrix determined (BlissSum; n ≥ 3). F, SKM-1 and MV4-11 xenograft models were treated (gray bar) with 5-Aza (8 mg/kg, Q7Dx3, IV), venetoclax (50 mg/kg, QDx14, PO), or venetoclax incombination with 5-Aza, and the effect on tumor growth determined. Data are presented as themean tumor volume� SEM obtained from eight mice per treatmentgroup.

Jin et al.


Bliss scores were calculated for each combination in the dose matrixand totaled to give a “Bliss sum” value.

ResultsChronic or acute 5-Aza treatment synergizes with venetoclax inpreclinical models of AML

5-Aza is a known DNA hypomethylation agent (40). To determineits hypomethylating potential in vitro, we treated AML cell lines withvarious doses of 5-Aza, replenished every 48 hours, over a total periodof 7 days. No difference in methylation was observed within the first24 hours of treatment of Kasumi-1 or SET-2 cells (SupplementaryFig. S1A). However, 5-Aza treatment for 7 days at the 300 nmol/L dosewas able to decrease global DNA methylation in four AML cell linestested (Fig. 1A). When these 5-Aza pretreated cells were then exposedto venetoclax for an additional 24 hours, OCI-AML5, Kasumi-1, andMV4-11 cell lines were found to be more sensitive to venetoclaxcompared with the DMSO pretreated cells (Fig. 1B). In contrast, theSHI-1 and SET-2 cell lines were not sensitized to venetoclax (Supple-mentary Fig. S1B). In humans, 5-Aza has an elimination half-life ofapproximately 4 hours, reaching maximum plasma concentrations ofapproximately 11 mmol/L, with intravenous dosing of 75 mg/m2 (38).To capture these acute clinical parameters in vitro in the context ofvenetoclax cotreatment, we measured the kinetics and number of cellswith active caspase-3/-7 (aCasp-3/-7þ) immediately following theaddition of the two agents to OCI-AML5 and MV4-11. The combi-nation of 5-Aza and venetoclax resulted in a rapid induction in thenumber of aCasp-3/-7þ cells that was greater than either agent alone(Fig. 1C). To further explore this observation, we evaluated the acutecombinatorial activity of venetoclax and 5-Aza in a panel of 14 AMLcell lines treated for 24 hours at various doses of either agent. In thissetting, 5-Aza significantly sensitized the AML cell lines to venetoclax-induced cell death (Fig. 1D; Supplementary Fig. S1C) and showedsynergistic activity in 12 out of 14 AML cell lines, as measured by theBliss independence model (ref. 39; Fig. 1E). To further validate thisobservation, we generated cell line–derivedmouse xenografts of SKM-1 and MV4-11 and treated the mice with either 5-Aza (once per weekfor 3 weeks), venetoclax (daily for 14 days), or the combination of5-Aza and venetoclax (Fig. 1F). In both xenograft models, the tumorgrowth inhibition caused by the combination of the two agents wasincreased as compared with the either agent alone, consistent with thereduction of cell survival in vitro.

5-Aza upregulates NOXA and PUMA in AMLTo identify a molecular mechanism of 5-Aza in promoting apo-

ptosis in combination with venetoclax, we characterized the proteinexpression levels of both proapoptotic and antiapoptotic BCL-2 familymembers by Western blot following 5-Aza treatment for 24 hours.Through its previously reported target-binding activity (7), 5-Azacompletely depleted the DNMT1 protein. This was associated with aconcomitant dose- and time-dependent increase inNOXAandPUMAprotein expression (Fig. 2A; Supplementary Fig. S2). To examinewhether the upregulation of PUMA and NOXA was a cause orconsequence of cell death, we pretreated the cells with the pan-caspase inhibitor Z-VAD-fmk to abrogate the induction of apoptosis.In the presence of Z-VAD-fmk and 5-Aza, a robust upregulation ofNOXA and PUMA was detected (Fig. 2B). Further, qPCR indicatedthat 5-Aza was able to significantly increase both PMAIP1 (NOXA)and BBC3 (PUMA) transcripts in a dose-dependentmanner as early as6 hours after treatment,with longer treatment periods of up to 24hoursbeing associatedwith a further increase inBBC3 expression in theOCI-

AML5, Kasumi-1, and MV4-11 cell lines (Fig. 2C). Interestingly,synergy between venetoclax and 5-Aza significantly correlated withthe 5-Aza induction of PMAIP1 and BBC3 transcripts in a broaderAML cell line panel, and was independent of TP53 status (Fig. 2D;refs. 41, 42). Similarly, primary AML patient cells treated withincreasing doses of 5-Aza resulted in an upregulation of BBC3 (3/4patient samples) and PMAIP1 (4/4 patient samples) after 6 hours, withelevated BBC3 expression maintained at 24 hours after treatment in 2of 4 patient specimens (Fig. 2E). Chronic 5-Aza treatment did not haveany significant effect on the gene expression or methylation, as thepromoter regions for both PMAIP1 and BBC3 genes were unmethy-lated (Supplementary Fig. S3A) and their transcript levels wereunaltered (Supplementary Fig. S3B) in a sample of AML cell lines.The rapid induction of these two transcripts at 6 hours, coupled withthe low baselinemethylation of either PMAIP1 orBBC3 and absence ofmethylation changes following 5-Aza treatment, collectively indicateda nonepigenetic mechanism for transcriptional induction.

5-Aza primes the antiapoptotic proteins with NOXA and PUMATo determine the functional consequence of NOXA and PUMA

upregulation, we measured the binding of these two proteins to theantiapoptotic proteins BCL-2, BCL-XL, and MCL-1 in AML cell linesafter 5-Aza treatment, by immunoprecipitation. The amount ofNOXA bound to BCL-2 in Kasumi-1 and MV4-11 cells, and NOXAbound to MCL-1 in Kasumi-1, OCI-AML5, and MV4-11 cellsincreased in a dose-dependent fashion following 5-Aza treatment(Fig. 3). Although to a lesser degree than MCL-1, PUMA binding toBCL-2 and BCL-XL was also increased. These results collectivelyindicate that 5-Aza enhances the amount of BH3-only proteins foundin complex with the antiapoptotic BCL-2 family members.

The ISR induces NOXA following 5-Aza acute treatment5-Aza has been shown to also inhibit protein synthesis through

RNA incorporation (11). The ISR pathway is induced by various stresssignals, including proteotoxic stress. During conditions of severestress, the ISR's main effector, activating transcription factor 4 (ATF4),can tip the cell toward death through transcriptional induction ofproapoptotic targets (43). As the transcriptional upregulation of bothBBC3 and PMAIP1 appeared to occur in a p53-independent manner(Fig. 2D), we postulated that the transcription factor ATF4may play arole in the induction of these transcripts. ATF4 has been reported tobind to the PMAIP1 promoter to induce its transcription (44, 45). Theprotein levels of ATF4 increased significantly when the AML cell lineswere exposed to 5-Aza for 24 hours (Fig. 4A). This was indicative ofISR pathway activation and was confirmed by upstream phosphory-lation of serine 51 (S51) on the alpha subunit of the eukaryoticInitiation Factor 2 (eIF2a; Fig. 4A). In addition, we observed signif-icant induction of a transcriptional target of ATF4, CCAAT-enhanc-er–binding protein homologous protein (CHOP). In agreement withthe upregulation ofCHOP at the protein level (Fig. 4A, SupplementaryFig. S4A), its transcript,DDIT3,was also induced in a dose-dependentmanner by 5-Aza after 6-hour treatment of AML cell lines (Fig. 4B)and primary AML patient samples ex vivo (Fig. 4C), although the levelof DDIT3 induction was reduced after 24-hour 5-Aza treatment(Fig. 4C). The level of DDIT3 induction also correlated significantlywith the synergy observed between venetoclax and 5-Aza combinationin 14 AML cell lines (Fig. 4D). Finally, to explore whether the ISRpathway may be responsible for the induction of PMAIP1 and BBC3,we used ISRIB, a small molecule that acts as a potent inhibitor of thispathway by reversing the effect of eIF2a phosphorylation (46). OCI-AML5 cells were treated for 6 hours with ISRIB, 5-Aza, or ISRIB and



Figure 2.

5-Aza upregulates NOXA and PUMA in AML cells independent of cell death.A,AML cell lineswere treatedwith 5-Aza for 24 hours, and the effect on the expression ofBCL-2 family members, caspase-3, PARP, DNMT1, and b-actin determined byWestern blot analysis. Approximate protein molecular size calculated from protein sizestandards. B,OCI-AML5, MOLM-13, and MV4-11 cells were pretreated with 40 mmol/L Z-VAD-fmk for 1 hour prior to treatment with 5-Aza for a further 24 hours, andthe impact on MCL-1, PUMA, NOXA, caspase-3, PARP, DNMT1, and b-actin determined byWestern blot analysis. C, AML cell lines were treated with 5-Aza for 6 and24 hours, and the effect on PMAIP1 (NOXA) and BBC3 (PUMA) gene expression was quantified using qPCR (n ¼ 3). D, Fold change in PMAIP1 and BBC3 geneexpression by 5-Aza (3 mmol/L, 6 hours) was correlated with the Bliss sum of the 5-Aza and venetoclax combination for a panel of AML cell lines. The cell lines wereclassifiedby TP53 status labeled inblack, forwild-type, and in red, formutated/null TP53gene (n¼ 3, dotted line: 95%confidencebandsof the best-fit line).E,Primarycells from patients with AML were treated ex vivo with 5-Aza at the indicated concentrations and timepoints, and the effect on PMAIP1 (NOXA) and BBC3 (PUMA)gene expression was quantified using qPCR. Reported percent blasts from pathology and treatment (Tx) status.

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5-Aza in combination. As expected, 5-Aza induced the PMAIP1,BBC3, and DDIT3 transcripts; however, in the presence of ISRIB, the5-Aza–mediated induction of all three transcripts was significantlysuppressed (Fig. 4E, Supplementary Fig. S4B). ISRIB partially rescuedAML cells from 5-Aza–induced death (Supplementary Fig. S4C).ISRIB also induced significant resistance to the venetoclaxþ5-Azacombinatorial cell killing (Fig. 4F), enhancing the venetoclax AUC(Fig. 4G) and reducing the synergy between these two agents (Sup-plementary Fig. S4D).

Deletion of PMAIP1 abrogates 5-Aza and venetoclaxcombinatorial activity

The 5-Aza–mediated induction of NOXA and PUMA, as well astheir subsequent binding to the antiapoptotic BCL-2 family members,suggested a role in priming the AML cells for apoptosis. To understandthe importance of NOXA and PUMA in this context, CRISPR-Cas9technology was employed to delete PMAIP1 (Fig. 5A) and BBC3,respectively (Supplementary Fig. S5A). Following deletion of thePMAIP1 gene, no consistent changes were observed in the expressionof other BCL-2 family members across cell lines (Fig. 5A). The

OCI-AML5, Kasumi-1, and MV4-11 PMAIP1�/� cell lines were eachmore resistant to venetoclax-induced cell death compared with theirrespective parental cell lines. The Kasumi-1 and MV4-11 PMAIP1�/�

cell lines were also more resistant to 5-Aza–induced cell death(Fig. 5B). In agreement with the cell viability data, the kinetics ofinduction and number of aCasp-3/-7þ cells in PMAIP1-deficient celllines were substantially reduced in response to either venetoclax or5-Aza treatment (Fig. 5C). Because venetoclax and 5-Aza inducedbroad synergistic cell killing in AML cell lines, we assessed the impactof PMAIP1 deletion on this combination. PMAIP1 deletion reducedthemagnitude of cell death resulting from combined venetoclax/5-Azatreatment in MV4-11, OCI-AML5, and Kasumi-1 cells (Fig. 6A),which was associated with a loss of venetoclax potency (EC50) in thepresence of increasing concentrations of 5-Aza (Fig. 6B) and theabrogation of synergy (Fig. 6C).

Lastly, we hypothesized that PUMA might also play a role inpriming the cells for apoptosis. The gene encoding PUMA, BBC3,was deleted in the parental and the PMAIP1�/� cell lines, OCI-AML5andMV4-11. The crRNAwas designed to target both BH3-containingisoforms of PUMA, alpha and beta. Western blot analysis indicated

Figure 3.

5-Aza enhances the amount of NOXA and PUMA in complex with BCL-2 and MCL-1 in AML cell lines. OCI-AML5, Kasumi-1, and MV4-11 cells were pretreated with40 mmol/L Z-VAD-fmk for 1 hour prior to treatment with 5-Aza for an additional 24 hours. The association of PUMA or NOXA with MCL-1, BCL-2, or BCL-XL wasdetermined by immunoprecipitation (IP) and subsequent Western immunoblot (IB) analysis as described in the Materials and Methods.



Figure 4.

5-Aza activates the ISR pathway to transcriptionally induce PMAIP1 and BBC3. A, OCI-AML5, Kasumi-1 and MV4-11 cell lines were treated with increasing doses of5-Aza for 24 hours, and the ISRpathway activationwasmeasured by immunoblotting for CHOP, ATF4 proteins, and phosphorylation of eIF2a at serine 51. Total eIF2awas used as control for total protein loading. # denotes nonspecific band. Approximate protein molecular size calculated using protein size standards. B, AML celllineswere treatedwith 5-Aza for 6 and 24 hours, and the effect on PMAIP1 (NOXA) andBBC3 (PUMA) gene expressionwas quantified using qPCR (n¼ 3). C, Primarycells from patientswith AMLwere treated ex vivowith 5-Aza at the indicated concentrations and timepoints, and the effect onDDIT3 gene expressionwas quantifiedusing qPCR. Reported percent blasts from pathology and treatment (Tx) status. D, Fold change in PMAIP1 and BBC3 gene expression was correlated with the Blisssumof the 5-Aza and venetoclax combination for a panel of 15 AML cell lines (n¼ 3). E,OCI-AML5 cells treatedwith 5-Aza alone (3mmol/LPMAIP1, DDIT3,or 10mmol/L for BBC3) or in combination with ISRIB (200 nmol/L) for 6 hours followed by quantification of PMAIP1, BBC3, andDDIT3 transcripts using qPCR (n¼ 3; �, P < 0.05).F,Venetoclax dose–response curve treatedwith increasing doses of 5-Aza in thepresenceofDMSOor ISRIB (200nmol/L;n¼ 3).G,Area under the curve of the dose–response curves from F for OCI-AML5, Kasumi-1, and MV4-11 cell lines (n ¼ 3; � , P < 0.05).

Jin et al.


complete absence of PUMA at the protein level (SupplementaryFig. S5A). Unlike the PMAIP1�/� cell lines, the BBC3�/� OCI-AML-3 and MV4-11 cell lines responded similarly to venetoclaxand/or 5-Aza as the parental cell lines (Supplementary Fig. S5B andS5C). In addition, BBC3 deletion in the PMAIP1�/�AML cell lines didnot further affect the response of PMAIP1�/� cells to the venetoclax/5-Aza combination as measured by cell viability (SupplementaryFig. S5B). Taken together, our findings show that, although bothNOXA and PUMA are induced by 5-Aza treatment of AML cell lines,onlyNOXA is critical in sensitizing theAMLcells to 5-Aza/venetoclax-induced apoptosis.

DiscussionVenetoclax has been approved by the FDA for use in combination

with 5-Aza in the treatment of elderly patients with AML (aged ≥ 75years) who are ineligible for induction therapy based on the significantimprovement in ORRs (34). Herein, we utilize preclinical models of

AML to providemechanistic insights into the clinical activity observedbetween 5-Aza and venetoclax. At physiologically relevant concentra-tions (38), we demonstrate that 5-Aza combines with venetoclax inAML cell lines via nonepigenetic mechanisms requiring the inductionof NOXA via the ISR pathway.

Treatment of AML cell lineswith the combination of venetoclax and5-Aza induced the rapid induction of apoptosis that was greater thaneither 5-Aza or venetoclax as single agents, both in terms of rate andmagnitude of response. This corresponded with broad synergistic cellkilling observed across a panel of AML cell lines that was also reflectedin vivo, where the cotreatment of MV4-11 and SKM-1 xenograftmodels with 5-Aza and venetoclax inhibited tumor growth superiorto either agent alone. The rapid kinetics (<24 hours) of apoptosisassociated with the combinatorial activity between venetoclax and5-Aza indicate that although DNMT1 expression is eliminated, theresulting cell death was independent of changes in DNA methylation,which required a week of continuous treatment. Similarly, acute celldeath was previously reported in cells of patients with AML treated

Figure 5.

Deletion of PMAIP1 in AML cell lines abrogates venetoclax and 5-Aza activity.A,Western blot analysis of BCL-2 family expression in OCI-AML5, Kasumi-1, andMV4-11cell lines deficient in PMAIP1 compared with the respective parental cell line. Approximate protein molecular size calculated using protein size standards. B, Cellviability following 24 hours of treatment with either 5-Aza or venetoclax was compared for the parental and PMAIP1 knockout cell lines (n¼ 3; � , P < 0.05). C, Cellswith active caspase-3/-7 (aCasp-3/-7þ) over time following venetoclax or 5-Aza treatment of the parental and PMAIP1�/� cell lines OCI-AML5, Kasumi-1, andMV4-11(n ¼ 3).



ex vivo for 24 hours with the venetoclax/5-Aza combination (47),aligning with the rapid blast clearance observed within 24 to 72 hoursafter treatment in patients with AML treated with this combinationtherapy (48).

In the acute treatment setting, 5-Aza did not affect the expressionof antiapoptotic proteins, but it significantly induced the expressionof the proapoptotic BH3-only proteins PUMA and NOXA, inde-

pendent of their respective gene methylation status. Severalmechanisms beyond hypomethylation have been proposed to beresponsible for 5-Aza's activity (12–14), including DNA dam-age (11). However, although PMAIP1 and BBC3 are both p53response genes (21, 22, 49, 50), they were both induced independentof TP53 status following 5-Aza treatment, and were not associatedwith changes in p53 phosphorylation status (data not shown). We

Figure 6.

PMAIP1 deletion in AML cell lines abrogates combinatorial activity of venetoclax and 5-Aza. A, Dose–response curves for venetoclax and 5-Aza combination in theOCI-AML5 parental andPMAIP1�/�cell lines (n > 3).B, EC50 for the 5-Aza and venetoclax combinations in the parental andPMAIP1�/�OCI-AML5, Kasumi-1, andMV4-11 cell lines (n > 3; � , P < 0.05). C, Synergy between venetoclax and 5-Aza drug combination as measured by the Bliss independence model for the parental andPMAIP1�/� cell lines (n > 3; �, P < 0.05).

Jin et al.


subsequently hypothesized that the induction of these BH3-onlyproteins was not a result of DNA incorporation by 5-Aza (7, 40),but rather a consequence of RNA incorporation (8–10) andinhibition of protein synthesis (11), which may result in activationof ISR. The regulatory region of PMAIP1 contains the binding sitesfor over 40 different transcription factors and coactivators includ-ing those mediated by DNA damage, hypoxia, epigenetic regula-tion, metabolic stress, proteasome inhibition, autophagy, and ISR.One of these transcription factors and ISR component, ATF4,induces PMAIP1 transcription and enhances the expression ofNOXA protein in a p53-independent manner, following treatmentof cells with proteotoxic agents (44, 45). Exposure of AML cell linesto 5-Aza resulted in the activation of the ISR pathway, as evident byCHOP and ATF4 induction coupled with phosphorylation ofeIF2a (S51). Subsequent pharmacologic inhibition of ISR pre-vented the 5-Aza–mediated induction in PMAIP1, BBC3, andDDIT3. Importantly, the degree of PMAIP1, BBC3, or DDIT3expression induced following 5-Aza treatment correlated with thedegree of synergy observed between venetoclax and 5-Aza in AMLcell lines in vitro. A recent study identified that the venetoclax and5-Aza combination killed leukemic stem cells from patients withAML by decreasing amino acid uptake and mitochondrial respi-ration (47). Interestingly, amino acid deprivation activates the ISRthrough general control nonderepressible-2 kinase signaling (43),offering a potential mechanism for the ISR activation observed inour study.

The capacity of the antiapoptotic BCL-2 family members tosequester or buffer elevations in their proapoptotic counterpartsthrough direct protein–protein interactions functions to thwart theexecution of apoptosis (16). In this context, MCL-1 functions as aresistance factor to venetoclax, operating in part as a sink tosequester BH3-only proteins released from BCL-2 upon venetoclaxbinding (51). 5-Aza enhanced the association of NOXA withMCL-1 in a dose-dependent fashion, potentially neutralizing theantiapoptotic capacity of this protein similar to one of severalmechanisms ascribed to the activity of bortezomib in combinationwith venetoclax in multiple myeloma (52). Although full-lengthNOXA binds MCL-1 with a Kd of 3.4 nmol/L, it also possesses adissociation constant of 250 nmol/L for BCL-2 (53). Reflecting thesedata, 5-Aza treatment also consistently enhanced the interactionsbetween NOXA and BCL-2 in AML cell lines, providing an additionalmechanism bywhich this hypomethylating agentmay potentially primeAML cells for apoptosis induction by venetoclax. 5-Aza–mediatedPMAIP1 induction correlated with venetoclax/5-Aza synergy in AMLcell lines in vitro; the failure to induce PMAIP1 associated with the poorcombinatorial activity observed between these two agents in THP-1 andSET-2 cells.

To further understand the impact of NOXA or PUMA on thecombinatorial activity between venetoclax and 5-Aza, we utilizedgene editing technology to delete PMAIP1 and/or BBC3, respec-tively, in OCI-AML-5, Kasumi-1, and MV4-11 cells. PMAIP1deletion, but not BBC3 deletion, significantly inhibited the synergybetween venetoclax and 5-Aza, additionally reducing the kineticsand magnitude of caspase-3/-7 activation when compared with theparental cell lines. BBC3 deletion did not inhibit apoptosis inducedby either venetoclax or 5-Aza and did not further enhance theresistance to venetoclax activity mediated by PMAIP1 deletion.Although these data contrast with DNA damage–induced apopto-sis, where PUMA drives most of the apoptosis with partial con-tributions from NOXA (54), our observations that PMAIP deletion

restricts apoptosis induced by venetoclax single-agent treatmentalign with recent studies demonstrating that elevated PMAIP1expression is responsible for enhanced sensitivity to venetoclax inpreclinical models of diffuse large B-cell lymphoma (55) andneuroblastoma (56). What is more, loss of PMAIP1 inhibited5-Aza–induced cell death in two of three AML cell lines assessed,complementing data that demonstrate NOXA peptides can dis-criminate the clinical responses of patients with AML, MDS, andMDS/PMN treated with 5-Aza (13).

Collectively, these data indicate that NOXA expression is a keydeterminant of venetoclax activity, both as a monotherapy and inthe combination setting with 5-Aza. In addition, this work demon-strates that, at pharmacologically relevant concentrations, 5-Azainduces NOXA through the ISR pathway to sensitize AML cells tovenetoclax in preclinical models of this malignancy. These dataprovide a rationale behind the ongoing randomized phase IIIclinical trial evaluating the activity of venetoclax in combinationwith 5-Aza in treatment-na€�ve subjects with AML who are ineligiblefor induction therapy (M15-656, NCT02993523). Consequently,understanding the association of NOXA and CHOP expression inpatients with AML in relation to venetoclax clinical activity is ofsignificant interest.

Disclosure of Potential Conflicts of InterestS. Jin is an employee of AbbVie. D. Cojocari is an employee of AbbVie. J.J. Purkal is

an employee of AbbVie. R. Popovic is an employee of AbbVie. N.N. Talaty is anemployee of and holds ownership interest (including patents) in AbbVie. Y. Xiao is anemployee of AbbVie. E.R. Boghaert is an employee of and holds ownership interest(including patents) in AbbVie. J.D. Leverson is an employee of and holds ownershipinterest (including patents) in AbbVie. D.C. Phillips is an employee of, and holdsownership interest in AbbVie, and is listed as a coinventor on a patent regarding adiagnostic test to predict sensitivity of non-Hodgkin's lymphoma (NHL) patients toBCL-2Antagonists andABT-199 (Venetoclax) that is owned byAbbVie. No potentialconflicts of interest were disclosed by the other authors.

Authors’ ContributionsConception and design: S. Jin, D. Cojocari, R. Popovic, N.N. Talaty, L.R. Solomon,E.R. Boghaert, J.D. Leverson, D.C. PhillipsDevelopment ofmethodology: S. Jin,D.Cojocari, J.J. Purkal, R. Popovic,N.N.Talaty,Y. Xiao, L.R. Solomon, D.C. PhillipsAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): S. Jin, D. Cojocari, J.J. Purkal, N.N. Talaty, Y. Xiao, E.R. BoghaertAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Jin, D. Cojocari, J.J. Purkal, R. Popovic, N.N. Talaty,Y. Xiao, L.R. Solomon, E.R. Boghaert, J.D. Leverson, D.C. PhillipsWriting, review, and/or revision of the manuscript: S. Jin, D. Cojocari, J.J. Purkal,R. Popovic, N.N. Talaty, Y. Xiao, E.R. Boghaert, J.D. Leverson, D.C. PhillipsAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): S. Jin, D. Cojocari, N.N. Talaty, D.C. PhillipsStudy supervision: E.R. Boghaert, D.C. Phillips

AcknowledgmentsThe design, study conduct, and financial support for this research were provided

by AbbVie Inc. AbbVie Inc. participated in the interpretation of data, review, andapproval of this publication.

We would like to acknowledge LorenM. Lasko from AbbVie Oncology Discoveryfor his technical expertise in single-cell sorting of the knockout cells.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 12, 2019; revised December 13, 2019; accepted February 10, 2020;published first February 13, 2020.



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