Identiﬁcation of CCR2 and CD180 as Robust …Tammie C.Yeh1, Greg O'Connor1, Philip Petteruti2,...

Cancer Therapy: Preclinical

Identification of CCR2 and CD180 as RobustPharmacodynamic Tumor and Blood Biomarkersfor Clinical Use with BRD4/BET InhibitorsTammie C. Yeh1, Greg O'Connor1, Philip Petteruti2, Austin Dulak2, Maureen Hattersley2,J. Carl Barrett1, and Huawei Chen2

Abstract

Purpose: AZD5153 is a novel BRD4/BET inhibitor with adistinctive bivalent bromodomain binding mode. To support itsclinical development, we identified pharmacodynamic (PD) bio-markers for use in clinical trials to establish target engagement.

Experimental Design: CCR2 and CD180 mRNAs, initiallyidentified from whole transcriptome profiling, were further eval-uated by quantitative PCR in hematologic cell lines, xenografts,and whole blood from rat, healthy volunteers, and patients withcancer. MYC and HEXIM1 mRNAs were also evaluated.

Results: RNA-sequencing data showed consistent decreases inCCR2/CD180 expression across multiple hematologic cell linesupon AZD5153 treatment. Evaluation of dose dependence inMV4,11 cells confirmed activity at clinically relevant concentra-tions. In vivo downregulation of CCR2/CD180 mRNAs (>80%)was demonstrated in MV4,11 and KMS-11 xenograft tumors at

efficaciousAZD5153doses. Consistentwith in vitro rat blooddata,an in vivo rat study confirmed greater inhibition of CCR2/CD180mRNA in whole blood versus MYC at an efficacious dose. Finally,in vitro treatment of whole blood from healthy volunteers andpatients with cancer demonstrated, in contrast to MYC, almostcomplete downregulation of CCR2/CD180 at predicted clini-cally achievable concentrations.

Conclusions: Our data strongly support the use of CCR2 andCD180 mRNAs as whole blood PD biomarkers for BRD4 inhi-bitors, especially in situations where paired tumor biopsies areunavailable. In addition, they can be used as tumor-based PDbiomarkers for hematologic tumors. MYC mRNA is useful as ahematologic tumor-based biomarker but suboptimal as a wholeblood biomarker. Utility of HEXIM1 mRNA may be limited tohigher concentrations. Clin Cancer Res; 23(4); 1025–35. �2017 AACR.

IntroductionThe number of epigenetic targets for cancer drug research and

development is increasing as more encouraging data emerge (1).Inhibitors of DNA methyltransferase (i.e., azacitdine and decita-bine) and histone deactylase (i.e., vorinostat and romidepsin) arealready approved drugs for hematologic malignancies. Anotherepigenetic target that has elicited high interest recently is theBRD4/BET (Bromodomain and Extra-Terminal) protein family(2, 3). These "epigenetic readers" include BRD 2, 3, 4, and T andare characterized by the presence of two tandem bromodomains.The binding of acetylated histones by the bromodomains pro-motes the recruitment of BET proteins to chromatin, resulting inthe activation of gene transcription through their subsequentrecruitment of positive transcription elongation factor (p-TEFb).Small molecules that bind to these bromodomains can block thisinteractionwith chromatin, resulting in significant transcriptionalchanges with biological consequences. "BRD4 inhibitors" are a

highly active area of research in recent years for indicationsranging fromcardiovascular disease to inflammation to oncology.For oncology, there are currently several BRD4 inhibitors, e.g.,iBET762, OTX-015 (MK8628), being evaluated in the clinicalsetting with promising results. Early clinical data have demon-strated proof of concept in indications such as acute myeloidleukemia (AML), lymphoma, and NUT-midline carcinoma(NMC) solid tumors (4–9).

AZD5153 is a potent small-molecule bromodomain inhibitorof the BET family, which is poised to enter the clinic (10). It hasdemonstrated impressive preclinical efficacy across multiplexenograft models at doses and schedules determined safe andtolerable (11–13). To support the clinical development of thiscompound in the oncology setting, our goal was to identify anddevelop pharmacodynamic (PD) biomarkers that could be usedin clinical trials to evaluate and confirm target engagement. Fromthe literature, there are at least two well-supported candidatesdescribed for tumor-based biomarkers for BRD4 inhibitors, i.e.,MYC and hexamethylene bis-acetamide-inducible protein 1(HEXIM1). MYC is a well-known oncogene whose protein prod-uct has been difficult to target pharmacologically. In fact, it isbecause of its striking inhibition by BRD4 inhibitors in multiplepreclinical tumormodels that has triggered the recent enthusiasmto evaluate BRD4 inhibitors for cancer treatment (14–16). Fur-thermore, its direct transcriptional regulation by BRD4 (via super-enhancers in some contexts) has been demonstrated (17, 18).Upregulation of HEXIM1 mRNA and protein has also beenreported in tumor cell lines treatedwith different BRD4 inhibitors(15, 19). Whereas BRD4 promotes the recruitment of P-TEFb tochromatin to activate RNA polymerase II, HEXIM1 sequesters

1Translational Sciences, Oncology Innovative Medicines Unit, AstraZeneca R&DBoston, Waltham, Massachusetts. 2Biosciences, Oncology Innovative MedicinesUnit, AstraZeneca R&D Boston, Waltham, Massachusetts.

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

Corresponding Author: Tammie C. Yeh, AstraZeneca R&D Boston, 35 Gate-house Drive, Waltham, MA, 02451. Phone: 781-839-4177; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-16-1658

�2017 American Association for Cancer Research.

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P-TEF away from chromatin (20). Given its interplay with BRD4,upregulation of HEXIM1mRNA is also an attractive biomarker toconfirm PD responses in the tumor setting.

In addition to tumor-based biomarkers, wewere also interestedinwhole blood PDbiomarkers. Paired biopsies can sometimes bechallenging to obtain and, evenwhen available, can be difficult tointerpret due to absence of sufficient tumor cells and/or tumorheterogeneity. Consequently, the inclusion of whole blood bio-markers to complement paired biopsies can increase the success-ful interpretation of biomarker data by allowing assessmentacross multiple time points. Furthermore, in clinical situationswhere paired biopsies can be difficult to attain, e.g., dose escala-tion in all-comers, having whole blood biomarker data to estab-lish target engagement early on in a trial setting is invaluable.

There are fewer reports on whole blood biomarkers forBRD4 inhibitors. Increases in circulating apolipoprotein A1,high-density lipoprotein (HDL) cholesterol, and large HDL par-ticle size in healthy volunteers as well as cardiovascular patientstreatedwith RVX-208, a less potent BRD4 inhibitor, were reported(21, 22). Although the evaluation of these biomarker readoutsmay still be warranted, they are not necessarily ideal for theoncology setting because they are based on lipogenesis pathways.In addition, mRNA-based biomarkers are more desirable as theyare direct readouts of BRD4 function. Although modulation ofMYC and HEXIM mRNA by BRD4 inhibitors has predominantlybeen described for the tumor setting, we have evaluatedand reported reasonable modulation of these readouts inAZD5153-treated human whole blood (13).

In this study, we have identified and characterized twoadditional biomarkers for demonstrating target engagement/pathway modulation in whole blood. We believe they will havehigh probability of success in the clinic due to their largedynamic range and robust responses at clinically predictedexposures. Our preclinical studies also support the use of thesereadouts, in addition to MYC and HEXIM1 mRNA, as tumor-based biomarkers in the hematologic setting, e.g., AML andacute lymphoblastic leukemia (ALL). Furthermore, our datasupport the potential overarching utility of these biomarkers toother BRD4 inhibitors.

Materials and MethodsCell lines

All cell lines were originally purchased from ATCC or DSMZand cultured according to the provider's instructions. Cell lineswere revived from AstraZeneca Central Cell Bank, where everycell line was authenticated and tested using DNA fingerprintingshort-tandem repeat (STR) assays at IDEXX before banking. Allcell lines were used within15 passages and less than 6 months.Cell lines were not re-authenticated by the authors before use.Cells were maintained in 10% FCS for RNA-sequencing anddose response studies.

Generation and analysis of RNA-sequencing datasetAfter AZD5153 (200 nmol/L) or vehicle (0.1% DMSO)

treatment of 15 hematologic and 13 solid tumor cell lines (Sup-plementary Table S1) for 24 hours, RNA isolation, cDNA librarygeneration, and sequencing to 12M reads on the IlluminaHighSeq was carried out at Q2 Solutions (http://www.q2labsolu-tions.com/). RNA fromMV4,11 tumors (see below) from vehicleand 5mg/kg AZD5153-treatedmice (n¼ 3/group) at 2 hours and8 hours after dose were also included. Bcbio-nextgen (https://github.com/chapmanb/bcbio-nextgen) was used to processFastQ files, perform quality control, alignment to genome buildhg19, and quantify transcription expression based on Ensemblannotation. EdgeR version 3.12.0 was used to identify differen-tially expressed genes using trimmed mean of M values fornormalization and the exactTest method for differential expres-sion. Analysis of a subset of these data has been previouslydescribed (11, 13).

Dose–response curves in MV4,11 cellsMV4,11 cells were treated with indicated concentrations of

AZD5153 for 4 hours. RNA was extracted from 1 � 106 cellsusing an RNAeasy mini kit according to the manufacturer'sprotocol (Qiagen; cat# 74104). RNAwas converted to cDNAwitha High-Capacity RT kit following the manufacturer's protocol(Applied Biosystems; cat# 4368814). Quantitative PCR was per-formed using Taqman Gene Expression Master Mix (AppliedBiosystems; cat# 4369016) with 300 ng of cDNA and taqmanprobes (Supplementary Table S2) on an ABI Prism 7900HTinstrument. Data were normalized to 18S as the housekeepinggene (HKG) and converted to relative signal (2�DCt) � 100 tocalculate the percentage of control.

PK/PD evaluation in in vivo xenograft modelsFemale CB17 SCIDmice were obtained from Charles River. All

animals were used in compliance with protocols approved by theInstitutional Animal Care and Use Committees of AstraZeneca.Tumor cells (107)were implanted into the right flank ofmice, andtumor volumewasmonitored twice weekly. Threemice per groupwere used per group for PD studies. Mice were treated with eithervehicle (0.5% hydroxymethylcellulose, 0.1% Tween80) orAZD5153 by oral gavage for 1 day (MV4,11) or 3 days (KMS-11). For each animal, tumors were collected and then snap frozen;blood samples were stabilized in EDTA. Plasma concentrationswere determined by liquid chromatography/tandem mass spec-trometry methods. Tumors fragments were thawed and placed inLysing Matrix D tubes (MP Biomedical, #116913050) with 1 mLof RLT lysis buffer from the RNA easymicro kit (Qiagen, #74004).Tumors were then ground up using the Fastprep-24 (MP Biomed-ical, #116004500) at 5.0m/s for 1minute. Tubeswere spun down

Translational Relevance

As more targeted agents enter the clinic and the pressure toprioritize programs increases, the need to provide clinicalevidence that the molecule under investigation is acting onits target becomes essential to justify continued clinical inves-tigation. We have identified CCR2 and CD180 mRNA asattractive clinical biomarkers for demonstrating target engage-ment of AZD5153, a novel BRD4/BET inhibitor. In addition tousing these biomarkers for hematologic tumors, our resultssupport high probability of success for the use of these bio-markers in humanwhole blooddue to the largedynamic rangeand sensitivity at predicted clinically achievable concentra-tions. Whole blood biomarkers can be especially informativein clinical trial situations where obtaining paired tumor biop-sies may be difficult and/or where evaluating pharmacody-namic effects acrossmultiple time points is desired. Finally, weshow downregulation of CCR2 and CD180 extends to otherBRD4/BET compounds.

Yeh et al.

Clin Cancer Res; 23(4) February 15, 2017 Clinical Cancer Research1026




at 14,000� g for 5minutes and 500 mL lysate removed to be usedin the RNA easy microextraction protocol. Reverse transcriptionand qPCR reactions were run as described above for cellular RNAsamples. Data were normalized to GAPDH as the housekeepinggene and converted to relative signal (2�DCt)�100 to calculate thepercentage of control.

In vitro and in vivo evaluation of AZD5153 in rat whole bloodWhole blood was collected from each of three female Han

Wistar rats (Charles River) into 1 mL EDTA tubes (Becton Dick-inson), divided into 2 � 500 mL volumes, and incubated withDMSO (final¼ 0.1%) or 1 mmol/L AZD5153 for 2 hours at 37�C.For in vivo studies, three female Han Wistar rats were orally doseddaily with 1.08 mg/kg AZD5153 for 7 days. Blood was collectedvia tail vein at pre-dose, 2 and 6 hours post dose on days 1 and 7.Whole blood (0.5 mL) was then transferred to tubes containing1.38 mL stabilizing reagent from PAXgene RNA tubes (PreAna-lytiX), incubated for 2 hours at room temperature, and stored at�20�Cuntil RNA extraction. RNA extractionwas performed usingPAXgene Blood RNA kits (Qiagen) according to the manufac-turer's instructions. Total RNA was then quantified, reverse tran-scribed, and used as a template for quantitative PCRusing species-specific primers (ABI) for the genes of interest (SupplementaryTable S2). Data were normalized against RPLP2 (selected fromhousekeeping gene optimization studies) and converted to rela-tive signal (2�DCt) � 100 to calculate the percentage of control.

In vitro evaluation of compound-treated whole blood fromnormal healthy volunteers

Whole blood was collected from healthy donors in 10 mLEDTA vaccutainers (Becton Dickinson) and incubated (3 mL/condition) with DMSO, AZD5153, iBET762, and OTX-015 atindicated concentrations for 2 hours at 37�C. Afterwards, 2.5 mLwas transferred to PAXgene RNA tubes (PreAnalytiX), allowedto sit for 2 hours at room temperature for RNA stabilization,and then stored at �20�C until RNA extraction, reverse transcrip-tion, and qPCR analysis, as described above. Data were normal-ized using GAPDH or RPL13A as the housekeeping gene andconverted to relative signal (2�DCt) � 100 to calculate thepercentage of control.

In vitro evaluation of AZD5153-treated whole blood frompatients with cancer

Whole blood was collected in 10 mL EDTA vaccutainers(Becton Dickinson) from two control healthy volunteers and11 patients with cancer with solid tumor or hematologiccancers through Conversant Bio. Compound incubation andsample processing was carried out at Conversant Bio within2 hours of blood collection. Briefly, 3 mL aliquots of bloodwere incubated with DMSO or serial dilutions of AZD5153 for2 hours at 37�C. Afterward, 2.5 mL from each condition wastransferred to PAXgene RNA tubes (PreAnalytiX), incubated for2 hours at room temperature for RNA stabilization, and storedat �80�C until shipment. RNA extraction was performed atQiagen Genomic Services using PAXgene Blood RNA kits (Qia-gen) on a QIAcube platform; RNA samples were shipped toAstraZeneca. Samples were processed and analyzed by qPCR asdescribed above. Data were normalized using RPL13A as thehousekeeping gene and converted to relative signal (2-DCt) �100 to calculate the percentage of control.

ResultsMining the RNAseq dataset for biomarker candidates

AZD5153 is a potent small-molecule bromodomain inhib-itor of the BET family. In contrast to other previously describedcompounds, it has a unique bivalent binding mode that allowsone molecule to bind to the tandem bromodomains concur-rently, resulting in high affinity and cellular potency (11, 13,23). In order to better understand the downstream biologythat results from this novel binding mode, we performeda whole transcriptome profiling on cancer cell lines and MV4,11 (AML) xenograft tumor samples that were exposed toAZD5153 (11, 13).

We tapped into this rich dataset to identify potential leadsfor pharmacologic biomarkers that could be used in theclinical setting. A flow diagram summarizing these activitiesis depicted in Fig. 1A. Initial analyses of the hematologic dataresulted in the identification of many candidate genes thatwere strongly and consistently modulated with compoundtreatment (13).

A subset of these genes was selected for further confirmationin a number of hematologic cell lines and xenograft studies.Candidate genes were further prioritized based on the robust-ness of their modulation by AZD5153 in rodent and humanblood. In brief, these biomarker-focused studies led us to theselection of C-C chemokine receptor type 2 (CCR2) and CD180as tumor and whole blood biomarker readouts for possibleclinical use. In addition, we also included MYC and HEXIM1,which have been previously described, in our evaluations.

The RNAseq data for these four readouts are shown in Fig. 1B.Both hematologic and solid tumor cell lines were treated with200 nmol/L AZD5153 for 24 hours before mRNA isolation. Thebottom left panel shows the impact of compound treatment onMYC. Its modulation in solid tumor cell lines was varied,whereas downregulation was consistently observed in themajority of the hematologic cells lines, as expected. The topleft and top right panels show similar data for CCR2 andCD180, respectively. Consistent with their immunologic roles,their baseline levels were minimal in the solid tumor cell linesbut often detectable in many of the hematologic cell lines.Notably, the downregulation of CCR2 mRNA levels was strik-ing in that its inhibition was often down to minimal levels ofdetection, regardless of initial baseline levels. Finally, thebottom right panel shows the data for HEXIM1. Unlike theother three readouts, upregulation of HEXIM1 by AZD5153was observed in all hematologic and solid tumor cell lines.

To understand earlier transcriptional responses, a subset of thehematologic cell lines was incubated with 200 nmol/L forAZD5153 for only 4 hours as part of the RNAseq study. Supple-mentary Fig S1 shows that even at this earlier time point,AZD5153 treatment could result in downregulation of CCR2,CD180, MYC and upregulation of HEXIM1.

Confirmation of CCR2 and CD180 as tumor biomarkers fromcellular and xenograft studies

To confirm results from the RNAseq study, which was per-formed at only one concentration of AZD5153, follow-up cellularstudies were performed. Figure 2 shows AZD5153 dose–responsecurves in theMV4,11AML cell line. These data not only confirmedthe downregulation of CCR2 and CD180 mRNAs by AZD5153but also supported the likelihood that robust modulation (>80%

CCR2 and CD180 mRNA as PD Biomarkers for BRD4 Inhibitors

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inhibition) of these transcripts would be detected at predictedclinically achievable concentrations. As expected, concentration-dependent decreases in MYC mRNA and increases in HEXIM1mRNA were also observed (data not shown).

Downregulation of CCR2 and CD180 mRNA in an in vivosetting was also evaluated. Figure 3A shows data from a MV4,11(AML) xenograftmodel wheremice (n¼ 3/group) were given onesingle oral dose of 5mg/kgAZD5153, a dose thatwhengivendailyresults in tumor regression in an efficacy study (11, 13). Tumorswere harvested at 4 hours after dose for RNA analysis. Consistentwith cell line data, CCR2 and CD180mRNAwere strongly down-regulated, i.e., >90% inhibition (top left, top right). MYC mRNAwas also inhibited�80%, andHEXIM1mRNA increased three- tofourfold (bottom left, bottom right).

The impact of sustained BRD4 inhibition from repeat dosingwas evaluated in a KMS-11 (ALL) xenograft model where mice(n ¼ 3/group) were repeatedly dosed for 3 days at 2.5 mg/kg b.

i.d., a dosing schedule that results in tumor regression (datanot shown). Tumors were harvested 4 hours after the lastdose for mRNA analysis. Significant downregulation of CCR2and CD180 mRNA was detected after six doses (Fig. 3B), withinhibition near or over 90%, compared with 60% to 70%for MYC (top left/right vs. bottom left). HEXIM1 mRNA wasupregulated by three- to fourfold (bottom right).

Altogether, these preclinical results support the monitoring ofCCR2 and CD180 transcripts, along with MYC and HEXIM1, todemonstrate PD impact of AD5153 in tumor biopsies fromhematologic malignances.

Evaluation of CCR2, CD180,MYC, andHEXIM1 aswhole bloodbiomarkers in rat

Given the limitations and challenges of paired patient tumorbiopsies, wewanted to find robust PDwhole blood biomarkers tocomplement the tumor biomarkers. Although many AZD5153-

Generate and analyze RNAseq data

Evaluate subset of candidates in heme cell lines/tumor models

Evaluate top candidates in whole blood

Treat cell lines and xenograft mice with AZD5153

Develop human whole blood PD biomarker assays

A

BFigure 1.

CCR2 and CD180 mRNAs areidentified as biomarker candidatesusing RNA-sequencing (RNAseq).A, Flowchart describing theprocess from generation ofRNAseq samples to identificationof biomarkers and developmentof whole blood assays for clinicaluse. B, RNAseq data from 15hematologic and 13 solid tumor celllines showing the downregulationof CCR2, CD180, and MYC and theupregulation of HEXIM1 mRNAupon 24-hour treatment of200 nmol/L AZD5153. Relativegene expression is displayed onthe y-axis as FPKM (Fragments PerKilobase of transcript per Millionmapped reads). Mean of triplebiological replicates is shown.Cell line cancer type is color codedas indicated at top of the figure.

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dependent transcripts were identified in the tumor RNAseq data-set, we prioritized CCR2 and CD180 because in whole blood, theextent of downmodulation by AZD5153 was striking, especiallywhen compared with MYC.

Initially, we looked at blood samples frommice thatwere orallydosed with AZD5153. Assay requirements and small mouseblood volumes that limited the ability to collect serial samplesconstrained the experiment to one mouse per time point.Although we did see a trend in the expected responses of all 4biomarker genes, the inter-mouse variabilitywas toohigh to showstatistical significance (data not shown). Because we had previ-ously demonstrated tumor growth inhibition in a rat xenograftMV4,11 model (data not shown), we shifted our efforts to ratblood.

Whole blood from normal, non–tumor-bearing rats were col-lected and treated in vitro with DMSO control or 1 mmol/LAZD5153 for 2 hours. RNA was isolated, reverse-transcribed, andanalyzed by qPCR. Figure 4A shows the impact of AZD5153treatment on CCR2, CD180, MYC, and HEXIM1mRNA in wholeblood. CCR2 and CD180 transcript levels were inhibited to agreater degree compared with MYC (75%–90% vs. 50%).HEXIM1 was increased by almost fourfold.

Next, we evaluated the four transcripts in an in vivo setting usingrats where multiple pretreament and posttreatment samplescould be collected from the same animal. Normal non–tumor-bearing rats (n ¼ 3) were treated with 1.08 mg/kg, an efficaciousdose resulting in tumor growth inhibition in the rat MV4,11xenograft model. After the first dose, blood samples were takenat pre-dose, 2 hours post, and 6 hours post-dose. In addition, tounderstand the impact of repeat dosing on these readouts, wecontinued to dose the rats daily for a total of 7 days. On the lastday, blood sampleswere also taken at pre-dose, 2 hours post and6hours post-last dose. Figure 4B shows that AZD5153 led to in vivodownregulation of CCR2, CD180, MYC, and upregulation ofHEXIM1 mRNAs in whole blood after one dose. As in the in vitrosetting, the impact on CCR2 was greater than that of MYC, with agreatermaximum inhibition of�80%at 2hours post-dose onday1 (top left). In addition, CCR2 continued to be downregulated tothe samedegree at 2 hours post-dose on day 7, demonstrating thatthe impact on CCR2 is maintained after both acute and repeatdosing of AZD5153. Myc modulation was modest and stayed ataround50% inhibition, even onday7 (bottom left).HEXIM1was

similar to both CCR2 andMYC in that its modulation was similarat days 1 and 7, with a greater induction of two- to threefold at 6hours post-dose (bottom right). Interestingly, the data suggestthat the impact on CD180 might be greater upon repeat dosing.

Prioritization of CCR2 and CD180 as human whole bloodbiomarkers for use in clinical trials

The overall goal of these studies was to identify biomarkers thatcan be used in the clinical setting to help demonstrate thatAZD5153 is engaging BRD4/BET proteins appropriately. Withthat goal, we proceeded to evaluate human whole blood. As aninitial pilot, we obtained blood from six healthy volunteers (HV)and treated the samples in vitro with DMSO control or 1 mmol/LAZD5153 for 2 hours. To parallel the planned processing at theclinical sites, 2.5 mL of blood was transferred and processed inclinically relevant PAXgene blood RNA tubes which enable easyblood collection and RNA stabilization. Figure 5A shows thatthe results were remarkably similar to what we had obtained forthe rat in vitro blood studies. AZD5153 treatment resulted indownregulation of CCR2, CD180, MYC, and upregulation ofHEXIM1 transcripts (three- to fourfold). As in rat blood, CD180and especially CCR2 were significantly more downregulatedcompared with MYC (80% inhibition vs. 60%). Next, we per-formed dose–response curves. Blood from another three HVs wasincubated with increasing concentrations of AZD5153, rangingfrom 1 nmol/L to 10 mmol/L. Figure 5B illustrates that CCR2/CD180 downregulation is greater than that of MYC, not just attotal blood concentrations �1 mmol/L but also at concentrationsas low as 100 nmol/L, supporting the likelihood to detect robustPD changes during a clinical dose escalation.

To build confidence that these biomarker readouts are sim-ilarly modulated in patients with cancer, we collected wholeblood from 11 patients with tumor indications ranging fromchronic lymphocytic leukemia (CLL), multiple myeloma(MM), lung, breast, and ovarian. To match the patient popu-lation to that of a clinical trial, we initially requested samplesfrom patients who were not currently receiving cancer treat-ment (i.e., pretreatment or follow-up visit) and not takingNSAIDS or steroids which might affect the transcripts indepen-dently. Due to these requirements, we were not able to collectsamples from any patients with AML. As a control, blood fromtwo HVs was also included in this study.

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CCR2 and CD180 gene expression issensitive to AZD5153 treatment inMV4,11 cells. Confirmation of RNAseqdata and evaluation of doseresponses. Cells were incubated for 4hours with increasing concentrationsof compound. RNA was isolated andanalyzed by qPCR methods. Mean �SD is shown for three biologicalreplicates.






Blood samples were treated in vitro with increasing concentra-tions of AZD5153 for 2 hours and processed externally as wasanticipated for the clinical setting. Dose–response curves for allfour transcripts were similar between these two HVs (Fig. 5C,dotted lines) and the previous three HVs (Fig. 5B), even thoughthe blood collection, compound treatment, and RNA isolation

steps were done at different places (externally vs. in-house). Inaddition, given the similarity of the curves between the HVs andthe first five patients with cancer, we decided to relax the patientrequirements for the remaining six patients so that there were norestrictions on co-medications or ongoing treatments; conse-quently, four patients on active cancer treatment were also

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CCR2 and CD180 mRNAs are robustlydownregulated in AML and ALL xenografttumors when AZD5153 is given at efficaciousdoses.A,Mice bearingMV4,11 xenograft tumorswere dosed with vehicle (n ¼ 3) or anefficacious dose of 5 mg/kg AZD5153 (n ¼ 3).At 4 hours post-dose, tumors were harvested.RNA was isolated and analyzed by qPCR (bars,left y-axis). Plasma samples were collected inparallel for PK analysis. Calculated freeconcentrations are shown here (diamonds,right y-axis). B, Mice bearing KMS11 xenografttumors were dosed twice daily for 3 dayswith vehicle (n ¼ 3) or an efficacious dose of2.5mg/kg AZD5153 (n¼ 3). At 4 hours after thelast dose, tumors and plasma samples werecollected and analyzed as above. In bothstudies, MYC and HEXIM1 mRNA were alsoevaluated, as indicated.

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included (Supplementary Table S3). Figure 5C shows the dosecurves for the 11 patients and the two HVs (dotted lines) for allfourmRNAs. The dose–response curves and IC50 values for the 11cancer patient samples were in line with what we observed forhealthy donors, further supporting the use of CCR2 and CD180mRNAs as whole blood PD biomarkers and providing additionalevidence that these readouts are likely more informative thanMYC mRNA in this setting.

Potential utility ofCCR2andCD180asPDbiomarkers for otherBRD4/BET inhibitors

To address whether the modulation of CCR2 and CD180mRNA is specific to AZD5153 or equally relevant to other BET/BRD4 inhibitors, whole blood from three HVs was treated with 1and 10 mmol/L of AZD5153, iBET762, or OTX-015 for 2 hours.Although AZD5153 has greater cellular potency, Fig. 6 shows thatother BRD4 compounds, when dosed at higher concentrations,can also modulate CCR2 and CD180, in addition to MYC andHEXIM1 mRNA, in the expected directions. Consistent withAZD5153 data, CCR2 is themost robust and sensitive biomarker.For iBET762 and OTX-015, 1 mmol/L treatment led to little or nomodulation ofCD180 (top right),MYC (bottom left), orHEXIM1(bottom right) mRNA, whereas inhibition of CCR2 (top left)mRNA was �50%. At 10 mmol/L for all three compounds, incontrast to MYC and CD180, inhibition of CCR2 mRNA wasalmost complete.

We also have data from the RNAseq dataset, which supportthe modulation of these readouts by other BRD4 inhibitors.For three of the 15 hematologic tumor cell lines, we included a24-hour incubation with JQ-1 (500 nmol/L) and iBET762(2000 nmol/L), in addition to AZD5153 (200 nmol/L). Theseconcentrations were chosen to reflect similar cellular prolifer-ation activities in two cell lines, MV4,11 and MM1.S. At thesematched concentrations, CCR2 and CD180 transcriptionalresponses to JQ-1 and iBET762 were similar to AZD5153(Supplementary Fig. S2).

Overall, these data support that downmodulation of CCR2 andCD180 mRNA is not specific to AZD5153 and is, instead, areflection of downstream biology from BRD4/BET inhibition. Wewould thus expect that these readouts could also be used todemonstrate target engagement/pathway modulation for otherBRD4 inhibitors.

DiscussionWe identified CCR2 and CD180 mRNA as PD biomarkers for

AZD5153with high potential for clinical utility withwhole bloodand hematologic tumor samples. CCR2 and CD180 are bothproteins with immune functions. The downmodulation of theirtranscripts with AZD5153 treatment is consistent with multiplereports describing the anti-inflammatory effects of BRD4 com-pounds (24–26). For example, disruption of BET protein function

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In vitro and in vivo data from ratstudies support the use of CCR2 andCD180 mRNAs as whole blood PDbiomarkers. A, In vitro incubation ofwhole blood from three individualrats with 0.1% DMSO or 1 mmol/LAZD5153 for 2 hours, followed byRNA isolation and qPCR analysis. B,Analysis of blood samples from ratsorally dosed with an efficacious dose(1.08 mg/kg). Consecutive bloodsamples from normal female HansWistar rats (n¼ 3) were taken at pre-dose, 2 hours post-dose, and 6 hourspost-dose on days 1 and 7 of repeatdaily dosing. Mean � SD is shownhere.






by siRNA or JQ-1 resulted in reduced macrophage inflammatoryresponses, as demonstrated by the strong inhibition of LPS-induced cytokines in cellular and in vivomodels (27). CCR2, alsoknown as chemokine (C-C motif) receptor 2, is a receptor for themonocyte chemoattractant protein-1 (CCL2), which serves torecruit monocytes to sites of inflammation (28). Interestingly,suggestionof its role in tumorprogression leads to the speculationthat CCR2 downregulation might help contribute to the overallantitumor activity of AZD5153 andother BRD4 compounds (29).CD180 is related to the Toll-like receptor family involved in innateimmunity and can collaborate with TLR4 to promote inflamma-tory responses to LPS (30).

Consistent with their immune roles, we observed adequate-to-high expression levels of CCR2 and CD180 mRNA in manyof the hematologic cell lines, whereas their expression was low-to-undetectable in the solid tumor cell lines, emphasizing that

their use as tumor PD biomarkers is most applicable to hema-tologic cancers. Because the most direct impact of BRD4 inhi-bition is on gene transcription, we focused our efforts onidentifying mRNA transcripts as PD biomarkers. Our limitedevaluation of CCR2 and CD180 protein levels in AZD5153-treated cells did not show any results as striking as the mRNAlevels, although we did not look at longer time points beyond 4hours or optimize antibodies. Further investigation is requiredto understand the exact relationship between BET bromodo-main inhibition and CCR2/CD180 transcriptional regulation.Interestingly, CD180 was also previously identified as one ofthe top 20 downregulated genes in JQ-1-treated LP-1 and Rajitumor cells (14).

In addition to CCR2 and CD180, we observed consistentinhibition of MYC mRNA across multiple hematologic tumorcell lines, but not solid tumor cell lines. These results, along with

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In vitro studies with whole blood fromhealthy volunteers and patients withcancer support the use of CCR2 andCD180 mRNA in the clinical setting. A,Whole blood from six healthy donorswas incubated with 0.1% DMSO or 1mmol/L AZD5153 for 2 hours. Blood wastransferred to a PAXgene tube,followed by RNA isolation and qPCRanalysis using GAPDH as HKG.Mean � SD is shown here.B, Dose–response curves weregenerated in blood from threeadditional healthy volunteer donors.Whole blood was incubated withincreasing concentrations of compoundfor 2 hours and processed as above.Each curve corresponds to one donor.Data were normalized to GAPDH.(Continued on the following page.)

Yeh et al.





prior reports from ourselves and other groups, support the use ofMYC mRNA as a tumor PD biomarker for hematologic tumors(11, 13–15). Interestingly, in the MV4,11 and KMS11 models,inhibition of CCR2 and CD180 mRNA was greater than that ofMYC.We also saw reasonable induction of HEXIM1mRNA, up tothree- to fourfold, inAZD5153-treated tumor cells and xenografts.Increased HEXIM1 is consistent with disruption of BRD4 bindingin that both events result in decreased P-TEFb recruitment andsubsequently, reduced RNA polymerase II transcriptional activity(20). Importantly, this increase occurs in both hematologic andsolid tumor cell lines,makingHEXIM1mRNA an attractive tumorPD target that could be used across all tumor types. However, themain challenge in the clinical setting will be the degree ofinduction observed in paired biopsies and whether the signal isdetected above baseline HEXIM1 levels.

Our primary goal was to identify and develop assays forrobust PD biomarkers that could be used with whole bloodsamples in the clinic, especially for the solid tumor settingwhere robust tumor PD biomarkers are not so apparent. Fromthis perspective, CCR2 and CD180 are extremely attractivebecause in contrast to MYC, near-complete inhibition of thesetranscripts was observed at concentrations of AZD5153 that arepredicted to be clinically achievable. In addition, the analysis ofwhole blood mRNA is more clinically amenable in that patientblood can be directly drawn into a vaccutainer tube, i.e.,PAXgene and stored with minimal need for additional proces-sing at the clinical sites.

To address whether CCR2 and CD180 would remain infor-mative in the oncology setting, we evaluated the impact ofAZD5153 on these transcripts in blood samples from patientswith cancer. Baseline transcript levels as well as dose–responsecurves were generally overlapping with those from healthy

volunteers. What was especially insightful was the observationthat even blood samples taken from patients who were onactive anticancer treatment and/or potentially taking multipledifferent types of medications for comorbidities all respondedsimilarly, especially for CCR2. These results also include bloodfrom CLL and MM patients, where significant tumor cells werelikely to be present, which is consistent with our observationsthat these transcripts are similarly modulated in hematologictumors as in whole blood. Because MYC downregulation is verysensitive in hematologic tumor cell lines (but not in wholeblood), a high percentage of tumor cells in the whole bloodsample may explain the potent MYC IC50 observed in onepretreatment CLL patient (patient 7; Supplementary Table S3).These results strongly support monitoring CCR2 and CD180mRNA in patient blood samples (i.e., for both solid andhematologic tumors), as well as MYC mRNA when significanttumors cells are likely present (i.e., certain hematologic indica-tions). HEXIM1 mRNA could be informative but more likely athigher concentrations where transcript increases are greaterthan twofold. Finally, the in vivo rat study, which showed robustdownregulation of CCR2 and CD180 even after 7 days of repeatdosing, supports their utility in whole blood beyond the firstday of dosing in the clinic.

In summary, we confirmed downregulation of MYCmRNA asa tumor PD biomarker for hematologic tumors and upregula-tion of HEXIM1 mRNA as a tumor PD biomarker for bothhematologic and solid tumors. We also evaluated their rolesas PD biomarkers in whole blood. More importantly, we iden-tified CCR2 and CD180 mRNA as superior whole blood PDbiomarkers with high potential for clinical success due tothe magnitude of downregulation by BRD4 inhibition and,consequently, the likelihood to overcome intrapatient sample

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(Continued. ) C, Dose–responsecurves were generated in wholeblood from two healthy volunteers(HV, dotted lines) and 11 oncologypatients with different cancer typesas indicated. See SupplementaryTable S3 for more patient details.Whole blood was treated withcompound within 2 hours ofcollection. Data was normalized toRPL13A after HKG optimizationstudies were conducted for theclinical setting.






variability. The use of CCR2 and CD180 mRNA as PD biomar-kers can also be extended to hematologic tumors. Finally, weshowed that downregulation of CCR2 and CD180 mRNAextends beyond AZD5153 and is a direct consequence of BETbromodomain inhibition. The clinical utility of these biomar-kers for other BRD4 compounds will largely depend on indi-vidual cellular potencies and clinically achievable concentra-tions, but the preclinical data clearly support that CCR2 inwhole blood would be the best biomarker to prioritize todemonstrate target engagement. Altogether, our studies dem-onstrate the utility of evaluating CCR2 and CD180 mRNA,along with MYC and HEXIM1, as whole blood and tumor PDreadouts for BRD4 compounds in the clinical setting.

Disclosure of Potential Conflicts of InterestT.C. Yehhas ownership interests (including patents) inAstraZeneca. A.Dulak

reports receiving commercial research grants from AstraZeneca. H. Chen hasownership interest (including patents) in and reports receiving commercialresearch grants from AstraZeneca. No potential conflicts of interest were dis-closed by the other authors.

Authors' ContributionsConception and design: T.C. Yeh, G. O'Connor, A. Dulak, J.C. Barrett, H. ChenDevelopment of methodology: T.C. Yeh, G. O'Connor, M. Hattersley,J.C. BarrettAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T.C. Yeh, G. O'Connor, P. Petteruti, M. Hattersley,J.C. BarrettAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T.C. Yeh, G. O'Connor, P. Petteruti, A. Dulak,M. Hattersley, J.C. Barrett, H. ChenWriting, review, and/or revision of the manuscript: T.C. Yeh, G. O'Connor,P. Petteruti, A. Dulak, M. Hattersley, J.C. Barrett, H. ChenAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): T.C. YehStudy supervision: T.C. Yeh, M. Hattersley, J.C. Barrett, H. Chen

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received July 4, 2016; accepted August 22, 2016; published OnlineFirstJanuary 10, 2017.

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Other BRD4/BET compounds can alsodownregulate CCR2 and CD180mRNAs in whole blood. Whole bloodfrom three healthy volunteers wasincubatedwith0.1%DMSO, 1mmol/L or10mmol/LOTX-015/MK8628, iBET762,and AZD5153 for 2 hours. RNA wasisolated and analyzed for CCR2,CD180, myc and HEXIM1 transcripts byqPCR, using RPL13A as HKG. Mean �SD is shown here.


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2017;23:1025-1035. Published OnlineFirst January 10, 2017.Clin Cancer Res Tammie C. Yeh, Greg O'Connor, Philip Petteruti, et al. InhibitorsTumor and Blood Biomarkers for Clinical Use with BRD4/BET Identification of CCR2 and CD180 as Robust Pharmacodynamic

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