CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY

AZD4573 Is a Highly Selective CDK9 Inhibitor ThatSuppresses MCL-1 and Induces Apoptosis in HematologicCancer Cells A C

Justin Cidado1, Scott Boiko1, Theresa Proia1, Douglas Ferguson2, Steven W. Criscione1,Maryann San Martin1, Petar Pop-Damkov2, Nancy Su3, Valar Nila Roamio Franklin4,Chandra Sekhar Reddy Chilamakuri4, Clive S. D'Santos4, Wenlin Shao5, Jamal C. Saeh5, Raphael Koch6,David M. Weinstock7, Michael Zinda1, Stephen E. Fawell1, and Lisa Drew1

ABSTRACT◥

Purpose: Cyclin-dependent kinase 9 (CDK9) is a transcriptionalregulator and potential therapeutic target for many cancers. Mul-tiple nonselective CDK9 inhibitors have progressed clinically butwere limited by a narrow therapeutic window. This work describes anovel, potent, and highly selective CDK9 inhibitor, AZD4573.

Experimental Design: The antitumor activity of AZD4573 wasdetermined across broad cancer cell line panels in vitro as well ascell line- and patient-derived xenograft models in vivo. Multipleapproaches, including integrated transcriptomic and proteomicanalyses, loss-of-function pathway interrogation, and pharmaco-logic comparisons, were employed to further understand the majormechanism driving AZD4573 activity and to establish an exposure/effect relationship.

Results: AZD4573 is a highly selective and potent CDK9inhibitor. It demonstrated rapid induction of apoptosis andsubsequent cell death broadly across hematologic cancer models

in vitro, and MCL-1 depletion in a dose- and time-dependentmanner was identified as a major mechanism through whichAZD4573 induces cell death in tumor cells. This pharmacody-namic (PD) response was also observed in vivo, which led toregressions in both subcutaneous tumor xenografts and dissem-inated models at tolerated doses both as monotherapy or incombination with venetoclax. This understanding of the mech-anism, exposure, and antitumor activity of AZD4573 facilitateddevelopment of a robust pharmacokinetic/PD/efficacy modelused to inform the clinical trial design.

Conclusions: Selective targeting of CDK9 enables the indirectinhibition of MCL-1, providing a therapeutic option for MCL-1–dependent diseases. Accordingly, AZD4573 is currently beingevaluated in a phase I clinical trial for patients with hematologicmalignancies (clinicaltrials.gov identifier: NCT03263637).

See related commentary by Alcon et al., p. 761

IntroductionCyclin-dependent kinases (CDK) are a family of closely related

serine/threonine kinases known for playing crucial roles in regulatingeither cell-cycle progression (CDK1, 2, 4, 6) or gene transcription(CDK7, 8, 9, 12, 13; ref. 1). As integral nodes in these regulatorynetworks, CDKs have been studied extensively as possible targets forcancer therapy, resulting in the development of nonselective CDKinhibitors (2) that show signs of clinical activity (3–6). Despitepharmacologically inhibiting multiple CDKs, the observed clinicalactivity of nonselective CDK inhibitors has been primarily attributedto their CDK9 activity (7–9).

CDK9 is crucial for the proper regulation and progression oftranscription. Through phosphorylation of serine 2 (pSer2) in theheptapeptide repeats within the C-terminal domain of RNA polymer-ase II (RNAP2), CDK9 releases RNAP2 from its paused state to enabletranscription elongation (10). Acute inhibition of CDK9, therefore,results in transient transcriptional suppression and preferential deple-tion of short-lived transcripts and proteins (11), providing a thera-peutic approach to indirectly target key driver oncoproteins with shorthalf-lives. Nonselective CDK9 inhibitors, such as dinaciclib andflavopiridol (alvocidib), have been repurposed to explore this oppor-tunity but have not progressed clinically as monotherapies due tonarrow therapeutic indices (6, 12). However, clinical trials are ongoingto evaluate these molecules in combination with other therapeutics.

Despite clinical development of multiple inhibitors with CDK9activity, the precise mode of action driving antitumor effects has yet tobe fully elucidated, although their preclinical activity has been attrib-uted predominantly to depletion of MCL-1 protein and subsequentinduction of tumor cell death (13–15). MCL-1 is an antiapoptotic,BCL-2 family protein whose high expression has been associated withincreased cancer cell survival that translates to chemotherapy resis-tance and poor patient prognosis (16–18).

This work describes themechanismof action and preclinical activityof the novel and highly selective CDK9 inhibitor, AZD4573. Anagnostic approach utilizing integrated transcriptomic and proteomicanalyses revealedMCL1 as one of the top oncogenes most significantlyand robustly downregulated at themRNAand protein level upon acuteAZD4573 treatment. Subsequent pharmacologic studies further sup-ported an MCL-1–mediated mechanism, including derivation of apharmacokinetic (PK)/pharmacodynamic (PD)/efficacy model

1Bioscience, Oncology R&D, AstraZeneca, Boston, Massachusetts. 2DMPK,Oncology R&D, AstraZeneca, Boston, Massachusetts. 3Discovery Sciences,BioPharmaceuticals R&D, AstraZeneca, Boston, Massachusetts. 4CRUKCambridge Institute, Cambridge University, Cambridge, United Kingdom.5Projects, Oncology R&D, AstraZeneca, Boston, Massachusetts. 6Departmentof Hematology and Medical Oncology, University Medical Center Goettingen,Goettingen, Germany. 7Department of Medical Oncology, Dana-Farber CancerInstitute, Boston, Massachusetts.

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

Current address for M. Zinda: Repare Therapeutics, Cambridge, MA.

Corresponding Author: Lisa Drew, AstraZeneca, 35 Gatehouse Drive, Waltham,MA 02451. Phone: 1-781-839-4836; E-mail: lisa.drew@astrazeneca.com

Clin Cancer Res 2020;26:922–34

doi: 10.1158/1078-0432.CCR-19-1853

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linkingCDK9 inhibition,MCL-1 depletion, and induction of apoptosisin response to AZD4573. Application of this mechanistic understand-ing using short-term treatment and intermittent dosingwithAZD4573revealed broad antitumor activity across preclinical hematologic can-cer models. Collectively, this work supports the clinical evaluation ofAZD4573 in patients with hematologicmalignancies (NCT03263637).

Materials and MethodsChemicals

AZD4573 and AZD5991 were synthesized at AstraZeneca. Vene-toclax (ABT-199) was purchased from MedKoo. For in vitro assays,compounds were solubilized to 10 mmol/L in DMSO. Compoundswere dosed to yield a final DMSO concentration of�0.3%. For in vivostudies, AZD4573 was prepared in a 2%/30%/68% mix of N,N-dimethylacetamide, PEG-400, and 1% (v/v) Tween-80, AZD5991 wasformulated in 30% 2-hydroxylpropyl-beta-cyclodextrin (HPBCD) atpH 9, and venetoclax was solubilized in a 10%/30%/60% mix ofethanol, PEG-400, and Phosal 50 PG.

Enzyme assaysAZD4573 was screened at 0.1 mmol/L against 468 kinases and

relevant variants using the KINOMEscan assay (DiscoveRx), andkinome selectivity images were generated using TREEspot SoftwareTool. SignalChem compound selectivity profiling was utilized toscreen AZD4573 against select CDKs and non-CDK kinases at5 mmol/L (high) ATP concentration for IC50 determination.

Immunoprecipitation and immunoblottingCell and tumor lysates and immunoprecipitation (IP) samples were

prepared and subjected to immunoblotting as described previouslyusing antibodies listed in Supplementary Table S1 (19).

In vitro caspase and viability assaysAssays were conducted as previously described (19) and according

to themanufacturer's protocols for Caspase-Glo 3/7 and CellTiter-Glo(Promega). For cell panel screens, cells were plated in 384-well platesand dosed the following day with 10-point, 1/2 log dilutions of com-

pound, whereas combination treatments used 5-point, 1/2 log dilutions.Automated dosing was performed using an Echo 555 acoustic liquiddispenser (Labcyte). To calculate percent caspase activation, lumines-cence values were normalized against maximum (100%, mixture ofMCL-1, BCL-2, and BCL-XL inhibitors) and minimum (0%, DMSO)controls. Percent growth inhibition was normalized to untreated cellsat the time of dosing, whereas percent viability was normalized againstmaximum (100%,DMSO) andminimum (0%,mediumonly) controls.Dose–response curves were generated and half-maximal effective(EC50) and growth inhibitory (GI50) concentrations calculated usingGeneData Screener and GraphPad Prism.

Washout assayCells were seeded the day before treatment with the indicated

AZD4573 dose. At the indicated times, cells were washed twice incomplete media and plated at 1� 103 cells/well in 96-well plates. Cellswere incubated until 12 or 24 hours after the time of initial dosing, atwhich point Caspase-Glo 3/7 and CellTiter-Glo readouts were per-formed, respectively.

RNA sequencing library preparation and analysiscDNA library construction for RNA sequencing (RNA-seq) was

performed at Novogene using an NEBNext Ultra mRNA Library PrepKit for Illumina (New England Biolabs) according to the manufac-turer's protocol. RNA-seq gene expression quantification was con-ducted using Sailfish against hg38 Ensembl transcripts (version 79),and differential expression analysis was done using Voom and LimmaR package. A detailed description of experimental procedures can befound in the SupplementaryMethods. The data have been deposited inNCBI's Gene Expression Omnibus and are accessible through GEOSeries accession number GSE129012.

Mass-spectrometry proteomicsTrypsin digestion, Tandem Mass Tag labeling, and LC-MS/MS

analysis were conducted as previously described (20). A detaileddescription of experimental procedures can be found in the Supple-mentary Methods. The data have been deposited to the ProteomeX-change Consortium via the PRIDE partner repository with the datasetidentifier PXD013796.

Real-time PCRMV411 cells (1 � 106) treated with vehicle or 0.1 mmol/L

AZD4573 were collected at the indicated timepoints. MV-4-11tumors were also harvested, placed in a Lysing Matrix D tube(Millipore) with Buffer RLT (Qiagen) supplemented with 1%b-mercaptoethanol, and mechanically disrupted using the MilliporeFastPrep. RNA isolation and cDNA conversion were conductedusing RNAeasy Mini (Qiagen) and High Capacity cDNA RT(Applied Biosystems) kits, respectively, following the manufac-turers’ protocols. Relative MCL1 mRNA levels were quantified onan ABI Prism 7900HT Real-time PCR system (ThermoFisher) usingTaqman Assays (Applied Biosystems, MCL1: Hs01050896_m1, 18srRNA: 4319413E). Relative MCL1 expression at each timepoint wascalculated using the -DDCT method by normalizing cycle thresholdvalues to the 18s rRNA reference.

In vivo studiesSubcutaneous xenografts

Studies were conducted at AstraZeneca in accordance with animalresearch guidelines from the NIH and the AstraZeneca Institutional

Translational Relevance

Multiple cyclin-dependent kinase 9 (CDK9) inhibitors, bothnonselective and selective, have been evaluated in clinical trials.Despite signs of clinical activity and ongoing combination studies,monotherapy development was ultimately not pursued due tonarrow safety margins. Contributing factors could include a lackof compound selectivity for CDK9, the absence of a clear patientselection strategy, and suboptimal choice of dose and/or schedule.The development and full preclinical mechanistic, pharmacoki-netic, and pharmacodynamic understanding of a highly selectiveand potent CDK9 inhibitor, like AZD4573, could mitigate thoselimitations. Accordingly, pharmacologic evaluation of AZD4573demonstrated that it represses MCL-1 through acute transcriptioninhibition and results in rapid induction of apoptosis broadlyacross preclinical hematologic cancer models. This work estab-lished a quantitative relationship between extent and duration ofCDK9 inhibition, depletion of MCL-1, and rate of apoptosisinduction that was used to estimate the efficacious dose range andhopefully maximize the therapeutic window in clinical studies.

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Animal Care and Use Committee and as previously described (19).Injected cell numbers for cell line xenografts are as follows: 5� 106 forMV-4-11, MOLP-8, and OCI-LY10, and 1 � 107 for all others.AZD4573 was dosed i.p.) at either 5 or 15 mg/kg, twice daily witha 2-hour split dose (BID q2h), on a 2-day on/5-day off or biweeklyschedule. Venetoclax was dosed daily at 100 mg/kg by oral gavage.AZD5991 was dosed i.v. at 60 mg/kg. For combination studies,venetoclax was administered first, and AZD4573 was administeredwithin 30 min.

AML disseminated patient-derived xenograftsStudy was conducted at Champions Oncology using nine mod-

els (21). Following 4 weeks of AZD4573 therapy, mice (n ¼ 3 permodel) were euthanized for end of study analysis on the spleen, bonemarrow, and peripheral blood by FACS analysis to detect humanCD45þ/CD33þ/CD3� acute myeloid leukemia (AML) blasts.

T-cell lymphoma patient-derived xenograftStudy was conducted under Dana-Farber Cancer Institute Animal

Care and Use Committee protocol #13-034 and Public Health Serviceanimal assurance #A3023-01. A full characterization of the model,DFTL-78024, is available online at www.PRoXe.org. Animal work wasperformed in NSG mice purchased from Jackson Laboratories aspublished previously (22). When sufficiently engrafted, mice weretreated for four cycles of AZD4573 and monitored daily for clinicalsigns of disease and humanely euthanized when they reached a clinicalendpoint. Annexin V was assessed in human CD45þ tumors isolatedfromperipheral blood 6 hours after the second day 2 dose as previouslydescribed (23).

PK/PD/efficacy model developmentModel parameters are listed in Supplementary Table S2, and full

methodology and derivation are available in the SupplementaryMethods.

Data sharing statementFor original data, please contact justin.cidado@astrazeneca.com.

ResultsIdentification of a highly selective and potent CDK9 inhibitor,AZD4573

Understanding the binding of a high-quality probe compound toCDK9 helped guide optimization of a selective and potent CDK9inhibitor with suitable physicochemical properties that would resultin a short compound half-life and enable intravenous administra-tion in humans. This structure-based drug discovery effort ulti-mately led to the identification of AZD4573 (Fig. 1A; ref. 24), whichpotently inhibits the CDK9 enzyme (IC50 at KM and high ATPconcentrations are <0.003 mmol/L and <0.004 mmol/L, respectively).Kinase selectivity profiling measured the affinity of AZD4573 acrossa set of 468 kinases and relevant variants using the KINOMEscanplatform. At a screening concentration of 0.1 mmol/L, which is morethan 100-fold above the measured IC50 in a CDK9 biochemicalassay, AZD4573 resulted in �90% reduction of binding to thecognate ligand capture matrix for 16 kinases (Fig. 1B). For 14 ofthe top hits, the IC50 for AZD4573 was determined at a physio-logically relevant concentration of ATP, revealing >10-fold selec-tivity for CDK9 compared with 13 of 14 kinases and >100-foldselectivity compared with 8 of 14 kinases (Fig. 1C). Similarly,AZD4573 exhibited >25-fold cellular selectivity for CDK9 over

other CDKs upon short-term treatment of MCF7 cells (Fig. 1D;Supplementary Fig. S1A). MCF7 is a favorable model for assessingAZD4573 selectivity and mechanism of action because a frameshiftmutation in the CASP3 gene results in the absence of functionalcaspase-3 protein, preventing potentially confounding apoptosisinduction and cell death.

Kinetic analysis of CASPASE-3 cleavage (“caspase activation”), ahallmark of committed apoptosis, and loss of viability in cell linescultured with AZD4573 over 24 hours showed that potent andselective CDK9 inhibition can kill cancer cells. Though resultsvaried slightly, they highlighted a rapid response to AZD4573 insensitive models and a direct correlation of caspase activation withloss of cell viability (Fig. 1E; Supplementary Fig. S1B and S1C).Conversely, the insensitive cell lines exhibited no caspase activationnor loss of viability until prolonged transcription inhibition causednonspecific cytotoxicity (Supplementary Fig. S1D). This observa-tion underscored the need to determine the requisite duration oftarget engagement to achieve maximal effect in tumor cells whilemitigating unwanted toxicity in normal tissue. Therefore, the AMLcell line MV-4-11 was pulsed with AZD4573 for various lengths oftime, washed out, and then assessed for caspase activation and lossof cell viability at 12 and 24 hours, respectively. This study showedthat approximately 6 to 8 hours of CDK9 inhibition was sufficient toachieve maximal cell death equivalent to continuous exposure ofAZD4573 (Fig. 1F).

CDK9 inhibition by AZD4573 results in rapid turnover ofMCL-1

In previous studies, MCL-1 overexpression rescued CDK9inhibitor–mediated apoptosis, seemingly validating MCL-1 deple-tion as the mechanism driving the antitumor effects (25, 26).However, overexpression studies relying on supraphysiologicalprotein levels come with known caveats, especially in systemsrequiring a delicate balance between multiple proteins (i.e., apo-ptosis regulation by BCL-2 family proteins; ref. 27). Therefore, anagnostic approach utilizing integrated transcriptomic and prote-omic time-course experiments was employed to elucidate thismechanism. For reasons listed above, the AZD4573-insensitivecell line, MCF7, was used for these experiments to ensure cell deathdid not introduce artifact into the assay. Cells were treated withvehicle or AZD4573 for short periods of time (0, 4, and 8 hours)based upon target engagement requirements (Fig. 1F). Biologicaltriplicates were subjected to RNA-seq or LC-MS/MS, which dem-onstrated high reproducibility based upon principal componentanalysis (Supplementary Fig. S2A).

A cancer-associated gene list emphasizing driver oncogenes, includ-ing the antiapoptotic BCL-2 and IAP family proteins, was curated frommultiple sources to identify candidate genes whose rapid mRNA andprotein depletion could drive AZD4573-mediated cell death (Supple-mentary Methods and Supplementary Table S3). Through this agnos-tic approach, the only oncogene and antiapoptotic protein withsignificantly diminished mRNA and protein levels after 4 and 8 hoursof treatment with AZD4573 compared with vehicle treatment wasMCL-1 (Fig. 2A andB; Supplementary Tables S4–S6). Conversely, theother antiapoptotic BCL-2 and IAP family proteins were quite stable(BFL-1 and BCL-B were not detected) despite reports that XIAP wasrapidly modulated upon CDK9 inhibition (28). Many other onco-genes, despite rapid mRNA turnover, either had prolonged proteinhalf-lives or were not detected (Supplementary Table S5). This sup-ports MCL-1 modulation as a major driver of AZD4573-mediatedtumor cell apoptosis.

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Figure 1.

Identification of a highly selective and potent CDK9 inhibitor, AZD4573. A, Chemical structure of the CDK9 inhibitor, AZD4573. B, TREEspot image of 468kinases screened with AZD4573 using the KINOMEscan platform. Percent control corresponds to the amount of kinase bound to its cognate ligand followingAZD4573 treatment and relative to vehicle control. C, Biochemical selectivity of 0.1 mmol/L AZD4573 at 5 mmol/L ATP for CDK9 compared with 14 top kinasehits from KINOMEscan. AZD4573 IC50 values for each kinase were determined and plotted as fold change over the CDK9 IC50. Selectivity thresholds aredepicted by dashed lines and shaded regions. D, Cellular selectivity for multiple CDKs was determined in MCF-7 cells using putative phosphorylation endpointsfor the respective kinase. Lysates of MCF-7 cells treated with a dose response of AZD4573 for 6 hours were immunoblotted and quantified by densitometry toderive IC50 values. E, MV-4-11 cells treated with 0.1 mmol/L AZD4573 were assessed for caspase activation and loss of viability at multiple time points relativeto controls. F, MV-4-11 cells treated with 0.1 mmol/L AZD4573 were washed out at the indicated times and assessed for caspase activation at 12 hours (red) orviability at 24 hours (blue).

AZD4573, a Selective CDK9 Inhibitor, Targets MCL-1

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Figure 2.

Acute inhibition of CDK9 with AZD4573 induces apoptosis through rapid depletion of MCL-1. A and B, Differential transcriptomic (A) and proteomic (B) analysis ofMCF-7 cells treated with 0.1 mmol/L AZD4573 for 4 hours (left) or 8 hours (right). Changes in transcript and protein levels are defined as not significant (gray, FDR >0.05), significant (yellow, FDR < 0.05), and significant with a strong effect (orange, FDR < 0.05 and two-fold change). Other key, measurable antiapoptotic proteinsare highlighted in the volcano plots. C, Lysates from MV-4-11 cells treated with a range AZD4573 concentrations for 6 hours were immunoblotted for the indicatedproteins.D,Protein andRNA lysates fromMV-4-11 cells treatedwith0.1mmol/LAZD4573 for 24 hourswere harvested at the indicated times. MCL1 transcript levelwasmeasured by RT-qPCR, whereas pSer2-RNAP2, MCL-1, BCL-2, BCL-XL, and cleaved caspase-3 protein levels were evaluated by immunoblot. Transcript and proteinlevels are normalized to the pretreatment time point.

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Transient exposure to AZD4573 induces apoptosis upondepletion of MCL-1

Effect of acute AZD4573 treatment on MCL-1 was measured in thesensitive MV-4-11 cell line. AZD4573 treatment for 6 hours reducedpSer2-RNAP2 and MCL-1 in a dose-dependent manner (IC50 ¼ 14nmol/L), and upon sufficient MCL-1 depletion, cleaved caspase-3levels increased. Although caspase activation only occurred in sensitivecell lines, suppression of pSer2-RNAP2 and MCL-1 occurred in allassayed cell lines, which shows that some cancers rely on survivalfactors other than MCL-1 (Fig. 2C; Supplementary Fig. S2B–S2D).Cleaved CASPASE-3 levels decreased at higher doses in the MV-4-11and MOLP-8 models, but this is likely due to the more rapid onset ofcell death at those concentrations and, therefore, earlier loss of signalthat is consistent with the relative kinetics of apoptosis induction forthose cell lines (Supplementary Fig. S1B–S1D).

Temporal effects ofAZD4573 on relevant biomarkerswere examinedto understand the relationship between rapid biomarker modulation,caspase activation, and loss of cell viability. When MV-4-11 cells weretreated with AZD4573, pSer2-RNAP2was reduced >80% within 1 hourand MCL1 mRNA and protein levels were reduced by >80% by 2 and4 hours, respectively (Fig. 2D; Supplementary Fig. S2E). Cleavedcaspase-3 levels began to increase between 4 and 6 hours. Importantly,levels of other antiapoptotic proteins, BCL-2 and BCL-XL, remainedrelatively unchanged over the same dose range (Fig. 2C) and time frame(Fig. 2D), consistent with the transcriptomic and proteomic findings.

Because caspase-3 cleavage can occur through nonapoptoticmechanisms (29), experiments were performed to ensureAZD4573-mediated caspase activation and subsequent cytotoxicityresulted from engagement of the intrinsic apoptosis pathway. Indi-vidual knockdown of apoptosis effector molecules, BAK and BAX, inthe diffuse large B-cell lymphoma (DLBCL) cell line OCI-LY10 onlypartially reduced caspase activation following 6 hours of AZD4573treatment, but simultaneous knockdown completely suppressed cas-pase activation (Supplementary Fig. S2F). InMV-4-11 cells, AZD4573rapidly decreased mitochondrial outer membrane potential andincreased caspase activation and phosphatidylserine exposure asmeasured by tetramethylrhodamine ethyl ester staining, Caspase-3/7Glo, and Annexin V staining, respectively, consistent with MCL-1inhibition kinetics and triggering of intrinsic apoptosis (19). Theseevents were closely followed by loss of cellular ATP, signaling the losscell viability (Supplementary Fig. S2G).

AZD4573 induces rapid cell death across a diverse panel ofhematologic cancer cell lines

With MCL-1 reportedly essential for the survival of varioustumor types, particularly leukemias and lymphomas (30, 31),AZD4573 was screened in a diverse panel of cancer cell lines toassess the breadth of anticancer activity. Caspase activation and lossof viability were measured following 6 and 24 hours of treatmentwith AZD4573, respectively. Greater activity was observed withAZD4573 across hematologic cancer cell lines (geometric meanEC50 ¼ 0.166 mmol/L, GI50 ¼ 0.023 mmol/L) compared with solidtumor cell lines (geometric mean EC50 ¼ 6.871 mmol/L and GI50 ¼0.774 mmol/L), although a subset of solid tumor cell lines wassensitive (Fig. 3A and B; Supplementary Table S7). Importantly,there was good concordance between caspase and viability data.Nonselective CDK9 inhibitors also exhibited robust activity acrossboth hematologic and solid tumor lineages, although longer-termreadouts were employed (32). Likewise, when viability was assessedafter 72-hour continuous AZD4573 exposure, equipotency wasobserved across all cell lines suggesting that prolonged transcrip-

tional repression results in general cytotoxicity or cytostasis (Sup-plementary Fig. S3A), consistent with data from the time-courseassays (Fig. 1E; Supplementary Fig. S1B–S1D).

The same panel of cancer cell lines was also assessed for 6-hourcaspase activation when treated with the selective MCL-1 inhibitor,AZD5991, one of multiple MCL-1 inhibitors in clinical develop-ment (19, 33). There was a strong correlation between the activityobserved with AZD4573 and AZD5991 (r2¼ 0.76; Fig. 3C), providingadditional evidence that depletion of MCL-1 is the primary mecha-nism driving AZD4573-mediated cell killing. Therefore, when MV-4-11 cells were treated with a combination of AZD4573 and AZD5991in vitro and assayed for caspase activation, no combination benefit wasobserved across the dose matrix (Supplementary Fig. S3B). Further-more, measurement of MCL-1 and BCL-XL protein levels for a subsetof AZD4573-sensitive and -insensitive cell lines revealed that higherlevels of BCL-XL corresponded to decreased sensitivity to AZD4573(Supplementary Fig. S3C). This is consistent with a lack of BCL-XLdepletion upon acute AZD4573 treatment (Fig. 2D) and previousreports of the MCL-1:BCL-XL expression ratio predicting response toCDK9 inhibitors and BH3 mimetics (13, 34).

Intermittent dosing of AZD4573 drives regression of MV-4-11tumor xenografts

To determine if observed in vitro activity translated to in vivoefficacy, AZD4573 was evaluated in a hematologic subcutaneousxenograft model using an intermittent dosing schedule that wasselected to provide the requisite acute target inhibition identified fromin vitro studies (�6 hours). Mice bearing MV-4-11 tumors weretreated weekly with 5 or 15 mg/kg AZD4573, dosed IP BID q2h onconsecutive days (5/5 and 15/15 mg/kg), resulting in a tolerated dose-dependent response. The 15/15 mg/kg dose led to regressions for allmice that were sustained out to more than 125 days (Fig. 4A;Supplementary Fig. S4A). Notably, when this study was repeated toexplore other biweekly schedules, similar robust and durable single-agent activity was observed (data not shown).

AZD4573 treatment was also compared head-to-head withAZD5991 in the sensitive MV-4-11 model and relatively insensitiveOCI-AML3 model. Both the 15/15 mg/kg AZD4573 regimen anda single i.v. dose of 60 mg/kg AZD5991 resulted in regressions ofMV-4-11 tumors and minimal tumor growth inhibition (TGI) ofOCI-AML3 tumors (Supplementary Fig. S4B and S4C). These strik-ingly similar effects in vivo for the two inhibitors provide furtherevidence of the primarily MCL-1–dependent mechanism of action foracute AZD4573 treatment.

A mechanistic PK/PD/efficacy model relates CDK9 inhibition,MCL-1 depletion, and induction of apoptosis in response toAZD4573 treatment

Using a series of studies withMV-4-11 tumor xenografts, a PK/PD/efficacy model was developed to quantify the dynamic relationshipbetween AZD4573 PKs and tumor pSer2-RNAP2 and MCL-1 PDs(Fig. 4B). Model parameter values were estimated by fitting the modelto compiled in vivo PK/PD study individual animal data (Supplemen-tary Table S2), and an example model fit is shown in Fig. 4B. The freeAZD4573 concentration that results in half-maximal inhibition ofpSer2-RNAP2 production rate was estimated to be 0.011 to 0.021mmol/L, which was consistent with the IC50 derived from in vitrostudies (Fig. 1D). The kinetics of MCL-1 mRNA and protein mod-ulation were also consistent with in vitro data, demonstrating anestimated mRNA and protein half-life of 5 and 17 minutes,respectively.

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Themodel was extended to quantify the relationship between extentof MCL-1 protein suppression and rate of cell death induction in vivoas measured by reduction in MV-4-11 tumor xenograft volume orincrease in cleaved caspase-3 in PK/PD study tumor samples. Theparameter values describing this relationship were estimated by fittingthemodel to sets of mean tumor volumes frommultidose level efficacystudies (Supplementary Table S2). The rate of tumor cell deathinduction wasmodeled as a saturable first-order process that exhibiteda steep apoptotic response to the suppression of tumor MCL-1. Theestimated tumor MCL-1 protein level associated with half-maximalrate of cell death induction (Mcl50) was consistent across studies andestimated to be approximately 25% of baseline. The estimated max-imum first-order rate constant for cell death induction (Kmax) was alsoconsistent across studies and estimated to be 0.16 to 0.19 hr�1.Satisfactory model fits to observed tumor cleaved caspase-3 kineticdata were also obtained using the same Mcl50 and Kmax values todescribe the relationship between MCL-1 protein suppression and therate of caspase activation from the pool of tumor proCASPASE-3.

Modeling suggested that each daily split dose results in the death of aconsistent fraction of xenograft tumor cells, underscored by the similaroverall efficacy for 2-day on/5-day off and biweekly dosing schedules

(Fig. 4B). Each 15 mg/kg split dose was modeled to reduce pSer2-RNAP2 below 20% of baseline levels for approximately 4 hours. Thisextent and duration of pSer2-RNAP2 reduction caused suppression ofMCL-1 below the determined Mcl50 for approximately 4 hours, whichsubsequently resulted in approximately 55% MV-4-11 tumor volumereduction that was sufficient to sustain progressive reduction in tumorvolume on a biweekly dosing schedule (Fig. 4B). Application of thisderived exposure–effect relationship to other preclinical models,including OCI-LY10, estimated equivalent parameters and predictedthe in vivo response (Supplementary Fig. S4D; SupplementaryTable S2).

In vitro sensitivity to AZD4573 accurately predicts in vivoefficacy in hematologic tumor xenograft models

Having established a detailed understanding of the relationshipbetween AZD4573 exposure, MCL-1 suppression, and apoptosisinduction in the MV-4-11 xenograft model, in vivo efficacy analysiswas expanded to include ten more hematologic models. Based onin vitro 6-hour caspase activity parameters used to define sensitivity(EC50 � 45 nmol/L based on modeled MV-4-11 EC99; maximumcaspase activation � 60%), certain models were predicted to be

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AZD4573 induces rapid cell death across a diverse panel of hematologic cancer cell lines.A,AZD4573was screened for caspase activation at 6 hours across a panel ofhematologic (n¼ 110) and solid (n¼ 105) tumor cell lines. Each dot represents the EC50 of individual cell lines, and cell lines are grouped by indication. The geometricmean EC50with 95% confidence interval is depictedwithin each indication. The dotted line represents the EC50 value forMV-4-11 as a comparison, and an � representsa significant P value < 0.0001. B, A similar screen was conducted for viability at 24 hours after AZD4573 treatment in hematologic (n¼ 99) and solid (n¼ 86) tumorcell lines. GI50 values are plotted with that of MV-4-11 represented by the dotted line. C, A Spearman correlation between the caspase EC50 values for AZD4573 andAZD5991, the MCL-1 inhibitor, in hematologic cancer cell lines (n ¼ 98).

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A mechanistic PK/PD/efficacy model relates CDK9 inhibition, MCL-1 depletion, and caspase activation to in vivo response with AZD4573 treatment. A, An MV-4-11subcutaneous xenograft model was treated with three cycles of vehicle or AZD4573 (5 mg/kg or 15 mg/kg, BID q2h, 2 days on/5 days off). Tumor volumes arepresented as geometric mean � SEM (n ¼ 5). Shaded area indicates treatment period. B, The top portion depicts a schematic of the established PK/PD/efficacymodel. A three-compartment PKmodel was used to describe the observed plasma and tumor AZD4573 concentration data. Tumor pSer2-RNAP2 andMCL-1 PDwasmodeled using a series of linked indirect responsemodels. Unperturbed tumor growthwasmodeled as afirst-order process, andMCL-1wasmodeled as inhibiting theinduction of apoptosis in the MV-4-11 cells. A series of transit compartments were used to bridge the time delay between cell death (hours) and observed tumorshrinkage (days). On the bottom, graphs show themodel fit (lines) to observed PK and PD data (individual data points) where darker lines indicate populationmeanwith lighter lines indicating 10% to 90% percentiles expected based on estimated interindividual variability. Efficacy graphs show the model fit to observed tumorvolume data. All modeling data are based upon MV-4-11 subcutaneous tumor xenograft studies.

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relatively insensitive (KG1a, OCI-M1, and OCI-AML3), borderlinesensitive (Karpas422), or mostly sensitive (MV-4-11, SU-DHL-4,AMO-1, NOMO-1, EOL-1, MOLP-8, and OCI-LY10). In these mod-els, intermittent dosing of AZD4573 for 2 or 3 cycles produced a rangeof responses, from tumor regressions to modest or no TGI (Fig. 5A;Supplementary Figs. S4B and S4C; S5A–S5I). The eight models pre-dicted to show in vivo sensitivity to AZD4573 did achieve eitherregressions or extensive TGI, whereas minimal response was observedin the three models predicted to be relatively insensitive.

AZD4573 demonstrates antitumor activity in disseminatedleukemia and lymphoma patient-derived xenograft models

In addition to subcutaneous cell line xenograft models, in vivostudies were expanded to include i.v. disseminated patient-derivedxenograft (PDX) models of AML and T-cell lymphoma. Nine AMLPDX models were evaluated for the ability of AZD4573 to decreasetumor burden in the bonemarrow. By the end of study, five of the ninemodels exhibited greater than 50% reduction of leukemic blasts in thebone marrow (Fig. 5B). The angioimmunoblastic T-cell lymphomaPDXmodel, DFTL-78024, which exhibited strongMCL-1 dependencebased upon BH3 profiling (Supplementary Fig. S5J; ref. 35), was alsoevaluated. Treatment with AZD4573 led to significantly more CD45þ

tumor cells undergoing apoptosis, as measured by increases inAnnexin V staining following the second dose on day 2, whichultimately led to a significant survival benefit compared with vehicle(P value ¼ 0.0018; Fig. 5C). Together, these data establish thatAZD4573 is efficacious in models of disseminated disease.

AZD4573 combinations with venetoclax are tolerated andefficacious

Despite broad monotherapy activity for AZD4573 across hema-tologic tumor models, several models exhibited little to no response(Supplementary Fig. S5A–S5C). Furthermore, in some sensitivemodels, AZD4573 resulted in extensive TGI, yet tumors regrewimmediately following cessation of dosing, suggesting either thepresence of an underlying refractory cell population or insufficienttumor cell death during the dosing period. To produce moredurable responses, combinations with the selective BCL-2 inhibitor,venetoclax, were explored given the observed benefit for AZD5991combinations with venetoclax (19).

AZD4573monotherapy treatment in the SU-DHL-4 (GCB-DLBCL)tumor xenograft model exhibited robust antitumor activity, althoughoverall regressionswere not achieved (Supplementary Fig. S5F). In vitro,SU-DHL-4 cells were sensitive to single-agent AZD4573 (EC50 ¼ 16nmol/L; max caspase activation ¼ 79%) and relatively insensitive tovenetoclax (EC50 ¼ 94 nmol/L; max caspase activation ¼ 24%).Combination treatment, however, increased the extent of caspaseactivation (EC50¼ 10 nmol/L; max caspase activation¼ 118%), whichwas also observed in vivo (Fig. 6A and B, left). Monotherapy treatmentwith venetoclax was minimally efficacious, consistent with the in vitroresponse, and single-agent AZD4573 again exhibited extensive TGI butnot regressions. The combination of AZD4573 with venetoclax, how-ever, produced highly durable regressions in 100% of treated animals,with all 8 mice remaining tumor-free out to day 63. Similar effects wereobserved in the OCI-AML3 model, which was intrinsically resistant toboth monotherapies (Fig. 6A and B, right). Notably, minimal bodyweight loss was observed, highlighting that this could be a toleratedcombination regimen (Supplementary Fig. S5K and S5L).

The combination benefit in cancer cell line models insensitive toinhibition of BCL-2 or both BCL-2 and MCL-1 was striking. Previousstudies have shown that venetoclax displaces proapoptotic molecules

like BIM from BCL-2, which can then become sequestered byMCL-1 (36). Similar BCL-2 family protein dynamics were observedhere. In vitro treatment with venetoclax revealed a rapid (�3 hours)increase in MCL-1 for both models (Fig. 6C). Similarly, SU-DHL-4tumor xenograft PD samples treated with a single dose of venetoclaxexhibited an increase in MCL-1 protein by 6 hours after dose withmaximal levels achieved by 24 hours (Fig. 6D). In OCI-AML3 cellstreated with venetoclax and coimmunoprecipitated with either anti–MCL-1 or anti-BIM antibodies, Bim was displaced from BCL-2 andthen sequestered byMCL-1 (Fig. 6E), likely stabilizing the protein andcreating an MCL-1 dependency, lending further credence to thereported combination mechanism.

DiscussionSeminal work with nonselective CDK inhibitors showed that

induction of tumor cell death required activity against CDK9 (37),which resulted in the rapid depletion of prosurvival factors, likeMCL-1 (7, 38). Despite signs of clinical activity, development of thesemolecules as monotherapies was suspended due to narrow therapeuticwindows (2). However, trials investigating their combination potentialare currently ongoing. Recently, more selective CDK9 inhibitors havebeen developed to overcome the noted limitations of nonselectiveCDK inhibitors (15, 39, 40). This work describes the novel CDK9inhibitor, AZD4573, that was optimized for high selectivity andpotency against CDK9 as well as physicochemical propertiesenabling intravenous infusion and a short PK half-life. Theseattributes allow for intermittent dosing of AZD4573, resulting inperiods of acute yet sufficient CDK9 inhibition that drives rapidtumor cell apoptosis without causing prolonged transcription inhi-bition that could erode the therapeutic index. AZD4573 is currentlyin phase I clinical trials for evaluation in patients with hematologicmalignancies (NCT03263637).

There are various studies that have successfully associatedCDK9 inhibition with MCL-1 depletion and subsequent inductionof tumor cell death (41). However, many come with study designcaveats such as prolonged CDK9 inhibition (15, 42), a narrowfocus on one indication and/or a limited number of preclinicalmodels (8, 43), and use of nonselective compounds (14, 44) thatmake it difficult to draw firm conclusions. This study overcamethose limitations by utilizing the selective CDK9 inhibitor,AZD4573, and applying multiple approaches to interrogate themechanism of action of CDK9 inhibition. First, with the agnosticand integrated transcriptomic and proteomic analysis, MCL1 wasidentified as the most robustly and significantly modulated cancer-associated gene in cells treated acutely with AZD4573. Interest-ingly, several transcripts and proteins were upregulated aftertransient AZD4573 exposure, which could result from CDK9inhibition driving stress responses or repressing another repres-sor (45). Although inclusion of more than one cell line in thesemultiomic experiments would have been preferred for hit valida-tion, focused evaluation of MCL-1 depletion in multiple othermodels revealed a high degree of consistency across tumor typesand ranges of AZD4573 sensitivity. This work also highlighted therequirement of an intact intrinsic apoptotic pathway to achieveAZD4573-induced cell death, a high concordance between theactivity observed in vitro and in vivo for AZD4573 and the novelMCL-1 inhibitor AZD5991 (19), and a lack of benefit for thecombination of AZD4573 and AZD5991. Collectively, these datasupport MCL-1 depletion as the primary mechanism drivingtumor cell death upon acute CDK9 inhibition.

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AZD4573 demonstrates antitumor activity in disseminated leukemia and lymphomaPDXmodels.A,Other subcutaneous tumor xenograftmodels fromAML,multiplemyeloma (MM), andDLBCL cancer cell lineswere treatedwithAZD4573 at 15mg/kg, BIDq2h, 2dayson/5daysoff for three cycles (� representsmodels receiving onlytwo cycles). Thepercent change from tumor volumeat timeoffirst dose is depicted. Redbars denotemodels predicted from in vitro caspase sensitivity parameters toshow signs of efficacy in vivo. B,Human disseminated AML PDXmodels grown in NOG-EXLmice were treated with four cycles of vehicle or AZD4573 (15 mg/kg, BIDq2h, 2-day on/5-day off; n¼ 3). FACS analysiswas used to detect leukemic blasts in the bonemarrow (humanCD3�/CD33þ) and presented asmean percent change� SEM frompretreatment counts.C,Kaplan–Meier survival curves for theDFTL-78024model treatedwith four cycles of AZD4573 (n¼ 5; left). PD response to vehicleor AZD4573 treatment assessed 6 hours after a single 15 mg/kg dose. Tumors from 3 mice per group were assessed for Annexin V staining in human CD45þ tumorcells via flow cytometry (right).

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Despite the strong correlation between AZD4573 and AZD5991activity in vitro, there were some hematologic cancer cell lines sensitiveto AZD4573 but not AZD5991. Although prolonged AZD4573 expo-sure would expectedly cause turnover of numerous proteins that couldcause general cytotoxicity, these were short, 6-hour assays. Therefore,

AZD4573 is likely rapidly depleting another factor necessary for cancercell survival where MCL-1 inhibition alone is insufficient to induceapoptosis.

A quantitative AZD4573 exposure/effect relationship was alsoestablished that further substantiates MCL-1 as the downstream target

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AZD4573 combinations with venetoclax aretolerated andefficacious.A,SU-DHL-4 (left)and OCI-AML3 (right) cells treated in vitrowith a dose-response of AZD4573, veneto-clax, or the combination were assessed forcaspase activation. B, SU-DHL-4 (left) andOCI-AML3 (right) subcutaneous xenograftmodels were treated with three cycles ofvehicle, venetoclax (100 mg/kg QD),AZD4573 (15 mg/kg, BID q2h, 2-day on/5-day off), or the combination. Shaded areaindicates treatment period. Tumor volumesare presented as geometric mean � SEM(n ¼ 5). C, Lysates from SU-DHL-4 (left)and OCI-AML3 (right) cells treated with0.1mmol/L venetoclax, 0.1mmol/LAZD4573,or the combination for various times wereimmunoblotted for the depicted proteins.D,SU-DHL-4 tumor lysates from mice treatedwith a single dose of vehicle or venetoclax(100 mg/kg) for the indicated times (n¼ 3)were immunoblotted for the indicated pro-teins. E, OCI-AML3 cells were treated with0.1 mmol/L venetoclax in vitro for the indi-cated times. MCL-1 and Bim were immuno-precipitated from whole-cell lysates (input)and immunoblotted for the indicatedprotein.

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mediating the induction of apoptosis. Reduction in pSer2-RNAP2occurs in a concentration- and time-dependentmanner that precedes aproportionate but delayed depletion of MCL-1 protein, and caspaseactivation occurs onceMCL-1 levels reach a critical value inAZD4573-sensitive cancer cells. The relationship between PD response and rateof cell death induction, as assessed by reduction in tumor volume,in vivo was similar to that observed in in vitro studies. At toleratedAZD4573 doses in mice, the extent and duration of MCL-1 suppres-sion was sufficient to drive MV-4-11 subcutaneous tumor xenograftsinto complete regression when using twice-weekly dose schedules. Amechanistic PK/PD/efficacy model was developed that relatesAZD4573 exposure to the extent and duration of pSer2-RNAP2 andMCL-1 suppression necessary to induce caspase activation and reducetumor size. The derived parameter values for the in vivo model wereconsistent with the observed in vitro target engagement requirement(�6 hours) in MCL-1–dependent cell lines. Given the applicability inmultiple tumormodels, this PK/PD/efficacymodel was used to informdesign of the current phase I trial. With this greater mechanisticunderstanding and quantitative modeling of AZD4573, the hope is tooptimize the dose and schedule to maximize the therapeutic window.

Venetoclax is now approved for treatment of adult patients withchronic lymphocytic leukemia based upon high response rates withmonotherapy. In other hematologic cancers, the response rates weremuch lower, but combinations with standards of care yielded betterresults and led to the approval of venetoclax in combination withhypomethylating agents or low-dose cytarabine for the treatment ofselect patients with AML who cannot receive intensive inductionchemotherapy (46, 47). Despite these promising responses and excit-ing approvals, acquired resistance to venetoclax is beginning toemerge, and one mechanism identified preclinically is compensationby increased levels of MCL-1 (48). Although this could result fromselection of subclones with preexisting MCL-1 overexpression, ourdata suggest that venetoclax can directly induce stabilization ofMCL-1by favoring its binding of BIM. Using models intrinsically resistant tovenetoclax, this study showed that adding AZD4573 to the venetoclaxregimen resulted in significant and tolerated combination benefit,which is in linewith venetoclax combination studies using other CDK9andMCL-1 inhibitors (14, 49, 50). This suggests that the combinationcould be used to increase depth and duration of response as well asprevent or overcome the emergence of resistance.

In conclusion, this study has identified AZD4573, a potent andselective CDK9 inhibitor, that induces cancer cell death across pre-clinical hematologic cancer models at tolerated, intermittent dosesprimarily through transient depletion of MCL-1. This mechanistic

understanding enabled the development of a quantitative PK/PD/efficacymodel that can aid in the selection of an optimized clinical doseand schedule to maximize the therapeutic index. These findingscollectively supported progression of AZD4573 as a clinical candidatefor the treatment of patients with hematologic malignancies.

Disclosure of Potential Conflicts of InterestJ. Cidado, S. Boiko, T. Proia, D. Ferguson, S.W. Criscione, P. Pop-Damkov,

W. Shao, J.C. Saeh, S.E. Fawell, and L. Drew are employees/paid consultants forAstraZeneca. D.M. Weinstock is an employee/paid consultant for Travera, Ajax, andBantam Pharmaceuticals, reports receiving commercial research grants from Astra-Zeneca, Abbvie, Aileron, Daiichi Sankyo, and Verastem, holds ownership interest(including patents) in Ajax and Travera, and reports receiving other remunerationfrom Genentech/Roche. M. Zinda is an employee/paid consultant for, reportsreceiving commercial research grants from, and holds ownership interest (includingpatents) in AstraZeneca. No potential conflicts of interest were disclosed by the otherauthors.

Authors’ ContributionsConception and design: J. Cidado, S. Boiko, D. Ferguson, S.W. Criscione, S.E. Fawell,L. DrewDevelopment of methodology: J. Cidado, S. Boiko, D. Ferguson, P. Pop-Damkov,L. DrewAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): J. Cidado, S. Boiko, T. Proia, M. San Martin, N. Su, V.N. RoamioFranklin, C.S. D'Santos, R. Koch, D.M. WeinstockAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Cidado, S. Boiko, T. Proia, D. Ferguson,S.W. Criscione, N. Su, C. Sekhar Reddy Chilamakuri, C.S. D'Santos, R. Koch,S.E. FawellWriting, review, and/or revision of the manuscript: J. Cidado, S. Boiko, T. Proia,D. Ferguson, N. Su, W. Shao, J.C. Saeh, R. Koch, D.M. Weinstock, M. Zinda,S.E. Fawell, L. DrewAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): J. Cidado, D. Ferguson, J.C. Saeh, M. Zinda, L. DrewStudy supervision: J. Cidado, T. Proia, W. Shao, J.C. Saeh, S.E. Fawell, L. Drew

The Fusion Lumos Orbitrap mass spectrometer was purchased with the supportfrom a Wellcome Trust Multi-user Equipment Grant (Grant #108467/Z/15/Z).

AcknowledgmentsThe costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 11, 2019; revised September 27, 2019; accepted November 4, 2019;published first November 7, 2019.

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2020;26:922-934. Published OnlineFirst November 7, 2019.Clin Cancer Res   Justin Cidado, Scott Boiko, Theresa Proia, et al.   MCL-1 and Induces Apoptosis in Hematologic Cancer CellsAZD4573 Is a Highly Selective CDK9 Inhibitor That Suppresses

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